Regras Entre Assembléias De Espécies: Relação Entre Biodiversidade E Funcionamento Do Ecossistema

Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Open letter. DOUTORADO EM CIÊNCIAS

BIOLÓGICAS

REGRAS ENTRE

ASSEMBLÉIAS DE ESPÉCIES:

RELAÇÃO ENTRE

BIODIVERSIDADE E

FUNCIONAMENTO DO

ECOSSISTEMA.

GIOVANNI AMADEU PAIVA DOS SANTOS

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Sub-Cover. DOUTORADO EM CIÊNCIAS BIOLÓGICAS Universidade Federal de Pernambuco Doutorado em Ciências Biológicas Centro de Ciências Biológicas

Regras entre assembléias de espécies: Relação entre biodiversidade e funcionamento do ecossistema.

Species assembly rules and the biodiversity- ecosystem functioning relationship.

Giovanni Amadeu Paiva dos Santos

Tese apresentada ao Doutorado de

Ciências Biológicas da UFPE, como requisito necessário para o recebimento do título de Doutor em

Ciências Biológicas.

RECIFE, JULHO DE 2007.

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007.

1.1.1 1.1.2

1.1.3 Santos, Giovanni Amadeu Paiva dos Regras entre assembléias de espécies: relação entre biodiversidade e

funcionamento do ecossistema / Giovanni Amadeu Paiva dos Santos. – Recife: O Autor, 2009.

263 folhas: fig., tab.

Tese (doutorado) – Universidade Federal de Pernambuco. CCB. Departamento de Ciências Biológicas, 2009.

1.1.3.1.1.1 Inclui bibliografia.

1. Biodiversidade 2. Funcionamento dos ecossistemas I Título.

574 CDU (2.ed.) UFPE 1.2 577 CDD (22.ed.) CCB – 2009- 043

COMISSAO EXAMINADORA

"Regras entre assemblfHas de especies: rela~ao entre biodiversidade e funcionamento do ecossistema"

TITULARES

,Ore c'~~ l .l;/'>1i( Ji /',- C'"'-~" -- ia Tereza dos Santos Correia (OrientadorIlJFPE)

/~-/z:; , ~ Prof. L Ricardo Coutinho - (IEAPM/RJ)

om Gilbert Willem Moens (Ghent UniversityIBelgica)

SUPLENTES

, " ' '/l{ ) c::< \, /,_k".. t,,, (/~/, C . c::, c, ;'v'" ("", '!1 ","" '" pror,. Dra Vera Lticia de Menezes Lima (UFPE)

~~~ pror, Dra Veronica Gomes da Fonseca Genevois (UFPE) Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Sub-Cover. DOUTORADO EM CIÊNCIAS BIOLÓGICAS Universidade Federal de Pernambuco Doutorado em Ciências Biológicas Centro de Ciências Biológicas

Regras entre assembléias de espécies: Relação entre biodiversidade e funcionamento do ecossistema.

Aluno: Professor Orientador: Giovanni Amadeu Paiva dos Santos Dra. Maria Tereza dos Santos Correia Departamento de Bioquímica -UFPE

Professores Co- Orientador: Dra. Verônica da Fonsêca- Genevois Departamento de Zoologia – UFPE

Dra. Luana Cassandra Breitenbach Barros Coelho Departamento de Bioquímica –UFPE

Dr. Tom Moens Marine Section, University of Gent, Bélgica.

RECIFE, JULHO DE 2007.

III

Thankfullness

A DEUS…

TO GOD…

…POR TODAS AS BENÇÃOS QUE ELE ME OFERTOU NESTA JORNADA.

…FOR ALL BLESSINGS THAT HE PROVIDE ME IN THIS JURNEY.

Acknowledgements

It is a motive of pride and satisfaction to have arrived to this point in my career and that is the reason why I would like to thank from the bottom of my heart those who have helped me in my journey, without no exception, I’m thankful to the one’s how gave me their hands or even their back, but in the end all of you made me stronger.

I am very grateful to God, who gave me strength to get ahead in life.

Thanks to my parents, João Adolfo e Eliane Paiva, for helping me to achieve personal fulfilment. Thanks for making it possible my stay in Europe and thanks for those uncountable nights we spent talking side to side by the computer.

Thanks to my brothers, Gustavo Adolfo and George Augusto, and their wives and sun for being always concerned about me and for turning the distance into a minor gap.

My heartily gratitude to my cousins, for being there when I needed them, and always remember me.

Thanks to my family, who has always supported me in everything I have done. Without their help and their wise pieces of advice I could not have been able to get here.

I would like to express my gratitude to Prof. Dr. M. Vincx, Head of the Marine Department and all my colleagues for sharing with me good and crucial moments.

I would like to extend my gratitude to all Professors and Lecturers during the first year of this PhD.

My special thanks for the incomparable friendship capacity of Prof. Dr. Tom Moens, for his uncountable valuable bits of advice during the process of my thesis.

Thanks for his patience, his constructive critics and his kindness. This extends to his family for the comprehensive capacity of everyone.

I do not have to forget to thank the technicians, some of you know me more then my own colleagues, thanks for everything you did things easier to me.

My sincere thanks to my co-promoters and promoter by sure your names mean much more than you can imagine, you have been guidance of new scientific carrier. Thank you for encouraging me to take this PhD and thanks for your valuable pieces of advice, and especially to the fully trust before during and after.

I would also like to thank all the Brazilians friends with no exception for having been always willing to help me, and also for the incredible moments away from home.

Thanks also to Tania Nara and Pierre Misseghers for the good moments spent in Belgium and for their willingness to help in everything.

Thanks my Brazilian colleagues helping me on my away labs needs.

To Gustavo Fonseca and Fabiane Gallucci for the funny and scientific moments shared.

Thanks to the ‘Portuguese Colony’ in Gent, especially to Maria, for being a real help in the lab, as much as a good friend.

Thanks to all my friends in Brazil, your e-mails, letters, MSN messages and telephone calls were really helpful. I will not say names because I don’t want to forget anyone of you.

Thanks to my teacher-friends in Brazil for the memories and candidly advice, they were always there for whatever I wanted to.

I am very grateful to all the students of the new PINC for their continuous enthusiasm helping me dialling with the distance away from home and also for the old PINC friends especially Caroline Mouton I just receive a strength message in the last moment of preparations.

A special thanks to the one’s that close or far helped me. Some of you I would like to keep anonymous for personal purposes.

Thanks to Luiz Naveda, Tatiana Maria, Isabel Eyre, Yirina Valdes, Mariuxe, Lucia and Julie Reveillaud for corrections and advices on this work. Tatiana has been an incredible friend and helping me in most of the work. Thanks for everything, you have taught me a lot in life and feelings.

Thanks also to all my friends in Gent: all the Spaniards with whom I lived, the Italians, Greeks, Belgians, Hungarians, Austrians, etc. They opened my mind to other cultures, other realities and other ways of life.

I have left for least but not least to the Latin community, all of you have been very helpful to my wellness. In special to Yirina Valdes for my inclusion on the Cuban community and all the incredible and special moments shared.

VII

Thought

UM DIA VOCÊ APRENDE QUE...

"DEPOIS DE ALGUM TEMPO VOCÊ APRENDE A DIFERENÇA, A SUTIL DIFERENÇA, ENTRE DAR A MÃO E ACORRENTAR UMA ALMA. E VOCÊ APRENDE QUE AMAR NÃO SIGNIFICA APOIARSE, E QUE COMPANHIA NEM SEMPRE SIGNIFICA SEGURANÇA. E COMEÇA A APRENDER QUE BEIJOS NÃO SÃO CONTRATOS E PRESENTES NÃO SÃO PROMESSAS. E COMEÇA A ACEITAR SUAS DERROTAS COM A CABEÇA ERGUIDA E OLHOS ADIANTE, COM A GRAÇA DE UM ADULTO E NÃO COM A TRISTEZA DE UMA CRIANÇA. E APRENDE A CONSTRUIR TODAS AS SUAS ESTRADAS NO HOJE, PORQUE O TERRENO DO AMANHÃ É INCERTO DEMAIS PARA OS PLANOS, E O FUTURO TEM O COSTUME DE CAIR EM MEIO AO VÃO. DEPOIS DE UM TEMPO VOCÊ APRENDE QUE O SOL QUEIMA SE FICAR EXPOSTO POR MUITO TEMPO. E APRENDE QUE NÃO IMPORTA O QUANTO VOCÊ SE IMPORTE, ALGUMAS PESSOAS SIMPLESMENTE NÃO SE IMPORTAM... E ACEITA QUE NÃO IMPORTA QUÃO BOA SEJA UMA PESSOA, ELA VAI FERILO DE VEZ EM QUANDO E VOCÊ PRECISA PERDOÁLA POR ISSO. APRENDE QUE FALAR PODE ALIVIAR DORES EMOCIONAIS. DESCOBRE QUE SE LEVA ANOS PARA SE CONSTRUIR CONFIANÇA E APENAS SEGUNDOS PARA DESTRUÍLA, E QUE VOCÊ PODE FAZER COISAS EM UM INSTANTE, DAS QUAIS SE ARREPENDERÁ PELO RESTO DA VIDA. APRENDE QUE VERDADEIRAS AMIZADES CONTINUAM A CRESCER MESMO A LONGAS DISTÂNCIAS. E O QUE IMPORTA NÃO É O QUE VOCÊ TEM NA VIDA, MAS QUEM VOCÊ É NA VIDA. E QUE BONS AMIGOS SÃO A FAMÍLIA QUE NOS PERMITIRAM ESCOLHER. APRENDE QUE NÃO TEMOS QUE MUDAR DE AMIGOS SE COMPREENDEMOS QUE OS AMIGOS MUDAM, PERCEBE QUE SEU MELHOR AMIGO E VOCÊ PODEM FAZER QUALQUER COISA, OU NADA, E TEREM BONS MOMENTOS JUNTOS. DESCOBRE QUE AS PESSOAS COM QUEM VOCÊ MAIS SE IMPORTA NA VIDA SÃO TOMADAS DE VOCÊ MUITO DEPRESSA, POR ISSO SEMPRE DEVEMOS DEIXAR AS PESSOAS QUE AMAMOS COM PALAVRAS AMOROSAS, POIS PODE SER A ÚLTIMA VEZ QUE AS VEJAMOS. APRENDE QUE AS CIRCUNSTÂNCIAS E OS AMBIENTES TEM INFLUÊNCIA SOBRE NÓS, MAS NÓS SOMOS RESPONSÁVEIS POR NÓS MESMOS. COMEÇA A APRENDER QUE NÃO SE DEVE COMPARARSE COM OS OUTROS, MAS COM O MELHOR QUE VOCÊ MESMO PODE SER. DESCOBRE QUE SE LEVA MUITO TEMPO PARA SE TORNAR A PESSOA QUE QUER SER, E QUE O TEMPO É CURTO. APRENDE QUE NÃO IMPORTA ONDE JÁ CHEGOU, MAS ONDE ESTÁ INDO, MAS SE VOCÊ NÃO SABE PARA ONDE ESTÁ INDO, QUALQUER LUGAR SERVE. VIII

APRENDE QUE, OU VOCÊ CONTROLA SEUS ATOS OU ELES O CONTROLARÃO, E QUE SER FLEXÍVEL NÃO SIGNIFICA SER FRACO OU NÃO TER PERSONALIDADE, POIS NÃO IMPORTA QUÃO DELICADA E FRÁGIL SEJA UMA SITUAÇÃO, SEMPRE EXISTEM DOIS LADOS. APRENDE QUE HERÓIS SÃO PESSOAS QUE FIZERAM O QUE ERA NECESSÁRIO FAZER, ENFRENTANDO AS CONSEQÜÊNCIAS. APRENDE QUE PACIÊNCIA REQUER MUITA PRÁTICA. DESCOBRE QUE ALGUMAS VEZES A PESSOA QUE VOCÊ ESPERA QUE O CHUTE QUANDO VOCÊ CAI É UMA DAS POUCAS QUE O AJUDAM A LEVANTARSE. APRENDE QUE MATURIDADE TEM MAIS A VER COM OS TIPOS DE EXPERIÊNCIA QUE SE TEVE E O QUE VOCÊ APRENDEU COM ELAS DO QUE COM QUANTOS ANIVERSÁRIOS VOCÊ CELEBROU. APRENDE QUE HÁ MAIS DOS SEUS PAIS EM VOCÊ DO QUE VOCÊ SUPUNHA. APRENDE QUE NUNCA SE DEVE DIZER A UMA CRIANÇA QUE SONHOS SÃO BOBAGENS, POIS POUCAS COISAS SÃO TÃO HUMILHANTES E SERIA UMA TRAGÉDIA SE ELA ACREDITASSE NISSO. APRENDE QUE QUANDO VOCÊ, ESTÁ COM RAIVA TEM O DIREITO DE ESTAR OM RAIVA, MAS ISSO NÃO LHE DÁ O DIREITO DE SER CRUEL. DESCOBRE QUE SÓ PORQUE ALGUÉM NÃO O AMA DO JEITO QUE VOCÊ QUER QUE AME, NÃO SIGNIFICA QUE ESSE ALGUÉM NÃO O AMA, POIS EXISTEM PESSOAS QUE NOS AMAM, MAS SIMPLESMENTE NÃO SABEM COMO DEMONSTRAR ISSO. APRENDE QUE NEM SEMPRE É SUFICIENTE SER PERDOADO POR ALGUÉM, ALGUMAS VEZES VOCÊ TEM QUE APRENDER A PERDOAR A SI MESMO. APRENDE QUE COM A MESMA SEVERIDADE COM QUE JULGA, VOCÊ SERÁ EM ALGUM MOMENTO CONDENADO. APRENDE QUE NÃO IMPORTA EM QUANTOS PEDAÇOS SEU CORAÇÃO FOI PARTIDO, O MUNDO NÃO PÁRA PARA QUE VOCÊ O CONSERTE. APRENDE QUE O TEMPO NÃO É ALGO QUE POSSA VOLTAR PARA TRÁS. PORTANTO, PLANTE SEU JARDIM E DECORE SUA ALMA, AO INVÉS DE ESPERAR QUE ALGUÉM LHE TRAGA FLORES. E VOCÊ APRENDE QUE REALMENTE PODE SUPORTAR... QUE REALMENTE É FORTE, E QUE PODE IR MUITO MAIS LONGE DEPOIS DE PENSAR QUE NÃO SE PODE MAIS. E QUE REALMENTE A VIDA TEM VALOR, E QUE VOCÊ TEM VALOR DIANTE DA VIDA! NOSSAS DÚVIDAS SÃO TRAIDORAS E NOS FAZEM PERDER O BEM QUE PODERÍAMOS CONQUISTAR, SE NÃO FOSSE O MEDO DE TENTAR."

(EXCERTO DE TEXTO DE AUTOR INCERTO. ATRIBUÍDO A VERÓNICA A. SHOFFSTALL, JUDITH B. EVANS OU MESMO WILLIAN SHAKESPEARE)

ONE DAY YOU’LL LEARN...

AFTER SOME TIME YOU LEARN THE DIFFERENCE, THE SUBTLE DIFFERENCE BETWEEN HOLDING A HAND AND CHAINING A SOUL. AND YOU LEARN THAT LOVE DOESN'T MEAN LEANING, AND COMPANY DOESN'T ALWAYS MEAN SECURITY. AND YOU BEGIN TO LEARN THAT KISSES AREN'T CONTRACTS, AND PRESENTS AREN'T PROMISES. AND YOU BEGIN TO ACCEPT YOUR DEFEATS, WITH YOUR HEAD UP AND YOUR EYES AHEAD, WITH THE GRACE OF A WOMAN, NOT THE GRIEF OF A CHILD. AND YOU LEARN TO BUILD ALL YOUR ROADS ON TODAY, BECAUSE TOMORROW'S GROUND IS TOO UNCERTAIN FOR PLANS, AND FUTURES HAVE A WAY OF FALLING DOWN IN MIDFLIGHT. AFTER A WHILE YOU LEARN, THAT EVEN THE SUN BURNS IF YOU GET TOO MUCH, AND LEARN THAT IT DOESN'T MATTER HOW MUCH YOU DO CARE ABOUT, SOME PEOPLE SIMPLY DON'T CARE AT ALL. AND YOU ACCEPT THAT IT DOESN'T MATTER HOW GOOD A PERSON IS, SHE WILL HURT YOU ONCE IN A WHILE, AND YOU NEED TO FORGIVE HER FOR THAT. YOU LEARN THAT TALKING CAN RELIEVE EMOTIONAL PAIN. YOU DISCOVER THAT IT TAKES SEVERAL YEARS TO BUILD A RELATIONSHIP BASED ON CONFIDENCE, AND JUST A FEW SECONDS TO DESTROY IT. AND THAT YOU CAN DO SOMETHING JUST IN AN INSTANT, AND WHICH YOU WILL REGRET FOR THE REST OF YOUR LIFE. YOU LEARN THAT THE TRUE FRIENDSHIPS, CONTINUE TO GROW EVEN FROM MILES AWAY. AND THAT WHAT MATTERS ISN'T WHAT YOU HAVE IN YOUR LIFE, BUT WHO YOU HAVE IN YOUR LIFE. AND THOSE GOOD FRIENDS ARE THE FAMILY, WHICH ALLOWS US TO CHOOSE. YOU LEARN THAT WE DON'T HAVE TO SWITCH OUR FRIENDS, IF WE UNDERSTAND THAT FRIENDS CAN ALSO CHANGE. YOU REALIZE THAT YOU ARE YOUR BEST FRIEND, AND THAT YOU CAN DO ANYTHING, OR NOTHING, AND HAVE GOOD MOMENTS TOGETHER. YOU DISCOVER THAT THE PEOPLE WHO YOU MOST CARE ABOUT IN YOUR LIFE, ARE TAKEN FROM YOU SO QUICKLY, SO WE MUST ALWAYS LEAVE THE PEOPLE WHO WE CARE ABOUT WITH LOVELY WORDS, IT MAY BE THE LAST TIME WE SEE THEM. YOU LEARN THAT THE CIRCUNSTANCES AND THE ENVIROMENT HAVE INFLUENCE UPON US, BUT WE ARE RESPONSIBLE FOR OURSELVES. YOU START TO LEARN THAT YOU SHOULD NOT COMPARE YOURSELF WITH OTHERS, BUT WITH THE BEST YOU CAN BE. YOU DISCOVER THAT IT TAKES A LONG TIME TO BECOME THE PERSON YOU WISH TO BE,

AND THAT THE TIME IS SHORT. YOU LEARN THAT IT DOESN'T MATTER WHERE YOU HAVE REACHED, BUT WHERE YOU ARE GOING TO. BUT IF YOU DON'T KNOW WHERE YOU ARE GOING TO, ANYWHERE WILL DO. YOU LEARN THAT EITHER YOU CONTROL YOUR ACTS, OR THEY SHALL CONTROL YOU. AND THAT TO BE FLEXIBLE DOESN'T MEAN TO BE WEAK OR NOT TO HAVE PERSONALITY, BECAUSE IT DOESN'T MATTER HOW DELICATE AND FRAGILE THE SITUATION IS, THERE ARE ALWAYS TWO SIDES. YOU LEARN THAT HEROES ARE THOSE WHO DID WHAT WAS NECESSARY TO BE DONE, FACING THE CONSEQUENCES. YOU LEARN THAT PATIENCE DEMANDS A LOT OF PRACTICE. YOU DISCOVER THAT SOMETIMES, THE PERSON WHO YOU MOST EXPECT TO BE KICKED BY WHEN YOU FALL, IS ONE OF THE FEW WHO WILL HELP YOU TO STAND UP. YOU LEARN THAT MATURITY HAS MORE TO DO WITH THE KINDS OF EXPERIENCES YOU HAD AND WHAT YOU HAVE LEARNED FROM THEM, THAN HOW MANY BIRTHDAYS YOU HAVE CELEBRATED. YOU LEARN THAT THERE ARE MORE FROM YOU PARENTS INSIDE YOU THAN YOU THOUGHT. YOU LEARN THAT WE SHALL NEVER TELL A CHILD THAT DREAMS ARE SILLY, VERY FEW THINGS ARE SO HUMILIATING, AND IT WOULD BE A TRAGEDY IF SHE BELIVED IN IT. YOU LEARN THAT WHEN YOU ARE ANGRY, YOU HAVE THE RIGHT TO BE ANGRY, BUT THIS DOESN'T GIVE YOU THE RIGHT TO BE CRUEL. YOU DISCOVER THAT ONLY BECAUSE SOMEONE DOESN'T LOVE YOU THE WAY YOU WOULD LIKE HER TO, IT DOESN'T MEAN THAT THIS PERSON DOESN'T LOVE YOU THE MOST SHE CAN, BECAUSE THERE ARE PEOPLE WHO LOVE US, BUT JUST DON'T KNOW HOW TO SHOW OR LIVE THAT. YOU LEARN THAT SOMETIMES IT ISN'T ENOUGH BEING FORGIVEN BY SOMEONE, SOMETIMES YOU HAVE TO LEARN HOW TO FORGIVE YOURSELF. YOU LEARN THAT WITH THE SAME HARSHNESS YOU JUDGE, SOME DAY YOU WILL BE CONDEMNED. YOU LEARN THAT IT DOESN'T MATTER IN HOW MANY PIECES YOUR HEART HAS BEEN BROKEN, THE WORLD DOESN'T STOP FOR YOU TO FIX IT. YOU LEARN THAT TIME ISN'T SOMETHING YOU CAN TURN BACK, THEREFORE YOU MUST PLANT YOUR OWN GARDEN AND DECORATE YOUR OWN SOUL, INSTEAD OF WAITING FOR SOMEONE TO BRING YOU FLOWERS. AND YOU LEARN THAT YOU REALLY CAN ENDURE. YOU REALLY ARE STRONG . AND YOU CAN GO SO FARTHER THAN YOU THOUGT YOU COULD GO. AND THAT LIFE REALLY HAS A VALUE. AND YOU HAVE VALUE WITHIN THE LIFE. AND THAT OUR GIFTS ARE BETRAYERS, AND MAKE US LOSE

THE GOOD WE COULD CONQUER, IF IT WASN'T FOR THE FEAR OF TRYING.

SOME CREDIT TO SHAKESPEARE; HOWEVER, AUTHORSHIP IS DISPUTED BETWEEN VERONICA A. SHOFFSTALL AND JUDITH B. EVANS.

XII

Título: Regras entre assembléias de espécies: Relação entre biodiversidade e funcionamento do ecossistema.

Giovanni A. P. dos Santos

Index

THANKFULLNESS ...... IV

ACKNOWLEDGEMENTS...... V

THOUGHT...... VIII

TÍTULO: REGRAS ENTRE ASSEMBLÉIAS DE ESPÉCIES: RELAÇÃO ENTRE BIODIVERSIDADE E FUNCIONAMENTO DO ECOSSISTEMA...... 13

INDEX ...... 13

ABSTRACT...... 16 RESUMO ...... 19 RESUME ...... 23 RESUMEN ...... 27

CHAPTER 1: INTRODUCTION ...... 31

CHAPTER 2: LITERATURE REVIEW ...... 34

FREE-LIVING NEMATODES ...... 34 FEEDING STRATEGIES...... 42 TROPHIC INTERACTIONS...... 47 DIRECT VS INDIRECT ECOLOGICAL INTERACTIONS ...... 53 DIRECT EFFECTS...... 53 INDIRECT EFFECTS ...... 55 TOP-DOWN/BOTTOM-UP REGULATION...... 56

CHAPTER 3: AIMS...... 59

CHAPTER 4: JUSTIFICATIONS...... 61

GENERAL INFORMATION...... 61 BACTERIVOROUS NEMATODES ASSOCIATED FOOD SOURCES...... 62 CULTIVATION OF BACTERIVOROUS NEMATODES ...... 63 BACTERIA ...... 64 THE MODEL SYSTEM...... 65 REFERENCES: ...... 67

CHAPTER 5: DO NEMATODE MUCUS SECRETIONS AFFECT BACTERIAL GROWTH? ...97 ABSTRACT...... 98 INTRODUCTION...... 100 MATERIALS AND METHODS...... 102 RESULTS AND DISCUSSION...... 105 ACKNOWLEDGEMENTS...... 112 LITERATURE CITED...... 113

CHAPTER 6: DIFFERENTIAL EFFECTS OF FOOD AVAILABILITY ON POPULATION DEVELOPMENT AND FITNESS OF THREE SPECIES OF ESTUARINE, BACTERIAL-FEEDING NEMATODES...... 117

ABSTRACT...... 118 INTRODUCTION...... 120 MATERIALS AND METHODS ...... 126 NEMATODE CULTURE ...... 126 EXPERIMENTS...... 127 DATA ANALYSIS ...... 129 RESULTS...... 131 PELLIODITIS MARINA...... 131 DIPLOLAIMELLOIDES MEYLI...... 135 DIPLOLAIMELLOIDES OSCHEI...... 139 DISCUSSION...... 144 ‘ENRICHMENT OPPORTUNISTS’ VS ‘GENERAL OPPORTUNISTS’ ...... 144 FOOD THRESHOLDS ...... 145 LIFE HISTORY, POPULATION INCREASE AND FITNESS...... 147 CONCLUSIONS...... 151 ACKNOWLEDGEMENTS...... 152 REFERENCES...... 153

CHAPTER 7: 1 + 1 + 1 = OR ≠ 3 ? THREE BACTERIAL-FEEDING NEMATODE SPECIES INDIRECT INTERACTIONS AND THEIR IMPLICATIONS FOR POPULATION DEVELOPMENT...... 159

ABSTRACT...... 160 INTRODUCTION...... 162 MATERIALS AND METHODS ...... 165 NEMATODE CULTURE ...... 165 POPULATION DEVELOPMENT ...... 165 TAXIS TOWARDS ESCHERICHIA COLI ...... 167 DATA ANALYSIS ...... 168 14

RESULTS...... 170 PELLIODITIS MARINA...... 170 DIPLOLAIMELLOIDES MEYLI...... 172 DIPLOLAIMELLOIDES OSCHEI...... 174 COMMUNITY ANALYSIS: JUVENILES ...... 177 COMMUNITY ANALYSIS: ADULTS...... 178 TAXIS EXPERIMENT ...... 179 DISCUSSION...... 182 CONCLUSION ...... 190 ACKNOWLEDGEMENTS...... 191 REFERENCES...... 192

CHAPTER 8: INTERFERENCE AND COMPETITION IN TWO CONGENERIC SPECIES OF BACTERIVOROUS NEMATODES (DIPLOLAIMELLOIDES MEYLI AND D. OSCHEI): IS FOOD A KEY FACTOR?...... 200

ABSTRACT: ...... 201 INTRODUCTION...... 203 MATERIALS AND METHODS ...... 205 NEMATODE STOCK CULTURES AND HARVESTING...... 205 POPULATION GROWTH EXPERIMENT ...... 205 TAXIS EXPERIMENTS WITH A SINGLE FOOD OPTION ...... 209 TAXIS EXPERIMENTS WITH MULTIPLE FOOD OPTIONS...... 211 FEEDING EXPERIMENT ...... 212 RESULTS...... 215 POPULATION GROWTH EXPERIMENT ...... 215 TAXIS EXPERIMENTS WITH A SINGLE FOOD OPTION ...... 218 TAXIS EXPERIMENTS WITH MULTIPLE FOOD OPTIONS...... 219 FEEDING EXPERIMENT ...... 222 DISCUSSION...... 224 ACKNOWLEDGEMENTS...... 228 REFERENCES: ...... 229

CHAPTER 9: TOP-DOWN EFFECTS OF A PREDATORY NEMATODE ON THE POPULATION DEVELOPMENT OF TWO PREY NEMATODES SPECIES...... 235

ABSTRACT...... 236 INTRODUCTION...... 238 MATERIALS AND METHODS ...... 240 NEMATODES...... 240 EXPERIMENTS...... 241 DATA ANALYSIS...... 241 RESULTS...... 243 DIPLOLAIMELLOIDES OSCHEI ...... 243 DIPLOLAIMELLOIDES MEYLI...... 246 DISCUSSION...... 250 CONCLUSION ...... 255 ACKNOWLEDGEMENTS...... 256 REFERENCES...... 257

Title: Species assembly rules and the biodiversity- ecosystem functioning relationship.

Abstract The biological diversity of ecosystems has important implications for their functioning. However, in order for reliable predictions to be made on the effects of species extinctions and/or additions on particular ecosystem functions, accurate knowledge is required on the ways in which species interact. These interactions in turn are important drivers of community structure and diversity. Direct interspecific interactions involve predator-prey interactions and competition for resources, whereas facilitation and inhibition are examples of indirect interactions between species. The latter type of interactions is crucial to an adequate understanding of the degree of redundancy among species within a given trophic guild. By now the concomitant contribution of bottom-up (i.e. resource-controlled) and top-down (i.e. predation-controlled) forces on community structure and dynamics has been widely accepted. But due to the great variability in experimental circumstances and test organisms, the results and conclusions are far from consistent. Nematodes are the most abundant and speciose metazoans in almost all marine soft sediments. Their communities are typically characterised by a high local diversity, and many species belonging to single functional guilds tend to co-occur. They are potentially important consumers of primary producers as well as of primary decomposers (bacteria), and are prey to a variety of predators, including other nematodes. It is hitherto unclear whether nematode community structure and dynamics tend to be controlled predominantly by resource availability or by predation. The importance and role of horizontal interactions, i.e. within a trophic level, such as competition and the indirect interactions (mentioned above), are even less well understood. The rules underlying species assembly in complex and speciose communities are a sum of direct and indirect interactions, occurring simultaneously in any direction: top-down, bottom-up and horizontal. Life history theory emphasizes that the impact of a stressor (for instance resource 16

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Abstract. DOUTORADO EM CIÊNCIAS BIOLÓGICAS limitation, presence of predators,…) on populations of organisms is determined by its effect on the fitness of a given population. More specifically, the concept of maximizing fitness is the key to gaining insight into the demographic effects of such stressors at the population level. We have performed microcosm experiments addressing bottom-up, top-down and horizontal effects on the fitness of populations of bacterivorous nematodes. A first experiment investigated whether and how nematodes may affect colonization of substrata by bacteria through their production of mucus trails. We were able to demonstrate that nematode mucus secretions consistently become colonized by a very low-diverse subset of the bacterial community present in the environment, and that this effect is nematode-species-specific. This microbial ‘gardening’ strategy provides novel insight into recently reported species-specific impacts of bacterivorous nematodes on bacterial communities, and may well have important implications for interspecific interactions among bacterial-feeders. Subsequently, we investigated the effect of resource availability on the population dynamics of three species of bacterial-feeding nematodes which typically occur in sympatry in their natural habitat. Resource availability was defined as bacterial (E. coli) density, and the response of each nematode species was studied in monospecific culture. Each nematode species showed a differential response to food availability, differences among species becoming manifest in parameters like reproduction rate, development time, maximal densities and individual as well as population biomass. One nematode species had an optimal fitness at the highest food availability, whereas the other two preferred intermediate food densities. Based upon these results, we constructed a hypothetical curve of how a community simultaneously comprising these three species would be expected to respond to food availability, and tested this in a parallel microcosm experiment in which the three species were now inoculated together. Our results did not follow the pattern expected from the individual species’ responses, and this as a result of three different horizontal interactions acting simultaneously within this artificial nematode community: facilitation, inhibition and competition for food. In a following set of experiments, we elaborated upon the inhibitory and

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Abstract. DOUTORADO EM CIÊNCIAS BIOLÓGICAS competitive interactions between the two species showing the most similar food-density dependence. In addition to population development, we now also studied food-finding (through chemotaxis) and food assimilation (following the fate of a 13C tracer). We confirmed the inhibitory effect between both species in a more complex feeding environment comprising a multitude of bacterial strains, but found it to become asymmetric (only one species negatively affecting the other, not vice versa), in contrast to the results from the previous experiment where the effect was largely symmetric. Surprisingly, food-finding and food assimilation of both species showed clear evidence of interspecific facilitation, the opposite of inhibition. Evidently, behavioural parameters studied at the short-term may yield different conclusions than population parameters studied over a longer term. However, there was concordance in the fact that food (i.e. bacterial) diversity weakened the observed interspecific interactions. Finally, we studied top-down effects, resulting from the introduction of a predaceous nematode, on the population development of these same two species in monocultures. The predaceous nematodes exerted a significant effect over total densities, age and size distribution and even sex ratio of the prey species. This effect was strongly dependent on, but not linearly proportional to, predator density. As such, we have shown that all three types of interactions (bottom-up, top-down and horizontal) are important drivers of population dynamics and community structure within the trophic guild of bacterivorous nematodes. We conclude that the indirect horizontal interactions are of particular relevance to our understanding of how species diversity within this trophic guild may affect ecosystem functions related to microbial activity, but that these indirect interactions in turn are bottom-up and top-down affected. key-words: biodiversity – ecosystem functioning – species assembly rules – bottom-up control – top-down control – succession – predation – detritus food webs – estuarine – nematodes – bacteria – trophic interactions – facilitation – chemotaxis.

Título: Regras entre assembléias de espécies: Relação entre biodiversidade e funcionamento do ecossistema.

Resumo A diversidade biológica tem importantes implicações no funcionamento dos ecossistemas. Porém, para realizar prognósticos sobre o efeito da extinção de espécies e/ou adição no funcionamento do ecossistema, requere-se conhecimento preciso sobre a interação das espécies. Estas interações por sua vez são importantes direcionadoras da estrutura e da diversidade das comunidades. Interações diretas: interações entre predadores vs presas, assim como, competição por recursos e interações indiretas: facilitação e inibição entre as espécies.. Essas duas últimas interações são cruciais para um entendimento adequado do grau de redundância entre espécimes dentro de um determinado grupo trófico. Até o presente momento a contribuição concomitante de bottom-up1 (i.e. controle de recursos) e top-down2 (i.e. controle predacional), forças sobre a estrutura e dinâmica das comunidades, vem sendo amplamente aceita. No entanto, os resultados e conclusões ainda são bastante inconsistentes devido à grande variabilidade das circunstâncias experimentais e dos organismos testados (cobaias), . Nematódeos são os metazoários mais abundantes e diversos em quase todos os ecossistemas sedimentares marinhos. Suas comunidades são tipicamente caracterizadas por uma alta diversidade local (α-diversidade), nas quais várias espécies pertencentes ao mesmo nível funcional tendem a co-ocorrerem. Esses organismos são importantes consumidores de produtores, assim como de decompositores (bactéria), e são presas de uma ampla variedade de predadores, incluindo outros nematódeos predadores. Até o presente momento, ainda é questionado se a estrutura da comunidade de nematódeos e sua dinâmica tendem a serem controladas, exlusivamente, pela disponibilidade de recursos ou por predação. A importância e o papel das

1 Bottom-up: Relação ecológica entre níveis tróficos inferiores afetando níveis tróficos superiores. 2 Top-down: Relação ecológica entre níveis tróficos superiores afetando níveis tróficos inferiores. 19

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumo. DOUTORADO EM CIÊNCIAS BIOLÓGICAS regras de relações horizontais, i.e. relações dentro do mesmo grupo trófico, como competição e interações indiretas, mencionadas anteriormente, são ainda pouco conhecidas. As regras que suportam as assembléias de espécies em comunidades complexas e diversas, são o resultado da soma das interações diretas e indiretas, que ocorrem simultaneamente em qualquer direção: top-down, bottom-up, e horizontais. A teoria da história de vida, enfatiza que o impacto causado por um agente (e.g. limitação de recursos, presença de predadores, etc.) na população, é determina pelo seu efeito sobre o fitness3 de uma determinada população (mais especificamente, o conceito de maximização do fitness) é a chave para o ganho de discernimento dentro de efeitos demográficos, como os impactos a nível populacional. Realizamos experimentos de microcosmos para direcionar o efeito entre relações verticais (Bottom-up e Top-down) e horizontais no fitness das populações de nematódeos bacterívoros. O primeiro experimento investigou quando e como os nematódeos afetam a colonização do substrato por bactérias através da produção de traços de muco. Demonstramos que a secreção de muco torna-se consistentemente colonizado por uma baixa diversidade de bactérias existentes no ambiente, e esse efeito é nematódeo espécie-específico. Essa estratégia de gardening4 microbiano nos permite uma melhor percepção do impacto espécie-específico de nematódeos bacterívoros sobre a comunidade bacteriana, e tem importante implicações para interações inter-específicas entre bacterívoros. Subsequentemente investiguei o efeito da disponibilidade de recursos sobre a dinâmica de populações de três nematódeos bacterívoros, que normalmente ocorrem em simpatria no seu habitat natural. A disponibilidade alimentar foi definida pela densidade bacteriana (Escherichia coli), e as respostas de cada espécie5 de nematódeos foram estudadas em culturas monoespecíficas. Cada espécie mostrou uma resposta diferente à disponibilidade de alimento. As diferenças entre as espécies se tornaram mais evidentes nos seguintes parâmetros: nível reprodutivo, tempo de desenvolvimento, densidades máximas,

3 Fitness: Adaptação ou aptidão de uma espécie ao meio ambiente em questão e níveis alimentares oferecidos. 4 Gardening: Capacidade de uma espécie cultivar sua presa através da liberação de secreções nutritivas específicas a determinada espécie (presa) e não a outras (não presas), assim como capacidade seletora mediante liberação de metabólitos secundários do metabolismo inato da espécie em questão. 20

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumo. DOUTORADO EM CIÊNCIAS BIOLÓGICAS biomassa individual e populacional. Uma espécie teve preferência a altas densidades alimentar enquanto as outras duas preferiram densidades intermediárias. Baseados nesses resultados, construímos a curva hipotética de como compreender as comunidades que contenham simultaneamente essas três espécies5 e como as espécies poderiam responder a disponibilidade alimentar presente no ecossistema. Isso foi testado em um experimento paralelo, no qual as três espécies foram inoculadas juntas. Essses resultados não seguiram o padrão demonstrado anteriormente, nos experimentos com as espécies inoculadas separadamente, e isso ocorreu devido ao resultado das três diferentes interações horizontais (facilitação inibição e competição por alimento) atuando simultaneamente dentro da comunidade artificial de nematódeos construída. Em experimentos subsequantes, elaboramos interações inibitórias e competitivas entre duas espécies6 mostrando a dependência mais similar de densidade alimentar. Adicionalmente, para o desenvolvimento populacional, estudamos a preferência alimentar (através de quimiotaxia) e assimilação alimentar (seguindo de 13C marcador radioativo). Isso confirmou o efeito inibitório entre ambas as espécies em um ambiente com maior complexidade alimentar, composto por múltiplas cepas bacterianas, mas a assimetria (apenas uma espécie afetou a outra e não vive-versa) foi a conseqüência final desse experimento. Contário aos resultados prévios do nosso segundo experimento, onde o efeito foi altamente simétrico. Surpreendentemente, tanto a capacidade alimentar, quanto a assimilação alimentar das duas espécies, mostraram evidências claras de facilitação inter-específicas, ao contrário da inibição. Evidentemente, o padrão de comportamento foi estudado em um período curto de tempo, o que pode levar a conclusões completamente diferentes para os parâmetros populacionais em experimentos realizados a longo prazo. Todavia, houve concordância no fato de que a diversidade alimentar (i.e. bactéria) enfraqueceu as interações inter-específicas observadas. Finalmente, estudamos o efeito top-down, resultante da introdução de um nematódeo predador7 sobre o desenvolvimento

5 Diplolaimelloides oschei, D. meyli (Monhysteridae) e Pellioditis marina (Rhabditidae). 6 D. oschei, D. meyli (Monhysteridae). 7 Enoploides longispiculosus (Enoplolaiminae). 21

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumo. DOUTORADO EM CIÊNCIAS BIOLÓGICAS populacional das mesmas espécies estudadas nas monoculturas. O nematódeo predador exerceu um efeito significativo sobre a densidade, a distribuição do estágio de desenvolvimento e o tamanho, assim como em relação à proporção sexual das presas. Este efeito foi altamente dependente, no entanto, não foi linearmente proporcional à densidade de predadores. Semelhantemente, mostramos que todos os três tipos de interações (bottom- up, top-down e horizontais) são direcionadores importantes da dinâmica populacional e da estrutura da comunidade dentro do mesmo grupo trófico de nematódeos bacterívoros. Concluímos, que as relações horizontais indiretas têm uma relevância particular para o entendimento de como a diversidade de espécies dentro do mesmo grupo trófico afetam o funcionamento do ecossistema, relacionado com a atividade microbiana. Contudo essas interações indiretas, uma após a outra, são relações afetadas pelos efeitos bottom-up e top-down do ecossistema.

Palavras Chaves:s Biodiversidade – Funcionamento dos ecossistemas – Regras entre assembléias de espécies – Controle ecológico ascendente – Controle ecológico descendente – Sucessão ecológica – Predação – Cadeia trófica de detritos – Estuários – Nematódeos – Bactéria - Relações ecológicas – Facilitação – Quimiotaxia.

Titre: Règles d'assemblages d'espèces et relations de biodiversité-functions d'écosystèmes.

Résumé La diversité biologique des écosystèmes a d’importantes implications pour leur fonctionnement. Cependant, afin d’émettre des prédictions certaines sur l effet d extinctions et/ou additions d espèces sur les fonctions d’un écosystème en particulier, une connaissance précise sur la façon dont chaque espèce interagit est requise. Ces interactions sont à leur tour d’ important facteurs responsables de structure et de diversité de communautés. Les interactions interspécifiques directes mettent en jeu prédateurs et proies et une compétition pour les ressources, tandis que facilitation et inhibition sont des exemples d’interactions indirectes entre espèces. Ce dernier type d’interactions est crucial afin de comprendre de façon adéquate le degré de redondance entre espèces a l’intérieur d’un même réseau trophique. A ce jour la contribution concomitante de forces de ‘bottom-up’ (contrôlé par la ressource) et ‘top-down’ (contrôlé par la prédation) sur la structure et la dynamique de communautés a largement été acceptée. Mais due à la grande variabilité des conditions expérimentales et organismes testés, les résultats et conclusions sont loin d’être cohérents. Les nématodes sont les métazoaires les plus abondants et diversifiés des ‘soft’ sédiments marins. Leurs communautés sont typiquement caractérisées par une grande diversité locale, et de nombreuses espèces appartenant à une fonction unique d’un réseau trophique sont représentées. Ils représentent d’importants consommateurs potentiels de producteurs primaires mais aussi de décomposeurs primaires ( bactéries), et sont les proies d’une variété de prédateurs,incluant d autres nématodes. Il est à ce jour encore ignoré si la structure et la dynamique des communautés sont contrôlées majoritairement par la disponibilité des ressources ou par la prédation. L’importance et le rôle d’interactions horizontales, c.a.d a l’intérieur même d’un réseau trophique, comme la compétition et les interactions indirectes mentionnées ci-dessus sont même moins bien comprises. Les règles déterminant les associations d’espèces

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Résumé. DOUTORADO EM CIÊNCIAS BIOLÓGICAS dans des communautés complexes et diversifies sont la somme d’interactions directes et indirectes, apparaissant dans toutes les directions: top-down, bottom-up et horizontal. La ‘théorie de l’histoire de la vie’ souligne que l’impact d’un facteur de stress (comme la limitation des ressources, la présence de prédateurs) sur les populations d’organismes est déterminée par son effet sur la fitness d’une population donnée. Plus spécifiquement; le concept du maximum de fitness est la clef pour comprendre plus en détails l’effet démographique de tels facteurs de stress au niveau de la population. Nous avons réalisé des expériences en microcosme adressant les effets de bottom- up, top-down et horizontaux sur la fitness de populations de nématodes bactérivores. Une première expérience cherchait à savoir si oui et comment les nématodes peuvent altérer la colonisation de substrats par les bactéries, via leur production de mucus. Nous avons pu démontrer que les sécrétions de mucus des nématodes étaient colonisées par une très petite diversité de communautés bactériennes présentes dans l’environnement, et que cet effet etait spécifique à l’espèce de nématode. Cette stratégie microbienne offre de nouvelles données sur les impacts ‘espèces-spécifiques’ rapportés de nématodes bactérivores sur les communautés de bactéries. Par ailleurs, nous avons étudié l’effet de la disponibilité des ressources sur la dynamique de population de trois espèces bactérivores qui sont typiquement présent en sympatrie dans leur environnement naturel. La disponibilité des ressources fut définie comme la densité de bactéries (E.coli), et la réponse de chaque espèce de nématode fut étudiée dans une culture mono spécifique. Chaque espèce de nématode a montré une réponse différente à la disponibilité de la ressource, qui semblait se manifester en fonction de paramètres comme le taux de reproduction, le temps de développement, la densité maximale d’individus ainsi que la biomasse de la population. Une espèce de nématodes montrait une fitness optimale avec une disponibilité de nourriture maximale, tandis que les deux autres montraient une préférence pour une densité de nourriture intermédiaire. Basé sur ces résultats, nous avons construit une courbe hypothétique représentant comment une communauté comprenant simultanément ces trois espèces répondait a la disponibilité de nourriture. Nous avons par la suite testé cela via une expérience de microcosme

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Résumé. DOUTORADO EM CIÊNCIAS BIOLÓGICAS parallèle dans laquelle les trois espèces étaient inoculées ensemble. Nos résultats n’ont pas donné le motif escompté avec les réponses individuelles de chaque espèce. Ceci est le résultat de trois différentes interactions horizontales agissant simultanément à l’intérieur d’une communauté artificielle de nématodes : facilitation, inhibition et compétition pour la nourriture. Dans une expérience suivante, nous avons testé les interactions d’inhibition et de compétition entre les deux espèces présentant la dépendance d’inhibition et de compétition la plus proche. En plus du developpement de populations, nous étudions aussi aujourd’hui la recherche de nourriture (au travers d’une chemotaxis) et l’administration de nourriture (en suivant un traceur 13C). Nous confirmons l’effet inhibitif entre les deux espèces dans un environnement plus complexe comprenant une multitude de souches de bactéries, mais trouvons qu’il est asymétrique (seulement une espèce affectant négativement l’autre, pas vice versa), à l’inverse des résultats d’expériences préalables où l’effet était largement symétrique. De façon surprenante, la recherche et l’assimilation de nourriture des deux espèces a démontré l’évidence d’une facilitation inter-spécifique, l’opposé d’une inhibition. De façon évidente, les paramètres de comportement étudiés pendant un laps de temps court peuvent induire des conclusions différentes que les paramètres de population étudiés pendant un temps plus long. Cependant, il y avait une concordance dans le fait que la diversité de la nourriture (bactérienne) diminuait les interactions spécifiques observées. Finalement, nous avons étudié les effets top-down, résultant de l’introduction d’un nématode prédateur, sur le développement de populations de ces deux mêmes espèces en monoculture. Les nématodes prédateurs exercent un effet significatif sur la densité totale, sur la distribution de l’âge et de la taille et même sur un sexe ratio des espèces de proies. Cet effet était fortement dépendant, mais non proportionnel, à la densité de prédateurs. Ainsi, nous avons montré que les trois types d’interactions (bottom-up, top-down et horizontal) sont d’importants facteurs de dynamique et structure de populations à l’intérieur d’un réseau trophique de nématodes bactérivores. Nous concluons que les interactions indirectes horizontales sont particulièrement intéressantes pour notre compréhension de comment la

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Résumé. DOUTORADO EM CIÊNCIAS BIOLÓGICAS diversité d’espèces au sein d ‘un réseau trophique peut affecter les fonctions d’un écosystème par rapport à l’activité microbienne, mais que ces interactions indirectes sont en revanche bottom-up et top-down affectées.

Título: Reglas de asociaciones de especies: Relación entre biodiversidad y funcionamiento del ecosistema.

Resumen La diversidad biológica tiene importantes implicaciones en el funcionamiento de los ecosistemas. Sin embargo, con el objetivo de pronosticar el efecto de la extinción de especies y/o la adición de una función particular del ecosistema, se requieren conocimientos precisos sobre la interacción entre las mismas. Estas interacciones a su vez son importantes directrices de la estructura y diversidad de las comunidades. Las interacciones interespecíficas directas involucran interacciones entre depredadores y presas, así como, competencia por los recursos mientras que la facilitación e inhibición son ejemplos firmes de interacciones indirectas entre las especies. Estos últimos tipos de interacciones son cruciales para un adecuado entendimiento del grado de redundancia entre especies dentro de un determinado grupo trófico. Hasta el momento la contribución concomitante de fuerzas bottom-up8 (e.g. control de recursos) y top-down9 (e.g. control predacional) en la estructura y dinámica de la comunidad van siendo ampliamente aceptadas. Debido a la gran variabilidad en circunstancias experimentales y a los organismos probados, los resultados y conclusiones son muy inconsistentes. Los nematodos son los más abundantes y diversos metazoarios presentes en casi todos los sedimentos marinos. Sus comunidades están típicamente caracterizadas por una alta diversidad local y muchas especies pertenecientes a un mismo nivel funcional tienden a coexistir. Estos organismos son consumidores potenciales de productores primarios así como de descomponedores primarios (bacterias) y a su vez son presas de una amplia gama de depredadores, incluyendo otros nematodos. Actualmente no queda claro si la estructura y la dinámica de la comunidad de nematodos tiende a ser controlada exclusivamente por la disponibilidad de recursos o por la depredación. La importancia y el papel de las interacciones horizontales (relaciones dentro de un nivel trófico) como son las

8 Bottom-up: Relación ecológica entre niveles tróficos inferiores afectando niveles tróficos superiores. 9 Top-down: Relación ecológica entre niveles tróficos superiores afectando niveles tróficos inferiores 27

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumen. DOUTORADO EM CIÊNCIAS BIOLÓGICAS competencias y las interacciones indirectas mencionadas anteriormente, son aun poco entendidas. Las reglas que soportan el agrupamiento de las especies en comunidades complejas y diversas son el resultado de interacciones directas e indirectas que ocurren simultáneamente en cualquier dirección: top- down, bottom- up y horizontal. La teoría de la historia de la vida enfatiza que el impacto causado por un agente (por ejemplo limitación de recursos, presencia de depredadores) en poblaciones de organismos es determinado por su efecto sobre el fitness10 de una población dada. Más específicamente el concepto de maximización del fitness es la clave para discernir dentro de los efectos demográficos, por ejemplo, los impactos a nivel poblacional. Se realizaron experimentos de microcosmos para dirigir el efecto entre relaciones verticales y horizontales en el fitness de las poblaciones de nematodos bacteriovoros. El primer experimento investigó cuando y como los nematodos afectan la colonización del sustrato por bacterias a través de la producción de trazos de moco. Se demostró que la secreción del moco fue consistentemente colonizada por una baja diversidad de bacterias existentes en el ambiente y que este efecto es nematodo- especie-específico. Esta estrategia de Gardening11 micriobiano permite una mejor percepción del impacto especie–específico de nematodos bacteriovoros. Subsecuentemente se investigó el efecto de la disponibilidad de recursos sobre la dinámica de la población de tres especies de nematodos bacteriovoros que normalmente ocurren en simpatria en su hábitat natural. La disponibilidad de recurso fue definida como densidad bacteriana (Escherichia coli), y la respuesta de cada especie de nematodos fue estudiada en monocultivos. Cada especie de nematodos mostró una respuesta diferente a la disponibilidad de alimento. Las diferencias entre las especies se tornaron más evidentes en los siguientes parámetros: nivel reproductivo, tiempo de desarrollo, densidades máximas así como en biomasa individual y poblacional. Una especie de nematodo tuvo preferencia por las altas densidades alimentarias, mientras las otras dos prefirieron densidades

10 Fitness: Adaptación o aptitud de una especie al medio ambiente con relación a los niveles alimentarios ofrecidos a especies. 11 Gardening: Capacidad de una especie de cultivar su presa a través de la liberación de secreciones nutritivas específicas a determinada especie (presa) y no a otras (no presas), asi como la capacidad selectora mediante la liberación de metabolitos secundarios del metabolismo innato de la especie en cuestión. 28

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumen. DOUTORADO EM CIÊNCIAS BIOLÓGICAS intermedias. Basado en estos resultados se construyó una curva hipotética para comprender como una comunidad que integra tres especies respondería a la disponibilidad de alimento. Esto fue probado en un experimento paralelo de microcosmo en el cual las tres especies fueron inoculadas juntas. Estos resultados no siguieron el patrón demostrado anteriormente en los experimentos con las especies inoculadas separadamente, y eso ocurrió debido al resultado de las tres diferentes interacciones horizontales (facilitación, inhibición y competencia por el alimento) actuando simultáneamente dentro de la comunidad de nematodos construida artificialmente. En experimentos subsecuentes, se elaboraron interacciones inhibitorias y competitivas entre las dos especies mostrando la dependencia más similar de densidad alimentaria. Adicionalmente para el desarrollo poblacional se estudió la preferencia alimentaria (a través de quimiotaxia) y asimilación alimentaria (siguiendo el marcador radiactivo C13). Esto confirmó el efecto inhibitorio entre ambas especies en un ambiente con mayor complejidad alimentaria, compuesto por múltiples cepas bacterianas, pero la asimetría (solamente una especie afecto a la otra y no viceversa) fue una consecuencia final de este experimento, en contraste con los previos resultados del segundo experimento, donde el efecto fue altamente simétrico. Sorprendentemente, tanto la preferencia alimentaria como la asimilación alimentaria de las dos especies mostraron claras evidencias de la facilitación interespecífica, lo opuesto a inhibición. Se evidenció que el patrón de comportamiento estudiado en un periodo corto de tiempo, puede arribar a conclusiones totalmente diferentes si se compara con los parámetros poblacionales estudiados en experimentos realizados a largo plazo. Sin embargo hubo concordancia en cuanto a que la diversidad alimentaria (bacteria) debilita las interacciones inter-específicas observadas. En el último experimento realizado se estudió el efecto top-down resultante de la introducción de un nematodo depredador sobre el desarrollo poblacional de las mismas especies estudiadas en monocultivos. Los nematodos depredadores ejercieron un efecto significativo sobre la densidad, la distribución del estado de desarrollo, tamaño, así como en la relación de la proporción sexual de las presas. Este efecto fue altamente dependiente pero

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter: Resumen. DOUTORADO EM CIÊNCIAS BIOLÓGICAS no fue linealmente proporcional a la densidad depredadora. De manera similar fue mostrado que los 3 tipos de interacciones (botton-up, top-down y horizontal) son conductores importantes de la dinámica poblacional y de la estructura de la comunidad dentro de un mismo grupo trófico de nematodos bacteriovoros. Finalmente se concluyó que las interacciones horizontales indirectas tienen una relevancia particular para el entendimiento de como la diversidad de especies dentro del mismo grupo trófico afectan el funcionamiento del ecosistema, relacionado con la actividad microbiana, pero estas interacciones indirectas son afectadas por efectos bottom-up y top- down de manera alternada.

Chapter 1: Introduction

Biological diversity is defined at different levels, ranging from habitat/ecosystem diversity over species to genetic diversity. Biological communities provide a variety of functions or ‘services’ important to mankind: production of (harvestable) biomass, decomposition of organic matter, nutrient mineralization, bioremediation of pollutants, etc… In order to ensure a continual survival of species and natural communities, and hence the provision of all these important ecosystem services, all levels of biological diversity may be important (UNESCO, 1992). A large part of the world’s species’ diversity is situated in soils and sediments. Indeed, even at very small spatial scales, species diversity in soils and aquatic sediments is typically very high (Groffman 1997, Wall & Moore 1999). This high biological diversity raises two fundamental ecological questions. The first deals with the functional implications of diversity. Put simply: does species diversity affect the performance of ecosystems with respect to crucial ecosystem services? And if so, how? A number of possible diversity-function relationships have already been proposed, but none can be generalized over different communities and habitats (Schlacher et al. 1999). The second inquires about the mechanisms which structure such diverse communities. This does not only involve ‘historical’ aspects like speciation and evolution, but also – and especially – those mechanisms responsible for the assembly of communities in which multiple species with (seemingly) similar ecological function coexist. Indeed, how is it possible that species with similar ecological roles coexist, and – coming back to the first question – how important is it for system functioning that there is more than one species in a given ecological role (are those species redundant or not? (Walker 1992). Concepts explaining such coexistences can be divided in two categories (Ekschmitt & Griffiths 1998): (1) deterministic models, which emphasize niche partitioning and imply that each species has its own niche, and (2) stochastic models, which allow for the coexistence of multiple species with identical or overlapping functions by taking into account temporary niche gaps resulting from unpredictable factors leading to species-specific mortality. As such, competitively inferior species

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 1: Introduction. DOUTORADO EM CIÊNCIAS BIOLÓGICAS may permanently coexist with superior competitors in (spatially structured) habitats subject to stochastic disturbance. A number of mechanisms with different spatial and temporal dimensions affect biodiversity. On large scales, speciation, dispersal, etc… are important determinants of biodiversity. On a small or local scale, interactions between organisms – predation, grazing, competition, facilitation… - and between organisms and their local environment are more important (Huston 1999, Lawton 1999, Godfray & Lawton 2001). Resource availability often shows a unimodal relationship with species diversity: diversity, then, increases with increasing resource availability up to a point where competitive exclusion overturns this positive effect, allowing but a limited number of species to dominate the system at the expense of other, competitively inferior species (Huston 1979, Kondoh 2001). However, ‘top- down’ impacts such as selective predation on dominant species may interfere with these ‘bottom-up’ (= resource-driven) trends so as to generate more complex diversity patterns (Worm et al. 1999). Vice versa, the resistance of populations to predation-induced (or other) mortality increases with increasing resource availability (Proulx & Mazumder 1998, Hillebrand et al. 2000). Hence, both ‘bottom-up’ and ‘top-down’ effects and their mutual interaction are essential in structuring biological communities (Hillebrand 2003). Actually biologists deal with a complex number of interactions among trophic biodiversity, organisms assimilation compounds, general direct and indirect interactions resulting in many different ecosystem functioning process. Few people are aware of the most numerous of all soil, marine and brackish-water dwelling animals, the nematodes. These minute, unsegmented roundworms are the most dominant members of the meiobenthos (organisms that can pass through a 1 mm mesh but will be retained by a 45 μm mesh, living on the sea botton) in different sediment types, in all water depths and in different salinities, attaining densities of up to several million individuals. m-² (Heip et al., 1985). Research on the role of local factors in shaping complex communities, and on the relationship between biodiversity and ecosystem functioning has hitherto largely focused on terrestrial and – to a lesser extent – freshwater systems,

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 1: Introduction. DOUTORADO EM CIÊNCIAS BIOLÓGICAS with particular emphasis on primary producers and their grazers (Loreau et al. 2001, Duffy 2002, Naeem & Wright 2003; but see Duffy et al. 2003). Much fewer studies have dealt with detritus foodwebs (but see Wardle et al. 1997, 1999), and topical studies on marine/estuarine benthic systems are lacking. This is problematic since aquatic sediments typically harbour highly diverse communities (Groffman 1997, Snelgrove et al. 1997). Heterotrophic bacteria are the predominant primary consumers of detritus. They, in turn, are eaten by a variety of grazers, among which nematodes are typically by far the most abundant metazoan representatives, and flagellates and ciliates the predominant protozoan ones. Nematodes and ciliates, then, are prey for other nematodes and ciliates, and for other meio- and macrofauna such as turbellarians and polychaetes (Watzin 1985, Moens & Vincx 1997). All these organisms are typically characterized by a high species diversity on a (very) small – and hence manipulable – spatial scale. This high small-scale diversity suggests that local factors (see above) are of prime importance in structuring detritus communities. Moreover, the interactions between bacteria and their grazers affect ecosystem processes such as organic matter decomposition, nutrient mineralization, etc. (Johannes 1965, Bonkowski et al. 2000, De Mesel et al. 2003, De Mesel et al. 2006).

Chapter 2: Literature Review

Free-living nematodes

Our knowledge of free-living nematodes dates from ancient times; already in the seventeenth century (1656) Borel knew the vinegar eel (Anguillula aceti). Müller (1756) later described several free-living species from freshwater; however, special attention was not paid to them until 1849 when Czernay published his monograph. During the succeeding years Schneider (1866) published his “Monographie der Nematoden” and nematodes were more intensively studied by scientists such as Dujardin (1845), Diesing (1851, 1861), Carter (1859) and Eberth (1863) so by 1865 about 80 species of free- living nematodes were known. Greater interest was focussed upon the species from soil and freshwater in the work of Bastian (1865) who described 20 new genera and about 100 new species. Subsequently, several authors published investigations (mainly morphological) on nematodes. An important impetus was given by Bütschli (1873) who described 61 species from soil and freshwater, about 30 of the species being new to science, afterwards he published several other papers about nematodes. De Man opened a new epoch when, in 1876, he published a treatise on soil nematodes. He mentioned 50 species, the greater part being new. Two other important investigations were published by him (1880, 1881) before his great monograph (1884). In this pioneer work 143 species were described, keys were given for the determinations and most of the descriptions were accompanied by figures, greatly facilitating identification. It must be stressed that a wealth of biological and ecological information is presented, so that this work may be considered the first ecological treatise of the soil and freshwater nematodes. The monograph was later followed by a long series of shorter papers (especially 1889, 1906, 1907, 1912, 1917). Oerley’s (1880) compilation paper contains the description of 202 nematode species grouped in 27 genera, providing a first good classification that gathered related genera into families, most of which still stand today. Diem (1903) inaugurated the study of the soil fauna as an entity and was

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS aware of the nematodes as a proper component of the soil fauna; however, his quantitative methods were far too crude to give a true picture of the detailed composition of the microfauna, although many important facts were stressed by him. From the point of view of nematodes, the quantitative results are without value because only the larger species (mainly Dorylaimus spp.) were obtained by his methods (direct manual sorting out of the fauna). During the following years the study of the arthropod component of the soil macro- and microfauna was advanced considerably through the technical improve- ments of Berlese (1905) which were further elaborated by several others (see Forsslund, 1943). However, the study of the nematode fauna of the soil lagged very much behind, because these animals were not isolated by means of the apparatus used for arthropods. In spite of the lack of automatic methods for extracting the nematode fauna of the soil, the interest in this group of soil inhabitants did not decrease. In arthropods the main interest was focussed upon the qualitative and quantitative distribution, while in nematodes the attention of the investigators was especially attracted by the fact that certain nematodes were able to become severe pests in cultivated plants. Especially the papers of Marcinowsky (1906, 1909) are important in this respect and they were followed by a large number of investigations, the interest of the authors especially being directed towards the Tylenchinae. Today this group is already massively studied. We shall not go further into details in this special branch of nematode biology which to a large extent has its own problems and methods, but will only mention, that much of our present knowledge has been summarized in several manuals among which may be mentioned those of Chitwood & Chitwood (1937) and Filipjev & Schuurmans Stekhoven (1941). The line developed by De Man was not neglected by zoologists even if the study of the plant parasitic species was mainly carried out by agricultural laboratories. The purely free-living species were the subject of many smaller studies, biological as well as taxonomic. Ditlevsen (1911) recorded 31 species from Danish soils, some of them being new to science. Brakenhoff (1913) found 52 species (2 new to science) in the neighbourhood of Bremen, Menzel (1914) studied the nematode fauna of alpine areas (Switzerland) and

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS especially the fauna of moss cushions and rosette plants also giving valuable information as to nutrition and other biological problems. In 1920 he published a paper on the nutrition of free-living nematodes, in which it is shown that Mononchus and others are predators. Steiner (1913, 1914) especially investigated the free-living nematodes of Switzerland, but later he published several zoogeographical papers containing material from very remote parts of the world, revision of this subject is targeted by Wieser (1953) and one of the most used feeding classification for marine nematodes. Micoletzky (1921) wrote a very large monograph of the free-living nematodes of soil which is still of value to workers on this subject. Besides presenting his own material (11 767 specimens) he reviewed as far as possible the European species and extra-European genera, giving keys for their identification. Much biological information is given which, however, is rather scattered, difficult to find and difficult to synthesize. It is regrettable that he does not state how his samples were collected and how large they were, with the result that his quantitative statements are of restricted value. In the taxonomic part of the paper he regards the absolute and relative measurements as significant specific characters and on this basis several species and different degrees of sub-species are described which cannot be maintained but must be considered individual modifications. In addition to this work he published several others, amongst which one (1925) is devoted to freshwater and marsh nematodes. Here he gives an important study of the permanently water-logged localities at borders of lakes etc. From this type of locality he enumerates many freshwater species as well as other species which where also occurring in soil. In Belgium, De Coninck, the predecessor of Coomans, was the one who started the studies in nematology. He published a large number of valuable papers most of them concerned with smaller faunas or single species of nematodes, of especial ecological interest, is his paper of 1930, where qualitative and quantitative differences between different biotopes are discussed. Franz & Beier (1942) and Franz (1942) have studied the soil nematodes in Austria. Franz’s papers are outstanding in that he was especially investigating

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS the quantitative distribution of nematodes, thus obtaining the largest numbers per unit area recorded up till then and of the same order of magnitude as those given by Stockli (1943) The seasonal variation of the nematode fauna was studied by Seidenschwarz (1923), he reached the conclusion that marked fluctuations in nematodes numbers occurred. Goodey from the U.K. wrote two important books: "Plant-parasitic nematodes and diseases they cause" (1933) and "Soil and Freshwater Nematodes" (1951). The latter work was reviewed by his son Goodey (1963) and was instrumental to the development of nematology in Great Britain. In Brazil, works on free-living nematodes began in the mid-20th century, with some records of nematode species, morphometrics of organism, distribution and ecological survey, given by Gerlach (1954, 1956a, 1956b, 1957a, 1957b). Studies then ceased until recently, when a new group of investigators began work in a most broad spectrum as this study, but we can regard specially relating the occurrence of nematodes to ecological process as well as some taxonomical aspects (Netto & Gallucci, 2003, Esteves et al., 2003, 2004, Genevois et al., 2004, 2006, Esteves, 2004, Netto et al., 2005, Da Rocha et al., 2006; Pinto et al., 2006, Fonseca et al. 2006, Venekey et al., 2005, Castro et al., 2006, Botelho et al., 2007, Maria et al. 2008) and this view shows that Brazilian researches start to give an useful number of information. Some other aspects of the biology of nematodes must be mentioned. Most of the quantitative investigations mentioned above (for the old publications) were based upon a simple sedimentation and decantation technique which in most cases yields highly unreliable results, as is seen from the small numbers. This method has been especially elaborated by Franz (1942) and Stöckli (1946a), so as to be completely satisfactory although time-consuming, but some recent and more useful techniques are summarized on the most important survey for marine nematodes in Heip et al. (1985). For the history of the quantitative concept of nematode populations of the soil and freshwater see Stockli (1946b) and Abebe et al. (2006), respectively. Special features of the activity of nematodes have been studied by several authors. Maupas gives a very valuable study of the postembryonic development (1900) and reproduction (1901) of some soil species, Schiemer

(1982, 1983, et al. 1983) have made some extremely important studies in the bioenergetics and reproduction of nematodes, followed by Vranken et al. (1981, 1984, 1988), in more recent years the studies rely on works done by Moens with collaborations (1996a,b, 1998, 2000a,b). The nutrition has been studied systematically in Rhabditis, Monhysteridae and other families (Hilgermann & Weissenberg (1918), Dotterweich (1938), Ludwig (1938), Vranken et al. (1984), Schiemer 1982, Schiemer et al. 1990 and Moens et al. (1999a, b). It was found that the food consists of bacteria, in most other nematodes (except the plant parasitic species) the question of nutrition has still not been satisfactorily investigated, consequently the modern concepts of the importance of nematodes in nature remain speculative (Heip et al. 1985), but there is a broad number been used as bioindicators (Bongers & Ferris, 1999, Nombela et al. 1999) ecological biodiversity (Boucher & Lambshead, 1995; Warwick & Clarke, 2001) functional biodiversity and ecosystems functioning (Heip, 2002, Somerfield et al. 2002). From an ecological point of view we know from Heip et al. (1985) that nematodes occur in the soil in numbers ranging from some thousands to some millions per sq. m., but how they live, and what they feed upon is practically regarded by few works: Vranken (1986), Moens & Vincx (1997) and Moens et al. (2005) in most species and the same applies, to their importance, if we are not to resort to mere speculation. Research involving nematodes was restricted at first to treating nematodes as a single taxonomic unit of the meiofauna, also considered to be a functional unit. We know now that nematodes are ecologically very heterogeneous and that they occupy different trophic positions in benthic food webs. These differences may be as large between families of marine nematodes as they are between orders in macrobenthic groups. This trophic diversity has been examined by Wieser (1953) who divided nematodes into four groups according to the structure of the buccal cavity, by postulating that morphological differences are linked to different feeding mechanisms: selective deposit-feeders; non-selective deposit- feeders; epigrowth feeders; predators and omnivores. The validity of this classification has been questioned since several important exceptions exist (Wieser, 1960, Boucher, 1972; Ward, 1975), but Wieser's proposition has

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS remained an important tool in the interpretation of nematode assemblages. However, more direct methods have become available and in the last decade important ecological and systematic advances have been made (Moens & Vincx, 1997, Moens et al. 2004). Regarding geographical distribution an overwhelming difficulty is met with, namely that the fauna of Europe, Africa and parts of Asia was mainly investigated by zoologists using European literature based on the standard works mentioned above, while the fauna of America and Australia was investigated by people using Cobb's papers on the “American fauna". It is greatly to be regretted that Cobb disregarded the European literature, thus created a complete confusion Micoletzky (1921) has proved that several of Cobb's species are identical to species described by European authors, later several others have correlated the two nomenclatures for other species, however, this has been solved for some phylogenetic studies such as Riemann et al. (1971) and Lorenzen (1979,1985, 1994) and molecular taxonomy (Blaxter et al. 1998, De Ley & Blaxter 2002, De Ley et al. 2005). Cobb (1917) has enhanced the possibilities of light microscopic observations by introducing the ingenious yet simple aluminium slide-holder with two cover slips, allowing observation of nematode specimens from both sides. He surrounded himself with a nucleus of people who were the real founders of the science of nematology in the U.S. such as Chitwood who's book "An introduction to Nematology" (Chitwood & Chitwood, 1950) is a classic. Thorne (1961) with his book "Principles of Nematology" was a specialist of Dorylaims and Tylenchs. Christie gave great contribution for crob protection science with a book on Plant Nematodes Their Bionomics and Control (1959). Steiner replaced Cobb after 1932 and provided important contributions on marine nematodes, and Taylor in collaboration with Sasser, wrote an important work on "Biology, identification and Control of Root-knot nematodes"(1978). Much has still to be done on nematology, and the whole problem of the geography of free-living nematodes is very difficult to discuss. Some indications are at hand, that most species are cosmopolitan, as showed by e.g. Soetaert et al. (1995) among different European estuaries and Johnston (1938) show that several species are common to soils of Europe and

Australia. Nevertheless it is good to be thinking that studies have mostly been done with an European based species key e.g. Platt & Warwick (1983, 1988) and Warwick et al. (1998). On the other hand some well defined and characteristic species have never been found outside the American continent, European continent, Australian continent, etc. Molecular evidences start to make a new overall history as well (Derycke et al. 2006). Summing up it appears that the qualitative composition of the soil fauna is well known in some localities e.g. Europe, but far from this in other continents e.g. South and Central America as well as Africa, while many quantitative statements in the literature are unreliable, the numbers most times are in disagreement being much too low. In addition, the quantitative relations have not been stated in terms of weight, apart from rather loose suggestions by Cobb and a few others (Stöckli, 1946a). Seasonal variations were distinct in many localities (Reiss & Kröncke, 2005). The faunas of different localities have been compared by several authors, however, an interpretation of the facts is difficult because edaphic as well as vegetational differences are involved. However it seems that certain species are confined to definite types of locality while most species are eurytopic (Warwick and Platt, 1998). The vertical distribution has been especially studied (Vanderborght & Billen, 1975); some new publications are trying to solve this unsolved problem (Steyaert et al. 1996, 1997, 1999) which seems to correspond to that of most other soil microorganisms (see e.g. Franz (1942) and Heip et al. (1977)). The moisture relations are known in principle but often neglected. The respiratory metabolism has been poorly investigated and the nutrition is only known from occasional observations, like culture experiments with defined food have mostly been performed with plant parasitic species, Rhabditis spp. and Diplogaster spp. (Dougherty & Calhoun, 1948), the nutrition elsewhere being unknown or statements being unreliable. The reproductive rate is known for a limited subset of species from experimental conditions (Maupas 1901, Vance, 1973; Geraert, 1980; Moens & Vincx, 2000a,b). The importance of nematodes has been discussed on the basis of unreliable suppositions. Field work has progressed both in methodology and in the number of habitats and geographical areas which have been investigated. In systematics, the number

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS of people involved was higher in the seventies than ever before and about 42 papers were published each year in the period 1971-1975 by about 50 marine nematologists, describing about 111 new taxa yearly (Gerlach, 1980). Currently the number of publications is over five hundred per year (ISI Web of knowledge, 2006) including free living and parasitic nematodes, but the lack of scientists for the most abundant eukaryotic organism “the nematodes” in the world still exists (Warwick, 1979). Experimental work involving the cultivation of species in the laboratory started in the late sixties and permitted much new information to be obtained on different aspects of nematode ecology, moreover the nematode trophic interactions and functional diversity is rarely reported. .

Feeding strategies

The feeding strategies of free-living nematodes have been touched upon by some authors partly as stray observations pertaining to the feeding biology of single species or genera and partly as general considerations on the whole group, and some times even including the parasites ones. It makes impossible to review the entire literature on this problem, as it is scattered all over the nematode literature, However, some of the more important references are given here. Bastian (1865) proposed that the free-living nematodes almost exclusively feed on material of plant origin. On the other hand, in some freshwater species (e.g. Cyatholaimus and Spilophora) he found diatoms and algae in the gut. He believes the food to be ingested by sucking. De Man (1884) was of opinion that the food of nematodes is of plant origin except for Dorylaimus spp. (observation of a Dorylaimus sp. which had pierced the cuticle of a Cephalobus sp. by its stylet). Also Mononchus sp. may feed on animals (in one case a Dorylaimus sp.). Oerley (1886) stated that the free-living Rhabditids feed on decaying materials of plant and animal origin. Diem (1903) found nematodes to be especially abundant near plant roots and suggested accordingly that they generally feed on fresh plant material. Marcinowsky (1906, 1909) comes to the conclusion that the semiparasites (can live in the soil as independent plants for a time, but are not vigorous or may not flower if they do not become attached to a suitable host), i.e. Cephalobus among others, are polyphagous. Hofmänner (1913) found diatoms in the gut of Theristus setosus; Potts (1910) believe that Rhabditis and Diplogaster generally feed on decaying substances of plant and animal origin. Conversely, they can be reared in cultures of bacteria and are able to feed on bacteria or their products, and the species having a stylet (Tylenchus, Dorylaimus etc,) feed on plant juices. Menzel (1914) state that Aphelenchoides parietinus may feed on the alga Chlamydomonas nivalis which diet causes a red coloration of the gut. Heinis (1916) mentioned that he has seen a Dorylaimus sp. in an empty test of Nebela collaris this have been “poorly” interpreted by assuming that the nematode has eaten the rhizopod

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS and taken over its test (for protection), In another case he saw a Dorylaimus sp. attacking and devouring a rhizopod. Steiner (1916) states the food of Rhabditis marina can be bacteria and unicellular organisms e.g. flagellates. Steiner & Heinly (1922) give a summary of the nutrition of nematodes expressing nice results but also the doubtful condition of feeding habits for some genera. The results of these show that some forms such as Rhabditis, Cephalobus etc. feed on decaying matter, that contained bacteria or bacterial products; others, such as Tylenchus, Heterodera, Aphelenchus etc, feed on plant tissues or fluids; others such as Dorylaimus etc. feed probably on both plants and animals; and still others such as Mononchus papillatus, described in the same paper, are apparently exclusively predatory, desagreen with Yeates and collaborators (1993). Later they stated that nematodes play a very important role as a factor in the humification of the soil, in transformation and removal of all kinds of organic matter, decaying bodies, and others organic material. From this Steiner (1925) concludes that Subelongatus may therefore be regarded as omnivorous and well fitted for varied life conditions. Discussing Dorylaimus he wrote about the importance of this nematode to be studied by experimental feeding, and by a study of the intestinal contents. Relating the advantages and disadvantages of these two mechanisms. Steiner (1928) wrote about Plectus granulosus has already been claimed by several authors as feeding, at least partly, in plants. It has been mentioned from seedlings of cereals and Micoletzky (1921) found chlorophyll as intestinal content and on P. communis feeds on fungus mycelium, at least partly digested mycelial thread was distinctly seen in the intestine. Menzel (1920) reached the following conclusions: Mononchus spp. feed on nematodes, rotifers and oligochaeta, Tripyla papillata on nematodes, rotifers and tardigrades and Trilobus gracilis on rotifers. The results were obtained by investigating the gut contents and by culture experiments. Apart from these reliable analyses he suggested without proving it that all species with a bulbus oesophagi (Rhabditis, Diplogaster, Cephalobus, Plectus etc.) ingest liquid food only, while the genera without a bulb are predators (Mononchus, Tripyla, Ironus and

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS many marine species), Dorylaimus and all the Tylenchinae feed on juices of plant and animal origin, which they suck by means of the stylet. Micoletzky (1921) agreed that Mononchus and Tripyla papillata generally feed on other animals, however, universally valid statements cannot be made as he has found T. papillata feeding exclusively on diatoms in the Lunzer lakes. It is a well known fact that several parasitic nematodes spend the larval stages free in the soil; this is the case in Ancylostomum caninum which spends the two first larval stages in faeces from dogs on the soil surface. During this period the larva feeds actively, two ecdyses occur and the length is doubled. Loos (1911) came to the following conclusion with regard to the nutrition of the free-living larva: The normal food of Ancylostomum larvae consists of the finest solid constituents of the faeces, perhaps also of certain substances contained in them in solution. Soon after he stated that bacteria have not a sufficient nutritive value for the larvae. McCoy (1929) studied the same problem in the same species. For this very nice investigation he obtained sterile eggs from the faeces, transferred them to agar plate cultures of different bacteria, where the larvae hatched normally and reached the infective stage in about 7 days on bacteria as sole food. In normal suspensions of bacteria in salt solutions they would also reach the infective stage. On heat killed bacteria and sterile filtrates of salt suspensions normal development did not occur. A single experiment with the yeast Torula rosea was negative. He concludes that the sum total of the evidence from the experiments indicates that living bacteria are the essential food utilised by hook-worm larvae in developing to the infective stage. Lapage (1937) states that many free-living nematodes of to-day eat algae, diatoms, bacteria and fungi and some of them are predatory, devouring rotifers, tardigrades and other nematodes (Mononchus, Trilobus), Other free- living nematodes seek environments rich in dissolved organic substances, such as manure and decaying plant and animal material, in which they live a saprozoic life. Those who have studied them are not, however, agreed about their diet, Filipjev & Schuurmans Stekhoven (1941) were the fist to try a general group classification including most of the free-living ones, and distinguished three groups, 1. plant parasites (Tylenchidae), 2. those using

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS decaying organic matter as a substratum (Rhabditis, Diplogaster, Cephalobus etc, and perhaps also Monhystera) "Many of these species do not eat the putrefying vegetable or animal material itself, but the bacteria, which live upon the same material and can be reared not without great difficulty in sterile media. Decaying plant material affords favourable conditions to the development of quite a fauna of saprozoic nematodes, among which species of Cephalobus and Acrobeles form the bulk," A third group comprises the predators. From there on a reasonable number of publications on nematode feeding classification became available, one of the most used, even if it is by far too generalist, for free-living nematologist are the one given by Wieser (1953), he divided nematodes in two main groups according to their mouth apparatus: 1. without buccal armature and 2. with buccal armature, and every one of these two are also subdivided in two: 1A. selective deposit feeders; 1B. Non- selective deposit feeders; 2A. Epsitrate feeders and 2B. Predators/Omnivores. Jensen (1987) made a relevant review on the subject including some information on D.O.M trans-epidermal uptake as well as resource partitioning. For freshwater nematodes important classification was given by Traunspurger et al. (1997), classifying nematodes in: Deposit feeders; epistrate feeders; suction feeders and chewers. Most of the freshwater studies are based on the different classifications (for review see: Nielsen 1949, Paramonov 1962, Wasilewska 1971, Yeates 1971), but the most relevant detailed classification on feeding habits and overview for soil nematodes was given by Yeates et al. (1993) classifying nematodes in 8 different categories and some of then with subdivisions: 1. Vascular plant feeding: (requires stomatostyle or odontostyle includes) 1a. sedentary parasites; 1b. migratory endoparasites; 1c. semi-endoparasites; 1d. ectoparasites; 1e. epidermal cell and root hair feeders and1f. algal, lichen or moss ‘piercers’. 2. Hyphal feeding (requires a stomatostyle or odontostyle and includes obligate and facultative hyphal feeders as well as alternate life cycles of some invertebrate parasites (e.g., Deladenus). 3. Bacterial feeding (wide range of mouth openings, but with different feeding modes, relatively selectively and some times predation on protozoa (Moens, pers. comm.). 4. Substrate ingestion this category

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS includes: diplogasterids; many (aquatic) Xyalidae (Daptonema, Theristus, etc) and normally they are selected based on particle size and shape, but some discussion on this shows that it can cause occasionally predation. 5. Animal predation: 5a. ingesters (Mononchus, Diplogaster, etc) and 5b. piercers (stylet or ‘revertible’ tooth) but some species combine both strategies (Oncholaimidae) and/or use poisons (Calyptronema). 6. Unicellular eukaryote feeding: digesting the whole-cell; piercing cells using stylet or tooth and cracking cells using tooth or teeth, nevertheless, they can make combinations of these strategies. 7. Dispersal or infective stages of animal parasites and 8. Omnivores: originally conceived as mainly encompassing dorylaims or ingesting sediment (e.g. Metoncholaimus (Myers, 1960), other oncholaims) and nematodes or agar (e.g. Diplenteron (Moens, pers. comm.)). Some literature makes clear that nematodes feed on dissolved organic matter (D.O.M.) directly and indirectly uptake (Chia & Warwick, 1969; Lopez et al., 1979, Moens et al., 1999a; Moens and Vincx, 1997; Moens et al. 2004.). Various environmental constraints on nematode feeding demonstrate that nematodes live in an informational society, food and abiotic conditions are often highly patchily distributed; moreover nematodes have to decide whether to feed or to search for a better feeding patch and these informations available to them are highly complex and can lead to conflicts (Dusenbery, 1996; Moens et al. 2006). Several statement have been built after Moens et al. (1999a,c; 2004) like: similar buccal structures may serve very different feeding habits, very different buccal structures may serve similar feeding habits and different buccal structures may serve to feed on similar food sources but in very different ways The concepts of these authors seem perfectly reliable even nowadays most of the old publications are useful and suggests that nematodes do not feed on an exclusive food source but on a range of different resources.

Trophic Interactions

Bottom-up and Top-down community regulation or ‘control’, is the alternative view of community regulation, was the implicit partner of succession theory as developed in most of the cases by plant ecologists. This standpoint held that since plant primary production fuelled the animal biota from primary consumers onward, plants (along with nutrients and light) regulated communities from the bottom of the food chain upward to higher trophic levels (e.g. White, 1978). This viewpoint was not extensively embraced by all ecologists, perhaps because, in branches, plant and animal ecologists interpreted the term ‘control’ in a different way, but also because examples could be cited that supported either perspective. Fretwell (1977, 1987), however, put a new twist on the idea of bottom-up community regulation. He combined the food-chain outlook ‘order vs. chaos’ (OxC) conundrum in community ecology by Hairston et al. (1960) vs. the idea that food chains varied in length as a consequence of variable environmental gradients of nutrients and productivity. Fretwell planned that, on landscape scales, gradients of primary production were the primary factor underlying communities of changing food chain length. In unproductive environments, food chains were just one association in length, consisting only of producers. Consumers might be present, but were not sufficiently abundant to have negative influences on producers. In environments of superior productivity, production in the long run would reach levels capable of supporting an abundant herbivore level (adding a second-association to the food chain), with increasingly strong negative feedback on characteristics of plant assemblages. Following the same interpretation, increased productivity would first amplify herbivore abundance, leading to amplified herbivore pressure, and would in due course add a predator level (third-association), and then a secondary predator level (fourth-association). Ecological energetics dictated that, even though five- or six-association food chains might be possible, the generally low efficiency of transfer of energy to higher trophic levels meant that most communities would possible collapse in the range ofmore than one- to four-association food chains (Menge 2000) in

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS marine environment. In moving along a gradient of increasing productivity, the ‘food-chain dynamics’ hypothesis thus predicted that consumers would be regulated by competition (one-association food chain), grazers (two association), competition (three association), and grazers (four association). At least for three-association food chains, Fretwell (1977, 1987) thus envisioned an ‘order vs. chaos’ like scenario, with Top-Down controlling community structure, and replacement between control by competition– predation–competition at predator, herbivore, and consumers trophic level. The emphasis, however, was on the gradient in productivity as the prime determinant of food-chain dynamics. This perspective was later formalized in a mathematical model (Oksanen et al., 1981, see further). Some evidence has been reported empirically bearing on these different viewpoints was slow to accumulate and, as noted earlier, controversy regarding modes of control of communities persists to the present. It wasn’t until the 1980’s that observational evidence more or less consistent with Fretwell vs Oksanen perspectives was offered from terrestrial (e.g. Oksanen, 1983, 1988) and freshwater environments (e.g. Carpenter et al., 1985). Experimental evidence for OxC-like dynamics has, however, only appeared relatively recently. By altering productivity levels in a food web in a northern California river, Wootton & Power (1993) observed results consistent with the Oksanen et al.’s (1981) prediction of alternating control by trophic level, and between food chains of length three and four links (Trophic cascades). Similarly, experiments in white oak forest demonstrated that birds indirectly enhanced plant growth by reducing insect herbivore abundance (Marquis & Whelan, 1994). In northern England, pesticide and turf transplant experiments at sites along a productivity gradient were consistent with the predictions of the Fretwell vs Oksanen model (Fraser, 1998; Fraser & Grime, 1997). At low productivity sites, invertebrate herbivores had little effect on vegetation, and experimental assays of the effects of top-down control suggested that predation was weak. At successively more productive sites, the impact of grazers first increased (intermediate productivity) then decreased (high productivity) while the impact of predators steadily increased. Therefore, abundance of vegetation was most strongly affected by bottom-up factors at

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS sites of low productivity and by top-down factors at sites of high productivity. Finally, in Kentucky, adding detritus to a forest litter community increased the abundance of all trophic groups, including fungivores, omnivores, and predators (Chen & Wise, 1999). Hence, increasing the bottom-up resource base of the food web led to increases at all trophic levels. Some long-term studies have shown convincingly that community structure depends on the interdependence of small-scale, short-term ecological processes (including top-down effects such as grazing), and larger-scale, long-term processes that create variation in bottom-up effects (such as El Niño-related nutrient depletion; Dayton et al. 1984, 1992, 1999, Dayton & Tegner 1984, Tegner & Dayton 1987, Tegner et al. 1997). Beyond this important ongoing work, evidence for bottom-up influences on community dynamics in marine environments was limited for a long period. In part, this was due to the success of experimental research in shallow marine near- shore environments in repeatedly demonstrating strong top-down and competitive effects on local community structure (Paine 1966, 1971, 1974, Connell 1961, 1970, Dayton 1971, Luckens, 1970, 1975, Menge & Sutherland 1976, Menge et al 1999, 2002 Menge 2000, Lubchenco & Menge 1978, Ebling et al. 1964, Kitching et al. 1959, Peterson, 1979, Garrity & Levings 1981, Lubchenco et al. 1984, Menge & Lubchenco 1981, Menge et al. 1986a, 1986b, Dungan, 1986, 1987, Lively & Raimondi 1987, Castilla & Duran 1985, Duran & Castilla 1989, Paine et al. 1985, Fairweather 1985, Fairweather et al. 1984, Underwood et al. 1983, Fawcett, 1984, Robles & Robb, 1993, Robles et al. 1995). All of these works have elucidated the interaction at community level but missing the importance of population effects in the marine environment. In addition, in intertidal habitats at least, oceanographic variation was generally thought to occur at scales far greater than the variation documented by ecologists (e.g. Paine, 1986, Branch et al., 1987, Menge, 1992), so there was little impetus to study possible influences of bottom-up effects on benthic community structure. By the late 1980’s, this perspective had begun to shift. Intertidal communities on offshore islands were observed to have high algal abundances, low abundances of grazing limpets, and dense colonies of seabirds. On mainland shores, birds and algae were

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS scarce, and limpets were abundant. Researchers hypothesized that high algal abundance on islands resulted from two effects of birds: predation on the limpets and nutrient inputs from guano. Results of field experiments suggested joint strong influences of increased nutrients and bird predation on algal production and abundance on offshore islands harbouring seabird nesting colonies (Bosman et al., 1986, Bosman & Hockey, 1986, Branch et al., 1987). That is, at least on a local scale, top-down and bottom-up effects evidently combined to regulate the structure of algal assemblages. In intertidal habitats Wootton et al. (1996) manipulated nutrients and molluscan grazers in an effort to determine the relative influence of each factor on biomass of algae and on density of micrograzers, in this case larval chironomids. Experiments were done during an El Niño year and two non-El Niño years. Results suggested that addition of nutrients (fertilizer inside porous clay pots) had no effect on algal biomass, but led to increased abundance of micrograzers. In contrast, exclusion of molluscan grazers led to large increases of algal biomass and abundance of micrograzers. This experiment thus suggested that grazing (on algae) and competition (molluscan grazers vs. micrograzers) had strong effects. Although addition of nutrients seemed to have little effect, Wootton et al. (1996) did not test if nutrients actually were increased by their method or if nutrients were naturally limiting to algal growth, so firm conclusions regarding bottom-up influences in this system await further study. Recruitment of species at the basal level of food webs can be considered a bottom-up effect when it increases the abundance of prey organisms (e.g. Menge et al., 1999). Most early analyses of intertidal community dynamics were conducted in regions having high rates of recruitment of sessile invertebrates. However, studies focused on recruitment demonstrated that recruitment densities could vary dramatically on local to geographic scales (Denley & Underwood, 1979, Underwood et al., 1983, Caffey, 1985, Gaines & Roughgarden, 1985, Raimondi, 1990, Menge, 1991). Such variation can significantly influence population and community structure, both directly and indirectly (Underwood et al., 1983, Fairweather, 1988, Sutherland, 1990, Menge, 1991, Caley et al., 1996, Minchinton & Scheibling, 1991, 1993,

Robles et al., 1995). For example, barnacle recruit abundance was a major determinant of barnacle density on the upper shore (Minchinton & Scheibling, 1993). Lower on the shore, a post recruitment factor, predation, controlled barnacle density. Collectively, these studies suggested that, in contrast to conventional wisdom, bottom-up factors can have a strong influence on population and community structure and dynamics in marine benthic communities. Bottom-up effects vary on larger spatial scales and over longer temporal scales, so further progress in understanding the regulation of marine communities depends on expanding the scope of research to a greater range of scales and to a larger suite of ecological processes (Dayton & Tegner, 1984, Levin, 1992, Menge et al. 1996, Menge & Olson, 1990, Wiens, 1989). During a long time, several workers have initiated studies to explicitly integrate top-down and bottom-up effects in analyses of community and/or ecosystem dynamics. Although few, these studies illustrate the general approach and suggest that, communities are sensitive to changes in trophic interaction, trophic interactions are normally seen as a unique process, and some important processes among populations and species have been drastically ignored and many controversial concepts still playing a role on bottom-up vs top-down models. The already mentioned Bottom-up model of Oksanen and colleagues (1981) and the top-down model by Menge and co-authors (Menge & Olson, 1990, Menge & Sutherland, 1987) contrast in predicting where in the food chain predation and competition are most important. These concepts predicts: (1) the control by these factors (bottom-up; nutrient/productivity) alternates with trophic level and that length of food chains increases with increases in productivity. The environmental stress (i.e. predation, top-down) increases in importance and competition decreases in importance from high to low trophic levels. Moreover, food chain length decreases with increases in environmental stress. Most of these vary among habitats and environmental conditions, depending on whether nutrients and productivity, or environmental stress are the ‘dominant’ environmental gradients (Menge & Olson, 1990). Additionally, discussion on the interaction between top-down vs bottom-up can be easy

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS exemplified by the ‘omnivory’ behavior of some species and how some model addressed it, e.g. the top-down theories assumes that omnivory are ecologically important, while the bottom-up concept assumes that omnivory is ecologically insignificant, the directions who drives the ecosystem function are normally dictated by the environmental condition and not by the animal feeding strategy. These make discussions over some controversy concepts keep in course. Rising evidence suggests that omnivory can be a powerful structuring force (Persson et al., 1988, Diehl, 1992, Lawler & Morin, 1993, Menge et al., 1986a, Morin & Lawler, 1996, Persson, 1999, Polis, 1999). In addition, within- and between-trophic level heterogeneity, size-structured interactions, and behaviourally-mediated interactions can all guide to departures from the alternating-control expectations of the bottom-up model (e.g. Osenberg and Mittelbach, 1996, Persson, 1999, Strong, 1992). Supported on such facts, alternative conceptual models have been proposed (e.g. Menge et al. 1996, Osenberg & Mittelbach, 1996, Persson, 1999, Polis, 1999). The strongest evidence that top-down and bottom-up processes interact to produce community structure has accumulated in freshwater environments but evidence from both terrestrial and marine ecosystems is also increasing (Persson, 1999). In the current reading both interactions will be metioned as trophic cascades (Mikola & Sëtalä, 1998a; Schmitz et al., 2004). Top-down effects are normally invoked to explain such variation on affected populations, studies focused first on predation and grazing. The Top-down effects of predators on microbivore community structure and diversity was studied for a better understanding of predator stress driving prey species functioning and the ecosystem structure on meiobenthos and mostly on nematode community. As well, Bottom-up experiment allowed the elucidation of several food density dependent parameters such as: egg, juvenile, adult and total developmental time of three nematodes species. Additionally, their fitness under different food availability permitted we test in further experiment species direct and indirect interactions.

Direct vs indirect ecological interactions

Direct effects

Many studies try to give a comprehensive explanation on ecological interactions, for marine environment they are based mostly on macrofauna association, but a good compilation of concepts is given by Menge (1995, 2000), brief summary will be described below. More the 35 years ago May (1973) have described according to ‘mathematical signs’ direct interactions for their effects on each interactor. For example, for interactors between X and Y, a positive (+) negative (-) interaction (term used as predation∗), udestanding as X having a negative effect on Y, and Y having a positive effect on B. Interference competition is a (-,-) interaction, in which X and Y have straight, reciprocally negative effects on each other. In theory, all possible combinations of positive (+), negative (-), and neutral (0) effects can occur in pairwise interactions (May 1973). We have to carefully address some field experiments, because they do not always document mutual effects. For instance, assays of predation might test the negative effect of a predator over its prey, other than typically the positive effect of a prey on its predator is supposed. This statement could often be suitable, but it is believable that a predator can have a powerful negative effect on a prey that gives little to its overall food intake (Abrams 1992). Likewise, interference competition is usually asymmetric (i.e., stronger in one way than another; Schoener (1983), and mutual effects are not at all times confirmed experimentally. Most of these reasons, and for the reason that single indirect effects result from a unidirectional series or chain of direct effects, we will discuss all treat mutual effects as a separately one, it provides a most comprehensive correlation between species process and ecosystem function. As a consequence, mutual effects like predator-prey interaction (+,-) are food (+) and predation (-). Interference competition effects (-,-) possibly will be whichever interference competition (-) and interference competition (-), or, if

∗ = Lubchenco (1979) use mostly like general term that includes carnivory, herbivory, and parasitism 53

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS the reverse effect is neutral (0) or not recognized or easily incidental, simply interference competition (-) with no reciprocal effect. Some of the most commons direct effects observed in ecological webs will be described below: Predation (considered as a trophic interaction between levels), and Interference competition (considered as a nontrophic interaction, within levels), both defined as above. These are the most intensively studied direct interactions (Connell 1983, Schoener 1983, Sih et al. 1985), and it is reasonable to expect that many if not most indirect effects will involve these interactions. Other types of interactions exist, of course, and one interesting result of this study (some of the interactions described below was not reported in our results) is the relatively high importance of these less-studied, "nontraditional" types of interactions. And such interactions are trophic and nontrophic effects but can occur both between trophic levels as well as within levels. Inhibition of recruitment (a negative effect), an established inhabitant of a habitat, whether mobile or sessile, decrease the rate of successful invasion of the habitat by recruiting stages of an additional species. This relation is considered as passive, distinguishing it from interference competition, which involves directed aggression or overgrowth. The "recruitment inhibition" have been mostly used rather than "preemptive competition," which is regard as a more specific form of passive inhibition resulting when a sessile species displaces other sessile species by colonizing, establishing, and monopolizing space previous to other colonists can settle. Inhibition of feeding (a negative effect), an interactor, whether mobile or sessile, decreases the feeding activity of an additional species, usually mobile. This is normally an interaction connecting established specimens. Enhancement of recruitment (a positive effect), a prior inhabitant of a habitat, whether mobile or sessile, intensifies the rate of successful invasion of the habitats by contributing favoured attachment sites or otherwise raising the probability of colonization. Like recruitment inhibition, this is a relation between juvenile colonizing stages and established specimens.

Provision of habitat or shelter (a positive effect), a specimen increases the survival of partners, or attracts migrants to itself, for the reason that it presents a more attractive microhabitat than exists away from the other organism. This is a relation between established species. Facilitation (a positive effect) is a definition that normally describes positive species interactions, at least to one of the related participant and should not harm to neither. Positives (facilitation) interactions descriptions have been given above, but the principal ones are given below: Mutualism (+,+) and commensalism (0,+) are not reported as direct effects for a simple reason that they can be effects by similar interactions already mentioned, and these are described by more specific names (enhance recruitment, provide habitat or shelter) rather than use the more broad term commensalism.

Indirect effects

A number of prior studies have recognized some models of indirect effect sequences (e.g., Kerfoot & Sih 1987, Fairweather 1990 and ref. therein): Keystone predation, where a predator indirectly increases the abundance of competitors of its prey via consumption of that prey (e.g., Paine 1966, 1969, and many others), is possibly the earliest interaction recognized as having an important indirect effect and playing a fundamental role on ecosystem function. Tri-trophic interactions, or an increase in one species (plant) abundance in consequence of the control of it predator (herbivores) by another predator (carnivore) (Hairston et al. 1960), have been considered as trophic cascades (Paine 1980, Carpenter et al. 1985), this term are recognized as an significant sort of indirect interaction (Strauss 1991). Exploitation competition is a decrease in a consumer density resulting from the decrease of its prey density by another consumer species. Even though, it has been the most important focus of ecology for a long time, exploitation competition was not accepted as an indirect effect until some extensive discussions. (e.g., Kerfoot & Sih 1987, Strauss 1991).

Apparent competition, or decrease of a species resulting from increases in a second species which enhances predation by a shared enemy, this relation was prior postulated as an indirect effect by theorists (Levine 1976, Holt 1977) and demonstrated in nature by Schmitt (1987). Indirect mutualism, or positive simultaneous changes in two species consequential from predation by each on the competitor of the other's main prey, was first defined by Vandermeer (1980), who had suggested it to be important in natural communities. Inspired by Vandermeer's model, Dethier & Duggins (1984) recognized a variant of indirect mutualism in some field assays in a marine plant-herbivore system, which they termed indirect commensalism, and claim reasons may be more frequent than indirect mutualism. Indirect commensalism results when one possible indirect mutualist is more generalized in diet and also feeds on the main prey of the other indirect mutualist. Finally, Habitat facilitation happens when one specimen indirectly improves the habitat of a second by modifying the abundance of a third interactor (Fairweather 1990). These samples of models are unquestionably not the only types of indirect effects. However, all have been documented in real communities, and several (keystone predation, trophic cascade, exploitation competition) are extensively supposed to be common. A remainig questions is: how many of these interactions (types of indirect effects) had being neglected and remain not well described for ecologist when we compare to direct effects (ecological relationships), how these indirect interaction affect the comprehension and significance in structuring communities comparative of their occurrence in natural communities?

Top-down/bottom-up regulation

The already mentioned Bottom-up model of Oksanen and colleagues (1981) and the top-down model by Menge and co-authors (Menge & Olson, 1990; Menge & Sutherland, 1987) contrast in predicting where in the food chain predation and competition are most important. These concepts predict: (1) the control by these factors (bottom-up; nutrient/productivity) alternates with

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS trophic level and that length of food chains increases with increases in productivity. The environmental stress (i.e. predation, top-down) increases in importance and competition decreases in importance from high to low trophic levels. Moreover, food chain length decreases with increases in environmental stress. Most of these vary among habitats and environmental conditions, depending on whether nutrients and productivity, or environmental stress are the ‘dominant’ environmental gradients (Menge & Olson, 1990). Additionally, discussion on the interaction between top-down vs bottom-up can be easily exemplified by the ‘omnivory’ behaviour of some specimens and how some model addressed it, e.g. the top-down theories assumes that omnivory are ecologically important, while the bottom-up concept assumes that omnivory is ecologically insignificant, the directions who drives the ecosystem function are normally dictated by the environmental condition and not by the animal feeding strategy. Rising evidence suggests that omnivory can be a powerful structuring force (Persson et al., 1988, Diehl, 1992, 1993, Lawler and Morin, 1993, Menge et al., 1986a, Morin and Lawler, 1996, Persson, 1999, Polis, 1999). In addition, within- and between-trophic level heterogeneity, size- structured interactions, and behaviourally-mediated interactions can all guide to departures from the alternating-control expectations of the bottom-up model (e.g. Osenberg & Mittelbach, 1996, Persson, 1999, Strong, 1992). Supported by such facts, alternative conceptual models have been proposed (e.g. Menge et al., 1996; Osenberg & Mittelbach, 1996; Persson, 1999; Polis, 1999). The strongest evidence that top-down and bottom-up processes interact to produce community structure has accumulated in freshwater environments but evidence from both terrestrial and marine ecosystems is also increasing (Persson, 1999). In the current reading both interactions will be mentioned as trophic cascade (Mikola & Sëtalä, 1998a). The indirect interactions are based in “three-species food chain” in a consequence of direct interaction, and we have been assessing it isolating a simple three-species (or trophospecies) linear food chain in which the middle species (consumer) adaptively responds to the density of the top stressor and/or the resource (trophic cascade). Consequently the middle (reacting) species transmits effects of either or both of the two fundamental mechanisms

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 2: Literature Review. DOUTORADO EM CIÊNCIAS BIOLÓGICAS and the resulting trait mediated indirect interactions (TMIIs) can constitute either top-down or bottom-up effects. Nontrophic links and Trait mediated indirect interaction will take place after we have explored TMIIs due to phenotypic responses associated with trophic links. However, TMIIs can arise from nontrophic links, especially various forms of interference competition. In a three-species shared-predator web, interference competition can occur between the two prey species. The causal path of the TMII here is from one prey to the behaviour (or other traits) of the other prey to the predator. For example, a superior interference competitor can exclude another from refuges and thereby increase the vulnerability of the latter to a predator. The three-species shared-predator interaction happens when consumers share a predator, on indirect effects, this is the food web that incorporates some likewise competition interactions (Holt 1977, Abrams 1987). Although often conceived of as a density mediated indirect interaction from one to the other consumer through predator population density, the presence of one prey species clearly can induce changes in predator traits that alter the interaction of the predator with the other prey species. Abrams (1987, 1992) has shown, in theory, the capacity of an adaptive behaviour of both predator and prey in trophic cascades can influence the nature of the predator’s functional response, which in turn clearly affects population growth rates of the prey. Subsequently, there are various potential TMIIs in this trophic-web that can take on any combination of positive and negative species effects. If a changing environment prevents populations from coming to equilibrium, the indirect effects transmitted through the predator’s functional responses to other prey can be more important than those transmitted through its density (Abrams 1987).

Chapter 3: Aims

The current project has two main aims: (1) to assess effects of species diversity of bacterivores on ecosystem functioning, and (2) to investigate species assembly rules responsible for structuring multispecies bacterivore communities. A major purpose of the present study will be to assess levels of redundancy among species with supposedly similar trophic strategies and ecological roles. A better integration of research on diversity-function relations with work on foodweb interactions is primordial in ecological research (Duffy 2002). For the first aim, we draw upon existing nematode culture collections to perform experiments in which the diversity and functional diversity is studied in the presence of bacteria and different combinations of bacterivores in different levels of species diversity. Previous experiments have yielded insight into how individual nematode species affect bacterial communities and decomposition in such microcosm experiments (De Mesel et al. 2003, 2004, 2006). This information served as a basis for novel experiments in which species diversity is the prime variable. Indeed, in addition to a possible effect of diversity by itself, recent experiments show that both species-specific and combination-specific effects exist. There are clear indications that species affect each other’s success, both positive (facilitation) and negative (inhibition) interactions between closely related and apparently coexisting species already having been illustrated (De Mesel et al. 2003, 2006, Moens et al. 2005.). Hence, in addition to direct interspecific interactions involving competition (for space and/or resources), indirect interactions probably have important structuring effects on communities of bacterivores. The extent of, and mechanisms behind, such indirect interactions remain to be elucidated, and this will be an important aim of the present study. Therefore, while experiments mainly addressing our first major aim will be performed at one resource-level and with all species introduced simultaneously, subsequent experiments to elucidate species assembly rules will introduce the following elements of variability:

(1) One set of experiments was focused on ‘bottom-up’ effects of resource availability on the population structure and combinated population diversity of bacterivores nematodes. To this end, the highest-diversity treatment from the previous set of experiments is to be repeated but now at varying levels of resource (= nutrient) supply. A varying resource supply yields variable food availability for bacterivores. This affects interaction strengths between competing species and/or favour certain species over others. (2) Another set of experiments will look into ‘top-down’ effects of predators on the structure and diversity of populations of bacterivores. Carnivorous nematodes are important predators of bacterivores; their predation also tends to be selective (a.o. Moens et al. 2000, Hamels et al. 2001). Predator-induced species-specific mortality is a potentially crucial factor underlying local species diversity (see introduction), and this experiment (parallel to the previous one, but now with and without predators) will provide insight into the structuring role of predation. Moreover, theoretically, predation affects not only prey species but in turn also the prey’s prey (here bacteria). (3) Finally, while virtually all experiments on diversity-function relationships have hitherto introduced all species simultaneously, this obviously neglects natural species successions, both deterministic (and therefore based on life history and colonization potential of species) and stochastic (based on dispersal). We will therefore study species assembly under different colonization ‘histories’, from simultaneous introduction of all species, over life- history-based reconstruction of ‘most plausible’ successions, to haphazard sequential introductions of species.

Chapter 4: Justifications

General information

Nematode densities in marine and estuarine sediments are typically in the range of 105 to 107 ind.m-2, and tend to be particularly high in intertidal and shallow coastal sediments. Correspondingly, biomass ranges between 0.2 and 2 g C m-2 (Heip et al. 1985, 1995). In addition to a high density, a high species diversity is also often characteristic of nematode communities (Heip et al. 1985). Much more than 10 species per 10 cm2 is rule rather than exception, and this usually encompasses a number of supposedly confunctional species (see below). Free-living nematodes feed on a range of particulate sources including detritus, diatoms, bacteria, ciliates and other meiofauna (as predators or scavengers). Bacteria and microalgae are generally considered major food sources (Montagna 1995, Moens & Vincx 1997). Marine nematodes are often classified into trophic or functional groups based on the morphology of their buccal cavity (e.g. Wieser 1953). However, within functional groups, heterogeneity and plasticity in feeding strategies and food sources may be substantial (Moens & Vincx 1997). Moreover, as opposed to a high species diversity, only four to six trophic groups are recognized, hence the robustness of feeding type classifications is unlikely to accurately reflect true functional diversity. As a result of the limited understanding of nematode feeding ecology, their roles in benthic foodwebs remain poorly resolved. For a long time, nematodes were considered as a ‘black box’ taking energy from lower trophic levels but not transferring it in any significant way to higher levels (McIntyre & Murison 1973). This view has since changed drastically and nematodes are now thought to play a potentially crucial role in fluxes of energy and matter through the benthos (Kuipers et al. 1981, Coull 1999).

Bacterivorous nematodes associated food sources.

Nematode communities on macrophyte detrital deposits are generally characterized by a somewhat lower (hence also more easily manipulative) species diversity than typically benthic communities. Trophic diversity in particular tends to be much smaller. In fact, representatives of only two nematode orders, Monhysterida and Rhabditida, are worldwide truly characteristic of such habitats (Warwick 1987). Within the Monhysterida they are mostly Monhysteridae, and within the Rhabditida, Rhabditidae and Panagrolaimidae12, which are typical ‘Aufwuchs’species (i.e. species associated with decaying (phyto) detritus at the sediment surface). In the above-mentioned feeding type classifications, rhabditid and monhysterid nematodes are listed as bacterivores or deposit-feeders, but the latter term is a bit misleading when applied to nematodes and mostly implies feeding on micro-organisms. Controlled lab experiments and culture trials indeed prove that most of these nematodes are mainly bacterivorous (o.a. Vranken et al. 1988, Moens & Vincx 2000b). It is much less clear whether and to what extent these nematodes feed selectively. Their often very similar mouth morphology and dimensions suggests major overlap in resource spectra for the different species involved; in the framework of existing feeding type classifications, this equally implies substantial redundancy of species. In other words, it should not matter much to phytodetritus decomposition which or how many species are present, since species would be functionally ‘interchangable’. By contrast, recent experiments have shown that closely related bacterivorous nematode species respond in highly species-specific and resource-selective ways to bacterial food (Moens et al. 1999b), and affect bacterial community activity and structure in an equally species-specific way (De Mesel et al. 2003, 2004, 2006).

12 The Rhabditida comprise mostly terrestrial, freshwater and insect-parasitic species and only a few typical marine/estuarine ones. Several Panagrolaimidae, however, are very halotolerant and perform well under brackish to almost marine conditions. 62

Cultivation of bacterivorous nematodes

Inspite of their high diversity, no more than ca. 30 marine nematode species have hitherto been cultured in laboratory conditions, and nearly 90 % of those are monhysterids and rhabditids from ‘Aufwuchs’communities (Moens & Vincx 1998). These nematodes can relatively easily be isolated into monospecific cultures after inoculation of small pieces of phytodetritus or sediment on agar media. Nematode densities in monospecific, xenic cultures amount to hundreds or even thousands per ml of agar medium, and can be harvested either individually (by handpicking) or in mass (by a rinsing procedure). An overview of culture and handling procedures is given in Moens & Vincx (1998). Currently, the following species are available in permanent monospecific cultures at the Marine Biology Section of Gent University: Pellioditis marina, P. ehrenbaumi, Panagrolaimus paetzoldi, Geomonhystera disjuncta, Diplolaimelloides meyli, D. oschei and Diplolaimella dievengatensis (the first three are rhabditids, the others monhysterids), other species have been cultivated but not used for this study. P. marina appears a complex of several cryptic species which are now all being set up in separate cultures (Derycke, 2006). All these species stem from, or are common in, the polyhaline reach of the Westerschelde Estuary (SW Netherlands) and belong to the typical fauna of phytodetrital deposits in intertidal areas within this estuary.

Contrary to the bacterivorous nematodes used in our experiments, no permanent cultures of any predacious nematodes are currently available. However, several oncholaimid species (particularly Oncholaimus oxyuris and species of the genus Adoncholaimus) are abundant at the same sampling sites from which the bacterivore cultures originate. These sites are within a 1- h drive from the laboratory in Gent, and the named species can easily be isolated alive and remain active for up to several months in laboratory conditions (Moens & Vincx 1998). While predation is not the only feeding strategy of these nematodes (Moens et al. 1999c), recent stable-nitrogen-

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 4: Justifications. DOUTORADO EM CIÊNCIAS BIOLÓGICAS isotope evidence shows that they are indeed mainly feeding on microbivorous meta- and perhaps protozoans (Moens et al. 2004).

Bacteria

Most experiments were performed using a ‘bacterial inoculum’ in the stock. The Marine Biology Section of the Biology Department at Gent University currently has some 30 strains from the Westerschelde Estuary. However, the BCCM/LMG Culture Collection of the Laboratory for Microbiology at Gent University disposes of many more relevant strains. We could easily select a variety of strains which are easily culturable on ‘general’ growth media (such as Marine Agar) and which are typical for estuarine habitats, and make mixtures of a few to many strains. A relevant part of these strains have been generated during the first practical part of the first publication annex here.

The model system

Foodweb interactions, their effect on ecosystem processes, and the role of diversity herein are key topics in current-day ecological research (Duffy 2002). The core questions of the current proposal thus have much broader relevance than merely for the model system highlighted here. However, the choice of our model system is far from arbitrary, and based on considerations of (1) scientific relevance and (2) feasible. As a model system we have chosen communities of bacterivorous nematodes associated with phytodetritus in estuarine, intertidal habitats. Nematodes are the most abundant metazoans in soft sediments; they are potentially important consumers of both microalgae and bacteria (Montagna 1995), and are capable of affecting decomposition processes through interactions with bacteria (Johannes 1965, Abrams & Mitchell 1980, Findlay & Tenore 1982, Alkemade et al. 1992, De Mesel et al. 2003, 2004). Benthic nematode communities also tend to be very species-rich. The sections of Marine Biology and Aquatic Ecology of the Biology Department, Gent University, have ample experience with taxonomic, ecological and experimental research on nematodes and other taxa, with the culturing of selected (bacterivorous nematodes). This research group co-operate with the Laboratory of Microbiology at the same university. A shortlist of arguments in favour of this research model includes the following: - Marine/estuarine research is hitherto underrepresented in studies trying to link biodiversity and ecosystem functioning. - Population processes, on detritus foodwebs: many authors emphasize that neither single diversity-function relationship nor any universal set of species assembly rules exists; indeed all depends on the community and ecosystem function studied. Obviously, productivity of vegetations is a function of worldwide relevance, but it strongly depends on nutrient mineralization and decomposition processes. The relevance of up taken and assimilation processes are a core for well

understanding on ecosystem function, and can be therefore similarly used to predict species productivity itself. - Heterotrophic bacteria are nearly always the major decomposers and mineralisers of organic matter in aquatic sediments. Nematodes are among the major consumers of bacteria, and therefore likely to have important impacts on bacterial communities. For nematodes, the major groups which predominate on phytodetritus in aquatic sediments – rhabditids and monhysterids – are also characteristic of enriched freshwater and terrestrial habitats. Carnivorous nematodes are important predators of other nematodes and ciliates, not only in estuarine sediments, but also in terrestrial soils. - The relative ease with which a sufficient diversity of bacteria and nematodes can be cultured and maintained under lab conditions render controlled laboratory experiments with all major representatives of this model system feasible, even at short term. - The spatial scale at which the biodiversity of these ‘Aufwuchs’ communities, and by extension of micro- and meiobenthic communities, becomes manifest (mm2 tot cm2) allows manipulation and experimenting in a feasible microcosm approach. - The short generation times of all bacterivorous species we used (and intend to use) all ≤ 14 days. - The absence of studies on nematode population fitness processes, as well as the relative importance of population biomass measurement values different environment stress, has not been extensively studied up to now. - Top-down process on population as well as at community levels, are being used as an explanation of diversity increases under ecological pressure, and it can be easily tested under microcosm experiment.

References:

Abebe, E.; Traunspurger, W. and Andrássy, I. (2006). Freshwater Nematodes: Ecology and Taxonomy, CABI, Wallingford, UK, 572p.

Abrams, P. (1987). Indirect interactions between species that share a predator: varieties of indirect effects. Pages 38-54 in W. C. Kerfoot and A. Sih, editors. Predation: direct and indirect impacts on aquatic communities. University Press of New England, Hanover, New Hampshire, USA.

Abrams, P. (1992). Predators that benefit prey and prey that harm predators: unusual effects of interacting foraging adaptations. American Naturalist 140:573-600.

Abrams, B. I. and Mitchell, M. J. (1980). Role of bacterial nematode interactions in heterotrophic systems with emphasis on sewage sludge decomposition. Oikos 35: 404-410.

Alkemade, R.; Wielemaker, A.; de Jong, S. A., and Sandee, A.J.J. (1992). Experimental evidence for the role of bioturbation by the marine nematode Diplolaimella dievengatensis in stimulating the mineralization of Spartina anglica detritus. Mar Ecol Prog Series 90: 149–155.

Bastian, C. H. (1865). Monograph on the Anguillulidae, or free nematoids, marine, land, and freshwater. Trans. Linn. Soc. London 25.

Berlese, A. (1905). Apparecchio per raccogliere presto e in gran numero piccolo arthropodi. Redia 2.

Blaxter M. L.; De Ley P.; Garey, J.R.; Liu, L.X.; Scheldeman, P.; Vierstraete, A.; Vanfleteren, J. R.; Mackey, L.Y.; Dorris, M.; Frisse, L.M.; Vida, J.T. and Thomas, W. K. (1998). A molecular evolutionary framework for the phylum Nematoda. Nature. 392:71-75.

Bongers, T. and Ferris, H. (1999). Nematode community structure as a bioindicator in environmental monitoring. Trends in Ecology & Evolution 14: 224-228.

Bonkowski, M.; Cheng, W. X.; Griffiths, B. S.; Alphei, G. and Scheu, S. (2000). Microbial-faunal interactions in the rhizosphere and effects on plant growth. Eur. J. Soil Biol. 36: 135-147.

Borel, P. (1656) De vero telescopii inventore etc. the Hague.

Bosman, A.L.; Dutoit, J.T.; Hockey, P.A.R. and Branch, G.M. (1986). A field experiment demonstrating the influence of seabird guano on intertidal primary production. Est. Coastal Shelf Sci. 23, 283–294.

Bosman, A.L., Hockey, P.A.R., (1986). Seabird guano as a determinant of rocky intertidal community structure. Mar. Ecol. Prog. Ser. 32, 247–257.

Botelho, A.P.; Da Silva, M. C.; Esteves, A.M. and Fonseca-Genevois, V., (2007). Four new species of Sabatieria Rouville, 1903 (Nematoda, Comesomatidae) from the Continental Slope of Atlantic Southeast. Zootaxa (1402): 39-57

Boucher, G. (1972). Distribution quantitative et qualitative des nematodes d'une station de vase terrigene côtière de Banyuls-sur-Mer. Cahiers de Biologie Marine 13:457-474.

Boucher, G. and Lambshead, P. J. D. (1995). Ecological biodiversity of marine nematodes in samples from temperate, tropical and deep-sea regions. Conservation Biology 9(6):1594-1604.

Brakenhoff, H., (1913) Beitrag zur Kenntnis der Nematodenfauna des nordwestdeutschen Flachlandes. Abh. Naturw. Ver. Bremen 22.

Branch, G.M.; Barkai, A.; Hockey, P.A.R. and Hutchings, L. (1987). Biological interactions: causes or effects of variability in the Benguela ecosystem? S. Afr. J. Mar. Sci. 5, 425–445.

Bütschli, O. (1873). Beitrage zur Kenntnis der freilebenden Nematoden. Nova Acta Acad. Leopold.-Car. 26.

Caffey, H. M. (1985). Spatial and temporal variation in settlement and recruitment of intertidal barnacles. Ecol. Monogr. 55, 313–332.

Caley, M.J.; Carr, M.H.; Hixon, M.A.; Hughes, T.P.; Jones, G.P. and Menge, B.A. (1996). Recruitment and the local dynamics of open marine populations. Ann. Rev. Ecol. Syst. 27, 477–500.

Carpenter, S. R.; Kitchell, J. F. and Hodgson, J. R. (1985). Cascading trophic interactions and lake productivity. BioScience 35, 634–639.

Carter, H. J. (1859). On Dracunculus and microscopic Filariidae in the Island of Bombay. Ann. Mag. Nat. Hist. 3. ser. 4.

Castilla, J.C. and Duran, L. R. (1985). Human exclusion from the rocky intertidal zone of central Chile: the effects on Concholepas concholepas (Gastropoda). Oikos 45, 391–399.

Castro, F. J. V; Bezerra, T. N. C.; Da Silva, M. C. and Fonseca-Genevois, V. (2006). Spirinia elongata, sp. nov. (Nematoda, Desmodoridae) from Pina Basin, Pernambuco, Brazil. Zootaxa, 1121: 53-68.

Chen, B. and Wise, D.H., (1999). Bottom-up limitation of predaceous arthropods in a detritus-based terrestrial food web. Ecology 80: 761–772.

Chia, F. S. and Warwick, R. M. (1969). Assimilation of Labelled Glucose from Seawater by Marine Nematodes. Nature 224:720 – 721.

Chitwood, B. G. and Chitwood, M. B. (1937). An introduction to nematology. Baltimore.

Chitwood, B. G. and Chitwood, M. B. (1950). An introduction to nematology. Baltimore, University Park Press.

Christie, J.R. (1959). Plant Nematodes. Their bionomics and control, 256p, Un. Fla. ed. Gainesville, USA.

Cobb, N. A. (1917). Notes on nemas. Contrib. Sci. Nematol. 5:117-28

Connell, J.H. (1961). Effects of competition, predation by Thais lapillus, and other factors on natural populations of the barnacle Balanus balanoides. Ecol. Monogr. 31, 61–104.

Connell, J.H. (1970). A predator–prey system in the marine intertidal region. I. Balanus glandula and several predatory species of Thais. Ecol. Monogr. 40: 49–78.

Connell, J. H. (1983). On the prevalence and relative importance of interspecific competition: evidence from field experiments. American Naturalist 122: 66 1-696.

Coull, B. C. (1999). Role of meiofauna in estuarine soft-bottom habitats. Austr. J. Ecol. 24: 327-343.

Czernay, A. (1849). Monographie des Essigalchens. BulL de Moscou 22.

Da Rocha, C.M.C.; Venekey, V.; Bezerra, T.N.C. and Souza, J.R.B. (2006). Phytal marine nematode assemblages and their relation with the macrophytes structural complexity in a Brazilian tropical rocky beach. Hydrobiologia, 553: 219-230.

Dayton, P.K., (1971). Competition, disturbance, and community organization: the provision and subsequent utilization of space in a rocky intertidal community. Ecol. Monogr. 41, 351–389.

Dayton, P.K.; Currie,V.; Gerrodette, T.; Keller, B.D.; Rosenthal, R. and Ven Tresca, D., (1984). Patch dynamics and stability of some California kelp communities. Ecol. Monogr. 54, 253–289.

Dayton, P.K. and Tegner, M.J., (1984). Catastrophic storms, El Ninõ, and patch stability in a southern California kelp forest community. Science 224, 283–285.

Dayton, P.K.; Tegner, M.J.; Edwards, P.B. and Riser, K.L. (1999). Temporal and spatial scales of kelp demography: the role of oceanographic climate. Ecol. Monogr. 69, 219–250.

Dayton, P.K.; Tegner, M.J.; Parnell, P.E. and Edwards, P.B., (1992). Temporal and spatial patterns of disturbance and recovery in a kelp forest community. Ecol. Monogr. 62, 421–445.

De Coninck, J., (1930). Over de oekologische verspreiding van vrijlevende nematoden in Belgie. BoL J aarb. 22.

De Ley, P. and Blaxter, M. (2002). A new system for Nematoda: combining morphological characters with molecular trees, and translating clades into ranks and taxa. Nematology. 4(2):141-142.

De Ley, P.; De Ley, I; Morris, K.; Abebe, E.; Ocampo, M.; Yoder, M.; Heras, J.; Waumann, D.; Rocha-Olivares, A.; Burr, A.; Baldwin, J. and Thomas, W. (2005). An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. phil trans r soc B360:1945-1958.

De Man, J. G., (1876). Onderzoekingen over vrij in de aarde levende nematoden. Tijdschr. Nederl. Dierk. Vereen. 2.

De Man, J. G., (1880). Die einheimischen frei in der reinen Erde und im siissen Wasser lebenden Nematoden. Ibid. 5.

De Man, J. G. (1881). Ueber einige neue oder noch unvollstandig bekannte Arten von frei in der reinen Erde lebenden Nematoden Ibid. 5.

De Man, J. G. (1884). Die frei in der reinen Erde nnd im siissen Wasser lebenden Nematoden der niederlandischen Fauna. Leiden.

De Man, J. G. (1889). tlber zwei in der feuchten Erde lebende Arten der Gattung Oncholaimus Duj. Ibid. (2) 2.

De Man, J. G. (1906). Observations sur quelques especes de Nematodes terrestres libres de l'ile de Walcheren. Ann. Soc. Zool. Malacol. Belg. 41.

De Man, J. G. (1907). Contributions a la connaisMnce de nematodes libres de la Seine et des environs de Paris. Ann. BioI. Lac. 2.

De Man, J. G. (1912). Helminthologische Beitliige. Zool Jahrb. Abt. Syst. 15.

De Man, J. G. (1917). Beitrag zur Kenntnis der in Norwegen frei in der reinen Erde lebenden Nematoden. Tijd. Ned. Dierk. Vereen. 16

De Mesel, I.; Derycke, S.; Moens, T.; Van der Gucht, K.; Vincx, M. and Swings, J. (2004). Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study. Environ. Microbiol. 6: 733-744.

De Mesel, I.; Derycke, S.; Swings, J.; Vincx, M. and Moens, T. (2003). Influence of bacterivorous nematodes on the decomposition of cordgrass. J. Exp. Mar. Biol. Ecol. 296: 227-242

De Mesel, I.; Derycke, S.; Swings, J.; Vincx, M. and Moens, T. (2006). Role of nematodes in decomposition processes: does within-trophic group diversity matter? Mar. Ecol. Progr. Ser. 321:157-166.

De Troch, M.; Van Gansbeke, D. and Vincx, M. (2006). Resource availability and meiofauna in sediment of tropical seagrass beds: Local versus global trends. Marine Environmental Research 61:59-73.

Denley, E.J. and Underwood, A.J. (1979). Experiments on factors influencing settlement,. survival and growth in two species of barnacle in New South Wales. J Exp Mar. Biol. Ecol. 36:269–273.

Derycke, S. and Backeljau, T. and Vlaeminck, C. and Vierstraete, A. and Vanfleteren, J. and Vincx, M. and Moens, T. (2006). Seasonal dynamics 72

of population genetic structure in cryptic taxa of the Pellioditis marina complex (Nematoda: Rhabditida) Genetica 128 (1) 307-321.

Dethier, M. N. and D. 0. Duggins. (1984). An "indirect commensalism" between marine herbivores and the importance of competitive hierarchies. American Naturalist 124:205-219.

Diehl, S. (1992). Fish predation and benthic community structure: the role of

omnivory and habitat complexity. Ecology 73, 1646–1661.

Diehl, S. (1993). Effects of habitat structure on resource availability, diet and growth of benthivorous perch, Perca fluviatilis. Oikos 67:403-414.

Diem, K. (1903). Untersuchungen iiber die Bodenfauna in den Alpen. Jb. St. Gall. Natur-wlss. Ges. 234--414.

Diesing, K. M. (1851). Systema Helminthum U1. Vindobonae., vol. 2, 588 pp.

Diesing, K. M. (1861). Systema Helminthum II und Revision der Nematoden. Sitz.ber. d. math. naturw. CL d. Wiener Akad. 42.

Ditlevsen, H. J. (1911). Danish heeliving nematodes. Vid. Medd. D. Nat. For. 63.

Dotterweich, H. (1938). Die Ziichtung von Rhabditis teres (A. Schn.) für physiologische und genetische Untersuchungen. Zool. Anz. 122.

Dougherty, E. S. and Calhoun, H. G. (1948). Experiences in culturing Rhabditis pellio (Schneider (1866) Biitschli (1873, Nematoda: Rhabditidae) and related soil nematodes. Proc. Helm. Soc. Wash. 15.

Duffy, J. E. (2002). Biodiversity and ecosystem function: the consumer connection. Oikos 99:201-219.

Duffy, J. E.; Richardson, J. P. and Canuel, E. A. (2003). Grazer diversity effects on ecosystem functioning in seagrass beds. Ecol. Lett. 6: 637-645.

Dujardin, F. (1845). Histoire naturelle des helminthes ou vers intestinaux. Paris.

Dungan, M.L. (1986). Three-way interactions: barnacles, limpets, and algae in a Sonoran Desert rocky intertidal zone. Am. Natur. 127, 292–316.

Dungan, M.L. (1987). Indirect mutualism: complementary effects of grazing and predation in a rocky intertidal community. In: Kerfoot, W.C., Sih, A. (Eds.), Predation: Direct and Indirect Impacts on Aquatic Communities. University Press of New England, Hanover, New Hampshire, pp. 188–200.

Duran, L.R. and Castilla, J.C. (1989). Variation and persistence of the middle rocky intertidal community of central Chile, with and without human harvesting. Mar. Biol. 103, 555–562.

Dusenbery, D. B. (1996). Life at small scale: the behavior of microbes. New York: Scientific American Library.

Eberth, C. J. (1863). Untersuchungen ueber Nematoden. Leipzig. 77 pp.

Ebling, F.J.; Kitching, J.A.; Muntz, L. and Taylor, C.M. (1964). The ecology of Lough Ine XIII. Experimental observations of the destruction of Mytilus edulis and Nucella lapillus by crabs. J. Anim. Ecol. 33, 73–82.

Ekschmitt, K. and Griffiths, B. S. (1998). Soil biodiversity and its implications for ecosystem functioning in a heterogeneous and variable environment. App. Soil Eco. 10 (3): 201-215.

Esteves, A. M. (2004). Free-living marine nematodes from Coroa Grande tidal flat (Sepetiba Bay, Rio de Janeiro, Brazil). Biociências, 12 (2): 185-186.

Esteves, A.M.; Maria, T. F. and Wandeness, A. M. (2003). Population structure of Oncholaimus cobbi (Kreis, 1932) in a tropical tidal flat. J. Mar. Biol. Ass. U. K. 83: 903-904.

Esteves, A.M.; Maria, T. F. and Wandeness, A. M. (2004). Population structure of Comesoma arenae Gerlach (Nematoda: Comesomatidae) in a 74

Brazilian tropical tidal flat, Rio de Janeiro, Brazil. Rev. Bras. Zool. 21 (4): 775-777.

Fairweather, P.G. (1985). Differential predation on alternative prey, and the survival of rocky intertidal organisms in New South Wales. J. Exp. Mar. Biol. Ecol. 89: 135–156.

Fairweather, P.G. (1988). Consequences of supply-side ecology: manipulating the recruitment of intertidal barnacles affects the intensity of predation upon them. Biol. Bull. 175: 349–354.

Fairweather, P G. (1990). Is predation capable of interacting with other community processes on rocky reefs? Australian Journal of Ecology 15: 453-464.

Fairweather, P.G.; Underwood, A.J. and Moran, M.J. (1984). Preliminary investigations of predation by the whelk Morula marginalba. Mar. Ecol. Prog. Ser. 17, 143–156.

Fawcett, M.H. (1984). Local and latitudinal variation in predation on an herbivorous marine snail. Ecology 65, 1214–1230.

Filipjev, I. N. and Schuurmans Stekhoven, J. H. (1941). A manual of agricultural helminthology. Leiden.

Findlay, S. and Tenore, K. R. (1982). Effect of a free-living marine nematode (Diplolaimella chitwoodi) on detrital carbon mineralization. Mar. Ecol. Prog. Ser. 8: 161-166.

Fonseca, G.; Decraemer, W. and Vanreusel, A. (2006). Taxonomy and species distribution of the genus Manganonema Bussau, (1993) (Nematoda: Monhysterida). Cah. Biol. Mar. 47: 189-203.

Forsslund, K.H. (1943). Studien liber die Tierwelt des Nordschwedischen Waldbodens,. Medd. Statens Skogsforsoksanst. 34.

Franz, H. A. (1942). Untersuchungen liber die Kleintierwelt ostalpiner Boden. 1. Die heilebenden Erdnematoden. ZooL J ahrb. Abt. Syst. 75.

Franz, H. A and Beier, M. (1942). Zur Kenntnis der Bodenfauna im pannonischen Klimagebiet der Ostmark. L Die freilebenden Erdnematoden. Zool Jahrb, Abt. Syst. 75.

Fraser, L. H. (1998). Top-Down vs Bottom-Up Control Influenced by Productivity in a North Derbyshire, UK, dale. Oikos, 81 (1): 99-108.

Fraser, L.H. and Grime, J.P., (1997) Primary productivity and trophic dynamics investigated in a North Derbyshire, UK, dale. Oikos 80, 499– 508.

Fretwell, S. D. (1977). The regulation of plant communities by food chains exploiting them. Persp. Biol. Med. 20, 169–185.

Fretwell, S. D. (1987) Food chain dynamics: the central theory of ecology? Oikos 50, 291–301.

Gaines, S.D. and Roughgarden, J. (1985). Larval settlement rate: a leading determinant of structure in an ecological community of the marine intertidal zone. Proc. Natl. Acad. Sci. USA 82: 3707–3711.

Garrity, S.D. and Levings, S. C. (1981). A predator–prey interaction between two physically and biologically constrained tropical rocky shore gastropods: direct, indirect, and community effects. Ecol. Monogr. 51, 267–286.

Genevois, V. F.; Somerfield, P.J.; Neves M. H. B.; Coutinho, R. and Moens, T. (2006). Colonization and early succession on artificial hard substrata by meiofauna. Marine Biology 148: 1039–1050.

Genevois, V.; Santos, G. A. P; Castro, F. J. V.; Almeida, T. C. M. and Coutinho, R. (2004). Biodiversity of marine nematodes from an atypical

tropical costal area affected by upwelling (Rio de Janeiro, Brazil). Meiofauna Marina, 13: 37-44.

Geraert, E. (1980). The female reproductive system in nematode systematics. Annales Soc. r. Zool. Belg.110.2:73-86.

Gerlach, S. A. (1954). Freilebenden Nematoden Aus Der Lagoa Rodrigo De Freitas (Rio de Janeiro). Zoologischer Anzeiger¸ 153: 135-143.

Gerlach, S. A. (1956a). Brasilianische Meeresnematoden I. Boletim do Instituto Oceanográfico da Universidade de São Paulo, 5: 3-69.

Gerlach, S. A. (1956b). Die Nematodenbeseiedlung Des Tropischen Brandungsstrandes Von Pernambuco, Brasilianische Meeres Nematoden II. Kieler Meeresforschungen, 12(2): 202-218.

Gerlach, S. A. (1957a). Marine Nematoden Aus Dem Mangrove-Gebiet Voncananéia (Brasilianische Meeres-Nematoden III. Jahrbuch Der AkademieDer Wissenschaften Und Der Literatur In Mainz, 5: 129-176.

Gerlach, S. A. (1957b). Die Nematodenfauna Des Sandstrandes Na Der Küste Von Mittelb (Brasilianische Meeres-Nematoden IV. Mitteilungen Aus Dem Zoologischen Museum In Berlin, 33(2): 411-459.

Gerlach, S.A. (1980). Development of marine nematode taxonomy up to 1979. Veröff. Inst. Meeresforsch. Bremerh.18:249-255.

Godfray, H. C. J. and Lawton, J. H. (2001). Scale and species numbers. Trends Ecol. Evol. 16: 400-404.

Goodey, T. (1933). Plant Parasitic nematodes and the disease they cause. London

Goodey, T. (1951). Soil and Freshwater Nematodes. John Wiley and Sons, Inc. New York, N.Y.

Goodey, T. rewritten by Goodey J.B. (1963). Soil and Freshwater Nematodes. Methuen, London. 544 pp.

Groffman, P.M. (1997). Global biodiversity: is it in the mud and the dirt? Trends Ecol. Evol. 12: 301-302.

Hairston, N. G.; Smith, F.E.; Slobodkin, L.B. (1960). Community structure, population control, and competition. Am. Natur. 94: 421–425.

Hamels, I.; Moens, T.; Muylaert, K.; Vyverman, W. (2001). Trophic interactions between ciliates and nematodes from an intertidal flat. Aquatic Microb Ecol 26: 61-72.

Heinis, F., (1916). Über die Mikrofauna am Bolchen (Liestal). Tiitigkeitsber. Nat. Ges. Baselland, 1911-16.

Heip, C. (2002). Data requirements for biodiversity indicators. In VLIZ special publication: The colour of ocean data. International symposium on oceanographic data and infromation management with special attention to biological data.11: 17.

Heip, C.; Willems, K.A.; Goossens, A. (1977). Vertical distribution of meiofauna and the efficiency of the Van Veen grab on sandy bottoms in Lake Grevelingen (The Netherlands). Hydrobiological Bulletin. 11. 35-45.

Heip, C.; Vincx, M. and Vranken, G. (1985). The ecology of marine nematodes. Oceanogr. Mar. Biol. Annu. Rev. 23: 399-489.

Heip, C. H. R.; Goosen, N.K.; Herman, P. M. J.; Kromkamp, J.; Middelburg, J. J. and Soetaert, K. (1995). Production and consumption of biological particles in temperate tidal estuaries. Oceanogr. Mar. Biol. Annu. Rev. 33: 1-149.

Hilgermann, R. and Weisseenberg, R. (1918). Nematodenziichtung auf Agarplatten. entralbL Bakt. Abt. I, Orig. 80. Hofmanner, B., (1913) Beitriige zur Kenntnis der freilebende Nematoden. Zool. Anz. 42.

Hillebrand, H. (2003). Opposing effects of grazing and nutrients on diversity. Oikos 100: 592-600.

Hillebrand, H.; Worm, B. and Lotze, H. K. (2000). Marine microbenthic community structure regulated by nitrogen loading and grazing pressure. Mar. Ecol. Prog. Ser. 204: 27-38.

Hofmäner, B. (1913). Beitriäge zur Kenntnis der freilebende Nematoden. Zool..Anz. 42.

Holt, R. D. (1977). Predation, apparent competition, and the structure of prey communities. Theoretical Population Biology 12: 197-229.

Huston, M. (1979). A general hypothesis of species diversity. Am. Nat. 113: 81-101. Importance. Ecological Monographs, Vol. 65, No. 1, 21-74.

Huston, M. (1999). Local processes and regional patterns: appropriate scales for understanding variation in the diversity of plants and animals. Oikos 86: 393-401.

Jensen, P. (1987). Feeding ecology of free-living aquatic nematodes. Mar. Ecol. Prog. Ser. 35: 187-196.

Johannes, R. E. (1965). The influence of marine protozoa on nutrient regeneration. Limnol. Oceanogr. 12: 189-195.

Johnston, T. H., (1938). A census of the freeliving and plant-parasitic nematodes recorded as occurring in Australia. Trans. Roy. Soc. S. Austr. 62, 1.

Kerfoot, W. C. and Sih, A. editors. (1987). Predation: direct and indirect impacts on aquatic communities. University Press of New England, Hanover, New Hampshire, USA.

Kitching, J.A.; Sloane, J.F. and Ebling, F.J., (1959). The ecology of Lough Ine VIII. Mussels and their predators. J. Anim. Ecol. 28, 331–341. 79

Kondoh, M. (2001). Unifying the relationships of species richness to productivity and disturbance. Proc. R. Soc. Lond. B 268: 269-271.

Kuipers, B. R.; de Wilde, P. A. W. J. and Creutzberg, F. (1981). Energy flow in a tidal flat ecosystem. Mar. Ecol. Prog. Ser. 5: 215-221.

Lapage, G., (1937). Nematodes parasitic in animals. London. X, 172 p.

Lawler, S.P. and Morin, P.J., 1993. Food web architecture and population dynamics in laboratory microcosms of protists. Am. Natur. 141: 675–686.

Lawton, J. H. (1999). Are there general laws in ecology? Oikos 84: 177-192.

Levin, S.A. (1992). The problem of pattern and scale in ecology. Ecology 73, 1943–1967.

Levine, S. H. (1976). Competitive interactions in ecosystems. American Naturalist 110:903-910.

Lively, C. M. and Raimondi, P.T., (1987). Desiccation, predation, and mussel– barnacle interactions in the northern Gulf of California. Oecologia (Berl.) 74, 304–309.

Loos, A. (1911). The anatomy and life-history of Anchylostoma duodenale Dub. II. The development in the free state. Rec. Schl. Med. Min. Educ. Egypt. 4.

Lopez, G.; Riemann, F. and Schrage, M. (1979). Feeding biology of the brackish-water oncholaimid nematode Adoncholaimus thalassophygas. Mar. Biol. 54 (4): 311-318.

Loreau, M. (2000). Biodiversity and ecosystem functioning: recent theoretical advances. Oikos 91:17.

Loreau, M.; Naeem, S.; Inchausti, P.; Bengtsson, J.; Grime, J. P.; Hector, A.; Hooper, D. U.; Huston, M. A.; Raffaelli, D.; Schmid, B.; Tilman, D. and

Wardle, D. A. (2001). Biodiversity and ecosystem functioning: current knowledge and future challenges. Science 294: 804-808.

Loreau, M. and Olivieri, I. (1999). Diversitas: an international programme of biodiversity. Science 292 (1):2-3.

Lorenzen, S. (1979). New and known gonadal characters in free-living nematodes and the phylogenetic implications. Annales Soc. r. Zool. Belg.108. 75.

Lorenzen, S. (1985). Phylogenetic aspects of pseudocoelomate evolution. The origins and relationships of lower invertebrates. 28. 210-223.

Lorenzen, S. (1994). The phylogenetic systematic of freeliving nematodes. The Ray Society, London, UK. 383p.

Lubchenco, J. and Menge, B.A., (1978). Community development and persistence in a low rocky intertidal zone. Ecol. Monogr. 48, 67–94.

Lubchenco, J.; Menge, B.A.; Garrity, S.D.; Lubchenco, P.J.; Ashkenas, L.R.; Gaines, S.D.; Emlet, R.; Lucas, J. and Strauss, S. (1984). Structure, persistence, and role of consumers in a tropical rocky intertidal community (Taboguilla Island, Bay of Panama). J. Exp. Mar. Biol. Ecol. 78, 23–73.

Luckens, P.A. (1970). Breeding, settlement and survival of barnacles at artificially modified shore levels at Leigh, New Zealand. New Zealand J. Mar. Freshw. Res. 4, 497–514.

Luckens, P.A. (1975). Predation and intertidal zonation of barnacles at Leigh. New Zealand. New Zealand J. Mar. Freshw. Res. 9, 355–378.

Ludwig, H. (1938). Die Variabilitiit von Rhabditis teres (A,. Schn) unter veranderten Erniihrungsbedingungen. Z. Wiss, ZooI. 151.

Marcinowsky, K. (1906). Zur Biologie und Morphologie von Cephalobus elongatus de Man und Rhabditis brevispina Claus nebst Bemerkungen

iiber einige andere Nematodenarten,. Arb. bioI. Anst. Land- und ForstwiItsch. 5.

Marcinowsky, K. (1909). Parasitisch und semiparasitisch an Pflanzen lebenden Nematoden. Ibid. 7.

Maria, T. F.; Silva, N. R. R.; Wandeness,, A. P. and Esteves, A. M. (2008) Spatio-temporal study and population structure of Daptonema oxycerca (Nematoda: Xyalidae) in Coroa Grande, Rio de Janeiro, Brazil. Brazilian Journal of Oceanography 56 (1): 41-50.

Maria; T. F., Esteves, A. M.; Smol, N.; Vanreusel, A. & Decraemer, W. Chromaspirina guanabarensis sp. n. (Nematoda: Desmodoridae) and a new illustrated dichotomous key to Chromaspirina species. Zootaxa (in press).

Marquis, R. J. and Whelan, C.J. (1994). Insectivorous birds increase growth of white oak through consumption of leaf-chewing insects. Ecology 75, 2007–2014.

Maupas, A. S. E. (1901). Modes et formes de reproduction des nematodes, Ibid. 8.

Maupas, A. S. E. (1900). La mue et l'enkystement chez les nématodes. Arch. Zool. Exp, et Gén 3. Sél. 7.

May, R. M. 1973. Stability and complexity in model ecosysterns, Princeton Monographs in Population Biology 6. Princeton University Pres\, Princeton, New Jersey, USA.

McCoy, O. R., (1929). The suitability of various bacteria as food for hookworm larvae. Amel. J. Hyg. 10.

McIntyre, A. D. and Murison, D. J. (1973). The meiofauna of a flatfish nursery ground. J. Mar. Biol. Ass. UK 53: 93-118.

Menge, B.A. (1991) Relative importance of recruitment and other causes of variation on rocky intertidal community structure. J. Exp. Mar. Biol. Ecol. 146, 69–100.

Menge, B.A. (1992). Community regulation: under what conditions are bottom-up factors important on rocky shores? Ecology 73, 755–765.

Menge, B. A. (1995). Indirect Effects in Marine Rocky Intertidal Interaction Webs: Patterns and Importance. Ecological Monographs 65(1): 21-74.

Menge, B. A. (2000). Top-down and bottom-up community regulation in marine rocky intertidal habitats. J. Exp. Mar. Biol. and Ecol. 250: 257–289.

Menge, B.A.; Daley, B.A.; Lubchenco, J.; Sanford, E.; Dahlhoff, E.; Halpin, P.M.; Hudson, G. and Burnaford, J.L., (1999). Top-down and bottom-up regulation of New Zealand rocky intertidal communities. Ecol. Monogr. 69, 297–330.

Menge, B. A.; Daley, B. A and Wheeler, P. A . (1996). Control of interaction strength in marine benthic communities. In G. A. Polis and K. O. Winemiller (eds.), Food webs: Integration of pattern and dynamics, 258– 274.

Menge, B.A. and Lubchenco, J., (1981). Community organization in temperate and tropical rocky intertidal habitats: prey refuges in relation to consumer pressure gradients. Ecol. Monogr. 51, 429–450.

Menge, B.A., Lubchenco, J., Ashkenas, L.R., Ramsey, F., (1986a.) Experimental separation of effects of consumers on sessile prey in the low zone of a rocky shore in the Bay of Panama: direct and indirect consequences of food web complexity. J. Exp. Mar. Biol. Ecol. 100, 225– 269.

Menge, B.A., Lubchenco, J., Gaines, S.D., Ashkenas, L.R., (1986b). A test of the Menge–Sutherland model of community organization in a tropical rocky intertidal food web. Oecologia (Berl.) 71, 75–89.

Menge, B.A., Olson, A.M., (1990). Role of scale and environmental factors in regulation of community structure. Trends Ecol. Evol. 5, 52–57.

Menge, B. A.; Olson, A. M. and Dahlhoff E. P. (2002). Environmental Stress, Bottom-up Effects, and Community Dynamics: Integ. And Comp. Biol. 42:892–908.

Menge, B. A.. and Sutherland, J. P. (1976). Species diversity gradients: synthesis of the roles of predation, competition and temporal heterogeneity. American Naturalist 110:351- 369

Menge, B, A, and Sutherland, J, P. (1987). Community regulation: variation in disturbance, competition, and predation in relation to gradients of environmental stress and recruitment, American Naturalist. 130:730-757.

Menzel, R. (1914). Dber die mikroskopische Landfauna der schweizerischen Hochalpen. Arch" Naturgesch. A.

Menzel, R., (1920). Dber die Nahrung der heilebenden Nematoden und die Art ihrer Aufnahme. Verh, Natur£. Ges. Basel 31.

Micoletzky, H., (1921). Die heilebenden Nematoden, Arch Naturgesch. 87 A.

Micoletzky, H., (1925). Die heilebenden Süsswasser- und Moornematoden Dänemarks. K.. D. Vid. Selsk. SkL Nat. og Mat. Afd. 8. Rk.. 10, 2

Mikola, J. and Setälä, H. (1998a). No evidence of trophic cascades in an experimental microbial-based soil food web. Ecology 79: 153-164.

Mikola, J. and Setälä, H. (1998b). Relating species diversity to ecosystem functioning: mechanistic backgrounds and experimental approach with a decomposer food web. Oikos 83: 180-194.

Minchinton, T.E. and Scheibling, R.E., (1991). The influence of larval supply and settlement on the population structure of barnacles. Ecology 72, 1867–1879.

Minchinton, T.E. and Scheibling, R.E. (1993). Free space availability and larval substratum selection as determinants of barnacle population structure in a developing rocky intertidal community. Mar. Ecol. Prog. Ser. 95, 233–244.

Moens, T.; Herman, P. M. J.; Verbeeck, L.; Steyaert, M. and Vincx, M. (2000). Predation rates and prey selectivity in two predacious estuarine nematodes. Mar. Ecol. Prog. Ser. 205: 185-193.

Moens, T.; Santos, G. A.P. dos; Thompson, F.; Swings, J.; Fonsêca- Genevois, V.; Vincx, M. and De Mesel, I. (2005). Do nematode secretions affect bacterial growth? Aquat Microb Ecol 40:77-83. Moens, T., Bergtold, M., Traunspurger, W., 2006. Chapter 6: Feeding ecology of free-living benthic nematodes. In: Eyualem, A., Andrássy, I., Traunspurger, W. (Eds.), Freshwater nematodes: ecology and taxonomy. CAB International Publishing, Cambridge, USA, pp. 105-131.

Moens, T.; Van Gansbeke, D.and Vincx, M. (1999a). Linking estuarine intertidal nematodes to their suspected food. A case study from the Westerschelde Estuary (south-west Netherlands). J. Mar. Biol. Ass. U.K. 79: 1017-1027.

Moens, T.; Verbeeck, L.; De Maeyer, A.; Swings, J.and Vincx, M. (1999b). Selective attraction of marine bacterivorous nematodes to their bacterial food. Mar. Ecol. Prog. Ser. 176: 165-178.

Moens, T.; Verbeeck, L.and Vincx, M. (1999c). Feeding biology of a predatory and a facultatively predatory marine nematode (Enoploides longispiculosus and Adoncholaimus fuscus). Mar. Biol. 134: 515-523.

Moens, T.; Vierstraete, A.and Vincx, M. (1996a). Life strategies in two bacterivorous marine nematodes: Preliminary results. PSZN I Mar. Ecol. 17: 509-518.

Moens, T.; Coomans, A. and De Waele, D. (1996b). Effect of temperature on the in vitro reproduction of Aphelenchoides rutgersi. Fundam. appl. Nematol. 19 (1): 47-52

Moens, T. and Vincx, M. (1997). Observations on the feeding ecology of estuarine nematodes. J. Mar. Biol. Ass. U. K. 77: 211-227.

Moens, T. and Vincx, M. (1998). On the cultivation of free-living marine and estuarine nematodes. Helgoländer Meerseunters. 52: 115-139.

Moens, T.and Vincx, M. (2000a). Temperature and salinity constraints on the life cycle of two brackish-water nematode species, Pages 115-135. J. Exp. Mar. Biol. Ecol. 243: 115-135.

Moens, T. and Vincx, M. (2000b). Temperature, salinity and food thresholds in two brackish-water bacterivorous nematode species: assessing niches from food absorption and respiration experiments. J. Exp. Mar. Biol. Ecol. 243: 137-154.

Moens, T.; Yeates, G. W. and De Ley, P. (2004). Use of carbon and energy sources by nematodes. Nematology Monographs and Perspectives - Proceedings of the fourth International Congress of Nematology, 8-13 June 2002, Tenerife, Spain 2:529-545.

Montagna, P. A. (1995). Rates of metazoan meiofaunal microbivory: a review. Vie Milieu 45: 1-9.

Morin, P.J.; Lawler, S.P. (1996). Effects of food chain length and omnivory on population dynamics in experimental food webs. In: Polis, G.A., Winemiller, K.O. (Eds.), Food Webs: Integration of Patterns and Dynamics. Chapman & Hall, New York, NY, pp. 218–230.

Müller, O. F., (1756). Vermium terrestrium et fluuiatilium, seu Animalium infusoriorum, helminthicorum et testaceorum, non marinorum, succincta historia. V.4 2: 1773-1774.

Myers, BJ (1960). On the morphology and life history of Phocanema decipiens (Krabbe, 1878) Myers, 1959 (Nematoda: Anisakidae). Can. J. Zool. 38: 331–344.

Naeem, S. and Wright, J. P. (2003). Disentangling biodiversity effects on ecosystem functioning: deriving solutions to a seemingly insurmountable problem. Ecol. Lett. 6: 567-579.

Netto, S. A. and Gallucci, F. (2003). Meiofauna and macrofauna communities in a mangrove from the Island of Santa Catarina, South Brazil. Hydrobiologia, 505: 159-170.

Netto, S. A.; Gallucci, F. and Fonseca, G. F. C. (2005). Meiofauna communities of continental slope and deep-sea sites off SE Brazil. Deep Sea Research, 52: 845-859.

Nielsen, C.O. (1949). Studies on the Soil Microfauna II: The Soil Inhabiting Nematodes Naturhistorisk Museum. 1-131.

Nombela, G.; Navas, A. and Bello, A. (1999). Nematodes as bioindicators of dry pasture recovery after temporary rye cultivation. Soil Biol and Bioch 31:535-541.

Oerley, L., (1880). Az anguillulidak maganrajza. A kir. m. termeszettudom. tersulat altal a bugatdijjal jutalmazott palyamii. Termeszetr. Fuz., 4 : 16-50.

Oerley, L.. (1886). Die Rhabditiden und ihre medizinische Bedeutung "Berlin" 47p.

Oksanen, L. (1983). Trophic exploitation and arctic phytobiomass patterns. Am. Natur. 122, 42–52.

Oksanen, L., (1988). Ecosystem organization: mutualism and cybernetics or plain Darwinian struggle for existence? Am. Natur. 131, 424–444.

Oksanen, L.; Fretwell, S.D.; Arruda, J. and Niemela, P. (1981). Exploitation ecosystems in gradients of primary productivity. Am. Natur. 118, 240–261. 87

Osenberg, C.W. and Mittelbach, G.G. (1996). The relative importance of resource limitation and predator limitation in food chains. In: Polis, G.A., Winemiller, K.O. (Eds.), Food Webs: Integration of Patterns and Dynamics. Chapman & Hall, New York, NY, pp. 134–148.

Paine, R. T. (1966). Food web complexity and species diversity. American Naturalist 100: 65-75.

Paine, R. T. (1969). The Pisaster-Tegula Interaction: prey patches, predator food preference, and intertidal community structure. Ecology 50: 950-96 1.

Paine, R.T. (1971). A short-term experimental investigation of resource partitioning in a New Zealand rocky intertidal habitat. Ecology 52, 1096– 1106.

Paine, R.T. (1974). Intertidal community structure: experimental studies on the relationship between a dominant competitor and its principal predator. Oecologia 15, 93–120.

Paine, R. T. (1980). Food webs: linkage, interaction 'strength and community infrastructure. Journal of Animal Ecology 49: 667-685.

Paine, R.T. (1986). Benthic community–water column coupling during the 1982–1983 El Ninõ. Are community changes at high latitudes attributable to cause or coincidence? Limnol. Oceanogr. 31, 351–360.

Paine, R. T.; Castila, J. C. and Cancino, J. (1985). Pertubation and recovery patterns of startfish dominanted intertidal assemblages in Vhile, New Zeland, and Washigton State. Am Nat 125: 679-691.

Paramonov, A. A. (1962). Principles in Phytonematology. Russian. Nauka, Vol. 1 Moscow. 31p.

Persson, L., (1999). Trophic cascades: abiding heterogeneity and the trophic level concept at the end of the road. Oikos 85, 385–397.

Persson, L.; Andersson, G.; Hamrin, S.F. and Johansson, L. (1988). Predator regulation and primary production along the productivity gradient of temperate lake ecosystems. In: Carpenter, S.R. (Ed.), Complex Interactions in Lake Communities. Springer-Verlag, New York, NY, pp. 45–68.

Peterson, C.H. (1979). The importance of predation and competition in organizing the intertidal epifaunal communities of Barnegat Inlet, New Jersey. Oecologia (Berl.) 39, 1–24.

Pinto, T. K.; Austen, M. C. and Benvenuti, C. E. (2006). Effects of macroinfauna sediment disturbance on nematode vertical distribution. Mar. Biol. Ass. U. K., 86: 227-233.

Platt, H. M. & Warwick, R. M. (1983) Free-living Marine Nematodes. Part I British Enoplids, Cambridge, Cambridge University Press, 307p.

Platt, H. M. & Warwick, R. M. (1988) Free-living Marine Nematodes. Part II British Chromadorids, Cambridge, Cambridge University Press, 502p.

Polis, G.A. (1999). Why are parts of the world green? Multiple factors control productivity and the distribution of biomass. Oikos 86, 3–15.

Potts, F. A. (1910). Notes on the free-Jiving nematodes. Q. J. MicL Sc. 55.

Proulx, M. and Mazumder, A. (1998). Reversal of grazing impact on plant species richness in nutrient-poor vs. nutrient-rich ecosystems. Ecology 79: 2581-2592.

Raimondi, P.T. (1990). Patterns, mechanisms, and consequences of variability in settlement and recruitment of an intertidal barnacle. Ecol. Monogr. 60, 283–309.

Reiss, H. and Kröncke, I. (2005). Seasonal variability of infaunal community structures in three areas of the North Sea under different environmental conditions. Est Coast Shelf Sci. 65. 253-274.

Riemann, F.; Von Thun, W. and Lorenzen, S. (1971). Über den phylogenetischen Zusammenhang zwischen Desmoscolecidae und Leptomaimidae (freilebende Nematoden). Veröff. Inst. Meeresforsch. Bremerh. 13.147-152.

Robles, C. and Robb, J., (1993). Varied carnivore effects and the prevalence of intertidal algal turfs. J. Exp. Mar. Biol. Ecol. 166, 65–91.

Robles, C.D.; Sherwood-Stephens, R. and Alvarado, M. (1995). Responses of a key intertidal predator to varying recruitment of its prey. Ecology 76, 565–579.

Schiemer, F. (1982). Food dependence and energetics of freeliving nematodes I. Respiration, growth and reproduction of Caenorhabditis briggsae (Nematoda) at different levels of food supply. Oecologia 54:108- 121.

Schiemer, F. (1983). Comparative aspects of food dependence and energetics of freeliving nematodes. Oikos 41:32-42.

Schiemer, F.; Jensen, P. and Riemann, F. (1983). Ecology and systematics of free-living nematodes from the Bothnian Bay, northern Baltic Sea. Ann. Zool. 20:277-291.

Schiemer, R.; Novak, R. and Ott, J. (1990). Metabolic studies on thiobiotic free-living nematodes and their symbiotic microorganisms. Mar Biol 106:129-137.

Schlacher, F.; Schmid, B. and Seidl, L. (1999). Expert estimates about effects of biodiversity on ecosystem processes and services, Oikos 84 (2): 346- 352.

Schmitt, R. J. (1987). Indirect interactions between prey: apparent competition, predator aggregation, and habitat segregation. Ecology 68: 1887-1897.

Schmitz, O. J.; Krivan, V. and Ovadia O. (2004). Trophic cascades: the primacy of trait-mediated indirect interactions. Ecology Letters, 7: 153– 163.

Schneider, A. (1866). Monographie der Nematoden. Berlim, Druck und Verlag. In Editor 1-357.

Schoener, T. W. (1983). Field experiments on interspecific competltlon. American Naturalist 122: 240-285.

Seidenschwarz, L. (1923). Jahreszyklus heilebender Erdnematoden einer Thaler Alpenwiese. Arb. ZooL Inst. Univ,. Innsbruck 1, 3.

Sih, A.; Crowley, P.; McPeek, M.; Petranka, J. and Strohmeier, K. (1985). Predation, competition, and prey communities: a review of field experiments. Annual Review of Ecology and Systematics 16: 269-3 11.

Snelgrove, F. V. R.; Blackburn, T. H.; Hutchings, F. A.; Alongi, D. M.; Grassle, J. F.; Hummel, H.; King, G.; Koike I.; Lambshead, P. J. D.; Ramsing, N. B. and Soliweiss. V. (1997). The importance of marine sediment biodiversity in ecosystem processes. Ambio 26(8):578-583.

Soetaert, K.; Vincx, M.; Wittoeck, J. and Tulkens, M. (1995). Meiobenthic distribution and nematode community structure in five European estuaries. Hydrobiologia. 311. 185-206.

Somerfield, P. J.; Yodnarasri, S.and Aryuthaka, C. (2002). Relationships between seagrass biodiversity and infaunal communities: implications for studies of biodiversity effects. Mar. Ecol. Progr. Ser. 237:97-109.

Steiner, G . (1913). Freilebende Nematoden aus der Schweiz, Larch Hydrob 9, 259-276.

Steiner, G. (1914). Freilebende Nematoden aus der Schweiz II. Arch. Hydrobiol, (Plankt.) 9, 420-438.

Steiner, G. (1916). Neue und wenig bekannte Nematoden von der Westküste Afrikas I. Zool. Anz. 47: 322-351.

Steiner, G. (1925) On some plant parasitic nemas and related forms J. Agr. Res. 28.

Steiner, G., (1928). Tylenchus pratensis and various other nemas attacking plants. J. Agr. Res 35.

Steiner, G. and Heinly, H., (1922). The possibility of control of Heterodera radicicola and other plant-injurious nemas by means of predatory nemas, especially by Mononchus papillatus Bastian. J. Wash. Acad. Sc. 12.

Steyaert, M.; Garner, N.; Van Gansbeke, D. and Vincx, M; (1999). Nematode communities from the North Sea: environmental controls on species diversity and vertical distribution within the sediment, Jorn. Mar. Bio. Ass. UK 79 (2): 253-264.

Steyaert, M. and Vincx, M. (1996). The vertical distribution of meiobenthos in coastal sediments (Belgium). Bull. Société Royale des Sciences de Liège. 65-1. 155-157.

Steyaert, M. and Vincx, M. (1997). The vertical distribution of meiobenthos in coastal sediments (Belgium). Proc. Prog.Bel. Ocean. Res.: 155-157.

Stöckli, A. (1943). Ober Methoden zur quantitativen Bestimmung der im Boden freilebenden Nematoden Ber. Schw. Bot. Ges. 53 A.

Stöckli, A. (1946a). Der Boden als Lebensraum. Viertelj"schr. Naturf. Ges" Zlirich 91.

Stöckli, A. (1946b). Die biologische Komponente der Vererdung, der Gare und der Niihrstoffpufferung" Schw" Landwirtsch. Monatsh 24.

Strauss, S. Y. (1991). Indirect effects in community ecology: their definition, study and importance. Trends in Ecology and Evolution 6: 206-210.

Strong, D. R. (1992). Are trophic cascades all wet? Differentiation and donor- control in speciose ecosystems. Ecology 73: 747-754.

Sutherland, J.P., (1990). Recruitment regulates demographic variation in a tropical intertidal barnacle. Ecology 71, 955–972.

Taylor, AL. and Sasser, J.N. (1978). Biology, Identification and Control of Root-knot Nematodes Meloidogyne species). Raleigh, NC: United States Agency for International Development.

Tegner, M.J. and Dayton, P.K. (1987). El Ninõ effects on Southern California kelp forest communities. Adv. Ecol. Res. 17, 243–279.

Tegner, M.J.; Dayton, P.K.; Edwards, P.B. and Riser, K.L., (1997). Large- scale, low-frequency oceanographic effects on kelp forest succession: a tale of two cohorts. Mar. Ecol. Prog. Ser. 146, 117–134.

Traunspurger, W.; Bergtold, M. and Goedkoop, W. (1997). The effects of nematodes on bacterial activity and abundance in a freshwater sediment. Oecologia 112 (1): 118-122.

Underwood, A.J.; Denley, E.J. and Moran, M.J., (1983). Experimental analyses of the structure and dynamics of mid-shore rocky intertidal communities in New South Wales. Oecologia (Berl.) 56, 202–219.

UNESCO, (1992). Agenda 21. UNCED Conferência, Rio de Janeiro.

Vance, R.R. (1973). On reproductive strategies in marine benthic invertebrates. The American Naturalist 107: 339-352.

Vanderborght, J.P. and Billen, G. (1975). Vertical distribution of nitrate concentration in intersistial water of marine sediments with nitrification and denitrification. Limnol Oceanogr 20(6):953-961.

Vandermeer, J. (1980). Indirect mutualism: variations on a theme by Stephen Levine. American Naturalist 116:441-448.

Venekey, V.; Lage, L. M. and Fonsêca-Genevois, V. (2005). Draconema brasiliensis and Draconema fluminensis (Chromadorida, Draconematidae): two new species of free living nematodes from a rocky shore affected by upwelling on the Brazilian coast. Zootaxa, 1090: 51-64.

Vranken, G. and Heip, C. (1986). The productivity of marine nematodes. Ophelia. 26: 429-442.

Vranken, G; Herman, P. M. J. and Heip, C. (1988). Studies of the life-history and energetics of marine and brackish-water nematodes. I. demography of Monhystera disjuncta at different temperature and feeding conditions. Oecologia 77:296-301.

Vranken, G.; Thielemans, L.K.; Heip, C.and Vandycke, M. (1981). Aspects of the life-cycle of Monhystera parelegantula (Nematoda; Monhysteridae). Mar. Ecol. Prog. Ser. 6:67-72.

Vranken, G.; Van Brussel, D.; Vanderhaeghen, R. and Heip, C. (1984). Research on the development of a standardized ecotoxicological test on marine nematodes. I. Culturing conditions and criteria for two Monhysterids, Monhystera disjuncta and Monhystera microphthalma. In: Persoone G, Jaspers E, Claus C (Eds.), Ecotoxicological testing for the marine environment, Vol. 2. State University of Gent and Inst. mar. scient. Res., Bredene: 159-184.

Walker, B.H. (1992). Biodiversity and ecological redundancy. Con. Biol. 6: 18- 23.

Wall, D. H. and Moore, J. C. (1999). Interactions underground - Soil biodiversity, mutualism, and ecosystem processes. Bio. 49 (2): 109-117.

Ward, A.R. (1975). Studies on the sublittoral free-living nematodes of Liverpool Bay. II. Influence of sediment composition on the distribution of marine nematodes. Marine Biology 30:217-225.

Wardle, D. A.; Bonner, K. I.; Barker, G. M.; Yeates, G; W.; Nicholson, K. S.; Bardgett, R. D.; Watson, R. N. and Ghani, A. (1999). Plant removals in perennial grassland: Vegetation dynamics, decomposers, soil biodiversity, and ecosystem properties. Ecol. Monogr. 69: 535-568.

Wardle, D. A.; Bonner, K. I. and Nicholson, K. S. (1997). Biodiversity and plant litter: Experimental evidence which does not support the view that enhanced species richness improves ecosystem function. Oikos 79: 247- 258.

Warwick, R.M. (1979). The taxonomic requirements of ecologists. Annales Soc. r. Zool. Belg.108:91.

Warwick, R. M. (1987). Meiofauna: Their role in marine detrital systems. In: Moriarty D.J.W. & R.S.V. Pullin (Eds.), Detritus and microbial ecology in aquaculture. ICLARM Conference Proceedings 14. International Center for Living Aquatic Resources Management, Manila: 282-295.

Warwick R. M.; Platt, H. M. and Somerfield, P. J. (1998). Free-living marine nematodes. Part 3. British Monhysterids. The Linnean Society of London and the Estuarine and Coastal Sciences Association, London. 296pp.

Warwick, R.M.and Clarke, K.R. (2001). Practical measures of marine biodiversity based on relatedness of species. Oceanography and Marine Biology: an Annual Review 39 :207-231.

Wasilewska, L. (1971). Trophic classification of soil and plant nematodes. Wiad Ekol 17:379-388

Watzin, .M. C. (1985). Interactions among temporary and permanent meiofauna: observations on the feeding and behaviour of selected taxa. Biol. Bull. Mar. Lab. Woods Hole 169: 397-416.

White, T.C.R. (1978). The importance of a relative shortage of food in animal ecology. Oecologia 33:71-86.

Wiens, J.A., (1989). Spatial scaling in ecology. Funct. Ecol. 3, 385–397.

Wieser, W. (1953). Die Beziehung zwishen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden. Ark. Zool. 4: 439-484.

Wieser, W. (1960). Benthic studies in Buzzards Bay. II. Meiofauna. Limnol Oceanogr 5: 121-137.

Wootton, J.T. and Power, M.E., (1993). Productivity, consumers, and the structure of a river food chain. Proc. Natl. Acad. Sci. USA 90, 1384–1387.

Wootton, J.T.; Power, M.E.; Paine, R.T. and Pfister, C.A. (1996). Effects of productivity, consumers, competitors, and El Niño events on food chain patterns in a rocky intertidal community. Proc. Natl. Acad. Sci. USA 93, 13855–13858.

Worm, B.; Lotze, H. K.; Boström, C.; Engkvist, R.; Labanauskas, V. and Sommer, U. (1999). Marine diversity shift linked to interactions among grazers, nutrients and propagule banks. Mar. Ecol. Prog. Ser. 185: 309- 314.

Yeates, G. W. (1971). Feeding types and feeding groups in plant and soil nematodes. Pedobiologia 11 : 173-179.

Yeates, G.W.; Bongers, T.; de Goede, R.G.M.; Freckman, D.W. and Georgieva, S. S. (1993). Feeding habits in nematode families and genera - a guide for soil ecologists. J Nematol 25, 315-331

CChhaapptteerr 55::

DDoo nneemmaattooddee mmuuccuuss sseeccrreettiioonnss aaffffeecctt bbaacctteerriiaall ggrroowwtthh??

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Abstract: Do nematodes muçus secreations affect bacterial growth? DOUTORADO EM CIÊNCIAS BIOLÓGICAS Do nematode mucus secretions affect bacterial growth?

Tom Moens1,*, Giovanni Amadeu Paiva dos Santos1,2, Fabiano Thompson3,4, Jean Swings3,4, Veronica Fonseca-Genevois2, Magda Vincx1 & Ilse De Mesel1,3

1 Ghent University, Biology Department, Marine Biology Section, Krijgslaan 281/S8, B-9000 Gent, Belgium 2 Federal University of Pernambuco, Centre for Biological Sciences, Zoology Department, Avenida dos Reitores 1235-Cidade Universitaria, 50670-420 Recife-Pernambuco, Brasil 3 Ghent University, Laboratory of Microbiology, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 4 BCCMTM/LMG Culture Collection, Laboratory of Microbiology, K.L. Ledeganckstraat 35, B- 9000 Gent, Belgium

ABSTRACT Many aquatic nematodes secrete mucus while moving, and prominent microbial growth on nematode mucus tracks has been observed. This has been interpreted as a mutualistic interaction where nematodes may feed on the micro-organisms that colonize their tracks (i.e. the mucus-trap hypothesis). Because of recent evidence that nematodes can affect bacterial community composition, we tested whether bacterial communities growing on nematode mucus differ from extant communities. We characterized the bacterial epigrowth of tracks produced on agar by two estuarine nematode species (the facultative predator Adoncholaimus fuscus and the bacterivore Geomonhystera disjuncta), and compared it to that of artificial tracks and to the bacterial inocula. The experiment lasted 8 days with bacterial community analyses (using Fatty Acid Methyl Ester – FAME analysis) after 2, 4, 6 and 8 days. Although our experimental design promoted a low-diversity bacterial community, multidimensional scaling generally separated communities on nematode tracks from inocula, artificial track communities typically being

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Abstract: Do nematodes muçus secreations affect bacterial growth? DOUTORADO EM CIÊNCIAS BIOLÓGICAS intermediate and highly variable. In a total of 6 bacterial inocula spotted with A. fuscus, only one bacterial strain was recorded on nematode tracks, compared to 6 on artificial tracks and 7 in the inocula. In addition, colony morphology of this particular bacteria, Pseudoalteromonas tetraodonis, was less diverse on nematode tracks than on artificial tracks or inocula. Treatments with G. disjuncta yielded similar yet less consistent and less pronounced results. Our results suggest that nematode mucus may affect colonization and succession patterns of bacteria. This may have important implications for foodweb interactions and ecosystem functions involving both bacteria and nematodes.

KEY WORDS nematodes – bacteria – nematode mucus – microbial community composition – facilitation – microbial foodwebs

INTRODUCTION Decomposition and nutrient mineralization by microbial communities in aquatic sediments often proceed faster and/or more efficiently in the presence of microbial grazers such as flagellates, ciliates and nematodes (Johannes 1965, Findlay & Tenore 1982). Such stimulatory effects are usually attributed to (1) grazing, which hampers immobilization of limiting nutrients in microbial biomass (Ingham et al. 1985, Ferris et al. 1997), and (2) bioturbation, which enhances fluxes of oxygen and nutrients through sediments (Alkemade et al. 1992b, Aller & Aller 1992). Nematodes are usually the predominant metazoans in estuarine and marine soft sediments (Heip et al. 1985), and have indeed been shown to affect microbial activity and/or decomposition processes (Findlay & Tenore 1982, Alkemade et al. 1992a,b, LillebØ et al. 1999, De Mesel et al. 2003). Grazing and bioturbation are not, however, the only mechanisms through which grazers interfere with microbial communities. Many nematodes also secrete mucus which, a.o., has been shown to agglutinate sediment and detrital particles (Riemann & Schrage 1978). These authors also observed prominent microbial growth on the tracks of marine nematodes and argued that (1) the mucus tracks provide bacteria with optimal growth conditions, and (2) micro-organisms trapped in the mucus serve as food for the nematodes; this is commonly referred to as the mucus-trap hypothesis. Recently, the functional implications of this phenomenon have been re-interpreted against the background of an extensive historical treatment of relevant observations (Riemann & Helmke 2002), but the general fact that nematode tracks – at least in artificial substrates – tend to become heavily colonized with micro- organisms remains. The mucus trails of an estuarine nematode became covered with monopopulations of a unicellular microalga, suggesting that micro-organisms growing on nematode tracks may differ from extant microbial communities (Warwick 1981). De Mesel et al. (2004) demonstrated that bacterial community composition and diversity can be top-down affected by nematodes, different nematode species differing in their effects on microbial

100

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Introduction.: Do nematodes muçus secreations affect… DOUTORADO EM CIÊNCIAS BIOLÓGICAS communities. Little work has been done to further elucidate this kind of nematode-microbial interactions. These observations raise the question as to whether nematode mucus may affect the composition and diversity of microbial communities. Therefore, we have performed a laboratory experiment to characterize the bacterial epigrowth of nematode tracks and compare it to that in the inoculum and in artificial tracks.

101

MATERIALS AND METHODS We spotted microbial inocula, obtained from various microhabitats (different types of sediment, different types of macrophyte detritus) in the Paulina salt marsh and on the adjacent intertidal flat, in the centre of Petri dishes (5 cm inner diam.) coated with an agar layer (Fig. 1).

Figure 1. Schematic representation of the experimental setup, with illustration of time zero and 4-day situation. The microcosms are 9 cm diam. Petri dishes coated with an agar layer.

The Paulina site is located in the polyhaline reach of the Westerschelde Estuary (SW Netherlands). The agar medium (0.7 % final concentration, salinity of 25) consisted of a mixture of bacto- and nutrient agar (DIFCO) in a 3/1 ratio (w/w). We made artificial tracks by carving the agar surface from the central inoculum towards the edges with the tip of a fine, sterile Tungsten wire needle. We then introduced nematodes in the centre of the plates and allowed them to move freely. For reasons of comparability with previous work 102

(Riemann & Schrage 1978, Riemann & Helmke 2002), we chose a species of the omnivorous/facultatively predatory nematode genus Adoncholaimus, i.e. A. fuscus, for our experiments. In addition, we performed experiments with the bacterivorous Geomonhystera disjuncta, a species typically associated with decaying macrophyte detritus and producing conspicuous mucus tracks on agar media (Moens unpubl.). G. disjuncta was obtained from synxenic laboratory cultures (Moens & Vincx 1998), whereas A. fuscus was obtained from Paulina intertidal flat sediment by a simple decantation procedure. Each Petri dish received two adult A. fuscus or five adult G. disjuncta (A. fuscus being considerably larger and more motile than G. disjuncta). Plates were incubated in the dark at 17°C for an 8-day period. Every second day, we used sterile forceps to take small samples of (1) the central inoculum, (2) the artificial tracks, and (3) the nematode tracks. These were subsequently inoculated in broth (0.2 g of Bacto beef extract (DIFCO) and 0.33 g of Bacto peptone (DIFCO) in 1 l of artificial seawater), and we characterized the dominant bacterial strains. All spots to be sampled over the entire 8-day period were marked upon the first sampling, such that (1) at later sampling occasions we were always sure to sample from a nematode track generated in the beginning of the incubation, and (2) a single spot could never be sampled twice. For the characterization of bacteria, aliquots of bacteria grown on broth were first diluted and spread on ‘Marine Agar’ plates (37.4 g Marine Broth (DIFCO) + 10 g Bacto agar (DIFCO) in 1 l of water) and frequencies of different colony types were scored. All pure cultures obtained at the highest dilution, representing the different colony types observed, were characterized by fatty acid methyl ester (FAME) profiling (Bertone et al. 1996), following the procedures detailed in Huys et al. (1994). Prior to FAME profiling, bacterial isolates were grown on Trypticase Soy Agar (Difco) supplemented with 2% NaCl for 24h at 28°C. The isolates were identified using our own FAME database that contains 52,000 profiles covering most currently known bacterial genera. All our isolates had profiles very similar to those of known species in the reference database and are therefore referred to here as putative species; however, other, molecular analyses such as 16S rDNA

103

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Mat. and Met.: Do nematodes muçus secreations affect ... DOUTORADO EM CIÊNCIAS BIOLÓGICAS sequencing and DNA-DNA hybridizations would be required to confirm their exact species identity. All treatments were set up in triplicate, but samples from the three replicates of a single treatment were pooled together in broth. Colony plating was always performed in duplicate, but these duplicate plates consistently gave (nearly) identical results. Similarity between bacterial communities from inocula, artificial and nematode tracks was analysed with Multidimensional Scaling (MDS) and group-average linking on presence/absence data using the Dice similarity coefficient, which is identical to the Bray-Curtis coefficient when this is calculated on presence/absence data (Clarke & Warwick 1994).

104

RESULTS AND DISCUSSION A total of 12 and 11 colony ‘morphotypes’ were found and isolated from the experiments with Adoncholaimus fuscus and Geomonhystera disjuncta, respectively. FAME analysis assigned these to 15 different putative bacterial species, 7 in the treatments with A. fuscus (Table 1) and 8 in those with G. disjuncta (not shown). Two (in one treatment) to 7 bacterial species were found per bacterial inoculum. Even though this experimental setup generally yielded very low microbial diversity, we almost consistently found lower bacterial diversity on nematode tracks than in the central inoculum or on the artificial tracks (Table 1). In fact, all bacterial strains isolated from A. fuscus tracks could be assigned to a single species, Pseudoalteromonas tetraodonis. Moreover, while there were always 3 or 4 colony morphotypes of this bacteria in the inocula and artificial tracks of the A. fuscus treatments, only one to (mostly) 2 were recovered from A. fuscus tracks. MDS and group-average linking always separated nematode tracks from central inocula, artificial track communities typically being interspersed between central inoculum communities and, to a lesser extent, nematode track communities (Fig. 2). Geomonhystera disjuncta tracks were always colonized by Photobacterium angustum/Vibrio anguillarum and by Pseudomonas pertucinogena. In just one of four G. disjuncta treatments, a third bacteria, Pseudoalteromonas nigrifaciens, was recovered from nematode tracks. Multivariate analysis generally yielded similar but less consistent and less pronounced results as in A. fuscus treatments (Fig. 2).

105

Table 1. Identity and diversity of bacterial strains found on nematode (Adoncholaimus fuscus) tracks, artificial tracks and central inocula over an 8 d incubation period with samplings every second day. Numbers refer to the day(s) on which bacteria werefound (A: absent over the whole incubation). Bacterial diversity (number of bacterial species found over the entire 8 d incubation) is indicated within parentheses

106

Figure 2. MDS of bacterial community composition (based on presence/absence data) in inocula, artificial tracks and nematode tracks after 48, 96, 144 and 196h. (A), (B) and (C) examples of treatments with the nematode Adoncholaimus fuscus, bacterial inocula derived from (A) muddy salt marsh sediment, (B) fine to medium sandy sediment on a tidal flat, and (C) decaying Ulva lactuca; (D) example of treatment with Geomonhystera disjuncta, inoculum derived from muddy salt marsh sediment.

The above results suggest that nematode mucus secretions can influence bacterial community composition. Hitherto, nematode impacts on microbial 107

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Res. and Disc.: Do nematodes muçus secreations affect… DOUTORADO EM CIÊNCIAS BIOLÓGICAS communities have mostly been viewed from the angle of grazing effects. Recently, De Mesel et al. (2003, 2004) observed variable impacts of nematode grazers on microbial activity and community structure, depending on nematode species identity. They also found that structuring effects may even show up at low grazing pressure, implying selective grazing and/or other, indirect interactions. Nematode mucus may be at the basis of one such type of indirect interactions. It is not a novel observation that free-living aquatic nematodes can produce mucus. Certain species from intertidal mudflats build tubes consisting of sediment and small organic particles glued together by mucus secretions from, a.o., the ventral gland, affecting sediment stability and at the same time probably offering shelter against both hydrodynamic impacts and predation (Nehring et al. 1990, Nehring 1993). Mucus production from ventral and caudal glands is widespread. Riemann & Schrage (1978) hypothesized a gardening mechanism, where the nematodes stimulate growth of bacteria and then use them as food; this is not necessarily restricted to interactions with bacteria (Warwick 1981, Jensen 1996). Species of the genus Adoncholaimus, do not rely substantially on bacteria as a food source (Moens et al. 1999), but this does not exclude the possibility that Adoncholaimus utilizes certain bacteria-derived nutritive compounds. Alternatively, Adoncholaimus and perhaps other nematodes may produce exo-enzymes which can start the initial decomposition of complex molecules. This would promote establishment and growth of bacterial populations which take over the organic matter decomposition, both nematodes and bacteria then feeding on the nutritious ‘soup’ of dissolved (DOM) and small particulate organic matter released from this shared use of enzymes (Riemann & Helmke 2002). Geomonhystera disjuncta is a bacterivore typical of detrital microhabitats in intertidal areas and is relatively easily culturable on agar media with bacteria as food (Vranken et al. 1988, Moens & Vincx 1998). We have often observed this species to produce prominent mucus tracks on the surface of agar layers and to deposit its eggs along these tracks. As a bacterivore, this mechanism may ensure that eggs hatch in a food-rich micro-environment. Alternatively, as a typical inhabitant of phytodetrital deposits in (often wave-washed)

108

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Res. and Disc.: Do nematodes muçus secreations affect… DOUTORADO EM CIÊNCIAS BIOLÓGICAS intertidal environments, the mucus may simply help G. disjuncta in attaching eggs to the substrate, much like many nematodes use secretions from the caudal glands for adhesion. The dominant bacteria associated with nematode tracks in our experiments were quite similar for both nematode species: Pseudoalteromonas tetraodonis was associated with Adoncholaimus fuscus tracks, and Pseudomonas pertucinogena with Geomonhystera disjuncta tracks. In addition, the latter always contained Photobacterium angustum/ Vibrio anguillarum and on one occasion Pseudoalteromonas nigrifaciens. Note that only one bacterial inoculum used with G. disjuncta contained Pseudoalteromonas tetraodonis. The bacterial strains isolated in this study are commonly found in a variety of aquatic environments (see e.g. Ivanova et al. 2003, Thompson et al. 2004, Skovhus et al. 2004). Much like for the exact benefits of this nematode-microbial relationship for the nematodes, it is not clear whether and how nematode mucus stimulates bacterial growth. In addition to benefitting from any exo-enzymatic activity in the mucus (Riemann & Helmke 2002), bacteria may simply use it for attachment or feed on it. The composition – and potential nutritive value for bacteria – of the nematode mucus is poorly known, but it does apparently contain a substantial share of acid mucopolysaccharides (Nehring et al. 1990). In addition, C/N-ratios of nematodes tend to be higher than those of bacteria (Anderson et al. 1983, Ferris et al. 1997), so nematodes feeding on bacteria obtain N in excess of their N-requirements. Nematodes thus typically excrete N-rich products and, depending on their nutritional status and on environmental conditions (esp. oxygen regime), void a variety of organic molecules including amino acids, glycerol, L-lactate, acetate etc… (Bolla 1980, Foll et al. 1998), which may serve as a source of food or nutrients (N) for bacteria. In addition, a variety of dissolved organic and inorganic molecules have been shown to sorb to microbial exopolymer secretions (Decho 1990); similar sorption of nutritious compounds to nematode mucus may occur, depending on the chemical composition of the nematode mucus. It is indeed unlikely that the nematode mucus in itself would provide sufficient carbon and nutrients for the dense microbial growth observed on the

109

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Res. and Disc.: Do nematodes muçus secreations affect… DOUTORADO EM CIÊNCIAS BIOLÓGICAS nematode tracks. In turn, bacteria growing on mucus tracks may be a food source for nematodes or provide nematodes with access to nutritious compounds (see above). In another peculiar form of mutualism in aquatic environments, snails of the genus Littoraria facilitate growth of fungi on live cordgrass (Spartina) through their grazing activity and faecal pellet production, while benefitting themselves from feeding on fungal hyphae (Silliman & Newell 2003). Furthermore, free-living aquatic nematodes are generally more mobile than bacteria and may therefore act as vectors transporting bacteria to new resource patches. Nematodes display chemotaxis along cues informative of suitable microhabitats (Riemann & Schrage 1988, Höckelmann et al. 2004). Oncholaimid nematodes are particularly mobile and aggregate efficiently on fresh deposits of organic matter (Lorenzen et al. 1987, Prein 1988). Neither A. fuscus nor G. disjuncta is prominently coated with bacteria, and there are at present no indications that specific microbiota associate with these nematodes, as in some nematode-bacteria symbiotic relations (Polz et al. 2000, Ott et al. 2004). No consistent temporal trends in bacterial community composition were found, but while composition and diversity of bacteria in inoculum and artificial tracks typically varied with time, they were constant over the 8-day incubation on nematode tracks. We had anticipated one or a few opportunistic bacteria to colonize nematode tracks instantly and other strains to develop during the course of the experiment, but the latter was never the case. On artificial tracks and in the inoculum spot we often found temporal shifts in community structure. This suggests that nematodes not only promote substrate colonization by one or a few opportunistic strains, but may also facilitate these strains to maintain their dominance. A relevant observation in this respect is that – esp. in A. fuscus experiments – there were two distinct types of nematode tracks: large main tracks and narrower side tracks. In addition to a time effect (bacteria start spreading from the nematode tracks thus widening them), the two types of tracks in our study originate from the fact that A. fuscus moves along a main track but regularly makes side excursions, then moving backwards again into the main track. As such, main tracks – or at

110

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 5: Res. and Disc.: Do nematodes muçus secreations affect… DOUTORADO EM CIÊNCIAS BIOLÓGICAS least parts of them – are visited several times while side tracks are visited only once. If the nematodes produce mucus continuously, main tracks will receive substantially more of it than side tracks, and as a result the mucus effect may be more pronounced in main tracks than in side tracks. In the present study, we only sampled from main tracks. The results presented here have to be interpreted with caution since the experimental design – with extensive utilization of nutrient-rich growth media – promoted the growth of a low-diversity microbial community dominated by readily culturable bacteria. The need for visible and easy-to-‘handle’ nematode tracks make initial trials on artificial media inevitable, but additional ‘culture’ steps could in future work be avoided by using microbial fingerprinting methods. In a next step, artificial media should be replaced by natural substrates such as sediment. However, while the identity of the bacteria colonizing nematode tracks will likely be different under more natural conditions, the fact that differences in community diversity and composition between artificial tracks and nematode tracks were found even in such low- diversity communities strongly suggests that nematode mucus secretions do affect bacterial community structure. Elucidation of the underlying mechanisms will require, a.o., a compositional analysis of the nematode mucus. Bacteria, for instance, also grow on nematode mucus tracks in nutrient-poor agar media (Riemann & Helmke 2002), but in our study they did so at a much slower rate and in lower densities. Although the exact nature and implications of these intricate nematode – microbial interactions remain to be elucidated, their effects for the functioning of microbial foodwebs are potentially far-reaching. Nematodes may transport specific bacteria to resource patches and importantly affect their colonization and succession patterns, which may well have implications for important ecosystem functions such as decomposition and mineralization. Such ‘founder effects’ may be quite widespread, and should be taken into account in future research on meiofauna-microbiota relations.

111

Acknowledgements - This work was financially supported by Ghent University through BOF-projects 011060002 and 1205398 and by the Flemish Fund for Scientific Research (FWO) (project 31521704).Giovanni dos Santos are granted by CAPES Brazil. Christine Van der heyden skilfully assisted in manuscript preparation. T.M. is a postdoctoral research fellow of the FWO.

112

LITERATURE CITED Alkemade R, Wielemaker A, Hemminga MA (1992a) Stimulation of Spartina anglica leaves by the bacterivorous marine nematode Diplolaimelloides bruciei. J Exp Mar Biol Ecol 159: 267-278 Alkemade R, Wielemaker A, de Jong SA, Sandee AJJ (1992b) Experimental evidence for the role of bioturbation by the marine nematode Diplolaimella dievengatensis in stimulating the mineralization of Spartina anglica detritus. Mar Ecol Prog Ser 90: 149-155 Aller RC, Aller JY (1992) Meiofauna and solute transport in marine muds. Limnol Oceanogr 37 : 1018-1033 Anderson RV, Gould WD, Woods LE, Cambardella C, Ingham RE, Coleman DC (1983) Organic and inorganic nitrogenous losses by microbivorous nematodes in soil. Oikos 40: 75-80 Bertone S, Giacomini M, Ruggiero C, Piccarolo C, Calegari L (1996) Automated systems for identification of heterotrophic marine bacteria on the basis of their fatty acid composition. Appl Env Microbiol 62: 2122-2132 Bolla R (1980) Nematode energy metabolism. In: Zuckerman BM (ed) Nematodes as biological models, volume 2. Academic Press, New York, p 165-192 Clarke KR, Warwick RM (1994) Change in marine communities: An approach to statistical analysis and interpretation. Plymouth: Plymouth Marine Laboratory, 144 pp. Decho AW (1990) Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. Oceanogr Mar Biol Annu Rev 28: 73-153 De Mesel I, Derycke S, Swings J, Vincx M, Moens T (2003) Influence of bacterivorous nematodes on the decomposition of cordgrass. J Exp Mar Biol Ecol 296: 227-242 De Mesel I, Derycke S, Moens T, Van der Gucht K, Vincx M, Swings J (2004) Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study. Environ Microbiol 6: 733-744

113

Ferris H, Venette RC, Lau SS (1997) Population energetics of bacterial- feeding nematodes: carbon and nitrogen budgets. Soil Biol Biochem 29: 1183-1194 Findlay S, Tenore KR (1982). Effect of a free-living marine nematode (Diplolaimella chitwoodi) on detrital carbon mineralization. Mar Ecol Prog Ser 8:161-166 Foll RL, Pleyers A, Lewandovski GJ, Wermter C, Hegemann V, Paul RJ (1999) Anaerobiosis in the nematode Caenorhabditis elegans. Comp Biochem Physiol B Biochem Mol Biol 124: 269-280 Heip C, Vincx M, Vranken G (1985) The ecology of marine nematodes. Oceanogr Mar Biol Annu Rev 23: 399-489 Höckelmann C, Moens T, Jüttner F (2004) Odor compounds from cyanobacterial biofilms acting as attractants and repellents for free-living nematodes. Limnol Oceanogr 49: 1809-1819 Huys G, Vancanneyt M, Coopman R, Janssen P, Falsen E, Altwegg M, Kersters K (1994) Cellular fatty acid composition as a chemotaxonomic marker for the differentiation of phenospecies and hybridization groups in the genus Aeromonas. Int J Syst Bacteriol 44:651-658

Ingham RE, Trofymow JA, Ingham ER, Coleman DC (1985) Interactions of bacteria, fungi and their nematode grazers: effects on nutrient cycling and plant growth. Ecol Monogr 55: 119-140 Ivanova EP, Sawabe T, Hayashi K, Gorshkova NM, Zhukova NV, Nedashkovskaya OI, Mikhailov VV, Nicolau DV, Christen R (2003) Shewanella fidelis sp. nov., isolated from sediments and sea water. Int J Syst Evol Microbiol 53: 577-582 Jensen P (1996) Burrows of marine nematodes as centres for microbial growth. Nematologica 42: 320-329 Johannes RE (1965) Influence of marine protozoa on nutrient regeneration. Limnol Oceanogr 10: 434-442

LillebØ AI, Flindt MR, Pardal MA, Marques JC (1999) The effect of macrofauna, meiofauna and microfauna on the degradation of Spartina maritima detritus from a salt marsh area. Acta Oecol 20: 249-258 Lorenzen S, Prein M, Valentin C (1987) Mass aggregations of the free-living

114

marine nematode Pontonema vulgare (Oncholaimidae) in organically polluted fjords. Mar Ecol Prog Ser 37: 27-34 Moens T, Verbeeck L, Vincx M (1999) Feeding behaviour of a predatory and a facultatively predatory marine nematode (Enoploides longispiculosus and Adoncholaimus fuscus). Mar Biol 134: 585-593 Moens T, Vincx M (1998) On the cultivation of free-living estuarine and marine nematodes Helgoländer Meeresunters 52: 115-139 Nehring S (1993) Tube-dwelling meiofauna in marine sediments. Int Revue Ges Hydriobiol 78: 521-534 Nehring S, Jensen P, Lorenzen S (1990) Tube-dwelling nematodes: tube construction and possible ecological effects on sediment-water interfaces. Mar Ecol Prog Ser 64: 123-128 Ott J, Bright M, Bulgheresi S (2004) Symbioses between marine nematodes and sulfur-oxidizing chemoautotrophic bacteria. Symbiosis 36: 103-126 Polz MF, Ott JA, Bright M, Cavanaugh CM (2000) When bacteria hitch a ride. ASM News 66: 531-539 Prein M (1988) Evidence for a scavenging lifestyle in the free-living nematode Pontonema vulgare (Enoplida, Oncholaimidae). Kieler Meeresforsch. 6: 389-394 Riemann F, Helmke E (2002) Symbiotic relations of sediment-agglutinating nematodes and bacteria in detrital habitats: The enzyme-sharing concept. PSZN I Mar Ecol 23:93-113 Riemann F, Schrage M (1978) The mucus-trap hypothesis on feeding of aquatic nematodes and implications for biodegradation and sediment texture. Oecologia 34:75-88 Riemann F, Schrage M (1988) Carbon dioxide as an attractant for the free- living marine nematode Adoncholaimus thalassophygas. Mar Biol 98: 81-85 Silliman BR, Newell SY (2003) Fungal farming in a snail. Proc Natl Acad Sci USA 100: 15643-15648 Skovhus TL, Ramsing NB, Holmstrom C, Kjelleberg S, Dahllof I (2004) Real- time quantitative PCR for assessment of abundance of Pseudoalteromonas species in marine samples Appl Env Microbiol 70: 2373-2382

115

Thompson FL, Iida T, Swings J (2004) Biodiversity of Vibrios. Microbiol Molec Biol Rev 68: 403-431. Vranken G, Herman PMJ, Heip C (1988) Studies of the life-history and energetics of marine and brackish-water nematodes. I. Demography of Monhystera disjuncta at different temperature and feeding conditions. Oecologia 77: 296-301 Warwick RM (1981) In: Jones NV, Wolff WJ (Eds.), Feeding and survival strategies of estuarine organisms. Plenum Press, New York: 39-52

116

CChhaapptteerr 66::

DDiiffffeerreennttiiaall eeffffeeccttss ooff ffoooodd aavvaaiillaabbiilliittyy oonn ppooppuullaattiioonn ddeevveellooppmmeenntt aanndd ffiittnneessss ooff tthhrreeee ssppeecciieess ooff eessttuuaarriinnee,, bbaacctteerriiaall-- ffeeeeddiinngg nneemmaattooddeess..

117

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Abstract:: Differential effets of food availability on poplation… DOUTORADO EM CIÊNCIAS BIOLÓGICAS Differential effects of food availability on population development and fitness of three species of estuarine, bacterial- feeding nematodes.

Santos, G. A. P. dos1,2; Derycke S.1; Genevois V. G. F.2; Coelho, L. C. B. B.2; Correia, M. T. S.2 & Moens, T.1

1- Marine Biology Section, Biology Department, Ghent University, Krijgslaan 281 (S8), 9000 Ghent, Belgium. 2- Universidade Federal de Pernambuco, Av. professor Moraes Rêgo, s/n, 50.000.000. Recife, Pernambuco, Brazil.

Abstract The significance of bottom-up controls on biological communities has been a long-standing topic of interest in ecology. However, before environmental effects on communities can be properly assessed, a thorough knowledge of the individual species’ responses is required. We studied effects of food availability (= bacterial density) on population development and on different life-history traits in three species of bacterial-feeding nematodes, Diplolaimelloides oschei, Diplolaimelloides meyli (both Monhysteridae) and Pellioditis marina (Rhabditidae), which co-occur on macrophyte detritus in the Westerschelde Estuary (SW Netherlands). The bacteria Escherichia coli was offered in five food availability treatments ranging from 3 x 1010 cells ml-1 to 3 x 107 cells ml-1. The three bacterial-feeding nematode species studied here showed differential responses to food density, which agreed with the general idea that Rhabditidae have extreme colonization abilities under very high food availability, while Monhysteridae tend to have a somewhat slower population 118

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Abstract:: Differential effets of food availability on poplation… DOUTORADO EM CIÊNCIAS BIOLÓGICAS development and comparatively lower food requirements. Several life history traits, including juvenile mortality and development time, did not exhibit a clear food-density dependence. Results on the F1 generation may, however, be affected by strong maternal effects on life history traits of their progeny. Patterns of food-density dependence of population increase and size at maturity were similar in P. marina. Both Diplolaimelloides species, however, exhibited a large size at maturity but a very low population increase at the highest food availability, suggesting a trade-off between biomass and reproduction. Comparison with published data on other nematode species reveals that nematode responses to food availability as well as to other environmental factors are highly species-specific.

Keywords: Bacterial-feeding nematodes; Bottom-up control, Fitness; Food availability; Marine; Population development

119

Introduction

The significance of resources in controlling populations and communities of organisms has been a topic of interest in aquatic and terrestrial ecology for many years, with debates having long been polarized between hypotheses on predator-based (‘top-down’) and resource-based (‘bottom-up’) regulation of different trophic levels (Hairston et al., 1960; Murdoch, 1966; McQueen et al., 1989; Power, 1992; Menge et al., 1999, and many others). By now the concomitant contribution of bottom-up and top-down forces has been widely accepted (Worm et al., 2002). A majority of studies on bottom-up controls have focused on the causes and consequences of biomass variation at different trophic levels (Rosemond, 1993; Hillebrand, 2002; Hillebrand et al., 2002), while fewer studies have addressed resource effects on community assembly, composition and diversity (Menge and Sutherland, 1976; Menge et al., 2002). Nematodes are the most abundant and species-rich taxon in marine and estuarine benthic communities, with densities in soft sediments typically ranging between 105 and 107 ind. m-2 (Heip et al., 1985). Estimates of the total species diversity within the phylum span several orders of magnitude (105 to 108 species worldwide – Coomans, 2000; Lambshead, 1993), and may still be underestimated as a result of a high cryptic diversity (Derycke et al., 2005, 2007a). The local (= sample) diversity of nematodes varies as a function of sediment texture, organic matter availability and quality, and other environmental factors, but is typically substantial, several tens of species m-2 being common (Heip et al., 1985). It is rule rather than exception to find several ‘confunctional’ (i.e. belonging to the same trophic guild) and congeneric species together, but the factors responsible for the structuring of these diverse local communities remain largely unknown (De Mesel et al., 2006). Top-down control by predatory nematodes (Moens et al., 2000; Gallucci et al., 2005) and macrofauna (Coull, 1985; 1986; Jian Li and Vincx, 1993), as well as bottom-up control through food availability and quality (Heip et al., 1985; Coull 1999) have been suggested as important forces affecting density, biomass and species composition of nematode communities, but the

120

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Introduction:: Differential effets of food availability on… DOUTORADO EM CIÊNCIAS BIOLÓGICAS available evidence is still scanty and does not allow for generalizations to be made. Moreover, information on food thresholds and functional responses of nematodes is still limited (Schiemer, 1985; Herman and Vranken, 1988; Moens and Vincx, 2000b). Permanent laboratory cultures of only few marine nematode species have ever been established (Moens and Vincx, 1998), but the ones that are easy to cultivate typically (1) have short generation times and produce many progeny (Warwick, 1981; Vranken and Heip, 1983; Vranken et al., 1988; Moens and Vincx, 2000a), (2) stem from the same type of habitat: the surfaces of decomposing macrophytes, (3) belong to one and the same trophic group: bacterial feeders, and (4) belong to only a few families, mainly Monhysteridae and Rhabditidae (Warwick, 1987; Moens et al., 1996). Since it is common to find several of these species co-occurring, these communities present interesting model systems to study effects of disturbance and resource availability on community assembly, and they are easily amenable to experimental manipulation under controlled laboratory conditions (Moens and Vincx, 1998). However, before environmental effects on (artificially assembled) communities of species can be assessed, a thorough knowledge is required on the individual species’ responses. According to life history theory, the impact of environmental stress on organisms is determined by its effect on fitness (Sibly and Calow, 1989). At the population level, fitness can be defined as the intrinsic rate of population increase (Murray, 1985; Nur, 1987), but at the individual level, different traits relating to, among others, growth, biomass, reproduction and feeding may all affect fitness (Kammenga et al., 1996a, b, 1997; Schiemer, 1982a,b; Herman and Vranken, 1988; Moens and Vincx, 2000b). The aim of the present paper is to study the effects of food availability (= bacterial density) on different life-history traits and on population development of three species of bacterial-feeding nematodes, Diplolaimelloides oschei, Diplolaimelloides meyli and Pellioditis marina, common on several types of macrophyte detritus in the Westerschelde Estuary.

121

Materials and methods

Nematode culture

Nematodes for our experiments were obtained from established monospecific, agnotobiotic cultures in exponential growth phase, with unidentified bacteria from the habitat as food (Moens and Vincx, 1998). The culture inocula of Diplolaimelloides oschei, D. meyli and Pellioditis marina originate from the Westerschelde Estuary (SW Netherlands). All three species co-occur on decaying macroalgae and macrophytes in the Paulina saltmarsh in the polyhaline reach of this estuary. Each species had been in permanent culture under identical temperature (17 °C) and salinity (25) conditions for many generations prior to the start of our experiments. In view of the recent discovery that P. marina is in fact a complex comprising different, often sympatrically occurring cryptic species (Derycke et al., 2005, 2006), we verified the identity of our laboratory population applying PCR- RFLP of the nuclear ITS region. Ten nematodes were randomly collected from the culture, DNA was extracted and the ITS region was amplified according to Derycke et al. (2005). A 5 µl aliquot of each PCR-product was digested with 0.5 µl AluI restriction enzyme (10 units/µl RE), 1 µl 10x Y+ tango buffer and 3.5 µl distilled water for two hours at 37 °C. The digested PCR- products were subsequently submitted to electrophoresis on 2 % midi agarose and bands were stained with ethidiumbromide. The P. marina culture used for the present experiments belongs to the Pm I lineage, which is the dominant species at the Paulina field site and in the Westerschelde Estuary in general (Derycke et al., 2005, 2006). Monhysterids mitochondrial (COI) as well as nuclear (18S) DNA markers have been sequenced using methodology fallowed by Derycke et al (2005). GenBank accession numbers of: D. meyli mitochondrial partial (COI) gene for cytochrome oxidase C subunidad 1 (Accession number AM748756); the 18S sequence of D. meyli was obtained from Blaxter et al. (1998) (Accession number AF036644).; D. oschei mitochondrial partial (COI) gene for cytochrome oxidase C subunit 1

126

(Accession number AM748757) and D. oschei partial 18S rRNA gene (Accession number AM748761).

Experiments

Experiments were conducted in Petri dishes (5 cm i.d.) with 4 ml of a 0.75 % bacto-agar medium, prepared with artificial seawater (ASW, Dietrich and Kalle, 1957) with a salinity of 25. The pH of the medium was buffered at 7.5–8 with TRIS–HCl in a final concentration of 5 mM, which raised the salinity of the agar substrate by ca. 1.2 units. The low nutrient content of the agar and the washing procedure of the nematodes (see below) ensured that growth of bacteria, cotransferred with the nematodes, in the experimental units was minimal. We further supplied the agar medium with 100µl l-1 cholesterol as a source of steroids, because nematodes are incapable of synthetizing sterols from a purely bacterial food source (Vanfleteren, 1980). Frozen-and-thawed Escherichia coli (strain K12) were used as a food source. These were offered in five different cell densities corresponding to each of 10 -1 9 five food availability treatments (C1, C2,…, C5): 3 x 10 cells ml , 3 x 10 cells ml-1, 1.65 x 109 cells ml-1, 3 x 108 cells ml-1, and 3 x 107 cells ml-1, respectively. Cell densities were obtained through dilution in ASW from a stock density of 3 x 1011 cells ml-1. Preliminary experiments were run to ensure that nematode growth rates were similar on E. coli as on the bacteria present in the nematode cultures. For experiments, adult male and female nematodes were manually picked up from stock cultures, rinsed twice in ASW to minimize transfer of bacteria, and left in ASW for 24 h at 5 °C before the start of the experiment. All experimental treatments were performed with the three nematode species separately. Five male and five gravid female P. marina were randomly collected and inoculated per experimental unit. For the monhysterid species D. meyli and D. oschei we used ten males and ten gravid females; these numbers are based on previous experiments with the same or similar species (Moens and Vincx, 2000a; Vranken et al., 1988). The different numbers of P.

127

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Mat and Met.: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS marina vs the monhysterid species relate to differences in generation time and rate of population increase (Vranken and Heip, 1983; Vranken et al., 1988; Moens et al., 1996; Moens and Vincx, 2000a). After the above described rinsing procedure, nematodes were transferred individually on the tip of a fine Tungsten wire needle to a 50 µl drop of sterile ASW on the agar substrate. 50 µl of the E. coli suspensions of the respective densities were added as food and spread over the agar surface. In treatments with P. marina, a second and equal amount of bacteria was added on the fifth day of the experiment, in treatments with monhysterid nematodes this was done on the tenth day. There were 8 replicates per treatment and species, four of which were used to study nematode population growth, the remaining four to harvest animals for morphometric and biomass analyses. In treatments with P. marina, nematode population (total numbers and number of adults) and life cycle parameters (fecundity, embryonic, postembryonic and total development time, adult sex ratio, mortality) were quantified daily during the first 11 days of the experiment and then every 48h until the 17th day; in treatments with Monhysteridae, counts were performed every 48h during the first 14 days and then every 92h until 46 days of incubation. Embryonic development time was defined as the time from egg deposition to hatching. Since reproduction in all species and treatments started almost immediately after inoculation, the minimal embryonic development time was taken as the time from T0 to the appearance of the first juvenile(s). Mean embryonic development time was approximated by taking the difference in the X-axis intercepts of the linear regressions of numbers of juveniles vs time and numbers of eggs vs time. The mean postembryonic development time was similarly approximated by taking the difference in the X-axis intercepts of the linear regressions of numbers of adults vs time and numbers of juveniles vs time (Moens and Vincx, 2000a). Total development time, then, is the sum of mean embryonic and mean postembryonic development time. Approximately 30 (15 males and 15 females) F1 adult nematodes per replicate were used for morphometric and biomass analyses. These were randomly collected and preserved in a hot (70°C), neutral formaldehyde

128

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Mat and Met.: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS solution (4 %), on day 8 for the P. marina experiments and on day 24 for the Diplolaimelloides experiments. The timing was based on the development time of each species, and was chosen such that F1-adults of both sexes were present in sufficient numbers in all food treatments. When less than 30 F1 adult individuals were present, all adults were picked out. Nematodes were mounted on glass slides for measurements of total body length (for biomass calculations in D. oschei, the length of the tail was subtracted from the total body length) and maximal body width. These measurements were performed using a Quantimet 500+ image analyser. Biomass was calculated from these measurements following a slightly adapted version of Andrassy’s formula (1956):

-6 Biomass (in µg wet weight) = (LW²/1.7) NRd .10 where: L = Total length (µm), omitting the elongated, filiform tail in D. oschei. W = Width (µm), maximum body diameter, avoiding vulva in female.

NRd= Nematode relative density, estimated at 1.13 for marine nematodes (Somerfield et al., 2005)

Data analysis

Differences between treatments, time and time x treatment were analyzed using repeated measures analysis of variance (ANOVA), or with one-way ANOVA in case of morphometric and biomass data. Data were first checked for normality and homogeneity of variances using Levene’s ANOVA-test on the deviation of scores and Bartlett’s χ² test. Where necessary, they were log10(x+1) transformed to fit the assumptions of normality and homogeneity of variances. Pairwise a posteriori comparisons were performed using Tukey’s Honest Significant Differences (HSD) test. If, after transformation, the data still did not match the criteria of normality and/or homoscedasticity, they were analysed with a Kruskall–Wallis non-parametric one-way ANOVA on the

129

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Mat and Met.: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS separate effects of treatment and time. All analyses were performed using the Statistica 6 software (Statsoft).

130

Results

In the following, the effects of food availability on fecundity, total population development, adult population development, sex ratio, embryonic, postembryonic and total development time, and adult biomass, are presented for each species separately.

Pellioditis marina

Eggs were visible from the first day onwards in all treatments, but their numbers remained low because of a largely ovoviviparous reproductive strategy; egg numbers were typically higher at lower food availabilities (C3 – C5), but these differences were not significant. Juveniles appeared from the first day onwards in all treatments. The first F1-adult females and males appeared on the 5th day in all treatments and replicates.

Surprisingly, fecundity of F0 P. marina was lowest at the highermost food availability, while differences between other food treatments were small (Table 1). Total population (including all vermiform stages but not eggs) development of P. marina was, however, considerably higher in C1 than in all other treatments (p<0.0001), indicating that the food effect on fecundity was completely different between F0 and subsequent generations. Population development was also significantly higher in C2 than in C5 (p<0.04). The interaction factor treatment vs time was also highly significant (F= 11.21; p<0.0001, Fig 1A).

131

Table 1: Total (i.e. for the first 14 days of the experiment for both Diplolaimelloides species and for the first 4 days for P. marina) and daily fecundity (eggs + juveniles) per F0 female at five different food availabilities. Data are means ± 1stdev of four replicates. Total Fecundity Daily Fecundity P. marina D. meyli D. oschei P. marina D. meyli D. oschei Treat ± 1 stdev ± 1 stdev ± 1 stdev ± 1 stdev ± 1 stdev ± 1 stdev 30,95 ± 19,14 ± 2,21 ± 1,37 ± C1 26,65 ± 5,70 16,28 1,63 6,66 ± 3,41 1,16 0,28 23,58 ± 11,10 ± 2,90 ± 1,68 ± C2 44,39 ± 8,74 40,59 ± 9,58 1,96 3,02 0,68 0,40 46,85 ± 22,65 ± 3,35 ± 1,62 ± C3 32,91 ± 4,70 28,57 2,15 8,23 ± 2,60 2,04 0,70 29,61 ± 19,45 ± 12,43 ± 2,11 ± 1,39 ± C4 49,72 ± 7,79 10,50 1,52 2,41 0,75 0,26 57,27 ± 11,59 ± 14,32 ± 1,22 ± 0,83 ± C5 16,45 17,02 ± 7,75 0,93 7,69 0,55 0,13

Adult population development differed significantly between treatments (interaction effect of treatment vs time: F= 5.76; p<0.0001). Numbers of adults started increasing from the 5th day onwards. The highest food availability yielded the fastest (Fig. 1B) and highest (Fig. 2A) population development, with an abrupt increase in adult density from the 11th day onwards.

1400 600

1200 A B 500

1000

400 800

600 300 Total Population 400 Adult Population 200

200

100 0

C1 C1 -200 C2 0 C2 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T13 T15 T17 C3 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T13 T15 T17 C3 C4 TIME C4 C5 TIME C5

Figure 1: A: Total P. marina population development , and B: P. marina adult population development over time at five different food availabilities. Data are averages of four replicates per treatment and time.

132

At lower food availabilities, adult densities remained low throughout the experiment (F= 19.80; p<0.0001, Figs. 1B and 2A). While abundances of adult P. marina tended to decrease with decreasing bacterial densities at C2 to C5, these differences were not statistically significant. The sex ratio was consistently female-biased and was highest at C3 with 66 ± 23.36 % (mean ± 1 stdev) females; however, differences between treatments were not significant (Fig. 2B).

0140 3.6

3.4 0120 A B 3.2

0100 3.0

2.8 080

2.6

060 N° Adult P.M. N°

Sex Ratio P.M. Ratio Sex 2.4

040 2.2

2.0 020 1.8

00 Mean 1.6 Mean C1 C2 C3 C4 C5 ±SE C1 C2 C3 C4 C5 ±SE TREATMENT TREATMENT Figure 2: The effect of food availability on A: numbers of adult P. marina; and B: adult sex ratio (expressed as the female/male ratio). Data are means ± 1 sterror of 4 replicates per treatment averaged over the total duration (17 days) of the experiment.

No significant differences between treatments were found for total development time of F1-progeny. Male development times tended to be longer than female development times, but differences were not significant (Table 2).

133

Table 2: Total development time of P. marina at five different food availabilities. Data are means ± 1 stdev of four replicates. Development Time Trea Sex Minimum Mean ± 1 stdev t. ♀ 3.85 4.02 ± 0.17 C1 ♂ 3.64 4.07 ± 0.35 ♀ 1.85 3.26 ± 0.98 C2 ♂ 2.97 4.34 ± 1.69 ♀ 3.54 5.01 ± 1.17 C3 ♂ 3.31 4.03± 0.64 ♀ 1.92 3.71 ± 1.66 C4 ♂ 3.47 4.56 ± 0.76 ♀ 2.22 2.80 ± 0.64 C5 ♂ 3.22 4.05 ± 0.91

Individual biomass of adult P. marina differed significantly between treatments (F= 38.31; p≤0.0001), which was largely due to the highest food availability treatment (mean adult wet weight 8.99µg compared to 2.95µg at the lowest food availability (Fig. 3A)). Both body length and maximal body width were significantly affected by food availability, yet according to somewhat different patterns. At the highest food availability, nematodes grew significantly longer (p<0.0001) than at all other food concentrations (Fig. 3B). Maximum body width decreased with decreasing food densities: C1 differed significantly from all other treatments (p<0.0001), but in addition, body width of nematodes at C2 was significantly higher than at C4 and C5 (p<0.0001), and C3 differed significantly from C5 (p<0.0001, Fig. 3C Y axis: individual biomass). Adult population biomass as calculated from numbers and mean individual biomass of adult P. marina differed significantly between treatments (F= 59.13, p<0.0001), time (F= 51.49, p<0.0001) and treatment vs time (F= 3.78, p<0.0001). Again, the highest food concentration (3 x 1010 cell ml-1) differed significantly from all others (p<0.0001), and the second highest (C2) differed

134

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Results: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS significantly from the lowest two food availability treatments (p<0.0001, Fig. 3D Y axis: population biomass ).

10 2000

9 A B 1900 8

7 1800

6 1700 5 Length Biomass (µg)

4 1600

3 1500 2

1 1400 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment

1400 80

C 1200 D 75 1000

70 800

65 600 Witdth Biomass (µg) 60 400

55 200

0 50

C1 C2 C3 C4 C5 Mean -200 C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 3: A- Individual biomass (µg), B-Body length (µm) and, C-Maximum body width (µm) of P. marina adults on the 8th day of the experiment. D- Population biomass of adult P. marina. Data are means ± 1 sterror of 4 replicates per treatment.

Diplolaimelloides meyli

Egg deposition occurred from the first counting day onwards, with a high increase in numbers after 92h at the higher food availabilities. Fecundity was highest at C2 and C3, but only the pairwise differences with C5 were significant (p<0.03) (Table 1). Juveniles appeared from 8 or 8.5 days onwards in all treatments, but in lower numbers at the lower two food availabilities. The total population of D. meyli (including all vermiform stages but not eggs) increased continuously and monotonously with time in all treatments, densities typically being higher at intermediate food densities (C2 and C3) (F=

135

4.73, p<0.01); the interaction factor treatment vs time was also highly significant (F= 2.22; p<0.0001, Fig. 4A). th The first F1-adult females appeared on the 16 day in all treatments, except th th at the lowest bacterial density. F1-adult males appeared from the 18 to 26 day onwards. Numbers of adults abruptly decreased from 30 days onwards at C2 to C4, and more gradually from 34 days onwards at the other bacterial densities. Peaks in D. meyli adult densities occurred fairly simultaneously at all food availabilities except the lowest one, where this peak was delayed by about 4 days (Fig. 4B).

1000 340 320 900 A 300 B 280 800 260 700 240 220 600 200 180 500 160

Total Population 400 140 Adult Population Adult 120 300 100 80 200 60

100 40 C1 20 C1 C2 C2 0 C3 0 C3 T2 T6 T10 T14 T18 T22 T26 T30 C4 T2 T6 T10 T14 T18 T22 T26 T30 T38 T46 C4 TIME C5 C5 TIME Figure 4: A: Total D. meyli population development, and B: D. meyli adult population development over time at five different food availabilities. Data are averages of four replicates per treatment and time.

Bacterial density had a significant effect on the average densities of adult D. meyli in our microcosms (F= 8.55; p<0.0001, Fig. 5A). Adult densities increased with increasing food availability up to a bacterial density of 3 x 109 cells ml-1; however, at the highest bacterial availability, adult D. meyli densities remained low and were comparable to the treatment with the lowest food availability. Pairwise differences were highly significant between C1 and C2 and between C2 and C5 (both p<0.0001). The adult sex ratio was highly female-biased at C1 and C3, with 65 ± 13.44 and 66 ± 12.35 % (mean ± 1 stdev) females, respectively, and differed significantly between treatments (p<0.03, Kruskal-Wallis test, Fig. 5B).

136

0140 3.6

3.4 0120 A B 3.2

0100 3.0

2.8 080

2.6

060 N° Adult D.M. Sex Ratio D.M. Ratio Sex 2.4

2.2 040

2.0

020 1.8

00 1.6 Mean C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 ±SE ±SE TREATMENT TREATMENT Figure 5: The effect of food availability on A: numbers of adult D. meyli; and B: adult sex ratio (expressed as the female/male ratio). Data are means ± stderror of 4 replicates among experimental time per treatment averaged over the total duration (46 days) of the experiment.

Embryonic development time, postembryonic development time and total development time did not differ significantly between treatments, although they were on average shorter at the intermediate food availabilities (C2 – C4) than at the higher- and lowermost ones (Table 3) Male postembryonic development time and total development time were considerably longer than in females (p<0.0001).

137

Table 3: Minimum and mean embryonic development time, postembryonic development time and total development time of D. meyli at five different food availabilities. Data are means ± 1 stdev of four replicates. Embryonic Dev. Time Postembryonic Dev. Time Development Time Trea Minimu Mean ± 1 Mean ± 1 Mean ± 1 Sex Minimum Minimum t m stdev stdev stdev ♀ 9.02 9.83 ± 0.70 11.89 12.99 ± 0.96 C1 2.87 3.16 ± 0.34 ♂ 9.77 13.81 ± 1.66 15.51 16.97 ± 1.51 ♀ 5.38 7.69 ± 1.73 10.30 11.01 ± 0.73 C2 2.40 3.32 ± 1.12 ♂ 9.40 10.89 ± 2.17 12.70 14.20 ± 1.90 ♀ 7.22 8.40 ± 0.80 10.90 12.50 ± 1.53 C3 3.12 4.10 ± 1.18 ♂ 8.79 10.52 ± 1.53 12.50 14.62 ± 1.75 ♀ 6.66 7.89 ± 0.87 11.10 11.16 ± 1.28 C4 2.95 3.27 ± 0.51 ♂ 8.70 9.91 ± 1.53 11.70 13.18 ± 2.00 ♀ 8.05 9.85 ± 1.58 11.60 12.98 ± 1.82 C5 2.49 3.14 ± 0.79 ♂ 8.51 11.89 ± 2.78 11.80 15.02 ± 2.53

Biomass of individual adult D. meyli showed a pronounced and monotonous dependence on food availability, being highest (0.57 µg) at the highest food availability and lowest (0.25 µg) at the lowest food availability (p< 0.0001; Kruskal-Wallis test, Fig. 6A). Nematode biomass is proportional to body length and to the square of the maximal body width. Both parameters were significantly affected by food availability, yet according to a slightly different pattern: At the highest food availability, nematodes grew significantly longer (p<0.0001; Kruskal-Wallis test), but not wider than at lower food densities. Body width only differed significantly between C5 and all other treatments, and between C2 and C3 + C4 (both p< 0.0001, Fig. 6B and 6C). Adult population biomass as calculated from numbers and mean individual biomass of adult D. meyli differed significantly between treatments (F= 13.86, p<0.0001) and time (F= 59.01, p<0.0001); the interaction factor treatment vs time was also highly significant (F= 5.85, p<0.0001). Adult population biomass was highest in C2 and showed a pattern that was overall more similar to the abundance vs food-availability pattern than to the individual biomass vs food-availability pattern (Fig. 6D).

138

0,80 1000

0,70 A B

900 0,60

0,50 800 Length 0,40 Biomass (µg) 700

0,30

600 0,20

0,10 500 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment 50

32 C 45 D 30 40

28 35

26 30

Witdth 24 25 Biomass (µg)

22 20

20 15

18 10

5 16 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 6: A- Individual biomass (µg), B-Body length (µm) and, C-Maximum body width (µm) of adult D. meyli on the 24th day of the experiment. D- Population biomass of adult D. meyli. Data are means ± 1 sterror of 4 replicates per treatment.

Diplolaimelloides oschei

Eggs appeared from the first counting day onwards in all treatments, with a steep increase in numbers after 92h at the highest food availability. Fecundity was significantly different between treatments (F= 3.78, p<0.03), the only significant pairwise difference being between C2 and C5 (p=0.02) (Table 1). Juveniles appeared from 6 to 7.5 days onwards in all treatments. Total population (including all vermiform stages but not eggs) development had a continuous and monotonous increase with time in most treatments, with higher numbers of individuals at intermediate food concentrations (C2-C4) (F= 4.61, p<0.01); the interaction factor treatment vs time was also significant (F= 1.57; p=0.01, Fig. 7A).

139

The first F1-adult females appeared on the 16th day in all treatments and replicates. The first F1-adult males appeared on the 16th to 22nd day. Numbers of adults started increasing from 16 days onwards at C2, and from 18 days onwards at the other food availabilities; they decreased again from 24 days onwards at C1-C4 and more gradually from 26 days onwards at C5. Peaks in D. oschei adult densities occurred fairly simultaneously at C2-C4 (Fig. 7B).

1000 340

900 320 A 300 B 800 280 260 700 240 220 600 200 500 180 160

Total Population Population 400

Adult Population 140 120 300 100

200 80 60 100 40 C1 C2 20 C1 0 C3 C2 T2 T6 T10 T14 T18 T22 T26 T30 C4 0 C3 C5 T2 T6 T10 T14 T18 T22 T26 T30 T38 T46 C4 TIME C5 TIME Figure 7:A: Total D. oschei population development, and B: D. oschei adult population development over time at five different food availabilities. Data are averages of four replicates per treatment and time.

Food availability significantly impacted D. oschei adult densities (F= 4.33; p=0.03, Fig. 8A), with a pattern similar to that in D. meyli. Adult densities increased, albeit discontinuously, with increasing food availability up to a bacterial density of 3 x 109 cells ml-1; however, at the highest bacterial density, D. oschei densities remained low and were comparable to those at 3 x 108 cells ml-1. Pairwise differences were only significant between C1 and C2 (p<0.02). The sex ratio was strongly female-biased in all treatments, with highest values of 70 ± 11.70 % and 71 ± 10.08 % (mean ± 1 stdev) females at C2 and C3, respectively, but did not differ significantly between treatments (Fig. 8B).

140

0140 3.6

3.4 0120 A B 3.2

0100 3.0

2.8 080

2.6

060 N° Adult D.O. Adult N°

Sex Ratio D.O. 2.4

040 2.2

2.0 020 1.8

00 Mean 1.6 C1 C2 C3 C4 C5 ±SE C1 C2 C3 C4 C5 Mean ±SE Treatment TREATMENT Figure 8: The effect of food availability on A: numbers of adult D. oschei; and B: adult sex ratio (expressed as the female/male ratio). Data are means ± stderror of 4 replicates among experimental time per treatment averaged over the total duration (46 days) of the experiment.

Embryonic development time was significantly longer at C5 than at all other treatments (p=0.01; Kruskal-Wallis test). Postembryonic development time and total development time did not differ between treatments (Table 4). Male postembryonic development time and total development time were considerably slower than in females (p<0.0001).

Table 4: Minimum and mean embryonic development time, postembryonic development time and total development time of D. oschei at five different food availabilities. Data are means ± 1 stdev of four replicates. Embryonic Dev. Time Postembryonic Dev. Time Development Time Tre Minimu Mean ± 1 Se Mean ± 1 Mean ± 1 Minimum Minimum at. m stdev x stdev stdev ♀ 9.74 10.65 ± 0.68 12.25 14.27 ± 1.41 C1 2.51 3.62 ± 0.98 ♂ 12.49 13.73 ± 0.84 15.00 17.35 ± 1.75 ♀ 7.84 9.76 ± 1.29 10.33 12.14 ± 1.21 C2 2.06 2.39 ± 0.31 ♂ 11.97 12.79 ± 0.79 14.30 15.17 ± 0.95 ♀ 9.24 10.88 ± 1.42 11.55 13.10 ± 1.81 C3 2.31 2.82 ± 0.59 ♂ 13.51 14.16 ± 1.48 14.40 16.08 ± 2.51 ♀ 9.29 10.33 ± 0.89 10.97 12.13 ± 0.88 C4 1.64 1.81 ± 0.26 ♂ 12.07 13.67 ± 1.33 13.75 15.48 ± 1.46 ♀ 8.50 9.39 ± 0.96 11.15 12.00 ± 0.99 C5 2.50 2.61 ± 0.09 ♂ 11.60 12.49 ± 0.77 14.25 15.10 ± 0.75

141

Individual biomass of adult D. oschei showed a pronounced and monotonous dependence on food availability, with highest biomass (0.37 µg) at the highest food availability and lowest (0.13 µg) at the lowest food availability (p<0.0001; Kruskal-Wallis test, Fig. 9A). Both length and maximal body width were significantly affected by food availability, yet according to a slightly different pattern. At the highest food availability, nematodes grew significantly longer (p<0.0001) than at all other treatments (Fig. 9B). Width decreased continuously and monotonously from the highest to the lowest food concentration (p<0.01). Moreover, the lowest food availability had a more pronounced effect on body width than on length (p≤0.0001, Fig. 9C). Biomass of the adult D. oschei population differed significantly between treatments (F= 13.63, p<0.0001), time (F= 221.95, p<0.0001) and treatment vs time (F= 1.70, p<0.0001), being higher at C2 and C3 than at all other food treatments (Fig. 9D).

0,80 760

740 0,70 A 720 B

700 0,60 680

660 0,50 640

0,40 Length 620 Biomass (µg) 600

0,30 580

560

0,20 540

520 0,10 Mean 500 C1 C2 C3 C4 C5 Mean ±SE C1 C2 C3 C4 C5 Treatment ±SE Treatment 50

32 C 45 D 30 40

28 35

26 30

Witdth 24 25 Biomass (µg) Biomass

22 20

20 15

18 10

16 Mean 5 C1 C2 C3 C4 C5 C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 9: A- Individual biomass (µg), B-Body length (µm) and, C-Maximum body width (µm) of adult D. oschei on the 24th day of the experiment. D- 142

Population biomass of adult D. oschei. Data are means ± 1 sterror of 4 replicates per treatment.

143

Discussion

‘Enrichment opportunists’ vs ‘general opportunists’ The three nematode species studied here show a differential response to food availability. The strongest difference is between the rhabditid, Pellioditis marina, and the two monhysterid species, the former clearly having highest fitness at the highest food concentration, whereas the monhysterids peaked at intermediate food concentrations. This is a very good illustration of the distinction terrestrial nematologists make between ‘enrichment opportunists’ (mainly rhabditids and diplogasterids) and ‘general opportunists’ (mainly Cephalobidae and to some extent Plectidae) (Bongers and Bongers, 1998). The former group consists of species with extremely short generation times and large numbers of progeny under optimal conditions, but these optimal conditions involve very high food levels (Vranken and Heip, 1983; Bongers and Bongers, 1998; Moens et al., 1996; Moens and Vincx, 2000b). Rhabditidae populations in general respond rapidly to bottom-up processes such as nutrient and organic matter loading through the stimulatory effects these have on bacterial cell density and production. When microbial activity and densities decrease below optimal values – perhaps in part through overgrazing of bacterial populations by the Rhabditidae (De Mesel et al., 2004, 2006; Moens et al., unpubl.) – these r-strategists form 'dauer larvae', a metabolically dormant yet motile juvenile stage which is able to survive food depletion and/or to disperse to new food-rich spots (Bongers, 1990; Bongers and Bongers, 1998; Ferris et al., 1995, 1997). Under field conditions, P. marina typically reaches high abundances on rotting macroalgae (Derycke et al., 2007b), especially in the earlier stages of decomposition, when labile components are leaching from the algae and bacterial growth is extremely high. However, P. marina populations typically decline quite fast. Monhysterid populations increase somewhat more slowly but also do not crash so rapidly (Moens et al., 1996; Moens and Vincx, 2000a). In contrast to rhabditid nematodes, monhysterids have no dauer larvae. Moreover, the species we have studied are typically abundant on more refractory detritus from vascular plants such as Spartina, where

144

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Discusion: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS decomposition is slower and bacterial densities are not as high as on macroalgae (Moens and Vincx, 2000b, and references therein). The fundamental niche of monhysterids with respect to food availability appears to be broader than in P. marina (Moens and Vincx, 2000b), yet the present results show that optimal fitness of the monhysterids is also restricted to a fairly narrow range of food availabilities.

Food thresholds

With respect to bioenergetic performance, Schiemer (1987) discriminated the following three food availability ranges: (1) below-threshold densities, where assimilation rates can at most support maintenance metabolism, (2) above- threshold densities where bioenergetic traits like production and assimilation show a pronounced increase with food supply, and (3) surplus food densities where the same bioenergetic traits display no or only a weak response to further increases in food availability. Previous studies with different species of Rhabditidae have shown that these nematodes typically require a threshold for feeding of close to 109 bacterial cells ml-1 (Nicholas et al., 1973; Schiemer, 1982a,b, 1983; Moens and Vincx, 2000b). Above those thresholds, feeding rapidly increases with increasing food level. However, assimilation efficiency may decrease at very high food densities (Moens et al., 2006), which is at odds with Schiemer’s (1987) proposed bioenergetic responses to surplus food. Comparing with the present results, it is surprising that even at a food concentration as low as 3 107 cells ml-1 there was still population growth, albeit very limited. In the presence of sufficient densities of food particles, rhabditid nematodes typically show a random food ingestion, a behaviour which is likely to be energetically inefficient at low food densities (Vranken et al., 1988). We also have to be careful when comparing the food densities in the present experiment with those in previous studies, because we applied 50 µl volumes of food at a given density. This implies that food density was not uniform over the entire surface of our microcosms, and therefore our bacterial densities may correspond to yet lower densities in other studies. This would

145

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Discusion: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS bring the optimal feeding conditions of our experiment closer to the optimal food density for assimilation found for P. marina (Moens et al., 2006). The two Diplolaimelloides species required somewhat lower food thresholds and densities than P. marina, which may relate to a generally more selective food uptake in Monhysteridae compared to rhabditid nematodes (Vranken et al., 1988). The threshold for active feeding in D. meyli was 2.5 x 108 cells ml-1 (Moens and Vincx, 2000b), while in another monhysterid nematode, Monhystera disjuncta, mortality and generation time increased significantly at bacterial densities of 108 cells ml-1 and lower (Vranken et al., 1988). Optimal bacterial densities for assimilation of food by D. meyli and for survival and shortest generation time in M. disjuncta were reached from 5 x 108 cells ml-1, and 10 (in D. meyli) to even 200 (in M. disjuncta) times higher bacterial densities did not significantly further improve monhysterid ‘fitness’ (Vranken et al., 1988; Moens and Vincx, 2000b). In the present experiment, however, the optimal bacterial density for the population development of both Diplolaimelloides species was 3 x 109 cells ml-1, and both higher (in both species) and lower (esp. in D. meyli) bacterial densities negatively impacted population development. This appears to be at odds with the above- mentioned broad range of ‘optimal’ food densities. However, the parameters determined in the studies referred to may not be adequate fitness parameters: Moens and Vincx (2000b) determined food assimilation, which was measured over a very short (1-h) incubation. Vranken et al. (1988) focused on mortality and development times, parameters which in our experiment also did not yield significant differences between most food availability treatments. A study on the movement of nematodes towards bacterial spots further indicated that D. meyli had a preference for food densities around 3 x 109 cells ml-1 (Moens et al., 1999), in line with the present results. D. oschei, however, had a bimodal pattern, preferring cell densities of >109 cells ml-1 as well as very low densities (107 cells ml-1).

146

Life history, population increase and fitness

Fitness, when defined as the intrinsic rate of population increase (rm), cannot properly be calculated from the current experiments, because they were not designed to determine age-specific mortality and reproductive output (Vranken and Heip, 1983; Kammenga et al., 1996a). In exponentially growing populations, rm can also be approximated from the ln of population increase divided over time (Warwick, 1981), but this approach is equally inapplicable to our experiments, since we started from an adult-only inoculum and did not achieve stable age distribution in our experimental populations. Hence, our assessment of nematode fitness is based on multiple traits which all contribute to the rate of population increase, such as fecundity, development time, and sex ratio. Biomass is included as a variable since population increase can also be approximated from population biomass instead of number of individuals, particularly when viewed from the angle of energy transfer through a population (Fenchel, 1974). Fecundity was generally low in our inoculum when compared to published values on P. marina (up to 600 eggs female-1 (Vranken and Heip, 1983)), but comparable to previous reports on D. meyli (Moens and Vincx, 2000a). However, in making such comparisons, one should be aware that our parental individuals (a) were not followed from the beginning of their reproductive period, and (b) deposited substantial numbers of eggs during the 24h incubation in ASW prior to their inoculation into the experimental microcosms. Trends in fecundity of parental adults resembled those of total population development reasonably well in both Diplolaimelloides species, but not at all in P. marina. A complete understanding of food availability effects on life history traits and fitness requires that effects be tested in subsequent generations, which is very time-consuming and technically difficult because of overlap between subsequent generations. Surprisingly, the only development-time related parameter exhibiting a significant difference between food treatments was embryonic development time in D. oschei. Postembryonic and total development time, however, were not significantly affected by food availability in any of the species studied

147

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Discusion: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS here. These results are in agreement with observations on Monhystera disjuncta, where food density also had only a very minor effect on minimum generation time (Vranken et al., 1988). The absence of a strong relation with food density in P. marina is, however, at odds with data on another rhabditid, Caenorhabditis briggsae, where minimum generation time increased threefold when bacterial densities dropped from 1011 to 2 108 cells ml-1 (Schiemer, 1982b), and on the freshwater bacterial-feeding Plectus palustris (Schiemer et al., 1980). Development times in bacterivorous nematodes are typically highly dependent on abiotic environmental factors such as temperature and salinity (Tietjen and Lee, 1972, 1977; Schiemer et al., 1980; Warwick, 1981; Moens and Vincx, 2000a). Embryonic development time of F1 in our experiments was likely more determined by the feeding history of the females than by the food availability surrounding the deposited eggs (see above). This is supported by observations that food supply affects egg size (Schiemer, 1982a). The lack of food availability effects on postembryonic development time, however, may suggest that juvenile maturation (postembryonic development) also still relies importantly on energy reserves transferred from female to progeny (Herman and Vranken 1988; Moens and Vincx, 2000a,). It is interesting to note that in both monhysterid species studied here, males had longer development times than females, irrespective of food availability. This agrees well with a previous study on D. meyli, where male development times were longer than female development times over a range of temperatures and salinities (Moens and Vincx, 2000a), but is at odds with observations on Monhystera. disjuncta. In the latter species, however, the type of food offered differentially affected male and female development times (Vranken et al., 1988). Juvenile mortality has been found to be a highly sensitive life-cycle trait with respect to different kinds of pollution (Kammenga et al., 1996 b, 1997). It also increased strongly at E. coli densities below 109 cells ml-1 in C. briggsae (Schiemer, 1982b). In our experiment, however, F1-juvenile mortality was negligible in all treatments and species. When we assume similar food-dependent effects on juvenile survival and development times in F2 and later generations as in F1, it is difficult to explain

148

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Discusion: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS the different trends in monhysterid individual biomass and population development vs food availability. Both monhysterid species grew larger but showed a reduced fecundity and population development at the highest food density. M. disjuncta exhibited even much more pronounced food-density effects on individual adult biomass, log (body mass) being a linear function of log (bacterial density). Its reproductive output, however, remained constant at surplus food densities (Vranken et al., 1988). Trade-offs between investment in biomass and reproduction are common in invertebrates (Bennett and Boraas, 1989; Ernsting et al., 1993; Clarke, 1993). In planktonic cladocerans like Daphnia, larger mothers are expected to either produce more or larger progeny (Lampert, 1993; Boersma, 1995). Our data show that the former definitely does not hold in the two Diplolaimelloides species. We did not measure egg sizes and hence cannot assess the latter option. However, the rationale for producing larger offspring is that these are typically more resistant to starvation and hence have better chances for survival. Juvenile mortality in our experiments was negligible in all food treatments. Resistance to starvation would seem more relevant under conditions of food limitation and/or crowding, both of which were more likely to apply to other than the highest food-availability treatments. The causes for the observed trade-offs between biomass and reproduction in both Diplolaimelloides species at high food availabilities remain unclear. In contrast, food density effects on P. marina adult biomass parallelled effects on population development, nematodes growing larger at the highest food density. Food availability also affected size at maturity in C. briggsae, but following a more gradual pattern (Schiemer, 1982a). The observation that P. marina had equally short development times at lower food densities, but a smaller size at maturity, may be interpreted as a strategy to cut down the metabolic costs required to attain sexual maturity (Schiemer et al., 1980). Sex ratio did not vary significantly between food treatments for any of the species studied here. There was generally a strong bias in favour of females (62%≤), which may be linked to the generally faster maturation of females in our experiments. For P. marina, moderately (Vranken, 1985; Moens and Vincx, 2000a) to strongly (66-75% females, Tietjen et al., 1970) female-

149

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 6: Discusion: Differential effets of food availability on … DOUTORADO EM CIÊNCIAS BIOLÓGICAS biased sex ratios have been reported in North Sea + Westerschelde and in a North-American population, respectively. Note, however, that these populations may actually have been different cryptic species. In contrast, Diplolaimelloides bruciei (Warwick, 1981) and D. meyli under optimal temperature conditions (Moens et al., 1996) had a 66% male dominance . This is at odds with the sex ratio of 2:1 in favour of females, as found in the present experiment. However, sex ratios in these nematodes vary importantly with environment, including e.g. temperature and salinity (Moens and Vincx, 2000a), and at least part of the discrepancy between this and previous studies may be explained by differences in the abiotic regime.

150

Conclusions

The three bacterial-feeding nematode species studied here showed differential responses to food density, which agree with the general idea of Rhabditidae having extreme colonization abilities under very high food availability, while Monhysteridae tend to have a somewhat slower population development and comparatively lower food requirements. Several life history traits, including juvenile mortality and development time, did not exhibit a clear food-density dependence. Care has to be taken, though, when extrapolating from results on the F1 generation to subsequent generations, because feeding history of the parental adults may strongly affect life history traits of F1 individuals. A population assay, focusing on total and adult population development, may be the easiest way to approach food effects on fitness in these nematode species. Comparison with published data on confamiliar nematode species further reveals that responses to food availability as well as to other environmental factors are very species-specific and may relate to responses in different life history traits. The present results can be used to generate hypotheses on total community development as well as on community composition in multispecies assemblages under different conditions of food availability.

151

Acknowledgements

This work was financially supported by a CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) grant to the first author, and by Ghent University through BOF projects 0110600002 and 01GZ0705. T.M. is a postdoctoral fellow with the Flemish Fund for Scientific Research. Tatiana Maria’s helpful hands saved the authors many hours of nematode sorting during the set-up of the experiments.

152

References

Andrassy, I., 1956. The determination of volume and weight of nematodes. Acta Zool. Acad. Sci. Hung. 2, 1-15. Bennett, W.N., Boraas, M.E., 1989. A demographic profile of the fastest growing metazoan: a strain of Brachionus calyciflorus (Rotifera). Oikos 55, 365-369. Boersma, M., 1995. Offspring size and parental fitness in Daphnia magna. Evol. Ecol. 11, 439-450. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Bongers, T., Bongers, M., 1998. Functional diversity of nematodes. Appl. Soil Ecol. 10, 239–251. Clarke, A., 1993. Reproductive trade-offs in caridean shrimps. Funct. Ecol. 7, 411-419. Coomans, A., 2000. Nematode systematics: past, present and future. Nematology 2: 3-7. Coull, B.C., 1985. Long-term variability of estuarine meiobenthos: an 11 year study. Mar. Ecol. Prog. Ser. 24, 205-218. Coull, B.C., 1986. Long-term variability of meiobenthos: value, synopsis, hypothesis generation and predictive modelling. Hydrobiologia 142, 271- 279. Coull, B.C., 1999. Role of meiofauna in estuarine soft-bottom habitats. Austr. J. Ecol. 24, 327-343. De Mesel, I., Derycke, S., Moens, T., Van der Gucht, K., Vincx, M., Swings, J., 2004. Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study. Env. Microbiol. 6, 733-744 . De Mesel, I., Derycke, S. Swings, J., Vincx, M., Moens, T., 2006. Role of nematodes in decomposition processes: Does within-trophic group diversity matter? Mar. Ecol. Prog. Ser. 321, 157–166. Derycke, S., Remerie, T., Vierstraete, A., Backeljau, T., Vanfleteren, J., Vincx, M,. Moens, T., 2005. Mitochondrial DNA variation and cryptic speciation

153

within the free-living marine nematode Pellioditis marina. Mar. Ecol. Prog. Ser. 300, 91-103. Derycke, S., Backeljau, T., Vlaeminck, C., Vierstraete, A., Vanfleteren, J., Vincx, M., Moens, T., 2006. Seasonal dynamics of population-genetic structure in cryptic taxa of the Pellioditis marina complex (Nematoda: Rhabditida). Genetica 128, 307-321. Derycke, S., Backeljau, T., Vlaeminck, C., Vierstraete, A., Vanfleteren, J., Vincx, M., Moens, T., 2007a. Spatiotemporal analysis of population genetic structure in Geomonhystera disjuncta (Nematoda, Monhysteridae) reveals high levels of molecular diversity. Mar. Biol., in press. Derycke, S., Van Vynckt, R., Vanoverbeke, J., Vincx, M., Moens, T., 2007b. Colonization patterns of Nematoda on decomposing algae in the estuarine environment: Community assembly and genetic structure of the dominant species Pellioditis marina. Limnol. Oceanogr. 52, in press. Dietrich, G, Kalle, K., 1957. Allgemeine Meereskunde. Gebrüder Borntraeger, Berlin, 492 pp. . Ernsting, G., Zonneveld, C., Isaaks, J.A., Kroon, A., 1993. Size at maturity and patterns of growth and reproduction in an insect with indeterminate growth. Oikos 66, 17-26. Fenchel, T. (1974). Intrinsic rate of natural increase: the relationship with body size. Oecologia 14:317-326. Ferris, H., Lau, S., Venette, R., 1995. Population energetics of bacterial-

feeding nematodes: respiration and metabolic rates based on CO2 production. Soil Biol. Biochem. 27, 319–330. Ferris, H., Venette, R., Lau, S., 1997. Population energetics of bacterial- feeding nematodes: carbon and nitrogen budgets. Soil Biol. Biochem. 29, 1183–1194. Gallucci, F., Steyaert, M., Moens, T., 2005. Can field distributions of marine predacious nematodes be explained by sediment constraints on their foraging success? Mar. Ecol. Prog. Ser. 304, 167-178. Hairston, N.G., Smith, F.E., Slobodkin, L.B., 1960. Community structure, population control, and competition. Am. Nat. 94, 421–425.

154

Heip, C., Vincx, M., Vranken, G., 1985. The ecology of marine nematodes. Oceanogr. Mar. Biol. Annu. Rev. 23, 399- 489. Herman, P.M.J., Vranken, G., 1988. Studies of the life-history and energetics of marine and brackish-water nematodes. II. Production, respiration and food uptake by Monhystera disjuncta. Oecologia 77, 457-463. Hillebrand, H., 2002. Top-down versus bottom-up control of autotrophic biomass: a meta-analysis on experiments with periphyton. J. N. Am. Benth. Soc. 21, 349–369. Hillebrand, H., Kahlert, M., Haglund, A.L., Nagel, U.G.B.S., Wickham, S, 2002. Control of microbenthic communities by grazing and nutrient supply. Ecology 83, 2205–2219. Jian Li, Vincx, M., 1993. The temporal variation of intertidal nematodes in the Westerschelde. I. The importance of an estuarine gradient. Neth. J. Aquat. Ecol. 27, 319-326. Kammenga, J.E., Busschers, M., Van Straalen, N.M., Jepson, P.C., Bakker, J., 1996a. Stress induced fitness reduction is not determined by the most sensitive life-cycle trait. Funct. Ecol. 10, 106–111. Kammenga, J.E., Riksen, J.A.G., 1996b. Comparing differences in species sensitivity to toxicants: phenotypic plasticity versus concentration- response relationships. Environ. Toxicol. Chem. 15, 1649–1653. Kammenga, J.E., Van Koert, P.H.G., Koeman, J.H., Bakker, J., 1997. Fitness consequences of toxic stress evaluated within the context of phenotypic plasticity. Ecol. Appl. 7, 726–734. Lambshead, J., 1993. Recent developments in marine benthic biodiversity research. Oceanis 19, 5–24. Lampert, W., 1993. Phenotypic plasticity of the size at first reproduction in Daphnia: The importance of maternal size. Ecology 74, 1455-1466. McQueen, D.J., Johannes, M.R.S., Post, J.R., Stewart, T.J., Lean, D.R.S., 1989. Bottom-up and top-down impacts on freshwater pelagic community structure. Ecol. Monogr. 59, 289-309. Menge, B.A., Sutherland, J.P., 1976. Species diversity gradients: synthesis of the roles of predation, competition, and temporal heterogeneity. Am. Nat. 110, 351–369.

155

Menge, B.A., Daley, B.A., Lubchenco, J., Sanford, E., Dahlhoff, E., Halpin, P.M., Hudson, G., Burnaford, J.L., 1999. Top-down and bottom-up regulation of New Zealand rocky intertidal communities. Ecol. Monogr. 69, 297–330. Menge, B.A., Sanford, E., Daley, D. A., Freidenburg, T. L., Hudson, G., Lubchenco, J., 2002. Inter-hemispheric comparison of bottom-up effects on community structure: Insights revealed using the comparative- experimental approach. Ecol. Res.17, 1-16. Moens, T., Vierstraete, A., Vincx, M., 1996. Life strategies in two bacterivorous marine nematodes: preliminary results. PSZN I Mar. Ecol. 17, 509–518. Moens, T., Vincx, M., 1998. On the cultivation of free-living estuarine and marine nematodes. Helgol. Meeresunters. 52, 115–139. Moens, T., Verbeeck, L., de Maeyer, A., Swings, J., Vincx, M., 1999. Selective attraction of marine bacterivorous nematodes to their bacterial food. Mar. Ecol. Prog. Ser. 176, 165–178. Moens, T., Vincx, M., 2000a, Temperature and salinity constraints on the life cycle of two brackish-water nematode species. J. Exp. Mar. Biol. Ecol. 243, 115–135. Moens, T., Vincx, M., 2000b. Temperature, salinity and food thresholds in two brackish water bacterivorous nematode species: assessing niches from food absorption and respiration experiments. J. Exp. Mar. Biol. Ecol. 243, 137-154. Moens, T., Herman, P.M.J., Verbeeck, L., Steyaert, M., Vincx, M., 2000. Predation rates and prey selectivity in two predacious estuarine nematode species. Mar. Ecol. Prog. Ser. 205, 185–193. Moens, T., Bergtold, M., Traunspurger, W., 2006. Chapter 6: Feeding ecology of free-living benthic nematodes. In: Eyualem, A., Andrássy, I., Traunspurger, W. (Eds.), Freshwater nematodes: ecology and taxonomy. CAB International Publishing, Cambridge, USA, pp. 105-131. Murdoch, W.W., 1966. Community structure, population control, and competition - A critique. Am. Nat. 100, 219–226.

156

Murray, B.G. Jr., l985. Population growth rate as a measure of individual fitness Oikos 42, 323-326 Nicholas, W.L., Grassia, A., Viswanathan, S., 1973. The efficiency with which Caenorhabditis briggsae (Rhabditidae) feeds on the bacterium, Escherichia coli. Nematologica 19, 411-420. Nur, N., 1987. Population growth rate and the measurement of fitness: a critical reflection. Oikos 48, 338-341. Power, M.E., 1992. Top-down and bottom-up forces in food webs: Do plants have primacy? Ecology 73, 733-746. Rosemond, A.D., 1993. Top-down and bottom-up control of stream periphyton: effects of nutrient and herbivores. Ecology 74, 1264-1280. Schiemer, F., 1982a. Food dependence and energetics of free-living nematodes. I. Respiration, growth and reproduction of Caenorhabditis briggsae (Nematoda) at different levels of food supply. Oecologia 54, 108– 121. Schiemer, F., 1982b. Food dependence and energetics of free-living nematodes. II. Life history parameters of Caenorhabditis briggsae (Nematoda) at different levels of food supply. Oecologia 54, 122-128. Schiemer, F., 1983. Food dependence and energetics of free-living nematodes. III. Comparative aspects with special consideration of two bacterivorous species Caenorhabditis briggsae and Plectus palustris. Oikos 41, 32-43. Schiemer, F., 1985. Bioenergetic niche differentiation of aquatic invertebrates. Verhandlungen der Internazionale Vereinigung für Theoretische und Angewandte Limnologie 22, 3014-4018. Schiemer, F., 1987. Chapter 6: Nematoda, In: Vernberg, J.F., Pandian, T.J. (Eds.), Animal energetics, Vol. 1. Academic Press, New York, pp. 185- 215. Schiemer, F., Duncan, A., Klekowski, R.Z., 1980. A bioenergetic study of a benthic nematode, Plectus palustris de Man 1880, throughout its life cycle. II. Growth, fecundity and energy budgets at different densities of bacterial food and general ecological considerations. Oecologia 44, 205-212.

157

Sibly, R., Calow, P., 1989. An integrated approach to lifecycle evolution using selective landscapes. J. Theoret. Biol. 102, 527-547. Somerfield, P.J., Warwick, R.M., Moens, T., 2005. Chapter 6: meiofauna techniques, In: Eleftheriou, A., McIntyre, A. (Eds.), Methods for the Study of Marine Benthos, 3rd Edn. Blackwell Science Ltd, Oxford, pp. 229–272. Tietjen, J.H., Lee, J.J.,1972. Life cycles of marine nematodes. Influence of temperature and salinity on the development of Monhystera denticulata Timm. Oecologia 10: 167-176. Tietjen, J.H., Lee, J.J.,1977. Life history of marine nematodes. Influence of temperature and salinity on the reproductive potential of Chromadorina germanica Bütschli. Mikrofauna Meeresboden 15, 263-270 Vanfleteren, J.R., 1980. Nematodes as nutritional models. In: Zuckerman, R.M. (Ed.) Nematodes as biological models. Vol. 2. Acad. Press. New York, pp. 47-80. Vranken, G., 1985. Een autoecologische studie van brakwater nematoden in laboratoriumomstandigheden (in Dutch). Ph.D. Thesis, State Univ. Gent, pp. 281 + 203. Vranken, G., Heip, C., 1983. Calculation of the intrinsic rate of natural increase with Rhabditis marina Bastian 1865 (Nematoda). Nematologica 29, 468–477. Vranken, G., Herman, P.M.J., Heip, C., 1988. Studies of the life-history and energetics of marine and brackish-water nematodes. I. Demography of Monhystera disjuncta at different temperature and feeding conditions. Oecologia 77, 296–301. Warwick, R.M., 1981. The influence of temperature and salinity on energy partitioning in the marine nematode Diplolaimelloides bruciei. Oecologia 51, 318–325. Warwick, R.M., 1987. Meiofauna: their role in marine detrital systems. Detritus and microbial ecology in aquaculture. ICLARM Conference Proceedings 14, 282–295. Worm, B., Lotze, H.K., Hillebrand, H., Sommer, U., 2002. Consumer versus resource control of species diversity and ecosystem functioning. Nature 417, 848–851.

158

CChhaapptteerr 77::

11 ++ 11 ++ 11 == oorr ≠≠ 33 ?? TThhrreeee bbaacctteerriiaall-- ffeeeeddiinngg nneemmaattooddee ssppeecciieess iinnddiirreecctt iinntteerraaccttiioonnss aanndd tthheeiirr iimmpplliiccaattiioonnss ffoorr ppooppuullaattiioonn ddeevveellooppmmeenntt..

159

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode species indirect interactions and their implications for population development.

Giovanni A. P. dos Santos1,2,*, Tom Moens1, Eline V. Puyvelde1, Verônica G. F. Genevois2, Luana. C. B. B. Coelho2, Maria T. S. Correia2.

1Marine Biology Section, UGent, Krijgslaan 281 (S8), 9000 Ghent, Belgium. 2Universidade Federal de Pernambuco, Av. professor Moraes Rêgo, s/n, 50.000.000. Recife, Pernambuco, Brazil.

* Corresponding author. E-mail: [email protected]

Abstract

For a better understanding of local community structuring and local diversity, species effects and interactions on system functioning were tested under microcosm experiments using bacterial feeders marine nematodes under five different food concentrations of Escherichia coli. Effect of species diversity on ecosystem function were done with three species belonging to the same trophic group, one Rhabidtidae with a very opportunistic feeding behaviour, while the other two cogenerics Monhysteridae, which are more general opportunistic. In a set of experiments, the monoculture of every species has tested the food densities dependency and the population developmental 160

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS parameters that were compared to another set of combination experiment where all species have been placed together and interacted during 46 days. Chemotaxis experiments between monhysterids cogeneric species were done to establish alelochemical interaction pattern. Interspecific interactions among species, within a single nematode trophic group depending on food availability, effects directly the population development and community structure following different patterns between species even for cogeneric species. Crucial availability of food sources drives a succession of species in combination experiment with direct and indirect interactions. At high food density rhabditids over grazes, facilitating monhysterids population development, while food densities decreases competitions playing an important stress over the most opportunistic species. Different population developmental peaks were observed for monoculture and combination cultures. No clear negative alelochemical interactions were observed in chemotaxis experiments. A high complexity and problematic species interactions render predictions on the outcome and functional consequences of changes in within-trophic-group.

Keywords: Biodiversity, Ecosystem function; Bottom-up effect; Bacterivorous nematodes; Diplolaimelloides oschei, Diplolaimelloides meyli, Pellioditis marina; Chemotaxis; Ecological indirect interaction; Facilitation and Food density dependence.

161

Introduction

In the context of growing concern that biodiversity losses may both increase vulnerability of communities to invasive species and jeopardize ecosystem functioning (Loreau et al. 2001; Solan et al. 2004), a better understanding of the factors structuring local communities, and the effects of species interactions on system functioning, is direly needed. Species tend to interact with each other and many of these interactions are not neutral (Tilman 1999; Loreau 2001). More attention has traditionally been focused on direct interspecific interactions than on indirect interactions (Huston 1997; Tilman 1999; Worm et al. 2002), although the impact of facilitative and inhibitory interactions on the diversity-functioning relationship can be very pronounced (e.g. Cardinale et al. 2002; Jonsson & Malmqvist 2003). Unknown and insufficiently understood interspecific interactions may, for instance, be largely responsible for the mostly idiosyncratic diversity-functioning relationships found in marine communities (Emmerson et al 2001). The effect of species diversity on ecosystem functioning is usually considered to be more pronounced when species are functionally more dissimilar (Heemsbergen et al. 2004), whereas diversity within functional groups would improve ecosystem reliability rather than functioning (Naeem 1998). On the other hand, several studies have demonstrated that species within predefined functional groups do not necessarily perform identical functions (Chapin III et al. 1997; Bongers & Bongers 1998; Naeem & Wright 2003) but, when occurring in sympatry, strongly interact (Pfennig & Murphy 2002; Detrich & Wehner 2003). Communities of free-living nematodes are well-known for their high local taxonomic diversity (Heip et al. 1985; Ettema et al. 1998; Wall & Moore 1999). The functional diversity of nematodes is usually summarized in a limited number of feeding types (Yeates et al 1993; Moens & Vincx 1997; Moens et al. 2004), life-history groupings (Bongers, 1990; Bongers et al. 1991; Bongers & Ferris 1999) or combinations of both (Ferris et al. 2001), one recent study on marine nematodes also having included various size- and shape-related parameters into a multiple functional trait analysis (Schratzberger et al. 2007).

162

It is common to find multiple species belonging to the same trophic guild and/or life-history group in sympatry on a sample scale, more often than not including several congeneric species (Moens et al. 1996; Warwick 1987; Fonseca et al. 2006) This is certainly the case for opportunistic bacterivorous nematodes associated with phytodetritus decomposition in various terrestrial and aquatic settings. While most studies aiming to model the role of these nematodes in carbon and nutrient mineralization focus on the functional group level, several studies have demonstrated (1) differential feeding preferences (Venette & Ferris 1998; Moens et al. 1999), even among congeneric nematode species, (2) differences in life history characteristics (Ferris et al. 1998; Santos et al. subm.), (3) different roles in, and contributions to, organic matter decomposition and nutrient cycling (De Mesel et al. 2003, 2006; Postma-Blaauw et al., 2005; Ferris et al. 1997), and (4) a differential impact on bacterial community activity (De Mesel et al. 2003, Fu et al. 2005) and composition (De Mesel et al., 2004). Hence, nematode species which belong to the same functional group, or even to the same genus, do not necessarily perform identical functions. The significance of such functional differences may be related to the degree to which the life histories of the species differ (Postma-Blaauw et al. 2005) Moreover, different species within functional groups may interact and affect each other’s population development and/or activity (Ilieva-Makulec 2001; Postma-Blaauw et al. 2005; De Mesel et al. 2006), rendering any relationship between within-guild species diversity and ecosystem functioning highly idiosyncratic. The most likely cause of interspecific interactions between species feeding on the same type of food would be competition for food or space (Ilieva-Makulec 2001; Debenham et al. 2004). However, facilitative (Cardinale et al. 2002; Jonsson & Malmqvist 2003) and inhibitory (De Mesel et al. 2006) interactions may be equally important. Such interactions often also relate to changes in access to food and/or in physical structure of the habitat (Chandler & Fleeger, 1987; De Mesel et al. 2006; Arroyo et al. 2007). The aims of the present study were to elucidate whether interspecific interactions among species within a single nematode trophic group (1) affect population development and community structure; (2) depend on food

163

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Introduction: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding… DOUTORADO EM CIÊNCIAS BIOLÓGICAS availability, and (3) are characterized by interaction strengths which increase with increasing differences in life-history characteristics of the species involved.

164

Materials and methods

Nematode culture

Nematodes were harvested from established agnobiotic, monospecifc cultures in exponential growth phase. Unidentified bacteria from the nematodes’ natural habitat serve as food in those cultures (Moens and Vincx, 1998). The original culture inocula of Diplolaimelloides oschei, D. meyli (Monhysteridae) and Pellioditis marina (Rhabditidae) all originate from the Westerschelde Estuary (SW Netherlands). Each species had been in permanent culture under identical temperature (17 °C) and salinity (25) conditions for many generations prior to the start of our experiments. The P. marina culture used for the present experiments belongs to the Pm I lineage, which is the dominant (cryptic) species of the P. marina species complex in the Westerschelde Estuary (Derycke et al., 2005, 2006). Diplolaimelloides oschei, D. meyli and P. marina are opportunistic colonizers of different types of decaying organic matter, and have partly overlapping (micro)habitat preferences (Moens, unpubl.). As a result, these species often co-occur on decaying macroalgae in the polyhaline reach of the Westerschelde Estuary.

Population development

We used the same basic experimental design as described by Santos et al. (subm.) for parallel experiments with single-species treatments. Briefly, experiments were performed on a 0.75 % bacto-agar medium with a salinity of 26 and pH 7.5-8 in Petri dishes (5 cm i.d.). The agar medium contained cholesterol at a concentration of 100 µl l-1, because nematodes on a bacterial diet cannot synthesize sterols (Vanfleteren, 1980). We used frozen-and- thawed Escherichia coli (strain K12) as food in five different food availability 10 -1 9 -1 9 treatments (C1, C2,…, C5): 3 x 10 cells ml , 3 x 10 cells ml , 1.65 x 10 cells ml-1, 3 x 108 cells ml-1, and 3 x 107 cells ml-1, respectively. In a previous paper (Santos et al., subm.) we have reported on the population development of each of these three nematode species when grown

165

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Introduction: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding… DOUTORADO EM CIÊNCIAS BIOLÓGICAS separately under the same range of food availabilities. Here we report on an experiment where the three nematode species were simultaneously inoculated to the same microcosms. Nematodes for this experiment were harvested from stock cultures, rinsed twice in ASW to minimize transfer of bacteria, and left in ASW for 24 h at 5 °C before the start of the experiment. Nematodes were then transferred one by one on the tip of a fine Tungsten wire to a 50 µl drop of sterile ASW on the agar substrate. Five males + five gravid females of P. marina and ten males + ten gravid females of both monhysterid species were used as inoculum. The reason for the smaller P. marina inoculum is its very short generation time and often explosive population increase (Vranken and Heip, 1983; Moens and Vincx, 2000a). 150 µl of E. coli at the respective densities was added as food. An equal amount of bacteria was added on the tenth day of the experiment. There were 4 replicates per treatment. Counts were performed every 48h during the first 30 days and then every 92h until 46 days of incubation. Results of adult counts are shown for the 4 to 18 day interval for P. marina, and for the 16 to 46 day interval for both species of Monhysteridae. Because nematodes in our experiments were quantified by life counts, in which it is almost impossible to routinely identify first and second stage juveniles to species, in this experiment we mainly focus on adult population development, development time (from egg to adult) and sex ratio. Since F1 (first generation progeny) adults started appearing from 4 days onwards in P. marina, and from 16 days onwards in both monhysterid species, and since the P. marina population collapsed at around 17 days, the adult counts presented and analysed statistically pertain to the 4 to 17 days interval in P. marina and to the 16 to 46 days interval in both Monhysteridae. In addition, we also show the total population development, which encompasses all individuals (adults as well as juveniles) of all three species.

166

Taxis towards Escherichia coli

Since results of the population development assays suggest that both Diplolaimelloides species affect each other’s population growth, we hypothesized that they may influence each other’s foraging activity. For instance, Moens et al. (1999) demonstrated that the presence of Diplolaimella dievengatensis affected the food (i.e. bacteria) preference of D. oschei in taxis experiments. Taxis is a directed (so non-random) movement, and in the case of nematode taxis to food probably involves chemotaxis (Moens et al., 1999). We prepared a dozen microcosms by pouring a 0.75 % bacto-agar medium in ASW into12 cm x 12 cm square Petri dishes. Care was taken to create a perfectly flat and smooth agar surface. We marked 6 equally wide zones (A – F) on the bottom of the microcosms. Nematodes were inoculated in the centre, with two control spots and two bacterial spots at the corners of a square surrounding this central inoculum. One control spot and one bacterial spot were located on the line separating zones A and B, and one each on the line separating zones E and F. The two bacterial spots were placed at opposite ends of a diagonal, and so were the two control spots (Fig. 1). All spots measured 1 cm x 1 cm. Bacterial inocula consisted of a 50 µl drop of E. coli at a density of 3 109 cells ml-1 in ASW (Santos et al. subm), whereas the controls consisted of a 50 µl drop of sterile ASW.

D E. coli - Inocula

E ASW - Control Nematodes F

Figure 01: Schematic diagram of the taxis microcosm.

167

We inoculated 100 individuals of D. oschei in a drop of ASW onto 4 of these microcosms, 100 D. meyli onto 4 others, and a mix of 100 D. oschei and 100 D. meyli onto the remaining 4 Petri dishes. Microcosms were incubated in the dark at 17 °C, and nematodes inside E. coli and control spots were counted after 1, 2, 3, 7 and 9 days.

Data analysis

We compared the individual species population parameters (adult density and development time) obtained from the present combination culture experiment (henceforth denoted as CC) with those from the parallel monospecific culture experiment (MC, Santos et al., subm.) using repeated measures analysis of variance (ANOVA) with the factors experiment (CC vs MC), treatment (five food availability treatments), and time. We also compared total adult nematode densities in the CC experiment with the summed densities of the three individual species obtained from the MC experiment. Because P. marina in the MC experiment was only counted until day 18, this comparison was only possible for the first 18 days of the experiment. Sex ratio data per species were averaged over time and compared by two-way ANOVA with experiment (CC vs MC) and treatment (five different food densities) as factors. Data were checked for normality and homogeneity of variances using Levene’s ANOVA-test on the deviation of scores. Wherever necessary, data were log10(x+1) transformed. Tukey’s Honest Significant Differences (HSD) test was performed for a posteriori pairwise comparisons. Analyses were performed using the Statistica 6 software (Statsoft). In the taxis experiment, we first assessed whether both Monhysteridae actively moved towards bacterial spots using a replicated G-test for goodness of fit (Sokal and Rohlf, 1995), based on the null hypothesis of no taxis (and hence only random movement) which should result in equal nematode numbers inside bacterial and control spots. We also compared the nematode densities in both bacterial spots and assessed heterogeneity among replicates using the same statistical test.

168

Densities of either Diplolaimelloides species inside bacterial or control spots were compared between the mono-species experiment (MSE) and the combination experiment (CE) using two-way ANOVA with treatment (MSE vs

CE) and time as factors. Data were log10(x+1) transformed when necessary. We also compared the ‘total community’ response by summing the numbers of D. meyli and D. oschei from bacterial or control spots in the MSE with the respective counts in the SSE.

169

Results

Pellioditis marina

Adult population development in the CC experiment differed highly significantly between treatments (F=190.5; p<0.0001) and treatment x time (F=27.34; p<0.0001). Numbers of adults started increasing from 4 days onwards. The highermost food availability yielded the fastest and highest population development. In between 6 and 22 days, numbers of adults remained fairly constant, then rapidly decreased until complete extinction after 34 days. At the lower two food availabilities, population densities remained comparatively low and collapsed already from the 16th day onwards. At intermediate food availabilities (C2 and C3), population dynamics resembled those at the highest food availability, yet with lower abundances and generally slightly later population extinction (Fig 2A). In the MC experiment, the adult population development also differed highly significantly between treatments (F=18.31; p<0.0001) and treatment x time (F=7.58; p<0.0001) (Fig. 2B). Differences between treatments, however, only became pronounced from 12 days onwards, and no adult extinction was apparent at any of the food availabilities tested in the first 17 days of the incubation.

CC Experiment MC Experiment 3,0 3,0

2,5 2,5

2,0 2,0

1,5 (x+1) Dendities 1,5 (x+1) Dendities 10 10

1,0 1,0 Adult Log Adult Log

0,5 0,5 P. marina P. marina

0,0 0,0 C1 C1 C2 C2 -0,5 C3 -0,5 C3 4 8 12 16 20 24 28 34 C4 4 5 6 7 8 9 11 12 13 15 17 C4 C5 C5 Time in days Time in days Figure 2: A: Densities of P. marina adult population development (from 4 to 34 days) at five different food availabilities in the CC experiment. B: Densities of P. marina adult population development (from 4 to 17 days) at five different food availabilities in the MC experiment. Data are log-transformed averages

170

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Results: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS of four replicates per treatment and time. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Time-averaged densities of adult P. marina in the CC experiment were highly food-density dependent. All pairwise comparisons, except that between the lowest two food availabilities (p=0.40), were highly significant (p<0.005) (Fig 3A). In the MC experiment, time-averaged densities of adults were also highly food-density dependent, but with somewhat smaller differences between C2 and the lower food availabilities (Fig. 3B). The interaction term experiment x treatment was significant (F=3.29; p<0.05).

MC Experiment CC Experiment 160 160 A

140 140 A

120 120

100 100 P. marina P. marina 80 80

60 B 60 Adult Average of Adult average of B 40 40 B,C C D C C 20 20 D

0 0 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 3: Effect of food availability on time-averaged (4 to 18 days) densities of adult P. marina. A: CC experiment, B: MC experiment. Data are means ± 1 sterror of 4 replicates per treatment. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Adult sex ratio did not differ between CC and MC experiments (F=0.02; p=0.90) However, sex ratio did not differ significantly between food treatments in either experiment (MC experiment: F=3.00; p=0.05; CC experiment: F=1.28; p=0.32), and showed a similar, albeit not identical, trend over treatments for both experiments (experiment x treatment: F=2.47; p=0.06) (Fig. 4). The P. marina sex ratio was consistently female-biased and did not vary significantly with food availability in either experiment.

171

P. marina 74

60 Sex Ratio (%)

50 C1 C2 C3 C4 C5 CC MC Treatment Figure 4: P. marina adult sex ratio (expressed as % females) as a function of food availability in the CC and MC experiment. Data are averages ± 1 sterror of 4 replicates per treatment (time-averaged from 4 to 17 days.

Diplolaimelloides meyli

Adult population development in the CC experiment differed highly significantly between treatments (F=37.51; p<0.0001) and treatments x time (F=8.33; p<0.0001). Numbers of adults started increasing from 16 days onwards. At the highermost food availability, adult densities increased monotonously with time, reaching maximal densities of ca. 1600 ind. after 46 days. At the lowest food availabilities (C4 and C5), population densities did not change significantly until the 22nd day, where after a small monotonous increase was observed until the end of the experimental incubation. At intermediate food availabilities (C2 and C3), D. meyli reached much lower densities than at both higher and lower food densities (Fig. 5A). No population collapse was observed in any treatment. In the MC experiment, adult population development also differed highly significantly between treatments (F=6.12; p=0.004) and treatments x time (F=2.56; p<0.0001), but both intermediate food densities sustained the highest population development (C2 and C3), and substantial population decreases were observed from 30 days onwards (Fig.5B).

172

CC Experiment MC Experiment 2,8

2,6 2,6

2,4 2,4

2,2 2,2 2,0 2,0 1,8 (x+1) Dendities 10 1,6 (x+1) Dendities 1,8 10

1,4 1,6 Adult Log

1,2 Adult Log 1,4 1,0 D. meyli D. 1,2 0,8 D. meyli 1,0 0,6 C1 C1 C2 C2 0,4 C3 0,8 C3 16 18 20 22 24 26 28 30 34 38 42 46 C4 16 18 20 22 24 26 28 30 34 38 42 46 C4 C5 C5 Time in days Time in days Figure 5: A: Densities of D. meyli adult population development (from 16 to 46 days) at five different food availabilities in the CC experiment. B: Densities of D. meyli adult population development (from 16 to 46 days) at five different food availabilities in the MC experiment. Data are log-transformed averages of four replicates per treatment and time. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Adult D. meyli densities thus differed significantly between experiments (CC vs MC: F= 6.35; p=0.017) and experiment x treatment (F=9.38; p<0.0001), highlighting the different food-density dependence pattern in both experiments. In the CC experiment, all pairwise comparisons, except those between the two intermediate (C2 and C3) and the two lowest (C4 and C5) food concentrations (p≥0.86), were highly significant (p<0.003) (Fig. 6A). In the MC experiment, only the differences between C1 and C2, and between C2 and C5 were statistically significant (p≤0.02) (Fig. 6B).

CC Experiment 600 MC Experiment 400 A

500 300

D. meyli D. D. meyli D. 400 200

B Adult average of C C Adult average of 100 100 A,B A,B

B A A B

0 0 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 6: Effect of food availability on time-averaged (16 to 46 days) densities of adult D. meyli. A: CC experiment (Y-axis braked), B: MC experiment. Data 173

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Results: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS are means ± 1 sterror of 4 replicates per treatment. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Sex ratio did not differ significantly between experiments (F=0.00; p=0.99), treatments at CC (F=1.77, p=0.19) and MC (F=2.61, p=0.08) separately were also not significant and nor for the interaction term experiment x treatment (F= 1.54; p=0.22). Sex ratio in both experiments with D. meyli was strongly female-biased and did not vary significantly with food availability (Fig. 7).

D. Meyli 74

60 Sex Ratio (%)

50 C1 C2 C3 C4 C5 CC MC Treatment Figure 7: D. meyli adult sex ratio (expressed as % females) as a function of food availability in the CC and MC experiment. Data are averages ± 1 sterror of 4 replicates per treatment (time-averaged from 16 to 46 days).

Diplolaimelloides oschei

Adult population development in the CC experiment differed highly significantly between treatments (F=96.23; p<0.0001) and treatments x time (F=17.42; p<0.0001). Numbers of adults started increasing from the 18th day onwards. The highermost food availability yielded the highest population development, reaching an average density of approximately 800 adult ind. per microcosm at the end of our experiment. Intermediate food concentrations (C2 and C3) yielded extremely low densities of adult D. oschei, considerably lower than in treatments with lower food availability (C4 and C5) (p≤0.02),

174

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Results: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS where population densities remained low until the 22nd day and then gradually increased up to ca. 200 adults per microcosm (Fig. 8A). Population development in the MC experiment exhibited much less pronounced differences between treatments, intermediate food densities (C2 and C3) now generally yielding the highest densities of adult D. oschei and the highermost food density scoring lower adult densities (Fig. 8B).

CC Experiment MC Experiment 2,5 2,5

2,0 2,0

1,5 1,5 (x+1) Dendities (x+1) Dendities 10 10 1,0 1,0 Adult Log Adult Log

0,5 0,5

C1 C2 C1 C3 C2 0,0 C4 0,0 C3 16 18 20 22 24 26 28 30 34 38 42 46 C5 16 18 20 22 24 26 28 30 34 38 42 46 C4 C5 Time in days Time in days Figure 8: A: Densities of D. oschei adult population development (from 16 to 46 days) at five different food availabilities in the CC experiment. B: Densities of D. oschei adult population development (from 16 to 46 days) at five different food availabilities in the MC experiment. Data are log-transformed averages of four replicates per treatment and time. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Time adult average D. oschei densities differ significantly between experiments (F=219.7; p<0.0001) and experiments x treatments (F=57.8; p<0.0001). Stressing the different food-density dependence pattern in both experiments. In the CC all pairwise comparisons, except those between the two intermediate (C2 and C3) and the two lowest (C4 and C5) food concentrations, (p≥0.12) were significant(p≤0.02) (Fig. 9A). In the MC experiment no pair wise differences between treatment was significant (Fig. 9B)

175

MC Experiment CC Experiment 300 300 A

250 250

200 200

AA D. oschey D. D. oschei D. 150 150 AA C A 100 C 100 Adult average of Adult average of

50 50

B B 0 0

C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 9: Effect of food availability on time-averaged (16 to 46 days) densities of adult D. oschei. A: CC experiment, B: MC experiment. Data are means ± 1 sterror of 4 replicates per treatment. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Sex ratio of D. oschei differed significantly between experiments (F=41.74; p<0.0001), being generally more female-biased in monoculture than in combination culture (Fig. 10). However, sex ratio did not differ significantly between food treatments in either experiment (MC experiment: F=0.44; p=0.78; CC experiment: F=1.7; p=0.20), resulting in a non-significant interaction term experiment x treatment (F=1.65; p=0.19).

D. oschei 74

60 Sex Ratio (%)

50 C1 C2 C3 C4 C5 CC MC Treatment Figure 10: D. oschei adult sex ratio (expressed as % females) as a function of food availability in the CC and MC experiment. Data are averages ± 1 sterror of 4 replicates per treatment (time-averaged from 16 to 46 days).

176

Community analysis: juveniles

Juvenile community both differ significantly between treatment MC (F=4.22, p=0.017) and CC (F=13.84, p<0.0001). Comparing densities of juvenile nematodes in the combination culture with the summed densities from the three monocultures), it is striking that total juvenile densities were considerably higher in the CC experiment (F=12.27; p<0.0001). The pattern of food-density dependence also differed between both experiments (experiment x treatment: F=14.02; p<0.0001, experiment x time: F=45.17; p<0.0001; and experiment x treatment x time: F=18.97; p<0.0001), with a general decrease with decreasing food densities in the CC experiment compared to a low food-density dependence in the MC experiment (Fig. 11).

Juvenile community 1000

900 A

800

700

600 B 500

400

Juveniles average Juveniles B 300 BB

200 b a, b aa, ba, b 100

0 CC C1 C2 C3 C4 C5 MC Treatment

Figure 11: Effect of food availability on time-averaged (during the first 8 days) densities of all larval stages for the three species. In red square label CC experiment; In green diamond label MC experiment. Data are means ± 1 sterror of 4 replicates per treatment. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

177

Community analysis: adults.

The general species trend over time are less informative in relation to food availability, but showed a good representation how population probably develop under food patched distributed environment. Species at both experiment tend to have a reverse behave between D. meyli vs P. marina, considering the different period in P. marina for each experiment and the late response of the monhysterids been more pronounced at the second half of the experimental time (Fig. 12). Difference were not significant between experiments (F=0.51; p=0.48) but highly significantly between species (F=7.00; p≤0.002). Pairwise species comparisons were highly significantly (p<0.01), except between monhysterids (p=0.83).

MC Experiment CC Experiment 450 450 400 400 350 350 300 300 250 250 200 200

150 150

Nematodes adult average Nematodes 100 Nematodes adult average Nematodes 100

50 50

0 0

-50 D. meyli -50 D. meyli T2 T6 T10 T14 T18 T22 T26 T30 T38 T46 D. oschei T2 T6 T10 T14 T18 T22 T26 T30 T38 T46 D. oschei P. marina P. marina Time in days Time in days Figure 12: Densities of adult population development for the three species in the CC experiment. B: of adult population development for the three species in the MC experiment. Data are averages ± sterror of four replicates for all food treatment during experimental time.

Total adult nematode densities averaged over the experiment time differ significantly between experiment (F=37.60, p<0.0001) experiment x treatment (F=44.15, p<0.0001) a different food-density dependence pattern between both experiments. All pairwise comparisons for the CC experiment were significant, except between the two intermediate food densities (p=0.79) and between the two lowest food availabilities (C4 and C5) (p=0.99). In the MC experiment, the only significant pairwise differences were between C2 and the two lowest food densities (C4 and C5) (both p<0.02) (Fig 13).

178

CC Experiment MC Experiment

600 A 600

500 500

400 400

300 300

Community adult average Community 200 Community adult average Community 200 A,B A,B CC B B 100 100 B B

0 0 C1 C2 C3 C4 C5 Mean C1 C2 C3 C4 C5 Mean ±SE ±SE Treatment Treatment Figure 13: Effect of food availability on time-averaged (total experimental time) densities of adult for the three species. A: CC experiment, B: MC experiment. Data are means ± 1 sterror of 4 replicates per treatment. C1: highest food availability, C5: lowest food availability (for details, see materials and methods section).

Taxis experiment

Both Diplolaimelloides species exhibited a significant response towards bacteria (GT>30, p<0.005), inspite of substantial heterogeneity among replicate microcosms (GH>6, p<0.05). Within microcosms, differences in nematode numbers between both bacterial spots were never significant in the

MSE (GT>10, p<0.05 for both species), while they became significant from 5 days onwards in the CE (GT>10, p<0.05). Numbers of nematodes inside control spots were consistently heterogeneous in all treatments (GT>15, p<0.005). Overall, numbers of D. oschei inside both bacterial and control spots tended to be higher than numbers of D. meyli, but these differences were not significant (Mann-Whitney U-test, p>0.05). Numbers of D. oschei reaching bacterial spots were significantly higher in CE with D. meyli than in the MSE (F = 67.9, p<0.001), especially from 3 days onwards (Fig. 14).

179

Taxis experiment: D. oschei vs D. oschei + D. meyli 30

27,5

22,5

17,5

12,5

10 Nematodes densities 7,5

2,5

0 12379DO Time in days DO + DM

Figure 14: Numbers of D. oschei (DO) inside E. coli spots as a function of time in MSE (red bars) and CE (green bars) treatments. Data are means ± 1 stdev of 4 replicates each. DM = D. meyli.

Vice versa, D. oschei also facilitated the response of D. meyli towards bacterial spots (F=3.95, p<0.003), an effect which again became significant from 3 days onwards (Fig. 15).

Taxis experiment: D. meyli vs D. oschei + D. meyli 55

Nematodes densities 15

0 12379DM Time in days DM + DO

Figure 15: Numbers of D. meyli (DM) inside E. coli spots as a function of time in MSE (red bars) and CE (green bars) treatments. Data are means ± 1 stdev of 4 replicates each. DO = D. oschei.

180

As a result, summed numbers of both Diplolaimelloides species inside bacterial spots in the CE were significantly higher (from 3 days onwards) than the sum of the responses of both species in the MSE (Fig. 16) (F=113.3, p<0.0001). The mutual facilitation is also illustrated by the significantly positive correlation between the densities of both nematode species (Spearman rank test, r = 0.71, p<0.0001). Nematode numbers inside control spots also tended to be higher in CE than in MSE, but these differences were never significant (data not shown).

Taxis experiment: D. oschei + D. meyli separatly vs D.oschei + D. meyli together 75 70 65 60 55 50 45 40 35 30 25

Nematodes densities 20 15 10 5 0 12379 Time in days DO + DM separately DO + DM together

Figure 16: Comparison of the summed response of D. meyli (DM) and D. oschei (DO) from MES (red bars) and CE (green bars) treatments towards E. coli spots. Data are means ± 1 stdev of 4 replicates each.

181

Discussion

Accurate prediction of the biodiversity – ecosystem functioning relationship requires that (a) the ecological roles of, and (b) the interactions among the component species in a community be adequately understood (Johnston et al. 1996). The question whether individual species effects can simply be summed to arrive at an adequate understanding of how the total community will function, largely depends on how these species affect each other’s behaviour, activity and population dynamics. Ecological roles of free-living nematodes at genus or species level are generally insufficiently documented. Consequently, an adequate assessment of the functional diversity of nematode communities is difficult. Commonly used functional traits in nematodes pertain to their feeding (use of feeding types of trophic guilds) (Wieser 1953, Yeates et al. 1993, Moens & Vincx 1997, Traunspurger 1997, Moens et al. 2004), life history (Bongers 1990, Bongers et al. 1991), or a combination of both (Ferris et al. 2001). We are aware of only one study further involving additional traits relating to body size and shape as well as tail shape into a multiple trait analysis and defining a high diversity of potentially relevant functional groupings (Schratzberger et al. 2007). However, the groupings that can thus be made do not have straightforward links to specific ecosystem functions or roles. In a previous paper (Santos et al. subm.), we have studied the population dynamics of three species of bacterivorous nematodes typical of decomposing macroalgal wrack in estuarine littoral habitats, as a function of food availability. Based upon food source, all three species belong to one and the same trophic guild, bacterivores. Based upon feeding habit, a distinction may be made between Pellioditis marina, a rhabditid frequenting patches characterized by very high food density where it feeds almost continuously, and the two Monhysteridae, Diplolaimelloides meyli and D. oschei, which prefer somewhat lower food densities and feed more intermittently (Moens & Vincx 2000b, Moens et al. 2004). Based upon life-history characteristics, the distinction between the rhabditid and both monhysterid nematodes also holds, the rhabditid being classified as an enrichment opportunist and the

182

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Discussion: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding … DOUTORADO EM CIÊNCIAS BIOLÓGICAS monhysterids as general opportunists (Bongers & Bongers 1998, Santos et al. subm.). Enrichment opportunists are characterized by extremely short generation times and high reproductive rates, but requiring high bacterial densities. They also usually possess a resistant survival stage called dauer larvae (Bongers et al. 1995). General opportunists have comparatively longer generation times (albeit still much shorter than average for free-living nematodes) but similarly high numbers of progeny, and require somewhat lower food levels. They are typically expected to follow after the enrichment opportunists in a succession on organic matter enrichment (Bongers & Bongers 1998). Hence, depending on which functional trait is considered, our three test species comprise one or two distinct functional groups, and the seemingly high functional similarity among the two Diplolaimelloides species suggests that they may be mutually redundant. This would imply that the role of this functional group will not depend on species identity, nor on its species diversity. However, this prediction may not hold if individual species affect each other’s activity and/or population dynamics. Such interspecific interactions may involve competition for resource and/or space, but also more indirect interactions such as facilitation or inhibition. As an example, Cardinale et al. (2002) demonstrated that the particle filtering function of a community of 4 species of caddisfly larvae was dependent on species identity as well as diversity, because one species facilitated the filtration activity of the others, resulting in a community function which significantly exceeded the sum of the individual contributions of the component species. The intensity of resource competition would be expected to be food-density dependent, while facilitation and inhibition may operate through different drivers. From the population experiments with each of these three bacterivorous nematode species grown separately on a range of food availabilities (Santos et al. subm.), we developed the following hypotheses: (1) At the highest food availability, there would likely be no important interspecific interactions, esp. not between P. marina and both Diplolaimelloides species, because such food availability supports optimal growth of the former but not of the latter species.

183

(2) At intermediate food availabilities, competition could arise between all three species, since such feeding conditions support optimal growth of both Diplolaimelloides species, while at the same time allowing moderate population growth of P. marina. (3) At the very lowest food availabilities, any interspecific interactions would likely be pronounced if resource competition is important, because such low food availabilities support very limited growth of all species considered. The rhabditid nematode P. marina largely followed these predictions. Its food- density dependence pattern did not differ between the mono- and combination culture experiment, with a pronounced preference for the highest food availability in both. The main difference between both experiments was in the fact that the lowest two food density treatments resulted in a complete collapse of the adult population within two weeks after the start of the CC experiment, whereas some – albeit very limited – population growth was observed at the same food densities in the MC experiment. In a previous paper (Santos et al. subm.), we have reviewed literature evidence that such low bacterial densities support but a very low and inefficient feeding activity of most Rhabditidae. We suggest that the presence of even a small population of Monhysteridae feeding more efficiently on the same limiting resource (Moens & Vincx 2000b) prohibited P. marina survival, in accordance with our third hypothesis. A completely different pattern emerged, however, for the two species of Diplolaimelloides. Compared to the MC experiment, their optimal food density shifted from the range of 1 – 3 109 cells ml-1 to a density of 3 1010 cells ml-1. Because we only added food at the start of the experiment and after 10 days, we did not control food density throughout the experiment. Previous experiments have shown that some enrichment opportunists, such as Panagrolaimus paetzoldi, are capable of down-regulating bacterial activity and diversity, albeit at population densities exceeding those of P. marina by up to two orders of magnitude (De Mesel et al. 2003, 2004). Since there was only one abundant strain of bacteria in our microcosms, and since most of the added bacteria were cells which had burst open after freezing and thawing,

184

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Discussion: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding … DOUTORADO EM CIÊNCIAS BIOLÓGICAS the parameters activity and diversity are not directly relevant to the present study, but the top-down effect over activity is likely the result of a depression of cell numbers. Similarly, Moens et al. (in prep.) found a substantial decrease in bacterial activity in 9 cm diam. microcosms containing a few thousand P. marina, suggesting that active populations of rhabditid grazers may down- regulate bacterial densities to a level which provides favourable growing conditions for the Monhysteridae. An alternative explanation may be linked to the fact that Rhabditidae are known to digest only part of the absorbed fluid they ingest, releasing a substantial fraction back into the environment after passage through their pharynx (Avery and Shtonda, 2003). This fraction may be larger at higher food densities, because these cause lower absorption efficiency (Moens et al. 2004). Gut passage may make some bacteria more readily available to consumers (Avery and Shtonda, 2003). However, this explanation is unlikely in the present study, because most of the added bacteria were already broken cells. In any case, our data provide clear evidence that P. marina facilitates the growth of both Diplolaimelloides species at high food densities. Interspecific facilitations are a key mechanism in enhancing ecosystem function (Fridley 2001). At the lowest two food densities, D. oschei performed as in the MC experiment, whereas D. meyli also showed a similarly slow and moderate population increase, but without the decrease noted after 42 days in the MC experiment. It is possible that the higher total amount of food available at the start of the experiment (the CC experiment summed the ingredients of the three separate MC experiments, implying that the food inoculum was three times larger in the CC experiment), and/or the comparatively somewhat lower monhysterid nematode densities obtained in the CC experiment, guaranteed a longer availability of sufficient food to support population growth. The most unexpected outcome of the present experiment was the completely opposite behaviour of the intermediate food-availability treatments in CC vs MC experiments. In the latter, both species of Diplolaimelloides exhibited (near) optimal population development at intermediate food densities, whereas in the CC experiment these intermediate densities yielded poorer results than the lowest food availabilities. There are basically two potential

185

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Discussion: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding … DOUTORADO EM CIÊNCIAS BIOLÓGICAS explanations for this phenomenon, which are not necessarily mutually exclusive. The first parallels the explanation for the facilitative effect of P. marina on the Monhysteridae at high food availability. P. marina likely also depleted the available resources at intermediate food levels, and could have done so down to levels which no longer allowed complete maturation of monhysterid progeny, as in D. oschei, or only to a limited extent, as in D. meyli. In the CC experiment, a collapse of the adult P. marina population occurred from 18 – 20 days onwards, which at the current culture conditions is the timing of maturation of a substantial portion of the monhysterid F1 progeny to adults. Food depletion around this time may have both caused the population collapse of P. marina and prohibited maturation of monhysterid juveniles. However, population densities of P. marina at these food densities remained consistently much lower than at the highest food availability, rendering it questionable whether the population was large enough to cause substantial food depletion. An important next step in this research will thus be to adequately determine the magnitude of the top-down effect of P. marina on bacterial food availability at different food densities. Alternatively, the extremely low population growth of the monhysterid species at intermediate food levels may be the result of a mutual inhibition. Similar results were found in other studies bringing different bacterivorous nematode species together in microcosms (Ilieva-Makulec 2001, Postma-Blaauw et al. 2005, De Mesel et al. 2006, Santos et al. in prep., next chapter). Whereas Ilieva-Makulec (2001) attributed such negative interspecific effects to resource competition, De Mesel et al. (2006) and Santos et al. (in prep.), working with combinations of Monhysteridae, argued that resources (food as well as space) were not limiting in their microcosms. This inevitably implies that another, indirect form of interaction must underly this interspecific inhibition. At this point, we can only speculate on the nature of that interaction, which may potentially involve some form of allelochemistry. A strong argument in favour of this indirect mechanism is that the mutual inhibition is consistently observed in the absence of enrichment opportunists (De Mesel et al. 2006, Santos et al. in prep.), albeit that the inhibition is not always symmetric. In the present experiment, both Diplolaimelloides species

186

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Discussion: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding … DOUTORADO EM CIÊNCIAS BIOLÓGICAS had a negative effect on each other’s growth, although this effect was yet more pronounced on D. oschei than on D. meyli. Extreme caution is due when comparing the absolute densities of nematodes obtained in the CC experiment with those of the MC experiment, because these two experiments were not run simultaneously. This implies that the stock cultures from which the experimental nematode inocula were obtained were not the same. Even small differences in the timing of harvest may cause important changes in the reproductive output of these populations (Moens & Vincx 1998, 2000a). However, when comparing densities of adult nematodes between both experiments, the only obvious differences which were large enough probably not to result from differences in the ‘fitness’ of the inoculum, were the comparatively higher monhysterid densities at the highest food availability, and their comparatively lower densities at the intermediate food availabilities. Densities of adult P. marina were almost identical over both experiments. This is in contrast with results of comparable experiments on soil nematodes where lower Rhabditidae densities were found in combination experiments with Cephalobidae (general opportunists) than in monocultures (Blanc et al. 2006). Densities of juvenile nematodes over the first 8 days of our experiment were, however, consistently higher in the CC experiment than in the MC experiment. At the highermost food density, this obviously relates to the comparatively much higher population increase of the Diplolaimelloides species in the CC experiment. At the other food densities, however, because these data only span the first 8 days of the experiment, this difference might be related to the threefold higher initial food addition in the CC experiment. Alternatively, again, the explanation may be in indirect interspecific interactions resulting in a higher yield at community level (CC) than at the level of monocultures (Loreau 2000, Loreau et al. 2002). In any case, when linking the comparatively high initial reproduction in the CC experiment to the nearly identical adult densities in CC vs MC, we have to conclude that a much smaller fraction of juveniles matured to adulthood in the CC experiment, which again may be taken as evidence of interspecific competition, inhibition or both.

187

As expected from previous studies (Grewal & Wright 1992, Moens et al. 1999, Shtonda & Avery 2005), the bacterivorous nematodes in our experiments exhibited significant taxis (probably a chemotaxis) to bacterial spots in their immediate environment. The response to different E. coli spots within the same microcosm was fairly homogeneous throughout the first week of the experiment. The relatively low response percentages in the present study agree with results from previous studies using the same nematode species (Moens et al. 1999 and unpubl.), and are linked to the facts that (1) many nematodes are simultaneously inoculated into the centre of the microcosms, where they tend to cluster, and (2) that the nematodes were not completely axenic, and were hence potentially exposed to bacterial cues in the inoculum itself. A remarkable result from this experiment was that both Diplolaimelloides species facilitated each other’s response to bacterial spots, resulting in considerably higher numbers of both species inside bacterial spots in CC treatments than in MC treatments. This effect was not fully symmetric, in that D. oschei exerted a somewhat more pronounced and rapid effect on D. meyli than vice versa. Moens et al. (1999) similarly observed that the presence of another monhysterid species, Diplolaimella dievengatensis, promoted the response of D. oschei (in that paper wrongly identified as Monhystera sp.) towards a gram-positive bacterium to which it was otherwise not attracted. These authors reported that the D. dievengatensis responded first to the presence of the gram-positive strain, and observed a response by D. oschei only after several D. dievengatensis had reached the bacterial spot. In the present experiment, D. oschei showed the fastest response towards E. coli spots, and had a proportionately larger effect on the slower-growing D. meyli than vice versa. We hypothesize that this ‘behavioural’ facilitation is a result of the (sloppy) feeding activity of the nematodes colonizing the bacterial spots. Through their feeding, they secrete waste products and metabolites which may diffuse through the agar and act as infochemicals for other nematodes. The increased response over time is in line with this argument, since the more nematodes feed in the bacterial spots, the more such infochemicals would be produced (Bilgramai and Jairajpurmi, 1988; Rodger et al. 2004). At the same time, the taxis experiments did not yield any evidence

188

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 7: Discussion: 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding … DOUTORADO EM CIÊNCIAS BIOLÓGICAS that either nematode species would negatively affect the activity of another species through allelochemical interactions.

189

Conclusion

The population growth in two of the three bacterivorous nematode species tested here showed significantly different patterns of food-density dependence in combination culture compared to monocultures. At very high food availability, the rhabditid nematode facilitated growth of both monhysterid species, probably as a result of down-regulation of bacterial density. At the lowest food densities, the presence of even low densities of monhysterid nematodes lead to exclusion of the rhabditid, which at such low food availability has a very inefficient food uptake. At intermediate food densities, abundances of both Diplolaimelloides species were strongly depressed in the combination culture, as a result of competition for food, indirect inhibitory interactions, or both. Interestingly, results of behavioural analyses, performed over comparatively shorter time intervals than the population development assays, also yielded evidence for indirect interspecific interactions between both Diplolaimelloides species, but these interactions were now facilitative rather than inhibitory. The complexity of the species interactions, and the incongruence between different parameters studied, render predictions on the outcome and functional consequences of changes in within-trophic-group diversity highly problematic.

190

Acknowledgements

This work was financially supported by a CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) grant to the first author, and by Ghent University through BOF projects 0110600002 and 01GZ0705. T.M. is a postdoctoral fellow with the Flemish Fund for Scientific Research. Tatiana Maria’s and Tania Bezerra had made some constructive comments in the initial part of the manuscript.

191

References

Arroyo, N. L.; Aarnio, K. and Ólasfson, E. (2007). Interactions between two closely related phytal harpacticoid copepods, asymmetric positive and negative effects. J. Exp. Mar. Biol. and Ecol. 3 (2): 219-227.

Avery, L. and Shtonda, B.B. (2003). Food transport in the C. elegans pharynx. J. Exp. Biol. 206, 2441-2457.

Bilgramai, L. and Jairajpurmi, S. (1988). Attraction of Mononchoides longicaudatus and M. fortidens (Nematoda : Diplogasterida) towards prey and factors influencing attraction. Revue Nématol., 11 : 195-202.

Blanc, C. ; Sy, M. ; Djizal, D. ; Brauman, A. ; Normand, P. and Villenave, C. (2006). Nutrition on bacteria by bacterial-feeding nematodes and consequences on the structure of soil bacterial community. Eur. J. Soil Biol. 42: 570-578.

Bongers, T. and Bongers, M. 1998. Functional diversity of nematodes. Appl. Soil Ecol. 10, 239–251.

Bongers, T.; De Goede, R.G.M.; Korthals, G. and Yeates, G. W. (1995). Proposed changes of c–p classification for nematodes. Russ. J. Nematol. 3, 61–62.

Bongers, T. and Ferris, H. (1999). Nematode community structure as a bioindicator in environmental monitoring. Trends Ecol. Evol, 14 : 224–228.

Bongers, T. (1990). The maturity index: an ecological measure of environmental disturbance based on nematode species composition, Oecologia 83: 14–19.

Bongers, T.; Alkemade, R. and Yeates, G. W. (1991). Interpretation of disturbance-induced maturity decrease in marine nematode assemblages by means of the Maturity Index. Mar Ecol Progr Ser 76: 135-142.

192

Cardinale, B. J.; Palmer, M. A. and Collins, S.L. (2002). Species diversity enhances ecosystem functioning through interspecific facilitation. Nature 24: 426-429.

Chandler, G.T. and Fleeger, J.W. (1987). Facilitative and inhibitory interactions among estuarine meiobenthic harpacticoid copepods. Ecology 68:1906–1919.

Chapin III, F. S.; Walker, B. H.; Hobbs, R. J. Hooper, D. U.; Lawton, J. H.; Sala, O. E. and Tilman, D. (1997). Biotic Control over the Functioning of Ecosystems. Science 277, 500-5004.

De Mesel, I.; Derycke, S.; Swings, J.; Vincx, M. and Moens, T. (2003). Influence of bacterivorous nematodes on the decomposition of cordgrass. J. Exp. Mar. Biol. Ecol. 296: 227-242.

De Mesel, I.; Derycke, S.; Swings, J.; Vincx, M. and Moens, T. (2006). Role of nematodes in decomposition processes: does within-trophic group diversity matter? Mar. Ecol. Progr. Ser. 321:157-166.

Debenham, N. J.; Lambshead, P. J. D.; Ferrero, T. J. and Smith, C. R. (2004). The impact of whale falls on nematode abundance in the deep sea. Deep-Sea Research 51: 701–706.

Derycke, S.; Remerie, T.; Vierstraete, A.; Backeljau, T.; Vanfleteren, J.; Vincx, M. and Moens, T. (2005) Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Mar Ecol Progr Ser 300: 91-103.

Derycke, S.; Backeljau, T.; Vlaeminck, C.; Vierstraete, A.; Vanfleteren, J.; Vincx, M. and Moens, T. (2006). Seasonal dynamics of population genetic

193

structure in cryptic taxa of thePellioditis marina complex (Nematoda: Rhabditida). Genetica 128: 307-321.

Dietrich, B. and Wehner, R. (2003). Sympatry and allopatry in two desert ant sister species: how do Cataglyphis bicolor and C. savignyi coexist? Oecologia 136(1): 63-72.

Emmerson, M.C.; Solan, M.; Emes, C.; Paterson, D.M. and Raffaelli, D.G. (2001). Consistent patterns and the idiosyncratic effects of biodiversity in marine ecosystems. Nature, 411, 73–77.

Ettema, C. H.; Coleman, D. C.; Vellidis, G.; Lowrance, R. and Rathbun, S. L. (1998) Ecology 79 (8): 2721-2734.

Ferris H., Venette, R.C., Lau, S.S. (1997). Population energetics of bacterial- feeding nematodes: carbon and nitrogen budgets. Soil Biol. Biochem.29 (8):1183-1194.

Ferris, H.; Venette, R.C.; Van der Meulen, H.R and Lau, S.S (1998). Nitrogen mineralization by bacterial feeding nematodes: verification and measurement. Plant Soil 203:159–171.

Ferris, H.; Bongers, T. and de Goede, R . G. M. (2001). A framework for soil fod web diagnostics: extension of the nematode fauna analysis concept. App. Soil Ecol., 18: 13-29.

Fonseca, G.; Vanreusel, A. and Decraemer, W. (2006). Taxonomy and biogeography of Molgolaimus Ditlevsen, 1921 (Nematoda: Chromadoria) with reference to the origins of deep sea nematodes. Antarct. Sci. 18 (1): 23–50.

Fridley, J. D. (2001). The influence of species diversity on ecosystem productivity: How, where, and why? Oikos 93: 514–526

Fu, S.; Ferris, H.; Brown, D. and Plant, R. (2005). Does the positive feedback effect of nematodes on the biomass and activity of their bacteria prey vary

194

with nematode species and population size? Soil Biol. Biochem. 37: 1979- 1987.

Grewal. P. S. and Wright, D. J. (1992). Migration of Caenorhabditis elegans (Nematoda: Rhabditidae) larvae towards bacteria and the nature of bacterial stimulus. Fundam. Appl. Nematol. 15:159–166.

Heemsbergen, D. A.; Berg, M. P.; Loreau, M.; Van Hal, J. R.; Faber, J. H. and Verhoef, H. A. (2004). Biodiversity effects on soil processes explained by interspecific functional dissimilarity. Science 306, 1019-1120.

Heip, C.; Vincx, M. and Vranken, G. (1985). The ecology of marine nematodes. Oceanogr. Mar. Biol. Annu. Rev. 23: 399-489.

Huston, M. A. (1997). Hidden treatments in ecological experiments: re- evaluating the ecosystem function of biodiversity. Oecologia 110: 449-460.

Ilieva-Makulec, K. (2001). A comparative study of the life strategies of two bacterial-feeding nematodes under laboratory conditions. II. Influence of the initial food level on the population dynamics of Acrobeloides nanus (De Man, 1880) Anderson, 1968 and Dolichorhabditis dolichura (Schneider, 1866) Andrássy, 1983. Pol J Ecol 49:123–135

Johnson, M. and Malmqvist, B (2003). Ecosystem process rate increases with animal species richness: evidence form leaf-eating aquatic insects. Oikos 89: 519-523.

Johnston, K. H.; Vogt, K. A.; Clark, H. J.; Schmitz, O. J. and Vogt, D. J. (1996). Biodiversity and productivity and stability of ecosystem. Trends Ecol. Evol. 11: 372-377.

Loreau, M. (2000). Are communities saturated? On the relationship between alpha, beta and gamma diversity. Ecology Letters 3:73-76

195

Loreau, M. (2001). Microbial diversity, producer–decomposer interactions and ecosystem processes: a theoretical model. Proceedings: Biological Sciences 268: 303-309.

Loreau, M.; Naeem, S.; Inchausti, P.; Bengtsson, J.; Grime, J. P.; Hector, A.; Hooper, D. U.; Huston, M. A.; Raffaelli, D.; Schmid, B.; Tilman, D. and Wardle, D. A. (2001). Biodiversity and ecosystem functioning: current knowledge and future challenges. Science 294: 804-808.

Moens, T.; Van Gansbeke, D. and Vincx, M. (1999). Linking estuarine intertidal nematodes to their suspected food. A case study from the Westerschelde Estuary (south-west Netherlands). J. Mar. Biol. Ass. U.K. 79: 1017-1027.

Moens, T.; Vierstraete, A.; Vanhove, S.; Verbeke, M. and Vincx, M. (1996). A handy method for measuring meiobenthic respiration. Journal of Experimental Marine Biology and Ecology 197: 177-190

Moens, T. and Vincx, M. (1997). Observations on the feeding ecology of estuarine nematodes. J. Mar. Biol. Ass. U. K. 77: 211-227.

Moens, T. and Vincx, M. (1998). On the cultivation of free-living marine and estuarine nematodes. Helgoländer Meerseunters. 52: 115-139

Moens, T. and Vincx, M. (2000a). Temperature and salinity constraints on the life cycle of two brackish-water nematode species, Pages 115-135. J. Exp. Mar. Biol. Ecol. 243: 115-135.

Moens, T.; Yeates, G. W. and De Ley, P. (2004). Use of carbon and energy sources by nematodes. Nematology Monographs and Perspectives -

196

Proceedings of the fourth International Congress of Nematology, 8-13 June 2002, Tenerife, Spain 2:529-545.

Naeem, S. (1998). Species redundancy and ecosystem reliability. Conserv. Biol. 12:39-45.

Naeem, S. and Wright, J. P. (2003). Disentangling biodiversity effects on ecosystem functioning: deriving solutions to a seemingly insurmountable problem. Ecol. Lett. 6: 567–579.

Pfennig, D. W. and Murphy P. J. (2002). How fluctuating competition and phenotypic plasticity mediate species divergence. Evolution, 56(6), 2002, pp. 1217–1228

Postma-Blaauw, M. B.; de Vries, F. T.; de Goede, R. G. M.; Bloem, J.; Faber, J. H. and Brussaard, L. (2005). Within-trophic group interactions of bacterivorous nematode species and their effects on the bacterial community and nitrogen mineralization. Oecologia 142: 428–439.

Rodger, S.; Griffiths, B.S.; McNicol, J.W.; Wheatley, R.W. and Young, I.M. (2004). The impact of bacterial diet on the migration and navigation of Caenorhabditis elegans. Microb. Ecol. 48, 358–365.

Schratzberger, M.; Warr, K. and Rogers, S. I., (2007). Functional diversity of nematode communities in the southwestern North Sea. Marine Environmental Research 63 (2007) 368–389

Shtonda, B. and Avery, L. (2005). CCA-1, EGL-19 and EXP-2 currents shape action potentials in the Caenorhabditis elegans pharynx. J. Exp. Biol. 208, 2177-2190.

197

Solan, M; Dawning, A. L.; Engelhardt, A. M.; Rueskin, J. L. and Srivastava, D. J. (2004). Extinction and Ecosystem Function in the Marine Benthos. Science 12 306: 1177 – 1180.

Tilman, D. (1999). The ecological consequences of changes in biodiversity: a search for general principles. Ecology 80(5): 1455–1474.

Traunspurger, W.; Bergtold, M. and Goedkoop, W. (1997). The effects of nematodes on bacterial activity and abundance in a freshwater sediment. Oecologia 112 (1): 118-122.

Venette and Ferris (1998). Influence of bacterial type and density on population growth of bacterial-feeding nematodes. Soil Biol. Biochem. 30: 949–960.

Vranken, G. and Heip, C. (1983). Calculation of the intrinsic rate of natural increase, rm, with Rhabditis marina Bastian 1865 (Nematoda). Nematologica 29: 468-477.

Wall, D. and Moore, J. C. (1999). Interactions Underground. BioScience 49 (2): 109-117.

Wieser, W. (1953). Die Beziehung zwishen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden. Ark. Zool. 4: 439-484.

Worm, B., Lotze, H.K., Hillebrand, H. and Sommer, U. (2002). Consumer versus resource control of species diversity and ecosystem functioning. Nature 417:848-851

198

Yeates, G.W.; Bongers, T.; de Goede, R.G.M.; Freckman, D.W. and Georgieva, S. S. (1993). Feeding habits in nematode families and genera - a guide for soil ecologists. J. Nematol. 25: 315-331.

199

CChhaapptteerr 88::

IInntteerrffeerreennccee aanndd ccoommppeettiittiioonn iinn ttwwoo ccoonnggeenneerriicc ssppeecciieess ooff bbaacctteerriivvoorroouuss nneemmaattooddeess ((DDiipplloollaaiimmeellllooiiddeess mmeeyyllii aanndd DD.. oosscchheeii)):: IIss ffoooodd aa kkeeyy ffaaccttoorr??

200

Interference and competition in two congeneric species of bacterivorous nematodes (Diplolaimelloides meyli and D. oschei): Is food a key factor?

Tom Moens1, Eline Van Puyvelde1, Giovanni dos Santos1,2,*.

1Marine Biology Section, UGent, Krijgslaan 281 (S8), 9000 Ghent, Belgium. 2Universidade Federal de Pernambuco, Av. professor Moraes Rêgo, s/n, 50.000.000. Recife, Pernambuco, Brazil.

* Corresponding author. E-mail: [email protected]

Abstract:

The importance of species interaction affects and shapes ecosystem diversity and function. Interactions within trophic group have been poorly reported and their implications for ecosystem function are still unclear. Nematodes communities are characterized by a substantial local diversity and co- occurrence of several species within single trophic functional group. Chemotaxis among different or the same bacterial strain, as well as, real food preference with 13C tracer, had been experimented to test nematodes interaction. The species Diplolaimelloides oschei and D. meyli were affected in combination treatment by the incubation timing of: nematodes species, inoculation timing and species densities. Some food preference was observed in treatment with species combinations. Furthermore, the clear observation of significant interspecific interaction, more than direct competition for food,

201

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Abstract:: Interference and competition in two congeneric … DOUTORADO EM CIÊNCIAS BIOLÓGICAS among species belonging to the same trophic group was discerned. In addition, food heterogeneity decreased interspecific interactions between species. Finally, the inconsistent and different observation found among studies on: population development, taxis and food absorption, can be related to negligence of species indirect interaction.

Keywords: Trophic Interaction; Interspecific interaction; Facilitation; Competition; Chemotaxis; Bacterivores Nematodes; Food preference; Diplolaimelloides oschei and D. meyli

202

Introduction

Interspecific interactions importantly affect the production and biodiversity of ecosystems (Levin et al., 2001), as well as the shape and predictability of the diversity-ecosystem functioning relationship (Johnston et al. 1996). There is a multitude of studies dealing with interactions between different trophic levels, and their effects on the ecosystem (Okuyama & Bolker, 2007, Smith et al. 2007). Fewer studies have addressed interactions among species within a trophic level and their implications for ecosystem functioning (Cardinale et al. 2002, Postma-Blaauw et al. 2005, De Mesel et al. 2006). Within a community, species belonging to one and the same trophic guild are more likely to be functionally redundant than species belonging to different guilds (Walker 1992). However, within-guild diversity may not only improve ‘ecosystem reliability’ (Naeem 1998) but also functioning per se, as a result of positive interspecific interactions such as facilitation (Cardinale et al. 2002, Jonsson & Malmqvist 2003). Positive interactions may generally have a more significant imprint on community structure than ‘negative’ interactions such as competition (Hutchinson 1975), as they may allow coexistence of more species on a local scale as well as a comparatively higher local community production (Tilman 1996, Weigelt et al. 2007). Nematodes are the most numerous and among the most speciose metazoans in marine as well as terrestrial sediments/soils (Heip et al. 1985, Blaxter, 1998). Their communities are often characterized by a substantial local diversity, and by a co-occurrence of several to many species within single trophic guilds (Moens & Vincx, 1997). Several recent studies have demonstrated that species within a trophic guild may both facilitate and inhibit each other’s population development as well as behavioural parameters (Ilieva-Makulec 2001, Postma-Blaauw et al. 2005, De Mesel et al. 2006, Santos et al. in prep.). Particularly striking was the finding that sympatrically occurring congeneric monhysterid species may inhibit each other’s population development (De Mesel et al. 2006, Santos et al. in prep.), whereas they facilitate each other’s food-finding behaviour (Santos et al. in prep.). It is, however, unclear to what extent food itself is a driving factor behind these

203

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Introduction: Interference and competition in two congeneric… OUTORADO EM CIÊNCIAS BIOLÓGICAS negative interspecific interactions. Moreover, the longer-term outcome of such interactions remains unclear. Michiels and Traunspurger (2005) argued that competition among meiobenthic species for a limiting resource may cause strong population fluctuations and chaos, preventing the competitively dominant species from driving less abundant species to extinction. Since several recent microcosm experiments have indicated that population growth rates of some monhysterid nematode species are hampered when in sympatry with confamiliar species, we performed a population growth experiment with the congeneric species Diplolaimelloides meyli and D. oschei, which co-occur on decomposing salt marsh vegetation in the polyhaline reach of the Schelde Estuary, SW Netherlands (De Mesel et al. 2003). Both species were grown in monospecific and in combination treatments on decomposing Spartina anglica leaves inoculated with a mixture of bacteria from the habitat. We tested whether any interspecific effects in the combination treatments were (a) affected by the timing of inoculation of both nematode species (simultaneously or with a 1-week interval, the latter potentially leading to priority effects), and (b) density-dependent. We also investigated whether any observed species interference could be linked to competition for food. To this end, we first performed taxis experiments in which the response of both named monhysterid nematode species towards a single food source, i.e. Escherichia coli, was quantified in monospecific and combination treatments. We then diversified food options by simultaneously offering four different bacterial strains, isolated from Spartina detritus, in microcosms with either single or both species of Diplolaimelloides. We tested whether Diplolaimelloides species had differential preferences for the bacterial strains offered, and if so, whether this affected the interspecific interaction strengths observed in the first taxis experiment with E. coli as the only food. Finally, we investigated whether preferential responses towards specific bacterial strains also implied true feeding preferences. To this end, we inoculated microcosms with 13C-labelled bacteria from each of the four strains of the second taxis experiment, and studied uptake of bacterial carbon by both nematodes in monospecific (one nematode species) and combination (both nematode species inoculated together) experiments.

204

Materials and methods

Nematode stock cultures and harvesting

Nematodes for the present experiments were harvested from monospecific, agnotobiotic cultures of either Diplolaimelloides species on sloppy agar layers. Permanent cultures of both Diplolaimelloides species have been maintained in our laboratory for more than 10 years (Moens & Vincx, 1998). Nematodes for all experiments were harvested from ‘old’ cultures near carrying capacity. In such cultures, the agar has typically turned almost completely liquid, and high densities of juveniles are present which do not fully mature anymore, probably as a result of food shortage. However, addition of aliquots of a frozen-and-thawed E. coli suspension at a density of 3 x 109 cells ml-1 leads to complete maturation of a majority of these juveniles within ca. 1 week, and even to novel reproduction, such that cultures are again dominated by active adult and older (J4) juvenile nematodes (Moens & Vincx, 1998). Nematodes from such cultures can be harvested ‘en masse’ by density centrifugation in sucrose at a final concentration of 40% (modified after Sulston & Brenner, 1974). This removes agar remains and a majority of the associated bacteria, and does not inflict mortality provided the sucrose washing is done fast and immediately followed by transfer of the nematodes to artificial seawater of suitable salinity. Repeated washing of the nematodes through centrifugation and resuspension in fresh artificial seawater ensures that sucrose remains are effectively eliminated from the final nematode suspension. This suspension can then be diluted or concentrated to the desired density and pipetted in small aliquots onto microcosms, provided the suspension is adequately homogenized at all times.

Population growth experiment

We prepared 108 microcosms, consisting of 9 cm diam Petri dishes filled with a thin layer of sediment, and to which fragments of Spartina anglica leaves and stems were added. Each microcosm received 15 ± 0.5 g of dried and

205

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric… DOUTORADO EM CIÊNCIAS BIOLÓGICAS homogenized sediment. This sediment consisted of a mixture (50/50 weight/weight) of fine sediment (particle size fraction of 180 – 250 µm) and silt (particle size < 63 µm), and had been precombusted in a muffle furnace at 550°C for 3 h to remove organic carbon. As such, the added phytodetritus was the only basal carbon source in our microcosms. This sediment was rehydrated until saturation with ca. 8 ml artificial seawater (ASW, Dietrich & Kalle, 1957) containing F2 nutrients (Guillard 1975; 20 ml F2 medium added to 980 ml ASW). The addition of F2 medium served to avoid early nutrient limitation of bacterial growth and hence food shortage for the bacterivorous nematodes, since Spartina detritus has a relatively high C:N ratio (Newell et al. 1985, De Mesel et al. 2003). Each microcosm further received 0.6 ± 0.02 g (dry weight) of Spartina leaves and stems (not homogenized). The exact dry weight in each microcosm was noted before and after the experiment, in order to assess the decomposition rate and how this was influenced by (a) nematodes and (b) any interspecific effects of nematodes. However, no significant differences were found in the decomposition rate between any treatments, and these results are therefore not reported in this paper. The Spartina tissue used in this experiment were fresh green stems and leaves collected from the Paulina saltmarsh (Schelde Estuary), which were treated as follows prior to their inoculation into the experimental microcosms. They were first rinsed thoroughly under tap water to remove most adhering fauna, and subsequently dried for 2 h at 110°C, then overnight at 60°C, to kill all associated proto- and metazoans which had not been removed by the rinsing procedure. The Spartina detritus was then immersed for 48 h in ASW to which we added a few drops of 0.8 µm filtered habitat water. As such, a strong bacteria-dominated decomposition was initiated, and compounds potentially negatively affecting nematode population growth (De Mesel et al., unpubl. results) largely leaked out. A positive side effect of this pre-treatment is that when the Spartina fragments are eventually inoculated into the experimental microcosms, the initial microbial growth is less explosive, which is favourable for the growth of monhysterid nematodes (Moens & Vincx, 2000, Santos et al. subm.). After this pre-treatment, the Spartina stems and leaves were again thoroughly rinsed under tap water and

206

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric… DOUTORADO EM CIÊNCIAS BIOLÓGICAS dried at 60°C till constant weight, then cut into pieces, weighed and distributed over the experimental microcosms. Aliquots of the ASW in which the Spartina had been immersed were filtered three times over 0.8 µm Millipore filters to remove any contaminating flagellates and fungal spores, and then used as bacterial inoculum for the experimental microcosms. This bacterial inoculum was mixed with several drops of filtrate obtained from both D. meyli and D. oschei cultures, to ensure that all bacterial strains present in either nematode culture were present in the bacterial inoculum of all experimental treatments and that any differences between treatments could therefore not be linked to the introduction of specific bacterial strains associated with only one of these nematode species. After addition of the bacterial inoculum to the microcosms, they were left for 72 h before addition of nematodes. There were 9 treatments in this experiment, each of which was checked at three time intervals (14, 28 and 42 days after the first nematode addition) and in 4 replicates per time and treatment, yielding 108 independent microcosms (Tab. 1).

Table1: Schematic representation of the 9 different treatments. At T0 nematodes were inoculated at experimental start and T1= nematodes inoculation happens one week after the experimental start. Experiment Code N° of invidious

Treatment A T0 = 200 ± 30 D. oschei + 200 ± 30 D. meyli

Treatment B T0 = 200 ± 30 D. oschei + 600 ± 60 D. meyli

Treatment C T0 = 200 ± 30 D. oschei + 66 ± 10 D. meyli

Treatment D T0 = 200 ± 30 D. oschei

Treatment E T0 = 200 ± 30 D. meyli

T0 = None Treatment F T1 = 200 ± 30 D. oschei

T0 = 200 ± 30 D. Meyli Treatment G T1 = 200 ± 30 D. oschei

T0 = None Treatment H T1 = 200 ± 30 D. meyli

T0 = 200 ± 30 D. oschei Treatment I T1 = 200 ± 30 D. Meyli

207

The first three treatments served two purposes: (1) to detect interspecific effects of D. meyli on D. oschei and vice-versa when both species were simultaneously inoculated, and (2) to assess any density-dependence of the effect of D. meyli on D. oschei. Treatment A received 200 ± 30 D. oschei and 200 ± 30 D. meyli. Treatments B and C received the same number of D. oschei, but, respectively, 600 ± 60 and 66 ± 10 D. meyli. Both species were inoculated simultaneously in treatments A, B and C. These treatments were compared with the ‘control treatments’ D and E, in which, respectively, 200 ±

30 D. oschei alone and 200 ± 30 D. meyli alone were inoculated at T0. Treatments F and G served to test the effect of a prior inoculation of D. meyli on D. oschei. For this purpose, treatment F received no nematodes at T0 and 200 ± 30 D. oschei after 1 week. Treatment G received 200 ± 30 D. meyli at

T0 and 200 ± 30 D. oschei after 1 week. Vice-versa, treatments H and I received, respectively, no nematodes at T0 and 200 ± 30 D. meyli after 1 week, and 200 ± 30 D. oschei at T0 and 200 ± 30 D. meyli after 1 week. All microcosms had a salinity of 25 and were incubated at a constant temperature of 18 ± 1°C in the dark. 14, 28 and 42 days after the start of the experiment, 4 replicates of each treatment were randomly taken out. The Spartina fragments were picked up by forceps and immersed for 30 min in an isotonic MgCl2.6H20 solution to sedate attached nematodes and facilitate their release from the detritus by rinsing (Hulings & Gray 1971). Rinsing was done with tap water over a 20 µm mesh size sieve, and the nematodes retained on that sieve were collected and added to the sediment. The sediment of the microcosms was then collected and stored in a 4% formaldehyde solution until further treatment. This treatment consisted of a double centrifugation-flotation in the colloidal silica-gel ludox HS40 at a specific density of 1.18. The supernatant after each centrifugation was washed over a 20 µm sieve, stained with Bengal Rose to facilitate detection of nematodes under a binocular, and stored in 4% formaldehyde until counting and identification under a compound microscope. Discrimination of both Diplolaimelloides species is easy, even under low magnification, because D. oschei has a long filiform tail and D. meyli has not.

208

Effects of D. meyli on the population development of D. oschei in simultaneous inoculations were tested using Kruskal-Wallis ANOVA comparing D. oschei densities in treatments A, B, C and D at each of the three counting days. The experimental design essentially fits a two-way ANOVA, but different data transformations failed to render the dataset normally distributed and homoscedastic, hence we decided to eliminate the effect of time and perform non-parametric variance analysis on each time separately. A posteriori pairwise comparisons followed Conover’s (1980) procedure. Similarly, effects of D. oschei on the population development of D. meyli in simultaneous inoculations used Kruskal-Wallis ANOVA on the D. meyli densities in treatments A, B, C and E, with special focus on the comparison of treatments A and E. Priority effects of D. meyli on D. oschei were investigated with a Mann- Whitney U-test on the D. oschei densities in treatments F and G. Vice versa, priority effects of D. oschei on the population development of D. meyli used a Mann-Whitney U-test on the D. meyli densities in treatments H and I.

Taxis experiments with a single food option

Previous experiments have demonstrated that bacterivorous nematodes, including both Diplolaimelloides species used here, can exhibit a directed movement (i.e. taxis) towards bacterial spots in their immediate environment, probably following chemical cues emanating from the bacteria (Moens et al. 1999 and refs. therein). In this experiment, we tested whether both Diplolaimelloides species would move towards spots of E. coli, and whether they would affect each other’s response when in sympatry. These experiments were carried out in square 12 x 12 cm Petri dishes coated with a thin layer of a 0.75 % bacto-agar medium in ASW. The low nutritious value of this medium limits growth of bacteria cotransferred with the nematodes. On the bottom of the microcosms, a grid was drawn dividing the microcosms into 6 sectors (A – F) of equal width. At the intersect of sectors A and B and of sectors E and F, two 1 x 1 cm squares were delineated (Fig. 1), and inoculated crosswise with 50 µl of an E. coli K12 suspension in ASW at a

209

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric… DOUTORADO EM CIÊNCIAS BIOLÓGICAS density of 3 x 109 cells ml-1, or with a 50 µl drop of sterile ASW as a control. In the centre of each plate, at the C-D intersect, 100 µl of a suspension containing 100 ± 10 nematodes in ASW was inoculated. Four microcosms were inoculated with D. oschei and four with D. meyli. In four other microcosms, 100 ± 10 D. meyli and an equal number of D. oschei were mixed and inoculated together. Microcosms were incubated in the dark at 18 ± 1°C and nematodes inside bacterial and control spots were counted under a compound microscope after 1, 2, 3, 7, 9, 12 and 16 days of incubation (Fig. 1).

D E. coli - Inocula

E ASW - Control Nematodes F

Figure 01: Schematic diagram of the taxis experiments with a single food option.

Nematode taxis towards E. coli spots was assessed in each of the three treatments using a replicated G-test for goodness of fit (Sokal & Rohlf 1995), starting from the null hypothesis of no taxis and only random dispersal, which should result in equal numbers of nematodes inside bacterial and control spots (Moens et al. 1999). This G-test calculates three test-statistics: (1) GH (where H stands for heterogeneity), which tests for heterogeneity among replicates, and is compared to a χ² distribution with (a-1)(b-1) degrees of freedom (where a = the number of treatments and b = the number of replicates), (2) GP (where P = pooled), which effectively tests the null hypothesis of equal distribution on the pooled data of all replicates, and is compared to a χ² distribution with a-1 degrees of freedom, and (3) GT (with T for total) which takes into account GH and GP and tests whether, inspite of potential heterogeneity among replicates, the null hypothesis of equal 210

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric… DOUTORADO EM CIÊNCIAS BIOLÓGICAS distribution can be accepted or rejected. GT = GH + Gp and is compared to a χ² distribution with b (a-1) degrees of freedom. Effects of one Diplolaimelloides species on the taxis of the other species towards E. coli were assessed using a two-way ANOVA (treatment and time) on the non-transformed nematode counts of either species in the monospecific and combination treatment. Finally, we also used two-way ANOVA to compare the summed numbers of D. oschei and D. meyli inside E. coli spots in the combination treatment with the summed numbers of both species inside E. coli spots in the respective monospecific treatments, to assess whether the species combination resulted in a higher or lower ‘community’ response than could be expected on the basis of the monospecific responses.

Taxis experiments with multiple food options

In a second taxis experiment, we introduced food heterogeneity in the microcosms by inoculating one spot each of four different bacterial strains, isolated from the natural habitat of the Diplolaimelloides species. Isolation, culture and purification procedures of these bacteria were as described in Moens et al. (2005). The strains are currently awaiting identification by FAME and related procedures (Huys et al. 1994, Bertone et al. 1996). Bacteria of each strain were harvested from liquid cultures on marine broth using three consecutive centrifugation-resuspension cycles in ASW. Microcosm experiments used the same type of square Petri dish and agar medium as in the previous taxis experiment. One spot of each bacterial strain and two control spots were inoculated equidistantly from the central nematode inoculum (Fig. 2). Nematodes were grown and harvested as described for the previous experiments.

211

= ASW (Control spots)

= Strain P1

= Strain P2

= Strain P3

= Strain P4

= Nematodes

Figure 2: Schematic diagram of the taxis experiments with multiple food options.

Four microcosms were inoculated with D. oschei only (100 ind.), four with D. meyli only (100 ind.), and the remaining four with a combination of 100 D. oschei and 100 D. meyli. Microcosms were incubated in the dark at 17 °C. Counting followed the same procedure as in the previous experiment but with a slightly different timing: after 1, 2, 3, 5 and 9 days. Statistical analysis followed the same procedures as described for the previous taxis experiment. Additional focus was now on the assessment of any preferences of nematodes for specific bacterial strains.

Feeding experiment

The aim of this experiment was twofold: (1) test whether any preferences for specific bacteria found in the second taxis experiment were translated in preferential uptake of those bacteria, and (2) assess whether and to what extent D. meyli and D. oschei may affect each other’s feeding activity. To this end, we used three of the four bacterial strains (P2, P3, P4) of the second taxis experiment, and prepared 36 3.5 cm i.d. microcosms filled with 3 g of a 50:50 (weight/weight) mixture of sterile fine to medium sediment (180 – 250 µm) and silt (< 63 µm). We buried a small piece of glass fibre paper in the sediment, because preliminary experiments indicated that grazing on bacteria by monhysterid nematodes may be improved when they can browse bacteria attached to, or associated with, filamentous structures (Moens unpubl.). 212

Each bacterial strain was first grown for 72 h at room temperature in a liquid marine broth medium. Bacteria were then harvested by centrifugation, washed several times in ASW, and resuspended in a 10 x dilution of the original growth medium, but with addition of 13C-glucose. They were then allowed to grow for 24 h in this labelled medium. This protocol optimizes label uptake and, depending on specific growth conditions, may yield bacteria with up to 65% 13C (Moens unpubl.). Bacteria were harvested from this medium by repeated centrifugation-resuspension in ASW to remove non-incorporated label, and subsequently freeze-dried. 1 mg (± 50 µg) portions of these freeze- dried bacteria were resuspended and homogenized in 1.5 ml of ASW and inoculated into the microcosms. Nematodes were harvested from stock cultures as described before, and added at a density of ca. 250 ind. per microcosm. They were allowed to feed on the bacteria for 48 h at 17 °C in the dark. For each bacterial strain, 4 replicate microcosms were inoculated with D. oschei, 4 with D. meyli and 4 with a combination of equal numbers of both species. Incubations were stopped by the addition of a 4% formaldehyde solution. Nematodes from each microcosm were subsequently elutriated with ludox HS 40 (colloidal silica) and collected over a 20 µm mesh size sieve. For each microcosm, ca. 150 nematodes of either species were handpicked on the tip of a fine Tungsten wire, rinsed in sterile ASW, and transferred to a drop of milliQ water in 2.5 x 6 mm aluminium cups (Elemental Microanalysis Ltd.). These cups were dried, pinched close and stored under dry atmosphere until stable carbon isotope analysis. We performed isotope ratio mass spectrometry (IRMS) on a Flash 1112 element analyser (ThermoFinnigan) coupled via a ConfloIII interface to a DeltaplusXL IRMS (ThermoFinnigan). Results are expressed in the δ notation relative to Vienna Peedee Belemnite. δ13C (in units of ‰) is calculated as:

Upon analysis, we integrated blanks (empty aluminium cups) and standards 13 13 (IAEA-CH6 with δ C = -10.5 ‰ and IAEA-309A, with δ C = +93.4 ‰, and 213

IAEA-309B, with δ13C = +535.4 ‰) into the sample run after ca. every 8th sample. We adjusted the obtained nematode δ13C for the δ13C of the aluminium cup based upon the measured δ13C and mean C-signal of the blanks, and on the C-signal of the nematode samples. We then expressed our results as specific uptake (Δδ13C, also in ‰), which is the difference between the δ13C of the ‘enriched’ sample and that of an unenriched sample. For the latter, we used nematodes from stock cultures which had never been in contact with any 13C-enrichment. δ13C and Δδ13C are relative measures and can only be used for comparison among organisms with very similar body mass. Hence, the results which we present here should not be used to compare absolute 13C-uptake between both Diplolaimelloides species, but rather to compare uptake within each species among treatments. Absolute uptake can be calculated from Δδ13C and nematode biomass as explained in Moens et al. (2002). Data from this experiment were analysed with two-way ANOVA, using nematode treatment and bacterial strain as fixed factors. Data were log(x+1)- transformed prior to analysis to meet the assumptions of normality and homoscedasticity.

214

Results

Population growth experiment

D. oschei attained significantly lower densities in treatments where it was simultaneously inoculated with D. meyli than in monoculture (P < 0.0001,

F(3,16) = 39.1 ; P < 0.0001, F(3,16) = 64.2; P < 0.0001, F(3,16) = 30.5 after 2, 4 and 6 weeks, respectively) (Fig.3). After 2 weeks, the only non-significant pairwise difference was that between treatments A and B. After 4 weeks, the only non-significant pairwise difference was between treatments C and D. After 6 weeks, the densities of D. oschei in the three treatments where it was in sympatry with D. meyli (A, B and C) converged and did no longer significantly differ from each other; however, they did differ significantly from the control treatment (D).

D. oschei 20000

15000

10000

5000 Average densities permicrocosm 0 Do+niets Do+Dm/3 Do+Dm Do+3Dm 2 weeks

Treatment 4 weeks 6 weeks

Fig. 3. Abundances of D. oschei in a control treatment (D, left bars), and in combination treatments with D. meyli, with increasing initial D. meyli density from left to right (treatments C, A and B). Blue bars represent data after 2 weeks of incubation, red bars after 4, and yellow bars after 6 weeks. Data are averages of 4 replicates ± 1 stdev.

215

D. oschei densities in microcosms to which D. meyli had been inoculated 1 week earlier also were consistently lower than in microcosms without D. meyli

(P < 0.001, F(1,8) = 41.8 after 2 and 4 weeks, P < 0.0001, F(1,8) = 107.4 after 6 weeks) (Fig. 4).

D. oschei 15000

10000 2 weeks 4 weeks

5000 6 weeks

Average densities per microcosm per densities Average 0 Controle+Do Dm+Do

Treatments

Fig. 4. Abundances of D. oschei in a control treatment (F, left bars), and in a combination treatments where D. meyli had been inoculated 1 week prior to inoculation of D. oschei (G). Blue bars represent data from 2 weeks after the start of the experiment, red bars after 4, and yellow bars after 6 weeks. Data are averages of 4 replicates ± 1 stdev.

When we compare the densities of D. oschei in treatments A, D and I (the last treatment first received D. oschei and 1 week later a similarly large inoculum of D. meyli), no significant differences were obtained after 2 weeks (Kruskal-

Wallis ANOVA, P > 0.05, H (2,12) = 2.0), but D. oschei densities were significantly lower in monoculture than in both combination treatments after 4 and 6 weeks (P < 0.05, F(2,12)= 14.2 after 4 weeks, P < 0.05, H (2,12) = 8.2 after 6 weeks) (Fig.5). D. oschei densities did not differ significantly between treatments A and I.

216

D. oschei

20000

15000

10000

5000

Average densities per microcosm per densities Average 2 weeks 0 4 weeks Do+niets Do+Dm Do+Dm(samen) 6 weeks Treatments

Fig. 5. Abundances of D. oschei in treatments D (control), I (with later inoculation of D. meyli) and A (with simultaneous inoculation of D. meyli). Comparison Between the average abundance of D. oschei (Do) on 2, 4 and 6 weeks. Blue bars represent data from 2 weeks after the start of the experiment, red bars after 4, and yellow bars after 6 weeks. Data are averages of 4 replicates ± 1 stdev.

Comparison of treatments A, E and F yielded marginally significant differences between the control treatment E (D. meyli alone) and both combination treatments with D. oschei, irrespective of whether D. oschei was inoculated simultaneously or after a lag of 1 week (P < 0.05, F(1,8)= 51.1 after

4 weeks and P < 0.05, F(1,8)= 8.1 after 6 weeks) (Fig. 6).

D. meyli 15000

10000

2 weeks 4 weeks 6 weeks 5000 Average densites per microcosm Average

0 Dm+niets Dm+Do Do+Dm(samen) Treatments

217

Fig. 6. Abundances of D. meyli in a control treatment (E), a treatment with later inoculation of D. oschei (F), and a treatment with simultaneous inoculation of D. oschei (A). Blue bars represent data from 2 weeks after the start of the experiment, red bars after 4, and yellow bars after 6 weeks. Data are averages of 4 replicates ± 1 stdev.

No significant priority effects were found of D. oschei on the population development of D. meyli, with the exception of a higher D. meyli density in the monoculture after 4 weeks (P < 0.05, F(1,8) = 35.2) (Fig. 7).

D. meyli

15000

10000 2 weeks 4 weeks 6 weeks 5000 Average densities per microcosm per densities Average

0 Controle+Dm Do+Dm Treatment

Fig. 7. Abundances of D. meyli in microcosms without D. oschei and where D. oschei had been inoculated 1 week earlier. Blue bars represent data from 2 weeks after the start of the experiment, red bars after 4, and yellow bars after 6 weeks. Data are averages of 4 replicates ± 1 stdev. Furthermore, it is interesting to note that D. meyli in monoculture (treatment E) reached maximal abundances only half those of D. oschei.

Taxis experiments with a single food option

The previous chapter (Santos et al. in prep), showed results of Diplolaimelloides oschei and D. meyli toward E. coli. Avoiding redundancy of information the author and co-authors preferred cite to previous chapter, and results of this topic will not be repeated, even if data expressed here are a little extended in time (1-16 days) there is no main changes in results and in 218

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Results: Interference and competition in two congeneric… DOUTORADO EM CIÊNCIAS BIOLÓGICAS statistical analysis. Argumentation and comparison with the previous chapter will be given in our discussion as “Taxis experiments with a single food option”.

Taxis experiments with multiple food options

In all treatments, D. oschei and D. meyli were always significantly heterogeneously distributed over the different spots (all GT > 40, P < 0.05,

20df). Replicates were also often heterogeneous (GT > 25, P < 0.05, 15df). Overall, D. meyli in the monospecific treatment exhibited a preferential response towards strain P3 (all GT > 25, P < 0.005, 4df). D. meyli numbers inside spots of P4 were also higher than inside spots of P1 and P2 (both GT > 15, P < 0.05, 4df). D. oschei initially (days 1 and 3) exhibited a preference for strain P1, but this preference shifted to strain P4 from day 5 onwards (all GT > 15, P < 0.005, 4df). In the combination treatment, D. meyli still exhibited a preference for strain

P3, but also P2 (GT > 15, P < 0.005, 4df). D. oschei, by contrast, showed only a marginally significant preference, mainly for P2 and to a lesser extent P3

(GT > 21, P < 0.05, 12df). D. meyli moderately affected the taxis of D. oschei, although only from 5 days onwards, with a significantly higher response of D. oschei towards P1 (all P <

0.05, F(1,8) 7 > 25.3), P3 (all P < 0.05, F(1,8) > 23.9) and P4 (P < 0.05, F(1,8) > 14.4 at days 5 and 15 only) in the combination treatment compared to the monospecific treatment. The response of D. oschei towards P2 did not differ among treatments (Fig. 8).

219

Fig. 8. Comparison of the response of D. oschei to different bacterial strains in monospecific and combination treatments. Data are means ± 1 stdev of 4 replicates per treatment.

D. oschei affected the response of D. meyli towards bacteria as follows: in the presence of D. oschei, significantly more D. meyli reached spots of P1 (P <

0.05, F(1,47) = 6.0) and P3 (P < 0.05, F(1,48) = 9.8), but not of P4 (P > 0.05,

F(1,48) = 0.03), than in the monospecific treatment. The response of D. meyli towards bacterial strain P2 showed significant differences between monospecific and combination treatment, but these differences were not consistent over time (Fig. 9).

220

Fig. 9. Comparison of the response of D. meyli to different bacterial strains in monospecific and combination treatments. Data are means ± 1 stdev of 4 replicates per treatment.

Inspite of the above-mentioned species-specific differences, the summed abundances of both Diplolaimelloides species inside bacterial spots did not exhibit any consistent significant differences between monospecific and combination treatments (P > 0.05, F(1,48) < 1.0) (Fig. 10).

Fig. 10. Comparison of the summed response of D. meyli and D. oschei to different bacterial strains in monospecific and combination treatments. Data are means ± 1 stdev of 4 replicates per treatment.

Spearman Rank tests demonstrated significantly positive (all P < 0.05) correlations between the numbers of both Diplolaimelloides species in all spots, including controls (data not shown). These correlations were strongest inside control spots and spots of strain P2, suggesting that random walk behaviour is highly similar in both species.

221

Feeding experiment

Δδ13C of D. oschei showed no preferential uptake among the three bacterial strains tested (P > 0.05, F(2,18) = 0.6). However, D. oschei absorbed significantly more 13C when in sympatry with D. meyli than when alone (P <

0.05, F(1,18) = 10.1) (Fig. 11). The interaction term treatment x strain was not significant.

Fig. 11. Δδ13C of D. oschei feeding on three different bacterial strains (P2, P3, P4) in monospecific and combination treatments. Data are means ± 1 stdev if 3 or 4 replicates.

Δδ13C of D. meyli, by contrast, showed a significant preferential uptake of strain P3 (P < 0.0005, F(2,18) = 18.6), but no effect of treatment (monospecific vs combination treatment) (P > 0.05, F(1,18) = 0.01) or treatment x strain (Fig. 12).

222

Fig. 12. Δδ13C of D. meyli feeding on three different bacterial strains (P2, P3, P4) in monospecific and combination treatments. Data are means ± 1 stdev if 3 or 4 replicates.

223

Discussion

Meiobentic communities are typically characterized by a high local diversity, usually encompassing several to many species of the same trophic level. This study addresses the question of what factors structure the coexistence of these multiple ‘confunctional’ species. The most prominent result was a significant negative influence of D. meyli on the population development of D. oschei. This effect increased with increasing densities of D. meyli. D. meyli also negatively affected population development of another monhysterid, Diplolaimella dievengatensis, and like in the present study, this effect was asymmetric (De Mesel et al. 2006). This contrasts with the results by Santos et al. (in prep.), who found a largely symmetric negative effect of D. meyli on D. oschei and vice versa, which was strongly food-density dependent. Postma-Blaauw et al. (2005) reported mostly asymmetric interactions between three terrestrial bacterivorous nematode species. The interaction between Bursilla monhystera and Acrobeloides nanus was asymmetrically competitive (0, -), as in our present study with both Diplolaimelloides species, whereas the interactions between B. monhystera and Plectus parvus and between A. nanus and P. parvus were contramensal (+, -). These authors contended that the strongest interactions can be expected between species with more divergent life histories, but the present results, in conjunction with those of De Mesel et al. (2006) and Santos et al. (in prep.), suggest that even congeneric species may exert a pronounced impact over each other’s population growth. There are, however, no indications in the present results that D. meyli could drive D. oschei to extinction; in fact, the observed negative asymmetric effect rather promotes evenness between both species, since the yields of D. oschei in monospecific cultures were twice those of D. meyli, whereas in combination culture both species attained similar densities. Our present experiments also provided some indications of priority effects, because the negative interspecific influence was bigger when D. meyli was inoculated one week prior to D. oschei. However, the reverse inoculation sequence (D. oschei one week before D. meyli) did not reveal any evidence of priority effects. The time lag (1 week) between inoculation of both species

224

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric … DOUTORADO EM CIÊNCIAS BIOLÓGICAS was, however, probably quite short to find more pronounced priority effects, since the generation time of these nematodes at the current experimental conditions typically exceeds 2 weeks. The negative impact of D. meyli on D. oschei could theoretically be related to competition for food and/or space. The latter explanation was already discarded by De Mesel et al. (2006) with the argument that joint densities of both Diplolaimelloides species in sympatry are no higher than the densities of D. oschei in monoculture. Competition for food is possible, as also indicated by Santos et al. (in prep.) based upon experiments with a single food source. However, in the present growth experiment, a multitude of bacterial strains was present, and the results of our taxis experiment indicate that the preferences of both species for different members of this bacterial community differ. Such heterogeneity of food sources should normally limit competitive interactions (Vetter et al. 2004). Moreover, De Mesel et al. (2006) found generally higher bacterial activity in microcosms with combinations of different monhysterid nematode species than in monospecific treatments, contradicting that bacteria as a whole would become limiting. Furthermore, the results of our feeding experiment demonstrated a higher carbon uptake by D. oschei when in sympatry with D. meyli, which is again not in line with the idea of competition over a limiting resource. In this respect, it is important to note that even these closely related bacterivorous nematode species may differentially affect the activity (De Mesel et al. 2003), community composition and succession of bacteria on decomposing Spartina detritus (De Mesel et al. 2004). Such impacts on bacterial communities may result from microbioturbation (Aller & Aller 1992), selective effects on bacterial settlement (Riemann & Helmke 2002, Moens et al. 2005), or selective grazing (De Mesel et al. 2004, Salinas et al. 2007). Whatever the underlying mechanism, De Mesel et al. (2004) found that the bacterial community composition in a combination treatment with D. oschei and D. meyli in sympatry more closely resembled the community composition from monospecific treatments with D. meyli than with D. oschei, suggesting D. meyli has a stronger effect of the bacterial community than D. oschei. Population development of bacterivorous nematodes is very strongly

225

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric … DOUTORADO EM CIÊNCIAS BIOLÓGICAS dependent on the types and diversity of bacterial strains present (Venette & Ferris 1998), so shifts in bacterial community composition may significantly affect nematode growth. An alternative explanation for the observed interspecific interference is allelochemical interactions (Huettel 1986), but there are no published studies proving that the production of semiochemicals by nematodes may affect the population development of other nematodes. Nevertheless, this type of interaction was also suggested to (partly) explain the negative effect of Pseudostenhelia wellsi on Cletocamptu deitersi, two harpacticoid copepod species (Chandler & Fleeger 1987). In this case, the chemicals were suggested to be secreted together with the mucus production. Many nematodes also produce copious amounts of mucus (Riemann & Schrage 1978, Nehring et al. 1990). The prime goal of the two (chemo)taxis experiments was to study the influence of D. oschei and D. meyli on each other’s response towards food (bacteria). We wanted to know if nematodes showed a directed movement (taxis) towards bacteria, whether this attraction is selective, and whether it is the same for both Diplolaimelloide species. Finally we wanted to know if such (selective) taxis of individual species would be affected by the presence of another species. Both D. oschei and D. meyli migrated towards all bacterial strains offered. When only a single bacterial strain (E. coli) was offered, both species facilitated each other’s response, the effect of D. oschei on D. meyli being more pronounced than vice versa. A possible explanation is that nematodes are ‘sloppy feeders’ and release certain metabolites into the medium upon feeding. The stronger effect of D. oschei may relate to the suggested higher motility of this species (Gallucci et al. 2005). These metabolites would act as signal molecules enhancing the response of the other nematodes (Moens et al. 1999). When we introduced four different bacterial strains, we observed differential preferences for both Diplolaimelloides species. Moreover, we no longer observed interspecific facilitation. This indicates that a differential preference for food weakens the strength of interaction between species in the same trophic level (Paine 1980, Neutel et al. 2002). In spite of the weakened interaction at the level of food-

226

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 8: Discussion: Interference and competition in two congeneric … DOUTORADO EM CIÊNCIAS BIOLÓGICAS finding, the negative interspecific effects on population growth remained in the food-diverse environment. Selective chemotaxis of bacterivorous nematodes towards bacterial cues has been demonstrated several times before (Andrew & Nicholas 1976, Grewal & Wright 1992, Moens et al. 1999, Zhang et al. 2005, Shtonda & Avery 2006, Blanc et al. 2006, Salinas et al. 2007). Nematodes can both taste and smell food (Croll & Sukhdeo, 1981, Bargmann et al., 1993), albeit that taste and olfactory receptors differ (Bargmann & Mori, 1997). In the feeding experiment, we studied the influence of D. meyli and D. oschei on each other’s food absorption using 13C-labelled bacterial strains (the same as used in the second taxis experiment). In contrast to the results of the taxis experiment, D. oschei did not show clear preferences for a specific bacterial strain. D. meyli on the other hand exhibited the same preference as in the taxis experiment. Salinas et al. (2007) observed a discrepancy between bacteria eliciting a highly preferential response of nematodes, and strains allowing optimal growth of these same nematodes. While we did not test for growth on individual bacterial strains, D. meyli exhibited the same preference in both taxis and feeding experiments. It should be checked in future work whether this preference is a true preference or may have been related to the recent feeding history of the experimental animals (Shtonda & Avery, 2006). In combination treatments, D. oschei consumed more bacterial carbon than in the monospecific treatments. This may be explained by the availability or digestibility of bacteria previously consumed but not digested by the other nematode species (i.e. D. meyli). In conclusion, significant interspecific interactions, other than direct competition for food, among species belonging to the same trophic guild may be rule rather than exception in intertidal meiobenthic communities. Food heterogeneity appears to weaken interspecific interactions. However, different end points (population development, taxis, food absorption) often yield inconsistent responses, rendering predictions on the outcome of the observed interactions difficult.

227

Acknowledgements

This work was financially supported by a CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) grant to the first author, and by Ghent University through BOF projects 0110600002 and 01GZ0705. T.M. is a postdoctoral fellow with the Flemish Fund for Scientific Research. Luiz Naveda had made some appreciable and constructive comments for some software utilization for optimization of editing time.

228

References:

Aller, R.C. and Aller, J.Y. (1992). Meiofauna and solute transport in marine muds. Limnology and oceanography 37:1018-1033. Andrew, P.A. and Nicholas, W.L. (1976). Effect of bacteria on dispersal of Caenorhabditis elegans (Rhabditidae). Nematologica 22, 451-461. Bargmann, C.I. and Mori, I., (1997). Chemotaxis and thermotaxis. C. elegans II, 717–737. Bargmann, C.I., E. Hartwieg, and H.R. Horvitz. (1993). Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74:515–527. Bertone, S., Giacomini, M., Ruggiero, C., Piccarolo, C. and Calegari, L. (1996). Automated systems for identification of heterotrophic marine bacteria on the basis of their fatty acid composition. Appl Environ Microbiol 62, 2122–2132 Blanc, C., Sy, M., Djigal, D., Brauman, A., Normand, P. and Villenave, C., (2006). Nutrition on bacteria by bacterial-feeding nematodes and consequences on the structure of soil bacterial community. European Journal of Soil Biology 42, 70-78. Blaxter, M., 1998. Caenorhabditis elegans is a nematode. Science 282: 2041–2046 Cardinale, B.J., Palmer, M.A., Collins, S.L., (2002). Species diversity enhances ecosystem functioning through interspecific facilitation. Nature 415, 426-429. Chandler, G.T. and Fleeger, J.W., (1987). Facilitative and Inhibitory Interactions Among Estuarine Meiobenthic Harpacticoid Copepods. Ecology 68, 1906-1919. Conover, W.J., (1980). Practical nonparametric statistics. New York; John Wiley & Sons, Tablas, 493 p. Croll, N.A. and Sukhdeo, M.V.K., (1981). Hierarchies in nematode behavior. Plant Parasitic Nematodes 3, 227–250. De Mesel, I., Derycke, S., Swings, J., Vincx, M. and Moens, T., (2003). Influence of bacterivorous nematodes on the decomposition of cordgrass. Journal of Experimental Marine Biology and Ecology 296, 227-242.

229

De Mesel, I., Derycke, S., Moens, T., Van der Gucht, K., Vincx, M. and Swings, J., (2004). Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study. Environmental Microbiology 6, 733-744. De Mesel, I., Derycke, S., Swings, J., Vincx, M. and Moens, T., (2006). Role of nematodes in decomposition processes: Does within-trophic group diversity matter? Mar Ecol Prog Ser 321, 157-166. Dietrich, G. and Kalle, K., (1957). Allgemeine Meereskunde. Gebrüder Borntraeger, Berlin, 492. Gallucci, F., Steyaert, M. and Moens, T., (2005). Can field distributions of marine predacious nematodes be explained by sediment constraints on their foraging success? Marine Ecology Progress Series 304, 167-178. Grewal, P.S. and Wright, D.J., (1992). Migration of Caenorhabditis elegans (Nematoda: Rhabditidae) larvae towards bacteria and the nature of the bacterial stimulus. Fund. Appl. Nematol. 15, 159-166. Guillard, R.R.L. (1975). Culture of phytoplankton for feeding marine invertebrates. Culture of marine invertebrate animals. Plenum, 29–60. Heip, C., Vincx, M. and Vranken, G., (1985). The ecology of marine nematodes. Oceanography and Marine Biology 23, 399-489. Huettel, R.N. (1986). Chemical communicators in nematodes. J. Nematol 18, 3-8. Hulings, N.C. and Gray, J.S. (1971). A manual for the study of meiofauna. Washington, DC: Smithsonian University Press, pp. 84. Hutchinson, G.E., (1975). Variations on a theme by Robert MacArthur. Ecology and evolution of communities. Belknap, Harvard University Press, Cambridge, Massachusetts, USA, 492–521. Huys, G., Vancanneyt, M., Coopman, R., Janssen, P., Falsen, E., Altwegg, M. and Kersters, K.(1994). Cellular fatty acid composition as a chemotaxonomic marker for the differentiation of phenospecies and hybridization groups in the genus Aeromonas. International Journal of Systematic Bacteriology 44, 651-658. Ilieva-Makulec, K. (2001). A comparative study of the life strategies of two bacterial-feeding nematodes under laboratory conditions. II. Influence of

230

the initial food level on the population dynamics of Acrobeloides nanus (De Man, 1880) Anderson, 1968 and Dolichorhabditis dolichura (Schneider, 1866) Andrássy, 1983. Pol J Ecol 49:123–135 Johnstone, R.A., Reynolds, J.D. and Deutsch, J.C. (1996). Mutual Mate Choice and Sex Differences in Choosiness. Evolution 50, 1382-1391. Jonsson, M. and Malmqvist, B. (2003). Mechanisms behind positive diversity effects on ecosystem functioning: testing the facilitation and interference hypotheses. Oecologia 134: 554-559. Johnson, K.H., Vogt, K.A., Clark, H.J., Schmitz, O.J. and Vogt, D.J. (1996). Biodiversity and the productivity and stability of ecosystems. Trends Ecol. Evol., 11, 372–377. Levin, L.A., Boesch, D.F., Covich, A., Dahm, C., Erseus, C., Ewel, K.C., Kneib, R.T., Moldenke, A., Palmer, M.A. and Snelgrove, P. (2001). The function of marine critical transition zones and the importance of sediment biodiversity. Ecosystems (New York, NY. Print) 4, 430-451. Michiels, I.C. and Traunspurger, W. (2005). Benthic community patterns and the composition of feeding types and reproductive modes in freshwater nematodes. Nematology 7, 21-36. Moens, T. and Vincx, M. (1997). Observations on the feeding ecology of estuarine nematodes. Journal of the Marine Biological Association of the United Kingdom 77, 211-227. Moens, T. and Vincx, M. (1998). On the cultivation of free-living marine and estuarine nematodes. Helgoländer Meeresuntersuchungen 52, 115-139. Moens, T. (1999). Voedingsecologie van vrijlevende estuarine nematoden. Een experimentele benadering Ugent, Ghent, pp. 302. Moens, T., Verbeeck, L., de Maeyer, A., Swings, J. and Vincx, M. (1999). Selective attraction of bacterivorous nematodes to their bacterial food. Marine Ecology Progress Series, 176: 165-178 Moens, T. and Vincx, M. (2000). Temperature, salinity and food thresholds in two brackish-water bacterivorous nematode species: assessing niches from food absorption and respiration experiments. Journal of experimental marine biology and ecology 243 :137-154

231

Moens, T., Herman, P., Verbeeck, L., Steyaert, M. and Vincx, M., (2000). Predation rates and prey selectivity in two predacious estuarine nematode species. Marine Ecology Progress Series 205, 185-193. Moens, T., Luyten, C., Middelburg, J.J., Herman, P.M.J. and Vincx, M. (2002). Tracing organic matter sources of estuarine tidal flat nematodes with stable carbon isotopes. Marine Ecology Progress Series 234, 127-137. Moens, T., Bouillon, S. and Gallucci, F. (2005). Dual stable isotope abundances unravel trophic position of estuarine nematodes. Journal of the Marine Biological Association of the UK 85, 1401-1407. Mori, I. (1999). Genetics of chemotaxis and thermotaxis in the nematode Caenorhabditis elegans. Annual review of genetics 33, 399-422. Naeem, S. (1998). Species redundancy and ecosystem reliability. Conservation biology 12, 39-45. Nehring, S., Jensen, P. and Lorenzen, S. (1990). Tube-dwelling nematodes: tube-construction and possible ecological effects on the sediment-water interfaces. Marine Ecology Progress Series 64, 123-128. Neutel, A.M., Heesterbeek, J.A.P. and De Ruiter, P.C. (2002). Stability in Real Food Webs: Weak Links in Long Loops. Science (Washington) 296, 1120-1123. Newell, S.Y., Fallon, R.D. Cal Rodriguea, R.M and Groene, L.C (1985). Influence of rain, tidal wetting and relative humidity on release of carbon dioxide by standing-dead salt-marsh plants. Oecologia 68 :73-79 Newell, S.Y. (1996). Established and potential impacts of eukaryotic mycelial decomposers in marine/terrestrial ecotones. Journal of Experimental Marine Biology and Ecology 200, 187-206. Okuyama, T. and Bolker, B.M. (2007). On quantitative measures of indirect interactions. Ecol Lett 2007, 264-271. Paine, R.T. (1980). Food Webs: Linkage, Interaction Strength and Community Infrastructure. The Journal of Animal Ecology 49, 666-685. Postma-Blaauw, M.B., de Vries, F.T., de Goede, R.G.M., Bloem, J., Faber, J.H. and Brussaard, L. (2005). Within-trophic group interactions of bacterivorous nematode species and their effects on the bacterial community and nitrogen mineralization. Oecologia 142, 428-439.

232

Riemann, F. and Helmke, E. (2002). Symbiotic Relations of Sediment- Agglutinating Nematodes and Bacteria in Detrital Habitats: The Enzyme- Sharing Concept. Marine Ecology 23, 93-113. Riemann, F. and Schrage, M. (1978). The mucus-trap hypothesis on feeding of aquatic nematodes and implications for biodegradation and sediment texture. Oecologia 34 :75-88. Salinas, K.A., Edenborn, S.L., Sexstone, A.J. and Kotcon, J.B. (2007). Bacterial preferences of the bacterivorous soil nematode Cephalobus brevicauda (Cephalobidae): Effect of bacterial type and size. Pedobiologia 51, 55-64. Santos, G.A.P.dos, Moens, T., Puyvelde, E.V., Genevois, V.G.F. and Correia, M.T.S., in preparation. 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode species indirect interactions and their implications for population development. Santos, G.A.P.dos, Derycke, S., Genevois, V., Coelho, L., Correia, T. and Moens, T., submitted. Differential effects of food availability on population development and fitness of three species of estuarine, bacterial-feeding nematodes. Journal of Experimental Marine Biology and Ecology. Shtonda, B.B. and Avery, L. (2006). Dietary choice behavior in Caenorhabditis elegans. Journal of Experimental Biology 209, 89-102. Smith, W.O., Ainley, D.G. and Cattaneo-Vietti, R. (2007). Trophic interactions within the Ross Sea continental shelf ecosystem. Philosophical Transactions of the Royal Society B: Biological Sciences 362, 95-111. Sokal, R.R. and Rohlf, F.J. (1995) Biometry: The Principles and Practice of Statistics in Biological Research. Freeman, New York Tilman, D. (1996). Biodiversity: Population Versus Ecosystem Stability. Ecology 77, 350-363. Venette, R.C. and Ferris, H. (1998). Influence of bacterial type and density on population growth of bacterial-feeding nematodes. Soil Biology & Biochemistry 30, 949-960. Vetter, S., Fox, O., Ekschmitt, K. and Wolters, V. (2004). Limitations of faunal effects on soil carbon flow: density dependence, biotic regulation and mutual inhibition. Soil Biology & Biochemistry 36, 387-397.

233

Walker, B.H. (1992). Biodiversity and ecological redundancy. Conservation Biology 6, 18-23. Weigelt, A., Schumacher, J., Walther, T., Bartelheimer, M., Steinlein, T. and Beyschlag, W. (2007). Identifying mechanisms of competition in multispecies communities. Journal of Ecology 95, 53-64. Zhang, Y., Lu, H. and Bargmann, C.I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438, 179-184.

234

CChhaapptteerr 99::

TToopp--ddoowwnn eeffffeeccttss ooff aa pprreeddaattoorryy nneemmaattooddee oonn tthhee ppooppuullaattiioonn ddeevveellooppmmeenntt ooff ttwwoo pprreeyy nneemmaattooddeess ssppeecciieess..

235

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Abstract: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS Top-down effects of a predatory nematode on the population development of two prey nematodes species.

Giovanni A. P. dos Santos1,2,*, Maria T. S. Correia2, Luana. C. B. B. Coelho2,Verônica G. F. Genevois2, Tom Moens1.

1Marine Biology Section, UGent, Krijgslaan 281 (S8), 9000 Ghent, Belgium. 2Universidade Federal de Pernambuco, Av. professor Moraes Rêgo, s/n, 50.000.000. Recife, Pernambuco, Brazil.

* Corresponding author. E-mail: [email protected]

Abstract

Ecosystem stress factor causes changes in: species abundance, population dynamics and interaction between organisms. The top-down control plays a very important rule in ecosystem relations through direct predator prey interaction. Predators can drastically cause shifts in prey densities and species composition, moreover, predator removal causes pronounced ecosystem disturbance. The comparison of two cogeneric prey nematodes species (Diplolaimelloides meyli and Diplolaimelloides oschei) under Enoploides longispiculosus predation stress was done over 4 different predator densities. All predator treatments were compared to a control treatment without the stressor. Experiments were performed during 20 days after prey population establishment was completed. Top-down stress effects differently prey population development even in relatively low predator

236

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Abstract: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS densities. The age/size distributions are strongly predator-density dependent, and uncommonly effects some juvenile stages. Predatory nematodes are diverse and abundant in many ecosystems, our results suggest that they are important drivers of prey (i.e. nematodes) functional community structure and population densities.

Key words: Top-down, Ecological interactions, Predatory nematodes, Marine nematodes, Sex ratio, Enoploides longispiculosus, Diplolaimelloides meyli, Diplolaimelloides oschei, Population development, Predator prey interactions, Species function and Ecosystem process.

237

Introduction

A continuous purpose of studying population biology is to explain the spatiotemporal variations in abundance of organisms by understanding factors that cause changes in abundance, population dynamics and interactions between species. In general, species can interact through direct interactions such as predation and competition (Finke & Denno 2005, Abrams 1987, Abrams & Matsuda 1996, Cahill 1999, Hixon & Jones 2005), and through indirect interactions such as facilitation and inhibition (Menge 1995, Levine 1999, Santos et al. in prep.). The present study investigates potential top-down control through direct predator-prey interactions (PPI) on populations of bacterivorous nematodes. Top-down forces are a dominant ecological determinant of community biomass and structure (Hairston et al. 1960, Paine 1966, Hassell & May 1986, Murdoch & Briggs 1996). Predators can cause drastic shifts in prey densities and species composition, and vice versa, predator removal can cause pronounced ecosystem disturbance (Frank et al. 2005). PPI are often important in determining the stability of biodiversity and ecosystem function (Ives et al. 2005). Top-down controls are important in many marine communities (e.g. Ambrose 1984, Wilson 1990, Connolly & Roughgarden 1999, Menge 2000), where they have been particularly well-documented in relation to fisheries impacts (Cury et al. 2003, Chassot et al. 2005, Gascuel 2005), but also in terrestrial habitats (Branch & Griffiths 1988, Lubchenco & Gaines 1981). Nematodes are the most abundant and diverse metazoans in marine ecosystems (Heip et al., 1985), feeding on a range of different sources (Wieser 1953, Moens et al.1997). While many nematodes are considered bacterial- and microalgal feeders, there is a large variety of species which, judging from stoma morphology, are candidate predators of other meiobenthos, including nematodes, copepods, oligochaetes and ciliates, as well as settling larvae of larger organisms (Watzin 1983, 1985, Kennedy 1994, Moens & Vincx 1997, Hamels et al. 2001). A limited number of observational (Moens & Vincx 1997) and stable isotope studies (Moens et al.

238

2004) largely support the predatory ecology of various nematode genera abundant in intertidal and shallow subtidal sediments. Some short-term (from minutes to 48h) experiments have been performed on the predation rates of two marine (Moens et al. 2000, Gallucci et al. 2005) and several terrestrial nematode species (Nelmes 1974, Bilgrami & Jairajpuri 1989, Khan et al. 1991, Bilgrami & Gaugler 2005, for review see Khan & Kim (2007)), and have lead to suggestions of potentially significant top-down control of predatory nematodes over their prey populations. To our knowledge, however, there have been no studies investigating predation effects of predatory nematodes on prey nematode population dynamics apart from Mikola & Setälä (1998). In the present study we compare two different but closely related prey nematode species (Diplolaimelloides meyli and Diplolaimelloides oschei), which occur sympatrically on decomposing Spartina detritus in salt marsh sediments. These two bacterivorous species negatively affect each other’s population development (De Mesel et al. 2006, Santos et al. in prep). Although they typically live in different sediments than the well-studied predator genus Enoploides, they have been proven to be very suitable prey for this predator, Enoploides moreover exhibiting a preference for D. oschei over D. meyli. We performed microcosm experiments in which Enoploides were introduced into established populations of either prey species at 4 different predator densities and compared to a control treatment without predators. We aimed to assess (1) top-down effects of Enoploides on the population development and age/size distribution of both prey species, and (2) predator-density dependence of such top-down effects.

239

Materials and methods

Nematodes

The prey nematode species Diplolaimelloides meyli and D. oschei (Monhysteridae) were harvested from permanent monospecific, agnotobiotic cultures fed unidentified bacteria from their original habitat, i.e. decaying attached Spartina anglica leaves in the Paulina salt marsh in the polyhaline reach of the Westerschelde Estuary, SW Netherlands (Moens & Vincx 1998). Each prey species had been in culture for several years prior to the present experiment and under the same temperature (17 °C) and salinity (25) conditions. For the experiments, prey nematodes were handpicked on the tip of a fine Tungsten wire, rinsed twice in artificial seawater (ASW) to remove most attached bacteria, and stored in ASW for 24 h at 5 °C to allow fairly simultaneous inoculation into all experimental units. The predatory nematode species Enoploides longispiculosus was sampled non-quantitatively from an intertidal flat bordering the Paulina salt marsh upstream, by collecting the upper 2 cm of the sediment at low tide. Nematodes were extracted alive from freshly collected sediment by repeated decantation with tap water over a 63 µm mesh sieve. Nematodes retained on the sieve were collected with ASW and subsequently sorted at genus level under a compound microscope. They were stored overnight in ASW prior to their inoculation into the experimental microcosms. Only adult and fourth stage juvenile (J4) Enoploides were used for this experiment. A sufficient number of Enoploides were sorted from the sediment so that in addition to the animals inoculated into microcosms, 15 randomly picked specimens could be used for microscopical identification to species level after serial transfer through a graded series of ethanol:glycerin solutions and subsequent preparation of Cobb slides (De Grisse 1969, Cobb 1917).

240

Experiments

Microcosms consisted of Petri dishes (5 cm i.d.) bottom-covered with 4 ml of 0.75% bacto-agar medium with a salinity of 25 and a pH of 7.5-8. 50µl aliquots of frozen-and-thawed E. coli (Strain K12) at a density of 3 x 109 cells ml-1 were added as a food source for the prey nematodes, since previous experiments have demonstrated that this food source and density supports optimal population growth of both Diplolaimelloides species (Santos et al. sub.). Cholesterol was added to the agar medium at a concentration of 100µg l-1 because nematodes are incapable of producing some essential fatty acids on a purely bacterial diet (Vanfleteren 1980). Twenty adult prey specimens (10 males + 10 gravid females) of either species were transferred one by one to a 50 µl drop of sterile ASW on the agar surface in the experimental units. The microcosms were then incubated for 20 days to allow population establishment. Subsequently, predatory nematodes were added in much the same way as the prey specimens, in four different predator-density treatments: 5, 15, 25, and 40 predators per microcosm, respectively. A fifth treatment without predators served as control. Since occasional mortality of predators occurred during or after their inoculation into the microcosms, all dead predators encountered throughout the experiment were immediately replaced by new individuals. There were 3 replicates per treatment. Counts of total prey nematode numbers, early stage juveniles (J1-J2), late stage juveniles (J3-J4), adult males, adult females and dead prey were performed 5, 10, 15 and 20 days after inoculation of the predators.

Data Analysis

Prey variables were compared among treatments using repeated measures analysis of variance (ANOVA). No data transformation was needed to meet the assumptions of normality and homogeneity of variances. A posteriori pairwise comparisons used Tukey’s Honest Significant Differences (HSD)

241

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Mat. and Met.: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS test. Adult sex ratios were compared using time-averaged data per treatment and analysed with one-way ANOVA. All statistical analyses were performed using the Statistica 6 software (Statsoft).

242

Results

Diplolaimelloides oschei

Total prey nematode densities (including adults and all larval stages) differed highly significantly between treatments (F=37.34; p<0.0001) and between treatments vs time (F=9.80; p<0.0001). In the control (0 predators) treatment, we observed a monotonous increase over time until the 15th day after predator inoculation (i.e. day 35), followed by a clear population decline at the latest counting. Treatments with low predator densities (5 and 15) followed a similar population development but with significantly (p≤0.01) lower densities at all but the last counting. The highest predator density yielded significantly lower prey densities than all other treatments (p≤0.04), whereas the 25-predator treatment had an intermediate effect throughout the duration of the experiment (Fig. 1A, B).

3000

2800 2250 B 2600 A

2400 2000

2200 1750 2000

1800 1500 1600

Total Population Densite 1250

1400 Total Population Densite

1200 1000 D. oschey D.

1000 oschey D. A 800 750 B 600 0P 5P 500 C 400 15P 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment Figure 1: A: D. oschei total population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B: Predator density effects on total D. oschei densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Densities of fist and second stage juveniles (J1 and J2) differed highly significantly between treatments (F=15.48; p<0.001) and treatments vs time (F=5.12; p<0.0001), this difference being largely attributable to the treatment

243

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Mat. and Met.: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS with the highest predator density and emerging from the tenth day after predator inoculation (Fig. 2A, B).

1800 1200

1600 A B ) Densities ) Densities 2 2 1000 1400 and J and J 1 1200 1 800 1000

800 600 600 A 400 Ealy Stage Juveniles Stages (J Ealy Stage Juveniles Stages (J 400

200 B B 0P D. oschei D. 5P oschei D. C 0 15P 200 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment

Figure 2: A: D. oschei early stages juveniles (J1 and J2) population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B:

Predator density effects on early stages juveniles (J1 and J2) D. oschei densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Densities of third and fourth stage juveniles (J3 and J4) differed highly significantly between treatments (F=28.71; p<0.0001) and treatments vs time (F=9.88; p<0.0001). Controls had higher J3 and J4 densities throughout the experiment, even though they started decreasing after a peak at day 30 (i.e. 10 days after predator addition). J3 and J4 densities at the lower two predator densities remained stable until day 30 and then increased with a peak at 35 days, whereas at the higher two predator densities, there was a gradual but slow increase until 35 days. Overall, however, densities of J3 and J4 did not differ significantly between treatments with different predator densities (Fig. 3A, 3B).

244

1100 800 B 1000 A ) Densities ) Densities

4 700 4 900 and J and J 3 3 800 600 700

600 500 500

400 Late Stage Juveniles Stages (J Late Stage Juveniles Stages (J 400 A 300 0P B D. oschei D. D. oschei D. 5P 200 15P 300 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Times (days) Treatment

Figure 3: A: D. oschei late stages juveniles (J3 and J4) population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B:

Predator density effects on late stages juveniles (J3 and J4) D. oschei densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Adult D. oschei densities differed highly significantly between treatment (F=90.78; p<0.0001) and treatments vs time (F=16.53; p<0.0001). Densities of adults in the control treatment peaked 5 days after the start of the predation trials as a result of full maturation of F1-progeny, and declined at the last counting (which is 40 days after inoculation of the parent prey specimens) (see Santos et al. subm.). All other treatments yielded significantly lower adult densities throughout the experiment, and all pairwise comparisons except that between the highest two predator densities were significant (all p≤0.04) (Fig. 4A, B).

350 260 240 B 300 A 220

250 200 180 200 160 140 150 120

Adult Population Densite 100 100 Densite Population Adult 80 60

D. oschey D. 50 D. oschey D. 40 A B 0 20 C 0P 0 5P D -50 15P -20 20 25 30 35 40 25P 0P 5P 15P 25P 40P 40P Time (days) Treatment

245

Figure 4: A: D. oschei Adult population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B: Predator density effects on adult D. oschei densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Adult sex ratio was consistently female-biased and differed highly significantly between treatments (F=9.86; p=0.01), with the lowest percentages of females in the control and the five-predator treatments (Fig. 5).

62 Sex Ratio (%) 60

52 AABB A,B 50 0P 5P 15P 25P 40P Mean ±SE Treatment Figure 5: D. oschei adult sex ratio (expressed as percentage of female) for all different predator treatment. Data are mean percentage ± sterror of 3 replicates per treatment averaged over the total population from the 20th to the 40th day.

Diplolaimelloides meyli

Total population densities differed highly significantly between treatments (F=14.77; p<0.001) and between treatments vs time (F=5.99; p<0.0001). In the control treatment, densities increased monotonously until 15 days after the start of the predation trials and subsequently started declining. D. meyli densities in the controls exceeded those in all predator treatments throughout the experiment. Among the predator treatments, only that with the highest 246

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Mat. and Met.: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS predator density yielded significantly lower prey densities than the other treatments, except that with 25 predators per microcosm (Fig. 6A, B).

1800 2400

2200 A 1600 B 2000 1400 1800

1600 1200 1400

1200 1000

1000 Total Population Denities Population Total 800

Total Adult Population Densities Population Adult Total 800

600 meyli D. 600

D. meyli D. A 400 B 400 200 0P 5P C 0 15P 200 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment Figure 6: A: D. meyli total population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B: Predator density effects on total D. meyli densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Densities of fist and second stage juveniles (J1 and J2) differed highly significantly between treatments (F=7.46; p<0.01) and treatments vs time (F=4.67; p<0.0001), controls yielding consistently higher densities than all other treatments throughout. With the exception of the 5-predator treatment, there was general tendency of decreasing J1-J2 densities with increasing predator density, but only few pairwise differences proved significant (Fig. 7A, B).

1800 1200

1600 A B 1000 ) Densities ) Densities 2

1400 2 and J 1200 and J 1 1 800 1000

800 600 600

400 400 200 Ealy Stage Juveniles Stages (J Juveniles Stages Ealy Stage Ealy Stage Juveniles Stages (J Juveniles Stages Ealy Stage AA 0 0P 200 B D. meyli D. 5P meyli D. -200 15P 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment

Figure 7: A: D. meyli early stages juveniles (J1 and J2) population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B:

247

Predator density effects on for early stages juveniles (J1 and J2) D. meyli densities. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Densities of J3 and J4 differed highly significantly between treatments (F=21.40; p<0.0001) and treatments vs time (F=2.54; p<0.01). Control and 5- predator treatments yielded on average significantly higher J3-J4 densities than all other treatments (Fig. 8A, B).

550 360 500 A 340 B 450 320

300 400 280 350 260

300 240

220 250 200

200 180

160 A Late Stages Juvenile (J3 and J4) Densities 150 Late Stages Juvenile (J3 and J4) Densities 140 B 100 0P 120 5P 50 15P 100 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment

Figure 8: A: D. meyli late stages juveniles (J3 and J4) population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B: Predator density effects on for late stages juveniles (J3 and J4) D. meyli densities. Data are time- averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Densities of adult D. meyli differed highly significantly between treatments (F=64.23; p<0.0001) and treatments vs time (F=11.18; p<0.0001), increasing substantially on in the control treatment. Increasing predator densities yielded decreasing adult prey densities, most pairwise comparisons being significant (Fig. 9A, B).

248

300 260

A 240 B 250 220

200 200 120

150 100

80 Adult Densities 100 Densities Adult 60 D. meyli D. D. meyli D. 50 40 A 20 B 0 0 C 0P 5P D -50 15P -20 20 25 30 35 40 25P 0P 5P 15P 25P 40P Mean 40P ±SE Time (days) Treatment Figure 9: A: D. meyli Adult population development with time in four different predator treatments and a control without predators. Data are means of three replicates per treatment. B: Predator density effects on adult D. meyli densities, note Y-axis break. Data are time-averaged (from counts on days 25, 30, 35 and 40) means ± sterror of three replicates.

Adult sex Ratio was consistently female-biased and did not exhibit any significant differences among treatments with a slight decrease at higher densities (Fig. 10).

Sex Ratio (%) 60

50 0P 5P 15P 25P 40P Mean ±SE Treatment Figure 10: D. meyli adult sex ratio (expressed as percentage of female) for all different predator treatment. Data are mean percentage ± sterror of 3 replicates per treatment averaged over the total population from the 20th to the 40th day.

249

Discussion

There is a long history in ecological literature of studies on predator-prey interactions and top-down control over community assembly, structure, diversity, biomass and functioning, a.o. through trophic cascades (Hairston et al. 1960, Pace et al. 1999, Pinnegar et al. 2000, Charcraft & Resetaris 2003, Finke & Denno 2005, Ives et al. 2005). Top-down impacts on marine benthic communities have been tested in both predator inclusion and exclusion trials (Leber 1985, Fairweather & Underwood 1991 add review by Olafsson (2003)). While many meiofaunal taxa are considered predators, facultative predators or omnivores (Coull 1973), the importance of predation among meiobenthos is hitherto poorly known. There is a general lack of knowledge on feeding habits and even more so on feeding rates. Nematodes are typically by far the most abundant metazoan meiofauna in marine soft sediments, and their trophic position is usually inferred from stoma structure (Wieser 1953, Moens et al. 2004), even though observational studies have demonstrated discrepancies between mouth structure and feeding habits, as well as flexibility in feeding behaviour (Romeyn & Bouwman 1983, Moens & Vincx 1997). Natural stable isotope abundances of C and N may help to reveal food sources as well as trophic position of dominant nematode genera, but routine application of this methodology to nematodes is hampered by biomass requirements for isotopic analyses (Carman & Fry 2002, Moens et al. 2005). The only dual isotope study hitherto published which covered several estuarine nematode trophic groups, largely supported the predatory position of genera belonging to Wieser’s (1953) 2B group of predators/omnivores, or to the predators and facultative predators sensu Moens & Vincx (1997), and even suggested a trophic position for a genus for which this was not expected based on stoma morphology (Moens et al. 2005). There is thus clearly an abundance of nematode genera with at least partly predaceous feeding modes, and the question is whether their predation rates can be sufficient to exert top-down control over prey communities. High predation rates of predaceous nematodes on nematode prey have been

250

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Discussion: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS reported for several terrestrial and/or freshwater species, e.g.: Diplenteron colobocercus (Yeates 1969), Monochoides fortidens and Monochoides longicaudatus (Bilgrami & Jairajpuri 1989), Aporcelaimellus nivalis (Khan et al., 1991), Laimydorus baldus and Discolaimus major (Bilgrami & Gaugler 2005), and for the marine Enoploides longispiculosus and Adoncholaimus fuscus (Moens et al. 2000). With the exception of the work by Mikola & Setälä (1998a, b) on soil nematode communities comprising the predator Prionchulus punctatus, we are, however, unaware of any studies which have looked at population-level responses of prey nematodes to predation. Our data show a clear top-down effect of the predatory nematode genus Enoploides over total population development of both monhysterid prey species, in accordance with the clear negative correlation between densities of Enoploides and of other (prey) nematodes in sediments at the field site where Enoploides for our experiments were collected (Gallucci et al. 2005). The observed effect was smaller than what we could have expected based on the predation rates found in short-term experiments (Moens et al. 2000). There may be several reasons for this: (a) agar is not an optimal medium for Enoploides, which is very sensitive to the matrix in which it forages (Gallucci et al. 2005). However, in short-term experiments, predation rates on agar were very similar to those in sediments (Gallucci et al. 2005). Moreover, we observed virtually no predator mortality in our experiments and, for the first time, we obtained reproduction of Enoploides in our microcosms, all suggesting that our experimental conditions were fairly favourable. (b) Predators could have been partially feeding on bacterial food, as demonstrated for some predaceous nematodes from soil (Yeates et al. 1969, 1987). Moens et al. (1999) however, found no measurable bacteria uptake by Enoploides. (c) Rates of short-term experiments may yield overestimates as a result of pre-experimental conditions (e.g. starvation). However, we always used freshly collected and non-starved predators. (d) Predator functional responses (see below). (e) Finally, if perhaps some of the differences between different predator treatments were not very high, this may relate to negative density-dependent effects in the predator population, as observed in

251

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Discussion: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS short-term experiments with Enoploides feeding on nematode and ciliate prey (Hamels et al. 2001). Based upon per capita predation rates obtained in laboratory microcosm experiments, Moens et al. (2000) suggested that in fine- to medium-sandy sediments where Enoploides reaches high relative abundances, this predator either had to use additional feeding strategies or would be subject to starvation for a majority of the time under field conditions. These authors also contended that, based on a simple extrapolation of the observed per capita predation rates, Enoploides could drive prey populations to extinction. They did, however, mention that the low efficiency of predation at low prey densities provided a window of opportunity for rare prey species to avoid extinction. The functional response described for Enoploides feeding on monhysterid prey implies that this predator requires relatively high prey densities for optimal predation rates (Moens et al. 2000). The densities of adult prey in the present experiments were close to the optimal densities at the start of the experiment, but as a result of predation rapidly dropped far below those optimal densities. This would obviously result in lower than expected per capita predation rates, unless the abundant small juveniles are also suitable prey. Our study clearly shows that this is not the case, and thus provides a novel mechanism through which prey populations may avoid extinction even at very high predator densities: size-selective predation. Enoploides had little effect on small (J1 and J2) juveniles of both Diplolaimelloides species, except at the highest predator densities, and was only successful at catching older juveniles and, mainly, adults. We observed many encounters of predators with small juveniles, and these consistently remained without a predator-prey interaction. The lower time-averaged small- juvenile prey densities at the two highest predator densities did not result from direct predation on juveniles, but from a strong top-down control over densities of adults and hence over the rate of population increase. Size- selective feeding is a common strategy in marine environments (e.g. Micheli, 1995, Posch et al. 2001, Lehtiniemi & Lindén 2006), and is at the basis of the often inverted biomass-based trophic pyramids in aquatic habitats (Begon et al. 2005). Size is also a common food selection criterion in meiofaunal

252

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Discussion: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS organisms grazing bacteria (Salinas et al. 2007) or microphytobenthos (Moens & Vincx 1997, De Troch et al., 2006a,b). But while predatory nematodes tend to be comparatively large, several species are capable of catching prey larger than themselves (Moens & Vincx 1997), implying that trophic structure in (meio)benthic communities does not have to be reflected in size structure. Gallucci et al. (2005) further demonstrated that temporal (e.g. tide-induced) fluctuations in sediment water content, and spatial heterogeneity in sediment texture, provide prey with temporal and spatial refuges against predation. Size-selectivity, in combination with a strong functional response and negative density-dependent effects among the predators (see further), and with habitat structure, may thus allow prey populations to survive under conditions of high predator density. If so, we could expect that in sediments with high predator abundance, there would be many prey species occurring at very low adult densities. If small juvenile nematodes would more generally be less susceptible to predation by other nematodes, one could expect to find higher proportions of small juvenile prey nematodes in samples with a high relative abundance of predators. There is currently little evidence for this, but perhaps this is due to sampling bias, smaller juveniles having a high chance of passing through the mesh sizes typically used in meiobenthic research (de Bovée et al. 1974). Size-dependent predation was not considered an important driver for the prey selectivity observed in a previous study with Enoploides, which contended that encounter probabilities together with the capacity of prey species for vigorous (escape) movements were the dominant drivers behind selectivity (Moens et al. 2000). Indirectly, however, prey size may be generally more relevant because larger nematodes may tend to be more mobile and hence will have higher encounter probabilities. With respect to selective predation, short-term (24 – 48 h) incubations have shown a clear preference of Enoploides for D. oschei over it congener D. meyli (Gallucci et al. 2005). No substantial differences between predator effects on the adult populations of these two prey species were found in our experiment. However, knowing the preference of Enoploides for D. oschei over D. meyli, the results of the

253

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007. Santos, G.A.P. dos UNIVERSIDADE FEDERAL DE PERNAMBUCO Chapter 9: Discussion: Top-down effects of a predatory nematode… DOUTORADO EM CIÊNCIAS BIOLÓGICAS present experiments allow to formulate a double working hypothesis for a next experiment, in which Enoploides is introduced in microcosms where both Diplolaimelloides species occur in sympatry. We would then (a) expect that prey populations rapidly become dominated by D. meyli, but (b) we may hypothesize that selective predation will reduce the strength of the negative (competitive and/or inhibitory) interactions between both congeneric prey species (Santos et al. in prep. a; b). (c) This top-down structuring effect would likely be strongly linked with bottom-up controls, because these have been shown to have a major effect on the interaction between D. oschei and D. meyli. Hence, integration of predator-prey dynamics with resource effects on within-guild diversity and structure is likely to yield complex patterns of species assembly (Worm et al. 2002). A final point of interest from the present results pertains to predator effects on the sex ratio of its prey populations. Direct trophic relationship, e.g.: top-down interactions, between invertebrate are expected to shape the benthic community structure and reflect in changes of sex ratio with an increase in species richness (Michiels & Traunspurger 2005). Sex ratio in our prey populations was consistently female biased for all treatments and for both prey species, but with a somewhat different patter between the two prey species. High predator densities increased the female dominance in D. oschei, whereas there was a – statistically not significant – tendency towards the opposite trend in D. meyli. The results on D. oschei are consistent with the idea that males are more motile than females and have therefore a higher probability of encounter with the predators (Moens 1999). On the other hand, the larger Enoploides are more motile than their smaller monhysterid prey, implying that encounter probabilities are more determined by predator than by prey motility (Moens et al. 2000). An alternative explanation for the increased female bias under high levels of predation is that under top-down pressure from a size-selective predator incapable of catching small juveniles, it may be advantageous for a population to invest more in offspring (and hence in a higher proportion of females).

254

Conclusion

We conclude from the present study that even relatively low predator densities have significant effects on the population dynamics of nematode prey. These effects are strongly predator-density dependent, and the observed predation is size-selective. This size-selectivity, together with the predator’s functional response and negative density-dependent effects on predation, create possibilities for prey species to avoid going extinct even under high predation pressure. Since predatory nematodes are quite diverse and sometimes abundant in aquatic and soil systems, we suggest that they may be important drivers of nematode community structure and density. The present results on Enoploides allow to generate testable hypotheses for future research. However, it remains to be investigated whether other nematode predators have similar behavioural characteristics and hence, whether the results from this model system can be generalized for models including other predaceous nematode species.

255

Acknowledgements

256

References

Abrams PA (1987) Indirect interactions between species that share a predator: varieties of indirect effects. Predation: Direct and Indirect Impacts on Aquatic Communities:38–54 in W. C. Kerfoot and A. Sih, editors. Predation, direct and indirect impacts on aquatic communities. University Press of New England, Hanover, New Hampshire, USA. Abrams PA, Matsuda H (1996) Positive Indirect Effects Between Prey Species that Share Predators. Ecology 77:610-616 Ambrose RF (1984) Food preferences, prey availability, and the diet of Octopus bimaculatus Verrill. Journal of Experimental Marine Biology and Ecology 77:29-44 Begon M, Harper JL, Townsend CR (2005) Ecology: from individuals to ecosystems. Blackwell Publishing Bilgrami AL, Gaugler R (2005) Feeding behaviour of the predatory nematodes Laimydorus baldus and Discolaimus major (Nematoda: Dorylaimida). Nematology (Leiden) 7:11-20 Bilgrami AL, Jairajpuri MS (1989) Resistance of prey to predation and strike rate of the predators, Mononchoides longicaudatus and M. fortidens (Nematoda: Diplogasterida). Rev. Nematol. 12:45-49 Branch GM, Griffiths CL (1988) The Benguela ecosystem: 5. The coastal zone. Oceanography and Marine Biology 26:395-486 Cahill Jr JF (1999) Fertilization Effects on Interactions between above-and Belowground Competition in an Old Field. Ecology 80:466-480 Carman KR, Fry B (2002). Small-sample methods for δ13C and δ15N analysis of the diets of marsh meiofaunal species using natural- abundance and tracer-addition isotope techniques. Mar. Ecol. Prog. Ser. 240: 85-92. Chalcraft DR, Resetarits Jr WJ (2003) Predator identity and ecological impacts: Functional redundancy or functional diversity? Ecology 84:2407- 2418

257

Chassot E, Gascuel D, Colomb A (2005) Impact of trophic interactions on production functions and on the ecosystem response to fishing: a simulation approach. Aquatic Living Resources 18:1-13 Cobb NA (1917) Notes on Nemas. Waverly Press. Contrib. to a Science of Nematology V, pp 117-128. Connolly SR, Roughgarden J (1999) Theory of Marine Communities: Competition, Predation, and Recruitment-Dependent Interaction Strength. Ecological Monographs 69:277-296 Coull BC (1973) Estuarine meiofauna: a review: trophic relationships and microbial interactions. Estuarine microbial ecology 499-511 Cury P, Shannon L, Shin YJ (2003) The functioning of marine ecosystems: a fisheries perspective. Responsible Fisheries in the Marine Ecosystem,. Ed. by M. Sinclair, and G. Valdimarsson. CAB International, Wallingford. 103-123 de Bovee F, Soyer J, Albert P (1974) The Importance of the Mesh Size for the Extraction of the Muddy Bottom Meiofauna. Limnology and Oceanography 19:350-354 De Grisse AT (1969) Redescription ou modifications de quelques techniques utilisées dans l’étude des nématodes phytoparasitaires. Mededelingen Rijksfaculteit Landbouwwetenschappen Gent 34:315-359 De Mesel I, Derycke S, Swings J, Vincx M, Moens T (2006). Role of nematodes in decomposition processes: does within-trophic group diversity matter?. Marine Ecology Progress Series 321:157-166. De Troch M, Chepurnov V, Gheerardyn H., Vanreusel A, Ólafsson E (2006a). Is diatom size selection by harpacticoid copepods related to grazer body size?. Journal of Experimental Marine Biology and Ecology 332:1-11 De Troch M, Van Gansbeke D, Vincx M (2006b). Resource availibility and meiofauna in sediment of tropical seagrass beds: Local versus global trends. Marine Environmental Research 61:59-73. Olafsson E, (2003) Do macrofauna structure meiofauna assemblages in marine soft-bottoms? Vie Milieu 53:249-265

258

Fairweather PG, Underwood AJ (1991) Experimental removals of a rocky intertidal predator: Variations within two habitats in the effects on prey. Journal of Experimental Marine Biology and Ecology 154:29-75 Finke DL, Denno RF (2005) Predator diversity and the functioning of ecosystems: the role of intraguild predation in dampening trophic cascades. Ecol. Lett 8:1299–1306 Frank KT, Petrie B, Choi JS, Leggett WC (2005) Trophic Cascades in a Formerly Cod-Dominated Ecosystem. Science 308:1621-1623 Gallucci F, Steyaert M, Moens T (2005) Can field distributions of marine predacious nematodes be explained by sediment constraints on their foraging success? Marine Ecology Progress Series 304:167-178 Gascuel D (2005) The trophic-level based model: A theoretical approach of fishing effects on marine ecosystems. Ecological modelling 189:315-332 Hairston NG, Smith FE, Slobodkin LB (1960) Community Structure, Population Control, and Competition. The American Naturalist 94:421-425 Hamels I, Moens T, Muylaert K, Vyverman W (2001) Trophic interactions between ciliates and nematodes from an intertidal flat. Aquatic Microbial Ecology 26:61-72 Hassell MP, May RM (1986) Generalist and Specialist Natural Enemies in Insect Predator-Prey Interactions. The Journal of Animal Ecology 55:923- 940 Heip C, Vincx M, Vranken G (1985) The ecology of marine nematodes. Oceanography and Marine Biology 23:399-489 Hixon MA, Jones GP (2005) Competition, predation, and density-dependent mortality in demersal marine fishes. Ecology 86:2847-2859 Ives AR, Cardinale BJ, Snyder WE (2005) A synthesis of subdisciplines: predator-prey interactions, and biodiversity and ecosystem functioning. Ecology Letters 8:102-116 Kennedy AD (1994) Predation within meiofaunal communities: description and results of a rapid-freezing method of investigation. Mar. Ecol. Prog. Ser 114:71-79

259

Khan Z, Bilgrami AL, Jairajpuri MS (1991) Some observations on the predation ability of Aporcelaimellus nivalis (Altherr, 1952) Heyns 1966 (Nematoda: Dorylaimida). Nematologica 37:333–342 Khan Z, Kim YH (2007) A review on the role of predatory soil nematodes in the biological control of plant parasitic nematodes. Applied Soil Ecology 35:370-379 Leber KM (1985) The Influence of Predatory Decapods, Refuge, and Microhabitat Selection on Seagrass Communities. Ecology 66:1951-1964 Lehtiniemi M, Linden E (2006). Cercopagis pengoi and Mysisspp. Alter their feeding rate and prey selection under predation risk of herring (Clupea harengus membras). Mar. Biol.149: 845–854. Levine JM (1999) Indirect Facilitation: Evidence and Predictions from a Riparian Community. Ecology 80:1762-1769 Lubchenco J, Gaines SD (1981) A Unified Approach to Marine Plant- Herbivore Interactions. I. Populations and Communities. Annual Review of Ecology and Systematics 12:405-437 Menge BA (1995) Indirect Effects in Marine Rocky Intertidal Interaction Webs: Patterns and Importance. Ecological Monographs 65:21-74 Menge BA (2000) Top-down and bottom-up community regulation in marine rocky intertidal habitats. Journal of Experimental Marine Biology and Ecology 250:257-289 Micheli F (1995) Behavioural plasticity in prey-Size selectivity of the Blue crab Callinectes sapidus feeding on bivalve prey. The Journal of Animal Ecology 64:63-74 Michiels IC, Traunspurger W (2005) Benthic community patterns and the composition of feeding types and reproductive modes in freshwater nematodes. Nematology 7:21-36 Mikola J, Setala H (1998a) Relating Species Diversity to Ecosystem Functioning: Mechanistic Backgrounds and Experimental Approach with a Decomposer Food Web. Oikos 83:180-194 Mikola J, Setala H (1998b). No evidence of trophic cascades in an experimental microbial-based soil food web. Ecology 79:153 -164

260

Moens T (1999) Voedingsecologie van vrijlevende estuarine nematoden. Een experimentele benadering In. Ugent, Ghent, p 302 Moens T, Bouillon S, Gallucci F (2005) Dual stable isotope abundances unravel trophic position of estuarine nematodes. Journal of the Marine Biological Association of the UK 85:1401-1407 Moens T, Herman P, Verbeeck L, Steyaert M, Vincx M (2000) Predation rates and prey selectivity in two predacious estuarine nematode species. Marine Ecology Progress Series 205:185-193 Moens T, Vincx M. (1998). On the cultivation of free-living marine and estuarine nematodes. Helgoländer Meerseunters. 52: 115-139 Moens T, Vincx M (1997) Observations on the feeding ecology of estuarine nematodes. Journal of the Marine Biological Association of the United Kingdom 77:211-227 Moens T, Yeates GW, De Ley P (2004). Use of carbon and energy sources by nematodes. Nematology Monographs and Perspectives - Proceedings of the fourth International Congress of Nematology, 8-13 June 2002, Tenerife, SpainVolume 2:529-545 Murdoch WW, Briggs CJ (1996) Theory for Biological Control: Recent Developments. Ecology 77:2001-2013 Nelmes AJ (1974) Evaluation of the feeding behaviour of Prionchulus punctatus (Cobb), a nematode predator. The Journal of Animal Ecology 43:553-565 Pace ML, Cole JJ, Carpenter SR, Kitchell JF (1999) Trophic cascades revealed in diverse ecosystems. Trends in Ecology & Evolution 14:483- 488 Paine RT (1966) Food Web complexity and species diversity. The American Naturalist 100:65-75 Pinnegar JK et al. (2002) Trophic cascades in benthic marine ecosystems: lessons for fisheries and protected-area management. Environmental Conservation 27:179-200 Posch T et al. (2001) Size Selective Feeding in Cyclidium glaucoma (Ciliophora, Scuticociliatida) and Its Effects on Bacterial Community

261

Structure: A Study from a Continuous Cultivation System. Microbial Ecology 42:217-227 Romeyn K, Bouwman LA (1983) Food selection and consumption by estuqrine nematodes. Hydrobiological Bulletin 17:103-109 Salinas K, Edenborn S, Sexstone A, Kotcon J (2007) Bacterial preference of the bacterivorous soil nematode Cephalobus brevicauda (Cephalobidae): Effect of bacterial type and size. Pedobiologia 51:55-64 Santos GAPdos, Moens T, Puyvelde EV, Genevois, VGF, Correia MTS, in preparation. 1 + 1 + 1 = or ≠ 3 ? Three bacterial-feeding nematode species indirect interactions and their implications for population development. Santos GAPdos, Derycke S, Genevois V, Coelho L, Correia T, Moens T (submitted) Differential effects of food availability on population development and fitness of three species of estuarine, bacterial-feeding nematodes. Journal of Experimental Marine Biology and Ecology Vanfleteren JR (1980) Nematodes as nutritional models. Nematodes as Biological Models 2:47–79 Watzin MC (1983) The effects of meiofauna on settling macrofauna: meiofauna may structure macrofaunal communities. Oecologia 59:163- 166 Watzin MC (1985) Interactions among temporary and permanent meiofauna: Observations on the feeding and behavior of selected taxa. Biological Bulletin, Marine Biological Laboratory, Woods Hole 169:397-416 Wieser W (1953) Die Beziehung zwischen Mundhöhlengestalt, Ernährungsweise und Vorkommen bei freilebenden marinen Nematoden. Ark. Zool 4:439-484 Wilson WH (1990) Competition and Predation in Marine Soft-Sediment Communities. Annual Review of Ecology and Systematics 21:221-241 Worm B, Lotze HK, Hillebrand H, Sommer U (2002) Consumer versus resource control of species diversity and ecosystem functioning. Communities 82:3295-3308 Yeates G.W. (1969). Three new Rhabditida (Nematoda) from New Zealand sand dunes. Nematologica 15: 115-128

262

Yeates GW (1987) Nematode feeding and activity: the importance of development stages. Biology and Fertility of Soils 3:143-146

263

TÍTULO: Species assembly rules and the biodiversity-ecosystem functioning relationship CCB, Recife-PE, 2007.