UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS

Cephalopods as vectors of harmful algal bloom toxins: a toxicokinetic and ecophysiological approach

“Documento Definitivo”

Doutoramento em Ciências do Mar

Vanessa Sofia Madeira Lopes

Tese orientada por: Professor Doutor Rui Afonso Bairrão da Rosa Doutor Pedro José Conde Reis Costa

Documento especialmente elaborado para a obtenção do grau de doutor

2018

UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS

Cephalopods as vectors of harmful algal bloom toxins: a toxicokinetic and ecophysiological approach

Doutoramento em Ciências do Mar

Vanessa Sofia Madeira Lopes

Tese orientada por: Professor Doutor Rui Afonso Bairrão da Rosa Doutor Pedro José Conde Reis Costa

Júri: Presidente: ● Doutora Maria Manuela Gomes Coelho de Noronha Trancoso, Professora Catedrática e Presidente do Departamento de Biologia Animal da Faculdade de Ciências da Universidade de Lisboa Vogais: ● Doutor Alexandre Marnoto de Oliveira Campos, Investigador Auxiliar no Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR) da Universidade do Porto ● Doutor Mário Emanuel Campos de Sousa Diniz, Professor Auxiliar da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa ● Doutor João Manuel de Figueiredo Pereira, Investigador Auxiliar do Instituto Português do Mar e da Atmosfera (IPMA) ● Doutora Ana de Jesus Branco de Melo de Amorim Ferreira, Professora Auxiliar da Faculdade de Ciências da Universidade de Lisboa ● Doutor Rui Afonso Bairrão da Rosa, Investigador FCT de nível de desenvolvimento da Faculdade de Ciências da Universidade de Lisboa

Documento especialmente elaborado para a obtenção do grau de doutor

Fundação para a Ciência e Tecnologia (FCT)

2018

Agradecimentos

Ter chegado a este momento não foi certamente apenas trabalho meu. Devo agradecimentos incondicionais a tantas pessoas, e não só!

A palavra agradecimento não chega para expressar a minha profunda gratidão ao meu orientador e “mestre”, Doutor Rui Rosa. Obrigada por estes 8 anos, por teres acreditado no meu potencial, por ainda me contagiares com o “bichinho” da ciência, por nunca te acabarem as ideias sobre o que fazer a seguir, por todos os conselhos, ensinamentos e desabafos. Por me inspirares a querer ser mais e melhor, por mostrares o que é ser mais e melhor, pela jovialidade, pelos bons exemplos, pelos “raspanetes” merecidos, por seres mais do que cabe em ti. Ao Doutor Pedro Costa, que não me conhecia de lado nenhum e que me aceitou como sua aluna. Por me teres mostrado o que este mundo da toxicologia tem para oferecer, por toda a motivação e conselhos que me deste, por seres sempre bem- disposto e aceitares todas as ideias que propus. Por me teres ensinado e guiado para que fosse autossuficiente.

Aos meus polvos, por mais vezes que me tenham fugido dos aquários e por mais dores de cabeça que me tenham proporcionado, encheram-me o coração naqueles dias menos bons, fizeram-me ver que há todo um mundo novo e inexplorado, são uma fonte inesgotável de conhecimento e inspiração. Por isso, fizeram-me querer ser melhor para os conhecer mais um pouco, ir um pouco mais além e fascinaram-me todos os dias com aqueles olhos lindos e fugas impossíveis! Obrigada por me deixarem entrar no vosso mundo.

Agradecer ao Laboratório Marítimo da Guia inclui também agradecer ao próprio edifício, por conter conhecimento centenário, por inspirar qualquer pessoa que lá entre, como aconteceu comigo, há “tantos” anos atrás. Foi o sítio que escolhi como segunda casa e no qual passei incontáveis momentos, bons e maus, como tudo na vida. Espero aqui continuar, por muitos anos.

Quatro anos é muito tempo, e muitas pessoas cruzaram a sua estadia na Guia comigo. Aos alunos de Erasmus que estiveram pouco tempo, mas ficaram tempo suficiente para deixar a sua marca. À “croatian woman” mais espectacular, Meri, por teres sido uma companhia tão presente, por teres entrado na minha família do coração, lindezas! Às minhas “awesome” espanholitas, Sara e Lídia, por terem sido tão preciosas, divertidas e por todos os momentos que passámos. To the “little Italian” Luanni, for all the insane car rides and countless laughs!

À Matilde Costa, a moça da mochila amarela, por me ter salvo a sanidade e ter incorporado uma parte considerável do trabalho em 2015, por partilhar o gosto dos polvinhos comigo.

À Catarina Vara, que foi um presente caído do céu, que veio para estudar imunologia e acabou por ser raptada para o mundo da toxicologia de polvos. Por toda a competência, desenrasque, mangas arregaçadas, alegria, motivação, ajuda e incentivo, por me fazer ter saudades dela quando se for embora, por estes últimos meses, tão críticos!

No IPMA, agradecimentos são devidos à Sara Costa e a todos do Laboratório das Biotoxinas Marinhas, por todas as vezes que perguntei onde estavam coisas e pacientemente me ajudaram.

Agora os agradecimentos aos residentes do “meu” forte à beira-mar. Toda a equipa com quem vivi o meu dia-a-dia, com quem desabafei os males e celebrei as alegrias. À “minha” Catarina Santos, Cat, por me mostrar tantas vezes o lado cor de rosa da vida, pela voz doce e pela insanidade inata. Nunca a percas, por favor. Por ter sido sempre uma presença constante no meu percurso e por todas as gargalhadas descontroladas e viagens de ida e volta que nunca mais foram as mesmas. Por seres tão real, que é desarmante, obrigada. Ao Vasco, Baskins, que chegou tão calmamente, mas que mudou os dias na Guia, com uma presença tão positiva que tornou todos à sua volta felizes, o mundo precisa de mais Vascos.

Ao Francisco Carvalho, por seres a alegria de viver em pessoa. Por todos os momentos parvos partilhados, por estes anos de coexistência, que tornaram a minha existência mais feliz! Ao Francisco Borges, um pequeno génio, de aparência séria, mas que no fundo é tão louco como os demais. Obrigada pelas viagens, foram mais leves contigo por perto.

Ao Zé, que é um investigador brilhante, e que é como um miúdo numa loja de brinquedos, de olhos a brilhar com o mundo científico. Obrigada pela motivação e encorajamento, és um exemplo a seguir!

À Cátia e Tiago Grilo, que contribuem tanto para a alegria generalizada naquele laboratório. Por terem sempre conversas interessantes e por toda a genuinidade!

A todos os alunos de Mestrado neste momento na Guia, que tornam aquele espaço num sítio mais feliz, que nos renovam e nos tornam mais sábios, que permitem que observe o vosso percurso e que me orgulhe nos vossos sucessos, Cláudia, Érica, Ricardo.

A todos os outros membros do nosso grupo na Guia, Maria Rita (Maryland) obrigada pela tua simplicidade, genuinidade, e nossa partilha de memes e expressões tolas; Marta, que bom que as condições se proporcionaram para nos aproximarmos, sou mais feliz assim; Ricardo Cyrne, obrigada por todos os contributos para a boa disposição, Miguel e Tiago Repolho por terem estado lá no início e por saber que posso sempre contar convosco.

Ao Eduardo, por ser eu meu mano cefalopódico, por termos o mesmo tipo de humor e as mesmas horas de almoço. Por todas as correcções preciosas que fizeste, por teres tomado tanto do teu tempo, tão pacientemente e bem-disposto para me ajudar no último trabalho desta dissertação. Por me tranquilizares e ajudares a que tudo tivesse corrido bem, por todo o companheirismo e pela nossa honestidade um com o outro.

À Rita, a “minha” Pipoks, que percorreu este caminho comigo, que partilhou as alegrias e momentos menos bons, em quem me pude apoiar tantas vezes, que me ajudou com os prazos e burocracias, senão se calhar teria falhado algum prazo importante e não estaria aqui. Obrigada por teres sido como uma irmã para mim, através de todas as adversidades que passei, estamos quase!!

Ao Sr. Ramos, pois sem ele, conseguir os meus polvinhos teria sido uma tarefa muito complicada, obrigada por poder contar consigo há tanto tempo.

A ambas as instituições de acolhimento que tornaram possível a execução desta dissertação, Marine Environmental Sciences Centre (MARE – ULisboa) e Instituto Português do Mar e Atmosfera. À Fundação para a Ciência e Tecnologia (FCT) pelo financiamento da minha bolsa de Doutoramento (SFRH/BD/97633/2013).

À minha família, que me permitiu sempre sonhar mais alto, não encaixar no molde e ser eu própria. Por todas as vezes que tiveram curiosidade sobre o que estava a fazer, aqui está o resultado! Obrigada!

Ao Jorge, por teres aparecido tão inesperadamente na minha vida e teres mudado o meu mundo para melhor. Por todos os imensos incentivos que me deste, por todas as vezes que me ajudaste a “digerir” resultados, desenvolver o meu sentido crítico e pensar mais além. Por te orgulhares de todos os meus sucessos, pequenos e grandes. Por todo o amor incondicional e compreensão, por partilhares comigo esta experiência.

Às duas pessoas que foram a maior parte da minha vida, que sem as quais esta dissertação teria sido feita pois não estaria no percurso em que estou. Ao menino que vi nascer e que já está mais alto que eu, que orgulho imenso em ti. Obrigada por veres em mim um exemplo e por me teres feito crescer, por me perdoares as minhas falhas. Obrigada Mô. À pessoa mais forte que conheço, e que tenho a certeza que há poucas como tu, se é que há. Sem dúvida que sou o que sou hoje quase exclusivamente por tua causa. Por tornares possível eu enveredar por este caminho que é mais difícil do que fácil, pelo orgulho que sentes por mim, por perceberes quase tanto de cefalópodes como eu. Por todos os dias que fui menos do que podia ser, teres sido sempre tão tu. Esta dissertação também é tua. Obrigada Mamma.

TABLE OF CONTENTS

List of abbreviations and units 1 List of figures 5 List of tables 9 Abstract and Keywords 13 Resumo e palavras-chave 16 Resumo alargado 18 List of papers 25

Chapter One General Introduction 29 1.1. Overview on Harmful Algal 31 Blooms 1.2. Routes of toxin exposure 32 1.3. Paralytic shellfish toxins 34 1.4. Amnesic shellfish toxins 35 1.5. HAB-toxin effects on marine 36 organisms 1.5.1. Paralytic shellfish 36 toxins 1.5.2. Domoic acid 46 1.6. Thesis objectives and outline 49 1.7. References 50

Chapter Two Cephalopods as vectors of harmful algal bloom toxins in 69 marine food webs

Chapter Three Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris). 99

Chapter Four Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains. 133

Chapter Five Feeding behaviour and chromatophore activity of octopus paralarvae are not affected by the harmful algal toxin domoic acid. 156

Chapter Six General discussion and final considerations. 186

LIST OF ABBREVIATIONS AND UNITS

α Net accumulation effiency AIC Akaike information criterion AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA Analysis of variance ASP Amnesic shellfish poisoning B1 Gonyautoxin 5, also designated as GTX5 B2 Gonyautoxin 6, also designated as GTX6 BrH/BH Branchial hearts BW Body weight C1+2 N-sulfocarbamoyl-gonyautoxin 1 and 2 Ca2+ Calcium

CDG Toxin concentration in digestive gland (DG)

Cfeed Toxin concentration in food

Ci Toxin concentration in the i-th compartment CNS Central nervous system

CV Toxin concentration in octopus viscera DA Domoic acid dcNEO Decarbamoyl neosaxitoxin dcSTX Decarbamoyl saxitoxin DG Digestive gland DgT Digestive tract DSP Diarrhetic shellfish poisoning F Feeding rate Gill/GL Gills GLM Generalized linear model Gon Gonad GPx Glutathione Peroxidase GST Glutathione-S-transferase GTX2+3 Gonyautoxin 2 and 3 GTX5 Gonyautoxin 5, also designated as B1 GTX 6 Gonyautoxin 6, also designated as B2 HAB Harmful algal bloom HPLC-FLD High-performance liquid chromatography with

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fluorescence detection IC Intracoelomic IP Intraperitoneal IPMA Instituto Português do Mar e Atmosfera k el V Elimination rate from viscera kel DG Elimination rate from digestive gland kel i Elimination rate from i-th compartment Kid/KD Kidney Transfer coefficient from digestive gland to KT i the i-th compartment LC-MS/MS Liquid chromatography with mass spectrometry detection LTP Long-term potentiation Man/MT Mantle mDG Wet mass of digestive gland mKD Wet mass of kidney ML Mantle length MRI Magnetic resonance imaging MS Maturity stage N North n Number of individuals nd Not detected NEO Neosaxitoxin NMDA N-methyl-D-aspartate NW Northwest OCI Occurrence index PSP Paralytic shellfish poisoning PST Paralytic shellfish toxin p-value Probability of statistic test Toxin concentration at the initial conditions q0 of the depuration period Toxin concentration in digestive gland at qDG initial conditions of depuration Toxin concentration in the i-th compartment at qi initial conditions of depuration Toxin concentration in kidney at initial qKD conditions of depuration

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ROS Reactive oxygen species Sal/SaG/SG posterior salivary glands SD Standard deviation SE Southeast SPE Solid phase extraction SPM Suspended particulate matter St Stomach St+Inst+Ce Pooled stomach, caecum and intestine STX Saxitoxin STX eq Saxitoxin equivalents SW Southwest t Time UOM Unidentified organic matter V Volt(s) W West WW Wet weight

% Percentage ± Approximately °C Degrees Celsius µg Microgram(s) µl Microliter(s) µm Micrometer(s) cm Centimeter(s) g Gram(s) h Hour(s) kg Kilogram(s) L Liter(s) mg Miligram(s) ml Mililiter(s) ng Nanogram(s) nm Nanometer(s) pg Picogram(s)

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LIST OF FIGURES

CHAPTER ONE – General introduction

Figure 1. Foodborne and waterborne exposure to HAB-toxins. Solid lines illustrate the well documented route of dietary exposure; dashed lines illustrate the less studied routes of dissolved toxins exposure.

Figure 2. Schematic view of HAB-toxins food web transfer.

CHAPTER TWO – Cephalopods as vectors of harmful algal bloom toxins in marine food webs

Figure 1. Weight (%W) of prey in the stomach contents of jumbo squid (Dosidicus gigas) from the Gulf of California during 1998–

1999 (data from Markaida and Sosa-Nishizaki, 2003). %W is defined as the weight of a certain prey relative to the total weight of all prey, expressed as a percentage. Legend: UOM - unidentified organic matter.

Figure 2. Cephalopod coastal diversity and respective life strategies. A, B and C - nektobenthic common cuttlefish, Sepia officinalis; D - benthic common octopus, Octopus vulgaris, E and

F - benthic sepiolid, Sepiola atlantica, G – semi-pelagic squid,

Loligo vulgaris; H and I – planktonic paralarvae of common octopus, O. vulgaris. (photo credits: Rui Rosa).

Figure 3. Total concentration (median, 25 and 75 quartiles, non- outlier range and outliers) of paralytic shellfish toxins (PSTs,

µg STX equiv. kg-1) in the tissues of common octopus, Octopus vulgaris (data from Monteiro and Costa, 2011) collected in the

NW Portuguese coast. Abbreviations: DG- digestive gland; BrH –

- 4 - branchial hearts; Gill – Gills; Kid – kidney; St+Inst+Ce – pooled stomach, caecum and intestine; Sal - posterior salivary glands.

Figure 4. Concentration of paralytic shellfish toxins (C1 + 2 N- sulfocarbamoyl-gonyautoxin-2 and -3; dcSTX dicarbamoyl saxitoxin; GTX2 + 3 gonyautoxin-2 and -3, B1 gonyautoxin-5 or

GTX5; STX saxitoxin; NEO neosaxitoxin) in several tissues of common octopus, Octopus vulgaris (data from Monteiro and Costa,

2011) collected from the NW Portuguese coast. Abbreviations: DG- digestive gland; BrH – branchial hearts; Gill – Gills; Kid – kidney; St+Inst+Ce – pooled stomach, caecum and intestine; Sal - posterior salivary glands.

Figure 5. Concentrations of paralytic shellfish toxins detected in Humboldt squids (Dosidicus gigas) from stranding events in

British Columbia, Canada. *STX eq. = STX + dcSTX*0.51 (data from

Braid et al. 2012). Abbreviations: DG – digestive gland; St – stomach; Man – mantle.

Figure 6. Schematic illustration of the maximum levels (µg g-1) of amnesic shellfish toxin, domoic acid (DA) found in common octopus (Octopus vulgaris) tissues.

Figure 7. Domoic acid levels (DA, µg g-1; median, 25 and 75 quartiles, non-outlier range and outliers) detected in the tissues of: A) common octopus (Octopus vulgaris), collected in the NW and South Portuguese coast, B) common cuttlefish (Sepia officinalis), collected in the NW Portuguese coast, and C)

Humboldt squid (Dosidicus gigas) collected in British Columbia,

Canada (data from Braid et al., 2012; Costa et al., 2004; Costa et al., 2005b).

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Figure 8. Domoic acid distribution in the DG cell fractions of the common octopus (Octopus vulgaris; median, 25 and 75 quartiles, non-outlier range and outliers) collected in the NW

Portuguese coast (data from Lage et al., 2012).

Figure 9. Domoic acid levels (DA; μg kg−1; median, 25 and 75 quartiles, non-outlier range and outliers) detected in the DG of

Eledone cirrhosa (from NW, SW and South Portuguese coast) and E. moschata (from South Portuguese coast) (data from Costa et al.,

2005a).

Figure 10. Stranded Humboldt squids (Dosidicus gigas) in

Californian (top panels, and bottom left and middle panels) and

Mexican coasts (bottom right panel).

CHAPTER THREE - Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris)

Figure 1. Schematic view for the five compartments of Octopus vulgaris. The solid lines represent the transfer coefficients from the digestive gland to the other compartments. The dashed lines represent the elimination rates of the five compartments.

The other parameters used are described in Table 2.

Figure 2 Concentration (µg g-1) of (a) GTX5, (b) dcSTX, (c) C

1+2, (d) dcGTX2+3, (e) GTX2+3 and (f) STX in octopus digestive gland throughout the experiment. Dots and error bars represent experimental data (mean based on three replicate samples). The dashed line represents the outputs of the best fit model.

Figure 3 Concentration (µg g-1) of (a) GTX5, (b) dcSTX and (c)

C1+2 in octopus kidney throughout the experiment. The dashed line represents the outputs of the best fit model.

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CHAPTER FOUR - Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains

Figure 1. Domoic acid (DA) concentration in mg kg-1 in octopus brain tissue (A), digestive gland (B) throughout the sample period, and Pseudonitzschia sp. abundance (C) between January and October 2016.

CHAPTER FIVE - Feeding behaviour and chromatophore activity of octopus paralarvae are not affected by the harmful algal toxin domoic acid

Figure 1. Box plot showing data distribution (median, 25 and 75 quartiles, non-outlier range and outliers) in all treatments used as a function of time. (A) The number of paralarval mantle contractions (jets, n=9 per each treatment at each sampling point) (B) the number of Artemia consumed (n= T1: 20; T4: 18;

T8: 16; T24: 14) (C) paralarva dorsal area covered by chromatophores, expressed in percentage (n=6 per each treatment at each sampling point).

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LIST OF TABLES

CHAPTER ONE – General introduction

Table 1. The most common toxins produced by marine phytoplankton.

Table 2. Documented cases of bivalves exposed to and affected by paralytic shellfish toxins (PSTs).

CHAPTER TWO – Cephalopods as vectors of harmful algal bloom toxins in marine food webs

Table 1. Diet of common octopus (Octopus vulgaris) in the

Portuguese coast, namely in Viana do Castelo (North region),

Cascais (Centre region) and Tavira (South region). Occurrence index (OCI) of prey found in the octopods’ stomach contents.

[data from Rosa et al. (2004)].

Table 2. Maximum levels of HAB-associated toxins in cephalopods.

CHAPTER THREE - Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris)

Table 1. Toxin profile of Donax clams given to octopus (mean ±

SD).

Table 2. Parameters used in kinetics equations.

Table 3. Net accumulation efficiency (α, %), initial concentration in digestive gland at beginning of depuration

-1 (qDG), elimination rate (kel DG d ) (standard deviation) and coefficient of determination R2 for each PST determined in octopus digestive gland during uptake and depuration. Asterisks indicate values within the confidence limit (P < 0.05).

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Table 4. Toxin transfer rate from digestive gland to kidney (KT.

-1 -1 d ), elimination rate (kel KD, d ) (standard deviation) and coefficient of determination R2 for each PST determined in octopus kidney during uptake.

Table 5. Toxin concentration at initial depuration conditions

-1 (qo), elimination rate (kel V, d ) (standard deviation) and coefficient of determination R2 for each PST determined following one compartment model, asterisks indicate values within the confidence limit (P < 0.05).

CHAPTER FOUR - Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains

Table 1. Domoic acid concentrations (mg DA kg-1) in the brain and digestive glands (DG) of other cephalopod species. (nd – not detected).

Table 2. Spearman’s rank order correlations between total weight (g), mantle length (ML, mm), gender, maturity stage (MS), domoic acid concentrations (mg DA kg-1) in the brain and digestive glands (DG) of Octopus vulgaris. Marked correlations in bold with asterisks are significant at p<0.05.

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ABSTRACT

Harmful algal blooms (HABs) are natural occurrences that can severely impact coastal marine food webs. Depending on the species, they can produce a wide array of toxins, which may elicit devastating effects on marine life. Paralytic shellfish toxins (PSTs) and domoic acid (DA) are the most common neurotoxins occuring along the Portuguese coast. It is established that cephalopods accumulate these water-soluble toxins in their tissues, mostly in the digestive gland, through ingestion of contaminated prey. Here, the uptake and transfer kinetics of PSTs between octopus (Octopus vulgaris) body tissues is described. It was shown that octopuses present low toxin conversion (regarding toxin concentrations between the contaminated prey and cephalopod predator), transference between tissues and elimination rates. The uptake period was better characterized using an exponential growth model, suggesting swift biotoxin uptake. PSTs were found to be present in kidney tissue at much lower concentration than in the digestive gland. Throughout the experimental period (16 days) the specimens did not display outward signs of intoxication after ingestion of high dosages of a paralytic toxin (up to 1249.2 µgSTX eq kg-1 at the end of the uptake period). In order to determine if HAB related neurotoxins reach cephalopod’s central nervous system, wild octopuses and cuttlefish were collected and brain tissue was analysed for DA. This neurotoxin causes amnesic shellfish poisoning in vertebrates and it is known to accumulate in cephalopods’ digestive gland. Here, it is also shown that octopus and cuttlefish accumulate this potent toxin in their brain tissue for at least four months (in octopus), suggesting that they can selectively retain and possibly tolerate this toxin. Concomitantly, there was no information on the effects of DA at cephalopod’s early stages of development when exposed to dissolved toxins in seawater. For that, 2-day-old paralarvae were exposed to ecologically relevant DA concentrations (150 µg DA L-1), and activity patterns, feeding behaviour and chromatophore activity was determined. These results show that DA did not impair or alter the feeding behaviour or

- 12 - chromatophore activity of octopus paralarvae. In conclusion, these studies show that cephalopods possess very high potential for HAB-toxins accumulation and may have developed mechanisms to protect them against potent neurotoxins that are frequently available in the marine environment.

Keywords: cephalopods, biotoxins, domoic acid, PSTs, central nervous system, kinetics, accumulation, depuration.

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RESUMO

Os “blooms” de algas tóxicas são fenómenos naturais que podem afetar distintamente vários níveis das teias tróficas marinhas. Dependendo da espécie, estas algas, tanto diatomáceas como dinoflagelados, podem produzir uma vasta gama de toxinas, que em casos extremos podem causar efeitos devastadores na vida marinha. As toxinas paralisantes (PSTs) do grupo saxitoxina e as toxinas amnesicas do grupo ácido domóico (AD) são as neurotoxinas mais comuns observadas ao longo da costa Portuguesa. Sabe-se que os cefalópodes acumulam estas toxinas hidrofílicas nos seus tecidos, com especial ênfase na glândula digestiva, através da ingestão de presas contaminadas. Na presente dissertação é examinada a cinética de “uptake”, transferência e eliminação de PSTs entre tecidos de polvo comum (Octopus vulgaris). Verificou-se que os polvos apresentam taxas de conversão (entre as toxinas presentes presa contaminada e no predador), transferência e eliminação reduzidas. Para além de se detectar elevadas concentrações na glândula digestiva, estas toxinas foram também encontradas no rim, mas em concentrações consideravelmente inferiores, o que evidência a lenta eliminação das toxinas. A exposição de polvos a alimento altamente contaminado (2665 ± 330 µg STX eq kg-1) durante 6 dias e apesar das elevadas concentrações de toxina paralisante presentes nos seus tecidos não se observou qualquer efeito/alteração comportamental. Por forma a determinar se neurotoxinas derivadas de “blooms” de algas tóxicas atingem o sistema nervoso central, foram capturados polvos (O. vulgaris) e chocos (Sepia officinalis), e o seu tecido cerebral foi analisado para a quantificação de AD. Esta toxina actua ao nível dos recetores glutamatérgicos, inibindo a ligação normal do neurotransmissor glutamato, desencadeando a permanente ativação de recetores AMPA, cainato e NMDA, resultando na despolarização da membrana neural e a degeneração celular no sistema nervoso central de vertebrados e está documentada a sua acumulação na glândula digestiva de cefalópodes. A presente dissertação demonstra que esta potente neurotoxina ultrapassa a barreira hematoencefálica e atinge o tecido cerebral de polvos e chocos, podendo ser

- 14 - acumulada no caso dos polvos por períodos de pelo menos quatro meses. Os resultados obtidos permitem inferir que os polvos retêm esta toxina e possivelmente toleram a sua presença. Concomitantemente, e até à data, eram desconhecidos os efeitos de biotoxinas em estados iniciais de desenvolvimento de cefalópodes, estados estes que são considerados como sendo fases mais vulneráveis a contaminantes externos, como as biotoxinas marinhas. Com vista a preencher esta lacuna, foram expostas paralarvas de polvo, dois dias após a sua eclosão, a 150 µg DA L-1 dissolvido na água, e foram determinadas as taxas de atividade, alimentação e atividade cromatofórica. Os resultados aqui apresentados demonstram que a presença de AD na sua fração dissolvida não afeta nenhum dos parâmetros testados. Em suma, estes estudos revelam que os cefalópodes possuem um elevado potencial de acumulação de toxinas provenientes de blooms de algas tóxicas e poderão ter desenvolvido mecanismos que os protegem contra a ação destas potentes neurotoxinas.

Palavras-chave: cefalópodes, biotoxinas, ácido domóico, PSTs, sistema nervoso central, cinética, acumulação, depuração.

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RESUMO ALARGADO

As comunidades fitoplanctónicas são tipicamente benéficas para o ecossistema, uma vez que constituem fonte de alimento para inúmeros organismos. De todas as espécies de fitoplâncton conhecidas, há uma pequena percentagem produz toxinas. No entanto, quando as condições do meio são consideradas ideais, estes organismos atingem elevadas taxas de crescimento e elevadas densidades, dando origem aos blooms de algas tóxicas. De um ponto de vista ecológico, as toxinas provenientes de blooms de algas tóxicas, podem causar efeitos devastadores nas comunidades marinhas, uma vez que, dependendo da espécie produtora, as toxinas podem atuar no sistema nervoso central, a vários níveis, ou no trato gastrointestinal. As biotoxinas, toxinas produzidas por microalgas, são principalmente acumuladas pela base da teia trófica, por organismos planctónicos filtradores, peixes planctívoros e bivalves. As toxinas produzidas por estas microalgas podem ser transferidas para níveis mais elevados da teia trófica marinha, como os cefalópodes. Os cefalópodes ocupam uma posição importante em todos os ecossistemas onde ocorrem, uma vez que são o alimento de predadores de topo, mamíferos e aves marinhas. Na dieta dos cefalópodes constam moluscos, crustáceos e pequenos peixes, podendo assim constituir um elo de ligação entre a acumulação por consumidores primários (e.g. bivalves), os predadores de topo, e até mesmo o Homem. Sabe-se que os cefalópodes são capazes de acumular grandes concentrações de ficotoxinas, no entanto, muitos fatores envolvidos nesta dinâmica permanecem incompreendidos. Assim, a presente dissertação teve como objetivo principal estudar a relação entre toxinas provenientes de blooms de algas tóxicas, nomeadamente toxinas paralisantes (PSTs) e ácido domóico (AD) e a sua subsequente acumulação em cefalópodes, mais concretamente em polvos (Octopus vulgaris). Em primeiro lugar examinou-se a cinética de acumulação e eliminação de PSTs. O principal órgão de acumulação foi a glândula digestiva (GD), seguido do rim, não se detectando PSTs em nenhum outro tecido. A reduzida taxa de eliminação calculada traduz a acumulação exponencial destas toxinas na GD. A transferência de

- 16 - toxinas entre compartimentos (GD e rim) também se verificou reduzida, com uma taxa mais baixa do que a taxa de eliminação, sugerindo que na excreção destas toxinas estejam também envolvidas outras vias ou mecanismos. A acumulação, transferência e eliminação são processos simultâneos, logo, são inerentemente difíceis de quantificar. Assim, ao serem utilizados modelos de cinética de primeira ordem, podem ser inferidas as taxas a que cada processo é efetuado. Na presente dissertação é apresentado um modelo de crescimento exponencial, utilizando dois compartimentos (GD e rim) para caracterizar a fase de acumulação, enquanto que, na eliminação de toxinas, o modelo com melhor encaixe foi um modelo de decréscimo exponencial utilizando apenas um compartimento, o total da víscera. Um facto relevante observado no decorrer deste estudo foi a aparente falta de resposta comportamental, por parte dos polvos, apesar das elevadas concentrações de toxina presentes nos seus tecidos. De forma a averiguar se estas toxinas causam algum tipo de efeitos comportamentais, foi necessário investigar a presença das mesmas em tecido cerebral de polvo, choco e lula. Foram capturados espécimes de polvo (O. vulgaris), choco (Sepia officinalis) e lula (Loligo vulgaris, L. forbesi e Todarodes sagittatus), cujo tecido cerebral e GD foram removidos para serem analisados para quantificação de AD, uma das neurotoxinas em estudo na presente dissertação. Verificou-se que apenas os polvos e os chocos apresentam AD no tecido cerebral. Este facto prende-se, possivelmente, com o estilo de vida dos diferentes cefalópodes e, por consequência, com as suas dietas. As lulas são predadores pelágicos, que se alimentam principalmente de pequenos peixes, que por sua vez poderão acumular toxinas durante curtos períodos de tempo. Por outro lado, cefalópodes bentónicos ou nectobentónicos, como os polvos e chocos, respetivamente, alimentam-se principalmente de bivalves e crustáceos, organismos mais passíveis de acumular toxinas. No caso dos polvos, onde foi feita a uma amostragem mais extensa, o AD foi detectado em todas as amostras de cérebro analisadas. No entanto, dados provenientes do programa de monitorização

- 17 - implementado e executado pelo IPMA, de periodicidade semanal, mostram que a acumulação de AD em bivalves nas épocas coincidentes com a amostragem de polvos foi muito restrita no tempo e aconteceu em baixas concentrações. Os dados aqui apresentados demonstram, pela primeira vez, que cefalópodes, mais concretamente polvos e chocos, acumulam a neurotoxina AD, produzida por diatomáceas, no tecido cerebral e que retém esta toxina no seu sistema durante longos períodos de tempo (pelo menos 4 meses). No entanto, os efeitos destas toxinas em estados iniciais de desenvolvimento de cefalópodes, fases essas consideradas por diversos autores como fases mais susceptiveis aos contaminantes ambientais incluindo contaminantes naturais como as biotoxinas marinhas, eram desconhecidos. Com o objetivo de determinar se os polvos são afetados pela presença desta toxina nas suas fases ontogenéticas iniciais, ao nível do comportamento, foram expostas paralarvas de polvo a AD na fração solúvel, com concentrações ecologicamente relevantes (150 µg DA L-1). No decorrer da exposição aguda realizada (24 horas) foram monitorizadas e registadas as taxas de atividade, medidas através da quantificação do número de contrações do manto (propulsões), o número de presas ingeridas e a área da superfície dorsal das paralarvas coberta por cromatóforos. Ao final de 24 horas de exposição foi verificado que a presença de AD dissolvido na água dos tanques experimentais não causou efeitos alguns sobre os parâmetros analisados. Após o final do período experimental (24h), a água dos tanques foi recolhida para averiguar a concentração de AD presente e confirmar a exposição, e constatou-se que não houve degradação da toxina, uma vez que as concentrações da mesma se mantiveram semelhantes aos níveis inicialmente adicionados. Este estudo permite inferir que as paralarvas de polvo não são afetadas pela presença de uma neurotoxina dissolvida no meio, uma vez que, as taxas de atividade, número de presas ingeridas e atividade dos cromatóforos permaneceram semelhantes tanto no grupo de exposição como no grupo controlo.

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Os estudos aqui apresentados constituem a primeira tentativa de compreender a relação entre cefalópodes e toxinas produzidas por microalgas. Na presente dissertação é descrita a acumulação e eliminação de um complexo de neurotoxinas hidrofílicas (derivados de saxitoxina), e é demonstrado que os polvos possuem baixas taxas de eliminação destas toxinas, que seriam, à partida, rapidamente eliminadas. As reduzidas taxas de eliminação permitem uma acumulação com tendência exponencial, e principalmente concentrada num tecido/orgão, a GD. Até à data, não se sabia se as neurotoxinas produzidas por blooms de algas tóxicas e acumuladas em cefalópodes poderiam atravessar a barreira hematoencefálica e atingir o sistema nervoso central. Na presente dissertação verificou-se que o AD atinge o tecido cerebral e, que esta acumulação ocorre durante, pelo menos, 4 meses. Este facto sugere uma retenção seletiva destas toxinas e enfatiza a aparente ausência de efeitos negativos na fisiologia destes organismos. De fato, após a exposição de paralarvas de polvo a uma neurotoxina na fração solúvel, este estado inicial do ciclo de vida, que é, por definição, mais sensível, uma vez que estes organismos ainda não possuem todo o corpo desenvolvido e são tipicamente mais vulneráveis a contaminantes externos, revelou ausência de efeitos negativos nos parâmetros testados. As paralarvas apresentaram padrões de atividade comparáveis ao grupo controlo, assim como o número de presas ingeridas e a atividade dos cromatóforos ao longo de 24 horas de exposição à toxina dissolvida no meio. Os estudos aqui apresentados demonstram que em O. vulgaris o “uptake” de neurotoxinas ocorre em taxas elevadas, em contraste com a sua eliminação, que, sendo reduzida, permite a acumulação exponencial destes compostos. Adicionalmente, verificou-se, pela primeira vez, que neurotoxinas provenientes de blooms de algas tóxicas penetram na barreira hematoencefálica e são retidas durante longos períodos de tempo. Por fim, após a exposição de paralarvas de O. vulgaris à toxina dissolvida constatou-se que esta não altera nenhum dos parâmetros estudados. Assim, a presente dissertação evidencia que os cefalópodes são organismos “imunes” a biotoxinas marinhas, que podem em casos extremos

- 19 - causar efeitos devastadores em muitos outros organismos marinhos e até em humanos. Em estudos futuros, será crucial compreender os mecanismos de defesa que estes invertebrados excecionais poderão possuir, que os permite acumular concentrações elevadas destas neurotoxinas, assim como investigar se estas toxinas serão acumuladas na hemolinfa e em que zonas do cérebro ficam acumuladas.

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LIST OF PAPERS

I hereby declare, as author of the present dissertation, that I participated and was responsible for the conception and experimental design of each chapter presented here. I was also responsible for sample collection, animal rearing, laboratory analysis, data analysis and writing of each manuscript. The other authors contributed in some of the tasks described above. The present dissertation is comprised of five scientific outputs, four scientific papers, of which 3 are published, and one book chapter accepted for publication, that can be found from Chapter One to Five.

Chapter One Lopes, V.M., Costa, P.R., Rosa, R. (In press). Effects of Harmful Algal Bloom Toxins on Marine Organisms. In Duarte, B., Caçador, I. (Eds.), Ecotoxicology of Marine Organisms, CRC Press, ISBN-13: 978-1138035492.

Chapter Two Lopes, V.M., Lopes, A.R., Costa, P., Rosa, R., (2013). Cephalopods as vectors of harmful algal bloom toxins in marine food webs. Marine Drugs 11, 3381-3409. doi:10.3390/md11093381.

Chapter Three Lopes, V.M., Rosa, R., Costa, P.R. (2018) Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains. Marine Environmental Research 133, 45–48. DOI: 10.1016/j.marenvres.2017.12.001.

Chapter Four Lopes, V.M., Baptista, M., Repolho, T., Rosa, R., Costa, P.R., 2014. Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris). Aquatic Toxicology 146, 205-211. DOI: 10.1016/j.aquatox.2013.11.011.

Chapter Five

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Lopes, V.M., Sampaio, E., Vara, C., Costa, P.R., Rosa, R. (in preparation). Feeding behaviour and chromatophore activity of octopus paralarvae are not affected by the harmful algal toxin domoic acid. Marine Environmental Research.

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“I firmly believe that the art of storytelling will never change.

If you tell a good story, people will hang on your words.”

- Sir David Attenborough

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CHAPTER ONE

GENERAL INTRODUCTION

The material in this chapter is adapted from:

Lopes, V.M., Costa, P.R., Rosa, R. (accepted) Effects of Harmful Algal Bloom

Toxins on Marine Organisms. In Duarte, B., Caçador, I. (Eds.), Ecotoxicology of Marine Organisms, CRC Press, ISBN-13: 978-1138035492.

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1.1. Overview on Harmful Algal Blooms Algal blooms are natural occurrences, defined as the sudden overgrowth of microscopic algae under optimal environmental conditions, reaching up to millions of cells per litre

(Hallegraeff, 1993). These blooms are typically beneficial for the ecosystem, increasing feeding opportunities for countless organisms. However, if toxin-producing microalgae undergo this sudden overgrowth, it can lead to harmful algal blooms (HABs).

Despite the fact that approximately 2% of microalgae species produce toxins (Hallegraeff, 2014; Smayda, 1997), HABs can significantly impact marine communities.

In the marine realm, the majority of HAB-toxins are produced by dinoflagellates and diatoms (Table 1). Biochemically, phycotoxins are secondary metabolites that can have a wide range of effects. They can act on the nervous system, which can induce permanent short-term memory loss (domoic acid) or cause sensorimotor impairment, leading to death (paralytic shellfish toxins) and act on the digestive tract, inducing gastrointestinal distress. During the last decades several new toxins and new toxin derivatives, such as gymnodimines, azaspiracids, pterotoxins, pinnatoxins and hydroxybenzoate saxitoxin, okadaic and domoic acid analogues have been described, mostly due to scientific and technological advances

(Cruz et al., 2006; Miles et al., 2000; Negri et al., 2003;

Satake et al., 1998; Takada et al., 2000; Zaman et al., 1997).

In addition, changes on global climate conditions and anthropogenic pressures have been causing several tropical and subtropical endemic HAB-toxins, namely ciguatoxins, palytoxins

- 31 - and brevetoxins to expand geographical range into temperate waters (Botana et al., 2015; Villareal et al., 2007).

Table 1. The most common toxins produced by marine phytoplankton

Toxin Toxin Toxic species Mode of action family Alexandrium sp., Inhibition of Gymnodinium Paralytic voltage-gated Saxitoxins catenatum, shellfish sodium channels Pyrodinium toxins in neural cells bahamense Binding to glutamate Pseudo-nitzschia receptors in Amnesic spp. Amphora Domoic acid neural cells shellfish coffaeaiformis, causing toxins Nitzschia sp., constant influx of Ca2+ Karenia brevis, Karenia sp., Binding to Chatonella cf. voltage- verrucosa, C. sensitive Neurotoxic antiqua, C. Brevetoxins sodium channels shellfish marina, causing toxins Fibrocapsa membrane japonica, depolarization Heterosigma akashiwo Inhibition of Okadaic acid activity of Diarrhetic and Dinophysis sp., protein shellfish dinophysistox Prorocentrum sp. phosphatase 1 toxins ins and 2

1.2. Routes of toxin exposure

Toxin transfer can be foodborne or waterborne, i.e. via food web transfer or through exposure to toxins dissolved in the water after their excretion or cell release (Fig. 1). The most likely pathway of toxin transfer is when toxin-producing species bloom, thus achieving massive concentrations in the water column.

However, there are many potential toxin vectors (Fig. 2), depending mostly on the ecology of the toxin producer (pelagic

- 32 - or epibenthic) and the organism’s likelihood of exposure to the toxin.

Figure 1. Foodborne and waterborne exposure to HAB-toxins. Solid lines illustrate the well documented route of dietary exposure; dashed lines illustrate the less studied routes of dissolved toxins exposure.

Legend: SPM – Suspended Particulate Matter.

If an organism is exposed to a sudden bloom of toxin-producing microalgae, the toxin concentrations will certainly trigger immediate physiological and behavioural alterations and ultimately cause the death of the organism. In addition, the continuous exposure to low HAB-toxin concentrations can lead to chronic effects (Landsberg, 1995).

Here, the pathways of exposure will be divided into direct and indirect contact with the toxin-producer. Through ingestion of

- 33 - toxic phytoplanktonic cells by filter-feeding organisms, such as bivalve molluscs, zooplankton and planktivorous fish, the toxins present inside the cell can accumulate in the predator’s viscera. This can create a chain of vectors throughout the food web, potentially eliciting adverse effects in marine communities. Depending on the vector, these toxins can be transferred to humans and cause a variety of shellfish poisonings, due to the ingestion of contaminated shellfish, such as Paralytic Shellfish Poisoning (PSP) or Amnesic Shellfish

Poisoning (ASP), among other syndromes (Table 1).

Some microalgae species produce exotoxins, or exudates, that are released into the water column, causing other organisms to come inadvertently in contact with these compounds. Similarly, when the bloom becomes senescent, the cells lyse and release the toxins to the surrounding environment (Lefebvre et al., 2008), opening another possible pathway to the organisms’ direct contact with the toxins. Lastly, there are other HAB-species which segregate the toxin on the outer surface of their cells, potentially inducing damage upon contact (Matsuyama et al.,

1997; Kamiyama and Arima, 1997).

The present dissertation is focused on paralytic shellfish toxins (PSTs) and domoic acid (DA) and their effects on marine organisms, due to the fact that these are hydrophilic neurotoxins and are the most relevant in Portugal’s socio- economic context (Sampayo et al., 1997; Vale and Sampayo, 2001).

1.3. Paralytic shellfish toxins

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PSTs are one example of phycotoxins produced by dinoflagellates, and one of the most abundant and toxic in oceans worldwide.

Dinoflagellates from three genera, Alexandrium, Gymnodinium and

Pyrodinium, produce saxitoxin or a suite of over 50 derivatives

(Anderson et al., 2012), which the most frequent can be divided according to their chemical structure into carbamoyl, decarbamoyl and sulfamate toxins. These compounds block the conduction of electrical impulses in neural cells through the inhibition of voltage-gated sodium channels on these cell’s membranes. This leads to membrane hyperpolarization and results in paralysis in muscle cells as determined in laboratory animals

(Kao and Nishiyama, 1965; Ritchie and Rogart, 1977). PSTs have shown to elicit a wide range of effects on marine organisms, from sublethal and recoverable effects to events of mass mortality in fish, marine mammals and seabirds (Gill and Harris,

1987; Ives, 1987; Reyero et al., 2000; Yan et al., 2001; 2003;

Shumway et al., 2003; Lefebvre et al., 2005; Samson et al.,

2008; Escobedo-Lozano et al., 2012).

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Figure 2. Schematic view of HAB-toxins food web transfer.

1.4. Amnesic shellfish toxins

DA is a potent neurotoxin produced by some species belonging to two genera of diatoms, Pseudo-nitzschia and Nitzschia. This toxin is known to cause Amnesic Shellfish Poisoning (ASP), and the attention on this toxin and its possible consequences was focused after an incident involving the death of 3 people in

1987 following ingestion of mussels contaminated with DA

(Quilliam and Wright, 1989). Afterwards, most coastal countries developed monitoring programs, analysing regularly bivalve tissue for DA and other phycotoxins in order to prevent foodborne illnesses. These monitoring programs have been successful at avoiding further human casualties.

DA acts in neural cells, competing for the same receptors as glutamate, an excitatory neurotransmitter. By having less affinity for these receptors, glutamate fails to bind normally, causing excessive concentrations of glutamate outside the

- 36 - synapses, triggering AMPA, kainate and NMDA receptors’ activation, permanently opening the neural cell’s membrane, leading to excessive influx of Ca2+ (Berman and Murray, 1997).

This causes membrane depolarization and subsequent degeneration of neural cells. Being glutamate receptors directly involved in controlling synaptic regulation in memory and learning processes

(Lynch and Baudry, 1984; Massicote and Baudry, 1991), it is the most likely cause for the memory loss.

1.5. HAB-toxin effects on marine organisms

1.5.1. Paralytic shellfish toxins

PSTs producers inhabit the pelagic realm, the same habitat as most planktonic species. Therefore, plankton species come in contact with PSTs through contact with PST-producing cells or their exudates. It has been shown that many planktonic organisms can be affected by these toxins. In some cases, PSTs induce disruptions in swimming behaviour leading to death and limited egg production in ciliates (Hansen, 1989; Hansen et al., 1992).

Diatoms and haptophytes had reduced growth rates after being placed in water previously conditioned by PSTs producer A. lusitanicum, presumably releasing the water-soluble toxins into the culture medium (Blanco and Campos, 1988).

Regarding the effects of PSTs on planktonic grazers, there are several studies indicating that some species can selectively avoid ingestion of toxic dinoflagellates, while others do not

(Teegarden, 1999; Teegarden et al., 2001; Turriff et al., 1995;

Turner & Tester, 1997). The latter group can present different effects, with some species presenting lower fecundity rates

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(Colin and Dam, 2004; Dutz, 1998; Gill and Harris, 1987), lower

hatching success (Frangópulos et al., 2000), lower activity and

feeding rates and high mortalities (Bagøein et al., 1996; Colin

and Dam, 2004; Ives, 1987; Sievers, 1969).

Direct exposure of bivalve molluscs to PST-producers has been

shown to elicit negative effects, as summarized in Table 2.

Exposure to dinoflagellate cells increased shell valve closure

in many bivalve species (e.g. Crassostrea virginica, Mytilus

edulis), leading to decreased filtration rates, potentially

impacting the animal’s normal feeding behaviour. A. minutum and

purified saxitoxin (STX) exposure in C. gigas resulted in

decreased phagocytic activity and ROS production in oyster

hemocytes (Mello et al., 2013), leading to higher susceptibility

of contracting an infection. Also, the presence of the toxic

dinoflagellates decreased byssus production in M. edulis and

Geukensia demissa. However, byssus production in mussels (M.

edulis) that have been previously exposed to these toxins was

less affected. Similar experiments performed on greenshell

mussels (Perna canaliculus) showed that mussels presented oxygen

consumption and clearance rates similar to the control group

after 24h exposure to A. tamarense (Marsden and Shumway, 1992).

Table 2. Documented cases of bivalves molluscs exposed to and affected

by paralytic shellfish toxins (PSTs).

Target PST source Route of Levels of Effects Reference species exposure exposure s Larvae of A. tamarense Exposure to Up to 11 Activity and (Yan et Argopecten A. pg STX eq growth al., irradians tamarense cell-1 inhibition, 2003) concentricu cells A. lower s tamarense attachment cultures rates and reduced

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climbing rates

Larvae of A. tamarense Exposure to Up to 5.9 High (Yan et Chlamys A. x 109 g mortality al., farreri tamarense STX L-1 rates, lower 2001) cells A. hatching tamarense rates cultures Crassostrea G. Exposure to 5 x 104 Reduced (Dupuy gigas washingtonen A. cells L-1 pumping and sis (now A. catenella activity, Sparks, catenella) cells increased 1967) valve activity A. minutum Exposure to Up to 12 x Decreased (Lassus A. minutum 104 cells valve et al., cells L-1 activity, 1999) clearance and filtration rates A. tamarense Exposure to Up to 12 x Reduced (Laabir and A. dinoflagell 108 cells clearance and minutum ate cells L-1 rates Gentien, 1999) A. minutum Exposure to 5 x106 Mono and (Haberkor A. minutum cells L-1 diacylglycero n et al., cells ls reduced in 2010) digestive gland, inflammation of gastrointesti nal tract, modified spermatozoa and mitochondria A. minutum Exposure to 15 x 106 Altered (Tran et A. minutum cells L-1 circadian al., cells rhythm 2015) Larvae of A. tamarense Exposure to Up to 108 High (Matsuyam C. gigas A. cells -1 mortality a, et tamarense rates al., cells 2001) A. taylori Exposure to Up to 108 High (Matsuyam A. taylori cells -1 mortality a, et cells rates al., 2001) C. G. monilata Exposure to Up to 1.2 Inhibition of (Sievers, virginica (now A. A. x 106 byssus 1969) monilatum) monilatum cells L-1 production cells and shell closure Brachiodont G. monilata Exposure to Up to 1.2 Inhibition of (Sievers, es recurvus (now A. A. x 106 byssus 1969) monilatum) monilatum cells L-1 production cells and shell closure

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Geukensia P. Exposure to 2.5-5.5 x Inhibited (Gainey & demissa tamarensis A. 105 cells cardiac Shumway, (now A. tamarense L-1 activity 1988) tamarense) cells A. tamarense Exposure to 105 cells Reduced (Lesser A. L-1 clearance and tamarense rates Shumway, cells 1993) P. Exposure to 5 x 105 Reduced (Shumway tamarensis A. cells L-1 clearance & Cucci, (now A. tamarense rates, 1987) tamarense) cells increased mucus production P. Exposure to 106 cells Inhibition of (Shumway, tamarensis A. L-1 byssus et al., (now A. tamarense production 1987) tamarense) cells Mercenaria A. tamarense Exposure to 105 cells Reduced (Lesser mercenaria A. L-1 clearance and tamarense rates Shumway, cells 1993) Mya P. Exposure to 2.5-5.5 x Inhibited (Gainey & arenaria tamarensis A. 105 cells cardiac Shumway, (now A. tamarense L-1 activity 1988) tamarense) cells A. tamarense Exposure to Up to 77 x Naïve (MacQuarr A. 104 µg STX populations ie and tamarense eq kg-1 in had higher Bricelj, cells viscera toxicity and 2008) mortality, reduced clearance rates, oxygen consumption rates and burrowing capacity A. tamarense Exposure to 105 cells Reduced (Lesser A. L-1 clearance and tamarense rates Shumway, cells 1993) P. Exposure to 5 x 105 Reduced (Shumway tamarensis A. cells L-1 clearance & Cucci, (now A. tamarense rates 1987) tamarense) cells A. excavata Exposure to Up to 30.4 Burrowing (Bricelj, and A. Alexandrium x 104 µg incapacity et al., tamarense spp. cells STX eq kg- 1996) 1 Mytilus P. Exposure to 2.5-5.5 x Inhibited (Gainey & edulis tamarensis A. 105 cells cardiac Shumway, (now A. tamarense L-1 activity 1988) tamarense) cells Dissolved Intramuscul 3330 µg Higher GST (Gubbins, STX ar STX kg-1 activity et al., injection 2001) A. tamarense Exposure to 105 cells Reduced (Lesser A. L-1 clearance and tamarense rates Shumway, cells 1993)

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P. Exposure to 5 x 105 Increased (Shumway tamarensis A. cells L-1 mucus & Cucci, (now A. tamarense production 1987) tamarense) cells P. Exposure to 106 cells Inhibition of (Shumway tamarensis A. L-1 byssus et al., (now A. tamarense production 1987) tamarense) cells Ostrea P. Exposure to 2.5-5.5 x Decreased in (Gainey & edulis tamarensis A. 105 cells heart rate Shumway, (now A. tamarense L-1 1988) tamarense) cells

P. viridis A. tamarense Exposure to 105 cells Reduced (Lesser A. L-1 clearance and tamarense rates Shumway, cells 1993)

Placopecten P. Exposure to 5 x 105 Increased (Shumway magellanicu tamarensis A. cells L-1 clearance & Cucci, s (now A. tamarense rates 1987) tamarense) cells Spisula A. tamarense Exposure to 105 cells Reduced (Lesser solidissima A. L-1 clearance and tamarense rates Shumway, cells 1993)

Exposure of M. chilensis to A. catenella for 21 days (Navarro

and Contreras, 2010) resulted in lowered clearance rates,

organic matter intake and absorption efficiency at the start of

the experiment, followed by an increase to levels similar to the

ones presented in the control group. STX uptake steadily

increased throughout the experiment, similarly to mussel’s

excretion rates. Oxygen consumption rates seemed unaffected by

the ingestion of this toxic species, revealing that it may

possess defence mechanisms that allow them to feed safely on

this dinoflagellate.

On the other hand, some scallop and clam species presented

negative effects when exposed to this toxin. The scallop

Nodipecten subnodosus presented paralysis of the adductor muscle

while maintaining the digestive tract functioning after

receiving intramuscular injections of GTX 2+3. Also, hemocyte

- 41 - number decreased and they presented mantle retraction and their shells remained open up to 40 days after the exposure (Estrada et al., 2010). In a different study on the same species (Estrada et al., 2007), exposure to G. catenatum cells resulted in increased production of mucus, pseudo-faeces, melanisation and hemocyte aggregation in gill tissue. Biochemically, an antioxidative stress response to the toxin was shown in gill tissue, being this tissue the first to come in contact with the toxin. There was an increase in glutathione peroxidase (GPx) activity and lipid peroxidation, along with a decrease in superoxide dismutase activity, indicating oxidative and cellular damage. Similar results were obtained when feeding G. catenatum cultures to scallops (Argopecten ventricosus) (Escobedo-Lozano et al., 2012). The scallops presented paralysis of the adductor muscle, lower feeding activity, increased pseudo-faeces production, increased number of hemocytes in gill, mantle and adductor muscle tissue and epithelial melanisation in gill and mantle tissue. These results indicate that scallops have efficient mechanisms that protect them against lethal effects from external toxicants. Studies conducted in Ruditapes phillipinarum feeding on A. tamarense cultures for 6 (Li et al.,

2002) and 15 days (Choi et al., 2006) showed reduced scope for growth, decreased absorption efficiency, clearance and growth rates after 6 days of exposure, and increased activity of GPx in hepatopancreas and gill lipid peroxidation with increasing toxin burden after 15 days of exposure.

PST effects on bivalve early stages are comparatively less understood. Bricelj et al. (2010), addressed this issue by

- 42 - exposing larvae, post-larvae and juveniles of Mya arenaria to A. tamarense. The authors showed that larvae were not significantly affected by the dinoflagellate due to the fact that the cells were too large for prey capture, thus the larvae did not accumulate the toxin or displayed any intoxication symptoms. On the other hand, the post-larvae presented decreased burrowing capacity, used as proxy of sensitivity to PSTs. Also, the post- larvae had increased mortality rates, especially in populations that are not usually exposed to A. tamarense blooms, while juvenile were less susceptible.

PST effects on bivalves are species-specific and seem to differ geographically within the same species and life stages. Species that are usually exposed to toxic dinoflagellate blooms seem to be more resistant and appear to have developed defence mechanisms that allow them to cope with high PST levels, unlike other species in areas less affected by blooms.

These studies reveal that bivalves are not immune to the effects of PST contamination, and there are some species with higher sensitivity to these toxins. This may pose additional concerns over the ecosystem’s health and elicit negative economic impacts, since some of these species are commercially farmed in shellfish aquacultures, and blooms may occur in farmed areas.

PSTs have long been associated with fish kills. Fish can be directly exposed to the toxins, as is the case of planktivorous fish such as sardines, herring and anchovies, or indirectly through feeding on vectors, affecting many levels of the marine food web, from groupers and hake to sturgeons and artificially fed fish, such as farmed salmon.

- 43 -

Only a few events have been directly linked to PST contamination, since these events are unpredictable and sporadic, many times leading to inconclusive data. For a complete list of fish kills associated with PSTs refer to Costa

(2016, Table 1).

When studying PST’s effect in fish, it is procedurally simpler to inject the toxin intracoelomically (IC) or intraperitoneally

(IP), in order to closely control the given concentration.

Standard STX is the toxin most commonly administered. However, despite the benefits, these methods are less ecologically relevant, since the toxins do not enter directly in the coelom, and STX is but a fraction of the toxins produced by the dinoflagellate species. Nevertheless, these studies provide windows into the symptoms presented by fish and insight into the effects of these neurotoxins.

STX effects in killifish (Fundulus heteroclitus) were quantified regarding the expression of c-Fos protein (Salierno et al.,

2006), responsible for regulating neural cell’s survival, and is associated with long term memory (Sadananda and Bischof, 2002).

It was shown that the expression of this protein decreased, and the fish presented behavioural alterations including paralysis, lethargy and loss of balance. STX most likely affects the neural pathways responsible for swimming. In Atlantic salmon (Salmo salar) it was shown that STX crosses the blood-brain barrier and that sublethal doses of this toxin affect the activity of brain subregions in the central nervous system (CNS), possibly affecting the organism’s cognitive abilities (Bakke & Horsberg,

2007). Intracoelomic injections of STX in white seabream

- 44 -

(Diplodus sargus) resulted in an increase of glutathione-S- transferase (GST) activity, an enzyme responsible for removing xenobiotics, among many other roles. STX also induced DNA damage

(chromosome breaks or loss) and increased erythrocyte nuclear abnormalities (Costa et al., 2012).

In order to simulate bloom conditions, milkfish (Chanus chanus) fingerlings were exposed to STX extract and A. minutum cells in increasing concentrations and cell density, respectively (Chen and Chou, 2001). After 24h, the fish presented oedema, hyperplasia and necrosis in gill lamellae. The exposure also resulted in increased mortality rates in the treatments with higher cell density and STX concentrations, due to increasing oxygen demand following gill damage. Similar results reporting gill damage and high mortality rates following fish exposure to

PST-producing dinoflagellate cells were found in salmon, trout

(Mortensen, 1985) and sheepshead minnow (Cyprinodon variegatus,

Sievers, 1969). White (1981) reported high mortality rates after

20 to 60 minutes in Atlantic herring (Clupea harengus harengus),

American pollock (Pollachius virens), winter flounder

(Pleuronectes americanus), Atlantic salmon (S. salar) and cod

(Gadus morhua) when dosed IP or orally with toxin extracts from

A. tamarense cultures. Prior to death, the fish presented loss of balance, immobilization and arrhythmic breathing, consistent with the symptoms described here in adult fish.

In early stages of development, some fish species present different ecologies than the adults, starting out as planktonic larvae, and thus occupying the same niche as the pelagic dinoflagellate PST-producers. Also, earlier stages of

- 45 - development are likely more vulnerable to the effects of these toxins as they possess higher mass-specific metabolic rates and they lack fully developed detoxification systems (Vasconcelos et al., 2010).

Overall, when fish early stages are exposed to bloom simulations in experimental conditions, it resulted in extremely high mortality rates, nearing the totality of the experimental population, besides the sublethal effects displayed by the young. Fish early stages can be exposed to the toxin through feeding on zooplanktonic vectors, such as copepods, or through direct exposure to the dissolved toxins. Recently settled flounders (P. americanus), sheepshead minnow (C. variegatus) and killifish larvae (F. heteroclitus) were fed with contaminated copepods, acting as vectors of A. fundyense (Samson et al.,

2008). After consuming 6-12 contaminated copepods, the fish died. In this study, the fish were also fed with fewer copepods, resulting in a variety of effects, such as reduced swimming abilities, prey capture success, predator avoidance and overall activity.

Gosselin et al., (1989) exposed capelin and herring larvae to three different treatments to ascertain the effects of PSTs through different routes of exposure, recurring to both direct exposure through feeding the larvae with A. tamarense cells in increasing densities, and placing toxin extracts in the experimental tanks. Indirect exposure was achieved by feeding the larvae with contaminated microzooplankton. Capelin and herring larvae fed on A. tamarense swam erratically, lost motility and sank to the bottom paralysed, dying after 20

- 46 - minutes of exposure, contrarily to the lack of effect when exposed directly to the dissolved toxin. Feeding these larvae with contaminated zooplankton elicited similar results as feeding directly on the dinoflagellate, resulting in paralysis and high mortality rates. However, exposure of herring larvae to dissolved STX resulted in a reversible dose-dependent suite of sensorimotor impairments (Lefebvre et al., 2005), such as spontaneous swimming and tactile response inhibition. Also, it was shown that older larvae were more susceptible to the dissolved toxin, likely due to the degree of gill and body maturation leading to higher toxin uptake.

Monk seal populations have been greatly impacted by PST outbreaks. In the late 1990’s in Cape Blanc peninsula over 100 monk seals died following PST intoxication. Tissue analysis revealed PSTs in brain tissue, suggesting that these toxins were present in the seal’s nervous system (Costas and Lopez-Rodas,

1998; Reyero et al., 2000). The cause has been attributed to

PSTs since there were high levels of these toxins in many fish species that the seal’s prey upon (Reyero et al., 2000). Dying organisms presented many behavioural alterations, lethargy, paralysis and sensorimotor discoordination (Hernández et al.,

1998).

Earlier, in 1987, over a dozen humpback whales washed ashore dead along Nantucket Sound. The cause of the stranding was ascertained by analysing fish and whale tissues. It was determined that one of the fish species analysed, Atlantic mackerel (Scomber scombrus) presented high levels of STX, and stomachal content analysis revealed that the whales were

- 47 - previously feeding on this species. Worth noting was the time- lapse between the onset of the first symptoms and death

(approximately 90 minutes, Geraci et al., 1989), suggesting a very quick process, characteristic of severe STX intoxication.

Seabird deaths due to HAB-toxins have been comparatively overlooked. However, there have been countless events where many seabird species died following ingestion of contaminated fish and shellfish. Shumway et al., 2003 extensively review all seabird deaths registered that were linked with HAB-toxins, including PSTs. PSTs were reported to cause loss of motor coordination and paralysis, resulting in the bird’s inability to feed and death by starvation. Female terns presented inability to lay eggs due to sublethal onset of paralysis, resulting in the egg breaking inside the body and causing fatal haemorrhages.

Other species presented severe inflammation of the gastro- intestinal tract and haemorrhages in the intestines and brain.

Understanding the effects of PSTs, toxins produced by many dinoflagellate species occurring worldwide is of vital importance, since, as reviewed here, the range of possible consequences is very wide. In some cases, the toxins affect directly the species, causing high mortalities, and in other cases the toxin accumulates and is transferred throughout many levels of the marine food web, causing indirect damage to the ocean’s health, communities and human populations.

1.5.2. Domoic acid

- 48 -

Bivalves are common vectors of this toxin and the effects of this toxin seem somewhat overlooked. Domoic acid (DA) seems to affect haemolymph chemistry (Jones et al., 1995a), increase the number of hemocytes (Dizer et al., 2001; Jones et al., 1995b), as well as increase cholinesterase activity and DNA damage

(Dizer et al., 2001). On the other hand, exposure to this toxin decreased phagocytic activity (Dizer et al., 2001), growth and survival rates (Liu et al., 2007, 2008). In some cases, the effects of exposure to the DA-producing diatoms were reversible and the organisms recovered after a short period of time (up to

24h), emphasizing the notion that bivalves are quite resilient to DA and other toxins.

Information regarding the effects of DA in wild animals is very scarce and limited to marine mammals and seabirds. Domoic acid’s effects on fish have been studied through IC injection. This technique allows the use of known DA concentrations without dispersal throughout the organism’s body. However, this method does not always allow for ecologically relevant DA concentrations to be used, or for natural DA uptake and transfer between body tissues to take place naturally. Regarding the effects of DA in fish, most studies concluded that it causes abnormal swimming behaviour, including spiral, circle and upside-down swimming, as well as physiological effects, such as increased cortisol levels and protein expression, and, ultimately death (Bakke et al., 2010; Lefebvre et al., 2007;

Nogueira et al., 2010; Wang et al., 2008). Other effects that can escalate DA toxicosis are inability to school in Engraulis mordax (Lefebvre, et al., 2001), possibly making the fish easier

- 49 - targets for predators, disrupting the balance of the food web during diatom blooms.

Killifish (F. heteroclitus) injected intracoelomically (IC) with up to 9 mg DA kg-1 showed that c-Fos activity, a protein associated with long term memory (Sadananda and Bischof, 2002), increased in several brain regions, indicating neuronal stress following exposure. Variations in c-Fos expression can lead to effects at the behavioural levels, as observed in Salierno et al., (2006), such as disorientation and loss of equilibrium.

One of the main groups affected by domoic acid are marine mammals, more specifically sea lions. There is an extensive record of sea lion deaths going back nearly two decades, when over 400 sea lions (Zalophus caifornianus) were found stranded or displayed neurological symptoms associated with DA intoxication, later confirmed by detecting DA in sea lion’s tissues (Scholin et al., 2000). The cause of death was attributed to ingestion of contaminated anchovies, a common food source for these mammals. Behavioural tests and magnetic resonance imaging (MRI) performed on sea lions displaying intoxication symptoms revealed abnormal behaviours, such as head weaving, ataxia and severe disorientation. The MRIs showed hippocampal lesions damaging hippocampal-thalamic networks.

Also, DA has been detected in the stomach contents of premature pups and shown to elicit premature births, abortions and death of pregnant sea lions (Brodie et al., 2006) due to consumption of contaminated prey, possibly endangering this species’ populations. Throughout the years, many other events of marine mammal’s mortality have been attributed to DA intoxication

- 50 -

(Bargu et al., 2011; Fire et al., 2009, 2010, Lefebvre et al.,

1999, 2010; Zabka et al., 2009).

It is worth noting, there have also been many seabird deaths attributed to DA toxicosis, with the birds presenting epileptic seizures, haemorrhage, tissue necrosis leading to death of many species (Sierra-Beltrán et al., 1997; Fritz et al., 1992; Perez-

Mendes et al., 2005; Work et al., 1993).

DA, as mentioned above, acts on glutamatergic receptors, mainly present in organisms with developed brains, possibly explaining the discrepancy between the effects caused in vertebrates and invertebrates. Invertebrates are likely less affected by this toxin, since they mostly lack complex brains and possess effective elimination systems, as in the case of bivalve molluscs. The fact that bivalves seem to be less affected and are efficient at eliminating DA does not exclude the sublethal effects that it may cause in other organisms higher up the food web, through chronic ingestion of contaminated prey.

1.6. Thesis objectives and outline

Throughout this chapter, it can be concluded that HABs cause a wide range of effects on marine organisms - from innocuous transient effects to sublethal effects and ultimately events of mass mortality in higher vertebrates. Previously to the present dissertation, there was no information regarding the dynamics of accumulation/elimination or the effects these neurotoxins may elicit on cephalopods, a class of very peculiar invertebrates.

Therefore, the main goal of the present dissertation was to shed light on what occurs when cephalopods are exposed to HAB-toxins,

- 51 - namely PSTs and DA, either through dietary route or direct exposure to the dissolved toxin. These toxins were chosen since they represent the major neurotoxins produced by phytoplankton along the Portuguese coast (Vale et al., 2008; Vale, 2011).

Cephalopods have highly developed central nervous systems, and are key organisms connecting the various links of marine food webs worldwide (Boyle and Rodhouse, 2005). Thus, studying the impact and characterizing the dynamics of these neurotoxins on these “intelligent” molluscs is of outstanding importance. More specifically, it was aimed to understand how PSTs are transferred between octopus’ tissues and the rate that they are eliminated from the organism, after ingestion of contaminated prey. Moreover, to better understand what kind of effects these toxins may elicit on cephalopods, it was investigated if DA crosses the blood-brain interface and reaches the central nervous system, and if so how does it affect the feeding behaviour of the early stages of development. Overall, these studies aim to contribute to the relatively unknown relationship between cephalopods and biotoxins and pave the way for future research.

The present thesis is composed of five chapters, which include four scientific articles (three published and one submitted to peer-reviewed international journals) and one book chapter (in press), all of which can be found from chapter one to five.

The specific objectives of each chapter are as follows:

Compilation of all available information on the effects

that neurotoxins, PSTs and DA, have on marine organisms

(Chapter one);

- 52 -

Compilation of the available information regarding these

toxins’ (PSTs and DA) accumulation in cephalopods (Chapter

two);

Description of PSTs’ uptake in octopus tissue, the

transfer rates to other body compartments and

quantification of accumulation and elimination rates

(Chapter three);

Investigate the accumulation of DA in brain and digestive

glands of cephalopods (octopus, squid and cuttlefish) from

different geographical areas of the Portuguese coast

(Chapter four);

Determination of the effects that exposing octopus

paralarvae to dissolved domoic acid have on activity,

feeding rates and chromatophoric activity (Chapter five).

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Science is a way of thinking much more than it is a body of

knowledge.

- Carl Sagan

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CHAPTER TWO

CEPHALOPODS AS VECTORS OF HARMFUL ALGAL BLOOM TOXINS IN

MARINE FOOD WEBS

The material in this chapter is adapted from:

Lopes, V.M., Lopes, A.R., Costa, P., Rosa, R., 2013. Cephalopods as vectors of harmful algal bloom toxins in marine food webs.

Marine Drugs 11, 3381-3409. doi:10.3390/md11093381.

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Cephalopods as vectors of harmful algal bloom toxins in marine food webs

Vanessa M. Lopes1, Ana Rita Lopes1, Pedro R. Costa2, Rui Rosa1

1Guia Marine Laboratory, Center of Oceanography, Faculty of

Sciences, University of Lisbon, Av. Nossa Senhora do Cabo, 939,

Cascais 2750-374, Portugal;

2IPMA—Portuguese Institute for the Sea and Atmosphere, Avenida de Brasília, Lisboa 1449-006, Portugal.

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Abstract

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These molluscs have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg−1 and palytoxin reaching 115 μg kg−1 (the regulatory limit for PlTXs is 30 μg kg−1 in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area.

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Keywords: marine toxins; harmful algal bloom; cephalopods; Octopus vulgaris; Dosidicus gigas; Sepia officinalis; strandings

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Introduction

As reviewed in the previous chapter, primary producers constitute the basis of marine food-webs and phytoplanktonic blooms are typically beneficial to food-web processes. However, under certain conditions, harmful algal blooms (HABs), a natural phenomenon, can become harmful to other forms of life. Toxins may cause heavy damage to marine animals and even death [e.g., behavior alterations (Kvitek et al., 1991), development impairments in early stages of life (Lefebvre et al., 2004,

2005), abortion and premature birth of marine mammals (Goldstein et al., 2009)]. They can also affect human populations, through the consumption of contaminated sea products, mostly shellfish.

As a result, many coastal countries conduct monitoring programs for marine toxins in shellfish (Landsberg, 2002).

Marine toxins are transferred to higher levels of the food web by predation on organisms that have been in direct contact with the toxins and have accumulated them, thus, creating a chain of vectors. This way, the toxin produced by microscopic algae can be passed onto the top predators of the marine food web (and even humans) and cause events of mass mortality (Bourdelais et al., 2002; De La Riva et al., 2009; Fire et al., 2010;

Flewelling et al., 2005; Geraci et al., 1989; Glibert et al.,

White 1977, 1980, 1981).

Cephalopods occupy an important position in the marine food web.

They are known to be preyed upon by marine mammals (Clarke and

Goodall, 1994; Daneri et al., 2000; Pauly et al., 1998), top predatory fish (Brock, 1985; Stilwell and Kohler, 1982) and seabirds (Croxall and Prince, 1996). On the other hand, they

- 81 - prey mostly on crustaceans, small fish and other molluscs, which are known vectors of marine phycotoxins (Hanlon and Messenger,

1996; Nixon, 1985; Rodhouse and Nigmatullin, 1996). Although these organisms play a key role in the link between primary production and higher trophic levels, there are still few studies on the transference and accumulation of marine toxins.

This review summarizes, for the first time, the current knowledge of HAB-related toxins in these highly versatile and opportunistic mollusc predators.

Cephalopod life strategies and feeding ecology

The class Cephalopoda is a very diverse group of exclusively marine molluscs that is currently represented by octopods, squids, sepiolids, cuttlefishes and the pre-historic Nautilus.

There are around 800 species of cephalopods inhabiting the ocean from the poles to the tropics, from intertidal pools to the abyssal trenches, exhibiting various life strategies (Rosa et al., 2012, 2008a, 2008b). Cephalopods are considered to be the most evolved invertebrates, since they can be compared to vertebrates in many features, such as their panoply of complex behaviours, well-developed senses and highly developed nervous system with a complex brain (Eno, 1994; Wells, 1988; Wells and

Wells, 1983, 1986). They are also known for their ability to mimic the surrounding environment with changes in colour and texture of their skin in order to avoid predators, to capture prey or even to communicate with each other (Hanlon et al.,

2009).

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Regarding the feeding ecology, cephalopods are voracious carnivores with many different feeding strategies (including cannibalism, Figure 1) that enable them to feed opportunistically on a wide range of prey (e.g., Table 1).

Nektobenthic cephalopods such as cuttlefish, can swim in the water column through the constant undulation of the fins but spend considerable amounts of time buried in the sediment to rest or to ambush their prey (sit-and-wait strategy; Figure 2A-

C). On the other hand, benthic cephalopod fauna, mostly represented by Octopoda, (Figure 2D) and Sepiolida species

(Figure 2E, F), spend all of their lives on or near the bottom.

Figure 1. Weight (%W) of prey in the stomach contents of jumbo squid

(Dosidicus gigas) from the Gulf of California during 1998–1999 (data from Markaida and Sosa-Nishizaki, 2003). %W is defined as the weight of a certain prey relative to the total weight of all prey, expressed as a percentage. Legend: UOM - unidentified organic matter.

After some external digestion, the flesh is consumed, and the exoskeleton rejected. Newly hatched octopods (paralarval stage;

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Figure 2H,I) feed on planktonic crustaceans and when they settle to the seabed they switch the diet to nektobenthic crustaceans and benthic molluscs (Villanueva, 1994).

Table 1. Diet of common octopus (Octopus vulgaris) in the Portuguese coast, namely in Viana do Castelo (North region), Cascais (Centre region) and Tavira (South region). Occurrence index (OCI) of prey found in the octopods’ stomach contents. [data from Rosa et al.

(2004)].

Occurrence index (OCI) Prey category Viana Cascais Tavira ANNELIDA 1.85 2.52 - Polychaeta 1.85 2.52 -

unidentified CRUSTACEA 38.27 67.31 36.9 Isopoda unidentified - - 0.18 Decapoda Natantia 1.93 6.04 2.57 Natantia unidentified 1.93 3.43 2.57 Caridae unidentified - 2.61 -

Decapoda Reptantia 36.34 61.27 34.14 Anomura - - 0.33 Paguridae unidentified - - 0.26 Pagurus prideauxi - - 0.07 Brachyura 36.34 61.27 33.81 Brachyura unidentified 18.96 39.33 17.51 Portunidae 10.84 21.94 10.17

unidentifiedLiocarcinus sp. 5.17 - 5.03 Polybius henzlow 1.36 - 1.1

MOLLUSCA 7.13 14.89 23.52 GASTROPODA 1.49 1.87 0.61 BIVALVIA 1.75 7.43 16.56 unidentified

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Carditidae - - 1.07

unidentifiedCardiidae unidentified - - 0.73 Mytilus sp. - - 1.72 Venus sp. - - 1.48 Bivalvia unidentified 1.75 7.43 11.56 CEPHALOPODA 3.9 5.59 6.35 Sepiolidae - - 1.39

unidentifiedSepia sp. - - 0.73 Illex coindetii 0.71 - - Octopus sp. 0.18 0.83 0.36 Cephalopoda 3.01 4.75 3.87

unidentified OSTEICHTHYA 52.74 15.28 39.59 Clupeidae unidentified 16.7 3.29 12.94 Gobidae unidentified 5.8 4.53 1.43 Osteichthya 30.24 7.46 25.22

unidentified The juveniles of coastal squids (Figure 2G) and cuttlefish

(Figure 2A-C) mostly prey on crustaceans but shift their diet to fish and cephalopods as they grow. The sepiolids (Figure 2E,F) feed almost exclusively on mysids and decapods crustaceans, neglecting crabs and fish (Boyle and Rodhouse, 2005).

- 85 -

Figure 2. Cephalopod coastal diversity and respective life strategies.

A, B and C - nektobenthic common cuttlefish, Sepia officinalis; D - benthic common octopus, Octopus vulgaris, E and F - benthic sepiolid,

Sepiola atlantica, G – semi-pelagic squid, Loligo vulgaris; H and I – planktonic paralarvae of common octopus, O. vulgaris. (photo credits:

Rui Rosa).

In the oceanic region, in order to support their particular life-style (e.g., high growth rates, short lifespan, semelparity) and physiological traits (e.g., high metabolic rates associated with the energetically-inefficient jet propulsion), most pelagic squids are well adapted to the seasonality and spatial patchiness of food resources.

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Figure 3. Total concentration (median, 25 and 75 quartiles, non- outlier range and outliers) of paralytic shellfish toxins (PSTs, µg

STX equiv. kg-1) present in the tissues of common octopus, Octopus vulgaris (data from Monteiro and Costa, 2011) collected in the NW

Portuguese coast. Abbreviations: GD - digestive gland; Kid – kidney;

St+Int+Ce - pooled stomach, caecum and intestine; BHr – branchial hearts; Sal - posterior salivary glands. ; Gill – Gills

To maintain their growth and maturation rates, they are known to make extensive migrations to exploit the latitudinal differences in productivity (Rosa et al., 2013a, 2013b, 2008b). Some oceanic squids also undertake sporadic (or recurrent) migrations over the continental shelves to feed (e.g., Todarodes sagittatus,

Dosidicus gigas). Moreover, most of these squids exhibit daily vertical migrations, from the darker and colder waters of the mesopelagic zone to the warm, well-lit and more productive epipelagic zone. One of the most well-known examples is the

- 87 -

Humboldt squid, D. gigas, a large ommastrephid squid that lives around 300 m during the day and migrates to the surface at night to feed (Rosa and Seibel, 2010), mainly on planktivorous fish, like myctophids, anchovies and sardines (Markaida and Sosa-

Nishizaki, 2003). It, thus, represents an important link in the exchange of energy throughout the water column.

The wide variety of life (and feeding) strategies makes cephalopods an important link in the marine food web (Figure 2), by connecting the primary production to the higher trophic levels. Consequently, they also play a key role on the transference and accumulation of marine toxins.

HAB-toxins in cephalopods

Paralytic Shellfish Toxins

Studies on the accumulation and tissue distribution of PSTs in cephalopods are limited and only focused in three species, namely the common octopus (O. vulgaris), the Humboldt squid (D. gigas) and the Australian octopus (Octopus (Abdopus) sp. 5)

(Braid et al., 2012; Monteiro and Costa, 2011; Robertson et al.,

2004) (Table 2). The highest PST levels are observed in DG because this organ is the primary site of digestive absorption and intracellular digestion in cephalopods (Rosa et al., 2005,

2004). Thus, it may act as PST reservoir, as it is for other substances (other toxins, lipids, contaminants, etc.) (Grisley and Boyle, 1988). PSTs accumulated to the greatest extent in DG

>> kidneys > stomach > branchial hearts > posterior salivary glands > gills of O. vulgaris (Figure 3). Toxin concentrations, in terms of saxitoxin equivalents, ranged from 390 to 2680 μg

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STX equivalents kg−1 in the DG; from 44 to 390 μg STX equivalents kg−1 in the kidneys; from 21 to 210 μg STX equivalents kg−1 in the stomach, from 14 to 140 μg STX equivalents kg−1 in salivary glands and from not detected to 180 μg STX equivalents kg−1 in branchial hearts (Monteiro and Costa, 2011).

Table 2. Maximum levels of HAB-associated toxins in cephalopods.

Maximum levels Toxin Species Tissue/organ Reference found Octopus Digestive Costa et 166.2 μg DA g-1 vulgaris gland al., 2004 Eledone Digestive Costa et 127 μg DA g-1 moschata gland al., 2005a Eledone Digestive Costa et 18.8 μg DA g-1 cirrhosa gland al., 2005a Sepia Digestive Costa et DA 241.7 μg DA g-1 officinalis gland al., 2005b Doryteuthis Bargu et 0.37 μg DA g-1 Stomach opalescens al., 2008 Dosidicus Digestive Braid et 0.23 μg DA g-1 gigas gland al., 2012

Monteiro Octopus 35 µg STX equiv. Digestive and Costa, vulgaris g-1 gland 2011 Dosidicus 4.83 µg STX equiv. Digestive Braid et PSTs gigas g-1 gland al., 2012 Octopus Robertson, 2.46 µg STX equiv. (Abdopus) Arms et al., g-1 sp.5 2004

Toxin profile on the DG of the O. vulgaris, collected from the

Portuguese coast where Gymnodinium catenatum is the main PST producer, was constituted by C1+2, dcSTX, GTX2+3, B1, STX and dcNEO (Figure 4). The toxin profile of the remaining organs, with the exception of organs with excretory functions was

- 89 - limited to B1, dcSTX and STX. In addition to the DG, dcNEO was found in kidneys and branchial hearts. Small amounts of C1+2 were also present in kidneys. B1 and dcSTX were the most abundant toxins in all tissues analysed. They comprised about

70% of the PSTs molar fraction in the DG. In the remaining organs B1 and dcSTX justified more than 90%. Decarbamoyl saxitoxin was the most abundant toxin detected in all tissues except for the organs with excretory function, where B1 was the dominant toxin. Selective elimination of PSTs with higher elimination of B1 and retention of dcSTX is suggested in this study for the common octopus (Monteiro and Costa, 2011). It is worth noting that the PSTs regulatory limit for closures to shellfish harvesting is 800 μg STX equivalents kg−1.

Analyses undertaken in Humboldt squids also revealed a wide range of PST levels in both stomach and DG (Figure 5). The PST levels in the stomach ranged from 470 to 7700 μg STX equivalents kg−1 and in the DG ranged from 2910 to 4830 μg STX equivalents kg−1, the latter being up to 6 times greater than the regulatory limit of PST in shellfish. The toxin profile was predominantly

STX, but low levels of dcSTX were also detected (Figure 5)

(Braid et al., 2012).

Unlike O. vulgaris and D. gigas, which accumulate PSTs in their viscera (especially the DG) and not in the edible part (i.e., mantle and arms), the Australian Octopus (Abdopus) sp. 5 is known to accumulate and retain STX in the arms, with no measurable levels of other derivatives. Since this study was only focused on the edible portion of the octopus, no other organs and tissues were analysed.

- 90 -

Figure 4. Concentration of paralytic shellfish toxins (C1 + 2 N- sulfocarbamoyl-gonyautoxin-1 and -2; dcSTX decarbamoyl saxitoxin; GTX2

+ 3 gonyautoxin-2 and -3, B1 gonyautoxin-5 or GTX5; STX saxitoxin; NEO neosaxitoxin) in several tissues of common octopus, Octopus vulgaris

(data from Monteiro and Costa, 2011) collected from the NW Portuguese

- 91 - coast. Abbreviations: GD- digestive gland; Kid – kidney; St+In+Ce – pooled stomach, intestine and caecum; BrH – branchial hearts; Sal – posterior salivary glands; Gill – Gills.

Here, and unlike most cases where this toxin has a dinoflagellate origin, the contamination seemed to be caused by the red algae Jania sp. (Robertson et al., 2004). The risk of human poisoning by the consumption of cephalopods is considerably lower for the common octopus and the Humboldt squid, since the PST storage is made exclusively in the viscera, which is usually inedible and removed for human consumption

(Braid et al., 2012; Costa et al., 2009; Monteiro and Costa,

2011). On the other hand, the consumption of the Australian octopus presents high risk of paralytic shellfish poisoning, since the allocation of the toxin is within the most edible portion of this small octopus, the arms (Robertson et al.,

2004).

Amnesic Shellfish Toxins

Non filter-feeding organisms accumulate DA through transfer from lower to higher trophic levels, since Pseudo-nitzschia diatoms can be preyed upon by both benthic and pelagic members of the food web (Landsberg, 2002).

Cephalopods have been reported to accumulate and store DA in their tissues (Figures 6 and 7). Highly variable DA levels, ranging from 1.1 to 166.2 μg DA g−1 were found in the digestive gland (DG) of the common octopus (Octopus vulgaris) off the

- 92 -

Portuguese coast (Figure 7A; Table 2). As with PSTs, DA accumulated mainly in DG.

Ontogenetic studies on the accumulation of DA in the DG of O. vulgaris also showed that younger octopus presented higher DA levels (Costa and Pereira, 2010) due to their faster growth and food conversion rates (Garcia and Gimenez, 2002).

- 93 -

Figure 5. Concentrations of paralytic shellfish toxins detected in

Humboldt squids (Dosidicus gigas) from stranding events in British

Columbia, Canada. *STX eq. = STX + dcSTX*0.51 (data from Braid et al.

- 94 -

2012). Abbreviations: DG – digestive gland; St – stomach; Man – mantle.

It has also been shown that younger octopus choose smaller mussel size classes (Smale and Buchan, 1981), which are known to concentrate higher levels of toxins.

Figure 6. Schematic illustration of the maximum levels (µg g-1) of amnesic shellfish toxin, domoic acid (DA) found in common octopus

(Octopus vulgaris) tissues.

Moreover, female octopus accumulate higher DA levels in the DG than males (Costa and Pereira, 2010), probably because females have greater energy requirements (and subsequent higher metabolic and food conversion rates) for egg production (Garcia and Gimenez, 2002).

Typically, DA levels are detected during, or shortly after, blooms of DA producing algae, but, although in relatively low concentrations (1.0 to 26.6 μg DA g−1), DA was detected in the DG

- 95 - of O. vulgaris 4 to 5 months after the bloom, suggesting a retention capability of DA in the octopus’ system for long

A

B

C

periods of time (Lage et al., 2012).

- 96 -

Figure 7. Domoic acid levels (DA, µg g-1; median, 25 and 75 quartiles, non-outlier range and outliers) detected in the tissues of: A) common octopus (Octopus vulgaris), collected in the NW and South Portuguese coast, B) common cuttlefish (Sepia officinalis), collected in the NW

Portuguese coast, and C) Humboldt squid (Dosidicus gigas) collected in

British Columbia, Canada (data from Braid et al., 2012; Costa et al.,

2004; Costa et al., 2005b). Abbreviations: DG- digestive gland; BrH – branchial hearts; Gill – Gills; Kid – kidney; Gon – gonads; St+Ins – pooled stomach, caecum and intestine; SaG - posterior salivary glands;

DgT – digestive tract; Man – mantle.

Sub-cellular partitioning of DA in the soluble and insoluble fractions (nuclei, mitochondria, lysosome, microsome) showed that nearly all DA (92.6%) is found in cytosol (Figure 8), irrespective of toxin levels. The distribution of the remaining

DA in each fraction was found in the following decreasing order: nuclei > lysosomes > mitochondria > microsomes (Lage et al.,

2012, Figure 8). This favours the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The branchial hearts also accumulate highly variable levels of DA, ranging from 3.0 to 67.1 μg DA g−1, surpassing, in some cases, the values detected for the DG

(Figure 7A,B). The branchial hearts are located at the base of the gills, receiving deoxygenated blood from body tissues. From there, blood is then sent to the gills where it is oxygenated.

In addition to this pumping function, the branchial hearts have an excretory role (Schipp and Hevert, 1981). Moreover, it has been demonstrated that these organs are able to accumulate high concentrations of some heavy metals (Miramand and Guary, 1980;

- 97 -

Nakahara et al., 1979; Nakahara and Shimizu, 1985), for this reason, they have also been called “kidneys of accumulation”

(Martin and Aldrich, 1970).

However, DA concentrations in the kidney are substantially lower

(0.2–3.5 μg DA g−1) (Figure 7A,B) (Costa et al., 2004). Further studies were performed on horned octopus (Eledone cirrhosa), the musky octopus (E. moschata) and common cuttlefish (Sepia officinalis) off the Portuguese coast. Again, all these studies showed that the DG is the main organ of DA accumulation (Figures

6 and 7; Table 2) (Costa et al., 2004; Costa et al., 2005a;

Costa et al., 2005b; Costa and Pereira, 2010).

Figure 8. Domoic acid distribution in the DG cell fractions of the common octopus (Octopus vulgaris; median, 25 and 75 quartiles, non- outlier range and outliers) collected in the NW Portuguese coast (data from Lage et al., 2012).

- 98 -

It is worth noting that DA levels in the DG of E. moschata were higher than in E. cirrhosa (ranging from 0.8 to 127 μg DA g−1;

Figure 9). Although these species are very similar and their geographic and bathymetric range partially overlaps, E. moschata generally inhabits shallower waters in the Portuguese Southern coast and, therefore, has a different feeding regime (Costa et al., 2005a). The horned octopus feeds mainly in crustaceans, but other types of prey have been reported as well, like fish, cephalopods, polychaetes and gastropods (Boyle, 1983; Grisley et al., 1999; Sanchez, 1981). Unfortunately, there is no available information about the diet of the musky octopus, except for laboratory studies that have shown that this octopus readily feeds on crabs, and thus the possible vector of DA remains unclear (Mangold, 1983).

Figure 9. Domoic acid levels (DA; μg kg−1; median, 25 and 75 quartiles, non-outlier range and outliers) detected in the DG of Eledone cirrhosa

(from NW, SW and South Portuguese coast) and E. moschata (from South

Portuguese coast) (data from Costa et al., 2005a).

- 99 -

The only study performed on the common cuttlefish, S. officinalis, revealed high DA levels in the DG and branchial hearts, ranging from 1.1 to 241.7 μg DA g−1 and 5.1 to 140.8 μg

DA g−1, respectively (Figure 7B). This study also showed that levels of DA persist through long periods of time after the bloom of Pseudo-nitzschia, and that the toxic profile suggests that there is degradation of DA into less neurotoxic isomers by the branchial hearts (Costa et al., 2005b).

The available information on the accumulation and tissue distribution of DA is very scarce for squid. The loliginid

Doryteuthis opalescens has shown to accumulate much lower DA levels than the other cephalopod groups mentioned above. The highest levels of DA were found in the stomach (0.37 μg DA g−1), while the viscera (including DG) presented values around 0.10 and 0.19 μg DA g−1 (Bargu et al., 2008).

Figure 10. Stranded Humboldt squids (Dosidicus gigas) in Californian

- 100 -

(top panels, and bottom left and middle panels) and Mexican coasts

(bottom right panel).

Additionally, stranded Humboldt squids (Figure 10) have also been shown to present low levels of DA in the stomach, gonad, DG and mantle. As expected, the highest concentrations of DA were found in the DG (up to 0.23 μg DA g−1) (Figure 7C) (Braid et al.,

2012). For decades there have been records of periodic events of massive strandings of Humboldt squids (Figure 10), mostly juvenile specimens, on the beaches of the Eastern Pacific Ocean.

However, their frequency has increased, and the geographical profile has changed, since there are now records from the

Mexican shore to Alaska. One of the explanations for the occurrence of these strandings is the uptake of DA through consumption of planktivorous fish, like the pacific sardine, during blooms of Pseudo-nitzschia spp. (Costa and Garrido,

2004). This implies important cascading effects in the marine food web, since Humboldt squid make up a large portion of the diet of many top marine predators (Davis et al., 2007). The

Humboldt squid may act as DA vectors to dolphins, whales and sea lions’ populations, which may be in jeopardy considering the devastating effects DA has, as reviewed above.

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(Lamarck, 1798) (Mollusca, cephalopoda) dans la mer

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the east coast of South Africa. Mar. Biol. 65, 1–12.

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estimates of daily ration of the shortfin mako (Isurus

oxyrinchus) in the Northwest Atlantic. Can. J. Fish. Aquat.

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cephalopod paralarvae. Aquaculture 128, 143–152.

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The most beautiful thing we can experience is the mysterious. It is the source of all true art and science - Albert Einstein

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CHAPTER THREE

UPTAKE, TRANSFER AND ELIMINATION KINETICS OF PARALYTIC SHELLFISH

TOXINS IN COMMON OCTOPUS (OCTOPUS VULGARIS)

The material in this chapter is published as:

Lopes, V.M., Baptista, M., Repolho, T., Rosa, R., Costa, P.R.,

2014. Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris). Aquatic

Toxicology 146, 205-211. DOI: 10.1016/j.aquatox.2013.11.011

- 115 -

- 116 -

Uptake, transfer and elimination kinetics of paralytic shellfish toxins in common octopus (Octopus vulgaris)

Vanessa M. Lopesa,b, Miguel Baptistaa, Tiago Repolhoa, Rui Rosaa, Pedro Reis Costab

a Laboratório Marítimo da Guia, Centro de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, Av. Nossa Senhora do Cabo, 939,2750-374 Cascais, Portugal b IPMA – Instituto Português do Mar e da Atmosfera, Avenida de Brasília, 1449-006 Lisboa, Portugal

- 117 -

- 118 -

Abstract

Marine phycotoxins derived from harmful algal blooms are known to be associated with mass mortalities in the higher trophic levels of marine food webs. Bivalve molluscs and planktivorous fish are the most studied vectors of marine phycotoxins.

However, field surveys recently showed that cephalopod molluscs also constitute potential vectors of toxins. Thus, here we determine, for the first time, the time course of accumulation and depuration of paralytic shellfish toxins (PSTs) in the common octopus (Octopus vulgaris). Concomitantly, the underlying kinetics of toxin transfer between tissue compartments was also calculated. Naturally contaminated clams were used to orally expose the octopus to PSTs during 6 days. Afterwards, octopus specimens were fed with non-contaminated shellfish during 10 days of depuration period. Toxins reached the highest concentrations in the digestive gland surpassing the levels inthe kidney by three orders of magnitude. PSTs were not detected in any other tissue analyzed. Net accumulation efficiencies of 42% for GTX5, 36% for dcSTX and 23% for C1+2 were calculated for the digestive gland. These compounds were the most abundant toxins in both digestive gland and the contaminated shellfish diet. The small differences in relative abundance of each toxin observed between the prey and the cephalopod predator indicates low conversion rates of these toxins. The depuration period was better described using an exponential decay model comprising a single compartment – the entire viscera. It is worth noting that since octopuses’

- 119 - excretion and depuration rates are low, the digestive gland is able to accumulate very high toxin concentrations for long periods of time. Therefore, the present study clearly shows that

O. vulgaris is a high-potential vector of PSTs during and even after the occurrence of these toxic algal blooms.

Keywords: saxitoxin, octopus, harmful algae, accumulation, depuration, marine toxins, neurotoxin, PSP.

- 120 -

1. Introduction

Marine toxins produced by harmful algal blooms (HAB), generally as secondary metabolites, alter cellular processes of other organisms from plankton to humans. Bioaccumulated toxins can be transferred up through the marine food web and ultimately cause events of mass mortality of top predators, such as marine mammals and sea birds [see reviews in Landsberg, 2002; Lopes et al., 2013]. Bivalve molluscs and planktivorous fish are traditionally the most studied vectors of toxins in marine food webs. Recent field observations have reported cephalopods as highly potential toxin vectors as well (Costa et al., 2004,

2005a, b, 2009; Costa and Pereira, 2010; Monteiro and Costa,

2011). These molluscs are voracious and opportunistic predators that occupy a key position in the coastal marine food webs.

Cephalopods prey mostly on crustaceans, small fish and other molluscs, including cannibalism (Hanlon and Messenger, 1996;

Nixon, 1985; Rodhouse and Nigmatullin, 1996). At the same time, they constitute important items in the diets of marine mammals

(Clarke and Goodall, 1994; Daneri et al., 2000; Pauly et al.,

1998) and top predatory fish (Brock, 1985; Stillwell and Kohler,

1982).

Among the panoply of known marine toxins, the highly potent and neurotoxic paralytic shellfish toxins (PSTs), produced by three dinoflagellate genera, Gymnodinium, Pyrodinium and Alexandrium, have been reported to accumulate in three different cephalopod species, namely the common octopus (Octopus vulgaris), the

Humboldt squid (Dosidicus gigas) and the Australian octopus

[Octopus (Abdopus) sp. 5] (Braid et al., 2012; Costa et al.,

- 121 -

2009; Monteiro and Costa, 2011; Robertson et al., 2004). Mass mortality events attributed to PSTs were also shown to affect top oceanic predator cephalopods, namely the Humboldt squid.

Evidence points toward marine toxins (PSTs and/or domoic acid) as the cause of strandings of these voracious predators (Braid et al., 2012; Lopes et al., 2013).

PSTs block conduction of electrical impulses in axons by reversely binding to voltage-gated sodium channels, inhibiting neuronal transmission (Henderson et al., 1973). In other marine organisms, such as fish, PSTs are known to cause impairments of sensorimotor function and decreased larval and adult survivals

(Lefebvre et al., 2005; Robineau et al., 1991; Samson et al.,

2008; White, 1981). Numerous compounds belong to the PST family, which are most commonly divided into three groups, based on their chemical structure: carbamoyl (saxitoxin – STX, neosaxitoxin– NEO, gonyautoxins – GTX 1–4), (ii) decarbamoyl

(derivatives of STX, NEO and GTX), and (iii) sulfamate (C-toxins

1–4, B1 – GTX5, B2– GTX6) toxins. A hierarchy of PST toxicity was established based on their neurotoxicity potential with the carbamoyl group containing the most toxic compounds [STX, neosaxitoxin (NEO), gonyautoxins (GTX 1–4)], followed by the decarbamoyl group that includes the decarbamoyl derivatives of

STX, GTX1-4 and NEO. The sulfamate group is the least toxic and comprises the four C-toxins and also B1 (GTX5) and B2 (GTX6)

(Oshima, 1995).

From the few PST-related studies conducted in cephalopods so far, it is known that the PST levels are highest in the digestive gland (DG). For instance, in O. vulgaris, PSTs

- 122 - accumulate to the greatest extent in DG (ranging from 390 to

2680 µg STX equiv. kg−1) >> kidneys > stomach > branchial hearts

> posterior salivary glands > gills. The respective toxin profile was constituted by C1+2, dcSTX, GTX2+3, B1, STX and dcNEO (Monteiro and Costa, 2011). As for the Humboldt squid,

PSTs were detected only in the stomach and DG, with the values of latter tissue ranging from 2910 to 4830 µg STX equiv. kg−1.

The most predominant toxin was STX, but low levels of dcSTX were also found (Braid et al., 2012). Last, it is worth noting that significant levels of STX were detected in the arms of the

Australian octopus, which may represent health hazards for human consumption (Robertson et al., 2004).

Toxin kinetics and depuration dynamics in cephalopods are still unknown. On the other hand, they are well documented in bivalve molluscs (Blanco et al., 2003; Botelho et al., 2010; Bricelj and

Shumway, 1998; Galimany et al., 2008; Li et al., 2005; Silvert and Cembella, 1995; Yu et al., 2007) and some fish (Costa et al.,2011; Kwong et al., 2006). The uptake, transformation and elimination are simultaneous processes, thus, it is difficult to directly measure them. One- and two-compartment kinetic models have been developed to describe changes in phycotoxins concentration, including the PSTs, in shellfish based upon the balance between input and output rates (Blanco et al., 2003;

Silvert and Cembella,1995; Yu et al., 2007). The one-compartment model assumes that toxin elimination occurs at a constant rate following an exponential decrease throughout the depuration period. The second category uses a multi-compartment distribution kinetic with a rapid initial phase, usually

- 123 - associated with an organ/tissue with high elimination rates, followed by a period of slower toxin loss as a result of residual toxin concentrations that may be retained or bound to particular organs/tissues, thus a two-compartment model. Within this context, here we assessed, for the first time, the time course of accumulation and depuration of PSTs under laboratory conditions in O. vulgaris and determined the kinetics behind the transfer of toxins between compartments.

2. Materials and methods

2.1. Collection and laboratorial maintenance of octopus

Thirty-three specimens of juvenile octopus (O. vulgaris; ranging from 115 to 331 g weight and from 5.1 to 10.5 cm mantle length) were obtained from traps employed by local fishermen between

February and March 2013 in Cascais, Portugal. After collection, organisms were transferred to the aquaculture facilities of

Laboratório Marítimo da Guia (Cascais). They were placed in individual 9 L seawater aquaria connected in parallel to a 270 L sump equipped with a wet-dry filter with bioballs (assuring biological filtration), a protein skimmer and one 36 W ultraviolet sterilizer. Natural sea-water was 1 µm filtered, with salinity being maintained at 34 ± 1 through the regular addition of freshwater purified by reverse osmosis and temperature was kept stable through the use of Hailea heating/cooling systems. The tanks were illuminated from above with a photoperiod of 14 L:10 D. Ammonia and nitrite were monitored every other day and maintained below detectable

- 124 - levels. Nitrate and pH showed average values (± standard deviation, SD) of 7 (± 2.5) mg L−1 and 8.1 (± 0.1), respectively.

Table 1. Toxin profile of Donax clams given to octopus (mean ± SD).

Concentration µg g-1 Molar Fraction % Toxin (SD) (SD) dcGTX2+3 0.23 (0.06) 3.2 (1.1)

C1+2 0.92 (0.13) 9.1 (2.2)

dcSTX 1.35 (0.20) 24.3 (1.2)

B1 5.19 (0.71) 63.1 (2.2)

STX 0.02 (0.00) 0.3 (0.1)

2.2. Preparation of contaminated octopus diet

Naturally contaminated clams (Donax sp.) were used to expose octopus to PSTs through a dietary route. Clams (0.64 ± 0.16 g) were collected in Olhão (South coast of Portugal) in September

2012 during a bloom of Gymnodinium catenatum. Clams were checked for the presence of PSTs by means of liquid chromatography.

Thirty clams were divided in 3 samples containing 10 clams each.

The PSP toxicity measured was 2665 ± 330 µg STX equiv. kg−1.

Toxin profile is presented in Table 1.

- 125 -

Figure 1. Schematic view for the five compartments of Octopus vulgaris. The solid lines represent the transfer coefficients from the digestive gland to the other compartments. The dashed lines represent the elimination rates of the five compartments. The other parameters used are described in Table 2.

2.3. PST exposure experiments

Previous to the exposure experiments, three specimens were selected for PSTs analysis. Having checked that octopuses were not naturally contaminated with PSTs, octopuses were fed with 10 contaminated clams every day for 6 days. Daily ingestion rates were 3.2 ± 0.9 % octopus body weight. In the following 10 days non-contaminated shellfish replaced the toxic diet. Every 24 h after feeding three specimens were randomly sampled and the digestive gland (DG), kidney, branchial hearts, salivary glands, gills and mantle were carefully dissected, weighed and prepared for toxin analysis. Although PST has been only associated with organs/tissues from the viscera of this octopus species, a

- 126 - portion of the mantle from each specimen was analysed to confirm whether toxins reached this tissue.

2.4. Toxin extraction and quantification

Toxins from the organ homogenate were heat-extracted in 1 % acetic acid, vortexed, and centrifuged (15,000 × g) for 10 min.

Extracts followed a solid-phase extraction (SPE) with octadecyl bonded phase silica (Supelclean LC-18 SPE cartridge, 3 mL,

Supelco, USA). Periodate and peroxide oxidations of PSTs were carried out and toxins were immediately quantified by high performance liquid chromatography with fluorescence detection

(HPLC-FLD) based on the precolumn oxidation method developed by

Lawrence and Niedzwiadek (2001). The HPLC-FLD equipment consisted of a Hewlett-Packard/Agilent Model 1050 quaternary pump, Model 1100 in-line degasser, autosampler, column oven, and

Model 1200 fluorescence detector. The PSTs oxidation products were separated using a reversed-phase Supelcosil LC-18, 15 ×

4.6, 5 µm column (Supelco, USA). The mobile phase gradient consisted of 0 - 5% B (0.1 M ammonium formate in 5% acetonitrile, pH 6) in the first 5 min, 5 – 70% B for the next 4 min and back to 0% B in the next 2 min. Then 100% mobile phase A

(0.1 M ammonium formate, pH 6) used for 3 min before the next injection. Flow rate was 1 mL min−1 and the detection wavelength set to 340 nm for excitation and 395 nm for emission.

Instrumental limits of detection (S/N = 3) were 5 ng g−1 dcSTX, 9 ng g−1 STX, 12 ng g−1 B1, 19 ng g−1 for dcGTX 2+3 and GTX 2+3, 34 ng g−1 C 1+2. Certified calibration solutions for PSTs were purchased from the Certified Reference Materials Program of the

- 127 -

Institute for Marine Biosciences, National Research Council,

Canada (STX-e, NEO-b, GTX 2+3-b, GTX 1+4-b, dcSTX, dcGTX 2+3,GTX

5-b (B1), C 1+2 and dcNEO-b). For the digestive gland and mantle three replicates per day were used, resulting in individual analyses for each specimen, however, for the other tissues pooled samples were used due to their reduced mass.

Table 2. Parameters used in kinetics equations. DG – digestive gland,

KD – kidney, BH – branchial hearts, SG – salivary glands, GL - gills,

MT – mantle.

Paramete Description Unit r µg C Toxin concentration in digestive gland (DG) DG g-1 Toxin concentration in the i-th compartment µg C i (KD, BH, SG, GL, MT) g-1 α Net accumulation effiency % g d- F Feeding rate 1 µg C Toxin concentration in food feed g-1 -1 kel DG Elimination rate from digestive gland d -1 kel i Elimination rate from i-th compartment d Transfer coefficient from digestive gland to K d-1 T i the i-th compartment Toxin concentration in digestive gland at µg q DG initial conditions of depuration g-1 Toxin concentration in the i-th compartment at µg q i initial conditions of depuration g-1 t Time days

2.5. Modelling

The model developed for the present study was based on the knowledge that the DG is the prime site of digestive absorption and storage of numerous substances, including PSTs (Monteiro and

Costa, 2011). Toxins are then transferred to the other tissue compartments (i.e. kidney, branchial hearts, salivary glands, gills and mantle). The model included six compartments, the DG

- 128 - being the first and the other five the remaining tissue compartments (i = kidney – KD, branchial hearts – BH, salivary glands – SG, gills– GL and mantle – MT) shown in Fig. 1. The biokinetics of uptake and depuration of the six compartments were modelled by first-order kinetic models. The equations used to fit experimental data of PSTs in octopus tissues are:

Uptake in digestive gland:

(1)

Uptake in the i-th compartment:

(2)

Depuration in digestive gland:

(3)

Depuration in the i-th compartment:

(4)

The description of the parameters involved in Eqs. (1) – (4) and

Fig. 1 is provided in Table 2. Octopus growth was assumed to be negligible due to the short span of the experimental period (16 days). The experimental data were fitted using the non-linear curve fit program of SigmaPlot 11.0 (Systat Software Inc.). The exponential growth model for best fit was selected after calculating the determination coefficients, R2, and examining the residuals.

3. Results

3.1. Feeding behavior, survival and toxin distribution

- 129 -

The naturally contaminated clams provided to octopus as feed were always consumed within the first hour and there was no mortality or behavioral changes during the experimental period.

Not surprisingly, the highest concentrations of PSTs were detected indigestive gland (DG) (Fig. 2). A clear exponential increase of toxin concentrations in the DG was observed during the uptake period, i.e., the first 6 days (Fig. 2). The sum of all toxin analogues at the end of uptake period was 52.2 µg g−1, corresponding in terms of toxicity to 1249.2 µg STX equiv. kg−1.

The toxin profile of the DG was dominated, in terms of molar fraction, by GTX5 (53%), followed by dcSTX (35%), C1+2 had intermediate abundance (8.5%), and the least abundant toxins were dcGTX2+3 (2.2%), GTX2+3 (1.1%) and STX (0.3%). This toxin profile resembles that of the clams ingested (Table 1). The occurrence of GTX 2+3 in octopus DG, a toxin which was not detected in the donax clams, is noteworthy. Afterwards, the shift from contaminated diet to non-contaminated one resulted in an exponential decay of toxin concentrations, although toxins were not completely eliminated during the depuration period. The relative abundance of PSTs was similar to that in the uptake period.

Table 3. Net accumulation efficiency (α, %), initial concentration in digestive gland at beginning of depuration (qDG), elimination rate (kel

-1 2 DG d ) (standard deviation) and coefficient of determination R for each PST determined in octopus digestive gland during uptake and depuration. Asterisks indicate values within the confidence limit (P <

0.05).

- 130 -

Toxin Uptake Depuration -1 2 -1 -1 2 α Kel DG (d ) R qDG (µg g ) Kel DG (d ) R dcGTX2+3 23% 0.311 0.991 1.1 (0.02)* 0.131 0.995 (0.018)* (0.008)* C1+2 23% 0.243 0.924 5.6 (0.62)* 0.229 0.958 (0.040)* (0.052)* dcSTX 36% 0.269 0.895 11.4 (0.83)* 0.193 0.9788 (0.054)* (0.029)* GTX2+3 - 0.292 0.941 0.2 (0.03)* 0.113 0.846 (0.042)* (0.045) GTX5 42% 0.336 0.948 40.6 (3.29)* 0.220 0.979 (0.048)* (0.035)* STX 18% 0.244 0.913 0.1 (0.01) 0.099 0.945 (0.043)* (0.072)

Figure 2 Concentration (µg g-1) of (a) GTX5, (b) dcSTX, (c) C1+2, (d) dcGTX2+3, (e) GTX2+3 and (f) STX in octopus digestive gland throughout

- 131 - the experiment. Dots and error bars represent experimental data (mean based on three replicate samples). The dashed line represents the outputs of the best fit model.

Paralytic shellfish toxins were also found in the kidney, although with levels three orders of magnitude lower than in the digestive gland (Fig. 3). The progressive increase of these toxins throughout the uptake period did not show a clear exponential growth as in the DG. The kidney profile of toxins was restricted to three toxins, namely GTX 5, dcSTX and C1+2, which were the dominant compounds in DG. The kidney toxin profile was not dominated by GTX5, as in the DG, since it only accounted for 11% in terms of molar fraction. In contrast, dcSTX was the most abundant toxin (45%) followed by C1+2 (39%). Toxin concentrations showed an exponential decay trend during the depuration period, and at the end of this period they were still detectable. PSTs were not detected in the remaining tissues analyzed, namely branchial hearts, salivary glands, gills and mantle.

3.2. Model fitting

The experimental data fitted to the dynamic model regarding PST accumulation in octopus DG provided a good description of toxin kinetics (Table 3, Fig 2). Using the calculated toxin concentrations at the initial conditions of the depuration period (qDG, Table 2, 3) and the elimination rate (kel DG, Table

2, 3) of the uptake period, the net accumulation efficiency (α) for each toxin was estimated (Table 2, 3). Net accumulation

- 132 - efficiency ranged between 18 and 42% for STX and GTX5, respectively. The toxin transfer coefficient from DG to the kidney (KT) was calculated using the obtained and the above

α values in equation 2, and considerably low values of transference for each toxin were observed. This finding together with the poor fit of the experimental data to the exponential models used (Table 4) lead us to adopt another approach to describe the accumulation/depuration dynamics of PST in octopus.

Instead of two compartments model, we assumed a single- compartment model in which all tissues analyzed correspond to the viscera of octopus (where the toxins are allocated).

- 133 -

Figure 3 Concentration (µg g-1) of (a) GTX5, (b) dcSTX and (c) C1+2 in octopus kidney throughout the experiment. The dashed line represents the outputs of the best fit model.

If the whole viscera are considered to be one single compartment, toxin concentrations during depuration can be calculated by:

(5)

- 134 - with mDG and mKD being the wet mass of the digestive gland and kidney, respectively, and qDG and qKD being toxin concentration in the digestive gland and kidney, respectively. Equation 4 can be simplified to describe the depuration of the single compartment:

(6)

where CV is the toxin concentration in octopus viscera, q0 is the toxin concentration at the initial conditions of the depuration period. The k el V denotes the toxin elimination rates from viscera (Table 5).

-1 Table 4. Toxin transfer rate from digestive gland to kidney (KT. d ),

-1 elimination rate (kel KD, d ) (standard deviation) and coefficient of determination R2 for each PST determined in octopus kidney during uptake.

Uptake Toxin -1 2 KT Kel KD (d ) R 0.148 C1+2 0.0927 (0.199) 0.349 0.147 dcSTX 0.0207 (0.099) 0.424 0.165 GTX5 0.0018 (0.124) 0.388

Table 5. Toxin concentration at initial depuration conditions (qo),

-1 elimination rate (kel V, d ) (standard deviation) and coefficient of determination R2 for each PST determined following one compartment model, asterisks indicate values within the confidence limit (P <

0.05).

- 135 -

Depuration Toxin -1 2 qo Kel V (d ) R 0.245 C1+2 4.9 (0.6)* (0.056)* 0.958 10.4 0.205 dcSTX (0.6)* (0.026)* 0.986 36.6 0.233 GTX5 (2.5)* (0.031)* 0.986

4. Discussion

Bioaccumulation of marine toxins in cephalopods is still poorly understood. Analyses of PSTs performed in specimens of O. vulgaris collected during blooms of G. catenatum suggested a great capacity for accumulation of these compounds (Costa et al., 2009; Monteiro and Costa, 2011). Here we provided the first conclusive evidence of such capacity under laboratory

(controlled) conditions. More specifically, we found very high

PST levels in octopus DG after six days of ingestion of contaminated clams. The toxin concentrations, in terms of saxitoxin equivalents, reached similar or higher values than those reported in the field observations (Costa et al.,2009;

Monteiro and Costa, 2011). The DG is the largest octopus organ

(with the exception of female gonads at the peak of maturity)

(Grisley and Boyle, 1988), and the prime site for substance storage (Rosa et al., 2004, 2005). For this reason, PSTs as well as other marine toxins, such as domoic acid, have been found at high levels in this tissue (Costa et al., 2004, 2009; Costa and

Pereira,2010; Lage et al., 2012; Monteiro and Costa, 2011). The profile of PSTs in the DG was dominated by the same toxins as present in the naturally contaminated clams provided here as prey to the octopuses. This indicates low toxin conversion between the bivalve prey and the cephalopod predator. On the

- 136 - contrary, biotransformation of the toxins was perceptible after feeding experiments involving fish (white seabream Diplodus sargus) and contaminated cockles (Cerastoderma edule) (Costa et al., 2011). In this case, conversion of C1+2 into their analogs, namely GTX5 was suggested (Costa et al., 2011). C1+2 are the less stable toxins and were the foremost dominant toxin analogues in fish feed with a molar fraction of about 84%, while in the present study C1+2 molar fraction did not reach 9%.

Interestingly, the octopus DG revealed, although low, levels of

GTX2+3, a toxin not detected in the clams. GTX2+3 was probably present in clams at levels below the HPLC-FLD detection limit

(19 ng g−1) and was then concentrated in the octopus digestive gland after ingestion of the 10 clams provided daily.

The fact that the toxin concentrations in the kidney were three orders of magnitude lower than in the DG, confirms low toxin transference between these tissues. The presence of PSTs in kidney, even if in low levels, may indicate that the renal processes are a route for toxin excretion, as was suggested in field studies (Monteiro and Costa, 2011). Three toxins were detected in the kidney, namely: GTX5, dcSTX and C1+2. They were the ones in highest abundance in the DG. PSTs were not detected in any other tissue, suggesting that longer periods of exposure and depuration are required to detect toxins in the remaining tissues (branchial hearts and salivary glands). As in previous studies, PSTs were not detected in the mantle indicating that toxins are allocated exclusively in the visceral tissues and are gradually released without being assimilated (Monteiro and

Costa, 2011).

- 137 -

First-order linear differential equations governing the kinetics of uptake and depuration are commonly used to predict the concentration of a given compound in marine organisms, both in invertebrates (bivalves, cephalopods) and vertebrates (fish)

(Barber, 2008; Bustamante et al., 2002; Kwong et al., 2006; Liet al., 2005; Yu et al., 2007). Here we show that the accumulation and depuration of PSTs in the common octopus can effectively be described by such models. Net accumulation efficiencies for the

DG ranged between 18 and 42% for STX and GTX5, respectively.

Comparing to other studies, O. vulgaris has shown to have higher accumulation efficiencies than fish [D. sargus – accumulating

1.7 and 5% of GTX5 and dcSTX (Costa et al., 2011)], and lower than bivalves [mussels; 67% of the GTX4 ingested (Blanco et al.,

2003)]. Elimination rates were calculated for both uptake and depuration period, with the uptake period presenting slightly higher values than depuration. With these results, sequestration or binding of the toxins can be suggested (Yu et al., 2007), which is in accordance with the elevated capacity for the common octopus to retain PSTs. On the other hand, when higher elimination rates are obtained during depuration, one may suggest the occurrence of conversion of toxins into their analogues (Costa et al., 2011; Yu et al., 2007). This was not the case in this study, in addition, identical profile of toxins was found in clams and octopuses.

After describing the toxin distribution profile, it was not surprising to observe low toxin transfer coefficients from the

DG to the kidney (Table 5; Eq. (2)). It is noteworthy that toxin transfer coefficients were considerably lower than toxin

- 138 - elimination rates (calculated for the DG), which suggests that other routes or mechanisms are associated with their elimination. Although the toxins were found in the octopus DG and in the kidney, the anatomic distribution of PSTs may not be restricted to these two tissues. It is supposed that toxin transfer may occur from digestive gland to other compartments, such as branchial hearts and salivary glands, but at even lower rates than that calculated for kidney. Therefore, discrepancies between elimination rates and toxins transfer coefficients to the kidney can be explained, in part, by elimination through transfer to other compartments at rates lower than those measurable by the analytical detection methods used. Moreover,

PSTs may be metabolized through biotransformation enzymes that are usually found in higher concentrations in digestive gland of octopus (Tang et al., 1994). The activity of detoxification enzymes has been investigated in amphipods and fish liver exposed to PST, showing increasing activities of glutathione-s- transferase (GST, Costa et al., 2012; Gélinas et al., 2013;

Gubbins et al., 2000).

One- or two-compartment exponential decay models are usually used in toxin depuration (Bricelj and Shumway, 1998; Costa et al., 2011; Kwong et al., 2006; Li et al., 2005; Mafra et al.,

2010; Yu et al., 2007). In this study, a two-compartment model was initially assumed to quantitatively predict the uptake and depuration dynamics of PSTs, based on the fact that the DG would be the primary site of absorption, storage and then release of the toxins to other visceral tissues, as was evident from previous field studies. However, during the experimental period,

- 139 - the transfer of PSTs from DG to the other tissues was not observed, except for the kidney where very low toxin concentrations were detected. In this case, we were left with the one-compartment model, which, instead of having a toxin flow from a rapidly detoxifying compartment 1 to a compartment 2 characterized by slow detoxification, has the foremost amount of the toxins in the octopuses remaining in the DG. Therefore, toxin depuration was better described with an exponential decay model from a single compartment. This model resulted from the negligible toxin transfer coefficients from digestive gland to kidney resulting in negligible toxin concentrations detected in kidney. The use of this model is only valid because there is a tissue accounting for the greatest majority of the toxin content, which in this case is the digestive gland. Similarly,

Li et al. (2005) suggested a one-compartment model after calculating negligible transfer coefficients of PSTs between hepatopancreas and other tissues of the green-lipped mussel

(Perna viridis). Silvert and Cembella (1995) also recognized that the extreme degree of simplification, i.e. one-compartment model, is often the only appropriate model to use. Although

Blanco et al. (1997) compared the two models for PST detoxification kinetics in mussels showing marginally better results with the two-compartment model, the anatomical distribution of the toxins was not examined, and the second compartment was hypothesized from the model output results.

After exposing oysters and mussels to domoic acid-producing diatoms, Mafra et al. (2010) observed that a two-compartment

- 140 - model resulted in comparable fits, but with higher degree of uncertainty.

Predictions inferred from the use of first-order kinetic models can become useful in understanding the movement of toxins throughout the various links of marine food webs, especially in cephalopods, because they play a key ecological role linking lower to higher trophic levels of the coastal marine food webs.

Octopuses showed low depuration and excretion rates, allowing the DG to accumulate PSTs at considerably high levels for longer periods of time. Thus, O. vulgaris is a potential vector of PSTs to higher levels of marine food webs.

This study points out the need to identify the mechanisms providing the octopuses with the ability to metabolize and detoxify HAB-toxins. Despite of the remarkably high levels of toxins detected no apparent harm neither signs of behavioural changes were observed. Coastal octopuses have probably evolved to acquire additional mechanisms that enable them to tolerate the HAB-toxins.

Acknowledgments

The Portuguese Foundation for Science and Technology (FCT) supported this study (in part) through project grant PTDC/BIA-

BEC/103266/2008, Programa Ciência 2007 to R. Rosa and Programa

Ciência 2008 to PR Costa. We also appreciate the valuable comments given by the two anonymous reviewers.

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The World is full of wonders, but they become more Wonderful,

not less Wonderful when Science looks at them.

- Sir David Attenborough

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CHAPTER FOUR

PRESENCE AND PERSISTENCE OF THE AMNESIC SHELLFISH POISONING

TOXIN, DOMOIC ACID, IN OCTOPUS AND CUTTLEFISH BRAINS

The material in this chapter is published as:

Lopes, V.M., Rosa, R., Costa, P.R. (2018) Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains. Marine Environmental

Research 133, 45–48. DOI: 10.1016/j.marenvres.2017.12.001

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- 154 -

Presence and persistence of the amnesic shellfish poisoning toxin, domoic acid, in octopus and cuttlefish brains

Vanessa M. Lopes. 1,2*, Rui Rosa 1, Pedro R. Costa 2,3

1 MARE – Marine Environmental Sciences Centre, Laboratório Marítimo da Guia, Faculdade de Ciências da Universidade de Lisboa, Portugal.

2 IPMA – Instituto Português do Mar e da Atmosfera, Avenida de Brasília, 1449-006

Lisboa, Portugal

3 CCMAR - Centre of Marine Sciences, University of Algarve, Campus of Gambelas, 8005-139

Faro, Portugal

Abstract

- 155 -

Domoic acid (DA) is a neurotoxin that causes degenerative damage to brain cells and induces permanent short-term memory loss in mammals. In cephalopod molluscs, although DA is known to accumulate primarily in the digestive gland, there is no knowledge whether DA reaches their central nervous system. Here we report, for the first time, the presence of DA in brain tissue of the common octopus (Octopus vulgaris) and the European cuttlefish (Sepia officinalis), and its absence in the brains of several squid species (Loligo vulgaris, L. forbesi and Todarodes sagittatus). We argue that such species-specific differences are related to their different life strategies (benthic/nektobenthic vs pelagic) and feeding ecologies, as squids mainly feed on pelagic fish, which are less prone to accumulate phycotoxins.

Additionally, the temporal persistence of DA in octopus’ brain reinforces the notion that these invertebrates can selectively retain this phycotoxin. This study shows that two highly- developed invertebrate species, with a complex central nervous system, where glutamatergic transmission is involved in vertebrate-like long-term potentiation (LTP), have the ability of retaining and possibly tolerating chronic exposure to DA, a potent neurotoxin usually acting at AMPA/kainate-like receptors.

Here, we filled a gap of information on whether cephalopods accumulated this neurotoxin in brain tissue, however, further studies are needed to determine if these organisms are neurally or behaviourally impaired by DA.

Keywords: Domoic acid; brain; cephalopod; octopus; cuttlefish; bioaccumulation; Algal toxins; Toxicity

- 156 -

- 157 -

Domoic acid (DA) is a phycotoxin produced by two genera of diatoms, Pseudo-nitzschia and Nitzschia, and is responsible for the Amnesic Shellfish Poisoning (ASP) in humans (Quilliam and

Wright, 1989). DA acts as an analogue of glutamate, an excitatory neurotransmitter, binding to the same receptors as the latter, i.e. ionotropic glutamate receptors in particular

AMPA receptor subtypes. This process leaves the receptors permanently open, causing an excessive influx of calcium ions to the cells (Hampson and Manalo, 1998). This leads to neural membrane depolarization and subsequent neurodegeneration, inducing permanent short-term memory loss in vertebrates

(Bejarano et al., 2008). This neurotoxin has also been linked to several events of mass mortality in fish, seabirds and marine mammals (see Pulido, 2008 and references therein). In the latter animal group, DA is known to elicit a variety of negative impacts, from behavioural alterations (leading to mass strandings) to abortions and premature births (Kirkley et al.,

2014; Scholin et al., 2000; Silvagni et al., 2005). It is worth noting that although stranded marine mammals (namely sea lions

Zalophus californianus and Pacific harbor seals Phoca vitulina richardii) never presented with DA in their brains, the lesions found in the hippocampus were characteristic of exposure to an neuroexcitatory toxin such as DA (Cook et al., 2015; Gulland et al., 2005; McHuron et al., 2013).

Among the invertebrate DA vectors in coastal food webs, it is known that cephalopod molluscs, in particular the common octopus

(Octopus vulgaris) and European cuttlefish (Sepia officinalis), accumulate high DA concentrations in the digestive gland (Lopes

- 158 - et al., 2013), their main storage site (Rosa et al., 2004). DA is found at much lower levels in other organs and it is undetectable in the muscle (Costa et al., 2004b). Yet, there is no information regarding DA accumulation in the brain of these cognitively skilled invertebrates.

To fill this knowledge gap, specimens of the common octopus (O. vulgaris), European cuttlefish (S. officinalis) and three squid species, namely the European squid (Loligo vulgaris), veined squid (L. forbesi) and European flying squid (Todarodes sagittatus) were collected in the NW (Peniche; 39°21′N 9°22′W) and SE (Olhão; 37°1′30″N 7°50′30″W) Portuguese coast from May to

September 2016. The detailed information about cephalopod sample size, sampling period, biometry, sex and maturation stages is provided in the Supplementary Material (Table S1). The digestive gland (DG) and brain (i.e. the supra-, sub-oesophageal masses and optic lobes) were removed, homogenized and 4 and 1 g aliquots of DG and the brain sections combined, respectively, were used for DA analysis via liquid chromatography with mass spectrometry detection - LC-MS/MS (all details in the

Supplementary Material). To compare cephalopod DA brain levels with DA- producing diatom abundance, Pseudo-nitzschia sp. data

(Jan–Oct 2016) was obtained in the Portuguese Institute for the

Sea and Atmosphere online database (IPMA, 2016).

Table 1. Domoic acid concentrations (mg DA kg-1) in the brain and digestive glands (DG) of other cephalopod species. (nd – not detected) captured in 2016.

Species n Sampling Brain DG

- 159 -

period (mg DA kg-1) (mg DA kg-1) Sepia officinalis 5 May 0.03-0.29 2.99-75.91 Todarodes 5 August nd nd sagittatus Loligo vulgaris 5 July nd nd Loligo forbesi 6 July nd nd The present study shows, for the first time, the presence of DA in brain tissue of O. vulgaris (Fig. 1A; n = 54) and S. officinalis (Table 1; n = 5), revealing that it permeates into cephalopod's central nervous system. Even though these species possess a tight blood-brain interface (Abbott et al., 1985;

Bundgaard and Abbott, 1992), the presence of DA in brain tissue of O. vulgaris and S. officinalis was consistently observed, as it was present in every single octopus and cuttlefish brain, reaching concentrations up to 2.14 and 0.29 mg DA kg−1 wet weight

(WW), respectively. Regarding other tissues, and as already observed in our previous studies (Costa et al., 2004b, 2005), DA was found at higher concentrations in the DG of O. vulgaris

(1.38–22.19 mg DA kg−1 WW; Fig. 1B) and S. officinalis (Table 1).

In contrast, there was no detectable DA content in the brain or

DG of the myopsid and ommastrephid squids (Table 1).

Accumulation of a toxin depends on the dynamic between accumulation and elimination, and the organism's feeding ecology. There are no studies regarding DA accumulation and elimination kinetics in squids. On the other hand, small pelagics are known to accumulate DA exclusively during bloom periods (Costa and Garrido, 2004; Lefebvre et al., 2001). Thus, we may argue that squids may be less prone to feed on contaminated fish, and accumulate this toxin.

- 160 -

In higher vertebrates, such as sea lions, DA toxicosis has been associated with brain lesions, seizures and memory deficits with implications for strandings (Silvagni et al., 2005; Cook et al.,

2015). In cephalopods, which have a complex and vertebrate-like central nervous system, the effects of DA are not known. The learning and memory area in octopus brain have been pointed out as similar to vertebrates with glutamate as a central neurotransmitter (Hochner et al., 2003).

- 161 -

A

B

C

Figure 1. Domoic acid (DA) concentration in mg kg-1 in octopus brain tissue (A), digestive gland (B) throughout the sample period, and

Pseudo-nitzschia sp. abundance (C) between January and October 2016.

- 162 -

Moreover, the excitatory effect of glutamate and kainic acid has already been demonstrated in octopus (Andrews et al., 1983,

1981). In fact, administration of these neurotransmitters, structurally related to DA, into the blood stream of an octopus through an aortic perfusion technique, revealed strong but transient chromatic and motor effects in the arms and mantle.

Upon injection with L-glutamate, orange and black chromatophores expanded and muscle tone increased in the injected area and mantle. Concomitantly, the presence and abundance of L-glutamate in cephalopod's brain, peripheral nervous system and muscle

(Messenger, 1996), suggest the presence of glutamate receptors in cephalopods central nervous system. The apparent lack of DA effects on octopus' brain suggests that cephalopods may possess some protective factors or defence mechanisms against DA neurotoxicity.

Another relevant result is the ability of octopus and cuttlefish to retain DA, which is a water-soluble compound usually found in marine organisms only during the toxic algae bloom or shortly after the bloom died out (Lefebvre et al., 2007). One exception to this rule are scallops, namely the king scallop, Pecten maximus, which is known to accumulate DA for long periods of time (Bresnan et al., 2017; Costa et al., 2004a).

Similarly, O. vulgaris accumulates DA for longer periods of time in the brain (and DG) implying the octopus’ ability to selectively retain DA in these tissues. As we previously showed

(Lage et al., 2012), DA is mostly present in the soluble fraction of the digestive gland, the cytosol. Thus, although the pathways for DA uptake are not known, we may argue that upon

- 163 - ingestion, DA is dissolved in the cytosol and is carried to the brain cells through haemolymph penetrating de neural cell membranes.

During the sampling period, the abundance of Pseudo-nitzschia sp. was higher in June in both sampling locations (and also in

August in Peniche; Fig. 1C). However, DA concentrations in shellfish, according to the official control for marine biotoxins carried out by IPMA (IPMA, 2016), was only found between April and June in bivalves. The presence of DA in shellfish from the Portuguese coast occurs typically during late spring-early summer, as described in Vale et al. (2008). In bivalves, DA is accumulated sporadically, and it is rapidly eliminated from most shellfish species. Scallops, as previously mentioned, are known to accumulate DA for long periods of time, and could act as a consistent source of DA for cephalopods. Yet, these organisms are not abundant along the Portuguese coast.

Thus, one can argue that the higher concentration of DA in the brain of octopuses from Olhão in June/July is likely due to the higher abundance of DA-producing diatoms, and its rapid transfer through the food web. Once DA is accumulated in octopus and cuttlefish, it is retained and not easily eliminated, contrarily as what happens in most bivalve species. Moreover, detection of

DA in cephalopod tissues was consistently found, but not in bivalve molluscs that may have acted as DA vectors.

As previously observed in octopus's DG (Costa and Pereira,

2010), a negative correlation between DA accumulation in octopus brain tissue and total weight was also found (Table 2). Such finding could be due to the fact that smaller octopuses feed on

- 164 - smaller bivalves, which contain higher toxin concentrations, since their DG accounts for a greater percentage of their body weight than in larger bivalves (Moroño et al., 2001; Novaczek et al., 1992).

Although important findings are here reported from field observations, further studies are needed to investigate whether brain-accumulated DA has any neurodegenerative effects or elicit behaviour impairments in these skilful invertebrates.

Table 2. Spearman’s rank order correlations between total weight (g), mantle length (ML, mm), gender, maturity stage (MS), domoic acid concentrations (mg DA kg-1) in the brain and digestive glands (DG) of Octopus vulgaris. Marked correlations in bold with asterisks are significant at p<0.05.

Weight ML Sex MS DG Brain (g) (mm) (mg DA kg- (mg DA kg-1) 1) Weight (g) 1.00 ML (mm) 0.40* 1.00 Sex -0.01 0.05 1.00 MS 0.48* 0.31* -0.30* 1.00 DG (mg DA kg- 0.11 -0.07 -0.14 0.11 1.00 1) Brain (mg DA -0.30* -0.23 -0.04 -0.17 0.62* 1.00 kg-1)

Conflicts of interest

None.

Authors’ contributions

PRC conceived the study, RR and PRC designed the experiment, VML and PRC performed the sample collection and toxin analysis, VML,

RR and PRC interpreted the data, statistical analysis and wrote

- 165 - the manuscript. All authors reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The research leading to these results has received funding from

Portuguese TOXMASS project (Operational Fisheries Program –

Promar 31-03-01-FEP-194). This work contributes to project

UID/Multi/04326/2013 from the Portuguese Foundation for Science and Technology (FCT). The authors would like to thank the

Portuguese Foundation for Science for the “Investigador FCT” grants to RR and PRC and the Ph.D. scholarship to V.M. Lopes

(SFRH/BD/97633/2013). We would also like to thank Pedro da

Conceição for his technical assistance.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.marenvres.2017.12.001.

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ANNEX A: SUPPLEMENTARY MATERIAL

Collection and preparation of cephalopods samples A total of 54 specimens of Octopus vulgaris, 5 Sepia officinalis, 5 Loligo vulgaris, 6 L. forbesi, 5 Todarodes sagittatus were captured in Peniche and Olhão, northwest and southeast Portuguese coast, respectively, from May to September (Supplemental Table 1). The specimens were frozen (-20 °C) for up to two weeks before analysis and the dissection of digestive glands and brain tissue (sub- and supra-oesophageal mass and optic lobes) was carried out under partially defrosted conditions. After dissection, gelatinous tissue surrounding the brain sections was removed. For each animal, the following parameters were recorded: body weight, mantle length, sex and maturity stage (Supplemental Table 1).

Domoic acid extraction and determination The extraction was carried out by blending 1 and 4 g of unwashed brain and digestive gland tissue, respectively, in 50% methanol (ratio 1:4) using a homogeniser probe, followed by 10 minutes centrifugation at 3000 g. The supernatant was filtered (0.22 µm) into a screw-cap vial and the equivalent of 1.0 mg extract (5 μl) was injected onto the column without any further clean-up. The LC-MS/MS equipment consisted of an Agilent 1290 Infinity coupled to a triple quadrupole mass spectrometer Agilent 6470. The chromatographic separation was conducted using an Zorbax SB- C8 RRHT (2.1 x 50 mm, 1.8 µm), protected with a guard column (2.1 x 5 mm, 1.8 µm). Elution was achieved using a binary eluent system: eluent A was water with 2 mM ammonium formate and 50 mM formic acid, and eluent B was 95% acetonitrile with 2 mM ammonium formate and 50 mM formic acid. A binary elution gradient was used at a flow rate of 0.4 mL min-1 as follows: 0- 0.3 min 95 % A, gradient from 95 to 40 % eluent A at 2.5 min; and 95 % eluent A from 2.51 to 3.00 min. Three MRM transitions from the protonated DA ion were monitored: m/z 312>266, m/z 312>248, and m/z 312>161. The optimized source settings were as following: gas temperature 225 °C, gas flow 12 L min-1, nebulizer

- 172 -

45 psi, sheath gas temperature 375 °C, sheath gas flow 11 L min-1 and capillary voltage 400 V. Domoic acid certified reference standard (CRM-DA-g) purchased from the National Research Council, Halifax, Canada was used for quantification. DA calibration standard solutions included the following concentration levels: 0.5, 1, 5, 25 and 50 ng mL-1. The limit of detection was 0.17 ng mL-1, which corresponds to 0.9 x10-3 mg kg-1 in tissue.

Supplemental Table S1. Summary of specimen collection, sample size, sample period and site, biometry, sex and maturity stages. Sample Total Period Peniche Olhão ML Species n (2016) (n) (n) BW (g) (mm) Males Females MS June- 781 - 120 - O. vulgaris 54 September 39 15 3922 230 28 26 II-IV S. 428- 150- officinalis 5 May 5 - 1144 233 4 1 III T. 285- sagittatus 5 August 5 - 574-875 327 0 5 II 183- L. vulgaris 5 July 5 - 182-383 232 0 5 III-V 180- L. forbesi 6 July 6 - 211-312 215 2 4 II

Supplemental Figure 1: LC-MSMS analysis of a) domoic acid standard and b) extract of octopus brain tissues. Multiple reaction monitoring in positive polarity was used to identify the toxins. Three ion transitions were monitored: m/z 312 > 266 (blue line), m/z 312 > 248 (red line) and m/z 312 > 161 (blue line).

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Planet Earth is blue, and there’s nothing I can do.

- David Bowie

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CHAPTER FIVE

FEEDING BEHAVIOUR AND CHROMATOPHORE ACTIVITY OF OCTOPUS

PARALARVAE ARE NOT AFFECTED BY THE HARMFUL ALGAL TOXIN DOMOIC

ACID

The material in this chapter is submitted as:

Lopes, V.M., Sampaio, E., Vara, C., Costa, P.R., Rosa, R.

(submitted). Feeding behaviour and chromatophore activity of octopus paralarvae are not affected by the harmful algal toxin domoic acid. Marine Environmental Research.

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- 179 -

Feeding behaviour and chromatophore activity of octopus paralarvae are not affected by the harmful algal toxin domoic acid

Vanessa M. Lopes1,2, Eduardo Sampaio1, Catarina Vara1, Pedro R.

Costa2,3, Rui Rosa1

1MARE – Marine Environmental Sciences Centre, Laboratório Marítimo da Guia, Faculdade de

Ciências da Universidade de Lisboa, Portugal.

2IPMA – Portuguese Institute for the Sea and Atmosphere, Avenida de Brasília, 1449-006

Lisboa, Portugal

3CCMAR- University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal

Abstract

- 180 -

Domoic acid (DA) is a phycotoxin that induces a wide range of lethal and sublethal effects on marine organisms. Although it has recently been shown to be accumulated at a great extent in cephalopods’ digestive gland and to reach their central nervous system, no studies have measured DA effects on the behavioural ecology of these cognitively skilled invertebrates. Because early ontogenetic stages still do not possess fully developed detoxification systems, they are also expected to be more susceptible to harmful algal toxins. Thus, the aim of the present study was to investigate, for the first time, whether acute exposure to ecologically relevant DA concentration (150 µg

DA L-1) affects activity rates, feed intake and chromatophore expansion of recently-hatched Octopus vulgaris paralarvae. After

24 hours of DA exposure, no mortalities occurred and there were no impairments in paralarvae swimming and feeding activities and chromatophoric patterns. Overall, our results suggest that cephalopod early stages may be quite resilient and tolerate this potent neurotoxin during the associated harmful algal bloom events.

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Keywords: Domoic acid, phycotoxin, paralarvae, octopus, behaviour, harmful algal blooms

Introduction

Marine phycotoxins are only produced by approximately 2% of phytoplanktonic species (Hallegraeff, 2014; Smayda, 1997).

However, when environmental conditions are optimal, these organisms show pronounced increases in growth rates, creating harmful algal blooms (HABs). HABs have been documented to elicit a wide range of effects on marine life, from sublethal effects to events of mass mortality in fish and marine mammals

(Landsberg, 2002, Costa 2016).

One of the most studied phycotoxins is domoic acid (DA), a water-soluble neurotoxin produced by Pseudo-nitzschia and

Nitzschia diatoms, known to cause Amnesic Shellfish Poisoning

(ASP) in vertebrates (Quilliam and Wright, 1989). DA acts on the central nervous system (CNS) as a glutamate agonist (Hampson and

Manalo, 1998). Glutamate is the most abundant excitatory neurotransmitter in the CNS, especially in the hippocampus, the brain area associated with learning ability and memory acquisition (Bliss and Collingridge, 1993). Upon reaching neural cells, DA binds to AMPA, kainate and NMDA receptor subtypes

(ionotropic glutamate receptors), leading to excessive concentrations of extracellular unbound glutamate and triggering the activation of said receptors (Berman and Murray, 1997).

Thus, DA permanently opens these receptors, resulting in an excessive influx of cations (Ca2+) to the cells, which, in this case, leads to membrane depolarization, as well as cellular degeneration and death (Hampson and Manalo, 1998). In

- 182 - vertebrates, this process causes permanent short-term memory loss, among other symptoms, such as abortion and premature birth in marine mammals (Goldstein et al., 2009), disorientation

(Lefebvre et al., 2001; Scholin et al., 2000) and behaviour alterations (Nogueira et al., 2010).

Marine organisms are usually exposed to DA through food web transfers. However, when the bloom becomes senescent, toxin- producing cells die and their cells lyse, releasing the toxin to the surrounding environment (Costa et al., 2010; Lefebvre et al., 2008). Thus, marine organisms can be directly exposed to toxins dissolved in the water. Over the years, hundreds of marine mammals (especially sea lions) strandings (Fire et al.,

2010; Lefebvre et al., 2010; Silvagni et al., 2005) and seabirds massive die-offs (Sierra-Beltrán et al., 1997; Fritz et al.,

1992) have been attributed to DA contamination through prey.

Notwithstanding, information regarding the effects of this toxin in other marine organisms is somewhat limited. Exposure of bivalves to DA-producing diatoms induced, in some cases, reversible effects (Jones et al., 1995) on bivalve haemolymph chemistry and haemocyte physiology, whereas in fish (through intracoelomic injections), it affected their ability to school, caused disorientation, abnormal swimming behaviour or even death

(Lefebvre et al., 2007, 2001; Nogueira et al., 2010).

Nevertheless, the effects of DA in early stages of development of marine organisms remain fairly unknown.

Cephalopods, as invertebrates with complex brain architectures, possess brain areas associated with learning and memory comparable to those of vertebrates. Octopuses have been shown to

- 183 - have vertebrate-like long term potentiation (LTP), a process relying on glutamatergic transmission, through which an organism strengthens the connection between neurons, enabling memory storage and learning abilities (Glanzman, 2008; Hochner et al.,

2003). Moreover, they are known for their camouflage abilities, enabled by a complex system of chromatophores directly controlled by the nervous system. Chromatophores are small multi-coloured pigment sacs embedded in the skin that are surrounded by radial muscles which contract or expand, concealing or exposing the pigment, respectively (Messenger,

2001). When glutamate, considered one of the main neurotransmitters in octopus CNS (Hochner et al., 2003), and kainic acid (both DA agonists), are injected into their blood stream, rapid expansion of the octopus’ chromatophores are provoked, resulting in general darkening of the animal (Andrews et al., 1983, 1981).

Coastal cephalopods accumulate DA in their tissues (Costa et al., 2004, 2005a,b), especially in their primary storage site, the digestive gland. Moreover, the common octopus (Octopus vulgaris) and European cuttlefish (Sepia officinalis) were recently shown to accumulate DA in brain tissue (Lopes et al.,

2018). DA persists in octopus brains for at least four months, during which bivalves, their most preferred prey (Ambrose and

Nelson, 1983), presented very low or no levels of DA in their tissues. This unexpected finding suggests that octopus and cuttlefish may accumulate and retain DA with no visible adverse effects. However, as mentioned above, there is no information regarding the effect of DA in marine organisms’ early stages,

- 184 - including for cephalopod species. Within this context, the aim of the present study was to describe for the first time, the effects of dissolved DA exposure (over 24 hours) on the feeding behaviour (activity levels, feed intake) and chromatophoric activity (mantle dorsal side area coverage) of O. vulgaris recently-hatched paralarvae.

Materials and methods

Specimen collection

Octopus embryos (O. vulgaris) were collected in Fuzeta island,

Algarve, South Portuguese coast (37°3′N 7°45′W) by local fishermen in November 2017. Upon collection, eggs were transported to the aquaculture facilities in Laboratório

Marítimo da Guia in Cascais, Portugal. There, the embryos were carefully transferred and distributed between three 20 L aquaria connected in parallel to a 150 L sump, equipped with a wet-dry filter, assuring biological filtration and a protein skimmer.

Natural sea-water was 1 µm filtered, with salinity being maintained at 35 ± 1 (± standard deviation), temperature was kept stable at 18 °C through Hailea heating/cooling systems and pH kept at 8.1 (± 0.1). The tanks were illuminated from above with a photoperiod of 14 L:10 D. Ammonia and nitrite were monitored every other day and maintained within recommended levels (see Iglesias and Fuentes, 2014).

DA exposure

After hatching, 2-day-old octopus paralarvae were transferred to the experimental tanks manually, using pipettes with wide tips,

- 185 - and were randomly distributed between nine 5 L containers. 2- day-old paralarvae were chosen because it had been previously demonstrated that the highest feeding rates were attained after

48 hours post-hatching (Iglesias et al., 2006). Each container was filled with 3 L of with filtered (1 μm) and UV-irradiated seawater, and the room was carefully maintained at 18 °C.

A nominal dissolved DA concentration of 150 µg DA L-1 was used for the exposure experiment. The selected concentration aims to reflect the natural conditions, as measured at the peak of a bloom (Trainer et al., 2007). Exposure lasted for 24 h, with sampling points at after DA inclusion (T1), 4 (T4), 8 (T8) and

24 (T24) hours, as, in most studies symptom reversion occurs under 24 h (e.g. Nogueira et al., 2010).

Activity and feed intake

The tanks were closely monitored throughout the experimental period and were filmed after DA inclusion (T = <1 h), at four (T

= 4 h), eight (T = 8 h) and at twenty-four (T = 24 h) hours of exposure. A recording camera was placed on the side of each tank, to fully observe the jet-propelling movements of the paralarvae. Five minutes were recorded for each tank at each sampling period. To assess activity rates, for every video, the number of “jets” (or mantle contractions), here used as a proxy of animal activity, was counted during 1 minute for three different paralarvae within each tank.

At every sampling occasion, juvenile Artemia sp. (~10 days post- hatching, 3.86 ± 0.31 mm total length) were placed in the experimental tanks at 1 Artemia : 1 paralarvae ratio. The tanks

- 186 - were filmed from the side for 10 minutes, and the number of paralarval strikes were registered. After 10 minutes all remaining Artemia were removed from the tanks.

Chromatophore analysis

At each sampling time, two paralarvae were removed from each tank (i.e. 6 paralarvae per treatment) and placed under a dissecting microscope and filmed, one at a time, for 5 minutes, following a similar methodology as used in Nande et al. (2017).

The animals were allowed to acclimate to these new conditions for over 4 minutes. Video analysis consisted in measuring paralarvae’s total dorsal area and the area covered by chromatophores in three still photographs of the last 30 seconds of each video.

DA extraction and quantification

To verify the continuous exposure of paralarvae to dissolved DA, an aliquot of seawater was taken for toxin quantification at the end of the experiment (after 24 hours).

Due to the minute size of paralarval tissue, DA quantification was not possible, as the method used was developed and optimized for larger samples, consequently impairing any accurate measurement.

A 2 ml aliquot of seawater was collected from each tank and acidified with 0.7 mL of 2% formic acid/ 5% methanol/ water

(2:5:93, v/v/v) to yield 0.5% formic acid in the sample. After homogenization, samples were desalted and extracted for DA by solid phase extraction (SPE) using Oasis HLB cartridges (Waters,

- 187 -

USA). SPE columns were conditioned with methanol and ultrapure water using a vacuum manifold. Then, 2 mL of the acidified seawater sample was loaded to the SPE column, followed by 5 mL of ultrapure water to rinse the sample tube and the SPE column.

The DA was eluted dropwise with 2 mL of methanol into a glass vial and analysed by LC-MS/MS (Wang et al., 2007).

The LC-MS/MS equipment consisted of an Agilent 1290 Infinity coupled to a triple quadrupole mass spectrometer Agilent 6470.

The chromatographic separation was conducted using an Zorbax SB-

C8 RRHT (2.1 x 50 mm, 1.8 µm), protected with a guard column

(2.1 x 5 mm, 1.8 µm). Elution was achieved using a binary eluent system: eluent A was water with 2 mM ammonium formate and 50 mM formic acid, and eluent B was 95 % acetonitrile with 2 mM ammonium formate and 50 mM formic acid. A binary elution gradient was used at a flow rate of 0.4 mL min-1 as follows: 0-

0.3 min 95 % A, gradient from 95 to 40 % eluent A at 2.5 min; and 95 % eluent A from 2.51 to 3.00 min. Three MRM transitions from the protonated DA ion were monitored: m/z 312>266, m/z

312>248, and m/z 312>161.

Domoic acid certified reference standard (CRM-DA-g) purchased from the National Research Council, Halifax, Canada was used for quantification. DA calibration standard solutions included the following concentration levels: 0.5, 1, 5, 25 and 50 ng mL-1. The

Limit of detection was 0.17 ng mL-1.

Statistical analysis

Generalized Linear Models (GLMs) were used to determine if DA affected activity and feeding rates, as well chromatophore

- 188 - coverage, throughout the exposure period, with statistical significance set to α = 0.05. We chose DA treatment and time of exposure as fixed effects, and GLMs were used to test for eventual random effects that different tanks (i.e. replicates) could elicit on the results, using lmerTest package. GLMs with

Poisson distribution family were used to analyse activity and feeding rates, whereas a Gamma distribution family GLM was used to analyse dorsal area covered by chromatophores. Model fitting was performed using the Akaike Information Criterion (AIC), an estimator providing the best fit using the simplest model possible (Quinn and Keough, 2002), and factors that did not affect data distribution were removed. Lastly, ANOVA tests were used to obtain general factor significance, and post-hoc tests

(lsmeans package) were used for pairwise test comparisons.

Statistical analyses were performed on R Studio (RStudio Team,

2016).

Results

At the end of the exposure period, DA was present in tank water without degradation, presenting concentrations of 148.9 ± 11.5

µg DA L-1. Also, there were no mortalities in both treatments

(control and DA) after such period.

- 189 -

A

B

C

1

Figure 1. Box plot showing data distribution (median, 25 and 75 quartiles, non-outlier range and outliers) in all treatments used as a function of time. (A) The number of paralarval mantle contractions

(jets, n=9 per each treatment at each sampling point) (B) the number of Artemia consumed (n= T1: 20; T4: 18; T8: 16; T24: 14) (C) paralarva dorsal area covered by chromatophores, expressed in percentage (n=6 per each treatment at each sampling point).

- 190 -

Exposure treatments (Control and DA groups, Figure 1A) did not, in general, affect the number of jets (Treatment, p = 0.724;

Treatment*Time, p = 0.062, see Supplemental Table 1), whereas time of exposure elicited significant differences (Time, p =

0.032, see Supplemental Table 1). However, GLM analysis revealed that the number of jets was significantly affected by exposure to DA and time of exposure on some occasions (Treatment; T24;

Treatment*T4; Treatment*T8; Treatment*T24, p < 0.05 for all, see

Supplemental Table 2). More specifically, the number of jets was lower under DA exposure, compared to the control group after DA inclusion (T1: Control - DA, p =0.028, see Supplemental Table

3). Additionally, paralarvae seemed to recover at 4 hours of exposure, with the number of jets under DA exposure increasing to levels comparable to the control group (T4: Control – DA, p =

0.208, DA: T1 – T4, p = 0.037, see Supplemental table 3).

Exposure to dissolved DA and time of exposure did not significantly affect the number of paralarval strikes (Figure

1B) in any occasion (p > 0.05 for all, see Supplemental Tables

4-6).

On the other hand, dorsal side chromatophore coverage (Figure

1C) was affected by exposure period and DA (T24, p = 0.005;

Treatment*T4, p = 0.001, see Supplemental Table 7; Time, p <

0.001; Treatment*Time, p = 0.001, see Supplemental Table 8).

In fact, time of exposure mostly increased the percentage of area covered by chromatophores in both treatments used (Control:

T1 - T24, p = 0.020; DA: T1 – T4, p = 0.021; T4 – T8, p = 0.023;

T4 – T24, p < 0.001, see Supplemental Table 9). Regarding

- 191 - differences between treatments, at 4 hours of exposure, dorsal chromatophore coverage was significantly lower under DA exposure than the Control group (T4: Control – DA, p = 0.001, see

Supplemental Table 9).

Discussion

Initial stages of development are expected to be more impacted by HAB-toxins (or any other contaminant) since their detoxification systems may not yet be fully developed

(Vasconcelos et al., 2010). However, here we report that exposure to dissolved DA did not impair/alter the feeding behaviour or chromatophore activity of octopus paralarvae.

Moreover, presence of DA did not affect survival – i.e. all paralarvae exposed for 24 hours survived. Thus, these results suggest that paralarvae are not lethally affected by dissolved

DA and can withstand acute exposures to this neurotoxin.

To date, there are but a few studies regarding the effect of DA in larval stages of marine organisms. Fertilized zebrafish

(Danio rerio) eggs, when injected with 0.12-17 mg DA kg-1 presented greatly decreased hatching rates (< 40% surviving embryos), with dead embryos displaying spinal deformities and other morphological impairments. Surviving embryos were less responsive to touch stimuli throughout the embryogenesis with increasing DA levels and exhibited convulsions (strong contractions of the whole body) at dosages higher than 0.4 mg DA kg-1 (Tiedeken et al., 2005). DA exposure also hindered normal development in king scallop (Pecten maximus) larvae, reducing scallop’s growth and survival rates (Liu et al., 2007).

- 192 -

Octopus paralarvae are negatively buoyant, therefore they must maintain their vertical position through jet propulsion, which is known to be energetically costly (O’Dor and Webber, 1986;

Rosa et al., 2009; Rosa and Seibel, 2008). Moreover, metabolic

(or activity) rates are known to increase in response to an external toxicant (Calow, 1991), triggering physiological processes in order to protect the individual from deleterious effects the toxicant may induce, and to promote its excretion

(Rowe, 1998; Rowe et al., 2001). Thus, in the present study, the lower number of jet propulsions in DA exposure group at the moment of inclusion, with the subsequent increase and stabilization throughout the experimental period was most likely stimulated by an increase of metabolic rates caused by the exposure to ecologically relevant DA concentrations.

Nonetheless, feeding activity was not affected by neither DA concentration nor time of exposure

It has been previously shown that DA, glutamate and kainic acid injections caused adult squid and octopus chromatophores to expand (Andrews et al., 1981; Messenger et al., 1997). Here, the fact that the average percentage of dorsal chromatophore coverage (i.e. the dorsal surface covered by chromatophores) remained mostly unaffected under DA exposure suggest that this neurotoxin may not elicit the same effects in hatchlings than it does in adult specimens at the concentrations tested. Yet, it is worth noting that substance uptake from seawater and direct injections in muscular tissue or blood stream may elicit different effects. Moreover, Hanlon and Messenger (1988) revealed that the brain region associated with chromatophore

- 193 - control (chromatophore lobes) were undifferentiated at hatching in Sepia officinalis.

In summary, the present study constitutes the first attempt at understanding how these highly developed invertebrates can accumulate a potent neurotoxin in their tissues (namely brain tissue, Lopes et al., 2018), when it elicits such devastating effects in other marine organisms, both vertebrates and invertebrates. Further studies are also needed to assess potential DA effects in cephalopod cognition-related features, such as learning and memory.

Acknowledgements

The authors would like to express gratitude to João Pereira and

Sónia Olim, who provided us the means to obtain the octopus eggs and Dr. Fátima Gil for providing live Artemia. This work contributes to project UID/Multi/04326/2013 from the Portuguese

Foundation for Science and Technology (FCT). The authors would like to thank the Portuguese Foundation for Science for the

“Investigador FCT” grants to R. Rosa and P. R. Costa and the

Ph.D. scholarship to V. M. Lopes (SFRH/BD/97633/2013) and E.

Sampaio (SFRH/BD/131771/2017).

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ANNEX: SUPPLEMENTARY DATA

Table S1. Summary of ANOVA results evaluating the effect of DA concentration and Time of exposure on mantle contractions (jets). Marked values in bold indicate p < 0.05. Resid. Df Deviance Resid. Dev p value NULL 68 95.499 Treatment 2 0.125 67 95.374 0.724 Time 3 8.821 64 86.553 0.032 Treatment*Time 6 7.340 61 79.213 0.062

Table S2. GLM analysis of the effects the treatments used, and time of exposure had on number of paralarvae jets (4 levels within the treatments, 2 levels within sampling times). Model formula on top, family and respective model AIC at the bottom. Std Error – Standard Error. Marked values in bold indicate p < 0.05.

Estimate Std Error z value p value GLM: jets in function of DA concentration * time (Intercept) 4.822 0.037 131.605 < 0.001 Treatment -0.106 0.048 -2.195 0.028 T4 -0.043 0.048 -0.910 0.363 T8 -0.056 0.048 -1.164 0.245 T24 -0.114 0.048 -2.356 0.018 Treatment*T4 0.160 0.064 2.478 0.013 Treatment*T8 0.146 0.065 2.255 0.024 Treatment*T24 0.129 0.066 1.961 0.049

Family = Poisson AIC = 550.7

Table S3. Detailed table of results from post-hoc tests for pairwise comparisons between DA concentration and Time of exposure effects on mantle contractions (jets). Marked values in bold indicate p < 0.05. Rate z Contrast ratio SE ratio p value T1 Control - DA 1.112 0.054 2.195 0.028

T4

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Control - DA 0.948 0.040 -1.258 0.208

T8 Control - DA 0.961 0.041 -0.926 0.355

T24 Control - DA 0.977 0.044 -0.512 0.609

Control T1 - T24 1.121 0.054 2.356 0.086 T1 - T4 1.044 0.050 0.910 0.799 T1 - T8 1.057 0.051 1.164 0.650 T4 - T8 1.012 0.044 0.282 0.992 T4 – T24 0.932 0.041 -1.605 0.376 T8 – T24 0.943 0.042 -1.324 0.548

DA T1 - T24 0.985 0.044 -0.333 0.987 T1 - T4 0.890 0.039 -2.683 0.037 T1 - T8 0.914 0.040 -2.070 0.163 T4 - T8 1.026 0.043 0.614 0.928 T4 – T24 0.903 0.039 -2.350 0.087 T8 – T24 0.927 0.040 -1.737 0.304

Table S4. GLM analysis of the effects the treatments used, and time of exposure had on number of paralarvae strikes (4 levels within the treatments, 2 levels within sampling times). Model formula on top, family and respective model AIC at the bottom. Std Error – Standard Error. Marked values in bold indicate p < 0.05. Std z p Estimate Error value value GLM: strikes in function of DA concentration * time (Intercept) 0.511 0.447 1.142 0.235 Treatment 7.163E-16 0.632 0.000 1.000 - T4 -0.223 0.670 0.333 0.739 - T8 -0.511 0.730 0.699 0.484 - T24 -0.916 0.837 1.095 0.273 - Treatment*T4 -1.386 1.285 1.079 0.280 Treatment*T8 0.693 0.949 0.731 0.465 - Treatment*T24 0.693 1.378 0.503 0.615

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Family = Poisson AIC = 72.31

Table S5. Summary of ANOVA results evaluating the effect of Time of exposure on successful strikes to the prey. Marked values in bold indicate p < 0.05. Resid. p Df Deviance Resid. Dev value NULL 23 31.097 Treatment 1 0.037 22 31.060 0.847 Time 3 5.1723 19 25.887 0.160 Treatment*Time 3 3.250 16 22.638 0.355

Table S6. Detailed table of results from post-hoc tests for pairwise comparisons between DA concentration and Time of exposure effects on paralarvae strikes to prey. Marked values in bold indicate p < 0.05. Rate Contrast ratio SE z ratio p value T1 Control - DA 1.0 0.632 0.000 1.000 T4 Control - DA 4.0 4.472 1.240 0.215 T8 Control - DA 0.5 0.354 -0.980 0.327 T24 Control - DA 2.0 2.449 0.566 0.571

Control T1 - T24 2.5 2.092 1.095 0.6925 T1 - T4 1.3 0.839 0.333 0.987 T1 - T8 1.7 1.217 0.699 0.897 T4 - T8 1.3 1.018 0.377 0.989 T4 – T24 0.5 0.433 -0.800 0.854 T8 – T24 0.7 0.609 -0.444 0.971

DA T1 - T24 5.0 5.478 1.469 0.456 T1 - T4 5.0 5.478 1.469 0.456 T1 - T8 0.8 0.505 -0.301 0.991

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T4 - T8 0.2 0.180 -1.659 0.346 T4 – T24 1.0 1.414 0.000 1.000 T8 – T24 0.2 0.180 -1.659 0.346

Table S7. GLM analysis of the effects the treatments used, and time of exposure had on dorsal chromatophore coverage (4 levels within the treatments, 2 levels within sampling times). Model formula on top, family and respective model AIC at the bottom. Std Error – Standard Error. Marked values in bold indicate p < 0.05.

Std t Estimate Error value p value GLM: area covered in function of DA concentration * time (Intercept) 0.080 0.013 6.059 < 0.001 Treatment -0.017 0.018 -0.950 0.348 T4 -0.025 0.016 -1.580 0.116 T8 -0.021 0.016 -1.293 0.198 T24 -0.042 0.015 -2.885 0.005 Treatment *T4 0.097 0.030 3.278 0.001 Treatment *T8 0.009 0.022 0.404 0.687 Treatment *T24 0.014 0.019 0.723 0.471

Family = Gamma AIC = 1070

Table S8. Summary of ANOVA results evaluating the effect of DA concentration and Time of exposure on chromatophore dorsal area coverage. Marked values in bold indicate p < 0.05. Resid. Resid. Df Deviance Df Dev p value NULL 140 100.972 Treatment 1 0.053 139 100.919 0.743 Time 3 12.890 136 88.029 < 0.001 Treatment*Time 3 7.817 133 80.211 0.001

Table S9. Detailed table of results from post-hoc tests for pairwise comparisons between DA concentration and Time of exposure effects on the percentage of dorsal area covered by chromatophores. Marked values in bold indicate p < 0.05.

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z p Contrast Estimate SE ratio value

T1

Control - DA 0.017 0.017 0.950 0.342 T4 - Control - DA -0.081 0.024 3.360 0.001 T8 Control - DA 0.008 0.128 0.612 0.541 T24 Control - DA 0.003 0.009 0.297 0.766

Control

T1 - T24 0.042 0.015 2.885 0.020 T1 - T4 0.025 0.016 1.596 0.381 T1 - T8 0.021 0.0163 1.293 0.567 - T4 - T8 -0.004 0.013 0.327 0.988 - T4 – T24 -0.017 0.011 1.520 0.426 - T8 – T24 -0.021 0.012 1.816 0.266

DA T1 - T24 0.028 0.0128 2.183 0.128 - T1 - T4 -0.072 0.0250 2.869 0.021 T1 - T8 0.012 0.014 0.874 0.818 T4 - T8 0.084 0.0238 3.538 0.023 - T4 – T24 -0.100 -0.100 4.335 <0.001 - T8 – T24 -0.016 0.010 1.529 0.420

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I wonder if, in the dark night of the sea, there, within its own sphere of instinct, the octopus dreams of me. - N. Scott Momaday

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CHAPTER SIX

GENERAL DISCUSSION AND FINAL CONSIDERATIONS

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The present dissertation constitutes the first attempt to understand the underlying mechanisms that allow cephalopods to accumulate HAB-toxins without displaying any symptoms of toxicosis.

Marine biotoxins, produced as a secondary metabolite by certain phytoplankton species, can alter cellular processes of other organisms, from plankton to top predators. Phytoplankton cells, including HAB species, are the main nutritional source of zooplankton grazers and filter-feeding shellfish. The ability to metabolize and detoxify HAB toxins is critical to their survival, which most likely evolved to acquire resources that enable them to tolerate these toxins. Filter-feeding shellfish and zooplankton grazers, although susceptible to biotoxins, often act as potent toxin vectors in the marine ecosystem. On the other hand, top predators at higher trophic levels that are less frequently exposed to HAB-toxins, may experience more severe effects. Accumulation, biotransformation and transfer throughout the marine food web can affect different organisms causing a wide range of effects, from innocuous transient to sublethal or lethal effects, that in extreme cases generate events of mass mortality in higher vertebrates. The discrepancy between the severity of effects displayed by vertebrates and invertebrates is presumably attributed to the fact that the toxins here mentioned act on the central nervous system, and most invertebrates lack complex brains. Yet, cephalopods are the

“exception to this rule”, since they can be compared to vertebrates regarding neural development, since they possess long term potentiation (LTP) similar to vertebrates (Hochner et

- 215 - al., 2003), possess complex behaviour repertoires (Hanlon and

Messenger, 1996) and are considered the world’s most intelligent invertebrates.

Upon reviewing the existing information on how these toxins affect marine communities and at what extent cephalopods accumulate them, the next steps were to: i) study the dynamics of accumulation, ii) discover new accumulation sites and iii) determine what potential effects these toxins may elicit in cephalopods. Within this context, the main questions to be answered with the present dissertation were:

- How do cephalopods accumulate these toxins and at what

rate are they eliminated?

- Do the toxins reach their central nervous system?

- Are they affected behaviourally when exposed directly to

dissolved toxins?

In chapter three, it was shown that the common octopus presented low conversion, transfer and elimination rates. As expected, the toxins presented in the clams were accumulated in the DG, the major site for substance storage in cephalopods. No other tissues besides the DG and kidney were positive for toxin presence, i.e. accumulation in other tissues was negligible, as were transfer and elimination rates. Also, toxin elimination rates were very low, allowing the digestive gland to exponentially accumulate these toxins. Similarly, toxin transfer rates between the DG and kidney were lower than the elimination rates, suggesting that other pathways or mechanisms may be responsible for toxin excretion.

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The uptake, transference and elimination are simultaneous processes, thus, it is difficult to directly measure them. The use of first-order kinetic models is widely used to estimate substance uptake and elimination in bivalves. Here, toxin uptake was better characterized as two-compartment (DG and kidney) exponential growth model, whereas the depuration had better fit using one compartment (whole viscera) exponential decay model.

Estimating accumulation-depuration of a given toxin is very relevant to track the movement of toxins throughout the various links of food webs and identify potential vectors. The fact that the specimens did not display any obvious sign of toxicosis throughout the experiment) with such high levels of toxins found in the DG) led to the need to investigate whether the toxins reach their brain tissue.

The results present in Chapter Four showed that octopus and cuttlefish do accumulate HAB-toxins in brain tissue. Moreover, information on cell density of HAB-toxin producer and toxin presence in bivalves was collected before, during and after octopus sampling period (June-September). It was found that, although phytoplankton cells were abundant in June and August, the toxin was only present in bivalve tissue between April and

June, suggesting swift elimination of the toxin. These results pointed to a rapid transfer throughout the food web, with octopuses and cuttlefish retaining this hydrophilic toxin, rather than eliminating it. Toxin accumulation greatly depends on feeding ecology and the kinetics of accumulation-elimination.

Thus, the fact that squid (both myopsid and ommastrephid groups) did not present detectable levels of this toxin, in either brain

- 217 - tissue or DG, is likely due to their feeding ecology. Squids, as mentioned above, feed mostly on small fish, most of which accumulate HAB-toxins exclusively during bloom periods and rapidly eliminate them, making squid less susceptible to accumulate and retain these toxins in their tissues.

Both studies (Chapter Three and Four) suggest slow elimination rates and high potential of toxin accumulation during and/or after bloom events, as toxins are accumulated at superior rates than they are eliminated. Furthermore, both studies also suggest an absence of toxic effects in cephalopods. Consequently, to ascertain whether these toxins do elicit any effect on these molluscs, specially during early ontogenesis, octopus paralarvae were exposed to one of these neurotoxins in its soluble fraction.

Chapter Five presents the effects of this exposure in terms of feeding and chromatophoric activity. After 24 hours of exposure there were no mortalities registered, and a general lack of effect was observed. The number of prey consumed by the paralarvae was not affected by toxin exposure or time of exposure. On the other hand, the number of jets (mantle contractions) was lowered after the toxin was added and returned to levels comparable to the control group afterwards, suggesting that the organisms may have increased metabolic rates, in order to protect themselves from an external toxicant. Regarding chromatophoric activity, there was a general increase in dorsal surface covered by chromatophores throughout the exposure period, regardless of toxin presence, indicative of paralarval aging and chromatophore differentiation and development.

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The results showed that octopuses accumulate these toxins with high efficiency and are slow at eliminating them. The fact that these toxins were found in brain tissue, and exposure to the dissolved toxin did not affect feeding and activity levels, as well as chromatophore activity, suggest that octopuses are immune to the negative effects these toxins elicit in many marine organisms.

It is predicted that many changes in the world’s oceans will occur. Increasing temperature and CO2 concentrations are but two of the many factors affecting HAB distribution, frequency and intensity. HAB ecology is complex, and it is dependent on the interaction of many factors, including ocean stratification, oceanic currents, nutrient availability and precipitation.

Currently, there are a number of studies on the effect of climate change in HABs (Fu et al., 2012; Tatters et al., 2013;

Band-Schmidt et al., 2014). However, the interactions simulated are scarce and do not allow for species adaptation and plasticity. Temperature fluctuations affect directly phytoplankton communities. Typically, with increasing temperatures, phytoplanktonic species tend to have higher growth rates until a species-specific temperature threshold is met

(Hallegraeff, 2010; Wells et al., 2015). There is growing evidence that HABs are increasing in frequency and intensity throughout the globe (Hallegraeff, 1993), and further studies are needed to better understand the shifts in HAB ecology and physiology in these new conditions.

Cephalopods (e.g., coastal octopus and cuttlefish) are good candidates to track the occurrence of harmful algal blooms and

- 219 - marine toxins. Due to the fact that their complex nervous system is comparable in certain aspects to those of the vertebrates, they are excellent model organisms to study the effects HAB- toxins. Since these organisms have been found to accumulate considerably high levels of toxins without apparent harm, it is of great value to identify the mechanisms that provide them the ability to metabolize and detoxify HAB-toxins, which is certainly critical to their survival. These organisms have probably evolved to acquire additional resources that enable them to tolerate the HAB-toxins. Further studies are needed to assess potential toxin effects in cephalopod cognition-related features, such as learning and memory.

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