Biomasa, distribución y metabolismo del zooplancton en aguas de la Península Antártica

ｾＳ＠ TESIS DOCTORAL Biomasa, distribución y metabolismo dell zooplancton en aguas de la Península Antártica ｾ＠ ' Lehette Pascal : LAS PALMAS DE GRAN CANARIA de Biología Diciembre 2011 Las Palmas de Gra11 canarta Nº 76 Curso 2011/12 UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA SUBDIRECCIÓN DE TERCER CICLO Y POSTGRADO Reunido el día de la fecha, el Tribunal nombrado por el Excmo. Sr. Magfco. de esta Univesidad, y finalizada la defensa y discusión de esta tesis doctoral, los señores miembros del Tribunal, emiten la siguiente calificación global: Votos favorables: Las Palmas de Gran Canaria, a 26 de jun·o de 2012 El Presidente: D. Javier Arístegui Ruiz El Vocal: D. Melchor González Dávila El Vocal: D. Álvaro Morales Ramírez El Vocal: D. Sergio Hernández Trujillo El Doctorando: D. Pascal Lehette J UNIVERSIDAD DE AS PAAS DE GRAN CANARIA DEPARTMENTO DE BIOLOGÍA JUAN LUIS GÓMEZ PINCHETTI, SECRETARIO DEL DEPARTAMENTO DE BIOLOGÍA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, CERTIFICA, Que el Consejo de Doctores del Departamento de Biología, en sesión extraordinaria tomó el acuerdo de dar el consentimiento para su tramitación a la Tesis Doctoral titulada "BIOMASA, DISTRIBUCIÓN Y METABOLISMO DEL ZOOPLANCTON EN AGUAS DE LA PENÍNSULA ANTÁRTICA" presentada por el Doctorando D. PASCAL LEHETTE y dirigida por el Prof. Dr. Santiago Hernández León. Y para que así conste, a efectos de lo previsto en el Art. 73.2 del Reglamento de Estudios de Doctorado de la Universidad de Las Palmas de Gran Canaria, firmo la presente en Las Palmas de Gran Canaria a 20 de enero de 2012. Campus de Tafira _ Edificio de Ciencias Básicas -35017 Las Palmas de Gran Canaria -(España)-r 34 -928 45 29 11 -Fx: 34 -928 45 29 22 AD DE AS PAAS DE GRAN CANARIA Depatamento de Biología Prorma de doctorado en Ocenoraía. Bienio 2005-2007. Título de la Tesis: Biomasa, distribución y metabolismo del zooplancton en aguas de la Península Antártica Tesis Doctoral presentada or D Lehette Pascal pra obtener el rado de Doctor por la Univesidad de Las Palmas de Grn Cnaria. Diriida por Dr. D. Sntiago Hemndez León Ela Director/a Ela Doctorando/a A mis padres ..\GR \DEC'l\llEVTOS AGRADECIMIENTOS Habitualmente, los agradecimientos se redactan en la última fase de escritura del manuscrito, pero he preferido empezarlos antes (¿y porqué no?) y así evitar olvidar a alguien. Cuando miro hacia atrás, me doy cuenta de cuantas personas han compartido directamente o indirectamente momentos importantes en esta fase de mi vida, es decir en la realización de mi tesis doctoral. La etapa en la isla de Gran Canaria fue sin duda la más intensa de mi vida, tanto por mi formación académica como por mi formación profesional. Estos formidables viajes hacia el continente blanco acompañados de unas personas tan buenas, simpáticas y divertidas, pero a la vez serias, se quedaran gravados para siempre. La aventura como investigador fue también una aventura lingüística (particularmente con el catalán) durante mi estancia en la BAE Juan Carlos Primero, transformando y forjando muchos aspectos de mi vida. Por estos motivos, quiero darle todas las gracias a Santiago Hernández-León por haber confiado en mí valía para participar en este formidable proyecto. Aunque seguramente se ha arrepentido, en más de una ocasión, de haber elegir a un belga de origen vietnamita para realizar en las Islas Canarias una tesis sobre el mundo polar antártico. Y como empezaré por lo que más quiero, doy las gracias a mi familia, a los amigos presentes y lejanos, a mis compañeros y ex-compañeros de piso, de barco, de casa, de música, de fiesta, etc. He pasado momentos inolvidables en canarias gracias a todos ustedes. Especialmente, dedico este trabajo de investigación a mis amigas y amigos: Yolanda, Ana, lnes, Pere, Severin y Romaric, Thierry, Agnese, Dora, Brian, Javier, Thomas, Fernando, Elena, Nuria, Isabel, Carla, Alicia, Maria, Marta, Paola, Damaris y the last bus no least Laura ... Quiero agradecer a mis ex-compañeros de laboratorio Sebastien Putzeys, Lidia Nieves, Marta Moyano, Claire Schmoker que aguantaron mi presencia en el mismo, y me ayudaron a superar las dificultades de este proyecto. No puedo olvidar Philippe Grosjean y Kevin Denis por haberme acogido en el laboratorio de Ecología numérica de la Universidad de Mons-Haintaut durante unos meses. 7 \GR.\l>H l\llE'\TOS También quiero agradecer al Plan Nacional Antártico, de la CICYT (Comisión lnterministerial de Ciencia y Tecnología) que financiaron este proyecto ICEPOS durante 4 años, a las tripulaciones y a los comandantes del "R.V. 810. Hespérides" y del buque "810. Las Palmas", así como a todos los miembros de la base Antártica Juan Carlos l. En particular doy las gracias a José Antonio Pozo Prados y Joaquim Llinas del Torrent i Casanovas por su apoyo a la hora de colectar las muestras en la bahía Johnson de la isla de Linvingston. Esta investigación ha sido totalmente financiada por los proyectos ICEPOS (Ren200204165) y ConAfrica (CTM2004-02319) de la CICYT. Y por último, quiero agradecer la asistencia en la mar del personal de la UTM (Unidad de Tecnología Marina). "Lo que sabemos es una gota de agua; lo que ignoramos es el océano." Newton, Isaac. 8 Í"DICE ÍNDICE PRESENTACIÓN DE LA TESIS PRIMERA PARTE CAPÍTULO 1: INTRODUCCIÓN •••••••••.•••••••••••• ••• ••••••••••••••••••••••••• •• ••••••••••••••••••••••• •• •• •••.•.••••••.•••••••••.• •• ••••• 1.1. EL ENTORNO OCEANOGRÁFICO DEL OCÉANO SUR Y LA CIRCULACIÓN DE LAS MASAS DE AG UA .. . 1.2. EL ECOSISTEMA MARINO ANTÁRTICO Y SU PARADOJA ................................................. . 1.3. CICLO ANUAL DE LA PRODUCCIÓN PRIMARIA ................. . ..................................... .... .. . 1.4. EL ZOOPLANCTON DEL OCÉANO SUR ............. . ........... . ..................................... . ... . ..... . 1.4. I COPÉPODOS 1.4.2 EUF AUSIÁCEOS 1.4.3 TUNICADOS 1.5. LA IMPORTANCIA DEL P AST AJE Y DE LA OMNIVORÍA ............................. . .................. .. 1.6. LA DISTRIBUCIÓN DE LOS PRINCIPALES GRUPOS ZOOPLANCTONICOS EN LA ANTÁRTIDA 1.7. EL CICLO GLOBAL DEL CARBONO Y LA BOMBA BIOLÓGICA EN EL OCÉANO ........................ .. 1. 7 .1 EL CICLO GLOBAL DEL CARBONO ....................................... . ............................ . .......... ... .. 1.7.2 LA BOMBA BIOLÓGICA 1.8. LOS FLUJOS DE MATERIA A TRAVÉS EL ZOOPLANCTON ..... . .......................................... . 1.8.1. LA RESPIRACIÓN DEL ZOOPLANCTON .. .. .. .......... .. ... ... ..... . ........................ . .... . 1.8.2. LA EXCRECIÓN DEL ZOOPLANCTON BIBLIOGRAFÍA PRIMERA PARTE SEGUNDA PARTE CAPÍTULO 11: OBJ ETIVOS DE LA INVESTIGACIÓN CAPÍTULO 111 : CONSIDERACIONES ADICIONALES SOBRE LA METODOLOGÍA •• ••••••••..... •• ••..•.....••••••••••• 3.1. METODOLOGÍA PARA EL ESTUDIO DE LA BIOMASA .. .. ......................................................... . 3.1.1. CALIBRACIÓN Y ESTUDIO DE LA BIOMASA .. .. ........ . ... ...... ................... .. .......... ........ . 3.1.2. MÉTODO PARA LA IDENTIFICACIÓN, EL CONTEO Y LA ESTIMACIÓN DE LA BIOMASA . .. . PLANCTÓNICA EN MUESTRAS ............ . ........... . .............. . .. . ... ... ...... . ..................... . ..... .. 3.1.3. PLANTEAMIENTO DE LA METODOLOGÍA .... ... ..... . ............... ... .............................. . 3.2. METÓDO DE RESPIRACIÓN COMUNITARIA . ....................................................................... . 3.2.1. PRINCIPIOS DEL METODO: SISTEMA DE INTERCAMBIADOR LIQUIDO-GAS ............... . 3.2.2. CONSIDERACIONES DEL SISTEMA DE MEDIDA DE DIÓXIDO DE CARBONO 3.2.3. PLANTEAMIENTO DE LA METODOLOGÍA PARA EL ESTUDIO DE LA RESPIRACIÓN COMMUNIT ARIA BIBLIOGRAFÍA SEGUNDA PARTE ............................. . .......... . ........................................................ . 9 , ' /\ l>H I TERCERA PARTE CAPÍTULO IV: Pascal Lehette and Santiago Hemández-León. Zooplankton biomass estimation from digitized images: a comparison between subtropical and Antarctic organisms. Limnology and Oceanography Methods (2009) 7:304-308. CAPÍTULO V: Pascal Lehette and Santiago Hemández-León. Carbon dioxide production rates of marine epipelagic copepods in subtropical waters during the late winter bloom. Journal o( Experimental Marine Biology and Ecology (2010) 385:38-43. CAPÍTULO VI: Santiago Hemández-León, Pablo Sangra, Pascal Lehette, Luis Lubián, Carlos Almeida, Sébastien Putzeys, Pierrick Bécognée and Maria Paz Andrade. Zooplankton biomass and metabolism in the frontal zones of the Bransfield Strait (Antarctica), submitted to Deep-sea-research l. CAPÍTULO VII: Pascal Lehette and Santiago Hemández-León. Zooplankton community structure along the Western Antarctic Peninsula, submitted to Polar research. CAPÍTULO VIII: Lehette Pascal, Tovar-Sánchez Antonio, Duarte Carlos and Hemández-León Santiago. Krill excretion and impact on primary production during austral summer 2005 along the Western Antarctic Peninsula, submitted to Marine Ecology Progress Series. CUARTA PARTE CAPÍTULO IX: DISCUSSIÓN GENERAL 9.1. ESTIMACIÓN DE LA BTOMASA POR MÉTODOS ÓPTICOS 9.2. RESPIRACIÓN COMUNTT ARTA POR PRODUCCIÓN DE DIÓXIDO DE CARBONO 9.3. DISTRIBUCIÓN DEL MACRO Y MESOZOOPLANCTON EN LA REGIÓN OESTE DE LA PENÍNSULA ANTÁRTICA 9.4. ACOPLAMIENTO ENTRE LOS SISTEMAS FRONTALES DEL ESTRECHO DE BRANSFIELD Y LA BIOMASA ZOOPLANCTÓNTCA 9.5. APORTES DE AMONTO POR PARTE DE LA COMUNIDAD ZOOPLANCTÓNTCA EN LA REGIÓN OESTE DE LA PENÍNSULA ANTÁRTICA CAPÍTULO X: CONCLUSIONES BIBLIOGRAFÍA CUARTA PARTE FUTURAS LÍNEAS DE INVESTIGACIÓN ANEXOS 10 Además, las altas concentraciones de amonio pueden ejercer un efecto inhibidor (Flynn et al. 1997) de la utilización del nitrato como fuente de nitrógeno. Recientemente, se ha observado una posible regulación del grado de inhibición del amonio por la disponibilidad en hierro (Armstrong 1999). Además, se ha detectado que el amonio permite reducir el efecto inhibidor de las radiaciones UV y así restablecer el crecimiento fitoplanctónico (Agustí et al. 2009). El amonio, principal producto de excreción del zooplancton (Hirche 1983, Miller y Glibert 1998, Conover y Gustavson 1999), proviene mayoritariamente de los organismos heterótrofos como el micro-, mesoy macrozooplancton. Los enjambres de krill Antártico tendrían un papel predominante pues su excreción debería generar importantes cantidades de amonio para el desarrollo de un bloom fitoplanctónico en pocos días, además de liberar cantidades importantes de hierro (Tovar-Sánchez et al. 2007). Así, la cuantificación de esos aportes de amonio podría aclarar el papel del krill y de otros organismos mesoy macroplanctónicos sobre las proliferaciones masivas de fitoplancton. Parte de esta tesis doctoral se enfocara sobre los aportes específicos de amonio a través del proceso de excreción de los organismos mesoy macroplanctónicos más importantes del Océano Sur. 1.3. EL CICLO ANUAL DE LA PRODUCCIÓN PRIMARIA La presencia de la cubierta de hielo estacional en las regiones polares juega un papel extremadamente importante ya que se encuentra estrechamente relacionada con una intensa actividad biológica. Cada año el deshielo produce una fuerte estabilidad de la columna de agua, la cual puede ser observada en distancias de hasta 150 km de la costa, lo que provoca la formación de intensas floraciones de fitoplancton. Se ha observado que cuanto más rápidamente se derrite la capa de hielo, el "bloom fitoplanctónico" se desarrollará con menos intensidad. Este deshielo libera, además de una gran cantidad de amonio necesario para el crecimiento de las células fitoplanctónicas, grandes cantidades de micro-algas en las estables capas de las aguas superficiales (Garrison et al. 1986). La red trófica herbívora (Legendre y Rassoulzadegan 1995) inicia este proceso de proliferación fitoplanctónica primaveral con unas células fitoplanctónicas superiores a 20 µm (Smith y Nelson 1986) (Tabla 1). El microfitoplancton (>20µm como las diatomeas formando colonias o las cadenas de los géneros Nitzchia spp. y Chaetoceros spp.) abundan cuando la producción primaria aumenta (Froneman y Pakhomov 2000, Froneman et al. 2001). No obstante, estas proliferaciones fitplanctónicas pueden presentar tanto variaciones espacio-temporales como también una composición taxonómica con especies de mucho menor tamaño (<20 µm). Sucesivamente, se 18 l'ITROOlCCIÓ"i desarrolla la actividad heterotrófica con el pastaje por parte del krill y de los copépodos que puede conllevar a una cambio importante en las comunidades microfitoplanctónicas (Schultes et al. 2006). Así, el nanofitoplancton (2-20 µm) como los flagelados o las pequeñas diatomeas y el picofitoplancton (0.45-2.0 µm) como las cianobacterias, dominan las zonas menos productivas del Océano Sur (Xiuren et al. 1996, Froneman et al. 200 l ). Las pequeñas células fitoplanctónicas (picoy nanofitoplancton) pueden soportar más fácilmente las bajas concentraciones en micro y macro nutrientes (Balarin 1999, Froneman et al. 2001). Su ventaja reside en su favorable relación superficie-volumen, permitiéndoles usar la luz solar disponible así como los nutrientes de forma más eficaz que el microfitoplancton (Fogg 1991). Tabla l. Espectro de talla de los diferentes compartimentos taxonómicos del plancton. Tabla modificada según Sieburth et al. (1978). Fuente: www.com.univ-rnrs.fr. PHYTOPLANCTON - 1 1 También, las zonas oceánicas menos productivas tienen tendencia a presentar células nanoº picoplanctónicas mientras las zonas costeras o cercanas a las zonas de deshielo desarrollaran importantes blooms de diatomeas (Weber y El-Sayed 1987, Varela et al . 2002). Este pulso anual en la productividad puede mantener perfectamente el crecimiento sostenido de la población de copépodos (Pakhomov et al. 2002) pero cuando esa biomasa microfitoplanctónica disminuye, la importancia del nano y picoplancton aumenta (V arela et al. 2002). En realidad, se ha visto que los copépodos y el krill tienen preferencias alimenticias por las grandes diatomeas (Olsson et al.1992) pudiendo cambiar la composición taxonómica de un bloom fitoplanctónico compuesto de grandes células de diatomeas a una composición de flagelados, especies de tamaño inferior (Kopczynska 19 ｬ｜ＱＱｾＱＱ＠ ll i l lll' 1992, Smith et al. 1996, Bode et al. 2002) fuera del rango de tamaño óptimo tanto para el krill como para los copépodos (Burkill et al. 1995). A lo largo de la transición temporal del bloom, el viento induce la mezcla de esa capa de agua estable de superficie con aguas más profundas, llevando las células fitoplanctónicas fuera de la capa eufótica y limitando su crecimiento (Smith et al. 2000). También se ha observado que esos procesos fisicos promocionan la aparición de fitoplancton de pequeño tamaño (Varela et al. 2002, Garibotti et al. 2003). El comportamiento nutricional del krill provocaría este cambio tanto en las poblaciones fitoplanctónicas como en las poblaciones zooplanctónicas según la sucesión estacional del bloom al post-bloom (Hemández-León et al. 2001, 2008). También, el hundimiento de las cadenas de diatomeas ocasiona las bajas concentraciones de clorofila (Cabal et al. 2002). Esas condiciones son las típicas de un escenario de post-bloom. Progresivamente aparece una comunidad microplanctónica herbívora (principalmente ciliados y dinoflagelados) favorecida por este cambio, ya que pueden aprovechar esas células fitoplanctónicas de pequeño tamaño (Burkill et al. 1995). Así, en un escenario de post-bloom, una gran mayoría de los copépodos pueden completar su dieta consumiendo este microzooplancton (Dam et al. 1993, Ro man y Gauzens 1997, Atkinson y Shreeve 1995, Froneman et al. 1996). Además del importante efecto del pastaje selectivo por parte del krill sobre la biomasa de diatomeas (Granéli et al. 1993, Haberman et al. 2003 ), el krill también es capaz de consumir el mesozooplancton (Price et al. 1988). Atkinson y Snyder ( 1997) sugieren que el krill posee una cierta preferencia hacia los copépodos de mayor tamaño (1-3 mm). Así, la hipótesis de la preferencia alimenticia del krill empezaría con la depredación sobre copépodos de gran tamaño como Calanus acutus o Calanus propinquus. Sucesivamente, los copépodos de menor tamaño representarían la mayor proporción de su dieta y acabaría con los copépodos migrantes como Metridia gerlachei (Hemández-León et al. 2008). 1.4. EL ZOOPLANCTON DEL OCÉANO SUR Las comunidades zooplanctónicas del Océano Sur pueden estar dominadas por tres grupos taxonómicos: las salpas, el krill o los copépodos (Siegel y Piatkowski 1990, Siegel y Loeb 1995, Loeb et al. 1997, Hosie et al. 2000, Nícol et al. 2000). Aunque el krill es el taxón dominante en las comunidades macrozooplanctónicas (ver Tabla 1) en la Antártida (Marr 1962, Laws 1985, Hopkins 1985), se ha observado que los copépodos pueden dominar tanto en biomasa como en abundancia 20 1 'iTROOl C CIO'i (Voronina 1998, Chojnacki y Weglenska 1984, Hopkins y Torres 1989, Ward 1989, Boysen-Ennen et al. 1991, Voronina et al. 1994, Cabal et al. 2002). También, existen otras especies de zooplancton como los poliquetos, ostrácodos, apendicularios, anfipodos y quetognatos, pero su contribución no llega al l 0% de la biomasa total de zooplancton (Knox 2006). 1.4.1. COPÉPODOS Estos organismos, probamente los organismos metazoarios más numerosos del planeta (Longhurst 1985, Mauchline 1998), están considerados como el mayor componente de la biomasa mesozooplanctónica del Océano Sur (Boysen-Ennen et al.1991 ). Las principales familias están representadas por Oithonidae, Oncaeidae, Pseudocalanidae, Calanidae y Metrididae (Cabal et al. 2002). Los copépodos forman un grupo taxonómico muy diverso con más de 70 especies en los 100 primeros metros de la columna de agua (Hopkins y Torres 1989) y pueden constituir entre el 40 y 98% de la densidad total del mesozooplancton (Conover e Huntley 1991, Pakhomov y Perissinotto 1997, Pakhomov et al. 1997b, Pakhomov y Froneman 1999, Pakhomov et al. 2000, Bemard y Froneman 2002, Cabal et al. 2002, Pakhomov y Froneman 2004a). Rhincalanus gigas, Calanoides acutus, Calanus propiquus y Metridia gerlachei representan las especies más comunes de la ACC y a veces dominan con más de el 90% de la biomasa de copépodos en ausencia del krill (Chiba et al. 2002). También, el ciclo de vida de los copépodos esta íntimamente relacionado con la variabilidad de las condiciones ambientales (Chiba et al. 2002). Así, durante el periodo productivo, el mesozooplancton de gran tamafio ha sido observado en las estaciones situadas en el límite de la ACC, mientras que las estaciones costeras y de la plataforma continental presentan valores mínimos de abundancia (Hemández-León et al. 2008). Los copépodos más comunes suelen realizar migraciones verticales diarias y ontogéneticas (Atkinson 1998) quedándose en capas profundas durante el invierno y reproduciéndose en superficie durante el breve verano austral. Eso explicaría por qué la capa de mezcla y la termoclina presentan picos de máxima biomasa en verano (Ward et al. 1995). La gran mayoría de los copépodos realizan migraciones estacionales según la evolución de su estadio de desarrollo, subiendo a la superficie en estado adulto (Atkinson 1998). También, algunas especies como Rhincalanus gigas pueden realizar migraciones verticales diarias más intensas y al parecer evitan de esta forma a depredadores como el krill Antártico (Atkinson et al. 1999). 21 ·'1' ｾＭ ' 1.4.2. EUFAUSIÁCEOS 1\1 fl 1· 11 ( ( 1 ,, La importancia del krill en la transferencia de energía hacia los niveles tróficos más altos (Laws 1985, Everson 2000) y su biomasa global del orden de cientos de millones de toneladas (Miller y Hampton 1989) hacen que Euphausia superba sea un organismo clave en el ecosistema antártico (Laws 1985). Presente en altas abundancias, pero disperso en enjambres, la densidad de krill dentro de una agregación puede alcanzar una densidad de 30.000 individuos·m-3 (Hamner 1984 en Watkins y Murray 1998). En algunos casos, los eufausiáceos pueden dominar la comunidad zooplanctónica (Pakhomov et al. 2000) y representar entre el 1 O y el 50 % de la biomasa total de zooplancton en el Océano Sur (Knox 1994). Sus frecuentes agregaciones hacen que estos organismos, dispersos en manchas de hasta 450 km2, puedan constituir comunidades de hasta 2.1 millones de toneladas (Macaulay et al. 1984 ). Tanto las zonas cubiertas de hielo como los frentes y el talud continental son propicias para las poblaciones de krill antártico debido a las altas concentraciones en clorofila (Lascara et al. 1999). Las variaciones en abundancia que se han podido observar se atribuyen a las migraciones estacionales u ontogéneticas (Siegel 1988) pero también al grado de extensión de la cobertura del hielo del invierno anterior (Brierley et al. 2000, Hewitt et al. 2003). Los cambios de masas de aguas (Priddle et al. 1988) y las corrientes oceánicas (Murphy et al. 1998) potencian estas migraciones. Por ejemplo, la intrusión del "Agua Circumpolar Antártica Profunda" (UCDW o Upper Circumpolar Deep Water) en la plataforma continental provoca una mejora de las condiciones nutricionales debido a un mayor crecimiento del fitoplancton que favorece el desarrollo embrionario y larvario (Hoffman y Husrevoglu 2003). Sin embargo, el ciclo de vida de Euphausia superba depende estrechamente de la dinámica de los hielos. De forma sencilla, el ciclo de vida del krill (Fig. 3a, b y c) se desarrolla en varias fases geográficamente bien separadas (Siegel 2000) y relacionadas con su estado de desarrollo. Durante el verano, el krill antártico deposita sus huevos en las aguas abiertas para optimizar la supervivencia de sus larvas (Quetin y Ross 2001 ). Después de haber realizado una larga migración vertical desde los 1000 metros de profundidad, las larvas llegan a las capas superficiales de hielo por el sistema circulatorio de la "Corriente Costera Antarctica" y "Corriente Circumpolar Antarctica". 22 • a Sp aw11ng re males Adull populallon - - -- - ·I - 1 .. (}! Developmental as.:ent ｾ ＬＬＮﾡ＠ 1 - o ｉ ｾ＠ - . ｬｾＬｴ＠ Sprtng bloom e --- -- -- - -- - - -- Krlll In summe r Juveniles Figura 3. Representación simplificada del ciclo de vida del krill antártico a lo largo de las estaciones. Extraído de Nicol 2006. 23 l:'liTROOl CCIÓ:'ll 1' 11' oJll 1 ( 1< ., Las larvas y el krill juvenil están estrechamente asociados a la capa de hielo donde se encuentra su alimento, principalmente las algas pero también donde pueden encontrar una protección contra la depredación (Daly 1990). Contrariamente a las larvas, el krill adulto puede utilizar otras fuentes de alimentos hundiéndose en la columna de agua en busca de alimento, lo que evita una cierta competencia con las larvas. Así, el krill se caracteriza por su migración vertical en los primeros 200 metros de la columna de agua (Pakhomov 1995) dispersándose en superficie durante la noche para alimentarse y regresar a estratos más profundos y agregarse durante el día (Everson y Ward 1980, en Pakhomov 2004). En verano, los juveniles se encuentran cerca de la costa y al pasar a estado adulto, realizan a su vez una migración ontogénetica hacía la plataforma continental. Así, los tres estadios de desarrollo del krill optan por una configuración en su distribución espacialmente bien separada que mejora las oportunidades de supervivencia (Quetin y Ross 2001 ). 1.4.3. TUNICADOS Las salpas (Salpa thompsoni) son organismos pelágicos de aguas más calidas y oligotróficas (Voronina 1998, N ishikawa et al. 1995 en Pakhomov 2004a ). Tienen un comportamiento alimenticio, un ciclo de vida y una dinámica distinta a crustáceos planctónicos. Debido a su capacidad de reproducción asexual rápida, las salpas son capaces de formar densas agregaciones (Perissinotto y Pakhomov l 998a, b, Pakhomov et al. 2002) que pueden dominar el macrozooplancton en las diferentes regiones de la Antártida (Park y Wormuth, 1993, Hosie 1994, Nishikawa et al. 1995, Dubischar y Bathmann 1997, Chiba et al. 1998). Recientemente, el estudio de la distribución de S. thompsoni ha mostrado una extensión de su distribución hacia el Sur (Pakhomov et al. 2002, Atkinson et al. 2004). Esta observación podría relacionarse con el calentamiento de las masas de agua o con la intrusión local de masas de agua más calidas (Pakhomov y Fronemann 2004a). La distribución espacial de las salpas estaría influenciada por las características biológicas y fisicas de dichas masas de agua (Huntley et al. 1989, Nishikawa et al. 1995, Alcaraz et al. 1998). 1.5. LA IMPORTANCIA DEL PASTAJE Y DE LA OMNIVORÍA A pesar de las condiciones climáticas extremadamente dificiles, el zooplancton puede consumir e ingerir, de forma selectiva o no, parte de la producción primaria. Las consecuencias del pastaje dependen globalmente de la biomasa del zooplancton, de las tasas de ingestión de las 24 l"\TROOl (CIÓ:\ diferentes especies y su selección sobre el fitoplancton en un rango de tamaño especifico. Así, el impacto del pastaje de las salpas puede controlar entre el 10 y el 100% de la producción primaria diaria dependiendo de la zona del Océano Sur (Dubischar y Bathmann 1997, Perissinotto y Pakhomov 1998a). Además de poseer unas tasas de filtración muy altas (Fortier et al. 1994, Perissinotto y Pakhomov l 998a, b ), las salpas pueden alimentarse de partículas en un rango de tamaño de 1 a 1000 µm, siendo importantes competidores de otros tipos de zooplancton herbívoro (Loeb et. al. 1997). Se ha visto que las salpas pueden alimentarse tanto de algas muy pequeñas (Kremer y Madin 1992 en Nishikawa et al. 1995) como de larvas de Euphausia superba (Huntley et al. 1989, Nishikawa et al. 1995). En las comunidades planctónicas de la Antártida, el pastaje del mesozooplancton es más dificil de evaluar, ya que en esta comunidad existen muchos ciclos de vida y distintos comportamientos. Según el estudio de Hernández-León et al. (2000) en el Estrecho de Bransfield, el impacto del pastaje sobre la producción primaria ha sido estimado a menos de 5%, mientras que ha llegado al 56% en Georgia del Sur (Ward et al. 1995). Los copépodos pueden participar activamente en los flujos de energía y nutrientes a través de la producción primaria y de los depredadores de mayor tamaño. Así, éstos pueden consumir hasta 8 veces la producción primaria consumida por el krill (Conover y Huntley 1991, en Chiba et al 2002). Aunque los copépodos no tengan una tasa de ingestión individual diaria muy alta, (Perissinotto 1992, Atkinson et al. 1996, Pakhomov y Perissinotto 1997, Pakhomov et al. 1997a, Urban-Rich et al. 2001, Pakhomov y Froneman 2004b), estos organismos producen a menudo un importante impacto sobre la producción primaria debido a su abundancia. En realidad, los eufausiáceos tienden a tener una tasa individual de ingestión diaria superior a la de los copépodos (Perissinotto 1992, Pakhomov y Perissinotto 1997, Pakhomov et al . 1997a, Pakhomov y Froneman 2004b). El microzooplancton, por ejemplo, puede consumir entre 21 y 27% de la producción primaria (Burkil et al. 1995 en Knox 2006) y el zooplancton herbívoro entre 25 y 56% (Ward 1995, Knox 2006). Se sabe que los copépodos pastan principalmente sobre las diatomeas en condiciones favorables de fitoplancton (Hopkins et al. l 993a) pero también pueden adoptar una tendencia más omnívora (Hopkins et al. 1993b). También, se ha observado una preferencia alimenticia del krill sobre copépodos en presencia de fitoplancton (Granéli et al. 1993). Atkinson y Snyder (1997) han sugerido una cierta preferencia del krill hacia los copépodos de mayor tamaño. Este comportamiento encuentra su explicación en el contenido lipídico y proteico más rico de estos organismos en comparación con las diatomeas. Así, Atkinson et al. ( 1999) han demostrado que la 25 l'.'\TRODL CCIÓ'I! distribución del krill no solo esta relacionada con las masas de agua sino que su abundancia esta asociada también con las bajas concentraciones de copépodos. Las migraciones más intensas de los copépodos hacia capas más profundas en presencia de krill podrían explicar esas bajas biomasas de copépodos (Atkinson 1999). Sin embargo, se ha sugerido que el buen estado nutricional y metabólico de los copépodos hacen que la depredación por parte del krill sea la explicación más probable a las bajas biomasas de mesozooplancton comúnmente encontradas en las aguas antárticas (Hemández-León et al. 2008). 1.6. LA DISTRIBUCIÓN DE LOS PRINCIPALES GRUPOS ZOOPLANCTONICOS EN LA ANTÁRTIDA El comportamiento ontogenetico, la migración vertical, el estado de desarrollo, las condiciones climáticas, las características biofisicas de las masas de agua, la producción primaria, la competencia interespecífica y la depredación son factores que influyen en la distribución espacial y vertical de los organismos planctónicos. Según la escala del estudio (micro-, mesoo macroescala) unos factores jugaran un papel más importante que otros. Por ejemplo, el krill Antártico y las salpas se distinguen claramente por presentar una separación biotópica bien marcada (Nishikawa et al.1995, Pahkhomov 2004) pero en algunas zonas de la Península Antarctica, las salpas y el krill pueden estar en competencia directa (Loeb et al. 1997). Al igual que el comportamiento migratorio diario del krill, también se ha observado migraciones verticales de salpas hasta las capas superficiales durante la noche (Pakhomov 1991 ), llegando hasta los l 000 m de profundidad durante el día (Pakhomov 2004). Así, las salpas pueden participar intensamente en la bomba biológica transfiriendo el carbono desde la superficie hacia las capas más profundas (Perissinotto y Pakhomov l 998a). En ocasiones, las altas concentraciones de partículas en suspensión provocan la obstrucción de su sistema de filtración y limitan así el desarrollo de sus poblaciones (Harbison y Glimer 1976). Se sugiere que por esa misma razón, las mayores biomasas de salpas se han podido observar en las zonas oceánicas con bajas concentraciones en partículas (Harbison et al. 1986 en Nishikawa et al. 1995). Por tanto, la zona de muestreo sobre el talud, en la zona costera o en mar abierto influye sobre la composición de cada grupo taxonómico (Siegel y Piatkowski 1990, Ross et al. 1996). En general, los copépodos se han adaptado a las condiciones ambientales dificiles pero presentan sus óptimas abundancias durante el bloom primaveral que es cuando empieza el periodo reproductivo (Atkinson 1998). Así, la mayor parte de los copépodos se encuentran en la capa de 26 l'iTRODl C'CIO"' mezcla y en la termoclina durante el verano (Ward et al. 1995). Sin embargo, la repuesta al bloom fitoplanctónico puede variar según las especies de copépodos (Atkinson et al. 200 l ). La gran mayoría de los estudios se han enfocado en la distribución de las especies zooplanctónicas a gran escala. No obstante, no se sabe aún como se distribuyen los copépodos en la columna de agua en relación con el krill durante el periodo del post-bloom tanto en zonas oceánicas como costeras. Según la depredación, la disponibilidad de alimentos, la estructura y la fisica de las masas de agua, la distribución de las especies en el Océano Sur es potencialmente muy variable. El krill antártico es un claro ejemplo que demuestra esta variabilidad. Podemos entender gran parte de los parámetros que dirigen tanto su distribución espacial como su comportamiento migratorio o su abundancia pero aun así, quedan considerables incertidumbres para desentrañar totalmente las razones que explican esta variabilidad (Nicol 2003). También, el muestreo estacional a mesoescala es recomendable para poder determinar las variabilidades existentes (Huntley y Niiler 1995). Por ejemplo, las condiciones ambientales de las masas de aguas y su nexo con la distribución del mesozooplancton aún no esté claramente establecido (Catalán et al. 2008). Dado que ya se ha observado una estructura en la distribución de la biomasa micronectonica que se atribuye a las características hidrológicas (Catalán et al. 2008), parece razonable pensar que exista también un acoplamiento a mesoescala con la biomasa mesozooplanctonica. No obstante, no se ha encontrado ninguna evidencia de un posible acoplamiento entre las características hidrológicas y los cambios de biomasa mesozooplanctonica (Cabal et al. 2002). La explicación podría residir en una escala de muestreo superior a la escala de las variaciones características de las aguas en latitudes polares Así, se espera, al reducir la distancia entre las estaciones, que en las zonas específicas de alta biomasa fitoplanctónica como por ejemplo los frentes hidrográficos, exista un acoplamiento entre la fisica compleja de las masas de agua del Estrecho de Bransfield y la red trófica que daría lugar a una estructura específica en la distribución de las comunidades zooplanctónicas. 27 l"\TRODl C'C'IÓN Los copépodos, a pesar de su enorme abundancia, tienen un papel menor en el flujo gravitacional. Sus heces son pequeñas y ligeras y por tanto tienen tasas de hundimiento más bajas, las cuales son rápidamente colonizadas por bacterias (Frangoulis et al. 2005). Muchas especies del zooplancton que viven por debajo de la zona eufótica durante el día, viajan por la noche hacia la superficie para alimentarse. La proporción del zooplancton migrante varía de una zona a otra y los esquemas de migración vertical también son diversos. En zonas polares existen especies como Rhincalanus gigas o Calanoides acutus (Ross et al. 1996) que migran estacionalmente y presentan un ciclo de vida acoplado a la migración vertical, por lo cual se le denomina migración ontogenetica. 1.8.1. LA RESPIRACIÓN DEL ZOOPLANCTON La respiración es una medida de la mineralización de la materia orgánica ingerida por el organismo y es altamente dependiente de la temperatura y de la biomasa individual (Vidal 1980, Ikeda et al. 2001). Un cálculo reciente de la respiración global de la comunidad mesozooplanctonica en la zona epipelágica integrada en todas las latitudes muestra un consumo de 10.4 Gigatoneladas de carbono al año (Tabla 2). La parte de la producción primaria respirada por la comunidad mesozooplanctonica se ha estimado entre el 17% y el 32% (Hemández-León e Ikeda 2005). Tabla 2: Respiración del mesozooplancton epipelágico (media y desviación estándar) obtenido por cada rango de latitudes utilizando biomasas y tasas de respiración específicas. Extraída de Hernández-León e Ikeda 2005. Latitude ranga Araa (1 a9 km2) Biomass (mg C m21 Respiration (mg C m2 a1) Global respiration Nurrber of data (Gt e .,ear-11 50-SOºN 19.58 1445.6 ± 245.5 47.39 ± 17.40 0.3±0.1 209 10-5fr'N 68.14 664.6 ± 183.1 97.52 ± 39.73 2.4 ± 1.0 301 1crN-1o·s 60.20 707.0 ± 100.2 144.48 ± 21.49 3.2 ± 0.5 96 10-50"5 101.64 816.2 ± 300.0 98.37 ± 51.90 3.7 ± 1.9 41 50-SOºS 41.05 1509.2 ± 396.4 52.20 ± 9.71 0.8 ± 0.2 191 Total 290.61 10.4 ± 3.7 Otros factores afectan las tasas de respiración tales como los factores químicos (saturación en oxígeno, pH, salinidad), fisicos (afloramientos, luz, turbulencia en las masas de agua) y biológicos (condiciones alimenticias, estado de desarrollo). También existe una cierta variabilidad temporal (diapausa, ritmos diarios o estacionales) y espacial (variabilidad horizontal, migración vertical, variabilidad a mesoescala, ... ) que influyen en las tasas de respiración del zooplancton. Una 34 l'iTRom : cc IÓS estimación de la respiración en el océano indica que éste consume más materia orgánica que lo que parece producir (del Giorgio y Duarte 2002). Entre otros procesos fisiológicos, la respiración del zooplancton en términos de oxígeno ha sido intensamente estudiada pues es una medida directa de las tasas metabólicas o un índice mínimo de la demanda energética en términos de carbono. Sin embargo y hasta el día de hoy, no existe un método que pueda medir rápidamente y de forma rutinaria la producción de dióxido de carbono con organismos mesozooplanctonicos, y particularmente con copépodos. Además, la contribución del mesozooplancton al ciclo global de carbono esta reconsiderándose como un proceso más importante de lo que previamente se había calculado (Zhang y Dam 1997, Steinberg et al. 2000). Sin embargo, los métodos de medición directa de producción de dióxido de carbono con el meozooplancton marino son aún incipientes y requieren un mayor esfuerzo para determinar con exactitud la contribución de la respiración de esos organismos al ciclo del carbono. 1.8.2. LA EXCRECIÓN DEL ZOOPLANCTON La materia orgánica es convertida por los heterótrofos en un compuesto inorgánico como el amonio que pueden utilizar directamente los productores primarios. Esa producción de nitrógeno ha sido denominada como producción regenerada (Dugdale y Goering 1967). El amonio es el primer producto del catabolismo del zooplancton y constituye entre el 59% y el 80% del nitrógeno excretado ( Quetin et al.1980, Le Borgne 1986, Miller y Glibert 1998). La excreción del zooplancton es una fuente importante de nitrógeno para el fitoplancton (Ketchum 1962) y puede alcanzar, junto con otros heterótrofos como los protistas y las bacterias, hasta el 95% de las necesidades del fitoplancton (Eppley y Peterson 1979). El microzooplancton y el bacterioplancton tienen habitualmente un papel más importante en la regeneración del amonio, pero también, el macro y mesozooplancton tienen un papel no despreciable en la regeneración del nitrógeno (Gilbert et al. 1992, Miller et al. 1998). Dado que el nitrógeno es un importante nutriente para la producción primaria, conocer las tasas de excreción tiene unas consecuencias relevantes en el balance del ciclo global de nitrógeno. Al igual que la respiración, la excreción presenta una serie de factores que alteran sus tasas. 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Polar Biology 16: 1-11 . Zhang, X., Dam, H. G. (1997) Downward export of carbon by di el mi grant mesozooplankton in the central equatorial Pacific. Deep-Sea Research II 44: 2191-2202. 51 SEGUNDA PARTE CAPÍTULO 11: OBJETIVOS DE LA INVESTIGACIÓN CAPÍTULO llI: CONSIDERACIONES ADICIONALES SOBRE LA METODOLOGÍA OB.IETI\ O OF: L \ I"\ F:STIG \( 10' CAPÍTULO Il: OBJETIVOS DE LA INVESTIGACIÓN Los océanos que rodean la Antártida juegan un papel clave en el ciclo global del carbono. Las altas concentraciones de nitrato y fosfato que se pueden encontrar, sus comunidades planctónicas dominantemente autotróficas hacen que este ecosistema tendría que presentar una mayor producción primaria. Por lo tanto, no lo es. Excepto las bajas disponibilidad de luz y de hierro, las profundas capas de mezcla, el estrés de las radiaciones UV, una posible explicación complementaria a esta paradoja podría relacionarse con la alta presión de los consumidores sobre la producción primaria. En este sentido, el zooplancton de pequeñ.o tamañ.o (principalmente los copépodos) y el krill podrían ejercer un control importante sobre dicha producción. Se conoce también que el krill tiene preferencias por el alimento animal en presencias de fitoplancton y zooplancton pero que puede controlar de forma directa e indirecta las concentraciones de fitoplancton. Este control por pastaje y predación puede suministrar aportes importantes de nitrógeno, y particularmente de amonio a la comunidad autotrófica. Las interacciones existentes entre los diferentes niveles de la cadena trófica originarían varios escenarios que pueden provocar tanto la desaparición momentánea de los copépodos como la del fitoplancton. Los enjambres de krill también que pueden provocar un efecto de retroalimentación, a través del proceso de la excreción, creando condiciones idóneas para la producción de una proliferación fitoplanctónica. En este sentido, la determinación dela composición taxonómica y la biomasa de los grupos planctónicos constituyen unos parámetros fundamentales. En particular, la peninsula antártica es una región con una dinámica hidrológica compleja con procesos de deshielo, variaciones batimétricas importantes, masas de aguas diferentes (salinidad y temperatura principalmente) donde dominarían unas intensas y diversas interacciones tróficas. En este sutil paisaje hidrográfico, la circulación de las masas de agua puede provocar unas interacciones complejas entre el fitoplancton, mesozooplancton y macrozooplancton. Para poder desentrañ.ar esas interacciones entre los diferentes niveles de la cadena trófica, el estudio de la distribución de la biomasa y de las estructuras marinas, y el metabolismo de estos organismos son unos parámetros esenciales para conseguir y estudiar la magnitud de los cambios en estas comunidades. 53 <1 1:1111 1•1 111 11 1" 1, 11 1, \ 1 111 ' Según este esquema descriptivo donde las interacciones entre los diferentes componentes de la cadena trófica son complejas, se plantearon los objetivos siguientes: 1. Diseñar un nuevo método óptico para estimar de forma más rápida y precisa la biomasa de las comunidades zooplanctónicas, 2. Extraer los parámetros que permiten determinar la biomasa de los grandes grupos taxonómicos a partir de imágenes digitales 2D, 3. Diseñar un nuevo método para determinar de forma directa y instantánea la respiración del zooplancton en término de carbono, 4. Estudiar las variaciones en las tasas respiratorias en relación con el estado de inanición con el fin de determinar el rango de metabolismo, 5. Describir la distribución vertical y horizontal así que la estructura de tamaño del zooplancton en distintas zonas de la península antártica, 6. Examinar las posibles interacciones entre los diferentes grupos zooplanctónicos, 7. Estudiar el acoplamiento físico-biológico en aguas del Estrecho de Bransfield con un enfoque sobre la distribución tanto vertical como horizontal del zooplancton, 8. Estudiar la biomas y la actividad específica en los diferentes sistemas frentales, 9. Determinar los aportes de amonio por las comunidades zooplanctónicas y la contribución de la excreción de esas comunidades a la producción primaria en las diferentes zonas de estudio. 54 CO:'\SH>F:R \( 10'\F:S ｾｏｂｒｅ＠ 1 \ \IFTOOOl.OGI \ CAPÍTULO III: CONSIDERACIONES ADICIONALES SOBRE LA METODOLOGÍA 3.1. METODOLOGÍA PARA EL ESTUDIO DE LA BIOMASA POR MÉTODO ÓPTICO 3.1.1. CALIBRACIÓN Y ESTUDIO ESPECÍFICO DE LA BIOMASA Tanto la determinación de la biomasa zooplanctónica como la identificación de las especies son parámetros claves, indicadores de las condiciones ambientales marinas en la base de cualquier análisis espacio-temporal de las comunidades planctónicas. En muchos casos, es imprescindible aumentar la densidad de las estaciones de muestreo para poder identificar las causas subyacentes a la gran heterogeneidad en la distribución del zooplancton. La obtención de datos de biomasa de las distintas especies siempre ha implicado una labor previa costosa en taxonomía y un importante consumo de tiempo ya que tradicionalmente origina largas sesiones de identificación bajo la lupa binocular (Postel et al. 2000), pues la cantidad de muestras es proporcional a la amplitud del muestreo. Debido a estos problemas, hemos realizado el mayor esfuerzo para una estimación de la biomasa zooplanctónica más rápida y precisa a través de métodos ópticos. Desde el nacimiento de los métodos ópticos se han descubierto nuevos horizontes tanto en el análisis de la abundancia zooplanctónica como en la composición taxonómica. Esto ha fomentado la creación de nuevos métodos para identificar y numerar automáticamente el zooplancton a través de programas informáticos especializados. Estos métodos han abierto una nueva rama de investigación en la que se puede también estimar la biomasa. Sin embargo, dado que dicha estimación es complicada además de necesitar factores de conversión (Billones et al. 1999, Postel et al. 2000), se deben obtener aún estos factores para poder estimar la biomasa del zooplancton desde imágenes en dos dimensiones (Hemández-León y Montero 2006). Con el fin de determinar la biomasa de las principales especies o de los principales grupos taxonómicos, Hemández-León y Montero (2006) realizaron un ejercicio de calibración con un microscopio estereoscópico acoplado a una cámara digital. Los resultados mostraron que la extracción de la biomasa a partir de imágenes en dos dimensiones es precisa. Estudiaron los parámetros de regresión que relacionan el peso individual con la superficie cotporal de algunas especies de crustáceos zooplanctónicos del Océano Sur. Por otro lado, apuntaron que es necesario determinar la relación peso-superficie coiporal de los organismos gelatinosos, dado que el contenido hídrico es muy diferente y así abrieron estas nuevas líneas de trabajo. ¿Existen 55 l O'SI DER \( ｉｏｾｅｓ＠ SOBRE 1 \ \IETOllOI OGI \ diferencias entre la relación peso-superficie corporal de los organismos gelatinosos y los crustáceos? ¿Existe una similitud en las relaciones peso-superficie corporal entre los grupos taxonómicos de las aguas polares y subtropicales? ¿Es posible una generalización hacia una ecuación global relacionando todos los organismos planctónicos marinos y es suficientemente precisa? 3.1.2. MÉTODO PARA LA IDENTIFICACIÓN, EL CONTEO Y LA ESTIMACIÓN DE LA BIOMASA PLANCTÓNICA 3.1.2.1. Consideraciones previas A pesar de la existencia de múltiples sistemas ópticos in situ cuya potencialidad para el estudio de la biomasa es enorme, las pescas con redes siguen siendo el método de muestreo más común y rápido para recolectar el zooplancton (Bell & Hopcroft 2008). Sin embargo, la calidad del reconocimiento automático de los organismos depende de la calidad de la muestra. El tipo de red, la forma de arrastrarla y de recuperar la muestra influye sobre el estado fisonómico de los organismos y consecuentemente sobre el análisis de los resultados. Así, es aconsejable utilizar redes que no dañen a los organismos (redes Bongo, WP-2, Bioness,. .. ). Por ejemplo, las redes planctónicas en continuo tipo Longhurst-Hardy Plankton Recorder (LHPR) son redes que aplastan los organismos entre las mallas y causan daños irreversibles que complican su identificación y así mismo la determinación de la biomasa por métodos ópticos. Por otro lado, es recomendable limitar la velocidad de recogida de las redes para evitar el aplastamiento o la ruptura de los organismos en los colectores. El problema general e inherente de los organismos cortados o deformados por la técnica de pesca con redes es el mayor inconveniente de estimación de biomasa por métodos ópticos. El cálculo de la biomasa de los organismos dañados es problemático. Dado que existe una relación potencial entre la superficie y el peso, dos partes de un mismo organismo tienen un peso menor que el mismo organismo entero. Hemos asumido que ese error es despreciable dado que el arrastre de las redes se realizó con precaución limitando así los daños sobre el zooplancton. Igualmente, la evaluación visual confirmó la buena calidad de las muestras. 56 CO'.\SIDF:R \CIO:-;ES SOBRE 1 \ \IF.TOOOI OGÍ \ 3.1.2.2. Preparación de la muestra y adquisición de imágenes El sistema de adquisición de imágenes de zooplancton está descrito en el artículo de Lehette y Hemández-León (2009). No obstante, para el estudio de la biomasa de una muestra o submuestra procedente de un proceso de separación (por ejemplo, con un separador Folsom), se necesita una adaptación de la técnica. El uso de un separador es imprescindible para obtener una densidad óptima en la célula de digitalización. Esta densidad óptima se obtiene cuando los organismos preparados para la digitalización ni se agrupan, ni se superponen en la muestra o submuestra. La muestra se deposita de forma homogénea sobre la célula transparente de digitalización (23x19cm). Para evitar que el programa considere los márgenes de la bandeja como partículas, además de la deformación óptica debida a los meniscos creados por los márgenes de la bandeja de digitalización, se toman una serie de 8 fotografías (representando aproximadamente un 30% de la célula de digitalización) seleccionadas al azar fuera de los márgenes con una resolución de 14.7 µm por píxel. Para asegurarse de que no haya efecto de heterogeneidad en la distribución de los organismos al seleccionar sólo una parte de la muestra, se realizó un "test de homoestaticidad" (p<0.05) confirmando la homogeneidad de las muestras. 3.1.2.3. Factor de corrección El número de partículas realmente digitalizado por fotografía es aún inferior al número presente en el tercio de la célula de digitalización. En realidad, las partículas cortadas por los márgenes de las fotografías no deben considerarse en el análisis de abundancia y de biomasa. Es necesario introducir un factor de corrección que permita evitar una subestimación de la cantidad de partículas presentes en la totalidad de la muestra. Para determinar este parámetro, se seleccionaron tres muestras en cada zona de estudio y se fraccionaron con un filtro de 1000 µm. Cada fracción (superior e inferior a 1000 micras) se digitalizó con un escáner (Epson perfection 4990) y con el sistema de macrofotografía descrito en el artículo de Lehette y Hemández-León (2009). Así, se obtuvo el número total de partículas realmente presente en cada fracción y el número de partículas capturado por cada fotografía. La diferencia en el conteo de partículas proporciona un factor de corrección (anexo 1) cuyos parámetros son de 0.865 ± 0.003 (fracción mayor de 1000 µm) y de 1.01 ± 0.007 (fracción menor de 1000 µm). Es decir la digitalización de la muestra por fotografía captura realmente 86.5% de las partículas para la fracción superior a 1000 µm. En el caso de las partículas inferiores a 1000 micras, la técnica de fotografías no es necesario aplicar dicho factor de corrección. 57 ｃｏＧｩｓｉｄｅｒＮ｜ｃｉｏｾｅｓ＠ SOBRE L \ \lf:TODOl.OGI \ 3.1.2.4. Método de estimación de la biomasa planctónica de forma rutinaria 3.1.2. 4.1. Programa de identificación, conteo y estimación de la biomasa planctónica El programa Zoolmage es un programa específico de reconocimiento de formas, de conteo y de cálculo de la biomasa del plancton. Zoolmage presenta varios pasos que el usuario debe cumplir para poder pasar de una sección a la siguiente. Para nuestro estudio, se seleccionaron 20 muestras representativas de toda la zona de estudio en cada profundidad. Zoolmage procesa todas las imágenes de una misma muestra y extrae las viñetas de todas las partículas encontradas, con sus características morfológicas, almacenando toda la información pertinente en un fichero de metadatos. La siguiente etapa engloba la sección de aprendizaje del programa (supervisado por un taxónomo ), siendo el paso clave para un reconocimiento adecuado de los organismos. El especialista clasifica las viñetas (con sus metadatos ), correspondientes a las partículas de interés (copépodos, larvas de krill o quetognatos, ... ) en un árbol de clasificación según las categorías taxonómicas adecuadas y definidas por el usuario. Además de clasificar los organismos de interés, todas las otras partículas (nieve marina, apéndices, fibras, burbujas, otros objetos ... ) deben ser clasificadas y reconocidas por el programa para evitar la contaminación de estas partículas en las diferentes categorías taxonómicas. Se crearon varias ｾ｡ｴ･ｧｯｲ￭｡ｳ＠ conteniendo cada una un mínimo de 20 a 30 partículas. Las de interés ( copépodos en posición dorsal o lateral, larvas de krill, quetognatos, parte de quetognatos, ... ) y las que constituyen las partículas de rechazo (nieve marina, fibras o burbujas ... ) representan la base de datos de entrenamiento para el clasificador. Una vez completadas a través del clasificador, estas categorías se comprobaron con un algoritmo de clasificación con el objeto de discernir los parámetros de cada categoría entre ellos y así obtener las mejores clasificaciones de las categorías con un mínimo de error. Existen varios algoritmos de aprendizaje automático. Los mejores resultados se obtuvieron con el algoritmo KNN (K-Nearest Neighbor) para las dos fracciones (inferior y mayor de 1000 micras). La clasificación de cada categoría se evaluó utilizando una matriz de confusión (Gislason y Silva 2009) con una validación cruzada aplicada 10 veces ("10-fold cross validation" en inglés) determinando el nivel de error entre la clasificación manual y automática. Este nivel de errores para cada categoría se refleja en los valores que aparecen fuera de la diagonal en la matriz. Dicha matriz permitió ver cuales de las categorías creadas (krill, copépodos, etc.) presentaban un porcentaje de 58 ＨｬＩ｜Ｌｬｬ＾ｬｬｾ｜ｬｬｃＱ｜ｩＧＢｊｬＺｬＧｩ＠ 1 \lll l1>1111i<J1.I\ error demasiado alto (falso positivo o falso negativo) con otras categorías. Así, los copépodos en posición dorsal y lateral y los quetognatos y apéndices de krill se confundieron en más del 25% de los casos. Al aceptar un porcentaje de error máximo del 25%, fue necesario unir las dos categorías de copépodos en una para disminuir el error en el reconocimiento. Asimismo, las categorías "quetognatos" y "apéndice de krill" han sido descartadas del análisis debido a la gran abundancia de esos apéndices comparada con la escasa abundancia de los quetognatos. Además, el error asociado a esta categoría hubiera inducido a un error de clasificación demasiado importante para poder estimar correctamente la abundancia de este grupo taxonómico. En resumen, combinar algunas categorías en un nivel de clasificación más general y bajar la calidad de la clasificación fue una tarea delicada. Por otro lado, la eliminación de las especies o grupos taxonómicos escasos es una etapa esencial para maximizar la capacidad predictiva del programa. La tercera parte del programa consiste en la creación de unas "series de análisis", es decir el análisis de todas las muestras y la creación de una tabla de datos con la abundancia y la biomasa de cada grupo taxonómico por profundidad en cada estación. El cálculo de la biomasa desde el área se realizó a partir de las ecuaciones obtenidas en los artículos de Hemández-León y Montero (2006) y Lehette y Hemández-León (2009). 3.1.2.4.2. Machine Learning El primer paso del método consistió en la determinación de la biomasa individual a partir de la imagen de una especie manualmente identificada (ver párrafo 3.1.1.). El siguiente paso para poder determinar la biomasa de una muestra entera de forma semi-automática a través de un programa especializado en el reconocimiento del zooplancton (Zoolmage) fue enseñar al programa a identificar y a clasificar los organismos en grupos o taxones de interés (Machine Learning). La rapidez y la eficiencia del método de identificación semi-automático de los organismos asistido por los programas Zoolmage o Zooprocess ya ha sido demostrada (Bell e Hopcroft 2008, Gorsky et al. 20 l O). La biomasa de cada organismo identificado y clasificado se calculó según los parámetros de conversión específica entre la superficie corporal y su peso individual (ver artículos de HemándezLeón y Montero 2006, y Lehette y Hemández-León 2009). 59 BIBLIOGRAFI \ SEGDiO.\ PARTE Lehette, P. and S. Hemández-León (2009) Zooplankton biomass estimation from digitized images: a comparison between subtropical and Antarctic organisms. Limnol. Oceanogr.: Methods 7: 304-308. Lehette, P. and S. Hemández-León (2010) Carbon dioxide production rates of marine epipelagic copepods in subtropical waters during the late winter bloom Joumal of Experimental Marine Biology and Ecology 385 (1-2): 38-43, [doi:l0.1016/j.jembe.2010.01.006]. Hemández-León, S., and l. Montero (2006) Zooplank.ton biomass estimated from digitized images in Antarctic waters: a calibration exercise. J. Geophys. Res., 111, C05S03, [doi: 10.1029/2005JC002887]. Lewis, G. N. and M. Randall (1961) Thermodynamics. 2nd. ed. revised by Pitzer, K. S., Randall, M. McGraw-Hill. O'Sullivan, D. W., Millero, F. J. (1998) Continual measurement of the total inorganic carbon in surface seawater. Mar. Chem. 60, 7583. Pilson, M. E. Q. (1998) An Introduction to the Chemistry of the Sea. Prentice-Hall Inc., New Jersey. Postel, L., Fock, H. and W. Hagen (2000) Biomass and abundance, p. 83-192. In Harris R. P., P. H. Wiebe,J. Lenz, H.R. Skjoldal, and M. Huntley. [eds.], ICES zooplank.ton methodology manual. Academic Press, London. Riley, J. P. and G. Skirrow (1965) Chemical oceanography, v. l. Academic. Weiss, R. F. (1974) Carbon dioxide in water and seawater: the solubility of a non-ideal gas. Mar. Chem. 2, 203-215. 66 TERCERA PARTE CAPÍTULO IV: Zooplankton biomass estimation from digitized images: a comparison between subtropical and Antarctic organisms. CAPÍTULO V: Carbon dioxide production rates of marine epipelagic copepods in subtropical waters during the late winter bloom. CAPÍTULO VI: Zooplankton biomass and metabolism in the frontal zones of the Bransfield Strait (Antarctica). CAPÍTULO VII: Zooplankton community structure along the Western Antarctic Peninsula. CAPÍTULO VIII: Krill excretion and impact on primary production during austral summer 2005 along the Western Antarctic Peninsula CAPÍTULO IV: ZOOPLANKTON BIOMASS ESTIMATION FROM DIGITIZED IMAGES: A COMPARISON BETWEEN SUBTROPICAL AND ANT ARCTIC ORGANISMS Pascal Lehette1* and Santiago Hernández-León1 1Biological Oceanography Laboratory, Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain. Pascal Lehette and Santiago Hernández-León, (2009) Zooplankton biomass estimation from digitized images: a comparison between subtropical and Antarctic organisms. Limnol. Oceanogr. Methods, 7: 304-308. 68 ZOOPL ｜Ｚ｜ｾｔｏＧ｜＠ BIO\I ｜ｓｾ＠ F.STl\I \TIO' ABSTRACT The measurement of mesozooplankton biomass in the ocean requires the use of analytical procedures which needs the destruction of samples. Altematively, the development of methods to estimate biomass from optical systems and appropriate conversion factors could be a compromise between the accuracy of analytical methods and the need to preserve the samples for further taxonomic studies. The conversion of the body area recorded by an optical counter or a camera was suggested as a suitable method to estimate total biomass by converting the digitized area of an organism into individual biomass. In this study, crustacean mesozooplankton from subtropical waters were analysed and individual dry weight and body area were compared. The obtained relationships agreed with other measurements of biomass obtained from a previous study in Antarctic waters. Gelatinous mesozooplankton from subtropical and Antarctic waters were also sampled and processed for body area and biomass. As expected, differences between crustacean and gelatinous plankton were highly significant. Transparent gelatinous organisms have a lower dry weight per unit area. Therefore, to estimate biomass from digitized images, pattem recognition disceming, at least, between crustaceans and gelatinous forms are required. Running head: zooplankton biomass by digital images INTRODUCTION Zooplankton plays a central role in structuring pelagic food webs and mediating biogeochemical cycles. Understanding their biomass and distribution within the world ocean is a requisite to predict their contribution to the global organic matter and energy fluxes (Banse 1995). Analytical measurement of biomass using standard methods (see Postel et al. 2000) requires the destruction of the sample (dry weight, ash-free dry weight, elemental analysis of carbon, nitrogen ... ). To avoid these procedures in order to allow further taxonomical and ecological studies, non-destructive methods should be used. However, adequate methods for a suitable estimation of mesozooplankton biomass are still not a standard. In this sense, several optical imaging techniques were developed over the past decades to examine zooplankton organisms. The use of an electronic flash source was the first tool to capture 69 /IHll'I 1'-kl11'. 1;1111¡1,,¡ ,jlll1llli'. instantaneously the silhouette of living plankton (Ortner et al. 1979; Edgerton 1981 ). An in-si tu silhouette camera system was also designed for zooplankton genera identification and abundance estimation (Ortner et al. 1981 ). Other systems based on video cameras were developed to classify zooplankton. For instance, a video camera was interfaced to extract the silhouette of preserved organisms and further classify them into different taxonomic groups (Jeffries et al. 1984). Microscope image processing systems were also used to classify and identify different stages of copepods (Oietrich and Uhlig 1984) and to study the size distribution of zooplankton samples (Rolke and Lenz 1984). A modified method was used to extract body area and size of copepods but only preliminary results conceming analysis and treatment of samples were presented (Gorsky et al. 1989). A submersible 35-mm camera system was also used to compare plankton density from insitu silhouette photographs with concurrent preserved net collections (Olney and Houde 1993). A recent method based on the digitalization of a net sample by a scanner (ZOOSCAN) was also developed in order to identify and automatically detect zooplankton organisms (Grosjean et al. 2004 ). The enumeration and measurement of a thousand specimens of zooplankters can be done in a short time and various morphological parameters like body length, shape and area can be extracted. One of the major attributes of the ZOOSCAN is rapid sample processing (Gorsky and Grosjean 2003). Because of the important evolution of image acquisition techniques of zooplankton, it is now possible to indirectly estimate zooplankton biomass using digital image processing. A high performance CCO camera mounted on a tripod was used to study Daphnia magna population growth using ellipsoid conversion factors to estimate its body volume (Frernvig et al. 2002). Similarly, a CCO video camera installed in a stereomicroscope and connected to a computer was used to measure the length of various zooplankton organisms and to calculate the biovolume of the whole sample (Alcaraz et al. 2003). The relationship between the biovolume and biomass was extracted from the integrated samples, although changes in the taxonomical composition of the sample can modify the relationship obtained. In order to estimate biomass of the most common species and groups of zooplankton in Antarctic waters, Hemández-León and Montero (2006) used a CCO camera connected to a stereoscope microscope to compare individual biomass with body area. The conversion of the body area spectrum into the biomass spectrum allowed the estimation of total and size-fractionated biomass. This method gave comparable results to previous measures carried out in the same region (Hemández-León and Montero 2006). However, gelatinous organisms like salps and chaetognaths 70 ZOOPI "KTO" 810\I \SS F.STl\I \TIO:'\ were not considered in this study due to poor representation in the samples. Biomass of chaetognaths is estimated to be 10-30% of that of copepods in the world oceans (Bone et al. 1991) and the tunicate Salpa thompsoni is among the most important filter-feeding metazoans of the Southern Ocean, ranking only after copepods in terms of total biomass (see Pakhomov et al. 2002). Thus, the gelatinous forms play a significant role in the transfer of energy to higher trophic levels (Bone et al. 1991) and their biomass estimation is of paramount importance. The objective of the present study was first to extract the relationship between biomass and digitized body area of the most common taxa of mesozooplankton in subtropical waters around the Canary Islands and then to compare ours results with those obtained in the Southern Ocean by Hernández-León and Montero (2006). Secondly, we tested the suitability of the method for gelatinous organisms in order to extract biomass. Results indicate that the body-area and biomass relationship for subtropical crustaceans fits well with measurements obtained in Antarctic waters for those organisms. As expected, a different relationship for gelatinous plankton was obtained. MATERIALS AND METHODS In order to compare body area with individual biomass, different specimens of copepods, chaetognaths, siphonophores and euphausiids were sampled around the Canary Islands in vertical hauls from 200 m to the surface during April-May 2006 using a WP-2 net (UNESCO, 1968) equipped with a 200 µm mesh. Samples from the net were size fractionated into 200-500, 5001000 and > 1000 µm size classes. The organisms of the different size fractions were gently washed to remove particles and immediately digitized for image processing and analysis. Salps (Salpa thompsoni) were captured in the shelfwaters of the Bransfield Strait (Antarctic Península) during January-February 2005. A BIONESS (Bedford Institute of Oceanography Net and Environmental Sensing System) net was deployed in oblique hauls from 400 m depth to the surface. We were unable to digitize them on board and, therefore, organisms were then frozen at - 20ºC for further processing and image analysis. To obtain the relationship between individual body area and dry weight, the body shape of each organism was photographed and body area was measured on a computer. Salps were gently defrosted from -20ºC and body area measured by image analysis. The nucleus of the organism was also measured in addition to body area because of its sharper and quite more visible form for optical devices. Therefore, we also tested the relationship between the nucleus and the biomass of the 71 whole organism. Organisms were dried using standard procedures (Lovegrove 1966). Except for salps, all specimens were first digitized and then stored at -20ºC before drying at 60ºC for 24 h, allowing the sample to reach room temperature, avoiding humidity, and then weighed using an ultra microbalance (Sartorius supermicro, ±0.2 µg). To generate a digital image of the silhouette of organisms, a standard digital camera with a CCD sensor was used (Nikon D l 00 equipped with a 55 mm Nikkor macro lens). In this study, each organism was individually photographed whatever its natural position and the specific dry weight measured. The camera was positioned at the smallest vertical distance to the organism to have exactly the right focus and maximum resolution. Three neon lamps (8 watts each) were placed at 5 cm below the transparent tray (Nunc ™) containing the organisms in a thin layer of distilled water. A frosted glass of 5 mm was used to attenuate and diffract homogeneously the beam. Diaphragm opening diameter, shutter speed and film sensitivity were controlled to obtain the best contrast in order to distinguish the silhouette from the background. lmage files were stored as Tagged lmage File Format (tiff) and processed with a personal computer using the image analysis software Global Lab® Image/2. After correcting for all band level thresholds of the image of each organism, the silhouette was extracted by the program-generated area and stored. The system was calibrated with a micrometer graduated ruler (Leica) to a resolution of 7.8 µm pixel-size for copepods, chaetognaths, siphonophores and euphausiids and 33.3 µm for the salps. Adopted resolutions were suitable for morphometric measurements and recognition to taxonomic group. Finally, body area of organisms can be considered as an independent variable because the error in measuring the individual body area here is much less than the measurement of the individual body mass, allowing the use of model l regression for predictive purposes (Legendre and Legendre 1998), avoiding the use ofthe model ll regression (Ricker 1973). RESULTS Our results showed that individual dry mass and body area was best fit by a power function (see Figure 1) for subtropical copepods and euphausiids showing a high correlation coefficient (r=0.967 and r=0.955, respectively). Similar slope coefficients were found in these taxa {l .59 ± 0.027 vs 1.47 ± 0.078, see Table 1). As differences between both regression curves were not significant, we pooled ali the data for a general subtropical crustacean relationship between body area and their respective dry weight. A high correlation coefficient was also obtained (r=0.968). 72 1000000,0 100000,0 10000,0 ,....., ' "C .s 1000,0 ｾ＠ copepods "C 00 :i 100,0 '-' .E 00 ·a; ｾ＠ >. 10,0 "C o '° 1,0 0,1 0,0 0,0 0,1 Subtropical crustaceans siphonophores euphausiids ... 1,0 .· .· 10,0 . ...... ZOOPL.A.'iKTO'i BIO;\l.\SS ESTl\IATIO'i .· .· '.. Antarctic crustaceans* •o.f • ... .. • . .,.;. . . · ........ .. ·fl' y Antarctic S.thompsoni chaetognaths 100,0 1000,0 10000,0 Figure l. Relationship between individual body area and individual biomass (as dry weight) of zooplankton in subtropical and Antarctic waters. Dashed line represents the relationship obtained by Hemández-León and Montero (2006) excluding Rhincalanus gigas (see text). Subtropical euphausiids showed similar slope coefficients of the area-biomass relationships to those found in Antarctic Waters (Hemández-León and Montero, 2006), but the intercept values of the regression curve (see Table 1) were significantly different (p<0.01). Similar intercept values of the area-biomass relationships were also observed for the Antarctic and subtropical mesozooplankton, but the slope coefficients were significantly different (p<O.O 1 ). A general areabiomass relationship extracted from our results and data from Hemández-León and Montero (2006) for mesozooplankton, euphausiids and both were also obtained showing good correlation coefficients (r=0.947, r=0.987, r=0.972, respectively). As expected, gelatinous zooplankton, however, showed quite different regression parameters (Figure 1 ). Relationships between individual biomass and area for salps, siphonophores and chaetognaths showed slightly lower correlation coefficients than for crustaceans (r=0.902, r=0.926 and r=0.840, respectively). Significant differences for slope coefficients and intercepts were found within these gelatinous organisms. Regression results for the different zooplankton are given in Table 1. 73 -...) ｾ＠ Table l . .Regressi.on (DW = a S 11 ) and conelation pamneten (a is the intercept, SE the standaJd mor ofthe regression coefficient, b the slope, r the coirelation coefficient. p the degree of significance, andn the nmiler oforganismsmeasured) obtained between body area (Sin mn:) and individual chy mass (DW in µg) for Subtropical :mdAntarctic orga!Usms. Body area shl'ws the r:mge observed. Organism a b±SE r p D Body .Ana, m.m 1 So urce 45.72 1.19±0.14 0.886 <0.001 23 0.5288.64-t Hemindez-León :md Montero, 2006 --- --- 56.43 1.44±026 1 0.777 <0.001 22 3.101-6.144 Hemández-León :md Mo!!!ero_ , 2006 -· -,- -- --- - , 0.797 <0.001 19 1.061-3.009 Hemindez-León 31!d Momem , 2006 22.44 1.78 = 0.26 1 76.71 0.63 ±0.28 0518 <0.001 16 .5912-1 7.402 _ ｈ ｾ ｾｮ､ＡｺＺｌ･￳ｮ＠ ｡ｮ､ ｟ ｾｾｴ･ｭ Ｌ＠ 2006 -- -- -- · -- -- -. - -- _, Ostracods 99.46 1.28 ±0.19 0.885 <0.001 1:5 1.10.$-4338 Hemández-León and Montero, :!006 -- - - - E hausia S UJ!.•rha. 87.45 134 ±O.O'! __ 0.967 <0 .001 71 1 7 .248-369 .1 Bemandez-León md fl.íol_!tero_ , 1006 ...._ - · - Antamic mesozoopmitton* 1 4238 1.47 ±0.08 __ 0,828 <0.001 89 0.5288.6..i.t ｈ･ｭ￡ｮ､･ｺ Ｍ ｌｾ￳ｅ＠ :md _Montem_, 2006 ｟ｑｾ ｌｊ＠ 1.52::1:0.04 Ｍｾ ＸＱ＠ --. Antardic crustaceans* <0.001 160 0.:528369.1 ｈ･ｭｩｮ､･ｺＭｌ･￳ｾｭ､＠ Montero, 1006_ . Alldata ＳＶ Ｎ ＶＱ ｟ｲ ＱＭＮＺｾ Ｑ ｟ ］ＰＮＰＵ＠ _0.961 <0.001 176 0.518-369.1 ｈｾｾｺＭｌ･￳ｮｾ｟ＡＡ＠ ｾｾｮｴ･ｭ Ｌ＠ 2006 ! 1 - - -- - · 23.45 1.19±0.13 o.uo <0.001 33 24 . 9-18 7.5 This study 112 4::1:0.08 ..__ 4.03 0.901 <0.001 11 170.19-997. 49 Tbisstwiy 67.66 0.78 ::::0 .07 0.940 <0.001 21 10. 70-9: 5.5 7 Thisstud v j---------' - · 43.17 1.02±038 0.916 <0.001 9 2.64-59.51 Thisstud y 43.81 1.47±0.08 0.955 <0.001 17 1. 67 -9.99 Thislludy 45"_2 51 1..59:0.03 0.967 <0 .001 138 0.10-8.31 Thisstudy 1.56±0 .02 0.968 <0.0 .Q.!_ • 155 0.10-9.99 Thisstud v 1-- -- -. lhia llludy and Heman"'dez-León and General mesazoo.elankton ＭＭﾺ Ｓ Ａ｟｟｟ ｾ ＴＺＺｴＺｏ Ｎ ｏ Ａ｟＠ _0.947 <0.001 227 0 .1 0 - 8. 6J4 Mottero, 2006 - [ 49.58 1.48±0.05 "Ibis study and Hmiández-León and General usiids 0.98j <0.001 88 1.67369.1 Momero, 1006 -- This study and Hemández-León and General aust.aceans* 43.97 1.51 ::1::0.0 2 0.972 <0.001 315 0.10-369.1 ?\fottero, .::!006 * -without .Rlli1ralanus gigas (stt text). - N o '"" ｾ＠ r- , z ;r, -l o ,,,_ = o .:::: ;... '-" ,.,, l"'l '-" -l 2 ;¡;.. -l o "..l. ZOOPLA:'liKTO'.'i 810.\l.\SS ESTl'.\t\TIO:'li In order to compare the methodology for assessment of individual biomass in salps, we obtained a better relationship between the area of the nucleus and their total dry weight (see Table 1) than measurement of the entire body area for the same organisms. The same conclusion was drawn in a previous study (Alcaraz et al. 2003). This relationship will allow establishing a more precise estimation of the dry weight of those transparent organisms. DISCUSSION Our results show the usefulness of extracting precise information on individual biomass from direct body area measurements. However, the question of whether or not to pool crustaceans into a general relationship from the two different regions is open to debate. In this sense, when all data for subtropical crustaceans (mesozooplankton and euphausiids) are combined, the intercept of the potential regression curve for crustaceans from subtropical waters was significantly different (p<0.01) to those from the Southern Ocean, but the slope coefficient was highly similar. However, excluding the large soft bodied copepod Rhincalanus gigas from the general relationship resulted in subtropical and Antarctic crustacean's regression parameters being highly similar. Therefore, the problem of extracting a general relationship between biomass and body area depends on the taxonomical composition of the sample. Thus, a general area-biomass relationship for crustaceans (r=0.972) can be obtained for these two quite different ecosystems ( excluding R. gigas, see Table 1) whatever its dorsal or lateral position. The two regressions obtained for mesozooplankton and euphausiids or even the one observed for the entire range size of crustaceans can be applied in both areas of the ocean. Gelatinous organisms showed a lower individual biomass per unit area than crustacean zooplankters (Figure 1 ). Moreover, the regression parameters of gelatinous organisms showed a higher standard deviation than crustaceans. The main reasons for this are the high amount of water and high variability in organic content of their tissues, as well as the difficulty of the software to accurately detect the edges ofthese transparent planktonic organisms. Differences between the regression parameters of salps, siphonophores and chaetognaths were significant (p<0.01). Thus, a general body area-biomass relationship cannot be applied for the gelatinous groups. Therefore, in order to estimate biomass from body area-biomass relationships, the crustaceans and gelatinous forms should be discerned. Although a universal relationship is envisaged for crustaceans, this will not be the case for all mesozooplankton as observed for salps, 75 C \RBO' DIOXIDE PRODL CTIO' R.\ TES MATERIALS AND METHODS Epipelagic copepods were captured by vertical hauls performed with a WP-2 net equipped with a 500 µm mesh size. The most important copepod genera observed were Clausocalanus and Paracalanus. The net was hauled from 100 m to the surface with a closed codend in order to maintain the organisms in good conditions (Steinberg et al., 2000). The net was towed at low speed (10-15 m·min-1) during the night. Dates of collection and position of sampling sites are indicated in Table 1. After the catch, all individuals were immediately transferred into new aquaria with surface seawater adjusted to in situ temperature (18ºC). Injured and dead organisms, large crustacean larvae, gelatinous organisms and other camivorous invertebrates such as thaliaceans and chaetognaths were removed. Table l. List of sampling dates, geographic positions and depth of the sampling stations around the Canary Islands during early spring. Station Date Local time (GMT+ 2h) Latitude (N) ｌｯｮｾｩｴｵ､･＠ (S) Bottom depth (m) STll 24/03/2006 2130 27°35.76 13º37.16 752 ST15 25/03/2006 2045 28°07.63 13º17.05 109 ST27 27/03/2006 2150 27°34.83 13°59.37 1814 ST39 29/03/2006 0320 27°07.50 14°24.19 2135 ST35 29/03/2006 2200 27°44.50 14º06.19 1867 ST43 30/03/2006 2130 27°35.10 14°21.41 2166 ST42 31/03/2006 2030 27°25.54 14º25.84 2287 ST53 01/04/2006 0310 27°15.97 14º41.57 2600 ST56 02/04/2006 2215 27°25.47 14°54.38 2622 ST62 03/04/2006 2105 27º52.34 14°58.32 2012 ST61 04/04/2006 0315 28º00.27 14º54.67 1272 ST72 05/04/2006 0350 27º15.88 15°32.26 2945 All the experiments were derived from the "water-bottle" method (Omori and Ikeda, 1984). Less than 5 minutes were sufficient to select and place healthy copepods into the bell-jar incubation bottle (600 ml). The experiments were performed in filtered seawater (GF/F Whatman filters) in a thermostatic bath at 18°C, close to the in situ temperature and in dim light. The number of copepods varied between 50 and 150 individuals depending on incubation time in order to have a significant response between the control and the experiment Copepod densities during experiment were far from the levels affecting metabolic rates (Christou and Moraitou-Apostolopoulou, 1995; Ikeda et al., 2000). At the end of the incubation, the respiration rate was calculated from differences between the control and experimental bottles. For very short experiments (ca. 0.5 h), we incubated 82 ｃａｒｒｏｾ＠ OIOXIDE PRODl CTIO'i R .\TES the largest number of animals in order to produce a measurable response in the carbon dioxide production (Peck and Prothero-Thomas, 2002). In order to avoid low oxygen saturation levels, low quantities of copepods were added in the incubation chamber for a longer incubation time. All copepods recovered at the end of the experiment were dried at 60ºC during 24 hours and weighed on a micro balance following the procedure of Lovegrove ( 1966). The health condition of all individuals was controlled before and after the experiments because injured or died animals would lead to an underestimation of respiration rates. In sorne replicates, only a few individuals were found motionless or injured and were not considered for the calculation ofbiomass. 3 7 6 OUTFLOW 2 ｄ ｄｄ ｾ＠ IRGA Figure l. Schematic diagram of the flow-trough system used for the determination of PC0 2 in seawater. 1. EGM-4 infra-red gas analyser. 2. Interna) air pump (200 ml·min-'), thermometer and pressure manometer. 3. Peristaltic pump with controlled water flow rate. 4. Drying agent tube. 5. Therrnostatic bath (fixed at 18ºC ± 0.1 ºC) . 6. Bell-jar bottle (650 ml). 7. Liqui-Cel® Membrane Contactor. 8. Air calibration gas (C0 2-free and 55 l ppm). 9. Air tubing system protected with synthetic foamjacket. JO. Water tubing system protected with synthetic foamjacket. 11. Four-way valve. 12 . Pressure regulator. 13. Personal computer. The pC0 2 was determined using a flow-through system coupled to an equilibrator (LiquiCel® MemObrane Contactor) to transfer seawater C0 2 to the gas phase and then to measure it by infra-red spectrometry (Robert and Smith, 1988; Kortzinger et al., 1996). Among the various methods to assess seawater pC0 2, the direct one using an air-flushing equilibrator coupled to a nondispersive infra-red gas analyser (EGM-4) is presently recognized as the best experimental approach (Dickson and Goyet, 1994). This pC0 2 system follows the design principle of the laminary flow type (Poisson et al., 1993). The pC0 2 in dry gas was measured using the standard gas 83 calibration (Legg and Parkinson, 1968). At each measurement, the IRGA was recalibrated to a zero level using zero C0 2 gas mixture, and every l O measurements recalibrated using a C0 2 molar fraction of 549 ppm. The system consists in a continuous flow of renewed seawater through a gasliquid exchanger in which a closed loop of air dried with a drying agent ( drierite<Ri) is circulated in a counter-current flow (Fig. l ) . 800 ,-.., a o.. o.. '-" i:: .9 - u ce c.!:: il) ........ o 600 a N o u 400 o • ' I• •I I• ;'. 1!111. • 1 1' • 1 1 1 1 I 1 ﾷＧＮｾ＠ ,. ' 1 ,. ,\ I I 1 1 j; • !•. • ;._\ '• 1( ｾＦＭ ... ﾷＭﾷｾﾷＭＭﾷ＠ :.•··-·---·-·-·----·-·-·-· -oControl l ·-o-- Control 2 <> Control 3 - • - Experiment 1 -• -- Experiment 2 --•- Experiment 3 • '. ""'&... ·-·-·-· ; /. ..... -i.......- ..... - Ｍｾ󰿃ＺＺｦＺｴＭＺＮｴＭ］ＭｬＭＭＭＱ］ＴＭ󰿃ＭﾷＭﾷＭﾷＭﾷＭﾷＭﾷＭﾷＭＭＭＭＭＭＭＭＭﾷＭＭﾷ＠ .¡ !'¡ "' <>··-<>· <> , . · ... o .. º .. º ·o .. -0 .. -0 ... 0 .. o .. o· o· º:.a·::fr·:a:· 4 0.o--o-D . 0--De: .oo· ·º o p o o d 5 10 15 20 25 30 35 Time (minutes) Figure 2. Example of different equilibration times for experiments and controls performed using filtered seawater. Observe that 20 minutes is enough to perform a measurement. The overall system was especially designed to avoid any air bubbles formation in the equilibrator at the seawater interface. This closed and fixed volume of air is re-circulated continuously during the measurement until it reaches the equilibrium with the constantly renewed seawater. The water volume of incubation bottles was the key parameter limiting seawater flow to low values (14.5 ml·min-1 ). Nevertheless, response time was such that 20 to 40 minutes were enough to reach the equilibrium without siphoning all the water in the incubation bottle (Fig. 2). The counter-current flow direction is supposed to facilitate the equilibrium (Kortzinger et al., 1996) and this equilibrium is reached after approximately twenty minutes, depending of the air and liquid fluxes and the differences of C0 2 partial pressure between the two phases. Incubations were made 84 CARDO'\ DIOXIDE PRODl ｃｔｉｏｾ＠ RA TES to obtain, at least, a difference of about 100 ppm between the control and experimental measurements. The equilibrator consists of a polycarbonate cylinder (MiniModule® lx5.5 inch.) where seawater run at 14.5 ml·min-1• The air circuit was maintained by the internal pump ofthe gas analyser at a flow rate of 200 ml·min-1• The seawater temperature was monitored using a thermostatic chamber fixed to 18±0.1 ºC. The flow-through system was protected with a synthetic foam jacket to minimize temperature changes. The accuracy of gas analyser EGM-4 was less than 1 ppm (or 0.034 µmol C0 2 at experimental conditions) and the linearity of the signal was better than 1 % throughout the calibrated range. The barometric pressure inside the equilibrator was close to the atmospheric pressure. Barometric pressure and temperature were monitored respectively in the air loop and in the water flow, and both water and gas circuits were checked for leakages. Because, the IRGA worked with dry air, correction for water vapour pressure was applied following the procedure described by Dickson and Goyet (1994). The correction factor for water vapour pressure was 0.98 at the experimental conditions (S=36.5; Tº=18.0ºC; P= 758mmHg.). C02 partial pressures in the water and in air were calculated assuming 100% saturation for the water vapour pressure (Keir et al ., 2001 ). The overall equilibration efficiency of the equilibrator was calculated from Weisler (1996), Grant ( 1998) and Minfang et al. ( 1996), and for experimental conditions was estimated to be 98.8%. lf we assumed that by far the greatest proportion of C02 is present in the form of.fC02 rather than H2 C0 3 since seawater is very close to the pH at which rates of formation of C02 hydrate are minima (Pilson, 1998), we can relate, using Henry' s law, the pC02 in the liquid portion as follows: pC0 2 (µmol) = Kh x ppmC02 x /atm where ｾ＠ is the Henry's solubility constant which depends on seawater temperature and salinity (Weiss, 1974), and fe.tm is the correction factor to adjust pC02 measurement at 1 atmosphere. The uncertainty of the whole pC0 2 system was estimated to be ±3.7 ppm (standard deviation calculated from 8 sets of control bottles). All data were converted to pC02 in wet air at 1 atm. Also, it is well known that ammonium is the dominant form of nitrogen excreted by zooplankton (Nemazie et al., 1993; Miller and Glibert, 1998; Conover and Gustavson, 1999; Atkinson and Whitehouse, 2000) and is likely to change alkalinity. However, a recent study (Davies et al., 2003) showed that changes in nitrogen (N0 3-and NRi 1 of an order of 500 µM did not affect substantially the alkalinity. Thus, we may assume alkalinity remaining constant because of the 85 1 \IU:il\. tllll\!lil f'I\ 1 11 1 l 11'\ 1< \ J ! " insignificant changes ( 1-1 O µM) m ammonium concentration at the end of the incubations (unpublished results ). RESULTS Carbon dioxide production rates of copepods showed, as expected, a significant decrease with time of incubation (Fig. 3). Standard metabolic rates were reached after only 4-6 hours of incubation (about 0.06±0.03 µmolC02·mg-1 dw·h-1 from4 to 14 h ofincubation). 1,0 'on s N o 0,6 u o s ::t '--' 11) "'¿;j ¡..... i:: o Ｍｾ＠ ¡..... ﾷｾ＠ 11) ｾ＠ 0,4 0,2 05 2 3 4 5 6 7 8 9 10 11 12 13 14 Incubation time (hours) Figure 3. Respiration rates of copepods in starving conditions over 14 hours of incubation in filtered sea water. Vertical bars represent standard errors. Observe the sharp decrease in carbon dioxide production rates after the catch and the values of standard metabolism after six hours of incubation. 86 Table 2. Review of specific respiration rates (µmol0 2· mg-1 dw· h-1) in mesozooplankton of tropical and subtropical waters with a special emphasis on \ organisms having similar size (µm), temperature of incubation (ºC) and experimental conditions. Refereace Regio• Zoopluktom Respintioa rate Size Temperatare Time of Eapsed time Acclimatioa - -1 -1 µm ·e iacalatim after captare ,_aolOnag dw·.11 2ertod May1.3Ud and Dallot Meditcrn.nean mesozoo 0,()1) -0,19 >300 12° -13° 4b -22h 12h (1973b) sea Nival et .al. North African copepods o. os -0,75 >690 15° 4h-6h llh (1974) Upwelling Smith and Whitledge North African meso zoo 0,37 223 -505 17 °-22 ° 2h-4h 3h-12h (1977) Upwelling LeBorgne Atlantic mesozoo 0.17 -0.26 >200 17° -20º 15h -21h no no (1977) equatorial LeBorgne Atlantic copepods 0,17 <500 (600/o) 20º 3h -24h no no (1979) equatorial 95"/o Oo LeBorgne North African copepods 0,13 -0,51 200-5000 17º 19h -23h -.....¡ no no (1982) Upwelling 95"/o Gandy and Boucher Indian Ocean copepods 0,04 -0,24 >500 20:: 24h lh -2h no (1983) Equatorial Hem ández-León Canary meso zoo 0,18 -0,41 >25rf 20º 24h no no (1986) Islands Hemá.ndcz-León lit al _ North Central mesozoo 0,26 >500 20" -24° no data no data no data r') ,. (1999) Atlantic " = Ikedaetal_ Subtropical copepods 0,15 -0.36 >200 22° - 24° no data no data no data o /. (2001) o are a o May zaud et a/_ North Atlantic copepods 0,04-0,25 >200 1 7" -18" lOh -15h lh no ｾ＠ o (2005)' Ocean l"'l "'O " This study Canary copepods 0.15-0,30c >500 18ª 0,5h -14b no no o o Islands ,.... lj ....¡ o ªPersonal communication. b C02 measurement. e lntegrated value over 4 and 14 h assuming a RQ of 0.87. '/. Al ｾ＠ ....¡ l"'l (IJ Maximum rates (average of 0.68±0.37 µmolC02· mg1 dw· h1) were measured only 0.5 h after the catch and were 11-fold higher than standard rates. ln order to compare our respiration rates with previous studies in tropical and subtropical waters, we converted our data into oxygen consumption using a RQ of 0.87 (Mayzaud et al., 2005). Our experimental respiration values were integrated for 0-4 h and 0-14 h periods (Table 2). Overall, these values of respiration rates in terms of carbon dioxide were similar to those found in tropical and subtropical regions by different authors. Analysis of gut content at the begining of incubation showed a range of total pigments between 24.7 to 74.0 ng·mg-1 protein. These values of gut content indicated that copepods were feeding on phytoplankton at normal rates before the capture (Hemández-León et al. , 2002). DISCUSSION The decrease in respiration rates by mesozooplankton during starvation was observed in many studies about mesozooplankton metabolism (Nival, 1974; Mayzaud, 1976; Skjoldal et al., 1984; Blazka et al., 1982; Abou Debs, 1984; Ikeda, 1977; Tsuda, 1994; Thor, 2003). This decay in metabolic rates is due to the effect of food shortage during incubation rather than the effect of stress after capture (Ikeda and Skjoldal, 1980). Thus, results obtained with a previous standard acclimation period in filtered seawater is debatable and immediate measurement after the catch is thought to be a more realistic approach to assess field rates (Satomi and Pomeroy, 1965; Le Borgne, 1979; Webb and Johannes, 1967; Biggs, 1977; Bamstedt, 1985; Omori and lkeda, 1984) Because the effect of starvation is rapid in time (Le Borgne, 1979), experiments performed during prolonged periods lead to an underestimate of respiratory activity (Schneider and Lenz, 1991 ). Respiration rates showed a decrease of 50% within 6 h for Acartia clausi (Mayzaud, l 973b) at l 3°C , and within 1 O to 20 h of incubation in filtered sea water in Acartia tonsa at l 8°C (Ki0rboe et al. , 1985). A decrease between 40% and 70% was also observed in respiration rates in Euchaeta norvegica during the 10 first hours after capture at 6ºC (Skjoldal et al. , 1984). Mayzaud (1976) observed that Acartia clausi reached standard metabolic rates after only 6 hours of starvation at 5°C. Usually, the nutritional status in the environment affect zooplankton metabolism (Comer and Cowey, 1968; Mayzaud, 1973a,b; Mayzaud and Dallot, 1973; Mullin et al ., 1975; Bamstedt, 1985; Alcaraz et al. , 1998; Gaudy et al., 2003; Gaudy and Thibault-Botha, 2007). Thus, a high variability is normally expected in short incubations. In fact, Comer et al. ( 1965) observed on Calanus sp. a more drastic decrease of metabolic rates when organisms were actively fe eding before the experiment. The same argument holds for our experiments. 88 C \RBO:\ OIOXIDE PRODl CTIO"I R\TES The ratio between active metabolism (maximum respiration rates in aerobic conditions) and standard metabolism is the so-called factorial scope, which ranges between 2 and 1 O in marine invertebrates (Hernández-León and Ikeda, 2005). In fishes this quotient is higher (10-20, Brett, 1964) but is normally rather low in marine zooplankton (2-4, Gaudy and Thiebault-Botha, 2007; 3 in Euphausia pacifica, Torres and Childress, 1983; 4 in Acartia tonsa, Ki0rboe et al., 1985; 6 in Diothona oculata, Buskey, 1998). Perhaps, these rather low values were related to the length of incubation normally used to assess respiration rates in marine zooplankton. By opposite, high values were also observed in the literature. Abou Debs (1984) found a 22-fold increase in respiration rates of the copepod Temora stylifera after incubation at increasing food concentrations, showing that well-fed organisms have rather high rates compared to starving animals. Conover and Lalli (1974) also observed a 20-fold increase in the pteropod C/ione limacina. The very short incubation of 0.5 h associated with recent feeding activity is probably the main reason for the high value between standard and maximum rates observed in the present work (11-fold). Because zooplankton respiration in the field is never maximal (Hernández-León and Ikeda, 2005) and the factorial scope of zooplankton can be as high as 20 or 22-fold, we suggest that respiration rates obtained after only 0.5 h of incubation in the present work are near the routine metabolism for copepods. Most of the difficulties arising from the measurement of metabolism in zooplankton are derived from their small size and the lack of sensitive techniques to measure respiration in shortterm experiments (Hoegh-Guldberg and Manaban, 1995; Marsh and Manaban, 1999). The method presented here based on infra-red spectroscopy and equilibration techniques allowed to measure C02 production rates (avoiding the assumption of standard RQ values) in rather short experiments (near the natural feeding conditions), and using relatively large incubation volumes (minimizing crowding and bottle effects ). This method has a wide application for both field and lab measurements of marine and possibly freshwater samples when relative changes in pC0 2 are quite important (at least > 100 ppm). Set up in a laboratory or on small research vessels is relatively easy. Nevertheless, the decrease in the performance of the membrane contactor by collapsing when working with turbid water may limit the applicability of the method to filtered water samples (at least <lOµm, Liqui-Cel® Membrane Contactor operating guideline). The manifold is simple and easy to use for gaseous samples, and can discriminate changes in C0 2 concentration with highprecision. The direct determination of carbon released by zooplankton in less than an hour using the traditional water bottle incubation method is a real advantage because of the rapid starvation effect in those organisms (Mayzaud, 1973b, 1976). Thus, the evolution of respiration rates can be also easily monitored as shown in this work. Routine metabolic rates are mostly associated with normal 89 activity level (Swadling et al., 2005) and to estimate this level is not simple. Therefore, it is also suggested that realistic values of respiration rates in epipelagic copepods at ca. l 8ºC can be achieved in rather short experiments (0.5 h), but also in starving conditions for ca. 10 h in order to assess their routine and standard rates, respectively. Acknowledgements This research was financially supported by projects !CEPOS (Ren2002-04165), ConAfrica (CTM2004-023 l 9) and Lucifer (CTM2008-03538) from the CICYT (Spanish Commission for Science and Technology). We acknowledge the assistance at sea of the members of the UTM (Unidad de Tecnología Marina) and the crew of the R/V Hespérides. The authors are indebted to S. Putzeys and J. C. Gómez Femández for their help and advice. 90 C \RBO'i 010:\IDE PROOL CTIO'i R \TES References Abou Debs C., 1984. Carbon and nitrogen budget of the calanoid copepod Temora stylifera: effect of concentration and composition of food. Mar. Ecol. Prog. Ser. 15, 213-223. Alcaraz M., Saiz E., Femández J.A., Trepat l., Figueiras F., Calbet A., Bautista B., 1998. Antarctic zooplankton metabolism: carbon requirements and ammonium excretion of salps and crustacean zooplankton in the vicllúty of the Bransfield Strait during January 1994. J. Mar. Syst. 17, 347-359. Atkinson, A. and Whitehouse, M. J., 2000. Ammonium excretion by Antarctic krill Euphausia superba at South Georgia. Limnol. Oceanogr., 45, 55---63. Bamstedt U., 1985. Seasonal excretion rates of macrozooplankton form Swedish west coast. Limnol. Oceanogr. 30 (3), 697-617. Biggs D.C., 1977. Respiration and ammonium excretion by open ocean gelatinous zooplankton. Limnol. Oceanogr. 22, 108-117. BlaZka P., Brandl Z. and L. Prochazkova, 1982. Oxygen consumption and ammonia and phosphate excretion in pond. Limnology and Oceanography 27 (2), 294-303. Buskey E.J., 1998. Energetic costs of swarming behavior for the copepod Dioithona oculata. Mar. Biol. 130, 425-431. Brett, J. R., 1964. The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Bd Can. 21, 1183-1226. Christou E.D., Moraitou-Apostolopoulou M., 1995. Metabolism and feeding ofmesozooplankton in the eastem Mediterranean (Hellenic coastal waters). Mar. Ecol. Prog. Ser. 126, 39-48. Cole J. J., Caraco N. F., Kling G. W. and T. Kratz, 1994. Carbon dioxide supersaturation in the surface waters of lakes. Science 265, 1568-1570. Conover, R. J., Lalli, C. M., 1974. Feeding and growth in Clione limacina a pteropod molusc. Il: assimilation metabolism and growth efficiency. J. Exp. Mar. Biol. Ecol. 16, 131-154. Conover, R.J., Gustavson, K.R., 1999. Sources of urea in Arctic seas: zooplankton metabolism. Mar. Ecol. Prog. Ser. 179, 41-54. Comer E.D.S., Cowey C.B., 1968. Biochemical studies on the production of marine zooplankton. Biological Reviews 43, 393-426. Comer E. D. S., Coweys C. B. and S. M. Marshall, 1965. On the nutrition and metabolism of zooplankton. III. Nitrogen excretion by Calanus. J. mar. biol. Ass. U .K. 45, 429-445. 91 ZOOPI \M<TO'I "THE FRO'ff\I . ZO\ES ABSTRACT Biomass, abundance, gut fluorescence and electron transfer system activity (ETS) of zooplank:ton were studied during a post-bloom scenario in the Bransfield Strait (Antarctic Peninsula). Two well defined frontal systems were observed. (l) The so-called Peninsula front between the Transitional Bellingshausen Water (TBW) and Transitional Weddell Waters (TWW), and (2) the Bransfield front related to the Bransfield Gravity Curent flowing northeastward along the slope of the South Shetland lslands. As expected, a typical pattem of plank:ton distribution was observed with higher phytoand mesozooplank:ton in the TBW. However, our more detailed study of the Peninsula front between the TBW and TWW showed the sinking of phytoplank:ton and a higher abundance of large copepods in the TBW side of the front, while krill and small copepods were observed in the opposite side, in the TWW. Secondary ageostrophic circulation around the front supported a striking food web at both sides of the front. This pattem is suggested to drive a relatively important flux of carbon due to the sinking of phytoplankton and the production of fast sinking fecal pellets by large copepods and krill. Keywords: zooplank:ton, biomass, metabolism, fronts, Antarctica INTRODUCTION The coupling between frontal systems, zooplankton biomass and metabolism is scarcely documented in the literature. In Antarctic waters this lack of knowledge is even more important. Most of the research in the Southem Ocean was performed in the study of the Polar Front where high chlorophyll and primary production are normally observed (Allanson, 1981 and many others). The relationship between the Subtropical Convergence (STC) and biological production is also relatively known. In this frontal area, high values of chlorophyll (Froneman and Perissinotto, 1996), primary production (Delizo et al., 2007), mesozooplank:ton (Pakhomov et al., 1994), and mesopelagic fish (Barange et al., 1998) are normally observed. This frontal area show high interannual variability with high values of chlorophyll coinciding with years of increased frontal 98 ZOOPI \ 'KTO'\ I'\ THF: FRO"H \I ZO'\F:S intensity (see Weeks and Shillington, 1996), but forming blooms with limited spatial and temporal scales as observed from remote sensing (Llido et al., 2005). Q) "O ::::::1 _. -62 + 27CTD o 21 XBT C> e: o _J -63 " • 20 3+ 40 T1 s+ 60 7+ 21+ 20C) 19+ T2 180 • .. 31+- BF _, • 380 24+ 250 36+ 260 27+ T3 2so 3s+ 290 39+ 40C) 41+ T4 420 ＴＳｾ ｐｆ＠ 45+ _-460 47+ Ｓ ｾＱＰ＠ 17+ 3'1-B20 33+ 480 49+ 160 ｴｾ Ｚ＠ u+ 80 . .. . ｾ＠ -60 9+ IOC) -59 • ｾ Ｍ - 58 -57 Latitude -56 Figure l.- Location of stations in the Branfield Strait (Antarctic Peninsula). Black cross are CTD stations while empty dots are XBT deployments. The dashed polygones depicted the position of the Bransfield Front (BF) and Peninsula Fronts (PF, see text). 200 m isobtah is also drawn. Near the Antarctic continent, the most studied area is the Antarctic Peninsula. Perhaps, the waters around the South Shetland, Elephant and South Orkney Islands, and the Antarctic Península are the better sampled areas. Here, the Bransfield Strait located between the South Shetland Islands and the Antarctic Península shows two well defined frontal systems (see Sangra et al., 2011): (1) A Península Front (PF) between the so-called Transitional Zonal Water with Bellingshausen influence (TBW, warm and fresh, flowing from the Bellingshausen Sea and the Gerlache Strait), and the Transitional Zonal Water with Weddell Sea influence (TWW, cold and salty), entering from the 99 eastem area, and (2) the Bransfield Front (BF) located along the South Shetland lslands slope. This front is related with the Bransfield Current that behaves as a gravity current flowing northeastward along the South Shetland lsland slope. In Figure l we show the position of both fronts along our observational domain. Between both fronts a system of mesoscale anticylonic eddies was observed. These mesoscale structures, dynamically connected, form part of the Bransfield Current System which is described in detail in Sangra et al. (2011). The Peninsula front is known to promote meanders (Sangra et al., 2011), but relationships between the frontal area and planktonic organisms are scarce. Even the biological differences between water masses were not always clear and different results are normally observed in the literature. While Basterretxea and Arístegui (1999) found differences in chlorophyll between the TBW and TWW, no significant differences were observed by Corzo et al. (2005). Similarly, Hemández-León et al. (1999, 2000) did not find significant differences in mesozooplankton biomass between the TBW and TWW. However, the latter authors found differences in the enzymatic activity of mesozooplankton related to metabolism and growth. Similarly, differences in the abundance of meroplankton (Vázquez et al., 2007) and fish larvae (Catalán et al., 2008) were related to these two main water masses. Therefore, the biological effect of a frontal system could be characterized as differences in abundance or biomass between water masses, and as differences in the rates of feeding, metabolism and growth. Frontal systems normally show increases m plankton biomass as the effect of (1) convergence promoting the accumulation of organisms, or (2) horizontal shear giving rise to the increase of nutrients developing the food web bottom-up (Lutjeharms et al., 1985). Curiously, those increases in biomass were not found in the frontal systems near the Antarctic Peninsula. Only Arístegui and Montero ( 1995) found a rather high plankton community respiration related to the Peninsula front. This lack of biomass signal in Antarctic frontal systems seems to be related to the change in the plankton community structure from the bloom to post-bloom periods in these waters. During melting in spring, water column stability promotes a bloom of large cells, mainly diatoms. After the bloom, strong mixing by wind gives rise to dominance of small phytoplankton cells, such as autotrophic flagellates (Holm-Hansen et al., 1989; Varela et al., 2002; Garibotti et al., 2003). Most of the large diatoms sink out the euphotic zone or are grazed by large copepods and krill. Moreover, during the post-bloom not only large phytoplanktonic organisms are scarce. Mesozooplankton is observed in rather low numbers over the Antarctic shelf and areas near the continent such as the Bransfield Strait (Hemández-León et al., 1999, 2000, 2001 ). Besides, 100 ZOOPI \'h:TO'li l'i THE FRO'IT \1 ZO'IES Hemández-León et al. (2008) found very low numbers of small and large copepods over the Antarctic shelf during the post-bloom, suggesting an important top-down control on copepods by krill before their seasonal migration towards the slope waters where they spawn in summer (see Siegel, 1988; Siegel et al., 1997). Therefore, the non-significant differences in biomass between water masses or, even, no differences in biomass over frontal zones could be due to the extremely low biomass of phytoand mesozooplankton during the post-bloom as a consequence of sinking and grazing of large cells, and the predation by krill on copepods. Meroplankton (V ázquez et al., 2007; Catalán et al., 2008) and zooplankton indices of grazing, metabolism and growth reflect the differences between water masses (Hemández-León et al., 2000) or the biological activity inside fronts (Arístegui and Montero, 1995). However, there is no evidence of increased abundance, biomass or activity of mesoor macrozooplankton in those frontal systems of Antarctic waters. In order to study this effect of fronts on zooplankton, we made an intensive survey along the Bransfield Strait at the end ofthe bloom period, in order to look for the poorly documented effect of different frontal zones on biomass and biological activity. A close relationship was observed between the Peninsula and Bransfield frontal zones and mesozooplankton biomass, suggesting that these mesoscale structures are of importance in the patchy distribution and productivity of the Antarctic realm. MA TERIALS AND METHODS Sampling was performed from 30th December 2002 to 7th January 2003 on board the R.V. Hespérides. Four transects crossing the Bransfield Strait allowed to identify the position of the Peninsula and Bransfield fronts and the different water masses (Fig. 1 ). Temperature, salinity and fluorescence were recorded using a CTD (SeaBird 911 plus) mounted in a rosette sampler. In order to study the biological effect of frontal areas with higher resolution, one of the four transects (transect T3, Fig. 1) was sampled every 5 nautical miles for temperature, combining CTD stations and expendable bathytherrnograph probes (XBTs). In this transect, we also performed seven biological stations, coinciding with CTD stations and hence separated 1 O nm, in order to study phytoand zooplankton distribution. Four biological stations were also made in the other three transects. Phytoplankton was collected from the Niskin bottles of the rosette and about 200 m1 of seawater were preserved for counting using a solution of lugol. Small cells were measured using a flow cytometer (F ACScalibur, Becton and Dickinson) and by direct counting under a microscope 101 , ' ZOOPI ｜ｾｋｔｏＧｩ＠ '" THF. FRO'iT \I ZO...,ES for the large ones. Mesozooplankton was obtained using a BIONESS net (Bedford Institute of Oceanography Net and Environmental Sensing System) in oblique hauls from 300 m depth to the surface. On board, a few individuals of the most representative species were picked up along transect T3 and preserved in liquid nitrogen (-l 96°C) for later analysis of gut fluorescence as an index of grazing (Mackas and Bohrer, 1976), and electron transfer system (ETS) as an index of metabolism (Packard, 1971 ). The samples were then counted and sized using a Lab Optical Plankton Counter (OPC) and later preserved in a 4% solution of seawater and buffered formalin. In order to obtain biomass values from the OPC, the equivalent spherical diameter (ESD) representing the body area of each organism was converted to biomass using the equations given by HemándezLeón and Montero (2006) for Antarctic organisms. The spectrum of body areas was converted to the body biomass spectrum, allowing the estimation of total biomass for the entire sample (see Hemández-León and Montero, 2006 for details). In the laboratory, the samples were counted, very large euphausiids sized in order to know their biomass, and copepods identified to species level using a dissecting microscope (Leica MZ 9.5). The individuals frozen in liquid nitrogen were later homogenized at 0-4ºC and subsamples were taken for protein analysis, gut fluorescence and enzyme activity measurements. Protein content was determined using the method of Lowry et al. (1951) or the method of Peterson (1983) for samples with very low protein content and using bovine serum albumine (BSA) as the standard. An aliquot of the homogenate made for the analysis of protein was placed in a test tube with 10 ml of 90% acetone and stored at -20°C (24 hours) for gut pigment analysis. Fluorescence of the samples was measured before and after acidification in a Turner Design fluorometer, previously calibrated with pure chlorophyll (Y entsch and Menzel, 1963 ). Pigments were calculated with the equations given by Strickland and Parsons (1972) slightly modified to Chlorophyll = k·(F0-Fa)·mg-1 protein Pheopigments = k-(R· Fa-F 0 )· mg1 protein where k is the instrument calibration constant, F 0 and Fa are the fluorescence readings before and after acidification and R is the acidification coefficient. The addition of chlorophyll and pheopigments was used as the gut pigment concentration. Electron transfer system activity was measured according to Kenner and Ahmed (1975) with the modifications introduced by Gómez et al. (1996) for zooplankton samples. Details of the 102 ZOOPI \:\KTO'\ 1' THF. FRO\T \I ZO:\F.S procedure are also given in Hemández-León and Gómez (1996). ETS activity was recalculated to in situ temperature using the Arrhenius equation andan activation energy of 15 Kcal·mor 1 (Packard et al., 1975). RESULTS (a) Section-Tl BF •ta. number ﾰＪＭｾ＠ 3 4* 678:'\ 10 11 f * ..._ ..._. * .. º" ｾ＠ * * ,,. 50 o 'I?.,. J "'" :- -1. :'; .. 1 g o 1 .!. '5 150 c.. " o ';" 200 ';' ｾ＠ !.. o ';' 250 ｾ＠ 300 -- o 20 40 60 80 100 Distance (km) Section-T3 (e) BF PF 8ta. number ｾｉ＠ ｾ＠ ｾｾﾡ＠ ｾ＠ -1 ｾ＠ 4 - .. C>., :e t o <¡< 9 !!! \\__ l 1- --- 9· -0.s 'e. ｾ＠ 1t .so g '5 fr '· o 1 \ .!. 200 ｾ｜Ｌ＠ ｾＱ＾＠ l :- <; 250 1! 9 ';' 300 - .!, o 20 40 60 80 100 Distance (km) (b) Section-T2 BF PF sta. number ｾｾＴＡｾ ｪ＠ ' "l'""l"' i ' -o ｾＮｉ＠ 50 ll ｾＢＺ＾＠ ... 100 ; :- g 1 '5 150c.. " o 200 1 ';' o., 2 50r <j .. ' 30lh--- ｾ｟｟［＠ 20 40 60 80 Distance (km) Section-T4 (d) BF E.E ata . number oi38 39 40 ｾ＠ 42 43 44 -446 9 * * -* * * * * ... Ｚﾺ Ｍ ｦＩｾ＠ o ... -- - 50 .. ¡:,!> 100 ';" g 1 .:; 150 fr i1) o 200 ｾ＠ :l· 1 l 1 1\ ·' o 20 40 60 80 100 Distance (km) 103 Figure 2.- High resolution (5nm) In situ temperature sections obtained combining CTD and XBT data along the four surveyed transects. Larger symbols at the top axes indicate the location of the station where zooplankton community was sampled. The location of the Bransfield Front (BF) and the Peninsula Front (PF) are also indicate on the figures top axes. Contour interval is 0.25ºC ZOOPL ｜ｾｋｦｏＧ｜＠ l'i THE FRO'T \I ZO'\ES Figures 2 and 3 show high resolution temperature vertical sections and low resolution potential density and chlorophyll sections along the four sampled transects. The Bransfield Front signal is clearly recognizable from the stepper and packed isotherms and isopycnals in those stations close to the South Shetland Islands (see Fig. 1). 200 -o 1 y. Section-Tl 40 60 80 100 Distance (km) (b) 250 Section-T2 BF PF sta.number 40 60 80 Distance (km) Figure 3.- Low resolution (lOnm) potential density (kg m3, thin contours) and fluorescence (volts) vertical section along the four surveyed transects. The location of the Bransfield Front (BF) and the Peninsula Front (PF) are indicated on the figures top axes. Contour interval for potential density is O.OS kg m3 (e) .6.E Section-T3 P F sta. number (d) 37 o Section-T4 a-PE 41 43 ｾ＠ ahl.number 47 49 24 36 27 35 30-34 33 60 50 1 6 100 100 15 g 14 1 3 .,s 150 f I 12 Q. " 11 Cl 200 09 ｾＩｬｴ＠ º' 07 06 260 1 0.5 0.4 0.3 20 40 60 80 100 (volts) 20 40 80 80 100 Distance (km) Distance (km) 104 ZOOPI ｜ｾｨＺｔｏＧＮＧｩ＠ l'\' THE FRO,T\L ZO'.'iF.S Close to the Antartic Penisnsula we observe a much shallower frontal region; the so-called Peninsula Front, which separates well stratified, relatively warm and fresher TBW from the homogeneous, relatively cold and saltier TWW. In transect Tl, TBW occupies the whole width of the Strait at surface layers being the signa! of the PF only noticeable in transects T2, T3, and T4. Comparison of high resolution temperature section and low resolution density sections shows that spatial resolution is critica! to properly resolve those mesoscale structures. For example in transect T2 the low resolution density sampling is not able to well resol ve the PF (compare Figure 2b and Figure 3b). BF 24 36 o"' * 50 IO!i ,,..__ s '-' .s 150 ¡; a 200 2 50 900 o 20 27 * Section-T3 35 JO * * 40 60 Distance (km) P F sta. number 80 34 * 1 100 (volts) 4.75 4.7 4.65 4.6 Figure 4.- Potential density (kg m-3, thin contours) and transmittance (volts) vertical section along transect T3. The location of the Bransfield Front (BF) and the Península Front (PF) are indicated on the figures top axes. Contour interval for potential density is 0.05 kg m-3 As already mentioned before, a detailed descripction of the above frontal regions which form part of the Bransfield Current System may be found in Sangra et al. (2011). In this study, we will pay attention to observational evidences suggesting the existence of a secondary ageostrophic circulation (SAC) vertical cell related to a frontolytic episode of the PF along transect D as discussed in next section. In this regard, Fig. 3c shows that at the northern edge of the PF (station 105 ZOOPI \:-0.KTO"'i I'\ THF. FRO\T\l. ZO"'iES 30) there are relative higher values of fluorescence at l 00 m, well below the euphotic layer. This suggests a cross isopycnal downward flow of phytoplankton that may be related to the occurrence of a downwelling in the northem edge of the PF. As depicted in Fig. 4, the transmisometer data along this transect also shows a subsurface relative maximum of particles at station 30, also suggesting a downward flow at the northem edge of the PF. Transmisometer data also shows a relative minimum of particles at station 34 which may be an indication ofupwelling at the southem edge ofthe PF. In this region of the ocean, density is well correlated with salinity but not so well with temperature, suggesting that temperature may partly behave as a passive tracer and therefore its distribution may give us sorne indications about the secondary circulation. A close inspection to isotherms in the PF region in transect T3 reveals that in the northem part of the front the isotherms deepen, whereas close to its southem edge the isotherms shoal (Fig. 2c). This is noticeable by a local deepening of the isotherms at station 30 anda shoaling of the -0.75 ºC isotherm at station 34. - E ｾ＠ +-' a. Q) Cl -20 -40 -60 -80 -100 t -20 -40 -60 -80 -100. 38 t 38 f . Station number %'T ::1<11 · 30 34 t t . . "· 4000 '\ \ \ 27 35 . 30 34 f t . . 150· ｾ＠ .$' $. ｾ＠ 33 t . 33 t 106 8000 7000 A 6000 5000 4000 3000 2000 1000 1soo B 1000 500 o Figure 5.- Vertical distribution of (A) abundance of phytoplankton (cells·mr1, <20 µm) and (B) abundan ce of cryptophyceas (cells·mr1 ). Observe the deepening of cells around station 30, coinciding with the downwelling of isotherms in the Peninsula front. ZOOPI \:\t.:TO'i I' THE FRO\T\I ZO,ES This suggests the existence of downwelling in the northem part of the front and upwelling at its southem boundary. Notice also that density section do not show a corresponding shoaling or deepening of the isopycnals (Fig. 3c) indicating a cross-isopycnal flow. Therefore, fluorescence, transmisometer and temperature sections suggest the existence of downwelling in the northem part of the PF and upwelling at its southem edge along transect T3, and hence the development of a counter-clockwise (facing eastward) SAC vertical cell. During sampling there was a clear predominance of small autotrophic flagellates (Fig. 5) and a rather low abundance of picoeucaryotes and diatoms (not shown). The latter feature indicated that we were sampling at the demise of the bloom. (a) 10-40m 40-70m 70-100m 100-200m 200-300m (e) 10-40m 40-70m 70-100m 100-200m 200-300m - T1 mesozooplankton abundance (índ.m3) •>10 •a 06 04 ｾＭＭＭ ＭＭＭ ＭＭＭＭＭＭＭ ｄ＼Ｒ＠ 5 9 12 T3 mesozooplankton abundance (índ.m"') • >10 • 8 D s D 4 ＭＭＭＭＭＭＭｾＭＭＭＭＭｾｄ＼Ｒ＠ 33 (b) 1040m 40-70m 70-100m 100-200m 200-300m (d) 10-40m 40-70m 70-100m 100-200m T2 mesozooplankton abundance (lnd_m3) ->10 B 06 04 ｾＭＭＭＭＭＭＭＭＭＭＭＭＭｾｄ＼Ｒ＠ 23 19 15 13 T4 mesozooplankton abundance (índ.m" 3) 200-300m ->10 8 06 04 ｾＭＭＭＭＭＭＭＭＭＭＭＭＭｾｄ＼Ｒ＠ 37 41 45 49 Station number Figure 6.- Zooplankton abundance (individual·m-3) at the four transects. Observe the higher biomass related to the Peninsula front at transects T3 and T4. 107 ZOOPI \'1KT0'1 l'\THEFRO'\T\I ZO'iES reproducing there. Other small copepods such as Oithona sp appeared in the 70-100 m layer at the TWW side of the Peninsula front. All these organisms were distributed around the front, at different depth strata. The vertical distribution of specific gut fluorescence and ETS activity also suggests that larval krill and copepods performed short vertical excursions in the water column. Larval krill located in the Bransfield front were observed at depth but the higher gut content was observed in the 40-70 m depth layer (Fig. l lA). The metabolic proxy used here showed higher values at depth suggesting that organisms migrate to the upper layers to feed (as observed from gut fluorescence) and metabolized at depth (as observed from ETS activity). Copepods also seemed to perform those vertical movements as specific gut fluorescence was high in the upper layers (see Fig. lOA), and decreased with depth as expected for organisms which defecated during their downward migration. ETS activity showed no clear differences with depth as expected of organisms that are performing excursions to the upper layer to feed. Therefore, the pattern of mesozooplankton distribution in the Bransfield Strait is driven by (1) the presence of the Bransfield and Peninsula fronts, and (2) the vertical distribution of the different species. As described by Sangra et al. (2011) and drawn in Figure 12A, we can distinguish four different hydrographical environments along the sampled transect: the Bransfield Front (BF) and related Bransfield Current region, the TBW inter-frontal region where an intense mesoscale eddy field has been reported (Corzo et al., 2005, Sangra et al., 2011), the Peninsula front (PF) region and the TWW region. Vertical sections of phytoplankton, transmittance and temperature suggest the development at the Peninsula Front region of a vertical counter-clockwise (facing eastward) secondary ageostrophic circulation vertical cell. A plausible hypothesis for the generation of this cell is that it is driven by a frontolytic episode and respond to the cross frontal balance of potential vorticity. In Figure 12A, adapted from Pollard and Regier (1992), we sketch the balance of potential vorticity and the related secondary ageostrophic circulation (SAC) across a front during a frontolytic event in the southern hemisphere (negative planetary vorticityf-). Adapting the discussion of Pollard and Regier (1992) to those circumstances, as water moves outside the frontal axis in the positive relative vorticity side Ｈｾ＠ +, anticyclonic, southern hemisphere) of the front, its absolute vorticity increases, and in order to preserve its potential vorticity the thickness between pairs of isopycnals must also increase, and hence a downward vertical velocity must result. Similarly, on the negative relative vorticity side ＨｾＭＬ｣ｹ｣ｬｯｮｩ｣Ｌ＠ southern hemisphere) of the front, the velocity must be upward. A SAC 114 ZOOPI \ 'KTO'i 11'i THE FRO'IT \l. ZONES counter-counter-clokwise close cell results, in which light water in the anticyclonic side downwells and relatively heavy water in the cyclonic side upwells. Near the surface, water from the cyclonic side cross the front toward the anticyclonic side, while in the deeper layers the flow is in the opposite direction. .s:: E. CI> o ... .. light (warm) ｾＫ＠ ｦＫｾ＠ increases Ap increases ... Frontolytic flow ... ... .. ... . .. r ... "' frontal jet heavy ( cold) isopycnals ｦＫｾ＠ decreases Ap decreases Figure 12A.- Schematics of the crossfrontal potential vorticity balance in the southern hemisphere during a frontolytic event (adapted form Pollard and Regier, 1992). In response to potential vorticity balance a vertical counter-clockwise (facing eastward) secondary geostrophic circulation vertical cell devellops (see details in text). ｾＮ＠ f and p denotes the relative vorticity, the planetary vorticity and the density respectively . A To be consitent our hypothesis needs a frontolytic (divergent) large scale flow; unfortunately we do not have reliable ADCP data to infer the horizontal cross-frontal velocity. However, we have sorne observational evidences that suggest that section T3 cross a meander trough which can leads to a frontolytic event as recently observed by Thomas and Joyce (2010) in the Gulf Stream. As depicted in Figure 9 of Sangra et al. (2011 ), a high resolution fast survey conducted with an undulating vehicle just after transect T4 sampling indicated an intense meander activity at the Peninsula Front with a meander trough crossing transect T3. Evidence of such trough can also be inferred from the position of the Peninsula Front along the four transects as shown in Figure l. The Peninsula Front is located in a more southem position in transect T3 than in transect T2 and T4 suggesting the passage of a meander trough. Evidence of frontolysis can be also extracted when comparing vertical sections of temperature and potential density along transects T2, T3 and T 4. Figures 2 and 3 shows a relative decrease of the intensity of Peninsula Front in transect T3. Near 115 ZOOPI ｜ｾｬ＼ｔｏＧ＠ I'\ THF. FRO'iT\I. ZO"':S surface cross-frontal temperature gradients where 2 and l .5ºC in transects T2 an T4 respectively whereas at transect T3 it was only 1. 25 ºC. Potential density sections also show how isopycnals where also more packed in transect T 4 than in transect T3 . BF TBW Eddy freid PF TWW Large E. superba . / J Small E. superba Small copepods Large E superba 49 SmaU phytoplankton .1 . So"" SheUaOd Shel l t ® - ｜ ｾｙｲ＠ / \ ' . / 1 1 ｾ＠ Large Large · copepo s copepods and nauplia 1 ) 1 1 1 1 Legend : / ) - Abundan ce !t Vertical excurs1ons OVerturning circulation B ® Flow ínto the paper Figure 12B.- Conceptual model of mesozooplankton and krill distribution in relation to hydrography in the transect crossing the Bransfield Strait. The large arrows indicate the overturning circulation around the front. Observe the phytoplankton sinking, the location of large and small copepods and krill (see text). BC stands for Bransfield Current, TBW for Transitional Bellingshausen Water, TWW for Transitional Weddell Water, and PF for Peninsula front. As detailed next and sketched in Fig. l 2B we hypothesize that the SAC promotes a unique biological effect around the front. The larger biomass of phytoplankton at the TBW sinks in the northem side of the front (Figs. 4 and 5) and large copepods accumulate there at the deeper layers of the proposed overturning circulation (Figs. 8 and 12B). It is plausible that large copepods benefits from the sinking phytoplankton biomass (and perhaps microzooplankton) above them as observed by their higher gut fluorescence (see below). They also seemed to reproduce there as observed from the high abundance of nauplia (Fig. 8B). Small copepods also seemed to drift into the area of high krill biomass in the upwelled TWW as observed by the presence of Oithona sp 116 ZOOPL\:\KTO_, I" THE FRO..,T\L ZO'íES below the krill (Fig. 8C). Therefore, the Peninsula front, which is a permanent feature all along the summer moving southwards, is suggested to hold a rather stable phytoplankton-zooplankton-krill food web. This pattern should be considered in further research as a mechanism increasing plankton at frontal systems in addition to the convergence and horizontal shear proposed by Lutjeharms et al. (1985). As also sketched in Figure12B at the Bransfield front (BF), large copepods and larval krill are able to perform vertical excursions to feed in the upper rich phytoplankton layers. These excursions are unmasked dueto the higher gut fluorescence oflarge copepods (Fig. lOA) and small krill (Fig. 1 lA) in the upper layers. However, ETS activity showed a rather homogeneous activity in the water column (Fig. lOB) or even an increase at depth (Fig. 1 lB) denoting that organisms feed phytoplankton at the shallower layers but metabolize, or ingest non-pigmented food, at depth. 1t is therefore suggested that large copepods and larval krill remain at depth to avoid predation by large krill at the upper layers. In this sense, Hernández-León et al. (2001) observed krill and large copepods distributed in two layers, the former in the phytoplankton rich layer and the latter (M gerlachei) at mesopelagic depths. They explained this distribution as a strategy to avoid predation. Copepods remained at depth during the day migrating to the shallower layers at night to feed on phytoplankton. Krill spent the day feeding on phytoplankton during the day and migrated toward deeper layers to feed on the only species of large copepod at depth (M gerlachei) as observed by the decrease in gut fluorescence of krill and the increase in crustacean pieces in their guts while they were at depth. Hernández-León et al. (2008) also observed this pattern of vertical migration using field and acoustic sampling. In the present work, we observed a similar pattern with large krill in the upper layers and large copepods at depth but performing excursions to the phytoplankton rich waters. Thus, the interplay between the physical structure of water masses and the avoidance of predation by large krill seemed to drive the mesozooplankton distribution in the Bransfield Strait. Mesozooplankton biomass, gut fluorescence and ETS activity were quite variable along the transect following the changes imposed by the physical frame and the vertical distribution of the organisms. However, there was rather marked differences between the gut fluorescence of the different species with higher specific values in the smaller organisms. This is a normal result in the study of gut content of Antarctic zooplankton as observed in a previous study (see Hernández-León et al., 2008). In the present work, copepods showed higher specific gut fluorescence values than euphausiid larvae (see Table 1). However, metabolic rates assessed through ETS activity showed similar values in both organisms. Community grazing (Table 2) estimated assuming a C/Chl=50 117 ZOOPI "K 10' 1' THE FRO'\T \I ZO"iES and a gut evacuation rate for copepods of 0.012 min-1 (Hemández-León et al., 2000), and also assuming that all the biomass were copepods showed values ranging from 1.2 to 43.9 mgC·m-2· d1 (average 15.1±13.9). Assuming a gut evacuation rate for small euphausiids of 0.011 min-1 (Daly, 1990) and that all the biomass was euphausiid larvae, community grazing ranged from 0.8 to 17 .O mgC·m-2·d-1 (average 3.9±5.9). However, total ingestion (1) estimated from respiration (R) and assuming a R/ETS ratio of 0.5 (Hemández-León and Gómez, 1996), and that 1=2.5·R (Ikeda and Motoda, 1978) showed values rather similar for copepods (20.0±15.5 mgC·m-2· d1) and euphausiid larvae (17.7±13.7 mgC·m-2 ·d -1 ), denoting that the latter presented a more omnivorous feeding than copepods. In any case, the impact of this mesozooplankton on primary production was quite low as autotrophic production in Antarctic water ranged normally between 300 and 1000 mgc-m-2·d-1 (Holm-Hansen and Mitchell, 1991; Basterretxea and Arístegui, 1999; Vareta et al., 2002) and our higher value (Table 2) is less than 18% of the lower range given above for primary production. Table 2.- Estimated values of grazing and ingestion by copepods and larval krill. Biomass as carbon was assessed as 40% of dry weight (dw). Biomass as proteins was estimated assuming dw=5.2·protein. Grazing was calculated from grazing rates obtained from gut fluorescence measurements (see Table 1), while potential ingestion (1) was obtained from activity of the electron transfer system (ETS) converted to the respiration (R) and assuming that 1=2.5· R (see text). Station Biomass Copepod Copepod Copepod Krill Krill Krill as carbon grazing potential pigmented grazing potential pigmented ingestion ingestion ingestion ingestion mgc-m·2 mgc-m-2·d-1 mgC·m·2·d·1 % mgC·m·2 ·d" 1 mgC·m·2 ·d1 % 24 440.8 10.9 15.3 71.4 1.6 11.3 13.8 36 528.2 19.2 18.6 103.1 17.0 21.3 80.0 27 109.8 1.2 4.0 30.3 0.8 2.5 33.2 35 352.7 10.9 14.6 74.2 2.1 10.1 20.6 30 306.9 13.8 17.0 81.1 1.3 25.0 5.1 34 1097.7 43.9 53.5 82.1 3.5 43.6 8.0 33 241.5 5.7 16.8 33.6 1.1 10.3 10.7 Although those biomass accumulations observed in the frontal zones of the Bransfield Strait are not able to promote an important control on primary production, the values of grazing and ingestion in the frontal system are an order of magnitude higher than outside (Table 2). Curiously, large 118 copepods showed a large contribution of pigmented food in their diet at the Peninsula front, while non-pigmented (animal) diet was found for larval krill there (Table 2). This feature also supports the proposed phytoplankton-zooplankton-krill food web around the front as krill would be ingesting rmpigmcntt:d food tbcre in contrast to tbeir pigmented diet in tbe Bransfield livnt Ｈｔ｡｢ｬ･ｾ＠ Fig. J J). Because the Peninsula frontis a permanent physical structure, its importance in driving the carbon flux in the area should be considered. First, the important sinking of phytoplankton in the TWB side ofthe front and, secondly, the higher biomass of copepods and krill around the front would explain the high organic carbon flux due to fast-sinking fecal pellets observed in sediment traps in the Bransfield Strait (Bodungen, 1986; Kim et al., 2004 ). It is suggested that those pellets could be produced by those accumulations of large mesozooplankton and krill around fronts. In summary, during a post-bloom scenario in the Bransfield Strait a typical pattem of plankton distribution was observed with higher phytoand zooplankton in the TBW as expected. However, our more detailed study of the Peninsula front between the TBW and TWW showed the sinking of phytoplankton and an accumulation of copepods in the TBW side of the front, and of krill in the TWW si de. The generation of this local food web around the front needs further research as it could be a recurrent structure developing through the summer, supporting a striking food web at both sides of the front. This pattern is suggested to drive a relatively important flux of carbon due to the sinking of phytoplankton and the production of fast sinking fecal pellets by large copepods and krill. Acknowledgements The authors are indebted with all the technical staff and crew of the RV Hespérides. This research was financially supported by projects Breddies (REN2001-2650) and Coupling (CTM2008-06343-C02-0l) from the CICYT (Spanish Commission for Science and Technology). 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The Bransfiled Current System. Deep-Sea Research 1, 58, 390-402. Siegel, V. (1988). A concept of seasonal variation of krill (Euphausia superba) distribution and abundan.ce west of the Antarctic Peninsula. In: Sarhage, D. (Ed.), Antarctic Ocean and Resources Variability. Springer, Berlin, Heidelberg, pp. 219-230. Siegel, V., de la Mare, W.K., Loeb, V. (1997). Long-term monitoring of krill recruitment and abundance indices in the Elephant Island area (Antarctic Peninsula). CCAMLR Sci. 4, 1935. Strickland, J. D. H. and T. R. Parsons (1972). A practical handbook of seawater analysis. Fish. Res. Bd. Ganada, Bull., 167 pp. Thomas, L.N, Joyce, T.M. (2010). Subduction on the Northem and Southern Flanks if the Gulf Stream. Joumal of Physical Oceanography, 40, 429-438.Vázquez, E., Ameneiro, J., Putzeys, S., Gordo, C., and P. Sangra (2007). Distribution of Meroplankton Communities in the Bransfield Strait, Antarctica. Marine Ecology Progress Series, 338: 119129. Vareta, M., Femández, E., Serret, P. (2002). Size-fractionated phytoplankton biomass and primary production in the Gerlache and south Bransfield Straits (Antarctic Peninsula) in Austral summer 1995-1996. Deep Sea Res. ll 49:749-768. Weeks, S. J. and Shillington, F. A. (1996). Phytoplankton pigment distribution and frontal structure in the subtropical convergence region south of Africa, Deep-Sea Res. 1, 43, 739-768. Yentsch, C.S. and Menzel, D.W. (1963). A method for the determination of phytoplankton, chlorophyll and phaeophytin by fluorescence. Deep-Sea Res., l O: 221-231. 123 ZOOPI \'il..:TO' CO\l\ll 'In STRlCTLRE depth, whereas they were constantly present in all the water column of the Bransfield Strait and constituted more than 50 % of the total biomass, with quite high abundance at the subsurface layer (<lOOm depth, Fig. 3E). A m¡z d"'m·' B mg ｌｬ｜｜ﾷｭﾷｾ＠ e mg dw ·m·l o 0,5 10 o 2 4 6 8 10 14 o 10 100 120 10-40 10-40 10-40 :=J111 111 111 11> 11> 11> en 40-70 en 40-70 (/) 40-70 e: e: e: CD 70-100 11> 70-100 ti> ID U> .. 70-100 :> :> ::> .,, <O 111 1 ｾ＠ 100-200 ｾ＠ 100-200 .§. 100-200 1 !J) O> e: e: e: ｾ＠ 200-300 ｾ＠ 200-300 ｾ＠ 200-300 ID ID ID 300-400 300-400 300-400 m¡zdw·w·' mgdw·m··' mg d \\ ·m·l o 0.5 1 1.5 10 o 2 4 6 8 10 14 o 1 2 120 10-40 10-40 10-40 -40-70 40-70 ·¡¡ 40-70 ｾ＠ ·g ｾ＠ 70-100 en 70-100 70-100 "O "O u q; a; a; 'tii 100-200 1j¡ 100-200 'ti 100-200 e e: e: <O 111 111 iii 200-300 iii 200-300 iii 200-300 300-400 300-400 111 p.dw· a1 J mg tl \\ ·m ª3 m¡t d\\·m·' o 2 4 6 8 10 o 2 4 6 8 10 14 o 20 120 10-40 10-40 10-40 u 40-70 e: -e -o 40-70· ::> e: 40-70· c: o 70-100 :J :J en o o 70-100 <.> rn 70-100 en 1! 100-200 <.> <.> ｾ＠ 100-200 Ｍ ｾ＠ 100-200- <O e: ｾ＠ "' ｾ＠ e e: ｾ＠ ｾ＠ Figure 3A, B and C. Vertical distribution oflarval krill (A), small krill (B) and large krill (C) in the different regions of the WAP. X-axe: biomass (mg dry weight·m-3 ). Y-axe: depth (m). 130 "' Ql (/) D o 10-401 ｏ ｾ＠ 40-7 ｾ＠ ｾ＠ 70-100 '...:_j;¡ o o -,•-;:- my.dw·ní3 0.5 2 3 10-40 .- 1 .- . ｾ ﾷ＠ ...., -g 4070 ﾷＢｾ＠ ｾ］ｾ＠ ｾｧ＠ :-,¡¡ ｲＺｾ＠ ﾡｾ＠ :::l bl 70-100 \:, ｾ＠ ｾ＠ ｾｾＮ＠ " ' tl 100 200 :"id '''!15!.'- ｾＮ［ＺＺ＠ ［ＧｬＧｬｴＧＮ Ｍ ｾ＠ ｴｾｾ＠ :- ' ｾＭＭＮＧ＠ lnf,i'. 1 lii - ""'l!B; ""° ﾷｾＧﾷ＠ .. ..,, >:º ﾷｾﾷ Ｑ＠ e: <( 4 4 5 5 5 "' E o 10-40 ｾ＠ 40-70 e: ｾ＠ 70-100 m ｾ＠ 100-200 Cl e: ｾ＠ 200-300 . CD 300-400 o 10-40 ｾ＠ 40-70 :::l ｾ＠ 70-100 (J ｾ＠ 100-200 J!l e: <( o mQdw·ru·l - 0,1 m¡idw·ru-1 10 0,1 0,2 ZOOPf. \.'l/KTO'\ COi\l\ll '\'IT'r STRL CTl RF. 25 25 25 Figure JA, B and C. Vertical distribution of larval krill (A), small krill (B), large krill (C), copepods (D) and salps (E) in the different regions of the WAP. X-axe: biomass (mg dry weight·m·3). Y-axe: depth (m). In the Antarctic Sound, a reverse trend was observed for the larval k:rill and copepods distribution. Higher catches of these organisms were obtained at surface layers (Fig. 4A). Neverthless, larval k:rill and copepods were homogeneously and numerously present at all depth layers in the Antarctic Sound (Fig. 4A and D). By opposite, salps were insignificant in this area (Fig. 4E). Results also indicated a general trend of increasing copepod individual biomass with depth for all the sampling stations (Fig. 5B). Relative to the southem sampling stations, the Antarctic Sound exhibited much smaller copepods and larger specimens of larval k:rill. Pooling the biomasses of larval k:rill and copepods for all net samples at all depths, we observed a significant coupling (Spearman rank correlation, rs=0.73, p<0.0001, n=38) between the distribution of both copepods and larval k:rill biomasses (Fig. 6). 131 t D.:usity tN · I 000 ni-1¡ 2000 15000 A o 10-40 ｾＭＭＭＭ ＭＭＭ ｾ＠ C/J 40-70 e ｾ＠ 70-100 "' 1! 100-200 Cl e ｾ＠ 200-300 IIl 300-400 o Density !N· 1000 ｭ ＭｾＩ＠ 2000 15000 10-40 ﾷ ｾ＠ 40-70 ｾ＠ 7 0. 100l !i ll 1 a:; ｾ＠ 100-200 ｾ＠ 200 -3 00 u e: :::J 300-400 o 10-40 40.70 ｾ＠ 70-100 o .,,, ｾ＠ 100-200 ｾ＠ D ｉ ｫｬｬｬｬￍｾ＠ (N· 1000 m-'1 5000 10000 15000 Density (N· l 1100 m- ·• ¡ u ___ __,_1=- 00.00 100000 ZOOPI \ '"'Tº' e 0\1\ll '\'IT\ TRl e Tl RE B o "' 10-40 j Uensity (N· IOOO m· 3¡ 1000 2000 ｾ＠ ＴＰＭＷＰ ｾｾｾｾｾ＠ ｾ＠ Ｗ ＰＭＱＰ Ｐｾ＠ "' ｾ＠ .§. 100-200 ｾ＠ e: ;¡;¡ 200-300 IIl 300-4001 Uensily (N · 11100 m··'J 3000 o 1000 2000 3000 ＱＰＭＴＰ ｾｾ＠ "" ＴＰＭＷＰｾｾｾ＠ ｾ＠ = C/J ＷＰＭＱＰＰ ｾ＠ :2 ｾ＠ QJ ｾ＠ 100-200§ "' 1 di 200-300¡ u e: :::J 300-400¡ o 10-40 40-70 ál 70-100 o !! 100-200 ｾ＠ <( "' ., E o 10-40 (/) 40-70 e ｾ＠ 70-100 ., ｾ＠ 100-200 Cl e Density tN IOOO ｭﾷｾﾡ＠ 1000 2000 3000 Ue n• 1t y (N · IUOO m-3) 100 2000 e 10-40 "' ｾ＠ 40-70 e: ｾ＠ 70-100 "' ｾ＠ 100-200 c:n e: ｾ＠ 200-300 IIl 300-400 o o 10 -40 . ·iij 40-70 §) u 70-100 · ｾ＠ l ｾ＠ 100-200 ) "' rn 200-300 300-400 =1 o 10-40· ｾ＠ 40-70· ::;;¡ ｾ＠ 70-100· o ｾ＠ 100-200, !! ¿ ｔ＾ｴｬｮ󰿃ￍｬｾ＠ (N· I 000 m-3) 200 400 600 800 1000 Dcn'lltytN 111oom · '1 20 980 1000 Uensny 1N· ｬｬｾｉｕ＠ m·' 1 100 200 1000 = 200-300 ｾ＠ . Demlity (N· I 000 m-3) º =- __ __ 5_0000 100000 Ｍｾ＠ tñ u -¡; 10-40 : ｾ＠ 100-200 " :: ｾ＠ 200-300 ﾷＺＺｾ［ﾡﾷ ｉ＠ 1 300-400 o Densi ty (N· 1000 m3¡ 50000 4 01 o ｲＭＮＮｯ ＺＧＭ ｾ Ｍ ］ Ｇ ］ ＺＬＮ ＢＭＧ ［ｾ ＧＭＢＢ ｾ ＢＭＭＭ ［＠ Ｇ ＧＭＧ ＧＧＭＭﾷ ｾ ｾ ＭＭＭＢＧ＠ 100000 300-400 o ±! !! U5 u Qj 0-40 ｾ＠ 100-200 ¡ij di 200-300 30()...400 o 10-40· ｾ＠ 40-70 · :::J ál 70-100 · o ﾷ ｾ＠ 100-2oof E <( Dcnsity(N·IOOO ｵｦｾＩ＠ 1000 2000 Density tN· 1000 m·3¡ 100 132 2000 Figure 4A, B, C, D and E. Vertical distribution of larval krill (A), small krill (B), large krill (C), copepods (D) and salps (E) in the different regions of the W AP. Xaxe: density ind .- 1000 m3• Y-axe: depth (m). ZOOPI \"iKTO' CO.\l\<ll "ilT\ STRLCTl RF: Indiúdual hiomass (fLgDWJ TndiYidual hiomass ( ftgDWJ o 200 400 600 800 1000 o 50 100 150 200 10-40 "' ｾ＠ 40-?0ml e ｾ＠ 70-100 - "' ｾ＠ 100-2oo 1111111111111111111111111 ｾ＠ 200-300 911: [IJ 300-4001111111 "O e :J o (/) 5 lndi,idual biomass (µgl)W) o 200 400 600 800 1000 Ind1l'idual biomass ! µgDWl 200 400 600 800 1000 "' Q¡ (1) ｾＴ＠ [J a ｾ＠ [J :E O) ·¡; 3 [J 3:: [J ｾ＠ a -e O) a É. 2 a a "8 o.. a cu 81 a c::P <.> r:P o a 10-40 . 40-70 ＷＰ ＭＱ ＰＰ ｾｾｾ＠ lnctindual bioma¡¡s (µgVWl o 50 100 150 200 10-40 '.··· "' ., J TnJi, iJuat hiomasx (¡1gDW¡ o 50 100 150 200 10-40 ===--. [J a o 1 2 3 4 5 6 krill larval (mg dly weight .m3) 133 7 Figure SA and B. Vertical distribution of individual biomass of larval krill (A) and copepod (B) in the different regions of the WAP. X-axe: Individual biomass (µg dry weight·ind-1 ). Y-axe: depth (m). Figure 6. Copepod versus larval krill biomass (r,=0.54, p<0.0001, n=38) from data obtained in ali the layers sampled along the Antarctic Peninsula. 7.00Pl <\""k'.TO' C0\1'll Nll \ STRLCTl RF: DISCUSSION Krill distribution Krill sampling carried out along tbe W AP region indicated a global biomass in the lower range of previously reported values (Siegel and Loeb 1995, Hemández-León et al. 1999, Shreeve et al. 2002, Siegel 2000, Siegel et al. 2004, Quetin and Ross 2003). As krill occurred more frequently in the top 100 m (Ross et al. 1996, Lascara et al. 1999, Atkinson et al. 1999, Hemández-León et al. 2001), sampling restricted to this deptb range could overestimate krill density relative to studies with sampling conducted over greater depth ranges (Siegel et al. 2004). This pattem of large krill distribution was rather clear in the Bellingshausen Sea area where 98.9% of the krill biomass was found in the upper 100 m. In contrast, both small and large specimens in the Antarctic Sound (Figs. 4A and B) exbibited a location at depth layers (>lOOm depth) This vertical distribution sbould find an explanation in the predation pressure by marine top predators. In the midshelf off Bellingshausen Sea, migrant mesopelagic fishes (Nishikawa et al. 2001, Hemández-León et al. 2001 and references therein) should drive krill distribution at surface layers while land-based predators of coastal waters off Antarctic Sound (Croxall et al. 1985, Fraser et al. 1989, Ritz 1994, Nevitt 1999, Zhou 2004). Nevertheless, small specimens (Fig. 2) were particularly located at onshore stations (Nishino and Kawamura 1996, Ichii et al. 1998).This seems to be a common result since the migration of large specimens into tbe waters north of the South Shetlands (Nisbikawa et al. 1995) during the progression of the productive period (Siegel 1988, Lascara et al. 1999) induce an increase of the proportion of small specimens as occured in the present work (Fig. 4B). This reason sbould explain the high abundances of juvenile krill generally found in the South Shetlands Islands and the Antarctic Sound (Siegel 1988, Trathan et al.1993). Similary, this ontogentic migration of larger krill in the midshelf off the Bellingshausen Sea after tbe bloom drive the krill size distribution in this offshore area (Trathan et al. 1993, Ichii et al. 1998). Salp distribution Our results seem to confirm the elevated salp biomass in the Bransfield Strait (Witek et al. 1985, Huntley et al. 1989, Pakhomov et al. 2002, Kawaguchi et al . 2004). In this region, salps were found in the entire water column with similar densities. No correlation was found with copepods. In contrast, the Antarctic Sound exbibited a much lower salp biomass tban the Bransfield Strait (Fig. 2) in agreement with the structure of the zooplankton community previously found in the area (Alcaraz et al. 1998) but also with the relatively higher levels of suspended particulate matter close to the marginal ice zone (Perissinotto and Pakhomov l 998a, Pakhomov et al . 2003). Also, it is reasonable to consider the permanent mesoscale structures of the Bransfield front precluding salps 134 m the southeastern reg1on off the Bransfield Strait (Weddell waters ), while large crustacean zooplankton appeared in the northwest (Bellingshausen waters). Salps generally inhabit the warmer and oligotrophic waters (Park and Wormuth 1993, Nishikawa et al. 1995, Voronina 1998), but the presence of salp in the 100-300 depth range off the Bellingshausen Sea, even at low density (Fig. 4E), may support the intrusion of deep warm water (Upper Circumpolar Deep Water) in the region allowing salp population to maintain its population (Pakhomov 2006). Larval krill distribution Larval krill densities m the Bransfield reg10n were in the normal range (Siegel 1989, Huntley and Brinton 1991, Nicol et al. 2000). Nevertheless, important differences were observed with the Antarctic Sound where high larval biomasses were found. Capella et al. (1992) suggested Weddell Sea as an important source of krill larvae for the Bransfield Strait region. Much of the Weddell Sea waters spread out into Bransfield Strait (von Gyldenfeldt 2002) and could promote passive transport of larval krill into the Strait flowing throug the Antarctic Sound. Our higher individual biomass, result of a faster developmental rate (Brichta and Belem 2002) in the inner shelf stations off the Antarctic Sound (see Fig. 5A) has to be related to a higher food quality and concentration associated with the proximity of melting sea ice cover (Pakhomov et al. 2004 ). Indeed, the bloom scenario of growing diatoms, protozoans and detritus corresponding to the typical diet of larval krill (Daly 1990) could also explain the significant higher larvae individual biomass observed in the Antarctic Sound (ANOV A test, p>O.O 1 ). Copepod distribution Copepod biomass reported here was generally low but within the range of previous studies in the region off the Bransfield Strait (Robins et al. 1995, Hernández-León et al. 1999, Cabal et al. 2002, Calbet et al. 2005) and Bellingshausen Sea (Robins et al. 1995). The lower copepod biomass in the Bellingshausen Sea, compared to other sectors of the W AP has been observed previously (Hernández-León et al. 1999). In contrast, the Antarctic Sound exhibited from far the higher densities (Fig. 50) in agreement with the higher chlorophyll concentrations found in these waters (Arístegui and Montero 1995). This result is also consistent with the one observed previously in an adjacent area (Hernández-León et al. 1999). The comparison of large and small specimens of krill and copepod reavealed a contrasting localization in the water column (Fig. 5B, C and D). This result put forward the scarcity of copepods at surface layers as a consequence of predation by krill (Hernández-León et al. 1999, 2000, 2001, Calbet et al. 2005). Indeed, in the Bellingshausen Sea, the analysis of the zooplankton 135 ZOOPI \ 'iKTO' C0\1\ll 'ilT\ Sl Rl CTl RE communities suggest a regulation of copepods population by the feeding of krill (small or large specimens) since their higher krill densities corresponded to lower copepod densities. In the Antarctic Sound, krill in depth coincided with the minimal copepod densities suggesting a top-down impact by krill on the vertical distribution of copepod (Fig. 4C and E). Also, phytoplankton may be the main food for both krill (Quetin and Ross 1991, Hopkins et al. 1993a) and copepods (Schnack 1985, Hopkins et al. l 993a, Atkinson and Shreeve 1995). Therefore, they should compete for food. However, Antarctic krill feeding activity of krill is generally based on phytoplankton but is also known to be an effective predator of copepods (Price et al. 1988, Hopkins et al. l 993a, Atkinson and Snyder 1997, Atkinson et al. 1999, 2001). Out content of small individuals yet indicated an omnivorous diet, mainly composed of diatoms and dinoflagellates (Daly 1990), but also protozoans and copepods during the summer period (Nishino and Kawamura 1996). Thus, it is also reasonable to consider that small krill in the Bransfield Strait may also perform a top-down control on copepods (Figs 3B and 30) as stated by Hernández-León et al., 2001. Interaction between copepods and larval krill The relationship between larval krill and copepods biomass (Fig. 6) is highly significant. The vertical distribution of both taxonomical groups indicated a strong coupling probably due to a similar diel vertical migration or feeding behavior. Indeed, krill larvae can feed across a large size spectrum, which includes particles size from diatoms to protozoans, nauplii of metazoans and small copepods (Daly 1990, Meyer et al. 2002 and 2009). Summer diet of the most important epipelagic copepods showed that they feed mainly on diatoms but also on protozoan and metazoan microzooplankton (Hopkins 1987, Hopkins et al. l 993b ). Thus, copepods and larval krill should directly compete for food. But, when larvae densities are important, their diet can comprise a substantial proportion of the mesozooplankton assemblage (Meyer et al. 2003). In this sense, the decreasing copepod biomass at higher larval krill densities (see Fig. 5) suggests a combination of competition and predation by larval krill at higher densities. lt also is probable that depending on their developemental stage, larval krill would migrate like sorne copepods. Unfortunately, very few records exist in the literature. It is only known that early-stage larvae do not appear to migrate vertically, but post-larval euphausiids are likely to perform vertical migration (Carr 2003 and references therein). Thus, the similarity of feeding behaviour of both taxa but the localization in the water column by diel vertical migration should enhance their interaction. 136 ｾ＠ f ZOOPl .\'.\KT0:-1 (0\1\ll 'iln STRl CTl RF Bloom and post-bloom scenarios and influence on zooplankton communities Despite the low biomass observed in the W AP region, zooplankton showed clear differences in the spatial distribution with scenarios depending mainly on nutrients, water mass properties, the proximity of the ice melting zone, and the local distribution and feeding activity of predators. The progressive melting of the pack ice impulse the drastical increase of primary production (Nelson et al., 1987) and successively the growth of heterotrophs (Kawall et al., 2001). The particular protection from storms favouring water column stability, the local hydrography (Park et al. 1999), and the rich phytoplankton Weddell water mass (Aristegui and Montero 1995, Agustí et al. 2004) tlowing in the area promoting a higher zooplankton biomass (Hernández-León et al. 1999) should induce the significant higher biomass per individual in krill larvae (Fig. 5) and the markedly higher zooplankton biomass (Fig. 2) as observed in the Antarctic Sound (Hernández-León et al. 2000). lt must be also noted that this marginal ice zone with ice-edge bloom influence displayed a krill population structure mainly composed of small individuals (Fig. 2) toward a dominance of larger specimens in the open waters off Bellingshausen Sea characterized by post-bloom conditions. This distribution agreed with the ontogenetic movement during the transition from bloom to post-bloom conditions proposed by Siegel (1988) with smaII specimens Guveniles) inhabiting the coastal waters (Siegel 2005) as observed in the Antarctic Sound and Bransfield Strait towards larger specimens in the open waters off Bellingshausen Sea. This ontogenetic migration across the W AP shelf region and the feeding behaviour of krill is suggested to induce a progressive decrease of copepod biomass from the coast to the offshore zone. lndeed, the low copepod individual biomass (Fig. 5B) observed in the coastal zone with ice melting influence (Antarctic Sound) to the open waters off the Bellingshausen Sea confinn the pattem of distribution previously reported by Ashjian et al. (2004) and Hemández-León et al. (2008). lndeed, krill may deplete the standing stock of large phytoplankton but during the progression of the bloom may switch its selectivity to copepods (Price et al . 1988, Graneli el al . 1993). As the 1arger copepods are firstly selected near the surface during summer (Atkinson and Shreeve 1995, Atkinson and Snyder 1997, Hemández-León et al. 2008), the proportion of smaII copepods becomes higher (see Fig . 5B) in the vicinity of the marginal ice zone (Hopkins et al . l 993a, Hernández-León et al. 1999). In the post-bloom scenario off the Bellingshausen Sea, the scarcity of mesozooplanktonic organisms (Fig. 40) seems to be a general pattern over the shelf (Hemández-León et al. 2008). Besides, sorne other larger copepods such as Metridia gerlachei do migrate at depth (Fig. SB) to avoid predation by krill during the day (Hemández-León et al. 2001). 137 ZOOPI \ "'KTO\ C 0\1\ll 'iln STRl C Tl Rf. CONCLUSION An essential prerequisite to better understand the role of the different groups of zooplankton in the flux of energy and matter is the correct biomass estimation. The present study is an example of the usefulness of digital image processing to accurately estimate the zooplankton biomass. The low mesozooplankton biomass observed in this study could be also a consequence of the sampling procedure discarding particulate matter that overestimates the abundance or biomass using the Optical Plankton Counter (Herman 1992) or the procedure described by Lovegrove (1966), respectively. Thus, our approach using image analysis software should give better estimates of mesozooplank:ton biomass. As a whole, the source of variability in the distribution of Antarctic krill was found in the onshoreoffshore gradient but also the progression of bloom during the productive period. This ontogenetic migration across the W AP region, through feeding behaviour is suggested to induce a progressive decrease in copepod biomass from the coast to the offshore zone. Thus, this substantial decrease of copepod biomass observed from the coastal zone with ice melting influence (Antarctic Sound) to the open waters off the Bellingshausen Sea confirm the pattem of distribution previously reported by Ashjian et al. (2004) and Hemández-León et al. (2008). Also, the pattem of copepods vertical distribution suggested a top-down effect by both large and small krill and possibly by krill larvae. Nevertheless, the magnitude of this predation impact by larvae and small specimens of krill is poorly known and further studies are needed to fully understand their control and impact on the distribution of copepods along the Antarctic Peninsula. Acknowledgements This research was financially supported by project !CEPOS (Ren2002-04165) and Coupling (CTM2008-06343-C02-0l) from the CICYT (Spanish Commission for Science and Technology). We acknowledge the assistance at sea of the members ofthe UTM (Unidad de Tecnología Marina) and the crew of the R/V Hespérides. The authors are indebted to S. Putzeys for bis help during the cruise. 138 ZOOPL\'XKTO:'\I (0\1\ll '\lf\ STRlCTl-RE References Agustí, S., Sarta, M.P., Mura, M.P. (2004) Summer community respiration and pelagic metabolism in surface summer Antarctic waters. Aquatic Microbial Ecology, 35 197-205. Alcaraz, M., E. Saiz, J.A. Femandez, l. Trepa, F. Figueiras, A. Calbet et B. Bautista (1998) Antarctic zooplankton metabolism: carbon requirements and ammonium excretion of salps and crustacean zooplankton in the vicinity of the Bransfield Strait during January 1994. Journal of Marine Systems, 17: 347-359. 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(1998) Comparative abundance and distribution of major filter-feeders in the Antarctic pelagic zone. Joumal of Marine Systems 17, 375-390. Witek, Z., Kittel, W., Czykieta, H., Zmijewska, M.I., Presler, E. (1985) Macrozooplankton in the southem Drake Passage and Bransfield Strait. Polar Research 6, 95-115. Zhou M., Dorland R.D. (2004) Aggregation and vertical migration behavior of Euphausia superba. Deep-SeaRes 1151:2119-2137. 147 h.1{11 1 ! \1 Ｌｾﾡ＠ · 11 ¡'\ \, !1 l' 1' \' 1' ' l'l'J \I \!{\ 1'1 11 'll 1 114 ¡".., CAPÍTULO VIII: KRILL EXCRETION ANO IMPACT ON PRIMARY PRODUCTION DURING AUSTRAL SUMMER 2005 ALONG THE WESTERN ANTARCTIC PENINSULA Lehette Pascal1, Tovar-Sánchez Antonio2, Duarte Carlos M.2 J, Hernández-León Santiago1 1 Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, España 2 Department of Global Change Research, IMEDEA, CSIC-UIB, Instituto Mediterráneo de Estudios Avanzados, Esporles, Mallorca, España 3 The UWA Oceans lnstitute, University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia Pascal Lehette1, Tovar-Sánchez Antonio2, Duarte Carlos M.2'3 and Santiago Hernández-León1, Krill excretion and impact on primary production during austral summer 2005 along the Western Antarctic Península. In revision at Marine Ecology Progress Series. 148 t.:Rll 1 ｦｩＺｘｃｒｆＮｔｉｏｾ＠ \ '\D 1\1 r \CT º' PRl\I \R' PRODl ( TIO'\ ABSTRACT During the austral summer, zooplankton excretion along the Western Antarctic Peninsula was studied in a contrasting hydrographic regime including coastal and oceanic waters. Ammonium supply by mesozooplankton indicated a low contribution to fuel primary production in coastal waters. In oceanic waters, however, Antarctic krill sustains a significant percentage of the nitrogen requirements of primary producers. Thus, the ontogenetic migration of adult krill during summer should be a key factor regulating the regenerated ammonium for primary production. A significant coupling of ammonium concentration in the water column and in situ krill biomass supported the significant role of krill excretion in the epipelagic realm. Besides, results from short-term experiments with Euphausia superba indicated much higher ammonium excretion rates than previously found. Because the use of metabolic rates close to field rates is more adequate, it is suggested re-assess the ammonium supply by other taxa of the epipelagic marine biota. Moreover, the outcomes of experimental krill excretion rates, in situ measurements of ammonium and data compilation on primary production suggest that Antarctic krill should sustain a high proportion of the daily phytoplankton production. Keywords: krill, ammonium, primary production INTRODUCTION The Southern Ocean generally presents rather low values of primary production. Nevertheless, a high spatial variability in the phytoplankton biomass and production is normally found, especially along the Antarctic Peninsula where sorne regions, associated with stationary phytoplanktonic blooms exhibit high productivity (Park et al. 1999, Garibotti et al. 2003, Varela et al. 2002). Light limitation (Dortch 1990), the low affmity of phytoplankton for nitrate due to the low temperature (Reay et al. 1999) and the higher energetic cost of nitrate uptake compared to ammonium uptake (Dugdale, 1976), and/or the effect of iron limitation (Martín and Fitzwater 1988) have been proposed to explain the low productivity of the Southern Ocean. Iron availability has been pointed out as a limiting factor for the development of primary production (Timmermans et al. 1994, De Baar et al. 1995, Boyd et al. 2000, Holm-Hansen et al. 2005). However, as coastal waters, particularly in the Antarctic Peninsula area, are probably iron149 KRILI EXC'RETIO\ \'iO l\IP \CT O:\ PRI \I \R' PROOl. C'TI0:-0 replete {Martin et al. 1990, Sullivan et al. 1993, Moore and Abbott 2000, Varela et al. 2002), critical concentrations of ammonium have been argued as a potential limiting factor as phytoplankton production is mainly sustained by this nutrient (Priddle et al. 1997, Whitehouse et al. 1999, Reay et al. 2001, Atkinson et al. 2001, Bode et al. 2002). Indeed, mesocosm experiments in Antarctic waters showed that ammonium additions greatly stimulated phytoplankton growth and biomass (Agustí et al. 2009). Nitrate and ammonium are similarly important for primary production but arnmonium has been reported to be a preferred source of nitrogen (Dugdale and Goering 1967, McCarthy et al. 1977). Bacteria! recycling and zooplankton activity through excretion are critical sources of ammonium for phytoplankton growth. Whilst nitrate commonly dominates nitrogen concentrations in Antarctic waters (Priddle et al. 1997; Bode et al. 2002), primary production based on ammonia excreted by zooplankton is thought to be important (Priddle et al. 1997, Alcaraz et al. 1998, Hernández-León et al. 2008) and, in fact, at least 50% to 93% of the nitrogen is assimilated as ammonium in the continental shelf waters (Koike et al. 1986). Previous studies showed that ammonium is the principal form of dissolved nitrogen excreted by crustacean zooplankton (Bfunstedt 1985, Miller and Glibert 1998, Conover and Gustavson 1999). Zooplankton should then exert an important control on primary production through both grazing and excretion. For instance, swarms of Antarctic krill represent important sources of regenerated nitrogen (Atkinson and Whitehouse 2000) since larger nitrogen concentrations were found within the aggregation (Johnson et al. 1984 in Priddle et al. 1997). Swarm-forming krill should cause through grazing and predation a temporary exhausting of both copepods and phytoplankton but at the same time should induce optimal phytoplankton bloom forming conditions through the release of ammonium (Tovar-Sánchez et al. 2007). Resolving zooplankton excretion rates is, therefore, critical to estímate the contribution of ammonium to nitrogen demand for Antarctic phytoplankton growth. Here we experimentally quantify the contribution of krill NRi-N excretion in the different hydrographical zones of the Western Antarctic Peninsula in arder to assess the role of krill in ammonium regeneration in these waters. MATERIALS AND METHODS Sampling and data processing A survey of the Western Antarctic Peninsula (W AP) region was conducted during !CEPOS 2005 cruise (January to February) on board R/V Hespérides. Three regions with distinct 150 h:RILI EX( RETIO' \'ID 1 \IP \CT O:'I: PRl\l\R' PRODl C TIO' hydrographic regimes were sampled along the W AP region. Several stations were sampled in the Bransfield Strait and in the Bellingshausen Sea, and one station in the Antarctic Sound (Fig. 1 ). ＶＰＤ Ｍｲ Ｇ ｾｾｾｾｾｾｾｾｾｾｾｾｾｾｾｾＭＭＭＭＬ＠ - Figure l. Survey area and location of 62 s64 s66 sSSI South Shetland Island Drake Passage the tows in the different regions during AS .r"';.: the ICEPOS 2005 cruise conducted on -·· f._/C the Antarctic Peninsula. Solid and B .L ｾ［Ｅ｛Ｎ ｾ ［Ｌ ｾ ﾷ＠ ｾＭ _ dashed contours show 500and 1000- ﾿ｻﾡｾｾｾ＠ R_ "!'.,_, ---..,, m isobath. Antarctic Sound ｾ ﾷｾ＠ "' ,¡ ＷＭＧｾ Ｈｉｃ＠ ... ; Ｉ ｾ＠ ｦＬ ｾ ｾｾ ｾｾｾ ＱＱ＠ ,,. CJ! () - 4t 'AS /) ｾ＠ -¿,.(-:· \ - ｾ＠ . ｊｾ＠ D •. 10 (\,B e!1 () .·rJ ｾｲＺ｟ｹ＠ ·( /{) () 0 8e4e r. "'e2 j 'J ;.-/Be 3'i4- --J ::: .-/ 1 /- ·. ,, ( 1 ' 68 sｂ･ｬｬｩｮｧｳｾｮ＠ ｾｾ Ｑ＠ ° 70 s- ;;; !(; ? "_¿? Weddell Sea / ) 80W 72W 64W 56W Sampling was conducted during daytime for all stations (Table 1). Zooplankton was collected using a BIONESS net (Sameoto et al. 1980) equipped with 6 nets of200 µm mesh size. Table 1. List of sampling dates, geographic position and depth of the sampling stations along the Antarctic Peninsula during austral summer 2005. Date of Local time La ti tu de Longitude Bottom depth Category Sampling (GMT-3h) (S) (W) (m) Be2 03/02/2005 1441 66º34.14 69º59.27 436 midshelf Be3 04/02/2005 1448 66º37.28 70º09.31 464 midshelf Be4 05/02/2005 1422 66°10.65 69°17.64 340 midshelf Be5 06/02/2005 1306 64°45.39 65°42.27 387 midshelf Asl 1 11102/2005 1817 63°23.73 56°44.33 289 coastal Br12 12/02/2005 1538 62°24.87 58°48.56 1222 coas tal Br14 14/02/2005 1247 62°44.35 60º32.27 298 coastal Br18 22/02/2005 1623 63°00.73 62°55.91 629 coas tal 151 KRll L F.:\( RF.TIO'.\ \:\D 1 :\IP \(TO:\ PRI \I \R' PROOl CTIO'\ The net was deployed in oblique hauls from 400 m depth or within 50 m from the bottom to the surface (10 m depth) for shallower stations. The water volume filtered by the nets was obtained by a calibrated General Oceanic flowmeter. All zooplankton was preserved in buffered 4% formaldehyde solution just after collection. Zooplankton was size-fractionated, counted using a standard digital camera with a CCD sensor (see Lehette and Hernández-León 2009 for details) and sorted in five taxonomic categories. Adopted resolutions (14.7 µm pixel-size for copepods and larval krill and 33.3 µm pixel-size for salps, small and large euphausiids) were suitable for morphometric measurements and for semi-automated plankton recognition and classification. Image processing and machine-learning methods followed those described by Grosjean et al. (2004). Ammonium experiments Krill (Euphausia superba) was captured in oblique hauls with an IKMT net equipped with a 1-cm mesh size. The net was towed from 100 m depth to the surface at a speed of 2-3 knots during the night. All individuals captured were immediately transferred into new on-deck aquaria with constantly renewed surface seawater. All the experiments were derived from the "water-bottle" method (Omori and Ikeda 1984) with 1 or 2 individuals by flask. Two series of experiments were carried out in filtered seawater and designed in order to have a significant response between the control and the experiment. The first series of experiments was carried out with surface seawater pumped through acid-cleaned Teflon tubing coupled to C-flex tubing (for the Cole-Parmer peristaltic pump head), filtered through an acid-cleaned polypropylene cartridge filter (0.22 µm, MSI, Calyx®), and collected in 2 L Low Density Polyethylene (LDPE) bottles for experiments. Experimental and control bottles were incubated in the dark in an incubation chamber set at surface water ambient temperature (±1 ºC). In a class-100 High Efficiency Particulate Air (HEPA) hood, water samples from the experimental bottles were collected at 2 b intervals, from 1 b up to 11 h from the onset of each experiment. At the end of the experiments, specimens were dried to constant weight in a drying oven set at 60°C and weighed to the nearest mg. The second series was performed in filtered seawater (GF/F Whatman filters) bell-jar incubation bottles (2L) in a thermostatic bath at l ºe ± 0.1 in dim light during one to 7 days. At the end of the incubation, excretion rates were calculated from differences between the control and experimental bottles. The health condition of all individuals was checked at the end of krill individuals were then digitized for dry weight determination (Lehette and Hernández-León 2009). 152 h:Rll 1 F.XC RF.TIO'i \'iD 1\1 P \( T º' PRl\l \Rl PROnt C'TIO' Determination of ammonia by jluorometry Ammonia determination based on the reaction with orthophtaldialdehyde (OPA) and sulphite followed the procedure and recommendations described by KeroueI and Arninot ( 1997). Incubations of 50 min were made in PVC flasks in a thermostatic bath fixed at 37°C ± O. l. The solution was then exposed to a specific excitation wavelength (365 nm) and produced a fluorescent isoindole fluorophore detected at a specific wavelength of 425 nm by the spectrofluorometer (Shimadzu RF-5301 PC). After calibration, final concentration is defined by the difference between the sample and the blank obtained with recent milli-Q water. This automated analysis method is accurate, easy to use, and highly acurate (<0.5 nanomolar) for ammonium determination of discrete marine samples (Kerouel and Aminot 1997). RESULTS Zooplankton biomass showed sharp differences in the vertical distribution of zooplankton biomass between the different regions of the W AP (Fig. 2). In the Bellingshausen Sea, the high zooplankton biomass was mainly related to large specimens of krill in the surface layer (Fig. 2A). A Biomass ( mg dw·m-3) 10 20 30 40-10 70-40 ., .. "' e 100-70 .. !! ., .t:: ｾ＠ 200-100 ｾ＠ ID 300-200 400-300 Bi om ass (m g dw·m-3) 10 20 30 70-40 -g ｾ＠ 100-70 ｾ＠ ｾ＠ 200-100 40 110 • 1 salp ocopepod •larval 11111smallkrill •largekrill 40 110 salp ocopepod •larval •smallkrill •largekrill B 40-10 70-40 ｾ＠ 100-70 "O ¡¡; 1200-100 ID 300-200 400-300 153 Biomass (mgdw ·m-3) o 10 20 30 40 110 ＬＮＭＭＭＭ ｾ ｾｾ ｾｾ ｾ ｾｾ ｾ ｾ ｾ＾ＭＭＭＭＭＭ salp E'J copepod •larval •smallkrill • large krill Figure 2. Vertical distribution of the different zooplank:tonic groups in terrn of biomass (mg dry weight·m-2) in the Bellingshausen Sea (A), Bransfield Strait (B) and Antarctic Sound. KRIU f.XCRF.TIO'i \ 'iD l'.\IP \( T O'i PRl\I \R\ PRODl CTIO'li 10, 1 A 8,0 6,0 4,0 y= 0.298 * xo,3098 • ..-.. • "" ·s o 2,0 ! 8 0,8 ::3 ·= 0,6 o § 0,4 • < 0,2 • • 0,5 1,0 5,0 50,0 500,0 Total zooplankton biomass (mg dw·m-3) B 10, 8 ,0 6 ,0 4,0 y = 0,303 * :x° .4 29 8 • ..-.. 13 ｾ＠ 2,0 • ! • • • 8 •• • ·= 0,8 Cl 0,6 o j 0,4 • • 0,2 • • o7 T.O - -- 5,0 50,0 500,0 ' Krill biomass (mg dw·m-3) Figure 3. Relationship be tween ammonium concentrations (mmol·m-3) and (A) total zooplankton biomass (mg dw ·m-3) , and (B) krill biomass (mg d w·m -3) .. Values are ploted on a Log-Log scale. Salps were abundant in the Bransfield Strait (Fig. 2B) with a biomass peak in the 70-100 m depth layer. In the Antarctic Sound (Fig. 2C), copepods and larval krill dominated the upper 100 m depth layer. A coupling between the overall biomass of zooplankton in the water column and the in 154 KRILL EXCRETIO:'\ \'\O l\IP \(T 0' PRl\I \R' PROOl ( TIO'i situ ammonium concentration was observed (r2 = 0.72; Fig. 3A) with slightly better coefficient of determination for the relationship of ammonium concentration to krill biomass (r2 = 0.78; Fig. 3B). As expected, the compilation of our experimental krill ammonium excretion rates with the results of Huntley and Nordhausen ( 1995) and those of Atkinson and Whitehouse (2000) indicated a decreasing krill excretion rate with the length of incubation (Fig. 4). Maximum rates (30.0±6.3 nmol NH/·mg1dw·h-1) were measured during the first hour of incubation and were approximately 12-fold higher than standard rates measured after 24 hours (2.45±1.1 nmol NH/·mg1dw·h-1 ). The relationship between incubation time (h) and excretion rates was given by the expression: NH4 Excretion (nmol NH/·mg1• h1) = 17.04 h -0.49 r2 = 0.76 Combination of the biomass of the main taxonomic groups at each sampling station (Fig. 5A) and excretion rates (Table 2) indicated that the sampling stations of the Bellingshausen Sea exhibited rather low ammonium production rates (Fig. 5B). In contrast, salps in the Bransfield Strait provided high ammonium excretion rates in the top 100 m depth layer. Applying the experimental excretion rates by krill during the first hour of incubation (17.04 nmol NH/·mg1dw·h-1 ), the Bellingshausen Sea (Be2, Be4 and Be5) exhibited a much higher ammonia excretion rates (Fig. 5C). Table 2. Range of ammonia excretion rates by each taxonomic group extracted from the literature and from experimental results in this study. Organism Dry weigth range Excretion rates (nmol References N"4+·ind-1·h-1) Salps 10.6 -14.4 mg 59.78 -80.23 Alcaraz et al. 1998 Copepods 61.9-138.1 µg 0.33 -0.74 Ikeda et al. 2001 1 Larval krill 233.3 -544.6 µg 1.94 -4.54 Meyer et al. 20022 Small krill 3.6 - 27.9mg 11.65 -59.85 Atkinson and Whitehouse 2000 Large krill 104. 7 - 186.3 mg 172.16 -272.83 Atkinson and Whitehouse 2000 Small and large krill 3.6-186.3 mg 61.34 -475.42 This sudy3 (1) Assuming equal proportion ofthe following copepods Ca/anus Acutus, Ca/anus Propinquus, Metridia Ger/achei and Rhinca/anus.gigas; (2) using the average value oflarval krill excretion rate; (3) using experimental close to field excretion rates about 17 nmol NH/· mg1dw· h1• 155