Is the Trail-Following Behavior of Juvenile Physa acuta Influenced by Kinship and Familiarity?

Trail-following behavior is exhibited by many species ranging from insects to gastropods. In gastropods, this behavior serves many functions such as the facilitation of movement, homing, mate and conspecific location, organization, aggregation, and protection. Kin recognition is also a behavior that is exhibited by a wide variety of species including both vertebrate and invertebrate animals. Kin recognition serves many functions including social interactions and status, discrimination, which can lead to outcrossing, and cooperative behaviors that can increase the likelihood of survival. Familiarity with individuals may also contribute to survival through protection and enhanced cooperation. Kin recognition can involve familiarity in conjunction with the detection of genetically-mediated cues or markers, and trail-following mechanisms may be linked to the chemical components of the trail itself. Experiments were conducted to determine whether kinship and/or familiarity influence the trail-following behavior of juvenile Physa acuta, a freshwater snail. The degree of trail-following was determined by tracing the trails of marker and tracker snails and by using the lengths of the trails and lines of overlap to calculate a coincidence index. The total length of the tracker trail and marker trail were analyzed to detect differences in marker and tracker trail lengths. There was no significant effect of kinship and familiarity on coincidence index and trail length. There was a pattern for trackers to follow trails of unfamiliar individuals and a pattern whereby tracker snails left longer trails when exposed to the trails of familiar marker snails. Possible benefits for following the trail of an unfamiliar individual may include colonizing a new hospitable environment with other conspecifics or leading the snail away from an inhospitable region. Increase in crawl distance for tracker snails may have been motivated by exploration of new environments, as the presence of a familiar individual would be associated with familiar locations that have already been explored. This study provides insight into the possible role of familiarity in trail-following and locomotive behavior in Physa acuta

Fighting for mates: the importance of individual size in mating contests in rocky shore littorinids

Studies of mating contests have reported how traits (e.g. body size) related to resource holding potential (RHP) and strategies to assess RHP and resource value influence contest outcome in many taxa but are rare in the Gastropoda. The influence of male size (as an index of RHP) and female size (as a measure of resource value) on contest outcome were investigated in two littorinid snails, Echinolittorina malaccana and E. radiata, in Hong Kong during May－June 2013. In these snails, contests between males take the form of a 'challenger' attempting to take over the copulation position occupied by a 'defender'. Both challengers and defenders were, generally, smaller than the females in both species. In both species, the larger the challenger relative to the defender, the more likely he would replace the defender in the copulation position. The challengers were, however, more successful in E. radiata, as they generally challenged defenders that were smaller than themselves, suggesting an ability to detect rival size before entering into a contest in this species. When sizes of the contestants were similar, defenders were more likely to win contests in E. malaccana but not in E. radiata. Evidence for pure self-assessment of RHP and the ability to assess resource value in challengers was found in E. malaccana. Different fighting strategies appear to have evolved in these congeneric marine snail species and decisions based on male and female sizes play an important role in determining male reproductive success

Interactions between two invasive crab predators, Carcinus maenas and Hemigrapsus sanguineus, and consequences for the native community

With continued globalization, species are being transported and introduced into novel habitats at an accelerating rate. As invasive species become more common, interactions between invasive species will also increase and may alter the way that these species impact invaded communities. The European green crab Carcinus maenas is an aggressive predator that was introduced to the east coast of North America in the mid 1800s and often has detrimental impacts on prey communities. A newer invasive predator, the Asian shore crab Hemigrapsus sanguineus, was first discovered on the Atlantic coast in the 1980s, and now inhabits many of the same regions as C. maenas within the Gulf of Maine. It too can have significant negative impacts on prey communities. Interactions between these species are often aggressive and may alter their influences on native prey. I used field and laboratory experiments together with spatial patterns in the field to investigate the impacts of these species and examine how interactions between them alter these impacts. My study focused on three interrelated areas of community ecology: multiple predator effects, prey dependent vs. ratio dependent foraging, and species redundancy. I demonstrate that aggressive interactions between and within these species strongly influence their impacts on native communities. The result is that when both species are present, their combined impacts are less than the sum of their individual impacts. However, the strength of interference varied with habitat type, prey density, predator density, and size of predators that interacted. Interference also affected predation by the two species differently, resulting in very different community impacts in areas where C. maenas dominates (northern Gulf of Maine) and areas where H. sanguineus has replaced C. maenas as the dominant predatory crab (Long Island Sound and southern Gulf of Maine, but progressing northward). Both conspecific and heterospecific interference strongly affected C. maenas, likely limiting its population size and subsequent impacts. In contrast, interference had little influence on predation by H. sanguineus. The lack of interference effects likely have allowed H. sanguineus to achieve very high densities observed in many areas, resulting in large population impacts on the native community

Distribution of epibionts and their effects on the marine snail Littorina littorea in northern New England

Epibiosis, or the growth of one organism on another, is a common life history strategy in marine environments where space is at a premium. Many epibiotic organisms live on or in biotic hard substrates, such as shells. In the spatially competitive rocky intertidal of New England, hard substrate surface area is greatly increased by the presence of the non-native marine periwinkle snail Littorina littorea. The snail\u27s shell can be exploited by epibiotic organisms such as barnacles, bryozoans, and encrusting calcareous algae. However, an in-depth examination of the prevalence and impact of epibionts on Littorina littorea in New England has not been done to date. In Chapter 1 of this thesis, I examine the distribution and prevalence of epibionts on Littorina littorea and other gastropod species in northern New England at four different tidal heights from Maine to Massachusetts. I then focus on local scale factors influencing patterns of distribution and abundance in the most common taxa of epibionts, encrusting calcareous red algae, in Chapter 2. In the final chapter, I examine the impact of algal fouling on Littorina littorea\u27s physiological parameters, grazing rates, movement patterns, predation susceptibility, and survival

Venomic Characterization of the Terebridae and Novel Terebrid Neuropeptides

Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow\u27s therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and Terebra subulata. De novo assembly and analysis were performed using next generation sequencing technology and state of the art bioinformatics tools to identify the cocktail of peptides, teretoxins, produced by the venom duct. These disulfide rich peptides often show a remarkable specificity for subtypes of ion channels and ligand-gated receptors, giving them therapeutic potential, but they are only available in vanishingly small amounts from the natural source. As a result, identification of teretoxins using next generation sequencing is a prelude to synthesizing them in sufficient quantities to test for bioactivity. Here recombinant expression and solid phase peptide synthesis have both been utilized for the synthesis of four different teretoxins, with a special focus on recombinant synthesis to design a reproducible strategy for synthesizing disulfide rich peptides greater than 40 amino acids in length. Preliminary characterization of bioactivity was performed by injecting synthesized toxin into the polychaete N. virens. A teretoxin identified from Terebra guttata, Tg77, has produced promising results in this assay, with repeated trials showing the effects of uncoordinated movement and rigid paralysis. Further testing on Tg77 and other teretoxins will be carried forward, with an evident need for high throughput assays to provide an efficient means for the testing of novel compounds with a variety of potential molecular targets

Impacts of ocean acidification and other (global change) stressors on marine invertebrates

The hypervariable nearshore marine ecosystem is home to intrinsic stressors for marine organisms. Global climate change and human activity are already affecting this aquatic environment and are expected to intensify over the next decades. These diverse changes include a rise in global temperature and a decrease in ocean pH. More carbon dioxide dissolves in the sea, lowers the pH, and makes the ocean more acidic. Now the main scientific challenge is to understand and predict the response of marine life to the predicted environmental change in the ecosystem. These challenges include a better understanding of the marine life’s response to changes in distribution and tribute to evolutionary changes or extinction. Species can respond to global change in a variety of ways. They can move from the environment of tolerance zone to newly available zones, can extend tolerance by summarizing and/or modifying their physiology or behaviour, and can demonstrate that the range shrinks when the environment of certain variable environmental factors is exceeded. Most marine species can respond to transformations, including behavioural or generational genetic selections that can improve perception, individual morphology, physiology, and/or performance in changing environmental conditions when undergoing periodic and rapid changes in the environment. Familiarity and adaptability to a changing environment is a type of environmental reaction due to the plasticity of an organism. In this doctoral dissertation, studies have been carried out to understand the potential physiological or biochemical effects of some global change stressors on five important marine and estuarine invertebrates: the anthuroid isopod Cyathura carinata (Krøyer, 1847), the benthic ragworm Hediste diversicolor (O.F. Müller, 1776), the sea snail Tritia neritea (Linnaeus, 1758), the most versatile amphipod Ampelisca brevicornis (Costa, 1853), and the pseudodiaptomid copepod Calanipeda aquaedulcis (Kritschagin, 1873) from the Iberian Peninsula (SW of Spain), because all of these invertebrates have been abundantly occupying and performing dynamic roles in the estuarine food chain for a long time, they have served as a viable food source for avifauna and ichthyofauna in those areas. In Chapter 3 we investigated the physiological or biochemical plasticity of OA in isopod C. carinata, and in Chapter 4 we describe how OA and ocean warming can alter the physiological and biological changes in a ragworm H. diversicolor. The effects of temperature rises and contamination of emerging pollutants such as lithium on the sea snail are discussed in Chapter 5. To determine whether OA includes propagating effects in the ecotoxicological study, the amphipod A. brevicornis is described in Chapter, 6 and finally, whether intragenerational plasticity can offset the negative effects that OA had on the life cycle of the copepod C. aquaedulcis is described in Chapter 7. To determine the tolerance and pH threshold that C. carinata could tolerate in future acidification scenarios, estuarine isopod was exposed to four pH treatments (control: 7.9; 7.5, 7.0, 6.5). Seawater acidification had a significant impact on the longevity of C. carinata, where the population density decreased significantly when treated at the lowest pH. The longevity, survival, and swimming activity of these isopods decreased with decreasing pH. Also, the swimming activity, Na+/K+-ATPase activity, and the RNA:DNA ratio of two populations of C. carinata, one in a stable environment (pH 7.5-8.0) and the other in a variable pCO2 regime (pH 3.3-8.5), were measured to assess the probable metabolic adaptability of this species. Populations in environments with a high pCO2 regime not only showed tolerance to pH 6.5 but had higher life spans and metabolic plasticity compared to habitat populations with little pCO2 conditions. These results indicate that C. carinata populations in stable environments may be susceptible to ocean acidification and can have a detrimental effect on survival and growth. Nevertheless, ocean acidification has limited effects on the energy budget and survival of C. carinata populations in highly variable habitats, indicating that they can cope with the elevated energy demand. Differences between the indicated populations probably indicate genetic differences in resistance to ocean acidification, possibly related to local adaptation, which may provide the raw materials needed to adapt to future conditions. In addition, our results indicate that population changes and metabolic responses should be considered when evaluating the response of marine crustaceans to changes in the global environment. Ragworm H. diversicolor was exposed in the laboratory to multi-stressors effects of elevated temperature and carbon dioxide levels mimicking the future OA and GC and we assess the physiological, behavioral, and biochemical changes in this species. The temperature rise exacerbated the negative effects of OA on the survival of the ragworm, delayed the excavation, and amplified the negative effects of lowering the pH on the feeding behavior of this polychaete. This is the first time this species has been shown to reduce its feeding capacity through the acidification of seawater. Wound healing and blastemal formation were slowed by these two climatic factors, which interfere with the regeneration process of the ragworm. Current results also show that even if polychaetes' metabolic capacity increases under stress conditions, organisms can still increase or maintain their energy reserves. Our results are of great importance for the environment, given that predictive conditions for climate change will affect the life, ecological and physiological capabilities of the species. This can lead to a decrease not only at the individual and population level but also at the diversity of microbes and endofauna, waste disposal in the estuary, and biochemical cycles at the ecosystem level. Therefore, the conservation of the H. diversicolor population is very important for the normal functioning of the estuary ecosystem. Sea snail Tritia neritea was exposed to lithium (Li, 0.08 mM) contamination and the rising seawater temperature (21 °C). We investigated the survival and trophic interactions (foraging behavior, success, search time, carrion preference, feeding time, and tissue consumption) of this intertidal scavenger. Trophic interactions were assessed using a Ymaze design using the same amount of two carrion species (Solen marginatus and Mytilus galloprovincialis) given simultaneously to all snails. Lithium pollution and synergistic warming reduce the survival rate of T. neritea, triggering a scenario for potential global change. Lithium contamination changes foraging behavior and increases the time it takes for snails to reach their carrion. Although T. neritea did not show a preference for the proposed carrion species in the control group, it shifted its foraging behavior to a more energetic carrion when contaminated with Li, which may represent a strategy that compensates for the high energy use required for survival. Results showing changes in the foraging activity of coastal mollusks in a global change scenario indicate potential changes in complex nutritional interactions between marine food pathways. Estuarine amphipod A. brevicornis was examined to study the physiological behavior and biochemical effects of the amphipod under OA. Wild harvested ovigerous females were reared in the laboratory and we started the experiment with 7 days old juveniles in a simulated OA scenario with four different levels of pH for a life cycle. Amphipods were incubated for up to 22 weeks to go through F1 production, successful reproduction, and hatching, and the length of F1 progeny compared to F0. The data obtained show that as the pH value of seawater decreases, mortality increases. The fertility rate reduced to 66.1% at pH 7.5 compared to the control group. The survival rate was higher in F1 juveniles than in F0 juveniles, but growth showed the opposite tendency to F1. These physiological parameters may be related to oxidative stress caused by climate-changing conditions as free radical generation interferes with cellular function, affecting the biochemical and physiological properties of the species, including burrowing, locomotory and ventilatory behaviours. This study is critical to assessing the impact of OA and providing baseline data that can be used as a guide for developing long-term strategies for delivering manageable and sustainable solutions. Copepods are an integral part of the marine food network due to their high biomass production and nutrient turnover compared to other zooplanktons in the marine ecosystem. Despite its enormous ecological role in the oceans, little is known about the effects of OA from increasing planetary carbon dioxide emissions in the future. Little information is available on the impact of OA on European copepod C. aquaedulcis. The purpose of this study was to investigate the impact of OA across multiple generations (F1 and F2) on survival, maturity, and fertility (hatching success, nauplii formation, and total adult population). C. aquaedulcis were exposed to three different pH gradients to simulate future seawater acidification scenarios. The survival rate of the copepod from nauplius to adult was significantly reduced in pH reduction and across generations. Results have also shown to have a marked effect on fertility, represented by a much smaller number of eggs per female in each generation. Similarly, hatching success showed a downward trend towards lower pH, and F1 females had significantly lower hatching success rates than F0 females. The results presented here appear to be ecologically important as the decline in the fertility of these animals can negatively affect marine feeding pathways. This is because the nutrition and growth of ichthyofauna are highly dependent on this component in the food web

Trematodes in Mediterranean coastal habitats: Transmission, life cycles and detection Methods

1. Introducción general Los metazoos trematodos (Platyhelminthes) son componentes ubicuos de los ecosistemas naturales entando temporal o permanentemente presentes en más de la mitad de las especies animales (Bush y col. 2001, Prietrock & Marcogliese 2003). Los parásitos trematodos pueden encontrarse en un amplio rango de condiciones y hábitats, teniendo un gran impacto en los procesos ecológicos: mediante la regulación de las poblaciones de hospedadores (Hudson y col. 2006) y afectando a la estructura de las comunidades y redes tróficas (ej. Mouritsen & Poulin 2002, Thompson y col. 2005, Lafferty y col. 2006, 2008). Son sobre todo, los parásitos que son transmitidos a través de la cadena trófica los que afectan a un gran número de especies de hospedadores, condicionan la estabilidad de las redes tróficas, sus interacciones, longitud y flujo de energía (Thompson y col. 2005, Lafferty y col. 2008). .En el Mediterráneo Occidental existe una gran riqueza de especies, encontrándose la mayor diversidad de peces e invertebrados en aguas de la plataforma continental. Las masas de agua de la plataforma continental constituyen el 20% del Mar Mediterráneo, por lo que tienen un papel importante en los procesos ecológicos (Bartoli & Gibson 2007, Coll y col. 2008). Los platelmintos constituyen el 6,7% del total de la diversidad de especies del Mediterráneo, siendo sin embargo poco conocidos, con menos del 50% descritas (Coll y col. 2008). Bartoli & Gibson (2007) aumentaron en gran medida nuestro conocimiento sobre platelmintos trematodos lagunares, al recopilar información sobre 72 especies nominales de lagunas del Mediterráneo Occidental, de las cuales aportaron el ciclo vital completo de 56. Los trematodos son los parásitos más comunes en hábitats intermareales de sedimento blando (Sousa 1991, Mouritsen & Poulin 2002, Poulin & Mouritsen 2006), siendo además de gran importancia en lagunas (Bartoli & Boudouresque 2007). El hábitat intermareal se caracteriza por fluctuaciones extremas de los factores abióticos (Palacín y col. 1991, Koutsoubas y col. 2000), con pequeños cambios en el nivel del agua que, por la evaporación, pueden exponer a los moluscos a altas temperaturas y salinidades elevadas (6°C a 30°C y de 21‰ a 38‰, respectivamente, Solé y col. 2009). Los moluscos, que constituyen del 15 al 25% de la macrofauna bentónica (Coll y col. 2008), son hospedadores de una gran variedad de parásitos, normalmente estados larvales de trematodos digeneos (Lauckner 1980). Las lagunas suelen ser utilizadas como zonas de alimentación y cría de aves y peces, por lo que la transmisión de los estados vitales de los trematodos entre los diferentes hospedadores podría verse facilitada gracias a su proximidad (Bartoli & Prévot 1986, Bouchereau & Guelorget 1998¸ Koutsoubas y col. 2000, Soppelsa y col. 2007). Durante el siglo pasado, las actividades pesqueras han evolucionado desde la producción a pequeña escala a la explotación industrializada y extensiva. Esta evolución de las pesquerías también ha sido evidente en el Mediterráneo. Por otra parte se sabe que tanto las costas marinas como la biodiversidad del Mediterráneo se ven amenazadas por el impacto antropogénico, como el cambio climático o la sobreexplotación (IPCC 2001, Coll y col. 2008). Debido a la mortalidad de organismos intermareales a causa de cambios extremos de temperatura (Harley y col. 2006) y su efecto sobre la abundancia y distribución de estos organismos (Harley y col. 2006, Helmuth y col. 2006), estos hábitats han sido propuestos como sistemas tempranos de alerta para la detección del cambio climático. Además, la alta sensibilidad de los parásitos a los cambios de temperatura y la contaminación también podrían ayudar a monitorizar el impacto ecológico del cambio climático (Marcogliese 2001, Poulin & Mouritsen 2006). Además, los descartes de las pesquerías facilitan el acceso de las aves marinas a grandes cantidades de comida, particularmente peces demersales que de manera habitual serían inaccesibles (Tasker y col. 2000, Oro & Ruiz 1997). Por lo tanto, aunque se conoce el impacto de estas prácticas sobre las poblaciones de hospedadores (ej. en láridos, Oro 1996, Oro y col. 1995, 1996), los efectos sobre la transmisión de los parásitos no se ha estudiado en profundidad. La subclase Digenea (Trematoda) comprende alrededor de 18.000 especies nominales (Cribb y col. 2001, Bartoli & Gibson 2007), lo que representa el mayor número de endoparásitos presentes en todas las clases de vertebrados (Bush y col 2001). La mayoría de los trematodos digeneos tienen un ciclo vital complejo que involucra tres hospedadores y diferentes estados de desarrollo (Galaktionov & Dobrovolskij 2003).Los hospedadores definitivos son por lo general vertebrados (aves o peces). Los huevos maduros se liberan al agua, normalmente a través de las heces, emergiendo una larva ciliada, el miracidio, que infecta al primer hospedador intermediario, por lo general un gasterópodo. Dentro del gasterópodo, los trematodos se reproducen de manera asexual en un esporocisto o redia, dando lugar a un gran número de cercarias que son liberadas al medio una vez maduras. Estos estados de vida libre no se alimentan, y parasitan al segundo hospedador intermediario, generalmente otro molusco o un pez, en un corto período de tiempo (menos de 24 horas). Después de la penetración en el segundo hospedador intermediario, la cercaria se enquista y se transforma en un metacercaria. De manera general, el hospedador definitivo adquiere las metacercarias infectivas a través de la cadena trófica. Cada etapa del ciclo vital del parásito muestra una estrategia de transmisión diferente y específica para completar con éxito el ciclo vital (Combes y col. 2002). Las etapas de vida libre de trematodos, es decir, los huevos, miracidios y cercarias, dependen de sus propios recursos energéticos y están directamente expuestos a los cambios ambientales, lo que condiciona la interacción entre los parásitos y sus potenciales hospedadores. Por lo tanto, los factores ambientales abióticos y bióticos influyen en el éxito de transmisión de los estados larvales de vida libre, en especial de las cercarias durante la búsqueda del siguiente hospedador, alterando su supervivencia o infectividad (Fingerut y col. 2003, Pietrock & Marcogliese 2003, Thieltges y col. 2008). Cabe destacar que los hábitats lagunares están caracterizados por grandes cambios ambientales y una alta heterogeneidad interna. La comprensión de los efectos de estos factores ambientales sobre la emergencia de cercarias es importante para estimar el impacto de las condiciones cambiantes en la transmisión y el ciclo vital de un parásito. Las instalaciones de acuicultura y los descartes provocan que diferentes grupos de hospedadores (peces, Bozzano & Sarda 2002; aves, Oro & Ruiz 1997 y gasterópodos, Morton & Yuen 2000) se concentren en la misma zona, por lo que la transmisión del parásito se ve facilitada. La atracción hacia instalaciones de acuicultura de potenciales hospedadores definitivos, como aves piscívoras (Witt y col. 1981, Arcos y col. 2001, Christel y col. 2012), puede, por lo tanto, intensificar la transmisión de parásitos. El impacto ambiental de las actividades pesqueras e instalaciones de acuicultura hacen que sea primordial estudiar posibles ciclos vitales que podrían verse favorecidos bajo estas condiciones. La coexistencia de dos organismos en un sistema parásito-hospedador se caracteriza por la obtención de beneficios por parte del parásito (ej. energía o espacio) a expensas del hospedador, por lo que un parásito, por definición, afectará a su hospedador de diferentes maneras (Lauckner 1980, 1983, Price 1980). Por lo general, el tamaño del hospedador es un factor importante para los trematodos, existiendo un compromiso entre el número de cercarias que emergen de un gasterópodo y su tamaño, proveyendo más espacio y nutrientes aquellos hospedadores más grandes (Loker 1983, McCarthy y col. 2002, Poulin & Morand 2004). Asimismo, se ha observado en varias especies la existencia de un efecto acumulativo de parásitos relacionado con el tamaño del hospedador (ej. Thomas y col. 1995, Karvonen y col. 2003, Osset y col. 2005). La mortalidad inducida por la acumulación de parásitos, por la cual los individuos más infectados son eliminados de la población (Rousset y col. 1996, Poulin 2001), se ha descrito tanto en peces como en invertebrados. Dada la corta vida de las cercarias, la sincronización de la emergencia tiene que estar optimizada para facilitar la transmisión al siguiente hospedador intermediario (Combes y col. 1994). La distribución de los hospedadores es igualmente importante, existiendo incluso parásitos que manipulan el comportamiento del hospedador en el que se encuentran para aumentar las probabilidades de alcanzar al siguiente hospedador (Poulin 2007). En cualquier sistema, la combinación de varios factores influye en la emergencia del parásito, así como a su transmisión y ciclo vital, teniendo que tener en cuenta la interdependencia de los factores a la hora de estudiar un sistema. La combinación de diferentes técnicas, como el empleo de herramientas moleculares, el desarrollo de experimentos y la implementación de técnicas nuevas para la detección de infecciones, es necesaria para obtener una visión holística del sistema parásito-hospedador estudiado. 2 Justificación y objetivos La presente tesis tiene cuatro propósitos generales: (i) describir un sistema parásito-hospedador común en lagunas del Mediterráneo que se ajusta a las condiciones en el hábitat intertidal, (ii) aumentar el conocimiento sobre los factores que juegan un papel importante en la emergencia, transmisión y desarrollo de las diferentes etapas larvales, (iii) proporcionar nuevas evidencias sobre la mayor precisión de los métodos moleculares para detectar infecciones en gasterópodos, teniendo también en cuenta sus desventajas, y (iv) ampliar nuestro conocimiento sobre el rango de hospedadores y la transmisión de las fases del ciclo vital de Cardiocephaloides longicollis, un parásito digeneo que está altamente presente en el Mediterráneo y que está integrado en sus redes tróficas. Los objetivos específicos a desarrollar son los siguientes: I.- Identificación morfológica y molecular de dos morfotipos diferentes de cercarias emergidas del caracol Gibbula adansonii, común en el hábitat intermareal de la laguna del Delta del Ebro, determinando las tasas de infección, así como su relación filogenética con especies de trematodos cercanas. II.- Evaluación experimental del efecto de los cambios en la temperatura, la salinidad, el fotoperiodo y el nivel de agua sobre la emergencia de larvas de Cainocreadium labracis y Macvicaria obovata, desde su primer hospedador intermediario G. adansonii, mediante técnicas in vitro, en el contexto de sus estrategias de encuentro del siguiente hospedador intermediario. III.- Comparación de métodos tradicionales de detección de infecciones en el primer hospedador intermediario, por emergencia y herramientas moleculares, con una PCR dúplex específicamente diseñada para la detección de estados larvales en gasterópodos, tanto en infecciones simples como dobles. Se utilizarán dos sistemas de parásito-hospedador presentes en el hábitat intermareal: i) dos especies de trematodo, C. labracis y M. obovata, que co-habitan en gasterópodo G. adansonii, desarrollándose en esporocistos y cuya interacción interespecífica no se ha estudiado, y ii) dos especies de trematodo, Maritrema novaezealandensis y Philophthalmus sp. , que co-habitan en el gasterópodo Zeacumantus subcarinatus, desarrollándose en esporocistos y redias, respectivamente, del cual se conoce su competencia interespecífica. IV.- Diferenciación morfológica y cuantificación de los estados larvales de desarrollo en esporocistos, y subsecuente descripción de los patrones de maduración en esporocistos de C. labracis y M. obovata infectando a G. adansonii durante los meses de primavera, cuando este gasterópodo se encuentra en el hábitat intermareal de la laguna del Delta del Ebro. Se determinará si existe un vínculo entre la maduración de las larvas y la migración del gasterópodo. V.- Detección a gran escala de potenciales hospedadores (moluscos, peces y aves) para documentar el espectro de hospedadores y la carga parásita de C. longicollis en el Mediterráneo, determinando también el microhábitat marino al que se dirigen las cercarias, comparando los niveles de infección en peces de diferentes hábitats. VI.- Cuantificación de las metacercarias de C. longicollis en los encéfalos de espáridos para analizar el efecto del tamaño de los peces en su acumulación a lo largo del tiempo, y explorar el papel de los factores antropogénicos como la pesca y la acuicultura, que puedan intensificar la transmisión de metacercarias al hospedador final. 3. Materiales y métodos generales En este resumen los materiales y métodos se describen por separado para cada una de las secciones correspondientes. A continuación se resumen los estudios realizados, estructurados por capítulos. 4. Identificación molecular y morfológica de opecoélidos larvales (Digenea: Opecoelidae) parasitando caracoles prosobranquios en una laguna del Mediterráneo Occidental 4. 1. Introducción Las hábitats costeros de las lagunas mediterráneas son áreas donde se lleva a cabo la reproducción y crecimiento de muchas especies de peces (Franco y col. 2006, 2011, Ribeiro y col. 2006, Verdiell-Cubedo y col. 2007). Además, los parásitos trematodos representan una parte importante de las comunidades marinas de los hábitats intermareales, ya que la naturaleza confinada de las lagunas permite que existan las condiciones propicias para la transmisión de los parásitos con ciclos vitales complejos, es decir, con dos o más hospedadores intermediarios. A pesar de que Bartoli y Gibson (2007) dilucidaron, en su revisión bibliográfica sobre digeneos del Mediterráneo Occidental, el ciclo vital de un total de 56 especies de trematodos, entre las referencias empleadas no existen muchos datos sobre los rasgos morfológicos que podrían ayudar a la identificación de las larvas en futuros estudios. Opecoelidae es una familia de parásitos cosmopolita, con al menos 25 especies parasitando peces marinos en el Mediterráneo Occidental (Bartoli y col. 2005). Sin embargo, sólo se conoce el ciclo vital completo de cuatro de ellas, probablemente por la falta de empleo de técnicas moleculares. Entre los ciclos conocidos se encuentra el de la especie Cainocreadium labracis (Dujardin, 1845) (Maillard 1971, 1974, 1976). En un estudio sobre los digeneos en moluscos de la laguna de “Els Alfacs” en el Delta del Ebro (España), se encontraron infecciones en dos moluscos: Gibbula adansonii (Payraudeau, 1826) (Prosobranchia, Trochidae), que contenía esporocistos con dos morfotipos de cercarias cotilocercas, y Cyclope neritea (L., 1758) (Prosobranchia, Nassariidae), que albergaba metacercarias. Durante el presente trabajo presentamos evidencias moleculares y morfológicas para la identificación de los estados larvales de dos especies de opecoélidos encontrados en dos moluscos. 4. 2. Materiales y métodos Se recogieron un total de 514 G. adansonii y 129 C. neritea en la laguna de “Els Alfacs” (Delta del Ebro, España) desde marzo a julio de los años 2010 y 2011. Después de recoger manualmente los caracoles del sedimento fangoso y sobre las hojas de Zostera noltii Hornemann, 1832, se transportaron al laboratorio en agua marina aireada. Para la extracción de ADN se utilizaron de dos a cuatro cercarias de cada morfotipo y dos metacercarias, fijadas en etanol 100% y disueltas en 300µl de tampón de extracción TNES-urea. Estas muestras se disolvieron durante la noche a 55ºC en 100µg ml-1 de proteinasa K, siguiendo después un protocolo fenol-cloroformo para extracción de ADN (Holzer y col. 2004). El ADN se resuspendió y disolvió en 40µl de agua nanopura durante la noche. Las amplificación del ADN por reacción en cadena de la polimerasa (PCR) se llevó a cabo con un termociclador en un volumen final de 30µl, conteniendo ≈0.5 unidades de polimerasa ThermoPrime Plus DNA y el correspondiente tampón (10x), que contiene 1.5mM MgCl2 (ABgene), 0.2 mM de cada dNTP, 0.5µM de cada primer, y aproximadamente 100ng de la muestra de ADN. Se amplificaron y purificaron dos fragmentos de ADN ribosomal: la secuencia completa de la región ITS (ITS1+5.8S+ITS2) y la secuencia parcial de la región 28S. Después de ensamblar las secuencias con Bioedit v7.0.5. (©1997–2005; Hall 1999), las secuencias resultantes de ITS se alinearon con secuencias de otros opecoélidos obtenidas en GenBank (Mafft v5.531 (Katoh y col. 2002), Gblocks v 2.0 (Castresana 2000)). Para estimar la posición de las secuencias generadas en relación a otros miembros de la familia Opecoelidae se llevaron a cabo análisis de Neighbour-joining (NJ) con MEGA5 (Tamura y col. 2011) e inferencia filogénica con máxima verosimilitud (ML) con RAxML (Stamatakis 2006). Se usó el programa FigTree v1.3.1 (Rambaut 2009) para la visualización de los árboles generados. Después de la emisión espontánea de cercarias de los caracoles G. adansonii, se estudió inicialmente la morfología de las cercarias con la tinción in vivo rojo neutro. Después, las muestras se fijaron en etanol al 70% y se tiñeron con carmín alumínico o acetocarmín férrico, se deshidrataron en series de etanol y se clarearon en dimetilftalato, para ser montados en una preparación con bálsamo de Canadá. Las metacercarias encontradas en C. nerita se sacaron del quiste y se estudiaron bajo el microscopio. Se tomaron fotografías con una cámara Leica DC300 acoplada a un microscopio DMR. Para el estudio de cercarias en el microscopio electrónico de barrido (SEM), se fijaron muestras en glutaraldehido al 2.5% o en etanol al 70%. Después de lavar los especímenes con tampón fosfatos 0.1M (pH 7.4), se deshidrataron en series de acetona, se secaron hasta punto crítico con CO2 y se revistieron por bombardeo iónico con oro. Las muestras se estudiaron con un voltaje de aceleración de 4kV (JEOL JSM 7401-F). 4. 3. Resultados Se obtuvieron secuencias completas de ITS ADNr y parciales de 28S ADNr de los dos morfotipos de cercarias emergidas de G. adansonii y de las metacercarias encontradas en C. neritea. Después del alineamiento de las 20 secuencias disponibles de ITS de otros opecoélidos, se seleccionaron las regiones no ambiguas para llevar a cabo el análisis filogenético (con NJ y ML). Los dos análisis revelaron la misma posición relativa de las secuencias nuevas respecto a las más cercanas filogenéticamente: las secuencias del morfotipo pequeño y de la metacercaria encontrada en C. neritea formaron un clado diferenciado, junto a muestras de Macvicaria obovata. El morfotipo grande también formó un clado monofilético separado, junto a secuencias de Cainocreadium labracis, siendo la divergencia en ambos casos inferior a la divergencia interespecífica de los géneros Macvicaria spp. (2,8–11,9%) y Cainocreadium spp. (5,3-9,4%). Se tomaron medidas para la descripción morfológica de los dos morfotipos de cercarias, así como las de los esporocistos que las contenían, y de la metacercaria encontrada en C. neritea. Además, se describió el comportamiento de las cercarias y su patrón de receptores sensoriales, utilizando fotografías con microscopía electrónica de barrido (SEM) para destacar algunos rasgos morfológicos de las dos cercarias, como por ejemplo las papilas que forman parte del sistema sensorial. Dependiendo del mes de muestreo, la prevalencia de C. labracis fue de 17,6-30,8%, mientras que la de M. obovata fue de 0,9-23,1%. Tanto los esporocistos como las cercarias de M. obovata resultaron tener un tamaño menor que los de C. labracis (M. obovata, longitud esporocitos (µm) 600-1650, longitud cercarias (µm) 196-296 (media 248); C. labracis, longitud esporocitos (µm) 900-2377, longitud cercarias (µm) 208-402 (media 294). Ambas cercarias, al tener una cola de tipo cotilocerco, se sujetaron al sustrato después de su emergencia desde G. adansonii, mostrando C. labracis movimientos laterales pendulares, más vigorosos en presencia de luz. Macviacria obovata, con movimientos más lentos, contraía y expandía el cuerpo, balanceándose en presencia de luz. 4. 4. Discusión La identificación de digeneos se suele basar en la morfología de los adultos, siendo la dilucidación de sus ciclos vitales una tarea difícil. Por lo tanto, el uso de herramientas moleculares es de gran ayuda para la identificación de estados larvales. Los estudios de Jousson y otros autores (Jousson y col. 1999, 2000, Jousson & Bartoli 2000, 2001, Bartoli & Jousson 2003) aportaron 17 secuencias de opecoélidos adultos encontrados en peces del Mediterráneo con los que comparar e identificar estados larvales de esta familia. La detallada descripción morfológica junto con los análisis moleculares que se han llevado a cabo, han permitido la identificación de dos opecoélidos que infectan al caracol G. adansonii como primer hospedador intermediario como Macvicaria obovata (Molin, 1859) (morfotipo pequeño) y Cainocreadium labracis (Dujardin, 1845) (morfotipo grande). Además, la metacercaria de C. neritea se identificó como M. obovata, por lo que también sabemos que este parásito especie utiliza a este gasterópodo como segundo hospedador intermediario. Se recopilaron datos de otras cercarias cotilocercas descritas en el Mediterráneo y las costas del Atlántico nororiental, para comparar las principales características. Las cercarias de C. labracis diferían del resto al presentar glándulas de penetración mucho mayores, coincidiendo además con la descripción de Maillard (1971) y Dufour & Maillard (1974). Aunque el número de glándulas de penetración de M. obovata coincide

Genetics and thermal biology of littorinid snails of the genera Afrolittorina, Echinolittorina and Littoraria (Gastropoda: Littorinidae) from temperate, subtropical and tropical regions

With the anticipated effects of climate change due to global warming, there is concern over how animals, especially ectotherms, will respond to or tolerate extreme and fluctuating environmental temperature stress. Littorinid snails are intertidal ectotherms that live high on the shore where they experience both extreme and variable conditions of temperature and desiccation stress, and are believed to live close to their tolerance limits. This study investigated the thermal biology of littorinid snails of the genera Afrolittorina, Echinolittorina and Littoraria from temperate, subtropical and tropical regions in South Africa and Brunei Darussalam using thermal tolerance, heart function, and proteome approaches. The effects of conditions, such as rate of change in temperature, acclimation, heat shock, season and starvation were also tested. In addition, the evolutionary relationships and genetic diversity between and within the South African Afrolittorina spp. were investigated using mitochondrial and nuclear markers. Genetic results confirmed that these are two distinct species, with the brown to black A. knysnaensis predominant in the cool-temperate region of South Africa and the pale blue-grey A. africana in the subtropical region. There was low genetic variation and differentiation within each species, suggesting high gene flow among populations as a result of the effects of ocean currents on the dispersal of their planktotrophic larvae. Tests using exposure to high temperatures revealed differences in the thermal tolerances, heart performance and protein profiles of species from different latitudes, regions and zones on the shore. Thermal tolerance conformed to expectations, with clear, statistically significant trends from high tolerance in subtropical species to lower tolerance in temperate species. However, for Afrolittorina spp., there were no significant differences in the thermal tolerances of conspecifics from different regions, though there was a significant difference in thermal tolerance between juveniles and adults. Overall, adults of all species showed higher thermal tolerances than juveniles. Although lethal temperatures for these species were higher in summer than winter, laboratory acclimation had no effect on heat coma temperatures. All species showed some regulation of heart rate, with a degree of independence of heart rate from temperature across mid-range temperatures. The tropical species showed quick induction and good regulation of heart rate followed by the subtropical and temperate species, which displayed mixed responses including regulation, partial regulation and lack of regulation. Overall, tropical Echinolittorina spp. showed good regulation, while the subtropical E. natalensis and Littoraria glabrata exhibited a mixture of partial regulation and regulation. The subtropical/temperate Afrolittorina spp. showed high individual variability, some animals exhibiting regulation, while others did not. These effects seem to be largely phylogenetically determined as there were no differences in the heart rate responses of Afrolittorina spp. from different regions. The temperatures at which heart rate became independent of temperature (thermoneutral zone) were within the range experienced under natural conditions. In addition, there were differences in Arrhenius breakpoint and endpoint temperatures, showing a trend from higher in tropical animals to lower for temperate animals. Conditions such as acclimation, heat shock and starvation had little or no effect on heart performance. However, a slow increase in temperature induced good regulation of heart rate with noticeable shifts of breakpoints and endpoints for Afrolittorina spp. Lastly, there were differences in the proteome responses between and within Afrolittorina spp. as a function of species, size and treatment. Although both large and small A. knysnaensis had a greater number of protein spots in their proteome than A. africana (though the difference was not significant), the later showed significantly higher differential expression of certain proteins following heat stress. In addition, juveniles of both species displayed greater numbers of protein spots in their proteome than adults. The results indicate a difference in the physiological and biochemical responses (i.e. adaptations) of these snails to temperature, and this seems to relate to differences in biogeography, phylogeny, species identity and ecology. The ability to regulate heart rate is phylogenetically determined, while thresholds and lethal limits correspond to biogeography and species ecology. The proteome seems to correspond to species ecology. The results also indicate that these littorinids can tolerate high temperature stress and in this respect they are well suited to life in the intertidal zones or habitats where temperature and other stresses or conditions are extreme and can change abruptly. However, the limited ability of these snails to acclimate to different temperatures suggests that they are already living close to their tolerance limits with small safety margins or narrow thermal windows and so may be vulnerable to small rises in substratum temperature and/or solar radiation

The Proteomic Response of Gill Tissue in Tidally and Subtidally-Acclimated California Mussels, Mytilus californianus, to Acute Emersion-Induced Anoxia

Intertidal mussels regularly experience emersion-induced anoxia, in contrast to normoxic conditions experienced during submersion. We therefore hypothesized that acclimation to a tidal rhythm, as opposed to a rhythm of constant submersion, preconditions the proteome of the California mussel, Mytilus californianus, to respond differently to emersion-induced anoxia. Following acclimation, mussels either continued to receive the acclimation conditions (control) or were exposed to 100% nitrogengas (anoxia) during aerial emersion. We collected gill tissue for subsequent analysis of protein abundance with 2D gel electrophoresis and protein identification with tandem mass spectrometry. Relative to subtidally-acclimated mussels, tidally-acclimated mussels showed a greater propensity to respond to distrupted protein homeostasis during emersion through higher levels of several small heat shock protein isoforms, while they showed lower levels of several chaperones involved in redox-sensitive protein maturation in the endoplasmic reticulum during acute anoxia. Several metabolic proteins showed elevated levels in tidally-acclimated mussels, suggesting a compensatory response to reduced feeding times. However, changes in the abundance of several tricarboxylic acid cycle enzymes (e.g. aconitase, succinate dehydrogenase) suggest that tidally-acclimated mussels are also primed to sense reactive oxygen species (ROS) and limit their production, respectively. These findings are further supported by higher abundances of several aldehyde dehydrogenases and thioredoxin peroxidase, which function as scavengers of aldehydes and ROS, common products of lipid peroxidation. Finally, tidally-acclimated mussels are also more responsive to changes in cytoskeletal and vesicular trafficking dynamics in response to acute anoxia. Together, our analysis showed that proteostasis, energy metabolism, oxidative stress and cytoskeletal and trafficking processes are all involved in priming tidally-acclimated mussels to respond more dynamically to acute emersion-induced anoxia in Mytilus gill