Physiological and Behavioural Responses to Noxious Stimuli in the Atlantic Cod (Gadus morhua)

In the present study, our aim was to compare physiological and behavioural responses to different noxious stimuli to those of a standardized innocuous stimulus, to possibly identify aversive responses indicative of injury detection in a commercially important marine teleost fish, the Atlantic cod. Individual fish were administered with a noxious stimulus to the lip under short-term general anaesthesia (MS-222). The noxious treatments included injection of 0.1% or 2% acetic acid, 0.005% or 0.1% capsaicin, or piercing the lip with a commercial fishing hook. Counts of opercular beat rate (OBR) at 10, 30, 60, 90 and 120 min and observations of behaviour at 30 and 90 min post-treatment were compared with pre-treatment values and with control fish injected with physiological saline, an innocuous stimulus. Circulatory levels of physiological stress indicators were determined in all fish at 120 minutes post-treatment. All treatments evoked temporarily increased OBR that returned to pre-treatment levels at 60 minutes (saline, 0.005% capsaicin, hook), 90 minutes (0.1% acetic acid, 0.1% capsaicin), or 120 minutes (2% acetic acid), but with no significant differences from the control group at any time point. Fish treated with 0.1% and 2% acetic acid and 0.1% capsaicin displayed increased hovering close to the bottom of the aquaria and fish given 2% acetic acid and 0.1% capsaicin also displayed a reduced use of shelter. The only effect seen in hooked fish was brief episodes of lateral head shaking which were not seen pre-treatment or in the other groups, possibly reflecting a resiliency to tissue damage in the mouth area related to the tough nature of the Atlantic cod diet. There were no differences between groups in circulatory stress indicators two hours after treatment. This study provides novel data on behavioural indicators that could be used to assess potentially aversive events in Atlantic cod

Detection of host plant odorants in the herbivore weevils Pissodes notatus and Anthonomus rubi: a comparative study, using electrophysiological recording linked to gas chromatography and mass spectrometry

Abstract - Each herbivore in the course of its evolution became associated with particular plants. The maintenance of host specializations requires the ability to find and recognize plants, which in natural habitas often grow in mixed and complex vegetations. The process of host selection is mediated by integration in the central nervous system of various sensory inputs, including olfactory cues originating in the plant. Odours from plants are complex blends, comprising a wide scope of constituents, which vary in time, space, and between individual sources. Odours are in most organisms, including insects, detected by olfactory receptor neurones (RNs). The detection of olfactory stimuli begins with the interaction of odour molecules with odorant receptor proteins, which determine the specificity of the RNs [i.e. its molecular receptive range (MRR)]. Substantial knowledge about olfactory mechanisms in insects stems mainly from studies of species of moths, cockroaches, the honey bee and the fruit fly. This thesis has contributed to elucidate the mechanisms of olfactory detection in another major group of herbivores, the weevils (Curculionidae). The family Curculionidae includes some of the most important pest insects in agriculture, forestry and stored products. Historically, searching for pheromones has been the preferred way to find potent attractants for pest insects. However, more than 20 pheromones have been chemically identified in weevils, and after decades of development, only relatively few have found successful application for control. It has been suggested that the reason for the disappointing results might be the poor understanding of which plant compounds interact with the pheromone attraction. Two oligophagous weevil species were chosen as modal organisms for the present study, the pine weevil, Pissodes notatus F. (Coleoptera, Curculionidae) feeding on pines (Pinaceae), and the strawberry blossom weevil, Anthonomus rubi Herbst, (1795) (Coleoptera, Curculionidae), feeding on strawberry (Rosaceae). Both species are economically important pests in their areas of distribution. The work in this thesis focuses on how plant odour information is detected and encoded by the RNs of weevils. The aims were: (1) To identify host and non host plant produced compounds detected by single olfactory receptor neurones (RN) in the two species; (2) To characterise the plant odour RNs by their molecular receptive ranges, specificity and sensitivity, and elucidate whether the single RNs can be classified into distinct functional. types; (3) Compare RN types across the two weevil species living on different hosts; (4) Investigate whether the odorants are constitutive in the host plant or induced by insect feeding; (5) Select the relevant compounds for behavioural studies. Electrophysiological recordings from single RNs were performed during stimulation with plant volatiles separated by gas chromatography (GC-SCR), followed by chemical identification of the active components, by GC mass spectrometry (GC-MS). Collection of volatiles from headspace of host and non-host plants was dope by entrainment techniques, using adsorbents and by Solid phase micro extraction (SPME). The thesis is organized in two parte. The first part includes the introduction to the topics and the methods, a presentation of the aims of the investigation, and a survey of the results followed by a general discussion with concluding remarks. The second part presents the original publications that form the basis of the thesis. The weevils investigated in this study, detect a wide range of plant volatiles present both as major and minor constituents of the headspace of plants. The odorants are monoterpenes, sesquiterpenes, aromatic compounds, and also aliphatic alcohols, aldehydes and esters, many of which are produced by the hosts of both species. The compounds identified as odorants in this thesis are ubiquitous in the plant world. Thus, in general the single compounds do not seem to provide a basis for host recognition. These findings support the hypothesis that insects use a blend of many compounds in a certain ratio to recognize a host plant. The RNs were narrowly tuned, responding consistently and selectively to one primary odorant (eliciting the strongest response) and a few secondary odorants (eliciting weaker responses), and could be classified in types. Both in A. rubi, and in P. notatus, functional types of RNs may appear in stereotyped pairs, as found in studies of other weevils, moths and other insects. Both in A. rubi and in P. notatus, was observed some overlap of the MRR of the narrowly tuned RNs, showing that information about one odorant is conveyed to the brain by more than one RN type. Thus the results indicate that the odour quality is partly coded in a combinatorial manner. The majority of the responses elicited in the RNs were excitatory. However, one RN type in A. rubi showed excitatory responses to α-pinene and an inhibitory response to linalool. This exception raises the interesting question of whether integration of olfactory stimuli starts in the periphery. The importante of chirality for bioactivity is well known. The use of GC-columns that separate enantiomers has allowed demonstrating that RNs tuned to plant odorants can also show enantioselectivity. In. A. rubi, separate RNs tuned to (-)- and to (+)-linalool exist. This means that each of the two enantiomers may carry different messages to the insect. In contrast, only one type of RN tuned to (-)-germacrene D was identified. #### /RESUMO / - A maioria dos insectos fitófagos são especialistas, i.e. monófagos ou oligófagos. A conservação das especializações alimentares dos insectos, requer a capacidade de seleccionar a planta adequada, de entre as muitas existentes na natureza. O metabolismo secundário das plantas dá origem a numerosos compostos, muitos dos quais são voláteis e cuja principal função parece ser a de conferir à planta resistência aos factores, do meio, incluindo o ataque por insectos fitófagos. As espécies fitófagas, por sua vez, adaptaram-se a estas barreiras químicas, por vezes utilizando-as em beneficio próprio. Disto é exemplo a capacidade de utilização de compostos voláteis da planta hospedeira como sinais de orientação para a encontrar. O processo de selecção da planta hospedeira é mediado pela integração, a nível do sistema nervoso central, de estímulos sensoriais de diversa natureza. De entre estes, destacam-se os estímulos olfactivos, com origem nas plantas hospedeira e não hospedeiras. A especialização ao nível da detecção e integração olfactiva desempenha um papel fundamental na estabilização das associações insecto-planta. Em todos os animais, os estímulos odoríficos são detectados por neurónios receptores olfactivos (M). Nos insectos, os RNs encontram-se localizados sobretudo nas sensillas olfactivas das antenas. O reconhecimento de um estímulo olfactivo, começa com a interação das moléculas odoríficas com as proteínas receptoras que residem na membrana das dendrites dos RNs. Estas proteinas, determinam a especificidade RNs, isto é, a gama de compostos a que o mesmo responde. Uma das questões fundamentais colocadas à investigação das interacções insecto-planta e dos mecanismos olfactivos, reside na defmição de estímulos e identificação de odores com significado biológico. Este tipo de questão tem vindo a ser elucidada particularmente em insectos que possuem um sistema olfactivo facilmente acessível à análise electrofisiológica. Trabalhos que combinam estudos electrofisiológicos com análise química têem permitido identificar moléculas para cuja recepção os insectos adquiriram sensores especializados ao longo da evolução,. Os resultados apresentados nesta tese contribuem para o conhecimento sobre os mecanismos da detecção olfactiva num dos grupos de herbívoros mais diversificados, os gorgulhos (Curculionidae). Algumas das mais importantes e destrutivas pragas agrícolas e florestais pertencem a esta família. Historicamente, a procura de feromonas, tem sido a via preferida para encontrar atraentes eficazes no controlo de pragas. No entanto, já foram identificadas, mais de 20 feromonas de curculionídeos e, após décadas do desenvolvimento experimental, relativamente poucas foram bem sucedidas no controle de pragas agrícolas. Uma das razões avançadas para estes resultados decepcionantes é o défice de conhecimento sobre os compostos da planta envolvidos no processo de identificação do hospedeiro, e de como estes interagem com as feromonas. Duas espécies de curculionídeos oligófagos foram escolhidas como modelo para este estudo: o gorgulho pequeno do pinheiro Pissodes notatus F. (Coleoptera, Curculionidae) cujo hospedeiro preferencial é o pinheiro bravo, Pinus pinaster (Pinaceae), e o gorgulho da flor do morangueiro, Anthonomus rubi Herbst (Coleoptera, Curculionidae). Ambas as espécies constituem pragas economicamente importantes nas suas áreas de distribuição. Embora sendo este um estudo de investigação fundamental, os resultados obtidos são projectáveis no plano da aplicação, nomeadamente em protecção integrada de povoamentos de pinheiro bravo e da cultura do morangeiro. O objectivo geral foi o de investigar os mecanismos de detecção e codificação de odores das plantas pelos RNs de P. notatus e A . rubi, e assim contribuir para o conhecimento de quais os compostos importantes para a localização do hospedeiro nestas espécies. Definiram-se os seguintes objectivos específicos: (1) Determinara gama de compostos emanados por plantas hospedeiras e não hospedeiras, detectados por RNs de P.notatus e A. rubi; (2) Caracterizara especificidade e sensibilidade dos neurónios receptores olfactivos, agrupando-os em classes de acordo com os diferentes compostos constituintes do odor das plantas hospedeiras e não hospedeiras, que detectam; (3) Investigar a indução da produção de compostos voláteis na planta hospedeira; (4) Pesquisar aspectos particulares do olfacto em curculionídeos, através da abordagem comparativa entre espécies; (5) Seleccionar compostos para estudos comportamentais Para a prossecução destes objectivos foram utilizadas, principalmente, técnicas de registo extracelular de potenciais de acção de neurónios receptores olfactivos, quando estimulados com compostos voláteis produzidos pelas plantas. Os registos electrofisiológicos foram executados durante a estimulação com os voláteis da planta, separados por cromatografia em fase gasosa (GC-SCR). Para a identificação química dos componentes das misturas que geraram resposta electrofisiológica, utilizou-se a técnica de cromatrografia em fase gasosa acoplada a espectrometria de massa (GC-MS)

Physiological and Behavioural Responses to Noxious Stimuli in the Atlantic Cod (Gadus morhua)

In the present study, our aim was to compare physiological and behavioural responses to different noxious stimuli to those of a standardized innocuous stimulus, to possibly identify aversive responses indicative of injury detection in a commercially important marine teleost fish, the Atlantic cod. Individual fish were administered with a noxious stimulus to the lip under short-term general anaesthesia (MS-222). The noxious treatments included injection of 0.1% or 2% acetic acid, 0.005% or 0.1% capsaicin, or piercing the lip with a commercial fishing hook. Counts of opercular beat rate (OBR) at 10, 30, 60, 90 and 120 min and observations of behaviour at 30 and 90 min post-treatment were compared with pre-treatment values and with control fish injected with physiological saline, an innocuous stimulus. Circulatory levels of physiological stress indicators were determined in all fish at 120 minutes post-treatment. All treatments evoked temporarily increased OBR that returned to pre-treatment levels at 60 minutes (saline, 0.005% capsaicin, hook), 90 minutes (0.1% acetic acid, 0.1% capsaicin), or 120 minutes (2% acetic acid), but with no significant differences from the control group at any time point. Fish treated with 0.1% and 2% acetic acid and 0.1% capsaicin displayed increased hovering close to the bottom of the aquaria and fish given 2% acetic acid and 0.1% capsaicin also displayed a reduced use of shelter. The only effect seen in hooked fish was brief episodes of lateral head shaking which were not seen pre-treatment or in the other groups, possibly reflecting a resiliency to tissue damage in the mouth area related to the tough nature of the Atlantic cod diet. There were no differences between groups in circulatory stress indicators two hours after treatment. This study provides novel data on behavioural indicators that could be used to assess potentially aversive events in Atlantic cod

Physiological and Behavioural Responses to Noxious Stimuli in the Atlantic Cod (Gadus morhua)

In the present study, our aim was to compare physiological and behavioural responses to different noxious stimuli to those of a standardized innocuous stimulus, to possibly identify aversive responses indicative of injury detection in a commercially important marine teleost fish, the Atlantic cod. Individual fish were administered with a noxious stimulus to the lip under short-term general anaesthesia (MS-222). The noxious treatments included injection of 0.1% or 2% acetic acid, 0.005% or 0.1% capsaicin, or piercing the lip with a commercial fishing hook. Counts of opercular beat rate (OBR) at 10, 30, 60, 90 and 120 min and observations of behaviour at 30 and 90 min post-treatment were compared with pre-treatment values and with control fish injected with physiological saline, an innocuous stimulus. Circulatory levels of physiological stress indicators were determined in all fish at 120 minutes post-treatment. All treatments evoked temporarily increased OBR that returned to pre-treatment levels at 60 minutes (saline, 0.005% capsaicin, hook), 90 minutes (0.1% acetic acid, 0.1% capsaicin), or 120 minutes (2% acetic acid), but with no significant differences from the control group at any time point. Fish treated with 0.1% and 2% acetic acid and 0.1% capsaicin displayed increased hovering close to the bottom of the aquaria and fish given 2% acetic acid and 0.1% capsaicin also displayed a reduced use of shelter. The only effect seen in hooked fish was brief episodes of lateral head shaking which were not seen pre-treatment or in the other groups, possibly reflecting a resiliency to tissue damage in the mouth area related to the tough nature of the Atlantic cod diet. There were no differences between groups in circulatory stress indicators two hours after treatment. This study provides novel data on behavioural indicators that could be used to assess potentially aversive events in Atlantic cod

Ethogram presenting the categorization of behaviors in the present study. Behavioural data are expressed as the percentage duration (%) or counts of episodes (#).

<p>Ethogram presenting the categorization of behaviors in the present study. Behavioural data are expressed as the percentage duration (%) or counts of episodes (#).</p

Whole blood parameters and plasma cortisol levels in Atlantic cod at 120 min post-treatment.

<p>Data are expressed as mean (±S.E.) for normally distributed data and as medians (±IQR) for non-normally (*) distributed data. There were no statistically significant differences between treatment groups for both normally distributed data (one-way ANOVA) and not normally distributed data (Kruskal Wallis). N = 7 fish per group except for 0.005% Capsaicin (N = 6) and 0.1% Capsaicin (N = 8).</p

Occurrence of Shelter and Hovering on the bottom behaviours in Atlantic cod before and after saline, acetic acid, capsaicin, and fishing hook treatments.

<p>The data are expressed as mean percentage of time (%,<b>±</b>S.E.) the behaviour was displayed during 15 min segments at 30 min and 90 min after treatment. For each time point, identical letters denote a statistically significant (p≤0.05) difference between treatment groups (repeated measures GLM followed by post-hoc test using the Bonferroni correction). N = 7 fish per group except for 0.005% Capsaicin (N = 6) and 0.1% Capsaicin (N = 8).</p

Opercular beat rates (beats×min⁻¹; mean OBR±S.E.) for each treatment group at 20 min pre-treatment (−20 min) and at 10, 30, 60, 90 and 120 min post-treatment (i.e. corresponding to the data presented in Table S1).

<p>There were no statistically significant differences (p>0.05) in OBR evidenced between treatment groups at any time-point (two-way repeated measures ANOVA with a Greenhouse-Geisser correction followed by post-hoc test using the Bonferroni correction). Asterisks (*) denote a statistically significant (p≤0.05) within-group difference in OBR compared to its respective pre-treatment recording (one-way repeated measures ANOVA with a Greenhouse-Geisser correction followed by post-hoc test using the Bonferroni correction). N = 7 fish per group except for 0.005% Capsaicin (N = 6) and 0.1% Capsaicin (N = 8).</p