Three New Zealand marine species, hoki (Macruronus novaezealandiae), kahawai (Arripis trutta) and trevally (Pseudocaranx dentex) were used to develop model fermented fish sausage. The formulation comprised fish mince, carbohydrate, minced garlic and salt in a mass ratio of 1 (fish): 0.15: 0.05: 0.03, respectively. The carbohydrate source was cooked rice or glucose. (Endogenous lactic acid bacteria (LAB) failed to ferment rice). Folate was also added to the mixture as a factor. The mixtures were extruded into 50 mL plastic syringes, where the needle end of the barrel had been excised by lathe. The lubricated barrel was overfilled to 60 mL, capped with a layer of ParafilmTM and aluminium foil, sealed tightly by rubber band and incubated at 30°C. Over time the piston was progressively advanced to yield samples for microbiological, physical, and chemical analysis. Over 96 hours an increase in the LAB count was observed with a concomitant decrease in pH. After fermentation was complete, the samples contained around 8.77 log cfu LAB g-1 with the pH range from 4.38 to 5.08. The microbiological and pH behaviour of each species varied between preparations. Hardness, adhesiveness, springiness and cohesiveness of the treatments increased with fermentation, except for hoki. The treatments showed different colour characteristics with fermentation. The light reflectance (L* values) of the trevally and kahawai treatments increased, while the a* (redness) and b* (yellowness) values decreased. Hoki exhibited smaller colour changes except for yellowness, which increased markedly. Proteolysis, measured colorimetrically by soluble peptide bonds, was greatest for trevally. Lipid oxidation, measured by the thiobarbituric acid method, was least for hoki, notably the species with the lowest fat content. Biogenic amines, which are a general quality indicator of fermented products, increased during fermentation. The trevally treatment generated the highest concentration of amines, but these values were lower than those reported for fermented fish sausage in Southeast Asia. Notably there were no important difference between folate treatments and those without folate. The results point to commercial opportunities and further research with New Zealand marine species, especially trevally. To improve the product quality and to show geographical exclusivity, further research could be done by using starter culture, and a New Zealand staple carbohydrate source such as kumara and potato, and spices and herbs which are commonly used in New Zealand, such as rosemary, thyme and sage or specific to New Zealand, such as horopito. In addition, sensory studies should also be performed before the products could be tested in the market.

The social brain hypothesis predicts that larger group size and greater social complexity select for increased brain size. In ants, social complexity is associated with large colony size, emergent collective action, and division of labor among workers. The great diversity of social organization in ants offers numerous systems to test social brain theory and examine the neurobiology of social behavior. My studies focused on the Australasian weaver ant, Oecophylla smaragdina, a polymorphic species, as a model of advanced social organization. I critically analyzed how biogenic amines modulate social behavior in ants and examined their role in worker subcaste-related territorial aggression. Major workers that naturally engage in territorial defense showed higher levels of brain octopamine in comparison to more docile, smaller minor workers, whose social role is nursing. Through pharmacological manipulations of octopaminergic action in both subcastes, octopamine was found to be both necessary and sufficient for aggression, suggesting subcaste-related task specialization results from neuromodulation. Additionally, I tested social brain theory by contrasting the neurobiological correlates of social organization in a phylogenetically closely related ant species, Formica subsericea, which is more basic in social structure. Specifically, I compared brain neuroanatomy and neurometabolism in respect to the neuroecology and degree of social complexity of O. smaragdina major and minor workers and F. subsericea monomorphic workers. Increased brain production costs were found in both O. smaragdina subcastes, and the collective action of O. smaragdina majors appeared to compensate for these elevated costs through decreased ATP usage, measured from cytochrome oxidase activity, an endogenous marker of neurometabolism. Macroscopic and cellular neuroanatomical analyses of brain development showed that higher-order sensory processing regions in workers of O. smaragdina, but not F. subsericea, had age-related synaptic reorganization and increased volume. Supporting the social brain hypothesis, ecological and social challenges associated with large colony size were found to contribute to increased brain size. I conclude that division of labor and collective action, among other components of social complexity, may drive the evolution of brain structure and function in compensatory ways by generating anatomically and metabolically plastic mosaic brains that adaptively reflect cognitive demands of worker task specialization and colony-level social organization.

In the wolf spider Rabidosa punctulata, upon encountering a female, males use one of two distinct strategies: (1) they court the female in an attempt to elicit a mating, or (2) they engage in a direct-mount tactic that involves extensive grappling with the female until a mating is achieved. The latter tactic appears more sexually aggressive, and both tactics come with the risk of being cannibalized. We explored the physiological mechanisms underlying this behavioural variation by assessing the relationship between circulating levels of the biogenic amine octopamine (OA), a neuromodulator suggested to play a role in 'fight or flight' responses of arthropods and male mating tactic expression. We predicted, and found support for, a relationship between OA levels and tactic expression, with males adopting the direct-mount tactic expressing higher OA levels than courting males. Male mating tactic and mass also showed a significant interaction, with a negative trend in direct-mounting males and no relationship in courting males. Males had considerably higher levels of OA circulating in their haemolymph than females and female OA level increased with female mass. Our experimental design cannot disentangle cause from effect, but our results are consistent with the hypothesis that OA plays a role in regulating mating tactic expression in R.punctulata.

Die Honigbiene <i>Apis mellifera</i> gilt seit langem als Modell-Organismus zur Untersuchung von Lern- und Gedächtnisvorgängen sowie zum Studium des Sozialverhaltens und der Arbeitsteilung. Bei der Steuerung und Regulation dieser Verhaltensweisen spielt das Indolalkylamin Serotonin eine wesentliche Rolle. Serotonin entfaltet seine Wirkung durch die Bindung an G-Protein-gekoppelte Rezeptoren (GPCRs). In der vorliegenden Arbeit wird der erste Serotonin-Rezeptor aus der Honigbiene molekular charakterisiert. <br><br> Durch die Anwendung zwei verschiedener Klonierungsstrategien konnten drei cDNA-Sequenzen isoliert werden, die für potentielle Serotonin-Rezeptoren kodieren. Die Sequenzen weisen die größte Ähnlichkeit zu dem 5-HT7- und 5-HT2-Rezeptor von Drosophila melanogaster bzw. dem 5-HT1-Rezeptor von <i>Panulirus interruptus</i> auf. Die isolierten Serotonin-Rezeptoren der Honigbiene wurden dementsprechend Am(<i>Apis mellifera</i>)5-HT1, Am5-HT2 und Am5-HT7 benannt. <br><br> Das Hydropathieprofil des Am5-HT1-, Am5-HT2- und Am5-HT7-Rezeptors deutet auf das Vorhandensein des charakteristischen heptahelikalen Aufbaus G-Protein-gekoppelter Rezeptoren hin. Die abgeleiteten Aminosäuresequenzen zeigen typische Merkmale biogener Amin-Rezeptoren. Aminosäuren, die eine Bedeutung bei der Bildung der Liganden-Bindungstasche, der Rezeptor-Aktivierung und der Kopplung eines G-Proteins an den Rezeptor haben, sind in allen drei Rezeptoren konserviert. Interessanterweise ist jedoch das in den meisten biogenen Amin-Rezeptoren vorhandene DRY-Motiv in dem Am5-HT2- und Am5-HT7-Rezeptor nicht konserviert. Das Vorhandensein einer PDZ-Domäne in dem Am5-HT1- und Am5-HT7-Rezeptor lässt vermuten, dass diese Rezeptoren als Adapterproteine fungieren, die Signalmoleküle zu einem Signaltransduktionskomplex vereinigen. <br><br> RT-PCR-Experimente zeigen die Expression der Rezeptoren in verschiedenen Geweben der Honigbiene. Auffallend ist die hohe Expression im Zentralgehirn. Des Weiteren konnte die Expression der Serotonin-Rezeptoren in den optischen Loben, Antennalloben sowie in der Peripherie, d.h. in der Flugmuskulatur und den Malpighischen Gefäßen nachgewiesen werden. Durch in situ Hybridisierungen wurde die Expression in Gefrierschnitten von Gehirnen adulter Sammlerinnen im Detail untersucht. Transkripte der Rezeptoren sind in den Somata von intrinsischen Pilzkörperzellen, Neuronen der optischen Loben und Neuronen der Antennalloben vorhanden. <br><br> In einem heterologen Expressionssystem wurde der intrazelluläre Signalweg des Am5-HT7-Rezeptors untersucht. Die Aktivierung des stabil exprimierten Rezeptors durch Serotonin führt zur Bildung von cAMP. Der 5-HT7-Rezeptor spezifische Agonist 5-CT zeigt eine mit Serotonin vergleichbare Fähigkeit, die intrazelluläre cAMP-Konzentration zu erhöhen. Am5-HT7 gehört daher funktionell zu der Gruppe der 5-HT7-Rezeptoren. Der EC50-Wert von 1,06~nM (5-HT), ist im Vergleich zu anderen 5-HT7-Rezeptoren äußert niedrig. Des Weiteren wurde gezeigt, dass das basale cAMP-Niveau in den transfizierten Zellen im Vergleich zu nicht transfizierten Zellen deutlich erhöht ist. Das heißt, dass der Rezeptor auch in der Abwesenheit eines Liganden aktiv ist. Diese konstitutive Aktivität ist auch von anderen biogenen Amin-Rezeptoren bekannt. Methiothepin wurde als wirksamer inverser Agonist des Am5-HT7-Rezeptors identifiziert, da es in der Lage ist, der konstitutiven Aktivität entgegenzuwirken.<br><br> Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass die Serotonin-Rezeptoren in verschiedenen Regionen des ZNS der Honigbiene an der Informationsverarbeitung beteiligt sind. Es kann eine Beeinflussung von Lern- und Gedächtnisprozessen sowie des olfaktorischen und visuellen Systems durch diese Rezeptoren vermutet werden. Mit der Klonierung und funktionellen Charakterisierung des ersten Serotonin-Rezeptors der Honigbiene ist eine Grundlage für die Untersuchung der molekularen Mechanismen der serotonergen Signaltransduktion geschaffen worden. / The honeybee <i>Apis mellifera</i> is a model organism for studying insect division of labor, learning and memory. An important substance that has been implicated in the control and regulation of these phenomena is the indolalkylamine serotonin (5-hydroxytryptamine, 5-HT) Pharmacological and functional studies indicate, that serotonin activates various receptor subtypes which predominantly belong to the family of G protein-coupled receptors (GPCRs).<br><br> Using a homology based screening approach on a brain-specific cDNA library of the honeybee and a PCR-based strategy we have isolated cDNAs encoding three putative serotonin receptors. The deduced amino acid sequences of these putative honeybee serotonin receptors show the highest homology to a 5-HT7 and a 5-HT2 receptor from <i>Drosophila melanogaster</i> and a 5-HT1 receptor from <i>Panulirus interruptus</i>, respectively. We have studied the distribution of the respective 5-HT receptor mRNAs in several tissues of the honeybee by RT-PCR. The analysis revealed a high amount in the central brain. By using in situ-hybridization we detected receptor encoding mRNA in cryostat sections of honeybee brain. We could prove receptor transcripts in neurons of the optic lobes, intrinsic mushroom body neurons, and deutocerebral neurons. <br><br> In a HEK 293 cell line, stably expressing the 5-HT7 receptor protein, we investigated the intracellular signalling pathway. When activated by serotonin, the heterologous expressed 5-HT7 receptor induces an increase in intracellular cAMP levels ([cAMP]i). The stimulation with other biogenic amines (octopamine, tyramine, and dopamine) did not induce a significant change in [cAMP]i Furthermore, Am5-HT7 causes a significant increase in the non-agonist stimulated cAMP levels relative to those of non-transfected cells. Therefore, the Am5-HT7 receptor displays agonist-independent (constitutive) activity which has also been demonstrated for many other GPCRs. <br><br> A specific affinity-purified anti-Am5-HT7 antibody detected a protein band of the expected size of ~66 kDa in homogenates of honeybee brains and HEK 293 cells expressing the Am5-HT7 receptor.

An animal’s ability to navigate through its natural environment is critical to its survival. Navigation can be slow and methodical such as an annual migration, or purely reactive such as an escape response. How sensory input is translated into a fast behavioral output to execute goal oriented locomotion remains elusive. In this dissertation, I aimed to investigate escape response behavior in the nematode C. elegans. It has been shown that the biogenic amine tyramine is essential for the escape response. A tyramine-gated chloride channel, LGC-55, has been revealed to modulate suppression of head oscillations and reversal behavior in response to touch. Here, I discovered key modulators of the tyraminergic signaling pathway through forward and reverse genetic screens using exogenous tyramine drug plates. ser-2, a tyramine activated G protein-coupled receptor mutant, was partially resistant to the paralytic effects of exogenous tyramine on body movements, indicating a role in locomotion behavior. Further analysis revealed that ser-2 is asymmetrically expressed in the VD GABAergic motor neurons, and that SER-2 inhibits neurotransmitter release along the ventral nerve cord. Although overall locomotion was normal in ser-2 mutants, they failed to execute omega turns by fully contracting the ventral musculature. Omega turns allow the animal to reverse and completely change directions away from a predator during the escape response. Furthermore, my studies developed an assay to investigate instantaneous velocity changes during the escape response using machine based vision. We sought to determine how an animal accelerates in response to a mechanical stimulus, and subsequently decelerates to a basal locomotion rate. Mutant analysis using this assay revealed roles for both dopamine and tyramine signaling. During my doctoral work, I have further established the importance for tyramine in the nematode, as I have demonstrated two additional roles for tyramine in modulating escape response behavior in C. elegans.

[pt] Uma sonda fotoluminescente do tipo turn-on foi avaliada para a detecção de putrescina usando pontos quânticos de grafeno (GQDs). Diferentes nanopartículas de carbono fotoluminescentes foram preparadas usando a abordagem bottom-up, usando ácido cítrico como precursor, sozinho ou misturado com outros compostos contendo heteroátomos (N e/ou S) visando funcionalização da nanoestrutura. Observou- se que as nanopartículas preparadas com ácido cítrico e tioureia (GQDs-TU) apresentaram melhor perfil aumento da fotoluminescência (resposta analítica) na presença dessa amina biogênica. As condições experimentais foram ajustadas para o melhor perfil de resposta e para obter os parâmetros analíticos de mérito. No ensaio em batelada, a curva analítica normalizada (concentração L-L0 versus PUT) foi linear (R2 = 0,9498) até 90 mg L1 . O limite de quantificação (LOQ) foi de 15,1 mg L-1 e o limite de detecção (LOD) foi de 4,5 mg L-1 . Além disso, a estratégia proposta para a determinação indireta de putrescina foi adaptada para um sistema de análise por injeção em fluxo (FIA) a fim de aumentar a frequência analítica, diminuir os resíduos e automatizar a quantificação do analito. Após a otimização do ensaio, com a faixa linear cobrindo o intervalo até 50 mg L-1 (R2 = 0,9980), os valores de LOD e LOQ foram 3,0 mg L1 e 9,9 mg L-1 , respectivamente. / [en] A photoluminescent turn-on probe was evaluated for putrescine detection using graphene quantum dots (GQDs). Different photoluminescent carbon nanoparticles were prepared using a bottom-up approach, employing citric acid as a precursor, either alone or mixed with other compounds containing heteroatoms (N and/or S) for nanostructure functionalization. It was observed that the nanoparticles prepared with citric acid and thiourea (GQDs-TU) exhibited a more pronounced increase in photoluminescence (analytical response) when exposed to this biogenic amine. Experimental conditions were fine-tuned to optimize the response profile and obtain the analytical parameters of merit. In the batch assay, the normalized analytical curve (L-L0 concentration versus PUT) was linear (R2 = 0,9498) up to 90 mg L-1 . The limit of quantification (LOQ) was 15,1 mg L-1 and the limit of detection (LOD) was 4,5 g L-1 . Furthermore, the proposed strategy for the indirect determination of putrescine was adapted for a flow injection analysis (FIA) system to increase the analytical frequency, reduce waste, and automate analyte quantification. After optimization of the assay, with the linear range covering the range up to 50 mg L-1 (R2 = 0,9980), the LOD and LOQ values were 3,0 mg L-1 e 9,93 mg L-1 , respectively.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3.Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción.El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas.Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad).La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%).Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina).La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%).La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC.El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados. / Low-acid fermented meat products (final pH, 5.3 to 6.2) are a group of traditional Mediterranean products with a great diversity within the regions.To control the microbial quality of this kind of sausages is necessary to use rapid methods able to produce results quickly and reliably. In this study, a highly sensitive PCR-based method to detect and quantify L. monocytogenes in fermented sausages was developed. This method combined the high sensitivity of the most-probable-number method with a L. monocytogenes specific PCR assay. Also a multiplex-PCR based method for the simultaneous identification of L. monocytogenes and Salmonella spp. was designed. The study of the microbial quality of the slightly fermented sausages was followed by the characterization of the microbial communities of this kind of products. Lactic acid bacteria (LAB), enterococci and Gram-positive catalase-positive cocci (GCC+) were identified at species level. RAPD-PCR and plasmid profiling were used to evaluate the genetic diversity within species and to identify identical isolates of the same strain. Safety and hygienic properties were also studied in order to characterize the isolates in detail. With this aim, bacteriocin production, biogenic amine production and antibiotic susceptibility were determined. By species-specific PCR, Lactobacillus sakei was identified as the predominant LAB (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). The production of biogenic amines was mainly related to the species L. curvatus (66% of the isolates were biogenic amine-producers).Species-specific PCR and partial sequencing of sodA gene were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species (51.9%) followed by Enterococcus faecalis (14,2%). All the E. faecalis strains carried virulence genes. E. faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides).Species-specific PCR and amplification of the 16S-23S rDNA intergenic region were used to identify GCC+ population. Staphylococcus xylosus was the predominant species (80.8%) in this kind of sausages.Tyramine was the most intense biogenic amine produced, although by only 4.6% of the GCC+ isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. A low percentage of isolates (4.6%) showed mecA genes displaying also resistance to multiple antibiotics. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene.The study of the safety and technological properties of the isolates allowed to select 2 strains of L. sakei and 2 strains of S. xylosus on the basis of their technological and safety characteristics. To evaluate their suitability as starter cultures two types of low acid fermented sausages, fuet and chorizo, were manufactured. Batters were inoculated with L. monocytogenes, S. enterica and S. aureus. Starter cultures were able to control the growth of L. monocytogenes, Enterobacteriaceae, Enterococcus and the biogenic amine content. Salmonella spp counts decreased significantly during ripening independently of the use of starter culture and product.High hydrostatic pressure treatment was necessary to assure absence of Salmonella spp. in final products.