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Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

This study presents evidence from mitochondrial and nuclear loci that there is genetic divergence among and within geographic populations of Lagenorhynchus obscurus. The effect of seasonal variation on the genetic structure within New Zealand was examined with mitochondrial DNA control region sequences from 4 localities. Analysis of nested haplotype clades indicated genetic fragmentation and at least 1 historical population expansion within New Zealand. AMOVA and Fst values from nuclear and mitochondrial DNA sequences suggested significant divergence between New Zealand, South Africa, Argentina, and Peru. Dispersal via the west-wind drift was not supported by patterns of population structure among regions. Alternatively, these data support reciprocal exchange among all four regions with 100% posterior probability for a root of origin in the Indian/Atlantic Oceans. The degree of divergence between Peru and other regions indicates the isolation of Peruvian stock is temporally correlated with the constriction of Drake’s passage in the Plio-Pleistocene. There is evidence that the Plio-Pliestocene paleoceanography of the Indian and Southern Atlantic Oceans influenced phylogeography with shifts of temperate sea surface temperatures northward ~5º of latitude, disrupting the dispersal corridor between New Zealand and Atlantic populations. A preference for temperate waters along continental shelves is proposed as an explanation for lack of contemporary genetic exchange among regions. This study supports the polyphyly of the genus Lagenorhynchus. North Atlantic species form a monophyletic Lagenorhynchus. In the Southern Hemisphere, L. australis/L. cruciger and L. obliquidens/L. obscurus do not form a monophyletic group. I discuss the taxonomic implications and propose taxonomic revision of the genus based on these results. Measures of character interaction indicate that combined evidence from nuclear and mitochondrial genes provide better phylogenetic resolution among delphinid lineages than any data partition independently, despite some indications of conflict among mitochondrial and nuclear data.

Foraging theory suggests that hungry animals balance a complex set of costs and benefits when determining what and how to eat. Prey distribution, patch size, and the presence of conspecifics are important factors influencing a predator's feeding tactics, including the decision to feed individually or socially. Dusky dolphins (Lagenorhynchus obscurus) in New Zealand employ different feeding tactics in varying habitats and seasons. I used programmed survey routes and opportunistic sightings to examine the habitat use and feeding mechanics of dusky dolphins in Admiralty Bay, New Zealand, a protected shallow-water environment frequented by wintering dolphins. I encountered 253 dolphin groups, of which 58.5% were engaged in food-acquisition activities. Photographic efforts revealed a total of 177 individually-recognizable dolphins, 100 of which were returnees from previous seasons. Thirty-seven feeding groups and 70 bouts of feeding behavior were followed. Two-minute interval sampling as well as active acoustic sonar were used to test the hypothesis that diurnally-feeding dolphins would work in a coordinated manner to bring schooling fish to the surface. Feeding tactics observed in Admiralty Bay were then compared to foraging by some of the same animals in the unprotected, deep-water environment off Kaikoura, where large numbers of dusky dolphins feed during the night on organisms associated with a vertically-migrating scattering layer. Evidence supporting coordinated surface feeding was not statistically significant, but indicative of behavioral flexibility in feeding styles as part of a larger feeding repertoire. A potential shift in prey distribution from previous years may also explain some observed patterns. Feeding groups were positively correlated with seabirds and New Zealand fur seals (Arctocephalus forsteri). Mean group size of 6.1 (± 8.23 S.D., n=253) in Admiralty Bay is dramatically less than groups observed off Kaikoura, a variation likely reflecting differences in prey number and distribution, as well as differences in predation risk by deep-water sharks and killer whales. Behavioral flexibility likely confers an adaptive advantage for species subject to environmental fluctuation, whether due to natural or anthropogenic sources. Further research is necessary to evaluate prey distribution in Admiralty Bay and its possible effects on feeding dusky dolphins.

This thesis examines the characteristics of dusky dolphin (Lagenorhynchus obscurus) nursery groups and ontogeny of dusky dolphin calves. Data were collected via boat-based group focal follows of nurseries from October 2006-May 2007. A total of 87 nursery groups were encountered. Data were analyzed according to age category (infant or yearling) and season (early or late). Nursery group membership was lowest in the early season and when yearlings were present. The average number of yearlings in a nursery group was less than that of infants. The predominant activity of calves was rest. Early infants rested the most, while travel seemed most important for late infants, and early yearlings were most likely to forage. With the exception of early infants, all calves were more likely than adults to interact with boats. When taking month into account, yearlings were more social in general than infants. Infants showed a positive trend in sociality, while yearling sociality remained relatively stable. Nursery groups are markedly segregated by calf age, and 80% of nursery groups contained calves of only one age group. Dusky dolphin calves show a similar trend in preference for position in relation to the mother as that in bottlenose dolphins (Tursiops sp.), with echelon swim decreasing with age. However, all calves appear to prefer echelon swim when nursery groups are traveling. Calves were more likely to swim independently in the late part of the season and while foraging or socializing, and were more likely to be in close proximity to their mothers while resting or traveling. Calves learned noisy leaps, followed by clean, coordinated, and acrobatic leaps, in that order. There was no clear relationship between behavioral state and types of leaps performed by calves. Early infants leapt less often than older calves, but leap frequency did not differ among the older calves. The overall pattern in the ontogeny of dusky dolphin leaps indicates that the physical development of leaps is learned individually, while the context in which the leaps are performed is learned from conspecifics. These results indicate that nursery groups represent an important environment for healthy physical and social development of calves.

This thesis examines the characteristics of dusky dolphin (Lagenorhynchus obscurus) nursery groups and ontogeny of dusky dolphin calves. Data were collected via boat-based group focal follows of nurseries from October 2006-May 2007. A total of 87 nursery groups were encountered. Data were analyzed according to age category (infant or yearling) and season (early or late). Nursery group membership was lowest in the early season and when yearlings were present. The average number of yearlings in a nursery group was less than that of infants. The predominant activity of calves was rest. Early infants rested the most, while travel seemed most important for late infants, and early yearlings were most likely to forage. With the exception of early infants, all calves were more likely than adults to interact with boats. When taking month into account, yearlings were more social in general than infants. Infants showed a positive trend in sociality, while yearling sociality remained relatively stable. Nursery groups are markedly segregated by calf age, and 80% of nursery groups contained calves of only one age group. Dusky dolphin calves show a similar trend in preference for position in relation to the mother as that in bottlenose dolphins (Tursiops sp.), with echelon swim decreasing with age. However, all calves appear to prefer echelon swim when nursery groups are traveling. Calves were more likely to swim independently in the late part of the season and while foraging or socializing, and were more likely to be in close proximity to their mothers while resting or traveling. Calves learned noisy leaps, followed by clean, coordinated, and acrobatic leaps, in that order. There was no clear relationship between behavioral state and types of leaps performed by calves. Early infants leapt less often than older calves, but leap frequency did not differ among the older calves. The overall pattern in the ontogeny of dusky dolphin leaps indicates that the physical development of leaps is learned individually, while the context in which the leaps are performed is learned from conspecifics. These results indicate that nursery groups represent an important environment for healthy physical and social development of calves.

I characterized dusky dolphin (Lagenorhynchus obscurus) underwater bait-balling behaviors and acoustic signals, and compared data between Argentina and New Zealand (NZ) to investigate the roles of ecology versus social learning. I quantified prey herding and capturing behaviors from video footage, and I analyzed acoustic signals from narrowband recordings. In both locations, I related bait-balling behaviors and acoustic signals to group and prey ball sizes. In NZ, I also related dolphin behaviors to prey ball escape behaviors and acoustic signal parameters to examine proximate functions. Observed herding behaviors typically involved dolphins swimming around or under a prey ball using a side body orientation, while dolphins typically captured fish from the side of a prey ball using a ventral orientation. Coordinated prey-capture behaviors may have made it easier for dolphins to capture fish by trapping fish between dolphins. Signals were categorized as click trains, burst pulses, and combinations due to a bimodal inter-click interval distribution. I observed 3 whistle-like chirp-screams, but no whistles. Sequences of burst pulses also occurred that contained 2-14 burst pulses that aurally and visually appeared closely matched. Similarities between locations suggest that ecological context related to broad behavioral and acoustic parameters, while social learning differences may occur on a finer scale. In NZ, prey balls exhibited horizontal and vertical movements, but the only behavior that preceded escape was “funneling”, the brief formation of a ball shape where the height was at least twice the width. Dolphin behaviors that related to prey balls ascending were type of herding pass, location of prey-capture attempts, and body orientation during attempts. These behavioral parameters may also be used to counter vertical prey escape behaviors. In NZ, all signal categories had a direct or indirect role in capturing prey. Click train-burst pulses were likely used for echolocating on prey, burst pulses and sequences appeared to have communication roles, and the role of click trains was ambiguous. No signal categories appeared to have a herding function, but the sheer number of signals emitted may have caused fish to cluster together more tightly and therefore facilitated capture.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T13:04:46Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EstudoAspectosClinicos.pdf: 2259793 bytes, checksum: 716dcd79ff35a0b14f3d83f54b23c3d9 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-07T14:11:03Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EstudoAspectosClinicos.pdf: 2259793 bytes, checksum: 716dcd79ff35a0b14f3d83f54b23c3d9 (MD5) / Made available in DSpace on 2017-11-07T14:11:03Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EstudoAspectosClinicos.pdf: 2259793 bytes, checksum: 716dcd79ff35a0b14f3d83f54b23c3d9 (MD5) Previous issue date: 2015-11-27 / O escorpionismo ou acidente por escorpião é um problema de saúde pública em todas as regiões brasileiras. Na Região Norte, é o Estado do Pará o que mais notifica a ocorrência de casos. Os municípios da Região Metropolitana de Belém (RMB) são os que mais têm apresentado estudos sobre estes acidentes, porém em poucos casos há comprovação do escorpião agressor. O objetivo principal deste estudo foi determinar os aspectos clínicos e epidemiológicos dos acidentes comprovadamente causados por escorpiões na RMB. Foram realizadas a identificação, a caracterização e a avaliação da importância médica dos escorpiões responsáveis por escorpionismos, doados ao Centro de Informação Toxicológica de Belém (CIT-Belém). Após notificação e atendimento médico das vítimas, foi traçado o perfil clínico e epidemiológico dos acidentados entre janeiro de 2007 a junho de 2015. Os municípios da RMB que apresentaram 55 acidentes comprovadamente causados por escorpião foram Belém (50,9%), Ananindeua (47,3%) e Benevides (1,8%). Os escorpiões identificados são espécies nativas na região: o Tityus obscurus Gervais, 1843 (76%), de cor escura que atinge até 87,95 mm de comprimento e o Tityus silvestris Pocock, 1897 (24%), de cor amarelada com manchas escuras que atinge até 45,84 mm de comprimento. As duas espécies acidentaram as pessoas preferencialmente no ambiente domiciliar e durante o período diurno, as vestimentas eram o local preferencial que ocultava o escorpião T. silvestris antes de ocasionar o acidente (53,8%), enquanto o abrigo mais frequente de T. obscurus foram os frutos (35,5%). Os casos ocorreram com maior frequência em pessoas entre 30 e 44 anos de idade, sendo as mãos o sítio anatômico preferencial da picada, não houve predominância de gênero nos acidentados. A maioria das vítimas de T. obscurus apresentou manifestações locais de envenenamento (83,3%), algumas evoluíram com manifestações sistêmicas (11,9%), todos os acidentados por T. silvestris tiveram manifestações locais e destes 23% evoluíram para quadro clínico de envenenamento sistêmico. Todas as vítimas após atendimento evoluíram para alta com cura total. Concluímos que ocorre escorpionismo na RMB por duas espécies nativas na Amazônia, o T. obscurus e o T. silvestris com diferenças morfológicas e epidemiológicas. É a primeira vez que se relata sintomatologia sistêmica nos envenenamentos por T. silvestris revelando a importância médica desta espécie. Estudos da peçonha desta devem ser realizados para verificar sua real importância em saúde pública. / The scorpionism or accident by scorpion is a public health problem in all Brazilian regions. In the North Region, is the State of Pará which more notifies the occurrence of cases. The municipalities in the Metropolitan Region of Belém (RMB) are that more have presented studies about these accidents, but in a few cases there was evidence the scorpion aggressor. The main objective of this study was to determine the clinical and epidemiological aspects of accidents caused by scorpions in RMB. Were performed the identification, characterization and evaluation of medical importance of scorpions responsible for scorpionisms, donated to the Center for Toxicological information of Belém (CIT-Belém). After notification procedure and medical care of victims, it was traced the clinical and epidemiological profile of the accident victims between January 2007 to June 2015. The municipalities of RMB that presented 55 accidents demonstrably caused by scorpion were Belém (50.9%), Ananindeua (47.3%) and Benevides (1.8%). The scorpions identified are native species in the region: the Tityus obscurus Gervais, 1843 (76%), dark in color that reaches up to 87,95 mm in length and the Tityus silvestris Pocock, 1897 (24%), yellowish with dark spots that reaches up to 45,84 mm of length. The two species injured people preferably into domicile and during the day, the garments were the common location that hid the scorpion T. silvestris before cause the accident (53.8%), while the most frequent shelter of T. obscurus were the fruits (35.5%). The cases occurred with greater frequency in individuals between 30 and 44 years of age, being hands the most common anatomical site for stings, there was no predominance of gender in victims. The majority of victims to T. obscurus presented local manifestations of poisoning (83.3%), some have evolved with systemic manifestations (11.9%), all the victims by T. silvestris had local manifestations and of these 23% evolved to clinical condition of systemic poisoning. All victims after answer evolved to high with total cure. We conclude that occurs scorpion envenomation in RMB by two native species in the Amazon, T. obscurus and T. silvestris with morphological differences and epidemiological characteristics. It is the first time that reports systemic symptoms in poisoning by T. silvestris revealing the medical importance of this species. Studies of the poison of this should be carried out to check their real importance in public health.

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