Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

Vertebrate extraocular muscles show a highly complex anatomy, which differs in many respects from skeletal muscles. Furthermore, there is a considerable variation among different species with regard to the presence of proprioceptive organs. Whereas muscle spindles and Golgi tendon organs are well developed in sheep and pig, neither are found in cat, and only poorly developed muscle spindles are present in human and monkey. In all vertebrates studied so far cuffs of nerve terminals around multiply innervated muscle fibers of the global layer, termed palisade endings (PE) are present at the myotendinous junction. Palisade endings (PE) are specialized nerve endings unique to extraocular muscles. There is still an ongoing debate on PE function. A proprioceptive function is supported by their ultrastructural morphology, and the location of the majority of their terminals within the muscle tendon. A motor function of PEs is suggested by the expression of different cholinergic markers, and the binding of α-bungarotoxin to at least a small proportion of PE nerve endings. So far the location of the somata giving rise to the PEs is unknown. After eye muscle injections with different tracers, and the investigation of retrogradely labeled cells after three days of survival, there are two possible options for the location of the cell bodies of PEs: The trigeminal ganglion (TG), where almost all of the sensory afferents of eye muscles come from, or the motor neurons of the abducens (nVI), trochlear (nIV) and oculomotor nucleus (nIII) in the brainstem, where the motor neurons of the extraocular muscles are situated. In the first part of this thesis the histochemical and morphometric properties of TG cells projecting to the extraocular muscles were studied and related to different nerve fibers and terminals in these muscles, to obtain more information about their function. Retrogradely labeled TG neurons and eye muscle terminals were processed for the presence of substance P (SP), nitric oxide synthase (NOS), calretinin (CR) and cholinacetyltransferase (ChAT). Injections of the tracer were placed into the medial rectus (MR) or the lateral recuts muscle (LR), either in the belly or in the distal, myotendinous part of the muscle. PEs were only labeled by CR or ChAT and none of the other markers that were used. Furthermore, CR positive retrogradely labeled tracer cells in the TG were rarely found (under 1 % of all tracer cells). The results indicate that the possible function of TG afferents could be mainly vasodilatation or nociception whereas the probability of proprioception via PEs is very low. These results point to the fact, that the source of the PE cell body is probably localized in the brainstem. In a second attempt to solve this problem, rhesus monkey received tract-tracer injections (WGA or CT) into the oculomotor nucleus, which contains the motor neurons of the medial, inferior and superior recti and inferior oblique muscles, as well as the trochlear nucleus, which contains the motor neurons of the superior oblique. All extraocular muscles were processed for the combined immunocytochemical detection of the tracer and non-phosphorylated neurofilament for the visualization of the complete muscle innervation. In all muscles (except lateral rectus) numerous anterogradely tracer labeled PEs were found, as well as tracer-filled tendon organs. In addition the en plaque and en grappe motor endings, were also strongly tracer positive. Double immunolabeling revealed that all types of nerve endings including tendon organs were anterogradely labeled, except the thin tyrosine hydroxylase positive autonomic nerve fibers of the sympathetic system. No anterograde labeling was found within the trigeminal ganglia. These results suggest that the somata of palisade endings are located within the brainstem, in or around the oculomotor nuclei, and confirm several previous studies. In how far the multiple nerve endings of non-twitch muscle fibers and the PEs form an anatomical entity with one parent soma in the periphery of the oculomotor nuclei was studied by morphological and histochemical analysis of the peripheral neurons around the motor nuclei. This data revealed two populations: one group of cholinergic multipolar neurons represent the motor neurons supplying the multiple motor innervation, one group of round calretinin positive cholinergic neurons giving rise to PEs. If the palisade endings do have a sensory function, then their cell body location amongst the non-twitch motor neurons would be an ideal location to control the tension in the non-twitch extraocular muscle fibers, and the data brought together in this thesis points strongly in this direction. To fully establish this hypothesis more physiological experimental data is required.

Microalgae have higher growth rates and higher lipid content than terrestrial plants and the yield per unit area is even higher by several orders of magnitude. Furthermore, the production of microalgae does not compete for fertile land for food production. Therefore, microalgae are in the focus of research for biodiesel production, nutritional supplements and aquaculture approaches. However, after almost half a century of research the full promise of microalgae as a feedstock for biofuel production has remained largely unfulfilled. My research was motivated by the obvious gaps in the application of ecological pros of microalgae. DIVERSITY-PRODUCTIVITY RELATIONSHIPS: THE ROLE OF DIVERSITY FOR MICROALGAL LIPID PRODUCTION The relationship between diversity and productivity within terrestrial and algal primary producers has been well documented in ecology. However, the importance of diversity for lipid production for biofuel remains limited. Hence, I set out to investigate, experimentally, whether diversity may also affect lipid production in microalgae. Microalgae from all major algal groups were grown in a large number of treatments differing in their diversity level. Additionally, I compared the growth and lipid production of laboratory communities with the lipid production of natural lake and pond phytoplankton communities along a diversity gradient. This comparison showed that the lipid production of selected laboratory monocultures was not significantly higher than that of natural phytoplankton communities. The lipid production in general increased with increasing diversity in both natural and laboratory microalgal communities. The underlying reason for the observed ‘diversity-productivity’ relationship seems to be resource use complementarity. Additionally, a very important observation was that diversity also influences the specific lipid production of each microalgae in the high diverse communities. DIVERSITY- LIGHT- LIPID RELATIONSHIPS: LIPID PRODUCTION IN THE RIGHT LIGHT The knowledge about the relationship between diversity and biomass/lipid production in primary producer communities for biofuel production is underestimated. However, basic ecological research studies on the growth of microalgal communities provide evidence of a positive relationship between diversity and biomass production and show that the observed positive diversity-productivity-relationships are related to an increase in the efficiency of light use by diverse microalgal communities. I cultivated microalgae from all major freshwater algal groups in treatments that differed in their species richness and functional group richness. Polycultures with high functional group richness showed higher light use and algal lipid content with increasing species richness. Additionally, I could show a clear correlation between light use and lipid production in functionally diverse communities. Therefore, a powerful and cost effective way to improve biofuel production might be accomplished by incorporating diversity related resource-use-dynamics into algal biomass production. DIVERSITY AND FOOD QUALITY: ADVANTAGES FOR AQUACULTURE FOOD WEBS Determining the factors that control the energy transfer at the plant-animal interface is a key issue in ecology, because this transfer is highly variable and despite its global importance it is still not well understood. Food quality of primary producers seems to be a crucial factor influencing the transfer efficiency towards higher trophic levels. One major aspect of food quality is the biomass fatty acid composition in terms of essential ω3-polyunsaturated fatty acids (ω3-PUFAs) of primary producers, because all animals are incapable to synthesize them de novo. However, the influence of diversity on phytoplankton food quality in terms of lipid composition (e.g. ω3-PUFAs) remains unclear. I tested via a series of experiments controlled for diversity how the diversity of microalgal communities influences their fatty acid composition. My study shows the significant influence of diversity of primary producer communities on their fatty acid composition; especially on essential ω3-PUFA content. MICROALGAL BIOMASS CONTROL VIA GRAZING: IMPACT OF MICROALGAL SIZE The direction and strength of phytoplankton community responses to zooplankton grazing most probably depend on the size of phytoplankton species. To examine the influence of migrating (diel vertical migration, DVM) and non migrating zooplankton communities on different sized phytoplankton communities, I designed an experiment where I manipulated the size distribution of a natural phytoplankton community a priori in field mesocosms. Comparison of “migration” and “no migration” zooplankton treatments showed that nutrient availability and total phytoplankton biovolume were higher in “no migration” treatments with phytoplankton communities comprising mainly small algae and in “migration” treatments with phytoplankton communities of a broader size spectrum of algae. Additionally my results showed experimentally that food size selection and migration behavior of Daphnia hyalina can cause a shift from small sized microalgae towards larger species. NEW CULTIVATION TECHNIQUES FOR BIOMASS AND LIPID YIELD OPTIMIZATION IN MICROALGAE For the installation of infrastructure for the large-scale production of biofuel from microalgae is essential to establish cultivation methods that maximize lipid production but which are also economically viable in terms of energy demand and resource supply. For this purpose, I compared different cultivation systems (semi-batch, continuous) to optimize simultaneously growth and biomass lipid content of Botryococcus braunii. To enhance both, biomass accumulation and lipid production at the same time I further investigated a two-stage cultivation method to replace one stage semi-batch cultivation systems. In the first step of this cultivation method a full growth medium allows an enhancement of biomass accumulation. In the next step, the culture was transferred into nitrogen limited growth medium, where a further accumulation of photosynthetic products in the form of lipids occurred. Two-stage cultivation cultures resulted in higher nutrient specific biomass production and lipid content of B. braunii compared to one stage cultivation. If a continuous cultivation of cultures with high biomass in stage one can be assured, an almost constant supply of huge amounts of algae with even high lipid content in the second step could be guaranteed. My results clearly show that a better understanding of general ecological principles for biomass and lipid production of microalgae provides a cost effective and environmental friendly way to cultivate high yielding microalgal communities for commercial approaches. The enhancement of the yield efficiency of lipid production in diverse microalgal communities would be difficult to do only by technical means such as increasing resource supply. In addition, increasing the supply of resources is usually correlated with high energy requirements and therefore cost intensive. It is therefore important for biomass production systems to utilize all possible ecological options to increase the efficiency of the use of the supplied resources by integrating basic ecological principles into the cultivation systems.