Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Information, risk and trust in the food chain : Ex-ante valuation of consumer willingness to pay for beef quality information using the contingent valuation method

The safety of food has become an increasingly interesting issue to consumers and the media. It has also become a source of concern, as the amount of information on the risks related to food safety continues to expand. Today, risk and safety are permanent elements within the concept of food quality. Safety, in particular, is the attribute that consumers find very difficult to assess. The literature in this study consists of three main themes: traceability; consumer behaviour related to both quality and safety issues and perception of risk; and valuation methods. The empirical scope of the study was restricted to beef, because the beef labelling system enables reliable tracing of the origin of beef, as well as attributes related to safety, environmental friendliness and animal welfare. The purpose of this study was to examine what kind of information flows are required to ensure quality and safety in the food chain for beef, and who should produce that information. Studying the willingness to pay of consumers makes it possible to determine whether the consumers consider the quantity of information available on the safety and quality of beef sufficient. One of the main findings of this study was that the majority of Finnish consumers (73%) regard increased quality information as beneficial. These benefits were assessed using the contingent valuation method. The results showed that those who were willing to pay for increased information on the quality and safety of beef would accept an average price increase of 24% per kilogram. The results showed that certain risk factors impact consumer willingness to pay. If the respondents considered genetic modification of food or foodborne zoonotic diseases as harmful or extremely harmful risk factors in food, they were more likely to be willing to pay for quality information. The results produced by the models thus confirmed the premise that certain food-related risks affect willingness to pay for beef quality information. The results also showed that safety-related quality cues are significant to the consumers. In the first place, the consumers would like to receive information on the control of zoonotic diseases that are contagious to humans. Similarly, other process-control related information ranked high among the top responses. Information on any potential genetic modification was also considered important, even though genetic modification was not regarded as a high risk factor.

Beasts on Fields. Human-Wildlife Conflicts in Nature-Culture Borderlands

Human-wildlife conflicts are today an integral part of the rural development discourse. In this research, the main focus is on the spatial explanation which is not a very common approach in the reviewed literature. My research hypothesis is based on the assumption that human-wildlife conflicts occur when a wild animal crosses a perceived borderline between the nature and culture and enters into the realms of the other. The borderline between nature and culture marks a perceived division of spatial content in our senses of place. The animal subject that crosses this border becomes a subject out of place meaning that the animal is then spatially located in a space where it should not be or where it does not belong according to tradition, custom, rules, law, public opinion, prevailing discourse or some other criteria set by human beings. An appearance of a wild animal in a domesticated space brings an uncontrolled subject into that space where humans have previously commanded total control of all other natural elements. A wild animal out of place may also threaten the biosecurity of the place in question. I carried out a case study in the Liwale district in south-eastern Tanzania to test my hypothesis during June and July 2002. I also collected documents and carried out interviews in Dar es Salaam in 2003. I studied the human-wildlife conflicts in six rural villages, where a total of 183 persons participated in the village meetings. My research methods included semi-structured interviews, participatory mapping, questionnaire survey and Q- methodology. The rural communities in the Liwale district have a long-history of co-existing with wildlife and they still have traditional knowledge of wildlife management and hunting. Wildlife conservation through the establishment of game reserves during the colonial era has escalated human-wildlife conflicts in the Liwale district. This study shows that the villagers perceive some wild animals differently in their images of the African countryside than the district and regional level civil servants do. From the small scale subsistence farmers point of views, wild animals continue to challenge the separation of the wild (the forests) and the domestics spaces (the cultivated fields) by moving across the perceived borders in search of food and shelter. As a result, the farmers may loose their crops, livestock or even their own lives in the confrontations of wild animals. Human-wildlife conflicts in the Liwale district are manifold and cannot be explained simply on the basis of attitudes or perceived images of landscapes. However, the spatial explanation of these conflicts provides us some more understanding of why human-wildlife conflicts are so widely found across the world.

Participation or further exclusion? : Contestations over forest conservation and control in the East Usambara Mountains, Tanzania

This Ph.D. thesis Participation or Further Exclusion? Contestations over Forest Conservation and Control in the East Usambara Mountains, Tanzania describes and analyses the shift in the prevailing discourse of forest and biodiversity conservation policies and strategies towards more participatory approaches in Tanzania, and the changes in the practises of resource control. I explore the scope for and limits to the different actors and groups who are considered to form the community, to participate in resource control, in a specific historical and socio-economic context. I analyse whether, how and to which extent the targets of such participatory conservation interventions have been able to affect the formal rules and practices of resource control, and explore their different responses and discursive and other strategies in relation to conservation efforts. I approach the problematic through exploring certain participatory conservation interventions and related negotiations between the local farmers, government officials and the external actors in the case of two protected forest reserves in the southern part of the East Usambaras, Tanzania. The study area belongs to the Eastern Arc Mountains that are valued globally and nationally for their high level of biodiversity and number of endemic and near endemic species. The theoretical approach draws from theorising on power, participation and conservation in anthropology of development and post-structuralist political ecology. The material was collected in three stages between 2003 and 2008 by using an ethnographic approach. I interviewed and observed the actors and their resource use and control practices at the local level, including the representatives of the villagers living close to the protected forests and the conservation agency, but also followed the selected processes and engaged with the non-local agencies involved in the conservation efforts in the East Usambaras. In addition, the more recent processes of change and the actors strategies in resource control were contextualised against the social and environmental history of the study area and the evolvement of institutions of natural resource control. My findings indicate that the discourse of participation that has emerged in global conservation policy debate within the past three decades, and is being institutionalised in the national policies in many countries, including Tanzania, has shaped the practices of forest conservation in the East Usambaras, although in a fragmented and uneven way. Instrumental interpretation of participation, in which it is to serve the goals of improving the control of the forest and making it more acceptable and efficient, has prevailed among the governmental actors and conservation organisations. Yet, there is variation between the different projects and actors promoting participatory conservation regarding the goals and means of participation, e.g. to which extent the local people are to be involved in decision-making. The actors representing communities also have their diverse agendas, understandings and experiences regarding the rationality, outcomes and benefits of being involved in forest control, making the practices of control fluid. The elements of the exclusive conservation thinking and practices co-exist with the more recent participatory processes, and continue to shape the understandings and strategies of the actors involved in resource control. The ideas and narratives of the different discourses are reproduced and selectively used by the parties involved. The idea of forest conservation is not resisted as such by most of the actors at local level, quite the opposite. However, the strict regulations and rules governing access to resources, such as valuable timber species, continue to be disputed by many. Furthermore, the history of control, such as past injustices related to conservation and unfulfilled promises, undermines the participation of certain social groups in resource control and benefit sharing. This also creates controversies in the practices of conservation, and fuels conflicts regarding the establishment of new protected areas. In spite of this, the fact that the representatives of the communities have been invited to the arenas where information is shared, and principles and conditions of forest control and benefit sharing are discussed and partly decided upon, has created expectations among the participants, and opened up opportunities for some of the local actors to enhance their own, and sometimes wider interests in relation to resource control and the related benefits. The local actors experiences of the previous government and other interventions strongly affect how they position themselves in relation to conservation interventions, and their responses and strategies. However, my findings also suggest, in a similar way to research conducted in some other protected areas, that the benefits of participation in conservation and resource control tend to accrue unevenly between different groups of local people, e.g. due to unequal access to information and differences in their initial resources and social position.

Food intake, growth and social interactions of signal crayfish, Pacifastacus leniusculus (Dana)

The knowledge about the optimal rearing conditions, such as water temperature and quality, photoperiod and density, with the understanding of animal nutritional requirements forms the basis of economically stable aquaculture for freshwater crayfish. However, the shift from a natural environment to effective culture conditions induces several changes, not only at the population level, but also at the individual level. The social contacts between conspecifics increase with increasing animal density. The competition for limited resources (e.g. food, shelter, mates) is more severe with the presence of agonistic behaviour and may lead to unequal distribution of these. The objectives of this study were to: 1) study the distribution of a common food resource between communally reared signal crayfish (Pacifastacus leniusculus) and to assign potential feeding hierarchy on the basis of individual food intake measurements, 2) explore the possibilities of size distribution manipulations to affect population dynamics and food intake to improve growth and survival in culture and 3) study the effect of food ration and spatial distribution on food intake and to explore the effect of temperature and food ration on growth and body composition of freshwater crayfish. The feeding ranks between animals were assigned with a new method for individual food intake measurement of communally reared crayfish. This technique has a high feasibility and a great potential to be applied in crayfish aquaculture studies. In this study, signal crayfish showed high size-related variability in food consumption both among individuals within a group (inter-individual) and within individual day-to-day variation (intra-individual). Increased competition for food led to an unequal distribution of this resource and this may be a reason for large growth differences between animals. The consumption was significantly higher when reared individually in comparison with communal housing. These results suggest that communally housed crayfish form a feeding hierarchy and that the animal size is the major factor controlling the position in this hierarchy. The optimisation of the social environment ( social conditions ) was evaluated in this study as a new approach to crayfish aquaculture. The results showed that the absence of conspecifics (individual rearing vs. communal housing) affects growth rate, food intake and the proportion of injured animals, whereas size variation between animals influences the number and duration of agonistic encounters. In addition, animal size had a strong influence on the fighting success of signal crayfish reared in a social milieu with a wide size variation of conspecifics. Larger individuals initiated and won most of the competitions, which suggests size-based social hierarchy of P. leniusculus. This is further supported by the fact that the length and weight gain of smaller animals increased after size grading, maybe because of a better access to the food resource due to diminished social pressure. However, the high dominance index was not based on size under conditions of limited size variation, e.g. those characteristic of restocked natural populations and aquaculture, indicating the important role of behaviour on social hierarchy.

Communities of wood-inhabiting fungi : Ecological requirements and responses to forest management and fragmentation

Despite much research on forest biodiversity in Fennoscandia, the exact mechanisms of species declines in dead-wood dependent fungi are still poorly understood. In particular, there is only limited information on why certain fungal species have responded negatively to habitat loss and fragmentation, while others have not. Understanding the mechanisms behind species declines would be essential for the design and development of ecologically effective and scientifically informed conservation measures, and management practices that would promote biodiversity in production forests. In this thesis I study the ecology of polypores and their responses to forest management, with a particular focus on why some species have declined more than others. The data considered in the thesis comprise altogether 98,318 dead-wood objects, with 43,085 observations of 174 fungal species. Out of these, 1,964 observations represent 58 red-listed species. The data were collected from 496 sites, including woodland key habitats, clear-cuts with retention trees, mature managed forests, and natural or natural-like forests in southern Finland and Russian Karelia. I show that the most relevant way of measuring resource availability can differ to a great extent between species seemingly sharing the same resources. It is thus critical to measure the availability of resources in a way that takes into account the ecological requirements of the species. The results show that connectivity at the local, landscape and regional scales is important especially for the highly specialized species, many of which are also red-listed. Habitat loss and fragmentation affect not only species diversity but also the relative abundances of the species and, consequently, species interactions and fungal successional pathways. Changes in species distributions and abundances are likely to affect the food chains in which wood-inhabiting fungi are involved, and thus the functioning of the whole forest ecosystem. The findings of my thesis highlight the importance of protecting well-connected, large and high-quality forest areas to maintain forest biodiversity. Small habitat patches distributed across the landscape are likely to contribute only marginally to protection of red-listed species, especially if habitat quality is not substantially higher than in ordinary managed forest, as is the case with woodland key habitats. Key habitats might supplement the forest protection network if they were delineated larger and if harvesting of individual trees was prohibited in them. Taking the landscape perspective into account in the design and development of conservation measures is critical while striving to halt the decline of forest biodiversity in an ecologically effective manner.

Harmful algae in the planktonic food web of the Baltic Sea

This study deals with algal species occurring commonly in the Baltic Sea: haptophyte Prymnesium parvum, dinoflagellates Dinophysis acuminata, D. norvegica and D. rotundata, and cyanobacterium Nodularia spumigena. The hypotheses are connected to the toxicity of the species, to the factors determining toxicity, to the consequences of toxicity and to the transfer of toxins in the aquatic food web. Since the Baltic Sea is severely eutrophicated, the fast-growing haptophytes have potential in causing toxic blooms. In our studies, the toxicity (as haemolytic activity) of the haptophyte P. parvum was highest under phosphorus-limited conditions, but the cells were toxic also under nitrogen limitation and under nutrient-balanced growth conditions. The cellular nutrient ratios were tightly related to the toxicity. The stoichiometric flexibility for cellular phosphorus quota was higher than for nitrogen, and nitrogen limitation led to decreased biomass. Negative allelopathic effects on another algae (Rhodomonas salina) could be observed already at low P. parvum cell densities, whereas immediate lysis of R. salina cells occurred at P. parvum cell densities corresponding to natural blooms. Release of dissolved organic carbon from the R. salina cells was measured within 30 minutes, and an increase in bacterial number and biomass was measured within 23 h. Because of the allelopathic effect, formation of a P. parvum bloom may accelerate after a critical cell density is reached and the competing species are eliminated. A P. parvum bloom indirectly stimulates bacterial growth, and alters the functioning of the planktonic food web by increasing the carbon transfer through the microbial loop. Our results were the first reports on DSP toxins in Dinophysis cells in the Gulf of Finland and on PTX-2 in the Baltic Sea. Cellular toxin contents in Dinophysis spp. ranged from 0.2 to 149 pg DTX-1 cell-1 and from 1.6 to 19.9 pg PTX-2 cell-1 in the Gulf of Finland. D. norvegica was found mainly around the thermocline (max. 200 cells L-1), whereas D. acuminata was found in the whole mixed layer (max. 7 280 cells L-1). Toxins in the sediment trap corresponded to 1 % of DTX-1 and 0.01 % PTX-2 of the DSP pool in the suspended matter. This indicates that the majority of the DSP toxins does not enter the benthic community, but is either decomposed in the water column, or transferred to higher trophic levels in the planktonic food chain. We found that nodularin, produced by Nodularia spumigena, was transferred to the copepod Eurytemora affinis through three pathways: by grazing on filaments of small Nodularia, directly from the dissolved pool, and through the microbial food web by copepods grazing on ciliates, dinoflagellates and heterotrophic nanoflagellates. The estimated proportion of the microbial food web in nodularin transfer was 22-45 % and 71-76 % in our two experiments, respectively. This highlights the potential role of the microbial food web in the transfer of toxins in the planktonic food web.