1. Principal investigator, title of research project, site of research, duration of project

Principal investigator (PI): Dr. Kaisa Haukka

Title of the project: Cyanobacteria in Tanzania – from harmful mass occurrances to a valuable natural resource

Site of research:

1. Division of Microbiology, Department of Food and Environmental Sciences, University of Helsinki, Finland

2. Faculty of Aquatic Sciences and Technology, University of Dar es Salaam, Tanzania3. Department of Veterinary Medicine and Public Health, Sokoine University of

Agriculture, Tanzania

Duration of the project: 48 months, 1.9.2013 – 31.8.2017

2. Background

significance of the research nationally and internationally previous research pertaining to the topic (in brief) and how the research project links

to it

how the research project links to other research by the PI or research team

Tanzania remains among the least developed countries in the world, despite fifty years of political independence, with two thirds of its citizens living below the poverty line on less than $1.25 a day. The country, however, fully subscribes to global efforts, such as the Brussels Programme of Action (BPoA) for the Least Developed Countries, of eradicating poverty and alleviating the social conditions of the poor in terms health, education, good governance, gender and environmental conservation. Tanzania is one of the Finland's seven long-term partner countries in development cooperation (source: Ministry of Foreign Affairs of Finland, http://formin.finland.fi). In Tanzania, Finland has concentrated on the natural resources, local government reform, and the development of economic activities. Reducing poverty requires balanced economic growth that also benefits poor and vulnerable population groups, as well as the sustainable use and utilisation of natural resources. Our project will target some of these questions.

I our project we study cyanobacteria that cause harmful mass occurrances in Tanzania. These so-called cyanobacterial blooms have been occurring at increasing frequency and endanger some of the most important income sources for the local people. Also, toxins

produced by cyanobacteria can cause direct adverse health effects to humans and domestic as well as wild animals. Toxins produced by cyanobacteria include the hepatotoxins microcystin and nodularin, the cytotoxin cylindrospermopsin, the neurotoxins anatoxin and saxitoxin and the dermatotoxin lyngbyatoxin (Dittmann et al. 2012). On the other hand, the secondary metabolites produced by cyanobacteria are rising interest as a source of diverse bioactivites of unique structure with potential pharmacological implications (Welker & von Döhren, 2006). A major part of the cyanobacterial secondary metabolites are peptides or possess peptidic substructures. The majority of these oligopeptides are synthesized by NRPS non-ribosomal peptide synthetase or NRPS/PKS (polyketide synthase) hybrid pathways, but also new synthetic pathways are currently being discovered. Marine cyanobacteria, especially those belonging to genus Lyngbya (Moorea) have recently been found to be a vast source of potentially important natural products (Gerwick & Moore, 2012). Fresh water environments have gained less interest in this context, but due to the great natural variation in their environmental conditions, they need to be explored more closely. Also terrestrial cyanobacteria have recently been found to produce a variety of peptides (Kaasalaineen et al. 2012). In the proposed project we will concentrate our studies on two important Tanzanian aquatic environments, where the toxic cyanobacterial mass occurrences cause considerable harmful effects but which, on the other hand, could turn out be real hot spots for cyanobacteria producing useful bioactive compounds.

Seaweed farming is an important source of income for women and families living on Zanzibar along the Western Indian Ocean coast line and it contributes approx. 25% to Zanzibar’s Gross Domestic Product. Seaweed Eucheuma denticulatum is harvested from the inter-tidal area of the sea shore and sold for production of carrageenan, which is used e.g. as a gelling-agent in food industry and cooking. Recently proliferation of cyanobacterial species of Lyngbya majuscula has caused serious harm to this local activity. In February-March 2012 between villages of Paje and Jambiani the Lyngbya (mashava in local language) blooms were so extensive that women could not attending their seaweed farms for fear of the inflammatory effect of the cyanobacterium. Some people claimed that even the air during this time particularly in the evening hours was irritating. Though they have been encountering mashava in the past years the women claimed that the cyanobacterium is appearing in extensive quantities in recent years compared to before. We speculate that this is due to e.g. overloading of coastal waters with nutrients from the many tourist hotels. Following fromation of annual Lyngbya blooms would give us information on the causes for their proliferation and information needed to prevent their formation.

Lyngbya has indeed been reported to produce many toxic substances. Lyngbyatoxin is a highly inflammatory and vesicatory substance (Cardellina et al. 1979). Also, Lyngbya is a well-known producer of complex natural products such as barbamide (Chang et al. 2002) and curacin A (Chang et al. 2004). Previously obtained 8.5 Mbp draft genome supports approximately 293 kbp of DNA sequence related to secondary metabolism, far too low to

encode the full suite of reported Lyngbya metabolites of approximately 200 different compounds (Jones et al. 2011). On the other hand, Lyngbya majuscule has been found to contain several phylogenetically distant species and recently strains in one main of the lineages has been renamed as Moorea producens and Moorea Bouillonii (Engene et al. 2012). These results suggest that the rich complement of natural products in Lyngbya is actually strain specific and studies on Lyngbya from various environments might bring out yet new compounds.

The second area of interest in our study will be the Momela lakes in Arusha National park, in the northern part of Tanzania. The Momela lakes are shallow alkaline saline lakes. They are made up of seven lakes, being big Momela, small Momela, El Kekhotoito, Kusare, Rishateni, Lekandiro and Tulusia. All seven lakes are mainly fed by separate underground water sources. Due to the varying mineral content of these underground sources (the salinity values vary from less than 5 to more than 20 ppt), each lake supports a different type of algae growth, resulting in uniquely differently colored lakes. Because these lakes are alkaline, the water is not utilized by animals for drinking, but they do however attract a wide variety of African bird life, particularly flamingos. The Lesser Flamingo occur in the area in spectacular assemblages, however, in recent years several mass mortalities of these birds have been reported (Nonga et al. 2011). Several environmental factors, such as heavy metals and pesticides have been suspected as causes of the mortalities, but also microcystins produced by cyanobacteria have been indicated. Nonga et al (2011) conducted pathological, microbiological and microcystin analyses to the 11 Lesser Flamingo carcasses and found high levels of microcystins in the livers, variants MC_LR, -YR and -RR. Several known toxic cyanobacteria genera such as Anabaena, Anabaenopsis, Oscillatoria and Nostoc have been reported from the saline lakes. We believe that due to the extraordinary growth conditions in the alkaline lakes, the cyanobacterial strains produce interesting variety of secondary metabolites. The gradient of salinity and alkalinity conditions on the Momela lakes will also offer us a unique insight in studying the ecological factors influencing the growth and secondary metabolite production of phylogenetically related cyanobacterial isolates. Besides a possibility of being a hot spot for new cyanobacterial bioactive compounds, the lakes are an important study site for their importance to the local human activities. Natural parks offer an important source of income to the locals and the causes of mass mortality of birds and other wildlife needs to be researched thoroughly in order to prevent them in the future.

Cyanobacteria research has been conducted in Tanzania previously but it has been sporadic. In their PhD thesis projects Drs. Charles Lugomela (2002), Karolina Bauer (2007) and have analysed cyanobacterial diversity and productivity in terms of carbon and nitrogen fixation in Zanzibar coastal waters. In addition, Dr. Mariam Hamisi (2010) studied the ecology of

epiphytic cyanobacteria on seagrasses and their role as nitrogen fixers in the same area. Dr. Hezron Nonga, on the other hand, has studied in his PhD thesis (2011) the lake ecosystems in northern Tanzania and the occurrence of microcystins produced by cyanobacterial blooms. Thus in Tanzania there are scientists, who have experience in cyanobacterial research. However, they all have their current teaching and x activities, C. Lugomela and T. Lyimo at the University of Dar es Salaam, M. Hamisi at the University of Dodoma and H. Nonga at the Sokoine University of Agriculture and very limited resources to continue their cyanobacterial research activities. Funding for our proposal would mean that they would be able to form a joint cyanobacterial resource initiative in Tanzania, joining the sources of expertise existing in the country and bringing it to a new internationally recognized level.

The Finnish partner in the program comprises the Cyanobacterial study group at the Department of Food and Environmental Sciences at the University of Helsinki. The group is led by Prof. Kaarina Sivonen, who has twice held the position of the Academy Professorship and been a co-leader in two Academy Centres of Excellency (other group members are described in chapter 6). Her group has a long reaching history in studying the taxonomy, phylogeny, physiology and ecology of cyanobacterial strains isolated from the Finnish lakes and the Baltic Sea. Molecular tools have been used to study the toxins, their production systems and detection techniques, such as PCR, quantitative-PCR as well as microarrays. In recent years the emphasis of the research has shifting towards studying the variety of bioactive compounds produced by cyanobacteria and their biosynthetic pathways. This work combines the use of mass spectrometry and genome mining. This work has led into discovery of new families of protease inhibitors and antifungal peptides as well as new enzymatic machinery for making cyclic peptides. E.g. new methods to screen cyanobacteria for protein kinase inhibitors or modulating activities has been developed and several interesting compounds have been detected (Fewer et al. 2011, Wang et al. 2011, Jokela et al. 2012, Leikoski et al. 2012). The Principal Investigator (PI) of the proposed project is Dr. Kaisa Haukka, who works as a senior scientist in the Cyanobacterial group and has plenty of previous experience on collaborative projects with the scientists from the developing countries (see chapter 6 for details).

3. Objectives

research objectives hypotheses

justifications for how the proposed research ties in with the call and its objectives, if the call has a specific objective (e.g. research programme calls)

In our research we will concentrate on detection, isolation and characterization of cyanobacterial strains and detection and identification of their secondary metabolites in two distinctive aquatic ecosystems plus selected terrestrial hotspots in Tanzania. For the newly identified secondary metabolites the genetic constitution and the regulation mechanism are looked for.

The detailed objectives for the study are to determine:

1. the phylogenetic diversity of a) Lyngbya (Moorea) growing in the marine environment (shallow coastline of Zanzibar), b) cyanobacterial community present at different times of the year in the alkaline, saline lakes (Momela soda lakes), and c) terrestrial isolates.

2. the secondary metabolites produced in the aquatic cyanobacterial blooms and by the isolates from different environments.

3. the genes that are responsible for production of newly discovered metabolites. 4. the physic-chemical factors that affect the cyanobacterial growth and production of the

secondary metabolites.

Hypothesis 1: The cyanobacteria isolated from the extra-ordinary environmental conditions in Tanzania produce bioactive compounds new to science.

Hypothesis 2: The occurrence of the bioactive compounds is correlated with the cyanobacterial species present and with the physico-chemical conditions in the environment.

The research is proposed under the Development research call since:1. we want to conduct high standard science on a very actual topic together with the

scientists from a developing country.2. we want to promote human resources development in a developing country by

educating local students and scientists and offering them a chance to retain the property rights when valuable natural resources (e.g. pharmalogically interesting bioactive compounds) are detected.

3. we want to increase understanding of the local environmental problems (cyanobacterial mass occurrances) inorder to find ways of combatting them thus improving also the human health and income possibilities.

Since the main emphasis of our project on Tanzanian aquatic environments, our project relates excellently also to the theme of the targeted funding “Water and development”.

4. Research methods and material, ethical issues

research methods, described so as to explain how they will contribute to answering the research questions/confirming the hypotheses or how they will support the chosen approach

research material

materials management plan (incl. electronic material): 1) how the research material will be obtained, 2) how it will be used, 3) how it will be stored and protected, 4) how its subsequent use will be facilitated, and 5) the rights of ownership and use

ethical issues (e.g. ethical governance procedures, informed consent, anonymity of subjects) and research permits or information on pending permit applications

risk management: critical points, alternative ways to implement the project

Research material includes samples of cyanobacterial blooms, which will be used for isolation of pure cyanobacterial strains and for direct measurement of bioactive compounds. The samples will be collected from the following sites:

- The Indian Ocean near the coast line of Zanzibar (benthic + net samples). The sites of the study will be the Villages of Bwejuu, Paje and Jambian in Unguja Island, Zanzibar. These villages are famous for seaweed farming and tourism on the Island.

- Momela Lakes in Arusha National Park, some 25 km from Arusha town will be the site for sampling cyanobacteria from Soda Lakes. Also some fish will be collected and dissected and their intestines will be monitored for the potentially toxic compounds.

- Additionally some interesting sites will be sampled to isolate symbiotic cyanobacteria, this includes collecting lichens growing in the Arusha or Zanzibarian forests/mangrove forests or water plants, macroalgae, tunicates, mussels or molluscs, which have been reported to be a rich source of new bioactive compounds (Gerwick & Moore, 2012).

The sample materials are taken to the laboratory at the University of Dar es Salaam, where a suitable cyanobacterial laboratory and culture collection will be established. Cyanobacterial samples from different environments can be very challenging to grow and the growth conditions need to be adapted to resemble the natural conditions, e.g. the salt concentration of the media can be varied. A following general method (Herfindal et al. 2005) will be modified accordingly: Cyanobacterial samples will be suspended in a solution of 0.9% NaCl (90 ml), with 0.114% Na5P3O10 and 0.002% Tween 80. After vigorous agitation (100 rpm for 1 h at 20 °C), 0.1 ml of the suspension is plated on agar plates (Z8X and Z8XS) without nitrogen source. The plates are incubated under continuous light (15 µmol m-2 s-1) and colonies are picked and streaked on fresh plates until pure cyanobacterial cultures are

obtained. We aim to obtain 200 pure cultures (at least unialgal if not axenic) during the project. The strains are identified based on the morphology according to Castenholz (2001). These strains will serve as a starting point for a new cyanobacterial culture collection in Tanzania that can be later amended and used for further studies both nationally and internationally.

To obtain enough cyanobacterial biomass for the analysis of the secondary metabolites, mass cultivation is done in 5-l Erlenmeyer flasks that contain 3 l of liquid medium and 1% of inoculum. Flasks are aerated with filter-sterilized compressed air and illuminated with day-light lamps 15 µmol m-2 s-1 at 20 °C. The cells are harvested by centrifugation or filtration after 20–60 days of culturing and freeze-dried. In the first instance, the freeze-dried samples will be sent to Finland for further analysis. The freeze dried cells are extracted with methanol and homogenized by shaking. The extracts are analyzed with high-performance liquid chromatography (HPLC) combined with a mass spectrometer (MS) (Agilent 1100 series LC/MSD with Ion trap XCT Plus and electrospray ion source). This set-up has been used widely in the Helsinki laboratory for detection and analysis of new chemical products produced by cyanobacteria (e.g. Jokela et al. 2012, Leikoski et al. 2012). Also the compounds can be measured directly from the bloom samples when the expression levels are high (viite?) The Tanzanian students will get familiar with all the LC-MS techniques during their visits to the Cyanobacterial group in Helsinki after which the system to analyze further samples is set up in Tanzania. At the Sokoine University of Agriculture (SUA) Tanzania, there is a HPLC which is currently used to analyze alkaloid compounds from plants. Fitted out with a photodiode array (PDA) detector, the machine can be used to precisely identify and quantify different types of bioactive compounds from cyanobacteria. Dr. Nonga from SUA has previous experience on analyzing microcystins from natural samples (Nonga et al. 2011, Miles et al. 2012).

When interesting new cyanobacterial compounds are detected, their exact formula will be solved by xxx. Based on the analysis of genetic structures responsible for similar kind of chemical compounds, the genes and expression systems behind the new compounds will be revealed. This can be done either using degenerative primers to search for related genes in isolated DNA or by subjecting the interesting strains for whole-genome sequencing, which allows screening for a variety of potential biosynthesis genes. As the price of the whole-genome sequencing keeps getting lower, an “unfinished” draft genome sequence might be the most cost effective way to get the necessary level of information for further studies (MacLean et al. 2009).

For the ecological analyses on the succession of the cyanobacterial populations in their natural conditions we will use both traditional detection methods, such as microscopy, and

molecular detection methods. Molecular methods that have been used in the past in the Cyanobacterial laboratory in Helsinki include use of e.g. PCR, qPCR, DGGE and microarrays (Rajaniemi et al. 2008, Rantala et al. 2008, Sipari et al. 2010). In addition, measuring the physico-chemical variables at each sampling point will make it possible to look for correlations between the environmental variables, cyanobacterial communities and the secondary compounds measurable in the environmental samples. This analysis will help in devising preventive actions for limiting the occurrence of harmful cyanobacterial occurrances.

No special ethical issues are involved in the project. All the necessary permissions for sampling will be obtained from the Arusha National Park administratives… etc. Considering the risk management we do not envisage any unsurmountable risks. Several research lines are included in the plan, thus securing that interesting results will be obtained. The culturing of pure cyanobacterial strains can be tricky and thus in the beginning of the project we need to put great emphasis on optimizing the culture conditions in order to make sure obtaining numerous isolates for the secondary metabolite studies.

5. Implementation: timetable, budget, distribution of work

timetable for the research justifications for the total cost estimate specified on the application, by type of

expenditure (budget table with justifications). Costs that do not pass through the books of the site of the research shall not be included in total project costs.

names, tasks and salary costs (with justifications) of persons working within the project. If the names are not known, enter N.N. Also include an estimate of the PI’s working hours on the project.

if funding (max. 12 months) is applied for to cover the salary of the principal investigator: give research-specific reasons for the funding

salary plan for the PI, covering the funding period, if the PI does not have a permanent employment relationship

6. Researchers, research environment and mobility 

merits of research team members write about the Tanzanian team site of research and any tangible support it offers the project, including available

equipment write about the Tanzanian site(s)

the research plan shall also describe the significance of the following items for project implementation (a detailed account on these items is also asked in the online application):

o key national and international collaboration and distribution of work (“Partners” on the online application)

o other partners (e.g. Strategic Centres for Science, Technology and Innovation), form of cooperation, description of how the project will benefit from the cooperation (“Partners” on the online application)

o use of international and national research infrastructure, description of how the project will benefit from it (“Infrastructures” on the online application)

o justifications for mobility: how the visits or work periods elsewhere contribute to research plan implementation.

Under “Mobility” on the online application, give a detailed description of possible mobility within the project: to and from Finland or between organisations in Finland. The description shall include information on the objectives and duration of the visits and on whether the visits have been agreed.

Below is a copy-pasted text from another application, I will shorten it considerably. You can add a short description on your own careers (see Charles’s below).

The applicant Kaisa Haukka has versatile experience on microbiological and microbial ecology research. After receiving her PhD in 1997 she studied the genetic diversity of aquatic microbial communities in Finland as a post doc in Prof. Sivonen’s laboratory. In 2000 she also received a post-doctoral grant from the Academy of Finland for her project. Retrospectively, viewing the articles published from the work done at that time, the publications have been well cited (up to 49 times). These publications have been highlighted in the attached list of publications. The h-index reflecting the citations for all the publications by the applicant, was last year 11, but it is showing a strong increasing trend.

After the Academy post-doc funding and two maternity leaves Kaisa Haukka moved to work at the National Public Health Institute (KTL), currently called National Institute for Health and Welfare (THL). There she worked on reference functions concerning enteropathogenic bacteria and also conducted research on the topic. Together with her unit leader Research Prof. Anja Siitonen she did well in attracting research funding to THL. As a principal applicant she obtained a four-year project funding (2008 - 2011) from The Academy of Finland to lead a project called “Epidemiology of water- and foodborne bacterial infections

in Burkina Faso (West Africa) - strategies for sustainable national public health management”. This project was a success and several manuscripts from the project are still under preparation. However, according to the new strategic guidelines and the strong budget constraint, microbiological research activities at THL have been reduced and limited to certain main research lines most important for the public health protection. Under these circumstances, the applicant could not see any continuation possibilities for her research career at THL and she returned to the University of Helsinki in the beginning of 2012. She is now continuing her aquatic microbial research line as a University Researcher in the Cyanobacteria research group, where she is especially keen on expanding her technical repertoire to the very latest techniques of the molecular microbiology, namely the proteomics, transcriptomics and whole-genome analysis. Next, her aim is set a research group of her own at the Department of Food and Environmental Sciences, University of Helsinki.

In her own research group Kaisa Haukka would like to combine the expertise she has gained from the different fields of microbiology during her career. Her group would concentrate on environmental health related microbiological issues, especially on the harmful micro-organisms in the water environments. These microbes include the cyanobacteria as well as the enteropathogenic bacteria. Cyanobacterial growth in the natural waters is suspected to support the survival of many enterobacteria, such as campylobacter and pathogenic E. coli. These bacteria can also form resting cells, known as viable but not culturable (VBNC) cells (Oliver, 2010). In the future, the applicant would like to use the same methods as to akinetes to study the survival strategies of the pathogens in aquatic environments. Currently she is still supervising, together with Prof. Anja Siitonen from THL, the PhD thesis work of an ABS Graduate school grantee, Outi Martikainen. At the moment, in Outi’s work we are undertaking a whole genome sequencing of 6 enterohemorrhagic E. coli genomes in order to study the diversity and genomic location of some unusual virulence genes that might expand their potential virulence ability. Sequencing will be done in collaboration with Dos. Petri Auvinen’s genome sequencing laboratory at the Institute of Biotechnology. The wealth of genomic information that can be anticipated will offer a good chance to continue fruitful collaboration with THL also in the future.

In the future Kaisa Haukka would also like to continue her collaboration with the researchers from the developing countries. She is planning to submit a new Academy of Finland developing project application, this time concentrating on the occurrence and potential health risks posed by cyanobacteria, but also looking for possibly useful new compounds in local cyanobacteria. Similarly to the water- and foodborne enteric bacteria her project studied previously, also cyanobacteria can cause serious threat to people forced to use poor-quality water sources in the tropical climate. In the developing country projects,

besides obtaining scientifically sound results, it is of great importance to have emphasis on the education and close collaboration with the local scientists.

As can be seen from above, Kaisa Haukka has a long and versatile research experience, which is reflected in her CV and list of publications. However, now back to the University of Helsinki, she has to start from the scratch in establishing her own research group. She is actively searching for a more permanent position as a lecturer or professor at the University. The University three-year research funding would form a solid foundation to these endeavours. This application concentrates on the high-tech research, which can lead into new discoveries and innovations in science. The applicant is very excited about the future research prospects in microbiology offered by the new technical development and by the great development to increase collaboration, core facilities and support networks at the Viikki campus and elsewhere at the University of Helsinki. Therefore she truly wishes to have a chance to resettle and develop her own, strong, career-long research line specifically at the University of Helsinki.

Dr. Charles Lugomela, PhD, is a Senior Lecturer at the University of Dar es Salaam. He has over 15 years experience in tropical marine ecology. His PhD dissertation was on Cyanobacterial diversity and productivity in coastal areas of Zanzibar, Tanzania, obtained from Stockholm University in 2002. Since then he has maintained his interest in the areas of are on nutrient dynamics, food-web interactions, plankton and microphytobenthic eco-physiology. His duties as a senior lecturer at the University of Dar es Salaam requires him to teach, conduct research and provide consultancy services, the latter two obligations also entailing travelling within the country as well as abroad.

Also, we are interested in the akinete differentiation and germination in filamentous cyanobacteria. These resting cells are likely to play an important role in the life cycle of cyanobacterial blooms. We want to use proteomics and transcriptomics techniques in combination with whole-genome analysis to reveal the cellular changes during the cell differentiation.

7. Researcher training and research careers (when the funding applied for goes to a research team)

researcher training, including arrangements for teaching and supervision write about the situation in Tanzania considering the MSc/PhD students involved in the project

doctoral studies within the PI’s team

promotion of careers of researchers to be hired to the project

promotion of gender equality within the project

8. Expected results and possible risks

expected scientific and societal impact of the research potential for scientific breakthroughs and for increasing the capacity for renewal of

science and research, identification of risks I have many ideas to write here but you can add even more!

applicability and feasibility of the research results

publishing of results and raising awareness among potential end-users, the scientific community and the general public

However, Lyngbya research offers also great opportunity to turn the nuisance substance into a valuable natural resource. the factors that affect the Lyngbya growth. We measure the physic-chemical parameters and look for correlations. This could help in giving some suggestions on the primary target areas in devising preventive actions. Protein kinase inhibitors show promise in cancer treatments and they could also lead into development of new drugs against parasitic protozoa causing tropical diseases such as leishmaniasis and human African trypanosomiasis (Charles Jaffe’s work).

9. Key literature or bibliography

Bauer, K. (2007) Diazotrophy and diversity of benthic cyanobacteria in tropical coastal zones. PhD Thesis, Stockholm University, Sweden.

Cardellina II JH, Marner FJ, Moore RE (1979) Seaweed dermatitis: structure of Lyngbyatoxin A. Science 204: 193-195.

Castenholz, R.W. (2001) Cyanobacteria, oxygenic photosynthetic bacteria. In: Boone, D.R., Castenholz, R.W. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Springer, New York, pp. 473–599.

Dittmann E, Fewer DP, Neilan BA (2012) Cyanobacterial toxins: biosynthetic routes and evolutionary roots. FEMS Microbiol Rev. doi: 10.1111/1574-6976.12000.

Engene N, Rottacker EC, Kastovsky J, Byrum T, Choi H, Ellisman MH, Komarek J, Gerwick WH (2012) Moorea producens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropical marine cyanobacteria rich in bioactive secondary metabolites. International Journal of Systematic and Evolutionary Microbiology 62: 1171–1178.

Fewer, D. P., J. Österholm, L. Rouhiainen, J. Jokela, M. Wahlsten and K. Sivonen. 2011. Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152. Appl. Environ. Microbiol. 77: 8034-8040.

Gerwick WH, Moore BS (2012) Lessons from the past and charting the future of marine natural products drug discovery and chemical biology. Chemistry & Biology 19, 85-98.

Hamisi M (2010) Genetic variability and nitrogenase activity of cyanobacterial communities associated with tropical seagrass meadows (western Indian Ocean). PhD Thesis. Stockholm University, Sweden.

Herfindal L, Oftedal L, Selheim F, Wahlsten M, Sivonen K, Døskeland SO (2005) A high proportion of Baltic Sea benthic cyanobacterial isolates contain apoptogens able to induce rapid death of isolated rat hepatocytes. Toxicon 46: 252–260.

Jokela J, Oftedal L, Herfindal L, Permi P, Wahlsten M, Døskeland SO, Sivonen K (2012) Anabaenolysins, novel cytolytic lipopeptides from benthic Anabaena cyanobacteria. PloS ONE 7: e41222.

Jones AC, Monroe EA, Podell S, Hess WR, Klages S, Esquenazi E, Niessen S, Hoover H, Rothmann M, Lasken RS, Yates III JR, Reinhardt R, Kube M, Burkart MD, Allen EE, Pieter C, Dorrestein PC, Gerwick WH, Gerwick L (2011) Genomic insights into the physiology and ecology of the marine filamentous cyanobacterium Lyngbya majuscule. PNAS, 108: 8815–8820.

Kaasalainen U, Fewer DP, Jokela J, Wahlsten M, Sivonen K, Rikkinen J (2012) Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis. PNAS 109: 5886-5891.

Leikoski N, Fewer DP, Jokela J, Alakoski P, Wahlsten M, Sivonen K (2012) Analysis of an inactive cyanobactin biosynthetic gene cluster leads to discovery of new natural products from strains of the genus Microcystis. PLoS ONE 7: e43002.

Lugomela, C. (2002) Cyanobacterial diversity and productivity in coastal areas of Zanzibar, Tanzania. Ph.D. thesis, Stockholm University, Sweden.

MacLean D, Jones JDG, Studholme DJ (2009) Application of `next-generation’ sequencing technologies to microbial genetics. Nature Reviews Microbiology, 7:287-296.

Miles CO, Sandvik M, Nonga HE, Rundberget T, Wilkins AL, Rise F, Ballot A (2012) Thiol derivatization for LC-MS identification of microcystins in complex matrices. Environmental Science and Technology. In press.

Nonga HE (2011) Impact of human activities in selected lake ecosystems in Tanzania and occurrence of microcystins and potential microcystin-producing cyanobacteria. Ph. D. thesis. Norwegian School of Veterinary Science, Oslo, Norwey.

Nonga HE, Sandvik M, Miles CO, Lie E, Mdegela RH, Mwamengele GL, Semuguruka WD, Skaare JU (2011) Possible involvement of microcystins in the unexplained mass mortalities of Lesser Flamingo (Phoeniconaias minor Geoffroy) at Lake Manyara in Tanzania. Hydrobiologia 678: 167–178.

Rajaniemi, P., A. Rantala, K. Haukka, P. Kuuppo, L. Lepistö, and K. Sivonen. 2008. Cyanobacterial community composition in shallow, eutrophic Lake Tuusulanjärvi studied by microscopy, strain isolation, DGGE and cloning. Algological Studies 126: 137-157.

Rantala, A., Rizzi, E., B., Castiglioni, G. de Bellis and K. Sivonen. 2008. Identification of hepatotoxin-producing cyanobacteria by DNA-chip. Environmental Microbiology 10(3): 653-664.

Sipari, H., A. Rantala-Ylinen, J. Jokela, I. Oksanen, and K. Sivonen. 2010. Development of a chip assay and qPCR for detecting microcystin synthetase gene E expression. Appl. Environ. Microbiol. 76(12): 3797-3805.

Wang, H., D. P. Fewer and K. Sivonen. 2011. Genome mining demonstrates the widespread occurrence of gene clusters encoding bacteriocins in cyanobacteria. PLoS ONE 6(7): e22384.

Welker M, von Döhren H (2006) Cyanobacterial peptides – Nature’s own combinatorial biosynthesis. FEMS Microbiol Rev. 30:530-563.