Progress 09/01/08 to 08/31/13

Outputs
Target Audience: Numerous undergraduate, graduate and postdoctoral researchers from UC Davis, UC Berkeley, UC Santa Cruz and UC San Francisco as well as Indonesian institutions have received extensive training as part of the laboratory and field research. Significant outreach efforts have been made by the AP4 team to local, regional and national stakeholders and policy makers in Indonesia. These efforts are having significant impact towards the local government’s policy and commitment to having the Mekongga Mountain area declared as a National Park, while ensuring that locals can sustainably utilize forest resources and services. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Numerous undergraduate, graduate and postdoctoral researchers from UC Davis, UC Berkeley, UC Santa Cruz and UC San Francisco as well as Indonesian institutions have received extensive training as part of the laboratory and field research. LIPI scientist Sigit Wiantoro (Chiroptera specialist) was brought to the US and worked with UCD zoologists on the determinations of all bat specimens collected. He visited the Field Museum, American Museum of Natural History, and Smithsonian during his training and determination phases. He presented a talk at the Texas A&M on Indonesian bat diversity and studies. UCD and LIPI arranged for Herpetologist Dr. Jimmy McGuire (UC Berkeley) to visit LIPI to work with Hellen Kurinati (LIPI Scientist) on training for the determination of Mekongga-collected frogs and lizards. This training took place in Cibinong the first two weeks of January 2014. Dr. Michael Ivie Associate Professor and Curator of Entomology, Department of Plant Sciences and Plant Pathology, Montana State University. He works on the beetles on a worldwide basis and knows them better than almost anyone else. He determined and trained LIPI scientists ion a trip 16 – 30 May 2013. Dr. Matthew Buffington, Research Entomologist, National Museum of Natural History, Washington, D.C., studies a group of parasitic wasps called the Cynipoidea. He traveled to Cibonong on 01-16 July training LIPI scientists in determinations and taxonomy of these groups of wasps. In the March 2014, Dr. Steve Heydon, Bohart Museum of Entomology will travel to work with taxonomistst in the Entomology Department at LIPI. He will be accompanied by Larry Bezark, a retired person who worked with the Department of Food and Agriculture in Sacramento. He is an expert on the long horned beetles (Cerambycidae) and the robber flies (Asilidae). Atit Kanti, LIPI microbiologist, is completing her PhD at Bogor Agricultural University in Indonesia, with ICBG research as one of her research elements. Several undergraduate students at Indonesian institutions received training in insect taxonomy as part of their undergraduate theses. Postdoctoral fellows Tyler Johnson, Laura Milan-Lobo and Johan Sohn received postdoctoral training. How have the results been disseminated to communities of interest? AP4 team, led by FORDA ecologist Hendra Gunawan presented numerous workshops and socialization events to share research results and promote sustainable use of the forest resources. Banners, flyers, and T-shirts were distributed. The AP4 team published an Academic Paper in Bahasa Indonesia to promote the research results of the ICBG project and the conservation of the Mekongga site. The AP4 team will also publish a compilation of selected photographs of ICBG expeditions in the Mekongga. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Central Operations: Agreements between UC and Indonesian institutions were signed, including an MTA and MOU. Biological collections of insects, plants, vertebrates and microbes in the US and Indonesia were greatly expanded through this project. Curators and staff in Indonesia received training in specimen collecting and preservation, identification, biological collection management, database operation. Collaborations were established and nurtured. AP1: The biodiversity of the Masembo Watershed, Mekongga Mountains was thoroughly documented from 2009 through 2011. Over 3,000 vertebrates (266 species, 18 new species), 1,100 plants (427 species, 8 new species) and 500,000 insects (with numerous new species) were collected along an elevation gradient from sea level to 2,500m. We are modeling diversity in the watershed and have developed a sampling protocol that can be used anywhere in the country in habitats under threat or being considered for conservation. The biodiversity survey information was used to support a proposal to have the research site declared a National Park. If and when this passes, it will be the first national park in Indonesia to be declared on the basis of biodiversity data. AP2: Over 60 novel yeast species were discovered. Yeasts able to convert waste biomass including corn stover hydrolysate into oil were discovered. Seventeen new oleaginous (lipid-accumulating) yeast species were discovered. AP3: Therapeutic activities from plants and microbes were detected for several GPCR targets, including mu and delta opioid receptor agonists and NK1 receptor agonists. Several derivatives of the lead molecules have been examined for their structur-activity relationships. There are two manuscripts in preparation from these studies, one describing the opioid receptor leads and the other describing the NK1 receptor leads. AP4: The AP4 team has worked to ensure that the local and national stakeholders understand the significance of designating the Mekongga area as a conservation area. The AP4 team has also encouraged the District and Province governments to have a strong commitment to propose and declare the Makongga as a national park.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Dewi, K. and H. Hasegawa. In press. Two new species of Syphacia (Nematoda: Oxyuridae) in endemic murid rodents from Sulawesi, Indonesia. J. Helminthology (2012): 1-9.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Huylebrouck, J., R. K. Hadiaty, and F. Herder. In press. Two new species of viviparous halfbeak (Atherinomorpha: Beloniformes: Zenarchopteridae) endemic to Sulawesi Tenggara, Indonesia. The Raffles Bull. Of Zoology.
• Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Trochet, J.A., I. Mohammad, A.T. Hitch, T. Haryoko, H. Ashari, D. Dwi Putra, and A. Engilis, Jr. In Review. Range Expansion of Sooty-headed Bulbul Pycnonotus aurigaster and Lemon-bellied White-eye Zosterops chloris to South-Eastern Sulawesi, Indonesia. Submitted to Forktail 18 March 2013.
• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Kartonegoro, A. and, D. Potter. The Gesneriaceae of Sulawesi Vi: The Species from Mekongga Mts.: with a New Species of Cyrtandra. Submitted 13 January 2014 to the Journal Reinwardtia.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Kurniati, H., A.T Hitch, J. McGuire, W. Tri Laksono & A. Engilis, Jr. in Prep. General herpetofauna survey results of the Masembo Watershed in the Mekongga Mountains South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Wiantoro, S., A.T. Hitch, and A. Engilis Jr. in prep. Report on the bats (Chiroptera) collected from the Masembo watershed, Mekongga Mountains, South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Wiantoro, S., A.T. Hitch, and A. Engilis Jr. Report on the bats (Chiroptera) collected from lowlands of South East Sulawesi, including several significant range extensions.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Sulistiarini, D., Irawati, and D. Potter. Flora of Orchidaceae of Mekongga Mountains, South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Sunarti, S. and D. Potter. Flora diversity of Myrtaceae of the Mekongga Mountains, South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Hidayat, A., and D. Potter. Diversity of fern of the Mekongga Mountains, South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Engilis Jr, Andrew, J.A. Trochet, M. Irham, H. Ashari, T. Haryoko, D. Dwi Putra, and A.T Hitch. A report on the avifauna collected and observed from the Masembo Watershed,Mekongga Mountains, South East Sulawesi.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Hitch, A.T., E.A. Widjaja, R. Ubaidillah, S. Wiantoro, A. Achmadi, T. Haryoko, M. Irham, H. Ashari, H. Kurniati, T. Laksono, Darmawan, J.A. Trochet, D.P. Dadang. Estimation and patterns of biodiversity along an elevation gradient in the Masembo Watershed, Mekongga Mountains, South East Sulawesi,Indonesia.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Irnayuli R. Sitepu a,b, Luis A. Garaya, Ryan Sestricc, David Levinc, David E. Blockd,e, J. Bruce Germana, and Kyria L. Boundy-Mills1*REVIEW: Oleaginous Yeasts for Biodiesel. 1. Current and future trends in biology and production. Manuscript in preparation.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Irnayuli R. Sitepu?ab, Mingjie Jinc, J. Enrique Fernandeza, Leonardo Sousac, Venkatesh Balanc, Kyria L. Boundy-Millsa Lipid accumulation by oleaginous yeasts in synthetic and authentic AFEXTM corn stover hydrolysate . Manuscript in preparation.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Atit Kanti1,2, Nampiah Sukarno3, Latifah K Darusman4, Endang Sukara5, Irnayuli .R. Sitepu6,7 and Kyria Boundy-Mills7 Species Diversity of Yeast Inhabiting Leaf Surfaces and Leaf Litter in Secondary Rain Forests inSouth East Sulawesi, Indonesia. Manuscript in preparation.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Irnayuli Sitepu1,2, Tylan Selby1, Shirley Zu1, Ting Lin1, Kyria Boundy-Mills1* Carbon source utilization and toxin tolerance of oleaginous yeasts. Manuscript in preparation.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Hitch, A.T., S. Wiantoro, and A. Engilis Jr. Estimating bat species richness and their community habitat relationships using hierarchical models with presence/absence data in southeast Sulawesi, Indonesia. 16th International Bat Research Conference/43rd North American Symposium on Bat Research. 14 August 2013 Costa Rica
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: MEKONGGA: A NEW SPOT FOR BIODIVERSITY CONSERVATION IN WALLACE ZONE. Presentation Paper , presented on  Second International Conference on Alfred Russel Wallace and The Wallacea,, Wangi-Wangi, Wakatobi , 10-13 November 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: MEKONGGA: HARAPAN BARU BAGI PELESTARIAN KERAGAMAN HAYATI SULAWESI. Presentation paper, presented on National Seminar on Ecology and Conservation, Makassar 20-21 November 2013, Organized by Bantimurung Bulusaraung National Park and Faculty of Forestry, Hasanuddin University, Makassar

Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: The biggest challenge of the current reporting period was a dispute between LIPI and UCD. LIPI administration officials expressed their concerns, primarily over coauthorship, in a letter mailed to UC Davis and other parties in April 2012. Shipment of ICBG specimens from LIPI to the US was halted for four months. Issues expressed in this letter were successfully resolved in a series of communications between LIPI and UC Davis. Boundy-Mills wrote a protocol for internal review of manuscripts before submission. The project is now back on track, with a successful resumption of all research activities. AP1 Biodiversity Surveys: Our focus since completing field work in December 2011 was identifying the specimens that we had collected, and developing habitat diversity models. Initial identifications of most of the vertebrate specimens was completed. We sequenced selected mammal specimens and found many interesting lineages and possible new taxa. When compared to other collections from Sulawesi, the specimens from Mekongga are very distinct, demonstrating the uniqueness of this watershed. Large scale multi-taxa surveys are rare and information from the Mekongga will allow us to apply new quantitative techniques on a unique and comprehensive data set. AP2 Microbe Surveys and Bioenergy Screening: The AP2 team exceeded the target goal of collecting 1800 microbes by several hundred strains. Screening for cellulase stability was expanded with industrial advice to include tolerance of high temperature, high and low pH, and other conditions. Next generation sequencing tools including a reference yeast ribosomal DNA sequence database are being developed and tested to characterize fungal and bacterial communities in beetle guts. AP3 Therapeutics Screening: Since March 2012, the AP3 team has received 81 new Indonesian microbial ethyl acetate extracts and screened them in the MTT and NFkB assays. Four lead compounds from 4 priority microbial extracts are under investigation, along with one new microbial extract. Bioassay of nearly all 228 extracts of Indonesian plant specimens have been evaluated in our AP3 assays. We have focused our efforts on pursuing bioactive plant extracts in this final year because they typically contain sufficient material to allow for multiple bioassays, scale up isolation and/or identification of the lead compound(s). Currently, the team is pursuing 2 lead compounds based on 5 priority bioactive extracts. The team now has 1 priority project from microbial extracts and 5 priority projects from plant extracts with active components characterized by their LC/MS properties. Structure assignment of these therapeutic leads has been completed or is in process. We have screened all 228 Indonesian plant extracts in the Whistler lab at UC San Francisco (Gallo Center) in neuro-bioassays (Orexin-1, NK1-R, NPS-R) and have identified 7 priority extract leads (3% hit rate), which are now under investigation. PARTICIPANTS: The primary institutions participating in the project are: in the US: UC Davis, UC Berkeley, UC San Francisco; in Indonesia: the Indonesian Institute of Sciences (LIPI), the Indonesian Ministry of Forestry (MoF), and the Technology Institute of Bandung (ITB). The lead US participants in the project include the following: Kyria Boundy-Mills (overall PI and AP2 leader); Daniel Potter (former PI); Len Bjeldanes (AP3 leader); Andrew Engilis, Jr. (AP1 leader), Ellen Dean (AP1 - botany); Steven Heydon (AP1 - entomology); Lynn Kimsey (AP1 - entomology); Nathan Schiff (AP 2 researcher); Selena Bartlett (AP3 researcher); Alan Hitch (AP1 researcher); John Trochet (AP1 researcher). The lead Indonesian participants in the project include the following: Elizabeth Widjaja (AP1 co-leader) Hari Nugroho (AP1 researcher) Eko Baroto Waluyo (AP4 researcher) Rosichon Ubaidillah (AP1 co-leader) Mohammad Hanafi (AP3 researcher) Atit Kanti (AP2 researcher) Agus Joko Nugroho (AP2 co-leader) Hendra Gunawan (AP4 leader) Isaac Kubo (AP1 researcher) Heddy Julistiono (AP3 co-leader) Leonardus Kardono (AP3 researcher) Endah Sulistiowati (AP4 researcher) Four post docs (Sitepu, Hitch, Johnson and Sohn) are being trained in this project. Seven UC Davis undergraduate students (Elaine Chow, Enrique Fernandez, Ngoc-Lien Nguyen, Panachon Lor, Neil Isip, Ain Mobeen and Pazao Lor) and one recent UC Davis graduate working as a Junior Specialist (Laura Ignatia) were trained in the Boundy-Mills lab working on AP 2. Four Indonesian students have received training as part of this project working on APs 2 and 3. Three Indonesian undergraduate students worked with Atit Kanti in Microbiology Division LIPI Laboratory. Caesario Nugroho and Adawiyah Pratiwi are students of State University Jakarta, and Eka Yulia Pertiwi is a student of Andalas University, West Sumatra. TARGET AUDIENCES: Significant outreach efforts by the AP4 team to local, regional and national stakeholders and policy makers are described in the sections above. These efforts are having significant impact towards having the Mekongga Mountain area declared as a Conservation Area, while ensuring that locals can sustainably utilize forest resources. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
AP1 Biodiversity Surveys: The results from the biodiversity analyses were used in conservation programs and management actions by the AP4 team. Plans are being made for AP1 member Sigit Wiantoro, LIPI zoologist, to visit several US biological collections in 2013. The UC Davis Bohart Museum of Entomology hosted over 80 tours of the museum facilities, at which ICBG specimens and the ICBG project were presented. AP2 Microbe Surveys and Bioenergy Screening: The AP2 team published a fast, inexpensive Nile Red fluorometric assay to evaluate yeast cell oil content, which will enable high-throughput screening of the oil content of many yeast strains, or the same yeast grown under multiple conditions. A manuscript is in preparation describing the survey of oil content of over 60 potential oleaginous yeast species, the first broad survey of its kind. AP2 member Atit Kanti of LIPI visited UC Davis, to characterize new yeast species in the Boundy-Mills lab. AP3 Therapeutics Screening: Plans are being made for AP3 team member Arif Nurkanto to work for four weeks in Len Bjeldanes' laboratory at UC Berkeley in 2013. AP4 Conservation Outreach and Education: A workshop was conducted in May 2012 to disseminate ICBG research results to over 80 policy makers and stakeholders at the national level, especially to the relevant institutes in biodiversity management [Ministry of Forestry, BAPPENAS (National Planning and Development Agency), Ministry of Environment, South East Sulawesi Province Government and NGOs] and to seek input for long-term conservation goals for the Mekongga region. A presentation was made by the AP4 leader to explain the Academic Paper, a document required as part of the proposal to designate Mekongga as a conservation area. Plenary discussions explored suggestions, comments, perspectives and input from participants for improving the next project and academic paper. AP4 leader Hendra Gunawan met with local, regional and national leaders in over a dozen meetings and seminars, to share goals and results of the ICBG project.

Publications

• I.R. Sitepu, L. Ignatia, A.K. Franz, D.M. Wong, S.A. Faulina, M.Tsui, A. Kanti, K. Boundy-Mills. In Press. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J. Microbiol. Methods. PMC Journal - In press 2012 Vera Budi Lestari Sihotang, Elizabeth A. Widjaja & Daniel Potter. Medicinal Plant Knowledge of Tolaki and Toraja in Tinukari Village and Its Surrounding. Proceeding of the International Seminar: Strategy and Challenges of Bamboo and Potential Non-Timper Forest Products (NTFPs) Management and Utilization, Centre for Forest Productivity Improvement Research and Development, FORDA, Department of Forestry, Bogor, September 2011 (in press).
• Chow, E., I. Sitepu, N. Schiff, L. Ignatia, A. Kanti, S. Faulina, A. Joko Nugroho, N.-L. Nguyen, K. Boundy-Mills. Filamentous fungi and yeasts collected from beetle-infested trees in Indonesia. UC Davis Undergraduate Research Symposium, 18 April 2012, Davis CA.
• Kyria Boundy-Mills, Nathan Schiff, Irnayuli Sitepu, Ain Mobeen, Laura Ignatia, Sarah Faulina, Agus Joko Nugroho, Atit Kanti. Identification of novel xylanase genes from fungi in wood and beetle guts in Indonesia. Society for Industrial Microbiology and Biotechnology 34th Symposium on Biotechnology for Fuels and Chemicals, April 30-May 3, 2012, New Orleans, LA.
• Irnayuli R. Sitepu, Laura Ignatia, Ryan Sestric, Sarah Faulina, Atit Kanti, Agustinus J. Nugroho, Kyria Boundy-Mills. Characterization of oleaginous yeast species from Phaff Yeast Culture Collection. Society for Industrial Microbiology and Biotechnology 34th Symposium on Biotechnology for Fuels and Chemicals, April 30-May 3, 2012, New Orleans, LA.
• Elaine Chow1, Irnayuli Sitepu1,2, Nathan Schiff3, Laura Ignatia1, Atit Kanti4, Sarah Faulina2, Agus Joko Nugroho4, Ngoc-Lien Nguyen1, Kyria Boundy-Mills1. Fungi Associated with Wood-Feeding Beetles in Indonesia. American Society for Microbiology 112th General Meeting, June 16-19, 2012, San Francisco, CA
• Irnayuli R. Sitepu, Laura Ignatia, Ryan Sestric, Sarah Faulina, Atit Kanti, Agustinus Nugroho, Kyria Boundy-Mills. Characterization of oleaginous yeast species. American Society for Microbiology 112th General Meeting, June 16-19, 2012, San Francisco, CA
• Laura Ignatia, Irnayuli Sitepu, Ain Mobeen, Sarah Faulina, Atit Kanti, Agus Joko Nugroho, Nathan Schiff, Kyria Boundy-Mills. Diversity of xylanase gene sequences from filamentous fungi associated with wood-feeding beetles in Indonesia. American Society for Microbiology 112th General Meeting, June 16-19, 2012, San Francisco, CA

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: This project underwent a major reorganization in early 2011 to streamline the operation of the project and involve more Indonesian participation:(1) The PI is now Kyria Boundy-Mills rather than Dan Potter.(2) The AP structure was simplified, reducing from six APs down to four. The Year 3 project meeting was held in February 2011 at UC Davis. Attendees included Flora Katz of NIH, two scientists from Indonesia, twenty participating scientists from UC Davis, UC Berkeley, UC San Francisco and UC Santa Cruz, and four members of the Advisory Board. The Year 4 project meeting was held in July 2011 in Cibinong, Indonesia. Attendees included Flora Katz, 8 scientists from UC Davis, UC Berkeley, and UC Santa Cruz, over 40 Indonesian scientists from LIPI and the Ministry of Forestry, and four members of the Advisory Board. The AP1 team (Macro-Organisms Surveys) performed fieldwork in December 2010 and July 2011. Specimens collected include plants, insects, and vertebrates, 5-kg samples of 70 plants for therapeutics screening, vertebrate gut contents and soil for microbe isolation by the AP2 team. The AP1 and AP4 teams worked together to run a Parataxonomy Training Workshop at Universitas Haluo Oleo in Kendari in July 2011, attended by 70 policy makers, students, local villagers, and forest rangers. The AP2 team (Microbe Surveys and Bioenergy Screening) preserved 2009 and 2010 microbes in all three culture collections: LIPI, Ministry of Forestry, and UC Davis. The AP2 team identified several new oleaginous yeast species, which are under further investigation for conversion of agricultural and forest waste to lipids for use as oleochemicals including biodiesel. Over 80 filamentous fungi were analyzed for xylanase activity and gene sequences. Between July 2010 and October 2011, the AP3 team (Discovery of Human Health Solutions) examined bioactivity of a total of 193 microbe extracts and 144 plant extracts. They identified 22 microbial and 19 plant leads, and produced natural product libraries of 15 microbial and 19 plant extracts. Two microbial and three plant lead compounds are under investigation, replication and elucidation. They identified potential anticancer agents. Cytostatic compounds from seven plants are being purified and identified. Of 225 extracts were assayed for anti-inflammatory activity, two plant and four microbial extracts showed significant activity, and are being examined further to identify active compounds. Extracts were also examined for neurological activities using an in vitro cell based calcium bioassay. Analysis of the plant extracts identified several that are mu or delta opiod receptor agonists and antagonists. The AP4 team (Local Conservation Education and Outreach) communicated with stakeholders at 13 institutions in the Mekongga mountain area and throughout Southeast Sulawesi, including universities, NGOs and government agencies. The AP4 team performed a Participatory Rural Appraisal with 30 participants, primarily farmers, from four tribes in Mekongga regarding their use of forest resources, their understanding of the forest ecosystem, and needs for improving agricultural production. PARTICIPANTS: The primary institutions participating in the project are: in the US: UC Davis, UC Berkeley, UC San Francisco; in Indonesia: the Indonesian Institute of Sciences (LIPI), the Indonesian Ministry of Forestry (MoF), and the Technology Institute of Bandung (ITB).The lead US participants in the project include the following: Kyria Boundy-Mills (overall PI and AP2 leader); Daniel Potter (former PI); Len Bjeldanes (AP3 leader); Andrew Engilis, Jr. (AP1 leader), Ellen Dean (AP1 - botany); Steven Heydon (AP1 - entomology); Lynn Kimsey (AP1 - entomology);Nathan Schiff (AP 2 researcher); Selena Bartlett (AP3 researcher); Alan Hitch (AP1 researcher); John Trochet (AP1 researcher). The lead Indonesian participants in the project include the following: Elizabeth Widjaja (AP1 co-leader); Hari Nugroho (AP1 researcher); Eko Baroto Waluyo (AP4 researcher); Rosichon Ubaidillah (AP1 co-leader); Mohammad Hanafi (AP3 researcher); Atit Kanti (AP2 researcher); Agus Joko Nugroho (AP2 co-leader); Hendra Gunawan (AP4 leader); Isaac Kubo (AP1 researcher); Heddy Julistiono (AP3 co-leader); Leonardus Kardono (AP3 researcher); Endah Sulistiowati (AP4 researcher). Seven current UC Davis undergraduate students: (Paul Park, Elaine Chow, Shelly Sianita, Shierly Mayasari, Mary Stump, Jessica Wijaya, Angela Pratiwiharja) and one recent UC Davis graduate working as a Junior Specialist (Laura Ignatia) have been trained in the Boundy-Mills lab working on AP 2. Four Indonesian students have received training as part of this project working on APs 2 and 3. Sarah Faulina, a recent graduate of Bogor Agricultural University, is working in the Forest Microbe Collection lab in the Ministry of Forestry. Three Indonesian undergraduate students are working with Atit Kanti in Microbiology Division LIPI Laboratory. Caesario Nugroho and Adawiyah Pratiwi are students of State University Jakarta, and Eka Yulia Pertiwi is a student of Andalas University, West Sumatra. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The AP1 Parataxonomy Training Workshop resulted in transfer of knowledge of biological specimen preparation to students in Sulawesi, which is now being used to generate a research and teaching collection of plant, insect and vertebrate specimens at the local university. The AP2 postdoc, Irnayuli Sitepu, has received extensive training in microbe isolation, identification and preservation, as well as culture collection management. At the conclusion of this project in 2013, she will return to her position as manager of the Forest Microbe Culture Collection at the Forestry Research and Development Agency, Ministry of Forestry, prepared to take the microbe collection to a new level of productivity. The AP2 teams at the Ministry of Forestry and the Institute of Science (LIPI) in Indonesia have received training in culture collection database management. The new database system has allowed much more efficient data management and stock tracking. The AP3 team The AP4 team (Local Conservation Education and Outreach) made great strides in reaching out to individuals at 13 local and regional institutions in the Mekongga mountain area and local cities throughout Southeast Sulawesi, including universities, NGOs and government agencies. The AP4 team performed a Participatory Rural Appriasal, to assess the training needs in the region. They interviewed 30 participants, primarily farmers, from four tribes close to Mekongga. Detailed information was obtained regarding their use of forest resources, their understanding of the forest ecosystem, and needs for improving their agricultural production. They also helped organize the Parataxonomy Training Workshop in Kendari in July, and have posted banners, posters, and distributed pamphlets to stakeholders in the region.

Publications

• Wu QX, Crews MS, Draskovic M, Sohn J, Johnson TA, Tenney K, Valeriote FA, Yao XJ, Bjeldanes LF, Crews P. Azonazine, a novel dipeptide from a Hawaiian marine sediment-derived fungus, Aspergillus insulicola. Org Lett. 2010 Oct 15;12(20):4458-61.
• P. Crews, T. Johnson, J. Sohn, W. Inman, S. Estee, S, Loveridge, H. Vervoort, K. Tenney, J. Liu, K.-H. Ang, J. Ratnam, W. Bray, N. Gassner, Y. Shen, S. Lokey, J. McKerrow, K. Boundy-Mills, A. Nurkanto, A. Kanti, L. Bjeldanes. 2011 In Press. Natural product libraries to accelerate the high throughput discovery of therapeutic leads. Journal of Natural Products.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: No significant changes have been made to the objectives or structure of the project. Major accomplishments over the past year include the following: 1) Our first annual project meeting was held at UC Davis Nov. 30 - Dec. 2, 2009, and was attended by representatives from all three Indonesian and all three US institutions involved in the project, as well as by Drs. Josh Rosenthal and Flora Katz from the Fogarty International Center. 1) A second field trip to Sulawesi by a team of the Indonesian scientists was undertaken in Dec., 2009. The expedition included specimen collections for APs 1 (Macro-organism Surveys, focused on vascular plants and insects), 2 (Microbial Surveys), and 5 (Vertebrate Survey and Conservation) at our field site in the Mekongga Mountains near the village of Tinukari, as well as meetings with local government officials in Kendari and Kolaka related to AP 6 (Conservation Partnerships, Training, and Ethics). 2) Significant progress has been made on website and database development and on-line communication among project participants, through collaborative work of personnel from Central Operations and AP6. 3) US-based activities in APs 2 and 3 (Discovery of Energy Solutions) have included training of UC Davis undergraduate students in isolation, identification, and screening of microbes and validation of screening protocols for bioenergy applications. 4) The AP4 (Discovery of Human Health Solutions) team, based at UC Berkeley and UC San Francisco, has validated the therapeutic screening methods using Phaff collection yeasts (provided by AP 3), while awaiting availability of Indonesian microbes. The team confirmed its ability to detect microbial production of compounds with anti-cancer and anti-inflammatory activities, and potential neurological activities against addiction. 5) Sampling strategies for field work on APs 1, 2, and 5, which will allow us to be able to survey the watershed along an altitudinal gradient and to estimate biodiversity in multiple taxonomic groups at a landscape scale, have been developed, under direction of AP5 personnel. 6) AP6 personnel have made significant progress in increasing the number of Indonesian collaborators for AP6, developing the project "virtual office" and online resource library, refining the project listserve, initiating SmartSite, Skype, and Elluminate software, teleconferencing, and webinar training, initiating a project-wide dialogue on overarching questions relating to our multi-agency collaboration, and surveying approximately 40 representatives from newly identified collaborating agencies and stakeholders in SE Sulawesi. 7) A third field trip, involving researchers from both US and Indonesian scientists from APs 1, 2, and 5, was undertaken beginning in mid-June, 2010. Additional field trips by US and Indonesian scientists are planned for fall 2010 and winter-spring 2011. Screening activities for APs 3 and 4 will continue, both in Indonesia and the US, throughout Year 3. Biodiversity data analysis for AP5 will begin in fall 2010. Website and database development in Central Operations will continue, as will both on-the-ground and on-line outreach and training efforts by AP6. PARTICIPANTS: The primary institutions participating in the project are: in the US: UC Davis, UC Berkeley, UC San Francisco; in Indonesia: the Indonesian Institute of Sciences (LIPI), the Indonesian Ministry of Forestry (MoF), and the Technology Institute of Bandung (ITB). The lead US participants in the project include the following: Daniel Potter (overall PI); Kate Scow (AP2 leader); Kyria Boundy-Mills (AP3 leader); Len Bjeldanes (AP4 leader); Andrew Engilis, Jr. (AP5 leader), Jeanine Pfeiffer (AP6 leader); Ellen Dean (AP1 - botany); Steven Heydon (AP1 - entomology); Lynn Kimsey (AP1 - entomology); Nathan Schiff (APs 2 and 3 researcher); Selena Bartlett (AP4 researcher); Alan Hitch (AP5 researcher); John Trochet (AP5 researcher). Three current UC Davis undergraduate students (Pamelia Hadiwidjaja, Sam (Bai) Zeng, and Abraham Woldemariam) and two recent UC Davis graduates working as Junior Specialists (Jaspreet Kaur and Vanessa Morales) have been trained in the Boundy-Mills lab working on APs 2 and 3. Four Indonesian students have received training as part of this project working on APs 2 and 3. Sarah Faulina, a recent graduate of Bogor Agricultural University, is working with Irnayuli R. Sitepu in her lab in the Ministry of Forestry. Three Indonesian undergraduate students are working with Atit Kanti in Microbiology Division LIPI Laboratory. Caesario Nugroho and Adawiyah Pratiwi are students of State University Jakarta, and Eka Yulia Pertiwi is a student of Andalas University, West Sumatra. For AP5, we have coordinated with several researchers that have assisted us in refining survey methodologies and techniques as well as curation and collection protocols. These collaborations have included researchers at several institutions in the US and Indonesia such as; The Celebes Birding Club - Sulawesi, Jack Dumbacher and Jen Vindum at the California Academy of Natural Sciences and Jim McGuire at the Museum of Vertebrate Zoology, University of California at Berkeley. February 1, 2010 marked the arrival at UC Davis of Alan Hitch, AP5 post-doctoral researcher who has been and will be responsible for the design of the vertebrate surveys as well as the organization and completion of the field surveys, the development of the habitat relationship models and collection of museum materials and tissue collections of vertebrates that are collected. TARGET AUDIENCES: Collaborations with Indonesian scientists have continued and increased, as have communication and exchange of information with local residents in the field site area. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Here, we report results for APs 2, 3, and 5, which are the portions of the project receiving direct funding from USDA. Results for the other APs may be found in the annual report submitted to NIH in April, 2010. First, we report on results of the December, 2009 field trip to Sulawesi by Indonesian scientists. For AP2, there were two major objectives. The first was to select a forest site for rearing of wood-feeding insects in wounded trees and to wound the trees. We selected a site in the Papalia Protected Forest near Kendari. A microbiologist at nearby Haluo Oleo University is granting access to his lab, which will make it much easier for us to isolate microbes from larval guts. We selected six trees of economic or ecological importance, collected herbarium specimens for tree species identification, and recorded GPS coordinates, soil pH and temperature, and dbh. The six trees were wounded by girdling. As they die, they release volatiles that attract certain wood-feeding insects. In July, we will cut down the trees, section the trunks into logs, and sample microbes from larvae found in alternate logs. The remaining logs will be placed in rearing cages to allow adult insects to emerge, which we can match to the species of larval tissue samples by DNA bar-coding. The second major objective was to collect soil, leaf litter, and fresh leaf samples for microbe analysis at Papalia and at Mekongga. Sixteen samples including 6 soil, 6 leaf litter, and 4 leaf were collected in the former, and 30 samples (20 soil and 10 litter) were collected from the latter. The total number of yeast collections was 118 isolates. Of those, 24 were isolated from leaf, 90 were isolated from litter and 4 were from soil sample. The total number of filamentous fungi collected was 69 isolates. Of those, 19 were isolated from leaf, 28 were isolated from litter and 22 were from soil sample. The total number of actinomycetes collected was 152 isolates. Of those, 66 were isolated from litter and 86 were from soil samples. The total number of bacteria collected was 320 isolates, of which 284 were selected for further characterization. Of those, 41 were isolated from leaf, 123 were isolated from litter, and 120 isolates were obtained from soil samples. All of the selected microbes were preserved both in LIPI Microbial Collection (LIPIMC) and Forest and Nature Conservation Research and Development (FORDA CC) Indonesia. Samples were frozen in glycerol solution and slant agar for storage. For AP5, bird specimens collected during this field trip included 28 specimens (12 species). Fifteen endemic species of birds were observed, while only six of these were collected. Three species listed under CITES were observed but not collected. Furthermore 5 species of bird protected under the Indonesian law were observed, of which one, listed as a "near threatened" species, could be collected. For AP3, progress was made during Year 2 in validating protocols for screening microbes for cellulase activity, identifying yeasts with potential to produce high levels of lipids, and refining protocols for preparing microbial extracts for therapeutics screening by AP4.

Publications

• Boundy-Mills, K. (2009) Bioprospecting for biofuels applications: Tapping biodiversity in Indonesia and the Phaff Yeast Culture Collection. Presented at the BIO Pacific Rim Summit on Industrial Biotechnology and Bioenergy, Honolulu, Hawaii.
• Boundy-Mills, K. (2009) Analysis of wood-feeding beetle gut microbes for novel cellulase activities. Presented at the BIO Pacific Rim Summit on Industrial Biotechnology and Bioenergy, Honolulu, Hawaii.

Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: The project has six main objectives: 1) to perform biodiversity surveys at low-, mid-, and high- elevation forests of southeastern Sulawesi, Indonesia; 2) to explore the potential of organisms from those sites as sources of natural products useful for novel bioenergy applications; 3) to explore the potential of those organisms as sources of novel therapeutics for treatment of cancer, addictions, and immune system disorders; 4) to compare levels of biological diversity in several taxonomic groups among lowland and upland forest sites and to develop recommendations for strategies to conserve biodiversity in the region; 5) to develop local conservation education and outreach efforts; and 6) to develop effective international agreements related to sharing and development of biogenetic resources. The project is organized into six Associate Programs. AP1, Macro- organism Surveys, conducts inventories of vascular plants and insects in order to characterize the ecosystems and document diversity present at the study sites. AP 2, Microbial Surveys, isolates yeasts, filamentous fungi, and bacteria from the field sites. AP3, Discovery of Energy Solutions, screens microorganisms from AP2 for use in biofuels applications. AP4, Discovery of Human Health Solutions, seeks to isolate agents potentially useful as anticancer therapies, immune enhancers, and treatments of neurological disorders. AP5, Conservation Research and Vertebrate Surveys, conducts inventories of lesser faunal elements and combines the data with information from APs 1 and 2 in order to develop multi-metric diversity models that will provide an ecological basis for watershed reserve design and guide the development of new reserves in this region. AP6, Conservation Partnerships, Training, and Ethics, facilitates interactions among all participants in the project, with the over-riding aims of creating effective partnerships and promoting best practices for international collaborative research. The first nine months of the project were devoted primarily to developing required international and inter-institutional agreements, obtaining permits and visas, planning for the first field trip to Sulawesi, and developing protocols for health and bioenergy screening. In November, 2008, PI Potter traveled for 3.5 weeks to Indonesia, where he met with researchers and administrators from all three Indonesian institutions involved n the project to discuss goals and plans. In late July and early August, 2009, a team of eight of the US researchers, along with about 20 Indonesian scientists, made the first expedition to the proposed field site in the Mekongga Mountains region in Southeast Sulawesi. The objectives of the trip were: 1) to provide opportunities for US and Indonesian scientists to meet and exchange ideas; 2) to confirm that the proposed field site would be appropriate for the project and identify logistical challenges that would need to be dealt with; 3) to make initial collections of the target organisms for the biodiversity surveys; 4) to make contact with local community residents and other stakeholders; 5) to isolate and export microbial cultures; 6) to plan for future field expeditions. PARTICIPANTS: The primary institutions participating in the project are: in the US: UC Davis, UC Berkeley, UC San Francisco; in Indonesia: the Indonesian Institute of Sciences (LIPI), the Indonesian Ministry of Forestry (MoF), and the Technology Institute of Bandung (ITB). The lead US participants in the project to date include the following: Daniel Potter (overall PI); Kate Scow (AP2 leader); Kyria Boundy-Mills (AP3 leader); Len Bjeldanes (AP4 leader); Andrew Engilis, Jr. (AP5 leader), Jeanine Pfeiffer (AP6 leader); Ellen Dean (AP1 - botany); Steven Heydon (AP1 - entomology); Lynn Kimsey (AP1 - entomology); Nathan Schiff (APs 2 and 3 researcher); Selena Bartlett (AP4 researcher); Alan Hitch (AP5 researcher); John Trochet (AP5 researcher). AP3 leader Boundy-Mills wrote a training and lab methods manual, detailing microbe isolation and identification methods as well as culture collection management policies. As part of the July-August trip to Indonesia by the US team, she ran a training session held at MoF and attended by 10 LIPI and MoF scientists. TARGET AUDIENCES: As mentioned above, collaborations with Indonesian scientists have been initiated, as has communication and exchange of information with local residents in the field site area. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Progress on agreements and permits: 1) A Letter of Agreement among the three Indonesian institutions involved in the project was signed by representatives from LIPI, MoF, and ITB. 2) Materials Transfer Agreements (MTAs) required for export of plant, animal, and microbial specimens from LIPI to UC Davis were finalized. 3) Research permits and visas were obtained for the eight US researchers who participated in the first field trip. Progress was made on developing laboratory protocols for screening microbes for bioenergy and therapeutic applications by the leaders of APs 3 and 4, respectively. Outcomes of the July-August field trip: AP1: Botanists explored forests up to an elevation of about 400 m, documenting common tree species and making about 30 collections of herbarium specimens of fertile material. Entomologists took 211 specimens of various Lepidoptera and 9 bags of sweeping samples. These were exported and are currently being sorted and mounted at UC Davis. AP2: At the field site, the following samples were collected for microbial isolation: 42 insect larvae, 18 soil, 18 leaf litter samples and 18 leaves. Microbial isolation methods in a field setting were tested. Over 130 microbes (bacteria, yeast and molds) were isolated from insect guts and infested wood, purified, and placed in cryostorage at LIPI and MoF. Identification of microbes by PCR amplification and sequencing of ribosomal regions is underway. The first four microbes were identified and imported to UC Davis in August. An additional 150 microbes are being isolated and identified from soil, leaf litter, and leaf surfaces. In addition, two alternate sites were identified that will be more suitable for the insect rearing method, which involves cutting trees and leaving them in cages in the field for up to a year to allow infestation by wood-feeding insects. AP5: The goals were to investigate the field site by visiting a low elevation camp for three days and cover logistics for future, more concentrated efforts. The team ran 5-8 mist-nets for three days and nights and 15 mammal traps nightly. The team netted six birds representing five families and species, observed and/or heard 29 species of birds, netted about 80 Pteropid bats representing 6 species in 5 genera, of which 45 specimens were collected and the rest released. Two murid rodents, two lizards, and three frogs were collected. AP6: The team conducted 30 hours of interviews and small discussion groups with ten individuals (aged 30-100+) representing the two major ethnic groups in the area. Relevant village archives were also consulted. Based on these activities, the team was able to reconstruct detailed histories of the settlement, agriculture, and forestry in the area, to document traditional uses of native plants and animals, and to provide records of visits by previous research teams to the area. Their findings were compiled in a detailed report that was provided to all project participants.

Publications

• No publications reported this period