In memory of my father Khamphanh Vongsombath To my family Vongsombath Phienthong, Daliphone, Manolom and Somphinith
Cover page pictures Clockwise from left to right: Croton roxburghii Wall.; fruit of Litsea cubeba (Lour.) Pers.; fruit of Sapindus rarak DC.; Tadehagi triquetrum (L.) H.Ohashi
List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I. De Boer, H.J., Vongsombath, C., Pålsson, K., Björk, L., Jaenson, T.G.T. (2010) Botanical repellents and pesticides traditionally used against haematophagous invertebrates in Lao People’s Democratic Republic: A comparative study of plants used in 66 villages. J Med Entomol, 47: 400-414; DOI: 10.1603/ME09273. II. Vongsombath, C., Pålsson, K., Björk, L., Borg-Karlsson, A.-K., Jaenson, T.G.T. (2011) Efficacy of essential oils, of traditionally used plant species, tested for repellence against mosquitoes (Diptera: Culicidae) at Vientiane, Lao PDR. Manuscript. III. Vongsombath, C., De Boer, H.J. (2011) Traditional use of plants to repel or kill blow fly larvae (Diptera: Calliphoridae) in traditional fermented fish production in Lao PDR: Evaluation of fly repellency and larvicidal activity. Submitted to PLoS ONE IV. Vongsombath, C., Pålsson, K., De Boer, H.J. (2011) Keeping leeches at bay: Field evaluation of plant-derived extracts against terrestrial blood-sucking leeches (Haemadipsidae) in Lao PDR. Submitted to Acta Tropica.
Paper I: CV, HdB and LB designed the field study and collected all field data. CV and HdB wrote the manuscript. All co-authors participated in the fieldwork. TJ revised the manuscript and was the corresponding author. Paper II – III: CV designed the experiments, collected the data and drafted the manuscripts. All authors analyzed and interpreted the results and revised the text. Paper IV: CV designed the study. All authors and TJ analyzed and interpreted the data. CV drafted the manuscript, which was revised by TJ and HdB.
The author is grateful to the Entomological Society of America and to Dr Alan Kahan, Director of Communications & Publications for permission to reprint Paper I that was recently published in Journal of Medical Entomology.
Contents
Introduction ...... 11 Traditional botanical repellents and pesticides...... 12 Mosquitoes, fly larvae and leeches ...... 13 Mosquitoes ...... 13 Myiasis ...... 13 Blood-sucking leeches ...... 14 Plants used in the experiments ...... 15 Aims ...... 17 Materials and Methods ...... 18 Study Sites ...... 18 Interviews (I) ...... 20 Steam distillation (II) ...... 20 Identification of chemical compounds (II) ...... 20 Extraction of plants (IV) ...... 21 Field experiments ...... 21 Experiment on mosquitoes (II) ...... 21 Experiment on fly larvae (III) ...... 22 Experiment on terrestrial blood-sucking leeches (IV) ...... 22 Identification of parasites and vectors ...... 23 Data analysis ...... 23 Results and Discussion ...... 25 Interviews on the use of plant-based repellents and pesticides (I) ...... 25 Repellence test on mosquitoes (II) ...... 28 Efficacy test on blow fly larvae (III) ...... 29 Repellence tests on terrestrial leeches (IV) ...... 31 Concluding remarks ...... 33 Lao Summary ...... 34 Sammanfattning (Swedish summary) ...... 37 References ...... 41
Abbreviations
CMPE Center of Malariology Parasitology and Entomology DEET diethyl-methyl-benzamid DF Dengue Fever DHF Dengue Hemorrhagic Fever GC-MS Gas Chromatography-Mass Spectrometry Lao PDR Lao People’s Democratic Republic US 622 Swedish commercial mosquito repellent containing 19% DEET NIST National Institute Standard and Technology NUOL National University of Laos SAREC Swedish Agency of Research Cooperation with Developing Countries SIDA Swedish International Development Cooperation Agency SPME Solid Phase Micro Extraction WREA Water Resources and Environment Administration
Introduction
Lao PDR (Laos) is located in the lower Mekong Basin and covers an area of 236,800 km2. Eighty percent of the country is mountainous while the remaining 20% comprises mostly flat flood plains along the Mekong River. The climate is tropical monsoon with two distinct seasons, the rainy season from the beginning of May to October and the dry season from November to April. Temperatures are highest in April and May (ranging from 30 – 39 C) and lowest in December and January (down to below 15 C). In the north and some mountainous areas elsewhere, mean temperatures can decrease to 10 C. Monsoon disturbance can cause heavy rains and flooding. Average annual rainfall ranges from 1300 – 3000 mm per year (WREA 2009). Laos is a country of high biodiversity. However only little research has been done on the use of plants or plant products for control of blood-sucking insects, ticks, mites and other disease-carrying or otherwise pestiferous invertebrates. Many diseases in Laos, e.g., malaria, dengue, Japanese encephalitis, and scrub typhus are transmitted by blood-sucking arthropods. They are problems for many people in both rural and urban areas, and may also affect the well-being of livestock. However, the national and international efforts, progress in control of malaria and other vector-borne diseases throughout the country, particularly the impregnated bed net project has been successful in the control of malaria. Thus, during the past 10 years, malaria infection rates have dropped considerably (Kobayashi et al. 2004, CMPE 2007). However, other infections, e.g., dengue has increased, especially during 2008 – 2010 dengue fever and dengue hemorrhagic fever (DF/DHF) were rapidly increasing; the total number of cases recorded were 4,149 in 2008, 7,214 in 2009 and 18,078 in 2010, respectively (WHO 2010). Dengue fever has been particularly severe in the urban areas and much more so than in the rural areas. This is a great problem for the country and the possibilities for controlling and treating dengue are insufficient. Thus, research concerning prevention of dengue and other vector-borne infections should be based on control of the vectors and is considered very important. This is a main reason why this research was initiated. It began in 2006 and has been funded throughout by Sweden and Lao PDR. The project has been a bilateral collaboration between the Swedish International Development Cooperation Agency (SIDA) and the National University of Laos (NUOL). The study program was run between NUOL as the applying institution and Uppsala University (UU) as the co-operating institution.
11 Traditional botanical repellents and pesticides
The main aim is to investigate which botanical repellents and pesticides are used traditionally against haematophagous invertebrates in Lao PDR. Thus, I attempted to investigate and to describe which types of local resources and indigenous knowledge that are used by the Lao people. Also, I wanted to study common vectors and parasites in each study area. What did they use for preventing, treating and how to use it? For many people in Lao PDR the use of plants or plant products is an important and economical alternative to the use of artificial, synthetic, usually expensive chemicals aimed at repelling or killing blood-feeding organisms. Thus, plant products may be used as natural insect repellents and biopesticides to repel or to kill biting insects and other blood-feeding invertebrates. Traditional medicines are largely plant-based and available at low cost, but the majority of Lao people do not use plant-based repellents against blood-feeding invertebrates, and there has been no government- initiated research on plant-based arthropod repellents until this study began. However, indigenous knowledge on traditional use of single plants or mixtures of plants as insect repellents or biopesticides is very diverse, and influenced by the specific vector or parasite to be targeted, and also by cultural aspects. A few examples of the various "pest control methods" used on the Lao countryside are: burning of dry plants, hanging plants inside the house, rubbing the plant directly on clothes or skin to repel mosquitoes and other biting insects, putting the plants under a bed or under the floor to repel bed bugs, putting the plants in the chickens’ nests to repel mites, putting the plants in the earthenware jars of fermented fish, putting a plant in or on the wound of cattle to repel or kill fly larvae, or rubbing the plants onto the shoes to prevent bites of land leeches. In the examples mentioned in the previous paragraph essential oils or chemical compounds in the plants are released or transferred and recognized by the pest organism. A corollary would be that the use of isolated essential oils from particular plants is relevant for scientific study. This research started by field investigations throughout the country. However, we wanted to focus mainly on rural areas in which there were problems with important infectious, vector-borne diseases, areas located far from urban areas; and we also wanted to focus on different ethnic groups with differences in knowledge about the use of plant-based arthropod repellents, insecticides, acaricides and similar pest control methods (Paper I). The information and the plant samples were analyzed.
12 Mosquitoes, fly larvae and leeches
As targets for our investigations we selected mosquitoes (Diptera, Culicidae), meat-infesting fly larvae (Diptera, Calliphoridae) and blood- feeding, terrestrial leeches (Hirudinea, Haemadipsidae).
Mosquitoes Mosquitoes are by medical entomologists considered to be the most important group of organisms for study; there are over 3,500 species worldwide (Eldridge 2005). Mosquitoes belong to the family Culicidae, order Diptera. The majority of mosquito species are members of two subfamilies: Anophelinae and Culicinae. Adult female mosquitoes are bloodsucking. Among important pathogens transmitted by mosquitoes are the malaria parasites (Plasmodium spp.) and several arboviruses including the yellow fever virus and the dengue viruses. The transmission of filarial nematodes causing human lymphatic filariasis by mosquitoes was the first vector-borne human pathogen to be identified. This discovery was done by Sir Partrick Manson in 1876 (Eldridge 1993). Other mosquito-borne pathogens were subsequently discovered, e.g. the malaria parasites in 1898, and that mosquitoes transmitted yellow fever in 1900, and dengue in 1902, and western equine encephalomyelitis in 1933 (Philip and Rozeboom 1973). In our field work in Laos, we found mosquitoes belonging to five genera: Aedes, Anopheles, Armigeres, Culex and Mansonia. Aedes mosquitoes are among the main vectors of arboviruses including dengue and yellow fever viruses. Anopheles mosquitoes mainly serve as vectors of malaria protozoa (Plasmodium spp.). Armigeres mosquitoes transmit the nematode Wucheria bancrofti (Al-Tawil 1979, WHO 2007). Some species of Culex, Aedes, Anopheles and Mansonia are also considered to be vectors of W. bancrofti.
Myiasis Myiasis is the condition when larvae of flies parasitize tissues or organs of living vertebrates. Such myiasis-causing fly larvae usually feed on tissues and/or body fluids of its living vertebrate host. Myiasis may be divided into three groups: obligatory, facultative, and accidental (Zumpt 1965). 1) Obligatory myiasis is when fly larvae infect wounds or epidermis and develop inside of the host. Examples of fly larvae causing obligatory myiasis are Cochliomyia hominivorax (the primary screw worm) and Chrysomia bezziana (the old world screw worm) (James 1969). 2) Facultative myiasis can be of two types, traumatic and cutaneous. Both types are frequently due to calliphorid blow fly larvae (Calliphoridae: Calliphora, Chrysomyia etc.). Myiasis is common in livestock in tropical areas (Zumpt 1965), but there is
13 also human myiasis, which is a common medical problem throughout the world, particularly in subtropical and tropical regions (Delhaes et al, 2001, Sukontason et al. 2005). 3) Accidental enteric myiasis is mainly due to fly larvae of the genera Musca and Calliophora. These fly larvae may invade the human digestive track after ingestion of food or liquid contaminated with fly larvae or fly eggs. However, in this type of myiasis the fly will not reach the adult stage (Sucharit 1998, Riley 1939). Adults of chrysomyines are important flies market places, for instance Cochiliomyia macellaria which lays eggs on meat; other common bazaar fly species are Ch. marginalis and Ch. megacephala (Greenberg, 1971). The waste, wet faecal material in latrines are suitable sources for breeding of Ch. putoria and Ch. megachela (Crosskey and Lane 1993). Flies are not only harmful in terms of public health but they also affect people's economy. Thus, Ch. megachela is a major cause of loss in the salted and dry fish industry in Southeast Asia (Esser 1991). The feeding activity of the flies are most obvious in places with garbage and organic waste from where it is easy for the flies to become infested with human pathogens which may easily be spread to other places. For example the common housefly, Musca domestica and Ch. megachela are important vectors for the dissemination of pathogens (e.g. Shigella sp., Vibrio cholerae, Escherichia coli, Staphylococcus aureus, Salmonella spp., and taenid tapeworm eggs) that cause numerous diseases in humans and animals (Greenberg et al. 1970, De Jesus et al. 2004, Forster et al. 2007, Sukontason et al. 2007, Lawson & Gemmell, 1990).
Blood-sucking leeches Blood feeding terrestrial leeches belong to the family Haemadipsidae which is subdivided into the duognathous and trignathous groups (Sawyer 1986). Their distribution patterns are rather distinct so that the Australian region is inhabited mainly by duognathous species while most leeches in the Sino- Japanese and Indian regions belong to the trignathous group, which includes the genus Haemadipsa (Jayaram 1949, Mann et al. 1962, Fogden and Proctor 1985, Enguang 2008). Leeches in this genus are particularly common in Laos and neighbouring countries of South-East Asia (Mann et al. 1962, Ngamprasertwong et al. 2007). Terrestrial leeches take blood meal from vertebrates mammals including humans. Haemadipsa spp. feed usually infrequently but when they feed they will usually take one large blood meal. The amount of blood ingested is usually 6-10 times the initial fresh weight of the leech (Mann et al. 1962, Fogden and Proctor 1985). In South-East Asia people are generally aware of the serious inconvenience and blood loss that may be caused by terrestrial Haemadipsa leeches. The bite of a leech does in general not threaten the life of the infested person. However, after removal of the leech, the bite wound
14 should not be scratched as this may cause secondary bacterial infection and delay healing (Heukelbach et al. 2009).
Plants used in the experiments
A total of 9 species was used in my studies (II-IV). Short descriptions of these plants are available in Newman et al. (2007), Gardner et al. (2000), and De Boer et al. (2010). Croton roxburghii Wall. (Euphorbiaceae) is a deciduous shrub or a small tree, growing in secondary and semi-open forest and widely distributed throughout Laos and neighbouring countries. The leaves of C. roxburghii are used in traditional medicine and the bark is used to repel mosquitoes by ethnic groups in Northern Laos and Thailand (De Boer et al. 2010, De Boer and Lamxay 2009). Hyptis suaveolens (L.) Poit. (Lamiaceae) originates from South America. It is regarded as an invasive alien weed in Southeast Asia (Maroon et al. 2004). The plant has a strong smell and grows preferably in open places with disturbed secondary vegetation and is found throughout Laos. In Africa it is used as a mosquito repellent (Sears 1996, Pålsson and Jaenson 1999, Seyum et al. 2002, Jaenson et al. 2006). In Lao PDR, this species is rarely used as a mosquito repellent. The only report of this plant in our interviews was in a single village in Southern Laos in which people had begun to use this plant by an incident: they discovered that as they cut down this weed for other purposes and burnt it, mosquitoes and gnats disappeared from their buffalos. This is the reason why H. suaveolens was selected for this study. Litsea cubeba (Lour.) Pers. (Lauraceae) is a small tree growing in young forest and in open places and is widely distributed in Laos. In large parts of South East Asia the plant is used in traditional medicine and for consumption. Crushed leaves, flowers, fruits and bark has a smell similar to that of Cymbopogon nardus (L.) Rendle. In some studies in Thailand this species shows potential as mosquito repellent (Santiwitchaya 2004, Trongtokit et al. 2005, Tawatsin et al. 2006) and it is traditionally used as an insect repellent by local people in northern Laos (De Boer et al. 2010). Tadehagi triquetrum (L.) H. Ohashi (Fabaceae) is small shrub, native to Southeast Asia where it grows on grassland, on edges of paddy rice fields, in gardens and on river banks. It is widely distributed, and is well known by the Lao name ˝Ya Non Nai", which means "killing fly larvae". Thus, people use the leaves and branches by placing them macerated in a jar with fish. It is also used in folk medicine for treatment of liver flukes (Libman et al. 2006). Uraria crinita (L.) Desv. ex DC. (Fabaceae) is a small shrub similar to T. triquetrum and grows on shady localities. This species was reported to be traditionally used as a wormicide, and to kill fly larvae in jars with
15 fermenting fish in Northeast Thailand, and Lao PDR (Chuakul et al. 2002, De Boer et al. 2010). Bambusa multiplex (Lour.) Raeusch. ex Schult. & Schult. f. (Poaceae). Bamboo shoots are used as food, especially in Asia. However, most species of fresh bamboo shoots are inedible due to their bitterness and toxic content of taxiphyllin. This cyanoglucoside has antifeedant properties and is present in all four species of bitter bamboo (Jones 1998). Informants also reported that shoots of bitter bamboos have an advantage as insect repellent compared to other insect-repellent plant species. Catunaregam spathulifolia Tirveng. (Rubiaceae) like other species of Catunaregam, is a deciduous thorny shrub or small tree, widely distributed through Asia and Africa (Kulakkattolickal 1989). Runyoro et al. (2006) reported that most species of this genus are known in folk medicine for their anti-inflammatory, antispasmodic and anti-dysenteric properties. Kulakkattolickal (1989) reported the use of C. spinosa as a piscicide and Lemmich et al. (1995) reported the use of C. nilotica as a molluscicide. Sapindus rarak DC. (Sapindaceae) is a tall tree that originates from Southeast Asia. It is not common although widely distributed throughout Asia and Africa (Wine et al. 2005). The fruit of Sapindus is rich in saponins, which have haemolytic and foaming properties (Pelegrini et al. (2008), Kamra et al. (2006) and Duke (2011). We found that local people used the fruit sap as soap for washing clothes and to protect themselves from bites of land leeches. Another common use of the fruit extract is as a molluscicide (Pelegrini et al. 2008, Duke 2011). Vernonia elaeagnifolia DC. (Asteraceae) grows as shrubs or vines and is widely distributed in Africa, Asia and Latin America (Georgewill 2009, Robinson et al. 2006). Belewu et al. (2009) reported that the major groups of chemical compounds in this plant are tannins, saponins, alkaloids, sterols and flavonoids. In Africa V. amygdalina is used orally as a folk medicine against leeches (Georgewill 2009, Belewu et al. 2009).
16 Aims
The specific aims of this thesis were:
1. To investigate by interviews what types (methods, plants, etc.) of botanical repellents and pesticides that is traditionally used against haematophagous invertebrates in Lao PDR.
2. Evaluate in the field the potential of essential oils, of different concentrations, from selected plant species to repel mosquitoes.
3. To evaluate selected plants to repel or to kill fly larvae infesting fish to be used for human consumption.
4. To evaluate selected plant extracts to repel terrestrial leeches.
After having completed the aims for my thesis as listed under # 1 – 4 in the previous paragraphs I intend to continue to work on the further development of plant-based products to obtain effective and optimal formulations of plant-based repellents to be used for protection of people against mosquitoes and leeches. One aim would be to introduce into the rural Lao villages, production methodologies by which the useful native plants can be grown, and processed with low technology. A second, later aim for me is to produce a popular scientific ‘easy-to- understand’ hand-book mainly to be used for people living in rural areas of South-East Asia. This village hand-book should provide information about how people can cultivate particularly useful plants, or where they can obtain potentially useful wild plants, and how they can thereafter prepare the plants to obtain repellents or pesticides to be used for control of disease-causing insects and other invertebrates.
17 Materials and Methods
Study Sites
Research data for this thesis were collected during multiple field research periods from June 2006 to June 2010. Interviews were carried out in a total of 66 villages with knowledgeable people from different ethnic groups. Twenty-three villages were located in the Northern provinces: Houa Phanh, Xieng Khouang, Luang Prabang, and Luang Namtha; thirty villages in the Central provinces: Vientiane, Bolikhamxay, and Khammouan; and another thirteen in the Southern provinces: Salavanh, Attapeu, and Champasack. All villages were geo-referenced using a Garmin E-trex Venture GPS receiver. The field experiments were done at three different locations based on the prevalence of the parasites or vectors (Fig. 1). Field experiments on mosquito repellence took place during March and May 2009, at Hauy Yang Forest Reserve (18° 04' 17.60" N, 102° 40' 20.08" E), Saythany District, and Vientiane Prefecture. This location is situated at an elevation of 178 m above sea level and is about 12 km from downtown Vientiane. The site was chosen because of its high abundance of Culex and Aedes mosquitoes, which is probably due to the nearby presence of suitable habitats for mosquito breeding such as small standing water bodies inside the forest, and paddy rice fields and other irrigated agricultural fields surrounding the forest. The study on fly larvae infesting fermenting fish was run during January and February 2010 at Ban Phonsavang, (18° 00' 9.55" N, 102° 37' 32.67" E), Chanthabouli district, Vientiane capital where the respondent, Chanda Vongsombath, and his family live. This place was chosen for logistic reasons including the convenience of observing and checking the experiment at all times throughout the experimental period. The experiment on terrestrial blood-sucking leeches was carried out at two sites during July and August 2009. Study site 1, Ban Mak Feuang (17° 50' 24.66" N, 105° 21' 30.45" E), Nakai district, and Khammouan province. The location is on an ancient point bar of the Nam Theun River, and is on a flat plain large enough to accommodate the village and some surrounding fields; the elevation is approximately 560 m above sea level. The village and the study site are located within the Naka-Nam Theun National Protected
18 Area (NPA). Study site 2, Ban Vang Lieng, (15° 14' 11.60" N, 106° 15' 15.09" E), Paksong district, Champasack province, the more commonly used name of which is the Bolaven Plateau, is on a highland plateau at about 1000 - 1200 m above sea level and is covered by grass, shrubs, and pine forests. The area is also rich in coffee plantations, and is important to Lao agriculture.
Figure 1. Map of Lao PDR. Villages located in this study are marked with yellow squares, number 1-66 corresponding to Table 1 paper I, Green squares with letter A, Ban Don Tiew, Field test on mosquitoes; B. Ban Phonsavang, located of fly larvae tested; C. Ban Mak Feuang, field test on leeches site 1 and D. Ban Vang Lieng, Field tested on leeches site 2. Map modified from Google Earth map by Anders Larsson, Uppsala University.
19 Interviews (I)
In each district, the district government and the district health care facility were contacted, and the research was introduced. Subsequently, villages were selected, and the research team together with representatives of the above authorities contacted the leader of the village (the village head). He would then consent to the research and identify a group of four to eight informants to be interviewed for the study. These informants were frequently elderly villagers, and would either be male or female, with no significant differences in gender ratio over the total of informants. Recording of interview data focused on extensive descriptions of the plant species used, the method of employment as a repellent or as a pesticide (to kill a pest), including the situations when it was used, distributions of the plant(s) and prevalence of each parasites or vector(s) in the village area as well as other neighbouring communities. All interviews were carried out in Lao and were either simultaneously translated to English, or during data entry on the same day. Data elicitation using structured interviews was used, with a fixed set of questions based on the most commonly recognized groups of blood-feeding invertebrates: mosquitoes, ticks, mites, leeches (subdivided into land leeches, nasal leeches, and aquatic leeches), lice, bedbugs, and fly larvae (subdivided into larvae-infesting wounds and larvae-infesting fermenting fish [pa dek]).
Steam distillation (II)
To obtain essential oils of plants, the oils were isolated by hydro-distillation. Fresh bark of C. roxburghii, the whole herb of H. suaveolens and fresh fruits of L. cubeba, 1 – 1.5 kg of each, were cut into small pieces, and put into a distillation flask with approximately 1.5-2 litres of water. Boiling was done during 3 hours and the essential oils separated from the condensed water. Isolated oils were stored in glass vials in darkness at 4 °C until used.
Identification of chemical compounds (II)
To identify the substances in the headspace above the H. suaveolens, L. cubeba and C. roxburghii oils, 100 l of the oils diluted 1/100 (v/v) in acetone, were added to filter papers and placed in separate glass beakers (50 ml) after evaporation of the solvent. After 15 min equilibration the volatiles
20 were collected by Solid Phase Micro Extraction (SPME) fiber coated with 65 m polydimethylsiloxane - divinylbenzene (Supelco, Bellefont, Pennsylvania) for 1 min and identified by GC-MS. A Varian 3400 GC connected to a Finnigan SSQ 7000 instrument with electronic ionization and o the ion source at 150 C was used. for volatile extraction. A SPB-1 capillary column (Supelco, Bellefont, Pennsylvania) was used (30m x 0.25 mm i.d.; film thickness, 0.25µm) for the separation of volatiles. For H. suaveolens the o o o temperature programming was 40 C (1 min) increase 5 C min-1 – 220 C (12 min). Initially L. cubeba and C. roxburghii were analysed using the same temperature programme. However, more separation of the compounds was needed for correct identification. The program was modified as follows: o o o initial temperature 40 C (1 min) increase 2 C min-1 – 100 C (2 min), and o increase 5 C min-1 – 220 (2min), the injector temperature was 215 oC and helium was the carrier gas with an inlet pressure of 10 psi (69 kPa) for the three oils. The compounds were identified by means of their retention times (GC-MS) and/or mass spectral data (MS) from authentic samples and the reference data bases Nist Library and Massfinder data bases.
Extraction of plants (IV)
Extraction of plant tissues was carried out to obtain plant extracts, with potentially active compounds, to be used for impregnation of stockings to investigate if such treatment would prevent terrestrial blood-sucking leeches’ from biting. Extracts of three plant species were tested. The extracts were from the dried fruits of S. rarak and C. spathulifolia and dried leaves of V. elaeagnifolia. A total of 200 g of each plant sample was sliced into small pieces, infused with 1000 ml cold distilled water and kept in darkness for 48 hrs. Each extract was filtered 3 times by using a poplin cloth to remove coarse particles. The solutions were stored in brown glass bottles at 4 °C until used.
Field experiments
Experiment on mosquitoes (II) The oils of C. roxburghii, H. suaveolens and L. cubeba were prepared as repellents in three concentrations: 5% (v/v), 10% (v/v) and 19% (v/v) oil dissolved in ethanol 95% (v/v). The white mosquito bed net cloths (30 x 50 cm) to be tied to the lower legs were sprayed 30 minutes before the test with either 0.5 ml/net of one of the oils in ethanol, or 0.5 ml/net of Myggspray US
21 622® (a Swedish commercial mosquito repellent containing 19 % diethyl- methyl benzamide (DEET)). Half a ml of the concentrations 5%, 10% and 19% corresponded to 1.7, 3.3 and 6.7 µg/cm² oil of the nets, respectively. In total, five volunteers assisted in each test: three persons had both their lower legs covered with net; one person served as negative control (used untreated nets), and the fifth person served as positive control (use DEET 19% treated nets). The volunteers were sitting 10 m apart and all mosquitoes landing on the nets on the legs were collected. The treatment on each assistant was changed every evening, so that everyone tested all concentrations of the oils and served as both negative and positive controls during the test. The whole test period lasted 15 evenings. Each day's test started at 6 pm and ended at 9 pm.
Experiment on fly larvae (III) Larvae of the latrine blow fly, Chrysomyia megacephala (Fabricius) (Diptera: Calliphoridae) were raised from eggs collected from a bait trap during the late morning (8:00 - 11:00), which was ample time for the female blow flies to locate the bait and to oviposit. The bait tray was moved to a hermetically sealed enclosure, and left for 36 hrs for egg hatching and larval development. Larvae were then collected and placed in the experimental containers. They consisted of 24 transparent plastic jars with a volume of 850 ml. Thirty fly larvae and 100 grams of fermented fish, as the feeding medium for the fly larvae were placed in each jar. Each jar was then covered with cloth netting to prevent other flies from entering, but still allowing larvae to escape from the jar. All jars were placed in a water bath to prevent entrance or disturbance by other predators. All containers were kept in one room for the whole experiment period. Three test plant species (T. triquetrum, U. crinita, B. multiplex) were used for two treatments (fresh or dried plant material), three amounts of plant weights added (20, 30 or 40 g), in triplicate, plus controls. Each 24 hours (at 24, 48 and 72 hours of incubation) the number of escaped larvae was recorded for each sample. After 72 hours the jars were emptied and the total numbers of dead larvae and surviving larvae were counted.
Experiment on terrestrial blood-sucking leeches (IV) The stockings used for the field tests were made of white cotton cloth (90 x 60 cm; 2 per pair). Three pairs of stockings were impregnated for 24 hrs with 100 ml of plant extract: S. rarak, C. spathulifolia, and V. elaeagnifolia, each corresponding to 1.9 mg/cm2. One pair was used as a positive control at Site 1 and was impregnated with 100 ml of deltamethrin solution, corresponding to 0.037 mg/cm2. At Site 2 DEET-treated (instead of deltamethrin-treated)
22 stockings were used as the positive control. This substance was sprayed directly on the stockings (1 ml per stocking or approximate 0.035 mg/cm2), and the stockings were treated 24 hrs before start of the field test (Nath et al. 2002). The experiment at each study site was conducted during 5 consecutive days between 9 and 12 a.m. The trail selected was based on a pilot study in which random quadrates were used to select areas with >5 leeches/m2 at both sites (Ngamprasertwong 2001), and inside each area a 170 m long trail was set out. The field work was carried out by six adult male assistants. Their daily treatment was assigned to them randomly with one of the following treatments: stockings impregnated with one of the three plant extracts or a positive control, untreated stockings as a negative control, or no stockings at all. The participant without stockings would walk in front with the purpose of arousing the leeches, based on the local traditional ecological knowledge that the person walking in front always gets the least bites. Each trial consisted of five replicates in which the six individuals walked the 170 m trail spaced roughly 10 m apart in 5 minutes. Following each replicate all leeches were collected, counted and stored in 40 % ethanol for later identification. At the end of the fifth day all 5 test persons had completed 5 different trails with 5 differently impregnated pairs of stockings (included the unimpregnated one).
Identification of parasites and vectors The samples of terrestrial blood-sucking leeches, mosquitoes and adult flies were labelled and brought to the laboratory for species identification using a dissection microscope and relevant identification keys. For identification of leeches the keys used were (Moore 1935, Sawyer 1986, Nesemann et al. 2001, Ngamprasertwong et al. 2007, and Borda et al. 2008). For identification of flies the key used was Sucharit (1988). Captured mosquitoes were identified using keys for female Anopheles according to Centre of Malariology, Parasitology and Entomology (CMPE 2000). Other mosquitoes were identified using a digital key called KeyMosq98 (Illustrated Key of Mosquitoes in Thailand, developed by the Department of Medical Science, Ministry of Public Health, Thailand 1998). Mosquitoes that remained uncertain after identification were taken to the CMPE in Vientiane to be compared with their mosquito voucher specimens.
Data analysis
Data of study I were analyzed and interpreted as expected species accumulation curves of the number of plant species reported as repellents to
23 assess the coverage of the sampling by using the Sobs (Mao Tau) function (Colwell et al. 2004) in EstimateS 8.0 (Colwell 2006). To test for consensus in repellents use among the villages Anthropac 4.98 was used (Borgatti 1996). Data were dichotomized, tested for similarity positive matches, and plotted by using non-metric multidimensional scaling (Kruskal and Wish 1976) following Puri and Vogl (2005). Distance measure and variance- regression to test for correlation between the species used per village and the independent variables, province, region, and ethnic group were analyzed by Bray-Curtis ordination in PC-ORD v4.25 (McCune and Mefford 1999). A study of scientific literature on the reported repellent use of all species was conducted to substantiate the traditional use found in the field study. Relevant scientific literature was located through Google Scholar. StatSoft Statistica 9.0 was used for statistical analyses of data of II-IV. The repellent activities of the plant extracts and oils were evaluated through a one-way ANOVA (Analysis of Variance) on the log-transformed [log(x+1)] total numbers of parasites or vectors recorded (collected) with a particular treatment. If significant differences among treatments were found the William’s mean [Mw = inv log ( log(x+1) / N)] were tested by Tukey’s Honestly Significant Difference (HSD) post hoc test to show the effect of each substance in relation to that of the control and each other. Percentage efficacy of each plant in Paper III was calculated as: % efficacy = (Mw repelled + Mw killed)/(Mw control survival) x 100. Percentage repellence for each plant extract (Paper IV), and distillation (Paper II)) was calculated using the following formula: % repellence = [(Mw no. parasites or vectors collected by untreated person – Mw no. parasites or vectors collected by treated person) / Mw no. parasites or vectors collected by untreated person] x 100.
24 Results and Discussion
Interviews on the use of plant-based repellents and pesticides (I)
The goals of this study were to document traditional knowledge on plants that are used to repel and/or kill haematophagous invertebrates, and to select some plants species to test with some common ectoparasites under field conditions. This is the first indigenous inventory on the traditional use of plants to repel and/or kill ectoparasites in Lao PDR. The interviews were carried out throughout the country, mostly in the rural areas. The baseline data of each village was listed in I. We recorded a total of 92 species in 123 different plant-ectoparasites combinations. The use of plants as repellents was found for 87 of the 92 species. The number of plants used was divided into two functions: repellents and/or for killing ectoparasites. Some plants were used for both functions, i.e., both as repellent and to kill. The selected plants used to repel and /or remove each parasite and local preparation mode are presenting in Table 1. The number of plant species per ectoparasite group is presented in Table 2. The saliency of each plant was analysed by using the total number of times each repellent species was mentioned. The most frequently reported species were Nicotiana tabacum L., S. rarak, and T. triquetrum, which were reported 50, 34, and 26 times, respectively. N. tabacum was used as both repellent and as pesticide to almost all ectoparasite. Villagers mentioned that smoking cigarettes was the best protection against mosquitoes and other biting insects. However, using tobacco may affect the user, especially women, children and other people. Common nuisance organisms mentioned in the interviews were mosquitoes, fly larvae, fleas (Siphonaptera), bedbugs (Hemiptera, Cimicidae), human lice (Anoplura, Pediculidae), mites and ticks (Acari) and leeches (Hirudinea, Gnathobdellida). Mosquitoes are well-known as important vectors of diseases. Fly larvae are a problem in fermenting fish for human consumption. Fermented fish is as a main traditional food for Lao people. This has the potential to cause intestinal myiasis when the food is infested with fly larvae. Many cases of ulcerative myiasis occur in livestock. Terrestrial leeches are common in the rural areas; these leeches are mainly of
25 inconvenience, annoyance and risk of secondary bacterial infection both to local people and visitors but also for livestock.
26 Table 1. List of plants tested as repellents and/or pesticides against blood-feeding organisms
No Lao name Scientific name Family Property Plant part Mode of preparation of plant by local used people 1 Ee Too Phee Hyptis suaveolens Lamiaceae Mosquito repellent Twigs, Burning dry plants leaves 2 Pao dong Croton roxburghii Euphorbiacea Mosquito repellent Bark Burning dry bark e 3 Si khai ton Litsea cubeba Lauraceae Mosquito repellent Fruits Rubbing the fresh plant to the cloths or skin 4 Sangphai (Nor) Bambusa multiplex Poaceae Larvicide Shoot Slice to small pieces, put in the jar of fermented fish, in wound of livestock 5 Ya Non Nai Tadehagi triquetrum Fabaceae Fly larvae Leaves Put in the jar of fermented fish, in repellent/pesticide wound of livestock 6 Nguan None Uraria crinita Fabaceae Fly larvae Leaves Put in the jar of fermented fish, in repellent/pesticide wound of livestock 7 Mak Keung Catunaregam spathulifolia Rubiaceae Leech Fruits bark Rubbing the fresh/dry plant to the repellent/pesticide shoes 8 Mak Sak Sapindus rarak Sapindaceae Leech Fruits bark Rubbing the fresh/dry plant to the repellent/pesticide shoes 9 Nguan Thaak Vernonia elaeagnifolia Asteraceae Leech Leaves Rubbing the fresh plant to the repellent/pesticide shoes/stockings
Table 2. Number of plant species used to repel and/or to remove (kill) ‘pest’ invertebrates per each pest group Number of plant Ectoparasites Repel Remove (Kill) species Bed bug (human) 16 16 0 Fly larvae (in fermented fish and wounds in cattle) 15 2 13 Lice (human) 15 0 15 Mites (poultry) 15 0 15 Mosquitoes (human) 15 15 0 Tick (human) 15 9 12 Pond leeches (human) 15 15 2 Terrestrial leeches (human) 11 7 11 Nasal leeches (human and pets) 6 0 6
Repellence test on mosquitoes (II)
The essential oils of the plants C. roxburghii, H. suaveolens and L. cubeba were field-tested in two experiments for repellence against mosquitoes. Numbers of mosquitoes collected were 644 in the first test and 549 in the second test. Approximately 91.5% of captured mosquitoes were identified to species. Seventeen species found, belonging to 5 genera: Aedes 4 species (11.9%), Anopheles 5 species (8.4%), Armigeres 1 species (33.9%), Culex 6 species (30.3%), Mansonia 1 species (7.0%) and unidentified (8.5%). There were significantly reduced biting activity between the oil treatments and that of the negative control (P<0.001), when combining the data from March and May. The repellencies were more than 50 % for all plant oils. There were no significant differences in mosquito numbers between the different collectors and plants species tested with the same oil concentration. DEET at 6.7 µg/cm2 was used as a positive control for each plant concentration and provided repellence of 82-85%. Combining the data of the first and second test showed that mosquito repellence (%) was similar in the two lower concentrations and increased in the higher concentration for all plant oils. All treatments were highly significantly different when compared to the negative control (P<0.001). The final estimated reductions in total number of mosquitoes (March and May combined) are presented in Table 3.
28 Table 3. Estimated % repellence, for each plant oil and oil concentration tested against mosquitoes
% Repellence for each oil concentration Preparations 1.7µg/cm2 3.3µg/cm2 6.7µg/cm2 Croton roxburghii 62.7 62.5 76.3 Hyptis suaveolens 56.8 56.7 67.0 Litsea cubeba 64.7 64.1 75.3 Pos. control (DEET) 85.5 84.5 82.5 Negative control 0.0 0.0 0.0
Efficacy test on blow fly larvae (III)
The three plant species, e.g. T. triquetrum; U. crinita and B. multiplex were tested for repellence and larvicidal activity on blow fly larvae infesting fermented fish in a controlled experimental setup. The analysis was based on separate and cumulative effects (repellence and killing) by the plant material on the fly larvae. Larval repellence was evaluated by the average number of larvae that escaped from and were found dead in the experimental container each day. For all three plant species, repellence increased with the plant’s weight. The repellence of fresh plant material was greater than that of dried plant material. Most fly larvae in the control jar remained in the jar and only a few escaped. Both types of plant materials (fresh and dried) were significantly repellent when compared to the control (Ffresh (11,24) = 46.553, p < 0.001 and Fdry (11,24) = 110.30, p < 0.001). Larval mortality was determined by counting the number of dead larvae in each jar after completing the repellence experiment (after 72 h). The mean numbers of larvae killed for each treatment were significantly different from the controls (Ffresh (11,24) = 78.942, p < 0.001 and Fdry (11,24) = 45.669, p< 0.001). Average mortality, with the fresh material was significantly higher for all species (29.6 %) than for dry material (19.0 %) (p < 0.001). In general, mortality increased when the treatment dose increased, especially with B. multiplex for which mortality was greater than for U. crinita and T. triquetrum (for both fresh and dry plant materials). The overall results show that fresh B. multiplex was the most effective larvicidal plant species to add to fermenting fish; (p<0.02 compared with T. triquetrum and U. crinita). The average larval mortality using B. multiplex, U. crinita and T. triquetrum was 25-51 %, 21-33 % and 18-24 %, respectively.
29 The total repellence is here defined as the total number of larvae repelled plus the total number of larvae killed, and is relevant in the context of the traditional use of these species to prevent fermenting fish from spoilage. The total repellence effect (repelling and larvicidal effect combined per sample) of T. triquetrum, U. crinita, and B. multiplex was 60-83 %, 77-90 %, and 60- 93 %, respectively. The repellence of T. triquetrum and U. crinita was significantly (p< 0.001) greater than that of B. multiplex, but B. multiplex had a greater larvicidal effect than that of T. triquetrum and U. crinita (p< 0.02). The total repellencies for all three species were similar, and no significant differences were found between the species, whether fresh or dry material. This shows that all three plant species were approximately equally efficacious as fermenting fish additives. Comparison of the use of fresh or dry material for repellent effect, larvicidal effect or total repellence shows that fresh material is significantly more effective in all cases. This corroborates the traditional use of adding fresh material on top of the fish in the fermentation jars. I found some reports on the traditional use of T. triquetrum, U. crinita and other species of Fabaceae to exhibit larvicidal effects in the traditional fermented fish production, and to prevent maggots to grow in the preparation of pickles in East and Southeast Asia (Lwin and Tu 1968, Liu 1952, Chuakul et al. 2002). We reported on the common traditional use of these plants by placing them on top of the fish in the mouth of the earthenware fermentation jars (I). The mechanism of repellent or larvicidal action is not known, but recent chemical and clinical studies of T. triquetrum from China reported that prenylated isoflavonoids are responsible for a mild anthelminthic effect in rabbits (Li et al. 2002a, Xiang et al. 2005) goats (Li et al. 2002b) and humans (Guangdong 1971). However, a clear relation between bioactivity of secondary metabolites and the traditional use to repel fly larvae from fermenting fish is absent. During the survey (I) informants suggested that the repellent effect was mainly due to the numerous scales on the leaves that would deter the fly larvae from crawling to the fish. Anyway this mechanical mode of action hypothesis does not account for the larvicidal effect of U. crinita, and it seems likely that so far unidentified bioactive constituents are responsible for its repellent and larvicidal effects. Bamboo shoots are use as food, but almost species of fresh bamboo shoots are inedible due to their toxicity and bitterness. Jones (1998) reported that the toxicity of bamboo shoots is caused by taxiphyllin, a cyanoglucoside with antifeeding properties. Hydrogen cyanide is the aglycone of taxiphyllin and is released through enzymatic hydrolysis when the vacuole of the cell is disrupted. It is highly toxic, and the lethal dose for humans is about 50-60 mg (Satya et al. 2010). The taxiphyllin antifeeding mechanism is present in all four common bitter bamboos, and informants reported that all species were equally suitable to repel fly larvae in fermenting fish production. Literature on the use of bamboo shoots in the preparation of fermented fish
30 in other countries in East Asia has not been found, nor have we found any previously published studies on this topic.
Repellence tests on terrestrial leeches (IV)
The evaluation of aqueous extracts of S. rarak, C. spathulifolia and V. elaeagnifolia was conducted in the field. The aim was to evaluate the efficacy of extracts of these plant species to repel terrestrial leeches. The efficacy of the extracts was determined by the mean number of leeches attached to stockings worn by men. Three species of leeches were found at the two study sites: Haemadipsa sylvestris, H. zeylanica, and H. trimaculosa. A total of 738 terrestrial blood- sucking leeches were collected: 249 specimens from Site 1 and 489 from Site 2. The results of tests showed that the plant extract-treated stockings were repellent to the leeches. Thus, untreated stockings attracted significantly more leeches than the treated stockings at both sites (p<0.01). For instance, S. rarak-extract-treated stockings attracted means of 0.7 and 1.9 leeches while the untreated stockings attracted means of 4.2 and 10.4 leeches at test site 1 and test site 2, respectively. C. spathulifolia and V. elaeagnifolia- treated stockings also had lower numbers of leeches compared to the untreated stockings. The number of leeches recorded on stockings among persons was not significantly different. However, for site 1 the mean number of leeches recorded per day during the 5-day test period dropped significantly (F(4, 120) = 3.86, P = 0.005) suggesting that the repellent activities of the substances were partly lost during the test period. Probably, this was due to the very wet and muddy conditions at this test site. This made the impregnated stockings wet and dirty. Thus, we conjecture that microbial or other degradation and/or a “washing-out effect” of the active ingredients may have caused the more rapid activity decline of the repellents at Site 1. At study site 2, the mean number of leeches recorded during the different days was not significant different F(4, 120)=0.11049, p=0.979. This suggests that the persistence of the different active substances did not decline markedly during the 5-day test period. Site 2 may be described as grassland with little mud on the ground and with less moisture than at Site 1. The impregnated stocking got less wet and dirty at Site 2. Thus, the active compounds did not degrade as rapidly at Site 2 compared to Site 1. The final results suggested that stockings impregnated with aqueous extracts exhibited moderate to high leech repellent activity: C. spathulifolia (62.6 %), V. elaeagnifolia (63.0 %), S. rarak (82.6 %), deltamethrin (73.1%) and DEET (88.4%).
31 Previous studies have reported on the potential repellent activity against terrestrial leeches of both synthetic and plant-derived chemicals (Walton 1956, Nath et al. 1993, 2002, Kirton 2005, Tawatsin et al. 2006, Enguang 2008). The plants used in our study have, to our knowledge, not been previously tested as leech repellents. Our tests showed that terrestrial blood- sucking leeches can be repelled from human's feet by wearing stockings impregnated with water extracts of S. rarak, C. spathulifolia or V. elaeagnifolia. The extracts exhibited high degrees of repellence to leeches, and persisted for at least two to three days. In particular, S. rarak extract seems to be highly effective. Stockings impregnated with extract of this plant can be used to substantially reduce the risk of being bitten by leeches. All field staff used all test substances; none of these volunteers noticed any adverse dermatological or other adverse effects from the substances. The plant materials can be found locally and can be obtained at very low or no cost. Thus, the method appears highly effective, cheap and safe. After detailed investigations of the plant extract(s) concerning potential toxicity to humans and/or other organisms, including potential environmental side- effects it may be possible to recommend this method to people working in locations infested with blood-feeding leeches.
32 Concluding remarks
By interviewing people in many parts of Lao PDR, we obtained data on which plant species that were traditionally used as repellents and pesticides. It is the first study of this kind in Laos. The data on the use of botanical repellents in Laos are representative for each region and reveal differences of indigenous knowledge among ethnic groups. Nine different plant species were used for more intensive research in my investigations. They appear to have significant arthropod-repellent and pesticidal properties. In view of their efficacies they may be used to prepare repellents and/or pesticides against mosquitoes, fly larvae and terrestrial leeches. The repellence of C. roxburghii and L. cubeba oils may be due to their contents of -pinene and -pinene in Croton oils; and sabinene, - phellandrene, 1,8-cineol and -pinene in Litsea oils. These compounds have previously been identified as insecticidal, pesticidal and insect repellents. This explains why local people use both C. roxburghii and L. cubeba to repel mosquitoes. The Hyptis oil is also effective as a mosquito repellent, but its use as a mosquito repellent in Laos is rare. This plant grows as a weed and can be found throughout Laos. Thus, it would be appropriate to teach Lao people how Hyptis might be used to repel mosquitoes. The efficacy of T. triquetrum, U. crinita and B. multiplex to repel and/or to kill fly larvae in this study is confirmed. T. triquetrum and U. crinita are highly effective. Regarding B. multiplex, bitter bamboo shoots from other species may be more effective compared to B. multiplex. S. rarak, C. spathulifolia and V. elaeagnifolia exhibited high degrees of repellency to terrestrial leeches. By using stockings impregnated with water extracts of these plants an option is given for people to prevent leech bites. In particular, S. rarak extract has high potential for use as a practical prevention method in order to substantially reduce the risk of being bitten by leeches. Natural plant-based repellents for impregnation of stockings would be good alternatives to synthetic chemicals. Especially since the latter are usually more expensive and less easily available to people in rural areas. Further research in order to develop more effective formulations of plant- based repellent is needed. Production of plant-based repellents could be introduced into the Lao villages where the native plants can be grown, and subsequently processed by villagers, initially under scientific guidance from the National University of Laos.
33 (Lao Summary)
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35 . , 80% ( ) Deltamethrin 25% 3-4 ( ).
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36 Sammanfattning (Swedish summary)
Blodsugande ryggradslösa djur såsom blodiglar, fästingar, vissa kvalster och stickmyggor samt myiasis-orsakande flugor är mycket vanliga och betydelsefulla medicinska problem i Laos (Lao Peoples Democratic Republic, Lao PDR) och andra regioner av Sydostasien. Ett av huvudsyftena med min forskning under åren 2006 till 2010 var att dokumentera den traditionella kunskap som finns hos olika etniska folkgrupper i Laos, speciellt den kunskap de besitter om hur man kan använda växter till att förhindra att insekter, spindeldjur och iglar kommer åt att suga blod av människor och husdjur. Detta kan man åstadkomma med växter, växtextrakt, essentiella oljor, torkade blad eller andra produkter av växter, som innehåller ämnen som avvisar (repellerar) eller dödar de blodsökande organismerna. Vi intervjuade människor i 66 olika laotiska byar tillhörande 17 etniska folkgrupper (I). Vi fann att människorna av tradition brukar använda åtminstone 92 olika växtarter (i 123 olika växt – "skadedjurs"- kombinationer) med syftet att repellera och/eller döda de ryggradslösa skadedjuren (= djur som generellt sett betraktas som oönskade). I vetenskaplig litteratur fann vi belägg för att 74 av växtarterna används mot insekter eller andra evertebrater. Vid litteratursökningen fann vi ytterligare 13 växtarter, som är närbesläktade med arter vi noterat under intervjuerna. En slutsats av denna undersökning är att landsbygdsbefolkningen i Laos i hög utsträckning använder växtbaserade repellenter och pesticider för att kontrollera eller bekämpa ryggradslösa s.k. skadedjur. Vissa av växtarterna från enkätstudien undersökte vi experimentellt; det rörde sig om arter som av landsbygdsbefolkningen i Laos används på grund av att derivat av dem påstods ha potential att repellera och/eller att döda stickmyggor (Culicidae), fluglarver som orsakar myiasis (Diptera, Calliphoridae) eller landlevande, blodsugande iglar (Hirudinea, Haemadipsidae). Tre växter som traditionellt används för att repellera stickmyggor är Croton roxburghi (Euphorbiaceae), Litsea cubeba (Lauraceae) och Hyptis suaveolens (Lamiaceae) (II). Hyptis används i Sydamerika och Afrika mot stickmyggor, men detta sätt att använda växten är ovanligt i Laos. Essentiella oljor av de tre växtarterna undersöktes i fältförsök nära huvudstaden Vientiane, i syfte att studera oljornas förmåga att repellera stickmyggor. De vanligaste stickmyggorna i försöksområdet tillhörde släktena Armigeres och Culex. Vi fann att relativt låga koncentrationer (1-7 ug/cm2) av oljorna från de tre växtarterna reducerade
37 stickmyggaktiviteten med åtminstone 50 % under den första timmen av experimentet, som började vid skymningen. Oljorna undersöktes med avseende på innehåll av terpener. Huvudkomponenterna i oljan av H. suaveolens var 1,8-cineol, sabinene och -pinene. I oljan från färska frukter av L. cubeba påvisade vi sabinene, -phellandrene, 1,8-cineol och -pinene; och i Croton-olja fann vi -pinene och -pinene. En slutsats av studien är att det vore värdefullt att lära ut bland befolkningen i Laos att Hyptis, som växer allmänt i landet, kan användas för att avvisa stickmyggor. I ett annat experiment undersökte jag den flugrepellerande och flugavdödande effekten av tre andra växtarter (III). Här var syftet att förhindra fluglarver att utvecklas i fisk som för att kunna användas till mänsklig föda, först tillreds genom jäsning i stora kärl. Spyflugor attraheras till fisken där flughonorna lägger sina ägg. De tre växter som jag undersöktes var Tadehagi triquetrum (Fabaceae), Uraria crinita (Fabaceae) och Bambusa multiplex (Poaceae). Alla tre hade signifikanta, repellerande och avdödande effekter på larver av spyflugan Chrysomyia megacephala. I ett tredje fältexperiment undersökte jag den potentiellt repellerande effekten mot landlevande blodiglar av vattenextrakt av tre växtarter, nämligen Sapindus rarak (Sapindaceae), Catunaregam spathulifolia (Rubiaceae) och Vernonia elaeagnifolia (Asteraceae) (IV). Extrakten impregnerades på olika par av strumpor som därefter bars på fötterna och nedre delen av underbenen av personer i två områden där blodiglar är vanliga. Som positiva kontroller använde vi de i handeln vanligt förekommande, syntetiska myggrepellerande ämnena diethyl-methyl- benzamid (DEET) och permethrin. Växtextrakten av de tre arterna samt DEET och permethrin uppvisade alla signifikanta blodigelavvisande effekt. Jag kommer inom kort att fortsätta detta forskningsarbete i Laos för att kunna ta fram mer optimala, dvs. effektivare, billigare och mindre energikrävande repellenter, insekticider, acaricider och blodigelavvisande medel och metoder som baseras på kemiska ämnen, extrakt eller oljor från växtriket. Målsättningen är alltså att utveckla metoder, som baseras på naturliga ämnen, som är lämpligare att använda jämfört med de syntetiska kemikalier som vanligtvis används, för kontroll eller bekämpning av blodsugande och sjukdomsöverförande organismer. Enkla och billiga växtbaserade kontrollmetoder mot blodsugare skulle kunna introduceras för användning och egen produktion i byarna i Laos. Råmaterialet – växterna – kan vanligtvis utvinnas från "vilda" växtpopulationer eller från sådana som odlas i eller i närheten av byarna. Initialt tänker jag mig att framställningen av dessa produkter och metoder, för användning mot blodsugande organismer, kan och bör ske under ledning från NUoL, Vientiane. I ett senare skede bör produktionen kunna ske av människorna i byarna - utan behov av bistånd utifrån.
38 Acknowledgements
Accomplishment is not easy. I was born in a country that was the subject of a secret war between powerful nations fighting or supporting communism in neighbouring Vietnam. A large part of my youth I spent living and hiding in the forests of Houa Phanh province, Laos. We would have a makeshift bamboo hut in the forest that would serve as school, a volunteer as teacher, but no teaching materials. During my teenage years stability came to Laos with the independence from colonial powers. Growing into my adult years I have always believed that education would be a key to development and progress, and have done my best to teach students in Vientiane. Nearly halfway my life, I got an opportunity to pursue doctoral studies at Uppsala University in Uppsala, in a country far away from my hometown, with a radically different environment, culture, language and academia. All of these aspects were presented as challenges in my life throughout the period that I was studying at the Department of Systematic Biology, EBC Uppsala University. I have always thought how can I be successful in my work? Without much confidence, but with a strong belief that diligent learning would get me there, I adapted to new conditions through learning by doing. I have been lucky to have met so many kind and friendly new colleagues at the department, all of whom sincerely supported me, especially my close friends Hugo de Boer and Anneleen Kool. Our strong friendship has been of key importance to success during my studies, from beginning to end. During my studies in Uppsala, I have had to confront many obstacles, both linguistic and academic, but also domestic, relating to the health of my son. Through good fortune, and with guidance and advice from all of my supervisors, I have overcome these challenges and can defend my dissertation this day. Therefore, I would like to thank all of those who kindly helped me here in Uppsala, and let you all know that I will remember you forever. First of all, I would like to express my special and deepest thanks to Professor Thomas Jaenson, an outstanding supervisor, for his scientific advice, practical insights, invaluable support, and steady encouragement for all my scientific endeavours. Secondly, I would also like to thank my co- supervisors: Associate Professor Dr. Lars Björk for his practical advice and good company during fieldwork in Laos; Assistant Professor Dr. Katinka Pålsson for help with field experiment design, statistics and chemical
39 analyses; Professor Anna-Karin Borg-Karlsson for fruitful cooperation and facilitation on chemical analyses; and last but not least Hugo de Boer, both as co-researcher and co-supervisor, for his role in initiating this PhD project, help during fieldwork, and inexhaustible scientific exchange on data analysis and manuscripts. My PhD project was funded by the Swedish International Development Agency – Swedish Agency of Research Cooperation with Developing Countries (SIDA-SAREC) through its bilateral program with Laos, and I hope that this thesis shows that their investment and trust has paid off. Through their support, and similar initiatives, we are creating better education and futures for many students in developing countries. I am very grateful to the Faculty of Science of the National University of Laos to give me this opportunity to study, and also to the kind cooperation and help from former Biology students, district governors, and leagues of villagers who shared their time and knowledge during fieldwork in Laos. I would like to thank the Department of Systematic Biology, EBC, Uppsala University for providing supervision, space and facilities during my studies. Thanks to all members of EBC, its technical staff and PhD students for your kindness, friendship and help. In particular I would like to thank Håkan Svensson and Stefan Ås for their great help every time I had any problems with my computers (more than once!), and to Anders Larsson for help creating maps. I would like to thank to all friendly and helpful people at the Lao Embassy in Stockholm for logistical and cultural support, as well as their kind friendship during my visits to the Embassy; and my Lao friends in Stockholm and Uppsala with whom I shared all difficulties and happiness in a country far away from home. Lastly, and most importantly, I am very proud and grateful to my beloved wife Phienthong Vongsombath, who in my frequent and prolonged absences during these years has been responsible for our whole family; thanks to my daughters and son for being the best children a father could wish for; and all my extended family. Thank you all for supporting me and helping me overcome all challenges in fulfilling my work.
Uppsala, April 10th 2011 Chanda Vongsombath
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