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 o
SSQ 7000 instrument with electronic ionization and
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
o
of volatiles. Foro H. suaveolens the
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.
o
The program
o
was modifiedo
as follows:
initial temperature -1
40 C (1 min) increase 2 C min – 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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34
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35
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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

40
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