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THEVETIA PLANT ECONOMIC POTENTIAL:

CHEMISTRY’S KEY POSITION
COURTESIES
The Vice Chancellor,
Deputy Vice-Chancellors (Academic & Administration),
Other Principal Officers of the University,
Provost, College of Health Sciences,
Deans of Faculties, Postgraduate School and Student Affairs,
Professors and other members of Senate,
My Academic and Professional Colleagues,
Non-Teaching Staff of the University,
My Lords, Spiritual and Temporal,
Great Unilorin Students,
Distinguished Invited Guests,
Gentlemen of the Print and Electronic Media,
Ladies and Gentlemen.
INTRODUCTION
I give honour, adoration and glory to God Almighty for
the unique opportunity to serve this great University as a pioneer
member of academic staff and and for seeing me through today
to give this inaugural lecture. I am doubly grateful to God and
the University authorities for honouring me to give this lecture
today, during a historic event, the convocation ceremony.
Ordinarily, the inaugural lecture should have been shifted, but
because the University Management and I believe the governing
Council wish to honour me for whatever reasons they may have
adduced. I remain particularly very grateful for this honour.
One common characteristic of plants and animals is life,
similarly both plants and animals feed for survival. In most
cases, animals feed on plants, this simple mode of feeding interrelationship
is referred to as herbivorous.
God at creation provided a model for the study of
science and the perfect food chain model. God created the very
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basic essentials for life first i.e. air, water and light. Thereafter,
He created plants and finally animals. Man was created as the
king of animals and given the authority to take control over all
He had created. Man exercises the control in progression as his
knowledge of the environments, particularly plants, increases. As
the population increased, survival challenges increased,
adaptation and utilization of the plants for his good also
increased. These conditions for survival called for better
knowledge i.e. study of the plant, became more and more
challenging. There arose a steady need to explore plants around
him to his economic advantage.
Farming, in the simple concept, is the multiplication of a
plant to meet the need of man, consequently increase in
population demands increase in scope of farmland and farming
technology. Farming also calls for selection of crop with
preferred qualities, this accounts for man’s activities whereby a
crop native to a location readily gets migrated by man to a
foreign land. The colonial explorers and traders who migrated in
the early history contributed very largely for easy spread of
plants from one region of the world to others.
Virtually all plants cultivated in plantations today were
once plants that grew in the wild, in their natural environment.
We are all familiar with fruit trees, e.g. mango and orange, palm
trees, and other economic tress, how man has cultured each to
maximize their plantations. Plantations today have developed
beyond just large scale farming; care is given to selecting best
variety for propagation.
Thevetia Plant
Thevetia plant is a tropical shrub which grows in the
wild and remains ornamental, despite the abundance of the plant
around our homes, schools and other buildings. The plant is
grown as hedges and kept for its bright and attractive flowers.
Thevetia plant is recorded to be more than 2000 years in its
native countries – West Indies, Brazil and Mexico. It was taken
to Europe about three hundred years ago, and today it has
naturalized in virtually all countries in the tropics. Thevetia plant
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thrives very well in all the climatic and vegetation belts of
Nigeria, it is readily found in Port Harcourt and in Maiduguri or
Sokoto. To date, thevetia plant remains a plant of no economic
value whereas it has a lot of potentials. This is the focus of this
lecture.
Before I go into serious business of the lecture, Mr. Vice
Chancellor sir, please allow me to introduce you to the plant of
the lecture.
Thevetia Plant Morphology
Thevetia plant is a dicotyledon which belongs to the
Aponaceae family. It is a composite, evergreen shrub, which is
found to have a milky sap. It is native to West Indies, Mexico
and Brazil. It is known as yellow oleander (nerium), gum bush,
bush milk, exile tree in India, cabalonga in Puerto Rico, ahanai
in Guyana, olomi ojo by Yorubas in Nigeria. The plant is a
shrub, reaching a height of 3 to 3.9metres. The plant is perennial;
the leaves are linear, narrow, sword-like and green. The flower is
yellow flute which develops to a fruit which has a pair of
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follicles or drupes, it has one to four compartments, each
containing a seed. The fruit when unripe is hard and green but
gradually turns black as it ripens. The fruit has varying masses
(2-6.1g) which are dispersed by man and propagated by seed or
stem. The plant fruits virtually ten out of the twelve months of
the year. The seed contains about 60 – 64% oil on dry matter
basis. The plant produces white latex (sap) that is highly
poisonous. The seed also is highly poisonous. This attribute
accounts solely for the lack of interest in the development of the
plant. In spite of the toxicity of the plant, it has found useful
applications in several spheres of life. Its latex is used as an
analgesic for toothache when the stem part is chewed in Juccata;
and as an insecticide, the latex or extract of the stem as vesicant
and the bark as a febrifuge and effective abortificacent. The
wood is used as axe handle.
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THE CHEMIST IN ACTION ON THE POTENTIALITY
OF A PLANT
Curiosity is one dominant factor a natural product
chemist possesses to explore his environment, particularly the
plants. The plant everybody does not pay any attention to could
readily engage the research interest of the natural product
chemist. It is an in-built trait for a chemist to notice a peculiarity
of a plant for his research and kick- start the study of the plant.
On the other hand, a plant scientist of any discipline say,
herbalist/pharmacist, agronomists/plant breeder, a nutritionist etc
may, by questions in his area of interest on a certain plant
material, stimulate interest of a chemist to pay attention on the
plant. Even in the event of the latter, the chemist will only accept
the challenge and do something meaningful if he is curious and
zealous about the problem.
Every plant is a stored-up treasure (nature) God has
provided for the exploration of man. It is like the inorganic
mineral and crude oil deposits. The deposit remains a thing of
potentiality until man ventures to explore it and analyze it for its
constituent and adapt these to the advantage of man. The
petroleum deposit in Nigeria did not get there after our
independence. Indeed had the British government a good
knowledge of the deposit and had explored this, our
independence would have been delayed. All the same the
exploration and development did not start until some people
showed interest, invested money and energy. The Obasanjo
administration had shown interest in the solid mineral
exploration, this is the initial impetus; there remains a need for
good implementation by all concerned. For now, most solid
minerals in Nigeria still remain potential as far as their
contribution to the economy of the country is concerned.
Like the solid minerals and crude petroleum deposits,
every plant is a treasure that must be explored to give man
maximum benefit of the plant. The plant, as we have studied at
the elementary school, is made up of the leaves, stem, root and in
most common plants, flowers and fruits. Many of the plants
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around us are used in their natural forms, deriving only basic and
minimium benefits. Plants that have been explored by all
scientific input have been developed at varying degree for
varying level of benefits to man. It is common knowledge that a
fruit tree produces fruits at its season, so its product is available
for a short period even when the fruit provides valuable nutrients
we need throughout the year. Unless the fruit is processed, the
services derivable from the fruit shall be limited.
Plants influence the good life of man in various spheres
such as in medicine, food and industry (agro allied industry). The
plant however shall remain potential or partly developed until
the chemist makes his contribution on the constituents of the
plant. For instance, malaria was a deadly disease until the
discovery of cinchona plant which contains quinine. The plant
even after man’s initial knowledge of its herbal proficiency
certainly required the input of the chemist to identify the active
ingredient and eventually work to supply and meet demand of
the drug, quinine.
Scurvy was a disease that was ravaging among sailors
and treated by taking fresh fruits. With the input of the chemists,
vitamin C (ascorbic acid) was mass produced and put on hold the
disease henceforth.
The role of the chemist goes beyond identification of the
active ingredient in a plant; he is also saddled with the
responsibility to synthesize the compound as a true replicate of
that which the plant biosynthesizes. By this singular role, he can
supply the populace beyond what the plant can, even if
plantations are developed. Ability to synthesize helps to provide
improved variety and new drugs and other chemicals in other
applied fields.
Plants Position in the World Economy
Plants in various forms provide the raw materials for
every agro—based industry. This sector accounts for a high
percentage of the economy of every nation. Countries not
endowed with petroleum deposit or mineral deposits have no
option other than develop its agricultural sector to supply raw
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materials for the agro-based industries. Nigeria was in this
position until the 70s when crude petroleum accounted for the
foreign exchange of the nation.
Plants may be classified to include oil seed plants, which
today contribute tremendously to the economy of all nations
even the developed countries. Malaysia and Indonesia invested
in the development of palm tree in the mid 50s, today this single
crop/plant gives the nation a high percentage per capital and
foreign exchange earnings.
Oils and fats are products of plants which produce fruits
and majority of plant fruits/seeds contain oils/fats in varying
quantities. A plant is referred to as an oil/fat seed plant when its
oil/fat is worthy of extraction for one use or another.
There are about twenty major oil seed plants recognized
worldwide today as oil seed plants. Nigeria is rated
underdeveloped judged by the state of the development of its oil
seed plants. Nigeria NIFOR has it on record to have provided the
first set of palm oil seedling to Malaysia in the 50s. Nigeria was
once rated as a world leading palm oil producing country up till
the 80s. Since the discovery of the crude petroleum, Nigeria had
abandoned the palm tree plantation and turned to import palm oil
for its industries requiring vegetable oils. Nigeria climate and
vegetations are best suited to develop almost all the following
major oil seed plants - palm tree, coconut, groundnut, cotton,
castor, sunflower, jathropha, melon, soybean, beniseed and shea
butter. It is sad to note that Nigeria is not noted in the production
of any of these among the first twenty leading producing
countries of the world.
Basic requirements to the emergence of a plant as an
economic plant
Every plant in the first instance grows in the wild. When
man discovers one use for the crop or product of the plant, he
invests energy, time and his saving to explore the potentialities
of the crop. To achieve early dividend in the exploration, a
combined efforts of each of the following agents must be
coordinated: the research team (universities and research
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institutions), crop farmers association, government agents,
industries and bankers. The emergence of a few selected oil seed
crop plants from wild to economic status is highlighted in this
section. Each plant is selected for at least one peculiar property
worthy of development for an existing application. Whereas in
the distant past, the emergence of a plant as an economic plant
was basically dependent on the quantity of the oil, today oil yield
is secondary, whereas individual characteristics now become
prominent, as will be seen in the following four oilseed plants.
Jojoba1
Jojoba is a slow-growing perennial plant, native to the
Sonora Desert of Arizona; California and Mexico. Its oils
composed of liquid esters with major components 40 and 42
carbon wax esters. The plant was first commercially harvested in
the US in 1982. Its plantations are being established in India,
Australia, South Africa and the Middle East. Jojoba oil is
specialty oil for use in cosmetic, this prospect excited researchers
to reach higher heights for the promotion of the plant; its oil and
oleochemicals. There are multi-tract approaches on the
production of jojoba seed and oil. Productivity average in 1991
was 226 pounds per acre and it is projected to quadruple by
2010, going by the investment on the plant production in 2004.
Lesquerella1
The plant belongs to the mustard family and is native to
North America. It is a potentially valuable source of hydroxy
fatty acid, supplementing castor seed oil. It is seen as a potential
drought – tolerant oilseed crop that could produce fatty acids for
lubricants, plastics, protective coatings, surfactants and
pharmaceuticals. One of its fatty acids is lesquerolic acids (14 –
hydroxy cis – 11- eicoseneoic acid) a hydroxy fatty acid, similar
to the castor oil derived recinoleic acid but two carbon atoms
longer.
Meadow foam1
Meadow foam is a slow growing herbaceous winter,
annual, wild flower, native to the Pacific North West. It is still at
experimental and developmental stage as a good alternate seed
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oil. Its emergence success as a world oil-seed is hampered by the
competition its oil faces in the world market. The high price of
the oil and the need to increase yield is a major deterent. Good
funding to farmers and research will facilitate early market
impact.
Cuphea1
Cuphea belongs to the family lyathraceae, a wild plant
which still must be domesticated to gain commercial value. Its
oil is rich in medium chain fatty acids. It competes favourably in
the market with coconut and palm kernel oils. The latter two are
tropical plants. The oils are well adapted to the manufacture of
soaps, detergents, surfactants, lubricants and related products. Its
oil is a good source of capric acid which is currently obtained
principally from petroleum source.
Mr. Vice Chancellor Sir, I wish to mention in passing at
this point that for any plant to transform from wild to economic,
concerted, collaborated efforts of various bodies must be
supported by government policies and adequate funding.
Contributions of oils and fats in the world economy
Each oil seed crop contributes it quota into the world
pool of oils and fats. Invariably oils and fats have uses first in the
domestic sector as part of food, and then conversion to other
products now called– oleochemicals – in the industrial sector.
Soybean is one major oil in the oleochemical industry and has
one of the largest adaptations and conversion products. Scheme 1
gives at a glance major new uses of soyabean in the
oleochemical sector.
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Scheme 1 New uses of soybean oil
New uses
Products Soy ink Soy toners Plasticisers Soy protein Biolubricant soy oil soy polyols
Competitizers adhesives
Products Printing copiers PVC, Plastic wood transports cosmetic plastics
Markets Inks & laser adhesive
printers
Consumptions
(M. Bush/Y) 100 20 100 150 100 1 100
Source: INFORM (2005) 16 (10) 660
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Oils chemists and oleochemicals manufacturers with
higher concern are ever becoming more and more anxious over
rates of production vis-a-viz consumption and demand for oil
and oleochemicals. The concern and anxiety comes from the
ever increasing demand by the oleochemical sector for oils that
primarily were once for food. If prices of food containing
vegetable oils will not fly out of reach of the common masses,
there must be matching new sources of oils that shall specifically
be oleochemical concern only. Towards this general concern,
Frank Gunstone2 in his lecture at the Stephen Chough Award by
AOCS in May, 2006 attempted to answer the question “will oil
and fat supply meet oil and fat demand in 2007?” He used
available figures for the 15 years (1990 to 2005 to consider what
supply and demand will be in the next 15years i.e 2005 – 2020.
He classified oils and fats uses into three categories. These three
classifications have oils and fats distribution as 80:14:6. The
ratio is changing rapidly, principally due to high demand for
biodiesel alone. Table 1 presents the supply and uses of oils and
fat in 1990 and 2005 based on 4 animal fats and 13 vegetable
oils.
Table 1: Oils and fats: supply and usage (MMT)
Supply Food Oleochemicals Others
1990
2005
80
135
64
108
11
19
5
8
Source: INFORM (2006) 17 (18) 541.
World supply of vegetable oils and fats shall ever be on
the increase, and with widening gap between supply and
demand, because of the environmental advantages of
oleochemicals over petro-chemicals. Furthermore, there is the
threat that petroleum resource decline with daily pumping of
crude pteroleum whereas there is ever encouraging increase in
terms of man input in acreage available for oil seed crops
production. Oleochemicals have a bright future, because of the
belief in many quarters that after a while, quantities of
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oleochemicals will be cheaper than the petrochemicals.
Furthermore, oleochemicals are environmentally friendly.
The greatest challenge for oils and fats in the
oleochemicals demand remains biodiesel. It is Gunstone’s
forecast that the world demand for production of biodiesel in
2020 may be 40-50MMT; this in turn will demand from the
world vegetable and animal oils and fats. Gunstone is his
lecture also considered the world production and use of oils and
fats in terms of population and quantities of oils and fats, he
viewed the world as made up of three categories. The developed
nations with a strong oleochemical industry but use of their oils
and fats in food are less than the global average. New Zealand
was taken to represent developed countries with strong
agricultural activity but probably with no oleochemicals
industry. Nigeria represents developing nations with populations
greater than 100million. In this third category, there is growing
demand for food fats and oils. Table 2 presents per caput use of
oils and fats (kg/yr) for all purposes in selected countries in 2005
(the world average is 21 MMT for 6,454 million persons.
Table 2: Per caput use of oils and fats (kg/yr)
Country Population
(Million)
Kg Country Population kg
EU-25 456 50.8 China 1,299 19.6
USA 380 49.0 India 1,097 11.7
New Zealand 4 38.2 Indonesia 225 18.2
Russia 141 22.2 Brazil 184 25.1
Mexico 106 25.9 Pakistan 161 19.4
Nigeria 130 13 Bangladesh 152 7.5
Source: INFORM (2006) 17 (18) 54-2
Biodiesel: a major consumer of world oils and fats
Biodiesel is methyl ester of fatty acid. It may be
produced by batch or continuous process represented in the two
equations for reactions involved.
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(a)(i) Fat/oil + NaOH RCOONa + glycerol
soap
(ii) RCOONa + MeOH RCOOMe + NaOH
biodiesel
(b) Fat/oil + MeOH RCOOMe + glycerol
Biodiesel
Biodiesel production at the present day volume is
relatively recent, but that not withstanding, it is experiencing
very dramatic expansion in the developed countries for
classification of national development, it is almost an index. The
need for the commodity, which is preferred to diesel, serves as a
great driver for success in the sector. The drive is greatly
supported by high price arising from artificial scarcity and fear
for future real scarcity of petroleum diesel.
According to the US National Biodiesel Board, the
number of active and proposed biodiesel plants grew by more
than 67% in six months in 20052. Projected production capacity
for 2005 was 545 million gallons per year. As the capacity of
biodiesel production increases, there shall be a corresponding
increase in demand for oils and fats. In USA, soybean is the
favorite oil, because it is easily available and the ease of
processing it into biodiesel. Even in the developed nations, there
is an aggressive drive for alternate seed oil feedstock for
biodiesel, in particular. This is in anticipation of a major need of
biodiesel by internal combustion engines; a justification for
source in unorthodox new oil seed crops world over.
Palm oil and palm kernel oil: the plant oil wealth neglected
by Nigeria
Malaysia purchased its first palm oil seedling in mid 50s
from NIFOR. In less than 50 years after, Malaysia and
Indonesia led the world in the production of palm oil and palm
kernel oil. In 2002, Malaysia produced 37MMT crude palm oil
and 11.9 MMT of crude palm kernel oil3. Today Malaysia has
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the largest oleochemicals capacity of any country in the world
and her capacity represents 25% of the world capacity in 2002.
16 oleochemical companies were in operation in Malaysia with a
total production capacity of 1.756 MMT. About 1.4MMT of
these oils were processed in to oleochemicals and 1.27 MMT of
these products (89%) were exported. Major oleochemicals
exported were fatty acids, fatty esters, fatty alcohols and
glycerine. Oleochemicals from Malaysia have been exported to
over 100 countries, including north America, European Union
countries, Japan and China. It is worthy to note at this point that
the volume of trade to Nigeria on the oleochemicals and crude
vegetable oils is negligible, not worthy of mention, yet Nigeria
imports a lot of its oleochemicals for the few oleochemical
industries in the country possibly mainly from Malaysia and
Indonesia. Furthermore, Mr. Vice Chancellor Sir, it is pertinent
to remark at this point that Nigeria has greater potentialities to
have been producers of oleochemicals which now compete
effectively with petrochemicals. We have potentials to produce,
but painfully we have neglected this ability. In no distant future,
we shall have to import biodiesel to supplement if not replace
our petroleum diesel then at a high price and a major drain on the
economy of the nation, what a pity this will be to us then as a
nation.
THE JOURNEY IN THE STUDY OF THEVETIA PLANT
The plant grows with widespread in Kwara state. The
abundance attracted my attention to consider what could be
made of the plant; so literature review was conducted to obtain
information on quantity of studies done so far on the plant. In
all, literature report was very scanty, which means very limited
work has been reported on the plant; even then, the little reports
available were on the toxicity of the latex, and the seeds.
The first experiment4 was organized to study the impact
of fertilizers on the seedlings, this involved pots experiments on
effect of NPK, calcium, potassium, and phosphorus. The result
of the studies revealed that application of NPK, single
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superphosphate, calcium nitrate, and murate of potash had effect
on the uptake of nitrogen, potassium and calcium, particularly
after seven weeks of application. Whereas the fertilizers rich in
calcium tended to depress uptake of potassium and nitrogen,
application of CAN, however, tended generally to enhance, more
significantly, uptake of calcium.
The plant was thereafter studied for the properties of the
seeds which the plant produces abundantly yearly. The seed is
60-65% oil and 40-45% protein. These two parameters
stimulated us to embark on aggressive studies particularly on
introducing it as a good substitute to orthodox supply of oils
used by the commercial soap making industries.
The oil physical properties, particular its saponification value,
120-124, and unsaponifiable matters, 0.24-0.41% prompted us to
study it in soap making. Tables 3a - c present some information
on the oil compared with other orthodox commercial oils.
Table 3a: Sterol content of a few selected seed oils (%)
Fat/Oil Sterol content (%)
Coconut 0.06 – 0.08
Cotton 0.20 – 0.31
Linseed 0.37 – 0.42
Palm 0.23 – 0.31
Palm kernel 0.06 - 0.12
Groundnut 0.19 - 0.25
Rapeseed 0.35 - 0.50
Soybean 0.09 - 0.11
Wheat gem 1.3 - 1.7
*T. Peruviana 0.44 -1.40 (plus phospholipids)
* Fadipe V.O., 1992 MSc. Thesis
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Table 3b: Oil seed mean oil content of a few selected seeds.
Type\source AOCS Ac – 44 Exhaustive extraction AOCS 2-93
Soybean 19.35 21.98
Rape seed 43.74 43.5
Cotton seen 18.17 20.61
Sun flower 12.71 46.10
Safflower 37.99 38.0
* Groundnut 21.24
* Lasquerella 25
* Varnonia 40
- T. peruviana 60-64
Source: INFORM (1997) 8 (10), 1048; Ibid (1998) 9(8) 749-835;
(1999) 2 (5) 686 – 691.
- Result in the department
Table 3c: Oil composition of selected seeds
Type/Source Sat Mono unsat Poly unsat
Soybean 15 24 67
Cotton seed 24 26 50
Palm 52 38 10
Palm kernel 86 12 2
Sun flower 11 20 69
Coconut 92 6 2
Ground nut 18 49 33.5
* T. peruviana 30-45 46-51 1-3
Source: INFORM (1990) 11 (4) 250
* Ibiyemi et al5.
The first and oldest6 oleochemical is soap. This is a
product of saponification of vegetable oils and fats. It is
believed to have been produced by the ancient Egyptians through
Phoenician into the Rhone valley in France as early as 600BC.
In 23-79 AD, Plinus, described soap as hair pomade which was
made from a combination of fats and ashes. At the early uses,
soap was not a cleansing agent. About 130-200 AD, Galenos, a
Roman doctor, discovered the cleansing property of soap. In 17th
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Century, the general use of soap for cleansing and bathing first
started when Justus V.Liebig (1803-1873) gave a citation that
“soap is an index of the prosperity and culture of any nation”.
This simply means that soap in the days was used to compare
two nations with the same population and to ascertain which one
is richer, more prosperous and better cultured.
The first type of soap in Nigeria is the black soap6 which
was milder, creamer and foam better than some imported soaps
as at the time under review.
The basic principle and process of soap making remains
practically unchanged for the past 1000 years. This involves
saponification of oils and fats with alkali and salting out of the
soap.
O
CH2-O-CR CH2OH
O
CH-O-CR + 3NaOH CHOH + 3RCOONa
O
CH2-O-CR CH2OH
Oil/fat glycerol Soap
Guided by the high level of the oil in the seed coupled
with the abundance of the plant with its widespread in the
country, the team including Mr. S.A. Akanji decided to study the
oil of the seed starting with saponification reaction. Towards
this goal, several kilograms of the fruit/seeds were processed to
obtain some gallons of the oil. In the first instance, samples of
the oils were made available for undergraduate students at their
practicals to conduct saponification reaction to obtain soap. The
performance of the oil at this level prompted us to provide the
Production Manager of Lever Brothers Nig. Ltd, Apapa (now
Unilevers Nig. PLC) one gallon of the oil for soap trial
production in 1981. The Production Manager was very
enthusiastic in accepting the oil and indeed, in a very short time
reported back to us his findings. The oil was very good in
production of soap that was suitable for bar and toilet soap.
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There was no need to bleach the oil before use, thereby saving
processing cost, and the lathering property was appropriate. We
considered the finding as good grounds for the department’s
breakthrough in its interaction with an industry.
In 1978, December 4-7, a symposium sponsored by
UNESCO was held in Toronto, Canada, and a team of twelve
chemists led by Prof. Ekong DEU, attended. The theme was
university – industry interaction in chemistry in Africa. The
useful thrust of the symposium is for members of the university
chemistry departments to forge a close relationship with
chemical-based industries in Africa. So the finding by the
production manager was to have signaled an opportunity to
record success of the aims and objectives of the Toronto
symposium. But low and behold this was not the case as the
relationship broke down with the Production Manager questions,
“what quantity (kilogram or gallons) of the oil we expected will
be produced from one hectre of the crop plantation”? We have
never thought of this because we saw ourselves concerned with
the chemistry of the oil not the agriculture and economics. That
not withstanding, however, to keep the relationship alive, we
suggested that we needed one thousand naira to engage the
services of labourers to keep one acre of farm to be planted at the
beginning of the next rains. The production manager was frank
and quick to let us know that such request is not common and
would require management decision. All other follow-up proved
abortive and so terminated the much cherished and anticipated
celebration of break-through with an industry.
The disappointment was taken as a challenge and we
sought alternative means to determine the yield of oil per acre, if
the plant was grown on a semi large scale. The team of chemists
did not see itself handling this assignment effectively, so talked
with our colleagues in the crop production department, Faculty
of Agriculture to accept to place final year project students on
the scheme. All our efforts yielded no response. So we ventured
into acquiring a plot in the Faculty demonstration farm for the
project, there was no success recorded.
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In 1992/93 I was away on sabbatical leave in ABU,
Zaria, and I enjoyed the favour and cooperation of Prof Daniel
Sarror, the Vice Chancellor, who was good to grant me a special
fund for the project. The farm management, under the
chairmanship of Prof Duro Olarewaju, made a plot available for
my project. At the beginning of the rains, 1000 seedlings were
planted and kept until the expiration of my sabbatical leave in
September 1993. There was no staff collaborating and the plants
were to flower in 1994, it was not convenient for me to maintain
the project. Thereafter I moved to Ilorin consequently and
painfully the farm was lost to weeds.
During the period I was in Zaria, I organized an MSc
student and an academic staff, Mr. J.O. Ojokuku, together with
Mr. V.O. Fadipe, an MSc student of University Ilorin to do a
comparative study of the plant seed oil obtained from seeds
collected in Ilorin, Edidi, Zaria and Enugu. I was briefly in
Enugu on WAEC assignment and I noted a variety of the plant
with purple flower rather than yellow. This peculiarity attracted
my attention for comparative studies of its oils with others with
yellow flowers. The results of analysis of the seeds on the
varietals based on the number of kernel per seed and
geographical location are presented in table 5.
Table 4a: Summary results of analysis of the four varieties
of seeds
Analysis Data Oneseed
Twoseeds
Threeseed
Fourseed
% oil yield 56-61.1 59-63.1 54.58.8 54-59.3
Saponification value 121-140 124-186 120.181 120-196
Unsaponifiable
matters (%)
0.27 0.21 0.42 0.38
Free fatty acids 0.488 0.434 0.564 0.513
Acid value 0.97 0.86 1.12 0.02
Peroxide value 16.46 14.47 19.97 19-41
Iodine value 76-. 81.2 80.2 79.2
Refractive index 1.461 1.462 1.462 1.462
Specific gravity 0.926 0.913 0.927 0.937
Viscosity (cent) 19.00 21.36 15.80 17.04
Source: Ibiyemi et al5
20
Table 4b: Common fatty acids present in oils of the varieties of thevetia
seeds based on kernel number and
geographical locations.
Source: Ibiyemi et al5
Location
Variety
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 Sat Unsat
Enugu 1
2
3
4
0.31
0.37
0.48
0.43
17.7
19.3
18.4
20.9
0.10
0.35
0.18
0.30
5.2
6.02
6.31
7.00
46.1
40.38
39.94
36.74
-
15.57
13.12
8.47
0.7
0.7
0.9
-
23.35
25.72
25.10
28.29
46.86
57.98
56.16
79.57
Edidi 1
2
3
4
0.56
0.17
0.72
0.31
14.2
20.6
16.0
19.81
0.3
0.3
0.20
0.27
5.01
7.75
5.48
6.56
34.06
40.85
34.12
36.58
13.85
8.85
10.76
7.95
1.35
0.12
0.16
1.34
19.78
28.22
22.65
76.05
51.58
50.11
45.21
46.15
Ilorin 1
2
3
4
0.31
0.17
0.17
0.32
20.42
18.34
21.00
15.97
0.25
0.21
0.91
0.21
7.35
5.28
7.20
5.80
45.06
30.44
43.39
33.76
12.58
14.05
14.08
16.97
0.44
1.33
0.45
0.71
28.90
23.79
28.27
22.09
58.33
46.03
58.71
57.65
Zaria 1
2
3
4
0.14
0.16
0.22
0.38
20.81
23.70
20.12
20.21
0.24
0.23
0.35
0.28
8.01
8.97
7.39
7.63
45.79
40.96
47.00
40.00
16.81
12.65
14.77
16.00
0.36
0.60
0.47
0.49
28.96
34.93
27.73
28.22
56.78
54.44
62.49
62.71
21
Enugu has the highest rainfall over 10 months, Zaria
with lowest rainfall, while Edidi and Ilorin lie in-between in
amount of rainfall, and period of rains. Seeds from Ilorin and
Edidi record highest oil content (62-64%), Zaria 58% and Enugu
61%. In terms of quality of the oil, Zaria seeds contain highest
unsaturation (59.3%) and Enugu lowest value (51.1%).
Basic research, in general, in most purposes and intent,
provides data that would guide the applications of the material
being investigated. This principle guided our plan of actions on
the analysis of thevetia seed oil, i.e. to accumulate as much data
on its physical and chemical properties, peradventure, some day
this will adapt the oil for an industrial use. Towards this goal,
the oil was studied for its thermal stability7. The result obtained
showed characteristic changes in physicochemical properties
when heated at 180oC, 200oC, and 220oC, over a period of 15
hours. There was no significant difference in the five parameters
(iodine value, acid value, peroxy values, saponification value
and amount of polar compounds) studied for the three working
temperatures. The study proved that the seed oil would be stable
to heating, therefore could be good for cooking, if adopted for
use in frying food. This property also shows justifications for
the oil to be used or converted for use in the making of lubricants
and greases.
Synthetic Lubricants: Thevetia seed oil and its chance.
Synthetic lubricants came into use during the World War
II in Germany, primarily due to lack of petroleum. However,
their use today is based on specific applications where
conventional petroleum base-lubricants fall short of the desired
requirements, primarily in the area of low and high temperature
performances. Table 5 presents some compound classes that fall
into the category of lubricants from vegetable oils and fats.
22
Table 5: Types of synthetic lubricants
The combination of chemically compactable thickening
agents with either petroleum oil or synthetic fluids results in
greases. There are greases which are soaps and non-soap types.
The soap-types contain the metal salt of organic acids, while the
fatty acids (non-soap) greases are usually palmitic acid, and
other saturated fatty acids.
Thevetia seed oil was therefore investigated with a view
to establish its suitability in preparing a lubricant or grease since
it has good thermal stability properties. Several project students
were placed on the modifications of the seeds oil. Like any other
seed oils, thevetia seed oil responded favourably to several
chemical reactions but analysis of the products to establish the
products was truncated by lack of facilities even as elementary as
separation techniques beyond plate and column chromatography.
At this point it may be mentioned that the university had
purchased an equipment vital for our studies of vegetable oils,
GLC, but the equipment is not adequately equipped to perform,
for lack of standards. All efforts made since 2004 to acquire the
standards directly from SIGMA in UK, and then through ZAYO,
the sole agent in Nigeria, based in Jos, is yet to yield fruit,
despite payment for the standards in pound sterling and naira.
Thevetia seed oil, like any organic liquid, could be
considered worthy to be tried for production of emulsion.
Emulsion could be cosmetic or lotions, depending on various
factors that may include the presence of more than one phase,
stability, or the phenomena of surface activity. Thevetia seed oil
was investigated peradventure it will make good varnishing
creams, an essentially oil-in-water emulsion with its fatty acid
Class Typical compound
Fatty acid esters Ethylstearate
Dibasic acid esters Dibutylphythalate, di (-2-ethyl hexyl
tricresy phosphate)
Silicone esters Tetraethylsilicate, hexa(-2-ethyl butoxyl disiloxane
Polyglycols Poly(propylene glycol)
Poly alkyl and aryl ethers Poly phenyl ethers
23
salts as the emulsifying agent. Stable emulsions of the oil were
prepared but the qualities other than stability could not be
established.
Thevetia biodiesel: Its chances as a seed oil
The world is daily seeking to substitute petrochemicals
in general, but most specifically biodesel and other engine
combustion fuels. The first use of vegetable oils8 in internal
combustion engines dates back to about 1900 when Rudolf
Diesel (1858-1913) experimented with groundnut oil and Fujio
Magao achieved operation with pine oil in 1948. The two oilprice
sharp increases (1973) sparked world-wide desire in
developing energy sources as alternate to petroleum in internal
combustion engines, boilers and other combustors. Effort since
then, has stimulated investigations in favour of use of ethanol in
Brazil, sunflower oil in South Africa9, rape seed oil in Europe,
and currently, and on a large scale spent cooking oils and
vegetable oil esters are gaining acceptance and use at a
surprising scale. For almost ten years, Malaysia and Indonesia
tend to lead the world by the nose by virtue of its success story in
biodiesel investments and production. Malaysia in 2005
announced a national biodiesel policy to stimulate the
development of the biofuel industry by four-prong strategy that
encompases the production of a biofuel blend of 5% processed
palm oil and 95% petroleum diesel (B5), encouraging the use of
B5 among the public and establishing biodesel plants in
Malaysia for the export market10.
Our research team which kept changing as the research
students graduated made coordinated planning very weak and
inefficient. This not withstanding, thevetia seed oil has been
studied to provide data on its conversion to biodiesel.
Production of its biodiesel involved both batch and continuous
processes. All available analytical techniques have been used to
establish a level of success in the esterification reactions. The
most reliable confirmation of the success should have come from
NNPC; however we have lately approached LUBCON for this.
Samples of our biodiesel were made available to NNPC Kaduna
24
in 1993 for analysis. The “no response” was taken as negative
results, but at the same time we needed their response on what to
do to achieve success. Of recent, our effort, whereby we
investigated the effects of catalysts on our production of
biodiesel has taken us to LUBCON to determine our level of
success. Samples have been presented to LUBCON; the result
of analysis is being awaited. Since LUBCON is close and more
cordial, there will be close interaction and we hope to have
success through this in the near future.
Thevetia seed oil in livestock feed formulation
The oil, if freed of the toxins of the plant, and the
plantation developed, and the oil is not used as cooking oils, it
may serve as oil component in the livestock feed meal
formulation. The team led by Prof. J.O. Atteh12 had the first trial
on the replacement of palm oil by thevetia oil in broiler chick
diets. In the experiment, the effects of replacing 0, 25, 50 or
100% of the dietary palm oil (5%) with oil of Thevetia peruviana
seed was investigated using broiler chicks 0-3 weeks old.
Increasing the content of thevetia oil in the diet reduced average
feed in-take and weight gain (P<0.05) and decreased protein and
fibre retention (P<0.05) and fat retention (P<0.01). However,
there was no significant effect on feed: gain ratio or mortality
rate. It was concluded that there was a need for further
processing of the seed oil before it can be used effectively as an
ingredient in broiler feed.
On our part, we have established that the oil indeed
requires refining. Charcoal treatment removes all taint colour
and recently we have confirmed the presence of the plant toxins
in the oil. A repeat of the trial feed experiment with the refined
seed oil is desirable, depending on the cooperation of our
colleagues in the animal science department.
Thevetia seed oil in the paint industry
The utility value of the seed oil is unlimited, so the seed
oil was considered possible alternate oil for the paint industry.
D.N. Meyer, sometimes in 2004, invited the department
to a round-table discussion on what the department could do to
25
source a local alternative alkyd, the major ingredient in the paint
formulation; an item if replaced by local sources would reduce
production cost. The alkyd accounts for about 50% of all
ingredients compounded to make gloss paints.
Table 6: Typical recipe for gloss paint formulation
Ingredients % Weight
Alkyd resins 49.6
Talcum powder 21.9
Turpentine 15
Titanium dioxide 17
Cobalt naphthenate 6.6
Source: Turner13
Soybean oil accounts for one of the major ten seed-oils
currently in use by major paints manufacturing companies
world-wide. Nigeria, to-date, imports every bit of its alkyds, a
vegetable oil polyester. The chemical reactions basic to the
preparation of alkyds are presented in the following equations.
1. Esterifications:
R’COOH + ROH R’COOR + H2O
2. Etherification of polyols (a possibility):
ROH + R’OH ROR’ + ROH
3. Transesterification:
RCOOR1 + R2COOR3 R2COOR1 + RCOOR3
The alkyd is a resin, synthesized from a dicarboxylic
acid such as phthalic anhydride or its acid and a polybasic
alkanol e.g. glycerol, pentaerythritol etc in the presence of a
suitable catalyst at about 220-240oC. The reaction is a
condensation reaction and produces polymers. It is believed that
the properties such as rapid drying, good flow, and excellent
weathering. They have almost completely outstripped drying
oils e.g. linseed oil, as binder for paints e.g. enamel, varnishes,
wood primers and lacquers.14
The percentage of oil can be varied giving rise to short
alkyd resins when the oil content is low and long alkyd resins
26
when oil content is high. Typical alkyd resins’ ingredients
contents are presented in table 7.
Table 7: Classification of alkyds based on the amount of oil
content
Parameter Long alkyd Medium alkyd Short alkyd
(%) oil >70 50-70 <50
phthalic
anhydride (%)
20-30 25-35 35-60
Viscosity (Sec) 90-120 120-150 150-700
Acid value 12 max 30 max 32 max
Source: Martens C.R. (2003) Alkyd Resins. Reinhold Publishing
Corp. N.Y. Pp 108.
The impact of the properties of the oil and performance
of the paint is presented in tables 8 (a) & (b)
Table 8a: Effect of oil on alkyd properties
Source: Kirk–Othmer (1963) Encyclop of Chem Techn.2nd Ed-
Intersci. Publ. JohnWiley & Sons Inc. NY Pp851-882.
Oil Type Coating Properties
Iodine
value
Speed drying Colour retention Gloss retention
Linseed 180
Tuna 170
Dehydrated castor 155
Safflower
Segregated
140
Conton seed 130-140
Cotton 110
Tall oil 125
Soybean 135
Ground nut 108
Castor 85
Olive 85
Coconut 90
27
Table 8b: Division of each drying and semi drying oils into
short, medium and long oil resins categories
Source: Deffar D and Soucek MD (2001) Journal Coat Techn 73
(919) 95
Thevetia seed oil has properties comparable with the
common oils being used in the preparation of alkyds; we
therefore embarked on the preparation of the alkyds, if only in
response to D.N. Meyer’s challenge. There are three teams of
members of staff in the department who have accepted the
challenge and have studied at least four seed oils in alkyd resin
preparations and characterization of the alkyds. Alkyds have
been prepared from thevetia seed oil, jathropha seed oil,
dehydrated castor oil, tobacco seed oil and parinary seed oil.
Tables 9a-c presents findings on the alkyds of thevetia and
jathropha seed oils.
Drying oil
resins
short oil resins Cure at elevated temp, give very hard, glossy
finishes
Used in finishes for appliances, signs and toys.
Drying oil
resins
Medium oil resins May be air dried or heated, give durable glossy
finishes.
Used for farm hardwares and metal furniture
Drying oil
resins
Long oil resins Have good brushing characteristics, dry rapidly
in air, reasonably durable, glossy film
Used in house-hold paints.
Semi-drying
resins
No division based on
oil length
Give film with improved resistance to yellowing
on ageing.
Used particularly for high gloss white finishes
Non-drying
resins
Short oil resins Used mainly in conjunction with amino resins.
Give improved adhesion and flexibility.
Used in storing finishes for appliances.
Non-drying
resins
Medium oil resins Used mainly as plasticizers for cellulose nitrate
for furniture finishes.
28
Table 9a: Solidification time of thevetia seed oil and
jathropha seed oil
Alkyds solidification time (sec)
(i) (ii) (iii) (i) (ii) (iii)
JAK-1 T-AKD-1 P-AKD-1 150 132 109
JAK-2 T-AKD-2 186 180 -
JAK-3 T-AKD-3 294 207 -
JAK-4 T-AKD-4 300 240 -
JAK– Jathropha alkyds; T-AKD - Thevetia alkyds; P-AKD –
Parinary alkyd.
Table 9b: Viscosity of Alkyds
(i) (ii) (iii) (i) (ii) (iii)
JAK-1 T-AKD-1 P-AKD-1 140 210 190
JAK-2 T-AKD-2 97 100 -
JAK-3 T-AKD-3 92 105 -
JAK-4 T-AKD-4 86 85 -
Table 9c: Drying characteristics of alkyd resins containing
no drier
Out-door
DFT
Indoor
DFT
Outdoor
STTT
Indoor
STTT
JAK-1 T-AKD-1
5.3* 16.8/28+
JAK-1T-AKD-1
11.6* 20/31+
JAK-1 T-AKD-1
51.8 4.8/15+
JAK-1 T-AKD- 1
70.1 8.7/19+
JAK-2 T-AKD- 2
10* 21/38+
JAK-2 T-AKD-2
16.5* 28/45+
JAK-1 T-AKD-1
99 10/24+
JAK-1 T-AKD- 1
125 14/27+
JAK-3 T-AKD-3
19.8* 39/50+
JAK-3 T-AKD-3
25.3* 44/55+
JAK-3 T-AKD-
113 12/23+
JAK-3 T-AKD-
149 16/34+
JAK-4 T-AKD-4
26.5* 41/55+
JAK4 T-AKD-4
31.7* 48/65+
JAK-4 T-AKD-4
16.1 18/37+
JAK-1 T-AKD-1
17.5 19/42+
P-AKD
19.2
P-AKD
24
P-AKD
5.67
P-AKD
8.5
DFT = Dust-free Time, STTT = Set-to-touch-time
Source: Akinwatimi (2006). B.Sc.Project.
Seeds processed all over the years would have produced
testa, if allowed to accumulate, would have amounted to several
kilograms, which if it is not properly disposed could readily be
29
an environmental nuisance of the type similar to saw dust in
sawmills and palm kernel shell in palm kernel processing
industry. On careful consideration, we thought that the testa
could be considered for board-particle production. Samples
therefore were sent to the Africa Timber and Plywood, Sapele in
1987. The product made out of the material was very hard and
brittle. The industry indicated no further interest in the
experiment, otherwise we would have wanted to have a blend of
the hard material with a soft material, particularly, baobao fruit
testa. (See appendix for the letter from the MD).
Thevetia seed: providing protein in animal feeds
The seed on the basis of its protein content (40-45%)
should be preferred to most orthodox protein sources in the
formulation of animal feeds. Brain et al15 and Bisset16 were
among the first few to report on the seeds for its toxins, cause of
death as recorded for two children, horses and other animals.
Compounds I to IV below serve as representatives of the
toxins.
OH
CH3
CHO
OH
CH2
OH
OH
OH
H
H
H
H
H

O
O
O
O
CH2OH
H
OH
OH
H
OH
H
H
H
O
O
CH3
OMe H
OH
H
H
HH
O
Thevetin A
30
O
CO2R
OH
CH2OH O- ß - D - glucose
(Theveside: R = H; Theveridoside: R = Me)
OH
O
O
CH3
H
CH3
O
O
H
CH3
OH
H
H
H
H
OMe
OH
Neriifolin
HO
OH
O
O
Digitoxigenin (Thevetigenin)
31
The seed is shown to contain between 3.6 and 4%
thevetin17, the major glycoside of the seed, and the most lethal
toxin. It is cardiotonic. Other compounds that have been
identified are cerberin, ruvoside, perusitin and neriifolin18. Paper
chromatography reveals that fresh seed of the plant contains
fifteen compounds. Some of the glycosides have been subjected
to clinical trials, especially in the treatment of congestive heart
failure19 and cardiac insufficiency. However the margin between
toxic and the therapeutic doses have been found to be too small
for many of the glycosides, especially thevetin, to be useful
therapeutically until further research is done in this regard. Some
of the compounds have been commercialized by ALDRICH
chemical company. This already paves way for possible use of
the extract when the seed will eventually be processed in large
quantities.
In favour of the plant emerging as a possible commercial
plant is the fact that other useful compounds have been isolated
from the plant, these include flavonones and flavonols
glycosides, extracted from the leaves of T.peruviana
The presence of anti-nutrients in oil seeds is not
sufficient reasons to neglect a plant with prospect as the case
with thevetia plant. There are not many seed that are free of antinutrients
or toxins of any one type. The quantity and lethal level
of the agent and ease of removal matter. Soybean, cotton seed
and castor seed in their raw forms are all intolerable to majority
of animals, particularly monogastric animals. Processing each
under specified conditions have been adopted in the
detoxification of such seeds. Irradiation is well established and
properly utilized to effect genetic re-engineering, thereby
producing improved variety of plant and animal types. This
could lead to variations of contents of seeds and could be
effective detoxification technique.
In the first set of detoxification treatment, dilute strong
alkaline solutions and dilute hydrochloric acid were used
separately and detoxification monitored by the level of bitterness
of the cake. The cake with minimum bitterness was used to
32
compound broilers meals containing 0,5, 10 15% thevetia cake.20
Inclusion of thevetia cake in broiler diets, irrespective of level of
inclusion, drastically reduced feed intake and weight gain
(P<0.01) at both the starter and finisher stages. The results
showed that both methods of detoxification are not efficient and
sufficient. In pursuant of effective detoxification of the seed,
other methods adopted include acid leaching using solutions at
pH 6-9, organic solvents extraction, followed by aqueous ethanol
extraction. Another method employed activated charcoal and
boiling at varying periods ranging from 1 to 5hrs. Table 10
presents cardiac glycoside contents of the raw cake and cakes
after various treatments.
Table 10: Cardiac glycosides content of thevetia cakes
Sample Total cardiac glycoside
Raw seed cake
Acid treated cake
Ethanol treated cake
Charcoal treated cake
4.27 ±0.44% or 4.27g/kg
0.22 ±0.71% or 2.25kg/kg
0.08 ± 0.25% or 0.83g/kg
0.24 ± 0.22% or 2.4g/kg
Source: Oluwaniyi et al21.
Charcoal treatment and ethanol extraction have been
found to be more efficient and effective in the
detoxification/debiterisation of the seed cakes. Acid hydrolysis
prior to ethanol extraction also proved to be efficient but residual
acid tended to leave a sharp taste that may not be desirable. The
work of Finnigan and Lewis22 using acid hydrolysis followed by
ethanolysis to remove glucosinolates as food component in the
rapeseed provides support that our results are as reliable and
efficient methods of detoxification process21. The detoxification
effectiveness was further established by a measure of the
remains of the cake by monitoring the quality and quantity of
protein in the cake, peradventure, protein may also have been
extracted, along with the glycosides. Loss of protein would
negate the primary objective of securing a good alternate protein
source.
Variations in the time of extraction, volume of ethanol
and quantity of cake were investigated and products obtained
33
analyzed for the protein content23. Results obtained are
presented in tables 11a & b.
Table 11a: Glycoside (%) extracted using varying ethanol:
cake ratio
Time (hrs) 10:1
Ethanol: Cake
15:1
Ethanol: Cake
20:1
Ethanol: Cake
0.00 5.44 5.44 5.44
0.75 0.61 0.48 0.58
24 0.56 0.48 0.48
48 0.52 0.45 0.24
72 0.46 0.32 0.24
Table 11b: Moisture and Protein content of cake after
extraction.
Time (hrs) 10:1
Ethanol: Cake
15:1
Ethanol: Cake
20:1
Ethanol: Cake
Moisture
Protein
Moisture
Protein
Moisture
Protein
0.75 17.24 64.92 12.10 69.09 15.80 65.92
24 16.95 65.76 11.13 68.91 15.63 66.05
48 16.09 63.45 12.80 68.91 15.80 66.34
72 18.00 65.77 12.89 68.58 15.46 65.91
Source: Oluwaniyi & Ibiyemi23
Table 12c: Effect of varying water in ethanol on the quality
of cake using 15:1 ethanol: cake for 72 hrs
% Ethanol in
water
Moisture Protein
Content Content
(%) (%)
Glycoside
Content
(%)
50 25.89 62.13 5.44
60 22.82 65.35 0.49
70 15.35 67.98 0.45
80 13.39 68.95 0.32
90 14.44 65.50 0.32
100 15.89 62.35 0.32
Source: Oluwaniyi22
Results in graphic form in Fig. 1(a) and (b)
34
0
1
2
3
4
5
6
0.00hrs 0.75hrs 24hrs 48hrs 72hrs
Time
Percentage glycoside
10: 1 solvent: meal ratio
15: 1 solvent: meal ratio
20: 1 solvent: meal ratio

Fig 1a: Extraction of cardiac glycosides from thevetia seed

meal by 80% aqueous ethanol/methanol
mixture.
0
1
2
3
4
5
6
Crude 50 60 70 80 90 100
% (v/v) Aqueous alcoholic solution
Percentage glycoside
Fig 1b: Extraction of thevetia glycosides using varying concentrations of
aqueous alcohol at 15: 1 solvent to meal ratio for 72 hrs.
35
The best adjudged treated cake was formulated as feed
meals and fed to chicks to establish the efficacy of the
detoxification technique. The feed experiments results are
presented in figures 2a & b.
0
20
40
60
80
100
120
140
Feed Intake /Bird/Week Weight Gained /Bird/Week Gain: Feed Ratio (Feed
Efficiency Ratio)
Mortality (%)
Control
T1A
T1B
T1C

Fig 2a: Performance Pattern of birds fed acid detoxified TSM

0
20
40
60
80
100
120
140
160
Feed Intake /Bird/Week Weight Gained /Bird/Week Gain: Feed Ratio (Feed Efficiency Ratio)
Control
T2A
T2B
T2C

Fig 2b: Performance pattern of alcohol detoxified TSM

36
Result obtained from detailed analysis of the blood
samples for birds fed each of the meals corroborated the result
presented above from chemical analysis of the meal
performance.
The qualities of the detoxified seed cake were further
established by the analysis of the seed cake for the protein
content and amino acids profile.
Table 13 presents the amino acids profile of the raw
cake, and each of the two treated cakes. The efficiency of this
ethanolysis by all standards is confirmed even by this parameter
to be more efficient and effective in the detoxification exercise.
Table 13: Amino acids profile of the seed cakes
Seed Cak e
Amino Acid
Raw Acid treated Ethanol treated
Alanine
Arginine
Aspartic acid
Cystine
Glutamic acid
Glycine
Htistidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threomine
4.49
4.48
19.85
1.69
14.21
3.63
1.62
2.94
5.49
4.47
0.88
3.38
4.24
3.93
2.61
3.04
4.25
21.86
1.05
20.10
2.24
1.39
2.09
4.88
3.97
0.64
3.22
3.85
3.12
2.04
4.56
5.19
20.34
1.69
15.67
3.70
1.65
2.97
5.59
5.65
0.90
3.70
4.49
4.00
2.67
Source: Oluwaniyi22
The result agrees with the report by El-Adawy and El-
Kadousy24 that ethanolysis led to an increase in the total,
essential, basic, and aromatic amino acids of the seed cake
detoxified.
37
Irradiation has been put into divergent uses in various
aspect of life including agriculture; it has been widely adopted
for genetic re-engineering to produce improved varieties in plant
and animals. This technique is imagined could influence genetic
variation that may effect the reduction and possibly elimination
of the glycosides-toxins of thevetia plant.
Thevetia seeds and kernels were subjected to 100 rads,
300 rads and 500 rads treatment (100 rad=I Gray when (GY) is
equal to IJK/ kg. Rad = radiation absorbed dose). Some were
analyzed, some planted and plants produced monitored within
the limit of chemists abilities for effect of the irradiation. Result
obtained on chemical analyzed of the seeds is presented in Table
14.
Table 14: Monitoring effects of Co-60 gamma rays on the oil
content of four sets of seeds based on kernel number
variety.
Oil yield (%) of the types based on number of kernel/seeSeed Type d
One-seed Two-seed Three seed Four seed Average
Untreated /raw
kernel
41.00 43.30 58.80 54.20 54.32
100 rad kernel 68.40 61.54 61.30 63.63 63.71
300 rad kernel 50.80 50.00 50.80 48.80 50.50
500 rad kernel 40.00 59.60 46.80 41.87 60.00
Source: Akinduro (1999) BSc. thesis
This result seems to suggest that seeds treated at 300 rad
contain lowest oil content, an undesirable result, if the plant
would be selected and nurtured after irradiation. The study of
the plant morphology for seeds irradiated at the three doses
however, suggests contrary performances. Table 15 presents
results of the seeds harvest from plants grown from seeds with
each of the three doses. Furthermore, the canopy size of the
plants right from the 6th month till the 24th month provided
convincing evidence in favour of 300 rad treatment. Plants from
500 rad seeds were dwarf, majority grew to about half the size of
38
others and such were never able to flower and fruit, even in the
fourth year.
Table 15: Summary results of the profile of fruits and seeds
from thevetia plants (treated and untreated seeds).
Seed-type (i) unirradiated (ii)300rad treated
(iii)500rad treated (using 20-30 plants per plot)
Average
(i) (ii) (iii)
Number fruits /plant 610 876 300
Weight of total fruit
/plant (g)
230.5 204.3 198.69
Weight of kernel 5.72 7.25 4.86
Weight of fruit 11.45 14.00 12.31
Seed population
distribution based on
kernel/seed
Type1
Type2
Type 3
Type4
13
16
17
4
12
15
16
4
15
14
15
6
oil yield (Pet ether
extract of seed cake (%) 54.46 62.40 47.00
Chloroform extract of
Seed cake (%) 2.10 1.70 2.00
Ethanol extract
of seed cake (%) 1.57 1.30 1.49
Source: Alabi (2004) B Sc thesis.
The apparent contradiction in the results obtained by
workers in 1999 and 2004 lends credence to the team conviction
that as chemists we should limit our concerns with analysis of
products provided by plant scientists and / or agronomists.
Furthermore, irradiation effects on plant may not manifest and
stabilize in the first or even second filial. i.e the changes
39
anticipated may emerge in latter generation. The desired
modifications may therefore manifest slowly.
Another monitoring conducted on the effect of the
irradiation of the seed with 300 rads was to analyse cakes
prepared from seeds harvested as first filial generation of plants
treated and untreated seeds. The results obtained for the amino
acids analysis are presented in tables 16a & b
Table 16a: Amino acid analysis of thevetia seed cakes
Amino acid T1 T2 T3 T4 T5
Alanine
Arginine
Aspartic acid
Glysteine
Glutamic acid
Glycine
Hitistidine
Isoleucine
Leucine
Methiomine
Phenylalanine
Proline
Threomine
Tyrosine
Valine
4.49
4.48
19.85
1.69
14.21
3.63
1.62
2.94
5.49
0.88
3.38
4.74
2.61
2.49
4.01
3.04
4.25
21.50
1.05
20.10
2.24
1.39
2.09
4.88
0.64
3.22
3.85
2.04
1.94
3.57
4.56
5.19
20.34
1.69
15.67
3.70
1.65
2.97
5.57
0.90
3.70
4.49
2.67
2.49
4.01
2.90
2.99
144.88
1.37
16.77
2.00
1.03
1.78
3.84
0.50
2.28
2.95
1.91
1.66
2.48
4.39
4.87
19.17
1.61
15.01
3.59
1.69
3.00
5.7
0.79
3.30
4.30
2.50
2.49
4.01
Table 16b: Distribution of amino acids on basis of group
Analysis T1 T2 T3 T4 T5
Total Amino acids 84.41 83.35 89.27 65.54 84.88
% Difference - - 0.66% + 5.76% -22.36% + 0.56%
Essential amino acids (%) 35.4 31.07 36.22 32.00 35.65
Acid amino acids (%) 40.35 50.04 40.34 48.29 40.27
Basic amino acids (%) 17.55 16.05 19.02 15.91 19.34
Sulphur amino acids (%) 3.04 2.73 2.90 2.85 2.83
Aromatic amino acids (%) 5.87 5.16 6.19 4.64 5.79
Source: O. O. Oluwaniyi (2007) Ph.D thesis (in progress)
40
T1 = crude (untreated thevetia seed cake)
T2 = acid detoxified seed cake
T3 = ethanol detoxified seed cake
T4 = Charcoal detoxified seedcake
T5 = 300 rad treated seed cake
Table 16(b) reveals that detoxification processes by
ethanolysis and radiation are worthy of further and detailed
studies particularly irradiation effect on toxin level. Amino acids
make up proteins. There are different members of protein
depending on the sequence of the amino acids. Major
classifications are globulin, albumin, prolamin and to lesser
extent and of limited occurrence is gluten, majorly sourced from
wheat. Solubility properties of the protein members are
presented in Table 17.
Table 17: Protein classification based on solubility.
Proteins of the seed cakes have been analyzed for the
various protein types; globulin and albumin have been
established in various proportions. There are indications, subject
to confirmation, that gluten may be present in an appreciable
quantity that may justify intense research of the seed for its
protein content, for gluten in particular.
Table 18a: albumin and globulin content of thevetia seeds
Cake
HCl treated
Cake
NaOH treated
Cake
Ca(OH)2 treated
Cake
NaCl treated
Extractant: protein(%)
Alb Glb
Extractant: protein(%)
Alb Glb
Extractant: protein (%)
Alb Glb
Extractant: protein (%)
Alb Glb
0.5M 0.6 0.50 0.2M 0.26 0.21 0.5M 2.63 2.61 0.5M 0.98 0.76
Protein Soluble medium
Albumin Water
Globumin Salt solution
Prolamin 70% ethanol
Gluten Alkaline solution
41
Table 18b: Composition of individual member proteins in
seed cake
Type/member Content %
Untreated Irradiated Detoxified Irradiated & detoxified
Crude total
protein
53.3 53.11 54.25 53.14
Albumin 11.70; 10.41* 12.89 - 13.88; 16.06* 10.24
Globulin 1.07; 7.65* 5.98 8.84; 5.66* 2.06
Gluten 15.62; 13.94* 14.38 12.54; 10.28* 15.84
Prolamin 4.56; 2.81* 2.38 0.99; 0.93* 0.85
* Result from two different procedures
In the event that gluten is unequivocally confirmed to be
present as indicated in table 18b and in a quantity comparable to
its presence in wheat, this singular parameter may be sufficient
to achieve the vision for the emergence of thevetia plant as an
economic plant. Table 18a provides good information on the
protein isolates of thevetia seed. This prompted us for further
study of the protein fractions.
CONCLUSION
We have studied Thevetia peruviana plant seed from as
many points of view, believing we have accumulated as many
pieces of information and data that should encourage scientists
of related disciplines, particularly agronomists to show sufficient
interest and work to bring out the potentialities of the plant. The
plant deserves to be studied to establish it valuable prospects
other than ornamental.
Mr. Vice Chancellor sir, I wish to inform this august
gathering that I have presented a poster paper at the annual
conference of American Oil Chemists Society held in Quebec,
Canada, May 12-15th 2007 with the purpose to stimulate interests
of chemists worldwide on the study of the seed for its oil and the
seed cake. The plant, if given the right quantum of research and
funding, could by 2015 be on field trials and its oil and cake
available in commercial quantities as a major feedstock for any
of the oleochemical and livestock feed meal industries.
42
RECOMMENDATIONS
Mr. Vice Chancellor sir, I wish to let you know that I
have specifically sent special invitation letters to persons who
should be in good knowledge of the research efforts on Thevetia
peruviana plant for one reason or another. Invitees include the
presidency, Raw Material Development Council, a few selected
paints industries, Soap /detergent industries, cosmetic industries,
flour mills; the Permanent Secretary of Federal Ministry of
Education, Secretary of NUC and MD of NNPC and LUBCON.
These people I have specially invited with belief that each, if
sufficiently informed and convinced of my vision for the plant,
are strategically placed to promote:
(i) research with focus on the plant,
(ii) promote the right relationship for healthy interaction
between industries and university research on vegetable
oils and fats in general and the plant in particular,
(iii) fund research in a satisfactory quantum,
(iv) appreciate the need for good and guided policies,
political will, and drive in Nigeria to invest NOW
specifically in the study of thevetia plant and in general
in the production of vegetable oils and fats in Nigerian to
make Nigeria contribute its quota effectively in the
world struggle for production of oleochemicals.
To achieve the stated objectives, Nigeria research needs
good and well articulated polices of the Federal Government and
NUC shall steer the ship of research to ensure the right drive for
good production of vegetable oils and fats in Nigeria. If the
federal government shall popularize and provide necessary
incentives to the farmers as has been done for cassava in the last
five years, the country doors to foreign investors are certainly
wider and more attractive in the production of oleochemicals
than we got on cassava. A very major drive in processing any
crop is production cost of the crop within a short period. Nigeria
is well suited to produce vegetable oil to storm the world market
within five years if it can be focused on the well established
43
annual oil crops- e.g. soybean, sun flower, melon in addition to
thevetia seed. If Malaysia and Indonesia have excelled as world
leaders on two major oils- palm oil and palm kernel oil, within
20years, Nigeria with the same oil plants and at least ten others
has better potentials that can readily make it be the leading not
just one of the leading oleochemicals producers by 2020, a
period oleochemicals may take over from petrochemicals, when
the latter may be less relevant.
Mr Vice Chancellor, Sir, please allow me to suggest the
following specific recommendations that I belief will put the
economy of Nigeria on a sound footing and rank Nigeria
economy at par with what obtains in the developed nations.
1. Government should provide policies and legislations for
the creation of:
(a) Oil farmers association to ensure production of a target
volume of the commodity in the short, medium and long
terms,
(b) A governing council to work for the establishment of a
viable oleochemicals production in Nigerian with a
target time for its product to be available in the Nigeria
and subsequently the world markets,
(c) A fund that derives its source directly from the crude
petroleum sale to foster the activities set up in (a) and (b)
above
2. National Universities Council should:
(i) Create and enforce compliance of a new policy on
interdisciplinary research in preference over and above
individual research in the universities. It should also provide
guidelines whereby a minimum of say 10% of fund allocated
to Universities shall be devoted to research studies,
(ii) Constitute a monitoring unit to ensure compliance in the
investment of research grants in every university,
(iii) Work discretely through a committee of NUC/ industries to
promote university research and industries interaction
through healthy collaborations between the two bodies and
with adequate funding by the industries,
44
(iv) Fill the gap between Raw Material Development Council,
and the universities through a committee of the Governing
Council for the RMDRC and NUC. The committee should
give premium to collaborative interdiscipline research in the
universities with sharper focus on direct and immediate
relevance on the life and developmental efforts of the nation.
3. The universities: Universities in Nigeria should create
a special central account specifically designated for equipment
that shall function and be properly maintained thereafter to
render useful services. In addition:
i. every University shall have a virile consultancy division to
foster a healthy and productive University-Industrial
relationship and attract necessary funding for research in the
university,
ii. every university senate shall constitute its committee to seek
and develop appropriate links with universities outside
Nigeria for exchange of research information and
collaboration on uses of facilities that make up for short falls
in Nigerian universities. The committee shall mandatorily
give reports, at least once, every academic session to the
senate on its progress.
In my final submission sir, Mr. Vice Chancellor, the
primary objective of the University is to provide unbiased
intellectual leadership for the development of the nation.
Thevetia plant is a challenge for the University of Ilorin to place
Nigeria in the world map as a pacesetter in the studies of thevetia
plant so that by 2015 it shall be an economic plant. It is an
achievable target and I sincerely plead with the University of
Ilorin management and Governing Council to accept the
challenge to act positively. Towards this goal, sir, distinguished
ladies and gentlemen and particularly my special invitees,
captains of industries, please accept this very special
recommendation as worthy of immediate implementation. That
the University of Ilorin Governing Council should establish
THEVETIA PLANT SPECIAL RESEARCH FUND into which
every stakeholder in Nigeria shall subvent generously. The fund
45
shall ensure success of thevetia plant research studies such that
the University of Ilorin shall be “World Centre of Excellence on
Thevetia Plant Development”. It is an achievable feat worthy of
careful consideration.
Thank you and God bless you for your attention.
ACKNOWLEDGEMENT
To God be glory, honour and adoration for His
goodness, mercies and for making today a reality.
God has made so many people to work to make today a
reality. I am sincerely grateful to all the people that God has
directed to contribute in any one form no matter how small.
I am particularly grateful to all undergraduate students
who have done their project studies with me. My sincerely
gratitude also goes to all my M.Sc. and Ph.D students. I say God
bless you all. Your own day of honour will not elude you.
I register my sincerely appreciation to the University
authorities who have given me employment, peace of heart and
the facilities and challenge of “publish or perish” that have
stimulated the research studies I have the opportunity to give
account of today.
Relations, social associates, Christian friends
particularly and many Muslim friends have been supportive in
very many ways.
Members of my family beginning with my wife have
endured so much, I thank you for your understanding and
endurance when you have missed me physically and when I have
been tight financially because I must succeed in my studies. The
honour and joy of today belong to all us.
Thanks to all who have come today in response to my
special invitation. You are great and the benefits that will accrue
from the challenges of the lecture certainly remain your great
reward.
God bless you ALL.
46
REFERENCES
1. Features (1991); INFORM 2 (8) 678-692.
2. Frank Gumstone (2006); INFORM 17 (8) 541-543.
3. Salmiah Ahmad (2003); Malaysia: the hub for plantbased
oleochemicals. INFORM 14 (10) 604-606.
4. Ibiyemi S.A. and T. Faloye (1988); Potassium, Nitrogen
and Calcium uptake by T. peruviana seedlings as
affected by various nutrient sources. Nig. J. Agronomy.
3 (2) 68-73.
5. Ibiyemi S.A., Fadipe V.O., Akinremi O.O. and Bako
S.S., (2002); Variation in Oil Composition of Thevetia
peruviana juss (Yellow Oleander) Fruits Seeds. J. Appl.
Sci. Environ Mgt. 6 (2) 61-65.
6. Fagbule, M.O. & O.A. Sosanwo (1983); Nig. J. Sci. &
Tech. 1 (11) 31-36.
7. Ibiyemi S.A., S.S. Bako, G.O. Ojokuku, & V.O. Fadipe
(1995); J. Am. Oil Chem. Soc. 72 (6) 745-747.
8. Thomas Mielke (2003); The world outlook for major
oilseeds; INFORM 14 (12) 712-713.
9. Maruyama T. (1994); INFORM 5 (10) 1338-1140.
10. INFORM staff reporters (2006); Ten years of oilseed, oil
and meal forecast. INFORM 17 (5), 290.
11. Ibiyemi, S.A and Oluwaniyi, O.O. (2003); Efficacy of
catalysts in the batch esterification of the fatty acids of
T. Peruviana seed oil. J. Appl. Sci. Environ Mgt. 7 (1)
15-17.
12. Oluwaniyi, O.O. (2007). Ph.D. thesis (in progress)
13. Turner G.P. (1988); Introduction to paint chemistry and
principles of paint chemistry and paint technology. 3rd
ed. Chapman and Hall, pp 108-169.
14. Algers, S. M. (2001); Polymer Science dictionary;
Elsevier Science Publ. Co. N.Y. 140 – 145.
15. Brain Author, A Fox, Altan G. Cameron, a textbook on
Food Science and Health, 5th ed. pp 172.
47
16. Bisset, N. G. (1963); Cardiac glycosides. IV,
Apocynaceae: a preliminary paper chromatographic
studies of the glycosides from T. peruviana. Ann. Bogor
4(2) 145-152 (Chemical Abstract 58:14438h).
17. Sun N.C. and N.I. Libizor (1965). The glycosides of T.
Peruviana. Chemical Abstract 6: 20496.
18. (a) Perez – Amador, M., E.A. Bratoeff and S.B.
Hernandez (1994); Thevetoxide and digitoxigenin,
cardenolides from two species of Thevetia
(Apocynaceae). Chemical Abstract 120: 319441t.
(b) Lang, H.Y. and N.C. Sun; (1964); The cardiac
glycosides of T. Peruviana II. Isolation and
identification of cerberin, ruvoside and a new cardiac
glycoside, perusitin. Yao Hsueh Hsueh Pao 11(7) 464-
472 (Ch.) (Chem. Abstr. 62: 9465a, 1965).
(c) Huang, C.C., K.H. Hung and S.H. Lo (1965);
Pharmacology of the glycosides of T. Peruviana I.
Thevetin. Yao Hsueh Hsueh Pao 12(2) 824-826 (Ch.)
(Chem. Abstr. 64: 18275d, 1966).
19. Arora, R.B., J.N. Sharma, and M.C. Bhatia, (1967);
Pharmacological evaluation of peruvoside, a new cardiac
glycoside from T.neriifolia with a note on its clinical
trials in patients with congestive heart failure; Indian J.
Exp. Biol. 5(1) 31-36 (Chem. Abstr. 67: 20362f, 1967).
20. Atteh, J.O., S.A. Ibiyemi, and A.O. Ojo (1995);
Response of broilers to dietary levels of thevetia cake. J.
Agric. Sci Cambridge. 125, 307 – 310.
21. Oluwaniyi, O.O., S.A. Ibiyemi and A.L. Usman, (2007).
Effect of detoxification on the nutrient content of T.
Peruviana seed cake. Res. J. of Applied Sci. 2 (2) 188-
191.
22. Atteh, J.O., S.A. Ibiyemi, F.O. Onadepo and
O.O.Ugboma, (1990); J. Agric. Sci. Cambridge. 115,
141-145.
48
23. Oluwaniyi, O.O. and S.A. Ibiyemi (2007); A study of the
extractability of thevetia glycosides with alcohol
mixture. African Journal of Biotechnology (Accepted).
24. El-Adawy, T.A. and El-Kadousy, S.A. (1995); Changes
in chemical composition, nutritional quality, physicochemical
and functional properties of peach kernel meal
during detoxification. Food Chemistry 52: 143 – 148.

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+ Show full citationThis is the first page of the item you requested.

• Morphology and Embryology of Holarrhena antidysenterica Wall.

• C. S. Lattoo
• Botanical Gazette, Vol. 135, No. 3 (Sep., 1974), pp. 173-180
(article consists of 8 pages)
• Published by: The University of Chicago Press
• Stable URL: http://www.jstor.org/stable/2474236
Morphology and Embryology of Holarrhena antidysenterica Wall., by C. S. Lattoo © 1974 The
University of Chicago Press.

Abstract
The wall of the microsporangium in Holarrhena antidysenterica Wall. consists of an epidermis,
endothecium, one or two middle layers, and a secretory tapetum. The endothecial cells develop
fibrous thickenings in later stages. Division of microspore mother cells is successive, and cytokinesis
takes place by cell-plate formation. Isobilateral, decussate, T-shaped, and linear tetrads of
microspores are formed; the pollen grains are two-celled at anthesis. The ovule is hemianatropous
and tenuinucellate. The development of the embryo sac conforms to the Polygonum type. Synergids
are long with prominent hooks, and antipodals are ephemeral. Endosperm development is of the
nuclear type and ultimately becomes cellular. Embryo development follows the Caryophyllad type
(Johansen 1950). The seed coat formed by the integument consists of three regions: an outermost
epidermis, a thick-walled middle region, and thin-walled inner cells. The epidermal cells contain
starch and tannin.

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1Current Address: Bridgewater College, Box 1568, Bridgewater, Virginia 22812, U.S.A.
J. Bot. Res. Inst. Texas 2(1): 489 – 493. 2008
CHROMOSOME NUMBER OF THEVETIA AHOUAI
(APOCYNACEAE: RAUVOLFOIDAE: PLUMERIEAE) WITH DISCUSSION
ON THE GENERIC BOUNDARIES OF THEVETIA
Justin K. Williams and Julia K. Stutzman1
Department of Biological Sciences
Sam Houston State University
Huntsville, Texas 77341-2116, U.S.A.
abstract
The mitotic chromosome count (2n = 20) for Thevetia ahouai is the first reported chromosome count for Thevetia sect.
Ahouai. The count
together with a previous count in Thevetia sect. Thevetia (also 2n = 20) provides an additional synapomorphy that further
supports
the monophyly of Thevetia as traditionally recognized. A discussion on the proposal to recognize Thevetia sect. Thevetia
as the genus
Cascabela is provided.
Key Words: Thevetia, Apocynaceae, chromosome number, Cascabela, Cerbera, Plumerieae
resumen
El recuento cromosomático mitótico (2n = 20) de Thevetia ahouai es el primero para Thevetia sect. Ahouai. Este recuento
junto con otro
previo en Thevetia sect. Thevetia (también 2n = 20) aporta una sinapomorfía adicional que apoya la monofilia de
Thevetia como se ha
reconocido tradicionalmente. Se aporta una discusión de la propuesta de reconocer Thevetia sect. Thevetia como el
género Cascabela.
Thevetia L. belongs to the Apocynaceae subfamily Rauvolfioideae tribe Plumerieae and
comprises eight species
of shrubs occurring from Central Mexico to northern South America (Gensel 1969; Williams
1996; Allorge
1998; Endress et al. 2007). According to various specialists in the Apocynaceae, the generic
boundaries of
Thevetia vary. In the concept of Thevetia sensu K. Schum. (Schumann 1895; Gensel 1969;
Williams 1996;
Allorge 1998; Alvarado-Cardenas 2004), eight species are sub-divided between two sections:
sect. Ahouai
K. Schum. with three species—T. ahouai (L.) A. DC., T. amazonica Ducke, and T. bicornuta Mull.
Arg.—and
sect. Thevetia K. Schum. with five species—T. gaumeri Hemsl., T. ovata (Cav.) A. DC., T.
peruviana (Pers.) K.
Schum., T. pinifolia (Standl. & Steyerm.) J.K. Williams, and T. thevetiodes (H.B.K.) K. Schum. In
the concept
of Thevetia sensu Lippold (Lippold 1980; Alvarado-Cardenas & Ochoterena 2007) the three
species of
sect. Ahouai are retained in Thevetia; the other five species of sect. Thevetia are segregated
into the genus
Cascablea Raf. Although Lippold (1980) and Alvarado-Cardenas and Ochoterena (2007)
segregate Thevetia
sensu K. Schum. into the two genera Thevetia and Cascablea, at no point do they argue
against the genera’s
“close morphological relationship” (Alvarado-Cardenas & Ochoterena 2007). In fact, a recent
morphological
cladistic analysis (Fig. 1, Alvarado-Cardenas & Ochoterena 2007) nests all eight species of
Thevetia sensu K.
Schum. in a clade supported by six synapomorphies. In short, Thevetia sensu K. Schum. is
clearly shown to be
monophyletic. Nevertheless, Alvarado-Cardenas and Ochoterena (2007) argue for its paraphyly
and choose
instead to recognize the two sub-clades of the clade (Fig. 1) as distinct genera: Thevetia and
Cascabela.
Chromosome numbers have proven useful in resolving generic relationships in the
Apocynaceae (Van
der Laan & Arends 1985; Williams 2007). To date, 73 of the 179 genera of the Apocynaceae
s.str. (subfamilies
Rauvolfioideae and Apocynoidoideae) have been counted (Van der Laan & Arends 1985;
Goldblatt & Johnson
2003; Williams 2007). Although previous chromosome counts for sect. Thevetia (T. peruviana;
2n = 20) exist,
none have been reported for sect. Ahouai. The present paper provides the first reported
chromosome count
for a species of sect. Ahouai and discusses the utility of chromosome numbers in interpreting
the systematic
relationship of Thevetia and Cascabela.
490 Journal of the Botanical Research Institute of Texas 2(1)
Fig. 1. Morphologically constructed dendogram of “Plumerieae” clade (from Alvarado-Cardenas &
Ochoterena 2007). a = Thevetia sensu K. Schum. and/
or “Cascabela-Thevetia” clade of Alvarado-Cardenas and Ochoterena 2007. This clade is supported by
seven synapomorphies. b = Thevetia sect. Ahouai
and/or Thevetia sensu Lippold. This clade is supported by two synapomorphies. c = Thevetia sect.
Thevetia and/or Cascablea Lippold. This clade is supported
by four synapomorphies.
Williams and Stutzman, Chromosome number of Thevetia ahouai 491
materials and methods
Roots tips and voucher specimens were collected from a greenhouse specimen of Thevetia
ahouai housed
at the greenhouse of the Department of Biological Sciences, Sam Houston State University
(Table 1). The
root tips were fixed and analyzed for chromosome number using standard procedures (Raffauf
1964; Van
der Laan & Arends 1985). A voucher specimen of the greenhouse plant was made and is
preserved in the
Warner Herbarium (SHST).
results
The format used for reporting chromosome numbers in this article follows that established by
Strother and
Nesom (1997). A mitotic chromosome number of 2n = 20 was recorded for Thevetia ahouai.
Van der Laan
and Arends (1985) reported chromosome lengths in the Apocynaceae to be between 0.5–4.0
μm, with the
average chromosome length between 1–2 μm. The length of the chromosomes in T. ahouai
varied between
1–3 μm, consistent with most other chromosomes in the Apocynaceae. The base chromosome
number (x =
10) for Thevetia ahouai is consistent with previous reports of x = 10 for Thevetia (T. peruviana
2n = 20; Gadella
1977; Ugborogho 1983; Van der Laan & Arends 1985; Santhosh & Omanakumari 1997).
discussion
Van der Laan and Arends (1985) postulated a base chromosome number of x = 11 for the
Apocynaceae s.
str. based on its prevalence in the family and on the observation that many of the
plesiomorphic taxa possess
a base number of x = 11. A base chromosome number of x = 10 is found in four genera
representing
three of the 11 tribes recognized in the Rauvolfioideae (Endress et al. 2007): Hunterieae
(Gonioma E. Mey.),
Plumerieae (Cerbera L., Thevetia), and Vinceae (Ochrosia Juss.). Based on tribal
circumscription (Endress
et al. 2007) along with molecular evidence (Simóes et al. 2007), x = 10 is reconstructed as
having evolved
independently at least three times in the Rauvolfioideae.
A cursory review of chromosome counts for the Apocynaceae (Van der Laan & Arends 1985;
Goldblatt
& Johnson 2003) reveals that at present the only chromosome counts for genera in the
Plumerieae, and thus
relatives to Thevetia (2n = 20), are for Allamanda L. (n = 9; 2n = 18), Cerbera (2n = 40),
Himatanthus Willd. ex
Schult. (2n = 18), Mortoniella Woodson (2n = 32), and Plumeria L. (2n = 36). At present there
are no chromosome
counts for the remaining four genera in the Plumerieae: Anechites Griseb., Cameraria L.,
Cerberiopsis
Vieill. ex Pancher & Sébert, and Skytanthus Meyen., and it is suggested here that effort be
made to obtain
count of these taxa. Figures 1 and 2 present cladograms of the Plumerieae constructed from
morphological
(Alvarado-Cardenas & Ochoterena 2007) and molecular evidence (Simóes et al. 2007),
respectively. Diploid
counts for the respective genera included in the phylogenies are presented for both Figures 1
and 2.
Alvarado-Cardenas and Ochoterena (2007) presented six synapomorphies that described the
Thevetia
sensu K. Schum. clade (Fig. 1, branch a). The diploid count of 2n = 20 presented here adds a
seventh synapomorphy.
When interpreting their data Alvarado-Cardenas and Ochoterena (2007) state that “(t)here is
still no consensus regarding the question of whether one should recognize one genus with two
(sections)
or two distinct genera (Lippold 1980), given that Cascabela and Thevetia are sister taxa.” We
would argue
that the consensus in evolutionary systematics is to assign generic boundaries that reflect
both monophyly
and shared ancestry. The decision to divide a well supported clade into two separate genera
may support
monophyly; however, it excludes shared ancestry. Without prior knowledge, most botanists
would be unaware
that Thevetia and Cascabela are sister taxa that share seven synapomorphies. Instead, an
evolutionarily
more meaningful interpretation of the clade would be that Thevetia sensu K. Schum. is
monophyletic and
support is provided for the recognition of two sections as defined by Schumann (1895). In
order to maintain
systematic consistency as pertains to current trends in phylogenetic nomenclature we
recognize Thevetia
sensu K. Schum. and regard Cascabela and all taxa pertaining to the genus as synonyms of
Thevetia.
492 Journal of the Botanical Research Institute of Texas 2(1)
Table 1. Voucher specimen for the chromosome number of Thevetia ahouai.
Taxon Voucher specimen Chromosome
number (2n)
Thevetia ahouai TEXAS: Sam Houston State University 20
Department of Biological Sciences
Greenhouse specimen, 22 Jan 2008
Williams 2008-1 (SHST).
Fig. 2. Molecularly constructed dendogram of “Plumerieae” clade (from Simóes 2007).
acknowledgments
We thank Tami Cook for providing us with access to her digital light microscope, and Bob
Rhodes for
mixing the Carnoy’s solution and aceto-orcein. Sibyl Buceli and an anonymous reviewer
provided valuable
editorial comments.
references
Allorge, L. 1998. Les Thevetia, compagnons des succulentes. Succulentes 21(1):23–32.
Alvarado-Cardenas, L.O. 2004. Apocynaceae. Flora del Valle de Tehuacan-Cuicatlan 38:1–57.
Alvarado-Cardenas, L.O. and H. Och oterena. 2007. A phylogenetic analysis of the Cascabela-Thevetia
species complex
(Plumerieae, Apocynaceae) based on morphology. Ann. Missouri Bot. Gard. 94:298–323. 2007.
Endress , M.E. and P. Bruyns. 2000. A revised classification of the Apocynaceae s.l. Bot. Rev. 66:1–
56.
Endress , M.E., S. Liede-Sch umann, and U. Meve. 2007. Advances in Apocynaceae: The
enlightenment, an introduction.
Ann. Missouri Bot. Gard. 94:259–267.
Gadella, T.W.J. 1977. IOPB chromosome number reports LVI. Taxon 26:257–274.
Gensel, W.H. 1969. A revision of the genus Thevetia (Apocynaceae). Masters Thesis University of
Connecticut.
Goldblatt, P. and D.E. Johnson. 2003. Index to plant chromosome numbers 1998–2000. Monogr.
Syst. Bot. Missouri
Bot. Gard. 94.
Lipp old, H. 1980. Die Gattungen Thevetia L., Cerbera L. und Cascabela Raf. (Apocynaceae).
Feddes Repert.
91:45–55.
Raff auf, R.F. 1964. Some chemotaxonomic considerations in the Apocynaceae. Lloydia 27:288–
298.
Santhosh , B. and N. Omanakumari. 1997. Karyomorphological studies on two varieties of Thevetia
peruviana. J.
Cytol. Gene. 32:95–98.
Williams and Stutzman, Chromosome number of Thevetia ahouai 493
Sch umann, K. 1895. Apocynaceae. In: A. Engler and K.A. Prantl, Die Natürlichen Pflanzenfamilien.
Wilhelm Engelmann,
Leipzig. 4(2):109–189.
Si móes, A.O., T. Livsh ultz, E. Conti, and M.E. Endress . 2007. Phylogeny and systematic of the
Rauvolfioideae (Apocynaceae)
based on molecular and morphological evidence. Ann. Missouri Bot. Gard. 94:268–297.
Strother, J.L. and G.L. Nesom. 1997 Conventions for reporting plant chromosome numbers. Sida
17:829–831.
Ugborogho, R.E. 1983. IOPB chromosome number reports LXXIX. Taxon 32:321.
Van der Laan, F.N. and J.C. Arends. 1985. Cytotaxonomy of the Apocynaceae. Genetica 68:3–35.
Williams , J.K. 1996. A new combination in Thevetia (Apocynaceae). Sida 17:185–190.
Williams , J.K. 2007. Documented chromosome numbers 2007: Chromosome number of
Laubertia contorta
(Apocynaceae: Apocynoideae) and its phylogenetic importance. J. Bot. Res. Inst. Texas 1:431–
435. 2007.

Biological Journal of the Linnean Society (1998), 63: 553–577. With 6 figures

The timing of insect/plant diversification:

might Tetraopes (Coleoptera: Cerambycidae)
and
Asclepias (Asclepiadaceae) have co-evolved?
B. D. FARRELL*
Museum of Comparative Zoology, Harvard University, Cambridge, MA 02138,
U.S.A.
C. MITTER
Department of Entomology, University of Maryland, College Park, MD 20742,
U.S.A.
Received February 1997; accepted for publication 24 October 1997
Ehrlich and Raven’s essay on coevolution has stimulated voluminous work on the mechanisms
of insect/plant interaction, but few explicit tests of their model’s prediction that the
evolutionary success of entire insect and plant clades is governed by their putative reciprocal
adaptations. This paper begins an inquiry into possible coevolutionary diversification for
North American milkweeds of the genus Asclepias and one of their few major herbivores, the
longhorn beetle genus Tetraopes, focusing first on the historical duration and continuity of the
interaction. A phylogeny for Tetraopes and relatives, estimated from morphology and
allozymes,
shows evident similarity to a morphology based hostplant cladogram synthesized from the
literature, though the significance of the correspondence under heuristic statistical tests
depends on the treatment of one beetle species reported (without certainty) from multiple host
species. Fossils and biogeography support the interpretation that cladogram correspondence
reflects synchronous diversification of these two clades, hence opportunity for coevolution,
rather than beetle ‘host-tracking’ of previously-diversified plants. Cladogram correspondence
is more evident at higher than at lower levels, as expected under Ehrlich and Raven’s model.
An apparent phylogenetic progression in the potency and location of milkweed cardenolides,
seemingly related to species diversity of both Asclepias and Tetraopes subclades, provides
further
suggestive evidence for that model. The phylogeography of the Tetraopes/Asclepias
assemblage
suggests that extant species evolved largely in their current, often quite localized biomes,
facilitating potential experimental tests for hypotheses of adaptation and counteradaptation
and their importance to diversification.
Ó 1998 The Linnean Society of London
ADDITIONAL KEY WORDS—coevolution – herbivores – plant–animal interactions –
phylogeny – cardenolides – escalation.
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . 554
*Correspondence to: B. D. Farrell. Email:bfarrell@oeb.harvard.edu.
553
0024–4066/98/040553+25 $25.00/0/bj970207 Ó 1998 The Linnean Society of London
B. D. FARRELL 554 AND C. MITTER
Natural history of Tetraopes and allies . . . . . . . . . . . . . 555
Relationships among hostplants . . . . . . . . . . . . . . . 559
Material and methods . . . . . . . . . . . . . . . . . . . 560
Morphological analysis . . . . . . . . . . . . . . . . . . 560
Allozyme electrophoresis . . . . . . . . . . . . . . . . . 561
Phylogenetic analyses . . . . . . . . . . . . . . . . . . 563
Comparisons of phylogeny estimates . . . . . . . . . . . . . 563
Results . . . . . . . . . . . . . . . . . . . . . . . . 564
Allozyme data . . . . . . . . . . . . . . . . . . . . 564
Phylogenetic analyses . . . . . . . . . . . . . . . . . . 564
Concordance of insect and plant cladograms . . . . . . . . . . 566
Discussion . . . . . . . . . . . . . . . . . . . . . . . 567
Ages of associated beetles and hosts . . . . . . . . . . . . . 568
Coevolution? . . . . . . . . . . . . . . . . . . . . . 569
Concluding observations . . . . . . . . . . . . . . . . . 572
Acknowledgements . . . . . . . . . . . . . . . . . . . . 573
References . . . . . . . . . . . . . . . . . . . . . . . 573
Appendix . . . . . . . . . . . . . . . . . . . . . . . . 577
INTRODUCTION
Among explanations offered for the great diversity of phytophagous insects
and
their hostplants, none has been more influential than Ehrlich & Raven’s
(1964)
theory of coevolution. For three decades this proposal has stimulated work on
the
physiological, genetic and ecological mechanisms of insect/plant interactions,
focused
especially on the role of plant secondary chemistry (Futuyma & Keese, 1992).
Only
recently, however, has there begun comparably rigorous investigation of the
model’s
macroevolutionary implication, namely that these interactions have
promoted diversification
of associated insect and plant groups.
This study opens an inquiry into the possibility of coevolutionary
diversification
for the North American longhorn beetle genus Tetraopes and its primary
hosts, the
milkweed genus Asclepias. We first present a phylogenetic analysis of
Tetraopes, based
on morphology and allozymes. In conjunction with published information on
the
phylogeny, biogeography and secondary chemistry of Asclepias, we then use
these
results to examine several questions raised by Ehrlich & Raven’s theory.
The main focus of this paper is on how long and continuously these beetle
and
plant species, and their ancestors, have interacted: what has been the
opportunity
for coevolution? One expectation, if insect and plant lineages have diversified
in
association, is that the phylogenetic order of divergence among extant host
taxa
should correspond in some way to that among their associated herbivores.
For
example, if the insects never switched hosts, the association could be
continuous
over time, yielding an essentially perfect match of speciation sequences. In
contrast,
the ‘escape and radiation’ process envisioned by Ehrlich and Raven should
produce
an only imperfect correlation of speciation sequences, but a closer match of
insect
phylogeny to the evolutionary succession of plant defences.
The few explicit studies so far suggest great heterogeneity in the
correspondence
between insect and hostplant phylogenies. In a review of 14 assemblages for
which
at least partial cladogram comparison can be made (Mitter & Farrell, 1991;
Farrell,
Mitter & Futuyma, 1992), the mean fraction of groupings on which insects
and
hostplants agreed was about 50%, suggesting that some degree of parallel
diversification
might be common. However, in only about a quarter of the comparisons
TIMING OF INSECT/PLANT DIVERSIFICATION 555
was the match significant or nearly so under heuristic statistical tests, and in
about
the same number there was no correspondence at all.
A biological pattern to this variation was suggested by a study of
Phyllobrotica leaf
beetles, which show the most detailed match to host phylogeny of any
herbivorous
insect group yet reported (Farrell & Mitter, 1990). These beetles exhibit
unusually
intimate dependence on their hosts, mostly herbaceous mints in the genus
Scutellaria.
Each beetle species is restricted to a single host species; the larvae are
internal
feeders, in the roots, and the adults both feed and mate on the larval host.
The adults, moreover, appear aposematically coloured, suggesting that like
other
herbivores of plants that contain iridoid glycosides (Bowers, 1988), they
might rely
on host-derived toxins for protection from their predators. All these traits
have been
argued to enforce long-term host fidelity (Feeny, 1987), and may make
parallel
diversification especially likely.
Tetraopes and its hosts provide a test of this conjecture, because the life
history of
these beetles is strikingly similar to that of Phyllobrotica. We will present
evidence
that phylogenetic relationships within Tetraopes are also strongly concordant
with
those of their hosts. Phylogeny concordance by itself, however, is not
sufficient to
demonstrate parallel phylogenesis: the entire insect clade could instead be
younger
than any of the host species, but have undergone colonization and speciation
in a
sequence dictated by features that mirror host phylogeny. For example,
derived
plant species might often bear unusual secondary chemistries, causing them
to be
colonized later than their less distinctive, more primitive relatives.
We will present initial evidence consistent with such ‘escape and radiation’:
Asclepias shows a phylogenetic progression in the potency and location of
cardenolides,
suggesting escalation of defence, which appears related to species diversity
of both
milkweeds and beetles.
Natural history of Tetraopes and allies
Tetraopes and its apparent nearest relatives, the genus Phaea, belong to the
monophyletic tribe Tetraopini, subfamily Lamiinae, of the Cerambycidae. The
tribes
Tetraopini, Hemilophini and Phytoecini together form the apparently
monophyletic
‘Phytoeciides vrais’ of Chapuis (1872), distinguished by bifid tarsal claws, and
constitute nearly 25% of the 50 000 described cerambycid species. Larvae of
these
tribes invariably bore in stems or roots of their hostplants, which are typically
herbaceous, while the adults consume the foliage and flowers. A preliminary
morphological phylogenetic study treating all 40 genera of Tetraopini plus
outgroups
confirmed that Tetraopes and Phaea together form an entirely New World
clade,
defined by appendiculate tarsal claws. This clade in turn appears most closely
related
to the Philippine genus Eustathes, with which it shares a pronotal umbone.
Tetraopes
is distributed from Guatemala to Canada, and is most diverse in the Sonoran
region (Chemsak, 1963; Chemsak & Linsley, 1979). Phaea, currently under
revision
(Chemsak, 1977; J. Chemsak, pers. comm.), appears to contain about 30
species
distributed from northern South America (Colombia and Venezuela) to the
central
United States, and is most diverse in Central America.
The following account of life histories in Tetraopes is based largely on the
revision
B. D. FARRELL 556 AND C. MITTER
TABLE 1. Distributions and host affiliations of Tetraopes species, plus the subset of species in
the sister
genus Phaea included in this study. An asterisk follows the name of each species sampled for
allozymes
Taxa Hosts Distribution
Phaea jucunda Ipomoea pandurata Southeastern U.S.
P. canescens I. leptophylla Midwestern U.S.
P. mirabilis* Thevetia ovata S. Mexico
P. maryannae* Stemmadenia palmeri S. Mexico Nicaragua
P. biplagiata Stemmadenia palmeri S. Mexico to Guatemala
Tetraopes mankinsi Honduras, Guatemala
T. melanurus* Asclepias tuberosa Eastern U.S.
T. cleroides Central Mexico
T. paracomes Matelea quirosii Guatemala to Costa Rica
T. comes S. Mexico to Costa Rica
T. elegans Baja California
T. ineditus Marsdenia lanata Western Mexico
T. batesi Western Mexico
T. umbonatus* A. glaucescens S. Mexico to Nicaragua
T. linsleyi A. linaria AZ, TX, NM
T. discoideus* A. subverticillata AZ, TX, NM
A. curassavica Mexico to El Salvador
T. annulatus* A. sullivantii (MO) TX, NM, AZ, North to Canada
A. subverticillata (AZ)
A. speciosa (UT)
T. pilosus* A. arenaria TX, KS, NE, OK
T. tetraophthalmus* A. syriaca Northeastern U.S.
T. varicornis* A. notha S. Mexico
T. texanus* TX, OK
T. mandibularis* A. latifolia TX, OK
T. quinquemaculatus* A. amplexicaulis Midwestern U.S., to S. Ontario
T. subfasciatus Central Mexico
T. thoreyi Central Mexico
T. sublaevis* A. erosa S. California
T. thermophilus SE TX, S to El Salvador
T. basalis* A. eriocarpa CA, S.Oregon
T. femoratus* A. speciosa Central and western North America
by Chemsak (1963), supplemented by observations from fieldwork by the
senior
author across North and Central America (Tables 1, 2).
Tetraopes larvae attack roots, while the adults, whose emergence coincides
with
hostplant flowering, feed on upper foliage and flowers. The adult females of
Tetraopes
tetrophthalmus (Forster) oviposit 8–20 eggs inside grass stems within a few
centimeters
of a milkweed plant, gaining access via a hole chewed for the purpose. About
10
days later, the hatchling larvae emerge from the oviposition access, fall to
the ground
and immediately burrow downward, in presumed search for host roots.
Hartman
(1977) documented extensive damage to the roots of Asclepias syriaca by
larvae of
Tetraopes tetrophthalmus, while Chemsak (1963) presented evidence of
similar damage
to the roots of A. erosa by its herbivore, Tetraopes sublaevis Casey; to A.
eriocarpa by T.
basalis LeConte; and to A. speciosa by T. femoratus LeConte. Similar damage
is caused
by other species of Tetraopes. Tetraopes larvae feed both inside and outside
of the root
system and there seems to be a correlation of adult body size with the size of
the
larval resource (Price & Wilson, 1976; Hespenheide, 1973). Larval root
feeding is
unique to Tetraopes in the subfamily Lamiinae; Phaea and other genera mine
stems
(Linsley, 1961).
With few confirmed exceptions, individual Tetraopes species or their
subspecies are
TIMING OF INSECT/PLANT DIVERSIFICATION 557
TABLE 2. Localities from which population samples of Tetraopes and Phaea species were
obtained for
protein electrophoresis
Taxa Localities sampled
Phaea mirabilis MEXICO: Estado Morelos, Zumpango del Rio
P. biplagiata MEXICO: Estado Guerrero, Iguala
P. maryannae MEXICO: Estado Guerrero, Iguala
Tetraopes discoideus (1) AZ: Portal MEXICO: (2) Taxco (3) Puebla, Atlixco
T. umbonatus MEXICO: (1) Guerrero, Xochichalca Ruins (2) Oculixtlhuacan (3) Iguala (4)
Puebla, Atlixco
T. melanurus MD: Prince Georges Co., Patuxent Wildlife Research Center
T. quinquemaculatus MO: (1) Clay Co., Flemington; (2) Le Petite Grande Prairie; (3) Neiwathe Prairie;
(4) KS: Reno Co., Hutchinson
T. texanus (1) MO: Clay Co., Flemington (2) TX: Cooke Co., Gainesville
T. annulatus NM: (1) McKinley Co. Mesita [16] (2) Gallup [19] (3) AZ: Apache Co., Houck
T. pilosus (1) KS: Reno Co., Sandhill State Park, Hutchinson (2) TX: Ward Co., Monahans
T. tetropthalmus MO: Clay Co.: (1) Independence; (2) Flemington; (3) VT: Chittenden Co.:
Colchester; (4) MD: Prince Georges Co. Patuxent Wildlife Research Center
T. mandibularis TX: Dickens Co., Dickens
T. varicornis MEXICO: Puebla, Rio Frio
T. femoratus (1) NV: Reno (2) CA: Graegele
T. basalis CA: Plumas Co.; Quincy
T. sublaevis CA: Riverside Co., Blythe
affiliated with single, differing species of milkweeds in the subgenus
Asclepias (Asclepias).
The following brief account is summarized in Table 1. Tetraopes umbonatus
LeConte
is affiliated with A. glaucescens throughout southern Mexico (Chemsak, 1963;
Farrell,
1991). Tetraopes paracomes Chemsak and Tetraopes ineditus Chemsak are
associated with
Marsdenia and Matelea, respectively, vining milkweeds in the lowland forests
of Central
America and Mexico (B. Farrell, unpublished). Tetraopes discoideus LeConte
is the
only species confirmed to use two hostplant species (Chemsak, 1963; Farrell,
1991).
It ranges from southern Mexico, where it feeds on A. curassavica (Chemsak,
1963;
Farrell, 1991), to the southwestern United States, where it attacks A.
subverticillata
(Chemsak, 1963; Farrell, 1991), and is absent from the area north of Mexico
City
to approximately the U.S. border. Both of these hostplant species are in
Woodson’s
(1954) series Incarnatae and their cardenolide profiles are similar (Roeske et
al.,
1976). Tetraopes melanurus Schonherr attacks A. tuberosa throughout the
eastern United
States (Chemsak, 1963; Farrell, 1991). Tetraopes tetrophthalmus feeds on
Asclepias syriaca
throughout its range (Farrell, 1991; Hartman, 1977), though an isolated
population
in a disturbed site in Illinois was reported on A. verticillata, where the adults
may
suffer reduced fitness (Price & Willson, 1976). Tetraopes femoratus is
affiliated with A.
speciosa throughout the western United States (Chemsak, 1963; Farrell,
1991), but
has been reported in very low numbers from A. syriaca at the eastern edge of
its
range (Lawrence, 1982). Tetraopes pilosus Chemsak and its host, A. arenaria,
are
restricted to the Quaternary sandhills of the midwestern U.S. (Farrell, 1991).
Both
beetle and host are clothed in white pubescence, possible adaptations
against moisture
loss and overheating in their xeric dune habitats. Tetraopes mandibularis
Chemsak is
affiliated with Asclepias latifolia in the Llano Estacado region of northwestern
Texas
and adjacent Oklahoma (Rice, Turnbow & Hovore, 1985; Farrell, 1991).
Tetraopes
varicornis Castelnau uses A. notha in southern Mexico (Farrell, 1991).
Tetraopes sublaevis
and its sole host A. erosa are confined to the lower Colorado Desert
(Chemsak, 1963;
Farrell, 1991).
B. D. FARRELL 558 AND C. MITTER
Two additional species of Tetraopes have been reported only from single host
Asclepias species, and we have no direct observations that contradict these
associations
though we cannot confirm them first hand. Tetraopes quinquemaculatus
Haldeman was
reported to attack A. amplexicaulis in the midwestern U.S. (Price & Wilson,
1979)
and we have collected several specimens of this species in the vicinity of A.
amplexicaulis
but have not observed adult or larval feeding. Tetraopes linsleyi Chemsak
reportedly
uses A. linaria, the only milkweed to occur in its dry Chiricahuan canyon
habitats
(Hovore, 1983; pers. comm.). We provisionally accept these associations
pending
confirmation. In contrast, while Tetraopes elegans Horn was inferred to use
A. subulata
by Chemsak (1963) on the grounds that this is one of very few milkweeds
that occurs
throughout its range in Baja California, the distribution of Asclepias albicans
is also
very similar (Woodson, 1954) to that of T. elegans and two other Asclepias
species,
very closely-related to A. albicans, also occur in Baja. Therefore, we regard
the host
of T. elegans as unknown.
The hosts of T. annulatus LeConte are uncertain, but this may be among the
very
few Tetraopes species to use more than one host. While adults of this species
have
been found on A. subverticillata in Arizona, they have also been collected
from A.
speciosa in Utah, and from the closely-related A. sullivanti in Missouri (M.
Rice, pers.
comm.). The remarkable range in body size of this species (Chemsak, 1963;
Farrell,
1991) also suggests larval feeding on different hostplants, although this
remains
unconfirmed.
Like other herbivore groups popular with amateur collectors, Tetraopes are
sometimes
recorded from plant species other than those supporting growth and
reproduction.
For example, while Tetraopes tetrophthalmus adults most commonly feed
on and oviposit near Asclepias syriaca, we have also seen this beetle feeding
on flowers
of A. incarnata (swamp milkweed), which often occurs in the wet margins of
fields
occupied by A. syriaca and flowers slightly later. However, we have never
observed
oviposition near A. incarnata, and it is very unlikely that the beetle larvae
could
survive the combination of very wet soil and the very shallow and fine root
system
offered by this milkweed species (Hartman, 1977). We have also occasionally
observed
adults of several other species on non-host milkweeds, at the edge of the
beetles’
range, and after peak adult emergence. Thus, at least some accidental
records may
be attributable to post-reproductive dispersal of adults, which sometimes
undertake
long-distance flights (Davis, 1980a,b, 1984; D. McCauley, pers. comm.).
With one exception, adult oviposition and larval habits of Phaea species have
been
heretofore undescribed. The genus appears to consist of two morphological
subgroups
which are affiliated respectively with Convolvulaceae and Apocynaceae,
members
of related orders in the subclass Asteridae (Olmstead et al., 1992) which
share by
convergence the presence of latex canals. Phaea jucunda has long been
known to bore
in the stems of the convolvulaceous vine Ipomoea pandurata (Craighead,
1923; Rice
et al., 1985), and other morphologically similar species also attack Ipomoea
species.
For example, P. canescens, the only other North American species of Phaea,
attacks
the shrubby Ipomoea leptophylla (M. Rice, pers. comm.), while the Central
American
P. acromela is affiliated with an as yet unidentified species of arborescent
Ipomoea (F.
Hovore, pers. comm.). The remaining Central American species in this
subgroup
seem likewise affiliated with woody Ipomoea species ( J. Chemsak, F. Hovore,
E.
Giesbert, pers. comm.). In southern Mexico, at least some of the species in
the other
Phaea subgroup oviposit in stems of arborescent Apocynaceae. For example,
Phaea
maryannae and P. biplagiata both attack Stemmadenia palmeri, while P.
mirabilis oviposits
TIMING OF INSECT/PLANT DIVERSIFICATION 559
in Thevetia ovata (Farrell, 1991). Verbal descriptions of the hosts of other
Phaea species
indicate that these are also latex-bearing trees with tubular flowers, strongly
suggesting
either Apocynaceae or Convolvulaceae, but these await identification.
Adults of species of Phaea and Tetraopes bear apparently aposematic, bright
orange
or red markings of varying extent. Aposematism is unusually widespread
among
herbivores of the Apocynaceae and Asclepiadaceae, and many of these
specialized
insects, including Tetraopes, sequester toxins from their hostplants for
defense against
predators (Brower & Brower,1964; Scudder & Duffy, 1972; Rothschild, 1973;
Isman,
Duffy & Scudder, 1977; Marsh et al., 1977; Nishio, Blum & Takahashi, 1983;
Brower
et al., 1984a,b; Berenbaum & Miliczsky, 1984; Ackery & Vane-Wright, 1984).
Variation in adult coloration and flight among Tetraopes species may be
correlated
with host chemistry. The more primitive beetle species, affiliated with
apparently
less toxic hostplants (see below), have much less surface area brightly-
colored
(Chemsak, 1963), are smaller in body size on average, and are also more
difficult
to capture.
Relationships among hostplants
The following review is summarized in Figure 1. The two main hostplant
families
of Tetraopes and relatives, Asclepiadaceae and Apocynaceae, form a group
sometimes
termed the Apocynales. Its monophyly is supported by, among other derived
characters, the possession of latex canals and cardiac glycosides (Cronquist,
1981).
A recent molecular phylogenetic study (Sennblad & Bremer, 1996) has shown
the
Apocynaceae to be paraphyletic. Of the two apocynaceous hosts of Phaea,
Thevetia
and Stemmadenia, Thevetia is closest to the monophyletic Asclepiadaceae,
while the
group that includes Tabernaemontana, the closest relative of Stemmadenia
(not included
in the study), is more basal (Sennblad & Bremer, 1996). Among the three
hostgenera
of Tetraopes, all in Asclepiadaceae, Marsdenia is closer to Asclepias than
Matelea
(Woodson, 1941).
Asclepias is a North American genus of about 120 species, most recently
revised
by Woodson (1954). Most of the known Tetraopes hosts are in the nominate
subgenus,
the largest of nine subgenera recognized by Woodson. Woodson grouped the
72
species of this subgenus into eight ‘series’, among which he postulated the
phylogenetic
relationships shown in Figure 1.
Striking, independent support for Woodson’s arrangement comes from
subsequent
studies of the distribution of cardenolide types among milkweed species
which are
host to the monarch butterfly. These have included 20 species representing
all but
one of Woodson’s series (Nelson, Seiber & Brower, 1981). Series 5–8 are
unique in
producing cardenolides of the labriniformin type, whereas series 1–4 produce
cardenolides of only the calotropogenin type, which are widespread across
Asclepiadaceae
and presumably primitive (Brower et al., 1984a,b). Within the chemically
advanced group of series, the levels, locations and identities of characteristic
cardenolides
can be further arranged into transformation series consistent with Woodson’s
arrangement (Fig. 1). As detailed in the Discussion, these features, in
addition
to supporting Woodson’s phylogeny, also suggest a sequence of increasingly
effective
defenses. In summary, while Woodson’s phylogeny needs re-examination, it
provides
a credible first estimate to which the independent estimate for Tetraopes can
be
compared.
B. D. FARRELL 560 AND C. MITTER
Figure 1. Taxonomic arrangement of hostplants used by Tetraopes and Phaea following
Woodson (1954,
1941) within Asclepias; and Sennblad & Bremer (1996) and Leeuwenberg (1994) outside of
Asclepias.
MATERIAL AND METHODS
Morphological analysis
Adult morphological characters were scored for all species of Tetraopes;
seven
species of Phaea, including all those with known hostplants; and the
Philippine genus
Eustathes, nearest relative of Tetraopes+Phaea. Morphological features were
examined
under a Wild dissecting microscope. Dissections were performed with
sharpened
insect pins and stored in alcohol for further examination. Soft tissues were
cleared
from genitalic dissections in 10% KOH. The 26 features showing essentially
fixed
TIMING OF INSECT/PLANT DIVERSIFICATION 561
TABLE 3. Morphological characters in Tetraopes and Phaea scored for phylogenetic analyses.
Numbers
following descriptions are ordered character state codings
HEAD
1. First antennomere longer than scape 0; shorter than scape 1
2. Mandible sexually monomorphic 0; dimorphic 1
3. Labial palpi long, narrow 0; subovoid 1; ovoid 2
4. Antennal annulation absent 0; present 1
5. Scape lateroapical carinae absent 0; present 1
THORAX
6. Pronotal umbone absent 0; present, weakly developed 1; strongly developed 2; strongly developed with
lateral
ridge 3
7. Umbonal lateral sulci absent 0; present at frontal 1/2 1; sulci along length of umbone 2; sulci continuous
around umbone 3
8. Lateral macula absent 0; present 1
9. Umbonal maculae absent 0; present 1
10. Procoxal cavities widely separated by prosternum 0; cavities contiguous or nearly so 1
11. Lateral umbone absent 0; present, weakly developed 1; strongly developed 2
ELYTRA
12. Elytral disc coarsely punctate 0; smooth 1
13. Discal chevron absent 0; present 1
14. Apical chevron absent 0; present 1
15. Humeral maculae absent 0; present 1
16. Discal maculae absent 0; present 1
17. Apical maculae absent 0; present 1
18. Elytral form mesally constricted, subparallel, slender 0; stout, parallel 1
19. Lateral macula absent 0; present 1
APPENDAGES
20. Claws appendiculate 0; bifid 1
21. Male metatrochanter spur absent 0; present 1
ABDOMEN
22. Female sternal sulcus present 0; absent 1
23. Aedeagus dorsal piece subtending ventral 0; overlapping ventral 1
24. Aedeagal apex lanceolate 0; explanate 1
25. Aedeagus with dorsal piece lateral explanation absent 0; present 1
OVERALL HABITUS
26. Exoskeleton with scattered, sparse golden pubescence 0; with dense, white pubescence 1
differences among species were coded as discrete characters for
phylogenetic analysis
(Tables 3,4).
Allozyme electrophoresis
Fresh material of three species of Phaea and 13 species of Tetraopes,
including all
those with confirmed host affiliations, was fresh-frozen in liquid nitrogen in
the field,
and stored at -85°C. Whenever possible, electrophoretic analyses for each
species
included samples from several geographic regions (Table 2).
Starch gel electrophoresis was performed using methods modified from Hillis
&
Moritz (1990). Frozen individual beetles were homogenized in 7ml of ice-cold
homogenization buffer (200mM Tris-HCL, pH 8.0, 26mM sodium metabisulfite,
10mM MgCl2 1.5mM EDTA, 5% w/v PVP-40, 0.05% 2-mercaptoethanol;
modified
from Futuyma & McCafferty, 1990), and spun for 5 minutes in a refrigerated
microfuge. Half the supernatant was frozen for subsequent runs. The rest was
loaded
B. D. FARRELL 562 AND C. MITTER
TABLE 4. Matrix of morphological and allozyme characters states for all species of Tetraopes
plus a subset of Phaea species, following their respective orderings in
Table 3 and the Appendix
P. canescens 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
P. jucunda 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
P. biplagiata 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 7 1 ? 3 ? 2 3 5 5 2 2 5 2 3 2 ? 2 4 2 2 ? 6
P. maryannae 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 4 2 1 5 2 3 2 4 6 5 3 5 7 3 1 3 2 6 6 2 2 ? 4
P. mirabilis 0 0 0 0 1 2 2 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 7 2 6 1 ? 2 6 6 5 2 7 2 3 2 1 2 1 3 2 2 8 5
T. mankinsi 0 0 0 0 0 2 2 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. paracomes 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. cleroides 0 0 0 0 0 3 2 0 0 0 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. comes 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. ineditus 0 0 0 0 0 3 2 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. elegans 1 0 1 0 0 3 2 0 0 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. batesi 1 ? 0 0 0 3 2 0 ? 0 0 0 1 1 0 0 1 1 1 0 1 1 0 0 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. discoideus 0 0 0 0 0 2 2 0 1 0 1 0 1 0 1 0 1 1 0 0 0 1 0 0 0 0 4 2 1 7 1 3 3 3 6 1 1 4 8 2 3 8 9 5 4 1 1 7 6
T. umbonatus 0 1 1 0 0 2 2 0 1 0 1 0 1 1 0 0 1 1 0 0 0 1 0 0 0 0 4 2 1 7 1 4 1 2 3 1 1 4 8 2 3 ? 5 5 1 1 1 7 6
T. melanurus 0 1 2 0 0 2 2 0 1 0 1 0 1 1 0 0 1 1 0 1 0 1 1 1 1 0 3 4 1 1 1 1 1 1 1 2 1 4 7 4 3 ? ? 5 1 1 1 ? 1
T. texanus 1 1 1 0 0 1 1 0 1 0 2 0 0 0 1 1 1 1 0 1 1 1 0 0 0 0 3 4 1 1 1 2 3 5 4 2 1 ? 6 4 3 3 ? 6 1 1 1 5 1
T. linsleyi 1 0 2 0 0 2 2 0 1 1 0 0 0 1 1 0 1 1 1 0 1 ? ? ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. thermophilus 1 1 2 1 0 2 2 0 1 1 0 1 0 0 1 ? 1 1 1 0 1 1 1 ? ? 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. quinquemaculatus 1 1 2 0 0 2 2 0 1 1 1 0 0 0 1 0 1 1 0 1 0 1 1 1 1 0 3 4 1 3 1 2 3 5 4 2 1 6 8 4 2 2 ? 7 1
1152
T. annulatus 1 1 1 1 0 1 1 0 1 1 2 1 0 0 1 1 1 1 0 1 0 1 1 1 0 1 3 ? 1 1 1 1 1 1 1 2 1 4 4 4 4 2 4 7 1 1 1 5 1
T. pilosus 1 1 1 1 0 1 1 0 1 0 2 1 0 0 1 1 1 1 0 1 0 1 1 1 0 1 5 4 1 1 1 1 1 1 1 2 1 4 4 4 4 7 4 7 1 1 1 5 1
T. tetropthalmus 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 1 1 0 1 1 1 0 0 3 3 1 ? 1 2 1 5 4 2 1 6 3 4 3 7 5 4 1 1 1
41
T. mandibularis 1 1 1 1 0 1 2 1 1 1 2 1 0 0 1 1 1 1 1 1 0 1 1 1 1 0 3 3 1 1 1 1 1 5 4 3 1 6 3 4 3 7 5 4 1 1 1 4
1
T. thoreyi 1 1 1 1 0 3 2 1 1 1 2 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. subfaciatus 1 1 2 1 0 3 2 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
T. varicornis 1 1 1 1 0 3 2 1 1 1 2 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 5 3 1 4 1 1 3 5 4 2 1 6 3 4 2 7 5 7 5 1 1 6 3
T. femoratus 1 1 2 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 0 1 1 1 1 1 0 0 5 3 1 ? 1 1 3 5 4 2 1 6 3 4 2 5 5 7 5 1 1 6 1
T. basalis 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 0 1 1 0 1 1 1 1 1 0 0 2 3 1 1 1 2 3 5 4 2 1 6 3 4 2 5 7 7 3 1 1 6 1
T. sublaevis 1 1 1 1 0 2 2 1 1 1 2 1 0 0 1 1 1 1 0 1 1 1 1 1 0 0 5 3 1 4 1 1 3 5 4 2 1 6 3 4 2 5 7 7 3 1 1 6 1
TIMING OF INSECT/PLANT DIVERSIFICATION 563
into cold gels (11% starch from Connaught Laboratories) using 5mm square
wicks
of Whatman #2 filter paper. All enzymes were run for 14–15 hours on small
(200 ml
total volume ) or large (450 ml total volume) gels, in one of seven buffer
systems
determined by an initial survey to be optimal for allele separation and
resolution
(Appendix). Trays of ice were placed on top of the gels, which were run in a
refrigerator at 0–4°C. Gels were sliced two or three times and stained
following
recipes in Hillis & Moritz (1990). Electromorph differences and identities were
confirmed through multiple, side-by-side comparisons.
Phylogenetic analyses
Morphological characters with more than two states (5 of 26 characters) were
coded to reflect transformation series inferred from morphological similarity
(Table
3). For the allozyme data, each locus was treated as a character, with alleles
as the
unordered states (Table 4). Polymorphism was treated as ambiguous.
Morphological and allelic data were first analysed separately for the subset of
16
taxa on which both were obtained, which includes nearly all species with
known
hosts. As these analyses yielded largely concordant groupings, a third
phylogeny
estimate was obtained for these species by combining the two data sets. This
tree
was then compared to that for all 29 species based on both data sets, as a
test for
the possible effects of missing taxa on the estimate for the reduced set.
Finally, a
tree was calculated for all taxa with known hosts, using both character sets,
and
compared to the host phylogeny.
Most parsimonious trees were generated in each analysis using the heuristic
search
routines in PAUP 3.1.1 (Swofford, 1993), with 100 random addition sequences
and
successive weighting using mean retention indices. Decay analyses used
AutoDecay
1.1 (courtesy of N. Erikkson), with 10 random addition sequences for each
reverse
constraint tree analysis. Similarity clustering was also performed on the
allozyme
allele frequencies, using the Manhattan, Nei’s and Roger’s distance
coefficients and
the UPGMA routine in NTSYS-pc (Rohlf, 1990).
Phaea mirabilis was deleted from the data matrix for calculations based on
distances
because it showed 100% divergence from the most derived Tetraopes
species, rendering
Nei’s distance infinite.
Comparisons of phylogeny estimates
The problem of measuring correspondence between host and parasite
phylogeny
estimates has been recently reviewed by Page (1995). Our earlier work
(Farrell &
Mitter, 1990) applied quantitative methods developed for similar problems in
biogeography (Page, 1990) and in evaluation of cladograms generated by
different
data sets for the same taxa (Shao & Sokal, 1986). Recent conceptual
advances
permit more realistic evaluation of phylogeny correspondence when host-
parasite
associations may result from several underlying processes (Page, 1995). We
therefore
applied cospeciation analysis as implemented in the TreeMap program
package of
Page (1995). In this approach, one maps the number of cospeciation events
that
explain the distribution of the observed insect species across hostplant
species entirely
through parallel phylogenesis, minimizing the total number of host-shifts,
sorting
B. D. FARRELL 564 AND C. MITTER
and duplication events. Statistical significance is evaluated by comparing the
observed
cospeciation count to the distribution of values for randomly generated
parasite
and/or host phylogenies (Page, 1995). TreeMap permits generation of trees
under
either Markov or proportional-to-distinguishable models (Page, 1995). We
selected
the more conservative Markov model of tree generation, which consistently
produced
higher (i.e. of lower significance) P values. We also chose to randomize the
beetle
phylogeny estimate as this contains fewer taxa for the purposes of this study.
When
comparing trees of unequal size, randomization of the smaller tree will
produce
higher P values for any given number of cospeciation events, and thus is
more
conservative.
Using this approach, the one species apparently associated with multiple
Asclepias
hosts, Tetraopes annulatus, was scored for association with each host
separately, and
scored for association with all three hosts. The strikingly allopatric Arizonan
and
Mexican populations of Tetraopes discoideus were treated as separate
entities, each
associated with a different host, as these populations are also divergent in
morphology
and allozymes (see Results). We did not include the Tetraopes species for
which there
were no direct observations of host-use.
RESULTS
Allozyme data
Twenty-three apparent loci were resolved (Appendix), all of which showed
interspecific variation . These include all the loci analysed in Tetraopes
tetrophthalmus
by McCauley & Eanes (1987), except that we did not assay leucine amino
peptidase
or hexokinase. Intra-population polymorphism was uncommon, and there
were few
instances where more than one allele was shared with other species. Thus,
very
little phylogenetic information was lost in the discrete coding. In no instance
of
polymorphism was there significant departure from Hardy–Weinberg
genotype
proportions.
Nei’s distance between the populations of T. femoratus, T. annulatus and T.
tetropthalmus
ranged between 0.007 and 0.044, suggesting moderate levels of divergence
accompanying
geographic isolation. For each of these species, mean distances were
used in the phenetic analysis reported below. The small samples of T.
discoideus from
southern Mexico and Arizona differed more strongly. Chemsak (1963) also
noted
consistent (though not invariant) differences in head color between these
populations.
Phylogenetic analyses
For the 16 species scored for both morphology and allozymes, and for which
hostplants are known, the morphological data alone yield four most
parsimonious
trees of which the strict consensus is shown in Figure 2a. The allozyme data
alone
TIMING OF INSECT/PLANT DIVERSIFICATION 565
Figure 2. (a) Strict consensus tree of the four most parsimonious trees based on morphology
alone,
for the subset of Tetraopes species for which allozyme data are also available. Morphological
characters
do not resolve relationships among T. femoratus, T. sublaevis, and T. basalis, and are
equivocal about
placements of T. melanurus and T. quinquemaculatus. Length is 42 steps, rescaled consistency
index=0.5.
(b) Strict consensus tree of two most parsimonious trees for subset of Tetraopes and Phaea
species based
on allozyme data, coded as in Table 4. Length is 90 steps, rescaled consistency index=0.7.
Allozymes
resolve all relationships but are equivocal about the placements of Phaea biplagiata and P.
maryannae,
which are almost completely divergent from Tetraopes species. (c) Single most parsimonious
tree for
Tetraopes and Phaea species for which allozyme data are available, based on Table 4 (all
data). Length
is 140 steps, rescaled consistency index=0.6.
resulted in two most parsimonious trees, whose strict consensus tree is
shown in
Figure 2b. UPGMA clustering on allele-frequency distances specifies an
almost
identical tree (Fig. 3). The allozyme-based estimate is slightly more resolved
within
B. D. FARRELL 566 AND C. MITTER
Figure 3. UPGMA phenogram of Nei’s distances for Tetraopes and Phaea species specifies
nearly
identical relationships to cladistic analysis of same data. UPGMA clustering under Manhattan,
Rogers’
and Prevosti’ distances specifies same tree.
Tetraopes than that based on morphology, but less resolved for the outgroup
Phaea
species. The two disagree only by minor re-positioning among nearby taxa,
namely
T. quinquemaculatus, T. mandibularis, and the pair T. annulatus/T. pilosus.
The two data
sets analysed in combination yield six most parsimonious trees. Following
successive
weighting by the mean retention indices of these, a single most parsimonious
tree
results (Fig. 2c) which is completely resolved, with at least one disagreement
resolved
in favor of each data set. The relationships among these taxa are little
changed in
the most parsimonious trees resulting when the 13 species scored only for
morphology
are added to the combined data set (Fig. 4). This tree confirms the
monophyly of
Tetraopes and suggests that Phaea is paraphyletic, with the Apocynaceae-
feeding group
most closely allied to Tetraopes.
Concordance of insect and plant cladograms
The phylogeny for the beetle species with recorded hosts, extracted from the
phylogeny for all species based on the combined data sets (Fig. 4), is
compared to
the literature-synthesized estimate of host relationships (Fig. 1) in Figure 6.
There
are several points of disagreement, of which the most striking involve T.
pilosus and
T. mandibularis, both of which appear to represent colonization of the
advanced host
series Roseae from ancestors affiliated with more primitive milkweeds.
Overall,
however, the phylogenies appear to match fairly well, an impression
supported by
heuristic statistical analyses. Under cospeciation analysis as implemented in
the
TreeMap package of Page (1995), 13 cospeciation events explain the
distribution of
beetle across host species when Tetraopes annulatus is scored for
association with either
TIMING OF INSECT/PLANT DIVERSIFICATION 567
Figure 4. One of the six most parsimonious trees for all species of Tetraopes plus a subset of
Phaea
species, based on all available data. Trees were obtained under heuristic search routine and
100
random addition sequences in PAUP (vers. 3.1.1:courtesy of D. Swofford). Length is 173 steps,
consistency index=0.62, retention index is 0.79. Character changes were optimized on
branches under
DELTRAN routine, which favors parallelisms over reversals. The numbers adjacent to internal
nodes
are the numbers of unambiguous changes followed by the maximum number of changes for
each
grouping, with the decay index in parentheses.
Asclepias sullivantii or A. speciosa (P=0.01). Ten cospeciation events result in
the
analysis when Tetraopes annulatus is scored for association with A.
subverticillata or all
three plant species (P=0.07). When Tetraopes annulatus is omitted from the
analysis,
on the grounds that its affiliations are ambiguous, the estimate is 12
cospeciation
events (P=0.038). If Phaea is also removed, confining the analysis to
herbivores of
Asclepiadaceae, the estimate is 10 cospeciation events. Ten or more
cospeciation
events are significant when the host or both host and beetle phylogeny
estimates
are randomized (P<0.05).
DISCUSSION
The agreement between morphological and allozyme analyses suggests that
the
Tetraopes phylogeny estimate is reasonably robust, but addition of other
molecular
B. D. FARRELL 568 AND C. MITTER
character sets, now in progress, is needed to settle several weakly-resolved
regions
of the tree. The need for modern re-examination of Asclepias phylogeny, also
underway, is even more evident, and dictates caution about any conclusion
to be
drawn here.
Nonetheless, the correspondence between the beetle and plant phylogenies
appears
stronger than expected by chance, and seems more likely to increase than
decrease
as those independent estimates are improved (i.e. unless error in the current
estimates
produced this pattern, correspondence should become more clear as error
decreases).
The match is inexact, suggesting that pairs of associated species have
typically arisen
most immediately by beetle colonizations from a related host rather than
parallel
speciation, but it is consistent with broadly synchronous diversification
between
lineages, providing the opportunity for long-term coevolution.
Ages of associated beetles and hosts
The earliest fossils of Apocynaceae are Paleocene (Muller, 1984), with extant
genera appearing in the Eocene. The divergence between Apocynaceae+
Asclepiadaceae and Convolvulaceae, which are in different orders, was
probably
considerably earlier. The earliest fossils of Asclepiadaceae appear in the
Oligocene
and Miocene (Muller, 1984); as these already represent extant genera,
including
Asclepias, the family is likely to be older. Fossil datings of subgroups within
Asclepias
are not available.
Tetraopini are represented by a single, Oligocene fossil, which cannot be
further
placed (Statz, 1938). Biogeography suggests that the Phaea+Tetraopes clade
itself is
this old or older: the disjunction between this New World lineage and its
Southeast
Asian sister group Eustathes mirrors the distributions of many pantropical
groups for
which the fossil record suggests Late Eocene to Early Oligocene origins
(Tiffney,
1985; 35–>45 Mya), before northerly dispersal routes were cut off by
Oligocene
cooling.
Mapping of species distributions on the phylogeny estimate (Fig. 5) suggests
a
tropical lowland origin for Tetraopes, followed by more recent occupation of
temperate
upland savannah and most recently, Sonoran desert and midwestern
Sandhills. A
similar biogeographic history is suggested for Asclepias by Woodson’s
arrangement.
Most species in the relatively primitive series 1–4 occupy mesic tropical
habitats
and are distributed from Mexico south, while most species in the derived
series 5–8
occupy grassy temperate savannah habitats. Species within the highly-
derived Roseae
(series 8) have invaded the youngest, most severe habitats, the Sonoran
Desert and
central Sandhills of the United States.
These sequences of habitat occupation parallel the order of appearance of
the
various habitats in the plant fossil record (Axelrod, 1979; Wolfe, 1978, 1985;
Tidwell
& Nambudiri, 1989), as expected if endemic beetle and milkweed lineages
and their
habitats differentiated synchronously. However, they also parallel the order
of
severity of these habitats with respect to moisture. It is conceivable that both
beetle
and milkweed colonization would have been constrained to follow a similar
sequence,
even if they occurred entirely after differentiation of the habitats, by the need
for
successive pre-adaptations to successively harsher environments. This
alternative
seems less parsimonious to us, but we cannot entirely rule it out. If we
provisionally
accept the time scale implied by our habitat datings, the origin of association
with
TIMING OF INSECT/PLANT DIVERSIFICATION 569
Figure 5. Biogeographic distributions optimized on tree from Figure 4 imply tropical origin
followed
by colonization of more temperate latitudes, and and at least one secondary invasion of the
tropics
(by the ancestor of T. thermophilus, T. subfasciatus, T. thoreyi, and T. varicornis).
Asclepiadaceae, corresponding to the split between Tetraopes and Phaea, is
dated to
40–47 Mya; association with subgenus Asclepias at more than 20 Mya (pre-
dating
divergence of T. discoideus); and exclusive association with the advanced
series (5–8)
of Asclepias (T. mandibularis through T. basalis in Figure 6) at >7 Mya. These
projections, like the sparse fossil evidence, are at least consistent with the
hypothesis
that Tetraopes and its subgroups have diversified in approximate synchrony
with their
asclepiadaceous hosts.
In contrast, the divergence between the Phaea groups feeding on
Convolvulaceae
versus Apocynaceae is probably not as old as that between their more
distantly
related host groups. Transfers between these families are relatively common,
and
may reflect both shared placement in the subclass Asteridae and the
convergent
possession of latex canals, which together account for a majority of the insect
faunal
connections between Apocynaceae/Asclepiadaceae and other plant families
(Farrell
& Mitter, 1993).
Coevolution?
The phylogenetic and temporal evidence adduced above suggests that
Tetraopes
and Asclepias, particularly subgenus Asclepias, have been associated during
much of
their respective histories, providing abundant opportunity for evolution in
response
B. D. FARRELL 570 AND C. MITTER
Figure 6. Comparison of most parsimonious tree, based on all data, for Tetraopes and Phaea
species
with known hosts, to hostplant phylogeny of Figure 1. There are 10–13 cospeciation events,
depending
on which reported host of T. annulatus is scored. Correspondence is significant under 2/3
scorings
(P<0.01), and if T. annulatus is omitted on the grounds that its host affiliations are ambiguous
(P=
0.038: see text for details). *T. annulatus is here depicted opposite its host A. sullivantii,
though adults
of this species have also been collected from A. subverticillata and A. speciosa (see
Introduction).
to their interaction. Direct evidence for coevolution has not been sought, but
several
suggestive observations point to directions for future study.
Reciprocal adaptation between particular pairs of plant and phytophagous
insects
is considered rare (Futuyma & Keese, 1992; Farrell & Mitter, 1993), and has
been
reported in just a few cases involving long-standing, highly specific
interactions such
as exclusive plant/pollinator associations (Thompson, 1994). However, such
pairwise
coevolution has rarely been directly looked for in antagonistic interactions.
For
Tetraopes and Asclepias, pairwise coevolution should most profitably be
sought in
paired endemics of distinctive, extreme habitats, such the affiliation of
Tetraopes pilosus
and Asclepias arenaria in the Central Sandhills. Such species seem especially
likely to
have differentiated together, and the harshness of their environment may
both
restrict the number of other herbivores with which the plant must contend,
and
place a premium on effective defense (Coley, Bryant & Chapin, 1987).
Tetraopes is
clearly capable of impairing milkweed fitness (see earlier references), and its
combination
of larval root feeding and adult feeding on reproductive parts may impose
especially severe selection on hostplants (Brown, 1990).
Diffuse coevolution as embodied in Ehrlich & Raven’s model (1964)—
evolution
of plant lineages in simultaneous response to suites of herbivore species and
vice
versa—has been accorded a wider influence on the structure and diversity of
insect/
plant communities (Futuyma & Keese, 1992). However, there are as yet few
plausible instances, let alone convincing demonstrations, of such coevolution
effects.
Berenbaum (1983) spelled out the predictions of Ehrlich & Raven’s (1964)
model,
and built a persuasive case for stepwise elaboration of coumarin compounds,
in
Apiaceae and other plant families, in response to counter-adapting
herbivores.
However, the phylogenies needed to secure this inference are not yet
available
TIMING OF INSECT/PLANT DIVERSIFICATION 571
(Miller, 1987). Coevolutionary ‘escape’ may also explain the supplementation
or
replacement of glucosinolates by other, very different secondary compounds
in some
lineages of crucifers (Feeny, 1977). Perhaps the strongest case for Ehrlich &
Raven’s
scenario is provided by secretory canals containing latex or resin. These
structures
are effective defenses against a spectrum of enemies, and no other
hypothesized
function for them seems plausible (Dussourd & Eisner, 1987). The multiple
lineages
in which they have independently arisen show consistently elevated
diversification
rates (Farrell, Dussourd & Mitter, 1991). They have in turn evoked
characteristic
counter-adaptations by some insect herbivores, some of which circumvent
the canals
by severing them before feeding (Dussourd, 1993).
The subgenus Asclepias presents a prima facie case for coevolution sensu
Ehrlich
and Raven with enemies including Tetraopes, that parallels on a smaller scale
the
examples just cited. There is, first, a phylogenetic progression in the types
and tissue
distribution of cardenolides, alluded to earlier (Fig. 1), which can be plausibly
read
as a stepwise escalation of defense (Nelson et al., 1981). Cardenolides in the
more
primitive of Woodson’s series (1–4), like those in other subgenera and genera
of
asclepiads, are of the simpler, presumably primitive calotropogenin type.
Series 5–8
are unique in producing cardenolides of the structurally complex labriniformin
family. These compounds are among the most emetic and toxic cardenolides
known
(Detweiler, 1967; Brower et al., 1982, 1984a,b). In series 5 (Syriacae, host to
T.
tetropthalmus and T. linsleyi ), the labriformin-type cardenolides are present
in only
trace amounts; they increase in successive series to a maximum in series 8
(Roseae,
host to T. sublaevis, T. pilosus, T. basalis and T. mandibularis). In Roseae,
moreover,
cardenolides are confined principally to the latex, where their deterrent
effects on
herbivores should be maximal (Nelson et al., 1981). Species in this series
have the
largest laticifers in the subgenus (Nelson et al., 1981), and the highest
concentration
of cardenolides known in any milkweed. Perhaps the ability of these plants to
invade
harshly xeric habitats unoccupied by other milkweeds derives in part from
enhanced
defences.
While this phylogenetic pattern suggests escalation of defence,
demonstrating that
such plant traits evolved as defences at all, let alone as a response to any
particular
enemy, has proven notoriously complex (review in Futuyma & Keese, 1992).
There
is some evidence that cardenolides are toxic or repellent to insects and
vertebrates
that do not feed on plants containing them (Detweiler, 1967; Cohen, 1983),
but
there has been little systematic attempt to compare the defensive
effectiveness of
the varying cardenolide profiles and deployments within subgenus Asclepias.
However,
preliminary field and lab observations suggest that milkweeds in series
Roseae are
free from the assemblages of oligophagous ctenuchine arctiid moths and
chrysomelid
beetles that attack chemically and phylogenetically more primitive
congeners. Indeed,
Tetraopes and the monarch butterfly are the only known folivores of Roseae.
Thus,
enhanced defences may have allowed these plants to escape former
enemies, including
most insects. Support for this inference, for example, would come from
demonstration
of negative effects of advanced cardenolides on the fitness of primitive
beetles.
Extending this scenario to the limit, one could further interpret Woodson’s
phylogeny as supporting Ehrlich and Raven’s conjecture that origin of novel
defenses
promotes diversification. That is, the three nested Asclepias subgroups
characterized
by the successive defence innovations postulated above are each more
species-rich
than their apparent sister groups (Fig. 1). In turn, the Tetraopes clade
associated with
the chemically advanced Asclepias series (circumscribed by T. linsleyi and T.
basalis in
B. D. FARRELL 572 AND C. MITTER
Fig. 5) is likewise more diverse than its sister group, suggestive of beetle
radiation
following colonization of a newly-diverse host clade.
Tests of this scenario for Asclepias faces the difficulty that each of its
component
adaptations has evolved only once. This makes unavailable the criterion of
repeatability
across independent lineages, a powerful form of evidence on both the
adaptive value of traits (Williams, 1992), and their consequences for
diversification
(Heard & Hauser, 1995). On the other hand, the most derived, and apparently
most toxic, series Roseae has been colonized by beetles three times (T.
pilosus, T.
mandibularis and the common ancestor of T. sublaevis and T. basalis), thus
providing
opportunity for comparative study of the necessary adaptations and possible
advantages
involved in use of these plants. Moreover, the phylogeography of the
Tetraopes/Asclepias assemblage suggests that extant species and their
adaptations
evolved largely in the habitats, often quite restricted, that they currently
occupy.
The ecological circumstances under which putative reciprocal adaptations
have
arisen in these relatively young, still-localized lineages may be better
preserved than
those surrounding the origin of older, now widespread traits such as the
possession
of cardenolides per se. This should permit correspondingly more compelling
experimental
tests of hypotheses about both the adaptive origins of traits, and the
mechanisms whereby improved adaptation translates into increased
diversification
(Sanderson & Donoghue, 1994). Experimental studies of examples such as
Roseae
are needed for rigorous evaluation of Ehrlich and Raven’s model, now that
broad statistical approaches have provided initial, presumptive evidence for
diffuse
coevolution between insects and plants.
Concluding observations
Hostplant use in Tetraopes is unusually conservative: our evidence suggests
that
these beetles’ exclusive association with Asclepiadaceae dates to the mid
Tertiary,
perhaps to as long as 40 Mya. Moreover, unlike that of most insect groups
which
have been similarly examined, the phylogeny of the Tetraopes/Phaea lineage
shows
significant concordance with that of its hosts. This concordance is plausibly
ascribed
to approximately synchronous diversification, at least between Tetraopes and
Asclepias.
These findings parallel results of an earlier analysis of Phyllobrotica leaf
beetles (Farrell
& Mitter, 1990). Thus, they support the conjecture that parallel phylogenesis
with
hostplants, with its attendant opportunity for long-term coevolution, is
promoted by
shared features of these two assemblages which probably reinforce
specificity and
conservatism of their habits. These traits include larval endophagy, adult
feeding
and mating on the larval host, and ‘toxic’ host chemistry on which the
apparently
aposematic adult beetles may be dependent for defence.
The hostplants of Tetraopes and Phyllobrotica, Asclepiadaceae and
Lamiaceae, like
the coumarin-rich Apiaceae (Berenbaum, 1983), belong to the derived
angiosperm
subclass Asteridae s.l. (Olmstead et al., 1992), which is characterized by an
exceptional
prevalence and diversity of toxic and repellent compounds (Cronquist, 1981).
All
three families are derived relatively recently (mid-Tertiary) from woody
tropical
ancestors, and consist partly (milkweeds) to mostly of temperate herbs that
have
presumably diversified with the spread of open, seasonal habitats since the
Eocene
( Judd, Sanders & Donoghue, 1994). For reasons advanced earlier, such plant
TIMING OF INSECT/PLANT DIVERSIFICATION 573
lineages should provide test cases for the prevalence and detectability of
escape and
radiation coevolution, rigorous study of which has barely begun.
ACKNOWLEDGEMENTS
For help with obtaining beetles and collecting locales for populations of
Tetraopes
and Phaea species we thank Marlin Rice, John Chemsak, James Farrell, Ed
Wappes,
Ed Riley, Ed Giesbert and Frank Hovore. We also thank the Patuxent Wildlife
Research Center for permitting access to field populations of Tetraopes and
Asclepias,
and Felipe Noguerra for aid in locating beetles at the Chamela Field Station of
UNAM. Special thanks to Irina Ferreras and Eva Silverfine for providing
essential
and enthusiastic help with protein electrophoresis, and to Doug Futuyma and
Dave
McCauley for providing recipes for allozymes. For loan of specimens used in
morphological studies, we thank the Museum of Comparative Zoology
(Harvard
University), the Bishop Museum, The Field Museum of Natural History, The
American Museum of Natural History, the Essig Museum (U.C. Berkeley), the
California Academy of Sciences, the Sacramento Dept. of Food and
Agriculture,
and the National Museum of Natural History.
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APPENDIX
Enzyme loci scored for phylogenetic analyses. The suffix 2 indicates a cathodal locus. 2. TBE:
Trisborate-
EDTA; TM: Tris-maleate; LiOH: discontinuous Lithium hydroxide; TC7, 8: Tris citrate; TVB;
Salb: Salamander B (Hillis & Mortiz, 1990; Richardson et al., 1986; Werth, 1985). The matrix of
allele
frequencies will be supplied on request by the senior author.
Enzyme E. C. # Locus1 Buffer2
1. Aconitase 4.2.1.3 Aco-1 1/2 LIOH
2. Aconitase 4.2.1.3 Aco-2 TVB
3. Aldolase 4.1.2.13 Ald SalB
4. Catalase 1.11.1.6 Cat SalB
5. Fructose-1,6 diphosphate 3.1.3.11 Fdp TBE
6. Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 G3p TC7
7. Glucose-6-phosphate dehydrogenase 1.1.1.49 G6p TC8dil
8. Aspartate aminotransferase 2.6.1.1 Got1 TC8dil
9. Aspartate aminotransferase 2.6.1.1 Got2 TBE
10. Hydroxybutyrate dehydrogenase 1.1.1.30 Hbd TC8dil
11. Alpha-glycero-phosphate dehydrogenase 1.1.1.8 Agpd TC8dil
12. Isocitrate dehydrogenase 1.1.1.42 Idh TC7
13. Malate dehydrogenase 1.1.1.37 Mdh-1 TC7
14. Malate dehydrogenase 1.1.1.37 Mdh-2 TC7
15. Malic enzyme 1.1.1.40 Me TC7
16. Mannose-6-phosphate isomerase 5.3.1.8 Mpi TC8dil
17. Phosphoglucomutase 5.4.2.2 Pgm TM
18. Glucose phosphate isomerase 5.3.1.9 Phi 1/2 LIOH
19. 6-phosphogluconate dehydrogenase 1.1.1.44 Gp6 TC8dil
20. Sorbitol dehydrogenase 1.1.1.14 Sdh TBE
21. Superoxide dismutase 1.15.1.1. Tox TBE
22. Xanthine dehydrogenase 1.1.1.204 Xdh LIOH
23. Adenylate kinase 2.7.4.3 Ak LIOH
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Cardenolide Glycosides of Thevetia peruviana and Triterpenoid Saponins of Sapindus
emarginatus as TRAIL Resistance-Overcoming Compounds
Abstract
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Takashi Miyagawa†, Takashi Ohtsuki†, Takashi Koyano‡, Thaworn Kowithayakorn§
and Masami Ishibashi*†
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-
ku, Chiba 263-8522, Japan, Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-
0012, Japan, and Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002,
Thailand
J. Nat. Prod., 2009, 72 (8), pp 1507–1511
DOI: 10.1021/np900202n
Publication Date (Web): July 13, 2009
Copyright © 2009 The American Chemical Society and American Society of
Pharmacognosy
* To whom correspondence should be addressed. Tel: +81-43-290-2913. Fax: +81-43-
290-2913. E-mail: mish@p.chiba-u.ac.jp., †
Chiba University.
,‡
Temko Corporation.
,§
Khon Kaen University.
Abstract
A screening study for TRAIL resistance-overcoming activity was carried out, and
activity-guided fractionations of Thevetia peruviana and Sapindus emarginatus led to the
isolation of four cardenolide glycosides (1−4) and four triterpenoid saponins (5−8),
respectively. In particular, cardenolide glycosides (1 and 2) from T. peruviana were
shown to have a significant reversal effect on TRAIL resistance in human gastric
adenocarcinoma cells, and real-time PCR showed that thevefolin (2) enhanced mRNA
expression of death receptor 4 (DR4) and DR5. In addition, 1H and 13C NMR
characterizations are shown for thevefolin (2) for the first time.
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Published In Issue August 28, 2009
Article ASAPJuly 13, 2009
Received: March 30, 2009
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African Journal of Pharmacy and Pharmacology Vol. 4(2). pp. 087-089, February, 2010
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 © 2010 Academic Journals
Short Communication
Anti-termite and antimicrobial
properties of paint made
from Thevetia peruviana (Pers.)
Schum. oil extract
P. G. Kareru1*, J. M. Keriko1, G. M. Kenji2 and A. N. Gachanja1
1Chemistry Department, Jomo Kenyatta University of Agriculture and Technology, Nairobi,
Kenya.
2Food Science and Technology, Jomo Kenyatta University of Agriculture and Technology,
Nairobi, Kenya.
Accepted 5 January, 2010
Thevetia peruviana (Pers.) K. Schum. seed oil was used to make a surface
coating with antifungal,
antibacterial and anti-termite properties. The paint exhibited inhibitory
activity against Escherichia coli,
Staphylococcus aureus, Bacillus subtilis and Candida albicans in a
concentration dependent manner.
The antibacterial activities were statistically significant (p = 0.05). The
repellent action of paint against
subterranean termites (Microtermes spp.) was significant (p = 0.03). From
these results, it was
concluded that the Thevetia peruviana-based oil paint was self-preserving
against microbes and
substantially protected wood from subterranean termite attack.
Key words: Thevetia peruviana, anti-termite, antifungal.
INTRODUCTION
Thevetia peruviana (Pers.) K. Schum. (commonly known
as Yellow oleander) is an ornamental plant which grows
in Kenya and other parts of the World, such as tropical
America, Western Asia, Southern Europe, India and
tropical Africa. Extracts from T. peruviana plant species
contain glycosides, whose toxicity against snails, slugs
(Panigrahi and Raut, 1994), bacteria (Obasi and
Igboechi, 1991), insects (McLaughlin et al., 1980) and
humans (Langford and Boor, 1966) has been documented.
T. peruviana plant extracts have also been reported
to have antifungal properties against Cladosporium
cucumerinum (Gata et al., 2003).
Toxicity and repellent effects of medicinal plant extracts
on subterranean termites (Isoptera: Rhinotermitidae)
have also been demonstrated (Verena-Ulrike and Horst,
2001). The presence of unsaturated linoleic acid in
Yellow oleander oil (Obasi et al., 1990), which has drying
properties (Cecilia et al., 2005), makes Yellow oleander
oil suitable for making a surface coating such as paint.
The aim of this study was to formulate an oil-based paint
using crude Yellow oleander oil and to determine its
insecticidal and antimicrobial properties.
*Corresponding author. Email: pgkareru@yahoo.com,
patgkareru@gmail.com. Tel: +254-6752-223. Fax: +254-6752-
446.
MATERIALS AND METHODS
Collection of Yellow oleander kernel seeds and extraction of oil
Yellow oleander kernel seeds were collected from Jomo Kenyatta
University of Agriculture and Technology campus. After removing
the kernel, seeds were macerated using a blender. Oil was extracted
with methanol. Filtered crude oil was stored in a refrigerator at
4°C till used.
Paint formulation
Commercial grade long oil (alkyd resin), titanium dioxide, anti-skin
agents, white spirit and paint dryers were purchased from a local
chemical supplier, Industrial area, Nairobi. Five kilogrammes (5 kg)
of paint was made by mixing appropriate ingredients. Yellow
oleander oil extract was used to make paint batches whose oil
concentration ranged from 0.0 to 80.0%.
Antibacterial and antifungal assays
The procedure followed was as described by Cheesbrough (1984).
Inhibition zone diameters for paints were determined against E. coli,
S. aureus, B. subtilis and C. albicans. The results were presented in
Figure 1.
Anti-termite activity
Labeled dry plywood plates (6 x 6 inches) were painted on both
sides, (in triplicate) with the formulated paints. One set of control
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088 Afr. J. Pharm. Pharmacol.
Figure 1. Inhibition zone diameters (mm) of oleander paint.
Figure 2. Repellent activity of Oleander paint towards Microtermes spp.
plates was painted with neat oleander oil, while the other was
painted with a paint in which Yellow oleander oil was not added.
After drying to constant weight in the laboratory environment, each
plate weight was determined. The wooden plates were then placed
side by side and covered with foliage under a termite (Microtermes
spp) nest and left for a period of one month. Moisture was
constantly maintained by pouring water on the foliage within the
exposure period, so as to maintain appropriate environmental
conditions favourable to termites. After the exposure period, the
wooden plates were washed with clean water to remove soil and
debris, and dried in the oven at 50°C to a constant weight. The
mass of each plate was then determined and the average weight
loss calculated.
RESULTS AND DISCUSSION
The Oleander paint inhibited the tested microbes in a
concentration dependent manner. The control paint
(containing 0.0% oil) did not inhibit the test bacteria and
fungus. From these results, it was concluded that
oleander paint was self-preserving against bacterial and
fungal attack. Antibacterial and antifungal activity of T.
peruviana plant extracts had been earlier established
(Obasi and Igboechi, 1991; Gata et al., 2003) and
collaborates with the present findings. From Figure 2 it
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_
was evident that the oleander paint repelled Microtermes
spp. The repellent action was highest when pure
oleander oil was used. However, no termite deaths were
reported in this study. Insecticidal and toxicity of Yellow
oleander oil has been reported (McLaughlin et al., 1980;
Panigrahi and Raut, 1994; Langford and Boor, 1966).
Also anti-termite activity of medicinal plant extracts has
been documented (Verena-Ulrike and Boor, 2001). The
present findings demonstrate that paint made from T.
peruviana plant oil extract could substantially protect
timber from termite attack.
Conclusion
Yellow oleander paint possesses antimicrobial and antitermite
activities. T. peruviana oil extract would serve as
an environmentally friendly bactericide and fungicide for
oil based paints.
ACKNOWLEDGEMENTS
The authors wish to thank Jomo Kenyatta University of
Agriculture and Technology for funding this study. Thanks
goes to Prof. Rosabella Maranga for identification of the
termite species used in this investigation.
Kareru et al. 089
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