------ Raf. J. Sci., Vol. 23, No.

4 pp 23-32, 2012------

Extract Bioefficiency of Five Euphorbia spp. (Euphorbaceae) on

Crimson-Speckled Moth, Utetheisa pulchella L. (Lepidoptera, Acritidae)
Growth and Development

Atallah F. Mekhlif
Department of Biology
College of Education
University of Mosul
[email protected]

(Received 6/ 2/ 2012 ; Accepted 11 / 6 / 2012 )

ABSTRACT
In the course of screening for novel naturally occurring insecticides from plants, the
ethanol extract of the Arial parts of 5 Euphorbia weeds was found to show insecticidal activity
against Utetheisa Pulchella L. Extracts from E. granulata L., E. petiolata L., E. peplis L., E.
nutans Lag. and E. heliscopia L. possessed bioinsecticidal activity against immature stages
with LC50 values between 84.0 and 480 ppm. Although all could be viable insecticides,
extracts of E. granulata, E. petiolata and E. peplis are by far the most likely to succeed.
Significant observations about wings malformations were reported, they varied between
fair and heavy curly wings and eclusion failure. Histological effect of the applied Euphorbia
extracts was tested by E. granulata extract, the extract induces separation of peritrophic matrix
and necrosis of epidermal cells. The extract affects the metabolic activities of prepupa midgut
which is represented by notable variation in chromatin colour of epidermal nuclei.

Keywords: Euphorbia spp., Utetheisa Pulchella, mortality, malformations, sensitivity.

‫ــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
(Euphorbaceae) Euphorbia ‫ﺘﺄﺜﻴﺭ ﻤﺴﺘﺨﻠﺹ ﺨﻤﺴﺔ ﺃﻨﻭﺍﻉ ﻤﻥ ﺍﻟﺠﻨﺱ‬
(Lepidoptera, Acritidae) Utetheisa pulchella L. ‫ﻓﻲ ﺘﻁﻭﺭ ﻭﻨﻤﻭ ﺍﻟﻌﺜﺔ‬

‫ﺍﻟﻤﻠﺨﺹ‬
‫ ﻓﻘﺩ ﻜﺎﻥ ﺍﻟﻤﺴﺘﺨﻠﺹ‬،‫ﻤﻥ ﺨﻼل ﺍﻟﺘﻭﺠﻪ ﺇﻟﻰ ﻤﺴﺢ ﺍﻟﻨﺒﺎﺘﺎﺕ ﻟﻠﺤﺼﻭل ﻋﻠﻰ ﻤﺒﻴﺩﺍﺕ ﺤﺸﺭﻴﺔ ﻁﺒﻴﻌﻴﺔ ﻤﺴﺘﺨﺭﺠﺔ ﻤﻨﻬﺎ‬
‫ﺍﻷﻴﺜﺎﻨﻭﻟﻲ ﻟﻸﺠﺯﺍﺀ ﺍﻟﻬﻭﺍﺌﻴﺔ ﻟﺨﻤﺴﺔ ﺃﺩﻏﺎل ﻤﻥ ﺠﻨﺱ ﺃﻡ ﺍﻟﺤﻠﻴﺏ )ﺍﻟﻌﺎﺌﻠﺔ ﺍﻟﺴﻭﺴﺒﻴﺔ( ﺫﺍﺕ ﻓﻌﺎﻟﻴـﺔ ﺃﺒﺎﺩﻴـﺔ ﻟﻌﺜـﺔ ﺍﻟﺤﻨـﺎﺀ‬
E. petiolata ‫ ﻭ‬Euphorbia granulata ‫ ﻜﺎﻨـﺕ ﺘﺭﺍﻜﻴـﺯ ﻤﺴﺘﺨﻠـﺼﺎﺕ ﻜـل ﻤـﻥ‬.Utetheisa pulchella
‫ ﻤﻥ ﺍﻷﻁﻭﺍﺭ ﻏﻴﺭ ﺍﻟﺒﺎﻟﻐﺔ ﻟﻶﻓﺔ ﻗﺩ ﺘﻔﺎﻭﺘـﺕ‬%50 ‫ ﺍﻟﺘﻲ ﺘﺴﺒﺒﺕ ﻓﻲ ﻗﺘل‬E. heliscopia‫ ﻭ‬E. nutans‫ ﻭ‬E. peplis ‫ﻭ‬
E. ‫ ﻓﺎﻥ ﻤﺴﺘﺨﻠﺼﺎﺕ‬،‫ ﻭﻋﻠﻰ ﺍﻟﺭﻏﻡ ﻤﻥ ﻓﻌﺎﻟﻴﺔ ﻫﺫﻩ ﺍﻟﻤﺴﺘﺨﻠﺼﺎﺕ ﺠﻤﻴﻌﺎﹰ‬.‫ ﺠﺯﺀ ﻓﻲ ﺍﻟﻤﻠﻴﻭﻥ‬480.0 ‫ ﺇﻟﻰ‬84.0 ‫ﻗﻴﻤﺘﻬﺎ ﺒﻴﻥ‬
.‫ ﻜﺎﻨﺕ ﺫﺍﺕ ﻓﻌﺎﻟﻴﺔ ﺃﻜﺜﺭ ﻻﺴﺘﺨﺩﺍﻤﻬﺎ ﺒﻨﺠﺎﺡ‬E. peplis ‫ ﻭ‬E. petiolata ‫ ﻭ‬granulata

23
24 Atallah F. Mekhlif

‫ﺴﺠﻠﺕ ﻤﻼﺤﻅﺎﺕ ﻤﻤﻴﺯﺓ ﺒﺎﻟﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺍﻟﻀﺎﺒﻁﺔ ﻋﻥ ﺘﺸﻭﻩ ﺍﻷﺠﻨﺤﺔ ﺒﺩﺭﺠﺎﺕ ﻤﻥ ﺍﻟﺘﺠﻌـﺩ ﻭﻓـﺸل‬
‫ ﻭﻗـﺩ ﺃﺤـﺩﺙ‬،E. granulata ‫ ﻓﻘﺩ ﺍﺨﺘﻴﺭ ﻤﻨﻬﺎ ﻤﺴﺘﺨﻠﺹ‬،‫ ﺃﻤﺎ ﺍﻟﺘﺄﺜﻴﺭ ﺍﻟﻨﺴﻴﺠﻲ ﻟﻤﺴﺘﺨﻠﺼﺎﺕ ﻨﺒﺎﺘﺎﺕ ﺃﻡ ﺍﻟﺤﻠﻴﺏ‬.‫ﺍﻟﺒﺯﻭﻍ‬
‫ ﻭﻜﺎﻥ ﻤﻥ ﺘـﺄﺜﻴﺭ‬.‫ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻨﻔﺼﺎل ﻏﺸﺎﺀ ﺤﻭل ﺍﻟﻐﺫﺍﺀ ﻭﺘﺂﻜل ﺨﻼﻴﺎ ﺍﻟﺒﺸﺭﺓ ﻓﻲ ﺍﻟﻘﻨﺎﺓ ﺍﻟﻬﻀﻤﻴﺔ ﺍﻟﻭﺴﻁﻰ ﻓﻲ ﺍﻟﻴﺭﻗﺎﺕ‬
‫ ﺇﺫ ﻴﻜﻭﻥ ﺍﻟﻠﻭﻥ ﻋﻤﻴﻘﹰﺎ ﻓﻲ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺍﻟـﻀﺎﺒﻁﺔ ﻭﺍﺯﺭﻕ‬،‫ﺍﻟﻤﺴﺘﺨﻠﺹ ﻓﻲ ﻁﻭﺭ ﻗﺒل ﺍﻟﻌﺫﺭﺍﺀ ﺘﻐﻴﺭ ﻟﻭﻥ ﺍﻟﻤﺎﺩﺓ ﺍﻟﻜﺭﻭﻤﺎﺘﻴﻨﻴﺔ‬
.‫ﻓﺎﺘﺢ ﻓﻲ ﻤﺠﻤﻭﻋﺔ ﺍﻟﻤﻌﺎﻤﻠﺔ‬

.‫ اﻟﺤﺴﺎﺳﻴﺔ‬،‫ اﻟﺘﺸﻮهﺎت‬،‫ ﻧﺴﺒﺔ اﻟﻘﺘﻞ‬،Euphorbia spp. ,Utetheisa Pulchella :‫ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﺩﺍﻟﺔ‬

‫ــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
INTRODUCTION
The injudicious use of chemical pesticides has resulted in multiple problems such as
increase in the insect resistance to insecticides, emergence of new pests, minor pestes
becoming major pest and pesticide pollution to the environment (Kannaiyan,1999). Many
plants provide an alternative to currently used pesticides for the control of plant pests, and
they have bioactive chemicals (Kim et al., 2005). Certain plants by nature act as
antifeedants, oviposition deterrents, ovicidal, inhibition of egg hatchability, larvicidal and
insect growth regulators (David, 2008). Jacobson (1982) stated that Dioscorides (A.D.4
0‫ـ ـ‬90) listed the juices of Euphorbia spp. as plant poisons. Many Euphorbia spp. are used in
folk medicine and over the past twenty years, they have received considerable
phytochemical and biological attention (Shi et al., 1999; Wu et al., 2009).
The family Euphorbaceae (Spurge family) consists of 300 genera and 5000 species
(Webester,1994). The genus Euphorbia is the largest genus in the family Euphorbaceae with
over 2000 species (Jassbi, 2006). The Euphorbaceae is well known in Iraq and represent 5
genera and 46 species, Euphorbia genus is dispersal with more 40 wild species, the famous
species are E. heliscopia, E. peplis, E. prostarta and E. granulate, all species of Euphorbia
are the very broad Arabic names; the lubbaina, om alhalib, Khanag aldajaj… because of
their poisonous milky latex which protects these plants from herbivorous animals( AL-
Musawi, 1987; Al-Sultan and Hussein, 2006; Haba et al., 2009).
Euphorbia extracts possess some insecticidal activities, Vanderplank (1945) study their
effect on adult Diptera, E. peplus extract was proved to be toxic to a mosquito larvae (Gayar
et al., 1971). E. fischeriana has antifeeding activity against stored-product insects (Geng et
al., 2011). E. heterophylla extract affects the Bonacroftian filariasis vector; Culex
quinquefasciatus ( Kuppusamy and Murugan, 2008).
Numerous ingredients had been isolated from Euphorbia species extracts, Jain et al.,
(2008) and Al- Younis and Abdullah (2009) identified flavinoids and phenolic acids from
several species of Euphorbia genus including E.granulata and E.heliscopia. Different
triterpenoids and diterpenoids were isolated from various Euphorbia species (Kurpadanam
and Srim, 1999; Shizuri et al., 1983 ; Abdel-Monem et al., 2008).
Crimson-speckled moth, Utetheisa pulchella (Acritidae) is a common migrant pest
between plains and middle heights in Iraq, it has been known to attack cultivated plants and
may defoliate their host plants with multiple generations (Wiltshire, 1957). U. pulchella is
 Extract Bioefficiency of Five……… 25

polyphagous leaf feeder pest and its host plants range are: Heliotrobium ramosissimum
(Boraginaceac), Launaea cassiniana (Asteraceae), Gossypium sp. (Malvaceae), Ricinus
communs (Euphorbaceae), Lawsonia incamis (Lgthraceae), Medicago sativa (Fabaceae) and
the host plants of solonaceae are lycopersicum esculentum, Solanum melongenia and
Wathania somnifera (AL-Ahmadi and Salem, 1995).
In the present study an attempt has been made to develop a new botanical insecticide
throughout evaluating the insecticidal activity of five medicinal plants from euphorbian
family namely E. granulata, E. petiolata, E. peplis, E. nutans and E. heliscopia.

MATERIALS AND METHODS

Insects
In the late August, the larvae and egg patches of the migrant crimson-speckled moth,
Utetheisa pulchella was seen in the fields near Mosul city/ north Iraq. They were observed
on helitropic weed, Heliotropium spp. and clover, Medicago sativa. Parts of the host plant
with pest larvae and egg-patches were collected and introduced to the laboratory and
incubated eggs and the growing larvae kept in the incubator under 27±1ºC, relative
humidity 65±5 and photoperiod 8:16. For refresh the host plant foliages, they were replaced
every two days.
Extraction Method
All the Euphorbia spp. in this study are herbal weeds, the whole plants were collected
through May till July and dried in a shadow place, then, crushed and grounded with electric
mill. 50 gm of each powdered plant species were macerated with 150ml of 96% ethanolic
alcohol for 48 hrs., then, the extract was filtrated with goose cloth, as well as filter papers
No.1 for two times. By rounding; the solvent was released. For chlorophyll separation as
possible, the ethanolic extract was dissolved again with v/v petroleum ether and ethanol in
the separation funnel and repeated for two times. The ethanol extract for each Euphorbia sp.
was rounded and dried until experimental treatment, the stock solution was prepared in 1000
ppm with ethanol solvent. Extraction was modified after Kaushik and Siani (2008).
Design of the experiments
New foliages of the wild host plant, Heliotropium ramosissimum were dipped in the
applied concentration for 20 sec. and dried in open place, then, the treated foliages
implanted in 100 cm3 bottles filled with distilled water. To prevent water from evaporation,
the bottles were sealed with cotton plugs, the bottles were kept in 500 ml flasks to prevent
larvae escaping, the flask's opening was shut with a goose cover.
According to the primary experiments, the following concentrations: 40,80,200 and
300 ppm were chosen, the experiments were designed with three replications for each
concentration and five of the pest larvae transformed for each replicate. Fourth instar larvae
were applied to the plant extracts.
After seven days of exposure time, dead and alive larvae are counted and the alive
ones kept to complete their development for separating normal and malformed adults. In
addition to dead larvae, dead prepupae, pupae and permanent larvae are represented dead
immature stages and total mortality.
26 Atallah F. Mekhlif

Histological preparation
To investigate the extract effect on the midgut of the immature stages, the 7 day 5th
instar treated larvae and prepupae were fixed in Boin solution for six days. Slides
preparation and staining had been done after Mekhlif (2004).
Sensitivity ratio
Sensitivity ratio is equal 1.0 for the lowermost Lc50 plant extract or positive control.
Then, relative sensitivity for all the last plant extract by consider any one of them as
denominator and Lc50 for the lowermost numerator (Mekhlif, 2004).
Statistical analysis
Mortality of the treated pest was corrected using Abbott´s formula (Abbott, 1925),
Lc50 (Lethal concentrations causing 50% mortality) was calculated using probit analysis
(Finney, 1971). Data from dead immature stages were subjected to analysis of variance,
two-way ANOVA. Also Duncan test for multiple comparisons is carried out.

RESULTS AND DISCUSSION

Bioactive effect of extracts
Mortality
In table 1, death of the immature stages (larvae, prepupae and pupae) of the pest
Utetheisa pulchella were significantly increased as the concentration of the applied
euphorbian leaf extract and positive control increased from 40-200 ppm, but immature
stages death were not significantly different as the concentration increased from 200 to 300
ppm. However, Euphorbia spp. extracts and the positive control (neem oil) were behaved as
phytoecdysteroid hormones with sigmoid curves, whereas, calibration curves were resulted
through application of the synthetic insecticides (Baker et al., 2000; Mekhlif, 2007).
Moreover, the death of the immature stages and their mortality percentage were
depended on Euphorbia species (Table 1 and 2). Thus, extracts of the weeds E. granulata
and E. petiolata can be canditated for preparing effective bioinsecticidies
Sensitivity ratio
It was found that the mortality of the pest varied according to the extract source. Table
2 and fig. 1 were illustrated this result, neem oil induces growth disturbances as insect
growth regulator (Zebitz, 1984). Fundamentally to comparison between Schmutterer and
Sing (1995) and the present plates, it can be reported that the active metabolites in
Euphorbia spp. extracts are insect growth regulators.
Table 2 shows the differences in phytoinsecticidal activity between the extracts of
Euphorbia spp. However, E. nutans and E. heliscopia are nearly 10 times less toxic than
neem oil. Also, neem oil more toxic than E. granulate, E. petiolata and E. peplis by 2 , 3
and 5 folds receptivity.
 Extract Bioefficiency of Five……… 27

Table 1: Number of Utetheisa pulchella immature stages death, their host plant is
treated with Euphorbia species.

Concentration (ppm)
Plant extract
40 80 200 300 Mean
1.0 1.33 2.0 2.0 1.58
E. heliscopia
h-j g-i e-h e-h D
0.67 1.67 2.33 2.33 1.75
E. nutans
i-k f-h d-g d-g CD
1.33 2.0 2.33 3.0 2.17
E. peplis
g-i e-h d-g b-d C
1.67 2.67 3.33 3.33 2.75
E. petiolata
f-h c-f a-d a-d B
2.0 2.67 3.33 3.67 2.92
E. granulata
e-h c-f a-d a-c B
2.67 3.33 4.0 4.33 3.56
Neem oil
c-f a-d ab a A
0.33 Zero Zero Zero 0.08
Control
j k k k E
1.38 1.95 2.48 2.67
Mean
b ab a a
* Each replicate 5 larvae.
** Numbers with the same letters are not significantly different (p>0.05, Duncan's multiple
range test).

Table 2: Immature stages mortality percentage and sensitivity ratio, host plant of
the pest, Utetheisa pulchella applied with Euphorbia spp. extracts.

Mortality at the concentrations (ppm) Sensitivity

Plant extract Lc50(ppm) ratio to
40 80 200 300
Neem oil
Neem oil* 49.9 64.3 78.6 85.7 42.0 1.0
E. granulata 35.7 49.9 64.3 71.4 84.0 0.5
E. petiolata 28.5 42.9 57.1 57.1 135 0.31
E. peplis 21.4 35.7 42.9 57.1 240 0.18
E. nutans 7.1 28.5 42.9 42.9 420 0.1
E. heliscopia 14.3 21.4 35.7 35.7 480 0.09
* neem oil is positive control.

Histological effect
To evaluate the effect of the Euphorbia spp. extracts on the internal organs and
morphology of U. pulchella, the extract of E. granulata as more effective was chosen. For
this purpose, sections of the midgut were prepared:
Larva midgut
One of the first systematic effects of the bioactive metabolites of the extract is the
gradual histolysis of epidermis cells and peritrophic matrix of the midgut. Priorly,
peritrophic matrix separates from the epidermis and it lyses later by increasing the
concentration to 200 ppm (Fig. 2). Peritrophic matrix is permeable to the nutrients forward
the epidermal cells, and protects them by avoiding the fraction with food particles
28 Atallah F. Mekhlif

(Wigglesworth, 1974). Also, the extract gradually degrades the epidermal cells.
Therefore, food absorbance is significantly decreased and that will affect the pest
metamorphosis and growth parameters.

Prepupa midgut
The present study is the first attempt to emphasize the internal organs of prepupal
stage, due to the importance of this stage in Noctuid family. However, full grown larva
prevents feeding and seeks for substratum shelter for contrast pupal cell, through that time
and later prepupa passes with dramatic changes before pupation. Effect of sublethal
concentration of the extract transforms into prepupa. Therefore, it was found bioactive
molecules of the extract interferes with cellular function of midgut epidermis. Fig. 3a
represents section in control prepupa midgut which indicates an active protein synthesis
through dense chromatin staining of the nuclei of the epidermal cells, in comparison pale
colour staining in Fig. 3b.

CONCLUSIONS
In view of the results mentioned above, it was concluded that plants produce diverse
phytochemicals which mostly secondary metabolites synthesized by the plant for defensive
purposes. These phytochemicals were applied to IPM programmes through direct death and
interfere with growth and development (Rembold, 1995).
The investigated Euphorbia spp. are weeds and can be easily cultivated with high
production with low agricultural operation. the spurge plants; E. granulate, E. peplis and E.
nutans are endemic weeds in farms, whereas E. heliscopia and E. petiolata are grown in rain
fed aired area.
Foliages treatment with Euphorbia spp. extract is reducing Utetheisa pulchella
population by significant mortality of immature stages and induction of adults malformation
(Fig. 4). The present finding gives promise for development of new generations of
unharmful and degradable bioinsecticides.

CONCENTRATION (PPM)

Fig. 1: Toxicity of the extracts of five Euphorbia spp. against the pest
Utetheisa pulchella L. in compared with neem oil.
 Extract Bioefficiency of Five……… 29

E
E E
F
Pm
a b c
Fig. 2: Light microscope section through the midgut of five- day 5th instar Utetheisa
pulchella larva: a – untreated control, b- treated with 40 ppm Euphorbia
granulate extract, C-treated with 200 ppm, E – epidermis,
F- food, Pm – peritrophic matrix. 100x.

a b

Fig. 3: section of Utetheisa pulchella prepupa midgut, diet in the larva stage treated
with 40 ppm Euphorbia granulate extract, a- control, b- treated, N- nuclei of
epidermal cells. 100x.
30 Atallah F. Mekhlif

a b

c d

e f

Fig. 4: Adults of Utetheisa pulchella cannot close their wings completely, they
developed from larvae treated with 40 ppm Euphorbia granulate extract
a- normal closed wings, b , c- mild corrugated, d- diverged wings,
e- very curly wings , f- eclusion failure. 4x.
 Extract Bioefficiency of Five……… 31

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