Insecticidal, biological and synergistic activity of 8 essential oils on Culex pipiens larvae (Diptera:Culicidae)

Insecticidal, biological and synergistic activity of 8 essential oils on


Culex pipiens larvae (Diptera:Culicidae)


Salam S. Teleb


Zoology Department, Faculty of Science, Zagazig University, Egypt.

ABSTRACT

The insecticidal properties of 8 essential oils were screened on Culex
pipiens larvae. The most effective oils were; Foeniculum vulgare (LC50 =
20.0 ppm) followed by Triticum vulgare, Rosa gallica, Salvia officinalis,
Mentha spicata, Rosa damascene , Matricharia chamomilla and Ammi majus
with LC50 values of 29.7, 30.0,30.3, 36.0, 78.0, 100.7and 210.0 ppm,
respectively.

The tested mixtures of the highest potentiating effect was for mixture
of co-toxicity factor equaled 60.0( Foeniculum vulgare + Rosa damascene).
The lower potentiating effects were obtained from mixtures of Rosa gallica
+ Foeniculum vulgare and Foeniculum vulgare + Ammi majus.

The Rosa gallica oil exerted zero hatchability (100% mortality) at
1000,500 and 250ppm. , till 500 ppm oils from Salvia officinalis ,
Matricharia chamomilla and Foeniculum vulgare whereas oils from Rosa
damascene and Mentha spicata at 1000ppm. Ammi majus oil reduced egg hatch
by 33.0%at 1000ppm.

Rosa gallica yielded 100% complete inhibition of emergence of adults
at 62.5 ppm and125 ppm for Mentha spicata and Matricharia chamomilla. Oils
from Ammi majus and Rosa damascene yielded also100% complete inhibition of
emergence at 250 ppm except Salvia officinalis and Foeniculum vulgare
yielded 30.0%of emergence at 250ppm

Key words: Insecticidal, synergistic , biological,essential oils , Culex
pipiens ,Diptera Culicidae.


INTRODUCTION

Mosquito species belonging to culicidae is a widespread insect pest with
medical importance; is responsible for the appearance of West Nile virus
(Lundström 1999; Dauphin et al., 2004).and vectors for the pathogens of
various diseases like malaria, filariasis, Japanese encephalitis,dengue and
dengue haemorrhagic fever, yellow fever, etc. The control of mosquito at
the larval stage is necessary and efficient in the integrated approach to
mosquito management. Essential oils are renewable, non-persistent ,have
strong aromatic components ,give distinctive odour, flavor or scent to a
plant and relatively safe to natural enemies, non-target organisms and
human beings (Halder et al., 2010). These are products of plant metabolism
and are commonly referred to as volatile plant secondary metabolites (Koulo
et al., 2008). many indigenous aromatic plants such as Fennel, Foeniculum
vulgare has been evaluated against a number of stored product insects.
,Sahaf et al., (2007) found the strong insecticidal activity of essential
oil from Carum copticum (Apiaceae) on S. oryzae and Tribolium castaneum
.Labiatae is a large family containing many useful plants with potential
therapeutic activity, especially due to their essential oils. There is
limited information concerning the activity of Mentha, Salvia, and Melissa
essential oils and their major constituents against Cx. pipiens mosquito
species (Ansari et al., 2000; Trabousli et al., 2002; Amer and Mehlhorn,
2006 and Cetin et al., 2006). Recently, essential oils have received
considerable attention as a potentially useful bioactive insecticide, with
their low mammalian toxicity and rapid degradability in the environment .
Many researchers reported on the effectiveness of plant extracts or
essential oils against mosquito larvae ( Teleb and Farag, 1999; Amer and
and Mehlhorn, 2006 ; Rahuman et al., 2009 and Sezgiet et al ., 2011 ).

In present study, attempt was made to assess the larvicidal, biological
and synergistic activity of Ammi majus, Rosa gallica Rosa damascena ,
Mentha spicata, Triticum vulgare L., Salvia officinalis, Foeniculum vulgare
and Matricharia chamomilla essential oils against Cx. pipiens larvae.

MATERIALS AND METHODS

1- Mosquito rearing

Mosquito larvae were collected from a colony that has been maintained in
the laboratory. Adults were kept in wooden framed cages (33×33×33 cm) with
32×32 mesh at 25±2°C and 80±5% relative humidity. Cotton wicks saturated
with 10% sucrose solution were used as food source for the mosquitoes.
Females laid eggs in round plastic cups (10 cm diameter×10cm depth) filled
with 150 ml of dechlorinated tap water. Egg rafts were removed daily and
placed in cylindrical enamel pans (with diameter of 35 cm and 10 cm deep),
in order to hatch. Larvae were reared under the above mentioned conditions
of temperature and humidity and were fed daily with fish food (TetraMin,
Fish Food) of water until pupation. Pupae were then collected and
introduced into the adult rearing cages.

2- Larvicidal bioassays

A series of aqueous solution with different concentrations of the
tested essential oils (ppm) was made and tested under previous conditions
of temperature and humidity.The larval mortality bioassays were carried out
according to the method of larval susceptibility as suggested by the World
Health Organization (WHO 1996). Twenty five numbers of early 4th instars
larvae were placed in plastic cups containing serial concentrations of each
oil with few drops of emulsifier (Tween 80). Mortality was recorded after
24 h. Four replicates were made per concentration and a control treatment
with dechlorinated tap water and emulsifier was included in each bioassay.







3- Biological studies :

Ovicidal activity : The method of Su and Mulla (1998) was followed for
the ovicidal activity. Ten replicates of egg rafts (0-6h.old )were counted
and transferred each in a separate plastic cups( 10cm height) half filled
with different concentrations of Ammi majus, Rosa gallica ,Rosa damascena ,
Mentha spicata, Triticum vulgare L., Salvia officinalis, Foeniculum
vulgare,and Matricharia chamomilla essential oils and observed daily for
hatching .Egg hatchability was expressed as the percentage of hatched eggs
from total oviposited eggs .

Number of hatched larvae x 100

Total number of eggs

For pupicidal action :Ten replicates of newly emerged pupae were
transferred into plastic cups 10cm height (4/ cup) containing different
concentrations from each oil. The number of emerging adults was observed
,calculated daily and compared with control (untreated ).

4 - Essential oils:

The following 8 essential oils used in the present study belonging to
6 families are shown in Table 1: Ammi majus ( Seeds), Rosa gallica(
Flowers), Rosa damascena ( Flowers), Mentha spicata(Leaves), Triticum
vulgare L.(Greens), Salvia officinalis(Leaves), Foeniculum vulgare( Seeds),
and Matricharia chamomilla ( Flowers), were purchased from El-captain
Company (CAP. PHARM., Egypt) for extracting natural oils, herbs and
cosmetics, Cairo, Egypt.

5- Mixtures toxicity (Joint action)

Paired mixtures of essential oils with each others were freshly prepared
at concentration levels of their respective LC25 values. Each mixture was
tested in four replicates along with controls, and the tests were carried
out as mentioned above. Mortality percentages were determined after 24h.
and the combined (joint) action of the different mixtures was expressed as
Co-toxicity factor according to Sun and Johnson( 1960) to differentiate
between potentiation, antagonism and additive, using the following formula:

Co - toxicity factor = (% observed mortality –% expected mortality) x 100


%expected mortality.

The co-toxicity factor differentiates the results into three categories.
A positive factor of+ 20 indicates potentiation, a negative factor of -20
indicates antagonism, and the intermediate values of >-20 to < 20 indicate
an additive effect. Because obtained LC25 values are mathematically
estimated, they were tested again against larvae to determine the accurate
expected mortality. The expected mortality of the combined pair is the sum
of the mortalities of single compounds at the given LC25 concentrations and
the observed mortality is the recorded mortality obtained 24 h after
exposure the mixture.

6- Data analysis

Data obtained from each dose - larvicidal bioassay (total mortality)
were subjected to probit analysis (Finney, 1971)and LC25,LC50 and LC90
values were calculated . All results were expressed as mean ± standard
error, and the data were analyzed using student T-test. Results with p Matricharia
chamomilla > Rosa damascene > Mentha spicata > Salvia officinalis > Rosa
gallica > Triticum vulgare > Foeniculum vulgare. majus with LC50 values
of 210.0 , 100.7, 78.0,36.0,30.3, ,30.0, 29.7 and20.0 ppm, respectively.

At the level of LC90 values, Rosa gallica oil showed the highest larval
toxicity (42.7 ppm), and Ammi majus oil was the lowest (833.0ppm). The
slope of regression lines ranged between 1.5 for Mentha spicata oil and 5.3
for Foeniculum vulgare, indicating different degree of homogeneity or
heterogeneity of the tested insects against the bioassayed oils.
Koliopoulos et al., (2010) showed that the most effective oils were M.
suaveolens (major constituent piperitenone oxide, 62.4%), M. spicata
(piperitenone oxide,35.7% and 1,8-cineole, 14.5%) and M. longifolia on
Culex pipiens larvae. Zahra et al.,(2009) showed that all essential oils
had significance repellency effect against P. interpunctella. moths ;The
strongest repellency in Anethum graveolens (100%), Thymus vulgaris (100%)
and Rosmarinus officinalis (93.33%) and the weakest repellency in Hyossopus
officinalis (7.69%) and Petroselinum sativum (9.48%). Gün et al.,(2011)
showed that extract of Salvia tomentosa Mill. was the most toxic, followed
by those of S. sclarea L., S. argentea L. and S. syriaca L. with LC50
values 60.61, 62.05, 107.40 and >200 ppm, respectively on Culex pipiens
larvae.. Kumar et al., (2011)showed that the essential oil from Mentha
piperita possed excellent larvicidal efficiency against Aedes aegypti and
LC50 and LC90 value of 111.9and 295.18ppm,respectively after 24 H of
exposure. Kandaswamy et al., (2012)indicated that the mortality rates at
80, 100, 200 and 400 ppm of M. piperita, Z. officinale, C. longa and O.
basilicum concentrations were highest amongst all concentrations of the
crude extracts tested against all the larval instars and pupae of A.
aegypti. Rana and Singh(2012 )reported that oil of Tagetes patula (T.
patula) and F. vulgare gave more than 90% mortality within 40 min.on Culex
quinquefasciatus larvae. LC50 values calculated after 60 min of treatment
were (84.80 ±7.65) and (24.69 ± 1.24) ppm for T. patula and F. vulgare,
respectively. At the same exposure period positive control temephos yielded
(22.13±2.39) ppm LC50 value. At 50 ppm T. patula, F. vulgare and temephos
exhibited LT50 values (113.71 ±10.29), (11.02 ±3.31) and (38.15 ± 5.90)
min., respectively. Presence of quantity of trans anethole in the essential
oils of F. vulgare was confirmed by TLC and HPL.

Combining of 8 essential oils resulted in paired mixtures of different
degrees of joint action towards 4th larval instar of larvae of Cx.
pipiens. The calculated "co-toxicity factor" exceeded 20; a result
accounting to "potentiation effect" (Table 4).The highest potentiating
effect was for mixtures of co-toxicity factor equaled 60.0( Foeniculum
vulgare + Rosa damascene). The lower potentiating effects( 20)were obtained
from mixtures of Rosa gallica + Foeniculum vulgare and Foeniculum vulgare
+ Ammi majus. Most of the tested mixtures induced antagonistic effects;
Rosa gallica+ Rosa damascene, Rosa gallica + Rosa damascena, Matricharia
chamomilla + Triticum vulgare, Matricharia chamomilla + Rosa damascene,
Triticum vulgare + Salvia officinalis , Triticum vulgare + Rosa damascene
, Triticum vulgare + Mentha spicata , Triticum vulgare + Salvia
officinalis , Triticum vulgare + Rosa damascene , Foeniculum vulgare+
Mentha spicata, Foeniculum vulgare + Triticum vulgare and Foeniculum
vulgare+ Matricharia chamomilla while mixtures of Rosa gallica + Ammi
majus, Rosa gallica + Triticum vulgare , Rosa gallica + Salvia officinalis
, Matricharia chamomilla + Mentha spicata ,and Foeniculum vulgare+ Salvia
officinalis induced additive effects.

Tripathi et al., (2011)have stated that F. vulgare has toxic action
against larval stage of An. Stephensi. The property was attributed to the
synergistic combination of essential oil derived from medicinal plants .

2 - Biological activities:


2-1 - Ovicidal activity . Results on the ovicidal effects of essential
oils were reported in the present study and confirmed their potential for
populations control of the mosquito( Table 5) . The Rosa gallica oil
exerted 100% mortality(Zero hatching) at 1000,500 and 250ppm, at 500 ppm
for oils from Salvia officinalis , Matricharia chamomilla and Foeniculum
vulgare whereas oils from Rosa damascene and Mentha spicata at 1000 ppm.
Ammi majus oil reduced egg hatch by 33.0% at 1000 ppm conc. the
hatchability increased by diluting with water. All tested conc.
significantly different from control. Pushpanathan et al.,(2006) evaluated
the essential oils extracted by steam distillation from Cymbopogan citratus
larvicidal, ovicidal and repellent activities against the filarial mosquito
Culex quinquefasciatus. The LC50 values calculated for the 2nd, 3rd and 4th
larval instar were 144.54 ± 2.3, 165.70 ± 1.2 and 184.18 ± 0.8 ppm
respectively. Hundred percent ovicidal activity was observed at 300 ppm.
Radhika et al.,(2011) revealed that the eggs laid in the water with 100%
essential oils did not hatch at all, whereas when 10% oils were used, only
the R. officinalis oil resulted in 28% egg hatch. At lower concentrations
(1%), the oils of M. piperita, O. basilicum, and C. nardus showed complete
egg mortality while those of A. graveolens and R. officinalis resulted in
71% and 34% egg hatches, respectively. When used at 0.1%, the O. basilicum
oil was found to be the only effective oil with 100% egg mortality, whereas
other oils resulted in 16–76% egg mortality, the least mortality caused by
the A. graveolens oil.


2-2 – Pupicidal activity. Table (6) shows that essential oils from Rosa
gallica yielded 100% completely inhibition the emergence of adults at
62.5ppm,at125ppm for Mentha spicata and Matricharia chamomilla . Oils from
Ammi majus and Rosa damascene yielded also100% completely inhibition the
emergence of at250ppm except Salvia officinalis and Foeniculum vulgare
yielded 30.0%of emergence at 250ppm .The emergence of adults increasing by
dilution. Qiao et al.,(2010) indicates that essential oils from leaves of
Cedrus deodara and Pinus massoniana had highly larvicidal and pupicidal
activity to Aedes albopictus and Culex pipiens quinquefasciatus.

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Table (1): Plant species and their families used in the present study
"English name "Scientific name "Family "
"Bishop's weed "Ammi majus L. "Apiaceae "
"Damask rose "Rosa damascena "Rosacea "
" "Mill. " "
"Red rose "Rosa gallica "Rosaceae "
" "Linn " "
"Spearmint "Mentha spicata "Labiatae "
"Wheat "Triticum vulgare"Poaceae "
" "L. " "
"Sage "Salvia "Labiatae "
" "officinalis " "
"Fennel "Foeniculum "Umbelliferae "
" "vulgare " "
"Chamomile "Matrichari " Asteraceae "
" "chamomilla " "

Table 2: Evaluation of essential oils at 500and 100ppm conc. against the
4th instar larvae of Culex pipiens

"Essential oils "% Mortality "
" "Mean* ±S.E "
" "500 ppm "100 ppm "
"Ammi majus "80.0 ± 4.0 "32.5± 2.9 "
"Rosa damascena "100.0± 0.0 "75.0± 2.9 "
"Rosa gallica "100.0± 0.0 "100.0± 0.0 "
"Mentha spicata "100.0± 0.0 "100.0± 0.0 "
"Triticum vulgare "100.0± 0.0 "100.0± 0.0 "
"Salvia officinalis "87.5± 2.5 "75.0± 2.9 "
"Foeniculum vulgare "100.0± 0.0 "100.0± 0.0 "
"Matricharia "82.5± 2.5 "55± 2.9 "
"chamomilla " " "


* Results are the means of 4 replicate.


Table 3: Toxicity data for the tested essential oils against 4th
instar larvae of Culex pipiens


"Essential "Toxicity values (ppm) Mean ±S.E "
"oils " "
" "LC25 "LC50 "LC90 "Slope "
"Ammi majus "82.0 "210.0 "833.0 "2.3 "
" "(88-108)"(180-250)"(700-1000)"±0.1 "
"Rosa "30.3 "78.0 "503.3 "4.4±0.6"
"damascena "(27-34) "(68-88) "(450-560) " "
"Rosa gallica "18.3 "30.0 "42.7 "1.6±0.1"
" "(17-20) "(24-36) "(40-46) " "
"Mentha "26.7 "36.0 "64.0 "1.5±0.1"
"spicata "(23-28) "(32-40) "(60-72) " "
"Triticum "15.7 "29.7 "100.0 "2.3±0.1"
"vulgare "(15-17) "( 27-32) "(90-110) " "
"Salvia "15.3 "30.3 "126.7 "4.4±0.2"
"officinalis "(14-17) "(24-40) "(125-130) " "
"Foeniculum "6.6 "20.0 "170.0 "5.3±0.0"
"vulgare "(5.6 -8)"(17-23) "(150-180) "3 "
"Matricharia "43.3 "100.7 "680 "4.2±0.2"
"chamomilla "(40-50) "(94-108) "(500-900) " "


Values between brackets are 90% fudicial limits of the corresponding
toxicity values. The latter values are estimated from their respective
regression lines (LC-P lines).

Table 4: Joint action of binary mixtures of the tested essential oils
against 4th instar larvae of Culex pipiens
"Mixtures "Lc25,s(ppm"%Obse"Co-toxi"Joi"
" ")for "rved "city "nt "
" " "Morta"factor "Act"
" " "lity " "ion"
" "Compo"Comp" " " "
" "und "ound" " " "
" "(1) "(2) " " " "
"Rosa gallica1 + Ammi "18.3 "98.7"40.0 "-40 "Ad."
"majus2 " " " " " "
"Rosa gallica 1 + Rosa "18.3 "30.3"10.0 "-80 "Ant"
"damascena2 " " " " ". "
"Rosa gallica 1+ Mentha "18.3 "25.7"10.0 "-80 "Ant"
"spicata2 " " " " ". "
"Rosa gallica 1 + Triticum "18.3 "15.7"40.0 "-20 "Ad."
"vulgare 2 " " " " " "
"Rosa gallica 1+ Salvia "18.3 "15.3"30.0 "-40 "Ad."
"officinalis 2 " " " " " "
"Rosa gallica 1+ Foeniculum"18.3 "6.7 "60.0 "+20 "Po."
"vulgare 2 " " " " " "
"Rosa gallica 1+ "18.3 "43.3"70.0 "+40 "Po."
"Matricharia chamomilla 2 " " " " " "
"Matricharia chamomilla 1 + "43.3 "15.7"10.0 "-80 "Ant"
"Triticum vulgare 2 " " " " ". "
"Matricharia chamomilla 1 +"43.3 "25.7"40.0 "-20 "Ad."
"Mentha spicata 2 " " " " " "
"Matricharia chamomilla 1+ "43.3 "15.7"10.0 "-80 "Ant"
"Triticum vulgare 2 " " " " ". "
"Matricharia chamomilla 1 +"43.3 "30.3"10.0 "-80 "Ant"
"Rosa damascene 2 " " " " ". "
"Triticum vulgare 1 + Mentha"15.7 "25.7"0.0 "-100 "Ant"
"spicata 2 " " " " ". "
"Triticum vulgare 1 + Salvia"15.7 "15.3"10.0 "-80 "Ant"
"officinalis 2 " " " " ". "
"Triticum vulgare 1 + Rosa "15.7 "30.3"10.0 "-80 "Ant"
"damascene 2 " " " " ". "
"Foeniculum vulgare 1 + Ammi"6.7 "98.7"60.0 "+20 "Po."
"majus 2 " " " " " "
"Foeniculum vulgare 1+ Rosa "6.7 "30.3"80.0 "+60 "Po."
"damascene 2 " " " " " "
"Foeniculum vulgare1 + "6.7 "25.7"40.0 "-20 "Ant"
"Mentha spicata2 " " " " ". "
"Foeniculum vulgare1+ Salvia"6.7 "15.3"50.0 "0.0 "Ad."
"officinalis 2 " " " " " "
"Foeniculum vulgare 1+ "6.7 "15.7"20.0 "-60 "Ant"
"Triticum vulgare2 " " " " ". "
"Foeniculum vulgare1+ "6.7 "43.3"30.0 "-40 "Ant"
"Matricharia chamomilla 2 " " " " ". "


Po.= positive factor of 20 indicates potentiation.
Ant .=negative factor of -20 indicates antagonism,
Ad .= >-20 to < 20 indicate an additive ef

Table 5: Ovicidal action of the tested essential oils
"Conc. (ppm) "Essentia"
" "l oils "
"31.25 "62.5 "


15.125 "31. 25 "62.5 "125 "250 "500 "Control " " "90.0±5.8* "75.0±5.0*
"60.0±8.2 "30.0±5.8* "0.0±0.0 "0.0±0.0 "100±0.0 "Ammi majus " "*85.0±5.0
"70.0± 5.8* "50.0± 5.8 * "30.0± 5.8 * "0.0±0.0 "0.0±0.0 "100±0.0 "Rosa
damascena
" "75.3± 5.0* "30.0± 5.8 * "0.0±0.0 "0.0±0.0 "0.0±0.0 "0.0±0.0 "100±0.0
"Rosa gallica
" "85.0± 5.0* "70.0± 5.8 * "30.0± 5.8 * "0.0±0.0 "0.0±0.0 "0.0±0.0
"100±0.0 "Mentha spicata
" "90.0±0.0* "70.0.± 5.8 "45.0± 5.0* "27.5± 5.7 * "0.0±0.0 "0.0±0.0
"100±0.0 "Triticum vulgare
" "100.0± 0.0 "85.0± 10.0* "65.0± 5.0* "45.0± 9.6* "30.0± 5.8*
"0.0±0.0 "100±0.0 "Salvia officinalis
" "100.0± 0.0 "90.0± 5.8 * "60.0± 8.2 * "50.0± 5.8 * "30.0± 5.8*
"0.0±0.0 "100±0.0 "Foeniculum vulgare
" "85.0± 5.0* "50.0± 5.8 * "35.0± 5.0 * "0.0± 0.0 "0.0± 0.0 "0.0±0.0
"100±0.0 "Matricharia chamomilla
" "*Significant at p=0.05 **Results are
the means of 4 replicates

الأنشطة الإبادية والبيولوجية والتآزرية لثمانية أنواع من الزيوت الأساسية على
يرقات بعوض الكيولكس بيبينز (ثنائية الأجنحة : كيوليسيدي).

سـلام سالم طلـب

قسم علم الحيوان، كلية العلوم، جامعة الزقازيق، مصر.

تم عرض خصائص السمية لثمانية أنواع من الزيوت الأساسية هي زيت جنين القمح
والورد البلدي و الميرامية و النعناع و الورد الأحمر والبابونج والخلة والشمر
على يرقات بعوض الكيولكس بيبينز.

وكان زيت الشمر الأكثر فعالية وكانت الجرعة نصف المميتة 20جزء في المليون
يليها زيت جنين القمح والورد البلدي و الميرامية و النعناع و الورد البلدي
والبابونج والخلة و وكانت الجرعة نصف المميتة 29,7، 30,3،30، 36,0، 78,0،
100,7 و210,0 جزء في المليون، على التوالي.
أثبتت المخاليط المشتركة إضافة عالية في التأثير ذو معامل السمية60 لكل من
(الشمر+ الورد البلدي) بينما كانت المخاليط المشتركة لكل من (الورد الأحمر +
الشمر والشمر + الخلة) اقل في التأثير الإضافي.

اثبت زيت الورد البلدي نسبة فقس صفر(100% موت)عند تركيزات 1000و500و250جزء
في المليون وحتى500جزء في المليون لزيت الميرامية و البابونج و الشمر كما
أبدت زيوت الورد البلدي والنعناع مانعات للفقس عند 1000جزء في المليون بينما
أبدى زيت الخلة اقل نسبة فقس 33,0%.عند1000جزء في المليون .

أظهر زيت الورد البلدي تثبيطا كاملا لظهور الحشرة الكاملة عند تركيز
62.5جزء في المليون وعند125 جزء في المليون لزيت النعناع والبابونج.كما أبديا
زيت الخلة والورد البلدي مانعا لخروج الحشرة الكاملة 100%عند تركيز 250 جزء
في المليون ماعدا زيت الميرامية والشمر حيث أبديا نسبة30% لظهور الحشرة
الكاملة عند250 جزء في المليون.