Page 9 - Volume 4, Issue 2, July 2011

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-menthane type compounds against mosquito

37

titative data were obtained electronically

from FID area percent data without the use

of correction factors. Qualitative analysis of

the essential oils was performed using the

same conditions with GC, in a Hewlett Pack-

ard II 5890 gas chromatograph equipped

with Hewlett Packard II 5972 mass selec-

tive detector in the electron impact mode

(70 eV). Most ingredients of the essential

oils were identified on the basis of compari-

son of GC relative retention times and mass

spectra with those of pure standards. As for

the rest components, tentative identifica-

tion was based on the comparison of their

mass spectra and elution order with those

obtained from the NIST 98 and Wiley 275 li-

brary data of the GC-MS system and from

Adams CD computer library (1).

Isolation of Essential Oils Major Ingredi-

ents

Ten (10) g of the extracted crude essential

oils EOpul1, EOpul2, EOpul3, EOpip and EOsp

were fractioned by column chromatography

on silica gel and eluted with a gradient of sol-

vents of increasing polarity (pentane + dieth-

yl ether: 100 + 0, 97 + 3, 95 + 5, 93 + 7, 90 +

10 and 80 + 20). Column fractions were moni-

tored by thin layer chromatography (TLC) with

pentane + diethyl ether (9.5 + 0.5). Fractions

with similar R

f

values on the TLC plates to au-

thentic co-eluted compounds were pooled.

Spots were detected under UV lamp and af-

terwards by spraying with a mixture of 1%

vanillin and 5% sulfuric acid solution (in eth-

anol) and heating at 120

o

C and/or PMA solu-

tion (phosphomolybdic acid 7.5%mgl

-1

in eth-

anol) and charring on a hot plate. The chosen

fractions merged to one and elution solvents

removed by a rotary flash evaporator. The dis-

tillation was stopped when the volume of sol-

vents was reduced to ~10 ml and completed

by flushing through nitrogen.

Mosquito Rearing

The colony of the species

Cx. pipiens

bi-

otype

molestus

has been maintained in the

laboratory of Benaki Phytopathological In-

stitute, Kifissia, Greece for more than 25

years. Adult mosquitoes are kept in wood-

en framed cages (33x33x33 cm) with a 32x32

mesh at 25±2

o

C, 80±2% relative humidi-

ty and photoperiod of 14:10 (L:D) h. Cotton

wicks saturated with 10% sucrose solution

are used as food source. Females lay eggs in

round, plastic containers (10 cm diameter x

5 cm depth) filled with 150 ml of tap water.

Egg rafts are removed daily and placed in

cylindrical enamel pans (with diameter of 35

cm and 10 cm deep), in order to hatch. Lar-

vae are reared under the same conditions of

temperature and light and are fed daily with

baby fish food (TetraMin, Baby Fish Food) at

a concentration of 0.25 gl

-1

of water until pu-

pation. Pupae are then collected and intro-

duced into the adult rearing cages.

Larvicidal Bioassays

Stock solutions were prepared in etha-

nol with a concentration of 1% mgl

-1

. A se-

ries of aqueous solutions of the tested ma-

terial, expressed as mgl

-1

, were made and

tested under laboratory conditions. Prelim-

inary experiments (data not shown) were

performed to evaluate toxicity and subse-

quently different concentrations were em-

ployed ranging from 10 to 200 mgl

-1

for each

tested material.

The larval mortality bioassays were carried

out according to the test method of larval sus-

ceptibility as proposed by the World Health

Organization (24). Twenty 3

rd

to 4

th

instar lar-

vae of the species

Cx. pipiens

biotype

moles-

tus

were collected from the colony. They were

placed in glass beaker with 250 ml of aqueous

suspension of tested material at various con-

centrations and an emulsifier was added in

the final test solution (less than 0.05%). Four

replicates were made per concentration and a

control treatment with tap water and emulsifi-

er was also included. Beakers with larvae were

placed at 25±2

o

C, 80±2% relative humidity

and photoperiod of 14:10 (L:D) h.

Data Analysis

Larvicidal effect was recorded 48 h after

treatment. Data obtained from each dose–

larvicidal bioassay (total mortality, mgl

-1

concentration in water) were subjected to

probit analysis in which probit–transformed