Page 46 - Volume 2,Issue 2, July 2009

Papachristos & Stamopoulos

92

pours on

A. obtectus

development, longev-

ity and fecundity.

Materials and methods

Insects and essential oils

Second instar larvae and 3-day-old pu-

pae were obtained from laboratory cultures

of

A. obtectus

according to the procedure

described by Papachristos and Stamopou-

los (18). The essential oils tested were ex-

tracted from the following plants:

L. hybrida

(flowers),

R. officinalis

(leaves) and

E. globu-

lus

(unripe fruit). Plant samples were collect-

ed in mid July 1999 from an experimental

farm in the region of Thessaloniki, Northern

Greece, where they were cultivated at adja-

cent sites. Bulk samples of about 1 kg were

collected randomly from 20–30 lavender or

rosemary plants and three eucalyptus trees.

Samples were transferred to the laborato-

ry and 100 g subsamples were subjected to

hydro distillation using a Clevenger appara-

tus (Winzer

®

) within 24 h. The distilled es-

sential oils were dried over anhydrous sodi-

um sulphate and stored in a refrigerator at

5°C. The chemical composition of the essen-

tial oils used in the present study has been

reported by Papachristos

et al.

(17).

Exposure to essential oil vapours

Gastight glass jars of 710 ml volume with

screwedmetallic capswere used as exposure

chambers. A small piece of woven dental

cotton (diameter = 8 mm, height = 3.5 mm)

was attached to the undersurface of the cap

to serve as an oil diffuser following the ap-

plication of the appropriate amount of pure

essential oil. Dose levels were selected to

cause 10, 40 and 70% mortality according to

our findings in a previous study (19). The ex-

act doses of each essential oil applied were:

lavender: 0.2, 1.4, 2 μl/l air for larvae and 21,

36.8, 57.9 μl/l for pupae; rosemary: 1.4, 2.1,

2.8 μl/l air for larvae and 26.3, 52.6, 73.7 μl/l

for pupae; eucalyptus: 1.4, 4.2, 7.1 μl/l air for

larvae and 57.9, 73.7, 89.5 μl/l for pupae. For-

ty bean seeds containing about 30-40 sec-

ond instar larvae or 3-day-old pupae of

A.

obtectus

were placed in each jar. The same

methodology was followed for the control

without the application of essential oils. Six

replicates were used for each dose and in-

sect stage. After 48 h of exposure to essen-

tial oil vapours, the beans were transferred

to clean vials for four days and subsequently

the beans in half of the replicates were dis-

sected in order to count the dead larvae and

pupae. The beans from the other three rep-

licates remained intact and were observed

daily until the adult survivors emerged. The

developmental time from the end of expo-

sure to adult emergence was also recorded.

After emergence the beans were dissected

and dead larvae and pupae were counted.

Sublethal effects on adults

The effect of the essential oil vapours

on adult insects derived from the survived

treated larvae and pupae was also studied.

For this purpose male or female survivors

from each dose tested were weighted just

after their emergence and subsequently, 20-

30 of them were placed individually in plas-

tic Petri dishes (9 cm in diameter) in order to

record their longevity.

To study the fecundity and fertility of fe-

male survivors, about 15 females, emerged

from each dose tested, were placed individ-

ually with two untreated one-day-old males

taken from the mass culture in plastic Petri

dishes (9 cm in diameter) containing 10 beans

and allowed to mate and oviposit until their

death. The number of eggs laid was counted

daily. The eggs were kept in the Petri dishes

until the end of hatching and the number of

hatched and unhatched eggs was recorded.

Experimental conditions

All trials were conducted at 65 ± 5 r.h, 25

± 1 °C and a 12 h photoperiod.

Statistical analyses

All mortality data were corrected by us-

ing Abbott’s formula (1). T-test was used in

order to compare the average mortality cal-

culated four days after exposure to essen-

tial oil vapours with that recorded after the

emergence of the adults.