Page 18 - Volume 4, Issue 2, July 2011

Anagyrus

sp. near

pseudococci

on

P. ficus

in Greece

47

merase. PCR was performed with a thermo-

cycler (Primus 25, PeqLab) with the following

amplification conditions: initial denaturation

phase at 97°C for 4 min, 35 cycles of denatur-

ation at 96°C for 25 s, annealing at 48°C for

25 s and extension at 68°C for 90 s were fol-

lowed by a final extension step at 68°C for

10 min. PCR products were purified using the

QIAquickTM 108 Kit (QiaGen) and directly se-

quenced with both primers described above

on an ABI 3770 capillary sequencer (Applied

Biosystems), in order to exclude cases of

base misincorporation due to PCR error (20)

and visualised using CHROMAS LITE (37).

The 818 bp long sequences were aligned

by eye and were put into the GenBank (36).

There, the similar sequences were deter-

mined using the Basic Local Allignment

Search Tool (BLAST) by nucleotide. In ad-

dition, twelve nucleotide sequences of

three

Anagyrus

species already submit-

ted to GenBank were used in the construc-

tion of a phylogenetic tree. Specifically,

An-

agyrus pseudococci

(DQ667743, DQ667745,

DQ667744, DQ667746, DQ667747)

Anagyrus

sp.

near

pseudococci

(DQ667737, DQ667738,

DQ667739,DQ667740,DQ667741,DQ667742)

and

Anagyrus dactylopii

(DQ667736) se-

quences were used. A Neighbor-Joining (NJ)

approach (25) was applied to construct a

tree from the pairwise distances that were

estimated using the substitution model

of Tamura and Nei (29) as implemented in

MEGA version 3.1 (21).

Bioassay

The primary culture of the mealybug

P.

ficus

was established in the insectary of the

Biological Control of Pesticides Laboratory

at Benaki Phytopathological Institute from

individuals which were collected in an in-

fested vineyard at the region of Helia-Pelo-

ponnese. Taxonomic identification of the

species was done according to Cox and Ben-

Dov (6) key (P. Milonas and F. Karamaouna,

personal communication). The culture was

maintained on sprouted potatoes of the va-

riety ‘Marfona’ in sandwich boxes (17x11x5

cm: Length x Width x Height) with net cov-

ered openings (d= 1.5 cm) for ventilation.

The boxes were kept in a Gallenkamp CΟ

2

growth chamber at 26

o

C and constant dark.

All biological stages of the mealybug were

present in the culture.

The parasitoid colony was reared on

sprouted potatoes from the

P. ficus

mass

culture in plexiglass cages [50x40x40 cm

with two net covered ventilation openings

(30x20 cm)] in the insectary at 28 ± 1

o

C and

L16:D8 h. In order to schedule parasitoid

emergence for the experiment, parasitoids

of both sexes from the colony were released

in sandwich boxes (5-10 individuals/box)

and were let to parasitise mealybugs on the

infested potatoes every week.

Individuals of the pest were transferred

from the mass culture on leaf sections (cen-

tral part) of

Nerium oleander

(Apocynaceae),

which is a host plant of the mealybug (Sca-

lenet database), a few hours before the exper-

iment. The leaf sections were kept with their

lower surface upwards on top of a layer of 8 g/l

Agar, which had previously been autoclaved,

in Petri-dishes of 9 cm diameter. Size classes of

the host were used rather than host stages but

they were selected so that each size class com-

prised mostly one stage according to sampled

sizes ofmealybugsmeasured aftermoults. The

size classeswere 0.5-0.9mm(2

nd

instar nymph),

1-1.5mm(3

rd

instar nymph), 1.6-2.3mm(young

female adult), >2.3 mm (preovipositing female

adult) [Classification by Karamaouna and Cop-

land (21)]. Only female mealybugs were used

in the experiments apart from the two small

size classes where males and females could

not be distinguished. Five mealybugs of each

size class (20 in total) were placed randomly

in each Petri dish.

Every 1-3 days, females of

A.

sp.

near

pseudococci

were collected fromthe sandwich

boxes of the mass parasitoid culture, in which

both female and male wasps were present.

These 1-3 days old females were assumed

to have mated and were used in the experi-

ments. During the experiments, wasps were

released individually in the Petri-dishes for 24

hours. At the end of the experiment, the ovi-

positing female wasps were measured (head-

width) and dissected to confirm that they had

been inseminated (17). The Petri-dishes were