Basic HTML Version
Journal of Mosquito Research, 2013, Vol.3, No.4, 21
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32
ISSN 1927-646X
http://jmr.sophiapublisher.com
22
stress or infection, its concentration can either rise or
fall, depending upon the animal species. Mosquitoes
and all other insects belonging to different orders so
far examined have an abundant haemolymph
transferrin (Tsf). Transferrin up-regulated upon
bacterial challenge of
A. aegypti
was well
characterized at both the biochemical and molecular
level. No information is available on its up regulation
in many other mosquitoes. Studies of bioassay and
analysis of protein profile of the larval
Cx.
quinquefasciatus
infected with entomopathogenic
nematodes
H. bacteriophora
(HB) and
S. carpocapsae
(SC) showed that the mosquito larvae were
susceptible to the infection with HB and immune to
infection with SC. Also, HB could successfully
establish in the host body and complete its life cycle
while SC was melanized (in press). The aim of this
study was to detect the activation of transferrin gene,
as an immune response of
Cx. quinquefasciatus
larvae
infected by entomopathogenic nematodes towards
bacteria by cloning and expression of Tsf protein and
to test the activity of the recombinant Tsf, as well as to
prove the activity of native protein on the bacterial
growth.
2 Materials and Methods
2.1 Infecting mosquito larvae with nematode
The mosquitoes used in this study,
Culex
quinquefasciatus
were supplied by Prof. Dr. Fatma
Kamel Adham, Faculty of Science, Cairo University,
Giza, Egypt. The nematode species were supplied by
Prof. Dr. Muhammed Mostafa Shams El-dean,
Zoology and Nematology Department, Applied Center
of Entomonematode, (ACE), Faculty of Agriculture,
Cairo University, Giza, Egypt.
For the semi-quantitative RT-PCR assay of transferrin
(Tsf) transcript abundance, the 4
th
instar mosquito
larvae were subjected to sub-lethal concentrations of
nematode infective juveniles of
H. bacteriophora
(HB,
a representative of nematode species that completed
its life cycle within host and caused high host
mortality value) and
S. carpocapsae
(SC, a
representative of nematode species that were
melanized and did not cause host mortality).
Transferrin transcripts were detected in control, HB-
and SC-treated insects after 3, 6, 9, and 12 hr post
infection. Ten 4
th
instar larvae were placed in a 5-cm
Petri dish half-filled with distilled water containing 50
nematode infective juveniles from each nematode
species. There were 3 replicates per treatment. Larvae
were starved one day before starting the experiment
and were fed on cat food during the experiment to
ensure ingestion of nematodes together with food.
Controls (no nematodes) were also treated the same
way. Larvae were collected after 3, 6, 9 and 12 hrs
post treatment and their surface was washed off with
distilled water to remove any nematodes on their
bodies. These larvae were subjected to RNA
extraction.
2.2 RNA isolation and cDNA synthesis
Total RNA of the 4
th
instar larvae of the control and
treated insects of
Culex quinquefasciatus
was
extracted from whole-body larvae using Wizard
®
Plus
SV Total RNA isolation System. Fresh or frozen tissue
(−80 ) was ground into a fine powder in liquid N2
℃
before the extraction buffer was added (1 mL of buffer
to 1 g tissue). This method was rapid and produced a
large amount of high quality and undegraded RNA. To
judge the integrity and overall quality of a total RNA
preparation, the total RNA extracted was examined on
native agarose gel electrophoresis (1% agarose gel)
and on denaturing formaldehyde/agarose gel (1.2%
agarose in MOPS buffer) (Lehrach et al., 1977). First
strand cDNA was synthesized using RevertAid
TM
H
Minus First Strand cDNA synthesis kit (Fermentas, cat
# K1632). First strand cDNA was synthesized using
random primer and 1 μg of total RNA.
2.3 Amplification of Transferrin transcript
cDNA region corresponding to transferrin protein was
amplified by PCR using two degenerate primers, p1
(5’-GAMCCYAAGGAYATGTAYGTRGC-3’) and p2
(5’-YCWCKYTCWATIACITCYKTGTA-3’), (Bioneer);
which were already described as the conserved
regions of insect transferrin sequences(Yoshiga et al.,
1997). One step PCR kit (Biobasic, cat # 705, Canada)
was used as the PCR reaction mixture. The
amplification reaction was carried out using the
following PCR reaction conditions: (1) an initial
denaturation step at 95 (3min); (2) 35 cycles of
℃
denaturation step at 95
℃
(30 sec); (3) primer-specific