Abstract |
Insects
have
a
protective
exoskeleton
made
up
of
a
multi-‐layered
cuticle
that
helps
to
withstand
various
environmental
and
pathogenic
challenges.
Against
pyrethroid
insecticides,
Anopheles
gambiae
mosquitoes,
have
developed
multiple
resistance
mechanisms,
such
as
target
site
and
metabolic
resistance.
A
newly
investigated
and
proposed
mechanism
concerns
of
the
thickening
and
the
composition
alteration
of
the
leg
cuticle,
resulting
in
slower
rates
of
insecticide
penetration.
According
to
previously
published
data,
cuticle
analysis
indicated
that
resistant
mosquitoes
had
a
thicker
epicuticular
layer
and
a
significant
increase
in
cuticular
hydrocarbon
content.
Furthermore,
in
a
very
recent
study
(Balabanidou
et
al,
unpublished),
the
leg
proteome,
analyzed
by
gel
based
proteomics,
consisted
of
1,120
proteins,
with
energy
production
and
lipid
metabolism
proteins
being
enriched.
Comparative
proteomic
analysis
between
legs
from
insecticide
resistant
and
susceptible
Anopheles
gambiae
mosquitoes,
showed
that
the
most
up-‐regulated
proteins
were
the
structural
chitin-‐binding
cuticular
proteins,
in
agreement
with
a
leg-‐thickening
phenotype.
It
was
hypothesized
that
this
differential
expression
of
these
proteins
may
play
a
role
in
the
chitin
biosynthetic
pathway,
as
chitin
is
closely
associated
with
them.
In
the
present
study,
CPR125,
CPR140,
CPR151
and
the
CPCFC1
cuticular
proteins
were
confirmed
over-‐
expressed
in
resistant
legs
validating
the
proteomic
data.
Immunohistochemical
analysis
of
CPR125
and
CPCFC1
proteins,
revealed
an
overexpression
in
endocuticle
of
resistant
legs,
in
contrast
to
the
susceptible
ones.
In
addition,
chitin
content
was
successfully
measured
in
the
legs
and
found
increased
in
resistant
mosquitoes.
Finally,
key
enzymes
of
the
chitin
biosynthetic
pathway
(Chitin
Synthase
1
and
Trehalose-‐6-‐
phosphate
phosphatase)
were
found
up-‐regulated,
indicating
a
possible
up-‐regulation
of
the
chitin
biosynthesis
pathway.
These
changes
in
the
resistant
Anopheles
gambiae
leg,
at
cuticular
protein
and
chitin
biosynthesis
level,
could
possibly
be
related
to
the
resistant
phenotype
and
penetration
resistant
mechanism.
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