Studies on the role of cuticular function-related genes in insecticide resistance mechanism in red flour beetles, Tribolium castaneum.

概要

報告番号

※

第

主

号

論

文

の

要

旨

Studies on the role of cuticular function-related genes in
insecticide resistance mechanism in red flour beetles, Tribolium
castaneum.（ 昆 虫 体 内 へ の 殺 虫 剤 の 侵 入 抑 制 に お け る

論文題目

表皮クチクラ関連遺伝子の機能解析）

ONGSIRIMONGKOL Paratthakorn
氏

名

論 文 内 容 の 要 旨
Insect body is covered with the exoskeleton, which consists of epidermal cells and
cuticular layer. The main components of insect cuticle are chitin microfibrils and cuticular
proteins. Chitin is the polymer of N-acetylglucosamine; in chitin biosynthesis in epidermal
cells, UDP-N-acetylglucosamine is incorporated into polymerized chitin by an enzyme called
chitin synthase 1 (CHS1). Cuticular proteins are another components of insect cuticle. A
number of cuticular proteins have been identified and characterized in model insects such as
the fruit fly, mosquitoes and the red flour beetles.
In insect molting, biosynthesis of new cuticle proceeds. After ecdysis (i.e. shedding
the old exoskeleton), newly synthesized cuticular layer undergoes pigmentation and
sclerotization process, which is called tanning. Enzymes such as tyrosine hydroxylase (TH),
dopa decarboxylase (DDC) and laccase 2 (Lac2) have been reported to be involved in the
tanning process. Through the tanning process, insect integument becomes more rigid with
darker color.

In general, insect epidermal cuticle has been regarded to be crucial for defense
against xenobiotics such as entomopathogenic microbes and chemicals. Previous studies
on the red flour beetle Tribolium castaneum revealed that some cuticular proteins are
essential for the defense against the attack of entomopathogenic fungi Beauveria bassiana
(Sirasoonthorn et al., 2021). However, the role of cuticle as the defense against
insecticides has not yet been examined.
In this study, I examined the role of cuticular function-related genes in terms of
the resistance (or defense) against topically applied insecticides, using the red flour beetle
T. castaneum. After knockdown of a gene of interest by RNA interference (RNAi), the

beetles were topically applied with insecticides, and the short-term neurotoxic effects
were evaluated. Their external morphology and the cross sections of the cuticle of
abdominal sternite were also observed. In this study, neonicotinoid insecticides, which
inhibit neurotransmission by acting on nicotinic acetylcholine receptor (nAChR) in
synapses, were used as the test chemicals with the sublethal concentration. This thesis
includes the two studies as described below.
Study 1. Functional analysis of cuticular proteins and Chitin synthase 1 (CHS1) in
the insecticide resistance mechanism in T. castaneum.
RNAi-mediated knockdown of major adult cuticular proteins (CPR4, CPR18, and
CPR27) was performed. By observing cross sections of abdominal sternites, it was
confirmed that CPR knockdown caused thinner and disorganized cuticle. The
knockdown of any of the CPR genes, either singly or in combination, resulted in
enhanced susceptibility of the beetles to insecticides. These results suggested that these
CPRs are indispensable in organizing the insect cuticular structure as the defense
against exogenous insecticides.
Mild knockdown of CHS1 also caused disorganized cuticle. However, it did not
affect the susceptibility of the beetles to the insecticides significantly.
Study 2. Functional analysis of genes in the tanning process in the insecticide
resistance mechanism in T. castaneum.
RNAi-mediated knockdown of genes in the tanning process ( TH, DDC, and Lac2)
was performed. Mild knockdown of TH, DDC, and Lac2 genes resulted in defects in

cuticle coloration as well as disorganized cuticle of the abdominal sternite. However,
it did not affect the susceptibility of the beetles to the insecticides significantly.
In summary, this study revealed that major adult cuticular proteins (CPR4, CPR18,
and CPR27) are indispensable for the defense against exogenous insecticides. These CPRs
have been reported to be essential for the

defense against the attack from

entomopathogenic fungi (Sirasoonthorn et al., 2021). It was concluded that these CPRs
are essential for the defensive function of the cuticle against both insecticides and
microbes. Knockdown of other factors (CHS1, TH, DDC, and Lac2) caused disorganized
cuticle, but did not have obvious effect on the susceptibility of the beetles to the
insecticides. In future studies, it needs to be examined whether more severe knockdown
affects the susceptibility of the beetles to the insecticides. These experiments will help to
elucidate the factors that are essential for the defense of the cuticle against exogenous
insecticides.