REVIEWER 2:
The manuscript has been significantly improved including the English quality; however,
it still has a few issues. I suggest that the manuscript should be accepted after minor
revision.
Major comments:
1) The Material and Methods sections has been greatly improved however, a few issues
still need to be addressed.
- To better explain how the susceptible strain of T. cinnabarinus was reared, I believe
authors should add the following paragraph (provided by the author in the “response to
the reviewer” file).
“The glass slide-dipping method was performed as follows. Pesticides were diluted in
sterile distilled water containing 1 % Tween 80 (Sigma-Aldrich, St. Louis, MO, USA)
and 0.6 % DMSO (Sigma-Aldrich, St. Louis, MO, USA). Sterile distilled water with 1 %
Tween 80 and 0.6 % DMSO was used as the control. Female adult mites were affixed
onto to one end of a 10 × 2 cm glass slide using double-sided adhesive tape. The mites
were individually dipped into pesticides solutions for 5 s. Once the slide was removed,
the extra solution was absorbed with filter paper. All treated mites were maintained at
a temperature of 27 ± 2°C, 60% RH, and a photoperiod of 16:8 h. Mite mortalities were
determined 24 h after treatment. The tested mites were considered dead if they did not
move when touched gently with a camel hair brush”.
Authors’ response: Thank you for your valuable suggestion. This paragraph was added
into the manuscript. Please see the details in the manuscript.
- The number of eggs, larvae, nymphs and adults that were used for the quantitative
RT-PCR still need to be fully explained. In the Manuscript, it is written, “The adult
mites were placed on the leaves. Soon after the females laid their eggs, the adult mites
were removed, and 1000 eggs were collected. After the eggs hatched, 500 pink larvae
with three pairs of legs were collected (Sangeetha et al., 2011). When they had four
pairs of legs, were slightly larger, and reddish (i.e., nymphs), 250 of them were
collected (Sangeetha et al., 2011). Subsequently, 200 female adult mites were collected.”
From that, I understood that the authors used 500 eggs, 250 pink larvae, 50 nymphs and
200 adult females in the qPCR. However in the “Response to the reviewers file”, the
authors said that they used 1000 eggs, 500 pinkish larvae, 250 reddish nymphs and 200
females. Please clarify this issue.
Authors’ response: Thank you for your valuable suggestion, and I am sorry for my
unclear expression. This paragraph was revised to make the meaning more clearly as
follows:
The adult mites were placed on the leaves. Soon after the females laid their eggs,
the adult mites were removed, and 1000 eggs were collected for RNA extraction. Other
eggs hatched, and then 500 pink larvae with three pairs of legs were collected for RNA
extraction (Sangeetha et al., 2011). Other larvae developed into nymphs, which were
slightly larger and reddish with four pairs of legs. 250 of nymphs were collected for
RNA extraction (Sangeetha et al., 2011). Other nymphs subsequently developed into
adults, and 200 female adult mites were collected for RNA extraction. After the samples
were collected, they were immediately frozen in liquid nitrogen and stored at -80ºC for
RNA extraction.
- Still there is not enough information about the quantitative RT-PCR to ensure that it
is a reliable assay (lines 198 – 234). Please add the following information to the text:
Cq (quantification cycle) of the Non-Template Control, calibration curves with slope
and y intercept, PCR efficiency calculated from slope, r2 of the calibration curve, linear
dynamic range.
Authors’ response: Thank you for your valuable suggestion. There is no signal
amplification in the Non-Template Control wells.
The standard curve has a slope of -3.365 and an intercept of 31.82 with an r2 of
0.988. PCR efficiency was calculated as 98.2% from the slope.
-2) Authors give too much emphasis to T. urticae in the discussion section when they
should emphasize T. cinnabarinus, which is the specie they studied. Please rewrite the
third paragraph of the discussion section (lines 344- 381) emphasizing T. cinnabarinus
rather than T. urticae.
Authors’ response: Thank you for your valuable suggestion. This paragraph was
revised as follows:
Tetranychus has been reported to have developed resistance to almost all acaricides
used for its control, and cross-resistance within the organophosphate family is a
common phenomenon in Tetranychus (Voss et al., 1964; Tsagkarakou et al., 1996; Van
et al., 2005). Enhanced detoxification and/or target-site insensitivities have been found
to be responsible for this resistance (Voss et al., 1964; Van et al. 2005). Three of four
AChE1 mutations (F331W, G328A, A201S) in the resistant strains of T. urticae may
be involved in resistance to organophosphate and carbamate insecticides (Khajehali et
al., 2010). A previously study found that F331W was consistently present in recently
resistant spider mite strains from different geographical regions, as well as in a strain
that developed resistance in the late 1960s (Khajehali et al., 2010). The phenylalanine
331 residue is involved in substrate guidance and in the hydrolyzing process by
interacting with the catalytic histidine (H440) (Millard et al., 1999). The 331 position
in AChE1 has been associated with organophosphate resistance in many species, which
has also clearly been illustrated by functional expression (Khajehali et al., 2010). The
T. urticae AChE1 3D structure suggests that G328A substitution may indirectly
influence the catalytic process by affecting position 331 located in the same α-helix
(Khajehali et al., 2010). Similarly, various insect species have been reported to have
resistance-associated target-site mutations. We also identified these resistanceassociated target-site mutations (A235S, G362A, F365W, numbered after the signal
peptide) by comparing AChE amino acid sequences of the insecticide-susceptible
strains of T. cinnabarinus and T. urticae (TKD strain, from Takeda Chemical Industries,
Ltd, Genbank: AAO73450.1) to the organophosphate-resistant strain ATHRos-Pm of
T. urticae. Structural alignment of AChE between the susceptible strains of T. urticae
and T. cinnabarinus, and the organophosphate-resistant strain of T. urticae showed that
these substitutions may modify the catalytic properties of AChE1. The A235, G362 and
F365 (numbered after the signal peptide) sites in AChE of T. cinnabarinus may be also
involved in its resistance to organophosphate and carbamate insecticides. AChE
mutations and their combinations can result in varying levels of resistance to many
organophosphate and carbamate insecticides. Different AChE mutations may be
selected by the insecticides used locally in pest-control programs (Khajehali et al.,
2010).
Minor comments/suggested changes:
Authors’ response: Thank you for your valuable suggestion. We have corrected each
mistakes in the manuscript according to the reviewer’s suggestion.
-Lines 253 - 254: I believe there is something wrong. The amino acid sequence of
Homo sapiens was cited twice. Is this correct? “AChE amino sequence identities were
obtained with Homo sapiens AChE (PDB: 4EY7; identity: 46%), Homo sapiens
AChE (PDB: 1B41; identity: 41%), Drosophila”
Authors’ response: Thank you for your valuable suggestion. We do compare with two
amino sequences of Homo sapiens. Maybe, we should change “AChE amino sequence
identities were obtained with Homo sapiens AChE (PDB: 4EY7; identity: 46%), Homo
sapiens AChE (PDB: 1B41; identity: 41%)” into “AChE amino sequence identities
were obtained with Homo sapiens AChE (PDB: 4EY7, identity: 46%; PDB: 1B41,
identity: 41%)”.
REVIEWER 4:
Comments:
This revised manuscript has an improved level of clarity. The points described in my
previous review were adequately addresses by the authors. I have not specifically
checked the points raised by other reviewers.
I have two suggestions that may improve the clarity of the manuscript:
1. The paragraph beginning at line 412 is long, and has more than one main topic. I
would suggest breaking this paragraph into two or three parts, with a stronger
conclusive paragraph.
Authors’ response: Thank you for your valuable suggestion. This paragraph was
broken into 3 paragraphs, and was revised as follows:
In the past decades, four AChE crystal structures have been elucidated, and an
increasing number of aces have been sequenced (Lang et al., 2012). The amino acid
identities of AChEs from different species and different aces gene types are not high,
but their 3D structures are similar (Lang et al., 2012). In the present study, some
interesting substitutions were found in the active-site gorge and in the sites interacting
with gorge residues of T. cinnabarinus AChE. There were Y477E substitutions at the
choline-binding sites, G154S at the oxyanion hole, F322V at the acyl-binding pocket,
and especially peripheral anionic site substitutions at the entrance of the catalytic pocket,
which may imply different catalytic properties of T. cinnabarinus AChE compared to
human AChE. These differences between T. cinnabarinus AChE and human AChE can
help to elucidate the active-site gorge characterization of target species AChE and nontarget species AChE.
There are also significant differences between the insect and vertebrate AChEs near
the opening of the gorge of AChEs, which are potential targets for designing selective
inhibitors (Lang et al., 2012). A cysteine residue is present at the active sites of insect
AChEs, but absent at active sites of mammalian AChEs. Based on this insect-specific
cysteine residue, a methanethiosulfonate-containing molecule was developed, at 6.0
mM, significantly inhibits AChE activity from nine insect species including greenbug
aphid (Schizaphis graminum), the mosquito vector of malaria (Anopheles gambiae), the
yellow fever mosquito (Aedes aegypti L.), the northern house mosquito (Culex pipiens
L.), cockroaches (Blattella germanica and Periplaneta americana), the flour beetle
(Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis),
the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons) without any
measurable inhibition of human AChE (Pang, 2007; Pang et al., 2009a; Pang et al.,
2009b; Polsinelli et al., 2010). According to this insect-specific cysteine residue, phenyl
methylcarbamate-containing molecules were also screened with 130-fold selectivity for
inhibition of A. gambiae AChE relative to human AChE (Carlier et al., 2008). There
are also structural differences between L. cuprina and human AChE in or near the active
sites, which have led to the identification of 19 previously unknown non-carbamate,
non-organophosphate inhibitors with IC50 values below 200 nM, which are up to 335fold more potent on the L. cuprina enzyme than on the human AChE (Ilg et al., 2011).
Thus, the different sites between the insect and vertebrate AChEs, such as insectspecific cysteine residue, can be used as targets for designing selective inhibitors.
A number of studies have indicated that there are distinct differences between insect
and human AChEs, and mites and human AChEs. These differences may offer new
insights into the insecticide resistance problem and the insecticides toxicity problem to
non-target species.
2. Figure 5 is included for a comparison. I'm having a difficult time
comparing/contrasting the two parts of this figure. I suggest combining both lines on
the same coordinate system, with (perhaps) two differently colored lines so that the
differences are obvious.
Authors’ response: Thank you for your valuable suggestion. Both the T. cinnabarinus
line and Homo sapiens line were showed on the same coordinate system. The figure 5
was revised as follows:
4
Score (Hphob. / Kyte & Doolittle)
3
2
1
0
5
105
205
305
405
505
-1
-2
-3
-4
Position
T. cinnabarinus
Homo sapiens
605