Identification and transcription profiling of NDUFS8 in Aedes taeniorhynchus (Diptera: Culicidae): developmental regulation and environmental response

Figures & data

Figure 1 A phylogenetic tree for NDUFS8 orthologs.

Notes: The phylogenetic tree was constructed using the Neighbor-joining tree-making method for NDUFS8 nucleic acid sequences of NADH dehydrogenase-ubiquinone Fe-S protein 8 subunit from other orthologues using the MEGA 5.05 program. The scale bar indicates the number of changes inferred as having occurred along each branch. The accession number of nucleic acid sequences of NDUFS8 orthologues used in this analysis are FJ458415.1, AY432654.1, AY440457.1, XM_001868794, XM-321378.5, XM_0021032227.1, XM_002030974, NM_0799980.3, XM_001980072.1, XM_002097653, XM_001990316, XM_002016993, XM_001954946, XM_002056337.1, XM_001998624.1, XM_002074083.1, BX933502.2, XM_002755626.1, XM_002400792, NM_002496.3, NM_0011131881.1, XM_003468313, BT075206.1, CR711559.2, XM_0031222435.1, XM_002921294.1, DQ885653.1, NM_001071780.1, M58717.1, DQ885652.1, XM_001104103.2, AB125184.1, XM_003206148.1, and XM_002196863.1.

Figure 2 AetNDUFS8 mRNA expression levels in eggs, larvae, pupae, and adults quantified by qPCR, with SD for three replicates.

Notes: Ages of eggs, 1 d, and 3 d, respectively; First instar, 5, 21, 29, 44, and 53 h posthatch, respectively; second instar, 69, and 77 h posthatch respectively; third instar, 93, and 101 h posthatch, respectively; fourth instar, 117, 125, 141, and 149 h posthatch, respectively; pupae, 165, and 173 h posthatch, respectively; and adults, and 1 d old male, (M) designated male, (ie, 8 d posthatch); 5 d old male (ie, 13 d posthatch); and 10 d old male (ie, 18 d posthatch); and 1 d old female, (F) designated female (ie, 8 d posthatch); 5 d old female (ie,13 d posthatch); and 10 d old female (ie, 18 d posthatch).
Abbreviations: d, day; h, hours; mRNA, messenger RNA, qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

Figure 3 AetNDUFS8 mRNA expression levels in 5 d old female treated topically with permethrin/acetone at 2.5×10⁻⁵ μg (high dose, HD), and 1.25×10⁻⁵ μg (low dose, LD) per mosquito, and acetone (Ac) quantified by qPCR, with standard deviation (SD) for three replicates. Please note X-axis is not to scale. Five-day-old adult postexposure to permethrin at 0, 5, 15, 30, 60, 180, and 360 min, and 24 h.

Abbreviations: d, day; h, hours; min, minutes; mRNA, messenger RNA; qPCR, quantitative real-time polymerase chain reaction.

Figure 4 AetNADH5 mRNA expression levels in eggs, larvae, pupae, and adults quantified by qPCR, with SD for three replicates.

Notes: Ages of eggs, 1 d, and 3 d, respectively; First instar, 5, 21, 29, 44, and 53 h posthatch, respectively; second instar, 69, and 77 h posthatch respectively; third instar, 93, and 101 h posthatch, respectively; fourth instar, 117, 125, 141, and 149 h posthatch, respectively; pupae, 165, and 173 h posthatch, respectively; and adults, and 1 d old male, (M) designated male, (ie, 8 d posthatch); 5 d old male (ie, 13 d posthatch); and 10 d old male (ie, 18 d posthatch); and 1 d old female, (F) designated female (ie, 8 d posthatch); 5 d old female (ie,13 d posthatch); and 10 d old female (ie, 18 d posthatch).
Abbreviations: d, day; h, hours; mRNA, messenger RNA; qPCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

Table S1 Expression of AetNDUFS8 in different developmental stages of Aedes taeniorhynchus

Sample stage	Sample name	Sample time	Mean cycle threshold (Ct) ± SD		Relative AetNDUFS8 expression level
			rR40s	AetNDUFS8	ΔCt-1	ΔCt-2	ΔCt-3	100×2−∆^Ct ± SD
Eggs	Egg 1	1 d	14.089±0.041	19.156±0.045	5.064	5.069	5.067	2.984±0.071
	Egg 2	3 d	14.840±0.013	19.509±0.039	4.669	4.649	4.688	3.931 ±0.074
1st instar larvae	Larvae 1	5 h^a	11.588±0.209	17.044±0.127	5.398	5.515	5.462	2.279±0.132
	Larvae 2	21 h^a	10.852±0.101	16.585±0.143	5.704	5.763	5.733	1.880±0.054
	Larvae 3	29 h^a	12.815±0.182	18.121 ±0.038	5.461	5.149	5.305	2.544±0.386
	Larvae 4	44 h^a	12.815±0.182	17.532+0.057	5.093	5.242	5.167	2.787±0.203
	Larvae 5	53 h^a	13.060±0.115	17.326±0.023	4.331	4.199	4.265	5.205±0.334
2nd instar larvae	Larvae 6	69 h^a	12.933±0.012	17.211 ±0.111	4.192	4.365	4.278	5.162±0.439
	Larvae 7	77 h^a	13.100±0.055	17.310±0.036	3.919	3.939	3.929	6.564±0.062
3rd instar larvae	Larvae 8	93 h^a	12.339±0.034	16.054±0.005	3.742	3.726	3.688	7.617±0.202
	Larvae 9	101 h^a	12.427±0.021	16.148±0.013	3.725	3.715	3.720	7.586±0.039
4th instar larvae	Larvae 10	117 h^a	12.386±0.034	16.131±0.033	3.747	3.742	3.744	7.459±0.015
	Larvae 11	125 h^a	13.228±0.060	17.005±0.077	3.789	3.765	3.777	7.295±0.086
	Larvae 12	141 h^a	13.845±0.064	17.803±0.129	3.821	4.095	3.958	6.464±0.864
	Larvae 13	149 h^a	14.210±0.007	15.316±0.044	4.211	4.216	4.214	5.390±0.013
Pupae	P1	165 h^a	14.191 ±0.020	18.717±0.011	4.518	4.534	4.526	4.341 ±0.034
	P3	173 h^a	15.216±0.026	19.568±0.043	4.401	4.303	4.352	4.901 ±0.235
Adults	A1 (M)^b	1 d	15.887±0.021	17.480±0.048	1.545	1.642	1.593	33.158± 1.573
	A2 (M)^b	5 d	18.641 ±0.076	21.423±0.034	2.811	2.751	2.781	14.549±0.426
	A3 (M)^b	10 d	17.961 ±0.055	21.257±0.190	3.200	3.391	3.296	10.204±0.954
	A1(F)^c	1 d	15.787±0.073	18.637±0.226	2.959	2.742	2.850	13.904± 1.469
	A2 (F)·	5 d	15.273±0.026	19.226±0.006	3.976	3.931	3.953	6.4571±0.143
	A3 (F)·	10 d	15.987±0.039	20.389±0.046	4.397	4.407	4.402	4.729±0.023

Notes:

a Hours post hatch;

b males;

c females.

Abbreviations: d, day; F, female; M, male; SD, standard deviation.

Table S2 Paired t-test data for comparison of relative AetNDUFS8 gene transcription between female (F) and male (M), as well as different ages between the same sex (either female or male) in Aedes taeniorhynchus

Sexes and ages	N	df	t-value	P-value
F1-d and M1-d	3	2	−15.841	0.004*
F5-d and M5-d	3	2	−70.216	<0.001*
F10-d and M10-d	3	2	−14.410	0.005*
F1-d and F5-d	3	2	14.583	0.005*
F1-d and F10-d	3	2	15.851	0.004*
M1-d and M5-d	3	2	22.810	0.002*
M1-d and M10-d	3	2	90.879	<0.001*

Note:

* Statistical significance (P<0.05).

Abbreviations: d, day; df, degrees of freedom.

Table S3 Paired t-test data for comparison of relative AetNDUFS8 gene transcription between eggs, larvae, pupae, and adults in Aedes taeniorhynchus

Stage and ages	N	df	t-value	P-value
Egg I and egg 3	3	2	−34.939	<0.001*
Egg 3 and larvae 1	3	2	−71.951	<0.001*
Larvae 1 and larvae 2	3	2	12.861	0.006*
Larvae 2 and larvae 3	3	2	3.691	0.066
Larvae 3 and larvae 4	3	2	−1.012	0.418
Larvae 4 and larvae 5	3	2	11.008	0.008*
Larvae 5 and larvae 6	3	2	0.136	0.904
Larvae 6 and larvae 7	3	2	9.026	0.012*
Larvae 7 and larvae 8	3	2	−9.897	0.010*
Larvae 8 and larvae 9	3	2	−0.462	0.689
Larvae 9 and larvae 10	3	2	12.522	0.006*
Larvae 10 and larvae 11	3	2	−5.675	0.030*
Larvae 11 and larvae 12	3	2	2.139	0.166
Larvae 12 and larvae 13	3	2	−3.088	0.091
Larvae 13 and pupae 1	3	2	120.587	<0.001*
Pupae 1 and pupae 2	3	2	5.095	0.036*
Pupae 2 and M1-d	3	2	−17.856	0.003*
Pupae 2 and F1-d	3	2	−38.296	<0.001*

Note: *Statistical significance (P<0.05).

Abbreviations: d, day; df, degrees of freedom, F, female; M, male.

Table S4 Expression of AetNDUFS8 under permethrin stress conditions in Aedes taeniorhynchus

Time point	Mean cycle threshold (Ct) ± SD		Relative AetNDUFS8 expression level
	rR40s	AetNDUFS8	ΔCt-1	ΔCt-2	ΔCt-3	100×2−∆^Ct ± SD
Ac-0 min^a	13.287±0.022	17.822+0.063	4.374	4.697	4.536	4.3117+0.685
Ac-5 min	13.687±0.064	17.879+0.040	4.175	4.209	4.191	5.4717+0.091
Ac-15 min	12.679±0.153	17.526+0.289	4.751	4.943	4.847	3.4826+0.326
Ac-30 min	12.647±0.077	17.325+0.120	4.538	4.817	4.678	3.9255+0.535
Ac-1 h	12.217±0.012	17.296+0.136	4.973	5.183	5.078	2.9678+0.305
Ac-3 h	12.693±0.290	17.191 +0.025	4.686	4.311	4.498	4.4627+0.816
Ac-6 h	13.229±0.261	16.688+0.072	3.591	3.325	3.458	9.1385+1.186
Ac-24 h	11.654±0.120	17.045+0.194	5.176	5.605	5.390	2.4106+0.504
LD-0 min^b	13.285±0.027	17.721+0.037	4.374	4.497	4.436	4.6212+0.280
LD-5 min	13.458±0.174	17.761+0.137	4.328	4.277	4.303	5.0686+0.127
LD-15 min	13.085+0.042	17.292+0.223	4.079	4.336	4.208	5.4341+0.681
LD-30 min	13.213±0.036	18.355+0.015	5.127	5.157	5.142	2.8318+0.042
LD-1 h	13.233±0.013	17.153+0.109	3.833	4.006	3.919	6.6212+0.561
LD-3 h	13.389±0.012	17.251+0.064	3.825	3.899	3.862	6.8820+0.249
LD-6 h	12.497±0.007	17.174+0.039	4.655	4.699	4.677	3.9098+0.086
LD-24 h	12.172+0.084	16.181 +0.010	3.943	4.076	4.010	6.2151+0.404
HD-0 min^c	13.086±0.119	17.721+0.047	4.473	4.797	4.636	4.0229+0.639
HD-5 min	11.385+0.237	15.483+0.127	4.356	3.840	4.098	5.9338+1.483
HD-15 min	12.164+0.086	16.093+0.093	3.802	4.056	3.929	6.5909+0.817
HD-30 min	12.098+0.099	16.956+0.082	4.730	4.987	4.858	3.4611+0.434
HD-1 h	12.621 ±0.071	16.749+0.012	4.087	4.170	4.128	5.7196+0.234
HD-3 h	12.120+0.091	16.809+0.023	4.769	4.609	4.689	3.8822+0.306
HD-6 h	12.770+0.175	17.057+0.081	4.354	4.221	4.287	5.1275+0.334
HD-24 h	12.639+0.025	17.875+0.221	5.105	5.369	5.237	2.6631+0.343

Notes:

a Acetone treatments in 5 d old female Ae. taeniorhynchus;

b pemethrin LD treatment in 5 d old female Ae. taeniorhynchus;

c pemethrin HD treatment in 5 d old female Ae. taeniorhynchus.

Abbreviations: Ac, acetone; h, hours; min, minutes; SD, standard deviation; LD, low dose; HD, high dose.

Table S5 Paired t-test data for comparison of relative AetNDUFS8 gene transcription between acetone, low dose (LD), and high dose (HD) permethrin treatments in Aedes taeniorhynchus

Permethrin treatments	N	df	t-value	P-value
LD and AC 0 min	3	2	−1.776	0.218
LD and AC 5 min	3	2	8.200	0.015*
LD and AC 15 min	3	2	−13.405	0.006*
LD and AC 30 min	3	2	11.273	0.008*
LD and AC 1 h	3	2	−34.620	<0.001*
LD and AC 3 h	3	2	−5.586	0.031*
LD and AC 6 h	3	2	10.039	0.010*
LD and AC 24 h	3	2	−94.119	<0.001*
HD and AC 0 min	3	2	−16.233	<0.004*
HD and AC 5 min	3	2	−0.666	0.574
HD and AC 15 min	3	2	−15.466	0.004*
HD and AC 30 min	3	2	−11.273	0.008*
HD and AC 1 h	3	2	−88.490	<0.001*
HD and AC 3 h	3	2	2.722	0.113
HD and AC 6 h	3	2	11.467	0.008*
HD and AC 24 h	3	2	−3.938	0.059
LD and HD 0 min	3	2	3.943	0.059
LD and HD 5 min	3	2	−1.515	0.269
LD and HD15 min	3	2	−20.865	0.002*
LD and HD 30 min	3	2	5.394	0.033*
LD and HD 1 h	3	2	6.700	0.022*
LD and HD 3 h	3	2	13.264	0.006*
LD and HD 6 h	3	2	−7.119	0.019*
LD and HD 24 h	3	2	141.984	<0.001*

Note: *Statistical significance (P<0.05).

Abbreviations: AC, acetone; df, degrees of freedom, h, hours; min, minutes.

Table S6 Expression of AetNADH5 in different developmental stages of Aedes taeniorhynchus

Sample stage	Sample name	Sample time	Mean cycle threshold (Ct) ± SD		Relative AetNADH5 expression level
			rR40s	AetNADH5	ΔCt-1	ΔCt-2	ΔCt-3	100×2−∆^Ct ± SD
Eggs	Egg 1	1 day	12.849±0.101	9.079±0.054	−3.925	−3.615	−3.769	1364.1 ± 146.9
	Egg 2	3 day	13.624±0.120	9.065±0.041	−3.716	−3.636	−3.676	1278.1 ±35.84
1st instar larvae	Larvae 1	5 h^a	10.512±0.447	10.051 ±0.151	−0.310	−0.612	−0.461	137.66± 14.43
	Larvae 2	21 h^a	10.676±0.333	10.458±0.269	−0.154	−0.282	−0.218	116.28±5.156
	Larvae 3	29 h^a	11.406±0.084	11.198±0.037	−0.293	−0.198	−0.245	118.52t3.911
	Larvae 4	44 h^a	9.1631 ±0.908	8.8999±0.162	−0.101	−0.425	−0.263	120.01 ± 13.53
	Larvae 5	53 h^a	11.542±0.112	9.3146±0.092	−2.048	−2.027	−2.228	468.37±6.672
2nd instar larvae	Larvae 6	69 h^a	11.375±0.098	9.3639±0.074	−1.988	−2.035	−2.011	403.18±6.582
	Larvae 7	77 h^a	11.769±0.029	9.143±0.003	−2.660	−2.591	−2.626	617.16± 14.74
3rd instar larvae	Larvae 8	93 h^a	10.874±0.057	7.909±0.099	−3.006	−2.924	−2.965	780.74±22.32
	Larvae 9	101 h^a	10.719±0.388	7.929±0.065	−2.856	−2.724	−2.789	691.51 ±31.63
4th instar larvae	Larvae 10	117 h^a	10.245±0.259	7.675±0.118	−2.451	−2.687	−2.569	593.58±48.66
	Larvae 11	125 h^a	11.198±0.034	8.122±0.027	−3.045	−3.101	−3.076	843.54± 18.27
	Larvae 12	141 h^a	12.446±0.069	10.019±0.018	−2.514	−2.339	−2.427	537.61 ±32.46
	Larvae 13	149 h^a	12.835±0.092	11.022+0.008	−1.913	−1.712	−1.813	351.25±24.49
Pupae	P1	165 h^a	13.134±0.003	11.055±0.069	−2.011	−2.146	−2.078	422.33±19.69
	P3	173 h^a	13.962±0.124	9.167±0.012	−4.658	−4.931	−4.795	2775.9±236.1
Adults	A1 (M)^b	1 day	12.939±0.073	8.079±0.197	−5.056	−4.661	−4.859	2903.4±398.4
	A2 (M)^b	5 day	16.684±0.019	12.056±0.013	−4.634	−4.622	−4.628	2472.7± 10.71
	A3 (M)^b	10 day	15.954±0.045	13.012±0.196	−3.138	−2.746	−2.942	768.64±104.9
	A1(F)^c	1 day	13.845±0.027	9.221 ±0.359	−4.958	−4.292	−4.625	2467.0±575.6
	A1 (F)^c	5 day	14.752±0.538	11.145±0.083	−3.690	−2.985	−3.607	1218.4±269.8
	A2 (F)^c	10 day	13.838±0.006	11.252±0.095	−2.674	−2.497	−2.587	600.26±37.06

Notes:

a hours post hatch;

b males;

c females.

Abbreviations: h, hours; F, female; M, male; SD, standard deviation.

Table S7 Paired t-test data for comparison of relative AetNADH5 gene transcription between female (F) and male (M), as well as different ages between the same sex (either female or male) in Aedes taeniorhynchus

Sexes and ages	N	df	t-value	P-value
F1-d and M1-d	3	2	−3.989	0.057
F5-d and M5-d	3	2	−8.497	<0.014*
F10-d and M10-d	3	2	−4.399	0.048*
F1-d and F5-d	3	2	6.908	0.020*
F1-d and F10-d	3	2	6.142	0.025*
M1-d and M5-d	3	2	1.999	0.184
M1-d and M10-d	3	2	12.669	0.006*

Note: *Statistical significance (P<0.05).

Abbreviations: d, days; df, degrees of freedom.

Table S8 Paired t-test data for comparison of relative AetNADH5 gene transcription between eggs, larvae, pupae, and adults in Aedes taeniorhynchus

Stage and ages	N	df	t-value	P-value
Egg 1 and egg 3	3	2	−1.175	0.361
Egg 3 and larvae1	3	2	39.295	<0.001*
Larvae 1 and larvae 2	3	2	3.276	<0.022*
Larvae 2 and larvae 3	3	2	6.000	0.438
Larvae 3 and larvae 4	3	2	−2.242	0.075
Larvae 4 and larvae 5	3	2	12.000	0.844
Larvae 5 and larvae 6	3	2	−2.242	0.031*
Larvae 6 and larvae 7	3	2	−28.819	0.001*
Larvae 7 and larvae 8	3	2	14.259	0.005*
Larvae 8 and larvae 9	3	2	5.385	0.033*
Larvae 9 and larvae 10	3	2	2.394	0.139
Larvae 10 and larvae 11	3	2	−14.178	0.005*
Larvae 11 and larvae 12	3	2	10.427	0.009*
Larvae 12 and larvae 13	3	2	40.517	<0.001*
Larvae 13 and pupae 1	3	2	−2.776	0.109
Pupae 1 and pupae 2	3	2	−28.885	0.001*
Pupae 2 and M1-d	3	2	−0.526	0.652
Pupae 2 and F1-d	3	2	0.933	0.449

Note: *Statistical significance (P<0.05).

Abbreviations: d, day; df, degrees of freedom.