Composition of An. gambiae s.l. (Tiassalé strain) mosquitoes

Species identification showed that An. gambiae s.l. samples used in the study consisted of two species; An. gambiae s.s. (26.53%) and An. coluzzii (23.47%). A hybrid of the two species constituted 50.00% (Table 2).

Table 2 Composition of An. gambiae s.l. mosquitoes

WHO insecticide susceptibility

Mortality in An. gambiae s.l. mosquitoes after exposure to pyrethroids

The results of insecticide susceptibility of An. gambiae s.l. (Tiassalé) to deltamethrin and permethrin insecticides are presented in Fig. 1. Adult mosquitoes that emerged at 36 °C died within the first 24 h post-emergence, and no adults emerged at 38 or 40 °C. Therefore, bioassay and molecular tests were restricted to only adults that emerged from 25, 28, 30, 32, and 34 °C. Mortality in the control replicates at 32 °C exceeded 5%; therefore, mortality was corrected, consistent with prescribed guidelines using Abbott’s formula [43].

Fig. 1
figure 1

Insecticide susceptibility of An. gambiae s.l. (Tiassalé strain) mosquitoes reared at different temperature regimes

Overall, deltamethrin insecticides induced higher mortality compared with permethrin insecticides irrespective of the temperature regime. Female mosquitoes showed high mortality (100%) to deltamethrin at 28 °C and mortality decreased at temperature above 28 °C (Fig. 1). Upon exposure to permethrin, mortality in An. gambiae s.l. (Tiassalé) mosquitoes decreased with increasing temperature from 28 (81.61%) to 32 °C (43.06%). All susceptible An. gambiae s.l. (Kisumu) mosquitoes exposed to permethrin and deltamethrin insecticides died irrespective of the rearing temperature (Additional file 1:Table S2).

Knockdown resistance ratio

The time at which 50% of the mosquitoes were knocked down (KDT50) after exposure to insecticides was assessed under different temperature regimes, 25, 28, 30, 32, and 34 °C. The KDT50 of An. gambiae s.l. (Tiassalé) mosquitoes exposed to both permethrin and deltamethrin insecticides decreased with increasing temperature from 25 to 34 °C. For mosquitoes raised at 25 °C and exposed to permethrin insecticides, the KDT50 was higher (888.70 min) than in the other temperature regimes (Table 3). Furthermore, the KRR based on KDT50 for deltamethrin decreased with increasing temperature from 25 to 32 °C. With permethrin, the resistance ratio was highest at 25 °C (51.31), followed by 32 °C (4.12), 28 °C (3.87), and 30 °C (3.59) (Table 3). Generally, all the mosquitoes in this study had developed a certain level of resistance to permethrin and deltamethrin insecticides, which enabled them to survive the knockdown effect for some time.

Table 3 Knockdown resistance ratio of An. gambiae s.l. mosquitoes (Tiassalé strain) at different rearing temperature regimes

Influence of temperature and insecticide on the expression of metabolic enzymes

MFO level

The level of MFO was assessed in mosquitoes reared at 25, 28, 30, 32, and 34 °C and not exposed to pyrethroids. The results showed that the MFO level was more elevated in mosquitoes reared at 32 °C [4.55 × 10–9 (IQR, 4.13 × 10–9) mol cytochrome P450/min/mg protein] compared to those reared at 34 °C [1.94 × 10–9 (IQR, 3.80 × 10–10)], 30 °C [1.49 × 10–9 (IQR, 2.26 × 10–9)], 28 °C [8.85 × 10–10 (IQR, 1.50 × 10–10)], and 25 °C [7.21 × 10–10 (IQR, 1.43 × 10–10) mol cytochrome P450/min/mg protein] (Additional file 1: Table S3). The levels decreased in the order of 32 > 34 > 30 > 28 > 25 (Fig. 2). Generally, levels of oxidase increased significantly (Kruskal–Wallis H-test, H = 144.42, df = 4, P < 0.001) with increasing temperature. Dunn’s multiple range test showed that all but 30 vs 28 °C (P = 0.113) and 34 vs 30 °C (P = 0.010) showed a statistically significant difference in oxidase level (Additional file 1: Table S4).

Fig. 2
figure 2

Median MFO level in An. gambiae s.l. mosquitoes reared at different temperature regimes. NB: no mosquito reared at 34 °C survived after being exposed to pyrethroids; hence, no enzyme level was measured

The effect of rearing temperature on MFO level was also assessed in mosquitoes that survived after being exposed to pyrethroids. Mosquitoes reared at 30 °C had higher [3.15 × 10–9 (IQR, 6.40 × 10–10) mol cytochrome P450/min/mg protein] levels compared to those reared at 32 °C [2.40 × 10–9 (IQR, 1.07 × 10–9)], 25 °C [2.07 × 10–9 (IQR, 7.50 × 10–10)], and 28 °C [1.34 × 10–9 (IQR, 2.50 × 10–10) mol cytochrome P450/min/mg protein] (Fig. 2). There was a significant difference (Kruskal–Wallis H-test, H = 68.18, df = 3, P < 0.001) in MFO levels among the different temperature regimes. Further tests using Dunn’s multiple range test showed a significant difference (P < 0.008) in the various temperature regime comparisons (Additional file 1: Table S4).

The MFO levels in mosquitoes reared at 25 °C (Mann–Whitney U-test, z = −7.72, P < 0.001), 28 °C (Mann–Whitney U-test, z = −5.33, P < 0.001), and 30 °C (Mann–Whitney U-test, z = −4.68, P < 0.001) and not exposed to pyrethroids were significantly lower than those that were exposed to pyrethroids. However, mosquitoes reared at 32 °C and not exposed to pyrethroids had significantly higher (Mann–Whitney U-test, z = 5.12, P < 0.001) MFO levels than those exposed to pyrethroids (Fig. 2).

GST level

The level of GST enzyme was assessed in mosquitoes reared at 25, 28, 30, 32, and 34 °C and not exposed to pyrethroids. The results showed that the GST enzyme level followed a trend (32 > 34 > 30 > 28 > 25) similar to that of the MFO level (Fig. 3). Statistically, there was a significant difference (Kruskal–Wallis H-test, H = 89.06, df = 4, P < 0.001) in the GST levels in mosquitoes among the different temperature regimes. However, further analysis using Dunn’s multiple range test showed statistically significant differences for 32 vs 25 °C (P < 0.001), 34 vs 25 °C (P = 0.002), 32 vs 28 °C (P < 0.001), 32 vs 30 °C (P < 0.001), and 34 vs 32 °C (P < 0.001) (Additional file 1: Table S4).

Fig. 3
figure 3

Median GST level in An. gambiae s.l. mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 °C survived after being exposed to pyrethroids; hence, no enzyme level was measured

With mosquitoes that were exposed to pyrethroids, the level of GST was highest at 32 °C [3.55 × 10–3 (IQR, 2.63 × 10–3)], followed by 25 °C [2.72 × 10–3 (IQR, 1.56 × 10–3)], 30 °C [2.62 × 10–3 (IQR, 1.69 × 10–3)] and the lowest recorded at 28 °C [1.35 × 10–3 (IQR, 6.59 × 10–4) mol cDNB/min/mg protein] (Additional file 1: Table S3). There was a statistically significant difference (Kruskal–Wallis H-test, H = 18.26, df = 3, P < 0.001) in the GST level in mosquitoes among the different temperature regimes. Further statistical tests using Dunn’s multiple range test showed statistically significant differences for 28 vs 25 °C (P = 0.002), 32 vs 28 °C (P < 0.001), and 32 vs 30 °C (P = 0.005) (Additional file 1: Table S4). In addition, mosquitoes in the temperature regimes exposed to pyrethroids (except for those reared at 32 °C) had higher GST levels compared to those that were not exposed to pyrethroids (Fig. 3). However, all but mosquitoes reared at 28 °C (Mann–Whitney U-test, z = −0.52, P = 0.605) showed a statistically significant difference in GST levels (Additional file 1: Table S5).

Alpha-esterase level

The level of α-esterase enzyme was assessed in mosquitoes reared at 25, 28, 30, 32, and 34 °C and not exposed to pyrethroids. The highest median α-esterase level was recorded at 32 °C [1.32 × 10–6 (IQR, 9.41 × 10–7)], followed by 34 °C [4.04 × 10–7 (IQR, 1.56 × 10–7)], 28 °C [2.83 × 10–7 (IQR, 4.32 × 10–7)], 25 °C [2.52 × 10–7 (IQR, 7.30 × 10–8)] and 30 °C [2.12 × 10–7 (IQR, 4.10 × 10–7) mol α-naphthol/min/mg protein] (Additional file 1: Table S3). Median α-esterase level did not increase with increasing rearing temperature; however, there was a statistically significant difference (Kruskal–Wallis H-test, H = 99.46, df = 4, P < 0.001) in α-esterase level in mosquitoes among the different temperature regimes. Further statistical tests using Dunn’s multiple range test showed a statistically significant difference for 32 vs 25 °C (P < 0.001), 34 vs 25 °C (P < 0.001), 32 vs 28 °C (P < 0.001), 32 vs 30 °C (P < 0.001), 34 vs 30 °C (P < 0.001), and 34 vs 32 °C (P < 0.001) (Additional file 1: Table S4).

The level of α-esterase in mosquitoes that were exposed to pyrethroids was also assessed. The results showed that α-esterase level was highest at 32 °C [3.13 × 10–7 (1.81 × 10–7)] and lowest at 28 °C [1.44 × 10–7 (2.40 × 10–7) mol α-naphthol/min/mg protein] (Additional file 1: Table S3). There was a significant difference (Kruskal–Wallis H-test, H = 11.31, df = 3, P = 0.010) in α-esterase levels in mosquitoes among the different temperature regimes. According to Dunn’s multiple range tests, a statistical difference existed only for 30 vs 28 °C (P = 0.003) and 32 vs 28 °C (P = 0.001) (Additional file 1: Table S4). Overall, mosquitoes that were not exposed to pyrethroids had higher α-esterase levels than those exposed to pyrethroids (Fig. 4). A Mann–Whitney U-test showed that all but mosquitoes reared at 30 °C (z = −1.53, P = 0.127) showed a statistically significant difference in α-esterase level (Additional file 1: Table S5).

Fig. 4
figure 4

Median α-esterase level in An. gambiae s.l. mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 °C survived after being exposed to pyrethroids; hence, no enzyme level was measured

Beta-esterase level

The level of the β-esterase enzyme was assessed in mosquitoes reared at 25, 28, 30, 32, and 34 °C and not exposed to pyrethroids. The level decreased in the order of 34 °C [3.56 × 10–7 (IQR, 1.54 × 10–7)] > 32 °C [2.87 × 10–7 (IQR, 3.21 × 10–7)] > 28 °C [1.38 × 10–7 (IQR, 1.82 × 10–7)] > 25 °C [1.36 × 10–7 (IQR, 6.10 × 10–8)] > 30 °C [1.30 × 10–7 (IQR, 2.93 × 10–7) mol β-naphthol/min/mg protein] (Additional file 1: Table S3). Generally, there was a statistically significant increase (Kruskal–Wallis H-test, H = 48.28, df = 4, P < 0.001) in β-esterase levels in mosquitoes with increasing temperature. However, post hoc analysis using Dunn’s multiple range test showed a statistically significant difference for 32 vs 25 °C (P = 0.001), 34 vs 25 °C (P < 0.001), 34 vs 28 °C (P < 0.001), 34 vs 30 °C (P < 0.001), and 34 vs 32 °C (P = 0.003) (Additional file 1: Table S4).

With regard to mosquitoes that were exposed to pyrethroids, β-esterase levels ranged from 1.11 × 10–7 (6.85 × 10–8) at 28 °C to 2.64 × 10–7 (1.63 × 10–7) mol β-naphthol/min/mg protein at 32 °C (Additional file 1: Table S3). There was a statistically significant difference (Kruskal–Wallis H-test, H = 37.91, df = 3, P = 0.001) in β-esterase levels in mosquitoes raised at different temperatures. Multiple comparisons using Dunn’s test showed statistically significant differences for 28 vs 25 °C (P < 0.001), 30 vs 28 °C (P < 0.001), 32 vs 28 °C (P < 0.001), and 32 vs 30 °C (P = 0.007) (Additional file 1: Table S4). Furthermore, β-esterase levels in mosquitoes reared at 25 and 30 °C and exposed to pyrethroids were higher than in those that were not exposed to pyrethroids. On the other hand, β-esterase level in mosquitoes that were not exposed to pyrethroids was higher than in those that were raised at 28 and 32 °C and were exposed to pyrethroids (Fig. 5). However, only mosquitoes reared at 25 °C showed a statistically significant difference (Mann–Whitney U-test, z = −6.03, P < 0.001) in β-esterase levels (Additional file 1: Table S5).

Fig. 5
figure 5

Median β-esterase level in An. gambiae mosquitoes reared at different temperature regimes. NB: No mosquito reared at 34 °C survived after being exposed to pyrethroids; hence, no enzyme level was measured

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