Screening results of tea infusions that attract mosquitoes to lay eggs
A preliminary egg-laying effect test was carried out on teas with different fermentation degrees (nonfermented, fully fermented and postfermented teas) and infusion times (approximately every 3 days) at 5 g/L. The results showed that postfermented tea at 3 d ( t = 6.87, p = 0.002 ), 6 d ( t = 8.43, p = 0.001 ), 9 d ( t = 16.77, p < 0.0001 ), 12 d ( t = 9.64, p < 0.0001 ) and 18 d ( t = 5.95, p = 0.004 ) had statistically significant effects on mosquito-egg laying compared with the control ; the average OAIs at 3, 6, 9, 12, and 18 d were 0 .45, 0 .72, 0 .62, 0.35, 0.74, 0.36, and 0 .38, respectively, and at each time period the OAI was greater than 0.3 (Fig. 3).
For the other two types of tea, there were no significant differences between the experimental group and the control group in oviposition (Fig. 3). These data indicated that postfermented tea infusion is a good botanical infusion candidate for attracting Ae. albopictus oviposition.
Furthermore, we refined the research on the oviposition effect on Ae. albopictus by Liu-pao tea and reduce the concentrations of Liu-pao tea used for this purpose, that is, we examined the doses of Liu-pao tea at 5 g/L, 1.5 g/L and 0.5 g/L at the same time every 7 days. The results showed that, except for the 5 g/L Liu-pao tea soaked for 7 d ( T = 14.37, P < 0.0001 ) and 14 d ( T = 13.65, P < 0.0001 ) and 1.5 g/L Liu-pao tea soaked for 7 d ( T = 7.48, P < 0.0001 ), 14 d ( T = 6.05, P = 0.004 ) and 28 d ( T = 5.15, P = 0.0004 ), there were significant differences between the experimental group and the control group in oviposition (Fig. 4). The average OAI except for those of the treatments with 0.5 g/L and 1.5 g/L soaked for 1d and 0.5 g/L and 1.5 g/L soaked for 21 d, were less than 0.3; the average OAIs of other doses soaked for 28 days at different time points were all greater than 0.3 (Fig. 4). It is worth noting that the average OAIs of 5 g/L Liu-pao tea soaked for 7 d and 14 d were 0.73 and 0.67, respectively, which were higher than the average OAIs of 1.5 g/L Liu-pao tea soaked for 7 d and 0.5 g/L soaked for 14 d. In summary, it can be seen that the higher the concentration of Liu-pao tea up to 5 g/L, the better the effect of attracting Ae. albopictus to lay eggs, and soaking the solution between 7 d and 14 d resulted in the best mosquito-attracting effect.
Furthermore, we tested the effect of soaking 0.5 g/L Liu-pao tea for 28 days on the egg laying of Ae. aegypti and found that compared with the control, soaking for 21 d and 28 d significantly attracted Ae. aegypti to lay eggs. The average OAIs were 0.89 (T = 9.10, P = 0.0008) and 0.63 (T = 6.33, P = 0.003), respectively. Compared with Ae. albopictus, the best soaking time for oviposition attraction in Ae. aegypti was obviously backward (Fig. 5).
Effects of microbes in Liu-pao tea infusion on attracting mosquitoes to lay eggs
Except for the results at 7 d, the average OAI was a slightly higher than 0.3 when comparing the preautoclaved and postautoclaved sterilization results, while at 15 and 21 d, the average OAI with postautoclaved sterilization was higher than the average OAI with the preautoclaved conditions (Fig. 6). Therefore, it can be preliminarily speculated that the bacteria in Liu-pao tea infusion may not be a key factor in attracting mosquitoes to lay eggs, and it may be the odor of the Liu-pao tea infusion or the odor produced by the microbes in it that attracts mosquitoes to lay eggs.
GC-MS identification of putative oviposition volatiles from Liu-pao tea infusion
GC-MS was used to analyze and identify the volatile components in the Liu-pao tea extract, combined with computer retrieval technology to identify the identified compounds, and a total of 223 peaks were separated (Fig. 7). Among them, there were 7 compounds with a relative content greater than 1% in the 0.5 g/L sample soaked for 1 d, 8 compounds with a relative content greater than 1% in the 5 g/L sample soaked for 7 d, and a relative content greater than 1% in the 5 g/L sample soaked for 14 d. There were 8 kinds of compounds, which were classified into carbon and oxygen compounds, alkanes, ketones, benzenes and nitrogen according to their chemical compositions. Carbon dioxide, octamethylcyclotetrasiloxane, toluene and nitrogen were all present in the samples at 3 time points. With further combined analysis of the aromatic substances of Liu-pao tea, it was found that the main aromatic substances linalool and cedrol were also present in the relevant samples. The ion chromatograms of these two substances in Liu-pao tea extracts after 7 days are shown in Fig. 8. The specific information and relative content of these two substances are shown in Table 3.
Table 3
Relative contents of aroma substances in GC-MS samples
Sample number | Linalool oxide (Linalool) % | Cedrol % |
C 1–1 | 0 .0194 | 0. 0 073 |
C 1–2 | 0 .0212 | 0.0085 |
C 1–3 | 0.0336 | 0.0148 |
C 7 − 1 | 0.1465 | 0.0216 |
C 7 − 2 | 0.190 | 0.0335 |
C 7 − 3 | 0.3536 | 0.0389 |
C 14 − 1 | 0.0588 | 0.0279 |
C 14 − 2 | 0.0266 | 0.0133 |
C 14 − 3 | 0.0235 | 0.0135 |
NOTE: C1 was 0.5 g/L soaked for 1 d; C7 was 5 g/L soaked for 7 d; C14 was 5 g/L soaked for 14 d, l-3 correspond to 3 repeats. |
Effects of the main aroma compounds in Liu-pao tea on mosquito oviposition
We tested the effects of cedrol and linalool oxides on Ae. albopictus, and the results showed that when cedrol was added at 1 mg/L, the average OAI was 0.38, indicating that it has a significant effect in attracting Ae. albopictus to lay eggs. However, for each concentration of linalool oxide tested, we did not observe an obvious effect on Ae. albopictus oviposition (Fig. 9).