Our results showed that in the presence of both light and kairomone the number of Diptera dominated by the Culicidae family was the highest (69% of captured insects) compared to the numbers of other orders of insects trapped in the Ambohidray site. This can be explained by the positive phototropism of nocturnal Diptera in relation to the light of the CDC trap. The results showed that the number of individuals in the control and baited traps is different for insects, mosquitoes and Anopheles spp. In general, a shift of about 1000 individuals separates the two traps. The numbers of individuals recorded in the baited trap are always greater than those of the control trap. This difference is due to the effects of the products tested on the insects present in the trapping areas.
Six mosquito species (Cx. decens, Cx. tritaeniorhynchus, Cx. giganteus, An. coustani, An. squamosus, An. mascarensis) and the Anopheles gambiae complex were captured during this study. This complex consists of eight sibling species of mosquitoes, An. arabiensis, An. bwambae, An. melas, An. merus, An. quadriannulatus, An. gambiae s.s, An. coluzzii and An. amharicus22, that are morphologically indistinguishable23,24. Among these species, An. gambiae s.s and An. arabiensis are known as the principal malaria vectors in the complex25. In this experiment, we suppose that these mosquitoes were the dominant species captured in the complex and especially An. gambiae s.s, known to have a high degree of anthropophilic behavior26, because the traps were placed next to human habitations. In addition, exclusive zoophilic behaviour of An. arabiensis reported in Madagascar seems to support this occurs27. However, this suggestion is not confirmed by molecular identification which was not conducted. On the other hand, it is reported that An. arabiensis (an opportunistic species, predominantly zoophilic) is the species that occur at higher densities among species in the An. gambiae complex in several African countries28,29.
The presence of Cx. decens and Cx. tritaeniorhynchus indicates probably that the site is closer to the forest because they are well-known as forest mosquitoes30. An. squamosus and An. coustani are widely present in this site because of the large areas of paddy fields which are favorable to the development of their larvae31,32. These species were known as zoophilic species33, the presence of livestock, especially zebu in this locality also explains their presence.
The number of insects in the traps declines rapidly depending on the progress of the trapping period, indicating the important relation between sampling, population density and trap efficiency. Our results showed that the numbers of insects and mosquitoes trapped in the control and baited traps decrease progressively based on the trapping session. At the beginning of the experiment, we captured 3714 insects and 2406 mosquito individuals for all the traps. This number decreases to 173 insect individuals and 128 mosquito individuals at the end of the experiment. By comparing the number of mosquitoes caught in the baited traps of different products, the results showed that the number of trapped individuals decreases linearly during the 16 days of capture. In the case of Anopheles spp., this number remains stable until the fourth day of trapping, and then it gradually decreases. A similar observation was recorded for An. gambiae s.l, and An. mascarensis together. Thus, a significant decrease in the mosquito population, Anopheles spp. and vector species was recorded at the site compared to the number of individuals caught in control particularly for malaria vectors (Fig. 5). These results offer an important research avenue on the possibility of selective eradication of one or more dangerous species in a given area from the use of baited attractant traps. But a longitudinal study should be done to measure their long-term effectiveness.
Curve showing the variation in the number of mosquitoes, Anopheles spp. and malaria vectors: An. gambiae s.l + An. mascarensis caught in the baited traps according to the trapping day. Curve in orange shows the number of An. gambiae s.l + An. mascarensis caught in the control traps.
According to the results, the temperature and relative humidity do not vary too much throughout the experiment. This assumes that these factors did not influence trapping in this study. However, the low number of mosquitoes caught for the last two trapping periods may be due to the decrease in temperature. These results were consistent with the findings of Khan et al.34 who reported a negative relationship with temperature, relative humidity and Anopheles abundance in Jeddah, Saudi Arabia. Bashar and Tuno35 reported also not finding any significant correlation between temperatures with mosquito density, but they found a positive association with relative humidity. Other authors found similar findings in their study36,37. In contrast, it was reported by Opayele et al.38 that fluctuations in relative humidity were not significantly correlated with mosquito vectors abundance in Ibadan, Nigeria. Minakawa et al.39 found also that the influence of temperature on mosquito density was significant for malaria vectors in Kenya. In case of our experiment, we were unable to detect a significant relationship between temperature, relative humidity and mosquito density because there was little variations of these factors during the trapping period. However, longitudinal studies on mosquito trappings through different climatic seasons will be important to observe the influence of these abiotic factors on malaria vectors density in Madagascar.
The results showed that the total number of Anopheles spp. recorded in the trap baited with: 4-hydroxycoumarin (10 mg), 4-hydroxycoumarin (10 mg) plus octenol (2 mg) and 4-hydroxycoumarin (10 mg) plus octenol (2 mg) and CO2 is higher than that found in the control. This suggests that these products have an attractant effect on Anopheles spp. compared to other genus of mosquitoes. The effectiveness of the blend of attractants especially in the presence of CO2, a standard attractant, to attract mosquitoes has been reported by other authors40,41,42,43. Sriwichai et al.44 demonstrated that CO2 significantly attracts the Anopheles mosquito by using the CDC light trap. Other studies showed that the use of CO2 as bait is not effective in capturing mosquitoes45,46.
According to our results, traps with 4-hydroxycoumarin (2 mg) has a high number of malaria vectors, An. gambiae s.l and An. mascarensis compared to control. But the percentage is low on the order of 25% or only a quarter of all captured Anopheles spp. A similar observation was also recorded in the case of the blend 4-hydroxycoumarin (10 mg) and octenol (2 mg). This can be explained by the low population density of these mosquitoes and the high abundance of other Anopheles species such as An. squamosus and especially An. coustani in the study site.
Regarding the efficacy of products with respect to Anopheles species, significant attractiveness effect was reported for 4-hydroxycoumarin (2 mg) on An. gambiae s.l but also on An. mascarensis which is a vector of malaria with anthropophilic behavior47,48. However, the average number of captured individuals was quite low in the case of An. mascarensis with 6 individuals per trap for four trapping sessions. By increasing the amount of 4-hydroxycoumarin to 10 mg or 5 times compared to the previous one, this attractiveness effect is statistically no more observed towards the two species mentioned above. However, results show that the difference in the average number of malaria vectors in control trap and bait traps is quite significant if the confidence interval is estimated at 90%. In this case, 4-hydroxycoumarin with a mass of 10 mg has also an attractive effect on An. gambiae s.l, and An. mascarensis. But an increase in the number of repetitions of the tests should be done to have a good reliability of the results.
In this study, octenol at a dose of 10 mg/ml and with a mass deposited of 2 mg did not have a selective attractive effect on An. gambiae s.l, and An. mascarensis. On the opposite it had a synergistic effect with 4-hydoxycoumarin (10 mg) to attract An. gambiae s.l even though the abundance of captured individuals was low. By adding CO2 in this blend, the attractiveness effect on An. gambiae s.l is no longer observed. This suggests that the addition of CO2 in the blend 4-hydroxycoumarin (10 mg) and octenol (2 mg) appears to reduce the selective attractive effect on An. gambiae s.l. This is difficult to explain because of the mean number of captured mosquitoes was very low. It would be useful to test this combination of products in the presence of high density of mosquitoes to really observe its effect.
The number of individuals trapped for each species is different depending on the products. It was shown that the number of An. gambiae s.l, An. mascarensis and An. squamosus obtained with 4-hydroxycoumarin (2 mg) was significantly higher compared to the other products that have been tested. This implies that this product at the used dose and quantity has a kairomonal property on these species. Thus, it is important to determine if the product is closer to a human or animal kairomone. In the case of octenol, it presents itself as an animal kairomone because it attracts much more An. coustani which has a strong tendency to bite animals especially cattle49.
According to the results, the kairomone index for An. gambiae s.l, and An. mascarensis together in the presence of 4-hydroxycoumarin (2 mg) and 4-hydroxycoumarin (10 mg) plus octenol (2 mg) combination was the most higher (KI = 70%) compared to the kairomone index of the other products. In addition, the kairomone index for each species shown in the results was very high with KI > 65% especially for An. mascarensis. This implies that these products have a high selectivity for An. gambiae s.l, and An. mascarensis. A synergistic effect was observed for the blend 4-hydroxycoumarin (10 mg) and octenol (2 mg) in this experiment. We reported similar results with Aedes using the sentinel-type trap20. It seems that it is 4-hydroxycoumarin that plays a major role in synergy because even without octenol the selectivity remains high compared to other products. We assume that octenol simply amplifies the attractively effect, but not selective, in this synergism. Thus, it attracts more mosquitoes and any species of Anopheles especially An. coustani and An. squamosus. This reduces its selectivity towards An. gambiae s.l, and especially An. mascarensis (KI = 0). The results have shown that by increasing the amount of 4-hydroxycoumarin to 10 mg, the kairomone index for An. gambiae s.l and An. mascarensis decreases to 48% and 0% respectively, meanwhile those of mosquitoes and Anopheles spp. increase significantly. It implies likely that at this quantity, the product may not be optimal to be selective for the malaria vectors and/or that the ratio of malaria vectors in the mosquito population is already much lower than the previous one. According to our results, the kairomone index of blend 4-hydroxycoumarin, octenol and CO2 for malaria vectors is about 30%. We can suppose that the CO2, a broad-spectrum attractant50, is responsible for the decrease of the selectivity for this blend. It is important to note that we discussed about the results of kairomone index of products, regardless of the attractive effect of light trapping.
The CDC light trap allowed for the evaluation of the attractiveness activity of products tested on mosquitoes. However, the difficulty of the attractiveness evaluation using this trap lies in the light because it was reported that the latter has also an attractive effect on bloodsucking insects51, especially in regard to the Anopheles mosquito. Almost all species belonging to this genus have a nocturnal activity52,53. Therefore, the efficacy of the products as attractants is not determined in this study. For this, it is necessary to measure this attractiveness and selective efficacy on mosquito vectors with a much more specific trap without light nor CO2, which are known as effective kairomone but without selectivity.
To conclude, this study allowed us to show the attractiveness property of 4-hydroxycoumarin on mosquitoes. 4-hydroxcoumarin at a dose of 10 mg/ml and with a mass deposited of 2 mg generates a significant attractant effect on Anopheles spp. and especially on the malaria vector species, An. gambiae s.l, and An. mascarensis in Madagascar. A significant kairomone selectivity of 4-hydroxycoumarin is also observed on these mosquitoes. In addition, this compound has a synergistic effect with octenol (2 mg) to attract Anopheles spp. and also selective on An. gambiae s.l, and An. mascarensis with kairomone index 70% and 100%, respectively. The selectivity of the products with respect to one or more mosquito species is very important in order to evaluate or even to eliminate them in an area at risk of epidemics. Moreover, in 16 days of experimentations with baited CDC light traps the number of malaria vectors seems to have decreased to almost none and this may be the most important point even if it makes the results analysis more difficult. The use of these selective attracting molecules may be highly ecological in vector control against malaria in Madagascar. However, using 4-hydroxycoumarin at different doses, in areas with high Anopheles density where the three main malaria anthropophilic vector species, An. gambiae s.s, An. mascarensis and An. funestus are present, a longitudinal study should be carried out to measure its effectiveness as mosquito control tools and a molecular identification must be performed for the An. gambiae complex.
Source: Ecology - nature.com
