Oviposition response of Culex pipiens pallens
Adult mosquitoes chosen for mosquito oviposition preference test were mosquitoes which had been 72 h after eclosion. 24 hours after blood feeding, ovitrap 1 (filled with 200 ml chlorine-free tap water + 20 ml PACl suspension, denoted as O1) and ovitrap 2 (filled with 220 ml chlorine-free tap water, denoted as O2) were placed in the cage. Every 24 hours O1 and O2 were observed, mosquito eggs in O1 and O2 were removed as well.
In the experiment, female mosquitoes laying eggs in two cups (d = 11 cm, h = 5 cm, transparent plastic material) were considered as valid oviposition. After each biting, all of the eggs laid by one single female Culex pipiens pallens would gather together as an egg raft and float on the water [35], thus we can recognize the number of valid oviposition times easily. Each observation and removal of all mosquito eggs in O1 & O2 is considered as an independent observation.
In order to exclude the influence of the placement order of ovitrap on the experimental results, the experiment was divided into two parallel control groups which only differed in the placement of O1 and O2, remaining other environmental factors consistent. Apparatus is shown in Fig. 1.
Table 1 Results of two replications in mosquito oviposition preference test
In this test, O1 and O2 had equal oviposition opportunities for female mosquitoes, and finally with a total of 320 valid oviposition times (Table 1). Mosquitoes showed the same oviposition preference in the two replications, so we take the results of R1 and R2 calculated in combination. Of the two replications, O1 had 182 valid oviposition times, which is 31.88% more than that of O2 (138 valid oviposition times), indicating that O1 was more attractive to oviposition, but not significant (n = 15, p = 0.345, p > 0.05).
Among the 15 independent observations with a duration of about 30 days (data not shown), there were 11 times to prove mosquitoes prefer O1 as their breeding habitat, only in 3 times there were more mosquitoes chose O2 rather than O1 to lay eggs, remaining one time equally. This suggests that the attraction of O1 to female mosquitoes’ oviposition was persisted and didn’t decrease over time.
Numerous studies indicated that the process of selecting breeding sites for female mosquitoes is mediated by multiple factors such as vision [36], smell [37], and pheromone [38]. We speculated in this study mosquitoes’ oviposition behavior is mainly influenced by vision. Studies have shown that yellow is more attractive to Culex pipiens pallens for laying eggs than white [39], which in this experiment after coagulation-flocculation, yellow alum floc deposited at the bottom of the ovitrap, which is considered to be nutrient-rich for larvae growing, therefore more female Culex pipiens pallens mosquitoes preferred O1 as breeding sites [40].
Effects of coagulation-flocculation on mosquito egg hatching performance and subsequent development of mosquito larvae
This phase was carried out in a jar containing 1L chlorine-free tap water. 0.01 g sterilized rat food was added in chlorine-free tap water in advance, for it would provide basic nutrition for larvae after egg hatching. The surface scums were scoured by deionized water, until most of scums dropped down to the bottom.
Pre-experiment confirmed that the appropriate dosage of PACl suspension was 60 ml/L, which means the mass concentration of PACl to water was about 0.6 mg/L. It was normal dosage in water treatment [41, 42]. The complete coagulation-flocculation operation includes the addition of coagulants and the mixing process, so the experiment was divided into four parallel control groups, group1 ~ 4 (Fig. 2). Groups could be divided into the coagulating groups and the non-coagulating groups, according to the addition of coagulant or not. Experiment started after mosquito eggs were added in Group 1 ~ 4, and then treated with stirring and/or coagulant adding.
Table 2 shows the comparison of water quality parameters of each group before (t = 0) and after (t = 30 min) treatment. No significant changes occurred in water quality parameters within non-coagulating groups before and after the treatment; group 3 exhibited obvious flocculation 30 minutes after the coagulation, while pH decreased slightly (7.36 ± 0.49 to 7.21 ± 0.43), and turbidity increased significantly (1.23 ± 0.18 NTU to 2.99 ± 0.38 NTU), for without stirring, alum floc produced by coagulation didn’t have enough opportunity for collision and aggregation [43], resulting in the size of some flocs being too small to settled, these floc fragments suspended in supernatant; the pH of group 4 decreased slightly (7.37 ± 0.48 to 7.11 ± 0.36) after treatment, since other factors were basically unchanged. The addition of coagulant increased the salinity (Sal) in the water, and the Electronic Conductivity (EC) and Total Dissolved Solids (TDS) of the coagulating groups (group 3 & group 4) were significantly improved after treatment.
Table 2 Water quality parameters of each group when t = 0 and t = 30 min
The pupation time of larvae in 4 groups (Fig. 3 (b)) were compared. The first pupa in coagulating groups (group 3) appeared significantly later than that of non-coagulating groups, except for there was no pupa in Group 4. The delay time varied in different batches of experiment, but all exceeded 7 days. Moreover, the pupation time of last larvae in coagulating groups (group 3) was also later than that of the non-coagulating groups, suggests that coagulation-flocculation treatment may delay the development of mosquito larvae. It may be a response of food-lacking, which could delay larvae development [44, 45].
Table 3 Comparison of pupation rate, lethal time of 50% (LT50) and lethal time of 90% (LT90) in 4 groups
In table 3, differences between pupation rate, LT50 and LT90 in 4 groups indicated what degree could coagulation-flocculation affect larvae survival in this experiment. The pupation rate of non-coagulating groups was up to 50% in average, while in coagulating groups was below 10%, suggests that by treating with PACl coagulation-flocculation, the number of larvae that become pupae would be definitely reduced. In addition, LT50 of coagulating groups were 2d and LT90 were over 7d, indicating that PACl coagulation-flocculation had a stronger killing effect on newly hatched 1st instar larvae and a limited killing effect on late instars.
7d-continuously cultivation to explore the main reason coagulation-flocculation cause 1st ~ 2nd instar larvae death
To explain why PACl coagulation-flocculation could definitely kill 1st ~ 2nd instar larvae, there are two possible reasons: 1) Culex pipiens pallens larvae may be sensitive to the water-soluble products produced by PACl coagulation, and died due to toxicity; (Ⅱ) alum floc layer hide the food from immature mosquitoes, increases the difficulty of larvae feeding, results in larvae starving to death, moreover, these little floc particles could adhere to the surface of larvae, which prevent larvae to float upward to breathe.
In order to find out which from above the main reason is caused the high mortality of 1st ~ 2nd instar mosquito larvae, a 7d-continuously cultivation of Culex pipiens pallens was conducted. From the egg stage, Culex pipiens pallens were raised in ordinary chlorine-free tap water (control group, recorded as “group C”), chlorine-free tap water after coagulation-flocculation (coagulation-flocculation group, recorded as “group C-F”), and filtered supernatant of group C-F (recorded as “group SC-F”), respectively.
Results revealed that survival of the larvae in group C and group SC-F was consistent and in good condition, while the number of larvae in group C-F reduced by 75% within 2d after the hatching, and made zero within 5d (Table 4). Therefore, the possibility of Culex pipiens pallens larvae died for the water-soluble products produced by PACl coagulation can be excluded, similarly the changes of pH, for larvae favor a neutral pH or slightly alkaline environment [46]. By observing the distribution of the dead larvae in group C-F, it can be found that the location of dead larvae is mainly in the surface and upper part of the floc sediments, and there is no food remains in the digestive tract of larvae (Fig. 4). This phenomenon illustrates that alum floc layer is the main cause of 1st ~ 2nd instar larvae’s death.
Table 4 Results of 7d-continuously cultivation in different groups