Anopheles arabiensis oviposition site selection in response to habitat persistence and associated physicochemical parameters, bacteria and volatile profiles

Abstract

A better understanding of the oviposition behaviour of malaria vectors might facilitate the development of new vector control tools. However, the factors that guide the aquatic habitat selection of gravid females are poorly understood. The present study explored the relative attractiveness of similar artificial ponds (0.8 m² ) aged at varying lengths prior to opening in such a way that wild Anopheles arabiensis could choose between ponds that were freshly set up, or were aged 4 or 17 days old, to lay eggs. Physicochemical parameters, bacterial profile and volatile organic compounds emitted from ponds were investigated over three experimental rounds. Fresh ponds contained on average twice as many An. arabiensis instar larvae (mean 50, 95% confidence interval (CI) = 29-85) as the ponds that had aged 4 days (mean = 24, 95% CI = 14-42) and 17 days (mean = 20, 95% CI: 12-34). Fresh ponds were associated with a significantly higher turbidity combined with higher water temperature, higher nitrite levels and a lower pH and chlorophyll level than the older ponds. Round by round analyses suggested that bacteria communities differed between age groups and also that 4-heptanone, 2-ethylhexanal and an isomer of octenal were exclusively detected from the fresh ponds. These characteristics may be useful with respect to developing attract and kill strategies for malaria vector control.

Keywords: Malaria; denaturing gradient gel electrophoresis; oviposition; physicochemical parameter; volatile compounds.

Figure 1:

Experimental set up: distribution of open field ponds shown for a round.

Figure 2:

Investigating position effect on larval distribution. A, B and C show round 1, 2, and 3 respectively of larval distribution in all 15 replicates per treatment. The red bar represent fresh ponds left open for colonisation after set up, whilst green bars represent ponds that had aged for 4 days and blue bars represent ponds that aged for 17 before opening for colonization.

Figure 3:

Daily rainfall during the 3 rounds of experiments. The green bars indicate the duration of aging (for 17 and 4 days) prior to opening the ponds on day 0. Larvae were sampled 4 days later.

Figure 4:

Partial least square – discriminant analysis (PLS-DA) score plot (A) of fresh pond 0 day old; age 1 (red dots), 4 day old; age 2 (green dots) and 17 day old; age 3 ponds (blue dots) and loading plot (B) based on physicochemical and bacterial viable count data at pre-colonization. Physicochemical data (green dots) included were: Conductivity (μS/cm); Carbonate hardness (mmol/l); Total hardness (mg/l); Phosphate (mg/l); Turbidity (NTU); Nitrite (mg/l); Nitrate (mg/l); Temperature (°C) ; pH; Oxygen dissolved (ppm); Biochemical oxygen demand (ppm) and Chlorophyll (μg Chl/l).

Figure 5:

Principal component analysis (PCA) of volatile compounds detected in pond water of different age group. Axis 1 and 2 explains 33% of the total variation in data. Compounds C15 = 4-heptanone, C44 = 2-ethylhexanal and C90= an octenal isomer associated with age 1 ponds (in red circle) were detected in up to 80% of the water samples from age group 1. Ages 1, 2 and 3 represented 0, 4 and 17 day old ponds respectively.

Figure 6:

A: Denaturing gradient gel electrophoresis (DGGE) of samples from pre-colonization pond water. The numbers 1, 2 and 3 represented different rounds while Ages 1, 2 and 3 represented 0, 4 and 17 day old pond water samples respectively. (B): Principal component analysis (PCA) of presence/absence of DGGE bacteria bands (Spp) in the three age groups at pre-colonization in all rounds combined. Axis 1 and 2 together represent 23% of the total variation in the data. (C): PCA of presence/absence of DGGE bacteria bands in the three age groups at pre-colonization round by round.