Study area
The study was carried out in Mvomero District in east-central Tanzania (5°47’09’’-7°23’40’’ S, 37°11’09’’- 38°01’33’’ E), between March and June 2012. This area has typical tropical characteristics: temperatures oscillating between 19 and 31°C, RH >80%, and annual rainfall of 1,146mm (based on data collected from Mtibwa meteorological station, 2008-2013). The area has a bi-modal type of rainfall with with long rainy season from March to June and a short one from October to December, with a relatively short dry spell between July and September.
Digoma village was selected for the field experiments; the village borders the Nguu mountains and receives water from rivers which flood the valleys. This enables irrigated rice production in the river basin throughout the year. In addition to rice production, therefore, the area has favourable environmental conditions for mosquito production. Malaria and lymphatic filariasis are the most common mosquito-borne diseases in the area [43]. The most abundant mosquito vectors in the area include An. gambiae s.s., Anopheles arabiensis, Anopheles funestus and Cx. quinquefasciatus. Anopheles gambiae s.s. and An. arabiensis are genetically related and morphologically indistinguishable, and are here grouped as An. gambiae unless otherwise mentioned.
Oviposition containers
Containers used for oviposition in this experiment included clay pots, plastic bowls, aluminium pans and plates which were either blue or transparent in colour. With the exception of aluminium plates, which had a diameter of 27 cm and a depth of 4 cm, all other containers were of similar size (average diameter of 25 cm and a depth of 7 cm).
Distilled water
Distilled water was used in the experiment to dilute chemicals and obtain the desired dosages, and also to dissolve oviposition substrates before setting up the experiments. Distilled water was also used for rinsing all washable items used in the experiments. It was produced and packed by LAL Laboratories, Tanga, Tanzania. Distilled water was used alone in the early experiments as oviposition substrate and in the control arm. For experiments involving nonane, a control solution was used, which consisted of 55% v/v distilled + 40% v/v methanol + 5% v/v tween20. Previous studies had not found any behavioural or larvicidal effect of this mixture [40].
Soil
Clay soil originated from a natural breeding site in Mvomero that contained early-stage larvae of An. gambiae. After collection, soil was air dried before further use. Two hundred gram of dry soil was added to each container to simulate natural conditions of breeding sites. Previous studies have shown that volatile emissions associated with microbial organisms in the soil mediate the location of potential mosquito habitats [44]. Therefore, a fraction of the dried soil samples was autoclaved twice for 15 min. at 130°C and 1.4 kg/cm2 pressure and allowed to cool down to kill any organisms that might be involved in the production of volatile chemicals [44, 45]. When the containers were filled with the treatment solution, the maximum depth was 8 cm.
Chemical cues
Nonane (Lot and filling code: 132995235107188, ≥ 99.0%; Sigma Aldrich Chemie BV, Zwijndrecht, The Netherlands) was selected as chemical cue to lure gravid mosquitoes (Schoelitsz et al In Press). Nonane is insoluble in water and, therefore, it was dissolved in methanol and tween20 in the following ratio: 55% v/v of nonane + 40% v/v methanol + 5% v/v tween20. The mixture was further diluted with distilled water to achieve a nonane concentration of 5.5 x 10-5M. In experiments with nonane, the compound was tested at a concentration of 5.5 x 10-5M and it was paired with a control solution of distilled water + methanol + tween20 (see above).
Selection of artificial oviposition containers
To simulate natural breeding sites, experiments were conducted to search for the most preferred artificial breeding site for a natural population of mosquitoes. A range of man-made liquid containers was evaluated in the field in order to identify the most suitable oviposition container for mosquitoes in the area. These included plastic bowls (blue and transparent), aluminium plates and pans, and clay pots. To explore possible colonization of artificial habitats by wild mosquitoes, 25 containers were placed randomly in an open sunlit field. Five lines, separated by 3 m, each composed of five containers that were placed at 3 m distance from each other in the ground, with the top of the container being at ground surface level. Containers were filled with distilled water to capacity and were checked daily for the presence of larvae/pupae for a period of 10 days. Evaporated water was replenished with an equal amount of water in each container daily. Distilled water had already been successfully used as oviposition substrate for mosquitoes in the laboratory and semi-field environment [40]. Therefore, the aim of this experiment was to test if a natural population of mosquitoes would oviposit in these simulated breeding sites containing distilled water. The container that produced the highest number of larvae was selected for the behavioural experiments.
Site selection for oviposition trial
Four sites (north, south, east and west) were selected for the dual-choice oviposition trial in an area covering a total of 4 hectares on both sides of a river near Digoma village, N.E. Tanzania. This area was chosen based on the following criteria: proximity to the river basin, presence of rice fields, absence of flooding, open to sun and proximity to human settlements. Rice growing is the main economic activity. All sites were surrounded by a wire mesh to prevent humans, animals or frogs from interfering with the experiments. In addition, a local field worker was hired to oversee the site during the entire study period.
Clay pots
As clay pots gave the best result as oviposition container (see above), they were selected for the remainder of the study. The pots had an average diameter of 200 mm and a depth of 100 mm was used as artificial breeding sites for the field trial. They were made locally from clay soil, moulded by hand to make a bowl-shaped pot and left to dry, where after they were cured by fire. Clay pots were positioned in the ground so that the margins of the pots were level with the surrounding ground (Fig. 1). The pots were placed in the valley plain, within a rice field in the vicinity of a village.
Design of oviposition experiments
Clay pots were placed at selected sites in the field 72 h before the start of the experiment and filled to capacity with distilled water; water was replenished until the clay reached saturation. Prior to the start of the experiment, the clay pots were emptied and immediately filled with 1L of the oviposition substrate (treatment or control) one h before sunset. Oviposition pots were left undisturbed for five days and from the 6th day, pots were inspected every morning and larvae were collected and recorded daily from 06:30 am for the next 10 days. Whenever the water level decreased in the pots, distilled water was added to maintain the water level. Collected larvae were then transferred to a temporarily established local laboratory together with the water from the pot and reared under controlled conditions. This water was used as rearing substrate in the laboratory for the first 24 h. After that time, larvae from each oviposition pot were transferred to mosquito rearing bowls, which contained distilled water. Rearing bowls were placed below light bulbs and larvae were fed Tetramin® fish food twice daily. Larval growth and development were observed and recorded until pupation and adult formation. Oviposition pots containing experimental substrates remained in the field for 15 d after which the substrates were removed; the pots were cleaned and replaced. For each pair of treatment and control, the pots were oriented facing East and West positions, and these positions were switched for each replicate.
Dual-choice tests
The effect of substrate on oviposition choice of wild mosquitoes was tested in a dual choice test, where one clay pot contained the treatment substrate and the other pot the control substrate (Table 1). Treatment and control were placed 3 m from each other. Each treatment pair was replicated 40 times, 10 pairs at four different sites (see site selection above); pots with substrate were incubated in the field for five days, and then examined for the presence of larvae for 10 days. Newly emerged larvae were collected daily. For each replicate, the positions of the treatment and control were switched each time to counterbalance the effects of wind direction.
Influence of autoclaved soil from a natural breeding site
A total of 200 g of autoclaved soil from a natural anopheline breeding site + distilled water in a clay pot and tested against distilled water only. Pots were each filled with 1250 ml distilled water.
Influence of untreated soil from a natural breeding site
Clay pots were filled with 200 g of dried soil from a natural anopheline breeding site. To test whether soil produces chemical cues or acts only as a visual cue for gravid mosquitoes [26], the soil was tested against autoclaved soil. The pots were each filled with 1250 ml of distilled water.
Influence of nonane
1250 ml of the nonane solution + autoclaved soil was tested against 1250 ml of distilled water + autoclaved soil. Pots were filled with either 200 g autoclaved soil and distilled water or 200 g autoclaved soil and a nonane solution.
Influence of soil from a natural breeding site and nonane
To investigate the interactive effects of breeding-site soil and nonane, combinations of both candidate stimuli were tested alone or as a mixture: (a) nonane + breeding-site soil against nonane + autoclaved breeding soil and (b) nonane + breeding-site soil against distilled water + breeding-site soil. Pots were filled with 200 g of soil and distilled water or a nonane solution until capacity.
Mosquito species composition
All larvae collected were transferred to the insectary and reared until adult emergence. Newly emerged anopheline adults were identified to species level using morphological keys [46]. Anopheles gambiae specimens were preserved in Eppendorf tubes, which contained silica gel for further identification to distinguish between sibling species in the An. gambiae complex. Genotypic identification was conducted by using the ribosomal DNA-polymerase chain reaction (PCR) to separate An. gambiae s.s. from An. arabiensis [47]. Culicine mosquitoes were identified as Cx. quinquefasciatus or other culicines.
Data analysis
SPSS 14 for Windows® was used to conduct Wilcoxon signed-rank tests for paired samples in order to determine the difference in the number of larvae in each oviposition bowl as an indicator of number of eggs laid. A Friedman test for multiple samples was used to determine the oviposition preference among several containers. The preferences of mosquitoes for ovipositing on different treatments were evaluated based on container index (CI) (% bowls harbouring larva). All statistical tests were conducted by using absolute numbers of larvae in pots as a proxy for the number of eggs laid in the pot.
The larval density index (LDI) was defined as the total number of larvae found divided by the total number of oviposition containers with larvae.
The oviposition active index (OAI) was used to determine the attractiveness to the treated substrate compared to control. It was calculated according to the formula; OAI = Nt-Nc/Nt+Nc [48]. Where Nt = number of larvae on the test substrate and Nc = number of larvae on the control substrate. In this study, it was observed that anopheline eggs, which are black in colour, tend to stick to the surface of the clay pot, which is also black. This poses a challenge to accurately score the number of eggs as a measure of oviposition activity of gravid females. Therefore, the number of larvae was scored as a proxy for the eggs that were laid in respective pots.