Anopheles gambiae s.s. (MBITA strain; colonised since February 2001) mosquitoes from Mbita Point, western Kenya, were reared in a greenhouse 7 in water obtained from a natural ground pool colonised by anopheline larvae. Average temperatures and relative humidities were 31°C, 52 % during the day and 24°C, 72% at night. The mosquitoes were exposed to the natural photoperiod, 00° 25' South of the equator. A data logger (HOBO™) was used to record ambient conditions. Larvae were fed on Tetramin^® fish food. Adult mosquitoes were kept in standard mosquito rearing cages (30 × 30 × 30 cm) made of a metal wire frame with a solid metal base and covered with white nylon mosquito netting. They were offered a 6% glucose solution soaked in white paper towel wicks. Three-to-four-day-old females were offered two blood meals, one each day at 18.00 hrs, from the forearm of a human volunteer. The unfed mosquitoes were removed from the cage after each blood meal. Fully engorged females were left in the cages until they were gravid or hypergravid. Gravid mosquitoes are those that were provided with oviposition substrates on the third evening after their first blood meal. Hypergravid mosquitoes were provided with oviposition substrates one day later. Wild, indoor-resting, blood fed anopheline mosquitoes were collected during early morning hours from houses in Lwanda village of Suba district, western Kenya, by means of aspirators. They were immediately transported to the greenhouse, sorted out to obtain An. gambiae s.l. females and provided with 6% glucose solution. They were used in periodicity experiments on the second evening after collection, as described below.

Turbid water taken from a natural ground pool colonised by anopheline larvae (anopheline habitat water), yellow-brown water from a reed swamp colonised by culicine larvae (culicine habitat water), and distilled water were used as oviposition substrates. Presence of larvae was determined by making five random dips using a 350 ml standard dipper.

The experiments were carried out under greenhouse conditions in 25 cm cubic Plexi^®-glass cages, each fitted with a white netting top and a side sleeve opening. To determine oviposition substrate preference, individual gravid An. gambiae s.s. mosquitoes were exposed to 20 ml of each of the above substrates in a three-choice bioassay (n = 55). The substrates were held in black plastic oviposition cups (2 cm depth, 4 cm diameter), placed at equal distances from one another. Individual mosquitoes were released into the cages at about 17.00 hours and left overnight. The following morning, eggs oviposited on each substrate were counted under a dissection microscope. In subsequent replications, oviposition cups containing substrates were rotated such that they occupied different positions every time in the oviposition cages.

Daily oviposition patterns of An. gambiae female mosquitoes on test oviposition substrates, which were offered individually, were determined as follows. Groups of five greenhouse-reared gravid and hypergravid An. gambiae s.s. females were held in separate cages into which anopheline or culicine habitat water or distilled water were introduced. Each mosquito and substrate combination treatment was replicated four times on each experimental day and the experiment repeated on three different days. At the end of the experiment, the mosquitoes that had laid in each group were identified by dissecting each under a dissection microscope and examining their ovaries for the presence of either retained eggs, coiled or uncoiled tracheolar skeins 8.

Groups of five gravid and hypergravid An. gambiae s.s. (ten cages of each) were placed in separate cages and allowed to choose from the three types of oviposition substrates. Similarly, groups of five wild-caught An. gambiae s.l. mosquitoes were offered a choice of the three substrates and their daily oviposition patterns monitored. The experiment was replicated twice on each experimental day and repeated on five different days with new mosquito batches. Individual species within the wild-caught An. gambiae mosquitoes that had laid were identified using polymerase chain reaction (PCR) 9.

The effect of the time of blood feeding of An. gambiae s.s. on its daily oviposition pattern was determined as follows. Four groups of three-to-four-day-old females were given two blood meals, one each day at 06.00 hrs, 18.00 hrs, 22.00 hrs or at 00.00 hrs, respectively. Unfed females were removed from the cages after every blood meal. Gravid mosquitoes were then provided with oviposition cups on the third day at 06.00 hrs and their daily oviposition patterns monitored.

In all experiments, the oviposition cups were removed from the cages after every hourly interval, for 24 hours, starting at 18.00 hrs and replaced with freshly prepared ones. The eggs laid on each substrate were counted under a dissection microscope. To minimise disturbance that might have been due to exposure to white light, red light was used at night while replacing the oviposition cups.

Since oviposition trends for gravid and hypergravid females were similar, data for the two were pooled for analysis. The differences in the number of eggs laid on different oviposition substrates were compared statistically by analysis of variance using the General Linear Model (GLM) procedure. The effect of oviposition substrate on the number of either gravid or hypergravid mosquitoes contributing to the total egg number was similarly compared. Means were separated by the least significant difference (LSD) procedure. Data were subjected to log₁₀ (n+1) transformation to normalise their distribution. All the analyses were carried out using the SPSS^® statistical package, version 11.0.

The mean number ± standard error (39.4 ± 6.1) of eggs oviposited on anopheline habitat water was significantly higher than that on the culicine (16.1 ± 4.6; P = 0.01) or distilled water (23.7 ± 5.3; P = 0.02).

Daily oviposition patterns of An. gambiae s.s. on different substrates, offered in either no-choice or choice assays, are presented in Figures 1 and 2, respectively. In both cases, the main oviposition time was between 19:00 and 20:00 hrs, approximately one hour after sunset, followed by a steady reduction in the number of eggs laid as the night progressed. In the choice bioassays, the gravid mosquitoes showed significant preference for anopheline habitat water over distilled (P = 0.004) or culicine habitat water (P = 0.001) throughout the daily cycle. In the no-choice bioassay, although the total number of eggs laid throughout the cycle on the different substrates was different, this was not statistically significant (P = 0.4). However, during the peak oviposition time, the eggs laid on anopheline habitat water were significantly more than those on the culicine one (P = 0.01) but not significantly more than those on distilled water (P = 0.07). Egg-laying by mosquitoes of different ovary development stages was influenced considerably by the type of oviposition substrate (P = 0.02). The hypergravid/ anopheline habitat water combination had the highest average number of mosquitoes (4.4 ± 0.3) laying their eggs, whereas gravid/culicine combination yielded the lowest response (2.5 ± 0.4; Table 1).

Unlike the greenhouse-reared An. gambiae s.s., the wild-caught An. gambiae s.l., which consisted of 23.9% An. gambiae s.s.,71.7% An. arabiensis and 4.4% unidentified gravid females (n = 46), displayed two main oviposition times early in the night, between 19:00 and 20:00 hrs and between 22:00 and 23:00 hrs, respectively (Figure 3). These mosquitoes also showed significant preference (P = 0.01) for anopheline habitat water over distilled or culicine habitat water.

An. gambiae s.s. females that obtained their blood meals later in the night displayed a somewhat broader oviposition peak time interval, ranging from 19:00 hrs to 22:00 hrs (Figure 4), than those that had fed earlier on, whose peak oviposition time interval was narrower (19:00 hrs to 21:00 hrs).