Two sources of mosquitoes were used: 1) The Kisumu laboratory strain of A. gambiae s.s. originated from the Kenya Medical Research Institute (KEMRI) of Kisumu, Kenya. It was reared at Michigan State University according to Huang et al. 15. Cages of mosquitoes were held in an environmental chamber maintained at 28 ± 1°C and 80 ± 10% RH under a LD 12:12 h photoperiod. Indirect light of about 0.17 lx was provided during scotophase by a shaded 4 watt tungsten bulb; it was intended to mimic moonlight. Mosquitoes were offered blood via a membrane feeder 2–3 days before ovipositional tests. 2) Blood-fed feral females were aspirated from the walls of houses near the KEMRI compound. These females were held in laboratory cages under high humidity for 24 h before ovipositional tests. As previously reported, ca. 90% of the females were A. gambiae s.s. and the remainder were A. arabiensis as determined by PCR 15.

The method of egg collection may influence the oviposition rhythm of some Diptera 16. Use of a mechanized egg collector may be less disruptive to egg deposition than manually changing an ovipositional resource at hourly or two-hour intervals. Mechanized egg collectors have previously been utilized in the study of ovipositional rhythms for both D. melanogaster 516 and Agrotis segetum 17. In the current study, we developed a new automated mechanical apparatus to sample oviposition over time. This apparatus was used to compare ovipositional periodicity by A. gambiae individuals vs. groups.

Battery-powered wall clocks (DSA Incorporated, Farmington Hills, MI, U.S.A.) measuring 31 cm in diameter were modified to progressively present a unique section of dark and wet substrate onto which mosquitoes could oviposit over 12 h (Figure 1). Each clock was positioned horizontally and its original face and hands were removed. The clock body was filled with moist sand of particle size 250 – 425 μm. The sand was topped with Envision^® high-capacity brown paper towelling (Georgia Pacific, Camas, WA, U.S.A). This paper towelling appears light when dry, but dark when wet. Thus, it provided the two key stimuli (dark and wet) necessary and sufficient to strongly stimulate A. gambiae to oviposit 15. A new and removable clock face was fashioned from a circular piece of thin plastic, from which had been cut a section equivalent to one hour on the clock (Figure 1). The opening and perimeter of the face-plate were lined with Parafilm ^® (Pechiney Plastic Packaging, Menasha, WI, U.S.A.) flaps to prevent mosquitoes from depositing eggs on any unexposed section of the substrate. The clock face was mounted on the hour-hand driver, so that the opening in the face plate made one revolution every 12 h. The clock face was covered with white paper so as to maximize contrast between background versus the actual ovipositional site 18.

Prior to insertion into the clock apparatus, the paper towel substrate was divided by pencil marks into 12 equal wedge-shaped sections. After being exposed to gravid female mosquitoes for 12 h, a clock was removed from the cage of mosquitoes and another was immediately inserted so as to extend the study over a full 24 h. The face of an exposed clock was carefully removed and the paper towelling bearing eggs was carefully peeled off the sand for egg counting under a dissecting microscope. Clock sections open at the beginning and end of a given measurement were exposed to females for a total of 1 h. However, it took 1 h for each intervening section to fully open and another 1 h for each to fully close. Thus, some of the eggs on each intervening section could have been laid over a span of 2 h.

Clocks were presented in white BugDorm-2 insect rearing cages (Mega View Science Education Services Co., Taiwan) measuring 60 × 60 × 60 cm and containing approximately 500 laboratory-reared females of the Kisumu strain varying in reproductive stages. The light cycle in the environmental chamber was set at 12:12 LD, to approximate the natural light cycle found in Kisumu, Kenya. A small tungsten bulb continued to burn in the laboratory at night so as to provide the equivalent light from the night sky. Light levels during scotophase were slightly less than full moonlight (10^-3 W m^-2; 19). Ovipositional clocks were also presented to groups of house-collected gravid females as described above. In this experiment, the BugDorm-2 cages housing approximately 100 females were placed just inside a screened porch of a house in Kisumu, Kenya. Egg recording sessions for both house-collected and laboratory reared groups were replicated 8 and 6 times respectively, using a different set of females for each test. Each recording session began at 1700 h, one hour prior to the onset of scotophase, and continued for 24 h. At 500 h, a clock apparatus containing fresh paper towelling was exchanged for the loaded clock. The numbers of eggs laid within each hourly period were counted under a dissecting microscope and incorporated into frequency histograms.

The bottom of the enclosure for these tests was the clock apparatus over which sat a 12 cm high cylindrical wire frame. Nylon netting (18 intersections/cm) was placed over the frame and secured by a drawstring. Six 2 cm diameter wet cotton balls (Kendall, Mansfield, MA) were placed on the roof of the cage as a source of moisture. Blood meals were offered to females between 1200 and 1700 h three to four days prior to use. Three or four days after a blood meal, an individual female was gently transferred to the clock cage by aspirator before scotophase. After the female had been exposed to the ovipositional resource for 4–5 h, the female was removed from the cage and fresh paper towelling was substituted for the previously exposed paper towelling within the clock. Then, the female was carefully reinserted. The exchange of the ovipositional resource was repeated 11 h later. The numbers of eggs laid within each hourly period were counted and incorporated into a histogram. Correlation analysis (SAS software version 9.1) was used to test for a correlation between the length of the preoviposition interval (defined as the time interval between a female's first exposure to the clock and the time oviposition was initiated) and the length of the oviposition interval (time interval during which oviposition occurred). It was also used to test for a correlation between the length of the preoviposition interval and the total number of eggs deposited per female. The ovipositional periodicity was measured for a total of 56 individual females, all of the Kisumu laboratory strain.

The terms gravid and hypergravid as used by Sumba et al. 14 refer the condition of the female mosquito when they are presented with an ovipositional resource three and four days, respectively, after obtaining a blood meal. Differences in oviposition by gravid versus hypergravid females were examined by comparing the mean numbers of eggs oviposited per female per hour of each respective group using a paired t-test (SAS software version 9.1). After each trial, females were dissected under a dissecting microscope to check for residual eggs.

Engorged females were randomly selected from newly blood-fed cages of mosquitoes and placed in groups of 20 into 8 cages made from 15 cm high and 19 cm in diameter white cardboard cartons. The top of the cage was covered with white netting (8 intersections/cm) and a sleeve of the same netting was fitted to a 10.5 cm hole cut in the side for mosquito and ovipositional resource insertion. Females were provided with a constant source of 10% honey solution and six wet cotton balls (Kendall, Mansfield, MA) were placed on the top of the cage to provide extra moisture. Two days after blood-feeding, an ovipositional resource was provided to half of the cages approximately 2 h before the lights were turned off to record egg deposition during scotophase. The ovipositional resource was a 100 × 35 mm clear plastic Petri dish containing 20 mL of distilled water, placed over a circular piece of black paper. At 1200 h the following day, the loaded ovipositional resources were replaced with new Petri dishes containing fresh filtered water. Four ovipositional resources were also introduced into the 4 cages from which an ovipositional resource had been withheld. These resources, identical to those previously mentioned, were used to record oviposition by gravid females during photophase. After the initial introduction, ovipositional resources were changed hourly from 1200–1600 h in the latter half of the cages and all exposed ovipositional resources were examined for the presence of eggs. Using a small brush, eggs present were brushed into lines on a piece of white paper and counted.

A. gambiae of the Kisumu laboratory strain revealed two ovipositional pulses (Figure 2). The first occurred from 1800 to 0 h, peaked at 2100 to 2200 h, and accounted for 65% of the total eggs deposited. A second but smaller pulse occurred between 0 and 1000 h, and peaked at 400 h. It is notable that some oviposition by females in groups occurred throughout scotophase. Moreover, a few eggs were deposited in the early hours of photophase. The percent eggs deposited by the laboratory strain during the first peak (2100 h) was significantly greater than the percent eggs deposited at 0 h when oviposition greatly diminished (p < 0.0001; Tukey's HSD test). The proportion of eggs deposited during the second peak was not significantly different from the proportion deposited during the first peak at 2100 h (p = 0.3). The valley between these two peaks was marginally significant (p = 0.054).

Two discrete pulses of oviposition were recorded for house-collected A. gambiae groups (Figure 3). The first began at dusk, peaked between 1900 and 2000 h, and ceased after 100 h. Seventy-four percent of the total eggs were laid between 1800 and 200 h. The second pulse commenced near dawn, peaked around 800 h, and ceased before 1300 h. Unlike the laboratory strain, wild-caught females deposited a substantial portion (more than 25%) of their eggs after sunrise.

Ovipositional periodicity of gravid and hypergravid females held individually was similar, although more gravid females contributed to the second ovipositional pulse than did hypergravids. A paired t-test of the total mean number of eggs oviposited per A. gambiae female per each hr (1700–1900 and 2100–2200 h) revealed no significant difference between gravid and hypergravid treatments (p = 0.61). However, correlation analysis revealed a significant positive correlation between the length of the preoviposition interval and the total number of eggs deposited (p = 0.03).

The time at which individual females initiated oviposition was highly variable (Figure 4). The mean length of the preoviposition interval was 3.5 h with a variance of 9.5, and the mean length of the oviposition interval was 2.5 h with a variance of 1.0. A Levene's test, as modified by Brown and Forsythe, was used to compare the variances of the preoviposition and oviposition intervals; it yielded a p-value of < 0.0001. However, there was no correlation between the length of the preoviposition interval and the length of the oviposition interval (p = 0.51). Compiled individual oviposition was similar to patterns of groups (fig. 5); egg deposition occurred throughout scotophase and even during certain hours of photophase. Seven percent of individuals commenced egg deposition before lights off and one individual initiated oviposition after lights on. Interestingly, Figure 4 documents that most females oviposited without detectable interruption and those females spread their eggs continuously over a few consecutive clock intervals. Only one individual out of 56 exhibited two ovipositional pulses; she commenced at 1900 h, ovipositing 12 eggs, and then paused until 2300 h before depositing another 105 eggs.

Seven out of the 12 cages from which an ovipositional resource had been withheld until mid-afternoon produced eggs (Table 1). All of the 12 cages provided with an evening ovipositional resource produced eggs. Cages provided only with a mid-afternoon ovipositional resource produced 641 total eggs, which is equivalent to 6% of the eggs produced by cages provided an evening ovipositional resource. Eggs from cages provided only a mid-afternoon ovipositional resource were spread over the entire 4 hr period. Sixty-seven percent were deposited in the first 2 h, and approximately 24% were deposited between 1400 and 1500 h. The remaining 9% were deposited in the last hr. Two hundred and three eggs were oviposited between 1200 and 1600 h in cages previously exposed to an ovipositional resource the evening prior.