Prey – Predator Relationship by Using Poecilia Reticulata (Guppies)

The category for common predator that was used in this experiment was P.

reticulata (guppies). These guppies were collected in the drainage systems of Putrajaya and Kuala Selangor. All fish were recorded for their wet weights and lengths before and after experiment. Before start of experiment all fish used were acclimatised to laboratory conditions and were placed in plastic aquarium L 22 cm x H 13 cm x W11 cm. Within one week prior to the actual date of experimentation, all fish were provided with blood worm and fish food as a diet. Guppies were starved for 24 hours before introduction to the experimental aquaria, as the hunger level of fish is 24 hours. The experimentation aquaria contained 1L of pond water for the feeding efficacy experimentation. For this experiment the daily feeding rate of guppies towards three species of mosquito larvae were recorded.

The single fish of Poecilia reticulata was exposed to a total of 100 of 4^th instar larvae Ae.

albopictus, Ae. aegypti and Cx. quinquefasciatus. Therefore, three aquaria were setup for every mosquito species and three replicates of experiments were done on separated days.

The time of first attack of guppies against every mosquito larva was recorded and the daily feeding rate was recorded every 3 hour interval. The same mosquito larvae that were left uneaten at end of experiment and fish were not used in subsequent experiments. At every 3

80 hour interval, the water from experimentation aquaria was sieved and transferred to a white tray for counting the number of mosquito larvae not eaten to obtain the number of mosquitoes consumed by predator fish. After that the numbers of mosquito larvae consumed were replenished into the aquaria. The experiment was carried out within 24 hours from 05.00: 1700h for light on and 17:00-05:00h light off. From this setup the active periods of P. reticulata consuming mosquito larvae can also be determined, that is whether their active feeding times were during the day time or the night time.

The second experimental setup was to assess the relationship of feeding rate with the different water volumes contained in the aquaria, also with the number of predator and prey densities. In these experiments 4 aquaria were set up for every mosquito species. This experiment was also carried out with 3 replicates of experiment on separate dates. In one experiment 12 aquaria were setup were used, 4 aquarium for Ae. albopictus larvae, 4 aquaria for Ae. aegypti and another 4 for Cx. quinquefasciatus

1) Aquarium A Female fish (1× 1 ×100) – Single fish with 1L of water volume and 100 4^th instars of mosquito larvae

2) Aquarium B Female fish (1 × 2 ×100) - Single fish with 2L of water volume and 100 4^th instars of mosquito larvae

3) Aquarium C Female fish (2 × 1 × 100) – Two fishes with 1L of water volume and 100 4^th instars of mosquito larvae

4) Aquarium D Female fish (1 × 1 × 200) – Single fish with 1L of water volume and 200 4^th instars of mosquito larvae

5) Aquarium A Male fish (1 × 1 × 100) – Single fish with 1L of water volume and 100 4^th instars of mosquito larvae

81 6) Aquarium B Male fish (1 × 2 × 100) - Single fish with 2L of water volume and 100

4^th instars of mosquito larvae

7) Aquarium C Male fish (2 × 1 × 100) – Two fishes with 1L of water volume and 100 4^th instars of mosquito larvae

8) Aquarium D Male fish (1 × 1 × 200) – Single fish with 1L of water volume and 200 4^th instars of mosquito larvae

82 3.5.4 Prey – Predator Relationship by Using Dragonfly Nymph

The dragonfly nymphs species used in these experiments were Orthetrum chrysis, Orthetrum sabina and Neurothemis fluctuans which were the dominant species in both study areas. All the three species of dragonfly nymphs were measured for the body lengths of every single species used by using a digital calliper before and after experiments. The mosquito larvae and their predator dragonfly nymphs were being maintained in the laboratory separately. Three species of dragonfly nymphs were exposed with three species of mosquito larvae Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus in different aquaria. Before the experimentation the dragonfly nymphs were supplied with aquatic insect. Nine aquaria were used which contained pond water and were oxygenated using air pumps. Every aquarium was labelled with the name of predator and name of mosquito species. During the experiment three species of dragonfly nymphs O.chrysis, O. sabina, and N. fluctuans were allowed to feed on 100 4^th instar mosquito larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus.

The number of mosquito larvae consumed by the nymphs of dragonfly was counted every 3 hour interval for 24 hours. The duration of time taken (first attack) by each dragonfly nymph to attack or consumed mosquito larvae were recorded. The numbers of mosquito larvae ingested by the dragonfly nymphs were counted by pouring through a fine mesh sieve to collect all of the mosquito larvae and were transferred to a white pan for counting of the larvae not consumed. After each 3 hour interval, the aquaria were replenished with the number of larvae that were eaten, along with the same volume of water, to maintain the same prey density. This experiment was conducted three times on three separate days (n= 3) with the same number of nymph for accuracy. After 24 hours all remaining mosquito larvae and dragonfly were removed from the aquarium. These

83 mosquito larvae and dragonfly nymphs were not used in subsequent experiment. The active period of dragonfly nymphs consuming mosquito larvae were assessed in this experiment by setup the time with 12 hour in day time and 12 hour in the night time. This experiment was conducted to see the prey-predation relationship. Ae. albopictus, Ae. aegypti and Cx.

quinquefasciatus were used as prey for the dragonfly nymphs. This experiment conducted also provided data on the most preferred species by dragonfly nymphs, the active time for every predator and daily feeding rate.

In another experiment the aquaria were set up to assess the relationship of predation efficiency and other factors that influenced the predation activities. The 36 aquaria were set up with different predator and prey species. This experiment were repeated on 3 separate day

1) Aquarium A Orthetrum chrysis(1 × 1 × 100) – Single dragonfly nymph with 1L of water volume and 100 4^th instars of mosquito larvae

2) Aquarium B Orthetrum chrysis(1 × 2 × 100) – Single dragonfly nymph with 2L of water volume and 100 4^th instars of mosquito larvae

3) Aquarium C Orthetrum chrysis(2 × 1 × 100) – Two dragonfly nymph with 2L of water volume and 100 4^th instars of mosquito larvae

4) Aquarium D Orthetrum chrysis(1 × 1 × 200) – Single dragonfly nymph with 2L of water volume and 200 4^th instars of mosquito larvae

5) Aquarium E (1 × 1 × 100) Orthetrum sabina, – Single dragonfly nymph with 1L of water volume and 100 4^th instars of mosquito larvae

6) Aquarium F (1 × 2 × 100) Orthetrum sabina, – Single dragonfly nymph with 2L of water volume and 100 4^th instars of mosquito larvae

84 7) Aquarium G (2 × 1 × 100) Orthetrum sabina, – Two dragonfly nymph with 1L of

water volume and 100 4^th instars of mosquito larvae

8) Aquarium H (1 × 1 × 200) Orthetrum sabina, – Single dragonfly nymph with 1L of water volume and 200 4^th instars of mosquito larvae

9) Aquarium I (1 × 1 × 100) Neurothemis fluctuans – Single dragonfly nymph with 1L of water volume and 100 4^th instars of mosquito larvae

10) Aquarium J (1 × 2 × 100) Neurothemis fluctuans – Single dragonfly nymph with 2L of water volume and 100 4^th instars of mosquito larvae

11) Aquarium K (2 × 1 × 100) Neurothemis fluctuans – Two dragonfly nymph with 1L of water volume and 100 4^th instars of mosquito larvae

12) Aquarium L (1 × 1 × 200) Neurothemis fluctuans – Single dragonfly nymph with 1L of water volume and 200 4^th instars of mosquito larvae

85 3.6 Secondary Data

Data for the number of mosquitoes borne diseases cases were obtained from Putrajaya Health Office and Kuala Selangor Health Office. The environmental data for environmental temperature, humidity and rainfall data were obtained from Malaysian Metrological Department (MMD) for the months of January 2010 until February 2011 in Putrajaya areas and from May 2010 to February 2011 in Kuala Selangor. This environmental parameter was used to determine relationship between ovitraps surveillance with the environment conditions. Temperature was measured in degrees Celsius and is defined as mean average of maximum and minimum temperature. Relative humidity is the average monthly humidity based on daily records and is expressed as the percentage.

Rainfall, measured in millimeters, is the amount of rainfall in a month. The secondary data on medical examination for staff that handled with machine fogging and were involved in fogging activities also obtained from Ministry of Health (MOH). Data on chemical use in fogging activities were also obtained from MOH.