Introduction

Although dragonflies are often associated with ponds, streams, and swamps, they have rarely been studied as a potential biocontrol agent against malaria and other insectborne diseases. Dragonflies belong to the order Odonata, which includes dragonflies (suborder Anisoptera)

and damselflies (suborder Zygoptera). Worldwide, nearly 5,000 species of dragonfly are known (Borror & White, 1970). The principal methods of distinguishing the various dragonfly families are based on wing venation, eye structure, color, and size. B. pratense belongs to the Darner family (Aeshnidae) of dragonflies and is commonly known as the "hairy hawker dragonfly" because of the numerous long hairs on its thorax.

B. pratense feeds voraciously on insects during its larval stages as well as in its adult stage. Its larger size allows for greater consumption. The natural feeding behavior of the nymph is also innately effective. The nymphs of various dragonfly species generally feed using one of three techniques: passively feeding from burrows in the bottom sediment (burrowers), hunting from the bottom sediment surface (sprawlers), or actively stalking their prey among the vegetation. The nymphs of B. pratense have good vision and are vegetation stalkers that actively hunt prey.

The life cycle of dragonflies is relatively long, and the nymph stage, consisting of 10 to 15 nymphal instars between egg and imago, may last for one year or more (Tillyard, 1917). The presence of dragonflies is not seasonal, but the adults are found in greater numbers during summer and monsoon seasons (March through September). Dragonflies have some preference with respect to habitat (preferring mesotrophic habitats), but the factors responsible for this preference are not clearly understood. The presence or absence of wild vegetations in and around ponds and lakes may be one factor affecting the level of nutrients in the water.

Anopheles subpictus is a house-frequenting mosquito among the anophelines reported from various parts of the world. It has also been reported as a malaria vector in the Austral-Asian zone (Russel, West, Manwell, & Macdonald, 1963) and as a secondary vector of malaria in Sri Lanka. It has also been reported as a malarial vector in various parts of India (Kulkarni, 1983; Panicker, Geetha Bai, Bheema Rao, Viswam, & Suryanarayanamurthy, 1981; Roy, 1943). In the Tarakeswar area of West Bengal, India, this mosquito species is very prevalent and acts as a primary vector of malaria (Chatterjee & Chandra, 2000). It is resistant to chemical insecticides in Pakistan, Nepal, Sri Lanka, Indonesia, and Bangladesh, as well as in various parts of India such as Delhi state, Punjab, and Pondicherry (Das, Chandrahas, & Panicker, 1980; Rathor, Taqir, & Reisen, 1980; Sharma, 1996). Anopheles subpictus breeds mainly in the small ponds and submerged fields of the study area. Although the larvae are present throughout the year, more than 70 percent of them occur during the rainy season (July to October) (Chatterjee and Chandra, 2000).

Mosquitoes serve as a vector not only for malaria, but also for yellow fever, dengue, filariasis, encephalitis, and other diseases. Owing to growing resistance to commonly used organochlorine and organophosphate chemicals in the field, control of mosquitoes through eco-friendly biological means may be advantageous. Other benefits of biocontrol agents include an ability to kill the targeted species while being safe for nontargeted ones; ease of field application; inexpensive production; and the lack of infectivity and pathogenicity to mammals, including humans (Rathor et al., 1980; World Health Organization [WHO], 1975a; WHO, 1984). The alternative malaria control strategy of bio-environmental improvement techniques gives primary importance to antilarval operations.

Although the larvae of Toxorhynchites sp. (mosquito) and Diplonychus indicus (water bug) have been considered as potential biocontrol agents against mosquitoes (Sankaralingam & Venkatesan, 1989; Steffan & Evenhuis, 1981), information on the predatory instinct of dragonfly nymphs with respect to mosquito larvae is rare (Fincke, Yanoviak, & Hanschu, 1997; Hati & Ghosh, 1965; Pritchard, 1964). The purpose of the study reported here was to observe the daily feeding rate of the Brachytron pratense nymph on fourth-instar An. subpictus larvae. The efficacy of these nymphs in controlling mosquito larvae under field conditions also was observed.

Methods

Laboratory-Based Experiment

An. subpictus larvae, which had been obtained from various sources of clean stagnant water in Burdwan, West Bengal, were colonized and maintained from 2000 continuously for several generations in a laboratory free of exposure to pathogens, insecticides, or repellents. The laboratory colony was maintained at a temperature of 25-30[degrees]C, relative humidity of 80-90 percent, pH of 6.95-7.03, and dissolved oxygen of 5.5-6.1 mg/L in the insectary of the Mosquito Research Unit, Department of Zoology, University of Burdwan. Under these conditions, full development from egg to adult lasted about two to three weeks. Larvae were fed on finely ground dog biscuit. The adult colony was provided with 10 percent sucrose and 10 percent multivitamin syrup, and was periodically blood-fed on restrained rats. Fourth-instar larvae were continuously available for the experiments. The fourth-instar larval form was selected because it is the last prepupal instar, with the highest survival rate among all the larval instars and the lowest likelihood of being consumed by a predator because of its maximum size.

The nymphs of Brachytron pratense were also collected from shallow freshwater ponds and rice fields in Burdwan and were placed separately in 5-L glass jars and maintained with a diet consisting of midge larvae (family Chironomidae) and dried yeast powder for three days. In the laboratory, one B. pratense nymph (4.5 cm in length, as measured by a stereoscopic Olympus microscope [10x]) was allowed to feed on 100 fourth-instar An. subpictus larvae for 24 hours in a 5-L water bowl containing 3 L of pond water. The pond water, which was similar in quality to that of the insects' habitat, was sieved through a net (>500 mesh) to exclude larvae of other insects. The temperature of the water ranged from 21 to 23[degrees]C, the relative humidity ranged from 55 to 66 percent, the pH from 6.63 to 6.87, and dissolved oxygen from 5.23 to 6.19 mg/L during the experiment. The numbers of An. subpictus larvae consumed by nymphs of each dragonfly species during the lights-on phase (05:00-17:00, Indian Standard Time [IST]) and the lights-off phase (17:00-05:00, IST) were counted at 3-hour intervals for 24 hours. The authors poured the water through a fine-mesh sieve to collect all mosquito larvae, which were transferred to a white pan for counting. After each 3-hour interval, the glass jar was replenished with the number of larvae that had been consumed, along with the same volume of water, to maintain the same prey density. The lengths of the lights-on and lights-off phases were maintained by the application of artificial lights (tube fluorescent lights) set on the walls of the laboratory (six 40-watt bulbs). The lights-on phase in the laboratory was manually synchronized with the natural outdoor sunlight photophase and the lights-off phase with the dark phase of the night. The experiment was conducted three times on three separate days (n = 3) with the same nymph to generate an arithmetic mean.

Field-Based Experiment

To examine the efficacy of dragonfly nymphs in field conditions, the authors selected the communities of Kalna and Burdwan in the District of Burdwan, West Bengal, India, as study areas. In both areas, 10 outdoor open concrete tanks were selected, each with the capacity to hold approximately 300 L of water. These tanks are usually used in the processing of rice (paddy). They remain unused for a long time each year--that is, from after the rainy season to early spring--and play the role of a natural breeding place for local mosquitoes (mainly comprising An. vagus, An. subpictus, An. barbirostris, An. annularis, Armigeres subalbatus, Culex quinquefasciatus, Cx. tritaeniorhynchus, and Cx. vishnui).

Each water tank was manually sieved to ensure that it was free from any larvae or nymphs of larvivorous insects or fishes. This step used fine netting (with >130 mesh) that allowed only the passage of mosquito larvae. After filtering, the debris was collected and washed away to keep the standard quality of the water. Before the field experiments were conducted, the larval density in each of the tanks was assessed with a 250-mL dipper (WHO, 1975b). Each time, 30 dips (n = 30) were taken, and the mean per-dip larval density was calculated. Ten freshly collected nymphs of B. pratense were introduced into each of five tanks (tank numbers 1 to 5). No nymphs were released into the remaining five tanks (tank numbers 6 to 10), which were kept as controls. Larval densities in all the tanks were assessed 15 days after the introduction of dragonfly nymphs and on the day nymphs were removed. Larval densities in all the tanks were assessed again 15 days after the removal of nymphs. Data were analyzed statistically by the application of a two-tailed t-test.

The experiments (both in laboratory and in the field) were conducted in the months of August and September 2004. During the field experiments, the temperature of the water was 19-23[degrees]C, the pH 6.23-6.79, the relative humidity 55-65 percent, and dissolved oxygen 5.16-6.23 mg/L.

Results

B. pratense nymphs are aquatic; during the laboratory-based experiments, they crawled at the bottom of the water bowl and came to the surface to capture An. subpictus larvae with their modified labium, which was drawn out into a prehensile organ called the mask. While they were catching the prey, this mask was thrown forward, and with incredible swiftness the prey was transfixed and drawn into the mouth cavity. During a 24-hour period, a nymph of B. pratense consumed an average of 66 fourth-instar An. subpictus larvae released in a water bowl containing 3 L of water. The average larval consumption at different hours is presented in Table 1. Out of 66 mosquito larvae consumed (average of three experiments) by a single B. pratense nymph in the course of 24 hours, 47 were consumed during the day (the lights-on phase) and 19 during the night (the lights-off phase). The consumption rate during daylight was significantly higher (p < .05) than during the night (t = 2.15). The reason for this difference is that the nymphs are daylight stalkers--that is, they need light for visual prey acquisition.

Under field conditions, the per-dip larval density of mosquito was higher in Kalna (7.34) than in Burdwan (5.48), probably because more breeding sites are available in Kalna, which is a rural area, than in an urban slum like Burdwan. The changes in the per-dip density of a mixed population of mosquito larvae under field conditions in Kalna and Burdwan (shown in Figure 1 and Figure 2, respectively) were measured at a fixed time interval of 15 days after the introduction of 10 dragonfly nymphs in each of the treated tanks. In the nymph-treated tanks, the per-dip mean density of the mixed population of mosquito larvae was significantly reduced 15 days after the introduction of B. pratense nymphs (t = 43.55 and 35.23 in Kalna and Burdwan, respectively, in comparison with a tabulated value of 2.04, at .05 level of probability). But in the control tanks, where no nymphs were introduced, mean larval density did not differ significantly (t = 0.79 and 0.76 in Kalna and Burdwan, respectively). Again mean larval density increased significantly in tanks (tank numbers 1 to 5) after 15 days from the removal of the nymphs (t = 32.29 and 28.13 in Kalna and Burdwan respectively). The control tanks (tank numbers 6 to 10) did not show any significant difference in per-dip density, as expected (t = 0.46 and 0.32 in Kalna and Burdwan, respectively, compared with the tabulated value of 2.04 at .05 level of probability).

Discussion

Resistance to chemical insecticides during the late 1950s resulted in an expected turn toward a search for biocontrol agents against the mosquitoes associated with public health hazards. Jenkins (1964) reviewed the literature related to the use of natural enemies against all arthropods of medical importance, and by 1999 there had been a substantial increase in research into the use of biocontrol agents (Bay, 1974; Garcia & Legner, 1999; Murdoch, 1982; Sebastian, Sein, Thu, & Corbet, 1990; Service, 1983). Several controversies, however, arose regarding the application of dragonflies: 1) dragonflies can displace native species, 2) augmented dragonfly populations can upset the ecological balance in aquatic ecosystems, 3) increased dragonfly populations can cause localized extinction through destruction of food supplies, and 4) suitable habitat conditions may not be found in all geographical locations where mosquito control is necessary. In spite of these concerns, some biological control programs were successful in controlling mosquito populations (Batra, Mittal, & Adak, 2000; Wang, Hwang, & Lay, 1990; Wang, Chang, Wu, & Ho, 2000; Wu et al., 1987).

When mosquito control programs use biocontrol agents, it is highly desirable to have agents that will yield long-lasting control with one or few treatments or introductions so as to be cost-effective. During the study reported here, the average daily rate at which B. pratense fed on fourth-instar An. subpictus larvae was very high (66 larvae per day), and it was higher than the rate associated with larvivorous fishes like Gambusia affinis (48 larvae per day) and Lebistes reticulatus (32 larvae per day) (Chatterjee & Chandra, 1997). From both the laboratory and the field test results, it is apparent that mosquito larvae are a favorite food source for the dragonfly nymph, a result that agrees with the laboratory-based observations of Hati and Ghosh (1965) in Calcutta.

The field study indicated that the presence or absence of nymphs in these naturally mosquitogenic sites had considerable impact on larval densities. The potential for malaria control resulting from the reduction of mosquito larvae in their natural breeding sites after introduction of nymphs was demonstrated quantitatively. The finding that variations in larval density were insignificant in the control tanks during the period of experiment rules out the possibility that other factors influenced the reduction and confirms the role of the dragonfly nymph as an effective predator that decreases larval-mosquito density in treated tanks. The increase in larval density that occurred in the same tanks after removal of nymphs furthers confirms the biocontrol efficacy of B. pratense nymphs.

Further work is necessary, however, to determine the proper methodology for the mass rearing and augmentative release of dragonfly nymphs that would make this biocontrol procedure feasible for widespread application. One method would be to select bodies of water known to be breeding grounds for mosquitoes, to create miniature biotopes adjacent to these bodies of water, and to artificially introduce native dragonfly nymphs into the water body. This process would make it possible to further explore the biocontrol potential of dragonfly nymphs.

Conclusion

Mosquitoes are important vectors of diseases, especially in the tropics. Resistance to chemical insecticides is a growing problem, and increasing attention is being paid to alternative control methods. Among the weapons available for use against mosquito larvae, biological control may be advantageous. The research reported here provided new information about the potential use of dragonflies as biocontrol agents in the worldwide campaign to control the insect vector of malaria. The study demonstrated that B. pratense nymphs are dependable predators of mosquito larvae. B. pratense feeds best during daylight. Its stalking habits make it an effective predator of many species of mosquitoes common to India. Because of its relatively long nymphal life (approximately one year in tropical locales and up to two years in colder climates) and its recorded daily feeding rate, B. pratense can be expected to consume a good number of mosquito larvae. The authors conclude that B. pratense can be used effectively as a strong biocontrol agent for the control of mosquitoborne diseases, as well as for control of mosquitoes that are or that may become resistant to pesticides. Additional research needs to be conducted to determine optimal methods for cost-effective commercial rearing and targeted use of the nymphs in various field conditions.

Acknowledgements: The authors wish to thank Professor B. Seal of the Department of Statistics at the University of Burdwan for computing all the statistical analyses.

Corresponding Author: Goutam Chandra, Professor and Head, Department of Zoology, University of Burdwan, Mosquito Research Unit, Rajbati, Bardhaman, West Bengal, India 713 104. E-mail: goutamchandra63@yahoo.co.in.

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Although most of the information presented in the Journal refers to situations within the United States, environmental health and protection know no boundaries. The Journal periodically runs International Perspectives to ensure that issues relevant to our international constituency, representing over 60 countries worldwide, are addressed. Our goal is to raise diverse issues of interest to all our readers, irrespective of origin.

S.N. Chatterjee, M.Sc., Ph.D.

A. Ghosh, M.Sc., Ph.D.

G. Chandra, M.Sc., Ph.D.

TABLE 1 Average Consumption of Fourth-Instar Anopheles subpictus Larvae
Over 24 Hours by Brachytron pratense Nymph*

Number of Larvae Consumed During 3-Hour Intervals

                          Lights-on Phase

5:00 to 8:00     8:00 to 11:00     11:00 to 14:00    14:00 to 17:00
5 [+ or -] 0.41  12 [+ or -] 0.82  19 [+ or -] 0.65  11 [+ or -] 0.71

                         Lights-off Phase

17:00 to 20:00   20:00 to 23:00   23:00 to 2:00    2:00 to 5:00
9 [+ or -] 0.76  5 [+ or -] 0.75  2 [+ or -] 0.34  3 [+ or -] 0.56

Mean [+ or -] Standard Error

Lights-off Phase

8.25 [+ or -] 1.66

*n = 3

FIGURE 1 Per-Dip Larval Density in Treated and Control Tanks Under Field
Conditions in Kalna (10 Nymphs per Field Tank)

                    Density of Larval Mosquitoes per Dip
                                   15 days after     15 days after
              Before introduction  introduction of   removal of
Type of Tank  of dragonfly nymphs  dragonfly nymphs  dragonfly nymphs

Treated       7.34                 0.83              6.83
Control       7.12                 6.5               6.79

Note: Table made from bar graph.

FIGURE 2 Per-Dip Larval Density in Treated and Control Tanks Under Field
Conditions in Burdwan (10 Nymphs per Field Tank)

                      Density of Larval Mosquitoes per Dip
                                   15 days after     15 days after
              Before introduction  introduction of   removal of
Type of Tank  of dragonfly nymphs  dragonfly nymphs  dragonfly nymphs

Treated       5.48                 0.22              5.23
Control       5.67                 5.33              5.39

Note: Table made from bar graph.