Indian J Med Res 118, October 2003, pp R. Srinivasan & D. Dominic Amalraj. Vector Control Research Centre (ICMR), Pondicherry, India

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1 Indian J Med Res 118, October 2003, pp Efficacy of insect parasitoid Dirhinus himalayanus (Hymenoptera: Chalcididae) & insect growth regulator, triflumuron against house fly, Musca domestica (Diptera: Muscidae) R. Srinivasan & D. Dominic Amalraj Vector Control Research Centre (ICMR), Pondicherry, India Received March 26, 2003 Background & objectives: In fly management programme chemicals are extensively used. Combined use of insect growth regulator (IGR) and parasitoids would yield a better reduction in fly density, as exposure of fly larvae to sub lethal dose of IGR enhances pupal duration and thereby increases the exposure time for parasitism. The objective of the present study was to explore the additive effect of both Dirhinus himalayanus, an insect parasitoid and triflumuron, an IGR in controlling house fly, Musca domestica. Methods: In the field trial the study sites were allocated based on two-way ANOVA performed on one year pre-treatment survey. Parasitoid release and IGR treatment were initiated when the growth rate (λ) of M. domestica was in an increasing trend. Parasitoids were released at a rate of 6 number/m 2 for three months (February - April 2000), whereas IGR was applied at a dose of 10 mg/m 2 at fortnightly interval from February to December Control efficacy was assessed using Mulla s formula. Results: Reduction in puparia density was 59.29, and per cent respectively in areas, where parasitoids, IGR and combination of both IGR and parasitoids were used. Corresponding figures for adult population were 55.69, and per cent respectively. The per cent reduction in puparia and adult density was significantly (P<0.05) higher in the experimental areas than in the check. When the per cent reduction in adult and puparia density among different experimental areas was compared, it was observed that the reduction in fly density was significantly higher in the areas, where the combination of both parasitoid-igr (P= ) and IGR (P= ) were used, while in areas where parasitoid (P= ) were released the reduction in fly density was not significant. Similarly, there was a significant reduction in puparia density in areas where both parasitoid-igr combination (P=0.0001) or IGR (P=0.002) were used, whereas in parasitoid (P=0.0612) released areas it was not significant. Interpretation & conclusion: The results show that the combined use of parasitoid and IGR is effective in reducing puparia and fly density. Therefore, for sustenance of an effective fly control programme, both parasitoid and IGR may be used. Key words Dirhinus himalayanus - insect growth regulator - Musca domestica - triflumuron Musca domestica (Diptera: Muscidae), a synanthropic fly has a close association with man and his environment. Besides a disease spreading agent, house fly disturbs people at sleep/or work. Its annoyance results in the reduction of milk and or meat production in dairy, poultry and piggery farms. Application of ecological principle has many advantages over the use 158 of chemicals in managing house fly population 1-3. Such a measure causes minimal disturbance to the functioning of varied biotic components in the ecosystem 4. In nature the reproductive potential of house fly is checked by a variety of natural enemies, which include pathogens, predators and parasitoids 5-7. Therefore, exploitation of natural enemies against the pest population is desirable 8,9.

2 SRINIVASAN & AMALRAJ : EFFICACY OF IGR & PARASITOID AGAINST HOUSEFLY Release of pupal parasitoids in house fly management is one of the potential avenues, which keeps the fly population under check Insect growth regulators (IGR) are also being widely used in suppressing these pests 15. The IGRs have been reported to be safe and specific in action against vectors Though topical application of IGRs in fly control has been reported to curb beneficial and non target organisms, which co-exist in nature, the IGR, triflumuron, when used at a sub lethal dose against house fly not only delays the amergence of adult, but also increases the exposure time for parasitism In addition, the elimination of non target organisms was also avoided 18. No information is available on the additive effect of both parasitoids and IGR in house fly control. In order to understand the influence of additive effect of both Dirhinus himalayanus (Hymenoptera: Chalcididae), the puparial parasitoid and triflumuron, an IGR in controlling house fly the present study was carried out in certain urban agglomerations of Pondicherry. Material & Methods Triflumuron [1-(2-chlorobenzoyl)-3-(4- trifluoromethoxyphenyl)], a chitin synthesis inhibitor was obtained from Dow chemicals Company, Ag-organics Department, Midland MI, USA and the parasitoid D. himalayanus was obtained from rearing and colonization division of the Vector Control Research Centre, Pondicherry. Study areas: The study was carried out in the urban areas of Pondicherry, which has a homogeneous, tropical maritime climate, as it is located on the Coromandel Coast. Mean minimum and maximum temperature recorded during the study period ( ) was 28.7 and C respectively.the northeast monsoon has a pronounced impact in this area. It starts from September and ends in December with a maximum downpour in October. The study areas were scattered over 4 km away from the center of the town. Most of the inhabitants in these localities lived in concrete houses and a few occupied mud plastered huts with thatched roof. About 200 cattle were domesticated in these areas. Organic waste and garbage from dwellings and cattlesheds were dumped in garbage bins kept on streets. Cow dung heaps and organic waste of animal and plant origin were also dumped in yards of the cattle-sheds in many places, which supported prolific breeding of house flies. 159 Selection of study sites: A preliminary survey was carried out in 20 areas during November 1998 to select potential fly breeding sites. Density of flies was monitored using Scudder grill technique 20. A total of 12 study areas were randomly chosen from those with similar baseline densities. The density of fly (number/grill/30 seconds) was measured. A year round pre-treatment survey on both fly and puparia density was made and two-way ANOVA test was applied, which revealed no significant variation in the fly density among the selected sites, but there was significant monthly variation (P=0.0001).The puparia density varied significantly with sites (P=0.0037) and months (P=0.0004). The study areas were divided into four groups, each with three sites, based on randomized allocation as follows; group I (Natesan Nagar, Chavukupet and Vaiyapurimudhaliar Thottam), group II (Kuruchikuppam, Karuvadikuppam and Solai Nagar), group III (Vazhakulam, VOC Nagar and Parimalamudhaliar Thottam), and group IV (Vambekeerapalayam, Vanarapet and Iyathoppu) (Table 1). Study design: The control efficacy of parasitoids and IGR separately and their additive effect when used together was assessed. In group I pupal parasitoid, D. himalayanus was released, in group II triflumuron, and in group III both parasitoid and IGR were used. Group IV served as check. Baseline information on species composition and relative abundance of house flies was collected at fortnight interval prior to treatment from January to December Density of adult flies was monitored using Scudder grill 20 while immature density was determined using litre sampling technique 20. Puparia obtained from the samples were isolated using floating and skimming technique 20, dried at room temperature and confined in one litre plastic container for the emergence of flies or parasitoids. Species composition was also recorded. Surface area of different breeding habitats in each locality was also measured. Finite rate of increase (λ) of the M. domestica was computed from the innate capacity to increase (r m ). The r m was calculated from the seasonal change in relative density during different months 21. The λ was calculated as follows: The finite rate of increae (λ) = e rm

3 160 INDIAN J MED RES, OCTOBER 2003 Where, e = base of Naperian log r m = innate capacity to increase The r m was calculated from the changes in relative density as follows: In Nt 2 - InNt 1 r m = (t 2 - t 1 ) % reduction = (Cl x T2) x 100 (C2xT1) Where; C 1 =Pre-treatment density in check areas T 1 = Pre-treatment density in treatment areas C 2 =Post-treatment density in check areas T 2 =Post-treatment density in treatment areas where: In = natural log Nt 2 = population at time t 2 Nt 1 = population at time t 1 One way ANOVA was performed after transforming the percentage reduction in puparia and fly density to ARCSINE values and the significant difference in reduction of puparia and adult population due to parasitoid release, IGR treatment and/or additive effect of both was determined. Release/treatment period of both parasitoids and IGR was made when the finite rate of increase was in an increasing trend. Parasitoids were released at the rate of 6 number/m 2 fortnightly for three months (February- April 2000), whereas IGR was treated at a dose of 10 mg/m 2 fortnightly from February till December Based on the daily mean fecundity of D. himalayanus 14 and recommended dosage of IGR for non-target organism in fly breeding sources 18,19 the release rate of parasitoid and treatment dosage of IGR was decided. Mated gravid female parasitoids were released over the fly breeding sites and the number varied depending on the surface area. Density of puparia and adult flies were also monitored during the trial period i.e. till December 2000 in all the study sites to assess the control efficacy of parasitoid and IGR. To determine whether the parasitoid had established in the sites, where it was released and to determine the infection rate, puparia were collected from all the study areas, brought to the laboratory and confined for emergence of either flies or parasitoids, if any. Species composition of the flies was also determined in the posttreatment/intervention period. A life and fecundity table was constructed both for D. himalayanus and M. domestica under laboratory conditions (28±2ºC) to determine their reproductive potentials based on the methods described elsewhere 14,22. The control efficacy of parasitoids or IGR or parasitoid-igr combination against house fly was assessed using the following formula 23,24. Results Pre-treatment survey: A total of 4965 puparia of muscoid flies were collected from 12 sites at monthly intervals. A total of six species of flies had emerged from the puparia, when confined in the laboratory. M. domestica was found to be the predominant species, constituting 64.2 per cent, followed by Stomaxys calcitrans (13.1%), M. sorbens (8.2%), Sarcophaga sp. (5.9%), Calliphora sp. (4.2%) and Fannia sp. (1.8%). The remaining puparia (2.6%) yielded neither fly nor parasitoids. When puparia collected from field were held for emergence under laboratory conditions no parasitoid was recorded indicating that the parasitoids were absent in the study areas during pre-treatment period. Cow-dung heaps were the main fly breeding sources ( 43.7%), followed by garbage dumps (26.2%), garbage bins (19.3%), decaying organic matters (6.7%) and animal droppings (4.1%). During the pre-treatment survey the average density of house flies (no./grill/30 seconds) varied between 25.8±0.5 and 16.5±0.6 in group I, 26.0±1.2 and 18.0±1.1 in group II, 28.0±1.2 and 16.5±0.6 in group III and 27.5±1.7 and 18.5±0.6 in group IV (check area). The corresponding figures for the puparia density (no./l) were 26.0±1.2 and 17.5±0.6, 25.5±1.7 and 16.5±1.6, 27.5±4.0 and 18.3±0.5 and 26.0±1.2 and 19.0±1.2 respectively. The density of both adult and puparia were relatively high during summer and low during rainy or winter months (Figs 1, 2).

4 SRINIVASAN & AMALRAJ : EFFICACY OF IGR & PARASITOID AGAINST HOUSEFLY 161 Fig. 1. Puparia density (pre-treatment) at the four sites with different intervention measures. Fig. 2. Fly density (pre-treatment) at the four sites with different intervention measures.

5 162 INDIAN J MED RES, OCTOBER 2003 The finite rate of increase (λ) varied between and The population showed an increasing trend from February 1999 and declined (0.997) in July and thereafter again showed an increase. Perhaps, the maximum temperature in July and heavy rainfall in October affected population growth (Fig. 3). Since the population showed an increasing trend from February 1999, release of parasitoid and treatment of IGR were made during this month. Reproductive potential of house fly and parasitoid was computed under simulated field condition (Table II). Reproductive potential of parasitoid (R 0 = 79.42) was higher than that of the host (R 0 = 23.87), implying the usefulness of this biocontrol agent in keeping the host population under check. When the influence of climatic factor on density of M. domestica was assessed, it showed a significant negative correlation with rainfall (r= ; P=0.033) and positive correlation with temperature. (r=0.6355; P=0.026), while puparia population did not show any such correlation with rainfall and temperature. Post treatment survey: M.domestica was found to be the predominant species, constituting 67.3 per cent. The other species were Stomaxys calcitrans (12.7%), M.sorbens (6.7%), Sarcophaga sp. (8.2.9%), Calliphora sp. (3.1%), and the rest (2.0%) were found to be dead. Cow-dung heap (36.6%) was the predominant breeding habitat. Garbage dumps (23.2%), garbage bins (18.2%), decaying organic matters (8.2%) and animal droppings (13.8%) were also found to be the common breeding sources. Fig. 3. Finite rate of natural increase of the fly population. Table I. Density of fly in a preliminary survey Group Control Study area Density of adult fly measure (No./grill/30 sec) I Parasitoid Natesan Nagar 27.8±6.2 Chavukupet 28.3±7.5 Vaiyapurimudhaliar Thottam 31.2±5.1 II Triflumuron Kuruchikuppam 30.9±3.7 Karuvadikuppam 33.4±5.2 Solai Nagar 26.0±4.0 III Parasitoid and Vazhakulam 32.4±2.4 triflumuron VOC Nagar 31.5±2.5 Parimalamudhaliar Thottam 34.2±4.3 IV Check Vambakeerapalayam 31.0±3.8 Vanarapet 30.7±7.1 Iyathoppu 30.3±5.9 Data are mean±sd Table II. Life table statistics of house fly, M. domestica and its parasitoid, D. himalayanus Characteristics M. domestica D. himalayanus Net reproductive rate (R O ) Mean generation time (T) (days) (days) Intrinsic rate of increase (r m ) Finite rate of increase (λ) During the post-treatment period the average density of adult flies (no./grill/30 seconds) ranged from 9.5±1.3 to 20.9±1.0 in group I, 8.5±1.3 to 21.8±1.7 in group II, 6.5±0.8 to 22.0±1.8 in group III and 22.5±1.3.6 to 29.3±1.5 in group IV (check area). The puparia density (no./1) declined from 22.5±1.32 to 11.5±1.7, 24.3±1.3 to 10.5±2.1, 23.7±1.0 to 9.0±0.8 in groups I, II and III respectively, whereas in the check area it fluctuated between 29.5±3.1 and 23.0±0.8 (Figs 4 and 5). In experimental sites, where parasitoids, IGR and both parasitoid and IGR were used, the density of puparia and flies showed a gradual decline. While in check areas the density of adults as well as puparia did not show any such reduction, except the natural fluctuation due to climatic factors. The influence of climatic factors (rainfall and temperature) on fly and puparia abundance during post-

6 SRINIVASAN & AMALRAJ : EFFICACY OF IGR & PARASITOID AGAINST HOUSEFLY 163 Fig. 4. Puparia density (post-treatment) at the four sites with different intervention measures. Fig. 5. Fly density (post-treatment) at the four sites with different intervention measures.

7 164 INDIAN J MED RES, OCTOBER 2003 control survey was observed in the check area, whereas in the experimental sites no such correlation was observed, due to introduction of intervention measures. In the check area house fly adults showed a significant negative correlation with rainfall (r= ; P=0.035) and positive correlation with temperature (r= ; P=0.021). A total of 1650 puparia collected from the four study sites were held in containers under laboratory conditions for emergence of flies/parasitoids to determine the infection rate. D. himalayanus recorded two months after the introduction in the study areas indicated that the species got established in the released sites. Percentage infection of puparia varied from 9.5 to 67.4 (average 32.5%) in the site where parasitoids were released, and 2.1 to 57.8 (average 22.6%) in the site treated with both parasitoids and IGR. No parasitoids had emerged from puparia obtained either from IGR treated or check area, indicating that there is no natural infection in the field. Control efficacy: Mulla s test indicates that the reduction in muscoid fly density was high in the area, where combination of both parasitoid and IGR was used. Density of adult flies and puparia had declined to and per cent respectively. In parasitoid released area, the per cent reduction in adult and puparia density was and per cent respectively. The corresponding figures for the area treated with IGR were and per cent respectively. However in the check area, the density of adult flies/puparia did not show any reduction and the trend was similar to that of the pre-treatment survey. The per cent reduction in puparia and adult density was significantly (P<0.05) high in the experimental areas than the check. When the per cent reduction among different experimental areas was compared using ANOVA test, it was observed that the reduction in fly density was significantly higher in the areas where the combination of both parasitoids and IGR (P= ) or IGR alone (P= ) was used. In areas where parasitoids were released, the reduction in fly density was not significant. Similarly, there was a significant reduction in puparia density in areas where both parasitoid and IGR combination (P=0.0001) or IGR alone (P=0.002) was used, whereas in parasitoids released areas the reduction was not significant. Discussion The fly population was prevalent throughout the year in all the study sites. The density was high in summer season and low in rainy/winter months. During both preand post-treatment period M. domestica was found to be the predominant species and the cow-dung heap contributed the maximum in fly production. Earlier investigations carried out in a dairy farm in Pondicherry by Panicker and Srinivasan 25 confirm this finding. House fly density was influenced by rain fall and temperature; fly population dispersed indoors during the rainy months and hence the density showed negative correlation with rainfall in breeding habitats, whereas it showed a positive correlation with temperature as temperature favours the diurnal activity. Neither rainfall nor temperature showed any correlation with puparia density, perhaps puparia got buried into the manure were not washed away by rain and were protected from sun. The per cent reduction in adult fly density was the highest in the areas where the combination of both parasitoids and IGR was used. In the USA it was reported that an average of 5700 wasps per release per week only reduced the house fly population in a box stall by 40 per cent and a release of about 4800 wasps per week in a small poultry coop reduced the adult flies by per cent 26. In a study from Pondicherry 25 release of Pachycrepoideus vindemmiae at the rate of 5000 per week for three months in a confined dairy farm reduced the housefly population to 71.8 per cent. Insect growth regulators have been reported to be biodegradable and their formulations improve stability of the active ingredients. Other advantages include safety to environment, selectivity of action on target organism and low level of toxicity to warm blooded animals 17,18. The IGR is shown to be of low toxicity to vertebrates when mixed into the feed of poultry or pig to kill the larvae of fly in excrements 27. In a study carried out in Denmark, Pinto et al 28 reported that resistance development was faster when the IGRs were used as feed-through, whereas in topical manure spray of IGR no case of resistance was detected. Therefore IGR can effectively be used in fly control programme. When house fly larvae were exposed to sub lethal dose of triflumuron in present study, adult emergence was delayed, the rate of parasitism increased as the exposure time for parasitism has been increased. However no adverse effect on the parasitoid was noticed

8 SRINIVASAN & AMALRAJ : EFFICACY OF IGR & PARASITOID AGAINST HOUSEFLY with this dose exposed to host puparia. The parasitoid has emerged from treated puparia are normal with no morphological deformation. D. himalayanus has been reported to posses the desired potential for the control of house flies due to its high fecundity and longevity 14. Besides, this species has an obligatory association with the house fly population for its survival and propagation. Non haematophagous feeding habit, highest rate of parasitism, stinging capacity, ability to survive in various climate and to recycle in natural environment may enhance its potential for use as a biological agent. It has already been reported 14 that D. himalayanus exhibits high age specific fecundity and survival than that of other related species. This fact had also been supported by Pickens et al 26. Besides the host specificity, this organism did not affect the other non target organism, which ranks this species as a potential agent against house flies 12. In the present study the reproductive potential of the parasitoid was found to be higher than that of the host, which shows the usefulness of this biocontrol agent in keeping the host population under check. Though the parasitoids are host-specific and cause complete mortality of infected host 13, the per cent reduction in the density of adult fly or puparia was significantly low in parasitoid treated areas, compared with that of IGR or IGR-parasitoid combination treated areas. Probably, IGR showed immediate reduction in fly abundance, while the sudden effect was not there with the parasitoids. Topical application of IGR may be effective initially if larvae are buried in garbage, since garbage are being dumped over the treated surfaces, the efficacy of IGR may be reduced. With the IGR-parasitoid combination, the house fly population could be effectively controlled. It appears that the IGR prolongs the duration of puparia and enhances infection rate, the parasitoids also search and locate the puparia buried in garbage and infect them. At a low dose the IGR has shown no effect on the rate of infection and recycling capacity of the parasitoid in nature 18,19. Therefore, the combined use of both parasitoid and IGR would be effective in fly control programme. These agents are environmentally safe and may be effective in the sustenance of the programme. Acknowledgment The technical assistance rendered by Shri S. Agathewaran is acknowledged. References Pradhan S, Mookherjee PS, Pillai MKK, Bose BN. Chemical control of fly breeding in compost. Indian J Entomol 1960; 22 : Keiding J. Resistance in the house fly in Denmark and elsewhere. In: Watson DL, Brown AWA, editors. Pesticide management and insecticide resistance. New York: Academic Press; 1977 p Keiding J. Review of the global status and recent development of insecticide resistance in field population of the housefly, Musca domestica (Diptera: Muscidae). Bull Entomol Res 1999; 89 : Suppl. 1: Solomon ME. The natural control of animal population. J Anim Ecol 1949; 18 : Mc Coy CE. Suppression of housefly breeding in tomato cannery waste lagoon by means of polyethylene cover. J Econ Entomol 1958; 51 : Legner EF, Gerling D. Host-feeding and oviposition on Musca domestica by Spalangia cameroni, Nasonia vitripennis, and Muscidifurax raptor (Hymenoptera : Pteromalidae) influences their longevity and fecundity. Ann Entomol Soc Am 1967; 60 : Legner EF, Bay EC. The introduction of natural enemies in California for the biological control of noxious flies and gnats. Proc Pap Annu Conf Calif Mosq Control Assoc 1969; 37 : Keiding J. Control arthropods of medical and veterinary importance. New York and London : Plenum press, p Giliomee J. Houseflies successfully controlled with small wasp parasitoids. Pluimvee Poultr Bull 2000; 67 : Legner EC, Bay EC, Mc Coy CW. Parasitic natural regulatory agents attacking Musca domestica L. in Puerto Rico. J Agri Univ Puerto Rico 1965; 49 : Axtell RC. Integrated fly-control programme for caged-poultry houses. J Econ Entomol 1970; 63 : Srinivasan R, Balakrishnan N. Preliminary note on the parasitoids of Musca domestica (Diptera: Muscidae) in Pondicherry. Entomon 1989; 14 : Srinivasan R, Panicker KN. Studies on Dirhinus himalayanus (Hymenoptera: Chalcididae) a pupal parasitoid of Musca domestica (Diptera: Muscidae). Entomon 1988; 13 : Srinivasan R, Panicker KN. Life table and intrinsic rate of natural increase of Dirhinus himalayanus (Hymenoptera: Chalcididae), a pupal parasitoid of Musca domestica (Diptera: Muscidae). Biol Cont Ins Pests 1994; 99 : Awad TI, Mulla MS. Morphogenetic and histopathological effects induced by the insect growth regulator, cyromazine in Musca domestica (Diptera: Mucidae). J Med Entomol 1984; 21 : Breeden GC, Turner EC Jr, Beane WL. Methoprene as a feed additive for control of the house fly breeding in chicken manure. J Econ Entomol 1975; 68 :

9 166 INDIAN J MED RES, OCTOBER Retanakaran A, Granett J, Ennis T. Insect growth regulators. In: Kerkut GA, Gilbert LT, editors. Comprehensive insect physiology, biochemistry and pharmacology, Vol.12, Oxford : Pergamon Press; 1985; p Das NG, Vasuki V. Potential of four insect growth regulators in housefly control. Entomon 1992; 17 : Geetha Bai M, Sankaran, T. Parasites, predators and other arthropods associated with Musca domestica and other flies breeding in bovine manure. Entomophaga 1977; 22 : Scudder HI. A new technique for sampling the density of housefly population. Public Health Rep 1962; 62 : Southwood TRE. Ecological methods. London Mathuen; 1966; p Andrewartha HG, Birch LC. The distribution and abundance of animals. Chicago: The University of Chicago Press; 1954 p Mulla MS, Axelrod H. Evaluation of the IGR Larvadex as a feed-through treatment for the control of pestiferous flies on poultry ranches. J Econ Entomol 1983; 76 : Mulla M, Norland R, F Anane D, D Arwazeh H, M Eken D. Control of chironomid midges in recreational lakes. J Econ Entomol 1971; 64 : Panicker KN, Srinivasan R. Preliminary studies of Pachycrepoideus vindemmiae (Hymenopetera: Pteromalidae) and its potential for controlling houseflies. Indian J Med Res 1986; 84 : Pickens LC, Miller RW, Centala MM. Biology, population dynamics and host finding efficiency of Pachycrepoideus vindemiae in a box stall and a poultry house. Environ Entomol 1975; 4 : Keiding J. The housefly; Biology and control. Training and information guide, Geneva: World Health Organization; WHO/ VBC/1986; 86 : Pinto MC, do Prado AP. Resistance of Musca domestica L. populations to cyromazine (inscet growth regulator) in Brazil. Memo Inst Oswaldo Cruz 2001 ; 96 : Reprint requests : Dr R. Srinivasan, Senior Technical Officer, Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, Pondicherry , India vcrc@vsnl.com