Oviposition behaviour of Aedes albopictus in temephos and Bacillus thuringiensis israelensis-treated ovitraps W.A. Naznia#, H.L. Leea, W.M. Wan Rozitab, A.C. Liana, C.D. Chena, A.H. Azaharia and I. Sadiyaha a
Medical Entomology Unit, Infectious Diseases Research Centre, Institute for Medical Research, Jalan Pahang 50588, Kuala Lumpur, Malaysia b
Epidemiology Unit, Medical Resource Research Centre, Institute for Medical Research, Jalan Pahang 50588, Kuala Lumpur, Malaysia
Abstract This study was conducted to determine the response of dengue vectors to ovitraps treated with temephos and Bacillus thuringensis israelensis (Bti) compared with untreated ovitraps which only contained seasoned tap water. The study was conducted at two sites: the natural breeding habitats where the ovitraps were exposed directly to sunshine and rain, and under shelter beneath a cabin without direct exposure. The ovitraps were placed for a period of five days at six different sites each in both conditions. The experiments were replicated three times. The results indicated that all mosquitoes breeding in the ovitraps were Aedes albopictus. There was no significant difference in the ovipositioning behaviour in the natural habitat, but there was a significant difference in the number of eggs laid in the ovitraps placed under the cabins. Though the mean number of eggs laid in traps in response to each treatment varied among the sites, the overall ovipositioning activity was not significantly different between sites when egg densities in the test ovitraps were averaged over the six-week ovitrapping period (site versus treatment effect) at p<0.05. The study also indicated that there was no ovicidal effect of both control agents since hatching of eggs occurred. Although eggs hatched, all larvae died eventually. The male to female ratio was 1:1 for both the sites. The study also showed that larval index is as effective as pupal index and can be used as a surveillance tool for the dengue vectors. It is concluded that containers treated with larvicide are not repellent to Aedes. Keywords: Aedes albopictus; oviposition; repellent; temephos; Bacillus thuringiensis israelensis.
Introduction Dengue as well as chikungunya fever pose serious public health problems in Malaysia. Ae. aegypti is the predominant vector of dengue, #
a mosquito-borne arborvirus belonging to the family flaviviridae which is capable of causing dengue fever, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). [1] During 2008, a total of 36 991 cases with
E-mail: nazni@imr.gov.my; Tel.: +60-3-26162687, Fax: +60-3-26162688 209
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81 deaths due to dengue were reported in Malaysia till October.[2] In addition, there were 1703 cases of chikungunya reported from 12 August to 25 September that year.[3] With the rapid movement of people the number of cases may only increase with time. Ae. albopictus is also a known vector of dengue and chikungunya fever in Malaysia. The chikungunya virus, as a potential menace and responsible for epidemics transmitted by Ae. aegypti and Ae. albopictus , has been documented since 1960. [4] Current measures for the control of Ae. aegypti and Ae. albopictus are based on: (i) the physical elimination of larval development sites; and (ii) the application of 1% sand granules formulation of temephos to artificial containers. Recently, as an alternative to the use of temephos, the biological larvicide such as Bacillus thuringiensis israelensis (Bti) is also used as a bio-larvicide. The appropriateness of larvicides for use in vector control programmes depends on multiple factors such as persistency of larvicide in the breeding sites and the behavioural responses of vectors against the larvicide. It is in this context that the present study aimed to assess the possible repellent effect of temephos and Bti on the oviposition of gravid females that may influence vector control effectiveness for dengue and chikungunya control programmes in Malaysia.[5]
grass and bushes between them. The cabins are placed in the midst of a natural habitat. The temperature and humidity of the site throughout the study period was 25.9 °C±0.68 and 77.7%±4.51 RH for the natural habitat and 25.8 °C±0.57 and 77.50%±4.95 RH for the cabins.
Placement of ovitrap The ovitrap used in this study was based on the specifications as described by Lee.[6] Ovitraps were placed in: (i) open natural breeding habitat of Aedes sp with minimal exposure to sunshine and rain; and (ii) underneath the cabins without direct exposure to sunlight and rain. The studies were conducted concurrently.
Concentration of temephos and Bacillus thuringiensis Six spots (three in each habitat) were identified for the placement of ovitraps for the tests. In each site, three ovitraps were placed with one containing water treated with temephos (ABATE) sand granule formulation at the operational dosage of 1 mg/l , and another water treated with Bti (a Vectobac WG, wettable granule formulation with 3000 ITU/ mg) at the recommended concentration of 0.008 g/l. the third ovitrap contained only seasoned tap water serving as control. All these ovitraps were placed adjacent to each other and remained in position for five days. In total, for each experiment, 18 ovitraps were placed in six spots. The experiment was replicated three times. All ovitraps were collected after five days and brought to the laboratory. A five-day duration was used because longer trapping periods increase the risk of egg loss through
Materials and methods Study site This study was conducted in the vicinity of the Institute for Medical Research, Kuala Lumpur (latitude: 3°10.190’ N and longitude: 101°41.950’ E). The area consisted of several cabins (10 ft X 30 ft X 8 ft) with many trees,
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predation and hatching which could lead to the disruption of production of adults.[7] The number of positive ovitraps were recorded and the ovitrap index was determined. In the laboratory, the paddle sticks from the ovitraps were dried and the number of eggs laid was counted under a dissecting microscope. The total number of eggs per ovitrap was also calculated. The container was screened for larvae and the number was recorded. The paddle stick was transferred into the same plastic container (14.5 cm x 8.5 cm x 7.5 cm) and the water from the respective ovitraps was poured into the plastic container. A pinch of beef-liver powder was added into the containers. Observations were made for a period of 14 days post-collection. The species of the mosquito was identified at the third instar stage for the control ovitraps. The total number of eggs, the hatchability rate, the pupation and the adult emergence were counted and recorded.
Data and statistical analysis Field data was analysed by the analysis of variance (ANOVA) method. The preference of Aedes mosquito to oviposit on temephos, Bti or seasoned tap water was evaluated based on the positive ovitrap index (OI) (% positive ovitraps). The oviposition active index (OAI) was used to determine the Aedes attractant to the treated water compared with the control.[8,9] Kramer and Mulla[8] suggested that compounds with an OAI of +0.3 and above are considered as attractants, while those with –0.3 and below are considered as repellents.
difference in the mean number of eggs laid in traps placed at different sites (site main effect; F=0.34; P=0.89), demonstrating that the oviposition activity was comparable among all sites that we used. Although the mean number of eggs laid in traps in response to each treatment varied among the sites, the overall oviposition activity was not significantly different between sites when egg densities in the test ovitraps were averaged over the sixweek ovitrapping period (site versus treatment effect; F=0.67, P=0.75). Egg densities in ovitraps with water only were similarly varied, but found to be not significantly different (P>0.05) within or between sites. On the other hand, ovitraps placed under the cabin showed a significant difference in the mean number of eggs laid in traps at different sites (site main effect; F=4.44; P=0.003). Similarly, the overall oviposition activity was not significantly altered between sites when egg densities in the test ovitraps were averaged over the six-week ovitrapping period (site versus treatment effect; F=1.45, P=0.20). The ovitrap index and the oviposition active index are presented in Table 1. The positive ovitrap index was in the range of 67%–89%. Mosquitoes laid eggs on both treated and untreated paddle sticks. The highest number of eggs laid was in the ovitrap treated with Bti with 425 eggs while the highest number of eggs laid in the control and temephos-treated ovitraps were 400 and 306 eggs respectively. More eggs were laid in the ovitrap placed in the natural breeding habitat compared to the ovitrap placed under the cabin as shown in Table 1. This could be due to the ovitraps placed in the natural breeding sites being exposed to sunshine and rain which could have diluted the treatment effect of the larvicides, but was still equally effective in eliminating larvae that had hatched in the temephos- and Bti-treated ovitraps over the five-day period. Another factor could be that
Results and discussion The oviposition responses of the Aedes females to the treated ovitraps placed in the natural habitat showed that there was no significant
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Table 1: Ovitrap index and oviposition response of Ae. albopictus to field conditions with ovitraps treated with temephos and Bacillus thuringiensis israelensis Treatment a
Total no. of ovitraps
Positive ovitraps (OI in %)
Total no. of eggs laid
Mean no. of eggs per ovitrap ± SD
OAI of baited ovitraps
OT placed in natural breeding sites 18 18 18 12 (66.67) 14 (77.78) 16 (88.89) 306 425 400 17.00±4.99 23.61±6.85 22.22±5.40 –0.13 +0.04
Temephos Bti Control a
OT placed under a cabin 18 18 18 12 (66.67) 14 (77.78) 14 (77.78) 265 198 316 14.72±5.03 11.00±2.93 17.56±4.37 –0.09 –0.23
Temephos Bti Control a
OT = ovitrap
in the natural breeding site for Ae. abopictus, the high oviposition rate might be due to the high mosquito population in the sites as well as easy accessibility to the ovitrap by the oviposition females. All mosquitoes from the collected eggs were of the Ae. albopictus species. The mean number of eggs per ovitrap for temephos, Bti and control placed in the natural breeding habitat were 17.00±4.99, 23.61±6.85 and 22.22±5.4 respectively as shown in Table 2. On the other hand, the mean number of eggs per ovitrap placed under the cabin for temephos, Bti and control were 14.72±5.03, 11.00±2.93 and 17.56±4.37 respectively. No significant differences were found in the number of eggs laid in the ovitraps with the treatments and control placed in the natural breeding habitat and those placed under the cabin (P=1.000). In some ovitraps there was no oviposition activity and this accounted for 26 out of 108 (24.07%) ovitrap containers.
A study by Craig[10] has stated that conspecific eggs provide an attractive cue for gravid Ae. aegypti because they are a signal of a suitable breeding site. The authors considered an Allee effect[11,12] in which the presence of conspecifics is attractive and presumably beneficial to egg-laying up to a particular density. Mosquitoes integrate a wide range of stimuli prior to the act of ovipositioning. Photoperiod, colour and optical density of the water, oviposition substrate texture and moisture, temperature and reflectance, volatile and contact chemical cues have all been shown to affect the choice of oviposition site by mosquitoes.[13] An additional factor affecting the endpoint of oviposition behaviour is known as “skip oviposition”, [14] which occurs when females lay their eggs in several containers as opposed to laying their entire clutch in one container.[15,16,17] This behaviour increases the distribution of eggs in an area
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Table 2: Mean number of eggs, larvae, pupae and adults obtained from the ovitraps placed in the natural breeding site and under a cabin Mean number ± SD Treatment Eggs Larvae Pupae Adults Male Female Total
a
OT placed in natural breeding sites 17.0±8.28 23.61±9.03 22.22±9.06 10.06±6.77 12.39±6.59 22.17±9.01 0 0 21.34±8.86 0 0 9.50±9.40 0 0 9.78±10.79 0 0 19.28±8.16
Temephos Bti Control a
OT placed under a cabin 14.72±9.02 11.00±5.14 17.56±7.20 9.28±6.33 7.33±4.09 16.84±7.21 0 0 16.45±7.17 0 0 7.78±8.24 0 0 7.67±9.07 0 0 15.50±6.94
Temephos Bti Control a
OT = ovitrap
and may be increased by the tendency of gravid females to avoid ovipositing in sites where eggs of conspecific females had been laid.[17,18,19] The oviposition attractive index for the Ae. albopictus at the natural breeding habitat and under the cabin with treatment of temephos and Bti were –0.13, +0.04, –0.09 and –0.23 respectively as shown in Table 1. This showed that there was no attraction for the gravid female mosquitoes to oviposit in either treatment, indicating that neither temephos nor Bti appears to influence the choice of ovitrap for oviposition site. However, the number of eggs laid showed that Bti has some influence on egglaying. According to Gubler,[20] ovipositing female Ae. albopictus are attracted to darkcoloured water. Hence, the visual stimulus might have been a factor in oviposition site selection, rather than the presence of Bti or any other chemical.[21] Furthermore, very
few chemicals have been found to attract ovipositing Ae. albopictus. Studies by Sharma[22] showed that compounds such as hexadecyl pentanoate, tetradecyl heptanoate and tridecyl octanoate presented significant oviposition repellent activity against the two mosquito species. However, the compound propyl octadecanoate was found to attract Ae. aegypti to the treated oviposition substrate. Previous studies[23,24,25] have indicated that temephos was not repellent for oviposition behaviour of Ae. aegypti and this was in agreement with our study. The emergence ratio of male to female in the natural breeding habitat and under the cabin was 1:1 and no significant difference was observed in the emergence for both sites for sex (p=0.923, t=–0.103 and p=0.974, t=0.034, respectively). The life-table of the development of the Ae. albopictus in both the sites is shown in Table 2. There is no significant difference
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(p>0.05) for egg production for the temephostreated ovitraps and control ovitraps placed either in the natural breeding habitat or under cabin. More eggs were laid in ovitraps treated with Bti in the natural habitat compared to ovitraps under a cabin. In a study conducted by Santos,[26] it was suggested that Bti did influence the number of eggs and that this effect may depend on the infusion concentration. The resultant metabolites from the bacterial growth within the ovitrap may also be a contributing factor for increased oviposition and ovitrap attractiveness.[27] It could also be due to the exposure to sunshine which activates the Bti spores to yield the aroma that attracts ovipositioning. It is interesting to note that all larvae that hatched from the temephos- and Bti-treated ovitraps died due to the toxic effect of the larvicide. This indicated that both larvicides did not exhibit ovicidal properties
because eggs did hatch but the larvae were completely eliminated upon hatching. An extended analysis in the non-treated group (control), with ovitraps placed in natural habitat and under cabin, showed that there was no significant difference in the developmental stages from larvae to adults (P=0.69; P=0.83, respectively), as well as from pupae to adults (P=0.77; P=0.88, respectively). Hence, the number of ovitraps positive for larvae (larval index) is still as effective as the number of ovitraps positive for pupae (pupal index). Since it is quite difficult in surveys to find containers with pupae, containers with larvae can also be a proxy for the number of adults that emerge, as shown in Figures 1 and 2 for ovitraps placed in the two conditions. The result from this study is a model for what can transpire in an environment.
Figure 1: Survival of Ae. albopictus in each developmental stage in natural breeding sites
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Figure 2: Survival of Ae. albopictus in each developmental stage in ovitraps placed under a cabin
Table 3: Percentage mortality and survival of Ae. albopictus in the ovitraps placed in natural habitat and under a cabin Treatment Natural breeding sites Temephos Bti Control Under a cabin Temephos Bti Control 265 198 316 167 (63.00) 132 (66.64) 303 (95.90) 0 0 97.68 0 0 94.22 306 425 400 181 (59.18) 223 (52.48) 399 (99.77) 0 0 96.25 0 0 90.34 No. of survival cases in each developmental stage (percentage) Eggs Larvae Pupae Adults
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This study also showed that the survival rate into adult stages in breeding containers in natural habitat and in semi-covered buildings was 90.34% and 94.22% respectively (Table 3), which is a good survival rate in natural environmental conditions, indicating that dengue or chikungunya transmission can be highly efficient and effective. Our study also showed that Ae. albopictus was not repelled by the treatment of either temephos or Bti. Hence, we can conclude that if Bti was used in the control programme, dispersing Bti by ultra-low volume (ULV) would not influence the oviposition behaviour of
Ae. albopictus in Malaysia. This, in fact, will also serve as a good control measure because containers which contain the dispersed Bti will be attracted to oviposition and larvae that hatch will eventually be killed by Bti.
Acknowledgements We would like to thank the Director-General, Ministry of Health, Malaysia, and the Director, Institute for Medical Research, Kuala Lumpur, for allowing us to publish this paper. Thanks are also due to the staff of the Medical Entomology Unit for their assistance.
References [1] Gubler DJ. Dengue and dengue hemorrhagic fever: Its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G, eds. Dengue and dengue haemorrhagic fever. Wallingford: CAB International, 1997. pp. 1-22. [2] Yao. Dengue Fever claims 81 lives in Malaysia. China View. 2008 Oct 25. http:// news.xinhuanet.com/english/2008-10/25/ content_10249418.htm – accessed 30 June 2010. [3] Centre of Disease Control. Dengue fever 2007. http://www.svinfectologia.org/Dengue%20 CDC%202007%5B1%5D.doc – accessed 30 June 2010. [4] Marchette NJ, Rudnick A, Garcia R. Alphaviruses in Peninsular Malaysia: II. Serological evidence of human infection. Southeast Asian Journal of Tropical Medicine and Public Health 1980;11(1):14-23. [5] Canyon D. Irritancy and repellency of Aedes aegypti (Diptera: Culicidae) to insecticides and implications for vector control operations. (http://www.tropmed.org/rreh/rrehi.htm – accessed 30 June 2010). [6] Lee HL. Aedes ovitrap and larval survey in several suburban communities in Selangor, Malaysia. Mosquito Borne Diseases Bulletin 1992;9(1):9-15. [7] Ritchie SA. The production of Aedes aegypti by a weekly ovitrap survey. Mosquito News 1984;44(1):77-79. [8] Kramer WL, Mulla MS. Oviposition attractants and repellents of mosquitoes: oviposition responses of Culex mosquitoes to organic infusions. Environmental Entomology 1979;8:1111-1117. [9] Geetha I, Paily KP , Padmanaban V, Balaraman K. Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride. Mem Inst Oswaldo Cruz 2003;98(2):223-226. [10] Craig RW, Katherine JL, Natasha JW, Veronica RS. The Allee effect in site choice behaviour of egg-laying dengue vector mosquitoes. Tropical Biomedicine 2008;25(2):140-144. [11] Allee WC. Animal aggregations, a study in general sociology . Chicago: University of Chicago Press, 1931.
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[12] Stephens PA, Sutherland WJ, Freckleton RP. What is the Allee effect? Oikos 1999;87:185-190. [13] Bentley MD, Day JF. Chemical ecology and behavioral aspects of mosquito oviposition. Annual Review of Entomology 1989;34:401-21. [14] Mogi M, Mokry J. Distribution of Wyeomyia smithii (Diptera: Culicidae) eggs in pitcher plants in Newfoundland, Canada. Tropical Medicine 1980;22:1-12. [15] Fay RW, Perry AS. Laboratory studies of the ovipositional preferences of Aedes aegypti. Mosquito News 1965;26:531-535. [16] Rozeboom LE, Rosen L, Ikeda J. Observations on oviposition by Aedes (S.) albopictus Skuse and A. (S.) polynesienses Marks in nature. Journal of Medical Entomology 1973;4:397-399. [17] Apostol BL, Black WC, Reiter P , Miller BR. Use of randomly amplified polymorphic DNA amplified by polymerase chain reaction markers to estimate the number of Aedes aegypti families at oviposition sites in San Juan, Puerto Rico. American Journal of Tropical Medicine and Hygiene 1994;51:89-97. [18] Kitron U, Webb DDW, Novak RJ. Oviposition behavior of Aedes triseriatus (Diptera: Culicidae): prevalence, intensity, and aggregation of eggs in oviposition traps. Journal of Medical Entomology 1989;26:462-467. [19] Chadee DD, Corbet PS, Greenwood JJD. Egg-laying yellow fever mosquitoes avoid sites containing eggs laid by themselves or by conspecifics. Entomologia Experimentalis et Applicata 1990;57:295-298. [20] Gubler DJ. Studies on the comparative oviposition behavior of Aedes (Stegomyia)
albopictus and Aedes (Stegomyia) polynesiensis Marks. Journal of Medical Entomology 1971;8:675-682. [21] Trexler JD, Apperson CS, Gemeno C, Perich MJ, Carlson D, Schal C. Field and laboratory evaluations of potential oviposition attractants for Aedes albopictus (Diptera: Culicidae). Journal of the American Mosquito Control Assocication 2003;19:228-234. [22] Sharma KR, Seenivasagan T, Rao AN, Ganesan K, Agarwal OP, Malhotra RC, Prakash S. Oviposition responses of Aedes aegypti and Aedes albopictus to certain fatty acid esters. Parasitology Research 2008;103(5):1065-1073. [23] Mather TN, DeFoliart GR Repellency and initial toxicity of Abate and Dursban formulations to Aedes triseriatus in oviposition sites. Mosquito News 1983;43:474-479. [24] Beehler JW, Mulla MS. Effect of the insect growth regulator methoprene on the ovipositional behavior of Aedes aegypti and Culex quinquefasciatus. Journal of the American Mosquito Control Association 1993;9:13-16. [25] Pates H, Curtis C. Mosquito behavior and vector control. Annual Review of Entomology 2005;50:53-70. [26] Santos SRA, Melo - Santos MAV, Regis L, Albuquerque CMR. Field evaluation consociated with grass infusion and Bacillus thuringiensis var. israelensis to determine oviposition rates of Aedes aegypti. Dengue Bulletin 2003;27:156-162. [27] Benzon GL, Apperson CS. Reexamination of chemically mediated oviposition behavior in Aedes aegypti (L.) (Diptera: Culicidae). Journal of Medical Entomology 1988;25(3):158-164.
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