Laboratory and Field Evaluations of Mosquiron® 0.12CRD, a New Formulation of Novaluron, Against Culex Mosquitoes Author(s): Tianyun Su, Min-Lee Cheng, Alfonso Melgoza and Jennifer Thieme Source: Journal of the American Mosquito Control Association, 30(4):284-290. 2014. Published By: The American Mosquito Control Association DOI: http://dx.doi.org/10.2987/14-6433R.1 URL: http://www.bioone.org/doi/full/10.2987/14-6433R.1
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Journal of the American Mosquito Control Association, 30(4):284–290, 2014 Copyright E 2014 by The American Mosquito Control Association, Inc.
LABORATORY AND FIELD EVALUATIONS OF MOSQUIRONH 0.12CRD, A NEW FORMULATION OF NOVALURON, AGAINST CULEX MOSQUITOES TIANYUN SU, MIN-LEE CHENG, ALFONSO MELGOZA
AND
JENNIFER THIEME
West Valley Mosquito and Vector Control District, 1295 E Locust Street, Ontario, CA 91761 ABSTRACT. Laboratory and field evaluations were conducted to assess the activity and efficacy of MosquironH 0.12CRD, a new formulation containing 0.12% novaluron, against immature Culex mosquitoes. In laboratory bioassays, this formulation was highly active against Culex quinquefasciatus as indicated by low inhibition of emergence (IE) values (IE50 and IE90). When Mosquiron 0.12CRD was applied at 1 briquet per underground stormwater vault, counts of late instars showed a significant reduction on day 28 posttreatment. When the late instars and pupae collected from Mosquiron-treated water were brought back to the laboratory for posttreatment observation, emergence inhibition was .90% on day 7. When the late instars from a laboratory colony of Cx. quinquefasciatus were exposed to the Mosquiron-treated water, 99% and 95% emergence inhibition was noted on day 7 and day 14, respectively. When Mosquiron 0.12CRD was applied at 11 briquets per vault, significant reductions of larval populations were encountered on days 7 and 35 posttreatment for early instars, and on days 14, 21, and 35 posttreatment for late instars. Laboratory observation of late instars and pupae sampled from the treated vault water showed nearly complete emergence inhibition from day 7 to day 28 posttreatment. A similar trend was observed in laboratory-reared late instars of Cx. quinquefasciatus when exposed to the treated water. Preliminary evaluations indicated that Mosquiron 0.12CRD is a useful new tool to control Culex mosquitoes breeding in persistent sources. KEY WORDS
Mosquiron, novaluron, insect growth regulator, mosquito control
INTRODUCTION Larviciding is the most efficient method in mosquito control operations. However, there have been very few new active ingredients or formulations registered for larval control during the past decades. Today, Bacillus thuringiensis var. israelensis de Barjac, B. sphaericus Neide, methoprene, spinosad, oils, and a few others are the only active ingredients registered for mosquito control in the USA. Screening and development of new bioactive agents against mosquito larvae are urgently needed. Insect growth regulators, including juvenile hormone analogs and chitin synthesis inhibitors, could potentially play an important role in mosquito larviciding operations (Mulla 1995), but they currently do less than desired. Novaluron, a chitin synthesis inhibitor, was developed by Makhteshim-Chemical Works, Ltd. (Beer-Sheva, Israel) over a decade ago. Makhteshim-Agan of North America, Inc. (Raleigh, NC) registered 2 novaluron formulations in 2003 with the US Environmental Protection Agency (USEPA): RimonH Technical, a manufacturing use product, and Rimon 10 EC, an end-use product, containing 98.5% and 10.0% active ingredient, respectively. Rimon 10 EC is registered to control whiteflies, thrips, and leafminers. As a new chitin synthesis inhibitor, novaluron has been shown to have high levels of activity by both ingestion and contact against numerous arthropod pests of agricultural, forestry, and public health importance, including species in the orders Coleoptera (Cutler et al. 2005, 2007), Hemiptera (Barbour 2007), Lepidoptera (Ishaaya et al. 2003, Beuzelin et al. 2010, Kim
et al. 2011), and Diptera (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Cetin et al. 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Lohmeyer and Pound 2012, Rajasekar and Jebanesan 2012). Prior to the registration of commercial formulations of novaluron by the USEPA for mosquito control, all earlier laboratory and field studies on activity and efficacy against mosquitoes were based on either technical-grade active ingredient or various formulations registered for controlling pestiferous arthropods of agricultural, forestry, or greenhouse industries (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Rajasekar and Jebanesan 2012). A controlledrelease briquet formulation, MosquironH 0.12CRD (Makhteshim Agan North America Inc.), was first registered with USEPA in May 2012 and with Health Canada in 2014 for mosquito control. In the current studies, we conducted laboratory bioassays and field evaluations under Pesticide Research Authorization 1306039, Department of Pesticide Regulation, State of California, to determine the larvicidal and pupicidal activity and efficacy of Mosquiron 0.12CRD. MATERIALS AND METHODS Test materials Mosquiron 0.12CRD (Lot No. 1012818, EPA Reg. 66222-232-89382, 0.12% novaluron, average
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total weight: 13.8 g/each) was provided by Tumaini Controlled Release Technologies Inc., Hamilton, ON, Canada, on June 24, 2013, for laboratory and field evaluations against Culex mosquitoes. This formulation is a whitish, waxy briquet that measured approximately 35 3 35 3 17 mm. Mosquiron 0.12CRD can only be applied to sites that do not drain directly into natural water bodies. The label rate is 1 Mosquiron 0.12CRD briquet per 189 liters of water with low organic matter content. Rate is doubled when application is made to habitats with high organic matter content. Laboratory evaluation Laboratory evaluation was conducted against Culex quinquefasciatus Say using standard bioassay protocols described in Su et al. (2003). One Mosquiron 0.12CRD briquet was arbitrarily selected from the shipment, and was shaved off using a razor blade. Particles were evenly suspended in tap water by vortexing for 3 min. In each test, 4 concentrations within the activity range resulting in approximately 5–95% mortality (Su et al. 2003) were used with 3 replicates for each concentration. One untreated control was set up in each concentration. Each 180-ml Styrofoam cup contained 100 ml of tap water (not aged) and 25 early 4th instars. Approximately 0.1 g of rabbit chow was added to each cup as larval food after the larvae were introduced in the cups. Water lost due to evaporation was replenished every other day. In order to obtain time-specific mortality data and to minimize impact on mortality reading by surviving larvae potentially feeding on dead individuals and exuviae, as well as decomposition of dead individuals and exuviae, mortality at larval, pupal (including larval–pupal intermediates), and adult stages (adults still attached to pupal exuviae) was assessed on a daily basis until all individuals died or successfully emerged as adults. Successful adult emergence was evaluated by counting free pupal exuviae from the cups. Bioassays were conducted under a photoperiod of 16L:8D and room temperature 24–26uC in an insectary. The 1st underground stormwater vault study Field trial: In total, 15 underground stormwater vaults (manholes) with constant mosquito breeding were chosen in the city of Ontario, CA. Sampling of immature mosquitoes in underground vaults was accomplished by using a dipper attached to an extendable aluminum pole. For pretreatment sampling, 5 dip samples were taken from each vault at 3- to 5-min intervals to allow larvae and pupae to resurface. Collected immature mosquitoes were categorized as early (1st–2nd) instars and late (3rd–4th) instars and
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pupae. In this round of field study, Mosquiron 0.12CRD was applied at 1 briquet/vault (minimum manageable dose). To attempt determination of posttreatment residual weight, each briquet was placed in a nylon pouch (approximately 500-mm mesh) that was tied to a string for retrieval. The string was then tied to the ladder on the side wall or the lid of the manholes. Vaults were sampled weekly posttreatment in the same manner as the pretreatment until day 28 posttreatment when the inhibition of emergence declined considerably as indicated in laboratory bioassay. New cups for holding and transporting larvae and pupae were used on each sampling day to avoid cross contaminations. No vaults were left as untreated control because of concern of excessive adult emergence during hot summer. Species composition of larvae collected was determined by identification of 25–30 late instars on each sampling day. Laboratory observations: On each sampling day, variable numbers of late instars and pupae were collected from each treated site if available, and kept in 335 ml of treated water collected from the same vault in a 473-ml plastic cup. Samples were transported to the insectary in an ice chest with blue ice. Preliminary observations indicated that there was a great deal of variation in the size of vaults, the amount of water in the vaults, the visual quality and eutrophication levels of the vault water, as well as the presence of immature mosquitoes. In order to even out the Mosquiron 0.12CRD AI concentration and the differences in water quality of the water samples for laboratory observations, an equal aliquot (44 ml) was taken from each of the 15 cups containing the vault water, and was pooled to a 14.5-cm 3 6-cm 3 10cm plastic container, resulting in a total volume of 660 ml. Two plastic containers held the pooled vault water: one was used for assaying the pooled late instars and pupae collected from the 15 vaults, and the other for assaying the late instars from a laboratory colony of Cx. quinquefasciatus. On each sampling day, 100–150 late instars (and some pupae for field-collected samples) were assayed. Both plastic containers for bioassay were kept in a screened mosquito cage (30 3 30 3 30 cm) at 24–26uC. Larval and pupal mortality was recorded on a daily basis until all individuals either died or emerged as adults. New plastic containers were used in each bioassay to avoid cross contamination. The 2nd underground stormwater vault study Field trial: In the 2nd round of field study, 11 Mosquiron 0.12CRD briquets were applied to each of 4 underground vaults. The detailed procedures for treatment and sampling were the same as described previously.
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Fig. 2. Dead pupa and incompletely emerged adult in response to MosquironH 0.12CRD exposure in laboratory bioassay against Culex quinquefasciatus.
RESULTS Laboratory evaluation
Fig. 1. Cumulative mortality (upper) and stagespecific mortality with inhibition of emergence (IE) IE50 and IE90 with 95% confidence intervals (lower) against Culex quinquefasciatus by MosquironH 0.12CRD in laboratory bioassay.
Laboratory observation: The detailed procedures for laboratory observations against fieldcollected larvae and pupae or laboratory colonized larvae were the same as described previously, except that 4 replicates were used, and the water amount transferred from each cup was about 165 ml to make each of 2 pooled water samples (660 ml) used in bioassays against field-collected larvae and pupae, or laboratory-reared larvae. Data analysis To analyze data from laboratory bioassay, mortalities obtained at each of the 4 concentrations were subjected to probit regression analysis with POLO-PC (LeOra Software 1987) to determine inhibition of emergence (IE) IE50 and IE90 values and their 95% confidence intervals (CI). Average dip counts of immature mosquitoes on each sampling day were subjected to 1-way ANOVA for repeated measures to determine the significance in population densities among all sampling days posttreatment. The total mortality of immature mosquitoes from the field or laboratory colony exposed to untreated or treated water collected on each sampling day was analyzed by chi-square test to explore the significance in efficacy among all sampling days posttreatment (StatView + Graphics [Abacus Concepts, Inc. 1987]).
In laboratory studies against 4th instars from a reference colony of Cx. quinquefasciatus, Mosquiron 0.12CRD showed IE50 0.470 (95% CI 5 0.376–0.587) ppb, and IE90 2.359 (95% CI 5 1.703–3.681) ppb. The daily cumulative mortality increased gradually from days 1 to 4, went up rapidly during days 5 and 6, and reached a plateau on day 7 posttreatment. At that time, a concentration-dependent mortality trend was distinctively established: very few individuals died at the lowest concentration of 0.05 ppb, while very few individuals survived at the highest concentration of 2.5 ppb. The majority of the individuals died as incompletely emerged adults or at the pupal stage, while some died as larvae, particularly at higher test concentrations (Figs. 1 and 2). The 1st underground stormwater vault study Field dip sampling: A single Mosquiron 0.12CRD briquet applied to each vault became completely disintegrated in the nylon pouch when retrieved from water on day 7 posttreatment. The counts of early instars and pupae were low throughout the test period. The densities of late instars increased on day 7 posttreatment, followed by a gradual decline from day 7 to day 28 posttreatment. Densities on day 28 were significantly lower than those on days 7 and 14 posttreatment (df 5 1, 28, F 5 5.25, P , 0.05) (Fig. 3A). Among the larvae collected, Cx. quinquefasciatus was the predominant species (92.2–97.1%), while Cx. stigmatosoma Dyar (2.2–6.4%) and Culiseta incidens (Thompson) (0.7–1.4%) were encountered at much lower numbers. Laboratory observations: Laboratory observation on late instars and pupae collected on each sampling day along with the treated water showed negligible emergence inhibition pretreatment, but
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Fig. 3. (A) Population density changes of wild Culex populations (average no./dip, df 5 1, 28, F $ 5.25, P , 0.05) (upper) and overall mortality by treated water against in situ field-collected Culex larvae and pupae (middle) or Culex quinquefasciatus larvae from laboratory colony (bottom), in response to treatment by MosquironH 0.12CRD at 1 briquet/vault (x2 $ 3.83, P , 0.05). (B) Population density changes of wild Culex populations (average no./dip, df 5 1, 6, F $ 5.71, P , 0.05) (upper) and overall mortality by treated water against in situ fieldcollected Culex larvae and pupae (middle) or Culex quinquefasciatus larvae from laboratory colony (bottom), in response to treatment by MosquironH 0.12CRD at 11 briquets/vault (x2 $ 3.83, P , 0.05).
nearly complete inhibition on day 7, which significantly decreased to 72% on day 14, and 56% on day 21 and day 28 posttreatment (x2 $ 3.83, P , 0.05). The late instars from a laboratory colony of Cx. quinquefasciatus showed a slight emergence inhibition pretreatment, but nearly 100% inhibition on day 7, and 95% on day 14, and then significantly reduced to 21% and 13% on day 21 and day 28 posttreatment, respectively (x2 $ 3.83, P , 0.05). For stage-specific mortality, on day 7 posttreatment, more individuals died at the larval stage, where larval mortality accounted for 82.9% out of the total morality of 98.4% for the fieldcollected larvae, and 67.6% out of the 99.1% for the laboratory-colonized larvae. With posttreatment progression of time, more individuals died at transition from pupae to adults, where mortality at incompletely emerged adults was 49.2% out of the total mortality of 57.1% for the field-
collected larvae, and 10.7% out of the 13.4% for the laboratory-colonized larvae. Assays on field-collected larvae/pupae and on laboratorycolonized larvae showed overall similar patterns (Fig. 3A). The 2nd underground stormwater vault study Field dip sampling: At higher concentrations of Mosquiron (11 briquets of Mosquiron 0.12CRD per vault), it was also noticed that all the briquets disintegrated in the nylon pouch on day 7 posttreatment. The densities of early instars were significantly suppressed on days 7 and 35 posttreatment (df 5 1, 6, F $ 5.71, P , 0.05). The late instars were significantly reduced on days 14, 21, and 35 posttreatment (df 5 1, 6, F $ 8.01, P , 0.05). Pupal counts were low throughout the test period (Fig. 3B). The species composition was similar to that observed in the 1st
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occurred at larval stage for the field-collected larvae, and 94.0% out of the 100% mortality happened in larval stage for the laboratorycolonized larvae. On day 42 posttreatment, however, mortality at incompletely emerged adults was 39.6% out of the total mortality of 43.1% for the field-collected larvae, and 5.1% of the 23.2% for the laboratory-colonized larvae. Overall, the proportions of larval mortality were higher in the 2nd study at higher dose than in 1st study at the lower dose (Fig. 3B). It was noticed that some 4th instars of Cx. quinquefasciatus collected on day 21 posttreatment showed some opaqueness in the head, thorax, and some abdominal segments, indicating that the cuticle might have been detached. Furthermore, each abdominal segment also appeared to be compressed (Fig. 4). DISCUSSION
Fig. 4. A 4th instar of Culex quinquefasciatus collected from an underground vault on day 21 posttreatment by MosquironH 0.12CRD at 11 briquets/ vault.
round of study; Cx. quinquefasciatus remained the predominant species (90.8–94.7%), followed by Cx. stigmatosoma (4.4–5.2%) and Cs. incidens (0.9–4.0%). Laboratory observations: The larvae and pupae collected from the field sites pretreatment showed very low level of emergence inhibition, while nearly complete emergence inhibition was observed in the larvae and pupae collected from day 7 to day 28; efficacy declined significantly on day 35 and day 42 posttreatment when the test was concluded (x2 $ 3.83, P , 0.05). The late instars from a laboratory colony of Cx. quinquefasciatus exhibited a similar pattern of emergence inhibition. Larvae exposed to the water samples collected pretreatment pupated and emerged as adults with very low level of emergence inhibition; ones that were exposed to the treated water showed 91–100% inhibition from day 7 to day 35, then significantly declined to 23% on day 42 posttreatment (x2 $ 3.83, P , 0.05) (Fig. 3B). The stage-specific mortality pattern was similar to that in the 1st underground vault study, i.e., more larval mortality occurred during earlier days posttreatment, while more individuals died during adult emergence during later sampling days. Briefly, on day 7 posttreatment, all mortality
Novaluron is a newly recognized chitin synthesis inhibitor against insects belonging to Coleoptera, Hemiptera, Lepidoptera, and Diptera. Technical-grade novaluron or Rimon 10EC has been tested against the following mosquito species, including Cx. quinquefasciatus, Anopheles albimanus Wiedemann, An. pseudopunctipennis Theobald, Aedes aegypti (L.), and Ae. albopictus (Skuse) (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Rajasekar and Jebanesan 2012). Furthermore, novaluron showed high activity against Ae. aegypti that was resistant to conventional chemical insecticides such as organophosphates (Fontoura et al. 2012), or Cx. quiquefasciatus as highly resistant to the biopesticide spinosad (Su and Cheng 2014). Novaluron is recommended by the World Health Organization for use in drinking water (WHO 2008), which provides the opportunity of application to control Ae. aegypti and other mosquitoes breeding in water storage containers in tropical and subtropical regions. Mosquiron 0.12CRD is the first formulation of novaluron registered for mosquito control in the USA. The activity against immature mosquitoes observed in this study was significantly lower than the technical-grade novaluron published previously by Su et al. (2003), who reported 0.118 ppb for IE50 and 0.595 ppb for IE90 against 4th instars of Cx. quinquefasciatus, and by Mulla et al. (2003), who found 0.045 ppb for IE50 and 0.160 for IE90 in 4th instars of Ae. aegypti. Furthermore, the stage-specific mortality demonstrated by Su et al. (2003) against Cx. quinquefasciatus and Mulla et al. (2003) against Ae. aegypti showed that the majority of the exposed individuals died at pupal stage or incomplete emergence at lower concentrations, while mortal-
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ity at larval stage substantially increased at high concentrations. In the current study, however, vast majority of exposed late instars died as incompletely emerged adults or adultoids, even at the higher concentrations. The stage-specific mortality observed during bioassays seemed somewhat in contrast to that in chitin synthesis inhibitor, rather similar to that of a juvenoid such as methoprene and pyriproxyfen (Su and Cheng 2014). The briquets applied were observed to have disintegrated completely on day 7 posttreatment. The expected residual efficacy did not appear to depend on controlled disposal of the inert carriers together with the AI off the briquet. The product evaluated was labeled at 1–2 CRD per 189 liters of water in mosquito breeding sources depending on organic matter content. Without knowing the exact amount of water and unpredictable flushing events in the underground vault system selected for the studies, Mosquiron 0.12CRD was applied at the minimum manageable dose of 1 briquet/ vault, with the goal to explore the minimum effective dose. The efficacy level was limited as indicated by the moderate and gradual reduction of larval populations. However, moderate to high level of emergence inhibition was noticed in Culex larvae and pupae collected from the treated habitats, as well as in the laboratory bioassays against laboratory colony of Cx. quinquefasciatus. It seemed that the novaluron concentration when applied at 1 briquet/vault was inadequate to significantly suppress the natural Culex populations breeding in underground vaults in a timely manner. In the 2nd field evaluation, the formulation was applied at 11 briquets per vault in order to yield higher levels of control. In this study, the initial natural larval populations were considerably higher than in the 1st field study. The early and longer efficacy as indicated by counts of early and late instars was achieved. At the same time, high activity of emergence inhibition in treated water persisted for 35 days in the laboratory observation against field-collected larvae and pupae, as well as larvae from laboratory colony. It was noticed in this field study that some 4th instars of Cx. quinquefasciatus collected on day 21 posttreatment from all treated sites showed opaqueness in the head, thorax, and abdominal regions, which may signify cuticle detachment. Further, the segments in the head, thorax, and abdomen appeared compressed. It is possible that these morphological alterations resulted from intoxication of novaluron (Farnesi et al. 2012). The morphological changes mentioned above may have occurred earlier than 21 days posttreatment, which warrants further detailed studies in the future. Mosquiron 0.12CRD showed high activity of emergence inhibition against Cx. quinquefasciatus in the laboratory and field. A couple of other
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mosquito species were also among the targets in the field. Dose-dependent initial and residual efficacy was demonstrated in the field when Mosquiron 0.12CRD was applied at 1 and 11 briquets per vault. Larval and pupal isolation, i.e., holding field-collected larvae or pupae in treated water collected from the field sites and reading emergence inhibition later in the laboratory, is one of the commonly used methods to estimate control efficacy. Bioassay on the treated water against mosquito larvae from laboratory colony is another recognized way to determine the efficacy of the field treatment of insect growth regulator formulations (Mulla 1991, 1995). According to the observations in the current studies, field efficacy of Mosquiron CRD could be underestimated by dip sampling of immature mosquitoes when one compares dip counts with laboratory bioassays on field-collected or laboratory-reared larvae. ACKNOWLEDGMENTS Special thanks are due to the following industrial partners: Makhteshim Agan North America Inc., Raleigh, NC, USA; Tumaini Controlled Release Technologies Inc., Hamilton, ON, Canada; and Pestalto Environmental Products Inc., Guelph, ON, Canada. The general assistance received during the studies from operations and laboratory departments in West Valley Mosquito and Vector Control District is duly acknowledged. REFERENCES CITED Abacus Concepts, Inc. 1987. StatView + Graphics. Berkeley, CA: Abacus Concepts, Inc. Arredondo-Jime´nez JI, Valdez-Delgado KM. 2006. Effect of novaluron (Rimon 10 EC) on the mosquitoes Anopheles albimanus, Anopheles pseudopunctipennis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Chiapas, Mexico. Med Vet Entomol 20:377–387. Barbour JD. 2007. Insecticide resistance and efficacy of novaluron and flonicamid for control of lygus bugs in alfalfa seed. In: Ellsworth PE, Naranjo S, eds. Proceedings: 2nd International Lygus Symposium. 2007 April 15–19; Asilomar, CA. J Insect Sci. doi: http://dx.doi.org/10.1673/031.008.4901. Beuzelin JM, Akbar W, Me´sza´ros A, Reay-Jones FPF, Reagan TE. 2010. Field assessment of novaluron for sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), management in Louisiana sugarcane. Crop Prot 29:1168–1176. Cetin H, Erler F, Yanikoglu A. 2006. Larvicidal activity of novaluron, a chitin synthesis inhibitor, against the housefly, Musca domestica. J Insect Sci 6:50–54. Cutler GC, Scott-Dupree CD, Tolman JH, Harris CR. 2005. Acute and sublethal toxicity of novaluron, a novel chitin synthesis inhibitor, to Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Pest Manag Sci 61:1060–1068.
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Cutler GC, Scott-Dupree CD, Tolman JH, Harris CR. 2007. Field efficacy of novaluron for control of Colorado potato beetle (Coleoptera: Chrysomelidae) on potato. Crop Prot 26:760–767. Farnesi LC, Brito JM, Linss JG, Pelajo-Machado M, Valle D, Rezende GL. 2012. Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron. PLoS One 7:e30363. doi:10.1371/journal. pone.0030363. Fontoura NG, Bellinato DF, Valle D, Lima JB. 2012. The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo Cruz 107: 387–395. Ishaaya I, Kontsedalov S, Horowitz AR. 2003. Novaluron (Rimon), a novel IGR: potency and cross resistance. Arch Insect Biochem Physiol 54:157–164. Jambulingam P, Sadanandane C, Nithiyananthan N, Subramanian S, Zaim M. 2009. Efficacy of novaluron against Culex quinquefasciatus in small- and medium-scale trials, India. J Am Mosq Control Assoc 25:315–322. Kim SH, Wise JC, Go ¨ kc¸e A, Whalon ME. 2011. Novaluron causes reduced egg hatch after treating adult codling moths, Cydia pomenella: support for transovarial transfer. J Insect Sci 11:126. doi:10.1673/ 031.011.12601. LeOra Software. 1987. POLO-PC: a user’s guide to probit or logit analysis. Berkeley, CA: LeOra Software. Lohmeyer KH, Pound JM. 2012. Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae). J Med Entomol 49:647–651. Mulla MS. 1991. Insect growth regulators for the control of mosquito pests and disease vectors. In: Chinese Journal of Entomology Special Publication
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No. 6: Proceedings of the 4th National Vector Control Symposium. Taichung, Taiwan, ROC. p 81–91. Mulla MS. 1995. The future of insect growth regulators in vector control. J Am Mosq Control Assoc 11:269–273. Mulla MS, Thavara U, Tawatsin A, Chompoosri J, Zaim M, Su T. 2003. Laboratory and field evaluation of novaluron, a new acylurea insect growth regulator, against Aedes aegypti (Diptera: Culicidae). J Vector Ecol 28:241–254. Rajasekar P, Jebanesan A. 2012. Efficacy of IGRs compound novaluron and buprofezin against Culex quinquefasciatus mosquito larvae and pupal control in pools, drains and tanks. Int J Res Biol Sci 2:45–47. Sfara V, De Licastro SA, Masuh HM, Seccacini EA, Alzogaray RA, Zerba EN. 2007. Synergism between cis-permethrin and benzoyl phenyl urea insect growth regulators against Aedes aegypti larvae. J Am Mosq Control Assoc 23:24–28. Su T, Cheng ML. 2014. Cross resistances in spinosadresistant Culex quinquefasciatus (Diptera: Culicidae). J Med Entomol 51:428–435. Su T, Mulla MS, Zaim M. 2003. Laboratory and field evaluations of novaluron, a new insect growth regulator (IGR), against Culex mosquitoes. J Am Mosq Control Assoc 19:408–418. Tawatsin A, Thavara U, Bhakdeenuan P, Chompoosri J, Siriyasatien P, Asavadachanukorn P, Mulla MS. 2007. Field evaluation of novaluron, a chitin synthesis inhibitor larvicide, against mosquito larvae in polluted water in urban areas of Bangkok, Thailand. Southeast Asian J Trop Med Public Health 38:434–441. WHO [World Health Organization]. 2008. Novaluron in drinking-water: use for vector control in drinking-water sources and containers. Background document for preparation of WHO guidelines for drinking water quality. WHO/HSE/AMR/08.03/11. Geneva, Switzerland: World Health Organization.
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Journal of the American Mosquito Control Association, 30(4):284–290, 2014 Copyright E 2014 by The American Mosquito Control Association, Inc.
LABORATORY AND FIELD EVALUATIONS OF MOSQUIRONH 0.12CRD, A NEW FORMULATION OF NOVALURON, AGAINST CULEX MOSQUITOES TIANYUN SU, MIN-LEE CHENG, ALFONSO MELGOZA
AND
JENNIFER THIEME
West Valley Mosquito and Vector Control District, 1295 E Locust Street, Ontario, CA 91761 ABSTRACT. Laboratory and field evaluations were conducted to assess the activity and efficacy of MosquironH 0.12CRD, a new formulation containing 0.12% novaluron, against immature Culex mosquitoes. In laboratory bioassays, this formulation was highly active against Culex quinquefasciatus as indicated by low inhibition of emergence (IE) values (IE50 and IE90). When Mosquiron 0.12CRD was applied at 1 briquet per underground stormwater vault, counts of late instars showed a significant reduction on day 28 posttreatment. When the late instars and pupae collected from Mosquiron-treated water were brought back to the laboratory for posttreatment observation, emergence inhibition was .90% on day 7. When the late instars from a laboratory colony of Cx. quinquefasciatus were exposed to the Mosquiron-treated water, 99% and 95% emergence inhibition was noted on day 7 and day 14, respectively. When Mosquiron 0.12CRD was applied at 11 briquets per vault, significant reductions of larval populations were encountered on days 7 and 35 posttreatment for early instars, and on days 14, 21, and 35 posttreatment for late instars. Laboratory observation of late instars and pupae sampled from the treated vault water showed nearly complete emergence inhibition from day 7 to day 28 posttreatment. A similar trend was observed in laboratory-reared late instars of Cx. quinquefasciatus when exposed to the treated water. Preliminary evaluations indicated that Mosquiron 0.12CRD is a useful new tool to control Culex mosquitoes breeding in persistent sources. KEY WORDS
Mosquiron, novaluron, insect growth regulator, mosquito control
INTRODUCTION Larviciding is the most efficient method in mosquito control operations. However, there have been very few new active ingredients or formulations registered for larval control during the past decades. Today, Bacillus thuringiensis var. israelensis de Barjac, B. sphaericus Neide, methoprene, spinosad, oils, and a few others are the only active ingredients registered for mosquito control in the USA. Screening and development of new bioactive agents against mosquito larvae are urgently needed. Insect growth regulators, including juvenile hormone analogs and chitin synthesis inhibitors, could potentially play an important role in mosquito larviciding operations (Mulla 1995), but they currently do less than desired. Novaluron, a chitin synthesis inhibitor, was developed by Makhteshim-Chemical Works, Ltd. (Beer-Sheva, Israel) over a decade ago. Makhteshim-Agan of North America, Inc. (Raleigh, NC) registered 2 novaluron formulations in 2003 with the US Environmental Protection Agency (USEPA): RimonH Technical, a manufacturing use product, and Rimon 10 EC, an end-use product, containing 98.5% and 10.0% active ingredient, respectively. Rimon 10 EC is registered to control whiteflies, thrips, and leafminers. As a new chitin synthesis inhibitor, novaluron has been shown to have high levels of activity by both ingestion and contact against numerous arthropod pests of agricultural, forestry, and public health importance, including species in the orders Coleoptera (Cutler et al. 2005, 2007), Hemiptera (Barbour 2007), Lepidoptera (Ishaaya et al. 2003, Beuzelin et al. 2010, Kim
et al. 2011), and Diptera (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Cetin et al. 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Lohmeyer and Pound 2012, Rajasekar and Jebanesan 2012). Prior to the registration of commercial formulations of novaluron by the USEPA for mosquito control, all earlier laboratory and field studies on activity and efficacy against mosquitoes were based on either technical-grade active ingredient or various formulations registered for controlling pestiferous arthropods of agricultural, forestry, or greenhouse industries (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Rajasekar and Jebanesan 2012). A controlledrelease briquet formulation, MosquironH 0.12CRD (Makhteshim Agan North America Inc.), was first registered with USEPA in May 2012 and with Health Canada in 2014 for mosquito control. In the current studies, we conducted laboratory bioassays and field evaluations under Pesticide Research Authorization 1306039, Department of Pesticide Regulation, State of California, to determine the larvicidal and pupicidal activity and efficacy of Mosquiron 0.12CRD. MATERIALS AND METHODS Test materials Mosquiron 0.12CRD (Lot No. 1012818, EPA Reg. 66222-232-89382, 0.12% novaluron, average
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total weight: 13.8 g/each) was provided by Tumaini Controlled Release Technologies Inc., Hamilton, ON, Canada, on June 24, 2013, for laboratory and field evaluations against Culex mosquitoes. This formulation is a whitish, waxy briquet that measured approximately 35 3 35 3 17 mm. Mosquiron 0.12CRD can only be applied to sites that do not drain directly into natural water bodies. The label rate is 1 Mosquiron 0.12CRD briquet per 189 liters of water with low organic matter content. Rate is doubled when application is made to habitats with high organic matter content. Laboratory evaluation Laboratory evaluation was conducted against Culex quinquefasciatus Say using standard bioassay protocols described in Su et al. (2003). One Mosquiron 0.12CRD briquet was arbitrarily selected from the shipment, and was shaved off using a razor blade. Particles were evenly suspended in tap water by vortexing for 3 min. In each test, 4 concentrations within the activity range resulting in approximately 5–95% mortality (Su et al. 2003) were used with 3 replicates for each concentration. One untreated control was set up in each concentration. Each 180-ml Styrofoam cup contained 100 ml of tap water (not aged) and 25 early 4th instars. Approximately 0.1 g of rabbit chow was added to each cup as larval food after the larvae were introduced in the cups. Water lost due to evaporation was replenished every other day. In order to obtain time-specific mortality data and to minimize impact on mortality reading by surviving larvae potentially feeding on dead individuals and exuviae, as well as decomposition of dead individuals and exuviae, mortality at larval, pupal (including larval–pupal intermediates), and adult stages (adults still attached to pupal exuviae) was assessed on a daily basis until all individuals died or successfully emerged as adults. Successful adult emergence was evaluated by counting free pupal exuviae from the cups. Bioassays were conducted under a photoperiod of 16L:8D and room temperature 24–26uC in an insectary. The 1st underground stormwater vault study Field trial: In total, 15 underground stormwater vaults (manholes) with constant mosquito breeding were chosen in the city of Ontario, CA. Sampling of immature mosquitoes in underground vaults was accomplished by using a dipper attached to an extendable aluminum pole. For pretreatment sampling, 5 dip samples were taken from each vault at 3- to 5-min intervals to allow larvae and pupae to resurface. Collected immature mosquitoes were categorized as early (1st–2nd) instars and late (3rd–4th) instars and
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pupae. In this round of field study, Mosquiron 0.12CRD was applied at 1 briquet/vault (minimum manageable dose). To attempt determination of posttreatment residual weight, each briquet was placed in a nylon pouch (approximately 500-mm mesh) that was tied to a string for retrieval. The string was then tied to the ladder on the side wall or the lid of the manholes. Vaults were sampled weekly posttreatment in the same manner as the pretreatment until day 28 posttreatment when the inhibition of emergence declined considerably as indicated in laboratory bioassay. New cups for holding and transporting larvae and pupae were used on each sampling day to avoid cross contaminations. No vaults were left as untreated control because of concern of excessive adult emergence during hot summer. Species composition of larvae collected was determined by identification of 25–30 late instars on each sampling day. Laboratory observations: On each sampling day, variable numbers of late instars and pupae were collected from each treated site if available, and kept in 335 ml of treated water collected from the same vault in a 473-ml plastic cup. Samples were transported to the insectary in an ice chest with blue ice. Preliminary observations indicated that there was a great deal of variation in the size of vaults, the amount of water in the vaults, the visual quality and eutrophication levels of the vault water, as well as the presence of immature mosquitoes. In order to even out the Mosquiron 0.12CRD AI concentration and the differences in water quality of the water samples for laboratory observations, an equal aliquot (44 ml) was taken from each of the 15 cups containing the vault water, and was pooled to a 14.5-cm 3 6-cm 3 10cm plastic container, resulting in a total volume of 660 ml. Two plastic containers held the pooled vault water: one was used for assaying the pooled late instars and pupae collected from the 15 vaults, and the other for assaying the late instars from a laboratory colony of Cx. quinquefasciatus. On each sampling day, 100–150 late instars (and some pupae for field-collected samples) were assayed. Both plastic containers for bioassay were kept in a screened mosquito cage (30 3 30 3 30 cm) at 24–26uC. Larval and pupal mortality was recorded on a daily basis until all individuals either died or emerged as adults. New plastic containers were used in each bioassay to avoid cross contamination. The 2nd underground stormwater vault study Field trial: In the 2nd round of field study, 11 Mosquiron 0.12CRD briquets were applied to each of 4 underground vaults. The detailed procedures for treatment and sampling were the same as described previously.
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Fig. 2. Dead pupa and incompletely emerged adult in response to MosquironH 0.12CRD exposure in laboratory bioassay against Culex quinquefasciatus.
RESULTS Laboratory evaluation
Fig. 1. Cumulative mortality (upper) and stagespecific mortality with inhibition of emergence (IE) IE50 and IE90 with 95% confidence intervals (lower) against Culex quinquefasciatus by MosquironH 0.12CRD in laboratory bioassay.
Laboratory observation: The detailed procedures for laboratory observations against fieldcollected larvae and pupae or laboratory colonized larvae were the same as described previously, except that 4 replicates were used, and the water amount transferred from each cup was about 165 ml to make each of 2 pooled water samples (660 ml) used in bioassays against field-collected larvae and pupae, or laboratory-reared larvae. Data analysis To analyze data from laboratory bioassay, mortalities obtained at each of the 4 concentrations were subjected to probit regression analysis with POLO-PC (LeOra Software 1987) to determine inhibition of emergence (IE) IE50 and IE90 values and their 95% confidence intervals (CI). Average dip counts of immature mosquitoes on each sampling day were subjected to 1-way ANOVA for repeated measures to determine the significance in population densities among all sampling days posttreatment. The total mortality of immature mosquitoes from the field or laboratory colony exposed to untreated or treated water collected on each sampling day was analyzed by chi-square test to explore the significance in efficacy among all sampling days posttreatment (StatView + Graphics [Abacus Concepts, Inc. 1987]).
In laboratory studies against 4th instars from a reference colony of Cx. quinquefasciatus, Mosquiron 0.12CRD showed IE50 0.470 (95% CI 5 0.376–0.587) ppb, and IE90 2.359 (95% CI 5 1.703–3.681) ppb. The daily cumulative mortality increased gradually from days 1 to 4, went up rapidly during days 5 and 6, and reached a plateau on day 7 posttreatment. At that time, a concentration-dependent mortality trend was distinctively established: very few individuals died at the lowest concentration of 0.05 ppb, while very few individuals survived at the highest concentration of 2.5 ppb. The majority of the individuals died as incompletely emerged adults or at the pupal stage, while some died as larvae, particularly at higher test concentrations (Figs. 1 and 2). The 1st underground stormwater vault study Field dip sampling: A single Mosquiron 0.12CRD briquet applied to each vault became completely disintegrated in the nylon pouch when retrieved from water on day 7 posttreatment. The counts of early instars and pupae were low throughout the test period. The densities of late instars increased on day 7 posttreatment, followed by a gradual decline from day 7 to day 28 posttreatment. Densities on day 28 were significantly lower than those on days 7 and 14 posttreatment (df 5 1, 28, F 5 5.25, P , 0.05) (Fig. 3A). Among the larvae collected, Cx. quinquefasciatus was the predominant species (92.2–97.1%), while Cx. stigmatosoma Dyar (2.2–6.4%) and Culiseta incidens (Thompson) (0.7–1.4%) were encountered at much lower numbers. Laboratory observations: Laboratory observation on late instars and pupae collected on each sampling day along with the treated water showed negligible emergence inhibition pretreatment, but
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Fig. 3. (A) Population density changes of wild Culex populations (average no./dip, df 5 1, 28, F $ 5.25, P , 0.05) (upper) and overall mortality by treated water against in situ field-collected Culex larvae and pupae (middle) or Culex quinquefasciatus larvae from laboratory colony (bottom), in response to treatment by MosquironH 0.12CRD at 1 briquet/vault (x2 $ 3.83, P , 0.05). (B) Population density changes of wild Culex populations (average no./dip, df 5 1, 6, F $ 5.71, P , 0.05) (upper) and overall mortality by treated water against in situ fieldcollected Culex larvae and pupae (middle) or Culex quinquefasciatus larvae from laboratory colony (bottom), in response to treatment by MosquironH 0.12CRD at 11 briquets/vault (x2 $ 3.83, P , 0.05).
nearly complete inhibition on day 7, which significantly decreased to 72% on day 14, and 56% on day 21 and day 28 posttreatment (x2 $ 3.83, P , 0.05). The late instars from a laboratory colony of Cx. quinquefasciatus showed a slight emergence inhibition pretreatment, but nearly 100% inhibition on day 7, and 95% on day 14, and then significantly reduced to 21% and 13% on day 21 and day 28 posttreatment, respectively (x2 $ 3.83, P , 0.05). For stage-specific mortality, on day 7 posttreatment, more individuals died at the larval stage, where larval mortality accounted for 82.9% out of the total morality of 98.4% for the fieldcollected larvae, and 67.6% out of the 99.1% for the laboratory-colonized larvae. With posttreatment progression of time, more individuals died at transition from pupae to adults, where mortality at incompletely emerged adults was 49.2% out of the total mortality of 57.1% for the field-
collected larvae, and 10.7% out of the 13.4% for the laboratory-colonized larvae. Assays on field-collected larvae/pupae and on laboratorycolonized larvae showed overall similar patterns (Fig. 3A). The 2nd underground stormwater vault study Field dip sampling: At higher concentrations of Mosquiron (11 briquets of Mosquiron 0.12CRD per vault), it was also noticed that all the briquets disintegrated in the nylon pouch on day 7 posttreatment. The densities of early instars were significantly suppressed on days 7 and 35 posttreatment (df 5 1, 6, F $ 5.71, P , 0.05). The late instars were significantly reduced on days 14, 21, and 35 posttreatment (df 5 1, 6, F $ 8.01, P , 0.05). Pupal counts were low throughout the test period (Fig. 3B). The species composition was similar to that observed in the 1st
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occurred at larval stage for the field-collected larvae, and 94.0% out of the 100% mortality happened in larval stage for the laboratorycolonized larvae. On day 42 posttreatment, however, mortality at incompletely emerged adults was 39.6% out of the total mortality of 43.1% for the field-collected larvae, and 5.1% of the 23.2% for the laboratory-colonized larvae. Overall, the proportions of larval mortality were higher in the 2nd study at higher dose than in 1st study at the lower dose (Fig. 3B). It was noticed that some 4th instars of Cx. quinquefasciatus collected on day 21 posttreatment showed some opaqueness in the head, thorax, and some abdominal segments, indicating that the cuticle might have been detached. Furthermore, each abdominal segment also appeared to be compressed (Fig. 4). DISCUSSION
Fig. 4. A 4th instar of Culex quinquefasciatus collected from an underground vault on day 21 posttreatment by MosquironH 0.12CRD at 11 briquets/ vault.
round of study; Cx. quinquefasciatus remained the predominant species (90.8–94.7%), followed by Cx. stigmatosoma (4.4–5.2%) and Cs. incidens (0.9–4.0%). Laboratory observations: The larvae and pupae collected from the field sites pretreatment showed very low level of emergence inhibition, while nearly complete emergence inhibition was observed in the larvae and pupae collected from day 7 to day 28; efficacy declined significantly on day 35 and day 42 posttreatment when the test was concluded (x2 $ 3.83, P , 0.05). The late instars from a laboratory colony of Cx. quinquefasciatus exhibited a similar pattern of emergence inhibition. Larvae exposed to the water samples collected pretreatment pupated and emerged as adults with very low level of emergence inhibition; ones that were exposed to the treated water showed 91–100% inhibition from day 7 to day 35, then significantly declined to 23% on day 42 posttreatment (x2 $ 3.83, P , 0.05) (Fig. 3B). The stage-specific mortality pattern was similar to that in the 1st underground vault study, i.e., more larval mortality occurred during earlier days posttreatment, while more individuals died during adult emergence during later sampling days. Briefly, on day 7 posttreatment, all mortality
Novaluron is a newly recognized chitin synthesis inhibitor against insects belonging to Coleoptera, Hemiptera, Lepidoptera, and Diptera. Technical-grade novaluron or Rimon 10EC has been tested against the following mosquito species, including Cx. quinquefasciatus, Anopheles albimanus Wiedemann, An. pseudopunctipennis Theobald, Aedes aegypti (L.), and Ae. albopictus (Skuse) (Mulla et al. 2003, Su et al. 2003, Arredondo-Jime´nez and Valdez-Delgado 2006, Sfara et al. 2007, Tawatsin et al. 2007, Jambulingam et al. 2009, Farnesi et al. 2012, Fontoura et al. 2012, Rajasekar and Jebanesan 2012). Furthermore, novaluron showed high activity against Ae. aegypti that was resistant to conventional chemical insecticides such as organophosphates (Fontoura et al. 2012), or Cx. quiquefasciatus as highly resistant to the biopesticide spinosad (Su and Cheng 2014). Novaluron is recommended by the World Health Organization for use in drinking water (WHO 2008), which provides the opportunity of application to control Ae. aegypti and other mosquitoes breeding in water storage containers in tropical and subtropical regions. Mosquiron 0.12CRD is the first formulation of novaluron registered for mosquito control in the USA. The activity against immature mosquitoes observed in this study was significantly lower than the technical-grade novaluron published previously by Su et al. (2003), who reported 0.118 ppb for IE50 and 0.595 ppb for IE90 against 4th instars of Cx. quinquefasciatus, and by Mulla et al. (2003), who found 0.045 ppb for IE50 and 0.160 for IE90 in 4th instars of Ae. aegypti. Furthermore, the stage-specific mortality demonstrated by Su et al. (2003) against Cx. quinquefasciatus and Mulla et al. (2003) against Ae. aegypti showed that the majority of the exposed individuals died at pupal stage or incomplete emergence at lower concentrations, while mortal-
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ity at larval stage substantially increased at high concentrations. In the current study, however, vast majority of exposed late instars died as incompletely emerged adults or adultoids, even at the higher concentrations. The stage-specific mortality observed during bioassays seemed somewhat in contrast to that in chitin synthesis inhibitor, rather similar to that of a juvenoid such as methoprene and pyriproxyfen (Su and Cheng 2014). The briquets applied were observed to have disintegrated completely on day 7 posttreatment. The expected residual efficacy did not appear to depend on controlled disposal of the inert carriers together with the AI off the briquet. The product evaluated was labeled at 1–2 CRD per 189 liters of water in mosquito breeding sources depending on organic matter content. Without knowing the exact amount of water and unpredictable flushing events in the underground vault system selected for the studies, Mosquiron 0.12CRD was applied at the minimum manageable dose of 1 briquet/ vault, with the goal to explore the minimum effective dose. The efficacy level was limited as indicated by the moderate and gradual reduction of larval populations. However, moderate to high level of emergence inhibition was noticed in Culex larvae and pupae collected from the treated habitats, as well as in the laboratory bioassays against laboratory colony of Cx. quinquefasciatus. It seemed that the novaluron concentration when applied at 1 briquet/vault was inadequate to significantly suppress the natural Culex populations breeding in underground vaults in a timely manner. In the 2nd field evaluation, the formulation was applied at 11 briquets per vault in order to yield higher levels of control. In this study, the initial natural larval populations were considerably higher than in the 1st field study. The early and longer efficacy as indicated by counts of early and late instars was achieved. At the same time, high activity of emergence inhibition in treated water persisted for 35 days in the laboratory observation against field-collected larvae and pupae, as well as larvae from laboratory colony. It was noticed in this field study that some 4th instars of Cx. quinquefasciatus collected on day 21 posttreatment from all treated sites showed opaqueness in the head, thorax, and abdominal regions, which may signify cuticle detachment. Further, the segments in the head, thorax, and abdomen appeared compressed. It is possible that these morphological alterations resulted from intoxication of novaluron (Farnesi et al. 2012). The morphological changes mentioned above may have occurred earlier than 21 days posttreatment, which warrants further detailed studies in the future. Mosquiron 0.12CRD showed high activity of emergence inhibition against Cx. quinquefasciatus in the laboratory and field. A couple of other
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mosquito species were also among the targets in the field. Dose-dependent initial and residual efficacy was demonstrated in the field when Mosquiron 0.12CRD was applied at 1 and 11 briquets per vault. Larval and pupal isolation, i.e., holding field-collected larvae or pupae in treated water collected from the field sites and reading emergence inhibition later in the laboratory, is one of the commonly used methods to estimate control efficacy. Bioassay on the treated water against mosquito larvae from laboratory colony is another recognized way to determine the efficacy of the field treatment of insect growth regulator formulations (Mulla 1991, 1995). According to the observations in the current studies, field efficacy of Mosquiron CRD could be underestimated by dip sampling of immature mosquitoes when one compares dip counts with laboratory bioassays on field-collected or laboratory-reared larvae. ACKNOWLEDGMENTS Special thanks are due to the following industrial partners: Makhteshim Agan North America Inc., Raleigh, NC, USA; Tumaini Controlled Release Technologies Inc., Hamilton, ON, Canada; and Pestalto Environmental Products Inc., Guelph, ON, Canada. The general assistance received during the studies from operations and laboratory departments in West Valley Mosquito and Vector Control District is duly acknowledged. REFERENCES CITED Abacus Concepts, Inc. 1987. StatView + Graphics. Berkeley, CA: Abacus Concepts, Inc. Arredondo-Jime´nez JI, Valdez-Delgado KM. 2006. Effect of novaluron (Rimon 10 EC) on the mosquitoes Anopheles albimanus, Anopheles pseudopunctipennis, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Chiapas, Mexico. Med Vet Entomol 20:377–387. Barbour JD. 2007. Insecticide resistance and efficacy of novaluron and flonicamid for control of lygus bugs in alfalfa seed. In: Ellsworth PE, Naranjo S, eds. Proceedings: 2nd International Lygus Symposium. 2007 April 15–19; Asilomar, CA. J Insect Sci. doi: http://dx.doi.org/10.1673/031.008.4901. Beuzelin JM, Akbar W, Me´sza´ros A, Reay-Jones FPF, Reagan TE. 2010. Field assessment of novaluron for sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), management in Louisiana sugarcane. Crop Prot 29:1168–1176. Cetin H, Erler F, Yanikoglu A. 2006. Larvicidal activity of novaluron, a chitin synthesis inhibitor, against the housefly, Musca domestica. J Insect Sci 6:50–54. Cutler GC, Scott-Dupree CD, Tolman JH, Harris CR. 2005. Acute and sublethal toxicity of novaluron, a novel chitin synthesis inhibitor, to Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Pest Manag Sci 61:1060–1068.
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Cutler GC, Scott-Dupree CD, Tolman JH, Harris CR. 2007. Field efficacy of novaluron for control of Colorado potato beetle (Coleoptera: Chrysomelidae) on potato. Crop Prot 26:760–767. Farnesi LC, Brito JM, Linss JG, Pelajo-Machado M, Valle D, Rezende GL. 2012. Physiological and morphological aspects of Aedes aegypti developing larvae: effects of the chitin synthesis inhibitor novaluron. PLoS One 7:e30363. doi:10.1371/journal. pone.0030363. Fontoura NG, Bellinato DF, Valle D, Lima JB. 2012. The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo Cruz 107: 387–395. Ishaaya I, Kontsedalov S, Horowitz AR. 2003. Novaluron (Rimon), a novel IGR: potency and cross resistance. Arch Insect Biochem Physiol 54:157–164. Jambulingam P, Sadanandane C, Nithiyananthan N, Subramanian S, Zaim M. 2009. Efficacy of novaluron against Culex quinquefasciatus in small- and medium-scale trials, India. J Am Mosq Control Assoc 25:315–322. Kim SH, Wise JC, Go ¨ kc¸e A, Whalon ME. 2011. Novaluron causes reduced egg hatch after treating adult codling moths, Cydia pomenella: support for transovarial transfer. J Insect Sci 11:126. doi:10.1673/ 031.011.12601. LeOra Software. 1987. POLO-PC: a user’s guide to probit or logit analysis. Berkeley, CA: LeOra Software. Lohmeyer KH, Pound JM. 2012. Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae). J Med Entomol 49:647–651. Mulla MS. 1991. Insect growth regulators for the control of mosquito pests and disease vectors. In: Chinese Journal of Entomology Special Publication
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No. 6: Proceedings of the 4th National Vector Control Symposium. Taichung, Taiwan, ROC. p 81–91. Mulla MS. 1995. The future of insect growth regulators in vector control. J Am Mosq Control Assoc 11:269–273. Mulla MS, Thavara U, Tawatsin A, Chompoosri J, Zaim M, Su T. 2003. Laboratory and field evaluation of novaluron, a new acylurea insect growth regulator, against Aedes aegypti (Diptera: Culicidae). J Vector Ecol 28:241–254. Rajasekar P, Jebanesan A. 2012. Efficacy of IGRs compound novaluron and buprofezin against Culex quinquefasciatus mosquito larvae and pupal control in pools, drains and tanks. Int J Res Biol Sci 2:45–47. Sfara V, De Licastro SA, Masuh HM, Seccacini EA, Alzogaray RA, Zerba EN. 2007. Synergism between cis-permethrin and benzoyl phenyl urea insect growth regulators against Aedes aegypti larvae. J Am Mosq Control Assoc 23:24–28. Su T, Cheng ML. 2014. Cross resistances in spinosadresistant Culex quinquefasciatus (Diptera: Culicidae). J Med Entomol 51:428–435. Su T, Mulla MS, Zaim M. 2003. Laboratory and field evaluations of novaluron, a new insect growth regulator (IGR), against Culex mosquitoes. J Am Mosq Control Assoc 19:408–418. Tawatsin A, Thavara U, Bhakdeenuan P, Chompoosri J, Siriyasatien P, Asavadachanukorn P, Mulla MS. 2007. Field evaluation of novaluron, a chitin synthesis inhibitor larvicide, against mosquito larvae in polluted water in urban areas of Bangkok, Thailand. Southeast Asian J Trop Med Public Health 38:434–441. WHO [World Health Organization]. 2008. Novaluron in drinking-water: use for vector control in drinking-water sources and containers. Background document for preparation of WHO guidelines for drinking water quality. WHO/HSE/AMR/08.03/11. Geneva, Switzerland: World Health Organization.
