Marcia Reed, Paula A. Macedo, Gary Goodman and David Brown
Sacramento-Yolo Mosquito and Vector Control District, Elk grove, CA 95624, [email protected]
ABSTRACT: Periodic evaluation of insecticide susceptibility is part of the Sacramento-Yolo Mosquito and Vector Control District Integrated Vector Management Program and constitutes good pest management practice. In 2012 Culex pipiens populations from 13 locations were evaluated for susceptibility to two pyrethroids and one organophosphate insecticide. Mosquito populations with increased tolerance to permethrin and sumithrin were found in different areas in Sacramento and Yolo Counties.
INTRODUCTION
Insecticide resistance has been reported to every chemical class of insecticides (Brogdon and McAllister 1998a). Therefore, mosquito populations should be periodically evaluated for their susceptibility to the pesticides used in mosquito control programs. Current susceptibility data ensure timely decisions and should be considered when adjusting control strategies. Insecticide resistance may have severe implications in the mosquito control industry, such as limitations on products labeled or permitted for use in mosquito control, interference with the development of new products, and most importantly, the undesirable public health consequences of ineffective mosquito control in disease outbreak situations.
At the Sacramento-Yolo Mosquito and Vector Control District (SYMVCD), the laboratory conducts periodic evaluation of insecticide susceptibility of Culex tarsalis and Culex pipiens populations in Sacramento and Yolo Counties. In 2012 the authors reported a population of Cx. pipiens from Orangevale, CA, which showed increased tolerance to permethrin (Reed et al. 2012). Further, it was shown that applications of pyrethroids or pyrethrins to that area for mosquito control had been infrequent and therefore not likely to have caused the tolerance levels observed. No increased tolerance has been observed in Cx. tarsalis populations in Sacramento or Yolo Counties. In 2013 the authors increased the number of Cx. pipiens populations tested throughout both counties to try to determine if the observed insecticide tolerance was an isolated occurrence, or if it was a widespread issue for the District.
MATERIALS AND METHODS
Study Areas. Mosquito populations from twelve urban and one rural site were evaluated (Figure 1). The urban sites were located in residential neighborhoods in either Sacramento County (ten sites) or Yolo County (two sites), and the rural site was in proximity to a dairy in Sacramento County.
Field Mosquito Populations. Culex pipiens populations were sampled by collecting gravid females using modified Reiter gravid traps (Cummings 1992). Gravid females were transferred to screened cardboard containers and allowed to lay eggs. The larvae were raised to the adult stage, and adults between three and five days old were subsequently evaluated using bottle bioassays. Figure 1. Locations for 2013 collections of Culex pipiens populations for pesticide susceptibility testing in Sacramento and Yolo Counties, CA.
Reference Colonies. The susceptible population used for comparison was the Cx. pipiens quinquefasciatus (CQ1) colony originally provided to our laboratory by Dr. Anthony Cornel at the University of California, Davis. This is the recommended reference colony for use in California according to the guidelines of the Mosquito Pesticide Resistance Monitoring Working Group. These guidelines were developed in 2008 to provide recommendations to the Mosquito and Vector Control Association of California (MVCAC) regarding the implementation of a pesticide resistance monitoring program. Susceptible mosquitoes used in the bioassays were approximately three to five days old.
Adult Bottle Bioassays. Mosquito populations were evaluated using the time-mortality method by Brogdon and McAllister (1998a, 1998b). In this procedure, glass bottles are dosed with a known amount of a pesticide, adult mosquitoes are introduced to the bottle and time to death is recorded. For each bioassay, four replicates of 25 adult females were used. Mortality was then recorded every 15 minutes for approximately three hours.
Resistance testing is part of SYMVCD’s surveillance program and helps management make timely control decisions based on susceptibility data. Because of this, the products tested varied with the products that were used and needed in the field. In the beginning of the season, most of the bioassays were conducted using permethrin as a surrogate for any pyrethroid. As the season progressed, District personnel decided to evaluate the active ingredient that was actually being used in the field at that time, the pyrethroid sumithrin (d-phenothrin). Due to some low susceptibility issues observed in some field populations, some bioassays were then performed with the addition of piperonyl butoxide (PBO) to sumithrin to evaluate the effect that adding the synergist would have in those mosquito populations and to link the resistance to an oxidative mechanism to try to gain insight into the possible mechanism of resistance. For evaluation of the organophosphate class of pesticides, naled was used. Locations that were evaluated for each active ingredient are shown in Table 1. Doses for each active ingredient used in the bioassays are shown in Table 2.
table 1. Locations
of Culex pipiens populations and active ingredients tested.
table 2. Doses (µg)
of technical grade standards used per bottle for resistance testing in Culex pipiens populations.
Data Interpretation. Percent mortality at each time point was plotted, and response curves were generated. These response curves are not conducive to formal statistics, therefore testing and interpretation of results is based on comparison of field-collected populations with a susceptible reference colony over time.
RESULTS
Culex pipiens populations from 12 locations were
evaluated for susceptibility to naled. As shown in Figure 2, 90 to 100% mortality was achieved by 60 minutes for 11 of the 12 populations; this was 15 minutes after 100% mortality was achieved in the susceptible CQ1 colony. Culex pipiens from Southeast Sacramento showed a slower response with only 74% dead at 60 minutes, but all mosquitoes were dead at 135 minutes. All populations tested had greater than 80% mortality within a reasonable time frame when exposed to naled.
Response curves varied greatly when mosquitoes from 12 locations were tested for permethrin susceptibility (Figure 3). The greatest response was from Hood-Franklin, with 94% of the mosquitoes dead at 60 minutes and 100% mortality at 105 minutes. This was the only rural population included in this study, and it remains susceptible to permethrin. We did not observe 100% mortality in any of the other 11 populations tested, even at 180 minutes. At 60 minutes the lowest mortality observed was from Woodland 1 (only 9%), and at 180 minutes only 79% mortality was observed. The lowest mortality observed at 180 minutes was from Folsom 1 (61%). Three Cx pipiens populations were used in bioassays with sumithrin and sumithrin + PBO (Figure 4). All three showed marked tolerance to these active ingredients at the doses evaluated. Percent mortality was affected by the addition of PBO (Table 3).
Figure 3. Percent mortality of Culex pipiens populations over time (minutes) in bottle bioassays using permethrin.
Figure 4. Percent mortality of Culex pipiens populations over time (minutes) in bottle bioassays using sumithrin with and without
piperonyl butoxide (PBO).
table 3. Percent mortality observed for three Culex pipiens populations tested in bottle bioassays for susceptibility to sumithrin
DISCUSSION
In 2012 SYMVCD evaluated Cx. pipiens populations from 12 different locations in Sacramento and Yolo Counties, CA, against two pyrethroids and one organophosphate. Increased tolerance to pyrethroids was detected in all but one of the 12 populations. The levels of tolerance varied with location and active ingredient used. The addition of PBO to sumithrin resulted in increased mosquito mortality in the three populations tested, which may be an indication of an oxidative mechanism of resistance. As a next step the District will be evaluating these populations using microplate assays to confirm the mechanism of resistance, and it is currently investigating PCR-based diagnostic tests.
In addition to the data shown here, past mosquito control applications of pyrethroids in the same areas (data not shown) were also investigated to evaluate how much selection pressure our mosquito control applications were exerting on the mosquito populations evaluated. Applications were usually infrequent and should not have been a driving force for resistance in those mosquito populations. That fact is particularly significant for mosquito control programs because it would mean that, even if there were products available for mosquito control from a different chemical class that could be used to revert the resistance observed, the main driving force for resistance would still be unaffected because it is not from mosquito control. If true, this would render that class of chemicals ineffective in the fight to control mosquitoes and to interrupt transmission of the diseases they carry. Added to increasing regulations, potential loss of available chemicals and the general lack of availability of products for public health use can be devastating to the mosquito control industry and ultimately to public health.
REFERENCES CITED
Brogdon, W.G, and J.C. McAllister. 1998a. Insecticide resistance and vector control. Emerg Infect Dis 4: 605-613.
Brogdon, W.G, and J.C. McAllister. 1998b. Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles. J Amer Mosq Control Assoc 14:159- 164.
Cummings, R.F. 1992. The design and use of a modified Reiter mosquito trap for mosquito-borne encephalitis surveillance in Los Angeles County, California, 1987. Proc Papers Calif Mosq Vector Control Assoc 60: 170-176.
Reed , M., P.A. Macedo and D. Brown. 2012. Increased Tolerance to Permethrin in Culex pipiens Complex Population from Sacramento County, California. Proceedings and Papers of the Eightieth Annual Conference of the Mosquito and Vector Control Association of California.