, the yellow fever mosquito, is considered one of the world’s greatest health threats. One class of chemicals commonly used to control Ae. aegypti
larvae are insect growth regulators (IGRs) [1
]. IGRs disrupt insect growth and reproduction by interfering with insect development [1
]. Juvenile hormone analogs (JHAs) disrupt insect development and prevent insects from reaching the adult stage by providing increasing juvenile hormone in insects at a time these compounds do not normally occur [2
], therefore preventing proper mosquito development.
Pyriproxyfen, a juvenile hormone analog, is a relatively stable chemical which results in insects being unable to molt to the adult stage [3
]. It is approved by the U.S. Environmental Protection Agency for use in small containers to control Ae. aegypti
because of its relatively low toxicity to non-target organisms [4
], and safety to humans. The World Health Organization (WHO) has also approved pyriproxyfen at a rate of 10 PPB for use in potable water [7
Control of Ae. aegypti
is difficult because of different behaviors including daytime feeding habits, the ability to develop in a wide variety of water holding containers, and skip oviposition, where one female will lay her eggs in numerous containers [8
]. However, pyriproxyfen is effective at reducing populations of Ae. aegypti
] since Ae. aegypti
females are not deterred from laying eggs in pyriproxyfen-treated containers.
Although effective for mosquito control, pyriproxyfen is labeled for treating large bodies of water, complicating control in small containers. The objective of this study was to test the efficacy of mosquitocidal chips treated with slow-release pyriproxyfen formulation to decrease Ae. aegypti
populations. Use of pre-treated chips with a small dose of pyriproxyfen facilitates the use of the product by final users, especially those lacking formal education. The pre-dosed chip requires no preparation, dilution, or additional manipulation by the final user, who can apply the mosquitocidal chips to a variety of potential mosquito breeding containers. Although the use of pyriproxyfen in drinking water may not be advisable due to some health concerns [13
], its use in containers of water not destined to human consumption, or other natural or artificial water-holding vessels, can be an important tool in the control of mosquitoes around human dwellings. The slow-release formulation on the chips remains active for a minimum of 6 months through dry–wet cycles and can provide continuous control of mosquitoes for a whole mosquito for at least one season without reapplication of the product.
2. Materials and Methods
Aedes aegypti colony with no known resistance to insecticides was acquired from the Center of Medical, Agricultural and Veterinary Entomology (CMAVE) and the United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Gainesville, FL. The colony was maintained in 30 cm × 30 cm × 30 cm cages (BioQuip® Lumite Screen Collapsible Cages, Rancho Dominguez, CA, USA), provided with 10% sugar solution and tap water in a rearing room maintained at 28 ± 2 °C with a relative humidity of 36% ± 5% and a 12:12 (L:D) photoperiod. Female mosquitoes were blood-fed on live domestic chickens (IACUC Protocol #20163836_01).
Mosquito eggs were hatched by placing strips of dried egg sheets into 55 cm × 45 cm × 8 cm into plastic trays (Del-Tec/Panel Control Plastic Trays, Greenville, SC, USA) with clean unchlorinated water. Larvae were fed ground fish flakes (TetraFin® Goldfish Flakes, Blacksburg, VA, USA). Pupae were placed into polypropylene (450 mL) cups and put into adult rearing cages for emergence.
Technical grade pyriproxyfen (Nylar® Technical, MGK® Insect Control Solutions, Minneapolis, MN, USA) was dissolved in methanol and serial diluted as needed for experiments or formulated for application on mosquitocidal chips. A chip formulation, containing 0.01% pyriproxyfen, was prepared using a base formulation with 1% fumed silica, 5% Butyl-methacrylate polymer, and 94% acetone. The application of 100 µL of the 0.01% pyriproxyfen formulation delivered 8.4 μg of the active ingredient to the chip. The control formulation contained all the formulations ingredients but no pyriproxyfen.
Ceramic tiles (hexagonal with 8mm side) (American Olean Satinglo Hex Honeycomb Mosaic Ceramic Floor and Wall Tile, Birmingham, AL, USA) were removed from glue backing and cleaned with dish soap and warm water and dried before being treated with the mosquitocidal formulations using a micropipette. Mosquitocidal chips were treated using 100 μL of the stock pyriproxyfen formulations pipetted onto the chips. Control chips received formulation with no active ingredient. Formulations were applied to the non-glazed side of each chip to ensure treatments adhered to the tile. Mosquitocidal chips were allowed to dry for a minimum of 24 h in a chemical hood before being placed in bioassay containers.
Polypropylene cups (WNATM, Chattanooga, TN, USA, 450 mL) were filled with 350 mL of clean unchlorinated water and treated with pyriproxyfen-treated or control chips. Ten late 3rd to 4th instar Ae. aegypti larvae were added to each bioassay container.
The purpose of the water volume experiment was to determine whether mosquitocidal chips effects were affected by varying water volumes (250, 500, 750 and 1000 mL) of clean unchlorinated water. Treatment and control vases (1000 mL, Libbey® glass cylinder vase, Toledo, OH, USA) contained the same volumes of water and untreated chips, or 8.4 μg pyriproxyfen chips (0.01% pyriproxyfen), which were deposited on the bottom of the vases using large forceps. Ten late 3rd–early 4th instar mosquitoes were pipetted into each vase from their rearing cups. There were four replicates of each treatment and control. Larvae were fed 200 μL of a slurry of ground fish food every other day. Vases were maintained at ca. 31 °C and 15% relative humidity (RH) and inspected every 24 h for dead or live larvae, pupae and adults. Experiments were run for 4 d or until all mosquitoes had either died or emerged as adults.
Percent mortality of dead insects in experiments was calculated and then arcsin-transformed, an analyzed using repeated measures ANOVA using days after application as the repeated measure. Mean mortalities were compared using a Tukey’s Honest Significant Difference (HSD) pairwise comparison.
The purpose of the container material experiment was to determine whether mosquitocidal chips were affected by container material that simulated habitats where Ae. aegypti larvae typically develop. The materials used were wood (Artminds® wooden box, Southfield, MI, USA), metal (Ashland® Galvanized Metal Bucket, Ashland, Covington, KY, USA), clay (Indigo spice, studio décor, Irving, TX, USA), ceramic (Indigo spice, studio décor, Irving, TX, USA), plastic 450 mL polypropylene cups (WNATM, Chattanooga, TN, USA), and glass (Kimble® Wide Mouth Jars, Vineland, NJ, USA). Unchlorinated water (200 mL) was placed into each container with either an 8.4 μg pyriproxyfen treated (0.01% pyriproxyfen) or an untreated chip. Wood containers were tightly wrapped with a layer of parafilm in order to prevent leakage for the duration of the experiment. Ten late 3rd–early 4th instar mosquitoes were placed in each of four replicates of each container type. There were two replicates of each container type with control chips. Insects were maintained and checked as described above. Percent mortality of insects was calculated and analyzed, as described above.
The organic matter experiment was designed to determine if different percentages of organic matter in water would affect chip efficacy. Treatments included 350 mL of water containing either 0%, 10%, 30%, 50%, 70% and 90% of a leaf infusion prepared according to Reiter et al. [14
] and the 8.4 μg pyriproxyfen chip (0.01% pyriproxyfen), or control chip for control treatments. Ten late 3rd–early 4th instar mosquitoes were placed in each cup and four replicates were prepared for each treatment and control. Insects were maintained and checked as described above. Percent mortality of insects was calculated and analyzed as described above.
The effects of the presence of mosquitocidal chips on female oviposition preference and on the overall reduction of populations of Ae. aegypti were tested using cages (60 cm × 60 cm × 60 cm BugDorm Insect Tents, MegaView Science Co., Ltd., Taichung, Taiwan, China), containing 4 cups with 350 mL of clean unchlorinated water with an oak leaf sachet prepared with fillable tea bags (disposable, self-seal tea bags, Otter and Trout Trading Co, Gainesville, FL, USA), containing 0.5 g of ground field-collected oak leaves. Cups were lined internally with filter paper where female mosquitoes could oviposit eggs. There were 4 treatments were: (a) 3 untreated cups and 1 treated cup with an 8.4 µg pyriproxyfen chip (0.01% pyriproxyfen), (b) 2 untreated cups and 2 cups treated with pyriproxyfen chips, (c) 1 untreated cup and 3 cups treated with pyriproxyfen chips, and (d) 4 cups treated with pyriproxyfen chips. For the controls, all 4 cups were untreated. There were 4 replicates of each of the 4 treatments and control, and the experiment was repeated twice over a 2 month period.
Ten gravid female Ae. aegypti were put into each cage 48 h after blood feeding and were allowed to oviposit on filter paper for 72 h. After this time, egg sheets and adult mosquitoes were removed from the cage. Egg sheets were allowed to dry for 24 h and eggs were counted, removed from the papers and returned to their original containers. Chips were temporarily removed from the containers which were closed with lids and hand shaken for 1 min to stimulate egg hatching. After shaking, the lid was removed, and chips were placed back into original containers. Larvae in containers were fed 200 µL of ground fish food every other day. A 120 mL cup with 10% sugar solution was placed in each cage for emerging adult mosquitoes to feed on. After 10 d, emerged adults were counted. Experiments were kept in a greenhouse at ca. 35 °C ± 5 °C and 25% ± 5 °C RH with a photoperiod between 12:12 (L:D) and 14:12 (L:D). Percent emergence data was calculated by using the number of eggs laid and number of adults emerged and was arcsin-transformed for statistical analysis. Number of eggs laid and number of adults emerged were analyzed using a one-way ANOVA and percent emergence data were compared using a Student’s t-test.