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Toxicity and effects of mosquito larvicides methoprene and surface film (Agnique® MMF) on the development and fecundity of the tadpole shrimp Triops newberryi (Packard) (Notostraca: Triopsidae) Tianyun Su1*, Yonxing Jiang2, and Mir S. Mulla Department of Entomology, University of California, Riverside, CA 92521, U.S.A., [email protected] Received 18 June 2014; Accepted 22 July 2014 ABSTRACT: We investigated the interactions of tadpole shrimp, a mosquito biological control agent, with the juvenile hormone analog methoprene and a monomolecular surface film. In laboratory assays, the tadpole shrimp (TPS) Triops newberryi (Packard) was able to tolerate high concentrations of methoprene without negative impacts on its growth, longevity, and fecundity when exposed to 1 to 10 mg/liter, or 90-900 fold, of the IE90 levels against a laboratory colony of Culex quinquefasciatus Say. The same held true in field trials when the habitats were treated with Altosid® Liquid Larvicide (Altosid® LL, 5% methoprene) at 0.3-1.2 liters/ha. or 1-4 fold of the label rates for mosquito control. However, some significant impacts on the TPS occurred when they were exposed to Agnique® Monomolecular Film (Agnique® MMF) at the label rates for mosquito control ranging from 1.89-9.45 liters/ha. under laboratory and field conditions. To avoid the negative impact of Agnique MMF on tadpole shrimp, it appears that 1.89 liters/ha. would be the maximum rate when Agnique MMF is used to control mosquitoes in the habitats where the TPS is employed as a biological control agent, or prevailing in the aquatic habitats with potential for suppressing mosquito larval populations. Journal of Vector Ecology 39 (2): 340-346. 2014. Keyword Index: Tadpole shrimp, Triops newberryi, methoprene, Altosid® LL, Agnique® MMF.
INTRODUCTION Tadpole shrimp (TPS) are fresh water crustaceans (Notostraca: Triopsidae) and are widely distributed on all continents except Antarctica. There are two genera, Triops and Lepidurus, in the family Triopsidae, each comprising ten identified species. The common natural habitats of the TPS are temporary freshwater bodies that are characterized by extreme physical and chemical conditions. Triops newberryi (Packard), formerly called Triops longicaudatus Le Conte, the predominant species of tadpole shrimp in the southwestern United States, including the states of Washington, Oregon, and California (Sassaman et al. 1997, Su and Mulla 2002a), was considered as a natural enemy of immature mosquitoes in temporary waters decades ago (Maffi 1962). However, their potential utility in mosquito control was not available until recent publications by Tietze and Mulla (1989, 1990, 1991) and Fry and Mulla (1994). The characteristics of fast nymphal growth, early maturation, high reproduction capacity (Fry-O’Brien and Mulla 1996, Su and Mulla 2002b), and their predation on immature mosquitoes (Tietze and Mulla 1989, 1990, 1991) make them suitable for regulating mosquito populations that share the same ephemeral or semipermanent habitats with TPS. The practical use of TPS in controlling immature mosquitoes in specific habitats in the Coachella Valley, southern California, has been successfully attempted by introduction of desiccation-resistant eggs into some date gardens (Su and Mulla 2002c).
A successful mosquito control agent should have the attributes of being easily and permanently established with little or no augmentation, a high reproductive potential, and resistance to biological and physiochemical constraints imposed on it, as well as being compatible with most of the other mosquito control materials. TPS can be permanently established in ephemeral habitats by stocking desiccationresistant eggs (Su and Mulla 2002c). The eggs hatch and populations colonize quickly after each flooding, and they develop synchronously with some floodwater mosquito species. The TPS also have high reproductive capacity and their desiccation-resistant eggs can remain viable in the soil for long periods of time and undergo installment hatching after each hydration. However, little is known about the compatibility of TPS with some of the most commonly used mosquito larvicides. Currently, Bacillus thuringiensis var. israelensis (B.t.i.), B. sphaericus, spinosad, methoprene (IGR), larvicidal oils, monomolecular surface film mosquito larvicides, and a few others are the materials registered for mosquito control in California and most of the U.S.A. The toxicity and effects of B.t.i., B. sphaericus, and larvicidal oil on the development and fecundity of T. newberryi have been reported by Su and Mulla (2005). The objective of this study was to determine the compatibility of two commonly used mosquito larvicides, Altosid® Liquid Larvicide (Altosid® LL) and Agnique® Monomolecular Film (Agnique® MMF), with the TPS. The toxicity and effects of methoprene and monomolecular
Current address: West Valley Mosquito and Vector Control District, Ontario, CA 91761, U.S.A. Current address: City of Gainesville Mosquito Control Services, Gainesville, FL 32609, U.S.A.
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surface film on growth, survivorship, longevity, maturity, and fecundity of TPS were evaluated in the laboratory and earthen ponds under field conditions.
made among control and treatments by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (Abacus Concepts, Inc. 1987).
MATERIALS AND METHODS
Field mesocosm studies Field studies on the impact of methoprene and surface film on TPS were conducted in mesocosm habitats at the Aquatic and Vector Control Research Facility. In total, 12 earthen ponds were used, each with 27 m2 (3.7 x 7.3 m) surface area and water depth of 30 cm, and water levels were kept relatively constant by float valves on the water lines feeding each pond. These ponds in the past have been used for research on the biology and control of mosquitoes. Shrimp eggs occur in large numbers in the dry soil where an egg bank has been well-established. Egg hatch occurs soon after flooding when water temperature and photoperiod conditions are optimal. Prior to flooding, all pond bottoms were allowed to completely dry and 1,500 g of rabbit chow pellets were added in order to boost nutrient levels in the pond water when flooded for the experiment. Treatment was made on day 4 post-hydration when the TPS grew to the size of CL measured 4-5 mm (Su and Mulla 2001). Each aliquot of Altosid LL in the amount of 0.8, 1.6, or 3.2 ml was diluted to 120 ml in a 125 ml plastic bottle and applied by jet spray to the water surface of each respective pond, which achieved the rates of 0.3, 0.6, and 1.2 liters/ha, respectively. Agnique MMF in the amounts of 5, 12.5, and 25 ml were transferred to 125 ml plastic bottles and brought to a final volume of 120 ml with tap water and applied to the water surface of each pond in the same way as described previously. These application rates were approximately equivalent to 1.89, 4.73, and 9.45 liters/ha, respectively. For each test, three treatments and one untreated control were assigned randomly with three replicates each. Treatments were made four days after flooding. The density of TPS was determined on day pre-treatment and days 3, 7, and 14 post-treatment. For sampling the TPS, a long-handled D net (30 x 25 cm) was used. Two samples were taken by sweeping the bottom of pond along each long side of the rectangular ponds. Collected TPS along with any debris, sand, and soil were transferred to an enamel pan (40 x 24 x 6 cm) containing 1 liter of water from the pond. The exact number of TPS in each pan was counted if the number was lower than 100 or estimated by counting approximately half of the TPS if the number was greater than 100. Up to 20 shrimp, depending on the number of shrimp available in the pan, were randomly selected and preserved in 75% ethanol and kept in a refrigerator at 5-7° C for later processing in the laboratory. Size of the shrimp was estimated by measuring the CL, and the percentage gravid was determined by examining modified appendages of the 11th pair of limbs, the ovisacs, where reddish, shining mature eggs were present in gravid individuals. Average population densities (no. /net) and sizes (CL mm) of the shrimp as well as their standard errors were calculated and comparison was made among treatments and control by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05. Percentage of gravids was analyzed among treatments and the control by the Chi
Test materials Technical grade methoprene (98.1%, Lot: 326-28A) was purchased from Chem Service, West Chester, PA, and Altosid LL (5.0% AI, Lot#: 020619087) was provided by Wellmark International Schaumburg, IL. Agnique MMF (100% AI, Lot# U83D221132) was acquired from Cognis Corp., Cincinnati, OH. For methoprene tests, technical grade material was used in the laboratory studies, while the commercial product Altosid LL was used in the field mesocosms. The commercial product Agnique MMF was tested under both laboratory and field conditions. Laboratory studies Laboratory studies of the effects of technical grade methoprene and monomolecular surface film on the development and fecundity of the TPS were conducted according to the procedures previously described by Su and Mulla (2005). In brief, the TPS eggs in 200 g of soil collected from the Aquatic and Vector Control Facility at the Agricultural Experimental Station of University of California at Riverside were hatched in an enamel pan (40 x 24 x 7 cm) by adding 2 liters of distilled water and maintained at 2528° C with a photoperiod of 16:8 (L:D). Two to three days after hydration, one juvenile TPS with carapace length (CL) at carinal suture of about 2 mm was transferred from the hatching pan to an individual enamel pan (30 x 19 x 5 cm) containing 200 g of soil (sandy loam devoid of TPS eggs) as substrate (Su and Mulla 2001) and 1 liter of distilled water. Each enamel pan was provided with 0.5 g rabbit pellets (Brookhurst Mill, Riverside, CA) and 20 2nd instar mosquito larvae from a laboratory colony of Culex quinquefasciatus Say every other day. For each test, a total of 48 enamel pans were set up. Technical grade methoprene was diluted with acetone to 5% AI, and appropriate aliquot (20, 100, and 200 ml) of this dilution was added to each of the enamel pans, yielding concentrations of 1, 5, and 10 mg/liter AI, respectively. Three treatments and one untreated control were assigned randomly with 12 replicates (pans) each. For Agnique, 10, 24, and 48.5 ml was added to each enamel pan, approximately equivalent to the label rate range of 1.89, 4.73, and 9.45 liters/ ha. Similarly, three treatments and one untreated control were assigned randomly with 12 replicates each. After treatment, the rearing pans were held in the insectary at 25-28° C and a photoperiod of 16:8 (L:D). Carapace length as a growth parameter was measured and mortality was recorded daily until all shrimp died, and the size at death as indicated by CL was determined. After the death of all TPS, rearing water was slowly drained, soil at the bottom of the enamel pans containing fresh TPS eggs was dried at 28-30° C, and eggs in the dried soil were then counted according to the procedures of Su and Mulla (2001). The daily growth was charted, and comparisons in size at death, longevity, and fecundity were
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square test (Abacus Concepts 1987). In each test, a minimummaximum thermometer was placed in one of the ponds for measuring water temperature. The min-max temperature was read on every sampling day. RESULTS Laboratory studies Methoprene. Four biological parameters related to growth and development profiles of the tadpole shrimp were measured in the laboratory: the daily CL increase during life, the CL at death, average longevity, and fecundity (eggs/ TPS). Exposure of TPS to technical grade methoprene at concentrations of 1, 5, and 10 mg/liter had no noticeable effects on the growth rate, as the growth curves among various treatments fluctuated within a narrow range (Figure 1). Various treatments ranging from 1 to 10 mg/liter of methoprene did not reduce the maximum size at death; shrimp treated at 5 mg/liter actually reached a significantly greater size (df = 3,44, F = 31.9, P < 0.001). The average longevity was not adversely impacted by methoprene treatment. The lifetime fecundity as expressed as total eggs produced per individual did not decline significantly in all treatments. The shrimp exposed to 5 mg/liter methoprene produced even more eggs (df = 3,44, F = 20.6, P < 0.001)(Figure 2). Agnique MMF. The growth curves of TPS exposed to Agnique at the rates of 1.89 to 9.45 liters/ha seemed overall comparable with untreated controls in the laboratory (Figure 3). The TPS, however, reached significantly smaller size at death across rates applied (df = 3,44, F = 38.8, P < 0.001). The shrimp exposed to Agnique MMF at the higher rates of 4.73 and 9.45 liters/ha had a significantly shorter life span (df = 3,44, F = 10.3, P < 0.01), while the ones treated at the highest rate of 9.45 liters/ha produced significantly fewer eggs during their lifetime (df = 3,44, F = 28.4, P < 0.001) (Figure 4). Field mesocosm studies Altosid LL. Field studies showed that exposure of TPS to a single treatment of Altosid LL at the rates of 0.3, 0.6, and 1.2 liters/ha did not adversely impact their density, with an 10
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exception that a significantly lower density was encountered at the highest rate of 1.2 liters/ha on day 3 post-treatment (df = 3,8, F = 5.7, P < 0.05). The average size and percentage of gravid shrimp did not show significant differences among all treatments. The percentage of gravid shrimp was actually higher on day 14 post-treatment at the rate of 0.6 liters/ha (c2 ≥ 4.01, P < 0.05) (Figure 5). Agnique MMF. In field tests of Agnique MMF, the population density of the TPS declined in relation to treatment rates and time post-treatment. The population density was not impacted by treatment at 1.89 liters/ha, but it significantly declined on day 7 and day 14 post-treatment at the higher rates of 4.73 and 9.45 liters/ha. Treatment at the highest dose of 9.45 liters/ha significantly reduced the TPS density as early as day 3 post-treatment (df = 3,8, F ≥ 6.4, P < 0.05). Rate-dependent differences were evident among the four rates applied (Figure 6). The average size of the TPS was not impacted by the treatments. Interestingly, the TPS tended to reach larger sizes when their population densities were lower at the higher rates of treatment (df = 3,8, F ≥ 5.2, P < 0.05). On day 3 post-treatment, a proportion of gravid TPS prevailed at the low levels at all treatments (χ2 ≥ 10.4, P < 0.01), but reached higher levels when their densities were lower at the higher rates (χ2 ≥ 8.4, P < 0.01). At the rate of 9.45 liters/ha the TPS population densities were too low to assess the percentages of the gravid individuals (Figure 6). DISCUSSION Juvenile hormones have been considered as agents of insect development since the 1960s (Williams 1967). Methoprene, together with kinoprene, hydroprene, triprene, and others, were synthesized in the late 1960s and have been recognized as active juvenile hormone analogs, or juvenoids, against a wide variety of insects of agricultural, medical, and veterinary importance (Sparks and Hammock 1983). Immature mosquitoes are among the most common target species of methoprene, where successful emergence of adult mosquitoes is interrupted due to the early exposure during larval and pupal stages. One of the additional benefits of
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Figure 2. Growth, longevity, and fecundity of tadpole shrimp treated by technical grade methoprene at 1, 5, and 10 mg/liter in the laboratory. Significant difference in each parameter was indicated among control and treatments by different letters by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (Growth: df = 3,44, F = 31.9, P < 0.001; Fecundity: df = 3,44, F = 20.6, P < 0.001). methoprene is that exposed mosquito larvae and pupae remain available to the trophic web in the aquatic ecosystems when control efficacy is expected later (Lawler et al. 2000). Various products ranging from fast release emulsifiable concentrates to slow release briquets have been developed and used for mosquito control worldwide. Their laboratory activity and field efficacy for mosquitoes, and safety profile to non-target aquatic invertebrates, have been well documented (Mulla 1995, Lawler et al. 1999, 2000). It is a quite common practice to incorporate chemical control with biological control agents in integrated vector control programs to achieve high levels or extended periods of efficacy. The current study indicates methoprene products have a potential to be used with TPS, because exposure of TPS to technical grade methoprene at approximately 90–900 x IE90 against a laboratory colony of Cx. quinquefasciatus (11.1 mg/liter) (Su and Cheng 2014), did not have noticeable effects on the growth rate and did not impact the maximum size at death or average longevity and lifetime fecundity under laboratory conditions. Furthermore, application of 5% methoprene formulation of Altosid LL at
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the rates of 0.3, 0.6, and 1.2 liters/ha over mesocosm ponds, each measuring 27 m2 and 30 cm deep, resulted in about 4.75, 9.57, and 19.10 mg/liter methoprene, respectively, which exceeded the IE50 (0.583 mg/liter) and overlapped with IE90 (11.1 mg/liter) against susceptible Cx. quinquefasciatus from a laboratory colony (Su and Cheng 2014). In agreement with laboratory studies, the field tests also showed a good margin of safety of methoprene to the TPS. Both laboratory and field study results clearly indicate the compatibility of methoprene with the TPS. It is believed that methoprene is a true analog of juvenile hormone III (JH III) in mosquitoes, therefore its activity is relatively target specific. The TPS depend on the major terpenoid hormone, methyl farnesoate, an immediate precursor of insect JH III to regulate their growth and ovarian development (LeBlanc 2007). The reason that TPS were not adversely impacted by methoprene could be either because the TPS need higher titers of the hormone than immature mosquitoes, or because of dissimilarities of the juvenile hormones in mosquitoes as compared with ones in the TPS. This aspect warrants further study. Agnique® MMF is an alcohol ethoxylated surfactant: poly (oxy-1,2-ethanediyl), α-(C16-20 branched and linear alkyl)ω-hydroxy (100%), which has the greatest use in domestic detergents, household cleaners, and personal care products. This active ingredient is made from renewable plant oils and is biodegradable under aerobic and anaerobic conditions. Agnique MMF can be applied to any mosquito habitats with standing water using conventional spraying methods, an invisible monomolecular film rapidly spreads over the entire surface of the aquatic habitats. This film interrupts the critical air/water interface in the mosquito’s larval and pupal development cycle causing them to drown. Agnique MMF can persist in the field for up to 22 days under certain conditions, but results of most studies indicate that this product breaks down relatively quickly in the environment and is often undetectable within 48 h after application (Stark 2005). A significant advantage is that mosquitoes cannot develop resistance to Agnique MMF because control is achieved through a physical mode of action. Another advantage of the physical mode of action is its effectiveness on all mosquito species. Agnique MMF was demonstrated to have a good safety profile against the fresh-water green tree frog, mosquitofish, and other fresh water fish, in a six month study and against salt water fish in a seven day study when applied at ten times the rate against mosquitoes (Webber and Cochran 1984). The safety against other fresh water fish, shrimp, isopoda, amphipod, crab, and crayfish was also documented by 96 h static acute toxicity testing when applied at 100 times the application rates (Hester et al. 1991). However, water surface dwellers, such as water striders (Gerridae), or ones which must make contact with the air-water interface to breathe, such as water boatman (Corixidae) and other hemipterans, as well as diving beetles (Dytiscidae), are negatively affected by Agnique MMF (Stark 2005). This product was withdrawn from the market in 2013 but may become available again in the near future. In the current study, some significant impacts on the life events of the TPS occurred when they were exposed to
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Agnique MMF at rates ranging from 1.89-9.45 liters/ha under laboratory and field conditions. Interestingly, fecundity tended to increase when the TPS population density was decreased as a result of mortality caused by treatment, when intraspecific competition was relieved at a lower population density. When assessing the gravid individuals in the populations, the need to sex the shrimp is negated because of the nature of the TPS being hermaphroditic. Each individual can potentially become gravid if ecological factors are conducive to the growth, development, and maturation. The proportion of gravid individuals can be very high in a welldeveloped population. It appears that 1.89 liters/ha would be the threshold rate when using Agnique MMF to control mosquitoes in habitats where the TPS serve as a biological control agent or natural regulatory force. Tadpole shrimp swim upside down just underneath the water surface when the water temperature is warm, even though most shrimp stay for extended minutes at the bottom when the water temperature is low. Monomolecular film interrupts the air/ water interface and cause the shrimp to drown. Additionally, the TPS take dissolved oxygen through their gills, and the wetting of gills may also cause anoxia and mortality. In conclusion, the current study indicates that the juvenile hormone analog methoprene is highly compatible with the TPS when applied to control immature mosquitoes in habitats where TPS and mosquito species coexist. However, precaution should be taken when doing the same with Agnique MMF, which might be safe to the TPS only at the lowest label rate. REFERENCES CITED Abacus Concepts, Inc. 1987. StatView + Graphics. Abacus Concepts, Inc., Berkeley, CA, 234 pp. Fry, L.F. and M. S. Mulla. 1994. Field introductions and establishment of the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), a biological control agent of mosquitoes. Biol. Contr. 4: 113-124. Fry-O’Brien, L.L. and M.S. Mulla. 1996. Optimal conditions
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Figure 5. Effect of single treatment of Altosid® LL (5% methoprene) at 0.3, 0.6, and 1.2 liters/ha on population density, growth and fecundity of tadpole shrimp in field mesocosms. For TPS density and size, different letters indicate significant differences among control and treatments by oneway analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (df = 3,8, F = 5.7, P < 0.05). For the percentage of gravids, different letters indicate significant differences among treatments and control by Chi square test (c2 ≥ 4.0, P < 0.05).
Figure 6. Effect of single treatment of Agnique® MMF at 1.89, 4.73, and 9.45 liters/ha on population density, growth, and fecundity of tadpole shrimp in field mesocosms. For TPS density and size, different letters indicate significant differences among control and treatments by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (df = 3,8, F ≥ 5.2, P < 0.05). For percentage of gravids, different letters indicate significant differences among treatments and control by Chi square test (c2 ≥ 8.4, P < 0.01).
for rearing the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), a biological control agent against mosquitoes. J. Am. Mosq. Contr. Assoc. 12: 446453. Hester, P.G., M.A. Olson, and J.C. Dukes. 1991. Effects of Arosurf MSF on a variety of aquatic nontarget organisms in the laboratory. J. Am. Mosq. Contr. Assoc. 7: 48-51. Lawler, S.P., T. Jensen, D.A. Dritz, and G. Wichterman. 1999. Field efficacy and nontarget effects of the mosquito larvicides temephos, methoprene, and Bacillus thuringiensis var. israelensis in Florida mangrove swamps. J. Am. Mosq. Contr. Assoc. 15: 446-452.
Lawler, S.P., D.A. Dritz, and T. Jensen. 2000. Effects of sustained-release methoprene and a combined formulation of liquid methoprene and Bacillus thuringiensis israelensis on insects in salt marshes. Arch. Environ. Contam. Toxicol. 39: 177-182. LeBlanc, G.A. 2007. Crustacean endocrine toxicology: a review. Ecotoxicology 16: 61-81. Maffi, M. 1962. Triops granaries (Lucas) (Crustacea) as a natural enemy of mosquito larvae. Nature 195: 722. Mulla, M.S. 1995. The future of insect growth regulators in vector control. J. Am. Mosq. Contr. Assoc. 11: 269-273. Sassaman, C., M.A. Simovich, and M. Fugate. 1997.
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Reproductive isolation and genetic differentiation in North American species of Triops (Crustacea: Branchiopoda: Nortostraca). Hydrobiologia 359: 125147. Sparks, T.C. and B.D. Hammock. 1983. Insect growth regulators: Resistance and the future. In: Pest Resistance to Pesticides. G.P. Georghiou and T. Saito (eds.). pp. 615669. Plenum Press. Stark, J.D. 2005. Environmental and health impacts of the mosquito control agent Agnique, a monomolecular surface film https://www.health.govt.nz/system/files/ documents/ publications/agniquereport-october2005. pdf (accessed June 16, 2014). Su, T. and M.L. Cheng. 2014. Cross resistances in spinosad – resistant Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol. 50: 428-435. Su, T. and M.S. Mulla. 2001. Nutritional and edaphic factors affecting growth, longevity and fecundity of the tadpole shrimp Triops longicaudatus (Notostraca: Triopsidae), a potential biological control agent of immature mosquitoes. J. Vector Ecol. 26: 43-50. Su, T. and M.S. Mulla. 2002a. Spatial distribution and hatch of field eggs of the tadpole shrimp Triops longicaudatus Le Conte (Notostraca: Triopsidae), a biological control agent of immature mosquitoes. J. Vector Ecol. 27: 128137. Su, T. and M.S. Mulla. 2002b. Factors affecting egg hatch of the tadpole shrimp Triops longicaudatus (Notostraca: Triopsidae) Le Conte, a potential biological control agent of immature mosquitoes. Biol. Contr. 33: 18-26.
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Su, T. and M.S. Mulla. 2002c. Introduction and establishment of tadpole shrimp Triops newberryi (Notostraca: Triopsidae) in a date garden for biological control of immature mosquitoes in the Coachella Valley, southern California. J. Vector Ecol. 27: 138-148. Su, T. and M.S. Mulla. 2005. Toxicity and effects of microbial mosquito larvicides and larvicidal oil on the development and fecundity of the tadpole shrimp Triops newberryi (Packard) (Notostraca: Triopsidae). J. Vector Ecol. 30: 107-114. Tietze, N.S. and M.S. Mulla. 1989. Prey-size selection by Triops longicaudatus (Notostraca: Triopsidae) feeding on immature stages of Culex quinquefasciatus. J. Am. Mosq. Contr. Assoc. 5: 392-396. Tietze, N.S. and M.S. Mulla. 1990. Influence of tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), stocking rate on Culex tarsalis development in experimental field microcosms. J. Am. Mosq. Contr. Assoc. 6: 265-269. Tietze, N.S. and M.S. Mulla. 1991. Biological control of Culex mosquitoes (Diptera: Culicidae) by the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae). J. Med. Entomol. 28: 24-31. Webber, L.A. and D.C. Cochran.1984. Laboratory observations on some freshwater vertebrates and several saline fishes exposed to a monomolecular organic surface film (ISA20E). Mosq. News 44: 68-69. Williams, C.M. 1967. The third generation pesticides. Sci. Am. 217: 13-17.
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Toxicity and effects of mosquito larvicides methoprene and surface film (Agnique® MMF) on the development and fecundity of the tadpole shrimp Triops newberryi (Packard) (Notostraca: Triopsidae) Tianyun Su1*, Yonxing Jiang2, and Mir S. Mulla Department of Entomology, University of California, Riverside, CA 92521, U.S.A., [email protected] Received 18 June 2014; Accepted 22 July 2014 ABSTRACT: We investigated the interactions of tadpole shrimp, a mosquito biological control agent, with the juvenile hormone analog methoprene and a monomolecular surface film. In laboratory assays, the tadpole shrimp (TPS) Triops newberryi (Packard) was able to tolerate high concentrations of methoprene without negative impacts on its growth, longevity, and fecundity when exposed to 1 to 10 mg/liter, or 90-900 fold, of the IE90 levels against a laboratory colony of Culex quinquefasciatus Say. The same held true in field trials when the habitats were treated with Altosid® Liquid Larvicide (Altosid® LL, 5% methoprene) at 0.3-1.2 liters/ha. or 1-4 fold of the label rates for mosquito control. However, some significant impacts on the TPS occurred when they were exposed to Agnique® Monomolecular Film (Agnique® MMF) at the label rates for mosquito control ranging from 1.89-9.45 liters/ha. under laboratory and field conditions. To avoid the negative impact of Agnique MMF on tadpole shrimp, it appears that 1.89 liters/ha. would be the maximum rate when Agnique MMF is used to control mosquitoes in the habitats where the TPS is employed as a biological control agent, or prevailing in the aquatic habitats with potential for suppressing mosquito larval populations. Journal of Vector Ecology 39 (2): 340-346. 2014. Keyword Index: Tadpole shrimp, Triops newberryi, methoprene, Altosid® LL, Agnique® MMF.
INTRODUCTION Tadpole shrimp (TPS) are fresh water crustaceans (Notostraca: Triopsidae) and are widely distributed on all continents except Antarctica. There are two genera, Triops and Lepidurus, in the family Triopsidae, each comprising ten identified species. The common natural habitats of the TPS are temporary freshwater bodies that are characterized by extreme physical and chemical conditions. Triops newberryi (Packard), formerly called Triops longicaudatus Le Conte, the predominant species of tadpole shrimp in the southwestern United States, including the states of Washington, Oregon, and California (Sassaman et al. 1997, Su and Mulla 2002a), was considered as a natural enemy of immature mosquitoes in temporary waters decades ago (Maffi 1962). However, their potential utility in mosquito control was not available until recent publications by Tietze and Mulla (1989, 1990, 1991) and Fry and Mulla (1994). The characteristics of fast nymphal growth, early maturation, high reproduction capacity (Fry-O’Brien and Mulla 1996, Su and Mulla 2002b), and their predation on immature mosquitoes (Tietze and Mulla 1989, 1990, 1991) make them suitable for regulating mosquito populations that share the same ephemeral or semipermanent habitats with TPS. The practical use of TPS in controlling immature mosquitoes in specific habitats in the Coachella Valley, southern California, has been successfully attempted by introduction of desiccation-resistant eggs into some date gardens (Su and Mulla 2002c).
A successful mosquito control agent should have the attributes of being easily and permanently established with little or no augmentation, a high reproductive potential, and resistance to biological and physiochemical constraints imposed on it, as well as being compatible with most of the other mosquito control materials. TPS can be permanently established in ephemeral habitats by stocking desiccationresistant eggs (Su and Mulla 2002c). The eggs hatch and populations colonize quickly after each flooding, and they develop synchronously with some floodwater mosquito species. The TPS also have high reproductive capacity and their desiccation-resistant eggs can remain viable in the soil for long periods of time and undergo installment hatching after each hydration. However, little is known about the compatibility of TPS with some of the most commonly used mosquito larvicides. Currently, Bacillus thuringiensis var. israelensis (B.t.i.), B. sphaericus, spinosad, methoprene (IGR), larvicidal oils, monomolecular surface film mosquito larvicides, and a few others are the materials registered for mosquito control in California and most of the U.S.A. The toxicity and effects of B.t.i., B. sphaericus, and larvicidal oil on the development and fecundity of T. newberryi have been reported by Su and Mulla (2005). The objective of this study was to determine the compatibility of two commonly used mosquito larvicides, Altosid® Liquid Larvicide (Altosid® LL) and Agnique® Monomolecular Film (Agnique® MMF), with the TPS. The toxicity and effects of methoprene and monomolecular
Current address: West Valley Mosquito and Vector Control District, Ontario, CA 91761, U.S.A. Current address: City of Gainesville Mosquito Control Services, Gainesville, FL 32609, U.S.A.
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surface film on growth, survivorship, longevity, maturity, and fecundity of TPS were evaluated in the laboratory and earthen ponds under field conditions.
made among control and treatments by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (Abacus Concepts, Inc. 1987).
MATERIALS AND METHODS
Field mesocosm studies Field studies on the impact of methoprene and surface film on TPS were conducted in mesocosm habitats at the Aquatic and Vector Control Research Facility. In total, 12 earthen ponds were used, each with 27 m2 (3.7 x 7.3 m) surface area and water depth of 30 cm, and water levels were kept relatively constant by float valves on the water lines feeding each pond. These ponds in the past have been used for research on the biology and control of mosquitoes. Shrimp eggs occur in large numbers in the dry soil where an egg bank has been well-established. Egg hatch occurs soon after flooding when water temperature and photoperiod conditions are optimal. Prior to flooding, all pond bottoms were allowed to completely dry and 1,500 g of rabbit chow pellets were added in order to boost nutrient levels in the pond water when flooded for the experiment. Treatment was made on day 4 post-hydration when the TPS grew to the size of CL measured 4-5 mm (Su and Mulla 2001). Each aliquot of Altosid LL in the amount of 0.8, 1.6, or 3.2 ml was diluted to 120 ml in a 125 ml plastic bottle and applied by jet spray to the water surface of each respective pond, which achieved the rates of 0.3, 0.6, and 1.2 liters/ha, respectively. Agnique MMF in the amounts of 5, 12.5, and 25 ml were transferred to 125 ml plastic bottles and brought to a final volume of 120 ml with tap water and applied to the water surface of each pond in the same way as described previously. These application rates were approximately equivalent to 1.89, 4.73, and 9.45 liters/ha, respectively. For each test, three treatments and one untreated control were assigned randomly with three replicates each. Treatments were made four days after flooding. The density of TPS was determined on day pre-treatment and days 3, 7, and 14 post-treatment. For sampling the TPS, a long-handled D net (30 x 25 cm) was used. Two samples were taken by sweeping the bottom of pond along each long side of the rectangular ponds. Collected TPS along with any debris, sand, and soil were transferred to an enamel pan (40 x 24 x 6 cm) containing 1 liter of water from the pond. The exact number of TPS in each pan was counted if the number was lower than 100 or estimated by counting approximately half of the TPS if the number was greater than 100. Up to 20 shrimp, depending on the number of shrimp available in the pan, were randomly selected and preserved in 75% ethanol and kept in a refrigerator at 5-7° C for later processing in the laboratory. Size of the shrimp was estimated by measuring the CL, and the percentage gravid was determined by examining modified appendages of the 11th pair of limbs, the ovisacs, where reddish, shining mature eggs were present in gravid individuals. Average population densities (no. /net) and sizes (CL mm) of the shrimp as well as their standard errors were calculated and comparison was made among treatments and control by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05. Percentage of gravids was analyzed among treatments and the control by the Chi
Test materials Technical grade methoprene (98.1%, Lot: 326-28A) was purchased from Chem Service, West Chester, PA, and Altosid LL (5.0% AI, Lot#: 020619087) was provided by Wellmark International Schaumburg, IL. Agnique MMF (100% AI, Lot# U83D221132) was acquired from Cognis Corp., Cincinnati, OH. For methoprene tests, technical grade material was used in the laboratory studies, while the commercial product Altosid LL was used in the field mesocosms. The commercial product Agnique MMF was tested under both laboratory and field conditions. Laboratory studies Laboratory studies of the effects of technical grade methoprene and monomolecular surface film on the development and fecundity of the TPS were conducted according to the procedures previously described by Su and Mulla (2005). In brief, the TPS eggs in 200 g of soil collected from the Aquatic and Vector Control Facility at the Agricultural Experimental Station of University of California at Riverside were hatched in an enamel pan (40 x 24 x 7 cm) by adding 2 liters of distilled water and maintained at 2528° C with a photoperiod of 16:8 (L:D). Two to three days after hydration, one juvenile TPS with carapace length (CL) at carinal suture of about 2 mm was transferred from the hatching pan to an individual enamel pan (30 x 19 x 5 cm) containing 200 g of soil (sandy loam devoid of TPS eggs) as substrate (Su and Mulla 2001) and 1 liter of distilled water. Each enamel pan was provided with 0.5 g rabbit pellets (Brookhurst Mill, Riverside, CA) and 20 2nd instar mosquito larvae from a laboratory colony of Culex quinquefasciatus Say every other day. For each test, a total of 48 enamel pans were set up. Technical grade methoprene was diluted with acetone to 5% AI, and appropriate aliquot (20, 100, and 200 ml) of this dilution was added to each of the enamel pans, yielding concentrations of 1, 5, and 10 mg/liter AI, respectively. Three treatments and one untreated control were assigned randomly with 12 replicates (pans) each. For Agnique, 10, 24, and 48.5 ml was added to each enamel pan, approximately equivalent to the label rate range of 1.89, 4.73, and 9.45 liters/ ha. Similarly, three treatments and one untreated control were assigned randomly with 12 replicates each. After treatment, the rearing pans were held in the insectary at 25-28° C and a photoperiod of 16:8 (L:D). Carapace length as a growth parameter was measured and mortality was recorded daily until all shrimp died, and the size at death as indicated by CL was determined. After the death of all TPS, rearing water was slowly drained, soil at the bottom of the enamel pans containing fresh TPS eggs was dried at 28-30° C, and eggs in the dried soil were then counted according to the procedures of Su and Mulla (2001). The daily growth was charted, and comparisons in size at death, longevity, and fecundity were
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square test (Abacus Concepts 1987). In each test, a minimummaximum thermometer was placed in one of the ponds for measuring water temperature. The min-max temperature was read on every sampling day. RESULTS Laboratory studies Methoprene. Four biological parameters related to growth and development profiles of the tadpole shrimp were measured in the laboratory: the daily CL increase during life, the CL at death, average longevity, and fecundity (eggs/ TPS). Exposure of TPS to technical grade methoprene at concentrations of 1, 5, and 10 mg/liter had no noticeable effects on the growth rate, as the growth curves among various treatments fluctuated within a narrow range (Figure 1). Various treatments ranging from 1 to 10 mg/liter of methoprene did not reduce the maximum size at death; shrimp treated at 5 mg/liter actually reached a significantly greater size (df = 3,44, F = 31.9, P < 0.001). The average longevity was not adversely impacted by methoprene treatment. The lifetime fecundity as expressed as total eggs produced per individual did not decline significantly in all treatments. The shrimp exposed to 5 mg/liter methoprene produced even more eggs (df = 3,44, F = 20.6, P < 0.001)(Figure 2). Agnique MMF. The growth curves of TPS exposed to Agnique at the rates of 1.89 to 9.45 liters/ha seemed overall comparable with untreated controls in the laboratory (Figure 3). The TPS, however, reached significantly smaller size at death across rates applied (df = 3,44, F = 38.8, P < 0.001). The shrimp exposed to Agnique MMF at the higher rates of 4.73 and 9.45 liters/ha had a significantly shorter life span (df = 3,44, F = 10.3, P < 0.01), while the ones treated at the highest rate of 9.45 liters/ha produced significantly fewer eggs during their lifetime (df = 3,44, F = 28.4, P < 0.001) (Figure 4). Field mesocosm studies Altosid LL. Field studies showed that exposure of TPS to a single treatment of Altosid LL at the rates of 0.3, 0.6, and 1.2 liters/ha did not adversely impact their density, with an 10
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exception that a significantly lower density was encountered at the highest rate of 1.2 liters/ha on day 3 post-treatment (df = 3,8, F = 5.7, P < 0.05). The average size and percentage of gravid shrimp did not show significant differences among all treatments. The percentage of gravid shrimp was actually higher on day 14 post-treatment at the rate of 0.6 liters/ha (c2 ≥ 4.01, P < 0.05) (Figure 5). Agnique MMF. In field tests of Agnique MMF, the population density of the TPS declined in relation to treatment rates and time post-treatment. The population density was not impacted by treatment at 1.89 liters/ha, but it significantly declined on day 7 and day 14 post-treatment at the higher rates of 4.73 and 9.45 liters/ha. Treatment at the highest dose of 9.45 liters/ha significantly reduced the TPS density as early as day 3 post-treatment (df = 3,8, F ≥ 6.4, P < 0.05). Rate-dependent differences were evident among the four rates applied (Figure 6). The average size of the TPS was not impacted by the treatments. Interestingly, the TPS tended to reach larger sizes when their population densities were lower at the higher rates of treatment (df = 3,8, F ≥ 5.2, P < 0.05). On day 3 post-treatment, a proportion of gravid TPS prevailed at the low levels at all treatments (χ2 ≥ 10.4, P < 0.01), but reached higher levels when their densities were lower at the higher rates (χ2 ≥ 8.4, P < 0.01). At the rate of 9.45 liters/ha the TPS population densities were too low to assess the percentages of the gravid individuals (Figure 6). DISCUSSION Juvenile hormones have been considered as agents of insect development since the 1960s (Williams 1967). Methoprene, together with kinoprene, hydroprene, triprene, and others, were synthesized in the late 1960s and have been recognized as active juvenile hormone analogs, or juvenoids, against a wide variety of insects of agricultural, medical, and veterinary importance (Sparks and Hammock 1983). Immature mosquitoes are among the most common target species of methoprene, where successful emergence of adult mosquitoes is interrupted due to the early exposure during larval and pupal stages. One of the additional benefits of
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Figure 2. Growth, longevity, and fecundity of tadpole shrimp treated by technical grade methoprene at 1, 5, and 10 mg/liter in the laboratory. Significant difference in each parameter was indicated among control and treatments by different letters by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (Growth: df = 3,44, F = 31.9, P < 0.001; Fecundity: df = 3,44, F = 20.6, P < 0.001). methoprene is that exposed mosquito larvae and pupae remain available to the trophic web in the aquatic ecosystems when control efficacy is expected later (Lawler et al. 2000). Various products ranging from fast release emulsifiable concentrates to slow release briquets have been developed and used for mosquito control worldwide. Their laboratory activity and field efficacy for mosquitoes, and safety profile to non-target aquatic invertebrates, have been well documented (Mulla 1995, Lawler et al. 1999, 2000). It is a quite common practice to incorporate chemical control with biological control agents in integrated vector control programs to achieve high levels or extended periods of efficacy. The current study indicates methoprene products have a potential to be used with TPS, because exposure of TPS to technical grade methoprene at approximately 90–900 x IE90 against a laboratory colony of Cx. quinquefasciatus (11.1 mg/liter) (Su and Cheng 2014), did not have noticeable effects on the growth rate and did not impact the maximum size at death or average longevity and lifetime fecundity under laboratory conditions. Furthermore, application of 5% methoprene formulation of Altosid LL at
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the rates of 0.3, 0.6, and 1.2 liters/ha over mesocosm ponds, each measuring 27 m2 and 30 cm deep, resulted in about 4.75, 9.57, and 19.10 mg/liter methoprene, respectively, which exceeded the IE50 (0.583 mg/liter) and overlapped with IE90 (11.1 mg/liter) against susceptible Cx. quinquefasciatus from a laboratory colony (Su and Cheng 2014). In agreement with laboratory studies, the field tests also showed a good margin of safety of methoprene to the TPS. Both laboratory and field study results clearly indicate the compatibility of methoprene with the TPS. It is believed that methoprene is a true analog of juvenile hormone III (JH III) in mosquitoes, therefore its activity is relatively target specific. The TPS depend on the major terpenoid hormone, methyl farnesoate, an immediate precursor of insect JH III to regulate their growth and ovarian development (LeBlanc 2007). The reason that TPS were not adversely impacted by methoprene could be either because the TPS need higher titers of the hormone than immature mosquitoes, or because of dissimilarities of the juvenile hormones in mosquitoes as compared with ones in the TPS. This aspect warrants further study. Agnique® MMF is an alcohol ethoxylated surfactant: poly (oxy-1,2-ethanediyl), α-(C16-20 branched and linear alkyl)ω-hydroxy (100%), which has the greatest use in domestic detergents, household cleaners, and personal care products. This active ingredient is made from renewable plant oils and is biodegradable under aerobic and anaerobic conditions. Agnique MMF can be applied to any mosquito habitats with standing water using conventional spraying methods, an invisible monomolecular film rapidly spreads over the entire surface of the aquatic habitats. This film interrupts the critical air/water interface in the mosquito’s larval and pupal development cycle causing them to drown. Agnique MMF can persist in the field for up to 22 days under certain conditions, but results of most studies indicate that this product breaks down relatively quickly in the environment and is often undetectable within 48 h after application (Stark 2005). A significant advantage is that mosquitoes cannot develop resistance to Agnique MMF because control is achieved through a physical mode of action. Another advantage of the physical mode of action is its effectiveness on all mosquito species. Agnique MMF was demonstrated to have a good safety profile against the fresh-water green tree frog, mosquitofish, and other fresh water fish, in a six month study and against salt water fish in a seven day study when applied at ten times the rate against mosquitoes (Webber and Cochran 1984). The safety against other fresh water fish, shrimp, isopoda, amphipod, crab, and crayfish was also documented by 96 h static acute toxicity testing when applied at 100 times the application rates (Hester et al. 1991). However, water surface dwellers, such as water striders (Gerridae), or ones which must make contact with the air-water interface to breathe, such as water boatman (Corixidae) and other hemipterans, as well as diving beetles (Dytiscidae), are negatively affected by Agnique MMF (Stark 2005). This product was withdrawn from the market in 2013 but may become available again in the near future. In the current study, some significant impacts on the life events of the TPS occurred when they were exposed to
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Agnique MMF at rates ranging from 1.89-9.45 liters/ha under laboratory and field conditions. Interestingly, fecundity tended to increase when the TPS population density was decreased as a result of mortality caused by treatment, when intraspecific competition was relieved at a lower population density. When assessing the gravid individuals in the populations, the need to sex the shrimp is negated because of the nature of the TPS being hermaphroditic. Each individual can potentially become gravid if ecological factors are conducive to the growth, development, and maturation. The proportion of gravid individuals can be very high in a welldeveloped population. It appears that 1.89 liters/ha would be the threshold rate when using Agnique MMF to control mosquitoes in habitats where the TPS serve as a biological control agent or natural regulatory force. Tadpole shrimp swim upside down just underneath the water surface when the water temperature is warm, even though most shrimp stay for extended minutes at the bottom when the water temperature is low. Monomolecular film interrupts the air/ water interface and cause the shrimp to drown. Additionally, the TPS take dissolved oxygen through their gills, and the wetting of gills may also cause anoxia and mortality. In conclusion, the current study indicates that the juvenile hormone analog methoprene is highly compatible with the TPS when applied to control immature mosquitoes in habitats where TPS and mosquito species coexist. However, precaution should be taken when doing the same with Agnique MMF, which might be safe to the TPS only at the lowest label rate. REFERENCES CITED Abacus Concepts, Inc. 1987. StatView + Graphics. Abacus Concepts, Inc., Berkeley, CA, 234 pp. Fry, L.F. and M. S. Mulla. 1994. Field introductions and establishment of the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), a biological control agent of mosquitoes. Biol. Contr. 4: 113-124. Fry-O’Brien, L.L. and M.S. Mulla. 1996. Optimal conditions
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Figure 5. Effect of single treatment of Altosid® LL (5% methoprene) at 0.3, 0.6, and 1.2 liters/ha on population density, growth and fecundity of tadpole shrimp in field mesocosms. For TPS density and size, different letters indicate significant differences among control and treatments by oneway analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (df = 3,8, F = 5.7, P < 0.05). For the percentage of gravids, different letters indicate significant differences among treatments and control by Chi square test (c2 ≥ 4.0, P < 0.05).
Figure 6. Effect of single treatment of Agnique® MMF at 1.89, 4.73, and 9.45 liters/ha on population density, growth, and fecundity of tadpole shrimp in field mesocosms. For TPS density and size, different letters indicate significant differences among control and treatments by one-way analysis of variance (ANOVA) for repeated measures with an alpha = 0.05 (df = 3,8, F ≥ 5.2, P < 0.05). For percentage of gravids, different letters indicate significant differences among treatments and control by Chi square test (c2 ≥ 8.4, P < 0.01).
for rearing the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), a biological control agent against mosquitoes. J. Am. Mosq. Contr. Assoc. 12: 446453. Hester, P.G., M.A. Olson, and J.C. Dukes. 1991. Effects of Arosurf MSF on a variety of aquatic nontarget organisms in the laboratory. J. Am. Mosq. Contr. Assoc. 7: 48-51. Lawler, S.P., T. Jensen, D.A. Dritz, and G. Wichterman. 1999. Field efficacy and nontarget effects of the mosquito larvicides temephos, methoprene, and Bacillus thuringiensis var. israelensis in Florida mangrove swamps. J. Am. Mosq. Contr. Assoc. 15: 446-452.
Lawler, S.P., D.A. Dritz, and T. Jensen. 2000. Effects of sustained-release methoprene and a combined formulation of liquid methoprene and Bacillus thuringiensis israelensis on insects in salt marshes. Arch. Environ. Contam. Toxicol. 39: 177-182. LeBlanc, G.A. 2007. Crustacean endocrine toxicology: a review. Ecotoxicology 16: 61-81. Maffi, M. 1962. Triops granaries (Lucas) (Crustacea) as a natural enemy of mosquito larvae. Nature 195: 722. Mulla, M.S. 1995. The future of insect growth regulators in vector control. J. Am. Mosq. Contr. Assoc. 11: 269-273. Sassaman, C., M.A. Simovich, and M. Fugate. 1997.
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Reproductive isolation and genetic differentiation in North American species of Triops (Crustacea: Branchiopoda: Nortostraca). Hydrobiologia 359: 125147. Sparks, T.C. and B.D. Hammock. 1983. Insect growth regulators: Resistance and the future. In: Pest Resistance to Pesticides. G.P. Georghiou and T. Saito (eds.). pp. 615669. Plenum Press. Stark, J.D. 2005. Environmental and health impacts of the mosquito control agent Agnique, a monomolecular surface film https://www.health.govt.nz/system/files/ documents/ publications/agniquereport-october2005. pdf (accessed June 16, 2014). Su, T. and M.L. Cheng. 2014. Cross resistances in spinosad – resistant Culex quinquefasciatus (Diptera: Culicidae). J. Med. Entomol. 50: 428-435. Su, T. and M.S. Mulla. 2001. Nutritional and edaphic factors affecting growth, longevity and fecundity of the tadpole shrimp Triops longicaudatus (Notostraca: Triopsidae), a potential biological control agent of immature mosquitoes. J. Vector Ecol. 26: 43-50. Su, T. and M.S. Mulla. 2002a. Spatial distribution and hatch of field eggs of the tadpole shrimp Triops longicaudatus Le Conte (Notostraca: Triopsidae), a biological control agent of immature mosquitoes. J. Vector Ecol. 27: 128137. Su, T. and M.S. Mulla. 2002b. Factors affecting egg hatch of the tadpole shrimp Triops longicaudatus (Notostraca: Triopsidae) Le Conte, a potential biological control agent of immature mosquitoes. Biol. Contr. 33: 18-26.
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Su, T. and M.S. Mulla. 2002c. Introduction and establishment of tadpole shrimp Triops newberryi (Notostraca: Triopsidae) in a date garden for biological control of immature mosquitoes in the Coachella Valley, southern California. J. Vector Ecol. 27: 138-148. Su, T. and M.S. Mulla. 2005. Toxicity and effects of microbial mosquito larvicides and larvicidal oil on the development and fecundity of the tadpole shrimp Triops newberryi (Packard) (Notostraca: Triopsidae). J. Vector Ecol. 30: 107-114. Tietze, N.S. and M.S. Mulla. 1989. Prey-size selection by Triops longicaudatus (Notostraca: Triopsidae) feeding on immature stages of Culex quinquefasciatus. J. Am. Mosq. Contr. Assoc. 5: 392-396. Tietze, N.S. and M.S. Mulla. 1990. Influence of tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae), stocking rate on Culex tarsalis development in experimental field microcosms. J. Am. Mosq. Contr. Assoc. 6: 265-269. Tietze, N.S. and M.S. Mulla. 1991. Biological control of Culex mosquitoes (Diptera: Culicidae) by the tadpole shrimp, Triops longicaudatus (Notostraca: Triopsidae). J. Med. Entomol. 28: 24-31. Webber, L.A. and D.C. Cochran.1984. Laboratory observations on some freshwater vertebrates and several saline fishes exposed to a monomolecular organic surface film (ISA20E). Mosq. News 44: 68-69. Williams, C.M. 1967. The third generation pesticides. Sci. Am. 217: 13-17.
