Tadpole shrimp (TPS), Triops newberryi (Packard) (Notostraca: Triopsidae), is a predatory agent for immature mosquitoes breeding in aquatic habitats. This biological control agent could be used with other larvicides in mosquito control programs. In order to elucidate compatibility of the TPS and commonly used mosquito larvicides, studies were initiated to investigate effects of Bacillus thuringiensis ssp. israelensis (B.t.i.) de Barjac, Bacillus sphaericus Neide and Golden Bear-1111 larvicidal oil on growth, longevity, and fecundity of TPS in laboratory and field. The exposure of TPS to high dosages of B.t.i. and B. sphaericus in the laboratory or in the field did not have significant adverse effects on growth, longevity, and fecundity. These results indicate that TPS are compatible with microbial larvicides and can be used jointly in practical mosquito control programs. The larvicidal oil GB-1111, on the other hand, caused almost 100% mortality within 48 h after treatment in the laboratory even at the low dosage of 0.38 gallons/ac. The impact of oil in the field was less severe, as significant adverse impact on population density was noted at 1 gallon/ac when water temperatures were warmer, or 2 gallons/ac when water temperatures were cooler. Almost 100% mortality within 48 h was noted at 2 gallons/ac when water temperatures were warmer or at 4 gallons/ac when the water temperatures were cooler. These results indicate incompatibility between this larvicidal oil and TPS at the higher rates of the label range, and joint use of TPS and larvicidal oil at dosages of 1-2 gallons/ac or greater, depending on water temperatures, should be avoided.

Bacillus sphaericus (Bsph) Neide has been recognized as an effective mosquito larvicide since its discovery 20 years ago. Various strains of this agent, such as 2362, 2297, 1593, and C3-41, have been developed, formulated, and field-evaluated against mosquito larvae in different countries. Their high efficacy in controlling mosquitoes breeding in various habitats, especially those in polluted water, has been documented. However, resistance to Bsph has been reported in Culex pipiens complex in both laboratory colonies and natural populations. During our field trials on Bsph water-dispersible granules (WDG) against natural populations of Cx. quinquefasciatus Say in a low-income community, Nonthaburi Province, Thailand, control failure occurred within 4 months after 5 treatments using VectoLex WDG at the dosages of 50-200 mg/m2. The suspected Bsph-resistant Cx. quinquefasciatus mosquitoes were collected and colonized in the laboratory, from which the late stage larvae of generations F14 through F17 were used in bioassays to elucidate the resistance profile. A high level of Bsph resistance was documented in this colony as compared with the susceptible mosquitoes from the same area in Thailand or from California, USA. The resistance ratios (RR) at LC50, depending on reference colonies, were 21,100-28,100-fold against Bsph WDG or greater than 125,000-200,000-fold against Bsph technical-grade material. These Bsph-resistant mosquitoes, however, were completely susceptible to Bacillus thuringiensis var. israelensis, (Bti) preparations, LC50 ranging from 0.017 ppm for technical material with 7000 ITU/mg to 0.052 ppm for water-dispersible granules with 3000 ITU/mg. The RR to mixtures of Bsph + Bti in this highly Bsph-resistant mosquitoes increased steadily upon the increase of Bsph ratios in the mixtures from 50, 75, 90, 95 to 99%. The resistance levels to the mixtures with various ratios of Bsph and Bti, however, were substantially lower than that in Bsph alone, suggesting addition of Bti to Bsph substantially enhanced the mosquitocidal activity (synergism) against these highly Bsph-resistant Cx. quinquefasciatus. Moderate tolerance to low levels of resistance to Bsph/Bti recombinant (RR 7.29-12.75 at LC50 and 5.15-13.40 at LC90) was also noted in this Bsph-resistant population.

Limited laboratory and field studies have indicated that the insect growth regulator (IGR) novaluron exhibits good activity against larvae of pest species in the orders Coleoptera, Hemiptera (suborder Heteroptera), and Lepidoptera by both ingestion and contact. We completed laboratory and field studies to evaluate activity and efficacy of novaluron against Culex mosquitoes. In laboratory studies, novaluron was highly active against Cx. quinquefasciatus, as indicated by low levels of inhibition of emergency (IE), at the 50% level, IE50 (0.159 ppb for 2nd-stage larvae and 0.118 ppb for 4th-stage larvae) and IE90, at the 90% level, (0.604 ppb for 2nd-stage larvae and 0.595 ppb for 4th-stage larvae). In outdoor microcosm and mesocosm studies against natural populations, novaluron yielded excellent control of immature Culex mosquitoes for up to 14 days at 1.25, 2.5, and 5 ppb in microcosms, and for up to 7 days at the dosages of 1, 5, and 10 mg/m2 in mesocosms. Based on qualitative observations, novaluron seemed to have a favorable margin of safety for nontarget aquatic invertebrates cohabiting with mosquito larvae. Further large-scale field studies are warranted to evaluate initial efficacy and longevity of novaluron against various mosquito species, as well as its safety for nontarget biota.

Underground storm drain systems in urban areas of Orange County include thousands of miles of gutters and underground pipelines, plus hundreds of thousands of catch basins and manhole chambers, all of which drain runoff water from residential, business and commercial establishments as well as highways and streets. These systems serve as major developmental and resting sites for anthropophilic and zoophilic mosquitoes. Investigations on spatial and temporal distribution of mosquitoes in these systems were conducted during November 1999 to October 2001. Immature mosquitoes were sampled by dipper or dipping net and adult mosquitoes by nonattractive CDC traps in manhole chambers, catch basins and a large drain. Culex quinquefasciatus Say prevailed at all 15 structures of the study in 4 cities of Orange County as the predominant species (>99.9%). Larvae and pupae were present from April to October, peaking from May to September. The population density of adults was the lowest in February with 2 peaks of abundance occurring from May to July and from September to October. Manhole chambers and catch basins harbored more mosquitoes than did the large drain. Minimum and maximum temperatures during a 24 h sampling period was an important factor influencing adult mosquito activity and catches; more mosquitoes were caught in traps when it was warmer, especially when the minimum temperatures were higher. The proportion of females to males in general increased during winter and early spring and declined during summer. The proportion of gravid females to empty females was higher during the winter than in summer. Other dipteran taxa such as psychodid moth flies and chironomid midges exhibited somewhat similar seasonal patterns as did mosquito populations. Average water temperature was relatively stable throughout the year, and water quality in underground drain systems was characterized by low dissolved oxygen, coupled with above normal electrical conductivity and salinity levels that were more pronounced during the summer. The episodes of measurable rains that occurred during the winter months (February and March) reduced mosquito population density (both larvae and adults) and altered water quality in the underground drain systems.

In recent years, highly potent mosquitocidal strains of the microbial agent Bacillus sphaericus (Bsph) have been isolated and developed for the control of mosquito larvae around the world. Laboratory selection experiments with the most active strains and their use in large-scale operational mosquito control programs resulted in the emergence of resistance in larvae of the Culex pipiens complex. This generated great concern among vector control agencies around the world, who feared reduced efficacy of this highly active larvicidal agent. To address this issue, the current studies were started to find practical strategies for controlling resistant mosquitoes and more importantly to develop resistance management strategies that would prevent or delay development of resistance. We initiated field studies in 3 low-income communities in Nonthaburi Province, Thailand. In 1 of the communities, larvae of Culex quinquefasciatus that were highly resistant (>125,000-fold) to Bsph strain 2362 were successfully controlled with applications of Bacillus thuringiensis var. israelensis (Bti) alone or in combination with Bsph. To prevent or delay resistance to Bsph, 2 other sites were selected, 1 treated with Bsph 2362 alone and the other treated with a mixture of Bsph 2362 and Bti. Mosquitoes treated with Bsph 2362 alone showed some resistance by the 9th treatment and almost complete failure of control occurred by the 17th treatment. After 9 treatments with the mixture over a 9-month period at another site, no noticeable change in susceptibility to Bsph was detected. During this period, the site treated with Bsph alone required 19 treatments, whereas the site treated with mixtures took only 9 treatments because of slower resurgence of larvae at the site treated with the mixture than at the site treated with Bsph alone. This is the 1st field evidence for delay or prevention of resistance to microbial agents in larval Cx. quinquefasciatus by using mixtures of Bti and Bsph. Further studies on the use of mixtures for the management of field resistance are warranted.

Novaluron, a new chitin synthesis inhibitor type of insect growth regulator, was evaluated in the laboratory and field against larvae of the mosquito Aedes aegypti (L.). In the laboratory, the technical material showed a high level of activity against 2nd and 4th instar larvae. The inhibition of emergence (IE) was 100% at concentrations of 0.25 to 1.0 microg/L. Second instars were slightly more susceptible than fourth instars. At the high concentration (1 ppb), most of the mortality occurred in the larvae, but at sublethal concentrations (for larvae) mortality also occurred in pupae and adults (incomplete eclosion). An EC10 (emulsifiable concentrate containing 10% active ingredient) formulation of novaluron was evaluated against Ae. aegypti in water-storage containers in the field in Thailand. Two tests at high (0.05 to 1 mg/L) and low (1 to 20 microg/L) concentrations were carried out in clay jars (200 L) and plastic pails (75 L). At high concentrations in clayjars, we obtained 86-96% inhibition of emergence (IE) for about 190 d, but the mortality declined at low concentrations after 190 d. At much lower concentrations, in jars and plastic pails in the second experiment, the IE was 80-100% for 2 mo. The highest concentrations of this series (10 microg/L in jars and 20 microg/L in pails) were active for 75 and 68 d respectively, losing efficacy by 82 d post-treatment. In the experiment using low concentrations, all treatments in jars and plastic pails failed to provide adequate control 90 d after treatment. These studies show that novaluron, even as EC10, has exceptional long-term activity against Ae. aegypti in water-storage containers, with higher concentrations yielding greater long-term control than low concentrations.

Tadpole shrimp (TPS), Triops newberryi (Packard), has been reported to have a potential as a biocontrol agent for larval mosquitoes breeding in intermittently flooded habitats. To develop and promote this predator for controlling mosquitoes, a date garden devoid of preexisting TPS populations was chosen in the Coachella Valley, southern California in 2000 to receive introductions of TPS eggs and mature TPS. Mosquito control by TPS was assessed in the plots one year after their introductions. In a selected block on this ranch, 2 rows were stocked with TPS eggs, where soil containing approximately 2,000 eggs was spread over the surface of dry ground in each row before flooding. Another 2 rows were used for mature TPS introduction, where about 400 mature TPS were released into standing water in each row 1 day after flooding. After a single egg or mature TPS introduction, active TPS in water and viable eggs in dry surface soil were noted in increasing numbers during the 3-4 subsequent irrigations. Disking before irrigation, which turned the eggs over and mixed them into the soil column, reduced TPS egg populations at the soil surface and subsequent active TPS populations in standing water after irrigation. After one or two irrigations, viable eggs and active shrimp were found in centers adjacent to the introduced plots in the stocked rows. Ample evidence is presented to show that TPS populations were established after a single introduction of eggs or mature TPS. TPS eggs and/or newly hatched TPS were also carried into the neighboring rows across the borders by the overflowing irrigation water, and TPS populations became established there too, as active TPS were noted after each irrigation in the adjacent unstocked rows. Considering the ease and economical storage, transportation and handling, dessication--resistant eggs have advantages over mature TPS for field introductions. Mosquito control by TPS was assessed in rows with and without TPS in July 2001, one year after TPS introductions. Production of Psorophora columbiae Dyar and Knab and TPS populations were determined 4-5 days after each of 2 irrigations, when there was no or little vegetation inside the rows. As compared with the row without TPS, the presence of relatively high numbers of TPS reduced Ps. columbiae by 73 to 99% as based on the average numbers of larvae, pupae and exuviae per dip in the rows with versus without TPS.

The tadpole shrimp (TPS), Triops newberryi (Packard) (Notostraca: Triopsidae), is a potential biological control agent for immature mosquitoes breeding in ephemeral habitats. Studies were initiated in the laboratory to investigate the effects of some ecological and physiological factors on egg hatch. When subjected to successive hydrations, most of the hatch occurred in the first hydration with additional hatches noted in the second and third hydrations. In each hydration, most of the eggs hatched in 12 to 48 h. Temperature had a marked effect on egg hatch. Preconditioning treatment of eggs in soil at extreme temperatures lowered egg hatch in succeeding hydrations. Temperature during hydration also influenced egg hatch, with maximum hatch at 29°C. Length of photoperiod was another ecological factor affecting egg hatch; darkness during the hydration period reduced egg hatch. As a simulation of incomplete hydration commonly encountered in arid regions associated with light precipitation and scarce irrigation water, TPS eggs in soil were wetted and flooded for short time periods. These treatments enhanced egg hatch during succeeding full hydrations. The presence of soil during preconditioning and hatching did not affect egg hatch. High water salinity reduced or prevented egg hatch. Preconditioning treatment at high water salinity lowered egg hatch during succeeding hydrations with distilled water. Maximum egg hatch occurred at pH 7; preconditioning treatment at pH 3 lowered and at pH 11 eliminated egg hatch during succeeding hydrations with distilled water. TPS eggs from dead gravids (unlaid eggs) did not hatch after proper preconditioning and hydration.

The tadpole shrimp (TPS), Triops newberryi (Packard) (Notostraca: Triopsidae) is a potential biological control agent for immature mosquitoes breeding in ephemeral habitats. The occurrence of TPS eggs in soil and their hatch were investigated in 11 flood-irrigated date gardens in the Coachella Valley of southern California in 1999. Each garden was sampled several times after the rows were recently irrigated. All these date gardens harbored from very few to a large number of eggs in the soil. Overall, the average density of total eggs on ranches with clay loam soil was significantly higher than that on ranches with silt loam soil. The average densities of total eggs were significantly lower on the ranches that were disked compared to those on the ranches that were undisked before sampling. Two types of eggs were found and designated as "fresh" (yellowish to brownish) and "old" (blackish) eggs. This is the first time that these dimorphic eggs have been reported. The density of fresh eggs was lower than that of old eggs in most soil samples. The date gardens with high egg densities were sampled for determination of vertical occurrence, where soil was sampled up to 38.5 cm deep. Fresh eggs were recovered from soil in depths up to 25.6 cm, but the densities progressively declined with depth. The old eggs, however, were recovered from all soil depths studied, and there was no obvious relationship between soil depth and their density. This pattern of vertical occurrence of TPS eggs is the result of frequent disking for weed control and fruit harvest. Hatch of TPS eggs in surface soil samples ranged from 0 to 7.2 per 100 g dried soil. Hatch of viable eggs had an inverse relationship with soil depth. No TPS hatched out from the soil samples taken deeper than 15.4 cm. Fresh and old eggs distinguished by color were subjected to hatching tests. Fresh eggs exhibited high hatch, with hatching rates of 35.5-45.0% and 40.2-60.3% for the 1st and 1st plus the 2nd hydrations respectively. The old eggs, however, did not hatch at all. These findings provide quantitative information with regard to occurrence of natural TPS populations in flood irrigated agricultural fields, which could serve as a potential regulatory force of immature mosquito populations sharing ephemeral habitats with TPS.

Bacillus sphaericus (Bsph) strain 2362 has been recognized as a promising mosquito larvicide, and various preparations of this strain have been tested and used in mosquito control programs worldwide. This control agent has advantages of high efficacy, specificity, persistence, and environmental safety. However, resistance in Culex pipiens complex mosquitoes to Bsph has occurred in both laboratory and field populations, necessitating development of resistance management strategies Studies were initiated aiming at reversing previously established Bsph resistance in a laboratory colony of Culex quinquefasciatus Say by selections with Bti alone, Bti and Bsph in rotation, or mixture. Partial restoration of susceptibility to Bsph was achieved by selection of resistant colony for 10 generations with Bti alone at LC80. After this colony was switched back to Bsph selection for 20 generations, resistance to Bsph partially increased to a stable level. Selections of Bsph-resistant colonies with Bti and Bsph in rotation or mixture resulted in steady decline of resistance over 30 generations, with rapid decline in resistance noted in the initial 10-15 generations. It is interesting to note that selections with Bti and Bsph in rotation increased susceptibility to Bti in Bsph-resistant colony. It is promising that selection with Bti alone, Bsph and Bti in rotation, or mixture have a potential for developing practical strategies to overcome acquired resistance to Bsph in Cx. quinquefasciatus populations.

The notostracan tadpole shrimp (TPS) Triops newberryi Packard has potential to be used as a biocontrol agent of immature mosquitoes. Eggs, nymphal or adult shrimps are considered to be the stages for field introduction. To yield good growth of the shrimp and high production of shrimp eggs under artificial conditions, nutritional requirements of TPS for growth, survival and fecundity need to be elucidated. In the laboratory, we evaluated various nutritional and edaphic regimens, such as soil alone, mosquito larvae or rabbit pellets alone and various combinations of these three components for culturing. These factors influenced the growth, longevity and egg production profoundly. It was shown that the simulated natural conditions, i.e. full combination of all three factors, yielded the largest TPS with longest survival and highest egg production, followed by the combinations of any two components. Any single component, soil, mosquito larvae, or rabbit pellets, did not result in good growth, survival and egg production. By formulating optimal rearing substrates, this species of TPS will yield large numbers of all stages for experimentation and field introductions. Under optimal conditions, they mature in 7-8 days and survive for about one month. Each TPS is capable of producing up to 1,000 eggs during its lifetime. These studies developed nutritional regimens for TPS mass culturing procedures, where the eggs, nymphal and adult TPS can be mass cultured for field introduction and stocking in mosquito developmental sites.

During 1999 and 2000 several larvicidal treatments of Bacillus sphaericus strain 2362 water dispersible granular (WDG) formulations were made at 50 to 200 mg/m2 in mosquito developmental sites in low-income communities in Nonthaburi Province, Thailand to determine whether larviciding dense populations would results in a noticeable reduction of adult mosquitoes in small treated areas. In the treated area in 1999 (Soi Jumpa), immature populations were suppressed to extremely low levels for extended periods, especially at the higher dosages. This decline in immature populations was followed by a substantial decline in adult mosquitoes. There was a lag of 7 to 14 days post-larval treatments before maximum decline in adults was noted. Adults that emerged prior to treatments survived for 7-14 days or longer, thus no drastic reduction was noted soon after treatments. Despite a slight resurgence in adult mosquitoes during the middle of the experimental period, adult female mosquitoes (over 98% Cx quinquefasciatus), remained low during the 5-month period of trials. During the last 2 weeks (17 days post last treatment) of the experimental period, female populations reached the pre-treatment level. During the 2000 tests at Wat Pikul reduction in larvae was 87-98% for 7 weeks after first treatment at 200 mg/m2, resulting in a reduction of 24 to 73% (2 and 7 days post-treatment respectively) and 87 to 98 (2-6 weeks) in the adults. In the second and third treatments at 50 mg/m2, larval control and subsequent adult reduction were lower and shorter-lived than at the high dosage, and the fourth treatment at 100 mg/m2 did not yield a high level of reduction in the larvae (18 to 33%), but reduction of adults was still 80%. The final fifth treatment at 200 mg/m2 yielded only 18% control of larvae, suggesting tolerance to B. sphaericus at this site. It was shown that at both treated sites repeated treatments with a larvicide such as B. sphaericus could result in substantial reduction in adult mosquitoes. Vigilance for detection of resistance development should be practiced, as resistance could emerge in certain populations following a few treatments.

Culiseta incidens Thomson is distributed over most of the western USA and Canada northward to Alaska. Because this mosquito is difficult to colonize, its biology has not been well investigated. We colonized this species in 1998 and studied the effects of temperature on various aspects of its life cycle. The time required for egg melanization and the duration of the egg stage were negatively correlated with temperature. The proportion of fertile egg rafts was temperature-independent. An inverse relationship existed between temperature and egg hatch. Molting and stadium duration after hatching were temperature-dependent, with higher temperature accelerating development and molting. Larvae and pupae experienced lower mortality and higher molting success at lower temperatures. Survivorship of adult mosquitoes fed on sugar solution was inversely proportional to temperature, lethal times for 50% mortality (LT50) were greater at the lower temperature than at the higher temperature. Females survived longer than did males at all test temperatures. Because this species is eurygamous, mating only occurred in large cages. Mating success was also affected by temperature. At the test temperatures, 20 degrees C, 25 degrees C and 30 degrees C, mating started from 3-5 days after emergence and reached a peak on days 13-15 after emergence. Maximum mating rates at 20 degrees C and 25 degrees C were higher than at 30 degrees C. Blood feeding, as indicated by cumulative feeding rates, was affected by cage size, mosquito age and temperature. Mosquitoes in large cages exhibited a much higher feeding rate than in small cages. With age, the cumulative blood feeding rate increased, with the highest rate at 25 degrees C, followed by 20 degrees C and 30 degrees C. At all temperatures tested, most of the blood fed females were mated.

Neem (Azadirachta indica A. Juss) products have been shown to exert pesticidal properties against a variety of insect species. In mosquito control programs, such products may have the potential to be used successfully as larvicides. In exploring other advantages of neem products, we studied the oviposition responses of Culex tarsalis Coquillett and Cx. quinquefasciatus Say to two experimental azadirachtin (AZ) formulations, wettable powder Azad(TM) WP10 (WP) and emulsifiable concentrate Azad(TM) EC4.5 (EC). Gravid Cx. tarsalis exhibited a distinct preference for the neem suspension of the WP, where significantly more egg rafts were collected from the treatment than from the control. The minimum effective AZ concentration for this activity was 0.5 ppm. The aged suspensions from 1-7 days at 0.5 and 1 ppm AZ were more active in eliciting oviposition responses in Cx. tarsalis than the fresh preparations. This activity of the aged suspensions lasted up to 14 and 21 days at 0.5 and 1 ppm AZ, respectively. Negative ovipositional responses were indicated in the tests of the EC vs. Cx. tarsalis, as well as both neem formulations vs. Cx. quinquefasciatus. In the tests of the EC formulation, significantly less gravid females were trapped by oviposition cups in the treatment than in the control, and in the tests of the WP significantly less egg rafts were collected from the treatment than from the control. The minimum effective concentrations for oviposition avoidance activity were 5 ppm AZ for Cx. tarsalis and 10 ppm AZ for Cx. quinquefasciatus, which lasted up to 1 and 4 days for these two species respectively. Neem products potentially used as mosquito larvicides may have many additional benefits in mosquito control programs, the oviposition modification noted in the current studies is one such example.

Bacteria and mosquito abundance were studied in outdoor tubs unenriched and enriched with 0.04% rabbit pellets during the winter, 1999. The irrigation water used to fill the tubs contained a total bacterial count of 1.15-1.35 x 10(3) cells/ml. Adding rabbit pellets for enrichment yielded a total bacterial count of 5.50-7.63 x 10(5) cells/g. Bacterial densities in unenriched water were significantly lower than in enriched tubs on every sampling day. When bacterial densities in both enriched and unenriched regimens reached peak populations on day 3 post-flooding, their numbers in enriched water were 25-fold higher than the unenriched water. Under cool weather conditions, mosquito oviposition activity was low and larval development was very slow. Egg raft counts and larval densities in enriched water were nevertheless higher than those in unenriched water. After reaching peak populations on day 3 post-flooding, the natural decline in bacterial densities in the top portion of enriched water without mosquito larvae was lower compared with that in water with larvae. In water with larval present, the decline of bacterial levels in top water was greater than in bottom water on day 7 post-flooding. VectoBac G, a granular formulation of Bacillus thuringiensis subsp. israelensis caused a reduction in larval numbers of 80, 93, 73% at the rate of 5.5 lb/ac and a reduction of 94, 93, 86% at the rate of 10.6 lb/ac on days 1, 3, 7 posttreatment, respectively. After treatment, the reductions of bacterial densities in untreated tubs were greater than treated tubs. These results indicate that mosquito larvae play an important role in the decline of bacterial populations by their feeding activity.

A variety of formulations of Bacillus thuringiensis var. israelensis de Barjac (B.t.i.) and Bacillus sphaericus Neide (B.s.) have been studied for mosquito control under laboratory and field conditions. High efficacy, specificity, low risk of development of resistance, long shelf-life, and transportability, as well as the safety to nontarget organisms of these 2 microbial agents have been well documented. Some of the currently available formulations of B.t.i. and B.s. have low potency per unit mass. Research and development efforts are focusing on commercializing formulations with high potency and low minimum effective dosage that are suitable for long-distance shipment. To achieve this goal, new water-dispersible granule (WDG) formulations of both microbial agents were prepared and made available by Abbott Laboratories for evaluation. The newly developed WDGs of B.t.i. and B.s. with high potency dispersed readily in water with gentle agitation. These WDGs were evaluated and the minimum effective dosages were determined in microcosms against natural populations of Culex mosquitoes. The minimum effective dosage for B.t.i. WDGs with 4,000 International Toxic Units (ITU)/mg was 0.27-0.53 lb/acre which yielded significant control for up to 7-12 days. The minimum effective dosage for B.s. WDGs with 350-630 ITU/mg was 0.05-0.10 lb/acre, which yielded significant control of immature mosquitoes for up to 14-20 days.

Two new water dispersible granular (WDG) formulations of the microbial control agent Bacillus sphaericus (strain 2362) were extensively evaluated in polluted waters against Culex quinquefasciatus in Thailand. The studies were carried out in stagnant as well as flowing waters during August 1997 to July 1998. The trial period covered both rainy and dry seasons. The two WDG formulations of B. sphaericus tested were low potency (350 ITU/mg) and high potency (630 ITU/mg) products. Both formulations were used at various rates to determine initial efficacy and longevity. The high potency formulations provided excellent control (80 to 90%) of immature mosquitoes at the rates of 50 to 100 mg/ m2, while the less potent formulation yielded similar control at the rates of 89 to 250 mg/m2. Longevity of control was anywhere from one week to four weeks or longer depending on the dosage, habitat, and environmental conditions. Two treatments with low dosages of Bacillus thuringiensis ssp. israelensis WDG provided short-term control lasting for one week. Frequent episodes of heavy rains facilitated long-term suppression of immature mosquitoes in klongs by flushing out the larvae. In the absence of rain, the longevity of treatments in flowing waters was shorter than in the presence of rain. Rain, however, had some but not pronounced effect on longevity in the stagnant water habitats. Operation of floodgates controlling water flow from stagnant water habitats into the Chaophrya River had greater influence on the abundance of mosquito immatures in the ponded polluted waters under dwellings. Precipitation, adding large amounts of water, was probably responsible for diluting the control agents and also resulted in lower counts of immatures per unit volume of water. These variables could influence the efficacy (especially longevity) of treatments employing formulations of microbial agents in operational control programs. The WDG formulations are preferred over other formulations, such as granules, because the former contain larger quantities of toxins per unit mass than the latter and are easily applied as aqueous sprays. Potent formulations with greater content of active ingredients are less costly to ship and transport to distant areas.

Three tests were conducted during July 17 to October 30, 1998 to study the impact of two mosquitocidal microbial agents on mosquito larvae and their contribution to bacterial flora in aquatic microcosms. Formulations of Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus strain 2362 (Bsph) were applied at various rates to outdoor tubs enriched with rabbit pellets and filled with irrigation water from a reservoir. Mosquito larvae were effectively controlled by all treatments; the magnitude of initial and persistent control depended on materials and dosages applied. Bacterial flora were assessed in the irrigation water as well as water in the enriched tubs before and after treatment with the microbial agents. The irrigation water contained 800-1000 total bacterial cells/ml. The populations of total bacteria and spore formers peaked on day 3 after enriching and filling the tubs, then declined progressively to the low levels at the end of the tests. After treatment, the numbers of Bti and Bsph spores in treated tubs prevailed at a dosage-dependent manner, their populations peaked at three hours after treatment, and declined progressively thereafter. The contribution of Bti and Bsph spores to the total bacterial flora was negligible but significant to the counts of spore-forming bacteria. The gram-negative bacteria made up more than 80% of the total bacterial flora during the test periods; and, of these, gram-negative rods constituted the greatest proportion, gradually increasing from the time of flooding to the end of the tests. Gram-negative cocci also occurred in relatively great proportion, but showed a reverse trend as compared with the gram-negative rods, declining gradually from pretreatment to the end of the tests. Gram-positive rods (spore formers), including Bti and Bsph, occurred in low numbers in all the tests but increased slightly in treated tubs due to the addition of Bti and Bsph spores. Gram-positive cocci occurred occasionally in some water samples.

Bacillus thuringiensis ssp. israelensis de Barjac ( Bti ) and Bacillus sphaericus Neide are promising microbial mosquito larvicides currently employed in mosquito control programs. During the screening and evaluation of new formulations of these control agents, it was noted that productivity of some algal species was noticeably suppressed, and that certain water quality parameters were enhanced by the suppression of primary productivity with these agents. Quantitative studies on these aspects were conducted in microcosms where Culex mosquitoes bred naturally. Treatments with VectoBac G ( Bti ) at the high dosage of 48.1 kg/ha, B. sphaericus water dispersible granules at 3.1 kg/ha, and Bti water dispersible granules at 0.6 kg/ha yielded good control of immature mosquito populations. Two species of unicellular algae, Closterium sp. and Chlorella sp., existed in both the controls and treatments. Growth of these algae was suppressed by the microbial agents applied. Suppressed algal productivity and photosynthesis resulted in lower water turbidity and oxygen concentrations in the treatments than in the controls, especially during the hot season. Water in treatments was discernibly clearer than in the controls, which was further borne out by differences in the optical density readings.

When late 3rd or early 4th-instar larvae of Culex tarsalis Coquillett and Culex quinquefasciatus Say mosquitoes were treated with sublethal dosages of neem insecticide until pupation, the blood-feeding activity of the resulting adults was essentially the same as that of untreated controls. In contrast, blood-feeding activity was suppressed when newly emerged adults were fed continuously on 10 parts per million (ppm) or 50 ppm azadirachtin (AZ) in 10% sucrose solution for seven days. Fecundity was also reduced by the various neem treatments. When late 3rd or early 4th-instar larvae were treated with 0.010 ppm AZ to pupation, the resultant females had a lower rate of oviposition than did the untreated controls after a full blood meal. When late instar larvae were treated at 0.005 ppm and 0.010 ppm AZ, the resultant females produced smaller egg rafts after a full blood meal, as compared to the controls, but egg viability was not affected. In newly emerged adults feeding continuously on 10 ppm and 50 ppm AZ in 10% sucrose for seven days (before blood feeding), the oviposition rate, size of egg raft, and hatching rate of the eggs after a full blood meal were all reduced. When newly blood-fed adults were fed continuously on 10 ppm and 50 ppm AZ in 10% sucrose for five days, their oviposition rate was lower than controls in most cases, but the egg raft size and viability of eggs were not affected. In freshly blood-fed females topically treated with AZ with 1 or 5 micrograms/female, the oviposition rate and size of egg rafts were generally reduced. The females receiving topical treatment laid eggs and their hatching was not affected. The longevity of adult females feeding continuously on 10 ppm and 50 ppm AZ in 10% sucrose solution after emergence was reduced, whereas, the longevity of males was only affected at the higher concentration.