Sustainable control of mosquito larvae in the field by the combined actions of the biological insecticide Bti and natural competitors

Integrated management of mosquitoes is becoming increasingly important, particularly in relation to avoiding recolonization of ponds after larvicide treatment. We conducted for the first time field experiments that involved exposing natural populations of the mosquito species Culex pipiens to: a) application of the biological insecticide Bacillus thuringiensis israelensis (Bti), b) the introduction of natural competitors (a crustacean community composed mainly of Daphnia spp.), or c) a combined treatment that involved both introduction of a crustacean community and the application of Bti. The treatment that involved only the introduction of crustaceans had no significant effect on mosquito larval populations, while treatment with Bti alone caused only a significant reduction in the abundance of mosquito larvae in the short‐term (within 3–10 days after treatment). In contrast, the combined treatment rapidly reduced the abundance of mosquito larvae, which remained low throughout the entire observation period of 28 days. Growth of the introduced crustacean communities was favored by the immediate reduction in the abundance of mosquito larvae following Bti administration, thus preventing recolonization of ponds by mosquito larvae at the late period (days 14–28 after treatment). Both competition and the temporal order of establishment of different species are hence important mechanisms for efficient and sustainable mosquito control.     

Crustacean biodiversity as an important factor for mosquito larval control

Newly established ponds, which are highly dynamic systems with changing levels of biological interactions among species, are common larval mosquito habitats. We investigated the impact of crustacean abundance and taxa diversity on mosquito oviposition and larval development. The effects of the biological larvicide Bacillus thuringiensis israelensis (Bti) on mosquito larvae were monitored according to fluctuations in crustacean communities. Populations of the mosquito Culex pipiens colonized artificial ponds that contained crustacean communities at different time points of colonization by crustaceans: 1) ‘no colonization’ (no crustaceans), 2) ‘simultaneous colonization’ by crustaceans and mosquitoes, and 3) ‘head‐start colonization’ by crustaceans (preceding colonization by mosquitoes). All types of ponds were treated with three concentrations of Bti (10, 100, or 1,000 µg/liter). Colonization of all ponds by Cx. pipiens (in terms of oviposition, larval abundance, and larval development) decreased significantly with increasing diversity of crustacean taxa. The total abundance of crustaceans had a minor effect on colonization by Cx. pipiens. The presence of crustaceans increased the sensitivity of Cx. pipiens larvae to Bti treatment by a factor of 10 and delayed the time of recolonization. This effect of Bti was relevant in the short term. In the long term, the presence of Cx. pipiens was determined by crustacean biodiversity.     

Factors Affecting Residual Dosages of Two Formulations of Bacillus thuringiensis subsp. israelensis Tested in the Same Stream During a 3-Year Experiment

Although many field trials have been conducted using Bacillus thuringiensis subsp. israelensis (Bti)-based formulations, most have been in rivers with different biotic and abiotic conditions thus rendering the evaluation of their performance very difficult. Recently, results of a threeyear experiment using a new field procedure brought new insight into the behavior and the performance (carry) of two liquid formulations of Bti, Teknar HP-D and Vectobac 1200L, tested in the same lotic environment and under similar abiotic and biotic conditions. Factors such as discharge, water temperature and the hyporheic zone were identified as elements affecting the downstream loss of activity of both products. However, to better understand the reduction of black fly mortality along a stream (measured by using gutters), data of residual dosages of both products (measured by laboratory assay with mosquito larvae) were compared with reduction of black fly mortality. Bti toxic activity was monitored from water samples taken at different distances downstream from an application point, and from probes driven into the hyporheic zone, to study the effects of abiotic factors on the loss of the toxic crystals. Results showed that the loss of dosage was exponential for both products but more crystals were recovered from Vectobac 1200L along the stream than from Teknar HP-D. However, the latter was more efficient, i.e. less toxins were needed to kill 50% of black fly larvae both in temperate (16°C) and warmer (19.5-22°C) water. Also, a rise in water temperature had a greater effect in the kill induced by Vectobac 1200L compared to Teknar HP-D. For the same residual dosages present at the stations, longer carries of toxin activity (higher mortalities) were obtained in warmer water. Finally, the hyporheic zone was identified as a major source of loss of activity of Bti products. Large stream discharges decreased the effect of the hyporheic zone and that was reflected in longer carry of the products.     

Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.     

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.     

Indirect effects of the fairy shrimp,Branchipus schaefferi and two ostracod species on Bacillus thuringiensis var Israelensis-induced mortality in mosquito larvae

Bacillus thuringiensis var israelensis (BTI), when ingested by mosquito larvae, is highly toxic to them. Many other aquatic invertebrates feed on bacteria but, in general, BTI is not toxic to them. We tested in the laboratory the hypothesis that certain crustaceans indirectly benefit mosquito larvae by reducing mortality caused by BTI. We presumed the mechanism to be ingestion of the bacteria by the crustaceans resulting in a lower concentration available to the mosquito larvae.Mortality of Aedes aegypti larvae exposed to BTI was reduced in the presence of the fairy shrimp, Branchipus schaefferi and the ostracod, Cypridopsis vidua (only during summer trials and not autumn trials for the latter species) but was not reduced in the presence of the ostracod, Heterocypris incongruens. By contrast, H. incongruens preyed upon infected, though still-moving, larvae. Feeding on the bacteria by the crustaceans may not be an important mechanism; our data indicates that the crustaceans did not reduce BTI in the water. Moreover, B. schaefferi, introduced into water and then removed prior to the introduction of BTI and mosquitoes, also reduced mosquito mortality.The mechanism for the protective effect of B. schaefferi and C. vidua is unknown. However, these results suggest that the abundance of certain organisms co-occurring with mosquito larvae may partially explain why the effective concentration of BTI varies among habitats.     

Ovipositional and ovicidal effects of the microbial agent Bacillus thuringiensis israelensis on Culex quinquefasciatus say (Diptera: Culicidae).

The magnitude of oviposition as well as the size, shape and the number of eggs per of egg rafts egg raft were determined after gravid Culex quinquefasciatus Say oviposited on water treated with water dispersible granules (WDG) of Bacillus thuringiensis ssp. israelensis (Bti) and on untreated water. The mean number of eggs/raft was lower in the treated than in the untreated water. Bti concentrations from 0.5 to 2.0mg/L affected the shape of egg rafts and number of eggs in each raft. As the concentration of Bti increased from 0.5 to 2.0 mg/L the shape of egg rafts became more irregular with fewer eggs in each raft. Exposure to Bti at 2- and 26-h reduced the hatching rates, and fewer eggs hatched at 26-h of exposure to Bti. As the concentration of Bti WDG increased from 0.5 to 2.0 mg/L, the hatching rate decreased. Eggs exposed for 2-h to 2.0mg/ L Bti had a hatch of 30% after 24 h, the rate increasing to 57% after 72 h. In contrast, in 26-h exposed eggs to 2.0 mg/L Bti, the hatching rate after 24 h was only 12% and this rate increased to 39% after 72 h. In larvae from eggs exposed for 2 h, the survival rate was 40% at 2.0 mg/L Bti and 87% in untreated controls. In contrast, the survival rates of larvae from 26-h exposed eggs was 91% in controls while it was 30% at 2.0 mg/L Bti. As the concentration of Bti increased from 0.5 to 2.0 mg/ 1 the survival rates of larvae decreased. The combined effects of reductions of egg rafts, low number of eggs per egg raft, and reduced hatching and survival rates could have significant cumulative effects on the yield of adult mosquitoes, and this could result in a greater control potential of this microbial agent.     

Evaluation of various control agents against mosquito larvae in rice paddies in Taiwan.

A field test was conducted in rice paddies adjacent to Wufeng, Taichung County in Central Taiwan to evaluate the efficacy of control agents against mosquito larvae. The agents included Bacillus thuringienesis israelensis (Bti), two Lagenidium giganteum products (Lg product A and T), and temephos. The major mosquito species found in the rice paddies were Culex tritaeniorhynchus/vishnui and Anopheles sinensis. Compared to controls, a 7-day treatment with Bti or Lg products A and T caused overall reductions in the number of immatures (larvae and pupae) of Cx. tritaeniorhynchus/vishnui of 77.5%, 49.7%, and 21.9%, respectively, whereas temephos caused an increase of 66.9%. The overall reductions in An. sinensis were 85.4%, 8.6%, 44.6%, and 92.1%, respectively. There was no significant reduction in the number of mosquito larvae following 42 days of treatment with these agents. In summary, 1-week treatments with both biological control agents produced moderate overall reductions in mosquito larvae in rice paddies. The insecticide temephos, on the other hand, was very effective at suppressing the larvae of An. sinensis but significantly increased the number of Cx. tritaeniorhynchus/vishnui larvae in temephos-treated plots.     

Pulse-exposure effects of selected insecticides to juvenile Australian crimson-spotted rainbowfish (Melanotaenia duboulayi)

Laboratory toxicity studies were conducted in southeastern Queensland, Australia, to determine the acute lethal effects of a 1-h pulse exposure of selected insecticides to adult and juvenile (<72 h old) crimson-spotted rainbowfish, Melanotaenia duboulayi (Castlenau). In addition, to its ecological significance, this native fish is a predator of mosquitoes. Two organophosphate (OP) compounds (temephos and pirimiphos-methyl), an entomophathogenic bacterium (Bacillus thuringiensis variety israelensis [Bti]) and two insect growth regulators (IGRs) (s-methoprene and pyriproxyfen) were evaluated. Although none of the five insecticides were acutely toxic to adult M. duboulayi under the test conditions, temephos and pirimiphos-methyl were found to be toxic to juveniles, with 24 h pulse exposure LC50 values of 27 and 15 microg/liter (ppb), respectively. Of the two OPs, pirimiphos-methyl was the most toxic, with a lethal dose ratio (pulse exposure LC50 temephos/ pulse exposure LC50 pirimiphos-methyl) of 1.8 (95% CL 0.5-6.4). These pulse exposure LC50 values represented 40 and 4.5% of the estimated environmental concentrations (EEC) for a 15 cm deep water body, respectively. Bti and the two IGRs had no acute toxic effects at up to 10 and 12.5 times the EEC, respectively. Accordingly, in the interests of environmental conservation and integrated pest management (IPM), preference should be given to the latter three insecticides for control of mosquito larvae in juvenile M. duboulayi habitat.     

Effect of proteolytic and detoxification enzyme inhibitors on Bacillus thuringiensis var. israelensis tolerance in the mosquito Aedes aegypti

Bacillus thuringiensis var. israelensis (Bti) is highly pathogenic to mosquito larvae and is widely used for mosquito control. Its mosquitocidal activity however is relatively low compared to many chemical insecticides. The detoxification mechanisms in the mosquito, among other things, might neutralize the Bti activity, resulting in resistance or tolerance. We tested whether or not the detoxification mechanisms against chemical insecticides might also operate against Bti, rendering it less effective. We targeted four enzymes in Aedes aegypti larvae involved in detoxification with inhibitors that have been used in resistance studies in chemical insecticides and assayed their effects on Bti toxicity. Results revealed that phenylmethanesulphonyl fluoride (PMSF), diethyl maleate, phenobarbital (PB), and piperonyl butoxide (PBO) altered Bti toxicity to various degrees. PMSF is a serine protease inhibitor that prevents Bti digestion and improves Bti activity. PB that induces several detoxifying enzymes had two different effects depending on the method of treatment. Mortality was higher when treatment with PB was discontinuous (149%) whereas with continuous treatment it was lower (101%). PBO, a typical cytochrome P450 inhibitor, increased Bti effect (159%). The combination of discontinuous pretreatment of larvae with PB followed by PBO had a synergistic effect and showed increased activity (146%). It appears that the mechanism for Bti resistance in mosquitoes is similar to that of chemical insecticides. Our studies indicate that we may be able to increase Bti activity by inhibiting some of the detoxification systems as active as broad spectrum chemical insecticides.     

Factors influencing the toxicity of xenobiotics against larval mosquitoes

In order to examine the factors influencing xenobiotic toxicity against larval mosquitoes, the larvicidal performances of two conventional insecticides (temephos and Bacillus thuringiensis var. israelensis: Bti) and a new potential phyto-insecticide (decomposed leaf litter) were compared under different conditions against three detritivorous larval mosquito types. Bioassays performed under standard conditions indicated differential tolerance levels according to the xenobiotic and the larval type. Bioassays performed under different conditions of xenobiotic dose and geometry of the water column indicated differential effects of those parameters on mortality rates. This allowed us to distinguish the performances of temephos versus those of Bti and leaf litter. These toxicological performances were examined as indicators for analysis of xenobiotic bioavailability for mosquito larvae in environmental water, and also for their comparative interest in field mosquito control.     

Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.     

Flocculation of Bacillus thuringiensis var. israelensis suspension and its efficacy against mosquito larvae

Bacillus thuringiensis ssp. israelensis (Bti) is increasingly used as an ecologically friendly anti-mosquito agent. The bacterium cells undergo fermentation in dilute suspensions; before practical use, therefore it is necessary to concentrate the suspensions. Aggregation by polymers is a powerful tool with which to regulate the stability of suspensions. Typically, polymers at low concentrations destabilize and at high concentrations stabilize colloidal systems. Bti suspensions can be flocculated efficiently by either cationic or anionic polyelectrolytes. Cationic polyelectolytes were found to be the most efficient flocculants for bacterial suspensions. It was shown that the degree of toxicity of the flocculated Bti suspensions for biting mosquito larvae was in the same range than in non-flocculated suspension.     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Temporal and spatial habitat preferences and biotic interactions between mosquito larvae and antagonistic crustaceans in the field

Investigations on natural antagonists of mosquito larvae found that micro‐crustaceans (e.g., Cladocera) control mosquito populations under experimental conditions. However, their relevance for mosquito control under field situations remains widely unclear because important information about habitat preferences and time of occurrence of crustaceans and mosquito larvae are still missing. In order to fill this knowledge gap, a field study was undertaken in different wetland areas of Saxony, Germany, in different habitats (i.e., grassland, forest, and reed‐covered wetlands). We found negative interactions between larvae of Ae. vexans and predatory Cyclopoida (Crustacean: Copepoda), which both were dominant during the first two weeks of hydroperiod, at ponds located at grassland habitats. Larvae of Cx. pipiens were spatially associated with competing Cladocera, but they colonized ponds more rapidly. Populations of Cladocera established from the third week of hydroperiod and prevented Cx. pipiens colonization thereafter. Ostracoda were highly abundant during the whole hydroperiod, but their presence was restricted to habitats of reed‐covered wetland at one geographical area. Mosquito larvae hardly occurred at those ponds. In general, we found that ponds at the reed‐covered wetlands provided better conditions for the initial development of crustaceans and hence, mosquito larval colonization was strongly inhibited. Grassland habitat, in contrast, favored early development of mosquito larvae. This study showed that micro‐crustaceans are relevant for mosquito management but their impact on mosquito larvae varies between species and depends on environmental conditions.     

Effects of mosquito larvae removal with Bacillus thuringiensis israelensis (Bti) on natural protozoan communities

The protein crystals produced by Bacillus thuringiensis israelensis (Bti) are used against the larvae of pestiferous flood-water mosquitoes in ephemeral wetlands. Although mosquito larvae are considered important predators on protozoans and bacteria, it is not known how a distinct reduction of mosquito larvae density in natural wetlands caused by application of Bti may indirectly affect these microbial communities. Here we show, in a large scale experiment in six natural wetlands, that the densities of heterotrophic protozoans was on an average 4.5 times higher in wetland areas treated with Bti than in control areas. In addition, the taxonomic richness of heterotrophic protozoans increased on an average of 60% in areas with Bti application compared to control areas. The increase in protozoan density and richness was fairly consistent among sites of different wetland habitats. We discuss the potential implications of our results for other parts of the ecosystem. Handling editor: K. Martens     

Comparative histopathology of some Diptera and Crustacea of aquatic alpine ecosystems, after treatment with Bacillus thuringiensis var. israelensis

Species of Diptera (larval Culicidae, Chironomidae and Simuliidae) and Crustacea (cladoceran adults) that are representative of the fauna of aquatic alpine ecosystems are routinely treated with Bacillus thuringiensis var. israelensis (Bti) for mosquito control. The toxicity of Bti on these organisms was investigated. Bioassays indicated that Bti used at the concentration for operational field application is deleterious to all dipteran species, but not to Cladocera. Histopathological observations reveal that, in all cases, the midgut epithelium is affected by Bti treatment. However, the vulnerability of epithelial cells to the microbial insecticide is different from one cell-type to another, clear cells being more vulnerable than dark cells. Because of the concentration of clear cells within the anterior midgut of dipteran larvae, this region appears to be the first to show symptoms of intoxication through enhancing a natural process of cell turnover. The relative importance of such a vulnerable region in the midgut of dipteran larvae may account for the general efficacy of Bti to these species. Its harmlessness to Cladocera may be correlated to the relative scarcity of clear cells and their patchy distribution along the whole midgut together with important cellular renewal capacity of the epithelium.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.