Bacteria

C H A P T E R III-2

Bacteria: Laboratory bioassay of bacteria against aquatic insects with emphasis on larvae of mosquitoes and black flies LAWRENCE A. LACEY Yakima Agricultural Research Laboratory, USDA-ARS, 5230 Konnowac Pass Road, Wapato, WA 98951, USA.

1 INTRODUCTION The insect families Culicidae (mosquitoes) and Simuliidae (black flies) contain some of the most medically important insect species. Interest in the development and use of Bacillus entomopathogens as microbial control agents of black fly and mosquito larvae has been greatly increased with the discovery of varieties of Bacillus thuringiensis with elevated larvicidal activity against these insects (de Barjac, 1978; Lacey & Undeen 1986). Their activity against certain families in the suborder Nematocera is principally due to the production of Cry IVA-D and Cry IIA toxins. These are predominantly found in B. thuringiensis var. israelensis and the PG-14 isolate of B. thuringiensis var. morrisoni. Additionally, several isolates of certain varieties of Bacillus sphaericus and isolates of Clostridium bifermentans also have good potential for the microbial control of pest and vector mosquitoes (Lacey & Undeen 1986; Thiery et al., 1992). In order to assess and compare the efficacy of any MANUALOF TECHNIQUESIN INSECTPATHOLOGY ISBN 0-12-432555-6

bacterium against both mosquito and black fly larvae, the bacterial toxins responsible for larvicidal activity must be ingested by the larvae. Comparative efficacy testing must be conducted under repeatable conditions which permit normal or close to normal feeding rates and do not produce abnormally high mortality (above 10%) in control larvae. Although there are other aquatic Nematocera that are susceptible to bacteria and/or their toxins, I will emphasize methods for the testing of bacteria against larvae of mosquitoes and black flies. Readers interested in the bioassay of Bacillus thuringiensis var. israelensis against chironomid larvae can refer to procedures used by Ali et al. (1981) and other authors cited by Dejoux & Elouard (1990) and Lacey & Mulla (1990). A multitude of aquatic non-target organisms are not directly affected by ingesting bacteria or bacterial toxins active against mosquitoes and/or black flies, but they may be affected by formulation components or the consequences of removing target species from the ecosystem. Results of testing and a review of the literature on the effects of entomopathogenic bacteria on non-target aquatic insects are Copyright9 1997AcademicPressLimited All rights of reproductionin any formreserved

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presented by Dejoux Lacey & Mulla (1990).

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2 BIOASSAY OF BACILLUS PATHOGENS AGAINST MOSQUITO LARVAE The bioassay of bacteria against larvae of most mosquito species is a relatively straightforward procedure. Larvae of most pest and vector mosquitoes are filter feeders and readily ingest particulates in the size range of bacteria and bacterial inclusions (Pucat, 1965; Merritt & Craig, 1987). Some notable exceptions are predatory mosquitoes that eat aquatic insects including the larvae of other mosquito species.

A Factors affecting activity A number of factors, such as mosquito species and age, number of mosquito larvae per bioassay container, water quality and temperature, volume and depth of water, presence or absence of food and other particulates, shape of the bioassay container and factors related to the bacterial preparation (size of particles, affect of adjuvants) can significantly influence toxin activity and the results of bioassays. Many of these factors also influence feeding rate and hence the amount of toxin ingested. The following sections present information on various bioassay parameters and how they might influence bioassay results.

1. Effect of species All filter feeding mosquito species thus far tested are susceptible to B. thuringiensis var. israelensis. There is considerably more variability among mosquito species in susceptibility to B. sphaericus. Culex, Psorophora, Anopheles and Mansonia species are highly susceptible to B. sphaericus, but many Aedes species, especially Aedes aegypti are not susceptible or considerably less susceptible. Recently, high levels of resistance to this bacterium have been reported in certain populations of Culex quinquefasciatus, a species that is normally very susceptible.

2. Feeding behaviour In addition to other susceptibility factors related to species, the feeding behaviour of a particular species will influence the amount of inoculum with which the larvae will come into contact. Some species may feed predominantly at the surface of the water (Anopheles species), within the water column (some Culex spp.) or predominantly off the substratum (some Aedes spp.) or some species may combine column and substrate feeding. Species that feed mostly at the surface usually consume less toxin due to settling of toxic moieties from their feeding zone and thus appear to be less susceptible to entomopathogenic bacteria. Thiery (personal communication) has observed that Anopheles larvae exposed to B. thuringiensis var. israelensis or B. sphaericus in very shallow water (i.e. where settling of toxin has less influence) are still less susceptible to the toxin than are Culex or Aedes species. The major difference in standardized bioassays for isolates of B. thuringiensis and B. sphaericus wiU be the target species. Because Ae. aegypti is relatively easy to rear and its eggs can be stored dry between rearings, it is an ideal test animal for bioassays of B. thuringiensis varieties. Due to the lack of susceptibility in Ae. aegypti to B. sphaericus, Cx. quinquefasciatus is the preferred test animal for this bacterium.

3. Number of larvae~container, volume and quality of water, and features of the bioassay container Because mosquito larvae, especially those feeding within the water column and off the substratum can be very efficient in removing particulates, the number of larvae per container will influence the amount of toxic moieties that will be available to each individual (Sin~gre et al., 198 la). Crowding may also negatively influence normal feeding rates. The amount of toxin per larva will also be a function of the volume of water used for the bioassay. Five ml of water per larva has enabled consistent and repeatable bioassays in studies conducted at the USDA-ARS Medical and Veterinary Entomology Research Laboratory (MAVERL; Gainesville, FL, USA) on both B. sphaericus and B. thuringiensis against several mosquito species (Lacey & Singer, 1982; Lacey et al., 1988). The shape of the container and water depth may also affect the availability of bacterial

B a c t e r i a : L a b o r a t o r y b i o a s s a y of b a c t e r i a

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quito larvae is, for the most part, positively correlated with temperature (Sin~gre et al., 1981b; Wraight et al., 1987; Lacey et al., 1988). Many of the species most utilized for laboratory bioassay (e.g. Ae. aegypti, Cx. quinquefasciatus, An. albimanus and An. stephensi) are tropical in origin and thrive at 27 ~C. Lower temperatures may be required for other species from more temperate climes.

6. Effect of food and particulates on activity of bacterial toxins

Figure 1 Bioassay of bacteria against mosquito larvae at the USDA-ARS Medical and Veterinary Entomology Laboratory, Gainesville, FL. (Courtesy of A1Undeen).

toxin. Small disposable cups (depth ca. 4 cm) filled with 100 ml of chlorine-free water (well water or aged tap water), are used for bioassays at MAVERL (Figure 1). The quantity of water used for bioassay at the Pasteur Institute (Pads, France) is 150 ml of water. The presence of chlorine can significantly reduce the larvicidal activity of B. thuringiensis varieties (Sin~gre et al., 198 lb) and be detrimental to larvae. Twenty larvae per container are used at MAVERL and 25 larvae per container are used at Pasteur Institute. Q

Depending on its nature and quantity, particulate matter can reduce or enhance the feeding rate of mosquito larvae (Dadd, 1970) and hence the amount of toxic inclusions that are ingested by larvae (Ramoska & Pacey, 1979; Sin6gre et al., 1981a; Aly, 1988). Even the addition of excess food that is normally desirable could inhibit feeding, dilute the toxins in the gut or interfere with the activity of toxins. During the exposure/incubation period, the absence of food will enable an accurate and repeatable bioassay of bacterial pathogens. Late third and early fourth instars can withstand starvation with little or no effect on control mortality. In assays involving younger larvae that require more than 48 h exposure and incubation, a small amount of food (5 mg-'), such as finely ground lab chow, could be added to the assay cups in the second 24 h of exposure. In assays at Pasteur Institute, food is provided to older instars of Anopheles and Culex species during bioassays (Thiery, personal communication).

4. Larval age The larval age group that enables good survival in controls and consistent results are late third and early fourth instars. Younger larvae (first and second instars) do provide more sensitive targets, but tend to be less hardy and unable to survive much more than 24 h without food (Lacey & Singer, 1982). Older fourth instars may pupate before the end of the exposure period. With the exception of first instars, mosquito larvae imbibe very little water. If solubilized toxins are to be assayed, it will be necessary to use first instars or to encapsulate the toxin(s).

5. Temperature Within the range of temperatures tolerated for a given species, the activity of bacterial toxins in mos-

B Selection of dosage Almost invariably one observes a dose-dependent mortality response in mosquito larvae to B. thuringiensis and B. sphaericus varieties. The exception being when bacterial preparations contain such low amounts of Nematocera-active toxin that the particulate load required to induce mortality also inhibits feeding. Skovmand (personal communication) suggests that hypersensitivity to toxin might also inhibit feeding. A range of 5-7 concentrations of promising candidate bacteria should be selected, which produce mortality of 10-90% with two above and two below the 50% mortality level. Dosage is usually expressed in milligrams of

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spore powder or formulation per litre of water or in parts per million (ppm). The use of international toxicity units (ITU) to compensate for the day to day fluctuations in larval response to B. thuringiensis var. israelensis has been proposed (de Barjac & Larget, 1979, 1984). ITUs are calculated by comparing bacterial preparations to a standard and using the ratio of the LCs0 values of the standard and test sample times the assigned potency (in ITUs) of the standard. For example: Potency (ITU) of sample =

LCs0 for standard x potency (ITU) of standard LCs0 for sample

The potency of IPS-78, the first standard prepared by de Barjac, for example, was arbitrarily assigned a potency value of 1000 ITU mg -t (de Barjac & Larget, 1979). It has been replaced by IPS-82 since 1982 (de Barjac & Larget, 1984). Primary powders of B. thuringiensis var. israelensis were also proposed by Dulmage et al. (1985) as US standards. Various parameters that can influence potency of products based on B. thuringiensis var. israelensis are presented by Skovmand (1996). Several reasons for avoiding the use of spore count as a measure of dosage are presented by Dulmage et al. (1990). Nevertheless, if suspensions of fresh cultures (48-72 h growth on agar) are used in assays, such as in preliminary screening of recently isolated strains, spore count (after heat shock at 80~ for 12 min) may be the only indication of dosage available. Cultures should also be checked for the presence of parasporal inclusion bodies (where endotoxins of B. thuringiensis and some other Bacillus species are found). Autoclaved suspensions that produce mortality in larvae indicate the production of exotoxins, some of which may be harmful to vertebrates (Melin & Cozzi, 1990). As in the other assays, tests with whole cultures of cells should include a range of dilutions. Ultimately, preparation of primary powders would enable comparative bioassays against a standard.

C Number of replicates and tests For each concentration and control, there should be a minimum of four sets (i.e separate containers) per test. Although a test conducted on a single date with several sets of each dosage will usually provide a homogeneous set of data, it is advantageous to run

replicate tests over time to account for the variation in susceptibility that is observed in different cohorts of mosquito larvae obtained from the same colony. Three replicate tests on separate test dates are the usual minimum. In general, the higher the coefficient of variation (standard deviation divided by the mean) for mortality data, the greater the need to do more replicate tests.

D Bioassay protocol In order to have repeatable results the various bioassay parameters, as well as rearing conditions, should be standardized. Suggested parameters for standardized bioassay of varieties of B. thuringiensis and B. sphaericus against mosquito larvae are presented in Table 1. Several similar protocols have been recommended for the repeatable bioassay of B. thuringiensis varieties (de Barjac & Larget, 1979, 1984, McLaughlin et al., 1984; Dulmage et al., 1990) against mosquito larvae. The following is an eclectic combination of procedures for the preparation of inoculum and bioassay against mosquito larvae.

Table I Suggested parameters for bioassay of varieties of Bacillus thuringiensis and Bacillus sphaericus against mosquito larvae. Dosage of bacteria No. concentrations No. of cups/control & concentration/test Replicate tests (separate dates) Age of larvae Duration of test Volume of water Source of water Food

mg/1 (ppm)~ 5-7 ~ 4 3 late 3rd early 4th 24-48 hc 100 ml non-chlorinated d none'

a Weightof primarypowderor formulation; with fresh cultures of spores, spore count (after heat shock; 80~ for 12 min) may be the only indication of dosage available; several reasons for avoiding the use of spore count as a measure of dosage are presented by Dulmage et al. (1990). b Concentration should be chosen such that at least two produce mortality between 10-50% and at least two produce mortality between 50-90%. c 24 h for B. thuringiensis var. israelensis; 48 h for B. sphaericus. Well wateror dechlorinated (aged and/or aerated) tap water. 9 If very young larvae are used, addition of food may be necessary. Finely ground and suspended food (e.g. Purina Lab Chow, Tetramin) will be required for larval diet.

Bacteria: L a b o r a t o r y b i o a s s a y of bacteria

1. Preparation of inoculum Suspensions of inoculum for bioassay should be freshly prepared from primary powders, formulated product or fresh cultures just prior to each bioassay. Initial homogenates are prepared with standards and primary powders by suspending 50 mg of powder in 10 ml of deionized or distilled water and agitating for 10 min using a bead mill (20 ml penicillin flask with several 6 mm glass beads) or similar method. Addition of a wetting agent such as (0.1%) Tween 80 will improve wetting of primary powders. Subsequent dilutions can then be made from the homogenate using routine dilution procedures (see Chapter III-1). Dulmage et al. (1990) describe making a 'stock' suspension from the above homogenate by adding 0.1 ml of the homogenate to 9.9 ml of water. The stock is then resuspended using a Vortex agitator. Following the procedures of de Barjac & Thiery (1984) cited by Dulmage et al. (1990), subsequent dilutions are made in the container in which the assay will take place by adding 15-120 pl of stock suspension directly into assay cups containing 150 ml of water. The use of more dilute suspensions is advantageous because accuracy is increased when larger volumes are measured. McLaughlin et al. (1984) recommended making a series of dilutions to be used for treatments using a minimum of 10 ml of bacterial suspensions for making subsequent dilutions and for applying the diluted suspensions to the bioassay cups. Skovmand (personal communication) points out that commercial products are not highly homogeneous and suggests larger quantities (1 g) be used for preparing initial suspensions.

2. Handling of insects and application of inoculum Materials and methods for rearing various species of mosquitoes used for bioassays are presented by Gerberg et al. (1994) and are not covered here. When larvae are in the late third to early fourth instar they are removed from rearing trays by sieving and placed in chlorine-free water without food. If a chlorine-free source of water is not available, tap water that has been aerated for 48 h will suffice. Either 20 or 25 late third or early fourth instars are transferred to each bioassay container using a Pasteur pipette with the narrow tip removed or an eye dropper with a wide opening. Filing or fire polishing the tip may be required to avoid larval injury.

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Small (ca. 4 - 5 c m deep) containers holding 100 ml of water provide an adequate arena in which to bioassay bacteria against most species of mosquitoes. Four cups per concentration for each isolate and control per test date are recommended. It is easier to first add larvae and then fill with the balance of the 100 ml of water, rather than adding the water first and then subtracting the amount that was added along with the larvae. If 10 ml of bacterial suspension will be added to each container rather than p 1 amounts, the containers should only be filled to 90 ml prior to adding treatments. Total amount of liquid based on weight is another method of determining when the appropriate amount of water has been added to assay containers. One method of adding larvae without the addition of more water, is to use small sieves or nets for their transfer.

3. Incubation and assessment of mortality After the appropriate treatments are added to each container, the larvae are then incubated for 24 h at 25-27 ~C in bioassays of B. thuringiensis var. israelensis and 48 h with B. sphaericus. Temperature is evenly maintained at MAVERL by placing the cups with larvae in large trays containing 2-3 cm of water. The trays are then set on thermostatically controlled heat tapes within a closed cabinet. The sensor for the thermostat is placed in one of the trays under water. Incubators or climate cabinets can also be used to maintain constant temperature. After incubation, the larvae are assessed for mortality. If larvae fail to respond to tapping the side of the assay cup, they are probed with a needle. Lack of response or only very weak response (i.e. the larva is moribund) is scored as dead. It is best to base calculations of percentage mortality on the number of larvae originally placed in the containers and number of living larvae after the exposure/incubation period due to the fact that dead larvae are often consumed by the survivors.

4. Data processing If control mortality does not exceed 10% the data are kept and analysed by probit analysis after adjusting for control mortality using Abbott's (1925) formula: adjusted % mortality=

observed % mort. - ave. control % mort. 100%- ave. controlmort.

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A bioassay is the use of a living organism to assay, or measure the amount of a substance, such as a toxin, in an unknown sample. A single discriminating concentration of bacterial suspensions may also be used to study the effect of abiotic and/or biotic factors on larvicidal activity of candidate isolates or formulations. In such cases the amount of toxin is already known and some other biological effect, not the amount of toxin is being tested. Strictly speaking, these tests are not bioassays. Various statistical tests for analysis of variance and mean separation and greater detail on probit analysis are presented in Chapters II and IV-3.

E Considerations for predatory larvae The testing of efficacy of bacteria against predatory species of mosquitoes, notably Toxorhynchites species and certain species of Psorophora as well as others, will necessitate first feeding the bacteria to a prey species, such as Aedes aegypti, and then adding the predator. Procedures used for testing of B. sphaericus and B. thuringiensis var. israelensis against Toxorhynchites spp. are presented by Larget & Charles (1982) and Lacey (1983).

A Factors affecting activity Factors that affect the susceptibility of black fly larvae to B. thuringiensis var. israelensis are, for the most part, the same as those that affect susceptibility of mosquito larvae (species, larval age, temperature, presence of food and other particulates, etc.; Lacey et al., 1978; Molloy et al., 1981; Coupland, 1993). The major difference between bioassays of bacteria against black flies and mosquitoes is the requirement for running water for black fly larvae. A number of bioassay systems have been proposed for the laboratory evaluation of bacteria against larval black flies (Lacey et al., 1982). The current required for bioassay can be artificially created by using a magnetic stirrer and stir bar in a beaker of water (Undeen & Nagel, 1978), with air bubbles (Lacey & Mulla, 1977) (Figure 2), using orbital shakers or other artificial methods (Lacey et al., 1982) or more naturally in

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3 BIOASSAY OF BACILLUS THURINGIENSIS ISOLATES AGAINST BLACK FLY LARVAE Like mosquitoes, the larvae of black flies are aquatic. Their habitats include running water ranging from small creeks to huge rivers. Although they are normally thought of as filter feeders, a range of feeding strategies is observed for the family that also include grazing (scrapers), deposit feeders and predators (Currie & Craig, 1987). The larvae of most vector or pestiferous species (i.e. those that warrant control) are filter feeders for a significant amount of the time. Using their labral fans and mucous secretions, black fly larvae are capable of filtering particles ranging from 0.09 to 350 ktm from the water column (Curfie & Craig, 1987). The combination of their filtering efficiency and susceptibility make black fly larvae ideal targets for B. thuringiensis var. israelensis, even though the period of exposure is relatively brief.

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Figure 2 Schematic drawing of a flushing bioas~ay, system. (From Lacey & Mulla, 1977, J. Econ. Entomol. 70, 453-456, with permission.)

Bacteria: Laboratory bioassay of bacteria systems which utilize flowing water (Gaugler et al., 1980; Guillet et al., 1985; Coupland, 1993) (Figures 3 and 4). As with mosquito larvae, the most important factor is to enable normal larval feeding rates

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and avoid excess control mortality. Systems that create a current using magnetic stirrers, aquarium air pumps and the like will be the least expensive, but the current is not as laminar as that obtained with

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lOom Figure 3 Schematic drawing of flow-through bioassay system. A, reservoir tub; B, recirculation pump; C, recirculation valve; D, water supply pipe; E, supply valve; F, organdie cloth filter; G, delivery funnel; H, funnel support; I, tray reservoir section; J, tray attachment section; K, standing waste pipe; L, waste trough; M, cap. (From Gaugler et al., 1980, Can. Entomol. 112, 1271-1276, with permission.)

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Figure 4 Flow-through system utilized by GuiUet et al. for stream side bioassay of candidate B. thuringiensis var. israelensis formulations. (Courtesy of Pierre Guillet.)

flowing water. The systems used by Gaugler et al. (1980) and Coupland (1993) can be operated in both recirculation and flow-through modes. In addition to current, other environmental conditions may also have to be met. Species that are normally found in pristine, cold, fast flowing streams may be difficult to use for laboratory bioassays unless their environmental conditions are adequately simulated. Larval feeding rates can be highly influenced by particle concentration in the water. Feeding efficiency increases at lower particulate concentrations (Kurtak, 1978) and increasing particle concentrations up to an optimum will increase the quantity of food consumed, but an excessive amount of particulates will actually inhibit feeding (Gaugler & Molloy, 1980). If feeding is inhibited before or during exposure to B. thuringiensis, fewer toxic inclusions will be consumed. If inhibition occurs following ingestion of toxin, mortality can be increased due to the fact that ingested particulates remain stationary in the midgut and hence prolong contact time with receptors on the target epithelium (Gaugler & Molloy, 1980).

B Exposure period Length of exposure is another factor that will differ from mosquito bioassays. Under most operational conditions, the time interval during which B. thuringiensis var. israelensis formulations are in contact with black fly larvae is fairly brief because stream flow carries the material downstream quickly.

Initial application time can be short as in the case of aerial application, or extended to 10-30 min when applied at ground level. To approximate natural conditions more realistically, exposure of the larvae to the bacterium in the laboratory should not be too protracted. For a standard bioassay in closed systems, I suggest 30 min of exposure. For screening purposes only, varieties of B. thuringiensis that produce low amounts of toxins active against black flies will require a higher dosage and longer exposure time to produce significant mortality (Lacey et al., 1978).

C Bioassay protocol The preparation of bacterial suspensions and number of replicates is as described above for mosquito bioassays. Table 2 presents some suggested standardized parameters for bioassay of B. thuringiensis varieties against black fly larvae. 1. Handling of insects

Except in the few laboratories with a black fly colony, larvae have to be collected from a breeding site. Getting from the field to the lab with larvae of some species may require transport of the larvae in aerated containers of water under cool or cold conditions. Several species, including Simulium damnosum and S. vittatum, can be transported for short periods of time on the damp vegetation on which they were collected. After removal of excess water, the plants are placed loosely in plastic bags in a cool

Bacteria: L a b o r a t o r y b i o a s s a y of b a c t e r i a Table 2 Parameters for bioassay of Bacillus thuringiensis varieties against black fly larvae. Concentration of bacteria Duration of exposure (min) No. concentrations No. containers/control and concentration/test Replicate tests (separate dates) Age of larvae Duration of test Volume of water Source of water Food

mg/1 (ppm)" 30 5_7 ~ 4 3 penultimate and early last instar 24 h 1000 ml non-chlorinated c 5 mg/1 after exposure~

Weightof primarypowder or formulation; with fresh cultures of spores, spore count (after heat shock; 80~ for 12 min) may be the only indication of dosage available; several reasons for avoiding the use of spore count as a measure of dosage are presented by Dulmageet al. (1990). b Concentration should be chosen such that at least two produce mortality between 10-50% and at least two produce mortality between 50-90%. c Well water or dechlorinated (aged and/or aerated) tap water. d Following the 30 min exposure period and finely ground and suspended (5 mg-~)Purina Lab Chow, Hog Chow, Tetramin, and the like have been used for larval diet. a

place for transport to the lab. An insulated box, such as an ice chest, works well. A layer of ice separated from the bags of larvae by a sheet of styrofoam is ideal for ensuring cool, but not excessively cold temperatures. In the lab, the larvae should be removed from the bags and placed in trays in chlorine-free water. Penultimate instar larvae or young last instars (those which do not have melanized 'gill spots' (organs that will become the pupal respiratory filaments)) can be gently removed using a soft latex eye dropper by nudging the larvae with the dropper near the anal gills at the same time as drawing water into the dropper. A camel's hair brush can also be used. Immediately transfer the larva to the container or system in which bioassay will be conducted. Twenty larvae per replicate container provides sufficient test animals and will avoid the entanglement of larvae in their own silk that occurs when too large a number of larvae are in a container. The larvae should be allowed to acclimate for approximately 3 h before exposure to candidate bacteria. Before adding bacterial suspensions, dead, detached and pupating larvae should be removed.

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Usually if healthy penultimate instars are used, the number of larvae that are not attached and actively feeding will be low or zero. If larvae are removed, additional larvae should be added to bring the count to the desired number for each bioassay container. After acclimation, and when larvae are feeding (as evidenced by the opening of cephalic fans), add bacterial suspensions. In closed bioassay systems, including recirculating systems, the desired concentrations can be added all at once. If a flow-through system is used the suspension will have to be dripped continuously in the flowing water for the desired exposure period. Figure 4 shows the plastic bottles used by Guillet et al. (1985) to meter in bacterial suspensions into small gutters through which stream water flows. With isolates containing high amounts of Nematocera-active toxin(s) an exposure period of 30 min is recommended.

2. Termination o f exposure

After exposure in closed non flow-through systems, it is necessary to change the water in which the larvae are exposed. This usually causes some of the larvae to detach and care must be taken not to lose them when the water is decanted. Detachment will also result in temporary cessation of feeding, which can result in increased mortality. In the system used by Lacey & Mulla (1977) (Figure 2), the inoculum is flushed from the bioassay container by adding water to each container from individual taps while it drains from the bottom and overflows. Larvae are not exposed to air or cessation of current and hence, do not detach. Flushing of inoculum without larval detachment is ideal when using flow-through systems such as that of Gaugler et al. (1980). 3. Incubation and post-exposure care o f insects

After changing the water, it will be important to add food to enable as close to normal feeding rates as possible. Five ppm of finely ground lab chow or similar food added as a suspension should enable normal feeding rates. If feeding inhibition is noted (labral fans are held closed for longer intervals than normal) a lower concentration of food should be used. Larvae are then incubated for at least 24 h before assessment of mortality. A lower temperature (20~ than that used for mosquitoes is recommended to approximate stream conditions more

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realistically. After incubation, larvae are observed for signs of life. When in doubt regarding the condition of the larvae, use a probe. Data are treated as described previously for mosquito larvae. One exception may be necessary. Because black fly larvae do not always respond favourably to collection, handling and the laboratory environment, it may be necessary to accept a higher level of control mortality; 20% control mortality may have to be the cut off point for acceptable test results. Because of lower temperatures and shorter exposure times, it may be necessary to take a second reading 24 h after the first. Readers interested in field assessment of bacterial pathogens against simuliid larvae are referred to Undeen & Lacey (1982). A combination of field exposure and laboratory assessment of B. thuringiensis var. israelensis formulations against black fly larvae is presented by Undeen & Colbo (1980) and Lacey & Undeen (1984). The assay of bacteria against other aquatic organisms, especially those found in running water can be quite complex depending on their requirements. The bioassay system and methods that are chosen should simulate the aquatic system in which the organism is normally found as closely as possible.

4 CONCLUSIONS Data from laboratory bioassays can provide useful information on species susceptibility, including development of resistance, and for comparing isolates of bacteria, particularly when primary powders of bacterial strains are compared to a standard. However, care must be exercised when using laboratory-derived data to make predictions of relative activity under field conditions. For example, ITU ratings based on bioassays against Ae. aegypti do not always correspond to relative efficacy of B. thuringiensis var. israelensis formulations against field populations of mosquito larvae (Dame et al., 1981). Similarly, the ITU ratings for B. thuringiensis var. israelensis formulations as determined with mosquito larvae are often not correlated with activity against black fly larvae in laboratory bioassays (Molloy et al., 1984) nor under field conditions (Lacey & Undeen, 1984). Even laboratory bioassays of formulated B. thuringiensis var. israelensis conducted with black fly larvae could provide mislead-

ing information regarding relative efficacy under field conditions. Molloy et al. (1984) observed a positive correlation between particle size and efficacy in lab bioassays, yet under field conditions formulations with smaller particle sizes provide greater effective carry (i.e. kill black fly larvae for a greater distance downstream), probably due to more rapid settling of the larger particles. Once effective isolates are identified in laboratory bioassays, the most realistic evaluation of formulated bacterial microbial control agents will be under field conditions.

ACKNOWLEDGEMENTS I thank Ole Skovmand, Isabelle Thiery, A1 Undeen and James Coupland for their review of the manuscript. Photographs supplied by Randy Gaugler, A1 Undeen, Pierre Guillet and Mir Mulla are gratefully appreciated.

REFERENCES Abbott, W. S. (1925) A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265-267. Ali, A., Baggs, R. D. & Stewart, J. P. (1981) Susceptibility of some Florida chironomids and mosquitoes to various formulations of Bacillus thuringiensis serovar. israelensis. J. Econ. Entomol. 74, 672-677. Aly, C. (1988) Filter feeding of mosquito larvae (Diptera: Culicidae) in the presence of the bacterial pathogen Bacillus thuringiensis vat. israelensis. J. Appl. Entomol. 105, 160-166.

Coupland, J. B. (1993) Factors affecting the efficacy of three commercial formulations of Bacillus thuringiensis vat. israelensis against species of European black flies. Biocontrol Sci. Tech. 3, 199-210. Currie, D. C. & Craig, D. A. (1987) Feeding strategies of larval black flies. In: International symposium on ecology and population management of black flies

(eds R. Merritt & K. C. Kim), Pennsylvania State University Press, pp. 155-170. Dadd, R. H. (1970) Comparison of rates of ingestion of particulate solids by Culex pipiens larvae: phagostimulant effect of water-soluble yeast extract. Entomol. Exp. Appl. 13, 407-419. Dame, D., Savage, K., Meisch, M. & Oldacre, S. (1981) Assessment of industrial formulations of Bacillus thuringiensis var. israelensis. Mosq. News 41, 540-546.

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Skovmand, O. (1996) Parameters influencing the potency of products based on Bacillus thuringiensis var israelensis. J. Econ. Entomol. in press. Thiery, I, Hamon, S., Gaven, B. & de Barjac, H. (1992) Host range of Clostridium bifermentans serovar. malaysia, a mosquitocidal anaerobic bacterium. J. Am. Mosq. Control. Assoc. 8, 272-277. Undeen, A. H. & Colbo, M. H. (1980) The efficacy of Bacillus thuringinesis var. israelensis against blackfly larvae (Diptera: Simuliidae) in their natural habitat. Mosq. News 40, 181-184. Undeen, A. H. & Lacey, L. A. (1982) Field procedures for the evaluation of Bacillus thuringiensis vat. israelensis (serotype 14) against black flies (Simuliidae) and nontarget organisms in streams. Misc. Pub. Entomol. Soc. Am. 12, 25-30. Undeen, A. H . & Nagel, W. L. (1978) The effect of Bacillus thuringiensis ONR-60A strain (Goldberg) on Simulium larvae in the laboratory. Mosq. News 38, 524-527. Wraight, S. P., Molloy, D. & Singer, S. (1987) Studies on the Culicine mosquito host range of Bacillus sphaericus and Bacillus thuringiensis var. israelensis with notes on the effects of temperature and instar on bacterial efficacy. J. lnvertebr. Pathol. 49, 291-302.