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A novel bioassay system for evaluating the toxicity of Bacillus thuringiensis israelensis against mosquito larvae
JOURNAL
OF INVERTEBRATE
PATHOLOGY
59,
286-289
(19%)
A Novel Bioassay System for Evaluating the Toxicity of Bacillus thuringiensis israelensis against Mosquito Larvae’ DONALD W. MUCH Department
of
Biology, Coker Hall, CB 3280, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 275993280
DALE F. BURNSIDE Department of Biology, Lenoir-Rhyne
College, Hickory, North Carolina 28601
TAMERA L. CECIL U.S. Army Biomedical
Research & Development Laboratory,
Fort Detrick, Frederick, Maryland
21701
Received March 22, 1991; accepted August 15, 1991
A bioassay system employing acutely toxic concentrations of a spore-crystal mixture of Bacillus thuringieusis subspecies israelensis against fourth instar larvae of Aedes aeggpti (L.) is described. Individual larvae are separately exposedto toxin in glass-lined miniature wells or scintillation vials. This method is free from the deaths due to predation among larvae. Such larval deaths are commonly encountered in bioassay groups of 25 larvae as currently specified in the World Health Organization guidelines. Our method offers shortened testing time, increased accuracy, and improved statistical precision. 0 1992 Academic KEY WORDS: bioassay; mosquito larvae; Bacillus thuringiensis israelensis (L.); acute toxicity. ~SS,
hf.
INTRODUCTION
The ultimate test of any insecticidal material is its effectiveness in controlling natural populations. Because of the high cost and long time required for field testing, laboratory bioassays of toxicity are routinely employed for preliminary screening of candidate pesticides and are also useful for quality control. However, bioassays are notorious for their variability when used by different laboratories and may also show inconsistencies within the same laboratory. Standard bioassay procedures for mosquito larvae have been published by de Barjac and Larget-Thiery (1979) for the World Health Organization (WHO) and more recently by McLaughlin et al. (1984). Vorgetts et al. (1988) note the lack of uniformity in testing larvi1 The statements and opinions contained in this article are those of the authors and do not reflect official views of the Department of the Army. 286 oozz-2011/92 Copyright All rights
$4.00 0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
tides against filter-feeding invertebrates, including mosquito larvae, and have stressed the need for a more universally adopted bioassay protocol. In this paper we describe a modified bioassay system and the results of using it to test the toxicity of a standard spore-crystal suspension of Bacillus thuringiensis subspecies isruelensis against the fourth instar larvae of Aedes aegypti CL.). We compare our system with the currently recommended standard bioassay procedure and review the findings, which we believe make our system more precise and more reliable. MATERIALSANDMETHODS 1. Culture of A. aegypti. Eggs stored at 60-70% relative humidity were hatched in dechlorinated tap water (DTW) conditioned by allowing it to stand overnight or longer and to which a small amount of liver powder (ICN-Nutritional Biochemicals 900396) was added. Egg papers were removed after 4-5 hr. Newly hatched larvae were harvested the following day, distributed at approximately 200 per liter of DTW in 23cm-diameter enamel pans, and then grown under 14 hr of daily illumination at 27°C. They were fed a suspension of liver powder ad lib. Early fourth instar larvae were selected for use after 80 hr in culture. 2. Bioassay procedure. Individual larvae were tested in 24-well polystyrene tissue culture cluster dishes (Costar 3524). Preliminary testing revealed that B. thuringiensis israelensis material bound strongly to the plastic surface (D. W. Misch, unpublished observations). For that reason glass liners made by cutting off the bottom 33 mm of 16 x 125-mm test tubes were used to hold the larvae within the plastic wells. These glass liners could be removed for washing
B. thuringiensis
israelensis:
NOVEL
BIOASSAY
and reuse without detectable carryover of toxicity.2 Early fourth instar larvae were individually exposed to various concentrations IPS-82, an international standard spor+crystal mixture of B. thuringienesis israeZensis(courtesy of I. Larget-Thiery, Institute Pasteur, Paris). Group bioassays of 25 larvae together were performed in 150 ml of DTW in 11.5-cm glass finger bowls. As a convenient alternative to 4-hr bioassays of single larvae in glass liners, we placed individual larvae in 6 ml of DTW in standard scintillation vials (e.g., Fisher 03-337-4, 20-ml vials, 61 x 28 mm). The vials were arranged in white Styrofoam racks for easy viewing of the larvae. This proved to be the method of choice and was used in most of our experiments. When all the larvae needed for a test run had been set up in DTW, 2 l.~l of B. thuringiensis israelensis suspension of the appropriate concentration was added in sequence to all test larvae. Four different concentrations of B. thuringiensis israelensis were tested against 10 larvae each; 10 control larvae were used for each test. For acute toxicity tests, deaths were recorded at 15, 30, and 60 min and at 2 and 4 hr. For subacute tests, deaths were recorded at 24 and 48 hr. Each test was replicated three times. Deaths of unmoving larvae were verified by administering an 18-V battery shock with a device of our own design (Fig. 1). Larvae that gave no visible response were judged to be dead. In trial tests using electrical stimulation to judge the time of death, larvae that reacted to the brief shock were also able to be stimulated by mechanical means. By this test we believe we have established a more objective and accurate test of death. RESULTS
AND DISCUSSION
The numerical results of bioassays under all conditions tested appear in Table 1. Probit analysis was used to determine LCsc’s (Bliss, 1934). In our experiments the LC& was affected by three factors: length of time of the bioassay, number of larvae per container tested (single vs grouped), and volume of the bioassay medium. Acute bioassays of single larvae carried out over a 4-hr period resulted in LC&,‘s nearly three times as high as those carried out under the same conditions for a period of 24 hr (Table 1, columns 2, 3), whereas the LC50’s for 24- and 48-hr bioassay periods were essentially the same (Table 1, columns 3 and 4). These data are shown graphically in the unstippled bars of Fig. 2. The dosage in the 4-hr bioassay corresponds to what we
AGAINST
MOSQUITO
287
LARVAE
refer to as an acute dose compared with the lower subacute dosages which suffice in the 24- and 48-hr bioassays. When groups of 25 larvae were bioassayed together in the same container, the LC,c was reduced by about one-half (Table 1, column 2 vs 5,3 vs 6, and 4 vs 7; Fig. 2, stippled bars). These results indicated to us that some sort of interaction must be occurring to account for this unanticipated potentiation of the toxicity of B. thuringiensis israelensis. The data also show that control larvae never exposed to B. thuringiensis isrcdensis died in 24- and 48-hr bioassays when grouped (Table 1, columns 6 and 7). Control larvae never died when tested singly, nor when grouped and tested for only 4 hr (Table 1, columns l-4 and 5). Judging by WHO bioassay guidelines, which call for grouped bioassays, deaths among control larvae are not uncommon-5% mortality is suggested as acceptable and up to 10% is correctable by Abbott’s formula (de Barjac and LargetThiery, 1979). When we observed our grouped bioassays carefully, we noticed some larvae apparently browsing and nibbling on others and on moribund and dead larvae. Predation and cannibalism were apparently the explanation for the free-floating head capsules that could frequently be found in grouped bioassays. We believe that this behavior accounts for all deaths among controls as well as some inappropriately early deaths among larvae exposed to B. thuringiensis israelensis. Not only would some larvae be scored as dead too early due to cannibalism, but also those larvae eating others could consume B. thuringiensis israelensis material already highly concentrated by filter feeding. Misch et al. (1987) have shown that B. thuringiensis israelensis may be concentrated by as much as lO,OOO-fold by filter feeding of the larvae of A. aegypti. The larval interactions described above were regularly seen in 24- and 48-hr grouped bioassays, but never in 4-hr bioassays, an observation that is entirely consistent with the occurrence of deaths among control larvae. We have as yet found no evidence to indicate whether cannibalism occurs in natural populations of
PB = puahbutlon
battery
’ It would be very convenient to use the polystyrene culture dishes without glass liners. This may be done in laboratories that can afford to dispose of them a&r a single bioassay. For those wishing an alternative, we have found that sonication of the cluster dishes for 10 min in methanol can remove residual toxicity due to B. thuringiensis israelensis material.
FS = foam platinum
wire
stopper
electrodes
FIG. 1. Wiring diagram for battery electrode low voltage stimulator to test viability of mosquito larvae.
288
MISCH, BURNSIDE, AND CECIL
TABLE 1 Bioassay
of B. thuringiensis
Concn of B. thuringiensis ismelensis (pg/literY 500 300 200 150 120 100 80 60 50 40 30 25 20 10 5 Total deaths LC 50 @g/liter) Control deaths
and Grouped Larvae: Number of Larvae Times of Exposure to Various Concentrations of B. thuringiensis israelensis israelensis
against
Individual 1 ml, 4 hr 24 15 6
6 ml, 48 hr
-
-
-
2
18
12
9 -
641120 60 O/30
28
Killed
at Indicated
Grouped larvaebzd
6 ml, 24 hr
25
471120 305 0130
larvae”.’
6 ml, 4 hr
-
Individual
28
-
-
24
24
14 5
17 6
711120 21 0130
751120 19 0130
-
-
-
150 ml, 4 hr 249 204 176 128 757/1200 33 O/300
150 ml, 24 hr 296 296 230 224 98 68 1212/1800 11 10/300
150 ml, 48 hr 300 299 263 263 137 128 1390/1800 9 111300
“Bacillus thuringiensis, subspiecies israelensis spore-crystal mixture, IPS 82. Activity = 15,000 IU/mg. * Single larvae were tested in either a l-ml or a 6-ml volume. Grouped larvae (25) were tested in a 150-ml volume. ’ ForIndividual larvae, 30 larvae were used per test concentration. d For grouped larvae, 300 larvae were used per test concentration.
these larvae, but it has been reported in laboratory cultures (MacGregor, 1915). Mitchell (1907) noted a similar larval-larval predation in Anopheles. For these reasons we recommend strongly against grouped bioassays of mosquito larvae, since the error cannot be reliably estimated in larvae exposed to test chemicals. We suggest that the approximately two-fold greater sensitivity of larvae ‘to B. thuringiensis israelensis in grouped bioassays is due at least in part to these larval interactions. Other factors are not ruled out. Our experiments also revealed another potential source of error which we call the threshold phenomenon. B. thuringiensis israelensis tested against individual larvae for a period of 4 hr in a l-ml volume resulted in an LC,, of 305 pg/liter (Table 1, column 1; Fig. 3). B. thuringiensis israelensis tested at the same concentrations on single larvae in a 6-ml test volume resulted in an LC,, of only 60 pgiliter. We suggest that the total amount of B. thuringiensis isruelensis in the l-ml volume was insufficient to kill one larva during the test period. The total of 360 pg in the 6-ml volume test was sufficient, giving an LCso of 60 pg/liter. Similar threshold-volume effects have been noted by Vorgetts et al. (1988). Longer term bioassays of 24- and particularly 48-hr duration run the risk that filter feeding will slow or stop as a molt or pupation approaches (MacGregor,
60
-
60
-
40
-
1 LARVA
PER
6 ml
d 3g
50-
20
-
10 -
4 nn
24 HR
46 HR
FIG. 2. Effect of time and grouping of larvae on LC,, of B. thuringiensis isrtzelensis. Larvae tested together in groups of 25 (stippled bars) were twice as sensitive to 8. thuringiensis israelensis as were larvae tested singly (open bars). As expected, lethality occurred at lower concentrations of B. thuringiensis israeknsis when tested over the longer periods of exposure (24 and 48 hr).
B. thuringiensis
israelensis:
NOVEL BIOASSAY
AGAINST
MOSQUITO
289
LARVAE
TABLE 2 300
-
LC,,
of B. thuringiensis israelensisToxin Against the Fourth Instar of A. aegypti Under Various Bioassay Conditions
Condition 200
-
Individual larva 1 ml, 4 hr 6 ml, 4 hr 6 ml, 24 hr 6 ml, 48 hr
2 38 =: s
100
Number of larvae
w50 (pg/liter)
150 150 150 150
Grouped larvae (25) 150 ml, 4 hr 1500 (60 groups) 150 ml, 24 hr 2100 (84 groups) 150 ml, 48 hr 2100 (84 groups)
-
-t-2 SD
Range
305 60 21 19
2 99 -c 41 t2 r4
219415 29-111 19-23 15-23
33 11 9
t 19 ‘- 6 26
15-53 7-19 4-15
ACKNOWLEDGMENTS 1 LARVA I PER ml
1 LARVA / PER 6 ml BIOASSAY
25 LARVAE I PER 150 ml
CONDITIONS
FIG. 3. Effects of volume and grouping
of larvae on LC,, of B. (4-hr acute assay). Larvae tested in only 1 ml of medium required much more B. thuringiensis israetensis for an L&o than did larvae tested in a volume of 6 ml. We suggest this difference was due to insufficient total B. thuringiensis israelensis in the smaller volume for the short 4-hr test period. The lower LC,, for the grouped larvae may have been due to larval-larval interactions. thuringiensis
israelensis
1915). The smaller amount of B. thuringiensis Zensisingested could skew the results. According
isrue-
to the WHO bioassay guidelines, pupation during the 48-hr test period is expectable, and pupae are to be excluded from the bioassay results. There is, however, no way to correct for reduced larval intake of those larvae that have not quite started the molt. It is well known that mosquito larvae in general and those of A. aegypti in particular do not develop synchronously (Christophers, 1960). For this reason, selection of either a 4- or a 24-hr bioassay test period is preferred over a 48-hr bioassay. Our proposed bioassay system offers advantages over the WHO bioassay: (1) increased precision by obviating cannibalism and thereby the major source of deaths among controls and inappropriate deaths among the experimentals, (2) significantly shortened test time of 1 or 2 working days compared with 2 or 3 working days, and (3) increased statistical precision-bioassay of 10 individually contained larvae provides 40% more statistically valid “points” than six groups of 25 larvae.3 A summary of our findings is presented in Table 2. 3 The use of four groups of 25 larvae in each container provides only 4 statistically valid replications, not 100. Such pseudoreplications are discussed in Hurlbert (1984).
Portions of this investigation were carried out in the U.S. Army Biomedical Research & Development Laboratory, Fort Detrick, Frederick, Maryland. We thank Dr. James H. Nelson and Cpt. Lewis R. Boobar for use of the facilities and for support. Mr. L. M. Anderson and Dr. Michael Perich provided useful suggestions and valuable discussions. This research was supported in part by a grant to D. F. Burnside from the Burroughs-Wellcome Corp., Research Triangle Park, North Carolina.
REFERENCES Bliss, C. I. 1934. The method of probits. Science 79, 38-39. Christophers, Sir S. R. 1960. “Aedes aegypti CL.),” pp. 246-247. Cambridge Univ. Press, London/New York. de Barjac, H., and Larget-Thiery, I. 1979. “Proposals for the adoption of a standardized bioassay method for the evaluation of insecticidal formulations derived from serotype H-14 of Bacillus thuringiensis,” VBC179.744. World Health Organization, Albany, NY. Hurlbert, Stuart H. 1984. Pseudoreplication and the design of ecological field experiments. Ecol. Monogr. 54, 187-211. MacGregor, M. E. 1915. Notes on the rearing of Stegomyia fasciata in London. J. Trop. Med. Hyg. 18, 193-196. McLaughlin, R. E., Dulmage, H. T., Ails, R., Couch, T. L., Dame, D. A., Hall, I. M., Rose, R. I., and Versoi, P. L. 1984. U.S. standard bioassay for the potency assessment of Bacillus thuringiensis serotype H-14 against mosquito larvae. Entomol. Sot. Am. Bull. 30, 2629.
Misch, D. W., Anderson, L. M., and Boobar, L. R. 1987. The relative toxicity of a spore preparation of Bacillus thuringiensis var. israelensis against fourth instar larvae of Aedes aegypti and Toxorhynchites amboinensis: Suspension feeding compared with enemas and forced feeding. Entomol. Exp. Appl. 44, 151-154. Mitchell, E. G. 1907. “Mosquito Life.” Putnam, New York. Vorgetts, J., Jr., Frommer, R. L., Gibbs, P. H., and Anderson, L. M. 1988. Interpretation of density-dependent data in comparisons of Bacillus thuringiensis var. israelensis formulations. J. Entomol. Sci. 23, 149-154.
OF INVERTEBRATE
PATHOLOGY
59,
286-289
(19%)
A Novel Bioassay System for Evaluating the Toxicity of Bacillus thuringiensis israelensis against Mosquito Larvae’ DONALD W. MUCH Department
of
Biology, Coker Hall, CB 3280, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina 275993280
DALE F. BURNSIDE Department of Biology, Lenoir-Rhyne
College, Hickory, North Carolina 28601
TAMERA L. CECIL U.S. Army Biomedical
Research & Development Laboratory,
Fort Detrick, Frederick, Maryland
21701
Received March 22, 1991; accepted August 15, 1991
A bioassay system employing acutely toxic concentrations of a spore-crystal mixture of Bacillus thuringieusis subspecies israelensis against fourth instar larvae of Aedes aeggpti (L.) is described. Individual larvae are separately exposedto toxin in glass-lined miniature wells or scintillation vials. This method is free from the deaths due to predation among larvae. Such larval deaths are commonly encountered in bioassay groups of 25 larvae as currently specified in the World Health Organization guidelines. Our method offers shortened testing time, increased accuracy, and improved statistical precision. 0 1992 Academic KEY WORDS: bioassay; mosquito larvae; Bacillus thuringiensis israelensis (L.); acute toxicity. ~SS,
hf.
INTRODUCTION
The ultimate test of any insecticidal material is its effectiveness in controlling natural populations. Because of the high cost and long time required for field testing, laboratory bioassays of toxicity are routinely employed for preliminary screening of candidate pesticides and are also useful for quality control. However, bioassays are notorious for their variability when used by different laboratories and may also show inconsistencies within the same laboratory. Standard bioassay procedures for mosquito larvae have been published by de Barjac and Larget-Thiery (1979) for the World Health Organization (WHO) and more recently by McLaughlin et al. (1984). Vorgetts et al. (1988) note the lack of uniformity in testing larvi1 The statements and opinions contained in this article are those of the authors and do not reflect official views of the Department of the Army. 286 oozz-2011/92 Copyright All rights
$4.00 0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
tides against filter-feeding invertebrates, including mosquito larvae, and have stressed the need for a more universally adopted bioassay protocol. In this paper we describe a modified bioassay system and the results of using it to test the toxicity of a standard spore-crystal suspension of Bacillus thuringiensis subspecies isruelensis against the fourth instar larvae of Aedes aegypti CL.). We compare our system with the currently recommended standard bioassay procedure and review the findings, which we believe make our system more precise and more reliable. MATERIALSANDMETHODS 1. Culture of A. aegypti. Eggs stored at 60-70% relative humidity were hatched in dechlorinated tap water (DTW) conditioned by allowing it to stand overnight or longer and to which a small amount of liver powder (ICN-Nutritional Biochemicals 900396) was added. Egg papers were removed after 4-5 hr. Newly hatched larvae were harvested the following day, distributed at approximately 200 per liter of DTW in 23cm-diameter enamel pans, and then grown under 14 hr of daily illumination at 27°C. They were fed a suspension of liver powder ad lib. Early fourth instar larvae were selected for use after 80 hr in culture. 2. Bioassay procedure. Individual larvae were tested in 24-well polystyrene tissue culture cluster dishes (Costar 3524). Preliminary testing revealed that B. thuringiensis israelensis material bound strongly to the plastic surface (D. W. Misch, unpublished observations). For that reason glass liners made by cutting off the bottom 33 mm of 16 x 125-mm test tubes were used to hold the larvae within the plastic wells. These glass liners could be removed for washing
B. thuringiensis
israelensis:
NOVEL
BIOASSAY
and reuse without detectable carryover of toxicity.2 Early fourth instar larvae were individually exposed to various concentrations IPS-82, an international standard spor+crystal mixture of B. thuringienesis israeZensis(courtesy of I. Larget-Thiery, Institute Pasteur, Paris). Group bioassays of 25 larvae together were performed in 150 ml of DTW in 11.5-cm glass finger bowls. As a convenient alternative to 4-hr bioassays of single larvae in glass liners, we placed individual larvae in 6 ml of DTW in standard scintillation vials (e.g., Fisher 03-337-4, 20-ml vials, 61 x 28 mm). The vials were arranged in white Styrofoam racks for easy viewing of the larvae. This proved to be the method of choice and was used in most of our experiments. When all the larvae needed for a test run had been set up in DTW, 2 l.~l of B. thuringiensis israelensis suspension of the appropriate concentration was added in sequence to all test larvae. Four different concentrations of B. thuringiensis israelensis were tested against 10 larvae each; 10 control larvae were used for each test. For acute toxicity tests, deaths were recorded at 15, 30, and 60 min and at 2 and 4 hr. For subacute tests, deaths were recorded at 24 and 48 hr. Each test was replicated three times. Deaths of unmoving larvae were verified by administering an 18-V battery shock with a device of our own design (Fig. 1). Larvae that gave no visible response were judged to be dead. In trial tests using electrical stimulation to judge the time of death, larvae that reacted to the brief shock were also able to be stimulated by mechanical means. By this test we believe we have established a more objective and accurate test of death. RESULTS
AND DISCUSSION
The numerical results of bioassays under all conditions tested appear in Table 1. Probit analysis was used to determine LCsc’s (Bliss, 1934). In our experiments the LC& was affected by three factors: length of time of the bioassay, number of larvae per container tested (single vs grouped), and volume of the bioassay medium. Acute bioassays of single larvae carried out over a 4-hr period resulted in LC&,‘s nearly three times as high as those carried out under the same conditions for a period of 24 hr (Table 1, columns 2, 3), whereas the LC50’s for 24- and 48-hr bioassay periods were essentially the same (Table 1, columns 3 and 4). These data are shown graphically in the unstippled bars of Fig. 2. The dosage in the 4-hr bioassay corresponds to what we
AGAINST
MOSQUITO
287
LARVAE
refer to as an acute dose compared with the lower subacute dosages which suffice in the 24- and 48-hr bioassays. When groups of 25 larvae were bioassayed together in the same container, the LC,c was reduced by about one-half (Table 1, column 2 vs 5,3 vs 6, and 4 vs 7; Fig. 2, stippled bars). These results indicated to us that some sort of interaction must be occurring to account for this unanticipated potentiation of the toxicity of B. thuringiensis israelensis. The data also show that control larvae never exposed to B. thuringiensis isrcdensis died in 24- and 48-hr bioassays when grouped (Table 1, columns 6 and 7). Control larvae never died when tested singly, nor when grouped and tested for only 4 hr (Table 1, columns l-4 and 5). Judging by WHO bioassay guidelines, which call for grouped bioassays, deaths among control larvae are not uncommon-5% mortality is suggested as acceptable and up to 10% is correctable by Abbott’s formula (de Barjac and LargetThiery, 1979). When we observed our grouped bioassays carefully, we noticed some larvae apparently browsing and nibbling on others and on moribund and dead larvae. Predation and cannibalism were apparently the explanation for the free-floating head capsules that could frequently be found in grouped bioassays. We believe that this behavior accounts for all deaths among controls as well as some inappropriately early deaths among larvae exposed to B. thuringiensis israelensis. Not only would some larvae be scored as dead too early due to cannibalism, but also those larvae eating others could consume B. thuringiensis israelensis material already highly concentrated by filter feeding. Misch et al. (1987) have shown that B. thuringiensis israelensis may be concentrated by as much as lO,OOO-fold by filter feeding of the larvae of A. aegypti. The larval interactions described above were regularly seen in 24- and 48-hr grouped bioassays, but never in 4-hr bioassays, an observation that is entirely consistent with the occurrence of deaths among control larvae. We have as yet found no evidence to indicate whether cannibalism occurs in natural populations of
PB = puahbutlon
battery
’ It would be very convenient to use the polystyrene culture dishes without glass liners. This may be done in laboratories that can afford to dispose of them a&r a single bioassay. For those wishing an alternative, we have found that sonication of the cluster dishes for 10 min in methanol can remove residual toxicity due to B. thuringiensis israelensis material.
FS = foam platinum
wire
stopper
electrodes
FIG. 1. Wiring diagram for battery electrode low voltage stimulator to test viability of mosquito larvae.
288
MISCH, BURNSIDE, AND CECIL
TABLE 1 Bioassay
of B. thuringiensis
Concn of B. thuringiensis ismelensis (pg/literY 500 300 200 150 120 100 80 60 50 40 30 25 20 10 5 Total deaths LC 50 @g/liter) Control deaths
and Grouped Larvae: Number of Larvae Times of Exposure to Various Concentrations of B. thuringiensis israelensis israelensis
against
Individual 1 ml, 4 hr 24 15 6
6 ml, 48 hr
-
-
-
2
18
12
9 -
641120 60 O/30
28
Killed
at Indicated
Grouped larvaebzd
6 ml, 24 hr
25
471120 305 0130
larvae”.’
6 ml, 4 hr
-
Individual
28
-
-
24
24
14 5
17 6
711120 21 0130
751120 19 0130
-
-
-
150 ml, 4 hr 249 204 176 128 757/1200 33 O/300
150 ml, 24 hr 296 296 230 224 98 68 1212/1800 11 10/300
150 ml, 48 hr 300 299 263 263 137 128 1390/1800 9 111300
“Bacillus thuringiensis, subspiecies israelensis spore-crystal mixture, IPS 82. Activity = 15,000 IU/mg. * Single larvae were tested in either a l-ml or a 6-ml volume. Grouped larvae (25) were tested in a 150-ml volume. ’ ForIndividual larvae, 30 larvae were used per test concentration. d For grouped larvae, 300 larvae were used per test concentration.
these larvae, but it has been reported in laboratory cultures (MacGregor, 1915). Mitchell (1907) noted a similar larval-larval predation in Anopheles. For these reasons we recommend strongly against grouped bioassays of mosquito larvae, since the error cannot be reliably estimated in larvae exposed to test chemicals. We suggest that the approximately two-fold greater sensitivity of larvae ‘to B. thuringiensis israelensis in grouped bioassays is due at least in part to these larval interactions. Other factors are not ruled out. Our experiments also revealed another potential source of error which we call the threshold phenomenon. B. thuringiensis israelensis tested against individual larvae for a period of 4 hr in a l-ml volume resulted in an LC,, of 305 pg/liter (Table 1, column 1; Fig. 3). B. thuringiensis israelensis tested at the same concentrations on single larvae in a 6-ml test volume resulted in an LC,, of only 60 pgiliter. We suggest that the total amount of B. thuringiensis isruelensis in the l-ml volume was insufficient to kill one larva during the test period. The total of 360 pg in the 6-ml volume test was sufficient, giving an LCso of 60 pg/liter. Similar threshold-volume effects have been noted by Vorgetts et al. (1988). Longer term bioassays of 24- and particularly 48-hr duration run the risk that filter feeding will slow or stop as a molt or pupation approaches (MacGregor,
60
-
60
-
40
-
1 LARVA
PER
6 ml
d 3g
50-
20
-
10 -
4 nn
24 HR
46 HR
FIG. 2. Effect of time and grouping of larvae on LC,, of B. thuringiensis isrtzelensis. Larvae tested together in groups of 25 (stippled bars) were twice as sensitive to 8. thuringiensis israelensis as were larvae tested singly (open bars). As expected, lethality occurred at lower concentrations of B. thuringiensis israeknsis when tested over the longer periods of exposure (24 and 48 hr).
B. thuringiensis
israelensis:
NOVEL BIOASSAY
AGAINST
MOSQUITO
289
LARVAE
TABLE 2 300
-
LC,,
of B. thuringiensis israelensisToxin Against the Fourth Instar of A. aegypti Under Various Bioassay Conditions
Condition 200
-
Individual larva 1 ml, 4 hr 6 ml, 4 hr 6 ml, 24 hr 6 ml, 48 hr
2 38 =: s
100
Number of larvae
w50 (pg/liter)
150 150 150 150
Grouped larvae (25) 150 ml, 4 hr 1500 (60 groups) 150 ml, 24 hr 2100 (84 groups) 150 ml, 48 hr 2100 (84 groups)
-
-t-2 SD
Range
305 60 21 19
2 99 -c 41 t2 r4
219415 29-111 19-23 15-23
33 11 9
t 19 ‘- 6 26
15-53 7-19 4-15
ACKNOWLEDGMENTS 1 LARVA I PER ml
1 LARVA / PER 6 ml BIOASSAY
25 LARVAE I PER 150 ml
CONDITIONS
FIG. 3. Effects of volume and grouping
of larvae on LC,, of B. (4-hr acute assay). Larvae tested in only 1 ml of medium required much more B. thuringiensis israetensis for an L&o than did larvae tested in a volume of 6 ml. We suggest this difference was due to insufficient total B. thuringiensis israelensis in the smaller volume for the short 4-hr test period. The lower LC,, for the grouped larvae may have been due to larval-larval interactions. thuringiensis
israelensis
1915). The smaller amount of B. thuringiensis Zensisingested could skew the results. According
isrue-
to the WHO bioassay guidelines, pupation during the 48-hr test period is expectable, and pupae are to be excluded from the bioassay results. There is, however, no way to correct for reduced larval intake of those larvae that have not quite started the molt. It is well known that mosquito larvae in general and those of A. aegypti in particular do not develop synchronously (Christophers, 1960). For this reason, selection of either a 4- or a 24-hr bioassay test period is preferred over a 48-hr bioassay. Our proposed bioassay system offers advantages over the WHO bioassay: (1) increased precision by obviating cannibalism and thereby the major source of deaths among controls and inappropriate deaths among the experimentals, (2) significantly shortened test time of 1 or 2 working days compared with 2 or 3 working days, and (3) increased statistical precision-bioassay of 10 individually contained larvae provides 40% more statistically valid “points” than six groups of 25 larvae.3 A summary of our findings is presented in Table 2. 3 The use of four groups of 25 larvae in each container provides only 4 statistically valid replications, not 100. Such pseudoreplications are discussed in Hurlbert (1984).
Portions of this investigation were carried out in the U.S. Army Biomedical Research & Development Laboratory, Fort Detrick, Frederick, Maryland. We thank Dr. James H. Nelson and Cpt. Lewis R. Boobar for use of the facilities and for support. Mr. L. M. Anderson and Dr. Michael Perich provided useful suggestions and valuable discussions. This research was supported in part by a grant to D. F. Burnside from the Burroughs-Wellcome Corp., Research Triangle Park, North Carolina.
REFERENCES Bliss, C. I. 1934. The method of probits. Science 79, 38-39. Christophers, Sir S. R. 1960. “Aedes aegypti CL.),” pp. 246-247. Cambridge Univ. Press, London/New York. de Barjac, H., and Larget-Thiery, I. 1979. “Proposals for the adoption of a standardized bioassay method for the evaluation of insecticidal formulations derived from serotype H-14 of Bacillus thuringiensis,” VBC179.744. World Health Organization, Albany, NY. Hurlbert, Stuart H. 1984. Pseudoreplication and the design of ecological field experiments. Ecol. Monogr. 54, 187-211. MacGregor, M. E. 1915. Notes on the rearing of Stegomyia fasciata in London. J. Trop. Med. Hyg. 18, 193-196. McLaughlin, R. E., Dulmage, H. T., Ails, R., Couch, T. L., Dame, D. A., Hall, I. M., Rose, R. I., and Versoi, P. L. 1984. U.S. standard bioassay for the potency assessment of Bacillus thuringiensis serotype H-14 against mosquito larvae. Entomol. Sot. Am. Bull. 30, 2629.
Misch, D. W., Anderson, L. M., and Boobar, L. R. 1987. The relative toxicity of a spore preparation of Bacillus thuringiensis var. israelensis against fourth instar larvae of Aedes aegypti and Toxorhynchites amboinensis: Suspension feeding compared with enemas and forced feeding. Entomol. Exp. Appl. 44, 151-154. Mitchell, E. G. 1907. “Mosquito Life.” Putnam, New York. Vorgetts, J., Jr., Frommer, R. L., Gibbs, P. H., and Anderson, L. M. 1988. Interpretation of density-dependent data in comparisons of Bacillus thuringiensis var. israelensis formulations. J. Entomol. Sci. 23, 149-154.