Journal of the American Mosquito Control Association, lO(I):51-55,1994 Copyright @ 1994 by the American Mosquito Control Association, Inc.

EFFICACY OF BIFERMENTANS SEROVAR MALAYSIA ON TARGET AND NONTARGET ORGANISMS

M. YIALLOUROS,T V. STORCH,: I. THIERYT erp N. BECKERI

ABSTRACT. Clostridium bifermentansserovar malaysia (C.b.m.) is highly toxic to mosquito larvae. In this study, the following aquatic nontarget invertebrateswere treated with high C.b.,,?.concentrations (up to 1,600-fold the toxic concentration for Anophelesstephensi) to study their susceptibility towards the bacterial toxrn: Planorbis planorbis (Pulmonata); Asellus aquaticzs (Isopoda); Daphnia pulex (Cla- docera);Cloeon dipterun (Ephemeroptera);Plea leachi (Heteroptera);and Eristalis sp., Chaoboruscrys' tallinus, Chironomus thummi, and Psychodaalternata (Diptera). In addition, bioassayswere performed with mosquito Larvae( aegypti, stephensi, and Culex pipiens). Psychodaalternatalamae were very susc€ptible,with LCro/LCro values comparable to those of mosquito larvae (about 103-105 spores/ml). The tests with Chaoboruscrystallinus larvae showed significant mortality rates at high con- centrations,but generallynot before 4 or 5 days after treatment. The remaining nontargetorganisms did not show any susceptibility.The investigation confirms the specificityof C.b.m.lo nematocerousDiptera.

INTRODUCTION strains of Aedesaegypti (Linn ) larvae, which are about I 0 times lesssensitiv e than A nophe I e s. The For several years, 2larvicidal bacteia, Bacil- LCr' (48 h) rangesfrom 5 x 103to 2 x lOscells/ lus thuringiensls Berliner var. israelensis (B.t.i.) ml (Thiery et al. 1992b).Larvae of Simulium and Bacillus sphaericus Neide, have been used speciesseem to be less susceptible(de Barjac et successfully for mosquito and blackfly control all al. 1990).Toxicity was not found for lepidop- over the world. Nevertheless, in consideration of teran, coleopteran, and dipteran (Brachycera) the increasing resistance to chemical insecticides larvae or for Biomphalaria glabrata Say snails and the need for alternative control agents (Gastropoda) (Thiery et al. 1992b). The safety (Anonymous 1987), there has been an ongoing for vertebrateswas demonstratedby a classical search for new mosquitocidal pathogens (Thiery seriesoftests recommendedby the World Health et al. 1992b). Organization (Thiery et al. 1992a). About 3 years ago, a Clostridium bifermentans Our objective was to extend the investigations Weinberg and Seguin strain toxic to mosquito concerning specificity and thus environmental and blackfly larvae was isolated from soil sam- compatibility of the bacterial toxin(s) by study- ples of a Malaysian mangrove swamp. Further ing possiblee ffects of C.b. m. on severalnontarget characterization and the existence of a specific organismsthat inhabit the larval habitatsofmos- H-antigen allowed it to be individualized as a quitoes and would, therefore, be directly con- serovar-malaysia(C.b.m.). It is the first strictly cernedby mosquito control operations.The se- pathogen anaerobic mosquitocidal that has been lection of the nontarget organismswas made in (de characterized Barjac et al. 1990). respectto their significanceand frequencyin the The larvicidal activity of C.b.m. is closely habitat. Another aspectof selection was the re- partly linked to the sporulation stage and is due presentationof different systematicgroups of or- to the existence of parasporal inclusion bodies ganisms and different feeding strategies. (Charles et al. 1990, de Barjac et al. 1990). The toxic proteins have not yet been clearly charac- terized, but they are very different from B.l.i. and MATERIAL AND METHODS B. sphaericus crystal toxins (Nicolas et al. I 993). Bacterial culture: The C.b.m. was grown un- The determination of the host range shows a der anaerobic.conditionsas describedin Nicolas specificity for larvae of the Culicidae and the et al. (1990)and wasstopped at t, ofsporulation Simuliidae. Susceptibility varies according to the (16 h culture).A lyophilizedpreparation ofthis mosquito species, wit}:, Anopheles species being whole culture (700/ocells contained spores,spore the most susceptible, followed by Culex and all density: 1.6 x 108spores/ml) was used for all assays.The sample was suspendedin deionized water prior The I to the start ofthe tests. suspen- German Mosquito Control Association(K.A.B.S.), sion was either used immediately or frozen in LudwigstraBe99, D-67 165 Waldsee,Germany. aliquots at -2O"C to prevent a loss of toxicity. 2 University of Heidelberg (ZoologielMorphologie gained l), Im Neuenheimer Feld 230, D-6912O Heidelberg, Appropriate concentrationswere by fur- Germany. ther dilution of the whole culture. 3 Unit6 des Bact6ries Entomopathog6nes,Institut Bioassaysof mosquitolarvae: a) Culicini lar- Pasteur,25 Rue du Docteur Roux, 75724 Paris Cedex vae: Laboratory-reared larvae of Ae. aegypti 15, France. (strain Bora-Bora) and Culex pipiens Linn. were 5l 52 Jounxer or nrE AunruceN Moseurro Cor.rrnol Assocr,q,rroN

Table l. Larvicidal activity of Clostridium bifermentans ser. malaysia on Culicidae larvae and on other Nematocera (Diptera).

Species LC5o(24 h)l LCeo (24 h) LCso (48 h) LC,o (48 h) Aedesaegypti 600 20.2 + 1r.32 143+ 8l 10.9+ 7.9 42.7 + 20.8 (Bora-Bora) 3.2+ 1.83 22.9+ t3 1.7+ 1.2 6.9+ 3.4 Anophelesstephensi 420 1.63.4 7.8 0.5 1.8 An. stephensi 525 0.7 + 0.2t.s 1.6+ 0.3 0.4+0 l.l + 0.1 Culex pipiens 600 92.3 + 19.92 263.3+ 89.6 65 -r 26 195+ 55 14.7+ 3.13 42.0 + t4.5 10.6+ 4.4 3l+9 Psychodaalternata 350 8.0+ 3.1' 52.O+ 27 2.O+ 1.9 7.3+ 6.2 1.3+ 0.53 8.3 + 4.2 0.3 + 0.3 1.2+ 1.0 Chaoborus crystal linus 300 3,577+ 359 22,355 + 9562'6 572+ 58 3,577+ 1533 ' Mean of 3 experiments + SE (P < 0.05). 'lJthal concentrationexpressed in l0 5 x dilution ofC.b.z. whole culture. 3 lrthal concentrationexpressed in lOa x spores/ml. 4 Result of one experiment. 5 Resultsobtained by Thiery et al. (1992b) (P < 0.05). 6 Mean of2 experiments + SE (P < 0.05), LC values on 7th day. testedagainst the above-mentionedpreparation lated for Anophelesstephensi (1.1 x l0 4 dilu- at dilutionsof 0.5,l, 2,4,8,16,and 33 x l0-o tion : 1.76 x 104spores/ml) served as a basis (spores/mlranging from 8 x 103to 5.3 x 105). for the choiceofthe bacterialconcentrations used Twenty-five early 4th-instar larvae were placed for nontargetorganisms: for Planorbisplanorbis, in plasticcups containing 150 ml of the appro- Cloeon dipterum, Plea leachi, Asellus aquaticus, priate bacterial suspension.Three cupswere pre- Dapnia pulex, and,E ristalis sp.: 3.5 x I 06spores/ pared per dilution and testswere run in triplicate ml (: 200-fold concentration); for the larvae of at24 + 2oC.Three cups served as controls. Mor- Chironomusthummi s.l.: 4.4 x 106spores/ml (: tality was determined 24 and 48 h after appli- 250-fold concentration); for Chaoboruscrystal- cation of the C.b.m. preparation. The LCr,, and linus: up to 2.8 x 107spores/ml (: 1,600-fold LCro values (P < 0.05) were calculatedby log concentration), and finally, for Psychodaalter- probit analysis by use of a computer program nata, concentrations varying between 103 and (Raymond1985). 105 spores/ml. To exclude nonspecific effects b) Anophelini larvae: Three cups containing basedon the activity of exotoxinsor metabolite 20 early 4th-instar lawae of Anophelesstephensi toxins, autoclaved C.b.m. mateial was used in Liston were prepared per dilution (series of 6 sometests. dilutions;rangeT x 10-6to 2.2 x l0 a, corre- Twenty to 25 individuals were placed in each spondingto l.l x 103to 3.5 x 104spores/ml), of the test dishescontaining 100-150 ml water 3 cupsserved as controls. The testwas run once. (mixture of original and spring water) and the Nontarget organisms: The following species appropriateC.b.m. concentration. Two to 3 rep- served as test organisms: Planorbis planorbis licates were prepared for each concentration; a Linn. (Pulmonata); nymphs of Cloeon dipterum respective number served as a control. Small Linn. (Ephemeroptera);Plea leachi MacGregor amounts of food were added meeting the special (Heteroptera);Asellus aquaticus Linn. (Isopoda); requirementsof the organisms(Tetra Min, plant Daphnia pulex De Geer (Cladocera);larvae of material,or preyorganisms, i.e., Daphnia pulex). Eristalis sp. (Brachycera,Diptera); and 2nd-4th- Experiments were conducted at 20'C. The du- instar larvae of Chaoboruscrystallinus De Geer, ration of the experiment was up to 2 wk, obser- Chironomusthummi s.1.,and Psychodaalternata vations were made every I or 2 days. Tests were Say (Nematocera, Diptera). The animals were run in duplicate or triplicate. Test results were collectedfrom their natural habitats, transferred interpreted by calculation of the percent mor- to the laboratory, and allowed to adapt to lab- talities observed.Deviations betweencontrol and oratory conditions (20'C; day-night rhythm: 14: treated batches were checked for their signifi- l0 h) for I or 2 days.Psychoda alternatalawae canceby meansof multiple rangetests according remained in their substrate until the beginning ro Duncan(1955). ofthe test, then they werewashed out with small- Psychodaalternata: LC5o/LCeotests were car- meshedsieves. ried out with this speciesdue to its susceptibility Toxicity tests.' The LCnn value (48 h) calcu- towards C.b.m. Dead and living animals were M,c,RcH1994 Ctosranotutt anexuanrt rs Senoven MAr/ffsIA

Table 2. Larvicidal activity of Clostridium bifermentans ser. malaysia on nontarget organisms. o/o Dose Test Species Order n (spores/ml) (days) Test Control Planorbis planorbis Pulmonata 60 3.5 x lOe 12 0 0 Cloeon dipterum Ephemeroptera 60 3.5 x 106 8 0 2.5+ 0.8 Plea leachi Heteroptera 60 3.5 x 106 7 0 6.7+ 0 Asellus aquaticus Isopoda 60 3.5 x 106 8 J.U+ t.7 4.3+ 1.0 Daphnia pulex Cladocera 75 3.5 x 106 6 4.3 I 2.3 J + I Eristalis sp. Diptera 60 3.5 x 106 3 20 5 Chaoborus crystallinus Diptera 50 t.76_29x 106 7 35+7-87+L 5+5 Chironomusthummi Diptera 60 4.4 x 106 6 12.5+ 7.5 ll.5 + ll.5 Psychodaalternata Diptera 50 103x 105 2 72.3+ 27.2- 3.0+ 3.5 99.3 + O.92 ' Mean of 2 experiments. ' Mean of 3 experiments(P < 0.05). counted 24 and 48 h after the beginning of the cies are summarizedin Table l. After 48 h of test. The results were submitted to a log probit larval exposureto C.b.m.,500/o of the population analysisto obtain LCro and LCro values. was killed by 0.3 x lOaspores/ml and 900/owas killed by 1.2 x 104spores/ml. According to these resultsPsycfto da alternata is even more sensitive RESULTS to C.b.m. than Ae. aegypti (about 6 times) and Lamicidal activity on Culicidae: The larvicid- Cx. pipiens (about 30 times) and at least as sen- al activity of C.b.m. on the mosquito species sitive as An. stephensi. tested is summarized in Table l. Aedesaegypti Table 3 presentsthe results of 2 toxicity tests was the most susceptiblespecies with 500/oof the performed with Chaoborus crystallinzs larvae. population killed by 1.7 x 104spores/ml and The percent mortality in the treated batches is 900/okilled by 6.9 x 10ospores/ml 48 h afterthe slightly higher than in the control batchesfrom application of C.b.m. Culex pipiens was lesssen- the 3rd day onward. There is, however, no sig- sitive,with 500/0of the populationkilled by 10.6 nificant deviation between control and treated x 104spores/ml and 900/okilled by 3l x lOa batchesuntil the 6th day of the test run. Multiple spores/ml. The bioassay with An. stephensi range tests show that on the 8th day the devia- showedthat this speciesis even more susceptible tions between control batch and batches with towardsC.b.m. than the 2 mentionedabove, with 1,600- and 800-fold concentrationsare highly LCro and LCnovalues of 0.5 x lOa and 1.8 x significant (P : 0.001), the deviation between lOa spores/ml,respectively. As thesewere the control and 400-fold concentrationis significant resultsof one bioassayonly, the LCrnlLCnoval- (P : 0.01),and thereare slightly significantdif- ues obtained by Thiery et al. (1992b) with the ferences(P : 0.05) betweencontrol and.200-/ same lyophilized preparation are added for rea- 100-fold concentrations.Similar results were sonsof comparability(see Table l). achievedwith testsusing 100-and 500-foldcon- Toxicity to nontarget organisms: Table 2 centrations. In all experiments,Chaoborus crys- summarizesthe eftcacy of C.b.m. on the non- tallinus showeda somehowdelayed reaction, with target organismstests. Planorbis planorbis, Asel- significant mortality ratesgenerally not before 4 lus aquaticus, Daphnia pulex, Plea leachi, and or 5 days after treatment with C.b.m. It was ob- Cloeon dipterum nymphs, as well as the larvae servedthat in the courseofthe test, larvae were of the dipterans Eristalis sp. and Chironomus moreand moreweakened, with their movements thummi s./., did not show any susceptibility. and reactions increasingly slowing down. Such Clostridium bifermentansserovar malaysia weakeningof Chaoboruslarvae was never noted showed some effect, however, on Chaoborus in the control batches.To excludethe possibility crystallinus and Psychodaalternata larvae, with of the larvae dying due to altered water condi- both speciesreacting with increased mortality tions, several tests were made with autoclaved ratesafter treatment witt, C.b.m. spore material. and thus nontoxic material.These experiments The larvae of Psychodaalternata were shown did not show any difference between control to be very susceptibleto C.b.m. The resultsof batchesand batchestreated with autoclavedma- the bioassays(LCso/LCso values) with this spe- terial. Table I shows the LCro/LCnnvalues for 54 Jouru.rnr or rnr Aurnrcnx MosQuIro Con-rnor Assocnnol Vol. 10, No. I

Table 3. Efficacy of Clostridium bifermentans ser. malaysia on larvae of Chaoboruscrystallinus. x-fold LCno Anoph- o/omortality + SEt eles Dose stephensi (spores/ml) Day I Day 3 Day 5 Day 6 Day 7 Control 0 0 0 5+5 5+5 5+5 100 x 1.76x 106 +3 l3 +9 16+10 33+5 35+7 200 x 3.5 x 106 J +3 1l+7 24+lO 35+11 45+15 400 x 7x106 I +l t0+8 35+1 53+5 68+4 800 x 1.4x 10? I +l t7+15 34+12 60+4 82+8 1,600x 2.8 x 107 0 20+14 37+15 64+4 87+l ' Mean of2 experiments (P < 0.05). n : 50 per concentration and experiment.

Chaoborus crystallinus. On the 7th day after docera),Cloeon dipterum (Ephemeroptera),Plea C.b.m. application, 500/oofthe larvae were killed leachi (Heteroptera),and also for the dipterans by 5.95 x 106spores/ml and 90oloby 35.J x 106 Eristalis sp. and Chironomusthummi s./. Tox- spores/ml. A comparison of these results with icity tests with the latter group of specieswere those gained for the most susceptiblemosquito of special interest, as the Chironomidae, es- species(LCro [48 h] An. stephensi)shows that pecially the subfamily Chironominae, are known about 300-fold more bacteriaare required to kill to be susceptibleto Bacillus thuringiensis var. 500/oof the Chaoborus uystallinus larvae and israelensis(Ali 1981,Schnetteretal. 198 l, Mor- about 2,000-fold more bacteriato kill 900/oof the awcsik 19834).A similar susceptibilitytowards population within 7 days. C.b.m. could not be observed. To the contrary, Psychodaalternata is highly sensitivetowards C.b.m.,even at low bacterial DISCUSSION concentrations.Indeed, with LCrn value (48 h) This study shows the limited host range and of 1.2 x lOa spores/ml,Psychoda alternata is thus specificityof Clostridium bifermentar?^rsero- more susceptlbletoC.b.m. than most ofthe mos- var malaysia towards nematocerous Diptera. quito species.As the reaction of Psychodaalter- Apart from Culicidae larvae, only Psychodaal- nata is similar to what has been observed with ternata and Chaoborus crystallinus show some Culicidae larvae, it can be postulated that the reactionto C.b.m.application. death of Psychodalaruae is also due to a direct The bioassayswith le. aegypti (Bora-Bora), influence of the sporal toxin. With this result, An. stephensi,and Cx. pipiens confirm the high C.b.z. showssome similarity to B.t.i. as far as susceptibility of mosquito speciesdescribed be- the host range is concerned.Both show fore (de Barjacet al. 1990,Thiery et al. 1992b). (apart from their toxicity to mosquito larvae) The most susceptiblespecies was An. stephensi, toxic effectson Simuliidae and Psychodidaelar- followed by Ae. aegypti.Culex pipiens was about vae (de Barjacet al. 1981,de Barjacet al. 1990, 5 to 6 times lesssensitive than Ae. aegypti.These Beckerand Margalit 1993).The susceptibilityof results confirm those observedby other authors the latter group is interesting in view ofits close (Thiery et al. 1992b).This is true at least for An. relationship to the phlebotomines, representa- stephensiand Cx. pipiens, which show much the tives of which are known to be important disease same LCrolLCro values as the strains used by vectors (the leishmaniases)in the tropics. those authors. In contrast, the Ae. aegypti strain The effectsof C.b.m. on Chaoboruscrystallinus (Bora-Bora)used in theseexperiments was more are not easyto interpret. Five to 6 days postin- sensitive than the Bora-Bora strain Thiery et al. fection, significant mortality rates are observed tested (LCro: 3 times lower and LCno: 9 times amongthe larvae,which (astests with autoclaved lower). The differencein susceptibility might be material show) are not causedby altered water due to a genetic changein the Bora-Bora strain conditions (high turbidity, etc.), but are linked in different laboratories over a period of time. with the applicationof C.b.m.spores. However, The experiments with several nontarget or- ganisms show that, apart from Psychodaalter- nata and Chaoborus crystallinus, they are not a Morawcsik, J. 1983.Untersuchungen zur Wirkung affectedby C.b.m.,even at high concentrations. von Bacillus thuringiensis var. israelensisauf aqua- This is true for Planorbisplazrorbrs (Pulmonata), tische Nontarget-Organismen.Dissertation. Univer- Asellusaquatlcrzs (Isopoda), Daphnia pulex (Cla- sitat Heidelberg. Mencn 1994 C tosrnntuu anra,uz yr,lNs SEnovm. MALAvSTA 55 as high bacterial coircentrations are required to vicidal effect on mosquitoes. Res. Microbiol. l4l: cause elevated mortality rates and as the reaction 72t-"133. of the larvae is rather slow, the experiment does deBarjac, H., I. Largetand R. Killick-Kendrick. 1981. not suggest whether the effects on Chaoborus Toxicit6 de Bacillus thuringiensis var. israelensis, s6rotypeHl4, pour les phl6botomes, crystallinus are due to a very slow toxicity or to larves de vec- teursde Leishmanioses.Bull. Soc.Pathol. Exot. 74: spore germination in the larval digestive tract. 485-489. This study demonstrates the safety of Clostridi- de Barjac,H., M. Sebald,J.-F. Charles,W. H. Cheong um bifermentans serovar malaysia to aquatic and H. L. lee. 1990. Clostridium bifermentansse- nontarget invertebrates. rovar malaysia, une nouvelle bact€rie ana6robie pathog6nedes larves de moustiques et de simulies. C.R.Acad. Sci. Paris 310(Ser. 3):383-387. ACKNOWLEDGMENTS Duncan, D. B. 1955. Multiple rangeand multiple F-tests.Biometrics I l: l-42. This investigation received financial support Nicolas, L., J.-F. 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