ARTHROPoDS 47 MAROH 1990 B. spatpwcus AND NONTARGET
AND CONTROL OF CULEX PIPIENS BY BACILLUS SPHAERICUS ROLE Or, NoNfencET ARTHROPODSIN ITS RECYCLING rno J' COZI S. KARCH,' N. MONTENY,I J' L' JULLIEN" G' SINEGRE' in a water treatment Barillus sphnericuswas used to control Culer Pipien's.bre^edins ABSTRACT. h/a'e) of commgr:illl near ruro'tp"ir'rff, ri".". ro"lt.L"t-""i. *i* n'titers/ha.(3'6 settlins basin ir'" larvalpopulation of cr. pipien'swith 50-600 ;;;i'r;fr";:;""ii.;Liiiiiiijii"r-W*t"i".'i'"a*"a",r;#;;;;i;i;;;;;e;;li" rast th. t,""t"a i;;i"*;iih;ivater prant14 days-fo[owing the ffiilil;;;. place the water surface where treatment. Natural ,..V.iittg oilfte bacteria *"i .tt.*" to take -at forms of B' sphaericusmay be linked sermination of sporeswas evidenced.The appearancu-oi""!"i"tit" ?; ,il;;;c;iri...grt r"*"t guts of severilhlter-feeding arthropods'
spp' in INTRODUCTION cies found in association with Culer natural breeding sites. Two bacteria, Bacillus sphaericusNeide and B. thuringiensrs H-14, have been introduced MATERIALS AND METHODS commerci;lly and used as microbiological con- trol agents against mosquito larvae.^Bac.ill.us Mosquito breeding sites.'A regional program sphaeiicushas a narrower spectrum of activity for moiquito control is carried out every year in tian B. thuringiensis H-14, but it seemsto be the south of France. Between June and August more active ag;inst larvae of certain gener-aof 1988we were able to join the staff and to study culicine speci;s (Sin6gre et al' 1980, Davidson the dynamics of a Culex pipiens population in et al. 1981,Hertlein et al. 1981,Lacey and Singer the settling basin of a wastewater treatment 1982).Its sporesmay persist for a long time in plant situated at Fabregues,in the areaof Mont- the environment (Hertlein et al. 1979,Mulligan pellier.The basin is approximately1,60O m'and et al. 1980,Des Rochersand Garcia 1984)'An izO-rSO cm deep. A large variety of aquatic poten- attractive feature of this organism is its organismsoccur in associationwith Cr. pipiens tial to recycle in the larval gft of Culex spe-cr9: lar"vae(Table 1). The larval population of Cr. (Karch and Coz1986, Charles and Nicolas1986) pipiens is found under the vegetation which by .po." germination, vegetative multiplication grows 1-2 m in the width along the periphe-ryof andiesporulation after the death of the larvae' Ihe basin where the depth never exceeds40 cm. This recycling in the larval habitat may lead to This vegetation contains duckweed, algae, ar- the control of more than one generationof mos- thropod- and zooplankton' Three sampling sites ouitoes.Hertlein et al. (1979)recovered spores were chosenin the plant: site 1 correspondsto of B. sphaericus9 months after treatment in a the water supply of the Iagoon with permanent roadsideditch in southwestFlorida and Hornby replenishment, site 2 is lateral to the water flow et al. (1984) reported that it persisted several "nd.it" 3 is the point (directly opposite to 1) months in sewageplants. More recently, Karch where water dischargesby overflow into a sec- et al. (1988) reported a A-yearpersistance in a ond basin.Water Ievelremains constant; it cor- natural pond and discussed the possible role respondsto the overflow line. The secondbasin nontarget fauna could play in this proce-ss.In a (2,i00 m') remained untreated and was used as laboratiry study, Karch et al. (1989) observed control for nontarget organisms until mid-July germination of B. sphaericusspores in the larval when Iocal authorities were urged to put an end guts of Chironomtts sp. and Culiseta annulata to mosquito annoyance in that area. Observa- (Schrank), thus suggestingthe possibility that tions unfortunately were stopped at the end of these speciesmay have some influence in the July with the emptying of the first basin for mainte;ance and seasonal occurrence of B. cleaning. sphaericus. The main physical-chemicalcharacteristics of This study investigated the dynamics of a water in the treatment plant (and in the control Culex pipiens Linn. population after treatment plant) in June are summarizedas follows: acidic with Barillus sphaericusand the ability of the organic matter 64.5 (43.5) mg/O2/liter; alkaline arthropod spe- bacteria to recycle in nontarget organic matter 30.5 (15.9) mg/O z /llte\; -ammo- niacal nitrogen 36.0 (36.0)mglliter; NOz- 0.25 (0.20) mglliter; Cl- 106'5 (88'7) mglliter; CO3 H- 475.5(512.4) mglliter; pH 7.5 (7'8);pollution l Medical Entomology, ORSTOM, Department of index 212.2 (201.8)' Water temperature was 93140-Bondy, France. 70. Route d'Aulnay, taken at 0900 h with a thermometric sonde 20 2Entente Interd6partementale pour Ia Demousti- (E.I.D.), P.O. Box 6036, 34030-Montpellier, cm deep. cation sphaericus France. Bacterial treatments: Bacillus 48 JouRNlr, oF THE AlrrnrceN Moseurr:o ColrrRor Assocrarron VoL.6, No. I
il tl t.+ + + + strain 2362 (Vectolex@,from Abbott Laborato_ ll j++++ ries,U.S.A., containing tl vro '++++ 1.2x 10espores/ml) was il .39 usedat a final concentration of n.ti titers/tra (S.e il lb/acre,4.8 1012 tl RX ++t*# spores/ha).The treatmeni was tl fi+++' '+++ applgq with a pressurized hand .I sprayer in a 2 @tl m width around the periphery of th-ewater cO tl treat_ oll ment plant. -tl cd >1ll | | rti Sample collection:Samples (200 ml) of water 5tl TH f -il and arthropods were taken from the surface :s@ around trtl \,: 1-+ the basin throughout the observation 6ll I r+++ period at least once a ol ++ week and before each ,tl treatment. For the bacteriological analyses, 'ti sampleswere taken 2 h after each treatment o tl :^ +++++ at o tl @d +++++ the 3 sites,both on the surfaceand at the bottom 6 tl +++++ F tl s3 of the treatedzone. htl ^l Bac_teriolngical kI counts: After harvesting,water € +++{+ o tl r\ +++++ samplesand 10 arthropodsground in i'ml of =tl +++++ sterile water were split in 2 subsamples,one of !tl which was submitted to heat shock (-g0.Cfor q j5 1Z =il3 lo I t+++ min) to kill vegetativecells and nonspore_form_ qll t3c t> ing bacteria. B 4; lcd Then, 0.1 ml of eachsu6sample or 3llE E^ t! r++++ a dilution of it was plated <=l l-i on MBS solid ,rrediu* IY (Kalfon et al. 1983)in Petri dishes .:|i : o t= containing cd 100mg/liter of streptomycin. The colony counts ]ilB +++++t,' aiilq IT were recorded after a 24-h growth period at i .ill= T 35'C. Identification of B. sphaericuswas con_ €ilx -r++++o firmed by morphologicaland microscopicobser_ N BII (, I vations. In the 3 sampling sites of tht hgoon, 5ll.! I t- the concentration of B. sphaericuswas evaliated UIIF | | t++T by the mean '!ll + of 3 bacteriolosicalcounts. E B^ studies:Tests wereperformed all 4 . ,Laboratory with @il o *o (, \o filter feeders (Daphnia 6ll a | | t++ I pulex and Cypris sp.) to tr) I determine their ability to ingest and releaseB. boil a -:? I i sphaericus trll I otF t+t++ 'a as already described elsewhere for RY >l Culisetaannulata 5 ll c,l hl ; and Chironomussp. (Karch et o tl ts tl H al. 1989).Several -iO I r+++ samplesof 10 orginisms were placed :tl in 10 ml of a suspensionaf0.1 mg/liter.-li. tr ll After a contact period 5tl 6 @ of 2, 5 and 24 one €ll t++++ samplewas removed, rinsed with sterile @tl F a++++'++++ distilled o tl water, diluted 100-fold o tl and treated as above for a tl bacteriologicalcounts. After 2 h contact,2 other al . ++ sampleswere removed,rinsed and placedin 10 o ll + i i++ Q.q Tii; N ml sterile if water. Two and.24 h latei, they otl E were F tl submitted for bacteriological counts as well. ! tl a Counts were also made in 6ll sE the sterile water itself qtl I r+++ by plating 0.,1ml. o tl o tl € trtl @ 6 tl 2i8 +++++ o RESULTS trtl 6 o .Efficacy o/ B. sphaericusfor the control of Cx.
1050
900
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E 300
z 150
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1050
900 o o N 750
o 600
450
300
z 150
0
1050
900.
o o 750 N 600
450
300
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0 .-"JULY--. (2 days)
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o
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JUNE----
Fig. 1. Density of preimaginal population of Culex pipiens in the 3 sites of a lagoon treated with Bacillus sphaericusand mean water temperature. 1a : site 1; 1b : site 2; 1c : site 3 and ld : water temperature at 0900 h. Blank : lst and 2nd instars; punctate: 3rd and 4th instars; black : pupae;Ti : treatment number i. 50 Jounnll oF THEAunnrceN Moseurro ConrRor,AssocrlrroN VoL. 6, No. 1
efficacy observed at site 1 under treatment was in both ponds. Egg rafts were observedamong due to leaching by water replenishment but not the vegetation generally blown to one side bi to the absenceof the product or to its ineffec- the wind. tiveness.Oviposition occurred at the same level Arthropods found in the treatment and control
E :t o
ct) I
E ()
o, o
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---JUNE__-__
Fig. 2. Total number of bacteria and heat-resistant spores of B. sphaericus 2362 present in the 3 sites of a lagoon treated against Culex plpiens. Samples were taken at the surface (blank : total bacteria; black : spores) or at the bottom ofthe pond (punctate : total bacteria; stripes : spores); 2a : site J.; 2b : site 2 and 2c : site 3. MARCH 1990 B. spHAERIcus AND NoNTARGoTAnurnopops
6 o
Fig. 3. Total bacteria (blank) and heat resistant spores (black) of Bacillus sphnerirus present in some arthropods found in breeding site of Cul.expipiens; "" : no bacterium found in the sample;filter feeders:3a : Daphnia pulex and 3b : Cypris sp.; predators: 3c : Cleond,ipteriunr and 3d : Corixapunctata: bottom feeders: 3e: Chironornussp. JounNlr, oF THEAunnrcnx Mosqurro Cox.rnor,Assocrarrorl VoL.6, No. 1 ytond.s:Table 1 gives an evaluation of the popu- per ml. It was considerablyhigher at the end of lations ofsome arthropods found in the breidine the experiment where it ranged from about b00 sites under study. The abundancelevels of thes6 to 2,000spores per ml. This increaseis due not aquatic arthropods is nearly similar whether or only to sedimentation,as the concentration also not B. sphaerlcusis present.Moreover, the num- increasedat the surface. bers of all species increased gradually in the Countings of colony forming units (CFU) treated portions as well as in the controls after thermic shock of the subsamplesreveal throughout the observation period. In this re- that a nonnegligible amount of bacteria may be gion of France, other Cx. pipiens breeding sites vegetativeforms. This is observablemost of the have been treated with chemical insecticides.It time near the surfacebut to a lower extent also has been our observation that insecticides,par- on the bottom. ticularly chlorpyrifos, kill nonselectivelyand in Fate of B. sphaericusin nontarget arthropod so doing eliminate predators of mosquito larvae species:The different arthropod speciespresent which might otherwise contribute to their nat- in the breeding site could contribute to the dis- ural control. persion of B. sphaericusin the treated area, as Fate of B. sphaericus in the breeding site: severalof them ingest bacteria. We followed the Figure 2 shows that the concentration of B. presenceof B. sphaericusin someof them col- sphaericuswas nearly similar in the 3 sampling lected in the settling tank. Results (Fig. 3) show sites throughout the course of treatments. The that filter feeders (Daphnia sp. and Cyprls sp.) sedimentation of this bacteria leads to its dis- ingest B. sphaericusas long as the bacteria re- appearancefrom the feeding zone of the mos- mains present in their feeding zone which is quito larvae as early as the first 48 h following closely related to vegetation decaying in the eachapplication. water column. A small percentageof B. sphaer- The concentrationof the bacteriawas as fol- icus in their guts are vegetativeforms. The same Iows: processis observedfor the bottom feeder Chi- 1) On the surface:At the beginning of the ronomussp. which feedsessentially on bacteria. experiment, the number of spores 2 days after As they filter continuously, it is not possible to spreading ranged from about 0 to 50 per ml, determine where germination took place. A lab- while the bacterial population was 50-600 sp./ oratory test substantiates the idea of germina- ml 2 weeks after the 4th treatment. The disap- tion inside their guts (Table 2) and spreadingof pearance of the Iarval population observed in vegetative cells in the environment. The 24-h Fig. 1 is consistent with the results obtained by contact result is not really significant because Hornby et al. (1984)and thoseof Nicolas et al. the suspensionhad been exhaustedby continu- (1987)which show that a spore count ranging ous feeding.For predators (Cleondipterium and from 100 to 500 per ml is sufficient to control Corixa punctata), B. sphaericusis found when mosquito larvae. present in their prey. The number of CFU is 2) At the bottom (0.40m in depth): From the probably related to the number of larvae they second week following the first treatment, the ingest.However this would require an additional number of sporesranged from about 100 to 200 quantitative study of their feeding habits.
Table 2. Results ofa laboratory test concerning the number of Bacillus sphaericus ingested or releasedby 2 filter feeders in 24 h. t:-.:1"^:1* rn B. sphaericussuspension water --.:t-l-t:t-":::,1.1i,,water/lndrvloual-
Species 2h 5h 24h 2h 24ll, 2h 24h Total bacteria 8,100 15,700 100 7,000 100 1,630 1,000 Daphnia pulex Heat resistant 7,900 10,300 0 6,500 0 L,570 520 spores % sporesin to 97.5 65.6 92.8 96.0 b2.u tal bacteria Total bacteria 8,900 8,200 1,500 6,300 100 530 1,540 Cypris sp. Heat resistant 8,100 7,100 0 5.700 200 360 1,290 spores % sporesin to- 91.0 86.6 90.5 67.9 83.8 tal bacteria I All results were obtained with 100-fold dilutions of samples. ' Results were obtained from 0.1 ml of sterile water containing 10 individuals/lO ml. Mencu 1990 B. SPHAEilICUS AND NONTARGET ARTHROPODS 53
Nevertheless, the disappearance of mosquito ries. This study was supported by the UNDP/ larvae will diminish their participation in B. World Bank Special Programme for Research sphaericusrecycling. and Training in Tropical Diseases.
DISCUSSION REFERENCES CITED Our observationsare consistent with those of other workers (Davidson et al. 1984,Mulla et al. Charles,J. F. and L. Nicolas.1986. Recycling of Ba- 1984) and show that this bacterium used as a cillus sphaerian 2362 in mosquito larvae: a labora- Microbiol. (Inst. Pasteur) larvicide against mosquitoesin aquatic environ- tory study. Ann. 131B:191-201. ments does not disturb the natural equilibrium Davidson,E. W., A. W. Sweeneyand R. Cooper.1981. among the various populations of the natural Comparativefield trials of Bocillus sphaericusstrain fauna. The most important point is the role 1593 and Bacillus thuringiensis var. isroel'en'siscom- likely to be played by this fauna in relation to mercial powders.J. Econ. Entomol. 74:350-354. the larval population of. Cx. pipiens. On one Davidson,E. W., M. Urbina, J. Payne,M. S. Mulla, hand, some species (Cleon dipteriurn, Corixa H. Darwazeh,H. T. Dulmageand J. A. Correa.1984. punctata and Nepo cinerea) are predators or Fate of Bacillus sphnericus 1593 and 2362 spores killers of mosquito larvae and their activity is usedas larvicides in the aquatic environment. Appl. additive to that of the bacterium; a phenomena Environ. Microbiol. 47:125-129. Des Rochers,B. and R. Garcia. 1984.Evidence for which could not have been observedwhile using persistence and recycling of Bacillus sphaericus. chemicalinsecticides. On the other hand, several Mosq.News 44:160-165. species(Daphnia sp., Cypris sp.) contribute to Hertlein, B. C., J. Hornby, R. Levy and T. W. Miller, redistribute the bacterium in the water column. Jr. 1981.Prospects of spore forming bacteria for Therefore, this study suggeststhat periodicity vector control with specialemphasis on their local of treatments with B. sphaericuswill dependon productionpotential. Dev. Ind. Microbiol.22:53-60. the density of the nontarget arthropod species. Hertlein, B. C., J. Hornby, R. Levy and T. W. Miller, Considerable natural fluctuations will be ob- Jr. 1981. Prospects of spore forming bacteria for served in a breeding site, particularly if this vector control with special emphasison their local productionpotential. density is high. The 4 treatments applied at 4 Dev. Ind. Microbiol.22:53-60. Hornby, J. A., B. Hertlein (4 x C. and T. W. MiIIer, Jr. liters/ha each 5 x 1012spores/ha) were 1984.Persistent sporesand mosquito larvicidal ac- sufficient to control emergenceof adult Cx. pi- tivity of Bocillus sphaerictts1593 in well water and piens during at Ieast 14 days under the present sewage.J. GeorgiaEntomol. Soc.19:165-167. conditions. Unfortunately, observations were Kalfon, A., I. Larget-Thiery,J. F. Charlesand H. de stoppedbefore knowing how long the recycling Barjac. 1983. Growth, sporulation and larvicidal could be effective. Mulla et al. (1988) reported activity of Bacillus sphnerirus.Eur. J. Appl. Micro- 99% control during 49 days with a single treat- biol. Biotechnol.18:168-173. ment of a wastewaterfacility at 4.48 kg/ha (3.4 Karch, S. and J. Coz. 1986. Recycling of Barillus pipiens (Diptera- X 1014spores/ha) which in fact represents 10 sphaericusin dead larvae of Culex Culicidae).Cah. Entomol. quantity (2 x O.R.S.T.O.M.Ser, Med. times the 1013spores/ha) which Parasitol. 24:47-43. yielded 100-94% reduction for only 4-7 days in Karch, S., N. Monteny and J. Coz. 1988.Persistance a study reported earlier (Mulla et al. 1984) or de Bacillus sphaericusdans un gite d moustiques4 the quantity applied in the present study. The ans aprds son introduction en vue de lutte biolo- cumulative effect of treatments resulting in long gique. C. R. Acad. Sci. Paris, 307, Ser. lll:289-292. lasting mosquito control will depend on several Karch, S., N. Monteny, C. Toneatti and J. Coz. 1989. factors such as the type and density of species Intervention de I'entomofaune dans le recyclageet present (carnivorousversus filter feeders),the le potentiel d'action du complexe cristal/spore de physical-chemical characteristics of the water Bacillus sphaericuslarvicide anti-moustiques. Cah. O.R.S.T.O.M. Ser. Entomol. Med. Parasitol. In that might allow bacterial reproduction, the press. presenceof vegetation and the possible attrac- Lacey, L. A. and S. Singer. 1982. Larvicidal activity tion of the habitat for mosquito oviposition. of new isolates of Bacillus sphaericusand Bacillus thuringiensis (H-14) against anopheline and culi- ACKNOWLEDGMENTS cine mosquitoes.Mosq. News 42:537-543. Mulla, M. S., H. Axelrod, H. A. Darwazeh and B. A. We are very gratefulto Mr. Vigo from E.I.D. Matanmi. 1988. Efficacy and longevity of Bacillus for his contribution to the physico-chemical sphaericu.s2362 formulations for control of mos- quito larvae analysesof water samples.We alsothank Dr. J. in dairy wastewater lagoons. J. Am. Mosq. Control Assoc.4:448-452. Cousserans,Director of E. I. D., Dr. G. Chauvet, Mulla, M. S., H. A. Darwazeh,E. W. Davidson,H. T. Chief of Department of Medical Entomology Dulmage and S. Singer. 1984. Larvicidal activity and Dr. Tibayrenc (ORSTOM, Montpellier) for and field efficacy of Bacillus sphaericus strains useof the facilitiesat the Montpellier laborato- against mosquito larvae and their safety to nontar- JounNer, oF THE AMERTcANMoseurro CourRor, AssocreuoN VoL.6, No. 1
get organisms.Mosq. News 44:336-842. 2362spores in Culexquinquefasciatus breeding sites Mulligan, F. S. III, C. H. Schaeferand W. H. Wilder. in West Africa.Appl. Microbiol.Biotechnol.25:341- 1980.Efficacy and persistenceof Bacillussphaerirus 345. and Bacillus thuringiensis H-14 against mosquitoes Sin6gre,G., B. Gaven and G. Vigo. 1980.Evaluation under laboratory and field conditions. J. Econ. En- preliminaire de I'activit6 Iarvicide de la souche1593 tomol. 73:684-688. de Bacillus sphaericusvis-A-vis de quatre espdcesde Nicolas,L., J. Dossou-Yovoand J. M. Hougard.1g8?. moustiques du littoral m6diterran6en francais. Persistence and recycling of Bacillus sphaericus wHo/vBC.762.