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. <tl i56 rt+++ pipiens .tl ' '+++ under field conditions: Four treatments *ll tie using B. sphaericus oil were sufficient to limit the =ll preimaginal population of Cx. pipiens during the dtl .a f++++ Fll i OT O '++++ observation period of June-July 1988. The in- lll '6r YA terval between 2 treatments was chosen in re- tl -flPG PXE> lation to the occurrenceof a new larval popula- tl - --._ p6 tion of Cx. pipiens. Figure tl la 1 shows thal the :,-€ mxiRid6 dll effectiveness tl E:A of the product varies with the sam- il Fq" PP 9 >> pling sites. This difference II is due to their loca- --bhb tion with regard to the water flow. The lower 49 MARCH 1990 B. spHAERIcus AND NoNTARGET ARTHRoPoDS 1050 900 o o 750 N 600 o 5 ! 450 E 300 z 150 0 1050 900 o o N 750 o 600 450 300 z 150 0 1050 900. o o 750 N 600 450 300 z 150 0 .-"JULY--. (2 days) 30 o o l- o (E = 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.