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Zbl. Mikrobiol. 139 (1984), 49-55
[Department of Insect Pathology, Institute of Entomology, Acad. Sci., Prague, Czechoslovakia,and WHO Research Unit I, Kaduna, Nigeria]
Entomopathogenic Spore-Formers from Soil Samples of MosquitoHabitats in Northern Nigeria
J. WEISER and S. PRASERTPHON
With 2 Figures
Spore-forming bacteria, isolated from bottom soil samples of periodical mosquito-breedingplaces in Kaduna, N. Nigeria, were tested for mosquito pathogenicity. Culex pipiens autogenicuswas used for this screening. Five strains, all of the Bacillus thuringiensis group, were active in85 isolates from 32 samples. The isolates were from three different serotypes (H-14, H-5a5c, andH-8a8b). The activity, compared with the SIP standard, was in the known limits of B. thurinqieneis israelenei«.
Sporulierende Bakterien aus dem Bodensediment periodischer Stechmucken-Brutplatze in derUmgebung von Kaduna, Nord-Nigeria, wurden isoliert und auf ihre Pathogenitat gegeniiberStechmiicken getestet. Durch Verfiittern an Culex pipiens autogenicus-Larven wurden aus 85Isolaten von 32 Entnahmestellen 5 aktive Stamme gewonnen. Aile geh6rten zur Bacillus thuringiensis-Gruppe mit parasporalen Kristallen, davon 3 zum Serotyp H-14, israelensis, 1 zum Serotyp5a5c, canadensis, und 1 zum Serotyp 8a8b, morrisoni. Die 3 Isolate des H-14-Serotyps wiesen eineunterschiedliche Aktivitat auf, von LC50 gleich 500 Sporenjml bei Stamm 6-1 bis zu LC50 gleich500000 Sporenjml bei Stamm 8-4. Diese Stamme zeigten eine Lahmung (knock-down) der Larvenschon nach 3 Stunden, und diese fiihrte innerhalb von 24 Stunden zum Tode der Larven (knock-down nach Typ A). Die 2 anderen B. thuringiensis-Stamme fiihrten auf andere Weise zum Absterben der Larven (knock-down nach Typ B). Bei ihnen konnte Herzschlag noch nach 70 Stunden beobachtet werden, bevor sie starben. Beide Typen waren irreversibel. Die LC50-Dosis dieserbeiden Stamme betrug 3 X]06 Sporenjml. Die H-14-Stamme hatten parasporale Toxinkristallein Form von runden K6rnchen.
The detection of bacteria, pathogenic to mosquitoes in their aquatic habitat, isa rather complicated affair, at least, there are only few records of such isolates.Material can only be collected on visibly infected animals in the habitat, in larvaewhich are lying dead or dying on the bottom of the habitat. Bacterial infections withactive strains take only a few hours, as a maximum one day, then the effect disappears. Therefore the ephemerous stage of infection is difficult to find in nature.In breeding places, where mosquitoes appear every season, remains of dead animalsare concentrated on the bottom of the habitats. There they remain during the periodof the dry season and probably appear in the next year, infecting new victims. Thisis usually the case with spore-forming bacteria, which survive for long periods ofdrought and hot weather. In the reported case old habitats of Anopheles gambiae,which were periodically filled with water during the rainy season, were sampled fora search of spore-forming bacteria which were pathogenic to mosquitoes. The localities were in the system of pools, investigated by the junior author in the environmentof Kaduna, Nigeria.
4 ZbJ. MikrobioJ., Ed. 139
50 J. WEISER and S. PRASERTPHON
Material and Methods
Samples of bottom sediments in periodic breeding places of mosquitoes, mainly Anophelesgambiae and Culex quinquefasciatus, in the vicinity of Kaduna, Kaduna State, Nigeria, were collected during the dry season of 1980. The uppermost layer of 0.5~1 cm thickness of soil on thebottom, consisting mainly of red lateritic soil with minimum residues of organic materials, wascollected with a clean and germ-proof spoon by the junior author into sterile polythene bags(10 X 15 cm) and sealed with a vire closure (NASCO Whirl-Pak). The samples contained 20 to30 g of soil and were sent after closing to the WHO Collaborating Centre in Prague for evaluation.Samples were collected on 32 different localities.
In the laboratory the bags were opened and used for inoculating the nutrient agar plates. Asterile platin loop was dipped in a droplet of sterile water on asterile slide. The soil was taken fromthe depth of the sample, adhering to the moist loop, and stirred in the droplet. Dilution strokeswere made with this suspension on plates of Petri dishes. After 24 hours growth at room temperature, colonies of bacteria were typed and counted and the relative abundance of individual typeidentified. Spore-formers were selected and subcultured on new plates. After one week the isolateswere tested for mosquito pathogenicity. Smears of the same material were used for staining withGram and Giemsa for the identification of the morphology and sporulation of the germs. Pureisolates were maintained on nutrient agar slants and transferred to the Culture Collection ofEntomogenous Microorganisms for further taxonomic evaluation. (CCEB is a part of the Dept.Insect Pathology, Inst. of Entomology of the Academy, Prague, and' serves as depository for isolates of bacteria for the system of WHO Collaborating Centres.)
Mosquitoes used for testing were Culex pipiens autogenicus, a strain with continuous breedingmaintained in the Department. Informative mosquito tests were performed in plastic cups of15 ml size. Each of 30 L 2 of Culex were exposed to 3 X 106 germs/ml. The concentration was prepared from a stock with bacterial cells, taken from the surface of the colony, emulsified betweentwo slides, and counted in a haemocytometer. Behaviour of mosquitoes was recorded after 30 min.,24 and 48 hours, and a final count was performed after 72 hours. For the identification of LCso'groups of 50 L 2 of Culex were exposed to dilutions of bacterial suspensions in plastic cups with50 ml of water. Behaviour of mosquitoes was recorded after 30 min., 24 and 48 hours. Signs ofknock-down in mosquitoes, infected with Bacillus thuringiensis, were of two types:
In Type A larvae were fixed without motion with their siphons on the surface membrane of thewater in the cup. Usually they were concentrated in a group (pushed aside by yet active larvae,migrating from bottom to surface). They moved their mouth parts and their dorsal vein was beating. Such larvae died during 24 hours without recovery.
In Type B larvae were spread on the bottom, without need to take air, motionless, only thedorsal vein beating. They were activated by slight pressure of a forceps and tried to escape, butremained on the bottom. Such larvae died after 70 hours without recovery. On smears the morphological identification was used. Germs were assorted according to their sporulation and formationof parasporal crystals. No other identification was made during this phase of evaluation.
During this investigation 12 samples in the first series and 20 in the second wereinvestigated and isolates were tested on mosquito larvae. From the first series 61strains of spore-forming bacteria were isolated and from the second 24 strains. Of the85 solates five showed a relevant mosquito activity and four others showed only alow activity. Active isolates were of the following origin:
Isolate 3-7 was from a soil sample, collected in the Nassarawa village near Kaduna. Its grey, dry colony had later some rosy pigmentation, which remained alsoin the dry bacteria. In sporulating cells there were spherical parasporal inclusions.First signs of knock-down appeared 3 hours after exposure to Culex larvae. The KDeffect was of the A-type. In 24 hours all larvae were dead. The mode of action wasidentical with that known in H-14 serotype of B. thuringiensis. Identification of theserotype, which was performed by courtesy of Prof. DE BARJAC, Pasteur Institute,Paris, confirmed the identification of the isolate as a H-14 israeleneis serotype. Its
E n tom opa t hogenic Spo re -Form ers fr om Soil Sa m pl es 51
75 6Dosage loglml
~ 70~::; 50e:c: 30.,t~ 10
F ig. 1. Morta lity of L 2 of Culex p ipi ensaut ogenicus in suspensions of B. thuringiensis iso!. 84 , indicated in log /ml, r ead ing afte r 24 hours; 50 a nim a ls in 50 mlof suspension .
LCso was in the usual range of that serotyp e, as represented by the st rain isolated byG OLDBERG and MARGALIT .
Strain 6-1 was isolat ed fro III a soil sample collected at Dankande, loco 2, nearKaduna , from a road- side mosquito hab itat . Its colony was white wit hout any pigment ation , beside the spores there were inclusions. F irst signs of knock-down app eared3 hours afte r exposure of Culex larvae, t he LCso was 500 cells/ml (F ig. 1, 2). The knock-down was of the A-type. Serot yping identified t he st ra in as belonging t o serotypeH-14.
Strain 7-3 was isolated from a soil sample, collect ed in locality No. 3 at Dankandenear Kaduna. Its colony was white , dry , wit hout any pigmentation . Beside t he ovalspores there were parasp oral crys tals. F irst signs of immobilizati on of the larvae,ex posed t o t his bact erium, were visibl e afte r 30 minutes, but the pr ocess was progressi ng slowly and in 48 hours 66 % of the larvae were on the bottom with the type Bkn ock-down. Mortality after 72 hours reached 94 % at a dosage of 3 X 106 sporesjrnl.Serot yping ident ified the st rain as B. thuringi ensis, serotype 5a5c (close t o B . t . canadensis).
St rains 8-2 was isolated from sample No.4 fr om the locality Dankande near Kaduna . The colony was white, non-pi gmented , dr y , similar to the former . There wereparasporal crystals in sporulat ing rods. K noc k-down effect was not evident before24 hours, but only in 54 % of t he larvae, and no mort ali t y could be recorded before72 hours of exposure . At that t ime mort ali t y reached 72 % at a dosage of 3 X 106
F ig . 2. Mort al it y of L 2 of Culex p ipiensautogenicus in suspension s of B. thuringiensis isol. 61, reading afte r 24 h and 84,read ing a fter 48 h, in log jml. 50 a ni m alsin 50 ml of suspe ns ion .
~.;:: 70~:=s 50s~ 30<.>e,
3 fDosage log / ml
52 J. WEISER and S. PRASERTPHON
spores/ml. The knock-down effect was of the type B, with larvae on the bottom, moving only when squeezed in the forceps. Serotyping identified the strain as B. thuringiensis, serotype 8a8d (close to B. t. morrisoni).
Strain 8-4 was isolated from the same sample from Dankande No.4. The colonywas white, non-pigmented, dry. There were parasporal granules in sporulating rods.Knock-down effect was evident after 30 minutes, resembling the A-type. Mortality inthe orientative tests within 24 hours reached 100 %at dosage of 3 X 106 . In the dilutionseries the LCso was 5 X 105 germs/ml in readings after 24 hours or 2.5 X 103germs/ml
in readings after 48 hours (Fig. 1, 2). Serotyping identified the strain as belonging toserotype H-14. Strains with low activity were not tested in further experiments.Among the isolates there was no strain with spores, morphologically related to Bacillussphaericus. Several strains, belonging to the B. cereus-tharinqiensis group, but notactive for mosquitoes, were not considered here.
Table 1. Spore-formers, isolated from the bottom of mosquito-breeding places near Kaduna, N.Nigeria, I
2 3 4 5 6 7
Nassarawa Bridge 1 20 10Nassarawa Bridge 2 10 10 20 1Nassarawa Village 1 30 1 5 1Nassarawa Village 2 10 1 30Dankande No.1 30Dankande No.2 20 1 20 1Dankande No.3 10 10 10
Dankande No.4 20 5 5 3Sobawa No.1 10 2 3Sobawa No.2 ;30 1DIe No.1 20 2 15DIe No.2 10 1 3 1
Germs from isolates were compared as 24-h and 5-day colonies, sporulation was studied on smears.Types 1. White, dry, heavy cover (B. cereus group).Types 2. B. mycoides (no isolates prepared).Types 3. B. thuringiensis-type.Types 4. Gray, soft, shiny colonies.Types 5. Semitransparent, pink, glistening colonies.Types 6. White icy, soft border of colonies.Types 7. White, dense, oily colonies.
This investigation was performed to demonstrate the simplified ways of isolationof active bacterial strains, affecting mosquitos from soils. The suggested methods canbe used under conditions of an expedition without sophisticated equipment. The isolations could be repeated and isolates could represent hundreds or thousands of germswith a fair chance of producing active strains.
In this investigation mosquito activity was the main criterion, and further identification of isolates was not necessary, except in active strains. This type of search canbe performed under field conditions, lacking most of the special microbiologicalequipments. Preparation of nutrient media from dry stock and the use of pre-sterilizedplastic Petri dishes urges of the set of specialized microbiological equipment only anautoclave or an ordinary large pot for steam sterilization.
E n t omopa thogenic Spore- Formers from Soil Sa m ple s 53
T able 2. Spo re-F ormers, iso lated fr om t he bottom of m osqu it o-bre eding pl aces near K aduna , N .Ni geria , II (Numbers of colon ies on pl a te)
Sample 2 3 4 5 6 7 8 9
1 152 1 113 3 114 4 95 8 6 1 26 1 2 17 2 8 6 68 5 109 2 1 11
10 3 1 1 311 8 1 7 212 6 2 713 4 2 7 214 16 5 3 415 16 2 616 1 6 2517 8 14 218 819 50 4 20 320 25 15
Ge rms isolates wer e co m pared as 24 -h a nd 5-day colon ies, sporu la t ion was studied on smears .Types 1. ' Vh ite, d ry, hea v y cover (B . cerew~ gro u p, I so1. 1, 3, 7, 12,17).Types 2. B . mycoides (no isolat e p rep ared) .Types 3. B . thul'ingiensis-typc.Types 4. Gray, soft , shiny colon ies (Iso1. 16, 21, 20) .Types 5. Semitra nspa ren t , pi n k, glistening co lon ies (Isol, 2, 4, 9, 18).Types 6. White icy , soft bo rder colon ies (I so1. 6, 8, 13, 14, 15, 23).Types 7. White , dense, oi ly colon ies (Isol. 10, 24) .Types 8. Whit e, wa tery bli s ter , later paper -dry (Isol. 5, 11, 19, 22) .Types 9. Tea- b rown, undula t ed border (I so l. 18).
Strains t o be scr eened for activity against a given pest and their determinationshould be performed only on ac t ive isolates. The t est animal may be change d fromcase to case (mosquito, blackfly , snail, caterpillars etc.). In this case an eva lua t ion ona Culex and standard mater ial with the IPS 78 of B . thurinqiensis israelensis was usedintently . A comparison of the susceptivity of different strains of A . aegypti t o thestandard (DEMPAH and Coz 1979) showed a vide var iabilit y in response. The samemay be t he case with local strains of Culex pipiens auioqenicus. H owerer the use of alocal strain does not t ak e the risk of an escape of an important vector of infectionsin the tropi cs and subtro pics .
The cha nce of isolating an active st rain in the first or successive samplings dep endson the size of samples . Plastic bags, fresh from the producer , are sterile in side and maybe used in t he fi eld everywhere as an ea sy-to-handle ster ile container for storage ofsamples. Our samples were collected in dry temporary po ols wit h a very poor st rat if ication of t he bottom layers and with a few remains of organic materi als. The mi crobial fl ora was rather uniform and poor. Spore-formers were more common than nonspore-forming germs. Strains of the Bacillus cereus group were most common, followedby Bacillus mycoitles . Other germs were less common . Bacillus sphaericus did notoccur among the isolates, although it was present in the region. It took anotherecology of distribution.
54 J. WEISER and S. PRASERTPHOK
Table 3. Orientative tests of mosquito activity of 2-week-old colonies of active isolates from soilsfrom mosquito-breeding places in Kaduna, Nigeria. Tests on 2 X 30 L 2 Culex pipiens autogenicusin plastic cups with 15 ml water. Concentration 3 X 106 sporesjml
Isolate 30 min 24h 48 h 72 h
Locality A KD D A KD D A KD D D
Series I56 isolates 100 100 100Isol. 3-7 100 100 100 100Nassarawa V.Isol. 6-1 10 90 100 100 100Dankande 2Isol. 7-3 28 72 52 48 34 66 94Dankande 3Isol. 8-2 100 46 54 10 84 72Dankande 4Isol. 8-4 48 52 100 100 100Dankande
Series 220 isolates 100 100 100Isol. I 100 90 10 90 10 10Isol. 3 100 90 10 90 10 10Isol. 6 100 85 15 85 15 15Isol. 7 100 80 20 80 20 20
A = active, KD = knock-down, difficult motions, D = dead.
Table 4. Mortality of Culex pipiens autogenicus L 2, exposed to different concentrations of isolate6-1 and 8-4. 2 X 50 animals in plastic cups with 50 ml suspension
No. Isolate 30 min 24 h 48 h
Concentration A KD D A KD D A KD D
I :1 X 104 100 1002 I X 104 70 30 1003 5x 103 90 10 1004 2 X 103 100 1005 I X 103 100 10 90
6 5 X 102 100 35 65
7 :1X 102 100 71 29
8 I X 102 100 76 24
I 3;< 106 50 50 6 4 90
2 I X 106 90 10 16 8 76
3 6 X 105 100 30 70
4 3 X 105 100 90 10
5 I X 105 100 86 14
6 6 X 104 100 96 4
7 3 X 104 100 99 2
8 I X 104 100 100 100
9 5 X 103 100 100 5 95
10 2 X 103 100 100 61 39
II I X 103 100 100 93 7
Results in %, A = active larvae, KD = knock-down, D = dead.
Entomopat.h ogenio Spore-Formers from Soil Samples 55
Serological identification of the isolates with mosquito act ivit y , which was performed by Dr. DE BARJAC , confir med in th e cases of st rains 3-7, 6-1 , and 8-4 their serologicalidentity with serotype H-14. The isolates originated fr om different localities and mostprobably they represent onl y a minute re-isolation of already wide-spread ba ct eria.The active st ra ins , in spite of th eir serological class ificat ion in one group of H-14,possess different in secti cid al ac t ivities, and these could be fur th er developed. Duringthe evaluat ion of these stra ins it became ev ident that any dr ying (e.g. , t he acetonmethod , proposed by D ULMAGE and RHODES (1971) cha nges the flotation abilit y ofindividual germs . Therefore , fresh sporulated materials were used for testing, and t heirconcentrat ion in suspens ions was checked by counti ng of spores in a haemo cytometerin two su bsequent dilutions. Tests with the same t echnique are compara ble. Underthese cond it ions, the differen ces in ac t iv ity of 6-1 and 8-4 sho uld be noted.
The st udy of two other less efficie nt isolates, 7-3 and 8-2, showed evidence of somemosquito-pathogenicity in sero ty pes, which are less com mon mosquito pathogens.It was interesting to find there a different type of kn ock-down of larvae between3 to 70 hours of exposition t o infection. To some extent that may reflect a differentmode of action of individual microbes. These strains are poor mosquito killers, butthey may find active target s in ot her in sects. The experimental isolations from soilsshow the range of chance of local collect ors to find acti ve st ra ins.
A ck n o wl ed g em ent
The a utho rs wish t o ex press thei r t han ks for the det e rminat ion of th e ser o types of the isola testo Dr . DE B ARJAC, H ead of t he B. tliurinqiensis centre of t he Pas teur Insti tute, Paris . This in vesti gation received financi al sup por t from U N D P / World B ank/\VHO Specia l P rog ramme for R esearch a nd T ra in in g in Tropi cal Disea ses .
DE~IPAH, .1., a nd Coz, J.: E ssa is de Bacillus t hu r ingiensi s isra elensi s su r les moust iques. "VHO /VBC/7 9.7 21, rn irn, doc. , 1979.
D ULMAGE, H . T ., and RH ODES, R . A .: Produc t ion of p a thogens ill a r t if ic ia l m ed ia . In : Micro b ialContro l of Insec t s a nd Mites (H . D . BURGES a nd N . \ V . H USSEY, eds .). Academic Press, L ondon1971, pp. 507-540.
Au t hor 's a dd ress:
Dr. JA RQSLAV "VEISER, Depar t men t of Insect P athology, Inst itu te of E n to mology of the Czech oslovak Academy of Sciences, os - 16000 Prague, Cze choslovakia .