Computer based evaluation of laboratory data in the rearing of tsetse flies 29

Berechnungen wird ein Computerprogramm empfohlen, das auf Wunsch von den Autoren zur Verfiigung gestellt wird.

References

COALE, A. J., 1972: The growth and structure of human populations, Princeton: Univ. Press. JORDAN, A. M.; CURTIS, C. F., 1968: Productivity of Glossina austeni Newst. maintained on lop

KEYFITZ, N., 1972: Population waves. In: Population dynamics. Ed. by T. N. E. GREVILLE. New

SOKAL, R. S.; ROHLF, F. J., 1969: Biometry. San Franzisco: Freeman. TIMISCHL, W. 1981: Optimization of productivity in the rearing of tsetse flies (Diptera,

Glossinidae). Z. ang. Ent. 91, 301-309. - 1982: Growth rates, optimal harvesting and related topics in the mass rearing of tsetse flies. In:

Operations Research in Progress. Ed. by G. FEICHTINGER and P. KALL. Dordrecht: Reidel,

eared rabbits. Bull. Entomol. Res. 58, 399-410.

York: Academic Press, 1-38.

301-310. - 1983: O n the time to stabilization in the rearing of insect populations. Submitted to Biom. J.

VAN DER VLOEDT, A. M. V., 1981: 2nd/3rd Quarterly report - Entomology. Seibersdorf Entomology Lab., Intern. Atomic Energy Agency, Vienna, Austria.

- 1982: Recent advances in tsetse mass rearing with particular reference to Glosszna palpalis palpalis (Rob.-Desv.) fed in vivo on Guinea pigs. In: Sterile Insect Technique and Radiation in insect control, 223-253, International Atomic Energy Agency, Vienna, Austria.

Anschriften der Verfusser: Dr. W. TIMISCHL, Abt. fur Mathematik in den Naturwissenschaften und Mathematische Biologie, Technische Universitat Wien, A-1040 Wien, ArgentinierstraBe 8; Dr. A. M. V. VAN DER VLOEDT, Joint FAO/IAEA Division of Isotope and RadiationeApplications of Atomic Energy for Food and Agricultural Development, International Atomic Energy Agency, P. 0. Box 100, A-1400 Vienna

National Research Centre, Dokki, Cairo

Mutation in relation to sporulation and potency of Bacillus thuringiensis vs. cotton pests

By H. S. SALAMA, M. S. FODA and M. SELIM

Abstract

A series of mutagenesis experiments was carried out using Bacillus thuringiensis vars. kurstaki HD-251 and entonocidus HD-635, to obtain auxotrophic mutants as a means for elucidation of possible relation between the endotoxin potencies vs. some cotton pests and certain nutritional markers. The auxotrophic mutants did not differ materially in their growth pattern and sporula- tion titers from their parent wild types. Furthermore, the potencies of the mutant endotoxins vs. Spodoptera littoralis and S. exigua were comparable to those of the wild types. Although the auxotrophic mutants were partially characterized and irrespective to their different genetic lesions, no specific correlation could be detected between mutation towards auxotrophicity and endotoxin prodJction.

U.S. Copyright Clearance Center Code Statement: 0044-2240/84/9701-0029 $ 02.50/0 2. ang. Ent. 97 (1984), 29-36 @ 1984 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-2240 / Intercode: ZANEAE

30 H. S. Salama, M . S. Foda and M . Selim

1 Introduction

Inspite of the great developments made in the various aspects of fermentation technology and production of various toxins formed by Bacillus thuringiensis varieties, there is practically no information yet available concerning the genetic make-up of the organism that would lead to an understanding of the genetic markers responsible for determining the potencies, specificities, host range, and the chemical structures of such toxins. Attempts have been made to reveal such genetic factors using different approaches including mutagenesis and transduction with specific bacteriophages.

The mutational approach has received more attention (LECADET et al. 1975; KUZMANOVA 1976). The sporulation, endotoxin formation and possible rela- tionships were also investigated through mutagenesis and isolation of sporeless mutants (NISHIITSUTSUJI-UWO et al. 1975; YOUSTEN 1978; JOHNSON et al. 1980).

In the present study, a different approach has been used namely the auxotrophic mutants, in the hope to reveal some information regarding the genetic markers involved in the biosynthesis of the endotoxins produced by Bacillus thuringiensis and their potencies vs. the two cotton pests, Spodoptera littoralis and S. exigua.

2 Material and methods

The two B. thuringiensis strains namely var. entomocidus HD-635 and var. kurstaki HD-251 that are known to have high potencies against major cotton pests (SALAMA et al. 1981) were selected. The auxotrophic mutants were obtained through mutagenesis of actively growing cultures of the two tested varieties of B. thurzngiensis using N-methyl-N-nitrosoguanidin mutagen. The survivors after exposure to 5 mg/ml mutagen for 15 min (less than 0.01 %) were plated first on complete medium (MI) which consists of the following in g: peptone 5, yeast extract 2, casitone 2, glucose 2, K, HPO, 8.7. This medium components were dissolved in 1000 ml of tap water and had a final p H = 7.5. Agar was added at 2 % concentration when required. After appearance of growth, the colonies were replicaplated on minimal glutamate medium, minimal glutamate-amino acids medium as well as complete medium (MI) agar plates. The minimal glutamate medium (pH = 7) has the following composition in gms.

Monosodium L-glutamate 7.5 K,HPO, 4.35 MgSO,. 7H,O 0.12 CaCI, 0.11 MnSO, . H,O 0.01 FeSO, . 7H, 0.01 Distilled water 1000 ml

The minimal medium or mineral salt-glucose medium has the following constituents in g: glucose 6, K,HPO, 4.4, MgSO, 7 H,O 0.25, MnSO H,O 0.03, FeSO, 7H,O 0.02, ZnSO, 7H,O 0.02, CaCO, 2 and yeast extract 2.

The fodder yeast medium was prepared by using dried fodder yeast of Succharomyces cerevisrue at 2 % concentration. For media fortification the following ingredients were added in g: glucose 6, yeast extract 2, potossium phosphate dibasic 4.4 and all were dissolved in 1OOOml tap water.

For growth, the growing cultures of the tested variety, were used to inoculate 500-ml Erlenmayer flasks each containing 50-100 ml of the medium understudy. The flasks were incubated on rotary shaker (100 rpm) at 30 "C for 3-4 days. The recovery of the endotoxin was described by SALAMA et al. (1981). The growth was estimated in term of optical density units (OD) at 600 mn. Sporulation yields of cultures were determined in appropriate dilutions of the cultures after pasteurisation at 65 "C for 10 min (DULMAGE 1970).

Standard colonies of S. littoralis and S. exgua were raised in the laboratory on semiartificial diet described by SALAMA (1970). The assay procedure proposed by DULMAGE et al. (1971) was adopted. The mortality percentage was calculated after 7 days of the incorporation of the tested

Mutation in relation to sporulation and potency of B. thuringiensis 31

formulation at a level of 500 pg/ml of the diet. For bioassay, 4 replicates were made for each experiment with 25 third instar larvae of each species representing each replicate.

3 Results and discussion

3.1 Nutritional requirements of the wild type strains

Before subjecting the original wild type cultures to mutagenesis their nutri- tional requirements were determined inorder to devise the selective defined media for their growth. With respect to var. kurstaki HD-251, no specific amino acid requirements could be observed. This particular strain grew well on mineral salt-glucose medium in the presence of 0.5 % L-glutamate as an organic nitrogen source. O n the other hand, the other var. entomocidus seemed to require specific growth compounds since it did not grow on the previously mentioned simple medium. Therefore, this strain was subjected to further studies. Table 1 summarizes the growth data obtained upon the supplementa- tion of the minimal medium with one or more of five amino acids namely valine, phenylalanine, methionine, threonine and leucine. Only in the presence of all those five amino acids in the minimal glutamate medium that appreciable growth of this variety could be obtained. Therefore, in the subsequent mutagenesis experiments, this five amino acid-supplemented defined medium was used as a basic medium for differentiation of wild type and the suspected auxotrophic mutants.

Table 1. Determination pf the nutritional requirements of B. t. var. entomocidus HD-635. Minimal medium (M.M.) was used supplemented with one or more of the following amino acids (1) valine (2) phenyl alanine (3) methionine (4) Threonine (5) Leucine. Growth was estimated in the liquid

medium after 3 days of aerobic incubation

Composition of medium Growth parameters OD 6000 nm pH of culture

M.M. only M.M. + (1) M.M. + (2) M.M. + (3) M.M. + (4) M.M. + (5) M.M. + (1) + (2) + (3) M.M. + (1) + (2) + (4) M.M. + (1) + (2) + (5) M.M. + (1) + (3) + (4) M.M. + (1) + (3) + (5) M.M. + (1) + (4) + (5) M.M. + (2) + (3) + (4) M.M. + (2) + (3) + (5) M.M. + (2) + (4) + (5) M.M. + (3) + (4) + (5) M.M. + (1) + (2) + (3) + (4) + (5)

0.06 0.14 0.14 0.14 0.16 0.13 0.11 0.18 0.12 0.28 0.19 0.18 0.11 0.12 0.12 0.13 1.56

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 5.0

Through chemical mutagenesis, some 25 auxotrophic mutants were derived from var. entomocidus whereas fourteen mutants were recovered from var. kurstaki HD-251. However, a number of those mutants has already reverted to prototrophicity of their wild type, thus, losing their auxotrophic characters

32 H. S. Salama, M . S. Foda and M . Selim

upon further transfers on the complete medium. This phenomenon was particularly evident in the var. kurstaki HD-251 where quantitative growth measurements showed that more than 50 % of the auxotrophic mutants reverted to wild type.

For further grouping of the auxotrophic mutants, the cultures were streaked on agar plates of three media namely minimal-glutamate, yeast carbon base-glutamate (obtained from Difco Laboratories, Detroit, Michigan) and YCB-glutamate supplemented with eight amino-acids, tryptophan, leucine, threonine, methionine, phenylalanine, histidine, valine, arginine in addition to three nitrogenous bases, i.e. guanine, adenine and thymine. The growth was examined after two days of incubation at 30 "C. Results show that none of the three media composition could support the growth of any of the auxotrophs derived from HD-635. On the other hand three strains derived from HD-251 namely 251-1, 251-2 and 251-103-1 could grow on YCB L-glutamate medium irrespective to other amino acid supplementations (table 2). These results suggested the possibility of vitamin requirements for those three auxotrophs.

Table 2. Extent of growth of auxotrophic mutants from B. t. var. kurstakiHD 251 on yeast carbon base YCB-glutamate agar plates supplemented with eight amino acids

Strain designation Growth on minimal Growth on YCB glutamate medium acar As such Supplemented

HD-251 wild type 251-1 251-2 251-3 251-13 251-103-1

++ ++

+ f f

++

++ ++ f - -

+

This assumption was confirmed upon streaking the auxotrophs on agar plates of five different supplementations of minimal glutamate medium as shown in table 3 where the two auxotrophic mutants 251-1 and 251-103, could grow well on minimal medium supplemented with vitamin mixture. The growth requirements of those two mutants was more pinpointed as shown in table 4 where they gave good growth upon supplementation of the minimal medium with four vitamins namely B,, B,, PABA, and nicotinic acid. Further characterization has shown that those two mutants required nicotinic acic for growth.

3.2 Sporulation and potencies of endotoxins produced by auxotrophic mutants

The auxotrophic mutants derived from HD-635 and HD-251 were grown under standard conditions on fodder yeast-basal medium. At the end of the growth period, the growth parameters were measured: sporulation titers were estimated and the endotoxin was harvested and assayed against S. littorulis and S. exiguu at a level of 500 pg/ml diet. The results obtained are shown in table 5. It appears that the sporulation titers varied markedly among the various auxotrophs. However, the sporulation yields did not exceed those obtained with the wild types. O n the other hand, the potencies of the endotoxins produced by those auxotrophs were in most cases similar to those of the wild type strains. So, the larval mortality in S. exiguu on using the mutants derived

Mutation in relation to sporulation and potency of B. thuringiensis 33

Table 3. Classification of auxotrophic mutants derived from B. t. var. kurstaki HD-251 with respect to their nutritional requirements. Four supplementations of the minimal medium namely vitamins, amino acids, nitrogenous bases and a mixture of them were tested with minimal medium

as a control

Growth on minimal medium supplemented with Strain tested As such Vitamin mixture Amino acids Pyriner + All

mixture pyrimidine 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

HD-251 wild type f + f + ++ ++ f f ++ ++ ++ ++ f f f f

- - f f - - + + ++ ++ f ++ - -

- - f + + - - - - 251-1 251-2 251-3 251-13 251-103-1

- - - - - - - - - - - - - - - - - -

- - - -

Table 4. Growth of auxotrophic mutants, on chemically defined synthetic medium supplemented with amino acids and vitamins

Strain Growth on minimal medium supplemented with designation None 4v 3 aa 4V i 3aa 5 aa

635-wt. 251-wt.

251-2

251-13

635-15 635-16

251-1

251-3

251-103

635-17 635-18 635-19

635-22 635-23

635-25

635-27

635-32 635-34 635-35 635-36 635-37 635-38 635-39

635-21

635-24

635-26

635-31

None = No supplementation; 4V = Vitamin mixture of B,, B,, PABA and Nicotinic acic; 3 aa = Tryptophan, histidine and arginine mixture; 4 V + 3 aa = Mixture of the two supplementations; 5 aa = Five amino acids, mixture of valine, leucine methionine, threonine and phenylalanine

Growth on agar plates was examined after 2 days at 30 "C

from B. thuvingiensis var. entomocidus HD-635 or var. kuvstaki, HD-251 was the same as that obtained from the wild type strain. With S . littovulis some of the auxotrophs of both varieties showed a similar effect to the wild type strain.

34 H. S . Salama, M. S . Foda and M. Selim

Table 5 . Growth, sporulation liter, yield of 8-endotoxin complex and potency of the endotoxin produced from auxotrophic mutants derived from B. t. var. entomocidus HD-635 and var. kurstaki HD-251. The auxotrophs were grown on fodder yeast-BM medium under standard conditions

Strain No. pH at Sporulation Endotoxin Yo mortality of harvest titerlml yield 3rd inrtar larvae of

time g/L 5. httoralis 5. exigua

HD-635 wild type 635-15 635-16 635-17 635-21 635-24 635-25 635-26 635-31 635-32

635-36 635-37 635-38

635-35

7.5 8.0 8.0 8.0 8.0 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 8.0

37 x 107 33 x 107 11 x 107 39 x 107 39 x 107 27 x 107 29 x 107 32 x 107 28 x 107 18 x 107 27 x 107 40 x 107 14 x 107 29 x 107

7.1 4.4 4.6 4.9 2.1 6.5 5.6 4.1 4.5 4.9 6.2 2.2 6.4 2.8

85 100 85 86 28 68 100 100 100 89 100 89 51 85

100 100 100 100 100 100 100 100 100 100 100 90 100 100

HD-251 wild type 8.5 - 6.1 72 100 251-1 8.0 51 x 107 4.6 96 100 251-2 8.0 36 x 107 5.2 58 100 251-3 7.5 32 x 107 4.5 90 100 251-1 03-1 7.5 32 x 107 4.3 100 100 251-13 8.0 26 x 107 4.9 80 100

In the present study the approach of the auxotrophic mutants was used in the hope to reveal some information regarding the genetic markers involved in the biosynthesis of the endotoxins of two strains of B. thuvingiensis that are highly active against major pests of cotton and their potencies. At first a study was made to define the nutritional requirements of those two wild type strains namely vars. entomocidus HD-635 and kuvstuki HD-251. This step was followed by mutagenesis of the two wild types using N-methyl N-nitro-N- nitrosoguanidine and picking of survivors followed by replicaplating on selective media that differentiate between the wild type colonies and those requiring auxillary nutritonal factors not required by their parent strains. Large percentage of those auxotrophic mutants reverted to their wild type pattern within limited number of transfers. The stable auxotrophs were then subjected to nutritional characterization where some of them proved to be of multi-auxotrophic nature requiring more than amino acid whereas others were plain single step mutants requiring a single vitamin. The multi-auxotrophic mutants will require more nutritional characterization to define the mutants were further characterized with respect to their growth pattern, sporulation titers and potency of their endotoxins produced on complete fodder yeast-BM medium. The results obtained showed that those auxotrophic mutants did not differ materially in their growth pattern and sporulation titers from their parent wild types. Furthermore, the potencies of their endotoxins assayed against third instar larvae of 5. littoralis and S. exiguu were comparable to those of the wild types. NORMANSELL and BURGES (1982) could not observe a notable reduction of activity among a wide range of B. thuvingiensis auxo- trophic mutants as compared to their parent strains. They reported that six isolates of each of histidine, arginine and guanine-requiring mutants, examined

Mutation in relation to sporulation and potency of B. thuringiensis 35

in each case, exhibited no specific trend with respect to their endotoxin potencies. They detected, however, one guanine-requiring mutant that, in their opinion, appeared to be considerably more pathogenic and deserves further investigation. They also reported that some other classes of auxotrophic mutants had reduced pathogencity whereas none showed significant increase. The authors expressed the opinion that the effect of auxotrophy on pathogenicity is not yet revealed and it may be related directly to an altered metabolic pathway or alternatively due to a pleiotropic effect.

One of the earliest reports on the mutational approach application in B. thuringiensis is that reported by NISHIITSUTSUJI-UWO et al. (1975). They were able to isolate five sporeless mutants, two were derived from var. alesti and three from var. aizawai. The insecticidal activity of the mutants was in principal, the same as the original strains. YOUSTEN (1978) used another procedure to isolate asporogenic and oligosporogenic mutants of B. thuring- iensis vars. kurstaki (HDI) , thuringiensis, galleriae, alesti, toloworthi, and subtoxicus. The procedure is based on streaking the bacteria onto a solid medium, incubating at 42 "C and picking small raised areas of growth which appeared on the streak after two days of incubation.

JOHNSON et al. (1980) carried out a detailed study on the parasporal crystals produced by the oligosporogenous mutants of B. thuringiensis designated as (Spo-Cr+). The mutants used in their study were derived from B. thuringien- sis var. kurstaki NRRL-B3792 (HD-1) through the treatment with N-methyl- N-nitro-N-nitrosoguandine and the selection of the mutant colonies that formed translucent or less pigmented sectors devoid of spores. The authors reported the isolation of six of such mutants. The toxicity of the parasporal endotoxin, produced by those mutants and assayed by an in vitro technique using culture insect cells, was comparable with that obtained from the parent wildtype strain.

Zusammenfassung

Mutationen in Beziehung zur Sporulation und Wirkung von Bacillus thuringiensis-Varietaten bei Baumwollschadlingen

Es wurde eine Serie von Mutagense-Experimenten mit B. thuringiensis var. kurstaki HD-251 und entomocidus HD-635 durchgefiihrt, urn mittels auxotropher Mutanten mogliche Beziehungen zwischen der Endotoxinwirkung bei einigen Baumwollschadlingen sowie bestimmten Ernahrungsfakcoren aufzuhellen. Die auxotrophen Mutanten waren hinsichtlich des Materials, des Wachstums und der Sporulation von den Wildtypen nicht verschieden. Dariiber hinaus waren die Wirkungen der mutanten Endotoxine gegeniiber Spodoptera littoralis und S. exigua mit jenen der Wildtypen vergleichbar. Obwohl die auxotrophen Mutanten partiell charakterisiert waren und ungeachtet ihrer unterschiedlichen genetischen Veranderungen bestand keine spezifische Beziehung zwischen den Mutanten hinsichtlich ihrer Auxotrophizitat und Endotoxin-Produk- tion.

References

DULMAGE, H. T., 1970: Production of spore &endotoxin complex by variante of Bacillus thuringiensis in two fermentation media. J. Invertebr. Pathol. 16, 385-389.

DULMAGE, H. T.; BOENING, 0.; REHENBORG, C.; HANSEN, G., 1971: A proposed standardized bioassay for formulations of Bacillus thuringiensis based on the international unit. J. Invertebr. Pathol. 18, 240-245.

JOHNSON, D. E.; NIEZGODSKI, D. M.; TWADDLE, G., 1980: Paraspoial crystals produced by oligosporogenous mutants of Bacillus thuringiensis. Can. J. Microbiol. 26, 486-491.

KUZMANOVA, I. N., 1976: Relationship between exprotease activity spore and crystal formation and virulence of bacteria Bacillus thurzngiensis. Prikl. Biokhim. Mikrobiol. 12, 495-500.

LECADET, M.; KLIER, A. F.; RIBIER, J., 1975: Isolation and characterization of two asporogenous Rifampicin resistant mutants of Bacillus thuringiensis. Biochimie 16, 1471-1479.

36 H. S. Salama, M . S. Foda and M . Selim

NORMANSELL, P.; BURGES, D., 1982: Potency of chemically induced mutants of Bacillus thuring-

SALAMA, H. S., 1970: Rearing the corn borer Ostrinia nubilalis Hiibn. on a semiartificial diet. 2.

SALAMA, H. S.; FODA, M. S.; SHARABY, A., 1981: Potency of spore &endotoxin complexes of

YOUSTEN, A. A,, 1978: A method for the isolation of asporogenic mutants of Bacillus thuringien-

zensis B. T. Newsletter 6, 6-7.

ang. Ent. 61, 216-218.

Bacillus thuringiensis against some cotton pests. 2. ang. Ent. 92, 388-398.

sis. Can. J. Microbiol. 24, 492494.

Address of the first named author: Prof. Dr. H. S. SALAMA, National Research Centre, Laboratory of Pests and Plant Protection, Tahrir St. Dokki, Cairo, Egypt

Biology and host specificity of Rhinocyllus conicus (Froel.) (Col., Curculionidae), a successful agent for biocontrol of the

thistle, Carduus nutans L.'

By H. ZWOLFER and P. HARRIS

Abstract

The weevil species Rhinocyllus conicus has been widely and successfully used for hiocontrol of Carduus nutans and related thistles. An analysis of the taxonomic position of R. conicus shows that it belongs to a tribe where many taxa have developed close associations with Cardueae host plants. Within the genus Rhinocyllus, R. conicus, which predominates on C. nutans, is geographically the main species and the other Rhinocyllus spp. occupy restricted distributions on the southern fringe of the R . conicus range. The life history of R. conicus is summarized. An account of the natural control factors of the eggs, larvae, pupae and adults of R . conicus in Europe and North America and on the effect of R . conicus on C. nutans is given. Field records of a 20-year survey show that the basic host range of R . conicus is restricted to the closely related Cardueae genera Carduus, Cirsium and Szlybum. Within this overall range, C. nutans is the most important host, but many local or regionally distributed populations of R . conicus have developed preferences for species such as Carduuspycnocephalus, Silybum marianum, Cirsium vulgare or C. arvense. The results of various types of screening tests are summarized to demonstrate the specificity of R . conicus. It is shown that the host specificity in the field depends primarily on adult feeding on the host plant, as this determines oogenesis and the oviposition behaviour. Only with access to plants which elicit adult feeding is the female able to produce the protective cap which covers the egg. This is a prerequisite for the successful establishment of the young larva within the flower heads, although it does not necessarily result in establishment on non-preferred hosts.

1 Introduction

Rhinocyllus conicus is a well known European weevil, 3-7mm long, that attacks the flower heads of Curdzus nutuns and to a lesser extent other thistles. Features of its life history were reported by GOUREAU (1845) and MELLINI

' This publication forms part of the Sonderforschungsbereich der DFG 137 ,,Gesetzmaaigkeiten und Steuerungsmechanismen des Stoffumsatzes in okologischen Systemen", Teilprojekt Al.

U.S. Copyright Clearance Center Code Statement: 0044-2240/84/9701-0036 !$ 02.50/0 Z. ang. Ent. 97 (1984). 36-62 0 1954 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-2240 / Intercode: ZANEAE