efficacy of ‘safe’ levels of antimicrobial food additives to control microbial contaminants in a...

Ent. exp. & appl. 14 (1971): 297--309. North-Holland Publishing Co., Amsterdam

EFFICACY OF 'SAFE' LEVELS OF A N T I M I C R O B I A L F O O D

A D D I T I V E S T O C O N T R O L M I C R O B I A L C O N T A M I N A N T S

I N A S Y N T H E T I C D I E T F O R AGRIA AFFINIS LARVAE

BY

PRITAM SINGH* and G. E. BUCHER Research Institute, Canada Department of Agriculture, Belleville, Ontario, Canada

The ability of eight antimicrobial food additives to suppress the growth of seven microorganisms in a chemically defined diet for an insect, Agria aIfinis auct. nec Fall6n, was tested by inoculating a standard number of the microorganisms into the diet and determining the amount of microbial growth and the effects on the rate of growth and survival of the insect. The antibiotics, mycifradin sulfate, streptomycin sulphate, and tetracyn, suppressed the growth of bacteria, and aerosporin suppressed the growth of yeast at concentrations that were innocuous for the insect. Mold inhibitors were less effective, and failed to suppress the growth of a species of Aspergillus. Potassium sorbate suppressed the growth of yeast, bacteria, and species of Penicillium, but only at concentrations that adversely affected the rate of growth and survival of the insect.

Antimicrobial compounds that are added to synthetic diets of insects to control contamination by microorganisms must be chosen carefully with due regard for the species of microorganisms that are responsible for the most serious contamination, for the tolerance of the insect to the effective concentrations of the antimicrobials, and for the nutritional level of the synthetic diet.

In a previous paper, Singh & House (1970b) determined 'safe' levels of 21 antimicrobial food additives for larvae of the fly, Agria affinis auct. nec Fall6n, reared axenically on a chemically defined diet. The 'safe' level of a compound was defined as the concentration that does not reduce the yield of pupae and adults and does not prolong the time for larval development more than 25% (compared with that of the control). Above the safe levels, the compounds exerted detrimental effects in proportion to the concentration used; they prolonged larval life and increased mortality in larval and pupal stages. Singh & House (1970b) also defined the 'primary inhibitory' level as the concentration that prolonged the larval period more than 25% but allowed at least 50% of larvae to reach instar III by the tenth day. The severity of these effects was influenced by the concentration of nutrients in the diet (Singh & House 1970a).

The purpose of the present work was to determine whether 'safe' or 'primary inhibitory' levels of antimicrobials would control the contamination that might be expected to occur in synthetic diets of A. affinis if axenic techniques were relaxed or accidentally breached.

* Present address: Entomology Division, D.S.I.R., Private Bag, Nelson, New Zealand.

2,98 PRITAM SINGH AND G. E. BUCHER

This paper shows that microbial contamination of a synthetic diet is detrimental to larvae of A. affinis, and that antimicrobial compounds can control some contaminating microorganisms without adversely affecting the rate of growth and survival of the insect.

MATERIAL AND METHODS

Detailed methods of rearing A. affinis, of preparing the diet, and of administering the antimicrobials were described by Singh & House (1970 a and b). The com- position of the chemically defined diet used in this work was described by House (1967) as diet A, and is identical to the '50% diet' of Singh & House (1970a). Larvae were removed aseptically from gravid females and reared individually at 23 __+ 1 ° C in test tubes containing the diet with varied concentrations of antimicrobial compounds.

Eight antimicrobial compounds were tested to determine their ability to suppress or control the growth of microorganisms inoculated into the synthetic diet alone and into the diet containing larvae. They were tested at or below the concentrations determined as 'safe' or 'primary inhibitory' levels by Singh & House (1970b). The antimicrobia!s! included three mold inhibitors: potassium sorbate, sodium benzoate and sodium propionate; and five antibiotics: aerosporin, mycifradin sulfate, pencillin G potassium, streptomycin sulphate, and tetracyn.

Each antimicrobial compound was tested against seven microorganisms consisting of four molds, a yeast, and two bacteria. All microorganisms were isolated at the Research Institute, Belleville and therefore represent groups that are potential contaminants of synthetic diets of A. affinis at this laboratory. The yeast and the molds are common air-borne contaminants and were isolated by exposing tomato-agar plates to laboratory air. The bacteria were isolated from contaminated tubes of diet containing larvae of A. affinis about 2 months prior to the present work. Large numbers of larvae of A. affinis are reared individually on synthetic diet under axenic conditions at Belleville. Contamination occurs in less than 1 in 1000 tubes and is usually caused by a mold.

The yeast produced smooth, nonmucoid, pink colonies on Sabouraud maltose agar. No ascospores or pseudomycelium were observed. We tentatively assigned it to the genus Rhodotorula as it was nonfermenting and did not assimilate inositol (Beech & Davenport, 1968).

The molds consisted of three species of Penicillium (herein called A, B, and C) and one species of Aspergillus. No attempt was made to identify 'these to species level.

Bacterium A was identified as Serratia marcescens Bizio; it had all of the biochemical characteristics of this species, as given by Martinec & Kocur (1961). It produced white colonies on agar and no gas in fermentable carbohydrates, but

Regis te red n a m e s a re g iven here ; for chemica l n a m e s and sources , see Append ix , p. 307.

ANTIMICROBIAL FOOD ADDITIVES 299

achromogenic and anaerogenic strains of the species seem to be commonly as- sociated with insects (Bucher & Stephens, 1959). Bacterium B was identified as a typical member of the Providence group of the Enterobacteriaceae according to diagnostic reactions given by Kauffmann (1954). Several proposals have been made for the binomial nomenclature of the Providence group, but we are not aware that the Judicial Commission of the Nomenclature Committee of the International Association of Microbiological Societies has made a firm ruling. Bacterium B had characteristics identical with the subgroup for which Ewing (1962) proposed the new combination Providencia alcalifaciens (De Salles Gomes).

Four other microoorganisms were tested in preliminary experiments but were discontinued because they grew too poorly on the synthetic diet of A. a[finis to allow measurements to be made on antimicrobial inhibition. Three of these, a white yeast, a bacterium, and a nonsporulating mold were isolated from laboratory air and were not identified. The bacterium, Bacillus thuringiensis Berliner, was chosen from stock cultures to represent a known insect pathogen that was gram-positive and that produced spores.

Highly concentrated suspensions of all seven test microorganisms were prepared by washing the growth of pure cultures from agar plates with sterile water. Suspensions of the yeast and of the bacteria consisted of vegetative cells, those of the molds of mature spores. The concentrated suspensions were stored at 4 ° for the duration of the tests. The concentration of each suspension was determined by viability counts on agar plates and by visual enumeration in a bacterial counting cell. Visual and viability counts were repeated at the close of the experiments to indicate that the viability of the concentrated suspensions remained virtually constant for the test period.

Immediately before each experiment 1 ml was withdrawn from each of the concentrated suspensions and serially diluted to such an extent that a standardized bacteriological loop of the final dilutions would deliver approximately 100 cells. This standard inoculum of 100 cells was used for every microorganism in each test. This inoculum size was chosen to satisfy three requirements: that it be in the range of magnitude likely to occur when insect diets are contaminated by chance; that it be not so large that it might swamp the antimicrobiat action of a compound; and that it be: not so small that variation, caused by the Poisson distribution of organisms in dilution suspension; might result in some loop samples containing few if any cells. Random spot checks showed that the loop delivered between 70 and 140 viable cells of all seven microoorganisms, a degree of variation that was considered acceptable.

E X P E R I M E N T A L

A. Detrimental ef[ects of experimental contamination of the synthetic diet Each microorganism was tested separately by adding the standard inoculum to

each of 25 test tubes containing 1 ml of synthetic diet. After 24 hours incubation at

300 PRITAI~ SINGH AND G. E. BUCHER

27 _ 1 °, a neonate sterile larva was added to each tube and reared at 23 --_ 1° for 10 days. A set of 25 control tubes contained larvae but were not inoculated with microorganisms. Daily records were taken on every tube of the extent of

microorganismic growth and on larval survival and stage of development. The rate of larval development was measured as the time required for 50% of the

larvae to reach the 3rd and final instar (ETs0). The ETso values were compared with that of the control set and the effects of microbial contamination were expressed

as a percentage increase in time of larval development. Values of the ETs~. could not be calculated for sets in which 50% of the larvae failed to survive to instar III .

Larval survival was expressed as the percentage that had survived and catered the final instar by the tenth day of the test. Surviving larvae were placed in sawdust for

pupation and emergence. Survival of pupae and of adults was expressed as a percentage of the original number of 25 larvae in each set. The time in days for a

heavy growth of the microoorganisms to cover the complete surface of the slanted diet in the tubes was recorded.

The complete experiment was repeated after completing experiments in sections

B and C to verify the results and to confirm the viability of the stored suspensions

of microorganisms. As the results of both experiments were similar, they were combined (Table I).

TABLE I

Ef/ects of microorganisms on development o[ A g r i a a f f i n i s larvae on a synthetic diet*

Increase in Percent Survival Time of heavy ETa0, time of larval Instar III growth of

Instar III, development larvae, microorganisms Microorganism days % day 10 Pupae Adults days

Bacterium A - - - - 6 0 0 1--2 Bacterium B - - - - 20 8 8 1--2 Aspergillus sp. - - - - 0 0 0 1--2 Penicillium A 4.7 9 76 58 50 6--7 Penicillium B - - - - 40 0 0 2--3 Penicillium C - - - - 32 0 0 3---4 Yeast 4.8 10 96 76 66 2--3 Control 4.4 0 98 94 88 no growth

* 25 larvae tested/each microorganism; average of 2 tests

All species of molds seriously interfered with the normal development of larvae.

Aspergillus sp. interfered most with larval development; it produced heavy growth

within 48 hours that formed a thick mat over the surface of the medium and

prevented larvae from reaching their food. Most of the larvae stayed in the 1st instar,

and none reached the 3rd instar; all died within 10 days. Penicillium B and C grew

rapidly and formed hard mats on the surface of the medium, which later cracked.

Larvae developed slowly and remained small and inactive; few reached the 3rd

instar and none pupated. Penicillium A interfered least with larval development.


It grew slowly for the first 3--4 days. The larval period was slightly prolonged and survival was reduced.

Both species of bacteria prevented normal development of larvae. Most larvae died before reaching the 3rd instar and only rare individuals were able to pupate or emerge. The bacteria grew rapidly, discolored the diet and produced a sour pungent odor.

The yeast did not seriously affect larval survival, but prolonged the larval period and reduced pupation and adult emergence. It grew rapidly on the diet, which became slightly fluid.

B. Ef fec t ive concentrations of antimicrobials in diets wi thout A. affinis larvae.

To determine the range of antimicrobial concentrations that would reduce growth of the microorganisms on the synthetic diet of A . affinis, tests were performed on the diet in petri-dishes without the inclusion of larvae. The diet was prepared, divided into samples, and various concentrations of the antimicrobials were added to the samples. The samples were poured into petri-dishes (25 ml per dish), allowed to gel, and four microorganisms were inoculated on the surface of each dish, one to each quadrant. The dishes were sealed with tape to minimize drying and were incubated at 27 +--- 1 °. Microbial growth was recorded daily for 10 days and compared with the growth on control plates containing no antimicrobial compounds by measuring the width of the zone of growth.

The antimicrobial action was rated by the following terms: suppressed - - no evident growth of microorganisms during the 10 days of the test; inhibited - -

growth of microorganisms s/owed to half or more than half the rate on control plates; inef fect ive - - growth of microorganisms not influenced or not reduced to half the rate on control plates.

Each antimicrobial was tested at three or more different concentrations, but the concentration ranges varied. As there was no point in determining antimicrobial effects at concentrations toxic to A. affinis larvae, the maximum concentration tested for a given antimicrobial did not exceed its 'primary inhibitory' level as determined by Singh & House (1970b). More dilute concentrations were in the range of 1/2 to 1/10 of the maxima. Concentrations are expressed as milligrams of antibiotic per 100 ml of diet and herein are shortened to mg (e.g. a concentration of 200 mg means a concentration of 200 mg per 100 ml of diet).

Aerosporin suppressed the growth of yeast at 160 mg (the highest concentration tested) but was ineffective at 80 mg, and did not inhibit bacteria or molds a~ any concentration.

Mycifradin sulfate suppressed bacterial growth at 100 mg and inhibited bacteria at 50 mg. It inhibited yeast growth at 300 mg (highest concentration) but was ineffective against molds.

Penicillin G potassium inhibited bacteria at 900--1200 mg but was ineffective against the other organisms at the highest concentration of 1200 mg.

302 PRITAM SINGH AND G. E. BUCHER

Streptomycin sulphate suppressed bacterial growth at 200 mg but was ineffective against molds and yeast at concentrations up to 800 mg.

Tetracyn suppressed bacterial growth at 100 mg and inhibited at 20 mg. It was ineffective against molds and yeast at the highest concentration of 300 mg.

Potassium sorbate suppressed the growth of Penicillium spp. at 600 mg and inhibited yeast and bacteria. It was ineffective at 300 mg and had no effect on Aspergillus sp. at the highest concentration of 600 mg.

Sodium benzoate inhibited the growth of yeast and some molds from 200 400 nag and bacterial growth at 400 mg (highest concentration). It was ineffective at

50 mg. Sodium propionate was ineffective against all microorganisms at 100 mg and

against the yeast at all concentrations. It suppressed Penicillium A at 400 mg and inhibited the other molds. At the highest concentration of 800 mg it inhibited bacteria but did not totally suppress mold growth.

C. Effective concentrations of antimicrobials in diets with A. affinis larvae The ability of the antimicrobials to control experimental contamination suf-

ficiently to allow the development of A. a[finis larvae was tested as described in section A. Each test was conducted on 25 larvae of A. affinis in individual tubes containing 1 ml of the diet that incorporated a given concentration of the antimicrobial. Each tube was inoculated with the standard concentration of 100 cells of the microorganism, 24 hours before the larvae were introduced.

Observations were made as stated in section A and results compared with control sets of larvae reared without antimicrobials or experimental contamination. The concentrations of antimicrobials to be tested were chosen from the results of section B. Antimicrobials were tested only against those specific organisms that they had inhibited to some extent at 'safe' or 'primary inhibitory' levels. For example, aerosporin was tested only against the yeast and the antibioties were not tested against the molds.

The results of 84 individual tests are summarized in Table I[. Note that sodium benzoate, which had inhibited mold and yeast growth in petri-dishes (Section B), failed to produce observable inhibition in the small tubes of these tests. Such discrepancies resulted from the fact that inhibition was not measurable accurately in tubes with a small surface area.

Table II does not show the serious effects on larval development and survival that occurred when the antimicrobials failed to control the experimental contamination. For example, mycifradin sulfate suppressed Bacterium B at 50 rag, larval development was not slowed, 100% of the insects pupated and 96% emerged as adults. But mycifradin sulfate did not suppress Bacterium A at 50 mg; as a result of bacterial growth, larval development was slowed by 84%, only 40% of the insects pupated and 36% emerged as adults. Similarly sodium propionate suppressed Bacterium B at 600 mg, larval development was slowed by only 14%, 88% of the insects pupated and 60% emerged as adults; these relatively mild effects are


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a t t r ibu tab le to sod ium p rop iona t e alone. But when sodium p rop iona t e only in-

h ibi ted Bacter ium A at 600 mg, larval deve lopment was s lowed by 3 6 % , 52%

of the insects p u p a t e d and only 20% emerged as adults; these serious effects are

due pa r t ly to sod ium p rop iona t e a lone and pa r t ly to some growth of the bac ter ium.

Detai ls of the adverse effects of the b road - spec t rum compound , po tass ium sorbate,

on larval deve lopment and survival a re shown (Table I I I ) as fur ther examples of

the serious loss of insects when the an t imicrobia l fai led to cont ro l microorganisms.

Note the low values for pupa t ion and emergence when Penicillium B and C were

only inhibi ted by 400 m g and compare with the higher values when the bac ter ia

and Penicillium A were suppressed by the same concentra t ion.

TABLE n I

Effect of potassium sorbate on the growth of microorganisms and Agria affinis larvae in a synthetic diet*

Potassium Increase in Percent Survival

sorbate ETs0, time of larval Instar III mg/100 ml instarlII, development larvae, Growthof diet Microorganism days % day 10 Pupae Adults microorganisms

400 Baeterium-A 6.6 57 I00 72 64 O 600 Bacterium-A 9.2 119 56 4 4 O 400 Bacterium-B 6.9 64 88 68 60 O 600 Bacterium-B 9.6 129 60 4 0 O 400 Aspergillus sp. - - - - 24 0 0 + + 600 Aspergillus sp. - - - - 4 0 0 + + 400 Penicillium-A 6.6 57 88 80 72 0 600 Penicillium-A 9.1 117 68 12 8 0 400 Penicillium-B 6.6 57 100 16 8 + 600 Penicillium-B 9.5 126 56 0 0 O 400 Penicillium-C 6.6 57 100 24 12 + 600 Penicillium-C 8.9 117 76 0 0 0 400 Yeast 6.3 50 100 84 76 0 600 Yeast 8.9 117 80 4 0 0

0 Control 4.2 0 100 100 100 - -

* 25 larvae/test + + ineffective, growth not reduced to ~ of control + inhibited, growth reduced to ~ or more of control O suppressed, no evident growth in 10 days - - no calculation possible

The abi l i ty of the ant imicrobia ls to control exper imenta l con tamina t ion of a

synthet ic diet is summar ized (Table IV) and c o m p a r e d with the ' safe ' and ' p r i m a r y

inhib i tory ' levels for A. affinis de te rmined by Singh & House (1970b). W e infer

that the an t imicrobia l s would have the same act ion against con tamina t ion that

might occur by chance if no rma l axenic techniques were accidenta l ly b reached or


if attempts were made to rear the insects on synthetic diets without maintaining sterility. Bacterial contamination was controlled by three antibiotics without in- fluencing the development or survival of larvae. Similarly aerosporin controlled yeast contamination. Aspergillus sp. was not controlled by any of the antimicrobials tested. Potassium so rbate controlled the other molds, the yeast and the bacteria, but only at concentrations that increased developmental time and that produced significant mortality in A. aflinis cultures. Combinations of two antimicrobials at decreased concentrations did not show any synergistic effects.

TABLE IV

Lowest concentrations (rag per 100 ml diet) of antimicrobials that suppress microorganisms compared with 'safe' and 'primary inhibitory' levels Jor Agria affinis

= ..= =

~" ~ "-" ~ o " ~ ~ ~ o =

Safe leveP 160 100 200 200 40 200 100 400 Primary inhibitory 170-- 110-- 210-- 210-- 50-- 210-- 110-- 410-- leveP 250 400 1600 1000 150 600 400 800 Bacterium A I 75 900 50 25 400 400 I Bacterium B I 50 600 50 25 400 300 600 Aspergillus sp. I I I I I I I I Penicillium A I I I I I 400 200 400 Penicillium B I I I I I 600 I I Penicillium C I I I I I 600 I I Yeast 150 I I I I 400 I I

1 Determined by Singh & House (1970b) I Ineffective at primary inhibitory levels of concentration

DISCUSSION

The most common microbial contaminants encountered in artificial diets for mass rearing of insects are yeasts, bacteria, and molds belonging to the genera Aspergillus, Rhizopus, and PenicilIiurn. It is common to find five or more species of microorganisms attacking a food medium. A review of literature showed that the presence of microorganisms in artificial diets is detrimental to insects. For example, molds cause mortality or retard larval development (Hensly & Hammond, 1968; Kishaba et al., 1968; Ouye, 1962; Clark et al., 1961); bacterial contamination is also detrimental (Jaques et al., 1969; Toba et al., 1969; Clark et al., 1961; Afrikian, 1960). The present work confirmed these observations; microbial contamination of a synthetic diet produced mortality and prolonged development of A. affinis. The mechanisms by which the growth of microorganisms adversely af- fects insects on artificial diets have not been clarified. Most probably the food is


rendered unfit for consumption, but toxic and pathogenic mechanisms may also be involved.

In the present work the detrimental effects of mold contamination appeared to result from the rapid formation of heavy mat-like growths that many larvae were unable to penetrate. Thus larvae starved to death or were so poorly fed that they were unable to pupate. Penicillium A, a slow grower, allowed more feeding and therefore greater survival. The rapid death in Aspergillus-contaminated media also suggested some toxic action, but we found no evidence that any of the molds produced a true infection of larvae and so could be considered pathogenic. Bacterium A (Serratia marcescens) is a well-known insect pathogen, but Bacterium B (Providence group) is not (Bucher, 1963). As we found no evidence that these bacteria produced true infections in A. affinis larvae, it seems that their detrimental effects are produced by reducing the food value of the diet or by producing meta- bolites that are toxic to larvae. The relatively mild effects of the yeast also appear to result from altering the food value of the diet.

Stock cultures of A. affinis are normally maintained by allowing adults to ovi- posit on pieces of liver and by raising the resulting larvae under conditions that are far from axenic. Developing larvae can certainly tolerate a highly contaminated environment. Thus it was surprising that larvae did not tolerate contamination of a chemically defined diet better than they did. Therefore we presume that the deleterious effects of the microorganisms arose chiefly from changes in the nutritional level and consistency of the artificial diet.

It is a common practice to use antimicrobial compounds, separately or combined, to prevent microbial contamination of synthetic diets for insects. In some cases these compounds have been shown to be detrimental to the insects (Singh & House, 1970a, b, c; Greenberg, 1970; Bass & Barnes, 1969; Toba et al., 1969; Kishaba et al., 1968; Vail et al., 1968; Moore et al., 1967; Prokopy, 1967; Ouye, 1962; Clark et al., 1961). In other cases they have not been effective in suppressing the growth of microbial contaminants (Bass & Barnes, 1969; Kishaba et al., 1968; Ouye, 1962; Clark et al., 1961; Brazzel et al., 1961). The present work demonstrated that the bacteria and yeast were readily suppressed but that mold contamination was much more difficult to suppress. The antibiotics were ineffective against all the molds and the mold inhibitors did not suppress mold growth at concentrations that were innocuous to the insect.

This work also demonstrated that only the complete suppression of microbial growth was acceptable in a culture of A. affinis on a synthetic diet. When microbial growth was only retarded by the presence of antimicrobial agents, the insect suf- fered almost complete mortality before emergence of the adults. This is shown for potassium sorbate acting on Penicillium B and C (Table III) but high to total mortality also occurred in the other tests where microbial suppression was not complete.

The ideal antimicrobial food additive would suppress a wide variety of microorganisms at a concentration safe for the insect. We have not attained this ideal for


A. affinis cul tures on a chemical ly def ined diet, but the ideal an t imicrob ia l or

combina t ion of an t imicrobia ls m a y exist for o ther insects reared on synthet ic diets.

However , careful select ion of an t imicrobia ls mus t be m a d e with due regard for

the kinds of microorgan isms responsible for the most serious contamina t ion , the

to lerance of the insect species to ant imicrobia ls , and the nutr i t ional level of the

ar t i f ic ial diet. W e feel that fa r too often invest igators have a d d e d an t imicrobia l s to

ar t i f icial diets for insects before de termining the concentra t ions at which they

suppressed con tamina t ion wi thout ha rming the insects.

W e a re indebted to Dr. H. L. House for his keen interest in this work, and to

Mr. W. W. Batsch for technical assistance.

APPENDIX (after Singh & House, 1970b)

REGISTERED, CHEMICAL NAMES AND SOURCES OF ANTIMICROBIAL AGENTS USED

Registered name of agent Description

Aerosporin

Mycifradin sulphate

Penicillin G Potassium

Potassium sorbate Sodium benzoate Sodium propionate Streptomycin sulphate

Tetracyn

Polymyxin B sulphate, 500,000 units equivalent to 50 mg polymyxin standard Burroughs, Wellcome and Co. (Canada) Ltd. Bacillus polymyxa

Neomycin sulphate, 0.5 g, e.g. 350 mg neomycin base The Upjohn Co. of Canada Streptomyces fradiae

10 million, I.U. Ayerst Laboratories, Montreal Penicillium sp.; Aspergillus sp. Matheson, Coleman & Bell Ltd. Fisher Scientific Co. Fisher Scientific Co. l g British Drug Houses (Canada) Streptomyces griseus

Tetracycline hydrochloride, 500 mg Pfizer Co. Ltd. Streptomyces aureofacieus

I~SUM]~

EFFICACITt~ DE L'ADJONCTION A L 'ALIMENTATION SYNTHI~T1QUE DES LARVES D'AGRIA AFFINIS D'ANT1BIOTIQUES A DOSES NON-

NOCIVES POUR L'INSECTE

Ce travail montre que les contaminations exp6rimentales d'un milieu synth6tique pour l'~levage ax~nique des larves d'Agria affinis a des effets d6favorables ou noeifs, soit en retardant le d6veloppement, soit en provoquant une mortalit6 des divers stades. L'adjonetion d'antibiotiques au milieu d'61evage, peut avec des doses appropri6es, 6viter les contaminations, sans affecter la croissance ou la survie de l'insecte.


Les micro-organismes utilis& pour la contamination du milieu d'61evage sont : 2 bact6ries (Serratia marceseens Bizio et Providencia alcalifaeiens (De Salles Gomes), 1 levure (Rhodo- torula sp.) 4 moississures (Penieillium sp. A, B et C et Aspergillus sp.).

Les compos6s anti-microbiens utilis6s sont: 5 antibiotiques (Aerosporin, Sulfate de Myci- fradin, Penicilline G potassium, sulfate de Streptomycine, et Tetraeyn) 3 mycostatiques (so,rbate de potassium, benzoate de sodium et propionate de sodium). La mycifradine, la streptomycine et la tetracyne emp&hent bien la d6veloppement des deux baet6ries test6es, l 'aerosporine supprime celle de la levure (tout cela ~ des concentrations Ilon-nocives pour l'insecte). Les mycostatiques furent moins efficaces en particulier contre une esp6ce d'Asper- gillus. Le sorbate de potassium inhibe la croissance de la levure, des bact6ries et de Penicil- lium, mais seulement k des doses qui sont d6favorables ~ la croissance et A la survie de l'insecte. Les micro-organismes semblent exercer leurs effets noeifs en modifiant la eonsistan- ce ou la valeur nutritive du milieu alimentaire des larves, plut6t qu'en provoquant des infections directes.

Le choix des compos6s anti-rnicrobiens qui peuvent &re adjoints h un milieu alimentaire d'61evage pour des larves d'insectes doit donc &re r6alis6 avec soin, en tenant compte d'une part, de leur efficacit6 r6elle sur les germes dont on dolt 6viter la prolif6ration, d'autre part, de leur inocuit6 sur l'insecte aux doses utilis6es.

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Rece ived for publ icat ion: November 25, 1970.

efficacy of ‘safe’ levels of antimicrobial food additives to control microbial contaminants in a...

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