Development of aposymbiosis in larvae of Sitophilus oryzae (Coleoptera: Curculionidae) by dietary treatment with antibiotics

J. stored Prod. Res., 1973,

Vol. 9, pp. 241-245. Pergamon Press. Printed in Great Britain.

DEVELOPMENT OF APOSYMBIOSIS IN LARVAE OF SITOPHILUS OR YZAE (COLEOPTERA : CURCULIONIDAE) BY DIETARY TREATMENT WITH ANTIBIOTICS* J. E. BAKER and P. T. M. LUM Stored-Product

Insects Research

and Development Laboratory, Ga. 31403, U.S.A.

(First received 27 June 1973, and

infinal form

Agric. Res. Serv., USDA,

Savannah,

14 August 1973)

Abstract-The symbiotic micro-organisms in the larval mycetomes of Sitophilus oryzae (L.) were sensitive to the dietary incorporation of chlortetracycline or penicillin. The number of bacteroids in the mycetomes of larvae fed diets containing O-01 or 0.05 % chlortetracycline was reduced after 5 days and all were completely eliminated during the 4th stadium. The mycetomes of these larvae did not develop completely, reaching a mean of 268 f 53 pm in width compared with a mean width of 551 & 54 pm in untreated larvae. Streptomycin and bacitracin were not effective in eliminating the bacteroids and along with chlortetracycline were toxic at dietary levels of 2.0 per cent.

INTRODUCTION

THE NATURALoccurrence of symbiote-infected and asymbiotic strains of the granary weevil, Sitophilus granurius (L.), is well known (MANSOUR,1935; MUSGRAVE and MILLER, 1958). Although no asymbiotic strain of the rice weevil, S. oryzue (L.), has been reported, SCHNEIDER(1956) produced aposymbiotic S. grunurius and S. oryzae by maintaining the infected insects at 35°C for extended periods. When the growth of these weevils on oats, barley, maize and sorghum was compared with the growth of infected weevils, he found distinct differences. The development of symbiotic and aposymbiotic weevils was comparable on sorghum. However, the FI generation of aposymbiotic weevils reared on oats, barley or maize did not produce viable progeny. Schneider therefore postulated that the Sitophilus symbiotes provided some factor of nutritional benefit to the host insect, a factor that occurred naturally in sorghum but was lacking in the other grains. To determine whether a nutritional relationship exists between larvae of the Sitophilus weevils and their intracellular symbiotes, a holidic (or reasonably defined) larval diet must be developed and a procedure found to eliminate the larval bacteroids. For this latter aspect, the dietary incorporation of antibiotics has been one of the most useful and efficient methods of obtaining aposymbiotic insects (RICHARDSand BROOKS,1958; BUCHNER,1965). Using the meridic larval diet of BAKERand MABIE(1973a), the effect of dietary antibiotics on growth and survival of S. oryzae and its associated bacteroids was studied. The results are presented in this paper. * Mention of a commercial or proprietary product in this paper does not constitute a recommendation an endorsement

of this product by the U.S. Department 241

of Agriculture.

or

242

J. E. BAKERand P. T. M. LIJM MATERIALS

AND METHODS

The techniques used to obtain and rear lst-stage larvae of S. oryzae to the adult were identical to those used to rear S. grunarius (BAKER and MABIE, 1973b). The diet consisted of the following: Component

Amount

Casein, vitamin-free Cornstarch Brewer’s yeast Wheat germ Wheat germ oil Cholesterol H20, distilled

1.0 g 7.99 g 0.75 g 0.25 g 0.025 g 0.01 g 1.4 ml

The antibiotics, chlortetracycline (HCL), penicillin G (potassium), streptomycin sulphate and bacitracin (all purchased from Nutritional Biochemicals Corp.) were thoroughly ground and mixed with the dry components to insure a homogeneous distribution in the diet. Portions of each diet (cu. 100 mg) were placed in each of a series of small glass rearing tubes along with a single, newly hatched larva. The following criteria were used to evaluate the effect of the antibiotic treatments: (1) mean time to pupation; (2) survival to adult; and (3) the presence or absence of bacteroids in the larval mycetomes or the adult midgut caecae as determined by the procedures of MUSGRAVEand MILLER (1953) and LUM and BAKER(1973). In the 1st test (Table I), each antibiotic was tested at O-5 and 2.0% (wt/wt) in the diet. Twenty-five Is&stage larvae of S. oryzae were placed on each test diet, reared in continuous darkness at 30.0 & 1.0°C and 65 % r.h., and the bioassay parameters determined. TABLE 1. MEAN DEVELX)PMENTAL TIMES, SURVIVAL, AND PRESENCE (+) ORABSENCE (-) OF BACTEROIDS IN ADULTS OF S. oryzue THAT DEVELCIPED FROM BATCHES OF 25 LARVAE REAREDINDNIDUALLYAT 30°C AND 65% r.h. ON THE MERDIC DIET TREATED WITH ANTIBIOTICS

Antibiotic Chlortetracycline

Penicillin G

Bacitracin Streptomycin

Mean time to pupation

(days f SD)

Number of adults

0 0.5 2.0 0

14.9 f 1.3 27.0 0 13.6 f 1.0

21 1 0 22

+ -

qg 0 0.5 2.0 0 0.5 2.0

2.4 1.6 0.8 2.7 3.5 1.6 3.1

21 ;: 24 2 19 22 1

+ +

Concn (%bywt)

14.9 14.8 13.5 16-2 36.0 15.2 16.9 29.0

fh & & & f &

Bacteroids

+

J+ +

In the 2nd test (Table 2), 90 lst-stage S. oryzue were placed on untreated diet. After 5 days, 25 larvae were removed and transferred to a fresh diet containing 0.5 % chlortetracycline. Six days later another 25 larvae were transferred to the treated diet. The

Development

of Aposymbiosis

243

development of the larvae that were transferred to the chlortetracycline-treated diets was observed and compared with that of the larvae maintained on an antibiotic-free diet.

TABLE 2. MEAN DEVELOPMENTAL TIMES, SURVIVAL, AND PRESENCE (+) OR ABSENCE (-) OF BACTERO~ IN ADULTS THATDEVELOPED FROM LARvAi3 0~ S.oryzae REARED IN~ALLYONTHE HERIDIC DIET AND THEN TRANSFERRED AT TWO AGE INTERVALS ON To THE DIET TREATED WITH 0’5% CHLORTETRACYCLINE

Age of larvae at transfer (days)

Mean time to pupation

Not transferred 5

15.1 f 3.0 31.3 rt 6.0

37140 19125

+ -

11

18.3 & 7.2

24125

+*

* Several of the preparations microorganisms.

(days f SD)

in this group contained

Proportion surviving to adult

noticeably

Bacteroids

smaller numbers

of the

In the 3rd test (Fig. I), the effect of low levels of chlortetracycline (0*01,0.05 and 0.1 per cent) on larval development was determined. Thirty I&-stage larvae were placed on each test diet and their growth rates determined, In addition, mycetomes of 4th-instar larvae were removed from the alimentary tract using the procedure of LUM and BAKER (1973) and their sizes and degree of infection were compared with those from larvae reared on an antibiotic-free diet. In the 4th test, 30 1St-stage S. oryzae were placed on diets containing 0.01 and 0.05 % chlortetracycline. Five larvae were removed from each diet after 5, 8, 11, 13 and 16 days, and the degree of infection in each larval mycetome was determined.

FIG. 1. Cumulative percentage

pupation of S. oryzae reared on the basic diet (controls) and on the diet containing 0.01, 0.05, and 0.1% chlortetracycline. The presence (+) or absence (-) of bacteroids in the midgut caecae of emergent adults is indicated in parentheses.

244

J. E. BAKERand P. T. M. LUM RESULTS

Dietary treatments of chlortetracycline and penicillin during the larval stage eliminated the bacteroids that normally would be found in the midgut caecae of newly eclosed adults of S. oryzae (Table 1). Chlortetracycline at 0.5 per cent inhibited larval development although many larvae survived for 3-4 weeks. Dietary levels of chlortetracycline of 2-O per cent were toxic. Penicillin did not affect larval growth or development even at a concentration of 2-O per cent. Bacitracin and streptomycin did not eliminate the bacteroids although both were toxic at the 2.0 per cent concentration. When 5-day-old S. oryzae larvae were transferred from the control diet to a diet containing 0.5% chlortetracycline (test 2), the growth rate was significantly reduced although 19 out of 25 larvae completed development (Table 2). No bacteroids were present in the midgut caecae of the newly eclosed adults. When 11-day-old larvae were transferred to the treated diet, their development was delayed; however, bacteroids were present in all the preparations examined, although the number in some adults was noticeably reduced. When larvae were reared on diets containing 0.01 and 0.05% chlortetracycline (test 3), the bacteroids in the mycetomes were completely eliminated (Fig. 1). Although survival was not affected, the growth rates on diets containing O-1% chlortetracycline were much slower compared with the other treatments. The mycetomes of 4th-instar larvae that were reared on antibiotic-treated diets were smaller (Fig. 2) (mean width of 268 f 53 pm (SE) and height of 123 f 33 pm) than the mycetomes removed from 4th-instar larvae that were reared on antibiotic-free diets (mean width of 551 f 54 pm and height of 372 f 55 pm) in a previous study (LUM and BAKER, 1973). The numbers of the rod-shaped bacteroids found in the mycetomes from larvae in test 4 were significantly reduced by day 5. This reduction in numbers continued through 11 days on the diet. However, it was not until the 4th stadium (13th-16th day) that the bacteria were completely eliminated from larvae reared on both 0.01 and 0.05% chlortetracycline-treated diets.

DISCUSSION

The toxic effects of high concentrations of chlortetracycline, streptomycin, and bacitracin on larvae of S. oryzue were similar to those on S. oryzue adults reported by STEINHAUS and BELL(1953). However, these investigators did not study the specific effects on the bacteroids. SCHNEIDER(1956) found a reduction of bacteroids in larvae of S. oryzue that were reared in whole wheat grains that had been soaked in a O-2‘A aqueous solution of chlortetracycline or terramycin. In contrast, DANGGABRANI (1971) was unsuccessful in obtaining aposymbiotic S. oryzue with treatments of chlortetracycline or penicillin. Our results demonstrated conclusively that the bacteria-like micro-organisms(B~~T~~~m and MUSGFUVE,1970; DANG-GABRINI,1971) in S. oryzue were very sensitive to the dietary incorporation of both penicillin and chlortetracycline. A complete elimination of the bacteroids did not occur until the last larval stage, but the number of bacteroids was so reduced during the first three instars that this technique may be suitable for the development of aposymbiotic larvae to be used in comparative nutritional studies. The elimination of the bacteroids with penicillin did not affect the larval developmental time or survival. This would appear to negate the role of the symbiotes in supplying a

A

,

65~u

B FIG. 2. Comparative sizes of mycetornes in 4th-instar larvae of S. oryzae that were reared on a diet containing 0"01 ~ chlortetracycline (A, ×370) or on an antibiotic-free diet (B, x225).

[facing page 244

Development

of Aposymbiosis

245

useful or required nutrient. However, the larval diet used in these tests contained 7.5 % brewer’s yeast and 2.5 % wheat germ and generally would be considered nutritionally adequate. Therefore, the need for symbiote-supplied growth factors would not be critical and differences in the growth rates of aposymbiotic and symbiotic larvae would not be expected. Only under conditions of nutritional stress would the inadequacies develop in the aposymbiotic larvae, assuming that the antibiotic-induced reduction in the number of bacteroids occurs before they can contribute qualitatively and/or quantitatively to the developmental needs of the insect. Nevertheless, in the tests with 0.01 and 0.05% chlortetracycline there was a short but significant (P < O-05) delay in the mean time to pupation compared with the controls, a delay which was even more pronounced at a concentration of 0.1 per cent (Fig. 1). If the delay was due to feeding inhibition, the positive gustatory and olfactory stimuli known to occur in yeast and wheat germ (LOSCHIAVO, 1965) apparently could partly mask the effect of the low concentrations of chlortetracycline but not the 0.1 per cent or higher concentrations. Another possibility is that chlortetracycline concentrations of 0.1 per cent and lower may have a direct or indirect effect on the efficiency of processes which affect larval food utilization. As the antibiotic concentration is increased above 0.1 per cent it becomes toxic. The lack of growth of the mycetomes in larvae of S. oryzae reared on chlortetracyclinetreated diets supports the findings of BROOKSand RICHARDS(1955), working with Blattella germanica (L.), and LUM and BAKER(1973), working with S. grunarius and S. oryzae, that mycetome development occurs only in the presence of associated micro-organisms. REFERENCES BAKER, J. E. and MABIE, J. A. (1973a) Growth

responses of larvae of Sitophilus granarius (Coleoptera: Curculionidae) on a meridic diet. Ann. ent. Sot. Am. 66,723-726. BAKER, J. E. and MABIE, J. A. (1973b) Growth and development of larvae of the granary weevil, Sitophiius granarius (L.) (Coleoptera: Curculionidae), on natural and meridic diets. Can. Ent. 105, 249-256. BHATNAGAR, R. D. S. and MUSGRAVE, A. J. (1970) Cytochemistry, morphogenesis, and tentative identification of mycetomal microorganisms of Sitophilus granarius L. (Coleoptera). Can. J. Microbial. 16, 1357-1362. BROOKS,M. A. and RICHARDS,A. G. (19.55) Intracellular symbiosis in cockroaches: II. Mitotic division of mycetocytes. Science, Washington 122,242. BUCHNER,P. (1965) Endosymbiosis of Animals with PIant Microorganisms. Interscience, New York. DANG-GABRANI,K. (1971) On the functions of intracellular symbiotes of Sitophilus oryzae Linn. Experientia 27, 107. LOSCHIAVO,S. R, (1965) The chemosensory influence of some extracts of brewer’s yeast and cereal products on the feeding behaviour of the confused flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae). Ann. ent. Sot. Am. 58,576--5%X LUM, P. T. M. and BAKER,J. E. (1973) Development of mycetomes in larvae of Sitophi/us granarius and S. oryzae. Ann. ent. Sot. Am. In press. MANSOUR.K. (1935) On the micro-organism-free and the infected Calundru nrunaria (Lin.). Bull. Sot. R. ent. Egypt 1$,290-306. MUSGRAVE,A. J. and MILLER, J. J. (1953) Some microorganisms associated with the weevils Sitophilus granarius (L.) and Sitophilus oryzue (L.). I. Distribution and description of the organisms. Can. Ent. 85, 387-390. MUSGRAVE,A. J. and MILLER, J. J. (1958) Studies of the association between strains and species of Sitophilus weevils and their mycetomal micro-organisms. Proc. 10th int. Cong. Ent. 2, 315-318. RICHARDS,A. G. and BROOKS,M. A. (1958) Internal symbiosis in insects. A. Rev. Ent. 3, 37-56. SCHNEIDER,H. (1956) Morphologische und experimentelle Untersuchungen fiber die Endosymbiose der Korn- und Reiskiifer (Calandra granaria L. und Calandra oryzae L.). Z. Morph. dkol. Tiere 44, 555-625. STEINHAUS,E. A. and BELL, C. R. (1953) The effect of certain micro-organisms and antibiotics on storedgrain insects. J. econ. Ent. 46, 582-598. S.P.R.9/4-c