DNA gyrase inhibitors: Novobiocin enhances the survival
of Pimpla turionellae (Hym., Ichneumonidae) larvae reared
on an arti®cial diet but other antibiotics do not
K. BuÈyuÈkguÈzel
Department of Biology, Faculty of Sciences, University of Ankara, Turkey
Abstract: Eects of novobiocin, nalidixic and oxolinic acids, which are potent antibacterial agents, on the survival and development of the hymenopterous endoparasitoid, Pimpla turionellae L. (Ichneumonidae) were investigated alone by rearing the larvae aseptically on chemically de®ned synthetic diets. Survival of the ®fth instar larvae was not signi®cantly aected by diets with dierent levels of antibiotics tested. A diet containing the lowest level of novobiocin signi®cantly increased yields of pupae and adults. This level also signi®cantly shortened the developmental time for ®fth instar but had no signi®cant eect on complete development of the larvae up to adult emergence. Oxolinic acid at the lowest level did not aect the survival but prolonged development of the insect. In general, the rate of development was signi®cantly increased and the survival was decreased with high levels of the antibiotics. However, nalidixic acid caused a striking decrease in the survival at all tested levels. These results suggest that novobiocin and to a lesser extent oxolinic acid are compatible for addition to arti®cial diet for rearing P. turionellae, which is an important parasitoid used in biological control.
1 Introduction
Pimpla turionellae discharge an anal secretion which has antibiotic eects against bacteria and fungi to protect the larvae while developing in their natural host (FUÈHRERand WILLERS, 1986). Microbial
contam-inations are a serious problem during mass-rearing in the laboratory using an arti®cial diet. These contam-inations make rearing of the Pimpla larvae unsatisfac-tory and sometimes impossible, and often do not allow for normal development of larvae on the arti®cial diet. These undesirable eects have led to the addition of some antimicrobial agents alone or in combinations to the diet and it is necessary to determine whether the insects are aected (BUÈYUÈKGUÈZEL and YAZGAN, 1996,
1999). The results of this research showed that these agents at high levels had adverse eects on the survival and development of the larvae to adult emergence. The physical textures of diets were also aected when antibiotics were used at high levels. For example high levels of the antibiotics caused the diet to become more watery or conversely more gelatinous. High levels of antibiotics also caused formation of some aggregations thus destroying the homogeneity of the diet. This situation may interfere with the nutritional value of the diet and therefore reduce its uptake by larvae. For this reason, the present work has focused on determining the appropriate levels of antibacterial agents that do not aect insect development or survival. It is possible that low levels of antibiotics will minimize changes in the physical properties of the diet. This work focused on the eects of some bacterial DNA gyrase inhibitors such as novobiocin, nalidixic and oxolinic acids on the
survival and development of P. turionellae larvae reared on the arti®cial diet.
2 Materials and methods
Stock cultures of P. turionellae were maintained with their host, the pupae of the greater wax moth Galleria mellonellae L., at 23 1°C, 75 5% relative humidity, and a 16 h photoperiod per day. Adults of P. turionellae were fed with 50% ®ltered honey solution every day, and with host pupae every other day for sucking haemolymph to provide sucient protein.
In the arti®cial rearing experiments the synthetic diet described by YAZGAN(1981) was used for rearing the larvae
of P. turionellae. Further details on preparation of the diet, equipment, techniques for handling and rearing the larvae aseptically for nutritional experiments are described else-where (BUÈYUÈKGUÈZEL and YAZGAN, 1996, 1999). As in the
previous works (BUÈYUÈKGUÈZEL and YAZGAN 2001;
BUÈYUÈKGUÈZEL 2001), the tested diets contained linoleic acid
(a 55% concentrate of linolenic acid) from Nutritional Biochemical Corporation (NBC: Cleveland, OH, USA) instead of linolenic acid from British Drug Houses Chem-icals Ltd (BDH; Poole, England).
The antibiotics, novobiocin (sodium salt, micronized form, 907 lg/mg) and oxolinic acid (99%, micronized form), were gifts from EczacõbasËõ Medicine Company Ltd, Turkey. Nalidixic acid (99%) was purchased from Sigma Chemical Co., St. Louis, MO, USA. The concentrations of the antibiotics were expressed as mg/100 ml of the diet. The required amounts of the agents were dissolved in a certain volume of bidistilled water, solutions were then sterilized by ®ltering with 0.22 lm por-sized membrane ®lter and then predetermined volumes of the antibiotic solutions were J. Appl. Ent. 125, 583±587 (2001)
Ó 2001 Blackwell Wissenschafts-Verlag, Berlin ISSN 0931-2048
added to diets before gel formation. The total volume of the diet was established by adjusting the volume of water in the diet. These completed diets were dispensed into test-tubes (10 ´ 7.5 mm) with about 0.5 ml of diet per tube. This volume of diet is in excess of the nutritional requirements of the larvae for their normal development. A large surface area of diet was dispensed in a lump on the lower wall of reclining test tubes, so that larvae would not move o the diet. One newly hatched larva was placed into each test tube of diet. Ten larvae were used for each diet and each experiment was replicated three times. The feeding experiments were carried out under the same laboratory conditions as described for the stock culture.
The survival and development of the insects were assessed for each antibiotic in the diets. Four diets containing graded levels of each antibiotics were tested and compared with the control diet (without antibiotic): novobiocin at levels of 1.5, 3.0, 4.5 and 6.0 mg; nalidixic acid at 1.0, 2.0, 3.0 and 4.0 mg; oxolinic acid at 0.75, 1.50, 2.25 and 3.00 mg per 100 ml of diet. The eects of experimental diets with antimicrobial agents on the insect were measured by determining the rate of development (average time to reach ®fth instar, pupa and adult emergence) and survival (number of survivors in these stages). Data on the rate of development were evaluated by analysis of the variance (SNEDECORand COCHRAN, 1967). To
determine signi®cant dierences between means DUNCAN's
(1955) multiple range test was used. Data on survival were compared by v2test (SNEDECORand COCHRAN, 1967). When
F and v2 exceeded their 0.05 value the dierences were
considered signi®cant.
3 Results
First, novobiocin, nalidixic and oxolinic acids were tested on insects at levels of 5, 10, 15 and 20 mg. Larvae on diets with the levels higher than 10 mg of the antibiotics did not pupate. The diets were observed to become increasingly sluried during feeding by larvae. Diets with 15 and 20 mg antibiotic were more
watery and larvae placed onto such diets submerged and could not moult to subsequent larval instars. Moreover, the ®fth instar larvae were very small and their development was slow in comparison with those of controls. At 10 mg of novobiocin and 5 mg of nalidixic and oxolinic acids most of the larvae reached to pupal stage but most pupae did not emerge to adults. Based on these pre-experiments, levels lower than 10 mg of novobiocin and the levels lower than 5 mg of nalidixic and oxolinic acids were added to the diets. The eects of dierent levels of novobiocin, nalidixic and oxolinic acids on the survival and development of P. turionellae larvae are shown in the table 1.
None of the levels of novobiocin tested aected the survival of the ®fth instar larvae. Larval development in the diet with 6 mg of the agents was extended by 1 day compared with the control diet. However, this time was shortened by 1 day with the diet containing 1.5 mg of novobiocin. The pupal and adult yields were signi®cantly lower with high novobiocin levels (6 mg). However, the yields were signi®cantly increased by the diet with 1.5 mg of novobiocin. This diet produced the pupal yield of 64.3% and the adult yield of 62.2%. The post-larval development was signi®cantly pro-longed by only the highest level of this antibiotic, compared with the control diet.
Larval survival and development were not signi®-cantly aected by nalidixic acid. However, the yields of pupae and adults were signi®cantly decreased at all tested levels of nalidixic acid. The higher levels (3.0 and 4.0 mg) of this agent markedly decreased adult emer-gence to 13.7 and 13.0%, respectively. The lower levels of nalidixic acid had no signi®cant eects on the development of the insect.
Oxolinic acid had no signi®cant eect on the larval survival. However, larval development was signi®cantly
Table 1. The eects of some DNA gyrase inhibitors alone on the survival and development of the P. turionellae larvae
Levels of anti-biotics (mg of 100 ml of diet) Initial no. of larvae Survival to ®fth instar (%) Time to ®fth instar, (days) (mean1 SD)2 Survival to pupal instar (%) Time to pupal instar, (days) (mean1 SD)2 Survival to adult instar (%) Time to adult instar, (days) (mean1 SD)2 Novobiocin
Control 28 92.9a 11.3 0.2a,b 46.4a 24.4 0.5a 38.7a 32.7 0.8a
1.5 27 88.9a 10.3 0.5c 64.3b 25.0 0.4a 62.2b 33.0 0.0a
3.0 29 86.2a 10.8 0.7ac 20.7c 25.2 0.6a 17.2c 33.3 0.7a
4.5 26 63.2a 11.8 0.4bd 15.4c 25.0 0.0a 15.4c 33.3 0.7a
6.0 28 78.6a 12.3 0.5d 10.7c 26.3 0.6b 10.7c 35.3 0.7b
Nalidixic acid
Control 24 88.3a 10.7 0.5a 44.6a 25.0 0.8a 44.6a 33.7 0.6a
1.0 25 66.0a 11.1 0.3a 20.0b 24.7 0.7a 20.0b 34.2 0.7a
2.0 27 81.5a 11.0 0.9a 25.9b 25.2 0.6a 25.9b 34.2 0.7a
3.0 23 65.2a 11.6 0.4a 13.7c 24.5 § 13.7c 34.5 §
4.0 23 74.0a 11.3 0.4a 13.0c 25.3 § 13.0c 34.5 §
Oxolinic acid
Control 26 88.4a 8.1 0.1a 46.1a 21.1 0.1a 46.1a 29.7 1.8a
0.75 25 88.0a 8.0 0.0a 44.0a 23.0 0.0b 43.0a 32.5 1.9b
1.50 26 92.3a 8.0 0.0a 19.2b 23.0 0.0b 19.2b 33.1 2.1b
2.25 26 88.4a 8.1 0.1a 15.3b 23.0 0.0b 15.3b 33.0 0.0b
3.00 20 95.0a 9.1 0.1b 15.0b 23.0 § 15.0b 33.0 §
1Average of three replicates; 10 larvae per replicate.
2Values followed by the same letter are not signi®cantly dierent from each other, P > 0.05. §: From two replicates.
prolonged by the highest level of this agent compared with control. The higher levels signi®cantly decreased the yields of pupae and adults. However, the lowest level of this agent did not aect survival. Total development time from egg to adult stage was signi®-cantly increased at tested levels of oxolinic acid.
4 Discussion
This work showed that one antibiotic, novobiocin, has signi®cant positive eects on the survival of a parasitic hymenopterous species, P. turionellae. The lowest level of novobiocin enhanced survival and had no signi®-cant eect on the development of the insect. The positive eect of novobiocin seen on P. turionellae holds promise for increasing yields in other parasitoids as well. Novobiocin at low levels may improve the nutritional value of the diet or it may provide more ecient utilization of the diet by the larvae. As suggested by ZUCOLOTO(1987), if a diet is nutritionally
better or of good quality it follows that yields of adults parasitoid will increase. The production of high-quality parasitoids reared in arti®cial conditions is critical to the success of the augmentative release approach. On a chemically de®ned arti®cial diet, hymenopterous endoparasitoid Itoplectis conquisitor larvae produced more adults when compared with those reared on their natural host, Galleria pupae and they were capable of producing a normal second generation on the natural host (YAZGAN, 1972).
Similarly, P. turionellae larvae reared on another synthetic diet resulted in adults with good performance (YAZGAN, 1981). In this work, the post-larval survival
and development were signi®cantly aected by the levels and kinds of lipid mixture in the diets and linolenic acid was an essential nutrient for post-larval development of P. turionellae. Seemingly normal adults of about 60% for this parasitoid were obtained with the control diet containing linolenic acid (BUÈYUÈKGUÈZEL and YAZGAN, 1996). In the present
work lower yields of pupae and adults in controls may be attributed to the substitution of NBC linoleic acid for BDH linolenic acid in diets. The results from the present work are similar to those found in other work where NBC linoleic acid was substituted for BDH linolenic acid (BUÈYUÈKGUÈZEL, 2001; BUÈYUÈKGUÈZEL and
YAZGAN, 2001). Moreover, a meridic diet has produced
signi®cantly higher quality female of hymenopterous ectoparasitoid Catolaccus grandis (Burks) (ROJAS
et al., 1996). The weight, fecundity and survival of the females were higher than those developing on their host. Recently, eorts have been directed toward the use of antibiotics in arti®cial diet to rear adults in sucient numbers of adequate quality to be used in biological control programmes. There have been no studies to investigate the positive eects of antimicro-bial agents alone on parasitoid hymenopterous insects. However, one study with a parasitoid dipterous species Hypoderma lineatum (Villers) showed that a low level of chloramphenicol in the arti®cial diet signi®cantly increased pupation by approximately 70.0% (CHAMBERLAIN and SCHOLL, 1991). It was
demonstrated that novobiocin sodium, at the lowest tested level, caused a slight increase in the pupal and adult yields of another parasitoid dipterous insect, Agria anis auct. nec Fallen (SINGH and HOUSE,
1970a). Yields were decreased inversely but the deve-lopment was increased directly with the increasing levels of this antibiotic. Novobiocin had similar eects on both entomophagous insects but all levels tested on larvae of the present insect were lower than those tested on A. anis. This dierence may be attributed to the unconnected or blind gut of Pimpla larvae (BUÈYUÈKGUÈZEL and YAZGAN, 1996). As the larvae are
not able to defecate gut contents during the active feeding period all diet consumed remains in the insect and likewise the antimicrobial agent.
Negative eects were seen for all antibiotics at high levels. Only novobiocin had a positive eect at the lowest level. Nalidixic and oxolinic acids share more or less a similar chemical structure. Novobiocin also brings a novoise sugar moiety (DAWSON et al., 1986).
This structural property of novobiocin might be responsible for its detrimental eects on the insect when fed at high levels. Similarly, a macrolacton antibiotic, abamectin with a sugar moiety, was detri-mental at high levels to gypsy moth, Lymantria dispar L. (DEECHER et al., 1989). BUÈYUÈKGUÈZEL and
YAZGAN (1996) demonstrated that high levels of
streptomycin, which is an aminoglycoside antibiotic, had negative eects on the survival and development of P. turionellae. It is reasonable to suggest that the increasing levels of the antibiotics may cause a change in the nutritional balance of the diet. For example, with increasing levels of streptomycin in the larval diet of A. anis the rates of growth, devel-opment, and survival were decreased. However, these eects were reduced with increasing levels of nutri-ents (SINGH and HOUSE, 1970c).
It was observed that the diets with high levels of the antibiotics became increasingly more watery during the active feeding period of the larvae in comparison with control diets. These diets produced signi®cantly low yields of adults. The larvae fed on these diets were apparently smaller in size and consequently the adults were more feeble than those on control diet. SINGHand
HOUSE(1970b) demonstrated that most of the
antimi-crobial agents in the diet of A. anis resulted in abnormally small larvae when the levels were increased. It is well known that novobiocin can combine with magnesium ions (DOWNES et al., 1985)
and therefore not available to the insect. Another work suggests that the negative eects of tetracycline on a parasitic protozoan, Plasmodium falciparum may be due to its complexes with calcium ions in the diet (DIVO
et al., 1985). It is possible for the physical texture of the diet to be aected by such interactions. A change in the physical properties of the diet may interfere with its nutritional composition and thus with food intake of the larvae. It has been reported that a semi-liquid form is necessary to support the larvae on the surface and to provide a suitable environment for feeding activity of endoparasitoids Itoplectis conquisitor (Say) (YAZGAN,
1972) and Pimpla turionellae (YAZGAN, 1981) larvae. In
but they are also an environment for the larvae (GRENIERet al., 1986).
It might be expected that non-nutritive dietary additives could in¯uence the feeding activity of the larvae. Possibly, the dierences in development may be caused by dierences in the amount of diet consumed by the larvae. SINGHand HOUSE(1970b) suggested that
antimicrobial agents aected the feeding activity of insects by possible repellent eects. Therefore, dieti-tians stated that although they have no eects on the survival, the use of antimicrobial agents are often undesirable for control of microbial contamination, as even at low levels there is usually an eect on the rate of development in mass rearing of insects (OUYE, 1962;
GREENBERG, 1970; SINGH and HOUSE, 1970a;
BUÈYUÈKGUÈZEL and YAZGAN, 1996). The low level of
the antibiotic novobiocin had a positive eect on the insect, but it may be unable to control micro-organ-isms or their eectiveness may be dependent on the concentration of contamination. Further work is necessary to investigate this important question. It would be valuable to determine the physiochemical basis for these exceptional eects of the antibiotics tested at much low levels. As, it has been reported that these antibiotics inhibit many of the enzymes involved in the replicative process of DNA in eucaryotes (CASTORA et al., 1983; RUSQUET et al., 1984; SHEN
and PERNET, 1985). This aspect should also be look
at with regard to levels in diet. However, some antibiotics are known to have physiologically positive eects on some nutrients in economically important higher animals (ENSMINGERet al., 1990).
This work has demonstrated that novobiocin alone, at low level, enhanced the survival of P. turionellae whereas at higher levels it had a negative eect. Following this work, BUÈYUÈKGUÈZEL (2001) has also
shown that these adverse eects of the gyrase inhib-itors alone on survival and development were greatly overcome by using these antibiotics in combinations in the diets of P. turionellae larvae. These ®ndings were mostly supported by the suggestion of SLANSKY(1982)
in which most of the responses of an insect throughout its life occur within a nutritional context.
Acknowledgements
I am very grateful to Professor Dr SËEVK_I YAZGAN for his
supervision and criticism throughout this work, and to Dr JONBEDICKand Dr HASANTUNAZ(University of Nebraska,
Department of Entomology, Lincoln, NE, USA) for reading and providing useful comments on a draft of this paper. I am also grateful to EczacõbasËõ Medicine Company, Istanbul, Turkey for the gifts the novobiocin and oxolinic acid. References
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Karaelmas University, Science and Arts Faculty, Biology Department, 67100, Incivez, Zonguldak, Turkey. E-mail: buyukguzel69@hotmail.com
