Effects on growth and respiration due to the ingestion of the rapeseed meal glucosinolates in young larvae of Tenebrio molitor

Camp. Eiochem. Physiol. Printed in Great Britain

Vol. 103A,

No. 2, pp. 391-395,

1992 0

0300-9629/92 $5.00 + 0.00 1992 Pergamon Press Ltd

EFFECTS ON GROWTH AND RESPIRATION DUE TO THE INGESTION OF THE RAPESEED MEAL GLUCOSINOLATES IN YOUNG LARVAE OF TENEBRIO MOLITOR PASCALEPRACROS,*~ CHRISTINECOURANJOU* and ROBERT MOREAU: *INRA-Station de Zoologie, B.P. 81, 33883 Villenave d’Ornon Cedex, France. Tel.: 5684-3289; SURA CNRS 1138, Laboratoire Neuroendocrinologie Universit6 Bordeaux I, avenue des Facultis 33405 Talence Cedex, France (Received 7 February 1992; accepted 11 March 1992) Abstract-l.

We investigated the effects of the ingestion of naturally occurring glucosinolates in rapeseed meal on growth rate, metabolic efficiency and respiratory rate in larvae of the yellow mealworm, Tenebrio moliror L. 2. In our feeding studies, larvae were reared on one of seven different diets, including a whole ground wheat control diet and rapeseed meal from six rapeseed varieties. Dry weight gain of larvae and dry food assimilated were measured after 4 weeks of rearing, and the conversion of food into insect biomass was determined. The results may be explained by variations in the glucosinolates content of the diets. 3. The effect of glucosinolates on food consumption, larval growth, expired carbon dioxide, oxygen uptake and respiratory quotient were studied. 4. Glucosinolates did not reduce food assimilation or growth after 1 day of experimentation, but they

caused some inhibition of respiratory exchanges and increased the RQ ratio.

INTRODUCTION

All species of the Cruciferae family contain several allelochemical substances called glucosinolates. Included in this family are the rapeseeds Brassica napus L. and Brussica cumpestris L.; the latter is commonly termed turnip rape. Brassica oilseeds are important commercial crops and much of the seed meal is used as livestock feed. Man’s continued efforts to become more efficient in growing and processing food, have been confronted with the unpalatability and toxicity of oilseed meal from Cruciferae. Poultry and swine can be fed on rapeseed meal but the amount in the diet has to be limited to prevent growth depression and enlargement of thyroid, liver and kidney (Bell, 1984; Bourdon, 1986). The practical solution to this problem has been partly resolved by breeding new rapeseed varieties with low glucosinolates contents (Srivastava and Hill, 1976). Classical varieties such as Jetneuf and Bienvenu can be fed in limited amounts (5% in diet) to monogastric animals; new low-glucosinolate rapeseed varieties such as Darmor and Tapidor may be fed at levels up to 20% in the ration as a source of supplementary protein, instead of soybean meal. The selection pressure for the creation of varieties with low-glucosinolates contents has affected the nutritional value of the new rapeseed varieties for herbivores. Glucosinolates are allelochemicals that defend plants against pathogens and phytophagous invertebrates, especially insects. Such insects are classified into monophagous, oligophagous or tTo whom all correspondence

should be addressed.

polyphagous groups, based on their host-plant range (Chapman, 1974). Polyphagous pests such as Pupilio polyxenes L. (Erickson and Feeny, 1974) or Munducu sextu L. (Schoonhoven and Derksen-Hoppers, 1973) react to a much lower concentration of common glucosinolates than, e.g. insect pests of Cruciferae like Pieris brussicue L. (Rothschild, 1972; Aplin et al., 1976) or Brevicoryne brussicue L. (McGibbon and Allison, 1968), species which are well-adapted to the chemical composition of their host plant. These insects may be attracted to the plant by the glucosinolates or their hydrolysis products. Numerous studies have shown that such chemical attraction may serve in the detection of the plant, ovipositioning of the female and feeding by larvae, however, little is known about the effects of these allelochemicals compounds on the physiology of oligophagous insects. In studies on the possible effects of glucosinolates on the larvae of the swallowtail butterfly, P. polyxenes, Erickson and Feeny (1974) showed growth inhibition and high mortality and suggested that glucosinolates were toxic to larvae. This physiological effect was estimated by Blau et al. (1978) using a “new technique that permits a clearer distinction between inhibition of feeding and toxicity”. Their assay was the measure of the reduction of growth rate at constant consumption rate of larvae fed diets containing increasing amounts of glucosinolates. So, they obtained calibration curves for the effect of different rates of consumption on larval growth rate. If a compound was not toxic but inhibited feeding, the points (consumption, growth) should fall on the calibration line, although they would be concentrated in the range of low consumption rate. 391

PASCALEPRACROSet al.

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Yellow mealworm larvae can be considered as oligophagous insects because of their ability to feed on a large diversity of protein sources. Some studies have shown that larval growth and food utilization are related to plant allelochemicals like alkaloids (Pracros and Anglade, 1984; Pracros and Couranjou, 1986) or saponins (Pracros, 1988a,b). The purposes of the research described here were to determine the consequences of consumption of glucosinolates on growth and food utilization by oligophagous larvae of Tenebrio molitor L., and to assess the effects of these compounds on respiratory rate. MATERIALS AND METHODS

Insects

Mealworms, Tenebrio molitor L., were obtained from stock cultures, reared in whole ground wheat at 25°C and 80% relative humidity in a dark rearing room. We used 7-week-old larvae for this study and all experiments were conducted at 25°C and 80% relative humidity. Experiment diets Preparation of rapeseed meal. Rapeseed meal of six varieties Tapidor, Darmor, Tandem, DCHl, Bienvenu and Jetneuf were prepared by the laboratory of the CETIOM (Centre Technique Interprofessionnel des Oltagineux MCtropolitains) in Bordeaux, France. Rapeseed contains about 40% oil by fresh weight, which had been removed by crushing (SObars) and solvent extraction (six successive extractions with hexane). Removal of hexane (during 40 min) was achieved by injection of water at 105°C. Chemical analysis of the meal produced after oil removal for each variety were conducted by CETIOM laboratory to determine the content of protein, lipid, cellulose and glucosinolates. The chemical methods used are described by the Association Francaise de Normalisation (Afnor, 1985). Preparation of experimental diets, The components per 100 g of diet were: glucose, 76%; MacCollum-Davis salt mixture (No. 185, ICN Flow, Animal Research Diets, Orsay, France), 4%; cholesterol, 1%; rapeseed meal of each variety, 19% and vitamin B solution, lOm1, which contained carnitine and zinc chloride (Fraenkel et al., 1950). Control diets without glucosinolates contained 95% of whole ground wheat (which is the natural feeding of this insect), salt mixture, 4%; cholesterol, 1% and vitamin B solution with carnitine and zinc chloride. Following preparation, each diet was carefully mixed and IS g quantities weighed (five portions). Three portions (5 g each) were dried under vacuum (100 Pa residual pressure) at a temperature level of SO”C, to determine the amount of dried diet proposed to each batch of larvae. Alimentary test

Batches of 20 larvae in the weight range 305 & 2 mg were starved for 24 hr and reweighed before being put on the experimental diets for a period of 4 weeks. Three batches of larvae were dried in uacuo at 50°C (after the starvation period) to determine dried larvae weight before beginning the test. After the experimental period, survival was recorded and the batches of larvae were weighed and dried (in uacuo, at 50°C). The dry weight of larvae was determined and the average dry weight gains were estimated (DWG, mg per larvae). Residual amounts of each diet (with insect feces) were weighed and dried to determine the quantity of dry food assimilated by insects expressed in mg per larvae

[DFA = dry food given--(non-consumed dry food + feces)]. The biotransformation of food assimilated into insect body were estimated by the IS ratio (IS = DFA/DWG). Respiratory test

Two respiratory tests were carried out: the first one in an automatic respirometer to determine the quantity of carbon dioxide expired by the larvae and the second one in a Gilson respirometer to measure the 0, uptake, the CO, and to determine the respiratory quotient (RQ). Batches of 20 larvae were starved for 24 hr, weighed and put on experimental diets for 2 days. These diets were prepared in the same manner as for the alimentary test, but after preliminary tests we retained two rapeseed varieties for use: one with low glucosinolates content (Tapidor) and the second with a high level of glucosinolates (Bienvenu). Wheat control batches were separated in five batches of larvae in starved condition, and five other wheat fed. Firsr respiratory test. After 3, 24 and 48 hr, each batch of 20 larvae were weighed and placed into the “insect test chamber” of an infrared carbon dioxide analyser system (model Siemens, Ultramat 2, MS2014, Lyon, France). The non-ingested food was then weighed to determine the diet consumption. The expired CO, was automatically recorded by a detection system, which consisted of an insect chamber maintained at 25°C by a temperature regulated water bath. It received a continuous flow of air, dried by calcium chloride and cleared of CO2 by potassium chlorate. Results were obtained in pl/hr/mg of fresh weight and expressed in percentage variation of the wheat control diet. Second respiratory test. This study was conducted with a Gilson apparatus (Gilson medical electronics, Villiers-LeBell, France). Eight replicates of 20 larvae were used for this assay. After 24 hr, batches of larvae were weighed and put into the insect chamber of the Gilson respirometer maintained at the constant temperature of 25°C. The expired CO2 and the 0, consumed were measured. Results were obtained in pl/hr/mg of fresh weight, and expressed in percentage variation of the wheat control diet. Statistical analysis

Results of alimentary tests and first respiratory measurements were the average of five replicates; results of the second respiratory test were the average of eight replicates. Each mean value was obtained at 95% confidence level. Data were analysed by one-way analysis of variance and treatment means were compared by the Newman and Keuls test (Statitcf software). In Table I, two means followed by the same superscript are not significantly different at the 5% level.

Table I. Nutritional value of varieties of rapeseed for the yellow mealworm larvae, Tenebrio moliror L., estimated by the dry food assimilated (DFA). the dry weight gain of larvae (DWG) and the conversion ratio (IS).after-4 &eks of diets presentation

Variety Darmor DCH I

DFA DWG (mg/larvae)

IS

Tandem Bienvenu Jetneuf

121.2 (8.3)’ 117.6 (7.8)’ 106.7(7.W I ’ 5.4 (7.2)’ 71.9 (5.2)X 74.8 (6.l)f

22.4(1.2)1/ 22.1 (1.4)11 19.2 (1.5)!i 21.4(1.3)~~~l 11.2(1.2)” 10.8 (1.2)*’

5.4 (O.l)$i 5.3 (0.1):: 5.5 (O.l)$$ 5.4 (0.2)$f 6.4 (0.3)& 6.9 (0.2)#

Control diet

122.5(9.5)’

27.6(1.7#

4.4 (0.2)tt

Tapidor

Results are average of live replicates. The mean value is given with confidence limit at 95% confidence level. Two means followed by the same symbol are not significantly different at the 5% level (Newman and Keuls test).

Effects of glucosinolates on Tenebrio molitor

393

RESULTS

Alimentary

tests

Amounts of protein, lipid, cellulose and glucosinolates of each diet are presented in Table 2. Larvae of T. molitor fed and grew at different rates on the diets prepared with the rapeseed meals of various rape varieties. Results are presented in Table 1. On the basis of DFA and DWG; Darmor, DCHl, Tandem and Tapidor showed greater nutritional value than Bienvenu and Jetneuf, which were classified as poor nutritional quality rapeseeds for this insect. When we compared the results obtained for IS, two different groups could be identified: the group of four varieties with low levels of glucosinolates and one of two varieties which contain high levels of these substances. The biological data obtained with the control diet showed the non-deterrent effects of the poor glucosinolates diets and the greater nutritional value of the wheat which is the natural food of the yellow mealworm. In spite of the relatively moderate data obtained in this present study, coefficients of correlation of DFA, DWG and IS can be calculated in relation to the principal components of experimental diets presented in Table 2. No significant correlations were obtained between biological test data and protein, lipid and cellulose amounts. Nevertheless, correlation coefficients (r) between DFA, DWG or IS and glucosinolates content were significant (r = - 0.950 DFA, r = - 0.949 DWG and r = 0.947 IS). Respiratory

test

Regarding the respiratory exchanges, there were marked differences between the larval groups placed in the four selected experimental conditions. The rate of CO, expired by larvae was always greatest in the wheat control diet and lowest in starved larvae diet (Fig. 1). The effect of glucosinolates against larval respiration was examined. The larger interaction between glucosinolates content of the diets and rate of CO2 outlet was confirmed at all three times of measurement. The CO2 output was greater with larvae fed on Tapidor, than with larvae fed on the Bienvenu diet (Fig. 1). This result was obtained as early as the first 3 hr of feeding, although the differences in food consumption and weight gain between larvae were not significant at the same time (Figs 2 and 3). In comparison with the wheat control larvae, we observed a similar drastic decrease in the rate of O2

3h

24 h

48 h

Time

Fig. 1. EtTects of the different diets on CO, expiration in Tenebrio larvae determined 3, 24 and 48 hr after the beginning of the diet ingestion. The variations in CO, are expressed in percentage of the CO, outlets by the wheat control diet-fed larvae, which were respectively: 1.7 +_0.1, 1.6 & 0.2 and 1.5 + 0.2 pl/hr/mg. (w) Wheat control; (El) Tapidor; (n) Bienvenu; (U) starved larvae. Means of five replicates, *P < 0.05.

consumption: 18 and 50% decreases for Tapidor diet and Bienvenu diet, respectively. These decreases in respiratory exchange observed after glucosinolate ingestion were never as important as those observed in fasting insects: 60% CO2 and 56% O2 in comparison with the wheat control. A significant increase of RQ appeared after high glucosinolates diet ingestion (7%) and a significant decrease was observed with fasting larvae (16%) in comparison with the wheat control fed larvae. DISCUSSION

The biological data obtained using the different diets cannot be explained on the basis of different

Table 2. Amounts of protein, lipid, cellulose and glucosinolates in the experimental diets studied

Variety

Glucosinolates Protein Lipid Cellulose (pmol per IOOg (g per 100 g dry diet) dry diet)

I .4

Tapidor Darmor Tandem DCHl Bienvenu Jetneuf

7.0 7.0 1.7 7.9 6.8 8.1

0.38 0.38 0.38 0.28 0.48 0.48

2.3 2.7 2.2 2.4 2.2 2.2

2.8 5.6 5.9 25.9 26.2

Wheat control diet

9.8

I .9

21.9

0

24h

48 h

‘ime

Fig. 2. Effects of the different diets on larvae weight. The variation of weight were determined 3, 24 and 48 hr after the beginning of the diet ingestion and are expressed as percentage of the weight of wheat control diet-fed larvae which were respectively: 0.36 + 0.02, 1.77 f 0.04 and 2.50 f 0.05 mg/larvae. (m) Wheat control; (N) Tapidor; (B) Bienvenu; (0) starved larvae. Means of five replicates, lP < 0.05.

PASCALEPRACROS et al.

394 z: al

1

GlOO-

TT

6

-5 z 805 z 60g = : 40:

‘z= E zoiz 6 u 07

Tim8 24 h 48 h 3h Fig. 3. Effects of the different diets on larval food consump-

tion determined 3, 24 and 48 hr after the beginning of the diet ingestion. The variations in ingestion are expressed as percentage of the ingestion of the wheat control diet by larvae, which were respectively: 1.27f 0.09, 3.76 + 0.31 and 6.02 + 0.39 mg/larvae. (B) Wheat control; (LSI)Tapidor; (a) Bienvenu. Means of five replicates, *P < 0.05.

protein, lipid or cellulose contents. Larvae of T. m&or reacted to the different amounts of glucosinolates in their diets and they did not starve for any length of time, even on the higher glucosinolate content diets. The early effect of glucosinolates observed on the rate of COZ outlet by insects was an important difference between the results of this paper and previous studies, which investigated relations between insects and cruciferous plants. Another notable difference was the clear demonstration of the metabolic disturbances induced by glucosinolates after the first 3 hr of assay. It has been known for a long time that insect growth is more or less dependent on food quality. The present studies provide evidence that the glucosinate molecules, contained in the rapeseed meals, do not appear to be of great importance regarding the feeding behaviour of T. molitor. Indeed, we did not observe starvation for any of the rapeseed meals fed to the experimental insects. However, it is possible that the insects were able, via their taste receptors, to appreciate food quality (Schoonhoven et al., 1991), but in our experiments mealworm larvae had no possibilities to choose between different foods so, they could only eat or starve. In all cases they appeared to adopt the first solution and their growth, as a function of each of the rapeseed meals, appeared to be equivalent from the beginning of the experiment. The weight decrease of larvae fed with the higher glucosinolate concentrations, appeared to be significantly different only 24 hr after the beginning of the experiment. We may suppose that the mass of the ingested food was in all cases comparable at the beginning of the experiment, but it became progressively different in the case of high concentrations of glucosinolates in rapeseed meal. The insect larvae had no difficulties in ingesting the rapeseed meal but at later times (24-48 hr), they exhibited some assimilation or metabolic problems. However, as compared with the starving

batches of larvae, the insects fed on rapeseed meal grew with less weight gain than control insects. However, the weight gain of rapeseed-fed insects was sufficient for them to reach the next larval pupal and adult stages but more time than the control was required (3 months more). The estimation of respiratory metabolism was of great interest because it was the first physiological disturbance which could be recorded with an automatic detection system for respiratory gas. Indeed, the modifications of respiratory exchanges were significantly perceptible 3 hr after ingestion of the highest glucosinolate content rapeseed meal. The respiratory levels were maintained in the treated insects at higher levels than in the fasting larvae, but the highly concentrated glucosinolates meals induced a 50% decrease in 0, uptake, compared with the control insects. This phenomenon suggests poisoning of respiratory metabolism, but not like insecticide effects which generally induce a large increase in 0, uptake from the earliest time of action (Moreau et al., 1987). Thus, it seems clear that glucosinolates ingestion induces metabolic difficulties which do not kill the insects but require induction of detoxification enzymes. Brattsten and Wilkinson (1977) have shown induction of microsomal mixed-function oxidase in larvae of Spodoptera eridunia with 30 min of ingesting diets which contain glucosinolates molecules (10 or 20% of sinigrin). During the period of glucosinolates action, the growth of experimental insects is slowed but not stopped. Confirmation of respiratory metabolism problems is provided by the increase of respiratory quotients (RQ = 1.08) under the metabolic influence of glucosinolates. All the RQ values obtained are close to the unit. Such high values of RQ are not particularly surprising. Previous studies have shown this phenomenon in Tenebrio molitor larvae (Gourdoux, 1979) which could be explained by the high level of transformation of carbohydrates into lipids in young growing tissues (Trimolieres, 1969). Results obtained show that oxidative metabolism of the high glucosinolates diets fed insects is partially blocked and so insects use less oxygen for their catabolic pathways. We suggest that glucosinolates shift the insects’ metabolism so that there is more carbohydrate and amino acid degradation than lipid oxidation. The in oivo metabolic alterations produced by glucosinolates ingestion have been demonstrated in the ohgophagous insect Tenebrio molitor, and it will be of great interest in the future to study the cellular effects of these molecules by means of in z:itro tissue studies. Acknowledgements-The authors gratefully acknowledge the collaboration of J. Evrard (Centre Technique Interprofessionnel des Oleagineux Metropolitains) for providing the rapeseed meals and the chemical analysis. The authors wish to thank Miss M. H. Davant for typing the manuscript.

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