Starvation and refeeding in Tribolium castaneum ...

Starvation and refeeding in Tribolium castaneum (Herbst). I . Effect on some biochemical components of sixth instar larvae MUSHTAQ A. SALEEM AND A. R.

SHAKOORI'

Can. J. Zool. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/03/13 For personal use only.

Department of Zoology, University of the Punjab, New Campus, Lahore, Pakistan Received July 22, 1985 SALEEM, M. A., and A. R. SHAKOORI. 1986. Starvation and refeeding in Tribolium castaneum (Herbst). I . Effect on some biochemical components of sixth instar larvae. Can. J. Zool. 64: 1628- 1632. Five days of starvation led to a significant increase in the amylase activity of sixth instar larvae of Tribolium castaneum. Acid phosphatase, alkaline phosphatase, and lactate dehydrogenase activities and glucose, glycogen, free amino acids, and RNA contents showed a significant decrease. Other parameters such as glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, total protein, soluble protein, lipids, cholesterol, and DNA content did not show any significant deviation. On prolongation of starvation for 10 days all the biochemical parameters tested were significantly reduced, except for the amylase activity and cholesterol content, which showed highly elevated values. The total and soluble protein content remained unaltered. Refeeding of starved larvae tended to normalize some of the parameters such as glucose, lipids, glycogen, and DNA. On the other hand, lactate dehydrogenase, acid phosphatase, alkaline phosphatase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, free amino acids, and RNA decreased drastically, whereas the soluble protein and cholesterol contents increased considerably in refed larvae. SALEEM, M. A., et A. R. SHAKOORI. 1986. Starvation and refeeding in Tribolium castaneum (Herbst). I. Effect on some biochemical components of sixth instar larvae. Can. J. Zool. 64: 1628- 1632. Cinq jours de jeQneont entrain6 une augmentation significative de l'activitk de l'amylase chez des larves de sixieme stade de Tribolium castaneum. En revanche, l'activitk de la phosphatase acide, de la phosphatase alcaline et de la lactate dkshydrogknase de m$me que les concentrations de glucose, de glycogene, d'acides aminks libres et d'ARN ont diminuk significativement a la suite du jeQne. Les autres pararnetres, notarnrnent l'activitk de la glutamate-oxaloacktate transarninase et de la glutamate-pyruvate transaminase et le concentrations de protkines totales, de protkines solubles, de lipides, de cholestkrol et d'ADN n'ont pas enregistrk de variations significatives. La prolongation du jeQne pour 10 jours a entraink la diminution significative de tous les parametres mesurks, sauf l'activitk de l'amylase et le contenu en cholestkrol qui ont augmentk considkrablement. Le contenu en protkines totales et en protkines solubles est demeurk inchangk. L'alimentation des larves apres le jeQnetend a normaliser certains de ces parametres, tels le glucose, les lipides, le glycogene et I'ADN. Cependant, la lactate dkshydrogknase, la phosphatase acide, la phosphatase alcaline, la glutamate-oxaloacktatetransaminase, la glutamate-pyruvate transaminase, les acides aminks libres et I'ARN ont diminuk skrieusement, alors que les contenus en protkines solubles et en cholestkrol ont augmentk considkrablement chez les larves nourries de nouveau. [Traduit par la revue]

Introduction Tribolium castaneum (Herbst), commonly known as the red flour beetle, is considered to be one of the best known and most serious pests of farm stored grains (Parkin 1956; Munroo 1966). Our ability to control this and many other important insect pests is seriously threatened by widespread resistance to insecticides, particularly in the warmer regions of the world (Green 1975; Champ and Dyte 1977). Although a review of the literature has not indicated the development of resistance in T . castaneum in Pakistan, the phenomenon has been reported by several workers in various parts of the world against chlorinated hydrocarbons, e. g. , BHC/lindane (Bhatia and Pradhan 1972), DDT (Spiers et al. 1971); organophosphates, e. g . , malathion (Spiers et al. 1967); fumigants, e.g., methyl bromide and phosphine (FA0 1975); and some synthetic juvenile hormones (Dyte 1972). The residue and (or) contamination and resistance problems resulting from the use of persistent and other insecticides have, therefore, focussed an increased interest on various other methods of control. In this laboratory synthetic pyrethroids are being tested as potential insecticides for the protection of stored grain against T . castaneum in Pakistan. Most of these studies concern the biochemical effects of pyrethroids on starved and fed insects, as bothofthesenutritionalstatesarelikelytoinfluencethedegree of insecticidal toxicity. The present report aims at providing ' ~ u t h o rto whom all correspondence should be addressed.

base-line data on the biochemical changes induced by total starvation. Stored grain insects do not frequently face total lack of food except perhaps during periods between grain shipments, when the storage facilities are cleaned. In nature, however, the ability to withstand starvation could have important evolutionary consequences if food supplies in a locality were to be exhausted by the population and migrating individuals in search of other food sources were forced to go through some periods of starvation (Sverdlov and Wool 1975). Furthermore, although the response of higher animals to stress is fairly well understood (Scharrer and Scharrer 1963), very little information is available on the effect of environmental stresses (starvation is one such stress) on physiological and biochemical systems of insects (Dahlman 1973). In the absence of sufficient biochemical data on starved T . castaneum available in the literature (Sverdlov and Wool 1973, the present studies were necessary to generate the basic data under total starvation conditions. These data will be of help for planning further studies on application of insecticides. Materials and methods

Rearing Of beetles The master culture of the red flour beetle, T . castaneum, was obtained from the Food Storage Research Institute of the Pakistan Agriculture Research Council, Karachi, and was maintained in a tem~era~e-con~olledlaborafor~at30~1~Cwi~relativehumid 60 5%. The insects were reared in empty jam jars covered with muslin cloth and whole-meal flour was used as the culture medium. Sixth instar larvae, collected 28 2 1 days after egg laying, were used in the present study.

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Procedure adopted About 1500-1600 newly emerged sixth instar larvae, which originated from the same stock and were collected on the 23rd day after hatching, were deprived of food and were kept in 15- 16 covered Petri dishes. About two-thirds of the larvae were analyzed biochemically 5 and 10 days after starvation. The rest were refed for 5 days on whole-meal flour after 10 days of starvation and were then also analyzed biochemically. Another group of 500 insects from the above stock (and which were, just like experimental insects, collected on the 23rd day after hatching) were biochemically analyzed and labelled as control for all the experimental groups. Since the control larvae were analyzed on the same day as the experimental insects were put on starvation, these control insects were also labelled as day 0 insects. Larvae that died during experimentation were not included in the various tests, and only living insects were used for biochemical analyses. All the biochemical parameters in starved and refed groups were compared with those of day 0 insects. Record of mortality and loss of body weight To determine the loss in total body weight and mortality due to starvation, a group of 250 insects collected 23 days after hatching was placed in five Petri dishes without any food. A daily record of change in total body weight and mortality, if any, was maintained. Loss in total body weight was determined with reference to the weight of the insect on day 0 of the experimental period. Biochemical analyses About 60 larvae weighing a total of 117 to 120 mg in each replicate were crushed in saline (0.89%) with the help of a motor-driven glass homogenizer. The homogenate was centrifuged at 4900 X g for 45 min at 4°C. The supernatant thus obtained was used for the estimation of acid phosphatase (Ac.P; orthophosphoric-monoester phosphohydrolase; EC 3.1.3.2) and alkaline phosphatase (Ak.P; orthophosphoricmonoester phosphohydrolase; EC 3.1 .3.1) activities according to King and King (see Wootton 1964), lactate dehydrogenase (LDH; EC 1.1.1.27) activity by a method based on Cabaud and Wroblewski (1958), glutamate oxaloacetate transaminase (GOT; L-aspartate:2oxoglutarate aminotransferase; EC 2.6.1.1) and glutamate pyruvate transarninase (GPT; L-alanine:2-oxoglutarate aminotransferase; EC 2.6.1.2) activities according to Reitman and Frankel (1957), and amylase (1 ,4-a-D-glucan glucanohydrolase; EC 3.2.1.1) activity according to the procedure described by Wootton (1964). The saline extract was also analyzed for soluble protein contents by the method of Lowry et al. (195 I), free amino acids (FAA) according to Moore and Stein (1954), and glucose content by the 0-toluidine method of Hartel et al. (1969). For the estimation of cholesterol and lipid content about 30 larvae (total weight of 60 to 65 mg) in each replicate were crushed in ethanol and left overnight before centrifugation at 1300 x g for 15 min. The ethanol extract was used for estimation of cholesterol contents by a method based upon the Liebermann-Burchardt reaction and ethanolsoluble lipid content according to the method of Zollner and Kirch (see Henry 1964). For the estimation of glycogen by the anthrone method as illustrated by Consolazio and Iacono in Albanese (1963) only two larvae, weighing a total of 4-5 mg in each replicate, were used. Nucleic acids were extracted from about 30 larvae (weighing about 60 to 65 mg in all) according to the method described by Shakoori and Ahmad (1973). The DNA and RNA contents were estimated by using the Schmidt and Thannhauser method, which has been described by Schneider (1957). The total protein contents were estimated from the digested pellet left after the extraction of nucleic acids. This pellet was digested in 0.5 N NaOH and then used for colorimetric estimation by the method of Lowry et al. (195 1).

Results EfSect on total body weight and mortality Sixth instar larvae of T. castaneum under total starved conditions survived 18 days. The percent mortality and percent loss in total body weight of living larvae at optimum tempera-

TABLE 1. Mortality and loss in weight of sixth instar larvae of Tribolium castaneum at 30°C and 60% relative humidity after starvation

Days

Weight of larvae, mg (mean 2 SEM)

% loss in

weight

Numbers living

% mortality

ture of 30°C and 60% humidity are shown in Table 1. The weights of only living larvae were accounted for, whereas the dead larvae were discarded. The average larval weight decreased from 2.322 + 0.017 mg to 1.86 + 0.025 mg at day 5 and to 1.552 + 0.058 mg at day 10, showing a 19 and 33% weight loss, respectively. Refeeding 10 days starved larvae for 5 days resulted in a weight increase to 1.826 +- 0.1 17 mg, which was still 21% less than the initial weight. Fifty percent mortality occurred after about 8 days of continuous starvation, while 100% mortality was achieved in 20 days. The maximum weight decrease was found on the 12th day of starvation (38%), after which mean weight fluctuated, probably because of the elimination of weaker and susceptible larvae among the population.

Effect on some enzyme activities Total starvation for 10 days led to a significant reduction in almost all enzyme activities of sixth instar larvae except amylase, which showed an elevated activity (Table 2). Of all the enzymes tested in the present study, amylase, Ac .P, Ak.P, and LDH were found to be the most sensitive. In 5 and 10 days starved larvae, significant reduction in the activities of Ac.P (by 57 and 65%, respectively), Ak.P (47 and 8 1%, respectively), and LDH (65 and 77%, respectively) were recorded. When 10 days starved larvae were refed for 5 days, these enzymatic activities increased, but after refeeding the activities of Ac .P, Ak.P, and LDH were still 58,44, and 55%, respectively, lower than the normal values. The amylase activity, on the other hand, increased 79 and 19%, respectively, after 5 and 10 days of starvation, whereas it decreased 26% after refeeding. The GOT and GPT activities were not altered until day 10 of starvation when they showed a 36 and 34% decrease, respectively. The transaminases remained 22 and 33% lower than normal, respectively, even on refeeding. Effects on some other biochemical components The parallel biochemical changes in other parameters tested in the present investigation are also shown in Table 2. Glycogen and glucose content showed drastic decreases after starvation. On days 5 and 10 of starvation, the glycogen content decreased 68 and 72%, while the glucose content was reduced by 44 and 85%, respectively. After refeeding the glycogen content was apparently recovered, while the glucose content was still 5 1% below the normal level. Lipids were not affected by the initial starvation for 5 days, but were significantly depleted (42%) after 10 days of starvation. Lipid content attained the normal

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TABLE2. Effect of total starvation and starvation

+ refeeding on the various biochemical components of sixth instar larvae of T . castaneum


Parametersa

Control larvae ( n = 9)

5 days starved larvae (n=3)

10 days starved larvae (n = 3)

+

10 days starved 5 days refed larvae (n=3)

Glycogen (pgllarva) Glucose (pgllarva) Lipids (pgllarva) Cholesterol (pgllarva) Total protein (pgllarva) Soluble protein (pgllarva) FAA (pgllarva) DNA (pgllarva) RNA (pgllarva) Amylase (SUIlarva) Ac.P (IUIlarva) Ak.P (IUIlarva) GOT (IUIlarva) GPT (IUIlarva) LDH (IUIlarva) "Abbreviations used: Ac.P and Ak.P, acid and alkaline phosphatase; FAA, free amino acids; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; LDH, lactate dehydrogenase. bMean + SEM. 'Student's I-test; P < 0.01. dStudent's I-test; P < 0.001. 'Student's I-test; P < 0.05.

level after 5 days of refeeding. Cholesterol concentration was likewise unaffected after 5 days of starvation, but deprivation of food for 10 days led to an increase of 72%. On refeeding for 5 days, the cholesterol content showed a tendency towards normalization but was still 21% more than the normal values. The total and soluble protein content remained unaltered under all experimental conditions, except for the soluble protein content which was significantly elevated in the refed group. The FAA content, on the other hand, proved to be more sensitive to starvation. The 5 and 10 days of starvation resulted in a 27 and 49% decrease, respectively. During refeeding for 5 days the value remained 25% below the normal level. The DNA content, when considered in terms of micrograms per milligram body weight, did not show any change after starvation or starvation + refeeding. When the total DNA content per larva was taken into account, the 5 days of starvation did not produce any significant alteration, while 10 days starvation resulted in a 39% decrease. Refeeding for 5 days once again normalized the DNA level. RNA content was more drastically affected. When considered in terms of micrograms per milligram body weight, the RNA content remained unaffected after 5 days of starvation, but was significantly decreased (40%) after 10 days of starvation. On refeeding the RNA content (micrograms per milligram) still remained significantly lower (34%) than the normal level. A similar trend is maintained when total RNA content per larva is taken into consideration. The RNA and DNA content hold a ratio of 9.96, which is reduced to 6.41 after 5 days of starvation, and to 6.53 after 10 days. Refeeding does not improve the condition. The total protein contents in relation to RNA content hold a ratio of 21.63, which is increased 2 times after 5 days and 2.83 times after 10 days of starvation. Refeeding tended to normalize this relationship.

Discussion A temporary voluntary starvation or circumstantial deprivation of food is detrimental to the body, longevity, and fecundity

of animals (Cascarano et al. 1978). During this abnormal state, the organism has to depend entirely on its own reserves for sustenance of life (Shakoori and Haq 1982), which entails drastic molecular readjustments (Parrilla 1978a, 1978b). Hence various biochemical abnormalities developing in any insect pest because of total starvation could play an important role in controlling it with or without other methods of control. This preliminary study has provided some basic data that may help in integrating this stress reaction with some recently developed insecticides at the later stages of this study. Moreover, in our opinion and also as pointed out by Sokoloff (1977) Tribolium can prove to be a good model system for biochemical studies on the effects of different types of stress conditions in insects, for these beetles have advantages such as ease of culture, remarkable reproductive potential, and a short life cycle. Five and 10 days of total starvation led to a drastic loss in the body weight of sixth instar T. castaneum larvae. Refeeding did not cause an unusual increase in the body weight, which has been reported from different laboratories for other insects (Sverdlov and Wool 1975) and rats (Szepesi et al. 1975). The glucose and glycogen content decreased significantly 5 and 10 days after starvation. Lipids, on the other hand, remained unchanged initially but showed decreased levels during an extended period of starvation. Under total deprivation, food energy was, therefore, obtained by mobilization of carbohydrates during the initial stages, and later by mobilization of lipids, as evidenced by their significant decrease during starvation for 10 days. The FAA content is also utilized for the same purpose. The present results do not tally with the data of Mwangi and Goldsworthy (1977) for adult female Locusta migratoria migratorioides. They reported an inverse relationship between haemolymph lipid and carbohydrate during starvation. On the other hand, similar results of Hill and Goldsworthy (1970) have shown that the haemolymph carbohydrate content of fifth instar larvae of Locusta was maintained at a constant level during the initial phase of starvation, falling thereafter, and that fat body

SALEEM AND SHAKOORI


glycogen was utilized before fat body lipid as an energy source during starvation. Immediate rise of the amylase activity initially and its continuous reduction subsequently in the present experiments also probably indicate the metabolism and utilization of starch present in the body. With the depletion of starch, the amylase activity also tended to decrease in the larvae. An increased level of cholesterol after 10 days of starvation is rather unusual. Whether some other lipid metabolites have been routed in this direction cannot be determined in the present studies. After refeeding, the glucose, glycogen, lipids, cholesterol, and FAA contents either reached the normal level or were on their way to normalization. No overshoot of these biochemical components after refeeding was found. Refeeding, therefore, remedied and readjusted these anomalies. Total and soluble protein content remained unaltered under starved conditions reported in this work. Refeeding increased the soluble protein, but the total protein did not exhibit any significant variation when compared with the values of control larvae. Similar results were obtained by Jutsum et al. (1975) who suggested that the haemolymph protein content of Locusta migratoria did not change significantly under conditions of food deprivation. Lim and Lee (1981), however, have shown a significant decrease in the total haemolymph lipid, carbohydrate, and protein in adult 18-day-old grasshoppers deprived of food for 96 h. Starvation had some drastic effects on the nucleic acid content of T. castaneum larvae. RNA appeared to be more sensitive as it was significantly depleted during short as well as longer periods of starvation. The DNA content, on the other hand, was depleted only after prolonged starvation. On refeeding, the RNA content remained significantly reduced, although it was on its way to normalization. In contrast the DNA content reached a normal level in starved refed larvae. This finding in larvae contrasts with previous reports from other laboratories, which have shown decreased RNA and unaltered DNA contents after starvation (Goodman and Ruderman 1980; Laird et al. 1955; Stocco et al. 1977). Quite a number of previous studies have shown decreased enzymatic levels after starvation (Freedland 1967; Sober6n and SAnchez 1961). A similar gradual decrease in Ac.P, Ak.P, GOT, GPT, and LDH was recorded in the starved larvae. Refeeding could not make readjustments of the enzyme activities, which remained significantly reduced compared with the control larvae, although they were also on the route of normalization. It appears from the results of this study that larvae of T. castaneum make use of carbohydrate, lipid, and FAA during starvation for sustenance of life. This macromolecular change is also accompanied by drastic abnormalities in the enzymatic activities and other biochemical components, a condition which persists even after refeeding. This would presumably make the survivors more susceptible to exposure to insecticides under field conditions. Keeping in mind that a 10-day starvation period is sufficient for major biochemical alterations, which did not fully normalize in the refed larvae, we are at present studying the interaction of starvation with insecticides s o as to aid in the development of suitable future control programmes against this noxious pest.

+

Acknowledgements The assistance and cooperation of S. S . Ali, Department of Zoology, University of the Punjab, New Campus, Lahore, Pakistan, during the initial part of this study is gratefully acknowledged. The suggestions of three anonymous reviewers

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greatly improved the quality of this manuscript, which forms part of a Ph.D. research project of the first author.

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