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Intermediate metabolism during the ontogenetic development of Anastrepha fraterculus (Diptera: Tephritidae)
Comparative Biochemistry and Physiology, Part A 147 (2007) 594 – 599 www.elsevier.com/locate/cbpa
Intermediate metabolism during the ontogenetic development of Anastrepha fraterculus (Diptera: Tephritidae)☆ B.K. Dutra a , F.A. Fernandes a , J.C. Nascimento, F.C. Quadros b , G.T. Oliveira a,⁎ a
Laboratório de Fisiologia da Conservação, Faculdade de Biociências, PUCRS, Brazil b Laboratório de Entomologia, Museu de Ciências e Tecnologia, PUCRS, Brazil
Received 13 April 2006; received in revised form 30 June 2006; accepted 24 August 2006 Available online 30 August 2006
Abstract The fruit fly Anastrepha fraterculus is a major pest of native and exotic fruit trees in South America. Changes in weight, water content and metabolism were observed during its ontogenetic development in standard conditions (25 °C, RH = 60% and 14 h:10 h photoperiod). The metabolic variables glycogen, total proteins, triglycerides and total lipids were measured by means of spectrophotometric methods. The results were correlated with pupae metamorphosis, temporal pattern, and beginning of adult life. Pupae were observed daily, and a sub-sample of 10 individuals was collected and maintained at − 20 °C. The same procedure was performed with adults at 4 days after adult eclosion. Levels of total lipids and triglycerides were constant during pupal development, peaking in 312-h-old pupae. In 0-h-old pupae, glycogen levels were high, and decreased progressively until the insects were 312 h old. The peak in total proteins coincides with the post-histolysis period of the larval tissue (96–120 h). These results indicated that glycogen and proteins may be the principal sources of energy for metamorphosis. Total lipid and triglyceride contents remained steady during metamorphosis, and these were consumed in the first 4 days of adult life. © 2006 Elsevier Inc. All rights reserved. Keywords: Anastrepha fraterculus; Developmental biology; Tephritidae; Insect; Pupal metabolism; Metabolic changes; Lipids; Proteins; Carbohydrates
1. Introduction Fruit flies (Tephritidae, Diptera) are insects belonging to a family with a cosmopolitan distribution limited only by host availability. Species of this family are classified in two groups based on physiological and ecological characteristics (Steck and McPheron, 1996). The first group includes univoltine species that have diapause, occurring in temperate regions, such as the genus Rhagoletis. The second group comprises multivoltine species ☆ This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. Depto. de Ciências Fisiológicas, PUCRS, Av. Ipiranga, 6681 Pd. 12A, CP. 1429, CEP 90619-900, Porto Alegre, RS, Brazil. Fax: +55 3320 3612. E-mail address: [email protected] (G.T. Oliveira).
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that do not have diapause, and are distributed in tropical regions, such as the genera Anastrepha, Ceratitis and Bactrocera. In general, the genus Anastrepha is highly polyphagous, using hosts from 18 families of plants in 29 genera (Zucchi, 2000). These plants are native to South America and the Antilles, or are introduced from Europe, Africa or Asia. More than 80 host species have been recorded for Anastrepha fraterculus (Norrbom and Foote, 1989); however, these data may be an underestimate of the actual number of hosts. According to Norrbom and Kim (1988), only 50% of the hosts of the Brazilian species of Anastrepha are currently known. In southern Brazil, the dominant species infesting apples is A. fraterculus. The broad range of distribution of the family Tephritidae, and the high adaptative and colonizing capacities may be related to its reproductive potential, which is linked to morphological characteristics developed in the pre-imaginal stage (Morgante, 1991). The holometabolous insects are characterized by a clear separation between larval, pupae and adults, molting from larvae to pupae and complete metamorphosis to the adult during the pupal instar, without any energy uptake where the energetic balance is usually
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negative (Schmolz and Lamprecht, 2000). However, analyses of the ontogenetic development of insects of economic importance, such as A. fraterculus, are in their infancy. During metamorphosis in insects, a dramatic transformation of plan occurs, and most of the larval tissues degenerate and disappear over a very short period of time. This programmed tissue disintegration, called histolysis, occurs sequentially and involves a combination of cytolytic and phagocytic processes. The end-products of histolysis are used to build adult structures through the process of hystogenesis (Lockshin, 1969; Rabossi et al., 2000). Schmolz and Lamprecht (2000) showed for different species of holometabolous insects that the general metabolic rate, measured by calorimetry, usually follows a U-shaped curve. That is, energy consumption is high during the first stages of metamorphosis, declines toward the mid-pupal stage, and increases again toward the last phases of the larval–adult transformation (Langley, 1970; Agrell and Lundquist, 1973; Nestel et al., 2003). Although it is generally accepted that lipids (mainly triacylglycerols) and carbohydrates (mainly glycogen and trehalose) are the main sources of energy during metamorphosis, little is known about the differential utilization of all these energetic metabolites and the role of proteins in this process (Langley, 1970; Agrell and Lundquist, 1973; Nestel et al., 2003). According to Hernandez-Ortiz et al. (2004) A. fraterculus is a major pest of native and exotic fruit trees in South America. The present study investigated aspects of the intermediate metabolism during the ontogenetic development of A. fraterculus, in order to improve understand of the physiology of the A. fraterculus. 2. Materials and methods 2.1. Sampling Larvae of A. fraterculus were collected from infested guava fruit (Psidium guajava L.) near Porto Alegre, state of Rio Grande do Sul, Brazil. Pupae from the first generation (F1) obtained in the laboratory were collected at 24-h intervals and weighed on an analytical balance; one group was stored in at − 20 °C for biochemical analyses, and the water content of the others was determined according to Force et al. (1995). The insects were maintained in standard laboratory conditions (temperature 25 °C, humidity 70–80%, photoperiod 14:10 light/dark), following the protocol described by Nascimento and Oliveira (1996). Adults were fed ad libitum with soy protein, dark sugar, and wheat (1:3:1), and provided with water. The determinations of the different metabolites were repeated three times, in three cohorts of insects. 2.2. Biochemical analyses A pool of five pupae of the each cohort (15 animals) was homogenized with an Omni Mixer homogenizer in a solution of methanol-chloroform 2:1 (v/v), according to Folch et al. (1957). Total lipids were determined in this homogenate using the
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sulphophosphovaniline method (Frings and Dunn, 1970). Triglycerides were measured by means of the reactions of lipase, glycerokinase, 1-P-glycerol oxidase and peroxidase enzymes (Biodiagnostic kit/GPO Trinder). The results are expressed in mg/g of insect. Glycogen was extracted from a pool of five insects of the each cohort (15 animals), following the method described by Van Handel (1965), and determined as glucose-equivalent (glucose-oxidase method) in quadruplicates after acidic hydrolysis (HCl) and neutralization (Na2CO3) following the method of Geary et al. (1981). Glucose was quantified using a Biodiagnostic kit (glucose oxidase). The results are presented as mg/g of insect. Total proteins were quantified following the method described by Lowry et al. (1951), using bovine albumin (Sigma) as the reference. The results are presented as mg/mL. 2.3. Statistical analyses Analysis of variance (ANOVA) was used to analyze the results for intermediate metabolism; a Bonferroni test was performed for multiple comparisons. The weight of the insects and their water content were analyzed using linear regression analysis. All tests were run using the program Statistical Package for the Social Sciences (SPSS), version 11.5 for Windows. The results are expressed as mean ± standard error of mean. 3. Results At 25 °C the development of pupae until eclosion of the imago lasted approximately 336 h, 2 weeks of development within the puparium, with a delay of approximately 6 h. Data in Table 1 show that the average weight of pupae was very changeable during the pupal development, with a gradual reduction until 72 h (pupae), followed by an increase until 120 h and a decrease by 192 h, and then remained at this level until hour 312. Linear regression analysis indicated that pupal weight
Table 1 Wet weight, dry weight and water content Time of development
Wet mass (mg)
Dry mass (mg)
Water content (%)
Pre-pupae Pupae 0 h Pupae 24 h Pupae 48 h Pupae 72 h Pupae 96 h Pupae 120 h Pupae 144 h Pupae 168 h Pupae 192 h Pupae 216 h Pupae 240 h Pupae 264 h Pupae 288 h Pupae 312 h
18.09 ± 0.60 16.48 ± 0.51 16.63 ± 0.28 18.09 ± 0.50 11.96 ± 0.72 13.82 ± 1.04 16.05 ± 0.75 10.80 ± 0.57 11.34 ± 1.08 08.90 ± 0.61 11.29 ± 0.60 09.76 ± 0.86 10.97 ± 0.63 11.97 ± 0.32 11.29 ± 0.43
4.64 ± 0.24 3.88 ± 0.12 5.60 ± 0.12 6.42 ± 0.18 5.57 ± 0.12 6.62 ± 0.12 6.26 ± 0.22 5.28 ± 0.19 4.47 ± 0.36 4.15 ± 0.29 4.48 ± 0.21 4.43 ± 0.29 4.59 ± 0.32 3.90 ± 0.14 3.95 ± 0.13
74.46 ± 0.60 76.45 ± 0.76 66.33 ± 0.43 64.50 ± 0.34 51.99 ± 2.90 49.04 ± 4.93 60.14 ± 2.61 50.71 ± 1.15 57.78 ± 5.05 52.19 ± 3.94 59.29 ± 3.13 52.75 ± 3.06 58.08 ± 1.71 66.61 ± 0.99 64.74 ± 1.31
Results represent the mean ± standard error of the mean, and are expressed in mg (weight) and percentage of water (%).
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Fig. 1. Total lipid levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
was significantly dependent on pupal instar (R2 = 0.31; F = 218.91; p b 0.001; a = 16.46; b = − 0.32); i.e., the puparium loses weight as the adult differentiates. Water content decreased in the first 96 h of pupal development, and then oscillated until the end of development (hour 312) (Table 1). The weight of the animal and water content at day 4 of life were not determined after the eclosion of the adult. Levels of total lipids were constant until 48 hours of pupal development, and then increased ( p b 00.05) by about 100% by the time that the pupae were 72 h old. The levels then remained constant until hatching and imago emersion, but afterwards decreased ( p b 0.05) to 60% in 4-day-old males and females (Fig. 1).
Levels of triglycerides were elevated in animals in the prepupae stage, and decreased by about 55% in pupae at T0 ( p b 0.05). These levels remained constant up to hour 48 of pupal development. At hour 72, the triglycerides showed an increase ( p b 0.05), followed by a significant decline in 96-h-old pupae. The levels then remained constant until the imago emerged. In 4-day-old flies, triglycerides levels showed a significant decline, in both males and females (Fig. 2). Fig. 3 shows that from the pre-pupal stage (larvae) until 0-hold pupae, the levels of glycogen increased by about 120% ( p b 0.05). From 0-h-old pupae until hour 72 of development, glycogen decreased to a low level, then increased again to the initial level by hour 96 of pupal development (24-h-old pupae).
Fig. 2. Triglyceride levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
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Fig. 3. Glycogen levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals) and the vertical bars represent the standard error of the mean.
The glycogen levels then remained constant until the stage preceding the imago emersion (hour 312). In 4-day-old males, the levels of glycogen were similar to those observed during the pupal development; whereas in females, glycogen levels increased significantly. Fig. 4 shows that between the pre-pupae and 0-h-old pupae there was an increase in the levels of total proteins ( p b 0.05). At 72 h of development, pupae show a peak of total proteins, followed by a decrease in the level of proteins in 96- and 120-h-old pupae. Between 144 and 312 h of pupal development, the concentration of protein begins to increase ( p b 0.05). The levels of proteins are highest in 312-h-old pupae, compared with larvae or pre-pupae ( p b 0.05). At 4 days of adult life, males and females show levels of total proteins up
to 3500 times higher than those observed during pupal development. 4. Discussion Our results demonstrate the occurrence of changes in weight, water content and intermediated metabolism which are stagespecific for the differentiation of the adult. The time of development (330 h until 336 h) verified in this work confirm the time of development that was observed by Taufer (1995) studying A. fraterculus under the same experimental culture conditions. Water content decreased in the first 96 hours of pupal development (312 h) (Table 1). Langley (1970) and Nestel et al.
Fig. 4. Total protein levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
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(2003) showed that the Mediterranean fruit fly (C. capitata) undergoes a relatively large water loss during the pupation and pupal phases (outset of the pharate-adult stage). Considering that in this work, was observe an increase of the total lipids in 72 h of pupal development and an intense decrease in 4 days old in males and females (Fig. 1), these results partially coincide with those of Municio et al. (1980), Pagani et al. (1980) and Nestel et al. (2003), but confirm the differential lipid utilization during larval to adult transition. Langley (1970) observed a constant rate of utilization of total lipids during the entire metamorphosis. However, Municio et al. (1980) and Pagani et al. (1980) showed a small decrease in total lipids during what seemed to be the pre-pupae and pupal phases. Their data also show that this small decrease during the first half of metamorphosis is followed by a more drastic utilization of lipids just a few hours before adult emergence. Nestel et al. (2003) showed in Ceratitis capitata a significant increase in lipids between 8 hours before pupae formation and 20.5 hours after pupae formation; from this point on, lipids begin to be consumed. This decrease in lipid load coincides with the disintegration of the larval fat body, as evidenced by histological sections of pupal tissue. Levels of triglycerides decreased (55%) during pre-pupae to pupae (0 h) transition and this results suggest that triglycerides are used by the animals during metabolic adjustment when the larval more away from food. At 72 h, the triglicerydes showed an increase ( p b 0.05), decline in 96 h old pupae, and levels remained constant. The results obtained for total lipids and triglycerides suggest that these metabolites are stored by the animals during pupal development, to be consumed during the imago emersion (Figs. 1 and 2), in the first flight activities, and searching for food in the first 4 days of adult life. Nestel et al. (2004, 2005) showed in C. capitata that pupae seem to regulate lipid loads towards a certain optimum level for adult emergence, regardless of the larval diet history or lipids content. In Anastrepha serpentina, Jacome et al. (1995), observed an initial drops of lipid reserves between 4 days and 6 days after adult emergence, this results suggests that when newly emerged flies encounter a protein source, metabolic activity and requirement rise. During these initial days in A. serpentina, lipids reserves may be used as an energetic source for the development of reproductive and endocrine tissues, and for flight activities. Fig. 3 shows that from the pre-pupal stage (larvae) until 0-hold pupae, the levels of glycogen increased by about 120%. This increase may be related to a similar decrease in triglycerides reserves, suggesting that the glycerol obtained from the catalyses of triglycerides is used in the glyconeogenic pathway. Several studies have demonstrated this glyconeogenic capacity in insects (Wigglesworth, 1942; Ishay and Ikau, 1968; Storey and Baley, 1978). After this increase our results indicate that, for both sexes, glycogen may be the main source of energy for differentiation of the adult individual during the pupal stage, because between 0 h pupae until 72 h of development this polysaccharide decreased 10 times (Fig. 1). In females, probably glycogen is also used as a metabolic reserve for reproduction (production and release of eggs). The significant increase in glycogen levels observed in the first
4 days of adult life may explain its role in the reproduction of the females. The increase in this polysaccharide in the first four days of adult life of A. fraterculus may be determined by the feeding behaviour and/or food source and/or by glycerol, a subproduct of triglyceride degradation during this period. Webster et al. (1979) showed that in Rhagoletis pomonella, females consume more carbohydrates than males, probably because of their more intensive body metabolism and movements related to egg production and searching for a host. The ingestion of carbohydrates in females increased more in the first week of adult life, decreased in the second and third weeks, and then remained stable until the end of the experiment (45 days). Canato and Zucoloto (1998) observed in C. capitata that sugars are the most important nutrients for adult females since they somehow succeed in producing eggs without ingestion a protein source, although production of eggs increases with protein ingestion. The results observed in this work for glycogen (Fig. 1) agree with the data presented by Tolmasky (2001) and Nestel et al. (2003) for different phases of the metamorphosis of C. capitata. They too found an initial decrease in glycogen reserves in the metamorphosis (pre-pupae and onset of the pupal stage), followed by an increase in this polysaccharide at the end of the pupal stage. Fig. 4 shows that between the pre-pupae and 0-h-old pupae there was an increase in the levels of total proteins. Rabossi et al. (2000) observed in C. capitata an increase in total proteolytic activity between 40 and 44 h after immobilization of the larva. This occurred approximately 1 day after apolysis, which marks the onset of the pupation process (Rabossi et al., 1992). The 40–44-h period of metamorphosis corresponds to the transition from pre-pupae to pupae, when the outermost layers of cuticle begin to be deposited (Boccaccio and QuesadaAllué, 1994; Rabossi et al., 2000). It is probable that the carbon atoms derived from amino acids are incorporated into glycogen molecules. Results reported by Nestel et al. (2003) suggest that during the initial phases of metamorphosis of the Mediterranean fruit fly, a portion of the amino acid pool may be directed into the tricarboxylic acid cycle and may used for energetic needs or other metabolites with glycogen. Between 144 and 312 h of pupal development, the concentration of protein begins to increase. The levels of proteins are highest in 312-h-old pupae, compared with larvae or prepupae. At 4 days of adult life, males and females show levels of total proteins up to 3500 times higher than those observed during pupal development. Larval protein breakdown during the initial stages of Mediterranean fruit fly metamorphosis and the subsequent resynthesis of adult proteins has been described by Rabossi et al. (2000) and Nestel et al. (2003). Agrell (1961) found in Calliphora erytrocephala a sudden decrease of precipitable protein at pupation. Our results showed that during the pharate adult stage, the amount of total proteins was restored, reaching values higher than those recorded for the pre-pupae. This work demonstrate the occurrence of changes in utilization of the energetic reserves of A. fraterculus which are stage specific for the differentiation of the adult. During the histolysis of the larval tissues, the levels of glycogen and total proteins are
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high; however, after 72 h all this material is utilized, probably for synthesis of muscles, skin, and other body tissues of the adult fly. In contrast, the levels of lipids and triglycerides remain high during metamorphosis (specific in the pharate adult) to be used by the adult in its first activities (flight, searching for food, mating and egg production). The regulation of these energetic reserves during pupation and first days of adult life is an intriguing question that deserves to be further investigated. Acknowledgements The Pontifícia Universidade Católica do Rio Grande do Sul supported this work. We thank Fernando S. Zucoloto and the anonymous reviewers for their valuable contributions. References Agrell, I., 1961. Metabolic progresses during insect metamorphosis as reflected by changes in the dehydrogenases. Symp. Genet. Biol. Ital. 8, 563–580. Agrell, I.P.S., Lundquist, A.M., 1973. Physiological and biochemical changes during insect development. In: Rockstein, M. (Ed.), The Physiology of Insecta, vol. 1. Academic Press, New York, pp. 159–247. Boccaccio, G.L., Quesada-Allué, L.A., 1994. Synthesis and deposition of PCG100, the main cuticle glycoprotein of the medfly C. capitata. Arch. Insect Biochem. Physiol. 27, 217–234. Canato, C.M., Zucoloto, F.S., 1998. Feeding Behavior of Ceratitis capitata (Diptera: Tephritidae): Influence of carbohydrate ingestion. J. Insect Physiol. 44, 149–155. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Force, A.G., Staples, T., Soliman, S., Arking, R., 1995. Comparative biochemical and stress analysis of genetically selected Drosophila strains with different longevities Develop. Genetics 17, 340–351. Frings, C.S., Dunn, R.T., 1970. A colorimetric method for determination of total serum lipids based on the sulfophosphovanilline reaction. Am. J. Clin. Pathol. 53, 89–91. Geary, N., Langhans, W., Scharrer, E., 1981. Metabolic concomitants of glucagon-induced suppression of feeding in the rat. Am. J. Physiol. 241, R330–R335. Ishay, J., Ikau, R., 1968. Gluconeogenesis in the oriental hornet Vespa orientalis F. Ecology 49, 169–171. Jacome, I., Aluja, M., Liedo, P., Nestel, D., 1995. The influence of adult diet and age on lipid reserves in the tropical fruit fly Anastrepha serpentina (Diptera: Tephritidae). J. Insect Physiol. 40, 1079–1086. Langley, P.A., 1970. Physiology of the Mediterranean fruit fly in relation to sterile-male technique. In: Anon (Ed.), Physiology of the Mediterranean Fruit Fly in Relation to the Sterile-Male Technique. Proceedings of a Panel held in Vienna in 1969. International Atomic Energy Agency, Vienna, Austria, pp. 25–32. Lockshin, R.A., 1969. Lysosomes in insects. In: Dingle, J.T., Fell, H.B. (Eds.), Lysosomes in Biology and Pathology, vol. 1. North Holland Publishing Company, Amsterdam, The Netherlands, pp. 363–384. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 183, 265–275. Morgante, J.S., 1991. Mosca-das-frutas (Tephritidae): características biológicas, detecção e controle. Boletim Técnico de Recomendações para os Perímetros Irrigados do Vale do São Francisco, Brasília, vol. 2, p. 19.
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Municio, A.M., Pagani, R., Suarez, A., 1980. Turnover of the glycerol moiety of different lipid classes during development of Ceratitis capitata. Comp. Biochem. Physiol. B 67, 519–525. Nascimento, J.C., Oliveira, A.K., 1996. Embryogenesis in Anastrepha fraterculus (Diptera: Tephritidae). Interciência 21, 158–165. Nestel, D., Tolmasky, D., Rabossi, A., Quesada-Allué, L.A., 2003. Lipid, carbohydrate and proteins patterns during metamorphosis of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Physiology, biochemistry and toxicology. Ann. Entomol. Soc. Am. 96, 237–244. Nestel, D., Nemny-Lavy, E., Chang, C.L., 2004. Lipid and protein loads in pupating larvae and emerging adults as affected by the composition of Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) meridic larval diets. Arch. Insect Biochem. 56, 97–109. Nestel, D., Papadopoulos, N.T., Liedo, P., Gonzáles-Ceron, L., Carey, J.R., 2005. Trends in lipid and protein contents during Medfly aging: an harmonic path to death. Arch. Insect Biochem. 60, 130–139. Norrbom, A.L., Kim, K.C., 1988. A list of the reported host plants of the species of Anastrepha (Diptera: Tephritidae). USDA APHIS 81-52, 114 pp. Norrbom, A.L., Foote, R.H., 1989. The taxonomy and zoogeography of the genus Anastrepha (Diptera: Tephritidae). In: Robinson, A.S., Hooper, G. (Eds.), Fruit Flies: Their Biology, Natural Enemies and Control, vol. IA. Elsevier, Amsterdam, pp. 15–26. Pagani, R., Suarez, A., Municio, A.M., 1980. Fatty acid patterns of the major lipid classes during development of Ceratitis capitata. Comp. Biochem. Physiol. B 67, 511–518. Rabossi, A., Wapper, P., Quesada-Allué, L.A., 1992. Larva to pharate adult transformation in the Medfly Ceratitis capitata. Can. Entomol. 124, 1139–1147. Rabossi, A., Ación, L., Quesada-Allué, L.A., 2000. Metamorphosis-associated proteolysis in Ceratitis capitata. Entomol. Exp. Appl. 94, 57–65. Schmolz, E., Lamprecht, I., 2000. Calorimetric investigations on activity states and development of holometabolous insects. Thermochim. Acta 349, 61–68. Steck, G.J., McPheron, B.A., 1996. Fruit Fly Pests: A World Assessment of Their Biology and Management. St. Lucie Press, Delray Beach, Florida. 596 pp. Storey, K.B., Baley, E., 1978. Intracelular distribution of enzymes associeted with lipogenesis and gluconeogenesis in fat body of the adult cockroach, Periplaneta. Insect Biochem. 8, 25–131. Taufer, M., 1995. Aspectos biológicos e o efeito de esteróides e análogos no desenvolvimento pré-imaginal de Anastrepha fraterculus (Diptera: Tephritidae). Monografia de Graduação. Universidade Federal do Rio Grande do Sul, Porto Alegre. Tolmasky, D.S., 2001. Synthesis and mobilization of glycogen reserves during metamorphosis of the Medfly Ceratitis capitata. Arch. Biochem. Biophys. 392, 38–47. Van Handel, E., 1965. Estimation of glycogen in small amounts of tissue. Anal. Biochem. 11, 256–265. Webster, R.P., Stoffolano, J.G., Prokopy, R.J., 1979. Long term intake of protein and sucrose in relation to reproductive behavior of wild and laboratory cultured Rhagoletis pomonella. Ann. Entomol. Soc. Am. 72, 41–46. Wigglesworth, V.B., 1942. The storage of protein, fat, glycogen and uric acid in the fat body and other tissues of mosquito larvae. J. Exp. Biol. 19, 56–77. Zucchi, R.A., 2000. Espécies de Anastrepha, Sinonímias, Plantas Hospedeiras e Parasitóides. In: Malavasi, A., Zucchi, R.A. (Eds.), Moscas-das-frutas de Importância Econômica no Brasil. Holos Editora, Ribeirão Preto, pp. 41–54.
Intermediate metabolism during the ontogenetic development of Anastrepha fraterculus (Diptera: Tephritidae)☆ B.K. Dutra a , F.A. Fernandes a , J.C. Nascimento, F.C. Quadros b , G.T. Oliveira a,⁎ a
Laboratório de Fisiologia da Conservação, Faculdade de Biociências, PUCRS, Brazil b Laboratório de Entomologia, Museu de Ciências e Tecnologia, PUCRS, Brazil
Received 13 April 2006; received in revised form 30 June 2006; accepted 24 August 2006 Available online 30 August 2006
Abstract The fruit fly Anastrepha fraterculus is a major pest of native and exotic fruit trees in South America. Changes in weight, water content and metabolism were observed during its ontogenetic development in standard conditions (25 °C, RH = 60% and 14 h:10 h photoperiod). The metabolic variables glycogen, total proteins, triglycerides and total lipids were measured by means of spectrophotometric methods. The results were correlated with pupae metamorphosis, temporal pattern, and beginning of adult life. Pupae were observed daily, and a sub-sample of 10 individuals was collected and maintained at − 20 °C. The same procedure was performed with adults at 4 days after adult eclosion. Levels of total lipids and triglycerides were constant during pupal development, peaking in 312-h-old pupae. In 0-h-old pupae, glycogen levels were high, and decreased progressively until the insects were 312 h old. The peak in total proteins coincides with the post-histolysis period of the larval tissue (96–120 h). These results indicated that glycogen and proteins may be the principal sources of energy for metamorphosis. Total lipid and triglyceride contents remained steady during metamorphosis, and these were consumed in the first 4 days of adult life. © 2006 Elsevier Inc. All rights reserved. Keywords: Anastrepha fraterculus; Developmental biology; Tephritidae; Insect; Pupal metabolism; Metabolic changes; Lipids; Proteins; Carbohydrates
1. Introduction Fruit flies (Tephritidae, Diptera) are insects belonging to a family with a cosmopolitan distribution limited only by host availability. Species of this family are classified in two groups based on physiological and ecological characteristics (Steck and McPheron, 1996). The first group includes univoltine species that have diapause, occurring in temperate regions, such as the genus Rhagoletis. The second group comprises multivoltine species ☆ This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. Depto. de Ciências Fisiológicas, PUCRS, Av. Ipiranga, 6681 Pd. 12A, CP. 1429, CEP 90619-900, Porto Alegre, RS, Brazil. Fax: +55 3320 3612. E-mail address: [email protected] (G.T. Oliveira).
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that do not have diapause, and are distributed in tropical regions, such as the genera Anastrepha, Ceratitis and Bactrocera. In general, the genus Anastrepha is highly polyphagous, using hosts from 18 families of plants in 29 genera (Zucchi, 2000). These plants are native to South America and the Antilles, or are introduced from Europe, Africa or Asia. More than 80 host species have been recorded for Anastrepha fraterculus (Norrbom and Foote, 1989); however, these data may be an underestimate of the actual number of hosts. According to Norrbom and Kim (1988), only 50% of the hosts of the Brazilian species of Anastrepha are currently known. In southern Brazil, the dominant species infesting apples is A. fraterculus. The broad range of distribution of the family Tephritidae, and the high adaptative and colonizing capacities may be related to its reproductive potential, which is linked to morphological characteristics developed in the pre-imaginal stage (Morgante, 1991). The holometabolous insects are characterized by a clear separation between larval, pupae and adults, molting from larvae to pupae and complete metamorphosis to the adult during the pupal instar, without any energy uptake where the energetic balance is usually
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negative (Schmolz and Lamprecht, 2000). However, analyses of the ontogenetic development of insects of economic importance, such as A. fraterculus, are in their infancy. During metamorphosis in insects, a dramatic transformation of plan occurs, and most of the larval tissues degenerate and disappear over a very short period of time. This programmed tissue disintegration, called histolysis, occurs sequentially and involves a combination of cytolytic and phagocytic processes. The end-products of histolysis are used to build adult structures through the process of hystogenesis (Lockshin, 1969; Rabossi et al., 2000). Schmolz and Lamprecht (2000) showed for different species of holometabolous insects that the general metabolic rate, measured by calorimetry, usually follows a U-shaped curve. That is, energy consumption is high during the first stages of metamorphosis, declines toward the mid-pupal stage, and increases again toward the last phases of the larval–adult transformation (Langley, 1970; Agrell and Lundquist, 1973; Nestel et al., 2003). Although it is generally accepted that lipids (mainly triacylglycerols) and carbohydrates (mainly glycogen and trehalose) are the main sources of energy during metamorphosis, little is known about the differential utilization of all these energetic metabolites and the role of proteins in this process (Langley, 1970; Agrell and Lundquist, 1973; Nestel et al., 2003). According to Hernandez-Ortiz et al. (2004) A. fraterculus is a major pest of native and exotic fruit trees in South America. The present study investigated aspects of the intermediate metabolism during the ontogenetic development of A. fraterculus, in order to improve understand of the physiology of the A. fraterculus. 2. Materials and methods 2.1. Sampling Larvae of A. fraterculus were collected from infested guava fruit (Psidium guajava L.) near Porto Alegre, state of Rio Grande do Sul, Brazil. Pupae from the first generation (F1) obtained in the laboratory were collected at 24-h intervals and weighed on an analytical balance; one group was stored in at − 20 °C for biochemical analyses, and the water content of the others was determined according to Force et al. (1995). The insects were maintained in standard laboratory conditions (temperature 25 °C, humidity 70–80%, photoperiod 14:10 light/dark), following the protocol described by Nascimento and Oliveira (1996). Adults were fed ad libitum with soy protein, dark sugar, and wheat (1:3:1), and provided with water. The determinations of the different metabolites were repeated three times, in three cohorts of insects. 2.2. Biochemical analyses A pool of five pupae of the each cohort (15 animals) was homogenized with an Omni Mixer homogenizer in a solution of methanol-chloroform 2:1 (v/v), according to Folch et al. (1957). Total lipids were determined in this homogenate using the
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sulphophosphovaniline method (Frings and Dunn, 1970). Triglycerides were measured by means of the reactions of lipase, glycerokinase, 1-P-glycerol oxidase and peroxidase enzymes (Biodiagnostic kit/GPO Trinder). The results are expressed in mg/g of insect. Glycogen was extracted from a pool of five insects of the each cohort (15 animals), following the method described by Van Handel (1965), and determined as glucose-equivalent (glucose-oxidase method) in quadruplicates after acidic hydrolysis (HCl) and neutralization (Na2CO3) following the method of Geary et al. (1981). Glucose was quantified using a Biodiagnostic kit (glucose oxidase). The results are presented as mg/g of insect. Total proteins were quantified following the method described by Lowry et al. (1951), using bovine albumin (Sigma) as the reference. The results are presented as mg/mL. 2.3. Statistical analyses Analysis of variance (ANOVA) was used to analyze the results for intermediate metabolism; a Bonferroni test was performed for multiple comparisons. The weight of the insects and their water content were analyzed using linear regression analysis. All tests were run using the program Statistical Package for the Social Sciences (SPSS), version 11.5 for Windows. The results are expressed as mean ± standard error of mean. 3. Results At 25 °C the development of pupae until eclosion of the imago lasted approximately 336 h, 2 weeks of development within the puparium, with a delay of approximately 6 h. Data in Table 1 show that the average weight of pupae was very changeable during the pupal development, with a gradual reduction until 72 h (pupae), followed by an increase until 120 h and a decrease by 192 h, and then remained at this level until hour 312. Linear regression analysis indicated that pupal weight
Table 1 Wet weight, dry weight and water content Time of development
Wet mass (mg)
Dry mass (mg)
Water content (%)
Pre-pupae Pupae 0 h Pupae 24 h Pupae 48 h Pupae 72 h Pupae 96 h Pupae 120 h Pupae 144 h Pupae 168 h Pupae 192 h Pupae 216 h Pupae 240 h Pupae 264 h Pupae 288 h Pupae 312 h
18.09 ± 0.60 16.48 ± 0.51 16.63 ± 0.28 18.09 ± 0.50 11.96 ± 0.72 13.82 ± 1.04 16.05 ± 0.75 10.80 ± 0.57 11.34 ± 1.08 08.90 ± 0.61 11.29 ± 0.60 09.76 ± 0.86 10.97 ± 0.63 11.97 ± 0.32 11.29 ± 0.43
4.64 ± 0.24 3.88 ± 0.12 5.60 ± 0.12 6.42 ± 0.18 5.57 ± 0.12 6.62 ± 0.12 6.26 ± 0.22 5.28 ± 0.19 4.47 ± 0.36 4.15 ± 0.29 4.48 ± 0.21 4.43 ± 0.29 4.59 ± 0.32 3.90 ± 0.14 3.95 ± 0.13
74.46 ± 0.60 76.45 ± 0.76 66.33 ± 0.43 64.50 ± 0.34 51.99 ± 2.90 49.04 ± 4.93 60.14 ± 2.61 50.71 ± 1.15 57.78 ± 5.05 52.19 ± 3.94 59.29 ± 3.13 52.75 ± 3.06 58.08 ± 1.71 66.61 ± 0.99 64.74 ± 1.31
Results represent the mean ± standard error of the mean, and are expressed in mg (weight) and percentage of water (%).
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Fig. 1. Total lipid levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
was significantly dependent on pupal instar (R2 = 0.31; F = 218.91; p b 0.001; a = 16.46; b = − 0.32); i.e., the puparium loses weight as the adult differentiates. Water content decreased in the first 96 h of pupal development, and then oscillated until the end of development (hour 312) (Table 1). The weight of the animal and water content at day 4 of life were not determined after the eclosion of the adult. Levels of total lipids were constant until 48 hours of pupal development, and then increased ( p b 00.05) by about 100% by the time that the pupae were 72 h old. The levels then remained constant until hatching and imago emersion, but afterwards decreased ( p b 0.05) to 60% in 4-day-old males and females (Fig. 1).
Levels of triglycerides were elevated in animals in the prepupae stage, and decreased by about 55% in pupae at T0 ( p b 0.05). These levels remained constant up to hour 48 of pupal development. At hour 72, the triglycerides showed an increase ( p b 0.05), followed by a significant decline in 96-h-old pupae. The levels then remained constant until the imago emerged. In 4-day-old flies, triglycerides levels showed a significant decline, in both males and females (Fig. 2). Fig. 3 shows that from the pre-pupal stage (larvae) until 0-hold pupae, the levels of glycogen increased by about 120% ( p b 0.05). From 0-h-old pupae until hour 72 of development, glycogen decreased to a low level, then increased again to the initial level by hour 96 of pupal development (24-h-old pupae).
Fig. 2. Triglyceride levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
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Fig. 3. Glycogen levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals) and the vertical bars represent the standard error of the mean.
The glycogen levels then remained constant until the stage preceding the imago emersion (hour 312). In 4-day-old males, the levels of glycogen were similar to those observed during the pupal development; whereas in females, glycogen levels increased significantly. Fig. 4 shows that between the pre-pupae and 0-h-old pupae there was an increase in the levels of total proteins ( p b 0.05). At 72 h of development, pupae show a peak of total proteins, followed by a decrease in the level of proteins in 96- and 120-h-old pupae. Between 144 and 312 h of pupal development, the concentration of protein begins to increase ( p b 0.05). The levels of proteins are highest in 312-h-old pupae, compared with larvae or pre-pupae ( p b 0.05). At 4 days of adult life, males and females show levels of total proteins up
to 3500 times higher than those observed during pupal development. 4. Discussion Our results demonstrate the occurrence of changes in weight, water content and intermediated metabolism which are stagespecific for the differentiation of the adult. The time of development (330 h until 336 h) verified in this work confirm the time of development that was observed by Taufer (1995) studying A. fraterculus under the same experimental culture conditions. Water content decreased in the first 96 hours of pupal development (312 h) (Table 1). Langley (1970) and Nestel et al.
Fig. 4. Total protein levels during pupal development of Anastrepha fraterculus. Columns represent the mean (15 animals), and the vertical bars represent the standard error of the mean.
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(2003) showed that the Mediterranean fruit fly (C. capitata) undergoes a relatively large water loss during the pupation and pupal phases (outset of the pharate-adult stage). Considering that in this work, was observe an increase of the total lipids in 72 h of pupal development and an intense decrease in 4 days old in males and females (Fig. 1), these results partially coincide with those of Municio et al. (1980), Pagani et al. (1980) and Nestel et al. (2003), but confirm the differential lipid utilization during larval to adult transition. Langley (1970) observed a constant rate of utilization of total lipids during the entire metamorphosis. However, Municio et al. (1980) and Pagani et al. (1980) showed a small decrease in total lipids during what seemed to be the pre-pupae and pupal phases. Their data also show that this small decrease during the first half of metamorphosis is followed by a more drastic utilization of lipids just a few hours before adult emergence. Nestel et al. (2003) showed in Ceratitis capitata a significant increase in lipids between 8 hours before pupae formation and 20.5 hours after pupae formation; from this point on, lipids begin to be consumed. This decrease in lipid load coincides with the disintegration of the larval fat body, as evidenced by histological sections of pupal tissue. Levels of triglycerides decreased (55%) during pre-pupae to pupae (0 h) transition and this results suggest that triglycerides are used by the animals during metabolic adjustment when the larval more away from food. At 72 h, the triglicerydes showed an increase ( p b 0.05), decline in 96 h old pupae, and levels remained constant. The results obtained for total lipids and triglycerides suggest that these metabolites are stored by the animals during pupal development, to be consumed during the imago emersion (Figs. 1 and 2), in the first flight activities, and searching for food in the first 4 days of adult life. Nestel et al. (2004, 2005) showed in C. capitata that pupae seem to regulate lipid loads towards a certain optimum level for adult emergence, regardless of the larval diet history or lipids content. In Anastrepha serpentina, Jacome et al. (1995), observed an initial drops of lipid reserves between 4 days and 6 days after adult emergence, this results suggests that when newly emerged flies encounter a protein source, metabolic activity and requirement rise. During these initial days in A. serpentina, lipids reserves may be used as an energetic source for the development of reproductive and endocrine tissues, and for flight activities. Fig. 3 shows that from the pre-pupal stage (larvae) until 0-hold pupae, the levels of glycogen increased by about 120%. This increase may be related to a similar decrease in triglycerides reserves, suggesting that the glycerol obtained from the catalyses of triglycerides is used in the glyconeogenic pathway. Several studies have demonstrated this glyconeogenic capacity in insects (Wigglesworth, 1942; Ishay and Ikau, 1968; Storey and Baley, 1978). After this increase our results indicate that, for both sexes, glycogen may be the main source of energy for differentiation of the adult individual during the pupal stage, because between 0 h pupae until 72 h of development this polysaccharide decreased 10 times (Fig. 1). In females, probably glycogen is also used as a metabolic reserve for reproduction (production and release of eggs). The significant increase in glycogen levels observed in the first
4 days of adult life may explain its role in the reproduction of the females. The increase in this polysaccharide in the first four days of adult life of A. fraterculus may be determined by the feeding behaviour and/or food source and/or by glycerol, a subproduct of triglyceride degradation during this period. Webster et al. (1979) showed that in Rhagoletis pomonella, females consume more carbohydrates than males, probably because of their more intensive body metabolism and movements related to egg production and searching for a host. The ingestion of carbohydrates in females increased more in the first week of adult life, decreased in the second and third weeks, and then remained stable until the end of the experiment (45 days). Canato and Zucoloto (1998) observed in C. capitata that sugars are the most important nutrients for adult females since they somehow succeed in producing eggs without ingestion a protein source, although production of eggs increases with protein ingestion. The results observed in this work for glycogen (Fig. 1) agree with the data presented by Tolmasky (2001) and Nestel et al. (2003) for different phases of the metamorphosis of C. capitata. They too found an initial decrease in glycogen reserves in the metamorphosis (pre-pupae and onset of the pupal stage), followed by an increase in this polysaccharide at the end of the pupal stage. Fig. 4 shows that between the pre-pupae and 0-h-old pupae there was an increase in the levels of total proteins. Rabossi et al. (2000) observed in C. capitata an increase in total proteolytic activity between 40 and 44 h after immobilization of the larva. This occurred approximately 1 day after apolysis, which marks the onset of the pupation process (Rabossi et al., 1992). The 40–44-h period of metamorphosis corresponds to the transition from pre-pupae to pupae, when the outermost layers of cuticle begin to be deposited (Boccaccio and QuesadaAllué, 1994; Rabossi et al., 2000). It is probable that the carbon atoms derived from amino acids are incorporated into glycogen molecules. Results reported by Nestel et al. (2003) suggest that during the initial phases of metamorphosis of the Mediterranean fruit fly, a portion of the amino acid pool may be directed into the tricarboxylic acid cycle and may used for energetic needs or other metabolites with glycogen. Between 144 and 312 h of pupal development, the concentration of protein begins to increase. The levels of proteins are highest in 312-h-old pupae, compared with larvae or prepupae. At 4 days of adult life, males and females show levels of total proteins up to 3500 times higher than those observed during pupal development. Larval protein breakdown during the initial stages of Mediterranean fruit fly metamorphosis and the subsequent resynthesis of adult proteins has been described by Rabossi et al. (2000) and Nestel et al. (2003). Agrell (1961) found in Calliphora erytrocephala a sudden decrease of precipitable protein at pupation. Our results showed that during the pharate adult stage, the amount of total proteins was restored, reaching values higher than those recorded for the pre-pupae. This work demonstrate the occurrence of changes in utilization of the energetic reserves of A. fraterculus which are stage specific for the differentiation of the adult. During the histolysis of the larval tissues, the levels of glycogen and total proteins are
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high; however, after 72 h all this material is utilized, probably for synthesis of muscles, skin, and other body tissues of the adult fly. In contrast, the levels of lipids and triglycerides remain high during metamorphosis (specific in the pharate adult) to be used by the adult in its first activities (flight, searching for food, mating and egg production). The regulation of these energetic reserves during pupation and first days of adult life is an intriguing question that deserves to be further investigated. Acknowledgements The Pontifícia Universidade Católica do Rio Grande do Sul supported this work. We thank Fernando S. Zucoloto and the anonymous reviewers for their valuable contributions. References Agrell, I., 1961. Metabolic progresses during insect metamorphosis as reflected by changes in the dehydrogenases. Symp. Genet. Biol. Ital. 8, 563–580. Agrell, I.P.S., Lundquist, A.M., 1973. Physiological and biochemical changes during insect development. In: Rockstein, M. (Ed.), The Physiology of Insecta, vol. 1. Academic Press, New York, pp. 159–247. Boccaccio, G.L., Quesada-Allué, L.A., 1994. Synthesis and deposition of PCG100, the main cuticle glycoprotein of the medfly C. capitata. Arch. Insect Biochem. Physiol. 27, 217–234. Canato, C.M., Zucoloto, F.S., 1998. Feeding Behavior of Ceratitis capitata (Diptera: Tephritidae): Influence of carbohydrate ingestion. J. Insect Physiol. 44, 149–155. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Force, A.G., Staples, T., Soliman, S., Arking, R., 1995. Comparative biochemical and stress analysis of genetically selected Drosophila strains with different longevities Develop. Genetics 17, 340–351. Frings, C.S., Dunn, R.T., 1970. A colorimetric method for determination of total serum lipids based on the sulfophosphovanilline reaction. Am. J. Clin. Pathol. 53, 89–91. Geary, N., Langhans, W., Scharrer, E., 1981. Metabolic concomitants of glucagon-induced suppression of feeding in the rat. Am. J. Physiol. 241, R330–R335. Ishay, J., Ikau, R., 1968. Gluconeogenesis in the oriental hornet Vespa orientalis F. Ecology 49, 169–171. Jacome, I., Aluja, M., Liedo, P., Nestel, D., 1995. The influence of adult diet and age on lipid reserves in the tropical fruit fly Anastrepha serpentina (Diptera: Tephritidae). J. Insect Physiol. 40, 1079–1086. Langley, P.A., 1970. Physiology of the Mediterranean fruit fly in relation to sterile-male technique. In: Anon (Ed.), Physiology of the Mediterranean Fruit Fly in Relation to the Sterile-Male Technique. Proceedings of a Panel held in Vienna in 1969. International Atomic Energy Agency, Vienna, Austria, pp. 25–32. Lockshin, R.A., 1969. Lysosomes in insects. In: Dingle, J.T., Fell, H.B. (Eds.), Lysosomes in Biology and Pathology, vol. 1. North Holland Publishing Company, Amsterdam, The Netherlands, pp. 363–384. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 183, 265–275. Morgante, J.S., 1991. Mosca-das-frutas (Tephritidae): características biológicas, detecção e controle. Boletim Técnico de Recomendações para os Perímetros Irrigados do Vale do São Francisco, Brasília, vol. 2, p. 19.
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