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Energy status in the fat body of Trichoplusia ni parasitized by the insect parasite Hyposoter exiguae
JOURNAL
OFINVERTEBRATE
PATHOLOGY
‘t‘+,
46-Sl(1984)
Energy Status in the Fat Body of Trichoplusia ni Parasitized Insect Parasite Hyposoter exiguae S.N.THOMPSONAND Division
of Biological
Control
K.A.
and Department Riverside, California
by the
YAMADA
of Biology, 92521
University
of California,
Received October 3, 1983; accepted November 28, 1983 The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguue. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association. KEY WORDS: Trichoplusia ni; Hyposoter exiguae; adenosine triphosphate; adenosine diphosphate; adenosine monophosphate; adenylate kinase.
feed on the host hemolymph (Jowyk and Smilowitz, 1978). During this period, the relationship is characterized by complex endocrine, nutritional, and metabolic interactions. For example, parasitized host larvae display severely depressed growth rates and delayed development (Thompson, 1982). Their hemolymph and fat body protein levels, however, are increased. Moreover, the levels of fat body glycogen and hemolymph trehalose were increased, and metabolic studies indicated markedly elevated rates of gluconeogenesis in the fat body of parasitized individuals (Thompson and Binder, 1984). Alterations in nutritional physiology include increased rates of assimilation and altered conversion efficiency (Thompson, 1983b). Several studies have demonstrated that the overall nature of these changes which result from parasitization are markedly influenced by virus particles which were present in the fluid surrounding the parasite egg at the time of oviposition (Vinson et al. 1979; Stoltz and Vinson, 1979). Later in its 3rd stadium, the parasite begins to feed directly on the fat body and other host tissues and, at the time the fully developed parasite
INTRODUCTION
Host response to parasitic infection is complex and variable (von Brand, 1979). During many invertebrate host-parasite relationships the physiological and metabolic alterations which accompany infection are often of a highly integrative and nonpathogenic nature. In some cases, as a result of natural selection, these responses appear to have provided benefit for the parasite, ensuring the survival of the parasite-host complex and the success of the association. Such complex interactions have been described during invertebrate host relationships involving nematode, trematode, and cestode parasites, as well as insect parasites of the order Hymenoptera (Thompson, 1983a). Although the latter ultimately kill their hosts, many associations, such as that of the solitary insect parasite, Hyposoter exiguae (Ichneumonidae), and its host, Trichoplusia ni (Noctuidae), appear highly integrative during the early stages. The parasitic larvae of H. exiguae develop free within the hemocoel of the host larvae and Ist, 2nd, and early 3rd instars 46 0022-201 l/84 $1.50 coovripht0 I%‘4hv
x+-e= I”,.
ENERGY
STATUS
OF
larva emerges to pupate, only the host integument remains intact. The present study was carried out to determine whether the cumulative physiological alterations which occur in parasitized 4th-instar T. ni, prior to the direct destruction of host tissues by developing H. exiguae, are accompanied by changes in the overall energy status of the host, as indicated by adenylate nucleotide energy ratios. MATERIALS
AND METHODS
Culture. Stock colonies of H. exiguae were maintained in the laboratory at approximately 25°C. The host, T. ni, was reared in 6-0~ paper cups on the artificial diet described by Shorey and Hale (1965), with a photoperiod of 12 hr light: 12 hr dark at 30” 5 05°C. In the present studies host insects were parasitized late in the 3rd stadium. Developing host larvae were parasitized individually when the cuticular sutures on the head were stretched just prior to ecdysis. Following parasitization, insects were placed back in cups and maintained with cups of control larvae under the above conditions. Parasitized larvae were sacrificed at the end of the 4th stadium 3 days after parasitization. Non-parasitized larvae at the same stage of development served as controls. Tissue preparation and extraction. Dissections of fat body were made on paraffin blocks by making a longitudinal mid-ventral incision along the entire body length and pinning back the cuticle. The exposed fat body was carefully excised and immediately frozen on blocks of dry ice. Frozen tissue was then pulverized in a tissue grinder containing 0.5 ml of frozen 3.5% perchloric acid. Following homogenization, extracts were centrifuged for 5 min at 8730g in a Beckman microfuge and then neutralized with 10 N KOH. After setting in ice water for a few minutes, the salt precipitate was removed by centrifugation, and the samples were immediately frozen and stored at -70°C until analyzed. Due to the
PARASITIZED
T. ni
47
dispersed nature of the larval fat body of T. excise and obtain accurate estimates of the total tissue per animal. Moreover, as previously described (Thompson, 1982), great difficulty was encountered in uniformly removing excess fluid from tissue samples such that consistent wet weights of partial tissue samples could not be determined. The latter problem was compounded in the present study due to the labile nature of nucleotides and thus the necessity to quickfreeze tissue immediately upon removal. Tissue adenylate nucleotide concentrations, therefore, were based on the level per milligram of total fat body protein. Following perchloric acid precipitation as described above, the resultant pellet was redissolved in 1 N KOH, and, after neutralization with HCl, the protein content was determined by the dye-binding method of Bradford (1976). Chromatography. All chromatography was conducted on a Varian Vista 56 ternary chromatography system with a Varian UV5 fixed-wavelength and a UV-100 variablewavelength detector (Varian Associates, Palo Alto, Calif.) as described by Yamada and Sherman (1984). Samples were injected via a Rheodyne 7125 injector with a 20-pl sample loop, and then chromatographed through a 4.6 mm x 3.0 cm, lo-pm spherical octadecylsilane (ODS) guard column (Brownlee Labs, Santa Clara, Calif.), and a 4 mm x 15 cm Waters Resolve 5-p.m ODS analytical column (Milford, Mass.). Chromatography-grade acetonitrile was obtained from Mallinckrodt (Paris, KY.). Water was obtained using a Barnstad Nanopure water-purification system. Tetrabutyl ammonium hydroxide (TBA) was obtained from Aldrich Chemical Company (Milwaukee, Wise.). Adenylate nucleotides were identified by retention time, internal standard, and absorbance ratio (Fig. 1). Purines used as standards were obtained from Sigma Chemical Company (St. Louis, Missouri). Adenylate levels were expressed as nanomoles per milligram of total fat body ni, it was not possible to completely
48
THOMPSON
AND YAMADA
ADP and AMP. Moreover, the adenylate kinase mass-action ratio and the energy ratios were within physiological ranges (Yushok, 1971; Chapman et al., 1971; Newsholme and Start, 1973). Parasitization caused changes in the adenylate nucleotide levels. In general, the level of ATP was decreased and ADP and AMP increased in parasitized individuals. However, the ADP level was slightly reduced in replicate 4 and ATP was elevated and AMP reduced in replicate 3. This latter result remains unexplained, except to point out that, among the control treatments, this sample had the highest AMP level and it may be that some breakdown occurred during preparation. The above changes in nucleotide levels were reflected in the energy ratio values. In all cases the ATP/ADP ratio decreased in parasitized individuals as did the ATP/AMP and energy change ratios, with the exception of replicate 3. Energy change, however, was only slightly affected, and the average energy charge for all replicates and treatments was 0.82 ZL 0.04 (mean +- SD).
!.hu ADP
II
AMP
r
L
FIG. 1. Chromatogram tracings showing adenylate nucleotides in fat body extracts from Trichophsiu ni. Unparasitized 4th-instar larval fat body (bottom). Same sample “spiked” with ATP, ADP, AMP, GTP, GDP, and GMP (top). 0.03 AUFS.
protein as described above. Energy charge, described by Atkinson (1968), was calculated as follows: Energy charge = (ATP + t/2 ADP)/(ATP + ADP + AMP), and the adenylate kinase mass action ratio, K = ATP . AMP/ADP2. RESULTS
The individual and total adenylate nucleotide levels of T. ni fat body were highly variable (Table 1). In all cases, however, ATP was present at levels far in excess of
DISCUSSION
The adenylate nucleotide system, composed of ATP, ADP, and AMP, is of major importance in the control of energy flow in living systems. These metabolites, particularly ATP and AMP, regulate the activity of many enzymes which determine metabolic flux through anabolic and catabolic pathways. The concentration of these three nucleotides is partially regulated by the adenylate kinase-catalyzed reaction: 2 ADP it ATP + AMP. The ATP level is maintained much higher than that of AMP, such that a small change in ATP causes a greater fractional increase in AMP (Newsholme, 1970). The theory of metabolic regulation by fractional amplification has led to the use of concentration ratios as measures of the phosphorylation potential of the adenylate system. One such measure, the “energy charge,” is half the average number of anhydride-bound phosphate groups per ad-
ENERGY STATUS
ADENINE
NLJCLEOTIDE
OF PARASITIZED
TABLE 1 RATIOS IN THE FAT BODY OF 4th-INsTAR Trichoplusia AFTER PARASITIZATION BY THE INSECT PARASITE, Hyposoter exiguae LEVELS
AND
ENERGY
Nucleotide levels (nmol/mg fat body protein)
Replicate 1 Control (10) Parasitized (10) Percentage change Replicate 2 Control (9) Parasitized (9) Percentage change Replicate 3 Control (10) Parasitized (10) Percentage change Replicate 4 Control (10) Parasitized (10) Percentage change Note.
49
T. ni
ni 3 DAYS
Energy ratios Energy/ charge
Adenylate kinase mass-action ratio
13.01 5.29 -59
0.84 0.77 -8
1.17 1.73
6.57 3.14 -53
21.72 12.29 -42
0.89 0.83 -7
1.99 0.80
53.78 65.14 +21
3.32 3.17 -3
6.18 10.70 +73
0.79 0.82 +4
1.78 0.94
58.64 53.24 -9
3.77 2.98 -21
13.51 7.44 -45
0.85 0.79 -7
1 .os 1.19
ATP
ADP
AMP
Total
ATP/ADP
42.16 34.32 -19
10.81 11.35 +5
3.24 6.49 +100
56.22 52.16 -7
3.90 3.02 -23
58.65 36.49 -38
8.92 11.62 +30
2.70 70.27 2.97 51.08 + 10 -27
36.76 46.22 +26
11.08 14.59 +32
5.95 4.32 -27
43.78 36.22 -17
11.62 12.16 -5
3.24 4.87 +50
ATPIAMP
The number of individuals tested is shown in parentheses.
enine moiety (Atkinson, 1968), and the catalytic nature of many enzymes is affected by this parameter rather than the absolute concentrations of adenylate nucleotides (Swedes et al., 1975). Regulatory enzymes from metabolic sequences in which ATP is regenerated are active at low levels of energy charge and activity decreases rapidly above 0.75; those from ATP-consuming sequences are inactive at low energy charge and sharply increase in activity above 0.75 (Atkinson, 1970). The response curves of these two types tend to intersect at about 0.85. The mean energy charge value of 0.82 found in T. ni during the present study indicates a reasonable balance between anabolic and catabolic metabolism in the 4th larval stadium of both control and parasitized individuals. The adenylate energy charge of normal, intact cells from a variety of sources appears to be stabilized between 0.8 and 0.95, and decreases during metabolic and physiological stress (Chapman et al., 1971). For example, the energy charge of Escherichia
coli during growth was reported to be 0.8 and, during the stationary phase or starvation, slowly declines to 0.5 (Chapman et al., 1971). Between the above values all cells were viable and capable of forming colonies, but under continued starvation the charge decreased further and death occurred below 0.5. Energy charges below 0.65, however, may not occur in surviving eucaryotic cells (Atkinson, 1977). The strict use of energy charge in predicting specific metabolic responses has been criticized from several theoretical as well as practical viewpoints. The application of the amplification theory depends upon the maintenance of the mass-action ratio of the adenylate kinase reaction near equilibrium. However, Yushok (1971) reported mass-action ratios several-fold higher than equilibrium at high ATP concentrations. Moreover, the ratio is markedly influenced by factors such as the uncomplexed Mg*+ ion and the total adenylate concentrations (Purich and Fromm, 1973). Indeed, independent evaluation of
50
THOMPSON
AND YAMADA
the effect of energy charge is made more crease in energy charge of duck erythrodifficult because, as occurred in the present cytes infected with Plasmodium lophurae study, the adenylate concentration tends to occurred during the final 6 hr of the pararise and fall with the energy charge, re- site’s life cycle. sulting in an even greater increase in the In the present study, the energy charge magnitude of change in ATP concentration of T. ni larvae 3 days after parasitization which accompanies changes in energy decreased only slightly, despite the dracharge than would occur if the adenylate matic physiological and metabolic alteraconcentration remained constant. Although tions which occur concurrently, as dethe above in vitro effects can greatly alter scribed under Introduction. The result sugthe steady-state levels of adenylate nucleogests that the parasite-host complex at this tides, under normal conditions the energy stage represents a viable and distinct physcharge is generally maintained within 5- iological entity, although its fate has been 10% (Atkinson, 1977). Moreover, the va- markedly altered from that of its nonparlidity of some criticisms may not be appliasitized counterpart. Although the parasite cable to intact cells and tissues because the was very small at this point and represented major consideration in examining whole less than 1% of the total biomass of the parcell energy charge ratios is to qualify the asite-host complex (Thompson, 1983b), it overall energetic state of the cell or tissue nevertheless has completed a substantial and not to examine the specific metabolic part of its development. It would be of inresponses of the adenylate nucleotides as terest to determine energy charge levels effecters of enzymes. throughout the entire development of H. The energy charge ratio is a qualitative exiguae and to evaluate the importance of measure of the potential energy of the or- the virus particles which play an important ganism and should, therefore, provide a pa- role in the successful development of the rameter for evaluating the overall metabolic parasite. It appears that insect parasites stress induced by parasitization. Thus, the such as H. exiguae have highly integrated degree of integration characterizing the de- metabolic associations with their hosts that velopment of the parasite-host complex as are mechanistically similar to those dewell as the onset of pathogenesis and/or de- scribed for many helminth and protozoan velopment of disease should be reflected by parasites, although the ultimate result of changes in the energy charge of the affected the association is, in most cases, host tissue. During parasite development an death. equilibrium must exist between the parasite’s utilization of the host metabolites and ACKNOWLEDGMENTS the host’s requirements for the same factors This work was supported by grants from the USPHS that are necessary for maintenance and sur(AI05226) and the UNDP/World Bank/World Health vival. Parasitization must not inflict too Organization Special Program for Research and much stress on the host before completion Training in Tropical Diseases. of the parasite’s development. Indeed, through natural selection, it is likely that REFERENCES host death often occurs synchronously with completion of the parasite’s life cycle. For ATKINSON, D. E. 1968. The energy change of the adexample, in malaria-infected erythrocytes, enylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry, 7, 4030the host cell is maintained until completion 4034. of the parasite’s development, and only in D. E. 1970. Enzymes as control elements the last stages was notable metabolic stress ATKINSON, in metabolic regulation. In “The Enzymes” (P. D. observed (Yamada and Sherman, 1980). Boyer, ed.), Vol. 1, pp. 461-489. Academic Press, New York. Specifically, over half of the total 35% de-
ENERGY
STATUS
OF
D. E. 1977. “Cellular Energy Metabolism and its Regulation.” Academic Press, New York. BRADFORD, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248-254. CHAPMAN, A. G., FALL, L., AND ATKINSON, D. E. 1971. Adenylate energy change in Escherichia coli during growth and starvation. J. Bacferiol.. 108, 1072-1086. JOWYK,E. A., AND SMILOWITZ, Z. 1978. A comparison of growth and development rates of the parasite Hyposoter exiguae reared from two instars of its host, ATKINSON,
Trichoplusia 467-472.
ni. Ann.
Entomol.
Sot.
Amer.,
II,
NEWSHOLME, E. A. 1970. Theoretical and experimental considerations on the control of glycolysis in muscle. In “Essays in Cell Metabolism” (W. Bartley, H. L. Kornberg, and J. R. Quayle, eds.), pp. 189-223. Wiley-Interscience, New York. NEWSHOLME, E. A., AND START, C. 1973. “Regulation in Metabolism.” Wiley, New York. PURICH, D. L., AND FROMM, H. J. 1973. Additional factors influencing enzyme responses to the adenylate energy change. J. Biol. Chem., 248,461-466. SHOREY, H. H., AND HALE, R. L. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol., 58, 522-524. STOLTZ, D., AND VINSON, S. B. 1979. Viruses and parasitism in insects. Vir. Res. 24, 125- 171. SWEDES, J. S.. SEDO, R. J., AND ATKINSON, D. E. 1975. Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli. J. Biol. Chem., 250, 6930-6938.
PARASITIZED
51
T. ni
THOMPSON, S. N. 1982. Effects of parasitization by the insect parasite Hyposoter exiguae on the growth, development and physiology of its host, Trichoplusia ni. Parasitology 84, 491-510. THOMPSON, S. N. 1983a. Biochemical and physiological effects of metazoan endoparasites on their host species. Comp. Biochem. Physiol. B, 74, 183-211. THOMPSON, S. N. 1983b. The nutritional physiology of Trichoplusia ni parasitized by the insect parasite, Hyposoter exiguae, and the effects of parallelfeeding. Parasitology 87, 15-28. THOMPSON, S. N., AND BINDER, B. 1984. Altered carbohydrate metabolism and gluconeogenesis in Trichoplusia ni parasitized by the insect parasite, Hyposoter exiguae. J. Parasitol., in press. VINSON, S. B., EDSON, K. M., AND STOLTZ, D. 1979. Effect of a virus associated with the reproductive system of the parasitoid wasp Campoletis sonorensis, on host weight gain. J. Znvertebr. Pathol. 34, 133-137. VON BRAND, T. 1979. “Biochemistry of Parasites.” Academic Press, New York. YAMADA, K. A., AND SHERMAN, I. W. 1980. Plasmodium lophurae: Malaria induced nucleotide changes in duckling (Anas domestica) erythocytes. Mol. Biochem.
Parasitol.
1, 187-198.
YAMADA, K. A., AND SHERMAN, I. W. 1984. Rapid high performance liquid chromatographic separation of selected blood purines by dynamic modification of octadecylsilane. J. Chromatogr., in press. YUSHOK. W. D. 1971. Control mechanisms of adenine nucleotide metabolism of ascites tumor cells. J. Biol.
Chem.,
246,
1607-1617.
OFINVERTEBRATE
PATHOLOGY
‘t‘+,
46-Sl(1984)
Energy Status in the Fat Body of Trichoplusia ni Parasitized Insect Parasite Hyposoter exiguae S.N.THOMPSONAND Division
of Biological
Control
K.A.
and Department Riverside, California
by the
YAMADA
of Biology, 92521
University
of California,
Received October 3, 1983; accepted November 28, 1983 The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguue. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association. KEY WORDS: Trichoplusia ni; Hyposoter exiguae; adenosine triphosphate; adenosine diphosphate; adenosine monophosphate; adenylate kinase.
feed on the host hemolymph (Jowyk and Smilowitz, 1978). During this period, the relationship is characterized by complex endocrine, nutritional, and metabolic interactions. For example, parasitized host larvae display severely depressed growth rates and delayed development (Thompson, 1982). Their hemolymph and fat body protein levels, however, are increased. Moreover, the levels of fat body glycogen and hemolymph trehalose were increased, and metabolic studies indicated markedly elevated rates of gluconeogenesis in the fat body of parasitized individuals (Thompson and Binder, 1984). Alterations in nutritional physiology include increased rates of assimilation and altered conversion efficiency (Thompson, 1983b). Several studies have demonstrated that the overall nature of these changes which result from parasitization are markedly influenced by virus particles which were present in the fluid surrounding the parasite egg at the time of oviposition (Vinson et al. 1979; Stoltz and Vinson, 1979). Later in its 3rd stadium, the parasite begins to feed directly on the fat body and other host tissues and, at the time the fully developed parasite
INTRODUCTION
Host response to parasitic infection is complex and variable (von Brand, 1979). During many invertebrate host-parasite relationships the physiological and metabolic alterations which accompany infection are often of a highly integrative and nonpathogenic nature. In some cases, as a result of natural selection, these responses appear to have provided benefit for the parasite, ensuring the survival of the parasite-host complex and the success of the association. Such complex interactions have been described during invertebrate host relationships involving nematode, trematode, and cestode parasites, as well as insect parasites of the order Hymenoptera (Thompson, 1983a). Although the latter ultimately kill their hosts, many associations, such as that of the solitary insect parasite, Hyposoter exiguae (Ichneumonidae), and its host, Trichoplusia ni (Noctuidae), appear highly integrative during the early stages. The parasitic larvae of H. exiguae develop free within the hemocoel of the host larvae and Ist, 2nd, and early 3rd instars 46 0022-201 l/84 $1.50 coovripht0 I%‘4hv
x+-e= I”,.
ENERGY
STATUS
OF
larva emerges to pupate, only the host integument remains intact. The present study was carried out to determine whether the cumulative physiological alterations which occur in parasitized 4th-instar T. ni, prior to the direct destruction of host tissues by developing H. exiguae, are accompanied by changes in the overall energy status of the host, as indicated by adenylate nucleotide energy ratios. MATERIALS
AND METHODS
Culture. Stock colonies of H. exiguae were maintained in the laboratory at approximately 25°C. The host, T. ni, was reared in 6-0~ paper cups on the artificial diet described by Shorey and Hale (1965), with a photoperiod of 12 hr light: 12 hr dark at 30” 5 05°C. In the present studies host insects were parasitized late in the 3rd stadium. Developing host larvae were parasitized individually when the cuticular sutures on the head were stretched just prior to ecdysis. Following parasitization, insects were placed back in cups and maintained with cups of control larvae under the above conditions. Parasitized larvae were sacrificed at the end of the 4th stadium 3 days after parasitization. Non-parasitized larvae at the same stage of development served as controls. Tissue preparation and extraction. Dissections of fat body were made on paraffin blocks by making a longitudinal mid-ventral incision along the entire body length and pinning back the cuticle. The exposed fat body was carefully excised and immediately frozen on blocks of dry ice. Frozen tissue was then pulverized in a tissue grinder containing 0.5 ml of frozen 3.5% perchloric acid. Following homogenization, extracts were centrifuged for 5 min at 8730g in a Beckman microfuge and then neutralized with 10 N KOH. After setting in ice water for a few minutes, the salt precipitate was removed by centrifugation, and the samples were immediately frozen and stored at -70°C until analyzed. Due to the
PARASITIZED
T. ni
47
dispersed nature of the larval fat body of T. excise and obtain accurate estimates of the total tissue per animal. Moreover, as previously described (Thompson, 1982), great difficulty was encountered in uniformly removing excess fluid from tissue samples such that consistent wet weights of partial tissue samples could not be determined. The latter problem was compounded in the present study due to the labile nature of nucleotides and thus the necessity to quickfreeze tissue immediately upon removal. Tissue adenylate nucleotide concentrations, therefore, were based on the level per milligram of total fat body protein. Following perchloric acid precipitation as described above, the resultant pellet was redissolved in 1 N KOH, and, after neutralization with HCl, the protein content was determined by the dye-binding method of Bradford (1976). Chromatography. All chromatography was conducted on a Varian Vista 56 ternary chromatography system with a Varian UV5 fixed-wavelength and a UV-100 variablewavelength detector (Varian Associates, Palo Alto, Calif.) as described by Yamada and Sherman (1984). Samples were injected via a Rheodyne 7125 injector with a 20-pl sample loop, and then chromatographed through a 4.6 mm x 3.0 cm, lo-pm spherical octadecylsilane (ODS) guard column (Brownlee Labs, Santa Clara, Calif.), and a 4 mm x 15 cm Waters Resolve 5-p.m ODS analytical column (Milford, Mass.). Chromatography-grade acetonitrile was obtained from Mallinckrodt (Paris, KY.). Water was obtained using a Barnstad Nanopure water-purification system. Tetrabutyl ammonium hydroxide (TBA) was obtained from Aldrich Chemical Company (Milwaukee, Wise.). Adenylate nucleotides were identified by retention time, internal standard, and absorbance ratio (Fig. 1). Purines used as standards were obtained from Sigma Chemical Company (St. Louis, Missouri). Adenylate levels were expressed as nanomoles per milligram of total fat body ni, it was not possible to completely
48
THOMPSON
AND YAMADA
ADP and AMP. Moreover, the adenylate kinase mass-action ratio and the energy ratios were within physiological ranges (Yushok, 1971; Chapman et al., 1971; Newsholme and Start, 1973). Parasitization caused changes in the adenylate nucleotide levels. In general, the level of ATP was decreased and ADP and AMP increased in parasitized individuals. However, the ADP level was slightly reduced in replicate 4 and ATP was elevated and AMP reduced in replicate 3. This latter result remains unexplained, except to point out that, among the control treatments, this sample had the highest AMP level and it may be that some breakdown occurred during preparation. The above changes in nucleotide levels were reflected in the energy ratio values. In all cases the ATP/ADP ratio decreased in parasitized individuals as did the ATP/AMP and energy change ratios, with the exception of replicate 3. Energy change, however, was only slightly affected, and the average energy charge for all replicates and treatments was 0.82 ZL 0.04 (mean +- SD).
!.hu ADP
II
AMP
r
L
FIG. 1. Chromatogram tracings showing adenylate nucleotides in fat body extracts from Trichophsiu ni. Unparasitized 4th-instar larval fat body (bottom). Same sample “spiked” with ATP, ADP, AMP, GTP, GDP, and GMP (top). 0.03 AUFS.
protein as described above. Energy charge, described by Atkinson (1968), was calculated as follows: Energy charge = (ATP + t/2 ADP)/(ATP + ADP + AMP), and the adenylate kinase mass action ratio, K = ATP . AMP/ADP2. RESULTS
The individual and total adenylate nucleotide levels of T. ni fat body were highly variable (Table 1). In all cases, however, ATP was present at levels far in excess of
DISCUSSION
The adenylate nucleotide system, composed of ATP, ADP, and AMP, is of major importance in the control of energy flow in living systems. These metabolites, particularly ATP and AMP, regulate the activity of many enzymes which determine metabolic flux through anabolic and catabolic pathways. The concentration of these three nucleotides is partially regulated by the adenylate kinase-catalyzed reaction: 2 ADP it ATP + AMP. The ATP level is maintained much higher than that of AMP, such that a small change in ATP causes a greater fractional increase in AMP (Newsholme, 1970). The theory of metabolic regulation by fractional amplification has led to the use of concentration ratios as measures of the phosphorylation potential of the adenylate system. One such measure, the “energy charge,” is half the average number of anhydride-bound phosphate groups per ad-
ENERGY STATUS
ADENINE
NLJCLEOTIDE
OF PARASITIZED
TABLE 1 RATIOS IN THE FAT BODY OF 4th-INsTAR Trichoplusia AFTER PARASITIZATION BY THE INSECT PARASITE, Hyposoter exiguae LEVELS
AND
ENERGY
Nucleotide levels (nmol/mg fat body protein)
Replicate 1 Control (10) Parasitized (10) Percentage change Replicate 2 Control (9) Parasitized (9) Percentage change Replicate 3 Control (10) Parasitized (10) Percentage change Replicate 4 Control (10) Parasitized (10) Percentage change Note.
49
T. ni
ni 3 DAYS
Energy ratios Energy/ charge
Adenylate kinase mass-action ratio
13.01 5.29 -59
0.84 0.77 -8
1.17 1.73
6.57 3.14 -53
21.72 12.29 -42
0.89 0.83 -7
1.99 0.80
53.78 65.14 +21
3.32 3.17 -3
6.18 10.70 +73
0.79 0.82 +4
1.78 0.94
58.64 53.24 -9
3.77 2.98 -21
13.51 7.44 -45
0.85 0.79 -7
1 .os 1.19
ATP
ADP
AMP
Total
ATP/ADP
42.16 34.32 -19
10.81 11.35 +5
3.24 6.49 +100
56.22 52.16 -7
3.90 3.02 -23
58.65 36.49 -38
8.92 11.62 +30
2.70 70.27 2.97 51.08 + 10 -27
36.76 46.22 +26
11.08 14.59 +32
5.95 4.32 -27
43.78 36.22 -17
11.62 12.16 -5
3.24 4.87 +50
ATPIAMP
The number of individuals tested is shown in parentheses.
enine moiety (Atkinson, 1968), and the catalytic nature of many enzymes is affected by this parameter rather than the absolute concentrations of adenylate nucleotides (Swedes et al., 1975). Regulatory enzymes from metabolic sequences in which ATP is regenerated are active at low levels of energy charge and activity decreases rapidly above 0.75; those from ATP-consuming sequences are inactive at low energy charge and sharply increase in activity above 0.75 (Atkinson, 1970). The response curves of these two types tend to intersect at about 0.85. The mean energy charge value of 0.82 found in T. ni during the present study indicates a reasonable balance between anabolic and catabolic metabolism in the 4th larval stadium of both control and parasitized individuals. The adenylate energy charge of normal, intact cells from a variety of sources appears to be stabilized between 0.8 and 0.95, and decreases during metabolic and physiological stress (Chapman et al., 1971). For example, the energy charge of Escherichia
coli during growth was reported to be 0.8 and, during the stationary phase or starvation, slowly declines to 0.5 (Chapman et al., 1971). Between the above values all cells were viable and capable of forming colonies, but under continued starvation the charge decreased further and death occurred below 0.5. Energy charges below 0.65, however, may not occur in surviving eucaryotic cells (Atkinson, 1977). The strict use of energy charge in predicting specific metabolic responses has been criticized from several theoretical as well as practical viewpoints. The application of the amplification theory depends upon the maintenance of the mass-action ratio of the adenylate kinase reaction near equilibrium. However, Yushok (1971) reported mass-action ratios several-fold higher than equilibrium at high ATP concentrations. Moreover, the ratio is markedly influenced by factors such as the uncomplexed Mg*+ ion and the total adenylate concentrations (Purich and Fromm, 1973). Indeed, independent evaluation of
50
THOMPSON
AND YAMADA
the effect of energy charge is made more crease in energy charge of duck erythrodifficult because, as occurred in the present cytes infected with Plasmodium lophurae study, the adenylate concentration tends to occurred during the final 6 hr of the pararise and fall with the energy charge, re- site’s life cycle. sulting in an even greater increase in the In the present study, the energy charge magnitude of change in ATP concentration of T. ni larvae 3 days after parasitization which accompanies changes in energy decreased only slightly, despite the dracharge than would occur if the adenylate matic physiological and metabolic alteraconcentration remained constant. Although tions which occur concurrently, as dethe above in vitro effects can greatly alter scribed under Introduction. The result sugthe steady-state levels of adenylate nucleogests that the parasite-host complex at this tides, under normal conditions the energy stage represents a viable and distinct physcharge is generally maintained within 5- iological entity, although its fate has been 10% (Atkinson, 1977). Moreover, the va- markedly altered from that of its nonparlidity of some criticisms may not be appliasitized counterpart. Although the parasite cable to intact cells and tissues because the was very small at this point and represented major consideration in examining whole less than 1% of the total biomass of the parcell energy charge ratios is to qualify the asite-host complex (Thompson, 1983b), it overall energetic state of the cell or tissue nevertheless has completed a substantial and not to examine the specific metabolic part of its development. It would be of inresponses of the adenylate nucleotides as terest to determine energy charge levels effecters of enzymes. throughout the entire development of H. The energy charge ratio is a qualitative exiguae and to evaluate the importance of measure of the potential energy of the or- the virus particles which play an important ganism and should, therefore, provide a pa- role in the successful development of the rameter for evaluating the overall metabolic parasite. It appears that insect parasites stress induced by parasitization. Thus, the such as H. exiguae have highly integrated degree of integration characterizing the de- metabolic associations with their hosts that velopment of the parasite-host complex as are mechanistically similar to those dewell as the onset of pathogenesis and/or de- scribed for many helminth and protozoan velopment of disease should be reflected by parasites, although the ultimate result of changes in the energy charge of the affected the association is, in most cases, host tissue. During parasite development an death. equilibrium must exist between the parasite’s utilization of the host metabolites and ACKNOWLEDGMENTS the host’s requirements for the same factors This work was supported by grants from the USPHS that are necessary for maintenance and sur(AI05226) and the UNDP/World Bank/World Health vival. Parasitization must not inflict too Organization Special Program for Research and much stress on the host before completion Training in Tropical Diseases. of the parasite’s development. Indeed, through natural selection, it is likely that REFERENCES host death often occurs synchronously with completion of the parasite’s life cycle. For ATKINSON, D. E. 1968. The energy change of the adexample, in malaria-infected erythrocytes, enylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry, 7, 4030the host cell is maintained until completion 4034. of the parasite’s development, and only in D. E. 1970. Enzymes as control elements the last stages was notable metabolic stress ATKINSON, in metabolic regulation. In “The Enzymes” (P. D. observed (Yamada and Sherman, 1980). Boyer, ed.), Vol. 1, pp. 461-489. Academic Press, New York. Specifically, over half of the total 35% de-
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THOMPSON, S. N. 1982. Effects of parasitization by the insect parasite Hyposoter exiguae on the growth, development and physiology of its host, Trichoplusia ni. Parasitology 84, 491-510. THOMPSON, S. N. 1983a. Biochemical and physiological effects of metazoan endoparasites on their host species. Comp. Biochem. Physiol. B, 74, 183-211. THOMPSON, S. N. 1983b. The nutritional physiology of Trichoplusia ni parasitized by the insect parasite, Hyposoter exiguae, and the effects of parallelfeeding. Parasitology 87, 15-28. THOMPSON, S. N., AND BINDER, B. 1984. Altered carbohydrate metabolism and gluconeogenesis in Trichoplusia ni parasitized by the insect parasite, Hyposoter exiguae. J. Parasitol., in press. VINSON, S. B., EDSON, K. M., AND STOLTZ, D. 1979. Effect of a virus associated with the reproductive system of the parasitoid wasp Campoletis sonorensis, on host weight gain. J. Znvertebr. Pathol. 34, 133-137. VON BRAND, T. 1979. “Biochemistry of Parasites.” Academic Press, New York. YAMADA, K. A., AND SHERMAN, I. W. 1980. Plasmodium lophurae: Malaria induced nucleotide changes in duckling (Anas domestica) erythocytes. Mol. Biochem.
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