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Biochemical systematics of the genus Lipara using isoenzymes
Biochemical Systematicsand Ecology,Vol. 15, No. 6, pp. 653-659, 1987.
0305-1978/87 $3.00+0.00 Pergamon Journals Ltd.
Printed in Great Britain.
Biochemical systematics of the genus Lipara using isoenzymes L. DE BRUYN and T. BACKELJAU Laboratorium v o o r Algemene Dierkunde, Rijksuniversitair Centrum Antwerpen, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium
Key Word Index--Lipara; Chloropidae; Diptera; isoenzymes; systematics. Abstract--In the course of a systematic and population genetic study of the genus Lipara, a new approach was tested, involving the examination of isoenzymes using PAGE and isoelectric focusing (IEF). The enzymes from whole individual specimens of both larvae or adult flies were analysed. Twenty-five enzyme systems have been investigated with PAGE, 18 of which gave interpretable results. General proteins were analysed with both PAGE and IEF. By means of these techniques, the intra- and interspecific variation of structural proteins and different enzyme systems were examined. Possible changes in general proteins and enzymes during the development from larvae to adults were also investigated.
Introduction Lipara species are monophageous parasites on Phragrnites australis (Cav.) Trin. ex Steud., on which they induce typical cigar or spike like galls. The genus comprises nine described and at least one undescribed species [1]. All these are restricted to the Palaearctic region: four of them occur in the East Asiatic subregion [2], one has a pan-Palaearctic distribution [3] and the remaining four are confined to the western half of the Palaearctic region, [3, 4]. Three species of the latter group are known to occur in Belgium, viz. L. lucens Meigen, L. pullitarsis Doscko~il & Chv~la, and L. rufitarsis (Loew) [5]. Recently, one of us (L. De Bruyn) started a systematic and population genetic study of the genus. It became clear that approaches, other than classical morphological and anatomical observations, were needed. Therefore, we have evaluated the use of isoenzymes using vertical polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). In this communication we present our preliminary results using these techniques.
TABLE 1. ENZYME ESSAYS GIVING INTERPRETABLE RESULTS
Enzyme system Glucose-6-phosphate dehydrogenase (G6PD) 0c-Glycerophosphate dehydrogenase (0¢GPD) Phospho-glucomutase (PGM) Malic Enzyme (ME) 0c-Amylase (AMY) Glutamate-oxaloacetate transaminase (GOT) Malate dehydrogenase (MDH) Peptidase (substrate Leu-Ala) Isocitrate dehydrogenase (ICD) 6-Phosphogluconate dehydrogenase (PGD) Glucosephosphate isomerase (GPI) Acid phosphatase (ACP) Xanthine dehydrogenase (XDH) Hexokinase (HK) Superoxide dismutase (SOD) Peroxidase (PER) Leucine aminopeptidase (LAP) General Proteins
E.C. No.
Result adults larvae
Buffer
1.1.1.49.
*
*
TG
1.2.1.12. 2.7.5.1. 1.1.1.40. 3.2.1.1.
* * * 1-
* * * t
TBE TBE TG TG
2.6.1.1. 1.1.1.37. 3.4.x.x. 1.1.1.42.
* * 1*
t * 1" *
TG TC TG TC
1.1.1.44.
*
*
TC
5.3.1.9. 3.1.3.2. 1.2.3.2. 2.7.1.1. 1.15.1.1. 1,11,1.7, 3.4.1.1.
-* * * 1" -t *
* * 1" * * t t *
TC TC TC TG TG TG TG TG
*Clear activity, fLow enzyme activity. --Not yet investigated.
Results and Discussion Twenty-five enzyme systems have been investigated with PAGE, 18 of which gave interpretable results (Table 1). However, genetic and quantita-
tive interpretation of these results is reserved until more data are available. The seven other enzymes; viz. aldolase (ALD: EC.4.1.2.13), diaphorase (DIA: EC.1.6.2.2), esterases (ES: EC.3.1.1.1), fumarase (FUM: EC.4.2.1.2), glyoxylase (GLO: EC.3.1.2.6.), nucleoside phos-
(Received 20 March 1987) 653
654
phorylase (NP: EC.2.4.2.1) and sorbitol dehydrogenase (SDH: EC.1.1.1.14), revealed no or only very weak activity. The weak or undetectable activity of these latter enzymes may be due to several causes. One possibility is to assume that sample preparations and/or electrophoretic conditions could have destroyed or altered molecular structures in such way that certain labile proteins may have lost their enzymatic properties. In other cases, enzyme activities depend on age, developmental stage, feeding habits, climatological conditions, etc. These are all factors which are known to influence gene expressions [6-8]. For instance, the larvae processed here, showed no esterase activity (neither did the adults), probably as a result of the fact that they were collected in winter and thus were fully developed and in diapause. Moreover, they had already stopped feeding at the end of August. However, younger larvae, which were collected in July (1986) and which were thus still in their alimentary stage, did reveal a clear esterase activity. It is indeed well known that certain esterases may be diet induced [9]. Another example of developmental and sex dependent differences in gene expression is found in XDH. In the larval extracts the XDH bands are only slightly visible. Extracts from the adults, on the contrary, show a distinct enzyme activity. XDH is an important enzyme in the catabolism of purines and has also an important role in pterin metabolism [9]. As a consequence, it should be clearly recognised that when species are to be compared electrophoretically, it is important to use individuals at the same developmental stages. The IEF of general proteins revealed complex banding profiles for each species. But as in PAGE, no quantitative analysis could be performed due to the lack of sufficient material. Yet, a preliminary qualitative approach indicates that each species may be defined by a specific banding pattern (Fig. 1), even though there exists intra-specific variation among these patterns. When different developmental stages within the same species are compared (Fig. 2), slightly different patterns may be found: some protein bands have disappeared, while others have
L. DE BRUYN AND T. BACKELJAU
emerged. The variation between the two stages, however, is much less pronounced than the interspecific variation. General proteins were also analysed by means of PAGE (Fig. 3). Here the banding pattern is less complex than with IEF. The profiles may be divided in a number of banding zones. In agreement with the results of the IEF, species-specific patterns may be found which nevertheless show some intra-specific variation. In conclusion, it is obvious to us that in order to obtain information on the electrophoretic variation of enzymes and other proteins within and between Lipara species, it will be necessary to study a large variety of enzyme systems in both adults and larvae, collected from over a wide geographical range.
Experimental Galls were collected in the field and transported to the laboratory, where part of them were dissected to obtain pupae and larvae which were identified according to refs [4, 10]. In order to rear adults, pupae were transferred into small glass vials, which were kept humid by a layer of moist peat-mould. For extraction of the proteins, adult flies or larvae were homogenized in a 20% aqueous sucrose solution: 20-30 p,I sucrose solution per 10 mg tissue in case of larve and 40-50 p.I sucrose solution in case of adults. 5 p.I of a 0.1% saturated phenylthiourea-EtOH mixture in H20 was added per 100 pl of diluted sample in order to prevent oxidative tyrosinase activity. After homogenisation, samples were centrifuged at 15 000 r.p.m, for 40 rain at 4 °. The resulting clear supernatant was then stored at - 2 0 = until analysis. PAGE was performed in vertical gels (80 × 80 × 2.7 mm; gel strength 6%). 3-6 ~1 supernatant of each sample was applied in individual wells on top of the gel. Two types of PAGE experiments were carried out: (a) discontinuous PAGE (TG) with Tris-HCI (0.05 M) as gel buffer and Tris-glycine (0.01 M) as electrode buffer, both at pH 9,0. (b) continuous PAGE where electrode and gel buffers are identical. Here two buffer systems were used, viz a Tris-citric acid (TC) buffer (0.025 M, pH 8.0) and a Tris-EDTA-boric acid (TBE) buffer (0.025 M, pH 8.9). Electrophoresis was started at 25 V during 15 min, then for the next 15 rain, voltage was increased to 50 V and finally, a 150 V regime was established during 2 h ('I'C and TBE) or until the migrating front had reached the anodal side of the gel (TG). Experiments were performed at a constant temperature of +5 °. After electrophoresis, gels were stained for enzymes or general proteins according to refs [11, 12]. IEF was carried out in horizontal 1% agarose gels (240 × 110 × 0.6 ram) in which a 6.25% Pharmalyte "I'M ampholines solution was incorporated, creating a 4-6.5 pH gradient. LKB electro-focusing strips were used as electrodes. They were wetted with 0.05 M H2SO4 for the anode and 1 M NaOH for the cathode. Per sample 6 p,I supernatant was applied in individual wells nearly halfway between both electrodes. Runs
655
-I-
/
bm
~ m
i
~ml m N
m
1
2
3
4
5
6
7
8
9
10
11
12
13
FIG. 1. GENERAL PROTEIN PATTERNS IN LIPARA LARVAE .AS REVEALED BY I.E.F. 1-3 L. lucens; 4,5 L similis;6,7 L. vallicola;8-10 L. rufitarsis." 11-13 L. pullitarsis.
656
-t-
milton
1
2
3
4
5
6
7
8
FIG. 2. VARIATION IN GENERAL PROTEIN PATTERNS IN L. lucens (I.E.F.). 1-3 adults; 4-8 larvae.
o
iD
m
z
Z
o m
m~
-n
~O
~0
C~
C~
CA~
1
1
I
I
BIOCHEMICALSYSTEMATICSOF LIPARA were conducted under constant power. They were started at 500 V and stopped at approximately 1500 V (+75 min). Afterwards, gels were stained for general proteins according to ref. [13]. Acknowledgement--The present work was supported by a grant of the "lnstituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw' (I.W.O.N.L.). The second author is a research assistant of the Belgian National Fund for Scientific Research (N.F.W.O.).
References 1. Kanmiya, K. (1984) Regular session, XVII International Congress of Entomology, Hamburg, 1984. 2. Kanmiya, K. (1982) Kurume University Journal 31, 37. 3. Beschovski, V. L. (1984) Acta Zoo/. 8u/g. 24, 3.
659 4. Chvbla, M., Dosko~il, J., Mook, J. H. and Pokorny, V. (1974) Tijdschr. En~ 117, 1. 5. De Bruyn, L. (1985) Bull. Annls. Soc. R. Beige. Ent. 121, 485. 6. Storey, K. B. and Storey, J. M. (1981) J. Comp. Phys. 144, 191. 7. Johnson, G. B. (1974) Science 184, 28. 8. Wright, C. A. (1974) Biochemical and Immunological Taxonomy of Animals. Academic Press. London. 9. Gilmour, D. (1965) The Metabolism of Insects. Oliver & Boyd, Edinburgh. 10. Kanmiya, K. (1983) Mere. En~ Soc, Wash. 11, 370 pp. 11. Harris, H. and Hopkinson, D. A. (1976) Handbook of Enzyme Electrophoresis in Human Gene~'cs. North Holland, Amsterdam. 12. Backeljau, T. (1985) Basteria49,, 11, 13. Backeljau, 1"., Ahmadyar, S. Z., Selens, M., Van Rompaey, J. and Verheyen, W. (1987) ZooL Scr. 16, 209.
0305-1978/87 $3.00+0.00 Pergamon Journals Ltd.
Printed in Great Britain.
Biochemical systematics of the genus Lipara using isoenzymes L. DE BRUYN and T. BACKELJAU Laboratorium v o o r Algemene Dierkunde, Rijksuniversitair Centrum Antwerpen, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium
Key Word Index--Lipara; Chloropidae; Diptera; isoenzymes; systematics. Abstract--In the course of a systematic and population genetic study of the genus Lipara, a new approach was tested, involving the examination of isoenzymes using PAGE and isoelectric focusing (IEF). The enzymes from whole individual specimens of both larvae or adult flies were analysed. Twenty-five enzyme systems have been investigated with PAGE, 18 of which gave interpretable results. General proteins were analysed with both PAGE and IEF. By means of these techniques, the intra- and interspecific variation of structural proteins and different enzyme systems were examined. Possible changes in general proteins and enzymes during the development from larvae to adults were also investigated.
Introduction Lipara species are monophageous parasites on Phragrnites australis (Cav.) Trin. ex Steud., on which they induce typical cigar or spike like galls. The genus comprises nine described and at least one undescribed species [1]. All these are restricted to the Palaearctic region: four of them occur in the East Asiatic subregion [2], one has a pan-Palaearctic distribution [3] and the remaining four are confined to the western half of the Palaearctic region, [3, 4]. Three species of the latter group are known to occur in Belgium, viz. L. lucens Meigen, L. pullitarsis Doscko~il & Chv~la, and L. rufitarsis (Loew) [5]. Recently, one of us (L. De Bruyn) started a systematic and population genetic study of the genus. It became clear that approaches, other than classical morphological and anatomical observations, were needed. Therefore, we have evaluated the use of isoenzymes using vertical polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). In this communication we present our preliminary results using these techniques.
TABLE 1. ENZYME ESSAYS GIVING INTERPRETABLE RESULTS
Enzyme system Glucose-6-phosphate dehydrogenase (G6PD) 0c-Glycerophosphate dehydrogenase (0¢GPD) Phospho-glucomutase (PGM) Malic Enzyme (ME) 0c-Amylase (AMY) Glutamate-oxaloacetate transaminase (GOT) Malate dehydrogenase (MDH) Peptidase (substrate Leu-Ala) Isocitrate dehydrogenase (ICD) 6-Phosphogluconate dehydrogenase (PGD) Glucosephosphate isomerase (GPI) Acid phosphatase (ACP) Xanthine dehydrogenase (XDH) Hexokinase (HK) Superoxide dismutase (SOD) Peroxidase (PER) Leucine aminopeptidase (LAP) General Proteins
E.C. No.
Result adults larvae
Buffer
1.1.1.49.
*
*
TG
1.2.1.12. 2.7.5.1. 1.1.1.40. 3.2.1.1.
* * * 1-
* * * t
TBE TBE TG TG
2.6.1.1. 1.1.1.37. 3.4.x.x. 1.1.1.42.
* * 1*
t * 1" *
TG TC TG TC
1.1.1.44.
*
*
TC
5.3.1.9. 3.1.3.2. 1.2.3.2. 2.7.1.1. 1.15.1.1. 1,11,1.7, 3.4.1.1.
-* * * 1" -t *
* * 1" * * t t *
TC TC TC TG TG TG TG TG
*Clear activity, fLow enzyme activity. --Not yet investigated.
Results and Discussion Twenty-five enzyme systems have been investigated with PAGE, 18 of which gave interpretable results (Table 1). However, genetic and quantita-
tive interpretation of these results is reserved until more data are available. The seven other enzymes; viz. aldolase (ALD: EC.4.1.2.13), diaphorase (DIA: EC.1.6.2.2), esterases (ES: EC.3.1.1.1), fumarase (FUM: EC.4.2.1.2), glyoxylase (GLO: EC.3.1.2.6.), nucleoside phos-
(Received 20 March 1987) 653
654
phorylase (NP: EC.2.4.2.1) and sorbitol dehydrogenase (SDH: EC.1.1.1.14), revealed no or only very weak activity. The weak or undetectable activity of these latter enzymes may be due to several causes. One possibility is to assume that sample preparations and/or electrophoretic conditions could have destroyed or altered molecular structures in such way that certain labile proteins may have lost their enzymatic properties. In other cases, enzyme activities depend on age, developmental stage, feeding habits, climatological conditions, etc. These are all factors which are known to influence gene expressions [6-8]. For instance, the larvae processed here, showed no esterase activity (neither did the adults), probably as a result of the fact that they were collected in winter and thus were fully developed and in diapause. Moreover, they had already stopped feeding at the end of August. However, younger larvae, which were collected in July (1986) and which were thus still in their alimentary stage, did reveal a clear esterase activity. It is indeed well known that certain esterases may be diet induced [9]. Another example of developmental and sex dependent differences in gene expression is found in XDH. In the larval extracts the XDH bands are only slightly visible. Extracts from the adults, on the contrary, show a distinct enzyme activity. XDH is an important enzyme in the catabolism of purines and has also an important role in pterin metabolism [9]. As a consequence, it should be clearly recognised that when species are to be compared electrophoretically, it is important to use individuals at the same developmental stages. The IEF of general proteins revealed complex banding profiles for each species. But as in PAGE, no quantitative analysis could be performed due to the lack of sufficient material. Yet, a preliminary qualitative approach indicates that each species may be defined by a specific banding pattern (Fig. 1), even though there exists intra-specific variation among these patterns. When different developmental stages within the same species are compared (Fig. 2), slightly different patterns may be found: some protein bands have disappeared, while others have
L. DE BRUYN AND T. BACKELJAU
emerged. The variation between the two stages, however, is much less pronounced than the interspecific variation. General proteins were also analysed by means of PAGE (Fig. 3). Here the banding pattern is less complex than with IEF. The profiles may be divided in a number of banding zones. In agreement with the results of the IEF, species-specific patterns may be found which nevertheless show some intra-specific variation. In conclusion, it is obvious to us that in order to obtain information on the electrophoretic variation of enzymes and other proteins within and between Lipara species, it will be necessary to study a large variety of enzyme systems in both adults and larvae, collected from over a wide geographical range.
Experimental Galls were collected in the field and transported to the laboratory, where part of them were dissected to obtain pupae and larvae which were identified according to refs [4, 10]. In order to rear adults, pupae were transferred into small glass vials, which were kept humid by a layer of moist peat-mould. For extraction of the proteins, adult flies or larvae were homogenized in a 20% aqueous sucrose solution: 20-30 p,I sucrose solution per 10 mg tissue in case of larve and 40-50 p.I sucrose solution in case of adults. 5 p.I of a 0.1% saturated phenylthiourea-EtOH mixture in H20 was added per 100 pl of diluted sample in order to prevent oxidative tyrosinase activity. After homogenisation, samples were centrifuged at 15 000 r.p.m, for 40 rain at 4 °. The resulting clear supernatant was then stored at - 2 0 = until analysis. PAGE was performed in vertical gels (80 × 80 × 2.7 mm; gel strength 6%). 3-6 ~1 supernatant of each sample was applied in individual wells on top of the gel. Two types of PAGE experiments were carried out: (a) discontinuous PAGE (TG) with Tris-HCI (0.05 M) as gel buffer and Tris-glycine (0.01 M) as electrode buffer, both at pH 9,0. (b) continuous PAGE where electrode and gel buffers are identical. Here two buffer systems were used, viz a Tris-citric acid (TC) buffer (0.025 M, pH 8.0) and a Tris-EDTA-boric acid (TBE) buffer (0.025 M, pH 8.9). Electrophoresis was started at 25 V during 15 min, then for the next 15 rain, voltage was increased to 50 V and finally, a 150 V regime was established during 2 h ('I'C and TBE) or until the migrating front had reached the anodal side of the gel (TG). Experiments were performed at a constant temperature of +5 °. After electrophoresis, gels were stained for enzymes or general proteins according to refs [11, 12]. IEF was carried out in horizontal 1% agarose gels (240 × 110 × 0.6 ram) in which a 6.25% Pharmalyte "I'M ampholines solution was incorporated, creating a 4-6.5 pH gradient. LKB electro-focusing strips were used as electrodes. They were wetted with 0.05 M H2SO4 for the anode and 1 M NaOH for the cathode. Per sample 6 p,I supernatant was applied in individual wells nearly halfway between both electrodes. Runs
655
-I-
/
bm
~ m
i
~ml m N
m
1
2
3
4
5
6
7
8
9
10
11
12
13
FIG. 1. GENERAL PROTEIN PATTERNS IN LIPARA LARVAE .AS REVEALED BY I.E.F. 1-3 L. lucens; 4,5 L similis;6,7 L. vallicola;8-10 L. rufitarsis." 11-13 L. pullitarsis.
656
-t-
milton
1
2
3
4
5
6
7
8
FIG. 2. VARIATION IN GENERAL PROTEIN PATTERNS IN L. lucens (I.E.F.). 1-3 adults; 4-8 larvae.
o
iD
m
z
Z
o m
m~
-n
~O
~0
C~
C~
CA~
1
1
I
I
BIOCHEMICALSYSTEMATICSOF LIPARA were conducted under constant power. They were started at 500 V and stopped at approximately 1500 V (+75 min). Afterwards, gels were stained for general proteins according to ref. [13]. Acknowledgement--The present work was supported by a grant of the "lnstituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw' (I.W.O.N.L.). The second author is a research assistant of the Belgian National Fund for Scientific Research (N.F.W.O.).
References 1. Kanmiya, K. (1984) Regular session, XVII International Congress of Entomology, Hamburg, 1984. 2. Kanmiya, K. (1982) Kurume University Journal 31, 37. 3. Beschovski, V. L. (1984) Acta Zoo/. 8u/g. 24, 3.
659 4. Chvbla, M., Dosko~il, J., Mook, J. H. and Pokorny, V. (1974) Tijdschr. En~ 117, 1. 5. De Bruyn, L. (1985) Bull. Annls. Soc. R. Beige. Ent. 121, 485. 6. Storey, K. B. and Storey, J. M. (1981) J. Comp. Phys. 144, 191. 7. Johnson, G. B. (1974) Science 184, 28. 8. Wright, C. A. (1974) Biochemical and Immunological Taxonomy of Animals. Academic Press. London. 9. Gilmour, D. (1965) The Metabolism of Insects. Oliver & Boyd, Edinburgh. 10. Kanmiya, K. (1983) Mere. En~ Soc, Wash. 11, 370 pp. 11. Harris, H. and Hopkinson, D. A. (1976) Handbook of Enzyme Electrophoresis in Human Gene~'cs. North Holland, Amsterdam. 12. Backeljau, T. (1985) Basteria49,, 11, 13. Backeljau, 1"., Ahmadyar, S. Z., Selens, M., Van Rompaey, J. and Verheyen, W. (1987) ZooL Scr. 16, 209.