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Isolation of the primary intracellular symbiote of the pea aphid, Acyrthosiphon pisum
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Isolation Symbiote
of the
23, 237-241 (1974)
of the Pea
E. J. HOUK’ Department
Primary Aphid,
Intracellular Acyrthosiphon
pisum
AND D. L. MCLEAN
of Entomology, University Davis, California 96616
Received September
of California,
10, 1973
A method for isolating the primary intracellular symbiote of the pea aphid, Acyrthosiphon pisum, is presented. The purity of the preparation, based on an area symbiote to area contaminant and a number to number basis, averages 93%.
siphon pisum. A method
of isolation is necessary for one to investigate the nutritional significance and possible evolutionary origins of the bacteroid symbiotes.
INTRODUCTION
The morphology of the mycetomal symbiotes of aphids has been examined by several authors in the past few years (Vago and LaPorte, 1965 ; Lamb and Hinde, 1967; Hinde, 1971; Griffiths and Beck, 1973; McLean and Houk, 1973). Based on their morphological investigations, several of these authors have postulated the systematic classification of the mycetomal symbiotes. Systematic classification and physiological-functions of the intracellular symbiotes have been variously hypothesized (Lanham, 1968; Brooks, 1963). There has, however, been no experimental evidence presented to substantiate these hypotheses. The main problem encountered, in attempting to clarify the above parameters, has been the inability of the experimentors to quantitatively separate one organism from the other. Cell biologists have been successful in the isolation of subcellular organelles through the use of rate-zonal, density gradient centrifu&tion. It seemed feasible, therefore, that a modification of this method might be applied to the isolation of the intracellular symbiotes of the pea aphid, Acyrtho-
of dissected symbiotes in a gradient. Mycetocytes were dissected from adult pea aphids in a hypertonic medium (McLean and Houk, 1973) and disrupted in a modified mitochondrial isolation medium (Bruening et al., 1971) by gently blowing air through the solution. The medium was modified by the addition of 1% polyvinyl pyrollidone (PVP-40) (w/v). This was to prevent the cross-linking of proteins due to the oxidation of polyphenolics. Density gradient separation was performed on a 2 to 10% Ficoll@ gradient (Campbell,* pers. comm.) . The gradients were formed by dispensing Ficoll at four percentage concentrations on the bottom of 50-ml centrifuge tubes. The concentrations, in order, were 2, 4, 6, and 10% (w/w) Ficoll@ in 0.01 M phasphate buffer (pH 7.2). The ratio of the respective layers was 3:3: 5: 10, with a total volume of 31.5 ml. The gradients were allowed to become established overnight at 5°C.
‘Present University 53706.
*R. N. Campbell, Dept. of Plant Univ. of Calif., Davis, CA 95616.
METHODS AND MATERIALS
address: Department of Entomology, of Wisconsin, Madison, Wisconsin
237 Copyright All ri&ts
@ 19i4
by Academic in any
of reproduction
Press, Inc. form reserved,
Behavior
Pathology,
238
HOUK
AND
FIG. 1. Schematic of the comparative sedimentation of (a) primary symbiote [relative mobility, coli (Rm = 0.36) (Rm = 0.60)1, (b) Escherichia and (c) Streptococcus fuecalis (Rm = 0.20). Calculated as the distance the microorganism traveled from the meniscus divided by the distance from the meniscus to the bottom of the tube.
In an attempt to ascertain the ability of this gradient system to separate intracellular symbiotes from bacteria, two speciesof
MCLEAN
bacteria, Eschericia coli and Streptococcus faecalis, were dispersed in the modified isolation medium. The symbiote, E. coli, and X. faecalis preparations were then gently layered on the top of separate gradients. The gradients were centrifuged at 400 g for 10 min using a Sorvall RC-2 centrifuge with an HB-4 rotor (Fig. 1). Behavior of whole aphid preparations in a gradient. Aphids were wrapped in a double thickness of cheesecloth and ground with a pestle in a porcelain mortar, containing isolation medium in the ratio of 3 : 1 to the aphid material (v/w). The supernatant was collected and passed through Miracloth@ and then disrupted by grinding in a Teflon and glass tissue grinder with a wall clearance of 25-50 pm. The suspensionwas centrifuged at 5000 g for 15 min. The supernatant was discarded, and the pellet was resuspendedin a 1: 100 dilution of the isolation medium. The total volume of suspensionmedium used was that amount required to yield
Fro. 2. Photomicrograph of the banding pattern symbiotes (arrow) from whole aphid extracts.
obtained
in the isolation
of the primary
ISOLATION
OF
SYMBIOTE
239
Fro. 3. Transmission electron micrographs of (a) isolated primary symbiotes; (b) contaminating mitochondria (m) and secondary symbiotes (9); and (c) the increased contamination seen when too large a fraction is taken.
a 3 : 2 ratio with the original aphid mass (v/w). The suspension was then layered on Ficoll gradients and centrifuged at 400g
for 10 min. The amount o,f material layered on top of each gradient was equivalent to 2 g of aphids,
240
HOUK
COMPARISON
OF CONTAMINATION
Symbiote area (cm”)
Isolate no.
208 141 128 162 122
1
2 3 4 .5
FROM
AND
MCLEAN
TABLE
1
DIFFERENT
Contamination area (cm”)
ISOLATES
OF PRIMARY
Total area (cm”)
Percent contamination
10 10
218 lFj1
4.5 6.6
14 7
142
9.8
10
133
169
Average contamination
The symbiote region of the gradient (Fig. 2) was fractionated by piercing the wall of the centrifuge tube with a No. 20 hypodermic needle and carefully withdrawing a 3-ml aliquot into a sterile syringe. The composition of this material was determined by the examination of electron micrographs (Figs. 3a, 3b, and 3~). Electron microscopy. The gradient fractions obtained above were sedimented by centrifuging at 15,000 g for 10 min. The pellet was then prepared for electron microscopy as described by McLean and Houk (1973). RESULTS
AND
SYMBIOTES
seen in the preparation (Figs. 3a, 3b). In order to avoid any substantial contamination of the preparation, more than a 3-4-ml aliquot of the gradient material should not be fractionated at one time. A larger fraction results in increased contamination with mitochondria and secondary symbiotes (Fig. 3~). The chemistry and in vitro culture of the primary symbiote is now being undertaken. This will hopefully help clarify the systematic status of the symbiotes and establish the nutritional significance of the primary symbiote.
DISCUSSION
Dissected symbiotes, when compared to E. coli and S. faecalis, are more mobile in the gradient (Fig. 1). The symbiotes are approximately twice as large in their largest dimension (4 pm) (McLean and Houk, 1973) than either of the two bacteria. The results indicate that the symbiotes can be separated from these two bacteria and most likely from other bacteria in the same size range as E. coli and S. faecalis. Isolates from the whole aphid formed a distinct light-scattering band in the same region that the dissected symbiote prep was found to sediment (Fig. 2). Examination of this fraction with the electron microscopy indicated that the primary symbiote could be isolated with a high degree of purity (Table 1; Fig. 3a). Some contaminants, in the form of mitochondria, secondary symbiotes, and membrane fragments, were
4.1 7.5 32.5 6.5 yO
ACKNOWLEDGMENTS
The authors wish to thank the Department of Botany, University of California at Davis and especially Dr. R. K. Falk, for the use of their Electron Microscopy facilities and Dr. R. S. Criddle, Department of Biochemistry and Biophysics, University of California at Davis, for his assistance in the modification of the isolation medium. REFERENCES
M. A. (1963). Symbiosis and aposymbiosis in arthropods. Symp. Sot. Gen. Microbial., 13, 200-231. BRUENING, G., CRIDDLE, R., PREISS, i, AND RUDERT, R. (1971). “Biochemical Esperiments.” Wiley (Interscience), New York. GRIFFITHS, G., AND BECK, D. (1973). Intracellular symbiotes of the pea aphid, Acyrthosiphon p&m. J. Insect Physiol., 19, 75-84. HINDE, R. (1971). The fme structure of the mycetome symbiotes of the aphids Brevicoryne bra&cue, Myzus per&cue, and Macrosiphon rosae. J. Insect Physiol., 17, 2035-2050. BROOKS,
ISOLATION
K. P., AND HINDE, R. (1967). Structure and development of the mycetome in the cabbage aphid, Brevicoryne brassicne. J. Invertebr. Pathol., 9, 3-11. LANHAM, U. N. (1968). The Blochmann bodies: Hereditary intracellular symbiotes of insects Bid. Rev. Cambridge Phil. Sot., 43, 269-286. MCLEAN, D. L., AND HOUK, E. J. (1973). Phase LAMB,
OF
SYMBIOTE
241
contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum J. Insect Physiol., 19, 62&633. VAGO, C., AND LAPORTE, M. (1965). Microscopic electronique des symbiontes globuleux des aphides [Horn. Aphidoideal. Ann. Sot. Entomol. Fr., 1, 181-196.
OF INVERTEBRATE
PATHOLOGY
Isolation Symbiote
of the
23, 237-241 (1974)
of the Pea
E. J. HOUK’ Department
Primary Aphid,
Intracellular Acyrthosiphon
pisum
AND D. L. MCLEAN
of Entomology, University Davis, California 96616
Received September
of California,
10, 1973
A method for isolating the primary intracellular symbiote of the pea aphid, Acyrthosiphon pisum, is presented. The purity of the preparation, based on an area symbiote to area contaminant and a number to number basis, averages 93%.
siphon pisum. A method
of isolation is necessary for one to investigate the nutritional significance and possible evolutionary origins of the bacteroid symbiotes.
INTRODUCTION
The morphology of the mycetomal symbiotes of aphids has been examined by several authors in the past few years (Vago and LaPorte, 1965 ; Lamb and Hinde, 1967; Hinde, 1971; Griffiths and Beck, 1973; McLean and Houk, 1973). Based on their morphological investigations, several of these authors have postulated the systematic classification of the mycetomal symbiotes. Systematic classification and physiological-functions of the intracellular symbiotes have been variously hypothesized (Lanham, 1968; Brooks, 1963). There has, however, been no experimental evidence presented to substantiate these hypotheses. The main problem encountered, in attempting to clarify the above parameters, has been the inability of the experimentors to quantitatively separate one organism from the other. Cell biologists have been successful in the isolation of subcellular organelles through the use of rate-zonal, density gradient centrifu&tion. It seemed feasible, therefore, that a modification of this method might be applied to the isolation of the intracellular symbiotes of the pea aphid, Acyrtho-
of dissected symbiotes in a gradient. Mycetocytes were dissected from adult pea aphids in a hypertonic medium (McLean and Houk, 1973) and disrupted in a modified mitochondrial isolation medium (Bruening et al., 1971) by gently blowing air through the solution. The medium was modified by the addition of 1% polyvinyl pyrollidone (PVP-40) (w/v). This was to prevent the cross-linking of proteins due to the oxidation of polyphenolics. Density gradient separation was performed on a 2 to 10% Ficoll@ gradient (Campbell,* pers. comm.) . The gradients were formed by dispensing Ficoll at four percentage concentrations on the bottom of 50-ml centrifuge tubes. The concentrations, in order, were 2, 4, 6, and 10% (w/w) Ficoll@ in 0.01 M phasphate buffer (pH 7.2). The ratio of the respective layers was 3:3: 5: 10, with a total volume of 31.5 ml. The gradients were allowed to become established overnight at 5°C.
‘Present University 53706.
*R. N. Campbell, Dept. of Plant Univ. of Calif., Davis, CA 95616.
METHODS AND MATERIALS
address: Department of Entomology, of Wisconsin, Madison, Wisconsin
237 Copyright All ri&ts
@ 19i4
by Academic in any
of reproduction
Press, Inc. form reserved,
Behavior
Pathology,
238
HOUK
AND
FIG. 1. Schematic of the comparative sedimentation of (a) primary symbiote [relative mobility, coli (Rm = 0.36) (Rm = 0.60)1, (b) Escherichia and (c) Streptococcus fuecalis (Rm = 0.20). Calculated as the distance the microorganism traveled from the meniscus divided by the distance from the meniscus to the bottom of the tube.
In an attempt to ascertain the ability of this gradient system to separate intracellular symbiotes from bacteria, two speciesof
MCLEAN
bacteria, Eschericia coli and Streptococcus faecalis, were dispersed in the modified isolation medium. The symbiote, E. coli, and X. faecalis preparations were then gently layered on the top of separate gradients. The gradients were centrifuged at 400 g for 10 min using a Sorvall RC-2 centrifuge with an HB-4 rotor (Fig. 1). Behavior of whole aphid preparations in a gradient. Aphids were wrapped in a double thickness of cheesecloth and ground with a pestle in a porcelain mortar, containing isolation medium in the ratio of 3 : 1 to the aphid material (v/w). The supernatant was collected and passed through Miracloth@ and then disrupted by grinding in a Teflon and glass tissue grinder with a wall clearance of 25-50 pm. The suspensionwas centrifuged at 5000 g for 15 min. The supernatant was discarded, and the pellet was resuspendedin a 1: 100 dilution of the isolation medium. The total volume of suspensionmedium used was that amount required to yield
Fro. 2. Photomicrograph of the banding pattern symbiotes (arrow) from whole aphid extracts.
obtained
in the isolation
of the primary
ISOLATION
OF
SYMBIOTE
239
Fro. 3. Transmission electron micrographs of (a) isolated primary symbiotes; (b) contaminating mitochondria (m) and secondary symbiotes (9); and (c) the increased contamination seen when too large a fraction is taken.
a 3 : 2 ratio with the original aphid mass (v/w). The suspension was then layered on Ficoll gradients and centrifuged at 400g
for 10 min. The amount o,f material layered on top of each gradient was equivalent to 2 g of aphids,
240
HOUK
COMPARISON
OF CONTAMINATION
Symbiote area (cm”)
Isolate no.
208 141 128 162 122
1
2 3 4 .5
FROM
AND
MCLEAN
TABLE
1
DIFFERENT
Contamination area (cm”)
ISOLATES
OF PRIMARY
Total area (cm”)
Percent contamination
10 10
218 lFj1
4.5 6.6
14 7
142
9.8
10
133
169
Average contamination
The symbiote region of the gradient (Fig. 2) was fractionated by piercing the wall of the centrifuge tube with a No. 20 hypodermic needle and carefully withdrawing a 3-ml aliquot into a sterile syringe. The composition of this material was determined by the examination of electron micrographs (Figs. 3a, 3b, and 3~). Electron microscopy. The gradient fractions obtained above were sedimented by centrifuging at 15,000 g for 10 min. The pellet was then prepared for electron microscopy as described by McLean and Houk (1973). RESULTS
AND
SYMBIOTES
seen in the preparation (Figs. 3a, 3b). In order to avoid any substantial contamination of the preparation, more than a 3-4-ml aliquot of the gradient material should not be fractionated at one time. A larger fraction results in increased contamination with mitochondria and secondary symbiotes (Fig. 3~). The chemistry and in vitro culture of the primary symbiote is now being undertaken. This will hopefully help clarify the systematic status of the symbiotes and establish the nutritional significance of the primary symbiote.
DISCUSSION
Dissected symbiotes, when compared to E. coli and S. faecalis, are more mobile in the gradient (Fig. 1). The symbiotes are approximately twice as large in their largest dimension (4 pm) (McLean and Houk, 1973) than either of the two bacteria. The results indicate that the symbiotes can be separated from these two bacteria and most likely from other bacteria in the same size range as E. coli and S. faecalis. Isolates from the whole aphid formed a distinct light-scattering band in the same region that the dissected symbiote prep was found to sediment (Fig. 2). Examination of this fraction with the electron microscopy indicated that the primary symbiote could be isolated with a high degree of purity (Table 1; Fig. 3a). Some contaminants, in the form of mitochondria, secondary symbiotes, and membrane fragments, were
4.1 7.5 32.5 6.5 yO
ACKNOWLEDGMENTS
The authors wish to thank the Department of Botany, University of California at Davis and especially Dr. R. K. Falk, for the use of their Electron Microscopy facilities and Dr. R. S. Criddle, Department of Biochemistry and Biophysics, University of California at Davis, for his assistance in the modification of the isolation medium. REFERENCES
M. A. (1963). Symbiosis and aposymbiosis in arthropods. Symp. Sot. Gen. Microbial., 13, 200-231. BRUENING, G., CRIDDLE, R., PREISS, i, AND RUDERT, R. (1971). “Biochemical Esperiments.” Wiley (Interscience), New York. GRIFFITHS, G., AND BECK, D. (1973). Intracellular symbiotes of the pea aphid, Acyrthosiphon p&m. J. Insect Physiol., 19, 75-84. HINDE, R. (1971). The fme structure of the mycetome symbiotes of the aphids Brevicoryne bra&cue, Myzus per&cue, and Macrosiphon rosae. J. Insect Physiol., 17, 2035-2050. BROOKS,
ISOLATION
K. P., AND HINDE, R. (1967). Structure and development of the mycetome in the cabbage aphid, Brevicoryne brassicne. J. Invertebr. Pathol., 9, 3-11. LANHAM, U. N. (1968). The Blochmann bodies: Hereditary intracellular symbiotes of insects Bid. Rev. Cambridge Phil. Sot., 43, 269-286. MCLEAN, D. L., AND HOUK, E. J. (1973). Phase LAMB,
OF
SYMBIOTE
241
contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum J. Insect Physiol., 19, 62&633. VAGO, C., AND LAPORTE, M. (1965). Microscopic electronique des symbiontes globuleux des aphides [Horn. Aphidoideal. Ann. Sot. Entomol. Fr., 1, 181-196.