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Bacteria from the alimentary canal of the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera, Aphididae)
J. Inns. Phyriol., 1963, Vol. 9, pp. 435 to 438. Pergnma
Press Ltd.
Printed in Great Britain
BACTERIA FROM THE ALIMENTARY CANAL OF THE PEA APHID, ACYRTHOSIPHON PISUM (HARR.) (HOMOPTERA, APHIDIDAE) P. N. SRIVASTAVA*
and J. W. ROUATT
Research Station, Research Branch, Canada Department of Agriculture, Saint-Jean, Quebec, Canada, and Microbiology Research Institute,? Research Branch, Canada Department of Agriculture, Ottawa, Ontario, Canada (Received
4 February 1963)
Abstract-Bacteria isolated from the alimentary canal of the pea aphid, Acyrthosiphoff&sum, were identified mainly as species of the genera &r&a, Mtiococcus, Achmmobacter,and Flavobacteriunz.Starch was rapidly hydrolysed by all species of &WC& and Fkavobacteriumand by rriost species of Achmmobacterand slowly by some species of Microco~cus. All of the types of bacteria found in the alimentary tract of the pea aphid were represented in the bacterial flora on the leaves of the pea plants on which the aphids were reared. It is suggested that these organisms become established in the gut after being picked up from the leaf surface while the aphid ia probing for the phloem bundles. INTRODUCTION
MICRO-ORGANISMShave been reported from the alimentary canal of aphids (PAILLOT, 1931; SCHANDERLet al., 1949; SCHANDERL,1950) and also from the special organs of the body cavity known as mycetomes (BtiCHNER,.l953). STJZINHAUS (1942, 1946) lists and describes bacteria isolated from many other insects, and in a recent review RICHARDSand BROOKS(1958) deal with symbionts and microorganisms of insects and their role in insect metabolism. SRIVASTAVAand AUCLAIR (1962) reported the presence of bacteria in the alimentary canal of the pea aphid, Acyrthosiphonpiwm (Harr.), and demonstrated that they could hydrolyse starch in vitro. The present studies were undertaken as a supplement to the above work with a view to isolating these bacteria and studying them in detail. It was thought desirable also to study tie bacterial flora of the leaf of the pea plant on which the aphids were reared, and to compare it with that found in the gut. MATERIALS AND METHODS Apterous, parthenogenetic, young-bearing adults (uniform as to stock Rl, CARTIER,1959) reared in the greenhouse on pea plants (P~.wwzsativum L. variety Perfection), as described by AUCLAIR(1958), were used in this work. Dissections * Post-doctorate
Fellow of the National Research Council of Canada. t Contribution No. 553 from the Microbiology Research Institute. 435
436
P. N.
SRIVA~TAVA AND
J. W. ROUA-IT
were carried out in a u.v.-sterilized room using aseptic precautions and sterile instruments. The aphids were surface sterilized by dipping in 95% ethanol for 2 min and washing twice in sterile water. Dissected alimentary canals were also washed in sterile water and stored in a separate dish until the required number was collected. They were then ground in a micro Pyrex tissue grinder and the volume of the gut suspension so adjusted with sterile water that 1 ml of the suspension contained material from ten alimentary canals. The gut suspension was centrifuged at 2000 rev/mm for 10 min. Three dilutions were prepared from the supernatant and plated with Bacto-Pennassay Base Agar (Difco) containing O.1o/o glucose, and the residue streaked on plates of the same medium. All plates were incubated at 37°C for 3 days, following which colonies were picked. Washings from the leaves of the pea plants on which the aphids were reared were similarly plated and isolates obtained. Starch hydrolysis was determined by streaking the isolates on the above agar medium containing 2% soluble starch and flooding with an iodine solution following 3 days’ incubation at 37°C. Identification of the bacteria was carried out according to BREEDet al. (1957), MANUAL(1957), and GERMAN (1959). RESULTS
AND
DISCUSSION
Representatives of the following four genera were identified from both the gut suspensions and leaf washings: Sarczita, MicTococcus, Achromobacter, and Flawobacterium. No growth was observed on plates on which sterilized aphids had been placed, indicating that the surface sterilization was satisfactory. Morphological characteristics : Surtinu-spheres, occurring singly and in regular packets (Fig. l), Gram-positive, non-motile, approximately 2 p in diameter; Mirrococcusdpheres, occurring singly, in pairs, and occasionally in irregular clumps (Fig. 2), Gram-positive to Gram-variable, non-motile, approximately 1 ~1in diameter; Achromobacter-rods, approximately 1.5 by 4-O II, Gramnegative, occasionally forming chains, actively motile by means of peritrichous flagella (Fig. 3), or non-motile (Fig. 4); Fluvobucterium-rods, approximately O-51.0 by 2&2-S p, Gram-negative, actively motile by means of peritrichous flagella, characteristically produce a yellow pigment when grown on nutrient medium. The diastatic activity of the isolates is presented in Table 1. Of a total of 114 isolates, approximately equal numbers from the aphid gut and leaf surface, 81 or 71 per cent were capable of hydrolysing starch in oitro. All species of the genera Sarcina and Flavobacterium and all motile species of Achromobacter were active hydrolysers whereas some non-motile species of Achromobacter and two species of ll4icrococcus slowly hydrolysed starch. These results confirm the report by SRIVASTAVA and AUCLAIR(1962) that starch hydrolysis by the gut homogenate and honeydew of the pea aphid was due to bacterial action and not to starch-splitting enzymes secreted by the gut. The inconsistent results obtained in these earlier studies may now be explained by assuming that when starch hydrolysis was slow or negligible the dominant forms
FIG. 1. Sarcina s p . x 1450. F~o. 2. Micrococcus s p . x 1450.
FIc. 3..dchromobacter sp. m o t i l e x 1450. FIc. 4. Achromobacter s p . n o n - m o t i l e × 1450.
BACTERIA FROMTHE ALIMENTARY CANALOF THE PEA APHID
437
in the homogenate were those which did not hydrolyse or only slowly hydrolysed starch. TABLE I-_~MY~.ASE ACTZYI~ OF BACTERIAISOLATEDFROMAPHID GUT ANDSURFACEOFPEALEAVJE Genus
Total No. of isolates studied
No. hydrolysing starch
Sarcina Micrococcus Achromobacter (motile) Ac~?~o~ct~ (non-motile) F‘hvobacterium
26 11 23 42 12
26 2 23 18 12
It is noteworthy that all forms isolated from the alimentary canal of the aphid were also found on the leaf surface, that the dominant forms in the alimentary canal and also on the leaf surface varied from time to time, and that the numbers and types of bacteria isolated from the gut were relatively few. In view of this and the feeding habits of the pea aphid (assuming the phloem sap of the pea plant to be sterile) it appears likely that the insect does not have a specific bacterial flora in its alimentary canal. Rather, the bacteria present in the gut are picked up by the aphids from the leaf surface while they are probing for the phloem bundles. N for their Acknowledgements-Thanks are due to Drs. J. L. AUCLAIRand H. KATZNELSO keen interest and useful discussions during the work and to Mrs. K. ORLIK-RUCKEMANN for effective technical assistance. REFERENCES AWCLAIR J. L. (1958)
Honeydew excretion in the pea aphid, Acyrthiphon pisum (Harr.) (Homoptera: Aphididae). J. Ins. Physiol. 2, 330-337. BII~II~R. S., MURRAYE. G. D., and SMITH N. R. (1957) BERGY’SManuol of Determinative BucterioZogy. Williams and Wilkins, Baltimore. BUCHNBRP. (1953) Endosymbiose der The mit pflanalichen Mikmorganismen. Birkhauser, Baael. CARTIERJ. J. (1959) Recognition of three biotypes of the pea aphid from southern Quebec. g. e&m. Ent. 52, 293-294. MAFIIIAL(1957) Society of American Bacteriologists’ Manual of Mi~a~o~o~a~ Methods. McGraw-Hill, New York. PAILLOT A. (1931) Parasitisme et symbiose chez les Aphides. C.R. Acad. Sci., Paris 193, 300-301. RICKS A. G. and BROOKSM. A. (1958) Internal symbiosis in insects. Annu. Rev. Ent. 3, 37-56. SCHANDXU H. (1950) uber die Hefesymbiose der Cerambyciden und Aphiden. l’erh. dtsck ZuoL Mainz 1949, 252-256. SCHANDERLH., LAUFF G., and BECKERH. (1949) Studien iiber die Mycetom und Darmsymbionten der Aphiden. 2. Naturf. 4b, W-53. SKERMAN V. B. D. (1959) A Guide to the Identification of the Genera of Bacteria Williams and Wilkins, Baltimore.
438
P. N. SRIVASTAVA ANDJ. W. ROUATT
SRIVASTAVA P. N. and AUCLAIRJ. L. (1962) Amylase activity in the alimentary canal and honeydew of the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). J. Ins. Physiol. 8, 349-355. STEINHAUSE. A. (1942) Catalogue of Bacteria Associated Extracellularly with Insects and Ticks. Burgess, Minneapolis. STEINHAUS E. A. (1946) Insect Microbiology. Cornstock, Ithaca.
Press Ltd.
Printed in Great Britain
BACTERIA FROM THE ALIMENTARY CANAL OF THE PEA APHID, ACYRTHOSIPHON PISUM (HARR.) (HOMOPTERA, APHIDIDAE) P. N. SRIVASTAVA*
and J. W. ROUATT
Research Station, Research Branch, Canada Department of Agriculture, Saint-Jean, Quebec, Canada, and Microbiology Research Institute,? Research Branch, Canada Department of Agriculture, Ottawa, Ontario, Canada (Received
4 February 1963)
Abstract-Bacteria isolated from the alimentary canal of the pea aphid, Acyrthosiphoff&sum, were identified mainly as species of the genera &r&a, Mtiococcus, Achmmobacter,and Flavobacteriunz.Starch was rapidly hydrolysed by all species of &WC& and Fkavobacteriumand by rriost species of Achmmobacterand slowly by some species of Microco~cus. All of the types of bacteria found in the alimentary tract of the pea aphid were represented in the bacterial flora on the leaves of the pea plants on which the aphids were reared. It is suggested that these organisms become established in the gut after being picked up from the leaf surface while the aphid ia probing for the phloem bundles. INTRODUCTION
MICRO-ORGANISMShave been reported from the alimentary canal of aphids (PAILLOT, 1931; SCHANDERLet al., 1949; SCHANDERL,1950) and also from the special organs of the body cavity known as mycetomes (BtiCHNER,.l953). STJZINHAUS (1942, 1946) lists and describes bacteria isolated from many other insects, and in a recent review RICHARDSand BROOKS(1958) deal with symbionts and microorganisms of insects and their role in insect metabolism. SRIVASTAVAand AUCLAIR (1962) reported the presence of bacteria in the alimentary canal of the pea aphid, Acyrthosiphonpiwm (Harr.), and demonstrated that they could hydrolyse starch in vitro. The present studies were undertaken as a supplement to the above work with a view to isolating these bacteria and studying them in detail. It was thought desirable also to study tie bacterial flora of the leaf of the pea plant on which the aphids were reared, and to compare it with that found in the gut. MATERIALS AND METHODS Apterous, parthenogenetic, young-bearing adults (uniform as to stock Rl, CARTIER,1959) reared in the greenhouse on pea plants (P~.wwzsativum L. variety Perfection), as described by AUCLAIR(1958), were used in this work. Dissections * Post-doctorate
Fellow of the National Research Council of Canada. t Contribution No. 553 from the Microbiology Research Institute. 435
436
P. N.
SRIVA~TAVA AND
J. W. ROUA-IT
were carried out in a u.v.-sterilized room using aseptic precautions and sterile instruments. The aphids were surface sterilized by dipping in 95% ethanol for 2 min and washing twice in sterile water. Dissected alimentary canals were also washed in sterile water and stored in a separate dish until the required number was collected. They were then ground in a micro Pyrex tissue grinder and the volume of the gut suspension so adjusted with sterile water that 1 ml of the suspension contained material from ten alimentary canals. The gut suspension was centrifuged at 2000 rev/mm for 10 min. Three dilutions were prepared from the supernatant and plated with Bacto-Pennassay Base Agar (Difco) containing O.1o/o glucose, and the residue streaked on plates of the same medium. All plates were incubated at 37°C for 3 days, following which colonies were picked. Washings from the leaves of the pea plants on which the aphids were reared were similarly plated and isolates obtained. Starch hydrolysis was determined by streaking the isolates on the above agar medium containing 2% soluble starch and flooding with an iodine solution following 3 days’ incubation at 37°C. Identification of the bacteria was carried out according to BREEDet al. (1957), MANUAL(1957), and GERMAN (1959). RESULTS
AND
DISCUSSION
Representatives of the following four genera were identified from both the gut suspensions and leaf washings: Sarczita, MicTococcus, Achromobacter, and Flawobacterium. No growth was observed on plates on which sterilized aphids had been placed, indicating that the surface sterilization was satisfactory. Morphological characteristics : Surtinu-spheres, occurring singly and in regular packets (Fig. l), Gram-positive, non-motile, approximately 2 p in diameter; Mirrococcusdpheres, occurring singly, in pairs, and occasionally in irregular clumps (Fig. 2), Gram-positive to Gram-variable, non-motile, approximately 1 ~1in diameter; Achromobacter-rods, approximately 1.5 by 4-O II, Gramnegative, occasionally forming chains, actively motile by means of peritrichous flagella (Fig. 3), or non-motile (Fig. 4); Fluvobucterium-rods, approximately O-51.0 by 2&2-S p, Gram-negative, actively motile by means of peritrichous flagella, characteristically produce a yellow pigment when grown on nutrient medium. The diastatic activity of the isolates is presented in Table 1. Of a total of 114 isolates, approximately equal numbers from the aphid gut and leaf surface, 81 or 71 per cent were capable of hydrolysing starch in oitro. All species of the genera Sarcina and Flavobacterium and all motile species of Achromobacter were active hydrolysers whereas some non-motile species of Achromobacter and two species of ll4icrococcus slowly hydrolysed starch. These results confirm the report by SRIVASTAVA and AUCLAIR(1962) that starch hydrolysis by the gut homogenate and honeydew of the pea aphid was due to bacterial action and not to starch-splitting enzymes secreted by the gut. The inconsistent results obtained in these earlier studies may now be explained by assuming that when starch hydrolysis was slow or negligible the dominant forms
FIG. 1. Sarcina s p . x 1450. F~o. 2. Micrococcus s p . x 1450.
FIc. 3..dchromobacter sp. m o t i l e x 1450. FIc. 4. Achromobacter s p . n o n - m o t i l e × 1450.
BACTERIA FROMTHE ALIMENTARY CANALOF THE PEA APHID
437
in the homogenate were those which did not hydrolyse or only slowly hydrolysed starch. TABLE I-_~MY~.ASE ACTZYI~ OF BACTERIAISOLATEDFROMAPHID GUT ANDSURFACEOFPEALEAVJE Genus
Total No. of isolates studied
No. hydrolysing starch
Sarcina Micrococcus Achromobacter (motile) Ac~?~o~ct~ (non-motile) F‘hvobacterium
26 11 23 42 12
26 2 23 18 12
It is noteworthy that all forms isolated from the alimentary canal of the aphid were also found on the leaf surface, that the dominant forms in the alimentary canal and also on the leaf surface varied from time to time, and that the numbers and types of bacteria isolated from the gut were relatively few. In view of this and the feeding habits of the pea aphid (assuming the phloem sap of the pea plant to be sterile) it appears likely that the insect does not have a specific bacterial flora in its alimentary canal. Rather, the bacteria present in the gut are picked up by the aphids from the leaf surface while they are probing for the phloem bundles. N for their Acknowledgements-Thanks are due to Drs. J. L. AUCLAIRand H. KATZNELSO keen interest and useful discussions during the work and to Mrs. K. ORLIK-RUCKEMANN for effective technical assistance. REFERENCES AWCLAIR J. L. (1958)
Honeydew excretion in the pea aphid, Acyrthiphon pisum (Harr.) (Homoptera: Aphididae). J. Ins. Physiol. 2, 330-337. BII~II~R. S., MURRAYE. G. D., and SMITH N. R. (1957) BERGY’SManuol of Determinative BucterioZogy. Williams and Wilkins, Baltimore. BUCHNBRP. (1953) Endosymbiose der The mit pflanalichen Mikmorganismen. Birkhauser, Baael. CARTIERJ. J. (1959) Recognition of three biotypes of the pea aphid from southern Quebec. g. e&m. Ent. 52, 293-294. MAFIIIAL(1957) Society of American Bacteriologists’ Manual of Mi~a~o~o~a~ Methods. McGraw-Hill, New York. PAILLOT A. (1931) Parasitisme et symbiose chez les Aphides. C.R. Acad. Sci., Paris 193, 300-301. RICKS A. G. and BROOKSM. A. (1958) Internal symbiosis in insects. Annu. Rev. Ent. 3, 37-56. SCHANDXU H. (1950) uber die Hefesymbiose der Cerambyciden und Aphiden. l’erh. dtsck ZuoL Mainz 1949, 252-256. SCHANDERLH., LAUFF G., and BECKERH. (1949) Studien iiber die Mycetom und Darmsymbionten der Aphiden. 2. Naturf. 4b, W-53. SKERMAN V. B. D. (1959) A Guide to the Identification of the Genera of Bacteria Williams and Wilkins, Baltimore.
438
P. N. SRIVASTAVA ANDJ. W. ROUATT
SRIVASTAVA P. N. and AUCLAIRJ. L. (1962) Amylase activity in the alimentary canal and honeydew of the pea aphid, Acyrthosiphon pisum (Harr.) (Homoptera: Aphididae). J. Ins. Physiol. 8, 349-355. STEINHAUSE. A. (1942) Catalogue of Bacteria Associated Extracellularly with Insects and Ticks. Burgess, Minneapolis. STEINHAUS E. A. (1946) Insect Microbiology. Cornstock, Ithaca.