Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum

J. Insect Physiol.,

1973,Vol. 19, pp. 625 to 633. Pergamon Press. Printed in Great Britain

PHASE CONTRAST AND ELECTRON MICROSCOPY OF THE MYCETOCYTES AND SYMBIOTES OF THE PEA APHID, ACYRTHOSIPHON PISUM D. L. MCLEAN Department

of Entomology, (Received

and E. J. HOUK

University

1 February

of California, Davis, California 95616

1972;

revised 28 April

1972)

Abstract-Phase contrast, scanning electron, and transmission electron microscopy of the symbiotes of Acyrthosiphon pisum was undertaken. Some staining properties of the symbiotes were also studied. The symbiotes of the pea aphid were found to be coccoid bodies 2 to 5 p in diameter, gram negative, stained slightly by Fuelgen’s, and stained blue by Machiavello’s. The symbiotes appear to be surrounded by three membranes. Ribosomes may occur within the cytoplasm of the symbiotes. The cytoplasm of the mycetocytes contains large numbers of mitochondria, endoplasmic reticulum, ribosomes, and a large nucleus, and nucleolus. A discussion of the classification of the symbiotes is also presented. INTRODUCTION

SELL in B~~CHNER (1965), VAGO and LAPORTE(1965), LAMB and HINDE (1967), and HINDE(1971a-c) have reviewed much of the literature on aphid symbiotes. VAGO and LAPORTE(1965) working with Macrosiphum rosae L. suggested that the symbiotes of this aphid were L-form bacteria from 2 to 4 I_Lin diameter. LAMB and HINDE (1967) concluded that the symbiotes of Brevicoryne brassicue (L.) were in the family Rickettsiaceae. The size of these organisms was 2 p in diameter. HINDE (1971~) working with B. bras&e, Myxus persicae (Sulz.), and M. rosae revised her classification of the symbiotes. Her contention in this paper was that the symbiotes may have arisen as a result of the adaptation of a Rickettsella-like pathogen to a symbiotic relationship with the aphid, or, that the symbiotes arose from some free-living bacteria. HINDE (1971a) attempted to culture the symbiotes of B. brassicae. She reported that they survived from 2 to 16 weeks and probably multiplied slowly. HINDE (1971b) suggested that in viviparous aphids, the symbiotes infect the embryo following the establishment of the blastoderm. She further stated that some symbiotes could be seen to degenerate within the mycetocytes as the aphid develops. All of the above studies were conducted using light and transmission electron microscopy. This paper includes a description of light, scanning electron, and transmission electron microscopy of the symbiotes of the pea aphid, Acyrthosiphon pisum (HARRIS). Results with definitive stains are also discussed. 625

626

D. L. MCLEAN ANDE. J. HOUK MATERIALS

AND METHODS

Apterous, parthenogenetic pea aphids, Acyrthosiphon p&urn were used for all microscopic observations. They were reared in greenhouses on broadbean, Viciu faba L. variety Windsor. In all cases, fourth instar or young adults were used for the study. All dissections were done in a 0.3 M sucrose solution. Phase contrast microscopy Material for observation was prepared in each of the following ways: Whole aphids. The aphids were placed in Carnoy-Lebrun fixative for about 24 hr. At the end of this period, they were washed in distilled water and dein Paraplasf hydrated in a tertiary butyl alcohol series. They were embedded (m.p. 61°C) and sectioned at 10 t.~. All material was stained with Mallory’s triple stain. Excised mycetocytes. Mycetocytes were dissected from young adults and placed in Carnoy-Lebrun fixative for 8 hr. The preparation, embedding, and staining procedures were the same as listed above. However, sections of the mycetocytes were cut at 3 p. Squashes of mycetocytes. Mycetocytes were dissected in a 0.3 M sucrose solution and placed on a depression slide containing more sucrose solution. The sucrose solution was removed and the mycetocytes washed twice in distilled water. A few mycetocytes were transferred to a microscope slide with a pipette. The mycetocytes were crushed with a fine dissecting needle. The material was air or heat dried and stained with Feulgen’s (LILLIE, 1965), Macchiavello’s (CONN et al., 1962), or gram stain (HUNGATE and FLETCHER, 1962). Scanning electron microscopy Mycetocytes were dissected as described previously. Following removal from the aphids, the mycetocytes were placed in boiling absolute ethanol for fixation. After about 2 min the ethanol was replaced with amyl acetate. The mycetocytes were placed in a critical point apparatus where the amyl acetate was removed in a CO, atmosphere at high pressure of about 1600 lb/n?. The valve was opened to release the CO, rapidly. This served to rupture the mycetocyte membrane exposing the symbiotes. Transmission electron microscopy Mycetocytes were dissected from aphids and some immersed in 5% glutaraldehyde that was buffered to pH 7.2 with 0.1 M sodium phosphate. The mycetocytes were fixed for 1 hr on ice. They were post-fixed for 1 hr in 1% osmium tetroxide on ice. Other mycetocytes were fixed in l*S”h potassium permanganate for 20 min and post-fixed for 1 hr in 1% osmium tetroxide. These procedures were also carried out on ice. All mycetocytes were dehydrated in cold ethanol. They were embedded in Spurr’s plastic (SPURR, 1969). The sections were stained for 20 min in 576 uranyl acetate and for 5 min in 2% lead citrate.

627

(a)

FIG. 1. (A) A diagrammatic drawing from a parasagittal section of a developing embryo of A. pisum showing the distribution of mycetocytes (M). The mycetocytes are posterior to the mesenteron (G). (B) A drawing from a x-section of a developing embryo of A. pisun~ showing the distribution of mycetocytes (M) around the gut (G). (C) A drawing from a parasagittal section of an adult A. pisum showing the distribution of mycetocytes (M). (D) Air dried smear of a mycetocyte showing bacilliform organisms (B) and symbiotes (S).

628

FIG. 2. (a) Scanning electron micrograph of symbiotes within a mycetocyte of .4. ~LWNI. (b) Scanning electron micrograph of a mycetocyte of A. pisum showing the polar invagination. (c) S canning electron micrograph of symbiotes (S) showing their variable size and division (arrow). (d) Scanning electron micrograph of the nucleus of a mycetocyte. (e) Scanning electron micrograph showing trachea (T) on the surface of a mycetocyte.

629

FIG. 3. (a) A transmission electron micrograph of a symbiote of A. pisum treated with KMN04 showing the double unit membrane (M,, M,). (b) Ribosome-like bodies (Rb) within a mycetocyte and symbiote. The double unit membrane (M,, M,) and a cytoplasmic membrane (Ma) associated with a symbiote are shown. (c) Mitochondria (Mit) and the distribution of symbiotes (S) within a mycetocyte. (d) Symbiote undergoing division. (e) A polymorphic nucleus (N) of a mycetocyte. The nuclear membrane (NM) can be seen. (f) Bacilliform organisms associated with the mycetocytes of A. pisztm.

MYCETOCYTES

AND SYMBIOTES

OF THE PEA APHID

631

RESULTS

Phase contrast microscopy The symbiotes stain brown and the mycetocyte nucleolus stains red with Mallory’s triple stain. The developing embryos appear to have most of their body cavity filled with mycetocytes (Figs. lA, B). Following birth, and throughout the life of the pea aphid, mycetomes appear to be situated in two principal body regions. One mycetome surrounds the posterior portion of the foregut directly above the thoracic sternites. The other is located in the posterior region of the abdomen (Fig. 1C). The number of mycetocytes in the pea aphid diminishes as the agrees with those made by LAMB and aphid becomes older. This observation HINDE (1967) with the cabbage aphid. The heat and air dried symbiote smears stained with Feulgen’s showed a light pinkish-red colour. The symbiote smears treated with Macchiavello’s stained light blue. Gram staining resulted in the symbiotes being stained pink. Observation of the smears revealed several bacilliform organisms in aggregation (Fig. 1D). These organisms appeared to be in close association with the symbiotes. However, further observations with the light and transmission electron microscope revealed that the bacilliform organisms were outside of the mycetocytes. B~~CHNER (1965) reported the presence of ‘primary and secondary’ symbiotes within individual mycetocytes of some aphids. The primary symbiotes are coccoid and the secondary symbiotes are bacilliform. HINDE (1971~) using electron micrographs as evidence, stated that B. brassicae and M. persicae have only the primary symbiotes, while M. rosae has both primary and secondary symbiotes within a mycetocyte. It is not known at this time if the bacilliform organisms in the pea aphid are symbiotic. They do not appear to be as closely associated with mycetocytes as was suggested by B~~CHNER(1965) and HINDE (1971~) for other aphids. Scanning electron microscopy When the mycetocyte membrane is removed or ruptured, the coccoid-shaped symbiotes become visible (Fig. 2a). The mycetocytes containing these symbiotes are irregularly round to slightly oval in shape, and have a polar invagination (Fig. 2b). The symbiotes are closely packed in the cytoplasm of the mycetocyte and appear not to be consistent in size (Fig. 2~). In Fig. 2(c), some dividing pairs can be seen (arrow). The nucleus is found near the centre of the mycetocyte and it is shown in Fig. 2(d). Each mycetocyte is innervated with trachae (T) as shown in Fig. 2(e). Transmission electron microscopy For the most part, the descriptions presented by LAMB and HINDE (1967) of the symbiotes of the cabbage aphid agree with the symbiotes of the pea aphid. However, the symbiotes in the pea aphid range in size from 2 to 5 TVin diameter. The symbiotes appear to be bounded by three membranes; possibly a double unit membrane (Fig. 3a) and another outside membrane similar to the cytoplasmic

632

D. L. MCLEAN AND E. J. HOUK

membrane described by LAMB and HINDE (1967) (Fig. 3b). The cytoplasm of the symbiotes appears to be filled with ribosome-like bodies (Fig. 3b). The cytoplasm of the mycetocyte contains large numbers of mitochondria (Fig. 3~). Endoplasmic reticulum can also be found and many ribosomes can be The easily seen (Fig. 3b). Fig. 3(d) s h ows a symbiote in the process of division. nucleus of the mycetocyte is bounded by a double membrane and is polymorphic (Fig. 3e). The bacilliform organisms are shown in Fig. 3(f). They appear to have a cell They multiply by binary fission and are about wall similar to many bacteria. 0.5 p in width and about 1.5 p in length. DISCUSSION LAMB and HINDE (1967) suggested that the symbiotes of the cabbage aphid They based this conclusion on the results of belong to the family Rickettsiaceae. staining and on the morphology of the organisms. From our results, it appears as though the symbiotes of the pea aphid do not fit into the rickettsiae group. Although some rickettsiae are spherical or coccoid, most are nearly bacilliform. Most rickettsiae are minute, usually less than 1 p in diameter. Rickettsiae have a cell The symbiotes are spherical, 2 to 5 ,.L in wall and sometimes are pleomorphic. diameter, and have a membrane or membranes enclosing the cytoplasm. Macchiavello’s is a standard diagnostic stain for rickettsiae. Reaction to Macchiavello’s stain was negative with the pea aphid symbiotes. The reaction with The reaction by the symbiotes to the gram Fuelgen’s stain was inconclusive. stain was probably inconsequential since the membrane would be lacking reaction sites that a cell wall might have. Based on the size differential, morphology, and reaction to Macchiavello’s stain, we believe that the symbiotes of the pea aphid more properly belong to the Eubacteriaceae. Possibly, one would suspect the symbiotes may be similar to L-form bacteria as VAGO and LAPORTE (1965) suggested. Another alternative might be suggested as to the classification of the symbiote organisms. Other than the size, the symbiotes are morphologically similar to PPLO or mycoplasma-like organisms, and if so, are probably non-pathogenic. Biochemical studies with the symbiotes are currently underway to determine structure and function of the membrane. Acknowledgements-The authors wish to acknowledge with thanks Mr. M. G. KINSEYfor his photographs of the scanning microscope material and Mr. R. 0. SCHUSTERfor his help in the preparation of some of the material for the scanning microscope. REFERENCES BUCHNERP. (1965) Endosymbiosis of Animals with Plant Micro-organisms. Interscience, New York. CONN H. J., DARROWM. A., and EMMEL V. M. (1962) Staining Procedures Used by the Biological Stain Commission. Williams & Wilkins, Baltimore. HINDE R. (1971a) Maintenance of aphid cells and the intracellular symbiotes of aphids in vitro. J. invert. Pathol. 17, 333-338.

MYCETOCYTESAh'D

SYMBIOTES OFTHBPEA

APHID

633

R. (1971b) The control of the mycetome symbiotes of the aphids Brevicoryne brassicae, Myzus persicae, and Macrosiphum rosae. J. Insect Physiol. 17, 1791-1800. HINDE R. (1971~) The fine structure of the mycetome symbiotes of the aphids Brevicoryne brassicae, Myzus persicae, and Macrosiphum rosae. J. Insect Physiol. 17, 2035-2050. HUNGATE R. E. and FLETCHER D. W. (1962) Laboratory Manual of General Bacteriology. Scholar’s Library, New York. LAMB K. P. and HINDE R. (1967) Structure and development of the mycetome in the cabbage aphid, Brevicoryne brassicae. J. invert. Pathol. 9, 3-11. LILLIE R. D. (1965) Histopathologic Technic and Practical Histochemistry. McGraw-Hill, New York. SPURR A. R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. VACO C. and LAPORTB M. (1965) Microscope electronique des symbiontes globuleux des aphides [Horn. Aphidoidea]. Ann. Sot. Ent. FY. 1,181-196. HINDE