- Email: [email protected]
The mycetocyte symbiosis of aphids: Variation with age and morph in virginoparae of Megoura viciae and Acyrthosiphon pisum
.I. hsect Physiol. Vol. 33. ho. 2, pp. 109-I 13. 1987 Printed in Great Britain. All rights reserved
THE MYCETOCYTE
Copyright
SYMBIOSIS
OF APHIDS:
c
0022-1910/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
VARIATION
WITH AGE AND MORPH IN VIRGINOPARAE OF MEGOURA VICIAE AND ACYRTHOSIPHON PISUM A. E. DOUGLAS and A. F. G. DIXON School
af Biological
Sciences,
University
of East Anglia,
Norwich
NR4 7TJ, England
(Received 9 June 1986; revised 25 July 1986) Abstract-Prokaryotic symbionts of aphids, which are important to the survival of the insect, are housed in specialized cells, mycetocytes. In a study of the aphids Acyrthosiphon pisum and Megoura oiciae. the mycetocytes were found to exhibit a consistent pattern of variation in both size and number, both with developmental :age of the insect and between alate and apterous morphs. The number of mycetocytes ranged betweer. 7(t90 and 6&70 in l-day old larvae of Megoura viciae and Acyrthosiphon pisum, respectively, ansd tended to 0 in post-reproductive individuals of both species. with a decrease of 50% between birth and the time of the final moult (days 7-9) in alates and in the late-reproductive period (days 17-18) in apterae. The volume of mycetocytes of M. uiciue increased with age from 1.17 x 10-5mm’ in l-day old larvae to 7.63 x 10m5 mm3 and 5.09 x 10-5mm~ in apterous and alate teneral adults (day 8). respectively, and this difference between the morphs closely mirrors the difference between the relative growth rates of apterous and alatiform larvae. It is suggested that mycetocyte loss may represent an important means by which the symbiont population is regulated. The results can also be interpreted as evidence for substantial variation in the characteristics of nutritional interactions between the aphid and its symbionts aith age and morph of the aphid. Key Wcrd Index: Symbiosis,
aphids,
mycetocyte,
INTRODUCTION
All members date contain
of the family Aphididae prokaryotic symbionts
examined to within special-
ized cells, mycetocytes, in the haemocoel (Buchner, 1966; Houk and Grif!iths, 1980). The aphids exhibit a high degree of dependence on their symbionts, as is indicated by their poor survival, retarded growth and low fecundity when the symbionts are experimentally removed. Under natural circumstances, all individuals of most species are provided with symbionts, which are transferrecl directly into the eggs of oviparae and early embryos of viviparae by the process of “transovarial transmission”. The structural organization of aphid mycetocytes has been studied in considerable detail (e.g. Buchner, 1966; Ponsen, 1976). Their cytoplasm is packed with the symbionts, each within an individual membranebound vacuole. In other respects (e.g. composition and structure of organelles), the mycetocytes exhibit no unusual features. ‘There is some evidence that the condition of the mycetocytes varies with age of the aphid. In particular, the studies of Lamb and Hinde (1967) on Brevicoryne brussicae and Ponsen (1976) on Myzus persicae suggest that the size of mycetocytes increases with age of the insect. Ponsen (1976) also indicates that the mycetocytes in Myzus persicae “degenerate” and are lost and Ehrhardt (1966) reports that post-reproductive Aphis fubae contain few or no mycetocytes. However, these histological studies were conducted on small samples and over limited parts of the lifespan of the insects. As part of a study of the interactions between aphids and their mycetocyte symbionts, it was considered important to
Megoura viciae, Aqrthosiphon
pisum, cell size
document these changes more fully and to determine accurately when and at what rate such changes occur. This paper presents a detailed study of the size and number of mycetocytes in virginoparae from birth onwards. The mycetocytes of embryos are not considered and therefore all estimates of number or volume of mycetocytes per aphid refer exclusively to the cells lying free in the haemocoel of the insects. Two species, the vetch aphid, Megoura viciae, and the pea aphid, Acyrthosiphon pisum, are examined. The data indicate substantial differences in the condition of mycetocytes with developmental age and between the apterous and alate morphs.
MATERIALS
AND METHODS
Clonal cultures of Megoura thosiphon pisum were maintained
viciae and Acyron Vicia faba (var. The Sutton) at 20°C under 16 h light: 8 h dark photoperiodic regime, with light intensity 45 PEinsteins m-* s-I P.A.R. To obtain animals of uniform nutritional condition. the following procedure was adopted. “Routine cultures” were transferred once a week onto fresh plants, which had been raised to the two- or four-leaf stage in the greenhouse (ambient illumination, average temperature 18°C). The “test cultures” were produced by allowing adult apterae from a routine culture to deposit larvae over 24 h on fresh plants, at a density of 10 adults per pot of four plants. Thus, at the initiation of each test culture (day l), the aphids were O-l days old and were designated “l-day-old”. Once the aphids in the test culture reached adulthood, their offspring were 109
A. E. DOUGLASand A. F. G. DIXON
110
removed twice weekly. To obtain an appreciable number of slates in the test cultures, the routine cultures were always maintained at a moderately high density. The number of mycetocytes in the body cavity of aphids was determined by two methods: dissection and sectioning. In both procedures, the aphids were fixed in modified Bouin-Dubosq solution (Ponsen, 1976). For dissections, the fixed aphids were rinsed in 80% ethanol and dissected in a drop of distilled water at x 25- x 40 magnification using fine pins. The mycetocytes could readily be distinguished by their rounded shape and uniform size; embryos are more elongate and in chains of regularly changing shape. Material to be sectioned was double-embedded in celloidin-methylbenzoate and paraffin wax (Gurr) by the method of Molnar (1974) and longitudinal sections of thickness 10 pm (adults) and 5 pm (larvae) were stained with Ehrlich’s haematoxyhn and eosin. For ease of interpretation, the number of mycetocyte nuclei was scored; mycetocytes in larvae and adults are uninucleate. To score the size of mycetocytes, the abdomen of each insect was punctured and the mycetocytes isolated by gently shaking the animal in a drop of buffer (50mM Tri-HCI, pH 7.2, 25mM KCI, 10mM MgCI,, 0.25 M sucrose) [modified from Ishikawa (1982)] on an improved Neubauer haemocytometer slide. The preparation was examined under dark-field illumination at x 100 magnification; under these conditions, the mycetocytes could be distinguished from contaminating material by their bright white appearance and large size. For M. uiciue, less than 10% of the mycetocytes were broken. as determined from the number of mycetocyte fragments containing a nucleus. The percentage of broken cells in preparations from A. pisum was consistently greater, at 2&60%, and was not reduced by systematic variation of buffer composition. The area of all intact cells in each preparation from M. uiciae was determined by a Delta-T area meter. To obtain an estimate for cell volume, each cell was treated as a sphere, the radius (r) of which was calculated from the equation: cell area = x rz and the volume was calculated as 4/3 x r3. The median cell volume per individual was determined. Between 5 and 12 individuals were scored for each age and morph class and the volume of mycetocytes was expressed as mean f SE of the median values. The relative growth rate from birth to adulthood of the aphids and their mycetocytes was calculated from fresh weight and volume data, respectively, as described in Adams and van Emden (1972). The fresh weight of individual aphids was determined to an accuracy of f 1 pg. RESULTS General observations The condition of the mycetocytes in A. pisum and M. uiciae was found to conform largely with published accounts of these species and other aphids (see review of Houk and Griffiths (1980)). In young larvae, the mycetocytes were aggregated together as a single bilobed structure, the mycetome, within a cellular sheath and lying dorsal to the gut in the
abdomen. In older larvae and adults, the mycetocytes were dispersed throughout the abdomen. either singly or in groups of 2-7 cells, and the mycetome sheath was no longer apparent. The time and extent of separation of the mycetocytes varied between individuals, but not consistently between the two species. The symbionts in the mycetocytes were very similar in A. pisum and M. viciae: they were coccoid. 1.5-2.5 pm dia. and did not stain positively with Machiavello’s stain for rickettsias or the Gram stain in either smear preparations of mycetocytes or sectioned material. As previously reported (Buchner, 1966) A. pisum. but not M. riciur, also contains rod-shaped, “secondary” symbionts which. in the clone used here, were within the mycetome sheath of young larvae but only loosely associated with the mycetocytes after the break-up of the mycetome during the latter part of larval life. This change in the condition of the secondaty symbionts may resolve an apparent contradiction in the literature; Griffiths and Beck (1973) who studied first and second larval instars, describe the secondary symbionts as intracellular in the sheath, but McLean and Houk (1973) report that they are external to the mycetocytes in fourth-instar larvae and young adults. Preliminary studies on routine cultures of A. pisum and M. viciae identified no division stages of mycetocytes. suggesting that they do not divide. It was also found that the mycetocytes increased in size with age of the aphid and that old animals had relatively few mycetocytes. A detailed study of these characteristics in aphids of known age from test cultures at 20, C was therefore initiated. Under these conditions, the lifespan of the two species was very similar. Larval development took approx. 1 week, with the moult on day 7-8. Larviposition, which commenced at day 9-l 1, continued to day 2&23, and most animals lived to day 22-28, with few individuals dying before day 20 or surviving beyond day 30. The relative growth rate (mean _t SE) of A. pisum over larval development was 0.31 + 0.03 pg pg-’ dayy’ for apterae and 0.27 f 0.02 ~‘g~cg~’ day-’ for alates. Equivalent vaiues for M. viciae were 0.31 + 0.04 pgpg-’ day’ and 0.25 i 0.04 ,ug pg-’ day- I. Number of mycetocytes The number of mycetocytes free in the body cavity of the aphids could be scored rapidly by the dissection method. As a check for the accuracy of this procedure, groups of l-day old larvae and 8-day old adults of A. pisum were divided into two classes, which were assayed by the dissection and sectioning methods, respectively. In all experiments, no significant difference between the numbers of mycetocytes determined by the two methods was found (Table l), and thereafter the dissection method was used exclusively for both A. pisum and M. viciae. The variation in mycetocyte number with age and morph of A. pisum and M. viciae is shown in Fig. 1. As already indicated in Table 1, most l-day old larvae of A. pisum contained 60-70 mycetocytes. By contrast, those of M. viciae had 7&90mycetocytes. However, the changes in mycetocyte number in the two species were very similar. Over the period of larval development of the apterous morph, the mean number of mycetocytes per aphid declined slightly, by
Aphid mycetocytes Table
I, Number
(&
of mycetocytes
I
8 8
by dissection
Number of mycetocytes mean f SE (no. of aphids) dissection sectioning method method
Instarlmorph
I
in A. pisum, as assayed methods
111
69.2 k 1.7(10) 64.3 f 2.7 (8) 22.4 f 3.1(10)
Adult/apterous Adultialate
73.4 i 2.7(7) 63.9 k 2.7(7) 23.8 f 3.0(9)
and sectioning
t-value (do
P
1.39(15) O.lO(l3) 0.32(17)
>0.05 >0.05 >0.05
same age; and the mean number of mycetocytes declined to 50% of the measurement on day 1 at 7.4 days and 9.2 days for alates of A. pisum and M. viciue respectively, but at 17.4 and 17.6 days for apterae of the same species. To investigate the influence of culture conditions on mycetocyte number, further experiments xvere conducted on A. pisum at 20, 18 and 15°C (Table 2). Very similar results were obtained at the different temperatures; from birth to early adulthood, the number of mycetocytes per apterous aphid exhibited little change but that in alates declined by 5&60%. Size of mycetocytes
Because of the fragility of isolated mycetocytes of A. pisum (see Materials and Methods), detailed stud-
Time
(days)
-Adult
Instar
Reproductwe
period
Fig. 1. Variation in number of mycetocytes with age and morph of M. uiciue and /I. pisum at 20°C. The mycetocytes in samples of 5 individuals of first- and second-instar larvae (triangles), apterae (squares) and alatae (circles) of M. viciae (closed symbols) and A. pirum (open symbols) were scored, and values of mean + standard error are shown.
less than 5% in most experiments and never by more
than 10%. During adult life, the number of mycetocytes declined further and at an increasing rate, so that by day 20, up to 30% of apterae contained no identifiable mycetocytes and only rarely were individuals with more than 25-30 mycetocytes scored. Many post-reproductive animals (assayed on day 25 onwards) were mycetocyte-free, and the mycetocytes identified were flattened in appearance. The decrease in number of mycetocytes occurred considerably earlier in the lifespan of alates. In the experiment shown in Fig. 1,6-day old alatiform larvae contained approx 25% fewer mycetocytes than apteriform larvae of the -
Table 2. Number
of mycetocytes
ies on the size of mycetocytes were conducted exclusively on M. uiciue. Data were obtained for l-day old larvae to 15-day old adults from test cultures at 20°C. Considering all the data, the volume of mycetocytes varied widely, from 0.6 to 30 x lo-‘mm), and within each individual scored, the volume of the largest cell scored was 1.5-5.5 times greater than that of the smallest. In all animals, the distribution of cell size was unimodal and, in many cases, it was positively skewed. Therefore, the median value was adopted in preference to the mean as an index of the “average” value. In Fig. 2a is shown the mean of these median values for each age and morph class between days 1 and 15. The median size of the mycetocytes clearly increased regularly with age throughout larval instars in both apterae and alates. The size of mycetocytes in apterae increased more rapidly, over a longer period and to a greater maximal value than in alates. Thus, in apterae, the relative growth rate over larval development (i.e. days l-8) was 0.26 mm3 rnrne3 day-’ and the greatest median volume was recorded on day 11, when it was IO-fold greater than that on day 1. In alates, the relative growth rate between days 1 and 8 was 0.21 mm3 mm-’ day-’ and the maximal volume (on day 8) represented a 4.3-fold increase. The volume of the smallest and largest mycetocyte scored per
in A. pisum reared at different
temperatures
Number of mycetocytes mean f SE (n = 5) T:mperature PC) 20 18 I5
l-day old larvae
Adults within 24 hours of final moult alate aDterOuS
62.6 f 2.8 60.0 * 3.1 63.0 + 2.1
58.0 f 3.4 58.2 k I .8 63.8 + 1.5
Routine cultures of the aphids had been maintained 3 months.
Age of aphids at final moult (days)
27.4 f 3.0 28.0 f I .3 24.6 + 5.4 at the experimental
7-8 9-10 12-14 temperatures
for
A.
112
E. DOUGLAS and A. F. G. DIXON
alates, suggesting that, as with increase in median cell size (above), the decline is mediated largely by reduction of size of individual cells. By contrast, the minimal cell volume of apterae decreased by substantially less than the median value and the difference between the values on days 11 and 15 was not significant (Table 3). It is thus probable that preferential loss of large mycetocytes contributes to the decrease in median mycetocyte volume in reproductive apterae. Figure 2b shows the variation of total mycetocyte volume in the haemocoel of M. vi&e with age. It highlights the substantial differences in the characteristics of the symbiosis between apterae and alates. In apterae, the total mycetocyte volume increased regularly with age at least to the onset of larviposition (day ll), when the mean total mycetocyte volume was 7.61 x 1O-3 mm3 per aphid. Equivalent values for 1l-day old slates are 0.97 x 10m3mm3 per aphid and at this stage, the total mycetocyte volume was already in rapid decline.
;*O- (a) E P GIOe E 6 E” 6i 4E 1 s
&:IJ’ , /
4
2
0
6
9
“E
-
E
Time
*‘-
1
,
,;
;4
- 10
6 (days)
(b)
60-
//‘-----’
40.
./
DISCUSSION
1
0
2
4
6 Time
8
10
12
14
(days)
Fig. 2. Variation in mycetocyte volume with age and morph of M. viciae at 20°C (symbols as in Fig. 1). (a) median mycetocyte volume per individual (mean I SE); (b) total mycetocyte volume per aphid, calculated from “median volume” x “number of mycetocytes per aphid”.
individual increased with age in parallel with the median value, indicating that the increase in median volume was mediated largely or exclusively by enlargement of the mycetocytes and not by preferential loss of small cells.
In contrast to the situation in larvae, the median mycetocyte volume in reproductive adults decreased between days 11 and 15, by approx 25% in apterae and 50% in alates (Fig. 2a); the differences between the values on days 11 and 15 are significant for both morphs (Table 3). The change in minimal cell volume over this period mirrored that of the median value in Table 3. Values of median
and minimum
Day II
Morph Apterous Alate
The current study indicates that the condition of mycetocytes varies in a regular fashion with age and morph, and that this pattern of variation is very similar in M. vi&e and A. pisum. At all times, loss of entire cells contributed to a decrease in mycetocyte biomass and a decrease in cell size was also apparent in reproductive adults (especially slates). By contrast, the sole mode of increase in mycetocyte biomass was cell enlargement, which occurred particularly during larval development. All the available data are consistent with the view that the mycetocytes in these species do not divide after birth; the number of mycetocytes per insect did not exceed the values obtained for l-day old larvae (Fig. 1) and division stages (e.g. b&nucleate cells) were never observed in the thousands of cells screened. To a large extent, these conclusions are consistent with published information. The sole striking discrepancy relates to the study of Lamb and Hinde (1967) on Brevicoryne brussicae, in which mycetocyte number is reported to increase from 37 at 12-18 h after birth to 47 and 44 in adult apterae and alates respectively. One may hypothesize that increase in mycetocyte biomass by cell enlargement without recourse tb cell division, as recorded in M. viciae, is advantageous to the insect. This mode of growth permits the continuation of various cellular activities, e.g. transcription of DNA, without interruption by mitosis. A further consequence is the reduced requirement for membrane components for the synthesis of plasma membrane (due to the low surface area per unit volume of large cells); membrane components are mycetocyte
Parameter (onset larviposition) Median Minimum ‘Median Minimum
volume
in reproductive
M. oiciae
IO-’ x mycetocyte volume (mm’) mean + SE (no. of aphids scored) Day 15
12.24+ 0.41(12) 6.24+ 0.75 4.44f 0.3+2) 2.26+ 0.26
(mid-reproducdon) 9.32f 0.88(12) 5.44* 0.52 2.32f 0.62(9) 1.12+0.32
f
P
-3.451 1.022 3.601 3.190
o.os
Aphid mycetocytes probably in great demand for the synthesis of membrane to enclose each cell of the growing symbiont population. The process of mycetocyte loss may represent a means by which damaged or inferior cells are removed. Alternatively or additionally, mycetocyte loss may contribute to the regulation of the symbiont population. One could argue that if the biomass of symbioms become too great, then, through their nutritional requirements and the space they occupy, the cost to the insect of maintaining the symbiont population could outweigh the benefit. If, as suggested above, large size of mycetocytes is advantageous to the insect, then loss of entire mycetocytes may represent a particularly appropriate mode of regulation. Implicit in this interpretation is the view that mycetocyte loss is programmed, as has been documented for a range of insect cells, e.g. muscle, fat body, yolk cells and oocytes (Lockshin, 1985). A striking aspect of the data reported here is the marked difference between the apterous and alate morphs, in terms of both enlargement and loss of mycetocytes. The greater rate of cell enlargement in apterous than in alatiform larvae (Fig. 2~) can be interpreted in terms of the faster overall growth rate of the former. Thus, in both apteriform and alatiform larvae, the mean relative growth rate of mycetocytes (at 0.26 mm3 mme3 da;,-’ and 0.21 mm3 mmm3 day-‘, respectively) is 84% of the values for body weight (0.31 mg mg-’ day-’ and 0.25 mg mg-’ day-‘). These data indicate, first, that the rate of mycetocyte enlargement is closely linked to (possibly determined by) the growth rate of the insect; and, secondly, that the greater total mycctocyte volume per unit body weight in teneral apterous adults (2.11 x 10m3mm3 mg-‘) than in equivalent alates (0.76 x 10m3 mm3 mgg’) arises from the greater extent of cell loss in alatiform than in apterous larvae (Fig. 1). The significance of this difference remains to be established. However, pursuing the proposed regulative role of mycetocyte loss (see above), one may speculate that cell loss in alatiform larvae is correlated with the development of wing musculature and deposition of fat (A. F. G. Dixon, unpublished results), which would be expected to impose severe constraints on the space and nutrients available for maintenance of the symbionts. Alterna.tively, it may be related to the smaller number of embryos in alates than apterae, and hence reduced requirement for symbionts to infect the next generaton. As with other examples of programmed cell death in insects (Lockshin, 1985) one may anticipate endocrinal involvement in mycetocyte cell loss; perhaps the hormonal titres implicated in morth determination have a direct influence on the lifespan of the mycetocytes. It is well established for a number of aphids that the offspring of alates are smaller and take longer to develop than the offspring of apterae (Dixon, 1985). Furthermore, the growth rate of the largest embryos is 40-50% lower in alatiform than in apterous larvae (Tashev and Markova, 1983; C. Newton, pers. commun.) and the difference in growth rate becomes apparent at a similar point as the onset of rapid
113
decline in mycetocyte number in alatiform larvae (day 46; see Fig. 1). These differences in the reproductive performance of alates and apterae may be linked to the difference in the condition of the mycetocytes between the two morphs. There is evidence that the symbionts synthesize amino acids, various lipogenic factors and vitamins and a single protein “symbionin”, which are believed to contribute to the nutrition of the insect (Houk and Griffiths, 1980; Ishikawa, 1984). This raises the possibility that the nutritional interactions between the aphid and its symbionts differ substantially between alates and apterae and that this difference is significant to embryogenesis. Acknowledgemenfs-We thank Dr C. Hedley (John Innes Institute, Norwich), in whose laboratory the study of cell size was conducted, Mr M. Ambrose for his patient instruction and advice on the use of the area meter, and Mr G. P. Cleveland for his help and guidance with histological techniques. This study was financed by a grant from the Science and Engineering Research Council. REFERENCES
Adams J. B. and van Emden H. F. (1972) The biological properties of aphids and their host plant relationships. In Aphid Technology (Ed. by van Emden H. F.), pp. 48-104.
Academic Press,. London. Buchner P. (19661 Endosvmbioses of Animals with Plant Microorgar&ms: Interscience, New York. Dixon A. F. G. (1985) Aphid Ecology. Blackie, London. Ehrhardt P. (1966) Entwicklung und Symbionten geflugelter und ungeflugelter virgines von Aphis fabae Stop. unter dem einfluss kunstlicher Ernahrung. Z. Morph. Okol. Tiere 51, 295-319. Griffiths G. W. and Beck S. D. (1973) Intracellular symbiotes in the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 19, 75-84. Houk E. J. and Griffiths G. W. (1980) Intracellular symbiotes of the Homoptera. A. Rev. Ent. 25, 161-187. Ishikawa H. (1982) Isolation of the intracellular symbionts and partial characterizations of their RNA species of the elder aphid, Acyrthosiphon magnoliae. Comp. Biochem. Physiol. 72B, 239-247. Ishikawa H. (1984) Molecular aspects of intracellular symbiosis in the anhid mvcetocvte. Zool. Sci. 1. 509-522. Lamb K. P. and Hinde R. (1967) Structure and development of the mycetome in the cabbage aphid, Breuicoryne brassicae. J. invert. Pathol. 9, 3-l 1. Lockshin R. A. (1985) Programmed cell death. In Comprehensiue Physiology, Biochemistry and Pharmacology (Ed. by Kerkut G. A. and Gilbert L. I.), Vol. 2, pp. 301-317. Pergamon Press, Oxford. McLean D. L. and Houk E. J. (1973) Phase contrast and electron microscopy of the mycetocytes and symbiotes of the oea anhid. Acvrthosiphon uisum. J. Insect Physiol. 12, 1245-1254. . . . Molnar L. M. (1974) Double embedding with nitrocellulose and paraffin. Stain Tech. 49, 311. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Ed. by Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press. New York. Tashev D. and Markova E. (1983) Morphofunctional bases of aohid fecunditv (Aohidina vioioviuara). V. Growth of the biggest embryo. ‘Annuaire de i-Universite de Sofa “Kliment Ohridski” Faculte de Biologie I-Zoologie 72173, 23-38.
THE MYCETOCYTE
Copyright
SYMBIOSIS
OF APHIDS:
c
0022-1910/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
VARIATION
WITH AGE AND MORPH IN VIRGINOPARAE OF MEGOURA VICIAE AND ACYRTHOSIPHON PISUM A. E. DOUGLAS and A. F. G. DIXON School
af Biological
Sciences,
University
of East Anglia,
Norwich
NR4 7TJ, England
(Received 9 June 1986; revised 25 July 1986) Abstract-Prokaryotic symbionts of aphids, which are important to the survival of the insect, are housed in specialized cells, mycetocytes. In a study of the aphids Acyrthosiphon pisum and Megoura oiciae. the mycetocytes were found to exhibit a consistent pattern of variation in both size and number, both with developmental :age of the insect and between alate and apterous morphs. The number of mycetocytes ranged betweer. 7(t90 and 6&70 in l-day old larvae of Megoura viciae and Acyrthosiphon pisum, respectively, ansd tended to 0 in post-reproductive individuals of both species. with a decrease of 50% between birth and the time of the final moult (days 7-9) in alates and in the late-reproductive period (days 17-18) in apterae. The volume of mycetocytes of M. uiciue increased with age from 1.17 x 10-5mm’ in l-day old larvae to 7.63 x 10m5 mm3 and 5.09 x 10-5mm~ in apterous and alate teneral adults (day 8). respectively, and this difference between the morphs closely mirrors the difference between the relative growth rates of apterous and alatiform larvae. It is suggested that mycetocyte loss may represent an important means by which the symbiont population is regulated. The results can also be interpreted as evidence for substantial variation in the characteristics of nutritional interactions between the aphid and its symbionts aith age and morph of the aphid. Key Wcrd Index: Symbiosis,
aphids,
mycetocyte,
INTRODUCTION
All members date contain
of the family Aphididae prokaryotic symbionts
examined to within special-
ized cells, mycetocytes, in the haemocoel (Buchner, 1966; Houk and Grif!iths, 1980). The aphids exhibit a high degree of dependence on their symbionts, as is indicated by their poor survival, retarded growth and low fecundity when the symbionts are experimentally removed. Under natural circumstances, all individuals of most species are provided with symbionts, which are transferrecl directly into the eggs of oviparae and early embryos of viviparae by the process of “transovarial transmission”. The structural organization of aphid mycetocytes has been studied in considerable detail (e.g. Buchner, 1966; Ponsen, 1976). Their cytoplasm is packed with the symbionts, each within an individual membranebound vacuole. In other respects (e.g. composition and structure of organelles), the mycetocytes exhibit no unusual features. ‘There is some evidence that the condition of the mycetocytes varies with age of the aphid. In particular, the studies of Lamb and Hinde (1967) on Brevicoryne brussicae and Ponsen (1976) on Myzus persicae suggest that the size of mycetocytes increases with age of the insect. Ponsen (1976) also indicates that the mycetocytes in Myzus persicae “degenerate” and are lost and Ehrhardt (1966) reports that post-reproductive Aphis fubae contain few or no mycetocytes. However, these histological studies were conducted on small samples and over limited parts of the lifespan of the insects. As part of a study of the interactions between aphids and their mycetocyte symbionts, it was considered important to
Megoura viciae, Aqrthosiphon
pisum, cell size
document these changes more fully and to determine accurately when and at what rate such changes occur. This paper presents a detailed study of the size and number of mycetocytes in virginoparae from birth onwards. The mycetocytes of embryos are not considered and therefore all estimates of number or volume of mycetocytes per aphid refer exclusively to the cells lying free in the haemocoel of the insects. Two species, the vetch aphid, Megoura viciae, and the pea aphid, Acyrthosiphon pisum, are examined. The data indicate substantial differences in the condition of mycetocytes with developmental age and between the apterous and alate morphs.
MATERIALS
AND METHODS
Clonal cultures of Megoura thosiphon pisum were maintained
viciae and Acyron Vicia faba (var. The Sutton) at 20°C under 16 h light: 8 h dark photoperiodic regime, with light intensity 45 PEinsteins m-* s-I P.A.R. To obtain animals of uniform nutritional condition. the following procedure was adopted. “Routine cultures” were transferred once a week onto fresh plants, which had been raised to the two- or four-leaf stage in the greenhouse (ambient illumination, average temperature 18°C). The “test cultures” were produced by allowing adult apterae from a routine culture to deposit larvae over 24 h on fresh plants, at a density of 10 adults per pot of four plants. Thus, at the initiation of each test culture (day l), the aphids were O-l days old and were designated “l-day-old”. Once the aphids in the test culture reached adulthood, their offspring were 109
A. E. DOUGLASand A. F. G. DIXON
110
removed twice weekly. To obtain an appreciable number of slates in the test cultures, the routine cultures were always maintained at a moderately high density. The number of mycetocytes in the body cavity of aphids was determined by two methods: dissection and sectioning. In both procedures, the aphids were fixed in modified Bouin-Dubosq solution (Ponsen, 1976). For dissections, the fixed aphids were rinsed in 80% ethanol and dissected in a drop of distilled water at x 25- x 40 magnification using fine pins. The mycetocytes could readily be distinguished by their rounded shape and uniform size; embryos are more elongate and in chains of regularly changing shape. Material to be sectioned was double-embedded in celloidin-methylbenzoate and paraffin wax (Gurr) by the method of Molnar (1974) and longitudinal sections of thickness 10 pm (adults) and 5 pm (larvae) were stained with Ehrlich’s haematoxyhn and eosin. For ease of interpretation, the number of mycetocyte nuclei was scored; mycetocytes in larvae and adults are uninucleate. To score the size of mycetocytes, the abdomen of each insect was punctured and the mycetocytes isolated by gently shaking the animal in a drop of buffer (50mM Tri-HCI, pH 7.2, 25mM KCI, 10mM MgCI,, 0.25 M sucrose) [modified from Ishikawa (1982)] on an improved Neubauer haemocytometer slide. The preparation was examined under dark-field illumination at x 100 magnification; under these conditions, the mycetocytes could be distinguished from contaminating material by their bright white appearance and large size. For M. uiciue, less than 10% of the mycetocytes were broken. as determined from the number of mycetocyte fragments containing a nucleus. The percentage of broken cells in preparations from A. pisum was consistently greater, at 2&60%, and was not reduced by systematic variation of buffer composition. The area of all intact cells in each preparation from M. uiciae was determined by a Delta-T area meter. To obtain an estimate for cell volume, each cell was treated as a sphere, the radius (r) of which was calculated from the equation: cell area = x rz and the volume was calculated as 4/3 x r3. The median cell volume per individual was determined. Between 5 and 12 individuals were scored for each age and morph class and the volume of mycetocytes was expressed as mean f SE of the median values. The relative growth rate from birth to adulthood of the aphids and their mycetocytes was calculated from fresh weight and volume data, respectively, as described in Adams and van Emden (1972). The fresh weight of individual aphids was determined to an accuracy of f 1 pg. RESULTS General observations The condition of the mycetocytes in A. pisum and M. uiciae was found to conform largely with published accounts of these species and other aphids (see review of Houk and Griffiths (1980)). In young larvae, the mycetocytes were aggregated together as a single bilobed structure, the mycetome, within a cellular sheath and lying dorsal to the gut in the
abdomen. In older larvae and adults, the mycetocytes were dispersed throughout the abdomen. either singly or in groups of 2-7 cells, and the mycetome sheath was no longer apparent. The time and extent of separation of the mycetocytes varied between individuals, but not consistently between the two species. The symbionts in the mycetocytes were very similar in A. pisum and M. viciae: they were coccoid. 1.5-2.5 pm dia. and did not stain positively with Machiavello’s stain for rickettsias or the Gram stain in either smear preparations of mycetocytes or sectioned material. As previously reported (Buchner, 1966) A. pisum. but not M. riciur, also contains rod-shaped, “secondary” symbionts which. in the clone used here, were within the mycetome sheath of young larvae but only loosely associated with the mycetocytes after the break-up of the mycetome during the latter part of larval life. This change in the condition of the secondaty symbionts may resolve an apparent contradiction in the literature; Griffiths and Beck (1973) who studied first and second larval instars, describe the secondary symbionts as intracellular in the sheath, but McLean and Houk (1973) report that they are external to the mycetocytes in fourth-instar larvae and young adults. Preliminary studies on routine cultures of A. pisum and M. viciae identified no division stages of mycetocytes. suggesting that they do not divide. It was also found that the mycetocytes increased in size with age of the aphid and that old animals had relatively few mycetocytes. A detailed study of these characteristics in aphids of known age from test cultures at 20, C was therefore initiated. Under these conditions, the lifespan of the two species was very similar. Larval development took approx. 1 week, with the moult on day 7-8. Larviposition, which commenced at day 9-l 1, continued to day 2&23, and most animals lived to day 22-28, with few individuals dying before day 20 or surviving beyond day 30. The relative growth rate (mean _t SE) of A. pisum over larval development was 0.31 + 0.03 pg pg-’ dayy’ for apterae and 0.27 f 0.02 ~‘g~cg~’ day-’ for alates. Equivalent vaiues for M. viciae were 0.31 + 0.04 pgpg-’ day’ and 0.25 i 0.04 ,ug pg-’ day- I. Number of mycetocytes The number of mycetocytes free in the body cavity of the aphids could be scored rapidly by the dissection method. As a check for the accuracy of this procedure, groups of l-day old larvae and 8-day old adults of A. pisum were divided into two classes, which were assayed by the dissection and sectioning methods, respectively. In all experiments, no significant difference between the numbers of mycetocytes determined by the two methods was found (Table l), and thereafter the dissection method was used exclusively for both A. pisum and M. viciae. The variation in mycetocyte number with age and morph of A. pisum and M. viciae is shown in Fig. 1. As already indicated in Table 1, most l-day old larvae of A. pisum contained 60-70 mycetocytes. By contrast, those of M. viciae had 7&90mycetocytes. However, the changes in mycetocyte number in the two species were very similar. Over the period of larval development of the apterous morph, the mean number of mycetocytes per aphid declined slightly, by
Aphid mycetocytes Table
I, Number
(&
of mycetocytes
I
8 8
by dissection
Number of mycetocytes mean f SE (no. of aphids) dissection sectioning method method
Instarlmorph
I
in A. pisum, as assayed methods
111
69.2 k 1.7(10) 64.3 f 2.7 (8) 22.4 f 3.1(10)
Adult/apterous Adultialate
73.4 i 2.7(7) 63.9 k 2.7(7) 23.8 f 3.0(9)
and sectioning
t-value (do
P
1.39(15) O.lO(l3) 0.32(17)
>0.05 >0.05 >0.05
same age; and the mean number of mycetocytes declined to 50% of the measurement on day 1 at 7.4 days and 9.2 days for alates of A. pisum and M. viciue respectively, but at 17.4 and 17.6 days for apterae of the same species. To investigate the influence of culture conditions on mycetocyte number, further experiments xvere conducted on A. pisum at 20, 18 and 15°C (Table 2). Very similar results were obtained at the different temperatures; from birth to early adulthood, the number of mycetocytes per apterous aphid exhibited little change but that in alates declined by 5&60%. Size of mycetocytes
Because of the fragility of isolated mycetocytes of A. pisum (see Materials and Methods), detailed stud-
Time
(days)
-Adult
Instar
Reproductwe
period
Fig. 1. Variation in number of mycetocytes with age and morph of M. uiciue and /I. pisum at 20°C. The mycetocytes in samples of 5 individuals of first- and second-instar larvae (triangles), apterae (squares) and alatae (circles) of M. viciae (closed symbols) and A. pirum (open symbols) were scored, and values of mean + standard error are shown.
less than 5% in most experiments and never by more
than 10%. During adult life, the number of mycetocytes declined further and at an increasing rate, so that by day 20, up to 30% of apterae contained no identifiable mycetocytes and only rarely were individuals with more than 25-30 mycetocytes scored. Many post-reproductive animals (assayed on day 25 onwards) were mycetocyte-free, and the mycetocytes identified were flattened in appearance. The decrease in number of mycetocytes occurred considerably earlier in the lifespan of alates. In the experiment shown in Fig. 1,6-day old alatiform larvae contained approx 25% fewer mycetocytes than apteriform larvae of the -
Table 2. Number
of mycetocytes
ies on the size of mycetocytes were conducted exclusively on M. uiciue. Data were obtained for l-day old larvae to 15-day old adults from test cultures at 20°C. Considering all the data, the volume of mycetocytes varied widely, from 0.6 to 30 x lo-‘mm), and within each individual scored, the volume of the largest cell scored was 1.5-5.5 times greater than that of the smallest. In all animals, the distribution of cell size was unimodal and, in many cases, it was positively skewed. Therefore, the median value was adopted in preference to the mean as an index of the “average” value. In Fig. 2a is shown the mean of these median values for each age and morph class between days 1 and 15. The median size of the mycetocytes clearly increased regularly with age throughout larval instars in both apterae and alates. The size of mycetocytes in apterae increased more rapidly, over a longer period and to a greater maximal value than in alates. Thus, in apterae, the relative growth rate over larval development (i.e. days l-8) was 0.26 mm3 rnrne3 day-’ and the greatest median volume was recorded on day 11, when it was IO-fold greater than that on day 1. In alates, the relative growth rate between days 1 and 8 was 0.21 mm3 mm-’ day-’ and the maximal volume (on day 8) represented a 4.3-fold increase. The volume of the smallest and largest mycetocyte scored per
in A. pisum reared at different
temperatures
Number of mycetocytes mean f SE (n = 5) T:mperature PC) 20 18 I5
l-day old larvae
Adults within 24 hours of final moult alate aDterOuS
62.6 f 2.8 60.0 * 3.1 63.0 + 2.1
58.0 f 3.4 58.2 k I .8 63.8 + 1.5
Routine cultures of the aphids had been maintained 3 months.
Age of aphids at final moult (days)
27.4 f 3.0 28.0 f I .3 24.6 + 5.4 at the experimental
7-8 9-10 12-14 temperatures
for
A.
112
E. DOUGLAS and A. F. G. DIXON
alates, suggesting that, as with increase in median cell size (above), the decline is mediated largely by reduction of size of individual cells. By contrast, the minimal cell volume of apterae decreased by substantially less than the median value and the difference between the values on days 11 and 15 was not significant (Table 3). It is thus probable that preferential loss of large mycetocytes contributes to the decrease in median mycetocyte volume in reproductive apterae. Figure 2b shows the variation of total mycetocyte volume in the haemocoel of M. vi&e with age. It highlights the substantial differences in the characteristics of the symbiosis between apterae and alates. In apterae, the total mycetocyte volume increased regularly with age at least to the onset of larviposition (day ll), when the mean total mycetocyte volume was 7.61 x 1O-3 mm3 per aphid. Equivalent values for 1l-day old slates are 0.97 x 10m3mm3 per aphid and at this stage, the total mycetocyte volume was already in rapid decline.
;*O- (a) E P GIOe E 6 E” 6i 4E 1 s
&:IJ’ , /
4
2
0
6
9
“E
-
E
Time
*‘-
1
,
,;
;4
- 10
6 (days)
(b)
60-
//‘-----’
40.
./
DISCUSSION
1
0
2
4
6 Time
8
10
12
14
(days)
Fig. 2. Variation in mycetocyte volume with age and morph of M. viciae at 20°C (symbols as in Fig. 1). (a) median mycetocyte volume per individual (mean I SE); (b) total mycetocyte volume per aphid, calculated from “median volume” x “number of mycetocytes per aphid”.
individual increased with age in parallel with the median value, indicating that the increase in median volume was mediated largely or exclusively by enlargement of the mycetocytes and not by preferential loss of small cells.
In contrast to the situation in larvae, the median mycetocyte volume in reproductive adults decreased between days 11 and 15, by approx 25% in apterae and 50% in alates (Fig. 2a); the differences between the values on days 11 and 15 are significant for both morphs (Table 3). The change in minimal cell volume over this period mirrored that of the median value in Table 3. Values of median
and minimum
Day II
Morph Apterous Alate
The current study indicates that the condition of mycetocytes varies in a regular fashion with age and morph, and that this pattern of variation is very similar in M. vi&e and A. pisum. At all times, loss of entire cells contributed to a decrease in mycetocyte biomass and a decrease in cell size was also apparent in reproductive adults (especially slates). By contrast, the sole mode of increase in mycetocyte biomass was cell enlargement, which occurred particularly during larval development. All the available data are consistent with the view that the mycetocytes in these species do not divide after birth; the number of mycetocytes per insect did not exceed the values obtained for l-day old larvae (Fig. 1) and division stages (e.g. b&nucleate cells) were never observed in the thousands of cells screened. To a large extent, these conclusions are consistent with published information. The sole striking discrepancy relates to the study of Lamb and Hinde (1967) on Brevicoryne brussicae, in which mycetocyte number is reported to increase from 37 at 12-18 h after birth to 47 and 44 in adult apterae and alates respectively. One may hypothesize that increase in mycetocyte biomass by cell enlargement without recourse tb cell division, as recorded in M. viciae, is advantageous to the insect. This mode of growth permits the continuation of various cellular activities, e.g. transcription of DNA, without interruption by mitosis. A further consequence is the reduced requirement for membrane components for the synthesis of plasma membrane (due to the low surface area per unit volume of large cells); membrane components are mycetocyte
Parameter (onset larviposition) Median Minimum ‘Median Minimum
volume
in reproductive
M. oiciae
IO-’ x mycetocyte volume (mm’) mean + SE (no. of aphids scored) Day 15
12.24+ 0.41(12) 6.24+ 0.75 4.44f 0.3+2) 2.26+ 0.26
(mid-reproducdon) 9.32f 0.88(12) 5.44* 0.52 2.32f 0.62(9) 1.12+0.32
f
P
-3.451 1.022 3.601 3.190
Aphid mycetocytes probably in great demand for the synthesis of membrane to enclose each cell of the growing symbiont population. The process of mycetocyte loss may represent a means by which damaged or inferior cells are removed. Alternatively or additionally, mycetocyte loss may contribute to the regulation of the symbiont population. One could argue that if the biomass of symbioms become too great, then, through their nutritional requirements and the space they occupy, the cost to the insect of maintaining the symbiont population could outweigh the benefit. If, as suggested above, large size of mycetocytes is advantageous to the insect, then loss of entire mycetocytes may represent a particularly appropriate mode of regulation. Implicit in this interpretation is the view that mycetocyte loss is programmed, as has been documented for a range of insect cells, e.g. muscle, fat body, yolk cells and oocytes (Lockshin, 1985). A striking aspect of the data reported here is the marked difference between the apterous and alate morphs, in terms of both enlargement and loss of mycetocytes. The greater rate of cell enlargement in apterous than in alatiform larvae (Fig. 2~) can be interpreted in terms of the faster overall growth rate of the former. Thus, in both apteriform and alatiform larvae, the mean relative growth rate of mycetocytes (at 0.26 mm3 mme3 da;,-’ and 0.21 mm3 mmm3 day-‘, respectively) is 84% of the values for body weight (0.31 mg mg-’ day-’ and 0.25 mg mg-’ day-‘). These data indicate, first, that the rate of mycetocyte enlargement is closely linked to (possibly determined by) the growth rate of the insect; and, secondly, that the greater total mycctocyte volume per unit body weight in teneral apterous adults (2.11 x 10m3mm3 mg-‘) than in equivalent alates (0.76 x 10m3 mm3 mgg’) arises from the greater extent of cell loss in alatiform than in apterous larvae (Fig. 1). The significance of this difference remains to be established. However, pursuing the proposed regulative role of mycetocyte loss (see above), one may speculate that cell loss in alatiform larvae is correlated with the development of wing musculature and deposition of fat (A. F. G. Dixon, unpublished results), which would be expected to impose severe constraints on the space and nutrients available for maintenance of the symbionts. Alterna.tively, it may be related to the smaller number of embryos in alates than apterae, and hence reduced requirement for symbionts to infect the next generaton. As with other examples of programmed cell death in insects (Lockshin, 1985) one may anticipate endocrinal involvement in mycetocyte cell loss; perhaps the hormonal titres implicated in morth determination have a direct influence on the lifespan of the mycetocytes. It is well established for a number of aphids that the offspring of alates are smaller and take longer to develop than the offspring of apterae (Dixon, 1985). Furthermore, the growth rate of the largest embryos is 40-50% lower in alatiform than in apterous larvae (Tashev and Markova, 1983; C. Newton, pers. commun.) and the difference in growth rate becomes apparent at a similar point as the onset of rapid
113
decline in mycetocyte number in alatiform larvae (day 46; see Fig. 1). These differences in the reproductive performance of alates and apterae may be linked to the difference in the condition of the mycetocytes between the two morphs. There is evidence that the symbionts synthesize amino acids, various lipogenic factors and vitamins and a single protein “symbionin”, which are believed to contribute to the nutrition of the insect (Houk and Griffiths, 1980; Ishikawa, 1984). This raises the possibility that the nutritional interactions between the aphid and its symbionts differ substantially between alates and apterae and that this difference is significant to embryogenesis. Acknowledgemenfs-We thank Dr C. Hedley (John Innes Institute, Norwich), in whose laboratory the study of cell size was conducted, Mr M. Ambrose for his patient instruction and advice on the use of the area meter, and Mr G. P. Cleveland for his help and guidance with histological techniques. This study was financed by a grant from the Science and Engineering Research Council. REFERENCES
Adams J. B. and van Emden H. F. (1972) The biological properties of aphids and their host plant relationships. In Aphid Technology (Ed. by van Emden H. F.), pp. 48-104.
Academic Press,. London. Buchner P. (19661 Endosvmbioses of Animals with Plant Microorgar&ms: Interscience, New York. Dixon A. F. G. (1985) Aphid Ecology. Blackie, London. Ehrhardt P. (1966) Entwicklung und Symbionten geflugelter und ungeflugelter virgines von Aphis fabae Stop. unter dem einfluss kunstlicher Ernahrung. Z. Morph. Okol. Tiere 51, 295-319. Griffiths G. W. and Beck S. D. (1973) Intracellular symbiotes in the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 19, 75-84. Houk E. J. and Griffiths G. W. (1980) Intracellular symbiotes of the Homoptera. A. Rev. Ent. 25, 161-187. Ishikawa H. (1982) Isolation of the intracellular symbionts and partial characterizations of their RNA species of the elder aphid, Acyrthosiphon magnoliae. Comp. Biochem. Physiol. 72B, 239-247. Ishikawa H. (1984) Molecular aspects of intracellular symbiosis in the anhid mvcetocvte. Zool. Sci. 1. 509-522. Lamb K. P. and Hinde R. (1967) Structure and development of the mycetome in the cabbage aphid, Breuicoryne brassicae. J. invert. Pathol. 9, 3-l 1. Lockshin R. A. (1985) Programmed cell death. In Comprehensiue Physiology, Biochemistry and Pharmacology (Ed. by Kerkut G. A. and Gilbert L. I.), Vol. 2, pp. 301-317. Pergamon Press, Oxford. McLean D. L. and Houk E. J. (1973) Phase contrast and electron microscopy of the mycetocytes and symbiotes of the oea anhid. Acvrthosiphon uisum. J. Insect Physiol. 12, 1245-1254. . . . Molnar L. M. (1974) Double embedding with nitrocellulose and paraffin. Stain Tech. 49, 311. Ponsen M. B. (1976) Anatomy of an aphid vector: Myzus persicae. In Aphids as Virus Vectors (Ed. by Harris K. F. and Maramorosch K.), pp. 63-82. Academic Press. New York. Tashev D. and Markova E. (1983) Morphofunctional bases of aohid fecunditv (Aohidina vioioviuara). V. Growth of the biggest embryo. ‘Annuaire de i-Universite de Sofa “Kliment Ohridski” Faculte de Biologie I-Zoologie 72173, 23-38.