Ann. uppl. Bid. (1987), 111, 3 3 4 1 Printed in Grear Britain

33

The effect of previously infested spruce needles on the growth of the green spruce aphid, Elatobiurn abietinum, and the effect of

the aphid on the amino acid balance of the host plant

BY MARTIN FISHER* Department of Biological Sciences, Bayero University,

P.M.B. 301 I, Kano, Nigeria

(Accepted 22 December 1986)

S U M M A R Y

The effect of chlorosis induced in needles of Sitka and Norway spruce by the green spruce aphid on growth of the aphid is investigated, and the effect of infestation of the aphid on amino acid levels in Sitka spruce foliage is reported.

On both Sitka and Norway spruce green spruce aphids were heavier when reared on chlorotic (previously infested) needles than when reared on green (previously uninfested) needles. The effect was more pronounced on Sitka than on Norway spruce. Infestation of the aphid altered the amino acid balance of Sitka spruce foliage but not the concentration of total amino acids. Possible causes of chlorosis, the influence of individual amino acids on aphid growth, the potential effect of chlorosis on outbreaks of the aphid and the differences in susceptibility of Sitka and Norway spruce to damage by the aphid are discussed.

I N T R O D U C T I O N

The green spruce aphid, Elatobiurn abietinurn (Walker), feeds exclusively on the genus Picea (Carter & Nichols, 1985), and is probably a native of mainland Europe where its original host may have been Norway spruce (Picea ahies L.) (Kloft, Kunkel & Ehrhardt, 1964; Carter & Nichols, 1985). In the British Isles the green spruce aphid causes retardation of growth and severe defoliation of Sitka spruce (Picea sitchensis (Bong.) Carr.) (Carter, 1977). Sitka spruce is a native of the Pacific coast of North America, and is the most widely planted conifer in Britain (Fletcher & Faulkner, 1972). In the south of England and in mild parts of Denmark outbreaks of the green spruce aphid on Sitka spruce occur every 3.1 and 6.4 yearsrespectively, and in the rest of the British Isles and colder parts of Denmark every 5.6 and 20 years respectively (Carter, 1977).

When the green spruce aphid feeds on spruce needles a yellow spot often appears at the point of insertion of the stylet. The spot becomes a yellow band, which spreads over the whole needle and may lead finally to needle fall (Kloft & Ehrhardt, 1959; Parry, 1974). This chlorosis appears after 8 - 12 days on Sitka spruce, and takes 4 - 6 days longer to appear on Norway spruce (Kloft & Ehrhardt, 1959; Parry, 1974). An aphid feeding on one needle for 12 h is sufficient to induce chlorosis (Kloft & Ehrhardt, 1959; Parry, 1974).

Senescing leaves, which generally turn yellow, are favourable for aphid growth because of the high levels of soluble nitrogen that they contain (Dixon, 1970). Similarly the chlorosis induced in spruce needles by the green spruce aphid could influence the growth of the aphid. Previous authors have considered the effect of the aphid on the development of chlorosis

* Present address: Department of Biology, Sultan Qaboos University, P.O. Box 32486 AI-Khod, Muscat, Sultanate of Oman 0 1987 Association of Applied Biologists

34 M A R T I N FISHER

(Kloft & Ehrhardt, 1959; Parry, 1974) but not the effect of chlorosis on the growth of the aphid. In this paper the effect of aphid-induced chlorosis on growth of the green spruce aphid is compared on Sitka and Norway spruce, and the effect of infestation of the aphid on amino acid levels in Sitka spruce foliage is reported. Possible causes of chlorosis, the influence of individual amino acids on aphid growth, the potential effect of chlorosis on the development of outbreaks of the green spruce aphid and the differences in susceptibility of Sitka and Norway spruce to damage by the aphid are discussed.

MATERIALS A N D METHODS

The effect of chlororic and undamaged spruce needles on growth of the green spruce aphid Aphids used came from a culture that was started with green spruce aphids collected from

Coed Sarnau, mid-Wales, and kept in an outside insectary. Four dormant 2-yr-old Sitka spruce and four dormant 3-yr-old Norway spruce were used as hosts. To rear the aphids cages that confined one aphid on one spruce needle were used. The cage was a 2 cm tall, 0.75 cm diameter cylinder made from acetate sheet (the sort used on overhead projectors) with a circle of black card glued to one end to form the bottom. The black colour facilitated the observation of cast aphid exuviae. To fit the cage onto a spruce needle two small holes were melted into the cylinder with a hot pin, opposite each other, 0.5 cm from the bottom. The top 0.5 cm of the inside of the cage was coated with ‘Fluon’ to prevent aphids escaping.

The experiment was conducted in January and February 1981 at 15 f 2°C and a 16 h photoperiod/day. Twenty cages were placed on each of the eight host trees. In 10 of the cages on each tree aphids were reared from birth to adult on the same needle, while the other 10 cages on each tree and the aphids they contained were moved to uninfested green needles after the third moult (i.e. at the fourth instar).

To start the experiment one adult aptera from the culture was placed in each cage, allowed to produce one nymph, and then removed. If two nymphs were born in 24 h one was removed along with the mother. Observations were made daily for cast aphid exuviae. At the third moult nymphs were weighed and then returned to their cages, either on the same needle on which they were born, or on a fresh needle. Each aphid was reweighed at the fourth moult (i.e. when adult). If there was any uncertainty over the date of an aphid’s moult, or if an aphid escaped from its cage, it was excluded from the analysis. Aphids were weighed on a Cahn electrobalance. On needles which bore aphids from birth to adult the number of days from when adult apterae were placed in the cages to the first appearance of chlorosis was noted; the needles were not followed after the aphid’s fourth moult.

Amino acid analysis of spruce foliage In a separate experiment an analysis of the amino acids in the foliage of infested and

uninfested 2-yr-old Sitka spruce was carried out. One branch on each of 10 trees was enclosed in a ‘sleeve’ (see below), and infested with 30 fourth instar or adult green spruce aphids (from the culture described above). Two controls were used : one uninfested branch on each of the 10 infested trees, and one uninfested branch on each of 10 completely uninfested trees. All 20 control branches were enclosed in a ‘sleeve’ in the same way as the infested branches.

The ‘sleeve’ was a 10 cm long, 8 cm diameter cylinder of acetate sheet with two 7 x 5 cm muslin ‘windows’, and a 9cm long cylinder of muslin on each end. To prevent aphids wandering off the branch, needles were removed from 2 cm at the top and bottom of each enclosed branch and smeared with tree grease. This was also done in the two controls. The muslin at the ends of each sleeve was tied to the branch with cotton. Only second year foliage was enclosed within the sleeves.

Growth of’ the green spruce aphid 35

The experiment was conducted at 15 f 2°C and a 16 h photoperiod/day in February and March 1981. After 30 days all of the aphids were removed from the infested branches by hand, placed in alcohol, and later counted. The concentration of amino acids in spruce foliage can vary diurnally (Durzan, 1968) and therefore to minimise variation needles were removed randomly from amongst the three experimental treatments. All needles on each of the 30 enclosed branches were removed with scissors, washed in a 0.1% Brij 35 solution (a mild detergent) and then in two changes of de-ionised water. The needles were frozen overnight in a deep freeze, freeze-dried to constant weight (10 days), and then ground to a fine powder. Amino acids were extracted in a 2.2 pH buffer solution and analysed on the 25 cm column of a Locarte automatic amino acid analyser. Each sample was analysed for 18 amino acids (Table 3), nine of which are regarded as essential for insect growth, and nine as non-essential (House, 1965).

R E S U L T S

The effect of chlorotic and undamaged spruce needles on growth of the green spruce aphid The mean time to the appearance of chlorosis was 15.6 k S.E. 0.38 days (n = 23) on Sitka

spruce and 17.5 f S.E. 0.32 days (n = 23) on Norway spruce. The difference was significant (t44 = 2.248, P< 0.05). There was a negative correlation between adult weight and time for chlorosis to develop on Sitka spruce but not on Norway spruce (Fig. 1). Two needles of Sitka spruce (8%) and five needles of Norway spruce (18%) did not develop chlorosis by the time the aphids became adult. This difference was not significant (contingency 1: = 0.42, P> 0.05).

Adult apterae produced either one or two nymphs in 24 h. Sixty-six single and 14 pairs of nymphs were born on Sitka spruce, and 74 single and six pairs of nymphs on Norway spruce. The difference was not significant (contingencyz: = 2.80, P > 0.05). Nymphs were born 1 - 7 days after adults were caged on the trees (Table l), and there was no significant difference in the time taken to produce nymphs on the two hosts (contingency x: = 1.72, P> 0.1). The difference in the growth of the green spruce aphid on Sitka and Norway spruce (see below) was not therefore due to differences in either the numbers of nymphs produced in 24 h or in the length of time before they were born.

The developmental time (birth to adult) of aphids that were not transferred to fresh needles at the fourth instar was 18.9 & S.E. 0.33 days (n = 25) and 20.1 f S.E. 0.38 days (n = 28) on Sitka and Norway spruce respectively (t46 = 2.129, P< 0.05).

The mean weight of aphids at the fourth instar and adult stages and mean relative growth rate (MRGR) over the fourth instar (RGR = (log adult weight - log fourth instar weight)/duration of fourth instar in days) are given in Table 2. Fourth instar nymphs were significantly heavier on Sitka than on Norway spruce (between species Fl,6 = 5.98, P< 0.05). Overall there was a significant difference in the weight of fourth instar nymphs assigned to the two treatments (F1.86 = 4.18 P< 0.05) but interaction terms were not significant (treatment x species Fl.86 = 0.08, P> 0.05; treatment x trees within species F6.86 = 2.21, P> 0.05).

Adult aphids were also significantly heavier on Sitka than on Norway spruce (between species Fl,6 = 6.23, P< 0.05). Aphids which were not transferred to a fresh needle at their third moult (i.e. remained on chlorotic needles) were significantly heavier when adult than those which were transferred (F1.86 = 37.41, P < 0.001; treatment x species FI.86 = 4.54, P< 0.05; treatment x trees within species F6,86 = 0.41, P> 0.05). There was no significant difference in MRGR on the two hosts (between species Fl,6 = 1.07, P> 0.05), but MRGR was significantly greater in aphids that were not transferred than in aphids that were transferred (between treatments F1.86 = 121.03, P < 0.001 ; treatments x species F1.86 = 11.42, P< 0.01; treatments x trees within species F6.86 = 0.74, P> 0.05).

36

0.40-

0.38

0.36

0.34

0.32

0.30

0.28

0.26

0.241-

M A R T I N F I S H E R

0

0

O ! 0

- 0

0 - - 0

0

0 . - 0 0

0 0

2 0.20 v - E 0.16

3 0.14 d ‘0

0.32

0.30

0-241

t o 0.16

12 1 3 14 15 16 17 18 19 20 21 22 Number of days to development of chlorosis

o? , , , , , , , , , ,

Fig. 1. Relationship between the time taken for aphid-induced chlorosis to develop and the weight of adult green spruce aphids on (a) Sitka and (6) Norway spruce. (a) rz, = -0308, P < 0.05 (6) rT1 = -0.355, P > 0.05.

Table I . The number of adult aphids that bore nymphs on each of seven consecutive days ajier they were caged singly on Sitka and Norway spruce. Each adult was removed after it bore one

or more nymphs Number of adults that bore nymphs on each day

, Host plant Day I Day 2 Day 3 Day 4 Day 5 Day 6 Day 1

Sitka spruce 35 26 12 4 1 1 1 Norway spruce 31 29 9 9 2 0 0

Growth of the green spruce aphid 37

Table 2. Mean weights (with S.E.D.S) ofjburth instar nymphs and adults, and the mean relative growth rate (MRGR) over the fourth instar of aphids reared on Sitka and Norwa-v spruce at 15 "C

and a 16 h photoperiod Host plant

Weight of fourth instars't n Not transferred n Transferred S.E.D.

Norway spruce 28 -2.060 (0.1275) Sitka spruce 25 - 1,915 (0.1474)

S.E.D. 0,0652

28 - 1.994 (0. I 362) 0.0454 21 - 1.851 (0.1571) 0.0503

0.0685 Weight of adultst Norway spruce' 28 - 1.463 (0.2315) 28 - 1.596 (0.2028) 0.043 1 Sitka spruce 25 - 1.148 (0.2870) 21 - 1.528 (0.2169) 0.0478

S.E.D. 0.0677 0.0710

MRGR over fourth instar Norway spruce 28 0.05 I4 28 0.0350 0.0027 Sitka spruce 25 0.0598 21 0.0300 04030

S.E.D. 0.0034 0.0036

* Since variance of the weight data was roughly proportional to mean weight a log transformation was applied for analysis of variance. t Figures presented are for means of log-transformed weights (with back-transformed means in brackets); approximate S.E.D.s are calculated as for a split-plot analysis with comparisons between treatments (transfer or not) based on within-tree variance, other comparisons on a combination of between- and within-tree variance.

Amino acid anal.vsis of spruce .foliage Six essential amino acids (histidine, isoleucine, leucine, phenylalanine, threonine and

valine) were significantly more concentrated in infested than in uninfested foliage on the same trees and one essential amino acid (arginine) and one non-essential amino acid (aspartate) were significantly less concentrated in infested foliage (Table 3). Since total amino acid concentrations were not significantly different (Table 3) differences in individual amino acids were due to a change in the balance of amino acids. The concentrations of individual and total amino acids in the two controls was not significantly different (Table 3). The effect of aphid infestation on the foliar amino acid concentration was therefore localised ; the concentration of foliar amino acids in other branches was not affected.

The mean number of aphids removed from the 10 infested branches was 879 (range 677 - 1224). There were no significant correlations (Kendall's rank correlation coefficient) between the number of aphids in a sleeve and the concentration of total amino acids, of individual amino acids, of aspartate and arginine combined (amino acids less concentrated in infested foliage), or of histidine, isoleucine, leucine, phenylalanine, threonine and valine combined (amino acids more concentrated in infested foliage). The magnitude of the change in concentration of amino acids due to infestation was therefore independent of the final density of aphids on each branch. The development of chlorosis is irreversible (Parry, 1974) and therefore once the aphid density exceeded one/needle (as it did on all 10 infested branches by the end of the experiment) further changes in amino acid concentration would not necessarily be expected.

DISCUSSION

The beneficial effect of previously infested spruce needles (most of which became chlorotic) on growth of the green spruce aphid can be likened to the effect of aphid-induced galls on aphid growth. Galling of apple plants by Dysaphis devecta (Walker) improves aphid growth,

38 M A R T I N FISHER

Table 3. Concentrations of 18 amino acids and total amino acids (mean pg/g dry weight of foliage 2 s.E.) in Sitka spruce foliage infested with E. abietinum, in uninfested foliage on infested trees and in infested foliage on uninfested trees. n = 10 for all means except for asparagine, serine and total

amino acids in control 2 where n = 9 (the two amino acids ran together in one extract)

Amino acid

Non-essential Alanine amino acids Asparagine

Aspartate Cysteic acid y-amino butyric

acid Glycine Glutamate Serine Tyrosine

Essential Arginine amino acids Histidine

Isoleucine Leucine Lysine Phenylalanine Threonine Tryptophan Valine Total amino

acids

Infested foliage

x f S.E. 129.3 f 9.5

2251.5 f 417.58 179.7 f 15.1 24.7 f 4.6

407.6 f 52.3

13.1 f 0.6 376.8 f 43.3 43.0 f 7.1 39.6 f 3.4

174.5 f 109.5 90.2 f 8.3

129.6 f 10.4 104.6 f 12.5 37.4 f 7.0 60.4 4.7

127.0 f 15.6 132.2 f 18.9 229.8 f 20.0

4550.8 f 492.9

Uninfested Uninfested Infested foliage on foliage on foliage vs

infested trees uninfested trees control 1 (control 1) (control 2) (paired x f S.E. x f S.E. r-test)

136.4 f 9.0 140.1 f 7.4 0.550 2131.5 f 410.4 1723.1 f 443.8 0.297 412.3 f 27.3 459.2 f 49.4 7.561***

27.5 f 3.3 24.9 f 3.5 0.575 470.6 f 59.8 558.4 f 57.6 1.906

12.5 f 1.3 436.0 f 61.8

38.1 f 9.4 35.7 f 5.3

239.8 f 111.4 60.8 f 6.1 37.1 f 5.5 22.4 f 4.2 87.6 f 33.3 38.2 f 5.0 56.3 f 15.4 73.4 f 19.9 65.2 f 10.0

4381.5 f 379.9

13.3 f 0.9 484.2 f 67.4 41.8 f 14.2 45.6 f 4.1

319.6 f 98.1 75.8 f 9.7 47.8 f 5.2 23.1 f 2.0

104.7 f 25.6 40.9 f 3.7 48.1 f 14.3

102.6 f 19.0 92.3 f 9.7

4343.0 f 587.0

0.332 1.295 0.378 0.736 3.990** 2.762’ 8.197*** 6.231*** 1.706 5.092*** 2.668* 1.666 6.928*** 0.297

Control 1

control 2 (simple r-test)

vs

0.325 0.695 0.83 I 0.539 1.058

0.499 0.527 0.101 1.483 0.537 1.315 1.411 0.151 0.408 0.435 0.390 1.059 1.947 0.058

D.F. = 9 for all comparisons of infested foliage and control I , and D.F. = 18 for all comparisons of controls 1 and 2, except for serine, asparagine and total amino acids where D.F. = 17. *, **, *** = P < 0.05, P < 0.01 and P < 0401 respectively.

and honeydew of aphids feeding on galled plants has an increased amino acid content (Forrest, 1971). Even without galling aphid infestation can increase the ‘quality’ of plants (determined as the concentration of either amino acids or soluble nitrogen) for aphid growth (Dixon, 1971; van Emden, 1973).

The greater growth of green spruce aphids on chlorotic than on undamaged needles may have been due to the better ‘quality’and/or arrestant properties of the phloem sap of chlorotic needles. Although the recorded MRGR was greatest on chlorotic needles, if undamaged needles had lower arrestant properties and aphids took longer to settle on them, the actual MRGR may have been the same on both needle types. The weight loss of green spruce aphids starved for 24 h at 15 k 2°C a 16 h photoperiod/day and 70% r.h. (the r.h. inside the cages used to rear the aphids) was 9.7% (M. Fisher, unpublished), and the time taken for 50% of green spruce aphids to commence sap uptake on undamaged needles of Sitka and Norway spruce at 15°C and a 16 h photoperiod/day was 3 h 1 1 min and 5 h 38 min respectively (Parry, 1971). It follows that a weight loss sufficient to give the mean adult weights recorded on undamaged needles (24 and 12% lower than on chlorotic needles of Sitka and Norway spruce respectively) could not have occurred if fourth instar aphids commenced sap uptake within 6 h of being placed on needles. Although previous aphid infestation could alter the arrestant

Growth of’ the green spruce aphid 39

Table 4. Summary of the characteristics of aphid-induced chlorosis and of aphid performance on Sitka and Norway spruce

Characteristic

Host tree r 7

Sitka spruce Norway spruce L

Time taken for chlorosis to develop shorter longer

Negative correlation between adult weight and yes no

Length of aphid feeding time that causes 50% shorter longer

Difference between aphid weight on chlorotic greater lesser

Developmental time of aphid shorter longer Weight of adult aphids greater lesser

time for chlorosis to develop

of needles to fall

and green needles

Source

This study; Kloft & Ehrhardt (1 959); Parry (1974)

This study

Parry (1974)

This study

This study This study

properties of spruce needles it appears that chlorotic needles provided a better quality food source for aphid growth than did undamaged needles.

The amino acid analysis of infested foliage of Sitka spruce was separate from the experiment on aphid growth. Nevertheless, amino acid analysis indicated that the improved quality of chlorotic needles for aphid development may have been due to a shift in the balance of amino acids in favour of essential amino acids. Fertiliser-addition experiments indicate that a combination of certain amino acids, rather than an increase in the concentration of total amino acids, causes an increase in numbers of the green spruce aphid on Sitka spruce (Carter & Nichols, 1985). Two of the six amino acids that were significantly more concentrated in chlorotic needles (histidine and isoleucine) are essential for some aphid species (Dadd & Krieger, 1968; Leckstein & Llewellyn, 1973), and all six amino acids are amongst the 10 amino acids essential for aposymbiotic Myzus persicae Sulz. (Mittler, 1971).

The development of chlorosis could be due to physical damage of plant tissues by the aphid’s stylet and/or to a toxin in the aphid’s saliva. When the green spruce aphid probes needles of Sitka and Norway spruce, cells are damaged (Parry, 1971) and saliva is deposited to form a salivary sheath (Kloft & Ehrhardt, 1959; Parry, 1971). Seven amino acids were found in saliva deposited on filter paper during probing by the green spruce aphid (Kloft & Ehrhardt, 1959). In this and other aphid species, amino acids and amides in saliva (Auclair, 1963; Kloft & Ehrhardt, 1959) and plant growth hormones detected in whole aphid extracts (Auclair, 1963) have been held responsible for damage to plants. However, it is unclear how the amino acids, which are present in plants, could induce damage, and it is not known whether the hormones and amino acids originated from the plants or the aphids. Parry (1971 ; 1974) suggested that since the green spruce aphid deposits more saliva at its feeding point on Sitka than on Norway spruce, the presence of a salivary toxin could explain why chlorosis develops more quickly on Sitka spruce. However, although Kloft & Ehrhardt (1959) found that spruce cuttings in a solution of homogenised green spruce aphids became chlorotic, the presence of a toxin in the aphid’s saliva has not been demonstrated. Damage caused by the green spruce aphid was localised: an aphid only induced chlorosis in the needle on which it fed and the foliar amino acid balance was only disturbed in infested branches. This indicates that if a toxin is involved it acts only at the point of feeding. Further work is required to clarify the cause of the aphid-induced chlorosis of spruce needles.

In the green spruce aphid fecundity is positively correlated with weight at the final moult, and at 15°C a 24% decrease in adult weight on Sitka spruce (as observed when the aphid was

40 M A R T I N FISHER

reared during the fourth instar on undamaged needles) results in a 17% decrease in fecundity measured over the first 10 days of reproductive life (M. Fisher, unpublished). Therefore the green spruce aphid will, on average, be more fecund on chlorotic than on undamaged spruce needles. This will accelerate the development of outbreaks of the green spruce aphid on Sitka spruce, and is probably an important contribution to the pest status of this insect.

Norway spruce is less susceptible to the green spruce aphid than is Sitka spruce (Carter & Nichols, 1985). Table 4 summarises the characteristics of aphid-induced chlorosis of spruce foliage and aphid performance that could contribute to this difference in susceptibility. Sitka spruce was first introduced into the British Isles in 1831 (Harris, 1978) and the green spruce aphid was first reported in 1849 (Walker, 1849). Sitka spruce has therefore been in contact with the green spruce aphid for a relatively short time, and this may account for its lower resistance to the aphid. In general North American species of spruce are damaged more by the green spruce aphid than are those from Eurasia (Carter & Nichols, 1985). It would be of value to investigate the influence of chlorosis-induced variations in aphid performance on the susceptibility of spruce species in the different damage categories identified by Carter & Nichols (1985).

I am indebted to J. S. Fenlon for statistical advice and for carrying out the Anovar, to Professor A. F. G. Dixon and C. I. Carter for their help and advice, to J. F. A. Nichols for help with amino acids analysis and to C. Betterton and S. A. Ghazanfar for reading an early draft of the paper. This study was supported by a studentship from SERC and by the Forestry Commission.

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Growth of' the green spruce aphid 41

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(Rewiped 2 January 1986)