"Bionomics of the Alder Sawfly Fenusa Dohrnii (Tischbein)"

"BIONOMICS OF THE ALDER SAWFLY FENUSA DOHRNII (TISCHBEIN)"

Kulapu Arachchige Don Wilmot Senaratne, B.Sc.(Ceyl).

Thesis submitted to the University of London

for the degree of Ph.D.

East Mailing Research Station Maidstone

Kent October 1978. 1

ABSTRACT

Field and laboratory studies during 1975-77 have shown that Fenusa dohrnii is bivoltine in south - east England with adults emerging during May - June and July - August. Average fecundity was estimated to be about 62 eggs per female, the number being correlated with both weight and tibial length of adult. At 15°C and 21°C respectively, the embryonic period was 13 and 7.5 days whilst larval development took 26.6 and 16.9 days. Selection of a cocooning site in the soil by the sixth instar larva was random; no active burrowing was observed, but the larvae gained entry via cracks and patches of loose soil. Prepupal diapause was terminated to a varying degree when cocoons were chilled for about 62 days at

-1.1°C, 3.3°C, 10°C and field temperatures. The highest percentage emergence of adults was from cocoons exposed to field temperatures and progressively fewer emerged as the chilling temperature was lowered from 10°C to

-1.1°C. For all treatments mean time for adults to emerge after transfer to 21°C decreased with increasing duration of chilling. In the field, distribution of eggs of the second generation was correlated more closely with the negative binomial than Poisson , Neyman type A or logarithmic distributions. Studies of the population development indicated that the generation mortality varied from 95 - 98 percent of which mortality of larvae within the mines and mortality of larva'VI due to parasitism by Lathrolestes pictilis (Ichneumonidae) and

Ichneutes laevis (13raconidae) contributed most towards 2

the generation K value. Two eulophid parasites Cirrospilus vittatus (Wik.) and Chrysocharis nitetis (Wik.) were also found parasitizing the young larvae. There was also circumstancial evidence that predators were responsible for some mortality of larvae but this was not confirmed either by observations or electrophoretic techniques. 3

ACKNOWLEDGEMENTS The work for this thesis was carried out at East Mailing Research Station. For the first two years of this study I was in receipt of a Colombo-plan scholar- ship. I am grateful to the Director of East Mailing Research Station for providing me with the facilities to carry out this project, the Director of the Tea Research Institute, Sri Lanka, for granting me leave of absence and the British Council for making all necessary arrangements. I am also indebted to my supervisor Dr. G.H.L. Dicker, and my Director of studies Dr. G. Murdie for their guidance, constructive criticism and helpful discussions throughout this project. Thanks are due to Mr. G.P. Barlow for statistical advice and analysis of some of the data, Dr. R.A. Murray for electrophoresis of insect samples, Dr. K.A.D. MacKenzie for the preparation of leaf sections, Dr. M.G. Fitton, J.S. Noyes and T. Huddleston of the British Museum (Natural History) for identification of parasites, Mr. D.G. Richardson for printing of the thesis, and members of the photographic studio for preparing some of the photographs. Grateful acknowledgement is also due to Annals of Applied Biology for permission to reproduce Figure 24. I also thank Dr. B.H. Howard, Dr. J.J.N. Flegg and all other members of the Department of Zoology for their invaluable assistance. I am also grateful to Mrs. G. Squires for typing this thesis and last but not least I wish to record my thanks to my wife Shiranganie for her financial assistance which made it possible to continue this work beyond two years. 4

LIST OF FIGURES Figure Page 1 Cages used to collect laboratory reared adults. 15 2 Part of the experimental area with emergence cages and sticky boards in position. 17 3 a) A recently laid egg and one containing a well developed embryo curled within the chorion. b) Blisters on the dorsal surface of a leaf of Alnus Zlutinosa indicating the position of developing egg. c) Transverse section of a leaf showing an egg between the palisade and spongy mesophyll. 21 4 Transverse sections of leaves of Alnus glutinosa a) from plants grown in the field; b) from plants raised in the glasshouse. 25

5 Ovarioles of Fenusa dohrnii showing developing and mature oocytes. 28

6 Relationship between the number of ovarioles (OV) and the weight of the adult (W). 29

7 Relationship between, a) number of eggs (E) and hind tibial length (L); b) number of eggs (E) and weight of adult (W); c) weight of adult (W) and hind tibial length (L); for adults shortly after (0) and approximately ten days after (.) emergence. 32 8 Development of eggs at constant temperatures of 21°C and 15°C. 34 9 Leaves showing (a) single mines and (b) coalesced mines. 36 5

Figure Page

10 Frequency of head capsule widths of larval instars and the range for each instar. 38

11 Relationship between head capsule width (Y) and larval instars (X) plotted on a log scale. 40

12 Period of development of eggs and larvae when reared at a constant temperature of 21°C or 15°C. 43

13 Relationship between area of mine and the total leaf area. 46

14 Paths followed by mature sixth instar larvae on bare stabilized soil before either cocooning or leaving observational area of 100 x 100 cm on a) October 21st, b) October 22nd. 48

15 Paths followed by mature sixth instar larvae on bare stabilized soil before cocooning in two experiments on 27th October. 49

16 a) Single and b) aggregated cocoons of Fenusa dohrnii showing the uneven surfaces. 53

17 Numbers of F. dohrnii in emergence traps at three-day intervals, 1976. 57

18 Relationship between weight of adult (WA) and the weight of mature sixth instar larva (WL). 59

19 Relationship between number of eggs (E) and a) weight of adult (W); b) weight

of mature sixth instar larva (W1). 60

20 Percent emergence at 21°C in relation to period of chilling. (a) F. dohrnii

(b) Parasites. 63 6

Figure Page 21 Mean time to emergence of adults after transfer to 21°C in relation to period of chilling. (a) F. dohrnii (b) Parasites. 65 22 Variation of S.E. of mean time to emergence of adults of F. dohrnii after transfer to 21°C in relation to period of chilling. 66 23 Numbers of L. pictilis and I. laevis in emergence traps, 1977. 78

24 Electrophoretic slab gels showing A) anthocorid starved for 36 h (1); anthocorid starved and then fed on larva of F. dohrnii (2); larva of F. dohrnii (3); B) anthocorid starved for 36 h (1); anthocorid starved and then fed on alder aphid (2); alder aphid (3). 82 25 Relationship between observed and expected frequencies for leaves containing no eggs (zero variate). 88 26 Relation of k to mean for 36 samples from a population of Fenusa dohrnii eggs. 89 27 Relationship between Y1 and X1 where X1 = mean2 - varianc;a number of sampling units

and Y1 = variance - mean. 90 28 Relationship between variance mean (M) (V) and mean (M) for 36 samples from a population of F. dohrnii eggs. 96 29 Relationship between mean crowding(Mx) mean (M) and mean (M) for 36 samples from a population of F. dohrnii eggs. 99 7

Figure Page 30 Relationship between variance (V) and mean (M) of the number of eggs per leaf,

plotted on log scale. 102 31 Relationship between Lloyd's measure of mean crowding (Mx) and mean density (M)

for eggs of Fenusa dohrnii. 103 32 Early growth of Alnus glutinosa showing

individual leaf clusters. 106 33 Numbers of adults, eggs, larva I and VI per 576 clusters. (a) and (b) generation

I, 1976; (c) and (d) generation II, 1976. 110 34 Numbers of eggs and each larval instar

per 576 clusters. Generation II, 1977. 111 35 Totals of eggs and each larval instar per 576 clusters on each sampling occasion

in generation II, 1977. 112 36 Changes in mortality of F. dohrnii expressed as k values. 118 37 The k values of various mortalities of F. dohrnii plotted against the density on which they acted. (a) ki on eggs ; (b) k3 on larva I ; (c) k2 on larva I. 120

LIST OF TABLES

Table Page

1 Measurement of 20 eggs shortly after deposition. 20

2 Force (gm/mm2) required to penetrate leaves on shoots of Alnus glutinosa grown in the field or in the glasshouse. ~3

3 Number of eggs laid in leaves of Alnus glutinosa by adults in cages. 26 4 Relationship between number of mature oocytes, adult weight, and tibial length in females

shortly after, and 10 days after, emergence. 31 5 Width of head capsule, Dyar's constant, and

the percentage increase. 39 6 Variation in the area mined by single larvae of F. dohrnii in the field. 45 7 Distance travelled on the soil surface and speed of 18 larvae. 50 8 The speed (cm/mta) of larvae in relation to

distance travelled on the soil surface. 51 9 The number of cocoons at various depths beneath alder trees, 54

10 Percent cocoons with prepupae and pupae, 1975-76 56 11 Percentage of cocoons occupied by F. dohrnii and by parasites. 62 12 Numbers of Ichneutes laevis and Lathrolestes pictilis emerging in relation to period of chilling. 68 13 Variation of mean time to emergence of parasites from samples chilled for 182 days. 69

14 Mean temperatures (°C) at 5 cm below soil surface, East Mailing 1975-76. 71 15 Parasites of Fenusa dohrnii and their sites of pupation. 73 9

Table Page 16 Numbers of larvae and pupae of Cirrospilus vittatus and Chrysocharis nitetis found associated with each larval instar of F. dohrnii, 1977, 74 17 Numbers of eggs and larvae of Lathrolestes pictilis and Ichneutes laevis found associated with each larval instar of F. dohrnii, 1977, 75 18 The relative abundance of Lathrolestes pictilis and Ichneutes laevis. 77 19 Percent larvae of I. laevis and L. pictilis in host prepupa or in the fibrillar cocoons 8o 20 The significance levels between observed and expected frequencies for four theoretical distributions based on Chi-squared test, and estimates of mean density, negative binomial parameter (k), and total number of individuals in an aggregate (N) for each of the 36 quadrants. 84 V Mean crowding(Mx) 21 Mean density, )C2 for M 1' x Mean (M) and level of significance of M > 1 of egg counts for 36 quadrant-height combinations. 94 22 Slope coefficients and percent variance accounted for by regression of M on mean, and M on mean,of eggs observed in each of the 36 quadrant-height combinations. 97 23 Regression coefficients (- S.E.) and percentage variance accounted for by regression of log variance on log mean, and mean crowding on mean, for the number of eggs observed in each of the 36 quadrant-height combinations. 101

24 Life tables of F. dohrnii. 114 10

CONTENTS

Page Abstract 1 Acknowledgements 3 List of Figures 4 List of Tables 8

I INTRODUCTION 13 II MATERIALS AND METHODS 14 A Rearing in laboratory 14 (a)Cocoons 14 (b)Adults 14 (c)Parasites 14 B Field studies 16 (a)The experimental plot 16 (b)Insect records 16 (c)Characters used to identify Fenusa dohrnii and Heterarthrus vagans 16 III BIOLOGY AND DEVELOPMENT OF STAGES 19 A The egg 19 (a)Description and size 19 (b)Oviposition 19 (c)Leaf preference for oviposition 19 (d)Number of eggs laid 24 (e)Estimation of fecunditiy by dissection 27 (f)Rate of development 33 B The larva 33 (a)General observations 33 (b)Width of head capsules 37 (c)Duration of larval period 42 (d)Variation of size of mine 44 (e)Selection of cocooning site 44 (f)Construction of cocoon 52 (g)Depth of cocoon 52 C The prepupa and pupa 55 11

Page D The adult 55 (a)Emergence 55 (b)Sex ratio 55 (c)Relationship between size of mature larva and adult 58 IV DIAPAUSE 61 A F. dohrnii 61 (a)Effect of period of chilling on emergence 61 (b)Effect of chilling on rate of development 64 B Parasites 64 (a)Effect of period of chilling on emergence 64 (b)Effect of chilling on rate of development 67 C Phenology of emergence 67 V NATURAL ENEMIES 72 A Parasites 72 (a)Cirrospilus vittatus and Chrysocharis nitetis 72 (b)Lathrolestes pictilis and Ichneutes laevis 76 B Predators 79 (a)Field observations 79 (b)Laboratory experiments 79 VI DISTRIBUTION OF EGGS 83 A Type of distribution 83 B Type of aggregation 92 C Indices of aggregation 92 (a)Variance to mean ratio 93 (b)Ratio of mean crowding to mean density 93 (c)Constants of Taylor's power law 98 (d)Regression of mean crowding to mean 100 12

Page

VII POPULATION DEVELOPMENT OF F. DOHRNII IN THE FIELD 105 A Methods 105 (a) Sampling procedure 105 (b)Statistical analysis 105 (c)Estimation of totals of larva VI and adults 105 B Results 107 (a) Sources of variation 107 (b) Integration of populations 108 (c) Construction of life tables 109 VIII DISCUSSION 117 APPENDICES I - VI 122-135 REFERENCES 136 13

I. INTRODUCTION

In England and the Netherlands alder trees,

Alnus sp. are planted extensively as windbreaks to provide shelter for plantation of fruit trees. A common member of the insect fauna is Fenusa dohrnii (Hymenoptera:

Tenthredinidae ) , the alder sawfly. According to

Benson (1952) this species is considered to be a native of North America but now has a holarctic distribution.

The larva, like those of the related species, F. ulmi Sundewall and F. pusilla (Lepeletier), is a leaf miner, its preferred host being Alnus glutinosa (L.) Gaertner, but larvae are occasionally also found in leaves of

A. cordata Desf. and A. incana (L.) Moench when these are grown in proximity to A. glutinosa.

Although widely distributed in England, larval populations usually are low on trees growing in their natural habitat by streams or swampy places. In contrast very high populations may occur on windbreaks although numbers have been noted to vary greatly between sites at any one time or between successive generations.

The study reported below was carried out to provide details of the life cycle of F. dohrnii and to try and identify factors responsible for its fluctuating populations. 14

II, MATERIALS AND METHODS

A. Rearing in the laboratory

(a) Cocoons

Cocoons were obtained by placing leaves from

the field containing late instar larvae on the surface

of plastic trays (33 x 15 x 7.5 cm) which had been lined

with polythene sheets and filled to a depth of about

4cm with sterilized soil passed through a 3mm mesh sieve.

The tray was covered with a sheet of perforated polythene

to reduce loss of moisture from the leaves. The leaves

were changed at intervals of two days and, about a week

after the last change, the soil was again sieved to

recover cocoons. Those attached to the polythene liner

were removed by cutting the polythene with a cork borer.

If not needed immediately, cocoons were stored in moist

soil in a north facing outdoor shelter.

(b) Adults

Cocoons thus collected were buried in moist

sterilized soil to a depth of about 2.5 cm either in

pots (10 cm diameter and 9 cm high) or plastic cups

(6 cm diameter and 5 cm high). Glass cylinders with

gauze lids placed on pots and collecting tubes mounted

on the lids of the plastic cups (Figure 1) were used

to trap the adults.

Adults of Lathrolestes pictilis and Ichneutes

laevis emerging from the host cocoons were collected

in the same way as adults of F. dohrnii. The two

eulophid parasites, Cirrospilus vittatus and Chrysocharis ~5

FIGURE

Cages used to collect laboratory reared adults. 16

nitetis, which complete their life cycles within the host mine, were bred by placing parasitized host larvae and parasite pupae in glass tubes (3 cm diameter and

6 cm high) lined with moist filter paper.

B. Field studies

(a) The experimental plot

The plot (Figure 2) consisted of a single row of 186 trees of Alnus glutinosa planted 1.2 m apart in

1968 in a north-south direction. To the east was arable ground cropped by cereals, to the west a footpath and an apple orchard. In winter the east and west sides were pruned to reduce the width to about 1 m from the stem.

The ground beneath the trees was kept clean by an annual application of simazine and paraquat.

(b) Insect records

Mature larvae dropping from mined leaves were collected on boards (1.0 x 0.6 m) coated with tree- banding grease (Stikem). Adults of F. dohrnii and parasites emerging from cocoons were trapped in fibre- glass cages of similar size. The area of soil covered by each sticky board or emergence cage was about 25 percent of that covered by the average tree canopy, and their positions were changed at two-day intervals.

Heterarthrus vagans (Fall.).

Alnus glutinosa is a host for two closely related sawflies, Fenusa dohrnii and Heterarthrus vagans, the larvae of both being leaf miners (Benson, 1952). 17

FIGURE 2

Part of the experimental area with emergence cages and sticky boards in position. 18

The latter occured in very small numbers and could be distinguished from F. dohrnii by the following characters:

Fenusa dohrnii Heterarthrus vagans

Eggs Laid in the leaf through Laid in the leaf through

the upper epidermis; the upper epidermis;

opaque white with no changes colour from

changes in colour. white to brown.

Larva No black marking on the Prothorax with a black

prothorax or on the plate; the rudimentary

rudimentary leg. leg is surrounded by a

black crescent-shaped

plate.

Shape of Usually elongate and Approximately circular mine confined to the area and not confined to the

between two adjacent area between two lateral

lateral veins. veins.

Cocoon The sixth instar larva A disc-shaped cocoon

leaves the mine and formed within the mine.

cocoons in the soil.

Adult Black; M, and lm-cu Black; M, and lm-cu

subparallel. converge strongly

towards stigma. 19

III. BIOLOGY AND DEVELOPMENT OF STAGES

A. The egg

(a)Description and size Freshly laid eggs extracted from leaves were ovoid in shape, enclosed in a very thin, unsculptured, transparent, membrane and with measurements as in Table 1. As development proceeded the eggs became opaque white in colour and the embryo was visible (Figure 3a). (b)Oviposition When a female is ready to lay she walks about on the upper leaf surface, touching the epidermis with her antennae and probing with the ovipositor as she searches for a suitable site in which to oviposit. Having selected a site the ovipositor is inserted through the upper epidermis and the abdomen is tilted to one side to permit movement of the saw in the plane parallel to the leaf surface. An egg is inserted in the mesophyl]_ of the leaf (Figure 3c) with an end protruding into the epidermal cells in the slit along which the ovipositor is withdrawn. At first only a pale scar representing the oviposition slit is visible, but after two or three days a blister appears in the upper leaf surface (Figure 3b) due to enlargement of the egg as the embryo develops, a characteristic feature of many sawflies as noted by

Friend (1933). (c)Leaf preference for oviposition Eggs are laid only in young leaves which have

expanded.to about 75 percent of their final size or have 20

Table 1. Measurements of 20 eggs shortly after deposition

95% confidence Range (mm) Mean(mm) S.E. of Mean interval

Length 0.40-0.42 0.42 t 0.002 ± 0.004

Width 0.15-0.20 0.18 ± 0.003 t 0.006 5oNm

FIGURE 3 a) A recently laid egg and one containing a well developed embryo curled within the chorion. b) Blisters on the dorsal surface of a leaf of Alnus glutinosa indicating the position of developing eggs. c) Transverse section of a leaf showing an egg between the palisade and spongy mesophyll. U = upper epidermis, C = chorion, E = egg, .L = lower epidermis. 22

recently reached maturity. They are usually laid in the central leaf area with only an occasional one near the margin.

The choice of leaf for oviposition could be a behavioural factor, possibly arising from selection,

since Beckwith (1970) has shown that larval survival and fecundity of adults of some insect species are influenced

by food source, and with a leaf mining species such as Fenusa dohrnii the larval feeding position is predetermined

by the oviposition site. Alternatively, it could result from a chemotrophic response associated with young leaves, or a physical factor such as force required to insert the

ovipositor. Difficulty in obtaining satisfactory oviposition on small plants raised in a glasshouse

precluded a study of the first two factors. Leaf toughness was investigated using a penetrometer as described

by Topping (1978) but modified to reduce the speed of the platform movement and to take a pin of 0.825 mm diameter instead of the probe. To avoid inaccuracy due to bending

(Beckwith and Helmers, 1976) the leaves were placed between two perspex slides each with two holes of 1.5 mm diameter

through which the pin could pass. These were aligned by means of two screws attached to the lower slide on which the upper slide could be lowered.

The data in Table 2 representing the mean of 12 penetrations per leaf, show that material collected from the field required less force to penetrate a leaf classified as suitable for oviposition than other leaves on the same shoot but, when all shoots were considered the range of forces required to penetrate suitable leaves 23

Table 2. Force (gm/mm2) required to penetrate leaves on

shoots of Alnus glutinosa grown in the field

or in the glasshouse

Oviposition rating

Suitable Unsuitable No. of Mean force/ No. of Mean force/ Shoot no. leaves leaf leaves leaf

Field

1 1 51.2 4 68.3-94.3

2 1 42.2 5 69.5-81.3

3 1 39.9 5 49.1-68.1 4 1 53.3 2 81.6-90.0. 5 2 65.4,70.7 5 73.0-86.9 6 1 69.5 7 78.1-90.4

Glasshouse

1 2 32.6,35.7 5 35.2-45.8 24

overlapped the lower end of the range for unsuitable ones. On the above evidence alone it is not possible to conclude that leaf toughness is of major importance in determining the choice of the oviposition site, although it may be one facet of a more complex behavioural pattern. Less force was required to penetrate both categories of leaves from a plant raised in the glasshouse

and this could be correlated with leaf structure (Figure 4). Epidermal cells were greatly reduced and there were fewer cells in the mesophyll resulting in a thinner leaf. Similar differences in structure of apple leaves have been reported by Avery (1977) and may be the result of lower irradiance and higher temperatures.

Analysis of the data using the multiple range test of Duncan (1955) showed that the force required to penetrate the basal leaves was significantly greater than for the terminal leaves of the same shoot (P4:0.05) (Appendix 1). (d) Number of eggs laid Compared with the number of mature oocytes present in the ovarioles of adults shortly after emergence

(Table 4) rather less than 50 percent were laid when. newly emerged females were placed in cages containing

small plants of Alnus glutinosa raised in a glasshouse

or shoots from field-grown plants (Table 3). Egg

production was similar when the plants were enclosed in

glass cylinders 12 cm diameter and 20 cm high or in glass/gauze cages about 0.06 m 2but fewer eggs were laid in the constant temperature room than in the insectary. Although leaves from plants grown in the 25

50µm ~

FIGURE 4

Transverse sections of leaves of Alnus glutinosa a) from plants grown in the field; b) from plants raised in the glasshouse. UE = upper epidermis, LE = lower epidermis, PM = palisade mesophyll, SM = spongy mesophyll, VB = vascular bundle.

H = hypodermis. 26

Table 3. Number of eggs laid in leaves of Alnus glutinosa by adults in cages

Number of Eggs per Site females female S•d-

Glasshouse-raised plants

Insectary 7 15.0 8.10 Insectary 8 14.1 5.10 CT room 15°C 11 10.4 6.48 CT room 21°C 11 9.7 7.28

Field-grown plants

Insectary 10 14.6 6.94 27 insectary had far fewer cells than those grown in the field

(Figure 4) the adults laid a similar number of eggs in each kind of leaf (Table 3) which suggests that this factor was not responsible for reduced fecundity. Lower fecundity of females confined under laboratory conditions has been reported by Cheng and LeRoux (1966a) for a closely related species, Fenusa pusilla (Lep.), and Benson (1950) in a general comment on the biology of the sawflies states "there is however a discrepancy between number of eggs produced and those usually laid in captivity" which I interpret as indicating reduced fecundity. Among other factors this could be due to the condition of the host plant or to unfavourable environmental conditions. (e) Estimation of fecundity by dissection Ovarioles are of the polytrophic type and those of adults which had just emerged from cocoons contained some mature oocytes. These were recognised by the large size, the presence of a well developed chorion and the absence of associated nutritive cells (Figure 5), and the number present was used as a measure of potential fecundity. The number of ovarioles varied from ii to 30 with a mean of 22. Although a strong positive correlation (r = 0.84; P<0.001; b±SE = 4.85 ±0.73) was obtained between the number of ovarioles and adult weight (Figure 6), it was not possible to ascertain the productivity of each ovariole as dissection often resulted in the rupture of the oviduct and release of mature oocytes into the isotonic saline in which they were dissected.

FIGURE 5

Ovarioles of Fenusa dohrnii showing developing and mature oocytes. G = germarium, 0 = oocytes, N = nutritive cells, 0 = mature oocyte, 29

in 30 •• w -J 0 110.0011.1.5:7:15: cr • • •∎••• • 0 •~ • u_ 20 0 ec w OV = 4-85W +11.17 m • r=0.84 z 10

1 2 WEIGHT [mg]

FIGURE 6

Relationship between the number of ovarioles (OV) and the weight of adult (W). 30

As ovarioles are known to vary in productivity in some

insects (e.g. Webber, 1955) it was therefore not possible to assess the potential fecundity by relating ovariole number to size.

Body weight is a simple criterion of size and is likely to reflect the potential fecundity of adults, but is seldom static, being affected by feeding and by food reserves. Therefore, the length of the right hind tibia was also measured.

The number of mature oocytes present in females about three hours after emergence from cocoons and of others which had been kept in cages for 10 days and supplied with a solution of 2% sucrose is shown in

Table 4. Weight and tibial lengths were similar for both age groups but the older females contained significantly more oocytes, indicating that oocytes continued to develop as the adult aged. High correlations were found between 1) number of eggs and weight; 2) weight and tibial length;and the relationship between eggs and tibial lengths was represented by a quadratic model for both age groups (Figure 7). Similar relationships have been demonstrated for a range of insects (e.g. Hard, 1976). The regression models that fitted the results best were determined by the method described by Seal (1964)(Appendix II). The stability of the measurements on adults were confirmed by principle component analysis of the correlation matrix (Morrison,

1967) and the first principle component accounted for 93.25% and 97.62% of the total variance for the young and older females respectively and is clearly attributed 31

Table 4. Relationship between number of mature oocytes, adult weight, and tibial length in females shortly after, and 10 days after, emergence.

Shortly after 10 days after emergence emergence Number of adults 20 68 Number of Range 1-67 10-112 mature Mean 33.8 62.2 oocytes S.d. 19.7 24.2

Weight Range 1.04-3.58 0.93-3.05 (mg) Mean 2.24 2.07 S.d. 0.66 0.53

Tibial Range 0.84-1.17 0.85-1.22 length Mean 1.02 1.06 (mm) S.d. 0.09 0.09

Measurement "t" Level of significance Oocytes 4.769 *** ';weight 1.196 NS Tibial length 1.603 NS

*** = Significant at P4:0.001 NS = Not significant at 5% 32

120 a 0 w 80 E=177-495L+ 362 L2 'ē U- R=0.98 0 • %~% i cc • °o. 40 • • 2 • '•s .o E=151.4-427L+303L o .1.0.0•11 0~,00 o z •• 000/ R=0-93 o• o 0 o

09 1-0 1.1 12 TIBIAL LENGTH [mm] 120 b • , •• r0 • 0 io00 • 0 80 E=-29+44W w sVs• o U- r =0-96 •0• S. 0 • «~ o m 40 •s•* • 000 E_ -30.3+29SW .•j • ~000 r=0.93 .•~ o00■° 0 , o , 1 2 3 WEIGHT [mg] 4 C rn E 3 W=-39+6-0L o.• o o ..._•• r=0.95 F- o.o ° ~• s CD 2 •a w W=-3.9 +5-7L r=0.96 •00 •• • •

0.9 1.0 1.1 1.2 TIBIAL LENGTH [m m]

FIGURE 7

Relationship between, a) number of eggs (E) and hind tibial length (L); b) number of eggs (E) and weight of adult (W); c) weight of adult (W) and hind tibial length (L); for adults shortly after (0) and approximately ten days after (•) emergence. R = multiple correlation coefficient; r = correlation coefficient 33

to size (Appendix III).

Under natural conditions adults probably lay as soon as oocytes are mature, thus facilitating the accommodation of eggs that mature later in the otherwise compact distal ends of ovarioles and oviducts. Because adults laid few eggs in captivity (Table 3) the best estimate of fecundity was probably 62.2 oocytes per female, obtained by dissection (Table 4). In making an estimate of fecundity it should be more appropriate to use tibial length because the weight of the adult is seldom static and is affected by loss of food reserves and by oviposition.

(f) Rate of development at constant temperature

Because eggs are laid in the leaf information on hatching could be obtained only by dissection. Females were released in glass/gauze cages containing seedling plants of Alnus glutinosa, allowed to remain for 12 hours, and the plants were then transferred to constant temperature rooms maintained at 15°C or 21°C. Preliminary studies indicated that hatching at 15°C and 21°C began after 8 and 5 days respectively, so samples of 20 to 30 eggs were dissected daily after these intervals to determine the hatching periods which are shown in Figure 8. The times taken for 50 per cent of the eggs to hatch were respectively 13 and 7.5 days at 15°C and 21°C.

B. The larva

(a) General observations

Recently evacuated mines contained five cast skins, indicating the presence of six larval instars.

Rearing studies showed that instar VI represented a non- 34

100 •,~• •„.• 0 w ► /21°C /15°C 80 in 60 c) i w 40 F- z otil 20 • • w • —. ' 1 -• / t i l ....•4 • u 0 L 1 I I I 4 6 8 10 12 14 16 DAYS AFTER OVIPOSITION

FIGURE 8

Development of eggs at constant temperatures of 21°C and 15°C. 35 feeding stage which resembled the previous instar in size but could be recognised by its creamy, opaque colouration and the absence of food in the gut. The mouth parts of the first five instars are prognathous, becoming nearly hypognathous in the sixth instar. The body of the newly hatched larva is compressed dorso-ventrally and tapers towards the anal end; it is opaque white in colour but changes to yellowish white in the subsequent instars.

During the first instar the mine consists of a circular area close to the oviposition site which just accomodates the curled larva. Later the larva assumes a position with its ventral side uppermost and the mine increases in size as mesophyll around the periphery is consumed, retaining an approximately circular shape until reaching the adjacent lateral veins. These prevent lateral expansion and further feeding continues towards the leaf margin resulting in a broad, elongated mine

(Figure 9a). Neighbouring mines between the same lateral veins may coalesce and larvae then occupy a common feeding area, but there was no evidence of cannibalism.

During the second generation feeding by older

larvae occasionally caused mortality to unhatched eggs and to very young larvae where the egg density was high due to the small number of leaves suitable for oviposition.

These leaves became completely mined (Figure 9b) but it

was only towards the margins that mines coalesced between

lateral veins.

The mined area becomes brown and the mature

larva emerges through a 'V'-shaped slit in the upper

epidermis, falls to the ground and enters loose soil to FIGURE 9

Leaves showing (a) single mines and (b) coalesced mines, 37 make a cocoon. (b) Width of head capsule

The frequency of widths of head capsules of feeding larvae, for which the instar was determined by the number of exuviae, and of the final non-feeding stage is illustrated in Figure 10.

The head capsule widths of the first five instars (Table 5) follow a geometrical progression as found by Dyar (1890) for many lepidopterous larvae and by Brooks (1886) for the body length of successive instars of the crustacean, Erichthus minutus (Stomatopoda:Squillidae). The geometrical increase in sclerotized structures with each successive instar can be represented by

M = r(x-1)M x 1 where M is the head capsule width of instar number x, M1 the head capsule width of the first instar and r the common ratio. Taking logarithms, the above equation can be written as

Log10Mx = xLog1Or + k

The linear regression of (Log10) head capsule width with instar number using the method of least squares (e.g. Johnson and Leone, 1964) (r = 0.98, P.<0.001) indicates a close agreement between actual and fitted widths (Figure 11).

The absence of an increase in width after the fifth instar has been noted in other species (e.g. Dahlsten, 1961) and may be due to the non-feeding habit of the final instar, the moult resulting in only a 38

I I III IV V

0 .3 .4 .5 .6 .7 .8 .9 WIDTH OF HEAD CAPSULE [mm]

FIGURE 10

Frequency of head capsule widths of larval instars and the range for each instar (•■••.). Unshaded area represents the head capsule widths of the sixth instar, 39

Table 5. Width of head capsule, Dyar's constant, and the percentage increase No. of Mean width S.L. of M Increase in x+1 Instar larvae of head mean M width capsule bnm) x (%)

I 160 0.29 0.02 II 132 0.38 0.03 1.31 31.0 III 157 0.51 0.04 1.34 34.2 Iv 158 0.64 0.04 1.25 25.5 V 145 0.78 0.04 1.22 21.9 VI 5o 0.79 0.03 1.01 1.3 40

1.0 .9 Ē .8 •T E .7 •T 1

1 • 1 LOG Y=-0.64+0.11X r=0.98

.2 II III IV V LARVAL INSTAR

FIGURE 11

Relationship between head capsule width (Y) and larval instars (X) plotted on log scale. • = mean; = range 41

physiological change.

Dyar's Law is not universally applicable to

all taxa of insects (e.g. Taylor, 1931). Ghent (1956) has reported a regular but arithmetic increase in some species of sawflies. Enders (1976), using Dyar's constant in terms of percentages records a range of values from 69 percent for the lesser cornstalk borer to only 15 percent for species of roaches and attributes this marked difference to the feeding habit of the two species. According to Enders (1976) grazing holo- metabolous insects have the largest value of Dyar's constant whereas the larval stages of insects which

search for food have the smallest values. For F. dohrnii Dyar's constant is 21.9 - 34.2 percent (Table 5). Growth of arthropods and nematodes occurs stepwise so that weight increases continuously and power (skeleton and muscles) discontinuously. The marked decrease in Dyar's value from fifth to the sixth instar larva from

21.9 to 1.3 percent results in a greater agility due to greater power to weight ratio (Schmidt-Nielsen, 1972). This may be viewed as being advantageous in the case of F. dohrnii because greater power to weight ratio could result in faster movement and quicker selection of a cocooning site, therd:y decreasing the risks of predation and prolonged exposure to adverse environmental condition.

Przibram and Megusar (1912) working on Sphodromantis bioculata stated that the weight of each instar is double the previous one and at each instar the linear dimensions increase by 1.26 or the cube root of 2. This was considered to be due to each cell 42

dividing once and increasing to its original size. To explain the rates of increase greater than 1.26

Bodenheimer (1927) suggests extra or latent divisions to have occurred but subsequent cytological studies does not support this concept of simple dichotomous cell division (Wigglesworth, 1965). It is however interesting to note that apart from the sixth larval instar of F. dohrnii this ratio remains close to 1.26 as in many other insects. (c) Duration of the larval period Because eggs are laid in the leaf and the larvae feed within the mesophyll it was only possible to obtain the total duration of the larval period. Plants containing eggs used for determination of the hatching period were allowed to remain at the same temperatures,i.e. 15°C or 21°C,until there was no further emergence of mature larvae from the mines. Daily records of the number of mature larvae emerging were taken and Figure 12 shows the cumulative percentage emergence. The time taken for 50 percent of the larvae to reach maturity from oviposition was 39.6 days at 15°C and 24.4 days at 21°C. The time taken for 50 percent of the eggs to hatch under the same conditions was respectively 13 and 7.5 days at 15°C and 21°C and therefore the time taken for 50 percent of the larvae to reach maturity was respectively 26.6 and 16.9 days at 15°C and 21°C. 43

100

-'a 80 / 21% cc w z 60 L.0 CD w 40 () w ►• UJ 20 •► 0 21 24 27 30 33 39 42 DAYS AFTER OVIPOSITION

FIGURE 12

Period of development of eggs and larvae when reared at a constant temperature of 21°C or 15°C. 44

(d)Variation in size of the mine Measurement of the area mined by individual larvae which had developed to the sixth instar (Table 6) shows that there was almost a fivefold difference between the largest and the smallest mines. This was independent of the total leaf area (Figure 13). The area mined by 86 percent of the sample was in the range 1.5-3.0 cm 2 and the more extreme values could reflect differences in nutritional requirements of individual larvae, nutritional status of the leaf, or the inability of the larvae to cross tiv lateral vein. Small leaves were often completely mined and competition for food also occurred when larval density per leaf was high. Even under these conditions there was no evidence of mortality within the mine, but the restricted food supply may have affected survival of the prepupae or resulted in the production of smaller adults.

(e)Selection of cocooning site

On emergence from the mine the mature larva falls to the ground without the aid of a silken strand.

Observations on larvae dropping from a bunch of detached leaves onto a stabilized bare soil provided no evidence of active burrowing, instead they wandered over the surface, and entry occurred only where natural cracks or worm casts were present. In contrast, larvae dropping on to the loose surface of recently cultivated soil entered immediately. The behaviour of larvae in search of a cocooning site was studied by releasing specimens on an area of soil which had been kept bare for at least two years Table 6. Variation in the area mined by a single larvae of F. dohrnii in the field

Number of S.L. of the Area (cm2) Mean (cm2) mines mean

16o o.87-4.19 2.19 ± 0.06

c~i 4 E • . U .

• ZW3 . • • •••.• ; • •...... • ..y• • ••. _ . • • • 2 .~.• ~..;•r •• •...... 1... • 3.... a . 0_ ...r• •••• . • • ••~ •• • •• • r • • • • : • .• . • . • Q w1 • •• CC • Q

0 0 20 40 60 80 100 AREA OF LEAF [cm21

FIGURE 13

Relationship between area of the mine and the total leaf area. 47

by the use of herbicides. As a result the surface was

stabilized and in this respect it resembled the soil

beneath the alder windbreak on which field studies were

undertaken. By means of a grid placed above the surface the positionsof larvae were plotted at 2 min intervals

(Figures 14, 15). There was no evidence of orientation.

In general, movement continued in the same direction until an irregularity in the surface had to be negotiated, when the larva either changed direction or, if it attempted to climb at a steep angle it invariably fell back and

then set off in the direction in which it was facing.

There was no evidence that the larva could recognise

suitable sites until these occurred in its path, as the larvae often passed within 3-5 mm of cracks. The data presented in Table 7 show a progressive decline, during the period October 21-27, 1977, in

the distance travelled by larvae in search of a cocooning

site. Although detailed records were not taken it is

suggested that this is correlated with minor changes of the soil surface. About 10 mm rain fell shortly before the experiment on 21st October and this could have resulted in many small cracks being obliterated by soil expansion or by movement of particles on the surface.

There was no more rain until after the 27th October and, as the soil dried, it is probable that an increasing number of cracks appeared, thus increasing the chance of one being discovered by a wandering larva. Larvae averaged about 1.2 cm/min, but for those which travelled longer distances (Table 8) the speed tended to decline with increasing distance, suggesting an effect due to 48

[cm)

10 20 30 40 50 60 70 80 90 Icml

Icml

FIGURE 14

Paths followed by mature sixth instar larvae on bare stabilized soil before either cocooning or leaving the observational area of 100 x 100 cm on a) October 21st; b) October 22nd.

A-H = Release positions of the larvae; ❑ = Positions of cocooning. 49

[cm]

40 50 60 [cm]

Ic m]

FIGURE 15

Paths followed by mature sixth instar larvae on bare stabilized soil before cocooning in two experiments on 27th October. Observational area = 100 x 100 cm; J-U = release positions of larvae; positions of cocooning. 50

Table 7. Distance travelled on the soil surface and speed of 18 larvae

Date Larva Total Total Average speed distance time cm/min (cm) (min) 21 Oct. A 68 40 1.7 D 103 60 1.7 C 194 125 1.6 B 282 168 1.7

22 Oct. F 57 27 2.1 G 58 47 1.2 E 77 29 2.7 H 84 39 2.2

27 Oct. L 6 8 0.8 M 13 20 0.7 J 37 21 1.8 K 44 54 0.8

27 Oct. R 2 2 - S 6 6 1.o P 9 5 1.8 Q 11 8 1.4 T 28 28 1.0 u 34 20 1.7

Table 8. The speed (cm/min) of larvae in relation to distance travelled on the soil surface

Distance (cm) 0 51 101 151 201 251 Larva to to to to to to 50 100 150 200 250 300

A 2.4 1.0 B 1.5 1.9 C 2.1 1.9 1.5 1.1 D 3.1 2.1 1.7 1.5 1.3 1.2 E 3.3 2.6 F 2.1 G 1.2 II 2.1 2.3 J 1.8 K 0.8 L 0.8 M 0.7 P 1.8 Q 1.4 R - S 1.0 T 1.0 U 1.7 52

fatigue.

(f) Construction of cocoon

Mature larvae introduced into loose soil in

petri dishes often cocooned against the glass surface.

Observations on such a larva showed that it wriggled at

the end of its tunnel simultaneously smearing a

mucilaginous secretion from its mouth over particles of

soil and organic debris, cementing them together to form

a cocoon with an externally uneven surface (Figure 16).

Examination of cocoons under the microscope did not

reveal any fibrillar structures, whereas larvae of

parasites on completion of feeding on the host spin a

fibrillar cocoon within the host cocoon. The cocoons

of Fenusa dohrnii are generally formed singly but

occasionally as many as 5 to 6 may be found adhered

together even in the field.

The larva at this stage becomes quiescent,

shrinks in length and appears hump shaped due to its

head being tucked against the ventral surface of the

body. At this stage it has reached the pre-pupal stage.

(g) Depth of cocoon

Samples with an area of 900 cm2 were removed

at horizons of 2.5 cm from 10 sites beneath an alder

windbreak where the soil had been undisturbed since

planting, ten years previously, and cocoons were

recovered by washing through a sieve of 3 mm mesh.

About 90 percent of occupied and empty cocoons occurred in the top 5 cm and occasional empty ones below 7.6 cm (Table 9) . 53

FIGURE 16 a) Single and b) aggregated cocoons of Fenusa dohrnii showing the uneven surfaces.

Table 9. The number of cocoons at various depths beneath alder trees

Depth New cocoons Empty cocoons cm Mean - S .d. Mean - S .d.

0-2.5 1.3 1.25 123.5 124.12 2.6-5.0 1.2 0.92 50.8 85.70 5.1-7.5 0.3 0.48 8.7 7.75 7.6-10.0 0 0.8 1.14 10.1-12.5 0 1.5 3.10 55

C. The prepupa and pupa.

The prepupa forms the overwintering stage

which is spent within the cocoon. In the field in 1976 (Table 10) a small percentage had transformed to

the pupal stage by early April. The freshly formed

pupa is whitish and exarate. As development proceeds the colour gradually changes to brownish grey and finally to black.

D. The adult

Adults of Fenusa dohrnii are black with yellowish brown tibiae and measure about 2.5 mm in

length. They were not observed to utilize any water droplets or nectar under field conditions but fed on sugar solutions under laboratory conditions. (a)Emergence

Adults emerge through a circular hole cut by the mandibles at one end of the cocoon, and rest on the soil surface until the wings have expanded and the

cuticle has hardened. In the laboratory a resting period of about one hour was observed before further activity occurred. Field results (Figure 17) show that two generations occur each year with adults emerging in clay-June and in July-August. (b)Sex ratio

Benson (1952) states that the male is unknown and Van Frankenhuyzen (1970) obtained only females.

During this investigation in which approximately 1500 adults were reared, only one male was obtained, which suggests that this species is normally parthenogenetic, 56

Table 10. Percent cocoons with prepupae and pupae,

1975 - 76

Prepupae Pupae

November 100 0

January 100 0

Early April 83 17

40-

20 -

1 rl~ 1 1- I II r 7 1 16 31 15 30 15 30 14 29 May June July Aug.

FIGURE 17

Numbers of F. dohrnii in emergence traps at three-day intervals, 1976. 58

representing an example of obligate thelytoky. (c) Relationship between size of mature larvae and adults The feeding sites of the larvae are pre - determined by the position of the eggs, and therefore competition between larvae for food at high densities of larvae per leaf is inevitable. As the mines coalesce at high densities it is not possible to associate a larva with a particular mine, making a study of the effects of competition between larvae difficult. The weights of mature larvae ranged from about 3 to 7.8 mg with a mean of 5.5mg and the weight of adults that emerged from these

showed a linear relationship (r = 0.75; P<0.001; b±SE = 0.341-0.06) (Figure 18). The number of eggs obtained by dissection of adult females within 24 h of emergence also showed a linear relationship with weight of the adult females (r = 0.78; P.40.001; btSE = 23.17 ±4.19) (Figure 19a) and with the weight of the mature larvae (r = 0.72; PAC 0.001; b±SE = 9.95 ±2.17) (Figure 19b). The variation in size of mature larvae, adults and fecundity of adults

suggests that scramble competition may be taking place

(Hassell, 1976) . 59

4 E I- J • °Q3 • • w • • 0

• w WA = 0.34WL +0-67 r =0.75

3 4 5 6 7 WEIGHT OF MATURE LARVA [mg]

FIGURE 18

Relationship between weight of adult (WA) and the weight of the mature sixth instar larva (WL). 60

100 a • 0 80 cD w • • •~• ō 60 • •I•~•I • • • • w 40 • • • cc z 20 E=23.17W-6.86 r =0.78 0 2.0 25 3.0 3.5 WEIGHT mg] 100 b •

0 80 • w • ō 60 • • •~•• • • w 40 •• m • .00• E=9.95W-3.31 20 z r=0-72 0 3 4 5 6 7 WEIGHT [mg]

FIGURE 19

Relationship between number of eggs (E) and a) weight of adult (W); b) weight of mature sixth instar larva (W1). 61

IV. DIAPAUSE

Larvae collected from the field in October, 1975, were allowed to cocoon in sieved sterilized soil and placed in an open north facing shelter until 4th November when random samples of 25 cocoons were placed in plastic cups (140 ml) containing moist sterilized soil and sealed with lids. Twenty-four samples were each transferred to constant temperatures of -1.1, 3.3 or 10°C and a similar number remained in the open shelter exposed to field temperatures. At intervals of approximately 30 days, four samples from each treatment were transferred to 21°C. Collecting tubes were attached to the lids (Figure 1) and, after emergence had ended, the intact cocoons were dissected to record the contents. All these cocoons contained dead prepupae or evidence of parasitism. Those in which a parasite larva was present within the prepupa, or a silken cocoon occurred within that of the host, were classified as parasitized, but it was not possible to distinguish between the immature stages of Lathrolestes pictilis and Ichneutes laevis. The proportion of cocoons with parasites (Table 11) was approximately the same for each treatment.

A. F. dohrnii (a) Effect of period of chilling on emergence Figure 20a shows that the same pattern occurred with all treatments. Emergence increased from less than 40 percent after chilling for 30 days to 60-85 percent after 62 days. A further small increase in the numbers 62

Table 11. Percentage of cocoons occupied by F. dohrnii

and by parasites

Chilling Days in Cocoons occupied Cocoons occupied temperature storage by F. dohrnii by parasites C

30 70 30 62 63 37 -1.1 92 69 31 120 66 34 152 68 32 182 67 33 Mean 67.2 32.8 3o 68 32 62 73 27 92 65 35 3.3 120 61 39 152 65 35 182 61 39 Mean 65.5 34.5 30 67 33 62 68 32 92 67 33 10 120 67 33 152 72 28 182 66 34 Mean 67.8 32.2 30 62 38 62 65 35 92 67 33 Field 120 70 30 152 64 36 182 6o 4o Mean 67.7 35.3

a 100 • FIELD /•._.._•■111111111.1115 ~•

• •4_3.3•C • .....•,...... • ...... ,.• ~~....~~~.••.••••••~t 70 • o • 1 i C w 40 • zw CD w 10 w

~~•...... m :Pie:

Iwo • ~•, . • • ..... ,_T .- . :~~~~ ~~..„,„,,,,„„....n.%

30 0.0 \ • Iwo r "__J1• 0 30 62 92 120 152 182 PERIOD OF CHILLING [days

FIGURE 20

Percent emergence at 21°C in relation to period of chilling. (a) F. dohrnii (b) Parasites. 64

emerging occurred when the chilling period was extended

to 152 days. This indicated that for the majority of individuals diapause was terminated after chilling for about 62 days at 10°C.

The highest percentage emergence was from cocoons exposed to field temperatures and progressively fewer

emerged as the chilling temperature was lowered from 10°C to -1.1°C suggesting that the lower temperatures had an

adverse effect on survival. There was no evidence that increased periods of chilling, up to 182 days, affected mortality.

(b) Effect of chilling temperatures on rate of development

In all treatments the mean time for adults to

emerge after transfer to 21°C (Figure 21a) decreased with

increase in duration of chilling. As the variances differed significantly it was not possible to test the means for any significant differences but comparison of

the standard errors of the means (Figure 22) suggests differences between samples chilled at various temperatures.

The relatively low value of the mean time to emergence

obtained for samples chilled at 10°C and stored for 182 days (Figure 21a) may be due to a higher rate of morphogenesis occurring at this temperature.

B. Parasites

(a) Effect of period of chilling on emergence

Due to the inability to recognise the larval stages of individual species it has been necessary to present data which represents the combined totals for both species. If each reacted similarly to the treatments 65

50 :•. ■

••i -a 30 3~3~ ••.. •4 4—FIELD w 1-1t w 4-10t CD I 10 w

- b

ā LI 40 .~~ 3t

'~—FIELD 20 \~-10°C 30 62 92 120 152 182 PERIOD OF CHILLING [ days]

FIGURE 21

Mean time to emergence of adults after transfer to 21°C in relation to period of chilling. (a) F. dohrnii (b) Parasites.

E 60 C

B ~ MERGEN

E D C A I ABJ A T40 II B C D jli B

I C A I I B D A I C 20

30 62 92 120 152 182 PERIOD OF CHILLING (days]

FIGURE 22

Variation of S.E. of mean time to emergence of adults of F. dohrnii after transfer to 21°C in relation to period of chilling. A = -1.1°C; B = 3.3°C; C = 10°C; D = Field; S.E. of mean =} 67

it is clear from Table 12 that larvae of I. laevis and

L. pictilis occurred in the ratio of approximately 30:70.

At constant temperatures (Figure 20b) an increase in chilling period from 30 to 62 days resulted in the emergence of more adults, but the increase was less than for F. dohrnii. There was also an indication that chilling for periods in excess of 120 days at 3.3 and -1.1°C had an adverse effect on survival. The data for field temperatures is less consistent but follows the same general pattern. (b) Effect of chilling temperature on rate of development

The mean time to emergence after transfer to

21°C (Figure 2lb) decreased progressively as the period of chilling was increased, and followed a pattern which was almost identical to that noted for the host. The mean time for emergence of samples chilled for 182 days at 10°C was significantly lower than for other treatments

(Table 13). This effect was also observed with the host, and is probably due to a higher rate of morpho - genesis during the later part of the chilling period.

C. Phenology of emergence Because diapause was terminated after a short period of chilling (62 days) at a relatively high temperature (10°C) the prepupa of F. dohrnii can be described as having a "weak" diapause. The same may be said of the parasites. However, under natural conditions the prepupae were present in cocoons from November to about April the following year at which time metamorphosis to the pupa occurred (Table 10). Although the threshold 68

Table 12. Numbers of Ichneutis laevis and Lathrolestes pictilis emerging in relation to period of chilling

Chilling Chilling temperature period I. laevis L. pictilis oC (days) 30 4 7 62 5 13 -1.1 92 4 8 120 5 9 152 2 3 182 3 5 Total 23 45 3o 6 10 62 5 12 92 6 15 3.3 120 5 16 152 3 6 182 3 8 Total 28 67 30 4 9 62 5 13 92 4 13 10 120 3 14 152 3 10 182 4 11 Total 23 70 30 7 16 62 4 14 92 4 15 Field 120 5 15 152 4 10 182 4 13 Total 28 83

Table 13. Variation of mean time to emergence of parasites from samples chilled for 182 days

Temperature ( °c) Mean time to emerge (days)

10 20 3.3 28 1.1 28.12

Field 28.19

* Bar indicating non-significance at 5% level 70

temperatures for development are not known, the field temperatures at 5 cm below the soil surface (Table 14) during this period were low and would explain the prolonged dormancy of the prepupa under natural conditions. The decrease in the mean time of emergence with increase in the duration of chilling (Figure 21a, 21b) shows that diapause is not abruptly terminated after a given period of chilling and it may also be inferred that timing of emergence is not dependent simply on a constant heat sum for morphogenesis. This effect, which has been described as a "brake" on the rate of development (Cranham, 1972), would act to prevent premature emergence if the weather became unusually warm in early spring before foliage was present on the trees; similarly if cold weather was unusually prolonged emergence could occur faster when it did turn warm. 71

Table 14. Mean temperatures (°C) at 5cm below soil surface, East Malling,1975 - 76

Month °C Month °C October 9.4 February 3.0 November 4.5 March 4.0 December 2.7 April 9.8 January 3.8 May 16.5 72

V. NATURAL ENEMIES

A. Parasites

A brief comment by Van Frankenhuyzen (1970) of undetermined species is the only reference to parasites of Fenusa dohrnii.

In the present study four species of Hymenoptera were recorded (Table 15); all attacked the larva and development of two continued within the host prepupa. The immature stages of individual species could not be identified, but it was possible to distinguish larvae of the two eulophid species from the others. The comments below thus refer to the composite life cycles of each pair of species. (a) Cirrospilus vittatus and Chrysocharis nitetis

The eggs of these minute species were not observed The larva of C. vittatus is ectoparasitic and that of

C. nitetis is endoparasitic. Both species are poly- phagous and have been recorded from many species of leaf miners (Boucek and Askew, 1968; Gruys, 1975). Both generations of the host were attacked by each species which pupated within the mine without forming a cocoon. The presence of empty pupal skins midway through the host generation suggested that one or both species may have more than two generations per year. Larvae and pupae of these species were most frequently found associated with L.I or L.II of the host

(Table 16); none occurred with L.VI. The presence of parasite larvae attacking L.I throughout the sampling period indicated that oviposition occurred over a long 73

Table 15. Parasites of Fenusa dohrnii and their sites of pupation

Pupation Species Type site

Cirrospilus vittatus (Wlk.) (Eulophidae) Ectoparasite Mine

Chrysocaris nitetis (Wlk.) (Eulophidae) Endoparasite Mine Lath r olestes pictilis Hol.(Ichneumonidae) Endoparasite Cocoon

Ichneutes laevis (Wesm.) (Braconidae) Endoparasite Cocoon 74

Table 16. Numbers of larvae and pupae of Cirrospilus vittatus and Chrysocharis nitetis found associated with each

larval instar of F. dohrnii, 1977

Instar of host larva I II III IV V Date L P L P L P L P L P 18 Aug. 2 0 0 0 ------22 Aug. 2 0 0 0 0 1 - - - - 29 Aug. 1 0 0 0 0 0 - - - - 5 Sept. 4 0 0 3 0 0 0 0 - - 12 Sept. 2 3 2 0 0 0 0 0 0 0 19 Sept. 4 0 0 0 0 2 X 0 0 0 26 Sept. 1 2 2 6 0 1 0 1 0 0 3 Oct. 0 0 1 3 0 1 0 0 0 0 10 Oct. 1 2 0 0 1 1 0 1 0 1 18 Oct. 0 0 0 2 0 2 0 1 0 0 24 Oct. 2 4 0 0 1 0 0 1 0 0 Total 19 11 5 14 2 8 1 4 0 1

L = larva; P = pupa or pupal skin 75

Table 17. Numbers of eggs and larvae of

Lathrolestes pictilis and Ichneutes

laevis found associated with each

larval instar of F. dohrnii, 1977

Instar of host larva I II III IV V VI Date E E E L E L E L E L

18 Aug. 10 5 6 2 - 22 Aug. 13 16 5 1 0 2 29 Aug. 20 10 20 4 1 3 5 Sept. 20 5 10 1 1 6 1 3 12 Sept. 6 20 13 2 4 3 4 1 2 0 19 Sept. 8 3 5 1 4 2 2 0 1 0 26 Sept . 10 5 2 0 1 1 2 2 0 1 3 Oct. 0 1 1 1 1 2 1 2 0 2 10 Oct. 0 0 0 0 0 0 0 1 0 0 18 Oct. 1 0 4 0 1 0 0 1 0 1 24 Oct. 0 0 0 0 0 0 0 0 0 0 Total 88 65 66 12 13 9 10 10 3 4

E = egg; L = larva 76

period, and the occurrence of parasite pupae in mines containing only L.I suggested that larval development can be completed on a single instar of the host. Eveleens and Evenhuis (1968) reported that C. vittatus paralyses the larva of Stigmella malella prior to oviposition, so it is possible that the pupae associated with L.I of

F. dohrnii were of this species. Adults of both species varied considerably in size; it is possible that this is related to the amount of food available, the larger specimens arising from larvae which attacked older instars of the host. (b) Lathrolestes pictilis and Ichneutes laevis Comparison of the numbers listed in Tables 16 and 17 shows that these species occurred more commonly than the previous ones, and of the two (Table 18) L. pictilis was approximately twice as numerous as I. laevis. Both generations of F. dohrnii were attacked. Records, which are available only for generation II, 1977, suggest a similarity in periods of emergence (Figure 23), the shorter period illustrated for I. laevis possibly being due to smaller numbers. Eggs were usually found in the abdominal cavity and were present in all instars of the host larva (Table 17), being especially numerous in L.I - L.III; larvae were found in L.III - L.VI.

Although multiple parasitism was observed occasionally only one larva developed within a single host. Sometimes none of the parasite larvae were successful and dead eggs and larvae were found chitinized within the body cavity of the adults of F. dohrnii. 77

Table 18. The relative abundance of Lathrolestes pictilis and Ichneutes laevis

Diapause experiments, Field emergence, 1975 1977

Number Percentage Number Percentage

L. pictilis 265 72.2 28 63.6 I. laevis 102 27.8 16 36.4 78

L. pictilis 6-

4 I .laevis

0 .....L ...... I..I.....I , * 16 31 15 30 Aug. Sept.

FIGURE 23

Numbers of L. pictilis and I. laevis in emergence traps, 1977. 79

Development of these species of parasites continues within the prepupal stage of the host. On completion of feeding the parasite larva emerges through the prepupal skin and constructs a fibrillar cocoon within the host cocoon. Data from soil samples (Table 19) show that over 80 percent of parasites had made fibrillar cocoons by November, but a small proportion of larvae remained in the host prepupae until April. None had pupated when samples were taken in April. The data on diapause has been discussed in the previous section.

B. Predators

(a)Field observations The presence of empty mines with the epidermis torn, and of dead larvae of all ages within intact mines was suggestive of predation; the former by birds or mandibulate insects, and the latter by insects with stylet- type mouth-parts. Positive evidence was obtained only on one occasion, when an adult of the mirid,Ulepharidopterous angulatus (Fall.), was observed attacking a larva, and its stylets were not withdrawn until one hour after the initial sighting.

(b)Laboratory experiments A survey of the experimental area showed that three potential predators occurred regularly in samples obtained by tapping branches. These were Anthocoris nemoralis (F.), B. angulatus and Forficula auricularia L.

Repeated tests in which 5-8 specimens of a species were confined for 4-5 days in cages containing infested leaf 80

Table 19. Percent larvae of I. laevis and L. pictilis in host or in the fibrillar cocoons.

In host prepupa In fibrillar cocoon

November,1975 16.4 83.6 January,1,976 22.4 77.6 April,1976 4.7 95.3 81 clusters did not produce evidence that these species would prey on larvae of F. dohrnii in leaf mines. However, larvae extracted from mines were readily attacked by each species. Further tests were made with the electrophoresis method (Murray and Solomon, 1978). This involved comparison of esterases from predators and their prey obtained by electrophoretic separation on polyacrylamide slab gel. As shown in Figures 24A and 24B diagnostic bands for the larva of F. dohrnii (a) and the alder aphid

(Pterocallis alni)(b) were readily identified when

Anthocoris nemoralis was tested. Tests on 60 individuals from the experimental plot failed to produce any evidence of predation on larvae of F. dohrnii, but established that the main source of prey was the aphid, Pterocallis alni. Similar results were obtained with B. angulatus and

F. auricularia. 82

a b

1 2 3

2 3

FIGURE 24

Electrophoretic slab gels showing A) anthocorid starved for 36 h (1); anthocorid starved and then fed on larva of F. dohrnii (2); larva of F. dohrnii (3); 13) anthocorid starved for 36 h (1); anthocorid starved and then fed on alder aphid (2); alder aphid (3). a = characteristic esterase band for F. dohrnii b = characteristic esterase band for alder aphid 83

VI. DISTRIBUTION OF EGGS

A. Type of distribution The local distribution of a species is to a large

extent governed by environmental and congenital factors such as adult behaviour, feeding habits, age structure of the population and interactions between them. A knowledge of the spatial disposition of the insect can help

to identify hetergeneities in the habitat and aid the planning of a sample programme. To investigate the distribution of eggs of the second generation of F. dohrnii laid in the leaves of Alnus glutinosa three randomly selected trees each about 3 m high were divided into quadrants based on the cardinal points of a compass and from each quadrant 75 leaves were selected at random at heights of 0-1 m (H1), 1-2 m (H2)

and 2-3 m (H3 ). Frequency tables for the number of eggs per leaf were compiled for each of the 36 quadrants. Four known theoretical frequency distributions i.e. Poisson (Hoel, 1965), Negative binomial (Anscombe, 1950), Neyman Type A (Neyman, 1939) and Logarithmic series (Pielou, 1977) were fitted to the observed frequencies and various parameters estimated using the Rothamsted MLP

computer programme. Agreement between the observed and expected frequencies were tested by Chi-square (Table 20).

That the Poisson distribution does not describe the distribution of eggs (Table 20) is not surprising as it

would require 1) all leaves to be equally attractive and accessible to the adults and 2) for, the process determining the laying of each egg to be unaffected in any way by the Table 20. The significance levels between observed and expected frequencies for four theoretical distributions based on Chi-squared test,and estimates of mean density, negative binomial parameter (k), total number of individuals in

an aggregate (X) for each of the 36 quadrants

Mean Quadrant number Negative of eggs binomial and Negative Logarithmic Neyman Tree per leaf parameter Poisson number height and ± S.E. (k) and binomial series type A

I QAH1 0.8810.17 0.25810.06 4.7 *** NS * NS QBH1 0.5210.11 0.23610.07 7.4 *** NS NS NS QCH1 0.7910.14 0.30310.07 9.6 NS NS NS QDH1 0.4910.11 0.19310.06 8.7 NS NS NS QAH2 0.3310.09 0.14610.05 7.3 ** NS NS NS QBH2 O.36±0.09 0.172-0.06 7.o *** NS NS NS QCH2 0.65±0.12 0.26210.07 9.1 NS NS NS QDH2 0.2910.08 0.21310.09 4.7 * NS NS NS QAH3 1.25±0.22 0.27610.06 16.5 *** NS NS NS QBH3 1.7610.29 0.27410.05 23.0 *** NS NS ** QCH3 0.9610.19 0.20810.05 15.9 *** NS NS NS QDH3 1.85±0.30 0.357±0.07 19.6 * * * NS * * * * Table 20 (cont.) Mean Quadrant number Negative of eggs binomial Negative Logarithmic Neyman Tree and per leaf parameter Poisson binomial series A number height and ± S.E. (k) and ±S.E. type

II QAH1 0.7510.13 0.29610.07 9.3 *** NS NS NS QBH1 0.6910.15 0.17310.04 13.3 *** NS NS ** QCH1 1.0910.22 0.18710.04 20.1 *** NS NS NS QDH1 0.3910.10 0.14910.05 8.7 *** NS NS NS QAH2 0.2910.07 0.2392'0.09 4.3 ** NS NS NS QBH2 0.7310.11 0.489:0.144 6.o ** NS NS NS QCH2 1.0710.19 0.26610.06 14.4 *** NS NS NS QDH2 0.51±0.15 0.23810.09 4.0 *** NS NS NS QAH3 2.5710.45 0.28110.05 32.4 *** NS NS **

QBH3 3.7910.73 0.22510.04 59.7 *** NS NS QCH3 1.1510.21 0.273±0.06 15.3 *** NS NS ** QDH3 0.79±0.17 0.199±0.05 13.4 * * * NS NS NS III QAH1 0.3710.07 0.53410.21 2.8 NS NS NS NS QBH1 0.37±0.08 0.201±0.07 6.4 *** NS NS NS QCH1 0.32±0.07 0.26110.09 4.4 ** NS NS NS QDH1 0.19±0.05 0.18110.09 4.4 * NS NS NS

Table 20 (cont.)

: iean :tuadrant number Negative of eggs binomial Negative Logarithmic Neyman and Tree per leaf parameter Poisson binomial series type A number height and - S.E. (k) and ±S.E.

0.133±0.04 NS NS NS III QAH2 0.24±0.07 7.0 QBH2 0.24±0.05 0.449±0.24 2.1 NS NS NS QCH2 0.40±0.09 0.168±0.05 7.8 NS NS NS ** NS QDH2 0.19±0.05 0.188±0.09 3.4 NS NS 0.35±0.07 0.347±0.13 3.8 * NS NS NS QAH3 QBH 0.043±0.02 * * * NS NS NS 3 o.19±0.11 13.9 QCH3 0.36±0.06 1.239±0.87 1.6 I#S NS NS NS QDH3 0.23±0.04 1.378±1.43 1.0 NS NS NS NS

Q = Quadrant Suffix A, B, C, D = NW, SW, SE and NE quadrants H = Height Suffix 1, 2 and 3 = Heights (0-1 m), (1-2 m) and (2-3 m) NS = not significant at 5% 87

presence or behaviour of other individuals, so that the

probability of obtaining an egg on a given leaf would be

the same for all leaves. The oviposition behaviour of

the adults of the second generation makes this a rare

event. However, as the density of eggs per leaf in some

of the quadrants was very low (Table 20) the distribution

of eggs tended towards randomness (Figure 25),a feature

noted for many insects at low densities (e.g. Robles, 1969).

The observed frequencies are to a varying degree

in agreement with those predicted by the negative binomial,

Neyman Type A, and logarithmic distributions (Table 20)

and the distribution of eggs is best described by the

negative binomial as in many other insects (e.g. Anscombe,

1949; 1950; Harcourt, 1960; Lyons, 1964). The variation

in the mean and k, the two characteristic parameters of the

distribution is shown in Table 20. The reciprocal of the

negative binomial parameter k showed no relationship with

the mean (Figure 26) suggesting that the population as a

whole could be represented by a common negative binomial

parameter (Elliott, 1977). The approximate value of this,

calculated using the method described by Bliss and Owen

(1958) is 0.2565. The stability of the common value of

the negative binomial parameter k obtained (Figure 27)

implies that the degree of contagiousness in the population

was relatively constant over all quadrant-height combinations

and over all densities considered.

When k of the negative binomial approaches zero the distribution could be adequately described by the

logarithmic distribution (Southwood, 1966) and as the values of k obtained in the present study were fractional

60 8 0~ o O 00 0000 0 Q 50 00 0 O O 40 0 ōc a w N30 U w 0 u- 20 X w 10

0 10 20 30 40 50 60 70 OBSERVED FREQUENCIES FOR ZERO VARIATE

FIGURE 25

Relationship between observed and expected frequencies for leaves containing no eggs (zero variate). Negative binomial distribution (•); Poisson distribution (0). 8 9

10 • 8 r=-017 So 6 -% N 1 •• • • • • K • 4 • • 1 h 2 •• • •• 0 0 1 2 3 4 MEAN

FIGURE 26

Relation of k to mean for 36 samples from a population of

Fenusa dohrnii eggs.

28 Ma

24 •

20 OP

yi 16 Om

8 •

2 4 6 I X

FIGURE 27 2 variance Relationship between Y/ and X / where X/ = mean Number of sampling units

and Y" = variance - mean. 91 in most cases, the reasonable agreement shown between the observed and expected frequencies predicted by the logarithmic distribution is not surprising.

Neyman Type A could be regarded as a generalised distribution whose probability generating function

/(Z-1) -1 H(Z) = e~e e.g. Pielou, 1977

where the number of clusters and number of individuals per cluster are both randomly distributed with means A and)W respectively. It is therefore possible to imagine the eggs of F. dohrnii to be randomly distributed in batches and the number of eggs to have a random distribution within each batch which seemed applicable to only some of the quadrants (Table 20). Pielou (1977) shows the derivation of the Neyman

Type A by considering both individuals within a cluster, and clusters to have a Poisson distribtuion with different parameters and also shows how this would give rise to a negative binomial distribution, if the individuals within a cluster were considered to have a logarithmic distribution. Similarly, Anscombe (1950) describes several models from which the negative binomial distribution could be obtained. Natural insect distributions have also been described by many theoretical frequency distributions (Beall, 1940; McGuire et. al, 1957). Therefore,fitting of theoretical frequency distributions is never by itself sufficient to explain the pattern of the natural populations, but the suitability of the three contagious models considered 92

here emphasises the degree of aggregation in the population of F. dohrnii eggs.

B. Type of aggregation

Aggregation, recognised by the negative binomial, may be due to either active aggregation by the insects or due to some environmental factor, and Southwood (1966) describes a method by which this may be distinguished by calculating the total number of individuals in the aggregate

(A). In the present study the values of A that were obtained for a majority of quadrant/height combinations were greater than two (Table 20),and therefore clumping of eggs may be due either to active aggregation by the females or to the heterogeneity of the environment

(Southwood, 1966).

• C. Indices of aggregation

Elliott(1977) gives a summary of the many indices which have been proposed to calculate the degree of non- randomness in a population, and also their values of maximum regularity, randomness and maximum contagion. These values show that at maximum contagion most of the indices are affected by the mean, the number of sampling units, and the total number of individuals in the sample, and

therefore the applicability of most of the indices to measure the aggregation is limited to when these remain

constant for all samples, which is rare in nature.

However, the behaviour of some of the more

commonly used indices, 1) variance/mean ratio for agreement with the Poisson series (see Elliott, 1977); 2) index of 93 mean crowding to mean density (Lloyd, 1967); 3) exponent b of Taylor's power law (Taylor, 1961; 1971); 4) regression of mean crowding on mean density (Iwao, 1968), were examined. (a)Variance to mean ratio

It is important to distinguish between the use of the variance to mean ratio ( M ) as a test of non-randomness and its use as a relative measure of contagion. Whilst being a good statistical test for departure from randomness

(i.e. Poisson series) the R ratio is a poor measure of the degree of patchiness in a population since it is greatly influenced by the number of individuals sampled (Elliot,

1977). Chi-squared values for R ratio shown in Table 21 indicate significant contagion (P 95.93, PL 0.01, x2y1o5.59, P

However, individual AT ratios are of little value as a measure of relative contagion since R increases with mean (Figure 28). The linear relationship between V M and mean, the slope of which differed significantly from 0 (Table 22),is by itself a measure of contagion since in a Poisson series R approximates unity independent of the mean. (b)Ratio of mean crowding to mean density

The constant k or k of the negative binomial index has been used as a relative measure of contagion for a wide range of species and sampling conditions (e.g.

Waters, 1959). It is also related to Lloyd's measure of patchiness ( mean crowding, M* ) in the following mean, M

Table 21. Mean density, x2 for 01, MeanMeanw(M) (Mx) x and level of significance of M > 1 of egg

counts for 36 quadrant-height combinations.

TREE I

Mean 2 Mx Sig: diff: of density X M RMx>1 (t S.E.) (± S.E.)

QAH1 0. 8810. 17 250.8*** 4.8810.94 * * * QBH1 0.52-0.11 224.9*** 5.2411.20 * * * QCH1 0.79-0.14 250.1*** 4. 3010. 79 * * * DH1 0.4910.11 206.5*** 6.1811.51 ** * QAH2 0.3310.09 241.9*** 7.8512.28 ** QBH2 0.36±0.09 179.1*** 6.8111.95 * QCH2 0.65-0.12 235.3*** 4.8210.99 * * * QDH2 0.2910.08 229.4*** 5.6911.98 * QAH3 1.2510.22 301.9*** 4.6210.75 * * * QBH3 1.7610.29 415.8*** 4.6510.67 * * * RCH3 0.96-0.19 352.2*** 5.8111.06 ** QDH3 1.85-0.30 509.8*** 3.8010.53 * * *

TREE II

QAH1 0.7510.13 219.0*** 4.38+0.84 *** QBH1 0.6910.15 377.4*** 6.78*1.42 * * * QCH1 1.0910.22 428.4*** 6.35±1.17 * * * QDH1 0.3910.10 316.7*** 7.77±2.21 ** QAH2 0.2910.07 148.0*** 5.18±1.61 * * QBH2 0.73±0.11 190.1*** 3.04±0.57 * * * QCH2 1.0710.19 307.1*** 4.76±0.80 * * * QDH2 0.51+0.15 376.6*** 5.20±1.62 ** 717.0*** * * * QAH3 2.57±0.45 4.56±0.61 1184.0*** QBH3 3.79±0.73 5.44±0.73 QCH3 1.15-0.21 412.9*** 4.66±0.77 * QDH3 0.79-0.17 361.8*** 6.03±1.25 95

Table 21 (cont.)

TREE III

Mean '~/ 2 Mx Sig: Jiff: of density /L M M_ (- S.E.) (± S.E.) N 7 1

QAH1 0.3710.07 142.0*** 2.87±0.75 QBH1 0.37±0.08 164.2*** 5.96±1.65 QCH1 0.32-0.07 143.5*** 4.83±1.44 QDH1 0.19±0.05 1324*** 6.52±2.63 QAH2 O.2410.07 187.9*** 9.85±3.45 QBH2 O.24±0.05 105.8** 3.23±1.21 CIī2 0.40±0.09 261.2*** 6.95±1.83 QDH2 O.1910.05 120.6*** 6.32±2.61 (1AH3 0.35±0.07 143.5*** 3.88=1.10 QBH3 0.19±0.11 370.0*** 24.26=12.11 QCx3 0.36±0.06 90.2 NS 1.8110.57 Q03 0.23±0.04 83.6 NS 1.73±0.75

H1 = 0-1 m QA = NW quadrant H2 = 1-2 m QB = SW quadrant H3 = 2-3 m QC = SE quadrant QD = NE quadrant

SE (Lloyd, 1967) " var (k)

)(.2 = m (N-1) (Southwood, 1966) x >1 n = 75; P<0.05; t = 1.67;O.01 P<; t = 2.30; M P<0.001; t = 3.15

16 •

10 V M 8 M 3.62 M +1-29 6 r =0.92 •• • • • •• • 4 • • • ••• •.•i 2 • !i

0 0 1.0 2•0 3.0 4.0 M

FIGURE 28 variance (V) Relationship between and mean (M) for 36 samples mean (M) from a population of F. dohrnii eggs. Random expectation 97

Table 22. Slope coefficient and percent variance

accounted for by regression of M on mean, and N on mean of eggs observed in each of the 36 quadrant-height combinations.

percent Regression Regression Slope variance Coefficient accounted for

Sig. at V M on M 3.42 *** 84.96 0.92 (P < o.o01)

M* on M -0.82 NS 2.86 -0.12 (NS)

*** slope significantly different from zero (P<0.001) 98

manner

M* M 1 + k (Lloyd, 1967)

Standard errors of individual M could only be calculated on the assumption that the counts are a random sample from a negative binomial distribution (Lloyd, 1967), and since random samples of 75 leaves were removed from each of the quadrants, the populations of which were described by the negative binomial (Table 20),the ± S.E. for each of the quadrants are shown in Table 21. Individual estimates of M ranged from 1.73 to 24.26 but most fell between 3.23 and 6.95 . On average, the distribution of eggs were judged to be significantly different from random expectation * ( M 7 1, P< 0.001). Although in some instances contagion, measured by the related statistic k , has been found to increase with the mean density (e.g. Elliot, 1977), in the present study values of 1 or g were uncorrelated with the mean density (Figures 26, 29) and therefore M* values of M derived from low and high densities may be compared. (c) Constants of Taylor's power law Taylor (1961, 1971) found that regression of log10 variance on log10 mean for a series of samples proved consistently linear, and suggested that the slope of the regression line was potentially a characteristic of the given population and habitat,axrl the intercept, a function of the sampling method. The linear regression of log10 mean for the data from the present study showed that a

10 •

8 r = -012 • Mx 6 • _••• • •• • M •• f •11• ••• • • 4 • • • • • • 2 ••

00 10 20 3.0 40 M

FIGURE 29

mean crowding (Mx) Relationship between and mean (M) for mean (M) 36 samples from a population of F. dohrnii eggs. Randon expectation (#). 100 high proportion of the variance was accounted for by regression,and also indicated a significant deviation of the slope and intercept from 1 (random expectation) and 0 respectively (Table 23, Figure 30),thereby confirming contagion in the distribution of the eggs. (d) Regression of mean crowding (M*) on mean (M) Iwao (1968) suggests that for many species the estimate of mean crowding can be linearly related to mean density by M* = 0C+ aM, where the value O( ("Index of Basic Contagion") indicates that at infinitesimal density an individual would be expected to live together with 0( other individuals in the space defined by the sampling unit, and the slope p ("Density-Contagiousness Coefficient") regarded as reflecting the manner in which individuals or groups of individuals distribute themselves in the habitat. The applicability of this method has been tested for many natural populations (e.g. Iwao and Kuno, 1971). In the present study the values of these parameters,obtained by the linear regression of M* on M

(Figure 31), indicate that the slope ! differs significantly from 1 (random expectation) whereas the intercept O( shows no significant difference from zero (Table 23). In terms of the relative magnitudes of p(and p, Iwao (1968) describes many types of dispersion patterns, and it appears that the present data falls into the category where the individuals are distributed randomly within the confines of the sampling unit (leaf)(O( N 0), but at the same time are concentrated in specific areas of the environment 93>1) • Distribution patterns of this nature probably result from the animal's response to gross changes in the Table 23. Regression coefficients (± S.E.) and percent variance

accounted for by regression of log variance on log

mean, and mean crowding on mean for the number of eggs

observed in each of the 36 quadrant-height combinations

regression Intercept Sig. diff. Slope Sig. diff. of percent of intercept slope from 1 variance from zero (random expectatim) accounted

log10 Variance on 0.67±0.03 * * * 1.61±0. 07 * * * 93.03 log10 Mean

Mean crowding on 0.32-0.26 NS 4.41±0.25 ** 90.25 Mean

NS = not significant at 5% 102

•

LOG V=0.67+1.61 LOG M r=0.96

• _„___ • •• .... 80.0000100.0* • •

•1 - 1 1 I 1 1 1 1 •2 .3 .4 -5 •6 -7 .8 •9 1 2 3 4 MEAN (LOG SCALE]

FIGURE 30

Relationship between variance (V) and mean (M) of the number of eggs per leaf, plotted on log scale. Random expectation ("Ay).

103

20 •

15 Mx=0.32+4.41M r=0.95 CDz 0 0 ~ 10

z UJ

i 5 • •

Is • 0 0 1 2 3 MEAN

FIGURE 31

Relationship between Lloyd's measure of mean crowding (Mx) and mean density (M) for eggs of Fenusa dohrnii. Random expectation (or-4Y). 104 environment. If this interpretation of the parameters is valid, the gross change in the environment within the present study may be regarded as the decrease in the availability of young leaves for oviposition during the second generation. However in the absence of similar data for the first generation it is not possible to make comparisons with the second generation studied here. 105

VII. POPULATION DEVELOPMENT OF F. DOHP.NII IN THE FIELD

A. Methods (a)Sampling procedure A portion of the hedge of Alnus glutinosa was divided into six blocks, each containing 12 trees of approximately equal size. The basic sampling unit was a cluster (Figure 32) which carried about five leaves at maximum growth and contained all stages of Fenusa dohrnii up to the sixth instar larva. At weekly intervals two trees were sampled from each block, with four clusters being taken from each quadrant at heights of 0-1, 1-2 and 2-3 m, making a total of 576 clusters. The trees and clusters were both selected at random. As data obtained in 1976 showed only minor variations between quadrants, the sampling method was modified in 1977 and eight clusters were taken from each side, east and west, instead of four clusters per quadrant. (b)Statistical analysis At each sampling occasion the total number of eggs, larvae, dead eggs and dead larvae obtained for

each of the quadrants or sides were tabulated and analysed. The general model used and, as an example, the analysis of

variance of the total egg counts obtained for 3rd Nay,1976,

are shown in Appendix IV.

(c)Estimation of totals for larva VI and adults To prepare life tables it was necessary to calculate numbers of larva VI and adults arising from

a sample of the same size as used for other stages, i.e. 106

FIGURE 32

Early growth of Alnus glutinosa showing individual leaf clusters which are indicated by arrows on the right hand branch. 107 576 clusters. Since each sticky board or emergence cage covered an area of soil representing 25 percent of that covered by a tree canopy, the total number of clusters was counted on six randomly selected trees and the appropriate conversion factor applied.

B. Results (a) Source of variation

Results of the analysis of variance for the two generations in 1976 and for generation II in 1977 indicated that, with minor exceptions, variation between blocks was not significant. They further indicated that variation was largely due to inter-tree differences, to a lesser degree to heights,and still less to quadrants. For generation II, 1977, variation due to heights appeared to be less than that for the sides (Appendix V, VI).

Inter-tree variation has been shown to be a characteristic feature of many forest and orchard insect species (e.g. Morris, 1955; LeRoux and Reimer, 1959; Cheng and LeRoux, 1966a) and of many field crop insect species (e.g. Harcourt,1961). Although the trees used in this study were of fairly uniform size the number of leaves suitable for oviposition may have varied from tree to tree, and the response of the insect to this may account for the observed variation. The inter-tree variation observed in the present study also suggests that for sampling purposes considerable between-tree replication is necessary. As significant variation also occurred between quadrant/sides and heights, but to a lesser degree between trees, it would also be necessary to sample all quadrant/ sides and heights. 108

(b) Integration of populations As the available data was insufficient to apply the methods described by Dempster (1961), Kiritani and Nakasuji (1967), Richards and Waloff (1954), Richards, Waloff and Spradbery (1960) or Southwood and Jepson (1962), an adaption of that described by Cheng and Le Roux (196Gb) was used to estimate numbers entering the egg and each larval stage up to the fifth instar. Totals entering the sixth instar and the adult stage were obtained respectively by converting the totals of larvae caught on sticky boards,

and of adults from emergence cages, as previously described. Thus, for example, the total eggs (Et) per

sampling occasion may be represented by :-

Et = E + L1 + L2 + L3 + L4 + L5 + L6 + T

where E is the number of live and dead eggs, L1 to L6 are the numbers of live and dead larvae in each instar,

and T is the cumulative total of larva VI collected on sticky boards and equated to 576 leaf clusters. However, due to loss of data, numbers of individual larval stages

that were live and dead within the mines were not known. Therefore the total number of larva VI on each sampling occasion was considered to be the cumulative totals of larvae caught on sticky boards and equated to 576 leaf

clusters. This would tend to give an underestimate of the numbers entering larva VI as mortality of this stage within the mine was omitted. As mortality of eggs and all larval stages within the mine was taken into account in calculating the

numbers entering larva I-V the totals on successive 109

sampling occasions should increase to a maximum and remain at this figure when recruitment to a particular stage ceased. Figures 33 and 34 show that the totals for eggs, and to a lesser extent larva I, declined during the latter half of the sampling period. In all cases the totals for the stage was considered to be the maximum figure recorded on the graph. The general trend of populations of individual stages from egg to larva VI for all generations was similar to that obtained for generation II in 1977 (Figure 35). A striking feature was the progressive reduction in the numbers of dead eggs, as is illustrated in this example from early September onwards. This was mainly because the minute scars caused by oviposition became indistinguish- able from other blemishes as the leaves aged. In addition, as surviving larvae extended their mines, the sites of many unhatched eggs were destroyed. The first instar larva is also very small and a similar explanation would account for the reduction in totals observed for this stage in each generation in 1976 (Figure 33). (c) Construction of life tables Leopold (1933) was one of the first to appreciate the possibility of the application of life tables in the field of ecology which he termed life equations. Deevey (1947) illustrated three ways in which life tables may be developed: 1) where age at death is directly observed for a large and reasonably random sample; 2) where survival of a large cohort is followed at fairly close intervals throughout its existence; 3) where the age structure of a population is obtained from a random 110

a • ADULTS 20

0 r r r r r T r r

1200

1000

800

600 0 400 x Q 200 w

z 0

20 C w • _. .. -• ADULTS 10

0 .-./'I r

600 d

400 . EGGS / 200

1/ r.~.._. L VI 0 r r 1 r r T r 1 r r ~, r r 7 i 171 31 14 28 12 26 9 23 6 22 4 MAY JUNE JULY AUG. SEPT. OCT.

FIGURE 33

Numbers of adults, eggs, larva I and VI per 576 clusters. (a) and (b) generation I, 1976; (c) and (d) generation II, 1976. 111

400 w 300

w z 200 EGGS •L.1 °■' vi co 100 ,~r•''s. L.III L.IV ✓ 6 L. V •~~ _____•______-'—. L.VI ‚ 'i- 0 2/. 18 22 29 5 12 19 26 3 10 18 AUG. SEPT. OCT.

FIGURE 34

Numbers of eggs and each larval instar per 576 clusters. Generation II, 1977.

112

EGGS 200

150

• \\ \\• 100

i 111 50 •` I• 111 i • ` • ♦♦ 0

100 •/•` INSTAR. I • \ .••••

_•~• ~~• G 0 r r 1 N I

50 INSTAR II MPL A S

0 1 I I r I 1 T

50 INSTAR III

•._%.• • •.,,~~.•.~,~• ~•~~•~~•-•1 0 I 1 f 1 1 I 1 1 1 1.L W m 50 D INSTAR IV z .\mir...... \,.,_.,,_..

0 I I I

50 INSTAR V

1 1

INSTAR VI 5 •~

01 1 1 r 1 1 I 18 22 29 5 12 19 26 3 10 18 24 AUG• SEPT. OCT.

FIGURE 35

Totals of eggs and each larval instar per 576 clusters on each sampling occasion in generation II, 1977. Live (um); Dead (vi ). 113

sample and the numbers dying inferred from the reduction in numbers observed between successive age intervals.

Morris and Miller (1954) suggested a way in which the second method quoted by Deevey (1947) could be applied to natural insect populations. Since the survival of the same individual cannot be determined, they suggested that survival could be determined by sampling the same population but not the same individuals, and proposed the construction of life tables with the following headings :

x = age interval

lx = numbers living at the beginning of stage

in x column

dx = numbers dying within age intervals in x

column

dxF = mortality factor responsible for dx

100gx = percentage mortality

S = survival value within x x Life tables for both generations in 1976 and for the second generation in 1977,but omitting the dxF cōlumni are• shown in Table 24. In addition to the headings proposed by Morris and Miller (1954) the logarithmic difference between the initial density and numbers surviving at the end of the stage, represented by k (Varley and

Gradwell, 1960), together with the sum of all k values represented by K, were also calculated for each generation.

Due to loss of original data an estimate of the number of adults for generation II in 1976 and in 1977 was

obtained by subtracting from the number of larvae VI

114

Table 24. Life tables of F. dohrnii

GENERATION I, 1976 x Lx dx 100gx S k K Eggs 1131 306 27.0 S1o.73 k1o.14 Larva 825 I 700 84.8 S110.15 k20.82 1.75 Larva 125 VI 105 84.o s1110.16 k30. 79 Estimated 20 adults 3 15.0 s iv o. 85 k40.07 Adults 17 emerged Generation mortality = 98.5% Trend index = 53.7%

GENERATION II, 1976 x Lx d 100q S k K x x Eggs 607 256 42.1 510.58 k10.24 Larva 351 I 326 92.9 Stp.07 k21.14 1.73 Larva VI 25 14 56.0 51110.44 k30.35 Estimated 11 adults Generation mortality = 98.2%

GENERATION II , 1977 x Lx dx 100gx S k K

Eggs 379 173 45.6 S10.54 k10.26

Larva 206 I 171 83.0 S1p.17 k20.77 1.32 Larva 35 VI 17 48.6 S1110.51 k30.29 Estimated 18 adults Generation mortality = 95.3%

115 entering the soil the total parasitized by L. pictilis and I. laevis. This would tend to give an over- estimate since other sources of mortality have been omitted. Loss of the data also made it impossible to calculate numbers of larvae entering instars II-V in both generations in 1976. Therefore, mortality of larva I to larva VI within the mines was considered to be the difference between the numbers entering larva I and larva VI. The trend index (I), represented by :

number of eggs in generation N2 I = X 100 number of eggs in generation N1 where N1 and N2 are two successive generations was calculated for only generation I in 1976 as data for the first generation, 1977, was lost. The percentage mortality of eggs in generation

II was of the same order in 1976 and 1977 and was considerably higher than in generation I of 1977 (Table 24). If it is assumed that percentage mortality of eggs in generation I, 1977, was of the same order as in generation

I, 1976, this difference between generations could be due to the ovipositional behaviour of the adults. In August, when generation II eggs are being laid, few shoots are still producing young leaves which are selected for oviposition by the females. This may result in aggregation and part at least of the increased mortality of eggs in generation II may be due to larval feeding in areas of leaves which contained unhatched eggs.

The generation mortality varied from 95-98 percent (Table 24),and was calculated by taking into 116

account the actual number of eggs laid and the estimated number of adults except for generation I, 1976, where the actual number of adults was available. Comparisons can be made only between generations I and II of 1976 as these are the only consecutive generations for which data is available. Under laboratory conditions the mean fecundity estimated by dissection of adults shortly after emergence and ten days after emergence showed a significant increase (P.<0.001) from 33.8 to 62.2 eggs respectively (Table 4). On the assumption that a mean fecundity of 62.2 was the maximum that could be attained, 1057 eggs could have been laid in generation II, 1976, but the total number of eggs laid in this generation was only 607 (Table 24). Therefore it would appear that the maximum rate of oviposition was not achieved. 117

VIII. DISCUSSION

Many factors, biotic and physical, combine to determine how a particular population fluctuates in numbers, and long term studies of field populations are essential to understand the manner in which these operate. The cause of population change from generation to generation may be identified by analysing life tables to determine the stage which contributes most towards the population trend (Watt, 1963; Morris, 1963), or by determining the "key-factor" (Morris, 1959) using the method developed by either Varley and Gradwell (1960, 1963a, 1963b, 1965) or Podoler and Rogers (1975).

As data from only three life tables were available the method described by Varley and Gradwell was considered more suitable. Although the values of k obtained were not for successive generations, Figure 36 shows that k1 differs both in direction and magnitude from that exhibited by total K. Therefore k1 which represents mortality of eggs does not appear to be a major cause for the change in populations. Figure 36 also indicates that k3 appears to follow the shape of K more closely than does k2, but if generation I, 1976, is omitted from the analysis both k2 and k3 would appear to follow the shape of K. Therefore on the limited data available either k2 or k3 or both contribute largely towards K and hence are the key-factors. Mortality of eggs varied from 27 to 45.6 percent for the three generations under consideration (Table 24). 118

k • 1.6 •,„

1.2 K

.g •

_ _ k •4 ~ 3

IMO • • ~k1 0 GEN . I GEN. II GEN . II 1976 1976 1977

FIGURE 36

Changes in mortality of F. dohrnii expressed as k values. k l = eggs; k2 = larva I-VI within the mine; k3 = larva VI due to parasites; K = total. 119

The cause of this mortality was not known but it may have resulted from predation or sterility of the eggs.

Alternatively, as the eggs were laid in the mesophyll of young leaves (Figure 3C) mortality of the eggs may have resulted from the hardening of the leaf tissue. The total mortality of larvae in the mines varied from 83.0 to 92.9 percent (Table 24). The only cause of mortality that could be identified with certainty was that due to the eulophid parasites C. vittatus, and

C. nitetis. Dead larvae in the mines that were damaged was suggestive of predation by mandibulate insects or birds. Although many other predators (e.g. A. nemoralis, B. angulatus) were observed to be present in large numbers in the experimental area, it was not possible to determine whether they were responsible for the mortality of many larvae found dead within damaged mines. The mode of action of the mortality factors determined by plotting k values on log density on which it acts (Varley and Gradwell, 1970) suggests that egg mortality

(k1) was inversely density dependent and k3 was directly density dependent (Figure 37a,b). It further indicates that larval mortality (k2) was not related to larval density (Figure 37c) and hence density independent. No explanation could be found for the weak inverse density dependence shown for eggs. As k3 was that due to the two parasites,L. pictilis and I. laevis,it may be speculated that either one or both parasites concentrated on larvae of F. dohrnii when their numbers were high and either sought alternative hosts or areas with high densities when their numbers in the experimental area showed a

23 24 2.5 26 27 2.8 2.9 3.0 LOG. EGGS

•6 k3 •4 • •

23 24 2.5 26 27 28 2.9 30 LOG . LARVA

12 •

1.0t0 k 2 • •8 •

23 2.4 25 26 2.7 28 29 30 LOG. LARVA Ī

FIGURE 37

The k values of various mortalities of F. dohrnii plotted against the density on which they acted. (a) k1 on eggs; (b) k 3 on larva I; (c) k2 on larva I. 121

decrease. The qualitative effects of inverse density dependence, density independence, and direct density dependence, on the assumption that each acted in isolation, would result respectively in instability, fluctuations, and stability (Varley, Gradwell and Hassell, 1973). However the outcome of such contrasting and opposing factors acting in a sequence would depend on their relative strengths and such estimations would require more life tables than considered in the present study. The distribution of eggs laid in generation II,

1977, was of the negative binomial type. As the adults laid eggs only in young leaves the aggregation recognized by the negative binomial distribution was probably a result of the limited availability of young leaves during generation II, due to the normal growth pattern of the tree. If this interpretation is correct adverse climatic conditions during the summer months, and fruiting of the trees,may result in a further reduction in the number of young leaves available for oviposition during generation

II leading to much intense aggregation of eggs. Due to the restricted area available to the larvae for feeding this may result in higher mortalities of eggs and larvae during generation II. Therefore, it appears that apart from other factors success of F. dohrnii in nature would also depend on the physiology of the host plant.

Appendix I

Analysis of the mean forces required (gm/mm2) to penetrate each leaf

in a cluster of leaves using the multiple range of Duncan

Least significant range (L.S.R) = R S2 r

R = Significant studentized range for the residual degrees of freedom obtained from tables

r = Replication (12/leaf)

S2 = Residual sum of squares

5% significant Residual Leaves numbered Mean force required Number studentized to penetrate each of shoot range from mean fromrom the base of leaf arranged in tables square the shoot ascending order

5 51.22 3 68.28 I 2.83, 2.98, 39.75 4 72.96 3.08, 3.15 2 82.46 1 94.25

Appendix I (cont.) 5% significant Residual Leaves numbered Mean force required Number studentized to penetrate each of shoot range from mean from the base of square the shoot leaf arranged in tables ascending order 6 42.22 2.83, 2.98, 3 69.46 1 74.51 II 3.08, 3.14, 18.68 * 3.20 2 75.76 5 76.85 4 81.30 6 39.90 4 49.11 2.83, 2.93, 3.08, 3.14, 17.06 3 55.58 III 2 3.20 5 1 68.06 I 3 53.24 IV 2.83, 3.03 46.08 2 81.62 1 90.04 I 65.39 6 70.69 2.83, 2.97, 5 73.04 V 3.07, 3.13, 11.94 4 75.99 * 3.19, 3.23 2 80.52 3 81.53 1 86.91 Appendix I (cont.) 5% significant Residual Leaves numbered Mean force required Number studentized from the base of to penetrate each of shoot range from mean square the shoot leaf arranged in tables ascending order 8 69.53 I 6 78.10 2.81, 2.96, 5 83.95 VI 3.06, 3.13, 2 86.44 3.19, 3.23, 19.75 3 88.07 3.27 1 88.47 4 90.42 7 91.12 7 32.58 2 35.15 2.81, 2.96 6 35.69 VII 3.06, 3.13, 8.06 5 36.63 3.19, 3.23 4 42.40 1 44.66 3 45.80

* 5% significant differences in force (gm/mm2)

between leaves shown by unbracketed ones. Appendix II

Comparison of the linear and quadratic models for estimating the number of eggs (E)

using weight (W) and tibial length (L) of adults of Fenusa dohrnii

x - S.Sy) (q-s) F(q-s),(N-q)d.f. = (S.S • S.Sy I (N-q) •

S.Sx = Residual sum of squares for the linear regression model

S.S = Residual sum of squares for the quadratic regression model

q = Number of parameters of the quadratic model

s = Number of parameters of the linear model

N = Number of pairs of observations

F = Variance ratio with (q-s) and (N-q) degrees of freedom

a-h = constants in the linear and quadratic models Appendix II (cont.)

Residual Model Number of Number of Variance sum of Variance ratio squares parameters adults accounted

E = ao+a 1W 931.2 2 20 85.7 Adults 0.018(N.S.) E = bo W+b W2 shortly +b1 2 930.2 3 20 84.9 after E = c emergence o+61 L 996.6 2 20 84.7 2.052 (sig: E = do+d1L+d2 L2 862.5 3 20 86.8 at 20%)

E = eo+e1W 2696.0 2 68 93.0 Adults 1.153(N.S.) E = f W+f 2 10 days o+f1 2 W 2649.0 3 68 93.0 after E = go+g1L emergence 2470.0 2 68 93.6 20.03(***) E = ho+h1L+h2 L2 1888.0 3 68 95.0

NS = not significant at 5%

= significant at 0.1%

Appendix III

Principal component analysis of the correlation matrix of the number of eggs (E), weight (W) and tibial length (L) of adults of Fenusa dohrnii

Adults shortly after Adults 10 days after emergence emergence

Number of eggs 1.0000 1.0000 Correlation matrix Weight of adult 0.8701 1.0000 0.9650 1.0000 Tibial length 0.9394 0.8861 1.0000 0.9680 0.9600 1.0000 I II III I II III Number of eggs -0.5801 0.4785 0.6592 -0.5782 0.1516 -0.8017 Latent vectors Weight of adult -0.5684 -0.8174 0.0931 -0.5766 -0.7711 0.2701 (loadings) Tibial length -0.5834 0.3207 -0.7462 -0.5772 0.6185 0.5332 Latent roots 2.7974 0.1430 0.0596 2.9287 0.0404 0.0309 Percentage of total variance 93.25 4.77 1.99 97.62 1.35 1.03

`ew axis for adults shortly after emergence = -0.5801E -0.5684W -0.5834L

New axis for adults 10 days after emergence = -0.5782E -0.5766W -0.5772L 128

Appendix IV

General model used for the analysis of variance of eggs

and larval counts (from leaf samples) of F. dohrnii

with a worked example for the egg counts

on 3rd May 1976

Observed Source of Expected mean mean Variance variation square d.f. square ratio

62+gh62m + Blocks (b-1)=5 4.961 0.930 tqh B2 (b-1) i

Trees within 62+gh62m 1.121 blocks b(t-1)=6 5.333 Total 11 5.164 1.085

Quadrants 62+bth 2 or sides Tq--1)~4 k (q-1)=3 13.435 2.824*

Heights 6 2+bth 1)tH21 (h-1)=2 14.090 2.962

bt Quadrants 62+ • (q-1)(h-1) 6.831 1.436 or sides x (q-1)(h-1) = 6 heights •£ 2 1 kl

Sub-plot error 62 (bt-1)qh-1) 4.758 =121

Total 132 5.191 b = number of blocks = 6 t = number of trees sampled per block = 2 h = number of heights = 3 q = number of quadrants or sides per height = 4 or 2 Capital letters denote fixed parameters such that their sums are equal to zero and 6z and 6m are error variance due to main plots and sub-plots respectively.

* significantly different at 5%. Appendix V

Significance of variation due to blocks (B), trees (T), quadrants (Q), heights (H) and quadrants x height (Q x H) between samples of eggs and larvae of

F. dohrnii in two generations, a sample of five clusters being taken

from each of four quadrants, from each of 12 trees.

Date of Mean Stage sampling per - S.E. B: T: Q: H: Q x H: 1976 sample First generation Total eggs May 3 1.26 0. 19 - - - - Total eggs May 10 2.68 0.57 - * - - - Total eggs May 17 3.77 0.31 Total eggs May 24 4.90 0.73 _ * ** ** ** Dead eggs May 24 0.88 0.19 * ** * _ Total larvae May 24 2.26 0.40 Dead larvae May 24 0.15 0.05 Total eggs May 31 3.28 0.46 ** _ ** Dead eggs May 31 1.25 0.15 Total larvae May 31 3.34 0.24 - - * - - Dead larvae May 31 1.28 0.16 * - - - Total eggs June 7 2.46 0.17 Dead eggs June 7 1.12 0.08 * Total larvae June 7 5.32 0.95 *** Dead larvae June 7 2.72 0.48 ** Appendix V (cont.)

Date of Mean Stage sampling per - S.E. B: T: Q: H: Q x H: 1976 sample

Total eggs June 14 1.77 0.14 - - - - - Dead eggs June 14 1.29 0.14 - - - - Total larvae June 14 5.49 0.62 - - * - - Dead larvae June 14 3.52 0.52 - - * y - Total eggs June 21 1.12 0.11 - - - - air Dead eggs June. 21 1.05 0.09 - - - - - Total larvae June 21 4.75 0.75 - *** * I _ Dead larvae June 21 3.17 0.45 - * - * - Total eggs June 28 0.92 0.14 - * - - - Dead eggs June 28 0.86 0.13 - * - - - Total larvae June 28 3.50 0.32 - - * * - Dead larvae June 28 2.86 0.32 - - - * - Total eggs July 5 0.48 0.08 - - - - - Dead eggs July 5 0.43 0.07 - - - - - Total larvae July 5 3.08 0.44 - * - - - Dead larvae July 5 2.91 0.43 - * - - - Total eggs July 12 0.56 0.10 - - - ** - Dead eggs July 12 0.56 0.10 - - - ** Total larvae July 12 2.76 0.38 - * - *** Dead larvae July 12 2.6o 0.37 - - - *** ONO Total eggs July 19 0.36 0.07 - - - ** - Dead eggs July 19 0.36 0.07 - - - **' - Total larvae July 19 1.64 0.39 - *** _ *** _ Dead larvae July 19 1.62 0.38 - *** - *** _ Total eggs July 26 0.24 0.06 - * - _ Dead eggs July 26 0.24 0.06 - * - _ Total larvae July 26 1.74 0.39 - *** _ * _ Dead larvae July 26 1.72 0.38 *** * Appendix V (cont.) Date of Mean Stage sampling per - S.E. B: T: Q: H: Q x H: 1976 sample

Second generation

Total eggs Aug. 2 1.33 0.25 - - - - Dead eggs Aug. 2 0.08 0.03 - - - - - Total larvae Aug. 2 0.25 0.06 - - - - - Dead larvae Aug. 2 0.02 0.02 - - - - - Total eggs Aug. 9 1.95 0.54 - *** - - - Dead eggs Aug. 9 0..15 0.07 - ** - - - Total larvae Aug. 9 0.14 0.04 - - - - - Dead larvae Aug. 9 0.02 0.02 - - - - - Total eggs Aug. 16 2.10 0.64 - *** - - - Dead eggs Aug. 16 0.33 0.10 ** - - - Total larvae Aug. 16 0.93 0.29 - * - - - Dead larvae Aug. 16 0.19 0.05 - - - - - Total eggs Aug. 23 2.41 0.17 - - - - - Dead eggs Aug. 23 1.10 0.07 - - - - Total larvae Aug. 23 1.42 0.22 - - * - - Dead larvae Aug. 23 0.41 0.07 - - - - - Total eggs Aug. 30 1.85 0.22 - * - - - Dead eggs Aug. 30 1.21 0.21 - ** - - - Total larvae Aug. 30 2.35 0.33 - * - * - Dead larvae Aug. 30 0.87 0.20 - * * - - Total eggs Sept. 6 1.81 0.22 - * - - - Dead eggs Sept. 6 1.44 0.26 - *** - - - Total larvae Sept. 6 2.26 0.50 - *** - - - Dead larvae Sept. 6 0.73 0.32 - *** - - - Total eggs Sept. 13 1.38 0.39 - * - *** - Dead eggs Sept. 13 1.26 0.36 - * - *** - Total larvae Sept. 13 2.20 0.26 - - - *** * Dead larvae Sept. 13 1.08 0.11 - - - *** - 1""6. W Appendix V (cont.) Date of Mean Stage sampling per - S.E. B: T: Q: H: Q x H: 1976 sample

Total eggs Sept. 20 1.32 0.17 - - - - - Dead eggs Sept. 20 1.25 0.16 - - - - - Total larvae Sept. 20 1.71 0.29 - - - ** Dead larvae Sept. 20 0.96 0.15 - - * - Total eggs Sept. 27 1.28 0.25 ** - - ** - Dead eggs Sept. 27 1.24 0.24 - - - ** - Total larvae Sept. 27 1.73 0.19 - - - - - * Dead larvae Sept. 27 1.20 0.17 - - - - Total eggs Oct. 4 0.73 0.15 - - - - - Dead eggs Oct. 4 0.73 0.15 - - - - - Total larvae Oct. 4 1.71 0.32 - * - - Dead larvae Oct. 4 1.60 0.29 - - - - Appendix VI -

Significance of variation due to blocks (B), trees (T), sides (S),

heights (H) and sides x heights (S x H) between samples of eggs and larvae of F. dohrnii in the second generation 1977. A sample of eight clusters being taken from each of the

two sides from each of 12 trees

Date of Mean Stage sampling per - S.E. B: T: S: H: S x H: 1977 sample

Second generation

Total larvae Aug. 18 2.11 0.49 Dead larvae Aug. 18 0.44 0.10 Total eggs Aug. 18 2.76 0.39 Dead eggs Aug. 18 0.17 0.03 Total larvae Aug. 22 2.88 0.38 Dead larvae Aug. 22 0.40 0.11 411111. IMP Total eggs Aug. 22 2.51 0.42 Dead eggs Aug. 22 0.07 0.03 Total larvae Aug. 29 2.44 0.21 * Dead larvae Aug. 29 0.29 0.09 Total eggs Aug. 29 2.46 0.25 alb REM Dead eggs Aug. 29 0.94 0.18 * *

Appendix VI (cont.) Date of Mean Stage sampling per - S.E. B: T: S: H: S x H: 1977 sample

* Total larvae Sept. 5 2.76 0.42 Dead larvae Sept. 5 0.82 0.10 * * - Total eggs Sept. 5 2.46 0.34 - * - - Dead eggs Sept. 5 1.71 0.38 Total larvae Sept. 12 2.94 0.50 * - - - 0.17 Dead larvae Sept. 12 1.39 ** 1.96 0.54 *** _ Total eggs Sept. 12 * *** ** _ Dead eggs Sept. 12 1.60 0.53 - Total larvae Sept. 19 2.61 0.29 Sept. 19 1.74 0.24 Dead larvae * * * Total eggs Sept. 19 1.71 0.23 Dead eggs Sept. 19 1.10 0.27 0.29 ** Total larvae Sept. 26 2.51 ** ** Dead larvae Sept. 26 1.89 0.26 Sept. 26 1.43 0.22 *** Total eggs *** Dead eggs Sept. 26 1.25 0.21 Total larvae Oct. 4 2.49 0.22 * 2.24 0.25 Dead larvae Oct. 4 * * Oct. 4 1.18 0.20 *** Total eggs *** * * * Oct. 4 1.17 0.20 Dead eggs Oa Total larvae Oct. 10 2.03 0.28 Dead larvae Oct. 10 1.88 0.26 1.26 0.29 *- ** Total eggs Oct. 10 *** Dead eggs Oct. 10 1.26 0.29 Appendix VI (cont.) Date of Mean Stage sampling per ± S.E. B: T: S: H: S x H: 1977 sample

Total larvae Oct. 18 1.56 0.09 - - - - - Dead larvae Oct. 18 1.26 0.11 - - - - - Total eggs Oct. 18 '0.57 0.18 - - - - - Dead eggs Oct. 18 0.51 0.18 - - - - - Total larvae Oct. 24 1.90 0.29 - - - - - Dead larvae Oct. 24 1.86 0.28 * - - Om Total eggs Oct. 24 0.19 0.11 - - - - - Dead eggs Oct. 24 0.19 0.11 - - - - - 136

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