Studies on the Ecology of Thistle Lace Bugs; In

STUDIES ON THE ECOLOGY OF

THISTLE LACE BUGS; IN PARTICULAR TINGIS AMPLIATA4k+.87.(HETEROPTERA: TINGIDAE).

William Enotiemwomwan Eguagie, B.Sc.

A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Science of the University of London.

Imperial College of Science & Technology, Field Station, Silwood Park, Sunninghill, May, 1969. Ascot, Berkshire. 2

ABSTRACT

The population dynamics of Tingis ampliata HrS. was studied in a relativel

delimited area of thistles, Cirsium arvense (L.) at Silwood Park, Berkshire, in the three years 1965, 1966 and 1967. The relative roles of natality and

different ecological factors influencing seasonal and annual variations in Tingis population density were assessed. Population budgets are presented

for the period of study.

Various biological characteristics of the insect, including reprod-

uction, distribution, body weight, sex ratio, and overwintering were investigated in relation to its life cycle, population age and structure.

The rate of dispersal of adults in a natural habitat was determined.

An analysis of the distribution of dispersing individuals and of the

climatic factors affecting dispersal rate and numbers were made to partly

explain daily occurence and distribution of Tingis on a thistle patch.

Histological studies were carried out on the seasonal relationship between

flight musculature, reproductive physiological state and level of flight

activity of thistle lace bugs. These are discussed in relation to habitat

type, local and geographical distribution and abundance of the species.

A short account is given on the bioecology of Tingis cardui L. on

the spear thistle, Cirsium vulgare (Savi) Ten. 3 CONTENTS Ewa SECTION I : INTRODUCTION 7 1. Review of Literature 9 2. The Habitat and Host plant 13 (a) General description of the Habitat and Host plant 13 (b) The number of thistle plants in the study area. 19 (i) Estimation 19 (ii)Annual variation 21 (c) The average total number of primary leaf-buds in the study area 23 (i) Estimation 23 (ii)Seasonal variation 25 (iii)Annual variation • 26 3, Life history of Thistle Lace-bugs 29 (a)Tingis ampliata H.-S. 29 (b)Tingis cardui L. 32

SECTION II : STUDIES ON TINGIS AMPLIATA 34 1. Description of stages 34 (a)Adults 34 (b)Immature Stages 37 2. Aspects of the Biology of the adult 40 (a) Overwintering 40 Introduction 40 (i) Autumnal migration. 43

(ii)Survival ( mortality) during overwintering • • • • 46

(iii)Weight loss during overwintering. 56 (iv)Cold-hardiness. 60 (v) Seasonal variation in under000ling point ..... 65

(vi)Other factors affecting undercooling point. 70

4 Pane (a) Nature of the contact surface. 70 (b) Presence or absence of food in the gut. ... 71 (vii) Pre-emergence feeding 74 (b) Mating behaviour 76 (i) Season. 76 (ii) Time of day. 77 (iii) Body orientation of Tingis in copula 80 (iv) Frequency of copulation. 82 (v) Duration of copulation. 82 (c) Oviposition behaviour 84 (i) Parts of the plant where eggs are laid. 84 (a)In the field 84 (b)In the laboratory 84 Precise sites of oviposition in the field. 85 Types of oviposition in Tingis. 90 Periodicity of oviposition in the field. 93 Oviposition studies in the laboratory. 97 Changes in reproductive organs and body weight in the field. 101 Weight of females in Spring and number of egg rudiments. 119

State of ovaries at death of females. 122

An unusual ovarian condition in TinFis 124 (d) Dispersal 126 (i) Walking. 126 (a)Methods of study. 127 (b)Distances moved by adult Tingids. 128 (c)Effect of weather. 150 (ii) Flight. 158 (a)Methods. 158 (b)Reproductive state of females caught in suction trap. 162 (c)Seasonal changes in flight musculature. ...163 (d)Laboratory studies on flight activity. • ... 171

(iii) Discussion. 183

Page (e)Distribution on the host plant. 190 (f)Diurnal variation in the distribution of adults. 203 3. Aspects of the Biology of the Immature Stages 207 (a) Development of eggs and nymphs. 207 (i) Their size. 207 (ii)Weight of larvae. 207 (iii)Role of temperature in development. 209 (iv)Development in the field; (eggs & nymphs) 213 (v) Effect of humidity on developmental hatching of eggs. 216 (b) The distribution of eggs 219 (c) The distribution of nymphs 228 (d) The effects of feeding of nymphs on thistles. 233 4, Development of Life Budget 239 (a) Methods 239 (b) Analysis 253 (i) Survival and mortality of adults. 253 (ii)Sex ratio of adults. 2% (iii)Sex ratio of nymphs. 259 (iv)Estimation of recruitment and mortality in the immature stages. 261 (v) Fecundity. 264 (c) Mortality Factors 272 1. Causes of mortality in Adult Tingis 272 (i) Parasitism. . 272 (ii) Winter losses. 275 (iii)Predation. 278 2. Causes of mortality in Immature stages 280 (a) Egg mortality 1 280 (i) Predation 280 (ii)Parasitism 282 (iii)Sterility 288

Page (b) Nymphal mortality 289 (i) Predation 289 (ii)Fungal diseases 296 (iii)Ectoparasites 298 (d) Population budgets, 1965-67 299

5. Discussion 309

SECTION III: SUPPLEMENTARY STUDIES ON T. CARDUI 311 SUMMARY 321

ACKNOWLEDGEMENTS 325

REFERENCES 4 327 SECTION I : INTRODUCTION

This thesis is essentially an account of some ecological observations on Tingis ampliata 1L-S.and Tingis cardui L., the two species of lace bugs found on thistles in Britain. These lace bussoccur on different thistle species in nature; the former on the perennial creeping thistle Cirsium arvense

(L.) Scop. and the latter on the biennial spear thistle Cirsium vulgare (Sale

Ten. Of the two Tingis species; T. cardui was locally less abundant and in some years was virtually absent from thistles in Silwood Park. The information presented on this species (Section III), has therefore been limited to data on some aspects of its biology and the seasonal fluctuation in the population of a colony studied in 1966. Some attention has been given to details of the spatial and regional distribution of the insect on its host plant together with records of potential predators associated with the bug.

The main part of the thesis (Sections I - II) deals primarily with the seasonal and annual variations in a population of T. ampliata on a delimited area of C. arvense in the period from April 1965 to Nov. 1967, and with the factors accounting for such variations. An attempt has been made at a quantitative assessment and interpretation of natality and mortality in relation to population changes, with a view to constructing a population budget for the bug. Mortality factors were identified and their significance assessed quantitatively whenever possible. Serological techniques were used to determine the predators of immature stages. 8

In some instances, population parameters were estimated by different

methods which acted as mutual checks on one another. Seasonal and annual variations in host plant and vegetative bud density were estimated and related to insect population density in an attempt to demonstrate any influence of the carrying capacity of the food plant on the numbers of Tingis.

The biological and behavioural characteristics of a species as well as its environment constitute some factors that may affect its abundance (Andrewartha & Birch 1954). Information on the biology of T. ampliata has hitherto been inadequate in scope and content. A considerable part of the present studies has therefore been devoted to various aspects of the biology of the bug. These include, detailed investigations on adult distribution, reproduction, overwintering and dispersal.

In a species which overwinters as adults, the site of the post- hibernation population in spring depends partly on (a) the ability of the hibernating individuals to survive adverse, cold winter conditions and (b) the amount and rate of break down of their metabolic reserves, (Kiritani et al. 1966). In the present work much attention has been given to the determination of cold-hardiness and some factors affecting cold-hardiness in Tingis. The pattern of weight loss associated with breakdown of food reserves in winter is also examined.

The distribution, development and feeding habits of immature stages

were investigated in relation to population size and age. A brief account is given of the synchronisation of the life cycles of the Mymarid (Chalci- doidea) egg parasites of T. ampliata with that of their host. 9

1. Review of Literature Although some species of Tingidae are of considerable economic importance in different parts of the world (See Fink 1915; Tillyard 1926; Sweet- man 1936 and Roonwal 1952); yet comprehensive ecological studies of the group appear to have been neglected. Miller (1956) summarises the situation briefly; "On the whole, little is known about the ecology of the Tingidae". Indeed, with the exception of studies such as those of Connell & Beacher (1947) and Bailey (1951, 1962-63) on the phenology and seasonal occurence of some North American species, publications on the Tingidae to date, have in general dealt with outlines of life history, descriptions of immature stages and feeding habits. It has been suggested that the group has for long failed to attract extensive detailed investigations on account of the small size of its adults (Butler 1923) and eggs (Leston 1953).

Prior to the present studies, there appears to be no published works on details of the bio -ecology of T. ampliata. Most of the limited literature on this species appeared mainly in the last fifty years, although the bug has been recorded as early as the late half of the nine- teenth century from Britain (Douglas and Scott 1865) and the continent of Europe (Lomnicki 1881; mainly Poland and Germany). The bulk of these publications concerned, (a) its association with thistles (Reclaire1940; Cobben 1948; Singer 1952; Southwood & Leston 1959) and different overwintering habitats (Parshley 1923, Goddard 1934 and Massee 1963); (b) its geographical distribution in the U.K. (Butler 1923, Bedwell 1945, Massee

1963) and in Europe and Central Asia, i.e. in the Palearctic region corresponding to the natural range of C. arvense (Butler 1923; Southwood 10

& Scudder 1956). Butler (1923) gives its distribution as extending to central and aauth eastern Europe, Turkestan and Siberia. Its northern limit in Britain is given as Yorkshire. The bug is said to be absent from Ireland, Giglio Island (Mancini 1952), Jersey and Channel Islands (Le Quesne 1953); (c) outlines of its life history and descriptions of nymphs (Butler 1923) and eggs together with keys for the identification of its nymphal instars (Southwood & Scudder 1956). It is noteworthy that Butler (1923) described the eggs of thistle lace bugsas unknown. T. amnliata is said to be univoltine in Britain and the continent (Southwood & Scudder 1956); (d) its relative incapacity for flight (Southwood 1960).

There are only a few published accounts on T. cardui. The main ones made a brief reference to its association with C. vulgare in the U.K. (Mason 1898, Butler 1923, Massee 1954) and with C. vulgare and Carduus nutans L. (Strawinski (1954) and C. nutans and C. acanthoides L. (Fieber 1860) in the continent of Europe. Details of its geographical distribution are given by Southwood & Scudder (1956) and by Butler (1923) who mentions that it is found "apparently in almost all places where the food plant occurs." This includes some islands such as Giglio, Jersey, Channel and Ireland (Halbert 1935) from which T. ampliata appears not to have been recorded. General outlines of its life cycle and description of its immature stages are given by Southwood and Scudder 1956.

There are only four species in the genus Tingis Fabricius in Britain, and keys for their identification are given by Southwood and Leston (1959).

All except one, viz T. angustataF4-S., which has not been associated with 11 a definite host (Southwood & Leston 1959), are plant suckers. Other publications on the genus Tingis in the temperate regions have primarily dealt with records of species such as T. crispatalL -S., T. pilosa Humm., T. geniculata Fieb. and T. maculataH.-S. from different habitats in Germany (Singer 1952) and T. pyri Geoff from France (Gautier 1925). Occasionally, some reference has also been made in the literature to economic species such as T. pyri which are said to cause widespread defoliations in fruit gardens and pear orchards (Anon 1912-13; Gautier 1925). None of these papers have dealt with details of the ecology of the species concerned. It is therefore hoped that the work on the biology and population ecology of T..ampliata and T. cardui described herein would provide some useful basic information on the approach to simtlar studies of related species.

FIG. I IMPERIAL COLLEGE FIELD STATION

SILWOOD PARK HILL SUNNINGHILL FIELD OBSERVATORY RIDGE SCALE: I IN. TO 209 FT.

X STUDY AREA El THISTLES C.ARVENSE L. SCOP

C.VULGARE SA/I TEN. SiLk#000 • BUILDINGS BOTTOM 0 TREES OR i vE = ROADS 1411L ": PATHS

.. 1

... ,-..-.., v.:. %--..-, , GAI:E:i..'\--.,'.--::•:PRj:1 9Or' wom ---,, DRIVE it ,.... ), r , f; FIELD ...„ _ --...• - ' K-. -.." ,....,• ,..._— ---„:1--..., ) - C H weacroo. ,..... k‘. INEMEATH- 650A (.P

soum \ fj SOUTH LODGE-, ,....CANooNON o~rq C LFCIDGEELD C SOUT -j.-H 147-' Lop C7.

TO) SUNNtp.50 AL I • To 13 2. The Habitat and Host plant (a) General Description of the Habitat and Host plant (i) Habitat Studies on the lace bugs were carried out on the grounds of Imperial College Field Station, Silwood Park, Sunninghill, Berkshire. Fig 1. is a sketch map of the Field Station which shows the precise locations of a small

area of creeping thistle, C. arvense , on which T. pmpliata was studied. The area measures 110 x 220 ft. and occupies a large proportion of the stretch of land between Elm Slope on the west and Drive Hill on the east. Its northern boundry is marked by a foot path, (5 ft. wide) which was used intensively from October, 1963 to October, 1966. The path has been used less frequently since then and is gradually being recolo- nized by various grasses. At about 150 ft. north of this path, another path, 20 ft. wide, runs almost parallel to the former, from Silwood Bottom to Gun- nes's Hill. The area between and beyond the two paths was essentially similar to the study area in vegetation. On the South, the study area is again bounded by a foot path, 4-6 ft. wide. This path runs from Drive Hill, through Elm Slope to the Rookery and Marsh and terminates by a small brook, about 200 yards from the western limit of the study area. The path is used frequently from March to November of each year. To the south of this path, and extending for about 500 yards beyond it is the Rookery Slope, which has a typical grassland type of vegetation. Detailed descriptions of the structure and composition of the vegetation of that part of Silwood Park have been given by Danthanarayana, (1965).

The Drive Hill stands on a rolling topography and extends for about 150 yards beyond the Eastern border of the study area. On the north, it extends 14 beyond the northern limit of the study area by about 30 yards, and ends in Silwood Bottom which in the Spring and Summer of 1965, 1966 and 1967, was ploughed up and sown to plots of Brussels sprouts and potatoes etc. The vegetation of Drive Hill consists of a densely packed mixture of grasses (Holcus, Festuca and Agrostis) and creeping weeds, mown regularly to a height of about 1 inch.

Between the western boundry of the study area and the wood:on Elm Slope, there is a rectangular piece of land about 20 ft. in width. This strip of land is often shaded in the evening by the trees of the adjacent wood. Its vegetation is characteristically that of a modified grassland on the edge of a wooded area; with nettles, (Urtica dioica L.); the Umbellifer, Conopod- ium majus Gowan. and graminae, Dactylis and Holcus as dominant species. This area was cut and burnt in the Autumn of 1964 and Spring of 1965. The occasional C. arvense seen on this strip in 1966 and 1967 were eliminated. The wood on Elm Slope, consists of different species of which oak, Quercus robur L., and Elm, Ulmus 2.22. formed the upper tree canopy. Immediately below these, younger and smaller birch, Betula pubescens Ehrh. trees formed another canopy, about 12 ft. from ground level. The wood extends westwards to the Marsh, and its discontinuous herb layer consists mainly of heath grasses, Agrostis tenuis Sibth; Deschampsia flexuosa L; Poa pratensis (L.) and bramble, Rubus fruticosus agg.

The study area shows a gentle topographical undulation from South-West to North-East. It has always been an unmodified grassland, except for the influence of light grazing from 1951 to 1956 (Mr. J. M. Stringer, personal communication). During the present studies, its floristic composition was l ~

Fig. 2 General view of the study area.

A. In Summer

B. In Winter 16 still essentially graminaceous, with Holcus App. forming the predominant ground cover. The matrix of Holcus mollis (L.) was interspersed with other graminae including, Festuca pratensis Huds, A. tenuis and Dact- yais glomerata L. During Spring and Summer of each year many herbaceous flowering weeds appear among the grasses. The most prominent of these non- graminaceous species are the compositae: Achillea mellifolium L. (Yarrow); Senecio jacobaea L. (Ragwort); Taraxacum officinale agg. (Dandelion); Cirsium arvense (L.) Scop. (Creeping thistle), Leontodon ERR. (Hawkbit); and various species belonging to other families including Rumex crispis L. (Curled Dock); R. acetosa (Sorrel); Plantago lanceolata L. (Ribwort) and U. dioica- (Stinging nettle).

Besides flowering herbs, there is occasional occurence of small saplr ings of oak (Q. robur), elm (Ulmus ap.) and birch (B. pubescens) on a narrow zone along the western border of the area. These saplings are thin- stemmed(about 0.5 - 1.5 cm in diameter), with a few lateral branches and small leaves. They vary in height from 1 to 5 ft. During autumn, they shed their leaves which combine with the wilted remains of grasses and herbs to form a deep accumulation of litter. A general view of the study area in summer and winter is shown in Fig. 2.

(ii) The Host Plant Although, T. ampliata has occasionally been found on Marsh thistle, Cirsium palustre (L.) Scop. (Singer, 1952), it appears that its host plant

is the creeping thistle, C. arvense (Reclaire, 1940; Cobben, 1948; and Southwood & Scudder, 1956). The author did not find it on other thistles. 17 Creeping thistle, in a perennial, dioecious herb with extensive creeping underground roots which produce erect aerial shoots during the growing seasons. It belongs to the family, compositae. Its stems (v.. shoots) are light-greenish, often furrowed, unwinged, glabrous or cottony above. 'They vary in height from 1 to 3 ft. (Gill & Vear, 1958); 1 to 3 to 5 ft. (Clapham, Tutin & Warburg, 1957). First leaves are characteristically oblong-lanceo- late in outline and are not aggregated as in other thistles into a compact rosette. They have a narrow stalk-like base with a pronounced midrib. Leaves are arranged alternately on stems. Quite often but not invariably, leaves may be pinnatifid and undulate with triangular toothed and spiny- ciliate lobes. Leaves in the middle and upper regions of plants are usually sessile, semi -amplexicaul and may be slightly decurrent. From mid-May to September, primary leaves bear axillary buds, with smaller, densely-packed leaflets. Thus, during the main period of vegetative growth, thistle plants look like small bushes. However, a careful observation shows that each primary leaf-bud unit is easily identifiable.

C. arvense flowers from June to September.. The pale-purple or whitish flowers are short-stalked, solitary or arranged in an irregular corymb of 2 to 4 per cluster. Flower heads measure about 1.5 to 2.5 cm in diameter. They are honey-scented and are often visited by various species of insect pollinators. Involucres of female flowers are ovoid in shape and those of male flowers are spherical.

Underground stems of thistles are perennial, but aerial ones have one growth cycle a year. Emergence of aerial stems starts in Spring, about early or late in April. The date of start and rate of emergence varies 18 from one year to another and seems to depend on the climatic conditions of the preceeding winter and of the first months of Spring (Bakker, 1960). Emergence of plants is usually complete by the end of July. Vegetative growth attains a peak at about the time of peak emergence of plants. After July, and depending on earliness of adverse climatic conditions in autumn, wilting of vegetative tissue starts from the bases of plants and progresses upwards. Wilted leaves are not shed soon after death, but often remain attached to stems, until late in November when wilting of plants is often complete, and some leaves may fall. During winter, most of the dead stems collapse and drop on the ground vegetation. Growth is resumed in the following spring by the sprouting of buds from the dormant underground organs of propagation.

Creeping thistle, is native to England and it is widely distributed throughout the British Isles. Its distribution extends as far north as 68° 50' N; through Scandinavia in Western Europe to Asia. It grows as far South as North Africa (Clapham, Tutin & Warburg, 1957). Its main areas of occurence in Silwood Park, are indicated in Fig 1. 19

(b) The Number of Thistle Plants in the Study Area

(i) Estimation: The assessment of the absolute numbers of adult and immature stages of Tingis in the study area requires a determination of the density of thistle plants and vegetative tissues. In each of the three years, 1965, 1966 and 1967; the density of thistles was estimated by counting the number of plants in a number of randomly distributed, 3 x 3 ft. (= 9 sq. ft.) steel quadrats. For locating the quadrats, each of a pair of adjacent sides of the area was divided into 10 equal units. A series of random numbers,

(selected from tables) were then used as pairs of co-ordinates, taking the two adjacent sides as axes and omitting repetitions. The mean number of plants per quadrat, (1 ) was calculated from the samples taken on each occasion and by relating this to the area of the plot, an estimate of the total number of plants in the area was obtained.

In order to determine the adequate number of samples for plant density estimation in the 1965 season, various sample sizes; 30, 40 and 50 quadrats were taken on 3rd May. The means of the first and second and of the second and third sets of samples in the above series were then separately compared

(Table 1).

Table 1: Comparison of means obtained from different numbers of quadrat

samples on 3.5.65. 20

, r - . No. of Total No. of quadrats plants Mean ,t1 P

30 >0.1 (NS) 80 2.66 0.59076 D.F = 68 40 120 3.00 )0.1 (Ns) 0.04036 50 149 2.98 D.F = 88 4 t

There was neither significant difference between the means of 30 and 40 quadrats nor between th::04e of 40 and 50. A sample of 40 quadrats was therefore considered adequate. Estimates made using this sample size at various other times during that year did not vary in precision of estimation from those made in May (Table 2). In the 1966 and 1967 seasons, the adequate sample size was determined, using a similar method to that of 1965. Although 40 samples proved sufficient in either year, in routine estimates, 70 and 90 quadrats were respectively counted. It was considered advantageous to take as large a number of samples as possible in order to improve the efficiency of estimates, especially in the 1967 season when host plant density fell to about one-half of the values obtained in either of the two previous seasons. The reliability of quadrat method for estimating thistle density is shown in Table 2. For each year, accuracy of estimates tended to increase with the number of samples taken. In general, the ratio of standard error to the mean decreased with increase in the value of the mean (i.e. with increase in thistle density). 21

Table 2: Showing reliability of quadrat method for estimation of host plant.

_.. 1 Mean No. of plants No. of Total No. per quadrat Standard Standard error Date quadrats of plants + 95% Fiducial error as % of limits mean gag - Ar At , 3.5.65 5o 149 2.98 + 0.66 0.33 10.96 3.6.65 4o 208 5.2 + 1.24 0.62 11.94 3.7.65 4o 287 7.17 + 1.46 0.73 10.14 31.5.66 7o 437 6.24 + 0.98 0.49 7.87 30.6.66 4o 264 ' 6.6 + 1.24 0.62 9.34 1 31.5.67 90 I 271 3.01 + 0.6 0.30 10.09 18.7.67 90 389 4.32 + 0.66 0.33 7.72 , 017 Annual variation: In each year, host plant density was estimated at least twice a month starting from May until the end of September. Estimates were usually made in the first and last week of each month or in the first, second and last week as in when three determinations were made in the month. The averages of the estimates for each month of the three years, 1965, 1966 and 1967 are presented in Table 3. All values are to the nearest whole number.

121.212-2i Annual variation in density of thistles in the study area. 22 _ _ . _ . Estimated No. of thistles & Year Month • , _ - .... 1965 1966 1967

MAY 8,067 16,779 6,211 JUNE 13,982 17,747 8,550 JULY 19,293 18,231 10,527 AUGUST 18,822 18,150 9,949 SEPT. 18,822 18,123 9,949 . _

There was a marked annual variation in host plant density. Estimated peak numbers of plants in the 1967 season was lower than (about one-half of) estimates for each of the two preceeding seasons. In each year, host plant density increased from May to a peak in July and then became approximately constant afterwards. The high rate of increase of density in 1966 as compared with 1965 and 1967 seasons, could be explained by the relatively warm and wetter Spring of 1966, which hastened thistle above-grass emergence and growth. By contrast, February - April of 1967 was very dry and warm and plant emergence was delayed (see sections (c) and II, 2(a)). The significance of the annual variation in thistle density will be obvious when the average total number of leaf-bud units in the area are considered in section (c) below. 23 (c) The Average Total Number of Live Primary Leaf-bud units in the Study Area

(i) Estimation: The total number of leaf-buds in the area was estimated by taking counts on randomly selected plants at least once a month during the period, May to September when the immature stages of Tingis occur in the field. In the 1965 season, counts were made on 60 plants on each occasion. The number of samples was increased to 70 in the 1966 season except for August in which counts were made on 75 plants. A minimum of 80 samples were counted on each occasion in the 1967 season. The reliability of different numbers of samples for leaf-bud estimation is shown in Table 4.

On each date of counting, separate records of the number of live, primary leaf-buds in the upper (UH) and lower half (LH) of each plant sampled were made. The significance of the division of thistle material into two regions will be apparent in section II; 3(b-c) in which the distribution of eggs and nymphs is discussed.

Table 4: Reliability of estimates of number of leaf-bud units in the area

Total Mean No. Standard Standard Date No, of No. of of leaf-buds error as samples leaf-buds per plant + 95% F.L error % of e . . mean 22.6.65 , 60 1125 18.75 4- 13.26 6.77 36.1 31.5.66 60 890 14.83 + 10.42 5.32 35.87 23.6.66 70 1393 19.88 + 8.09 4.13 20.77

4.8.66 75 927 12.35 + 9.95 5.08 41.13 12.5.67 80 801 10.01 + 5.31 2.71 27.07

16.6.67 80 1035 12.93 + 6.64 3.39 26.21 . . FIG . 3. SEASONAL VARIATION IN MEAN NUMBER OF LIVE PRIMARY LEAF - BUD UNITS

24 .

20 .

' ...0 1965 ...... 1966 ''' '13 1967

1 1 li IL I I 0 10 20 30 40 50 60 70 80 90 100 1 0 120 130 DAYS (DAY 0 =30TH MAY

25 (ii) Seasonal variation: The seasonal variation of the average number of primary leaf-buds per plant during each of the three summers is shown in Fig. 3 . In Spring and early Summer the number of live leaf-buds per plant increases rapidly to a peak and then decreases gradually as the season ages. The post-peak decrease in the average number of primary leaf-buds per plant is due to the progressive wilting of vegetative tissue from the bottom upwards to the top of plants (see also Tables 5-7). Vegetative growth of thistles was similar in 1965 and 1966; in which peak of numbers of leaf-buds per plant was attained around mid-June. By contrast, there were significantly fewer numbers of leaf-buds per plant in 1967 season, and peak of growth was delayed till mid-July. Reference has been made to the relatively dryer Spring conditions of 1967, as compared with 1965 and 1966. The distribution of vegetative tissues between the two regions of plants also showed seasonal variation (Tables 5 - 7). There were more live, primary leaf- buds in the upper (UH) than in the lower half (LH) of plants. The proportion of leaf-bud units in the latter half decreased and that of the former increased with the age of the season.

Table 5: Mean No. of live leaf-bud units per plant at different levels during sampling in 1965, as % of total in bracket.

(TOTAL) DATE UH LH BH

NNW 30.5.65 8.42 (58.19) 6.05 (41.81) 14.47 22.6.65 10.5 (56.00) 8.25 (44.00) 18.75 13.7.65 9.8 (55.05) 8.0 (44.95) 17.8 3.8.65 9.0 (61.47) 5.64 (38.53) 14.64 2.9.65 6.5 (66.8 ) 3.23 (33.2 ) 9.73 26 Table 6: Mean No. of live leaf-bud units per plant at different levels

during sampling in 1966, as % of total in bracket.

(TOTAL) DATE UH LH BH

31.5.66 8.61 (58.0) 6.21 (41.87) 14.83 23.6.66 10.75 (54.07) 9.12 (45.87) 19.88 14.7.66 9.1 (52.29) 8.3 (47.7 ) 17.4 4.8.66 8.65 (69.64) 3.77 (30.35) 12.42 3.9.66 5.42 (72.26) 2.07 (27.6 ) 7.5

•Prer.orip.••••••••••••••••••••••••••ormoomr.WMP......

1212222i Mean No. of live leaf-bud units per plant at different levels during sampling in 1967, as % of total in brackets,

(TOTAL) DATE UH LH BH

12.5.67 5.62•( 56.17) 4.38 (43.82) 10,01 26;5.67 6.62 ( 54.35) 5.56 (45.64) 12.18 16.6.67 7.5 ( 57.97) 5.43 (42.02) 12.93 30.6.67 9.17 ( 60.41) 6.01 (39.58) 15.18 14.7.67 9.56 ( 61.05) 6.1 (38.94) 15.66 31.7.67 7.89 ( 78.9 ) 2.1 (21.09) 10.00 18.8.67 6.55 ( 88.69) 0.83 (11.3 ) 7.39 29.9.67 2.57 (100.0 ) 0.0 ( 0.0 ) 2.57

(iii) Annual variation: In order to obtain the average total number of live, primary leaf-buds in the area in each month, the average number of 27 leaf-buds per plant was multiplied by the total estimate of plants in the

area for the corresponding month. The results for the three years are shown in Table 8.

Table 8: Annual variation in monthly estimate* of live primary thistle leaf-bud units in the area.

Year and estimated average total No. of leaf-bud units

Month .1111...... 4. 1965 1966 1967

MAY 116,729 248,833 68,942 JUNE 262,163 352,810 ' 120,213 JULY 343,415 317,219 135,061

AUGUST 275,554 225,423 73,523 SEPTEMBER 183,138 135,923 49,546

*A11 values are to the nearest whole number.

Estimates of the average total number of live, primary leaf-buds for the entire period, May to September were calculated from data in table 8 . The results are presented in Table 9. Monthly and annual estimates of live primary leaf-bud units in 1965 and 1966 were similar and significantly higher

than estimates for the 1967 season. The estimated average total number of leaf-bud units in the latter year was slightly higher than one-third of the

estimate for the 1966 season. 28

Table 9: The average total number of live, primary leaf-bud units in the area during May to September in the years 1965-67.

F t P Total number of Year primary Average leaf-bud units . . 1965 1,180,999 236,199.8 1966 1,280,208 e56,041.6 1967 447,285 89,457.0 29 Ige History of Thistle Lace Bugs

(a) T. ampliata This species is univoltine. Oviposition occurs in Spring Summer and adults of both sexes overwinter. Thus, the life cycle falls under class II (la) of Woodward's,(1952) classification.

Teneral adults emerge from early to late August. The date of peak emergence varies from year to year according to environmental temperature. There is a variable period (usually one to three weeks) of intensive post- emergence feeding on thistles. Considerable amounts of fat and flight muscle are laid down and weight of the insect may increase by as much as 90,4% of that at emergence during the autumn feeding period. Females remain sexually immature, without differentiation of oocytes in the autumn.Sperm- atogenesis starts in autumn but is slowed down considerably during the winter months. With the onset of low temperatures in late August and September, there is cessation of feeding and the insects gradually move to the bases of thistles and eventually into grass tufts, roots and mixed floral litter in which they hibernate. The rate of this movement into hibernacula tends to vary from one year to another according to ambient conditions. It was more rapid in 1965 than in either 1966 or 1967. The insect may be found in litter at a depth of 1 inch during early winter, bUt they move further down with the severity of the winter, so that some may be found at depths of upto 5"

in March and April.

The bugs remain in a more or less inactive state (usually, just able to

move their appendages when disturbed) during the cold winter months. How•►

ever, when exposed to higher temperatures, they rapidly become active and walk about more readily. There is a substantial decrease in fat content and 30 body weight during the period of hibernation. Females remain in diapause until early April.

Post-hibernation emergence occurs in spring, usually early May. The dates of first appearance and of peak emergence varies with year according to the severity of the preceeding winter and the prevailing weather conditions in early spring. Emergence usually occurs when grass temperatures reach about 13 °C and when thistle shoots have emerged above the grass surface.

Copulation occurs mainly around noon although bugs 'in copula' may be found in the field at any time of the day. The time of copulation is from late April to the end of June with the exception of a single pair, found on 9.7.65.

The'emergence of thistles above grass level in spring is closely synchronised with and usually preceeds emergence of Tingis from diapause. How- ever, in a dry and relatively warm Spring, e.g. 1967, Tingis fed on etiolated thistles before the host plants grew above the surface of grass.(Table 28). Active feeding in the first one or two weeks after hibernation results in a considerable increase in fat content and body weight of bugs. Ovarian maturation starts from early April and most females are mature by the second week in May. There is a correlation between pre-oviposition body weight and

the stage of development of ovaries.

Oviposition begins in the first or second week of May (the date of commencement depending on that of emergence from hibernation). It may last for 6 -12 weeks; and usually stops by early or late in July. Consequently, the various stages overlap considerably. The rate of egg- 31 laying increases with temperature and attains a peak in late May or in early

June. Body weight remains roughly constant during the period of active oviposition and then decreases towards the end of this process. Many females die without completing oviposition and a few survive until the last week of

July. There is a variable period of one to two weeks, in which the overwintered (recognisable, by a thick deposit of white wax on brownish- black body cuticle) and new generation females (having little wax on brown cuticle) overlap in the field. This is probably way Butler, (1923) found the imago "practically all the year round". Post-hibernation longevity in males is usually less than in females. Most of the males die soon after copulation, although a few may be found in the field by late June and mid-

July.

Adult life is long, with a maximum of approximately 11 months. Eggs are laid in leaf mesophyll, mainly on the midribs and veins of the abaxial surfaces of primary thistle leaves. Leaves in the upper half of plants may contain eggs but usually more are laid in the lower half. Eggs are laid singly, but occasionally may be found in batches of 2 - 5; and tend to be concentrated in the first third Of the leaf length from the node. Incubation

period varies from 15 - 25 days. Within certain limits, the rate of develop-

ment of eggs increases with temperature. First instar nymphs begin to

appear from first week inJune.The date of first appearance varies from one

year to another, according to field temperatures and the time of oviposition.

On hatching, the first instar nymphs colonise the axillary buds and feed in

small aggregates. The degree of aggregation increases to a peak in the

second instar nymphs and then decreases in the older instars, 3 - 5. These

older instars move from one bud to another and were seldom found on stems. 32 By contrast, instars I and II remain relatively immobile in buds. Feeding

of nymphs produces yellowish-white mottlings, which later turn brown and

result in wilting of buds. Development from first to the fifth instar takes

50 - 60 days under field conditions and the instars increase in weight

considerably during this period.

(b) T. carded. ; The life history of this species is similar to that of T. ampliata. It is univoltine and adult males and females overwinter.

New generation adults start to emerge on spear thistle, Ci vulgare

from first week in August, with peak emergence occurring in the last

third of the month. Emergence is followed by a short period, usually about

two weeks of active feeding during which fat and flight muscles are accumu-

lated, but ovaries remain immature without differentiation of oocytes. Adults may be found on C. vulgare till about the middle of September. If

the adults emerged on thistles in their second year of growth, and which are

already wilting by August, a sudden pre-hibernation migration from the

plants may take place, as I have noticed in a colony on North Gravel in 1966.

Between 24th and 28th August 1966, the number of adults in the colony fell

by about 45 percent. It is probable that a few individuals may reach

thistles in their first year of growth in the autumn. The insects over-

winter (from September to May) in litter, under grass tufts or moss.

Post-hibernation emergence in spring, is immediately followed by a

spring, dispersal flight period of about 3 weeks. C. vulgare is often

locally sparse and many individuals die if they fail to reach a host. This

probably explains why only a few (usually 1 to 3) overwintered individuals

may be found in a field with many stands of thistles.' Those that succeed in 33 finding a host, mate and start feeding and accumulate fat. Oviposition takes place from May to June. It appears that many eggs are laid by a female.

Eggs are laid singly in the under surface of leaves, usually on either side of the midribs. There is a tendency for more eggs to be laid near the apices of leaves in the top half of plants close to thistle flower heads. Incuba- tion is about 15 - 21 days.

Nymphs occur in the field from June to the end of August. The last 5th instar nymph was seen on 28.8.66. Teneral or new generation adults were first seen in the field on 4.8.66. There is considerable overlapping of all stages. The possibility of two broods per year based on the evidence that nymphs occur in June and August as was suggested by Buchanan White (quoted by Butler, 1923) appears incorrect.

Adults and nymphs may be found occasionally on the main stems or axillary buds of thistle leaves. However, they are more often confined to the crevices at the bases of and between the bracts of the involucre and in a small region, of about 3.0 cm, below the latter where they feed in small aggregates. SECTION II: STUDIES ON T. AMPLIATA

1. Description of Stages (a) Adults: The type specimen of Tingis ampliata was first described as

Monanthia ampliata by Herrich-Schaeffer in 1839. Fieber, 1844 has also referred to the genus by the synonym, Phyllontocheila which has since been superseded by Tingis Fab. Taxonomic descriptions and key to the identification of Tingis species are given by Southwood & Leston (1959). The thistle lace bugs, T. ampliata and T. cardui were identified with the key of the latter authors. Furthermore, differentiation between the two species was facilitated by the fact that they colonize separate host plant species in nature (Southwood & Scudder, 1956; and the author's personal observation).

At emergence from the fifth instar, the adult T. ampliata is creamish- white in colour. It gradually turns light-brown and by the second day after emergence, the insect is brownish in colour, except for the presence of small irregular patches of brownish-black mottlings on the pronotum. With age,

varying quantities of a whitish powdery wax are deposited on the brown inte- gument so that the adult usually has a greyish-brown appearance. The upper 35 surfaces of the pronotal margins are covered with short curled hairs. There are four rows of meshes on the marginal areas of the fore wings and lateral margins of the pronotum. In the latter, the number of rows of meshes are reduced posteriorly to two. The adult has a pair of long occipital processes which extend anteriorly beyond the bases of the three (two long lateral and one short median) frontal processes. Details of other morphological characters of adult Tingis are given by Southwood & Leston, (1959).

As in some other terrestrial Heteroptera (Fewkes, 1958; Eyles, 1960; Woodward, 1961; and Anderson, 1961), there is a great variation in body size of individuals of each sex of Tingis. Size differential between the sexes is also marked. Females are generally larger than males, which are narrower across the hemelytra. The hemelytra of the females are more convex and wider. The results. of body length measurements of adults are shorn in Table

10. Bugs were measured under a binocular microscope, fittetith a Micto4 meter eye piece. Body length was taken as the distance (in mm) betweeWthe anterior margin of the rostrum and the tip of the abdomen.

Table 10 Size variation in the sexes of adult Tingis.

1 No. Mean Body length (mm) Range Sex measured + S. Deviation

Females 100 3.73 + 0.25 3.1 to 4.1 Males 100 3.32 + 0.2 3.0 to 3.7 . .

A 't' test showed that the mean body length of the sexes are significantly different, (t = 33.895; P4p.001). The frequency distributions of size in PERCENTAGE PERCE NTAGE FIG. 4. 30

DISTRIBUTION OFSIZEINADULTTNGIS 3 2

BODY LENGTH(MM) 34

44 36 37 the two sexes are compared in Fig.4.

As a result of the range of size found in each sex (Table 10 ); the two sexes overlap considerably in size (see also section II,2(d)iii for data wing length and breadth). Thus, size could not be used as a reliable criterion for separating the sexes in the field. Throughout the present . studies, use was made of the morphological differences in the terminal abdominal segments for distinguishing between the sexeo.Southwood & Scudder, (1956) have shown that the external sex organs of nymphs are reliable characters for separating males from females. In teneral females and in the 5th instar nymph the ovipositor is already developed and its position is marked externally by a raised ridge from the 8th to the 10th abdominal stern- ite. It is clearly visible to the unaided eye (Fig. Except for a small U-shaped groove at its tip, the general outline of the posterior margin of the abdomen of the female is oval. The male has a pair of stout, curved claspers, which bulge out on either side of the lower abdominal segments to form a characteristic shape, rather similar to. that of the lower half of a panduriform leaf (Fig. 5), During field studies on adults, the sexes were identified by carefully turning or tilting the leaf or other plant organ on which Tingis was found, so as to expose the ventral aspect of the insect. Alternatively, the bug was carefully brushed on a white sheet of paper, sexed and released.

(b) Immature stases: Description of diagnostic features and measurements of nymphs (Fig. 6 ) have been given by Southwood & Scudder (1956). The key of the latter authors was used for the identification of the nymphal instars of thistle lace bugs. Some account of the size and general characteristics of Tingis eggs (Fig. 7 ) are given in section II; 3 (a). Fig. 5 Ventral view of abdomen of adult

male female

I·AMPLIA TA

IN STAR I

11 2 T. C AR DUI

)) 4

II 5

VENTRAL VIEW J

Fi g . 6 Nymphs of thistl e lace bugs ( X 6.6) 39 Fig. 7. Eggs of T. ampliata

Unparasitised (X20)

Parasitised by mymarids (X20)

Embedded in midrib; Note operculum and faeces (X62.5)

3mb edded in lea f: Egg in late stage of develop ment. Note loss of operculum and swelling. ( X58.3) 40 2. Aspects of the Biology of the adult

(a) Overwintering Introduction: In the records of Butler, (1923); Reclaire, (1940);

Singer, (1952) and Southwood & Scudder, (1956), it is stated that T. ampliata is univoltine and overwinters in the adult stage. Observations made during the present studies confirm that this is so. Examination of various hibernacula in the winters of 1964/65; 1965/66 and 1966/67 showed that other stages of the insect do not occur in the field during the winter months. Some species of the genus Tingis overwinter in different habitats and sites, often close to or at short distances away from their host plants. For example, T. reticulata HTS. has been reported hibernating in moss (Singer, 1952; Southwood & Leston, 1959; Massee, 1963); T. maculata HTS., under moss and turf and T. geniculata Fieb. in grass tufts (Singer, 1952). Be- sides moss and grass tufts (Southwood & Leston, 1959; Massee, 1963; ), other overwintering sites listed for T. ampliata include, under leaves (Douglas & Scott, 1865; Massee, 1963); litter (Southwood & Leston, 1959; Massee, 1963) under bark (Butler, 1923) and at 'roots of grass' (Douglas & Scott 1865; Goddard, 1934).

Between 10th and 25th March 1965, an attempt was made to ascertain the overwintering sites of T. ampliata. Two types of habitat were selected for this investigation. The first included, The Marsh, Elm Slope, Garrison Ridge and North Gravel; all being areas on which C. arvense grows. The other type consisted of South Gravel and Wood bank from which the weed was absent and which were 41

about fifty yards from North Gravel and the Marsh respectively. The sampling technique adopted was similar to that used by Boyce & Miller, (1953) for the study of hibernating onion thrips, Thrips tabaci Lind. (Thysanoptera: Thripidae). A total of 40 grass/litter samples were taken from randomly selected sites from each habitat on a sampling date. Each sample consisted of the grass mat, litter, and soil contained in a cylindrical steel auger 5" in diameter, pushed down to a depth of 4". Each sample was collected in a 18" x 19" labelled polythene bag, the open end of which was tied to prevent escape of contents. Tingis were extracted from the samples collected on the 10th March by using an apparatus, a simple modification of Berlese funnel as described by Luff, (1964). Extraction was complete in 3 days. On the subsequent occasions, samples were hand-sorted on a white 75 x 75 cm. card board paper. Hand-sorting proved faster. The numbers of T. ampliata collected from the different habitats on four occasions are shown on Table 11.

Table 11: Number of Tingis collected from different habitats in

winter.

No.of Tingis and date Habitat 10.3.65 15.3.65 20.3.65 25.3.65 Total

A:C. arvense present The Marsh 2 1 5 3 11 Elm Slope 1 0 4 1 6 Garrison Ridge 0 0 0 0 0 North Gravel 0 1 1 0 2 B:C. arvense absent Wood Bank 0 0 0 0 0 South Gravel 0 0 0 0 0

42 Tingis was collected from those habitats(except Garrison Ridge) on

which the host plant grew in the Summer preceeding the winter of

observation. Examination of Garrison Ridge in the Summer of 1966,

showed that the host plants did not carry a population of Tingis.

Pearce,(1948) has suggested that the dense vegetation found

at the bases of grass tussocks may shelter from cold many overwinter-

ing insect species. In the next series of investigations, Dactylis

grass tussocks in Group A (see Table 11) habitats were sampled after

the method of Luff, (1964). An account of the microclimatic

conditions in and between Dacqlis tussocks has also be given by

Luff, (1965b). Table 12 shows the number of tussocks sampled and Tingis found on the 20th March,1965. Only tussocks of 4 - 5" in diameter were sampled. Temperature in the centre of the tussocks at

ground level was measured with a mercury in glass thermometer. T.

ampliata, hibernates in the thick mat of organic debris composed of

grass roots, moss and various leaf litter including those of C.

arvense (Tables 11 and 12).

Table 12: Number of Tingis found on sampling Dactylis tussocks from

different habitats in Silwood Park on 20.3.65

No. of No. of Mean No. Mean Habitat tussocks Tingis of Tingis tempt sampled found per tussock (°C)

The Marsh 15 6 0.4 11.31 Elm Slope 11 3 0.27 13.18 Garrison Ridge 10 0 0.0 13.4 North Gravel 18 1 0.05 12.86 43

The bases of grass tussocks in the immediate vicinity of host plants

may also be used for protection in the winter (Table 12).

During the winters of the present studies, the cracks in barks

of various trees; Quercus Epp., Ulmus app and Birch, fringing these

habitats were examined at different times. Neither T. ampliata nor T. cardi►i was found in them.

(i) Autumnal migration

It is now known that in many terrestrial insects, migration represen-

ts an evolved adaptation rather than a reaction to current adversity

(Johnson, 1964,1965, 1966). In some actively flying species, annual pre-

hibernation migration occurs often over considerable distances, from the

breeding to the overwintering sites (see Fedotov, 1944; Brown, 1965; and

Prokopy & Gyrisco, 1965). However, there are other species which have

little capacity for flight and which characteristically colonize more or

less permanent habitats. Since the level of migratory movement is depend-

ent on the degree of impermanence of the habitat, (Atkins, 1960; Southwood,

1962), it is not surprising that in these latter species, pre-hibernation

migration consists mainly of vertical movements to protective hibernacula

at, or close to the bases of their host plants. For example, the Tingids,

Corythuca arcuate Say Var.; Monanthia globulifera Walk; Gargaphia solani

nsp. and Corythuca mollicula Osborn & Drake are said to hibernate on or

close to their host plants (see Comstock, 1878; Sharga, 1953; Fink, 1915;

and Bailey, 1963 respectively). Such species are therefore not truly

migratory in the sense of Kennedy, (1951) and Johnson, (19600.. 44

T. ampliata colonizes the perennial weed, C, arvense, the aerial

shoots of which die annually in late autumn. Newly emerged adults feed on plants for one or two weeks and then start to migrate from the green top

parts of plants to the wilted, brown tissues at the base and eventually

into grass and ground litter. The proportions of total bugs found in different regions of host plants on separate sampling occasions in the

autumn months of the present study are shown in Fig. 8. From Mid-August onwards, the proportion of bugs found in the upper half of plants declined

progressively while that in the lower half increased correspondingly. By

the first week of October of each year, almost all the bugs were confined to the lower halves of plants.

As in many insect species, older individuals, (i.e. post-teneral adults with darkish-brownl hardened abdominal sternites) tended to migrate

before the younger ones, (i.e. those with brownish, hardening abdominal and other cuticles). Thus, on any of the sampling occasions, a majority of bugs found in the lower half of plants were older than those found on the top halves in which younger bugs predominated. It is noteworthy, that at the start, and throughout the early stages of movement towards hibernacula, host plants usually contained a large number of green vegetative tissues, which were generally more abundant on the upper than on the lower halves. It appears therefore, that such movements are not induced by the morphological condition or scarcity of the host. According to

Johnson, (196041,0966) migration is a feature of young, sexually immature individuals, which are not distracted by the need to rest, feed or lay eggs. It is probable therefore,that pre-hibernation movement of Tingis into overwintering sites constitutes an adaptive, ontogenetic process 45

FIG. 8. VARIATION IN THE DISTRIBUTION OF ADULT TINGIS BETWEEN DIFFERENT ZONES OF HOST PLANT IN THE AUTUMNS OF 1965-67 P 20 Ia: Z 15 Lf±-j < o i t 1967 5t

20 . 00 a: 151 NTS A 10 `1. -

PL Z F 5 1966 LF O A H R PE UP N O

1965 LTS ADU

AUG. SEP. OCT. 46 which is controlled by the endocrine system 'in response to environmental

factors' (see Johnson 1965, 1966). This behaviour has significant

survival advantage to the population, since it enables individuals to

reach more protective micro-habitats during early and late stages of the

winter. A similar autumnal movement into hibernacula at bases of host

plants, has been reported in many species including, the non-migratory

Pentatomid, Aelia furficula Fieb. (see Brown, 1965); the curculionid,

Sitona regensteinensis Hbst. by Danthanarayana, (1965) and in Spiders and

other arthropods (Allee et al; 1959).

(ii) Survival (mortality) during overwinterinK

In a species which overwinters before the reproductive phase in its life cycle, the level of the population density after hibernation is

determined partly by the density of the pre-hibernation population and et. al. partly by the mortality during winter (Kiritani,/1966). In the winters

of 1965-66 and 1966-67; Tingis were exposed to various field conditions

and their survival was assessed. Measurements were made of climatic

factors, mainly temperature, rain and snowfall. In the-1965-65 experi-

ments, Tingis were collected in the field on 13th October 1965. They were

weighed individually on a 5 mg•torsion.balance, calibrated in 0.01 mg

divieions; and put in (11+ 1 )pairs in hibernation cages. The cages

were labelled and the weights of the bugs in each .ere separately noted.

Experimental cages consisted of plastic petri dishes, the bottoms of which

were drained with numerous apertures of small diameters to prevent

flooding of cages and ingression of predaceous arthropods. The material of the lid of each cage was removed and covered with fine nylon netting. 47

A small quantity of leaf litter (10 mg) was put in each cage to provide hideouts. The two halves of each cage containing the bugs were held together with a rubber band. A total of 62 pairs were prepared and these were then divided at random into two groups. In one of these, (31 pairs) the cages were put one beside the other on the surface of grass mat on a site in the study area. The other group were similarly arranged at a level, 3" below the grass mat. At approximately monthly intervals throughout the winter the cages at the two levels were examined, mortality Was recorded, and the live bugs were reweighed; and returned to their respective cages. The daily maximum and minimum temperatures on the grass mat surface between cages were measured with a Six's thermometer.

Continuous temperature records at each level were made with weekly recording thermographs in a manner similar to that described by Holmquist,

(1931). Daily minimum temperatures in cages were read with a minimum mercury thermometer which was checked periodically with thermocouples connected to Doran recording potentiometer. Rainfall and snow records were made at the Station's meteorological unit, situated about 100 yards,.

South East of the experimental site.

In the 1966-67 experiment, bugs were collected early in November, weighed and placed for hibernation on grass mat surface in cages identical to those used in the preceeding winter. There were a total of 30 (11

+ 4) pairs. Unlike the 1965-66 experiments, cages were examined and bugs reweighed once, viz at the end of the hibernation period, in April

1967. Meteorological data were also collected during the entire period of the winter. 48 The survival of Tingis kept at the surface and at 3" below the grass surface are compared in Table 13. In both sexes, bugs which hibernated 3" below grass surface showed a higher percentage of survival at each stage and at the end of the experiment; than those which hibernated on the surface. About 74 percent of females hibernating below grass survived the experimental period, compared with only 10 percent of those kept on the surface. This suggests that cover is an important factor in the winter survival of these insects.

Table 13: Survival of male and female Tingis exposed to different field conditions in winter, 1965-66.

' ___...... , 3" below grass Exposed on grass surface Date No. surviving Av. Min. No. surviving Av. Min. Temp. oC - Temp. oC females ' males females males . ..., 13.10.65 31 31 31 31 (original) 0.72 -0.58 30.11.65 29 29 27 25 0.24 -2.97 30.12.65 28 25 26 21 -0.84 -3.55 3o. 1.66 28 24 20 15 2.72 -0.48 28. 2.66 28 21 16 15 30. 3.66 0.42 -5.00 30. 4.66 23 9 3 1 0 . - . % Surviving 29.03 period 74.19 1 9.6? 0.0 49

The difference in the survival of bugs kept at the two levels appears to be associated with variations in climatic conditions. A comparison of the average minimum temperature between consecutive dates on which mortality was recorded (Table 13) shows that the sites 3" below grass were relatively warmer than the surface. It is also probable, that bugs hibernating below the surface, were less exposed to the direct adverse effects of the heavy rains which fell in the last month of the experimental period (Fig. 11). The pattern of mortality (survival curves) of

Tingis exposed on grass surface in the 1965-66 winter is shown in Fig.9, which also includes a curve of the average minimum temperature between consecutive dates on which mortality was recorded. Mortality of Tingis took place in distinct steps which were closely correlated with minimum temperatures. Although the average minimum temperature between 30th Jan. and 28th Feb. 1966; was higher than that in the period from 30th Dec, 1965 to 30th Jan, 1966, there was some female mortality in the former period, probably due to the effect of age. Mortality rate of bugs was gradual from the start of the experiment to the end of February, but increased significantly between February and 30th April. In bugs at the surface, about 46 percent of the total mortality occured in the latter period, in which the daily minimum temperature averaged - 3.61 °C (Limits -13.3 to 5.5°C). The relationship between the percentage mortality per day

(i.e. the number of Tingis which died between any two consecutive occasions, divided by the number of days of the period and expressed as percentage of number of Tingis alive on the first of the two consecutive dates) and the average daily minimum temperature in the same period is shown inFig.10. A dotted line has been fitted to the points by eye.

50 FIG. 10.THE RELATION BETWEEN AVERAGE MINIMUM TEMPERATURE AND MORTALITY OF TINGIS HIBERNATING ON GRASS (WINTER 1965-66)

3.2

• FEMALES it 2. O MALES ••• >- 2.0 s •

1.2 0

0•8 • ,C.) 0 E a

0.0 -8 -6 -4 _2 0

AV. MN1MUM TEMPERATURE (°C.)

FIG. 9.THE SURVIVAL OF TINGIS KEPT IN CAGES z ON GRASS OVER WINTER 1965-66 LU

,,u

0 • • 8 c, _2 • • • • z 6 .q -4 < 0

100 •--• FEMALES 80 0-0 MALES

ce D 40 Lel

°43 20

0 40 so • Ilo DAYS (13 OCT. : DAY 0

51 FIG .11. MEAN MAXIMUM WEEKLY RAINFALL IN SILVVOOD PARK (AUTUMN - WINTER 1965 -66) ""is 60-

`1-

401-

LW 30'

• 20

E 10

0 AUG. S • T. NOV. DEC. JAN. FE R. PR. 1965 1966.

FIG. 12 ,DIFFERENTIAL MORTALITY IN THE SEXES OF TINGIS HIBERNATING IN DIFFERENT HIBERNACULA (WINTER 1965 —66) o ON GRASS • BELOW GRASS 0

80 >- NY : 6.7984 + 11314X 0-1 70

%N./ I— 60 • U

ISO-MORTALITY LINE

w • //f/ 30 • • o w EZci 20

• /0

30 40 50 60 70 80 FEMALE MORTALfTY PER CENT (ANGLES): X 52 Owing to the wide scatter of the points, which probably is a reflection of the effects of intrinsic and other extrinsic factors on Tingis mortality, no attempt was made to calculate a regression equation. However, inspite of the scatter of points, data on Fig.10 show that under the conditions of the experiment, the rate of mortality tended to increase as the minimum temperature decreased.

In Tingis hibernating in the winter of 1965-66 the females showed a higher percentage survival at each stage and at the end of the period.

This differential mortality of the sexes became more marked in the latter stages i.e. between the end of February and April, when both sexes suffered the highest mortality, A comparison of mortality in the two 46. sexes is given in Table 14. AZ squared test shows that mortality was higher in males than in females in bugs kept at 3" below the grass surface ( 2,2 = 5.507; P40.02) 0.01) .

Table 14: Mortality of the sexes of Tingis kept 3" below surface in winter

1965-66.

2 Sex Females Males Totals z No. at start 31 31 62 5.507 40.02;>0.01 No. dead at 8 22 30 end

Bugs kept on the surface in the same winter, 10% of the females survived the experimental period and all the males died. This difference was however, not statistically significant, (Table 15). 53 Table 15: Mortality of the sct-es of Tingis kept on grass surface in

winter, 1965-66.

Sex Females Males Totals

No. at start 31 31 62 >0.95 but 0.0782 <0.90 No. dead at end 28 31 59 NS.

In natural conditions Tingis hibernate in concealed hibernacula below the grass surface. The non-significance of the difference in mortality of the sexes in Table 15 seems to suggest that under conditions of exposure to long and severe climatic conditions, (e.g. the bugs kept at the surface), the survival rate of the two sexes tends to be identical. Thus, although there is differential mortality of the sexes in hibernating

Tingis, this difference tends to be reduced in severe winters in which climatic factors account for a very large proportion of the total winter mortality. The relationship between male and female mortalities are shown in Fig.12, which is based on the experimental data obtained in the winter 1965-66. Percentage mortalities were transformed to angles and used to calculate a regression equation; Y = 6.7984 1.1313662X (r

0.945; p = 4:0.001); where Y and X are male and female mortalities respectively. The fitted regression equation shows considerable departure from the iso-mortality line (broken line in Fig02), above which all the points lie. Even if female mortality had been zero, about 7 percent of the males would still have died under the conditions of the experiment.

It should be noted that this order of difference in mortality is much lower than that which may actually occur during a winter in a population 54 of naturally hibernating Tingis which move down to considerable depths within grass-litter (See Tables 13 and 14). Differential winter mortality of the sexes further results in an excess of females in the post-hibernation population in spring. Differential winter mortality of the sexes is known in some species of Heteroptera including Pentatomids (Fedotov, 1947; Brown, 1962; Kiritani, et al 1966) and Anthocorids (Anderson,1962). The relative roles of intrinsic and extrinsic factors in determining differential mortality of the sexes of some species of Het- eroptera are discussed in Fedotov, (1947) and Kiritani et al, (1966).

A comparison of the mortality of bugs kept on grass surface from 30th Nov. to 30 April in the two winters (Table 16) shows that survival was slightly higher in the 1966-67 winter.

Table 16: Survival of Tingis kept on grass surface from 30th Nov. to 30 April in two consecutive winters.

No. alive on No. alive on % Surviving on 30th Nov. 30th April 30th April Winter

1965-66 27 25 3 0 11.11 0.0 1966-67 30 30 7 3 23.3 10.0

The total rainfall (in mm), mean air and grass temperatures during the

period of experiment are shown for each winter in Table 17. 55 . Table 17: Rainfall, air and grass temperatures in Silwood Park.

min. Total rainfall Mean air Mean/grass Period (mm) temp. C temp. °C.

30th Nov. 1965-30th April 1966 261.3 5.7 ... 3.0 30th Nov. 1966-30th April 1967 262.9 6.08 2.85

The slight difference in survival of bugs kept out of doors in the two winters, (Table 16) is apparently associated with the similarity of climatic conditions in both seasons. Table 18 below, shows the number of days of snow and days on which the ground was frozen in each month of the two winters.

Table 18: Comparison of climatic conditions during winters 1965-66 and 1966-67.

No.e of days on which No. of snow days Month ground was frozen 1965-66 1966-67 1965-66 1966-67

Oct. 0 0 0 Nov. 7 0 0 0 Dec. 6 3 0 0 Jan. 9 11 Feb. 0 4 0 0 Mar. 0 0 0 0 Apr. 0 0 2 0

Total 22 18 9

Holmquist, (1931) and Luff, (1964) have demonstrated, that in the 56 absence of a long period of snow cover, microclimatic conditions in various hibernacula are closely correlated with those of the atmosphere. Although, snow was recorded on 9 occasions in the 1965-66 winter, it did not cover the ground or the insect cages for more than a few hours at a time before melting. It is probable, therefore, that climatic effects on bugs in hibernacula below the surface were identical in the two winters, It will be shown in section 1;4w that winter disappearance of adults was slightly higher in 1965-66 than 1966-67.

(iii) Weight loss during overwintering

The works of some authors (Anderson, 1962; El-Hariri, 1966) have shown that different unrelated insect species, lose a substantial amount of their autumn body weight during hibernation in winter. The mean live weights (in mg) of Tingis which. hibernated in cages 3" below grass surface in the 1965-66 winter are shown in Tablesl9 and 20. The pattern of weight loss in the two sexes is shown graphically in ig.13: Weight loss on any date was taken as the difference between the mean weight of bugs at the start and their mean weight on that date. The loss in weight is expressed as percentage of original weight. Both sexes progressively lost weight during hibernation. Weight loss occurred in distinct steps or stages which were also identical in the two sexes (rig. 13). 57 Table 19: Weight loss of 30 females hibernated in grass from /3.10.65 to 30.4.66.

Date Mean weight Weight loss Percent weighed (mg) + S.D. (mg) weight loss

13.10.65 (orig.) 1.88 + 0.12 - - 30.11.65 1.72 + 0.2 0.16 8.51 30.12.65 1.71 + 0.21 0.17 9.04 30. 1.66 1.66 + 0.16 0.22 11.7 28. 2.66 1.64 + 0.4 0.24 12.76 30. 3.66 1.63 + 0.13 0.25 13.29 3o. 4.66 1.6 +_ 0.17 0.27 14.36

Table 20: Weight loss of 30 males hibernated in grass from 13.10.65 to 30.4.66.

Date Mean weight Weight loss Percent weighed (mg) ± S.D. (mg) weight loss

13.10.65 (orig.) 1.43 ± 0.1 .11.• 30.11.65 1.31 ± 0.11 0.12 8.39 30.12.65 1.28 + 0.13 0.15 10.48 3o. 1.66 1.25 + 0.19 0.18 12.5 28. 2.66 1.23 + 0.15 0.2 13.98 30. 3.66 1.228 + 0.2 0.2 14.12 3o. 4.66 1.22 + 0.06 0.21 14.68

In both sexes the rate of weight loss was high from October to

January, and about 85.6% of the total weight loss in either sex, had occurred by 30th January. This high initial loss was probably mainly

FIG.13. THE PATTERN OF WEIGHT LOSS IN HIBERNATING TINGIS WINTER 1965-66 0.3

(34125

0 OOP 0.2 U, 1.7.1 z t 0.15

I5 -

Lj 40 80 120 160 200 240 280 DAYS (131065 :DAY 1) .59 due to dehydration of body tissues and/ or elimination of gut contents which are known to preceed and persist in the early stages of hibernation in some insect species (See Payne, 1929; Anderson, 1962). Loss in weight was small between the end of January and March, and then increased substantially in the period between this month and 30th April. These high losses in weight during the late stages of hibernation, apparently arose as a result of further breakdown of body fat especially at the relatively high temperaturesbetween March and April. Males tended to lose weight at a slightly faster rate than the females, as is indicated in Fig.13. The difference in percent weight loss in the sexes was not apparent until 48 days after the beginning of the experiment (Fig.13). In bugs hibernating on the surfade of grass in the winter of 1966-67, females and males lost 16.9 and 19.7% of their body weights respectively

(Table 21).

Table 21: Weight loss of 30 males and 30 female Tingis, collected on 30.11.66 and hibernated an grass till 30.4.67.

Mean wt. (mg) Mean wt. (mg) Wt. loss Percent Sex on 30.11.66 + S.D on 30.4.67 + S.D (mg) weight loss

Females 1.69 + 0.14 1.41 + 0.11 0.28 16.86 Males 1.33 + 0.12 1.07 + 0.08 0.26 19.71

Owing to the thermal stratification which occurs in grass (Section,

(ii) above; table 13) and other hibernacula, (Holmquist, 1931 ) it is

probable that the metabolic rates; and hence the rate and amount of

weight loss by non-feeding hibernating insects depends on the stratum of 60 the hibernacular profile at which the insects spend a large part of the

winter. The wide variation in depth of grass and/or litter from which

Tingle were collected in the winter months suggests that the optimum

stratum varies with the time of winter according to the severity and

adversity of climatic conditions.

In some Scutellerid speciesf e.g. Eurygaster integriceps Put. (See

Fedotov, 191+7; Biryukova, 1960; Brown, 1962) and Aelia rostrata (See Brown,

1962), differential winter mortality of the sexes has been associated with

a higher rate of exhaustion of metabolic reserves in males. In T.

ampliata, males lose a higher proportion of their body weight than females

during winter. Furthermore, the females are larger in size (see section

II, 1), and heavier than males (Tables 20 & 21). It is probable that the differential mortality of the sexes in Tingis (section ii above),is also

associated with the apparently higher proportion of body weight loss in

males, as has also been observed in some Coccinellid species (El-Hariri,

1966). Body size and weight constitute intrinsic factors which are known

to affect the winter mortality of the sexes of the green stink bug,

Nezara viridula L. (See Kiritani et al, 1966).

(iv) Cold Hardiness

In many temperate insect species, the size and quality of the field

population in spring is determined by the density and composition of the pre-hibernation population as well as the ability of the overwintering

autumn individuals to survive exposure to sub-zero temperatures over varying

lengths of time, (Brown, 1962; Kiritani et al, 1966). Such temperatures 61 are often less extreme and higher than the undercooling point; which is the temperature at which the insect instantaneously freezes to death;

(Salt, 1936).

The apparatus used for testing tolerance of low temperatures by adult

Tingis, consisted of a soft-wood cabinet; (30 x 20 x 20 cm) which was divided into four compartments with expanded polysterene cross-boards.

Compartments were lined with a thin layer of cotton wool, to improve insulation between them. The cabinet was attached to a corner of a deep freeze. A temperature gradient varying from - 2.0 °C at the top to

- 13.0 °C at the bottom compartment was thus set up. Intermediate compartments had temperatures of - 4° and-8.e °C respectively. Tingis of both sexes collected in the field on 19th October, were put in equal numbers in petri-dishes lined with moist filter paper in each,of the four compartments (two dishes per compartment). Thermocouples were attached to all four compartments and with the aid of a Doran Mini recording potentiometer, the corresponding temperature, (°C) in each compartment was separately read from a'Voltage-Temperature' table.

Daily records of mortality were kept. On each recording occasion, dishes were brought out of the cabinet and allowed to warm up to room temperature ( = about 20 °C) for an hour. Tingis which were still incapable of a withdrawal reaction after 10 and 5 prods with a fine brush at each tarsus and antenna respectively were recorded as dead. Dishes were returned to their appropriate compartments after mortality was recorded. The results of exposing bugs to the various temperatures for periods up to 32 days are expressed graphically in Figs 14 and 15. The FIG. 11+. SURVIVAL OF ADULT TNGIS AT SUB-ZERO TEMPERATURES A : FEMALES

100

0....<0 -•-•-•-•-• 0 '''' .

\ \• .1==11, • .=l1P . N . 80 \0-0 - \ 1 •N

0 \ 0 60 \

0-0 • • • • • • • , moo— 0s. b-o \ 0

\ 0 N C.,. • 0— 0--0— 0 ‘ 0 `oc•N 8 t _13°C. o- 0,- . , a • 1, I 4 8 12 16 20 24 28 32 TIME (DAYS) AG. 15. SURVI\AL OF ADULT TINGIS AT SUB-ZERO TEMPERATURES B 1(MALES)

m••• • ••• • am, • .i. • • i• •\%. 1 •.i• • • MEr • iM. , • Gm • ,r/••• 1 • 411•Ml• • 41• • MI• ..., • •••••• • 1 0 .\ \ • • N., , •\.,. 0—\ S • • • •

• 0—. 0 4\--.\\ • • • —4 et. 0.—.0—cos,

0-0‘

. 1 = 8 12 16 20 24 28 32

TIME (DAYS) 64 figures show that in each sex, mortality increased with decreasing temperatures from - 2.0° to - 13.0 °C. In Tingis kept at - 13.0 °C, mortality after one day was about one-third of that after 4 or subsequent days; thus suggesting that relatively short exposures to temperatures of this magnitude could cause considerable adverse effects on Tingis populat-

ions. In both sexes, mortality tended to be gradual and took place in distinct steps in the three higher temperature regimes. This suggests a tendency towards acclimatization. Although there are insects species which do not show acclimatization when conditioned at moderately low temperatures (Salt, 1956a; Atwal, 1960); the works of Payne, (1926b), Mellanby, (1939) and Colhoun, (1960) suggest that the behaviour is common to a wide variety of species. There appeared to be no obvious significant difference between the sexes in their ability to survive low temperatures in the range of - 2.0° to - 13.0 °C (Table 22).

Table 22: Survival of male and female Tingis exposed to - 13.0 °C for 4 days.

Sex (;' Totals P No. exposed 25 20 45 > 0.1 0.7962 but No. dead 21 19 40 4:0.5

However, in the bugs kept at - 13 00, all the males had died by the 9th day whereas 5% of females remained alive. Similarly, 5% of females kept at - 8 °C were alive on the 26th day of exposure while all the males died by the 23rd day. These observations suggest that a higher proportion 65 of females tend to tolerate longer periods of exposure than the males. The

duration of exposure may therefore be more important in determining differ-

ential survival of the sexes than the actual temperature to which bugs are

exposed.

(v) Seasonal variation in undercooling point

In order to find out to what extent adults can withstand and survive,

the low winter field temperatures, their cold death point was investigated.

In many insect species cold death arises from freezing of the body fluids.

It is known however, that some species can supercool below the true freezing

point of their body fluids, with death occurring instantaneously only when

they freeze at a temperature Called, the 'undercooling point', (Salt, 1936).

The undercooling point is therefore a reliable index of an insect's cold-

hardiness (Salt, 1956b).

lJndercooling point studies were carried out in the winter of 1965-66.

To find out whether there was any seasonal variation in the undercooling

point of Tingis, determinations were made once every month throughout the

winter. Data for individuals of each sex were recorded separately on each

occasion. Some methods of determining the supercooling points of insect

species, consist of measuring the insects body temperature thermoelectric-

ally as it freezes in alcohol or other similar liquid, cooled by a cooling

coil (Salt, 1956) or by adding solid carbon dioxide (Way, 1960; Sullivan

and Green, 1964; Stenseth, 1965). In the present studies, Tingis were

cooled thermoelectri lly on a 'frigistor' (De la Rue Frigistors Ltd.) as

described by Luff, (1964). On each day of determination of undercooling

point, bugs collected in the field were immediately tested in the laboratory 66 to avoid-lees in field--acquired cold-hardiness. Each test specimen was put into a dry, glass probe (3 mm, internal diameter), with its anterior region facing the rounded end of the probe. The thermo-couple wire was inserted into the small space between the hemelytra and the abdomen. Care was taken to avoid piercing the insect. This latter caution coupled with the use of a separate dry probe for each specimen, helped to prevent innoculative freezing. The probe containing the test insect was put in place on the frigistor, and as the bug cooled, its temperature was recorded on a Rustrak recording ammeter, which has a built-in D.C. amplifier. The lowest point on the cooling curve at which the temperature suddenly rose due to the release of the latent heat of the cooling insect was recorded as the undercooling point. With a current of 3 amps. passing through the frigistor, it took on the average about one minute to cool to - 15° and cooling to about - 35 °C was achieved. Salt, (1961, 1966a) has pointed out that the supercooling point of a species is not affected by a rate of cooling which is faster than about 1 degree per minute. Before making determinations on each occasion, the recorder and frigistor were calibrated by taking the temperatures of dry ice and the room, first with a standard mercury in glass thermometer and then with the thermocouple probe.

The mean undercooling points (based on 25 replicates per occasion) for the two sexes and for the winter months are shown in Table 23 and expressed graphically in Fig 16. Vertical bars in Fig 16 show the range of undercooling point observed on each date. Tingis can supercool to very low temperatures in winter; and there was a wide degree of variation in the cold-hardiness of individuals of either sex. A range of - 22.0 to - 31.5 o °C and - 22.2 to 31.4 C was recorded for females and males respectively 67 FIG. 16 .SEASONAL VARIATION IN UNDERCOOLNG POINT OF TINGIS

-10 FEMALES T o MALES

L.7 J-20 0 0 cc W e _25 z

-30

a NOV. DEC. a JAN. I FEB. MAR. APR. 1965 1966

FIG. 17.THE RELATIONSHIP BETWEEN UNDERCOOLING POINT OF TINGIS AND AIR TEMPERATURE -10-

au -15 Y - 24 8857 + 0 5373 X r: 0547 p< 001

G • N I - 20 • • RCOOL NDE U

Z -25

-30 -10 -5 0 5 10 15 MEAN AIR TEMPERATURE C3C : X 68 in December.

Table 23: Undercooling point of Tingis; winter 1965-66.

Undercooling points °C.

Mean Range Date Lowest highest

1g to 15.11.65 -23.4 -24.2 -29.0 -27.0 -19.0 -21.8 15.12.65 -27.8 -26.59 -31.5 -31.4 -22.0 -22.2 15. 1.66 -26.0 -25.5 -28.0 -27.0 -22.0 -21.0 15. 2.66 -20.0 -19.88 -24.5 -22.0 -16.5 -17.0 15. 3.66 -18.88 -20.0 -21.0 -22.0 -15.5 -16.0 28. 4.66 -20.5 -20.5 -22.5 -22.5 -18.0 -18.5 •

There was a distinct seasonal variation in cold hardiness of Tingis. Cold-hardiness increased in both sexes from November to a peak in December, then decreased progressively through the other winter months till the onset of spring. The small increase in cold-hardiness which was observed between March and April, was probably associated with the heavy snows of late-April which followed a relatively dry and warm March. Many authors, including Payne, (1926b); Yuill, (1941); Luff, (1964) and Agwu, (1967) have reported the occurence of seasonal variation in the cold-hardiness of widely unrelated insect species. The mean undercooling points of Tingis shown in Fig.16 were positively correlated with the mean air temperatures for those dates on which cold-hardiness was measured, (r = 0.946; P0.01) (Fig.17). The calculated regression equation, relating ambient temperature 69 to undercooling point, is: Y = -24.88572 4, 0.537399X; where Y = mean undercooling point (°C) calculated from several determinations, and X is the mean air temperature (°C) in the field for the same day as that on which the undercooling point determination was made. The data in Fig.17 are based on the undercooling points of females. Since there was no apparent significant difference in the cold-hardiness of the sexes (Table 24 below & Fig. 16). the regression equation above may also apply to males. As ambient temperatures are usually correlated (in the absence of a long period of snow cover)

Table 24: Comparison of the mean undercooling points of the sexes of bugs tested on 15.11.65.

No. Total Mean Sex undercooling undercooling Significance tested pt. (°C) pt. (°C)

Females 25 -586.4 -25.456 t = 1.2788 P710.1 Males 25 -605.2 -24.2 with temperatures in grass (See Mail, 1932; Luff, 1964), it is probable that a positive correlation may also be found between undercooling point of Tingis and the temperature of its grass hibernacula. The regression equation of such a correlation (if found) could prove even more important than the one calculated above. The results shown in rigs.16 and 17 suggest annual variations in the monthly minima mean undercooling points of the overwintering insect, since annual variations in monthly intensity and quantity of cold are common. 70 (vi) Other Factors affecting undercooling point

The factors now known to affect the undercooling point of a species include, (a) the rate of cooling (already referredto), (b) the moisture content of the insect, (Salt, 1956a), (c) the glycerol content of the insect (Lauritz, 1964; Somme, 1964; Sullivan, 1965), (d) the nature of the surface with which the insect is in contact, (Hudson, 1937; Johnson, N. E. 1967) and (e) the presence or absence of food in the insect's gut, (Salt, 1953). In the present studies, the effects of the two last factors on the undercooling point of Tingis were investigated.

(a) Nature of the contact surface Four sets of undercooling point determinations, (3 experimental and 1 control) were made on females on 18th February, 1966. In one of the three experimental sets, each test bug was placed in contact with a 2 x 5 mm piece of dry, dead Holcus leaf in the probe. It is mentioned in section t,260 that Holcus is the predominant grass species in the study area. In the second set, bugs were stood on live, green Holcus leaf and in the third set, they were placed on pieces of wet, dead leaf. Control insects were tested dry without any organic substrate. The results are shown in Table 25.

Table 25: Undercooling points of female Tingis in contact with different surfaces.

No. Mean Underooling 95% Confidence Treatment tested point (°C) interval

Controls 11 -20.0 -15.47 to .•24.53 Dry dead Holcus leaf 11 -19.88 -17.63 to -22.13 Living, green Holcus leaf 10 -18.19 -14.96 to -21.42 Wet, dead Holcus leaf 10 -15.22 -11.85 to -18.59 71

Neither dry, dead nor living, green Holcus seemed to have altered the undercooling point of bugs, as compared with control insects. It is note-worthy however, that contact with the latter surface i.e. living green leaf, has decreased the range, thus suggesting that inoculation occured in some determinations but not in others. There was a significant difference, (t = 5.9-

587; P4.0.001) between the mean undercooling point of the control bugs and those placed in contact with wet dead Holcus leaf during test,tt is of ecological significance that the latter type of surface raised the undercooling point of Tingis. In the field, the bugs are more likely to come frequently in contact with wet than dry organic surfaces. However, data prevented in section II,2(a)ii show that the minimum temperatures of hibernacula may not fall as low as the highest undercooling point that may arise through contact with wet surfaces.

(b) Presence or absence of food in gut

A number of bugs of each sex were placed for hibernation in grass in the 1965-66 winter; (section II,2(a)ii above. On 24th April, 1966, the hibernation cages were examined for survivors. 80 survivors of each sex were randomly divided into two groups. The 40 bugs in the first group were then randomly divided into two sets of 20, one of which was caged in pairs on potted plants in the field and the other left in similar cages without

C. arvense, as controls. The undercooling points of bugs that had access to food and those caged without food were determined after 24 hrs. In the second group of 40, half of the insects were caged on plants for 48 hrs and the other 20 were caged without food. After the 48 hn period, the under- 72 cooling points of individuals of both sets of bugs were determined on the frigistor. All experimental insects allowed to feed for either 24 or 48 hours, were dissected after their undercooling points had been determined. Bugs, that had chlorophyll in their guts were classed as fed and those without chlorophyll in their gut contents as unfed. The number and proportion of bugs of each sex which fed in the experimental periods are shown in Table 26.

Table 26: Proportions of hibernated Tingis which fed after spending various times on host plant.

Time spent No. Unfed No. fed % fed on plant (Hrs) Sex

24 4 16 80.0 6 14 70.0

48 0 20 100.0 1 19 95.0

Results of determinations of undercooling points of experimental and control insects are shown in Fig. 18. Open dots, indicate the mean and vertical bars, the range of undercooling point for each set of determinations.A t test has been used to compare the undercooling points of bugs which were provided with food and those which had no food (Table 27) below.

Table 27: Comparison of mean undercooling point of unfed female Tingis and

those caged on plants for different periods.

FIG.18. EFFECT OF FEEDING ON THE UNDERCOOUNG P0111 OF MPS _5

-10

_15

C.9

FED 0 _20 0 48 HOURS J. z

-25 UNFED FED UNFED 24 HOURS

11z

-30

TESTED . 25 466 26 4 66

35 74

Total Mean Tine spent on under - under- Sioilificnnce Treatment host plant cooling cooling (hrs) point (°C) point (c)c)

Food unavailable -410.8 -20.54 t = 2.1946 available 24 -387.8 -19.39 p < 0.05

Food unavailable -408.4 -20.42 t = 3.8412 " available 48 -270.0 -13.5 P 4; 0.001

Unfed, control bugs were more cold-hardy than bugs which were provided

with food. This difference was more marked in insects that had access to

food for 48 hrs.than in those caged on plants for 24 hrs.(Table 27). After

24 hours, 80% of the females and 70% of the males had fed compared with 100%

and 95% respectively for insects which had access to food for a 48 hour

period. It is probable that the higher significance of the difference in cold-hardiness of control insects and those which had access to food for 48

hours, was due to the higher proportion of individuals which had fed in the

latter period as compared with 24 hours (Table 26). Owing to the small

numbers of individuals not feeding among bugs caged on host plants, attempts

were not made to compare the undercooling points of those with and without

chlorophyll in their guts in each group of insects.

(vii) Pre-emergence feeding

Host plant emergence in Spring usually preceeds that of overwintered

adult bugs. However, when a spring is preceeded by a relatively warm and

dry winter, insect and host plant may emerge almost simultaneously. When

this happens, as in spring of 1967, some individuals may feed on litter- 75 covered, etiolated thistles. The guts of some of the bugs dissected on

23.4.67. (Table 28) contained light-yellowish ingesta and matter, probably chromoplasts or carotinoids.

Table 28: The condition of the guts of bugs collected in grass on 23.4.67.

Sex No. No. No. dissected unfed fed fed

Females 20 12 8 40.0 Males 20 9 11 55.0

The gut contents of TinRis that have fed on aerial shoots of thistles often include large quantities of chlorophyll, which give the faeces a characteristic dark-greenish hue. "The chlorophyll of plants, for the most part, passes unchanged through the gut of phytophagous insects", (Wiggles- worth, 1961). 76

(b) Mating behaviour

(i) Season Although males dissected in autumn showed evidence of spermatogenesis; mature spermatozoa were not formed until about March-April, i.e. some weeks be-

fore emergence from hitmtnntion LSection (c)below). In general, pairing adult' were often observed in the first two weeks of active feeding following post- hibernation emergence, but anatomical investigation of females indicated that mating may start long before activity is resumed in April May (section (a)vi below). Evidence of in situ feeding before emergence from hibernation in

some years is presented in section II,2(a)vii above. Such in situ feeding may provide individuals with extra energy for searching for mates. "A behaviour pattern for the acquisition of food may be in part transformed by evolutionary processes into acquisition of mates" (Allee et al, 1963, p. 690).

In the spring and summer of the present studies, different habitats on which C. arvense grows in Silwood Park were searched for mating TinEis. Searching on each habitat was limited to half an hour each day; usually in the period between, 9 a.m. and 8 p.m. Table 29 shows the first and last dates on which mating was recorded in the field.

Table 29: Dates of first and last pairing in the field.

Mating first Max. Temp Mating last Max. Temp Year observed C. observed C.

1965 6.5.65 14.25 9.7.65 16.75 1966 26.4.66 15.0 3.6.66 24.25 1967 28.4.67 16.5 20.6.67 18.5 77 Pairing was first recorded on Hill Copse at 10.30 a.m. in 1965, on the

study area in 1966 at 1 p.m. and in 1967 at 12 noon. Since pairing occurs

soon after the post-hibernation emergence of Tingis,annual variations in

dates of emergence appear to be closely associated with those of first

pairings. Post-hibernation emergence and pairing were earlier in 1966 and

1967 than in 1965 (Table 29). Close annual synchronization of Spring

emergence and pairing has also been reported in some other insect species

including Coleoptera (See Siew, 1963; Danthanarayana, 1965; Zwolfer &

Harris, 1966) and Heteroptera (See Grasse, 1951; Fewkes, 1958).

(ii) Time of day

Many authors, including Rendel, 1945; Wallace & Dobzhanskyi1946; Bates, 1949, p. 55; Spieth & Hsu, 1950 and Myers, 1952; have published works which

suggest that insects exhibit a daily copulatory rhythm during the mating

seasons. Most of these studies indicate that insects could be conveniently

grouped into two main categories according to whether or not their mating

takes place in light or in darkness. Indeed, Wallace & Dobzhansky, (1946)

have suggested that for a species which may engage in normal copulatory

activity under a wide range of light intensities, there is always an optimum

illumination.

During routine sampling of adults and of immature stages of Tingis in

1965-66, records were kept of the precise time of mating in the field.

Further observations were made on non-sampling days generally between 8 a.m.

and 9 p.m. The results are summarised in Table 30. Records of more than

one pair at anytime indicate that the insects were seen simultaneously on

the same plant or on two separate but adjacent plants inspected within ten

minutes of one another. 78 Table 30: No. of mating pairs recorded at different times of day during seasons, 1965-66.

Time of day and number of pairs seen a.m. p.m. Date 6.7.8.9.10.11.12.1.2.3.4.5.6.7.8.9 7-5-65 1 9-5-65 4 17-5-65 2 2-6-65 1 10-6-65 9-7-65 1 26-4-66 2 27-4-66 1 28-4-66 2 3-5-66 2 10-5-66, 1 3 18-5-66 19-5-66

3-6-66 1

The time of mating was identical in the two years,i.e. Tingis may mate in the field at any time of day between 6 a.m. and 9 p.m. During the mating season, considerable diurnal fluctuations occur in field climatic conditions, especially in light intensity, humidity and temperature. The extent of the hourly variation in temperature is illustrated in Fig.19 which is based on records for 9th May, 1965 and 10th May 1966; dates on which four and five mating pairs respectively were found. In general, temperatures tend to rise gradually from about 8 - 12 °C. at 7 a.m. to a peak at 12 coon or early afternoon and then drop progressively to 5 - 9 °C. at 12 p.m. Table 30 suggests that there is a tendency for incidence of mating to be highest 79

RG19..HOURLY FLUCTUATIONS OF FIELD TEMPERATURE IN MAY

I5 Zl

9565 a_ d

10 5 66 5

0 7 8 9 10 II 12 1 2 3 4 5 6 1 8 9 K) II 12 AM PM

Time of day

Fig • 29 Dorsal and ventral views of Tingis in copula 8o between 12 noon and 3 p.m. when temperatures were about 14 - 16 °C. It is possible, that this tendency is brought about by increased probability of contact between mates as a result of higher activity induced by relatively high ambient temperatures. It will be shown in section II12(f) that adult distribution on host plants shows diurnal fluctuations.

In these studies, no attempts were made to observe mating in Tingis at night in the field. However, mating was frequently observed in hours of light but not of darkness in the laboratory at 20 °C. and with 16 hours photoperiod. It is shown in section (v) below that the mating process in

Tingis lasts several hours in light. Thus, individuals may continue copulation from late afternoon until late at night.

(iii) Body orientation in copula Excellent records on the great range of variation in copulatory positions normally assumed by the sexes in Heteroptera in particular and arthropods in general have been published by Grassee, (1951) and Alexander, (1963) respectively. The latter author has pointed out that various groups within the Heteroptera show one or other of the different types of body orientations (with the exception of two) normally found in terrestrial pterygotes today. The two exceptions are: (1) female above; 9,venter tot' dorsum and (2) venter to venter, faring the same way. Unlike Richards, (1927) who reports that some kind of male above position is primitive in the pterygota, Alexander,

(1963) maintains that the primitiVe position was the female above; or male- side; in either alignment, with the male reaching under the female's abdomen.

During the spring - summers of the present studies, Tingis collected in 81 the field for laboratory dissections and experiments often paired in petri- dishes within periods up to 30 minutes or more. The relative positions of the wings, abdomen and general orientation of each sex of every pair was recorded. Positions of the frontal processes and median terminus of hemelytra of the sexes of a pair were marked on a sheet of paper, and the two points corresponding to the longitudinal axis of each sex were joined. The resultant line was produced in either direction and each of a pair of vertically opposite angles between two axes was measured with a protractor; and the average was recorded. Table 31 shows the number of pairs measured in each year and the mean of the average angle (in degrees) between each pair, together with their standard deviations. The mean angle between pairs was identical in the two years. It was 69.04° in 1965 and 68.33° in 1966.

Table 31: The angle between mating pairs and the relative positions of

their wings and abdomen.

. Position of male Mean angle No. of ) between Year (0 Abdomen Wings pairs pair + S.D. Dorsal Ventral Dorsal Ventral a ,,- 1965 25 69.04 + 3.24 0 25 23 2

1966 20 68.33 ± 5.00 0 20 20 ! 0 .

The dorsal surface of the tip of the male abdomen was always in contact with the ventral aspect of that of the female (Pig.20). During copulation, the short curved, pointed-tipped claspers are used to grip the female. A thin, whitish, threadlike aedeagus may be seen when the sexes are gently 82 teased apart under the microscope. In 55% of the pairs observed, the males

were to the right of the females. With the exception of two pairs in 1965,

the male hemelytra were always dorsal to the females.

The copulatory position described for T. ampliata above is maintained

from the start to the end of the process. It represents a step further in

the advancement of the primitive female-above position in Alexander's,

(1963) classification; in which venter to venter (facing the same way)

mating ranks highest in the evolutionary scale. Ghilarov, (1958) and

Brinck, (1958) have pointed out that 47 - side (reaching under 1g) position

as well as its modifications lends credence to the probable "derivation cf

pterygote mating from an act involving indirect spermatophore transfer via

the substrate and a ventral femabm genital opening".

(iv) Frequency of copulation It is shown above that Tingis mate from late April or early May till

the end of June (Table 30 ). This corresponds with the main period of

oviposition and this suggests that copulation may occur more than once

during the season. However, a large proportion of males dissected after May,

had no sperm. It is possible that individuals that emerge late also mate as

late as June. In males, post-hibernation death rate is very high and it is

probable that many mate only once, and die soon afterwards.

(v) Duration of copulation

In some Tingids, the process of sperm transfer from males to females

often takes several hours (Wigglesworth, 1961). In the Homoptera and

Heteroptera, this duration is variable (Grassee, 1951). An attempt was 83 made in 196, to ascertain the duration of mating in Tingis under laboratory conditions. Groups of adults collected in the field were kept in petri- dishes. As soon as a pair was formed, it was carefully transfered to a separate dish containing a mall thistle shoot, and the precise duration of copulation was recorded. Each mating pair was examined at one minute intervals. Termination of copulation was indicated by the separation of the sexes. These data on 18.5.65 are summarised in Table 32). It was impracticable to examine more than three pairs concurrently. Laboratory temperatures varied from 18 - 20 °C., and the relative humidity was about 67% during the experimental period.

Table 32: Duration of mating in Tingis.

Pair Time of start Time of end Duration (in hours) No. of mating of mating

1 12.40 p.m. 8.42 p.m. 8.03 2 1.00 p.m. 9.00 p.m. 8.00 3 2.00 p.m. 12.00 p.m. 10.00

Although only a few observations were made, data on Table 32 suggest that in Tingis, as in other Heteroptera, mating is of long duration (8 - 10 hrs). No attempt was made to estimatig the duration of mating in the field.

However, it appears improbable that it would be significantly different from that in the laboratory. 84

(i) Parts of the _plant in which eggs are laid

(a)In the field: There is considerable evidence to show that oviposition behaviour in the Tingidae is very similar to that of the.Miridae; the, smooth unsculptured eggs; are generally laid in plant tissue (Kullenberg,

1946; Leston, 1953; Southwood, 1956). T. ampliats is said to lay in thistle stems (Southwood & Scudder, 1956).

Between 13th May and 6th June, 1965 i.e. the period from the second week of oviposition to peak of egg population in the field, 10 thistle plants were randomly selected and harvested at ground level from the study area on each of 7 occasions, at approximately 3-day intervals. On each sampling date the leaves and stems of plants were carefully examined under the microscope for Timis eggs. A total of 146 eggs were found in the leaves, but none in the stems. On 29th June, towards the end of the period of oviposition, the area was again sampled: 12 eggs were found in leaves of 10 plants but none was found in stems. These observations were confirmed in the 1967 season. A total of 16 plants were again randomly removed from the study area on 4th June, at about peak of egg population in the field. A total of

108 eggs were found in leaves, none in stems.

(b)In the laboratory: Tingis may lay in thistle stems under laboratory conditions, especially at high adult densities per plant. In the 1965 season, 2, out of 200 eggs (1%), collected on one occasion from cages containing groups of 5 - 10 spring females per plant, were laid in stem.

All other eggs were laid in leaves. The 2 eggs laid in stem were inserted 85 in a short region below the apical meristem of the same plant. Room temp-

erature was variable, (25 - 27°C.) and light was provided for 16 hours in a 24 hour cycle. There was no humidity control.

Maximum densities of females observed per plant during the oviposition

periods of the 1965-67 seasons varied from 2 to 4; which were below the

levels at which the laboratory population was kept. These observations

suggest that T. ampliata prefers leaves for oviposition, although usually

high densities and/Or other environmental factors may induce it to lay in

other plant organs. It is noteworthy that Tingis laid readily in pieces of

thistle stem when about 100 females and 50 males were put in each of 13.5 x

\ 7.5 x 5 cm plastic cages in field and laboratory during attempts to obtain

large numbers of eggs for experimentation. By laying predominantly in

leaves rather than stems, T. ampliata conforms to a pattern of behaviour

which is common to most leaf-sucking Tingid species (See Comstock 1878;

Morril, 1903; Fink, 1915; Johnson, 19041We, 1937; Samuel, 1939; Usinger,

1946; and Bailey 1962-3 etc.)

(ii) Precise sites of oviposition in the field

During routine sampling of eggs (Section 1I,4(a) ) in the sprin: and

summer of 1965 and 1966, it was noticed that leaflets of azillary buds which

were harvested with primary leaves occasionally contained some eggs. A

quantitative estimation of the proportion of eggs laid between the two organs

was attempted in the 1967 season. Leaf-bud samples (150) collected at 4-

day intervals in the period between 28th May and 21st June, were examined

and eggs found in bud leaflets and the main primary leaves were recorded 86 separately. There was a total of 7 sampling occasions, covering the period just before and after peak of egg population in the field. The results are

shown in Table 33. The proportion of eggs laid on the main (= primary) thistle leaves varied between sampling dates from 77.6 to 99.0 per cent of

total eggs recorded on all samples. On six out of the seven sampling dates,

more than 90 percent of eggs.were found in main leaves. Unlike nymphs

which predominantly feed on leaflets of axillary buds, adults occur and

feed more often on main leaves. It is probable that there is an association

between the distribution of feeding adults on and eggs in the various organs

of the plant.

Table 33: The proportion of eggs laid in main leaves and leaflets of

axillary buds, 1967.

P No. of Eggs Date (a) as % '.:Of(b) Leaflets of Main' buds leaves (a) Total (b)

28.5.67 17 59 76 77.6 1.667 1 v 100 101 99,0 5,6.67 9 157 166 94.5 9.6.67 7 228 235 97.0 13.6.67 14 251 265 94.7 17.6.67 20 225 245 91.8 21.6.67 10 251 261 96.1

Eggs found in main leaves or leaflets were divided into four categories according to whether they were laid in one or other of the following sites: midribs; primary veins, secondary veins and interveinous spaces. The 87 results are presented in Table 34, which shows that more eggs were laid in midribs than in any other site.

Table 34:, The numbers of eggs laid in different sites - 1967.

No. of eggs recorded on: Date Total Prim. ' Sec. Interv. midribs veins veins spaces

28.5.67 61 10 0 5 76 1.6.67 37 14 15 35 101 5.6.67- 79 28 17 42 166 "7.6.67, 92 40 22 81 235 13.6.67 101 54 20 90 265 17.6.67 86 34 19 , 106 245 21.6.67 76 57 25 103 261

Total 532 237 118 462 1349

Mean 76.0 33.8 16.8 66.0 192.7 % total 39.43 17.56 8.74 34.24

The variation in the proporo of eggs recorded on the different sites bet4een s--Impling dates are shown graphical_Ly in. Yig, Eggs found on primary and secondary veins (i.e. veins other than the midribs) have been grouped together as 'other veins'. The proportion of eggs laid in midribs tended to decrease from over 80% in May to 29% in late June as egg density increased in the field.

Surface of leaves in which eggs are laid

On each of five sampling occasions, (from 5th to 21st June, 1967); the number of eggs laid in the abaxial and adaxial surfaces of leaf samples were FIG. 21. SEASONAL VARIATION IN PROPORTION OF EGGS RECORDED ON VARIOUS SITES OF THISTLE LEAVES AND LEAFLETS

•---• MIDRIBS w 100 a_ce to----n OTHER VEINS o— —0 INTER- VEINOUS SPACES & 90 LEAF MARGINS

50 ctNp z 0 ...... 0 40 .- .". 0 a `pu 30 6 LL, 20 cc 10 / tr9' 0 2 10 15 20 DAYS 28.5.67 : DAY 1 89 recorded separately. The surface of a leaf in which an egg is laid is defined here as that on which the operculum alone or together with any portion of the chorion (according to whether there is complete or partial embedding of the chorion), or the entire egg (as when the egg is not inserted into leaf mesophyll), is visible above the epidermis of that surface. This definition is necessary inorder to distinguish between ^ccasional situations, especially if the leaf is thin, when the anterior and posterior regions of an egg are visible on the opposite surfaces of a leaf (Fig. 22, A 1-4). Where eggs are laid in leaf margins (as in rig. 23 ), the oviposition surface was taken as that on which the larger area of the operculum was visible when leaf margin was viewed edge on. The proportion of eggs found in the abaxial surfaces of samples on each date is shown in Table 35.

Table 35: Proportion of eggs found in abaxial surfaces of leaves - 1967.

Eggs recorded on surface of leaf Total Date (b) as % of (c) Adaxial Abaxial (c) (a) (b)

5.6.67 ,,c4 142 ' 166 85.5 9.6.67 f 41 194 235 82.5 13.6.67 47 218 265 82.2 17.6.67 56 189 245 77.1 21.6.67 51 210 261 80.4 Total 219 953 1172 407.7 Mean 43.8 190.6 234.4 81.5

Eggs are laid on both surfaces of leaves but a significantly higher proportion (varing from 77.1 to 85.5%; an average of 81.5 percent) of total eggs 90 were laid in the abaxial surfaces. Eggs laid in adaxial surfaces were almo-

st entirely confined to inter-veinous spaces of laminae.

(iii) Types of oviposition in Tingis

T. ampliata has an externally visible, stout ovipositor and as

already indicated usually inserts its eggs in thistle leaf tissue. Fig.22 shows some of the oviposition types observed during the present studies.

Eggs were implanted in leaves to varying depths (Fig. 22 ; C(1.4), at the greatest depth (CI) the operculum of the egg may be sunk a few millimetres

below the epidermis of the oviposition surface. The most frequent position

is with the main body of the egg entirely embedded (Cu) but quite often, varying portions, roughly from one-third to three-quarters of the ohorion

may lie exposed above the epidermis ( IIZv). Very occasionally, an egg may be deposited entirely exposed, between the felty hairs of the abaxial

surface of the leaf, usually on a small rough depression that appears to

have been made by the tip of the ovipositor on the epidermis (C v1).

When eggs are laid in the adaxial (upper) epidermis of leaves (Fig. 22

A c, they may occupy several positions partly dependent on the thick- (1 - ) ) ness or thinness of the leaves. When the leaf is thin, the posterior pole of the eggs may (a) force out the lower epidermis into round, bulbous areas

(Fig. 22 A1 & 2 ) or (b) the lower epidermis may be pierced during-oviposition, and a small portion of the egg extruded (Fig. 22 A ) or '(e) 3 about one-third or one-half of the egg may be exposed (Fig. 22; A ). 4

In eggs laid in the lower epidermis, especially on the midribs and veins, a small oval ridge of sheared tissue may form round the egg (Fig.22 91

Fig. 22. Types of oviposition in Tingis.

Different positions of eggs laid in upper surfaces of leaves - the effect of varying thickness of leaves.

B (1) Raised ridge of dead and/or dying tissue formed round eggs laid in lower surfaces of leaves, especially midribs and veins.

(11) Two eggs in a group, each laid in a different surface. Concave sides of eggs facing the same direction.

(111) Normal, unraised tissues around eggs in lower surface of leaves. Also shows variation Ln angle of insertion of eggs.

CI _V Varying degrees of egg embedding in thistle.

Fig. 23. Egg l aid in leaf margin. 92 FIG. 22.

[03M M.

3 4

103 MM.

EPCERMIS

FIG. 23.

°---=11*.r 93

B I) or the area around the egg may remain normal and unraised, (Fig. 22 ;

B ). Hyprtrophy of leaf tissue is seldom noticed around newly laid eggs; 3 and seems to occur in cases in which a long or several long oviposition slits, with considerable extensive damage to leaf tissue have been made before oviposition. The epidermis fringing the slit often turns brown, probably due to dehydration and death of cells.

The angle of egg insertion into leaf tissue varies widely between 1 and 900 (Fig. 22 B ). 3

Tingis eggs are usually laid singly but occasionally a few eggs may be found close together. It was normally impossible to tell whether the eggs in a group were laid by one or more females. The tendency for eggs to occur in groups was most pronounced in 1967, when the density of females was highest: the average total number of host plants and leaf-bud units was in that year about one-third of that in 1966 (Table 9; section 1,2 (c), whilst the population of female Tingis was higher, Observations made on the number of eggs per grou.p found in samples LcIdec.,1 23th Nay and 21st ju-;a, 1967 are sAmixn ,lble 36, A group of Es;.!.,s r,2:'gan Of the ,-;1.%.1•.. Where there Rre two egg o grout), these r/IF:y be laid on the same surface in such a way that the concave sides of the eggs face the same or opposite directions ('ig. 22 ; A (2) & B (3) respectively). Occasionally, the two eggs in a group may be laid in different surfaces, with their concave sides facing the same direction (Fig. 22 ; B (2)).

Table Number of eggs per group found in samples in (1967). 94

,,. . ... No. of Group and No. of eggs Mean eggs Date groups of Total per eggs abode group . . 28.5.67 1 4 - - - 4 4.0 1.6.67 1 6 - - - 6 6.0 5.6.67 2 3 2 - - - 5 2.5 9.6.67 5 2 2 6 2 2 14 2.8. 13.6.67 3 3 2 4 - - 9 - - 3.0 17.6.67 1 3 - - 3 3.0 21.6.67 1 3 - - - - 3 3.0 Total 14 24 6 10 2 2 44 24.3 Av. No. ' t. of eggs per 3.4 group 1 1 (iv) Die]. periodicity of oviposition in the field The activities of many species are naturally correlated with the diurnal rhythm of might and day (Andrewartha & Birch, 1954; Harker, 1958). These activities may be connected with fundamental life processes such as feeding, mating and/or oviposition or movement from one place to another. Marked oviposition rhythms have been demonstrated in some species of Heteroptera by different authors, (See Smith, 1927; Beard, 1940; Johnson, 1942; Cumber, 1951 and Eyles, 1963). There does not appear to be any published work on oviposition rhythms in the Tingidae.

Tingis put in oviposition cages in controlled laboratory environments showed a distinct oviposition rhythm (Section (v) ). To find out whether

Tingis also exhibit diel periodicity in egg laying in the field, an experiment was made between 21st and 23rd June, 1967. 15 male and female pairs collected in the field were put into separate, 14 x 7.5 x 6 cm plastic cages. 95 An area of 50 sq. cm was cut off the lid of each cage and covered with nylon netting. Thistle shoots (from green house) were provided to insects for

food and oviposition. Wilting was prevented by wrapping cut ends of shoots in moisture-saturated cotton wool. Cages were put out in the field where

temperature was recorded in a thermograph. The food was changed at six hour intervals and the number of eggs laid within the period was recorded after examination of shoots under a binocular microscope. The results are summerised in Fig. 24. Temperature records were kept from six hours before the

beginning of the experiment and continued for a further 12 hours after to complete the pattern of temperature fluctuation. Tingis showed a definite oviposition rhythm (Pig. 24 ), which appears to be positively correlated

with the daily rhythm of physical conditions, especially temperature. Ovi-

position could take place at any time of day or night but most eggs were laid during the day. The total number of eggs laid rose from a minimum value between midnight (= 00 hrs.) and 06.00 hrs. to a peak between

12.00 hrs. and 18.00 hrs+ and then dropped again. The proportion of total females which contributed to the eggs laid within each six hour period also

fluctuated in the same way as the total eggs laid (Fig. 24 ). Owing to the heavy rains which fell in the afternoon ,Ind evening of the 23rd June, observations could not be continued.

The variation in the number of eggs laid per female in each six hour period and the total numbers of females laying during the 48 hour period of observation is shown in Table 37.

Table 37: Total frequency of females laying eggs in two, 6 hour periods. 96

FIG24. PERIODICITY OF OVIPOSMON OF FEMALES AT SIX HOURLY INTERVALS. (2123.6.66)

0 NO. OF EGGS LAID

• NO. OF coo LAYING EGGS rt. OUT OF 15) o—o MEAN TEMP

1200 18 06 1800 2400 o‘oo iloo Boo goo 0600 abo PERIOD (HOURS B.sT) 97

No. of females and time of day No. of eggs hour per 6 06.00 - 12.0n - 18.00 - 00,.00 period 12.00 hrs. 18.00 hrs. 24.00 hrs. 06.0o hrs

0 16 11 19 24 1 9 6 7 4 2 4 7 1 1 3 3

4 1 5 1 1 6 7 0 Total 30 30 i6 30

Not only did a higher proportion of females lay eggs in the 12.00 - 18.00 hrs. periods, compared with any other time in a 24 hour cycle, but also on the average, the number per female was higher. In the second 24 hour cycle of the experiment, about 55% of eggs were laid within the 6 hour peak period. Maximum oviposition in the afternoon has also been observed in the Coreid, Anasa tristis De Geer, the Pentatomid, Nezara viridula L. and the Lygaeid,Nysius huttoni White: (See Beard', 1940; Cumber, 1951 and

Eyles, 1963 respectively). Light and temperature are among various environmental factors which may determine the phase-setting of some insect physiological rhythms (See Choppard, 1930; Johnson, 1942; Harker, 1958; 1964) 98

(v) Laboratory studies on oviposition

A male and a female Tingis were caged on potted thistles with cylindrical celluloid cages, 12" high and 6" internal diameter. The top ends of all cages were covered with fine nylon netting, and their bottom ends were pushed about 1 inch deep into moist soil at bases of plants. Two small openings, (one about 2" wide) and also covered with net, were made on opposite sides of each cage in such a way that one was situated a short distance above soil level and the other close to the top end of the cage.

Bugs were transfered to fresh plants from the greenhouse at weekly intervals and counts of eggs laid within the week were made by microscopic examination of thistle materials. Plants were watered regularly. Of 40 pairs brought from the field before the start of oviposition in the spring of 1965, 20 were kept in cages in a 20°C. constant temperature room set at 16 hrs. photoperiod and 60% relative humidity. The other 20 pairs were kept in another room at 15°C. with identical light and humidity conditions. In the spring of 1966, 25 pairs were caged for oviposition in a constant temperature room set at 20°C. Since observation in 1965 and 1966, had shown that there was a correlation between fecundity in laboratory and field in each year females were not kept for laying in the laboratory in the 1967 season.

The oviposition records of "the laboratory females" for the two years are summarised in Table 38. Mean number of eggs laid per female was slightly more in 1966 (74 eggs) than in the 1965 (61 eggs) season. However, these means were not significantly different. There was a considerable variation in the number of eggs laid per female in each'seAson (Table 38). In o the 1965 season, the number of eggs laid per female at 15 C.was about one- third (average = 20 eggs and range 6-57) of that laid at 20°C. thus

indicating that a decrease of 5°C. in temperature may reduce total fecundity.

At both temperatures, the number of eggs laid per female per week tended to

show some periodicity, when plotted for the entire oviposition period (Fig.

25). Table 38: : Laboratory egg laying at 2000.

[No.of eggs per femalel Year No.of females r Mean Range

1965 20 61 13 - 101 1966 , 25 74 25 - 117 t 4

A similar oviposition rate curve is obtained for individual females as

exemplified by data on four randomly selected layers presented in Fig. 26 .

Weeks of high oviposition rate tended to alternate with those on which fewer eggs were laid; but in general four distinct periods are noticeable:

(1) the first 2-3 weeks following the start of oviposition when a few eggs are laid; (2) a variable period of 1-2 weeks of greatly reduced oviposition following period (1); a third period, about 2-4 weeks during which cviposit- ion rate usually attains its highest peak and most eggs are laid and a fourth, post-peak period, often of 2-3 weeks duration of decreasing oviposition rate and in which death of the insect occurs. When they die "laboratory females" still contained eggs as was the case in the field. That oviposition is more or less continuous in most females (Figs. 25&26) apparently, indicates that egg maturation and feeding are probably concurrent processes in this species. 100

FIG. 26 ANIPOSIDON OF FOUR FEMALES AT mt.

3Or

2C••

io 2b k 4b sb do sb DAYS (5.5.65 = acif

FIG. 25.OVIPOSITION OF FEMALES AT 20t,

10 20 30 40 50 60 70 90 90 DAYS(5.5.65=--DAY 0) 101

In females kept at 20°C, oviposition period varied from 6 to 10 weeks

(average of about 7.6) in 1965 and from 6-11 weeks (average of 8 weeks) in

1966 season. Females kept at 15°C in the 1965 season laid for 3 to 7 weeks (average = 5.2 weeks).

(vi) Changes in reproductive organs and body weight in the field

(A) The organs: Pendergrast, (1957) has given a general systematic description of the male and female internal reproductive organs of the

Heteroptera, and a detailed account of the morphology of the male and female genitalia of Rhynchota has been published by Pruthi, (1925) and

Dupius, (1955) respectively. In Tingis, the female reproductive organs

(Fig.28) consist essentially of:

(i) a pair of bilateral ovaries, each containing seven ovarioles.

Carayon, (1950) and Woodward, (1950) have published accounts of

the ovariole numbers of many species of Heteroptera. According

to the latter author, the most frequent number found in members

of the sub-order is seven. The ovarioles are of the acrotrophic

type. Each ovariole consists of a long, thin terminal filament;

a germarium; a row of oocytes in various stages of development,

(if ovary is in an active state) and a short pedicel.

(ii) a pair of long, often slightly curved lateral oviducts, into

which the pedicels open. The expanded bases of the lateral

oviducts converge and form a median common oviduct.

(iii)two saccular spermathecae, which are connected by short sperm-

athecal ducts; one to each of the dorsal wall of the base of a

lateral oviduct. 102

(iv) a long, thin accessory gland and

(v) an ectodermal vagina, which is continuous anteriorly with the

median common oviduct. In Tingis the cuticular lining of the

genital duct is continued for some distance into the lateral

oviducts.

In the male, (Fig.30); there are a pair of small, almost spherical testes, which are connected to a pair of long, wide vesiculaeseminales by thin vasa deferentia. There are a pair of mesodermal accessory glands, one close to the outer margin of each vesicula seminalisi The paired accessory glands and vesiculmseminales open into a short bulbus ejaculatorius, from the posterior region of which, a pair of small, saccular ectadenes arise.

The ductus ejaculatorius is short.

(B) The seasonal changes in organs and body weight: Changes in live weight and morpho-functional condition of adults were studied by weighing 20 males and 20 females and dissection of a minimum of 10 individuals of each sex at 7-10 day intervals. Bugs were weighed individually on a 5 mg. torsion balance calibrated in 0.01 mg. divisions. In making dissections, bugs were killed in ethyl acetate vapour, and stuck by their ventral surfaces to the top layer of white wax in a micro-dissecting dish. To stick an insect, the top of the wax was touched with a hot pin and the insect was quickly lowered into the thin layer of molten wax before the latter solid- ified. The hemielytra and the thoracic and abdominal terga were then removed with the aid of a thin, mounted blade and a pair of fine forceps.

Ovaries or testes and their associated structures were teased free and placed in a drop of distilled water on a clean slide. Note was made of the 103 following:-

1. Degree of maturation of ovaries: Shape and size of germaria;

presence or absence oocyte differentation and mature eggs.

2. Size of ovaries: A micrometer eyepiece was used to measure ovarian

dimensions. Both ovaries of each bug were measured. The volume of

an ovary was taken as the product of its length (= distance between

the top most apex of the germaria and the base of the pedicels) and

two maximum diameters, one at right angles to the other. Mean

volume (in mm3) of the two ovaries in individual bugs were calcul-

ated. The grand mean for all bugs on each occasion are

presented in Fig.27. Although, these values may not have absolute

meaning, they were nonetheless roughly proportional to the volume

of ovaries and provided a basis for comparing variation in ovarian

size from one date to another.

3. Total number of mature eggs in the ovaries, from Which number of

eggs per female was calculated (Fig.29).

4. Evidence of oviposition: Presence or absence of corpora lutea and/

or dilated empty pedicels.

5. Evidence of mating: Virgin unmated females usually have shrunken

and wrinkled, almost clear or translucent spermathecae. By cont-

rast, mated females were often characterised by turgid, pinkish

sperm-filled spermathecae (see Fig. 28(E)).

6. The amount, colour and condition of the fat.

The seasonal changes in body weight and ovarian volume of bugs dis- FIG. 27.SEASONAL CHANGES IN OVARIAN VOLUME 5 BODY WEIGHT AND FAT CONTENT

• • MEAN OVARIAN VOLUME 24 c•--o MEAN BOCK WEIGHT FAT CONTENT

— 2.0 9-0°- 0 cc 0.. 0 0`0-.0 o, 1.6 / -o-- o DEATH C.1.1* O d, 1.2 f!' Cr BIRTH DEATH

BR 0.0 MAR. APR. MAY JUN. JUL. AUG. SE OCT 1965 105 seated in the 1965 season are shown in Fig. 27 and Table 40. There was no oocyte differentiation and females were not fertilized in the autumn. However, a small, post-emergence increase in ovarian volume (from 0.2 to 0.7 mm3) occured during the few weeks of pre-hibernation feeding. This increase was mainly in the size of the germaria. A considerable amount of fat was laid down from August to September and body weight increased correspondingly. Bugs gained as high as 0.95 mgs. (approx. 90.4% of weight at emergence) during this period. In late autumn, and throughout the period from Nov. to March, the ovaries remained in the same condition as in early autumn, except that they become smaller in size and were shrunken to an almost spherical knob (Fig. 28(B)). Newly emerged adults kept in a constant temperature room_at 20°C, 60% R.H. and 16 hrs. photoperiod from August to November did not show any proliferation of oocytes. Body weight and fat decreased during hibernation (section II,2(a)iii above). From Mid-March, the germariaJ?egan to enlarge further. Constriction of each germarium into two or more bulbous lobes may occur in some females by the end of March. 9 out of 10 females dissected on 20.3.65 had large germaria which were already dividing in this way. It appears that these germarial units are the pre- cursors of tissues which later divide repeatedly to form egg rudiments. A large swelling usually appears on the middle region of each lateral oviduct, at about the same time at which germarial division begins. These swellings persisted in females throughout the period of active oogenesis (April to June). Their function was not quite clear to the author, but they appeared to be associated with the process of egg maturation.

Egg rudiments start to form by about early-April, long before post- 106

Fig. 28. Changes in female reproductive organs.

At Immature, prediapause condition in autumn.

Bt Immature and in diapause, in winter.

C and Dt Onset of ovarian maturation.

Et Mature but not laying.

F t Mature and laying (late stage of oviposition).

a.g. = accessory gland. b.l.o. = expanded base lateral oviduct. c.l. = corpus lutetun. c.o. = common oviduct. e.r. = egg rudiment. g. = germarium. 1.0. = lateral oviduct. m.e. = mature egg. o. = ovary. p. = pedicel. sp = spermatheca. sp.e = sperm. t.f. = terminal filament. v. = vagina. FIG. 28. B

"0717111.

DISSECTED1 ON j 5.965 3 f 66 0 4MM

DISSECTED1 .4.66 12 10-5.66 7.7.66 ON 108

FIG. 29. SEASONAL VARIATION IN NUMBERS OF EGGS IN FEMALES OF THE FIELD POPULATION OF TINGIS

30 •+ NO. OF EGGS PER FEMALE 0----0 OF FEMALES MATURE x--ItII )) MATED

-25 .100

020 I 4 80

LL 15 , 60 cx a.

Ln w 10 .40 LL O Ucc Lai O

DEATH

APR. MAY I JUN. I JUL. 1965 109 hibernation emergence. The ovaries develop rapidly from April onwards to the end of May, the rate of maturation depending on temperature. Percentage of females which were (a) mature, i.e. with at least one ripe egg in the ovaries; and (b) mated; are shown together with the number of mature eggs per female for the 1965 season in Fig. 29. By 19th May, all the females dissected were mature. A maximum of 26 ripe eggs per female were recorded on 29th May. Maturation rate varies considerably from one year to another.

Dissections made on 3,5.67; (Table 39) showed that all the females were already mature; 30% had mated and none had laid by that date.

Table 39: Stage of maturation of females dissected on 3.5.67.

Bug No.of mature No.of Mated (+) or Ovary Nog egg in ovaries oocytes Unmated (-) Stage 1 21- 15 + 3 2 11 10 It 3 12 9 + It 4 16 17 - U 5 19 12 - i, 6 22 15 + n 7 15 18 - ft 8 20 14 - rt 9 11 8 - u 10 2 4 - u

(+) = 4, Total 149 122 (-) = 7 (+) = 30% Mean 14.9 12.2

The structural changes which occur during ovarian development, egg maturation and oviposition are as characteristic of many Heteroptera, (See

Woodward, 1952). At maturity, the number of oocytes per ovariole of a female may vary from 2 to 5. As the ripe egg passes down into an already lengthened and dilated pedice1,- a dark yellow corpus luteum is left at the 110 proximal end of the vitellarium., A new corpus luteum is formed as the next ripe eggs descends into the pedicel and lateral oviduct. At oviposition, the oviduct becomes slightly longer and straighter in form.

On the basis of the above changes, four distinct stages in ovarian development and oviposition of Tingis were recognised as illustrated in Fig. 28.

1. Ovary in prediapause or diapause stage, with small germaria; short pedicels and lateral oviducts; no oocytes - immature (Fig. 28

( A & B )).

2. Large germaria already dividing into two or more lobes; middle of each lateral oviduct greatly dilated - onset of maturation (Fig. 28 ( C & D )).

3. Small germaria; 2-5 oocytes per ovariole; one or more mature eggs, with fully formed chorion; corpora lutea absent - mature but not laying (Fig. 28 (E)).

4. Numerous fully formed eggs; some in pedicels or lateral oviduct corpora lutea present - laying (Fig. 28 (F)).

From mid-April to the time of post-hibernation emergence in first week of May, and between emergence and the start of oviposition, body weight increased with ovarian volume and maturation. Between 19th April and 9th May, 1965, mean body weight rose from 1.35 to 1.88 mg.and mean ovarian volume from 0.52 to 0.91 mm3. The increase in body weight during this period is attributable to active feeding and accumulation of fat immediately before and soon after emergence. There was an initial drop in weight at the 111 beginning of oviposition in May, but this was followed by a further increase to a peak of about 1.9 mg.on 29th May; the same date on which ovarian volume attained a peak of 2.27 mm3. During the remaining period of oviposition in June, body weight fluctuated slightly and decreased progressively with ovarian volume as the bugs became senile except for a small rise noticed on 8th July.

The dates of start of oviposition varied from one year to another according to the rate of maturation. In 1967, a very early year a female with corpus luteum was first found on 4th May compared with 9th and 11th May for 1965 and 1966 respectively; slightly later years.

Changes in Male Reproductive organs

At emergence in autumn, the testes were small and the accessory glands and vesiculaeseminales, narrow and whitish (Fig. 30(A). Neither the testes nor the vesiculaaseminales of bugs dissected from August to October of the three years, 1965, 1966 and 1967, showed the presence of mature sperms.

There is however, a considerable increase in the size of the organs as the bugs age in autumn. Dissections made in early winter (= 18.11.65) showed that the seminal vesicles and the of the accessory glands had enlarged to about two times their size in August (Fig. 30 (B)). Active secretion of fluids had evidently started in the accessory glands by that date. The general outline of the organs on that date was smoother and the contents of the seminal vesicles had altered in colour from white to creamish-yellow; but the spermatozoa were not mature. By 30.3.66, the contents of the seminal vesicles were already pinkish or orange and ripe sperms were present. It appears that the process of spermatogenesis is 112

Fig. 30. Changes in male reproductive organs.

A, Immature in autumn.

Bt Mature but not mating.

Ct After mating in summer •.

a.g. = accessory gland. b.e. = bulbus ejaculatorius. d.a. = duct of accessory gland. e. = ectadene. e.d. = ductus ejaculatorius. t. = testis. v.d. = vas deferens. v.s. = vesicula seminalis. FIG. 30. A

t v.d

v.s

d.a b.e —e e.d

DISSECTED.) 12.8.65 18.11.65 30.6.66 ON ilk slowed down but not completely stopped during the winter. During the main copulatory season, (late April to May), the proportion of bugs with turgid accessory glands and seminal vesicles decreased. 6 out of 10 males dissected on 27.5.66 had thin, shrunken and twisted accessory gland and vesiculae seminales,obviously due to loss of spermatic fluids and sperms. By 30.6.66, all bugs dissected had reproductives in a shrunken condition with no sperms (Fig. 30 (C)).

The seasonal pattern of change in live weight of males was similar to that of females; but females were consistently heavier than males. Mean weight of males increased sharply, in the period between emergence and the onset of hibernation in autumn. In the 1965 season, this increase was of the order of three times the body weight at emergence. Mean body weight decreased throughout hibernation from September to April. From late April to about first week in May, when males emerged from hibernation, live weight increased; but soon decreased again in the late half of May. The high proportion of males copulating at that time of the year probably accounted

for the decrease. This decrease was followed by a short period (about two

weeks) of sharp increase, probably as a result of active post-copulation

feeding. From early June to the end of July, mean weight of males decreased

significantly. The senescence of bugs is probably associated with decrease in live weight as noticed towards the end of the reproductive season. Mean live weights of males and females, together with their standard deviations

are shown in Table 40. 115

Table 40: Seasonal changes in body weight of adults - (1965 season).

Mean weight of bug (mg) ± S.D. Date Females Males

19- 4-65 1.35 ± 0.05 0.835 ± 0.03 29- 4-65 1.75 ± 0.13 1.00 + 0.06 9- 5-65 1.88 + 0.02 0.95 ± 0.002 19- 5-65 1.69 ± 0.12 0.79 ± 0.01 29- 5-65 1.97 ± 0.012 1.69 ± 0.013 8- 6-65 1.85 ± 0.15 1.40 ± 0.02 18- 6-65 1.79 ± 0.02 1.085 ± 0.003 hO 28- 6-65 1.70 ± 0.01 1.25 ::1 0.005 0 1-1 8- 7-65 1.855 ± 0.04 1.25 ± 0.13 P.4 18-7-65 1.65 ± 0.01 1.20 ± 0.24 28- 7-65 1.575 ± 0.02 1.195 ± 0.11 7- 8-65 1.56 ± 0.02 1.03 ± 0.03

12- 8-65 1.05 ± 0.03 0.55 ± 0.01 19-8-65 1.125 ± 0.05 0.75 ± 0.03 26- 8-65 1.94 + 0.12 1.55 4. 0.1 2- 9-65 1.96 ± 0.1 1.52 ± 0.13 9- 9-65 2.00 ± 0.04 1.49 0.0u0 16- 9-65 1.9 ± 0.08 1.50 ± 0.05 23- 9-65 1.85 ± 0.02 1.50 ± 0.02 30- 9-65 1.8 ± 0.07 1.45 ± 0.03 7-10-65 1.8 ± 0.01 1.45 ± 0.07 116

Changes in Fat

The amount of fat was assessed by two methods. In the first method which was used from 20th March, 1965 to 31st March 1966, fat content of bugs was graded by eye and ranked, 1-5 (=0-4); on a scale as used by Waloff &

Richards, (1958) and Anderson, (1962). The results are shown as average fat

values in Fig. 27. The second method was used between October, 1965 and March 1966; i.e. from the start of and during the period of hibernation and diapause. Groups of bugs randomly collected in the field were weighed and then dried to a constant weight in an oven set at 110°C. The water content of bugs was determined by the difference between wet and dry weight. Dried, water-free bugs were ground up and then put into a thimble which had been weighed immediately after removal from a P205 dessicator; and weighed again.

The ether-soluble fat was extracted in a micro-soxhlet for about 6 hrs. After extraction, the thimble and fat-free residue were dehydrated to a constant weight. The weight of the ether-soluble fat was taken as the difference between the weight of dry thimble and Tingis material before extraction and the weight of dry thimble and fat-free residue. This difference method minimises errors of estimation which would have been large if the relatively small quantity of fat extracted under these conditions had been estimated by evaporating the extractant in the extraction flask. Fat-free solids was estimated by the difference between the dry weight of thimble and residue and that of dry thimble alone. The results are presented for females and males in Tables 41 and 42 respectively. Values are based on the average of two determinations on each occasion. Seasonal fluctuations in fat content (Fig. 27 ) were very closely associated with changes in body weight and reproductive organs. 117

Table 41: : Changes in the constitution of hibernating Tingis - females

STAGE Live Dry wt. 1 „, wt. wt. fat wt. i IN REPR. wt. wt. H2O 740 fat per -. f at% non-fat non-fat Date CYCLE (ms (mg) (m g) H2 (mg) (mg) splids solids ‘, L tilIeJ JUST BEFORE38.12 10.63 27.48 71.82 6.60 0.33 17.3 4.03 11.0 2-10-65 DIAPAUSE (20)* -,..

31.89 10.14 21.75 68.21 5.25 0.26 16.5 4.89 15.3 2-11-65 HI M- (20) NATION 31.60 9.0 22.60 70.75 4.0 0.2 12.7 5.0 16.6 2- 1-66 REPR. (20) DIAPAUSE 30.99 13.00 17.99 58.08 2.0 0.1 6.5 11.0 35.5 2- 3-66 (20) t

Table 42: Changes in the constitution of hibernating Tingis - males

1 -',. , -4 • P wt. , Dry wt. wt. wt. fat r 70, STAGE 1Live 0-% nonfat IN REPR. wt. wt. H2O fat per a: fat non-fat Date CYCLE , H-1.0 (mg.) solids, solids (mg.) (mg.) (mg.) 2 (mg.) (mg. ) . . .

JUST BEFORE 28.46 7.77 20.69 72.75 4.52 0.225 15.9 3.2k 11.4 2-10-65 DIAPAUSE (20)

...0 25.48 8.18 17.30 68.o 3.69 0.18 14.5 4.49 17.5 2-11-65 (20) HIBER- NATION 25.57 7.0 18.57 72.0 3.0 0.15 11.7 4.0 16.27 2- 1-66 & (20) REPR. DIAPAUSE 15.95 5.0 10.95 68.6 0.98 0.07 6.1 4.02 25.2 2- 3-66 (14)

* No.of bugs in brackets 118

Bugs contained a small amount of creamish-white fat at emergence into the adult stage. There was a considerable increase in the size of fat during the feeding period in autumn. Colour of fat altered from creamish-white to deep yellow as bugs aged. It also tended to be more compact as bugs grew older. Fat level dropped progressively throughout the winter; but bugs were not completely depleted. There was a further increase in amount of fat in the one to two weeks of post-emergence feeding. A peak fat score of 4.8 was reached by 19th May. From then onwards and throughout the remaining period of active oviposition, fat level decreased, becoming less variable by the end of oviposition in late June and early July. The decline in fat during oviposition is apparently due to conversion of reserves in the fat body to yolk. The general pattern of seasonal change in fat body was identical in both sexes. The only difference noted was that females contained more fat,

(probably on account of their larger size), except during oviposition period and at death in summer when males appear to contain slightly more, for their smaller size.

The results of Soxhiet extraction of fat confirm those of assessment by visual examination of dissected bugs. They show that in both sexes, percentage fat/live weight and water/live weight decreased during the hibernation period, 1st October, 1965 to 2nd March, 1966. In females, fat/live weight percentage decreased from about 17.2 to 6.5 while water/live weight fluctuated from 71.8 in October to 68.2 in November and then decreased to about 58.0 by 2nd March. In both sexes non-fat solid/live weight percentage increased in the period. The body constitution of males and females were identical. Besides the quantity and quality of fat (See Kozhantshikov, 119

1938), the water content and loss at the start of and during hibernation are

known to affect the level of cold-hardiness in many insect species.

It is probable that the decrease in water/live weight percentage of Tingis

is associated with supercooling and the development of cold-hardiness

(Section II2(a)iv).Although, Salt, (1936) found some species in which water

loss did not affect supercooling, Payne (1927) has pointed out that there

is considerable variation between different insect species in the amount of

water content and water loss during the overwintering season in which cold-

hardiness is developed. Decrease in fat during hibernation evidently arises from break down of reserves for the needs of basal metabolism of the insect.

(vii) Weight of females in Spring and Number of egg rudiments

A number of females were collected from beneath grass litter, a day

before the date on which the first emerging female was found in spring of

1967. They were weighed individually on a 5 mg. (1 Div = 0.01 mg;) torsion

balance and dissected in water under a binocular microscope. The total

numbers of oocytes and mature eggs in each female were recorded separately,

against the corresponding weight in mg. Note was made of the presence or

absence of,(a) food in the gut; (b) sperm in the spermathecae and (c)

corpora lutea. None of the dissected specimens had laid. Eight had

orange-coloured substances in their guts; (probably carotinoids or chromo-

plasts injested at feeding on etiolated, thistles); and five others had

mated. Since data on fed and/or mated females would introduce great vari-

ation in the relationship between body weight and number of, (a) oocytes

or (b) oocytes plus mature eggs, they were excluded from the results pres-

ented graphically in Fig. 31. The numbers of oocytes and eggs were FIG. 31 . THE RELATIONSHIP BETWEEN WEIGHT OF FEMALES IN SPRING AND NUMBERS OF EGGS AND OOCYTES

03) 40 Y2 : 29 63X —20 155 C2 : 0 8947 TES

CY P < 0 001 OO & 3

GS (A) G E • aC

Y1 : 212I8X — 20 095 co 2 - 0 8804 P< 0 001 ES T CY I0 00

F (A) O

N 0 0.0 0.5 1.0 1.5 2.0 25 LIVE WEIGHT OF FEMALE (MG.) : X 121 separately, and jointly, positively correlated with live weight. Regression lines were fitted to the data by eye. There is a considerable scatter of

points about the lines. This wide scatter obviously reflects the great individual variation in the extent to which fat and other metabolic reserves in hibernating Tingis had been converted to ovarian tissue. The calculated correlation coefficient between oocytes and weight i.e. r1 was 0.8803,

(P4:.001) and for oocytes plus eggs and weight i.e. r2 was 0.8947, (P40.001),

The equations for the regression of numbers of oocytes (Y1) and oocytes plus eggs (Y2) on weight were: Yl = 21.218X - 20.095 and Y2 = 29.63044X - 20.155 respectively. The quantitative relationship between some index of size e.g. body weight and numbers of oocytes has provided a satisfactory basis for the determination of the reproductive capacity in some lepidopterous species

(See Bevan & Paramanov 1957; Miller, 1957 and Dissescu, 1964) and Hymenop- tera (See Heron, 1966). In such species, females are often sexually mature at final moult or soon afterwards and oviposition is complete in a few days.

Moreover many lepidoptera do not feed after emergence. In Tingis, maturation and oviposition are continuous and lasts several weeks. Feeding also continues throughout adult life. Furthermore, females tend to die without completing oviposition. Thus, the number of eggs and oocytes per female at post-hibernation emergence may not reliably indicate the reproductive capacity of a natural population of Tingis. Therefore, the data presented in

Fig. 31 are only an indication of the extent to which weight variation in the female population could affect their potential reproductive capacity; and also the extent to which temperature and other environmental factors had affected maturation, (See Norris, 1963) of individuals by emergence date. The calculated regression coefficient of numbers of oocytes plus 122 mature eggs on weight at post-hibernation emergence was 29.63. This suggests that an increase of 1 mg. in the weight of a female would ideally increase its potential reproductive capacity by a factor of about 30 oocytes or eggs. Although of little predictive value, data in Fig. 31 illustrate that heavier females in a Tingis population have a tendency towards higher reproductive potentials than the lighter ones.

(viii) The reproduo'cive state of females at death in Summer

In some heterop'erous species, the end of oviposition period is often marked by morphological changes in the gonads. For example, Woodward,

(1952) observed that the end of the reproductive period in Stenodema laevi-

Aatum (L.) and Notostira erratica (L.) (Heteroptera: Miridae) was associated with absorption of egg rudiments and a great reduction in the size of germaria which also turned yellow. During laboratory and field studies of oviposition in Tingis; it was observed that females often died suddenly 'in oviposition'; with abdomen still conspicuously distended apparently with eggs. Some of the females found dead in laboratory oviposition cages and in field were dissected in order to examine the state of their ovaries.

The results are presented in Tables 43 and 44 respectively.

Table 43 : Number of ripe eggs in some females dying in the laboratory

1966 and 1967.

Date No Total No Germarial Dissected of ripe eggs Mean condition

25.5.66 2 25 12.5 +1- 2.7.66 5 4-6 9.2 +++++ 28.5.67 3 32 10.6 +4-

(4-) indicates white, large and (-) small germaria compared with those of living laying females. 123

Table 44: Number of ripe eggs in females dying in field (1967).

No Total No Date Mean Germarial Dissected of ripe eggs condition 16.6.67 5 54 10.8 ++44+ 21.6.67 7 62 8.8 +++4.--++

(4-) indicates white, large and (-) small germaria compared with those of living laying females.

In all these bugs, the ovaries contained ripe eggs, and oocytes of various sizes and stages of development. The only differences noticed between the ovaries of living, laying and dying females was that in those of the latter; all the component parts except the germaria were more yellowish and harder in consistency. These characteristic differences were in all probability due to the physiological and chemical changes which take place just before and at death of the insects. The observations in Tables 43 and 44 suggest that in Tingis, complete deposition of ripe eggs is not the rule but an exception. Johnson's (1936) observations on the Rhododendron lacebug, Leptobyrsa (= Stephanitis) rhododendri Horvath (Hemiptera: Tingidae) whose females are said to 'die in act of oviposition' and those of the author on the gorse Tingid, Dictyonota striehnocera Fieber and the broom

Tingid, Dictyonota fuliginosa Costa (Heteroptera), suggest that the habit may be common among many species of the Tingidae. The two latter species overwinter as eggs and are found as adults from June till October or Sept- ember (Southwood & Leston, 1959). Seven specimens (4 dead and 3 alive) of

D. strichnocera collected on the Heath on 4.10.65 contained on dissection an average of 8 and 10 ripe eggs per dead and living female respectively.

A single D. fuliginosa caught on broom on 29.9.65 was found to contain four 124 ripe eggs on dissection.

In T. ampliata, egg production and oviposition appears continuous as in some Mirids and Lygaeids(Michalk, 1935). However,unlike some members of the former family females die without completing oviposition. There is no appreciable post-oviposition survival period and consequently their gonads may not show many of the morphological post-oviposition changes normally associated with species with long post-oviposition life.

(ix) An unusual ovarian condition in Tingis In specimens dissected on 22.3.66; a single female with infected ovaries was found. The separate ovarioles and germaria were greyish-black and seemed to have coalesced into one solid, swollen mass of tumour-like 'growth& (Fig. 32B ). There was no evidence of these structures being either fungal, bacterial or viral in origin. 125

Fig. 32. A. Female reproductive organs (healthy ovaries) B. tf tI " (diseased ovaries) 126 (d) Dispersal

Many animals including insect species show some form of increased activity at one stage or another in their life histories. This activity or • movement may be restricted to the "favourable" part of their habitat or it may involve the movement of individuals out of it. In the latter situation, dispersal is said to occur if such movements lead to a scattering of population; an increase in the mean distance between the individuals, (Andrewartha

& Birch, 1954; Kennedy, 1961; Schneider, 1962; and Southwood, 1962). Thus, according to the definition of dispersal by Johnson, (19604)and Southwood

(1966) and the classification of animal movement by Provost, (1952) and

Southwood, (1962), insect movements which do not produce a scattering effect on population are regarded as 'trivial'. Some of the behavioural characteristics of dispersive and trivial movements have been discussed by Johnson,

(19601)and Kennedy, (1958) respectively. Drifting, walking and/Or flying are among ways by which some insects may achieve dispersal, (Andrewartha,

1961). In the Heteroptera, 'which may perhaps fly for only part of their life', the 'normal mode of locomotion is walking' (Southwood, 1960). Two possible mechanisms of dispersal, walking and flight in T. ampliata were investigated in the present studies.

(i) Walking In T. ampliata, walking on and between thistle plants, especially

adjacent plants in contact, is undertaken to some extent by late instar

nymphs and to a larger extent by the adults. The effectiveness of walking

as a dispersive mechanism in Tingis nymphs was not studied, but it is

probable that these movements have a considerable influence on the 127 distribution of individuals on various parts of the host plant (See Section

II3(c)). Active walking by adults is most noticeable at two distinct physio-

logical stages in Tingis. One is in late August and early September when

the new generation adults seek hibernation sites, usually in grass mats at

the bases of plants. The other is at post-hibernation

emergence in Spring, when the adults seek mates and the females search for

suitable oviposition sites within the habitat. The latter movement takes

place under conditions of increasing ambient temperatures and consequently

it appears to be more active and persistent (not in the sense of Kennedy,

1951), than movement in autumn which is often associated with the fall in

temperatures.

(a) Methods of study: Dispersal by walking was assessed by two methods.

In the first method; seven pairs of thistle "trap plants" were planted along

a South East direction, away from the study area. The two plants in a pair were 8 ft. apart, each being about 6 - 12" high. The pairs of trap plants were planted on a logarithmic scale of 2, 22 27, i.e. at a distance

of 2 - 128 ft. The first pair was 2 ft. from the study area. All plants

were watered daily until they were fully established. Planting was done in

the spring of 1965, as soon a sufficient number of emerging thistles was

found for transplanting. Since establishment of plants was not complete

until late summer of that year, they were not sampled in spring and in early

summer. However, during late summer of 1965, (beginning from end of July

when new generation adults began to emerge), trap plants were examined for

Tingids daily between 10 and 11 a.m. With the exception of a single male

caught on 14.8.65, on a trap plant 2 ft away from the study area, no bugs

were found on trap plants throughout the period of observation, (from 31st 128 July to 30th November). It appears that movement in autumn Is mainly

confined to within rather than between plants. The pre-hibernation migration

of adults to overwintering quarters a the bases of host plaabs was discussed

in section II, 2(a)(i).

During the winter of 1965-1966, the aerial shoots of all trap plants

died. In the spring of 1966; new shoots grew from the overwintered sub-

terranean thistle roots on the same sites as those on which the trap plants

were intially planted in 1965. In some sites, more than one shoot grew and

these had to be thinned out to one per plant. All the trap plants were

sampled daily as in the prececding autumn and any Tingis sliaken off each

pair was recorded and removed for dissection. No Tingis was caught on the

trap plants in the autumn months of 1966, The pattern of catch during the spring and summer of the same year is shown in Table 45.

Table 45: Catch of Tingis on trap plants in 1966 season.

Distance from study area (ft.): 2 4 8 16 32 64 128 Total

No. of Tingis caught: 3 0 0 0 0 0 0 3

A season's total of 3 specimens (2 .412 and 1-g ), were caught on trap

plants 2 ft away from the study area. Catches were made within the first

three weeks of post-hibernation period which preceeded peak of adult popul-

ation in the field. The two females caught had immature ovaries and one

had mated (i.e. there were spermatozoa in spermathecee).

(b) Distances moved by adults: In the second experiment, dispersal of

Tingis within its natural habitat was studied. The site chosen for the

experiment was a small, rectangular patch of thistles, about 100 x 120'; 129 situated on Elm Slope. Its northern limit was marked by a foot-path, 12'

wide, which runs from Silwood Bottom to South East corner of Gunnes's Hill.

On the South, it was bounded by another foot-path, 5' wide which was being used frequently. The topography, agronomic history and floristic composition

of the selected site was as described for the main study area (See Section

2(a).

A thistle plant was arbitrarily chosen in the middle of the site and

with that as centre, the area in the immediate vicinity was mapped out into

11 concentric circles at intervals of 1 ft., in a manner rather similar to

that used by Dobzhansky & Wright (1943, 1947) in their studies on Drosophila

pseudoobscura and by Norris, (1966) in his studies on the bushfly, Musca

vetustisgima Walk (Diptera: Muscidae). Thin bamboo canes (1 ft high) were

placed at intervals along the circumference of each circle and connected to-

gether with fine cotton thread. On each of the 11 circles, the points

corresponding to the four cardinal points were marked by taller bamboo stakes

(5 ft. high). The stakes forming each of the two axes, North - South and

East - West were then separately joined by thread. The number and distribution of thistles in each annulus of the mapped experimental site were determined on 13.5.66. These are shown in Table 46 and rig.33 respectively.

Table 46: The no. of thistle trap plants in each annulus of mapped site.

Annulus No. (Starting from centre) 1 2 3 4 5 6 7 8 9 10 11

No. of thistles 8 9 14 21 20 26 33 28 30 35

With the exception of annuli 8 and 9; the number of thistles in annuli

tended to vary in proportion to their area. According to Andrewartha, • 130

FIG • 33. DISTRIBUTION OF THISTLES ON MAPPED SITE USED FOR EXPERIMENT ON DISPERSAL OF TINGIS

S FIG. 34. THE DISTRIBUTION OF MARKED MALES ON 8 THREE SUCCESSIVE DAYS . DAY I 4

tel UJ 8 DAY 2

4 O •

cc wcc) 0 8 DAY 3 z

0 12 10 8 6 4 6 8 10 I 2 DISTANCE FROM POINT OF RELEASE (FEET) 131 (1961), this distribution is advantageous especially with low density species in which the accuracy of the estimate of density in each annulus may be improved by using more traps and catching many individuals.

Adults of both sexes were collected in the field in the morning of

15.5.66. They were marked out of doors with two contrasting colours of artist's oil paint (red for the females and yellow for the males). Muir, (1958) has used this type of paint for marking the mirid, Blepharidopterus angulatus (Fall)(Hemiptera) and Mellanby, (1939) found it suitable for marking the bed bug, Cimex lectularius L. (Hemiptera). The marking technique was simple and bugs were marked individually. A bug to be marked was first laid on its back, with its posterior half slightly hanging over the edge of a flat surface. This positioning enables one to avoid injury to the abdomen while picking up the bug by holding the anal tip of the hemelytra in a pair of fine forceps in the left hand. The hand was then turned through an angle of about 1800 to expose the dorsal surface of the bug. Using the right hand, a small quantity of the appropriate paint on a No. 16 entomological pin head was dabbed on the right fore wing. Each marked bug was isolated in a separate dish for a few seconds until the paint had completely dried. A preliminary experiment had shown that neither the red nor the yellow paint has any effect on the activity or survival of marked bugs. A total of 212 females and 60 males were marked and released on the 15.5.66. Since observations had shown that Tingis tended to be more active in the morning and afternoon than in the evening (see (f) belLow), marked bugs were released one by one on the central host plant between 5 and 6 p.m. in order to minimize dispersal due to the "excitation" caused by marking. The method of trapping, marking and releasing wild individuals has been used in studies 132 on _dispersal of_many insect .species `(See Quarterman, Mathis& Kilpatrick,

1954; Clark, 1962; Greenslade,.1964 and Rivard, 1965). Numbers of Tingis on

the central plant and on plants in the annuli were subsequently counted on

each of five consecutive days. These counts provided an approximate estimate

of the population in the centre and in annuli. Owing to the low recovery

rate and intensive walking on the site by the fifth day, further observations

were not made after that date. The mean density in each annulus was regarded

as the frequency with which individuals occurred at that distance from the

central release point. These frequencies were then arbitrarily divided

between two directions from the centre. The results of the first three days

counts for males and females are shown in form of a frequency distribution

(mean arbitrarily set a zero, the position of the central trap plant) in

Figs. 34 and 35; and for both sexes in Fig.36. The total numbers of Tingis

caught at the release point and in annuli at various distances from the

release point are shown for the five consecutive occasions in Table 47.

Table 47: Total no. of Tingis caught at the release point and in different

annuli on each day of recapture.

Total No. of Tingis caught at indicated distances (in ft.) from point of release Day No. Sex 0 1 2 3 4 5 6 7 8 9 10 11 12 Total

5 11 11 8 6 201 10000 45 5 4 4 0 1 0 0 1 0 0 0 0 0 15

5 5 15 19 18 6 3 2 2 0 0 0 0 75 4 6 2 2 2 1 0 0 0 0 1 0 0 18

1 15 19 28 8 2 5 0 1 0 1 0 88 6 1 5 2 2 1 1 1 1 1 0 0 0 21

4 4 8 9 19 4 4 0 3 0 1 1 0 57 2 1 3 1 0 0 0 1 1 0 0 0 0 9

4 2 8 8 12 8 3 2 0 3 1 0 0 51 4 0 2 1 2 0 1 0 0 0 0 0 0 10 133

FIG. 35. TI-E DISTRBUTION OF MARKED TINGIS FEMALES ON THREE SUCCESSIVE DAYS.

DAY I 12

4 1 1 i DAY 2

8 4

03 0 X 16 DAY 3

8 7

1›. 1 1 1 12 10 8 6 4 6 8 10 12

DISTANCE FROM POINT OF RELEASE (FEET) 134

FIG.36. THE DISTRBUT1ON OF MARKED TINGIS FEMALES & MALES ON THREE SUCCESSIVE DAYS.

16 DAY I 12

0 IA t7 I DAY 2 Z I- 12 U- uice zco mz

DAY 3

I I I I 1 IIll, 12 10 8 6 2 z 4 6 8 10 11

DISTANCE FROM POINT OF RELEASE (FEET) 135 The numbers and percentage recovery of males and females for each of the five days of recapture are shown in Table 48.

Table 48: The daily percentage recovery of Tingis out 212 females and 60

males originally marked and released.

Percentage Recovery Day No. recovered No. Females Males .Females Males

1 45 15 21.23 25.0 2 75 18 35.37 30.0 3 88 21 41.51 35.0 4 57 9 26.88 15.0 5 51 10 24.05 16.66

With the exception of the first day of recapture on which 21% and 25% were respectively recorded for females and males, percentage recovery was generally higher in females. This difference in percentage recovery was probably a reflection of the differential mortality of the sexes at that time of the year (females have a higher post-hibernation survival rate) rather than an indication of the slightly higher rate of dispersal in the females. In both sexes, recovery was highest on the third day. This suggests that in the two days following release, many of the bugs were in transit in grass and so were missed during counts. The date of highest percentage recovery of the females corresponded with that on which the longest distance from the point of release at which an individual was found. In general the low level of recovery was due to the high mortality of bugs caused by predators. Many spiders and harvest men, mainly Xysticus 136 and Platybunus spp. were observed taking advantage of the concentration of

Tingis by feeding on them. Some mortality could also have resulted from intensive walking on the small experimental area. Day to day variation in numbers of Tingis caught were caused by differences in levels of activity on different days and differences in activity of the two sexes. In order to find out whether Tingis showed any heterogeneity with respect to the rate of dispersal of marked individuals, kurtosis (Ku) (the departure from the normal curve of the frequency curve of numbers with distance from the point of release) was calculated for each day, according to the formula:

N44 np Ku = Fr1=7-0-ri, (in Southwood, 1966) ( 4dp2 np)2

P`t•-• 0 3rt. where N = total Tingis caught in all traps, dp = distance of the recpature

point (p) from the point of release and np = total number caught on plants at recapture points the same distance from the point of release. Table 49 shows the values of kurtosis obtained for males and for females on each of the five days of these observations.

Table 49: Values of (Ku) calculated for male and female Tingis on five

consecutive occasions. . 137

Kurtosis (Ku) Day No. Females Males

1 2.72 3.77 2 1.86* 4.44* 3 2.03 2.22 4 2.29 2.54 5 2.02 1.59*

*Suggests heterogeneity with respect to rate of movement.

Values of Ku which are significantly greater or smaller than 3 (= value for normal curve) are said to indicate some heterogeneity with respect to

dispersal. With the exceptions of day 2 on which kurtosis values for females

and males were 1.86 and 4.44 respectively and day 5 on which a value of 1.59

was obtained for males, there was no evidence of possible heterogeneity in

the dispersal rate of individuals of the two sexes. The slight indication

of heterogeneity in day 2, is also reflected in the frequencies

near the tail of the distribution in Figs. 34 and 35. In both sexes, kurtosis varied only slightly from day to day. The apparent lack of heter-

ogeneity in movement of individuals was in females probably due to their

reproductive state, (many were mature and ready to lay) and probably

reluctant to move.

Inactivity of the males probably resulted from the availability of an

.unusually large and concentrated number of potential males under the condi-

tions of this experiment. 138 The data presented in Table 47 were analysed in a manner described by Dobzhansky & Wright,(1943) and in Andrewartha, (1961); and the results are shown in Tables 50 and 51 below. Estimates of variance of the distance moved by Tingis were based on the theory of the normal curve in males and normal and non-normal curves (leptokurtic distribution) in females.

Table 30: The dispersal of male Tingp.

Day Mean Temp. Variance Daily increment S. Deviation No. (°c) (in sq ft.) in variance X 0.67 (in ft.)

1 14.2 2.8161 - 1.12 2 13.75 6.2005 3.38 1.66 3 11.9 8.0751 1.87 1.9 4 9.55 9.4207 1.34 2.05 5 10.5 4.2159 -5.2 1.37

Table 31: The dispersal of female Tingis.

2% Variance (C ) in Daily increment Day Mean Temp. ft in variance S: Deviation No. (°C) (x 0.67) in ft. normal non-normal normal non-normal normal non-normal

1 14.2 6.779 5.656 1.74 2 13.75 11.445 12.914 4.665 7.257 2.26 3 11.9 11.127 9.303 -0.318 -3.361 2.23 4 9.55 15.432 14.627 4.305 5.324 2.63 5 10.5 16.693 15.954 1.261 1.324 2.73

Tingis did not move at a constant rate (see columns 5 & 6 Table 51 & column 4 Table 50 ). The daily increment in variance was higher at the start 139 (especially in males) than at the end of the experiment. This seems to suggest that the rate of dispersal was dependent on the degree of crowding.

In column 5, Table 50 and 7 Table 51 , the standard deviations have been multiplied by 0.67 in order to obtain for each day the distance (in feet) away from the centre that half the Tingis population would have reached or passed.

It appears that females moved at a slightly faster rate than males.

However, since males tend to emerge from hibernation, before the females, the slight difference in the rate of dispersal of the sexes may have been a reflection of "physiological age" than of sex. The maximum distance away from the release point at which an individual of either sex was recovered on each day of count is shown graphically in Fig. 37. A male moved a highest maximum distance of 10 ft. in two days and a female 11 ft. in three days.

Since an estimate of the rate of dispersal could be made from the known distance between release and recapture points; and the time interval between release and recapture (lishida, 1966), these would give maximum rates of 5.0 and 3.6 feet per day respectively. Maximum possible dispersal rates have been calculated for each of the five days in Table 52 below.

Table 52 : The maximum dispersal rate of Tingis.

Maximum dispersal rate of an individual Sex in (ft/day) on occasions: 1 2 3 4 5 Female 8.0 4.0 3.6* 2.75 2.0 Male 7.0 5.0* 3.0 2.0 1.2

*Values corresponding to the highest maximum distance away from the point of release at which an individual was recovered in the five day period. 1 40

FIG.37. MAXIMUM DISTANCE OF RECOVERY OF MARKED TINGIS

10 FEMALES e^‘ i— Lu Lu •—, 8

w u z < 1-v, 8 6 MALES / / / /

rim il r■ t7rm t § 1 2 3 4 S DAYS AFTER RELEASE 141 The very high maximum dispersal rates observed for Tingis on the first day

following release of marked bugs, might have been due to the disturbance of the wild bugs. Clark, (1962) and Greenslade, (1964) have mentioned that

marked insects may show exceptionally high rates of dispersal in the first

day after release. After days two and three on which males and females

respectively attained a highest maximum distance from the release point,

movement tended to be slow and trivial with decrease and no further increase

in maximum distance covered.

The daily recovery of Tingis in each of the four sectors, North-east

(NE); North-west (NW); South-east (SE) and South-west (SW) of the gapped

site is shown in Table 53.

Table 5 Distribution of marked Tingis between the four sectors of the

experimental site.

Day after Sector and No. of Tingis recovered release NE NW SE SW Total 1 21 11 10 8 50 2 17 16 29 22 84 3 23 15 31 26 97 4 13 13 20 14 60 5 18 13 9 13 53 Total 92 68 99 83 342

The data in Table 53 suggest the absence of any persistent directional move-

ment of marked Tingis. Individuals seemed to have spread out of the release

point in all directions. 142

It is shown in Figs. 34 & 35 and Table 47 that the density of Tingis fell with distance away from the release point. An attempt was made to determine the most appropriate regression equation for describing this fall- off of density with distance. Southwood, (1966) has listed five regression equations which various ecologists have fitted satisfactorily to data on different dispersing insect species. These equations take the form:-

(1) Y = a + b log X ... . • • ... (Wolfenbarger, 1946) (2) Y = a + b log X + ( it 1946) (3) Log Y = log a1 + b X ... ( Kettle, 1951 )

(4) Log Y = log a + b log X ...... 0041 (MacLeod & Donnelly, 1963) ) (5) Y = a + A ( Paris, 1965 where Y is density at a distance X from the point of release and a, b and C are constants. The results of fitting four of the above equations and a fifth, Y = a + b X +()(14.,..to Tingis data are shown in Tables 54 and 55. In fitting the data, the total population of Tingis in an annulus on a given day has been divided by the number of plants in that annulus, so that density

Table 54 : Fall off of male density with distance away from the point of release.

Day Equation: Y = a + b log X .. ..(1) No. (Wolfenbarger, 1946) 1 Y = 0.421756 - 0.46527 log X 0.001 2 Y = 0.52022 - 0.570686 log X 0.001 3 Y = 0.307691 - 0.297895 log X 0.05 4 Y = 0.195312 - 0.20137 log X 0.05 5 Y = 0.109388 - 0.10199 log X NS

143

Day Equation: log Y = log a1 + b X .. (3) P No. (Kettle, 1951) 1 log Y = - 1.18563 - 0.222949 X 0.05 2 log Y = 0.206557 - 0.602809 X 0.01 3 log Y = 1.19809 - 0.56792 X 0.01 4 log Y = - 1.11925 - 0.478378 X 0.05 5 log Y = - 1.64096 - 0.460617 X 0.05

Equation: log Y = log a + b log X .. (4) (MacLeod & Donnelly, 1963) 1 log Y = - 0.158846 - 6.11859 log x 0.01 2 log Y = 1.13506 - 6.70034 log X 0.01 3 log Y = 1.16495 - 5.05741 log X 0.05 4 log Y = - 0.267941 - 5.47551 log X 0.05 5 log I = - 1.78174 - 3.9444 log X NS

C Equation: Y = a + X .. (5) (Paris, 1965) 0.62X7461 1 I = - 0.0755865 -. 0.001 802757 2 Y = - 0.100186 + 0. X 0.001 4. 0.284272 3 Y = 0.0186149 X NS

4 Y = 0.00807594 + 0.223218X 0.05 0716446 5 Y = 0.0170855 + 0. X NS 144 Day Equation: Y = a + b X + .. (6) No. 1 Y a 0.305829 - 0.0339433 X 0.05 2 Y = 0.372775 - 0.0408258 X 0.05 3 Y = 0.268013 - 0.0270473 X 0.05 4 Y = 0.15732 - 0.0165649 X 0.05 5 Y = 0.104128 - 0.0105408 X NS

Table 55 : The fall off of female density with distance away from the point of release.

Day Equation: Y = a + b log X .. ..(1) p No. (Wolfenbarger, 1946) 1 Y = 1.32608 - 1.41613 log X 0.001 2 Y = 1.38228 - 1.32875 log X 0.01 3 Y = 1.20066 - 1.06429 log X NS 4 Y = 0.87734 - 0.815229 log X 0.01 5 Y = 0.695187 - 0.617019 log X 0.05

Equation: log Y = log a1 + b X .. (3) (Kettle, 1951) 1 log Y = 1.55056 - 0.714914 X 0.001 2 log Y = 2.33624 - 0.743464 X 0.001 3 log Y = 1.32753 - 0.530088 X 0.05 4 log Y = 0.834421 - 0.459171 X 0.05 5 log Y = 1.3931 - 0.546079 X 0.01 11+5

Day Equation: log Y = log a + b log Y. .. (4) P No. (MacLeod & Donnelly, 1963) 1 log Y = 2.11194 - 7.2 log X 0.01 2 log Y = 2.45595 - 6.84613 log x 0.01 3 log Y = 1.42447 - 4.89729 log X 0.05 4 log Y = 1.19497 - 4.02447 log X NS 5 log Y = 1.44769 - 4.98244 log X 0.05

C Equation: Y = a + R ...... (5) (Paris, 1965) 1.75817 4 Y = - 0.152965 + x 0.001 1. 2 20004 NS Y = 0.110783 + X 4. 0.692075 3 Y = 0.251825 NS

4 Y = 0.0921098+ 0.756118 NS

4. 0.494926 NS 5 1 = 0.120871 X

Equation: Y = a + b X + .. (6)

1 Y = 1.02252 - 0.110896 X 0.01 2 Y = 1.24841 - 0.127277 X 0.01 3 Y = 1.18692 - 0.116327 X 0.05 4 Y = 0.799585 - 7.87603 x 0.01 5 Y = 0.66163 - 6.35032 x 0.01

(Y), represents the number of insects per plant.

These equations fitted Tingis data to varying degrees of accuracy. Tingis dispersed at a slow rate. For every 1 ft. distance away from the 146 release point, there was a fall of about 0.03 males and 0.1 females per plant

(equation 6; Y = a + b X + ..) and no Tingis was recovered outside the 11 ft. radius. The significance of regression of fall off of numbers within as short a distance as 11 ft., suggests that Tingis dispersed over short distances. The higher negative regression coefficients for females suggest that they might have moved at a slightly faster rate than the males. This confirms the results obtained on the rate of change of variance i.e. rate of dispersal (Tables 50 & 51) in which females tended to show higher r'tee• The best fits were obtained with the equations of Wolfenbarger, (1946); and MacLeod & Donnelly, (1963). In these equations, the regression of density on distance was highly significant in the first two days after release. Significance dropped in the last three days, probably as a result of mortality or disappearance. By expressing density as numbers of insects per plant, the 'dilution factor' component seemed to have been removed from the fall off in density with distance. This removal of the dilution factor is indicated in the Paris, (1965) equation for females which was significant on the first day only.

The closeness of fit of equations (1), (4) and (6) to Tingis data is shown in Figs 38 - 40 . In Fig.38, the density (Y) has been plotted against, the log of the distance X (in ft.) from the release point. For

purposes of comparison, Paris's, (1965) data (experiment 2, day 1) on the isopod, Armadillidium vulgare have been used to obtain values for a second curve (open circles, dotted line) which is super-imposed on Fig. 38. Before

plotting the log of density (Y) against the log of distance (X) (MacLeod & Donnelly' s 1963 equation) in rig. 39, unity was added to values of X, in

order to permit logarithmic transformation of zero values. The plot of • TINGIS DATA (MALES ) DAY 2) • ISOPOD i) MALES ) DAY 2 0.8 8 1); >- >- „ 0.7 7 1-•z .54 2 0.6 Y : 0.5202 - 0.5707 LOG. X 6 a- LOG Y : 1 1351 - 6 7003 LOG X P< 0,001 ce ge 0.5 5 18 P<001 8 0.4 4

0.3 3

O 2 9 0 2 z 01 1 < ND. LL 9 00 • 2 4 .6 Ile 1 12 4 g 10 I2 LOG. DISTANCE : X IN FEET LOG DISTANCE : X IN FEET

FIG.38 . THE FIT OF TINGIS DATA TO VVOLFENBARGER1 S FIG. 39. THE FIT OF TINGIS DATA TO MACLEOD 6946) EQUATION OF FALL-OFF OF DENSITY WITH & DONNELLY'S (1963) EQUATION OF FALL- DISTANCE FROM THE POINT OF RELEASE OFF OF DENSITY OF BLOWFLY WITH DISTANCE FROM POINT OF RELEASE FIG.40. FALL- OFF OF DENSITY OF TINGIS (MALES DAY 2) WITH DISTANCE FROM THE POINT OF RELEASE . THE EQUATION : Y : A + BX + IS NOT A GOOD FIT FOR THE DATA .

Y : 0.37277 - 0.04082X + . P<0.05

3 4 5 6

DISTANCE FROM POINT OF RELEASE : X IN FEET 149

density against distance according to the equation, Y = a + b X + (equation

6) is shown in Fig. 40. This equation does not seem to fit Tingis data over

the complete range of distance of observation. The fit was nonetheless

good for approximately half of the distance (i.e. for about 6 ft). Plots of

equations (1); (4) and (6) in rigs. 38, 39 and 40 respectively have been

made using data collected on day 2; one of the two days on which the regression of density on distance proved highly significant for equations (1); (4)

and (6). These equations suggest that the fall off in Tingis numbers is

related to a power of the distance. According to Wadley, (1957) this may

correspond to the 'loss factor' in insect dispersion. Southwood, (1966) has

suggested that equations (1)&(4) may apply to comparatively short-lived pop-

ulations which show some heterogeneity of movement and survival as in when individuals of mixed ages are released. In the present studies as well as in those of MacLeod & Donnelly, (1963) individuals of different ages have been marked and released.

Equation (5) has been used to describe the dispersal of woodlice (Paris,

1965). It suggests a linear dilution of density with the reciprocal of the distance from the release point. Although it has been applied to the dispersal of animals (such as Tingis) which spread out in almost all directions from the release point Gable 53 & Southwood, 1966) it did not fit

Tingis data as satisfactorily as equation (1). For example, it fitted data for females on day 1 only as compared with equations (1) and (4) each of which fitted the data on 4 out of 5 days (Table 55 )• 150

The equation of Kettle, (1951) fitted Tingis data for males and females,

but at a lower level of significance in the latter sex. This indicates that, numbers released (60$ ; 212 q!..) had no effect on the experiment. Higher orders of densities would have been needed to show any effects of initial

numbers. The equation indicates that density falls at a constant rate per

unit increase in distance. It was found applicable to the dispersal of

midges from, a breeding site (Kettle, 1951). The experiment shows that Tingis

are capable of short lateral movement. It is unlikely that undisturbed Tingis

in a natural habitat could attain dispersal rates of the order of magnitude

observed during this experiment.

dispersal of Tingis, records of various climatic factors were kept during the

period of observation in dispersal experiment discussed above

These factors were, the mean temperature of the 24 hours (from 6 p.m. on any day to 6 p.m. of the following day); preceeding each occasion on which records of dispersing Tingis were made; the maximum and minimum temperatures

in the same period; mean relative humidity (in %); rainfall, and hours of

sunshine ; Temperature was measured with a thermo-hydrograph; maximum and

minimum temperatures in the 24 hr. period were counterchecked with a Six's

thermometer. Other meteorological data were obtained from the Field Station

data, gathered on a site about 150 yards away from the experimental site.

The age of marked Tingis was also considered as one of the variables (Tables 56 and 57 ) that may affect the numbers of marked Tingis caught on plants

and the rate of their dispersal. The day of release of bugs was taken as

day O. Two separate analyses of the data were carried out on the college 151

computer, (IBM, 7090) by Dr. G. Murdie. One was a multiple correlation and

regression of numbers of marked Tingis caught on the other 7 variables. The other was a similar analysis, in which the daily increment in variance of population of marked individuals caught in mapped experimental site (Tables 48, 53 and 51 were correlated with the variables.

Table 56 : The mean and standard deviations of the variables. No. of pairs of observations = 5

Variable Mean S.D.

= 3.00 1.581 A Age of bugs (in days) . 3.00 1.581 B Mean temperature, °C. 11.99 2.006 C Maximum temperature (°C.) 16.55 3.441 D Minimum temperature (°C.) 6.54 1.567 E Mean Relative humidity (%) 75.21 3.897 F Rainfall (in ins.) 1.14 2.493 G Hours of Sunshine 9.99 4.346 9 m 63.19 17.838 HN Numbers of Tingia caught 7:"= 14.59 5.128

Table 57 : The means and standard deviations of variables involved in multiple correlation and regression analysis with daily increment of variance of dispersing Tingis population. No. of sets of observations = 4 152

Variable Mean S.D.

A = 3.5 1.291 Age of bugs (in days) = 3.5 1.291 B Mean temperature (0C.) 11.44 1.830 C Maximum temperature (°C.) 15.44 2.752 D Minimum temperature (°C.) 6.62 1.796 E Mean Relative humidity (%) 75.97 4.055 F Rainfall (in ins.) 1.42 2.783 G Hours of Sunshine 9.12 4.481 Hy Daily increment in variance = 2.47 2.409 of Tingis population = 0.34 3.797

The correlation matrices for numbers of females and males on the variables are shown in Tables 58 and 59 respectively.

Table 58 : Multiple correlation coefficients of variables (data for female numbers).

A -0.914* -0.813 0.946** -0.393 0.368 0.085 0.194 -0.504 -0.727 0.351 E 0.006 -0.039 -0.266 0.874* 0.456 -0.327 0.641 0.812 -0.163 -0.979*** -0.325 G -0.053 0.083 -0.209 0.874* 0.471 0.777 -0.317 HIVE

Level of significance: * = 5%; ** = 2%; *** = 1% 153 Table 59: Multiple correlation coefficients of variables (data for male numbers).

A -0.914* B -0.813 0.946** C -0.393 0.368 0.085 D 0.194 -0.504 -0.727 0.351 0.006 -0.039 -0.266 0.874* 0.456 F -0.327 0.641 0.812 -0.163 -0.979** ir -0.325 G -0.585 0.648 0.408 0.935** 0.039 0.689 0.161 HN. 4

Level of significance: * = 5%; ** = 2%; *** = 1%

The data in Tables 58 and 59, show that the numbers of Tingis found

on thistles on any day was significantly correlated with the minimum temper-

ature. There were also significant intercorrelations between some of the other variables. These were; between mean and maximum temperature; minimum

temperature and rainfall; and negatively, between age of bugs and mean temperature; and relative humidity and hours of sunshine.

A series of multiple regression analyses, each involving a pair of the

7 independent variables, A to G; was carried out in order to detect the most important factors affecting the numbers of Tingis caught. There was no significant correlation between the numbers of females caught and any pair of the other variables. The few degrees of freedom arising from the small number of observations made, might have accounted for the apparent lack of significance. Five pairs of variables were separately, significantly correlated with male numbers. The multiple correlation coefficients (R4 5); 154 and their significance levels are shown for the five analyses in Table 60 .

Table 60 : Multiple regression coefficients of numbers of males caught on variables.

Analysis 2 No. Variables R R Significance

(1) HNm on B and D 0.991 0.982 0.05,P,0.01 (2) II " C " D 0.992 0.984 0.05> P>0.01 (3) yl " E " D 0.985 0.971 0.0571:0'0.01 (4) 11 " G " D 0.988 0.976 0.05>P>0.01 (5) it " G " E 0.993 0.986 0.05)P> 0.01

The various values of R2 in Table 60 indicate for each analysis, the proportion of the total variability of the numbers of males caught that are accounted for by the two variables. The regression equations for predicting the number of males, (Y) on plants on any given day are shown in Table 61 .

Table 61: Regression equations of male numbers on variables.

Analysis No. Equation

(1) Y = 1304212 + 0.8989 B + 2.6352 D (2) Y = - 12.9736 0.4932 C 2.9672 D (3) Y = 24.8280 - 0.4350 E + 3.4397 D (4) Y = - 10.3450 + 0.3809 G + 3.2317 D (5) Y = - 524.5849 + 5.8078 G + 6.3959 E where B, C, D, E, and G refer to variables indicated in Tables56 ; 59 ; and

155 60 above.

The relative predictive value of each of a pair of variables is indicat-

ed by the p coefficients shown in Table 62 below.

Table 62 : coefficients of the predictor variable.

Analysis Variable and B coefficients No.

= 0.352; D = 0.806 C = 0.331; D = 0.907 E = - 0.331; D = 1.052 G = 0.322; D = 0.988 G = 4.922; E = 4.86

The numbers of males caught on plants on each day was affected by mean

temperature, maximum temperature, relative humidity, number of hours of sun-

shine and minimum temperature. Table 62 shows that it is the minimum temp-

erature that is probably the most important factor affecting numbers in the

field on any given day.

The results of the multiple correlation of daily increment of variance

of male population with variables, A to G, are shown in form of a matrix in

Table 63 below.

Table 63 : Correlation matrix of the variables; (data on daily increment of

variance of male population). No. of obs. = 4 156 A B -0.861 C -0.619 0.921 D -0.682 0.565 0.251 E -0.175 -0.336 -0.664 0.334 F -0.255 0.152 -0.123 0.878 0.397 G -0.014 0.517 0.788 -0.122 -0.974* -0.243 H -0.892 0.539 0.212 0.617 0.582 0.271 -0.431

Level of significance: * = 5•%.

A series of multiple regression analyses were carried out on pairs of variables and daily increment of variance of male population, (V) as described for numbers above. An analysis of variance of regression showed that in two of these analyses, the correlation coefficients were significant at the levels indicated in Table 64 below.

Table 64: Correlation coefficients and their significance levels.

Variables 2 Analysis correlated R R Significance No. with (V)

(1) A and G 0.997 0.995 100.99 0.1) Py 0.05 (2) C and G 0.997 0.995 103.196 0.1 ' P) 0.05

N.D.F. 1 = 2; N.D.F. 2 = 1

The corresponding regression equations relating daily increment of variance or rate of movement to the variables are as follows:

(1) V = 13.0468 - 2.6458 A - 0.3768 G (2) V = - 18.5651 2.0171 C 1.3426 G 157 The level of significance of correlation (Table 64) was low (slightly above, 5%), probably as a result of the few degrees of freedom. However, the results show that there are suggestions that the daily increment of variance, i.e. the rate of dispersal of males was negatively correlated with age of the bugs, and the number of hours of sunshine, but positively with maximum temperature. A comparison of the B coefficients of the variables, A, C and G

(Table 65 ) indicates that C and G (i.e. maximum temperature and number of hours of sunshine) were the most important factors affecting variability in the rate of dispersal.

Table 65 : The p coefficients of variables A, C & G.

Analysis No. coefficients

(1) A . 0.8993; G = - 0.4446 (2) C = 1.4619; G = - 1.584

The absence of a significant correlation between any pair of the variables and the daily increment of variance of female population, suggests a higher threshold than males for movement with respect to these climatic factors. However, some of the females in the field were laying (eggs were present in leaf bud samples) during this period. Thus, their physiological condition may have altered the thresholds of their activity. (ii) Flight 158 (a) Methods

The flight activity of Tingis was investigated by three methods, invol-

ving the use of insect traps. In Heteroptera, the trap population is a

relection of the flight activity of that part of the population which is

currently able to fly (Southwood, 1960).

Window traps: The model used was that described by Chapman & Kinghorn,

(1955). Essentially, it consists of a large rectangular glass sheet about 3 ft. wide and 2.5 ft. high; held vertically, with a trough

containing water and wetting agent below it. Flying insects hit the plane

of the glass and drop into the trough of water. Observations had shown that

Tingis tended to drop and lie helpless on their backs when suddenly released

from a height or blown against an obstacle. In order to trap bugs that come

or go out of the study area, two traps were arranged facing north-south and

east-west at the boundary. Bugs flying out of and into the area were

expected to fall in the water trough on the inner and outer planes of the

glass respectively. Traps were examined for catch at regular intervals

throughout the 1965-1967 seasons. No Tingis was caught, probably due to

lack of appreciable flight capacity or ineffectiveness of the traps arising

from the low numbers flying. It is noteworthy that these traps occasionally

caught some other species of Heteroptera including, Anthocoris nemorum (L.)

(Anthocoridae); Stenodema laevigatum (L.) (Miridae) and Heterogaster urticae

(Fab.) (Lygaeidae).

Suction traps: Two types of suction trap were used in these studies.

One was a 9 in. Vent-Axia ('Silent Nine'), (293 ft3/min.); equipped with a

disc-dropping mechanism for separating catch into hourly samples (See John-. 159 son, 1950a; Taylor, 1955). This trap was operated in 1965 and 1966 seasons over a small patch of thistles, C. arvense, situated about 150 ft. north- west of the study area. The other was the enclosed cone, 18 in. propeller type (2510 ft3/Min.) (See Johnson and Taylor, 1955a, b). Two of the latter type are run annually in 'Silwood Bottom' (See Fig. 1 ) at sites approximately 50 and 70 ft. away from the north eastern corner of the northern limit of the study area. One of the traps samples air at 30 ft. and the other at 4 ft. above ground level.

A single female T. ampliata was caught in the 9 in. Vent-axia trap between 12 noon and 1 p.m. on 8.5.65. Maximum air temperature on that date was 16.0°C and this was attained around mid-day. Tingis was absent from this trap in 1966 season, inspite of the fact that thistles as near as 1 ft. away were infested. Details of catch of T. ampliata and T. cardui in the 18 in. suction traps are shown in Tables 66 and 67 respectively.

Table 66 : No. of T. ampliata caught in 18" suction trap set at 4 ft. above ground level.

1965 1966 1967 Max.emp. Max. Date ;0 ,:. Date 1 t 00. a 1; 10.V. 1 0 15.0 - - - 11.V. 1 1 20,0 - - - - 14-16.V. 0 1 21.6* - - 3-5.VI. 0 1 22.25 17.VI. 0 1 18.5 - -

Season's 0 1 0 0 total 2 3

* mean maximum temperature 160 T. ampliata was not caught at 30 ft. above ground level; and was completely absent from traps in 1964 and 1967. The numbers caught in 1965 and 1966 were too small to merit correlation with climatic factors. However, it is probable that the higher number caught in 1965 was a reflection of the higher spring adult population in that year as compared with 1966 (See section II,4(b)). Of the five specimens caught in 1965, four were in May, during the period before peak of post-hibernation emergence. Southwood and Johnson, (1957) have pointed out that mass post-emergence flights in May are characteristic of many species of Coleoptera and Heteroptera which hibernate in concealing habitats.

Table 67: No. of T. cardui caught in 18" suction trap set at 4 ft. above ground level.

1965 1966 1967 Maxo Temp. Max. Temp. Date C. Date C.

11.V. 0 1 20.0 - - - - 12.V. 1 0 25.0 - - - - 13-15.V. 0 1 18.0* ' 14-16.V. 1 0 21.6* - - - - 21-23.V. 1 0 15.6* - _ - -

Season's 0 1 0 0 total 3 1

* mean maximum temperature

Although, T. cardui is less abundant locally, than T. ampliata; the

catch of the two species in the 1965-67 seasons was remarkably similar

(Tables 66 & 67 ). A single male T. cardui was taken on 10.V.64 (max. 161 Temp. = 16.5 °C.). In 1965, a single female was caught in the trap at 30 ft.

above ground level on 11th May, (max. temperature = 20 °C.). Neither T.

ampliata nor T. cardui was caught in suction traps in the autumns of the present studies.

Sticky traps: These traps were used to find out, (a) whether or not

Tingis fly in the habitat (b) the height of flight (if they fly) and (c) the

direction of such flights in and out of the study area. Several authors,

(Lewis, 1959; Cornwell, 1960) have used sticky traps similar to the cylindri-

cal type designed by Broadbent et al.(1948), for the study of various insect

species. The trap used in the present studies (Fig. 2 ) was a modification

of the latter design in which the adhesives (grease bands) were fastened to

12 thin, expanded steel plates each six inches square. Pairs of these plates were fixed at one foot intervals to a wooden support, 7 feet high and cubical in cross section. A base mark was made at a height of about 6" on the support so that when pushed into the soil to the level of the mark, the first

pair of plates would stand at exactly one foot above ground. The plates were

arranged in such a way that if the first pair faced opposite directions,

(north and south); the plates at 2 feet would face east and west and those at three feet, north and south and so on till 6 feet. This means that each of the four directions was sampled at three levels. Four of these traps

were set up in the habitat, at regular intervals along a north-south median

line running through the study area; in such a way that there was one trap

each on its northern and southern limits. Traps were examined daily for any

catches of Tingis.

Tingis was not caught on any of the traps in the 1966 season. It 162 appears that Tingis do not fly within the habitat, thus further suggesting that the little movement (See (b) above) from plant to plant that occurs takes place by walking. In March 1967, long before the emergence of Tingis from hibernation, new grease bands were put on all plates and the positions of the two middle traps were altered. They were put along an east-west axis, one at each boundry of the area. This arrangement was intended to give some information on flights into and out of the area along the east-west directions. Again, no Tingis was caught on any of the four traps.

(b) Reproductive state of females caught in suction traps

In Tingis, a proportion of the females may or may not be sexually mature at post-hibernation emergence in Spring. The physiological age of dispersing females caught in suction traps was therefore assessed by recording (a) the relative numbers of ripe egg and egg rudiments; (b) the presence or absence of sperms in the spermathecae and (c) evidence of oviposition as indicated by the presence of corpora lutea. The results are summarised in

Table 68 .

Table 68 : Reproductive state of T. ampliata females caught in suction traps

in 1965.

Corpora Date No. No. of No. of Sperms caught dissected ripe eggs oocytes lutea * * 8.V. 1 0 42 10.V. 1 0 35 11.V. 1 0 56

* (+) indicates presence and (-) absence. 163 Flying bugs were sexually immature and 2 out of the 3 bugs dissected were virgins (Table 68 ). A comparison between the state of maturation of females in the field population (Fig, 29 Section II2(c)) and that of females caught in suction traps (Table 68 ) shows that about 57% of the former group was already mature by 9th May; while all were mature by 19th May. This suggests that flight was undertaken on3y by the immature fraction of the field population; with reduced or complete cessation of inclination to fly after maturation. Migration is generally a feature of young animals (South- wood, 1962, 1966) and in many insects migratory flights are made by sexually immature and young females before or just after the teneral period (Johnson,

1960, 1963).

It is now known that in some insect species, incapacity for flight arises from morphological causes. Short-winged Heteroptera such as the females of Loricula elegantula (Baerensprung) (Microphysidae); Mecoma ambulans (Fall.) (Miridae), and most adults of Dolichonabis limbatus (Dahl.)

(Nabidae) cannot fly (Southwood, 1960). However, there are other species with well-developed membraneous wings which are relatively static. For example, the mirids, Capsus ater(L.) and Liocoris trinustulatus (Fab.) and the aquatic heteropteron, Ilyocoris cimicoides (L.) have membraneous hind wings and do not seem to fly. Poisson, (1924) has associated incapacity for flight in the latter species with anatomy of the insect - a complete lack of flight muscles. Indeed, Young, (1961, 1965) has observed a distin- ction based on the amount of flight muscle development, between flying and flightless morphs in some species of Corixidae and Notoneotidae, That a 164 reduction or complete antolysis of flight muscles is associated with a

decrease or cessation of flight activity has been demonstrated in some Cole-

optera (See Jackson, 1933, 1952, 1956; Chapman, 1956), ants (in Wheeler,

1928), mosquitoes (Hocking, 1952) and aphids (B. Johnson, 1953, 1957). Although, T. ampliata was common in the field, only a few specimens were

caught in suction traps. The decrease in the flight duration of bugs flying in the laboratory from late May to the-end of the ovipo-

sition period in June will be referred to in (d) below. Seasonal

changes in the state of flight muscle of T. ampliata was studied in the 1966- 1967 season.

According to Wigglesworth, (1956) the main propulsive forces in flight

of insects other than the Orthoptera, Dictyoptera, Odonata and Coleoptera, are developed by the powerful indirect flight muscles. These indirect muscles consist of the dorsal longitudinal which runs from an anterior

prephragma to a posterior postphragma and the dorso ventral which runs on either side of the gut from the thoracic tergum to the sternum. Two methods

- dissection and microtomy were used in the study of these muscles in Tingis.

Specimens for dissection (10 males and 10 females) were collected in the field, (the same habitat) at intervals, killed with ethyl acetate vapour, and dissected in water under the high power of a binocular microscope ( x 5 x 10). Bugs were categorised into the following five groups, (scored one to five) according to the condition and amount of flight muscle in their thorax:

1. Thorax predominantly filled with parenchymatous creamish-white

fatty tissues. 165 2. Hard, yellowish fatty tissue with a few muscle fibres.

3. Muscle fibres with dense fatty connective tissue.

4. Dense muscle fibres, with very little fatty connective tissue.

5. Dense, hard muscle fibres, no fat in connective tissue.

Although the presence or absence of each of the indirect muscles was noted, no attempts were made to score them separately in these studies. The number of bugs in each of the categories, one to five above, and the total score for the ten bugs dissected on each occasion are shown in Table 69.

The mean scores are presented as 'index of flight muscle development' in Fig. 41. At final moult from the fifth instar to adult insect, and a few days afterwards, the thorax of Tingis was predominantly filled with parenchymatous tissue saturated with creamish-white fat. There was a gradual occurence and development of flight muscles, especially, the longitudinals, during the autumn. The little amount of muscle built up during the autumn remained virtually unchanged throughout the winter months. During hibernation in winter, there was little fat in the thorax and the muscles became dense and compact. In the first two weeks of post-hibernation emergence in spring, there was a further development of muscles (iig, 41). Dissections made on the 3rd May 1967, showed that the dorso-ventrals had become considerably large and thickened relative to the longitudinals which had also increased appreciably by that date. From then onwards and throughout the period of oviposition in females, there was a gradual reduction in the amount of muscle. It is probable that this reduction in the flight musculature of Tingis is associated with the decrease in readiness to fly and in

166 Table 69:

No. Flight muscle Date Sex category and No. Total Mean Dissected of Tingis Score 1 2 3 4 5

5. 8.66 t 10 4 5 1 0 0 17 1.7 01144. 10 3 4 3 0 o 20 2.0

17. 8.66 10 0 2 4 4 0 32 3.2 10 0 2 5 3 0 31 3.1

31. 8.66 10 0 2 4 4 0 32 3.2 10 0 3 5 2 0 29 2.9

6. 9.66 10 o 3 2 4 1 35 3.5 0r1 10 0 3 4 2 1 31 3.1

20. 9.66 10 1 6 3 0 0 22 2.2 ve t 10 0 7 2 1 0 24 2.4

4.10.66 10 0 2 2 6 0 34 3.4 10 0 3 4 3 0 3o 3.0

10 0 2 6 2 0 30 3.0 18.10.66 10 0 1 4 5 0 34 3.4

0 5 4 1 0 26 2.6 3.11.66 $4 1010 0 7 3 0 0 23 2.3

18.11.66 lo 0 4 6 0 36 3.6 10 0 0 5 5 0 35 3.5

30.11.66 10 0 1 4 5 0 34 3.4 10 0 2 7 1 0 30 3.0

167 Flight muscle Date No. Total Dissected category and No. Mean of Tingis Score Sex 1 2 3 4 5 Q.. 10 20.12.66 TA 0 O 4 6 0 36 3.6 0 10 0 O 5 5 0 35 3.5

10 20.1.67 TA 0 O 4 6 36 3.6 vr 10 0 O 5 5 0 35 3.5

6. 2.67 9 10 0 0 3 6 1 38 3.8 10 0 o 3 7 0 37 3.7

8. 3.67 ? 10 5 0 35 3.5 cp 10 5 1 37 3.?

io 0 O 6 4 0 44 4.4 3. 5.67 10 0 O 3 6 1 38 3.8

10 0 30. 5.67 ?•• O 1 5 4 43 4.3 01 10 0 O 0 7 3 43 4.3

21.6.67 10 0 O 1 5 4 43 4.3 10 0 0 2 5 3 41 4.1

flight duration of bugs tested after Mid-May. There appears to be no diffe-

rence in the seasonal changes in muscles between the two sexes. However,

muscle development was slightly higher in females than in males. This diff-

erence is probably a reflection of the body size difference between the two

sexes (section II11(a)). There was no evidence of flight muscle polymorphism

since all the bugs dissected on each occasion after the autumn increment of

muscles (i.e. from 30th November onwards) were in a similar condition of

muscle colour, consistency and growth (Table 69).. It is noteworthy that

the build up of flight muscles which occurs in autumn and early spring, 6 w 10 1 U-

- VELOPMENT 1.5 MUSCLE DE 2.0 0.0 3.0 4.0 5.0 4.5 2 3.5 . 5 AUG. 'SEP. 'OCT.'NOV.DEC. 'JAN.'FEB.MAR.' APR.'MAY'JUN. 'JUL. FIG. 41.SEASONALCHANGESINTHEAMOUNTOFFLIGHTMUSCLE 1966

1967 0- -0MALES • -• FEMALES 169

<-0

UP'

soO o C•1 C; N

Fig. 42 Changes in flight muscles of T. ampliata DIi m Dorsal longitudinal muscle. DVM = Dorso-ventral muscle.

170

forms part of the general process of growth of the insect.

For microtomy„ Tingis were killed in ethyl acetate vapour and fixed in alcoholic-Bouin's. The hemelytra were not removed from bugs fixed between

August and December, 1966. After December, the hemelytra had to be removed

before fixation of bugs, since earlier observations showed that the presence

of the hemelytra tended to increase brittleness of sections and to cause

the head and thorax to break into separate pieces. After fixation, bugs

were double embedded in celloidin and paraffin after the method of Peterfi (See Pantin, 1948 p.29). Longitudinal sections (20 microns thick) of bugs were cut. Trial sectionings showed that thinner or thicker sections than

20 microns were unsatisfactory for the present studies. Sections were then

double stained in Heidenhein's Iron Haematoxylin and 1% Eosin and mounted

in Euperal. The results obtained are shown in Fig. 42. These results

appear to supplement those from dissections.

Flight muscle in T. cardui

Owing to the difficulty of finding enough numbers of T. cardui for

dissection or sectioning, detailed studies on seasonal changes of flight

muscles were not made. However, the results of a few dissections made on 8-9-66 (Table 70 below), show that flight muscle development also occurs in this species during the autumn months following emergence of new generation

adults.

Table 70: State of development of flight muscles of T. cardui dissected

on 8-9-66. 171

No. of bugs in flight Total Date No. Mean diss. muscle category Score

1 2 3 if 5 8-9-66 7 V 0 0 2 2 3 29 4.1 7 ir o 0 1 3 3 30 4.2

For purposes of comparison results of dissections made on T. ampliata on the same date are presented in Table 71 .

Table 71 : State of flight muscle of T. ampliata dissected on 8.9.66.

Date No. No. of bugs in flight Total Mean diss. muscle category Score

1 2 3 if 5 10 V 1 6 2 1 0 23 2.3 8.9.66 10 04 3 3 3 1 0 22 2.2

In both species, specimens dissected had moulted from a collection of fifth instar nymphs on 11.8.66 and were subsequently caged on host plants in the field until required for dissection. The data in Tables 70 and 71 are therefore directly comparable since they are based on individuals of identical age. A larger amount of flight muscle (about X 2) had built up in T. cardui than in T. ampliata by that date. As in the latter species, there appears to be no significant difference in the extent to which flight muscle is developed in the two sexes of T. cardui.

(d) Laboratory studies on flight activity The author did not observe Tingis flying in the field during these 172 studies. However, attempts made to induce the bugs to fly in the laboratory were successful. A pilot experiment was carried out with pre-hibernating individuals on 19-10-65. Bugs were warmed to varying degrees by separately exposing groups of 20 in a petri dish under a 60 watt electric bulb at different heights above the dish. When bugs were sufficiently warmed, some made attempts to fly by opening their wings. Others, simply walked to the edge of the container. The results are summarised in Table 72. The dish containing the experimental females was accidentally damaged and consequently, no data are presented for this sex when heat source was 33 cm above insects.

Bugs put at the latter distance below the light bulb were warmed to about 30°C; those at 22 and 11 cm below the height source were warmed on the average to 35° and 40°0.

Table 72: Number of flight attempts made by Tingis when warmed by 60 watt

bulb at different heights above insects.

Height of No. of flight attempts in Mean attempt bulb above in 10 mins dish 2 4 6 10 mine

Males

11 cm 0(0)* 2(0) 4(3) 6( 8) 0.3 22 cm 1(0) 6(2) 11(2) 19( 3) 0.95 33 cm 0(0) 0(4) 1(5) 5(11) 0.25

Females 11 cm 4(0) 5(0) 5(0) 5(1) 0.2 22 cm 3(0) 8(o) 11(0) 32(0) 1.6

* Values in brackets refer to numbers of bugs out of 20, crawling to

the edge of experimental container. 173 Mean attempt was highest when the bulb was at 22 cm than at either 11 or 33 cm above the dish. The number of attempts increased with the durE.tion of exposure (in minutes). When too warm i.e. when the bulb was at 11 cm or less from the dish and when not warm enough (when bulb was at 33 cm above dish) bugs resorted to active walking out rather than attempting to fly. Although no 'take offs' involving actual flight were observed in the autumn experiment, a few of the Tingis which were similarly warmed in the following spring did fly. The results of warming bugs for 10 mins. to about 35°C (60 watt bulb at 22 cm. above dish) are given in Table 73 and Pig. 43.

Table 73 : Flight attempts and actual flights made by warmed Tingis in a 10 min. period on 3.5.66.

Sex Females Males No. tested 28 13 No. of attempted flights 30 17 Mean no. of attempts 1.07 11.3 No. of bugs making attempts 4 3 % attempting flight 14.28 23.0 No. of take off/flights 4 1 No. of bugs flying 2 1 No. flying as % of No. attempting 50.0 33.3 % of bugs tested flying 7.14 7.3

Room temperature was about 20°C and relative humidity was approximately 57%. Take-off flight was usually preceeded by a fast walk with frequent unfolding of the wings. Then the hug would stop suddenly, raise its head and fore legs in the air and take off. Often the bug would alight a short distance from the point of take-off in the dish and commence walking. The 174 estimated horizontal distances covered during each such take-off are shown in Table 74.

Distance of flight (in cm.) Bug No. Total Mean 1 2 3 1 1.0 0.8 1.8 0.9 2 1.4 0.5 1.9 0.95 3 4.4 o.4 0.4

Height reached at take-off was not measured but appeared to be only a few millimetres.

In a second series of experiments carried out in spring 1966, individuals emerging from hibernation were collected in the field and maintained on thistles in a constant temperature room at 20°C; 60% relative humidity, and

16 hour photo period. At regular intervals from the date of collection (= 3 .5.66) samples of both sexes were taken from the stock of bugs and tested for flight. During tests, bugs were attached at the pronotum to a pinhead by means of a small quantity of artisis oil paint. The sharp end of the pin was pushed into one wooden tip of an ordinary bench lead pencil.

The other end of the pencil was then clamped at a height such that a test bug was about 35 cm above a dull brown desk. A 60 watt electric bulb was arranged at 22 cm above the'level of the test insect. Some bugs flew on being lifted by the pin attached to pronotum; and these were given a flight threshold value of (1) (Table 75). Others had to be subjected to 'a standard sequence of proceedures given one after the other' until flight was initiated. These proceedures were given relative threshold values

175

F1G.43. FLIGHT ATTEMPTS OF TINGIS COLLECTED ON 3.5.66 AND WARNED FOR DIFFERENT PERIODS.

I4 0- — -0 MALES -0-- -0 '-- 1.2 1 4,---• FEMALES I 1.0 I E 0.8 I < 6 0.6 /

$0 I

EXPOSURE TIME (MIN.)

3.5 FIG.44. THE LABORATORY FLIGHT ACTIVITY OF TINGIS . vs (.) •-----• FEMALES .....16i 3.0 ,0 o — —o MALES 02 17- 4( \i---- MATED j2. 0 1— \ I . (2 E.i 1 Z LI a

8 16 24 32 AGE OF TINGIS IN DAYS DAY 0 :3566) 176 varying from 2 to 10 as listed by Dingle, (1965). For example, bugs which neither flew nor opened their wings unaided by the author were given a threshold value of 10, while bugs which opened their wings to any of the proceedures 2 to 8 but never flew were given a value of 9. The duration (in secs) of the first six successive flights made by each flying bug were estimated 1 by means of a Smith's /5 sec. stop watch, and their average was recorded*

The means of these averages are presented graphically in rig. 44, and the range of flight durations observed is shown in Table 77. . After 9th June, flight durations had become so short that it was impossible to make further determinations without considerable errors. However, no bugs flew by 30th

June. Except for a short rise in males on day 7, flight duration decreased and threshold increased as the bugs aged from the date of start of experiment (Fig. 44 and Table 76). . The proportion of females which flew also decreased with age. It is significant to note that by 19th May, some of the females were already laying eggs (revealed by microscopic examinations of thistle leaves from bug cages). The main period of active flight appears to be between post-hibernation emergence and the initiation of oviposition in most females. These observations seem to support current theories on the role of physiological factors in insect migration by flight

(See Johnson, 1963). Other authors, (Young, 1965; Dingle,1965) have similarly observed that the cessation or reduction of flight activity of some other Heteroptera is closely associated with the onset of reproduction, oviposition. Two of the three males which flew in these tests copulated on the 13th May and the third, on the 16th May. After 19th May, there was no difference in the means of flight duration of the sexes. This was confirmed by results of two flight tests carried out on 22.5.67, on 177 Table 76 a Age of Tingis from start of experiment and flight threshold

Females Days from start Threshold 2 9 16 23 30 37 1 9 6 6 4 1 2 2 2 3 1 1 3 1 3 1 2 - - - - 4 3 3 1 2 2 1 5 ------6 - - - 1 1 - 7 - - - 1 - 8 - - 1 - - - 9 3 4 2 3 5 5 10 2 2 1 - 1 1 Total 20 20 12 12 13 10

Males 1 2 1 - - - - 2 - - 1 2 2 1 3 ------4 - - - - - 1 5 ------6 - 1 - - - - 7 - - 1 - - - 8 - - 1 - - - 9 3 2 1 10 7 8 4 6 7 8

Total 10 10 10 10 10 10 178

Table 77 : Age of Tingis, variation in numbers flying and range of flight duration (in secs.).

Range of Day Sample No. of bugs No. of flights (3.5.66 = Day 0) size Flight duration (secs.) flying over 5 secs.

Females 2 20 0 - 20.0 15 7 9 20 0 - 5.0 14 0 16 12 0 - 2.1 9 0 23 12 0 - 2.0 9 0 30 13 0 - 3.0 7 0 37 10 0 - 0.6 4 0

Males 2 10 0 - 4.0 2 0 9 10 0 - 8.0 2 2 16 10 0 - 2.0 3 0 23 10 0 - 3.0 2 0 30 10 0 - 2.5 2 0 37 10 0 - 0.4 2 0 samples of wild Tingis. The mean flight duration for females and males were 0.43 and 0.44 sec. respectively in the first experiment and 0.42 and 0.36 sec. in the second. A chi-squared test (Table 78 ) also showed that there was no significant difference in the proportion of flyers in the two sexes on that date. 179.

Table 78 : Proportion of male and female Tingis flying on 22.5.67.

Experiment I

Sex Non flying Flying Totals 42 p

Females 15 10 25 0.1138 .9 but 40.95 Males 11 9 20

Totals 26 19 45

Experiment II

Females 13 12 25 0.288 )0.5 but <0.9 Males 12 8 20

Totals 25 20 45

The small proportion of males which flew in the 1966 test was probably due to sampling errors or to variation in age distribution among individuals of the original sample taken from the field. Some individuals of both sexes did not fly throughout the test period and since they survived as well, and the females laid as actively as the flyers, it is suggested that behavioral polymorphism is probable. The results of measurements of Tingis wing length and maximum breadth are shown in form of frequency distributions in Fig. 45 (a & b). Measurements were made under a binocular microscope fitted with a micrometer eyepiece. These distributions are approximately unimodal and suggest the absence of polymorphism in wing size. eff....NOMIX11/0.•••• 180

RG.45. THE DISTRBUT1ON OF WNG LENGTH AM) BREADTH IN TINGIS 60- FEMALES MALES

30 AGE NT E

C 20 PER

0 095 1.0 104 109114. 08609 0951.04 1:0 164 169 114 I 2 09 695 164 lin

WING BREADTH (MM) 50

FEMALES MALES 40

30 GE TA N

E 20 PERC I0

2.2 2.4 2.6 2.8 3.0 3.2 22 24 26 28 30 3.2

WING LENGTH (M M) 181 In the 1966 experiment on flight duration (Fig. 44); first tests were made on 5.5.66, i.e. 48 hrs. after collection in the field.

In order to find out whether there was any difference in the flight activity of bugs tested immediately and those tested 48 hours after recovery from the field, an experiment was made in the spring of 1967. 80 females collected in the field were divided at random into two groups of 40. From each group 20 bugs were randomly selected and immediately tested for flight:

The other 20 were caged on plants at 20°C and tested for flight 48 hrs.later. In one of the two replicates, 8 out of 20 bugs flew when tested immediately and in those tested 48 hrs.later. In the other replicate, 9 out of 20 tested immediately flew; and of those caged, 3 died within the 48 hr. period and 5 flew out of the 17 bugs that survived and were available for flight tests.

For all flying insects in the two replicates and treatments, the duration

(in secs.) of each of six successive flights were measured and the average flight duration was calculated for each bug. An analysis of variance was carried out using the weighted difference method for obtaining interaction sums of squares as described by Rao, (1952) for correcting for unequal numbers of observations in cells. The result is shown in Table 79. An F-test shows that there was no significant difference in the mean of flight duration between replicates or between insects tested immediately after recovery and 48 hours later.

Table 79 : Comparison of flight duration (secs.) of bugs tested immediately after capture in the field and those tested 48 hours later. 182

D.P. S.S. M.S. F. Replicates (ignoring times) 1 0.0219 0.0219 1.2882 N.S. Time 1 0.0055* 0.0055 3.0909 N.S. R x T 1 0.0016

Between Cells 3 0.0290 0.0097 Within Cells 26 0.4434* 0.0170 Total 29 0.4724

* By difference.

Within cell variation is larger than interaction mean square and is used in testing replicates and times. The frequencies of occurence of bugs having each of the flight thresholds, 1 to 10 in bugs tested immediately (= coded, Al and A2) and in those tested 48 hours later (= coded, B1 and B2) are compared in Table 80 below.

Table 80 : Flight threshold of bugs tested immediately (A1 _ 2) and those

tested 48 hours (B1 - 2) after capture in the field.

Time of Flight threshold and No. of bugs: test & expt. No. 1 2 3 4 5 6 7 8 9 10 Total

A 1 2 2 0 0 6 1 0 0 3 6 20 2 2 0 1 3 0 1 1 0 8 20

B 1 3 3 0 0 3 1 1 0 3 3 17 2 4 2 0 0 5 0 0 0 3 6 20 183 Flight threshold appeared not to have differed between bugs tested on the

two separate- occasions; thus supplementing the results presented in Table 79.

(iii) Discussion on flight & dispersal of thistle Tingis.

Although dispersive movements may often lead to death of many individ-

uals of a population through inability to find a suitable habitat, it appears

that animals need to be constantly dispersing (Andrewartha, 1961). In many

terrestrial Heteroptera, the usual modes of dispersal are by flight and/Or

walking (Southwood, 1960). The results of the experiments on walking indic-

ated that T. ampliata, like other Heteroptera undertakes some degree of

locomotion within its natural habitat. However, unlike many non-migrating,

Mirids (See Southwood, 1960, 1962) and Pentatomids (See Brown, 1965) which

have relatively high rates of movement, of the order of several metres per

day, Tingis displacement rate was slow; about 2 ft. per day (round the release point). Dispersal rates of the above order were observed within the

two weeks following post-hibernation emergence, when dispersal was ideally

expected to be most active. It therefore seems unlikely that rates higher than 2 ft. per day could be attained later in adult life when oviposition and senescence of individuals became more prevalent. Although, the rate of movement was not constant from day to day, the data on Tables 50 & 31 shows that 50% of individuals of both sexes were clumped on plants within a radius of 2 ft. from the release point on each of the five consecutive days.

This suggests that although analysis of the data failed to inditate excessive density-induced dispersal, it is probable that the maximum distances moved in the first day after release must have been due to the high concentration of bugs on the release point. The highest density of Tingis per plant seen 184 under natural conditions in spring did not exceed 5, in all the three seasons

of this study. Under such conditions of low density, maximum dispersal rates

of the order observed in this experiment therefore seem unlikely. This view

is supported by the result of the 1965-1966 trap plant experiment, in which

the maximum distance of recovery of bugs from the study area was 4 ft. Increase in activity produces dispersal but some animal species are rarely

seen outside the shelter of the vegetation upon which they feed, (Lewis &

Taylor, 1967). This statement seems true of T. ampliata; and other Tingids

(See Johnson, 1936; Bailey, 1952) which may heavily infest a plant, while

leaving other host plants close by completely free. Thus, the movement of

Tingis, observed in these experiments could be regarded as trivial since move-

ments consisted mainly of displacements between plants in the immediate

vicinity of the point of release. It is noteworthy that repeated daily

search did not indicate the presence of marked Tingis on host plants outside

the area within the 11 ft. radius of the mapped site.

The flight activities of some speCies of Heteroptera are now known to be affected by climatic factors, especially maximum temperature (Southwood,

1960; Waloff and Bakker, 1963). In T. ampliata, the small numbers flying

(Table 66 ) did not permit any statistical analysis. Analysis of data on walking experiment provided evidence which suggests that the rate of movement

of males is affected by the age of bugs, number of hours of sunshine; and maximum temperature. In this respect, T. ampliata appears similar in behaviour to the Rhododendron lace bug, Leptobyrsa rhododendri Hovarth and the lantana bug, Teleonemia lantanae Distant which are said to spread more rapidly in warm dry weather than in cold, wet ones (See Johnson, 1936 and 185

Fyfe, 1937). The movements of T. cardui and the possible role of climatic factors in relation to the rates of such movements were not studied. How- ever the observation of the author and those of Cobben, (1948) who reported that it can be found on thistles 'even in bad weather' coupled with the fact that its distribution extends further north and south than that of T. ampliata (See Southwood & Scudder, 1956) suggests that it is naturally more tolerant of varied and severe climatic conditions.

Tingid species, like those of other Heteropteran families, vary considerably in their capacity for flight. Whilst some species such as the broom tingid, Dictyonota strichnocera Fieb. and the alder lace bug, Corythuca mollicula O. & D. are relatively static (See Southwood, 1960 and Bailey, 1962-3, respectively), others fly actively and may in fact engage in migratory movements at some stage in their annual reproductive cycle. Members of the latter category include species such as, Monanthia globulifera Walk.; Corythuca spp., Stephanitis and Teleonemia serupulosa Stel (= lantanae Distant) (See Sharga, 1953; Glick, 1939; Pierre - P Grasse, 1951; and Roon- wal, 1952 respectively). Although, T. ampliata was common on plants close to suction traps, only a few specimens were caught in the 1964-1967 period. This suggests that it does not engage in any significant dispersive flight in the field. It is noteworthy that in his conclusion on the flight activity of Heteroptera, Southwood, (1960), grouped T. ampliata among the static category. Under laboratory conditions, it was possible to induce flight in T. ampliata by warming bugs to high temperatures rarely attained in the field. Often such flights were mainly of relatively low thresholds and short durations (a few seconds). Working on the non-actively flying milk- 186 weed bug, Oncopeltus fasciatus (Dallas) (Reteroptera: Lygaeidae), Dingle,

(1965), concluded that low threshold, short duration flights represent non- migratory 'flits', whereas high threshold, long duration ones indicate migration. It appears that the low threshold, short duration flights observed in T. ampliata under laboratory conditions, could similarly be regarded as 'flits'. Although flitting may neither occur frequently in any single individual nor in many individuals in a season, it is highly probable that on a few warm days, some may become activated enough to engage in it.

According to Taylor, (1960) a small, flitting insect may become wind-bora:a

In Tingis, such wind-bourne vagrants, would account for the few specimens caught in traps. The observations of Sharga,(1953) and Slocock, (1934) suggest that other non-actively flying Tingids are occasionally blown from plant to plant by wind in a similar manner. Although such vagrants are of ecological significance in the colonization of new habitats, their loss does not seem to have any significant effects on the density and. balance of the parent population.

T. cardui, unlike T. ampliata is an active flyer (Southwood, 1960).

The observations made during the present studies confirm that this is so.

It is said to engage in diurnal post-hibernation flight activity(Southwood

& Johnson, 1957). In the spring of 1965; the numbers of T. cardui caught in traps were unexpectedly high (4 specimens; 3!.t and compared with

T. ampliata (5 specimens, 2 and 3 t), which was locally more abundant.

This discrepancy between trap catches and 'commoness' in the field, appears to be a common feature of some related or unrelated insect species (Thomas, 1938)-Southwood, (1960) has reported large catches of Calocoris norvegicus 187

(Gmelin); Lygus rugulipennis Popp. and Orthops kalmi (L.) which do not seem to reflect abundance in the field. The relatively high numbers of T. cardui caught, coupled with the fact that C. vulgare, its host plant, was absent from Silwood Bottom and adjacent habitats in the 1964-1967 seasons, suggests a higher level of frequency and intensity of flight as compared with T. ampliata. Thus, the occurrence of T. cardui in traps did not depend on the floristic composition of the immediate surroundings of traps.

Tingis with marked flight capacity may attain differing altitudes

during flight. Easley, (1962) has recorded catch of Tingtls at an elevation

as high as 50 ft., and Glick, (1957) has collected some species from an aeroplane. During the present studies, T. cardui was caught in a trap at 30 ft., thus further indicating that it is an actively flying migrant. In his review of animal migration in relation to habitat, (Southwood, 1962), concluded that the colonisation of new habitats is of great ecological as well as evolutionary significance to many species; and that in the Heterop-

tera, the level of migratory dispersive movement le governed by the degree of permanence or impermanence of their habitats. Thus, high dispersive movements are associated with inhabitants of temporary habitats, and inability

to fly, with those species that live on permanent habitats. Atkins's (1966) review on Fehavioural variation among Scolytids, also emphasised the role of the type of habitat as a determinant factor in the level of migratory

activity of the beetles.

The two thistle lace-bugs, T. ampliata and T. cardui colonize two

seperate host plants with different growth habits. The former species colonizes the perennial weed, C. arvense whilst the latter is confined to 188

the biennial, C. vulgare. Consequently, it is reasonable to assume that in

order to reach a host which may be locally rare at periods of greatest mig-

ratory tendency in its life cycle, T. cardui, must engage in active disper-

sive movements, - flights. Such flights may be of long duration and may

necessarily involve coverage of long distances if they are to be effective,

i.e. enhance the possibility of locating a new host plant. Such flight may

take place before and/or after hibernation. Although T. cardui was not

caught in traps in the autumns of the present studies, the records of South-

wood & Scudder, (1956) show that autumnal flights may occur. Autumn flights

serve as insurance against failure to reach a host in the following spring;

and they may be partly associated with the sudden death in the autumn of a

thistle in its second year of growth. It is worth mentioning here that, in

a colony of T. cardui studied on North Gravel in the 1966 season, the pop-

ulation dropped by about 45% between 8th & 12th August. Examination of the

bases of plants and other micro-habitats showed that the bugs had not

migrated downwards as adults of T. ampliata usually do (see section II,2(a)).

Since the number and composition of predators on the flower heads did not

increase or alter in the four day period, it seemed logical to assume that

the missing bugs had probably migrated by flight.

The relative flight capacity of T. ampliata and T. cardui appears to be

closely associated with the degree of development of flight musculature in

the two species. Anatomical studies showed that in the former species,

flight muscles are present, but developed to a lower extent than in the

latter, (at least during the autumn, post-emergence period). In this respect, the differential flight behaviour of thistle Tingis is similar to 189 that of the fully-winged; flying, Labia minor L. and non-flying, Furficula auricularia L. (Dermaptera: Furficulidae), in which incapacity for flight in the latter species is associated with a reduction in amount of longitudinal muscles and an absence of the dorso ventrals (Mercier and Poisson,

1923). T. ampliata, therefore enters the category of the 'chersodromian' process of Guenot and Mercier (1923) which is characteris'.ed by the persis- tence of wings and a more or less marked atrophy of flight muscles. How- ever, unlike F. auiicularia, T. ampliata has well developed dorso ventral muscles, at least during spring. T. cardui also shows a slight superiority over the latter species in having well developed dorso-ventral muscles even in autumn. Thus the differing flight capacity of the two Tingis species may be comparable to those of some corixids and notoneCtids, in which the essential anatomical characteristic distinguishing flying and flightless morphs is the amount of flight muscle present, rather than a presence or absence of flight muscle (Young, 1961, 1965). By having fully developed, membraneous wings but showing virtually no active flight in the field, T. ampliata conforms in behaviour to mirids like, Capsus ater (L.) and Liocoris tripustulatus.(Fab.) which inhabit more or less permanent habitats. In the absence of migratory movements by flight or walking, the cutting and convey- ance of plant tissues containing Tingis must be as in other non-migratory

Tingids (See Johnson, 1936), the main mode of dispersal of T. ampliata.

The relatively static nature of T. ampliata suggests that its population dynamics over a number of years may be studied and interpreted without the complications of immigration and emigration, provided the study area remains undisturbed and clearly delimited. Such a population study may be difficult to carry out with T. cardui, which is constantly migrating in response to 190 changes in the location of its habitat. Thus, whilst high migratory ability enables T. cardui to cover the wider range of distribution of its host plant (See Southwood & Scudder 1956), it is always locally less dense than T. ampliata in spring, probably as a result of the high mortality of migrants through failure of many individuals to reach a host plant.

Ce) Distribution of adult Timis in the field

As a result of the heterogenous nature of habitats, the. distribution of most animals in the field tends to be patchy, that is, aggregated rather than random or even (Elton 1949, Andrewartha, 1961). According to the latter author, the pattern of the distribution may often be explained either by the behaviour of the animals or some event in the history of the population. Information on the occurence of different stages of T. ampliata on various parts of thistles has been given by Southwood and Scudder, (1956), who mentioned that in early May, adults aggregate into localised colonies.

The spatial distribution or degree of aggregation of an insect species may be expressed in one of several statistical indices. A detailed account of some of these dispersion parameters and the techniques of their application is given by Southwood, (1966).

1. Taylor's Power Law: 10 plants were randomly selected from each of 8, (coded A - H) plots, (one, 55.4 x 55.0 ft.), into which the area was divided. Counts of adult Timis were made between•10 a.m. and 1 p.m. on each of several occasions at weekly intervals. From the data collected, the mean 191 number of bugs per plant and their variances were calculated for each plot. These were transformed into log ( X + 1); (where X = value of mean or variance) as shown in Tables 81 and 82 . Table 81 : The means and variances of Tingis counted on the different plots on 21st and 28th May, 1965.

Mean Nqfr of F Log. ( X + 1) transformation 211:11Z-Aer Plot Total plant Variance Mean Variance code Tingis (TC ) ( S2) (T) (s 2) . ' (28.5.65) A 9 0.9 1.7 0.27 0.44 B 6 0.6 1.15 0.2 0.33 C 8 0.8 1.78 0.25 0.44 D 6 0.6 0.45 0.2 0.16 E 9 0.9 0.93 0.27 0.28 F 6 0.6 0.45 0.2 0.16 G 3 0.3 0.1 0.11 0.04 H 2 0.2 0.1 0.07 0.04 A + B 15 0.75 1.35 0.24 0.37 C + D 14 0.7 1.22 0.23 0.34 E + F 15 0.75 0.93 0.24 0.28 G + H 5 0.25 0.19 0.09 0.07 A + H 11 0.55 0.99 0.19 0.29 B + G 9 0.45 0.68 0.16 0.22 C + F 14 0.7 1.06 0.23 0.31 D + E 15 0.75 0.93 0.24 0.28 / (21.5.65) A 6 0.6 1.15 0.2 0.33 B 9 0.9 1.65 0.27 0.42 c 7 0.7 0.9 0.23 0.27 D 7 0.7 0.45 0.23 0.16 E 2 0.2 0.17 0.07 0.07 F 8 0.8 1.51 0.25 0.39 G 3 0.3 0.41 0.11 0.16 H 0 0.0 0.0 0.0 0.0 # 192

Table 82 : The means and variances of Tingis counted on the different plots on 19th and 26th May, 1967.

Mean Notof Lgg. ( X + 1) transformation Tingis per Plot Total plant Variance Mean Variance code Tingis (I) ( s 2) (TC ) (S 2) (19.5.67) A 3 0.3 0.45 0.11 0.16 B 10 1.0 0.44 0,3 0.15 C 6 0.6 0.48 0.2 0.17 D 11 1.1 2.54 0.32 0.54 E 11 1.1 2.1 u.32 0.49 F 11 1.1 1.21 0.32 0.34 G 5 0.5 1.05 0.17 0.31 H , 3 0.3 0.23 0.11 0.09 A + B 13 0.6 0,55 0.21 0.19 C + D 17 0.85 0.97 0.26 0.29 E + F 22 1.1 1.46 0.32 0.39 G + H 8 0.4 0.62 0.14 0.21 A + H 6 0.3 0.33 0.11 0.12 B + G 15 0.75 0.77 0.24 0.25 C + F 17 o.85 0.87 0.26 0.27 D + E ' 22 1.1 1.67 0.32 0.43 (26.5.67) A 9 0.9 0.76 0.27 0.25 B 5 0.5 0.5 0.17 0.18 C 11 1.1 0.76 0.32 0.25 D 15 1.5 2.5 0.39 0,54 E 8 0.8 1.06 0.25 0.32 F 13 1.3 2.01 0.36 0.48 G 9 0.9 0.54 0.27 0.19 H 5 0.5 0.5 0.17 0.18

When log. variance, (S2) was plotted against log. mean (1), (Fig. 46 ) it was found that they increased together. This relationship has been demonstrated for many insect species (See Harcourt, 1961b, Hayman & Lowe, 1961; Harcourt, 1963); and according to Taylor, (1961, 1965), it obeys a power 2 - b law, generally expressed by, S =aX ,where'a'and'b'are constants;le is FIG. 46. THE RELATION BETWEEN VARIANCE AND MEAN FOR COUNTS OF TING IS ON DIFFERENT PLOTS

x ON 21.5.65 x ON 19.5.67 • n 28.5.65 • v 26.5.67 8 I BULKED SAMPLES ON 28.5.65 o } BULKED SAMPLES ON 19.5.67

Y: -0 05 + I 56X Y: -0 03 + 126X

b: 156 b: 13

•x x 0 0• • 0 • x

LOG. MEAN (3-( 194

said to be mainly a sampling factor, while'b'is an index of aggregation

characteristic of the species. Tingis numbers were generally low. Many plants harboured no bugs and of those which had bugs, none had more than 4 or 5 insects on any occasion. The various values of means and variances used to obtain the points in Fig. 46 and for calculating the regression equations for the fitted lines were collected on different sampling dates, and by bulking samples from adjacent plots to form different-sized sampling units and means. Southwood, (1966) states that this practice does not in- validate the application of the power law. There was considerable variation in the scatter of points around lines fitted in the different years. This

scatter appears to be a reflection of variation of habitat on the degree of aggregation (Southwood, 1966). The annual variation in the habitat and host density has been discussed in section I2(b-c). Table 83 shows the regression equations and coefficients calculated for the fitted lines for the 1965 and 1967 data in rig. 46 . Table 83 : The regression equations for lines in Fig. 46.

Year Equation 1965 0.868 < 0.001 Y =-0.05+1.56X 1967 n 0.779 <0.001 Y =-0.03+1.26X

There was no significant difference between the regression coefficients; (i.e. the values of 'b' obtained in the two seasons): a comparison of the two coefficients after the method described in Brownlee, (1960) gave a value

of 't' = 1.036 (P>>0.5) for 44 degrees of freedom. These observations indicate that adults exhibit a positive contagious distribution in the field, i.e. they are more aggregated than would be expected from a random 195

distribution; ('b' was significantly greater than unity).

Healey, (1964) found the index of aggregation, 'b' a useful parameter for comparing the distribution of species of grass-inhabiting Thysanoptera It is shown in section III that the value of 'b' obtained for T cardui was 1.63, which appears not different from that observed for T. ampliata. This similarity in the value of 'b' suggests aggregation behaviour in these, two related species which colonize separate host plants in nature.

2. Poisson distribution: Counts of new generation adults were made on each of 70; 3 x 3 ft. (=9 ft2), random quadrats taken from the study area on 25.8.66. Each plant in a quadrat was beaten three times on a 3 ft. square white canvas 'beating' tray, and the bugs counted and removed. The total number of bugs in a quadrat was taken as the sum of bugs beaten off all plants in that quadrat. The frequency of occurence of quadrats containing, 0, 1, 2, n bugs is shown in Table 84. . The observed variance, (S2) of this distribution was 5.17, which was almost twice as large as the mean, I (=2.59). The distribution was tested for agreement with the Poisson series. Since the expected variance of a Poisson distribution is equal to its mean, the observed variance multiplied by the degrees of freedom n, (= N 1; where N, is number of observations); may be divided by the 2 2 nS sample mean to obtain , = — (See Bliss & Fisher, 1953). On this basis, X 2 2 for Tingis counts was 138.061376, (P4:0.001), was significantly higher than its expectation; thus indicating that the overdispersion was too large for the Poisson series. The insects did not show random distribution:,

3. Negative Binomial distribution: Counts on insect populations which show 196

positive contagion may be adequately described by the negative binomial distribution (Bliss & Owen, 1958; Harcourt, 1965); which is also applicable to a wide variety of other biological data (Anscombe, 1949; Bliss & Fisher, 1953). Data on Tingis were used to calculate the expected negative binomial frequencies, 0 as described by Debauche, (1962). The values are presented in Table 84 . Table 84 : Fitting the negative binomial to Tingis counts (Beating Method) 25/8/66. . ... No.of Observed Negative binomial Tingis frequency expectations . (f - 904 per beat f 0 0 O 15 11.975000000 0.764144 x-- 9 15.074249750 2.447650 C V 16 13.355483793 0.523640 K 1

13 10.173005559 0.785598 3 7.116729498 2.381350 U

4.713338779 4 / 1 0 7 3.004423537 L%

- 1.861781177

C 14 11.776736922 0.419717 O

0' 0 1.128909634 2 0.677841430 0 r- '1141 4132D 0.395442365 Total 70 69.471205522 7.322099 J. r P 2 2 4 ( f 90 7.322099 - D.F = 4. P) 0.1(0.2

In order to carry out al 2test for the goodness of fit of the expected 197

negative binomial frequencies with the observed, f; small frequencies at the

bottom of each series have been pooled in order to avoid values of, ci less

than 5. For 4 degrees of freedom, 2 was 7.322099; (P = 0.2 approx. i.e.

about 20%). The fit can be considered as satisfactory; since P, would have

to be 5% or less before the hypothesis of agreement between the observed and expected distribution can be regarded as disproved (Brownlee, 1960). .A

similar analysis of Tingis data at lower population levels, showed that the

distribution tended towards random. The dispersion parameter, of the

negative binomial distribution may be computed by several methods (South-

wood, 1966). 'k'values obtained for Tingis by approximate methods, (1) and

(2) as listed by the latter author were 2.58 and 1.62 respectively. Both

methods give estimates of'k' that are reasonable for populations with small

means i.e. of 'low density'. Means calculated for Tingis data were generally

low and about 15 out of 70 (= 21.4%) samples were free of insects. The

estimates of,Ik'for Tingis by these methods are therefore reliable and,

according to Anscombe, (1949); wheniki >1; method (1); i.e. that based on 3t 2 the relation between variance and mean: k = ; gives about 90% S2 efficiency. Using the maximum likelihood approach (= method (3) in South- wood, 1966), which is the most efficient method and like method (2) involves

'iterative solution';'k' was found to be 2.45, which confirms that method

(1) gives a reasonable estimate.

The exponent,'10 is a good measure of the degree of aggregation; although it generally increases with the mean and is not constant for the species (Waters & Henson, 1959). Its value varies from, 0 (at highest aggregation levels) to infinity, (when the distribution is truly random). 198

In general, high values of'k' indicate a tendency towards randomness. The

'k'value of 2.4, estimated for Tingis autumn population suggests that the

insect is moderately aggregated in the field.

Aggregation of adult Tingis is partly a reflection of the distribution of its immature stages, i.e. of oviposition behaviour of females. It will

be shown in section II3(b-c) that eggs and nymphs are aggregated to varying

degrees. Field observations showed that autumn adults tended to be found

only on plants whioh had carried a population of eggs and nymphs. 'The

patchy distribution of thistles has been mentioned in section 112(a-c). Some

of the important biological basis of aggregation in insects have been

discussed by Waters, (1959) and the role of abiotic factors in aggregation

by Allee et al, (1959).

The aggregation of individuals often results in localised condensations

of population (See frequency distribution in Table 84 ); and consequently

observed variances are often greater than would be expected from a random

distribution. Thus, the total observed variance (S2) is composed of two

components: (1) an increment proportional to the degree of aggregation of

the population and (2), the normal variance resulting from habitual fluct-

uation about the mean, (3Z), (Debauche, 1962). In a negative binomial distribution; component (1) above is said to correspond to the term lc- , 2 2 -5- 2 in the relation, S = X+ is not aleatory and is often excluded Xk k from calculations of errors, In analysis of Tingis population data (See

section II,4(b)), this point has been taken into consideration.

4. Other measures.cil disyersive_Rattern: Green, (1966) has discussed the 199

.relative usefulness of the parameter,qd of the negative bin6mial distribution and other indices for the measurement of non-randomness in the spatial distribution of a population. He proposed the ex coefficient, generally S4 -1 2 presented as Cx = m (where S = variance; m = mean andi)(, is the den- fiC -1 sity„ i.e. total number of individuals sampled. This index is said to be appropriate for positive contagion in that its value varies from 0 to 1, for randomness to maximum positive contagion, regardless of variation in number of samples, sample size or insect density. Morisita's (1959, 1962) index of dispersion, 18 ; is also relatively independent of the type of distribution, the number of samples or of the size of the mean (Southwood, N 1966). IS may be presented as: IE - ,fx , where N = total ( .x)2 - 1x number of samples; X" = numbers in a sample and iox is the total number of individuals in all samples. Its value varies from less than 1 (regular distribution) to unity (random) and to greater than unity (when there is positive contagion). Both CL and IS. , are said to be good measures of aggregation for comparing sets of samples with equal numbers of observations.

They were calculated from data on counts of adults made at weekly intervals on randomly selected thistles in the study area in 1965 and 1966 seasons (section II, 4(a)). The seasonal pattern of distribution, as indicated by the two indices is shown in Figs. 47 and 48). Fluctuations in the values of both indices were similar in each season. In the two seasons, aggregation of Tingle tended to be highest during periods of high population in spring and autumn (i.e. when qt and I were respectively greater than

0 and 1). Soon after the start of spring and autumn emergence, when populations were small, the distribution of bugs tended towards random (i.e. 200

FIG. 47- SEASONAL CHANGES IN DISTRIBU TION OF ADULTS 1965 •-•-• Cx 0.036 0- -0 IE, 7.2

0.032 6.4

0.028 5.6

0.024 4.8

0.02 4.0 It. Cx 0.016 3.2

0.012 I 24

1 / 0 0.008 1.6

0 0.004 0.8

0.000-rimj 9- 0.0 ° T"--"7- 0° ._ AUTUMN i EMERGENCE MATING DYING PRE-HIBERNATION -0.004[ & DISAPPEARANCE t FEEDING LAYING 0.012 -0.012 201

FIG. 48. SEASONAL CHANGES IN DISTRIBUTION OF ADULTS 1966. 0.05 A cx o- -o Is 0.032 6.4

0.028 5.6

0.024 4.8

0.02 4.0 I Cx Ia 0.016 -3.2

0.012 24

0.008 1.6

0.004 0.8

0.000 0 0 0 0-0 o 0.0 M JY A 0 DYING (MATING UTUMN PRE-HIBERNATION 281H LAYIN EMERGENCE APR. & FEEDING DISAPPEARANCE —0.012• • I& • • —0.012 FIG50 SHCMING THE INDEPENDENCE OF GREEN'S (1966) Cx INDEX ON THE SAMPLE MEAN (R) • 065 FIG 9.SHOWNG THE INDEPENDENCE OF 16 O 1966 ON THE SAMPLE MEAN (g • 1965 01966 0 0 • 5.0

4.0

• 0 • 0 • • 0 • • 0 • • • • 0 0 • • 0 • • 0• • •• •• 0 • o • • • • 0 • 0 0 • 0 0 0 r° 2 4 .5 .6 ./ A MEAN 00 203

with Cx 4: 0; I = or K 1). Similarly, the degree of aggregation was low or near random during the few weeks which preceeded the last date of occurence of adults in spring and autumn. The relative independence of either IS or Cx on the sample mean calculated for Tingis data in the two seasons is shown in Figs. 49 and 50.

(f) Diurnal variation in the distribution of adults

Some insects (McDonogh, 1939) and mites (Rodriguez & Ibarra, 1967) are known to show marked diurnal activity rhythms which consist of either up and down movements on vegetation or of movements from one part of the habitat to another, (Dempster, 1957).

Diurnal changes in the vertical distribution of adults was studied in the summer of 1965; from 2nd - 23rd June. Each of 20 plants were infested with six males and a second set of 20 plants were similarly infested with six females in the field. The insects were caged on plants with modified W & D cages, the sides of which were made of nylon netting (except the top 1" and bottom 4"), supported with four thin, vertical steel plates. Each cage had a circular removable lid, with about two-thirds of its area covered with netting. To cage the insects, the bottom ends of cages were pushed to a depth of 4" below ground level. The general arrangement of cage and plant is as shown in Fig. 51. Counts of Tingle on the top and bottom halves of plants were made at 6 a.m.; 1 p.m., and 8 p.m. on each of several days following the date of the start of the experiment. The cage FIG. 51. CAGE USED FOR STUDYING DIURNAL VARIATION IN VERTICAL DISTRIBUTION OF ADULTS

- - UD

- - NYLON NETTING

- - THISTLE SHOOT

- THISTLE LEAF

- - - CIRCULAR TUNNEL

t *-1 1-f ff - - GRASS & WEEDS

I 205

Was removed before and replaced after counts on a plant. This was made possible by a 1" x 2" tunnel, which was dug round the limit of the outer circumference of each cage in position. The results of 12 days observation are summarised in Fig. 52, which shows the proportions of total insects found in the two regions of plants at different times of day. Although, more Tingis were generally found on the top than on the bottom half of plants, there was a marked diurnal variation in the proportions found in different regions of plants. The proportion of insects in the top half declined and that in the lower half increased progressively from 6 a.m. through 1 p.m. to 8 p.m. Besides innate cycles of activity, response to weather changes are said to account for diurnal fluctuations in the vertical distribution of some insects on vegetation (Dobzhansky &

Wright, 1943; Fewkes, 1961; and Southwood, 1966). There was no significant difference between the total numbers of Tingis recorded at the different times of day (Fig. 52X), The difference between the mean temperature of any two of the three different times on which counts of insects were made was about 5°C. 206

FIG -52. VARIATION IN THE VERTICAL DISTRIBUTION OF ADULTS

TOP HALF OF PLANTS MI BOTTOM HALF OF PLANTS

100- FEMALES MALES FEMALES & MALES

111•••••• 80 0

60 O

E 4° U 8 20 ONO 0 111 I i I I I 6 I 8 6 I 8 6 8 A.M P.M P.M A.M PM P.M A.M P.M P.M TIME OF DAY

FIG. 52X.NUMBERS OF TINGIS COUNTED AND MEAN TEMPERATURE 160 AT DIFFERENT TIMES OF DAY

140 •—• FEMALES o- - o MALES 120 x TOTAL AM_ MEAN TEMP

ca R 80

MBE .0— — -o 0 NU

40 TAL

TO 20 A.M PM PM TIME OF DAY 207

3. Aspects of the Biology of the Immature Stages (a) Development of eggs and nymphs (i) Their size. Tingid eggs are often of small dimensions (Lesion 1953) though they are large relative to the small size of the adults (Butler 1923). The results of measurements of 200 eggs of T. ampliata are shown in sable 85 . Eggs were measured under a binocular microscope fitted with a micrometer eye piece. To prevent shrinkage of eggs, all measurements were carried out on moist filter paper in petri dishes.

Table 85 : The length and maximum width of eggs.

Mean ± Standard Measurement (mm) deviation

Length 0.61 ± 0.017 Maximum width 0.23 4' 0.011

As in other Tingidae (Roonwal 1952), there is considerable variation in the size of the eggs of T. ampliata. This variation in egg size may mark the beginning of the variations in the sizes of nymphs; but this interesting subject now needs experimentation. Data on the size of nymphs of Tingis are given by Southwood & Scudder (1956).

(ii)Weight of nymphs. Thistle buds containing nymphs were collected in the field. Nymphs were separated from buds in the laboratory and weighed individually on a Cahn Gram Electrobalance. The results are shown in Table 86. 208

Table 86 : The mean weight of nymphs collected in the field on 2.8.67.

Instar No, Mean weight Range weighed (mg) (mg)

I 10 0.027 0.02* - 0.0372 II 17 0.047 0.03* - 0.0586 III 31 0.13 0.041* - 0.4548 Iv 18 0.496 0.425* - 0.642 if 18 0.459 0.406* - 0.602 ir 17 1.068 0.489* - 1.546 di 16 1.066 o.446* - 1.532

*Weight of an indiVidual immediately after hatching or moulting into that stage.

The most striking feature is the sharp weight increase between the third and fourth instars. This pattern of growth does not appear to be consistently related to the relative duration of instars (Table 87 ).

Table 87 : Comparison of ratios of increase of weight of instars and those of their durations.

IT ZIT IV

0.11•1/••• ••=11.1..• MOM.

II /II IV Ratio of increase of 1.74 2.76 :3.81 I. :2.15 weight of instars ct :3.53 of:2.32 mean:3.67 mean:2.23 Ratio of durations of instars 1.26 1.05 1.33 1.16

The efficiency of food conversion by a nymphal stage of a hemipteron may not depend on its duration (Fewkes 1958; Johnson 1960)0. It 209 is probable that Tingis instars also vary in their ability to metabolise plant materials into body tissues.

The lower value in each range of weights in Table 86 refers to the weight of an individual immediately after hatching or moulting into that stage. Weight of nymphs immediately after moulting was always less than before. This weight loss at moulting of the instar obviously arises from loss of exuvial material. A knowledge of the weight of nymphal instars is of great importance in the study of the energy ecology of a species.

(iii) Role of temperature in development. Tingis eggs were laid in leaves of caged thistles in a constant temperature room at 20°C and 60% relative humidity. Leaves were examined under the high power of the binocular microscope and eggs were removed by cutting off a small leaf area of about 4 mm square from the point at which each egg was laid. The eggs were randomly divided into five groups and each group was immediately transferred to and incubated in one of five constant temperature regimes;

10, 15, 20, 25 and 30°C. Incubation cages consisted of 7.5 x 1.5 cm plastic petri-dishes, the floors of which were lined with moisture-saturated cotton wool to a depth of about 0.3 cm. Leaf cuttings containing eggs were put on a filter paper which was spread over the cotton wool layer of each dish and the lid was put 'in place'. Relative humidity, (approx. 100%) in cages was tested at intervals with cobalt thiocynate paper after the method of Solomon (1957). Incubation cages were opened once each day and records of daily egg hatch were kept for the five temperature regimes.

Data on the incubation period of eggs at the various temp- 210

eratures are given in Table 88 and Fig. 53 which shows the conventional temperature-time curve.

Table 88 : The incubation period of eggs of Tingis at constant temperatures.

Temp. Incubation period (days) 2 ± Standard deviation S

10 Oa 15 24.27 ± 0.31 0.9697 20 12.37 ± 0.63 0.396 25 8.43 ± 0.5 0.254 30 7.17 ± 0.38 0.1477

A rise in temperature within the range 10-30°C, reduced the period of development, Y in days. The relationship between the rate of develop- _ 1Y ment, and temperature in °C is also shown in rig. 53 . The rate of

development of eggs also increased with temperature. Although Davidson, (1944) and Andrewartha & Birch, (1954) have suggested that a logistic curve should be fitted to growth data, works on various hemiptera (Johnson 19110, Muir 1966, Champlain & Butler 1967; Champlain & Sholdt 1967) and other insect species (Stephen, 1965) have demonstrated that within a temperature o range of 15-35 C a linear regression equation provides a satisfactory approximation. A regression line has been fitted to the points in Fig. 53 . The high coefficient of determination, R, of the relationship between temperature and rate of development, (R = 0.991; P (0.01) shows that the data was adequately described by a linear regression. Calculated values 1 of the reciprocal of the time of development, T at the various constant 211

FIG-53. EFFECT OF TEMPERATURE ON THE RATE OF DEVELOPMENT IN EGGS OF TIMIS

30- 0....0Period of devpt. -0.3 40...... 40Rate of devpt. 28

0.25 24 e•-.%

H r

16 T ENT

12 OPM L OPMEN DEVE EVEL 0.1 D r.=.4 0 F 8 O

0 rz 0 at 0.05 4

00 0 5 10 IS 20 25 30 35

TEMPERATURE (t.) 212 temperatures are presented in Table 89 . The data in Table 89 were used 1 1 to calculate a regression equation: Y = 0.00665X-0.05462, where Y is the inverse of time of development in days and X is temperature in °C. From the above regression equation, a prediction formula has been calculated thus: Y 1000 (6x - 54)

Table 89 : The rate of development ( Y ) of eggs at different temperat- urea.

Temp. Time of dev. Rate of dev. Average % dev. oC (days) ( Y ) ) per day (42)

10 - - - 15 24.27 0.0412 4.12 20 12.37 0.0808 8.08 25 8.43 0.1186 11.86 30 7.17 0.1395 13.95 where Y is the developmental time in days at the given average temperature X in °C. When the fitted regression line was produced backwards it cut the abscissa at 9°C. Although, there was evidence of embryonic development, eggs did not hatch at 10°C. The threshold temperature for the development of Tingis eggs was probably between 9 and 10°C. Percentage of eggs which hatched at each of the five different constant temperatures is shown in

Table 90 . Although egg hatch at 25°C was not significantly higher than at 30°C; the point corresponding to the latter temperature (in Fig. 53) deviated slightly to the right of the fitted line, thus suggesting that 213 developmental time at 30°C was longer than would be expected. It appears that 30°C is slightly above the optimum temperature for egg development. Table 90 : Percentage hatch of Tingle eggs incubated at various constant temperatures.

No.of eggs No.of eggs percentage Temp. hatched oC incubated hatched

10 31 MIN 15 31 22 70.96 20 31 27 87.09 25 32 30 93.75 30 31 29 93.54

(iv) Development in the field. The period of development of eggs and nymphs in the field was estimated by different methods based on times (in days) between (a) dates of first occurence (b) dates of last occurence and (c) dates of peak occurence of successive stages in field samples. These were supplemented by daily observations on individuals incubated and reared on thistle buds in 3 x 1" tubes fitted with hollow stoppers covered with muslin. Details of the results of an incubation experiment carried out in 1967 are given in Table 91 . Table 91 : Development of eggs incubated in the field, 1967.

Laid Hatched Mean incubation Mean Temp. period (days)

21.6.67 7-11/7 18.8 n* = 16 15.7 22.6.67 9-10/7 19.2 n = 5 15,3 23.6.67 12-13/7 17.1 n = 3 16.0 n* = no of individuals 211+

The mean duration of eggs and nymphs reared in tubes are compared with data computed by other methods in Table 92 & 93 Owing to sampling errors, some individuals may be missed, especially at the beginning and end of each season when insect density is very low. Data obtained on individual experimental insects are therefore more reliable than those of the other methods. Table 92: Period of incubation of eggs in the field.

Method Year and period of incubation in days 1965 1966 1967

1. Days between first 25 24 28 occurence of eggs and C=16.5 C=17.82 C=16.74 first instars D= 7.2 D= 6.39 D= 7.25 2. Days between peak 23 25 16 occurence of eggs and C=18.14 C=20.67 C=19.82 first instars D= 8.74 D= 9.51 D= 7.97

3. Individual observations 16.0* 17.4* (9-26)** (17-20)** E=14.3 E=13.7

C = mean maximum temperature, °C, and D = mean minimum temperature, C. E = mean temperature, °C. * = mean incubation period in days. ** = range of incubation period. Table : Duration of development of instars in the field (in days)

INSTARS 1965 1966 r 1967

Method I II III IV V I II III IV V I II IIT IV V

'1. Time between days n 10 20 of first occurence ' 8 15 6 7 12 13 20 8 4 12 8 21

2. Time between days of last occurence 7 7 9 10 20 7 8 10 11 16 7 7 8 14 14 J I 3. Time between days 8.o of peak occurence 13.0 13 10 20 4 7 14 17 19 8 4 12 8 18

4. Individual 6.7 8.5 9.0 observations* 12.2 14.0 6.0 7.1 10.0 12.0 15.8 7.0 7.1 11.0 12.0 13.2 .

*mean period of development (days) 216

The period of incubation in the field varied only slightly in the years, 1965-67 (Table 92). As can be expected, the period of development varied with the stage of instar; young instars (1st and 2nd) apparently developing at faster rates than older ones (3rd to 5th). The total time taken for nymphal development in the field in the three years are compared with average daily maximum and mean temperatures during May to September in

Table 94.

Table 94 : Time taken for nymphal development in 1965-67 compared with

average daily maximum and mean temperatures.

Days between start Av. daily Av. daily max. temp. mean temp. Year of hatch and appearance o o of first adults c c

1965 60 17.92 13.38 1966 58 18.92 13.84 1967 53 .1 19.00 14.16

The period of development of nymphs tended to vary from one yeear to the other according to field temperatures (Table 94).

(v) Effect of humidity on development and hatching of eggs. In insects, a "zone of optimum humidity as well as upper and lower fatal limits exist for each species and each stage of its development" (Uvarov,

1929). Among terrestrial Heteroptera the great variation in the range of humidity preference for egg development and hatching is often associated with variations in their egg structures and normal oviposition sites 217

(Kayumbo, 1963).

Eggs were laid by male and female pairs in a constant temperature room at 20°C. They were removed from thistle leaves as previously described (p. 209) and randomly divided into six groups within twelve hours of laying. Each group was incubated at 25°C in a separate 'micro-dessicator' in which relative humidity was controlled with a saturated solution of an inorganic chemical salt. Essentially, each dessicator consisted of two 3" x 1" tubes arranged one above the other in such a way that the open end of the upper tube fitted tightly to the top end of a hollow parallel-sided stopper on the lower which was about half full of solution. The bottom end of the stopper was covered with muslin. The saturated solutions used and their equilibrium relative humidities as given by Stokes & Robinson, (1949) and listed by Solomon (1951) are shown in Table 95 . A 10% R.H. was obtained by using a saturated solution of zinc chlOride (See Howe, 1956). Leaf cuttings containing eggs were put on the muslin and the relative humidity of the air in the egg compartment of each micro-dessicator was checked with cobalt thiocynate paper (See Solomon, 1957). The observed range of humidities in each tube is also shown in Table 95 . Table 95 : Chemicals used for humidity control and the relative humidities at 25°C

Chemical Expected R.H. Observed R.H. (%) range (%) Zn Cl 9-10 2 10.00 Mg C12. 6H20 33.00 32-35 Mg (NO3)2. 6H20 52.86 51-53 Na Cl 75.28 75-77 KC1 84.26 84-85 KNO3 92.48 91-93 218

Tubes were opened for about two minutes each day and records of hatched eggs in each group were made. Eggs which did not hatch after 60 days were regarded as failing to hatch. The results are summarised in Table 96 . Table 96 : Incubation period and hatching of eggs in absence of contact

moisture at various humidities at 25oC.

Relative No. of Humidity eggs No. Mean duration (%) incubated Hatched Hatch (days)

10 - 11 33 0 0.0 - 32 - 35 28 0 0.0 51 - 53 27 1 3.7 12.0 75 - 77 29 1 3.4 10.0 84 - 85 29 3 10.3 10.0 91 - 93 29 3 10.3 9.3

Since the number of eggs laid in a 12 hr. period was not sufficient for all

experiments, the eggs incubated in each humidity regime were bulked from oviposition on four consecutive occasions. Egg hatch was extremely low even in the high humidities. Percentage hatch tended to increase with humidity. Incubation period was shortest in eggs kept at 93% R.H. than at any other lower humidity.

Eggs which failed to hatch were examined under the binocular microscope. Many of those in the low humidity regimes (below 53% R.H.) showed

no evidence of embryonic development, the eggs having shrivelled and turned

dark-brown. By contrast, a majority of unhatched eggs in the high humidity range (76 - 93% R.H.0 showed evidence of partial development. There was 219 some degree of swelling and colour change from creamish-yellow to pale

brown. It appears that failure to hatch at both low and high humidities

was due to cessation of development of embryo as a result of either excessive loss or inadequate supply of moisture. It is shown in Table 90 that hatch of eggs laid by the same females and incubated in a saturated atmosphere on a continuously damp surface at 250C was as high as 93 per cent. This coupled with the low hatch of eggs at high humidity regimes in the humidity experiment suggest that Tingis eggs need to absorb moisture either by imbibition through direct contact with a free moist surface or from a saturated atmosphere in order to complete embryonic development and hatching. At laying, the eggs are pale-white and slim and may be partially or completely embedded in the succulent thistle mesophyll. During development, the chorion is considerably distended (probably due to the imbibition of contact moisture from the mesophyllic cells) and the eggs become notice-. ably increased in size (Fig. 7(D)). Absorption of moisture and consequent swelling during embryonic development seems to be a common feature of the eggs of some insect species including Heteroptera which oviposit in green plant tissues (See Johnson 1954, 1937; Kayumbo 1965).

(b) The distribution of eggs

An account of oviposition behaviour in Tingis and of the different sites of egg-laying in thistles has been given in section II2(0)0 Observat- ions made in the summer of 1965 and 1966, indicated that eggs tended to be more abundant in a short region close to the leaf base. On three egg sampling occasions in 1967, viz; 16.V; 20.V; and 24.V; the horizontal 220 distribution of eggs was investigated further. The length of each of 150 primary leaf samples collected in the study area on a sampling date (See section II4(a)was measured (in cm); and then divided arbitrarily into three equal regions, coded first-third (FT), middle third (MT) and last third

(LT). Leaflets of axillary buds harvested with each primary leaf were similarly divided into three regions. Records were made of the number of eggs found in each of the three regions of each leaf or leaflet. The results are summerised in Table 97 . More eggs (an average of about 50% of total) were found in the first third of leaf lengths, followed by the last and middle thirds in a decreasing order.

Table 97 : No of eggs found in different region of thistle leaves and

leaflets; percentage of total in brackets.

Number of eggs in: Date Total FT MT LT

16.5.67 11(40.7) 6(22.2) 10(37.0) 27 20.5.67 13(65.0) 3(15.0) 4(20.0) 20 24.5.67 24(47.4) 16(27.1) 15(25.0) 59

Observations on the vertical distribution of eggs were made in the period from 13th May to 6th June, 1965. On each of 6 occasions, 10 plants were selected at random and harvested at ground level from the study area. Each plant was divided into two equal regions; an upper (UH) and a lower half (LH). Leaves and leaflets in each half were examined individually under the microscope and the number of eggs found were recorded.

The results are presented in Table 98 . 221

Table 98 ; The number of eggs laid in different regions of thistles.

Date Total Eggs laid (LH) as % eggs of Total eggs (UH) (LH)

13.5.65 4 0 4 100 16.5.65 13 0 13 100 19.5.65 19 7 12 63.1 24;5.65 28 2 26 9248

30.5.65 30 5 25 83.3 6.6.65 23 2 21 91.3

In the 1966 and 1967 seasons, 75 plants were randomly selected from the study area and sampled. Each plant was divided by eye into an upper (UH) and lower (LH) half. Two leaf-bud samples were taken in separate polythene bags; one from each region of the plant. The four aspects, North, South, East and West of plants were sampled. Thus, if the two samples from the two levels of one plant were taken from the northern aspect, in the next plant, they would be taken from the eastern, and in the next, from the southern and so on. Samples were examined for eggs under the microscope in the laboratory. A 't' test has been used to compare the number of eggs laid in the two regions (Table 99 ). Data used for comparison in 1966 (Table 99 ) are based on 20 sampling occasions between 16th May and 6th August; which c_vers about two-thirds of the oviposition period. Those for 1967, were collected in the period from 16th May to 21st June, the date of maxima egg numbers in the field. Results obtained in the two season supplement one another and further support those presented for 1965 in Table 98. 222

Table 99 : Comparison of number of Tingil eggs in different regions of the host plant.

Mean Year Region of No,of Total plant °beery. eggs No of 't' Significance eggs

1966 vx 20 235 11.75 LH 20 412 20.6 2.08015 P4; 0.05

1967 UH 10 364 36,4 LH 10 1096 109.6 2.7317 0.02>134),_2:21_ 411.1111111101••••111.11.1.111•111M0•••••••••••••

These results show that significantly more eggs are laid in the lower than in the upper halves of plants. They therefore differ from the observations of Southwood & Scudder, (1956).

The vertical distribution of eggs indicated in Table 99 was studied in greater detail in 1967. A random sample 16 plants were harvested at ground level, from the study area on 4th June. In the laboratory, the height of each leaf-bud unit on the main stem was measured (in inches) and the number of eggs were counted under the microscope and recorded against the corresponding height. There was a total of 228 leaf-bud units and these were grouped according to height on stem at 1" intervals: 0-1; 1-2; 2-3 n-n4-1. A total of 108 eggs were recorded. Eggs found in leaf/ buds of the different height groups are expressed as percentages of total eggs in form of a frequency distribution in Eig. 54 . Most eggs were laid in leaf-buds standing at 1-2" above ground level. Egg density falls progressively from the latter level to the top of plants. Eggs were always absent from leaves in a region 2 to 4" below the apical meristems

FIG.5. THE VERTICAL DISTRIBUTION OF TINGIS EGGS

36-

32-

bn 24 (9w 20 __I 1--< 1-0 I6 6- 12 cep 8

0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 HEIGHT OF THISTLE LEAVES ON STEMS (IN INCHES) 224 of plants. This is probably due to the fact that adults seldom frequent the involucres of plants (Southwood & Scudder, 1956), some of which were already flowering by 4th June. There was very little variation in heights of plants (range, 7-10 ins). A large proportion of leaves at 0-1" level were mature and some were already turning yellow. They are among the first aerial leaves formed, and they are usually old before the onset of oviposition. It is probable that age and yellowing might have made them less suitable to bugs for feeding and oviposition. This may partly explain the small numbers of eggs found in that region.

The relationship between the density of eggs and mean number of leaf-bud units at the different levels of plants is shown for the 1965 and 1966 seasons in Table 100. There were more leaf-buds in the upper than Table 100: The relation between the vertical variation in numbers of eggs and mean number of leaf-bud units.

PART OF PLANTS ITEM YEAR UH LH

No.of eggs/150 leaves (as %) 18.07 81.93 1965 Mean No.of leaves/plant (as % of total) 57.095* 42.905*

No. of eggs/150 leaves (as %) 36.32 63.67 1966 Mean No.of leaves/plant (as % of total) 56.06* 43.87*

*Based on average for determinations in May and June, the main oviposition months. 225 in the. lower halves of plants. However, leaves in the lower halves were always longer and broader than those in the upper half. The total leaf area available for oviposition was probably identical in the two regions.

It, therefore appears that the difference in the proportion of eggs laid in the different regions was not due to variation in available oviposition site. Scarcity of oviposition site would be expected to have a marked influence on the distribution of eggs, when insect density per plant is high (See Kobayashi, 1966; Agwu, 1967). Since most of Tingis eggs are laid around noon (Section II2(aand there is a tendency for more adults to stay on the top half of plants about noon, (Section II2(fIl the vertical variation in the numbers of eggs laid in the two regions could not be explained on the basis of adult distribution on plants. The habit of laying in the lower regions of plants appears to be an innate behaviour such as is common to Leptoterna dolabrata (L.) and some other grassland Mirids (See Osborne, 1918; Jewett & Townsend, 1947).

The number of plants out of 10, with 0, 1, 2 n eggs per plant were arranged in a frequency series for each of the six occasions in 1965, on which vertical distribution of eggs was studied (see Table 98 ). The total frequency distribution is shown in Table 101.

This distribution did not fit a Poisson series. AZ2 test based 2 2 on the relation; = S (N-1) (See Bliss & Fisher, 1953), gave a. value of 2 2 A,e = 191.20511, (P4:0.001). The variance, S (= 6.319491) of the distribution was about three times as large as its mean, m (=1.95). The signi- 2 ficance ofX indicates that eggs were more grouped than would be expected if they were randomly distributed. The negative binomial expectations, g 226

Table 101 : Fitting the negative binomial distribution to data on Tinvis eggs.

No of Total Negative binomial 2 eggs per frequency expectations (f - 91) plant f 0 0

0 24 28.34 0.6646294 1 10 10.292 0.0082844 2 8 6.05645 0.6236964 3 6 4.01882 4 5 2.81764 5 1 2.038962 6 1 1.5061017 7 2 1.128657 8 1 0.854885 14.2086787 1.00116 9 1 0.652871 10 0 0.501862 11 1 0.387837 12 0 0.301043

o Total o 60 58.8971317 2.30821

have been calculated and fitted to the data as shown in Table 101. Good- 2 ness of fit was tested byiAte . For purposes of the test, frequencies smaller than 5 have been bulked. The value ofZ2 was 2.3082 (0.5 P>.0.1); thus indicating a satisfactory fit between observed and expected negative binomial distribution. Estimate of the parameter k, based on variance and mean was 0.870238. This fractional value of k indicates that eggs are aggregated in the field. Values of the S2 ratio, k and of Morisita's

(1959, 1962) Is (See p099) calculated from egg counts on leaf-bud samples for dates corresponding to maxima of egg numbers in 1966 and 1967 are shown in Table 102. 227

Table 102: Estimates values of aggregation indices for dates of maxims

number of eggs in field samples.

Indices and their values Date S2 m k Is

30.5.66 2.67 0.29 4.37 21.6.66 1.93 0.5 2.98 21.6.67 4.31 0.52 2.89

The values of the aggregation indices in Table 102 , tended to vary with the level of the plant. Table 103 shows the results obtained for the upper (UH) and lower (LH) halves of plants on two different dates in 1966. Table 103 : Variation of the values of aggregation indices with region of plants.

Values Date Index (LH) (UH)

52 3.04 1.29 30.5.66 k 0.36 0.86 Is 3.77 2.19 s2 m 2.23 1.28 21.6.66 k 0.45 1.34 Is 3.22 1.75

The degree of aggregation was slightly higher on the lower half of plants, probably as a result of the fact that more eggs are laid in that region 228 than in the upper half. It appears that the aggregation of eggs is associated with that of nymphs which is discussed in the next section.

Reference has been made in section I,2(b) to the distribution of nymphs on different parts of thistles. Numbers of nymphs found in the two halves of plants, on separate consecutive sampling occasions (see section II,

4(a)) during the 1966 and 1967 seasons are compared in Table 104 . Them was a distinct vertical variation in the distribution of nymphs; more being found in the upper (UH) than in the lower half (LH) of plants.

Table 104 : Comparison of occurence of nymphs at different levels on the

host plant. (1966 & 1967).

Year -3Vegion No. of Total No. Mean no. observ. nymphs nymphs

1966 UH 28 796 28.1+2 LH 28 64 2.28

1967 UH 22 988 44.9 LH 22 231 10.5

The proportion of nymphs of each stage (expressed as percentage of total of that stage) found in samples from the two regions of the plants on dates corresponding to peak occurence in the field is shown in Table 105 . 229

Table 105: Proportion of nymphs found in different regions of host on

dates of peak occurrence of nymphs.

Year Region of INSTAR plant I II III IV

1966 UH 100.0 100.0 100.0 100.0 61.5 LH 0.0 0.0 0.0 0.0 38.5

1967 UH 72.09 93.54 83.33 90.0 47.36 LH 27.9 6.45 16.66 10.0 52.63

The vertical distribution of nymphs may be partly explained by the variation in number of green leaf-bud units at the different levels of plants, (Table 106 ). Table 106 : The relation between vertical variation in numbers of nymphs and mean number of thistle primary leaf-bud units

ITDI PART OF PLANT YEAR (UH) (LH) No.of Nymphs in samples (as % of season's total) 61.48 38.519 1965 Mean No.of leaf-bud units/plant (as % of Total) 58.654* 41.345* No.of Nymphs in samples (as % of season's total) 92.55 7.44 Mean No. of leaf-bud units/plant 1966 (as % of Total) 59.044* 40.913* No.of nymphs in samples (as % of season's total) 81.1 18.9 Mean no. of leaf-bud units/plant 1967 (as % of Total) 63.48* 36.51*

*Based on averages for determinations in the period, May - September. 230

There were more leaf-bud units in the upper halves of plants in each of the three seasons. It is noteworthy that unlike egg's (See above), nymphs are more abundant in the upper half of plants. This seems to suggest that there is an active post-eclosion dispersal of first instar nymphs, probably in search of food and/or protective buds.

There was a tendency for the proportion of nymphs found in the two levels of plants to vary with the season i.e., the age of the instar (Fig. 55 ). Young instar nymphs (1st & 2nd) remains relatively inactive in leaf-buds. However, during the 3rd - 5th instar stage, nymphs become noticeably more active and movement from one bud to another may occur. This partly explains the progressive decrease in the proportion of nymphs found in the upper half of plants. Changes in distribution pattern with age of the instar appears to be a common feature of some Heteroptera, (see Kiritani, 1964; Nishida, 1966). It is noteworthy that this significant change in distribution occurs late in the season, (July to September), when deterioration of the habitat has started from the lower levels of plants (see section I, 2(c)).

From the data on 150 leaf-bud samples collected on each sampling 2 occasion; estimates of the ratio of variance to the mean (S ); k, and Is (See sections 1I,2(e) & II,3(b)) have been trade for each of five sampling occasions about the date of peak occurence of each instar in the field. Values obtained for dates of peak numbers are shown in Table 107 . The

mean and range (in vertical bars) of Is for the five determinations around the peak of each stage are shown for the 1966 season in Fig. 56. FIG. 55. SHOWING TIE EFFECT OF GENERATION AGE ON THE DISTRIBUTION OF FIG. 56 .SHOWING AGGREGATION OF IMMATURE INDIVDUALS STAGES N THE FIELD (1966) ♦ -♦ 965 -x 1966 o-o 1967

IN STARS INSTARS 232

Table 107 : The variation of degree of aggregation with stage (date of

maxima numbers).

Year Aggregation Instars and values index I II III IV I I • I 1 1 If I S2 2.05 1.6 2.37 2.24 1.17 1966 k 0.2 0.26 0.18 0.12 0.2 Is 6.04 4.89 6.5 9.?4 4.16

S2 in 1.89 2.1 2.6 1.22 1.01 1967 k 0.32 0.186 0.198 1.16 1.4 Is 4.15 6.45 6.1 1.73 1.06

For purposes of comparison, similar estimates for the egg stage are 2 included in Fig. 56 . In each instar nymph, the value of S was higher and that of k, less than unity; thus suggesting that nymphs are usually aggregated in the field (See sections II,2(e) & II,3(b)). Similarly, values of Is

vary from less than unity (under dispersion) to unity (random distribution) and greater than unity as when there is positive contagious distribution. The degree of aggregation varies with the instar. It was highest in the early instars, 1 to 3 and then decreases in the older instars, 4 and 5 (Fig. 56 ). The change in degree of aggregation with instar age, is probably associated with changes in the behaviour of instars from, one of relative inactivity in the young to that of pronounced activity and

redistributive movement on the host in older nymphs. On two occasions;

i.e. 6th and 12th August, 1966, Is values were found to be zero for 5th instars. This appears to suggest that the insects were underdispersed on

233 those dates. However, this may not be true since calculations made for data on a later date, 16th August gave a value of Is = 4.2. Furthermore,

Kiritani & Kimura (1966) have pointed out that clumping may remain undetect- able in a late stage of an insect owing to mortality of earlier stages and moulting of that stage into adults. At low population levels, the signif- 2 icance ofZ in a test of departure from randomness may be suspect (Healy, 1962). With the exception of the 5th instar, the degree of aggregation tended to be higher in nymph than in the egg stage (Fig, 56 ).

The seasonal change in 4 calculated from sampling data for adults and immature stages (eggs and nymphs); is shown as a composite diagram in Fig. 57.

Data for adults are based on 3 weeks running mean for Fig. +8; and those for immature stages on values obtained from calculations on randomly selected sampling occasions.

(d) Effects of feeding of ilymohs on thistles.

It has been shown in section II,3(c) that Tingle nymphs are mainly confined to axillary buds of thistle leaves where they feed in small aggregates. Early in summer, when only the first and second instar nymphs are in the field, the effects of feeding on buds may not be instantly noticeable. Later in the season, usually in the first half of July, older nymphs start to appear. These remove large quantities of sap from the leaflets of axillary buds. Sucked cells are depleted of chlorophyll, and the abaxial surfaces of leaflets become mottled with yellowish blotches indicating the feeding punctures made by nymphs. Several adjacent cells may be sucked so that eventually, the leaflets present a variegated, chlorotic appearance. Small dark-green spots of egesta are often scattered over the leaf surface. Teter on in the season, infested leaflets begin to 234

FIG. 57 COMPOSITE DIAGRAM SHOWING CHANGE IN II THROUGHOUT THE SEASONS, 1966 . • ADULTS 0 IMMATURE STAGES

0 0 0 0

0 0 o 0 0 • •0 0 • • 0 • • • • • 0 • • • • 0 MAY JUN . JUL. AUG. SEP OCT

Fig. 58, Top row: Thistle leafl ets and bud s howing effects of feeding of nymphs. Bottom row: Unattacked leaflets and bud. 235

wilt prematurely and turn brown, starting from their apices and margins.

At this stage, leaflets of buds curl and the bud itself may show signs of

'die back' (Fig. 58). Attacked buds may be seen as distinct yellowish-

brown tissue from a few yards away. The whole plant or patch of plants may

look as if suffering from the effects of drought. In this respect, the

effect of feeding of T. amuliata on thistles is similar to that of other

Tingids on their host plants (See Sharga, 1993; Fink, 1915; Usinger, 1946;

Roonwal, 1952). Unlike some of these, attacked leaves or leaflets do not

seem to drop; but remain attached to the vegetative bud long after wilting.

It is probable that Tingis may inject toxic saliva into feeding punctures,

- a behaviour which has been demonstrated in some mirids, (See Smith, 1920;

Urquhart, 1955),

In 1967 season, an attempt was made to assess quantitatively, the

effects of nymphal feeding on thistles. 25 thistle plants were selected

at random from a thistle patch on Silwood Bottom, adjacent to the

Observatory Ridge. From the upper half of each plant, two bud samples,

one green and the other yellowish-brown, were taken in separate polythene

bags. In the laboratory, the top one inch of each sample (measured from

the tip of the bud along its main axis) was taken. The total number of

nymphs and leaflets on each one-inch bud unit was counted and recorded

separately for both types of bud samples. The leaf area of the leaflets

of each bud sample was then determined in a Photo-electric Planimeter

(The EEL Large area Meter), which is designed to 236 measure areas up to 900 cm2. In this experiment, the unigalvo type 20, fitted with a 50 cm2 plate was used. The leaflets of each bud were carefully removed from the main axis and arranged on the plate of the planimeter. The galvanometer was zeroed before each measurement and readings 2 were converted to actual area (in cm ) by multiplying by a factor of 0.5. The mean leaf area of each type of leaflets was calculated. After determining the leaf area of samples, the leaflets and 'stem-bits' of each bud were put into small, (4 x 4 x 1") filter paper bags, labelled and dried to-a constant weight in an oven set at 100°C for 24 hours. Each dry bud was quickly weighed (5 secs.) in mg.in an optical balance (Stanton Inst. Ltd. Model B.20) and the mean dry weight of each bud type was calculated. The results of comparing, (a) the mean numbers of nymphs, (b) the mean area of leaflets and (c) the mean dry weight of the two groups of buds are shown in Tables 108-110 below. Table 108 : Comparison of numbers of nymphs in green (slightly attacked) and brown (heavily attacked) thistle buds.

Mean No. of ' Type of Bud Total No. of nymphs t (48) and P nymphs per bud , 10 BROWN 78 3.12 t = 2.47485 (a) GREEN 36 1.44 P <0.02 237

Table 109: Leaf area of leaflets of green and brown thistle buds.

r t = 5.387806078 Type of bud I Total Area of Mean Area of Leaflets (cm2) Leaflets (cm2) D.F. = 48 , fr I / , BROWN 125.85 5.034 (b) P < 0.001 GREEN ` 220.95 8.838 ,

Table 110: The dry weights of green and brown thistle buds.

-,. -.. Mean Dry wt. t = 4.293188 Type of bud Total Dry wt. (mgm) mgms D.F. = 48 1. , t BROWN 1481.0 59.24 Co) P< 0.001 GREEN 2417.9 96.716 E P I&

Although, there was no difference in the mean number of leaflets per bud in the two types of bud (Green = 6.88 and brown = 6.8); the tt' test , showed that brown buds contained a significantly higher number of nymphs than green ones. The mean leaf area and mean dry weight of brown buds were correspondingly significantly lower than those of green buds. These obser— vations indicate that Tingis may cause considerable damage to thistles, especially when a few plants carry a heavy population of the insect. By their feeding, they reduce growth of buds and in severe cases of infestation may cause pefmanent, premature wilting. It could be added here that the Tingid, Teleonemia lantanae Distant. has been tested for biological control of its host plant, Lantana spp. weed, because the nymphs of the insect cause appreciable damage through feeding (See Sweetman, 1936; Fyfe, 193?; Roonwal, 1952). Commenting on the general effects of Tingids on their 238 host plants, Tillyard, (1926) wrote "these very small, handsome bugs are plant feeders, sometimes damaging plants and fruit trees". 239

4. Development of Life Budgets

(a) Me*hods Population of adults was estimated by three sampling methods; viz direct count on randomly selected thistle plants; mark-recapture and by 'beating' thistles.

(i) Direct count on plants. The study area was divided into eight square plots (coded A - H), each measuring 55 ft x 55 ft. Each of a pair of adjacent sides of every plots was divided into five units

numbered 0 to 5. From each plot 10 plants were selected at random, (from

tables; using the pair of adjacent sides as co-ordinates and ommiting numbers higher than 5 and any repetitions). A separate series and set of random numbers were used for selecting plants in each plot. Essentially, this is similar to the restricted or partial random sampling described in Greig-Smith (1964) and which is ideally suited for sample plots which contain a slight variation of surface topography (Kershaw 1966). When a selected point fell on a thistle-free spot, the plant nearest to the point was taken. The plot code and plant number were written in black, weather- resistant 'gem marker' on a white 8 x 2 cm. rectangular cellulose nitrate label attached to the top of a 3 ft. high, thin, galvanized iron wire beside each selected plant. Observations later showed that plots did not vary significantly in insect density. Data from samples in all plots were therefore bulked on each sampling occasion.

Counts of adults were made at weekly intervals throughout the spring, summer and autumn of each year. In sampling a plant, leaves 24o and other vegetative structures were carefully examined one by one, starting from the bottom of the plant and working upwards. Numbers of adults of each sex found were recorded. Occasionally, a plant selected for routine sampling died from unknown causes. In such a situation, the dead plant was replaced. At the start of sampling in 1965 season, various sample sizes were taken and their means compared (Table 111 ).

Table 111 : Comparison of mean number of Tingis in different sample sizes taken on 7.5.65.

, No, of Total No. Mean No. samples of Tingis of Tingis Significance per sample

40 15 0.375 't' . 1.8; D.F. = 102 P y 0.05; N.S. 64 17 0.265 ,t, = 0.3413; D.F. = 142 80 20 0.25 P y 0.1; N.S.

There was no significant difference between the means of 40 and 64 samples. Similarly, the means of 64 and 80 samples were identical. The latter sample size was therefore taken as adequate. The reliability of sampling adults by this method at various times in each of the three seasons, 1965- 67 is shown in Table 112. Owing to the considerable labour and time involved in examining each plant, the number of samples were small. Adult density was generally low and missing of a few individuals would tend to lead to large errors in estimates. However, under such conditions, error margins are partly offset by the fact that samples are absolute, covering 241 the entire study area (Fewkes 1958). The total number of Tingis in the study area on any date was estimated by relating the mean number per plant on that date to the average total number of plants in the area in that month.

Table 112; Reliability of sampling of adults by direct counts on host plants.

, 4 . Date No. of Mean No. of Tingis per Standard Standard error samples plant + 95% F. Limits error as % of mean 4 14-5-65 80 0.45 + 0.16 0.078 17.44 21-5-65 8o 0.525 7 0.2 0.101 19.16 11-6-65 80 0.5875 7 0.19 0.094 15.93 2-7-65 80 0.187 70.12 , 0.059 31.69 21-8-65 80 0.287 7 0.13 0.067 23.35 20-9-65 80 0.375 + 0.16 0.081 21.66 12-5-66 8o 0.412 + 0.17 0.085 20.54 16-6-66 Bo 0.15 71.: 0.09 0.047 31.52 18-8-66 80 0.387 7 0.15 0.074 19.22 19-5-67 , 80 0.75 + 0.22 0.11 14.66 16-6-67 80 0.5375 7 0.17 0.087 16.19 11-8-67 8o 1.325 + 0.29 0.143 10.8 8-9-67 80 0.5125 7 0.19 0.096 18.85 .., . Accuracy of estimates tended to increase with sample mean. i.e. Tineis der sity.

(ii) Mark. Release and Recapture. A sample of Tingis was taken, marked on the right hemelytra with red artist's oil paint and released at, different points in the study area. Details of the method of marking Tingis have been described in section 11,2(d). 96 hours later, another sample was taken by beating method and the number of marked and unmarked bugs was recorded. They were then marked on the left hemelytra with yellow paint and released. On subsequent occasions, at 96 - 144 hr. intervals, samples were taken from the area and note was made of the number of bugs 242 .

which were unmarked and those which had been marked on each and both of the two first occasions. Population estimates were made by the formula of

Bailey (1952) as described in Richards (1953). From the periodic recaptures of bugs marked on either marking occasion, Jackson's (1939) 'positive' method was also used to analyse the data and.estimate Timis population size.

(iii) Beating methods. A minimum of 70, 3 x 3 ft (= 9 sq ft.) random quadrats were taken from the area. Every thistle plant in a quadrat was beaten three times on a 3 ft. square, white canvas 'beating' tray and

the Tingis which fell off were counted, sexed and removed. The sum of bugs beaten off all the plants in a quadrat was taken as the total number of bugs in that quadrat. Bugs were released after all plants in a quadrat had

been beaten. The number of Tingis per quadrat on each sampling day was calculated and by relating this value to the area of the site, the total

population in the study area was estimated. Beating method was used in the summer and autumn of 1966. Since the results obtained by this method were found to be identical to those from direct counts on plants (see Table 114), it was not used in 1967, except for two occasions in the autumn.

The beating method was quite suitable for sampling populat-

ions of adult Tingis, which when disturbed tend to drop readily and lie on their backs. One of the main disadvantages of the method for sampling of insect populations is that it involves a considerable disturbance of the

habitat. The reliability of the beating method for sampling adult Timis is shown in Table 113. The proportion of standard error to the mean varied only slightly between sample sizes or dates. 243

Table 113: The reliability of beating method for sampling adult Tingis

. . No of Mean No of Tin s Standard Date samples Total No Standard 1 of Tingis per sample + 9 Error Error as % beaten Fiducial limits of mean . 0 . . 30-6-66 40 24 0.6 + 0.24 0.12 20.41 7-7-66 40 22 0.55 7- 0.22 0.11 21.32 11 50 27 0.54 7 0.2 0.1 19.24 lt 7o 38 0.54 71: 0.17 0.08 16.21 14-7-66 70 20 0.28 -r- 0.12 0.06 22.36 21-7-66 70 15 0.21 + 0.11 0.06 25.83 25-8-66 7o 181 2.58 -T- 0.57 0.19 7.43 9-9-66 70 242 3.46 + 0.44 0.22 6.43 23-9-66 70 134 1.91 7 0.42 0.22 11.38 i . , . . ,

Comparison of Results from Different Methods of Sampling Adults

The estimates of Tingis population made by the three sampling methods are shown in Table 114, These results are in fairly close agreement. At low population levels, beating method gave slightly higher estimates than the method of direct count probably because it involved sampling a larger part of the habitat. However, at high densities, both methods gave similar estimates.

Population estimates by mark, release and recapture method were slightly higher than those by the direct count on plants, probably as a result the low recaptures on the second and third occasions of individuals marked on the first and second occasions respectively. The mark-recapture method was tried in autumn, when Tingis movement consisted mainly of vertical displacements towards hibernacula (section III2(0). Under such conditions, marked insects are less likely to mix sufficiently with the 244 rest of the field population and low recaptures would be the result of migration to hibernacula rather than of dilution of marked specimens by the unmarked population. K1; the fraction by which the population decreased from the first to the second occasion was 0,87 0.61. The estimate of K1 , though liable to errors (Richards 1953) suggests that about 13% of the population was leaving (apparently to hibernacula) and/or dying from the area every four days; i.e. an average of 3.25% per day. The mean population estimates obtained by analysing the capture-recapture data using the technique of Bailey (1952) and Jackson's 'positive' method compare favourably in magnitude.

Table 114: The estimates of the population of adult Tiugis by different methods. Capture Recapture 'Beating' Date Direct count method on plants *Bailey's Jackson's ** Method positive method 4.9.66 13,20o 15,18o 17,697 -1- 5,402 8.9.65 10,250 13,176 10,461 mow + 6,508 12.9.65 9,600 14,82o - ± 7,371 30.6.66 1,060 - - 1,614 1- 645 7.7.66 860 - - 1,459 .± 457 14.7.66 660 - - 688 .± 323 21.7.66 660 - - 576 1 596 28.8.66 6,800 - - 6,952 ± 995 23.9.66 4,279 - - 5,149 ± 1,129 * Fiducial limits calculated as described by Bailey (1952) ** Fiducial limits are based on negative binomial distribution. 245

Methods of sampling immature stages

At the start of the sampling programme in May 1965, 10 plants were randomly selected and harvested at ground level from the study area, at weekly intervals.Samples were taken in separate polythene bags. In the laboratory, leaves and vegetative buds were examined under a dissecting microscope for eggs and nymphs. Both the top and under surfaces of leaves and leaflets as well as stem tissues were examined. Separate records were made of eggs sucked or damaged by predators; and of intact eggs. Undamaged eggs were removed from samples by cutting leaf tissues, each about 0.5 cm2 from around the points of oviposition. They were then incubated on moist

filter paper strips in 1 x 7.5 cm tubes in the laboratory at 20°C for observations on hatching of nymphs, sterility and parasitism. By 6th June, 1965 and before the appearance of nymphs, the amount of vegetative tissue in samples had become so large that it was impossible to examine all samples within three days of collection in the field. The method of 'whole plant' sampling was therefore discontinued.

From 14th June 1965 onwards and throughout the summers and autumns of the 1966 and 1967 seasons, a new sampling technique based on a knowledge of the growth habit of thistles and of the distribution of the immature stages of Tingis (Section II,3(b-c)) was adopted. The host plant was taken as a unit of habitat and a primary leaf with its axillary bud as the sampling unit. Details of sampling of immature stages have been given in section II,3(b-c). Since immature stages tended to be absent from dead thistle tissues, only live leaf-buds were sampled. In periodic estimates of survivorship of an insect species, some authors (Morris 1954; Varley & Gradwell, 1967), sample 246 the same sampling plant. throughout a generation. Tingis nymphs colonize thistle buds and the maximum number of primary leaf-bud units per plant varied from 18.6 in 1965; 19.8 in 1966 to 15.8 in 1967. Since a minimum of two samples per plant were taken every week in 1965 and every four days in 1966 and 1967; it was impracticable to sample repeatedly the same plants throughout a season. On each occasion, sampling was therefore made of one of five closely adjacent plants on randomly selected sites within the study area. This method of sampling could be satisfactorily used in situations in which the immature stages of an insect are confined to special parts of the host plant (Kim et.al. 1965).

A minimum of 75 plants were sampled in the 1965-67 seasons; so that the minimum number of leaf-bud units examined on each sampling occasion was 150. The reliability of sampling of eggs and nymphs by this method is shown in Tables 115 and 116 respectively. On separate occasions in the 1966 and 1967 seasons, the number of samples was increased to 160 and 300. Although, the accuracy of estimates tended to increase with the number of samples, 150 samples proved to be the maximum number that could be examined in two days. Attempts were not made to store excess samples in deep freeze since such treatments would have affected egg hatchability. Population trends were expressed as numbers per 150 primary leaf-bud units. The nymphs found on each sampling date were dissected under the microscope to detect the presence of internal parasites. 247

Table 115: The reliability of egg sampling

Mean No. of No. of Standard Date Leaf-bud eggs per Standard leaf-bud -.E 95% error Error as units Fid. Limits % of mean ,23.5.66 150 0.373 ± 0.15 0.075 20.52 160 0.356 ± 0.142 0.071 20.15 30.5.66 150 0.5 ± 0.188 0.094 7.03 13.6.66 150 0.39 ± 0.13 0.065 16.75 4.7.66 150 0.25 4. 0.102 0.051 20.26 300 0.226 4" 0.072 0.036 16.08 16.5.67 150 0.18 ± 0.096 0.048 26.83 160 0.175 ± 0.09 0.045 26.08 24.5.67 150 0.413 + 0.146 0.0728 17.62 5.6.67 150 1.106 + 0.322 0.161 14.52 13.6.67 150 1.76 + 0.486 0.243 13.86 23.7.67 150 0.2 + 0.096 0.048 24.12 300 0.186 + 0.066 0.033 17.54

Table 116: Reliability of nymph sampling

No. of Mean No. of nymphs per Standard Date Leaf-bud Standard Error as units sample ± 95% error Fid. Limits % of mean 14.6.65 15o 0.026 ± 0.026 0.013 50.0 22.6.65 150 0.16 ± 0.07 0.035 22.18 6.7.65 150 0.34 + 0.141 0.0706 20.78 18.8.65 150 0.193 ± 0.08 0.04 20.91 21.6.66 150 0.33 ± 0.136 0.068 20.61 4.7.66 150 0.48 ± 0.258 0.129 26.87 2.8.66 150 0.2 + 0.088 0.044 22.18 17.6.67 150 0.16 + 0.086 0.043 27.36 300 0.1467± 0.06 0.03 20.54 23.7.67 150 0.746 ± 0.194 0.097 13.00 16.8.67 15o 0.23 ± 0.108 0.054 23.41 21+8

The seasonal trend in egg population for the 1966 and 1967 seasons are shown in Fig. 59 . As two different methods were used consecutively for sampling eggs in 1965, a similar curve for egg data in the latter year is omitted. Sampling data on the various instar nymphs in the three years,

1965-67 are presented in Figs. 60-62. . Since samples were taken once a week in 1965 season and the period of development of first instar nymphs was about 5-7 days, most of the individuals of that stage were missed.

Data for first instar nymphs have therefore been omitted from Fig. 60.

Hatching of eggs, moulting and mortality of nymphs apparently accounted for fluctuations of numbers, but changes in the host plant, e.g. the reduction of the number of leaf-bud units in early autumn may lead to estimates higher than the numbers of nymphs occuring in the field. The effects of this possible cause of variation has been taken into consideration and corrected for in estimation of absolute numbers of each stage in section (b) below. Weather conditions were less settled at the start of hatching in

1966 than in the 1965 and 1967 seasons. Consequently, hatching was less uniform; a fact which is reflected in wider fluctuation of numbers of each instar in the 1966 season. The annual variation in the quantity of thistle leaf-bud units might also have affected the accuracy of sampling, and so account for the differences in the degree of fluctuations of numbers between years. For example, numbers of each instar fluctuated less dram- atically in the 1967 season than in 1966; the average total number of thistle leaf-bud units in the former year being about one-third of that in the latter (Section I, 2(c)). 249

FIG.59. EGG SAMPLING DATA 1966 & 1967

100

D 60 /966

>- cc 240

200 0 'LC' I60

120 1967 v) 80 cc PI 40

0 10 20 30 9 19 29 4 19 29 8 18 28 7 17 27 MAY JUN. JUL. AUG SEP FIG. 60. OCCURENCE OF NYMPHS IN SAMPLES 1965

J z —•—•— INSTAR II —o— —o — ii co III LL' •o 0." IV

LU 48 —o—o— I V

>c2 40 /0\ o_ 32 0 — 24 c4 o_ 16 c ....0 '.. co • ..... / • • / ...... 0 • .* 4•14 •I" ...*al b'I :0 • • • • • • Or , o 0 ' • JUN. JUL. AUG. I SEP. II I— Z FIG. 61. OCCURENCE OF NYMPHS IN SAMPLES 1966 D

0 D ca --0----e-- INS T AR I LLI 4 ... • —•— II II < w -0--0- If III -J 0 0... If IV 40 .....00... II V

0 `12 24 / % ce • % / : '• Lu . .. 1 / ..•. a. b.. 16 1 o **. V

•O FIG. 62. OCCURENCE OF NYMPHS IN SAMPLES 1967

•-•••• INSTAR I

an. • ., • me II II

,.. 0 ... -0 ,... 56 I' III ••0 e. r1 Iv

•••0•••• IP/ V

>.. 40 cc < E R 32 a. I I 2 24 cc w / 1' a- 16 oi /

...... t, JUN. 1 253

(b) Analysis (i) Survival and mortality of adults. The general trend of fluctuation in adult numbers in the three years, 1965-7, as derived by the method of direct count is shown in Fig. 63 . Adult population in the field attains two distinct peaks each year; one in spring and the other in autumn. The autumn peaks are generally higher than those of the following spring. The difference between the two peaks provides an estimate of the winter mortality, which in the present studies was appreciably high and variable in magnitude. Since reproduction occurs in the spring generation; its size and the survival of its members are important determinants of the size of the new generation which occurs in autumn. Mortality of adults in spring did not occur at a constant rate. It was low at the start of the season, then increased progressively with age. This pattern of mortality is shown clearly when the logarithm of the total numbers of Tingle (Figs. 64 & 65) is plotted against time i.e. age of bugs in days. The relationship was curvilinear of the theoretical equations; the second order polynomial of the form: Y = a + bX + cX2; where Y, is the logarithm of the population estimate on day X; X being the day number.

Survivorship (lx ) curves obtained for the sexes in the three years are shown in Figs. 64 and 65. For purposes of comparison, the 5th of May has been taken on day 1 in the calculation of the corresponding equations for the different years. Population estimates on the last dates of occurence of the sexes in the 1966 season (marked qi in Figs. 64 & 65 were spuriously high, probably owing to sampling errors. Consequently,• they were not used in the calculation of equations for that year. The 254

FIG. 63. CHANGES IN ADULT POPULATION

16000 1965 12000

8000

4000

12000 1966

8000

1 4000 co

Z 0 16000 1967

12000

8000

4000

0 'APR I AkJUN I JUL ""AaGJU _ ° I tE; 1 8e-r 255

FIG. 64. SURVIVORSHIP Os) CURVES FOR MALES

• 1965 Y = 3.187 + 0.0I5X —0.00034X2 '-"2 LLI 4A o 1966 Y = 3.301 — 0.004X — 0.00021X2 I? < + 1967 Y= 3.226 + 0.008X —0.00034X2 II 4.0 X

.RI 3.2 I- + 0 1— 2.8

0 2.4 9 2.0 0 10 20 30 40 50 60 70 80 90 100 DAYS 28.4. ---. DAY I

FIG. 65. SURVIVORSHIP (ix ) CURVES FOR FEMALES

• 1965 Y= 3.057 + 0.037X —0.00049J o 1966 Y= 3.607 — 0.006X — 0.00015 X2 2 + 1967 Y= 3.266+ 0.019X — 0.00032X

J :ti1— 3.2 0 F- 2.8 do 2,4 _1 2.0 0 10 20 30 40 50 60 70 80 90 100 110 I 0 DAYS 28.4. ---. DAY I 256 equations of the survivorship curves are as follows:

1965 Y u 3.057 0.037X - 0.00049X2 Females 1966 Y r 3.607 - 0.006X - 0.00015X2 196? Y = 3.266 + 0.019X - 0.00032X2

1965 y . 3.187 + 0.015X - 0.00034X2 Males 1966 Y = 3.301 - 0.004X - 0.00021X2 196? Y u 3.226 + 0,008X - 0.00034X2

Differences in survival rates between the sexes and years are reflected in the values of the coefficients of X2 in the equations above. Except in 1965, survival rate was higher in females than in males. For both sexes, survival rate was higher in 1966 than in either 1965 or 1967. 1966 and 1967 were slightly earlier seasons than 1965. This is indicated by the annual differences in the dates of first peak occurence of each sex in the field (Figs. 63-65.

(ii) Sex ratio of adults. Besides factors of environment and the genetic constitution of a species, behavioural differences associated with seasonal changes may influence the relative proportions of its two sexes found in the field. Estimates of sex ratio based on individuals of a new generation are therefore less biased and more reliable (DanthanarAY- ans., 1965). Sex ratio values obtained during routine sampling of Tingis in the autumns of the present studies are shown in Table 117 257

Table 117: The sex ratio of Tingis.

Year and sex ratio Method 4 1965 I 1966 1967 -- I _ 1. Mark-recapture: 7.71:7.4 f (on 4th Sept) - 6.8e:6.3$ (on 8th Sept.)

2. Counts on plants (a)at peak of population - 5.04/:5.01. 6.844:6.2*, (b)autumn average - 6.34(:6.51r 2.581:2.5 1

3. 'Beating' 4.6t:4.4 I- 1.38$:1.54 L(on 9th Sept) (on 18th Oct) p

As is common with goat Heteroptera, the sex ratio was approximately 1 :1 . Ratios obtained by the different sampling methods were identical.

The changes in the sex ratio with age of adult population in Spring and Summer of 1965, 1966 and 1967 are shown in Fig. 66 . The proportion of females in the population are expressed as percentages of total Tingis. Variations in the proportion of females were similar in the three years. Percentage of females increased with population age. This was probably due to differential emergence (males tend to emerge before females) and/or mortality (females have longer post-emergence survival rates than males, many of which die soon after mating). The fall in the proportion of females on 18th June, 1965 may not be due to sampling errors since it occured again on 2nd June, 1966 and 9th June, 1967. These dates varied according to the earliness or lateness of the season of each year 258

FIG. 66. CHANGES IN SEX RATIO WITH POPULATION AGE -o--o- 1965 ••x••••x• • 1966 ...... 1967

1 259 and were closely correlated with dates of the second and smaller peak of male emergence. It is probable that the small increase in the proportion of males on those dates might have accounted for the fall in the ratio of females to total insects.

(iii) Sex ratio of nymphs. Using the external sex organs described by Southwood & Scudder, (1956) as diagnostic features, males and females of fourth and fifth instar nymphs in routine samples were easily distinguished under the microscope. The sex ratios obtained for the two instars in the 1966 and 1967 seasons were as follows: Year Instar and Sex ratio IVth Vth 1966 6.7 It :6.9 4.6 :5.0 1967 2.1 t :1.9 2.0 t :2.0

The numbers of males and females of fourth and fifth instar nymphs caught during routine sampling in 1967 are shown in Fig. 67A & B. Males tended to appear earlier than females in samples; a condition which was more marked in 1967 season in which the average mean and maximum temperatures for July, (the main month of first emergence of fourth and fifth instar nymphs) were 17.2°C and 22.7°C respectively, as compared with 14.7°C and 19.3°C in the 1966 season. It is probable that the two sexes develop at different rates.

Sex ratio of nymphs was approximately 1 1 S. It is shown above that adults have a similar ratio; thus suggesting that there is no differential mortality of the sexes in the nymphal stage. 260

FIG • 67. OCCURENCE OF THE TVVO SEXES N NYMPHAL STAGES (FELD DATA 1%7 -o--o- MALES --4,--6- FEMALES 28- A : 4TH NSTAR NYMPHS

24- 3 20,-

28,-

24- )t 20-

16-

12,-

80-

4- 261

(iv) Estimation of Recruitment and Mortality in the immature stages Various methods, (See Southwood 1966); based on different assump- tions have been developed for the estimation of recruitment and mortality in each stage of an insect population. These methods vary in degree of accuracy and some are more applicable to specific types of population data than others. In T. ampliata, the period of oviposition (see (v) below) and egg hatching in the field are long. Consequently, the immature stages overlap considerably. For a population of this sort, the number of individuals in a developmental stage concurrently suffers decimation through moulting and mortality and an increase by oviposition, hatching and moulting.

A graphical method, (Southwood & Jepson 1962) was used for the analysis of Tingis data. The number of individuals of each stage (eggs or instar nymphs) per 150 live primary leaf-bud units on each sampling date was plotted against time in such a way that one square was equivalent to one individual and one day. The points were joined and the total area, in squares was estimated with a planimeter, To obtain the value of the total population of the stage per 150 leaf-bud units, the total area was divided by the period of development in days, which is the number of days a given stage is present in the field. An estimate of the total numbers of each stage was then made by relating the resultant value to the average total number of primary leaf-bud units in the study area during the period of occurence of the stage.

Though there are wide fluctuations in the population curves (Figs. 59 - 62 ), the numbers of each stage build up to one highest peak. 262

Some idea of the size of the mortality in each stage is indicated by the difference between peaks of successive stages, excepting of course, between the second and third instar, in which the peak of the latter stage exceeds that of the former owing partly to the longer duration of the third instar relative to the second. This is in accordance with Richards (1940), who pointed out the numbers of each instar of an insect, found in a series of systematic samples should correspond with the time spent in that instar; any deviation from which represents the magnitude of the mortality in that stage. For the construction of provisional population budgets for Tingis, the mortality in each stage was estimated from the difference between the

total initial numbers of two successive stages. Thus, mortality in the egg stage is given by the difference between total initial numbers of eggs and that of first instar. Similarly, mortality in the first instar is given by the difference between total recruitment into the first instar and that of the second; and so on for subsequent instars. Mortality in the fifth instar was estimated by the difference between total initial numbers of 5th instar nymphs and the estimate of adult peak population which is ideally equal to the total number of adults recruited if peak of numbers is attained soon after the start of autumn emergence (Jag.- 63). The use of these peak numbers apparently gives a low estimate of adult population, and so could partly account for the rather high estimates of mortality in the 5th instar stage. For each year, the size of the mortality in the egg stage is also given by the ratio of the sum of the parasitised, unparasitised but unhatched and damaged or sucked eggs to the total number of eggs recruited. The estimates of the initial numbers of each stage in the three years are summarised in Tables 118 , 119 & 120 . 263

Table 118 : Estimates of initial numbers and mortality in immature stages (1965).

Stage No. Recruited Mortality (56) Eggs 358,560* 47.7 'not= 1 187,276s* 5.8 ft II 176,413 14.5 ft III 150,743 49.8 it IV 75,584 15.9 If V 63,525 79.2 Adults 13,150

* Calculated from mean fecundity of field females in Spring. ** Estimated by deducting numbers of parasitised, damaged, and unhatched eggs from total estimate of initial egg numbers.

Table 119 : Estimates of initial numbers and mortality in immature stages (1966).

Stage No. recruited Mortality in Stage (%) Eggs 313,779 39.5 Instar I 189,573 10.0 it II 170,597 4.3 I, III 163,116 57.2 1, IV 69,700 38.1 it V 43,143 76.5 Adults 10,100 264

Table 120: Estimates of initial numbers and mortality in immature stages (1967).

Stage No. Recruited Mortality in Stage (%) Eggs 320,683 72.33 Instar I 88,709 11.41 ii II 78,583 2.32 tt III 76,757 39.03 I " Iv 46,798 3.25 I, V 45,272 66.4 Adults 15,172

(v) Fecundity. Estimates of average fecundity in the field were obtained by three methods. In the first method, the fecundity, f was derived from the relation; f = ; where X is the total number of eggs laid in the study area in a season and n, is the initial number of females in the Spring population. The value of X for 1966 and 1967 was estimated from the egg sampling data (graphical method) and that of n from the survivorship curves. Thus, the fecundity in the years of study were as follows:

1965 = 54 eggs per female

1966 - It tf it 313,975'779 - 79

320,683 It tt 1967 = -4756- =70

The second estimate of fecundity was obtained from the weekly egg laying records of a minimum of 20 females, paired with males in cages over potted thistles in the field. Cages consisted 265

of cellulose acetate material, 12" long and 9" in diameter. For ventilat- ion, about two-thirds of the top three-quarters of the sides were cut open and covered with coarse nylon sheeting. Part of the lower quarter of cages was pushed into the moist soil in the pots. Plants were changed at weekly intervals and eggs laid by each female during the week were counted under the microscope in the laboratory. Males were replaced at death and records of oviposition of females were kept until they died. This provided an estimate of absolute fecundity of females, since they were caged before the start of oviposition in the field. The rates of oviposition and average fecundity of caged females in the 1965 and 1966 seasons are compared in Fig. 68 and Table 121 respectively. Table 121: The average fecundity of females in the field.

No.of eggs laid per female Year No.of females Mean* Range

1965 20 56 7 - 117 1966 25 65 12 - 141

* To nearest whole number.

There was a considerable variation in the number of eggs laid by individual females in each year. Oviposition rate and average fecundity were higher in 1966 than in 1965. Temperature and age are among other factors that affect the rate of oviposition of some insects in the field (Danthanarayana, 1965; Agwu, 1967). To explain the difference in oviposition rate and total

fecundity between the two years, partial regression analyses were carried out on egg laying data. The number of eggs laid per female per week (Y) 266

FIG .68. WEEKLY OVIPOSITION RATES ) 1965 & 1966

1:110 U 15 0 cu E w I-

5 Z IS E

1965

-20 00 -15

0 6 9 267 was taken as the dependent variable and the average mean temperature in the field in 0C. for that week (X1 ) and the age of bugs in weeks (X2) as the independent variables. To facilitate intepretation of results, each sea‘.. son's data were analysed in two stages;viz:from start to peak, and from peak to end of oviposition. The value of X2, corresponding to the first week of oviposition was taken as 1. Equations for the regression of Y on XI and X2, together with the regression coefficients of X1 and X2 and their standard errors are shown in Table 122. Table 122 : The regression of rate of oviposition on temperature and age of bugs.

Stage of oviposition Year Start to Peak Peak to end Regression Equation: Y.1.132X1*1.623X2 -13.997 Y=-0.702X1-3.367X2+45.354

1965 Regres =-0.702±3.42;b2=3.36±1.46 sionIts:b1=1.132,_+1:267;b 2=1.6210.383 b1 Standard Errors: 0.6468 ; 0.7055 1.7494 0.7453

Regression Y=2.769X1+1.315X -28.883 Y=-0.334X1-5.625X2+68.386 Equation: 2

Regression ** 1966 b -0.334+6 Coefficients:b1'=2.769±1.753-b 2 =1.314+1.885 1 = .98;b2ft5.4±.3..71 Standard Errors: 0.8767 ; 0.9423 3.1+908 1.8571

Level of significance: ** = 1%; * = 5; e NS

268

Regression coefficients which are about twice as large as their standard errors are significant at approximately 5% level (Brownlee 1960). The regression equations show that both temperature and age have a significant effect on the rate of oviposition„ especially in the pre-peak phase in which both factors are positively correlated with oviposition rate. That is, the rate of oviposition increased with increase in temperature and age. In the post peak phase, oviposition rate tended to be negatively correlated with , and less sensitive, to changes in either temperature or age. That is, oviposition rate decreased with increase in either age or temperature. The latter factor had no significant effect in this phase. The regression coefficient of XI in 1966 was more than twice as large as that for 1965 during the pre-peak oviposition period. Hence, it appears likely that temperature was partly responsible for the difference in fecundity between the two years.

From the regression analysis, the contributions of temperature and age to the total variation in rate of oviposition were obtained. These are expressed as percentages in Table 123 . Table 123: The proportion of total variation in rate of oviposition due to different factors.

Stage of oviposition Factor Year & % of Total variation 1965 1966 Temperature 42.05 87.64 Start to Peak age 36.98 4.04 Unknown 29.97 8.32

Temperature 44.64 26.62 Peak to end age 50.00 55.29 Unknown 5.36 18.09 269

These figures show that variation brought about by unknown causes in the pre-peak phase of oviposition was greater in 1965 than in 1966; with the effect of temperature being twice as large in the latter as in the former year.

Besides temperature and age, nutrition (Waloff & Richards, 1958) and size (weight) of individuals (Donia 1958) are among other factors that may influence the fecundity of insects. It has been shown in section II, 2(c) that at post hibernation emergence, heavier Tingis tended to have higher numbers of eggs or egg rudiments, and hence might also lay more eggs. The mean weight of 25 females in 1966 were significantly higher than that of 1965 (p 0.05).

Finally, Fig. 68 shows that on the average, oviposition period was about two weeks shorter in 1965 than in 1966; in which more eggs were laid in the pre-peak phase when the effect of temperature on rate of oviposition was often more significant.

The third method of estimation of fecundity was essentially similar to the second, except that it was based on the oviposition of females paired with males in the laboratory at 20°C. An account of laboratory oviposition behaviour has been given in section II2(cki). The average fecundity was derived from the relation: Total number of eggs laid ± 95% fiducial limits Number of laboratory females

On this basis, 61.25+10:27 and 74.05112.93 eggs were laid per female in the 1965 and 1966 seasons respetively. 270

Fecundity estimates by the three methods are compared in Table 124

Table 124 : Fecundity estimates by three methods.

Method and No.of eggs per female Year Leaf-bud sampling Egg laying in Egg laying in field laboratory

1965 54 56 61 1966 79 65 74

1967 70 ••• IMP

The estimates of fecundity by the three methods appear to support one another. The graphical method over-estimated the fecundity in 1966. All three methods point to a higher fecundity in the latter year than in 1965. Annual variations in average fecundity appear to be associated with variations of field temperature during oviposition period (Table 125); and post-hibernation longevity (page 125 ). Table 125: Average mean and maximum temperatures of oviposition period (May to July) in the three years, 1965-67.

Year Average meanmoan temp& Average maximum temp. 'C

1965 13.3 17.8 1966 13.76 18.7 1967 13.96 19.03

Weight of females and number of eggs and egg rudiments per female on emerg- gence in Spring (Fig. 31) are also possible factors affecting fecundity. 271

The estimates of total eggs laid in each season, as computed from the fecundities determined by the three methods (Table 124) are shown in Table 126. Table 126: Estimates of total egg population by three fecundity methods.

Total Number of Eggs Year No. of Leaf-bud Egg laying Egg laying in females sampling in the field the laboratory

1965 358,668 371,952 40,162 6,642 1966 314,025 258,375 294,150 3,975 1967 321,720 . - 4,596

These estimates of initial egg population are reasonably close particularly in 1965 and 1966. 272

1. Causes of mortality in adults (i) Parasitism Fourth and fifth instar nymphs and adults of T. ampliata are parasitised by a small red ectoparasitic mite in Silwood Park. Mr. W. O. Steel identified the mite as a species of the Trombioulidae. Early in May and June of each year, red mites apparently identical to those found on Tingis, begin to occur on Cercopids and Phalangids in the study area. Usually, the mites start to appear on Tingis by late June or early July. Microscopic examinations revealed that mites were not just clinging to but feeding in their hosts. The probosis of the parasite was often so deeply embedded in the host that dislodging from the latter was not easy. Small, almost circular feeding punctures were always left at the point of attach- ment to the host. Table 127 shows the numbers of adults collected from various habitats in Silwood and the percentage parasitised by the mite Fig. 69 in 1965 season. Table 127 : Parasitism of Tingis by Trombiculid mite, 1965.

No.of No. No. of No. Month parasitised parasitism collected parasitised parasitised collected May 98 0 o.o 71 0 0.0 June 118 1 0.8 55 0 0.0 July 49 4 8.16 35 2.85 August 70 2 2.77 38 2.63 September 98 3 3.06 52 6 11.53 October 112 17 15.17 go 9 10.0 November 195 3 1.53 163 3 1.84 December 45 0 0.00 45 O 0.0 Total 785 30 549 20 Mean 98.13 3.75 3.93 68.62 2.5 3.61 273 Fig. 69. Ectoparasitic mite (Trombiculid) on T. ampliata

Fig. 70. Cage used for studying predation on adults. 274

The number of mites on an individual varied from 1 to 3. In adults collected on different dates between 30th June and 17th August, 1966 9 out of 120 females (7.5%) and 4 out of 36 males (11.1%) had mites. More commonly, mites found on abdomina (AB), legs (L) and antennae (AN), sucked from the articular surface between sternites and segments respectively. Those which were caught on the head (H) and hemelytra (HE) frequently sucked in the region between the head and the prothorasic segment and in the grooves between lateral areas of bugs. Table 128 shows the distribution of 51 mites between various regions of the body of adults collected in the 1965 season.

Table 128 : Distribution of 51 ectoparasitic mites between different sites

of the host's body.

Site Part of body and number of mites Month AN HE H AB L Total

May 0 0 0 0 0 0 June 0 0 0 1 0 1 July 1 0 0 3 1 5 August 0 0 1 1 1 3 September 1 1 2 3 2 9 October 0 7 2 13 4 26 November 0 2 0 3 2 7 December 0 0 0 0 0 0

Total 2 10 5 24 10 51 % of all sites 3.9 19.6 9.85 47.05 19.6 100

Of the 13 mites found on individuals of both sexes in 1966, 8 were on abdomen, 4 on legs and 1 on hemelytron. More mites were caught feeding on the abdomen than any other site (Table 128 ). Dissections of =parasitised and parasitised specimens showed that mite feeding has no noticeable effect on the development of the gonads of the latter group of bugs. Thereforelthe

275

Trombiculid mite does not appear to cause the host any serious damage and

it probably does not constitute a significant mortality factor. Ecto

parasitic mites are known to attack other Tingids, especially Corythuca spp. (See Drake, 1922; Bailey, 1962-3).

(ii) Winter disappearance

Adult peak population estimate in autumn was always higher

than that of the following spring, when the bugs had hibernated (Fig. 63 ).

The difference in the values of the two peaks provided a measure of the

number of adults which disappeared during each winter (Table 129 ).

Table 129 : Numbers of Tingis disappearing during the winters of this study.

1965 1;22 1967 No., of Tingis in autumn 13,200 10,100 15,172

Nosof Tingis in Spring of the following year 5,900 7,556 Number that disappeared 7,300 2,544 Percentage that disappeared 55.3 25.18

Weather is an important factor which partly explains the large number of individuals disappearing in the winter. The ability to survive the cold

wintery conditions is dependent on the cold-hardiness of a species and the intensity and duration of cold to which it is exposed. Detailed accounts

of experiments on the cold-hardiness of Tingis and of the relationship between minimum temperature and mortality of adults have been given in section II,2(a), These results pointed to the fact that although Tingis have relatively low undercooling points, there is considerable variation in cold- 276

hardiness of individuals. Furthermore, they indicated that long exposure to o o low temperatures of the order of -13 to -2 C could have significant lethal

effects on adults; especially at the start of hibernation. The lowest daily

minimum temperature recorded on grass ranged from -0.5 in October to -10.75oC in December in 1965-66 and from -3.75 in October to -12.5°C in April in the 1966-67 winter. Inspite of these ranges of temperatures, the latter winter

was on the average less severe, than the former. The frequency of day on which grass minimum temperature fell below -2.0°C was 80 in 1965-66 and 57 in 1966-67. Minimum temperatures observed at 3" below grass in the 1965-66 winter ranged from 9.8°C to-9.5C. These data suggest that Tingis may be

exposed to lethal sub-zero temperatures at the start or end of hibernation when they are on the upper layers of hibemacula and when they are less cold hardy (Fig. 16

Another probable factor accounting for disappearance of adults in winter is predation by various species, some of which are active in early winter or spring. The most important groups of these potential predators are the Carabidae and spiders. According to Greenslade (1960) some species of Carabidae over winter as adults or larvae and may be caught in pit fall traps all the year round. In 32 regularly-spaced pitfall traps (3" x 6" jam jars sunk 2" below ground level) which were set in the study area and observed daily for catch in the period from October to December, 1965, 50% of total arthropods caught were Coleoptera; 25% were Araneids, while various species of Hymenoptera, Diptera, Dermaptera and Lepidoptera formed the remaining fraction of the catch. Carabids formed the highest proportion of the Coleoptera, the predominant species being, Pterostichus madidus Fab., 277

Carnbus problematicus (Hb.), Nebria brevicollis (F.) and Necrophorus humator (CZ). Staphyllinids formed the second most abundant group. The identity of these beetles was determined by Dr. B. Critchley. Of the total spiders caught, ground hunters, Lycosidael Gnaphosidae etc. formed 94.2% and foliage hunters, Thomisidae, Clubionidae etc. constituted the remaining fraction. 5 specimens of the vole, Microtue agrestis macgillivrall, B-H & Hinton, were also caught in traps. To identify some of the suspected predators, laboratory tests were carried out in which a known number of Tingis was supplied to potential predators for 48 hours. The results are given in Table 130. Species tested included those that are usually active in early Spring before the start of Tingis post-hibernation emergence. Table 130: No of adults taken by predators in 48 hours.

No. No. of Tingis No. Species tested caged supplied taken Carabidae Nebria brevicollis 12 16 0 Carabus problematic us 7 10 0 Pterostichus, caerulescens 2 14 5 Trechus obtusus Erichson 5 6 0 Abax sp. 2 6 1 Pterostichus madidus 10 15 0 Nechrophorus humator 7 7 0 Amara communis Pz. 5 10 0 Araneida Xysticus cristatus (Clk.) 3 9 4 Tibellus oblongus* (Walck.) 10 20 0 Tarentula sp.** 4 9 2 Trochosa terricola Thorell 5 11 0 Hemiptera Nabis rugosus L. (Nabidae) 4 15 0 Anthocoris nemorum L. (Anthocori- 5 12 0 dae) * Took a few bites but would not eat Tingis; resorted to cannibalism instead. 278

** Prey is generally killed a few minutes before the start of feeding.

Feeding lasted about five minutes. The internal tissues of the prey was dissolved and sucked up leaving the skeletal parts behind.

The list of predators on Table 130 is far from being exhaustive. For example, dense flights of insectivorous birds such as starlings, Sternus vulgaris (L) and Blue-tits, (Paramus caeruleus) (Prazak) which are said to be potential predators of a wide range of insect species (Smith, 1957; Agwu, 1967) were repeatedly seen on the study area in the mornings during March to May. The birds stir the herbage and grass intensively and may take up Tingis emerging from hibernation and so account for some mortality in the hibernating population. Large quantities of avian faeces were often strewn over the study area.

(iii) Summer Predation To assess the role of predators in the post hibernation mortality of adults, a predator exclusion experiment was made in the 1966 season. Two groups, each of 160 thistle plants were randomly selected from the study area. On 26.5.66, the numbers of adults on each group of plants were recorded and all the plants in one group together with their insect populations were separately covered with cylindrical muslin cages, 1.5 ft. in diameter and 3 ft. long (Pig. 70 ). The open ends of the cages were then tied firmly with cotton strings to the bases of the plants. On subsequent dates, the numbers of Tingis in the covered and uncovered plants were counted to compare their rates of decline. On each occasion the number of dead and missing insects in the covered plants were also recorded. 279

The results are presented in Table 131 . Insect numbers declined at a faster rate in uncovered than in covered plants. On the basis of the proportion of insects which died on the caged plants, about 42 out of the orig-

inal 63 adults (= 66.7%) in uncovered plants could be assumed to have died from natural causes, the remaining 33.3% (i.e. 21) being that part of the population lost through those causes responsible for the "missing" individuals in the covered plants (15.25%) & those taken by predators (19,04%, i.e.

12).

Table 131 : Comparison of rates of decline in numbers of adult Tingis on

160 covered and uncovered thistle plants.

Number of Tingis on:

Dates Uncovered Plants Covered Plants Living Living Dead Missing

26-5-.66 63 59 0 0 (Original) 9-6-66 44 50 5 4 23-6-66 13 43 7 0 7-7-66 7 32 10 1 21-7-66 4 23 6 3 4-8-66 1 20 3 0 11-8-66 0 11 8 1 , Net Balance 0 11 39 9 % of original 0.0 18.64 66.0 15.25

It is mentioned in sections II,2(d) & III4(0)(2b)that spiders and harvest spiders are the main predators on ampliata. 280

P. Cauges of mortality in immature stages (a) Egg mortality

(i) Predation Since Tingis has long oviposition and incubation periods, eggs often remain exposed to the sucking and/or damaging influence of predatory species for a considerable length of time. The chorions of sucked or damaged eggs were usually left on leaf-bud samples. Numbers of damaged eggs tended to increase with the total numbers of eggs in samples. The average percentages of total eggs damaged in samples in the three seasons,

1965-67 are shown in Table 132 .

Table 132 : The proportion of eggs damaged by predators in each year of

study

No. of Total Na of Year intact eggs damaged eggs eggs (a) as % of (b) (a) (b)

1965 224 12 236 5.08 1966 558 89 647 13.75 1967 2,056 173 2,229 7.76

From the average percentages in Table 132 , estimates of the total number of eggs damaged by predation in each of the three years were calculated

(Table 133 ).

Table 133: The numbers of eggs destroyed by predation, 1965-67

Estimated Total No.of % damaged Year Total No. by predators damaged of eggs eggs 1965 358,560 5.08 18,215 1966 313,779 13.75 43.145 1967 320,683 7.76 24,885 281

The proportion of eggs damaged in samples from the different regions of the host plant were apparently identical in any one season (Table 134).

Table 134 ; The proportion of damaged eggs on samples from different regions

of thistles, in 1967.

Region of No.of eggs No.of eggs (a) as % of (b) plant in samples=(b) damaged=(a)

Upper Half 554 47 8.48 Lower Half 1,675 126 7.52

However, a higher proportion of the total eggs damaged in each season were found in samples taken from the lower half of plants (Table 135 ). This distribution of proportion of damaged eggs is to be expected, since more eggs are usually laid in the lower half of plants (Section 11,2(c)).

Table 135 : Proportion of Total damaged eggs found in samples from lower

half of plants, 1965-67.

Eggs damaged Total eggs damaged Year in samples from lower (a) as % of (b) in samples = (b) half of plants = (a)

1965 12 8 66.6 1966 89 67 75.2 1967 173 126 72.8

To identify the insect predators of Tingis eggs, the following species; Orthotylus concolor Kirsch; 0. viriscens Douglas & Scott

(miridae); lie rugoaus; Dolichonflbis limbatus Dahlbom (Nabidap); A. nemorum and A. nemoralis (Anthocoridae) were separately provided 282

with Tingis eggs in petri-dishes in the laboratory. None of the above species sucked Tingis eggs. The coccinellids, Coccinella septempunctata L. and Adalia bipunctata L. which were common on thistles also failed to take Tingis eggs. The mite Anystis appears to be one of the predators on

Tingis eggs. When 12 individuals of the mite were supplied with 20 eggs for a 24 hour period, one egg was completely sucked and 4 were partially damaged. It is also probable that a large proportion of eggs damaged in samples were accidentally destroyed by the many phytophagous red and white mites which dug characteristic tunnels and grooves on the midribs of thistle leaves. Besides mites, some phytophagous coleoptera, viz Centorhynchus litura (F) (Curculionidae) and Cassida spp (Chrysom elidae) also attack

thistle leaves in Silwood Park. Although the occurence of these beetles on the study area was occasional, it is likely that their feeding might

have caused the destruction of some eggs.

(ii) Parasitism

A considerable mortality in the egg stage is caused by

parasitism by chalcid wasps, Anaphes fuscipennis (Hal.) and Parallelaptera

panis Enock (Mymaridae). The identity of these parasites was ascertained in the winter of 1966-67 by Professor, R. L. Doutt of the Division of

Biological Control, University of California, Berkeley.

In Britain, there is a relatively limited number of

Mymarid genera; the species of which are widely distributed (Hinks 1950).

The latter author records the genus Anaphes Haliday as parasitising eggs of

Dytiscidae, Chrysomelidae, Curculioindae; Miridae, Delphacidae, Coccidae,

Lepidoptera and Jassidae; the last family being mentioned as the common 283 host of the genus Parallelaptera Enock In the U.K. Prior to the present work, it appears that there was no recorded association of the latter genus with a Tingid in Britain, Southwood & Scudder, (1956) found a single specimen of a species of Anaphes, parasitising the egg of T. ampliata in

2cthamstead Expt. Station.

To determine the level of parasitism by mymarids, eggs collected in field samples were incubated on strips of filter paper moist- ened with 0.01% Nippagin (to inhibit fungal growth) in 1 X 7., mm tubes in the laboratory. Separate records were kept on hatching, not hatching and parasitised eggs. Owing to the very small size of the eggs of A. fuscipennis and P. panis, accurate detection of parasitism is impracticable in their egg stage. However, as parasite development progresses, the host's egg gradually changes colour from white to dull-yellow and subsequently to brownish-black.

By contrast, the unparasitised egg is pale-fawn at hatching (Fig. 7(a & b)).

Furthermore, a parasitised egg may be distinguished by the presence of a typical compound eye instead of the five separate facets characteristic of the Tingis first instar larva (Southwood & Scudder 1956). These methods of detection of parasitism obviates possible errors that could stem from estimates based solely on emergence of adult parasites, since some larvae and adults may die and so fail to emerge.

The seasonal trend in the parasitism of Tingis eggs is shown in Fig. 71 . The proportion of parasitised eggs tended to be higher towards the end than at the start and peak of egg occurence in the field.

However, these relatively higher values of percentage parasitism were more 284 FIG.71. PARASITISM OF TINGIS EGGS BY MYMARIDS.

o---o NO. OF EGGS KILLED BY PARASITES

`11. OF EGGS PRESENT -60 PARASITISED 50

16 1965 40

12 30

8 - 20 0 0 S / , / 0 \ 0 v) 0,0 0 0„. 1.7 0

ASITE 0

R 24 60 Q

PA Q 20 50 1966 >- co 16 -40 1— A Z 0 I2 ,0 A -30 v) LA.1 ‘0' 8 0 ,a *20 0 0 4 • II It I \ •10 0 VI L 0 0 L2 L9 I w 36 • 90 a 32 I 0 0 O 10 28 70

1 24 - I 60 < ERS o-0 1967 F- 0 20 50 w

NUMB o U 16 I •40 of '01 12 -30

8 I 0 • 20 I '0 0 4 I 0 0 \ I0 0 —0 10 20 30 40 50 60 70 80 90 100 110 120 DAYS (,ST. MAY: DAY I)

285

a reflection of the smaller numbers of host eggs available, rather than an

increase in the actual level of parasitism (Fig. 71 ). It is shown in

Table 138 that the annual variation in period of occurence of parasites in the field is closely associated with that of their host.

The average percentage parasitism in each year is shown in Table 136 .

Table 136: Parasitism of Tingis eggs by Mymarids 1965-67.

Year No. of eggs No, of eggs incubated parasitised parasitism

1965 224 33 14.73 1966 56o 125 22.32 1967 2,056 253 12.3

From the percentage parasitism and the number of intact eggs in the field,

the number of eggs killed by parasites in each year was estimated (Table 137).

Table 137: Numbers of eggs killed by parasites; 1965-67.

Estimated No. Calculated No. Year of eggs surviving parasitism of eggs killed predation by parasites

1965 340,345 14.73 50,133 1966 270,634 22.32 60,406 1967 295,798 12.3 36,383

The life cycles of the mymarid parasites appear to be

closely synchronised with that of Tingis. This is obvious when the dates 286 of first and last occurence of host and parasites in field samples are compared (Table 138 ). Table 138 : The occurence of mymarid parsites and of Tingis eggs in the field.

Year 1965 1966 1967 Dates of first Parasites 16.5. 23.5. 20.5. occurence Tingis eggs 13.5. 9.5. 8.5.

Dates of last Parasites 6.7. 29.7. 7.9. occurence Tingis eggs 14.7. 9.8. 7.9.

In each year, the host tended generally to preceed the parasites by a few days. Except in the 1967 season in which both parasites and host remained detectable in samples for the same period; the host appears to outlive the parasites by about a week. This suggests that the parasites probably move on to other hosts when Tingis eggs become scarce or completely absent in the field. Observations in 1967 (Table 139) showedthat of the two parasitic species, P. panis was the first to occur in and disappear from Tingis eggs. Table 139 : Comparison of dates of first and last occurence of P. penis and A. fuscipennis in samples (1967).

Date of Date of last Parasite Species first occurence occurence

P. penis 20.5. 16.8. A. fuscipennis 24.5. 7.9.

Since samples were taken at 4 day intervals, a rough idea of the period of development of each parasite from the egg to the adult may be obtained from the interval between date of first occurence of parasite in samples and 287 that of emergence of the adult bred from those samples. In the 1967 season, the first adults of P. panis and A. fuscipennis emerged on 6th June. This corresponds to a generation interval of about 16 and 12 days respectively.

Added to the long period over which the parasites occur in samples (Tables

138 and 139 ). These apparently short generation intervals suggest that several generations of the parasites are passed in Tingis eggs.

An attempt to assess the separate effects of the para-

sites on the host egg in the 1967 season was not entirely successful owing

mainly to the minute size of their eggs and larvae. However, of the total

parasites developing to the pupal and adult stages (and a majority of

parasites did), 54% were P. panis and the remaining 46%, A. fuscipennis.

Only one individual parasite (P. panis, or A. fuscipennis) developed per

parasitised host. It was not clear whether one or more eggs of each or both

parasite species were laid in one host. Assuming an equal survival rate in

the eggs and larvae of the two parasites, it appears that their effects on

the host population was identical; judging from the proportions of their

pupae and adults. Of the total adults of each parasite species bred from

samples in 1967, the sexes occured in the following ratios:

Parasite species Sex ratios

P. panis 0.79 : 0.57' A. fuscipennis 0.68 Q : 0.48 S'

Both species showed a slight excess of females over males.

One of the eggs found in samples on 24th August, 1967

was parasitised by a hymenopteron which Dr. T. News identified as a species 288 of Telenominae (Scelionidae), a family which are known as parasites of

Pentatomonorphan Heteroptera (Cumber 1954).

(iii) Sterility

The difference between estimates of total initial egg numbers and those of first instar nymphs (See section II,4(b)) gives an estimate of the total number of eggs lost in each season. This loss is due to the combined effects of three factors; predation, parasitism and egg sterility and developmental failure. The total egg loss less those due to the first two factors mentioned above provides an estimate of the total number of sterile eggs (including developmental failure) (Table 140 ).

Table 140 : The number of sterile eggs in the field 1965-67.

1965 1966 1967

Total egg loss 171,284 124,206 231,974 Predation /I 18,215 43,145 24,885 Parasitism 50,133 60,406 36,383 Sterility 102,936 20,655 170,706 Sterility loss as % of total initial eggs 28.7 6.6 53.2

There is some overlapping of the mortality factors. Some of the eggs destroyed by predators may have been sterile and/or parasitised by mymarids.

However, data in Table 140 show that sterility was the most important cause of egg mortality; especially in 1965 and 1967. The causes of the high proportion of sterile eggs were not certain, but hatching of eggs from field samples indicated that the level of sterility tended to be greatest in eggs collected in the first and last few weeks of egg occurence in the field. 289

(b) Mortality of nymphs

(i) Predation

Considerable mortality of nymphs occurs as a result of predation by insect and other species. The first and second instars remain more or less inactive in thistle buds and are therefore protected to some extent from predators. By contrast, third, fourth and fifth instar nymphs are more active and may wander from one bud to another. They are therefore more exposed to hazards in the environment and a large proportion of them are killed by predators. The period of development of instars increases with age or stage. This means that old instars remain vulnerable for longer periods than young ones to possible predator attacks.

The predator-prey relations of Tingis and its potential predators was studied by serological techniques, (the precipitin test after the method of Dempster, 1960). Essentially, the test consists of determining the presence or absence of interaction between the gut contents of a suspected predator and the antibodies in the blood serum of rabbits which had been innoculated with an extract of Tingis. Ideally, each stage of the prey should be tested separately. Different stages of Tingis overlap in the field. Furthermore, with small-sized, and light nymphs, it was impracticable to collect sufficient insects to form a minimum of 5 gm. freeze-dried antigen for injection of one rabbit. Collections of nymphs (mainly 3rd to 5th instars) were therefore supplemented with some adults. All specimens were collected from habitats other than the study area in Silwood Park. Speci- mens were starved for 24 hours to remove their gut contents and then killed with cyanide and crushed in 20 ml. saline solution (0.9% NaC1.), in a mortar 290 with pestle. A few crystals of 1000 WI, were added to the contents of the mortar and kept at 4°C for about 24 hours. It was then centrifuged at 9000 revs./min. for about 10 minutes. The supernatant, yellowish liquid was sterilized by passing it through a Seitz E.K. sterilizing filter pad. After filtering, the clear, sterile antigen was freeze-dried and stored in vials until required for reconstitution and injection into the rabbit.

To produce the antiserum, the antigen was reconstituted with distilled water and the soluble proteins were precipitated with 0.4% potassium alum. Its pH was then adjusted to 6.8; and 2.5 ml. of the suspen- sion was injected intra-muscularly into a rabbit by Dr. D. Pinnock. About

2 weeks after innoculation, the rabbit was bled from the ear and its serum tested against a Tingis extract. Sensitivity of the serum was increased by giving the rabbit further injectionsd50 ml of blood was then taken from the rabbit and the serum separated out. Lipoids were extracted from the serum with ether at a temperature below -25°C. The serum was then freeze-dried and stored in 1 ml amples.

In 1965, 1966 and 1967, specimens of potential predators were collected from thistles from June to September, the main period of occurence of nymphs in the field. On collection, specimens were killed, identified and their gut contents or whole insects (if small species) were smeared on labelled filter papers. The predator meals were then dried over phosphorus pentoxide, (P205) in a dessicator; and kept until required for test. Before tests, each predator meal was allowed to soak for 24 hours in 0.2 ml. normal saline in a 1 X 3 cm tube and centrifuged. To conduct a test, 0.02 ml. of the saline extract was first drawn into a serological 291 test tube, followed by an equivalent volume of the serum. Care was taken to minimise mixing of the two liquids. Test tubes were allowed to stand for about three hours after which the interphase between the liquids was viewed against a black background with an indirect light. A positive reaction was indicated by the presence of a ring of a yellowish-white precipitate at the point of contact of the two layers of liquid.

Before testing predator meals, the Tingis serum was tested for sensitivity agaiList ether Heteroptera, viz, Ste nodema laevigatum

L., viriscens (Miridae). N rugosus, N. ferns and D. limbatus (Nabidae) which occured in the habitat. None of these species showed any positive reaction to Tingis serum.

Table 141 : Predation on Tingis as indicated by the precipitin test.

Predator species No. No. Tested Reacting Reacting Araneidae Xysticus cristatus* 51 6 11.76 Dictyna spp.* 44 9 20.45 Pisaura pigmentata* P. mirabilis* (Cik.) 24 4 16.66 Evarcha arcuata (Cik.) 21 1 4.7 Araneus gnadratus (Clk.) 19 1 5.26 Linyphia triangularis 16 1 6.25 Imosa 5 - Meta segmentata (Clk.) 15 1 6.66 Enoplognatha ovatum (Cik.) 4 - - Tibellus oblongus 24 - - Clubiona reclusa (0.P.C.) 13 - - Misumena spp. 3 - - Trochosa terricola 12 - Platybunus trianallarie (Herbst.) 18 2 11.11 Mitopus morior Fabricius 16 1 6.25

Table141 continued on next page 292

Table 141 continued

Coccinellidae

Rhizobius litura L. 25 1,0 Coccinella peptempunctata (adults) 16 C. li (larvae) 12 C. 14 punctata 5 411m, U. 11:punetata 3 ••• Adalia bipunctata 13 .am Adalia hieroglifica L. 4

Carabidae Pterostichus madidus 17 QM. Calathus melanocephalus L. 13 ORO Abax parallelcmtpedus Phill & Mitt. 12 MO Amara sp. 23

Nabidae Nabis ru osus (L.) 16 1 6.25 N. ferns L.) 10 1 flavomarginatus Scholtz 3 Dolichonabis limbatus* (Dahlbom) 8 1 12.5 Stalia major Costa 1

Miridae Orthothylus viriscens 4 MI6 40

Anthocoridae Anthocoris nemorum 18 OM A* nemoralis 4 ••• Onus spp 5

Deimaptera Furficula anricularia L. 29

Neuroptera Chrysopa spp. 4 elm gIND Micromus variegatus (Fabricius) 12

Acarina

Anystis agilis Banks 8 Trombiculid mite 5 293

A total of 555 predator meals including Araneids, Phalangids, Mites, Nabids, Anthocorids, Mirids, Coccinellids, Carabids,

Dermaptera, and Neuroptera were tested against Tingis serum. The results are presented in Table 141 . Spiders, phalangids and mites were identified by Mr. D. J. Clark of the Natural History Museum, London; and Coleoptera by Mr. W. 0. Steel.

Data on Table 141 show that spiders, Phalangids and Nabids are the main predators of Tingis in the field. The most important predatory species which may affect the abundance of Tingis in nature are the spiders, X. cristatus; Dictyna spp.; P. pigmentata; P. mirabilis; E. arcuata; L. triangularis; the phalangids, P. triangularis and M. morior and the Nabid, D. limbatus. Most of the species listed above are herbage dwellers and are therefore likely to come in contact with Tingis frequently during each season. Some species, e.g. Dictyna spp; and L. triangularis web readily on thistles and Tingis nymphs were often caught in these webs in the field. X. cristatus; L. triangularis; P. triangularis and M. morior have been seen feeding on adults and nymphs in the field by the author.

The seasonal activity and occurence of spiders on thistles are shown for the 1965 and 1966 seasons in Fig. 72 . There were two main peaks of abundance of spiders, one in June and the other in Sept- ember; although different species vary in peak and period of occurence. The two main peaks of occurence of spiders corresponds closely to those of

Tingis (Figs.63 & 60-62).

An experiment was made in 1967 to study quantitatively,

294

FIG. 72. SEASONAL ACTIVITY OF SPIDERS ON THISTLES IN THE STUDY AREA IN—* ALL SPECIES °---0 XYSTICUS 24 1965 CRISTATUS

".•

< Ed 8 ... Jia...o % a 4 ono o—o co /% I\ i (L A 0,. I 'Ik 'ti o % / %o-o._ e c> a 0/ z 0 O

in ce w zco D Z 20 1966

12 r

4 o —o ... ° — ck. ..,,o,. .o --o o . o o. o i \ A 0-0. MAID1 °- 7tY. I a. Ali. 1 SEP. 1 OCT.° 295

Fig. 7i. Cage used for studying predation on nymphs

A. Olosed B. Open 296 the predation on Tingis nymphs. Each of 10, potted thistle plant was infested with 10 newly emerged third instar nymphs, and distributed randomly in the area. Another set of 10 plants were similarly infested with equal numbers of third instar nymphs and caged in a field cage 5 ft. high x 4 ft. x 3 ft., the sides of which were made of nylon netting (Fig. 73 ). Caged and uncaged plants were examined between 6 and 8 a.m. each day and numbers of adults moulting out were recorded and removed. The results are shown in Table 142 .

Table 142: No.of adults emerging from caged and uncaged third instars.

Plants exposed No. of 3rd instar No. of adults in nymphs at start emerging

Field 100 7

Nylon Cage 100 26

More adults emerged from the caged than uncaged nymphs. However the number of adults which emerged from the caged insects was low and this was probably due to invertebrate predators somehow entering the cage. On three occasions of recording emergence, viz. 28.viii; 27.viii and 2.ix, predaceous spiders,

Dictyna spp; P. mirabilis and L. triangularis were seen on separate plants in the cage. The spiders apparently entered into the cage during periods of recording Tingis emergence and the complete exclusion of these and.similar species from the cage was not always guaranteed.

(ii) Funimal Diseases

In the laboratory, some 3rd, 4th and 5th instar nymphs were occasionally attacked by a fungus, characterised by fine, whitish 297

mycelia and hyphae. The presence of the fungus was often detected at

moulting. Nymphs dying at moult may become covered with a dense mass of

fungal hyphae within a day of death. Pure cultures of the fungus extracted

from Tingis were identified by Dr. M. F. Madelin, of the Department of

Botany, Bristol University, as a species of Paecilomyces. This is a pathogenic hyphomycete belonging to the group Deuteromycetes (Fungi I mperfecti)

many different species and genera of which are parasitic on insects (Made-

lin, 1966). Dr. M. F. Madelin (personal communication) was not certain

whether the Paecilomyces isolated from Tingis was the same species as the

'rather weak parasite' already associated with other insects in Silwood

Park (See Eyles, 1962). However, the occurence of virulent species such as

Paecilomyces farinosus and P. fumoso-roseus on silkworms, Bombyx mori

(Madelin 1966) suggests that the members of the genus vary enormously in

degree of pathogenicity.

Another fungus, a Penicillium species of the Asymmetrica

section was also identified by Dr. Madelin. The fungus occured usually in

5th instar nymphs found dead or dying in field samples. The incidence was

extremely low; about one in 200 individuals (0.5%). Dr. Madelin says that

although a few species of the genus have been found on insects, they are

not often virulent primary pathogens. In the case of Tingis, the feeding

punctures left by ectoparasitic mites and the gustatory or exploratory bites

by predators such as some spiders may provide entry points for a weakly pathogenic fungus like Penicillium. For example, on two occasions, during the present studies, a live 3rd instar nymph with an amputated hind leg was seen in samples. On another occasion, a 4th instar with 1.5 antennae was found. 298

(iii) Ectoparasites

Nymphs like adults are parasitised by the Trombiculid mite. Out of 43, fourth instar nymphs collected in July 1965, two were parasitised and one out 60 fifth instars examined in August of the same year had a mite. 2 out of 20 fifth instars collected on 12th and 16th August 1966 were parasitised by the mite. Infested nymphs seem to moult as satisfactorily as uninfested ones. Out of 15 infested 5th instar nymphs kept for observation in the laboratory 12 moulted into adults compared with

13 out of 15 uninfested nymphs. It appears unlikely that the mite is an important mortality factor. 299

(d) Population budgets, 1965-67

Simple population 'budgets' of the Richards and Waloff (1961) type are presented for the three years, 1965-67 in Tables 143 , 144 & 145 . In general, mortalities have been expressed as percentages of total eggs laid; but in some instances as percentages of the numbers entering a stage. The highest mortalities occured (a) in the egg stage, (b) in the third to fifth instar when nymphs may wander from one thistle bud to another and so become exposed to predators and (c) during overwintering of adults in grass and litter.

If a species has an average fecundity of 100 eggs, and the two sexes occur in equal numbers, then a mortality of 98% would be needed for the population to remain stable (See Richards & Waloff 1961). The sex ratio of Tingis in the springs of 1965, 1966 and 1967 was approximately, 0.34: 1g; 0.5t: 1? and 0.6t: 1.0 respectively. As the corresponding average fecundities were 54, 79 and 70; mortalities of the order of 97.x+96; 98.1% and 97.7 would have produced stability in the population. Mortalities higher than these would have resulted in a decrease and those below an increase in the population. Changes in adult population are investigated in relation to estimated actual mortalities (Tables 143 144 & 145 ) and those expected to give rise to stability in Table 146 . In 1965-66, season percentage mortality was higher than that which would theoretically have produced stability in the following season. The fall in adult population in the spring of 1966 is a reflection of this fact. On the other hand,

actual mortality in the 1966-67 season was lower than that expected to Table 143: Population Budget - 1965.

Mortality of that Accumulated Total population No,dying % of that Stage of stage Stage which stage as % of mortalities as within stage died total egg No. % of egg No.

Adults in Spring 9,125 Eggs 358,560 171,284 47.7 47.7 47.7 Instals I 187,276 10,863 5.8 3.0 50.7 II II 176,413 25,670 14.5 7.1 57.8 tt III 150,743 75,584 49.8 21.0 78.8 I, IV 75,584 12,059 15.9 3.3 82.1 u V 63,525 50,325 79.2 14.0 96.1 Adults in Autumn 13,200 7,300* 55.3 2.0 98.1

* No. disappearing in winter 1965-66 Table 144: Population Budget - 1966.

Total population % of that Mortality of that Accumulated No. dying Stage of stage within stage stage which stage as % of mortalities as died total egg No. % of egg No.

Adults in Spring 5,900 Eggs 313,779 124,206 39.5 39.5 39.5 Instar I 189,573 18,976 10.0 6.0 45.5 " II 170,597 7,481 4.3 2.3 47.7 I, III 163,116 93,415 57.2 29.7 77.4 ,, Iv 69,70o 26,557 38.1 8.4 85.8 I, v 43,143 33,043 76.5 10.5 96.3 Adults in Autumn 10,100 .3,811* 37.7 1.2 97.5

* No. disappearing in winter 1966-67 Table 145: Population Budget - 1967.

Mortality of that Total population No. dying % of that Accumulated Stage of stage stage which stage as % of mortalities as within stage died total egg No. % of egg No.

Adults in Spring 7,556

Eggs 320,683 231,974 72,3 72.3 72.3 Instar I 88,709 10,126 11.4 3.1 75.4 ft II 78,583 1,826 2.3 0.5 75.9 it III 76,757 29,959 39.0 9.3 85.2 ti Iv 46,798 1,526 3.2 0.4 85.6 ff v 45,272 30,100 66.4 9.3 94.9 Adults in Autumn 15,172 303 give stability in the 1967 season. The adult population in Spring 1967 was correspondingly higher than that of 1966. Table 146: Annual deviations of mortality from those necessary for stability. Year 1965 1966 1967 Adults from eggs of 9,125 5,900 7,556 previous generation Mortality necessary for 97.4 98.1 97.7 stability (%) (including variations in fecundity) Actual mortality (%) 98.1 97.5 Difference (%) -0.7 +0.6 The population budgets indicate that the numbers of the new adults in each autumn were in general, always proportional to the initial egg numbers in that generation. This appears to be a common feature of the population dynamics of some insect species and according to Southwood (1966; 1967), there are cases in which variations in natality may 'almost entirely' account for the annual fluctuations in the size of the adult population.

A detailed analysis of the relative contribution of various factors to the total mortality in each generation is given in Tables 147, 148 & 149 in Varley and Gradwell (1960) type population budgets. To recognise the key factor/factors influencing population trend, the various k's i.e. ko;

km k1 and total K were separately plotted against generation (Fig. 74 ). The resultant curves of k's were then visually examined for any correlation with K. Since adults were not sampled in spring 1968, the values of k6 and K for 1967 are missing in Fig. 74 . Those of IC (kappa), Table 147: Analysis of budget - 165. Nos. Log Nos. k's Adults in Spring 9,125 Max. Pot. natality 524,718 5.71991 k (Variation in natality) 0.16542 0 Eggs laid 358,560 5.55449 k (Predation loss) 18,215 0.02263 1 Eggs surviving predation 340,345 5.53186 k (Parasitism loss) 50,133 0.06917 2 Eggs surviving parasitism 290,212 5.46269 k (Other egg losses) 102,936 0.19039 3 Total egg loss 171,284 0.28219 1st instar = Eggs hatching 187,276 5.27230 14 (kappa) = 1.43392 2nd " 176,413 3rd " 150,743 4th 75,584 k (nymphal mortality to) 0.46939 4 ( 5th instar ) 5th instar 63,525 4.80291 k (Predation and other ) 5 (nymphal mortality ) (from 5th instar to adults) 0.68234 Adults in Autumn 13,200 4.12057 (Overwintering loss) k6 0.34972 Adults in Spring, 1966 5,900 3.77085 Total K = 1.94906 Table 148 : Analysis of budget - 1966 Nos. Log Nos. k's Adults in Spring 5,900

Max. Pot. natality 313,779 5.49651 k0 (Variation in natality) 0.00000 Eggs laid 313,779 5.49651 k1 (Predation loss) 43,145 0.06420 Eggs surviving predation 270,634 5.43231 k2 (Parasitism loss) 60,406 0.10968 Eggs surviving parasitism 210,228 5.32263 k (Other egg losses) 20,655 0.04501 3 Total egg loss 124,206 0.21889 1st instar = Eggs hatching 189,573 5.27762 1 (kappa) = 1.49219 2nd " 170,597 3rd it 163,116 4th " 69,700 k4 (nymphal mortality to) 0.64274 ( 5th instar ) 5th instar 43,143 4.63488 k (Predation and other 5 (nymphal mortality 0.63056 (from 5th instar to adults) Adults in Autumn 10,100 4.00432 k6 (Overwintering loss) 0.12602 Adults in Spring, 1967 7,556 3.8783o Total K = 1.61821 Table 149; Analysis of budget - 1967

Nos. Log Nos. k's Adults in Spring 7,556 Max. Pot. natality 363,084 5.55991 k 0 (Variation in natality) 0.05396 Eggs laid 320,683 5.50595 k1 (Predation loss) 24,885 0.03510 Eggs surviving predation 295,798 5.47085 k2 (Parasitism loss) 36,383 0.05687 Eggs surviving parasitism 259,415 5.41398 k (Other egg losses) 170,706 o.46606 (kappa) = 1.32504 3 Total egg loss 231,974 0.65803 1st instar = Eggs hatching 88,709 4.94792 2nd " 78,583 3rd If 76,757 4th 1/ 46,798 k4 (nymphal mortality to) 0.29211 ( 5th instar ) 5th instar 45,272 4.65581 k (Predation and other ) 5 (nymphal mortality ) 0.47490 (from 5th instar to adults) Adults in Autumn 15,172 4.18091 k6 (Overwintering loss) Adults in Spring, 1968 ? rn 307 FIG. 74. THE CORRELATION OF VARIOUS k's WITH K ; RECOGNITION OF THE KEY FACTORS

0[ 2.0 K Imo.. wog. lc I 1.0 0 ...... 00 IC (KAPPA) 1 2 0 0.4 k 40.1.0. OVERWINTE RING 6 ammo. 01 LOSS 0.8i k PREDATION AND 5 OTHER NYMPHAL 0.4 MORTALITY FROM 5TH INSTAR TO 0 A DULTS

0.81 PREDATION AND OTHER NYMPHAL k 0.4 MORTALITY TO 5TH INSTAR 0 0.6- EGG STERILITY 0.3 ,..-±3___ 0 0.1ir , ...... „...... —•-•,...... EGG PARASITISM K 2 0 0.I .....„. EGG PREDATION ki.------0 0.2 NATALITY 1 k 0 0 1965 1966 1967 YEAR

308

the total mortality from eggs to autumn adults in each year are plotted (on an inverted scale) in Fig. 74. The order of magnitude of it for 1967, coupled with the more severe conditions of winter 1967-68 as compared with that of 1966-67, (Table 150) seem to suggest that a higher level of mortality might have occured in the former winter. That is, higher values of k6 and K, as are indicated by the dotted lines in Fig. 74. The results show that the key factors affecting Tingis population were variations in natality

(ko) egg mortality other than those due to parasitism and predation (k3) and possibly also overwintering loss of adults (k6). Table 150: Comparison of environmental conditions from October to the following April.

Winter 1966-67 1967-68

No.of days on which 0 12 snow covered ground

Average daily minimum8 3.12 2.22 air temperature, C. (1.9 - 7.1)* (-1.2 -7.6)*

Average daily grass Minimum temperature, -4.8 -6.7 C. (-10.8 -13.75)* (-12.25 -12.5)*

*range. 309

Discussion

As discussions of results on proceeding aspects of this work have been made in the relevant sections of the thesis, only the data on population budgets are commented on here. According to Richards and Southwood (1967), an ecological study of an insect species should attempt to explain its distribution, average level of population and the annual or seasonal deviations from the average; (i.e. "to recognize the conditioning, the regulating and the disturbing processes"). Usually, an insight into the mathematical relationships of the factors involved in these processes is gained from the analysis of life tables.

Hitherto, there have been no published population budgets on the Ting- idae, although some authors (Morril 1903, Sharga 19,3, Drake et al. 1922, Golfari 1937, Fyfe 1937 and Loan 1967) have indicated that various mortality factors may affect abundance of different members of the group in nature.

Life budgets of the Varley and Gradwell (1960) type (See Southwood 1966; p.

302-4) were constructed for T. ampliata for three years, 1965-1967. The'k' factor analysis was done although three years are much too short for certain recognition of the key factor or factors. A minnimum of five years is regarded as reasonably adequate for the formation of a comprehensive picture of the population dynamics of an insect species in Great Britain (Richards

1961). However, it does seem as if k0 (natality); k3 (egg losses other than those due to parasitism and predation) and probably also k6 (overwintering loss of adults) are the key factors and hence their variations are the main causes of the variation in adult population from year to year. It is not surprising that Tingis population levels are determined by a comb- 310 ination of factors for as Varley & Gradwell (1967) have pointed out "an

insect's mean density and thus its status as an abundant or rare animal is a function of all the factors acting upon it".

If life budgets were available for more years, the various k's could

be plotted against the density of population on which they are acting to

determine which factor or factors are regulating the population. That is,

to detect the presence and type of density dependent mortality factors.

These would generally be expected to differ from the key factor (Varley

1963, Varley and Gradwell 1965, Klomp 1966a) but they can be the same

(Varley 1953, Southwood 1967).

The present study has therefore shown the practicability of this type of study in a tingid and has indicated the approach towards the analysis of the results. Whether k0 (natality) and k3 (egg losses other than those due to parasitism and predation) are really the disturbing factors remains for future work. 311

SECTION III: SUPPLEMENTARY STUDIES ON T. CARDUI

Of four stands of C. vulgare on North Gravel in the summer of 1966, only one harboured a population of T. cardui. The host stand was situated about 20 ft. to the east of the first third of the length of a foot path which runs from Silwood House through North Gravel to the Observatory

Ridge (Fig. 1 ). It was composed of 10 main stems, 1 short and central and the remaining 9 were tall-stemmed; with the stems radiating in a circular formation from the centre. The vegetation at the bases and the immediate vicinity of the stems consisted of a mixture of grasses and herbs as described by Luff (1964).

Numbers

A female found on the stand on 10th May was marked with a dab of red artist's oil paint on the pronotum and released on the original flower head. She was recaptured on three subsequent occasions, the last being

31st May. The numbers of nymphs and autumn generation adults recorded on the stand at 4 day intervals are shown in Fig. 75. Owing to the relatively small size of first instar nymphs, it was impracticable to make direct visual stages. counts of that stage in the field. There is considerable overlapping of the/

Oviposition and fecundity

From each of the 9 tall stems of. the 1966 thistle stand, 2 leaf samples were taken on 15th May. A total of 4 eggs were recorded from the 18 samples. When incubated on moist filter paper at 20°C, 2 of the eggs hatched on 31st May, and a third on the 5th June. The fourth did not hatch. Eggs were absent from samples taken on 4th August. Oviposition

312

FIG. 75. OCCURENCE OF T.CARDUI ON L.VULGARE 1966 5 40.- cc 2ND INSTAR

ICL5 20- 1-4 Lo- 40- 3RD INSTAR 20- (7z2 0 D 40 0 4TH INSTAR 02 20- Z 0 Lt_ 40- 0 5TH INSTAR 220- W 40°- ADULTS z 20-

0 9 17 25 7 f5 23 JUN. JUL. I AUG. 4 SEP I 1 313 site, egg placement and distribution on leaves were as described by South- wood end Scudder (1956). Since only one female was found on the stand throughout spring and early summer, it appears that judging from the numbers of immature stages recorded, T. cardui has a high fecundity as compared with T. ampliata.

Development of nymphs in the field

The period of development of nymphs varies with the instar. Using the dates of last occurence of successive instars in the field, developmental periods have been estimated as follows:

Period of development Instar (days)

III 8.o

IV 12.0

V 12.0

Sex ratio: As in T. ampliata, the sexes of T. cardui differ in size and can be separated in the field by careful examination of the ventral aspect of the posterior abdominal segments for external sex organs (Section II,1(a))0

The relative proportions of the sexes in adults counted on thistles in autumn 1966 were as follows: 314

Date Sex ratio Q, l' 8.8.66 1.0 : 0.6 12.8.66 1.0 : 1.0 16.8.66 1.0 : 1.25 20.8.66 1.0 : 0.8

Distribution

(A) On different regions of host plant Phytophagous insects tend to prefentially select specific sites of their hosts for food or oviposition. Various regions of the 9 tall shoots of the 1966 stand of C. vulgare were examined on the 4th August, and the numbers of each stage of T. cardui encountered were recorded. The results are shown in Fig. 76 . Although, individuals were occasionally found on the main stems and axillary buds of thistle leaves, the highest proportion of insects were always confined to the involucres and the short region immediately below them. This distribution pattern did not vary throughout the autumn, except for the progressive increase in the numbers of adults on involucres as more 5th instar nymphs moulted to adults. The crevices between the bases of bracts were predominantly selected. It is probable that such micro-habitats may have a survival function since they tend to conceal the insects from large predatory species.

(B)Spatial It was shown in Fig. 76 that T. cardui is almost entirely confined to thistle involucres in the field. Although stems were not more than 0.5 - 1 ft. apart from each other, only the involucres of 4 — 7 out TOTAL NUMBERS REGION OF HOST PLANT o 4* r -I-

1NVOLUCRE

REG ION lil BELGA/ INVOLUCRE I I LEAVES ACAXIAL SURFACES

ABMIAL SURFACES

AXILLARY BUDS I

AXILS OF LEAVES

MAIN STEMS t...•73 0 I I z-I z D. > 73 73 316 of the 9 harboured Tingis. Counts of involucres and bugs on individual stems were made on the 4th and 12th August. The numbers of bugs on each stem are compared with the corresponding numbers of involucres in Fig. 77 . This scatter diagram shows the independence of the numbers of Tingis on a stem on the numbers of involucres on that stem. On some plants, some flower heads had as high as 6 bugs, whereas others on the same or near plant were completely free of bugs. Thus, T. cardui tends to form aggregates of different sizes on the host plant. This aggregation behaviour was clearly indicated when the mean number of bugs per stem ( X ) and variance ( S2) were calculated for counts on each of 8 consecutive sampling occasions (Table 151).

Table 151: Means and Variances calculated for Tingis recorded on 9 C.

vulgare stems on 8 consecutive occasions.

Occasion Total Tingis TMean No of Variance Log ( X+1 trgnsformation ingis per No. on 9 stems ( s2) 2 stem C X ) mean a ) Variance ( S )

1 32 3.55 8.27 0.550 0.918 2 27 3.00. 9.5 0.477 0.978 3 28 3.11 4.11 0.493 0.613 4 28 3.11 4.11 0.493 0.613 5 25 2.77 8.87 0.442 0.770 6 23 2.55 7.27 0.407 0.861 7 18 2.00 3.35 0.290 0.525 8 11 1.22 1.19 0.086 0.076

These value?s have been transformed to logarithms and plotted graphically in

6 2 LO CE(s cc

G. VARIAN 2 4

FIG. 78.PLOT OFVARIANCEvsMEAN ONALOG/LOGSCALE FIG77. SHOWINGTHEINDEPENDENCEOFINGISDENSITY FOR COUNTS OF I CLAMON THISTLES .Y1ficiabaE ON THENUMBEROFINVOLUCRESTHISTLESTEM 6 NO. OFINNOLUCRESONSTEM LOG. MEAN(g • 6 o

• • o b .4 • 0

• (2 14 • o

•

• • 0 r : Y :1.637X+00067 P t0.01

0.852 0 18 201224 • • o ON 4.8.66 f? 12.8.66 317 318

Fig. 78. With the exception of one sampling, occasion, variance was always much greater than the mean. There is considerable scatter of the points. A line has been fitted to the points by eye. The equation of regression of Log ( S2) = Y on log ( ) = X is Y = 1.637 X 0.0067. The regression coefficient of 1.63 is greater than unity and is a measure of the degree of aggregation of the species (Taylor 1961). This level of aggregation has been compared with that of T. ampliata (Section 11,2(e)).

Predators associated with T. cardui Between 4th August and 8th September, 1966, records of potential predators of T. cardui were made on thistle flower heads on the stand on North Gravel. The results are summarised in Table 152.

Table 152: Numbers of each predatory species found with T. cardui on thistle flower heads.

Date Species found Total 4/8 8/8 12/8 16/8 20/8 24/8 28/8 1/9 8/9 Xysticus cristatus 3 1 2 2 4 3 3 3 1 20 Tibellus oblongus 1 0 1 0 0 0 0 1 0 3 Mitopus morio 2 0 0 0 0 0 0 0 0 2 Lycosa spp 0 3 2 2 1 1 1 0 0 10 Platybunus triangularis 2 1 0 0 0 0 0 0 0 3 Coccinella aeptempunctata 1 1 1 0 1 1 0 0 0 5

Furficula auricularia 0 1 1 0 2 1 3 2 6 16 Abax parallelopipedus 0 0 0 0 0 1 1 0 1 3 FIG.79. THE RELATIONSHIP BETWEEN NUMBERS OF SPIDERS AND ICARDU1 ON SPEAR THISTLE .

.---• NO. OF ICARDUI 35 7 0.- -0 NO. OF SPIDERS

30 -6 % 5 % 0 2 /• .5 < X / • / % L u / • O H 2 0 0 .4

O- .3 o X z

___1 X0 I-- .I 1--0 I 1 i ) 10 15 0 5 35 45 DAYS 1.8.66= DAY I 320

Spiders (mainly Xysticus, app.) and earwigs (Dermaptera) were the commonest predatory species on flower heads. The relationship between the numbers of spiders and T. cardui on thistles in the period is shown in Fig. 79 . Although none of the species listed in Table 152 was observed feeding on T. cardui in the field, data in Fig. 79 suggest that there may be some correlation between numbers of Tingis and its potential predators. 321

SUMMARY

1. T. ampliata is univoltine and overwinters as adults in grass and litter. The onset of overwintering is marked by autumnal migration of adults to the bases of the host plant. Hibernation is associated with (a) differential mortality of the sexes (b) a moderate degree of cold-hardiness, and (c) a considerable loss in body weight and metabolic reserves. Cold- hardiness varies with the season, nature of the contact surface, and the presence or absence of food in the insect's gut. Undercooling point is positively correlated with ambient temperatures. Weight loss during winter does not occur at a constant rate, and males tend to lose a higher proportion of their autumn body weight than females. Numbers of eggs and oocytes are significantly correlated with weight of females at post-hibernation emergence, the earliness or lateness of which varies with the year according to pre-emergence spring climatic conditions. In a warm,,Ary year with delayed thistle above-grass emergence, some feeding may occur before insect and host emerge on the grass surface,

2. Reproductive organs and live weight undergo seasonal changes which are correlated with distinct physiological phases in adult life cycle. Mating lasts several hours and occurs at any time of the day in spring and early summer. Eggs are usually laid singly and mainly in the midribs and other veins of the undersurfaces of thistle leaves; most eggs being laid in the lower halves of plants. There is a distinct periodicity in oviposition, the peak of which occurs in the afternoon. Females die without completing oviposition. The rate of oviposition increases with temperature during the pre-peak phase of oviposition. Temperature has no significant 322 influence in the post-peak phase during which age of bugs is significantly negatively correlated with oviposition rate. The rate of egg development increases with temperature in the range, 100 - 30oC. Moisture is essential for the development and hatching of eggs.

3. Nymphs colonize thistle buds, especially those in the upper halves of plants in which they feed in small aggregates, causing chlorotic blotches, malformation and reduction in area of leaflets; culminating in reduced growth and wilting of buds. The live weight of nymphs increase with age and instar, most of the growth occuring between the third and fifth instar.

Adult feeding does not produce any noticeable effects on thistles.

4. T. ampliata is relatively static when compared with the more actively flying species, T. cardui. Under laboratory conditions, various isolated or combined stimuli such as high temperatures, tarsal contact and agitation would induce some sexually immature T. ampliata to fly in the few weeks immediately after post-hibernation emergence. Readiness to fly and duration of flight decreased with age and maturity of bugs. The level of flight activity in the two Tingid species is closely associated with their flight muscle content or anatomy and the degree of permanence of their habitats.

Adults of T. ampliata engage in trivial horizontal movements of about 2 feet per day in their habitat. Movement tends to be multi-directional. The main climatic factors affecting numbers of males occuring in the field on any given day are: mean temperature, maximum temperature, relative humidity, hours of sunshine and minimum temperature, The last of the above factors is the most important and is positively correlated with numbers caught. by age but The rate of movement of adults is affected/mainly by the hours of sunshine 323 and maximum temperature. The former factor was negatively and the latter positively correlated with the rate of movement. There is a seasonal variation in the amount of flight muscle which is closely associated with feeding and reproductive cycle of adults.

5. Nymphs and adults of T. cardui and all stages of T. ampliata show a moderate degree of aggregation in the field. The level of aggregation varies with population age and size. There is diurnal variation in the vertical distribution of adults in the summer. The proportion of insects in the upper halves of plants decreases and that in the lower half increases from morning, through afternoon to evening. Adults and nymphs of T. cardui are almost entirely confined to the involucres of C. vulgare L. The stages overlap considerably in the two Tingid species.

6. Females of T. ampliata generally have a higher post-hibernation survival rate than males, although most individuals of the latter sex tend to emerge earlier than those of the former. Males of fourth and fifth instar nymphs also tend to occur before females in the field. The sexes occur in virtually equal numbers in the nymphal stage and in adults of the pre- hibernation population. Adult population shows two peaks each year; one in May-June and the other in mid-August. The latter, i.e. that of the new generation adults is always higher than that of the former, (i.e. the overwintered population). There is a variable period of one to two weeks during which females of the pre and post-hibernation populations occur together in the field. Annual variations in fecundity are associated with differences in ambient temperatures during oviposition period, the average 324 weight of females of the reproducing population and the post-hibernation survival rate of females.

7. Mortality of eggs is caused by predation; parasitism by Mymarid (Chalcidoidea) parasites, A. fuscipennis Haliday and P. panis and by sterility. Nymphal mortality was mainly due to predation by (a) spiders, notably X. cristatus; Dictyna spp.; P. pigmentata and L. triangularis; (b) harvest spiders viz, P. triangularis and M. morior and (c) Nabids - D. limbatus and N. rugosus. The highest proportion of this mortality occurs in the third to fifth instar stage. Losses in adult population were caused mainly by winter deaths and predation by spiders and harvest spiders.

8. T. ampliata fluctuated in numbers far below the carrying capacity of thistles. Annual variations in size of adult population are associated with variations in natality and egg mortality (other than those due to parasitism and predation). Population budgets are presented for the three years; 1965-67. 325

ACKNOWLEDGEMENTS

I wish to express gratitude to Professor O. W. Richards for research facilities at Silwood Park.

This work was carried out under the joint supervision of Professor T. R. E. Southwood and Dr. N. Waloff. I sincerely thank them for their persistent interest and invaluable guidance throughout the period of the work; and especially for their constructive criticism of this manuscript.

I am also grateful to: Dr. R. L. Doutt, of California University, Berkeley for the identification of the mymarid parasites of Tingis eggs.

Dr. G. Murdie for statistical analysis of data on dispersal in relation to weather.

Messrs J. W. Siddorn (late) and H. Devitt for photographic assistance.

Dr. D. Pinnock for preparation of antiserum used in the determination of predators of Tingis nymphs.

Mr. D. J. Clark of Natural History Museum London, for identification of spiders and harvest spiders.

Dr. F. Madelin of Dept. of Botany, Bristol University, for identifying fungal parasites.

Mrs. M. F. Van Emden and Dr. H. Danks for translation of German and French papers respectively. 326

Mr. 0. W. Steel, for identifying Trombiculid mite and coleoptera.

Miss C. Collins for typing the thesis.

This work was carried out whilst I held a U. K. Technical Assistance- ship Award to the Federal Republic of Nigeria. I am greatly indebted to the relevant authorities of the scheme for grants and to the Members of the

Governing Council of the Cocoa Research Institute of Nigeria for offering me the opportunity for this study. 327

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