Th is dissertation has been microfilmed Mic 60-5331 exactly as received MCMAHAN~ Elizabeth Anne. LABORATORY STUDIES OF CRYFTOTERMES BREVIS (WALKER) (ISOPTERA: KALOTERMITIDAE): WITH SPECIAL REFERENCE TO COLONY j ~ DEVELOPMENT AND BEHAVIOR. )[ ...~

..~ University of Hawaii~ Ph.D., 1960 .;! /A Zoology ft (~ ~~iversitY ~Cbig~~ ..... Microfilms. Inc.• Ann Arbor. . . .. i LABORATORY STUDIES OF CRYPTOTERMES BREVIS (WALKER) (ISOPTERA: KALOTERMITIDAE)

WITH SPECIAL REFERENCE

TO

COLONY DEVELOPMENT AND BEHAVIOR

A THESIS SUBMITTED '.cO THE GRADUATE SCHOOL OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

July 1960

By

Elizabeth Anne McMahan ii

TABLE OF CONTENTS

LIST OF TABLES . ... . iv

LIST OF ILLUSTRATIONS .. .' ... viii

INTRODUCTION ...... 1

STUDIES OF COLONY ESTABLISHMENT AND DEVELOPMENT IN CRYPTOTERMES BREVIS (WALKER) 6

Swarming and Colony Establishment in Nature. 6

Laboratory Studies of Colony Development. . 8

Incipient Colonies Established by Primary Reproductives ...... 8

Incipient Colonies Established by Primary Reproductives Paired with Supplementary Reproductives ...... 30

Incipient Colonies Established in Termitaries of Differ ent Kinds of Wood...... 33

STUDIES OF CRYPTOTERMES BREVIS BEHAVIOR 51

An Investigation of Feeding Behavior Using a Radio- isotopic Tracer Technique. 51

Plan of Experiments ... 52

Equipment and Apparatus ...... 62

Experimental Procedure ...... 69

Reliability of Figures ...... 86

Results ...... 'I •••••••••• • 94 iii

An Investigation of Termite Behavior Through Direct Observation...... 164

General Procedure 167

Effect of Molting on Termite Activities 174

Behavior of Individual Termites 182

Time Spent in Food Solicitation...... 201

Inter-Termite Relationships...... 203

SUMMARY ... 212

BIBLIOGRAPHY 216 iv

LIST OF TABLES

Table Number Subj ect Page

I Comparison of survival of different primary reproductives from incipient colonies according to age of colony and whether or not self-paired or force-paired. ... 15

II Comparison of number s of eggs and nymphs present in incipient colonies of different ages...... 21

III Comparison of survival rates and egg and nymph production for the two combinations of reproductives ...... 32

IV Preliminary wood preference tests: Compari­ son by means of weighted scores of amounts of termite feeding on each type of wood. . 39

V Comparison of development of colonies from pairs of new dealat.:;s (D-D) in different types of termitary wood...... 43

VI Comparison of development of colonies from pairs of supplementary reproductives (5-5) in different types of termitary wood. 44

VII Compa.rison of developme n t of colonies from pairs of old established primary reproduc­ tives (E-E) in different types of termitary wood...... 45

VIII Comparison of development of colonies from pairs of new dealate-old established primary r eproductives (D-E) in different types of termitary wood ...... 45 v

IX Effects of wood preference on colony development 47

X Radioactive termitaries used in feeding experiments ...... 68

XI Pairing schedule for hot and cold termites .. .. 81

XII Errors at the 95 percent confidence level for five- minute counts of representative samples. .. 88

XIII Radioactivity picked up by donor sand recipients in Experiments 1-7...... 95

XIV Radioactivity picked up by donors and recipients in Experiment 8...... 96

xv Survival of the different termite types from one stage to another...... 99

XVI Termite survival according to partner for Experiments 1 to 7 ...... 102

XVII Mean radioactivity tranFfer percentages for each combination of termite types ...... 109

XVIII Comparison of radioactivity acquired by donor types having access to the same hot termitary (gut volume relationships) " 119

XIX Comparison of radioactivity lost through fecal pellets by isolated and non-isolated termites at various intervals following removal from hot termitaries ...... 130

XX Mean radioactivity transfer percentages for each combination of termite types (omitting sex) after gut corrections and log (x + O. 1) transformation...... 141

XXI Transfer percentages of Nand n termites in Experiment 8...... 146 vi

XXII Comparison of radioactivity acquired by molted and nonmolted recipients for all types of partnerships...... 151

XXIII Comparison of radioactivity acquired during Feeding Period A by molting and nonmolting donors a.ccording to termitary group. .. 155

XXIV Comparison of radioactivity acquired by re­ cipients paired with molted and nonmolted donors for all types of partnerships. ... 159

XXV Activity suspension interval for food solicita- tion, according to instar ...... 176

XXVI Activity suspension interv'al for food donation, according to instar ...... 177

XXVII Activi t y suspension interval for grooming. according to instar ...... 178

XXVIII Activity suspension interval for being groomed, according to instar ...... 179

XXI X Activity suspension interval for gnawing, according to instar ...... 180

XXX Comparison of food solicitation behavior exhibited by the termites of the two colonies observed...... '...... 185

XXXI Comparison of food donorship by the termites of the two colonies observed...... 186

XXXII Comparison of grooming behavIor exhibited by the termites of the two colonies observed. 187

XXXIII Comparison of frequency of being groomed for the termites of the two colonies observed. . 188

XXXI V Comparison of gnawing behavior for the termites of the two colonies observed...... 189 vii

xxxv Comparison of frequency of jerking behavior during molting and nonmo1ting intervals ...... 198

XXXVI Comparison of frequency of egg-tending and pellet-arra.nging for the different termites of the two colonies...... 200

XXXVII Comparison of time in seconds spent by each ter- mite in the act of proctodea1 feeding. .... 202

XXXVIII Comparison of frequencies with which each ter­ mite of Colony 1 was observed to solicit food from each of its colony mates...... 204

XXXIX Comparison of frequencies .with which each termite of Colony 1 was observed to groom each of its colony mates ...... 205

XL Comparison of frequencies with which each ter­ mite of Colony 2 was observed to solicit food from each of its colony mates...... 206

XLI Comparison of frequencies with which each termite of Colony 2 was observed to groom each of its colony mates ...... 207 viii

LIST OF ILLUSTRATIONS

Figure Number Subject Page

1 Cryptotermes brevis (Walker). 3

2 Dismantled termitary of birch tongue blades used in experiments on colony establishment. 12

3 Body length relative to hea.d width for nymphs representing different instars...... 27

4 Termitary composed of different types of wood veneer used in preliminary tests of wood preference ...... 35

5 External sex characteristics of G. brevis termites . 60

6 Termitaries used in feeding experiments 65

7 Immobilization chamber for examining termites 73

8 Compa:dson of gain in radioactivity of different types of termites coruined together in hot termitaries for one week...... 115

9A A comparison of rate of loss of radioactivity by isolated termite types ...... 125

9B A comparison of rate of loss of radioactivity by non-isolated termite types ...... 126

10 Observation termitary composed of the halves of three birch tongue blades ...... 171

11 Comparison of colony activities for the different termites of Colony 1...... 190

12 Comparison of activity frequencies for the different termites of Colony 2. .... 191 LABORATORY STUDIES OF CRYPTOTERMES BREVIS (WALKER) (ISOPTERA, KALOTERMITIDAE)

WITH SPECIAL REFERENCE

TO

COLONY DEVELOPMENT AND BEHAVIOR

INTRODUCTION

Cryptotermes, a genus of the family Kalotermitidae, is primarily tropical in distribution. It is represented in the Australian, Papuan,

Indomalayan, Ethiopian, Malagasy, and Neotropical zoogeographical regions of the world, ::md it also has one native temperate species in

Florida (Emerson, 1955). Probably none of the species is of greater economic importance than Cryptotermes brevis (Walker), the West

Indian termite, which is a pest not only in the Caribbean islands,

South and Central America, Florida, and Louisiana but also in Hawaii and South Africa.

A considerable number of paper s have been devoted to the species, but they have been concerned primarily with direct means of economic control. Life history studies have not been made of ~. brevis, nor has its behavior been closely investigated. The present work was 2 concerned primarily with observations on colony development and with behavior studieA.

The work was carried out in Hawaii where C. brevis is an introduced species. It has been unable to invade the native environment but remains confined to human habitations, infesting walls, floors, ceilings, furniture, books, and almost any other type of cellulose product (Zimmerman, 1948).

The colonies, which appear not to exceed about 300 individuals, live entirely within galleries formed as a result of feeding on the wood. In­ festation is detectable through the discovery of fecal pellets which the termites push out of the galleries.

~. brevis, like other kalotermitid species, has no true worke::- caste, the work of the colony being done by the nymphal forms of the soldier and reproductive castes. Figure 1 shows alates, a soldier, and two sizes of nymphs of the species.

The colony development studies were made on incipient colonies reared in the laboratory in special termitaries, each colony being descended from a single pair of reproductives. Rate of colony increase and mortality rate were studied, and comparisons of these colony characteristics were made between colonies begun with different combinations of reproductive types.

Other observations included sizes of different instars, the effect of molting on the suspension of various activities, and the effect of favored as compared with non-favored woods on colony development. 3

•

i ',r/'IT.. ' '-,;;.."." b

Fig. 1. Cryptotermes brevis (Walker). a. Alates, side and dort?a~ views. b. S:>ldier. c. Small nymph (third instar). d. Large nymph (fifth instar or olderj. 8. 5 X. 4

Termite behavior was investigated by means of two different techni­

ques: the use of radioactive tracers and direct visual observation. The isotopic tracer technique was applied primarily to a study of the transfer

of food from one individual to another.

Much of the soci~.l organization of the ~. brevis colony (as well as that

of other social insects) is based on food exchanges. The two youngest instars and the soldier cannot feed directly from wood and would starve

unless fed by colony mates. In addition, food transfer not only serves to refaunate the termite gut following a molt, but is also probably one of

the chief routes of distribution of the substances believed by many investi­

gators to govern caste formation (Castle, 1934; Light, J.942, 1943; Karlson

and Butenandt, 1959; Luscher, 1956). Wheeler (1923) first proposed the

concept of trophallaxis (exchange of nourishment) as an integrative

mechanism among social insects. La Masne (1953) regards food trans­

mission in the ant colony as an integral part of the adult-larva relationship

essential to social cohesion, and Ribbands (1953) suggests the possibility

that food transmission may be an important means of communication

among worker honey bees.

The present study was an attempt to investigate the food exchanges

between individuals of C. brevis cC'lonies to see whether or not consistent

relationships could be found based on specific combinations of sex, caste,

or instar . Evidence of such social organization might be of the nature 5 of a "dominance hierarchy" (reported for many vertebrate groups and some invertebrates, including wasps) but organized more along biological than along psychological lines .

The direct visual observation studies sought more evidence on the question of whether or not these termite colonies were organized accord­ ing to a dominance-subordinance kind of social hierarchy and whether division of labor, apart from caste-based functions, could be detected.

The completion of the work herein reported was greatly aided by the help and encouragement of a number of persons, particularly

Mr. Susumu Kato and Mr. Lester Zukeran. Thanks are also due

Mr. Ralph Hinegardner for help with photographic techniques. The

Diamond Match Company provided some of the materials used in the construction of termitaries, and I was able to purchase various supplies and equipment with two grants-in-aid given by the University of Hawaii

Chapter of the Society of the Sigma Xi. I appreciate the use of the facili­ ties at the University of Hawaii Marine Laboratory for carrying out the radioisotope studies. Most of the work was done in connection with

National Science Foundation Grant No. G958l awarded to Dr. L. D.

Tuthill. STUDIES OF COLONY ESTABLISHMENT AND DEVELOPMENT IN CRYPTOTERMES BREVIS (WALKER)

Swarming and Colony Establishment in Nature

In Hawaii the swarming or colonizing flight of~. brevis usually begins in April and continues through July, but infested furniture has been found to contain numerous alates as early as February and as late as August. Swarming occurs chiefly during the early evening hours, beginning around 7:00 o'clock, and seems not to be associated with rains. This lack of relationship between the swarming of dry- wood termites and rains has been noted by Harvey (1934, p. 220) and

Weesner (1960, p. 160).

Observations on C. brevis swarming behavior were usually made indoors. In most cases the emergence of alates (primary reproductives) took place at spaced intervals during the evening. For example, on the evening of May 3, 1959 dozens of alates were seen to emerge from an exit hole in an infested desk, beginning at 7:00 P. M. and continuing for about 30 minutes. No further emergences were noted until about

9:00 P. M. when activity was resumed, this time involving fewer alates.

Again swarming activity ceased for about two hours and was resum.ed 7 at about 11:00 P. M. There was a progressive decrease in numbers of emerging alates as the evening progressed. C. brevis_swarming has also been seen at hours up to about 3:00 A. M. (Matsui). Harvey

(1934, p. 221) has reported this same cyclic emergence of alates for the drywood species, Kalotermes minor Hagen.

In all the observed cases alate flight was relatively brief, due per­ haps to the fact that they were always indoors. Soon after alighting the alates shed their wings and began running about erratically. Many formed tandem pair s, with the female leading and the male following closely, behavior characteristic of new dealates of most termite species.

Pairs eventually found cracks or crevices into which they crept. On one morning following a n.ight of swarming, numerous pairs were found within the folds of the previous day's newspaper which had been left lying on a table overnight. Each pair already had made a small chamber for themselves by building up walls of a brown parchment-like material between the pages. When new dealates are introduced into artificial termitaries one of their fir st activities is to seal up all cracks and joints with the parchment-like material. The result is a snug chamber which effectively excludes ants, probably the chief enemy of termites.

Mating in~. brevis, as in other termite species, does not occur until after the pair have sealed themselves into their chamber. Mating 8 activity was not observed in the present investigation, but in other termite spl.7·cies it is reportedly accomplished end to end with heads of the pair pointing in opposite directions (Pickens, 1934, p. 162).

Laboratory Studi.es of Colony Development

Incipient Colonies Established by Primary Reproductives

A detailed study of the establishment and development of a colony from its beginning with a newly dea1ated pair has not been re­ ported previously for ~. brevis, although other species have been so studied. The latter include Zootermopsis angusticollis (Hagen)

(Castle, 1934), Tenuirostritermes tenuirostris (Desneux) (Light and

Wee sner, 1955a) Ka10termes flavicollis Fabr. (Goetsch, 1936),

Reticulitermes 1ucifugus Rossi (BuchU, 1950), and~. hesperus Banks

(Pickens, 1934; Weesner, 1956). In the present investigation a number of primary or incipient colonies of ~. brevis were studied with a view to finding rate of colony increase, sex and caste ratios after different time intervals, and other aspects of colony establishment and composition.

Experimental Procedure

During the 1959 swarming season from April 29 to

June 14 alates were captured and held (usually overnight) in plastic boxes (8" x 4" x 5") until they had lost their wings. These new dea1ates were used in setting up 240 potential colonies in 240 separate wooden termitaries, 20 of which were to be examined at each of 12 monthly interva.ls according to a fixed schedule in order to 9 study their development.

The termitaries were set up on 20 different days, the date and the number on each date being determined by the swarming of alates. In an attempt to control the effect of possible factors affect­ ing termite vigor that might have been associated with a given day and might have affected colony establishment, the schedule for opening the termitaries was worked out systematically so that a particular set-up date was represented equally, insofar as possible, in all the check-up dates.

In half the cases the dealates were paired by sexing the termites and arbitrarily placing a male with a female in a given termitary.

This procedure will be called "force-pairing." In the remaining half the dealates were allowed to choose their own mates, a proce­

dure to be called "self-pairing. II In these cases the termites were placed overnight in holding chambers containing slotted wooden strips which provided a number of small dark chamber 0 • By morning mo:::t c·f-the slots wert: found to contain a single termite pair (with an occasional instance of more than two individuals in a single slot).

These self-paired couples were transferred to the experimental termitaries. Light and Weesner (l955a) have reported that the selection of tandem pairs to be used in setting up incipient colonies of !: tenuirostris has not given reliable results under laborator.y 10 conditions, but has sometimes resulted in the pairing of two termites of the same sex. In the present investigation only one instance is known in which the self-pairing method gave this result. This was a case in which two females were paired for 9 months. When the termitary was opened, both were lively, and two eggs were present. The eggs were saved but did not hatch.

In accordance with the experimental schedule 20 termitaries, half containing force-paired and half containing self-paired dealates were opened and examined at each of 12 monthly intervals. (At the 8-month check-up the two dealate types were not 10 and 10 but were 8 and 12, and at the lO-month check-up the relationship W2.S reversed, 12 and 8). Be­ cause of the termitary construction (see below) it was .a simple matter to find every egg and every termite present. The following data were recorded for each termitary: state of vigor of the primary reproduc­ tives; number of eggs present; number of nymphs present; and sex, head width, and body length of each nymph (except where impossible to determine). Any special findings were noted in addition. After the data had been recorded the contents of each termitary were preserved separately in 70% alcohol.

Termitaries

The termitaries used in the colony establishment experiment were all alike: each wag constructed of six birch 11 tongue blades bolted together to form a unit and containing a central termite chamber. This construction permitted easy access to the termite inhabitants during later examinations and also appeared to repr esent a natural environment for C. brevis termites. C. brevis colonies, for example, are freqnently found at the University of

Hawaii infesting wooden slide boxes, which are made of thinner pieces of wood than the termitaries described.

Figure 2 is a diagram of Cl. dismantled termitary showing its construction. In preparing a termitary six unwarped large tongue blades (6" x 3/4" x 1/16") were chosen and fitted snugly together.

They were lettered from ~ to.! for ease in preserving proper se­ quence. A slot 1/2" x 1/4" was cut in the center of blades c and d by drilling two 1/4" holes side by side and cutting away the small bits of intervening wood. The 6 blades were then re-assembled and a 1/4" hole was drilled about 1" from each end of the assembled blades. A 1/4" bolt was inserted in each hole and secured with a wing nut, resulting in a "solid" block of wood with a center chamber about 1/2" x 1/4" x 1/8". Introduction of each termite pair into a chamber was accomplished by bolting one end of the termitary, not too tightly, and then sliding blades ~ and ~ slightly out of position so that a small opening into the center chamber was revealed. The termite pair were deposited by means of a camelis hair brush beside the 12

o o

Fig. 2. Dismantled termitary of birch tongue blades used in experiment on colony establishment. Actual size. 13 opening which they usually entered immediately. Blades a and b were then slid back into position, the bolt at the free end was inserted, and both nuts were tightened. On the a blade was recorded the termitary number, the date, and a designation indicating "self-paired" or "force-paired." Similar data were recorded in a notebook. The termitary was placed on a wire rack in a steel cabinet where it was left at room temperature until time for the later check. The termitaries were set on metal racks and not allowed to touch each other in order to decrease the po s sibility that pair s might tunnel out of their chambers and enter adjacent wood. This procedure was successful, for in only 2 instances were the primaries totally missing from their termi­ taries, although I'exit holes ll were found in most of the termitaries opened last. These holes were used for ridding the chamber of pellets.

Results

Survival of primaryreproductives. Survival

of the colonizing reproductives did not appear to be

related to whether the pair had been self-paired, or

force-paired, and except for the first 2 months it was

not markedly dependent upon the length of time that

had passed since they were placed in the termitary. 14

Table I shows the number of self-paired and force­ paired couples surviving at each of the 12 check-up periods. In 26 cases only one of a given pair survived to check-up time, and these, as well as the 57 cases in which both members died, were omitted from the present analysis. This left 157 termitaries in which both primary reproductives were alive at the time of check-up.

It is not known how well the survival percentages given in Table I represent thosE'\ for natural incipient colonies of C. brevis. Harvey (1934, p. 247) in his early attempts to study colonizing pairs of !5. minor using small 11block cells" and "dowel cells" in the laboratory found an extremely high mortality rate.

He believed that the following factors in his tests tended to increase the mortality of primary pairs above normal and to slow down egg production and individual development: 1) injury from handling; 2) ill effects from keeping alates in a dry atmosphere beyond the normal interval; 3) delayed entry into the wooden cell; and 4) abnormal factors in the laboratory environment such as large spaces to be sealed and unfavorable 15

TABLE I

Comparisons of Survival of Differ ent Primary Reproductives from Incipient Colonies According to Age of Colony and Whether or Not Self-paired or Force-paired

Age Self-Paired Force-Pair-ed Tot a 1 of Origi Surviv- Origi- Surviv- Origi- Surviv Per- Colony na1 ing nal ing na1 ing centage (Mo. ) pairs pairs pairs pairs pairs pairs Survival

1 10 8 10 8 20 16 80

2 10 8 10 9 20 17 85

3 . 10 5 10 7 20 12 60

4 10 6 10 8 20 14 70

5 10 6 10 4 20 10 50

6 10 7 10 7 20 14 70

7 10 5 10 6 20 11 55

8 8 6 12 7 20 13 65

9 10 6 10 7 20 13 65

10 12 7 8 6 20 13 65

11 10 5 10 8 20 13 65

~ 12 .I.U' 7 10 4 20 11 55

120 76 120 81 240 157 16 temperature and moisture conditions resulting from the sm.all size of the wooden cells used and their ex- posed location. When an attempt was made to eliminate these factors the mortality rate was greatly reduced: out of 38 pairs. 30 or about 79 percent were alive and active after 10 months. This result is better than that obtained in the present study in which only 65 percent survived 10 months. Perhaps some of the factors listed by Harvey operated here also. Nevertheless, the results are very much better than his earlier mortality figures and compare favorabl y with his later ones. Although the termitaries used were small they were probably not subjected to harmful fluctuations m temperature and humidity. Weather conditions in

Hawaii are not usually extreme. Furthermore, the termitaries were kept in a closed steel cabinet which was further insulated by being placed in a closed closet.

A more pertinent question concerns the adequacy of clean birch tongue blades as a termite diet. Hendee

(1934). Hungat e (1941). Light and Weesner (1947) and others have stressed the importance of fungi in the diet of termites. Feeding tests by these and other workers 17 have consistently showed that termites depend on fungi for certain food elements and that the use of sound wood in culturing these insects results in increased mortality. The tongue blades were used "as they werell with nO attempt being made to inoculate them with fungi.

They probably were not initially invaded, but contami­ nation of the termitaries with fecal pellets may soon have remedied such probable deficiences. In cases in which both primary reproductives were found dead in a termitary, after one month of pairing, the chamber usually contained fairly abundant growths of fungi. This indicates that fungal invasion soon occurred.

Most of the 157 surviving pairs were found to have lost portions of antennae and/or legs during their con­ finement together, a finding that may be related to diet defich~ncy. A tendency to cannibalism has been attributed by some workers (Cook and Scott, 1933) to a lack of suffi­ cient nitrogenous material, and Hendee (1934, p. 113) has found it to be excessive among Zootermopsis ter­ mites kept on fungus-free wood. Grass~ (1942) reports that in certain species, at least, mutilation of the antennae normally precedes coition, but Williams (1959) 18 has found no connection between such mutilation and sexual behavior. He considers it an example of ordinary cannibalism even though it occurs between fit and unwounded individuals. He suggests that the relative inactivity of the reproductive pair in the confined space of the early cell may make each imago appear sluggish to the other and result in cannibalistic attacks. He does not rule out the physiological state of the imago as a possible factor in these mutilations.

In assessing the extent to which the present find­ ings are representative of natural incipient~. brevis colonies it can be said only that individual vigor, wood excavations, egg and nymph production, and pellet production all indicated an environment that was con­ ducive to termite growth and development.

Rate of Colony Irlcrease. Termite colonies increase at a relatively slow rate initially. The primary repro­ ductives lay only a few eggs in the beginning. This first brood is then reared to an age at which the young termites are able to take over the task of caring for later broods and doing the other work of the colony, thus freeing the original pair for their main function of re­ production. In certain termite species the reproductive 19 pair have not been observed to feed during the period

of rearing the first brood which are fed on secretions from the bodies of the primaries. Skaife has reported

this fasting by young reproductives for Amitermes atlanticus Fuller (Family Termitidae), and Coaton (1'138) has reported it for Hodotermes mossambicus (Hagen)

(Family Hodotermitidae). Presumably because of

undernourishment during their growth period, first brood nymphs are usually undersized. C. brevis primaries, on the other hand, were observed in the present investigation to feed frequently on wood for at least 6 months after they were paired, and the young nymphs of the first brood obtained their nourishment by feeding proctodeally from the primaries. By the third instar they were feeding directly from wood, but also continued proctodeal feeding, C. brevis first brood nymphs might therefore be expected to be less under­ nourished than their counterparts among the higher termites.

Egg Production. C. brevis eggs resemble tiny jelly beans. When first laid they are pink and exhibit a

plump appearance but after about 4 weeks they become 20 progressively whiter and their surfaces become creased with tiny furrows. A group of 53 eggs re­ moved from termitaries containing supplementary reproductives were measured in order to obtain an average size characteristic of~. brevis eggs.

These supplementary eggs appeared in no way different from those produced by primary repro­ ductives. Both length and width were measured by means of an ocular micrometer in a dissecting microscope, with the following results: length =

1. 20 mm. + .09 and width = .50 mm. + .01.

Length varied from. 90 mm. to 1. 35 mm. and width from.45 mm. to .59 mm.

In the present investigation eggs were observed in termitaries by the end of one month and were found during every month of the year. By far the greatest number ocr.urred, however, during the first three months after colony establishment (see Table 11) when each reproductive pair averaged approximately

2, 4, and 4 eggs, respectively. These figures probably indicate that the first batch of eggs is usually laid within the first two months of pairing TABLE II

Comparison of Numbers of Eggs and Nymphs Present in Incipient Colonies of Different Ages

Age No. N y M P H A L I N S T A R S of of ( M. M. H EA D) I Colony Colo- Eggs First Second Third Fourth Nymphs (Mo. ) Dies Total Ave. .40-.45 .60-.65 .80-.85 .90-.95 1.0-1. 05 1.10-1.15 Total Ave.

1 16 29 l.8 2 17 74 4.4 3 12 52 4.3 9 6 1 16 1.3 4 14 22 1.6 6 13 15 2 3 2 41 2.9 5 10 10 1.0 2 5 12 0 23 1 43 4.3 6 14 9 0.6 1 3 25 3 35 3 70 5.0 7 11 4 0.4 0 2 7 27 21 2 59 5.4 8 13 5 O•.1, 0 0 1 23 29 5 58 4.5 9 13 6 0.5 0 1 3 27 33 0 64 4.9 10 13 6 0.5 0 0 1 10 37 0 48 3.7 11 13 9 0.7 0 0 1 4 38 1 44 3.4 12 11 12 1.1 2 2 0 6 26 1 37 3.4

N 157 480 ...... 22

and take approximately 2 months to hatch. Data ob­

tained from the direct observation studies indicate

this is the approximate time required for a ~. brevis

egg to hatch at ~oom temperature, although some have

taken at least 11 weeks. The data presented in Table II

show that the number of eggs in the terrnitaries de­

creased with hatching until about the ninth month when

they began again to increase, indicating that the pri­ maries had resumed egg production.

It may be of interest to compare the initial egg

production of paired primary reproductives with that

of new supplementary reproductives. In another ex­

periment, supplementaries were produced by isolating

10 large C. brevis nymphs together during August, 1958

in termitaries similar to those used in the present in­ vestigation. After two months the termitaries were

opened, the supplementaries removed, and the eggs

counted. The 24 termitaries in which supplementaries

were fOlmd contained 108 eggs or an average of 4.5 eggs per termitary. This is close to the average of 4.4 eggs

per termitary produced by the primary reproductives

after two months. In another supplementary-forcing 23 experiment the 10 nymphs were isolated for four months

(October, 1958 .- February, 1959), before the termitaries were opened. Again sllpplementaries were produced in

24 termitaries which contained 337 eggs or an average of 14.0 eggs per pair. This is considerably more than the 1. 6 average produced by the primaries after four months. Superiority in egg production on the part of supplementaries has been reported by other investigator s

(Light, 1934, p. 29), but at least some of the superiority in the present instance, may be due to the altered "social

circumstances. II Unlike the case of the pairs of young primary reproductives which apparently took Iltime out" to rea.r their first b.rood, the supp1ementaries did not appear to suspend egg production. They had the com­ panionship of large nymphs from the beginning and these extra termites may have been responsi.ble for more favorable :mtrition or some other factor conducive to egg production.

Several workers have reported that eggs of some ter­ mite species do not hatch if removed from the ministra­ tions of the termites (Michener and Michener, 1951;

Noirot, 1952/1953/ Skaife. 1953). This is not true for 24

C. brevis eggs. They were frequently removed from termitaries or from natural colonies and kept in covered watch glasses to protect them f!'om ants. Some took over two months in the watch glass to hatch but eventually hatching exceeded 80 percent in nearly every test.

Nymphs. In a study of an incipient colonyi s growth rate and development during the first year it is important that each instar be identifiable. Size, particularly head width, is the main criterion on which such identification is based. Published inforD1ation on this relationship of

head size to instar could not be found for C. brevis and was worked out in the present investigation chiefly on

the basis of daily observation of other colonies of ~. brevis nymphs whose individual development was followed from molt to molt. Only a few data were collected on

which instar head width relationships could be predicted.

They were as follows:

Observed Instar Head width cases (mm~

10 1 .40 - .45 4 2 .60 - .65 5 3 .80 - .85 1 4 1. 00 6 "large nymphs" 1.15 - 1.30 (froD1 natural colonies) 25

The data suggested that the first four instars would be easily distinguishable, and examination of the nymphs from the incipient colonies showed this to be true for the first three instars. Difficulties arose, however, when an at.tempt was made to identify later instars.

Among the larger nymphs, head widths of .90 mm. ,

. 95 mm., 1. 00 mm., 1. 05 mm., 1. 10 mm., and 1. 15 mm., were found. How were they to be classified as to instar? Judging on the basis of the known fourth instar from the observation colony, it seems certain that a 1. 00 mm. head width identifies a nymph as having reached at least the fourth instar, but how about one of

.90 mm... or one of 1. 10 mm.? Some termitaries con­ tained nymphs with both. 90 mm. and. 80 mm. head sizes for example, and the two seemed definitely to be different instars. Coaton (1958) believes, however, that intergradations in size found in established colonies of

Hodotermes mossambicus can be attributed to variations in amounts of nourishment given individual nymphs during their growth period.

In a fUrther attempt to clarify the instar picture, body length was considered in combination with head width, and 26

Figure 3 shows the result of plotting one against the other. Body length is to some extent dependent on stage in molting cycle as well as on nutrition and is therefore not as good an indication of instar as head width should be. Figure 3 tends to imply that head widths of .90 mm. and 1.10 mm. indicate different instars, but does not settle the question of precise instar identification.

The number of segments in the antennae have also been used in characterizing the various instar s in

Reticulitermes lucifugus Rossi var. santonensis Feytaud

(Buchli, 1958). but this method was not satisfactory with

C. brevis. Alates in this species"" verage 16 antennal segments, but the number varies from 15 to 17 and is often not equal for the two antennae of one individual. The younger the instar the more difficult it is to determine accurately the number of segments. This is because the region immediately following the third proximal segment, where new segments are added, is indistinct. For example, in the first instar. the two proximal and the four distal seg­ ments are fairly distinc.t. The number in the indistinct region appears to vary between 2 and 4. Furthermore: In­ st ars known to be different often gave what appeared to be 27

5

4 ...-----R13l ~ S (lO2l S 3 -..c.., / (66) b.Os:: - '0 2 0 CQ Q) b.O

I-l ""Q) ~ 1

.50-.40 .70-.60 .85-.80 .95-.90 1. 00­ 1.10­ 1. 05 1. 15 Head Width (mm. )

Fig. 3. Body length relative to head width for nymphs representing different instars. Mean lengths and standard deviations shown. Number of cases in each category given in parentheses. 28

identical segment counts 0 There is a general increase in segment number with age, but attempts to use this information in instar identification were unsatisfactory.

It was decided to present the "nymph development data." m the case of the larger nymphs, under three categories of head width: .90 - .95 mm., 1.00 - 1.05 mm.• and

1.10 - 1,15 mm.• without attempting to assign definite instar s to each.

Table II shows at each check-up the number of nymphs that fell into each head-width category. It bears out the egg production data in showing that these primary repro­ ductives during the first year of colony establishment produced initially only a few eggs representing the first brood. These were laid mostly within the first three months and hatched into nymphs that developed more or less as a group. By the sixth month almost all of the initial eggs had hatched and the nymphs had developed to at least the second instar. Most of them already had reached at least the fourth instar (which in some cases was attained within four months of the initial pairing of the reproductives).

By the ninth month virtually all nymphs had reached at least the stage of fourth instar. At about this time (or 29 perhaps somewhat later}, the primaries resumed egg production, and by the twelfth month first and second instar n)'lnphs were again present in the termitaries.

The largest number of nymphs per termitary was found in the seven-month-old colonies, and the number decreased for older colonies. The decrease may have been due to the cumulative effects of unfavorable condi­ tions of laboratory ::.-earing, such as inadequate diet.

Extrapolating from the date of Table II it would seem that an incipient colony of C. brevis can be expected to contain at the end of a year (in addition to the r eproduc­ tives) from four to eight nymphs of the fourth instar and older, the first batch of offspring, plus several younger nymphs representing later progeny

Other Observations. Sex was identified for 444 third instar and older nymphs from the incipient colonies (sex cannot be determined easily for younger nymphs). The sexes were found to be approximately equal, with 236 males and 208 females, a 1. 13: I ratio. The small dif- ference may represent chance variation.

Not one soldier was produced in an incipient colony within a year. In another experiment, involving wood 30

preferences, a soldier was found in a 14-month-old

incipient colony, which was confined in a termitary com-

posed of maple veneer. The colony at that time consisted

of the primary pair, 16 nymphs, 1 soldier, and 2 eggs.

It is not unusual in other termite speices to find that no

soldier is produced during an incipient colony's first

year. (Light and Weesner, 1955b).

In two termitaries in the colony establishment experi-

ment, each containing only one member of the primary

pair when opened, it was found that one of the nymphs

of the colony had become a supplementary reproductive.

In one case the colony was 10 months old, in the other,

11 months. The head widths of these su:::,plementaries

wer e both 1. 00 mm., indicating at least fourth instar

nymphs. Castle (1934, p. 293), reports that in

Zootermopsis angusticollis, nymphs of the fourth to

seventh instars may develop into supplementary repro-

ductives.

Incipient Colonies Established by Primary Reproductives Paired with Supplementary Reproductives

It has already been mentioned that ~. brevis supplementary repro- ductives appear to be more prolific egg layers than primary reproductives, at least under the conditions of the comparison. Another e:'periment 31 was carried out to study the success of colony establishment when one member of the original pair was a primary, the other a supplementary r eproductive.

During May 1959, 44 birch tongue blade termitaries were set up, half containing a male primary and a female supplementary reproductive and the other half containing a male supplementary and a female primary reproductive. The termitaries were opened and their incipient colonies examined one year later, in May 1960. Of the 22 cases in which the female was a supplementa.ry reproductive 14 pairs survived (64 percent).

Twelve of the 22 pairs involving female primary reproductives survived

(55 percent). The former type of pairing appeared to be favored not only from the point of view of survival, but also in terms of the number of nymphs produced per surviving pair: 3.5.±. 1.1 as compared with

2. 8 + 1.2. Termitaries with female primaries, however, contained more' eggs at the time of check-up.

Table III presents the data for the experiment and shows the number of eggs and the number of ny~phs (by instar) present in each termitary representing the two pairing types. There are some indications that the primary females laid a number of eggs at the beginning of the pairing interval (as had been generally indicated for the colonies containing two primary reproductives in the previous section) and then laid no more until the end of the year. This is implied by the lack of instars below TABLE III

Co:mparison of Survival Rates and Egg and Ny:mph Production for the Two Co:mbinations of Reproductives

Type N y M P H A L I NST A R S of Ori- Sur- % ( M. M. HEA D) Pair- ginal ving Survi- No. First Second Third Fourth Total per Standard ing pairs pairf val Eggs .4-.45 .6-.65 ,8-.85 .9-.95 1. 0-1. 05 l.o.uO+ Ny:mphs pair Deviation d"De- alate 22 14 64 5 2 1 2 0 43 1 49 3.5 + 1.1 ~ Sup- - ple:m. d'Sup- p1e:m 22 12 55 8 0 0 0 1 34 0 35 2.9 + 1.2 ~De- - alate

Total 44 26 59 13 2 1 2 1 77 1 84 3.2 , N 33 the fourth when the check-up at the end of the year was made and also by the number of eggs found at that time, indicating a recent resumption of egg-laying.

The data for the supplementary females, also given in Table III, may be interpreted as indicating that egg-laying was resumed sooner than in the case of the primary females. and may not, in fact, have been sus- pended. There were five nymphs representing first, second, and third instar s, in addition to those that had reached at least the fourth. The egg concentration was not as great as in the other group. The answer to whether or not the apparent differences shown in these aspects of colony establishment by the two types of reproductive combinations are real must await further experimentation. The above experiment does support the view that mating between primary and supplementary reproductives is not unusual. Snyder (1934, p. 192) also reports that mating between primary and supplementary reproductives occurs in old. established colonies of Reticulitermes £lavipes Kollar.

Incipient Colonies Established in Termitaries of Different Kinds of Wood

An. experiment involving WOt:)d preferences was related also to the investigation of colony establishment. Various combinations of r~produc- tive types were introduced into termitaries composed of different kinds of wood veneer ordinarily used in furniture manufacture. The untreated 34 veneer (presumably kiln dried) was obtained from a veneer factory m sheets approximately 1/32 inch, or a little more, in thickness.

Fifteen differ ent woods were included in the tests: (1) northern hard maple; 2) tupelo, 3) yellow poplar, 4) sycamore, 5) sweet gum,

6) white oak, 7) Spanish oak, 8) black walnut, 9) white pine, 10) yellow pine, 11) cherry, 12) ash, 13) African mahogany, 14) elm, and 15) korina.

They were not all tested equally or systematically, and consequently conclusions regarding their relative resistances to termite attack can be only approximate.

Preliminary tests were carried out to determine the relative att ractiveness for C. brevis termites of each kind of wood. In these pl'eliminary tests two groups of termitaries were used, one group con­ sisting of seven, the other of eight termitaries. In the fir st group, each of the seven was constructed of woods 1 to 7 listed above. In the second group, each of the eight termitaries was constructed of woods 8 to 15. A typical termitary is diagramed in Figure 4. In

1 group 1 it consisted of 14 sheets (3 x 6" x 1/32 '1 ) of veneer, two for each type of wood, and a top and bottom of plexiglass. The two veneer sheets nearest the plexiglass in each case were left entire, but the re­ maining 10 sheets had a 1'1 x 2" square center hole cut out, so that when the sheets were all assembled and bolted together a center chamber approximately 1" x 2" x 7/16 '1 was formed. The two sheets of each 35

6" ) ~\3" ~\

Fig. 4. Termitary composed of different types of wood veneer used in preliminary tests of wood preference. Top and bottom of plexiglass. 36 type of wood were always placed together in order to provide a total thick- ness that would permit a termite to tunnel completely within that type. The order of wood type relative to position in the termitary was different for each of the seven termitaries. The following schedule shows the composi- tion of each termitary of the first group in terms of the order of the veneer sheets used in its construction.

ORDER OF VENEER SHEETS IN TERMITARIES 1 - 7

Wood 1 2 3 4 5 6 7 ,... Spanish Oak ST M Iv B AW

Tupelo T M C BA WS

Hard Maple M C BAW S T

Cherry C BA WS TM

Black Walnut B AW STM C

Ash A W ST M C B

White Oak W S T M C BA

It should be noted that order of wood types relative to each other was not controlled by this schedule. The regular shift in composition was for the purpose of controlling the effect of-veneer position. relative to the termitary chamber. on amounts eaten by the termites.

The eight termitaries of the second group were identical in construction except that they consisted of the remaining eight types of veneer and 37 consequently of 16 veneer sheets bolted together.

In setting up a termitary 50 ~ brevis termites taken from infested plywood boards were placed in the center chamber,. the top veneer sheets and the plexiglass cover placed in position, and the termitary bolts tightened. The termites were all large nymphs probably represent­ ing at least the fifth instar. A few nymphs with large wing pads were in~luded in each termitary, but most of the nymphs ha.d only small wing pads or none.

Following the introduction of the 50 nymphs into a group of termi­ taries the latter were set on edge on the shelves of a steel cabinet at room temperature. The termitaries of group 1 were set up on February 3,

1958 and those of group 2 on June 29, 1958. The group 1 termitaries were left for 150 days and the group 2 termitaries for 140 days before they wer e opened.

The seven termitaries of group 1 had originally contained a total of

350 large nymphs. When opened after approximately 5 months they con­ tained 2 a1ates, 18 supplementary reproductives (usually a pair in each termitary), 3 soldiers: 185 nymphs (old and new nymphs were notre­ corded separately) and 151 eggs.

The eight termitaries of group 2, which had originally contained a

~otal of 400 large :nymphs, contained after approximately 4-2/3 months

16 supplementary reproductives, 2 soldiers, 180 old nymphs, 70 new 38 nymphs, and 307 eggs.

The chief interest in the experiment, of course, was the comparison

of relative amounts of wood eaten from the differ ent types of veneer

available. This was determined not by measuring the change in weight

of the veneer sheets (which would have been a good procedure if moisture

conditions could have been precisely controlled), but in a less quanti­

tative fashion to be described below. The purpose of the preliminary

tests was to provide a basis for the formulation of hypotheses regarding

preferences for the types of wood bei.ng tested, and this was done as

follows:

At the time of check-up the two sheets of each type of veneer were

considered in turn for each termitary and were judged according to a

scale involving 4 degrees of. apparent "feeding preference, " based on

amount of wood consumed. A designation of "0" meant that there was

not the slightest indication of an attempt by the termites to feed on the

11 particular sheets. A score of 111 meant that there were very few such

indications, consisting sometimes of a single hole (of termite diameter)

showing that the termites had me-rely tunneled through tha sheets on

thei.r way to another area. A score of 112" meant that evidence of

gnawing was definitely more than in the case of "l" samples, and a

score of "3" indicated extensive tunneling.

Table IV gives the results of this means of comparing relative 39

TABLE IV

Preliminary Wood Preference Tests: Comparisons by Means of Weighted Scores of Amounts of Termite Feeding on Each Type of Wood

Termitaries of Group 1 Total Score Wood 1 2 3 4 5 '6 7

Spanish Oak 3 1 0 2 0 0 2 8 Tupelo 3 3 2 3 3 2 3 19 Hard Maple 3 3 3 3 3 3 3 21 Cherry 2 1 1 2 1 0 0 7 Black Walnut 0 0 0 1 2 1 0 4 Ash 1 0 3 0 2 3 2 11 White Oak 0 0 0 0 0 0 1 1

Termitaries of Group 2 Total Score Wood 1 2 3 4 5 6 7 8

Yellow Poplar 3 3 2 3 3 3 3 3 23 Afr. Mahogany 1 1 0 0 0 2 1 1 6 Sycamore 3 3 0 3 3 3 3 3 21 White Pine 1 0 0 1 2 2 0 0 6 Yellow Pine 0 2 2 0 1 2 1 0 8 Elm 0 3 3 0 0 0 2 0 8 Korina 0 0 0 0 0 0 0 0 0 Sweet Gum 2 2 3 3 1 1 1 1 14 40 attractiveness of the different veneer types. On the basis of these re­

sults the woods may be arranged in descending order of " preference. II

Hard Maple (most "preferred")

Yellow poplar

Tupelo

Sycamore

Sweet Gum

Ash

Spanish Oak

Cherry

Yellow Pine

Elm

African mahogany

White Pine

Black Walnut

White Oak

Korina (least " preferred")

The rest of the investigation involved setting up incipient colonies in termitaries composed of these differerent types of wood to see if the above trends in wood preferences would show up in terms of differences relating to colony development. The hypothesis was that incipient colonies developing in termitaries made of the least favored woods 41 would have a higher mortality rate, fewer nymphs, and fewer eggs after a given interval than those in the more favored woods.

The termitaries were constructed on the same plan as that used in making the tongue blade termitaries (Figure 2) and a total of ten

small wooden sheets of veneer, each approximately 6" x I" X 1/32" were bolted together to form each termitary. All the veneer sheets of a given termitary represented one type of wood. The sheets were lettered from~ tol..and the central chamber (1/2 "x 1/4" X 5/16") was cut out of sheets ~ to Ii:

A reproductive pair was introduced into each termitary. These

pairs included the following types:

I- D-D : both members were new dealates (primary reproductives): 93 pairs.

2 - S-S both members were supplementary reproductives: 55 pairs.

3 - E-E: both members were old established primaries: 17 pairs.

4 - E-D: one member was an old established primary queen -­ the other a new male dealate: 5 pairs.

This made a total of 170 pairs of reproductives in the pre;;;el.1t ;"xperi- ment comparing wood types.

The S-S termitaries containing supplementary reproductives were

set up during the months from September 1958 to February 1959 when

primary reproductives were difficult or impossible to obtain. Supple-

mentary reproductives were obtained whenever needed by placing 42

together 10 to 15 large nymphs in birch tongue blade termitaries

and leaving them undisturbed usually for from two to four weeks.

At the end of this time most of the nymphal groups contained a pair

of supplementary reproductives. These were removed and placed alone in the S-S termitaries (or in proper combination with other

reproductive types in other termitaries).

Termitary types other than the S-S type were set up during the months {rom February to September during 1958 and 1959. The ter­ mitaries were opened after intervals varying from 9 to 23 months.

Tables V to VIn present the results for the different experi­ mental conditions according to two age groupings: colonies aged 9

to 15 months and colonies aged 18 to 23 months. Most of the compari­

sons will be made on the younger group which comprised 142 of the

169 colonies.

Survival rate was low for all combinations of reproductives, but especially so for paired supplementaries and for old established primaries. It would appear that such pairs cannot alone establish

incipient colonies: due perhaps to their dependence on colony mates for nourishment. In nature, of course, such isolated pairs would

not be expected to occur.

Incipient colonies are usually begun by new dealates, and their

survival rate in the present experiment was 29 percent for the younger TABLE V

Co:rnparison of Develop:rnent of Colonies fro:rn Pairs of New Dealates (D-D) in Different Types of Ter:rnitary Wood

Colonies Aged 9 to 15 Months Colonies Aged 18 to 23 Months Age of Ori- Survi- Aee of Ori- Survi- Colony ginal ving No. No. Colony ginal ving No. No. Wood (Mo. ) (Mo. ) pairs pairs Eggs Ny:rnphs , pairs pairs Eggs Ny:rnphs

Yellow Poplal- 13-15 7 4 1 11 20 2 0 0 0 Hard Maple 13-14 10 5 5 25 21-23 11 4 0 5 Syca:rnore 13 8 2 2 6 Tupelo L3-l4 3 2 2 8 22 7 5 2 17 Sweet Gun~ 13-14 11 6 2 21 Ash 13 2 0 0 0 Spanish Oak 13 1 0 0 0 Yellow Pine - - - - - 20 3 0 0 0 Cherry 14-15 3 0 0 0 Afr. Mahogany 13-14 6 0 0 0 White Pine 11 1 0 0 0 Black Walnut 12-13 11 1 0 0 Black Oak 13 7 0 0 0

~ VJ TABLE VI

Comparison of Development of Colonies from Pairs of Supplementary Reproductives (S-S) in Different Types of Terrnitary Wood

Colonies Aged 9 to 15 Months Age of Original Surviving Number Number Wood Colony (Mo. pairs pairs Eggs Nymphs

Yellow Poplar 9 - 15 11 0 0 0 Hard Maple 9 - 15 7 1 1 3 Sycamore - - - - - Tupelo 13 - 15 5 0 0 0 Sweet Gum 11 - 14 13 1 1 4 Ash 15 6 1 1 1 Spanish Oak 15 2 0 0 0 Yellow Pine 11 4 0 0 0 Cherry 15 2 1 0 1 Afr. Mahogan y - - - - - White Pine 11 3 0 0 0 Black Walnut 13 1 0 0 0 White Oak 13 1 0 0 0

H'­ H'- TABLE VII

Comparison of Development of Colonies from Pairs of Old Established Prima.ry Reproductives (E-E) in Different Types of Termitary Wood

Colonies Aged 9 to 15 Months Colonies Aged 18 to 23 Months Age of Origi- Survi- Age of Origi- Survi- Colony nal ving No. No. Colony nal ing No. No. Wood (Mo. ) pairs pairs Eggs N-yrnphs (Mo. ) pairs pairs Eggs Nymphs

Yellow Poplar 11-15 5 1 \1 1 20 1 0 0 0 Hard Maple 15 3 0 0 0 21 1 0 0 0 Tupelo 13-14 3 0 0 0 Sweet Gum 14 1 0 0 0 Yellow Pine 20 1 0 0 0 Cherry 9 1 0 0 0 White Pine 20 1 0 0 0

TABLE VIII

Comparison of Developrnent of Colonies frorn Pairs of New Dealate-Old Established Pri1llary Reproductives (D-E) in Different Types of Termitary Wood

Yellow Poplar 15 2 1 2 2 Hard Maple 15 1 1 1 6 Tupelo 14 1 0 0 0

Sweet Gum 13 1 0 0 0 ~ U1 46 colonies and 39 percent for the older. These percentages are not as good as those for the incipient colonies reared in birch tongue blades.

A relatively good showing in terms of survival was made by the 5 colonies begun with a new male dealate paired with an old established queen. These data are too few to permit conclusions but may suggest that the dealate feeds enough on the wood to feed them both.

The next step is to see if the hypothesized effects based on wood preferences did in fact emerge. The preliminary tests indicated an "order of termite preference" for the 15 kinds of wood used in the tests. Only 13 of the 15 types of veneer were included in the later experiment, elm and korina being omitted. If the three termitaries of Spanish oak, the wood that lies seventh in the descending order of preference, are omitted from the present analysis there remain six more-preferred and six less-preferred types of wood. How do these two termitary groups compare with regard to the colony establishment criteria of survival of repro­ ductives, of egg production, and of nymph production? Table IX summarizes the data. The results are given for the different pairings separately as well as pooled. The most meaningful com­ parison from the standpoint of effect of wood type on colony TABLE, IX

Effects .of Wood Preference on Colony Development

Repro- Colonies A£zed. 9 - 15 Months Colonies Ae:ed 18-23 Months duc- Origi.• Surviv- %Sur- Origi- Surviv- %Sur- tive nal ing vi- No. No. nal ing vi- No. No. pairs pairs pairs val Eggs Nymphs pairs pairs val Eggs Nymphs Preferred 'Woods *

D-D 41 19 46.3 12 71 20 9 45.0 2 22 5-5 42 3 7.1 3 8 - E-E 12 1 8.3 1 1 2 0 0.0 0 0 E-D 5 2 40.0 3 8 -

Total 100 25 25.00 19 88 22 9 40.9 2 22 Non-preferred Woods*':C

D-D 28 1 3.6 0 0 3 0 0.0 0 0 S-S 11 1. 9.1 Q 1 E-E 1 0 0.. 0' 0 0 2 0 0.0 0 0

Total 40 2 5.0 0 1 5 0 0.0 0 0

*Yellow Poplar, Hard Maple, Sycamore, Tupelo, Sweet Gum, Ash. ** Yellow Pine, Cherry, African Mahogany, White Pine, Black Walnut, White Oak. ~ --:J 48 establishment involves the D-D termitaries in which new dealates were paired and for which survival percentages were best. The

41 termitaries made from the more-preferred woods yielded 19 surviving pairs (46.3 percent), 12 eggs and 71 nymphs. The 28 termitaries made from the less-preferred woods yielded only one pair of survivor s (3.6 percent), and this pair had produced no eggs and no nymphs. These results show that the preliminary tests did indeed indicate favorable as opposed to unfavorable wood.

Kofoid (1934, page 11) has stated that "termites instinctively

seek environmental conditions favorable to their existence. II

Kofoid and Bowe (1934) suggest that the factors that determine what woods \'-'ill be selected by termites in nature include the moisture content of the wood, the amounts and chemical nature of the various wood extractives, the physical qualities of the wood resulting in hardness or other properties causing resistance to gnawing, the differences between sapwood and heartwood, the nature and amounts of substances favoring the growth of fungi in the wood, and the nature and extent of pre-existing fungus attack.

In a test of the relative resistivity of several kinds of wood to the attack of Kalotermes minor these authors found a notable difference between the different woods in the number of termites abandoning the test block. In other tests with the same termite species they 49 found that death rate and volume of excavations differed con$istently with the kinds of wood tested, redwood heartwood proving to be most resistive. Kofoid and Bowe suggest that the death rate of termites in redwood may be due to the lethal effect of redwood extractives on the protozoa of the termite gut. Sherrard and Kurth (1934) showed the detrimental effect of the extractives on fungal growth.

Wolcott (1953) working with ~. brevis reported further work in­ dicating that immunity of so- called termite resistant woods is due to the presence of specific chemical constituents (such as tectoquinone in East Indian teak) in relatively small amounts which cause them to be unpalatable to the termites. He found that these natural con­ stituents can be extracted and used in minute amounts to impregnate susceptible woods, making them immune also. He further reported

(1946) that some highly :resinous woods are termite resistant, as are woods with a high lignin content. Marchan (1946) reported a relationship between lignin, ash, and protein content of various kinds of wood and their resistance to termite attack. These considerations are probably involved in the present tests.

Some of the woods shown in the present study to be unfavorable to termite colony development (for example, oak and pine) have appeared to be favored in other studies with other termite species.

The combination of factors affecting selection of woods by termites 50

(mentioned by Kofoid and Bowe) and presumably relating to termite . survival and colony development were probably different for these different experiments. In the present tests these factors were detrimental. Whatever their basis, the wood preferences demon- strated by the g. brevis termites were shown to be related to colony survival and development. STUDIES OF TERMITE BEHAVIOR

AN INVESTIGATION OF FEEDING BEHAVIOR USING THE ISOTOPIC TRACER TECHNIQUE

Radioisotopes as tracers are playing an increasingly important part in entomological investigations. They have been us ed, for exarnp1e, to study insect metabolic proc~sses, to follow the movement and dispersion of insect pests, to clarify parasite-host and insect-plant relationships, and to study the path of food transfer among social insects. Summarie:s of some of this work and references can be obtained from the publication by the United States Army Quartermaster Corps (1957) and from articles by

Jenkins and Hassett (1950). and Lindquist (1952).

The food transmission studies carried out previously have concerned the transfer of nutritive materials within hymenopteran societies. Nixon and Ribbands (1952) studied the distribution of radioactivity among members of a colony of honey bees after training a few to feed from a food source

32 containing.. p Wilson and Eisner (1957) made a comparable study of

131 f00 d exchanges in. col'onles 0 f'SiX species.0 fants uSing . 1 . No one to my knowledge has heretofore investigated in a similar way the intracolony 52

feeding relationships among termites.

Plan of Experiments

Aims

Termites frequently solicit food from each other. In stomodeal

feeding one termite receives food from the mouth of another; in procto­

deal feeding the food is obtained from the anus of the donor. C. brevis

individuals in observation colonies were rarely seen to engage in stomo­

deal feeding, but proctodeal feeding was often observed.

In the present investigation the original intention was to look for food

exchanges between~. brevis individuals that might be related con­

sistently to specific combinations of caste, instar, or sex. For example,

younger instars might show a tendency to exchange food among themselves

. more frequently than with older instars, soldiers might solicit food more

readily from older instars than from younger, or male nymphs might

consistently feeci from female nymphs. If such relationships were found,

a search for their basic explanations might lead to better insights into the

social organization of termite colonies.

The radioisotopic tracer technique provided a means whereby trans­

fer of food from one termite to another might be traced. The experimental

plan was as follows. Termites representing different castes, sexes, and

lf instars were to be made radioactive (llhot ) by allowing them to feed on

wood that had been soaked in a raciioisotopic solution. Each was then to 53 be paired with a nonradioactive ("cold") termite for a week according to

a system that would permit all possible combinations of radioactive and nonradioactive types. If food was transferred from the "hoe' to the

"cold" partner through stomodeal or proctodeal feeding it could be traced by means of the radioisotope. The assumption was that the amount of

radioactivity transferred would be an indication of the willingness of the "cold" termite to feed from the "hot" termite, and of the latter IS

willingness to allow food solicitation by its particular partner.

Proctodeal feeding involves solicitation by the recipient termite and

some cooperation by the donor termite. A termite can prevent another

from feeding from it by walking away so rapidly that the solicitor cannot

keep up and finally desists. Termites in the premolting phase, for

example. have often been observed to employ this tactic. Usually the

donor cooperates by remaining motionless until the recipient has

stopped feeding. Grass~ (1949) in discussing proctodeal feeding has re­

ported that tactile stimulation by the recipient causes the release oi

the defecation reflex on the part of the donor whereby a portion of the

contents of the hindgut is voided. Observations in the present investi­

gation have suggested that the C. brevis recipient plays a more vigorous

role in proctodeal feeding than is implied in Grass~'s description.

Sometimes it appeared that the donor was trying to move away. but

that the recipient was actually restraining him. jiggling him about with 54 vigor of the feeding. The end of the feeding was usually signalled by a jerk or a "plucking" motion of the recipient's head, as though re­ leasing a definite hold.

In the present investigation it was hypothesized that the amount of radioactivity transferred would be correlated with feeding "performance", which might be recipient preference or donor preference or both. In general, however, it appeared that any preferences involved in a feed­ ing relationship were primarily on the part of the recipients, the donors playing a relatively passive role.

Scope

A comprehensive study of possible feeding relationships between castes, sexes, and instars would call for a study of food transmission between both sexes of all instars of all castes. It would necessitate the precise identification of each experimental termite, requiring more basic information than is at present available for f. brevis. Therefore the choice of types of individuals whose feeding relationships were to be studied in the feeding experiments was guided by practical considera­ tions.

Feeding relationships between castes. Food transfer between

castes was the first relationship considered. Caste determination at the

adult level is, of course, easy. C. brevis, like other species in the family 55

Kalotermitidae has no true worker caste. The work of the colony is performed by the immature stages of the reproductive and soldier castes. Adults of both are easily distinguished from each other" and from all nymphal stag,::s (See Figure 1). The most obvious characteristics of the primary reproductive are the dark-brown pigmentation of its entire body and the presence of wings, or, if the latter have been shed, of wing bases. Secondary reproductives, which arise from older nymphs when primary reproductives are lacking, are yellowish-tan and usually have small wing pads. The soldier is easily identified by its dark, enlarged, and trUi""Lcate head, its light body, and its lack of wings or of wing pads. Nymphs are uniformly light in color except for pigmentation on the mandibles.

The presence and size of wing pads depend primarily on age.

The primary reproductives available at the beginning of the experiments were new dealates ready to begin colonies of their own.

When two such primaries were placed in a wooden chamber together with nymphs or soldiers, both the latter types were severely bitten or even killed by the reproductives. In nature, of course, new dealates are not found in an established colony. Because of this fact, plus the difficulty of Obtaining a ready supply of old established kings and queens and of supplementaries, the idea of having adult reproductives in the initial feeding experiments was discarded. 56

In an old established colony there are, bsides the royal pair, nymphs in all stages and a number of soldiers. A study of the feeding relationships between nymph and nymph, soldier and nymph, and soldier and soldier seemed a practical first step and represented the caste relationship aspect of the feeding experiments.

Feeding Relationships Between Instars. The next con­

sideration was the feeding relationship between instars.

The details of the life history of~. brevis have not been com­

pletely determined. The number of instar s through which a

typical nymph passes in order to become an alate, for example,

is unknown, though the number probably lies between six

and eight. In these lower termites the number of molts is

not limited but is indefinite in number (Weesner, 1960). It

was impossible, therefore, to identify with certainty the exact

instar represented by a given indlvidual except for the first

four (distinguished by head size), the penultimate primary

nymph (with swollen wing pads), and the adult of each caste.

Wolcott (1948) has pointed out the difficulties of instar determi­

nation for the species.

Since I was unable to identify exactly the instar s beyond

the fourth(and even the fourth is uncertain), I decided to use

only nymphs falling into two size groupings: 1) small nymphs 57

apparently in the 3rd and 4th instars and 2) large

nymphs weighing about four times as much as the

small ones. If feeding relationships based on

instarexist I felt that the use of these widely

differing sizes of nymphs, representing early

c.nd late instars, would permit them to appear.

Feeding Relationships Between Sexes. The

remaining feeding relationship to be studied was

that based on sex, requiring a determination by

external examination of the sex of each type of

termite selected.

The external characteristics used for sex

identification in termites concern primarily the

last few abdominal sterna and their appendages.

The presence or apparent absence o~ these struc­

tures and their variations in a given sex depend

upon the species concerned. One of the best

descriptions of external sex morphology in

termites has been given by Imms (1919) for

Archotermo psis wroughtoni Desneux. Sumner

(1933) has studied similarly Zootermopsis

angusticollis Hagen, and other authorshave 58 mentioned external sex characteristics (chiefly for primary reproductives) for other species.

In determining the specific characteristics for

C. brevis, I used these earlier works as a starting point. The study has been reported e1sewher e (McMahan).

A typical termite has ten abdominal terga and nine sterna (the first sternum is atrophied).

Anal cerci are always present, arising from beneath the tenth tergum, and subanal styles a.re often present on the ninth sternum. Figure 5 is a lateral view of the abdomen of a C. brevis male showing the arrangement of terga, sterna, subana1 styles, and cerci.

Reproductives. The sex of primary repro­

ductives is easily distinguished along the

lines described by Imms and Sumner. In

the male, sterna 2- 9 are simple plates while

sternum 10 is divided into two subanal

plates. Subanal styles are present. The

female appears to possess fewer sterna than 59 the male due to the fact that the eighth and ninth are hidden by the much-enlarged seventh.

The tenth is divided as in the male. There are no subanal styles. Figure 5b and c show the ventral aspect of male and female abdominal apices.

Supplementary reproduc.tives have these

same distinguishing characteristics, as have also the penultimate instars .

Soldiers. Male C. brevis soldiers can be distinguished easily from female soldiers by the presence of subanal styles, which the

females lack. The sexes may also be differ61tiated on the basis of the shape of sternum 8. In females (Figure 5d) the posterior margin is

concave, making it appea.r as two lateral plates

posterior to and partially covered by sternum 7.

In males the margin is straight.

Nymphs. In the nymphs, subanal styles do not help to identify sex as they do in adults.

Both male and female nymphs with the exception

of females in the penultimate instar possess them. 60 Intersegmental membrane

T9 TIO V-...... -/.jE;:;~ cereus $/0 styles sternum 2 55

a. Ref

IUb-ancJl style Aternull 10 ~.,....~ ~ercus

S7

S6

b. Rei' c. R$

sub-anal style tergum 10 sternum 10 sternum 10 _ •..--"'"<:"00. ~:::cc~ cercus

57

56 56

a. A d. S~ e. N¥

Fig. 5. External sex characteristics of C. brevis termites. a. Lateral view of abdomen of male primary reproductive. b, c, d, e, Ventral views of abdominal apices of male primary reproductive, female primary reproductive, female soldier, and female nymph, respectively. 61

Sex in nymphs must be distinguished on the

basis of the posterior marginal contour of

sternum 8. In female nymphs, as in female

soldiers, the margin is notched, the degree

increasing with maturity. With every molt

the eighth sternum becomes more completel y

overgrown by an enlarging seventh sternum

under which it can be telescoped (Figure 5e).

In older females only a lobe of the eighth is

visible on each side, as in the female soldier,

Male nymphs exhibit a straight margin on the

eighth sternum like male primaries and male

soldiers.

Difficulties Encountered. Several experimental difficulties were recognized from the beginning. One was that paired indivi­ duals were able to exert no choice in their solicitation of food. Each had to feed from its partner if it solicited food at all. Providing a choice by allowing a cold termite to have two radioactive partners or a hot termite to have two cold partners would have complicated matters, since all three would have had solicitation access to each other, possibly feeding. in all combinations in a chain type of reaction and hopeles sly confusing the transfer picture. It seemed best to 62 work with pairs only, insuring that all the radioactivity picked

up by the cold termite came originally from its one partner.

Another difficulty was based on the caste difference in self­

feeding ability. Soldiers, unlik.e nymphs: are unable to scrape

food from termitary walls because of the modification of their mandibles for colony defense and must therefore be fed. One

aspect of the present work gave added evidence for this depen­

dence of soldiers upon colony mates for food. This was that

donor soldiers could be made radioactive only by placing them

with hot nymphs who fed them. Therefore their radioactivity was

always secondhand.

A third difficulty concerned differences in gut capacities of

termites of different types with corresponding effects on amounts

of radioactivity transferred in the various partnerships.

Equipment and Apparatus

The feeding experiments which involved the use of a radioisotope

were carried out at the Hawaii Marine Laboratory between August, 1959

and January, 1960, using the facilities there under the supervision of

Dr. S. J. Townsley, research director of AEC project No. AT(04-3)-56.

Isotope

The radioisotope selected for the experiment was strontium

85 in the form of carrier-free Sr C 12' Its selection was due partly to the 63

Terluitaries

In an attempt to keep the termites in a relatively natural environ­ ment during the experiments, wooden termitaries were constructed of short lengths of ash doweling. The doweling used was either 1/2 inch or 3/8 inch in diameter. The center of a length of doweling was cut out on a wood lathe to a depth of from 1/2 to 3/4 inches and then that section was cut off in such a way: as to produce a small wooden 64 cup. The use of dowels with a diameter of 1/2 inch or less allowed the termitaries to fit into the shell vials used in the scintillation well, thereby permitting measurement of the radioactivity of an entire termitary if desired.

All the experimental termitaries were similar, but some were made radioactive and some not, depending upon their use. The method of closing them also varied. The radioactive te~ mitaries had lids either of wood veneer or of flexible plastic. An insect pin driven through the edge of the lid and into the wall of the cup, pro­ vided a ll swinging door. 11 The pin served also as a handle for ease in removing the termitary f.rom its container vial whenever termites were to be inserted or removed. The non-radioactive termitaries were closed by means of small corks with insect pins for handles.

Figure 6 is a diagram of radioactive and non-radioactive termi­ taries.

Three different sets of radioactive termitaries for the production of donor termites were used in the course of the experiments. The first set. prepared in August, 1959, consisted of seven termitaries, each 1 inch high and 1/2 inch in diameter. Set 2, prepCt.t'~d in Septem­ ber, consisted of nine termitaries exactly like those of Set 1. Set 3, prepared in November, consisted of ten slightly larger termitaries

(1-1/2 inches high and 1/2 inch in diameter). 65 ____insect pin .",- - ~ :;=:.; glass --= vial- ~ I ~

plastic --- lid 3S Sr _.__ cork wooden 'S~t3 termitary- -- ./ ~

a b c

Fig. 6. Termitaries used in feeding experiments. a. Radioactive termitary used at Stage A, lid open. b. Radioactive termitary inside glass vial, lid closed. c. Nonradioactive termitary used at Stage B. Actual size.

Set 1. The isotope solutions used in preparing the first

and second sets were made up by Mrs. Della Reid, Junior Marine

Biologist at tlJ.e Hawaii Marine Laboratory. For Set 1 she pre­

pared a 10 ml. radioactive solution of Sr85 in distilled water from

the Sr85 available in laboratory stocks. One termitary was

soaked in the solution from August 5 to August 9. On. August 19

the remainder of the solution was poured into the six other

termitaries, each of which had been placed in a relatively close-

fitting shell vial. The wooden lids were then closed and the termi-

taries carefully inverted to wet the lids. The vials which contained the

termitaries with the Sr85 solution were then placed in a warming 66 oven at llOoC to evaporate the water.

The termitaries were later counted in the scintillation well in order to get an estimation of their radioactivity. Because of their high count rate the scaler could not handle them at the 10- volt window width or. the gain setting proper for the isotope.

Hence both settings were reduced. By counting standard Sr85 samples at the altered settings and at the proper settings and using the data to correct the termitary reading, it was possible to get an idea of the range of radioactivity exhibited by the seven termitaries. They varied from 4.5 x 106 counts per minute (for the lone termitary prepared on August 5) to a range of from approximately 6.4 x 105 to 1. 7 x 106 for those prepared on August 19.

The differences in radioactivity apparently were a result of slight size differences and of absorption of varying amounts of the radio­ isotopic solution. Experiments 1. 2. 3, and 5 were carried out using this fir st set of radioactive termitaries.

Set 2. On September 23 the second set of tern:litaries were pre­ pared. Because all the stock Sr85 in the laboratory had been ex­ hausted by the previous set, Mrs. Reid pooled all the isotope solutions available from completed research projects and made up another 10 m1. Sr85 solution. This was poured into nine newly made termitaries similar in every way to those of Set 1. Again 67 the water. was evaporated in the warming oven.

The counts per minette for these termitaries ranged from 5 5 1. 8 x 10 to 2.2 x 10 , giving a lower average rate than that

£01' Set 1. The Set 2 termitaries were used in Experiment 4.

Set 3. By October 15, 1959, decay had lowered the radio- activity of most of the termitaries to a level that was too low for effective tracing of food from donor to recipient. A new supply of Sr8"~ was therefore ordered, and on November 3, soon after its arrival, the ten termitaries in Set 3 were soaked in the strontium solution. The amount of radioactivity taken up by each was in this case more accurately controlled. The laboratory assay for the isotope was listed as 201)-lc.

This was diluted to a total of 10 ml. (20.l pc/ml.), and 0.8 mJ.. of the solution was pipetted into each termitar y, which was con- tained in a vial. Since the disintegrations per minute for l'p c 6 equals 2.22 x 10 and there wer e approximately l6,.u c in each termitary, the disintegrations in each were at the rate of nearl y

6 35 x 10 per minute. Not all this radioactivity was confined to the termitary however. After the O. 8 m!. of solution had been pipetted into a given termitary the pipette was rinsed by filling it four times with distilled water and emptying it into the termi- tary. This filled the cup to the brim and spilled over slightly, 68

resulting in a film of solution between the termitary and

the glass walls of the vial. Some of the radioactivity put

into the termitary, therefore, adhered to the vial when the

termitaries were later dried in the warming oven. When

this third set of termitaries were counted in the scintilla-

tion well they were found to be considerably more radio­ 6 active than Sets 1 and 2, varying between 5. 1 x 10 and 6 9.5 x 10 counts per minute. They were of course some-

what 1arger . Experiments 6 to 8 utilized this third set.

Table X summarizes the termitary data.

TABLE X

Radioactive Termitaries Used in Feeding Experiments

Prepa- No. Termitary Counts ration Termi- S i z e Per Minute Set date tad es Length Diam. Lowest Highest Average 6 1 Aug. 5 1 1 in. 1/2 in. 4.5 x 10 5 6 6 Aug. 19 6 1 1/2 6.4 x 10 2.6 x 10 1. 7 x 10 5 5 5 2 Sept. 23 9 1 1/2 1. 8 x 10 2.2 x 10 2. 1 x 10 6 6 6 3 Nov. 3 10 1-1/2 1/2 5. 1 x 10 9.5 x 10 8.0 x 10

Individual termites were handled with the aid of a camel's

hair brush in order to avoid injury. 69

Records of termite radioactivity were entered on

mimeographed sheets.

Experimental Procedure

In the descriptions and discussions of the feeding experiments to·follow ~ designates a large nymph, E. designates a small nymph, and ~ designates a soldier. The conventional sex symbols are used in combination with the letters in identifying a particular experimental termite.

Seven experiments were carried out according to the same basic plan, aimed at elucidating feeding relationships among six types of termites: N6' ,~~ ,r::..d' ,r::..~ ,scI' ,S!f An eighth experi- ment was similar except for features to be explained later. The originally radioactive member of a given pair is called the "donor 11 and the nonradioactive partner the "recipient". It is to be under­ stood of course that these terms are used merely for ease in designation, for the "donor" presumably could feed from the "reci­ pient" as well.

~ach experiment consisted of three stages, each stage occurring a week later than its predecessor. Stages herein refer only to the one-day periods, a week apart, when experimental manipulation of the termites occurred. The week-long intervals between these stages, 70 during which the termites were left to feed unmolested in their termi­

taries, will be called "Feeding Period A" and "Feeding Period B. II

A brief summary of the main features of these stages and feeding periods precedes their detailed discussion.

Stage A: Experimental termites selected, segregated

into types, and weighed. Half (donors) placed

in radioactive termitaries, half (recipients)

in nonradioactive termitaries.

Feeding Period A: Termites left undisturbed

in their respective termitaries for one week.

Stage B: Radioactivity of donor termites counted in

scintillation well. Donors paired with reci­

pients in cold termitaries.

Feeding Period B: Termite pairs left undis­

turbed in their respective termitaries for one

week.

Stage C: Both donor and recipient termites counted in

scintLllation well. Discarded.

Prior to Stage A a supply of C. brevis individuals was obtained, usually from a piece of infested furniture that had been kept intact un­ til one or two days before the beginning of a particular experiment.

The termites were collected from the furniture (typically of plywood 71 construction} by prying apart the plywood sheets or by breaking apart the pieces. Many termites were killed or injured during the process.

The survivors were placed on paper towels in a covered plastic box

11 11 or holding chamber measuring 8 x 5 X 4" and set aside for one or two days. This delay permitted the injured to die and gave the survi­

ll vors an lIadjustment interval. Usually two to six thousand nymphs of all sizes and from 20 to 60 soldier s were collected on a given occasion.

The original experimental plan called for only 72 termites, (12 each of the 6 types -- ~6' ,N~ ,nanimals, the low soldier ratio was in every case the limiting factor, for each experiment required the same number of soldiers as large or small nymphs.

Stage A:

On the day of beginning an experiment the first task

was the selection of experimental animals and their

separation into types. First, all the soldiers still

alive in the holding chamber were isolated. Next the

two groups of nymphs were selected according to

size. Only those nymphs were selected for a given

group that appeared on microscopic examination to 72 be active, the same size, and (in the case of the small nymphs) in the same instar. Large nymphs were chosen which had only slightly developed wing pads.

The selected termites were next segregated accord­ ing to sex. This required close microscopic examination, especially of the small nymphs, and the use of an I'immobilization chalnber ll constructed of two hinged microscope slides, thin wooden marginal strips, and a gauze backing on one side (See Figure 7).

The design for this device grew out of a conversa- tion with Professor Paul lUg and is similar to those used by Light and his coworkers. The termite was placed ventral side down on the glass panel, and the gauze panel was gently lowered. The wooden strips prevented crushing and the gauze gently held the termite immobile against the glass. The chamber was then inverted for observation under the binocular microscope. The external characteristics used in sex identification of termites were fairly easily observed when the termite was thus held motionless. Sexing was continued until about 132 termites had been 73 masking tape holding gauze I I gauze-lined "roof" bounded by wooden stri I f hinge of I masking I I tape I I I marginal wooden strips I I I ,I ======~- -- ~ L_ I _.1 I I _.J ~~~~!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!~==!

Fig. 7. Immobilization chamber for examining termites; constructed of two glass slides, thin wooden marginal strips, three thicknesses of gauze, and masking tape. Termite is placed in the center -of the bottom slide. and gauze-covered slide is lowered into position. Gauze holds termite snugly but gently against the bottom slide. Wooden strips prevent crushing. Chamber is then inverted and termite examined under dissecting microscope. Actual size.

selected. proportioned typically as follows:

IBNd' , IB~9, 24~cr, 24~~, 24.§6', and

24 S¥ . Sometimes a smaller number of soldiers

were available for use. These 132 termites repre-

sented the total number from which the 72 experimental

animals and their alternates were to be drawn for a

given experiment. Half were to be made radioactive

donors and the rest were to be used as recipient

partners. 74

The next step was the weighing of the termites.

They were weighed in 12 groups, six representing prospective donors and six prospective recipients.

A "group" consisted of individuals of one type. The six donor groups were constituted typically as follows:

9~o", 9~~, 12~d', 12!!~, 12ed" , and12S~

The six recipient groups were of similar size.

The 9 or 12 individuals of one type wer e wei ghed together in a stainless steel planchet on a Christian

Becker chain-o-matic scale, and the average weight of the individuals was then computed.

Since the termites comprising a particu.lar group had been selected for equality of size (and insofar as possible, identity of instar ) it was hoped that this procedure of getting average weight would yield fairly accurate information. A check. was made for one experiment by weighing each individual of a group separately and then comparing the weights wi th the average obtained from a weighing of the total group.

Nine large male nymphs together weighed a total of

81. 1 mg. or an average of 9.01 mg. each. Indivi­ dually they weighed 10.5, 10.2, 10.0, 9.5, 9.4, 75

9.3, 9.1 and 9.1 mg. The sum of these weights is 86.6 mg., indicating an increment error of

5. 5 mg. connected with separate weighings.

When the nine separate weights are corrected

(assuming an increment error of .6 mg. for each) they become 9.9, 9.6, 9.4. 8.9. 8.9, 8.8, 8.7,

8.5 and 8.5, with an average of 9.02 ± .5.

This is nearly identical with the average weight obtained from the group weighing. Similar re­ sults were obtained in the other instances in which group members were weighed together and separately. In spite of variations within a group, exceeding in a few cases, 10 percent, there was never an instance in which a small nymph's weight overlapped that of a large nymph. The average weights of the large nymphs varied from 9.50 mg. in Experiment 1 to 5. 83 mg. in Experiment 6, those of the small nymphs, from 3.42 mg. in

Experiment 1 to 1. 50 mg. in Experiment 7. Al­ together, the large nymphs averaged 8.02 mg. and the small nymphs 2.32 mg. Soldier weights ranged from 8.80 mg. in Experiment 8 to 4.60 mg. in 76

Experiment 2 and average 6.81 mg.

It will be seen that the weights of a given termite type varied fairly widely from experiment to experi­ ment. The particular size used in each experiment was primarily dependent upon the size that was ob­ tainable in sufficient abundance to provide a proper supply of experimental animals. The necessity of obtaining approximately 24 individuals of each of the

6 experimental types dictated the selection, for example, of third instar small nymphs for one experi­ ment and fourth instar nymphs for another.

Because of the relative scarcity of soldiers, it was usually necessary to use all healthy individuals available, regardless of size. Soldier s obtained from one item of furniture were usually found to be of equivalent size. Those taken from different sources, however, sometimes varied greatly, indicating per­ haps a nutritional element controlling size or the production of soldiers from different instars. Other workers have found that in general the older the c.o1ony the larger the soldiers . (Weesner, 19.60).

Whenever soldiers from such varying sources had to 77 be used in a single experiment they were weighed and evaluated according to the source.

After all the experimental termites had been weighed they were placed in termitaries. those to become donors in radioactive termitaries. those to become recipients in non-radioactive ones. In order to keep the sexes easily identifiable for later convenience in pairing, all the termites put into a particular termitary were of the same sex. The one experimental exception was the soldiers isolated togeth€l" in one radioactive termitary for testing self-feeding ability. This was not part of the study of intra-colony feeding relationships but was carried out conconlitantly. Here the sex ratio in the termi- tary was usually 1: 1.

The radioactive termitaries were filled first. The following schedule of termitary make-up was typical for the seven radioactive termitaries comprising Set 1. used in Experiments 1. 2. 3, and 5. 78

Radioactive Termitary IIColony" make up

#1 6 Nd' , 2S&' 2 6 ~~ , 2 S~ 3 9 n~ 2 Sd' 4 9 n~ , 2 S~ 4 Sd' , 4S~ 6 3 Nt!' , 2 Sd" 7 3 ~~ , 2S~

):CTermitary #5 represents the one in which soldiers were isolated; their number depended upon the number available.

A similar schedule was followed for the termi- taries of Set 2 (Experiment 4) and Set 3 (Experi- ments 6 - 8).

As each termitary was filled it was closed and returned to its vial which fitted into a plastic box kept behind a lead brick screen in the laboratory.

The non-radioactive termitaries were next filled with the remaining experimental termites, accord- ing to a schedule similar to the one given above for the radioactive termitaries, and set aside in their vials.

The termites were allowed to feed undisturbed for one week. Those in the hot termitaries were to 79

provide the 36 donor termites to be used one week

later in the final stages of the experiment. From the

cold batch would come the recipients which were to

be paired with the donors. Because both donors and

recipients had been taken from the same furniture

source, had 'been treated similarly through Stage A

and Feeding Period A, and were housed in termitaries

made from the same doweling, it was hoped that when

the termites were paired at Stage B after the week of

separation they would accept each other without the

antagonism that is characteristic of termites from

different colonies.

Stage B:

Exactly one week after the experimental termites had

been placed in their respective termitaries they were

again examined. On this day the 36 radioactive donors

to be used in the food-transfer portion of the experiment

were selected on the basis of their apparent vigor and

degree of radioactivity. Similarly, the 36 most lively

recipients were selected from the occupants of the cold

termitaries. Pairing of donors and recipients was to

follow a schedule that provided for one example of every 80 possible combination of the six radioactive and six non-radioactive types, a grand total of 36 pairs. Table

XI on page 81 shows the 36 pairings for a typical experiment. The originally radioactive member of the pair is always on the left. The numbers indicate the number of the Stage B termitary in which a given pair was placed.

The first step in setting up the pairings was the numbering of three dozen new cold wooden termi­ taries from 1 to 36. Into them were transferred the most vigorous of the recipient termites, one to a numbered termitary, following exactly the schedule indicated in Table XI. The extra te~mites were dis­ carded.

The next step was the counting in the scintillation well of every live donor termite from the radioactive termitaries. All the counts for Stage B were made on the same day and this was true also for Stage C.

At the beginning of each counting session and again after approximately every tenth termite, a standard sample containing a known amount of Sr85 was counted.

This constituted a check on the functioning of the .- .. ------

TABLE XI

Pairing Schedule for Hot and Cold Termites

Donors Recipients (non-radioactive) (radio- active) Ncr N~ nc! n~ sc1' S~ 1 7 1j 1'1/ 2:> 31 Nf! Nc1 Nd' N6' N~ Ncr nd' Ncr n~ Nc! sc! Ne5 S~ 2 8 14 2~ ~ 26 3~ ~ N~ N+ Ncf' N~ N~ N~ nd' n~ N~ sJ' S~

::s 9 15 21 27 33 nd' nc! Ncr nd' N~ nd" nc? n6 n~ nc:l' S6 nc? S~

16 28 '* 0 1~ 2~ ~ 34+ n~ n+ Ncf + N~ n~ ncf n~ n~ sci' n S~

5 II. 17 23 29 35 sci' sci' Nd' sc:?' N~ sci' nci' sc? n+ sci' sci' sif' S~

6 ~.2" 18 24 30 36 S+ S~ Nc:f' S~ N~ S~ nc1' S~ n+ s+ sc!' S+ st ex>..... 82 counting equipment and permitted comparisons to be made of data obtained on different days. Immediately after every standard count, an empty vial was counted in order to get a measure of background radiation for later data correction.

The donor termites were placed separately in clean vials which were inserted in turn in the scintilla- tion well for counting. Usually the scaler was set for a five-minute count for each. As soon as the count was recorded the termite was placed in an appropriate cold termitary already occupied by a recipient, according to the table on page 81. On the record sheet beside the counts recorded for the donor termite was written the number of the termitary in which it was subsequently placed. Whenever the counts per minute seemed unusually low, as though the termite had failed to feed as much as the others during its week in the radioactive termitary, or if the termite seemed sluggish, it was usually discard'2d. In some cases, however, it was necessary to use a terinite with a low count in order to have enough of a given type for the experiment. 83

It was not always possible to follow the pairing chart exactly in placing donor and recipient termite.s.

Frequently the schedule had to be altered for soldiers and sometimes for small nymphs because of their tendency to die. In such cases a nymph was substituted whenever possible, and a careful note was kept of all such substitutions.

Soldiers in the self-feeding experiment that had been isolated together in a hot termitary were counted individually and then paired with nymphs that were either already radioactive or had acces s to radio­ active wood. This served as a form of control on the soldiers' ability to become radi oactive under "normal" circumstances.

The procedure just described in Stage B was followed almost exactly in Experiments 1 - 7. Experi­ ment 8 was different only in the fact that the donor­ recipient pairs were placed not in wooden termitaries but in numbered glass vials lined at the bottom with fine nylon mesh to provide footing for the termites. This deprived them of food except that solicited from each other, and put soldiers on an equal basis with nymphs 84

from that point of view.

Stage C:

On the third week the 36 termitaries were opened

one by one, and the two occupants of each were placed

separately in clean vials for counting. In Experi­

ments 5 - 8 the fecal pellets that had accumulated

during the week were also removed from each termi­

taryand counted together, or the entire termitary

containing the pellets was counted. This permitted

a checl<. on the disposition of all the radioactivity

brought into a particular termitary by the donor, and

an analysis of rate of loss of radioactivity through

defecation.

Both termites from a given termitary, whether

dead or alive, were listed separately by type and sex

and counted in the scintillation well. A note beside

each indicated its state of vigor and whether or not

the individual had apparently molted during the preced­

ing week.

Molting information is important in assessing

feeding behavior; hence, the criteria for determining

whether or not a molt had recently occurred should be 85

explained. Positive proof was the presence in a

termitary of an exuvia, but unless two skins were

present or unless one of the termite partners was

a soldier (which could not molt), a decision still

had to be made as to which partner had shed its skin.

If the pair consisted of a large and a small nYmph,

exuvia size was a sufficient clue. When two small

nYmphs or two large nYmphs weI' e paired other

criteria were used. Newly molted small nymphs are

ordinarily whiter than are those not recently molted.

This is true also for large nymphs unless, as was

usually the case, a molt transformed the individual

into a supplementary reproductive. When this

happened, the yellow-tan color and, in the case of

females, the modified terminal sterna permitted no

doubt that molting had occurred.

It seems likely that most newly molted individuals

were detected. There is nevertheless the possibility

that a few es~aped notice. It is known that termites

sometimes eat the shed skin thereby. removing this

evidence. Collins (1959, p. 347) noted, however, that

"unlike other species of termites. Cl'yptotermes brevis 86

individuals do not seem to eat the exuviae cast

during an experiment or in the culture containers. 'I

My observations in general tend to support this view.

Almost invariably one was found in each termitary

containing a new supplementary reproductive,

instances in which molting was doubly proved. Never­

theless, I have also seen nymphs kept in an observation

chamber devour the skin of a just-molted colony mate.

The possibility remains, therefore, that in a few

cases in the feeding experiments the exuvia may have

been eaten and the molted nymph looked enough like

the non-molted to escape special notice.

At Stage C the soldiers in the self-feeding experi­

ment that had been isolated together at Stage A and

then paired with hot nymphs at Stage B were recounted

to see if their radioactivity had increased in the interval.

This ended a given experiment, and all the experi­

mental termites were then disposed of.

Reliability of Figures

Before the results of the feeding experiments are presented, an attempt should be made to assess the reliability of the figures upon which the analyses are based. 87

The primary data consist of counts -per -minute values which are a measure of disintegrations per minute of the strontium 85 present in the individually labelled termites.

The occurrence of nuclear disintegrations is, of course, a random phenomenon, and therefore the number of scintilla­ tions recorded per minute for a given sample will not be uniform from one determination to another. Accuracy in measuring the Ittrue disintegration ratell is proportional to the length of the counting interval. Furthermore, the gr eater the number of counts within a given interval (i. e., the "hotter II the sample), the smaller the likelihood of random error. The experimenter must choose, therefore, a counting interval that is small enough to be practicable from the stand­ point of time involved and yet long enough to give reasonable assurance of reprodueibilit,y. Nomograms, graphs, and other aids have been published to help determine adequate counting intervals for samples whose degrees of radioactivity vary.

In the feeding experiments a five-minute counting interval usually was chosen, partially because the scaler is based upon a circuit of preset time, but mostly because of time restric­ tions. Some idea of the amount of random variation 88

to be expected in the data can be obtained from a considera-

don of the expected error s for five -minute counts of selected

samples with representative amounts of radioactivity. Table

XII shows the error in counts per minute expected for samples

with counting rates of la, 100, and 1, 000 counts per minute

and gives the percentage error for each. (These figures were

derived from the nomogram on page 208 of Aronoff's Techniques

of Radiochemistry).

TABLE XII

Errors at the 95 percent Confidence Level for Five-Minute Counts of Repr esentative Samples

Counts per Random Sample Minute Error %

1 10 2.7 27.0

2 100 9.5 9.. 5

3 1000 21. a 2.1

Theoretically, samples with counting rates above 100 cpm

are not likely to vary more than 10 percent in separate five-

minute counts. In feeding experiments 1-7, the live donor

ter".. tites at Stage C averaged 754 counts per minute, varying

according to termite type (see Table XIII), but the live 89 recipients (with live partners) averaged only 61 cpm. The comparable figures for Experiment 8 were higher (see Table XIV).

It would have been preferable, of course, to have used a longer counting interval for termites with counting rates below 100 cpm, but the five-minute interval was chosen primarily in order to per- mit a stage of a given experiment to be completed in one day.

Even so, the average time needed to complete a stage was 14 hours.

It has been mentioned already that l} a vial containing a standard 85 Sr sample and 2) an empty vial for measilring background radiation were counted at intervals during a day1s work. The data obtained from these two vials may be used to estimate the amount of actual random variation within the experimental data at any given instance, because their characteristics were known quantities.

Both the standard and the background vials were usually counted six times or more during a single counting session. The back- ground data will be considered first.

Background counting error. A total of 102 separate background

counts were made during the course of the eight feeding experi-

ments, 78 for five-minute intervals, 3 for ten-minute inter-

vals, 19 for fifteen-minute intervals, and 1 each for thirty-

and sixty-minute intervals. The average number of counts

obtained for the five-minute interval (representing most of 90 the data) was 74 with a standard deviation of 8. 41. The fifteen- minute average count was 220 .±. 20. 2.

All the experimental data used in the analysis have been corrected for background radiation. In making these correc­ tions the figure representing the average background counts per minute, computed from all the background counts for a particular day, was subtracted from every individual termite count obtained for that day. There was a total of 16 counting days, representing Stages Band C for each of the eight feeding experiments - - hence, 16 average background figures.

Each average was rounded off to the nearest whole number

before being subtracted from the d ay l s sample counts, which were similarly rounded off. The composite average for the

16 rounded-off background figur es was 15 cpm .±. l.

It may be concluded that the error in deviation introduced into the experimental data through the use of background cor­ rections averages about 1 count per minute for any given sample.

Machine counting error. The chief purpose of

the standard Sr85 vial was to keep a check on the scaler

and analyzer to see that their functioning did not vary

excessively during the experiments and to permit

corrections to be made for such variations. The standard

data were also used in correcting for radioactive 91 decay. In the analyses the experimental data of Stages B and C were made comparable for each experiment by multi:. plying each individual datum of Stage C (already corrected f or background) by a correction constant ?btained by divid- irig the average standard cpm figure for Stage B by that for

Stage C. If, for example, the average cpm for the Stage B standards was 48 and that for the Stage C standards was 44, the correction constant was 48 =1.0909. 44

Two different standard vials were used: one, with low radioactivity (average cpm = 46 ±. 8), was used in Experiments

1 - 5; the other, with relatively high radioactivity (average cpm = 1608 + 33), was used in Experiments 6 - 8. Standard

#1 was counted 59 times (47 times for five-minute intervals and 12 times for ten minutes or more), and Standard #2 was counted 38 times (each for a five-minute interval), a total of 97 separate counts. It is unfortunate that the low-count

standard ,"vas used. Its random disintegration variability was

so' great as to disguise any machine variability. In consequence, the correction constants derived from the use of this standard may have caused error s as high as 20 percent. The correc- tion constants based on the high-count standard, on th.e . other hand, were off by not more than 2 percent. This is the 92 approximate amount of variation expected due to random disintegration fluctuations, indicating that any variation due to machine fluctuation was negligible.

In spite of the possibility of a large amount of err'or m determining accurately the constants used in correcting the

Stage C data for Experiments I- 5, in which Standard #1 was used, comparisons between types of termites can still be made with a fair degree of confidence. Each experiment was a complete unit in itself, with all termite types and all feeding combinations represented equally, insofar as possible. (Exceptions will be discussed later). If a correc­ tion constant applied to a given day1s figures was too high or too low, it was nevertheless applied to all the data, rais­ ing or lowering every figure, and in no way altering the relative standing of any given termite in the experiment.

One other bit of evidence can be used in helping to decide whether or not the counts per minute utilized in the experimental analyses were reasonably ,accurate indications of the true radioactivity of the labelled termites. If the figures are correct, the sum of the counts-per-minute figures for both members of the termite pair in a given termitary at Stage C, plus the counts obtained from the termitary itself, plus those 93

from the fecal pellets which had accumulated in the termitary

during the week, should give approximately the same figure as

that recorded for the donor alone at Stage B.

In three experiments, 6, 7, and 8, the radioactivity at

Stage C was measured not only for the donor and the recipient

of a particular termitary, but also for the termitary and its

contents. The total figure was compared with the amount of

radioactivity brought into th.e termitary by the donor at Stage B.

The analysis showed that 99 percent of the total amount of

radioactivity brought into all experimental termitaries at

Stage B was accounted for at Stage C. This figure represents

a total of 108 cases. When these cases were considered indi- vidually, and a percentage of radioactivity retention was ob­

tained for each, the result was an average of 98 percent + 13.

The relatively large standard deviation is due in large part to

one very atypical case in which only 18 percent of the original

radioactivity at Stage B (65 cpm) was recovered at Stage C

(12 cpm). If this case is omitted the average percentage re­

covered for the remaining 107 cases is 99 ±. 11.

In addition to the evidence this analysis gives to support the contention of accurate cpm readings used in subsequent

analyses, it also indicates that radioactivity was not lost by the 94

donor (or by the recipient) in any way other than by the

routes considered; i. e .• by food transfer from one

indvidual to another. by the defecation of fecal pellets. and

-by contamination of the termitary through the production of

the brownish material used by termites in sealing off their

chambers.

Results

General Results

1£ the original experimental plan could have been carried out

precisely. with no termite mortality. there would have been 36 donors

and 36 recipients in each of the eight experiments. a total of 288 partner­

ships (8 sets of 36 different donor-recipient combinations; see Table XI).

The lack of available soldiers. however. resulting in the substitution of

nymphs. plus mortality a:mong termites of all types altered the plan

and forced an imbalance in the donor-recipient combinations that were

eventually available for study.

All the data for Experiments I to 7 are summarized in Table XIII,

omitting only ter:mites that died and those whose partners died prior to

the counting day for the particular stage involved. The table shows the

average amounts of radioactivity in counts per minute picked up by the

different types of donors and recipients. 95

TABLE XIII

Radioactivity picked up by Donors and Recipients in Experiments 1 - 7

D 0 N 0 R S RECIPIENTS Type Stage B Stage C Stage C of No. Ave. No. Ave No. Ave. Termite Cases cpm Cases cpm Cases cpm

N~ 55 2287 48 1884 36 65

-N ~ 52 2132 39 1627 35 43 _.ntf 38 581 31 344 32 35

n ~ 41 546 29 306 40 38 -sci' 28 325 20 156 20 130 S~ 31 338 20 208 24 56

Total 245 1035 187 754 187 61

Table XIV presents comparable data for Experiment 8. The data

are presented separately from those of the other seven experi-

ments because, unlike them, no food in the form of wooden termi-

taries was available to any of the termites after Stage B. All food

had to be solicited from each other. On1v two soldier donors were

available for Experiment 8 and no soldier recipients. 96

TABLE XIV

Radioactivity picked up by Donors and Recipients in Experiment 8

D 0 N 0 R S RECIPIENTS Stage B Stage r.... StaR;e C Termite No. Ave. No. Ave. No. Ave Tvpe Cases cpm Cases cpm Cases cpm

-N d' 8 3957 4 1355 6 106 -N ~ 9 4384 9 2072 5 313 143 -n 6' 9 1183 6 397 7 -n !f 8 1355 5 800 7 128

-scf' 1 795 0 -- --- ~~ 1 1953 1 648 - --

Total 36 2271 25 1054 25 173

All the data are lumped together in these tables with no separation

of results for the different donor -recipient combinations upon which

feeding relationship studies were eventually based. Furthermore, the

lumped data do not necessarily indicate accurately the feeding rates

of the different termite types, for the amount of radioactivity taken up

by a given termite depended upon several factors: 1) its general st ate

of health, 2) its stage in a molting cycle, 3) the degree of radioactivity

of its particular food source -- the hot termitary in the case of the

donors and the hot partners in the case of the recipients -- and 4) the

termite I S feeding capacity. This information is not available from the 97 figures given in Tables XIII and XIV.

The rest of the Results section, therefore, will be devoted to more detailed analyses. Because analyses of feeding relationships included only termites that survived the various experimental stages, a dis­ cussion of termite mortality will precede that of other findings.

Termite survival

From the beginning, it seemed on the basis of general obser­ vation that soldiers had a higher mortality rate under the experimental conditions obtaining than had nymphs. An estimated one-third of the number removed from infested furniture died within a day, and many of the ones that survived long enough to be used in the feeding experiments died during the course of the two weeks of tests. Small nymphs seemed next most vulnerable, while large nymphs seemed relatively sturdy. The low soldier ratio' for C. brevis, resulting in a scarcity of soldiers, naturally made their survival of greater importance to the experiments and this conceivably could have colored :any casual comparison of

soldier and nymph survival rates. In order to test the general impression of greater soldier mortality an analysis was carried out in which each of the termites used in Feeding Experiments 1 - 7 was followed from Stage to Stage to see at what point a non-survivor died. Experiment 8 was omitted from this analysis because its experimental conditions were different. 98

Table XV shows the survival figures for all types of donors

(section a) and recipients (section b) separately at the various experi­ mental stages, and then pools the data for Stages Band C (section c).

Of the 389 donor termites placed in the radioactive termitaries at

Stage A, only 263 could be followed all the way to Stage C, two weeks later, after having been paired with recipients for a week. At Stage B only the 245 healthiest and "hottesttl of the survivors of the donor group were selected to be used in the feeding partnerships. An addi­ tional 18 large radioactive nymphs from the donor supply at Stage B were utilized in the Soldier self-feeding experiment, and these have been included in the analysis: making a total of 263 selected donor s.

The sixty-six excess survivors from the donor group at Stage A were discarded at Stage B. The donor data entered in Table XV at Stage B are therefore revised in order to get a true picture of termite survival to Stage C. For example, only 117 out of the 137 large radioactive nymphs surviving from Stage A were selected for use at Stage B, and

111 of the latter survived to Stage C.

Recipients could be followed only from Stage B to Stage C be­ cause no recor ds were kept on the excess individuals that survived out of the total number placed in the cold termitaries at Stage A. The health­ iest were selected and used in setting up partnerships at Stage B, as in the case of the donor termites, and the rest were discarded. TABLE xv

SURVIVAL OF THE DIFFERENT TERMITE TYPES FROM ONE STAGE TO ANOTHER*-

ST A G E A S T A G E B ST A GEC BEY a: D 0 N 0 R S Ii n S N n S liT n S Selected selected Selected Sur- % Sur- % Sur- '/0 vived vived vived (/' 72 81 42 68 94 68 84 29 69 ~ 72 81 41 69 96 64 79 31. 76 Total 144 162 83 137 95 :L32 82 fIJ 72 Selected Selected Selected Sur- % Sur- % Sur- % vived vived vived c! fiJ 41 29 58 97 83 22 76 ~ 57 45 31 53 93 ~ 84 23 74 Total 117 86 w III 95 72 /)4 45 75 b: RECIPIENTS Selected Selected Selected Sur- 'lo Sur- 'Jo Sur- G(.> vived vived vived ci' 41 49 29 41 100 33 67 23 79 ~ 41 48 37 40 98 41 85 27 73 Total /)2 97 66 /)1 99 74 76 50 76 c: DONORS AND RECIPIENTS POOLED Selected Selected pelected Sur- % Sur- % Sur- 'to vived vived vived 101 90 99 9/) 67 74 45 7/) ~I 98 93 ~ 93 95 79 85 50 74 Total 199 1/)3 126 192 96 146 00 95 75

*EXPERIMENT 8 OMI'lTED FROM ANALYSIS.

-.t:l -.t:l 100

Table XV breaks down the data according to termite type. The

survival percentages for a given type are very consistent from one

stage to another. For example, those for Nd" donors are 94 and 97

percent for Stages Band C, respectively, and those for ~ ~ donors are

96 and 93 percent. Other termite types show similar consistencies.

Sex appear s to have had no effect at all on survival. The only figure

that seems to be out of line is that for.:: eft recipients at Stage C. The

67 percent survival figure would seem to lie too low, judging from

comparable figures for the .:: ~ recipients (85 percent) and for

ncr' andE~ donors (79 - 84 percent).

Pooling donors and recipients for Stages Band C (Table XV,

section c) gives an overall picture of termite survival during one week

of experimentation. The survival percentages at Stage C for ~, '::' and S,

respectively, are 96, 80, and 75 percent, paralleling closely the

donor figures for Stage B (95, 82, and 72 percent), and bearing out the

earlier supposition of greater soidier mortality.

A further analysis of the data for Experiments 1 - 7 was made

to see if mortality rate at Stage C was related to the type of partner

with which a given termite was paired at Stage B. Ignoring sex, there

were six types of partnerships, representing combinations of the three

basic termite types used in the feeding experiments: ~ - N, ~ - ~

N- S, n - n, n ,.. S, and S- S. There were 72 cases in which two 101 large nymphs were paired ~ - ~L 69 ~ - n cases, 48 N-S cases,

62 ~ - n cases, 45 ~ - ~ cases, and 32 S-S cases.

Table XVI presents the survival percentages for each type of partner in each type of partnership. No significant survival differences attributable to partner was found. Large nymphs survived well regard­ less of partner; small nymphs gave lower but consistent survival percentages for all partner types; and soldiers survived at still lower, but fairly constant, rates.

Light and lllg (1945) point out that "all investigators who have attempted to use termites as experimental animals have encountered, and most have reported, the occurrence of relatively high mortality in experimental groups.·1 They discuss the causes of such mortality, which range from disease to excessive handling. In the present investi­ gation emphasis is on differences in mortality rate characteristic of the three types of termites used. Since all three were treated similarly, the group differences must have been due to innate factors. TABLE XVI

Termite Survival According to Partner for Experiments 1 to 7

Type of Type Partner- of Individuals at Individuals at Survival ship Partner Stage B Stage C Percentage

N-N N ( 36 ) 72 70 97 N ( 36 )

68 -N-n- -N 69 99 -n 69 57 83

--N-S -N 48 45 94 S- 48 34 71

( 3:1 ) -n-n- n 62 49 79 - ( 3:1 ) -n

36 80 -n -- S -n 45 -S 45 36 80

--S-S S ( 16 ) 32 25 78 -S ( 16 ) Total 490 470 86

,- o N 103

Food Transfer Relationships

One of the major aims of the radioisotope feeding experiments was the clarification of food transfer relationships between different types of termites. It was hoped that the technique of pairing radioactive with nonradioactive termites and subsequently measuring the radioactivity ac­ quired by the latter would help to answer the question of whether or not differential feeding relationships exist between different combinations of sex, caste, and instar. In comparing results for the various types of partnerships the assumption was made that the larger the amount of radioactivity acquired by the recipient by the Stage C check the stronger the indication that a feeding preference was exhibited. Conversely, the smaller the amount transferred, the smaller the likelihood that the parti­ cular feeding combination was a I'preferred" one.

Sources of Variation in the Data. Apart from possible effects

based on constant feeding relationships, the amount of radioactivity

acquired by a given recipient from its donor depended primarily

upon four factors: 1) the general state of vigor of both donor

and recipient, 2) whether or not either was preparing to molt,

3) the degree ,?f radioactivity exhibited by the donor, reflecting

degree of termitary radioactivity during Feeding Period A, and

4) the donor I s size, which determinedits capacity as a food source, 104

and the recipient I s size, which was correlated with its feeding capacity. Also involved was the length of time during which pairing occurred. This was related to the rate of re­ placement of donor radioactive gut content with nonradioactive food, thereby reducing the amount of radioactivity available to the recipient.

The first two factors (general vigor and molting phase) were controlled insofar as possible by selecting only termites that appeared healthy for use in the experiments and by omit­ ting from the analyses all cases in which one or both of the partner s in a combination either died oJ:' molted before the final check-up. (A discussion of the feeding behavior of molt­ ing nymphs is given elsewhere). The possibility remains uncontrolled that some of the nymphs were in the premolting phase but had not yet molted by Stage C. Furthermore, not only was a termite1s initial state of vigor difficult to deter­ mine but it might also have deteriorated during the experiment.

These factors could have accounted for much of the wide variation found in the data.

The second two factors (degree of donor initial radioactivity and termite size variations) may appear to be aspects of the

same problem. The donor IS initial radioactivity is, of course, 105 a function of its size. The point to be stressed in a con­

sideration of factor 3 is the degree of radioactivity of the donor's gut contents, which means the counts per minute per milligram. The gut contents of a donor nymph con­ fined in a termitary that gives 5, 000 cpm per milligram of wood should become more radioactive than those of another donor nymph, equivalent in every way, but confined in a

termitary that gives only 1, 000 cpm per milligram. A

small donor nymph froIn a very hot terInitary might give a

higher counts-per-Ininute figure than a large donor nyInph

froIn ales s radioactive termitary, and their respective

recipients could be expected to reflect these differences.

One Inethod of controlling this factor of differences in

degree of donor radioactivity is to express the cpm trans­

fers in terms of percentage of donor cpm. Such a procedure

would give as Inuch weight, for example, to a transfer of

100 cpm froIn a 400 CpIn donor as to the transfer of 200 CpIn

. froIn a 800 cpm donor, both being 25 percent. This still

. leaves differences in termite size as such to be considered.

The experiInental plan, calling for a comparison of feeding

relationships of different castes and instars, Inade unavoidable

the use of termites of different sizes. Each Ineasurement of 106 radioactivity was accomplished by placing the particular termite in the scintillation well and getting a cpm reading for the entire individual. Large termites would naturally be expected to give higher counts on the average than small nymphs on the ba~is of size aione.

Comar (1955, p. 196) discusses the problems involved in comparing amounts of radioactivity concentrated by animals of differ ent weights. He points out that lIthe con­ centration found in a tissue is a direct function of the dose administered to the biological systemll and recommends that amounts of radioactivity be expressed in terms of per­ centage of dose. This is in line with the procedure already mentioned of expressing recipient cpm as a percentage of donor cpm. In this case the initial Itdosel1 administered to the biological system is represcnted by the donor IS cpm, the "biological systemll itself is the donor-recipient com­ plex, and the litissue" in which the concentration is later measured is represented by the recipient. The dynamics of an inter-termite food exchange r elatio n ship involving the transfer of radioactivity from donor to recipient introduces complications into the results, many of them revolving around effects based on size differences of donor and recipient in the 107

various partnership combinations.

Comar further states that when animals of different sizes

are compared and body weight must be taken into consideration

it is desirable to compensate for such differences through the

use of what is called the "biological concentration coefficient. II

Application of this method to the present data would involve

finding the counts per minute of radioactivity acquired per

milligram for each recipient, computing this figure i s per­

centage of the donor I s tota.l cpm figure, and then multiply-

ing the percentage by the donor's weight in milligrams. In

other wor ds, the transfer of radioactivity in a given partner­

ship would be studied by finding the recipient's cpm per

milligram of body weight and expressing it as a percentage

of the donor I s cpm per milligram of body weight. Such a pro­

cedure aSSUInes that each donor termite's gut is full, that gut

size is directly proportional to body weight, and that all gut

contents show equal counts per minute per milligram. In

the actual feeding experiments these conditions were not

necessarily Inet, but variations in fullness of gut were probably

distributed randomly among all the types of donors compared

and the other factor s could be controlled sufficiently so that

any observed differences in transfer percentages may be inter­

preted as "feeding preferences. 11 108

The basic data, obtained by using the formula

Recipient cpm/mg x 100 will be presented first. The Donor cpm/mg formula was applied to the cpm figures of each of the 152 recipient-donor combinations, and all percentages

:t:'epresenting anyone of the 36 types of combinations were averaged to produce the results of Table XVII. The number of cases represented in each cell is given in the upper left corner, and the average transfer percentage is shown in the center of the cell. The results for a partnership com- posed of a particular donor and a particular recipient type is found by following the appropriate row and column to their interesecting square. These data are automatically corrected for degree of radioactivity of the donor and for termite size. The transfer percentages would doubtless have been greater if recipients could have maintained a stable radioactivity equilibrium with their donors. This of course was impossible because the latter were constantly replacing radioactive gut content with nonradioactive wood.

Rate of replacement determined the rate of decrease in radioactivity available to a recipient. 'Therefore the pairing interval prior to the measurement of recipient radioactivity TABLE XVII

Mean Radioactivity Transfer Percentages for Each Combination of Termite Types

Recipients Total Donors N n ::; Donors d' Q if ~ aT Q 8 8 4 7 6 I 4 37 rf 5.210 2.752 6.573 5.957 7.035 5.550 5.300 N 1--- 6 2 5 7 3 2 25 ~ 4.391 3.393 7.757 10.957 5.568 3.828 6.919 6 i 3 4 4 4 5 26 6' 2.201 2.263 14.767 19.026 5.583 3.968 7.590 n I 4 6 5 3 2 5 25 ~ 2.354 I 2.576 8.517 3.403 7.906 3.205 4.380 4 3 5 i 3 3 2 20 cr 4.842 16. III 14.708 I 2.882 20.481 29.228 13.490 S 3 4 4 4 I 3 19 !F 9.621 13.085 12.809 14.005 1. 258 27.486 14.325 ! Total 31 26 27 28 19 21 152 Recip- 4.480 5.836 10.797 9.621 8.408 9.840 8.012 ients

...... o -.0 110 was important. Although the transfer percentages in the present investigation were doubtless affected by donor loss of radioactivity, the figures probably reflect relative amounts of transfer.

Inspection of the table indicates there are no consistent differences between males and females. The greatest difference is that between the male and female donors of the n - n combination, but an analysis of variance con­ ducted on the data represented by these cells was found to be insignificant. The F value is 2. 10 which corresponds to a probability greater than 0.05. The conclusion reached on the basis of this analysis was that sex had no consistent effect on the amount of radioactivity transferred between donor and recipient in the present experiment. In subse­ quent analyses, therefore, sex was omitted, making the analyses much less complicated.

The radioactivity transfer percentages presented in

Table XVII were computed on the assumption that termite size (as expressed in milligrams of weight) was directly proportional to gut size. It was assumed that a termite weighing for example 4 times the weight of another would have a gut volume also 4 times as great. Dissections of 111

.f.: brevis nymphs and soldiers, however, showed this assumption to be false. Soldiers weighing exactly the same as nymphs had guts that were obviously much smaller in volume. Holmgren (1909) carried out morphological studies showing the same kind of gut size relationships for soldiers and nymphs of a number of termite species, including .!5. £1avicollis of the Kalotermitidae. Katzin and Kirby (1939) found that the weight of the digestive tract of nymphs of Zootermopsis angusticollis and Z. nevadensis is about 1/3 that of the termite while the weight of the gut of the soldier is only about 1/6 to 1/5 of the total. It appears certain, therefore, that the transfer percentages given in Table XVII should be corrected for differences in gut size for the different types of termites.

The problem lay in arriving at proper correction factors.

Assuming that most of a termite's radioactivity is in the gut, and assuming that the various termite types fed comparably, one way of estimating relative gut sizes would be to observe the amounts of radioactivity exhibited by hot termites which have come into equilibrium with their radioactive termitary.Such termites should exhibit radioactivity in amounts proportional to their relative gut 112 sizes. This is presumed to be true of soldiers, which cannot feed directly on wood, as well as of nyInphs, which can, if both soldiers and nymphs are confined together. It is a presumption based on the argument that after termites reach radioactivity equilibrium with their termitary and with each other the radioactivity entering any termite is approximately the same per unit voJ.ume of food as that leaving it as proctodeal food solicited by a termitary partner. This implies that eventually all the food circulating among termites in a given hot termitary is of equal radioactivity per unit volume. The time required for this point to be reached depends upon the length of time it takes:for radioactivity to reach the hind gut and for the contents of the latter to corne into equilibrium with the radioactivity of the food source.

In a test of the time required for radioactivity to be detected in fecal pellets, a large nymph placed in a hot termitary of approximately 5 x 105 cpm/mg produced with­ in 8 hours a fecal pellet that gave 300 cpm. This mayor may not indicate speed of food passage through the gut. The radioactivity may have found its way into the hemolymph 113 and thence into the hind gut by way of the Malpighian tubules. (Radioautographs made in connection with the

present study indicated a concentration of radioactivity in the Malpighian tubu.les). Regardless of m.ode of transfer, radioactivity may become detectable in the hind gut within 8 hours of the termites l feeding on radio­ active wood. Thereafter the proctodeal food provided by such a donor presumably increases in degree of radioactivity until equ.ilibrium is reached.

In order to obtain a ccurate data on which to base gut­

size corrections, it was necessary to find out how long it takes for termites to reach radioactivity equilibrium.

Rate of Radioactivity Gain in a Radioactive Termi­

tary. An experiment was carried out specifically to

find out the rate of gain in radioactivity for termites

confined in a hot termitary (as well as the rate of its

subsequent loss when they were removed). and to see

how long it took for radioactivity equilibrium to occur.

Two hot termitaries,A and B, approximately equal

in degree of radioactivity, were used in the radio­

activity-gain experiment. Into each were placed eight

termites - - a pair of supplementary reproductives, 114 a soldier, three large nymphs, and two small nymphs. Each termite was identifiable and was counted alone in the scintillation well at the same time each day for seven days. Following each counting session the two sets of eight termites were returned to their respective termitaries and left undisturbed until the next day when they were recounted individually. All the cpm figures have been corrected for background, for isotope decay, and for possible machine fluctuations in the way described earlier. Experimental conditions were the same in both termitaries, and the gain trends were very similar j therefore, the results have been pooled for each termite type to show its average gain in radioactivity over the course of the week.

The results, presented in Figure 8 indicate that feeding rate was relatively constant, radioactivity rising fairly steadily until it apparently reached a plateau on about the fifth or sixth day. The Nl s showed a fairly strong dip in radioactivity beginning on the fifth day and the other types showed a similar but less marked drop, the explanation of which is not • Soldiers

3,000 A Supplementary Reproductives

• Large Nymphs o Small Nymphs 2,500

QJ ~ ~ ~ •.-1 2,000 ~ I-l QJ flt (/) ~ ~ 1,500 ~ 0 0

1,000

500

..... i-' 1 2 3 4 5 7 U1 Days in Hot Termitary Fig. 8. Comparison of gain in radioactivity of different types of termites confined together in hot termitaries for one week. 116

known. In order to see how steadily the plateau is maintained one would need to extend this phase of the experiment longer than was done in the present instance.

It appears, however, that radioactivity equilibrium was reached for all termite types by the fifth day.

Relationship of Gut Volume to Body Weight. Figure 8 shows that gut size differences do appear to be reflected in differences in amounts of radioactivity acquired, large nymphs exhibiting more at every point than small nymphs. Soldiers, which weighed nearly as much as the large nymphs and considerably more than the small nymphs gave approximately the same radioactivity measure­ ments as the latter. These data are not sufficient, how­ ever, for determining gut correction factors. Feeding

Experinents I to 8 provide data on which an analysis cf gut size relative to body weight could be made. Experi­ ment 8 could be included because its experimental condi­ tions did not vary at this point from those of Experiments

I to 7.

It will be recalled that at Stage A of the feeding experi­ ments the termites to be used later as donors were placed in hot termitaries to feed on the hot wood during Feeding 117

Period A and to become radioactive. At Stage B the

radioactivity of each of these donor termites was mea­

sured in the scintillation well. These are the data used in the gut-volume analysis.

It was important in the gut-volume analysis that

comparisons of radioactivity be based on cases in which the termite types being compared had had access to food of the same degree of radioactivity. This condition is met in the present instance. Two termite types were

always present in each hot termitary during Feeding

Period A, and sometimes all three types were present

(see schedule on page 78). Comparison of the radio­ activity acquired by different types of donors, all having access to the same hot termitary, was therefore possible.

Cases were omitted in which only termites of a single

type survived in a given termitary.

The unit within which comparisons were made was the hot termitary. In most cases there were unetqual numbers of~, ~ and.E survivors in a single termitary,

so average cpm and average weight figures had to be ascertained for each termite type in each termitary. A total of 30 separate instances of hot termitaries containing 118 at least one survivor of each of two different types was obtained. Eight additional termitaries contained at least one of each of the three types, which meant that in these cases each type average was used twice in the present analysis. For example, the ~cpm for such a termitary was used once in the ~ -n comparison and once in the ~ -§. comparison for that termitary.

The total number. of individuals included in the analysis was 244 representing 112 different ~IS, 82 different n's, and 50 different SiS. The results of the analysis ar e given in Table XVIII. In the table the apparent total number of termites adds up to more than 244, but this is due to the fact that the data for some of the individuals (those in termitaries containing all three types) were used twice, as already explained.

The table pr esents the average weight of each ter- mite type and the average cpm acquired by each in every combination compared within a termitary. Cpm figures are then given in terms of cpm/mg, and finally the ratio of the two cpm/mg. figures is given for each combination, showing the gut size of the fir st termite type relative to that of the second. The table shows that TABLE XVIII

Comparisons o~ Radioactivity Acquired by Donor TYPes Having Access to the same Hot Termitary (Gut Volume Relationships)

~--:- TYPes Termite Cases Average Average cpm/mg. cpm/mg. S.D. o~ compared TYPe m. (mg.) cpm Ratio Ratio

-N 92 8.18 2:>85 254.9 N-S 3.85 ~ 3.4 -- S 34 7.18 475 66.2 n 71 2.49 512 2:>5.6 n-S - 2·92 + 4.2 -- S 25 7.17 505 70.4 -

N 38 8.42 3069 364.5 -N-n- 1.07 ~ 3.7 -n 31 2.54 867 341.3

....I-' -.0 120

if weights of the compared termites were equated,

~ termites in the present study had a gut size 3.85

times that of ~terInites, ~s had a gut size 2.92 times

that of ~ I s, and.!'J I s had a gut size 1. 07 times that of

n's. The large standard deviations associated with the

three relative gut size figures indicate rather wide

variations in these data, based no doubt on uncontrolled

molting and morbidity effects. Soldiers, of course, do

not molt, but they have a higher mortality rate than

nymphs. Therefore these effects, while not operating

equally for these termite types, probably tended to

cancel each other in any given type combination. The

results of the morphological studies, showing the same

general relative gut sizes, and the reasonably large

number of cases in the present analysis permit the tenta­ t ive conclusion that the figures obtained in the gut- size

analysis are probably representative of the true situation

in C. brevis termites. When data from termitaries contain­

ing only male t'lpes are analyzed separately from those

of termitaries containing only female types the same

general gut- size relationships are shown for both groups,

indicating that the effects are probably real. 121

The interpretation of the results in terms of gut volume differences assumes that most of the radioactivity exhibited by a termite was contained in its gut and therefore reflected fairly accurately gut volume. A portion of the radioactive isotope was doubtless incorporated into the termites' tissues, and some may have contaminated the body surfaces.

1£ these amounts were considerable, then the gut correction factors shown in Table XVIII may not be correct. 85 Metabolized Sr A more suitable isotope for use in the present investigation would probably have been one that was metabolically inert. Food exchanges between termites involve the transfer of a portion of gut content from one individual to another. Ideally, therefore, only the gut content itself should be labeled and traced. Radioactivity representing molecules of the radioisotope incorporated into termite tissues only complicates the transfer picture.

Strontium is not a metabolically inert element but behaves in a inanner similar to calcium and can be substituted for the latter. What is the extent of its utilization by termites? One of the factors affecting the 122 rate of uptake of an isotope by a tissue is the amount of the element present in the tis sue. This involves the concept of turnover, which in the present instance refers to rate of replacement of the strontium (or calcium) in termite tissues by new molecules of strontium. No chemical analyses were made to deter­ mine the amounts of these elements normally present in the various termite types; hence, the isotope I s specific activity, the radioactivity per unit weight of elenlent, and the turnover rate cannot be computed.

This exact information would be necessary in order to determine the relative amounts of Sr85 metabolized by the different termite types, and this in turn would be important in computing the relative amounts of the radioactivity exhibited by an individual termite that represented only gut content. If the different types of individuals being compared do not differ in their ratios of metabolized isotope to isotope present in the gut content, then the fact that an individual's total radio­ activity does not represent gut content alone does not greatly matter. This seems a rather logical assumption but it may be in error. A soldier termite, for example, 123 has a gut capacity considerably less than that of a nymph of comparable weight. Its larger and more sclerotized head and the possible relative increase or reduction of other tissues (e. g., reproductive tis­ sues), might conceivably result in a different ratio of isotope utilization from that of a nymph.

The view that the differences in the amounts of radio- activity acquired by the various types of donors in radio­ active termitaries probably reflected fairly accurately their differences in gut capacity was based chiefly on morphological studies and was not intended to imply that strontium is not appreciably metabolized by ter­ mites. On the contrary, the present studies indicate that it may have been so utilized as the next few para­ graphs explain. Unfortunately the data do not permit hard and fast conclusions regarding possible differen­ tial strontium metabolism by the different termite types.

The same 16 termites whose increase in radioactivity was plotted in Figure 8 were removed from their radio­ active termitaries after 7 days and placed in cold termi:' taries. Each of the 8 individuals from T ermitary A was isolated in one of eight cold termitaries, while the 8 124 from Termitary B were placed together in one cold termitary in order to see the effects of isolation versus non-isolation on rate of radioactivity loss,

Again the termites were measured individually for radioactivity over a period of 63 days. (See

Figure 9, A and B). Measurements were made on the first, third, fifth, seventh, twenty-ninth, forty­ ninth, and sixty-third days following their remova.l from the hot termitaries, At each of the 7 counting sessions the radioactivity of the individual termite, and also of the fecal pellets, from each termitary was measured. The amount of radioactivity lost through elimination could therefore be determined for each of the isolated A termites at each check, but only the total loss for the elltir e B group could be determined at each. check.

The isolated termites from Termitary A showed a higher rate of los s than did the non-isolated termites from Termitary B, doubtless because the radioisotope was not re-entering their 1;:>odies through reciprocal

feeding, as in the case of the B termites: This is shown by the relative steepness of the declines in Figure 9, A .100 .... , • Soldier .... , .... , 90 , , • Supplementary Reproductive ..... , ..... • Large Nymph 80 ' ....0 o Small Nymph

70

60 ~ 50 .-'"'a-_--...a..._ ------_ ---~~~~------.------40

1 3 5 7 29 49 63 Days After Removal from Radioactive Termitary Fig. 9A. Comparison of rate of loss of radioactivity by isolated termite types. I-' N Ql • Soldier

100 .. Supplem.entary Reproductive • Large Nyrn.ph 90 o Sm.al! Nyrn.ph

80 ------__ A ...... 70 ~------~------~ ~ ------1:------• -- - 60 ------...... -0------

50

40

1 3 5 7 29 49 63 Days After Rem.oval from. Radioactive Term.itary Fig. 9B. Com.parison of rate of loss of radioactivity by non-isolated term.ite types. ~ N C1' 127

and B). Measurements were made on the £irstl

thirdl fifth, seventh, twenty-ninth, forty-ninthl

and sixty-third days following their removal from

the hot termitaries. At each of the 7 counting

sessions the radioactivity of the individual termite,

and also of the fecal pellets, from each termitary w as measured. The amount of radioactivity lost

through elimination could therefore be determined

for each of the isolated A termites at each check,

but only the total loss far the entire B group could

be determined at each check.

The isolated termites from Termitary A showed

a higher rate of loss than did the non-isolated termites

from Termitary B, doubtless because the radioisotope

was not re-entering their bodies through reciprocal

feeding l as in the case of the B termites. This is

shown by the relative steepness of the declines in

Figure 9, A and B. The A termites als 0 died sooner,

an indication of the deleterious effect of isolation on

termite survival. All but ~two (~s) of the A termites

wer e dead by the twenty-ninth day check-up, following

isolation and only one of these nymphs survived to the 128 forty-ninth day. It was found dead on the sixty­ third day. Of the B termites, 7 were alive on the twenty-ninth day, 6 on the forty-ninth day, and 2

(N's) on the sixty-third day.

Figure 9A illustrates loss of radioactivity for the isolated termites from Termitary A, and Figure

9 B, shows the same for the non-isolated termites from Termitary B. The data are given in terms of ; average percentage of radioactivity retained by the different termite types in each group on each of the seven check dates. The same symbols are used to indicate type in both groups. The number of indivi­ duals represented by the various points across the graph decreased with time because of termite mortality.

When a dead termite was found on a given check date it was first counted for radioactivity and then discarded.

The e:xact date of its death, of course, could not be determined.

Figure 9 A and B indicates that in general there was a fairly rapid loss in radioactivity during the fir st

week following removal from the hot termitaries l after which the rate of loss was much slower. It will 129 be noted that the last three check dates represent­ ing a period of 8 weeks are not placed in prop~r distance relationship to the first four, which repre­ sent a period of one week. The broken lines indicate this telescoping of the graph data.

Another analysis showing this initial rapid loss of radioactivity involved a consideration of the fecal pellets collected on each check day from each termi­ tary. Table XIX presents these data separately for the A termites and for the B termites. Both the num­ ber of pellets and their total radioactivity are pre­ sented for each termitary check. The radioactivity for individual pellets is not known in most cases, since all from a single termitary on a given day were counted together in the scintillation w~ll.

Because of the fact that A termites died sooner than

B termites the results in order to be comparable are expressed in terms of "termite days". For example, at the ;twenty-ninth day check there were only 2 A sur­ vivors, whereas 7 B termites were still alive. The pellets in each termitary had accumulated for 22 days since the previous check. There were 2 x 22 = 44 termite TABLE XIX

Comparison of' Radioactivity Lost through Fecal Pellets by Isolated and Non-isolated Tennites at Various Intervals Following Removal f'rom Hot Tennitaries

A Tennites (Isolated) B Tennites (Non-isolated) I No. of1 Pellets Total Total CPII per- pays Sincll Days Since Ter-llites "Termite No. of Pellets Totall Total cpm per- iTermites "Termite I Pellets per Pellets cpm per- Pellet Alive Days" Pellets per Pellets cpm per- IPellet Removal last Alive Oays" Termite cpm Termite per Termite Cpll Termite per- fr-om Hot checkup Day Day Tar-mite I Day Day Termite lTer-mitary Day Day 1 1 8 8 1 0.12 79 9·9 82.0 8 8 1 0.125 554 69.2 554.0

3 2 8 16 15 0.94 1039 64.9 69.0 8 16 4 0.25 806 50.4 202.0 5 2 7 14 27 1.93 2578 184.1 95.4 7 14 4 0·29 277 19.8 68.3 7 2 6 12 9 0.75 384 32.0 42.7 7 14 4 0.29 23) 16.4 56.6 29 22 2 44 50 1.J.4 400 9.J. 8.0 7 154 34 0.22 427 2.8 12.7 49 20 J. 2) 4 0.2) J.7 0.8 4.0 6 J.2) 3J. 0.26 98 0.8 3.J. 63 14 0 0 0 0.00 0 0.0 0.0 2 28 13 0.46 39 1.4 3·0

Total. ll4 106 0.93 4497 39.4 42.4 I •312 91 0·29 2431 7.8 26.9

~ W o 131

days for the B termites. Some of the termites that died in Termitary B between check-ups may of course have contributed to the pellets found at the subsequent check. This seems particularly likely at the sixty-third day check (when the number of individuals had been reduced from 6 to 2) and is indicated by the increase in average number of pellets per termite day from less than 0.30 to 0.46.

According to Table XIX, where comparisons are made on the basis of "termite days", the average production of pellets per each A termite was nearly

I pellet per day (0.93) whereas, in the non-isolated termites the average production per day was much lower (0.29 per day). Thus it appears that procto­ deal feeding by partners reduces the number of pellets produced.

Table XIX also enables a comparison to be made of amounts of radioactivity lost via pellets by A and B termites. A valid comparison requires a knowledge of the average initial radioactivity of each group. The

A termites averaged 1341 cpm and the B termites averaged

842 cpm on the day they were removed from the radioactive 132

termitaries. The counts per minute of individuals

cannot be taken into account in this analysis because the B group was handled as a unit. In terms of ter­ mite days: A pellets averaged 41 cpm per termite day while B pellets averaged 27 cpm per termite day.

The ratio of A-pellet cpm to B -pellet cpm j /0) 1. 52: 1.

as compared with a ratio of 1. 59: 1 for the initial

average A-termite cpm to B -termite cpm. Apparently in each case the pellets were about equally representa­

tive of the termites I radioactivity. At the time of

check-up, it appeared that the average A pellet was

slightly smaller than the average B pellet. but no

quantitative measurements of pellet size were made.

Table XIX supports Figure 9 in showing that for both A and B termites most of the radioactivity

eliminated was in the fecal pellets produced during

the first 7 days. The total of 4497 cpm lost in fecal

pellets by A and the 2431 cpm lost by B represent

only about 40 percent of the total initial radioactivity

exhibited by the termites of the two groups. Does this mean that the strontium remaining in the termites

was incorporated into their tissues and no longer 133 present in quantity in the gut lumen? The results of the pellet analysis would seem to imply this since pellets produced after the first week were very low in radioactivity. There may be another expl anation, although it appears hard to postulate a mechanism that would permit the gut content to be markedly radioactive and yet prevent to a large extent the incorporation of Sr85 into the fecal pellets. One hypothesis might be that the protozoa, present chiefly in the hind gut (but usually not in the rectum wher e pellets are formed), take up strontium in relatively large amounts, thereby main­ taining it in the termite gut lumen. The technique of radioautography should allow a test of this hypothesis, but the results of the few radioautographs made so far are inconclusive. Additional tests are called for.

The experiment designed to show rate of decrease in radioactivity also provides a basis for estimating roughly the biological half-life of Sr85 in termites.

This is the time taken for activity exhibited by a termite to drop to half its original value as the result of elimination. In the case of vertebrates whose bodies hold strontium in the bone l the bio logical half life is almost equal to the half-life of the radioisotope

itself (Glasstonel 1950 1 p. 508). In the case of termites the biological half-life should perhaps be expected to be shorter.

In the analysis of the decrease in radioactivity illustrated in Figure 9, it is the data from the A termites that should be considered in est imating biological half-life. These A termites had no access

to radioactivity except their own fecal pellets l which are not usually eaten by C. brevis termites. B ter­

mites l on the other handl probably continued to cir­ culate their radioactivity through proctodeal feeding.

The small nymphs, represented by the light circles in Figure 9 are atypical in that they showed, after 3 days, a slight rise in percentage retained.

There is no way in which such an increase could have occurred except through error, and this apparent increase is probably due to random variation in dis­ integration rate. The. other termites generally decreased in radioactivity for as long as they lived or until dis­

carded at the end of the experiment. The soldier I 135

unable to feed, did not defecate after the third day

and therefore lost no more radioactivity. By the

seventh day, the supplementary reproductives and

the large nymphs ha:d lost approximately half their

original radioactivity. On the basis of this small

amount of data, it would appear that the biological

half-life of Sr85 in G. brevis is about a week. It must be reulembered, however, that metabolic turnover rate has not been determined, so the

estimation of one week is only approximate.

Surface contamination. The application of the isotope tracer technique to the study of food transfer

relationships among termites is based partly on the

assumption that acquisition of radioactivity by a

recipient termite is the result of its having solicited

and obtained part of the gut content of the donol" part­

ner as food. If appreciable radioactivity can be

acquired in other ways, then this method of studying

inter -termite feeding relationships is one of question­

able merit.

In the present investigation the possible ways in

which a recipient might have acquired radioactivity 136 was by ingesting part of the gut content of its donor partner, by feeding on the donor I s fecal pellets or on the plaster material produced by the donor, or by becoming contaminated with these materials through picking them up on the body surfaces. It has already been noted that C. brevis termites are rarely seen feeding on pellets. They were never observed in the course of the present investigation to attempt to feed on the plaster mater iaL

While there remains the possibility that a certain amount of radioactivity might occasionally have been acquired in this way, it seems unlikely that it could have represented more than a very insigni­ ficant portion.

Surface contamination as a source of radioactivity in a recipient was also shown to be unlikely. The hy pothesis in this case was that the recipient either ingested radioactive materials contaminating the sur­ faces of their partners during the process of grooming, or they themselves picked up the materials on the sur­ faces of their own bodies. In order to test the hypo­ thesis of surface contamination, termites that had 137 just been removed from a period of confinement together in a hot termitary were swirled about in a vial of water containing a few granules of laundry detergent as a wetting agent. If radioactive materials had been present on the body surfaces they should have been removed during the washing and left in the wash water.

Two tests were made with termites taken directly from hot termitaries where they had remained for

21 days. All the termites in a given test were first measured together in the scintillation well for total radioactivity. They were then placed in a small nylon mesh bag, immersed in the vial of detergent water (about 1 ml.), and gently agitated for 20 minutes. At the end of this time the bag containing the termites was lifted from the water, and drained into the vial. The vial containing the wash water was then placed in the scintillation well to see how

much of the termites I radioactivity, representing that contaminating the body surfaces, had been left behind in the wash water. The results of the tests are shown below, with each ~pm corrected for background counts. 138

No. of Ave. Ter- Original Ave. cpm Wash water cpm per Test mites cpm per termite cpm termite

1 5 18413 3682.6 52 10.4

2 6 20531 3421. 7 77 12.8

The 6 termites of Test 2 were returned to the

wash water in the vial and left for 13 hour s longer.

When re-tested in the scintillation well at the end

of that time, the wash water measured 560 cpm, or

93.3 cpm per termite. This rise in radioactivity

is interpretered as indicating that the prolonged

exposure of the dead termites to detergent water

permitted their radioactive body contents to diffuse

out. This may also have occurred to some extent

during the earlier 20-minute washing period, but

in any case the results indicate that little radio-

activity was to be found on the body surfaces of

the radioactive termites.

In summary; surface contamination was not an

important source of possible error in making gut

size corrections, but it is still not known if strontium

was metabolized differentially by the different types

of termites. Therefore gut size corrections for 139

this possible source of variation cannot be made.

Morphological studies indicate that the correction

factor s obtained in the gut-volume analysis by

comparing relative amounts of radioactivity ac­

quired in hot termitaries by the different termite

types are probably correct. In any case it appears

that differential strontium metabolism would not

greatly affect the final interpretations of results.

Analysis of Gut-corrected Data. The results of the gut­ volume analysis implied that if it were possible to have~, n, and

5 termites of the same weight, their respective gut volumes would vary according to termite type. If ~s gut volume is given a relative val ue of 1. 00 the comparable value for n would be O. 93 and for 5, 0.26. In other words, according to Table XVIII, in order to

correct the various cpm figures for gut volume, N figures must be multiplied by 1. 00, ~ figures by 1. 07, and 5 figures by 3.85.

These factors, the best estimates available, were used to correct the data represented by Table XVIII in order to permit more accurate comparisons of food transfer percentages for the different feeding combinations ..

Each of the 152 transfer percentage figures was multiplied by the appropriate factor obtained by making a ratio of the recipient 140 and donor gut figures. For example, the N-N percentage 1. 00 figures were multiplied by 1. 00 , the N-n percentage figures 1.07 3.85 by 1.00, the n-S percentage £igur.es by 1. 07 , the S-S per­ 3.85 centage figures by 3.85, and so on.

Inspection of the resulting data revealed that the means for the feeding combinations were correlated with the variances, and in orderpr..operly to apply analysis of variance to the data it was necessary to make a transformation to decorrelate them.

Several transformation methods were tried including arcsin, square root, and logarithmic as descr.ibed by Snedecor (1956).

It was decided that the best transformation, one that completely decorrelated the means and variances was the method using log

(x + O. l). The addition was to avoid the logarithm of zero.

Table XX presents the m.eans for these corrected and trans- formed data. The figure in parentheses in each cell represents the mean of the logarithms obtained with the transformation method. The figure immediately above the logarithmic mean is the transfer percentage resulting from the reconversion of the logarithm. The last row in the table and the last colt,lmn on the right give the weighted logarithmic means for each donor type and each recipient type, respectively.

The chief point of interest in the present investigation using TABLE XX

Mean Radioactivity Transfer Percentages for Each Coxnbination of Terxnite Types (Oxnitting Sex) after Gut Corrections and Log (x + O. 1) Transforxnation

R e c i pients Donor - Weighted Means Donors N n S (log) 24 23 15 62

2.5 3.7 16.3

N ( 0.407) ( 0.579) ( 1. 216 ) (0.666)

19 16 16 51

1.5 6.1 12.5

n ( 0.203) ( 0.794) (I. 104) ( 0.671)

14 16 9 39 1.7 2.0 15.6 S ( 0.257) ( 0.316) (1.197 ) ( 0.498 ) ..... 55 40 ~ 57 ..... Recipient Weighted ( 0.302) ( 0.565) ( 1. 167 ) Means (log) 142

radio-strontium lie5 in the possibility of finding differential feeding relationships based on specific combinations of sex,

caste, and instar. This would be disclosed as interaction in

an analysis of variance. Sex has already been shown to play no

consistent part in determining feeding relationships. The

pos sible effect of caste and instar in various combinations was next studied.

A preliminary analysis of variance was applied to the trans-

formed data with the following results.

Source of Degrees of Sum of Mean Variance Freedom Squares Square

Subclass 8 20.316

Donors 2 0.845

Recipients 2 17.895

Individuals 143 43.213 0.302

The mean square for individuals was used in combination

with a further analysis of variance which involved weighted

.means (Snedecor, 1956, Section 12. 17). The method is appro-

priate in cases like the present one in which the number of in-

dividuals in the subclasses are unequal and disproportionate, and

leads to an unbiased estimate of interaction if it is present. A

positive a"n.d statistically significant interaction would indicate 143 that there is a tendency for a specific recipient type to favor a specific donor type or vice versa.

The analysis of variance, using Snedecor I s method of fitting c'onstants gave the following results:

Source of Degrees of Sum of Mean Variance Freedom Squares Squares

Donors 2 0.702 0.351

Recipients 2 17.752 8.876

Interaction 4 1. 719 0.430

Individuals 143 43.213 0.302

F for interaction = 1.424; P > 0.05

F for donors = 1. 162; P > 0.05

F for recipients = 29.391; P < 0.01

There is no interaction (P) 0.05) so no differential feeding "preferences" involving specific combinations of caste and instar are indicated. The main effects, the differences between the types of donors and recipients, can next be examined. Since there is no evidence of interaction these differences may be considered to be unbiased estimates of the behavior of these different types, within the limits of the experimental conditions.

The P value of > 0.05 for donors indicates that the 144 different donor types did not differ significantly in the amounts of radioactivity (representing food) transferred from them to the recipients. In the case of the recipients, however, the analysis of variance shows that the different types differed significantly in the amounts of radioactivity acquired.

The next question with regard to recipients is: where do the differences lie? It is obvious from the weighted logarithmic means of Table XX that the soldiers acquired a much greater amount of radioactivity than either of the other recipient types.

This of course is to be expected in view of their complete depen- dence on colony mates for nourishment. Do the N and ~ recipients also differ to a significant degree? The significance of differences between means was tested using Tukey's method described by Snedecor (1956, section 10.6). It involved the use of the formula D = S;2 0, in which Sx =~ :;2. and 0 is obtained from Snedecor's Table 10.6.1. Because of the unequal number of individuals in the subclasses it was necessary to use the following formula for obtaining the best estimate of average number of individuals per subclass: 2 2 2 57 + 55 + 40 ~ = 1/2 (152 - ---:-1=52---- = 50.1

The D value was found to be 0.261, which means that the 145 difference between any two types of recipients had to reach this figure in order to be statistically significant at the 0.05 level. The.E weighted logarithmic mean of Table XX is obviously different from that of ~ and of ~ The difference between the means of Nand 12. is 0.263 which is just signifi­ cant. It indicates that small nymphs obtain relatively more radioactivity from their donors than do large nymphs. This also is not unexpected, for nymphs until the third instar ob­ tain all their food from colony mates and might be expected to be "weaned away" only gradually. The small nymphs in the present investigation were third and four instars.

How would N and ~ recipients compare with each other and with~ recipients in amounts of radioactivity acquired if they, like the soldier, were forced to obtain all their nourish­ ment from their partners? Experiment 8 was carried out in an attempt to answer this question.

Experiment 8

This experiment was conducted in the same way as the othersexcept that at Stage B the members of each pair were placed in a glass vial lined with nylon mesh and had no acces s to food other than that obtained from each o'ther. Unfortunately only a few soldiers were availa,ble for use in the experiment, 146

and only one (a donor) survived to Stage C. Molting and death

further reduced the nu.mber of pairs to 22.

The trap-sfer percentages were transformed by the logarithmic

method used previously. The one S donor was paired with a

~ recipient and the transfer percentage (reconverted from the

logarithm) was 6.1. This can be compared with the mean trans-

fer percentage of 1. 8 for the S-N cell of Table XX. The data for

!:! and n termites of Experiment 8 are given in Table XXI.

TABLE XXI

Transfer Percentages for Nand E. Termites in Experiment 8

Recioients Donors N n 4 7

N 2.6 16.7 (0.4l5) (I. 222)

4 6

n 7.7 20.3 (0.884) (1. 308)

Here it will be seen that for both N and ~ recipients the

transfer percentages tended, to be higher than was the case

when they were able to feed on wood (compare with Table XX). 147

In particular the transfer percentages were higher for the ~

recipients: exceeding even those for ~ recipients in Table XX.

From these data it appears that the small nymphs, when droied

other food sources, fed from partners to an even greater ex­

tent than did soldier s. Large nymphs, however, increased

their proctodeal feeding only moderately under these condi­

tions. The data are few, but they illustrate a means of obtain­

ing a " s tandard" with which extent of proctodeal feeding under

"normal" partnership conditions can be compared for the

three types of termites.

* *),'0:

Although a comparison of food transfer relations for

different termite types was the main purpose of the radioisotope

experiments, other effects also emerged. These included the

effect of molting on the transfer of radioactivity and the use

of the isotopic tracer technique to show that soldiers do not

feed directly from wood.

Feeding Behavior of Molting Nymphs

The number of feeding partnership~ that could be analyzed for food transfer relationships was depleted because of the fact that in many cases one or both members of a pair either died or molted during their week 148

together. In such cases the normal feeding l'elationships wer e dis­

1'upted.

It is a well-established fact that termites do not feed just prior to molting (Andrew 1930). The period of premolt fasting. according to data to be presented later. appears to vary with instar, but may begin in the case of older Cryptotermes nymphs at least as early as two weeks preceding the actual molting process. and feeding is usually resumed within two or three days after molting. The present investigation indicates not only that nymphs fast in the premolting stage but also that other termites do not feed from them during this interval. For these reasons when it was found that either member of a pair had molted during their partnership. the pair was excluded from the food transfer analyses. It was not surprising to find a high molting rate among the large nymphs. When nymphs of the fourth instar or older are isolated from reproductive individuals some of them llsually become supplementary reproductives within about two weeks. The change is acco~panied by a molt.

Fasting

Failure of termites to feed during the premolting in­

terval had been discovered through direct observation. The pre­

sent study presents further data of a quantitative nature to support

this finding. The pertinent analyses consisted in comparisons of 149 the amount of radioactivity (counts per minute) acquired by molting termites and that acquir eO. by nonmolting termites. Two different kinds of data were examined: a) the radioactivity figures for molted and non-molted recipients paired with hot donors, and b) the radio­ activity figures for molted and nonmolted donor s kept in hot termi­ atries. Usually only nymphs could be considered in the analyses, because adult soldiers do not molt, and Experiment 8 is not in­ cluded. The recipient data will be considered first.

When the paired termites were examined at Stage C after their week together some were found to have molted during the interval.

Unfortunately it could not be determined at what time during the week of pairing the molting had taken place -- whether, for example, it had occurred soon after the termites were placed together in the termitary or just before they were removed at the end of the week.

In the case of a molted recipient, if molting had occurred at the beginning of the week, then the recipient probably would have be­ gun soliciting food within a day or two thereafter and would have acquired some radioactivity by the end of the week. If molting had occurred at the end of the week the recipient would have had little opport,unity to feed from its partner, and would have acquired little or no radioactivity. This uncertainty regarding the exact time of molting affects the results of the present analyses but still permits 150 a consideration of the effect of molting on feeding behavior.

In all analyses comparing feeding behavior of molted and non­ molted termites, as in analyses for feeding relationships in general, cases were omitted in which either partner died or the partner whose premolting feeding behavior was not being considered molted.

At Stage C, 12 cases involving molted recipients and 98 cases involving nonmolted recipients met the criteria for inclusion in the present analyses. These included 17 cases in which the donor was a soldier. The inability of the latter to molt does not affect this particular analysis because only recipients are being compared.

Table XXII presents the data by partnership type for both molted and nonmolted recipients. In every type of partner combi­ nation the molted recipients acquired on the average a lower per­ centage of the radioactivity brought into the termitary by the donor than did the nonmolted recipient. It will be noted that there were no available cases of.§ donors paired with molted N re­ cipients, so the comparable nonmolted S-N cases are put in parentheses and omitted from the total results. The last row in the table shows the average results for molted and nonmolted cases regardless of partnership types. The average molted 151 recipient acquired less than 1 percent of the donor's radioactivity while the average j,1.onmolted recipient acquired 4 percent. It seems likely that if the molted recipients had been counted i.mmediately following their molt, they would have given approxi- mately zero counts per minute. The cases in which relatively high counts were obtained for molted recipients probably repre- sented nymphs that molted soon after being placed in the termi- tary, thereafter feeding normally during the remainder of the week.

TABLE XXII

Comparison of Radioactivity Acquired by Molted and Non­ molted Recipients for all Types of Partner ships

Molted Recipients Nonmo1ted Recipients Type of Re- Re- Partner- cip- cip- ship Donor ient Donor ient. (Donor- Ave Ave %Ac- Ave Ave % Ac- Recip. ) Cases cpm cpm quired Cases cpm cpm quired --N-N 3 2156 6 0.28 24 2665 95 3.56 N-n-- 2 4914 6 0.12 ~3 2186 47 2.15 n-N-- 4 867 24 2.77 19 599 52 8.68 Il-n-- 2 276 0 0.00 16 562 60 10.68 --S-N . - - - - ( l':!Pl< (234})l< (36})l<(lS. 38})l< S-n- 1 389 11 2.83 16 346 17 4.91 Total 12 1725 12 0.70 98 1430 57 4.00 )jCCases. omltted from total results. 152

The" other way of indicating the fasting behavior of molting termites in the present investigation involved a consideration of donors rather than recipients. Donors, it will be recalled, were made radioactive by being confined in radioactive termi­ taries during Feeding Period A. If a nymph entered the pre­ molting stage during this period it would be expected to feed less and to become less radioactive than its nonmolting tel'mi­ tary mates.

At the Stage B check-up (immediately following Feeding

Period A) the donor termites did show wide variation in the amount of radioactivity acquired. Termites with the lowest counts were omitted insofar as possible from further considera­ tion in the food transfer experiments and were discarded.

It seems very likely that the low count of many of them was due to their being in the molting phase, but no conclusion regarding this possibility can be reached because they were not followed further. The experimental set up required that at least the 6 most radioactive donors of each type be used for pairing with recipients at Sta'ge B (N and ~ donors sometimes had to be substituted for dead ~ donors) and there­ fore relatively nonradioactive nymphs sometimes had to be 153 used. It is hypothesized that the least radioactive of these donors entered the molting phase sometime after being placed in the hot termitary and after feeding for a longer or shorter time (as indicated by their degree of radio­ activity) on hot wood. When such a donor was transferred at Stage B to a cold termitary together with a cold recipient it was already in the molting phase. The actual molt then occurred during Feeding Period B and was first discovered at the Stage C check-up.

The analysis was made as follows. When a donor was found to be molted at Stage C its radioactivity, measured at Stage B was compared with that of donors that had not molted at Stage C and could scarcely have been in the molting phase while in the hot termitary. Furthermore, in order to control the variation in termite radioactivity, resulting from differences in degree of termitary radio­ activity, the molted donor was compared only with the nonmolted donors that had been present with it in the same termitary during Feeding Period A.

There were 17 different termitary groups in the present analysis, involving 27 molted and 67 nonmolted donors. 154

All the nymphs in a given termitary were either all ~

or all ~ donors (5 donors are omitted from this analysis).

The 27 molted donors averaged 964 cpm from their week

in the hot termitary; the 67 nonmo~ted donors averaged

1780 cpm. Table XXIII shows that the acquisition of radioactivity by molted nymphs is consistently lower than for nonmolted nymphs when the results are pre­

sented separately for the different termitary groups,

showing an opposite trend only in the case of termitary

Group C.

Termitary groups A-N consisted entirely of large nymphs; those from O-Q were made up of small nymphs

Far fewer small nymphs than large nymphs molted during

the course of the experiments. This is doubtless due not to an increased molting rate as such in older nymphs but rather to the fact that older nymphs separated from

reproductives develop into supplementary reproductives.

This transformation, at least in C. brevis nymphs, is

accompanied by a molt.

The results of these comparisons of molted and non­ molted termites show only that nymphs in the molting 155

TABLE XXIII

Comparison of Radioactivity Acquired During Feeding Period A by Molting and Nonmo1ting Donors According to Termitary Group

r Molted Nonmolted Termitary Donors.,------_._--I------Donors Group Cases Ave. cpm Cases Ave. cpm

A 2 801 4 1391 B 2 496 3 1418 '')7(... t: C 1 .... , - J 1273 D 1 1239 5 3271 E 1 262 5 962 F 2 430 4 885 G 3 398 3 574 H 3 328 3 557 I 1 472 1 571 J 1 748 I 4 1120 K 1 1382 5 2389 L 2 4.3:04 I 6 6172 M 3 2523 4 5589 N 1 896 2 2934 0 1 99 5 403 P 1 398 5 408 Q 1 328 3 351

! Total 27 I 964 67 1780 I 156

phase do not eat as much as do nymphs not in this phase. Visual observations in connection with another experiment indicate that feeding occurs rarely if at all at this time, and the fact that the molted nymphs did acquire c;L con­ siderable (though smaller) amount of radio­ activity is due doubtless to their feeding prior to or subsequent to the onset of the molting interval.

Denial of Partner Feeding

Direct observation of small termite colonies led to the hypothesis that termites in the premolting phase refuse to allow colony mates to

solicit food from them. If this is the usual situa­ tion one should expect to find that molting donors

in the present investigation lost less of their

radioactivity to their respective recipients than

did nonmolting donors. A test of this hypothesis

called for a different analysis from that just

presented. The original~mountof radioaCtivity·

available to or acquired by a given donor during 157

Feeding Period A was of no particular consequence in the new analysis. Of primary importance was the amount of radioactivity retained by a donor after its pairing with a' recipient during- Feeding

Period B. Comparisons~ therefore~ did not have to be restricted to nymphs occupying the same hot termitary, though cases of dead and molted partners were again omitted. The data are from Experi­ ments 1 - 7.

Twenty molted donorsand 113 nonmolted donor s ar e included in the present analysis. The former were nymphs found to be molted at the Stage C check-up~ immediately following Feeding

Period B, and the latter were nonmolted nymphs. The 20 molted donors had an average of 1139 cpm at Stage B and one of 1141 cpm at Stage C~ almost exactly the same count. The 113 nonmolted donors averaged 1580 cpm at Stage B and 751 cpm at Stage C, a reduction by about half. The only means by which the donor's loss of radioactivity could have occurred was by way of food transfer to its partner, through the production of fecal pellets, or through

the anal extrusion of the "plaster I.' material used by termites in

sealing their termitaries. It would seem on the basis of this analysis that the molted donors, having apparently lost none of

thei~ radioactivity, 'had carried out ,none oJ these activities. The nonmolted donors, on the other hand, lost approximately half their 158 radioactivity during the same week.

In order to study further the matter of premolt retention of gut content, a comparison was made of the amounts of radioactivity acquired by t~ respective recipients (5 recipients included) of these molted and nonmolted donors during the week of pairing. The re­ sults of the donor analysis just described would indicate that the recipients paired with the m.olted donors could not have acquired any radioactivity, in view of the fact that these donors appeared to lose none. In actuality, recipients paired with the molted donors did pick up a certain amount of radioactivity as shown in Table XXIV, acquiring an average of 16 counts per minute or 1. 40 percent of the donor I s radioactivity. The discr epancy in these data is due probably to random variation in isotope disintegration rate for individual termites. The donor s probably did lose some of their radioactivity, an amount small enough to be masked by the chance variation.

The number of recipients paired with nonmolted donors was re­ duced from 113 to 99 in the present analysis because the N-5 partnership represented only by nonmolteds was rlot included in the total results (though 'presented in parenthesis in Table XXIV).

These 99 recipients acquired an average of 68 cpm or 3.97 percent.

Table XXIV shows that all partnership types except N-n were con­ sistent in showing that less radioactivity was acquired by recipients 159

TABLE XXIV

Comparison of Radioactivity Acquired :By Recipients Paired with Molted .and Nonmolted Donor s for all Types of Partnerships

Molted Donor Nonmolted Don or Type Recip- Recip- of Donor ient Donor ient Partner Ave Ave %Ac- Ave Ave % Ac- ship Cases cpm cpm quired Cases cpm cpm quired

Ii:ll 6 2395 18 0.08 24 2665 95 3.56 N-n 7 758 20 2.64 23 2186 47 2.15 n-N 1 328 Q 0.00 19 599 52 8.68 n-n 2 55 0.5 1.00.. 16 562 60 10.68 N-S 4 664 15 2.26 17 20.07 86 4.16 n-S -- - - (14) (630) (119) (18.89) I

Total 20 1139 16 1. 40 99 1715 68 3.97

paired with molted donors than by those paired with nonmo1ted

donor s . Some of the figur e s in the right half of the table are

identical wi.th those of Table XXII. This is inevitable because the

nonmolte~ T1airs are the same.

Again, .le lack of precise information on,exact time of molting

prevents a mor.e accurate aegr.:egation of donor termites into "molted"

and "nonmo1te'd" categories. It s~ems likely that retention of radio-

activity by: just-molted dono~.s .'8()uldhevirfua.·iiy comp1etce ...... ' .. '.' ". . . 160

Nonproduction of fecal pellets

It has just been shown that molting donors lose little radioacti,;,"ity. This implies that they not only deny food to partners, but that they also fail to produce fecal pellets during the molting period.

In only three feeding experiments {5, 6, and 7L involving a total of 7 molted donor s: were the fecal pellets collected from each termitary at Stage C and counted. There are few data, therefore: from which to make comparisons of pellet production by molting and nonmolting termites. All of the 7 molted donors were large nymphs, so only the large nymphs among the nonmolted donors were selected for comparison.

At Stage C the number of pellets found in each of the termitaries housing a donor and a recipient was recorded: and the entire termi­ tary containing the pellets was counted in the scintillation well.

T he resulting counts-per-minute figure represented the portion of the originally-introduced radioactivity not accounted for by the bodies of the donor and recipient termites. Separate measurement of pellets and termitary .showed that usually the termitary itself was not radioactive but that the pellets were Y..ery much so. Occasionally a termitary was found to be fairlyradiQactive, and.in these cases inspection revealed that the junction between termitary and cork lid 161

had been sealed with the previously mentioned brownish plaster.

An average of 4.4 pellets were found in the 7 termitaries

containing a molted donor at Stage C. Thes e termitaries and

their pellet contents averaged 11 cpm or approximately 3 cpm

per pellet. Besides the molted donor, a recipient partner had

also been present in each termitary during the week preceding

the Stage C checkup, and this partner was probably responsible

for most of the pellets found. Termitaries containing non-molted

donors, a total of 31, averaged 17 pellets each. The average

counts per minute for termitary and pellet contents was 982,

approximately 58 cpm per pellet.

This analysis indicates that molting individuals do not defe­

cate, and in view of the fact that defecation is directly correlated

with feeding, this finding is not unexpected.

Fee~iing dependence of soldiers

Observation of soldier behavior led. early investigators to conclude that the soldiers of most termite species, and particularly those of species that attack relatively sound wood, are unable to feed directly from the wood itself but must obtain their nourishment from colony mates

(Grasse, 1939). This conclusion was presumably based on the fact that soldiers, with their defense-adapted mandibles, were never seen to feed from wood, that soldiers were observed soliciting food from other members 162 of the colony, and that soldiers isolated from nymphs died, apparently from starvation.

The radioisotopic tracer technique suggested a further method of

"establishing the dependenr.y of soldiers upon colony mates for food. If soldiers cannot feed directly from wood they will presumably remain nonradioactive when isolated in radioactive termitaries. Those placed in such termitaries with nymphs, however, should become hot from the radioactive wood they obtain secondhand from the feeding nymphs.

An experiment to test this hypothesis was designed to run concurrently with the general feeding experiments. At Stage A of each experiment,

2 to 8 soldiers representing both sexes were placed together in a radio­ active termitary (Termitary #5 in the schedule on page 78 ). They were isolated in groups in order to enable them to exchange proctodeal food and thereby to reduce the likelihood of their starving to death during their period of isolation from nymphs. One week later, at Stage B, the sur­ vivors were removed from the termitary and their radioactivity measured

separately in the scintillation well. This gave a measurement of each

soldier I s degree of radioactivity and demonstrated its ability or inability to feed from- the wooden termitary. Each soldier was then pdred for a week with a nymph that had access to radioactive wood. At Stage C the

soldiers were again "placed individually in. the scintillation well for a ·re­ count of their radioactivity. 163

There were 32 cases in which soldiers were isolated from nymphs for a week in.hot termitaries. Of these 32 soldiers, 19 survived.

Their radioa~tivity varied between a and 13 counts per minute with an average of 6 cpm after corrections for machine fluctuation and back­ ground radiation. This is only slightly above background and chance variation could have accounted for all counts. There is a possibility that some of the counts may have been due to a very slight surface contamination. The indication is that the soldiers had not fed during the interval except perhaps from each other.

The 19 soldiers were then placed with radioactive nymphs for a week, and at the end of that time, retested. Cases in which the soldier died or its partner died or molted, were discarded because of the possibility of a food transfer between nymph and soldier was thereby altered or prevented.

Ten soldiers with active, nonmolted nymph partners survived the week.. They showed a rise in radioactivity from a previous average of 5 cpm to 135 cpm. Their relative failure to acquire radioactivity during their isolation in a hot termitary and their rise in radioactivity when paired with radioactive nymphs are in line with past conclusions regarding the dependence of soldiers upon their colony mates for food. AN INVESTIGATION OF TERMITE BEHAVIOR THROUGH DIRECT OBSERVATION

The investigation of food transfer relationships among ~. brevis individuals through the use of the isotopic tracer technique was basically a study of the social organization of termites. It was designed to test the hypothesis that food transfer between termites is related to specific combinations of sex, caste, and instar. No such significant re1ation- ships were found, but feeding characteristics based entirely on caste and instar were confirmed.

Other types of inter-individual relationships may also exist in ter- mite colonies, relationships based not on such fundamental categoriza- tion.s as caste and instar but on individual reactions. It has been shown that animal groups of many species are organized into social hierarchies.

Schje1derup-Ebbe (1922) first described the phenomenon of social domi- nance in flocks of chickens, and later investigators have found similar examples in such widely diverse animals Cl;s cats (Winslow, 1958) and crayfish (Bovbjerg, 1953). Most of the work has been done with verte- brates (for bibiiography see Collias., 1950). : AUee· and Dickinson (1954) 165 note that although dominance- s:ubordinance patterns of behavior are reported for some of the more specialized invertebrates, including

Sepia, certain crabs, and lobsters, such social patterns are not the rule. When they do occur they appear to be based on size. These authors mention specifically (p. 363) that many invertebrate forms have been observed, among them fiddler crabs, ants, and termites, without finding any sign of dominance behavior. It has been suggested that "although hierarchical relations may influence the social system of primitive social insects, there is little to indicate much importance of such r elations in the more advanced insect societies. 11 (Allee,

Emerson, Park, Park, and Schmidt, 1949). According to these authors dominance hierarchies based on individual competition might in the course of the development cmd evolution of the societal system be re­ placed by the more cooperative types of social integration.

The only insect group in which dominance behavior has been reported is the order Hymenoptera, specifically Polistes wasps (Pardi, 1948 and

Deleurance, 1952). Pardi worked with the fertilized females of Polistes gallicus that associate in the spring in founding the same nest. He found that all the females on a given nest could be ranked according to a "domi­ nance scale" and assigned a IIsocial status. II Dominance order was re­ lated to a preferred position on the nest, to amore fr equerit transfer of· regurgitated· food from dominated to dominant individual, to egg laying and 166 other work at the nest as opposed to foraging, and to development of the ovaries. The dominance scale between worker s was foundtobe very closely related to age, which, as Pardi points out, is related in turn to ovarian development. Morimoto (1954) working with another

Polistes species confirmed the Pardi cor.relation between oviposition frequency and dominance order.

Animal groups are organized on the basis of both competitive and cooperative relations, and dominance hierarchies demonstrate aspects of competition. Pardi in his Polistes paper mentioned above quotes

Allee (1940) as saying in effect that competition between individuals of

/ a community with an underlying stratum of cooperation establishes de- grees of dominance and subordination which may furnish a basis for more effective community cooperation. In competition with other such organized

I communities the cooperating systems that are the most fit have the advantage. Pardi concludes that in Polistes the work distribution appears to be a consequence of the dominance system which in turn is the result of intrasocial competition, and this work distribution, he believes, repre- sents an advantage for the society. He expresses an interest in knowing if there are similar phonemena of competition to be found in other social insects. The present study is 'an investigation of the problem with r,ela- tion to te.rmites. 167

It was mentioned earlier that negative findings have been reported / with regard to a dominance hierarchy in termites (Allee and Dickinson,

1954). Kalshoven. (1958) has found that a division of labor occurs to some

extent among the workers of Hospitalitermes, with some collecting and.

others carrying the food pellets they roll up during foragfr~g activities.

Weesner (1960) states that such division of labor may be more general

among termites than is supposed, especially among species having di-

and tri-morphic workers and soldiers.

In the present study the matter was re-investigated to see if evidence

for a social hierarchy or at least for division of labor among .f. brevis

termites could be found.

General Procedure

If a social hierarchy exists within a particular animal group its

presence can be detected by an observer only if each individual animal

is identifiable. This necessity for identification of every individual

limits the number of animals whose behavior one can effectively observe

and record. A natural.f. brevis colony may contain several hundred

individual~, an impossibly large number for a study of this type. In

the present investigation, there~ore, two small"colonies" were studied,

one containing 8 the other 9 individuals.

.' ··.Colony 1 was an incipient colony drawn .from the colony establishment

experiment described earlier and consisting of primary reproductives and 168 their 6 progeny. Colony 2 was a fragment of a natural colony taken from infested furniture, It consisted for most of the observation period of a supplementary king and queen, a soldier, and 6 nY!llphs. In each case the colony was placed in a small observation chamber, to be described later, and observed daily for a period of several weeks, In order to prevent identification problems only one colony was s'et up and followed at a time, The following specific behavior, which included the more obvious daily activities of each colony, was recorded whenevp.r it was observed in any termite:

1. Feeding-:y

a. Food solicitation: record of solicitor and of donor, and length of feeding time.

b. Wood gnawing (including tunneling).

2. Grooming: record of groomer and termite groomed.

3. Work:

a, Licking and transferring of eggs.

b. Rearranging of fecal pellets in the chamber.

c. Plastering of cracks and joints in the chamber.

4. '!Aggressive" or "disturbed" behavior (jerking),

The'use of symbols and abbreviations permitted the behavior exhibited by any termite tc be recorded quickly~ Jror example, "4 g S" meant that the termite identified as 4 groomed the soldier, 169

Observations were made at any convenient time during the day (and

usually included a morning session). The length of a session was also determined by convenience. This complicated later al'l;alyses and inter­

pretations, and it would have been much better if regular observation periods of equal length, as discussed by Collias (1950). had been possible.

An average of 15 minutes a day for 86 days were spent in recording

the behavior of the termites of Colony 1 and an average of 24 minutes a day for 58 days for Colony 2. The daily times of observation, however, varied from a brief glance to see if any termite had molted or an egg

hatched, to 110 minutes. Because of the fact that the behavior of all the

termites was recorded. during a given observation session it is believed

that the data are a representative sample of the behavior characteristic

of these particular colonies. The method used for taking into account

the molting interval of each termite (during which the various activities

were suspended) will be discllssed later.

Analyses were based on the enumeration of the times each termite

member of a colony engaged in a particular type of activity. If a termite

exhibited one type of behavior, for example wood gnawing, th,roughout a

particular observation session without pause this behavior was counted

only once in'the analysis. If, on the other hand, it interrupted the parti­

cular activity to engage in another type~ and then returned te, the original·

activity, the latter was counted twice. Food solicitation by a termite was 170

noted every time the solicitor changed donors even if it returned even­ tnally to a previous donor in the same observation session.

The seconds spent in. proctodeal feeding were estimated in each instance by counting "a thousand and one, a thousand and two, II etc.

Observation Chamber

Each observation chamber was constructed of three birch tongue blade halves (3/4" x 3 11 X 1/ l6!1) held together with masking tape, one above the other (See Figure 10). A hole 1/2 inch in diameter was drilled through the top two blades, which made a shallow chamber when the three blades were assembled. A square of clear plastic, taped above the chamber, served as a transparent !lroofll through which the chamber's inhabitants could be observed. Masking tape was also used to seal the edges of the entire wooden block and to hold the blades closely against each other. The chamber itself filled almost the entire visual field of the binocular microscope when the magnification used was 3 x.

The observation chamber was usually kept in a closed box1 except when observations were being ma.de. Care was taken not to jostle it or otherwise to disturb the termites unduly during the course of the investigation. Microscopic examination was made in a room lighted by flourescent ceiling lights. The termites did not appear to be parti­ cularly disturbed by being transferred from dark box to lighted room 171

Fig. 10. Observation termitary compo8ed of the halves of three birch tongue blades. The chamber is covered with a transparent square of plastic and the termitary edges are sealed with masking tape. Actual size. each day. In a few instances when the chamber was accidentally tapped during the transfer the termites sometimes exhibited jerking behavior for a brief time thereafter which seemed definitely related to the disturbance.

This usually ceased within a minute and the termites resumed their usual activities.

Colony 2 was opened twice in the course of the observation period in order to remove the fecal pellets and to plug tunnels constructed by the termites that hampered observation. These major disturbances resulted in the termites exhibiting for several days more agitated behavior when first placed under the microscope than was normal for them.

Description of Colony 1

This was an incipient colony from the colony establishment experiment. The primary reproductives had been paired in a birch tongue blade termitary (See Figure 2) four months earlier and had produced, since that time, 6 ny~phs and2.eg~s.Fourdifferent instars ~ere represented among the nymphs: first (2 cases)~ second (lL third (21 and fourth (1).

They were studied carefully for di:fferences in size, antennal aberrations, 172 and other means of identifying each and were numbered from largest to smallest as a means of designation. The eight member s of this colony wer e de'signated as follows:

f1 (primary reproductive)

5? (primary reproductive)

1 (fourth-instar nymph)

2 (third-instar nymph

3 (third-instar nymph)

4 (second-instar nymph)

5 (first-instar nymph)

6 (first-instar nymph)

The two eggs were eventually eaten by the colony and no more were laid during the time of observation which was begun on September 16, 1959 and lasted until December 10, 1959.

Description of Colony 2

The original members of this colony were taken from an infested plywood drawer at about noon on December 4, 1959. They in- eluded a large. gravid supplementary female reproductive (whose abdo- men appeared pink from developing eggs), a male soldier, and 5 large . . nymphs. By the morning of December 5, the female reproductive had ciep~sit~d4:~ggs, and bytha.t afternoon had deposited: 3 more. On

December 6 there were 9 eggs in the chamber; on December 7 there were 173

13 eggs; on December. 8, 16 eggs; on December 9, 18 eggs; and on

December 10, at least 19 eggs. Some of them were eaten by members of the colony and some soon shrivelled. By Feb:t'uary 11 only 3 remained in the chamber. They hatched on February 24 and 25, 1960.

0:1 December 29, 1959, one of the large male nymphs molted to be­

come a supplementary reproductive. A new egg was laid on February 14 and one on March 6. Neither hatched during the remainder of the observa- tion period .

Daily observation.s of the ~)ehavior of the termites of Colony 2 were begun on February 24 and lasted until May 9, 1960. The composition of

the colony on February 24 was as follows:

(supplementary reproductive)

(supplementary reproductive)

S (soldier)

1

2 (large nymphs; at least fifth instars) 3

4

5

6 . (first instar nymphs)

7 174

One of the tiny nymphs disappeared on March 11 but the other two

(5 and 6) survived to the end of the observation period.

Effect of Molting on Termite Activities

The feeding experiments involving the radioisotope supported the work of other investigator s in showing that feeding did not occur (or. at least decreased) during the period surrounding a molt. It also showed that molting termites did not donate proctodeal food. The present study, in which direct observation of termite behavior was made, showed not only the suspension of gnawing, proctodeal feeding, and food donation, but also the cessation of grooming and being groomed. In fact it was found, not unexpectedly, that all the usual termite activities declined during the molting interval except jerking, an activity that increased to some extent.

An analysis was made to see whether or not instar was related to the length of time during which activity was suspended in connection with a molt, and also to see whether or not the various activities were suspended for equal intervals.

A total of 20 cases of molting were observed during th~ observation periods of Colony I and Colony 2; These cases provide relatively few data for analysis, especially in view of the fact that not all of them could be in-

'eluded be~ause of incomplete data. They are sufficient, however" to per- , , mit hypotl1esestegardirig the 'effect of instar on length of molting interval and the differential effect of molting on the cessation and resumption of the 175 different activiti~!L

Daily observation permitted every molt to be discovered. In a number of cas'es a single termite molted several times during the total period of. observation, and the particular instar resulting from a molt was always known exce pt in the case of the older nymphs of Colony 2.

In making the later analysis the date of each molt and the instal' resul t ­ ing were noted. In each case the records of colony activity were examined day by day just prior to and just following the particular molt in order to determine the last day prior to the molt that each of the five activities listed above had been observed and the first day after the molt that each had again been observed. In a few cases the interval of activity cessation apparently started before the beginning of the observation period or ended after its ending, and these cases are either omitted from the analysis, or as with fifth instars and older, they are included and the fact indicated.

The method of basing length of activity-suspension interval on actual ob­ servation doubtless results in longer apparent intervals than are actually correct. It is believed, however, that variations in intervals for different individuals, du~ to this error, are random in nature and that comparisons can still be made.

Tables XXV, XXVI, XXVII, XXVIII, and XXIX present the results of the analyses comparing the intervals of suspension of food solicitation, food donation, grooming, being groomed, and wood gnawing, respectively. TABLE XXV

Activity Suspension Interval for Food Solicitation, According to Instar

Suspension Interval Suspension Interval Ave. Length Prior to Molt After Molt of Activity- No.of Ave. No. Observed No.of Ave. No. Observed Suspension. Indivi- of Range ndivi- of Range Interval Instar duals days (days) duals days (days) (days) +. 5th 5 14.2 11 - 24 5 3.0 1 - 5 17.2·

4th· 4 8.0 5 - 12 4 1.5 1 - 3 9.5

3rd 4 6.8 4 - 10 5 1.8 1 - 3 8.6

2nd 3 5.7 1 - 9 3 1.3 0-4 7.0

Total 16 9.2 17 2.0 11. 2

..... -..J 0' TABLE XXVI

Activity Suspension Interval for Food Donation, Accord~ng to Instar

Suspension Interval Suspension Interval . Ave. Length Prior to Molt After Molt of Activity- No. of Ave. No. Observed No.of Ave. No. Observed .. Suspension Indivi- of Range Indivi- of Range Interval Instar duals days ( days) duals days (days) . (days)

5th+ 5 8:6 5 - 13 5 6.0 1 - 9 14.6

4th 4 4.2 o - 10 4 1.8 1 - 2 .. 6.0

3rd 5 6.4 2 - 16 5 3.6 2 - 6 10.0

Total 14 6.6 14 3.9 10.5

..... --J --J TABLE XXVII

Activity Suspension Interval for G:r.oom.ing, According to Jnsta.r.

Suspension Interval Suspension Interval Ave. Length Prior to Molt After Molt of Activity- No. of Ave. N:>. Observed No.of Ave. No. Observed . Susp.ension Indivi- of Range Indivi- of Range Interval !nstar duals days (days) duals days (days) (days) I I 5th+ 5 1 13. 4 11 - 17 5 5.0 2·-11 18..4

4th 4 8.0 5 .- 12 3 4.0 2-7 12.0

3rd 4 7.0 5 - 10 4 4.2 3-6 . 11. 2

2nd 1 11. 0 11 1 3.0 I 3 14.0

Total 14 9.9 13 4.4 14.3 ! I

...... -J 00 TABLE XXVIII

Activity Suspension Interval for 1.3 eing GroOlned, According to Instar

Suspension Interval Suspension Interval Ave. Length Prior to Molt After Molt of Activity.... r-- No. of Ave. No. Observed No. of ! Ave~ Observed Suspension:

Indivi- of Range Indivi- of Range Interval .. Instar dual days (days) dual days (days) (days) + 5th 5 7.6 3 - 19 5 2.0 I 0,- 9 9.6 I 4th 4 3.2 1 - 6 3 0 0 3.2

3rd 5 2.2 1 - 5 5 2.6 0-6 4.8

~ I 2nd 3 -.vn 1 - 6 3 0 0 3.0 ._.

Total 17 4.2 16 1.4 5.6 I

~ -...J ~ TABLE XXIX

Activity Suspension Interval for Gnawing, According to Instar

Suspension Interval Suspension Interval Ave. Length Prior to Molt After Molt of Activity- No. of Ave. No. Observed No.of Ave. No. Observed Suspension Indivi- of Range lndivi- of Range Interval Instar. duals days (days) duals days (days) (days) + 5th+ .5 13.0 11 - 5 5 I 9.2 5 - 13 22.2

4th .4 9.0 8 - 11 4 7.2 3+- 10 16.2

3rd . 1 9.0 9 4 6.0 5 - 7 1·5.0 .. Total 10 11. 0 13 7.6 18.6

00 -o 181

For activities in which the youngest instars were not seen to engage

(e. g. ~ food donorship) only third instar and older nymphs were con­ sidered. Instars above the fourth ·(5+ in the table.s) were grouped to­ gether in the analysis 'because the exact instar could not be determined.

The wide range in the various activity suspension intervals for a given instar probably reflects the fact that the samples of behavior observed happened not always to include the last example of the activity that occurred prior to a molt or the first that occurred following a molt.

The consistency of the general trends shown in the tables, however: permit the following hypothesis: nymphs representing older instars tend to cease a given activity sooner relative to their prospective molt than do nymphs representing younger instars. They also usually delay resumption of the activity after the molt for a longer time than do younger nymphs. This results in a total activity suspension intvrval that tends to become progressively longer with increased age of instar at time of ;molt.

Further study of the datc;l in T abIes XXV to XXIX indicates that the length of activity- suspension interval was related not only t~ instar but also to type of q.ctivity. The latter may be arranged inthe order of "most affected" to "least affected" according to the extent to which the average interval was lengthened about a molt. Regardless of instar the activity­ suspension interval was usually greatest for gnawing, next greatest for 182 grooming, next for food soliciation, next for food donation, and last for being groomed. This order seems a logical one. Gnawing involves a definite strain on the jaw musculature and on'the bpdy in general.' It - . . ... '.. '. might be expected to beo affected first due m'erely t'o the physiological' .' ,"' .. ' .' ., aspects of molting. Proctode<:tl £eeciing all;d grooming are'examples of less active behavior '. but they too require muscular effort. These acti- vities, then, would be next to go. Food donation and the process of being groomed are both relatively passive activities over which the termite being solicited or groomed has les s control. It can move away from the solicitor or groomer, but the initiative in these activities rests primarily with the other termite. These activities might be expected, then, to continue for a longer time into the molting interval. The actual shedding of th ' old skin, which usually takes only one to a few minutes, is usually accompanied by grooming of the molting individual by colony mates.

Behavior of Individual Termites

The question of a possible social hierarchy based on competition and resulting in a particular organization of the colony, c·an now be con- sidered. Is there,. f~r exa~ple, a ?etectable division of lahorwithhi .the , c~loIlY apart frotrJ. the .ob~iousdivlsi

comparison of the number of times each termite of a given colony en-

O' '.' .~: .. ga~ed in.a par.t'{~ulat typ~~f:.~(~i:li~Vior, In cases of behavior invo1v-

,;. ". .. ;.' .' '. ing two terrnIte~ '{gr90mirig'a,J;l.d.feedi.ng} the active or passive role .. :.. .

.:' .0 of each partner was considered. ~ ...~

mites of each colony was complicated in the present study by the fact

that periods of daily observation were not equa1'in length, as men-

tioned earlier. If, for example, it happened that a given colony was

observed for a relatively long total time while Nymph 1 was molting

as compared with observations during its non-molting phase. then

other things being equal. the data for Nymph I could be expected to

show an activity deficit in the various types of 1?ehavior r'elative to the

behavior of the other termites which molted at other times. In order

to take into account suc h possible sources of error the total minutes

of observation of each termite during its non-molting phase was

determined, A particular activity could then, be expr essed as the

number of examples observed per hour of nonmolting phase'for each

termite. and data for different termites could be comparee.:

It .has already been shown that length of activity-suspension'interval ...... Cl.s;s,b:cia~,e~>~ith molting differ ed with instar as well as with type of " ......

each instar for each activity was used to define the molting intervals 184

for each individual termite. The time spent in observation during such

intervals was subtracted from the total number of minutes comprising

the entire observation period to give the minutes representing that

particula~ termite' s no~-molting interval during which time the q..ctivity

u:nder' conSi~~er:ation had a chance of being observe~. ~t was hoped in

, . this way to make the behavioral data for the·different inrlividuals com-

parable insofar as the effects of molting on the different activities were

concerned.

Tables XXX to XXXIV present the data which bear en the existence

of possible social hierarchies in termite colonies. In each table each

termite from Colonies 1 and 2 is considered separately, and individual

frequencies of a single type of behavior are presented. The frequencies

are given first as the number of instances actually observed during the

entire observation period, and then in the form of number per hour of

observation of the particular termite's non-molting interval. The

latter mE(thod permit's comparisons between individuals.

, " ,,: ,.. :.~ub:~:'~:b~p'.ar,i~;ons may be ~acilitated'by a pl."esentation of these 'data o.0..o• • 0 .. 0 ..:., .'• •0: .:.. : 0.: '" • as ~is·tog;~ms. ~igure 11 give'S the results'for the termites of ,Co·lony. 1 ..

and Fig~re 12 for those of Colony 2. Colony 1 will be considered first.

Colony 1.

The histograms ox Figure 11 show that the male reproduc-

tive did not appear to participate as frequently in the usual ..

TABLE XXX . ., Co~pa.r~son of Food Solicitation Behavior Exhibited by the Termites o£"~he~'Two '. ." Colonies Observed ... ., '. ....: . ,.::~,. : • '.'. ,C 0 LON Y 1 COL 0 NY··.'<,2\:',:' pb'~·e.rva- Hrs. of Observa-, T~B£~:{f;.SoHci·,1 Solici·- Hrs.'q£ ITotal Soli- Solici­ . '. - -; •.\.~;;:.;:.••' I tioritime during citations Ob tations tion time during ::Ya:t:~·{o'ns Ob-~ations Termite nonrrioltin pHase! served per Hr. Termitel non-molb.ng phase :·'kcif..v~~~_!:T.E....:. I ~ .• • I I• ",' 0' 2.1.6 68 I 3. 1 a 29.7 1'-8S! . 2 9 . I 1 . : 1 . I ~ 21.6 5.1 ~ 29.7 I, 83 2 8 I III 1 . ! 1 .°17.3 103 6.0 1 19. 2 29 11.5 . .. I 2 16.2 45 2.8 2 23.6 28 11.2 I I 3 15.4 49 I 3.2 3 18.6 27 \ 11. 4 4 1~.0 I 50 I 3.9 4 22.4 14 1°·6 ; 5 10.5 I 67 6.4 17.8 48 2 7 %} 1 . 5.2 I 6 9.4 I 49 S 29.7 46 1. 6 'I I I I I I I I i

.­ 00 \,]1 TABLE XXXI

Com.parison ·of Food Donorship by the Term.ites of the Two Colonies Observed

COLONYl COLONY·2 Hrs. of Observa-! Total Do-I Do- Hrs. of. Observa- Total Do- Do- tion during non- ' nations nations tion during non- nations nations Term.ite m.olting phase Observed per Hr. Term.ite m.oltin~ phase Observed per Hr.

'21.6 26 1.2 , 29.7 68 d" I c!' ! I 2.3 I ~ 21.6 176 8.1 ~ 29.7 31 1.0

1 19.0 112 5.9 1 21. 3 18 0.8

2 ·17.~ 67 3.9 2 24.4 24 1.0 .. 3 13:7 59 4.3 3 21.8 75 3.4

4 12. 1 I 49 4.0 4 24.2 94 3.9 ! ... I 5 .. 12..0 38 3.2 5 20.7 19 0.. 9

6 11.4 15 1.3 6 28.5 0 0.0

S 29.7 30 1.0

.... CD C1' TABLE XXXII

Compar.ison of Grooming Behavior Exhibited by the Termites of the Two Colonies Observed

c 0 L 0 N Y 1 C 0 L 0 N Y 2 Hrs. of Observa- Total Groom- Hrs. of Observa- Total Groom- tion dur.ing non- Groomings ings tion during non- Groomi.ngs ings Termite molting phase Observed per Hr Termite molting phase Observed per Hr.

rf' 21.6 44 2.0 d" 29.7 186 6.3

~ . 21. 6 81 3.7 ~ 29.7 88 3.0

1 17.2' 83 4.8 1 18.6 11 0.6

2 14.8 52 3.5 2 22.7 18 0.8

I 3 13:2 35 2.6 3 18.3 25 1.4 4 ' 10.2 30 2.9 4 21.9 65· 3.0

5 7.6 9 1.2 5 16.4 3 0.2

6 3.8.. 4 1.0 6 25.0 0 0.0 S 30.0 1 0.0

...... ex> -.J TABLE XXXIII

Comparison of Frequency of Being Groomed for the Termites of the Two Colonies Observed

C O· L 0 NY 1 C 0 L 0 NY 2 Hrs. of Obser- Hrs. of Ob!'ler':' vationduring Total Times vatio~ during Total Times nonrnolting Times Groomed nonrno1ting Times Groomed Termite phase· Groomed pr Hr. Termite phase Groomed pr Hr.

cJ' 21. 6 ~. 57 2.6 d' 29.7 88 3.0

~ 21.6 64 3.0 ~ 29.7 . 78 2.6

1 . 19.8 32 1.6 1 23.8 20 0.8

2 18.2· 42 2.3 2 26.0 14 0.5

3 17.4 . 59 3.4 3 26.3 27 1.0

4 15.8 20 1.3 4 25.8. 29 1.1

5 16.6 . 37 2.2 5 23. 1 43 1.9

6 16.4 27 1.6 6 28.3 14 0.5 . I S 29.7 84 2.8 ...... 00 00 TABLE XXXIV

Compar:i:son of Gnawing Behavior for the Termites of the Two Colonies Observed

c 0 L 0 N y 1 C 0 L 0 NY 2 Hrs of:0bser- Toeall Hrs. of Obser- Total vatiori during Times IGnawing vation during Times Gnawing ~onrn.olting Gnawing I Times nonrn.olting Gnawing Times I Termite .phase Observed i per Hr. Ter:mite phase I Observed per Hr. ! ~ 2(.6 20 0.9 if 29.7 i 10 0.3 I I ~ ',21. 6 58 2.7 ~ 29.7 0 0.0

1 16.8 66 3.9 1 17.4 49 2.8 I I, I 2 10.6 58 5.5 2 22.7 I 84 3.7 I I 3 .11.9 61 5.1 3 17.2 I, 50 2.9 I 4 8. ·1 .52 I 6.4 4 20.9 130 6.2 I 5 n.7 23 I 2.0 5 19.3 4 0.2 I . I 0.0 6 11. 0 . 6 0.6 6 - 0 ..,

S 29.7 0 0.0 I .....

...... (Xl -.0 6

4

2

Food Donorship

4

2

6

4

2

: .. ·.·· ..:,:6: .'."':"

4

2

1 2 3 Termites Fig. 11. Comparisotl of Colony Activities for the Different Termites of Colony 1. 191

6 Food Solicitation

4

2

Grooming

Being. Groomed

." .. ' ~ ",'.', .:.•. .' :.' . 6

4 Gnawing

2

1 2 3 4 5 6 S Termites Fig. 12. Comparison of Colony Activities of the Different Termites of Colony 2. 192

colony activities as the other termites, although his general stage

of vigor appeared as good as that of any other . The male was

rarely approached in food solicitation, being as infrequent a . .' ' .. '. . ,.....~"."" .. " ' Ji,y:;~~~'~~J~~~J~~t~~rt";:&ek::~::'~:~:~~:b:~~~¥~::~:~k;:=~~"1;NF1k;!(;

.', ." the female reproductive as shown later), groomed, and was

groomed at an average rate for the colony, but was only rarely

observed to gnaw wood.

The female, on the other hand, seemed to be one of the more

active members of the colony. She was most frequently observed

engaged as a donor of proctodeal food. She was seen to gnaw at

the wooden walls and floor of the observation chamber over twice

as often as the Inale, although not as fr equently as the older

nymphs of the colony. She was observed to groom colony Inates

more often than any other terInite of Colony le:x;cept Nymph 1, and

she also received an above-average amount of grooming.

. . .., . Nytnph lwasth~)Cl.rgestari4·o1destoLtheI1yrnphs.It was .a;. £6lirthlristar:"'aftb"e'\je'i'i~iri'ng' 6:£'-tfi~'6'b~el'~~a:~i6;r':p~'~;i6d::'£Jc{:i'/'::"':'::::

Inolted once thereafter. It was the chief grooIner of the colony

and was second only to the queen in frequency of food donations.

It solicited food from colony mates Inore frequently than the

other large nymphs, but it exhibited gnawing behavior less frequently. 193

o This appeared tc be a genC::J:al effect: termit~s with the

highe,st fJ:'equency of food solicitation exhibited the least ~z~f!N,;,t;:!¥!:'c"'M';:;\i·P/c~~l~i~i:~'~~~~ig'f.

..... , .;...... , ,", Nymph 2, a third insta:c nymph that molted during the

observation period to a fourt~, and Nymph 3, a second instar

nymph that molted to a third, occupied relatively unexcep-

tional positions with regard to the frequencies of all their

observed activities. Both spend much of their time gnaw'-

ing the wood of the observation chamber.

Nymph 4 was the chief gnawer of the colony. It molted

twice during the observation period to third and then to

fourth instar. It was less frequently groomed than the other

member s o(the c'olony - - less even than the two younger

nymphs.

Nymphs 5 and 6 were first instars when first observed.

Nymph 5 molted three times to become eventually a fourth

ip.sJcir aI.ld Nymph 6 molted twice. Nymph 6 . disappeared from , : ~ '. . ,

the colony (probably eaten) before molting again; First~and

second-instar nymphs do not gnaw wood or groom other nymphs.

Neither are tr.ey fed from or groomed as often as are older

nymphs. This doubtless accounts for the relatively few ob-

servations of such behavior in the case of Nymphs 5 and 6.

For Colony 1 the general picture is one of individual 194 differences. Even when the older nymphs, which should be a rf~latively homog" .LOUS group, are considered alone, they are found to differ considerably in the frequency with which they exhibited different types of behavior. One might be

characterized as a "gnawer: II another as a "groomer, II and so on. Such individual differences, which do not appear to . . be based entirely on physiological and morphological differences of caste or instar, may indicate a sort of division of labor similar to that shown by Pardi's wasps and Kalshovenls termites.

Colony 2

The histograms of Figure 12 present the data for Colony 2.

The termites of Colony 2 apparently did not exhibit as much· food solicitation behavior as did .those of Colony 1, probably .. because of t~eir g';teater average age. The results agree with those of Colo:n.y l,in showing that t.ermites· doing most of the .. . . "I. ," : :," wood gnawing di~·';h~:';1,.~.~~~.. ·~:rPO~(.cit£obd:··~gii2:it~tion and vice- ", .. ' : . versa. The supplementary reproductives gnawed very little in contrast with the primaries of Colony 1.

The male supplementary reproductive was a relatively favored donor in Colony 2 and also spent much time in groom- ing activities. The female supplementary reproductive also in 195 contrast to her counterpart in Colony I, was not especially sought after as a donor. She was frequently groomed, how- ever, and spent a considerable amount of time in grooming other members of the colony. ·~i~t~ttSt~~ti!!w1I~IT,,~rc'~::::·':::i:er::::::::~g course of theobservatio.n period. Nymp.l 1 was .an infrequent donor and rarely engaged in grooming or being groomed. Nymph

2 was relatively ipactive but spent alarge part of its time in gnawing. Nymph 3 dId not gnaw as much as 2 but was a more frequent donor and groomer. It was also groomed more fre- quently than 2. Nymph 4 was primarily a gn"wer but it also participated rather f:requently in grooming activities and as a donor.

. The t~o s,~all n;ymph s, 5 and 6, were r.elatively·.inactive, d

passing from first instar to fourth. Nymph 6 molted twice..

The soldier was frequently groomed by th~ other termites.

It was never observed in a gnawing attempt and only once did it seem to be grooming another termite. As a food solicit0r it ranked fifth. 196

In Colony 2, as in Colony 1, there appeared to be individual

differences exhibited among the termites indicating an indivi-

dually-bas'ed division of labor. The data presented so far have

not been of a type that would permit the detection of a pos sible

;: .. :,', .. ..;~;'::'i>i.:··i;i.,,~:;,~S~~,¢~i.;~1;\~~:¢::~~a,~,~,h~:l.":'-'{P;~cedeht:et()£ood,often used in other ..::...~ ',.;' ····i... :.:·'::·>.:'· :':..;' ".': .'.' ~""~'.". ~;; ..' .: . studIes as an index to dominance, cannot be used in connection

with termite behavior without taking into accounfageCl:nd.cas.te .. ',' '-', ,"':." '.,'

. . '.; .,,",-;'.', of the individual, which are related to its degree ofenforce.cr,·

dependency on colony mates for nourishment.

Another criterion of dominance in other studies has been .

the relative frequency of threats or blows administered or re-.

ceived by each individual of a group, relative to everyother·

member. Among termites the only comparable behavior ab.:. ..

served is the jerking exhibited by disturbed individuals. If

apparently serves ordinarily to communicate alarm. Thisbe':'

havior not only occurs when the colony as a whol~ is disturbed,

hOwever, but is also exhibited sometimes by a termite that is

~b:rti:s}ie't:lhY·a:[J.(;jthertermite, by onewhClsepath is blocked by .. ' .. '. ',", - .... ,.. , ~ '-, ' ..- ' .

. ariother,or for no apparent reason other than a termite's proxi-

mity. It was apparently not directed. consistently agamst

specific individuals and was r s not .. .1 expression of a. social . ,

hierarchy. ,Jerking did appear to 'I-,~ associated in nymphs with 197

molting. A test of this hypothesis was made by counting the

number of times each nymph displayed this behavior during

the molting interval (defined her e as the interval during which

food solicitation was suspended) and comparing it with the

number of times it was displayed at other times. Table XXXV

presents the data for both Colony 1 and Colony 2. Jerking

or 2·.32 obse.rvations

.' ~ " .

...... : .. '... :,.,,:.•...,... per hour. Nymphs 1 and 3 of Co~ony 1 were seen to jerk much

more frequently than the other termites of that colony. It would

be interesting to know whether either of these nymphs was a

potential soldier. Perhaps the jerking behavior is a soldier

characteristic that appears before more obvious morphological

changes. The primary reproductives of Colony 1 jerked much

more frequently than the supplementaries of Colony 2. Perhaps ',t·'

-:,

...... ;"."

COD>parison of Frequencyo£ j~t;4~~~!i~~tt~~~c'~~:~~~~~~iNomnOlting mtervals

I C 0 L \J L'l, ,.:.1.', .,' ,,:.,.:,.:, .,>:~." • o NY 2 :<" ....:; ... ;.~'.' •. ::T:~ ..;~.; ;~:::'::~».:;;.~~~ ~~',":"., ,.I.J Molting Interval I .....;"...•.~" .• \Noninolting Inter_v:al Hrs. of' Cases I Hl."s. of ICases I T~r-IOb~er- o~ Jerkr' Cases ! O',~ser-lof Jerk Cases nl1te Ivatlon ln~ per Hr : vation In per Hr

" I cJ! 21 ., 6',:;£;b:/'~~:::'i:+,?;~:'0,;'T" . , j 29.7 I 5 I 0.2 I ~ 29.7 15 i 0.5 1 S 29.7 69 ! 2.3 1 1 I 1.9 9 .1 4.8 9.8 5 19.9 7 0.4

2 4 1.1 4 23.0 7 0.3 I 3.8 I I 3 I 5.9 16 I 2.7 1A 18.5 48 2.6

4 I 8.7 6 I 0.7 20.7 5 0.2

5 4.9 6 I 1.2 18.2 I 0 I 0.0

6 7.5 I 1 I G. 1 Total for 32.6 I 42 I 1.3 100.2 67 0.7 Nvnlphs 199

this behavior was related to the fact that no soldier was o present in Colony 1. This type of alarm-behavior may

prove to be characteristic of young primary reproductives

just beginning a colony. . ,:. Three other types

: '. "" " • t'

;,.":.::,':,.

':";i"," "':'."

...... ·.. <);::W;}~~~;::~~~~i;~~:~i~~$.;~~#j~~!g:.'Wa;$.:~~:W!~~ ~~"¢~~#l·9.i:l:~X·9l1:~.· .. ?e,'~:~si():n . <:':'''/:'}:.::' . :;:. """", ':"'.'---' -,.." , ,'. ~'<::':~:,::'~'~,.. ,

., : ..

..' :',

as evidenc edbythe'-£a¢tt1?-at joints wereal~ays1<.fe:ptse,~i.ed.

The only termite seen to engage in plastering was Nymph 3

of Colony 2 (a fifth instar or older). It sealed the spaces

between the plastic " roof ll and the wooden chamber by ex-

truding a fluid from the anus onto the junction area. It

began these plastering operations by running its antennae o

TABLE XXXVI

Comparison o:f Frequencyof':'~~I$.~t.¢P:iiing r:md Pellet-arranging :for the Dif:ferentTerilltl;$~$:';~#' Colonies

. ,'.. "

··C.9~P.~ :"!: ... 1 CO:L0.NY.· .. 2 .:t"·

>' Tending .El$gE?:<>tArf~,ingp"ellets Termite

, 10.2 I 5 0.2 29·7 .L 0.0 .;1. p.4- I 4 I 0.2 29·7 0 0.0 .1' I I 29·7 0 0.0 I 12 0.6 8 0.4 19·9 0 0.6 I 1 0.1 17 1.0 23·0 1 9. 0.4 I I 7 0.;3 4 0.2 18.5 I 8 ·14 0.8 1 0.1 2 0.2 CDo 7 1 .Q;.{)5 4 0.2 .,j' 0 000 0 000 1802 I 0 Q.'Q 0 0.0 0 0.0 0 0.0 .L;. ······:1"· 97·2 34.< ~\ O~l~ 40 0 .. 4 100.2 II ,'., ..o.~ i:'( . 40 0.4

N o o 201

l' () along the roof margins. At spots wher e the roof did not fit

snugly the an.tennae were passed to and fro over the area for

several seconds. The nymph then reversed direction and

brought the tip qf its abdomen into position at the exact point

,examined by the antennae. The tip was pressed firmly

against the area and the termite's entire body began a quiver-

i1'),gmovement that lasted s~v:~ral,s~c9.nds. Fluid was seen to

•• " '. l

. ';.:'-.""","" ' ..

- .. . ".: ", ,". .:::';':;';"-:.;" . . '.' ,:::~,9¢;,~:~:~gh.'$j··,·" .' ',<' . "'- -:',:: . ',::";:: ,_.;:-':;:", '. , "":"':~':::;.' .. ~.:> ".- -;.:. : .".:..:.: .,' " ' ':'.::',:... , ."". , ; ": '. '.- ... ". "~ .: .. , . ',' , ',',,:' ",;,:: .-: .. ~.:::

;h;~'",;i(i'i;,:';.;!~%;~~;i!i~~;f&;";?p.el.·let' rearrangin~':/ii~:~~'i:~~~" ~sTable XXXVI shows. In

. yo. ".;~ . ", .,'.

C,?lony 1 the primary reproductives played a much bigger role

in the work of the colony, but again the large nymph.s were the 11 chief workers.

Comparison of Time Spent in Food Solicitation

An analysis was carried out to see if length of time spent in the

act of food solicitation differed according to termite. Table XXXVII shows 202

TABLE XXXVII o o • Comparison of Time in Seconds '6pent by Each Termite in 0 o~f P.ioctod~a.1.' ~. o the Act. lf eoediI1:g 0: ••• 0 ,':.....

.' .~ ,

• • 203

the results by termite for Colony I and for Colony 2 separately. It is

obvious that the youngest instars spent a much longer time in the actual c

solicitation process than did the older instars, the soldier, and the

reproductives, The average solicitation times given for the youngest

o nymphs are actually shorter than they should have been; ter~ination of

feeding in these cases were usually brought about by the walking away . :

. ~ .~ .:~ ···;"'l·.r<~,·~·::}X:·:: ~~:.: '. .. 0 '-" ....'_:::,." ....::~:., ..:: .. ·of the donor before the solicitor had finished feeding, In the case of :·~::i',:,2~~'~!:;:;;)t;:':!/<::it:~;//i::. :.... '.,. - .' 0 :.:,:;: ~··:;:::;·;(::::;;(i.":.t::,:i~·\,:·:':t·h::e.J.arge· nYIPphs, soldier, and reproductives feeding was usually ter-

~eed~ng time did not seem to be related to o

:/:'..\: ,:",:,:,:",:,,:';::,;,:1. ':.:,,::.':::i:.:·.·:;-;I:nY~,r~;qt:er,m];te -Relatlonshlps r'.<:·~:·::~.. ::""':-:;:::>;":<::~'i'~ -:~::·\::;~;·:;:';f:;}':~~?;{;~i\l!J:.lf~tO:~lli~\·S-~p'~iri.e 'the, various termite activities have been analyzed ::~~~.::.:;:>'- \: :':~r·'·:~::;·:,::~~~~;~:~:~t,:~Y};:YI0!:f.~:~~~0-~B(~f f&0:r:E::;;:,:':~:/.':'~;~i: :':', :. '. '" . .;'.~L'.,,:,:·· ,", :.:·.<::·'.\;X::';§{{·::Wij:~i~~~;~~~gl~lm:(!h~·~aJoior exhibited by individual termites. Some of the ;'/:].~:\;:::'~~~:'-" ~);:A~~~~~j,;~?fKit~Wk~;~~/:.:':.: '~.i ::::\:.)) \::: >", J /.:" •• - '. ' -.~.):i,<;,~,,~·::~:(?\V::~,t.:fIi::~'r;Jt~M~~,~§~,':o.:~,~·~.~Yed r equired the interaction of two individt:tals (procto-

,:i};i]';:i!~7~~;~\~101~~gLI~~~~:~and graan:ing). . Is there evidence that the termites s hawed

consistent partner selections? These data would be the best place to

look for a possible social hierarchy in these termite colonies. Tables

XXXVIII and XXXIX show these relationships for Colony 1 and Tables XL

and XLI show them for Colony 2. The data for Nymphs 5 and 6 for

Colony 2 are pooled,

For Colony I every colony member except Nymph 1 selected the fe-

male reproductive most frequently as donor during food solicitation, and 204

TABLE XXXVJTI

Comparison of Frequencies with Which Each Termite of Colony 1 was Observed to Solicit Food from Each of Its Colony Mates

Donors Solicitors {Recipients ~ ~ 1 2 3 4 5 6 Total

1 2 24 17 28 12 11 9 103

0 . 2 0 18 10 2 11 3 1 45

3 1 15 11 8 7 5 2 49

4 1 18 13 6 3 8 1 50

5 5 40 8 3 5 5 1 67

6 2 28 12 3 1 2 1 49

Total 26 176 112 67 59 49 38 15 542 205

TABLE XXXIX

Comparison of Frequencies with which Each Termite of Colony 1 was Observed to Groom Each of Its Colony Mates

Termites Groomed IGroomerG ~ 1 2 3 ,." 4 5 6 Total c!' 20 3 8 8 1 3 1 44

~ 27 6 8 14 7 8 11 81

o • 1 12 22 10 18 4 9 8 83

2 8 6 13 12 3 7 3 52

3 6 9- 4 11 2 3 0 35

4 3 6 4 5 3 6 3 30

5 1 0 2 0 4 1 1 9

6 0 1 0 0 0 2 1 4

Total 64 57 32 42 59 20 37 27 338 I 206

TABLE XL 0

00 0 Comparison of Frequencies with which Each Termite of Colony 2 was Observed to Solicit Food from Each of Its Colony Mates

D 0 no r s Solicitors d'1 ~ S 1 2 3 4 5 & 6 ·l'obil

c'.,; t- 20 8 3 4

~ 4 5

S 5 0

1 7 9 3

2 1 5 9 6

.. 3 4 5 5 3 2 .. ,:, .. 4 3 4 1 1 0 2

:} 7 25 0 1 4 7 4 48

Total 31 68 31 18 24 75 94. 19 360 o 207

II 0 .. TABLE XLI 0 0 .. D ...... , _Compar.ison ai.Frequencies with Which Each Termite of Colony 2 was Ob.served to Groom Each of It s Colony Mates 0 0 "

"" - o· -...... :.", Termites Groomed .. : .:;;., 1 2 3 4· 5&6 Total

4 10" '19 20 186 0

o. 1 -4--- 3 0 88 '. 0 0 .1 0 1

'" 1 1 2 4 11

2 2 3 1 3 1.8 ~.'-": '.

00. ". 10:;--'. '.~ 0&,5 '.," : ~:-::'\' 2 '3 0 1 1 8 c···...... ,. ~ :: ';.-." ".,"; .:'... ..:';:; , 4 7 ·1 14 7 7 9 22 6.5 D :} 0 0 0 1 0 0 "2 3

Total 88 78 84 20 14 27 29 57 397 208 she was next most frequent donor for Nymph 1. The smallest nymphs were particu1a.r1y likely to solicit from her. Next most favor ed as donors by most colony members were the large nymphs, particularly

N l' The results for Colony 1 indicate that feeding relationships were probably based on biological considerations of caste and instar and perhaps on individual preference but were not motivated by a dominance type of hierarchy.

Grooming in Colony 1 appeared to follow similar lines. The repro-· o ductives spent the largest percentage of their grooming time on each other, and the nymphs also tended to groom the reproductlves.t

Nymph 2 concentrated its grooming activities on Nymphs 1 and 3, and 0

Nymph 3 concentrated on Nymph 2.

o In Colony 2 the female reproductive again appeared to be selected most frequently as dono,r by most of the other termites, but donor

"preference" varied considerably. Even the soldier appeared to be selected preferentially by one individual, Nymph 2.

In grooming, the reproductives of Colony 2 concentrated on each other and on the soldier. Nymphs 2 and 3 groomed the male reproduc- tive most frequently, and Nymph 4 tended to groom the small nymphs.

It appeared generally that a few specific individuals in each colony were favored in the food solicitation and grooming activities of most o 209

colony members. Other specific individuals tended to be missed by

most of the colony. This apparent selection was to some extent re-

~.ated to caste.and instar; for example, the reproductives were most

frequently groomed and the youngest instars were not favored as

donors. The older nymphs tended to do most of the grooming and

most of the donating of proctodeal food.

.. Behavioral differences did not follow caste and instar lines com- o pletely, however. They appeared to involve individual attributes and ° " .. "preferences" as well. One of the chief contributions of the direct o obOservation studies was this demonstration of individual differences

. • 0 . . o .. o in termite behavior. Individuals differed in the treatment they re- o 0 I) ceived from colony mates and in the relative frequency of the acti-

°vities in which they themselves engaged. In Colony 2, for instance, ..o • 0 " NYlnphs land 2 were to most outward appearances like Nymphs 3

and 4, yet the latter were seen much more frequently being

solicited for food than were the former. Did Nymphs 3 and 4 have

an especially attractive odor or a stronger one as compared with

Nymphs I and 2? What was the basis for the selectivity shown? The

answers to questions such as these may have an important bearing

on the problem of caste formation, particularly that of soldier

production. Caste differentiation in Kalotermitid termites appears

not to be genetically based, but is believed to be a phenomenon based 210 either on a sociohormone or on an inter-individual sensory stimula­ tion acting through the central nervous system on the humeral system (Brian 1957). The regulation of soldier production appears to be brought about through a direct inhibiting action of soldiers al­ ready in the colony. This does not answer the question of what stimulates the appearance of the first soldier in an incipient colony or explain why one nymph and not another becomes a soldier.

Weesner (1960) has suggested that a positive principle must be in­ volved as well as a negative one. Such a positive principle may be sought in direct observation experiments like the present one in which differences in treatment of the various nymphs from the first instar could be studied in relation to subsequent soldier production.

No particular dominance hierarchy was indicated by the investi­ gation. Apparent tendencies for particular termites to engage in certain activities did not seem to follow a dominance type of pattern, though it may indicate a sort of division of labor in the colony. In­ stead of showing a st:.i.'aight-line chain of behavior characteristic of social organiz ation based on dominance. a particular activity such as grooming or food solicitation was as likely to be reciprocated as not. ThE: present study. then. is in line with the earlier work men­ tioned by Allee and Dickinson regarding the lack of a dominance hierarchy among termites. It is also in line with Kalshoven' s finding 211 that there is an individually-based division of labor among members of a colony.

Do the studies of termite relationships based on direct observation of small coloni.es support the findings of the radioactivity studies?

Because only two colonies were followed in the former case the data for the different types of termites are too few for detailed com­ parisons to be made. However, both studies indicated that molting individuals do not feed and are not fed from, that soldiers do not feed dir ectly from wood, and that small nymphs tend to do more proctodeal feeding than large nymphs. In addition, both studies failed to indicate food transfer relationships based on specific combi­ nations of sex, caste: and instar. 212

SUMMARY

Colony development studies were made primarily on 240 incipient colonies of C. brevis, each colony produced by a pair of primary re­ productives reared in the laboratory in special termitaries. The colonies wer e examined after intervals varying from one month to twelve months. Survival of the original pair was at least 50 percent for the cases falling in every interval but was best for the younger colonies. Egg and nymph counts indicated that there was an initial period of egg laying that occurred within the first two months of pairing, after which egg production ceas ed until about the ninth month. The data suggest that a year-old colony can be expected to contain from 4 to 8 nymphs of at least the fourth ins tar, represent­ ing the first group of progeny, and several much younger nymphs.

No soldier is likely to appear during the first year. The male: female ratio in the incipient colonies studied was 1. 13: 1.

Egg produc.tior~ by supplementary reproductives confined with several large nymphs was usually greater than that for primary pairs but did not appear to show the definite spacing into first and subsequent batches. One-year-old incipient colonies produced as 213

the :t"esult of the mating of a primary and a supplementary reproductive

gave survival and progeny figures comparable to those of colonies

arising from a .. pair of primary reproductives. Colony development

characteristics, however, appeared to follow those of primary-or

sup,plementary-produced colonies depending on the type of female

reproductive.

Isolated supplementary reproductives and old prina ry reproduc-

tives isolated from established colonies rarely survived.

Colonies in termitaries made of woods designated as favored showed

better survival and more progeny than colonies in unfavored woods.

Behavior was studied with the radioisotopic tracer technique

8S (using Sr ) and by direct visual observation. The radioisotopic o studies investigated the possibility of consistent food transfer relation- o

ships based on caste, instal' or sex, or on specific combinations of

these biological catego ries. Large nymphs, small nymphs, and

soldiers of both sexes were made radioactive by having them feed

(either first or second hand) on wood soaked in a Sr8S solution. They

were then paired individually in nonradioactive wooden termitaries

with nonradioactive termites in all of the 36 possible combinations.

Strength of feeding " preference" was evalu.ated in terms of amounts

of radioactivity (counts per minute) transferred from radioactive donor

to recipient partner for all combinations of termite type s. o

214

Termites confined in a radioactive termitary tended to reach radio­ activity equilibrium in a week or less. Fecal pellets in at least one case became "hot" within eight hours. Biological half-life for Sr85 in termites appears to be about one week.

~. brevis soldiers have a gutv.:olume between 1/3 and 1/4 that of nymphs of comparable weight as indicated by radioactivity exhibited after a week in a hot termitary and by morphological evidence.

Data bearing on feeding relationships were corrected for degree of donor radioactivity and for relative gut s~ze. Soldier recipients ac- quired significantly more radioactivity from their donor partners than did nymphs, and small nymphs acquired significantly more than did large nymphs when size was taken into account. These results are related to the recipient's degree of dependency on colony mates for nourishment. No statistically significant feeding relationships based on specific combinations of sex, caste, and instar were found, al- though there appeared to be a tendency for small nymphs to obtain more radioactivity from other small nymphs than from large nymphs or from soldiers.

The study supported past observayion that nymphs in the molting phase do not feed from wood or from colony mates, do not allow other termites to solicit food from them, and do not defecate. 215

Soldier mortality rate under the. experimental conditions was o greater than that of small nymphs, which was greater than that of

large nymphs. The investigation provided quantitative data confirming

earlier reports that soldier termites are unable to feed directly on

wood but must depend upon colony mates for food.

In the direct visual observation studies of behavior two small

I'cclonies" of termites were observed daily for several weeks and o the behavior of every termite was recorded during the observation

periods. Behavior such as food soli.dtation and donation, grooming

activities, gnawin,g, and "jerkingll was studied to see if there was

evidence of a dominance- subordinance hierarchy similar to that

characteristic of other animal groups. Such was not demOnstrated, o although individual behavior differences not based entirely on caste

and instar were indicated.

The effect of molting on the length of the suspension ir.o.terval for

the different types of termite activities appeared to be related to

instar, the older the instar the longer the interval. Length of the

nonactivity interval also varied according to the activity involved. It

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