Activity rhythms in the American cockroach, Periplaneta americana

J. Insect Physici., 1970,Vol. 16, pp. 1555to 1566. Pergamon Press. Printed in Great Britain

ACTIVITY

RHYTHMS IN THE AMERICAN PERIPLANETA AMERICANA* G. R. LIPTON?

Department of Entomology (Received

COCKROACH,

and D. J. SUTHERLAND

and Economic Zoology, Rutgers University-The University of New Jersey 3 November

1969;

revised

12 February

State

1970)

Abstract-The American cockroach has been the object of much attention with respect to its biological rhythm of activity. Some authors have indicated the presence of both exogenous and endogenous activity rhythms in adult males. The object of the present study was to determine the variability of such rhythms in a laboratory population, as assayed with two types of recording instruments, one of which was a capacitance monitor. The second and most frequently employed instrument was a running wheel cage. Exogenous (entrained) rhythms were determined in an LD/12 : 12 lighting regimen, and endogenous (free-running) rhythms were determined in constant darkness. Under an LD/12 : 12 lighting regimen, 93.4 per cent demonstrated entrained rhythms or patterns (loose or weak rhythms), and 4.4 per cent displayed apparent random activity. The rhythms and patterns generally were similar to those previously described in the literature, namely the onset of activity occurring within the first few hours of darkness, followed by a relatively quiescent period in the latter part of the dark period and throughout most of the light period. However, a large proportion (31.8 per cent) also possessed a secondary active phase in the first hours of light. Of 75 adult male cockroaches examined in constant darkness, 34.7 per cent exhibited free-running rhythms, 40.0 per cent demonstrated freerunning patterns, and 25.3 per cent displayed apparent random activity. The possibility was discussed that a laboratory strain of cockroaches does not show a high percentage of free-running rhythms since the presence of these rhythms is of little survival value. The adult female cockroaches examined did not exhibit activity rhythms that were related to the lighting regimen. A possible relationship of activity to the reproductive cycle was suggested by some experiments, i.e. active when carrying an oiitheca and relatively quiescent after deposition. Only virgin females exhibited an entrained activity rhythm similar to that of the adult males. INTRODUCTION

THE ACTIVITYrhythms investigators

of biological

of the American

cockroach

have been studied

clocks using a variety of recording

by several

apparatuses : tilting cage

actographs (CLOUDSLEY-THOMPSON,1953 ; HARKER, 1956), indirect attachment of the cockroach to a kymograph (HAFXER, 1956), running wheel cages (ROBERTS, 1960, 1962), and an infrared detector (BRADY, 1967). There is general agreement that the adult male American cockroach has a rhythm o’f activity under 24-hr

* Paper of the Journal Series, New Jersey Agricultural Experiment Station, RutgersThe State University. This work was supported by Public Health Service Grant No.

CA-07299. t Present address: Department of Biology, Trenton State College, Trenton, New Jersey. 1555

1556

G. R. LIPTONANDD. J. SUTHERLAND

cycles of alternating light and darkness. Under these conditions, activity is largely confined to the first few hours of the dark period. However, there is disagreement concerning the persistence of these rhythms under constant conditions. CLOUDSLEY-THOMPSON(1953) found a gradual decline in total activity of isolated cockroaches in constant dark. HARKER(1956) reported that under constant conditions of light or dark the previous rhythm of activity (under alternating conditions) was carried over a period of 3 to 5 days, after which the activity became random or arrhythmic, When alternating conditions of light and dark were re-established, the cockroaches immediately exhibited a new rhythm of activity. However, a persistent rhythm under conditions of either constant light or constant dark was demonstrated in adult males by ROBERTS (1960). In constant dark the rhythm persisted for at least 3 months. Disagreement or uncertainty is also suggested in a few studies concerning the presence of activity rhythms in adult females. Bursts of activity following the production of an oijtheca have been noted (CLOUDSLEY-THOMPSON, 1953). HARKER (1956) indicated that the activity of female adults might be affected by a reproductive cycle, but later (HARKER, 1958) implied the existence of an activity rhythm. To our knowledge, no author has adequately stressed the possible importance of individual variation in the ‘strength’ of the biological clock. Although this variation may only be indicated by the clock’s visible expression, i.e. activity as recorded by various laboratory apparatus, such variation would be encountered and be important in investigations of the clock’s mechanism as well as the pathological effects of its interruption. In the latter regard, our failure, as reported by HAR~HBARGERand TAYLOR (1968), to induce tumours or lesions by repeated implantation of suboesophageal ganglia (HARKER, 1958) may have been due to such variation. The object of the studies reported here was to determine the incidence and variation of exogenous and endogenous activity rhythms in our laboratory strain of cockroaches, information which was necessary in studies of the cockroach hunger drive and its rhythm. MATERIALS

AND METHODS

Cockroaches Specimens of adult Periplaneta americana from a laboratory colony in culture for 30 years were, with the exception of certain adult females, entrained in battery jars on a LD/12 : 12 lighting regimen for at least 2 weeks prior to experimentation. This entrainment was initiated within 1 week after the larval-adult ecdysis. In studies with virgin as opposed to mated individuals, adult females in a newly ecdysed untanned state were isolated and after 24 hr considered ready for experiment. Individuals with defects, such as two or more legs missing, no antennae, or badly mutilated wings, were not included.

Conditioru of light and dark Experiments were conducted under two different environmental conditions : a clock-controlled light-dark cycle (LD) generally 12 hr each condition (12 : 12),

ACTIVITY

RHYTHMS

IN

THE

AMERICAN

COCKROACH

1557

to reveal exogenous activity rhythms (entrained), and constant darkness (DD) for endogenous rhythms (free-running). In the former set of conditions the cockroaches were exposed to the LD/lZ : 12 lighting regimen, with lights on at 10.00 hr and off at 22.00 hr. In some instances, the cycle was reversed, i.e. light from 22.00 hr to 10.00 hr, to determine that observed rhythms were relatively independent of other factors. Light was provided by a pair of 40 W fluorescent bulbs with a range of intensity of 160 to 300 lx, depending on location of the cockroach enclosure. In LD conditions no attempt was made to regulate laboratory temperature whose maximum fluctuation was 20 to 26°C. The condition of constant darkness was maintained in small light-proof individual cabinets in a subterranean room. A concrete floor assured a minimum of any vibrational disturbance, and temperature was maintained at 23 + 1°C.

Measurement and recording of activity Activity monitor. This activity recorder was similar in principle to that used by SCHECHTERet al. (1963) and based on the dielectric properties of the cockroach. The cockroach enclosure, or transducer (constructed by M. Rosengarten of Columbia Presbyterian Hospital, New York), consisted of two copper plates cut in a chequer board design, each being held between two sheets of Plexiglas, which served as the base and top of the enclosure. Plexiglas outer walls and inner partitions (height 1.9 cm) divided the enclosure into corridors. Cockroach passage into and out of an area between plates alternately increased and decreased the capacitance, the change being relayed by an FM transmitter, receiver, and a pulseshaping and trigger circuit, which yielded pulses suitable for registry and count on a digital recorder. Printing of the number of passages per unit time was controlled by a recycling timer. Running wheel cage. Cost of the above activity recorder per unit and its sensitivity to electrical interference were responsible for the selection of a running wheel cage as the major apparatus to monitor activity. In contrast to those described by ROBERTS (1960), the wheel cages were of plastic and metal construction as suggested by models of M. Loman of the Max Planck Institute, West Germany. Circuitry included electromagnetic switches (Hamlin Inc., Model DRS-5) and recorders (Esterline-Angus Operations Recorders, Model A620X). Activity on the fixed vertical metal support, which closed the plastic cage and amounted to one-quarter of the surface available to the cockroach, was not recordable. Food and water were offered continuously through portals in this support. RESULTS

Adult males In initial experiments with the activity monitor and adult male activity, 7 of 8 individuals, examined under LD/12 : 12 for intervals up to 28 days, exhibited rhythmic activity. An example 8-day actograph is shown in Fig. l(A) and consists

G. R. LIPTON ANDD. J. SUTHERLAND

1558

of seven successive 24-hr bar graphs with relative hourly activity indicated verticab. The 12-hr dark period is indicated by the horizontal black bar at the top of a graph series. The entrained rhythm was characterized by greater activity during the first 2 or 3 hr of darkness (days 3, 4, 5, 6), followed by a relatively quiescent period for the remainder of the dark period and by intermittent activity throughout most of the light period.

B

e

II I

7&

-’

I

7-

14&&&w

t I

I1 .21

FIG. 1. Actograph of male (A, 7 days) and female cockroach (B, 21 days) as recorded by the activity monitor in LD/12 : 12. Approximate time of appearance and deposition of oiitheca indicated by diamonds and x ‘s respectively.

In experiments employing the running-wheel activity monitors, greater numbers of individuals were examined, and it is necessary to establish and define terms and criteria prior to the presentation and discussion of the data. The term ‘rhythm’ is defined (NEILSON, 1959) as ‘movement marked by regular recurrence of, or regular alternation in, features, elements, phenomena, etc. ; hence regularity of recurrence or alternation, or an instance of it; periodicity’. Many researchers of activity rhythms employ the term essentially as defined and have limited its use to the onset of activity. Since the Esterline-Angus Operations Recorder precisely records the time of onset of activity, the term as used here refers solely to the onset and not to the activity interval itself. To be classified as a rhythm in these studies employing wheel cages, the activity record of an individual must satisfy the following criteria: (1) under LD conditions, the successive daily onsets of activity occur within 1 hr, and (2) under DD conditions, the successive daily onsets of activity occur in a fairly constant

ACTlVITyRKYTHMSIN THRAMERICAN COCKROACH

1559

time relationship to one another so that an ‘eye-fitted’ line may connect such onsets. Other terms are used to describe those rhythms which are restricted to the first hours of darkness (uniphasic) or also include an active phase in the first hours of light (biphasic). However, certain activity records do not indicate definite rhythms of onsets. It would be incorrect to simply group such individuals in an arrhythmic or random category since some do exhibit a pattern of activity. Therefore, the term ‘pattern’ is used to denote consistent intervals of activity occurring at a definite time within the 24-hr period, but not exact rhythms as described above. In such cases, the interval of activity rather than its onset was given greater value. A third category of ‘random’ activity was established for those individuals exhibiting neither rhythms nor patterns but only sporadic and inconsistent activity. The activity of 135 adult male cockroaches was recorded in running wheel cages under conditions of alternating light and darkness (LD/12 : 12) for 7 to 37 days ; a summary analysis of their activities appears in Table 1. Of these TABLE I-SUMMARY

OF ACTIVITY RHYTHMS OF 135 ADULTMALE COCKROACHES UNDERAN LD/12 : 12 LIGHTINGREGIMEN

ANALYSIS

Percentage of activities judged as Rhythm Total percentage

93.4

Of these: Uniphasic Biphasic

68.2 31.8

Total numbers

126

Pattern 2.2

Random 4.4

100 0

-

3

6

individuals, 93.4 per cent exhibited entrained rhythms. Examples of rhythms involving little, moderate, and great amounts of activity respectively are shown in Fig. 2(A), (B), (C). Each horizontal line on the actograph is a 24-hr period reading left to right, with succeeding days arranged chronologically below. A black horizontal bar at the top of the actograph indicates the 12-hr interval of darkness in the 24-hr period. In some following actographs, light-dark conditions were subsequently changed and are so indicated. Generally the onset of activity began soon after the onset of darkness, followed by a relatively quiescent period. Of the 126 entrained rhythms that were evident, 31.8 per cent were biphasic (Fig. 2C) and 68.2 per cent were of the more common uniphasic type. Entrained patterns were demonstrated by 2.2 per cent of the cockroaches and 4.4 per cent displayed apparent random activity. Of 75 of the above individuals (Summary analysis, Table 2) examined in constant darkness (DD), only 34.7 per cent exhibited free-running rhythms (Figs. 3A, 3B, 4A). These actographs indicate a 7-day initial examination under an LD/12 : 12 lighting regimen with time of change to constant conditions at the point

G. R. LIPTON AND D. J. SUTHERLAND

Actographs of 3 male cockroaches, recorded by running wheel ,cage (LD/12 : 12 7 days, DD 27 days) with little (A), moderate (B), and great (C) actrvrty.

FIG. 2.

FIG. 3. Actographs of 2 male cockroaches, (A) and (B) (LD/12 : 12 7 days, DD 27 days), exhibiting free-running rhythms in DD. Approximate periods of biphasic rhythm in (A) indicated by diagonal lines. FIG. 4.

Actograph of 2 male cockroaches, (A) and (B), conditions and rhythms similar to Fig. 3. Change of period of free-running rhythm occurs in (B).

FIG. 5. Actograph of male cockroach (LD/12 : 12 13 days, DD 26 days, LD 3 days) with continuity of secondary phase less evident than in Fig. 3(A).

ACTIVITY RHYTHMSIN THE AMERICANCOCKROACH

1561

indicated by DD. Of free-running rhythms exhibited, 25.3 per cent were biphasic (Figs. 3A, 5) the secondary phase of which was a continuation of that activity phase associated with the early hours of light under the previous LD/lZ : 12. Generally in DD, this secondary phase abruptly shifted, in the case of Fig. 3(A) 5 hr toward the main free-running phase, which is a continuation of the darkactive phase under LD/lZ : 12. With phases separated by approximately 7 hr, both phases shifted similarly. In some individuals, the continuity of the secondary phase from LD to DD was not as evident (Fig. S), or this phase was lost apparently in DD (Fig. 4B). Of other individuals examined in DD 40.0 per cent demonstrated free-running patterns (Fig. 6), whose shift in periodicity was definable on the basis of successive intervals of activity. The approximate periods of the onsets of activity in the free-running rhythms also varied (Table 2). A period less than 24 hr was exhibited by 42.3 per cent of the cockroaches, while a period greater than 24 hr was exhibited by 30.7 per cent. TABLE 2-SUMMARY ANALYSISOF ACTIVITY RHYTHMSOF 75 ADULTMALECOCKROACHES UNDER CONSTANTDARKNESS (DD) Percentage of activities judged as RhythlTlS

Pattern

Random 25.3

Total percentage

34.7

40.0

Of these: Uniphasic Biphasic

76.9 23.1

73.3 26.7

-

Period of: <24hr >24hr < >24hr 24 hr

42.3 30.7 15.4 11.6

26.6 10.0 20.0 43.4

-

Total number

26

30

19

< > , one or more shifts in period in DD.

Three cockroaches (11.6 per cent) demonstrated periods very close to 24 hr. Those individuals that had spontaneous changes of periods comprised 15.4 per cent of those with free-running rhythms (Fig. 4B). Cockroaches demonstrating free-running patterns also showed variations in the periods of activity. A period less than 24 hr was exhibited by 26.6 per cent of these cockroaches, and a period greater than 24 hr was exhibited by 10 per cent. Periods very close to 24 hr were demonstrated by 43.4 per cent. Spontaneous changes of periods were demonstrated by 20 per cent. Apparent random activity was displayed by the remainder (23-l per cent) of the individuals in DD (Fig. 7). The activities of most of these had been rhythmic in LD/12 : 12.

G. R. LIPTON ANDD. J. SUTHERLAND

c 2:..:.j ,_-‘.,. ;:‘:

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FIG. 6. Actograph of male cockroach (LD/12 : 12 37 days, DD 31 days); activity in DD judged to be free-running pattern. FIG. 7. Actograph of male cockroach, same conditions as Fig. 6; activity in DD judged to be random. FIG. 8. Actographs of 3 female cockroaches, (A), (B), (C) (LD/12 : 12 20 days). Approximate time of appearance and deposition of obtheca indicated by diamonds and x ‘s respectively. FIG. 9. Actographs of 4 virgin female cockroaches, (A), (B), (C), (D) (LD/12 13 days).

: 12

1563

ACTIVITY RHYTHMS IN THE AMERICAN COCKROACH

Adult females Initial studies of adult female activity were conducted with the activity monitor and examined 7 females in LD/12 : 12 for 6 to 28 days. Upon observation that the first two females examined did not exhibit entrained rhythms similar to those of the males, the possibility was considered that activity might be influenced by a reproductive cycle. Thereafter, visual observations were made at the beginning, middle, and end of the light period for ootheca production and deposition. The actograph of one such female is presented (Fig. lB), in which there appears to be no detectable daily cycle of activity such as that of the adult male cockroach. On days 3, 4, 6, 8, and 9 there was more activity in the light period than in the dark period. The activity instead seems to be influenced by the reproductive cycle which is approximately indicated. The black diamonds on the micrographs indicate that time when formation of an oiitheca was first observed and the x ‘s indicate the time its deposition was first observed. Four females appeared most active when carrying an oijtheca and relatively quiescent after egg deposition. The activity of only one female did not appear to be influenced by either the lighting regimen or the reproductive cycle. Female activity was also assayed in the wheel cage and oStheca formation and deposition noted. Of 10 females examined in LD/12 : 12 for 21 days, 9 exhibited entrained rhythms, the quality of which generally differed from that of males. Three individuals were light-active (Fig. 8A) instead of dark-active, while the activities of two others were biphasic, with most of the activity occurring in the light period (Fig. 8B). Only 3 females exhibited uniphasic rhythms with activity restricted to the dark period similar to most males. One female displayed random activity (Fig. 8C). In contrast to results with the activity monitor, there appears to be little or no relationship between activity and the reproductive cycle (Fig. 8A, C). Approximate times of oijtheca formation and its deposition are indicated as before. Intense activity occurred before and after deposition, and generally there was no quiescent period after deposition such as that observed with the activity monitor. Nor was the appearance of a second oijtheca (Fig. 8C) followed by increased activity. To further investigate the possible influence of the reproductive cycle on female activity, 8 young virgins were removed from the stock culture and their activity assayed. One individual (Fig. 9A) displayed sporadic activity while the other 7 exhibited entrained rhythms 3 of which were uniphasic (Fig. 9D) and similar to the dark-active rhythms of males. Four such rhythms were biphasic (Fig. SC) and a fifth (Fig. 9B) was uniphasic with activity extending into the light period. No oiithecae were produced during the experiment. DISCUSSION

Exogenous rhythms

of males

Previous investigators 1962)

have

already

(CLOUDSLEY-THOMPSON,

documented

an exogenous

1953; HARKER, 1956; ROBERTS, activity

rhythm

in the adult

male

1564

G. R.LIPTON

SUTHERLAND

American cockroach, and this study serves first as another confirmation of the presence of this rhythm. However, this study also has pointed out variations that exist in these exogenous rhythms, the most interesting variation being the portion of the adult males (31.8 per cent) exhibiting biphasic rhythms with two distinct periods of onsets, one at the beginning of the light period and the other at the beginning of the dark period. In a recent study (NISHIITSUTSUJI-UWO et al., 1967) cockroaches having biphasic (bimodal) rhythms were noted as being rare individuals. It is not known whether the light-active phase is a valid activity phase in this portion of the population or if it is an artefact due to a combination of negative phototropism and stimulation by the moving substrate (wheel cage). Although 72 per cent of biphasic individuals maintained this phase in total darkness, some such individuals lost it within 4 days indicating that the phase may be imprinted or entrained by the onset of light in LD. However, in the majority of cases this phase generally in 1 to 3 days in DD shifted closer to the main phase, and eventually both phases maintained equivalent periods as free-running rhythms. These results support the validity of this phase and its endogenous character, however secondary to the main activity phase, and justify its continued investigation.

Endogenous rhythms of males The one striking indication of the present study of endogenous cockroach activity rhythms is that only 34.7 per cent of the individuals examined demonstrated such rhythms in DD, and 40.0 per cent exhibited loose rhythms or freerunning patterns. Such values or even the implication of arrhythmic cockroaches have not been previously reported by most investigators, One author (HARKER, 1956) found that the endogenous rhythms did not persist under constant conditions of light or darkness, while others (HAMNERet al., 1962) concluded that some strains of cockroaches are arrhythmic. Such findings are supported by data presented here, i.e. that some cockroaches may not demonstrate free-running rhythms even It is entirely possible that certain though they may have entrained rhythms. individuals of,some strains of Periplaneta americana do not possess innate physiological clocks of sufficient strength to operate under constant conditions. The following explanation is offered for any particular strain of cockroaches not exhibiting a high percentage of free-running rhythms. The population of cockroaches used in these experiments has been cultured for at least 30 years with only occasional infusions of other strains. If the hypothesis and views of PITTENDRIGH and BRUCE (1957) are accepted, namely that all organisms possess an innate biological clock, it is not difficult to accept its adaptive value in the nocturnal cockroach. Since it would be advantageous to remain quiet and concealed from diurnal predators, this trait in wild populations would be continually selected even before sexual maturity and possessed by the vast majority of individuals. In a laboratory strain, this trait would not have such strong survival value nor be so selected. While the clock mechanism and its capability to be entrained might remain, its efficiency of operation might be reduced and thereby require a more sensitive

ACTIVITY

RHYTHMS IN THE AMERICAN COCKROACH

1565

device to measure than those used in such laboratory studies as these. Studies with field strains should clarify this aspect. The fact that arrhythmic cockroaches may occur within a Iaboratory population should now be considered by investigators. Such variability as to the presence of endogenous rhythms may have significance in the conflicting views as to the control centre. In addition, the demonstration of an entrained rhythm in a test animal does not necessarily prove the existence of a free-running rhythm in an individual. Adult females The presence of an activity rhythm in the female cockroach of a laboratory strain is still not clear. This is partially due to the differing results obtained in studies with two types of rhythm measurement, the activity monitor and the wheel cage. With the activity monitor, a 24hr rhythm in LD appeared to be missing, and instead activity seemed correlated with the reproductive cycle as suggested by HARKER (1956). With the wheel cage such a relationship was not indicated, and although a rhythm under LD/12 : 12 was evident, it was dissimilar to that of the male, in some consisting of a light- rather than dark-active phase. Although the majority of virgin females examined exhibited activity rhythms comparable to the adult male, the exact influence of the reproductive cycle is still not known since activity was not assayed after mating. This aspect is being further studied elsewhere (FABER, 1970). Since much of the study of activity rhythms depends on the sensitivity of methods to measure them, it is important to consider the differences between the electrical (activity monitor) and the mechanical (wheel cage) devices apparently responsible for the aforementioned variation in the female rhythm. The inherent nature of the activity monitor described in this report is such that it records incidental activity, such as cockroach self-cleaning movements and rubbing against upper surfaces, as well as ordinary locomotory activity. Just before deposition, the female examines prospective places for oijtheca placement (usually on or adjacent to the food tablet). The act of deposition itself generally involves some undulating movements of the abdomen which positions the oiitheca. Also involved are behavioural patterns that result in covering the oijtheca with saliva and debris from the immediate surroundings (ROTH and WILLIS, 1954). These supplementary movements may in fact be recorded by the activity monitor and interpreted as locomotory activity. The activity recorded by the wheel cage, however, is generally restricted to locomotory activity. It should be noted that the substrate (wheel) is movable and, therefore, in being moved by the cockroach might act as feedback to further stimulate movement. This could result either in an overemphasis of any actual rhythm, thereby making it more distinct, or in masking a secondary active phase (Fig. 7B). In our studies several female adults were observed to move almost continuously for more than 4 hr. This same type of feedback might have occurred in experiments involving tumour induction by repeated implantation of suboesophageal ganglia (SEG) (HARKER, 1958), wherein it is not

1566

G. R. LIPTONANDD. J. SUTHERLAND

clear whether activity was measured by a device involving direct attachment to the pronotum or by a tilting cage (HARKER, 1956). Possibly such devices respectively offered either high or low feedback enforcement of the activity rhythm and/or affected the SEG. Although the latter may not be the site of rhythm control (ROBERTS,1966) and the tumours probably are only lesions involving haemocyte encapsulation (SUTHERLAND,1967, 1969), the effect of epithelial hyperplasia and apparent haemocyte involvement was observed, nevertheless, and cannot be discounted. The effect might be explained if the SEG was the source of some factor (hormone or secretogogue) governing midgut holocrine secretion and regeneration and if the rate of regeneration was increased by the continuous presence of the factor due to repeated SEG implantation. Acknowledgement-The authors gratefully Mrs. J. M. CHILLSEYZN.

acknowledge

the valuable

assistance of

REFERENCES BRADY J. (1967) Histological observations on circadian changes in the neurosecretory cells of cockroach sub-oesophageal ganglia. J. Insect Physiol. 13, 201-213. CLOUDSLEY-THOMPSON J. L. (1953) Studies in diurnal rhythms-III. Photoperiodism in the cockroach Periplaneta americana (L.). Ann. Mag. nat. Hist. 6, 705-712. FABERB. (1970) Activity patterns, endogenous and under light-dark cycles, of last instar male and female nymphs and adult female American cockroaches. M.S. Thesis, Rutgers-The State University, New Jersey. HAMNER K. C., FLINNJ. C., JR., SIROHIG. S., HOSHIZAKIT., and CARPENTER B. H. (1962) Studies of the biological clock at the South Pole. Nature, Lond. 195,476-480. HAFXER J. E. (1956) Factors controlling diurnal rhythms of activity of Periplaneta americana L. J. exp. Biol. 33, 224-234. HARKER J. E. (1958) Experimental production of midgut tumours in Periplaneta americana L. J. exp. Biol. 35, 251-259. HARSHBARGER J. C. and TAYLORR. L. (1968) Neoplasms of insects. A. Reo. Ent. 13,159-190. NEILSON W. A. (1959) Webster’s New International Dictionary of the English Language, 2nd ed., unabridged. G. & C. Merriam, Springfield.

NISHIITSUTSUJI-UWO J., PETROPULOS S. F., and PITTENDRIGH C. S. (1967) Central nervous system control of circadian rhythmicity in the cockroach-I. Role of the pars intercerebralis. Biol. Bull., Woods Hole 133, 679-696. PITTENDRIGHC. S. and BRUCEV. G. (19.57) An oscillator model for biological clocks. In Rhythmic and Synthetic Processes in Growth (Ed. by RUDNICK D.), pp. 75-109. Princeton University Press, New Jersey. ROBERTSS. K. DEF. (1960) Circadian activity rhythms in cockroaches-I. The free-running rhythm in steady state. J. cell. camp. Physiol. 55, 99-l 10. ROBERTSS. K. DE F. (1962) Circadian activity rhythms in cockroaches-II. Entrainment and phase shifting. J. cell camp. Physiol. 59, 175-186. ROBERTSS. K. DE F. (1966) Circadian activity rhythms in cockroaches-III. The role of endocrine and neural factors. J. cell. camp. Physiol. 67, 473-486. ROTH L. M. and WILLIS E. R. (1954) The reproduction of cockroaches. Smithson. misc. Coll. 122, l-19. SCHJXHTER M. S., DUTKY S. R., and SULLIVANW. N. (1963) Recording circadian rhythms of the cockroach with a capacity sensing device. J. econ. Ent. 56, 76-79. SUTHERLAND D. J. (1967) The development of salivary tumours in Periplaneta americana (L.) as induced by duct ligation. J.. Insect Physiol. 13, 137-152. SUTHERLAND D. J. (1969) Nerve severance and tumor induction in the cockroach, Periplaneta americana (L.). Nat. Cancer Inst. Monogr. 31, 399-418.