Abstract--Food is an important extrinsic factor in the control of moulting as well as in the control of reproduction in colonies of Blattella germanica. Starvation following a moult or a parturition delays the initiation of another moulting cycle or female reproductive cycle. The initiation of a moulting cycle after a period of starvation requires only a short period of food availability (12 hr). To be able to postpone development until adequate food is available is advantageous to intermittent feeders and scavengers, such as cockroaches, which must forage for food. Synchronization of the development of colonies of cockroaches by controlling the availability of the food is a valuable research tool.
Development in apterygote and immature pterygote insects is dominated by the cyclical process in which an old exoskeleton is moulted and replaced by a new and larger one (WIGGLESWORTH, 1959; LOCKE, 1964). The basic endocrine events which control moulting are now well known (reviews by WIGGLESWORTH, 1964; GILBERT, 1964), but the extrinsic controls of the endocrine system are much less clear (DE WILDE, 1961).
While the basic endocrine events are shared by all insects investigated (SCHNEIDERMAN and GILBERT, 1964), the extrinsic controls vary widely from group to group and species to species. The recently described tanning hormone, bursicon (FRAENKEL and HSAIO, 1962, 1965; COTTRELL, 1962, 1964), although demonstrated to be present in a variety of orders of insects, is known to be extrinsically controlled only in the blowflies. Brain hormone is released by a variety of mechanisms which differ widely even at the level of the genus. In the blood-sucking bug, Rhodnius prolixus, the distention of the gut by a blood meal is the extrinsic signal for the release of brain hormone (WIGGLESWORTH, 1934). VAN DER KLOOT (1960), using electrophysiological techniques, showed that the blood meal stretches abdominal proprioceptors, which send nerve impulses to the brain, resulting in the secretion of brain hormone. Hormone secretion by the brain of diapausing pupae of saturniid silk moths is renewed in a number of ways. In the majority of silkmoths, including Hyalophora cecropia, Samia cynthia, and Antheraea polyphemus, chilling of the brain is the extrinsic signal for brain hormone secretion (WILLIAMS, 1956). However, in A. pernyi photoperiod is the dominant environmental signal to break diapause (TANAKA, quoted in LEES, 1956).
In contrast to the foregoing examples of positive extrinsic stimuli controlling moulting, the meal-worm Tenebrio seems to have no positive extrinsic controls of the moulting cycle since moulting occurs under constant conditions and total starvation (WIGGLESWORTH, 1953).
The vertebrate oestrous cycle provides a parallel in which extrinsic factors are of varying importance to different species in the control of an hormonal cycle (Scharrer and Scharrer, 1963).
This paper deals with food as an extrinsic factor in the control of development in cockroaches. Starvation has been shown to delay the reproductive cycle in a number of cockroaches, including Blattella, during the first preovipositional period (Scharrer, 1946; JOHANSSON, 1955; VON HARNACK, 1958; ROTH and STAY, 1962; ENGELMANN, 1964; ROTH and Barth, 1964), but no information has been published on the effects of starvation and feeding on the moulting cycle or on reproductive cycles following the initial one. The present study demonstrates that feeding has an integral role in the initiation of the moulting cycle as well as the reproductive cycle in cockroaches.
The German cockroach, Blattella germanica, has a typically orthopteroid gradual metamorphosis. There are normally six larval instars. The adult female lays an ootheca containing 30-40 eggs and carries it protruding from her genital opening until it hatches.
Colonies of cockroaches (35-1000) were raised in finger bowls and were fed a diet of unsupplemented Ken-L Biskit or Purina Rat Chow with water constantly available. The temperature was maintained constant in incubators (+/-0.5°). Maximal growth and development of larvae occurs at 30°. Reproduction, however, was optimal at 26°. During experiments the incubators were opened at 12-hourly intervals for a maximum of 15 min. At all other times the incubators were kept closed and dark. Preliminary experiments had shown that animals which were raised with a circadian activity rhythm did not moult during the activity period. In order to allow uniform moulting throughout the day, the animals were raised without a light-dark rhythm. If activity rhythms were present, they presented no difficulties in these experiments. A bowl of water was kept in the bottom of each incubator to maintain a high humidity; other than this precaution, humidity was neither rigidly controlled nor monitored.
The entire stock of B. germanica are progeny derived from a single ootheca from a female caught in New York City. In the initial breeding programme it was necessary to avoid close inbreeding to obtain high yields of viable oothecae. The adults derived from the first ootheca, for example, were of necessity interbred and this sibling mating produced only 50 per cent viable oothecae. After about four generations of inbreeding, however, crossing siblings yielded almost 100 per cent viable oothecae.
Oothecae about to hatch were recognized by the development of a green line on the ootheca a week before hatching (CLAYTON, 1959). Larvae could be sexed readily as early as the first instar (ROSS and COCHRAN, 1960). The larvae were quickly and conveniently handled without damage by the use of an aspirator. Weight measurements were taken of colonies of individuals (+/- 0.1 mg) without anesthesia, by transferring them via an aspirator to a weighing bottle with its rim lightly greased with petrolatum to prevent escape. Colonies of the American cockroach, Periplaneta americana, were maintained for comparative purposes, and in general the same methods of rearing larvae were used for both Blattella and Periplaneta. The initial oothecae for establishing the P. americana colonies used in this investigation were generously provided from the stock colonies of Professor EDWARD S. HODGSON.
Development and food availability
To determine whether the availability of food has
a role in controlling the onset of the moulting cycle in cockroaches, colonies
of Blattella were raised as described with only food as a variable.
Colonies of cockroaches, in which food is constantly available, soon become markedly asynchronous in their moulting cycle (see Fig. 1-O). If the moulting cycle were dependent on some extrinsic factor such as food for initiation, it should be possible to eliminate the additive factor in the asynchrony by controlling the availability of food.
In a pilot experiment a control colony of 35 larvae, hatched from a single ootheca, was raised at 29° with food constantly available (Fig. 1-O). Three other colonies were raised under similar conditions, but food was removed before the colonies started moulting and reinstated only after all the larvae had moulted (Fig. 1, A, B, C). Progressive asynchrony was eliminated in colonies in which the animals began feeding simultaneously in each instar.
The adults derived from colonies A, B, and C were interbred at 26° as indicated in Fig. 1, and oothecae were produced synchronously. After the oothecae hatched, the females were again fed simultaneously and allowed to mate with the males, and again a synchronous batch of oothecae was produced (not shown in Fig. 1). By controlling the availability of food to the adult females only, synchronous ootheca production and hatching can be repeated up to 3 or 4 times. Mating is not necessary during every reproductive cycle, but the maximum number of larvae per ootheca was obtained when males were available. A synchronous breeding colony of 30 females and 30 males can thus produce 900-1200 synchronously hatched larvae at regular intervals defined by the feeding schedule of the females.
To further substantiate the availability of food as an extrinsic factor in the control of the moulting cycle, five subcolonies (D, E, F, G, and X, Fig. 2) of 50 unfed fourth-instar larvae were isolated from a larger colony of larvae which all moulted on the same day. Subcolonies D, E, F, and G were placed in a 30° incubator and given food 1, 3, 5, and 7 days respectively after they had moulted to the fourth instar. Subcolony X was similarly treated but not given any food and did not moult. Figure 2 shows the direct dependence of the time of moulting on the availability of food.
Since initiation and maintenance of the moulting cycle could be associated with either the beginning, the entire length, or the termination of feeding, an experiment was designed to determine which phase of the feeding period was related to the initiation of moulting. Five colonies of 50 larvae each were isolated from a larger well-fed colony as they moulted to the fourth instar. These colonies of larvae were starved for a day at 30° and then were all fed at the same time. Food was removed 0.5, 1.0, 1-5, 2-0, and 2.5 days respectively after it had been presented.
Ninety per cent of the larvae fed for only 1 half. day moulted to the succeeding instar. Thus 1 half. day of food availability was sufficient to initiate a moulting cycle in these cockroaches. All larvae fed for 1 day or more moulted to the next instar. All the larvae which did moult underwent ecdysis at the same time, 5 days after the food was presented. This indicates that the initiation of the moulting cycle in cockroaches is associated with the early phase of feeding and is not dependent on sustained feeding.
Weight change during the larvae instar
Blattella approximately doubles its weight
in each succeeding instar, starting with a 1 mg egg and ending six moults
later with an unfed adult weighing about 64 mg (Woodruff, 1938).
Initially there is little difference in adult weight between the sexes.
The female, however, almost doubles her weight during oocyte development
whereas the male remains approximately constant.
Weight changes during individual instars have hitherto received little attention, especially weight changes early in the instar. By weighing entire colonies or individuals at 12 hr intervals before and after feeding, it was found that larvae raised at 30° gained 80-100 per cent in weight during the first 12 hr after food becomes available. As demonstrated before, the initiation of the moulting cycle can also be associated with the first 12 hr of food availability. The effects of the appearance of the food in the cockroach system in this first 12 hr, which result in moulting cyle initiation, are still uncertain.
Population density and the moulting cycle
Conflicting evidence has been presented on the effect of raising Blattella individually or in groups with various densities (CHAUVIN, 1946). In order to determine whether variations in population density have any significant effect on the moulting cycle of Blattella raised in colonies, various population densities were used in many experiments as controls (0.2-1.2/cm2 or 0.1-0.7/cm3 in 300ml finger bowls; 0-2-2.0/cm2 or 0.05-0.5/cm3 in 1500ml finger bowls). Although increasing density of larvae had the general effect of reducing the average weight gain in larval colonies, there was no effect upon the average length of the moulting cycle measured from the beginning of food availability to ecdysis.
Extrinsic controls of reproduction in Periplaneta and Blattella
Although the availability of food plays an identical role in the control of moulting in both Periplaneta and Blattella, they differ in the role which food plays in the control of reproduction. Starvation prevents the initiation of the reproductive cycle in the first preovipositional period in Periplaneta, but up to five additional oothecae per female may be oviposited after removing food from a group of females which had been fed ad lib. up to the time of food removal. This independence from feeding in each reproductive cycle is reflected in the weight relations during the female reproductive period. An unfed Periplaneta adult female weighs approximately 850 mg; after feeding ad lib. the female attains and maintains during its reproductive life a weight of about 1200 mg; with subsequent starvation the female will oviposit a total of four to five 50-60 mg oothecae at regular intervals and stop ovipositing with a weight of about 900 mg. Additional feedings result in comparable bursts of ootheca production. In the smaller cockroach Blattella, as seen above, oviposition must be initiated anew by feeding in each preovipositional period. This is also reflected in weight relations during Blattella's reproductive cycle. The unfed Blattella adult female weighs about 60 mg. A fully fed female carrying a 40 mg ootheca weighs about 105 mg. Thus, whereas Periplaneta diminishes its food reserves by 15 per cent with each ootheca oviposited, Blattella uses up (almost) all (90 per cent) of the food accumulated during the preovipositional period on a single ootheca.
Feeding has been shown to be an important extrinsic
factor in the control of the moulting cycle and reproductive cycle in the
cockroach Blattella germanica (Blattellidae) and also the distantly
related cockroach Periplaneta americana (Blattidae). In any event,
feeding provides a convenient method of obtaining and maintaining relatively
synchronous colonies of cockroaches for developmental studies. Previously,
in order to obtain cockroaches of comparable age and stage, considerable
effort had to be expended. First, oothecae oviposited on a single day were
isolated from mass cultures of breeding stock; then larvae from oothecae
which hatched on the same day were raised in mass cultures and at each
ecdysis the animals which moulted within the desired limits were isolated
for experimental study. As Fig. 1, O, demonstrates, continuous food availability
introduces progressively greater asynchrony in the later instars. With
the method described above (Figs. 1, A, B, C, and 2) the period during
which the animals are to moult or produce eggs can be planned by modifying
the feeding schedule.
Although considerable caution must be taken in comparing traits, such as feeding, which are very adaptable to specific environments, it is interesting to look at feeding in teleological terms. Insects which have an abundance of food in their natural environment do not need to adapt their hormonal events to the availability of food. Cyclical moulting and feeding behaviour can be intrinsically linked to the hormonal cycles. This type of control operates in Locusta (CLARKE and LANGLEY, 1962) and in Tenebrio (WIGGLESWORTH, 1953). The animals in which feeding is an extrinsic factor initiating the moulting cycle are intermittent feeders: the bedbug, Cimex, and the blood-sucking bug, Rhodnius, both need a single large meal in order to initiate a moulting cycle. A relative of Rhodnius, Triatoma needs two or more blood meals per moulting cycle. As shown above, non-circadian colonies of the cockroach B. germanica need a feeding period of 12 hr to initiate a moulting cycle. Clearly, these animals are making use of a mechanism to delay development until sufficient food is available to complete a moulting cycle.
The mechanism by which feeding initiates a moulting cycle in the cockroach is as yet unknown. SCHARRER (1958) concludes that it is more efficient to route stimuli from the external and internal milieu through some integration centre such as the central nervous system in order to form a 'definite message' to be sent to the target organ. Although other types of receptors are possible (DAVEY and TREHERNE, 1963), feeding may control moulting in cockroaches via the central nervous system and stretch receptors, as in Rhodnius. The 80-100 per cent increase in weight of Blattella within 12 hr of food becoming available represents an approximate doubling of the volume of the cockroach through feeding and drinking. This doubling of volume provides a 26 per cent increase in linear dimensions, which should be ample for a stretch receptor mechanism. Indeed, stretch receptors have been found associated with the dorsal abdominal region of the adult male cockroach, Periplaneta (FINLAYSON and LOWENSTEIN, 1958). ENGELMANN (1964) has presented evidence that stretch reception is the afferent stimulus in the feeding-induced yolk deposition in the roach, Leucophaea, and also suggests (ENGELMANN and RAU, 1965) that the rate of feeding is the important factor in the activation of the corpora allata.
As demonstrated in this study, feeding is a stimulus for ootheca production in Blattella and in Periplaneta. Whereas Blattella uses up all of its food accumulated in a preovipositional period in the production of a single ootheca, Periplaneta stores enough food during a single preovipositional period to produce a number of oothecae and only stops ootheca production when its food reserves are depleted. If stretching as a result of feeding mediates yolk deposition in Periplaneta then, according to the above observations, the state of stretching due to stored food is important rather than the rate of change of stretching due to actual feeding. This would require an extremely non-adaptive stretch receptor since it would be working over a period of a month or so, the length of time it takes to deplete the food reserves through ootheca production.
It is interesting that the two cycles, moulting and reproduction, could be controlled by the same extrinsic factor since in the apterygotes moulting and reproduction occur in the same instar. This primitive proximity of the two cycles could provide avenues for common control. In this respect it should be noted that in the apterygote Thermobia (Thysanura), although moulting and reproduction occur in the same instar, they never overlap (WATSON, in preparation). Finer coordinating links between moulting and reproduction must exist therefore in the apterygotes.
If common extrinsic control of moulting and reproduction has been retained at all in the pterygotes, then it is most likely to be seen in its clearest form in the hemimetabolous pterygotes, which have similar habits in the larval and the adult stages. Indeed the cockroaches (Dictyoptera), as one of the most ancient groups of pterygotes, provide ideal material for studies of primitive control mechanisms. The systematics of cockroaches has been recently revised (McKITTRICK, 1964). It is convenient that a selection of three commonly cultured cockroaches, Blattella (Blattellidae), Periplaneta (Blattidae), and Leucophaea (Blaberidae), includes the three major families of cockroaches, providing good comparative material for developmental studies.
Acknowledgments -- I am indebted to my teachers at Columbia University and especially the late Dr. F. J. RYAN, Dr. A. W. POLLISTER and Dr. E. S. HODGSON for their indulgence, inspiration, and generous supply of space and facilities. For the major portion of the criticism of the theoretical and technical aspects of this study and for facilities while at Western Reserve University, I am indebted to Dr. M. LOCKE. I am also grateful for critical reading and discussion of the manuscript by Dr. J. S. Edwards, J. Lai-Fook, and J. Collins. The author was supported during the course of this study by a University Scholarship from Columbia University, a Reagents Scholarship from the State of New York, and United States Public Health Service grant GM 09960.
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Figure 1. The effect of controlled food availability on the synchrony of moulting in colonies of the German cockroach, Blattella germanica. Each colony (A, B, C, and o) of 35 to 40 larvae hatched from a single o6theca. Adults of colonies A, B, and C were outbred as indicated for maximum viability. The production and subsequent hatching of o6thecae are indicated for the first ovipositional period only.
Figure 2. Dependence of moulting on the time of food availability in the German cockroach, Blattella germanica. Each colony of 50 larvae was isolated at the third moult from a larger colony kept synchronous as in Fig. 1A, B, C.