171 Activity of the Corpora Allata during Pupal Diapause in Mimas tiliae (Lepidoptera) By K. C. HIGHNAM (From the Department of Zoology, the University of Sheffield) With one plate (fig. 2) SUMMARY Histological observation indicates that the corpora allata of Mimas tiliae are secretory during pupal diapause, but become inactive by the end of the low-temperature period which terminates diapause. Removal of the corpora allata, together with the corpora cardiaca, from the dia- pausing pupa increases the thickness of the hypodermis and decreases the number of fat-body inclusions (compared with operated controls), but does not result in any visible signs of diapause break. The oxygen uptake of the pupa increases by about 60% by the end of the low- temperature period, compared with the diapausing pupa. It is probable that the corpora allata play some part in the maintenance of diapause, possibly by exercising some control over the fat-body metabolism. This role is sub- servient to that of the neurosecretory cells in the brain, together with their associated corpora cardiaca. INTRODUCTION N a previous paper on the problem of diapause 'break' in the pupa of I Mimas tiliae (Highnam, 1958) it was shown that the cerebral neurosecretory cells, together with their associated corpora cardiaca, play an important part in terminating diapause in this insect. The mechanism of this process, in which the prothoracic glands are also involved (Williams, 1947), is essentially similar to that controlling moulting and metamorphosis in most insects (Wigglesworth, 1955). The present paper is concerned with demonstrating that variations in the secretory activity of the corpora allata occur in the pupal stage of M. tiliae, which indicate that the glands are involved in the continuation of diapause and in the process of diapause development at low temperatures that brings diapause to an end. MATERIAL AND METHODS Samples were taken of M. tiliae in diapause, of pupae after 1, 2, 3, and 4 weeks or more at the temperature of 3° C which terminates diapause (High- nam, 1958), and of post-diapausing pupae until adult emergence. These were fixed in Bouin, Duboscq-Brasil, Susa, or Zenker, embedded in paraffin wax after stripping off the pupal cuticle, and sectioned at 6 /u.. Serial sections were stained with Weigert's haematoxylin and eosin, or with Mallory's or Masson's technique. In those series in which measurements of gland or cell-volumes were made, care was taken that in each the material spent an exactly similar [Quarterly Journal of Microscopical Science, Vol. 99, part 2, pp. 171-180, June 1958.] 172 Highnam—Activity of the Corpora Allata during time in the fixative (Bouin), and was embedded in paraffin wax at the same temperature for the same time. Glands were extirpated by a technique similar to that described by Williams (1947). The method is outlined on p. 176. Oxygen uptakes of chopped pupal tissues were measured in the Warburg respirometer according to directions given in Dixon (1951). RESULTS The histology of the corpora allata during diapause The general relationship of the corpora allata with the rest of the endocrine system has already been described (Highnam, 1958). Each corpus allatum is surrounded by a very obvious connective tissue coat, about 5 yx in thickness (fig. 1). The membrane stains blue or green with Mallory's or Masson's tech- nique. The thick membrane must be added at pupation since the larval corpus allatum is surrounded by a membrane which is very thin. It is probably formed by haemocytes (Wigglesworth, personal communication). Immediately beneath the membrane are a number of neurones with axons running parallel with the surface of the gland. From certain points on the periphery, nerve- fibres pass to the inside (fig. 1, A) and many neurones are present at these points. Besides these small nerves, a large nerve (the nervus allatum) enters each gland from the corpus cardiacum of the same side. The nervus allatum is the point of origin of the fibres running beneath the membrane. Some of the fibres form a very fine nerve which leaves the posterior face of each corpus allatum and runs in the direction of the aorta. The secretory cells which make up the bulk of each corpus allatum are large and irregularly shaped (fig. 1, A). The cell-membranes are often difficult to detect. The cytoplasm has a fibrous appearance with the fixatives used, and is weakly acidop'hil. Large numbers of vacuoles are present throughout the glands and contain very acidophil globules or granules (fig. 1, A), which seem to be secretory material elaborated by the cells, since in some cases acidophil bodies are found in the cytoplasm surrounding the vacuoles. This surrounding cytoplasm differs from that of the rest of the cell in its more homogeneous appearance, and greater acidophilia. The nuclei of the secretory cells are large, with an irregular shape (fig. 1, A). They are densely granular, with a single well-defined nucleolus. The nuclei are arranged more or less radially around the periphery of the glands, but are oriented at random in the centres. The corpora allata are richly supplied with tracheae. A large branch springs from each of the upper two tracheal trunks which enter the head from the pro- thorax and passes to the corpus allatum of the same side. As the branch enters the gland, it breaks up into a number of smaller branches, the nuclei of the lining epithelium of which can be seen in prepared sections. Histology of the corpora allata during diapause development at 3° C During the latter part of the low-temperature treatment, the histological appearance of the corpora allata undergoes a very marked change. For the first Pupal Diapause in Mimas tiliae (Lepidoptera) 173 3 weeks at 30 C, the glands are large (mean diameter 130 to 150 JX), with large nuclei separated by wide tracts of cytoplasm (fig. 1, B). The cytoplasm is vacuolated, and the vacuoles often contain acidophil inclusions (fig. 1, c). In general appearance, therefore, the corpora allata during the first 3 weeks at the 200/t 50M FIG. I. Camera lucida drawings of sections of corpora allata. A, in the diapausing pupa. Note the size of the gland, the distance apart of the nuclei, and the presence of vacuoles, some with inclusions. B, after i week at 30 C; features similar to A. c, detailed drawing of part of B to show the intracellular vacuoles and inclusions. D, after 4 weeks at 3° C. The gland is small, with few vacuoles and no inclusions. The nuclei are small and aggregated closely together. low temperature are very little different from the glands of the unchilled diapausing pupa (fig. 1, A). By the end of the fourth week at 30 C, the glands are considerably smaller (mean diameter about 100^.) with smaller nuclei aggregated more closely together (fig. i, D). Vacuoles are present in the cytoplasm but these are not sur- rounded by the differentiated cytoplasm seen in the diapausing and early low- temperature glands. There are no inclusions in the corpora allata at this time. No mitoses occur in the corpora allata during the diapause and low-tem- perature periods. It is possible, therefore, to compare directly the size of 174 Highnam—Activity of the Corpora Allata during the glands during diapause and after i, 2, 3, and 4 weeks or more at 30 C, either by measuring the volume of the glands, or their cell-volume, at these different times. As will be shown later, these are methods for determining objectively the secretory activity of the corpora allata. Volume of the corpora allata The volume of each corpus allatum was calculated from the mean section area. Assuming that the corpora allata are spherical, volume = (mean section area)3'2X2^/§7r. The mean section area was calculated by drawing every second section of a corpus allatum under a camera lucida, and measuring the areas of the drawn sections with an 'Allbrit' precision disk planimeter. The magnification being known, the values for the actual gland sections in square microns are easily obtained. The sum of the section areas divided by the number of sections drawn gives the mean section area. The gland-volumes can then be calculated. Table 1 gives the values obtained for diapausing and low-temperature pupae. TABLE I Volumes of corpora allata during diapause and diapause development Mean volume of corpora Series allata (X IO"8/A3) Standard error Comparison of means aa 1 34 ±0-09] P > o-i ^1 1-22 ±O-2O) P > o-i I-8I ±O-22] P > o-i a3 i"93 ±0-33/ P << 001 a4 o-75 ±0-OS] ±0-05/ P < o-i > 005 as 056 P > o-i a6 062 ±0-05 aa Represents a sample of pupae diapausing at 25° C. a,, a2, ..., o0 represent samples taken T, 2, ..., 6 weeks after transfer to 3° C. In each sample the volume is the mean of 10 corpora allata from 5 individuals. Means compared by the t-test for small samples. The difference between the volumes of corpora allata after 3 and 4 weeks respectively at 3° C is well marked and is statistically significantly different (table 1). The gland-volumes in the sequences a0 to <z3 (diapausing and for the first 3 weeks of the low-temperature period) and #4 to <z8 (after 4 to 6 weeks at the low temperature) are not significantly different from one another (table 1). The large standard errors of the means of the volumes of corpora allata for each sample taken during the diapause period and for the first 3 weeks at the low temperature indicate a large variation in volume size between individuals, which may be obscuring differences in mean volume between samples.