striction, constriction and deconstriction stages will be indicated in that order, separated by a slant line. Light microscopy. Larval segment patterns were analysed ...
/. Embryol. exp. Morph. Vol. 51, pp. 1-26, 1979 Printed in Great Britain © Company of Biologists Limited 1979
The specification of metameric order in the insect Callosobruchus maculatus Fabr. (Coleoptera) I. Incomplete segment patterns can result from constrictioninduced cytological damage to the egg By JITSE M. VAN DER MEER 1 From the Department of Zoology, Catholic University, Toernooiveld, Nijmegen, The Netherlands
SUMMARY Eggs of the pea-beetle Callosobruchus were divided into two at different stages of development. Both fragments were allowed to develop into partial larvae. The segment patterns of normal and partial larvae are described using cuticular markers of cell differentiation. To study the contribution of cytological damage to the segment gap phenomenon three different types of constriction were performed: complete and incomplete permanent constriction and complete temporary constriction. Changes in the structure of the egg can produce absence of segments resulting from two different effects. First, partial absence of segments results from a decreased egg circumference in the constriction region and involves the disturbance of a morphogenetic process (dorsal closure). Secondly, cytological damage can result in a gap between two arrays of segments. The loss of segments in the gap occurred in two different ways. In a spatial segment gap the two arrays of segments were physically discontinuous, whereas in a non-spatial gap the segments bordering the gap were juxtaposed in a physically continuous cuticle. The extent to which the gap phenomenon can be attributed to cytological damage is discussed. We also discuss, on the basis of certain dorsal defects, a possible stepwise specification of the dorsal transverse cuticular pattern. INTRODUCTION Pattern formation is the development of spatially integrated sets of differentiated cells. Insects are very suitable to study pattern formation because of their simple metameric organization. Moreover, each larval or adult segment can be distinguished from all others by cuticular markers (bristles, hairs, stigmata, etc.). Epigenetic metamerization in insects can be described either in terms of two morphogenetic signals (Krause & Sander, 1962; Kalthoff, 1976; Sander, 1976) or in terms of only one such signal (Meinhardt, 1977). Sets of blastoderm cells would become instructed to form particular segments by the position-dependent absolute value of one signal or by the ratio between two such signals. 1
Author's address: Division of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Centre. D-6900 Heidelberg, Federal Republic of Germany.
2
J. M. VAN DER MEER When an insect egg is divided into two fragments in an early stage, the resulting anterior and posterior partial larvae each lack a number of segments near the constriction region. As fragmentation is made at successively later stages the number of missing segments becomes smaller. This gap phenomenon is interpreted to result either from the interference by the constriction with the transport of morphogenetic signal(s) (Sander, 1976), or from signal redistribution after constriction and its interference with cellular commitment (Meinhardt, 1977). The result would be the loss on either side of the constriction of a number of specific instructions and their corresponding segments. However, the loss of segments may also be caused by cytological damage to the egg or to a putative mosaic of prelocalized segment determinants. Various arguments make this possibility unlikely (Herth & Sander, 1973). Moreover, development is often normal if the constriction is immediately released and the ooplasm of the two egg halves re-fuses (Alleaume, 1971; Sander, 1975, 1976; Yogel, 1977), or if re-fusion is forced by puncturing the barrier produced by constriction (Schubiger, Moseley & Wood, 1977). Since neither of these procedures is likely to reduce the disturbance of the egg by the constriction, it was concluded that the segment gap is not due to egg cell damage. In this paper, however, we describe that in Callosobruchus different types of constriction can damage the egg resulting in modified segment patterns. MATERIALS AND METHODS
Adults. Pea-beetles {Callosobruchus maculatus Fabr., syn. Bruchus quadrimaculatus Fabr.) were provided by the Pest Infestation Control Laboratories.1 A stock culture was kept on green peas {Pisum sativum) in the dark at 30 °C and 70 % relative humidity (r.h.) (Brauer, 1925; Brauer & Taylor, 1936; Howe & Currie, 1964). Development from egg deposition through four larval instars till the emergence of the adult then lasts about 4-5 weeks (Howe & Currie, 1964: table IV). Culture tubes and oviposition boxes were treated with 5 % of methyloxybenzoate (nipagine) in 96 % ethanol to prevent growth of fungus, which serves as food for the dust-louse {Liposcelis divinatorus Miill.) and for mites (Tyrophagusputrescentiae Schrank.). [A warning: prolonged exposure to beetles and/or pea powder may result in severe allergic reactions.] For description of adults (Fig. 1 a, b) and their egg laying behaviour see Brauer (1925), South gate, Howe & Brett (1957) and Howe & Currie (1964). Eggs. The egg's shape is that of one longitudinal half of a hen's egg (Fig. 1 d). The average egg dimensions are: 700 /on long, 200/mi wide and 150 jam largest height. Large numbers of eggs were obtained by collecting about a hundred young females in a box covered with 0-5 mm mesh plankton gauze. Males should be added to stimulate oviposition. Oviposition was initiated by adding brown beans {Phaseolus vulgaris) on which the beetles were placed with a brush. Staging accuracy was ±15min. The first egg collection could be used for 1
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Pattern formation in insect embryogenesis
Fig. .1. Dorsal views of a female (a) and male (b) of Callosobmchus: length about 3-4 mm. The female abdomen extends posteriorly from under the elytrae and shows a white median stripe while the elytrae are characterized by four dark spots (a). The males have a short abdomen and a less pronounced coloration (b). (c) Constricted egg seen through the window of the constriction apparatus. The anterior and posterior isolate each contain a partial embryo, (d) Cellular blastoderm stage: vertical bar shows the constriction levels, (
