1971 515 A Study of Haemocytes in a Centipede Scolopendra morsitans (Chilopoda: Myriapoda) G. Sundara Rajulu Department of Zoology, Madras University, Madras-5, India Received March 11, 1970 Introduction Bucherl (1939) and Shukla (1964) described two kinds of haemocytes, amoeboid haemocytes and lymphocytes, in Scolopendra viridicornis and Scolopendra morsitans. In addition the third type known as hyaline cell, filled with granules, was described from Lithobius forficatus (Gregoire 1955). These authors have taken into consideration the shape and size of the cells and the presence or absence of granules in the cytoplasm as the basis for distinguishing haemocytes in these chilopods. In recent years quite a good deal of information on haemocytes of various groups of arthropods have become available for distinguishing different types of haemocytes and a classification of haemocytes has been proposed based on certain basic morphological characters (see Jones 1962, Sundara Rajulu et al. 1969). In the light of recent observations, the criteria applied by previous workers to distinguish the haemocytes in centripedes is of little importance, since the characters taken into consideration are found in more than one type of blood cells and are shown to be variable in the same haemocyte during different growth stages. Therefore a new study of the haemocytes in chilopods would be of some interest. Material and methods The centipede, Scolopendra morsitans, collected from the Alagarkoil forest region, supplied the materials. The haemolymph was collected from manually immobilized, unaesthized centipedes, by piercing through an intersegmental membrane with a sharp needle (Jones 1967). The blood exuded freely and no clot plug was formed at the wound, in concordance with Sundara Rajulu (1969). The direct and capillary methods of McLauglin and Allen (1965) were used for placing the haemo lymph on a coverslip. The direct method consisted of touching a coverslip to the exuding haemolymph. The capillary method involved collection in a capillary tube made from a 1mm capillary tube finely drawn and fire polished. The haemolymph was expelled onto a coverslip. The coverslip was then inverted over a clean slide and supported by a thin ring of white petroleum jelly so that a deep layer of haemolymph, not compressed by the weight of 516 G. S. Rajulu Cytologia 36 the coverslip was formed. Smears of haemolymph were prepared as described elsewhere (Sundara Rajulu et al. 1969). The smears were stained either directly or after fixation in methanol. Giemsa and toluidine blue were the most useful stains. The haemocytes were examined with phase contrast and ordinary micro scopes and were classified using the nomenclature of Jones (1962). Phagocytic activity was examined 24 hours after injection of either 0.1% carmine or 1% methylene blue. The injections were effected from the dorsal side of the penultimate segment. The haemolymph was studied between the slide and the coverslip without subjecting it to any other treatment including fixation. Observations Six types of haemocytes were distinguishable in blood of Scolopendra morsitans based on the size, shape and nature of cytoplasmic inclusions and behaviour in vitro. Type 1: These are small ovoid or round haemocytes with large centrally located round nucleus (Fig. 1). The nucleus has extremely fine dark grey, granular chromatin material around a single, slightly eccentric round nucleolus. These haemocytes have a small amount of smooth or sometimes finely granular cytoplasm. In some cases, the nucleus is eccentric and ovoid; the nucleolus is irregular in shape. On a number of occasions this type of haemocytes have been seen to undergo amoeboid movements in vitro. During such movements, the nucleus was frequently constricted or otherwise distorted. On many instances these haemocytes have been seen to degenerate in vitro; the nucleus is generally ejected and the cytoplasm rounds up into a pale grey sphere with fine granules within and around it. They measure from 4 to 6ƒÊ in diameter. Mitotic divisions were seen only in cells slightly larger than the typical ones. These haemocytes were relatively rare and only in the young ones were they fairly numerous. This type of haemocytes strongly recall the prohaemocytes of insects (Yeager 1945). Type 2: The haemocytes of this type are characterized mainly by their morphological variability. The cytoplasm is granular, dense and unifrom; or it contains various larger dark granules, exhibits areas of different density and displays small vacuoles or inclusions which are either grey or highly refractive. The nucleus is usually quite large and finely punctate or granulated. On some occasions these cells become hyaline or highly refrac tive at their periphery. The most common variety is a round cell with a single, large and centrally-located round nucleus. The cytoplasm contains sharply-out lined round or ovoid granular inclusions (Fig. 2). The round cells measure 8.9 1971 A Study of Haemocytes in a Centipede Scolopendra morsitans 517 to 9.5ƒÊ in diameter and the nucleus measure 4.9 to 5.2ƒÊ. A common variant from this type is shown in Fig. 3. This is characterized by possession of one to several hyaloplasmic extensions. When movement of the blood caused the cells to be carried along, they would often temporarily attach to the surface by these extensions. This form is observed to change into a disc-like shape or to attach at one point and elongate into a fusiform, or spindle shaped cell. This type is highly unstable in vitro. Fjgs. 1-10. Haemocytes of Scolopendra morsitans. 1, prohaemocyte. 2, round plasmato cytes. 3, plasmatocyte having hyaloplasmic extensions. 4, fusiform plasmatocyte. 5, granular haemocytes. 6, disintegrating granular haemocytes. 7, spherule cell. 8, dis integrating spherule cells. 9, adipohaemocyte. 10, oenocytoid. The fusiform haemocyte (Fig. 4) is also seen upon immediate examination of a preparation, but is not as frequent as round cells or those with cyto plasmic extensions. The fusiform cell could also be formed directly from a round plasmatocyte. The cytoplasm varies from dense and uniform but finely granulated to a differentiated cytoplasmic area of slight refractivity and Cytologia36. 1971 35 518 G. S. Rajulu Cytologia 36 containing small vacuoles or dark granules. The fusiform cells often have tenuous, hyaline, filiform cytoplasmic extensions from either end which exhibit a searching motion. The type 2 cells may correspond to the plasmatocytes of insects (Jones 1962). Type 3: The cells of this type are round or ovoid and larger than the type 2 cells. They vary from 11 to 1411 in diameter. In freshly with drawn blood these cells often have a very pale, yellowish-brown cast and are characteristically filled with numerous round granular inclusions of a mostly uniform size from 0.5 to 111. The granules generally tend to obscure the relatively small, round centrally-located nucleus in fresh material (Fig. 5). In unfixed films of haemolymph, these haemocytes occur in two very distinct forms; those which remain intact for long periods in vitro and those which suddenly degenerate shortly after withdrawal of the haemolymph sample. Many of these haemocytes of various sizes have been watched as they degenerate in vitro (Fig. 6). The intact haemocyte seems to twist suddenly, contract or constrict along its longitudinal axis; and then collapses, releasing the nucleus and many fine granules. The granules thus releared are much more sharply outlined than in the intact cells and have a very faint greenish cast. The granules do not vanish quickly but tend to maintain their identity for a considerable time. In freshly fixed Giemsa stained smears, the granules in these cells appear pale to deep blue or slate grey occasionally very faint rose. The cytoplasm stains a pale blue; nuclei appeared pale rose and had a pale blue nucleolus. These haemocytes concentrated methylene blue into yellow or red vacuoles and also the carmine particles. In the foregoing features these haemocytes recall the granular haemocytes of insects (Jones 1965). Type 4: These conspicuous haemocytes are round or ovoid in shape and vary in diameter from 15 to 3011. Their nucleus, when visible, is very large and round and has a large nucleolus. These haemocytes contain few to many very large brilliant spheroid inclusions or spherules which fill up the whole cell (Fig. 7). The spherules vary in diameter from 3 to 511. These cells undergo a remarkable change in appearance in in vitro preparations. A sudden halo of cytoplasm appears about the thick mulbery shaped mass. A large round nucleus can be made out as the crystal-clear cytoplasmic halo continues to spread out; and as the nucleus grows clear all the spherules very suddenly vanish leaving behind clear round vacuoles in the cytoplasm with medium size granules scattered at their edges. Some of these cells break down without the formation of a cytoplasmic halo and others, on breaking down, develop enormous very thin clear blisters of cytoplasm (Fig. 8). In vitro break down of these cells is probably not due to coverslip pressure, for many of these cells were observed to remain intact for 10 minutes or longer in spite of repeated and deliberate pressure on the coverslip. 1971 A Study of Haemocytes in a Centipede Scolopendra morsitans 519 These haemocytes concentrate neither carmine nor methylene blue. These characteristics of these cells seem to recall the spherule cells of insects (Maclaughlin and Allen 1965). Type 5: On rare occasions haemocytes of a bubbly appearance with an eccentric nucleus and many brilliant droplets of various sizes have been encountered in the haemolymph (Fig. 9). Most of the droplets of these cells stain with rhodamine B and sudan IV suggesting that they are of the nature of fat. When treated with methanol, the droplets are dissolved. Such type of haemocytes were classed as adipohaemocytes by Jones (1962). They measure 8.5 to 9.5 in diameter. Type 6: The haemocytes of this type are large round cells measuring 20 to 30ƒÊ in diameter and with one sharp end.