The Direction of Evolution in the Drosophila Rnelanogaster Species Subgroup Based on Functional Analysis of the Crystal Cells T

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 212: 323-328 11980)

The Direction of Evolution in the Drosophila rnelanogaster Species Subgroup Based on Functional Analysis of the Crystal Cells T. M. RIZKI AND ROSE M. RIZKI Department of Zoology, Division of Biological Sciences, The University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT The crystal cells in the hemolymph of Drosophila melanogaster contain paracrystalline inclusions and phenol oxidase. Melanization of this type of blood cell can be induced by treatment of larvae with hot water. By using this functional criterion to establish cell homology we compared the shape of the inclusions in the crystal cells of the D. melanogaster species subgroup and a number of distantly related species of Drosophila. The crystal cell inclusions in four of the sibling species (simulans, erecta, mauritiana, yakuba) are the same as those found in D. melanogaster. In the fifth species, D. teissieri, the crystal cell inclusions resemble those found in the hemocytes of the other larval drosophilids that were examined rather than those of D. melanogaster. On the basis of these observations and the phylogenetic relationships deduced from analysis of chromosomal inversions by Lemeunier and Ashburner ('76), we propose that D. teissieri is closer to the ancestral form or is the ancestral species of the melanogaster species subgroup of Drosowhila. This is the first instance in which a hemocyte type has been used to determine the direction of evolution within a subgroup of insect species.

The larval hemolymph of Drosophila melan- in this mutant are melanized, the phenol ox- ogaster contains two classes of hemocytes, idase within them is destroyed; hence Bc lar- plasmatocytes and crystal cells (Rizki, '57a). vae lack phenol oxidase activity. Furthermore, Plasmatocytes differentiate into podocyte and melanization of the crystal cells in Bc+ larvae lamellocyte variants but the crystal cells, can be induced by hot water treatment, and comprising 5-1096 of the larval hemocyte pop- the distribution of heat-induced black cells in ulation, remain uniquely distinguishable by these larvae mimics the distribution of black their prominent paracrystalline inclusions. cells in Bc mutant larvae. From this series of Such cytoplasmic structures have not, to our observations it is clear that two methods can knowledge, been reported in the blood cells of be used to identify crystal cells in D. melano- other insects or even other species of Droso- gaster: structurally, on the basis of their par- phila. Yet the function ascribed to these cells, acrystalline inclusions; and functionally, by storage of the components involved in hemo- hot water treatment to blacken them. Utiliz- lymph phenol oxidase activity (Rizki and Riz- ing these criteria we examined the hemocytes ki, '59), is a common attribute of insect hem- of a number of sibling species of D. melano- olymph. gaster and some other members of the family Studies on a recently isolated mutant gene, Drosophilidae. For the latter group, no effort Black cells (Bc, 2-80.621, agree with the lo- was made to be exhaustive; we used species calization of hemolymph phenol oxidase activ- that were available in our laboratory. The five ity to the crystal cells (Rizki and Rizki, '79a; sibling species of D. melanogaster used are Rizki et al., submitted for publication). Blood those studied by Lemeunier and Ashburner cells with paracrystalline inclusions are ab- ('76). The present report summarizes these sent in Bc larvae that contain instead melan- findings on Drosophila hemocytes and sug- ized cells in the hemolymph and lymph glands, gests that analysis of hemocyte types may be suggesting that the blackened cells are a mu- useful in delineating phylogenetic relation- tant form of the crystal cells. When the cells ships in Drosophila.

0022-104X/80/2123-0323$01.40CG 1980 ALAN R. LISS, INC. 323 324 T.M.RIZKI AND R.M. RIZKI

MATERIALS AND METHODS from those found in D. melanogaster crystal, The sibling species used for this study were: cells, and representative crystal cells from D. melanogaster (Ore-R wild type), D. erecta these four species are included (Fig. 1).There (154.1), D. simulans (C135.20), D. yakuba are variations in size of the paracrystalline (115), D. teissieri (128.2), and D. mauritiana. inclusions, and the number of inclusions per The other species examined were: D. pseu- cell also differs among crystal cells taken from doobscura, D. virilis, D. novamexicam, D. wil- the same hemolymph sample. Cells similar to listoni, D. nebulosa, D. buskii, and Zaprionus the four cells in this plate, each taken from a uittiger. different sibling species, can all be found in a All larvae were grown on cream of wheat single sample taken from any one of the sib- medium with live yeast at 18°C except Za- ling species, including D. melanogaster. Treat- prionus larvae, which were grown on corn ment with hot water blackened the cells with meal-agar medium and D. buskii larvae, crystals in the four sibling species. which were grown on medium containing flax Figure 2 contains photographs of crystal seed. Mid-third instar larvae were washed cells from D. teissieri, another sibling species with 1.2% NaClO and rinsed well with dis- of D. melanogaster. The inclusions in these tilled H,O to remove extraneous food particles cells lack the general rectangular appearance from the body surface. Larvae were opened by found in the crystal cells of the other five tearing the body wall, taking care not to species of the melanogaster subgroup of Dro- rupture internal organs, and allowing the sophila. They appear chunky and globular. hemolymph to flow into a small drop of buff- The inclusions are quantitatively variable but ered formaldehyde (2.3% in phosphate buffer none of the inclusions resemble those found in at pH 7.2). After 4 minutes dilute Giemsa the other sibling species. That these cells are stain was added to the hemolymph sample, functionally similar to the crystal cells of the and the stained cells were examined. At least other melanogaster sibling species can be dem- five camera lucida drawings of crystal cells onstrated by immersion of the larvae in hot were made for each species to assure that water, for they respond to this treatment pre- variations in shape and size of the inclusions cisely as do the crystal cells. were recorded. In all the larvae from outside the melano- To establish the functional identity of the gaster species subgroup the hemocytes with cells with inclusions, larvae were immersed in inclusions resembled those found in D. teissieri hot water at 70°C. When blackening of hem- rather than those found in D. melanogaster ocytes became apparent, the treated larvae (Fig. 3). There were size and quantitative were examined either directly under trans- variations, but none similar to the melanogas- mitted light (compound microscope: 12.5 x 16 ter type were found. For each species the or 12.5 x 25 magnification), or the larvae functional relationship to the melanogaster were opened in Carnoy fixative and tissues crystal cell was established by hot water treat- with attached darkened cells were processed ment. and examined. The cytoplasmic inclusions are DISCUSSION still visible in the cells that are only lightly This report is the first attempt to use a tanned, so the cells were not permitted to functional criterion for classification of a spe- blacken fully. The inclusions in the melanizing cific hemocyte type in the genus Drosophila. cells and in the stained preparations were Classification of insect hemocytes relies pri- compared. marily on morphological characteristics, so categorization of cells to a common class RESULTS among different insects is possible only when The cytoplasmic inclusions in the crystal there are distinctly comparable structural cells of D. melanogaster have a strikingly traits. To draw analogies in the absence of regular shape. The two longer sides of the such similarity or in the absence of informa- structures appear parallel with ends that are tion on the functions of a given cell type is either rectangular or conical. Since photo- unwise, since hemocytes display morphologi- graphs of D. melanogaster crystal cells have cal variations in response to physiological been published previously (Rizki and Rizki, changes in the hemolymph; they also show '591, none are included here. However, the variations during their own stages of devel- paracrystalline inclusions in four of the sib- opment (Rizki, '78). D. melanogaster with its ling species, D. simulans, D. mauritiana, D. many mutant strains has been exceptionally yakuba, and D. erecta are indistinguishable useful for studying hemocyte interrelation- CRYSTAL CELIS IN DROSOPHILA 325

Fig. 1. Morphology of the paracrystalline inclusions in the crystal cells of: a, D. simulans; b, D. mauritiana; c, D. yakuba; d, D. erecta.

Fig. 2. Crystal cells of D. teissien. 326 T.M. RIZKI AND R.M. MZKI

Fig. 3. Crystal cells of: a, D. willistoni; b, D. psedoobscum; c, D.novamexicam; d, Zapriow uittiger.

ships: melanotic tumor strains provided the morphologically similar” although Rizki and material for studying the plasmatocytes and Rizki (’59) did not “analogize the crystalloid their morphological variants (Rizki, ’57b; Riz- cells ofD. willistoni” with the crystal cells in ki and Rizki, ’79b); the Bc mutant confirmed D. melanogaster. From the present study it is the crystal cell as the source of hemolymph clear that the crystal cells in D. melanogaster phenol oxidase activity and lead to a simple are analogous to the cells in D. willistoni technique for releasing melanization in the described as spheroidocytes (Rizki, ’53); there crystal cells of Bc’ larvae (Rizki and Rizki, is as yet no information suggesting a relation- ’79a). With the latter method hemocytes that ship, if any, between the crystalloid cells of D. are not otherwise readily comparable by mor- willistoni and the crystal cells of D. melano- phological criteria can be grouped into one gaster. category, and we are now able to unravel the The fragility of the crystal cells is a distinc- enigma in the literature concerning crystal tive feature of this hemocyte type in D. me- cells in drosophilids. Yeager’s (’45) terminol- lanogaster (Rizki, ’57a). When the body wall ogy was adopted for classification of the blood is opened or hernolymph is removed from the cells in D. willistoni (Rizki, ’53) with the hemocoel, the inclusions within these cells exception that one category of cells was des- begin to disintegrate and the cells often rup- ignated crystalloid cells. These hemocytes ture. We proposed that effective separation of contain highly refractile, birefringent rodlets phenol oxidase from its substrate is achieved and are found in young larvae. In subsequent within the crystal cell by storage of the latter studies on D. melanogaster (Rizki, ’57a) the in the paracrystalline inclusions (Rizki and term crystal cell was adopted for the cell type Rizki, ’59). Disturbances that influence the that is the subject of the present report. Crys- integrity of the paracrystalline inclusions will tal cells are found throughout larval life in D. allow contact of substrate with enzyme, thus melanogaster and are morphologically distinct resulting in melanization. Since the crystal from crystalloid cells. On the basis of our cell inclusions are unusually sensitive to ex- published work, Nappi (‘70) concluded that perimental manipulations, we cannot ignore “crystalloid cells of D. willistoni and the crys- the possibility that the morphological differ- tal cell of D. melanogaster are unquestionably ence observed between the melanogaster and CRYSTAL CELLS IN DROSOPHILA 327 non-melanogaster cell types is due to factors similar morphology of these structures where- in the cells or hemolymph that influence the as the remaining species, D. teissieri, differs stability of the inclusions, rather than a dif- from these and resembles the other Drosophila ference in the structural components of the species that we examined. inclusions themselves. Hopefully, biochemical We stress that the sampling of species out- and/or ultrastructural examinations of these side the melanogaster subgroup examined in cell types will answer this question. In the this study is very limited, and we do not imply meantime, the distinction between these two that the melanogaster-type crystal cell occurs groups is apparent by utilizing the fixation only in this subgroup. An extensive review of methods employed in the present study. the drosophilids and their phylogenetic inter- A brief summary of the species included in relationships is presented by Throckmorton the melanogaster subgroup of Drosophila is ('75). Aside from the melanogaster subgroup, presented by Lemeunier and Ashburner ('76). we examined the hemocytes of willistoni, ne- These authors analyzed the banding patterns bulosa, and pseudoobscum, representing at of the salivary gland chromosomes of the six least two distantly related species groups of sibling species, and on the basis of the over- Sophophorans, two species from the virilis lapping inversions established phylogenetic group, a species of the genus Zaprionus, and relationships among the species. Their scheme D. buskii in the subgenus Dorsilopha. The includes bidirectional arrows for, as stressed inclusions in the hemocytes of all these species by these authors, chromosomal inversion phy- are like those of D. teissieri. If we accept that logenies are seldom useful for determining the the widely occurring characteristic is the an- progression of evolution from primitive to cestral trait, then D. teissieri is closer to the more recent species. Additional distinguishing ancestral form or is the ancestral species of features must be sought, and these might then the melanogaster species subgroup. This sug- be combined with the information from inver- gestion is combined with the scheme of Le- sion phylogenies to delineate lines of descent. meunier and Ashburner ('76) in Figure 4. Among the morphological traits generally Such an evolutionary pattern supports the useful for distinguishing sibling species are conclusion of Tsacas and Lachaise ('74) and the structural features of the male genitalia. Throckmorton ('75) that the melanogaster spe- Interestingly, structural similarity of male genitalia does not necessarily indicate genetic closeness, for the male genitalia of the sibling speciesD. pseudoobscura and D.persimilis are distinguishable with difficulty (Rizki, '51) teissicri @ whereas those of D. simulans and D. melanogaster are quite distinct (Sturtevant, '29). In- I deed, the male genitalia of the six sibling I species of the melanogaster subgroup are suf- ficiently different to allow recognition of these species (Bock and Wheeler, '72; Bacas and David, '74; 'hacas and Lachaise, '741, but there do not appear to be differences and similarities in these organs that would be useful for ana- lyzing the direction of evolution. A recent analysis of polypyrimidine stretches in drosophilids including sibling species in the melanogaster subgroup shows a similarity of sequences in melanogaster, mauritiana, and simulans DNA, but erecta, yakuba, and teissieri do not have polypyrimidine sequences melanoga / ster simuians@ that will hybridize with D. melanogaster po- @----- lypyrimidines (Cseko et al., '79). Therefore, Fig. 4. The proposed direction of evolution in the D. evolutionary directions for this subgroup can- mehnogaster species subgroup deduced from analysis of not be assessed by these methods. We believe morphology of the inclusions in the crystal cells. This scheme is based on the phylogeny of inversions presented that the unique morphology of the inclusions by Lemeunier and Ashburner ('76); I, 11, and III are hyp in the crystal cells can be used for this pur- thetical inversion steps and the dotted lines represent an pose. Five of the six sibling species share a alternate scheme proposed by these authors. 328 T.M. RIZKI AND R.M. RIZKI cies subgroup arose in Africa since D. teissieri Cseko, Y.M.T., N.A. Dower, P. Minoo, L. Lowenstein, G.R. Smith, J. Stone, and R. Sedemff (1979) Evolution of is endemic to Africa (Tsacas, '71). E'urther- plypyrimidines in Drosophila. Genetics, 92:45%484. more, D. yakuba and D. erecta are also African Kaneshiro, K.Y. (1976) Ethological isolation and phylc- endemic species;D. mauritiana is known from geny in the planitibia subgroup of Hawaiian Drosophila. the island of Mauritius (Tsacas and David, Evolution, 30: 74&745. '74). Lemeunier, F., and M. Ashburner (1976) Relationships within the melanagaster species subgroup of the genus Mating preference has been suggested as a Drosophila (Sophophora). 11. Phylogenetic relationships means for determining phylogenetic relation- between six species based upon polytene chromosome ships among Drosophila species by assuming banding sequences. Proc. R. Soc. Lond. B, 193:275294. that females of an ancestral species discrimi- Nappi, A.J. (1970) Hemocytes of larvae of Drosophila euromtw (Diptera: Drosophilidae). Ann. Entomol. Soc. nate against males of a derived species (Ka- Am., 63:1217-1224. neshiro, '76). The success of reciprocal hybrid Rizki, T.M. (1951) Morphological differences between matings between mauritiana, simulans, and two sibling species, Dmophila pseudoobscurn and Dre melanogaster was measured by Watanabe and sophila persirnilis. Proc. Natl. Acad. Sci., 37: 156-159. Rizki, T.M. (1953) The larval blood cells of Drosophila Kawanishi ('79). These authors, however, sug- wzllistoni. J. Exp. Zool., 123:397-411. gest that mating preference is exerted by the Rizki, T.M. (1957a) Alterations in the haemocyte popu- females of the newly derived species and on lation of Drosophila meluw&. J. Morphol., 100437-458. the basis of this assumption propose the evo- Rizki, T.M. (1957b) Tumor formation in relation to met- amorphosis in Drosophila melanogaster. J. Morphol., lutionary scheme from melanogaster + si- 100:459-472. mulans + mauritiana. If Kaneshiro's ('76) Rizki, T.M. (1978) The circulatory system and associated assumption is accepted, then the same data cells and tissues. In: The Genetics and Biology of Droso- predict the direction of evolution as mauri- phila. M. Ashburner and T.R.F. Wright, eds. Academic Press, London, Vol. 2b, pp. 397-452. tiana melanogaster. Crystal cell morpholo- Rizki, T.M., and R.M. Rizki (1959) Functional signifi- gy together with inversion phylogeny sug- cance of the crystal cells in the larva of Drosophila gests that D. melanogaster is the more melanogaster. J. Biophys. Biochem. Cytol., 5:235240. recently derived species of this group. In line Rizki, R.M., and T.M. Rizki (1979a) Phenogenetics of Black cells (Bc). Genetics, 91:s103-104. with Throckmorton's ('75) reasoning on the Rizki, R.M., and T.M. Rizki (1979b) Cell interactions in wide spread distribution of D. melanogaster the differentiation of a melanotic tumor in Drosophila. and its association with man, it is interesting Differentiation, 12: 167-178. to consider that this association was first es- Sturtevant, A.H. (1929) The genetics of Drosophila simulans. Carnegie Inst. Wash., 339; 1-62. tablished in Africa. Thmkmorton, L.H. (1975) The phylogeny, ecology, and geography of Drosophila. In: Handbook of Genetics, Vol. ACKNOWLEDGMENTS 3. R.C. King, ed. Plenum Press, New York, pp. 421-469. Ihacas, L. (1971) Drosophila teissieri, nouvelle esee We are especially grateful to Dr. R. C. africaine du groupe mehogaster et note sur deux autres Woodruff of Bowling Green State University espkes nouvelles pour 1'Afrique (Diptera: Drosophilidae). for providing us with the sibling species of D. Bull. Soc. Ento. France, 76:35-45. Ihacas, L., and J. David (1974) Drosophila mauritiana melanogaster and to Dr. L. H. Throckmorton n. sp. du groupe melamgaster de I'Ile Maurice (Dipt.: of The University of Chicago for helpful com- Drosophilidae). Bull. Soc. Ento. France, 79:42-46. ments on the manuscript. This research was Tsacas, L., and D. Lachaise (1974) Quatre nouvelles es- phs de la Cote d'Ivoire du genre Drosophila, groupe supported by Grant No. CA-16619 awarded by melanogaster, et discussion de l'origine du sow-groupe the National Cancer Institute, DHEW. melanogaster (Diptera: Drosophilidae). Ann. Univ. Abid- jan,E7(1):193-211. LITERATURE CITED Watanabe, T.K., and M. Kawanishi (1979) Mating preference and the direction of evolution in Drosophila. Bock, I.R., and M.R. Wheeler (1972) The Dmsophila me- Science, 205:906-907. lamgaster species group. Stud. Genet. VII. University of Yeager, J.F. (1945) The blood picture of the southern Texas Publications, 7213: 1-102. armyworm (Prodenia eridaniu). J. Agric. hs., 71:l-40.