W&M ScholarWorks

VIMS Articles Virginia Institute of Marine Science

1976

Fine Structure of sydneyi sp. n.: Haplosporidan Pathogen of Australian Oysters

Frank O. Perkins Virginia Institute of Marine Science

Peter H. Wolf

Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles

Part of the Marine Biology Commons

Recommended Citation Perkins, Frank O. and Wolf, Peter H., Fine Structure of Marteilia sydneyi sp. n.: Haplosporidan Pathogen of Australian Oysters (1976). Journal of Parsitology, 62(4), 528-538. https://scholarworks.wm.edu/vimsarticles/1990

This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. THC JOURNAL OF PARASITOLOGY Vol. 62, No. •, August 1976, p. 528-538

FINE STRUCTURE OF MARTE/LIA SYDNEY/ SP. N.­ HAPLOSPORIDAN PATHOGEN OF AUSTRALIAN OYSTERS*

Frank 0. Perkins and Peter H. Waif Virgin ia Institute of Morine Scie nce, Gloucester Point, Virginia 23062 ond Fisheries Branch, Chief Secre tary's Deportment, Sydney Austrolio

AOSTnACT: A new species of oyster pathogen, Marteilia syd11eyi, from Australian oysters, Crassostrea commercialis, is described incorporaling light and electron micro~ropr obsrrvatious. The pathogen is a hnplosporidan which e,ists n.s a plasmodiwn in the oyster hepatopnncreas. Upon sporulation, 8 to 16 uninuclcate sporangial prlmordia are internally cleaved ( endogenously budded ) from each plasmodium; thus c.-onver:;ion lo a sporangiosorus occurs. Each spornngium enlnrsres and internally cleaves into 2 or 3 primordia each of which, in turn, internally cleaves into 3 uninuclcalc sporoplnsms of graded sizes, the largest containing the \ mailer 2 in u vacuole and the inte m1ediale-~izcd one containing the smallest in a vacuole. The spore wall Is continuous without an orifice ur upt•rculum.

W olf ( UJ72) recorded the presence of a Aus tralian coast, not in the cold er w aters south haplosp oridan in Syd ney Rock oysters ( Crassos­ of Richmond River in Australia. The other trca commcrcialis Ired ale a nd Roughley) from species o f Crassostrea, C. ecliinata Quoy and Moreton Day, Queensland, Australia, in which C aimard, fro m the eastern coast of Austra lia is he noted in the hc patopuncrcas. In also parasitizcd b y a species of Martcilia prob­ subsequent papers Crizcl e t al. ( 1974) and ably identical to the one d escribed herein Mi~ and Sprague (1974) noted its close simi­ ( W olf, unpublished ). larity to Marteilia refringen~ Grizcl, Comps, Bonarni, Cousserans, Duthoit, and LePennec, a MATERIALS AND METHODS p athogen of Linne in French Blocks of hepatopancreas, l to 4 mm" in ~ize, estuaries. C rizel et al. ( 1974 ) suggested that were obtained from Moreton Bay, Queensland oy~ler~ ( C. commercialcs) and fixed in 6% M . refringens is one of lhc lower fung i; how­ glutaraldchyde for 5 to 7 days during tramil from ever, Perkins ( 1976) noted haplosporidan Austrnlia to the U.S.A. Subsequent h·eatmenl has charac teristics in the form of haplosporosomes been described ( Perkins, 1976). Light micrographs and in the mechanis m of spore forma lion from were obtained from histological slides of oysters fi xed in Davidson's fluid and stained with Hanis' sporang ia. The organism has, therefore, been hemato;1.-ylin and eosin. placed in the Class H aplosporea. The present Conclusions concerning spore structure were paper shows tha t the pathogen of C. com­ ba~ed on electron microscope observations of serial mercialis is a h aplosporidnn and is in the same sections through 2 spores as well as observations of indh idual sections. Serial sections were mounted genus as the pathogen of Oslrl'a ed11lis, popu­ on a Fonnvar sub~tratc on Mason and Morton slot larly known as the agent of J\ h r r disease (2 X l mm ) d iscs. ( A ldcrman, 197 4 ). RESULTS The pa thog en has bcc11 found only in sub­ Marteilia s ydne yi sp. n. tropical and tropical regions of the eastern (Figs. 1-11 l llost: Crassostrea commercialis Iredale and Received for publication 14 October 1975. Roughlcy. • Contribution o. 74-l, Virginia institute of Locality: foreton Bay, Queensland, Australia. \lnrint• Sci('nce, Clouce~Ler Point, Virginia 23062. 11 a/Jitat: I lepatopancreas.

• l<1cu= 1- 11. Marte/Lia syd11cyl sp. n., pathogen of U1e Australian O)ster, Crassostrea commcrcialis. All Figs. are electron micrograph., llnless otherwise inclicntecl. l. Light micrograph of hematoxylin and eosin-stained plasmodium in oyster bepatopancreas. PlasmodiaJ nuclei ( ) ; oyster nucleus ( 0). x 2,000. 2. Plasmodial mitocbondrion. Crista ( C ). X 103,000. 3. Multi vesicular body of plasmodium. lJaplosporosomes ( JI) are b<'lievecl to arise from vesicles (V) in multivesicular body. X 100,000. 4. Plnsmod ial haplosporosomes. Nole internal membrane (arrow) and delimiting membrane ( double arrow) nf each organelle. X 154,000.

528 PERKINS AND WOLF- FINE STRUCTURE OF MARTE/LIA SYDNEY/, OYSTER PATHOGEN 529 530 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. •, AUGUST 1976

F1clT11E 5. Plasmoclium differentiating into sporangiosorus. A sporangial primorclium ( unlabeled arrows) has lwen cleawcl from the plasmoclial protoplasm. 'ote absence of haplosporo80mes (H) and multive~icular bodies ( MV } from primordium. Primorclium nucleus ( P ). X 22,000. lnset is light micrograph of a cell comparable to one in Fig. 5. Plasmodial nucleus ( N); sporangial primorclium nucleus ( PN ). X 1,500. PERKINS ANO WOLF-FINE STRUCTURE OF MARTEil/A SYDNEY/ , OYSTER PATHOGEN 531

F1curu: 6. Sporangium with two spore primordia. Limits of one primordium are indicated by unlabeled arrows. Primordial nuclei (Nu); mitochondrion ( M ) ; extraspore sporangial cytoplasm ( SC); haplo­ sporosomes in non-sporangial cytoplasm of sporangiosorus ( Ji); plasma lemma of sporangiosorus (Pl); oyster cell ( OC). Sporangial wall not yet fom1ed. X 26,000.

Description EarUest known presporulation stages are plas­ enlarges and becomes a sporangiosorus. Eight to 16 modia in hepatopancreas Jumens, in and between sporangia form within sporangiosorus by internal hepatopancreas cells, and in connective tissues sur­ cleavage, then each sporangium cleaves internally rounding hepatopancreas tubules. Plasmodial cyto­ to form 2 (rarely, 3) spores. Spores spheroidal with plasm contains large multivesicular bodies and greatest diameter 2.0 to 3.2 µm in Davidson's-and haplosporosomes. During sporulation plasmodium formalin, acetic acid, alcohol ( FAA )-fixed mate- 532 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO, 4, AUGUST 1976

F1cURE 7. Sporangium showing one immature spore in which the three uninucleate sporoplasms are visible. Nuclei of outermost (N,), intermediate (N,), and innermost (Na) sporoplasms; extraspure sporangial nucleus ( SN ) ; extrasporangial nucleus (EN) of sporangiosorus; refringent inclusion (I); oyster cell ( OC); multi vesicular body ( MV ) of extrasporangial cytoplasm. X 21,000. Inset is Ught micrograph of spomngiosorus in same stage of development as the cell in Fig. 7. Unlabeled arrows indicate limits of one immature spore in which two nuclei are visible as dark dots. Refringent inclusion ( I ) ; oyster nucleus ( 0). X 1,500.

rial [i ± (s;)(t.m) = 2.7 ± 0.05; N = 30]. Proto­ tains haplosporosomes. Spore wall continuous plasm of each spore consists of 3 uninucleate without orifice or operculum. Extraspore proto­ sporoplasms of graded sizes. Intermediate sporo­ plasm of sporangia degenerates leaving large plasm lies in a vacuole of the largest sporopJasm; numbers of spheroidal inclusion bodles 1.7 to 3.2 the smallest sporoplasm in a vacuole of the µm diameter. Spore cell wall enclosed by a myelin intermediate one. Only outer sporoplasm con- whorl, 140 to 420 nm ll,iclc. PERKINS AND WOLF-FINE STRUCTU RE OF MARTEil/A SYDNEY/, OYSTER PATHOGEN 533

F1cunE 8. Nearly mature spore in which nuclei of intermediate ( N.) and innermost ( N.) sporoplasms are visible. Haplosporosomes ( If) of outermost sporoplasm; double membrane-limited, flattened vesicles ( V); spore wall ( W); sporangial wall (SW); extraspore sporangial nucleus (SN ); refringent inclusion ( I); multi vesicular body ( MV) in extrasporangial cytoplasm; sporangiosorus plasmalemma (Pl ). Note reticulated cytoplasm ( R) of sporangial cytoplasm outside ~pores. X 30,000.

Biology Prespornlution stages consist of plnsmodia M. refri11ge11s, it appears that haplosporosomcs (Fig. 1) which contain scattered haplosporo­ nrise from vesicles inside the MV's which bud somes, large multivesicular bodies ( MV's) , from the periphery of the MV, thus acquiring a membrane-free endoplasmic reticulum (E.R. ) delimiting, second membrane (Fig. 3). Free cistemae, scattered ribosomes, and vesicular haplosporosomes vermiform or obla te, ranging mitochondria which contain few cristae (Fig. from 146 to 603 nm long by 29 lo 60 nm 2). As noted by Perkins ( 1976) in studies of diame ler ( N = 18) (Fig. 4) . Striated inclu- 534 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO 4, AUGUST 1976 PERKINS AND WOLF-FINE STRUCTU RE OF MARTEil/A SYDNEY/, OYSTER PATHOGEN 535

F,cunE 11. Marl. sydn. cont'd. Mature spore showing Lhick mantle of concentric membranes ( MW) around wall ( W ). Outermost sporoplasm ( S,); nucleus ( , ) of intermediate sporoplasm; flattened vesicle ( V); refringent inclusion (I). x 132,000. Inset is light micrograph of mature sporangiosorus showing high density of inclusion bodies ( I ). Spore (Sp). X 1,200.

~ F'ic:um,;s 9- 10. ~lurt. sydn. conl'd. 9. Cytoplasm of 011Lem1o~l ~poroplnsm in nearly muturc spore ~bowing presun1cd clevclop111c•ntul sequence ( 1• 4 ) for huplosporosome fom,ation. X 124,000. r-'ully clifforontiatecl haplosporomme~ (arrows) are shown in inset. Internal membrane is presumed to arise from collapsing and inpockeling of one side of vesicle membrane ( between stages 2 and 3) followed by refusing of leading edges. X 136,000. 10. Double-membrane limited, fl attened vesicles ( V ) of intermediate sporo­ plasm. Plasmalcmma of intem,ediate sporoplasm ( M,); vacuole membrane of outern1ost sporoplasm ( M.); pla:;malm1111a of outermost sporoplasm ( M, ); wall ( W ); clcgeneratfag extrwporo sporang ial cylo­ plnsm (SC). X 130,000. Inset contains cross-sectional views of flattened vesicles in which the pair of membrn,ws can be disliniuished. X 132,000. 536 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 4, AUGUST 1976 sion bodies as found in plasmodfa of M . refrin­ pocketing of the vesicle membrane yields the gt•11s not present in M . sydneyi. second, innermost membrane. Further accumu­ Sporulation is initiated by delimitation of lation of electron-dense material yields the internal, uninucleatt'cl segments of the plus­ mature form of the organelle (stages 3, 4; Fig. modial protoplasl us n result of link-up of fl at­ 9). Fully differentiated haplosporosomes are tened vesicles aligned end-to-end (Fig. 5). The pleomorphic with most being oblnt<', spheroidal, resulting pair of membranes in parallel orienta­ or vermiform (Fig. 11) . The longest axis is tion comprises the plasmalemma of a spo­ 148 to 288 nm with diameters of 57 to 163 rangial primordium and a vacuole membrane nm ( N = 10). Other cytoplasmic organelles around the primordium. Haplosporosomcs and consist of vesicular mitochondria, vesicles, E.R., MV's arc not included in the cytoplasm of the and membrane-free ribosomes. Lomasomelike primordium; only mitochondria, E. R. cistcrnae, extensions of the sporoplasm surface are found lttrgc n11mhers of ribosomes, and a si11gle in one or two convoluted groupings of tubulur nucleus. The ribosome density is higher than cytopla~m. A wall (27 to 49 nm U1ick; N = 17) that observed in the surrounding, nonprimordial is formed around the sporoplasm involving the cytoplasm. Prior to sporangial primordia de­ same mechanism as described for M. refringens umitalion the plasmodia are 8 to 15 µ.m long (Perkins, 1976) (Figs. 8, 11). by 5 to 10 µ.m wide ( = 20) (measurements The intennecliate sporoplasm lies in a vacuole from histological sections of Davidson's-fixed of the outermost sporoplasm and differs from material). From the time of spornngial primor­ the former in the lack of haplosporosomes and dia formation lo completion of spore fonnation the presence of flattened vermiform vesicles 440 the size of the former plasmodium increases to 1160 nm long and 50 to 77 nm ( = 15) to 21 lo 30 by 17 lo 22 µ.m ( 1 = 25). After diameter, which consist of two concentric mem­ thf' appearance of sporangial primordia through branes ( Figs. 8, 10, 11). One end of the spore maturation, the former plasmodia are vermiform vesicles is enlarged and filled with termed sporangiosori. electron-dense material within the innermost Nuclear multiplication within the spora11 gial membrane. \ ,Vhether these enlargements Inter primorclin followed by internal cleavage yields develop into haplosporosomes was not deter­ two (rarely, 3 ) uninucleated spore primorclia mined. The other end of the flattened vesicles and one or two nuclei in the remai11i11g ~po­ is closely opposed to the sporoplasm plasma­ rangial cytoplasm (Fig. 6 ) . The latter degen­ lemma with electron-dense granular material erates toward the end of sporulation while the bridging the gap between the two structures nuclei of spore primordia divide to yield three ( Fig. 10). The innem1ost sporoplasm has the nuclei per spore (Fig. 7). Presumably there least organelle diversity, consisting of a single is one followed by mitosis in only one nucleus, one to three mitochondria, and mem­ daughter nucleus since there is no evidence of brane-free ribosomes ( Fig. 8). a degenerate nucleus. Internal cleavage by Extraspore, sporangial protoplasm becomes vesicle link-up occurs lo yield three uniJ1ucleate vacuolated after spore primordium formation sporoplnsms of graded sizes, one within a (Fig. 8) then further degrades into groups of vacuole of the other. Hefringenl inclusions are myelin whorls ( Fig. 11). Each spore becomes formed in the extra-spore cytoplasm of the sporangium after spore primordium deljmitation covered by a whorl 140 to 420 nm thick (Fig. 7). ( = 15). Large ( 1.7 to 3.2 /tm diameter; The outermost sporoplasm is the only one N = 20) spherical inclusion bodies accumulate which acquires haplosporosomcs ( Fig. 8 ) . The in the sporangial cytoplasm during spore matu­ organelles disappear from the plasmoclia as they ration, ultimately becoming so numerous as to differentiate into sporangiosori then reappear mask the presence of spurns i11 histological during sporoplasm delimitation, apparently sections (Fig. 11 inset ). A 16 to 27 nm arising from vesicles scattered throughout the ( = 20) thick wall is formed around each cytoplasm ( Figs. 9, 10). Electron-dense mate­ sporangium about the time of spore primordium rial accumulates against the vacuole membrane formation and persists through spore matura­ ( stages 1, 2; Fig. 0) then presumably an in- tion (Fig. 8). PERKINS AND WOLF-FINE STRUCTURE OF MAR TEil/A SYDNEY/, OYSTER PATHOGEN 537

DISCUSSION not previously reported for , fungi, or M. sydneyi is considered to be a member of algae. The term endogenous budding from the genus because the mechanism of spore classical protozoology appears inappropriate in formation ( i.e. plasmodfal conversion via a denoting intem al cleavage because fine struc­ series of internal cleavages into a sporangiosorus ture studies show that budding of protoplasm containing sporangia and spores) and spore into an internal vacuole or cell surface depres­ structure is nearly identical to that of M. sion does not occur. In Marte i/ia spp. primordia refringens, the only other member of the genus. are delimited by vesicle fusion (Perkins, 1976 ), The reader should refer to the paper on M . as has been described in the Phycomycetes refringens (Perkins, 1976) for more complete (Cay and Greenwood, 1966) and some algae descriptions of structures or developmental ( Dodge, 1973). stages which are present in both species. The In the senior author's paper (Perkins, 1976) rationale for considering Marteilia spp. to be on Marleilia ref ringens and in the present paper haplosporidan is also presented in that paper. the botanical tem1s, sporangium and sporangio­ The Australian oyster pathogen is considered sorus, have been used ; however, the usage is to be distinct from M . refringens because not intended to imply that species of Marteilia striated inclusion bodies are not formed in and other Haplosporea are considered to be plasmodia, sporangiosori contain 8 to 16 spo­ fungi. They are clearly Protozoa. " 'e consider rangia rather than about 8, and sporangia con­ the distinction between botanical and zoological tain two or, infrequently, three rather than four terminology to be irrelevant at the phylogenetic spores. The sizes of M . syclneyi and M. level under consideration since we adhere to refringens mature sporangiosori are about the Whittaker's {1969) five classification same as seen in histological sections, the former in which most Protozoa and simple algae are having a mean length ( the longest axis) of 25 grouped in tl1e Kingdom Protista. µ.m (range: 21 to 30 ,...m; = 25) and the In the absence of strong evidence of sexu­ latter having a mean length of 21 {range: 16 ality, the classical botanical terms a) sporula­ to 27 ,...m; N = 25). Spores are the same diam­ tion, b ) sporangiosorus, c) sporangium, and eter [i ± ( s.;) ( t.oa) = 2.7 ± 0.05] for M . syd­ d ) spore have been tentatively selected because neyi vs. 2.6 ± 0.05 1.1m when measured from they most adequately denote asexual stages. histological sections [material fixed in David­ The most appropriate and equivalent proto­ son's or formalin, acetic acid, alcohol {FAA) zoological terms would be a) sporogony, b ) fixatives]. "Diameters" were considered to be sporocyst, c) spore, and d ) sporozoite; how­ the longest axis since the spores are not always ever, sexuality would be implied. At present perfectly spherical. Student " t" test indicates no haplosporidan life cycles have been eluci­ no significant difference in spore sizes within dated thus it is not known whether spore the 95% confidence limits. formation is a meiotic or mitotic process. lf Spl.)res of M. sydneyi differ from those in the the periodic nucleoplasmic structures described 0 . edulis p athogen in that a heavy layer of for M . refringens (Perkins, 1976 ) are poly­ concentric membranes surrounds mature spores complexes, then meiosis is indicated. Sporogony of the former whereas M . refringens spores and associated terminology would then be more generally lack such a covering, although loosely appropriate than the terminology now being applied whorls have been observed around a used. Protozoological terminology used t o de­ few spores. Otherwise the spores appear to note asexual development and cell types (i.e. be identical. schizogony, endopolyogeny, schizonts, and The two species of Marteilia have a unique merozoites) is not adequate in descriptions of mechanism of spore formation. Internal cleav­ spore formation in Marteilia spp. age of sporangia witl1in plasmodia, followed by internal cleavage of spore primordia within ACKNOWLEDGMENTS sporangia, then internal cleavage of two sporo­ The authors gratefull y acknowledge the plasms within each spore primordium, yielding technical assistance of Mrs. Deborah DeBiasi, a total of three sporoplasms, is a mechanism Miss Susan Fox, and Mrs. Patsy Berry. 538 THE JOURNAL OF PARASITOLOGY, VOL 62, NO. 4 , AUGUST 1976

LITERATURE CITED C'rl11lis Linne. Science et P~chc, Bull Inst Pt1chC', \larit 240: 7-30. A1 .1>tll\lA'I, D. H>74. For,•ign cli~t•tlst•~ litvard Mix, ~I. C. AI\ I> V. SP1v.c;u£. 1974. Occ11rrcnct.• British shellfish. New Sci,•ntist 63: 122-12<1. of a huplosporidiun in native oysters ( O~tri•a IJ01Jci::, J. D. HJ73. The fhw structure of ulgol /11 rlcla) from Yaq11inn Buy and Alsra Buy, c,•lls. Acadl•mic Press, ew York, p. 159-166. Oregon. J lnwrt Pathol 23: 252-254. GA,, J. L. A'ID A. D. Cnt:1.;,\\ orn>. I H(j(i. Struc­ PmK1 ~. F. 0. 1976. Ultraslnicture of sporula­ turnl aspects of 'l.OOspon• production in Sawo­ tion in the Europl'an flat oyster pathogen, lcµ, nia /el'a:O: willt pa,liculur reference to the Martellill re/ringc11s-ta,onomic implications. cell and vac11olar memhrones. fo M . F. J Proto. .mo l 23: 61-74. t.ladclin (ed.), The spore. Buller­ \V11rrrAKEn, H. 11. 1969. 'ew concepts of king- worths, London, p. 95-110. doms of organisms. Science 163: 150-160. Cnrzu., 11., M. CoMPS, J. R. l3o"1M11, F. WoLF, l'. H . 1972. Occurrence of a haplo­ Coussmv."ls, J. L. Du, 1101T, A'\U t-1. A. sporidan in Sydney Hock oysters ( Cmsso~trca L~;Pm,I\EC.. 1971. Rl•clwrchc M ir l'og,•nl de commercialls) from Moreton Bay, Qtll'cnslnnd, la muladic clc In glanclc• digcsliv,• de O.\trea Australiu. J lnvc11 Pathol 19: 416-417.

Suggestions to Prospective Authors In nddition to general instructions inside the article into the dummy and 11my result in earlier front cover of tho Journal, the following hints publication. huve been printed from lime to time: The author should 11111ke il clc,1r whclhcr mt1g- 11lflrnlions of plat<'s, as gi, en, arc for the pl.Ile On bibliographic style: set• notice on p. 300 as it will appear in the Journal, or must be recal­ vulu111e 61, and follow modeb in the Journal culated for the intended reduction. Where plates On the Hese:m·h Note: 1t7: .'.396. hnvc heen rlP.signerl for spPc-in l trPntmcmt, in con­ Hints on writing: 48: 176, 51: 553, 52: 232, ference with the editor, such as bleeding to edge 406, 54: 535, 55: 354,688, 58: 722, 59: 276. of page, this must be clearly marked bu the author Preparation of drnwings and plates: 52: 1024, on margin or back of plate. Otherwise such 53: tl<:l0, 54: 50, 358, 5(1: 264, 58: 554. arrangements 11re likely to be forgollC'n by tlw lime the manuscript goes to the printer. It is Authors are 110w charged ut the rate of $40.00 f,xilish to wnste word~ trying to describe features 1wr pagP for nil png1·~ over 11, l~XC't'ssiw rorrcct ions hPsl seen in the illuslrnlions. There is nothing in proof will be charged LO the nuthor. wrong with staling "as figured." All copy must be double spaced, including Excessive bibliographics are to be avoided, bibliogrnphies, legends lo plates, etc., und Lhe particularly references to procedures which bavt· master copy must be n ribbCJa copy 0 11 bond paver. hy now become standard (such as E/\1 techniques). Legends must be Lyped on II separate sheet and Twenty five entries should be a maximum. Re­ not tacked on to the illustrations. All illustrntions cently some research notes have been submitted must be numbered on the back for idcnlificntion. with ns mnny as five nuthorsl This would ht! Composite halftone plates should be mounted with t'Xcessive for n full hlown article, nnd on II research al lenst an inch margin lo protect edges of tho note approaches the ridiculous. prinls, and for ed itorial marking. When in doubt, authors should study recent l'latcs must ho designed for either single column numbers of the Journal for hrmdling of similar ( 16 picas), or double column (33 picas) width, as mul<'rinl. We cannot permit wide dc•partures from rcwod11ccd. Odd si'l.cs arC' not pcrmiltC'cl. Plates established Journal style since this greatly compli­ must be rectangular. Steps in margins are not cates lhe work of the editor and printer. \Vherr permitted. Indiviclunl prints must be accurately most measurements arc less than l mm, values squ,1red and butted, no white showing. The should be given "in microns unless otherwise ungravC'r will e11t a fine hairline of separation slated." between the figure~. It is advantageous, where The editor's office cannot answer trivial in­ convenient, to have Lhe plalt• as printed a little quiries unless n self-addressed stamped envelope shorter than page length, so that the l<'gend con or postcard is inclucled. be fitted below. This 111ak1·~ it c·asl(•r lo fit the THE EDJTOII