rechls SET, tre ore: bised * via ape ¥ ee ee, Rt ea pine , ; ‘ Digitized by the Internet Archive in 2010 with funding from University of Toronto http://www.archive.org/details/bulletin/v8toky O AC ; “OY ‘ =i ss 2 Kat K KR Ae 2S k fF B ion i, . = ee ae es = 3 BULLETIN OF THE ,COLELEGE OF AGRICUL PURE, TOKYO ImpEeRIaAL UNIVERSITY, JAPAN. Fs o, Vor. Vik-——C I906—1908 CONTENTS OF VOLUME VII. Some Catalytic Action of Platinum Black. By O. Loew and K. Aso. Ueber die Veranderung des Zellkerns durch kalkfallende Mittel. By Ere Netra cathe eee. I RI PEG ond = i oe ee Injurious Action of Acetates and Formates on Plants. By K. Aso .. Konnen kleine Dosen Kupfer eine chronische Kupfergiftung hervor- AppiesTig ea boyae Mem AN Ooma@a) caer late, oe. & ss: 2 was. 3 he On the Formation of Anthokyan in the Stalk of Barley. By S. SITES! 6 BOGE Sue ST Aa gee Cette 9a oe BN On the Influence of the Reaction of Manure upon the Field. By earencnaatiol Map ataG UG ae ca. os poe coy 63 by gs oS On the Manurial Value of Caleium Cyanamide. By K. Aso ...... The Efficacy of Calcium Cyanamide under Different Conditions. Jer nied lec nconhs ii Mele Beet AE ccs ee ee See fein aes On. the Lime-Factor for Flax and Spinach. By S. Namikawa ... Regeneration of Overlimed Soil. By S. Maki and S. Tanaka ...... The Manurial Value of Different Potassium Compounds for Barley PUT Sleericemment ss Vw Key MAISON Aas ahrigtetader ace ss Ste us nc v's cues ore eee On the Effect of Various Potassic Manures on the Growth of | Colocasia7anmquorum, “By .S, Namikawa..: 2... aemanae On the Application of Chilisalpetre as Top-dressing for some NigpanesewO Lopssy Uy tee NGOU. ce fl Nas oes oe Seeger eee On the Stimulating Action of Manganese upon Rice, IIT. By M. Olea tar teh Ann em eben ee tS Ss iT ve eee Stimulating Influence of Sodium Fluorid on Garden Plants. By K. JAS i. the os ten kate ht dine ey os ee el ge aA he On the Stimulating Action of Calcium Fluorid on Phaenogams. By Ge AGU Nr oot ts ot AEs ne Rie RE Ls tb 2.2 SR aes apgeee ME a On the Degree of Stimulating Action of Manganese and Iron Salts one bamlenem sown A, sKabavanila’ sts, Bsc vse othe ene in'S eiee mite tds aie Gaerne Hormation of umus.’ By iS: Suzuka . 2... ce. 5. ete ee A New Variety of Mycoderma Yeast as a Cause of a Sake Disease. Hive bee lealkcoihiaelal iy 0 < abt e hte). vsraar Gauge tang apatite ehege Note on Bacteria Pathogenic to Silk-Worm. By S. Sawamura On the Micro-Organisms of Natto. By S. Sawamura............ PAGE. 95 101 106 107 il PAGE On Wine from the Loquat Fruit. By T. Takahashi ............ 111 A Condensed Vegetable Milk. By T. Katayama .............-.--. 113 On the Preparation of Vegetable Cheese from the Protein of the Soy-bean. . By T. Watgamig 2... i eee os en 117 On the Composition of the Fibrous Part of the Japanese Orange. Bayi: Babar cer eee | Sateye aici oS 6 OG ey Ge econ ns 121 Fresh Water Algae as an Article of Human Food. By S. Nami- RROD 2 so. ws, 2c at reno ahs e is reed Sepa aoe Ste ree gee 123 Contributions to the Study of Silk-worms, II. On the Polygamous Hobits of thé Suk Waray Ko Toyama 2...) 65. ee ae 125 Contributions to the Study of Silk-worms, ITI. On the Parasitic Fly of the Domesticated Silk-worms of Siam. By K. Toyama. 247 Studies on the Hybridology of Insects, I. On the Silk-worm Crosses, with special Reference to Mendel’s Law of Heredity. By K. DP ePg EIAs tro eee ater Sa ed ysis st isos set's 5. tie betel a a ie oY 259 On Physiologically Balanced Solutions. By O. Loew and K. Aso. - 395 Jenzoesdure in Pinguicula vulgaris. By O. Loew and K. Aso.... 411 On the Action of Naphalene on Plants. By K. Aso ............ 413 Studies on Humus Formation, IJ. By S. Suzuki .............. 419 Konnen Phosphate Chiorose erzengen? By T. Takeuchi ........ 425 Does any Organic Silica Compound occur in Plants? By T. WaWeiGhl-- pee eS ales is he oS eee lel aloe een 429 Can Calcium Carbonate cause Loss of Ammonia by Evaporation from fees orl SB eee LE oo sea Sosa 'arn) cepa tah oe ree ee a eS 433 On the Relation of Plant Growth to Root Space. By S. Kumagiri. 437 On the Physiological Effects of an Excess of Magnesia on Barley. HSA oss a IR Een 3 Pst oteriolotsia sets =) «= & Bie wn aces ee salou 440 On Changes of Availability of Nitrogen in Soils, I. By O. Loew and | ST I Nr ees 9 ed ee LS 443 On the Stimulating Action of Manganous Chlorid on Rice, III. By MOP AGG 2 os Sealck, Gee ae wh wee ee aS Le 449 Observations on Stimulation of Plant Growth. By 8. Kakehi and K. SE chs 5 aa > x wpe giant cavehe ecto. Para ghee tomatoe he ae ea 454 On Different Forms of Phosphoric Acid in Press Cakes. By T. rae Sst 90's He SE eebpae Rees o's css larietese: aca Eaah ek apenas 457 On Bat Guano from Marianne Islands. By S. Kanamori ........ 461 On the Composition of the Shoots of Aralia cordata. By T. Wakeneits <2 2.4 .).). 4:3 Shree te she eS 2 oe es ee, 8 465 Note on the Composition of a Chrysanthemum Flower, serving as Mood sare he tsgmieinie toe irc es pier Note on Japanese Tabacco from Satsuma. By K. Baba.......--- Note on Bacillus methylicus. By Malevich ~ =) vosgear Ree Remy. « Ueber die chemische Zusammensetzung der japanischen Soya-Souce oder ‘“Shoyu.” By U. Suzuki, K. Aso und H. Mitaral ...-.. Ueber die Verbreitung von “ Anhydro-oxy-nethylen-diphosphorsauren Salzen”? oder “Phytin” in Pflanzen. By U. Suzuki und K. MiG aIMMIUUAY <2 tapers. -)-2-hen tee Lb a Pere aie eS Ueber ein Enzym “Phytase” das “ Anhydro-oxy-methylen-diphophor- siiure” spaltet. Von U. Suzuki, K. Yoshimura und M. Takaishi. Sindies on Humus Formation, III. By ee Suzys, sete Mois ae Studies on Diseases of Sake. By T. Wialeabashi-...:oa\e + ca cea tae On the Detection of Methyl-Lactate. By T. “Takalashits sees cree On Changes of Availability of Nitrogen in Soils, Il. By O. Loew iid ee ASO! se eo eee atari = = Hees. 8) aan he anes On Observation of Continuous Growth of Pea on the Same Soil. Bye So. Suzuki - jes Be Peete ttt astrel eats set eters On the Absorption of Varying -\mounts of Lime and Magesia by Pilates «Bye Va Watkememi years es: Monokaliumphosphat. | a a Um zu sehen, ab Kaliumnitrat und die Kaliumphosphate vielleicht bei hdherer Concentration solche Erscheinungen am Kerne hervorrufen kénnen, wie Kaliumoxalat und Natriumfluorid bei 0.5%, wurden Faden der gleichen Spirogyra in je fiinfprocentige Loesungen jener Salze gelegt. Es trat dann, wie vorauszusehen, Plasmolyse ein, welche nach mehreren Tagen zum Tode fithren musste.1 Nach 24 Stunden wurden folgende Erscheinungen beobachtet : Bei Monokaliumphosphat. In vielen Zellen normale 1 Nur bei Plasmolyse durch Zuckerloesungen bleiben die Zellen noch lange am Leben, wie aus den interessanten Studien von A7Zeds hervorgeht. > 10 Oscar Loew. Plasmolyse mit lebendem Cytoplasma und normalem Zellkern, in einer Zahl von Zellen ist der plasmolysirte Inhalt bereits abgestorben und der Zellkern liegt als rundliche Masse dem Cytoplasma an, in wieder anderen Zellen hat sich der tiberlebende Tonoplast aus der sich contrahirenden Cytoplasmahiille durch eine entstandene Oeffnung frei gemacht und liegt nun als eine straff gespannte Blase! neben der toten cytoplasmatischen Hiille mit dem Zellkern und Chlorophyllkérpern. In dieser Form hatte ich nie zuvor die anomale Plasmolyse beobachtet. In den finfprocentigen Loesungen von Dikaliumphosphat und von Kaliumnitrat waren unerwarteter Weise weit mehr der plasmolysirten Zellen abgestorben, als beim sauren Mono-Kaliumphosphat. Nirgends war eine seitliche Contraction des Zellkernes zu sehen, wie bei den 0.5 procentigen Loesungen von Kaliumoxalat und Natriumfluorid. Diese Erstarrung zu cinem Faden mit den Plasmodienstringen zz sz/w ist jedenfalls characteristisch fiir pl6tzlichen Tod unter besonderen Umstiinden, denn selbst bei Tétung mit 1 procentiger ,, Veberosmiumsaure”’ wird dieses nicht beobachtet. Es bleibt auch hier zwar Alles zz sz¢z aber der tote Kern hat sich nicht seitlich contra- hirt, er hat seine Spindelform noch beibehalten. In enger Beziehung zu der Giftwirkung kalkfallender Mittel steht die schon vor langer Zeit beobachtete Giftwirkung von Magnesiumsalzen auf Pflanzen. Nur eine geniigende Menge von Kalk kann, wie Atterberg, Ulbricht und der Schreiber dieses unabhingig von cinander beobachtet haben, diese Giftwirkung autheben? und die Magnesia kann dann ihre physiologische Function ausiiben. Fiir die niederen Algen und fiir die Pilze, welche ohne Kalk leben kénnen, fiir welche desshalb Oxalate kein Gift sind, ist auch ein Ueberschuss von Magnesiumsalzen nicht giftig. Dieser Parallelismus wurde zwar schon von mir hervorgehoben, aber ich musste nochmals darauf zuriickkommen, weil in einem kiirzlich erschienenem Buche? die ganze Frage wieder verdunkelt und von einem einscitigen Parteistandpunkt behandelt + In dieser Blase, welche sich hie und da in zwei geteilt hat, sind éfters k6rnige Ausscheidungen zu sehen, welche wahrscheinlich aus dem labilen Reserveeiweiss des Zellsaftes hervorgegangen sind. 2? Eine friihere Notiz von Boehm war unbeachtet geblieben. % Czapeck, Biologie der Pflanzen, II. Ueber die Verinderung des Zellkernes durch kalkfallende Mittel. II wurde. Es wurde in jenem Buche z. B. betont, ,,dass auch andere Salze und Salzgemische bei Mangel von Kalksalzen Erkrankungen bei Wasser- culturpflanzen erzeugen kénnen, die durch Kalksalzzusatz paralysirt werden kénnen.”” Beim rechten Lichte betrachtet ist dieses aber gar kein Einwand gegen meine Theorie tiber die Rolle des Kalks; denn bei Kalkmangel und Gegenwart verschiedener Kalisalze wird eben ein langsames Absterben in Folge mangelhafter Ernahrung, also quasi ein Tod durch Verhungern eintreten, welcher nur durch Calciumsalze aber nicht durch Magnesiumsalze aufgeschoben werden kann. In jenem Buche wurde ein ganz wesentlicher Unterschied totgeschwiegen, namlich der, dass die Wirkung von Magnesia- salzen bei Ausschluss von Kalk eine wahre Giftwirkung ist, die gar nicht zu verwechseln ist mit dem ebenerwahnten Tod durch Erudhrungsmangel. In 0.1 procentigen Loesungen von Magnesiumsalzen sterben z. B. Spirogyren in 4-5 Tagen ab, wahrend bei ebenso starken Loesungen anderer Nahrsalze, wie z. B. Kaliumnitrat der Dikaliumphosphat das Leben noch viele Wochen lang dauert.1 Selbst in 0.5% Loesungen des sauren Monokaliumphosphats mit 0.2% KNO, kénnen Spirogyren noch mehrere Wochen fortleben, bei Abwesenheit von Kalk. Aehnliche bedeutende Unterschiede existiren fiir Phanerogamen. In dem l6blichen Bestreben, nach weiteren Einwanden gegen meine Theorie der Kalkfunction zu suchen, erwahnt der Autor des obencitirten Buches noch Folgendes: ,,Es steht nicht ohne Analogie da, dass das Calcium-ion entgiftende Wirkungen besitzt. Loeb fand, dass die Eier des Teleostiers Fundulus in reiner, dem Seewasser isotonischer NaCl-Loesung rasch zu Grunde gehen, dass man aber durch cine Reihe mehrwertiger Kationen die schadliche Wirkung des Na-ion(!) aquilibriren kann. 1 Aequ. Ca-ionen entgiften 1000 aequ. Na-ionen.” Eine Erklarung, in was denn die q D>? » entgiftende Wirkung”’ des Kalks hier besteht, versucht der Autor gar nicht und doch halt er jene Beobachtung fiir einen ,,Einwand.”’ Bei jenen Seetieren handelt es sich jedenfalls darum, dass die Proteinstoffe des Cytoplasmas mit + Das zu diesen Versuchen dienende Wasser muss aus Glasgefiissen destillirt sein, d. h. darf keine Spur Kupfer enthalten ! 12 Oscar Loew. Kalksalzen locker verbunden sind! und dass, wenn diese in einer Chlor- natriumloesung verdriingt werden, schidliche Quellungsanderungen ent- stehen. Dieser fiir Seetiere ganz specielle Fall hat kein Analogon bei Siiss- wasser -und Landpflanzen, ebensowenig bei héheren Land - und den Siiss- wassertieren. Man kann Spirogyra wochenlang ohne Schaden in einer 0.2 procentigen Chlornatriumloesung aufbewahren. Eine concentrirtere bringt allerdings Schaden, aber dieser kann durch Kalksalze zzeht verhindert werden. Die Einwande in dem erwahnten Werke sind somit ganzlich un- begriindet. Erscheinungen beim Absterben von Sfzrogyra-Zellen. Die Chlorophyll- bander sind der Einfachheit halber weggelassen. a. Normaler Zellkern. b. Zellkern, get6dtet durch Kaliumoxalat oder Fluornatrium. c. Zellkern, getédtet durch verdiinnte Schwefelsdure oder Kochen. d. Zellkern, getédtet durch Schwefelkohlenstoff. e. Eigenartiger Fall von anomaler Plasmolyse, hervorgerufen durch eine 5% Loesung von Monokaliumphosphat nach 24 Stunden. 1 Es mag dieses nétig sein um der Magnesiamenge des Meerwassers gegeniiber eine gentigende Resistenz zu erziclen. Kobert (1903, Sitzgsber. Rostock) hat im Cephalopodenblut locker gebundenen Kalk nachgewiesen. Injurious Action of Acetates and Formates on Plants. K. Aso. Although free acetic and formic acid exert even in considerable dilution injury on lower and higher plants, it was not to be expected that the sodium and calcium salts of these acids would in moderate concentration also exert an injurious action on phaenogams. Various observations have convinced me, however, of this fact. The probable cause is that they undergo easily a hydrolytic dissociation in the living cells, whereby the base is absorbed by proteids and the acids are set free.!| The behavior of nitrate is evidently different and becomes dissociated in the cells only in the measure as the nitric acid can be reduced and its nitrogen assimilated. Moreover, the following observations show that the effect of various acetates and formates is very. different from that of oxalates and further that there exists quite a remarkable difference in the behavior to acetates and formates between the phaenogams and the algae. Experiment with an Alga. In the first experiment, a few threads of Spirogyra were put in 1% solutions of potassium oxalate, calcium acetate and calcium formate. The result was: 1 Calcium acetate as a source of lime may nevertheless be applied successfully in high dilution to plants growing in soil deficient in lime, as experiments by S. Suzuki in this college have shown, 14 kK. Aso. : Potassium oxalate Calcium acetate Calcium formate lime. 1% 19% 19% Eee > Pe oes arts tee, 3 : Aes” Chlorophyll bands con- | The whole appearance | The whole appearance | tracted, and its spiral] | of all threads was quite | was quite normal, also Atter 5 minutes. | form destroyed, Nucleus | normal. Nucleus was | nucleus normal, | in most cells contracted | also quite normal. to a thin thread. > = aay ~~ es oe r | ae. oH ape = 113 hour. Phenomena essentially Quite normal. Quite normal. z | the same as before. | In the next experiment, 0.5% and 0.1% solutions of potassium oxalate, calcium acetate and calcium formate were applied at a temperature of 10-16°C with the following results : Potassium oxalate. Calcium acetate. Calcium formate. Time. — ——-- ——— = — 0.5% 0.1% 0.5% o:1% 0.5% 0.1% In most } iter cells, chloro- | Normal. Normal. Normal. Normal. Normal. I hour. phyll- band | | contracted, Almost all | | chlorophyll - 2hours. | bands and =, a ae | “ | a nuclei con- | tracted. All cells | But few | Most cells died, spirals | threads still | alive, only a i aiens contracted, | alive. ; | few threads as oa de the color i se! 4 | dead. ” became pa- | ler. 6 weeks. All dead. All dead. | a = | x | p | The threads of Spirogyra in 0.19% and 0.5% solutions of the acetate and the formate contained all the time much starch and labil reserve protein as the reaction with coffein revealed. From these results, it will be clearly seen . that calcium acetate and formate are not poisonous for Spirogyra, while potassium oxalate is a decisive poison, which had been observed long ‘time ago by Loew. Experiments with Shoots. The results obtained with shoots of Sorghum, barley, onion and pea are seen from the following tables : 15 f Acetates and Formates on Plants, ion 0 Act jurious In " aa a ne en eee ——————— ee “UMO.IC 73 “ce “cc DUIVIOG ‘skup €4 SOAVD] [LV | | og ME ta, “ “ "pracy “ a ‘skp g ‘ 6 % ‘prac a ‘pracy “prod *patap sci. “prod "prac ‘sAvp “dn "1OD.1N} IOYJEM 0} ‘paroyyiM | porip sdy “Poot yl \\ O50] O} "103.10 }SO| PY2IUWOTUULOD | *sAup c *|VU ION *[VULION "| VULION, "| UULIONS SOAVI] ‘SaAvo] UO SOAVO] OIL J, POMUOLUWTOD ~— SOAVD] OUT, SOAVO] IOV poavadde SOAVO] OUT, jo sdiy, sjods UMOAg a7 077: OAC hire den f 5 rie ol 7010 me) % 10 26 S'0 or % 1'0 %S'0 ol | | OUT (jomuo) | aE oyeydyns ‘O}V}JOIV WINTpOS ‘OJLVJOOR wmni9[Vyy | “OLULIOZ. UINIOTVD) uM pos I OR ER A A AR I I RS EN A “Suoc] “ws of “WOAHOMOS JO SLOOHS ; OS) Ea ————————————————— OAye WS | are [IS ce “ “ « ‘“ ° IS z =“ ‘ . \ == *]RULION *|VULION *[RULION 0 “. 65°O Co (apAtpur) oe ; 0/Cp: aqnagtu oyeydyns 5 So'O Ty ) oa *[O.UOr eee OAH TNS OATES *MOTOK omy aurraaq sd oy, *[VULION *O}v}99NV MUA pag “prac “roped apy v ouwmeoaq SOATO'T ‘suoyT “uo Sy oF tc “OATTR “TNS | | | | | | ¢ *proac] “pracy | | | | ‘OP BV | *ystavoyod | poyed | O}7} *|VULIONT | | SOAR] JO posuryo | 1OJOD OY, | soAvory | %So'0 %1'O %S'o “OAT: TIS r « | | *proac] *proc] ‘royud | *YSIMo]pox oumndaq */RULION oureoaq SOAVIT JO SOALO'T AOJOD IU, 5 So'o , Hite) Nie) ‘O}VJOOV wUNIO]V VIAVIL AO SLOOTIS “OPVULIOT UUM TOP Ry ‘sAUp O7 *sAvp II ‘shup ¢ INV ‘OUT, LLL LLL LL LL TE CE SE A SS SS hh es ee ee ts f Acetates and Formates on Plants. ction 0 ijurious A hh Opp v podopaaoc] “IY U PomOULAY WPT “ 73 [ULUTON %S'0 (jo.4;U09) “OUI wunyva ‘OPE ® podopoaacy se ae *PEULION | ‘OL & podopaac] “cc *PEULLION “op v pod oJO Ao] « “pracy « “USEMOT[OA VYULODIC ce SOATO] ayy jo sdry, */VULIO\ *[ULUIO NS *SoyV}OOV. WNT pos “oy v podoyaaacy “cc *]VALION % |e) ‘OyVJOOV WNL “prod “prop JSOUUpY *105.1N} 4SO] SOAVO] AUWOG ‘suop ‘wo $ ‘NOINO AO SLOOHS OMT v podopoaacy “ “ “cc */ BULLION 2 So°o ee a podopaaocy 4 prvo¢ I 105.10} }SO] SOAVO'T %$0 “OPVULIO} CUNO Te) "sAup 0% *skup ¢ * OUT TY, Y “OAYL | | ve TS | ae es | | ad | . La proc] “Op wv ‘OMY V poIOULAY PAtOU PLAY *]RULION *UULION *[ BULLION “pro ] = | a | ee Pd | 4 %1 261 y = ‘aqyeydjns =| (‘apAyqur) DE I'O %S'O un pos | oyvayu tM D]V9 | | | UO: ) a a ee ee eT EY el a Res ee ale ‘OV IV Int poy (SNOGATALOD AO CAAA) *]VULION “ “e ee "prop | JSOULLY ‘ee e. “ee *pomoypEa *pro(] | *]VULION jvo] }SMO'T ’ 0/C- Jy. . ime) 7920 %oV'0 9650 ‘OPVJOOV WinloTVe) ‘suo, ‘wo z1 ‘OPRULLO} wuntoy ey) ‘VAd LO SLOOTIS ‘sAUp oO ‘sXvup 11 ‘skup § a0}; y ‘OWE, 19 Injurious Action of Acetates and Formates on Plants. From thrce results, it is quite clear that the injurious action on shoots of 0.5% solution of formates and acetates is not due merely to the cencentration itself, since sodium sulphate at 19 concentration exerted no such noxious influence. In the next experiments, the influence of oxalate, acetate, formate and nitrate of sodium upon shoots of barley and pea was compared. The results are shown in the following tables : SHOOTS OF BARLEY, 10 cm. long. Sodium Sodium oxalate, Sodium acetate, Sodium formate. nitrate. , (control) Time. = z = = - =a fe cov 9 Oo’ 07% -0/4 v7 ov | 0.5% 0.1% 0.5% 0.1% 0.5% 0.1% 0.5% | | | | fe lipsof | After leaves Normal, Normal. Normal, Normal. Normal. Normal. 2 days. | killed. | . | = ee | | | | Yellowing | Much Much 5 days. Dead. of leaves | 2 eo % injury. injury. ‘commenced. | | | | | | } One leaf | | Tip of Tips of dried up, | | a leaf a leaf 7 days. another | Dead. | Dead. » commenced | commenced | nearly | to yellow. to yellow. dried up. | | One leaf | j 4 “= | = still alive One leaf 4 weeks Dead. | Dead. became and a young shows its tip. | yellowish. 20 kK. Aso. SHOOTS OF PEA, 5 ¢.m. long, DEPRIVED OF THE COTYLEDONS. —————_—_—_— Se Sodium Sodium oxalate. Sodium acetate. Sodium tormate. nitrate. (control) Time. aes. oa 2 a 7 Sy ee -O/ av -0/7 0.5% 0.19% 0.5% 0.1% 0.5% 0.17% 0.570 Atter Leaves ; : i : Normal, Normal. Normal. Normal. Normal, Normal. 2 days. withered. Leaves Some Leaves 5 days. Dead. i withered e leaves withered. partly. withered. Lower leaves Almost 7 days. Dead. - 59 “s lost dead, turgor. Stull 2 weeks. Dead. = Dead, “3 nlive. These results show evidently that the injurious action of oxalates on phaenograms is much more pronounced than that of acetates and formates. Further the phenomenon was noticed that the plants killed by the 0.5% oxalate solution had still preserved the green color, while the plants which died in the acetate and the formate solutions had turned light brown. In this case the withering proceeding more slowly gave the oxidases time to act on a chromogen. Injuricus Action of Acetates and Formatcs on Plants. Experiments with branches. Young branches of Quercus acuta, Photinia glabra and Capsicum longum were placed in the solution mentioned with the following results : QUERCUS ACUTA. Calcium formate. Calcium acetate. Time. == = 1% 0.576 . 1% 0.5% After Normal, Normal. Normal. Normal. 2 days. Brown color|/Browu color [Brown spots appeared | appeared appeared 5 days. in the in the » on some | veins of veins of leaves. leaves. leaves. ‘Brown spots Almost Almost | | appeared S days dried dried | Pr on all up. up. leaves. | Dried Dried | Dried Dried 13 days up. up. up. up. Sodium Sodium acetate. sulphate. (control) 0” -07% / 1% 0.5! 0 1% Normal. Normal. | Normal. Brown spots appeared ) 33 on some leaves. ‘Brown spots appeared > 99 on all leaves. | Brown spots appeared | Dried on some | Still up. leaves, | alive. | but still . | alive. lo bo K. Aso. PHOTINIA GLABRA. i Sodium Calcium formate. Calcium acetate. Sodium acetate. sulphate. (control) Time. es = | Pd s -0 of 1% | 0.5% 1% 0.5% 1% 0.5/0 1% ——— + es! 2) 8 ee J : j After as ' ; z J a ; Normal, | Normal. | Normal. Normal. Nor Normal. Normal. 2 days, | Micribs_ | Youngest Brown spots of leaves | leaf appeared © days. “= - 3 2. : became | became on some dark brown,| dark. leaves. | | pe Brown spots Almost | Petioles i S | appearec 8 days. dried became 2. PP 3s m ; on all up dark. leaves. | Brown spots 5 | Still some Lower as ae appeared Be Dried | Still Dried Eee Sull 13 days. | leaves leaves : on some : c up. % ; : alive. up. alive. | alive. still alive. leaves, but still alive. CAPSICUM LONGUM. Calcium Calcium formate. Calcium acetate. Sodium acetate. nitrate. aay (control) Time. So » & 6 Bey = 0.5% 0.1% 0.5% 0.1% 0.5% 0.1% 0.5% Aft Commenced | After Ie B : : : : ae to lose | Normal. Normal. No-mal, Normal. Normal. Normal. 2 days. i - turgor. | j ' ' ' | Leaves Lost | eee - : : 5 days > Withered, Withered. withered 53 turgor. d ; a little, | | § days. Dead. | eA Dead. - Dead. i | Withered. lo Oo Injurious Action of Acetates and Formates on Plants. QUERCUS ACUTA. Sodium oxalate. Sodium acetate. Sodium formate. eee Time. be eee 0.5% | 0.8% | 0.59% 0.5% | Brown color ap- After 2 days. | peared on veins of Normal. Normal. Normal. certain leaves. All veins of the The color of The color of 5 days, | leaves became petioles turned petioles turned to brown. brown. brown. 8 days. ae a dried up. | Veins of leaves Veins of became brown, 2 weeks. Dried up. | leaves became and brown spots % brownish, appeared on leaves. An injurious effect of acetates and formates on young branches is therefore distinctly recognised ; the injurious action of the formates, however, was still more marked than that of the acetates. The action of both these salts, however, is much weaker than that of the oxalates. CONCLUSION. I. Acetates and formates of alkali metals and calcium act injuriously on phaenogams in solutions of 0.5% and over, while they are under the same conditions not injurious for higher algae, as Spzrvogyra. This forms a marked contrast to the action of neutral potassium oxalate which at the same concentration is not only a more powerful poison for phaenegams but exerts the same poisonous character also upon the higher algae, as Spirogyra, kK. Aso. 2. The poisonous action of acetates and formates very probably is caused by the hydrolytic dissociation of these salts into acid and base in the living cells, whereby the base is absorbed by proteids and the acid set free injures the living protoplasm. Konnen kleine Dosen Kupfer eine chronische Kupfervergiftung hervorrufen ? VON M. Toyonaga. Verschiedene Versuche haben gezeigt, dass kleine Dosen von Kupfer- verbindungen dem tierischen K6rper nicht schaden und Lehmann berichtet, dass selbst 20-30mg. Kupfer pro Tag nach Monaten thm nicht geschadet hat- ten. Doch spricht sich Tschirch! dahin aus, dass, um defintiv zu entscheiden, ob es eine chronische Kupfervergiftung giebt, Versuche Jahre lang fortgesetzt werden miissten und dass ,, manche Widerspriiche noch der Lésung harren.” Fir die Ungiftigkeit des Kupfers scheint auch zu sprechen, dass manche Tiere, sowohl Mollusken als Arthropoden, besonders aus dem Meere ein kupferhaltiges Haemoglobin, das sogenannte Haemocyanin, im Blut ent- halten. Dhéré fand in tooc.c. Blut von Octopus vulgaris 18.0-23.5 mg. Kupfer und in dem von Astacus fluviat. 4,0-8,0 mg. Kupfer. Kobert fand ferner, dass dieses Haemocyanin ohne Giftwirkung ftir Kaninchen ist. Ich beabsichtigte, Kaninchen Jahre lang mit kleinen Dosen Kupfer zu behandeln und falls sie dabei am Leben blieben, das Blut auf kupferhaltiges Haemoglobin zu untersuchen und begann meine Versuche mit vier Tieren, von denen zwei als Controll-Tiere und zwei als Kupfer-Tiere dienten. Jedes Tier erhielt vom 15 April ab pro Tag 50 g. Gerste, befeuchtet mit 10 g. Wasser. Bei den Kupfer-Tieren enthielt dieses Wasser anfangs 5 mg. Kupfer, welches in der Form von kohlensaurem Kupfer (Kupferchlorid mit kohlensaurem Natron in equivalenter Menge versetzt) dargereicht wurde. Jeden Tag um 12 Uhr wurde die iibrig gelassene Nahrung gewogen. Jeden zweiten Tag wurde das K6érpergewicht bestimmt und die Faeces von Zeit zu Zeit auf Spuren von 1 Das Kupfer. Stuttgart 1893, S. 114. 26 M. Toyonaga. Kupfer gepriift. In den ersten Monaten wurde indessen selbst mit der so empfindlichen Ferrocyan-Reaktion kein Kupfer in der Asche der Faeces eefunden; erst als spater (von 15 September ab) das cine Kupfer-Tier alltiglich 20 mg. Kupfer erhielt, wurde bald darauf Kupfer im Kote gefunden. Das zweite Kupfer-Tier erhielt von September ab 10 mg. Kupfer pro Tag. Der Tod saimmtlicher Tiere erfolgte leider zu frih, wahrscheinlich im Folge von Erkaltung. Die Autopsie ergab bei den Kupfer-Tieren ebenso wenig etwas Abnormales als bei den Controll-Tieren. Eine Priifung der Leber ergab einen geringen Kupfergehalt, dagegen war kein Kupfer im Gehirn vorhanden. In folgender Tabelle sind die taglichen Wagungen fiir das Monat berechnet zusammenfasst : a —————————————————————————————————————————————————————— (A) (B) (C) (D) Controll-Tier. | Controll-Tier, Kupfer-Tier. | Kupfer-Tier. | | = | : | . var Monat. KGrper- KGrper- | | Korper- | Korper- Nahr- gewicht Nah- gewicht Nah- | gewicht | Nah- gewicht _ungsauf- | am Ende | rungsauf- | am Ende | rungsauf- | am Ende | rungsauf- | am Ende | nahme jedes nahme. jedes nahme. jedes nahme. jedes Monats. Monats. | Monats. Monats, April | 506 g. 1508 ¢. 655g. 1498 ¢ 502 ¢ 1398¢. | 689 g. 1658¢ (15-30) _| ae Sal | | ame | Mai. 1421 1557 1330 1587 TAGOME | LSS EOS 1827 Juni 1680 1677. | 1706 1727 1806 1697 | 1761 1887 ve Ws roll o> Lele by, bred £ | | | Juli. 1596 1697 1639 AT TOA IOSO fl 8757 eos 2007 Aug 1229 pNvtyp || sell e) 1617 1492 1617, 1665 1917 icky a ae ey, ee: a Sept 565 1557-4 604. III7 1237 ae Tey 643 1427 Oct 579 1160 Am 14. verendet. 875 1320 99 1040 — aaa | — ae = ail Novy $72 1200 1121 | 1180 | Am 7, vérendet. Dec. | Verendet am Io, | Am 3. verendet. | Das Tier C hatte im ganzen aufgenommen 2035 mg. Cu, und das Tier I ) 39 3”? ” 1040 mg. Cu. Konnen kleine Dosen Kupfer eine chronische Kupfervergiftung heryorrufen 2? 27 Eine chronische Kupfervergiftung ist somit durch das Kupfercarbonat nicht herbeigefiihrt worden ; denn selbst bis kurz von dem Tode zeigten die Tiere keine Vergiftungssymptome. —_6 + o—____—___ Ein Mangan-Versuch. Zum Schluss sei auch noch ein Versuch kurz erwahnt, in welchem Kaninchen fast elf Monate lang Manganchlorid erhielten. Ich hatte die Vermutung, dass dadurch der Effect der oxidierenden Enzyme im Tier vermehrt wurde und wollte ferner priifen, ob das Haemoglobin dieser Tiere manganhaltig wiirde'!; denn Kobert erwahnt, dass das Pinnaglobin der Steckmuschel Mangan statt des Eisens enthalt. Doch vor allem wollte ich zuerst versuchen, ob die Kaninchen durch die Manganbehandlung vicelleicht etwas widerstandsfaihiger gegen Infectionskrankheiten werden kénnten, aber ein Versuch mit Milzbrandinfection entschied nicht in diesem Sinne. Ver- suche in Bezichung auf einen Mn-Gehalt der roten Blutkérperchen hoffe ich spiter wieder aufzunehmen. Ein Mangantier erhielt in clf Monaten im ganzen 27 g¢ Manganchlorid ohne irgend eine abnorme Erscheinung zu zeigen. Die folgende Tabelle giebt die Fiitterungsdaten,? sie diirfte wohl zeigen, dass eine chronische Manganvergiftung per os nicht enistiert. 3 Es wird Mangan nur in sehr geringen Mengen aus dem Darm resorbiert, aber von Riche wurden doch bis 2.5 milligramm Mn,04 in 100 g. normalem Rindsblut gefunden. Nach Debierre (1885) wird durch Mangan die Zahl der roten Blutkérperchen vermehrt. Mangan-Eisen-Pepton wird in neuester Zeit therapeutisch verwendet. 2 Die tiglich abgewogenen Futtermengen waren hier nicht immer gleich Oz1z GE ober oot FG NS areH SoSz gz C6cSr | ‘jOpudto, "cs wy yof pl aa ae oe alt Deas ~. £ as > ae (are = a ae | | l $Soz gia |) Sort ogtz gz | g6rSr Chiari cke \ eSor, \e obye oc | 9991 “un | | | | | == ae <7 oe Site ees Races . i © | z | ie —; vin a. Ree. 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During a series of experiments with barley in pot culture the writer had repeatedly observed that in certain pots the stalks of the barley plants showed a red coloration which according to the tests made with alkali and acid was due to the anthokyan. In other pots manured differently, however, the stalks were all of a normal green color. In the former case it was further observed that the lower leaves in dying off gradually did not show a normal yellow straw color, but a more reddish color here and there with violet spots. These phenomena were not restricted to one variety of barley but observed with four different varieties, some of the two lined and others of the six lined character, viz. Goldenmelon and the Japanese varieties Minoguro, Hozoroi and Kobizen. While not denying the possibility that there exist varieties which under all conditions produce normally some anthokyan in the stalks, it was in the cases I observed certainly an effect of the composition of the manure and therefore a special series of experiments was instituted in order to find the conditions which cause the formation of anthokyan. My observations had led me to the inference that it must be a deficiency of some manuring compound which brought on the formation of anthokyan, since these plants yielded always a smaller harvest than these plants which has a nice green stalk. I experimented with two kinds of soil, the one a sandy soil! of great natural fertility, the other a loamy c soil? of poor natural fertility. My determinations gave the following numbers :— 1 This soil came from Kawasaki, a place about 12 miles south of Tokyo. 2 This soil came from Komaba, a suburb of Tokyo. 30 S. Suzuki. The sandy soil contained 97.89% of fine earth ( | . 2600 Unmanured. 2.602 0.100 f _ | (orig. soil) XVII, Loamy soil} 1750 | 3 | 1750 58 0.768 0.044 | (orig. soil) 10 seeds were sown per pot on Jan. 13 (1905), and the young shoots reduced to 3 of nearly equal size on Febr. 3. On Febr. 9 (27 days after sowing) some difference was perceptible which gradually became still more marked. The stalks of those plants grown in pots with a small amount of phosphoric acid showed a red color and the less the content of phosphoric acid in a pot the deeper was also the coloration. Indeed such plants showed a sickly appearance—a phenomenon of phosphoric acid hunger—they re- * 2600 g. of the sandy soil, 1750 g. of the loamy soil and 2500 g. of the sand had almost the same volume. Li) S. Suznki. oP) mained short, the formation of new shoots was retarded, and the tips of the older leaves died off gradually with a reddish yellow color. The details of observations in different periods and the final harvest are shown in the follow- ing table :— Percent ~ AV a 3 hee No. of | Kind of ee Coloration of stalk. talk | length a phe sae ae, P.O, a | Bere Bebe ea sheet = the soil.| Febr. 9. Febr. 21. | March to. : c.m. = | I. Sandy soil | 0.010 red red red 3 9.9 20 | 0 i: a 0.017 slightly red a P 7 12.4 3-0 | Oo IIT 0.026 ; 5 12.3 6.0 | 0.6 I\ 0.051 3 9-5 75 | 2.0 Ve pa 0.100 re slightly red * 9 | 15.1 PAB S| | 6.4 VI a 0.102 green a; = 9 | 18.9 22.2 | 6.6 VI. - 0.108 o green ae I4 21.6 23-1 6.6 VII. Ae 0.183 4 = = green TO” *} 22014 24.8 | 73 IX. Loamy soil 0.005 red red red 23 9.1 1.0 | fe) X 0.008 | 9.1 1.6 | oO XT 0.018 ; | 8.9 230 toe SI. 0.044 = , 9.9 0.8 | o NI ; 0.045 : “= f 10.0 AAG) | 0.4. XT\ 0.046 , 9.8 Fed | x5 x. 5 0.056 a = 6 12.9 11.5 | 2.2 XVI. A 0.167 green deep green slightly red 12 21.0 19.0 pi XVII. Sandy soil 0.100 red red red 6 11.9 6.6 | 20 XVIII. Loamy soil 0.044 = = ie 3 9.7 0.2 | fo} Ot The observations do not quite agree with similar observations of Clausen on the stalk of oats. He observed on a very peculiar soil! that with the increase of phosphoric acid the number of stalks was increased but not the weight of grains and further that in these cases of the depression of the yield 1 A black peaty sandy-soil. On the Formation of Anthokyan in the Stalk of Barley. On oe) in grains also the stalks showed a bluish red color.! In order to observe whether also the deficiency in nitrogen or potassa can cause the formation of anthokyan in the stalk some further experiments were made, the plan of which is seen from the following table : ee Quantities of, General No, of Kind of Relative amount of available manure, ots. soil, Soi S; N ir e sol pots Soil, Sand. per pot. in the soil. g. g, pXIX. Sandy soil. 2600 fe) Sandy soil, 1 i 7 ‘ j cC 7) XX. 650 1875 is O25 5 a = oe 2 a |XX. 433 2083 ot es c.16 AS a2 Eh ae ne Og (XXII. Loamy soil. 1750 fe) 2-5 Loamy soil, I boa} 2 w . = 2 8 = 2 9 XXII. 438 1875 an jn (O25 ae. Pic ‘ Lexty, 292 2083 POLLO i = = ot General : - : No. of Kind of Relative amount of available 7a manure, pots. , soil. Soil. Sand ak potassa in the soil. per pot. oO ao S: S (EX. Sandy soil, 2600_—s#gy fe) Sandy soil, 1 XXXVI. 650 1875 = a) 0.25 Z ee 3 yg |XXVIL ; 433 2083 Bf as 0.16 as 3) ee 38 2 eS Bo eB 8 [XXVIIL. | Loamy soil. 1750 fo) 226 }oamy soil, 1 = =) 8 is = 5s Betas me = a aA eo ee 3 . ; ake available Fe. 9- nS | Sean pots. soil, Nin stalks, 58 | Slo | ws = a Febr. Febr. 21. |March to.| Poe | OO meee | the soil. | # 46 |RE | & 1 = | a | | - - . . t | | XIX. Sandy soil I | green | red |_ red 9 20/0) | Neses 0.7 £ Ixx al we 2)! Resss ” 0.25 A=) ” | ” 5 14.3 3-0 1.0 2 | E | | = |XXI. pce ce) a ambiguous - a 7 I4;0 | 22 dees Ki ieee sit © o | Ss S| aR 455 a ee eu = slightly | distinctly | | an 5 XXII. [Loamy soil! I S coal aes Al _ 6 | 15.0} 1.1 0.3 = AS) | cS) = Ss Ixy | S io) ; = XXIII. = | 0.25 re red | ss | . 8 13:5) 26 0.6 6 | a | ree | XXIV. _ 0.16 ; * By 5 11.4] 09 0.2 | 4 ea | Relative Cilaiat Be 2° 8 Ul bs o.9 se oloration of stalks alld No. of | Kind of amount N fy) a i = a: z NO, of} = A Ce (>) : ios eee 1 On a We sole available|"b- 9. a ea oO Cir || Ses pots. soil, K.0i stalks.| £73 |-s& | oe 2O in “= | oO tn | ee OO es | ese Febr. 16. | Febr. 21. March 1o. Zu | © Oa | |\ y Lotassiumer nitrate. 1.61,, ,, Magnesium sulphate (anhydr.). Few drops of ferric chlorid. IV. With phosphoric acid. Solution HI+1 per mille Monopotassium phosphate. O:02.. », Terric phosphate. V. Without potassa. 3 per mille Calcium nitrate (anhydr.). Ol. 3, Magnesmim sulphate ( 4, ””): 1 If in an unheated glass house during cold winter months young barley plants show some anthokyan even on well manured soil, this is easily explained by the low soil temperature while the air temperature in the house can reach on sunny days 16-12°C. Ina case I observed the pots holding 8 kilo soil showed in the afternoon in the center 15 c.m. below the surface only 6° C, which naturally depresses the function of the roots, becoming incapable to provide the shoots with a sufficient amount of mineral food, The root is too cold for the shoot exposed to the warm air, 36 S. Suzuki. 2 per mille Monocalcium phosphate (hydr.). Few drops of ferric chlorid. VI. With potassa. Solution V+1 per mille Potassium nitrate. Cts, ,, Monopotassium phosphate. VII. Knop’s solution. 3 per mille Calcium nitrate (anhydr.). I »» », Potassium nitrate. 1.61 ,, ,, Magnesium sulphate (anhydr.). I »» y, Monopotassium phosphate. 0.025, gaalierkic-phosphate. Young barley shoots were placed in these solutions! on Jan. 14 (1905) and kept in a glass house. From time to time a current of air was passed through the flasks. Seventeen days afterwards a very striking difference was noticed. The plants which could develop normally in the full nourishing solution, had also a deep green stalk, The plants in the incomplete culture solutions could of course not develop beyond a very limited size, but anthokyan was shown only in the case of deficiency of nitrogen? and of phosphoric acid, but not in the case of the deficiency of potassa. In this latter case, however, the first leaves dying off gradually developed some brown spots, a phenomenon which also was regarded by Wilfarth as characteristic for the deficiency of potassa.* Finally not only these soil experiments were repeated with several varieties of bariey with essentially the same results, but also a similar experiment was made with lettuce, which growing on poorly manured soil showed an abundant formation of anthokyan in the leaves and after transplanting in richly manured soil they developed soon a deep green color. My observations with barley will, I think, permit the conclusion : 1 During the first three weeks, I used dilute solutions, adding the same volume of distilled water, * The shoots developed in absence of nitrogen were characterised by their unusually long main roots. % Journal fiir Landwirtsch; 1903, p. 129. io) NI On the Formation of Anthokyan in the Stalk of Barley. The formation of anthokyan in the stalks of barley can be regarded as a sign of deficiency of available phosphoric acid or nitrogen or of both in the soil. Lo. : 7 - ¢ . 7 an - : 2 oa iT 2 ~ ven a W eeuveriln A tu oaltacyse'—® web atE a 7 ‘ 7 4 — - - . + og ge ae fea liini in, ito le nea ae - A - , P| %, s « i 7 o an ewe ru Pret fe, ~ ans < a 7k ae on ars om tyes? ? ie Pe Gre es os rane ° i ‘ ‘ ae 2 iy el. ee a ; — . iq - ' o Ose $ ae iy we : ical a. é ae : ane : : Se ; =I a ’ 7 ae . | " * = ae = « : : : a - tee ae pe x - a , =F a 7 - ist = = Ls ' - 1 ] ‘ . A De r nr < ‘ ® Ge a | y uae - ~ ; a" 7 ; the e ya » — 2 7 ‘ 7 2%; ea At . - eae 7 -Y « ee a - Fee eae : y oa 0 Aes * = mn On the Influence of the Reaction of the Manure upon the Yield. BY K. Aso and Rana Bahadur (Nepal). Various reasons have been adduced for the fact that on some soils sodium nitrate is superior to ammonium sulphate, while on other soils both forms of nitrogen are equally satisfactory. On some light soils again ammonium sulphate was found superior to sodium nitrate. A loss of ammonia may be caused by certain kinds of soil bacteria which more readily assimilate ammonia than nitrate, rendering thus the ammoniacal nitrogen partly insoluble in the form of bacterial protoplasm. Some loss of ammonia is further ascribed to the transformation of the sulphate into carbonate and volatilization of the latter, a process that may take place on soils rich in calcium carbonate (Wagner). Nitrification with following leaching may also cause losses; finally nitrate-nitrogen may be lost by denitrification. Some soils and manures favor one process more than another, hence the differences in the yield according to the forms of nitrogen can be partly accounted for. But there exist still other reasons. Loew has called attention to the fact that for the assimilation of nitrates! in the leaves a certain concentration of sugar is required, i.e., for the reduction to ammonia needed for the protein- formation. This condition is not so readily fulfilled in cool seasons and with clouded skies, than in warm sunny weather. In cool rainy seasons ammonia therefore will be better utilized than nitrates, ceteris paribus. 1 The absorption from the soil by the roots is sometimes called erroneously assimilation. 2 The absence of sufficient sugar in etiolated leaves kept in darkness accounts for the observation ox Laurent, that such leaves assimilate much more readily ammonium salt than nitrate. 40 K. Aso and Rana Bahadur. Another and powerful factor for the efficacy of one or the other form of nitrogen is the reaction of the manure. A mixture of superphosphate with the physiologically acid ammonium sulphate is generally unfavourable unless some carbonate of lime is applied to counteract the evil effect of the acid reaction on the roots (Wagner). In order to avoid the formation of too acid a reaction by the application of ammonium sulphate, it is further recommend- ed to use a mixture of it with the physiologically alcaline sodium nitrate (Kossowitsch). Such considerations have led us to compare the effects of the (neutral) disodium phosphate with the (acid) monosodium phosphate and ‘with calcium superphosphate in presence of ammonium sulphate or of sodium nitrate in sandculture and in soilculture ; the former was carried out by K. Aso, the latter by Rana Bahadur. Sandculture with pea and barley. Each pot containing 2.5 kilo well purified sand was manured as fol- lows : PGEASSUIN SURGE ANIC Ea as atetetl ans dnnnn di obs sabeeinn do 0.73 Sodium chlorid .....:::... peeeeebinny sae bs. care: eas

i ey ‘ = . = = Sat ) =v = P: 7 ie = o — a 7 nd . ' = : 7a = ies 3 _ | Gee x ; : ar". . ; , bay . Y - x 7 ari hie = } = ; eh 7 7 ry > Z n ete ee a a lyr rae adit amiparieres are t= = pete ee a . he oe ( mr Ly : eet: ae r 7 On the Manurial Value of Calcium Cyanamide. BY K. Aso. Since the manufacture .of calcium cyanamide had been established in Berlin, various authors have reported on the results of manurial experiments with this compound. M. Gerlach and P. Wagner! were the first who concluded that it has a manurial value similar to that of chilisalpeter for oats, barley, mustard and carrot, and that it did not injure plant growth in the quantity applied. Later on, Wagner? compared the action of chili- salpeter, ammonium sulphate and calcium cyanamide upon turnips and observed that calcium cyanamide yielded the best when applied before sowing but it exerted a very unfavorable action when applied in form of topdressing. A. Frank* also concluded that the manurial value of nitrogen in the form of calcium cyanamide is almost equivalent to those of ammonium salts and nitrates. But Br. Tacke* made a series of pot experiments with a peaty soil and noted an injurious action of calcium cyanamide on mustard on applying it 8 days before sowing, and that the injury was not observed on applying it 2} months before sowing. The manurial value on that kind of soil was behind that of chilisalpeter. Steglich® observed a retarding action of calcium cyanamide on mustard when applied 7 days before sowing, but not when applied 17 days before sowing; the result was even better than with chilisalpeter and ammonium sulphate. 1 Landw. Presse. 1903. 30, 367. nw Ebend. 1904. 30, 493. 3 Ill. Landw. Zeit. 1903. 23, 491. 4 Mitt. d. Ver. z. Forder. d. Moorkultur.?. D, R. 1903. 21. > Tatigkeitsber. d. Versuchsstation Dresden, 1993. 48 k. Aso. R. Otto! found that the value of calcium cyanamide is equivalent to those of nitrates and ammonium salts for spinach, lettuce, white cabbage and maize, and concluded that this manure seems to be suitable for garden plants, provided that it is applied a week or two before planting or else dug into a depth of 13-26 cm. H. von Feilitzen? observed that this new fertilizer gave poorer results than nitrate of soda and was also not equal to ammonium sulphate on certain soils with barley, oats, wheat and potatoes. Zielstorff$ showed by pot experiments, that when calcium cyanamide was applied at sowing time, its value was 88.4% of that of chilisalpeter and when the seed was sown 10 days after the application of the manure, the value of the latter was equal to 92.8%. A.D. Hall* concluded that calcium cyanamide is an effective nitrogenous manure, though more extended experiments are necessary to decide whether the unit of nitrogen is worth more or less than in sulphate of ammonia. Most recently Haselhoff*? published a series of the experimental results made in Marburg and arrived at the conclusion that calcium cyanamide has a retarding action on the germination of seeds, and as soon as the cyano-compounds are decomposed, all nitrogen contained in this manure assumes the form of ammonia and its value is almost equal with nitrogen in chilisalpeter. He noted also that the time required for the complete decomposition of this cyanamide is different according to the qualities of soils. It was for us of special interest to carry on similar manurial experiments with crops especially cultivated in Japan. One series of experiments was made in large zinc cylinders open on both ends, which were sunk into the ground and filled with a soil unmanured for six years. The area of each cylinder corresponded to 3545, hectar. For manuring the following ratios were applied: K,O=80 kg. per ha. as potassium sulphate, P,O,=100kg. per ha. as double superphosphate ; further N in the form of calcium cyanamide in cylinder I at the ratio of 100 kg. N 1 Gartenflora, 53, 19003. No. 20, 524-538. > 53; 3 524-53 te Abstr. in Exp. stat. Record. Washington. Vol. XVII. No.1. 17. 3 Bied. Centr.-Blt. f. Agrik.-chem. 1905. 34. 217-218. 4 Journ. of Agric. Science. Vol. J. PartI. 1905. 146-148. Landw. Jahrbiicher XXXIV. 1905. 597-616. o On the Manurial Value of Calcium Cyanamide. 49 per ha., while cylinder II received the equivalent quantity of N in the form of ammonium sulphate and cylinder III in the form of chilisalpeter. The calcium cyanamide was the commercial crude product, a fine black powder, containing 19.29% N. On April 19, 17.2 grams of calcium cyanamide were mixed with the soils to about 5 c.m. depth, while the other manures were applied April 28. Ten days after the application of calcium cyanamide, some buckwheat seeds were sown in each cylinder, which germinated well but were injured afterwards by insects. Hence, on June 109, 18 seeds of upland rice were sown and later on the young plants reduced to nine per cylinder, all of about equal height. On Oct. 13 the plants were harvested with the following result : | Number Weight of lw Weight of Total Cylinder. Form of N. of grains | straw yield shoots. air dry. | air dry. air dry. 3 | | | I, Calcium cyanamide ............ 48 91.5 g. 120.5 g. 212.0 g. Il. Ammonium sulphate ............ 39 68.5 92.0 160.5 III. Sodium nitrate: $22.4 cac.-s-ccene. 42 | 70.5 80.2 150.7 An analogous experiment was made with Sesamum. The seeds were sown May 25, two weeks after the application of calcium cyanamide and the plants in each cylinder were reduced to nine. On Sept. 15, the plants were harvested, but unfortunately all the leaves had already been dropped at the ripening stage. The result was as follows : Weight of | Weight of seeds stems without] Total yield. air dry. leaves. Average Form of N, length of Cylinder. each plant. dip Calcium cyanamide ............ : : 13.0 g. 54.0 g. 67.0 g. ar, Ammonium sulphate ............ : 14.8 59-5 74.3 I, Sodrammmittate: 23-0: ../0..20ente 42.1 4.5 43.2 47.2 In a third experiment, hemp seed was sown in such cylinders on June 26, six weeks after the application of calcium cyanamide. Later on, the 50 K.' Aso. young plants were reduced to six of equal size in each cylinder. On Oct. 13, the plants were harvested with the following result : | Weight of | Weight of Cylinder. | Form of N, leaves stalks Total yield. air dry. | air dry. — — —_—— —— es | ie Calcium cyanamide <.....:..... 47.5 g. | 36.5 g. 84.0 g. WE Ammonium sulphate ............ 29.9 34.1 64.0 III. Sodiam nitrate.2 tee 31.7 | 39.2 72.9 It is therefore quite evident that calcium cyanamide was generally superior to ammonium sulphate and to chilisalpeter, in the three experi- ments. Since the manurial value of calcium cyanamide is different according to the kind of soils, as several authors have shown, a series of pot experiments with two different soils was made, one of which was an alluvial sandy soil from Kawasaki near the river Rokugo, containing only 1% of humus, while the other was a diluvial loamy soil from Komaba, containing about 10% of humus. The experiments were carried out in pots, each containing about 8 kg. air dry soil and of an area of 55,55, hectar. The quantities of manures applied May 25 were as follows : Common superphosphate ....... -. 15 grm.) G : eneral manures. Kainit’..93.. 23 eee rtare nwa toa. 10 erm.| Calcium cyanamide... =s+-z:.. 2:6 “ern! Ammonium sulphate ........ .... 2.4 grm.}Special manures. Sodium) sitrate teen. a a 7 7 ome On the Lime Factor for Flax and Spinach. BY S. Namikawa. The lime factor, i.e. the best ratio of lime to magnesia differs with dif- erent plants, and even with ‘different organs of plants. In regard to the leaves this ratio is larger than for all the other organs. With the flax it is principally the stem, while with other plants the fruit, with others again the leaves (cabbage and tobacco) which are the most valuable parts of the harvest. Hence also the question of the lime factor depends to some extent on the special organs to be chiefly developed. In regard to flax the question was which ratio of CaO: MgO is the most favorable for the production of the strongest fibre ? For my experiments served the same soil from our College farm that served for other investigations mentioned in this Bulletin. This soil had been examined recently again by T. Katayama, whose determinations of the available amount of lime and magnesia yielded the following data : In the fine earth: Lime. | Magnesia. Fine Earth of the = seas soil = 76.33 % 0.60% 0.49% Hence, the original soil contained Metinte apace. 0.458% Magnesia.....ae 0.374% The Wagner pots used held 10 kg. soil. This amount contained therefore 45.8 gr. CaO and 37.4 gr. MgO. 58 On the Lime Factor for Flax and Spinach. By addition of finely powdered calcium carbonate and magnesite were procured the following ratios :! CaOpne ome Urn Oe I MgO = ie ; 8.4 gr. MgO= 17-5 Si. MgCoO, CaO" 28 x anaes II MeO 1 ) 29.0 gr. C20 — 51.8 gr. Cac@s Ca@ jes Sian nf aid ? = : Ill MeOlr ae Gon er, CaQ=118.5 er. CaCO, Each pot reeeived further the following general manure : 5 gr. potassium sulphate 10 gr. double superphosphate 10 gr. ammonium sulphate 5 gr. sodium nitrate. 20 seeds per pot were sown on Nov. 1. The number of young plants when 10-12 cm. high was reduced to 10 per pot, all of nearly equal height. The plants showed gradually a considerable difference in development, which will be noticed also from the photograph taken April 12 and repro- duced on Plate II. On May 20, the plants were cut and left to become air dry. My observations were as follows : anti. March 27. June 3. CaO MgO Height | Number |} Height | Number | Height | Number |Weight (g)|Weight (g) cm, of cm. of cm. of of of average. | branches.| average. | branches.| average. | branches.| fruits. | total crop, = 12 14 51 20 92 20 19 47 = fe) fe) 35 20 83 20 13 Yi a $8 o 34 20 80 20 2 37 1 Former experiments with flax by I. Kawakita had shown here that an increase of magnesia over lime produced a depression of yield. S. Namikawa. 59 CaO I It will be seen that the ratio ise was the most favorable. The increase of lime beyond that ratio depressed the total production from 47 gr. to 37 gr. =21%. Since phosphoric acid was applied here as double superphosphate, this depression cannot be due to a decrease of the avail- ability of phosphoric acid in the soil, as is the case when bone dust serves -as phosphatic manure. After removal of the brittle parts the fibres were subjected to a rough comparison as to strength and it was thus easily recognized that the fibers CaO 3 grown with the ratio MgO = were much weaker than those grown with the ratio =. The general result agrees well with the statement of Blomeyer! that soils rich in lime are not favorable for flax. In order to determine the best ratio of lime to magnesia for the growth of spinach, a sand culture served in which the lime and magnesia were applied in the form of very fine powder of the natural carbonates in such proportions that the ratios | were obtained. Cio: | The total quantity of CaO in pots No. I and II was two grams for 23 kilo. purified sand. Hence the proportions of powdered marble and powdered magnesite became as follows : I CaCO, _ _ 3-57 g- LaviscO- 8.36 g. ll CaCO, _ 3-57 g- " MgCO, 4.18 g. CaCO, 7-148. MeO, PA13'¢. Ca€OF 2 5G77 g. MeCO 7 13s. EV. 1 Die Cultur der landwirtschaftlichen Nutzpflanzen, II, p. 333. 60 On the Lime Factor for Flax and Spinach. Each pot received the following general manure : 0.6, g-1K SOR 1.0 g. Double superphosphate. 3.5 g. NH NG: 2.0 g. ferric hydrate. On April 24, ten seeds were sown in each pot and the young plants reduced to four of equal size after one month. The plants were cut June 24, and weighed in the fresh state. The results were as follows : a RS Limefactor, Height, cm. Number of stalks. Total weight (g). II | 16 Go. II 16 8.9 12 7 II 17 CaO ~ 13 16 13.2 MgO “1 II 10 II 8 10 14 CaO 2 a = 7.9 MgO ~1 8 “a 7 8 10 10 C20 3 e 7 5.1 MgO ~1 7 5 7 5 | It will be seen that the true limefactor for spinach=1; this ratio produced the best results, like with flax. ———+ + Regeneration of Overlimed Soil. BW; S. Maki and S. Tanaka. It has been repeatedly pointed out in these Bulletins, that a certain ratio of lime to magnesia entering into the plant body is among other things necessary to insure the best growth, and the experiments of Aso, Daikuhara, Furuta, Suzuki, Namikawa and Katayama here have furnished ample confirmation of the former experiments of Loew and May. Recently Nakamura! operated with a soil that contained seventeen times more lime than magnesia (1,76% CaO and 0,119% MgO). The absolute amount of magnesia would have been sufficient for a series of crops, but the ratio to lime was unfavorable. By manuring with magnesia the yield of barley in pot culture was increased by 69%. Since it occurs sometimes that poor sandy soils are injured by a heavy dose of ‘lime it was of value to.observe the restitution of overlimed soil to its former state by manuring with magnesia compounds. For the culture of cereals lime and magnesia should be present in equal quantities, provided that both these bases are present in such forms that their availability for the plant roots is equal. To this end the best form of magnesia for manuring an overlimed soil would be magnesite, since it is in regard to solvents similar to the lime compounds present in most soils. But this material is often difficult to procure and not easily pulverized. The cheapest soluble magnesium salt is the sulphate, but much less of the magnesia in this form is required than in the form of magnesite since this salt represents a much more available form of magnesia. It was therefore 1 Bulletin of the Imp, Central Experiment Station at Nishigahara, Tokyo, No, 1, 62 S. Maki and S. Tanaka. of importance to determine how much of this salt would be required for counteraction of the overliming. For the test served a loamy soil with 0.6% CaO and 0.59% MgO. It was mixed with twice as much lime as was present Originally, in order to overlime it in relation to barley, and would require now an addition of 1.39% MgO in the form of magnesite to procure the ratio 1:1 which had been also nearly the ratio in the original soil. But in the form of magnesium sulphate much less was required. Hence the overlimed soil was mixed with various quantities of crystallised magnesium sulphate in order to find the most favorable dose. The lime was applied as quicklime which was slaked before mixing it with the soil, and the magnesium sulphate was added after this lime had passed completely into carbonate and no trace of an alkaline reaction was perceptible. For the experiment served nine pots, each containing eight kilo soil. Pot I. contained the original soil, the other pots the overlimed soil. Pot II. received no dose of magnesium sulphate while the others rising doses of this salt, viz: III., 4 of the calculated amount of magnesia in the form of magnesium sulphate. * IV., 3535 Vs so; VL, 35; VIL, 3 VIL, 3; IX., 74, of that calculated amount of magnesia in the form of magnesium sulphate. These doses corresponded to: II. =120.78 grams or MgSO, +7 aq. IV, =',60.20, ss, 95 ’ Q V. =—295Sl ae, " iy y Vi=-2oage » ” ” VIL= D5:0ate. %9 9 ” VAIL. = * 53208 Si os y9 %» LX. = JG: 4a = 55 %9 %» The magnesium sulphate was added in high dilution, well mixing it with the soil. The general manure per pot was 5 St. .......Double superphosphate. 1O ts /s550 aceeeceeeeee Potassium sulphate. TO:Q0 sis ema tae t ee Sodium nitrate. Regeneration of Overlimed Soil. 63 This was applied after the addition of magnesium sulphate. Twenty seeds of sixsided barley were sown on Oct. 25 and after the young plants had reached 10—12 cm. in height, the number was reduced to 6 per pot, all of equal size. Early in April the plants began to show ears. The plants were cut June 17 and weighed in the air dry state with the following results. Total | Weight | Weight OL | sok e |) =0k Ears _ | Harvest) gears. | Straw! |\Grains, a em. £ g. g. g. I. (Original soil.) 34 33 63.5 | 76 30 46 25 | EEe( ) 29 | 29 61 Manis: 23 36 18 Ill. (4 mgO.) 30 | 34 62.5 | 66 27 39. | 22 = EVs ao) | 38 40 62.5 | 68 | 29 39. |../23 g V. (25 » ) 36 49 | 62.5 | 7° 30 | 40 24 Ets HM epomtueey: ||* Gaye)" 65° | eg ee aa ee Sey VIL Ge ay) 24 25 | 62 ca Pea aa | 20 | WATT (ty 57h!) | 25 27 62 60: Hi 25 Beer 39 Bee [hes |" 27a ze, | 6e 59 | 23 pee | 18 In comparing I. with II. it will be seen that overliming has depressed the yield in barley and in comparing IJ. with the other pots, that the addition of certain quantities of magnesia in the form of sulphate had a counteracting effect. The best result was obtained when 3), of the theoreti- cally necessary amount of magnesia as magnesite was added in the form of the crystallised sulphate. The harvest in grains did here most approach that on the original soil. In comparing therefore this amount of magnesium sulphate with the calculated amount of magnesite, we find that 14 parts of the former have accomplished as much as 100 parts of the latter; in other words: the agronomical equivalent of the crystallized magnesium sulphate on this soil is=14- While the former experiment was carried out on a rather poor loamy 64 S. Maki and S. Tanaka. soil it was a sandy soil of great natural fertility from Kawasaki which served for the next trial with two sided barley. This soil was overlimed by adding so much lime as slaked lime that the total quantity of the lime in the soil became three times as high as the amount of MgO present.! In order to produce now for barley the most favorable ratio, the MgO was applied in the form of crystallized magnesium sulphate in the following quantities : No. I. Original soil. No. II. Overlimed soil ; addition of 124g. CaO.? No. III. 4 of MgO as sulphate=134.6g. MgSO,+7H,O. |\No. IV. qo» 4p vs = O77gre “A Overlimed / No. Vist » ms a = 337s Es SoH Nod eae LA ' |'No.* ARIE 5 | = enGisee ; No.” Vea s 5 = @eAee 5 No. IX. sty» is jx RR " This magnesium sulphate was added to the soil after the lime had passed completely into carbonate. The general manure per pot of 10 Kilo soil was 5 Goi asavesie danpeemeeins Double superphosphate. 10 Gs, nse bseeeremse tes iO, LO Sac eteeebes eae NaNO... On January 15, 20 seeds of barley were sown. After about three weeks, the number of the young plants was reduced to 6 per pot, all of equal size. When the ears of barley developed, a great difference in the height was observed. Hence a photograph of some of the pots was taken, May 22, which is reproduced on plate II. 1 This soil contained 68.89% fine earth in which was contained 0.639% CaO and 0.80% MgO, soluble in 10% HCl. Ten Kilo soil contained therefore 43.4g CaO +55.0g MgO in available form. 2 In order to restitute the best ratio for barley e eo =) it would have been necessary to added 113g MgO in the form of finely powdered magnesite. Regeneration of Overlimed Soil. 65 At that time the height was measured with the following results. ‘Height. ‘ Height. £ g5 cm. aval 109 cm. INE OF; VIL. 107 IU. QO- s; VIII. 085 IV. TRO: #5 IX. OS | Mee IG.” The plants were cut June 22 and after becoming airdry they yielded the following weights : | Ears. | Total a ———— SSS Seeds, TEE Number, | Weight. ' | I. Original soil. 154g. 52 62.5 ¢. 50.5 g- Il, no MgO. 3215) on 52 62.0 ,, 5Ol0i ss Tl. 4 of MgO. IG | 57 73.5, 5, 60.0 ,, A Ver . 195 63 $0.0 (Sis) 3 Nee is + | 63 > 5 SB.s%s, WS Bess 5 Wie, ts ; 189 ,, 62 $5.0 ,, 680 ,, 2 ice ar by Mee 62 Seca 67.0 VITI G; 7/2) ee | 62 78.0 ,, 65.0 ,, IX. 160 ” 155 ” | 54 | 64.0 2? 52.6 33 It will be seen that ,!, of MgO as sulphate compared with MgO in the form of magnesite brought the best harvest; a decrease as well as an increase depressed the yield again. Hence the results on a sandy soil with two sided barley agree with the above result on a loamy soil with six sided barley.! 1 A peculiarity of this soil is however, that the increase of lime and magnesia together increased the yield above that on the original soil. i +; . a i a ees 08 - ’ Hl L * = > = = 3 ‘oe atid ae hth +¢ ah ce, ~ 3 Pigs "y Gut ox Wwe idee ‘ol ays Veale oft ed ces ve rg 22 SOs ae a oa IT, ( y TM IG, VAG, WALL. TR. mak LOL MKOVE,, ¢ ie ue BH ASE I arte Overlimed S IT, IV, mt | ee ale el es ll ~~ '@) Ol = | 50 i ie ©) FE Overlimed Soil + qo Of the calculated amount of MgO as sulphate, Same +54, of-MgO as sulphate. V. IX. Flax, On the Limefactor for Po page 58. Same +745 of MgO as sulphate. To page 64, _ Ait The Manurial Value of Different Potassium Compounds for Bariey and Rice. BY K. Aso. Many authors have compared the manurial effect of potassium chlorid, potassium sulphate and kainit. Several authors also compared the action of potassium silicate with other potassium compounds and Nobbe_ has already in 1870 compared potassium chlorid, sulphate and nitrate in their action on buckwheat in waterculture. The general result was that the same amount of potassa in different forms have not the same action with different crops. Thus, while a certain amount of potassium chlorid can depress the starch content of potato, it seems not to depress the starch content of the barley grain. Further, the chlorid! acts in other cases more favorable than the sulphate, and in combination with chlorids of sodium and magnesium in the form of kainit it has been found superior to the 40% potassium salts. The effect of potassium chlorid on the sugar-content of the sugar-beet is reported by several authors to be unfavorable. The nature of the soil and the absolute amount of potassium chlorid will however somewhat influence the result. Sebelien observed (igor) that potassium chlorid acts especially favorably on grain-production while potassium sulphate more on that of straw. Thus far, | have not encountered in the literature an experiment in which at the same time the four potassium compounds, namely, the carbo- nate, the sulphate, the chlorid and the silicate were applied to the same plants and further no comparisons at all have been made for plants growing 1 Wagner observed that potassium chlorid is not more easily absorbed by the plants than potassium sulphate as has been asserted. 68 kK. Aso. in paddy soil., I have therefore compared these four salts in three consecu- tive years in their manurial effect on barley and paddy rice. Experiment with Barley. Four pots containing 8 kilo air-dry soil were manured with 20 g. ammonium sulphate and 50.4 g. sodium phosphate (Na, HPO,+12 aq.) while the other four pots of equal size received 20 g. ammonium sulphate and 18.1 g. double superphosphate, the quantity of P,O,; being equal in each case. The potassium compounds were used in equivalent quantities : Per pot POlLAaSSOIMI Rea eBOUALE .....-.-.....50c0000. 9.6 g. igs esis. 02) 7 re 12.12. bya Riss rib sos) oe 23 AD: POEABSIARMOIG Ee ss coe wens seen cues 10.4 g. : On Nov. 27, 1903, 15 seeds of barley, soaked in water and heated at 45° C. for ten minutes,? were sown in each pot, and the young plants later reduced to eight of an equal size on Jan. 8, 1904. In the first stage ot development notable differences were not present; but later, in April the plants in the pots containing potassium silicate showed the best growth. The flowering commenced at first in the pots containing potassium chlorid and on April 13 the number of flowering stalks was counted : Potassium Potassium Potassium Potassium Chlorid. Silicate. Carbonate. | Sulphate. With Sodium phosphate. ............ 29 7 7 4 7 With Double superphosphate ...... 29 II 13 On June 3, the plants were cut ; the yield, weighed in the air-dry state, is shown below in the table. In the next experiment, 20 seeds were sown Oct. 26, 1994 and later on the young plants reduced to five per pot, all of an equal size. The growth in + This preparation contained 28%K,0. 2 This was done to prevent the smut disease of barley. The Manurial Value of Different Potassium Compounds for Barley and Rice. 6g the pots with potassium chlorid was at first a little behind the other plants, but neverthless the ears appeared earlier. On June 8, 1905 the plants were cut and the harvest weighed in the air-dry state. RESULTS. 1904. : = ah Quotient of Tot Straw Jars: Grains iV Se Total. Straw. Ear xrains. Wield . (| Potassium Sulphate ...... 124 77.0 47.0 31.0 40.3 S - i 39 , 3a |Potassium Carbonate ... | 119 74.0 45.0 30:5 4I.2 ce ag Potassium Chlorid......... 136 65.5 79.5 59-5 90.8 SS eee . Sey 5 ; 2 a Potassium Silicate ......... 134. 55.5 48.5 32.5 35.0 » & (Potassium Sulphate ...... 133 $0.0 53.0 38.5 48.1 se : 5 &|Potassium Carbonate ... 131 79.0 54-5 38.5 50.4 ag | ‘S © | Potassium Chlorid......... | 138 67.0 71.0 59.5 88.8 SS | i \Potassium Silicate ......... | 127 73.0 54.0 ALOT} 56.2 ° | 1905. : cs : ie: | Quotient of Tot; Straw 2ars rains ty: Lota. Straw. Ears. Grains. Yield. ee Potassium Sulphate ...... 116.5 71.5 45.0 52.0 73-0 2 ae c ; +3 |Potassium Carbonate ... 120.5 70.0 59.5 50.5 72.1 Be. N : < 8 Potassium Chlorid....... 131.5 68.0 63.5 53.0 78.0 ~ or =a 7 te > , F Pe as Potassium Silicate ......... 155.5 91.3 67.2 52.5 57-5 » & (Potassium Sulphate ...... 129.7 67.9 61.8 35.0 ids 22 35 &|Potassium Carbonate ... 126.5 68.5 58.0 45.0 79.1 ag ~ : . SS £/ Potassium Chlorid — ...... 111.0 48.0 63.0 52.2 108.7 = 2 A ‘Potassium Silicate ......... 121.0 61.5 59-5 52.3 85.0 Experiment with Paddy Rice. Experiments with rice were conducted under essentially the same conditions as with barley, except as to watering. On July 13, 1903, shoots of paddy rice were transplanted, three plants in a bunch and three bunches in each pot. The plants exhibited towards the end of August 31 great difference in development ; above all, it may be mentioned that the pots which received potassium carbonate together with calcium superphosphate produced much better plants than the pot that received potassium carbonate with the secondary sodium phosphate. Evidently, the alkaline reaction is in the latter case much stronger than in the former, which is unfavorable for the roots. On Sept. 9 the plants commenced to flower and on Nov. 6, they were cut and later weighed in the air-dry state. Another experiment was carried out in the summer of 1905 with paddy as well as upland rice under the same manuring conditions. On July 11, the young plants of paddy rice were transplanted. Of the upland rice 20 seeds were sown in each pot, and the plants reduced later on to eight. On Nov. 15, the plants were cut.! RESULTS. 1903. - Le | (2 | ; _ | Ouotien Total. | Straw, Grains, oo ull Empty of grains. | grains. | yieg oie LE a | = | | | oe). | lars g. g | « ec ~ (Potassium Chlorid......... 178.0 | 97.5 $0.5 76.0 4.5 $2.6 50 3 @ } Potassium Carbonate ... 268.5 145.5 123.0 118.0 50 86| SC 84.5 na. rE g Potassium Sulphate ...... 279.0 .; 151.0 128.0 2s 6.5 84.7 afer Ss = ~~ \Potassium Silicate ......... 299.5 | 160.5 139.0 134.0 sow | 86.6 Potassium Carbonate+ Double Superphospuate: pss eee 337-5 195.5 142.0 137.0 5.0 pei 1 The weather was too cool for the growth of rice plants through the whole summer in 1905, so that the ripening process was very much retarded, especially in the case of upland rice. The Manurial Value of Different Potassium Compounds for Barley and Rice. 71 1905. Paddy Rice. Upland Rice. | } i veers | | | a | | i j } —- = -_| Full | Empty] 3 = ae as Fa Ss ;, | Grains = re? | Total. | Straw. |Grains.| 3 ;= Total. Straw. Grains, | grains, | grains, jG | Ss | As | ee : fee | — : Z jeotassium | g. | g. g. g. g. g. g. g. Chiorid ...) 95.9} 709 | 250 | 39.1 | 5.9 | 35.2 |.770 | 73.8} 3.2 4.3 2 “0 uas7e8 482.2 6.3 | 605.5 | 1094.0 108 Zac get | 474.0 | 5.5 | 594.3 | | et ale me | | ezboeet | le e | | | | | 5 4 | 4420 | 5.0 | 5900 | A | 20 | 489.0 | [eSio 587.3 473.0 6.3 587.2 1066.5 105 | | 29 | 487.0 | 6.0 | 584.3 =A i gaa a a at. 6 | 516.0 | 60 | 684.8 | 185) 4 Be 505.0 | 5 580s |) -532.3 8.0 | 626.3 | 1166.6 115 30 | 477.0 8.5 | 604.8 | = = | = i _ —— | — —— iT. em 552.0 | 6.0 635.3 | | 9 | 30 506.0 | 7.0 Gogg | 510.3 6.5 | 606;5 | 1123.3 III 31 } 473.0 | 65 | 5785 | | | 4 x ae. |__- “8 | 478.0:), i105 lagers. | | 20 35 | 535.0 | 60 | 637.3 | 495.5 | 67 | 561.3 | 1163.5 115 | 32 | 483.0 5.0 | 595.3 | | | | 8 | 491.0 5.0 559-3 21 40 516.0 | 7.0 | 6208 500.3 6.7 586.5 | 1093.5 | 108 33 | | 494.0 8.0 | 580.3 | | mie 10 422.0 5.0 501.8 | 22 | 45 546.0 8.0 568.3 481 6.1 | 536.4 | 1023.4 IOI | 34 | 5750 | 55 | 5393 | { | | ; | Mn,O, Full Empty | Average. Noxo£ |, ae | Straw ee =. i , : | Comparative : | per ha grains | grains ' Total. | Frames. | | Full Fmpty Sea increase. | kg gr. or gr. | grains, | grains. | ~ Srp: } | ‘ KY 399.0 5.0 525.8 | 23 50 | 569.0 11.5 705.3 | 453.0 7.0 553.0 Ne LOLEE ICO 35 385.0; 45 | 428.3 | ist core Ca. 1 an re | 430.0 5.5 561.3 ; 24 | 55 424.0 | 4.5 | 5178 | 446.8 | 6.0 | 563.5 | 1016.3} — 100 36 | 486.5 | 80 | 6113 | The stimulating action led in this case to an increase of only 15 percent by the same dose of manganese that led two years before to one of 37%. This increased growth had of course drawn upon the store of mineral nutrients of soil and manure, hence the control plots were now in a much better soil condition, and in the interest of a fair experiment it would now have been necessary to procure equal soil-conditions again, before a fresh trial with manganese would be started. But it was the intention to compare in the following year the effect of manganese on the partially exhausted plots with the yield on the contro] plot. In the year 1905 the experiment was carried out in the same frames and under the same general manuring conditions as before. But the doses of manganese were no longer varied from 10-55 Kilo Mn,O, per ha; only one dose, namely that in the ratio of 25 Kilo Mn,O, per ha, which had proved to be the most favorable in the first year was applied, but in three different forms, namely as MnSO,+4 aq.; MnCl,+4 aq., and MnCO, respectively, corresponding per frame to 6¢., 5 g. and 3,06g. The yield (air-dry) is shown in the following table : 1 The manure was applied at the following ratio per ha: 109 Kilo N as ammonium sulphate 100 Kilo K,O as K,CO, and 100 Kilo P,O, as superphosphate. 80 M. Nagaoka. No Manganese. Manganous Sulphate, No. of No. of Frames. Frames. : Total. Grains. Straw. Total. Grains. Straw = — = I- — = = 2 $29 289 54 3 S06 258 548 14 877 352 525 4 656 215 441 26 855 291 564 5 647 243 404 40 SoI 291 510 6 760 270 490 4I 798 2858 510 7 552 210 ae 42 886 315 571 8 715 249 466 43 793 282 511 9 537 211 326 44 888 325 563 10 590 200 390 45 $53 312 54! II 612 182 430 46 $13 293 520 12 $16 298 518 Average.| $39.3 303.8 D300) Average. 672.1 235.6 438.5 | Manganous Chlorid. Manganous Carbonate. No, of » | No. of Frames. | — Frames. Total Grains. Straw. Total. Grains. Straw. = : z ad a= Bet § , g. g. g. g, g. “ g, 15 334 325 509 27 753 275 478 16 580 193 387 28 $36 308 528 17 $31 330 501 29 780 257 523 18 $43 290 558 30 821 280 541 19 893 339 554 31 355 303 552 20 351 302 549 32 994 355 639 21 $03 301 502 33 $62 302 560 22 748 270 478 34 945 313 632 23 670 228. 442 35 708 308 400 24 773 278 495 36 385 290 595 Average. 782.6 285.6 497.0 | Average. 843.9 299.1 544.8 The summer 1905 was very unfavorable for rice, on account of the prolonged rain a There was however no damage by fungi observed on our On the Stimulating Action of Manganese upon Rice. TIT. 81 plots. Some small damage by animals (rats) was reported by the assistant but unfortunately the number of the frame was not noted. It will be noticed that manganese sulphate and chlorid had this time depressed the yield, the greatest depression taking place on such plots as had in 1902 yielded a considerable plus-yield. Taking now inconsideration that the frames with manganese carbonate did not share in the depression, although a number of these frames had in 1902 received also large doses of manganese sulphate and had produced a considerable plus-yield, it will be save to conclude that the depression on the other manganese plots partly was due to increased acidity in the soil, which on account of its high humus content was some- what acid from the outset but could not be neutralised by the dose of potassium carbonate applied in the manure.t Superphosphate, ammonium sulphate and manganese sulphate united in increasing the acidity. The further fact, that manganese carbonate did not lead to an zucrease by stimulation must be ascribed to the partial exhaustion of the soil by the former three plus-yields. In continuing the experiments on the same plots, small doses of lime will be applied as a remedy of the increased acidity and care will be taken to restore all plots to the same manuring conditions. 1 Indeed the aqueous extract of these soils showed a strong acid reaction on litmus paper, more so than the control plot. The aqueous extracts also gave a stronger reaction for chlorine and sulphuric acid respectively than the carbonate and control plots did. Stimulating Influence of Sodium Fluorid on Garden Plants. BY K. Aso. Three pots each holding 8 Kilo air-dry soil were manured each with : Be CO ene ie eee Pons oe vle'ecn ace vetiees an Lav Peer ine Ne) a. er 6; (205 9 ets sy on ac A Sie Common superphosphate ..........6..-5:5- i One pot received 0.02 g. sodium fluorid and the other 0.2 g. of it ; the third served for control. On ‘March 14, seeds of Helichrysum bracteatum and Pedicellaria viscide were sown, and later on the young plants of both species reduced to three of equal size. Gradually a difference in height became noticeable. On July 2, a photograph was taken (see plate III.) and the following height observed : PEDICELLARIA. Control. 0.02 g. NaF. 0.2 ¢. NaF. cm. cm Cl 78 75 so 65 87 69 75 gI So ae A stimulating effect of sodium fluorid had therefore taken place. The flowers appeared first in the pot with 0.02 g. sodium fluorid. In regard to the size of flowers, however, there was hardly any difference observed.} As to Helichrysum, the influence of sodium fluorid was not so marked. Also here the size of the flowers was not affected. i Tn a former paper was mentioned by the writer that the size of the flowers of the plum-tree was considerably reduced under the influence of a certain amount of sodium fluorid. Small twigs with flower buds of plum-trees had been placed in a solution of 0.0019 sodium fluorid. The flowers, that opened after twenty one days, were all very much smaller than those of the control case. When the buds, however, are too far developed before they are placed into the fluorid solution, this peculiar effect can not be noticed. On a Stimulating Action of Calcium Fluorid on Phaenogams. BY K. Aso. I have shown in former communications that sodium fluorid acts on the one hand as a strong poison! on seeds and seedlings and on the other hand that it acts as a stimulant of development when highly diluted. The fact, however, that in soil cultures the sodium fluorid can easily pass into calcium fluorid whieh is exceedingly little soluble in water viz. 1 : 260007 renders probable the supposition that the stimulating compound in soil cultures is not sodium fluorid, but calcium fluorid. This salt can of course hardly exert any poisonous action, not only on account of being very difficultly soluble, but also because it can not precipitate the absorbed lime necessary in the cells. Indeed young onion plants remained alive for several weeks when they were placed in a concentrated suspension of well washed precipitated calcium fluorid. In order to observe whether calcium fluorid can exert any stimulating action, the following experiments were made. Experiment with Pea in Waterculture. On October 1, pea shoots (about 5 cm. long) were placed in the following suspensions of precipitated CaF, : 1 Of the enzyms, zymase and oxidases are much more easily injured than the other enzyms, 2 The freshly precipitated gelatinous calcium fluorid may show a somewhat higher degree of solubility, especially in presence of certain other salts. 86 K. Aso. Vege Gls C0 o> i ee 0.1% b “= A 0.01% e : MMe snd bk Sane see 0.001 % ad. | —Soee setae = etcoterareierols .0.0001 9% é. Check. These plants had to rely for the time being on the reserve stores of the cotyledons. On October 18, the following observations were made : | Length of each plant. Fresh weight of each plant. cm. germ. a. 38.5 1.52 b. | 34.5 | 1.40 e 35.0 1.45 37-7 1.60 é 34-5 1.25 . This result made some stimulating action of calcium fluorid on the growth of pea-plants probable. Experiment with Pea in Sotlculture. Porcelain pots, each holding 1.5 kg. soil served here for the experiments. Each pot received the following general manure : LS. pocaeeeeeeeeeeee Double superphosphate: Pe se re Potassium sulphate. 2. sepaeee Sea ees Sodium nitrate. Besides these compounds calcium fluorid was added in the following quantities : Cs ar I RE oes = os oo ns ws renee 0.006 grm. Oo ein, eee eo... see OOO CO. wongtind soetin nites ee ot OCA z 0.050 ,, 1: Ja. SoS ge. See eee: OOD 5, On a Stimulating Action of Calcium Fluorid on Phaenogams. 87 On October 19, 1904, five pea-seeds were sown in each pot and after- wards reduced to three of nearly equal size. was taken (see plate III.) and the following observation made : Average-height. cm. ee ACH RRA RIO OO0:0 U6 Co UG BETIS TCIH aera a 35 RS ei = Rs Bloc) te cic <= re eee en (Eo Fee pee ert te: olor nt S ERECT ee IOI OPER ah PNA EIT Si so. sb Here oe eee 105 (EE San DOE DOCS OSC CCOCISE 5 3.00 OCC ARE Ena ete 104 On February 13, a photograph Although the increase in height in @ and @ was striking, there was otherwise not any marked difference as to the development generally. On May 5, the ripe plants were harvested with the following result : Number of pods. Fresh weight of truits. Fresh weight of straw. grm. grm. a. 19 10.0 6.5 | b, 17 $8.5 6.0 ee 21 11.0 6.5 d, De 12.9 7h 2: 18 3.9 6.2 Some stimulating action in regard to fruit formation seems therefore probable to have taken place in pots ¢ and d. / Experiment with Barley in Waterculture. On October 18, a pair of barley shoots (about 10-11 cm. long.) were placed in the following solution : Galen nitratereee..-....<. Rey ee ee fart 0.2 % Potassium nitrateeen.......+- ea rere 0.15 % Monopotassium phosphate ............... 0.05 % Maonestum) sulphate: .......cic025 0.006.808 0.05 % JNU OMMUON SUMOMNALC! 2... 2ececagcass eanee 0.05 % Perroussulphaipemme. 25. i... ites)Jsacsedse% Trace. kK. Aso. ioe) a Calcium fluorid freshly precipitated and well washed was added in the following proportions : CLs a isiarel ae ee ee Pea x. 6 ala a a'evie s slaveleiels O. 1% Ganon ae soc slvane Gadenes 0.05 % Ee ee U PREG MI cco ciowescesischuigiaen 0.01 % D> SESS os ai. Se wets Sean eS 0.001 96 Oy dict ee ee Check. These solutions were renewed from time to time. The following obser- vations were made April 20: Average length. Total fresh weight. cm. grm. a. 108 170.0 b. 107 | 202.0 Bs 102 221.5 i d. 100 216.5 é. | 94 154-5 These results show that calcium fluorid can indeed exert a moderate stimulating action. The continuous application of the small dose of too grms. sodium fluorid per hectar would very probably even after many years not exert any injurious action whatever, as it passes into the calcium fluorid in the soil.1 It may in this connection be of some interest that Wein, in comparing Wiborgs phosphate with common superphosphate, observed a much more favorable action of the former. This beneficial influence may to some extent be due to the presence of 19% fluorine in Wiborg phosphate. + Whether potassium iodid, by application in the same ratio, would ever accumulate to an injurious amount is also doubtful, since rains would leach out the ‘small doses left after harvesting. Very small doses only might be retained in the soil by its absorptive powers, Ona Stimulating Actiou of Calcium Fluorid on Phaenoganms. 89 A favorable action of calcium fluorid on the yield was observed recently also by Ampola.t_ But his explanation can hardly be accepted. He assumes that calcium fluorid would be decomposed by carbonie acid, or weak organic acids with liberation of hydrofluoric acid, which then would act on complex. silicates of the soils and. render the potash assimilable’ Calcium fluorid is however not altered at all by carbonic acid and other weak acids: We must therefore assume that calcium fluorid being soluble a little in water can act as a stimulant of plant growth under favorable conditions, and that this salt is formed in the soil when sodium fluorid is applied. ? + Gazzetta chim. ital. 1904, 34, ii, 156-165. He applied CaF, at the ratio of rookilo. per ha. 2 There is not always an effect perceptible when sodium fluorid is applied in form of topdressing after the plants had reached a certain height, as an experiment with upland rice treated at the rate of 200 g. NaF per ha has shown, PEATE LE RULE COLE, AGK. VOL, VII. NaF Pedicellaria. 0.208. Control, 0.02g. NaF. To page 83. On the Degree of Stimulating Action of Manganese and Iron Salts on Barley. BY T. Katayama. Former observations in this college with oats, upland rice,! barley and wheat have shown that the stimulating effect of manganese salts on these Graminez is not so powerful as on the leguminous plants. Thus, an applica- tion of 0.015% manganous sulphate upon the soil has led with the pea to 50% increase of straw and 25% iucrease in seeds, while neither that amount of the sulphate nor a further increase to 0.04% produced more than about 10% total increase with cereals in pot culture. It seemed therefore of some value to determine that dose of a mangancse salt which would produce also with the common cereals such a favorable result as was obtained with the pea. The doses were therefore further increased, namely to I. 0.01% TID 010596 Fassett cot ow ewts .MnSO, +4aq. Ill. so For comparison, the action of ferrous sulphate in the same doses? was observed, and also a simultaneous application of a mixture of both those sulphates. 4 With paddy rice in field culture the result was much more favorable; several reasons might be given for this difference observed on dry and swamp land. 2 Several authors have recommended 200 kilo. green vitriol per hectar as a favorable dose, others 65-350 xilo. per hectar, but on unmanured soil Larbalétrier and Malpeau (Ann. Agr. 1896, p. 20) have not observed any effect of a dose of 150 kilo. green vitriol per hectar. Q2 T. Katayama. The salts were applied in the form of top dressing in fractional doses in high dilution (Dec. 7th and Jan. 25th). The pots contained 8 kilogramm soil which had not been manured for 5 years and was partly exhausted by yearly crops. The general manure for each pot was: double superphosphate 8 er., kainit 5 gr., ammonium sulphate 10 gr., sodium nitrate 5 er. 25 seeds of barley were sown Nov. 13 and when the young plants had reached 12-18 cm., they were thinned, taking care that the remaining plants, 8 per pot, were all of nearly equal size (17-18 cm. high) on Jan. 24th. The plants were cut May 26 and weighed in the air-dry state, with the following results : Straw. Grains, Control. Eek | eae 0.01% MnSO,, 4aq. } = 40.01% FeSO,, jag ee | 96.2 55-0 0.03% MnSO,, 4aq. } Bh +-0.03% FeSO, vag Vote oe $2.2 52.5 0.059% MnSO,, 4aq. ) > 40.05% Fes, PN aaa 85.5 48.1 0.1% MnSO,, 4aq.) = 2 + 0.1% FeSO, aan ee 79.2 37.5 Control. 93.9 50.7 0.01% FeSO, 96.5 55.0 0.05% FeSO, 100.0 50.7 o.E%% FeSO: | $5.7 | 49.5 0.01% MnS0O, $9.0 53-1 0.05% MnSO, 107.0 47.7° 0.1% MnSO, 95.0 44.0 SS On jhe Degree of Stimulating Action of Manganese and Iron Salts on Barley. 93 Weight of Grains. = aaa J = *[OUOr) ole] Sy Oy] O1,0 “UAT ‘UN? These results show that 0.019% of manganese and iron sulphates produced a moderate increase in harvest, 6.219% in straw, 7.219% in seed, and further that the increase of sulphates of manganese and iron beyond 0.01 % of the soil led to a general decrease of the yield. On the Formation of Humus. BY S. Suzuki. Many authors have examined the influence of calcium carbonate on the formation and decomposition of humus. But the results do not fully agree. Hilgard believes that lime promotes the decomposition of humus.! Kosso- witch and Tretjakoff, however, believe that the experiments of Petersen and Wollny are not very convincing and infer that the calcium carbonate in the majority of cases exerts a retarding influence on the decomposition of organic matters.? These Russian authors filled a glass cylinder of 0.5 litre capacity and of 34cm. height with oak leaves or hay with and without addition of 0.5% and 10% CaCO,. Each glass cylinder received 80g. leaves moistened previously with 160 cc. water and was infected with 1g. of a humous soil. From time to time a current of purified air was sucked through the apparatus, which was connected with a flask containing potassa, thus determining the CO, developed during the humification process. The experiments lasted from 97 to 112 days. Almost in every case it was found that calcium carbonate retarded the humification process. This result can be easily explained by assuming that the fungi necessary for the humification were not provided with soluble phosphates, since the excess of lime present must have rendered insoluble all phosphoric acid originally present in the organic matter, hence development of fungi was retarded. 1 Forschungen der Agricultur-Physik 1892, p. 400. ? The decrease of organic matter by the humification process was found by Ramann and Kostyt- cheff to amount to 559% in the first year, while by Henry in one year only 159. Various conditions can of course have influence on the progress of the changes. = 96 : - Suzuki. In the soil, the fungi producing the humus! find all their necessary mineral nutrients in a more or less available state. Since lime is not absolutely required by fungi, while magnesia is absolutely necessary and secondary magnesium phosphate is also more easily soluble than the calcium phosphate, it seems to me of interest to repeat such experiments under more favorable condition for fungi. Therefore potassium phosphate and magnesium sulphate were added in small quantities and the effect of calcium carbonate was now compared with that of magnesium carbonate upon the process of humification. My experiments were made in the following way: Some Erlenmeyer flasks (a. 1200 cc.) were filled with dry coarsely powdered (<6 mm.) leaves of Quercus serrata, Thunb. which commonly serve in Japan as litter. "Each flaskcontained 100 g. of these leaves, previously moistened with 200cc. of water and mixed with 1 grm. of humous soil taken from the fields of our college. The first flask served as check. To the second flask was added 5g. precipitated basic magnesium carbonate previously mixed with some water to a fine milk and further 0.5 ¢.K,HPO,. To the third flask 5 ¢. CaCO,, 0.5.¢. K,HPO, and 0.5 g. MgSO, were added, while to the fourth only 5g. precipitated CaCO. to compare the observations of Kossowitch with the behavior of the leaves under more favorable conditions. The second, third and fourth flask were infected with 1 g. soil as in the control. case. From time to time about Io litres of purified moistened air was sucked by an aspirator through the vessels and then through a Liebig’s bulb filled with caustic potassa, and the carbonic acid in this air thus determined. More importance than the determination of carbonic acid was the transfor- mation of organic matter into black humus. Therefore from time to time a small portion of the leaves was examined under the microscope. Into another flask (V) prepared like the flask II. was introduced the mycelium of a peculiar kind of Penicillium (Schokoladenfarbener Schimmelpilz?) after sterilisation of the flask. This fungus has the peculiarity of producing a black substance when cultivated in koji-extract. 1 On the destruction of organic matter by soil bacteria compare also: O, Bail, Centralbl. f. Bacteriol. II. Abt. Vol. IX. The formation and destruction of humus was here not a special object. 2 Cf. Lindner, Mikroscopische Betriebscontrolle, p. 243. On the Formation of Humus. 97 The general plan of the experiment is shown in the following table : Amount | No. of of | Water | Humous| MgSO, | Precipitated] Precipitated powdered K,HPO, | Fungus. flasks. | air-dry | added. | soil. | (anhyd.)| MgCO, | CaCo, | te leaves. g. ce | g. g. g ae g. I 100 | 200 I — = = ae = | | | Il. 2 > | = 5 = 0.5 | — lil | . 0.5 ast 5 0.5 | mas ave | 2 o | =: = = 5 h — aes Vi | s a 33 — 5 — 0,5 fungus. } . } | | The experiment was started on Febr. 7, 1905. Until the end of Nov. 36 determinations of carbonic acid were made with the following result : AMOUNT OF CO,, g. I. I. BEG IV. ae Date, ue a eee eee hee = Soil alone. | +K,HPO,- | +K,HPO,. Soil+ CaCQOg. | + Fungus. | Febr. 14. 0.4394 0.4116 | 0.4832 0.3428 0.0868 a 22: 0.3594 | 0.4216 | 0.4498 0.4880 | 0.1544 Mar. 3. 0.4628 0.6316 0.5746 0.4156 0.0254 ye or 0.4153 0.4708 0.4810 0.5286 0.1314 a 16. 0.4500 0.6282 0.5032 0.4462 | 0.0922 a 24 0.4216 0.5247 0.5003 0.5412 0.1424 “ 31 0.4148 0.5398 0.4612 0.4669 0.0973 April 7 0.4366 | 0.4812 0.4434 0.2974 0.1320 es 14 0.4292 0.5272 0.5304 0.5800 0.1478 2 21 0.4472 0.6151 | 0.4603 0.4958 0.1138 7 28 0.4876 0.4840 0.4650 0.4569 | 0.0701 May 5 0.4759 | 0.4944 0.4963 05073 | 0.1895 = 12 04701 | 0.5979 0.5018 0.4506 0.1054 = 19 0.4900 0.4989 0.5192 0.5336 0.1354 = 26 0.4752 0.5316 0.4524 0.4600 | 0.1049 98 S. Suzuki. l I, TI, HI. PVE V. aos =f Soil +MgU0 Ba serie ee as Heo. Soil alone. +K,HPO,: +K,HPO,. Soil +CaCO,. ut Fungus. June 2 0.4586 0.5880 0.4922 0.5204 0.1685 2 9. 0.4911 0.5065 0.4566 0.5169 0.4018 3 12, 0.4645 0.5378 0.4809 0.5032 0.3501 22° 0.4506 0.5093 | 0.4472 0.4302 0.0097 + 30 0.4541 0.4924 0.4775 0.4912 0.2966 July if 0.4552 0.5221 0.4671 0.4900 0.2685 14. 0.4578 0.5280 | 0.4714 0.4611 0.0769 21 0.4678 0.5032 0.4590 0.1715 0.0723 5 28. 0.4320 0.4522 0.4376 0.4252 0.4253 Aug 4. 0.4320 0.4678 | 0.4314 0.3604 0.04.00 m 26. 0.5198 0.6074 0.3190 0.0156 0.0074 Sept. 2. 0.3598 0.4130 0.4218 Gane? 0.0237 + 8. 0.4089 0.4798 0.4392 0.2203 0.0625 3 15 0.4110 0.4335 0.4160 0.1138 0.0502 = ie 0.4879 0.2895 0.3195 0.0799 0.0399 a 20. 0.4080 0.4549 | 0.4062 0.3095 0.0366 Oct 6. 0.3983 0.3601 0.1288 0.0432 0.0271 “3 1 0.3648 0.3998 0.4177 0.2220 0.0217 = 20. 0.3082 0.3102 0.3966 0.3900 0.2736 Nov 8. 0.5794 0.6735 0.6690 0.4468 0.3922 22, 0.4064 0.3894 0.4302 0.4206 0.3064 Sum. 15.8912 17.7770 16.3070 13.7559 4.8998 On the Formation of Humus. 99 If now the results in IIL, IJ]. and IV. are compared with those in the control case it becomes evident that magnesium carbonate (II.) promoted the development of carbonic acid,t while calcium carbonate (IV.) retarded it. Hence also the humification process is promoted by magnesium carbonate and retarded by calcium carbonate. In comparing III. with IV. we see that the addition of 0.5 g. potassium phosphate to the flask III. had a very esssential influence on the increase of the carbonic acid. The case IV. would confirm the experiment of Kossowitch, but in nature the case is different, since in the humification process of leaves in the soil these are in contact with soluble phosphates. Hence we can further conclude that the opinion of Hilgard corresponds more to the xatural condition of the humification process. I have also examined the physical properties of the leaves and their microscopical aspect and became convinced that the change in color and brittleness and also the development of mycelium go parallel with the development of carbonic acid. It remains further to be mentioned that after sterilisation and introduction of the peculiar kind of Penicillium known thus far only ” under the name of “ Schokoladenfarbener Schimmelpilz” the humification process proceeded much slower than under the original conditions. The amount of carbonic acid in flask V. was only about one third of that of control flask I. This experiment on the formation of humus will be continued so long until all the particles of leaves are transformed into real black humus. It is noticeable that eleven months have sufficed to transform the particles of leaves very considerably, the color has become very dark and the cohesion of the particles has been almost destroyed. 1 Supposing that some acid decomposition products would act upon the carbonate added to the flask II., [1] and IV. and liberate carbonic acid from that source, I subtracted the carbonic acid contained in the carbonate added from the amount of carbonic acid developed during this experiment with the following result : Bio" OF HaSkS, (MgCO:, 52.) (Cac, 52.) (CaCOz, 52.) CO, —developed. 17.7770 16.3070 13.7559. CO, —content of carbonates. 2.6079 2.1978 2.1978. Difference. 15.1691 14.1092 11.5581. Here also the amount was greatest in the case of the flask IT. ! z WE eG | s' i 4 ean al noe A New Variety of Mycoderma Yeast as a Cause of a Sake Disease. T. Takahashi. Saké, the Japanese rice wine or rice beer is not unfrequently altered by bacteria causing either acidity or a change of flavour and a turbidity. Several authors have reported on such sake diseases. Recently, however, a “turned” Saké was sent to me for investigation, which proved to be infested with a Mycoderma yeast.? The colonies of this yeast on koji-extract gelatine were white and showed concentric rings with radiations. A preliminary test, further, showed thal pasteurized sake, containing 17 vol. % of alcohol was attacked by this yeast on infection, whereby after 10 days cultivation at 20-28°C the alcoholic content was decreased to 9.37%. This observation led me to study this yeast purified by Lindner’s droplet culture. The following characteristics were then observed : 1. Form and usual size: Elliptic, filamental or sausage shape, rarely globular. Two or three fat globules are often seen in the large cells. 2. Growth. a. On koji-extract agar: pasty white colonies ; on plate culture : yellowish white cup shaped center filled with granular masses. On streak culture: folded and semitransparent on the margin; in 1 Atkinson, Chemistry of Saké Brewing, 63 ; K. Otani Journal of Tokyo Chemical Society XXII. Vol. 8; G. Torii. J6zdshikenjo hd ko ku. No, 2. 2 It came from the Niigata prefecture and contained 8.1 Vol. % of alcohol, 0.3696 acids, 1.5% of extractive matters, 102 F. Takahashi. stab culture: develops chiefly at the mouth forming a white granular pin head, altering to greyey after long culture. 6. On koji-extract gelatine: grayey-white and pasty coating, granular in the central part. Stab-culture: grayey white mesenteric growth chiefly on the mouth of the canal, gelatine is easily liquefied. 1 Gigantic colony (at 10-14°C) in plate culture shows a fine mesenteric structure and numerous concentric rings (see Plate IV.), which is one of the characteristics. c. On wort-agar: the surface culture (at room temperature) gave a white somewhat pasty and mesenteric coating ornamented by radiated lines on the margin. , ri ; fa & i = ‘ i a Pate ' ‘a Ses, ay - 1 ao a . . 7 = ‘ at Ts o " ! ‘ . ulti ei ; he al sll er 7 ; - ie 55 @ | $ : : :°)* Sane . ue a LPs, = per =a nee : r * Sins mr : i : —— - " ae fe i: : 7 a2 Be bet ky 4) ae ya ta ’ . ~ es . i J ne oh War ae i : S . a. ae ae ’ 7 ie : 4 i) ry i i OF) a ice Cr) y Vv Bey), » Sa ota \ “ae a i On the Micro-organisms of Natto. BY S. Sawamura. Watto is a kind of vegetable cheese prepared in Japan by fermentation of boiled soy-bean wrapped:in rice straw and left. for one or two days in a warm place. This product contains much mucilage filled with innumerable bacteria and it is the great viscosity that is especially esteemed with this cheese. Yabe! isolated from zatto three species of micrococci which formed yellow, orange and white colonies respectively, and a becillus which was immotile, liquefied gelatine, produced a green fluorescence and formed white colonies on soy-bean. According to Yabe the micrococcus of the yellow colonies produced the peculiar aroma of zatto when cultured on soy-bean, but thus far it was not decided which microbe gives rise to the viscous substance or mucilage. Yabe has found that a large part of the albumi- noids of soy-bean is decomposed to pepton and amido-compounds by the fermentation. The micro-organisms of xzatto consist in the beginning chiefly of bacilli, but on being kept for some time micrococci gain predominance. ‘The writer isolated various kinds of bacilliand micrococci from za¢to and observed their behavior in cultures on sterilised soy-bean. Two kinds of bacilli grew well on soy-bean and formed a product similar to zatto in regard to taste, aroma and viscosity. Other bacilli, however, and micrococci in developing on soy- bean did not produce a palatable product, nor did mixed cultures of them with the former two species. Also the behavior of Bactllus mesentericus vulcatus, Bac. mes. fuscus, Bac. subtilis, Bacterium filiforme and B. tyrothrix 1 This Bulletin Vol. II. No. 2. 108 S. Sawamura. filiformis was observed by the writer, but the products formed were of disagreeable taste and odor. The writer infered that although many bacteria can grow on soy-bean, the genuine wzatto can only be produced by certain bacilli thus for isolated from the zatto-cheese. Those bacilli show the followiog characteristics : Bacillus No. f. Form: Cells cultured on bonillon at 30°C for 24 hours are 1 w wide and 3-4 long. .The ends of the rod are rounded. The bacilli unite sometime to a long thread. Motility : The bacillus of young cultures is motile. Spore: An oval spore is formed in the middle of the cell. Gram’s method: Positive. Oxygen: Facultative zrobic. Bouillon culture: A light yellowish-brown scum of dry mealy appearance is formed at 30°C after 20 hours, Pepton-water culture: A white dry scum is formed at 30°C after 20 hours. Gelatine plate culture: Small round light brown colonies having a point in the centre and a feather-like divided periphery are formed. Gelatine streak culture: Gelatine is liquefied along the needle track. Gelatine stab culture: Gelatine is liquefied in the shape of a funnel. Agar plate culture: Light brown dry lipped colonies having a peculiar mealy appearance and sometimes with a point in the centre. Agar streak culture: ) fina = < E Sr =; aed vot hee he al On the Preparation of a Vegetable Cheese from the Protein of the Soy Bean, BY T. Katayama. The soybean which serves in Japan not only for the preparation of Miso! and of Shoyu sauce,? but also for the preparation of Tofu,* contains according to Osborne and Campbell+ as the chief proteid constituent® elycinin, a globulin similar in properties to legumin but of somewhat different composition, containing nearly twice as much sulphur, four tenths per cent more carbon, and a half per cent less nitrogen. The composition was found by these authors to be : [OFS 6.0) OR AS Sc eS ee ey PE eee: iy COREL. 55.5: Shae ee Se a rs 6.93 INGER 62. oe en ea neri ans stem mige is > 17-53 SUL NLULIT ot 3s Seow Eee Nee oases hl evs 0.79 eye, 25 fe. a0. en Se the ries ars 22-62 100.00 This protein can be extracted from the soybean by boiling water: on account of the presence of phosphate of soda and potassa. The liquid thus obtained resembles cow's milk in appearance, and yields a precipitate with 1 These Bulletins, Vol. I. No. 6. nls, = Vole I No: 3: See: is Vol. II. No. 4. » Journal of American Chemical Society, Vol. XX. No. 6, 1898. ou The small quantity of other proteins consists of legumelin, contained also in pea, vetch, horse bean and lentil ; a protein resembling phaseolin, and a proteose, 118 T. Katayama. calcium and magnesium salts, and thus a product is obtained which is in commerce !in Japan under the name of Tofu. It closely resembles freshly precipitated casein from milk which suggested the experiment to try whether it could not be transformed into a cheese similar to the well known Szzss Cheese. Some tabular pieces of freshly prepared Tofu were pressed in order to remove a portion of the water. 450g. of such pressed Tofu were mixed with 60 g. of common salt and with 50g. of pressed casein obtained from fresh milk by precipitation with acetic acid, and further 2g. of finely grounded swiss cheese. These addition of casein and cheese were made in order to introduce the characteristic microbes. This mass was wrapped in a linnen cloth saturated with a solution of common salt, and left for 5 months in a room with an average temperature of 15° C, moistening the cloth from time to time. After this time the mass had acquired a very compact consistency ; the cclor however was not white but grey, perhaps due to a trace of changed tannin. Further the numerous pores produced by gas development in the swiss cheese were absent in this tofu cheese, although some milk sugar (2¢.) had been added in the beginning to produce a fermentation similar to that of the swiss cheese. A crust was gradually formed on the surface, but its odor differed from that of swiss cheese. The taste of the vegetable cheese thus obtained was quite agreeable however and in no way recalling any putrefaction. A small portion was crushed in a mortar, extracted with water and filtered. The filtrate behaved as follows : On boiling some albumin was coagulated; the filtrate was slightly yellowish and neutral, but yielded a weak ammoniacal reaction by Nessler’s test; Biuret reaction was also very decisive. By saturation with ammonium sulphate, some albumoses were precipitated. The colorless filtrate gave with nitric acid on boiling a yellowish color, and phosphotungstic acid a voluminous precipitate in the presence of some sulphuric acid, indicating pepton and basic compounds produced by the bacterial enzyms from protein. On the Preparation of a Vegetable Cheese from the Protein of the Soy Bean. | 19 Second Experiment. Here the addition of fresh casein was omitted and the milksugar increased. Fresh Tofu was pressed in a linnen sac and divided into three parts and mixed with the following amounts of salt, cheese and milksugar : A B Cc Pressed Tofu, 700 &. 700 ¢. | 550.0 & NaCl. 105 g. 84 g. | $2.5 ¢ : { 55 Swiss Cheese, | 30g, | 10 g. 3-0 g. Milk-Sugar. 35 g. 35 g. 20.0 &. These masses were wrapped in linnen cloth moistened with common salt and kept in a warm room as in the former experiments. The rough surface of these mixtures became after few weeks smooth and the original yellowish white color changed to greyish ; also the characteristic smell of soybean entirely disappeared. It was considered ripe when a complete compactness, density and uniformity was reached. It was somewhat unexpected that here, notwithstanding the increased amount of milksugar, no formation of holes due to gasbubbles by fermentation, was observed. After removal of the crust the taste of this cheese was quite agreeable although different from that of Swiss Cheese. In consideration that 706f¢u is an exceedingly cheep material, and further that it has to be freshly prepared every day on account of easily undergoing putrefaction, the preparation of this cheese seems to me of some importance. ete ae ; be Same fac ij ars | ate mm 4 i a ie ire Gag a é 4 » a { ‘ gs ely 5 ey On the Composition of the Fibrous Part of the Japanese Orange. BY | Rana Bahadur. The juice of the orange has been an object of chemical investigation,! but not the fibrous part which remains behind when the juice has been pressed out and all the soluble parts completely removed. ‘This insoluble part, the fibrous pulp and the flesh-sac walls, resembling thin films, deserved however some chemical examination. It was above all of interest to see whether the polysaccharids of this material would also correspond to the sugars in the juice. Sometimes there exists not the expected relationship between these carbohydrates. Thus, e.g., with the Kaki fruit the juice contains invert sugar and canesugar, but no mannose, while the seeds are rich in mannan. Five Japanese oranges yielded 4.5 g. of this insoluble matter. After becoming air dry, a careful analysis was made, according to the usual methods.. For the estimation of galactan, 3.704 g. of the substance was boiled with HNO, of sp. gr. 1.15 until the mixture was reduced to one-third of its original volume. The mucic acid formed was after some time collected on a filter and further purified. It weighed 0.584g. From the quantity of mucic acid thus obtained, the amount of galactan was calculated. For the estimation of pentosan, 4.496 g. of the substance was distilled with HCl of 1 The juice of the European orange contains among others :-— 1.93% Free acid (citric acid). 5.319% Cane Sugar. 5-43.% Dextrose. 0.50% Mannitol and Pectin. 85.0499 Water. 122 Rana Bahadur. sp. gr. 1.06. The furfurol thus obtained in the distillate was precipitated with phloroglucin and from this precipitate amounting to 1.447 g., the amounts of furfurol and pentosan were calculated. Starch was not found by microscopi- cal test with iodine. Mannan was also found absent, when after hydrolysis with sulphuric acid of 39%, neutralization with baryta, evaporation to a syrup, phenyl-hydrazin acetate was added and the mixture stirred; mannose hydrazon was thus not obtained. 2.254¢. of the substance extracted with ether gave 0.029g. ether extract, and 1g. yielded after Kjehldahl 0.010245 g. NH, equal to 0.008439 g. Nitrogen. The final result of the analysis was :— Phy erescopiceamarear s....2.-..2-.+22640 12-16% - Protein. ia.6-- eee . . eee ina'oe epsearas Either Bria ereeetee . ....csc.ntaenes Lo Ga lalate ere eeneee® - « «ans ascstaeces 18.015, PeRtO Saat ce eres « ... oie e sane ese oe Celihilase apes eres. «5s: -s cssdeeeee 22s tie We notice here again the interesting fact that this insoluble part of the orange contains polyanhydrids of sugars that are not found in the juice. Fresh Water Algae as an Article of Human Food. BY S. Namikawa. Among the vegetable articles, of human food in Japan are not only sea water algae but also two kinds of fresh water algae. The former have repeatedly been the object of chemical investigation! but not so the latter, hence some chemical data suitable as a measure for the nutritive value of these eatable algae? may be welcome. The one kind is Nostoc Phylloderma of the group of the Shizophyceae, with the Japanese names : Suizenji-nori, kotobuki-nori. It is chiefly collected in the mountainous regions of the provinces of Higo and Chikuzen in Kiu-shu, and is extensively sent to Central Japan, where it forms a somewhat expen- sive delicacy, especially as an addition to soup. It is collected by small nets, ali the year, mostly however in June and July, and cleaned from adhering other algae. One man can collect only 1 litre a day. The mass is cut into small pieces, spread on briks and dried in the sun ; it forms thus thin cohering sheets. About 2 litres fresh mass give one sheet of 2 sq. feet. 5 sheets weigh go g. and cost 24 yen. The second kind of eatable algae is Prasiola japonica of the group of the Chlorophyceae with the Japanese names: Daiyagawa-nori, Nikko-nori. It is chiefly collected at Nikko in Central Japan. 1 These marine algae are frequently cultivated in shallow places on the coast. There are 6 kinds: Porphyra vulgaria (nori), Enteromorpha compressa (ao-nori), Cystophyllum Fusiforme (hijiki), Capea -elongata (arame), Ulothrix pinnatifida (wakame), and Laminaria japonica (kobu). Many species of -seaweed serve as food also in the Hawaii Islands. 2 Fresh water algae in general have but rarely been the subject of chemical tests. O. Zeew and Th. Bokorny (Journ. prakt. Chem. 36, 272) have analyzed a species of Zygnema and found fat, lecithin, cholesterin, starch, tannin and succinic acid, but not asparagin. In the dry matter was 28-32% protein, 6-9% fat and 60-669 cellulose and starch. 124 S. Namikawa. Nostoc Phylloderma. The dried product of commerce swells up considerably in warm water,. making the presence of certain hemicelluloses probable. Cold water extracts. a special coloring matter; after soaking for two or three days, the liquid shows dichromatic property : a pink red by transmitted ray, and a reddish violet by reflected ray. Mineral acids and also acetic acid turn the color to violet while alkalies decolorize it; on addition of acids the color is regenerated. Ether, benzene, chlorform, carbon disulfide do not dissolve it. Some galactan, starch and lecithin were present while mannan, sugar and tannin were absent. The following analytical data were obtained after the usual methods. In regard to determine the galactan 20¢. of the air dry matter were boiled for seven hours with 400 cc. dilute sulfuric acid of 5%, replacing the water lost by evaporation. The filtrate was neutralized with baryta and _ this. filtrate evaporated nearly to dryness, then boiled with nitric acid. Mucic acid soon separated as a crystalline powder. For determination of the total N, 3g. air dry substance (=2.4576g. dry” matter) yielded after Kjeldahl=o.0975 N. 3g. air dry substance yielded by extraction with ether =0.0168 g. fat. . (=4.096g. dry matter) were for the determination of pentosans boiled with hydrochloric acid of 1.06 sp. gr.; the distillate yielded with phloroglucin=0.394 g. precipitate (=0.204 g. furfurol). The final result was as follows : HyGtoscOpic MOmsMFe. .......-00-..25e5 18.07% In 100 parts of dry matter : (Crude eae tn casera ey « - -+- cine Sane ss 24.75% Crude fat 27. eo 5 eee 0:93)5; Grade fibres ere. .. -52> >. sseenacee 3:04 PeEntosSaris. ee eee... enn veeee Sane 4-50 5, Gala chan (ese eeerer ees. «se senaisc bee 1.86 ,, SEY on in one ene ee oD ss. bie ee 1228, Difference, chiefly: Starch ....:..223... . 58.40 ,, Contributions to the Study of Silk-Worms. Il. On the polygamous habit of the silk-worm. It is a general belief amongst our silk-worm breeders that when a male moth mates with more than one female, the health of the offspring thus produced is much affected, that is to say, the offspring of later copulations will be weak and unhealthy, and that the longer the copulation the better will be the health of the offspring. Moreover, it is said that if the copulation is not of sufficient duration, less eggs will be laid. From these considerations, silk-worms are kept in a monogamous state among Japanese raisers. It is, consequently, often the case that breeders can not get good pairings when the moths come out, and considerable pecuniary loss is the consequence, since in our races it is exceedingly difficult to distinguish the sex of the cocoons. The object of our experiments was to see if there exists any such relation between the habit of the parent and the health of the offspring. Series J. First of all, we will see what the effect of polygamous habit is on the eggs laid. Experiment a. One male was made to pair with nine females inthree days. “The moths used for the experiment belonged to the Siamese multivoltine yellow race. 126 kK. Toyama. | | Date of ; | z ; ae. fa Number aoe Date | Duration Number of eggs laid. hie ey J pried newly hatche os | iene | | Unferti worms, : wid : Unferti- Total copulation.| Q | 3 | pairing. aaa | lene Dead. otal. per 100 heads. i » Hie: | Ont | Oct: paaaees 2? mS | copulation. yth. | 7th. 7th. 2 hours. a. ” 173° 2 Ree Pom | 20 5 | 0225 OT b. ieee " 329 mela 331 | | Second. a. x | i 325 2 6 333. 4 ] | | ¢ 0.0322 o1 é. S 3 - 281 | 2 I 284 \ : | } | Third. : = 30* I II = a, ” ; 0 | {ooaor er 6. s3 55 | 363 4 fe) 367 Fourth. a. 2: 3 | 7a 28 ] a, 7 | 0.0310 gr, b. 4 oo! eee x 226 2 fo) 228 Fifth, a. 23 7 2? | 104* I Oo ma a : | 0.0312 gi - 6. * 33 ai | 322 8 4 334 Sixth. a. 5 5 Sth. 4 hours. 348 | 7 2 357 i 0.03C9 gr. b, 3 ” ey ” USA 10 6 — : Seventh. a, ” ” 3° ”? 316 4 I 321 0.0332 gr. b. ” " » r 296 i 389. he Eighth, | a. ” ” bb) ” 184 125 te) 309 6 0.03066 gr. b. ”? ” ” | ” 18 57 | Oo ag" | Ninth. | Oct. | ; a. gth. s goth. 6 hours. 396 3 I 400 0.0327 gr. 6 co ” ” oO Oo — * Those marked with an asterisk laid some of their eggs in the cells and the figures do not represent the total member of the eggs laid. Contributions to the Study of Silk-worms. Experiment b. Sy In this series, eight copulations were gone through by a single male. The moths were of the same race as before. ’ Date of | | : Number emergence, | Date | Duration Number of eggs. of a of of copu- | Sa [SEEN Gennaro , : : , | Unferti- copulation.| 92 | & | mating.| lation. ||Hatched. ised Dead. | Total. SRO cw | a ee ee aie ve | | | | | First June | June | June | 30 copulation,| 18th. | 18th.| 18th. | minutes. lameze2tje a2 | 3 |) 207 Second | | one hour | - Eee * ia : copulation.” 23 30 mt, | 2 fae Gila ae 3, 3 hours. e4Om al) » 2 9 357 Fourth, A 3 3 7 hours. AGO! =) ©) 3 ace Bit Ns » | 18=19th.| ro hours. | 404 | Eee $15 420 ~ | whole || | Sixth, | , | roth. | } day. | 389 | 3 R 395 oad eee { whole || | 2 Seventh. | 19th.| , 19-20th. ) night. | 337. | 108 | 16 461 of ;hole |} Mew |, bs, eee ee Hee 68 3 | 20 250 § | | |) day. | 3 3 | 39 Experiment c. In this series also, one male was made to pair with cight females, the moths being of the same race as before. “ | Date of é | : * RemaVER el emeroence Date | Duration Number of eggs. | = : Weight of newly of ot | of ee r ; = : F ie a atched worms, mating. | Q & | mating.| mating. ||Hatched. ee Dead aro set er | | & ee é 5 - | | | July | July | July | 30 I} 3rd. | 3rd 3rd. | minutes. 304 17 I 3228 First = red | x mating. ” ” ” ” 364 17 +t 355 0.033 gt en ae : | 2 297 10 3 310 | | diss Average, 355 14.6 2.6 339 one hour | ag 33 » |to minutes,| ° 58 : ae | Second.J 2} ,, - 5 Pe fo) 38 oO 35 Lo, gi 3 9 ” ” ” 12] 9 2 I I Average. fe) 34.3 0.66 35 | 128 K. Toyama. Date of i ‘ Number |} eaecene Date | Duration | Number of eggs. : : ; Weight of newly of of of i iets : a i- atched worms. mating, | Q & | mating.} mating. Itzatchea bapa Dead. | Total, ' | . I July | July | July si) 3rd. | 3rd. 3rd. 2hours. | 358 | 9 5 372 Third.J2) ,, ede, : 359° | ‘7 | .4 4° (380 |Yotosaemas | 3} 2 oe | ’ 332 28 4: 364 i ‘ } | | | | = | Average. 349.6 | 18 43, | 379 | | | | at | / = ; f | | | 1] 5» = 4 ie eee So fd | BW eee | | | | | Fourth.J2) ,, ” | ? | ” 195 | 6 69) - 4, “270 | 0.034 gr. | } | j 3 ’ | 7 | : 2 299 4! : 344 | ] Average. | | | 290 17 25-3 | 332-3 | | } | i | i He es > + 4hours. | 394 | 7 Be) 40058) | ; | | Bigths 2) 55) He: | oe) at 3 HH. | | 26 - 144 | 0.0315 gr. ; | } 3 | os » 99 304 ps A ee: 319) 4) Average. | 271.3 14.6 4.6 290 | | | E | AE ccs oe | a), 4th: “|| srd- 4th. | 4 hours. Bot 4 7 | 9 S37 awl Six , a a : 330 16 | 20 366 ©|\0.031 gr. H | 3, s - ¥ Oo 6 oO 6 i | | _ eee ae Average. | ; 217 | 9:6" j= G6 236 | seit ere i) ae kee | | i. es ee ‘ars 351 8 2 8 aay | | | Seventh. 2 2 23 | 2 | 23 Oo | 5 | o) 5 0.035 st. ; | 3] » | \~ 318 10 7 335 | Average. | | 223° | ~7.6 5 | 235.6 | 4 \ is | 4 hours. 1 z / = sa i. » — |10 minutes | a ers . | oe Fighth.J2] ., E. 4 ” |"zo2 | 9 46 4 357 0.035 gt | | | \3 > { 23 > | oO 31 s) | 31 | Average. | | 34 | 24 | 15.3 | 73-3 Contributions to the Study of Silk-worms. . 129 Irom these experiments, we sce that there is no considerable difference between the first and subsequent copulations either in the total number of eggs laid, or in the relative number of unfertilized or dead eggs. No difference can be detected also in the newly hatched worms. If we pick out particular cases, the best result is found in the ninth (a2) and fourth (4, c) copulations, not in the first or second. go As a general result, however, we may say that after fifth or sixth copulation, the number of unfertilized and dead eggs gradually increased, < which is not a good omen for breeding purposes. Serzes Jf. Let us now consider the effect of the age of the moths on fertilization. The worms used were of the Siamese cross-bred multivoltine yellow breed. Experiment a. WITH OLD FEMALES. Date of is | : . : | Tr - - ‘r of eggs, =e Number | emergence, Date | Duration | AGS Ss Weight of newly | of of of PP Tu al — p~ | hatched worms. “= oats oa nierti- | om Pairing. | 9 4 pairing. pairing. |/Hatched. ier. | Dead. | Total. per 100. | | oa ] ‘ 3 a) * ! | | | July | July | July | 30 | | | | / Bi 4th. | 5th. | 5th. minutes. |} © 4 215 | O | 215 '\\ a | | | fe) I ot | = 2 ” 9 ’ ” Oo | 78 i Oo ] 75 7S } | | a Lo. e er een Mae » Or 39) 2 O) SR S89r Ores =) } z AO dl bates Ys o 103 Ge 103 & | | | 5 | ” ” 93 ” Oo 31 j oO } 31 | Average. | o 93.2 OF i 98.2) | kK. Tovama. Number ot pairing. Date of | emergence. “ I = | 2 5 9 S) o 5 4 o a z > Average. to Vhird copulation, ' Average. = 2 = = = ota ee ae) ext j = - 4+ = 2) ‘ eo On | 2 / } | a } j | : 2? j | — ' j | 2 o: } ? 23 > 3 ” : | ” = | | oe ; | ” 2° | ” | | 7 ’ | ; : ‘s Average. fifth copulation. Average. ut July | July 6th. | 5th. } 2? > | | } : ts } } * : | | | EES Number of eggs =e = - Date Duration | Number of eggs. | Weight of newly of of \- = = ate | hatched worms. le alee / } ulerti- | i or pairing. | pe lee Dead. | Total. I g | pairing, peeeied: vcd: a | per 100. | ) | i | | July : 5th. t hour, || 380 9 II 4cor f, # % 286 5 II 302 | 3 | » 23 | 348 15 82 445 |\.0.032 g 33 3° 380 20 Ig 419 | | » ” ie) 43 o 43 | = a | 278.8 18.4 | 24.6 321.8 | | | || i ao ; ‘ BenoOursa ess 7 10 25 392 33 33 .e) 97 Oo 97 - é 307 3 20 420 io 033 gr. | * ; 361 3 27 391 | oO } » 139 13 6 158 = 250.8 25.2 15.6 291.6 | | zs 4 hours. 359 I3 19 Bw | : . 340 6 19 365 | i] . 302 17 9 328 |\.0.032 gr. 2» 59 360 13 J 380 | ! 3 : 342 24 34 400, 340.6 14.6 17.6 372.8 —| z : July 6th. 3798 10 37 425 , 396 5 51 452 | i , 386 13 Ss 407 |$0.033 gr. Es A 10 280 O 290 | | 29 33 i| 331 3I 27 389 1/ |} | i | 300 67.8 | 24.6 392.6 | Contributions to the Study of Silk-worms. Experiment WITH OLD MALES. b. The worms used for this experiment belonged to a between Japanese divoltine white and French univoltine *‘ Var.’ 131 cros-bred race a Date of | | | = P DiEBeS SIS co erconee | Date Deration | Number of eggs. | g : | Weight of sly £ | Weight of newly 18} | | of = | j | | |U en | | | Sa = = nferti- hatched worms. ° g. |\Hatched. Dex ota pairing. | & | pairing. | pairing | e | oe | Dead. | Total. | } } | | | / | = June | June} June I | 28th.| 26th.} 28th. | 6 hours. 413 4 6 423 — | i 2 | | 1a | 358 I 4 363 — | Cae: ma 415 3 2 420 = | Bea oie [se 3 429 fe) 2 431 — ! a 5 H Led ” 23 > 34 4 Oo SS ————d 6 ] | Ss } 2 2 3S 6 j . ! ” 397 Oo 3 400 eT a, ° he = 7 > | ” ” 2 409 oO 3 412 —— 2 = 5 = | 8 > s: ‘> 33 496 J I 500 | i | < 9 +E) | 2 2” 3 430 I 3S 440 ores 10 Br ” +3 427 Sy 5 438 tk é II PA 213 fe) 2 215 a n|) < ” Pe, ai oO £3) oy |. eee ae oo i | ' | mag > j | 2 or - | Average, | | 374 2 Du fe SA as a : man = i | | | une | June | June | I 28th. | 27th.| 28th. | 6 hours. 396 2 I 399 = 2 = %, I ax 2 = 4c6 2 fe) 408 — 3 ” oe hd * | 415 o + 419 == | il i} 2 : ps - + ” cy) ! 2? i 323 16 5 347 = a” 3 | | = | I a) 5 —| = ~ | 417 I 2 420 —— 2 3) 6 > 2 S 460 3 I 464 a= oe ro) 7 cy) ” 399 I 2 402 ——— >. = ois ane ‘ 8 ” ”? ” 351 20 2 asd ars 9 > >: > } 382 I 5 388 Ta 10 . ss > | 37 278 18) 0 278 —— II , + 7 , | 442 13 + 459 == Average. io) 132 kK. Toyama. : Date of % hs y i ears Number emergence. | Date Duration ean ae of of aie | | Ee | pees pairing, 2 | & | pairing. Number of eggs. | Weight of newly ! (Ginter | hatched worms. pairing. Hatched ea Dead. | Total. | re | | ized, | 1} | | | } | June |} June | june \ 1| 29th.| 26th.| 29th. | 6 hours. | 108 2A1 a3! |) are i ee = BZ irs 5) see a | 419 4 As 9) ee = = 3 | 4 | oO 36 oO ieee = Seas, 4 : 2 ? ” i} 366 73 | 23 i 462 j as = 9 | i| / i = = i| Ee Ms ? j ' = 5 i 5 | ’ 49 i 355 58 | 15 431 | ——— = | i . 6 Sy aaa : 340 16 10 | 366 | — | : | | : Bere | eae ; ; 473 9 55) -497 Soa z 1 | | | | Average. 307 62.4 10 380 — | | From experiment a, with old females, we see that the number of unfertilized eggs and especially of dead ones is much greater, when compared with the result obtained with new female and old male. (See series I and series II 3). Some analogous facts have been observed in Lepidoptera by Standfuss, ! who says that “female germs seem to be much more sensible to influences than the male.” We may say, therefore, that the process of fertilisation is affected by the condition of the female much more than by that of the male. Even with males which have been kept during three days, we can get a good result when paired with new healthy females ; but those which had been kept during four days did not give as good a result, as the table shows. Seses, IIT, In this series of experiments we have two cases; in the one case, the moths were paired during 30 minutes, and in the other, during four to six hours, which is a common Japanese custom. 1 Standfuss Synopsis etc. 190. Contributions to the Study of Silk-worms. 233 The result will show the effect of the duration of copulation upon fertilisation. The breed used for the experiment was a cross between Japanese and Siamese breeds. (A) ies j ] | Number Duration Total number | Number of Number of Number of of of hatched unfertilised of moth. copulatoin. eggs, worms. eggs. dead eggs. ———————— : ; 30 I minutes. 373 aii I I 2 : 345 331 7 7 3 340 32 6 ia) 4 385 350 zy, 6 5 , 393 293 3 7 6 A 513 311 2 ie) 7 372 366 : + : 385 378 5 2 S. 387 392 ~ 3 10 - 218 211 2 5 II iz 360 354 2 4 7 , 315 310 + I 13 ; 325 319 2 4 14 , 344 339 - 3 15 zs 352 350 I - Average. | 3453 334.8 2.8 3.6 lo (a5 4 k. Toyama. (A) at Number Duration Total number Number of Number of | Number of ot of hatched unfertilised of moth. copulation. eggs, worms. eggs. dead eggs. I 4 hours, 349 342 2 5 2 35 202 356 2 5 7 ; 128 127 I Oo 4 355 349 4 5 5 5 369 361 5 5 6 s 351 344 | 4 3 7 f 370 370 fe) fe) 8 365 354 5 6 9 » 355 339 8 5 10 29 353 345 I 7 It 29 315 397 3 5 i2 > 359 344 10 5 13 * 335 331 2 5 14 2 307 293 13 I 15 ; 249 247 I I : Bae : =| of = - Average, 328.6 310.6 4 3.9 * This does not represent the total number of the eggs laid by a moth, since some of the eggs were laid on the cell. Contributions to the Study of Silk-worms. i) io Ut a Number | Duration | Total number | Number Number of Number of of of of unfertilised of moth. copulation. | eggs. hatched worms. eggs, dead eggs. I 30 minutes. | 291 229 21 31 : : 246 Igo 29 27 3 * 204 255 3 6 4 > Pee 350 3 37 5 se 400 380 I 13 6 433 416 6 II 7 3 fo) oO oO fe) 8 414 382 2 30 9 : 373 342 3 25 IO ” ; 337 310 + 23 II 8 297 276 a I4 12 : 427 422 I 4 13 > 337 30! = 32 14 : 393 386 I 6 T5 : 355 351 o 7 if " 67 37 7 3 17 : 344 262 58 24 18 35 333 299 I 33 19 ; 319 253 5 or 20 , 204 250 5 29 21 : 332 285 fe) 37 22 a 124 107 | 10 / 7 23 ie 348 332 3 13 24 2 277 221 25 31 25 ; 381 341 | 29 Io 26 2 / 348 343 3 2 K. Toyama. wn @)) (B) le ee ET EEEEEEeene een | | | Number Duration | Total number Number Number of | Number of of of of unfertilised of - moth. copulation, eggs. hatched worms. eggs, | dead eggs. | | ie 27 30 minutes. A474 456 2 16 28 395 351 25 19 29 B70 375 ) I 30 » 391 390 oO I 31 , 417 390 6 21 3 > 344 322 5 17 33 , 246 215 3 25 34 : 356 335 7 11 35 , 324 295 7 19 2 | 36 8 fe) o o | fe) | 37 5 346 338 2 6 38 , 333 315 2 17 219) _ 263 194 44 25 40 ; 388 341 2 | 45 41 b 246 171 15 60 2 : 419 409 I 9 43 » | 338 | 319 | 2 17 | 44 + 308 | 270 12 17 45 105 | 75 22 8 if 46 a 286 230 31 25 47 4 366 275 24 67 45 5 405 | 396 3 60 49 377 363 5 9 | 50 | » 379 355 7 17 | | | 51 | a 286 | 209 66 | II 52 | bs | 351 | 261 12 | 78 : WW [_ 4 ae i 4 | | | | Average. 10.6 | 20 | Contributions to the Study of Siik-worms. 2 (B) Ze Number | Duration | Total number | Number Number of Number of of of of unfertilised of moth. copulation. eggs. hatched worms, | eggs. dead eggs. I 6 hours. fo) oO fo) fo) 2 . 326 319 2 5 3 % 360 357 I - 4 45 Sir 263 16 22 5 5 241 215 10 16 6 es 105 SI 21 3 7 a 50 fo) 50 o 8 53 358 298 15 45 9 5 fo) oO fo) oO 10 2 342 311 10 21 II ; 251 | 16 14 12 , oO oO Oo oO 13 ” 379 354 7 9 as ? 354 323 9 ie T5 33 351 348 3 o 16 : 346 335 3 9 17 oS fo) oO fo) oO 18 . 413 391 10 12 19 2s 323 fe) 322 oO 20 0 373 357 3 13 21 ” 391 319 57 15 22 sf 342 335 ce) 7 23 4 107 oO 107 o 24 | : 378 372 ° 6 25 5 168 148 17 | 4 26 : 371 337 | o 34 3 K. Toyama. lo a5 yx. (B) > rr Number Duration Total number Number Number of | Number of of of of unfertilised | of moth. copulation. eggs. hatched worms, eggs. | dead eggs. : peinesel 2 <3 ae = =a | | ae 6 hours. oO fe) oO fo) 28 33 oO fo) oO oO 29 Bs ra) oO oO | oO 30 360 344 7 9 21 i 152 46 838 18 32 : 275 227 26 | 22 33 , 371 351 3 | 17 34 5 469 456 2 II 35 A 21 oO 21 | Oo 36 , 315 273 | 19 | 23 37 398 358 3 37 38 ” fo) fe) Oo fo) 39 , 367 333 6 28 40 | 2 42 Oo 42} (a) AI 5 409 | 396 I 12 2 > 379 352 3 24 43 , 230 212 13 5 44 : 355 233 41 3! 45 ; 256 225 17 14 46 + 416 397 I 15 47 332 | 324 + 4 48 379 | 363 I 15 | 49 - 291 | 276 13 2 50 320 | 297 I .- 22 5! s 196 190 2 4 | | } 52 : 369 | 342 10 17 Average, 256 | 225 19.3 11.3 Contributions to the Study of Silk-worms. 239 Both series gave nearly identical results. The results of the preceding series of experiments also support this fact. It seems that the significance of the duration of copulation has been overrated. It is, however, to be borne in mind that if the copulation is too short, it sometimes occurs that all the eggs laid are unfertilised, most probably on purely mechanical grounds. Then the question naturally arises: What is the proper duration? The next series of experiment will give an answer to it. Series IV. In this series of experiment, a single white female was made to pair with two males, one white and the other yellow, the duration of the copulation being different in each case. As the white eggs fertilised by the white male will produce white worms while those fertilised by the yellow, yellow worms, the proportion of these two kinds of worms in the same batch will determine the proper duration of the pairing. The worms used for the experiments were a cross between the Japanese and Siamese yellow races. : Kind of & and duration of copulation. | Number of worms resulted. Number of anna = a nee tmale: | Ist pairing. | Second pairing. White. | Yellow. | I 30 minutes by yellow.) five hours by white. | 167 129 | | | a, 2 do. | do, | 359 Se =) | | 2 | op | do. do. | 146 50 z | | Ae! | = 4 do | do. 230 60 } | | 5 do. | do. 296 43 Average. | 239.6 56.4 240 K. Toyama. Seen mmmmmmmmmmmmmmmmmmmmmmmmmmmnem ae ¢ Kind of & and duration of copulation. Number of worms resulted. Number of are — = SS white female. é Bie < ar ay ts i Ist pairing. Second pairing. White. Yellow. sees Ll = 6 one hour by yellow. | four hours by white. 2 | 161 = 7 do, do, OI 2311 = io 8) - ra S ao. ! do, B22 IIo 3 9 do. do, 217 47 Ip. , Io do. do, 200 120 Average. 188.6 133.8 855 9 9 | | | | | | | | | | } 3 _ ‘ : 375 ' |) “ao |) Mio 782 | | | | | | | | \ | 4 5) = is 1,160 20 20 552 { | | | | | , (ee | | a Bs | ue , 1,116 i205) 12 | 600 ors | | | Ai | | | The worms used for the experiment were of the Siame Contributions to the Study of Silk-worms. Wes s:- third “ S Ps 7 4. fourth 3 Ayo the rearing. Difierence between the dry and wet bulbs. Perens ide 6 eee saz eS eaee. eee omy ou fhe: See eee ea ee pein ree fe tt 5S). saa dace Dee AGG: every group. Weight Weight of worms just after, Weight a cae f | fresh- se aie hatched eee | First Second | Third | Fourth | Matured| worms | PSF 5°: | moulting. moulting.) moulting.| moulting. ones. | per 100. | | } — | gram, gram, gram. gram. gram, gram. gram. 30.32 0.108 0.400 1.80 8.60 46.24 0.032 29.59 O.1C7 0.419 1.82 8.41 45-43 0.0315 | | | 30.60 0.107 0.416 1.85 8.03 45.97 0.0325 | | 29.56 0.102 0.419 1.84 $8.05 45.23 — | | } | ' | 29.71 a — —_—- — —— — — se multivoltine yellow race. i) QO 244 K. Toyama. As far as our experiments went, we could not find any deffinite difference between the offspring of the first copulation and later copulations. Let us next see the habit of some wild silk-worms. Theophila mandarina, m. which is considered to be one of the nearest allies! of the domesticated silk-worm is quite polygamous. The eggs laid, however, are well fertilised. A similar case is found in Azthcraca Yamamai, but in this species copulation lasts longer than in 7keophila. When reared Cricura trifenestrata of Burmah in 1903, we observed that during the night a male visited many females ; yet all the eggs laid by them are well fertilised. Thus we may say that polygamy is a normal habit in some wild silk- worms. There is an opinion that if the copulation is not sufficiently long the eggs will be imperfectly fertilised. This is the cause of the custom of preventing the polygamous habit of the domesticated silk-worm. On this subject, there is nothing better than to quote Prof. Weismann’s statement? which will give a final decision to the matter. He says: ‘‘ nowadays we are no longer justified in using such an expression as imperfect fertilisation. Whenever a living spermatozoon enters an egg, the latter becomes fertilised ; and an imperfect fertilisation could only be supposed to occur if the sperma- tozoon is abnormal—if, for instance, it contains too few idants.” Henking? observed cases in the silk-worm where several spermatozoa entered the egg, yet only one of them united with the egg-nuclens while the others degenerated. Thus we may safely determine the duration of copulation by the number of unfertilised eggs. When there are no unfertilised eggs or very few of them, we may conclude that the duration has been snfficient. 1 Concerning this point, Prof. Sasaki has published valuable observations. Annotations zoologicae ‘ Jap. Vol. II. Pars If. 2 Weismann’s Germ-plasm. 1893. 3 Henking—Untersuchungen tiber die ersten Entwicklungsvorginge in den Eiern der Insekten. 1892. Contributions to the Study of Silk-worms. ' ons Summary and Conclusion. Taking into account all the facts and considerations above referred to, it seems that there is no escape from the following conclusion, with which we will bring our discussion to a close. 1. Polygamy is a normal habit of the silk-worm; even when a male pairs with eight or more females in two or three days fertilisation is complete. bo Fertilization is much more influenced by the condition of the females. 3. For healthy males and females, copulation for thirty minutes may be considered sufficient. For practical purposes, however, it is advisable to allow two or three hours to avoid mechanical disturbances. 4. Where good pairs of moths can not be obtained, a male may be allowed to copulate twice or more according to the circumstances. If the males are more numerous than the females the former may be keep over until the next day when new females may emerge. To keep males in a healthy state, they should be removed from the cocoon baskets or trays before they have smelted the scent of the alluring glands. When once stimulated, they become much excited, fluttering their wings and walking about until they become nearly exhausted. Even a distance of two or three meters on the leeward side is not sufficient to prevent the oder from reaching the males. To keep the males in good order, there- fore, strict care should be taken to avoid the odor of the alluring glands. If the above precaution is carefully attended to, the males can be kept quietly lying in a dark cool place without injury to their health. a See SY Contributions to the Study of Silk-worms. Ill. On the parasitic fly of the domesticated silk- worms of Siam. BY K. Toyama. _ (With plate V.) During my stay in Siam, I had an opportunity of studying that pernicious tachina-fly, the larvae of which are parasitic in the body of the silk-worm, and make terrible havocs among them. In the following pages I shall give an account of the investigations made in Siam. the fegemeios. lI) Ti). The egg is laid on the skin of the silk-worm. It is milky white and is long and slender, cylindrical, and slightly tapering anteriorly. The ventral side with which it attaches itself to the skin of the worm is flat and membféanous, while the dorsal surface is convex, and the chorion is marked out into characteristic polygonal areas. Its average Size is 0.5 to 0.57 mm. by 0.2 to 0.22 mm. From its first deposition until hatching no change of color takes place. *. The larva. (Figs. IV, V, VI, VIL XD. The larva or young maggot emerges from the egg through a hole (Figs. IV, V, 0’) made in the chorion on the anterior portion of the ventral side of the egg, and makes its way into the body of the worm on which the egg was laid. At the same time, a round pore (Figs. IV, V, 0) is formed on the anterior portion of the dorsal side of the egg. It probably serves as a breathing pore for the developing maggot. 245 K. Toyama. Fig. IV represents a surface view of an egg and its maggot, the latter lying under the skin of a worm. In the anterior portion of the egg we see a round hole (0), under which we again see another depressed hole (0’). The latter is the opening on the ventral side of the egg, through which the young larva has passed into the body of the worm. A little in front of the. ege is seen the larva through the skin of the worm. Ina living specimen, it is seen as a translucent spot on the skin of the silk-worm. At this stage, the size of the young larva or maggot is ;°,; mm. long. A longitudinal section of an egg and maggot in a similar stage is shown in Fig. V. E. is the egg attached to the skin. On the right side of the figure we see clearly the dorsal (0) and the ventral (0’) holes already described in the surface view. The ventral hole is continued into a sac made partly of the cuticle and partly of the hypodermal epithelium of the host, in which the young maggot (m) lies. The wall of the sac near the opening is colored dark. This color gradually becomes more intense, and at last a black patch is produced in the skin of the host (Fig. II), which may be depended on as a certain guide to the presence of this parasite in the body of the silk-worm. The shape, size and position of this spot or patch are not constant. Some of them are nearly round, some pear-shaped while still others are irregular in form. It is a common fact, however, that in its middle or periphery there is a small round pore with rough edge, a little elevated above its surroundings. Rarely we meet with a vermiform patch, with a hole at either end. Sucha one is probably produced by two eggs laid side by side. The size of the patch is generally 1-1.5 mm. in diameter, but sometimes larger. A vermiform one may be 3 mm. long and 0.36 to 0.4 mm. broad. When the maggot gets within the body of the host, numerous large migrating cells may be observed near the hypodermal epithelium constituting a portion of the sac in whieh the maggot lies. They gradually arrange themselves around the wall of the sac and contribute to its enlargement, which is necessary for the growing maggot. Fig. V1 represents a maggot 3mm. long and 1mm. or a little more broad. It is enclosed within a fibrous sac (in the figure a portion of the sac is broken to show the maggot) with a black posterior end. With this Contributions to the Study of Silk-worms. 249 black end, it is firmly attached to the dermal cuticle and hangs down in the body cavity of the host, supported by some adipose tissue, tracheae etc. When the maggot has nearly attained its maturity, it gets free from the sac and passes into the body cavity of the worm. Then it makes a hole through the skin of the host, and keeps its posterior end always close to the hole, evidently in order to have a free access to the air. In the mean time, the infested portion of the body of the host gradually undergoes disintegration, while the other parts still remain alive. Such half disintegrating worms are often meet with in a culture. The number of maggots which may become full grown whithin the body of a worm is not constant, sometimes one, sometimes itwo or three or more. When the parasite becomes quite mature, it pierces through the skin of the host and leaves it in the form commonly called maggot. The full-grown larva or maggot. (Fig. XI). The adult maggot is yellowish white in color and cylindrical in form. It tapers anteriorly while posteriorly it is somewhat broad and abruptly truncated. Its size is 9-10 mm. long and 3.5-4 mm. broad. The body is composed of twelve segments, each of which is provided in its anterior portion with several transverse rows of minute brown setae. At the anterior end of the first segment (Fig. VII A.) a little ventrally there are two pairs of processes, and from the ventral side a pair of black mandibles project, near the base of which we again notice a small tubular process on either side. On the lateral line of the posterior edge of the second segment (Fig. VII C.) we meet with a process, on the apex of which open three round spiracles (Fig. IX A.). Each spiracles (Fig. IX B.) is surrounded by a thin chitinous ring of a brown color. Its diameter is 0.02 to 0.024 mm. On the posterior edge of the fifth segment on the same lateral line on which the spiracles of the second segment are situated, there is a round hole or spiracle (Fig. VIII). It is larger than those of the second segment and 250 K. Toyama. has the average diameter of 0.ogmm. Around this hole there are arranged many rows of setae. The anus (Fig. VII B.) opens on the ventral median line of the eleventh segment, and is surrounded by regular rows of setae. On the truncated end of the last segment we see two large spiracles as black spots. Under the microscope, they are not simple spots, but have a complicated structure. Fig. X represents the central portion of the trun- cated end of the last segment in which the two spiracles are situated. Each spiracle, as the figure shows, consists of a black chitinous disc, on which three vermiform thin portions having a complicated structure are present. In the ventral portion of the disc we find a round membranous space with a pore. As the maggot makes its exit from the body of the host or from the cocoon, it immediately seeks a dark place, and especially a moist ground by getting down through some cracks or fissures in the floor of the house where it comes out, and in about to hours it begins to pupate, regardless of whether it has found a good sheltered place or not. Thus after some hours, we find it as a cylindrical puparium tinged with dark brown. The puparium. (Vig. XI). The puparium is of cylindrical form rounded at both ends, and is dark brown to nearly dark in color. Its average size is 7.17 mm. by 3. mm. The same number of body-segments as in the larval stage may be observed, the first being much contracted. The stigmata on the second and fifth segments may be seen as black spots. On the ventral median line of the 11th segment the anus can be seen as a slightly elevated and pigmented area. On the end of the last segment we see those conspicuous spiracles as in the larva. The Imago or fly. (Figs. XI, XIV). A bristly greyish fly of a moderate size.! The average length of the body and the expansion of the wings are : in the female 10. mm. and 18 mm. and in the male 11.5 mm. and 20. mm. respectively. 1 The size of larva, puparium and imago differ according to the size of the host. Contributions to the Study of Silk-worms. 251 The head is somewhat triangular and bears large compound eyes which are dark reddish brown in color. In some specimens we may observe some short hairs on the surface of the eyes while in others they are wholly absent. The space situated between the compound eyes and the mouth parts is provided with sonie black bristles and is silver white in color. Dorsally the space gradually becomes of a light brownish shade. Three simple eyes of a reddish brown color are situated on the dorsal part of the head, making a triangle with one of the angles directed toward the front. The antenna (Fig. XVI) is club-shaped and greyish black in color. It consists of three segments, the terminal one, which is the largest and a little depressed, bears the dorsal bristle, about twice as long as the segment itself. It is two-jointed ; the proximal is very short and the distal is long, tapering to a whip-like shape. The segment and the dorsal bristle are covered with very fine hairs. In the second segment of the antenna we notice some short bristles on its dorsal portion. The manillary palpus is club-shaped, reddish brown in color, and covered with some black bristles. The dorsal surface of the thorax is brownish-grey and is marked with black stripes. On the proescutum we notice four stripes which extend to the scutum, the middle two terminating at the middle of the scutum, while the other two nearly reach the posterior edge of the scutum. Besides these stripes we may observe a median black stripe running through the scumtum ge. This marking is wanting on the from its anterior to the posterior ed preescutum. The spaces situated between those stripes are covered each with a single row of black bristles. The scutellum is nearly hemispherical. Its anterior portion has the same color as that of the preceding segment, while posteriorly it gradually assumes a brown tint, and is also provided with some bristles. Under the magnifier we can observe very fine black hairs on the whole surface of the thorax, besides those enumerated above. The wing (Fig. XV) is light greyish and transparent. The shape and the venation are represented in the figure. The costal vein bears short, black hairs on its frontal edge. Near the basal portion of the third longitudinal lo Wat i) k. Toyama. vein, we again observe a row of some short black hairs on the vein. The first hind-marginal cell becomes narrowed at the margin near the apex. In the hind angle of the first marginal cell, there is an elongation of the fourth longitudinal vein, but it is not well developed. The fifth longitudinal vein does not reach the margin; near the hind transverse vein it stops and the remaining portion of the vein is represented in an imperfect way. The 6th and 7th longitudinal veins also do not attain their full development except in the basal portion. The posterior lobe is well developed. The allulae are greyish white. The legs are of moderate size, and are provided with black bristles ; the femur is dark-grey, and the rest black. The pulvilli are membranous and are tinged with brown, The abdomen.1 The abdomen is long oval and is slightly shorter in the female than in the male. Four segments can be seen from above or below. The first is dark colored, while each of the rest is whitish-grey in its anterior portion and black in the posterior half, thus producing transverse striped markings on the abdomen. The entire surface of the abdomen is covered with small hairs. Those on the dorsal median line and on the posterior portion of the third and the last segments are long and stout. The ventral side of the abdomen is greyish-black and is provided with black hairs. Habit of fly and tts life history. The fly is very active in the magnanerie. When it frequents the worm- baskets, it does not stay in one place, but always flies from one worm or place to another, and cleverly finds some shade, such as is afforded by the leaves or the worms. Especially, when it is disturbed it soon hides itself in a shady corner. Even when a basket-frame was covered with a mosquito- net to prevent the flies, some were found after some time to have cleverly got in through some slit that may have been left. When it visits a worm, it bends its abdomen and stretches out the ovipositor, and no sooner than the end of the ovipositor touches the body 1 In some specimens we observe a brownish patch extending between the second and the third segments, on both sides of the body. bo al On. Contributions to the Study of Silk-worms. of the worm, than a white egg is laid there, and the fly then goes away to an other place or worm. Sometimes it stays on a worm and lays many eggs. It freely lays eggs in confinement, doing so even when it is put together with some worms in a large reagent bottle. If we put a fly together with two sets worms, one beginning to moult and the other just after moulting in a bottle, the latter is invariably first attacked by the fly. After visiting most of the second set of worms and laying eggs on them, it haunts the first set of worms, the skin of which is much more extended and harder than that of the former. In these worms, the eggs laid will be cast off before hatching together with the old skin. ~ It must also be mentioned that in the former cases, the fly lays eggs without any hesitation while in the latter case it takes a long time to lay an egg, moving its body many times. Hence it seems that it prefers softer skinned worms to lay eggs on. In the following, we give a note of the rearing of the parasitic fly :— Sept. Lith, 1.30 P.M. Oviposited on six worms. ea i2th, 1.30 P.M: No change has occurred. jae rath, FO A.M. All eggs hatched. We can see on each worm one or two small translucent spots on the skin, near the anterior end of the egg. (This section is repre- sented in Fig. V). 16th, 4 P.M. Those translucent spots have become black. Average size Imm. by 0.5mm. Some of the spots are round, some elliptical, most of them long pear- shaped. (Fig. II). Seth: Of the six worms, two are nearly dead. On one of these there is in the ninth segment a hole 1 mm. in diameter, through which the posterior end of the maggot may ‘be seen, all the segments from the 7th to the last have become rotten, while the anterior segments are quite healthy. Three other worms have nearly lost its apetite and become very sluggish. Sometimes they lift up their heads and bend the body considerably, as if suffering from pain. 254 K. Toyama. Sept. 18th, 1 P.M. All the worms nearly dead. From some of them maggots are coming out. a 13 3D Eee Maggots have begun to pupate. Toward evening they have gradually become light brown. 7... FQth. In the early morning, we got two maggots. - », 10.30 A.M. Their color has begun to change. = eee. Become dark brown. Oct. 29th. Since a few days, the color of the puparium has become considerably darker, and some flies emerged during the preceding night, and all the rest emerged by the next morning. Hence we may say that 1. The egg stage lasts about 30 to 40 hours ; parasitic life about 7 days. lo The larval stage lasts | free lite about 10 hours. 3. The pupal stage }, about 10 days. Total duration of a generation about 18 to Ig days. In the coldest season, viz. December or January, the pupal stage lasts longer, that is to say, two to three weeks according to circumstances, and consequently the total duration of a generation is increased to about 25-30 days. The fly is polyvoltine. According to our experience, at least 8 or 9 generations are gone through in a year. Oviposition takes place not only on the silk-worms, but on many other wild worms. Hence, when we first reared some silk-worms in Bangkok, where for some 50 or 60 miles around people have no custom to rear the worms we suffered from the attack of this fly. The puparium may be transported together with the cocoons or some implements to considerable distances. We have experienced such cases during our collection of cocoons from various districts of Siam. It is also an interesting fact that in the magnanerie belonging to the Royal Siamese Sericultural Department and situated at a locality surrounded on all sides by paddy fields, and at least 300-400 meters removed from any jungles or bushes, where the flies may be expected to haunt, no attack of the i) wt vi Contributions to the Study of Silk-worms. fly has been experienced during two years of our experiments, except once when the infection was supposed to have been due to a large importation of cocoons from Korat. This may serve to show that the flies do not wander to distant places. Distribution of the fly. This fly is widely distributed throughout the whole Indo-Chinese peninsula. In Siam, we find it in all districts where sericulture is practised. Thus, on the east we find it in great abundance in Korat plateau which is the principal silk-producing district in Siam, extending between about 14° 5 and 18° N.L. The worm-raisers of the district have suffered greatly from the injury of the flies. During my inspection there, I often met with cases where some 70 or 80% of a whole lot of worms reared by the natives were injured by the parasite. On the Cambodian frontier and also in Cambodia, we have been told by some Chinese residents there that a similar disease producing black spots on the body of the worm prevails there. In Muntong Rachaburi, the district situated along the mountain range separating Siam from Burmah we have observed the flies haunting the native huts where silk-worms were being reared. Even in the northern mountainous districts extending between 17° and 20° N. L., such as Muntong Pitsanulok, worms can not escape the attack of this fly. We are told that in Annam, a similar disease occurs among the worms reared by the natives. In Canton and Southern China, Chinese books! often refer to some flies doing injury to the worms. We have not yet in our hands specimens of these flies, yet we have good reason to believe that they certainly belong to the same species as that of Siam, since it is also found in northern China, where it frequents the magnanerie and deposits eggs on the silk-worms. _ Prof. Sasaki? identifies it as Zachina rustica L.,to which he also refers the flies we have obtained in Siam. 1 Chifushinsho. 2 On the parasitic fly on silk-worms in China, 1899, and Text-book of sericulture. 1905. 256 kK. Toyama. In 1903, November, I received, through the kindness of H.R.H. Prince Devawongse, Minister of Foreign Affairs in Siam and H. E. Phya Surisan- thorn, Undersecretary for the Ministry of Agriculture in Siam, samples of living cocoons produced in Burmah, from which we obtained some parasitic flies belonging to the above species. Now we may arrive at the conclusion that the distribution of this tachina-fly is very wide, extending from northern China to Burmah, including the whole Indo-Chinese peninsula. I am much inclined to believe that in India the same species exists besides that well-known parasite, Oestraea Bengalensts. Some methods for the prevention. The Siamese generally wrap some cotton cloth around each worm- basket. The Chinese, on the other hand, use mosquito-netted windows or doors to prevent the coming in of the fly to the room where worms are reared. The latter is one of the best methods, yet to do this we must take particular care to close up the slits in the floors, windows, doors etc. as carefully as possible. As already described, they cleverly come in through small fissures or slits to the room where worms are reared, so it is highly necessary to pay special attention to this habit. Even when we put a fine wire-gauzed cover on a basket, they cleverly creep in through some small slits between the cover and the basket. An indirect means of prevention is to kill the maggots, puparium and if possible the flies when we meet with them anywhere. December, 1905. Zoological Institute, College of Agriculture, Tokyo Imperial University. ies Mf. Fig. ep fi TE. ai, V. ov 1: a WI, a. WAIL. ge. Contributions to the Study of Silk-worms. i) a NI Explanation of plate V. Silk-worm laid with eggs of the fly ; on the third, six and ninth segments we see white eggs. 2x. Infected silk-worm. Dark patch on the 5th segment. Nat. SIZe. Eggs of the fly. x ‘faa zeiss. Surface view of an egg and a hatched larva or maggot. L., larva; E., egg; O., dorsal opening and O’, ventral opening ofthe egg. x '/aazeiss. Left two figures x !/B zeiss. Longitudinal section through an egg and a hatched larva together with the skin of the worm. x 3/B zeiss. E., egg; ch., chorion; V., vitelline membrane; O., dorsal pore; O’, ventral pore; G., gummy substance with which the egg is attached to the skin; C., cuticle; H., hypodermal epithelium ; M., young maggot. Young maggot enclosed within a sac, a portion of the sac is broken to show the maggot. Portion of a maggot. Much magnified. A., first segment bearing four pairs of appendages ; B., anus ; C., stigmata on the second segment. Stigma on the fifth segment. x 3/p zeiss. Stigmata on the second segment. A., entire view. x 3/pzeiss. B. two stigmata. x 3/D zeiss. Truncated end of the Iast segment of a maggot. Two stigma are represented. x 3/aa zeiss. Matured maggot. Ventral view Nat. size. Puparium, dorsal view. Nat. size. Imago 2... 2x2 Frontal view of the head of female fly. 2x. Left wing of Imago. Antenna of imago or fly. x 1!/aa zeiss. : > Ww) f . : * "e vj ine . re ha | u j bs ” & #e ! 4 7 Pine wid od ‘ocl oii ae > cee iio. trsnogse ley tsi wow eh aa . ort teh lial — aa be a ‘oyna 4c ee ne “> a / get . larttes's. tl) gia dias ete. ka <9), 28 0) lan . pad, (bly ea) AOI os (yh, ieypt, 4 3 1 diaertiielk & ito JAS ie Until He é ote i Tie nv he Larmts AY ont tee Mite nite SSA toal a atl y oye (Ay rhe 3 stn che ' ry “100 Sag + wie seine Jou gies les eet * enor aa ; RU oe ue ak. | a -witigtergese Dr: ae re 7 * i oe of ; ie r= - ‘ei a” n 4 yg ys E nto Whee Ly RoR: yt rik) (Pies regi Lh ee -. - < 7 “sz Ven . P a = : ‘ a : oe . t @ Ae ) MT Rca AeR ARERR. wc ores ia ; ; 2, p> pelea ute Helio tiet TUARE ! ¥ , Fi Kyuss hinw [ys iol) itl =e : S ? lo wiv Lalgnhs > esasrtd lp > ‘r ’ nana » > 7g a i iy = a = Ry, wee ‘ B a ia Z AI 4 i3 Studies on the Hybridology of Insects. I. On some silkworm crosses, with special reference to Mendel’s law of heredity. BY K. Toyama. (With plate VI—XTI.) CONTENTS. Page. MepREe Gbe fCHIArhS Ys) ee a ee wt VR ROE es ae A. New observations. Experiment Case I. Crosses between Siamese yellow and white breeds ... ... 264—280 seerone wn. Vhe first scncramom! ~-.. 2... 20% ee he ee 4: Bay oi «seconds. ee ee Ce oe eee 5 Ger 5e = third . OS, Pe el) a a re i} oa fourth 2 pap RP 2S ee aa, ae eae $: Bet 434 ith EA Et 20 SE Cee Oe ea ‘ Bae: oe secth se Bh poh 2s Gee ere eats ‘ Gi. ~seventh: = ha ee OR. ae ee ss EH: iy eleithy \ on Saleh ears MM relay” a umemolce gs E > minth: ss ae ad 218 Mr Tiel eos Hees a Summary PAR oR cA. nie mn nl Or ae TO beta abe a7 18 Experiment Case II. Crosses between Japanese annular “whites”? and Siamese munivoltne: “yenows: seme tes... LS ek oe ee Section 1. The first generation Bis) si: 228 Dest. 7 eee eee 4 as, Second. —e Be 3. | EE Se eens a |, Chird a Sea A NE Mee ee ee y ai? > fourth rf Ac cit col) ee eee ee eee 260 K. Toyama. Page. Section 5. The fifth generation j=. bes 2 *tn py ee 6. 3 (SESE “ nee wee we ee SUA tY_- op pon ep we Le eee Experiment Case III. Crosses between Japanese white and Siamese yellow breeds... ere eww. wk) ses See Section 1. The first generation ee a » second "2 Perr 3 2. ae Enea - LOU 2. ee Summary eet ree Experiment Case IV. Back-crossing a cross-bred yellow form with one of their parent-breeds 3.. “20a -:. =. ---: -+- at nepeyy. eee Section 1. The first generation ... «th Gos opety ites eeieeeeeenae * 2° |, second’ Be Treen ee Summary a mere bE Experiment Case V. Crosses between striped and “pale” breeds... .:. ..4) --. 326332 Section 1. The first generation wae’ sae Tpeiedy =2a0 Seen een Ee 2. » second = 2 Pee 5 2. So ethird Be PE Es ree ee Summary es kl ke a re Experiment Case VI. Crosses between pale breed spinning yellow cocoons and Striped breed Spine @yliteteacoons... ... fyss, =: 12 gee Section 1. The first generation Lec nde) nee dee, eee - 2; 3, $s8coudeaer, ws. age bee [llkoee ge ere 2 te 33 beat 3 o-- ceachliystel, eae syy tek een = 4. -,, > fourth and further generations, , «2. «j4!. 2h) SumMaLy..... 4. - >See EE ss cep eae ee =: Experiment Case VII. Crosses between Japanese common marked and Siamese striped breeds) Gaemeneeeener— =. 2 tg he ee ae ae ss > v7 Studies on the Hybridology of Insects. Page. Section 1. The first generation 348 ns 2. 55... SECON a memes 349 = ge) 4, athe ae x 4. » fourth 351 SIMMIary ss. 1.2 f:: 352 Experiment Case VIII. On a mosaic form derived from a cross between Japanese common marked white and European striped yellow breeds ... 53a oe Experiment Case IX. On the color of eggs 358—359 Experiment Case X. On the cocoon 359—304 Summary and general consideration. 1. Monohybrid 365 2. Back-crossing a cross-bred form with one of the parent-forms. 369 Ae fcr ei ee er 371 4. A modified case of dihybrids 374 5. Another case of dihybrids ... 277 6. Non-Mendelian characters 380 7. Summary ... 386 262 K. Toyama. INTRODUCTORY REMARKS. The present subject was started in the spring of 1900 when I reared various breeds of silk-worms for a comparative study in the Zoological Institute, College of Agriculture, Tokyo Imperial University. Since then I have continued this work in the Laboratory of the Royal Siamese Sericulture Department in Bangkok where I stayed from 1902 to 1905. A part of my work is now finished and the results arrived at seem to be not without interest. In the following pages I will try to give an account of them. In the spring of 1900, we made reciprocal crosses between a Japanese divoltine white race and a French univoltine yellow race called “ Var”, both of which races have ‘bred true since I first got them in 1885. The crosses thus raised, amounting to 2,300 heads in the case of “ white 2 + yellow @” and 968 in the case of “ white @ + yellow 9,” spun yellow cocoons without any exception. In the next generation paired zzter se, however, they displayed the white character in the following proportion :— Total number of worms reared Number of yellow cocoons. Number of white cocoons. from one parent. 118 89 29 (75-39% (24.696 On the contrary, when we crossed the female of the first cross gene- ration or mongrel-yellow form with the male of the pure white breed, one of their parent-races, there were produced two kinds of worms, yellow and white cocoon-spinners, their respective proportion being as follows :— Studies on the Hybridology of Insects. 263 No. of parentage. | Number of white worms. | Number of yellow worms. Total. (48.97% (51.0376) 1 143 149 291 (46.47% (53-54%) 2 216 249 465 (47-42% (52.57%) Average. 179.5 199 378.5 It is a proved fact that the colour of the abdominal legs of the caterpillar always corresponds to that of the cocoon it will spin ; so that, by observing these, we can exactly tell of what colour the cocoons will be. To avoid the loss of worms incidental to rearing the countings were mostly made during the larval stage, but sometimes also by the cocoons. Thus we see that on crossing a white and a yellow race, the offspring raised in the first generation exhibit only the yellow character, which in the next generation appears to split up into their parent-characters, the white and the yellow, according to a definite law. The following series of experiments will prove whether there is such a general law or not. 204 K. Toyama. A. NEW OBSERVATIONS. fomor., I. Crosses between Siamese white and yellow races. SECTION A. The first gencration. The silk-worm generally reared by the Laos in Siam is one of the multi- voltine races and may be divided into two breeds, one spinning white cocoons,! the other yellow cocoons. We have reared these two breeds for five generations without obtaining even a single different form. Then, we crossed the yellow females with the white males. The worms derived from each parent being reared in a separate basket gave the following results :— aes Date of laying Date of Date of Number of Color of Parentage.? e SIS = eggs. hatching. * mounting. cocoons, cocoons. May May June I 25-26 16 17-18 72 all yellow. 2 ~ 69 do 3 Ss 17-19 65 do 4 ~ = 75 do 5 i 17-18 79 do 6 ; 70 do Total. | | 400 do. Among the four hundred cocoons spun by the worms reared from these six parents, there was not a single white cocoon; that is to say, the yellow character predominates over the white. SECTION B. The second generation. The moths of the first generation emerged on the 26th and 27th June, 1 These are not pure white, but rather greenish white. 2 Throughout this paper each number represents all the eggs laid by a single parent. Studies on the Hybridology of Insects. 265 and the mating was made to take place between the offspring of the same parent. Each moth was made to deposit her eggs in a separate cell, as in the last case. We selected the eggs of four moths each from the offspring of those six parents or groups in the first generation, and reared them in the same D DS way as in the last generation. The following table gives the results obtained in this generation. Number of worms. Date of = Parent- Pees: laying Baa 10 date of Gocenae age. = eos hatching. F ] mounting ene White. Yellow. Total. June July | (27-77%) July I 26th 5th 90 239 329 28-29th | white and yellow. (30.60%) 2 o es 71 161 222 do. : (26.13%) 3 ” ” 75 212 287 do, (25.52%) 4 ” or) 85 248 333 55 do, (27.29%) | (72.70%) Total. 321 855 1,176 do. (22.22%) July 5 > » 64 224 288 28-30th do (27.45% 6 3 $5 oe 74 102 do 2 (21.53%) 7 9 x 6 204 260 do, (23.07% 8 » i 51 170 221 = do (22.84%) | (77-15%) Total. 199 672 871 do. (22.25%) 9 ” ” 77 3 269 346 3 do, (27.45% 10 + e 84°" 222 306 sf do. 3 (26.45%) II 9 3 CH] 214 291 = do. (24.05%) 12 ” 95 57 180 237 a do. (25.26) | (75-9) Total. 295 885 1,180 do. arn Neem Ce a NS SS 266 K. Toyama. Date of hatching. July 5th . Date of Parent- eee es a5e ggs aymng ay eggs. June 13 26th 14 aS 4 15 ” 16 x Total. 17 am 18 5 19 3” 20 3) Total. 21 3 22 = 6 23 a 24 3° Total, Grand total. Number of worms. White. Yeliow. Date of mounting. (28.51%) 67 (22.09%) 57 jeg ) (26.87%) 7 93 209 253 July 28-30th (25.20%) 280 (74.79%) 831 I,1II oe ) (22.5696 65 (26.54%) 90 (21.64%) 50 v7 (24.32%) 289 (75-67%) 899 1,188 (27.68% 67 (27.07%) 75 (25.50%) 63 (23.29%) 65 (25.17%) 1,654 (74.83% 4,917 Cocoons. white and yellow. do. do. do, do, do. The total number of worms derived from these twenty-four parents, as the table shows, was 6,571 of which 4,917 (74.82%) were yellow worms and 1,654 (25.17%) white worms, the former spinning yellow cocoons and the latter white cocoons. In single cases, however, the proportion of white and yellow worms varied from 30: 70% to 21: 79%. Studies on the Hybridology of Insects. 267 ' Thus, we clearly see that each parent produced offspring, some (about 25%) with white or grandfather’s character and others (about 75%) with yellow _or character grandmother’s. And, therefore we may say that in this case a simple reversion to their grandparents took place. SECTION | C. The third generation. The moths of the second generation came forth on the 6th and 8th August and those with similar characters were allowed to couple amongst themselves. Thus we have procured two kinds of eggs, one being laid by white cocoon-spinners and the other by yellow cocoon-spinners, and reared them by the same method as in the preceding generations. The hereditary phenomena displayed by these worms can be seen from the following table :— (A) Offspring of the white cocoon-spinners. Number of worms. Parent- Sa Date of Date of Fon ae a e88s- = ae White. | Yellow. Total. eo j Aug. Aug. Sept. I 7th 17th 301 fo) 301 I0-11th all white. 3 3 3 302 oO 302 , do 5 : 9 296 fo) 296 do 7 2 232 fe) 232 do 9 ; 5 ZTE fo) 271 do II > PA 195 fe) 195 do 13 : x 348 fo) 348 ; do. 15 a - 280 fo) 280 do 17 3 : 293 oO 293 ; do 19 » » 259 0 | 259 do 21 39 310 fe) 310 do 23 : 4 205 fe) 205 ae do, Total, 3,292 o 3,292 do, * Throughout this paper those marked with an asterisk show that some of the offspring are used in the next experiment. 268 K. Toyama. (B) Offspring of the yellow cocoon-spinners. ; Number of worms, Parent- : Date of Date of Date of . Eggs. laying | 4.:...|>>s : Cocoons. age. oe egos, (hatching. mounting. SSIESS White. Yellow. Total. Aug. Aug, Sept. 2 13 7th 17th fo) 189 189 1o-11th yellow. (18.27%) ; 4 14 5 = 36 161 197 x white and yellow. (28.61%) 6 ni 5 a3 87 217 304 55 do, 8 16 a as O 348 348 ee yellow. 10 17 3 $ fo) 317 317 u do. 12 18 a ss fe) 212 212 = do, 14 19 ” 3° oO 333 333 39 do, 16 20 3 fe) 291 291 f do. (24.9%) : 18 21 3 + 64 193 257 3 white and yellow. 20 22 a a fo) 322 322 ‘ : yellow. 22 23 is os fe) 228 228 x do. (27.38% ; 24 24 es a 69 183 252 Hs white and yellow. Now we see that the white form produced broods, all coming true to the parents, While the yellow ones split up into two kinds of broods, one entirely like the parents, and the other containing both white and yellow worms approximately in the proportion of one white to three yellows. Thus, we have now 3 kinds of parentages. A. White form derived from the yellow parents in the second genera- tion (Class I). B. Yellow form. 1. Those from mixed offspring (yellow 75% + white 25%). (Class ID). 2. Those from uniform offspring (Class III). Each of these forms was kept separate for experiment on the next generation. Studies on the Hybridology of Insects. 269 SECTION D. The fourth generation. In this generation, we have, as already stated, three classes of different parentage. The results of rearing can be seen from the following table :— CEASS: — Offspring from the white cocoon-spinners. Dateek Number of worms. Parent- E livin Date of Date of Cs age, 885. pe! = hatching. mounting, ie hrs 88S. White. | Yellow. | Total. Sept. Sept. Oct. I 20th 29th 245 fo) 245 27—29th all white. 2 ” oy 337 oO 337 26—28th do. 5 3 2 ” 241 12] | 241 ” do. 4 z - 305 fe) 395 + do. 5 ” » 209 o | 209 26-29th do. Total. 1b 7) fe) 1,337 | do. Of the one thousand three hundred and thirty-seven worms born from five parents, none spun yellow cocoons, that is to say, the offspring raised all came true to the parents. Ciass II. Offspring from the parent which produced two kinds of worms, white and yellow, in the last generation. 270 K. Toyama. (A) Offspring raised from the yellow cocoon-spinners. | 3 Number of worms, Cocoons. Parent- ee aoe of Date of Date of ges aying , . age. =e coos, (hatching. mounting. SSS. White. | Yellow. | Total. White. | Yellow. Sept. Sept. Oct. 2 2oth 29th fo) 178 178 26-27th fe) 130 (26.2%) 3 5 5 a 160 216 27-28th 52 141 4 - fe) 174 174 > o 158 5 3 fo) 202 202 $3 oO 187 (26.86%) 6 55 54 147 201 26-27th 30 134 "S (23.23% 9 , 9 69 228 207 27-28th 62 198 (25.4%) 10 > 63. 185 248 » 59 179 16 a fo) ZI1 air i {o) 207 17 As fe) 274 274 5 fo) 265 (23.826 18 ; : 45 144 189 55 32 135 (B) Offspring from the white cocoon-spinners. Dee Number of worms, Cocoons, Parent- E By ~ ° | Date of Date of age. 88 ae hatching. mounting, €885. White. | Yellow. | Total. White. | Yellow. Sept. Sept. Oct. I 2oth 29th 199 fo) 199 26-27th 181 oO } 2 i" 203 fo) 203 27-28th | 199 fe) | 15 3 i 172 fo) 172 4 | 166 oO 4 ¥ 3; 149 fe) 149 26-27th | 138 oO | 5 3 iy 208 fe) 208 24-28th | 199 o | | | Total. 931 oO 931 | 883 oO Studies on the Hybridology of Insects. 271 With the yellow form, we again find two kinds of offspring, one (Nos. 3, 6, 9, 10 and 18) producing white (about 259%) and yellow (about 75%) worms, the other (Nos. 2, 4, 5, 16 and-17) only yellow ones. The offspring of the white form, on the contrary, remain constant and uniform. In this group of worms, therefore, we see the repetition of the same phenomenon observed in the last generation. CEASS Itt Offspring from the parent which produced only yellow cocoon-spinners in the last generation. | | Number of worms. Cocoons. Parent- fees poe Date of | Date of | age. 238% pe “> |hatching,| l ‘mounting, | | S85. White. | Yellow. | Total. | | White. | Yellow. | | | j Sept. Sept. | | Oct. | I 20th 29th | Oo 308 308 | 27-28th 1 oP” 2os | | | 2 PU Ae Mae fe) 295) | 205 -| 26-27th | oOo | 298 | 20 3 y MY Oo 188 188 | a Gye ) 198 | | | 4 | » he oO | 253 253 x ro) | 236 5 : s oO | 221 221 27-28th | fo) 181 | Total. fe) 1,265 1,265 fo) 1,154 | Contrary to the yellow form of class Hl, they produce only yellow offspring. The results above described show that the white character after being crossed with the yellow, can easily be separated again as a pure strain. On the other hand, the yellow character is difficult to separate from the white, when once intermingled with it, and some of offspring always retain the white character lying dormant in them, which will reappear in succeeding generations. As to the question, whether this is to be looked upon as a constant rule or not, further series of experiments will show. to bo K. Toyama. SECTION ~ E. The fifth generation. In this generation, we made further breeding from the three classes of the last generation by the usual method. erAss” 1. Offspring from the white form. . Number of worms. Cocoons. Parent. | Date of Date of Date of : Eggs. laying Saeeumall —- —- - | Saeateoteetes age. == aan hatching. ! | mounting. | So” White. | Yellow. Total. White. | Yellow. | | SS eee Nov. Nov. |: I | I 6th 16th | | 2 | 2 , veal eo Sn *| o 951 — 850 fe) SU a TS » » | | | | Again, they produce uniform white offspring. Geass II. Offspring from the yellow parent which produced yellow and white worms in the preceding generations. (A) Deter Number of worms, Parent- Bees. | laying Date of | | Date of ' Cacnane age, SS ce jhatching. | | mounting, €88- White. | Yellow. | Total. | | Nov. Noy. | Dec. | 1} oth: 16th oO 359 |. 359 | 23-24thy yellow only. | 2 na 17th fe) 32 |) B32. we do. Miss (23.98%) | ; |2 3 Bt s 77 DAA) Sail 4s white and yellow. yr | a | 4 = 16th Oo nO: ||) | ronal if | yellow only. ; | = | 5 ” 17th oO 342 | 342 ” do. ~- | | - Sh aie: 16th | esas | = 7 - a 885 fo) o = white only. 8 | | | bb) ” Studies on the Hybridology of Insects. 273 | | Date of Number of worms. Parent- | ~ : Date of | Date of a age. | Eggs bane hatching mounting. | ae | SSS | White. | Yellow. | Total. | | z | Nov. Nov. 26.% - Dec: /0 : Qg- | .6th 16th 65 185 250 23-24th | white and yellow. 7G (25.66%) = 10 * omen ney 7/ 223 300 44 do, a2 | So a | rr | a Sat fo) 175 175 yellow only. Bel? .¥ = fe) 205 205 do. Pes. 33 oO 220 220 do. | pe = £ | (24.87% 14 17th 50 I5I 201 BS white and yellow. (26.38% ) ey re | 16th 95 265 360 do. = | | | 5) 16 7th 17th fa) 330 330 yellow only. = | 17 fo) 256 256 do, S ee) ee EE ee ee ee eee eee | 38 6th 16th ) | = 19 - 1,008 fo) T,008 white only. ha | / 20 > 2 i | | | 21 7th 17th fe) 399 399 <5 only yellow. (23.06%) 22 54 180 234 white and yellow. S 23 ; : fo) ZO 263 yellow only. > (19.24%) 24 71 298 369 white and yellow. Oo. F (26.45%) al 25 82 228 310 do, S as ee Ee eee 26 : | iS 27 j 677 fo) 677 white only. } 238 - as in the last generation. The phenomenon of segregation of parental characters is just the same It will be remembered, however, that some of the eggs (Egg Nos. g—13) were derived from the parents which produced only yellow offspring in the last generation, whilst others were derived from those 274 K. Toyama. which produced mixed offspring (white 259 +yellow 75%). The former is, therefore, an analogous case with those of class III and the latter with those of class II in the preceding generation. Both of them, however, give identical results, in this generation. (B) Offspring from the white cocoon-spinners. eee Number of worms. Date of Date of | Parent- a - : ee som : age. Eggs. bene hatching. mounting. = SFG White. | Yellow. | Total. Noy. Nov. | 2 I 6th | 16th 380 oO 380 — all white. 3 2 7th 17th } | 4 3 “ - 608 Oo 608 = do. 5 4 } | Total. 988 fe) 988 | do. a No coloured offspring was produced. @exss: Il. Offspring from the yellow parents which produced uniform offspring in the preceding generations. } Number of worms. Parent=||) 5.0 pee S Date of | Date of | = A : | Eggs. | laying igi page| Cocoons. age. 2 pane hatching. mounting. | Werte | White. | Yellow. Total. | =e . Tae Nov. | Nov ify I 6th | 16th fe) 330 330 “= all yellow. Dinh Ake A oO 400 400 ah do. sa 3 + . fo) 342 342 -- do, | } j Eo ens 55 i oO 288 288 — do. 5 | 5 oO 294 204.8 ie | do, Pana sn i | ice Total. | | | Beemet.054 | 1,054) do. | | | | The result is just the same as in the last generation. to Studies on the Hybridology of Insects. SECTION F. The stxth generation. In this generation, we kept only two kinds of worms: one, the white cocoon spinners of class I, and the other, the yellow cocoon-spinners derived from the parents which produced mixed offspring in the last generation. (Class IT). Cxiass_ I. Offspring of the white cocoon-spinners. Number of worms Date of Parent- Ros - Date of Date of - 6 ggs laying |. 5 —$—$$— : Cocoons. age. =3 SA hatching. r a mounting. 88°. White. | Yellow. | Total. Jan. Jan. Feb. ; Coal tt Sth 18th 146 fo) 146 26-28th all white. ip. eee 6 33 as 276 fo) 276 do. 7 ae a I4I oO 141 i do. Total. 563 fo) 563 do. They were again uniform and constant. Cweass . It Offspring of the yellow cocoon-spinners. Number of worms. Parent-| poos eee is Date of |_ Date of | eae age S85 | ne hatching. mounting. | i a | cao White. | Yellow. | Total. St ne (en Jan Jan (25.86%) | Feb. | I 6th 19th 30 86 116 29th white and yellow. 9 5 9 y 2 - a fo) 89 89 = only yellow. 3 3 oO 99 99 do. (29.74%) 4* 7th - 58 137 195 i white and yellow. (21.69%) 5 - or Ze [eeiti2.. 143 re do, The result is just the same as in the last generation. 276 K. Toyama. SEeETION G. The seventh gencration. In this generation, we reared only the yellow form, which gave rise to two kinds of offspring, white and yellow, in the last generation. eass Ii. 3 Number of worms. Cocoons. Parentzi|5 a.) pees | Date of | | Date of | Eggs. | laying - | —— — — : age. | °°" | Coos [hatching l mounting. |» eel White. | Yellow. | Total. | | White. | Yellow. | | | | | | March | March | April etre - | Tee Socrh oO 241 241 20-21st | a2 |) aes | : I (26.15 § 0) , 2* cs 68 | 192 260 : 32 156 | | (28.1 7% ) 1a ole wes : 71 181 252 42 166 } 4 o 283 283 oO 259 5 ; : ) 250 250 fe) 227 The result is similar to that obtained in the third generation. SEGTION ~H. The eighth generation. SGAss II: Yellow form derived from the parent which produced two kinds of worms. Studies on the Hybridology of Insects. 27 NI Number of worms. Date of | Parent: | = Date of | : | Date of = ‘ age. Eggs. pele hatching. mounting. | eS Songs} White. | Yellow. | Total. | | April May | June } Fit) || 2oth roth oO | 263 263 | 7-8th only yellow. al le ” o 319 319 # do. (25.21%) se s oe 30 89 lig | a white and yellow. to _ to ue fon) “I So i—) — 4* = re 76 232 308 he do. (25-67%) 5 30th re OR es 226 225 8-9th do. | (24.54%) 6 55 $5 67° | 206 2s a do. The result is again the same as in the preceding generations. SECTION © I. The ninth and further generations. As a conclusion to these series of experiments we endeavoured to rear all the yellow offspring derived from a single parent, for which purpose we chose the worms Nos. 1, 2,3 and 4. The former two produced exclusively yellow offspring in the last generation, while the latter two mixed offspring, white and yellow in the usual proportion. From No. 1 were obtained fifty-five batches of eggs, each representing the offspring from a single parent. Each batch of eggs was, as usual, reared separately in a single basket, and the result confirmed the fact that there was no hybrid, all being pure yellow cocoon spinners. Those derived from this group, as far as our experiments went, no longer produced any white worms in the succeeding generations. So we may say that they have become a constant breed. Those of No. 2, on the contrary, produce two kinds of offspring, one giving rise to uniform offspring and the other to mixed offspring (white 259% +yellow 75%). Among the 81 batches of eggs laid—each batch represent- bo NI io) K. Toyama. ing the offspring from a single parent—those! laid by sixteen mother-moths (19.75%) produced white and yellow worms in the usual proportion, while the rest (80.25 %) gave rise to yellow worms only. As seen above the parents of these two kinds of worms, Nos. 1 and 2, produced only yellow worms in the last generation; yet the offspring from No. 2 produced two kinds of worms in this generation, while those from No. 1 produced only yellow worms remaining constant through subsequent generations. Nos. 3 and 4. Both produced mixed offspring in the last generation and we may expect from the facts already obtained that further breeding will only give similar results. No. 3. The yellow form bred cuter se laid 16 batches of eggs, of which seven (43.74%) produced mixed offspring in the usual proportion, *while the others (56.26%) produced only yellow offspring. No. 4 gave an analogous result to the preceding. Amongst the fifty- three batches of eggs derived from the yellow form we found twenty-four (45.28%) to give rise to mixed, and the others (54.71%) to uniform, offspring. Now we see clearly that in the yellow form once separated as uniform offspring from the hybrid parent, there may be found some apparently yellow or hybrid ones in which the white character lies latent. The proportion of such hybrid forms in the offspring from a single parent is represented, in the former case (No. 2) to be about 20% (hybrid one) and 80% (uniform one), and in the latter (Nos. 3 and 4) about 50% : 50%. Résumé of the results so far obtained about the hereditary phenomena < regarding the two colours of cocoons, the white and the yellow :— io4 1 The total number of worms raised from these 16 parents was 3,945, of which 881 (23.3%) wer white cocoon-spinners and 3,064 (77.69%) yellow cocoon-spinners. Studies on the Hybridology of Insects. 279 A PEDIGREE REPRESENTING THE INHERITANCE OF COLOUR (WHITE AND YELLOW) CHARACTERS IN THE HYBRID. S (white) + 2 (yellow). J, First generation. all yellow. pee eee II. Second . ,, yellow(75%) # white(25%). i 2. aS = —_—_—— >» ay Sey Wf. Third rs only yellow yellow(75°¢) + white(25%). only white. | } ite 2. ——$—__—_—~ ee aa Re IV. Fourth F only yellow. | only yellow. yellow(75%)+white(25°%). only only Rh ae ] ! white. white. | Ie, Zs ie Zs “ ; ie Sarin V. Fifth i only yellow. yellow+white. yellow. yellow+white. only only yellow. (75%) (2596) (759%) (259%). white. white. i Le 2. VI. Sixth - yellow. yellow(75 0%) + white(25%). only white, | i 2. Witleeeseventh 5 yellow. yellow(75 % )+ white(25 %). | We 2. ——. —$ — VIII. Eighth yellow. yellow(75%)+ white(25 %). parent No. 2. parent No. 3. No. 4. No, 1. all 2 SS SS oh = ; =e (80.25%) (19.759) (56.25%) (43-74%) (54-71%) (45-2879). yellow. yellow white+ yellow yellow white+yellow yellow white + yellow only. (259%) (75%). only. (259%) (7596). only. (25%) (7570). EX: Nineth 1. Thusin the first cross generation between the yellow and white breeds, the yellow character predominates over the white, and the offspring raised, without any exception, spin yellow-cocoons. In the second generation paired zzter se, however, the yellow form breaks up into two forms, each with one of the parental characters, in the proportion of one to three. As far as our experiments went, the white character, when once separated from the yellow, persists, the yellow form never being produced from it. With the yellow form it was different. Although they were bred zener se, they never produced uniform offspring: Some of them produced only 280 K. Toyama. yellow offspring which remained constant, as in the case of the white ; others produced yellow offspring which, however, split up into the, two parental characters in subsequent generations, according to the following scheme : Seemingly yellow parent ie 2. yelllow. yellow (75%) + white (25 9%). The proportion of the uniform and the mixed offspring was not constant : ye in one case we got approximately the ratio 80% : 20%, in another about re OY - r OF 55/0: 45/70- 2. When the white and the yellow characters are brought together by crossing, they are transmitted to the offspring without fusion, but the white may be transmitted in the latent state for a generation or more in the yellow ones without losing its independent quality. The former may be called recessive and the latter dominant character, after Mendel. CASE II: Crosses between Fapanese univoltine white and Siamese multivoltine yellow breeds. SECELION «I. The first cross generation. Japanese white (2) by Siamese yellow (@). We obtained six batches of eggs by this cross, each batch representing all the eggs laid by a moth, as in the last case. Most of the eggs laid, however, turned out to be univoltine and did not hatch. In May, between the 25th and the 28th, 1903, twenty-nine worms hatched out from one of these batches. They spun twelve cocoons, all of which were pure yellow in colour, and spindle shaped. Thus we may say that in the first cross generation, as in the last series of experiments, the yellow character predominates over the white. Moths emerged on the 23rd _ to 26th, June, and laid eggs. Most of them were again univoltine and did not hatch, except those laid by moth No. 3, which furnished the material for experiment on the next generation. Studies on the Hybridology of Insects. 281 SECTION 2. The second generatton. The total number of eggs laid by moth No. 3 was four hundred and twelve, among which there were 218 darkly pigmented eggs (univoltine eggs), 120 light yellow ones (multivoltine eggs) and 74 lightly pigmented ones (some univoltine, some multivoltine). From these multivoltine worms, the following result was obtained :— ] i] | | Wate of ‘ Number of worms. i Cocoa laying Date of : d ___| Date of | ; : i. ; eee |hatching. | ; mounting. pips Wed ges. | | White. Yellow..| Total. | Yellow. ped i = sh} White. | | yellow. white. gee tipeh tegen 6252376) | (74778) July | (55.11%) (16.53%) | (18.89%) | (9.449%) 25th | 3-7th 35 103 | 135 24-30th |} 70 21 | 24 12 Now we see that the proportion of yellow and white worms well corresponds with the result of the second cross generation of the experiment, case I, and that each kind of worms could be divided into two; namely in the yellow worms there were yellow and pale-pinkish-yellow cocoon-spinners and among the white worms, white and greenish white cocoon-spinners. In this case, however, the worms raised were not the whole of the offspring derived from a single parent and consequently we are unable to draw an exact conclusion as regards the mutual relation between those various kinds of worms, yet it is one of the interesting phenomena of heredity, since both parent races hitherto have never spun any pale-pinkish-yellow or greenish white cocoons, as far as we have experimented. We may, therefore, conclude that as a result of the crossing new characters—which perhaps have lain hidden for ages—appeared in this case. SOCTION \ 3: The third cross generation. ra) In this generation, we have four kinds of worms derived from the last generation :— Group A. Those derived from the yellow cocoon spinners. -* P,P. yellow =pale pinkish yellow. 282 K. Toyama. Group B. Those derived from the pale-pinkish yellow cocoon spinners. Group C. Those derived from the pure white cocoon spinners. Group D. Those derived from the greenish white cocoon spinners. GROUP A. Offspring of the worms which spun yellow cocoons in the preceding generation. | ay! ee J Cocoons Take GE Dereae Number of worms. Date ot “ocoons, Eggs. | laying | jichine mount- }———} | = I ing. Se - 2 ess. | White. | Yellow. | Total. ees White. | Yellow. ae Total eee | eee | eee | Aug. Aug. | Sept. (22.839) (56.62%) (20.54%), aes 3rd 12th | 58 22008)" 287 I-2nd 50 124 45 | 219 . (74.1695) (25-83%) 3 | 4th | 13th fo) 375 375 2-5th fo) 221, |= 77a) ees | (23-07%) (76.92%) al lal 30 HOS 207 395 I-2nd 69 230 fo) 209 eee als oF 399 401 15th cd 239 5i |} 202 | | | (23.89%) | (76.1%) 6 | 5th » | 61 162 223 3 45 144 o 189 | | | (23.52%) (76.47%) y Up i7th 16-17th|} 50 17I 225 4-7th 44 143 fa) 187 (74.33%) (25-66%) 8* , 0 319 319 o 223 77 300 | oe ine (23.26%) (76.73%) 10 gth 18-I9th | 89 283 272 6-8th 77 254 fe) 331 Tey Es ct fo) 25a) 11) 1-253 zs oO 181 fe) 181 : (24.7%) (56-320) (18.9%) 72*\)) 7th 15-i7th 86 287 373 4-7th 86 196 66 348 In this group, we obtained four different kinds of offspring: the first, those producing only yellow cocoon-spinners ; the second, those producing yellow and white cocoon-spinners ; the third, yellow and pale-pinkish-yellow cocoon-spinners and the last, those producing yellow, pale-pinkish-yellow and white cocoon-spinners. The respective proportion of the various worms in the offspring is represented in the following :— ** Perhaps mixed from other groups. to CO ioe) Studies on the Hybridology of Insects. ite fisstikind: 22). Only yellow cocoon-spinners. f ; White cocoon-spinners 235 (23-35%): The second kind ...}__ ¢ Yellow cocoon-spinners 771 (76.64%) Total 1,006 heads. : - Pale-pinkish-yellow cocoon-spinners 154 (25.75%). Thethird kind ...... : , “A Yellow cocoon-spinners 444 (74.24%). Total 598 heads. * White cocoon-spinners 136 (23.98%). The fourth kind i oa cocoon-spinners 320 (56.43%). Pale-pinkish-yellow cocoon-spinners L1I (19.57%). Total 567 heads. This confirms that in the yellow form the various characters lay dormant. GROUP BB. This group of worms was derived from the worms which spun pale- pinkish-yellow cocoons in the last generation. | Date of Number of worms. | Cocoons, Ea ee Ate OF |e. ape ee Be Datewot aaa I I ges. | gee hatching, | | mounting. | pep. | 55° White. | Yellow. | Total. | | White. pets Total. | | | yellow. Aug, Aug, Sept. I |. 7th | 17=18th | 20, | OO. » 128 6-11th 25 85 110 | | | Ze i) | : 33 SSee DIS 6-10th 20 79 108 | Aas Ito Be i eee | | ; Dat ane | (25.2%) | (74-7%) (25.2%) | (74-726) Total. | 2 184 246 55 164 218 | | | | We obtained only pale-pinkish-yellow cocoons and white cocoons, no yellow ones. The respective proportion among the offspring from a single * Among these, we may distinguish the pure white and the greenish white cocoons. 284 K. Toyama. parent was about 75% of the former and 25% of the latter. It is analogous to a case which we met with in the cross between the white and yellow breeds. GROUP C. These are the descendants of the pure white cocoon-spinners of the last generation. a | Number of worms. Cocoons. " Date of : “fea Date of Xe Date of Eggs. |. : laying = —~—- z - |hatching. aaa | | mounting. | | ggs. White. | Yellow. | Total. White. | Yellow. | Total. | | | : == : ae pe = | | | Aug. Aug. Sept. ree ith 16th 206 | fe) 296 5-Sth 268} fo) | 268 | | a All the offspring spun white cocoons. GROUP D. Offspring of worms which spun greenish white cocoons in the preceding generation :— | | | lDatelor Number of worms. Cocoons. Bess. | daying | Date) a _| Date of | SS > |hatching mounting hs FOS ce = - | = > a —. eggs, White. | Yellow. | Total. te eee otal. | | greenish white. | | — |. a Aug. | Aug. |. Sept] ag th 15—16th 280° | fo) | 280 | 5-12th | 198 198 (Lie | | | 5 | 9 9 | } | | | 2 Sth |16-17th| 450 | o | 450 | Sr2th | 388 een | | | | } | | as ES 17-18th} 437 | ) 437. | 7-1oth 322 322 As the table shows, there are no deep coloured cocoons such as yellow or pale pinkish yellow. Ona closer examination, however, we could observe that some of the cocoons were pure white, while the others were light ereenish white, and the latter were always much more numerous than the former, but we failed to count them accurately because it was very difficult to distinguish exactly light greenish ones from some spoiled white ones. Studies on the Hybridology of Insects. 285 SECHION — 4: The fourth generation. In this generation, we have four kinds of silk-worms as in the last generation :— Group A. Offspring of group A of the last generation, which may be divided, as already described, into four kinds :— Kind 1. Worms which spun only yellow cocoons. Kind Kind Worms which spun yellow and white cocoons. lo 3. Worms which spun yellow and pale-pinkish-yellow cocoons. Kind 4. Worms which spun yellow, pale-pinkish-yellow and white cocoons. Among these four kinds of worms, the offspring of the third and fourth kinds were kept for experiment on this generation. Group B. Offspring of group B of the preceding generation, that is, those which spun only pale-pinkish-yellow and white cocoons. Group C. Offspring of group C of the third generation, that is, those derived from pure white cocoon spinners. Group D. Offspring of group D of the last generation which spun some white and some greenish white cocoons. Group: A. KIND 3. Parents: No. 8 of the third generation. 2£6 K. Toyama. a. Offspring of the yellow form. tye Number of worms. 3: Cocoons. Date of D : Date of : = ate of 8 ——————— : Fees | laying hatehiee - mount- |- = pegs. [UNE eevee rot | ie | white. |vatow [roses | eee ; rite. ellow. otal. : rite. ellow. | yellow. otal. c— Stel (74.8%) | Sept. Sept. Oct. Dupion 2) (25.1%) I 17th 26th OFM mein hae27y;. | 21-22th oO 183 | 63 250 (73-3526) (26.64%) D. 6 | 2 fe) 2320 330 «=| 21-23th fo) 178 | 65 259 3* ° 281 281 |21-24th| 0 212 | © 212 D. 4 4 fe) 253 253 + | 20-23th o 223 | fo) 231 (77-81%) ; D. 2° |(22,3895) 5 fe) 290 299 | 20-22th | fe) 230! TW ROE 275 | (70.49%) (29.59% D. 5 D. 6 6 i 4 fs) 357 357. |21-24th| o 197 75 294 (74-47%) (25-52%) D. 4 i Dap 7 o 298 298 . | 20-22th wee 205 7u | 286 | | | (74.9396) (25.0696 ;* £0 D. 4 8* fe) 407 407 | 21—23th fo) 261 86 375 D.7 | 9* fe) 366 366 =| 20-23th fo) 310 |, Oo | Piteen (74.16%) D. 13 (25.83%), 10* 3 - o | 368 368 =| 21-23th fo) 195 | _ 97 Al ees | j | Of the offspring from ten parents, those raised from parents No. 3, 4 and 9 spun uniform yellow cocoons, while the remainder, Nos. 1, 2, 5, 6, 7, 8 and 10, two kinds of cocoons, yellow and pale-pinkish-yellow, but no white. The total number of cocoons spun by these worms was 2,037 in’ which there are 1,513 (74.27%) of yellow and 524 (25.72%) of pale-pinkish-yellow cocoons. In the offspring from each parent, nearly the same proportion of yellow and pale-pinkish-yellow cocoons was obtained, as the table shows. NT Studies on the Hybridology of Insects. 2 6. Offspring of the pale-pinkish-yellow form. es | | Number of worms. Cocoons. poe | ee ~ | Date of | Date of | “85>. oo ns hatching. | mounting. PP €gss- White. | Yellow. | Total. | Whites |! 223 8 PFotal yellow. | Sept. Sept. | Oct” «| al Bh ey rou |-- 27th Oo SOM = 3 TO 22-23rd o 2OT-= A268 | i. on 2 | Oo 301 301 22-24th af 287 | ar D. 8 | 3 ; 84 179 179 23rd fe) 139 155 | mes Aes } fe} 206 206 22-23rd fe) 199 203 | | Des a | oO 318 318 23-25th o 259 269 | | | D.7 6 | He fe) 185 185 22-23rd | fe) 163 177 } | D: 13 | - | he 8 300 a | ae: 257 | 283 | | i | D. Leta 8 | 19th 28th fe) 334 334. 24—26th fo) 175 201 } | iD Rey S| ee * | fe) | 312 312 24-27th o |\~ 239 —-J~ «255 | | pola tie | } D. 59 Total. fe) | 2,451 | 2,451 fe) Zs 2,127 Other batches of eggs laid by nine moths gave similar results, only pale-pinkish-yellow cocoons being obtained. We see clearly that the hereditary relation between the yellow and the pale-pinkish-yellow character is quite the same as that between the white and the yellow breeds, before mentioned. Group “A. KIND 4. Parents: No. 12 of the last generation which spun three kinds of cocoons, yellow, pale-pinkish-yellow and white. * D. means dupion, ~ o] 20 k. Toyama. a. Offspring of the yellow form. | Number of worms | Cocoons Date of -NSumber or: worms, Date of ; Date of Eggs. | laying |hatching. — [eee atts ] | pp | 8s | | White. | Yellow. Total. we White. | Yellow. | yellow. Total. ec a a= | Sept. | Sept Oct. 1D. OF | I 17th 26th fo) | 223 | 323. |20-22nd; oO | 249 | fe) 263 | | | | | ee: 16° oss. S76) “a7. bes 2* it ak | 3A | 224 | 295 | | 58 | 240 | | | | | | | D. 14 3 | ete a. 307 307 | 2I-22nd | o 17 |) os 215 | | L | (76.30%) 23.69%) | Doa2: |) Dae 4 fe) 308 | 308 _20-22nd | Qo, |) oR 60 287 | | | . | | | | eras | | } EE 4 / ‘s 3 5 » - fo} 277 277 | o | 202 48 264 | | (70.5990) (62 1290)(17.2726) es ro B72 6 77 264 | 341 | 60.) 41@7s ae 301 A | | | | (74.7696) 25.23%) | | | - 20 ees tf gm eet fo) | 365 365 ae ree: 203 54 325 ) | | | M Kezr 19% |, Dee 3 8 | o | a2) |) 328. | 21-23rd Oo. |) xem 58 291 | i } | | | | | (22.49% | |) Disgaea: 2 9 18th | 27th 2 280 | 291 | 22-24th | 2 "|, aB5 | 52 249 | | | . | (24.59%) j | | es 5 1Dy § 10 % Oo 426 426 fe) 258 | 77 355 | | | | | | (25.90) | (75-%0) | D2 | Doane Ei? | 2th 26th 83 229 312 |20-22nd| 66 138° 4. 0 280 | | | | | | | D. 10 | 12* Oo 299 299 ©|21-22nd| oO 158 | o 178 | | os ; | (18.7576 (60-93%) (20.31) cal ee ob Te aS 75 221 296 | 20-22nd 46 100 40 256 * DP. means dupion. C \O \ Studies on the Hybridology of Insects. 28 Baiciot Number of worms. Date of Cocoons. Eggs, | layin Bee | mount- |— S85. ncaa hatching. ae | [i peptal 885. White. Yalow. Total. = | White. | Yellow. Flee Total. == 77 an | Sl SEA es ae fee —— | | (21.29%) (78.70%) Sept Sept | Oct. | Lop ba TS 14* 17th 26th 78 | 260 | 338 |20-22nd) 52 | 177 o 263 | | | | | pea | |) ieee eel 15 , fe em me 315 [> 0 aes 59 «| «286 | | | 4] ae | (25.11 %)|(62.336)(12-55%)| 16 5 us | 88 264 | 352 ze | -58 | 144 | 29 | 231 fal 2 > iat RE |e a | | + D. 13 | 17* 18th 27th fo) 394. 384 |23-24th{ oO | 231 oO | 257 (26.20%) | D. 20 D. 2 18 oe 39 Oo 394 394 22-23rd fe) | 236 94 | 374 | | | | | | D;, 22 19 Bs 53 o- | 396. “gg | 23-25th oO 281 o 225 (20.25 9 )(79-74.% IDE i bers 20 : ‘ 7O | 241 | “311 |22-23rd 56 198 fe) 286 | We again meet With the same phenomenon of the separation of the parental characters as that displayed by the worms, group A in the third generation, that is to say, (1) Some (Nos. 1, 3, 12, 17 and 19) of the parents producing only the worms which spin yellow cocoons only ; (2) Some (Nos. 11, 14 and 20) producing the worms spinning yellow and white cocoons, the total number of cocoons spun being 829, in which the white 184 (22.19%) the yellow 645 (77-80%) ; (3) Some (Nos. 4, 5,7, 8, 9, 10, 15 and 18) producing two kinds of worms, one spinning yellow cocoons and another spinning pale-pinkish- yellow cocoons, the total number of cocoons being 2,429, of which we find the pale-pinkish-yellow 590 (24.28%) the yellow 1,839 (75-71%) 290 K. Toyama. and (4) the remaining (Nos. 2, 6, 13 and 16) spinning three kinds of cocoons, white, pale-pinkish-yellow and yellow. The total number was 1,028, in which there are * the white 226 (21.98%) the yellow 627 (60.99% ) the pale-pinkish-yellow 175 (47-0200 B. Offspring of the pale-pinkish-yellow form. . Number of worms. Cocoons. a 2: 8 a Date of : Date of Eggs. | laying | ; — ; g aie natching| E mounting: yy PP d 55” | White. | Yellow, | Total. White. yellow Total. an a - 1,185\ fa) 1,185 | 13-14th | white without any exception. x = sf ee Studies on the Hybridology of Insects. 299 | } Cocoons, | Number of worms. eae eee zi | Date of Dhite.ok | Date of | Eggs. _ laying ihotehi i ia. = | mount= | = | eggs. rate 1n$.| ar = ea | ine | d S > P eae | White. | Yellow. | Total, =" | White. | Yellow. | oat | | | | | yell Ww. Ock | Nov. (27.31%)! elec: ID), i 1D 7 I Skies LOthies|) "62 HOSweee22 7, || l2=1th 45 oO 122 3g | (24.719%)| one: D. 15 5 2 ¥ 55 64 | 195 250 3-I4th | 54 fe) 163 2 s | { | = (23.79%) Di D. 14 z 3 69 | 221 290 49 fe) 171 3) | ae (23 07%) 1D). Hi IB). ae pS 4 fe) 200 260 41 fe) 162 ay D. 4 5 ; ; fe) 226 226 fo) fe) 154 | | ‘ | — = Ty 7 si | (aqunrof a | | IDG) | Dix I | 3 gth fe) 222 222 12-13th fe) 202 | 50 | (15.92%) (20.52%) Dy 2 D187) g 2* 71 275 346 43 7 fo) I (22.91%) m D. 9 1D), yi ~ 3 1oth fe) 217 2G) fe) 128 44 = 2} = = Nov. | 1D) yi = 4* Ist 11th fe) 253 253 14-15th fe) 99 fo) ID ie 5 x fe) 285 285 fe) 197 fe) 1. The worms reared from the white parents again spun no colored cocoons, as in the case of the other white breeds derived from the colored forms. 2. Those derived from the pale-pinkish-yellow parents produced two forms, one, spinning two kinds of cocoons, the white and the pinkish-yellow, their respective proportion being 25% and 75% and the other, spinning only pale-pinkish-yellow cocoons. 3. The third or those derived from the yellow parents produced three kinds of offspring :— a. Those (Nos. 1 and 3) producing two kinds of worms, one spinning yellow and the other pale-pinkish-yellow cocoons. 6b. Those (No. 2) producing white and yellow forms. c. Those (Nos. 4 and 5) producing only yellow forms. 309 K. Toyama. In this generation there were none of that kind which produced three kinds of worms, the yellow, the pale-pinkish-yellow and the white cocoon- spinners in the offspring from the same parent. (6). Offspring of the parents which spun two kinds of cocoons, the yellow and the white, in the preceding generation. Takeoor | Number of worms. lateoe Panes ta ag) | -s | Date of | : Parentage. | Eggs. | laying |,-)...) |—————__—__—_—___ ——}| mount- | Cocoons, : oo eee: hatching. | eae SERS White. | Yellow. |! Total. | > } | | | Fe | Oct. | Nov. leeecs | 2 A 31st _| oth fe) 284 | 284 | 12-13th all yellow. = | Nov. (26.44%) | ; D =o x 5s Nigar ete { white and yellow, e | = 2 2nd | t1ith 3 203 27 15-16th ) number missed, — es | | ! = | Oct Z g i), fi) Base oth |) ae 472 oO 472 | 12-13th all white. = 2 } , } j | | : | — I . 5 fe) 264 | 264 a all yellow. 2 4 | z3 oO 302 302 Ss do. = } i = 3.2) | fe) 346 346 re | do, > | '; 4 j ae 7 | re] 315 315 He do. So a ON! | One| 326 326 : | do, e | | | Xe} 5 | | j 4 I 2 ies | -o 274 all white. Nov. | 2 Ist roth | g | = 3 “5 | = 1] | = 1 1,313 fe) 1,313 | 13-14th | do. 4 | | | 1] Laat In this case, the offspring from the yellow parent No. 14 produced two kinds of worms, as is usual, while those from No. 11 produced only one kind, spinning yellow cocoons only. Those descended from the white ones spin again white cocoons in both cases. Studies on the Hybridology of Insects. ioe) (2) _ (c). Offspring from the worms which spun only yellow cocoons in the last generation. | emia teem: |e 5 ae umber of worms. | Date of | Parentage. | Eggs. | laying Date of ——| mount- | Cocoons > 25 esos, hatching. ae hdres | 385. | White. | Yellow.| Total. | >: | Pe th. Oct: Nov. f I I) SYS oth j\ tad 2 | | a5 | Dec. , ; ~ | | | - ; Ago (SS 5 , ee FI at alee ae oae 1 £206 |a221e8h | white and yellow, o = a te. | ‘ note | ae >" || number missed Ac | | } : [) ' = s | | beret u,2 | j ion | q | | | i | i | = | Nov. } | I 2nd rith ‘} | lt 82 | 442 524 |15-16th do. a os 2 | 2s 4 | Z a 3 3rd 12th | A 3 2 (y - only. numb 4 2 { yellow only, number 5 6 | 16- Diode 23 o | 645 45 [28 18th | missed. | 4 : | s: J | { ee | he ee They exhibited only the yellow character in the last generation, yet some of their offspring in this generation, as we see, again split up into two forms, the white and the yellow. In this series of experiments, however, we reared the worms descended from different mother-moths indiscriminately in one or two baskets, so we could not exactly determine which mother-moth produced mixed offspring, but from the experience hitherto obtained, we have good reason to believe that, in both cases one of the mother-moths produced mixed offspring, and consequently the respective percentage of the two forms in the offspring from one parent being about 25% and 75%, thus exactly corresponding with the results obtained in the series of the experiment, Case I and also of the preceding series. We shall, therefore, discontinue this and the preceding series of experi- ments in the next generation. K. Toyama. B. Offspring derived from the pale-pinkish-yellow form. j | | te c “ar - | | Date of | ae Number of worms. Date of | Parentage. ggs. | laying Of | —_________________| mount- | Cocoons. en aonam e l ie hatching. aes | cee White, | Yellow. | Total. = | Oct. Nov. Dec. | § White. I 28th 6th 361 oO 361 5-7th all white. a | S\ Pale- (25.25%)| | | ae FE sn P- 7 > some white, some ed 2 ” ” 54 159: bate * | pale-pinkish-yellow. 4 | W 2 is = 310 fo) 310 | all white. a | a 2 (23.35.20 hi Z, Sys ioe } ZO) 5 es | white and | Eee 4 i, ze | 250 334 pale-pinkish-yellow. W 5 30th oth | 349 Oo | 349 $-1oth | white only. e) (30.27%) =e Z r |! See white and P. P. Y., | _ : 7 oh hale ee) 2 377 number missed. / | | W: 7 -|, 28th ~|) SGk | 390 | 0 | 390 5-7th white only. < | | | | : s a bas, 8 rr | p. ao | 274 274 P. P. Y. only. is 9 29th 7th fe) | 247 | 247 6-Sth do. { | | | | | | ign | | W. |- 31st ane aoiyr | ‘oO 1,171 | 9-11th white only. is} | : 16 | | ~ | | Z [ (24-13%) | be a 17 | 9th 70 | 220 | 290 |12-313th| white and P, P. Y. yp aks roth o | 2255 | 2225 PPS OY, onlye PPX. | | 19 oe | Oo |} 283 283 “ | do. } | } 20 eee | 252 252 | 3. do. { Nov. | 26 5th 13th | W. . pS 424 | 15-16th white only. ~~ y 27 ” 9: ) | L | * 24 ks 15th Om |) 337 337. +| 18-20th all: P-P Ne PUP IY = . 25 ” ” Oo | 363 363 ” do, Studies on the Hybridology of Insects. | ate OF | a - Number of worms. | Date of Parentage, | Eggs. laying hatching, | eee Cocoons. | eses: | | White. (ees Total. | "8: | | | | Nov. Nov. | Sane) ocd i3th | Dec. | 1! 391 oO 391 15-17th all white. ; fe S54 \) S é Z i 3m (j E2th 7 see he 205 295 | 12-15th | all Pe ve | j | | | PPy.. 31 | 13th Gault 257 257 | 13-15th do. | | | 22 4th 14th | fe) 325 325 | 14-16th do. | | Oct. | | | i | - < | 24 | 3ist | 1oth Pa 22 | P ig 754 754 13-14th all: Pps G py ‘| | ee — | : | Nov. | = I ae | ae a 28 ard |) asthy || | a | Oo | 584 584 | 15-16th | do. ZA 2 | 29 ) | | We have become so familiar with these phenomena of heredity in the preceding series of experiments that any further explanation about them seems to be superfluous. YY: Offspring of the white form. Number of worms. : | Date of | Date of | | Date of | Parentage. | Eggs. | ae ihatching.| geass Cocoons, 55 | | White. | Yellow.| Total. | ~ = Oct. Noy Dec. I I | 29th 8th san |) oO 335 5-7th all white. ae { | 2 |} 2 | |; 28th 7th 438 fe) 438 7-9th do. eV ee s bi nes a, 6th $65-]) © 865 | 5-S8th do. (he | | | oc | | Total. | 1,628. he -o 1,638 do. Again white offspring. 304 kK. Toyama. GROUP B. Offspring of the pale-pinkish-yellow form which diverged from the yellow parents in the second crossed generation. (1) Darwer Number of worms. Date wt Parentage. | Eggs. | layin Date of mount- Cocoons. = es § hatching a €S8s. White. | Yellow.| Total, | ‘8: f Nov Novy | I Ist 11th | J 2 5 } Dec. ; = -1,094°| Oo | 3,094 |15-16th all white. 2 3 ” : | 4 33 th) 3 ea a Bs 5 2nd 12th Oo 287 287 =| 16-17th all P. P. yellow. 2) 2 6 x fe) 308 308 do, is (25 87%) > . ba 2 We F Fe 74 212 286 es white and P, P. Y. = (25% z 8* a ; 75 225, |||, 300 a do, = | ray 9 i A) oO 376 | 376 is all P. P. yellow. Io 3 5 | as (O25 |. Oo 603 | 17-18th all white. = I , uit) = | 12 3rd 13th 330 Oo 330 =| 18-20th do, 1 aaa (26.96%) = 1 2nd 12th 72 195 267 |17-18th| white and P. P. Y. = (25.64%) = 14 , F 80 232 312 a do. a 15 3rd 13th By | 331 331 |18-19th| P. P. yellow only. Ss 16 . . 2 17 i y ° 786 | 786 |19-23rd| all P. P. yellow. 4* z 18 - a tes 19 a 14th © o 584 584 93 do, rai 20 4th : * Compare Group A, Kind 4, a. ¢. last generation, Studies on the Hybridology of Insects. | = -| Number of worms. Date of | | Date of See 2 tage. Eggs. laying | = mount- Cocoons Parentag gg ae hatching. Ae t Cocoons. So” White. | Yellow. | Total. > = Oct. Oct. z : = 21 3Ist roth Dec. : = 3 { 72% ane She 14-16th white and P. P. yellow, - | =z 3 507 = =e 7) ie fe, | ae =e number missed, Hh } -_—_— 99 I ay; | : Nov. a | 22 TSE ty beh tee, 1 64* 518 582 15-16th do. a 24 : om We see clearly now that the hereditary phenomena displayed by this group correspond very well to those shown by the cross between the white and the yellow breeds in the fourth or further generations (see Case I). 2) Offspring from the white form which diverged from the pale-pinkish- rellow parent in the third generation. y g Number oi worms. ] | Parentage. | Eggs. | laying | Date of | : Date of | | Det ob bane WER [orc jist | ae S| ae Se eras jhatching,| a oe emia | f= | White. | Yellow. | Total. | aa 7 .oree 21 Noy. Nov. | 2 } 2nd 12th { Dec. li: ve | 14 2 5 - 953 fe) 953 18—19th white only. : = == 4 . asl iba Zz 3rd 13th 3 4 5 2275 fo) 2,375 all white. 6 | | / 4th i (0.9 Quite familiar phenonrenon observed in other white forms. 306 K. Toyama. SECTION 6. The sixth gencration. In this generation, we kept the following kinds of worms for experiment :— 1. Group A. Kind -2: a. 1. Offspring from the yellow form which spun only one kind of cocoons, that is to say, the yellow. Offspring of the yellow form which spun two kinds of i) cocoons, the yellow and the pale-pinkish-yellow. 6. Offspring of the pale-pinkish-yellow form which spun only pale-pinkish-yellow cocoons. Kind 4. a. Offspring of the yellow form which produced three kinds of worms in the fourth and fifth gererations. We kept now only the yellow form which produced (1) both the white and the yellow forms and (2) the yellow form only in the offspring from a parent. 6. Was no longer reared. ae, 5 8. In this group there were two kinds, one spinning only (1) pale- pinkish-yellow cocoons and the other (2) two kinds of cocoons. the pale-pinkish-yellow and the white. y. Offspring of the white form. 2. Group B. Offspring of the pale-pinkish-yellow form which diverged from the yellow parent in the second generation. Nn O N Studies on the Hybridology of Insects. GROUP A. KIND 3. (a). 1. Offspring of the yellow form which spun only one kind of cocoons, the yellow, in the last generation. vo TF. 3 x 7 a = & | BA Or LS | Number of worms. Tatene Cocoons, 5 | Eggs ne acca | Ph be | eggs, i 5" =e | ro at ing. aie - Ass cae Se. White. | Yellow. | Total. = White. | Yellow.| Total. | | | | | | Dee Jan, Ni Feb. 15) ii I 25th Sth OMS eee 157) | T3—r4the ee o 112 114 | Dz. I I 2 27th roth fe) M235 122 16-17th O 7. | 80 | 2 28th + fo) 146 | 146 fo) 94 94 Again produced uniform offspring. 2. Offspring of the yellow form which spun yellow and pale-pinkish- “~e yellow cocoons in the last generation. . | vu tf —— im = on Date e Number of worms. Date. of" Cocoons. oe : Date of é | Pace PA yIDe oe een so | count. | s eggs. 2) vay a h ing. = FaMteats | pee be 3 5 White. | Yellow.| Total. : White. | Yellow. ; ral yellow. | Jan. Jan. Feb. I 5th 17th fe) 221 221 |25-28th|} oo iff 24 2 Dita IB Pi 8. 9. 2 3rd 16th o 188 TSS 2i22nd | ao 81 24 | | D. I 3 5 15th fo) 203 203 ~=~| 20-22nd fo) 120 oO 4 4th 17th ges: 143 143 | 25-28th fo) 57 19 10! 2. 5 5 ts o Baal 83 26-28th fo) 32 8 | 6 2 oO 229 220 n fo) 94 fe) eu A repetition of the same phenomenon as observed in the preceding generations. 305 kK. Toyama. \ b). Offspring of the pale-pinkish-yellow form. 2 EA =A Number of worms. Date of | ~ Date of ee . | Date of | ‘ =e 2 Parentage. | Eggs. laying | en —J| monnt- Cocoons, =e ares, jhatching. | aes st | White. | Yellow. | Total. Fr Jan. Jan. Feb:) || Ist | 14th Oo 162 162 |20-23rd| all P, P. yellow. : , | ake | fe) 198 | 195 ~ do. | | : ae an te x o 224 224 Be do. a Quite the same as in the preceding generations. KIND 4. a). 1. Offspring of the yellow form which spun yellow and white cocoons in the last generation. SS LL : - Number of worms. : Cocoons, | Date of Ditent atl} pete Date of ~ ° < Bue Parentage, | Eggs. laying oS mount- : 3 aerator hatching. a | fiat | White. | Yellow. | ‘otal. >° White. Yellow. H end E . bat i aleaaen | Dee. || sheniin(25275.%5) = Beb: Dr “Saas I | 209th EILEh) | aeGo 173 233 | 18-10th 21 62 2 3s 3 o 66 66 x all yellow. cee ee ee ETN ETN ae 2. Offspring of the yellow form which spun only yellow cocoons in the last generation. eT j Number of worms. | Date of S| Date of ) } 1a 3 Date of | akg CAnaee Parentage. | Eggs. | laying itebade = ap OUR -ocoons. } Pog 2 = [hae > es ing. | “ss* | White. | Yellow. | Total. = ; eebec: Jan. | Feb. I 26th Sth fe) | 236 236 =| 13-16th all yellow. | | i 2 2 el Ome 213 213 it do. ; | = | 3 , o e255 255 > do. Studies on the Hybridology of Insects. 309 Quite the same phenomenon as observed in the case of the crossing between the white and the yellow breeds. 8. Offspring of the pale-pinkish-yellow form. 1. Those derived from the parent which produced two kinds of worms. | ; = x Number of worms | Date of piltagl umber of worms. DAiteve Parentage. | Eggs. | laying hatcl ale ———}| mount- Cocoons. aaeeaes natening,. csp eee = | ing erg . | White. | Yellow.| Total. a Rs Dec. Jan. | Feb. ie) I | 18th 2nd o 230 230 | 5-6th | P. P. yellow only. ee 2 o Y ) 230 230 a do. re (27.6%) | meretwiete | 3 69 181 250 white and P. P. yellow. es prih4 1) cas | E52 fe) 352 white only. yi nis _ | 5 , er = | z I 25th Sth ae || 197 197 | 13-14th all P. P. yellow. ~ | a | > 2) |} 126th oO 278 278 = do. 6] 6; | ome | ie | 2 _ fe) | 271 271 a do. } | i 2. Those derived from the pale-pinkish-yellow form which spun only one kind of cocoons. Number of worms. | Date of | | Date of Date of Parentage, | Eggs. | laying lnatching| = =| mount- | Cocoons. | | eggs. | White. | Yellow. Total | ee | ‘ees ae } Dec. | Jan. Feb, I 27th | oth fo) 207 207 13-14th| all P. P. yellow. INO 21, |, 52 $3 3 Oo 241 241 ell do. a | fe) 194 194 do | The same phenomenon as repeatedly observed in the preceding generations. IO K. Toyama. ios) Y- Offspring of the white-form. eerie remem weer ea | : N r of worms. - Date of | pabet of wort Date of Parentage Eggs laying pe nount Cocoon arentage. Lgas, ay : ——— S| i = oc s S Se cece Hatching. ; opr ; Ss ms 71: r te ed . SIRS White. | Yellow. | Total. = Dec Dee | I 17th 31st | | Janu. I and 2 | | 539 | =O 539 =| 28-30th all white. 2 | | They have retained uniform character from their first production. GROUP B. Offspring from the pale-pinkish-yellow form descended from the yellow parent in the second generation. (1). a. These spun two kinds of cocoens, pale-pinkish-yellow and white, in the last generation. We reared only the former in this generation. Se ae . Number of worms. Date of Pe ep Oe atetes . ae ; Date of eee oe Parentage. | Eggs. laying | : SSS —} mount- Cocoons. = oe |natching. aes — | White. | Yellow. | Total. oF | Jan. Jan. Heb: Fes Oley MSs 14th Gee) 127) «|| «8127 . 1 tq=2st all P. P. yellow. 3 | (24.65%), 2 , _ 36 GEO! ||| sky Oumy % white and P. P. yellow. | } | | &. These are descended from the parent which produced one kind of worms in the last generation, that is to say, the pale-pinkish-yellow. eee a | Number of worms. > | Date of | nate of | ae? Date of Parentage. | Eggs, laying Ih: ; i ———- | nr | mount- Cocoons. angen latching. oe E on one 55° | White. | Yellow.j| Total. | > | | | | 7 | Jan. Jan. | | | Feb: I 5th Totnes Ose 274 274 | 24-25th all P. P. yellow. | 4 | 2 | 17th fe) | 201 20% | ne | do. | | | | | | ; | | | | 3 18th | Oo 250 250) | od do, 3 | ] Studies on the Hybridolegy of Insects. 311 Such instances have been met with many times in the preceding generations. (2) 2. Offspring of the white form. | Date of poh. Number of worms. Date of ros } 71 | pak | Coc S Eggs. laying lhatching. Tae EEE, Neas-ieec White. Yellow. Total. eae | | 1! fan. Jan. I ie Ist 14th | | Feb. 2 + - any hese ay fo) 783 1g—2Ist | all white. io) SSS Again uniform offspring. Summary. Now we will sum up the hereditary phenomena of the various colours of the cocoons observed in this series of experiments, as follows :— When we crossed a female moth of a Japanese univoltine white breed, called Aobzki with a male moth of the Siamese multivoltine yellow breed, the offspring of the first generation spun yellow cocoons without any exception, that is to say, the yellow character predominates over the white. In the next generation paired zzter se (the second cross generation), the yellow form splits up into four different characters, the yellow, the white, the pale-pinkish-yellow and the light greenish-white, the latter two characters being quite new. Next we will describe separately the hereditary phenomena displayed by each of those forms produced in the second cross generation in the ; following order :— I. The yellow form. II. The pale-pinkish-yellow form. Ill. The greenish white form. IV. The white form: oP) _ i) k. Toyama. I. The yellow foriii. The yellow form paired among themselves produces four different kinds of offspring, that is to say, the first kind spinning only yellow cocoons, the cond kind, the yellow (75%) and the white (25%), the third, the yellow (75%) and the pale-pinkish-yellow (25%), and lastly the fourth, the yellow (56.2%), the white! (24.65%) and the pale-pinkish-yellow (19.13%). a. With the first and the second kind, we have every reason to believe that they will follow the same law which we repeatedly observed in the case of crossing between the white and the yellow breeds. So we have discontinued further experiments. 6. In the third kind, the yellow form produces some uniform (the yellow only) and some mixed [the yellow (75%) and the pale-pinkish-yellow (25%) in the offspring from a parent] offspring in the next generation ; the yellow offspring separated from both yellow forms again exhibit the same cycle of hereditary phenomena in subsequent generations. The pale-pinkish-yellow form, on the contrary, when once separated from the yellow form remains constant throughout. Thus the relation between the yellow and the pale-pinkish-yellow forms is the same as that between the yellow and the white characters. c. In the fourth kind, (1) the yellow form again produces four kinds of offspring, but in the next generation, we have missed that kind which produces three kinds of worms, the yellow, the pale-pinkish-yellow and the white, probably owing to the scantiness of the worms reared for experiment. (2) The pale-pinkish-yellow form splits up into the white and the pale-pinkish-yellow, displaying quite the same phenomena of heredity as those exhibited by the white and the yellow. (3) The white form, on the contrary, produces always uniform offspring through subsequent generations. Tis “Lhe pale-pinkish- vellow form. This form yields two kinds (the white 25% and the pale-pinkish-yellow 75%) of offspring, in the next generation. 1 In this, as already described, some greenish-white ones are contained. Studies on the Hybridology of Insects. 313 The white form thus raised remains constant in colour generation after generation, while the pale-pinkish-yellow form produces some uniform (producing only the pale-pinkish-yellow) and some mixed (the white 25% and the pale-pinkish-yellow 75%) offspring in each succeeding generation. Ill, The greenish-white form. In this form, as in the preceding, we have two kinds of worms, one spinning pure white cocoons and other light-greenish-white cocoons, in the next generation. Although we failed to count the number of both forms, yet we have good reason to believe that they will follow the same law governing the hereditary phenomena of the other colours mentioned above. LV. The white form. Not only this, but every white form since from its first production remains true to itself, as far as our experiments have gone. Thus we come to the conclusion that as a result of crossing, the yellow form splits up into four different forms, the pure yellow, the pale-pinkish- yellow, the white and the greenish-white. Among the four characters thus enumerated, the yellow is predominant over the others; next comes in succession, the pale-pinkish-yellow, the greenish-white and the white, that is to say, the white is absolutely recessive in this case. When these four characters are combined by crossing, the yellow appears as an active character in the offspring, while the others become latent. On the contrary, when the other three characters are combined, the pale-pinkish-yellow one becomes active character and the other two latent, and finally in the case of the greenish-white and the white ones, the former is active and the latter latent. The latent character, however, becomes active in the next generation, occupying one-fourth of the whole number of the offspring from a single parent. The relation of these four characters to one another, therefore, is quite constant, displaying always the same phenomenon in successive generations, which may be summarised as follows :— 314 K. Toyama. A=dominant character. B=recessive character. The first cross generation (A+B) Offspring A only. he second cross generation Offspring 2 -hi oats | ; 189 | o | 189 3 white only. 2 9 | | es | | BX 4) || and | 1ith 1300} ce) 130 53 do, | | | | | | { { J The yellow parents produced two kinds of offspring, and the white uniform offspring. As regards the hereditary phenomena of the colour of the cocoons, we again meet with similar facts as observed in the preceding series of experiments, thus Studies on the Hybridology of Insects. 319 SERIES I. The first generation : only yellow offspring. i The second __,, white(25%) ==—Ss=*«*~ Pure Siamese yellow breed. : Number of worms. Date of Dateet Eggs, laying Gio Cocoons. She se =a hatching. 8s. White. Yellow. Total. Nov. Nov I 6th 16th fe) 375 375 yellow only. 2 i ‘ Oo 382 382 do, 3 5 17th fo) 302 302 do, 4* E te fo) 381 | 381 do. | | | Re , ; oO 359 359 do 6* +5 $s fo) 390 390 do. 7 Sth 18th o 350 350 do. Total. fo) Dae? 2,53¢ do. 539 539) g& Pure Siamese yellow. : @& The yellow form of No. g, Class II, of the fourth generation. 1 A similar case has been observed with Japanese races: when we crossed divoltine striped worms which sometimes produce common marked worms with common marked breed, we got the following figures :— I. Il. common marked worms. 130 heads. 122, striped worms, 145 heads, 145. 322 K. Toyama. Tite or Number of worms. ae, Pa Date of | : Eggs. laying hatehaan Cocoons. €S85- ~ | White. Yellow. Total. Noy. Novy. I Sth 18th oO 412 412 yellow only. 2 Oo 410 410 do, 5 fe) 415 415 | di 4 é - oO 424 424 | do. 5 fo) 426 426 | do. Total. oO 2,087 2,087 do. In these reciprocal crosses, only yellow offspring were obtained. SECTION II. The second generation. SERIES. 1. (a). 5 We ete Number of worms. Parent- ee i oa | Date of C 4 age. on Ae | hatching. | a | Sor a | White. Yellow. | Total. Jan: | jana I 5th |. Toth 62 122 185 white and yellow. | | | all | = | ; : a el 2 | 6th roth 47 181 | * 228 do. ig | / Onell | A 2 | a 69 224 203 do a | | | 5 50 142 192 do. S 9 | | | ; | (25.38%) | (74.61%) | Total. | 228 | 670 | 898 | do, | | Studies on the Hybridology of Insects. 323 Date of Number of worms. Parent- E renee Date of anes LoS, ao iS = — = ~OCcoons age. 55 Ree hatching. | 36 “SS>- | White. Yellow. Total. | oe. 5 BY.9 a 2 ot es é Jan. Jan. . = I 6th 1oth | 48 149 197 white and yellow. a é e 2 & ” 38 72 IIo do. a = 5 51 195 246 do. | (24.77%) | (75-22%) Total. 137 416 553 do. Now we see that in the offspring from each parent which spun only yellow cocoons in the last generation, there may be found two kinds of worms, one spinning yellow and the other white cocoons, the respective Se DZ s arel one -% (the white percentage being 75% (the yellow) and 25% (the white). z Number of worms. Date of Parent- ee - Date of : Eggs. laying : Cocoons. age. 55 ee hatching. | 55°: | White. Yellow. Total. Jan. Jan. I 6th 19th a2 78 110 white and yellow. S 2 t. a4 47 155 205 do. <* Fi 3 61 192 253 do. a = 2 Z + ” F m7 139 176 do. =| > 5 5 ” a9 8 185 243 do. = 2 5 > 5 = z 6 93 x | 7 1534) he. Gees do. Oo | — ; : a (23.76%) | (76.23° 3 Total. 282 05 1,187 do. A) / ro Z = 7 - - 206 fe) 206 white only. 2) = 8 119 oO 119 do. = | _ | Total. 325 fe) 325 do. Asin the case of the last generation, the yellow form again produces two kinds of worms, white and yellow, yet their proportion in the offspring of a parent is quite different from that observed in the last generation, that K. Toyama. is to say, in the former generation one-half of the offspring from a parent consists of the white and the orher half of the yellow form, while in this generation three-fourths is yellow and one-fourth white. SERIES 2. (a). The offspring of the worms which spun only yellow cocoons in the last generation. Date of Number of worms. Parent- ices iS. ite Date of ‘ Cocoons age. AESES oy = hatching. l a 88s. White. Yellow, Total. . | | f = | Jan. Jan, I 6th 19th o 212 212 all yellow, 2 %s - oO | 265 265 do, No. 4 3 ra) 242 242 do, 4 x 5 a) | 193 | 193 do, 5 ; fe) | 254 | 254 do, 22, <——- 4 t / | 6 3 = fe) | aR 257 do. 7 3 35 fo) 120 131 do, No. 5 8 0 ©6—O| «S263 263 do, 9 (e) 152 152 do. | 10 oO 230, i) Sepa do. / + e. 3 II 5th 18th o | 205 205 do 12 a ‘ Oo 162 162 do | No, 6, i 6th 19th o 275 275 do 14 oa f Oo 189 189 do | 15 S - 9) 146 146 do } Total. fe) | 35177 \ 3azy do, A In this series, no white cocoons were found. In consequence of the scantiness of the leaves, we discontinued Studies on the Hybridology of Insects. 3 to UI this series of experiments, so we will give, as before, a short résumé as follows :— Back-crossing of a cross-bred yellow form with a pure parental white breed. The reciprocal crosses gave a quite parallel result, as follows :— some some parents : : 1. The first back-crossed generation: produced only yellow + white z yellow form. (50%). (50%). 2. The second ee “4 yellow + white yellow + white white (75%). (25%). (75%). (25%). only. SERIES 2. Back-crossing of a cross-bred yellow form with a pure parental yellow form. The reciprocal crosses gave a quite parallel result as follows :— 2. The first back-crossed generation : only yellow. a thesecond -_,, = again only yellow. Thus we see that when a cross-bred dominant form is back-crossed with the parental recessive breed, it produces some uniform and some mixed offspring of the dominant and the recessive forms, their proportion in the offspring from each parent being about 502% for each. In the succeeding generations, however, the two dominant forms which appeared in the first generation will split up into their parental characters according to the law already observed, when a yellow breed was crossed with a white breed, that is to say, 75% of the dominant character and 25% of the recessive. The white form produced in each generation will no more produce any coloured offspring. On the contrary, when we back-crossed a cross-bred dominant form with the pure parental dominant breed, the offspring showed only the dominant character. In the second generation, they again produced only dominant form. 326 K. Toyama. In consequence of the inability to continue this experiment for further generations, we could not draw any definite conclusion with regard to their further fate, yet we have every reason to believe that they will split up into their parental characters in the succeeding generations. The account will be dealt with subsequently in the chapter on general considerations. or Vv. In the preceding series of experiments, the inheritance of the various colours of cocoons was discussed. Now we shall pass on to the considera- tion of the various markings on the larval body. For this purpose, we selected two breeds, one ( Pl. X. Fig. 6, Pl. XI, I) having no markings on the body, except on the dorsal portion of the first segment where some very faint dark markings could be observed ; the other (Pl. X, Fig. a, Pl. XI, ID having a black stripe on each intersegmental region. The former breed will be called hereafter under the name “ pale breed ”’, and the latter ‘“ striped breed ”’. The pale breed remained constant for six generations, while the striped breed sometimes produced a few pale worms, in spite of our efforts to exclude all the pale forms in each generation. We can not say, therefore, that it is a pure race, but rather a cross-bred form between the striped and the pale ones. Both these breeds, however, always spun white cocoons, never yellow ones. The result of the reciprocal crossing between these two breeds will be given in the following pages. SeetION I. The first generation. SERIES I. The patewsreceas (PI. XT,’ I). The striped breed. (PI. XI, II): 0) 40 ww to NS] Studies on the Hybridology of Insects. Number of worms. Date of : gy ag Date of one Eggs. laying ee Cocoons. £ Fatty hatching. SEF Pale. Striped. Total. Sept. Sept. ae 21st 30th fe) 370 370 all white. ae fo) 376 376 do 4 186 198 384 do 5 fe 2 180 165 345 do. 6* 3 5 159 165 324 do. ie (49.9%) | (50.0%) Yotal. 525 528 1,053 do. Some parents (Nos. 2 and 3) produced only striped worms, while others (Nos. 4, 5 and 6) both pale (about 50%) and striped ones (about 50%). SERIES 2. 2 The striped breed. G The pale breed. Number of worms. |’ Date of : - Ms Date of | eta Eggs. laying : = Cocoons. = seine hatching. ecese= Pale. | Striped. Total. Sept. Oct. 3 | 22nd 2nd tose 4 1So 373 all white. Ae —— | 4 | aa aa oO 306 306 do. 5 o 342 342 do. 9 fe) 287 287 do 10 5 | 43 o 356 356° do. Quite the same as in the last series. Now the result of this reciprocal crossing reminds us of that of the back-crossing mentioned in the preceding series of experiments, Case IV. 32¢ K. Toyama. SECTION II. The second generation. SERIES I. a. Offspring of the parents which produced only striped-worms. | | Number of worms | Parent- | Ree gan Date of \ i | C Meal ae Eh ee hatching. | | i | SEO | Pale. | Striped. | ‘otal: | | | | | | | | | Nov. | Nov. 23.11%)" | 2D | 6th | 17th 49 | 163 212) eu white only. (25%) | 3 | 42 126 168 do. } | ' | | | (19.04%) No. 2 4 64 272 236 | do. | | | (22.42%) | 5 35 | Hs 50 173 223 do. \ | | (23.9%) 7 | | 60 191 251 do, | | | | | | (24.035) fe) + | 16th 56 177) |) S238 do, | | P (26.4%) , Non 3: ti 9 at Es | 47 131 178 do, | | | | (25.25 0) | 10 + | 49 145, | 194 do. | | | | —_ | | ae (23.2%) | (76.76%) | Total 417 [) Sco 7S seas do, | | | 6. Offspring of the parents which produced two kinds*of worms, striped and pale. Studies on the Hybridology of Insects. i>) i) Ne) iene Date of ae Number of worms. 4 Eggs laying Sats = Cocoons, age. ae panes hatching. 88s. Pale. Striped. Total. Nov. Noy. | II sth 18th 248 fe) 248 white only, | s 12 5 5 291 fo) | 291 do, a7 : } eS 13 + roth 275 oO ene 755 do, | 14° ss 319 fe) 219 do (26.22%) ie | 15 86 202 ee do (17.34%) | Se 16 - % 53 244 297 do S (25-55%) e 17 ” * 92 268 360 do. (26.67%) = 15 1sth go \) 247 337 do. = | =; | 5 19 33 Toth oO 254 254 do, (25.14%) | 20 bi 18th 85 QCard {| has do, (24.47%) | 21 60 185 | 245 do 22 fe) 287 287 do | OOOO ‘SERIES 9 a. Offspring of the parents which produced striped worms only. Number of worms. date Parent- rece pees Date of Cannas age, 25 pee hatching, bh eae SHEP Pale. Striped. Total. | ele Noy. Nov. I | Sth 18th 74 244 | 315 white only. : | 44 | 3 ) | 2 roth 69 189 255 do. | | 3 18th 74 264 | 335 do. | | : ; 4 roth 106 | *280° 1.1386 do, No. 4 | | 5 ns 103 307 | 410 do, 6 | ~ 56 203 | | - 246 do, 7 | if 18th 82 257) Bao de, | | 8 | sf : So goa 1) |- Banal do. | oP) oe) e) K. Toyama. Numl er of we rms, Date of Parent- Eogs ngs, laying Date of Cocoons. age. pace hatching. eggs, > 3 zs 7 Pale Striped. Total. Nov Nov. 9 Sth Igth 94 312 406 white only. Io 79 289 368 do. No. 5 II 59 197 256 do 12 81 206 287 do 13 - 100 280 380 do. No. 10. I4 z 72 202 274 do. T5 . = 71 200 271 do, Be (24.22%) (75-7770) Yotal. 1,200 3,754 4.954 do. 6. Offspring of the parents which produced two kinds of worms. Number of worms. Date of Parent- - : | Date of re Eggs. laying | I ate! | Cocoons, age. : ie hatching. =Sis i Pale Striped. Total. | Nov. Nov, I Sth 18th 286 oO 286 all white. 2 3 207 re) 297 do, E) 3 401 fa) 4OL do, 4 255 o 255 do. 5 301 oO 301 do, - Total. 1,540 o 1.540 do. Le (26.41%) 7 19th | 70 105 265 do, (24.06%, ) : 5 go 294 374 do. & (25.13%) = 9 94 280 374 do. 75) 10 Oo 227 227 do. 11 fo) 316 316 do wr) Ll Studies on the Hybrifology of Insects. a Now we learn that all the striped worms descended from the parents which produced only striped worms in the last generation split up into their parental forms, the striped and the pale ones. The proportion of these two kinds of worms appearing in the offspring from a parent is about 75% of the former and 25% of the latter. The striped form derived from the parent which produced the two kinds of worms, striped and pale, in the last generation, on the contrary, again produce two kinds of offspring, one uniform (striped only), the other mixed [the striped (75%) and the pale ones (25% )]. The former case may be seen in the second cross generation of pure races and the latter in the third cross generation. Seerion Ii. The third generation. SERIES [. Offspring of the striped form which produced two kinds of worms in the last generation. | Number of worms. | ] | if ) = ate of Parent- | Bae ty ie Date of | Cocoons age. eo | ae | hatching. -| ‘ea as bears | mueeale. Striped. Total. Aas | Jan. | 9 jam. | (24.3%) ; I 6th 19th 52 162 214 white only. 2 fe) 146 146 do 2 ay 3 . ° 154 154 do ay | (22.6%) : ci + A 39 133 172 o s | (30.5%) Zi 5 59 134 193 i2 198 (@) 19¢ ao. .* ' ' ~ | | a | 13 : | 165 oO 165 do. ox | | : 14 198 re) 195 ce) K. Toyama. SERIES 2. Offspring of the striped form which produced two kinds of worms in the last generation. Date of | Number of worms. Egg laying Date of ——__—__—_—— | Co S o> pe hatching, ae = ae | Pale: ||. Striped. | Total. Jan. Jan. I 7th 2oth fe) 229 229 white only. | (28.82%) 2 49 121 170 do, 3 Sth a fe) 219 } 21G do. 4 3 + fe) 262 262 do. 5 fe) 77 77 do 7 7th I51 oO 151 do 8 146 fe) 146 do ) 35 fo) 85 do 10 FS a 232 O 222 do. II = 55 137 fe) ei do. Now we have arrived at a familiar phenomenon repeatedly observed in the preceding series of experiments and may conclude with safety that this is a case of back-crossing between a cross-bred race and a pure parental race and also that various larval markings of the silk-worm will follow the same law as that governing the heredity of various colours of cocoons. The following is the summary :— some some parents 2 The first cross generation : produced only striped and pale striped worms. (75%). (22%). pale striped - Striped 7 (2a eee only The second ,, ze (25%). (75%). only. ues oF SE pale. a a a DE (25%). (7526)- The third - ,, r PAgGukestipe Se aay. pale (25%)+striped (75%). Hence the striped character is dominant and the ‘ pale’ recessive. Studies on the Hybridology of Insects. Os Us Os CASE. VI. In this series of experiments, we will try to consider, what will be the result when two races having many different characters are crossed. For this purpose, we crossed a pale breed spinning yellow cocoons with a striped breed spinning white cocoons. These may respectively be called “ pale-yellows”’ and “ striped whites.” Thus four different characters were brought together as the result of this crossing :— i (1. The character producing pale larvae. From one breed } eee (2. The character spinning yellow cocoons. 1. The character producing striped larvae. From the other eae . ae (2. The character spinning white cocoons. SECTION I. The first generation. SERIES I. Siamese pale race spinning yellow cocoons or ‘ pale yellows’. “> Siamese striped race spinning white cocoons or ‘striped whites’. Number of worms. Eggs. | Cocoons. Striped | Pale | Striped Pale at ae ese Pee Yotal. white. | white. | yellow. yellow. | | | I fo) oO 207 oO 207 yellow only. 2 oO ) 1. Se oO 327) | " do. 3 fo) fo) 308 o 308 do. 4 fo) fo) 281 fe) 281 do Total, oO fo) 1,123 fe) 1,123 do. ni K. Toyama. Number of worms. Cocoons. Striped Pale | Striped Pale Total white. white. | yellow. yellow. 2 | | o o 150 LSi 281 | yellow only. | | | | o oO 143 | 163 | 306 do | | | | | | Oo 1o) Oo 285 @) 288 | do fe) fe) 284 fe) 284 do. | e j | oO fe) 351 O a5r do O fo) 365 oO 365 do fe) fo) 405 fo) 405 do | | | | fo) fo) 164 | 202 366 do. | | | | } fe) o 360 oO 360 do. | fe) oO 307 fe) 207; do, } | | | oO Oo | 389 fe) 389s do | | | oO O | 352 | fe) 352 do | | | 2 o | 393 £ 393 ae. | fe) | fe) 20% 2| 207 410 do. | s - 29 d O O 170 154 324 0 O fo) 202 194 396 do | | | | | | | | | | fe) fo) do, j | | Studies on the Hybridology of Insects. On Os wa SERIES 2. Siamese striped race spinning white cocoons. “> Siamese pale race spinning yellow cocoons. Number of worms. Eggs. a aloes ; Cocoons. | Striped | Pale | Striped | Pale Total | white. | white. yellow. | yellow. Eis I oO | fo) 90 $5 175 yellow only. 2 fo) fe) -212 oO 212 do. 3 fe) o 120 ) 99 216 de 4 fe) fo) 122 | 107 229 dc 5 fe) | fe) 904 96 190 do 6 fo) o 330 oO 330 dc 7 fe) | fo) 381 fo) 381 d 8 o oO 332 o 332 di 9 fe) Oo 340 Oo 340 do C2 ea 356 ° 356 do II oO o 115 113 228 de i2 | Oo Oo 299 | oO 299 do 13 oO o 163 | 132 205 do i] AS ie} ie) | ioe) N =) OO ton f Lop) fon} Ne) From the result of this reciprocal crossing between ‘pale yellows’ and ‘striped whites’, we learn that in the first generation some parents produce only striped worms, spinning yellow cocoons or “striped yellows”, while others ‘striped yellows’ (about 50%) and “ pale-yellows’’ (about 50%), or pale worms spinning yellow cocoons but no white one, that is to say, in one case two dominant characters become active and in the other one dominant (the yellow) and one recessive (the pale) characters become visible. In general, the result arrived at in this generation agrees very well to that obtained in the two preceding series of experiments, Case IV and V. ae ioe) OV K. Toyama. SECTION IL. Lhe second generation. SERIES I. a. Offspring of the parent which produced only the striped yellow forms in the last generation. Parent- | | Number of worms, a Eggs Cocoons se Striped | Pale Striped | Pale | otal | white. | white. yellow. | yellow. | : : (25.4%) | (7.2% (52.7%) | (11.5%) ifs 84 } 24 174 | 38 | 320 white and yellow. | (18.8%) | (6.696) | (53.1%) | (21.3%) | No. 2. | 2 45 16 127 | 51 |. 239 do. | (18.89%) | (6.6%) | (58.26) | (16.5%) | & 57 20 176 50 | 393 do. | eee | er ee (21.57%) | (6.96%) | (55-3396) | (16.129) | Total. 186 60 477 139 862 do, | 4 60 20 154 50 | 284 do | 5 48 | 23 139 61 |, 2 27 do. | No! 7. | 6* | 83 22 | 256 76 | 437 do i | re | 7 55 22 164 47 | 288 do. | 8 65 23 227 So 395 do. A | aa | (18.56%) | (6.56%) | (56.11%) | (18.74%) | Total. 311 110 940 314 1,675 do. | | | | | 9 55 28 204 56 343 do | | 10% sj) () 908 Pee 236 | 72 | 420 do No. 8. II 04 20 204 go 408 do. | : | 12 65 18 QQ) MEGS) CAR eae do, 13 fe | 21 170 | 64 | 308 do. (19.72%) | (6.02%) | (55-46%) | (18.7896 Total. 357 109 1,004 | 340 1,810 do, Studies on the Hybridology of Insects. 337 Number of worms. Parent- E ai pes, == a Cocoons, 8°. | Striped | Pale | Striped | Pale Total. | | white. white. yellow. | yellow. aa | | | ) | | | | | 14 88 24 233 Si 426 | white and yellow. No. 11. 15 67 | | 26 230 O7 | gaa, | do. 16 69 18 154 62 | 303 | do. | | | j i | 224 68 617 210 | 1,119 | do. | (19.72%) | (6.34%) | (55-57%) | (18.3526) | | 1,07 | She Ser eeos5 1,003 | 5,466 do. } | | ! The total number of worms derived from sixteen parent-moths was 5,466, of which Tee’ Striped whiteaees.?-.<... ..1,078 (19.7296). 2. a4 Pale whitesieaa.----..- 347 (6.34%). 4. ect Stripednyellawsa |-.......- 3,038 (55-57%): A-s¢4 Pale yellompeee ¢2.....-- 1,003 (18.35%). The same relation obtains for the offspring of each parent. The relation between the colour of cocoons. Total number @fwormms-........4.....-. 5,466, of which 1. White cocoon spinners 1,425 (26.07%) in which (striped ones 1,078 (75-64%). { pale, 347 (24-35%): 2. Yellow cocoon spinners (striped ones: 3,038 (75.17%). 4,041 (73.92%) in which} / / 4,041 (73.926 | pale zs 1,003 (24.82%). The relation between the striped and the pale worms. Total number of worms........... ce Be 5,466, of which 1. yellow 3,038 (73.8%). 2. white 1,078 (26.1%). 1. yellow 1,003 (74.29%). white 347 (25-7%)- me otiped worms. ...:..:- APIO ZS -3,% ) to [prlicch alos) ee ane 1,350 (24.6%) | i) 338 K. Toyama. 6. Offspring of the parent which produced two kinds of worms in the last generation. a Number of worms. FE gr a Eggs. ; Cocoons, sg ce Striped | Pale Striped Pale Total white. white. yellow. yellow. as I 33 12 120 | 40 205 white and yellow. ES 2 26 II 105 27 169 do. 2) ay 3 18 6 EG | 18 98 do. ro) | = 4 45 19 166 58 288 do. = | 5 40" |) ene 133 39 226 do. } (16.42%) | (6.2896) | (58.829) | (18.4596) : Total. 162 62 580 182 986 do, oe Ae} ce, | A 6 fe) 64 fe) 215 279 do. > 7 fo) 2 o 202 274 do. & | 2 8 oO 38 oO 99 139 do, oO z 9 oO | g80 4 oO 116 154 do, 10 o 40 | Oo |) a Se tefl > MSS do. | | (25.22%) | (16.42%) Total. oO 252 (o) 162 999 do. | | ee The offspring from the striped parents, as in the last case, consist of four kinds of worms :— The total mumberor worms............2. 2:2: 986, of which —_ . 2. ‘Pale whites’ 3. ‘Striped yellows’ 4. ‘Pale yellows’ ‘Striped whites ’ _. ee 162 (16.42%). _. eee 62 ( 6.28%). 580 (58.82%). 182 (18.45%). The same relation holds good for the offspring of each parent. The relation between the colours of cocoons. Total number of worms of which Studies on the Hybridology of Insects. 339 1. White cocoon spinners I. Striped worms 162 (72.32%). 224 (22.71%) in which. ; a 2. Pale worms 62 (27.67%): 2. Yellow cocoon spinners I. Striped worms 580 (76.11%) 762 (77.28%) in which 762 (77.2874) in (2. Pale worms 182 (23.88%). The relation between the striped and the pale worms. Total number ofgvestes...-. .. 25... 22.2654 986, of which 1. yellow 580 (78.16%). I. Striped worms...742 (75.25%) in which! ? P ees: io’ los, white 162 (21.83%). I. yellow 182 (74.59%). Jae aeale Worms... |... 244 (24.74%) in which/ ; : 74-5970) white 62 (25.40%). On the other hand, those from pale worms spinning yellow cocoons split to up into two kinds, the ‘ pale whites’ (about 25%) and the ‘ pale yellows’ (about 75%). Thus of the nine hundred and ninety worms (to speak in round numbers), we find two hundred and fifty two (25.22%) pale worms spinning white cocoons and seven hundred and forty four pale worms spinning yellow cocoons (74.77%). The same relation holds good for the offspring of each parent. SERIES 2. Offspring of the parent which produced only striped worms spinning yellow cocoons in the last generation. Number of worms. Parent- Eggs. : Cocoons. eer Striped Pale Striped Pale | Total white. white. yellow. yellow. | ‘ I 45 17 164. ‘| 42 | 268 | white and yellow. Bic | 74 32 248 71 } 425 | do. No. 7. ae he 85 18 Zia (rn -oe aee = do. 4 | 75 12 228 71 386 do. 5 61 18 164 | 62 |, 395 do. | Rican (19.0596) | (5.43%) | (57-82%) | (17-82%) | | Total. 340 97 1,029 | 318 | 1,784 do. } 340 K. Toyama. Number of worms. Parent- Boos : age. ‘SE*- See ty: = Cocoons. > Striped Pale Striped Pale Total | white. white. | yellow. | yellow. Ses fe 2 : a || 60 De 205 59 347 white and yellow. 7 66 20 225 69 380 do. No, Q, Ss 2 - : ‘ 57 14 16m’) |} 68 300 do, 9 67 19 198 65 349 do. | % (18.16%) | (5.52%) | (57.33%) | (18.96%) Potal. 250 76 789 261 I 376 do =| pe 3 : 10 54 18 171 60 303 do, II 46 20 160 59 [or e2es do 2 by 2 2. 202 eae 12 7 27 212 | 75 392 do 13 68 26 210 | 67 371 do 14 71 | 18 i96 | 74 | 359 do. } ! | = Beak, (18.53%) | (6.3796) | (55-49%) | (19.59%) Total. ay 109 949 335 1,710 do, Granq | (18629) | (5.799%) | (56.81%) | (18.76%) fal, 907 282 2,767 gI4 4,870 do, The phenomenon of the segregation of the parental characters is the same as in the case of the last series. Total number of worms derived from fourteen parents...... 4,870, of which fr. We Striped wihttes eaeeemnore:......... 025.58 907 (18.62%). Zo Pale whiteSe Geers onsen Ss 282 (57 oR, I 3. , * Striped yellows spemere. =... 2... ener 2,767 (56.81%). A. ‘Pale yellows re eereere ee «6 oss nie ca ee 914 (17-76%). The same holds good for each litter. The relation between the colours of cocoons. 1. Yellow cocoon spinners {* striped worms 2,767 (75.16%). 2 0%) in whi 3081 (75-59 7G mee | pale worms 914 (24.83%). Studies on the Hybridology of Insects. 341 2. White cocoon spinners I. striped worms 907 (76. 28%). 189 (24.49%) in whi h| e289) (24-470) No. pale worms 282), (23.71%): Conversely, the relation between striped and pale worms was as follows :— I. Striped worms (I. yellow cocoon spinners 2,767(75.31%). oa 04 -hy 33074 (70-4474) of w Bie 2 pe white cocoon spinners —_.907(24.68%). 2. Pale worms 1. yellow cocoon spinners 914(76.42%). 1,196 (24.55%) of w hich, white cocoon spinners = 282(23.57%) It is very interesting to see that in this generation, the striped yellow form which appeared in the first cross generation splits up into four different kinds of worms, (1) the striped worms spinning white cocoons, (2) pale worms spinning yellow cocoons, (3) pale worms spinning white cocoons and (3) striped ones spinning yellow cocoons, the latter two being quite new combinations of the characters, already present. The respective number of these worms present in a brood was quite constant, always keeping the following proportion :— Iege StEtped. Whites: deer. - about 53, or 18.75%. Ze Pale: whites cere. about ;4, or 6.25%. 244 rale, yellows’ pie - about 3; or 18.75%. Ax i Striped) yellowsi apie... about ;9; or 56.25% With respect to the colours of cocoons and larval markings, they kept a constant relation independently of the other characters, that is, 1 25.9%. The recessive one about } o The dominant one about ? or 75%. SECTION III. The third generation. The four kinds of worms which appeared in the second generation were reared separately and they exhibited the foliowing phenomena of the segregation of the parental characters :— No, 42 K. Toyama. SERIES I. Number of worms. Parent- = : age Eggs. | ; Cocoons. Ts Striped Pale Striped Pale | Total white. white. yellow. Yellow. | oT a (25.%) I 301 99 oO fe) 400 white only. e (22.9% ez 2 252 75 o o | aay. | do. B hye, Fo 2 | © e383 fe) oO | oO | 383 do. ale) (22.5%) | lH 4 203 59 fo) fe) 262 do. | = 291 fe) fe) fo) | 291 do. | ) | | 6 | cage 2 oS fe) 1,380 fo) fe) | 1,380 do. bee 8 ‘| | | & | | 9 | ae ee / ; | | } (26.1%) (73-9%) = 10 93 o 262 oO 355 white and yellow. a | II | o | fe) 288 oO | 288 yellow only. hd | | | 2] | | | (74.48%) | (25.51%) |e 12 o oO 219 75 204 do. | z | (26.896 (73.2%) & 13 78 fe) 21 | fo) 294 ~| white and yellow. fe | | (67.2195) | (32.78%) 14 o fe) ae 79 241 yellow only. | | | | (15-44%) | (7.59%) | (56.2%) | (20.69%) 15 | 59 29 215 79 382 | white and yellow. == | | | 16 | oO oO o 356 | 5 Sa56 yellow only. | a 17 fo) | = © o 369 | 369 _do., hes | “(2Eergy | z 18 fe) 85 ) 312 4 487 white and yellow. 3 (21.0%) | i 19 fo) 69 o 259. |) 1328 do. | (24.3% | 20 fo) 101 fe) 314 415 do. | Studies on the Hybridology of Insects. 343 UTI Parent- Number of worms. age. Eggs. ; : Cocoons, | Striped Pale Striped Pale Total white. white. yellow. yellow. er (23.849) | 2 I 182 57 fo) fo) 239 white only. ‘ee | (21.5% | =e 2 157 43 fc) | Oo 200 do. 1S) Ay : | | F 3 225 {o) fo) fe) 225 do, | og 4 fo) 214 oO fe) 214 do, aS 5 oO 226 oO fe) 226 do. : (21.59 % S 6 fo) o 205, || 59 Pe) 264: yellow only. x. a z 7 fe) fo) 218 fo) 218 do. 2) (22.5% = 8 re) oO fy 187 53 240 do, B (27.9%) = 9 fo) fe) 186 7Z 258 do. ‘ (15.27%) | (6.89%) | (54.6796) | (23.1596) ; 10 31 14 PLT 49 203 white and yellow. Z II o Oo fe) 258 258 yellew only. = 12 oO fe) oO )_ 255 255 do, 7] | = 13 o 60 fo) 171 231 white and yellow. 2 I 93 fo) fo) fe) 93 white only, E | | (25.28%) = 2 130 44 fe) fo) } 174 do. 2 } | = 3 74 fo) fe) | ) 74 do. of | = = 4 fe) 212 (e) (e) 212 do. = = s | Eee ous . | (23.62%) | ae 5 ) 0 ii | 50 ipleay yellow only. o =| 6 0 : Ee eae 45 do. 2 = 7 fe) fe) 240 | Oo | 240 do. yi | . | | 3 8 o o oO II | II do, S Fl 344 K. Toyama. SERIES 2. Number of worms, Parent- _ = cae legs ; Cocoons. aa Striped Pale Striped Pale I : : : Total. white, white. yellew. yellow. (20.66% Bg I 96 25 o fe) 121 white only. * = 2 125 oO fa) fe) 125 do. oO st 3 fe) 185 o oO 185 do, = 2 4 = 83 oO oO 83 do. al (20.45% ; Be “ 5 45 o 7s) | oO 220 white and yellow, 3 S | Z, 2 (25.37% o 6 fo) fo) 150 51 201 yellow only. 3 : a 7 fe) fe) 143 fe) 143 do, 5 8. o o 135 o 135 do. 2 (27.65%) = 9 fe) 39 o | 102 14! white and yellow. - (22.44%) - 4 10 fo) 55 o 190 245 o. ay g II 225 fo) fo) fe) 225 white only. = : (20.78%) = 12 221 58 fo) | Oo 279 do, = (22.00%) a 13 156 44 oO oO 200 do. ) = 14 fa) 122 fo) fa) 122 do. = | = 15 oO 228 fe) fe) 228 do. ot = So ic * A o 16 fo) oO 185 68 253 yellow only. a (25-74%) ; = 17 69 fo) 199 oO 268 white and yellow. vo e. 18 fe) fe) 93 oO 93 yellow only. n : (23.1896) z 19 o 48 o | 159 207 white and yellow. ES 20 oO o fe) | aay yellow only. v | = 21 re) re) fe) 192 do, Studies on the Hybridology of Insects. 345 From the results of these reciprocal crossings we learn firstly, that the ‘striped whites’ give rise to two kinds of offspring, one producing two kinds of worms, pale (about 25%) and striped (about 75%), the other producing only one kind of worms, striped; secondly, that the ‘pale whites’ remain constant, as in the case of the white against the yellow ; thirdly, the ‘striped yellows’ produce four kinds of offspring, the first producing ‘striped yellows’ (about 75%) and ‘striped whites’ (about 25%), the second, ‘striped yellows’ only, the third, ‘striped yellows’ (about 75%) and ‘pale yellows’ (about 25%) and the fourth, four kinds of worms in a brood, ‘striped whites’ (about ;;), ‘pale whites’ (about. ;,), ‘striped yellows’ (about ;%,)-and ‘pale yellows’ (about ;3,). This is a repetition of what was found in the second generation. Lastly, the ‘pale yellows’ produce two kinds of offspring, one ‘pale whites’ (about 25%) and ‘ pale yellows’ (about 75%), the other ‘ pale yellows’ only. The phenomena of heredity observed in this generation remind us of those of the worms of Group A, Kind 4 of the Experiment Case I, and we believe that both cases are due to the same law, which will be discussed in the chapter on general consideration. 346 K. Toyama. SECTION IV. The fourth and further generations. In the fourth generation, we kept only those forms which produced one kind of worms in the last generation. Number of worms. Parent- = s age Eggs. mT = Cocoons, : Striped Pale Striped Pale Total | white. white. yellow. yellow. ae I 172 oO o o 172 white only, 2 (21.93%) | ac = 2 121 34 oO | o 155 do, cS a : (19.37%) , A iar 2 208 50 oO fe) 258 do. 7 | 4 183 fo) fe) fo) 183 do. 5 oO o 185 a) 185 yellow only. Re | | z 6 fe) oO 130 fo) 1320 do << wlero | re Hoes | e88 7 fo) oO 115 | oO 115 do Aa ee | 5 8 oO oO BSS Hc fe) 205 do P 9 oO fe) 138 fe) 138 do | { | | | A result already familiar to us. After weeding out all other forms during three successive generations we got an entire brood of the striped worms which spin yellow cocoons. We are now able to say that from the crossing between the pale breed spinning yellow cocoons and striped breed spinning white cocoons we can obtain two new breeds, the striped worms spinnings yellow cocoons and the pale worms spinning white cocoons. Now we will give, as usual, a diagram showing the inheritance of the various colours of cocoons and the larval markings. MN + in@) sects lology of In ic Studies on the Hybr oMy ‘sauo MOOA "(95 2) "(2eGx) WA (ete) MOTPOA + OIA MOT[AA oped ayud aped ——_—_—_—X—X— —, = ‘S aE ee aud ayy so dyin podrys ayy fF Stojonanyo ‘untoy MoT[AA padi.as aand jos JO SUOTVVUIGUUOD ANOJ “HOH RUTG UOS JO puby uo Ajuo souo SOUSA WOT MO OA OM “STYI WOT T podiajs ayy ‘ ap oped oy} Apsvy pur § SMOYS WRIBVIP dU) "(%Sz) *(O6S2) *AlUC G2) (%%Sz) *£[ UO Mo]PPA ayed + MOT[AA padiays = Mo]JaA podiays any podins + oy ayed aga pedis on EY Sa SS re ea i ae ————" ‘% Ou 'S On | Ooms) (WEwSS) (Gres) "(OFeber) (2,92) — *(lehe) “(O4SL) *(%Se) ‘Ayuo (% Se) *(%4S2) “A[UO a netod + MoTToA 4- ary + ary MOJOA + MOT[AA — MOjJOA + OITYAN —- MOTIAA Kuo OMY + aya aM ayed pedis ared pedis pedis aped pedis padias pedis any oped ajud = pedis padi.as —>———$— Se —— oe a — ——— = oY ft ‘% 3 a “(9gS2) “(96 Se) (%SL-g1) “(4 Se9S) +(9¢Sz°9) TOseBr) “(Se Os) Wese'9) —(9GS2"Bt) Xe) | CLS aanya MOT[OA + eat + ayn 4+ a7iyn MOT[aA + VR + oyn + aya oped aed oyed pedis aed podins ayed pods aed podins ) mood aped 4- a ae ‘OuUlOsS SCUAME «ALITA Gd t.1s (Ofos) MOT]OA padiiys > (INV ——— “ATUO Mo[[a& podiays ~_—_—_ “OULLOS MOTIAA W1IVds NAAMLAG DNISSOM)D SUO]P VAI WU09 SY : UOlwINUas \yanoj OUT, , UOWeaASU Par OqT, : UdlWBAIBUOS puoses OUT : uoneiauad SAY Ot T, 348 K. Toyama. CASE VII. As the last of our series of experiments, we will cite another instance of crossing between two impure breeds ; one parent being the common Japanese (PI. X, III ,c) breed, and the other striped Siamese breed (PI. X, II] a). Both of these breeds, however, contain the “ pale’’ character in a dormant state. SECTION I. The first generation. SERIES 1. 2 Common Japanese white race. (PI. X, Il, Fig. c.) @ Striped Siamese whiterace. (PI. X, Il, Fig: za.) Number of worms. Eggs. a ae TT Cocoons. | | Striped. Common, | Pale. Total, ig B75 o o | 375 all white. | | 2 316 fe) re 316 do. } | (56.19%) ; 138 177 oO aris do, (51.42%) 4 153 162 fe) 315 do (47.34%) (30.20%) (22.44%) : 3 i 74 55 245 ee. | (50.46%) (25.920) (23.61%) ; s 5 109 5 51 | 216 o. (47.21%) (24.53%) (28.25%) 7 127; 6 76 269 do (48.21%) (26.84% (24.93%) Total. 352 196 152 730 do. Studies on the Hybridology of Insects. 349 SERIES 2. Striped Siamese white. Ld & Common Japanese white. Number of worms. Eggs. _ - ——____—_——. | Cocoons. | Striped. | Common, | Pale. | Total. | | | | | | - | j A he 280 oO oO 280 all white. i | (54.3%) | 2 | IgI 202 fe) 393 do. | | | 3 | 364 ° cs) 364 | do. | } In this generation, as the tables show, we obtained three kinds of offspring : the first producing only striped worms; the second, striped (about 50%) and common marked worms (about 50% ; and the last, three kinds of worms, striped (about 50%), common marked (about 25%) and “pale” ones (about 25%). The last one is a new combination of characters appearing for the first time in our series of experiments. SECTION II. The second generation In this generation, we kept the offspring from the parent which produced only striped worms in the last generation. 350 K. Toyama. SERIES 1 Number of worms. Parentage.| Eggs. Cocoons. Striped. Common. | Pale. Total. | | | t | pa , | in % , | | (75-7296) | (78.93%) | (5.339%) | I 156 39 II 206 all white. | § | (72-976) | (21-9376) | (5.16%) 2 | 113 34 8 155 do, | (76.35%) | (17-569) | (6.08%) Be 226 52 18 296 do. (74-7726) | (18.14%) | (7.07%) 4 169 } 4l 16 226 do (76.07%) | (18.18%) | (5.74%) No. 1 5 | 159 38 | 12 209 do | (70.08%) | (23.169) | (6.2596) 6 157 ol 53 14 224 do | (76.53%) | (17.85%) | (5.619%) Fi 150 | 35 II 196 do | (73-93%) | (19-43%) | (6.63%) 8 156 41 14 211 do, | (77-29%) | (13-10%) | (9.60%) | | 9 177 30 22 | 229 do. | | | | | (74-94%) | (18.59%) | (6.39%) | | Total. | 1,463 263 126 1,952 do. | | | | | SERIES 2. 2 | Number of worms. Parentage. Eggs Cocoons. | Striped Common.| Pale. | Total. | | | (76.77%) | (17.229) | (5.99%) | I | ay 46 16 | 1 Gy - all white. | (76.209) | (16.269%) - (7.539) | 2 253 54 25 332 do. E (73-12%) | (22.34%) | (3-52%6) No. I eS 66 53 8 227 do. (72.28%) | (20.85%) | (6.829%) | ; 4 180 52 17 249 do. (71.73%) | (21.01%) | (7.249%) 5 99 29 10 138 do. : (74.44%) | (19.29%) | (6.2726) Tota!. 76 do 234 Studies on the Hybridology of Insects. 351 Here we again meet with a new combination of characters in the offspring from a litter, that is to say, each striped parent produced three kinds of worms, the striped (about +3), the common (,3,) and the pale (about ;';). SECTION III. The third gencration. We kept only striped worms for breeding in this generation. SERIES 1. Number of worms. | | Parentage. Eggs. | Cocoons. | | Strtped. | Common. Pale. Total. | | | | (73.819) | (19.04%) | (6.149%) Lt 155 40 15 210 all white. ae | (74.9%) | (25-20) | E 2 102 34 fe) 136 do. | | | (62.6%) | (18.29%) | (19.10%) | 3- a | i ae 45 47 246 do. | (62.92%) | (20%) | (17.0776) | 4* 129 4I 25 205 do. (76.8676) | (23.13%) 5 I | 103 fo) 31 | | They exhibited different proportion of various worms in a litter, as below :— (1) the striped (about +2), the common (about ,3;) and the pale (about ;1,); (2) the striped (about }), and the common (about }); (3) the striped (about 3) and the pale (about 1); (4) the striped (about 60%), the common (about 18%) and the pale (about 18%). SECTION IV. The fourth generation. We kept, in this generation, only the offspring of the striped and the common forms. 352 K. Toyama. : Number of worms. Parentage. | Eggs. | Cocoons. Striped. | Common. Pale. | Total. — a — — | | — I 175 fe) fe) 175 white only, Stripe. | (73-4376 (26.56%) | | , 2 07 34 oO 128 do, . | | Common. 2 fo) 261 oO 261 do “tripe. 4 | 269 fe) fe) 269 | do, | | | i.) (74.75 ) | (25.2 %) | 5 o 154 52 200 | do. Common. (78.57%) | (21.42%) 6 oO 143 39 182 | do. — et et (78.239 ) (21. 76%) | 7 115 fo) 32 | 147 do, 4. Stripe. | 8 | 156 fe) Oo | 156 do. Some of the striped parents produced only striped worms; others, striped (about 75%) and common (about 25%) worms; while the rest striped (about 75%) and pale (about 25%) worms. Similarly, some of the common marked parents produced uniform and others mixed (common worm, about 75% ; pale worms about 25%) offspring. This is a phenomenon very familiar to us in the previous setsei of experiments. We will give, as before, a short résumé as follows some. some. some. —— a ee The first striped striped+common striped+common+ pale generation: only. (50%). (50%). (50%). (25%): (25%): t The second striped + common + _ pale generation : 175%)- (18.75%). (6.25%). zy Ze 3 4. Q ——— ——. ———— a ‘The third striped+common+ pale striped+common striped +common + _ pale striped pale generation: (75%). (18.75%). (6.25%). (75%)- (25%). (57-8896). (19.19%). (18.12%). (about 752%).7 (252%). I | i The fourth striped (striped+-common) common striped (striped+common) common-+ pale ’ generation: only. (75%). (25%). only. only. (75%). (25%). (75%)= (25%)- Stadics on the Hybridology of Inscets. 353 Now we see with certainty that of the three larval markings under consideration, the striped character is first in the order of dominancy, the common ranks next and the *‘ pale” comes last. The respective hereditary relation is quite the same as observed in the case of the various colours of cocoons. It will be of some interest to note here that the striped form obtained from the crossing between the common Japanese form and the Siamese striped breed exhibits a pair of the common curved markings on the dorsal part of the eighth segment (PI. X, II.), where no such markings can be found in the striped Siamese breed. Some of them will remain true in the successive generations, and, therefore, it is possible to combine the two different markings into one form by crossing the two breeds. GAsk VIII. On a mosaic form obtained by crossing the common Fapanese white and the striped French yellow breeds. Before leaving the subject of the hereditary phenomena of the larval markings of the silk-worm, let us consider an interesting instance met with in one of the experiments before mentioned. In the Spring of Ig01, we reared some worms derived from the cross between the striped European breed (@) (Pl. VI, Fig. I, 4.) and the common Japanese one (@) (PI. VI, Fig. I, a2). Among them, we found two larvae (Pl. VI, Fig. II, a.) of great interest, that is to say, the left half of the body exhibited maternal striped markings and the right half the common paternal markings. The ‘mounting’ took place on the 29th, May, but one of them was destroyed by Ujimza. The other moth emerged on the 18th, July. In the following, we will try to give an account of the larvae and the moth above referred to. t. Phe lapyaee (Pl. -VI, Fig. IT, 2.) The body in the fifth age is creamy white in colour; the dorsal shield of the first segment is light pinkish-brown. On the dorsal portion of the 354 K. Toyama. first segment, we find a dark median line beginning at the middle of the second segment and gradually tapering towards the anterior end, where it nearly touches the base of the dorsal shield, and where there is a light dark spot on the left side but none on the right. The second segment possesses dorsally a pair of the eye-like markings and acentrally placed trapezoidal marking commonly found both in the common and the striped worms. The joints of the remaining segments are embroidered with a dark band in its left half, while the right half has the common markings. The dark dots or patches normally met with on the basal line of the body of the striped worm are present only on the left side, the one on the first segment being very faint, but nothing of this maternal character is to be found on the right side. Other markings common to the two parents can be observed on both sides of the body. 2. Thelmago. (Fig. II, 0.) When the moth emerged, it exhibited its male nature, fluttering its wings and trying to find a chance for pairing. On a closer examination, it was found that the two halves of the body showed different characters, as in the larval stage. The following description will serve to illustrate it :— a. The antennae. _ The colour of the right antenna was brownish on its dorsal side where it is covered with scales, while that of the left was grey or much lighter than the former. With respect to size, the former was larger than the latter ; the former consisted of thirty-five and the latter of thirty-six segments, excluding the basal joint. The pectinated branches arising from each of the antennal segments were longer and larger on the right than on the left. The figures representing the length of the pectinated branches on the tenth segment of both antennae will confirm it : , 27 mm. in the left ; ;7, mm. in the right. Studies on the Hybridology of Insects. 355 6. The wings. (Figs. II and III.) The wings had also a peculiar structure on either side. The size of the left fore wing (Fig. II, c,d) was larger than that of the right one (Fig. III, @,%); the length being 22mm. for the former and 20mm. for the latter. The left hind wing (Fig. III, @.) did not attain its normal development, exhibited an abnormal form, and was much smaller than its fellow. As regards the markings, the right wings (a, &), both fore and hind, had deeper and clearer markings than the left ones (c, 2), which had lighter and faint markings, showing only a trace of the eye-spot on the fore-wing and a dark spot on the inner margin of the hind wing. c. The abdomen. (Figs. IV, V.) In the left half seven somites could be counted and in the right, eight segments—these numbers are typical of the abdominal segments of the normal male and female. d. External genital armature. (Figs. IV, V and VI.) Before describing the mosaic form, it will be convenient for comparision to give a brief account of the normal form. 1. Female. (Fig. VII). The outer genital aperture is provided with several chitine-plates : dorsally it is guarded by a narrow and curved plate —the dorsal plate (a). This is arched down at both ends to surround an area, where a spherical process (0) covered with some short hairs—the ovipositor—projects. On the ventral side, there are two plates (c, d) placed one over the other; in the space situated between these two plates we find a pore, the copulating pore. In the centre of the space surrounded by those dorsal and ventral plates, the ovipositor is situated, as above mentioned. On its apex, there is a narrow median slit in which two openings, one dorsal (the anus) and the other ventral, (the ovipositing aperture) may be seen, and from both sides of its basal portion a pale-yellow bladder-like sac is everted in the same manner as the osmetrium in fafilio larvae. This is the alluring glands 356 K. Toyama. which secrete the odoriferous fluid to entice the male. And moreover, there is another thin plate situated at its ventral. 2. Male. (Fig. VIII.) It is surrounded dorsally by the thick margin (like a lip) (Fig. VII, a) of the integument of the last segment. This may be called “dorsal lip” and serves as a tactile organ. Ventrally, it is guarded by the chitinous expansion (?) of the integument as in -the case of the female. It has a triangular projection (7) which is well chitinised, and is situated on both sides. In the space guarded by these two portions of the integument, compli- cated armatures are present. First, we will mention the ana] armature. It consists of a dark square-shaped frame (d@), over which a well-developed chitine ‘plate—the cover plate (c)—is situated. The latter is joined to the former at the dorsal base, where it is supported by a dark plate (the basal plate) and may be lifted up at will. In the central soft space surrounded by the frame the anus is situated. Secondly, the sexual armature. At both sides of the anal area, we find a strong ventrally curved chitinous hook—the clasper (¢). Near the ventral base of the clasper, there is again a projecting triangular plate (/) slightly curved inward. These two are the accessory apparatus for copulation. Lastly, the sexual apparatus. It is situated in the median portion, under the anal area and projects as a needle-shaped organ—the penis, around the base of which a chitinous area is well developed. Having thus given a brief outline of the structure of the genital armature of the normal moth, we may now proceed to the observation of the structure of the mosaic form. Fig. IV represents the dorsal portion of the abdomen. In the left half, we Can enumerate seven and in the right, eight segments, the latter projecting a little beyond the former. In the terminal segment of the left half (Figs. IV and V) we ean observe the dorsal chitine plate (dc) and one-half of the ovipositor fev), from the left base of which an alluring gland (g) is prescat. On the other hand, we can sce those male apparatus, such as the clasper (Figs. IV, e, and V, ¢) the cover plate (Fig. IV, ¢) the triangular projection ( £) of the ventral plate etc. on the richt side. Lat N Studies on the Hybridology of Insects. ae The details concerning these various apparatus in the two halves are shown in Fig. VI. It represents the frontal view of the terminal portion of the abdomen. On the left side of the figure, the median line being the boundary, we see the dorsal portion (a) of the seventh scgment, under which the dorsal plate (c.) is situated. It ends abruptly in the median line, while on the left side it arches down around the basal portion of the ovipositor (g’). Near the middle of the median line, we find the half of the ovipositor (¢’), at the left base of which the alluring gland (@) projects. Ventrally situated to the ovipositor and the alluring gland there are two plates (¢. f) which also end near the median line. These are the two ventral plates commonly met with in the normal female moth (compare Fig. VU, ¢, @). Now we will turn to other half. First, we observe the well developed dorsal lip (4’) on the dorsal margin. The anal apparatus which can be observed only in the male come next. As the figure represents, the square frame (s), the cover plate (f) and the basal plate (c’) are developed only on the right side and exhibit one-half of the normal form. In the right side of the anal area, there is a strong and well developed clasper (@’) below which we find the triangular accessory plate (2). Different from the other apparatus it is represented on either side by a small triangular projection (%. £). In the Paedein portion below the ovipositor and the anal area, there is atrace of chitinous portion (1) guarded by a chitinous area (/) on its right side. There is the rudiment of the penis. On its left side, we meet with a thin plate (7) which commonly occurs in the normal female. Now we see clearly that in this case one half of the worm shows the maternal, and the other half the paternal, characters. Similar cases have been enumerated by Darwin, who says :-— “ According to Rengger, the hairless condition of the Paraguay dog ts either perfectly or not at all transmitted to its mongrel offspring; but I have seen one partial exception in a dog of this parentage which had part of its skin hairy, and part naked, the parts being distinctly separated as in a piebald animal.” He proceeds still further saying that “‘ when Dorking fowls with five toes are crossed with other breeds, the chickens often have 35¢ kK. Toyama. five toes on one foot and four on the other. Some crossed pigs raised by Sir. R. Heron between the solid-hoofed and common pig had not all four feet in an intermediate condition, but two feet were furnished with properly divided, and two with united hoofs.” With bees, similar cases have been observed by Kraepelin (°73) and others. ! From the above facts, we may safely conclude that when a commingling of two characters takes place as a result of crossing, it may occur that all the parental characters separately occupy one half of the body of the offspring, even sexual characters being no exception. On the colour of the eggs. When we got four different kinds of cocoons, white, light greenish white, pale-pinkish yellow and pure yellow, from a crossing of the Japanese white and the Siamese yellow, we kept them separately, as before stated. The following observations were then made. The eggs of each of these kinds have a characteristic colouration, that is to say, those of the pure white form are always light pale-yellow, while those of the yellow form are clear yellow. Curiously enough, however, those of the pale-pinkish-yellow are of a deep yellow colour, with some brownish shade. Those of the greenish white have a deeper colour than that of the pure white, but its difference from the latter is so slight that we can not always distinguish them clearly. During the whole series of our experiments, we paid particular attention to this point and have convinced ourselves that this relation holds good in every case. Thus it becomes very easy for us to foretell from the colour of the eggs what kind of cocoons the worms hatched would spin. 1 Standfuss describe analogous cases in some L-epidopetra in his “ Synopsis of Experiments in Hy bridization and temperature made with Lepidoptra.” Caspari IT. made a description regarding a similar case of Sat, Pavonia. Coutague (02) alse found three silk-worms which were black on one side and white on the other. Studies on the Hybridology of Insects. 359 Of these four colours of eggs, the yellow predominates over the other three. Next comes the brownish-yellow, and these colors when brought together by crossing are governed by the same law that regulates the hereditary phenomena of the various colours of cocoons. For instance, when we crossed the yellow breed with the pale-pinkish-yellow, the eggs laid by the cross-bred offspring were yellow, and these in the next generation split up into two, one yellow (75%) and the other brownish-yellow (25%). @reor, | X. On the cocoon. Leaving, then, the question of heredity as regards the colours of cocoons and eggs, and the larval markings, we shall next enquire how the construction of the cocoons is affected by crossing. The following statements which are an epitome of the results of experiments, Case II and III will give some idea about it. Before entering into the subject, it seems better to give an account of the cocoons of the parent-breeds. The cocoon of the Japanese breed (Pl. VII, I, a.) is white in colour ; cylindrical or oblong in shape, with rounded ends, and a constriction in the middle. The floss is very small, amounting only one or two milligrams in each cocoon; the texture is compact and strong, with nice wrinkles or “ grains’ on the surface. Its size, quantity of silken matter and fineness of the filament are as follows :— ; AVerasememoin.............-....2Gs3 mime Size of cocoons \averapemmecadth . ...>..-22..-- 15.1 mm. ma avetare SUKEN Matvemmee.........-.s-:05-t0c-5 0.19 gf. 2 ae epee 0.021 mm. Diameter of the filament Me. of cocoon...0.033 mm. | mrelette ’'....-.22-:- 0.020 mm. That of the Siamese breed (PI. VII, 1, 4.), on the contrary, is spindle shaped, with the two ends, or only one of them, pointed; without any constriction in the middle. The texture is very loose, showing no wrinkles 360 K. Toyama. on the surface. Around the cocoon there is abundant floss silk (0.02-0.03 gr.) which forms a loosely wound fibrous matrix in which the cocoon is embedded. The following figures give the size, quantity of silken matter etc. = average length ...... 28-30 mm. Size of cocoon (average breadth ...... 12-13 mm. Averace silken marten me: ........3: 0.07-0.10 gr. BYOSS. «20. c0r¢ 0.0195 mm. 2 middle of Diameter of the filament cocoon...0.0262 mm. Ptelette ’...0:0164 mm. We will now examine the cocoon of the cross-bred form. a. The form of the cocoon. In the first cross generation (Pl. VII, 11), most of the cocoons spun were spindle or conical in shape, very few of them being oval or ellipsoidal ; none of an oblong form with rounded ends, like the Japanese breed.! In the next generation (Pl. VII, I), however, we obtained various forms of which we may first mention those of conical or spindle shape, which are most abundant, next comes those of an oval or ellipsoidal shape. It was sometimes found that both forms had a trace of constriction in the middle. Besides these there were some intermediate forms which could not exactly be distinguished from one another. The various forms of cocoons appeared in the second generation, as far as we have experimented, have the inclination to separate from one another and segregate into several constant forms, when reared separately from one another. Thus after selection for three consecutive generations, the oval or ellipsoidal form became a constant form (PI. VIII, and IX, If); on the other hand, it is very difficult to get a pure spindle form, since some other forms always appeared among them (PI. IX, 1). With respect to the Japanese oblong form which appear very rarely as an active character, we have failed to trace its ultimate fate by inheritance. 1 In the reciprocal crossing between ‘ Changhai Blanc’ and ‘Jaune Var,’ Coutagne (’02) recorded some similar facts, Studies on the Hybridology of Insects. 361 Now we may say that of the forms enumerated above, the spindle or conical form predominates over the other forms. Next ranks the oval or ellipsoidal form, and lastly comes the oblong or cylindrical form with rounded ends. g. Eloss. The floss gave the following figures in the first cross generation :— Pure Japanese paremt.......... 0.00 I-0.002 gr. Pure Siamese. patetiv...:...... 0.020-0.030 er. Monerelpatent-esee-.......... 0.007-—0.017 gr. In the second and further generations, a great deal of individual differences among the worms reared from the same parent or among the worms derived from different parents, appeared. The following figures obtained in the second, third and fourth generations will bring out this point :— I. (0.0065) 2%. 2. 0.0045 gr. 3. 0.0085 gr. 4. 0.0035 gr. 5 0.0155 gr. 6: 0.02202. The second generation (From the samiegoapent.) .....:..<.-...- | H | | | 7. 0.0380 gr. 8. 0.02508 | Q: , OOL ES; 2t- \. 10: ‘OM110 S 0.012-0.026 gr. Parent, No. 1. average 0.0176¢gr. The third Seneratipn . 25. ts J 0.006-0.012 gr. Parent, No. znd average 0.009 gt. ; { 0.007—0.0195 gt. Parent, No. 3-+-j average 0.0127 gr. 0.009-0.019 gr. Parent, No. a ‘average 0.0162 gr. WwW oO’ i) K. Toyama. e 0.010 —0.0185 gr. Parent, No. 1..4 is laverage O.O1 52 gr. E 0.027 -0.009 ¢g Parent, No. 2. 5 average 0.0152 gr. Parent, .Ne: 35 { O.O0151—-0.029 gr. average 0.0183 | OF DS 0.014 —0.033 gt. Parent, * Noz 4: ‘i oO D> average 0.024 Although we have failed to establish a constant form from these crossings, yet we have good reason to believe that they will follow the same law regulating the hereditary phenomena of the various forms of cocoons, and that the character to spin much floss is a dominant one. c. The constriction. This is one of the most unstable characters of the cocoon. In the first cross, very few of the cocoons have a trace of it. In the second and further generations we have observed its rare occurrence as an active character, but it is always not well developed. In consequence of the scarcity of materials we could not arrive at a definite conclusion with respect to its hereditary phenomenon. d. The size of the cocoons. It is pretty constant as the following figures shows :— i length ...29.9-33 mm. The first generation...... average breadth...12.5-15.5 mm. ‘ average length ...29 -—36 mm. The second generation.. | average breadth...13 -—16 mm. From the third generation on, however, there were found some individual differences, which may be mentioned as follows Studies on the Hybridology 6f Insects. 363 i 3 ss > the Average : Average SOLES Sus 2 ly Length, 2 Breadth. : age worms reared : length. | breadth. from a parent. | o B | mm. mn, | mim. mim, | 1 > | | noo - No. 10 29-31 | 30 12.5-14.5 | Less - No, II. 30-34 32.3 14-15 14.5 | No. 12 | 28-31 29 | 1225s 12.6 No. 13. 27-31 29.1 12.5-13.5 13.16 EE ————— Ee eg. Silken matter. It is intermediate in quantity and remains in a pretty constant state. In the first generation, we got the average figure 0.1329 gr. which in the second and further generations varied between 0.1264 and 0.1028 gr. f. The diameter of the filament. This also showed an intermediate character between those of the parents. In the first and second generations we obtained the following average figures :— Diameter of Diameter of the Diameter of Generation. | the floss. filament in middle. | telette. | First. 0.0145-0,0266 0.0218-0,0303 0.0096-0.0218 Second. 0,01 21-0,0230 0.0218-0.0314 0.0096-0,0215 See Worms were kept until the fifth generation, when we got the following figures :— ww lon SS K. Toyama. Diameter Of outer nesses. ....... 0.012—0:022 mm: Diameter of the filament of the middle of cocoon....... 0.0218-0.030 mm. Diameter of ‘ telette’............... 0.010 — 0.020 mm. g- The texture of the cocoons. The texture of the cocoons becomes correspondingly compact and strong with the increase of the silken matter. h. \Nrinkles or ‘ grains’. This is also one of the most unstable characters. During the first three generations, a trace of it could be observed in some cocoons. In the fourth and fifth generations it nearly disappeared. Of these characters, those mentioned in the paragraphs e—/ are much influenced by the quality of the leaves given, management, climato- logical conditions etc., and we could not know exactly how much of the change produced was due to the crossing and how much to the influence of management, nutrition etc., and therefore, it is very difficult to draw any definite conclusion as regards the effect of the crossing. Studies on the Hybridology of Insects. 305 B. SUMMARY AND GENERAL CONSIDERATICN. I. Monohybrid. Looking back through the whole course of the hereditary phenomena of the various colours of the cocoons and _ larval markings of the silk-worm, we may summarise them, in the case ot monohybriad, as follows :— The first cross generation ............. CEE EERE tees Se daNinentetceead sche aacseneteneneee Uniform offspring (dominant) “IMINE: SSCL EA he ee OEE RRO aE dco. Sor dark eee eee eee Mixed offspring. (Dominant 75 % + Recessive 25%). A A = | The third ,, Aap ae ee ae * somet some! "al ae uniform, mixed. uniform. (ena ‘Dominant Recessive Recessive only. ) ( 75% : 25% ) ( only ) | | | The fourth ,, ,, some? some2 ~ “some! some? al = allies uniform, mixed. uniform. mixed. uniform. uniform. (D. only) (D 75% +R. 25%). (only D.) (D. 7596 +R. 25%). (only R.) (only R.) This shows such a close parallelism with the law of heredity first enunciated by Mendel (65) in Pzswm and Phaseolus, that without quoting his statement here we would not be able to get a better comprehension of the matter. In the first cross generation,* Mendel says: ,, das eine der beiden Stamm- merkmale ein so grosses Ubergewicht, dass es schwierig oder ganz unméglich ist, das andere an der Hybride aufzufinden. In the second generation, , treten nebst den dominirenden Merkmalen auch die recessiven in ihrer vollen Eigenthiimlichkeit wieder auf, und zwar in dem entschieden ausge- sprochenen Durchschnittsverhaltnisse 3:1, s0 das unter je vier Pflanzen aus dieser Generation drei den dominirenden und eine den recessiven Charakter erhalten.‘ He further says that ,, Jene Formen, welche in der ersten 1 These numbers are not constant, sometimes 55% : 459%, sometimes 75% : 25%, etc. 2 These are also not constant. We once got figures; 80% of uniform and 20% ot mixed offspring. ; 3 Our first cross generation corresponds with Mendel’s hybrid, his first generation with our second cross generation, Los) oO’ Orv K. Toyama. Generation den recessiven Charakter haben, variiren in der zweiten Genera- tion in Bezug auf diesen Charakter nicht mehr, sie bleiben in ihren Nachkom- men constant.“ With respect to the dominant form appearing in the second generation, he finds ,,dass von jenen Formen, welche in der ersten Generation das dominirende Merkmal besitzen, zwei theile den hybriden Charakter an sich tragen, ein theil aber mit dem dominirenden Merkmale constant bleibt.‘ And therefore, ,, Das Verhaltniss 3:1, nach welchem die Vertheilung des dominirenden und recessiven Charakters in der ersten Generation erfolgt, lést sich demnach flr alle Versuche in die Verhiltnisse 2: 1:1 auf.“ Thanks to the interest taken in the subject by such eminent botanists as De Vries (00, 03 Agvrostemma, Chelidonium, Hyoscyamus, Lychnis, Oeno- thera, Papaver, Zea, Datura, Trifolium etc.), Correns (’00, ’o1, Pisum, Zea), Vschermak (’oo, ’o1, Prsum, Phaseolus) and others a great deal of light has been recently thrown on this aspect of the problems of heredity. In animals, studies of poultry (Bateson ’o2), mice, rats (Cuénot ’o2, ’03, 04; Castle ’03; Allen 04; Davenport ’04), rabbits (Castle ’03), Helzx (Lang ’o4) etc. have yielded further evidences of this principle. Our results with silk-worm crosses, as Will be seen, correspond very well with the Mendelian principle above cited, the discrepancy being seen only in some points of detail and isto be attributed to the sexual union of different individuals. Firstly, Mendel and_ others proved that one-third of the dominant forms appearing in the second generation remain uniform and constant, and the remaining two-thirds split up again into the parent forms in subsequent generations. This is not the case with the silk-worm. We have not been able to get the constant proportion 1:2 between them. Secondly, in plants, the dominant form appearing as uniform offspring in the third generation remains true, generation after generation. With the silk-worm, on the contrary, some of the uniform offspring remain constant while others break up into the parent forms, like the dominant form derived from a mixed offspring. Such differences concerning the separation of the pure dominant and hybrid dominant forms may be illustrated as follows : Studies on the Hybridology of Insects. 367 In the second generation, as Mendel taught us, there are two kinds of dominant forms, one pure or D and other hybrid or DR. As there is no means to distinguish D from DR, mating between these two forms take place at random. From these random matings we may reasonably expect to get the three combinations. ie By BE 3.— Wea, 2, WR SOD Both Dx D and Dx DR produce only dominant offspring, while one of them (Dx DR), when mated zzter se, will produce mixed offspring. Similarly from such random matings it will not be expected to produce pure yellow or D and hybrid yellow or DR in a constant proportion. Now we may justifiably assert that the yellow character is dominant and the white recessive and the phenomena of the segregation of the two characters are quite the same as in the case of plants observed by Mendel and others. Cuénot (02) and Allen (’04) tested the pigmented mice of the second cross generation and found the Mendelian expectation 1D:2DR: 1R realised. It is highly interesting to note that Coutagne (’02) obtained a result diametrically opposed to mine by crossing white and yellow breeds, such gam blancs de’ Alpes’, ‘ Bagdad Jaune Var’, ‘ Jaune’ Defends’, “Petit blanc Pays’ etc. In most cases, the white colour dominates the yellow and the offspring is white, yet the resulting hybrid-white, when bred znfer se, produced both white and yellow offspring, in some cases in the ratio of 1: 3, im.obhers 1: 1 There are, however, many irregular cases recorded by him. For instance, in reciprocal crosses between ‘ Changhai blanc’ and ‘Jaune Var’, the yellow character behaved as dominant. While ‘Jaune Defend x Petit blanc Pays’ produced uniform white offspring, the same yellow race mated with ‘ Bagdad’ (a white race) yielded only yellow cocoons. In dihybrids, he observed more interesting phenomena of segregation and combination of the parental characters. In crosses between ‘ Blanc des Alpes (worms white, cocoons white) and “Jaune Var’ (worms striped, cocoons yellow), the first cross produced four kinds of worms : 308 K. Toyama. OQ’ =striped worms spinning yellow cocoons. 116. OQ” =white worms spinning yellow cocoons. 124. OQ’ =white worms spinning white cocoons. III. O’""=striped worms spinning white cocoons. 108. This is an instance of (D+R)R. In the next generation mated among similars, O’ yielded four kinds of worms in the following proportion : Striped worms spinning yellow cocoons 236= 53.75% Striped worms spinning white cocoons 80=18.22%, White worms spinning yellow cocoons 89=20.27% White worms spinning white cocoons 34= 7.74% O” yielded White worms spinning yellow cocoons 441, White worms spinning white cocoons 120=24.24%, O'” yielded White worms spinning yellow cocoons 140=25.97%, White worms spinning white cocoons 399, And lastly O””" produced Striped worms spinning yellow cocoons 109= 26.45%, Striped worms spinning white cocoons 180=43.63%, White worms spinning yellow cocoons 36= 8.73%, White worms spinning white cocoons 87=21.11%,. In the former two forms, O’ and O” the yellow character is dominant while in the latter two, O’” and O’/” the white dominant. Thus he says “les mnemons du caractére ‘Cocon blanc’ n’ont pas, dans toutes les races a cocons blancs, la méme force de transmission hereditaire. Les mnémons des races ‘ Blanc des Alpes’ et ‘ Petit blanc pays des Cévennes’ sont plus forts que les mnémons de la race, ‘Jaune Var’; mais inversement les mnémons de la race, ‘Jaune Var’ sont plus forts que les mnémons de la race ‘ Bagdad’. Similar cases may be quoted from the albino character (which is ordinarily recessive) of Mice and Matthiola etc. (Bateson and Saunders, ’02, Studies on the Hybridology of Insects. 369 Castle, ’03, Cuénot, ’o2, Allen, 03, '04 etc.), since it sometimes behaved as dominant. Hence we may say that such instances occur not rarely in animals and plants. The principal cause of the discrepancy between the results of Coutagne and of mine, however, seems to rest on the neglect of ancestry of the breeds, because in the former case the breeds used for experiments were derived from various breeds having different ancestry, even hybrid ones are used, while in the latter the lineage of the breeds is quite pure and simple. It is also very instructive to quote Castle and Allen’s hypothesis of “impure recessives’’ in which’ case the pigment-forming character (which is dominant) is either partially or completely latent. The white character studied by Coutagne might belong one of such instances. Il. Back-crossing. Wet us next consider a case of back-crossing a cross-bred form with ore of the pure parent-breeds, for which Experiment Case IV furnish a good illustration. a. First crosses of the cross-bred yellow with the pure white gave two kinds of offspring, one producing uniform yellow worms, another a mixture of yellow and white worms in the proportion of I : 1. In the next generation paired zzter se, all the yellow forms were disintegrated into their parent-forms in the proportion of three yellows to one white, while the white form remained true to the parents. A precisely similar result was obtained with the various larval markings of the silk-worm (Case V). Of the larval characters, Coutagne’s (’02) results also confirmed that both black colour and transverse striping are evidently dominants to the normal whitish colour. 6. On the other hand, from crosses between the cross-bred yellow and the pure yellow forms, uniform yellow offspring resulted through two consecutive generations. According to the Mendelian principle, they should be that: a. When cross-bred dominant forms are mated with pure recessives, The first generation, (D+R)R=one half hybrid dominant or yellow and the other half pure recessive or white. 370 K. Toyama. and The second generation mated among similars, 1. (D+R)x(D+R)=one-third pure recessive or white, two-thirds dominant or yellow. CART OSA 3 =all recessive or white. 6. When cross-bred dominant forms are mated with pure dominants, The first, generations yee): . (D+R)D=all dominant or yellow. The second generation paired infer se, (1), Daas (ia R x DR); (3) (DXDR; or some (1) pure yellow offspring, some (2) mixed offspring of white, and yellow, and the rest (3) a mixture of pure and hybrid yellow worms. Such aberrations may be explained as follows :— In our experiments, the cross-bred yellow forms were derived from a mixed offspring of the fourth cross generation between white and yellow breeds and consequently some of them were pure yellow or D while the others were hybrid yellow or (D+ R). By crossing with white or R, therefore, there will be produced the combinations, 1. DxR=hybrid yellow and 2. (D+R)x R=DR+R=a mixture of hybrid yellow and pure white in the proportion of I: 1. Thus the yellow forms raised from both formulae, when bred together will certainly break up into their components according to the normal monohybrid formula, (D+R)(D+R)=D+2DR+R=a mixture of three dominants and one recessive. The results actually obtained agree very satisfactorily with this assumption. | In the cross with the pure yellow, on the other hand, we expect to have the following combinations :— 1. Dx.D or pure yellow; 2. (D+R)xD=D+DR or uniform yellow consisting of one pure yellow and one hybrid yellow. Studies on t'e Hybridology of Insects. 371 The former combination DxD will remain constant through further generations, while the latter may produce the three combinations of the parental characters in the next generation : 1 DxDR=Dx(D+R)=D+DR=a mixture of pure yellow (50%) and hybrid yellow (50%). 2. DRxDR=D+2DR+D=a mixture of two hybrid yellows, one pure yellow and one pure white. 3- DxD or pure yellow. There might be chance oniy for the first and the third combinations to be reared in the second generation, if we keep for the experiment a portion of the offspring descended from the first generation. This assumption may serve as an illustration for the second case. From these considerations, we are led to the Mendelian principle cited before. Ill. Dikybrid. From the reciprocal crosses between the ‘ pale yellow’ and the ‘striped white’ breeds (the latter is not pure, sometimes pale worms appear), we have obtained interesting combinations and segregation of the parentai characters, in the offspring (Case VI). In the first cross there appeared two kinds of offspring, the first being all ‘ striped yellow’ worms, the second being a mixture of ‘striped yellow’ (50%) and ‘ pale yellow’ (50%) worms, which is a common phenomenon in a back-crossing already described. With respect to these two kinds of worms thus raised we learn that the ‘pale yellow’ form was disintegrated, in the next generation, into two forms, ‘pale yellow’ (75%) and ‘pale white’ (25%), as in the second generation of monohybrid, while the ‘striped yellow’ form produced four kinds of worms, ‘striped yellow’, ‘ pale yellow ’, ‘striped white’ and ‘ pale white ’ in the following proportion :-— ‘Striped yellow’ worms (new form). 55.57%. ‘Pale yellow’ worms (parent form). 18.35%. In the first group - ‘ A ‘Striped white’ worms(parentform). 19.72%. '‘Pale white’ worms (latent form). 6.34%. 372 K. Toyama. ‘Striped yellow’ worms (new form). 58.82%. ‘Pale yellow’ worms (parent form). 18.45%. In the second group} _ Past i | Striped white’ worms (parent form). 16.42%. ‘Pale white’ worms (latent form). 6.28%. In the third generation, each of these four forms produced the following kinds of offspring, when bred together with similars in the same brood. a. A mixture of ‘striped white’ (25%) and ‘striped yellow’ (75%) worms. 6. Only ‘striped yellow’ worms. 3 a eee A mixture of ‘ mg aa yellow (74-487) and ‘pale yellow’ (25.519) worms. d. A mixture of ‘striped yellow’ (56.2%), \ ‘striped white’ (15.44%), ‘ pale yellow ’ (20.6% ) and ‘ pale white’ (7.59%) worms. a. All ‘pale yellow’ worms. 2. From ‘pale yellow’ forms | A mixture of ‘ pale white’ (25%). and ‘ pale yellow’ (75%) worms. a. All ‘striped white’ worms. 3. From ‘striped % - A mixture of.‘ pale white’ (25%) white’ forms | I 5/0) and ‘striped white’ (75%) worms. 4. The ‘ pale white’ form produced only offspring like the parents. Results closely similar to these have been disclosed by Mendel in Pisum, and by De Vries, Correns, Tschermak, Saunders etc. in various kinds of plants. As. regards some discrepancies existing between our results and those of some botanists above referred to, the following formulae derived from the Mendelian conception would give a clear explanation and serve as a verification of our result. Let the characters be represented by A=yellow character, B=striped character, a =white character, 6 =pale character. Studies on the Hybridology of Inseets. CVA: Then the ‘ pale yellow’ breed would be represented by AJ, the ‘ striped white’ by @B. As the latter form is not a pure strain it may be either a Bor (a B+a B). . | In the first reciprocal cross between them we shall get the combinations, a. A&xaB or uniform ‘ striped yellow ’ offspring. 6. Ab(aB+ad)!=a mixture of ‘striped yellow’ and ‘ pale yellow’ in the proportion of 1: 1. In the second generation, (Ad+aB) or the ‘striped yellow’ form, when bred zxzter se, will give the combinations, (Aé+aB)(Ad +aB)=(A+2Aa +a)(6+26B4+ B)=Ab+ ab +AB-+ Ba+2Aaéd+2AaB + 2aBé + 2ABO + 4AaBé, From which we estimate the following figures :— ‘Striped yellow’ form (AB, 2A@B, 2ABd+4AaBd). 56.25%. Sealesyellow, forms (AG-b2NA)o.. <.s..0s. nese uece- 19-7506: Souniped, white * forms (Ba; @aiem).....2..-..0.._c.s2es-+0- 18.75, %- Signer witihe OTIS (27). eee: .-.5- 0 cence. (4 striped 12 or 75%. ~ 3 - - OY Common 3%, or 18.75%. 2 5 2 | Stele! 4 Pale (fy) Ot 0-25 %- This may serve to illustrate the phenomenon of the segregation of the parental characters in the second generation. The striped form raised from this brood when paired zxfer se produced many kinds of offspring, each of which has a different proportion of the various kinds of worms, as in the case of the colour-characters ‘of the cocoon before mentioned. In the latter case, however, the combination ‘ yellow + pale-pinkish-yellow +greenish white and pure white” is natural while in the former the combination “ striped +common-+ pale” is artificial, but the resulting phenomena are quite the same in both cases. Furthermore, we see that as a result of crossing between the Japanese common form and the Siamese striped form a new constant form having both parental characters commingled has been produced! (PI. X, II). Coutagne (’02) observed a parallel case in some crosses between “ Bagdad vers noirs”’ and striped “ Jaune Var” in which both black and striped characters appears as an active character. We observed the same phenomenon in crossing various Chinese races of the silk-worms. The facts above enumerated together with De Vries’ results with Antirrhinum may afford excellent illustrations for the hybridological analysis and synthesis of plants and animals. With regard to the spotted mice, Castle and Allen (’03, 04) developed the ‘mosaic’ theory of gametes, but whether it may be adopted to our worms we must wait the result of further experiments. These very facts and considerations referred to in the preceding paragraphs furnish a further welcome proof for the correctness of the Mendelian theory and confirm that his theory may be applied, with equal exactness, both for animals and plants. 1 Such an example is seen in the case of the ‘walnut’ comb of poultry, produced by crossing rose-comb with pea-comb poultry. (Bateson ’o5). 380 K. Toyama. VI. Non-Mendelian phenomena of heredity. Leaving the Mendelian cases, we come now to consider the non-Mendelian group of hereditary phenomena. Mendel (’69) has already confirmed in the Hzeraczum crosses that two or more different forms appear in the first cross; each of which when bred inter se come true to its parent in subsequent generations. Millardet’s false hybrids (94) which ordinarily show only the character- istics of one of their parents and come true to this type in later generations may be mentioned as another example. Recently a good number of non-Mendelian crosses together with Mendelian cases! have been discovered by many eminent naturalists. . De Vries (01, ’03,) who has done some epoch-making works on the physiology of heredity has observed many cases where the Mendelian law does not hold good and says that ,, Den Mendel’schen Spaltungsregeln folgen im Allgemeinen nur phylogenetisch jiingere Eigenschaften, sogennante Ras- senmerkmale ; von diesen aber wiederum nur ein Theil. Welcher Theil, weiss man aber auch jetzt noch nicht.“ Correns (o1,” 01”) has crossed various kinds of Zea mays and enumerates four different types of hereditary phenomena of various characters: the first is the pzsum type in which ,,das Merkmalspaar ist heterodynam und schizogon; the second, ,,das Merkmalspaar ist heterodynam und homéogon ; the third Zea type in which ,, das Merkmalspaar ist homédynam und schizogon “ and the last Méeractum type in which ,, das Merkmalspaar ist homodynam und hom6ogon “. Tschermak (’00, 01) has observed some similar phenomena in crossing various kinds of peas and beans. In crossing Lychuis, Saunder (02) also finds that the leaf-characters obey Mendel’s law while the colour of the seeds and corolla, the position of the capsule teeth do not follow it. Davenport (04) also enumerated some non-Mendelian cases and says: “while Mendelian principles seem applicable to some cases of crosses 1 A good illustration of blending inheritance is found among rabbits and guinea-pigs by Castle (705). Studies on the Hybridology of Insects. 381 between sports and the normal species, there seem to be others where neither Mendel’s nor Galton’s law of inheritance holds.” Our results with the silk-worm crosses may add another example to the above category,' since, as has been said before, some characters, such as the colour of the cocoon, the larval markings, are governed by Mendel’s law, whilst others, such as the form of the cocoon, exhibit quite different phenomena of heredity. The brood-character of the silk-worm, such as _ univoltine, divoltine, multivoltine etc. is another example of non-Mendelian characters. Thus when we crossed a multivoltine with univoltine breed, the eggs laid by the moth were either pure maternal or pure paternal, very rarely a mixture of both parents as the following table shows :? (a. Japanese univoltine @ x Divoltine 2. | Eggs laid were all divoltine or maternal. He Japanese univoltine & x Divoltine @. \ i Eggs laid were all univoltine or maternal. a. European univoltine yellow x Japanese divoltine @. All divoltine, or maternal. 9) 6. European univoltine yellow 2 x Japanese divoltine @. All univoltine or maternal. 3. Japanese univoltine 2 x Divoltine @. All divoltine or paternal. Japanese divoltine 2 x Siamese multivoltine @. All multivoltine or paternal. Japanese divoltine @ x Siamese multivoltine 2. All univoltine or paternal. Those forms raised from the first cross do not remain true to the parents in subsequent generations. Even when we selected multivoltine forms for five generations we failed to get any constant multivoltine breed. Summing up the results of this very hasty survey, we may assert that of various characters of a species or variety of animals and plants some are 1 According to the experiment of Coutagne (’03) the character ‘“‘ richesse de soic”” shows, to all appearances, a non-Mendelian instance, not undergoing any sharp gametic segregation. 2 The results of further experiments show that the first cross is always maternal in crossing pure breeds. (Note, added May, 1906). $2 K. Toyama. Oo governed by Mendel’s law, while others follow other laws which can not be so clearly formulated as Mendel’s. There are, however, many irregular cases observed by many naturalists. Even those characters which are governed by Mendel’s law exhibit apparent exceptions from the general rule, as Correns (’02) has observed it in Maize. From his extended experiments with poultry, Bateson (’02) also says ; ‘*even those characters which follow the Mendelian law, sometimes produce aberration or oscillation and that not only different individuals similarly bred may give different proportion, but that these proportions may change also at different times ”’. Some coat colours of mice possibly come under the similar category. While Cuénot (02) states that the gray and white characters follow Mendel’s law, Darbishire (’03) finds that in crosses between a peculiar race of partial albino mice and true albinos, the albinism does not entirely disappear in the offspring. Moreover, his results indicate that not all albinos breed alike when crossed with the same pigmented stock. Similar results are recorded by Castle (’03), Allen (’03, ’04) who distinguished two kinds of recessives, pure and impure. Spotted mice, guinea-pigs, rabbits! recorded by Castle and Allen (’03, ’04) and others may add another example, since spotted rats or mice are crossed either with gray individuals (dominants) or with albinos (recessives) the offspring are commonly all gray or black in colour, none spotted. Such an individual is called a ‘‘ mosaic” by Castle and Allen. A like result also is recorded by Davenport (’04). The result obtained by Wood ('03) with rabbits? is far from the Mendelian expectation. Much more complicated phenomena have been discovered by Saunders (02), who says that in the matthiola crosses the result obtained are so complex that it is difficult to draft statements which shall give a precise and comprehensive view of the phenomena. In some extreme cases, two 1 See Castle’s works “Heredity of Coat characters in guinea-pigs and Rabbits, and Recent discoveries in heredity and their bearing on animal breeding.” 1905. (Added May, 1906). - 2 Castle (’05) finds that there are some coat-characters which conform in their inheritance to Mendel’s law of heredity, while the lop-eared condition is probably a non-Mendelian character in its relation to normal ears (Note, added May, 1906). Studies on the Hybridology of Insects. 383 sister plants belonging to the half-hoary type (dominant form) when crossed with the glabrous form (recessive) -produced 72 plants all glabrous, although when crossed with another glabrous strain the same two individuals gave the usual results.? Furthermore, she says that with respect to the leaf-character and seed- colour the dominancy is not absolute in Matthiola and thus in crosscs of pure dominant forms intermediate or recessive sometimes appear. Examples may be increased when we quote the statement of Tschermak (o1) who says that in peas and beans ,,in der ersten Generation die Lang- form der Hiilse in dem einen Falle Dominanz, in anderen Gleichwerthigkeit, ahnlich die Schmalform. Die langspitze Form war gar in einer Combination dominant, in der Anderen (fast) recessiv. Die Walzenform des Samens (Zweiter Generation) einerseits dominant, anderseits recessiv, in einer dritten Verbindung gleichwerthig : die Langform das einemal recessiv, das andere- mal dominant : das Merkmal ,, gedriickt “‘ recessiv, beziehungsweise gleich- werthig.“ Correns (’00) also enumerates many non-Mendelian cases in Matthiola. Quite recently McCracken (’05) has published an interesting paper concerning some crosses of Liza Lapponica. Although he says that it shows no exact parallelism to the Mendelian result, yet when his result is compared with ours, we shall at once be struck with the similarity between them. The principal discrepancy lies in two points: firstly that in the first cross (Table I) the recessive parents produced some dominant offspring and secondly that in the expected case of (D+R)R (Table VIII) an unexpected proportion of recessive forms appeared. Such may happen from the impurity of the character chosen for the experiments. Lastly we shall consider the results of Standfuss (96,00), who has crossed various spccies of Saturnia, suchas S. pavonia, ‘s pyrt, S. spint. etc. He did not refer to the Mendelian principle, yet his illustrations (Plate II) 1 Bateson, Saunders and others (05) find that even ‘albino’ plants gave coloured offspring and they consider it to be reversion. We are inclined to believe that concerning the character ‘albinism’ there exists a close parallelism between the results obtained by Bateson and Saunder (with plants )and those obtained by Castle and Allen (with Mice and Rabbits). (Note, added May, 1906.) 384 K. Toyama. give us some notions concerning the hereditary phenomena of various characters of these insects. With respect to the larval markings, we see that the spzz7-form predomi- nates over those of favonza and pyri, and that of Pavonia over that of pyrz. The produced forms, however, do not exhibit uniform dominant character but certain variations are to be observed. A similar phenomenon has been observed by myself in the crosses between Bombyx mori and Theophila Mandarina. With regard to the structure of the cocoons we may again observe that the characteristic architecture for the exit-hole of the cocoon of Pavonia cr pyri predominates over that of spzmz (see his Figs. 1, 2, 3, 4, 5), while the general form is intermediate. Owing to the scantiness of the worms reared by him from a brood, we can not draw any exact conclusion with regard to this interesting problem. Phenomena of heredity being so complicated and irregular in some crosses, Weldon (02, 03) has advanced the opinion concerning the ambiguity of the Mendelian categories and says ‘that segregation of seed-characters is not of universal occurrence among cross-bred peas, and that when it does occur, it may or may not follow Mendel’s law. The law of segregation, like the law of dominance, appears therefore to hold only for races of particular ancestry. In special cases, other formulae expressing segregation have been offered, especially by De Vries and by Tschermak for other plants, but these seem as little likely to prove generally valid as Mendel’s formula itself”’. From our experience in silk-worm rearing we are struck, however, with the belief that the irregularity may be due to the impurity of the characters and strains chosen for the experiment, and to arrive at any satisfactory conclusion concerning the irregular cases, further extended investigations are needful. The facts and considerations above enumerated afford some help for the explanation of the well-known fact that the offspring of the first cross generation are generally uniform, while the subsequent generations produced by these hybrids display a diversity of characters. tudies on the Hybridology of Insects. 385 Darwin (’88) attempts an explanation of it and says: ‘“ hybrids in the first generation are descended from species (excluding those long cultivated) which have not had their reproductive systems in any way affected, and they are not variable; but hybrids themselves have their reproductive systems seriously affected, and their descendants are highly variable ”’. Weismann’s view (’92, 04) based on the phenomena of ‘ reducing division ’ deserves much attention. In “Germ plasm”, he says ‘as this halving of the germ-plasm occurs, in a different manner in different instances, we may presuppose that it will also exhibit differences with regard to the proportion of paternal and maternal idants which come together in each germ-cell in consequence of the reducing division; and this supposition is most satisfac- torily borne out by the facts, for it is well known that the offspring of hybrid plants produced by fertilization with their own pollen, become very variable in the following generation. It is evident, indeed, that they must vary greatly, according to whether each one has received a greater number of maternal or paternal ids, or an equal number of both, from the two germcells which combine in the process of fertilization to produce this particular individual ”. According to the Mendelian principle, we may ascribe them to the dominancy and recessiveness of the characters and their segregation. The dominance of some characters over other antagonistic ones certainly produce slight variability in the first cross, since in this cross only dominant characters appear as active, while when the cross-bred formis paired zuter se, most of the parent-characters appear as active components. Evenin the case of dihybrid we may get four different kinds of offspring. If plenty of different characters are mingled together we may safely expect to produce abundant combinations of characters according to the law of combination. The State of things would become much more complex and diversified when other characters which do not follow Mendel’s law are commingled. In addition to these cases, the frequent occurrence of compound charac- ters may be mentioned as a factor to produce a diversity of forms in the second or further generations. From the industrial point of view the results obtained with the silk-worm may have some economic importance, since they would give some help for 386 K. Toyawa the selection of cocoons and larvae which is one of the difficult and most important things for silk-worm breeders. To get pure white race from mixed yellow race, sixty-five years’ selection have been practised in France (after Darwin) and the white race called ‘‘Sina”’, by careful selection during the last 75 years, “ est arriveé a un tel’etat de pureté, qu’on net voit pas un seul cocon jaunes dans des millions de cocons blancs.” Hutton (64) met with a similar difficulty when he tried to select the dark brown or blackish brindled worms from the common race. Our silk-worm breeders have similar experiences concerning the selection of the cocoons and the larval markings. This difficulty will be overcome if we follow Mendel’s law, which is highly to be recommended to worm-breeders for the selection of their breeds. The writer wishes to express here his indebtedness to Prof. Ishikawa. He is also indebted to Messrs. Y. Takano and Y. Nagashima, assistant in the Royal Sericulture Department in Bangkok who helped him in rearing the worms. Summary. 1. Of the various characters of the silk-worm, some strictly follow Mendel’s law of heredity while others are governed by other laws. The colour of the cocoon and the egg and the various larval markings belong to the former:category, and the shape of the cocoon and the brood- characters such as uni, di and multivoltine ctc. to the latter. 2.1 Among the various larval markings which we have tested, the striped marking comes first in its dominancy, next the normal marking and last the “ pale” one. With regard to the colour of the cocoon, yellow comes first, next ranks pale-pinkish-yellow or flesh, then greenish white, and pure white? in succession. Hence in the highest dominant form, all the others may lie dormant for a generation, sometimes more. 1 A parallel case regarding the Coat-characters of guinea-pigs is recorded by Castle (’05). 2 That albinism is a recessive character in mice, rats, and guinea-pigs has been proved by many authors. Farabee’s observations (Castle, 03) indicate that the same is true even in Man. In plants the depigmented condition is generally recessive. It is probable, therefore, that the recessiveness of albinism is a general law of heredity in animals and plants. Studies on the Hybridoiogy of Insects. 387 3. Thus, the yellow form in which other recessive characters are lying latent, when paired zxter se, exhibit complicated phenomena of the segrega- tion of the parent-characters which possibly give a verification of the Mendelian principle : The first generation, Uniform yellow form. 1. Uniform yellow form. 2. A mixture of white (25%) and yellow 5/0 y (75%) forms. A mixture of pale-pinkish-yellow (25%) Ss] —s The second generation, +> and yellow (75%) forms. 14. A mixture of greenish white + white (23.98% ), pale-pinkish-yellow (19.57% ) and yellow (56.43% ) forms. The third generation, The yellow form derived from the mixed offspring which produced four kinds of worms again repeated the same phenomenon of the disintegration of the parental characters, producing the four kinds of offspring as the last generation. The same phenomenon has been repeated in subsequent generations. (see Case II). 4. Phenomena similar to those cited above have been observed in the crosses of various larval markings. (See Case VII). 5. Incrossing striped race with common one (both having the character ‘pale’ in the latent state), an interesting instance of modified dihybrids were obtained. It is quoted as follows : The first cross generation, Uniform striped offspring. The second cross generation, A mixture of striped (about }§ or 75%), common (about 53; or 18.75%) and * pale’ (about ;'; or 6.259%) forms. This may serve as an another verification of the Mendelian principle. 6. The crosses between the Japanese oblong and the Siamese spindle forms give in the first cross two kinds of forms, the spindle and _ the ellipsoid. Most of them, however, belong to the former. In the second generation 388 K. Toyaina. paired among similars, they split up into various forms, of which some become constant while others again produce various forms when they were mated with similars. But after selection, we can gradually sift out the inconstant ones. Of those various forms, the spindle or conical shape stands highest in dominancy, next comes the oval or ellipsoidal form and lastly the oblong or cylindrical form rounded at both ends. 7. With regard to the brood characters, the same character sometimes behaves as dominant, sometimes as recessive, according to the sex of the parent, and the order of segregation is not so regular as in other cases. 8. There are many compound characters! which behave exactly like a single independent one, while when crossed with new characters they are disintegrated into the component ones. Each component character thus produced behaves as an independent one, breeding true to the parent. g. Conversely two independent characters belonging to the parents or different breeds may be combined into one form when we cross them and the resulting form breeds true, as if it is single independent character. 10. When commingling of two characters takes place, as a result of crossing, sometimes it occurs that the characters of both parents or breeds even sexual characters occupy cach half of the body of an individual. (See Case VIII). 11. Those facts and considerations above referred to may serve to explain the well-known phenomenon of crossing: the slight variability of hybrid in the first cross and the greater variability in subsequent generations. (P. 384—385). June 20th, 1905. Zoological Institute, College of Agriculture, Tokyo Imperial University. 1 Morgan’s interpretation (05) concerning the yellow mice obtained by Cuénot deserves much attention. (Added May, £906). Studies on the Hybridology of Inseets. 389 LITERATURE CITED. 1. Allen, G. M. (’04) :—The heredity of coat-colour in mice. Contrib. fr. the Zoo]. Laboratory of the Museum of Comp. Zoology at Harvard College. 1904. 2.* Bateson, W. and Saunders, E. R. (’02) :—Experimental studies in the physiology of heredity. Reports of the evolution Committee. 1902. 3.**Castle, W. E., et Allen, G. M. (03) :—The heredity of albinism. 1903. 4. ——— ('03):—The heredity of sex. 1903. 5. Correns, C. (00) :—Gregor Mendel’s ,, Versuche uber Pflanzenhybrid- en“ und die Bestatigung tuber Ergebnisse durch die neuesten Untersuchungen. Bot. Zeits. Nr. 15, 1900. 6. —— (’00) -—Uber Levkojenbastarde. Bot. Cent. 1900. 7. ———— (or) :—Mendel’s Regel ttber das Verhalten der Nachkommen- schaft der Rassenbastarde. Ber. d.d. bot. Ges. tgo1. Bd. XVUL. 8. ———— (’o1") :—Uber Bastarde zwischen Rassen von Zea Mays. Ber. d.d. bot. Ges. Bd. XX. Igor. g. —— (01°) :—Bastarde zwischen Maisrassen, mit besonderer Beriick- sichtigung der Xenien. Bibliotheca botanica. 1gol. 10. ——— (’02) :—Scheinbare Ausnahme von der Mendel’schen Spaltungs- regel fiir Bastarde. Ber. d.d. bot. Ges. Bd. XX. 1902. 11. ——— ('04):—Ein typisch spaltender Bastard zwischen einer cinjah- rigen und zweijahrigen Sippe des Hyoscyamus niger. Ber. d.d. bot. Ges. 1904. 12. Coutagne (’02) :—Recherches experimentales sur Vherédité chez les vers asoie. 1902 Paris. 13. Cuénot, L. (0204) :—La loi de Mendel et ’hérédité de la pigmentation chez les souris. Arch zool. exp. et gene. 1902, 1903, I904. 14. Darbishire, A.D. :—Note on the results of crossing waltzing mice with European albino race. Biometrika Vol. Hl. 1903. * Bateson, Saunders, Punnet and Hurst (’05): Reports of the evolution committee. 1905. ** Castle, W. E. (’05) :—Heredity of coat characters in guinea-pigs and rabbits. 1905. = (05) :—Recent discoveries in Heredity and their bearing on animal breeding. 1905. 21; K. Toyama. Darbishire, A. D. (’03):—Second report on the result of crossing Japanese waltzing mice with European albino races. Biometrika Vol-H.- “1902; (03) :—Third report on the result of crossing Japanese waltzing mice with European albino races. Biometrika Vol. HW. 1903. Darwin, C. (°88) :—The origin of species. 6th ed. —— ('99):—The Variation of Animals and plants under domestication. 2nd Ed. Davenport, C. B. (04) :—Colour inheritance in mice. Wonder Horses and Mendelism. Science, 1904. De Vries, H. (’00) :—Das Spaltungsgesetz der Bastarde. Ber. d.d. bot. Ges. Bd. XVIII. -ag00: (00) :—Uber erbungleiche Kreuzungen. Ber. d.d. bot. Ges. Bd. XVIII. goo. —— (’03) :-—Die Mutationstheoric. Bd. Il. 1903. Farabee, W. .C. and Castle, W. E. (’03):—Notes on Negro albino. SCIENCE, 1903. Focke (81) :—Die Pflanzenmischlinge. 1881. Von Guiata, G. (98) ;—Versuche mit Kreuzungen etc. der Hausmaus. 1898. ; Hutton, Th. (64) :— On the reversion and restoration of the silk-worm. Transact. of the ents seeeseondon. Volo i. 1864 Kraepelin, K. (’73) :—Untersuchungen iiber den Bau, Mechanismus und die Entwickelung des Stachels der Bienenartigen Tiere. Z. f. w. zool. Bd. XX, weg. Lang (’04) :——Uber Vorversuche zu Untersuchungen iiber die Varietiten- bildungen von Helix hortensis m. und Helix nemoralis L. 1904. Mendel, G. (’65) :—Versuche tiber Pflanzenhybriden. 1865. (69) :—Uber einige aus kiinstlicher Befruchtung gewonnene Hieracium Bastarde. 1869. Millardet, A. ('94):—Note sur l’hybridatien sans croisement ou fausse hybridation. Mem. soc. sc. phys. et natur. de Bordeaux. 1894. Morgan, T. I. (’05) ;—The assumed purity of the germ cells in Mendelian results, Science, 1905. 39: 4O. Studies on the Hybridology of Insects. 391 Tschermak, E. (00) :—Uber kiinstliche Kreuzung bei Pisum sativum. Zeit. f.d. landw. Versuch. in Oesterreich. 1900. (or) :—Weitere. Beitrage uber Verschiedenwerthigkeit der Merkmale bei Kreuzung von Erbsen und Bohnen. Zeits. f.d. landw. Verschw. in Oesterreich. 1901. Standfuss, M. (96) :—Handbuch der palaarktischen Gross-schmetter- linge. 1896. (00) :—Synopsis of experiments in Hybridization. 1goo. Weismann, A. (’93):—The Germ-plasm. 1893. (04) :—Vortrage tiber Deszendanztheorie. 1904. Weldon, W. (’02) :—Mendel’s laws of alternative inheritance in peas. Biometrika. Vol. I. 1902. (03) :—On the ambiguity of Mendel’s categories. Biometrika. Vol. Ths 1903. Woods, T. A. ('03):—Mendel’s laws and some records in rabbit breeding. Biometrika. Vol. Il. 1903. ioe) Ke) to K. Toyama. EXPLANATION OF PLATES. Eee Vi. Fig. I. Represents larvae of Japanese common (a) and French striped (4) races. Nat. size. Fig. Hl. Represents mosaic larva (a) and moth (4) produced from crossing of above races. Nat. size. Fig. III. Represents wings of the same mosaic moth. a. J, right wings ; c. d, left wings. Little magnified. Fig. IV. Dorsal view of the same moth. 8th, eighth abdominal segment ; de, dorsal chitine plate ; ov, ovipositor ; .¢, alluring glands ; c, cover- plate ; e, clasper ; %, projection of ventral plate. Fig. V. End of the abdomen of the same moth, more magnified. Lettering as in preceding figure. Fig. VI. Frontal view of the sexual armature of above moth. Little magnified. a@, dorsal portion of the seventh segment; a’, dorsal portion of the eighth segment; c¢, dorsal plate; g’, ovipositor; d, alluring gland; e.f. two ventral plates; 6’, dorsal lip; p, cover plate; c’', basal plate; d@’, clasper; %, triangular accessory plates ; f, chitinous area; 1, rudiment of penis; x, thin plate. Fig. VII. Female genital armature. Little magnified. a, dorsal plate; 34, ovipositor ; ¢. d, ventral chitine plates. Fig. VII. Male genital armature. Slightly magnified. a, dorsal lip; 6, ventral plate; 4%, triangular projection of ventral chitine plate; c, cover plate; d, anal frame; ¢,clasper; dc. basal plate; /, triangu- lar plate ; /, penis. Poa Vii. No. I. Represents cocoons of Japanese (a) and Siamese (4) races; ¢, yellow cocoon; d, pale-pinkish-yellow cocoon. No. II. Represents cocoons (all perforated) produced by worms raised from a cross between above races. ~~ Studies on the Hybridology of Insects. 393 PEE VIII. No. I. Represents cocoons (yellow and white) spun by worms of the second cross generation of the above cross. Some cocoons are perforated. No. II. Represents cocoons of pale-pinkish-yellow form (fourtl: generation) which become constant. PEaIE IX. No. I. Represents cocoons of conical form which produce various kinds of cocoons. (Third generation). No. II. Represents cocoons of pale-pinkish-yellow form (third generation) which become constant after the selection. PEATE X. No. I. Represents yellow and white cocoons produced from a mother- moth. Each “ mounting basket” represents a whole brood from a parent. No. II. Japanese common marked worms and new worms derived from the crossing between striped and common worms. No. HI. .Represents three kinds of worms. a, Siamese striped worm ; 4, pale worm having no markings ; c, Japanese common worm. Peet XI. > Represents Siamese pale worms having no markings. B. Represents Siamese striped worms. Bull. Agric. Coll. Vol. VII. Fi. VIL. EL. EX. Bull. Agric. Coll. Vol. VII. Bull. Agrie. Coll. Vol. VII. PE 51 5 Pl. Xd. Bull. Agric. Coll. Vol. VII. On Physiologically Balanced Solutions. BY O. Loew and K. Aso. About half a century ago various authors have carried out experiments in order to find a solution in which plants could be grown to perfection which are cultivated in soil. After many failures Axzop succeeded to compose a culture solution of the desired qualities, it was superior to all others, that of Sachs not excepted. In other words, it was a physiologically balanced solution ; the injury by a one-sided nutrition was prevented by the proper quantity of other nutrients. It must have been doubtless recognised by Knop, altho it was not pro- nounced with emphasis, that the ratio of the different nutrients to each other zs of fundamental importance for the best development of the plants and that this principle of the water culture must hold good also in regard to soil and manure for the field crops.! In studying the cause of the toxic action of mag- nesium salts we were led to infer that special consideration is necessary for the regulation of the relative amounts of lime and magnesia available to the roots. Numerous experiments have shown beyond any doubt that the injurious action which magnesium salts exert on plants from the higher algze up- wards can only be prevented by lime salts and that the important function of magnesium salts can therefore only be realised in the presence of lime salts. 1 It is true, some few adhere to the opinion, only holding good for aquatic plants, that the osmotic laws determine the amount and kind of the necessary nutrients to be absorbed. But the current of transpiration plays a more important réle than that for the land plants and itibrings into the plant body much more mineral matter than needed. 396 0. Loew. and K. Aso. Our investigations have further demonstrated, that the most favorable develop- ment of plants depends among other things upon a certain quantitative ratio of lime to magnesia available to the root. ! We have proved by water—, sand—, and soil culture that an excess of lime as well as an excess of magnesia beyond that best ratio,—the lime factor —depresses the yield of various crops more or less and have pointed out that the determination of magnesia in partial soil analyses is as important as that of lime—but thus far not much attention was paid to this important principle. The law of physiologically balanced solutions was clear before our mind, and no doubt also was this law regarded by Godlewski, Schrétter and others when they tried to find by field experiment the best ratio of nitrogen to phosphoric acid and potassa for certain crops.? ‘“Heavy doses of strongly nitrogenous manures also necessitate heavy doses of phosphoric acid to annihilate the injurious effect of an excess of nitrogen,” is a statement copied from a book just before us ; similar utterances are numerous in agricultural reports. We must call attention to this, because that law of physiologically balanced solutions was recently claimed as a new discovery. There may be a slight distinction made between a _ physiologically balanced solution for the maintenance of life only and one which would insure the best development of plants ; only the latter is of course of importance. As that author further did not distinguish different phenomena relating to this subject, we must enter upon a further discussion. That there exist very intimate, special relations between lime and magnesia in their réle as plant nutrients becomes evident from the fact that 1 Cf. Flora, 1892 p. 381; ibid. 1903 p. 498 and 1905 p, 336; Landw. Vers.-Stationen 1892, vol. 41 p. 467; Landw. Jahrbiicher 1902 p. 561; ibid. 1905 p. 131 and 1906 p. 527; Zeitschrift fd, Lanaw, Versuchswesen in Oesterreich, 1905. Cf. further Loew and May, Bul. No, 1 Bureau of Plant Industry, Washington 1901 ; and the Bulletins of this College, vol. IV p. 361-381: ibid. V p. 495-502; ibid. VI, p. 97-124 and p, 347; ibid. VII’p. 8-12 and p. 57-65. 2 Also here at this College some years ago an experiment was made by Bahadur to find the most suitable ratio of N to P, O, for barley in soil culture (cf these Bulletins VI, No. 4). 3 As physiologically balanced solutions were mentioned by that author blood and sea water. On Physiologically Balanced Solutions, 397 magnesium salts are not poisonous at all for those lower forms of algz and fungi which do not require lime for life and propagation. ! In perfect accordance with this behavior is that to oxalates which only are poisonous for plant life from the higher alge upwards, but not for the lowest forms of alge, flagellata and fungi. The most characteristic property of oxalates being the withdrawal of lime from lime compounds? it becomes clear that lime must assume a very important position in the organised structure, as soon as a certain stage of differentiation to higher forms is reached. In regard to marine alge which doubtless belong to the higher alge Duggar? in a series of interesting investigations has observed that magnesium salts exert but a very weak toxic effect. But it must be taken into account that in his experiments magnesium sulphate was dssolvedin seawater which contains already /zme, further that a relatively small amount of lime can depress the toxic action of a considerably larger amount of magnesia and finally that the marine alge contain more lime than magnesia. ‘This sur- plus of lime in the plants can also depress the toxic effects of entering magnesia. It must be born in mind that sea water is richer in magnesia than in lime (ratio=3.8: 1) and that marine alge, in order to adapt themselves to this unfavorable condition, must accumulate lime in their cells, which may be done in the form of organic salts.® The theory of one of us as to the functions of lime and magnesia in plants assumes the existence of calcium-protein compounds in the tectonic of the > 1 Lower forms of algze do not require “ physiologically balanced solutions” since they can develop in a 4% solution of magnesium sulphate in presence of mere traces of N, K,O and P,O, (Pelmel/a, Ulothrix). These forms even can develop in a 5% solution of manganese sulphate and can adapt themselves gradually to a 4% solution of NaCl, 2 On the similar behaviar of sodium fluorid, cf, Flora 1905, p. 336. 3 Trans. Acad. Sc. of St. Louis, vol. XVI, No. 8. 4 Gédechens, Ann, Chem, Pharm, 1854. Also Bul. No. 1., Bureau of, Plant Industry, Washington I9OI, 5 We avoid bere the term “ion,” since this may confer a wrong idea. In regard to the electroly- tic dissociation theory compare the important investigations of Louis Kahlenberg.— 398 0. Loew and K. Aso. nucleus! and chloroplasts of the higher plant forms and ascribes to magnesia the role to mediate in the assimilation of phosphoric acid when nucleo- proteids and lecithin are to be formed from anorganic phosphates. The theory further has pointed out that a certain excess of magnesium salts will act on the lime compound in the nucleus, replacing calcium by magnesium and changing thereby the capacity of the nucleus for imbibition, leads to disorganisation and death, while on the other hand an undue excess of lime will retain the phosphoric acid and prevent the formation of magnesium phosphate, of this important compound for the assimilation of phosphoric acid.? We never had observed such intimate relations as evidently exist between the physiological functions of lime and magnesia, also to exist between potassa and magnesia. However recently not only a toxic action of potassium salts for plants was assumed to exist but also an antitoxic action of potassa to magnesia. These observations were, however, not made with phenogams but only with Sfzvogyra and gemme of Lunularia, further only with one potassium salt, the chlorid.* When one of us made his first studies in this line (1892) the behavior of magnesium salts to potassium salts and sodium salts was of course compared 1 The view of some authors that lime salts are only required for certain processes of metabolism in the plants cannot be upheld. It might be objected, e.g., that in this case strontium salts should be capable to replace calcium salts, which is however impossible ; these act injuriously, in absence af lime salts. Cf. O, Loew, The Physiological Réle of Mineral Nutrients, II Edition, pp. 46 and 54 U.S. Dept. of Agriculture, 1903 ; and U. Suzuki, these Bulletins IV, No. 1. Manganese salts act evidently in the same way poisonously as magnesium salts do. In accordance therewith a poisonous effect for all plants trom the higher a!gze upwards is noticed and no poisonous effect for lower algz and fungi. Thus Palmella-forms and Ulothrix-like filaments can grow in a 5% solution of manganese sulphate, while Spirogyra is killed by solutions weaker than 0.1%. 2 Since it was recently shown by Willstitter (Ann. Chem. 350, p. 46) that the molecule of chloro- phyll contiins magnesium, it follows that magnesium has still another function to perform. Willstiitter ascribes to it a réle in the assimilation of carbon. Since, however, potassa is also indispensable for the assimilation process, as has been shown long ago by Wodde, it may be possible that both these metals must be present in the transformation of CO, into organic compounds, It deserves mentioning, that Berthelot (1906) has observed, especially in the leaves, potassium compounds insoluble in water. 2 Cf V. Osterhout, vol. II, No. 11 of the Publications of the University of California, 1906.— On Physiologically Bzlinced Solutions. 399 with that to calcium salts. No toxic action of potassium salts had been observed however, while a retarding action of potassium salts was observed in one case and an accelerating action in an other, on the toxic action of magnesium salts. The experiment was the following. In a 0.2 per mille solution of magnesium sulphate Spirogyra communts died in 5-7 days, while upon addition of 0.1 per mille dipotassium phosphate in 15-18 days and on the other hand upon addition of 0.1 p.m. monopotassium phosphate in 3 days. In a solution of 0.2% monopotassium phosphate and even on further addition of 0.2% KNO, the alga can remain alive for a series of weeks.1_ But already at a concentration of 1% and a temperature of 12-20° various salts are injurious which are harmléss at 0.2-0.5 per cent. At 4-6° C the resistance power is greater, especially with the larger kinds. Further, a gradual adaptation may be reached. Spirogyra cells that had been kept in 0.5% Na Cl solution can resist a 19% solution longer than otherwise. Effects of physiologically wot balanced culture solutions on alge (Spirogyra) were observed years ago by one of us. Thus it was noticed that a considerable preponderance of lime over magnesia retarded the cell division ; an undue preponderance of phosphoric acid and nitrogen over potassa rendered starch accumulation in the chloroplast impossible, all carbohydrate produced by assimilation of carbon being at once transformed into protein required for the rapid growth ; on the other hand a surplus of potassa led to a considerable accumulation of starch, when nitrogen was present in a munimum amount, while an undue simultaneous maximum of nitrogen and potassa led to the accumulation of much protein? in vacuole and cytoplasm, and but little starch becomes visible. 1 The salts applied should be chemically pure. Often a very faint trace of copper is present, when the salts had been recrystallised from common distilled water. Only water distilled from glass vessels should serve for recrystallisation, In such distilled water Spirogyra can remain alive for a very long time. The flasks for the tests with Spirogyra should be first washed with hydrochloric acid, then with this distilled water. The amount of solution applied should not be too small, Generally 100 cc. served for a small number of filaments, because otherwise some dying filaments losing nutrient compounds by exosmosis can thus influence the resistance power of the neighboring filaments. 2 This protein is of yery labilnature. Cf, O, Loew and Th. Bokorny in ,,Die chemische Energie der lebenden Zellen” by O. Loew. 400 0. Loew and K. Aso. Under certain conditions the chloroplast grows more rapidly than the cytoplasm, finally filling this out entirely and rendering the nucleus invisible ; under other conditions again the cytoplasm grows more than the chloroplast, the latter changing its spiral form finally to a straight line.! Again in certain culture solutions the cytoplasm is rendered turbid from fine precipitates of phosphates, in others again the filaments break up into single cells, which remain perfectly healthy. This phenomenon is in many cases due to increased turgor.? Some of the many trials? may here be mentioned. As favorable culture solutions served the following : a b KON PO). 22s ap. mille-saae. ee on ae ND: , ae ey yy 6 ee ee ODin! ae Ca (NO, ) jvc:-cce2- ee ., 5) ees O:5 450s Mo'S0),: .tse-see ene ae 2, 5, 4a Os) is Fe SQ, 3. ee PeICe «=, eee trace MoH, (CO,),. ce ae — O:2 eee When in solution (a) the potassium nitrate was replaced by 0.3 p.m. mono-ammonium phosphate the development was somewhat abnormal, some 1 During such observations, attention must be paid to the presence of Chytri-déa, parasites which easily perforate the cell-walls of Spzvogyra. Brown has observed over 20 species attacking various alge, Sometimes Spirogyra is attacked also by Pseudospora, which is a mixomycet according to Zopf. These parasites may often be destroyed by placing the alge for 1-2 days ina 1 per mille solu- tion of phenol in well water. Parasites will doubtless become most abundant after a portion of the Spirogyra cells present had died, furnishing by exosmose from the vacuole organic nutrients tor the parasites outside and attracting them to the laments. The presence of infusoria is favorable as they (especially Vortice//a) devour Chytridia, 2 This phenomenon was also observed when Spirogyra was kept in very moist air, i.e, under a bell-jar spread on moss, thoroly moistened, Once it was observed by us also by touching the fila- ments with very dilute OsO,, W. Benecke made especial studies on this subject. J. w. Bot. 1898. 3 Spirogyra is very sensitive to ammonium salts, especially in weak alkaline culture solutions, while nitrates may serve well as source of nitrogen even at concentrations of 0.29. Mono- ammonium phosphate which is of acid reaction, may at a concentration not higher than 0.05% serve, however, as a source of nitrogen in weak acid culture solutions, On Physiologically Balanced Solutions. 401 cells reaching a great length before cell division took place. Also unusually much tannin accumulated. In the following solution some Spirogyra cells showed a change of the cylindrical shape to a barrel shape, globular formations appeared in the cells, and numerous rhizoids were produced. Death {resulted after a few weeks. That solution was: Nae-EL PO)... ys: ARMA inn os ES I per mille. NatiCO):, 1. Gee oes 00d O25 “seal Wis: SO), .. vce) FR ac Lee Orpen! ays CalQNOs ) 5 ici oes bos - oa Orlewnle: Be NOs. 2a. oes. Seas: ORG PLis,- Paes Be SO , th. TR Ss. eet lde trace It was of weak alkaline nature, and with lime in the minimum. Potassa did zot counteract the toxic effects of magnesia, as an increase of lime would have done, in accordance with our former observations. In the following solution the effect of an excess of lime on the srowth of the chloroplast became especially noticeable : KH, PO, . eee os ae 0.1 per mille. CaSO,” cceegeeeee es. oe ea cae EO. 55 Ca, (NO, 5°42 + sno eieoth iio eee 55 Ne SO) ad. ee Of 64) a Be SQOi, eee ee irace The growth of the cytoplasm and the cell division were here much retarded, the increase of the number of cells was slow but the chloroplast continued to grow so that it filled out all available space in the cytoplasm and in some cells it grew beyond that, causing an irregular form of the spiral by the pressure of growth. In the following sclution with a relative preponderance of potassa and nitrogen a great deal of protein was formed and stored in the vacuole and cytoplasm, the starch produced by assimilation of carbon being rapidly utilised for that purpose, therefore only little was seen of it in the chloroplast. Growth of the filaments was not very energetic, as phosphoric acid and magnesia were in the minimum. That solution was: 402 0. Loew and K. Aso. (OEOr ee... ... ee 0.5 per mille. Cac(NO 2), cos see eee Oy Se as WESC Geeteess 0... ee O:05 55. 55 i Ale laid OF kee: 4.0... re O.@55; 3: lx go\ 0 rer os... es trace We have recently also made further observations on the effect of imperfect solutions on Spirogyra nitida, one of the larger species. The concentrations of these solutions were mostly below 0.5% and did in no case reach 1%. A small number of filaments of 6-10 cm. length was placed in 100 cc. of the solutions prepared with water distilled from glass vessels. The temperature varied from 8-22° C. The flasks were exposed to direct sunlight, later on only to diffused but bright daylight.— The figures in parenthesis in the following table signify the percentage of anhydrous salt ; they stand mostly in simple relation to the molecular weights. Mg (NO ), (0.2) All cells killed in 2-4 days. Mg Cl, (0.2) K Cl (0.1) All cells heaithy after 10 weeks. All cells healthy for 3 weeks, then a gradual change of the chloro- K Cl (0.3) plast-spiral took place, it contracted, moved to the cell ends and formed no longer starch, Gradually also the nucleus suffered. 1 1 Such injured cells had still the normal turgor, but the nucleus and very probably also the chlorophyll body were killed. The nucleus had contracted to an irregular shaped mass and was lying on the side. Such cases were observed years ago by one of us when highly diluted solutions of oxalic acid acted on the cells. These cells with the cytoplasm alive and the nucleus killed recalled Gerassimows Spirogyra cells without any nucleus, obtained by the influence of low temperature or On Physiologically Balanced Solutions. 403 K NO, (0.15) All cells normal and rich in starch after 15 days; later on gradual , death; some cells alive after 42 days. A ber of cells still perfectly healthy after 50 days. Injury = K, SO, (0.3) A number o still perfec y ealthy after 50 day njury com meaced after 283 days. Parasites numerous, like in the former case. All cells alive after 80 days. In a few cells the nucleus has moved to Ca Cl, (0.2) the wall. Much starch, no parasites. Gradual death afterwards. Ca (NO,), (0.2) All cells healthy after 50 days, no parasites. ys sites. fi s Ca (SO,) (0.2) All cells healthy after 80 days, no parasites. Later on the filament became yellowish. Much starch. Most cells killed after 10 days; the still living show chlorophyll body K Cl (0.15) +Mg Cl, (0.1) attacked, its lobes being retracted and sometimes the spiral torn into fragments, But such injured cells were still alive 4 weeks later. All cells healthly for 30 days; half the cells dead after 50 days. The living cells have now received nutrients and perhaps also lime from K, SO, (0.3) + Mg SO, (0.2) the decaying dead cells, as was clearly evinced by the cell-division taking place here and there. Much starch noticed in these ceils after 60 days. Some rhizoids.1 anzsthetics after the cell division had madeastart, The substance contained in the chlorophyll body may serve to sustain the life of the cytoplasm in case the assimilation of carbon in the former had ceased. Such cells without nucleus are capable to live for six weeks (Gerassimow). 1 Thus far rhizoid formations were observed by us in solutions containing : Ca (NO,)2 + (NH), SO, (0.002%) or CaSO, + K, SO, or Mg SO, + K, SO, but in no case in any of these compounds alone. Sulphates seem to be essential for that phenomenon. It deserves to be mentioned that in the numerous cases of imperfect culture solutions we observed only in lime salts and in 0.19% K Cl solution that the filaments of Spirogyra showed the phenomenon of geotropismnt. Spirogyra sometimes shows the phenomenon of Az/iotaxis. One of us (L.) has seen once that Spirogyra filaments lying on the bottom of a flask moved with great rapidity into a nearly vertical position, when the first rays of the morning sun reached them. 404 0. Loew and K. Aso. K NO, (0.15) + Mg(NO,) (0.2) (1 Mol. : 1 Mol.) Most celis killed in 17 days; the injured cells have living cytoplasm but dead nucleus ; all cells killed after 30 days, K NO, (0.5)+Mg SO, (0.2) (3 Mol.: 1 Mol.) After 25 days healthy. After 50 days about half the cells killed, Chlorophyll body attacked, forming no starch in sunlight, hence probably dead. while the living cells show swollen nucleus, About 10% of the cells alive after 3 days, while without Na, SO, all killed in 3 days. K NO,(0.15)+ Ca (NO,), (0.2) K,SO, (0.3) + Ca SO, (0.2) Mg(NO3).(0.2) +Ca(NO 3) 2(0.2) Mg SO, (0.2) + Ca(NO 5), (0.4) Mg Cl, (0.1) +Ca Cl, (0.2) Mg SO, (0.2) + Ca SO, (0.2) Most cells alive after 50 days; nucleus normal in all cells. Many rhizoids had formed. Cells almost all alive after 50 days, they have grown in length more than in any one of the cases mentioned here ; nucleus in most cells normal but Chlorophyll body often some- what emaciated, with change of the spiral shape. Almost all the cells after 50 days perfectly normal and healthy, starch present. Very few Chytridia. All cells healthy for 30 days, a few filaments iniured after 50 days, showing emaciated and disrupted chlorophyll body ind displaced contracted nucleus. The healthy cells show starch. About 95 per cent of all the cells after 80 days perfectly normal. All cells normal after 50 days. Later on a yellowing set in. No rhizoids. No parasites. Mg(NO,).(0.2)+ K,SO,(0.01) +Ca (NO3),(0.04) Remained healthy for 32 days, but later on many cells died, and those cells that lived after 50 days showed injury to chloroplast and The displaced nucleus. No further starch formation was possible, effect of a relative excess of magnesia was evident. No rhizoids were observed. a 1 Tt must be not lost sight of in these experiments that a living cell can extract thru the separating wall, from a neighboring cell in a dying condition, various compounds of organic and anorganic nature and thus become able to a prolonged resistance under unfavorable conditions. On Physiologically Balanced Solutions. AOS It will be seen from this table that the cells remain alive and healthy in solutions of calcium salts at a concentration of 0.2% and further that the poisonous action of magnesium salts can only be prevented by certain doses of calcium salts. It will be further noticed that potassium salts can retard but not prevent the toxic action of magnesium salts, which influence is more noticeable when both bases (or one of them) are present as sulphates than in other cases. Jt would be, however, not be justified to give the same ex- planation for both cases of counteraction without close examination. One might, e.g., suppose that potassium-protein compounds! in the living matter can exchange their potassium against magnesium and that this might lead to a similar disturbance as by the substitution of the calcium of the nucleus or magnesium. Such an explanation would demand the proof that the assumed potassium protein-compound forms really on essential part of the tectonic of living matter; it might merely be loosely connected with the structural elements and in that case the substitution of its potassium by magnesium would not lead to a collapse of the tectonic, as is the case of the calcium-protein compound of the nucleus when its calcium is replaced by magnesium. Further, that hypothesis would necessarily imply that calcium salts must also act poisonously, which is not the case. The alge cells showed even much starch after 2 months in a 0.2% solution of Ca Cl,. It is much more probable that the retardation of the toxic action of Mg-salts by K-salts is due to the property of forming double salts with potassium. These double salts may exert less energy in a similar way as also Mg-bicarbonate exerts less toxical energy on Spirogyra than many other Mg-salts do.? It is stated (cf. Muspratt’s Chemistry) that a very stable double salt is formed by both the sulphates of Mg and K, but not by those 1 The existence of such compounds in the living cells was assumed by one of us long ago, cf: The Physiological Réle of the Mineral Nutrients, p. 27, Washington 1899, and Die chemische Energie der lebenden Zellen I Edition p. 32, foot note and 2d Edition p. 34. The assumption that such a protein compound would be necessary for the chemical condensation processes 7 a// ce//s does not exclude Willsattters view on the réle of Mg in the chlorophyllbody. 2 We have observed that magnesium-potassium sulphate acts on calcium carbonate at 90° much more slowly than magnesium sulphate alone does. 406 0. Loew and K. Aso. of Mg and Na.! This would explain, why the alge live longer in the mixture of Mg and K sulphates than in that of the nitrates or chlorids; in the latter cases so well defined double salts as with the sulphates have not been obtained but the existence in the solutions of the mixture is more pro- bable than for the mixture of magnesium and sodium salts. Still another hypothesis may be considered which however does not exclude the former. It is possible that potassium salts can attach themselves to the calcium protein compounds of nucleus and chloroplast and thus render- ing the calcium more negative diminish its faculty to be substituted by magnesium. Further investigations are necessary.2, So much follows from our various experiments with water and soil cultures that the action of potasstum salts, can not be identified with that of calcium salts tn counter- acting the injurious action of magnesium salts, altho that retarding action of potassium salts can also be observed with phenogams. Young barley plants of 8 cm. hight were carefully deprived of the endosperm in order to exclude the influence of stored up mineral matter, and placed into the following solutions (3 in each flask) : I 0.495 Me (NODE: We 04% Me (NO.)e 0.2% Ca SO,, Ill 0.4% Mg (NO,), + 0.2% K, SO,, TV O49, SOs After 7 days the plants in I were dead, after 15 days two of the plants were dead in III, after 30 days the third was perfectly yellow and 11 days later it died. In IV two ofthe plants died after 28 days, the last after 36 days, while in II (Ca + Mg) each plant had three green healthy leaves 1 A double salt of Mg and Na-sulphate can only be obtained in presence of much Mg Cl,, but as soon as the double salt is treated with water, it undergoes a splitting into the two simple sul- phates,—In coincidance therewith is the fact that sodium sulphate cannot essentially (a few days only) retard the toxic action of magnesium sulphate for Spirogyra, E ? In comparing the peculiarity observed in the mixture of KCl + MgCl, (see table), that the cytoplasma can remain alive long after the death of nucleus (and chloroplast) it seems probable that potassium salts can also increase the resistance power of the cytoplasm to disturbing influences in the cell. On Physiologically Balanced Solutions. 407 after 8o days, while the oldest leaves only had died off. The most remark- able difference was however the growth of the root in this case from 6 cm. to 14 cm. while in the other three solutions growth had stopped altogether. These plants were still alive five weeks later, the old leaves died, but young ones started anew. A similar experiment was made with young pea plants. Here only those plants developed branches and reached the flowering stage, which were placed in the solution II. These plants increased in height 20 cm., those in III only 6-8 cm., while those in I and IV stopped growth and died gradually. When the endosperm of barley shoots is not removed it will take much longer until the toxic effect of magnesium salts causes death. Thus such barley seedlings of 6-8 cm. height, placed in 0.209% Mg (NO,), were still alive after 18 days, altho the leaves had almost entirely turned yellow. By the simultaneous presence of 0.25% KNO, this yellowing had not yet develop- ed so far as in the former case, but it had spread over nearly one half of the leaf area ; the former plants died after 31 days, the latter after 40. As to the alleged toxic action of potassium salts it may be mentioned that when barley seedlings are deprived of the rest of the endosperm after they had reached 18 cm., they can remain 2-3 months alive, if they are placed ime@5)¢4 solutions of KNO,;, KE! or K, SO, a sure proof that the assertion of the toxic action of potassium salts is unfounded; the older leaves die, but new ones develop, utilising mineral food from the dying leaves. Similar experiments were made with seedlings of maize, which were still alive 7 weeks after being placed in a 0.5% solution of K, SO,. The antitoxic action of K to Mg is, furthermore, too weak to play any decisive rdle in manuring. We recognised, e.g., the law that the common ee cereals thrive best when the available amounts of lime and magnesia are about equal. If now potassium salts would exert any notable action in the sense mentioned, the maximum harvest would have been obtained with very much less lime in those cases where the potassium salts of the manure were increased. But asa matter of fact the same lime factor was observed at very different amounts of potassium salts in the manure. Some influence of potassium-sulphate can however be recognised so long as the plants are 408 ©. Loew and K. Aso. young. Six pots each holding 2 kilo of an exhausted loam soil received the following general manure: @.6g,K, SO, ; 05g. Na, HPO,;5 082 NEG NO,, while the special manure consisted in : I No further addition. Ik se: Ree III 1og. artificial magnesium carbonate. IW Ai * : sf + 10g. KCl. V ‘s 5, 53 7 + 59. Ko'S0%: VINES, 3 's 3 + 100. Ca 'COre Two pots, each with 5 barley plants served for each case. The seed was sown Oct. 30. The fresh weight of the young plants on March 16 yielded in average the following figures, g: Ils Ho V = 5.0 ares Vig e.7 It will be seen that the increase of the potassa in the form of sulphate exerted some counteraction on the depression by an excess of magnesia but only lime was able to counteract fully that injurious effect. Comp. Plate XI. A similar experiment was made with spinach. After one month the young plants had reached only 2 cm. in height at the excess of magnesia and at this excess + an extradose of 5g. KCl per pot, the average height was quite the same, while at the addition of calcium carbonate, the average height was 4.6 cm. Summary. 1. The view recently expressed that ‘ physiologically balanced solu- 39 tions have not been made use of by botanists,” can hardly be sustained, since Knop’s culture solution must be regarded as sucha solution. Lower forms of algz and fungi do not require physiologically balanced solutions. 1 This large quantity was required on account of the great availability of the magnesia in the artificial magnesium carbonate, The original soil contained 0,49§ MgO and 0.5% CaO, On Physiologically Balanced Solutions. 409 2. Potassium sulphate and nitrate are only injurious for plants when the concentration is abnormally high. Potassium chlorid at 0.39% exerts after several weeks a slow injurious effect on Spirogyra, but on phenogams not for many weeks, even at 0.5%. The final death of Spirogyra cells in dilute solutions of potassium sulphate or nitrate is merely due to the one sided nutrition and exhaustion.— 3. Potassium salts can retard but not prevent the toxic effects of magnesium salts. The cause of this retardation is entirely different from the prevention of this toxic action by calcium salts. 4. Some interesting observations may be made on Sfirogyra kept in emperfect culture solutions. Thus, e.g., in a solution containing only KCl and MgCl, the cytoplasm can remain long alive after the nucleus is killed, recalling Gerassimow’s cells without a nucleus ; in a solution containing only K, SO, and Ca SO, an abundance of rhizoids is formed. This rhizoid forma- tion depended in our cases only upon the salts in solution, while in other cases it depends upon the contact with an object, as Borge and Kny have observed. In saturated gypsum solution the tendency to show geotropism is strongly preserved and the cells continue to produce an abundance of starch even after the chloroplasts have gradually turned yellow. This starch formation can be considered as a proof that neither potassium nor magnesium of the chloroplast had been replaced by calcium. This yellowing is not observed in the solution of 0.2% Ca Cl, even after three months. 5. Interesting effects can be observed with Spirogyra kept in full, but not balanced culture solutions. ' 7 > ¥ 3 4; rT Tat « a yuan ae CEG ¥/ ee 7F- ' Ss bi (tae to T° dol eda jar cy at stevie 4 ¥ a j j as i sh at pel jee i A ah wit ‘ 4st j ig! | mith ify Stmue ills ag thea - or 4 r4 F (het) ais ehoeenae hu . 7 ' : es a. OF ; d =! = r> I Py Z 2 F 4 <4 BUERCOLE AGI VOLS VILL. PLATE X11, * “a ¥ < » oe e To page 414, Plate showing the influence of naphthalene on barley and pea. seer tery STUDIES ON HUMUS FORMATION, II. BY S. Suzuki. In a former paper! the writer has described an experiment which had demonstrated that not only carbonate of lime but also carbonate of magnesia promote the decomposition of moist leaves by fungi, judged by the amounts of carbonic acid produced. This production may, however, more relate to the respiration of the fungi grown on the leaves than to the humification proper, altho the production of dark black substances was observed in connection with it. W. Hilgard in his extensive studies on soils observed that carbonate of lime promotes the transformation of plant remnants into humus, which however in dry countries accumulates only in clayey soils, otherwise also in calcareous soils. Proteins as well as carbohydrates? are assumed to contribute to the formation of the black substance. However, the frequent nitrogen content of humus is not yet a sure proof that the humus is derived from proteins, as shown by Udransky? who heated sugar in presence of urea with sulphuric acid and obtained a humus-like compound containing nitrogen. The nitrogen content observed in crude humus may also often chiefly be due to an admixture of bacteria, of mycelia of fungi or of chitin parts of insects as was 1 These Bull, VII, No. 1, p. 95. 2 Pure cellulose does not yield any humus according to Hoppe-Seyler, but lignin substances will do so according to Lange, who gave in this regard, however, only a short note without any experi- ment being mentioned, (Zeitschrift fiir physiologische Chemie XII, p. 84), 3 Zeitschrift fiir physiologische Chemie XII, p. 42. 420 S. Suzuki. pointed out by P. E. Miiller,' v. Post and others. But this can not hold good for the humic acid precipitated from solution. André (1900) concluded that iime gradually transforms the amido nitrogen of humus into ammonia, and that the composition of the soluble nitrogenous compounds is very variable. H.I. Wheeler, Sargent and Hartwell (1898) observed that lime diminishes the humus centent of soils? and renders its nitrogen more available. The writer has recently carried out some experiments to reveal the nature of the nitrogen content of humus. This investigation is not yet finished, but so much can be regarded as certain, that it was in the case examined due to protein matters, perhaps more or less decomposed.* Humus was boiled with concentrated hydrochloric acid for eight hours and the amido-compounds thus obtained treated after E. Fischer’s method ; and from the fractions obtained by distillation of the esters, the amido compounds were regenerated and thus obtained: leucin, alanin, glutamic acid, aspartic acid and tyrosin. A full account of this investigation will be shown in the next Bulletin. Hoppe-Seyler denied the participation of bacteria in the humification process. According to Benni this process consists in a slow oxidation of proteins and carbohydrates.4 The writer has examined the behavior of several kinds of bacteria occuring in humus-particles. From a section of loamy soil freshly opened to the depth of about 12 feet rootlets were selected at the depth of 3 feet below the surface which rootlets had turned black by the humification process. These were transfered by means of a sterilized pincet into a test tube con- taining sterilized bouillon. After several manipulations three kinds of bacteria were isolated from this sample, and from another sample which also was derived from the same place, one kind, showing a greenish fluorescence of its culture. Starting from the fact that pentosans are capable to yield by » Natiirl. Humusformen, p. 173. 2 Déherain and Demoussy observed a slow oxidation o/ humus at 4¢—30°, and more so at 100° C, 3 Close investigation is necessary to examine the cause of this resistance to putrefaction, ~ Chem. Centralbl. 1897, J, 31. Stud’e: on Humus Formation, IT. 421 decomposition with hydrochloric acid about half their weight of furfurol which easily can be turned into black matters, the writer supposed that certain bacteria might be capable to accomplish a similar process. Hence the following culture media were prepared: Bouillon, containing : a. 1.0% Yeast nuclein (moist). B°0.5 ;, Stare é.. 0:5, Atabam @. 0.5, Xylan After sterilization these culture media were inoculated with the isolated bacteria above mentioned and the flasks left several weeks in an incubator at 17°C. But formation of a black substance could not be observed under these conditions. The possibility existed, however, that the degree of the access of air had an influence on the formation of humus. It had been observed that clay soils and loamy soils form much more humus than loose sandy soils. Hence, since a restriction of access of air seems to be favorable for humus formation, a clay soil was mixed with 10% of araban, xylan, starch, dry egg albumin, glucose, cellulose, tannin, fat (sesam oil), coniferin, vanillin,’ fine saw dust, and finely powdered straw and leaves respectively, and the mixtures left partly in Petri dishes (13 g. soil in each) and partly in test-tubes (each containing 9 g. of soil) in order to observe the influence of the extent of zration. The amount of water added was equal to that of the soil. In the cases where sugar and other carbohydrates had been added to the soil, and in some other cases two series of mixtures were observed, namely with and without addition of 0.19% of meat extract.2 The clay soil was selected from a locality in which the humus formation took place extensively, it was taken just below the stratum blackened by extensive humus formation. In order to observe whether with a decrease of clay also the humus formation would be decreased, a further experiment was made in which the same clay soil was mixed with fifteen times of its amount of coarse quartz sand, To this mixture 59% of saw dust and starch respectively with 0.19¢ of meat 2 Vanillin and coniferin have a close relation to lignin. 2 This was of course done ior the purpose of nourishing the soil bacteria. 422 S. Suzuki. 3 extract were added. In this test however the amount of water was only one half of the other cases\in order to imitate the looseness of a sandy soil. The mixtures were prepared Oct. 26 and left in a thermostate at a constant tem- perature of nearly 30°C. After three days all the tannin mixtures com- menced to blacken, but this is not surprising. Since, further, tannin on account of its relatively small quantity in most plants can not play any essential rdle in the humus formation, the tannin mixtures were not further considered. The phenomena observed after 90 days are recorded in the following table : | with meat extract.—Slightly black. I Crude araban without ,, » —No blackening. with meat extract.—Black, but only in the lower I] Xylan portion of the test-tube. without ,, », —No blackening. \ with meat extract.—Deeply blackened to a large ex- | tent in the test-tube, and at the Wh Starch ‘ bottom of the Petri-dish in iso- | lated spots. without ,, » —No blackening. ee meat extract.—Slightly blackened, both in the IV Glucose test-tube and in the Petri-dish. without ,, » —No blackening. VV Cellulose.—No blackening in either case. VI Saw-dust.—No blackening in either case. VII Straw.—Slightly blackened in both cases in the test-tube, but only at the bottom in the Petri-dish containing meat extract. VII Tannin.—Blackened throughout the whole mass. IX Vanillin and coniferin—No blackening in either case. X Egg-albumin.—Blackened to a large extent in the test-tube, and only slightly at the bottom but not at the surface in the Petri- dish, 1 A very peculiar odor was noticed there, however not that of putrefaction, to 2 Studies on Humus Formation, EI. 4 XI Sesam oil.—No reaction whatever. XII Leaves.—Particles became black in a!l cases. The blackening may be due in the first place to the tannin content of the leaves. XIII Saw-dust in Sand.—No blackening. XIV Control (soil alone).—No blackening. Summary. These preliminary tests show that protein, starch and _ pentosans can contribute to the black matters of humus but neither fat nor cellulose, rand that the restriction of the air: is very essential to the humus formation. . The nitrogen content of humus in two cases examined was found due to protein. LA. Konnen Phosphate Chlorose erzeugen ? VON T. Takeuchi. Bisher war bei Versuchen mit Wasserkulturen noch von Niemanden beobachtet worden, dass ldsliche Phosphate ungiinstig gewirkt hitten. Deshalb diirfte die kiirzlich von Crone gemachte Angabe (Bonner Inau- guraldissertation, 1904, (Dez.) und Biedermanns Centralbl, 1906, S. 30.), dass lésliche Phosphate Chlorose erzeugen kénnten, wohl einiges Bedenken her- vorgerufen haben. Vergleichen wir jedoch die N&ahrlésung, welche Crone anwandte mit der wohl bewahrten Knop’schen Nahrlésung, so findet sich, dass jene N&hrlosung nicht nur weniger Stickstoff, sondern auch _ be- deutendere Mengen von Sulfat enthielt. Sie enthielt ferner Dikalium- phosphat, was die Resorbirbarkeit des Eisens herabdriicken musste. An- gesichts der zalreichen bisherigen Erfahrungen mit Knop’s Loesung wird auch der weitere Schluss Crones kaum auf Zustimmung rechnen kénnen : ,,Die Voraussetzung, dass diejenige Nahrfliissigkeit die besten Erfolge versprechen musse, die alle ihre Bestandteile nur in gelésten Zustand enthalte und dadurch der Wurzel der Arbeit des Aufschliessens enthebe, muss jetzt als vollig irrig hingestellt werden.’ Losliche Phosphate sind im Gegenteil zu Crones Behauptung unerlass- lich, um Chlorophyllbildung hervorzubringen, wie O. Loew vor langer Zeit folgerte (Uber den Einfluss der Phosphorsaure auf die Chlorophyllbildung. Bot. Centralbl., 1891). Er experimentierte mit Algen, welche zunachst in eine mit destillirtem Wasser (2 L) hergestellte Nahrldsung gebracht wurden, welche o.2 p. mille Calciumnitrat und 0.02 p. mille Ammonium- sulfat enthielt. In die sehr geraumige Flasche wurde hie und da etwas 426 T. Takenchi. Kohlensaure eingeleitet. Nach sechs Wochen Stehen im zerstreuten Tages- licht bei 14-16° waren die Zellen gelb geworden, aber trotz der Unvollstandig- keit der Nahrlésung nur wenige Zellen abgestorben. Hierauf wurde 0.02 p. mille Ferrosulfat zugesetzt und die Loesung mit den Algen in zwei méglichst gleiche Portionen geteilt und zur einen Halfte noch 0.08 p. mille Dinatrium- phosphat gesetzt. Schon nach finf Tagen ergab sich ein héchst auffalliger Unterschied: Die Phosphat-Algen hatten eine intensiv griine Farbe ange- nommen, die Control-Algen aber hatten ihre gelbe Nuance behalten, trotz des Zusatzes eines Eisensalzes. Dieser Versuch beweist klar, dass trotz des Eisenzusatzes bei den Algen Chlorose fortdauerte, wenn Phosphate mangelten, wahrend bei Anwesenheit von Phosphaten sie schon griin erschienen, dass also lésliche Phosphorsaure hier unumganglich nétig war zur Chlorophyllbildung. Um nun zu beweisen, dass in dem Versuche Crone’s nicht Phosphorsaure es war, welche Chlorose hervorrief, verglich ich die Crone’sche Nahrloesung mit einer, in welcher das Calciumsulfat dieser durch die doppelte Menge Calciumnitrat ersetzt, und das Phosphat nur als Monokaliumphosphat gegeben war, nicht als Gemisch mit Dikaliumphosphat. Die N&hrlésungen (die Salzmengen beziehen sich auf den wasserfreien Zustand) enthielten im Liter ¢: Crone’sche Loesung. Control-Loesung. Kaliumnitrat:.:..¢90 22.2 52 See 10 1.0 Calciumsulfat. 0022) 2S ee 0.5 — Calenimnttrat)..8)). ee eee —— 1.0 Magnesiumsulfat (1. ee eee 0.5 0.5 fisensulfat...2 45) eee Roe. 0.005 0.005 Dikaliumphosphat:. 4:..2..51.5. seen 0.25 = Monokaliumphosphat . 2223. .a =. 0.25 os Als Versuchspflanze diente Weizen. In feuchten Sagespanen gekeimte und in Brunnenwasser gezogene Keimlinge von ca. t1ocm. Hé6he wurden am 19 Mirz in diese Loesungen, je 2} L. in einem Zylinder eingesetzt. Der geringe Niederschlag von Eisenphosphat wurde von Zeit zu Zeit aufgerihrt. Es zeigte sich schon nach 25 Tagen, dass die Blatter der Sprosse in der Crone’schen Nahrfliissigkeit ein gelbliche Farbung annahmen, wahrend in der Konnen Phosphate Chlorose erzeugen ? A427 Control-Loesung sie sch6n griin erschienen. Auch ein bedeutender Héhen- unterschied war bemerkLar. Die Beobachtung am 16 April ergab folgende Data : Crone’sche Loesung. | Control-Loesung. I II III I II Ill Langstes Blatt. ...... 21 cm. 2mlem. 22 cm. 28cm. 29cm. 28 cm. Zen der Blatter. ..... 5 5 5 | 7. 6 6 RAG OEY on)... gelblich gelblich gelblich | erin griin griin Wurzel-Liange ... 17 cml, Beem. 26 cm. 26/em. °35, CHL. 32) em. Da nun die Blatter in der Crone’schen Nahrloesung von Tag zu Tag blasser wurden, und infolge dessen das Absterben bald zu erwarten war, so wurde am 26 April zu samtlichen Nahrlosungen je 15 ccm. einer ziemlich concentrierten Aufschwemmung von kiinstlichem Ferriphosphat gegeben und wieder von Tag zu Tag der Niederschlag aufgeriihrt. Es zeigte sich schon nach wenigen Tagen, dass die jiingsten Blatter in der Crone’schen Loesung wieder griin wurden und die Pflanzen weiter wuchsen. Aber auch die Pflanzen in der Control-Loesung, obwohl griin, fingen an, noch etwas dunkler zu werden. Die Messung am 7 Mai ergab, cm. : Crone’sche Loesung. | Control-Loesung. I Il il | I II Ill Langstes Blatt.... .. 22 30 33 [Pog 52 51 Zahl der Blatter ... 8 8 8 | 14 9 9 Wurzel-Lange ...... 32 33 33 44 46 4I Am g Mai wurde was Frischgewicht bestimmt, g. : Crone’sche Loesung. Control-Loesung. I m Ui i| & II II | Frischgewicht ...... 2:01 2.52 2.47 6.51 6.48 7.46 ee 2.53 | 6.81 Der Versuch wurde nun als beendet betrachtet, da nun erwiessen war, 1. Dass Eisen nicht giftig wirkte, wie Crone meinte. 2. Dass die Nahrlésung Crone’s der Aufnahme von Eisen bei geringem Eisenzusatz Schwierigkeiten bereitete. 428 T. Takeuchi. 3. Dass entgegen Crone lésliche Phosphate keine Chlorose verursachen konnen, was allerdings langst bekannt war. 4. Dass entgegen Crone die Pflanzen durchaus normal gedeihen, wenn die Nahrstoffe in léslicher Form dargeboten worden, was ebenfalls bekannt war, seitdem Wasserkulturen mit Knop’schen Nahrloesungen ausgefihrt worden sind. Does Any Organic Silica Compound Exist In Plants? IDNG T. Takeuchi. It is a well known fact that various organs of the animals contain among the mineral constituents also some silica, but whether this silica is present in the form of an organic compound and whether it performs any special phy- siological réle, is not yet decided. Itis thus far only with the feathers that Drechsel (Centralbl. f. Physiol. 11, p. 361.) isolated by extraction with a mixture of alcohol and ether an organic silica compound in small quantities which he supposed to correspond to a cholesterin ester of silica. Whether, however, this compound has anything to do with the growth of the feathers, further whether it occurs also in other forms of keratin, as wool, hairs, hoofs, etc. and whether that organic silica compound is produced in the animal body or is derived as such from vegetable food, has not yet been decided. Drechsel died soon after his discovery and nobody has followed up thus far his observation. It may, in regard to that question, be of some interest to compare the amounts of silica found in various organs of animals and plants, One thou- sand parts of ash of egg yolk were found to contain 5.5-14.0 parts silica, while one thousand parts ash of egg white 2.8-20.4 parts (Poleck). The ash of feathers contains 10-30% silica. Young animals contain in the same tissues more silica than old ones. In the ash of the pancreatic gland 129% silica was found by Faulhaber. In regard to plants Wolf's tables contain the following data: Parts of SiO, in 1000 parts dry matter : 430 T. Takeuchi. Roots and tubers wee... .....seaeonp ee oe 0.6-16 Leaves OR TOO erGpsmeme.....:...... ROPE ic L-S-Losk Leaves oMGramittes merres.......-.......0 1 3.6-42.0 Grains atid Seed see eeeeeey .......... 52 Soe aeee 0.2-7.1 Elodea candclemuismememee.........-..ccceeeer 28.9 In order to decide whether in plants or in their products silica is present in an organic form, two oils were tested for the presence of silica, namely rape and sesam oil. 100 c.c. were burned in a platinum basin, finally the minute residue was fused with some sodium carbonate and the solution eva- porated to dryness with an excess of hydrochloric acid, but no trace of silica could thus be detected. Since the leaves of Graminez are especially rich in silica, one kilo of hay cut into small pieces was extracted for a week with ligroin, and after filtra- tion the ligroin distilled off. The small amount of fatty residue remaining was fused with some carbonate of soda. After adding now hydrochloric acid in excess to the solution and evaporating no trace of silica was observed, on treatment with water the solution obtained was perfectly clear. The extracted hay was now left for ten days with alcohol of 90% and this extract treated as before, but only a turbidity was obtained at the fina test. The hay was now extracted with water for ten days, the filtered extract after evaporation mixed with carbonate of soda and the mixture fused at red heat until all carbon had burned away. The fused mass after dissolving in warm water was evaporated with hydrochloric acid in excess and the eva- poration residue treated again with distilled water. This solution showed hardly a trace of turbidity, so that the presence of silica in this extract became improbable. The extracted hay was now left with a solution of 2% crystallised sodium carbonate for eight days, and the filtrate treated.as before mentioned. In this case silica was present clearly. The well washed silica, very pro- bably present as hydrate, obtained from this extract weighed 0.062 g. The small amount of silica observed in the above mentioned Alcoholic extract made a second test with another sample of hay desirable. This time the finely cut hay (500¢., air dry) was directly extracted with alcohol Does Any Organic Silica Compound Exist In Plants ? 431 of 90% (1.4 litres) for 15 days at the ordinary temperature and the filtered extract after evaporation fused with a mixture of potassium and sodium carbonate. After evaporation with HCl of the fused product and treatment of the dry residue with water a not inconsiderable amount of SiO, was obtained, it corresponded to 0.065% of the hay. Since anorganic silicates are insoluble in alcohol, it appears therefore that silica occurs in an organic form in Graminez! and that its quantity varies considerably. Further tests are necessary to clear up the nature of this organic silica compound. 1 The sample of hay serving for this test consisted chiefly of Graminez, : y ot wey wah ie i ‘ io < 7? ‘7. on es) 4 iS . re é = 1p. -* Al's we ,, = es ‘7 . mr vaily 3 ; “ Can Calcium Carbonate Cause Loss of Ammonia by Evaporation from the Soil ? 1eNG T. Takeuchi. It is often assumed in agricultural circles that in the manuring with ammonium sulphate of soils containing calcium carbonate a loss of ammonia can be caused by the production of ammonium carbonate and the partial volatilization of this compound. Thus differences are often explained when on comparative manuring with nitrate and ammonium salt, the nitrate has produced a higher harvest. However, Stutzer and also Pfeiffer have already called attention to another possible explanation of such a difference, namely to the rapid absorption of ammonium carbonate thus formed by bacteria, since it is a very ‘favorable source of nitrogen for their growth and multi- plication ; thus a portion of the nitrogen becoming inavailable, the harvest would become less favorable than expected. These authors further called attention to the fact that ammonium carbonate eventually produced would be saved from volatilization by the absorptive power of the soils. However, there are still other circumstances to consider. The reason why chili-saltpeter is often (not always) superior to am- monium sulphate might be that the alkalinity produced gradually by the decomposition of nitrate in the soil serves to neutralize the acidity of the superphosphate and thus will render the manure neutral, while ammonium sulphate would in contrary gradually increase to an injurious degree the acidity, caused by superphosphate, when calcium carbonate is absent. Considering the above question from the plain chemical standpoint, however, it seems very improbable that ammonium sulphate would be capable of reacting easily with calcium carbonate and passing thus into 434 T. Takeuch . ammonium carbonate, with the production of calcium sulphate. In contrary, the reverse is true: when calcium sulphate comes tu contact with ammonium carbonate, calcium carbonate is rapidly formed with the production of am- montum sulphate and Liebig has recommended therefore more than 60 years ago to spread some gypsum on putrefying stable manure to prevent the escape of ammonium carbonate. The reaction in the opposite direction may only be possible under special conditions, as e¢.g., at high temperature. In which degree this process may be realized at szmmer temperature deserved to be tested. To a solution of 10 g. ammonium sulphate in 5 c.c. distilled water were added 100g. precipitated calcium carbonate and kept at 24°C in a well closed flask. Soon after the mixture was made a weak alkaline reaction became noticeable on turmeric paper. After four weeks the flask was pro- vided with a double perforated stopper bearing two glass tubes! of which one was connected with a flask of dilute sulphuric acid and the other with 10 c.c. of titrated sulphuric acid. Through the whole apparatus was sucked air which, in passing through the first flask, was deprived of any trace of ammonia that might have been accidentally present in the air. Thus puri- fied air passed then through the main flask? and carried the ammonium carbonate into the titrated sulphuric acid, which after two nours was titrated again. A special test showed that the air passed through the apparatus after that time did not give any further reaction with Nessler’s reagent. Found by titration=o.0152 g. NH, corresponding to 0.15299 of the am- monium sulphate. The test was repeated 8 days later and found this time =0.0139 g. NH, =0.1399% of the ammonium sulphate. The third test after a week yielded 0.0123 g. NH,=0.123%% of the ammonium sulphate.* The mixture was now subjected to boiling whereby the reaction was accelerated. 1 This operation was so rapidly performed, that no loss of ammonia was possible. 2 The main flask was repeatedly shaken and kept in a water bath at 24-30°C, 3 10 c.c. of our titrated sulphuric acid =0,102125 g. NH3. Neutralized at the first test = 1.488 c.c. At the second test = 1.362 C.c. At the third test = 1,204 C.c. Can Calcium Carbonate Cause Loss of Ammonia by Evaporation from the Soil ? 435 After three hours 50 c.c. of the titrated sulphuric acid were already com- pletely neutralized. This is however not surprising at all and was expected. In order to test the rapidity of the normal reaction 20 g. of gypsum were mixed with 4 g. ammonium carbonate dissolved in 100 c.c. water. The smell of ammonia disappeared almost at once; and the alkaline reaction became weaker, but after a certain time it did not decrease any more. The ex- amination of the filtrate and washing showed by titration that there were still necessary 16 c.c. of our titrated sulphuric acid for neutralization. The supposition that this remaining alkaline reaction was due to the lime salt of carbamic acid, was confirmed by the not inconsiderable lime content of the solution. In a further experiment, therefore, the commercial ammonium carbonate which contains carbamate of ammonia was avoided. The normal ammonium carbonate, produced by a mixture of ammonium sulphate and_ potassium carbonate in equivalent quantities, was here applied. 5 g. ammonium sulphate dissolved in 10 c.c. water, were mixed with 5.23 g. potassium carbonate dissolved in 5 c.c., whereupon a great portion of potassium sulphate formed was separated as a crystalline powder. The whole mixture was exposed to very low temperature and then filtered. Half of the filtrate, containing 2.16 g. ammonium carbonate was diluted with water to 50 c.c. and the solution shaken with 10 g. of gypsum. This excess of gypsum was used in order to increase the surface of contact. After shaking for some time, and standing 20 hours the mixture was filtered. The filtrate with wash water was slightly alkaline and the titration with our titrated sulphuric acid showed that only 1.7 c.c. were neutralised corres- ponding to 0.01736 g. NH,=0.69% NH. of the ammonium sulphate present. We can therefore infer that while gypsum and ammonium carbonate at the ordinary temperature act rapidly upon each other with production of ammonium sulphate and calcium carbonate, the process in the opposite direction can be realized only in a very insignificant measure at average summer temperature, even under the most favorable condition present in our flask. 436 T. Takeuchi. Hence it may be concluded that there ¢s no danger of losing any signifi- cant amount of ammonia by manuring a soil with ammonium sulphate when calcium carbonate ts present. 1 This conclusion agrees well with the observation of Wagnik, that no loss of ammonia was ob- served on a sandy soil containing fully 109§ CaCO,. Relation of Plant Growth to Root Space. BY S. Kumakiri. The causes of the smaller yield of plants when grown in small pots compared with such grown in larger pots have been repeatedly discussed by various authors, most recently again by Lemmermann. The final conclusion at which this author has arrived is that the conditions of the soil nutrients, and especially of the water supply are less favorable in small than in large pots. It is a fact that pots kept in a glass house and manured at the same rates as is usual in the fields, will yield generally less harvest than fields for an equal number of plants. The increased supply of nitrogen by the rain can not fully explain the better growth on the fields—under otherwise equal conditions. f The roots of plants grown in small pots will run to a great extent along the walls of the pots, as Sachs had already pointed out, hence they are on one side not in contact with the soil from which they draw the nutrients. This unfavorable condition will not be so great ina large pot asin a small pot under otherwise equal conditions. It is clear that the differences will increase with the number of plants and size of the species. In order to obtain here some data, the yield of a small species, spinach, was compared with that of a larger, viz, barley. The soil serving for the experiment was a loamy humus soil and was manured per 10 kilo with: 5g. Double superphosphate. 6,, NaNO, A, (NH gesOy Gor IG SOEs S. Kamakiri. ts LoS) ia) The small pots held 2 kilo soil while larger pots 10 kilo. The manure was certainly abundant as the number of plants grown per pot were only two. The objection that there was not enough of mineral nutrient in the small pots would therefore have been impossible. On October 10, 15 seeds of spinach and 15 seeds of barley respectively were sown in each of the large pots, while the small pots received 8 of spinach seeds and 8 of barley grains respectively. The young plants were thinned October 28 to two plants of equal size in all the pots. The spinach plants showed at an early date a considerable difference in height. The measurements were, cm. : December 22. January 17. February 2. & 7°5 8.7 9-7 Small pots lee! | 8.1 10.0 10.6 Vera arse 7.8 9.3 10.1 (een 13.9 16.9 atce pots y-aae | 12.4 14.2 17.8 JAVELASE "ene 12.2 14.0 L763 These plants were harvested on February 2 with the following KESult WO Small pots. Large pots. 17.4 49.0 Total harvest (Asser f 23-0 49-3 Average see ouveemecas 20.2 49.15 An examination of the roots in both cases revealed an immense difference, as in the small pots a very great number of roots were growing along the walls, very much more so than in the large pots. The barley plants also showed a very marked difference in height, as will be seen from the following data in cm. : December 21. January 17. May 29. 24. 28.6 86.7 Large pots: ee | ae < | 23) 28.0 770 Relation of Plant Growth to Root Space. 430 December 21. January 17. May 29. v 14g 16.8 57.0 Smiall-pots =:.... | 16.1 L77, 63.7 The plants in the large pots flowered earlier and ripened earlier than those in the small pots. The plants were cut May 29 and weighed in the air-dry state : Small pot. Large pot. Jn bs ( 8 27 Number of Stalks .:...28 | 5 20 2007 91.0 3 / 9 eB ne othe as = Se 5's | { 4. 70.8 ; 8.2 41.0 Gaatiisw Ooo. ieee. bss: ee 6.0 36.0 Hence the plants in the large pots produced here 5.4 times more seed than in the small pots. The examination of the barley roots also showed a very great difference in regard to the amount of root growing along the walls. Conclusion. With barley the total yield in the large pots was 4.8 times of that in the small pots, while with spinach the former was 2.5 times that of the latter, hence the extent in which the roots can spread along the walls of the pots has a very great influence in diminishing the harvest. On the Physiological Effects of an Excess of Magnesia upon Barley. BY S. Kumakiri. It is a well known fact that an excess of magnesium compounds depresses the yield of many crops; it has further been shown that it is especially the ratio of magnesia to lime which determines the degree of depression. But it was also of interest to observe whether special phenomena would characterise the growth at an excess of magnesia in the soil. For this purpose barley was grown at a considerable excess of magnesia. Six Wagner pots holding 8 Kg loamy humus soil containing 0.5% CaO and 0.4% MgO soluble in HCl of 10% received the following manure per pot, g: 4 Double superphosphate. 4.8 NaNO, 3.2 (NH,), 50 Aven) Ke Oke Twenty five grains of barley were sown in each pot Nov. 28 and in the following month the young plants were reduced to 15 of equal size per pot. Two pots A served as check pots and did not receive any further addi- tion. Two pots B received 10 g. each of crystallized magnesium sulphate in high dilution.! 1 The doses of magnesium sulphate employed here in the pots B and especially in C would certain- ly have produced a much worse result on a light sandy soil than they did on our loamy humus soil. 442 ~S. Kumakiri. Two pots C received each 50 g. of the same salt. It was noticed in, the following month that in the pots C the plants remained in height far behind the plants in pots B and these again remained behind the control plants which showed also much earlier development of ears than the plants of B and C. On June 8, the check plants were deadripe while the plants in B were in the majority still green and the plants in C had not yet commenced yellowing at all. With these latter plants a very remarkable phenomenon Mas noticed, namely the sheathes of the leaves had separated from the stems in many Cases causing the leaves to hang downwards. In comparing the number of shoots, it was observed that in the pots C not a single original seed had developed more than one stalk while in the pots B were found 2 plants with 2 shoots, one plant with 3 shoots and one with 4 shoots. Inthe check pot, however, were observed ftve plants with two shoots and one plant with three shoots. The plants were now untied and left exposed to the wind, whereby it was noticed that the plants in C had week stalks, offering but little resistance and bending down heavily. Summing up it may be stated. 1. With an excess of magnesia over lime growth and ripening process are retarded, the more so, the greater that excess. 2. A moderate excess of magnesia does not diminish essentially the number of shoots, but a larger excess will.? 3. An excessive amount of magnesia in the soil diminishes the strength of the leaf sheathes and of the stalks. 1 If we now take into consideration that according to former experiments carried out here, 14 parts of crystallised magnesiumsulphate are as effective on this soil as 100 parts of magnesite in finest powder and assume the availability of magnesite to be the same as that of the natural magnesium compounds in that soil, the ratio of available lime to magnesia would be in that mixture now=;z4 or= 2 The number of stalks starting from one seed depends therefore not only upon peculiarities of race or variety, and further upon influenc2 of moisture and temperature, but also upon certain propor- tions of magnesia in a soil. On Changes of Availahility of Nitrogen in Soils, |. BY O. Loew and K. Aso. Altho the useful as well as the injurious activities of the bacterial flora in soils have been repeatedly investigated and discussed, there still remain various points requiring a further clearing up. Soluble nitrogen compounds often serve to a certain extent to rapidly increase bacterial life,! instead of benefitting the crops. A sort of contest takes place in the soil for the nitrogen compounds between the roots and most kinds of the soil bacteria. How does the nitrogen of these bacteria again become available for the roots? When the bacteria die, what agency renders them soluble and where are the probable enzyms derived from which split these bacterial proteids into amidocompounds? and ammonia ? _ The question whether bacteriolytic enzyms are produced by certain soil bacteria has thus far never entered into discussion, altho the existence of such enzyms has been proved for Bact. pyocyaneum and the related Bact. fluorescens liquefaciens.* The fyocyanase, obtained from cultures of the former was found capable to dissolve typhoid-, pest-, cholera-, anthrax-, and diphtheria-bacilli, gonococci and the cocci of meningitis. Ammoniacal] nitrogen is more favorable for soil bacteria than nitric nitrogen, as Stutzer has observed in several cases. 2 Various amido-compounds can be absorbed by the root as such and serve directly for nutrition. Beessler, e.g., has shown that for asparagin. Cf. also E. Schulze, Landw, Vers.-Stat. 1905. Loew and Bokorny have proved that principle for alge. 2 Rudolf Emmerich ani Oscar Loew, Zeitschr. f. Hygiene 1899. These enzyms were called nucleases, since they dissolve the nucleoproteids of microkes, 444 0. Loew and K. Aso. Recently it has been shown by Heinze! that a sterilised mass of Azoto- bacter furnished available nitrogen to mustard plants but how these bacteria became soluble or how the nitrogen of thcir protoplasm became available to the roots was not yet decided by this author. Since yeast cells behave in various chemical relations like bacteria, some observations with yeast cells may be mentioned, as they shed some light also on the behavior of bacteria. Excretion of protein from living yeast cells. Under certain conditions living yeast excret a not tnconstderable amount of albumin, as one of us has observed many years ago, namely on treatment with a current of air at 30-32°. An amount of beer yeast, corresponding to 2 g. dry matter, was suspended in 250 c.c. of a solution, containing : Canesusarzeeaeeere......... 28508 10% Ammonitiim@getaber........ 20s... 1% KH PO, eee :-- 2 Sage 2% MgSO), ipo mereers,....---ssieaene 0.02% CaCl eee eee a... .- 0 scptnrs 20.0826. and a swift current of air, filtered through cotton, passed thru?. After 15 hours some fibrin-like flocculi had separated on the wall of the flask and the filtrate yielded after acidulating with nitric acid and heating a mass of coagulated albumin, amounting in the dry state=7.6% ofthe dry matter of the yeast. Recently M. Miiler,+ studying the behavior of the microbes in the in- testinal canal of cattle, observed similiar facts and he declared: ,,the pro- tein compounds produced by those microbes (of the first stomach or pounch) form only to a small portion real protoplasm, since they are excreted in considerable quantities.” It remains to be investigated how far this con- 1 Landw. Jahrb, 1906, Heft VI. 2 The experiments are described in the work of C. Nigeli: ‘Theorie der Girung, p. 81 [1879]. 3 The efiect was about the same when the ammonium acetate of the above solution was re- placed by nitrate or carbonate of ammonia, while sodium nitrate was not utilized. 4 Pfliig. Arch, f. d. ges. Physiol. 112, p. 247 [1995]; also Chem. Ztg. 1995. On Changes of Availability of Nitrogen in Soils, I. 445 clusion would hold good also for the microbes of the soil.1 It would appear, however, that in soils the conditions are not so favorable for energetic protein formation as in the intestinal canal and that usually excretion of protein from the living soil bacteria would not take place to any great extent. Not only yeast and bacteria, but also green plants produce more protein than immediately needed. This excess, however, is not excreted by them but remains dissolved in the cell-sap, either in the original labile state, active albumin or protoprotein,? or in the changed, passive state, ordinary albumin, which is separated in flocculi upon addition of some nitric acid to the filtered juices and heating. The organised proteids of cytoplasm, nucleus and chloro- plasts are insoluble in water. Nitrogenous matters excreted by dying cells. \t is a well known fact of plant physiology that in the moment cells die, all soluble matter diffuses from the cells to the surrounding water outside. This is due to the fact, that in the moment of death not only the chemical but also the physical properties of the living matter change,* and the cytoplasm, playing the role of an osmotic membrane as long as the cells are alive, becomes a mere filter, thru the pores of which all soluble matters can make their way.+ The writers have proved with yeast cells that the amount of nitrogenous matters, partly consisting of pcftones, passing to the outside upon the death of the cells, is by no means inconsiderable. * Fresh, pure cultured beer yeast was suspended in water and then by filtration under moderate suction the adhering water removed as far as it was practicable. It contained in this state=22.8% of dry matter. 63.27 g. of this yeast=14.42 g. dry matter were mixed with 120c.c. water and 20C.c. bisulfide of carbon and with repeated shaking left for 17 hours, after which 1 Since many microbs excret an enzym and enzyms are closely related to proteins, the excretion of nitrogen in this case is not doubtful. 2 Loew and Bokorny, in: Loew, Die chemische eepene der lebenden Zellen 2d, Ed, Chapt. VIII. 3 Ibid. Chapt. II and IX. 4 This principle holds good also for the animal cells but the physiologists have thus far not paid sufficient attention to it. A suitable object for demonstration is Spirogyra, which, on being killed, gives up at once the tannin content, as can be shown by ferrous sulphate. 446 0. Loew and K. Aso. time the yeast had assumed the grayish color of dead yeast while the yeast in the control flask with water alone had preserved the normal yellowish color of living yeast. The filtrates from both flasks were now evaporated to dryness and the nitrogen determined in the residue. The result was: the killed yeast yielded 2.9618 g. extractive matters containing 0.2383 g. nitrogen, while the control yeast yielded 0.4106 g. soluble matters, containing 0.0133 g. nitrogen. From this it follows that the original yeast cells with 8.70% nitrogen, on being killed by bisulfide of carbon had lost 20.52% of the dry matter containing one fifth of the total nitrogen, while the living yeast had inthe same time and at the same temperature (10-15°) only excreted 2.84% of its dry matter, containing only 1.069% of the total nitrogen of the original yeast cells. In a second test with another sample of fresh beer yeast the amount of mineral matters excreted in the moment of death was determined. Also this sample lost, on being killed with bisulfide of carbon, over seven times the amount of soluble matters than was excreted by the living cells in the same time; from 8.46 g. dry matter of yeast was thus obtained 1.5878 g. extractive matters, while in the control case with living yeast only 0.2172 g. These extracts were incinerated and the ash determined. The original yeast contained 6.79% of ash in the dry matter; 5.64 parts=8o0 per cent of the ash were water-soluble phosphates. The matter excreted by the living yeast in 15 hours yielded 0.0514 g. ash=0.61% of the dry matter of yeast, while the matter excreted from the killed yeast yielded 0.3970 g. ash=4.69% of the dry matter of yeast=69% of the total ash of the yeast. This excreted ash constituents were entirely soluble in water and contained 0.167 g. phosphoric acid and 0.155 g. potassa hence consisted chiefly of potassium phosphate. Various authors have analysed the yeast ash ; their data vary from 44-59% P,O, and from 28-39% K,O. It is obvious from the above determination that dying yeast cells lose by filtration to the outside much nitrogen, potassa and phosphoric acid. This law holds good also for all plant cells, also for other species of fungi and hence also for bacteria. These facts throw now some light also on the favorable effects of the On Changes of Availability of Nitrogen in Soils, I. AA7 soil treatment with bisulfide of carbon and other bactericidal substances, observed by Nobbe, Hiltner, Richter and others. Moritz and Scharpe! write: ,,422ucreased nutrition with nitrogen and mineral matters scems to take place after treatment of the soil with bisulfide of carbon.’ Efforts were made further to discover whether bisulfide of carbon would be able to render mineral matters in the soil more available by forming some sulfuric acid on oxidation, but such was not the case. These last named authors observed also that sterzlising the soil by heat \ed about to the same tucrease of crops than the treatment with bisulfide did, what becomes quite intel- ligible under the point of view developed above. However soils rich in humus often form acid by sterilisation at 100-1257 as Schulze observed and in this case an addition of some lime is necessary to render visible the beneficial effect of sterilizing. By the killing of the microbes, however, not only a part of their nitrogen, potassa and phosphoric acid becomes available to the roots, but also the air in the soil will be less over-charged with carbonic acid, produced by the respiration of the microbes, and better conditions for the respiration of the roots are produced, what becomes more important for clayey soils than for loose sandy soils. According to the views of several authors small doses of bisulfide of carbon and of other volatile matters act also as stimulants for root growth. Such a view is perfectly correct, as it not only was proved recently by one of us (A) for naphthalene? but also observations by Mr. Daikuhara from the Exp. Station at Nishigahara have shown it to take place with bisulfide of carbon.* This acts beneficially in several ways. Summary. 1. Protein matters are excreted under favorable conditions of growth by yeast and bacteria. 1 Arbeiten aus der biolog, Abtei!ung am kaiser], Gesundheitsamt, 1V, Heft. 2 [1904]. 2 Cf. page 413 of this Bulletin. % These observations will be published later on by the author. 448 Q. Loew and K. Aso. 2. On the death of cells all soluble matters can pass thru the cytoplasm to the outside. Peptones and mineral nutrients are excreted largely by dying yeast cells and very probably also by the microbes of the soil. This phenomenon throws some light on the beneficial action on crops of bisulfide of carbon when applied to soils. On the Continuous Application of Manganous Chlorid in Rice Culture, Il, BY K. Aso. In a former communication! the writer had stated that an increase of one third in grains and about one half in straw was obtained with rice by the application of 25 kilo Mn,O, per hectar in the form of manganous chlorid. That experiment was continued in the following year on the same field under the same circumstances as before. Two plots, each of 30 sq. metres, used for the former experiment, served again. 27 kilo barnyard manure, 15.5 kilo rotten human excrements, 230g. double superphosphate and 570 g. wood ash were applied to each plot, and while one plot received 200g. crystallized manganous chlorid, the other served as control. On July 5, the young rice plants were transplanted from the seed-bed? and on Nov. 5, the plants were harvested and weighed in the air-dry state with the following result : Manganese plot | Control plot. Miotalpharvests ila) #ey..t.c; ces meeee 27-12 26.52 GrrainGh ses ase .e0 PARE POPC COr ona occ 12.42 | MPA) RUMI eariccias cam \ch/sleuec naicecesie saree 14.70 14.35 MAURO ANINS oe eos a. seeaeeece cet ece nae eee eae | 11.85 Empty grains ..... ais dia). Sige haere 0.30 | 0.32 1 Bul. College of Agric. Toky6 Vol. VI. p. 131-133. [1903]. 2 Each plot received 306 bundles of twelve equally developed individuals, There was caused no noticeable damage by fungi or insects during the season. According to Fraps((Texas Apr. Exp. Stat., 450 K. Aso. These differences (2% of total harvest) are so small that a stimulation by manganese can hardly be inferred. The cause is probably due to the fact that weather conditions during that year (1904) were exceedingly favorable for rice culture, so that the plants of the control plot reached the most favorable growth possible. The action of manganese as a stimulant proved therefore superfluous ; in fact stimulation was hardly possible. In the following year (1905) the experiment was repeated on the same paddy field. The quantities of barnyard manure, rotten human excrements and double superphosphate were the same as before ; but 500g. kainit were applied in this experiment instead of wood ash. The manganese plot received again 200 g. manganous chlorid. On July 7, the young plants were transplanted from the seedbed. The growth proceeded without any disturbances by parasites. Inthe year of 1905 the weather at the flowering time in August was cool and rainy and interfered with the fertilization process. The plants were cut Nov. 27, and weighed in the air-dry state with the following result : Manganese plot. | Control pot. | otal hanvestyikcilot: saseses a teseee sere 26.103 | 25,201 (Grains; sete. eee ee ee 9.343 | 9.001 SUUAW “asecsasedecccta sien s seeeeenecreeeteees 16.760 16.200 Bull oral ns veqcscst0.2.02 teeseaueeaeeteees | 8.511 8.220 Brmpty*erainssscsentt eee 0.832 | 0.781 These figures show in the first place that the stimulating effect of manga- nese was much smaller than in 1903; the increase in straw being only 3.5% Bull. No, $2) an average rice crop consumes 16 pounds P,O., 42 pounds N and 55 pounds potash per acre, Rice straw carries with it, when removed, 14 pounds N and 31 pounds of potash per acre. In burning the rice stubble nearly 5 pounds of nitrogen goes up in smoke and requiring about 70 pounds of rape cake to restore the loss, In burning rice straw 14 pounds of nitrogen per acre pass off (which are contained in about 200 pounds of soy-bean cake). The ashes of one acre of rice straw contains nearly 3 pounds of P,O, and 37 pounds of potash. An average rice crop consumes more nitrogen than an average crop of Cotton, oats or corn. Ifthe rice straw ashes are restored the loss of potash is only 5 pounds per acre, the amount contained in the grain. On the Continuous Application of Manganous Chlorid in Rice Culture I. A5I and that in grains 3.89 over the yield on the control plot. This result might have been due to the fact that by the experiment of 1903, much more of the mineral nutrients of the soil and manure were absorbed by the increased production on the manganese plot than on the control plot leaving the latter richer than the former. However the manganese plot was not only left poorer in nutrients, but also must have increased in acidity. In order to decide the question of the cause of that phenomenon, the experiment was modified in the following year (1906). The relative amount of manganous chlorid applied remained the same as in the previous years, but it was partly applied in conjunction with lime, and partly with an increase of manure. Six plots served for this trial,! each measuring 9.5 sq. metre. Three of them received crystallized manganous chlorid in high dilution, 66.6 g. each, July 11, while the other three served as check plots. Two plots were limed and two received 33% increase of manure. The plan of the experiment is seen from the following sketch : No increase of manure.2 A. 1D: Manganese chilorid. Check. No increase of manure. ; 7 Check » Manganese chlorid + lime. | ee 33% increase of manure. GC: | Be Manganese chlorid. Check. | 200 g. calcium carbonate were applied to cach of the two central plots Band E, May 20. The transplantation of the young rice plants took place 1 The former manganese plot was subdivided into three manganese plots, the former check plot into three check plots. 2 The general manure (applied July 10) consisted for the plots A, B, D and E of 166g, Kainit, 77 g. double superphosphate, 9 k, barnyard manure and 5 k. rotten human dung respectively, The plots C and F received one third more. 452 K. Aso. July 12, 126 bundles for each plot, each bundle of twelve healthy plants. The weather of this year was not favorable for rice growth, still not so inferior as the previous year. The plants were harvested on Nov. 21 and weighed in the air-dry state four weeks later. The result was in kilograms : Total harvest. | Grains, Straw. Full grains, Empty gr2ins A | 7.249 | 1.969 | 5.280 1.580 0.389 | B | 7,318 | 2.373 ! 4.945 2.000 0.373 | | | | ( | 7.300 | 2.490 4.810 2.140 0-350 D 5.868 | 1.361 4.507 0-890 0.471 13 7-000) 2.178 4,828 1.760 0.418 F 7.322 2.327 4.995 1.960 0.367 It will be learned from this table : 1. Liming was favorable, the total production being increased by 19% (compare the check plots D and E), 2. Increase of manure at 33% increased the total harvest for 24.8% (compare F and D). 3. On the manganese plots the increase was relatively greatest where the manuring conditions were the least favorable; the increase in total harvest being 23.5°% (compare the check plot D with the manganese plot A). 4. On the limed plots the manganese has led to an increase of 4.4% only of the total harvest and of 13.6% in full grains (compare the limed check plot E with the manganese plot B). 5. Onthe plot with increased manure the manganese exerted no effect as to the total harvest (Difference—0.3%), but as to full grains an increase of 9% took place, there being relatively more straw produced on the check plot F than on the manganese plot C. 6. Since the considerable effects of manganese of 1903 have not been reached again neither after liming nor after increase by manure, the experi- On the Continuous Application of Manganous Chlorid in Rice Culture II. 453 ments will be continued! until a satisfactory answer to the question is obtained, under which conditions the effects of manganese are the most favorable? Finally the increase of total harvest in the different years by the action of manganese may be compared : LQO3) ss... Sue oo ons deco sk wi ogee ee 41.89% 11012 Teas Total weight, ¢.......... 4 | 328 232 J [ 352 | 298 ( 49 ( 48.0 Weight of ears, g....... 4 47.8 49.0 1] Wie ‘| 465 ee This gives the following ratio : Average Of The 2aeMerie PGES ......-2serassaenpeceore = 100 Manganese sulphate in top dressing at the rate of 40 kalo per Mapes. .....- <2 sepgee aes fats Sodium fluorid, at the rate of 0.5 kilo per ha ... =107 Manganese sulphate had therefore in this case produced a better result than sodium fluorid ; however this may change on other soils.? 1 In certain soils sodium fluorid may be much more quickly transformed into the but little active calcium fluorid, than in others. On Different Forms of Phosphoric Acid in Press Cakes. BY T. Funatsu. Since refuse press cakes are frequently used as manure, it is of some im- portance to determine the amounts of phosphoric acid present in different forms, as the availability for plants differs very much in different compounds. A portion of the phosphoric acid in such cakes is soluble in water another in dilute acids while a further portion is dissolved by alkaline liquids. This last mentioned portion is due to lecithin and nucleoproteids. As to lecithin, it is probably easily decomposed in the soil by microbs, but a certain part of nucleoproteids might resist destruction and contribute a share to humus which contains not only some nitrogen but sometimes also some P,O, in a form not easily available to the plants. From this point of view the writer has determined in several press cakes and for comparison also in fish guano the amounts of phosphoric acid soluble in different solvents. Soybean cake: Soybean cake is imported from Manchuria to Japan where it serves extensively as manure. Soybeans contain about 1.6% lecithin (Schulze) and yield 14-169% of oil!. Soybean cake shows according to a report from Experiment Stations in average : N=6.5% P,0,= eee K,O =20%8 Our soybean cake contained 12% hygroscopic moisture. In determing total phosphoric acid 3 g. air dry cake were incinerated and the phosphoric | This oil serves in China for cooking purposes. 458 T. Funatsu. acid determined as usual ; obtained 0.057 g. Mg,P,0, =1.211% P.O; of the air dry cake. To determine phosphoric acid present in the form of lecithin 10 g. air dry cake served for extraction with ether and alcohol. After evoporating these extracts to dryness and fusing with KNO, and Na,CO, was obtained 0.027 g. Mg, P,0, =0.16% PO, of the air dry cake. ne determine phosphoric acid present in the form of nuclecproteid, the residue of this extraction was treated with dilute hydrochloric acid, (2) and the well washed residue incinerated with addition of carbonate and _ nitrate of soda. Obtained: 0.031 g. Mg,P,0,=0.197% P,O,. The acid liquid and wash water (a) were filled up to 250 c.c., one fifth of this liquid wes evaporated and the residue incinerated as before. Obtained : 0.31 g. Mg,P,0, =0.1979% P,O,, present in inorganic form and as so- called anhydro-oxy-methylen-phosphoric acid. Calculated for the cake, dried at 100°, the result is: Total phosphoric acid =1.38 % P,O, as lecithin =O Ag P,O, as nuclein = 0.226% P.O, soluble in 49 HCl=0.9809% Sum = 380%, The same determinations were carried out with cotton seed cake, rape cake and herring guano dried at 100° with the following result, to which we add for comparison the soybean cake analysis mentioned above. tton se : Soybean cake. ee ae Rape cake, Herring guano, | Total PLOY eee 1-38% | 2-25.96 252% 4.56.26 | ] | P,O, as lecithin ..4...... 0.17% 0.12.96 0.2096 0.35% ! | POO. as nuclein. ..7 22502 023% 0.30% 0.26% 0.66% P,O, sol. in dilute HCl 0.98% 1.80% 2.37% 3.55% | On Different Forms of Phosphoric Acid in Press Cakes. 459 The relative amounts total P,O, =100. | | 5 a, | Soybean cake. Score eo | Rape eake. Herring guano. . . . . | ; iz O-vasdlecithin) ves-.5.0- | 12.4 5.0 7.0 eal k ; ie On eaS Glenn. 702.2. | 16 5 | 13.2 | 9.0 14.4 P,O, sol. in dilute HCI 71.0 | 81.7 | 84.0 | 78.0 These results show that the amounts of phosphoric acid present in the form of nucleoproteids are comparatively small. As to the better availability of phosphoric acid of fish guano, observed by Nagaoka, this may be due to the forms soluble in water (K,HPO,) and in dilute hydrochloric acid. In seed cakes there exists much more phosphoric acid in organic com- binations than in fish guano. A special test was further carried out in order to observe whether a part of phosphoric acid would be easily split off from nucleoproteids by soil bacteria Nucleoprotein (from beer yeast) freshly prepared and correspond- ing to 9.9 g. dry matter was mixed with sand, infected with soil bacteria and moistened with a 0.5% solution of potassium acetate containing 0.2 g. MgSO,. After two months in the thermostete at 27° only 39% of the original phosphoric acid had become soluble. The mass had become almost black and had an ammonical and fecal odor, showing a certain degree of putrefaction. 1 Cotton seed cake and also poppy cake contain often more phosphoric acid than observed in the above samples of cake. ‘Thus in U.S, the avarage amount of P,O, in cotton seed cake is considered = 2.889%, and as to poppy cake, as much as 3.5% PO, was observe in one sample by Mach. On Bat Guano from Marianne Islands. BY S. Kanamori. The caves of Rota and Saipan in the Marianne Islands contain a loose dark brown mass, resembling somewhat dry peat, in strata of several meters in thickness.1 There occur small white particles and occasionally crusts of about the size of a walnut in that formation but whether these admixtures are spread uniformly through the strata is not yet known. Since the microscop reveals numerous particles of wings and legs of insects and even occasionally wing-scales of butterflies, there can hardly exist any doubt that this deposit represents bat-excrements which probably have undergone partial changes in course of time. Warm dilute caustic potassa extracts a humus like substance with evolution of some ammonia. ‘The solution be- comes at the same time very slimy, rendering filtration rather difficult. Hydrochloric acid precipitated from this solution a brown flocculent mass, which contained nitrogen. The extracted residue consisting mainly of chitin represented the chief mass of the manure ; it was digested with hydrochloric acid of 5% for one hour, washed and dried and served for some further bacteriological tests. The white particles and crusts contained in average 8.45% P,O, and contained nitrogen and carbonate and phosphate of lime. The latter might have derived and accumulated from the urine of the bats. The analysis of the loose brown mass yielded the following results: 1 A sample of this formation was kindly furnished us from Mr. Weinberger of Yokohama wh had received it from a German officer stationed at those Islands. 462 S. Kanamori. Hyeroscoepic Wateeeees.........2:--- 14.2% Organic matter’ ~22.2.- . ee. TA3 >, ASH? ts ae... .....- ac) eE, In the air dry mass of two samples?, % ... I II Total phesphorieacia, 2 5).......... «sso age Total nitreten 2 255. ee 14.89] 8.97 Potassa 2255.1 see EEE... « tee s “2A47\ 2:09 P,O,, soluble in. 1% citric acid ...... E50) 1-75 Nitric acid, <2 Seer eee... . >. ceca 0.22 N, soluble in acidulated water, chiefly amMnioOnia ees Petts Me re oan O14 (a) N in humus, soluble in alkali ...... 2.64 (6) N, liberated as ammonia by hot alkali... :2 225 seems ... Se sem eso, The aqueous extract contained besides some nitrate also traces of sul- phates and chlorides. The question arises whether this guano would be a readily available nitrogenous manure. This question can, however, not be answered directly since a good deal of the nitrogen is present in the form of chitin, a substance that putrefies not so readily as protein matters do.* Besides chitin, however, there are present doubtless also other nitrogenous compounds, which can 1 Analyses have been published of bat-guano, found in different parts of the globe, showing great differences in composition of this product. Since the water content was found between wide limits (20-67%) it can easily be explained that these guanos from different localities have undergone a very different degree of putrefaction and extraction. The nitrogen content varied from 2-14%, phos- phoric acid from 2-11%, potassa from 1.6-29§, nitric acid from 0.2-1.2%. 2 Sample I contained many of the white particles mentioned, Sample II was nearly free from them. 2 Whether common soil bacteria can gradually attack chitin was mot yet investigated. Benecke IV. Benecke, however, has isolated from putrefying chitin (tortoise-shell) a kind of bacterium which destroys chitin very easily, He named it Bact//us chitinovorus. It may be that this microb occurs also in soils occasionally which questicn I am now investigating. The writer has seen mouldfung attacking chitin, On Bat Guano from Marianne Islands. 463 easily be decomposed by boiling with a solution of 1% sodium hydroxid. Thus the air dry manure yielded after boiling for an hour and a half 2.879% N in the form of ammonia, determined by titration of the distillate.1 Very probably this nitrogen becomes more easily available to the plant than that of chitin. Chitin=C,,H,,N,0O,, contains 6.09% N and would yield after perfect decomposition 7.289% NH,. The question of the availability of that organic guano-nitrogen had to be decided by a practical test. Two pots A filled with 8 Kilo unmanured loamy humus soil were manured, each with : Potassium sulplatemmee. "aoas..<..-.--..t0-+ 0-20, 6g. PUNO, Stl PM ees 26% sass vce -ematsacenseeee iss Secondary calcium phosphate ............... Eee Two pots B received 11, 4 g. of the guano, corresponding to the nitrogen amount of pots A. The difference in P,O; and K,O inthe pots A and B was supplied to the pots B in the form of secondary calcium phosphate and potassium sul- phate. Hence the pots B received: BAEAGUANO cp IO cress = x 2anrevesvecas II.4¢. Potassium sulphate ..... 5 i a ea os Secondary calcium phosphate ............... O:7G; Two further pots C were prepared like A, except that no nitrogen manure was added. Barley, zo seeds per pot were sown Nov. 8. The young plants when 15 cm. high were thinned to Io per pot, all of equal height. During the vegetation it was noticed that only the plants with full manure were perfectly green while with the bat-manure and in absence of nitrogen the stalks were reddish, due to the presence of authokyan which, as S. Suzuki? has shown, appears when either phosphoric acid or nitrogen is wanting. In our case however it only can be ascribed to the lack of nitrogen. Already this phenomenon shows that the nitrogen of the bat- 1 Taken 2 g. sample; distillate required 2.8 c.c. titrated su!phuric acid. Io C.c. of this acid= 0.6982 g. H,SO,. 2 This Bulletin Vo'. VII. No. 1. 464 S. Kanamori. guano was not easily available, altho there existed bacteria in the soil which can attack it gradually, as my observation have proved.! Also a superficial comparison showed that the plants of the fully manured pot were far ahead of the other plants. Since there existed danger of attack by fungi, the plants were cut Jan. 29 and weighed in the fresh state. The observations were : | | Average number of stalks, Fresh weight, g. (average). A 42 53-5 Full manure. B “ 35 3g Bat-guano. C ee 36 28 No nitrogen added. This result shows that this bat-guano, containing undecomposed chitin, forms no readily available source of nitrogen. oD 1 Nitrogen from chitin, becoming thus nitrogen of the bodies of microbs, cannot at once become available for roots. On the Composition of the Shoots of Aralia cordata. BY T. Takeuchi. The shoots of Aralia cordata (Jap. Udo) serve as food in Japan. Its branched stems are eithet consumed in the fresh state with addition of some salt, or after boiling with soy souce. Some time ago, Dr. Fairchild,! agri- cultural explorer of the Department of Agriculture in Washington who on his exploring tours around the globe had also visited Japan, has warmly recommended these shoots for the American cuisine and proposed the cultivation of the plant in the United States. He declared that a fine salad may be prepared by cutting the shoots into long thin shavings and pouring over them the dressing composed in the usual way of oil, vinegar, salt and pepper. ‘‘ These slices of Udo are crisper than slices of celery and have none of the objectionable stringy fibres of the latter. They have a fresh taste, like the midrib of a lettuce leaf, with a slight but most agreable suggestion of pine flavor. The tenderest young shoots of celery could not be more brittle than these branched stems of Udo.” “From the first of October until the middle of May this vegetable is for sale in the markets of Japan, and in this winter character, aside from its being an excellent salad, lies its great value. It is comparatively cheap and is eaten by the poor Japanese as well as by the rich.” “From its adaptability to winter culture and its excellent quality, this plant deserves to become as well known as asparagus or. celery,” Indeed, in the cooked state its taste is at least as fine as that of asparagus. 1 Bulletin No. 42, Bureau of Plant Industry, Washington. 466 T. Takeuchi. There are two varieties of wdo, called respectively ‘ Kan-udo”’ and “ Moyashi-udo,” and these, though of similar appearance as they are placed on the market, are quite differently cultivated. My observations on the chemical composition relate to Kan-udo, the shoots of which are in average 45 cm. in height and 1.5 cm. in thickness. For investigation served only that part which also serves as food ; hence the lowest part and the tip, consisting in some small branches with leaflets, were cut away. The surface is reddish, due to the presence of antocyan to judge from the change into blue color by alkali. The juice has a slight acid reaction, and contains some soluble albumin, reducing sugar, tannin, further oxidase, peroxidase and catalase. Pentosans, and galactan were observed, but not mannan. The presence of starch was revealed by the iodine test. The fresh substance consists of 94.5% water and 5.5% dry matter. 100 c.c. of the juice required 8.5 c.c. of =", NaOH solution for neutralization. The analytical determinations were carried out after the usual methods. 2g. served for the determination of nitrogen. 25; 5 33 iss “: ,, protein nitrogen. LO; = sane “ ,, ethereal extract. 5 g. served for the determination of pentosans. In their determination 0.174 g. and 0.1705 g. of the furfurol precipitates by phloroglucin were obtained in two samples. In the determination of galactan, 5 g. yielded on oxidation with nitric acid in 2 texts: 0.0208 g. and 0.0204 g. of mucic acid. In the determination of tannin, 5 g. yielded after Wolff's method 0.039 g. and 0.0382 g. of CuO. Also citric acid is present. In the following table the results obtained are shown, and for comparison the average of 4 samples of Asparagus officinalis is added.1 1 Data from Kénig’s “Chemie der menschlichen Nahrungs-und Genuss-mittel.” Vol. II., p. 660. On the Composition of the Shoots of Aralia cordata. 467 Shoots of Aralia cordata. Shoots of Asparagus officinalis. | 6 of dry 9% of fresh 96 of fresh | matter (1o0°C,) | substance. substance. Wich elame dies na scans ceteaeesctte | fo) 94.50 Wiaterimse ee tcceat cnet: | 93-75 Grud Sproteimysce.s sce -.ce- | 19.97 1.10 (Crudempkoteinees see sceee 1.79 Grademibrescsstsceensecn tae | 15.38 0.85 Crude fibres eee tee 1.04 Btherealvextract®: v2... 7.67 0.42 Crud effa tance | 0.25 JENS] DL paca teRSt coco ee SEE CRE EE | 9.91 0.55 ASHP PRE eeat ad cores 0.54 Miotalimiteopenl) es....-2+-- | 3.20 0.18 Nitrogen free extract ... 2.26 Albuminoid nitrogen...... | 2.01 O.1II PS (Se gs} a A A ane 0:37 Non-albuminoid nitrogen | 1.19 0.05 | LENO} teaereoenee aeessoes | 12.56 0.69 | WD Exit OSGi eaeaccecereciacacs 4.41 0.24 | MUIGCLOSE! Wee sar sdcebacsncess | p20 0.07 SlamiGhie | oeweeese-eees-nsscrs 2.26 0.12 IBGHLOSANS ese). 22 gehe2-ataseee 7-47 0.41 Galactanky on. sche.c2tcsscess 0.53 0.03 MIFCTAININNM ase ae ss eee sees. aces | 5-95 0:33 Since the introduction of Aralia cordata into Europe and America, for the purpose of the cultivation of its shoots, might some day take place, we add the ‘following culture notes, taken from Fairchild’s Bulletin above mentioned. “ The Cultivation of Kan-udo. The seeds of this variety are sown broadcast in seed beds, prepared of rich garden earth, zz the month of March or April, and are allowed to grow there for one year. The following spring the individual seedlings are transplanted from this seed bed, after the tops, which have died during the winter, have been removed, and they are then set in rows 2 feet apart and 10 inches from each other in the rows. In these rows they are cultivated all summer, or until September, when the leaves begin to turn brown. The stems are then cut back close to the rootstocks 1 Besides fat and lecithin there was another substance present, which I intend to examine later on, 468 T. Takeuchi. and the earth is piled up in a mound 2 feet high above the latter. In forty days the new shoots, which begin to form as soon as the oid ones have been cut back, appear above the surface of the mound. They are then ready for cutting, and the mound is opened and the marketable shoots cut. Each rootstock produces about five of these blanched shoots, three of which are probably fit for the market at the first cutting, early in October. The remaining small shoots are covered up again and allowed to grow for a second cutting a week or so later. In removing these shoots for market care is taken to cut close to their bases, so as not to leave stubs, as the presence of the latter is said to prevent the rapid growth of the remaining young shoots. Generally only two crops of shoots are secured of the Kan-udo, but occasionally there are three. After the removal of the last crop the root- stocks are buried and allowed to remain over winter. In the spring the mounds are opened and rich manure is applied in trenches running on both sides of the plants. Thru-out the summer the plants are allowed to grow and are again cut down in autumn and treated in a similar way to that just described. The life of the Kan-wdo rootstock is more than ten years, but beyond that age its use ceases to be profitable. Altho generally grown from seed, this variety can be reproduced from root cuttings, though the latter method is considered less practicable, owing to the fact that the large root cuttings take up more space in the field. The season for Kan-udo is October and November, and being the earliest variety and occupying the fields to the exclusion of other crops it is also the dearest, sometimes selling for as much as 25 cents for a bundle of 16 shoots. It is not otherwise preferable in any way to the other variety, which first appears in the market toward the end of November.” Note on the Composition of a Chrysanthemum Flower, serving as food. BY T. Funatsu. In the province of Akita in Japan, the yellow flowers of a kind of Chrysanthemum are largely consumed as an addition to fish, or also soaked in hot water and prepared as a salad. These flowers are called ‘“ Hoshi- kiku,’ are of sweet taste and are considered as a delecacy. They are sold, either in the dry state pressed into sheets, or in the salted state. It was of some interest to determine the composition which was found to be as follows: Hygroscopic moisture ......... 2 i eae 10.20% EINE OSS Fa.) a... oo 5 32, EE) oon ain poe cmesemp ee 25,” LOO) i Wane SUCAL....<:--:.3:.05aeen Bees. ahd vacant meee 2:80; Grade PrOLeul . -..,. i: cee ears senses -nesndeteens-seene 10:82. SIUC gs... 95: in eae air 13:03 33 Baepermer CxXtraCt) 2 aemMED ts 5. oleic acess eames TENG = FRM Nas ico 155 sa oe Basic ten vee ao dee eee 5-O5r;; Se) ee Pe. eg Se E.O27, Extractive matters, pentosans, etc. from diff. ...... 25.38 ,, Note on Japanese Tobacco from Satsuma. BY K. Baba. The Tobacco raised in Japan has served thus far chiefly for the manu- facture of cigarettes. Moderate quantities were also exported for wrapper purposes but no tobacco variety suitable for fillers in cigar manufacture has thus far been produced. The heavy manuring applied in the United States for the production of “ gummy” leaves, has thus far not been applied, at least not in central Japan. Recently, however, some attention has been paid to the production of a suitable cigar leafand while formerly tobacco was only cured but not fermented, the fermentation in bulk has commenced to be practiced. Hence the question whether Japanese tobacco varieties are capable to develop a good cigar aroma has become of interest and the writer has carried out the following investigation in regard to the qualities of a tobacco (Nicotiana rustica) raised in Satsuma province, thus far the most famous tobacco in Japan. Since the aroma decreases with the increase of fat and protein, it was necessary to determine these constituents. Kissling holds that the resins of tobacco are important for a proper aroma; but also certain compounds, produced from nicotine by oxidation in a thorough fermentation process, may contribute to the formation of aroma.!__ Hence resins and nicotine also were determined, the resins after Kissling’s method. For analysis the middle vein of the leaves was discarded, the determinations were made according to the usual methods, the nitrogen after Kjeldahl, protein-N after Stutzer, nicotine by distillation and titration. 1 Thoms has obtained an ethereal oil from the smoke of tobacco, which results from a product of the fermentation process, by the dry destillation in smoking, 472 K. Baba. Four qualities served for the test, namely A, lowest or sand leaves (doha), B, lower middle leaves (chuha), C, upper middle leaves (honpa) and D, upper most leaves (tenpa). Here as in other countries the central leaves are considered superior to the lowest, oldest and to the youngest. The result of the analysis was : Hygroscopic water. EA oi inp OSE EEE... 05s ocd na iene 8.789% By oie _ 8.148, Creede one eG we oe. eonnaes Snes, EgOuU age Dads ete. . «sonst Ses 8.062 ,, The dry matter contained, % : A B E D Cradetiabar ht. 2. cee cndce | 6.302 11.549 14.004 12.616 Soluble in petroleum ether... 3.001 5-839 6.666 6,003 Resin 2 ay erethereese2 wl dase 0.365 0.432 0.824. 0.799 + 3. Praleoholl ts. 0.498 | 0.858 1,133 1,021 otal nitrogen 2 Ls hs ae 0,893 1.381 1.525 1.594 Protem nitrogen J.123540.0.252. 0.5438 0.7754 0.8080 0.8753 Protest sends ea tceeeec neces 3.4300 4.8463 5.0500 5.4706 Amido-nitrogen ............... 0.163 0.272 0.300 0.372 INICOLINE Meee scence eae enone 0.8897 1.4435 1.8112 1.4099 (corresponding to nicotine N) 0.1541 0.2500 0.3137 0.2442 INET ho eo eee noes 0,033 0,102 0.126 0.125 Nitriciacid te +. teen 0.1180 0.1723 0.1691 0.2012 Ash: 3 Sane el SEE eet cesses: 22.271 14.811 14.042 12.654 These numbers show a moderate content of nicotine and protein and a unusual high proportion of the resin fraction soluble in petroleum ether. The figures for crude fat in B, C and D are rather high, too high for a good cigar leaf. In addition I mention some analytical determinations of three other Japanese Tobacco varieties and add for comparison the numbers obtained with the Ibusuki tobacco above analysed. Note on Japanese Tobacco from Satsuma. 473 In dry matter, 100 parts: Total aoe Crude Nitric | 2 K COs in N. Nie fat, ae RE | the ash | | SHIEOUME ss ee 1,122 1372 7.821 0.140 14.552 | 1.990 B | 1.632 0.679 8.135 0.200 16,000 2.204 Takeda produced | cs Bee \C | 2.072 0.807 8.521 0.353 | 14.123 2.052 B 1.299 1.187 9.235 0.180 | 18.465 2.201 Daruma produced } ,, 2 Ss Pe a Te Cc 1.589 1.273 11.671 0.183 | 16,121 | 2.356 D 1.712 1,002 11.102 0.188 | 13.111 | 2,016 A 0.893 0.889 6.302 0.118 22.271 | 2.043 | ; Ibusuki produced B 1.381 1.443 11.549 0.172 | 14.811 2.406 in Kagoshima, 1.525 see 14.004 0.169 | 14.042 2.132 | 1.594 1.409 12.616 0.201 | 12,654 | 1.693 i - a i. i Note on Bacillus Metiylicus. T. Takeuchi. This microb occurs in a, reddish and ina colorless variety. Moreover the red variety when cultivated in bouillon develops in a colorless state. Since it seemed to me that the reaction of the solution might have an in- fluence on the formation of the color, a further experiment was made. The original culture solution of Loew contained sodium formate, which by its gradual conversion into sodium carbonate produced a tolerably strong alka- line reaction. In order to avoid this reaction I substituted magnesium formate! for sodium formate. After exposing the culture solution for several months in an open Erlenmeyer flask to the air,2 a considerable formation of white films was noticed and further at the bottom of the flask a crystallisa- tion of magnesium ammonium phosphate; the liquid itself had remained perfectly clear. An alkaline reaction was hardly perceptible. The films appeared to consist of a pure culture of Bacillus methylicus.* The composition was : Magnesium formate 0.5% Dipotassium phosphate 0.2,, Diammonium phosphate 0.1 ,, Magnesium sulphate 0,01,, 2 This microb also occurs, as Katayama (These Bulletins V p. 255 and VI p. 191) observed, in the soil at moderate depth, also in rivers and sea water, 3 This microb is far different from the Bacil/us formicicus of Omelianski ; the latter can not develop with formates as an exclusive food and shows other characteristics, as Katayama (Ll. c.) has already discussed. It is, further, different from B, methanicus which has cilia, is motile and can assimilate methan, 476 T. Takeuchi. In order to observe whether this color/ess culture of B. methylicus would assume a veddish color, when nourished with sodium formate, the films were shaken with sterilized bouillon-gelatine and a plate culture pre- pared. From one of the numerous colonies which all resembled each other a trace was inoculated in sodium formate culture solution and in sterilized bouillon ; further the growth on potatoes and in stab culture in bouillon gelatine was observed. . The result was that in sodium formate solution the microb showed again a ved color, while on the control media the development was quite charac- teristic for B. methylicus. Hence not only a red and white variety of B. methylicus must be dis- tinguished, but also it must be admitted that the red variety can grow in a colorless state when care for neutral reaction is taken. Ueber die chemische Zusammensetzung der japanischen Soja-Sauce oder “ Schoyu.” VON U. Suzuki, K. Aso und H. Mitarai. Da die japanische Soja-Sauce oder “ Schéyu”’ ein unentbehrliches Volks- Speise-Mittel ist, dessen Verbrauch in ganz Japan jahrlich iiber vier Millionen Hektoliter steigt, so bietet es physiologisch, wie technisch ein wichtiges und interessantes Problem, die chemische Zusammensetzung und auch die chemischen Vorgange, die wahrend des Reifeprozesses der Sauce vor sich gehen, genau kennen zu lernen. Zwar haben sich manche Chemiker bereits mit dicsen Fragen beschiaftigt, trotzdem bleibt aber noch vieles unklar, besonders das Schicksal der Stickstoff-Verbindungen, die im Ausgangs- material (Sojabohnen und Weizen) hauptsachlich als Eiweiss-Stoffe sich vorfinden, und wahrend des Reife-Prozesses entweder durch Enzyme oder durch Mikroorganismen eine energische Spaltung und Veranderung erleiden, was natiirlich zu einer ganzen Reihe verschiedener complizirter Ver- bindungen fiihren kann. Da die Trennung und das Identifizieren der Eiweiss- zersetzungs-Produkte eine schwierige Aufgabe ist, so hat man his jetzt in dieser Richtung keine befriedigenden Resultate bekommen. In neuester Zeit hat aber unsere Kenntniss tiber die Zersetzungs-Produkte der Eiweiss-K6rper durch die Untersuchungen von E. Fischer, E. Schulze, A. Kossel u. A. eine neue Gestalt gewonnen, und deshalb schien es uns auch méglich, durch ’ Anwendung dieser neuen Methoden das Problem der ‘“‘ Schoyu”’ etwas naher aufzuklaren. Die nach altbewahrten Regeln hergestellte Sch6yu-Probe, die wir ftir diese Untersuchung anwendeten, wurde uns von Herrn K. Mogi, Besitzer einer grossen Schoyu Brauerei in Noda, giitigst geliefert, wofiir wir ihm unseren besten Dank aussprechen. Diese Probe wurde aus 3 Maischen hergestellt :— 478 U. Suzuki, K. Aso und H. Mitarai. 7 April 1904 3 Hektoliter 7 April 1905 3 Hektoliter 27 October 1905 5 Hektoliter und die véllig ausgereifte Maische ausgepresst. Als Material wurde teils gewohnliche Sojabohne teils nackte erwendet und mit dem gleichen Volumen Weizen aus der Higo-Provinz gemischt. Das Kochsalz war aus England bezogen. Bei der Herstellung der Sauce werden zuerst die Soyabohnen 5 Stunden gekocht, dann nach dem Abkiihlen mit gerésteten Weizenkérnern gemischt und diese Mischung nach Infection mit den Sporen von Aspergillus Oryzae auf Gestellen in einer engen, meist halb-unterirdischen Kammer bei 30—40° 3 Tage lang belassen, wodurch ein weit verzweigtes Mycel um alle Kérner herum sich entwickelt. Diese nun Soya-koji genannte Masse, wird mit der Kochsalzlésung von 15-20% vermischt und in grossen Kufen 1-3 Jahre stehen lassen, wobei die auf der Oberfliche wachsenden Schimmelbildungen von Zeit zu Zeit in die Masse geriihrt werden. Jenes Mycel liefert die verschiedenen beim Reifeprocess nétigen Enzyme. Um eine bessere Qualitat Sauce zu erzielen, wird nach langeren Pausen zu der fermentirenden Sauce noch ein oder zweimal Soya-koji gesetzt. Diese Probe hatte folgende quantitative Zusammensetzung :— Karbe™ ..2. 5.3)... eee Dunkel braun. Realtion 5. 2.4.55... eee Ziemlich stark sauer. Sp2z; Gewicht - ) oes 1.197 Wasser: <.0....c-a -oe eee 67.15% Trockensubstanz (2..5-49->- 32.85% In 100 Theilen Trocken substanz. Organische Substanz......... 49.12 Roh-Asche ,:<<2b-7.2ee ee - 50.88 Chior — 3... ccacdece ee 27-24. Chlor als NaCl berechnet ... 44 94 In 100 gram Schoyu. In 100 c.c. Gesammtstickstoff ......... 1.249 1.488 Eiweissstickstom” geese 0.037 0.044 Ammoniakstickstoff ......... 0.140 0.169 Ueber die chemische Zusammensetzung der japanischen Soja-Sauce. 479 In 100 gram Schéyu. In 100 c.c. Durch Phosphorwolfram- | siure fallbarer Stickstoff -0.330 0.361 (Ammoniak ausgenommen). Stickstoff in anderer Form 0.742 0.914 In 100 Theilen Trocken substanz, Gesammt stickstoff als 100, Gesammt stickstoff, 2-2-6 3.802 100.00 UBaweiss stickstotiie ee - 0.113 2.96 Ammoniak stickstoff......... 0.462 11.16 Durch = Phosphorwolfram- | saure fallbarer Stickstoff 10.965 26.41 (Ammoniak ausgenommen). | Stickstoff in anderer Form 2.262 59.49 Der gesammte Stickstoff wurde nach Kjeldahl und der Eiweiss stickstoff ‘nach Stutzer bestimmt. Fur die Bestimmung des Ammoniak stickstoffs wurde 100 c.c. Schoyu mit 200c.c. Wasser verdiinnt, mit Natronlauge ‘beinahe neutralizirt, mit etwa 1-2g. Magnesia usta geschiittelt bis die Flissigkeit schwach alkalisch reagirte, und bei niederem Druck (15-20 mm.) bei 40-50 c.c. destillirt. Das Destillat wurde in normal Schwefelsaure- Lésung eingeleitet, und in gew6hnlicher Weise titrirt. Fiir die Bestimmung -des Stickstoffs in organischen Basen wurde to c.c. Schoyu mit 40 c.c. Wasser -verdiinnt, durch basisches Bleiacetat gefallt. Zum Filtrat von dem Bleiacetat- Niederschlag wurde nach Entbleien durch Schwefelsiure noch so viel “Schwefelsaure zugegeben bis die Flissigkeit ungefahr 5% der Sdure enthielt ‘und so viel Phosphowolframsaure-Lésung zugegeben bis kein Niederschlag mehr entstand. Nach 24 Stunden wurde der Niederschlag abgesaugt, mit 5% iger Schwefelsiure gewaschen bis das Waschwasser keine Reaktion auf ‘Chlor zeigte und gleich darauf der Kjeldahi’schen Methode der Stickstoff- bestimmung unterworfen. Wahrend des Kochens des Nicderschlags mit conc. Schwefelsiure muss man unbedingt 1-2g. Kupferoxyd oder Kupfer- sulfat zugeben, damit das heftige Stossen vermieden wird. Aus dem analytischen Resultate sieht man, dass in Schoyu nur 3% des gesammten Stickstoffs als Eiweissstoffe vorhanden ist d. h. die Spaltung ist so ~weit vorgeschritten, dass die resultirenden Produkte nicht mehr als Nahrung- 480 U. Suzuki, K. Aso und H. Mitarai. stoff betrachtet werden kénnen. Dies fithrt zum Schluss, dass Schdyu nicht als ein Nahrungsmittel sondern als ein Genussmittel aufzufassen ist, das- keinen direkten Einfluss auf die Ernahrung des Menschen hat. In folgenden Seiten teilen wir die Isolirung der einzelnen stickstoff- haltigen Bestandteile und auch der organischen Sauren mit. I. Organische Basen. Zwei Liter Schoyu wurden zuerst durch Eindampfen im Vacuum vom gréssten Teile NaCl befreit, dann mit etwa 2 Liter 10% iger basischer Bleiacetat Lésung versetzt, wobei ein dicker Niederschlag entstand. Das klare Filtrat wurde durch Zusatz von Schwefelsdure vom Blei befreit, und dann mit so viel Schwefelséure angesiiuert bis die Fliissigkeit ungefahr 5% Schwefelsiure enthielt, und dann mit einer conc. Lésung von Phospho- wolframsiure gefallt. (Man braucht dazu etwa 2 kilo Phosphowolframsaure.) Der Niederschlag wurde abgesaugt und mit 5% iger Schwefelsdure ge- waschen. Da die Entfernung von Kochsalz durch Waschen sehr langweilige Arbeit war, wurde der Niederschlag noch mal mit 5% iger Schwefelsaure verrieben, wieder abgesaugt und mit 5% iger Schwefelsaure gewaschen. Als- das Waschwasser keine Reaktion auf Chlor mehr zeigte wurde der Nieder- schlag in Wasser verteilt und mit Uberschuss von Bariumhydroxyd verrieben. Das Gemisch wurde 6fters umgerithrt und bei einer Temperatur von 25-30°C. 24 Stunden stehen gelassen und abgesaugt. Der Riickstand. wurde nochmals in Wasser verteilt und mit Baryt verrieben. Diese Opera- tion wurde dreimal wiederholt. Die vereinigten Filtrate wurden durch. Kohlensaure vom Baryt befreit und in Vacuum bis auf 1 Liter eingedampft. Das in der Flissigkeit vorhandene Ammoniak wurde dabei vollstandig entfernt. Die alkalisch reagirende Flussigkeit wurde jetzt mit Kohlensaure sesittigt und mit einer gesittigten wasserigen Lésung von Quecksilber- chlorid versetzt, bis die Fliissigkeit schwach sauer reagirte und kein Nieder- schlag mehr entstand. A). Der Quecksilberchlorid-Niederschlag wurde in Wasser verteilt,. mit Schwefelwasserstoff zerlegt. Die vom Schwefel-Quecksilber befreite Fliissig¢keit wurde in Vacuum eingeengt. Es blieb dabei ein hellbrauner Syrup, der nicht krystallisirte. Der Versuch, dicsen Syrup als Methylester Ueber die chemische Zuasammensetzung der japanischen Soja-Sance. 481 salzsaures Salz zur Krystallisation zu bringen hatte auch keinen Erfolg. Wir haben spater gefunden, dass wir hier eine polypeptidartige Verbindung in der Hand hatten. Nach den Untersuchungen von E. Fischer, A. Levene, Siegfried, Pick. u. A. geht die tryptische Spaltung von Eiweissk6érpern nie so weit, wie mit conz. Mineralsauren. Es giebt eine gewisse Gruppe im Eiweissmolekul, die gegeniiber der enzymatischen Wirkung sehr wieder- stand fahig ist und nur durch die Saure-Wirkung weiter gespalten wird. E. Fischer schlug fiir solche Verbindungen den Namen “ Polypeptide ” vor, weil sie mit den von ihm dargestellten kiinstlichen Polypeptzden eine grosse Aehnlichkeit haben und durch starke Saure in die einfacheren Aminosduren gespalten werden. E. Fischer hat auch darauf aufmerksam gemacht, dass in den Polypeptiden oft ‘Prolin und Phenylalanin vorherrscht d.h. diese beiden Aminosduren scheinen gegen die enzymatische Wirkung am meisten Wiederstand zu leisten. Wir haben einen Theil des Syrups mit 109% iger Salzsdure 6 Stunden gekocht und in der That haben wir gefunden, dass etwa 409% des Stickstoffs in Form von Monaminosduren abgespalten wurde. d.h. sie wurden nicht mehr durch Phosphowolframsaure gefallt und von dem Filtrat vom Phospho- wolframsiure Niederschlag nach der Entferunng der Phosphowolframsdaure durch Baryt und des Baryts durch Schwefelsaure und Verestern in bekannter Weise haben wir Monaminosaure-Ester bekommen. Leider geniigte die Menge nicht, um die Ester zu fraktionieren. Nach dem Verseifen durch Baryt wurde Prolin mit Sicherheit nachgewiesen. (Durch Alkoholléslichkeit der freien Monaminosaure und des Kupfersalzes) und ausser dem eine kleine Menge Krystalle, die wahrscheinlich Phenylalauin waren. Der Phosphowolframsaure-Niederschlag wurde wieder durch Baryt zersetzt, die alkalische Lésung, die freie Basen enthielt, wurde mit Kohlen- sdure gesattigt mit Quecksilberchlorid-Lésung gefallt und in folgender Weise behandelt. a). Der Quecksilberchlorid-Niederschlag. Der Niederschlag wurde in Wasser verteilt, mit Schwefelwasserstoff -zersetzt. Die von Quecksilbersulfid abfiltrirte Fliissigkeit gab nach dem ‘Eindampfen in Vacuum farblose Krystalle, die wir anfangs ftir Histidin- 482 U. Suzuki, K. Aso und H. Mitarai. chlorid gehalten hatten. Die nahere Untersuchung zeigte uns jedoch, dass kein Histidin chlorid vorlag. Wir haben namlich versucht, diesen Korper zu verestern, indem die Krystalle in Methylalkohol gelést, und trockenes Salzsiuregas bis zu Sattigung eingeleitet und dann in Vacuum eingedampft wurde. Die dabei ausgeschiedenen Krystalle waren aber kein Ester, sondern waren ganz unverandert geblieben ; es war also sicher, dass der Kérper keine Carboxy] gruppe enthielt. Die aus heissem Methylalkohol durch Zusatz von Methylalkohol und’ Aether ausgeschiedenen farblosen Krystalle betrugen ca 1.32¢. Fir die Analyse wurden sie nochmals aus Methylalkohol umkrystallisirt und im Vacuum bei 100°C getrocknet. Ausserdem wurde die Halfte der Krystalle aus wenig Wasser umkrystallisirt und analysirt. Die Krystallform: Schmelzpunkt so wie auch Chlorgehalt waren genau dieselben wie bei dem aus Methylalkohol umkrystallisirten Praparat. 0.100g. Subst : (Aus Wasser umkrystallisirt) gab 19.3 c.c. N(19° 748 mm.) 0.1490g. ,, ( ” ) 0.1953g- CO, O.14g90g. ,, 33 9 ) 0.0730g. H,O SiiTES. 1% i ef ) 0.0422¢. Cl ( ( 0.0980¢. ,, (Aus Methylaikohol umkrystallisirt) 0.036504g. Cl C H N Cl C,H,N.2HCl »Berechnet, 30.77 5-62 21.45 30.16 { 36.70 Gefunden 35.75 5.49 2153 : (37.20 Das aus Methylalkohol umkrystallisirte Praparat bestand aus tarblosem Prismen, schmolz bei 232-233°C (uncorr.) ziemlich scharf unter Zersetzung. Es zeigte aber keine lebhafte Schaumung wie es bei Estern der Fall ist. Das salzsaure Salz hat kein Krystallwasser, lést sich leicht in Wasser,. reagirt ziemlich stark sauer. In warmem Methylalkohol lést es sich ziemlich. leicht, in Aethylalkohol schwer, in Aether Benzol, Chloroform und Petrol- ether ist es unléslich. Das salzsaure Salz wurde in wenig Wasser gelést, die Salzsdure durch. normal Natronlauge genau neutralisirt und die etwa drei fache Menge Pikrinsdure in Substanz zugegeben und so lange erwarmt bis sie klar geldést wurde. Nach dem Erkalten schieden sich gelblich rothe seidenglanzende- Ueber die chemische Zusammensetzung der japanischen Soja-Sauce. 483 Krystalle in reichlicher Menge aus, die zweimal aus heissem Wasser um- krystallisirt und gereinigt wurden. Fir die Analyse wurden sie im Vacuum bei 100°C getrocknet. Subst. 0.16242. a 0.1035g. - 0.407 38- ” 0.16242. o2igee CO. 0.0480g. H,O 10.61eeeN(L7-. 755 mim) 0.3227¢. Pikrinsdure. € H N Pikrinsaure. (orien). (Cor. N.O7)o9 wets | 37.17 2.58 21.69 78.83 2 Gee 36.46 3.31 21.83 eae 78.98 Das Pikrat bestand aus orangegelben Prismen oder Tafeln und hat kein Krystallwasser. In Capillarrohr rasch erhitzt wurde es bei 200°C allmahlich braun und bei 230°C (uncor.) zersetzte es sich unter lebhaftem Schaumen zu einer schwarzen Fliissigkeit. Es lost sich schwer in kaltem Wasser, leichter in heissem Wasser, in Methylalkohol auch leichter als in Aethylalkohol und in Aether, Petrolaether und Benzol ist es unléslich. Das saJzsaure Salz wurde in wenig Wasser geldést, eine wasserige Losung von Platinchlorid in geniigender Menge zugesetzt und langsam eingedunstet. Es schied sich bald eine orangegelbe Krystallmasse aus, die aus kurzen Prismen oder Tafeln bestand. Fiir die Analyse wurde sie einmal aus heissem Wasser umkrystallisirt, mit Alkohol und Aether gewaschen und in Vacuum bei 100°C getrocknet. 0.1880g. Subst. 0.1880¢. 5 0.0903g. CO, 0.0461g. H,O 0.1465¢. * 0.0545g. Pt ¢ H Pt Ceri )otiCl. PtCl, Ber E3250 2.07 30.50 Gef. 13.10 2.72 37.20 Das Platindoppelsalz wurde gegen 250° vollstandig schwarz, schmolz aber bis 290° nicht. In heissem Wasser ziemlich leicht, in kaltem Wasser schwer, und in Alkohol und Aether ist es fast unléslich. Aus den Analysen kann man schliessen, dass die Base der empirischen Formel C,H,N, entspricht. Was die Strukturformel derselben betrifft, so 484 U. Suzuki, K. Aso und H. Mitarai. wissen wir bis jetzt nur drei synthetisch dargestellte Kérper, denen die Formel C,H, N, zukommt, namlich dem Nitril der a.a’. Imido-dipropionsaure (C. 1904. (I) 353), dem Di-cyanmethyl-athylamin, oder Nitril der Aethyl imido-diessigsaure. (B. 37. 4092) und drei isomere Amido-di-methyl-diazine (Amido-di-methyl-Pyrimidin). Nur die letzt genannten Amido-di-methyl- diazine haben nach der Beschreibung grosse Ahnlichkeit mit unserer Base, naimlich : sie bilden krystallinische Salze mit Quecksilberchlorid, Salzsaure (C,H,O,. 2HCl) Platinchlorid (C,H,N,. H,PtCl,) und auch ein Pikrat (G,H,N,-. C,H.N,O,. od: Cele. --4.(C,H,N,Q;),). Bloss*die Schmaetz- punkte solcher Verbindungen stimmen mit unserem Praparat nicht. Da noch andere Strucktur-Isomerien méglich sind so ist unsere Base wahrschein- lich eine isomere Form von diesem Diazin-derivat. Besonders interessant ware es, weil es mit den Nucleinbasen Uracil, Thymin und Cytosin in naherer Beziehung steht. N—C-CH, f. 183° - Il Cre Ce. CH Chloro platinat : Gelbe Nadeln. C,H,N,H,PtCl,. | N=C-NH, Pikrate (Gelbe Nadelbiischel.f.214°. Co Nee CH, weoe Quecksilberchloridsalz : feine weisse Nadeln. C,H ,N. Bee (B. 0359577: | C.greereia) 1236) 2: N=C-H f. 214-215° { NH,-C C-CH, Salzsaures Salz: lange gefiederte Stabchen. tt elt N—C-CH, Platindoppelsalz: rétlich gelbe Stabchen und Tafelchen f. 227° unter Aufschaumen schmelzend. (B. 34. 2819) 2 N—C(CH,) f. 150-152°. W4sserige Lésung stark alkalisch. WET. CE Chlorplatinat, gelbe Prismen bei 225°C schmelzend. het) | N=C-CH, Salzsaures Salz: C,H,N,. 2HCl. Stemmforuns gruppierte Nadeln f. 181°C., in Wasser und Alkohol leicht léslich. Pikrat. C,H,N,.(C,H,N,0,), gelbe Blattchen. L230 & Ueber die chemische Zusammensetzung der japanischen Soja-Sauce. 485 Es sei noch bemerkt, dass die Formel des Histidin C,H,N,O, mit der unserer Base C,H,N, eine gewisse Ahnlichkeit hat. Da wir keine Spur von Histidin in Schoyu fanden, obgleich diese Base in den sauren Spaltungs- produkten der Eiweisskérper von Soyabohnen und auch von Weizen vor- kommt, so diirfte man auch annehmen dass die Base aus Histidin durch Bakterienwirkung entsteht.1 Wir beabsichtigen spater, diese Base etwas naher zu untersuchen und zu entscheiden,'ob diese Base durch Saurewirkung aus Soya-oder Weizen-Eiweisskérpen gebildet wird, oder ob ein sekundares Umwandlungsprodukt vorliegt. B). Das Filtrat vom Quecksilberchlorid-Niederschlag (a) wurde durch Schwefelwasserstoff vom Quecksilber befreit und im Vacuum eingedampft. Nach dem das Wasser vollstandig ausgetrieben war, wurde es mit trockenem Methylalkohol versetzt, trockenes Salzsauregas bis zur Sattigung eingeleitet, im Vacuum eingeengt, und mit absolutem Alkohol und Aether versetzt. Die dabei ausgeschiedenen farblosen Krystalle betrugen ca 2.2g. Das einmal aus heissem Methylalkohol umkrystallisirte Praparat schmolz gegen 195°C unter lebhaftem Schaumen und enthielt 31.809 Cl, wahrend Lysin-Methyl- ester-salzsaures Salz 30.47% Cl enthalt. Die entsprechenden Salze von Arginin und Histidin enthalten noch weniger Chlor (27.2% bezw. 29.29% Cl), desshalb war anzunehmen, dass noch kein einheitlicher Kérper vorlag und wurde das Estersalz verseift und in das Chlorid und weiter in das Pikrat ver- wandelt. Inder Tat haben wir zwei verschiedene Pikrate bekommen, die durch ihr Léslichkeitsverhalten leicht von einander sich trennen liessen. Die Hauptmenge bestand aus Lysinpikrat. Es waren lange gelbe Prismen etwa 1.9g. mit genau denselben Schmelzpunkt und Krystallform wie das reine Lysin-Pikrat. Das andere bestand aus hellgelben Prismen, die viel schwerer léslich in Wasser waren, als Lysin-Pikrat. Nach zweimaligem Umkrystallisiren aus heissem Wasser betrug es o.8g. Fir die Analyse wurde es in Vacuum bei 80° getrocknet : 1 Noch zu bemerken ist, dass unsere Base eine starke dunkel rote Firbung mit Diazobenzolsulfo- sdure in alkalischer Losung giebt, was von Pauly als eigentiimliche Reaktion ftir Histidin und Tyrosin angegeben wurde, Wir sind aber sicher, dass unser Praparat nicht ein Gemisch von Histidin war, 486 U. Suzuki, K. Aso und 9. Mitarai. 0.1312g. Subst. 0.1726g. CO, + 0.0403¢. H,O O.EIQg. 54 20.2 c.c, N(13°, 766 mm.) 0.42378. ‘ 0.3444¢. Pikrinsdaure. G H N _ Pikrinsaure. (C,H,3N.) (C,HsN,Os)}oabee: 25007 3.30 20.52 83.88 (C.H,,N.,):(C,H,N 20a 30.43 3:57 20.00 81.80 Gef. 35 Das Pikrat bestand aus hellgelben Prismen, schwer léslich in kaltem 88 3.41 20.22 S25 Wasser. Im Capillarrohr erhitzt, fangt es von 200°C an allmiahlich braun zu werden, bei 230° dunkel braun und bei 260° (uncorr) zersetzt es sich pl6tz- lich unter starkem Schaumen zu einer schwarzen Flissigkeit. Aus dem Pikrat wurde das Chlorid dargestellt. Es bildete farblose Prismen, spielend leicht léslich in Wasser, schwer in Alkchol und unléslich in Aether. Im Capillarrohr erhitzt zersetzte es bis 290°C nicht. Erst bei hdherer Temperatur zersetzt es sich unter Schdumen. Da die Menge des Chlorids zu wenig war, haben wir es nicht analysirt und unmittelbar in das: Platindoppelsalz verwandelt, indem das Chlorid in wenig Wasser gelést und mit einer wasserigen Lésung von Platin-Chlorid versetzt wurde. Es schieden sich dabei hellgelbe Krystalle eines Platindoppelsalz es, aus die selbst in heissem Wasser ziemlich schwer léslich waren. Sie wurden abgesaugt, mit wenig kaltem Wasser, dann mit absolutem Alkohol und Aether gewaschen und in Vacuum bei 80°C getrocknet und analysirt : 0.1939g. Subst. 0.0701g. CO, 0.19398. $5 0.0552g. H,O 0.135I¢g. - 0.05 30g. Pt. C H PE. C,H. Ne 2HCLetice Ber. 9.68 2.82 39.00 CHa gNy 2HiGl Bea Ber. 11.76 3.14 38.00 Gef. 9.86 3-19 40.20 Das Platindoppelsalz bestand aus kleinen hellgelben Tafeln oder Prismen, die sich manchmal unregelmassig zusammen gruppiren. Im Capillarrohr erhitzt wurde es gegen 220°-230° schwarz, zersetzte sich aber bis 290°C nicht. Ueber die chemische Zusammensetzung der japanischen Soja-Sauce. 487 Die analytischen Zahlen stimmen also am besten mit der Formel C,H,,N,. Héchst wahrscheinlich handelt es sich hier um Tetramethylen- diamin oder Putrescin, Da es kein Arginin in Schoyu vorhanden war, so kann man erwarten, dass das Arginin durch Enzyme oder Bakterien in Ornithin und Harnstoff und das Ornithin weiter in Tetramethylendiamin gespalten war. Dass es tatsachlich aus Arginin durch Bakterien-Wirkung Putrescin entsteht, hat Ellinger nachgewiesen. B). Das Fiitrat vom Quecksilberchlorid-Niederschlag A. wurde durch Schwefelwasserstoff vom Quecksilber befreit, im Vacuum eingeengt, um Schwefelwasserstoff auszutreiben. Als die Fliissigkeit bis auf 4 Liter einge- engt war, wurde die vorhandene Salzsaure durch Silbernitrat gefallt. Zum Filtrat von Chlorsilber wurde ein Uberschuss von Silbernitrat und Baryt- hydrat zugegeben. Der dabei entstandene braune WNiederschlag wurde abgesaugt, mit Wasser gewaschen, und dann in Wasser verteilt, durch Schwefelwasserstoff zerlegt, abfiltrirt und in Vacuum eingeengt. Von der conzentrirten Flissigkeit wurde ein Pikrat dargestellt, das sich aus heissem Wasser erst 6lig ausschied, und nach 24 Stunden zu nadelférmigen Krystallen umwandelte. Die Analyse des gereinigten Praparats gab folgendes Resultat. C 15.21% H 3.22% N 18.50% Hochst wahrscheinlich handelt es sich hier um ein anorganisches Pikrat. Arginin war also nicht vorhanden. C). - Das Filtrat von dem Niederschlag B. wurde durch Salzsaure .vom Silber und durch Schwefelsdure vom Baryt befreit, und so viel Schwefelsaure zugegeben, bis die Fliissigkeit ca 5% derselben enthielt ; hierauf wurde es mit Phosphowolframsaure-Lésung gefallt. Der Niederschlag wurde ab- gesaugt, mit 5% iger Schwefelsiure gewaschen und durch Barythydrat zerlegt. Das Filtrat, nach dem es vom Baryt befreit war, wurde im Vaccum stark eingeengt, und von der stark alkalisch reagirenden conzentrirten Lésung wurde unmittelbar in bekannter Weise das Pikrat dargestellt. Es -hatte die charakteristische Krystallform von Lysin-Pikrat und betrug ca 5.5g. Das zweimal aus heissem Wasser umkrystallisirte Praparat fing bei 220°C an braunschwarz zu werden und gegen 245° zersetzte es sich unter Schaumen. Fiir die Analyse wurde es im Vacuum bei 80°C getrocknet : 488 U. Suzuki, K. Aso und Mitarai. 0.767g. Subst. gab 0.4678g. Pikrinsaure. O.154908- 24.5 c.c. N (4°C, 762.5 mm.) N Pikrinsaure. (C,H,,N,0,) (C,H,N,0,). Ber. 18.67 61.07 Geir 19.00 60.99 Aus dem Pitrat wurde das Chlorid und das Methylester-Salzsduresalz dargestellt. Das Chlorid hatte dieselbe Krystallform und den Schmelz- punkt wie das active Lysinchlorid, und das Methylester-Salzsauresalz krystallisirte in farblosen Prismen. Es schmolz gegen 195-196°C (uncorr.) unter Schaumen. Fiir die Analyse wurde es zweimal aus heissem Methyl- alkohol durch Zusatz von Aether ausgeschieden und im Vacuum bei 80°C getrocknet : 0.120g. Subst. gab 0.03615¢. Cl= 30.139 Cl, auf C,H,,N,O,. 2HC] berechnet 30.47% Cl Da wir aus der Fraktion A. etwa 1.9g. Lysin Pikrat gewonnen haben, so betrug die gesammte Ausbeute an Lysin-Pikrat 7.4¢. TT. Monaminosdéuren. 2 Liter Schoyu wurden im Vacuum eingeengt, von dem ausgeschiedenen Kochsalz befreit, und dann mit Wasser auf 4 Liter verdiinnt, und ca 2 Liter einer 10% igen basischen Bleiacetatlésung zugesetzt. Das Filtrat vom Blei- Niederschlag wurde durch Schwefelwasserstoff von Blei befreit und im Vacuum stark eingeengt. Um das vorhandene Wasser vollstandig aus- zutreiben, wurde der Syrup zweimal mit etwas absolutem Alkohol versetzt und in Vacuum eingedampft. Der Riickstand wurde jetzt mit 2 Liter absolutem Alkohol versetzt und trockenes Salzsduregas bis zur Sattigung eingeleitet. Das dabei ausgeschiedene Kochsalz wurde abgesaugt und mit absolutem Alkohol gewaschen. Um die Veresterung méglichst zu vervoll- standigen wurde das Filtrat vom Kochsalz wieder im Vacuum eingedampft. Der zuriickgebliebene dicke Syrup wurde nochmals mit 2 Liter absolutem Alkohol versetzt und trockenes Salzsauregas bis zur Sattigung eingeleitet, im Vacuum bis zum Syrup eingedampft und zwei Tage in einem kalten Zimmer stehen gelassen. Da hierbei salzsaurer Glycocollester sich nicht aus- geschieden hatte, so wurden nach der bekannten Ester-Methode von E. Ueher die chemische Zusammensetzung der japanischen Soja-Sauce. 489. Fischer die freien Aminosdurenester dargestellt und bei niederem Druck fraktionirt. Nach der Verseifung der einzelnen Ester-Fraktionen haben wir folgende Aminosauren isoliert : 4 Versuch 1. Versuch 2. Alanin 1.5 1.4 Alanin+Leucin 6.0 2 Leucin 4.0 6.0 Prolin 3.0 3-0 Asparaginsaure Vorhanden. Vorhanden. Phenylalanin Vorhanden ? ? Nach Glycocoll wurde mit besonderer Vorsicht gesucht, indem die erste Fraktion mit Salzsiuregas gesattigt und in der Kalte stehen gelassen wurde. Auch die Alanin und Leucinfraktion wurde ebenso behandelt um hier beigemengtes Glycocoll aufzufinden, aber in keinem Fall haben wir die Krystalle von salzsauren Glycocollester gefunden : Analyse des Alanin Praparats. o.1891g. Subst. 0.2956g. CO, +0.1334g. H,O 0.1782¢. 35 22.2 Gewen(i9’, 765 mm.) C H N C_lJNO; Ber. 40.45 7.87 13.93 Gef. 42.63 7.QI 15.10 [albp?? = 2 Leucin. 0.1780g. Subst. 0.3596g. CO, +0.1618g. H,O 0.1475¢. Fe 13.5 c.c. N(o°, 762 mm.) ' C H N Gin, .NO, Ber. 54.90 9-93 10.68 Gef. 55-04 10.10 11.02 [a]p°?°° = +14.84° 1 Die Operation wurde genau nach der von E, Fischer angegebenen Methode ausgefiiart. Man vergleiche das Buch von E, Fischer “ Untersuchungen itiber Aminosduren, Polypeptide und Proteine.” 490 U. Suzuki, K. Aso und H. Mitarai. Prolinkupfer (actives). 0.1280g. Subst. verlor bei 100°C in Vacuum getrocknet 0.0145g.=11.31% H,O O.1105g. ,, (wasserfrei) gab 0.029g. CuO od. 0.023159g. Cu=20.96% Cu. HzZ0 Ca Ci .H 1, Ne Op Ciegeeie@ Ber. 10.99 19.41 Gef. 11.32 18.59 Da wir noch keine gute Vacuumpumpe in unserem Institut zur Ver- fiigung hatten, konnten wir mit der Wasserstrahlpumpe héchstens 15-17 mm. Druck erreichen, was fiir die héher siedenden Ester noch zu hoher Druck war. Desshalb war das Vorhandensein von Glutaminsaure nicht leicht nachzuweisen, obgleich diese Saure in Schdyu sicher vorhanden sein wird. Die Ausbeuten an Asparaginsaéure und Phenylalanin waren so gering, dass wir die beiden Aminosduren nicht analysiren konnten. Die Abwesenheit von Serin und Aminovaleriansaure ist ziemlich sicher. Nach Oxyprolin und Tryptophan wurde nicht gesucht. Wir hoffen jedoch spater nochmals mit besserem Vacuumapparate diese Versuche zu wiederholen. Um das Vorhandensein von Tyrosin und Cystin nachzuweisen, wurden 200 c.c. Schoyu mit einer Lésung von basischem Bleiacetat versetzt. Die vom entstandenen Niederschlag abfiltrirte Flissigkeit wurde nach Entbleiung durch Schwefelwasserstoffgas im Vacuum eingeengt. Um das Chlor zu entfernen, wurde die Fliissigkeit mit einen Uberschuss von Silbernitrat gefallt. Das Filtrat von Chlorsilber wurde durch Schwefelwasserstoff vom Silber befreit, und in Vacuum eingeengt. Die so gewonnene Fliissigkeit gab sch6ne rote Farbung mit Millons Reagens. Da keine Eiweisskérper vorhanden waren, so ist diese Reaction auf Tyrosin zu bezichen. Vor Kurzem hat Pauly eine neue Reaktion auf Tyrosin empfohlen (Zeitschrift. f. physiol. Chem. 1904 s. 513) Dies beruht auf der Entstehung einer dunkelroten Farbung auf Zusatz von Diazobenzolsulfosaure zu einer soda-alkalischen Lésung von Tyrosin (Histidin giebt dieselbe Farbenreaktion). In unserem Fall haben wir hiemit ebenfalls eine schéne Farbenreaktion bekommen. Das Vorhandensein von Cystin wurde dadurch nachgewiesen, dass man die conc. Flissigkeit mit conc. Kalilauge kurze Zeit kochte und basisches Bleiacetat Ueber die chemische Zasammensetzung der japanischen Soja-Sauce. 491 zugab. Der schwarze Niederschlag von Schwefelblei wird nur in Anwesen- heit von Cystin hervorgerufen. ITl!, Organische Séuren. 100 c.c. Schoyu wurden mit roo c.c. Wasser verdiinnt, mit Schwelsaure angesduert und im Vacuum destillirt. Das Destillat wurde in normal Natron- losung eingeleitet. Die fliichtige Saure im Destillat neutralisirte 0.24304¢. NaOH. Als Essigsaure berechnet ergiebt sich 0.36456¢.=0.365 %. Fiir die Bestimmung der gesammten Aciditat wurden 1oo c.c. Schodyu mit Tierkohle entfarbt, mit Wasser verdiinnt und unmittelbar durch normal Natronlésung titrirt. Als Indicator verwendeten wir Phenolphthalein. Es wurde 1.008g. NaOH zum Neutralisiren gebraucht. Wenn man nun 0.365¢. (d.h. die Menge der Natronlauge die zum Neutralisiren der fliichtigen Saure notig ist) davon abzieht, so bleibt 0.765¢. NaOH fir die nicht fliichtig en ‘Sauren, welche als Milchsaéure berechnet 1.721g.=1.721% ergeben wiirden. Man darf aber nicht vergessen, dass diese Zahl auf keine Weise der totalen Menge der nicht fliichtigen Sauren entspricht, weil, wie wir schonSangegeben haben, in Schoyu nicht nur freie organische Saure, sondern an Ammoniak, organische Basen und andere Kérper gebundene vorkommt, was auf die Reaktion der Fliissigkeit grossen Einfluss ausiibt. Um die flichtigen Saiuren zu isoliren wurde 1 Liter Schoyu mit 13 c.c. conc. Schwefelsiure versetzt, und in Vacuum bei 50-60°C. destillirt. Das Destillat wurde in normal Natronlauge eingeleitet. Nachdem_ keine flichtige Saure mehr im Destillat nachweisbar war, wurde die Natron- lésung eingedampft, der Riickstand in wenig heissem Wasser gelést, mit Schwefelsiure angesduert und mit Aether wiederholt geschiittelt. Die ztherische Loésung hinterliess nach dem Verdampfen des Aethers einen nach Essigsaure riechenden Riickstand, der in wenig Wasser gelést und mit Uber- sschuss von Bleicarbonat am Riickflusskiihler mehrere Stunden gekocht wurde. Es wurde dann abfiltrirt, stark eingeengt und mit viel absolutem Alkohol versetzt. Es entstand dabei ein weisser Niederschlag, der mit absolutem Alkohol und Aether gewaschen und bei 100°C getrocknet wurde. Die Aus- beute betrug etwa 0.15g. Die Bleibestimmung gab folgendes Resultat : 492 U. Suzuki, K. Aso und H. Mitarai. 0.135g- Subst. gab 0.137g. PbSO, =0.09357g. Pb Pb C.H,O,Ps Ber. 69.70% Gef. 69.31% Die Analyse stimmt also ziemlich gut mit ameisensaurem Blei. Ferner war die Krystallform mit dem reinen ameisensauren Salz identisch. Der eigentiimliche Geruch der freien Séure und ihr Reduktions-Vermégen gaben uns keinen Grund zu zweifeln, dass es Ameisensdaure war. Das alkoholische Filtrat vom Bleiformiat hinterliess nach dem Einengen einen Riickstand, der etwa 0.77g. betrug. Dieser wurde aus wenig heissem Wasser umkrystallisirt. Die tiber Schwefelsdure getrocknete aus vierseitigen Prismen bestehende Krystallmasse wurde analysirt : 0.4495g-. Subst. gab 0.3519g. PbSO, (C,H,O,), Pb+3H,O Ber. = 54.50% Gef. 53.496 Der mit Schwefelsdure angesauerte und durch Abdestilliren von den fliichtigen Saéuren befreite Riickstand wurde wiederholt mit Aether ge- schiittelt. Die ztherische Lésung hinterliess nach dem Verdampfen des Aethers einen gelbbraunen Syrup, aus dem nach mehreren Tagen Stehen eine kleine Menge Krystalle sich ausschied, die wahrscheinlich aus Bernstein- siure bestanden. Leider reichte die Menge nicht aus fiir eine genauere Unter- suchung. Der Syrup wurde jetzt in Wasser gelést, mit Uberschuss von Zinc carbonat mehrere Stunden gekocht, heiss abfiltrirt. Das Filtrat wurde stark eingeengt und erkalten lassen. Es schieden sich allmahlich die Krystalle von Zinclactat in reichlicher Menge aus, die von der Mutterlauge getrennt und auf einer Tonplatte getrocknet wurden. Die Ausbeute an Rohprodukt betrug etwa 1.151g. Die Mutterlauge gab noch eine zweite und dritte Fraction, so dass die gesammte Ausbeute 2.517g.=1.61g. freie Milchsaure betrug. Das Rohprodukt wurde aus wenig heissem Wasser umkrystallisirt, iiber Schwefelsaure getrocknet und analysirt : ; 0.2620g. Subst. gab bei 110°C 0.227g.=0.035g. H,O 0.227g. | wasserfreie Subst. gab 0.077g. ZnO 0.092g. wasserhaltige Subst. gab 0.0275g. ZnO Ueber die chemische Zusammensetzung der japanischen Soja-Sauce. 493 Krystallwasser Zn (€,H1,0,),Z0+ 2H, OF ier. 12.9 23-41 ce , 23-6 Gef. Sa eae oe 2. 22557 Wie bekannt enthalt das Zinksalz der activen Milchsiure zwei Molekule Krystallwasser, wahrend das der inactiven Sdure drei Molekule davon enthalt, es war daher unser Praparat die active Saure. Die optische Drehung stimmte auch auf die active Saure ; es wurde gefunden : la)? eee Diese Zahl liegt ziemlich nah mit der Beobachtung von Y. Kozai, der mit den aus verschiedener Bakterien isolierten Sauren zwischen -5° bis —7° fand. Es sei hier bemerkt, dass das links drehende Zinksalz eine freie Siure gibt, die nach rechts dreht. Zusammenfassung der Resultate. Aus 2 Liter Schdyu wurden isolirt :— Alanin 1.6g.+5.0g. unreines Alanin. Leucin 6.0 Prolin 3-0 Lysin 2.6 Neue Base C,H,N 1.0 gttgits Base C,H,,N; o@ Ammoniak 4.2 Eiweissstoffe 5.4 (Nach der Berechnung). Ameisensaure _ 0.10 Essigsaure 0.40 Milchsaure 3.20 Vorhanden waren :— Tyrosin Asparaginsaure Polypeptidartige Stoffe Phenylalanin ? Cystin 494 U. Suzuki, K. Aso und H. Mitarai. Nicht vorhanden waren :— Glycocoll Histidin Arginin Serin Aminoisovaleriansaure ? Glutaminsaure ? ‘Ueber die Verbreitung von “ Anhydro-Oxy-Methylen-diphosphor- sauren Salzen” oder “ Phytin” in Pflanzen, VON U. Suzuki und K. Yoshimura. Die von Palladii, Schulze, Winterstein, Posternak, Patten, und Hart u. a. untersuchten so-genannten “anhydro-oxy-methylen-diphosphorsauren “Salze (von Ca und Mg) oder “ Phytin,” scheint im Pflanzenreich tberall verbreitet zu sein. Besonders im Samen scheint diese Substanz als Reserve- stoff wahrend der Keimung eine wichtige Rolle zu spielen, deshalb haben -wir uns mit der Frage nach der Verbreitung und der physiologischen Func- ‘tion derselben beschaftigt. Wir haben zuerst unsere Aufmerksamkeit auf die Reiskleie gerichtet, die sehr reich an Phosphor ist. In der Tat haben wir tiber 85% des gesammten Phosphors in der Kleie im Form des Phytins vorgefunden. Da die Darstellung desselben héchst einfach und das Praparat schr leicht zu reinigen ist, bietet die Reiskleie ein ausgezeichnetes Material dar fiir die Gewinnung des Phytins in gr6ésseren Mengen. Auch aus anderen Pflanzensamen und aus anderen Organen haben wir -das Phytin isoliert. Im Folgenden teilen wir das Resultat mit. 1 W, Palladin ; Beitriige zur Kenntniss pflanzlicher Eiweiss Stoffe Zeitschr. f. Biclogie 1894, p. 199. 2 Schulze und Winterstein; Uber einen phosphorhaltigen Bestandteil der Pflanzensamen, Zeitsch. f, Physiol Chem. 22.90. 3 Posternak ; Revue generale de Botanique 12. 5 & 65 (19CO). No. 3 (20. Iullet 1903). 3 Comptes rendus 137 eyes Aout ,, ). 4 O(a ra 4 A,I, Patten and E. B, Hart. The Nature of the principal phosphorous compound in wheat «bran. New York Agr.-Exp, Station, I. 1902-1904 Bull. No. 250. 496 U. Suzuki und K. Yoshimura. I. Reitskleze. Die sorgfaltig gereinigte Reiskleie, die zu unserem Versuche diente, hatte: folgende Zusammensetzung : Gesammt Phosphor als I ockensubstanz. : n Troc 100 berechnet. Gesammithos Pphoreees.ce-.es aeceeee eee 227) 100.00 Ihosphor in Wecithine 2c... .ss-ceeeseeeeee 0.02 0,80 Phosphor, léslich in 0.2% HCl ............ 1.92 $4.48 { anorganischer Phosphor ......... 0.13 5.89 Davon l organischer Phosphor ............ 1.68 74.17 Fir die Bestimmung des anorganischen Phosphors im 0.2% igen Salz-- sdure-Extracte wurde der Extract mit Ammoniak neutralisirt, mit Salpeter- sdure etwas angesauert, und durch Molybdanlésung gefallt. Wir haben verschiedene Methoden versucht, und fast immer tibereinstimmende Resultate- bekommen. Was hier als organischer Phosphor bezeichnet ist, wurde in der Weise- bestimmt, indem wir den (0.2% tigen) Salzsiure-Extract unmittelbar mit Bariumchlorid versetzten und von dem Phosphor in diesem Niederschlag den organischen Phosphor subtrahierten. Die Differenz wird annahernd den: Phosphor des Phytins reprasentieren. Fiir die Darstellung des Phytins wurden toog Reiskleie mit Aether extrahirt und dann zweimal mit 95% tigem Alkohol gekocht. Der Riick- stand wurde in 40 c.c. von 0.29 iger Salzsaure suspendirt, ofters geschiittelt,.. und bei gewdhnlicher Temperatur stehen gelassen. Nach sechs Stunden wurde abfiltrirt, und das Filtrat mit absolutem Alkohol vesetzt, wobei ein weisser flockiger Niederschlag in reichlicher Menge entstand, der sich allmahlich am Boden absetzte. Nach 24 Stunden wurde der Niederschlag~ sesammelt, mit 50% tigem Alkohol, dann mit absolutem Alkohol und zuletzt mit Aether gewaschen, und tiber Schwefelsdure getrocknet. Das so gewonnene Produkt war schon ziemlich rein, und die Ausbeute betrug etwa 7-8g, je nach Umstanden. Diese Menge entspricht iiber 809§ des gesammten Phosphors. Ueber die Verbreitung yon *‘ Anhydro-Oxy-Methylen-diphosphorsauren Salzen,”’ 4Q7 Fiir die Analyse wurde das Rohprodukt noch zweimal in 0.29 iver ‘Salzsaure gelést, durch absolutem Alkohol gefallt, und bei 100° c. getrocknet. Analyse des Phytin-Praiparats aus Reiskleie, Werlust: beim Glihen Sites int---.+-.22c2s00- 272 Ee EABOSDUGE © 52a. Sat ea ee cin s ss wos « sav aces ve 2RAG WEAETESSIUNY |. oe ee ons wees sen ensseo ec 17-45 5s PME BIC LUNN) 2325 J. > peed ao «sc wo dn dee ech EO. 3. oa cic I sn s 00a sevesanee spur I ECUTLT >.. . 25 cee ns sss onan t oa — NOt 9.2... Ae ace I 8 as os caanewaras — SG 4 6)| Eee eer — Dieses Praparat ist ein weisses Pulver, nicht hygroscopisch, lést sich in ‘kaltem Wasser, die wasserige Lésung reagirt schwach sauer, beim Kochen -entsteht ein weisser flockiger Niederschlag, der beim Erkalten wieder ver- schwindet. Es lést sich sehr leicht in verdiinnter Salzsaure, Schwefelsdure und Salpetersaure, nicht aber in Essigsdure. Durch Zusatz von verdiinnter ‘Kali oder Natronlauge wird es gallertartig, lost sich aber nicht. In Methyl- und Aethylalkohol ist es unléslich. Mit Molybdan-Lésung erwarmt giebt es eine weisse Fallung. Die wdsserige Lésung wird durch ‘neutrales Bleiacetat, Kupferacetat und Bariumchlorid gefallt. Silbernitrat -giebt ehenfalls einen weissen Niederschlag, der durch Zusatz von Salptersdure ‘verschwindet. 1.5g. Phytin wurden mit 15 c.c. einer 309 igen Schwefelsaure auf 130° C. 14 Stunden erhitzt. Nach dem Erkalten wurde mit Wasser verdiinnt, die Schwefelsaure durch Barytwasser quantitativ entfernt, und das Filtrat stark eingeengt, und mit viel absolutem Alkohol versetzt. Der dabei entstandene Niederschlag wurde schnell abfiltrirt, und das Filtrat stark eingeengt, mit einem Uberschuss von absolutem Alkohol und Aether gefallt. Der weisse ‘Niederschlag verwandelte sich allm&hlich in farblose Krystalle. Nach der Reinigung bestand es aus glanzenden rhombischen Tafeln, die bei 220° -schmolzen, Scherer’s Reaction gaben, und vollstandig das Verhalten des Inosits zeigten. U. Suzuki und K. Yoshimura. as \O fe) 2. Wetsen-Kleie. Die Weizen-Kleie hatte folgende Zusammensetzung : In 100 Teilen des rockensubstanz, : eeeckensubst Gesammt Phosphors. Gesammet Phosphor. s.3.c00.05-s esse eee 1.114 100.00 Phosphor im Lecithinty:.<4....:..ée: 20 eee 0,010 0.81 Phosphor, léslich in 0,29 HCl ............ 0,638 57.24 anorganischer Phosphor ......... 0,050 . 4.49 Davon { ‘organischer Phosphor ............. 0.579 52.00 Die Darstellung des Phytins war genan dieselbe wie aus Reiskleie, die- Ausbeute war etwa 29% der luftrockenen Weizen-Kleie. Das Produkt ent- hielt 16.819 Phosphor. Patten und Hart haben aus 1 Kilo Weizen-Kleie 7g Phytin isoliert, das 16.38% Phosphor enthielt, somit war bei uns die- Ausbeute etwa dreimal so hoch wie bei jenen Autoren. 3. Samen von. Sesamum tndicum. In 100 Teilen des In Trockensubstanz. Gesammt Phosphors. Gesammit (Phosphorses-pe sce. eee eee 0.772 100,00 Bhosphonineleecit hin ees eee eee eee 0.030 3.91 Phosphor, léslich in 0.29 HCl............ 0.144 18.61 anorganischer Phosphor ......... spur spur Davon organischer Phosphor ............. 0.125 16.24 4. Samen von Rieinus commuitis. In roo Teilen des In Trockensubstanz. Gesammt Phosphors. Gesammit Phosphor..i.0 2). 4ee eee 0.261 100,00 Phosphor im, Lecithin, -; sie.ecsceesa-aenees 0.013 5-13 Phosphor, léslich in 0.29% HCl ............ 0.110 42.29 anorganischer Phosphor ... ...... spur siplee Davon organiseher Phosphor ............ 0.1Ccg 41.61 Ueber die Verbreitung von “ Anhydro-Oxy-Methylen-diphosphorsauren Salzen.” 499 5. Ocl Kuchen von Brassica Napus. In 100 Teilen des In Trockensubstanz. Gesammt Phosphors, Cesium PHosphor.. ...<3...c-..2.7-.s eee 11.50. 7.50 (=) 4-00 Gesammt-Phosphor ..............-.0006 0.0341 0.0326 =) 0.0015 Lecithin-Phosphor. .. :.24ssce-censseeees 0.0005 0.0014 (+) 0.0009 In 0.2% HCI léslicher Phosphor ... O.0I5! 0.0259 | (+) 0.0108 | Anorg.-Phosphor ......... -- Spur. 0,0086 (+) 0,0086 Davon. 4 Org.-Phosphor, —<.. 2-2. --e--e2 0.0142 0.0145 (+) 0.0003 Ueber ein Enzym ,, Phytase.* 507 Gesammt-Phospher der Samen als roo berechnet. Samen. Keimlinge. (+) (—) Gecammicbhospuor ..!22....cesseceeese- 100.00 95.60 (—) 4.40 cert aE SPOR <2... se vs asccesacoerses 1.47 4.10 (+) 2.63 In 0.2% HCl ldslich Phosphor ...... 44.28 76.00 (+) 2172 ( Anorg.-Phosphor ............ Spor. 25.22 (+) 25.22 Davon, f @re-Phosphor=.... .ésan02ss0 41.€4 42.52 (+) 0.88 Versuch 2. Am gten October warden 200 von Brassica Napus in feuchten Quarzsand gesaét und keimen glassen. Am _ 18ten als die Keimlinge etwa 3 cm. erreichten, wurden sie getrocknet und analysirt : 200 Samen. 200 Keimlinge. (+) (—) SREY SUDSANZ, ... 0. caccesesso0-005 5.90 4.20 (=) 4 oe Gesamint=EBOspuOL §s-52.2.2.5<.c25a0---22<- 0.0029 0.0041 (+) 0.0012 In 0.2% HCl loslicher Phosphor ... 0.0269 0.0326 (+) 0.0057 § Anorg.-Phosphor ..,......... Spur. 0.0292 (+) 0.0292 Davon, Org-Phosphior <.)..:-2<<é-<.: 0.0259 0.0012 (-—) 0.0248 Gesammt-Phosphor der Samen als roo derechnet. Samen. Kleimlinge. (+) (-) Gesammt-Phosphor ...... Sassen sees 100,00 96.64 —) 3.36 Lecithin-Phosphor ............220.0s0000: 6.95 9.83 (+) 2.88 In 0.2% HCl léslicher Phosphor ... 64.51 78.18 (+) 13.67 Anorg.-Phosphor ............ Spur. 70 02 {+) 70.02 Davon, @re-Ehosphor, 22.52. +<5-2--<: 62.11 2.88 (—) 59.23 Versuch 3. Am sten October wurde Gerstensamen in feuchten 508 U. Suzuki, K. Yoshimura und M. Takaishi. Quarzsand gesat, und am 18ten, als die Keimlinge etwa g cm. erreicht hatten, wurden sie getrocknet und analysirt : 300 Samen. 300 Keimlinge. (+) (3 Trocken-Substanz <2... .2.- has shown that at least a part of nitrogen of the humic acid is present in the form of amino-acid, as free nitrogen is devolped when it is treated with nitrous acid. Dojarenko® made quantitative determinations of the different forms of nitrogen contained in the seven samples which were prepared from Russian black earths, by extracting them with ammonia or sodium carbonate, with the following result : Fotal nitrogen..........7ee =. 2-64=4.538.% Ammonia-nitrogen, ..77a.-.-.--- trace. Amino-acid-nitrogen OI-2 detec a 1 fo 1 er ee eseese ecco e 41 2.34 ” Bohmer’s method determined by simimo-nitrogen .../...-.0aee--s...- 0.22-0.48 ,, (gue peer 1 Meddelanden fran konigl, Landbruks-Akademiens-Experimentalfilt, Nr, 3. Stockholm, 1888. p. 1-66.—Biedermann’s Centralbl. f. Agrik, Chem. 1889. p. 75. 2 Agric. Science 8, 165.—Centr. Bl. Agrik. 1895. 24. 218. 3 D. landw. Presse, 1895. 490; ref. Centr.-Bl, Agrik, 1896. 25. 271. 4 Exper. Stat. Rec. 1898. 9. 632 (Minnesota Stat. Bul. 53. 12). 5 Landw. Versuchsst, 1899. 51. p. 153. 6 Landw, Versuchsst, 1902. 56. p. 311. 516 Shigehiro Suzuki. All experiments seem to show that a portion of nitrogen contained in the soil or especially in the humic acid is of the nature of an amide or amino- acid. But it is not yet clearly examined what kinds of amino-compounds or amino-acids are contained in the humic acid, or are formed as decomposition products when the humus is boiled with hydrochloric acid. In order to study these questions more clearly, the writer has examined three samples of humus, one was imported from Germany (A), Acid. humic., pur., von E, Merck-Darmstadt, while the second (B) was prepared from a soil not manured for seven years. The third (C) was derived from a kind of compost heap. In the two latter cases the humus was extracted by caustic soda! (2%) (the soil being at first repeatedly washed ona funnel with 19¢ hydrochloric acid to remove lime compounds) and precipitated by a weak solution of hydrochloric acid and after. well washing dried.2 Unfortunately, the origin and the method of preparation of the sample A was quite unknown to the writer, but according to its chemical behavior it is highly probable that it was derived from peat. These three samples were crushed in a mortar and after sifting with a sieve of 0.5 mm. meshes served for the following tests. On examination they showed the following nitrogen content : In per cent of Original In per cent of ash-free dry matter. substance. A TS oe a7 3.90 Beep ence oo see a eS CP. eee oe oo ee B OZ Se The chemical behavior of these samples was as follows : They were but little soluble in cold water, but on boiling partly decomposed with a dark brown color and had an acid reaction. When they were heated in a test-tube a strong development of ammonia was observed and more energetically so when mixed with soda-lime. At the ordinary temperature dilute hydrochloric acid attackes them a little, but on boiling with con- centrated hydrochloric acid they were decomposed. They dissolved com- 1 According to the investigation of Charles Rimbach (Report of the Agri. Ex. Stat. of the Univ. of California, 1898-1901. p. 43) the humic acid prepirel by the ammonia extraction method is widely different, from that obtained bv the caustic soda extract. The latter contained more nitrogen than the former. 2 The amount of yield was in the case B 3.92% of dry fine earth and in C 3.34%. Studies on Humus formation, III. 517 pletely in a caustic alkali solution and reappeared on neutralisation with acids as a flocculent brown precipitate. Dilute solution of sodium nitrite upon addition of a few drops of hydrochloric acid develops only a few bubbles of nitric oxid energetic development of gas was observed, perhaps owing to the presence gas, but on addition of some humic acid a most of NH, groups in humic acid. The work of the writer which clears up the nature of the nitrogen compounds present, is described in the following lines : iBive g. of humic acid were boiled for 10 hours with 50 c.c. concentrated hydrochloric acid in connection with reverted cooler. The extraction was repeated three times and after well washing the residue served for the elementary analysis. In the extract was determined besides total nitrogen (by Kjelhahl’s method) also ammonia-nitrogen (ammonia was expelled by an excess of magnesia, using a vacuum distillation apparatus) and the nitrogen in phosphotungstic acid precipitate. (For this purpose 50% solution served for precipitation and in the precipitate washed with a 5% sulfuric acid the nitrogen was determined by Kjeldahl’s method). The results were asuollows -— a). Residue. In per cent of dry matter, ae l l Total residue N | € | EE Ash jan | in in in in in | original) reside ova residue original residue | original | residue Humic acid A 63.20 3.73 | 1.78 ‘ 58.36 | 60.92 || 5.21 | 5.00] 4.40 3.34 pe) B 53.76 3-14 | 1.45 | 44.54 | 63.18 | 3-49 | 2.98 | 16.53 | 5.63 ee GC 58.86 3.02 | 1.79 || 45.58 | 66.55 | 4.16} 3.58 14.34 9.50 In per cent of ash-free dry matter. in original | in residue in original | in residue in original | in residue Humic acid A 3.90 1.83 61.05 63.03 5-45 5.17 » Ete ale 3.76 1.53 53-36 66.96 4.18 215 ” » © 3-53 1.97 53-21 73.10 4.86 3.95 ne ne ee 518 Hence, from 100g. of original dry matter, the following amounts of C, H Shigehiro Suzuki. and N were dissolved by boiling with concentrated hydrochloric acid. G H N Humic acid A: IN su rieneacenesee eceeeeee CORSO ME EAM. PS Seas ddade ines 0.80 , ASH) 53 5.dt.0cc.ssosceienaee GLB, ontaecb hago PROHORDACaROD Adoe = Altho the nitrogen content of residue by and by decreases, it is very difficult to remove it com- pletely. 2 Zeitschr. f. physiol. Chem, 33. p. 151. and 412 (1901). 520 Shigehiro Suzuki. and passed dry hydrochloric acid gas to saturation, the precipitate! formed was filtered off. The filtrate was again evaporated, mixed with absolute alcohol and saturated with hydrochloric acid gas and then the excess of hydrochloric acid removed by boiling away in vacuo. The syrupy residue was treated with caustic soda, with continuous cooling by ice, and anhydrous potassium carbonate was added to a consistency of a semi- fluid and the free ester thus formed extracted with ether, shaking out several times, until the extract did no more show alkaline reaction to test paper. Then, after evaporation of the ether, the residue was subjected to the frac- tional distillation with the following result : Fractions Temperature Weight of distillate (g.) ois, WL AAR DIN CC re 5.0 (colorless) Br Dee Snaeeeee CONGO MEM. <....<.5¢8 9.0 (slightly green) lente eles tins es 1OO—05 Giemieees...-.. 00003 12.1 (light yellow) eens Pee -co... eee Bae ee) Sum 29.6 The first two fractions were decomposed by boiling with water for 7-8 hours, while the third and fourth fractions by baryta water heating at 70-80? C on the water bath for 2 hours. Thus it was easy to decide that various amino-compounds were present and evidently were formed from the protein or some related substances attached to the humic acid. After decomposition, the solutions of free amino-acids were evaporated to a small volume and treated with absolute alcohol and the following products obtained : I). From first fraction (at 60°C) : OWS... eee ee (Substance I) 1 This precipitate was easily soluble in water, and the solution showed the presence of much ammonium salt by Nessler’s reagent. The precipitate contained 54.89 of crude ash which had the following composition, In per cent of crude ash; RErICente 10) Shee tees cee 32336 MnezOFe...-...... 0.63 KO eeiiecess: pny ea ey sete 13.31 C210) oA CeCe 12,02 NOs. sree TU 7 vO Pacers esc 0.29 EO). 0.20 PO. Mecvoroess 0.48 Sudies on Humus formation, Lil. 521 Il). From second fraction (60-100°C) : I). First crude prodmge..-.. 0.542. Purified by the recrystallisation. 1), "ODOR ae vee. 15 bai se tees (Sub. II) fi): (OfUACe pee cos 5 oe ocean ne cles (Sub. III) 2). Second crudeproduct...... 3.908. By the fractional crystallisation, 3 samples are obtained. 1) 0.4 7iRRR Pert eee gases ns (Sub. IV) li). OQ. L2Gge Re ee no's a sine nae ewes (Sub. V) lil) 2:55 Giese Sains oles Sahn wo eee (Sub. VI) Ill). From third fraction (100-150°C) : a) Water solution. i) Insoluble in alcohol...... Tee Senate (Sub. VII) li), Soluble, 3yamer. a) Copper salt, insol. in alcohol...0.53g. (Sub. VIII) 8). Aes SOL. Gy Doge 4 7 Te Guba) @)) Ether extract: PAGE... a rE ee ee (Sub. X) IV). From fourth fraction (150-200)°C : i) Insol. in all@ghol...........: OS OR ass. (Sub. XI) ii) Sol, in F? Coppemealt....<.....0% OLB SF. cer (Sub. XII) V). From residual part. Residue was treated by an excess of baryta and the filtrate therefrom just neutralized with sulfuric acid and the precipitate filtered while hot and the filtrate evaporated to a small volume and heated to boiling with copper hydroxyd to make copper salt and filtered. After evaporation copper salt was precipitated by absolute alcohol. The weight of product was 6.06¢g. (Sub. XIII). After purifying by the recrystallisation method, the identification of these products was carried out in the following manner :— Sub. I. Result: This product is not glycocoll. Water solution did not yield on evaporation characteristic needle crystals of glycocoll and also the preparation of ethylesterhydrochlorid gave negative result. 522 Shigehiro Suzuki. Sub. IT. Result: beucinsiG@iemi—o.28¢.) i) Melting point:..72-se5 291°C ii) Elementary analysis. 0.12772. Sub. gavel eee. 0.2570g. CO, +0.1c60g. H,O O:132002.9 95 9°" eee F3.5 C.c. NW(5., yo2 mn») &; H N Leucin €,H, NO; @alenlated: 55.80 10.07 10.85 Observed : 54.89 9.31 11.47 The flocculent crystaline mass was difficulty moistened with water and formed an insoluble copper-salt. Sub. III. Result : Aminovalerianic acid (yield=o0.14¢.) i) Elementary analysis. 0.1326¢. Sub. Save seer - 13.3 c.c. N14 [Os mama) N Aminovalerianic acid C,H,,NO, Calculated: 11.97 Observed : 11.98 Sub. IV. Result : Aminovalerianic acid (yield =0.47¢.) t) Melting Spoink. ee 290°C ii) Elementary analysis. 0.19442. Sub.-cave!y eee 19.8 c.c. N (13°, 766 mm.) 0.1808. __,, 5 .3412g. CO, +0.1546¢. HO c H N Aminovalerianic acid C,H,,NO, Calculated: 51.28 9.40 11.97 Observed :—° 51.47 0:50) i220 Sub. V. Result : Aminovalerianic acid +alanin (yield =o0.12¢. ) s Elementary analysis. 0.12048. Sub. gavel: see 13.4 c:c: Noi Gy Gormmans) Aminovalerianic acid C;,H,,NO, Calculated: 11.97 Alanin C2, NO, 5 - e768 Observed : 13.40 Sub. V1. Result: Alanin Geld 2.5 52.) i) Melting point... ae 2EaeC ii) Elementary analysis. Sudies on Humus formation, III. 523 @) 0.2009g. Sub. gave............25.7 c.c. N (20°, 755 mm.) 5) o.1996¢. ,, — 2EZi yyy se CEO GY Jaren ee) Ey POsIgi 22: \; —.. == 2A toy) hay AE ee AI gee) ByONO77E= 5; ae 0.2974g. CO, +0.1360g. H,O e H N Alanin C,H,NO, Calculated: 40.45 7.86 15-73 Observed: a) — ae 14.68 A : 6) — — 14.62 a €) — — 15-29 ae. 2). AT.03 Ta7k — Sub. VII. Result: Impure aspartic acid (?) (yield =1.51¢. i) Melting point............191-193°C ii) Elementary analysis of free acid. 0.1890¢- Sub. gave.......2a 19.5 c.c. N (13°. 762 mm.) O10742. 5, 1° 70 0.2620g. CO, +0.1170g. H,O & H N Aspartic acid C,H,NO, Calculated: 36.09 5.26 10.53 Observed : 38.05 6.98 12.3% iii) Analysis of copper salt. 0.2319g. Sub. (dried at 100°C in vacuo) gave 17.6c.c. N(17°, 762 mm.) 1570s. -.,; ( - } ye 00 76se-Ca) N Cu Copper aspartate C,H,NO,Cu, Calculated: 7.21 32.67 Observed: 8.83 28.88 Sub. VIII. Result: Impure copper salt of inactive prolin (yield =o.53¢.) 0.1216g. Sub. (dried at 100°C in vacuo) gave 0.0396g. CuO Cu Prolin copper salt Calculated: 21.80 Observed: 25.99 Sub. IX. Copper salt of active prolin (yield=o.71¢g.) No test was made. ‘Sub. X. Result: Leucin (yield =2.03¢.) i) Melting point............ 29190 ji) Elementary analysis. 4 Shigehiro Suzuki. @) 0.1966g. Sub. gave 18.4 c.c. N(13% 761 mm.) }) 0.206287 45, 4 0.4129¢. CO, +0.1906g. H,O €).0.19588- 4, 5 0.3888g. CO, +0.1764g. H,O @) 30702678" i 0.2562¢g. CO, +0.1080g. H,O (@ lel N Leucin'\C,Hj,NO; @Gatealated 55.80 10.07 10.85 Observed: a) — — 11.08 mee) 54:61 10.36 — es ic) (§4a8 10.10 = meee: 2) «55.15 9.50 — iii) Determination of the rotation power. 0.1845g. Sub. dissolved in to c.c. 209 HCl. In 10 c¢.m. tube ob- served, the angle of rotation was +0.34°. [@]p?°°= +18.43° Sub. XI. Result: Impure aspartic acid (yield=0.59¢.) i) Elementary analysis of free acid. 0.1682g. Sub. gave t2-3°c.c. N (13°, 761-mm.) O-16002."* 5, 3 0.2350g. CO, +0.0900g. H,O Cc H N Aspartic acid C,H,NO, Calculated: 36.09 5.26 10.53 Observed : 29-72 5-94 8.89 ii) Analysis of copper salt. 0.147 3g. Sub. (dried at 100°C in vacuo) gave 0.0580g. CuO Observed 31.43 Cu Aspartate of copper C,H;NO,Cu Calculated: 32.67 Sub. XII. Result: Copper salts of impure acids. (yield=o.8g.) 0.1496g. Sub. (dried at 100°C in vacuo) gave 0.0502g. CuO Vee eu = 20.81. Sub. XIII. Result: Copper salt of impure acids. (yield =6.06g.) i) Determination of copper. Dry substance (dried at 1c0°C in vacuo) contained 24.20% Cu. ii) Other qualitative tests were made as follows: The sample dissolved in water and the copper was precipitated by H,S and the filtrate evaporated to a small volume. The solution Sudies on Humus formation, IIT. 525 had an acid reaction. Biuret reaction negative. Mercuric chlorid and tannin gave no precipitate, Millon’s reaction distinctly. Phosphotungstic acid gave voluminous white precipitate, soluble in excess on warming. The remaining portion was boiled with five times of its volume conc. HCl for 6 hours. The filtrate did not show the Millon’s reaction and on evaporation long needle crystals appeared (perhaps hydrochlorid of glutamic acid). The yield was too little to study its nature. For the detection of tyrosin, another portion of humic acid A (20g.) was boiled with concentrated hydrochloric acid (300 c.c.) for five hours. The extract evaporated to a small volume in vacuo to remove an excess hydrochloric acid, and after neutralisation by NaOH tested for tyrosin with Millon’s reagent. A faint red coloration showed a presence of a minute quantity of tyrosin. From these results we can conclude that the nitrogen in humus is chiefly due to protein or some related compounds which are split up into several kinds of amino-acids when boiled with concentrated hydrochloric acid. But the true nature of that compound originally present in humic acid was not yet fully determined. And also the questions remained unsolved what would be the result when the humic acid was boiled with dilute hydrochloric acid or water instead of concentrated hydrochloric acid, and whether organic bases would be produced as an decomposition product of humus. To answer these queStions, further experiments were carried out. 10g. of the sample A was boiled with 200 c.c. of water for one hour and the extract filled up to 250 c.c. and the determination of total nitrogen, ammonia nitrogen and the nitrogen in phosphotungstic acid precipitate were made in the same way as mentioned above with the following result :— 5206 Shigehiro Suzuki. In 100g. original Total N calculated sample as 100 DotaliN co. ee ee 3.7298. 100.00 N, not dissolved in H,O (N in residue) .., 3-113 | 83.48 N, dissolved.in H,O .....2.... 3 eee 0,616 16.52 N in phosphotungstic precipitate | (ammonia excluded) ............ 0.315 8.45 N in ammonia ......06---2. eee 0.047 1.26 N in other forms ..,..78 Snes 0.254 6.81 Fresh portions of the sample A (150¢g.) were again split with 1.5L of 10% hydrochloric acid for one hour, and with a part of the extract determinations of nitrogen in different forms were made. The result was as follows : In 100g, original Total N calculated sample as 100 Total N) °322..501/2. 0034. ee 3.7298. cs N, not dissolved in HCl (N in residue) ... 1.423 38.16 N, dissolved in HCl (2221).2..23 eee 2.306 61.84 N in phosphotungetic acid precipitate (ammonia excluded) ............... 0.398 10.66 Nin ammonia... .s¢¢.005-502-0 0.309 . 8.30 Nan other forms 22..5..0.5:¢2<.4 ee 1.599 sees The main extract was treated with phosphotungstic acid, then this precipitate with baryta, and the excess of baryta removed in the filtrate by sulfuric acid and thus obtained a solution of a strong basic character. With a portion of the solution some qualitative tests were carried out. i) Phosphotungstic acid. After acidifying the solution, this reagent gave a strong white precipitate, which dissolved on warming or in excess of the reagent, but was insoluble in sulfuric acid. ii) Mercuric chlorid or nitrate, basic lead acetate, tannin and a mixed solution of KI and HgCl, in acetic acid produced a white précipitate. iii) Distinctly showed Biuret and Millon’s reaction. iv) White precipitate formed by a mixture of ether and absolute alcohol, but the filtrate therefrom did no more show Biuret reaction. = Sudies on Humus formation, III. B27, v) Nessler’s solution showed a slight turbidity but almost no precipitate. vi) Xanthoprotein reaction was ambigious and no picrate was formed. The remaining solution was evaporated in vacuo to a small volume and put into a vacuum desiccator and let it stand to dry at ordinary temperature. After three weeks a yield of 3.67g. was obtained. 2.5. of this sample was again boiled with 50c.c. of 20% HCl for 6 hours; and a portion of the extract served for the determination of the different forms of nitrogen with the following result : In total extract Total N calculated as 100 UCTS. Goats alee oats ae ea ae EEE A cioocinc, 0.348¢. 100.00 N, in phosphotungstic precipitate............ 0.186 53-45 IN abt BICSTEAO)OTE)) | = Ae Sao Aen BARARHRRBBBoGOCBED Es 0,000 0.00 INMROLMERMOLIMSS etetacewadtecsesecsecasacteeeee 0.162 46.55 Of the remaining portion, arginin, lysin and histidin were tested after Kossel’s method, but no organic bases were found, except only a trace of histidin. From these results we can say that the humic acid was also decomposed by dilute hydrochloric acid of 10% and more than half of its nitrogen dissolved. The nitrogen compounds contained in extract and pre- cipitable by the phosphotungstic acid seems to be of the compounds akin to protein substance, but they do not exactly coincinde. Altho in many cases they showed the same reactions, the former had a strong basic character and was partly soluble in absolute alcohol, and when boiled with more con- centrated hydrochloric acid decomposes partly into a form not precipitable by phosphotungstic acid, but produces no ammonia. Perhaps, resleisver simpler constitution than the ordinary protein matter. A further experiment was made in regard to the ash content with the following result : 100g. of original dry matter contained. Humic acid A B c Crude ash: 4.40¢. 16.538. °% 14.34g. 100 parts of dry ash contained : 528 Shigehiro Suzuki. Insoluble} Soluble » 2 residue | SiO, MgO | Fe,0, | Al,O; | K,0 | Na,O P,0; = 5 3 land Cl, Humic acid A} 33.92 0.90 | 12,07] 1.87 | 11.62] 7.94 | 1.82] 6.92 | 7.32 | 10.96 4.67 B} 19.82 1.08 O7E | O5r | 50.36] 7:00 | 057) 3.34 |] 7:93 | 167 2,02 39 = rt 41.95 0.42 0.74 | 0.90 | 13.84 | 26.15 | 0.37 si 8.09 | 1.25 212 | { This result shows that various mineral compounds accompany the humus or partly remain in intimate connection with the humus when it is dissolved and precipitated. Conclusion. This investigations show that the nitrogen in the humus is not present as amino compounds, but chiefly asa kind of protein which may be con- nected more or less intimately with the black substances. In my former communication! the observation was mentioned that not only starch but also proteins are blackened by the humification process. This, however, does not exclude that some of the protein is derived from soil bacteria, while another part from the decaying roots.—It seems that during the humi- fication process certain atomic groups in the protein molecule are considerably changed or also oxydized away and this becomes thus less suited as food for bacteria and mold fungi. According to this result, the writer is inclined to believe that Udransky’s artificial nitrogenous humic acid? would naturally differ from that of the natural one, because the protein-like substance or several kinds of amino-acids would not be formed when a mixture of glucose and urea is treated with boiling hydrochloric acid. It was further shown that of amino-acids as such only traces are present, and that such compounds are only obtainable after treating with hot con- centrated hydrochloric acid ; 500g. of dry humic acid yielded the following decomposition products :— 1 These Bull, Vol. Vil. No. 3. p. 419. 2 Zeitsch, f, physiol. Chemie. Bd. XII. p. 42. (1888). Sudies on Humus formation, III. PEMEERRTED, Uwe oc > 2 sw nn he EE oes oo dale aE AS wn ws 2.39g.4 CHENG So). 2.2525 Ee ts Se oe ccd wee aaees, oe 2.16 Aflanin+-aminovaleriamiG@ietl ......--- 2... /ccecceeveenee O.11 vammnovalerianic acid” GRMMM-<12...--.-.2-.s0seeces ede 0.57 : ae salt» of deems prolin ..........0020006 0.67 Prolin ; a 53. summers: § (2) ossecbe0dds ees 0.50 PRSBAEIC ACIC ... ... 222 RM oo av on do ire nese cee ee 0.06 inapare-aspartic acid!(2 ype coe 22-5 2.500. -cneet eee. 2.50 cleiiartic ACiC /..,.. 12:0 eee foes svaae ones auenet nace present. POON Sas ss 2 oe vino ae >» 5+ 2 Sao Se seae Aen aes ee trace. EASE a... 2 3 5) sn 3 a wc ese cutnar conse ne trace. PARIOOGIA, 2.2... ee... J MRRP Set Bey ne 1.90 Copper salts of unknown acids ............. tet Sa ae AOL O 529 1 Of course, these quantities show the minimum amount of the yield, as a part of these substances are lost during the purification process, The writer expresses his sincere thanks to Prof. U. Suzuki for his valuable advice thruout the progress of the work. Studies on Diseases of Saké. BY T. Takahashi. In a former report! the writer described a new variety of Mycoderma yeast causing a kind of Saké disease, but there remain still many questions regarding Saké diseases to be solved, altho various observers have made in- vestigations on this subject. The samples of the altered “Saké,” generally called here Hyochi-Sake, which were investigated by the writer amounted to fifty? in number, while the 48 factories of these samples were distributed over 17 prefectures. These samples could be classified by their flavor and taste as follows. 1. Characteristic “ Hyochi” flavor and somewhat strong acidic taste. 2. Taste merely sour. 3. Flavor of sea weed. 4. Odor putrefactive and fish-like. 5. . Strong volatile acid flavor. 6. Flavor after acetic acid and taste strongly sour. 7. Very strongly acid and bitter taste. 8. Characteristic “Hyochi” flavor but destitute of any distinguishable taste. g. No distinguishable flavor ; only sweet and sour taste. 10. Trace of Characteristic “‘ Hyochi” flavor and sweet taste. 11. Odor of acetic acid and acetic-ester emitted ; taste sour and bitter. 1 These Bulletins. Vol. 7. No. 1. 1906. 2 Two of these samples had altered already during their fermentation and must be distinguished from “ Hyochi,” which name is generally applied to the altered Saké after the fermentation, clarifica- yion and pastorisation, 532 T. Takahashi. 12. Flavor of “ Hyochi” Saké and of caramel. As to turbidity several cases can be distinguished. 1. A sediment forms easily, the liquid becoming clear. 2. The turbidity continues very long, sometimes even for several months. 3. The sediment shows a brown color. : 4. The formation of turbidity is succeeded by ‘ Hyochi” flavor.? By direct microspical observation it was observed that in the majority of cases a long non-motile bacillus was present and only in nine cases a motile bacillus? and in further forteen, those microbs were accompanied by yeast cells. Method of isolation. As culture medium for the isolation of the bacteria in these samples was used sterilised “Saké”-agar* in glas tubes. The dilution was made in three tubes and after producing an inclined surface in glas tubes sterilised “Saké” was poured in, whereupon the mouth of the tubes was sealed with a parafi- nized cotton. . The microbes thus isolated were :— 1. Mycoderma yeast from three samples. 2. Acetic bacillus accompanied with lactic bacillus from four samples. Acetic bacillus from four samples. Mycoderma yeast and Saprogenes (Hyochi) bacillus from 3 samples. 3 4 5. Lactic bacillus from six samples. 6. Mould and nothing else from one sample. “I . Saprogenic (to Saké) bacillus from 35 samples. 1 The white deposit is common with the “ Hyochi” Saké, 2 It is common that the “ Hyochi” flavor developes before a marked turbidity is produced. 3 This kind of bacillus was unfortunately not always found on the agar-culture plate prepared from these samples, 4 The original “Saké” for this preparation contained 16.49% Alcohol, 0.189% ext. matters, After yserilisation of the culture medium there was a certain amounts of alcohol retained. Studies on Diseases of Sake. 533 LT.) Bacillus saprogenes Saké (“ Hyocht” bacillus). For this bacillus it is characteristic that it makes a growth almost ex- clucively in Saké, and that it produces a peculiar flavor! of the “Saké” attacked by this bacillus. We can distinguish two chief varieties :? Bacillus saprogenes Saké. I. This variety implies those forms which make a growth in yeast-water as a nutrient. We must distinguish? further here motile and non-motile sub-varieties, which all are sporeless, and occur in 7 forms. Ll. Sub-variety. The bacillus of this sub-variety makes a growth only in Saké 149 and less alcohol,+ but not in stronger ; in Saké-agar it developes a colony composed of disks attached to each other in 3 directions, as will be seen from drawing or plate produced from the photograph.* But such a form is not quite constant. 1. Form and size: Very long bacillus, 4-7% in Saké, sometimes filanmentous, attaining to 15-20. (with a width of .5~). Frequently a long chain of cells or two cells combined. Non-motile. Colored well by com- mon anilin colors, and Gram’s staining. 2. Growth. a) Solid nutriment: Stab-culture on Saké-gelatine ; no growth ‘after two months (at 16°C), but grows well as round or rather de- formed pea shaped colonies in hydrogen atmosphere. Saké-agar streak culture: grows slowly along the track, but more quickly when covered with «Saké” or yeast-water. (5 days at 22-28° C). 6) Fluid nutriment®: Yeast-water: Only traces of developement 1 This flavor is called, in Japan, “ Hyochi Ka.” 2 A former observer, Torii, has reported only one variety of “ Hyochi” bacillus which from his description must belong to the first variety observed by myself, 3 The forth sub-variety is motile. 4 Alcohol 14.4%. Total acidity 0.137% as succinic acid. 5 The colonies of such a form are frequently observed with yeast, especially with mycoderma yeast on agar culture, but the growth is quicker in the case of yeast. & All the nutriments mentioned here were employed to all other bacilli isolated. 534 T. Takahashi. (a month at 28-30.5° C). No growth in tyrosin solution,! “ koji”’-extract, or “moto”-mash. More or less in yeast-water-galactose or yeast-water-glucose. Almost no growth in alcoholic-yeast-water,? or yeast-water-maltose. No growth in protein-free-saké.$ 2. Behavior to carbohydrate: Galactose is assimilated but neither glucose nor maltose and from galactose a non-volatile acid is formed. 3. Behavior to “Saké”: Duration of turbidity of ““Saké”4 is short (5-6 days) in general. In diluted “Saké” 0.0413% of non-volatile acid and 0.0156% of volatile acid® are formed after 55 days (summer time). Bitter taste is formed also. 4. Behavior to temperature: Delopes at 16-31° C favorably at 20- 28° C very slowly at 10-12° C. Death will result at 55-56° C during 15 minutes in diluted “ Saké.’’® 5. Behavior to an anticeptic. 0.213197 of salicylic acid in diluted Saké does not prevent the growth. Second sub-variety. The bacillus of this sub-variety grows easily in ‘“‘Saké”’ with even above 14% alcohol® compaired to the first sub-variety. The form of the colony is not constant, sometimes round and flat? in another case resembling the first variety and still in another case it appears as two plates in cross position. 1. Form and size: Very similar to the first one but in some cases!®° somewhat longer; 5-7-10y. larely 25u f° in ‘“Saké.’ Involution 1 Tyrosin 0,025g. glucose 8.0g. alcohol 48 c.c. maltose 2.0g. magnesium-sulphate o.2g. K- phosphate 2.0g. water 400 c.c. 2 Alcohol 160 c.c. yeast-water 300 c.c. sugar 2.0g. water 650 c.c. 3 After the evaporation of the volatile part of “Saké*” the excess of basic-lead-acetate is added to precipitate protein matters and the filtrate is passed enough H,S gas and the filtrate is made to the original volume, # and © Alcohol 12.4%. Total acidity 0.1713%. 5 The non-volatile acid was calculated as succinic acid and the volatile acid as acetic acid. 7 10.5 “momme” in one “ koku ” (180,39. L). 16.49%. But no growth in “Saké” containing 17.5% of alcohol, and by this character we can distinguish this from 4th and 5th sub-varieties. ® The bacillus of such colony assimilate non-albuminoid nitrogenous compound in “ Sake.” 10 Round flat colony. Studies on Diseases of Sake. 538 form! is found in “Saké” (20 days) culture especially in round flat colony. Stains by Grams method in the case of round flat colony. 2. Growth: a) Solid nutriment: Saké-gelatine stab-culture ; (16- 20°C) ; spheric colony appears along the stab canal. Better growth in hydro- gen atmosphere. Sake-agar stab-culture:? grows like the first sub-variety. 6) Fluid nutriment: No growth in tyrosin solution, koji-extract, “moto”-mash* and only traces in yeast-water-, and in yeast-water- glucose. Negative in alcoholic-yeast-water, yeast-water-maltose:° Gene- rally negative in protein-free-“ Saké.”® 3. Behavior to carbohydrate: The assimilation of sugars and acid formation not quite constant, according to the origin of the bacillus. 1. e. Assimilation of Acid formation from a I Origin,” galactose. glucose. galactose. glucose. ot. +++ - + -- B. ++ ~ ++ + Y: - — = = 8. ++ + at = PSF LS alate 2 ry +4 Par 4. Behavior to Saké: Turbidity increases for 7-10 days. The acid production is also not constant,§ but the fixed acid is surely lactic acid (Uffelmann’s test). 2 As shown Fig. 6, 2 In agar culture the death of the bacillus will be observed after 6 months ; while in gelatine culture it lives still longer. 3; 4; © Some of this sub-variety (round flat colony bacillus) show a good growth as an exception 5 With an exception. 7 Perhaps it will be good to make a further sub-division of this sub-variety according to their origin, but the above description is more simpler, 8 Duration of cult, Non-vol. acid. Volatile acid. a 4o days. +0.13806%. —0,0015 %. B. 22 i +0,00708%. —0,033%. 8. Spears +0.13098 %. +0.018%. N. Deh oes +0,0755%. —0,0134.%. 536 T. Takahashi. 5. Behavior to temperature: Developes at 16-31°C, favorably at 28-29° C1 or 20-21.5° C.2 Death temperature is the same as that of the first sub-variety. 6. Behavior to salicylic acid. Some (7. 8) grow in diluted Saké con- taining 0.21319 of salicylic acid, but others not. Third sub-variety. The bacillus of this sub-variety grows very well in “ Saké” containing 16.4.% of alcohol (coincide with second sub-variety) but differs by not forming acid in fluid containing galactose in spite of the assimilation being possible. The form of the colony is the same as that of the first sub-variety. 1. Form and size: Very similar to the second sub-variety. Staining by Gram’s method ts negative, but cellulose reaction observed. 2. Growth: Solid nutriment: Not distinguishable from the above varieties. Fluid nutriment: In all the above liquids negative, except in yeast-water and alcoholic-yeast-water. 3. Behavior to carbohydrate: Neither glucose nor maltose are as- similated but only galactose, but no acid is formed. 4. Behavior to Saké: Fixed and volatile acid are produced®, beside “Hoychi”’ flavor and an unpleasant taste accompanied by a bitter taste. 5. Behavior to temperature and salicylic acid: Grows at 16-32°C; optimum 25-26° Cnotvat fosi@we. . Dies at: 582586 €i(15 an) eee smaller quantity (0.2101 %) of salicylic acid is sufficient to prevent the growth forth sub-variety. The bacillus of this sub-variety is capable to make a growth in “Saké” with 17.5% of alcohol. This bacillus is motile and grows well in protein- Jree-“ saké,” containing some amido-compound. The form of the colony in agar culture is round and flat. The duration of turbidity of “Saké” is longer sustained than with other bacillus. 1 § and 7. 2 a, B, ¥. % Jod and concent. H,SO,. (Blue coloration) 4 This sub-variety grows unfavorably on “Saké ”-agar 5 During 4o days at 26-29°C, the fixed acid was increased by 0.1770% and the volatile acid by 0,0108% of the Sake. Studies on Diseases of Sake. 637 1. Form and size: Very long in “Saké” culture: 7-oy. seldom 17.5m. or 4-5. Similar form with above varieties. Involution forms in old “Saké” culture: Club shaped, curved, etc. Gram’s staining is negative. 2. Growth: Saké-gelatin: Same as the former varieties. Trace in yeast-water, yeast-water-galactose. Negative in tyrosin-solution, ‘“ koji’’- extract, mato-mash. Good in alcoholic yeast-water or protein-free-Saké. 3. Behavior to carbohydrate and “Saké.”” Assimilate glucose but not galactose or maltose. In diluted! “Saké” forms fixed acid and destroys the volatile acid? and alcohol. The duration of turbidity of ‘““Saké” continues from 20-58 days according to the nature of “Saké.’’3 4. Behavior to temperature and salicylic acid. Grows 16-31, optimum 23-28° C, not at 10-12° C.” Dies at 55-56° (15 minutes). 0.2101% of sali- cylic acid is sufficient to prevent the growth. fifth sub-variety. The bacillus of this sub-variety grows in “Saké” with 17.5% alcohol as the third sub-variety. The form of the colony in “Saké”-agar is round and flat. In general properties this bacillus has many similarities to the third sub-variety, but in regard to the assimilability of sugars distinction exists. 1. Form and size: Similar form as the first sub-variety: 2.5-4y. Gram’s staining ts negative. 2. .Growth: White (spheric) colony in ‘Saké’’-gelatine, better in H-atmosphere. Very energetic growth in “‘Saké”-agar.4 Trace in yeast- water, better in yeast-water-galactose. Not in the other nutrient fluids above mentioned. 3. Behavior to carbohydrate and “Saké.” Like the third sub-variety 1 Alcohol 12.4%. 2 After 40 days (at 26-29° C): +0.10856% of fixed acid and —0,0036% of volatile acid, — 1.679%. of alcohol. 3 Composition of Saké, Duration of turbidity. ay Peleohol 16.496; Hxt. mi. 1.89%) Payee Cesc diestecceccees 58 days. b). TRANG: | 5S ODGIE MI LED ao csecsecceet Cer SP AR EA ale RP SEA UCR cls 5o race de enenesees 20 5 4 Galactan, perhaps may be assimilated by this bacillus. 538 T. Takahashi. assimilates galactose, but not glucose or maltose. Trace of acid is produced from galactose. Destroys ‘“Saké,”1 4. Behavior to temperature and salicylic acid. Grows at 16-32°C; optimum 27-28° C, not at 10-12° C, Dies at 55-56 after 15 minutes. Szxrth sub-variety. The bacillus of this sub-variety grows as well in normal “ Saké”’ as the fifth sub-variety, but on regard to the assimilation of sugars and to acid for- mation just the reverse is observed. The form of the colony in “Saké” agar is the same as that of the first sub-variety. 1. Form and size: Similar to the fifth sub-variety ; commonly 2.54., 3-54, forming very long chains. Involution form not observed. Gram’s staining failed. 2. Growth: Glolular in “Sake’-gelatine; resembling a mouldy growth in the “Saké” of the “Saké”+‘“Saké”-agar culture. No growth in all the fluid nutriments above mentioned except in yeast-water. 3. Behavior to carbohydrate and “Saké.” Assimilates glucose and forms acid from it, but not so with galactose. Fixed and volatile acid are increased in Saké.? 4. Behavior to temperature and salicylic acid. Grows at 16-32°C, optimum 29-30° C, not at 10-12°C. 55-56°C (15 minutes) and 0.2101% of salicylic acid in diluted Saké are sufficient to cause the death of the cells. Seventh sub-variety. The bacillus of this sub-variety grows well in Saké? not diluted. The colony in ‘‘Sake”-agar appears generally in two types; one of them has likeness to the first sub-variety and the other one appears as two round plates intersecting each other vertically. This bacillus assimilates glucose, galac- tose, and maltose and thus is distinguished from the sixth-sub-variety. 1. Form and size: Long bacillus: 7.5, 124. Gram’s staining ts 1 During 17 days at 26-29°C the fixed acid was increased by 0.1298% of the Saké while the volatile acid was decreased by 0,0237% of the Sake, 2 0.25489 of fixed acid and 0,04659% of volatile acid were formed during 36 days at 26-29° C, 3 Alcohol :=17.5%. Studies on Diseases of Sake. 539 positive. Involution forms are observed in “ Saké” culture. 2. Growth: White, globular colony in Saké-gelatine, better in H.- atmosphere. Grows well in yeast-water, moto-mash, and protein-free-Saké, but not in the other fluid nutriments mentioned. 3. Behavior to carbohydrate and Saké. Assimilates the three sugars mentioned and forms fixed acid from them. Alcohol ts produced from glucose. The increase of acid in Saké somewhat large.1 4. Behavior to temperature and salicylic acid: Grows at 10-30° C,? dies at 55-56°C during 15 minutes. 0.2101% of salicylic acid in diluted Saké is sufficient to prevent the growth. Bacillus saprogenes Saké. 1. The bacillus belonging to this variety can not grow in yeast-water and by this character we can distinguish this from the first variety. Moreover, none of the sub-varieties show any growth in protein-free-Saké, except the first sub-variety. All die at 55-56°C in 15 minutes; 0.2101% of salicylic acid in diluted Saké? is sufficient to prevent their growth. In H.-atmosphere grow better than in the common medium. First sub-vartety. The characteristic property which distinguishes this sub-variety from the following, is its faculty of growing in protein-free-Saké. The form of the colony in Saké-agar is generally a combination of three discs developing in three directions. 1. Form and size: Short bacillus: 2-5. in Saké, often in long chain of cells. Gram’s staining negative. 2. Growth: White spheric or kidney-bean shaped colony in Saké- gelatine. No growth in all the fluid nutrients mentioned above, except in protein-free-Saké. 1 After 27 days at 26-29°C, the increase of fixed acid was 0.2383% and of the volatile acid 0.018%. 2 At 15-17° C it grows almost equally well as at 30° C. 3 Alcohol 12.49%. Total acid 0.1713%. 540 T. Takahashi. 3. Behavior to carbohydrate and Saké: Galactose was assimilated and oxidized to acid. In Saké, fixed and volatile acid were increased, but alcohol! was destroyed. 4. Favorably grows at 16-30°C; optimum 23-25; but not at 10-12° C. Second sub-variety. This bacillus grows well in Saké containing 16.49% of alcohol, and the developement in Saké-agar was very energetic; the forms of the colony in this medium are either the form of the first sub-variety or two plates inter- secting vertically with each other. 1. Form and size: Either long (5-7.5) or short (2.5-4-5y) cell. Two cells in combination are common. Involution forms were observed in old Saké-culture. (30 days culture). Stazus well by Gram’s method. 2. Growth: A white spheric or bean shaped colony in Saké-gelatine. An energetic growth in Saké-agar led me suppose that this bacillus has perhaps, the power to assimilate galactan. No growth in all fluid nutrients mentioned above. 3. Behavior to carbohydrate and Saké: Galactose is oxidized into acid. The acid was increased in Saké;? and the flavor of caramel was observed at the same time. 4. Temperature limit for the growth lays from 16-32°C; optimum 23-25° C, but not at 10-12° C. Third sub-vartety. The bacillus of this sub-variety grows well in Saké containing 16.49% of alcohol. The colony in Saké-agar is composed of five discs; three of them starting from a common line, the other two growing in other way forming certain angles to the former discs. (compare plate). 1. Form and size: Long bacillus; commonly 84, sometimes very 1 After 25 days at 26-29° C, there was the increase of the fixed acid by 0,03566% and of the volatile acid by 0.2024% ; the decrease of alcohol by 2.95 %. After 33 days (at 26-29° C) ; +0,0233% of fixed acid and +0.00849% of volatile acid, Studies on Diseases of Sake. . 541 long filaments. The involution forms appear easily.! Stains well by Gram s method.” 2. Growth: A spheric colony in Saké-gelatine. All fluid cultures failed, except in yeast-water-galactose. 3. Behavior to carbohydrate and Saké: Galactose was oxidized to acid. In Saké an increase of fixed acid and a decrease of volatile acid? was found. 4. Temperature limit for the growth lays 10-31°C but the optimum lays between 23-25° C. No growth at 10-12° C. Fourth sub-varzety. As the former sub-variety, this bacillus grows well in Saké containing below 16.4% of alcohol; while assimilation of galactose did not take place in this case and by this character we can distinguish it from the third sub- variety. The colony in Saké-agar has the same shape of the first sub-variety. 1. Form and size: Either short or long bacillus: generally 5-7y or sometimes 107. Involution forms were observed. Stains well by Grams method. 2. Growth: A _ white spheric colony in Saké-gelatine.4 The fluid cultures mentioned above, all failed as nutriment. 3. Behavior to carbohydrate and Saké: Galactose was not assimilat- ed. Fixed and volatile acid were increased in Saké.° 4. The optimum temperature for the growth was 29-30° C, though it can grow at 16-32. No growth at 1o-12° C. A bacillus was isolated which behaves very simillary in many respects to this bacillus, but the differences consisted in the size (3-5, chiefly), in its 1 It appears in 18 days culture already. 2 By this character we can distinguish this from the second sub-variety of B. saprog, s, II. 3 After 37 days at 26-29° C, there was an increase of fixed acid by 0.03068% and a decrease of volatile acid by 0.02373%. 4 A bacillus very similar to this bacillus, differing by its formation of round flat disc formed colony in Saké-agar, dies off more quickly, in agar-culture, than this bacillus. 5 After 2 months at 22-29° C there was an increases of the acid. PCA MREIA tei. 2. Meio 20 Ube by ¢.2147%. Molatile acidiecosn-2,.cs.ss020 »» 0.0108%, 542 T. Takahashi. failure by Gram’s staining and in a scarcely noticeable growth in yeast- water containing a trace of alcohol. Fifth sub-variety. This bacillus can grow in Saké containing alcohol up to 14.4%. Fur- ther, the faculty of developing in yeast-water containing a small quantity of alcohol is a characteristic property, which distinguish this from other sub- varieties. 1. Form and size: Long bacillus ; 5-8. or very long chains of cells. Involution forms were observed (Fig. 24) and these forms, when stained, show colored stripes. Gram’s staining failed. 2. Growth: A white spheric colony in Saké-gelatine. All the fluid nutriments mentioned above failed as nutrient media, except yeast-water containing a small quantity of alcohol, or galactose. 3. Behavior to carbohydrate and Saké. Galactose was oxidized to acid. In Saké fixed acid was increased but volatile acid was destroyed. ! 4. The optimum temperature lays near 30° C, though it can grow at 16-33° C. No growth at 10-12° C. Sixth sub-variety. This bacillus stands very nearly in its general properties to that of the fifth sub-variety. We can distinguish it from this by :— a) Size: generally shorter than the former one; 2.5~4-5v. 6b) Staining well by Gram’s method. c) The colony in Saké-gelatine having spines on tts surface. a) The surface? growth in H-atmosphere, when Saké-gelatine was used as nutrient medium. e) The weak growth in yeast-water containing a small quantity of alcohol. 1 After 66 days culture, there was an increase of fixed acid by 0.0799%% and a decrease of volatile ara é acid by 0.003%. 2 2 Such a property have not been met with in any other case. Studies on Diseases of Sake. unr aS os) Seventh sub-variety. This bacillus has closely related properties to the sixth sub-variety, but the differences consist in : @) The shorter size ; 2.5-5y. 6) The cellulose reaction. (Blue color made by Jod-Jodpotasium and strong H,SO,). c) The white spheric colony with smooth surface in Saké-gelatine. @) The volatile acid in Saké was destroyed. é) Involution forms were not observed. Etghth sub-variety. This bacillus has almost equal properties with that of the seventh sub- variety, but may be distinguished by the following points :— a) The longer size: 7-84. commonly. b) Negative behavior to Gram’s staining method. c) The colony in Saké-gelatine was semi-transparent and a_ spleen shaped. Ninth sub-varicty. This baciilus can grow in Saké containing less than 12.4% of alcohol (diluted Saké). The colony in Saké-agar consisted of three combined discs. 1. Form and size: Either short or long bacillus ; 2.5-3-5-7u4. some- times in long chains of cells. Involution forms were observed. Sfazus well by Gran’s method. 2. Growth: A white spheric or kidney-shaped colony in Saké- gelatine. A weak growth in yeast-water-galactose, but no growth in all other untrients mentioned above. 3. Behavior to carbohydrate and Saké. Galactose was assimilated, but no acid was found in the culture. In Saké an increase of fixed and volatile acid was observed. ! 1 After 2 months at 20-29? C, there was found an increase of the fixed ac'd by 0.191169 and that of the volatile acid by 0,0204.9¢. 544 ; T. Takahashi. A caramel flavor was found in the culture in Saké. Ll.) Lactic acid bacillus group. The bacilli belonging to this group form lactic acid! in Saké or other nutrients but we can not fined any trace of the so-called characteristic “ Hyochi’’-flavor. Six varieties must be mentioned. All of these are non- motile except one variety and sporless. All grow well in a H-atmosphere. All die at 50-52° C after 15 minutes. I. Bacillus panis fermentati. var Saké. 1. Form and size: Short bacillus and forms long chains of cells in diluted Saké.?_ In “ koji’’-extract a combination of two cells was found chiefly, or long irregularly curved cells are seldom found. 2. Growth: In Saké-agar plate culture there was found chiefly im- bedded in medium, a white, round and flat colony, with smooth surface. The surface, when observed under microscope, seems granular. Stad- culture: A white spheric colony was found in the inner part of Saké- gelatine. (16° C after 2 months). An energetic growth was observed along the stab-canal of koji-extract-agar ; when old there was found a pin-head like growth at the mouth of the canal accompanying gas production. A still more energetic growth was found in case of Saké-agar, better after pouring some Saké on the solid medium. Fluid culture: In yeast-water, bouillon, koji-extract, protein-free-Saké makes a good growth, especially in koji-extract; but there was no growth in “moto ’’-mash, Hayduck’s solution or tyrosin solution. 3. Behavior to carbohydrate and Saké. In the culture of diluted Saké, there was an increase of both acids (fixed and volatile)? ; while the alcohol of it was destroyed. This bacillus could not develope in Saké 1 The acid was isolated by Uffelmann’s method from the culture of these bacilli in yeast-water- galactose (10%). 2 Alcohol 12.4%, total acid 0.1713%. 3 In Saké containing 12.49% of alcohol and 0.1713%% of total acid, there was an increase of the fixed acid by 0.0413 % and tl.e volatile acid by 0.925%, where the decrease of alcohol was by 0.27% after 27 days at 26-209° C. Studies on Diseases of Sake. 545 containing 17.55% of alcohol and 0.17796 of total acid; while at 16.4%, it shows an energetic growth!. The acid formation from various sugars may be shown by the following table :— Substance, Bacillus panis fermentati.2 | Bacillus panis ferm. var Saké, ENGST CaM eter sarc ssc) vexndacéees os SS a5 sPsrPars NG LOSI ene orn Gerd sticeiels pe ao HE ae apse Galactose Et isthe ones aniiet sures te arse as (GUIGOSS: on seo at pene ae eee eee + SR aP ae a AGS CMM nes tere eee a otis.cia Sos -in ve ae == S535 } ANITA OSE eee ee eeneriace tose: Substance. | 3acillus Delbriicki, | Bacillus Delbriicki. var Saké. dATra bb INOSE piers dass ioeeeatenee toe — Staats ate shy EXVILOSE) casehac ses ee tew ee ie ach eeenee | - | SF cSt | (Galactose cacsai0 ein: Salta oes 55 | ae ameter GlUCOSE hc ea nt aon SP apes | air Tr Puctose yas. -cedeescncceonecere sees Saat oats RhamMMNoses. 2. cqsstesossos-nase sae = a Maltosetiea. Jasascterestnes aeneeees Saisti ats Se SE Sp a5 Saccharose sys psy soeeee sap seee ee + 7p ap ae ar TSA CtOSENcaenMeectaa. sas ocean ce meee | = = Ha. FIMOSE Was a. neeee ae nce eee eee = =F Wextrineg isciates eesaamee ce eee oe Sisk larChiccrgcton« sedate eee eae = ats Arians 33he3 fos Rave eeeeens eee = = IMATTTCI NAT eae Rees ee = es a-Methyl-glucoside .................. — | ++++ Thus the property of the forming acid from arabinose, xylose, starch, a-methyl-glucoside and raffinose forms a decisive difference from B. Delbriicki. Further, a trace of alcohol was formed from galactose and maltose. 4. Behavior to temperature and salicylic acid. Grows at 16-32° C; optimum 28-29, but not at 10-12°C. 0.2131% of salicylic acid in Sake prevent the growth, but not 0.2101 %. 1 After one month, there was an increase of volatile acid by 0.006% and a decrease of fixed acid by 0.0047%. Studies on Diseases of Sake. 549 IV. Bacillus Taetis acidi. var Saké. 1. Form and size: Either short or long bacillus: 3-4y-7.54. In- volution forms were observed in old culture. Revolving motion was observed. 2. Growth: A white pasty colony on the surface of koji-agar plate culture ; while in the inner part grows as white round disc!, which observed under microscope was found a granular surface. In yeast-water, bouillon, koji-extract grows very well especially in the latter one, but no growth in Hayduck’s solution, moto-mash, tyrosin-solution or protein-free-Saké. Stab-culturc. A white spheric colony with spines on the surface was found in Saké-gelatin. Grows only along the canal in Saké-agar or koji-extract- agar but not on the surface. °3. Behavior to carbohydrate and Saké: It grows in Saké containing up to 14.049 of alcohol? and 0.1416% of total acid, whereby an increase of fixed* and volatile acid and decrease of alcohol were observed. Further, unpleasant flavor and bitter taste developed after the growth of the bacillus. The acid formation from sugars is shown in the following table :— Substance. Bacillus lactis acidi. Bacillus lactis acidi. var Saké. JNCLUBYINGR=! apse conan PoReE eee — ap arse ar ESATO S” Soh Snipe cays os ae eae eee = a Galactose Pease racttarenic. seinaseee ace + Tre a GIBCO S es Ss ee Ss ee +++ Teast UGC OSCE RR Rea re eka saci +++ fat + AN AMINIGS EMME cect erees (ccc: waiaSoks = 25 JUL Sher Saar ee ae. eae +++ stale cimaly SHAG CEN eo a re i re +44 hehe charts NGWCLOSE: sgn. cose: “HE Sn SERS onoee ac Ste ahetate at 1 Or sometimes appears a second disc growing vertically to the first one. 2 B. lactis acidi loss its acid forming power and its propagating energy in 3% of alcohol. 3 After one month at 26-29° C, there was an increase of fixed acid by 0.0212% and volatile acid by 0.0273 %. 550 T. Takahashi. Substance. | 3acillus lact's acidi. Bacillus lactis acidi. var Saké, Ratinpse: 60000 Lee Py: are MERUEANG Tob ..a5, o2se ooo a ee ++ EE Stan) oo. sesee ace ce doe eee | = + Spal USS oe re ue == - Manuite a-methyl-glucoside = ned Thus the acid forming property of this bacillus from arabinose, xylose, rhamnose, starch, a@-methyl-glucoside identifies this from B. lactis acidi. Further, alcohol was formed from galactose and maltose. 4. Behavior to temperature and salicylic acid: Grows well at 16- 32° C ; optimum 23-24? C, but no growth at 10-12°C. Very weak growth in Saké containing 0.210199 of salicylic acid but not in 0.2131 %. V. Bacillus wortomani. var Saké. 7. Form and size: Very long bacillus: 2-2.5-34 commonly but in old culture: 204 as a filament. A flocculent or mouldy mass was formed in old Saké culture. 2.. Growth: The form of the colony in Saké-agar was almost tke same as Bacillus lactis acidi. var Saké. A mouldy mass was found also in Saké over Saké-agar culture, but when it grows in the inner part it was almost equal to that of B. Delbriicki. var Saké. In yeast-water, bouillon, protein-free-Saké, koji-extract it grows well especially in the latter one, and a trace in moto-mash but not in Hayduck’s solution or tyrosin solution. Stab-culture. A white spheric colony in Saké-gelatine but not on the surface. Only along the stab canal in Saké-agar, koji-extract-agar. 3. Behavior to carbohydrate and Saké. A very weak growth in Saké containing 16.4% of alcohol and 0.15% of total acid, but an energetic growth was observed at lower parentage of alcohol (13.163%) and acid (0.1228%). In Saké an increase of fixed acid and decrease of volatile acid Studies on Diseases of Sake. 5 at — were found.! The acid formation from carbohydrate will be shown in the following table :— Substances. Bacillus Maerkeu Bacillus cueame= Bacillus , | Bacillus Wor- | eris fermentati. | Wortomani. tomani var Sake. EXGADIMOSCw eres enc mec + | SP Se Sr +44 + +44 DMOVLOSE a reaneeae ena sein Vi | J | +44 Galactose boone eeee | + + + | Re ae SF 1 SS ap oe \ClGORS scronecsGonsscoder | SS ot | 5° 45 55 +++ = ISEMCEOSE eee ng antes sss | Hosp | +++ ++ fod Vebse ee ce INEST NORE Ooaseandeocse Ve | we ye + MictitaSe: Mokena echsacamcck +++ tit +f 4 tHit | SQCCMAOSE) 4. .Seescc ccc as =p Se Sr S552 55 | 42 55 4255 NEA CLOSE A inact ence + +++ +4 +ti44+ INatTMOSE = sen -eeeeseckeee ++-+ +++ ap oe | a= 45 Wax brine sco. je. Asear | + | + | ++ | gB ot ce ot ‘SLBUIRCIN, Ces ssaee ar neTeeene | fe | Wa | Va ++ INANEUIDY, Beecep eenceuose eee = | _ = _= Mannite ...... Lees fod | 4h ge eee | a-methyl-glucoside ... — | = -F | pipet te Thus, this bacillus do not form acid from glucose and is thereby distinguishable from the three closely related varieties. Further, the acid forming property from a-methyl-glucoside may be mentioned as a dis- tinguishing point from B. Maerkeri and B. cucumeris fermentati. Moreover, a trace of alcohol was formed from maltose, saccharosc, lactose, galactose and a-methyl-glucoside. 4. Behavior to temperature and salicylic acid. 0.2101% of salicylic acid in Saké is sufficient to prevent the growth. Grows at 16-32°; optimum, 28-29°C, but no growth at 1o-12°C. VI. . A-némplactic. bacillus. igetormiand size; Short baeilus 202.5 «1.254; 4X 2p, cately 75 oF 1 After 21 days at 26-20° C, there was an increase of fixed acid by 0.042489 and a decrease of 9 ? FAS) volatile acid by 0.018%. 552 T. Takahashi. in long chains of cells. Both ends of the cell have larger refractive power of light than other part. 2. Growth: A white disc in Saké-agar, which under microscope seems granular. Yeast-water, bouillon, koji-extract! are good untriment but Hayduck’s solution or tyros in solution not. Stwd-culture: A white spheric colony was found in Saké gelatine and a pin-head like growth was found at the month of the stab-canal. A good developement was observed in Saké-agar or koji-extract-agar. , 3. Behavior to carbohydrate and Saké. A weak growth was found in Saké containing 16.4% of alcohol and 0.153% of total acid, whereby an increase of acid was observed. The acid forming property from carbo- hydrat will be shown in the following table :— Substance. This bacillus. Arabmose: 222. .see EE... 5 soi se sees == Mylose!cec8 cca ee. 2 heme Sat as Galactose ..o.. 9 eeaee Me ee ce = Glucose ...:.5.5. 55a ee -.. SERRE Fructose... 2..2 see a eee _ I hatinose =... eee ee «~~ enue cao — Maltose cc... (bs ote aoe - «0. ssedeeaae - SACCHATOSE (fe.0 1) eee... - oneness a Wactose o.oo eee. oe ae Se RR ARINOSE.. 2.03 soe cos = I . «0 ans pete as Destine pee Pavaa eae BEER... . os .ceaeeee Se Rl 21 «meee A. SOME... os lcs svasee ve Inolin......2s.0:s seen .. - ee = Mannite?: o...cse S92 4220-5, 3 a ae a-methyl-gluceside, eperettes =... .s.ecsens= zie Thus, this bacillus behaves very similar, to sugars, as B. Wortomani. var Saké ; the difference between them is generally a weaker production of acid and the absence of this property in rcgard to fructose, rhamnose, maltose and galactose in the case of this bacillus. 1 Acidic fluid is better than neutral one. Studies on Diseases of Sake. 553 4. Behavior to temperature and salicylic acid: It grows well at 16-33°C ; optimum 28—20°C, but not 10-12°C. 0.2101% of salicylic acid in Saké is sufficient to prevent the growth. Ill. Acetic acid bacillus group. The bacillus of this group appeared in Hyochi Saké in two types. One of its type was a bacillus, which shows a surface growth on Saké without causing turbidity in Saké. The rather smooth film, with grey white coloration, was very brittle ; so that it sanks down to the bottom of the flask as soon as it reached to its maximum point of growth. Further, it was killed soon by its own product, so unfortunately the pure culture failed. The other type was a bacillus which must be divided into five varieties, all of them belonging to a species of Bacillus kittingianum. I. a.—Variety. t Form atid size: A sh@g bacillus: 2.5 .Su. 3.5. Involution form was not found in Saké even when old. Non-motile. 2. Growth: A grey round creamy colony on the surface of the plate culture of koji-extract gelatine. A grey white disc formed colony on the surface of the plate culture of Saké-agar, whereas in the inner part it appears as a combined three discs growing in three directions. Stad- culture: A smooth light-refracting growth at the mouth of the stab-canal, and a redish-yellow color at the most elevated part of the growth. A ring- like growth along the side of the tube of wort-gelatine. Chiefly a surface light refracting growth on Saké-agar culture, where the central elevation colored purplish-grey, moreover fine streams were found on its surface. Of such culture, after four months, a test with guaiac tincture and H,O, was made but no positive result. Further, such old culture contained large (even 27.5) involution forms of various shape. Chiefly upper part of the stab-canal in Saké-gelatine, where no sign of liquefying gelatine was observable. Surface-culture. A grey’ white light 1 The grey color was most dense among the five varieties, 554 T. Takahashi. refracting creamy coating after 10 days. (at 27-30°C). Flutd culture: A ring-like growth along the side of the test tube with certain turbidity and sediment. (10 days at 28-30°C). ‘This culture medium after one month loss its power of reducing Fehling’s solution. In hoji-extract containg 5% of alcohol, it does not form the film causing a turbidity and acetic acid flavor after three days, but after 4-5 weeks there was formed a grey white smooth film on the surface of the fluid and ¢hzs fiem stained blue by todine KI solution.1 There was no growth in koji-extract which containcd more than 10% of alcohol. A thin film was formed on the surface of a beer. In Saké?, a turbidity after 5 days (at 27-28°C), and after 15 days the fluid changed to the clear fluid with some sediment. 3. Behavior to carbohydrate* and temperature. Substance. After 5 days. Acid production, GINCOSe Ete ee eee Fluid clear, a trace of sediment. = Hrvctose: sich eeciess. ane risen Little turbidity » | as Gala ctoseste co. aseheeoseeeennee 2 ” = IATADINOSE Sector _.. | Ring onside of the tube, fluid clear, | — Saccharse eyo hese ree Fluid clear, little sediment. | = | Miles eee ee Little turbidity, little sediment. | = Nea ClOSE! ere acereioaccnt euceePeeee Fluid clear, » ” | => IR PHMOSE #4 5d one coscece ae eeeeeee Little turbidity. | = IDEXEGINE a waeceece snore teeta 9 ” | = AMUN eee ee ee ee Ring on side of the tube, fluid clear, = SEARED Oia. ac teases eee Thick film, little turbidity. | = Matimit@!s: cae cnacereacmcsesereeeer Little turbidity, little sediment. | 5 Glycerine 3c) cx oteteaae coe eee Fluid clear, ” + | = The optimum temperature lay near 27~-28°C. and grows difficultly above at 31°C. 55-56°C in 15 minutes kills the cell. 1 All other varieties described below gave the same reaction. 2 Alcohol 16.49%, and 14.49%, and these Sake were used to all varieties described below. 3 Carbohydrates were added into bouillon in another five experiments mentioned below. 4 Jn all cases used, glycerin was added instead of saccharose in Hayduck’s solution. gly ‘- Studies on Diseases of Sake. 5 ut tt II. p.—Varicty. The different points from the @.-variety are: 1. Form and size: Short bacillus: 24 in common; 4-5» rarely. 2. Growth: A white round creamy colony on Saké-gelatine plate culture. A yellowish round colony but sometimes a rhizoidal growth was found. Stab-culture. A smooth flat! growth with a yellowish-red colora- tion on beer-agar. A filmy growth ascending along the side of the tube on wort-gelatine. A flat and creamy growth with more or less purple colora- tion, on Saké-agar. A redish greyey white growth on Saké-gelatine. Surface-culture. A creamy but lightcr grey than a.-variety on Saké-agar. Fluid-culture. A very thin film was formed on yeast-water, but a thick film on koji-extract containing 5% of alcohol. 3. Behavior to carbohydrate and temperature. Substance. Growth after 5 days. Acid formation. Glucose...... Ese see cae: Thin film, turbid, sediment. = LD gate oy Fe eee » dense turbidity, sediment. + Galacteser 2b fot Turbid sédiment _ EAT ADINGSCs, or ecee2 o cece Thin film, fluid clear. — WAcCHATOSE= =. $2524.53005: ee turbid, sediment. eae Maltose: “Ei, ke... 2 9 > = Eactose 2... poets = A re = StHMIOSE 20s © 28 2.ch. sae. = fluid clear. _ Dextrine = ...-: Se Aer oe 9 » = Weta Lette ese 2d ce 3 * a STA? Ne a Thick film, a = iSO 2 = a Turbid, sediment. — Givceniners..c-2..--286. Thin film, turbid, sediment. _ 4. In koji-extract forms butyric acid and ethyl-alcohol in addition to a flavor of altered vingar, but not acetic—, formic acid, methyl-lactate or 1 This is a distinguishing point from a.-variety, and the three mentioned varieties be‘ow coinside in this point. 556 T. Takahashi. fusel-oil. (after 3 days at 28°C). It grows at common temperature and optimum lays at 30-32°C. Heating 15 minutes at 55—56°C is not sufficient to kill the cells. yee aricty. This variety may be distinguished from two above varieties by the following properties. I. Form and size: The shortest bacillus among five varieties: 1- 1.5 x 1, but when in the film 5 x 1p. 2. Growth: » i sles: JN!) UNO soca, MOODEOBECE Thin film, turbid. ++ | MA CCHALOSE] 2! sscs cece ee | Ringly growth, turbid, sediment. | teats Maltases och 2 s1eiieissees rf ~ a 1 Se MEACEOSE® ca ieecanasessavacs: re » ” ay Rafiimose .......... eeeeeeee | Lh) filmy turbid = Dextrineseens esse Mem eaeas » » a Artal, Sevens xs cesavsacsesse » » 7 1 The former 4 varieties could not. 560 T. Takahashi. Substance. After 5 days. Acid formation. Starch eee ase 2 |Ploidvelear - Niamn ite hoses way cass seee Thin film, turbid, sediment. - Ber eee NS Fluid tnrbid, : nis Thus the acid forming property has likeness rather to that of B. oxidans. ! Optimum temperature : 30-32°C. Heating 10 minutes to 55-56°C was not sufficient to kill the cells. IV). “ Kahm-hefe’”’ group. The “ Kahm-hefe ’-group found in Hyochi-Saké may be classified into two divisions : 1. Willia anomala (sacch. anomalus.) with hat-like spores and emiting a flavor of acetic ester. 2. Mycoderma group; with no spores but emit a flavor of altered vinegar. THE EXPERIMENT ABOUT THE BEHAVIOR OF KNOWN BACTERIA AGENT SAKE. Since a former observer Otani had reported that the microbs of “ Hyochi” belong to a certain group of well known bacteria, the writer has also infected steritised Saké (12.49§ of alcohol) with several kinds of known bacteria and observed the following result :— Bacteria, After 5 days. After 19 days. area citrina 5..-.-ssses: Ls ioe Sediment. Sediment increased. ‘ alba. ° — = = “a ¥ oe as ss | =S-é oe ire At eee eS aay 3 ee L@ 0% IX _— [ZENE ps gia) a ae | ei e-2)\ 23 0X pry = CEF AG fg Bo a, | Se | Re as 8 FSS SN Ee ae PAIN go SEI KUEN GES =» a IRIs HY — on ) he 7 f y/ VRR. SY ne AS] 4p _ B53 “= "991 , hoe Fe “ss. (ey Tey ; f rl ee Se a a1 be —_— saciid yy an! If lA On the detection of Methyl-lactate, BY T. Takahashi. In a former communication* the writer has described a mode of detec- tion and determination of fusel-oil by means of anisaldehyd and sulphuric acid. The fusel-oil, however, is accompanied in general by certain kinds of esters which fact induced me to apply the same test to the following esters. Compound. | Mery E-ACCtALG ..........2:.---5aem Buthyl-acetate Propyl-acetate Amyl-acetate SSS ar Iethyl-aceto-succinate Ethyl-succinate Propyl-tartrate BMyIEARECALE 222. ..2275: -scsarnede | Methyl-lactate Ethyl-lactate * These Bull. Vol. 6. No. 4. p. 437. Coloration at the surface of contact. Dirty yellow. Yellowish to a dirty crimson. Intensely yellow. Clear purple-red ; below this a yellow stratum. | Opaque blood red. Intenscly yellow. Dirty crimson red. Bluish-green layer, below crimson red. Clear yellow. Intensely bluish-green, blow this a grey-yellow. Milky white; below which a yellow- wish to crimson red stratum. £66 T. Takahashi. Thus the coloration produced by amyl-acctate and methyl-lactate is characteristic, especially that of the latter; hence in order to define the delicacy of the reaction, methyl-lactate in certain dilutions was tested, 3 c.c. of these solutions serving for the test’. % of methyl-lactate. a After 20 minutcs a weak but decidedly bluish layer formed. LOG After /-2 minutes a bluish-green stratum. Methyl-lactate in higher concentration yields on account of the opaque turbidity produced, a less delicate reaction. Further tests were made with whisky” and the distillate of Saké. The result was positive, hence an effort was made to prove the presense of lactate also in another way. A sufficient quantity of { normal sodium- hyroxide solution was added to these liquids and after saponification by heating the mixture for 4 hours, methyl-alcohol was found in the destillate, while in the residue lactic acid was identified after Ueffelmann’s method. It can therefore safely been concluded that anisaldehyd in conjunction with concentrated sulphuric acid can be applied as a test for methyl-lactate, a bluish green® colored stratum being the characteristic reaction. 2 To the solution contained in the test tube 3 to 4 drops of aleoholic solution of anisaldehyd were added and after shaking well, 3 c.c. of strong H,SO, was run carefully along the side of the test tube forming then the lower stratum. ? Sco'ch whisky and Glevivent whisky ; on account of the brown color these liquids were distilled before the test was applied. 3 The cause of this colorreaction is of course the lactic acid, resp. the group CHOH init. Indeed free lactic acid gives also a coloration, but this is purplish, It appears that methyl-lactate is more easily split than ethyl-lactate sinee this latter gives no bluish coloration, 4 The coloration always occurs above the purple-blue layer (fusel oi! layer) so that the observer can distinguish very clearly both substances, On Changes of Availability of Nitrogen in Soils, IT. BY O. Loew and K. Aso. The writers have, in their first communication on this subject? called attention to the existence of bDacteriolytic enzyms which probably play a réle in the soils when the nitrogen of bacteria is rendered available for the roots. It was desirable to observe whether common soil- bacteria produce such enzyms, as the B. pyocyaneus does, the specific enzym of which had been thus far alone be studied to some extent. Sterilised bouillon was inoculated with 8. mycoides, B. megatherium, B. subtilis, B. fluorescens liquefaciens and Proteus vulgaris. The cultures were kept in a thermostate at 20° and were gently shaken almost daily. The development was most rapid with B. fluorescens and after 7 days the bacterial sediment agelutinated to a compact ropy mass while the liquid itself turned very slimy. Only a small residue ‘remained insoluble after several weeks; the slimy character of the liquid did not change further even after 6 weeks. The slowest development was ob- served with B. subtilis, but after 3 weeks a considerable mass of floceuli had developed, which two weeks later were dissolved again, leaving only an insignificant sediment, showing that also this microb produces a bacteriolytic enzym. The solution did not become so slimy as with I’'Iuorescens, but somewhat less slimy. B. mycoides and B. megatherium formed voluminous floceulent masses. which even after 7 weeks did not decrease in volum; hence hacteriolytic enzyms are not produced by these microbs when cultivated in bowllon. *Bul. College of Agriculture, Tokyo, vol. VIT, No. 3. 568 0. LOEW AND XK. ASO. Proteus vulgaris produced no flocculi but an intense turbidity which decreased gradually after five weeks but had not entirely disappeared after six weeks. The microscopical examination revealed among amorphous particles also bacterial forms in all these cases and the inoculation into sterile bouillon proved that there were still living microbs also in the slimy sediment of Fluorescens and Subtilis. Perhaps they had been protected by the slime from the attack of the enzym which gradually increased in concentration in the measure as the dissolution of the microbs had proceeded. The dissolving process of bacteria is but the first step which will lead to a production of amidocompounds either by the same enzyms or by other proteolytic enzyms secreted by microbs. If, furthermore, the conditions for the growth of the microbs of putrefaction (Proteus and others) are favorable, a further decomposition with production of ammonia can result. When bacteria die off in the soil and are gradually dissolved with a further splitting of their proteins, the roots will no doubt reap a certain benefit from this process but a part of the available nitrogen produced will again be eagerly absorbed by new microbs. Thus probably the bacterial flora changes back and fro with the conditions changing from favorable to unfavorable and back. Moldfungi and certain microbs will also by oxidation destroy the products of dissolved bacteria, but it appears that a certain fraction of the nucleoproteidmelecule offers considerable resistance (perhaps after assuming an acid character by partial oxidation?) and remains unchanged for a long time associated with the humus? in the soil. We have further endeavored to obtain a sufficiently large quantity of bacteriolytie enzym for some special experiments. For this purpose served B. mycoides and B. fluoresceus liquefacieus. Our hope that B. mycoides, found unable to produce bacteriolytie enzym in bouillon, might form such an enzym in certain other solutions, was realised. ? Of. the communication of S. Suzuki on humusformation, in these Bulletins VII Neo, 3 and 4, and VIII, No 1, ™ ON CHANGES OF AVAILABILITY OF NITROGEN IN SOILS, IL. 569 Three liters of the following culture solution were distributed in six flasks, each of one liter capacity, sterilised as usual and after infection kept in a thermostate at 24°. The solution first infected contained: Glycerol .. 5 % NaNO, . Oa; LPO, 02s MgSO, . ii : 0.02 ,, NaCl Os... HeSO? 4 2s) ee es | Ra ae rae: Since after one week no growth was observed, 5g. pepton and 0.5g. Na.CO; were added to each flash and after sterilisation again infected. Gradully films developed which were distributed through the liquid daily by shaking. After six weeks two of the flasks developed not further any new film and the hquid assumed a lghtbrown color, while the sedi- ment was reduced to a minimum. It was evident that the great masses of bacteria that had grown were mostly dissolved again.? The liquid had not assumed any slimy consistency, showed still a weak alkaline reaction and had a weak not unpleasant odor The larger part of the two flasks in which further growth had stopped was now evaporated in vacuo to one fourth of its original volume and to 100cc., placed in a sterilised conical flask, added one gramm of soil from the depth of about one foot of a field that had received organic manures about 4 months previously. Another similar quantity of the concentrated bacterial culture quid was boiled for a few minutes and after cooling also mixed with one gramm ot soil from the same spot. After one week a very striking difference was noticed: the not boiled liquid was clear, neither film nor turbidity had developed and an inoculation in bouillon yielded after 5-6 days only some growth of Bac. subtilis, perhaps originating from spores that resisted the dissolving action of the enzgym—while the portion that had been *The sediment contained amorphous and crystalline particles, baterial remnants and but very rarely also barterial rods. 570 0. LOEW AND K. ASO. boiled, hence where the enzym was destroyed, developed a luxuriant film of microbs and the liquid became very turbid. After further four days a development of gas commenced, while even after four weeks there was no change noticed with the not boiled liquid’. This result leaves no doubt that also soil bacteria can produce a bacteriolytic enzym which gradually renders new bacterial growth difticult. Since this may happen also in the soil, some explanation can be furnished for the fact that bacteria] life does not increase infinitely in organically manured soils. Under certain conditions the nitrogen of the microbs absorbing free nitrogen” seems to become rapidly available for the crops, as the cultivation of rye on an experimental field near Halle showed. YT. Kiihn observed on that field no decrease of- harvest for twenty years during which no nitrogenous manure had been applied. It was caleulated that 25—30 kilo nitrogen per ha was annually gained by the azomicrobs. Similar obser- vations were made at the Experiment-Station of Rothamsted (by Hall) and recently also at Cracow (by S. and H. Krzemieniewski). How important would this be for the cntire agriculture if the necessary con- ditions could be produced in every soil! Beijerinck® believes that “the protoplasm of Azotobacter is easily changed to ammonia.” But this change can hardly be due to any thing else but to the action of an enzym, which perhaps also causes the involu- tion of the Azotobacter cells. An interesting experiment was recently carried out by Heinze.’ Mustard was successfully grown with a mass of Azotobacter as the only nitrogenous manure. This author recommends to work and cultivate the soil very well in order to render the conditions very favorable for *A portion of the not boiled liquid was exposed to air for some time, but no microbs, only a small red Torula, developed. *It might be suggested to call the group of “microbs assimilating free nitrogen” simply “azomicrebs” for the sake of abbreviation. This name does cf course not imply any botanical connection. * Centr. Bl. Bakt. Il. Abt. 9, p. 43. *“Landw. Jahrb. 1907 p. 910, ON CHANGES OF AVAILABILITY OF NITROGEN IN SOILS, II. 571 an abundant growth of algae which can furnish carbohydrates as carbonaceous food for Azotobacter. But it appears that not in every soil the conditions are favorable for a very abundant growth of Azotobacter. Gerlach and Vogel® report at jeast that a special inoculation showed no influence. Since Azotobacter loses gradually its power of assimilating free nitrogen, when cultivated in media containing assimilable nitrogen com- pounds it is not surprising that it shows on fallow fields more power of assimilating free nitrogen than on richly manured fields. Thus it may be explained why Azotobacter isolated from certain fields yields not very satisfactory results. A pure culture of Azotobacter derived from an experimental field near Cracow assimilated in three weeks only 1.33 milligrams free nitrogen,? in a solution of 4g. mannitol in 200cc. water. In 200ce. of a 1.2% glycose solution was assimilated only 3.2—4.9 milligrams nitrogen. Crude cultures assimilated more nitrogen than pure cultures, but in the former case always the bulyric bacillus (Clostridium pastorianum) was present, to judge from the odor emitted. Our own observations agree very well with those made in Cracow. The microb developing butyrie acid can however be avoided by using sodium malate as organic nutrient for the crude culture. Our solution eontamed: Sodium malate Me OS A we ee ES ete Osy . eee se a eae MeESOi ':.) ee 2s ae Oe CaCl, ‘ee Oe ee Be 2)! 5 ee a Ss ee aie After adding 10g of earth from 30cm. depth of a fallow field, a rich development was gradually taking place of Azotobacter but later on also a very fine mycelium developed. For pure culture of Azotobacter, however, sodium malate seems not very favorable. We have tried a variety of 8Centr. Bl. Bakt. II. Abt. 9, p. 887, Cf. their publications ibid. 8, p, 669 and 10, p. 636. ®°S. and H. Krzemieniewski, Bull. de Vetcad. Sc. Cracow, July, 1906. 572 ©. LOEW AND K. ASO. solutions but will mention only, that no growth at all was obtaimed when potassium oxalate (0.5%) was used as organic substance. This salt had the peculiar effect of dissolving some humus!’ from the earth, probably by mutual decomposition with calcium humate. The dark brown solution thus formed contaming potassium humate did not favor the growth of Azotobacter." In regard to Azotobacter it is a most remarkable fact that it re- quires lime, as Gerlach and Vogal had observed. We have made several observations confirming this observation. A mannitol culture solution as well as a sodium malate culture solution without nitrogenous compound were prepared, with and without lime, but growth was only observed where a few drops of a diluted solution of calcium chlorid had been added. This is a remarkable fact since it is an exceptional case with lower fungt and algae; should there exist a relation between this fact and the power of assimilating free nitrogen? Christensen’* proposes even to test with Azotobacter the presence of CaCO; in soils. He observed that that the occurence in different soils stands in close relation to the presence of calcium carbonate and to the basicity of soils. Whether Azotobacter prepares also a bacteriolytic enzym is not yet decided, but as to the nodule-forming B. radicicola it is quite evident that this does so, especially when cultured in pea leaves extract. After a maximal development a gradual decrease sets in and finally, after 6 weeks at 24° C the liquid is perfectly clear and only a small sediment at the bottom. It is well known that the microbs in the nodules of leguminous plants dissolve gradually; this is due evidently to their own bacteriolytic enzym, after this attains a certain concentration in the surrounding fluid. Views on the chemistry of assimilation of free mtrogen. The question as to the first product of transformation of the free nitrogen “The fallow field contained still 7% humus. 1 Since Heinze (l.c., page 909) mentions that humus can be utilised as food by azotobacter it may be that in the above case the exalate acted as a poison. ™ Centr. BI. f. Bakt. If Abt. 17 (1906). ON CHANGES OF AVAILABILITY OF NITROGEN IN SOILS, II. 573 in the living cells of the specific microbs is doubtless of profound interest. Altho there exists a great difference in the opinions and no safe ground has thus far been reached, so much is sure that the chemical energy of the living matter of those cells is absolutely necessary for the process. But since chemical energy is present in every living cell, there must still, in addition, exist a special condition in those microbs whereby the gaseous nitrogen is forced to yield a soluble compound. Winoegradski, Reinke and Stoklasa’® assume transformation of free nitrogen into ammonia by nascent hydrogen, which would be at once utilized in the process of protein-or asparagin formation. But the supposed development of hydrogen cannot be noticed in pure cultures of Azotohacter', altho it is observed with those of Clostridium. Gautier and Dronin hold that the free nitrogen is oxidised by the microbs and the nitrous acid or nitric acid thus formed is rapidly changed to other nitrogenous compounds. It may be mentioned that nitrate reaction is sometimes—by no means always—obtained with the nodules of Leguminosee but this may be due to absorption from the soil when nitrification had been going on. For the oxidation of nitrogen in the laboratory, however, a very high temperature or electric sparks are by no means absolutely necessary, for Tlosway has observed the formation of nitric oxid, when a mixture of nitrogen and oxygen was passed over platinum black at 180C°. Gerlech and Vogel!® assume that the free nitrogen combines directly with carbon-compound in the living cells, pointing out the absorption of free nitrogen by calcium carbid as an analogy. But the example seems to us not well suited, as the absorption in this case is more due to the calcium than to the carbon. Heinze’ entertains the 2 Stoklasa mentions the occurrence of small amounts of asparagin in the nodules of leguminous plants at a certain stage of development. “S. and H. Krzemieniewski, Bull. Acad. Sc. Cracow, July 1906. 4% Centr. BI. Bakt. 9, p. 817 and 881. %Landw. Jahrb. 1906, p. 907. This author also holds that the formation of carbamic acid may result from the assimilation of nitrogen, but this view seems to be identic with that of the formation of ammonia. 574 0. LOEW AND K. ASO. same view as those authors and mentions in support of it the formation of hydrocarbons of the acetylene series he observed in the crude but not the pure cultures of Azotobacter. Such a process, e.g., might be expressed by the following equations: t} + = See 615 ae) Acetylene Freenitrogen Hydrocyanic acid?’ ONH + 2H,O = HCOONH, Ammonium formate. Hydrocyanie acid can of course not be assumed to exist longer than for a moment in the cells on account of its poisonous action. Our own view holds as most probable the formation of ammonium nitrite, as represented by the following equation: N,+ 2H,.0 = NOLIN, which formation is rapidly followed by the reduction of the nitrous acid to ammonia. Indeed nitrous acid is found sometimes in the nodules of Leguminons plants, while we did not succeed yet to show the presence of ammonia in them. However it may be objected that traces of nitrous acid are formed by oxidation process. This view can be supported by the possibility of realising this process at the ordinary temperature by the action of very energetic platinum black upon free nitrogen in the presence of some alkali’. As diffrent as these four views are, in one point they agree, the formation of ammonia before the proteinsynthesis commences. 7 Berthelot has observed the formation of hydrocyanic acid from acetylen and nitrogen under influence of the induction current. 8 This view was discussed by one of us (LL) in the Ber. D. Chem. Ges. 23, p. 1443 (1890) where this action of platinum black was described, which was confirmed by L. Woehler, ibid. 36, p. 3879. The nodules of leguminous plants show often traces of ammonia. On Observation of the Continuous Growth of Pea on the Same Soil. BY Shigehiro Suzuki. Many observations have been made on the continuous cultivation of clover and pea on the same soil and in this case very often a surprising decrease of yield was obversed from year to year. This phenomenon called “TKleemiidigkeit”” and ‘“Erbsenmiidigkeit” have been very differently explained. K. K. Gedroiz' who has made extensive investigation with clover in this direction has especially determined the phosphoric acid content of clover in relation to its development and in comparing the requirement of clover for easily soluble phosphoric acid with that of other plants, he reached the conclusion that at least in certain cases it is the decrease of easily soluble phosphoric acid which causes the above phenomenon, but he admits that there may exist cases in which the phenomenon can be produced by the exhaustion of the soil in regard to potassa. There exist, however, no doubt also cases in which the pheno- menon is caused by nematodes? and since they increase immensely with each year of culture of pea or clover on the same soil it becomes intelligible why even full and rich manure may often not prevent the extension of the calamity®. Since that phenomenon with pea has been very often observed in Japan, the writer has also undertaken to study that pheno- menon. For four years peas have been grown in the same soil pots holding 8 kilo of humy loam soil which had not been manured for six years. The manuring and sowing was done between October 22 and December 3. In each pot were grown in the first year 7, in the second 5, in the third 3 and in the fourth 4 pea plants. The result was as foliows :-— ?Russisches Journal fiir Exp. Landwirtschaft, 1907, Heft. 1, p. 61. *Salfeld: Die Boden-Impfung, 1896, p. 99. *The writer has observed here in Tokyo similar phenomenon with the egg-plant and the examination made it evident that an increase of nematodes was the cause. 576 a4 SHIGEHIRO SUZUKI. t ty Weight of harvest, No. of pots Epo vi per pot in g. General manure, and a. = = Rema per pot in g. treatment of the |5 22) moray : soil. 9 ale eee EIctite! Seeds weight | uF 7 a eae S & | Double superphos....20 |All four pots con-| c.m. HT a Sl Compost .2.-.ceseeeek es 50 tained a frech soil. 120) 135.0) 72:41) 00:2 ae F = 60 average O ee 4 pots. = I (Fresh soil.) 131 | 321.5) 167.2 ah ees.) 137 | 325.9 169.44 — Average.) 134 | 323.7] 168.3 — Second years | — oe aay ) 13504) ,gSresisSal te VK v3 )) 147-5] 373-5) 192.5) — =o AYA KG a ) 147-5) 343.5 181.5, —| weighed 2 3 | Potassium sulfate....8.5 Average.| 143.3} 366.2) 187.5} in the 7 | Double superphos. ...18) S q spageeded —— f fresh = i Ammo. sulfate......... 8} Sor Dee | state. 2 2 | Sodium nitrate 6 ees zed ae ee ee yy) by steam at 1007) 135 37 | 180.0 — ra @ itor 4 hours) =) Se on consecutive 3 days. Second years VII4 soil, well wash-| 145 | 370.9, 176.9 —|J ft ed with water.) Qh o_—=— I (Fresh soil.) =| 7RO Bev 26.6/) II ( ue) —| 80.9 32.8] 30.1 Average.| Tex) = Shiouly ket! ae~ Second years \| 5 3 HII ( al ) — 94.0 AV) Spier! iy Same as the 2nd year. } on ee —| 96.9 39.2) 35-8)\in the air- an } i a at 95-5! 37-0; _33-4|fdry state. a jy V Third years eZ soil. ) =| 1074; 2.4) 39.0 WE > ) —, 103.7] 40.7} 37-0 Vil ¢ 9 )} ——— DRA) 742t4\= S016 f Average.| 107.5] 42. | 38-5)| iFresh — soil, | | ({ manured. ) =| 57) ee 30.7 | II ( > ) —| 63.2} 34.5] 32.0 Manured with Average. | 60.5} 34.2) 31.4 Potassium sulfate ...8.5 | Third years | =a Superphosph......... 45.04 III ( An experiment with spinach in sandculture had bee CaO MgO that plant which was found =1. In that case not such an enormous made a year ago by Namikawa to determine the lime factor for depression was noticed with the increase of carbonate of lime, probably on account of ammonium nitrate having served in this case as source of nitrogen. Our result above mentioned may therefore be connected 6 Of, Maki and Tanaka. ‘These Bul; Vol, VII. No. 1, p. Gl. 594 T. TAKEUCHI. partly with the reaction of the manure. In our case nitrate of soda served as source of nitrogen, a physiologically alkaline compound whose alkalinity was corrected in pots C. and D. by gypsum, but not by carbonate of lime in pots E, F and G. This circumstance would perhaps not have influenced so enormously the result, if the root hairs would secret a certain degree of acidity; but in regard to spinach it seems very probable that the root is of too weak acidity and in this case its absorptive power would be very much depressed by alkalinity of the manure. The writer has consulted various books on cultivation of special crops but nowhere he was able to find any information on injury to spinach by liming the soil. ence in order to collect further information on this point a new series of experiments with the same soil were made, con- taining in the general manure the same amounts of potassium sulphate and secondary calcium phosphate (Ca HPO,+2aq.) as in the first case, but in place of the 12 g. sodium nitrate the equivalent amount of am- 3 monium sulphate (=9.5 g.). The further additions were: A, Original soil. B, 0.2¢ Magnesia alba. es = 3 » + 0.8% Gypsum. De.- <3 - » + 0.5% Carbonate of lime (equiv.) In a third series the nitrogen was apphed in the form of ammonium nitrate in equivalent amounts (==5.63 ¢.), otherwise all conditions were the same as in the second series. 15 Spinach seeds were sown per pot April 16, and the young plants reduced to § of equal size on May 4. During the month of May con- siderable differences in development were noticed, caleimm carbonate depressing the development also in both these series, altho not so much as in the first case. The plants were cut?® June 12 and weighed in the fresh state with the following results: * The plants of these two series were cut at a younger stage than those of the first; besides the former developed flowers, the latter had not. GYPSUM AS A MANURE: 5095 Second Series. ee | Average height, cm. Total weight, g. Ratio. A, 21 47.5 87.1 B, | 19 40.0 73-4 oa 24 54.5 100.0 IDF, Ii 12.0 20.1 Third Series. Average height, cm. Total weight, g. Ratio. A, 19 34:5 91.3 Be 16 32.0 84.7 C; 18 37.8 100.0 iD) 6 4.5 12.0 The beneficial action of gypsum became here again very marked. It might be supposed that the manure of weak alkaline character de- pressed the availability of phosphoric acid. But it has been found by various authors that the availability of the phosphoric acid of dicaleium phosphate is not depressed by liming the soil, further Séderbaum™ has also compared the availability of various phosphates when the nitrogen was offered as sodium nitrate as well as a mixture of sodium nitrate with ammonium sulphate, and found the depression only between 5 and 8¢. Hence the alkalinity in itself must have acted injuriously on the activity of the living cells of the root. The need of spinach for lime seems not very great as may be followed from the following analytical data taken from Wolff’s Tables of plant- ashes Vol. I. p. 101. The total ash amounts in average to 16¢ (of the dry matter). “ Tandw. Vers.-Stat., Bd. 63, p. 252. 596 PAKEUCHI. In 100 parts of ash were found: K.0... ...23-.4— 9.69; MgO... ...7.4— 5.2; SO... | ---4-4—-0:3 Na,O — ...31.4—39.1; Fe,O, ...2.1— 4.6; SiO, ---3-I—5.8; CaO... ...10,6—13'5 PO, +. 9-5— 11.93 Cle sO — 77s An experiment with oats was next started Jan. 28 and with the same manure of an alkaline nature as above mentioned with the first spinach experiment. 15 seeds were sown and thinned to 8 per pot of about equal size, Feb. 24. The plants developed at first at about equal rate, but gradually increasing differences were noticed and the condition at the end of May was as follows: | Average ht. Number < et oa cm. of shoots. A. Original soil - Saeeee Developing flowers 93 3 5. Magnesia alba o. CNB |e No ears yet visible SI 3 e es +0.5%¢CaSO, ... Shows ears 86 3 D. +2%CaSO, ... | Ears just developing gI 3 E = +c.5 CaCO, ... About like B. 94 2 iz = +1%CaCO, ... | Yellowish and no ears 76 2 G 2 +22¢CaCO Yellowish and no ears 75 2 The plants were cut after the ears had completely developed and weighed in a fresh state, June 10; the results obtained were as follows: eee ed | Average ht., Total fresh | Wt. of root, 4 cm. weight, g. | air dry, g. A. Original soil... =... - sn. ape 105 110.0 9.6 B. Magnesia alba 0.293 ... 1... oss 104.5 9.1 C : » 9 0.5% CaSO, 114.0 8.5 D , » 9» t2 » 109 132.0 10.2 E > » » £055,, Ca COle IOI.0 7.8 ¥ 5s ees ey ss 80.5 4.5 2 eee 77.0 4.3 mr GYPSUM AS A MANURE. 597 This result shows again that gypsum may be applied with success in soils with a relative excess of magnesia, especially when the total manure led to an alkaline reaction in the soil. Summary. Gypsum is a very valuable addition to manure when sodium nitrate had been applied, or more generally when there is any alkaline reaction produced in the soil. If, however, an acidic manure, as superphosphate and ammonium phosphate, had been applied, gypsum has rather a depressing effect. Gypsum has also a favorable effect in overcoming the injurious action that @ certain relative excess of magnesia can exert on the plants. Spinach is injured considerably by carbonate of lime, but not by sulphate; provided the reaction of soil and manure is not acid. On the Depression of Growth by Large Doses of Lime. BY C. Kanomata. Depressions in harvest by liming soils have been observed to take place occasionally on poor sandy soils, further when bone dust or phosphatic rock had been used as phosphatic manure. On the other hand it has been observed by various authors, that the availability of super- phosphate and of dicalcium phosphate was not depressed by liming the soil. This would again confirm that carbonate of lime does not transform dicaleium phosphate into tricaleium phosphate. For a series of years, howeyer, it has been shown by experiments at this college that a depression of the harvest by liming can be caused when there is relatively but very little magnesia in the soil. Thus an unfavorable ratio of lime to magnesia may be caussed by excessive liming. In such a ease the remedy consists in the application of magnesium salts in order to change the unfavorable ratio again to the favorable one! “A case was recently reported by Kraus, in which all the plants, growing on the so ealled “Wellenkalk” soil in the Wiirzburg district of Bavaria are characterised by attaining only a very low growth, viz. / "/5—*/19 of the normal size, which phenomenon was attributed by that author to the deficiency of water caused by the looseness of the coarse soil. But this view was not supported by experiment and the real cause might be a deficiency of magnesia. In order to observe the degree of depres- sion caused by a great excess of lime over magnesia,” the writer observed *Compare these communication of Maki and Tanaka in these Bulletins Vol. VII. No. 1. *These experiments at this college have thus for only considered a moderate excess of lime over magnesia, 600 CG. KANOMATA. the development of seyeral plant species in sand culture, at a ratio CaO 0 / : : : TCs tas 1007, (Series A) which was compared with a check culture * 2 . = CaO — 1 . * a 3 containing the ratio “eo /, (Series B)?. Each pot held 2.5 kilo of quartz sand and received the following manures, &.: K SQOp*.c2 = eee) Lael te ope aD NE.NO: (cae ee ee OS Nas POD 12teuameeee.. |. ERT Oe: SIPIOES HeSO, 3 AG, Se sw eee Se eel Macnesitte 2.) Sn. ws ee ee ee The main pots A further contained 56 germ. of CaCO, (—28 germ. CaO), while the control pots B only 0.56 grm of CaCO, (0.28 grm CaO)*. For this experiment served barley, oats, rice, buckwheat, mustard and onion (small variety). 10 seeds of barely were sown Oct. 30th, 1906, and the young plants reduced to three of equal size, when they had reached 10—12 cm. Since there existed danger from attack by fungi, the plants were eut, Jan. 29th, 1907, and weighed in the fresh state with the following result : *Lime and magnesia were supplied as natural carbonates in the form of the finest powder (< 0.25 mm.) *These data would therefore correspond for 100 parts of sand: K.0 0.0202 P.O; 0,0039 N 0,0128 FeO 0.0012 MgO 0.0112 Gao J Series A 1,1200 | Series B 0,0112 ON THE DEPRESSION OF GROWTH BY LARGE DOSES OF LIME. 601 Se CaO Average height Biiniber of stalks Weight of shoots MgO in cm. i in grm. 100 f A, 31.3 30 27.5 I | A, 31.1 28 26.4 “¥ B, 34-2 43 38.9 I B, 33.5 35 37-2 This result shows that altho the plants were still very young, a considerable depression had already been caused by the excess of calcium carbonate. This depression would have increased later on, to judge from former similar experiments. The other plants above mentioned were sown in March and April; after five weeks a great difference in height was noticed with buckwheat and mustard. The results are seen from the following table: : CaO 100 CaO I pp Lime factor. “ae a MaOrr op Average Fresh Average Fresh height, cm. | weight, grm. | height, cm. | weight, grm. Buck wheat, 3 plants ... 10.1 3 23.2 17 Mustard, 2 plants... ... 4 2 13 8 In regard to the rice plants there was a yellow coloration of the leaves noticed in the series A, perhaps a case of so called lime-chlorosis but this phenomenon was not observed with oats under the same condition ; all oat plants showed a normal deep green. The oat plants were har- vested shortly before showing the ears; the result observed were as follows: Number of plants per pot = 3 Number of shoots developed as in A | ee in 5 Fresh weight of plants per po == 201 pera ® | oo Qo oO eH oe ds he =} ee) 602 CO. KRANOMATA. The shoots were measured and gave the following figures, em, A B = 136 = 93 = go oI 70 89 57 62 54 60 53 51 46 48 23 Total sum. 401 622 By the excessive dose of carbonate of lime, the weight was therefore reduced by 39% and the total length of shoots reduced by 354. It might be here pointed out that this depression would be simply due to a decrease of the availability of the phosphatic manure. But such a view can not be correct, because in our sand culture a soluble phosphate (viz. Na,HPO,) has been applied, and this salt, even after transforma- tion into CallPO, would not suffer any loss in the degree of availability. Furthermore it must be considered that high diluted secondary sodium phosphate acts at common temperature only gradually on calcium carbonate with the production of dicaleium phosphate, altho at boiling heat this decomposition is rapid. The rice plants were harvested June 3 with the following result: D ON TILE DEPRESSION OF GROWTH BY LARGE DOSES OF LIME. 603 Series A. Series B. BopMEEEC ss. sions) |) 520 utes OZ eich trol antS> CI. nsesu ieee es OMeeEESnCnS) sau s 06 Greene OD DOMME Mises. «4.5 | ss%). vase UeecOR Colorrok vplants: ....- ... ss. yelowism ... “0 Vc. si. .edarks green: ‘Number OlTSHOOts:.. ..._ ese s- ca. ess) tae) @ oe O BReSHeWelehre Gis. 205, ssseKAnCMMME as. ss) cuar any eer ZOOS On the onion plants. the following osbervations were made, June 17. Series A. Series Bb. Number of living shoots .. .. 6 12 Average height of shoots, em... 7.3 12.3 Various authors might here object that what causes the depression in all these cases there is merely the undue excess of a carbonate and not an unfavourable ratio of lime to magnesia. But that such an opinion is erroneous follows from many experiments made thus far at this college. A further proof that the depression by the large dose of CaCOs 1s not due to this large amount in itself, is furnished by the fact that an addition of so much pulverised magnesite that the ratio oS —— a too te produced, has again a favorable effect, as an experiment with buckwheat had demonstrated. The manure for each pot of 2.5 kilo sand was the same as above mentioned, and both the pots received 56 g. CaCO,; but one of the pots only 0,6 g. magnesite while the other 60 g. The result was that in the latter case the buckwheat plants showed after 3 weeks with the ratio 1°°/,9) the same height (20 em.) as the buckwheat plants had shown at 1/, while at 1°°/, the plants had reached only 8.5 cm. Hence the ratio 1°°/,9) was far superior to the ratio 1°°/,;. This experiment further shows that buckwheat is not injured by an excess of carbonate of lime in itself, which agrees very well with a former observation *In a former experiment with rice only one shoot was produced when the lime amount was increased to thirty times of that of magnesia and the ripening process was much retarded, phenomena also observed by Mr. Kumakiri (these Bul. VU, No. 1.) at an excess of magnesia. 604 C. KANOMATA. of T. Furuta,® who obtained, by adding to 8 kilo soil 42.6 g. of quick lime corresponding to 76.08 g calcium carbonate, a great increase of harvest, namely an increase from 190 g. in the check pot to 382 g. In a second series, the former pots yielded 240 g. while the check pots only 281 g. harvest of buckwheat. In a recent experiment described in the ‘Journal fiir Landwirthschaft (55, p. 82), a similar dose of calcium carbonate caused a depression of the yield. But in this case no deter- mination of the magnesia content of the soil was made; perhaps this aniount was relatively very small and in this case the liming would naturally produce a still more unfavorable ratio of lime to magnesia and hence the depression. The most favorable result with buckwheat in Furuta’s case was observed with the ratio ne =*/,, while the check pots showed the ratio */;. Experiment wilh soil culture. In another experiment with oats an exhausted loamy soil, contaming 0.6% CaO and 0.5¢ MgO, soluble in 10¢ HCl, received the following manure per pot, g. (NH) 250, 6 NO2NS 22 ./e eee. sa gp Uevenee cieee E Double superphosphaig . .. .. wm «- =a, © KSOe 5 he, + oe ea een ce 4 Pots, each holding 10 kilo soil served for the test. 2 pots A receieved each 786 g. CaCOs, corresponding to 440 g. CaQ, which in addition the available CaO already present in 10 kilo soil would make the total amount of CaO=500 g=10 times that of magnesia, while 2 pots B received only 10 g. CaCO, more than sufficient for correcting any acidity of the humus present. 15 Seeds were sown in each pot, and later on the young plants were reduced to 10 per pot, all of nearly equal size. After nearly 4 months a very great difference in development was noticeable, see Plate XIV on ®*These Bulletins, Vol. IV. No. 5. ON THE DEPRESSION OF GROWTH BY LARGE DOSES OF LIME. 605 which the photograph was reproduced. Shortly after developing the ears, May 30, the plants were ent and weighed with the following result (average of 2 pots), g. Feuer et : : Greatest Number Fresh weight Dry weight EE height, cm. of shoots. of stalks. of root. CaOiers oe 126.5 39 290.5 Ey) CaO _ I0 7MEOo IOI.0 28 140.7 8.2 Hence it will be noticed that the increase of the lime content in the soil’ from 0.64 to 5.04 caused a depression of harvest by nearly 48%. Also here the objection that the availability of phosphoric acid was depressed, would not be permissible. Tt was further very interesting to observe the great difference of the development of the roots. The roots were very carefully collected from each pot and well washed in order to remove every visible particles of soil, and well dried. Another experiment in a similar line and with the same soil had been made at the same time by Mr. H. Yokoyama of this college and from his results the following data are taken: Manure for 10 kilo soil, g: Wouble superphosphatewpe: .. °.. -« 9 i 18 K.SO, he as... a nriereme ec! CRUE Op. 2.6. ti gas oc oh) Se, s, ole, eee 15 Seeds of oat sown, reduced Jater on to 10. Harvested just at the a) beginning of showing ears. 7 Also in this case there was no trace of lime chlorosis noticed with oats. SIt deserves to be mentioned that the roots from the pot with the excess of lime showed a more intense order of fresh malt than those from the other pot. 606 CG. KANOMATA. Fresh weight from two pots, g. Lime ratio. sc ; CaO 1-2 ginal soil J.<. <:+, s:2 7 jeceneeens eS 3 Original soi MgO 771 153.0 Original soil+6g. CaCO, for 10 kilo... 153.2 Original soil--152g. CaCO, for 10 kilo. C207 4s , > 3 MgO == Pay 73-0 Finally the result recently obtained by Mr. H. Hamasaki at this College with oats under the influence of different ratios of lime to magnesia may be mentioned’. Each pot of 2.5 kilo quartz sand received as general manuring, g K50;,:..0 EGO Femeeeee =~ ee re ee ee Finest Powdered limestone... .. .. .. .. 43 Two pots A. received so much magnesite (5 g.), as a finest powder, that the amounts of lime and magnesia were equal. Two pots B recetved=s. 9. .. Oll.g. —-\ a D Sap... 016-2 -1 Paatitiers maenesium a ee Gee —. .. 09, . ” ” ” carbonate. Ppa oe bes. = aaa 5 ay, Ae 5s Ver | ok Deere 5 i. ee - *The composition, however, is subjected io little differences according to the conditions of the precipitation. While there exists a great difference as to_ the availability of natural and artificial magnesium carbonate, this is not the case in regard to the availability of natural and artificial calcium carbonate (provided the limestone is applied in an exceedingly fine powder), as experiments with oats in sand culture at this college have shown. There exists also not such a chemical difference here, as is the case with the natural and artificial magnesium carbonate. ON THE AGRONOMICAL EQUIVALENT OF ARTIFICIAL. 611 Six seeds of barley, previously soaked in water, were sown Noy. 15 in each pot. The young plants showed gradually a very great difference in development. The average height was on Dee. 20 as follows, em: ? mn = 13.8 ‘Ds 8:6 Be = 9.5 KY S82 Cra 195 P60 G*== 4.6 Since there existed danger from attack by fungi the plants were harvest long before flowering on Jan. 29th and weighed in the fresh state. The observative were as follows. | | Average height, Number of Average fresh | cm. stalks per pot. weight of a plant. A. magnesite 5g | | oe a \ average=7.01 0.18 25.8 24 t 2 (B O.1g | 29 2 J ” 8.95 Nae if | 29.9 33 \ r C -0.3¢ 7} | 28.1 36 ” 8.73 aes 28.5 31 1 . D.0.6g ( 26.3 24 ii ” 6.03 sai { magnesum4 Fo oe 25.6 23 \ . carbonate. 2.98 ( 24.6 18 » 4.05 14 7 F. 15g { Se A } 5.) G60 10.5 - 6 7 G.2.0¢ { 11.4 8 \ ” 0.33 A second experiment under the same conditions was made with oats. Six seeds were sown March 4 and the young plants reduced to 3 per pot later on. The differences in growth and the gradually increasing damage caused by the moderate increase of the doses of magnesia alba became also here soon very marked, and will be recognised from the photagraph of some of the pots reproduced on Plate XY. The plants were cut at the flowering time of the Check pot, and weighed in the fresh state with the following result, g.: 612 S. KANAMORI. Os1 CM... vce, ew LN Magnesia alba. {0.9 ,, mom NM NO Oo by Gd Go G2 G2 Gi NESS Et Gur CO: NV O0-ita ONO) OlGIrO MO) CDG &O) OMNI O OUE Lan! wn 2? 210) 3, oer Tt will be seen from this result, that doses of 0.1—0.6 g. magnesia ~ alba were agronomically equivalent to 5g magnesite; a further increase of magnesia alba led to a depression of harvest. The question will be further studied also in relation to seed-production. Bul. College of Agric, Vol. VII, No. 5. Plate XV. On the Agronomical Equivalent of Magnesia Alba. J. Magnesite 5 g.; II. Magnesia alba 0.1; III. Magnesia alba 0.3; IV. Magnesia alba 0.9 ; V.. Magnesia alba 1.5. aA g* Pi a a aS) Rey? ie po er elnamet re ne rhb j r dy i. Speen a © abe +) abbots Ae * fe} ao Kan : ee we 5 aes, (rl pe «Ne, 23: bk ni ae i # a3 = ’ sivatiy’: zi Topdressing with Magnesium Sulphate. BY J. N. Sirker. (Caleutta). Since a high lime content of the soil may cause some depression of the yield in barley and in similar crops it was desirable to be tested by a field-experiment whether magnesium sulphate in small doses would act favourably in overcoming the effects of a relative excess of lime. The experiment of the writer was carried out on the loamy soil of the eollege-farm which contains fine earth 70¢, CaO 0.6¢; and MgO 0.5¢ easily soluble in HCl of 10%: 2 qe) ne) te Eee Potassium sulphatemeeyeenw0)!:- ..- =<. .2 69g. Four pots A received 10 g. erystallized disodium phosphate, while four pots B 5 g. double superhosphate, the equivalent in P.O . Two pots A and two pots B received 20 seeds of oat each, further two pots A and two pots B received each 20 seeds of onion; Nov. 22nd. Later on the young oat plants were thinned to ten per pot, all of nearly equal size, while the young onion plants were reduced to five. After 4 months a considerable difference was observed with the oat plants, the superphosphate plants showing a greater height and a darker green color. The photograph taken on May 5th, reproduced on plate Pl. XVI. shows this difference. 1These Bul. Vol. VII. No. 1, 632 I. NAMBA AND C. KANOMATA. Between May 10 and 14 the oat plants were flowering. In this stage they were cut and weighed in the fresh state with the following result: yield. ne =2758. Sodium phosphate A, ee B, =280¢. Double superphosphate B, = 305g. With the onion plants a difference in height was hardly noticeable, altho also here a small difference in favor of the superphosphate was revealed, when the plants were cut (May 13th) and weighed in the fresh state. yieid. : A, =25 g. Sedinm) phosphate gaesipeete aes. ics Ties) Oe. ieee A,=22¢. Be 27 Double superphosphate ... B,=28 g. If now the average is taken and the yield with sodium phosphate taken ==100, we obtain the following result, to which we add for com- parison the result obtained a year ago by Inamura with Brassica. | Sodium phosphate+ Double superphosphate Lime-Nitrogen. + Lime-Nitogen. Avenaysativa’ 2.5 <3.) 2-5 ee 100 106 Allium, fistulosum... ... ... 1co 117 Brassica chinensis... ... ... 100 109 Altho these differences are not very great, they show that lime- nitrogen (an alkaline nyanure) yields a better result in conjunction with superphosphate, than in conjunction with a neutral phosphate, doubtless due to the former mixture coming nearer neutrality than the latter. In another experiment with lime-nitrogen, carried out by Mr. C. Kanomata, bonedust was applied in conjunction with it, and not super- phosphate as in the former case. ON THE EFFICACY OF CALCIUM-CYANAMID. 633 4 pots each holding 8 kilo soil, received the following manure, ¢: 2 Pots, A. each: KESO,.. Gi- See Bone; dust .. <<. ees «s .. Calcium cyanamid) WM .s s5 1. uk ee A 2 Pots, B. each: Ll i. - ine Bonewdish 2: Woe 3s we ore | TD (NH,),S0, es ee ae eras (Equivalent to N in 4 g. calcium cyanamid). The special maure, the limenitrogen or caleium cyanamid? was applied to the soil 2 weeks before the other general manures, and this mixture kept sufficiently moist in order to support the decomposition by microbs into calcium carbonate and ammonia. 10 Seeds of Brassica chinensis were sown Noy. 13, 1906, and when the young plants were about 15 em. high, they were reduced to 4 per pot, all of equal size. The weight in the fresh state, Jan. 25 was: JX) 5 f8 B=113.60¢g. CaCN, }esity.s0, t= 122:0155 Biz. Oo These differences with Brassica show that lime nitrogen compared with ammonium sulphate does not depress the availability of bonedust. A further experiment was made with oats. 20 seeds per pot were sown and later on when 15-18 em. high the number of plants was reduced to 10 per pot, all of equal size, April 18. The plants were cut soon after the flowering period with the following result: A B : 125 115 Greatest height, cm. { a ars Fresh weight, g. { 155°5 156.0 »$ mies 152.0 153.0 *The sample of calcium cyanamid at my disposition contained 18 per cent. nitrogen. r= . aie ; . se “ 634 ; . KANOMATA. b 4 = 2 i = a ’ : X 7 This result shows again that lime-nitrogen—unlike sodiumnitrate— 5 does not depress the availability of bonedust; it resembles ammonti sulphate in this regard. . 4 * i t ’ « . ‘ ‘. ' n -_ ° 7 , oe =e 4 : , eae . ; if = ™ - ) “ 4 a } mn ~ .? Om hin r sath i: am a ee ed ~ + 7 " [oat : Fs Pan o by ? ery) 4 a> hy »: ‘ Ps 4 S RY ty oy al ¢ ‘ J Z a) P : 7 re -» s hy F iy a : ' 4 7 Pah Se oY aad are. a . Th Pa * a a tn ee ee | eS Pee . nes : reer A 7 we ) 7" eLe See ¥ view, * = bd a oa “7 a <7? aT = =e — x - a a P y=, - . oe Wor? - €< ris Rae ' a ee ¥ j _ J wai's c , r~? os ~ oe hom - - 4 LY ee ‘ i 4 id - Al - - ~ - ~ wd — ‘ %¢ c* ( — a ~ a ~ . = i . — i) ‘ eae wit e 3 Fl “~~ ¢s ea a ™ re ’ Ss : e ea) A : ‘é A : af 7 : : ae 4 7 a. Ly . te Vk ) c GE ~é 7 a Bul. College of Agric. Vol. VII, No. 5. Plate XVI. Double/ Superphosphale On the Efficacy of Lime-nitrogen in Presence of Different Phosphates. ee ee ad eT) a ip BAS reel bse Lae io eect oh 5 oe Wi eee kat Cw ae ‘ >) n i oa ‘ - ‘ A! ‘ne - ' ? ’ é u ind . ' ; . ay i a 4 bs es . i A : a Ld , . 14 ae Young Bees as a Delicacy. BY M. Takaishi. In the province of Shinano in Japan a kind of wild bees (Japanese name jibachi or anabachi), which live in earth holes, serve as food, the young bees as well as their larve. Considered in the proper light, this dish is valued rather than as a delicacy than as a real food in that province, otherwise the high price would not be justifiable. The dish is prepared with sugar and shoyu-sauce; the larvee and bees thus prepared are now sold also in tin boxes, like canned meat, and about one thousand boxes are now exported annually to other provinces of Japan. One kilo of this preparation costs yen 2.50. The insects are caught in the autumn by firing some gun powder at the entrance to the nest; the smoke spreading throu the underground cavities stupifies the insects. The place is rapidly digged up and the insects caught in a basket which is then covered with cotton cloth and placed for a moment in hot water. For my analysis served the preserved content from a tin box 13 g of this content were ground in a mortar; 1 g. served for determination of total nitrogen, 1 g. for ash, and 1 g. for water content. The remaining 19 g. were washed well with water, to remove shoyu and sugar, and after drying served for determination of fat, which amount was calculated for the original dish. The wash water served for the determination of sodium-chlorid and sugar. The analytical results were as follows: ias* a? ha 42 : af 5 i: * z ; 6 4 . " — Water . - +. 28.1 % Crude protein. 13.6 4 Glucose .. 5.714 Cane sugar.. 5,81¢ % NaC? oe. pee * Ash .. .. 10.92¢ *The Shoyu sauce contains generally about 15% NaCl. = _ ¥ A | 7 - het 7 a 1a an 5 6 a aA Lie _v=q ” Ot Z a 7. > - 7 = ek . corresponds to 41.53 parts shoyu? R oy ff BK Bop kK # BULLETIN OF THE COELEGE OF AGRICULTURE, re Vow, VATE 1908-1909. INO T IGE; The Bulletin of the College of Agriculture, Tokyd Imperial University will be continued under the name of the ‘‘ Journal of the College of Agricul- to) ture, Imperial University of Tokyo,” Vol. VIII, No. 2 being the last number of the Bulletin. CON@EEN TS. No. 1, September, 1908. Miyake, T.:—A List of Panorpidae of Japan, with Descriptions of Ten New Species. With Plate I.... ae Oxasima, G.:—Contributions to the Study of Japanese Aphididae. I. On the Structure of the Antennae of Aphididae. With Plates IIJ—III. Oxasima, G. :—Contributions to the Study of Japanese Aphididae. II. Three New Species of Trichosiphum in Japan. With, Plate IV—V. Kusano, §.:—Biology of the Chrysanthemum-Rust. With one Figure in the Text Kusano, S.:—Notes on Japanese Fungi. V. Puccinia on the Leaves of Bambuseae. With Plate VI and one Figure in the Text oe Kusano, S.:—On the Parasitism of Siphonostegia (Rhinantheae). With five Figures in the Text ... 2... 11. cose Kusano, §.:—Further Studies on Aeginetia indica. With Plate NET No. 2, April, 1909. Kusano, S.:—A Contribution to the Cytology of Synchytrium and its Hosts. With Plate VIII—XI. Miyake, T. :—Description of a New Species of the Genus Lati os- trum, with Remarks on the Generic Character and the Signi- ficance of its Long Palpi. With one Figure in the Text Miyake, T.:—A Revision of the Arctianae of Japan. With six Figures in the Text ... PAGE. 13 59 A List of Panorpidae of Japan, with Descriptions of Ten New Species.’ BY T. Miyake, Rigahkushi. With Plate I. The Panorpidae hitherto known to occur in Japan are as follows :— 1. Panorpa japonica Thunberg, Wesrt., Trans. Ent. Soe. Lond., Vol. IV: p. 188 (1845); M’Lacu., Trans. Ent. Soe. Lond., 1878, p. 183. 2. Panorpa macrogaster M’Lach., Trans. Ent. Soc. Lond., 1162 pe lee: 3. Panorpa Klugi M’Lach., Trans. Ent. Soc. Lond., 1878, p. 185. 4. Panorpa Pryeri M’Lach., Trans. Ent. Soc. Lond., 1878, Ds £85. 5. Panorpa leucoptera Uhler, Trans. Ent. Soc. Lond., 1878, p. 186. 6. Panorpa Wormaldi M’Lach., Trans. Ent. Soe. Lond., 1878, p. 186. 7.. Panorpa Lewisi M’I.ach., Bull. Soc. Ent. Suiss., 1887, p. 402. 8.. Panorpa cornigera M’Lach., Bull. Soc. Ent. Suiss., 1887, p. 404. 9. Panorpa bicornuata M’Lach., Bull. Soc. Ent. Suiss., 1887, p- 403. * Contribution from the Zoological Laboratory. bo T. MIYAKE: 10. Panorpa communis L., Marscmvra. Senchiizukai (Thou- sand -Insects of Japan), Vol. I. p. 164, pl. XI, fig. 6 (1904). 11. Leptopanorpa Ritzemae M’Lach., Trans. Ent. Soc. Lond., 1878; sp: 2 8ae 12. Leptopanorpa Siebo!ldi M’Lach., Trans. Ent. Soe. Lond., 1878, p: 188. 12. Panorpodes paradoxa M’Lach., Trans. Ent. Soc. Lond., 1878, p. 189. 14. Panorpodes decorata \’Lach., Pull. Soc. Ent. Sniss., 1887, p. 405. 15. Bittacus sinensis Walk., M’Lacn., Bull. Soc. Ent. Suiss., 1887, p. 406; Marsumvra, Senchizukai (Thousand Insects of Japan), Vol, p. 165, pl. XI. fig. 5 (1904). Now, while engaged in examining the collections of the Agricultural College and of the Imperial Central Agricultural Experiment Station of Nishigahara, I have discovered a number of specimens belonging to the family, but which are apparently not referable to any of the above mentioned species. On studying them, I have come to the conclusion that they include at least ten species, all which I consider to be new to science. They shall therefore be described in the present paper. Tt is a well known fact that in the family Panorpidae the most important characters for the distinction of species are offered by the last. four abdominal segments, especially by the form of appendages of the 9th segment (cheliferous segment) of male individuals. The characters just referred to are however of no use for systematic purpose in cases In which only females are known but not males. Under such circumstances, the wing-markings might be utilized to a certain extent for the purpose of specific distinction. This holds good for at least the ‘better differentiated forms, in which the wing-markings, subject as they are to much individual variations, present to a greater or less extent features peculiar to the species, irrespective of the sexes. In fact, the A LIST OF THE PANORPIDAE OF JAPAN. 5) course indicated had been taken by several entomologists when they had oniy female specimens to deal with. So that, it seems to me justifiable if I describe in the following lines five new species on the basis of female specimens alone, laying weight principally on their wing-markings. The Panorpidae of Japan is rich in species, beautiful in colouration and remarkable in structure. Ali the following ten new species, like the rest of the family (excepting P. communis), show in common the well known peculiality mentioned by M’Lacuian, that “the subcosta in all the wings scarcely extends beyond the middle of costal margin.” So that this interesting and remarkable feature: may still be said to characterize the Japanese members of the Panorpidae. 1. Panorpa ochracea n. sp. (Kthada-shiriagemushi). (Pl. I. figs. 9, 9a 9b o%.) Body brownish ocuraceous; head black, ocelli and compound eyes brown; rostrum purplish brown; prothorax black except the hind margin; anterior half of the mesothorax blackish; a black line on the anterior margin of the metathorax. Antennae black; legs brownish ochraceous. Wings moderate; apex elliptical tinged with ochraceous the basal half more deeply coloured ; a narrow brownish black fascia rather beyond the middle, and a brownish black apical space with slight internal sinuation; two small spots (in the specimen these spots are indistinct in the right wing) before the fascia in the fore-wing and a small spot near the posterior margin in the hind-wing. Longitudinal veins blackish ; transwerse veins whitish. Abdomen moderately long, brownish ochraceous; a transverse black line on the Ist dorsal segment; two irregular black patches on the 2nd dorsal segment; the posterior margin of the 3rd dorsal segment produced into a short broad median lobe as in P. japonica, P. Klugi and its allies; 6th and 7th segment thick, cylindrical, truncate and equal in length; a slight prominence on either side of the two segments beyond the middle ; 4 T. MIYAKE: Sth slightly longer than the 7th, cylindrical; 9th (cheliferous segment) short; lateral pieces less stout than in LP. japonica and P. Klugi, the chelae brownish ochraceous with brown tips, longer in proportion than in the preceding species, the appendages ochraceous brown, linear, rather, short as in P. japonica but much more curved than in that species. Expanse 37 mm. A single male specimen captured by Mr. Tsucuripa at Yoshino, Aug., 1902. This species is closely allied to P. Klugi in the colouration of body and in the markings of wing, but is readily distinguished from the latter by the difference in size (wing-expanse in P. Klugi 27-30 mm.) and by the more elongated chelifercus appendages. In structural respect, it resembles P. japonica; but the colouration of body, wing- markings and the structure of the cheliferous segment well separates the two species. 2. Panorpa sinanoensis n. sp. (Usumon-shiriagemusht). (Bioeedies.-7, ‘Ta, Tp c-) Head and thorax black; rostrum and antennae also black; legs ochraceous yellow. Wings whitish; a very broad ereyish fuscescent (not deep) fascia beyond the middle, the apex also very broadly greyish fuscescent, so as to leave a very narrow untinged space between the fascia and the apical dark portion; an angulate spot on the posterior margin in this space; two small greyish spots before the fascia in the fore-wing, the smaller one of which is on the anterior, and the other larger and more irregular one on the posterior margin; the former spot absent in the hind-wing; another small spot near posterior margin in the fore-wing; longitudinal veins black ; transverse veins whitish. Abdomen rather slender, ochraceous fuscescent, all segments much as in P. japonica, the posterior margin of the 3rd dorsal segment produced A LIST OF THE PANORPIDAE OF JAPAN. 3) in the middle into a broad short lobe; 8th longer than the 7th; 9th (cheliferous segment) short as in P. japonica; lateral pieces less stout as in P. ochracea, the chelae long, the appendages as in P. ochracea but more linear, ochraceous brown. Expanse 36 num. A single male specimen captured at Sakaki in Shinano by the author on May 22, 1206. Allied to P. japonica in wing-markines and in structure, but differs from it in the appendages of the cheliferous segment, in lighter wing- markings and also in the colouration of abdomen, 3. Panorpa rectifasciata nu. sp. (Obi-shiriagemusht). (Pl. I: figs. 10, 10a, 10b, o%.) Varying from black to brown, the cheliferous segment reddish brown; rostrum black to reddish brown; antennae black to yellowish brown; legs ochraceous yellow. Wings rather narrow, whitish, the apex rounded; a rather broad black fascia (in some specimen not deeply coloured) beyond the middle ; a broad black apical space, this space is very slightly incurved on its inner margin; both with very sharply defined edges; neither lne nor spot anywhere present; longitudinal veins of basal half and the portion where they cross the markings black; the rest and transverse veins whitish. Abdomen black to brown; a broad median lobe on the 3rd dorsal segment as in the preceding species; 9th segient relatively smaller than in P. Klugi although somewhat larger in some specimens; lateral pieces rather stout, the chelae not longer than P. A/lugi, the appendages very short, yellowish brown, the divided portion much broader and shorter in proportion than in P. Aiugi and the preceding species and more curved. Expanse 28-30 mm. Collected by Mr. Tstcutpa at Chiizenji, Nikko on July 31, 6 T,. MIYAKE: 1885, a male; at Kominato, Aomori, on Aug. 20, 1902, two males; at Sanbogi, Aomori, on Aug. 15 and 29, three femuries. Allied to P. Klugi, but certainly distinct in the structure of the appendages of the cheliferous segment. A large female specimen (Exp. 32 mm.) obtained by me in Ki has apparently the same wing-markings as above mentioned form; only the inner margin of the apical black portion is straight and not incurved, the ground colour of wings very ochraceous and the whole body black in eolour. Whither this constitutes a variety of the present species or not cannot be exactly determined without an examination of the male. 4. Panorpa striata n. sp. (Suji-shiriagemusht). (Pi gigeae. 1. 1a, 1bpe.) } Body black, the cheliferous segment ochraceous brown; rostrum black; antennae black; legs fuscescent yellow. Wines with elliptical apex, the hind-wing somewhat shorter than the fore-wing; whitish, with black markings as follows:—the subcostal vein with a streak from base to end; a small elongated spot connected transversally on the end of the vein; three conjoined spots along the posterior margin, which are in the hind-wing less emphasized; an irregular fascia, broader than the others, beyond the middle of the wing; three elongated spots on the posterior margin between the two fasciae just mentioned; a curved line just before the apex; apex with a narrow dark pertion; longitudinal veims brownish black; transverse veins mostly whitish. Abdomen black, the posterior margin of the 3rd segment produced into a short median lobe; 6th segment larger than the others; 7th and Sth segment not so long as the others (except the 1st segment), 8th segment scarcely longer than the 7th; 9th segment stout; lateral pieces larger, fusecescent yellow, the chelae very short, the appendages of the segment black, rounded, short and very broad in proportion to same A LIST OF THE PANORPIDAE OF JAPAN. 7 of the species hitherto examined, the divided portions extremely short, the distal part of the appendages bent downwards between the two lateral pieces of the cheliferous segment (no such case in any other species) so that they represent a transverse ridge above. Expanse 27 mm. A single male specimen in the collection of the Imperial Central Agr. Exp. Station at Nishigahara without date of capture. This species I have supposed at first to be the male of P. Wormald M’L., of which only the female has been described by M’Lacuian in Trans. Ent. Soe. Lond., p. 186 (1875). But after a careful examination I have decided to consider the specimen as representing another and distinct species. There is a female specimen of P. Wormaldi in my collection, which very closely agrees with the original description of MTLacuian in all parts. Comparing this specimen with the present and taking into consultation the original description, the differences are as follows :—wing-markings of the present species are deeply blackish, while in Wormald: they are light coloured; beyond the middle of wings there are three fasciae in both species, of which the first fascia is nearly the same, but the second fascia of the present species is topograhpically the same as the third of Wormaldi, so that the second fascia of the last species 1s wanting in striata ; the third fascia of striata is to be recognized as a part of the apical black portion of Wormaldi; longitudinal veins of Wormaldi, where they do not cross the fascia, are mostly whitish, while the same of striata are mostly black. 5. Panorpa nihonensis n. sp. (Ko-shiriagemushi). (Ply We eg. 35, °3b; 5.) Body totally black; legs fuscescent. Wings rather broad, the apex rounded, whitish; a very broad blackish fascia beyond the middle; apex also broadly blackish, sinuated internally; two blackish spots before the fascia; longitudinal veins 8 T. MIYAKE: blackish; transverse veins whitish; markings mostly similar to those of P. japonica; the posterior margin of the 3rd abdominal segment produced into a short broad median lobe; 6th, 7th and 8th segment much as in P. japonica; 9th (cheliferons segment) stout, the appendages blackish, very long, slender, straight, the branched portions widely divaricate. Expanse 28 mm. A single male specimen captured at Nojiri in Shinano, by Mr. Tsucurpa on July 24, 1887. Allied in colouration of body and wing-markings to P. japonica, and in size to P. Klugi; but readily distinguishable from both by the structure of the cheliferous segment (see figs. 3a, 3b, 3c, 3d), from the former by size, and from the latter by wing-makings and colouration of body. 6. Panorpa pulchra n. sp. (Aya-shiriagemushi). Pig fe. 4, 2) Body totally black; legs vellowish. Wings. rather broad, whitish; apex somewhat elliptical; a very broad fascia (broader than in any other species) beyond the middle; the fascia furcare externally at 2 point a little lower than the middle, forming a narrow branch which terminates on the posterior margin, running obliqnely in a direction contrary to that of the fascia; apex also very broadiy black, its inner margin sinnated; between this black «pical space and the fascia a narrow untinged portion is left, diverging towards the posterior margin; another narrow fascia before the middle of wing, oblique in directon to that of the above stated broad fascia (this narrow fascia is reduced to two irregular spots in hind-wings); an irregular spot near the anterior margin between the two above mentioned fasciae ; a short basal streak along the posterior margin; longitudinal veins black especially in basal portion, those in the outer untinged porticn whitish ; transverse veins white, even in the apical black portion, so that they constitute two very fine white striae in it, A LIST OF TIE PANORPIDAE OF JAPAN. 9 Expanse 33 mm. A single female specimen captured in Tosa by Mr, Taxenovcnr (without date of capture). This species resembles P. japonica to a certain extent, but differs in the more pronounced wing-markings and in the presence of two fine white striae in the apical black space. 7. Panorpa trizonata n. sp. (Misuji-shiriagemushi). CPi tt. 9.) Body black; eyes yellowish; legs ochraceous yellow. Wings very narrow, the apex rounded, yellow; a broad black fascia before the middle of wings: a likewise broad fascia bevond the middle, in the fore-wing the fascia is fureate externally in the middle forming a narrow obliquely running branch; apex also broadly black, its mner edge slightly incurved; transverse veins ochraceous: longitudinal veins colourless. Expanse 32 mm.'; 3S mm.? 1) two females captured on Nachi in Kii by the author on July 24, 1906; 2) a female captured on Takaovama in Musashi by. Prof. Sasaxr, on Sept. 21, 1902. The Takaoyama specimen hag a black spot in fore-wing between the two black fasciae. 8. Panorpa brachypennis n. sp. (Maruhan~-shiriagemusht). (PL depen 6, 2 .) Body blackish or testaceous; yvostrum ochraceous in testaceous specimen and black in the blackish specimen; Icgs testaceous. Wings broad towards apex, broader in proportion than any of the other species, narrower in basal portion; apex rounded, ground colour * The Nacti specimen. ? The Takaoyama specimen. 10 T. MIYAKE: somewhat ochraceous; a narrow brownish black fascia rather beyond the middle, sinnated externally; apex also broadly brownish _ black, internally ineurved in the middle; (in a specimen a small spot near the anterior margin before the fascia present) ; longitudinal veins cchraceous; transverse veins whitish. Expanse 28-55 mim. Three female specimens obtained at Nikko by Mr. Tsventpa on Ang. 28, 1885; a female by Mr. Murata on Sept. 6, 1902. 9. Panorpa Takenouchii n. sp. (/Toshi-shiriagemush’). Gaile fic. 5, oQ) Body black; basal joints of antennae and rostrum ochraceous; prothorax with yellowish posterior margin (in one specimen obsolete) ; some yellowish patch on the dorsal sides of meso— and metathorax (in one specimen less emphasized); antennae black; legs ochraceous, Wings rather broad; apex rounded ; white ; four somewhat quadrate black spots along the anterior margin, of which the external one is largest; five likewise quadrate spots alone the posterior margin, of which the second spot from the base of wine is smallest; external second spot connected with the last external spot of the anterior margin; a very small linear spot on the posterior margin near the base in the fore-wing; apex black (originally consisted im two conjoined spots), with acute internal edge; main longitudinal veins blackish; the rest and transverse veins whitish. Expanse 35 mm.; 30 mm. Two female specimens captured in Tosa by Mr, Taxenowcnt (without date). A LIST OF THE, PANORPIDAE OF JAPAN. 11 10. Panorpa nikkoensis n. sp. (Viihd-shiriagemushi). (Pre ase 2, 9.) Head black; rostrum yellowish; thorax and abdomen ochraceous brown; a yellowish patch on the mesothorax; (antennae lost); legs ochraceous. Wings rather broad; apex elliptical; white with shght brown tinge; three small brownish black spots along the anterior margin; a small spot on the posterior margin beyond the middle in the fore-wing, and just at middle in the hind-wing; a small spot just at the apex. Expanse 52 mm. A single female specimen obtained at Chiizenji in Nikko by Mr. Mvgara, on Aug. 28, 1887. P.S.—There are two female specimens in the collection of the Agr. Exp. Station at Nishigahara, obtained at Chizenji in Nikko (June 13,1902 Coll. Murara), which bear a close resemblance to P. Pryeri. They are very large in size (exp. of wings in the two speci- mens 40 min.) and the wing-markings are strongly pronounced ; more- over the apex of wings is suffused with black (See Pl. L fig. 8). Whether they repsesented a new species or not, cannot be determined unless the male be obtained and examined. However, I consider the specimens to deserve being made into at least a variety of P. Pryert if not into a new and distinct species. I shall call them P. Pryert var, major. October, 1907. 12 T, MIYAKE: Explanation of Plate I. (Panorpa; a denotes apex of abdomen; b appendices. ) Fig. 1. Panorpa striata n. sp... (la, 1b). Fig. 2. P. nikkoensis n. sp. 9. Fig. 35. P. niphonensis n. sp... (3a, 3b). (3ce do. of P. japonica; 3d do of P. Kluge). Fig. 4... P» pulchre n.. sp, 9 9% Fig. 5. P. Takenouchi n. sp.. Fig. (. -P. smaneensis n. sps (ia; TB): io. Fig. 6. P. brachypennis n. sp.. 2. z fe) fig. 8. P. Pryert var. major 9. Fig. 9. P. ochracea“u. sp.,° go. (9a, 9b). Fig. 10. VW. rectifasciata n. sp., 7. (10a, 100). Ris, 1. PP. trieonata no sp.) eee liyake et Yokoyama del. % wv r ? “e Contributions to the Study of Japanese Aphididae.* I. On the Strueture of the Antennae of Aphididae. BY G. Okajima. With Plates II and III. The antennae of Aphides (Plant-lee) are, as is well known, long and filiform, and inserted m front of the eves, directly on the head or on raised protuberences, the so called frontal tubercles. The length is very variable, some being longer than the width of head while others are more than twice as long as the body. In the Subfamily Aphidinae, and especially im the Genus Siphonophora, the antennae are almost always very long, while in the Subfamilies Callipterinae, Lachninae, Erioso- matinae and Phylloxerinac, they are usually short. The antennal joints vary from three to six in number. The chief object of this paper is to discuss their number.’ Some give it as three to seven, others as five to seven, while still others give it as invariably six. Up to the present, the first of the above statements appears to have been regarded as true by many entomologists. J. F. Juprrcn and H. Nirscux* state in their work as follows: ‘Die altere Angabe, die Gattung Aphis L. habe sieben- elederige Fiihler, beruht darauf, dass bei ihr das Ghed sechs sich gegen das Ende zu plotzlich verdiinnt. Sie tragen an den letzten Gliedern kleine Riechgriitben.” And they add: “bei den jungen Larven 5-gliedrig, bei den erwachsenen Formen dagegen meist 6-gliedrig und oft langer als der Kérper, nur selten 5-gliedrig” Mr. E. Wrrnaczi’ after criticizing briefly the observations of Kavrensacu, Koci and Bucxroy, * Contribution from the Zoological Laboratory. * Lehib, der Mitteleurop. Forstinsekt, 1895. p. 1197. ® Zur Anatomie der Aphiden. 1882. p. 10. 14 G. OKAJIMA: says, “Wie ich schon bemerkt have, ist dies unrichtig. Ich fand bei den Aphiden allgemein sechs Fiihlergheder, Bei’ der ebenfalls als mit fiinf Fiihlerghedern unterschiedenen Gattung Pemphigus zeigen alle gefliigelten agamen Weibchen und die Mannchen sechs Fiihlerglieder. Allerdings nur im vollkommen ausgebildeten Zustande, indem die Larven derselben nur 4-5 Fihlerglieder besitzen, von denen aber oft eines einen Einschnitt in der Mitte zeigt, so andcutend, dass wir nach der nachsten Hautung ein Glied mehr zihlen werden.” Bucxron,' the eminent author, in the first volume of his celebrated Monograph states that: ‘in Aphis proper there are seven joints; Pemphigus and Schizoneura have six antennal joints, while in Phylloxera the number of joints is reduced simply to three.” Even Mr. D. Suarv? expresses the doubt “whether the antennae have ever really more than six joints, the apparent seventh joint beimg actually a sort of appendage of the sixth.” Thus it appears that the exact number of the antennal joints vemains still unsettled, but many authorities agree in regarding the number three, found in the Gen. Phyllowera, as the lowest limit. Unfortunately I have not been able to obtain any species with three jointed antennae, so I shall here ecoufine my discussion to forms having more than three jointed antennae. Many entomologists are of the opinion that most Aphides possess seven joints, while Juprrci, Nirscur, Wrriaczim and others affirm that the antennae are composed of only six joints. On this point Bucxron® says as follows: “the terminal joint of the antennae affords good discriminating characters. In Siphonophora and like genera, the seventh or the last joint is very lone and imbricated. In Lachninae. the seventh joint, although obvious, is much curtailed in lengtb, and here it shows the first tendency to abort.” It is not needless to make a further study of the number of primary antennal segments of Aphides. In the Subfamily Aphidinae (Fig. 23) the first and second joints of the antenna are usually short globous, and + Monogr. of the Brit. Aphides. Vol. I. 1876. p. 12. “Insects. “Part il .901gp pole * Monogr. of the Brit. Aphides. Vol. I. 1876. p. 12. CONTRIBUTIONS TO TEES STUDY OF JAPANESE APHIDIDAR. 15 freely movable. The third joint, usually the longest, is equal te or longer than the length of the two next combined. The fourth and the fifth, mostly short, are equal to each other and longer than half the length of the third joint. The sixth, which is usually longer than or «qual to the third in length, is divided into two portions, a short, stout, proximal and a long, slender, filiform or sometimes whip-like distal portion (Figs 1, and 8-15). In the Subfamily Aphidinae, the distal is, with certain exceptions, usually much longer than the proximal portion (Figs. 21-23 and 27). This sixth joint is the very point of discussion, for some regard the joint as one, while others look upon it as two combined. The two portions are not only different in form, but on the so-called node between them there lies a small tubercle. Tf this node be a true joint, then the two portions must be capable of bending towards each other: but as a matter of fact they are inflexible. Further there is no articulaz membrane which is characteristic of a true node (Figs. 1, 3 and 13-15). It is convenient to remark here on the tubercles or sensoria* of the antennae. They are usually small circular spots, but sometimes large and oval or irreenlar in shape, they are moreover, ring-shaped, so as to impart a rough surface te the antemnae (Figs. 5 and 16-19). They are covered with a membrane like drums 2nd look much like ocelli (Figs. 1, 18 and 20). These spots are present commonly on the third joint, and often on the fourth, fifth and sixth, but never on the first and second joints. Near the tip of the last joint and the one before the last, one or more of these scnsoria are invariably to be found. It is one of these sensoria that divides the sixth joint into two portions, and simulates a node between them (Fig. 15). Their number, size and arrangement are quite different in different species. They are sometimes arranged in a single row (Figs. 3, 7, 19, 21, 24 and 25), and sometimes irregularly aggregated (Figs. 22, 23 and 27). There are two forms of 2mnulated sensoria, one 1s merely linear and raised around the antenuae, or semi- circular (Fig. 7), the other ridged along the raised portion so that the ? OEsTLUND, O. W.. Synopsis of the Aphididae of Minnesota. 1887. p. 2. 16 G. OKAJIMA: antennae appear dentate on a side view (Figs. 16,17 and 19). Mr. O. W. Orstiunp, who adopted this character in describing species for the, first time, state that, “they are considered by entomologists to be organs of smell or hearing, or both. I have always found them present and they often give good specific characters, though very few writers have vet made use of them in describing species.” In fact, these sensoria afford a discriminating character for species as well as for genera. Moreover, the five subfamilies proposed by Mr. G. W. Kirxanpy’ can also be distinguished by this antennal character. 1. In Aphidinae, generally long, frequently reaching to twice the body length. Six jointed. Distal part of the sixth joint always very long. Small circular sensoria mostly at irregular distances, present on the third joint only or on third to fifth. 2. In Callipterinae, long, and more slender than in the preceding subfamily. Six jointed. Distal part of the sixth joimt usually short or sometimes equal to the proximal. Sensoria more or less oval in shape, present on the third joint alone. They are close together en the basal half of the joint and very few in number. 3. In Lachninae, short, mosty Jess than the body length. Six jointed. Distal part of the sixth joint shorter than the proximal, and looks like a nail-like process of the latter. Sensoria, round or oval in shape, arranged in a single row and very few in number. The presence of long hairs is peeuliar to this subfamily. 4. In Eriosomatinac, very short, not more than half the length of the body. Six or five jointed. The last joint with a short spur (distal). The third and following joints are annulated or furnished with transverse sensoria, at regular or sometimes irregular distazces. 5. In Phylloxerinae, exceedingly short. Five or three jointed, In the genns Chermes, the antennae are hardly epual to the width of the head. They are furnished with comparatively large sensoria one on each of the three distal joints. * Catalogue of the genera of the Hemipt. Fam. Aphidae. Can. Ent. 1906. CONTRIBUTIONS TO THE SRUDY OF JAPANESE APHIDIDAR. yi Conclusion. I. The antennae of Aphides are composed of not more than six joints. IT. Numerous sensory pits are present on the third and following joints, in particular they are never absent from the third. TIT. Near the tips of both the last jomt and the one before Jast, there are always one or more circular sensoria. IV. These sensoria divide the last jot apparently into two parts, of which the distal is usually slender than the proximal. V. This distal part may be ealled “flagellum” as in Crustacea and others. March, 1908. Explanation of Figures. Plate II. (Joints of the Antenna.) Fig. 1. Sixth and a part ef fifth joint of Aphis sp. a. sensoria on the sixth joint ; b, the same on the fifth. (4D. Zeiss.) Fig. 2. Schizoneura uimi L. a, sensoria on the sixth. A, 3-6 joints. B, side view of the sixth. (4xD.) Fig. 3. Lachnus sp. (4xA.) Fig. 4. Lachnus sp. Third to sixth joints. Fig. 5. Sehlechtendalia chinensis Bell. (1D.) Fig. 6. Schlechtendulia sp. (1xD.) Fig. 7. Pemphigus sp. (1xD.) Sixth joint with tip of fifth of Siphonophora sp. (IxA.) v2) Fie. Fig. 9. Fifth and sixth of Phoroden sp. (IxA.) Fig. 10. Pterocallis tiliae L. Fifth and sixth. (IxA.) Fig. 11. Callipterus castaneaec Buck. Fifth and sixth. (IxA.) Fig. 12. Welanoxraxthus sp. Third to sixth. (IxA.) Fig. 13. A part of sixth joint of Phorodon galcopsidis Kalt., showing the sensoria upon it. (4xD.) 18 Fig. Fig. 14. . Fourth to sixth of the same. (IxB.) — for) G. OKAJIMA: Parts of fifth and sixth joints of the above species. Plate III. (Antenna. ) Hamamelistes sp. (1xD.) . Astegopterix nekoashii Sasaki. . Schizoneura corni Fab. . Schizoneura lanigara Hans. . Chermes laricis Hartig. . Aphis sp. 2. Phorodon galeopsidis Kalt. . Aphis brassicae L. . Callipterus sp. . Callipterus sp. . Cryptosiphum artemisae Buck. . Megoura viciae Buck. (IxD.) rag ke a ene ee hip ee ae ge eee pS a eel aa : ns : : , ‘ \ i/ ; ‘ : ee ae ay i a ‘ie * - 7 ; “ = “ _ Png jy ce al — 5 ov. 2 e ieee ate a Ls) i heir a ae ' “ ’ a a a ia stay 7 . ue ‘ i LI oF ‘7 ry . ’ — > a i ‘ » f - ' ag? t — 7 ~ 6 / f - ‘ 7 s os & a . ’ } . , i ' wl ( = bs . I ¥ ‘ ) 7000 ———— ———— yd ULL. AGRIC COLL. VOL. VI. Contributions to the Study of Japanese Aphididae.’ If. Three New Species of Trichosiphum in Japan. BY G. Okajima. With Plates IV and V. Species of Trichosiphum, a new genus created by Mr. T. Percanps for one of cur plant-lice have lately been found in the entomological collection of the Imperial Agricultural Experiment Station at Tokyo. The plant-lice belonging to this genus are very peculiar in shape as well as in habit and deserve special attention. A plant-louse from Ceylon, which Prof. J. O. Wresrwoop? described and figured in 1890 under the name of Siphonophora Artocarpi, is in my opinion a_ species of Trichosiphum. In our Trichosiphum, the cornicles on the abdomen are hairy and exceedingly long (Figs. 16, 18, 20 and 21). The second oblique vein of the fore wing is curved in its outer half. The stigma is short and broad, but Prof. Wrsrwoop appears to have erroneously figured the antennae as being eleven jointed. My studies of the insects are still incomplete and require further examination, but a few results obtained thus far may be briefly given. Genus Trichosiphum Pergande, 1905.° Rostrum long, reaching to the coxa of hind legs. Antemnae nearly 1 Contribution from the Zoological Laboratory. ? Trans. Ent. Soc. Lond. 1890. p. 649. ® Duplicate specimens of some Japanese Aphides were sent to Mr. T. PERGANDE, U.S. Department of Agriculture, by Mr. I. Kuwana, Imperial Agricultural Experiment Station at Tokyo. To one of them, the American entomologist gave this generic name with reference to the remarkable character of the cornicles. As no original description is at hand, I drawn up the generic as well as a specific characters (Trichosiphum kuwanea) according to my own observations. This genus has a very remarkable character, so that it may perhaps represent a new subfamily. 20 G OKAJIMA: equal to the length of the body; first and second joints short, third joint longest, equal to the length of the three following taken together. The tubercles on the third joint are arranged in a single row along the entire length. Fourth and fifth equal in length. Sixth, the last, somewhat longer than the preceding two joints, and divided into two halves, of which the distal is slender and uswally longer than tke proximal. Compound eyes large. Supplementary eyes prominent. Wings laid vertically when at Test. Venation similar to that of the Gen, Aphis excepting the following points. Second oblique vein arising near the first and strongly curved for the last third of its length. Cubital vein Iving parallel to the second oblique vein, obsolete at the base. Infra- marginal cell very large. Cornicles exceedingly long, mostly straight, but sometimes slightly curved. Cauda blunt. Legs short and_ stout. The entire body and especiaily the long cornicles are covered thickly with hairs, which are never found in other genera, hence the generic name Trichosiphum. . Prof. J. O. Wersrwoop' states that, ‘a striking character of the species (Siphonophora Artocarpi) consists in the enlarged size of the cornicles. These tubes are stated by Mr. Green to be carried diverging and elevated at an angle of 45°; they are sometimes as long as the whole remainder of the insect, and are strongly setose, the fine bristles set on nearly at might angles...... When alarmed the insects suddenly dropped from the leaves to the ground. They are very active, and walk rapidly.” This description also applies to the genus before us. 1. Trichosiphum kuwanea Pere. (O-kebukaaiimakt). (Figs. 1-5, 15 and 76). a. Winged viviparous female. Expanse,,0t WaG@Speme 2s te oe seep ee Lenvth of, body = ot? ee ee Se ee 3 ) Salento. os) eee ee ee i, 5, COLMME TNE <> 5. a eee waa ee * Trans. Ent. Soc. Lond. 1890. p. 649. CONTRIBUTIONS TO THE SEUDY OF JAPANESE APHIDIDAE. 21 Head large. First antennal joint large, third joint longest, provided with about thirty tubercles of various sizes. Fourth and fifth equal in length, the latter with a single tubercle near the apical end. Sixth equal to the length of the preceding two taken together. The distal part of the sixth twice as long as the proximal. Ocelli distinet. Compound eves large, bright red. Supplementary eyes remarkably prominent. Rostrum long, reaching to the third coxa. Prothorax with a transverse black band. Mesothorax well developed, thoracic lobes large and black. Seutellem dark brown. Abdomen oval, with four blackish broad bands on the dorsal and ventral surface, and large black spots lving on the sides of each segment Cornicles very long, cylindrical, almost equal to the length of the thorax and abdomen. Cauda conical blunt. Wings ample. Cubitus brown. Stizma short, thickend, and grayish in colour. Hooklets on hind wings three or four. Legs short and stout, dark brown. Body looks dark brown and thickly covered with hairs. This may, perhaps, be the female of the second generation or migrant form of the species. It is found forming small colonies on the young shoots of Quercus serrata Thunb. and Q. acuta Thunb. Appear in June. Habitat, Tokyo. h. Apterous viviparous female (stem mother). Rencth of bedy ..2\ aaa gp alent See ce EB, 2 “|. cormiclemee: > os eg OF, Dark brewn. Body much swollen, round in shape, ventral surface flat. Head comparatively small. Antennae similar to those of the winged form. Compound eyes bright red. Supplementary eyes prominent. Rostrim long, reaching to the third coxa, thorax small, abdomen hemispherical, cornicles short, somewhat sickle-like in shape. Cauda conical and blunt. e. Pupa.* Oval and somewhat corpulent. Antennae and compound eyes same * Nymph in strict sense. 22 G. OKAJIMA: as in stem mother. Legs same as in the winged females. Cornicles short stick-like. Head, prothorax, wing case, legs and cornicles dark brown. Dorsal surface of the abdomen with numerous dark spots of variable size, symmetrically arranged on each side. Body hairy, pale brown with a slightly vellow shade. 9. Trichosiphum tenuicorpus noy. sp. (losonaga-kebuhkaarimah). (Figs. 6-10, 17 and 18). a. Winged viviparous female. Hixpanse Of WiInSeme .. 6s we =e, DL Lensth of body Spammer << + geet ee Ue Sw ee hk Ss S sy on 99 MOORING Kes Body dark brown or black, slender and almost linear. Head of moderate size. Antennae inserted upon a gibbous tubercle, nearly two- thirds as long as the body. First and second joint comparatively small. Third longest, as long as the three following taken together. Furth and fifth equal. Sixth, the last, divided by a tubercle into two nearly equal halves. Tubereles on the third joint arranged in a single raw, about twenty in number. Compound eyes small, bright red. Supplementary eyes prominent. Rostrum long, reaching to the third coxa. Rectangular band on the prothorax black. Thoracic lobes black. Abdomen some- what fusiform, and blackish. On the ventral surfee of the abdomen run two longitudinal lines, on the outside of which there are four dark spots on either side. Cornicles exceedingly long, nearly equal to the body, lying vertically, or sometimes horizontally. Legs short and black. Wings thin and delicate, venation as in the preceding species. Stigma black, very long, equal to two-third of the fore wing. First oblique vein comparatively broad and short. Infra-marginal cell large. Hind wings small and narrow, hookleis two. Found on the young shoots of Pasania cuspidata Oerst. This form, I think, may be a migrant, though it has been captured CONTRIBUTIONS TO THE SPUDY OF JAPANESE APHIDIDAE. 25 in September or later. In the colony I have found pupae and larvae of various stages, as well as a few apterous viviparous female; but no males or oviparous females have been met with. b. Apterous viviparous female. Tenzin of body .2 eee - -CtCts ee OP ee = ,, antenna 7.0) os “4 ; COMMiclo mene. ©. «2 of eT .. Body brown, pear-skaped, broader behind. Frontal tubercles, gibbous, first antennal joint big, third longest, equal to the length of the three following combined, fourth and fifth equal, sixth divided into two halves, with a knee-shaped angle between, giving rise to the appearance of two different joints. Compound eves bright red. Supplementary eyes prominent. Rostrum long reaching to the third coxa. Head and thorax equal in width. Abdomen broadens toward the caudal end. Canda blunt. Cornicles equal to the length of abdomen. Legs short and stout. Body much covered with hairs, dirty brown. e. Pupa. Body slender, hairy. Pale brown to brownish yellow. Antennae, legs, cornicles and anal plate dark coloured. Antennae and cornicles equal separately to the length of abdomen. Compound eyes bright red. Legs short and stout. 3. Trichosiphum pasaniae nov. sp.(Mo-hebukaarimaht). (Figs. 11-14, 19 and 20). a. Winged viviparons female. xoqnse-0f Wits eee ww ws | OTT, Beucib or body "yieee C L, re + antenna Ree Oe See aes, COMiclow———ee! .. -- «= O8 =, Body small, dirty brown. Head of moderate size. Antennae two- third as long as the body; first and second joints small, third longest, 24 G. OKAJIMA: equal to the two following jomts combined, fourth equal in length to the fifth of the six-jointed antennae of the preceding species, short, with a tubercle near the apical end, fifth, the last, almost twice as long as the preceding, and divided as usual into two halves by an intervening tu- bercle. Distal half twice as long as the proximal. Tubercles on the third joint arranged along its entire length, about twenty in number. Compound eyes prominent and bright red. Supplementary eyes very prominent. Rostrum long, reaching to the third coxa. Transversa band on the prothorax black. Thoracix lobes black. Adbomen oval with a large irregular black patch on the dorsal surface, the three lateral patches black. Cauda blunt. Ventral surface of thorax and fifth abdominal segment black. Cornicles long, as long as the 2bdomen, slightly thickened towards the base in the distal two-thirds of their length. Wings ample, venation same as in the previous species. Stigma very lone, almost equal to one-third of the costal margin of the fore wing. Hooklets of the hind wings two or three. Infra-marginal cell exceedingly broad. Legs short. b. Pupa. Oval, light brown or dirty yellow, tips of antennae, prothorax, cor- nicles, cauda, tibial end and tarsi darker coloured. Dorsal patches of the abdomen and the terminal antennal joimt similar. to those of Trichosiphum kuwanea. Cornicles somewhat sickle-shaped. Legs short. Found on the young shosts and underside of leaves of Pasania cuspidata Ocrst. Quercus serrata Tiinb. and Q. acuta Thunb. The colony is not so large and frequently found together with that of Trichosiphum Iewmanea. The antennae (Fig. 19) of this species are only five jointed. At first I was inclined to suppose that the species had more than five-jomted antennae, but careful examination of teri specimens (all in, my possession ) has made it clear that the antennae are normally 5-jointed in this species. CONTRIBUTIONS TO THE STUDY OF JAPANESE APTHIDIDAE. 25 Synopsis of the three Species. Body slender, cornicles equal to or longer than the body length | AL} ve veeses ee L€UICOTPUS. | Boay oval, cornicles shorter than the body length...............B. Antenne with five joints, stigma long........................pasaniae. Antenne with six joints, stigma short ......................../uwanea. The author expresses his cordial thanks to Prof. C. Sasaxt to whose instructions and permission of the free use of the valuable literature in his possession this study is chiefly due. Thanks are also due for the kindly encouragements given him by Profs. C. Isurkawa, 8S. Goro and K. Toyama. Moreover I haye great pleasure in acknowledging the friendly advices of Mr. I. Kuwana. March, 1908. Explanation of Figures. Plate IV. Fig. 1. Trichosiphum kwweanea Perg. Vivip. ¢. Fig. 2. Ditto. Ventral side. = Fig. 3. Stem mother. Fig. 4. Hook angle of the hind wing of the species, with four hooklets. Fig. 5. Pupa or the last larval stage. Fig. 6. Trichosiphum tenuwicorpus noy. sp. Vivip.&. Fig. 7. Ditto. Ventral side. Fig. 8. Viviparous wingless female. Fig. 9. Hook angle of the hind wing of the species. Fig. 10. Pupa. Fig. 11. Trichosiphum pasaniae noy. sp. Vivip. ? - Fig. 12. Hook angle with two hooklets. Fig. 13. Ventral side of the winged viviparous fema!e Fig. 14. Pupa. G. OKAJIMA: Plate V. 5. Antenna of Trichosiphum kuiranea Perg. . Cornicle of the same. . Antenna of Trichosiphum tenuicorpus. . Cornicle of the same. Antenna of Trichosiphuin pesaniae. . Cornicle of the same. . Cornicle of Siphenophora sp, probably the longest among the subfamily. BULL. AGRIC. COLL.VOL.VHI . aa PLATE IV. G. Okajima del. BULL. AGRIC. COLL. VOL. VIL. ; PLATE Ve -_ Biology of the Chrysanthemum-Rust. BY S. Kusano. With one Figure in the Text. Black Rust (Puccinia Chrysanthemi Roz.). Some years ago a most dreadful rust appeared suddenly in several countries of Europe and America, and furiously spread in a short interval of time, inflicting no inconsiderable damage upon chrysanthemum- cultivation.” Although it was thought at that time that the rust was imported from Japan, where the same rust had long been known to attack the same plant, yet the correct specific name of the fungus has remaimed undetermined. Some held the view that it was identical with Puccinia IMieracit Mart® while others took it for P. Tanaceti De. or P. Balsamitae (Str.) Rbh. Roze after a more careful study has, however, arrived at the conclusion that it was different from any known species, and consequently called it P. Chrysanthemi un. sp.* Subsequently Henxryxes described the Japanese species as new to science under the name of P. Chrysanthemi chinensis’ which, however, P. Sypow has identified with the known species, ?. Pyrethri Rab. This * Contribution from the Botanical Laboratory. A short account has already appeared in Japanese in Bot. Mag., Tokyo, Vol. XVIII, 1904, p. 99. * See Jacky, E., Der Chrysanthemum-Rost. Zeitschr. f. Pflanzenkrankh., Bd. X, 1900, p. 132. * Masse, G., Chrysanthemum-Rust. Gardener Chron., Vol. II, 1898, p. 269. * Roze, E., Le Puccinia Chrysanthemi, cause de la Rouille du Chrysanthemum indicum L. Bull. de la Soc. myc. de la France, T. XVI, 1900, p. 88. 5 Hennincs, P., Einige neue Japanische Uredineen. Hedwigia, Bd. XL, 1901, p- (26). * Sypow, P., Monographia Uredinarum, I, p. 46. 28 Bac USANO : confusion in the identification of the chrysanthemum-rnst seems to have arisen from the small morphological difference exhibited by the allied species of the genus. JacKky* then undertook a comparative study of Japanese and European resis. His infection-experiments proved that the rust of Chrysanthemum indicum L. did not infect other Chrysanthemum or other allied plants of Compositae, except Chrysanthemum sinense Sab., the natural host of the rust in Jiapan, and that the Japanese rust of Chrysan- themum sinense couid easily infect Chrysanthemum indicum, the host of the European rust. There being no morphological difference both in uredo and teleutopores taken from both hosts, he came to the conclusion that the Japanese rust was identical with the European, and hence he referred it to Puccinia Chrysanthemi Roz. But curiously enough, there exists a great difference between the Japanese and European rusts in their mode of development. The Euro- peal species repeats the uredogenerations through the vear, and the uredospores can winter en the young shoots of the host kept in the house. The formatien of the teleutospores is exceedingly rare. When they are formed, mesospores accompany them invariably. Further, we have in almost all cases variously formed and. two-celled uredospores in the European species. As Jacky noted, the formation of mesospores and two-celied uredospores, abnormal as it may be, is almost constant?. In the Japanese species such cases seem to occur very seldom. In Tokyo and xe its vicinity ihe uredogeneration is regularly followed by the teleutostag ? and the hibernating teleutospores germinate at once in the early spring. The following is the development of the rust observed by myself in Tokyo. The first appearance of the uredosori takes place at the end of May or at the beginnmg of June, when the host has attained the height of 30cm. or ihiereabout. Among several garden-varieties of the host-plant placed side Ly side a certain variety was first attacked by the rust. At the Botanic Garden of the Agricultural College, Komaba, it was observed * JACKY, loc, cit.: ——, Der Chrysanthemum-Rost. II, Centralbl f. Bakteriol., IT. Abt., Bd. X, 1903, p. 369. * Jacky, loc. cit. Centralbl. f. Bak. BIOLOGY OF THE CHRYSANTHEMUM-RUST. 29 that a variety called “Omihakkei’ first produced the uredosori on its lower leaves. The rapid spread of the rust later on to other varieties seemed to be due to infection from this source. At the beginning of July almost all the varieties were attacked by the rust. A most careful examination has failed to reveai any spermogonium in the first generation of uredosori, so that it is most probable that the fungus belongs to Hemipuccinia. In the autumn, when the host approaches its flowering season, the uredostage is followed by the teleutostage. I have found it at the be- ginning of October. Aiterwards the tcleutosori spread over most rapidly from the lower portions of the stem up to the bracts of the heads. It may be noticed that in the first generation of the teleutostage the sori form a ring of 1.5-3.0 mm. in diameter around each uredosorus of the last generation, so that there is no doubt that the teleutosori may originate from the mycelium around the uredosorus. Numerous isolated teleutosori developed afterwards seemed to originate from the urdeospores of the year. In early winter, wnen the stems begin to wither from injury by the frost, young shoots appear from the mother-stock, The fungus then invades these vigorous offsprings perhaps by means of the uredospores, and forming there the teleutosori it can winter on the living host. Though rare, a few new tcleutosori made their appearance even in the midst of the winter (end of January), but later the formation of the sori seemed to have ceased entirety. The teleutospores on the dead host of the last year germinated at once in April. The uredosori which, as stated above, suddenly appear at the end of May probably owe their origin to the sporidia thus produced from the teleutospores. Such being the course of development of the rust in Tokyo, I will now give a few observations made in a warmer region in Japan. At my request Mr. Yosw1nxsca has made some observations since a few years on the development of the rust in Proy. Tosa, and he has kindly put 30 5. KUSANO: the specimens he has collected at different localities from time to time at my disposal. An examination of these specimens shows beyond doubt that the rust produces in these warm coastal localities, as for instance Akimachi and Kochi’, only uredosori generation after generation through the year without the formation of teleutospores even in the midst of the winter. It suggests to us the possibility of the uredospores retaining in these localities their germinating power through the winter and becoming a new starting poimt for the rust in the next year. This must be admitted to be perfectly possible, as the uredospores exposed to -25°C. may retain, according to Jacky", their germinating power. As in this way the rust develops in the coastal region of Tosa just as in Europe without the formation of the teleutospores, I have given special attention to the abnormal formation of spores at the localities mentioned above. In the specimens collected at Kochi’, December 13, 1907*, which consisted entirely of vigorously developed uredosori, I could without difficulty find numerous abnormal and two-celled uredospores, as in the European specimens. Further, the formation of mesospores was ascertained at somewhat colder localities in Tosa. Mr. Yosurnaca has already found in November, 1901 abundant teleutospores at Sakawamachi’, 4 ri (ca. 10 miles) distant from the sea-shore, and quite recently he has collected them again in a mountainous region Ujimura, 1.5 ri west trom JXochi on December 15, 1907°. In both specimens the teleutospores were developed as much as in those of Tokyo, but their form was much irregular, and the sori contained mesospores. These facts show clearly that the abnormal formation of spores is not a characteristic of the rust introduced to Europe, but takes place in its native country, though not constantly. As regards the constant formation of abnormal spores in Europe 2 The frost is rare in these localities. 2 JACKY, loc. cit. Zeitschr, £ Pilkrkh., Bd. X, p. 141. ’ A town near the sea-shore. in February the host withered away without forming any teleutosorus. ° HENNINGS, Fungi japonici. V. Engler Bot. Jahrb., Bd. XXXIV, 1905, p. 595. At a place a little nearer to Kochi he found on the same day only uredosi. BIOLOGY OF THE CHRYSANTHEMUM-RUST. 31 several views may be proposed. Jacky remarked about this point, “es diirfte die Bildung derartig anormaler Sporen vom Standort und der Beschaffenheit der Nahrpflanze abhingig sein.”’ That the rust develops differently in Tokyo and Tosa makes it probable that the formation of abnormal spores is due to the influence of the locality where the fumgus oceurs, but the exact nature of this influence remains still unknown. In Japan the cessation of the development of the teleutospores in the coastal region of Tosa is probably due to the much warmer climate there, but we can not as vet conclude on that account that the climate is the cause of the abnormal formation of spores. Next we may ask whether the rust on Chrysanthemum indicum in Japan develops differently from that on C. smense, as it does in Europe. In Japan I endeavoured to get the specimen of the rust on C. indicum, but as most of the cultivated chrysanthemums belong to C. sinense, it was not easy for me to find abundant material for study of the rust on the desired plant. However, I was fortunate enough to find a few diseased leaves of this plant? in the herbarium in the Botanical Institute, Science College, Tokyo Imperial University. The plant was collected by T. Maxrno at Mt. Hie, Prov. Yamashiro on Nov. 7, 1904. The teleutospores were found abundantly as on C. sinense, but neither mesospores nor two-celled uredospores were found, showing that the rust may develop in the same way on C. indicum and C. sinense. Tn this connection I may mention the rust on a wild chrysanthemum, Chrysanthemum Decaisncanum Max. This isi a coastal plant limited to the warmer region. At the beginning of January, 1907, I collected the (rust on it at Hanemura, a coastal region in Proy. Tosa. At that time the host had nearly withered from frost, but the rust was present all over. On bringing it back to Tokyo T found that the sori were almost entirely occupied by uredospores, of which the form, structure of the wall, and 1 Jacky, loc. cit. Centralbl. f. Bakt., Bd. X, p. 375. 2 Chrysanthemum indicum TL, var, genuinum Max. 4 S. RUSANO: the number of the germ-pores did not differ from those of the uredospores of the typical Puccinia Chrysanthemi. Exceedingly rarely, I found a few teleutospores whose morphology coincided also with that of the ieleutospores of P. Chrysanthemi. Moreover, remarkably enongh I eould find the irregular and two-celled uredospores, and numerous 1 So that the rust on Chrysanthemum Decarisneanum is\ quite mesospores. the same in every respect as that on C’. indicum in Europe. . This gives a strong evidence for the assertion given above that the abnormal develop- mient 1s by no means characteristic of the European rust, but may take place in its native country. We see thus a creat variation in the development of the rust in different localities in Japan. In the coastal region of Tosa irregular and iwo-celled uredospores are very common, while the teleutospores develop sparingly. In inland localities the rust has a tendency to develop the teleutospores regularly, and inhibit the formation of abnormal uredospores. In Tokyo and its vicinity the formation of these abnormal spores ceases apparently, while the teleutospores follow the uredospores. It also seems probable that the chrysanthemum-ruct now so widely found originated from Chrysanthemum Decaisneanum,-on which the warmer | climate brought about the inhibition of the formation of the teleutospores; it then spread to the cultivated chrysanthemum, retaining still the character it has assumed on ©. Decaisneanum. As it spread wider and wider on the cultivated chrysanthemum in much colder regions it acquired the character of forming the teleutospores on the one hand, and inhibited the formation of abnormal spores on the other. This character has becomes nearly fixed in Tokyo and its vicinity, but in Tosa, where the occasional infection of C’. sinense by the rust of C. Decaisneanum is possible, the character of the rust can still be observed unchanged on the former. That the direct in- fluence of the environment is not concerned on this point may be partly demonstrated by Jacxy’s infection-experiments.? He sueceeded in getting 1 The difference between the sea-shore and inland forms was also described by ARTHUR in Uromyces acuninatus Arth. (Arriur, J. C., The Uredineae oceurring upon Phragmites, Spartina, &c. in Bot. Gaz., Vol. XXXTY, 1902, p..1), > Centlbl. 7. Bakt., Bd. X, p. 370. BIOLOGY OF THE CHRYSANTHEMUM-RUST. 33 abundant teleutosori by infeeting C. idicum with the spores of the Japanese rust,’ but not with the European rust, which shows that there is no immediate change in the developmental habit of the Japanese rust when transferred to Europe. The specific name of the host in Europe still remains a question for Japanese botanists. It is there assumed to be Chrysanthemum indicum, but if it has all been imported from Japan, we have a strong ground for believing it to be C. simense. The Japanese chrysanthemum with large Howers helongs exclusively to C. smmense, though some small-flowered forms belong to C. indicum, which is, however, cultivated very little. Hence it is very probable that the host of the rust is the same species in Japan and abroad, being either C. sinense or C. indicum, so that there is but little doubt about the identity of the Japanese chrysanthemmn-rust with the European White Rust (Puccinia Horiana P. Wenn.).? This is another chrysanthemum-rust recently made known botanieally, though gardeners seem to have been acquainted with it long since. It a. b. Pig. 1. a, teleutospores: 6. germination of sporidia on the leaf of the host (3 days after sowing). 400. 1 The materials were collected by N. Nambu at Urawa near Tokyo on December 4, 1900. ats, . , + . =p Ea “4 C 2 Heynincs, P,. Einige neue japanische Uredineen. Hedwigia, Bd, XL, 1901, p. 34 SemUSANO: is characterised by having large, white, waxy sori, 2-3 mm. in diameter on the undersurface of the leaf. The sori consist entirely of comparatively small thin-walled teleutospores which germinate as soon as they become mature (Fig. 1a). The rapid propagation of the rust is due to infection by the sporidia thus produced. Sown on a leat of the host kept in a moist chamber, they soon produce germinating tubes which penetrate through the outer wall of the epidermis, and send out swollen haustoria, or inter- cellular mycelia (Fig. 1b). As no spermogonium has ever been found, the fungus should be referred to Leptopuccinia. The development of this rust proceeds almost without interruption throngh the vear, though its activity varies more or less according to the condition of the host and the condition of the environment. In Tokyo the host-plant suffers most generally in May or June, while it is still young, and the propagation of the rust seems to be exceedingly rapid in a moist place, or where the host-plants are densely planted. The rainy seasons are especially favourable for the development of the fungus: in Tokyo they occur in the spring, early summer, and September. The intimate connection of the development of the rust with moisture is due to the circumstance that the teleutospores and sporidia germinate immediately after maturation. When the old stems of the host die in the autumn, the rust directly attacks the new shoots, and voung and old sori, though not vigorous in development, can pass the winter in the field. There is no doubt that low temperature checks the development of the fungus, but the ripe spores ean resist it, and germinate soon when favourable conditions return the next spring. The white rust is more injurious than the black rust because of its attacking young hosts and of its rapid propagation. However, many forms of Chrysanthemum sinense are quite free from its attack. -The edible variety of the host-plant called “Ryorigikw” is the one which is most commonly attacked. In the Botanic Garden, Koishikawa, Tokyo, a form ealled “Kiazami,” perhaps closely allied to “Ryorigiku,” is also attacked every year. Further, we have observed at several localities that BIOLOGY OF THE CHEYSANTHEMUM-RUST. 35 the rust is found on Chrysanthemum sinense Sab. var. japonicum Max, (“Rytndgikw’), the common wild chrysanthemum. In Tosa it is also found on C. Decaisneanum together with other rusts. Bordeaux mixture is very effective, when used repeatedly, in pre venting the propagation of the rust. Brown Rust (Uredo autumnalis Diet.)" This rust is characterised by its light brownish colour. The sori are very small, and appear densely scattered over the surface of the host. In coniradistinction to the rusts previously described the sori are pro- duced much more on the ‘wpper than on the under surface of the leaves. So far only the uredosori have been observed, but it is probable that they represent a stage of certain Puccinia. In external form the present rust is hardly distinguishable from Uredo Artemisiae japonicae Diet? widely distributed on Artemisia. The character of the uredospores is also lke that of the latter rust, having a faintly coloured thin wall. However, in the materials examined by me the sori are not provided with paraphyses, which occurs invariably in Uredo Artemisiae japonicae, as Dreren has already observed.* The mode of wintering has not yet been ascertained, but it is most probable that the uredospores can winter without losing their germinating power, as they do in Puccinia Chrysanthemi in Europe. This must be quite possible in warmer regions like Tosa where it occurs commonly. In the vicinity of Tokyo the rust is only known on Chrysanthemum sinense var. japonicum, but in Tosa it occurs on several chrysanthemums, viz. C. Decaisneanum, C’. sinense (cultivated form), and C’. indicum. From the communication of Mr. Yosuryaca we know that the cultivated chrysanthemums there suffer as much from this as from the black rust. 1 DizteL, P., Uredineae japonicae. VI. Engl. bot. Jahrb., Bd. XXXVII, 1905, p. 108. 2 Dieter, P., Uredineae japonicae. V. Engl. bot. Jahrb., Bd. XXXIV, 1905, p. 591. - 3 DrETEL, P., Uredineae japonicae. VI, p. 108. 36 Sa kUSANO: We see from the observations described above that all the rusts known io eceur on Chrysanthemum attack Chrysanthemum Decaisncanum in Tosa, where they also have a tendency to occur on C. sinense, but in Tokyo and its vicinity they show more or less specialisation among them- selves with regard to their hosts: thus Puccinia Chrysanthemi oceurs on C. sinense and C. indicum, P. Horiana on certain garden-varieties of C’. simense and on C. sinense var. japonicum, and Uredo autumnalis on C’, sinense var. japonicum only. We also sce that the rust on the eultivated chryvsanthemmn in Kurope and that on Chrysanthemum Decaisncanum in Japan show no difference so far as their mode of development is ecen- cerned. These facts Justify our asstunption that the chrysanthemun:rusts vow occurring on several hosts have originated from C. Decaisneanum. Notes on Japanese Fungi. V. Puecinia on the Leaves of Bambuseae.* BY S. Kusano. ; With Plate VI and one Figure in the Text. Five species of Puecinia have been known to occur upon the leaves ef bamboo-plants, of which two, viz. P. longicornis Pat. et THariot and P. Kusanoi Viet., are indigenous to Japan, while the rest are exotic.? My attention has been directed to this subject for some years and an exhaustive examination of all the species of bamboo to which I have been able to get aeces- has cnabled me to add two more species and one variety still un- described. To a description of these indigenous rusts I propose to append a complete list of their hosts, which now amounts to eighteen in number, belonging to three genera (Phyllostachys, Arundinaria, and Sasa), and thus place the relation of the rust to the several species of its host in a clear light. As is well known, the vegetative organs of the different species of bamboo exhibit hardly any distinctive features characteristic of each, while the floral organs, the most important for the study of affinity and specitic characters, are developed exceedingly rarely. On this account, although the study of our bamboo-plants made recently by Maxrvo and Suisara’ from the systematic and anatomical standpoints * Contribution from the Botanical Laboratory. * Puccinia Arundinaiiae Schw. in America (Syn. Fung. Car., 1822, p. 72; Sacce., Syll. Fung., Bd. XIV, p. 355; Bot. Gaz., Vol. XXXIV, 1902, p. 1; Sxp., Monogr. Ured I, p. 731), P. canthosperma Syd. in East India (Ann. Myc., Bd. IV, 1906, p. 437), and P. melanocephala Syd. in East India (Ann. Myc., Bd. V, 1907, p. 500). * Makino, T., Description des Produits forestiers envoyés 4 lExpostion uniyerselle de 1900 & Paris par le Ministére de Agriculture et du Commerce ;—, Bambusaceae Japo- nicae. Bot. Mag., Tokyo, Vol. XIV, 1900; Sumatra, K., Beitriige zur Wachstum- geschichte der Bambusgewiichse. Journ, of Coll. Sci. Tokyo Imp. Uniy. Vol, XII, 1900; Maxkrxo, T. and Suipara, K., On Sasa, a new genus of Bambuseae, and its Af- finities. Bot. Mag., Tokyo, Vol. XV, 1901, p. 1S. 38 S. KUSANO: has rendered the systematic position of each species very definite, it seems to me that an account of the specialisation of the rust on certain species of bamboo is important from a practical point of view for the determination of the genera and species, in case only their branchlets or leaves are accessible. In order to avoid any possible error regarding the specific name of the hosts, due to an examination of sterile specimens in a dried state, I have consulted as far as possible living specimens which had been identified by Mr. Maxino, the authority on Japanese bamboo. They are all cultivated in the Botanic Gardens both of the Science College and the Agricultural College in Tokyo, with the single exception of Sasa ramosa which is found flourishing in Séma in Proy. bwaki. I desire in this place to acknowledge my indebtedness to Prof. Miyase who has generously put at my disposal the rich herbarium speci- mens of the Museum of the Agricultural College of Sapporo, which have been particularly useful for a study of the distribution of the fungi concerned. Puccinia Phyllostachydis Kusano un. sp. (Figs. 1, 2, 10, 11.) Uredosori hypopivllous, isolated, early becoming naked, pulverulent, ferruginous ; paraphyses hyaline, swollen and thick-walled at the top, with a septum near the top’; spores obovate, 30-33x23-25 y 3; epispore brown, coarsely echinulate; pores + (rarely 5), equatorial. Teleutosori hypophyllons, isolated, scattered, prominent, round, com- pact, 0.5 mm. in diameter, dark brown or black; spores oblong, rounded at the upper end, slightly taperimg towards the lower end, constricted at the septum, wall scarcely or not thickened at the apex, finely dotted, brown, 45-70 x 15-20 #; pedicel persistent, slender, hyaline, more than 150 » long. Common in various places in Tokyo. * I can not yet exactly decide whether they are young sporophores or not. ae NOTES ON JAPANESE FUNGI. 39 On Phyllostachys bambusoides Sieb. et Zuce. (== Ph. megastachya Steud. Ph. macrantha Sich. et Suce., Ph. Quirioi Riv., Ph. Mazeli Hort.) B.G.*; Komaba; Kyoto*? (Y. Taxsuasut, July 4, 1895; N. Hiratsuka, dune 12, 1895). Phyllostachys bambusoides Sieb. et Zuce. var. aurca Makino (aurea Riv;). B.C. Phyllostachys bambusoides Sieb. et Suce. var. Marliacea Makino (=Ph. Marliacea Mitf.). B.G. Phyllostachys bambusoides Sieb. et Zuce. forma Nasirodake Makino. B.G. In general feature the teleutospores appear to resemble those of Puccinia Avundinariae Schw. with thin-walled apex, judging from the figures of J. C. Arrsvr,® but the present species iy distinguished by the presence of paraphyses in the uredosori. It is a very interesting fact that this species is confined to the four forms above mentioned, which according to Maxrno must al! be referred to Ph. bambusoides.4 As may be seen from the synonyms, these had been regarded as so many different species, but the specialisation of Puccinia Phyllostachydis on these bamboos points to a more close relationship among themselves in contrast to the others, and so Maxino’s revision appears justified from the mycological point of view. When the branchlets and leaves alone are taken into consideration, Phyllostachus bambusoides vay. aurea resembles very closely Ph. puberula Munro, but the occurrence of the rust, if any, will enable one to distinguish the two. * B. G.—Botanic Gardens, Koishikawa, Tokyo. ?.=Dried specimen. 2 J. C. Aprnur, The Uredineae occurring upon Phragmites, Spartina, and Arun- dinaria in America. Bot. Gaz., Vol. XXXIV, 1902, p. 1. * The genus Phyllostachys includes 4 species and a number of varieties. Bot. Mag., Tokyo, Vol. XIV. 1900, p. (61). 40 S. &USANO: Puccinia longicornis Pat. et Hariot. Buil. Soe. Myce. Franee, 1891, p. 143; Sacc., Syll. Fung., Bd. XI, p.. 200; Syp., Monogr. Ured., I, p. 784; —, Exic. Ured., n. 1314. This is a species of common occurrence. As far as my observations go, it is found every vear in the Botanic Garden at Koishikawa and other localities in Tokyo. The formation of the teleutospores at a certain season of the vear is so vigorons that they form solid, ferrnginous to dark brownish, orbicular pustules of 1 mm. or sometimes more in diameter, and cover densely the under surface of the leaves, doing much damage to the host. Tn general features the uredosori resemble those of the preceding species. They are however characterised by having clavate paraphyses (Fig. 4). The spores are globose or oval, and coarsely echinulate. Four or rarely five germ-pores are present in the equatorial plane. The typical teleutospores are elongated, fusiform, well constricted at the septum, and tapering at both ends. Some deviating forms resemble those of the typical spores of P. Kusanoi, or its variety to be deseribed below (Fig. 3a, b). The distinctive character of the spore is the extra- ordinary thickening of the wall of the apex so as to form a long papilla of 324 in length. The typical ones measure 80-100 x 15-20 4. Pedicel is hyaline, slender, and more than 200 y long. Only two species of bamboo have so far been ascertained as host:-— Sasa paniculata Makino et Shibata (—Bambusa tessellata Munro," B. palmata Marl., BP. paniculata Makino, B. senansis Franch. et Sav., Arundinaria palmata Bean, A. kurilensis var. paniculata Fr. Schw., ete). B.G.; Sapporo* (T. Mrvaxg, May 4, 1902). Arundinaria japonica 8. et Z. B.G.; Nagoya in Prov. Owari™ (Y. Taxanasnt, July, 1895). , Dieter, Uredineae Japonicae. T. Enel. bot. Jahrb., Bd. XXVIII, 1899, p. 568, NOTES ON JAPANESE FUNGI. AY The fact that the presenf rust is confined to the two hosts mentioned above seems to show the close relationship of the latter, though they are new inclided in different genera. The bamboos now included in Susa were formerly considered to belong to Bamlusa in the section Enbambuseze, but their anatomical as well as morphological characters necessitated 4 revision, and thus a new genus Sasa was erected conjointly by Maxryo and Sureara.* To this genus are now referred eight species which were all formeriv included in Bambusa, Taking the rust as our enide it appears that Sasa and Arundinaria are allied genera. The above authors have already remarked that Arundinaria japonica is a somewhat aberrant form in the genus, and stands rather close to Sasa in the character of the culm and leaves, and in having at times more than 3 stamens*: it is in fact a transitional form between the two genera. Hence it is not strange that Arundinaria japonica should harbour the rust apparently peculiar to Sasa, instead of the one which occurs commonly on several species of Arundinaria. Puccinia Kusanoi ict. (Figs. 5, 13, 14.) Engl. bot. Jahrb., Bd. XXVITT, 1899, p. 568; Sacc., Syll. Fooe:, Bd. XVI, p. 309; Sxe5 Monoer. Ured., I, p. 13825 —, Exic. Ured. n. 1313, 1373. (=Uredo Arundinariae Syd.). Wedwigia, 1898, p. (208) ; Sacc., Syll. Fung., Bd. XTV, p. 406; Syd., Exie> Ured. n. 1239. (=Puccinia Bambusae Selircet. in herb. ) This rust is most common owing to the wider distribution of its host. It is found mostly on species of Arundinaria, thus :— Arundinaria Simoni A. et C. Riv. B.G.; Tokvs* (S. Horr, March 19,1891; K. Mryase, Sept., 1889; K. Onxcma, April 30, 1883; K. Sencokrv, Nov. 25, 1895, Jan. 14 1896: T. Nisuipa, Oct. 24, 1899) ; + Makino, T. and Snrpata,. K., loe. cit. 2 Phyllostachys and Arundinaria have 3 stamens. while in Bambusa and Sasa they are 6 in number. 492 SeRUSANO: Kazusa* (K. Seneoxt, Jan. 1, 1896); Hakone* (S. Hort, April, Omiya near Tokyo* (N. TIcntKawa, Oct., 1886); Chiba in Prov. 1891; K. Mrvazse, Apri] 12, 1901): Gifu* (N. Toxusucnr, Oct; 1889, Dec. 3, 1898). A. Simoni A. et ©. Riv. var. Chino Makino. B.G.:- Komaha: Sendai* (K. Smncoru; Oct 5. 1895 ). A. Simoni A. et-C. Riv. var. variegata Hook fil. B.G. A. variabilis Makino var. viridi-striata’ Makino (=A. Forlinet fine 2 Ao. A. variabilis Makino var. Tanakae Makino. Womaba. {. variabilis Makino forma foliis pubescens Makino. Hirosaki ate in Prov. Mutsu* (N. Hrratsvca, Oct. 26, 1896): Shiroishi im Proy. Iwaki® (Y. Taxanasnt, Aug. 1, 1895); Nagoya in Prov. Owari* (Y. Taxanasur, July 19; 1895). A. variabilis Makino forma foliis glabris Makino, Tokyo* (Y. Taxanasnt, July 21,1895); A.: Narthira Makino. BiG. ;, Komaba. A. Narihira Makino forma YVashadake Makino. JKomaba. Sasa sp. OS. nipponica Mak. et Shib. 7). B.G.; Prov. Rikuchu* (Y. Taxanasui, Nov. 7, 1897, May 13, 1893). Thus, out of eight species of our native Arwndinaria? the fungus eceurs on three species and their varieties. Of these hosts, the first two are most widely distributed, from south to north, especially however in the central part of Japan, and are everywhere subject to attack by the rust. The uredosori do not differ essentially in appearance from those of the other species, except that they lack paraphyses which characterise two species deseribed before. The spores are obovate to subglobose, with coarsely echimulate wall. The size varies, more or less, according to the host. For instance, it is smaller on Arundinaria Simoni. measuring ZO nok oe Ioan 30 4, but on the other hosts it may attain * Engl. bot. Jahrb., Bd. XXVIII, 1899, p. 569. * Bot, Mag., Tokyo, Vol. XIV, 1900, p. (61). XOTES ON JAPANESE FUNGI. 43 to 27 x 28 or even 27*35 yw. There are apparently three or four germ- pores in the equatorial plane. The teleutospores form solid, firm, orbicular sorus just as in the preceding species. The typical ones are oblong or fusiform, rounded at both ends, the wall conically thickened at the apex. They measure 61x17 yp or thereabout, but there may occur even in the same sorus very short and broad spores measuring from 42 « 22 to 40 x 20 4. Moreover, slight variation in form and size may occur according to different hosts. For instance, on A. Varihira forma Yashadake large spores predominate wheh may measure as much as 88 x 16 er 70 « 15 4. Sometimes they are iinear oblong, not constricted at the septum and tapering gradually at both ends. On A. Simoni and its varieties Chino and variegata the constriction at the septum is somewhat conspicuous; on A. Varihira the apex of the spore is rounded, the constriction is more obvious, and the shorter ones are more predominant; and on A. variabilis var. viridi-striata and A. Narihira forma Yashadake larger ones with a slight constriction at the septum are more usual. With respect to the occurrence ct the rust upon a species of Sasa a question arises again concerning the affinity of Arundinaria and Sasa, and also concerning the specifie name of Sasa in question. The plant has been cultivated for a long time in the Botanic Garden of Tokyo but has never produced floral organs. Mr. Maxtyo inclines to the view that it is Sasa ramosa Makino et Shibata, though the sterile specimens do not show distinctly any character to distinguish it from Sasa nipponica Makino et Shibata. As the rust on the Sasa in question is somewhat different from that on the typical Sasa ramosa, which will be given below, I believe it better to assume at present the questionable Sasa as WS. nipponica. The rust on S. nipponica bears a more close resemblance to that on Arundinaria Narihira forma Yashadale (Fig. 5 ¢-f). The uredo and teleutosori are smaller than on other hosts; the oval uredospores measure from 40 x 20 to 42 x 25 4; the teleutospores are long and regular in form, well constricted at the septum; and the typical ones measure 44 Sa USANO’ from 72 x 19 to 65 x 20 p, but deviating ones may be 80 x 17 —- 55 290 v. The general characiers of the rust on S. nipponica are therefore quite the same as those of the rust on most -Lrundimaria. This singular fact indieates that some species of Sasa may be closely allied to the genus Arundinaria, As has been ascertained from ana- tomical as well as floral characters’ certain species of Sasa resemble Arundinarig Simoni, just in like manner as A. japonica ap- proaches to Sasa. In the present case it seems probable that S. nipponica stands next to S. ramosa which in turn is closely related to A. Simoni: S. nipponica is phylogenitically nearer to A. Simoni than to A. japonica’. Puccinia Kusanoi Diet. var. azuma [Kusano n. yar. (Figs. 6, 12, 15.) Uredosori hypophyilous, isolated, small, naked, pulvernlent, ferru- ginous, without paraphyses; spores oval to obovate, 25-28x20-25 rings epispore brown, coarsely echinulate, germ-pores +3, equatorial. Telentesori hypophyllous, isolated, scattered, prominent, round, compact, 0.5-1.0 mm. in diameter, black to brown; spores linear oblong, tapering towards both ends, not or rarely constricted at the septum, wall conically thickened at the apex, brown, finely verrucose, 55-87 12-20 » ; pedicel persistent, slender, hyaline, more than 200 / long. It has been found at Koganei near Tokyo (T. Maxryo, May, 1894)*, and S6ma in Proy. Lwaki. On Sasa ramosa Makino et Shibata (<= Bambusa ramosa Makino, Arundinaria ramosa Makino). In the general form of the teleutospores the present species resembles Puccinia iongicornis, but differs from it in having the wall ‘ at the apex less thickened. Generally the constriction at the septum is exceedingly slight, and the spores are longer than those of P. Kusanoz. At times we find some short and broad spores which resemble the typical 1 Makino and Surpara, lee. cif. 2 See Maxtno and Smteara, loc. cit., p. 30, NOTES ON JAPANESE FUNGI. 45 spores of P. Ausanoi, but since the above mentioned characters of the spores as well as others remaim constant on S. ramosa, I think it better io distingnish the rust 2s a new variety. This will seems natural when we take into consideration the relationship of the host to Arundinaria. The authors of the genus Sasa remark, “Sasa ramosa, which we include provisionally in our new genus, resembles, however, Arundinaria Simoni in its floral characters, and has often less stamens than 6. Further study will decide whether it represents the type of a distinct genus or not”' Thus, the host seems to be different from other Sasa, intermediate between Arundinaria and Sasa, and for this reason it is not strange that its parasite is closely related to P. Kusanot. Intection-experiments will perhaps decide whether the rust on S. ramosa is or is not specifieally identical with that on Arundtnaria Simoni. Puccinia Sasae Kusano un. sp. (Figs. 7-9.) Uredosori hypoplivllous, isolated pulverulent, terrugimous ; parapliyses hyaline, swoilen at the top; spores globose or subglobose, 32-35 4 ; epispore very thick, brown, coarsely cchinulate ; germ-spores 5, equatorial. Teleutosori hypophyllous, isolated, prominent, round, compact ; spores oblong, rounded at the upper end, tapering towards the lower, constricted at the septum, lower cell generally longer, wall not or shelhtly thickened at the apex, finely verrucose, brown, 37-80 x 14° 20 ws. On Sasa borealis Makino et Shibata. Nikko* (T. Makino, Aug., 1905). In a single dried specimen the rust was imperfectly preserved and almost all the teleutosperes had germinated. In general appearance the teleutospores resemble those of Puccinia Phyllostachydis, but the upper eell is mostly wider and shorter than the lower. Among a large number of longer, thin-walled, light brownish telentospores we find a few shorter * Makino and Snieara, loc. cit., p. 30. 46 S. KUSANO: and broader, thick-walled, dark brownish ones, The paraphyses resemble those of P. longiecornis. The uredospores are the largest and have the thickest wall of the rusts deseribed above, and they are also characterised by having 5 germ-pores. Although the specimen is somewhat imperfect, still these characters show beyond doubt that it can not be referred to any of the other species that are formd on the bamboo-leaves. General Characters and Development of the Fungi. All the known species of Puceinia on the leaves of our bamboo-plants present externally nearly the same characters and can scareely be distinguished from each other. The only differences which ean be regarded as of diagnostic value are the structure of the uredosori and Mext= bic sen Young shoot of Sasa paniculata. ad, at the time the teleutospores germinate; 4, at the end of May. the form of the teleutospores. In Puccinia Phyllostachydis, P. longi- cornis, and P. Sasae characteristic paraphyses oceur which are wanting NOTES ON JAPANESE FUNGI. 47 in P. Kusanov and its variety; and the thickening at the apex of the teleutospore is exceedingly slight mm P. Phyllostachidis and P. Sasae,, prominent in P. Kusanot and its variety, and most remarkable in P. longicornis. The most vigorous formation of the teleutospores takes place, so far as observed in Tokyo, in February and Mareh, and they come to germinate at the beginning of April, whenever moisture is supplied in sufficient quantity. The uredosori appear all at once on the same leaves on which the teleutospores are developed, or on the young leaves of new shoots, attaining their full development at the end of May. The portions of the young leaves on which thé uredosori first appear, are strictly those that were exposed exactly at the time the teleutospores were germinating (Text-fig. 1). It is therefore highly probable that the uredosori originate from the sporidia. The rust then proceeds to attack the voung leaves as they come ouf one after another, developing successively new uredosori upon them. The development, however, is exceedingly retarded in the summer and autumn, and so the leaves developed later are for the most part apparently healthy, though a few discoloured spots may be observed, which are points infected by the uredoypores. As winter approaches we recognize here and there a few teleutosori appearing from these diseased spots, but the number of the sori is not so numerous in midwinter as in the early spring. This is an ecologically interesting fact. Usually the teleutospores are the hibernating spores in many Uredineae, and they are formed at the beginning of winter, or sometimes when the host has completed its vegetation period, which occurs in some species in the summer, In bamboo-plants, the leaves are all perennial, and survive mostly till the end of the second year. Hence, if the cold season is concerned in the formation of the teleutospores: in the bamboo-rust, fit seems that the teleutospores must ali be formed in or before the winter, instead of in the next spring. The fact being, however, otherwise, we must aseribe this mmusual mode of development to another unknown cause, for which a further study is required. The unfavourable conditions of the autumn and winter check the 48 S. KUSANO: development of the fungus, and it is practically in a resting state durmg these seasons. The numerous fine discoloured spots which we find during the winter on the leaves are produced by the uredospores disseminated during summer, and the intercellular mycelium developed in these spots is mostly inhibited from further development till the spring of the next vear: the fungus can therefore winter in the form of sterile mycelium in the tissue of the host, as many perennial fungi do. The formation of the teleutospores in the spring appears to be a means of propagation for the fungus upon the young leaves by the formation of sporidia. As the — function of the teleutospores is thus somewhat different from that of many other rusts, it does not seem strange that the wintering mycelium in the infected spots does not always develop teleutospores, but uredospores as is often observed especially during warmer winter'. The mode of germination of the teleutospores is quite similar in all the species. Kept in a moist chamber they germinate in from 24 hours to a few days, the first noticeable change being the production from the upper cell of a short curved promyeelinm consisting of four cells each bearing a pyriform sporidimn. In water they send out a long germ-tube which may sometimes grow to 700 yp, usually with no septum (Fig. 11), or produce unusaully lone and thick sterigmata (175 »# ) with or without sporidia (Figs. 12, 13). The formation of abnormal promycelimm is common in the rust on Sasa nipponica, and appears to take place under the same ecnditions as in other species. Wlule the sterigma is usually produced near the upper septum, the second sterigma appears in this species constantly at the lower septum (Fig. 13). This is the character by which we can distinguish the rust on Sasa nipponica from Puceinia Kusanoi on Arundinaria, but it is of too little value to be taken as a specifie character of the rust on this problematic Sasa. March, 1908. * It was most remarkable in the spring of 1907 on Arwndinaria Simoni, A. Narihira f. Yashadake, and Pliyllosiachys baintbusoides, 4 NOTES ONS SAE ANESE FUNGI. Key to the Species. I. The wall at the apex of the teleutospore thickened very slightly. a, Uredosori with septate paraphyses ........... . LP. Phylostachydis Kusano. 4. Uredosori with non-septate paraphyses .................. P. Sasae Kusano. Ti. The wall at the apex of the teleutospore thickened conically. a. Teleutospore broad and constricted at the septum .... P. Azsanoi Diet. 4. Teleutospore long and not constricted at the septum P. Kusano Diet. var. azuma Kusano. Ill. The wall at the apex of the telentospore produced into a long papilla ; uredosori with paraphyses ......... P. longicornis Pat. et Hariot. Hest-Index. Arundinaria japonica Sieb. et Zuce... ... ... ... ... Puccinia longicornis Pat. et Hariot. maven Makino ... =. seeeweee--- «..... P. Ausana Diet. - A. Narihira Makino, forma Vashadake Makino ... ... P. Kusano Diet. eawrninE Aa et C. Riv. :.. “cee... ... ... 2. Kusanoz Diet. A. Simont A.et C. Riv. var. Chino Makino ... ...... P. Kusanoi Diet. A, variabilis Makino var. Tanakae Makino ... ... ... P. Kusanoi Diet. A. SimoniA.et C. Riv. var. variegata Hook fil ... ... P. Kusanoz Diet. A, variabilis Makino var. viridi-striata Makino ... .... P. Kusanoz Diet. A. variabilis Makino forma foliis glabris Makiro ... ... P. Kusanoi Diet. A. variabilis Makino forma folits pubescens Makino... P. Kusanot Diet. Phyllostachys bambusoides Sieb. et Zuce.- ... ... ... P. Phyllostathydis Kusano. Ph. bambusoides Sieb. et Zucc. var. auvea Makino... P. Phyllostachydis Kusano. Ph. bambusoides Sieb. et Zucc. var. AMlarliacea Makino... P. Phyllostachydis Kusano. Ph. bambusoides Siel. et Zucc. forma Kashirodake Makino. P. Pihytlostachydis Kusano. Sasa perealis Makino et Shibata eee. .. ...* £& Sasae Kusano: S. nipponica Makino et Shibata (?) ... 2... ... rath: P. Kusanot Diet. ” |S. paniculata Makino et Shibata... =: =... <.... .:. P. longicornis Pat. et Hariot. S: vaniosa Makino et Shibata’ ... 1... 22... 2... .... PF. Kusanoi Diet. var- azuma Kusano. 49 50 S. KUSANO: | Explanation of Figures. All figures except Figs 7—9 are drawn from fresh specimens and magnified 400 times. Fig. 1. Teleutospores of Puecinia Philostachydis. a, longer spore; b-e, typical spores. Fig. 2. Paraphyses in the wredosori of Puccinia Phyllostachydis. Fig. 3. Teleutospores of Puecinia longicornis, a. b. shorter spores: e-f. typicai spores. Fig. 4. Paraphyses in the uredosori of Puccin‘a longicoi nis. Fig. 5. Teleutospores of Puceinia Kusanoi. a,b. on Arundinaria Nimoni var. Chino; ¢-f. on Sasa nipponica ‘ 7). ‘ig. 6. Yeleutospores of Puccinia Nusanoi var. azuma. a, short and broad spore: ae h-e, typical spores. Fig. 7. Teleutospores of Puccinia Sasae. a-c, typical spores; d-e round forr. Fig. 8. Uredospores of the same. a, ripe spore; b, young spore. Fig. 9. Paraphyzes in the uredosori of Puccinia Sasae. Fig. 10. First stage of germination of Puccinia Phyllostachydis on Phyllostachys bLambusoides var. aurea (after a week). Fig. 11. Germination of the same in water (after a week). Fig. 12. Germination of Puccinia Kiusanoi var. azuma in water (after 24 hours). Fig. 13. Germination of Puceinia Kusanoi on Sasa nipponica (7) in water (after 24 hours). Fig. 14. Germination of the same in mo:st air. Fig. 15. Basidium and sporidia of Puccinia Nusanoi var. azuwina, —=——— ets = x —_— A omens: — in Ka wy Se ee =o OO , = i OSCE NAG OES yaa < ( nO Gece (aa 4 - BULL. AGRIC. COLL. VOL .VifT. On the Parasitism of Siphonostegia (Rhinantheae) .' LY S. Kusano. With five Figures in the Text. Since the discovery of the parasitism of Rhinantheae by Decatsnr? numerous genera belonging to this subfamily of Scrophulariaceae have been ascertained as hemiparasites*. Although there is but little doubt that the remaining genera may perhaps also contain such plants, yet no one appears te have examined closely their root-system; so that! many of them have stili to be studied on this point. Of our Rhinantheae we have two widely distributed but little studied indigenous genera: Monochasma aud Siphonostegia. Without detailed descriptions the former plant (J/. Shearcrt Max.) was already enumerated among parasites in Sutras “Diseases of Plant” written in Japanese (1894), and I can now confirm his statement by my own examination of its root-system, but nothing has vet been recorded about Siphonostegia, whether it is an autophyte or a hemiparasite. During my study of Japanese phanerogamic parasites, | became convinced already in 1898 that Siphonostegia chinensis Benth. was a hemiparasite, but at that time I thought it better to report this fact after studying its biology and physiology. Owing to various circumstances I have however been unable to carry my intention into effect, so that J shall give in this paper only an account of the structure of the haustorium. * Contribution from the Botanical Laboratory. A short account has already ap- peared in Japanese in Bot. Mag., Tokyo, Vol. XVIII, 1904, p. (144). 2 Decaisne, J., Ann. d. sci. natur., III Sér:e. t. VII, 1847, p. 5. * Welainpyrun, Tozzia, Huphrasia, Orthantha, Odontites, Bartschia, Alectrolophus (Rhinanthus), Pedicularis, ete. 3P SS eSA NO: Siphonostegia chinensis is 2 herbaceous plant common on grassy fields in the central part of Japan. Over its whole root-system, which is as well developed as that of an autophyte, there occur numerous haustoria of various size. Many of them appear as lateral swellings of the root. but oid haustoria occupy frequently a terminal position of the older roots, similarly as in Lathraca*, Buckleya*, and Santalum’. The form is mostly globular or ovai, bemg nearly sessile, and the size varies according to that of the mother-root, the largest one that has come under my observation being 2 mm. in diameter (Fig. 1). As to the anatomical structure the haustorimm of Siphonostegia is very different from the same organ of other Rhinantheae, so far as the arrangement and relative size of the three main parts are concerned, namely cortex (“Rinde”), nucleus (“Kern”) and sucker (“Saugfortsatz”). According to Prrrat, Serms-LavBacn”, Lrciterc pu Sasion®, Koon Voikart’, Sperricn’, an: others, these parts are not essentially different in strueture im all the Rnimantheae studied by them. From the in- vestigation of Speriicu we see that these three parts have each a function of its own and are important to the haustorium for its action as absorbing organ. But in the haustorium of Siphonostcgia it is scarcely possible to distinguish these important parts distinctly. The cortex, which SperLicH has been taken as a reservoir of carbohydrates, occupying the greater part ot the haustorinm, shows no peeuliar structure that seems especially adapted to play such a function: it consists simply of loosely connected parenchymatous cells, as in the cortex of the mother-root itself, and serving as a reservoir of carbohydrates in a merely subordinate way. + Hetxnicner, E., Cohn’s Beitr. z. Biol. d. Pfi., Bd. VII, 1895, p. 315. * Kusano, S., Journ. of the Coll. of Sci., Vol. XVII, 10, 1902. 1. 1906. * BaRBer, C. A., Memoirs of the Dept. of Agric. in India. ‘ Bot. Series. Vol. I, No. 1., 1906. 4 Pirra, A., Bot. Ztg., Bd. XEX, 1861. * Somrms Lavpacy, H. Gra¥ zu, Jahrb. ft. wiss. Bot., Bd. VI, 1865, p. 566. * LecLERC pu Sapion, Ann. d. sci. natur., VII. Série, t. VI, 1887, p. 90. * Kocu, L., Jahrb. f. wiss. Bot., Bd. XX, 1889, p. 1 and Bd. XXII, 1891, p. 1. S VotKart, A., Untersuchungen tiber den Parasitismus der Pediculasisarten, 1899. Ziirich. ° Sperricu, A\., Beibefte zum Bot. Centlbl., Bd. XI, 1902, p. 437. ON THE PARASITISM OF SIPILONOSTEGIA., 29 Remarkably divergent is the structure of the nucleus. Both from the illustrated descriptions of the same part by various authors and from my own observations in other Rhinantheae, we know that it shows the same characteristic in all the plants studied thus far in having a hyaline tissue of wide extent with a few tracheidal strands in its centre, and distinguished from the surrounding cortical parenchyma by having much smaller, larger-nucleated cells rich 12 plasm. To this tissue Sperrice attributed the elaboration of the formative materials for the parasite. The tracheids here appear in longitudinal sections mostly in two or three rows extending from the mother-root to the host. In none of the haustoria of Siphonostegia examined by me have I been able to detect such a distinct hyaline tissue in a corresponding position. In its place there occur massivety developed tracheids of just same extent. Numerous paren- chymatous cells are found scattered here and there among the tracheids, which seein to correspond to those of the hyaline tissue of other species. The tracheids are irregular in form but appear to retain the form of the parenchymatous cells from which they have been differentiated. Contrary to the great development of the tracheids in the nucleus we see in the neck, which lies between the haustorium and the mother-root, exceedingly few tracheids; so that the relative development of the characteristic tissues is the reverse of what we find in other Rhinantheae. They form in the neck a few isolated strings traversing the paren- chyma and cxtending from the vaseular strand of the mother-root to the nucleus of the haustorium. On the whole, the development of the tracheids in the haustorium is quite the same as in Buckleya (Fig. 5). In the rext place it is exceedingly difficult to point out a sharply defined part as sucker that usually represents the frontal portion of the haustorium in most of the parasites above quoted. In the adult haustorinm attached to a slender host-root the frontal portion of the nucleus seems to come directly in contract with the living tissue of the host, accompanied by the inner layers of the cortical parenchyma (Figs. 2, 3). The above account may hold true with a somewhat younger haustorium, though the relative size of the three parts differs slightly: the 54 S-ERUSANO: nucleus is narrower with much more parenchymatous cells, and the frontal] portion including the nucleus and ja portion of the cortex penetrates nore or less inte the cortex of the host-root, and assumes the appearance of the so-called sucker. To make the difference in structure between the haustorium ot Siphonostegia and the otier Rhinantheae still more clear I may take for comparison a figure of the haustorium of Alectrolophus (Rhinanthus) given by Sorms-Lavsacu’ (Fig. +), with which the haustoria of all the other Rhinantheae essentially agree in structure so far as the main parts above mentioned are concerned. Ilere the hyaline tissue composing the nucleus is very prominent, and the tracheids which traverse its median portion, very few as they are, developed in just the sufficient and necessary amount to convey the water taken up by the sucker to the mother-root. The differentiation of the frontal portion mto fhe suexer is also apparent. A similar disposition of parts is found also in the haustorium of some Orobanchaceae*. We can therefore distinguish the haustorium of Siphonostegia from that of other Rhinantheae by the following characters :— 1. The absence of the massive hyaline tissue in the nucleus which is sharply distinguishable from the cortex. 2. An abundant occurrence of the tracheids in the nucleus. 3. The oceurrence of very few tracheids between the mother-root and the haustorium, as compared with the nucleus. 4. The obseure demarcation of the sucker from the other part of the haustorium. These characters show that the haustorium of Siphonostegia presents a close resemblance in strueture to the same organ of Santalaceae (Fig. 5). Considering that the tracheids generally serve for the conduction of aqueous solutions, it will not be superfluous to add a few words here 1 Soums-LauBacn, Joc. cit., Pl. XXXIV. Fig. 2. * HEINRICHER, loc. cil. Cr Or ON THE PARASTTISM OF SIPILONOSTEGIA, regarding the probable function which the massive tracheids occurring in the haustoriuin ef Siphonostegia and Santalaceae must fulfil. For the mere purpose of conveying aqueous solutions from the host to the parasite their abundant occurrence in the nucleus strikes us at once as being dis- proportionate. As I have stated above in Siphonostegia and as I have mentioned elsewhere in Santalaceae’, the passage of aqueous solutions between the host and the haustorium as well as between the haustorium and the mother-root takes place through a few strings of tracheids. The widely spread tracheidal mass in the nucleus is too conspicuous to be regarded as a mere continuation of the former. Considered from the merely anatomical standpoint we may with good reason look upon this structure as an example of ‘“Speicheriracheiden.” This name was first proposed by Heryricuer for the tracheids which are found in some xerophilous leaves” and in the sealy leaves of Tozz7a. He regarded this tissue as: a water-reseryoir in a wide sense, and says, “Bei Pflanzen trockenen Standortes sind die Speichertracheiden als Vorrathsreservoire fiir Wasser angebracht, die eyentuell eimtretendem Wassermange!] begegnen sollen. Bei Vozzia ist soleh cin Mangel nicht zu beftirehten, bei ihr handelt es sich darum, einem Zuviel an Wasser abzuhelfen. Die Speichertracheiden sind auch hier Wasserbehiilter; aber micht als ftir die Reserve wichtiges Material wird das Wasser in ihnen gesammelt, sendern win eine Erfiillung der Intercellularriume mit Wasser 22 verhiiten, wird es in den Speichertracheiden, die hier gewissermassen als Stauwerk dienen, untergebracht, wenn die wasserausscheidenden Organe, die Hydathoden, nicht schnell genug arbeiten sollten. | Die Speicher- tracheiden erscheinen demnach bei Tozzia als eim die Hydathoden erginzender Apparat’’*. The same argument may be applied to the ease of Siphonostegia and Santalaceaec. Since the transpiring organ, the leat, may have different structure in the host and parasite, it 1s obyious that the demand of water is not equal in the two, notwithstanding that 1 INUSANO, S., loc. cil. Hernricuer, E., Bot. Centlbl., Bd. XXIII, 1885, p. 25. Herrxricuer, E., Jahrb. f. wiss Bot., Bd, XXXVI, 1901, p. G60. we oo O06 5. KUSANO: Fig. 1. Fig. 1. Root-system of an adult Siphonostegia, bearing numerous haustoria. Nat. size. Fig. 2. Longitudinal section of a lateral haustorium. im. mother-root; /. host-root. 2, nucleus of the haustorium. 950. Fig. 3. Longitudinal section of a terminal haustorium. Mother-root is shown also in longitudinal section. m, n, h, as before. 22. . Vig. 4. Longitudinal section of a haustorium of Rhinanthus (Alectrolophus). mt, mother-roct; m, hyaline tissue in the nucleus: /. host-root. (After Sorms- Laubach) . ; Fig. 5. Longitudinal section of a haustorium of Buckleya, m, mother-root; n, tracheids in the nucleus; /, host-root. 5 (After S. KUSANO). ~ ON THE PARASIRISM OF SIPHONOSTRHGIA. 57 it has to be supplied by the host-root alone. From the anatomical cha- racters of the leaves and using “eobalt-test,” we may be sure that the transpiration, for insiance, in Buckleya* surpasses that im any of its host— conifers and some foliage trees; and its variation during the day and night, and under different conditions of environment, may be greater than in its host, er the supply of water by the host-root may not necessarily accompany its loss by the parasite. It may therefore oceur that, under a condition which promotes transpiration in the parasite, the amount of water necessary to replace it may not be supplied by the host. At any rate it is certain that the demand and supply of water in the parasite is not always uniform under the different conditions of environment. Again, the anatomical characters of the leaves of Siphonostegia point out that it absorbs water from the host under similar condition as Buchleya. Therefore it is highly probable that in both parasites the massive tracheids in the nucleus of their haustoria may act as a reservoir of water, serving either to supply or retain it according to cireumbstances ; in short, they act as the “regulator” of water-supply for the parasites. The problem here presented may apply to all the other phanerogamic holo-and hemi-parasites, and I think the development of tracheidal element in the hansterium may be connected with the mutual relation of the parasite and the host with particular regard to the amount of water which they require. To ascertain how far this view is correct, therefore, com- parative and more extended studies of all of these parasites is necessary. Groom® has stated that parasites in moist soil have leaves so constructed as to enable them to get rid of anv excess of water absorbed. We may here remark that the structure of leaves of the plants under consideration is not ouly correlated to the environment under which they grow, but is also intimately associated with their capacity of absorbing water which, in turn, depends upon tie structure of the haustorium, March, 1908. ‘ For the anatomical structure of its leave see Ben, Beitriige zur anatomischen Charakteristik der Santalaceen. Dissertation. 1895. = Groom, P., Journ. Linn. Soca Vomex xX; 1895, p. 149; Ann, Bot., Vol. XI, 1897, i ae Further Studies on Aeginetia indica2 BY S. Kusano. With Plate VIT. In my former paper (’03) some accounts were given of the morphology, anatomy, and biology of Aeginetia indica. So far as my observations went, this parasite showed no special character in the manner of its development, which can be distinguished from that of Orobanche as thoroughly investigated by Kocu (’83). When I undertook during the past year a further study of this parasite, particularly as regarded the germination of the seeds and the development of the seedlings, T could show that at an early stage of development Aeginetia displayed many peculiarities, some of which are perhaps unique. As the results obtained appear not only interesting in themselves, but also to contribute something to the knowledge of phanerogamic parasites, I think it advisakte to give them briefly in the present paper. Very little has as yet been published on the early stage of develop- ment of the Orobanchaceae. In Lathraea Wrinricuer (794, 795) made some experiments on the germination of the seeds and the development of the seedlings. According to him, the seeds show no features during ger- mination and further development that are worthy of special mention. The vegetative organs sre very much reduced in form, but the embryo does not differ essentially in structure from that of most autophytic plants, being provided apparently with a pair of cotyledons and a radicle. In germination the radicle first grows into a filamentous root which soon branches into numerous rootlets. The rootlets then produce haustoria : j ; Me WAR eae where they esrae in coniact with the host-root (ITrmvricner. 94. p. 1 Contribution from the Botanical Laboratory. 60 S. KUSANO: ~ (128)). Further he ascertained that the seeds require in germination the presence of proper host-root which he believed to exert a chemical stimulus. Koon (’83) extended our knowledge on Orobanche, and succeeded in raising seedlings from the seeds laid on or near a proper host-root. In this plant the embryo is so much reduced in form jas to appear like a younger stage of a dicotyledonous embryo (Kocu, 78, p. 259), being merely an oval cell-mass, and the changes that take place during germina- iion show certain peculiarities. At first the radicular half of the embryo develops into a filamentous root (Kocu, *83, p. 189), while the plumular half remains throughout in the endosperm, acting as an absorb- ing organ. Differing from Lathracn-scedling the parasitism of this seedling is effected by the root-tip, provided it abuts on a host-root lying before its course. In his eulture-experiments Kocn (’83) assumed that in germination the seeds required a chemical stimulus from the host-root. Such being all that we know, at present, about the early stage of develop- ment of the Orobanchaceae, it appears to me to be not the less interesting to extend our study on Aeginetia which exhibits a close resemblance to the last mentioned species of Orobanchaceae, on account of the structure of the seeds as well as the vegetative organs, and to ascertain how far what was tound on the latter plant is applicable to the former. While the present study was carried out with this end in view, I have never undervalued the problem about the condition which the seeds of such holoparasite require in germination. Although it has been iascertamed by the above mentioned authors that the stimulus of the host-root is in- variably necessary to germination in plants of this family, the nature of the stimulus has not yet been studied with accuracy. Concerning this point I can not yet express any definite view, but as it seems to me that the results of a few incidental experiments are suggestive for a further study on this subject, I will note them briefly in the present paper. Methods. The seed of Aeginetia being very jine and pulverous, a special treat- ment is required in observing its germination. In order to observe easily the successive stages of germination, and of the development of the FURTHER STUDIES ON AEGINETIA INDICA. 61 seedlings, I iransplanted, a month or two previously, some vigorous host- plants in pots of 15-20 em. in diameter. These being kept sufficiently moist, the plants began to produce after a while young rootlets mainly traversing between the wall of the pots and the soil inside. When a thick meshwork was thus formed by the rootlets, I lifted up carefully the plants from the pots, laid the seeds of Aeginetia upon the meshes, and then put the plants again in the pots as before. By taking the plants from time to time out of the pots without disturbing the arrangement of their root-system on which the seeds were laid, I was able to follow in detail the changes that took place during the germination and subsequently. The seeds used in the experiment were collected in the preceding year and kept dry. Under favourable conditions they germinated within two weeks in the early summer. However I could observe no germina- tion to take place in seeds preserved in a dry state for two years. It has not yet been ascertained how long the germinating power can be kept intact in seeds kept moist. This is a practically important matter in connection with the protection of the cultivated plants (Ktvsano, 03), in case they should be invaded by this parasite. Embryo. The embryo is microscopically small and enclosed in the endosperm packed with starch. In order to take it in tofo out of the endosperm the seed was treated a day or more with a concentrated solution of chloralby- drate. If such a seed be gently pressed under the cover glass, the endosperm would escape easily from the testa, and the embryo from the endosperm. The mature embryo thus taken out consists simply of a few isodiametric parenchymatous cells of nearly equal size. It is somewhat oval in form with its narrow end directed towards the micropylar end of the seed. No morphological differentiation into plumule, radicle or cotyledons being visible, it represents, as it were, the younger stage of an embryo of a phanerogamic plant. Optical section shows that at most two but often a single row of cells in the direction of the long axis of the embryo is enclosed by the epidermal cells (Figs. 1, 2). Very simple 62 S. KUSANO: as 1t may be in structure, still it is not difficult to point out both the radicular and plumular ends in the embryo. These become evident in a germinating seed; the narrow end, which often consists of smaller cells, corresponds to the radicle, while the other end represents the plumule. As a whole the embryo of Aeginetia has quite the same structure as that of — ? Crobanche (Kocu °78, Smiru, 01, p. 118). Seedling. It is a noteworthy fact that in spite of a great similarity in structure of the seed in Aeginetia and Orobanche the mode of germination is very divergent. In Orobanche germination is brought by the multiplication of cells in the embryo, so that a filamentous seedling of 1-2 mm. in length is the result. Both the radicular and plumular ends are seen to consist in longitudinal section of four rows of cells enclosed by the epidermis (Kocn, 78, Figs. 17-19). The connection of the seedling with the host is effected by the tip of the radicle. The tip on coming in contact with any host sends out its epidermal cells in the form of papillae (Kocu, ’83, p. 189), and the subjacent iiitial cells then commence to proliferate and produce the tissue of the primary haustorium. In Aeginetia the changes are quite different. In the first place we can scarcely recognise multiplication of cells or longitudinal growth in the seedling before it finds out the proper host, and in the second place the development of the radicular end is very characteristic. The first change that can be observed as the sign of germination only consists im that two or three, large, hyaline globular cells appear outside the testa at the micropylar end of the seed (Figs. 3, 4). These are highly turgescent with abundant cell-sap. The nuclei are large and conspicuous, and the cytoplasm radiates from them. At an advanced stage the globular cells increase in number generally up to 15 approximately (Fig. 12). As can be seen in Figs. 1, 2 and 6, these are not_a new tissue, but only the epidermal cells of the radicle but swollen up to nearly 4 times the original diameter. Simultaneously with the change all the other cells swell up more or less, making the embryo much larger in size; and judging from the number of cells seen in an optical section of the FURTHER STUDIES ON AEGINETLIA INDICA. 63 embryo before and after germination (compare Figs. 2 and 6), it is very improbable that a multiplication of cells may partly concerned in the increase of size. An accumulation of starch more especially in the tissue under the globular cells is perhaps connected not with the cell divi- sion in this place, but with the further development of the globular cells. Now follows the outgrowth of the globular cells one by one. Their external wall protrudes so as to make them first conical and then papillar- like in form (Figs. 7, 9, 10). The outgrowths proceed further until they become slender hairs growing at times up to 1 mm. in length. The diameter of the hairs is much smaller than that of the globular cells, measuring 38 yon the average while the latter measure generally 115 4 in diameter. Although they belong morphologically to the category of trichomes, yet they are not identical in structure and even in function with the typical root-hairs; they are often septate or even branched (Figs. 8, 9, 14), resembling rather the rhizoids of some cryptogamic plants (Hazrrianpr, 04, p. 200). If undisturbed, they are all straight and radiate from the radicular end in all directions as shown in Fig. 8, but if one of them during its further prolongation should come in contact by its tip with a young host-root, it seems to attach itself firmly to the latter and then to coil or contract through its whole length, whereby the seedling is drawn closer to the host (Fig. 10). This is evidently a most advanta- geous contrivance for the parasite to facilitate its organic connection with the host, that is to say, ihe formation of the primary haustorium. In Fig. 9 is shcwn one of the hairs just adhering to a host-root, and about to bend itself, while in Figs. 10 and 12 are shown hairs in a much con- tracted condition with the radicular end brought much nearer to the host. By what means the tip of the hair fixes itself to the host has not yet teon made out exactly. It is not impossible that a cementing substance is secreted by the hair, bus there has actually cone under my observation such a case as is shown in Figs. 9 and 12, where the fixation was effected by a slight penetration of the tip of a hair between the epidermal cells. My observations, however, are not extended enough to justify the con- clusion that this is a general case with Aeginetia. 64 S. KUSANO: So far 2s I know, such an organ has not hitherto been described in phanerogamic parasites. Analogous but not homologous cases may per- haps be found in the root-hairs that develop previous to the formation of haustoria on the typical root of somc hemi-and holo-parasites, such as Melampyrum (Lecienc pu Savion, *S7), Lathraea (Hernricuer, 795, p- 381), Santalum (Basen, 06). In all these cases the root-hairs appear to serve simply for the fixation of the root of the parasite to the host. The cushion-cells in Cuscita (Price, 93) may be considered to perform the similar function. In Acginetia it is quite obvious, as already stated, +hat the hairs serve first of all as a “tentacle.” and after eontact sith the host as a “prehensile organ,” besides drawing the seedling closer to the host. In function, therefore, they possess all the characters of a typical tendril (i.e., in Cucurbitaceae), and hence I venture to propose for them the name of “hair-tendrils.” Tn the root-system a similar function has already been known to appertain to the so-called root-tendrils (see Prerrer, ’04, p. 416). They are not, however, identical morphologically with the hair-tendrils; for, in typical root-tendrils the entire root plays the part of a tendril, while in hair-tendrils an appendage of the radicle comes into play. In origin again, the hair-tendrils may be homologous to the papilla-like cells at the tip of the radicle in the seedling of Orobanche (Kocu ’83, p. 189). How- ever, in structure and function the latter organ seems to be different from the former, showing a rather close resemblance to the cushion-cells of Cuscuta. The kind of stimuli required in causing the curvature of the tendrils remains still unknown. ut on the basis of my culture-experiments it seems highlv probable that, unlike the true root-hairs (see Prerrer, ’04, p- 459), mere contact with sand or soil particles remains quite ineffectual, but that some chemical stimulus must be coneerned, to which the tip of the tendrils coming in contact with the host-reot must respond. That normal tendrils may respond to chemical stimuli has already been ascertained by Correns (’96, p. 16). FURTHER STUDIES ON AEGINETIA INDICA. 65 In almost all cases the globular cells do not appear to develop all into the hair-tendrils: some of them remain unchanged, while some are arrested from further development after reaching the conical or papillae stage. As for the most probable ground of such variable develop- ment of the globular cells, my observations of a number of seedlings have led me to the conclusion that the number of tendrils that are formed in a seedling must depend more or less upon the chances of meeting with an appropriate host. In fact I have found that when a seedling came in contact with a host by a premature development of some tendrils. the re- maining ones were more or less arrested from further development and the globular cells from forming further tendrils (Figs. 9, 10, 12); while when a scedling remained away from the host long enongh, many tendrils were observed to develep at once and in full length, or many globular cells to give rise to tendrils (Fig. 7). This fact makes it most probabie that the seedling develops as many tendrils as possible in several directions until it finds out a host, thus securing as many chances to meet with a desired host-root, but that as soon as one of the tendrils comes in contact with it, the seedling does not need the development of further tendrils. Usually only the apex of the tendril is responsive to the stimulus, but that the other portions may also react may be seen in Fig. 11, where a tendril is shown twining around a root-hair of a_ proper host-root (Zingrber). The tendril on coming in contact with the host seems to be retarded in growth as in the typical tendril (Frrrrye, 703, p. 604), and it seems to wither and die away, if kept indefinitely away from a proper host. In view of all these facts there can not be any doubt that the hair in Aeginetia-seedling is quite different both morphologically and physiolo- gicaily from the true root-hair, and that it most closely resembles the typical tendril in its function. While the changes described above are taking place at the radicular end, we can not find any notable change ‘at the plumular end except for 66 8. KUSANO:! a slight increase in size. The general form of the embryo at this stage is then as reproduced in Fig. 12. It is perhaps the last stage to which an embryo can develop withont coming in contact with the host-root. Much starch-granules remain in the embryo and endosperm, and serve as the reserve material for the further development of the seedling, Tubercle and Primary Haustorium. When a seedling as above described comes in contact with a host-root by means of a hair-tendril, further development follows immediately. By a rapid multiplication of cells the seedling grows so as to become visible to the naked eye. The newly produced tissue gives rise, besides a primary haustorium, to a tubercle from which the shoot and root-system of the plant are afterwards formed. What is remarkable is that the multiplication of cells does not take place, unless the seedling becomes attached by one of the tendrils to the host. Since the seedling is other- wise entirely incapable of further development, in spite of the presence of the reserve material left in the endosperm, it follows that the further development of the seedling is associated with the stimulus of the host. The multiplication of cells occurs under the tendril-cells. The parenchymatous tissue thus derived pushes and finally breaks the latter, and comes to lie in direct contact with the tissue of the host-plant. Until an organic connection becomes established between the seedling and the host-tissve, the multiplication of cells must be due to the reserve material in the seed. The maximal size to which the cell-mass can thus attain is less than 1 mm. in diameter, approximately the same as that to which the seedling of Orobanche can reach with the help of its endosperm alone (Kocu, 783, p. 189). The cell-mass thus formed becomes a tubercle generally of a spherical or oval form (Fig. 13). It forms a large part of the seedling, making the plumular end, globular cells, and tendrils highly inconspicuous. The formation of the tubercle has already been observed in Orobanche, in which however only one fifth of the whole length of the seedling is transformed into it. ——_—- FURTILER STUDIES ON AEGINETEA INDICA. 67 The frontal portion of the tubercle penetrates into the young cortex of the lost-root and becomes differentiated into a primary haustorium which is corspleted by the formation of tracheids in direct connection with the conducting system of the host-root. On the completion of the haustorium the tubercle derives nouvishment from the host, and there ensnes a vigorous development. The further development of the tubercle —formation of the shoot and root-system—is quite the same as in Orobanche (Kocn, °83). Germination-Experiments. As has been quoted above, there is no doubt that in the germination of the Orobanchaceae, as ascertained in Ovrobanche and Lathraea, a chemical stimulus comes into play. Still it has not been conclusively shown whether the stimulus in question is due to the character of the roots as such, or is entirely peculiar to the root of the proper host. Al though Kocr has expressed the opinion that, “die Samen der Orobanchen keimen nur im Anschluss an die Wurzel einer geeigneten Nahrpflanze” (Kocu, ’83, p. 188), it seems to me that a sufficient number of plants has not been tested with this point in view. Hertnricuer (794) succeeded in raising the seedling of Lathraea on the roots of a very few kinds of trees. Erom his experiments we ean not conclude that the roots of all trees can stimulate the seed to germination. A further study is also needed to decide whether the seed germinates on the roots of herbaceous plants. But when we consider that these parasites thrive only on certain plants,’ one xight consider himsecif justified in assuming that the germination takes place only on these plants. Likewise, as only :nonocotyledonous plants are, at present, known as the hosts of Aeginctia in the field?, one 1 Among more than 300 species enumerated by vON BECK (’90) as hosts of Orobanc he no monocotyledonous plant is mentioned decidely as the proper host. 2 So far the following plants have been ascertained to serve as the host. Canna indica V,. (Dandoku). Carex lanceolata Troott. (Hikagesuge). C. Morrowi Boott (Kansuge.) C. transversa Boott. (Ko-onisuge). Imperata arundinacea Cyr. var. Koenigii (Benth.) Hack. (Chigusa). Miscanthus sinensis (Anders.) (Susuki). MW. sacchariflorus Hack. (Ogi). Oryza sativa L. (Upland form) (Okabo). Panicunt mtiliaceunt V.. (Kibi). P. flumentaceus L. (Hie). Saccharum officinarum L. (Satokibi). Setaria ttalica Kth. var. germanica Trin. (Awa). 9 Zza_ Mays 1... (Tomorokoshi). Zingiber Mioga Rose. (Mysga). 68 Ss EUSANO: might be led to the same assumption. This has, however, bcen proved to he quite incorrect by the germination-experiments now to be described. As these expriments were originally planned to verify what we had as- suined, they were not so extended as were afterwards found desirable. 1. Germination of the seeds on pot-plants. Aeginetia-seeds were laid on the roots of several pot-plants. The experiments were made in July, and the germination took place within two weeks. The plants used comprised several species of Phanerogams and Cryptogams, two pets being prepared for each. Pteridophytes: Selaginella involvens Spring. (Iwahiba) and Aspidium rhomboidewm Wall. (Kanawarabi) have rather weakly de- valoned roots. After two weeks some of the seeds laid on them were seen hove produced a few elobular cells outside the testa, but no futher elopment took place even after four weeks cr more. Gynmosperms: Cryptomeria japonica Don (Sugi) and Thujopsis Jolabrata S. et Z. (Asunavo) were used. Although the roots are not very vigorously developed, yet a few of the seeds produced globular cells. Further development remained uncertain. Monocotyledons: Keeping in mind that Aeginetia grows im the field exclusively on plants of this group, I have used for my purpose several species from various families, comprising also the well-known hosts for control. * Juncaceae. Luzula campestris DC. var. capitata Mig. (Suzumenohie). Crperacene. Carex japonica Thunb. var. chlorostachys (Don.) Kik. (Shirasuge ). * C. Morrowi Boott. (Kansuge). Gramineae. . - Arundinaria Stinont Riy. (\Medake). Calamagrostis arundinacea Noth, (Chigusa). *= Miscanthus sinensis (Anders.) (Susuki). 1 The natural hosts are marked with an asterisk, FURTHER STUDIES ON AEGINETIA INDICA. 69 * Oryza sativa iL. (Upland form) (Okabe). * Panicum miliaceum I. (Wibi}. Setaria excurrens Mig. (Inuawa)}. * Zea Mays L. (Tomorokeshi). Araceae. Acorus gramineus Ait. (Sekisho). Coinmelinaceae. Pollia japonica Hornst. (Yabumyoga). Thick, soft and vigorous roots with densely developed root-hairs. Rhoeo discolor Hee. (Murasakiomoto). Liliaceae. “Allium fistulosum L. (Negi). Vigorous development of roots. Hemerocallis fulva L. (Yabukwanzo). Ophiopogon japonicus Ker. (4 anohige). Roots dense but not vigorous. Inidaceae. Tris tectorum Max. (Iehihatsu). Dioscoreaceae. Dioscorea sativa L. (Marubadokoro). The development of roots far less vigorous than other plants. | Zingiberaceac. | * Zingiber Mioga Rose. (Myoga). Root very vigorous. Caunaceae. ‘Canna indica L. (Dandoku). With the exception of Ophiopogon all the plants above mentioned gaye the required stimulus, and the seed «attained after two weeks to a stage similar to that shown in Fig. 4. The percentage of germination seemed to be larger on plants whieh produced vigorous reots. In Zingiber and Pollia young roots were constantly and Iuxurianily produced during the experiment, so that almost all the seeds laid on them came to germina- tion. As for Ophiopogon the roots were not very active during the ex- periment, and the necessary stimulus, if present, seemed to have been too feeble. 10 S. KUSANO : Dicotyledons: Only a few plants were taken here. ‘This was due to the ciremmstance that more plants had not been prepared as pot-plants tor my purpose. Phimbaginaceae, Lrmerta maritima Willd. Araliaceae. Fatsia japonica Dene. et Plane. (Yatsude). The roots were very few and not vigorous. Geramlaceae. Pelargonium Zonalle Willd. (Montenjikuaoe). Rosaceae. Pirus Marus L. var. tomentosa Koch. (Ringo). Roots very few, uot vigorous. Prunus Mume 8. et Z. (Mume). Roots very few, not vigorous. Solanaceae. Solanum tuberosum L. (Barcisho). Roots very scanty. Leguminosae. Pisum sativum L. (Endo). Compositae. Chrysanthemum sincuse Sab. (Wik). Solidago occidentalis Torr. et Gray. (Oawadachiso). Taraxacum officinale Wigg. var. glauscens Koch, (Tanpopo). Moots very few. Of these plants FMalsia and Taracacum did not bring the seed te germination. This might perhaps be due to a comparatively weak develop- ment of the roots as above noticed. On the other hand, the seeds laid on all the other plants mostly germinated just as they did on Monocotyledons. It must, however, be remarked that the seedlings thus produced did not all develop so far as to produce the hair-tendrils: stopping at the stage shown in Fig. 4, they ultimately came to death, mainly owing to mould fungi or other microorganisms. FURTHER STUDIES ON AEGINETIA INDICA. Ti The foregoing experiments show, contrary to our natural expectation, that the stimulus necessary for the germination of Aeginetia-seed is not peculiar to particular species of plants, but is given by all vigorously developing roots, whether of Phanerogams or Vascular Cryptogams. If it be admitted that a chemical stimulus is concerned here, it is most prob- able that the stimulant is an exeretion of the roots. The following ex- periments afford some evidence for this view. 2, Germination of the seeds wrapped in paper on pot-plants. This experiment was undertaken to ascertain whether direct contact of the seed with the host-root is necessary for germination or not. The seeds were wrapped in one-or several sheets of well-washed filter paper and laid among the root-meshes of the pot-plants. For control, seeds prepared in the same manner were kept at the same time in a moist chamber, and again unwrapped seeds were laid directly on the roots of the same pots. The seeds wrapped in 3-5 sheets of paper did not germinate about the time that the unwrapped seeds germinated vigorously. However, those wrapped in one sheet and laid on Zingtber and Polhia germinated partly. In the mean time the control seeds in the moist chamber remained entirely unchanged, From this experiment we see that direct contact of the seeds with the host-root is by no means an indispensable condition in bringing them to germination, and that the germination is associated with a certain sub- stance or substances excreted by the host-root and diffused into the surrounding inedium. That the percentage of germination is sinaller in the case of the wrapped seeds than in those laid directly on the root, and that it becomes less with the increase of the sheets of paper are strong evidences that the amount of the diffusible substance depends upon the nature of medium through which it must pass to reach the seeds. 3. Germination of the seeds without host-root. The seeds were kept in water (tap-water or distilled water) or in a moist chamber. They were also sown in soil without any visible plant. Jn either case I was not able to observe any sign of germination. If such (2 S. USANO: seeds were afterwards brought on the root of any plant, the germination took place easily. Hence it follows that the seed of Aeginetia always requires a stimulus from the roots for germination. 4. Germination of the seeds in chemicals. This is only 2 preliminary experiment to find out a stimulating substance among chemicals, and only a few substances were tested. Under a bell jar, one end of a piece of filter paper moistened pre- viously with distilled water, on which the seeds were placed, was immersed in a given solution of the substance to be tested in a small vessel. By capillary action the given solution diffuses up the paper, so that the seeds are acted on by the substance in various degrees of concentration at different parts of the paper. For control, tap-water and distilled water were tested in the sarae manner. The results were entirely negative, and no germinating seeds were observed aftcr two weeks or more. The seeds were attacked by mould fungi and destroyed. The chemicals tested and concentration in the vessels were the following: Hydrochloric acid .. .. 1/100 and 1/500 mol. Phosphoric acid See. 1/100¢and 1/5008 age Tartarie acid .. .. .. 1/100 and 1/500. mol. Citric acid eee. 1/100 amol. Formic acid “32 720%. 1/100’ and 1/500 mol Mahe acid eee. 1710 sandel/500 sank Mcnopotassium phosphate . 1/100 mol. Sodium hydroxide .. .. 1/100 and 1/1000 mol. It would be of great interest and importance to extend the above experiments and te determine, if possible, a chemical or chemicals that would stimulate the Aeginetia-seed to germination. If such a substance be found out, it is highly probable that it is one of the excretions of the roots. Much difficulty must certainly lic in the way of such a study. It is generally known that roots excrete acidie substances (CzAPEK, 05, p. 873), and recently Scurerner and Reep (’07) have found out that a very slight amount of substances is excreted by roots, which act de- leterious to their growth.! ‘The amownt is so exceedingly small that it 1 Vor the literature on root-excretions see SCUREINER and REED, loc. cif. FURTHER STUDIES ON AEGINETIA INDICA. 73 can not be detected by chemical analysis, but its presence is revealed by the chemotropism of roots. The method proposed by the last named authors is very ingenious, and it leads us to think that our germination- experiments, if extended further, might perhaps be applicable to the investigation of root-exeretions. Development of Tubercle and Selection of Host. Although it is clear from the foregoing accounts that all roots ean stimulate Aeqginetia-seeds to germination, still the facts obtaimed both from field-observation and enlture-experiments clearly show that Aeginetia can not grow on all plants. This is proved by the germination- experiments. By careful examination at intervals of the pot-plants on which the seeds were laid, we could ascertain that the germinating seeds did not develop equally well on Cryptogams, Gymonosperms and Dicotyledons. Again, among Monocotyledons different plants acted very differently. The plants of this group that induced the seedlings to form tubercles were Luzula, both species of Carex, Calamagrostis, Miscanthus, Setaria, Oryza,- Panicum, Pollia, Zingiber, and Canna, most of them being already known as natural hosts.* Further, the development of tubercles was not only very unequal on these plants, but even in the same species it was different on different individuals. After two weeks all the seedlings reached the size shown in Fig. 4, but the size of the tubercles during the next two weeks was very variable, some attaining to the size of poppy grains and others to that of the corn. The growth of the tubercles was especially vigorous on Zingiber and Pollia. Tt was also observed that the development of the parasite was less rapid on pot-plants than on those in the field: in September it was all in flower in the field, while the shoot seareely appczred above ground in the pots. It follows that the growth of the parasite is most intimately connected with that of the host, and m particular with the activity of its roots. * See foot-note in the preceding page (p. 67). 74 S. KUSANO: T have already remarked that the host-rest induces no germination when too feebly developed. The same cause must not be assigned for the non-develonment of tubereles on some of the plants used in the ex- periment. For instance, Al/7um, Iris, Acorus, Hemerocaitis, and others produced numerous vigerous rootlets and appeared always to be much more rapid in growth than some natural hosts such as Carex and VWiscanthus. It is certain, therefore, that there are fit and wnfit plants as the host of Aeqginetia. As for the intimate relation between the seedlings and the proper hosts, or plants unfit as the host, I have no evidence to bring forward. Tt may be that the roots of some plants are unfit for inducing the forma- tion of the hair-tendrils, the primary haustoria, or perhaps the tnbereles. Which of these assvimptions holds true must be settled by further in- vestigations. Af present I can go no farther than to state that the stimulus which causes the seeds to germinata and the stimulus which causes the seedlings to develop further are of a quite different nature. General Remarks and Summary. On leoking over what have been deseribed above, we see that Aeginetia presents many remarkable characters which must be due to ifs parasitic life. In the first place, the formation of the hair-tendrils is a most specialised contrivance for finding ont the host. It mav be that m Lathraca and Orobanche the seedlings can not easily reach the host, unless the seeds are placed close to the host-root so that their radicles lie against the latter. Otherwise, the tip of the radicles may diverge from the host- root more and more, as they grow further and further so as to make the development of the seedlings impossible, inst as the same organ of Viseum would do, if it should be insensitive to light, or the same organ of autophytic plants, if insensitive to light and gravity. In Aeginetia the formation of the hair-tendrils is alone suticient to avoid such a danger. FURTHER STUDIES ON AEGINETIA INDICA. 1D: In the second place, Aeginetia shows some transitional states be- tween autophytic and the most advanced parasitie life. In most hemi- parasites, or more strictly speaking, green parasites such as Santalaceae (Kusano, ’06, Barser, ’06, 707), Rhinantheae (Hrtnricuer, ’01, ’02), and Loranthaceae, the germination is neither associated with the presence of the host, nor have they any marked tendency to select their host. But some holoparasites, or at least Orobanchaceae hitherto studied, have acquired the habit of not developing and even of not germinating without the presence of the roots of their proper host. While thus the intimate relation of the parasite and host-root is in this case restricted to certain limited species of plants, -Aeginefia sows itself to be many-sided in this respect: in Orobanche and Lathraea the selection of the host takes place already at the period of germination, but it takes place in Aeginetia at a later period. Thus certain variations being observed to occur in the Orobanchaceae in their behaviour towards the host-roots, it may be remark- ed that a study of other species of the same family is very desirable. The chief results of the experiments described in the foregoing pages may be summarised as follows: 1. The germination of Aeginetia-seed does not take place in water, moist chamber, or soil. It requires always the stimulus of the root of other plants. 2. The seed kept dry for two years loses its germinating power. 3. The plants which stimulate the seed to germination may be Vascular Cryptegams, Gymnosperns, or Angiosperms. 4. The stimulant is an unknown substance that is perhaps excreted by the active roots of all higher plants. 3. The development of the seedlings takes place only on certain species of Monocotyledons. — Its conditions are entirely different from those that are necessary for the germination of the seeds, the former being fulfilled only by certain piants while the latter are found in the roots of all higher plants. 6. The first change that takes place during the germination is the swelling of the epidermal cells at the radicular end of the embryo and their transformation into the hair-tendrils. 76 S. KUSANO: 7. The seedlings are much reduced in form, and before they are connected with the host no multiplication of cells takes place. S. The seedlings develop, when attached to the host, spherical tubercles. They are formed by the meristematic tissue under the hair- tendrils. 9. For the multiplication of celis in the seedlings certain stimulus from the host-roots to which the hair-tendrils are sensitive seems to be required. 10. The tubercles become differentiated first into the primary haustorium at the frontal portion, and then into the stem and root-system _at the other portions. June, 1908. FURTHER STUDIES ON AEGINETIA INDICA. iq) Literature Cited. Baveer, C.A. (06): Studies in Root-Parasitism. The Haustorium of Santalum album. 1. Early Stages, up to Penetration. Memoirs of the Department of Agriculture in India. Bot. Series. Vol. I, No. 1, Pt. I. 1906. (07): The same. 2. The Structure of the Mature Haustorium and the _ Inter-relation between Host and Parasite. Ibid. Vol. I, No. 1, Pt. II, 1907. Beck von Mannacetra, G. (90): Monographie der Gattung Orobanche. Bibl. Bota- nica. 1890, Heft 19. CorkENS. C. (96): Zur Physiologie der Ranken. Bot. Ztg, Bd. LIV, 1896, p. 1. CZAPEK, F. (’05): Biochemie der Pflanzen, IT, 1905. Jena. Fittine. H. (03): Untersuchungen tiber der Haptotropismus der Ranken. Jahrb. f. wiss. Bot. Bd. XXXVIIE, 1903. p. 545. HABERLANDT, G. (04): Physiologische Pflanzenanatomie, 3. Aufl., 1905. Leipzig. HErNRIcHER, E. (94): Die Keimung von Lathraea. Ber. d. deutsch. Bot. Gesellsch., Bd. XII, 1894, p. (117). (95): Anatomischer Bau und Leistung der Saugorgane der Schuppen- wurz-Arten. Cohn’s Beitrg. z. Biol. d. Pflanzen, Bd. VII, 1895, p. 315. (98): Notiz tiber die Keimung von Lathraea squamaria. Ber. d. deutsch. Bot. Gesellsch., Bd. XVI, 1898, p. 2. (701): Die Griinen Halbschmarotzer II]. Jahrb. f. wiss. Bot., Bd. XXXVI, 1901, p. 665. —__ (92): The same iV. Ibid., Bd. XXXVIJI, 1902, p. 264. Kocn, L. (°78): Ueber die Entwicklung des Samens der Orobanchen. Jahrb. f. wiss. Bot., Bd. XI, 1878, p22use (’83): Untersuchungen uber die Entwicklung der Orcbanchen. Ber. d. deutsch. Bot. Gesellsch, Bd. I, 1883, p. 188. Kusano, 8. (703): Notes on Aeginetia indica Roxb. Bot. Mag., Tokyo., Vol. XVII, 1903; p. 1. (96): Studies on a Perennial Hemiparasite. Ibid., Vol. XX., 1906, p. (59). In Japanese. LECLERC DU SABLON (’87): Recherches sur les organes d’absorption des plantes para- sites (Rhinanthées et Santalacées). Ann. Sci. Nat., VII Serie, T. 6, 1887, p. 90. Petrce, J. (93): On the Structure of the Haustoria of some Phanerogamic Parasites. Ann. of Bot., Vol. VII, 1893, p. 291. PFEFFER, W. (04): Pflanzenphysiolog‘e, IJ, 2. Anfl., 1904. Scureryer, O. and Reep, H. S. (707): The Production of Deleterious Excretions by Roots. Torr. Bot. Club, Vol. XXXIV, 1907, p. 279. 78 3. KUSANO: Scuwarz, F. (°83): Die Wurzelhaare der Ptlanzen. Arb. aus d. Inst. Tiibingen, Bd. I, Heft 2, 1883. p. 135. — Saat, A. C. (°01): The Structure and Parasitism of Aphyllon uniflorum, Gr. Publ. of the Univ. of Pennsylvania. Contrib. from the Bot. Laborat., Vol. IT. 1901, Delile Explanation of Figures. All figures except Figs. 13 and 14 are drawn with the aid of the camera lucida from the fresh materials and magnified 150 times. Fig. 1. An adult embryo in a ripe seed. Fig. 2. The same shown in optical section. Fragments ef testa are attached to the radicular end. Fig. 3. A seed at the beginning of germination, with some swollen epidermal cells at the radicular end appearing outside the testa. Fig. 4. A seed at somewhat later stage. Fig. 5. An embryo in the germinated seed as shown in Fig. 3. Fig. 6. The same in the seed shown in Fig. 4. (Optical section). Starch- granules accumulate at the median, portion. Fig. 7. Radicular end of a seedling showing one of the globular cells protruded into a papilla. Fig. 8. The same with full grown tentrils. Fig. 9. The same showing one of the tentrils attached to a host (Zingiber). Tts apex is penetratins between two epidermal cells of the host. Fig. 10. The same with a much shrinked tendril. Fig. 11. The same with a tendril twined round a root-hair of a host (Zingiler). Fig. 12. Two seedlings at advanced stage. In the right is shown an entire embryo taken out of the endosperm. Fig. 13. A tubercle on the root of Zingiber. xca. 40. Fig. 14. The same at somewhat advanced stage. xca. 40. S : . A Contribution to the Cytology Introduction and its Hosts. BY S. Kusano. With Plate VIII—XI. * CONTENTS. Mode of infection of the swarm-spores in the host-cell Vegetative period I, The swarm-spore . Cytology of Synchytrium Puerariae... A. 2. Youngest form in the host-cell 3. Growing stage a of Synchytrium Vacuolation of the nucleolus and the formation of the secondary nucleoli 6. Condensation of the chromatin c. The nuclear membrane d. Dimension of the nucleus Reproductive period 1, Primary mitosis 2. The secondary nuclei and their division... Resting stage ... Prophase and metaphase Anaphase Telophase Formation of the membrane... Fate of the nucleolus ee Abnormalities eee see 80 S. KUSANO: 3. Formation of the sporangium .... Sp aoe daz we er he 4. Development of the sporangium and the swarm-spore a3 oa pee | FSGS IV. Cytology of Synchytrium decipiens 5s ae Ho 535 ies aS Bree lis V. Discussion... oe = ss 24 oe Bee Be = = = oh ZS: VI. Cytology of the host-cell ... oe es ied = se sa 4 a M25 VII. Summary ar e Soe ae ae sce er aes =e ies soo ee Postscript ae Sf Bo oes ore eh ot Ses jae am oe 133 Literature cited ee ee ae aa Aa, +e =- Bes Re odo, Ten Explanation of figures oe aes oF Ba ae ae ene = on oe I. Introduction. In the “Centralblatt fiir Bakteriologie, etc., Bd. XIX, 2. Abt., 1907” (Kusano, ’07b) I have published a short account on Synchytrium Puerariae and S. decipiens. The results of observations set forth in that communica- tion were chiefly concerned with certain cytological phenomena relating to their hosts. In the present article I intend to communicate the results of cytological studies on the fungus-bodies themselves, with a view to throw some light upon the structure of the nucleus and nuclear division in this group of fungi, which no one has heretofore exhaustively investigated. I may also be allowed to dwell upon the influence of these fungi upon the cytological phenomena of their hosts, of which certain points were not dealt with in detail in the former communication. The authors who have worked upon the cytology of Synchytrium are not so numerous as those who have devoted themselves to studies in similar directions in other genera of Phycomycetes, and moreover none of them seems to have attempted to follow throughout the whole life history of the fungus. This is perhaps due to the small size of the nucleus and to.the difficulty in getting serial stages of development. A comparatively full account of the successive stages of development was given by DANGEARD (790) in S. Tarazaci, in which he pronounced the nuclear division to be mostly amitotic, while mitosis occurred in rather exceptional cases. Later, A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 81 ROSEN (793) studied the same fungus and confirmed DaNncrarp’s view as to the existence of amitotic division, remarking, however, that in the secondary nuclei mitosis occurred much more frequently than amitosis. Further, he noted that the amitotic division was quite different from the tvpical form: the chromatic threads thickened, the nucleolus divided into two halves, each migrating into one of the daughter-nuclei, and the nucleus constricted itself in the middle without the aid of the usual achromatic structure. In his study on the cell-division in sporangia and asci, Harper (’99) dwelt upon the mode of the sporangium-formation in S. decipiens and S. Taravaci. He gave a very full account of the manner of cleavage-forma- tion in the fungus-body, by which the protoplast was successively cut off into smaller and smaller portions till each portion contained finally a single nucleus in 8. decipiens, or a few nuclei in S. Tararaci, and thus he came to the conclusion against DanararD (790) who maintained that the division of the primitive cell of S. Tararaci to form sporangia was a process of simultaneous fragmentation, whereby the multinucleate protoplasmic mass was at once cut into a number of polyhedral multinucleate portions. As the object of Harper’s work was to know the details of the formation of sporangia, no statements were made about the other cytological phenomena of the fungi. A considerable advance of our knowledge of the nuclear division in Synchytrium was made by Stevens (03). He took the primary nucleus of S. decipiens as the object of his study, and concluded, contrary to DANGEARD and Rosen, that the division was usually mitotic. Besides, he mentioned several characteristics of considerable interest in the nucleus, namely a sudden shrinkage of the notoriously large nucleus and marked diminution of chromatin previous to division, dissolution of the nuclear membrane into a yranular persistent halo around the karyokinetic figure, a peculiar form of the gspireme, etc. These results of his observations particularly attracted my attention and induced me to undertake the present work, intending on the one hand to verify such remarkable nuclear phenomena by my own ob- servations in the same fungus, and on the other to ascertain whether they hold true also in allied species. Papers referable more or less to the cytology of Synchytrium were pub- lished by LéwentHAL (05 a, b) and recently by Ryrz (07). The former $2 S. KUSANO: anthor described the structure of the nucleus in the resting condition in S. Taraxaci and in the. resting cell of S. Anemones, while the latter author uoted, during his morphological and biological studies on several species of Synchytrium, the structure of the resting nucleus and also briefly the formation of the sporangium in S. Succisae. These are all that we know at present on the cytology of Synchytrium. The results obtained by the authors mentioned above, of course valuable as they are, seem somewhat fragmental, and consequently we can not by any means draw from them a definite conclusion, as for instance, about the karyokinetic figures in the fungi under consideration. Regarding other nuclear phenomena a more extended comparative study seems to be very much desir- able. In my present work special attention has been paid upon the behaviour © of the nucleolus during the nuclear division, the formation of the nuclear membrane, and the ontogenetic origin of different elements in the daughter- nucleus. Relating to the nucleolar problem in general some interesting and important results have been obtained by Wacer (704), M. v. DERscHaU (706), GrorcGevircH (708), and others, but regarding the origin of the nuclear membrane and the other nuclear elements which enter into the composition o: the daughter-nucleus, there are several difficult questions still awaiting solution. These questions are certainly due to the difficulty of observing the finer details of the processes that take place during the reconstruction ox the daughter-nncleus. Now in my material, I have been fortunate enough to follow very closely the changes of the nuclear elements from the telophase stage to that of the reconstruction of the daughter-nucleus. Some of the important results on the nuclear phenomena obtained at these stages were already given in my preliminary note (’07 a). The authors who have studied Chytridiaceae have directed their atten- tion more or less upon the cytology of the host-cell. Macenus (797, 701, 702) reported several instances of the fact that Urophlyctis dissolves the cell-wall ef the host. Lowrntuat (705 b) mentioned the hyperchromic deformation ‘of the nucleus in the host-cell infested by Synchytrium Anemones, while the hypertrophy and ‘fragmentation of the nucleus took place, according to PotravLT (705), in the host-cell of Physoderma. Among other intracellular parasites Plasmodiophora and Dendrophagus A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 3 ~ show in their manner of development and in their nutritive relation to the host-cell many points of resemblance to Synchytrium, so that their pathological influence upon the host should be taken into consideration in the present sstndy. Nawascuin (99) came to the conclusion with Plasmodiophora that the cytoplasm of the host-cell somewhat increased in amount, the nucleus hypertrophied, and both were then consumed entirely by the parasite. At nearly the same time Toumry (700) studied Dendrophagus and arrived -at a similar conclusion. The material of the present study—Synchytrium Puerariae on Pueraria Thunbergiana and 8. decipiens on Amphicarpaca Edgeworthii var. japonica— ~was collected mostly at Komaba in the spring of several years and at dif- ferent times of the day. It was fixed chiefly in FLEMMING’s solution and ‘Ketser’s acetic sublimate, directly in the field, or after keeping for a few hours in a moist chamber at 20-25°C. Most of the karyokinetic stages were obtained from the warmed material. The sections were stained either with Fremmine’s triple stain, or with HEIDENHEIN’s iron haematoxylin. Sometimes fuchsin iodine-green was very satisfactory in differentiating the structure of small nuclei in sporangia. In this place I express my deepest thanks io my friend Dr. K. Miyake, Lecturer in our College, for his kind advices and criticisms during the pro- -gress of the present work. II. Mode of Infection of the Swarm-Spores in the Host-Cell. Almost all the authors who have undertaken the study of Synch ytrium agree in stating that the infection takes place exclusively on the epidermal cells -of the host. However, according to the illustrations of some tubercles caused by the fungus by Scunorrer (770), Ryvz (07), and others, it appears that the fungus infects the subepidermal cells, since host-cell is shown as lying beneath the surface of the host. Notwithstanding, evidences to the contrary have been given by many observers who have studied the develop- 1 ala! cane eae ment of the tubercles. Hence, taking the mature tubercle caused by 4 ason to believe, aS decipiens and 8. Puerariae into account, we have good re 84 S. KUSANO: its structure is in no wise different from that of the tubercles caused by other- Synchytrium, that the host-cell belongs also to the epidermis. This view seems to be especially applicable to the tubercles caused by S. decipiens on the stems, petioles, and veins of the leaves; but the comparative studies on the development of the tubercles in different portions of the host, made on the two fungi under consideration, afford a strong evidence that in no case the swarm-spore enters the epidermal cell. If inoculation should take place on the epidermis, it must be admitted, unless the swarm-spores are able to penerate the thick cuticula, that the possibility of inoculation be limited to the younger stages of development of the host, when the cuticular layer is not yet produced. The fact that inoculation may take place on older parts of the host makes it most probable that the swarm-spores enter the host through the stomata, or water-pores. Chemotactic experiments made with S. Puerariae offer a strong evidence for this view (Kusano, ’08). I found that certain tissues of the host contain a strongly attractive substance; for: instance, an adult but still living trichome, when its cut end is inserted in a drop of water in which the swarm-spores swim actively, attracts them most promptly at or near the given end, and also a piece of hyaline tissue exerts the same action upon the spores, while the green tissue repels them,. or even acts injuriously. Though not so striking as in S. Puerariae, the attractive action of hyaline tissue upon the swarm-spores of S. decipiens is quite evident. Hence, the conclusion to be drawn from these facts relating” to the mode of infection of the swarm-spores will be that a certain attrac- tive substance is diffuscd out through the stomata, or water-pores, from the inner tissue to the fluid on the surface of the host, in which the swarm- spores are liberated; it stimulates them to approach the source of stimulant after leading them to an intercellular space under the stomata. As I have stated elsewhere (07), the young fungus-body is always found in the hyaline cell, or the cell with very little chlorophyll, a fact entirely in accord with the result obtained by experiment that hyaline tissue attracts, while green tissue repels, the swarm-spores. } Let us now consider how the position of the host-cell is controlled by the development of the intercellular space. The size of the intercellular space has bearing upon the distribution of the swarm-spores after entering the subepidermal tissue. Generally, the host-cell of S. decipiens is found A CONTRIBUTION, TO THE CYTOLOGY OF SYNCHYTRIUM. 85 in the hyaline mesophyll lying between the palisade and the spongy tissue, -and in the hyaline tissue of the veins, petioles, and stems immediately below the epidermis, especially just under the stomata. So far as my observa- ‘tions go, the peripheral position of the host-cell, in all parts of the host- plant except the mesophyll, is correlated with the lesser development of intercellular space in the tissue of the given parts. For, it is evident that ‘the swarm-spores, after reaching the wide intercellular space developed under the stomata, are not capable, on account of the compact arrangement of the cells, to proceed further in the subepidermal tissue, so that they are com- pelled to infect any available cell in the immediate vicinity (Fig. 97). We find in this case very characteristic structure of the tubercle to be developed, as a rule, in the infected spot by the enlargement of the host-cell due to the subsequent growth of the fungus-body inside and by multiplication of ells around the host-cell. The epidermis is forcibly upheaved and con- sequently stretched. The guard-cells of the stomata, being then separated from each other, may leave between them a wide space, and may bring the wall of the host-cell to direct exposure through this space on the surface of the host. Cross sections or surface views of the tubercle thus developed reveals no different feature from that developed when the epidermal cell is infected, as is the case in other Synchytrium (Fig. 98). There is a remarkable difference in the structure of the tubercle caused by S. Puerariae. In the tubercle either in the mesophyll or other parts, we find in almost all cases many layers of cells between the epidermis and the host-cell, and no definite relation can be made out between the position of the host-cell and the stomata. This peculiarity of the tubercle is due, in my opinion, to the development of intercellular spaces in the subepidermal tissue, large enough to allow the swarm-spores more latitude of movement, and to give them more frequent occasions to attain the cells more removed from the stomata. The conclusion to be drawn from the results of my observations is that the swarm-spores of both S. decipiens and S. Puerariae enter the subepidermal tissue through the stomata, being responsive to chemical stimuli, and infect those cells which contain the stimulating substance, the depth of infection depending upon the degree of development of the intercellular spaces in different parte of the host. In the mesophyll of the leaf the swarm-spores 86 S. KUSANO: of both fungi can with equal easiness reach the proper host-cell, since the intercellular spaces necessary for their forward movement is sufficiently developed in the spongy tissue, while in other parts of the leaf and in the stem, S. decipiens develops near the surface and S. Puerariae in the deeper tissue according as the intercellular spaces are more or less developed. III. Cytology of Synchytrium Puerariae. After the swarm-spore enters the host-cell, it begins to grow rapidly in the protoplast of the host till it becomes a spherical, orange yellow,,. semifluid mass easily recognizable with the naked eye. When the fungus attains its maximal size, the feeding action of the host seems to be closed,. and now commences nuclear divisions in the fungus-body. The repeated divisions are followed by the formation of sporangia, in which the swarm- spores are formed by further successive nuclear divisions. Unlike other Synchytrium, no typical resting spore (“Dauerspore”) is found in this fungus. For the sake of mere convenience we will designate the uninucleate stage- the vegetative, and the subsequent stage the reproductive period. A. VEGETATIVE PERIOD. 1. The Swarm-Spore. For the sake of comparison with the youngest form of the fungus-body ceveloped in the host-cell I shall take here first the swarm-spore into con- sideration. The general shape of the swarm-spore is oval, with a long fiagellum at narrow end. At the broader portion of the body it has a few drops of orange yellowish oily matter, which stains intensely black with osmic acid (Fig. 80a). The nucleus is comparatively small, on which account we can not recognize distinctly the detailed structure (Fig. 80, 81). In well- stained preparation we find two or three deeply stained granules, all adhering to the indistinct nuclear membrane, and a hyaline space in the interior of the nucleus. Of the granules the largest represents the nucleolus, and the other, chromatin. Compared with another stage of development the A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 87 relative size of the nucleolus at this stage is very small, being more or less flattened and not spherical as usual. In the living state the nucleus is visible as a refractive speck. The refraction of light is due perhaps to the nuclear sap which makes up the main part of the nucleus, so that I can not agree with ZIMMERMANN (796, p- 33) who considers the similar speck visible in the swarm-spore of Chytridiaceae as nucleolus, which ScuHMItTz takes as chromatin-mass. 2. Youngest Form in the Host-Cell. The youngest form of the fungus in the host-cell, as observed by myself, is shown in Fig. 1, and on the basis of culture experiment of the swarm- spores, I estimate it to be nearly one day old after infection. In the given figure the fungus-body measures twice or more in diameter as the swarm-spore. The cytoplasm is dense and granular, being easily distinguished from the somewhat fibrillar protoplast of the host in which it lies imbedded. It is not always spherical, though usually so, and often it undergoes more or less deformation (Figs. 1-3). The visible change in the nucleus at such a stage is, above all, its increased size and the enlarged nucleolus, while the chromatin-granules remain as before in size and number, and the nuclear membrane is yet obscure. The enlarged nucleolus is now spherical, homogeneous and deeply stained. As a whole, there is no essential change in the structure of the nucleus except the increase of nucleolus in size (Figs. 1, 2). 9 3. Growing Stage. As the fungus undergoes further development, some changes can be seen in the growing nucleus. Accompanying its growth in size the chromatic substance increases gradually. It appears mostly as fine granules, lying close to the nuclear membrane. There occur moreover considerably 1. I was able to raise the swarm-spore to a certain stage of growth by inserting a capillary tube filled previously with the spores in the young shoot of the host, whereby the spores near the inserted end of the tube are nourished by the juice of the host, and after 24 hours they grew up to a spherical mass twice or more in diameter as compared with the spores at rest (KUSANO, ’08). 88 S. KUSANO: large granules, or globules, studding the nucleolus, which appear to be made up of the chromatin judging from their staining reaction (Figs. 3, 4). The Achromatic substance also increases in amount. It does not form a typical reticulum, but forms fine granules without any definite structure, or some- iimes of a faintly fibrous structure. It is a very remarkable fact that the quantity as well as the structure of the achromatic substance are not constant according to the fixing fluids, even in nuclei of similar stages of development. Fixed in KEtIsEr’s solution we always find a large amount of it in a finely granular (Figs. 31-37) or globular form (Fig. 5). When FLEMMING’s solution is used, it appears more or less fibrous and is very scanty (Figs. 4, 7, 26, 29). Careful observations appear to show that the globular mass produced by the former fixative occupies in the cavity of the nucleus the space left hyaline by the latter. This difference comes out constant whatever stains we may use. Hence, there is but little doubt that what we designate achromatin in this case on account of its staining quality comprises in reality two substances of quite different origin, one the true linin substance which is revealed dis- tinctly by FLewMrNe’s solution and the other a precipitate of the karyolimph, which is brought out more prominently by KEIsErR’s solution. The precipita- tion seems, I believe, to be due to the presence of certain protein substance. Davis (98, p. 268) found a similar structure of achromatin in the nucleus of the mother-cell of the tetraspore of Corallina, which he also assumed as due to a soluble protein substance in the karyolimph. It seems, however, that the protein substance in question of Synchytrium is not of the same nature as that of Corallina, since in the latter it is precipitated by FLEMMING’S solution, which is not the case in the former. The lightly stainable, finely granular mass found by L6WEeNTHAL (°05 b, Fig. 2) in the nucleus of Synchytrium Anemones is probably a precipitation product in the karyolimph closely similar to that of S. Puerariae; yor, it was also obtained from a material fixed in a solution of sublimate (hot alcoholic solution). Accompanying the increase of chromatin-granules in the cavity of the nucleus a remarkable change occurs in the enlarging nucleolus. Hitherto it has appeared as a compact mass, but now it becomes highly vacuolate. At first, a few comparatively large vacuoles occupy the centre, but finally A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 89 numerous small ones come to lie uniformly distributed in the peripheral portion. The time of the first appearance of the vacuoles can not be definitely made out, since the nucleolus about such a stage stains always deeply. It is however to be observed that the comparatively small nucleolus, in spite of its compact appearance, betrays a vacuolate structure, if a very thin section through its peripheral portion be examined. Hence it is safe to conclude that some vacuoles are present at an early stage, as shown in Figs. 3 and 4, but are invisible on account of the deep staining of the ground substance of the nucleolus. The vacuolation is a general occurrence in the nucleolus of plant as well as animal cells (WAGER, *04), and has been already ascertained in Chytridiaceae (Rosen, 793, STEVENS, 03, LOWENTHAL, 705 b, Ryvz, 707). In the latter family the most remarkable vacuolation was first noticed by STEVENS in Synchyirium decipiens. He distinguished two different structures imbedded in the granular matrix of the nucleolus in the primary nucleus—the homogene- ous globules of varying size (dissclution product) and ordinary vacuoles, both of which become more numerous in later stages. As regards the enclosed globules he did not mention their fate with accuracy. As the vacuolation is more prominent in S. Puerariae, I can not only confirm the view of STEVENS, but possibly show the behaviour of the nucleolus, which appears to have some bearing upon the vacuolation. a.—Vacuolation of the Nucleolus and the Formation of the Secondary Nucleoli. The increase of vacuoles in the nucleolus is accompanied by increase of the extra-nucleolar substances in the cavity of the nucleus, of which the large globules (Figs. 3-5) show at once their ontogenetic connection with the nucleolus by their attachment to the latter, though this is not always the case. At their first appearance at a young stage of the nucleus, these globules are comparativery small and compact, and in stained preparations look like the usual chromatin-granules. It is safe to say that, if they are not entirely made up of chromatin, they contain at last much more chromatic substance than those appearing at later stages. As the nucleolus becomes larger and larger, the globules that are presumably to be derived from it increase also in size, as shown in Figs. 6 and 7. In these figures these 90 S. KUSANO: globules are very numerous and are mostly detached from the nucleolus. In materials fixed in Ketser’s solution and stained with haematoxylin, they are always stained heavily like the nucleolus itself and are indistinguishable from the typical chromatin-granules that are also present at this stage (Fig. 6). But in materials fixed in FremMine’s solution and stained with haematoxylin, they take up less stain than the chromatin-granules or smaller globules, just like the nucleolus. I therefore believe that the large globules do not differ materially from the nucleolus, so that I propose to call them “secondary nucleoli” in distinction from the parent-nucleolus which may now be called “primary nucleolus.” The chromatin-lumps which STEVENS (703, Figs. 3, 4) mentions as occurring in a similar stage in S. decipiens seem to be homologous to, if not exactly same with my secondary nucleoli. Although the lumps figured by him are somewhat irregular in shape, it does not seem that they are invariably so. For, we have easily convinced our- selves that the secondary nucleolus in S. Puecrariae, though originally spherical, may become irregular in outline, when the chromatin of the nucleolus be- comes condensed on its surface as granules, which are nevertheless hidden from view by reason of the deep stain. According to my own observations on S. decipiens, which will be given fully later on, the lumps represent secondary nucleoli. Otherwise, they represent perhaps deformed nucleoli instead of being true chromatin-masses.* There is perhaps no need practically to dwell upon the problem whether the smaller globules are of strictly chromatic or more or less nucleolar nature, but with the larger ones it is a matter of importance to make clear their nature. If we take into consideration the occurrence of globules of con- siderable size, such as are represented in Fig. 9, it will be at once clear that they are certainly of nucleolar nature. In staining reaction the secondary nucleoli differ in varying degree from the primay nucleolus. Usually the oxyphile element predominates in the former, in consequence of which the secondary nucleoli take up the blue colouring matter. Stained with FLEMMING’s triple stain, therefore, they are violet, while the primary nucleolus is intensely red. Both nucleoli also 1. The differentiation of chromatin and achromatin in the secondary nucleolus appears less distinct with ELEmMiINc’s triple stain than with iion l:aematoxylin. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. oF behave differently towards haematoxylin: fixed in FLemMinG’s solution the secondary nucleoli are siained more intensely than the primary, and vice versa when fixed in KEIser’s solution (Fig. 13). In plant cells the occurrence of secondary nucleolus has not hitherto been paid much attention to as in animal cells. As Monrcomery remarked’ already (799, p. 515), “it is, in animal kingdom, quite frequent in many egg- cells, though it is infrequent in somatic cells of Metazoa.”* In the plant kingdom an instance was given by Fercuson (01) in the egg-nucleus of Pinus Strobus, but no additional cases have, as far as I know, been brought forward, which are at all comparable to it, nor has the nature of the secondary nucleolus been so throughly studied as in animal cells. Concerning the ontogenetic origin of the secondary nucleolus various views have been advanced by zoologists. Braver (791), FLoperus (796), and Montcomery (799) believe that the small nucleoli are simply budded off from the surface of the larger one, while ScHNEIDER (’83), MarsHAL (92), and McGitt (706) have brought forward evidences to show that the former are produced in the interior of the latter, and pass out through the pores in the cortical substance. FERGUSON confirmed in Pinws the latter view. In Synchytrium we can readily mention several facts to prove that the secondary nucleolus is derived from the primary one. Firstly some larger ones among the secondary nucleoli are frequently found attached to the surface of the primary nucleolus; secondly the more the secondary nucleoli increase in number, the more the vacuoles appear inside the primary nucleolus ; thirdly the relative amount of chromatin in the secondary nucleoli is ap- proximately equal to that contained in the primary nucleolus just at the time of their appearance; and lastly, when the primary nucleolus is highly vacuolate, the secondary nucleoli that are still attached to it are also equally vacuolate. All these facts irresistibly suggest that the secondary nucleoli have a genetic connection with the primary nucleolus. As regards the manner of their derivation I believe that they are preformed inside the primary nucleolus. Two large vacuoles in Fig. 14, which are found somewhat projecting from 1. He called the secondary nucleolus “ paranucleolus” and ordinary one, “ true nucleolus.” Witson designates them “accessory” and “ principal” nucleolus respectively. the surface of the primary nucleolus, seem to me not to be the ordinary vacuoles, but to be globules representing the secondary nucleoli, only slightly stainable with haematoxylin at this stage. In Fig. 13 one of the globules is In the way of passing out through the pore on the surface of the primary nucleolus in the cavity of the nucleus. This augument is strengthened by a similar process to be observed in the nucleolus of the allied form S. decipiens. In Fig. 93 two fissures may be found on opposite sides on the primary nucleolus, which were perhaps produced hy compression under the cover glass. Close to each fissure lies a large vacuolate secondary nucleolus, probably just pushed out through it. It is highly probable that the fissures were produced at the hollow space made after the emission of these secondary nucleoli. A large vacuole near the surface of the primary nucleolus shown in Fig. 92 would represent with certainty such a hollow space. The crescent- shaped depression on the surface of this vacuole is in all probability the place from which the secondary nucleolus had passed out. A quite similar figure was given by Fereuson (701, Fig. 36) in a nucleolus of Pinus Strobus, which, according to her, produces secondary nucleoli. The secondary nucleoli lying near, or attached to, the parent-nucleolus are usually larger than those already detached, and the vacuolation which may be seen in the attached nucleolus disappears in the detached ones. From these facts it may be concluded that the substance of the secondary nucleoli is being gradually condensed, and becoming more compact. We may further assert that the composition of the secondary nucleoli are more or Jess similar to that of the parent-nucleolus; while the latter contains more chromatin at its younger stage, the secondary nucleolus derived from it shows a stronger chromatic character, but when the parent-nucleolus becomes vacuolate and poor in chromatin, it produces also vacuolate and less staining secondary nucleolus. LOWENTHAL and Ryvz have already mentioned multinucleolate nucleus in Synchytrium, but they did not notice the origin of the smaller nucleoli, r their relation to the larger one. b.—Condenzation of the Chromatin. The chromatin-granules increase gradually, as the nucleus advances in growth, and they attain the maximal number in the full grown nuclelus in which numerous secondary nucleoli are present. From observations of nuclei A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 9S in several stages of development I have arrived at the conclusion that the chromatin-granules are mainly condensation products either of the secondary or primary nucleolus. Though many of these granules are freely distributed in the cavity of the nucleus when full grown or a little earlier, showing no apparent evidence of their genetic connection with the nucleoli, yet some granules are found always to stud the secondary nucleoli when a certain period of time has elapsed after their derivation from the primary nucleolus. That these granules are derived from the nucleolus is quite obvious when we observe the development of the latter. With all secondary nucleoli the staining reaction shows at once that at an early stage they contain chromatin uniformly distributed. The staining capacity by chromatic stains of the ground sub- stance gradually decreases ‘during the development of the nucleoli, while the peripheral portion becomes more heavily stainable. The irregular shape which is often observed in some secondary nucleoli is certainly due to the precipitation of chromatin-masses at the cortical layer of the nucleoli. An advanced step of such process is shown in Figs. 7 and 8, in which the chromatin- granules apparently adhere to the surface of the nucleoli whose ground substance has now lost considerably their staining capacity. The next step at which the granules are to be entirely detached from the nucleoli is represented in Fig. 9. The same process comes out most clearly in the primary nucleolus which has discharged all its secondary nucleoli. Its ground substance appears very faint in stained preparations, and small yacuoles which were previously distinct is now scarcely visible. There is perhaps a residue of chromatin, and it now commences to condense at the peripheral layer and then appears as a distinct cortical layer of the nucleolus (Fig. 15) or small granules studding the surface (Fig. 7). Some observers have already expressed the opinion that in the nucleolus of higher plants the chromatin occupies the peripheral portion, while the interior is filled with plastin (cf. STRASBURGER, 00, p. 138, 707, p. 75 and Korrnicke, 703, p. (111)). In Synchytrium it is, of course, not to be denied, from the facts above given, that the chromatin is contained in the nucleolus. As to its state of distribution in the nucleolar substance I can not yet advance any definite view as to whether it occupies always the peripheral portion or not. So far as the stained preparations 94 S. KUSANO: are concerned, the chromatin seems to be distributed uniformly in the plastin of the nucleolus in the younger stage of the nucleus. The relation of the chromatin and nucleolus is thus clearly demonstrated in the later stages of development of the nuclei, but there is some doubt about it when the nuclei are at the beginning of growth. It is then not practically apparent whether the globules derived from the nucleolus are chromatin-granules -or small secondary nucleoli rich in chromatic substance, in which the conden- sation process of the chromatin is not clearly exhibited. Perhaps in the smaller secondary nucleoli condensation process may be practically omitted, and they then originate as globules approximately of the nature of chromatin- granules. When we take into account the nature of smaller and larger globules derived at various successive stages of the nucleolus, it may be seen that the designation of secondary nucleolus does not accurately express its definite nature as distinctive from the parent-nucleolus. As its staining character, vor instance, is not constant in the smaller and larger, i.e. early and lately derived nucleoli, it can hardly be said that it corresponds strictly to the paranucleolus (WiILson, *00) or oxyphile nucleolus (McGrt1, 706). On the question of the derivation of chromatin from the nucleolus there is an immense literature, to which I can not refer in detail here. The critical reviews are found in an elaborate memoir of Montcomery (799) on the papers published up to 1897. On the botanical side WacrER (704) and vy. Derscuav (707) have dwelt upon this problem, and KoERNICKE (703, p. (108-109) ) and SrrasspurcER (707, p. 74-77) have published critical reviews. instances of the case im question are increasing in the plant kingdom, and are known in the nuclei not only of the lower, but also of the higher plants (GEORGEVITCH, “08, Nicnots, 08). We may now mention Synchytrium as affording an additional instance of the derivation of the chromatin from the nucleolus. Together with these changes there appear fine granules thickly besetting the inner surface of the nuclear membrane. They increase in number as the nucleus grows. A great variation is found in their size and staining character; some are like chromatin-granules in size and staining reaction, while others ere much smaller and less stainable (Figs. 3, 4, 7, 8). Re garding their origin I am of the view that they are derived directly from A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 95 cither the primary or secondary nucleoli, or that they are dissolution-products of chromatin-granules. Their peripheral situation in the nucleus is in favour of the view that they are passing out in a dissolved form into the surrounding cytoplasm through the nuclear membrane. The gradual diminu- tion of size and staining capacity is most probably an expression of the process of dissolution they are undergoing. Similar phenomena have already been known in animal egg-cells, in which the nucleolar substances are given off into the surrounding cytoplasm as deutoplasm or yolk-nuclei (W1Lson, 700). In the present case the chromatin developed in the growing nucleus is certainly more than sufficient for the formation of the chromosomes during karyo- kinesis, which, as will be given below, are exceedingly small and few in number. This fact renders it most probable that there are, as in animal egg-cells, two different components in the so-called chromatin, of which the essential com- ponent forms the chromosomes, while the other undergoes some chemical change and subserves another function, perhaps something related to nutrition. c.—The Nuclear Membrane. Various views have been expressed as to the structure ot the nuclear membrane in Synchytrium. In S. Taraxaci DANGEARD (790) mentioned the granular structure. According to STEVENS, the wall of the primary nucleus in S. decipiens is sharply defined, and at the end of growth the wall becomes very thick and is gelatinized finally. LOwENTHAL’s figure (705 b) of the nucleus of the resting spore of S. Anemones does not show the existence of a distinst membrane. Lately Rytz (’07, p. 814) succeeded in observing a somewhat sharply defined membrane in certain species of Synchytrium. The definite limitation of the nuclear membrane in S. Puerariae is easily observable in the full grown nucleus, when the fine nuclear granules are closely arranged along its inner surface. In surface view the presence of granules makes the membrane appear as granular (Fig. 8), as DANGEARD stated. The increase of the granules in later stages may easily lead us to think that the membrane is swelling into a granular form, but previous to the division of the nucleus these granules vanish entirely, while the membrane proper be- comes indistinct and disappears ultimately. ~ Though more or less obscure, the membrane consists invariably of intricated kinoplasmic fibres, and in no case have I been able to observe swelling at any stage. Neverthless there exists in fact a granular mass at certain stage of karyokinesis, exactly similar 96 S. KUSANO: to that of S. decipiens found by STEVENS as originating from the membrane. As to its origin I must, however, offer a diffcrent view from that of STEVENS, as will be given later on at full length. d.—Dimension of the Nucleus. STEVENS has already remarked that the primary nucleus of Synchytrium decipiens is exceptionally large (35 # with a nucleolus 11 # in diameter) among fungi, which are generally known to have exceedingly small nuclei. In S. Pucrariae the nucleus is still larger, measuring when full grown 70x52 4, with a nucleolus 14” in diameter. This is doubtless the largest nucleus known among fungi. Even in Phanerogams, so far as the vegetative cells are concerned, instances of such a large nucleus are perhaps very few. In sexual cells ZIMMERMANN (796, p. 11) mentioned among Angiosperms the embryosac-cell of Fritillaria imperialis (50x25y) as having the largest nucleus. The nucleus of the oosphere of Gymnosperms is much larger; thus according to IkeNo (°98, p. 583), it measures in Cycas revoluta 3704 in length, and according to my own measurement in a preparation of Mr. K. Mryake, it is in Zamia floridana 610x426. If we except these sexual cells, or those vegetative cells which have filamentous nuclei (KORNICKE, °03, p. (132)) in some higher plants, Synchytrium Pucrariae may be considered as one of the plants which possess the largest nuclei.* B. REPRODUCTIVE PERIOD. t. Primary Mitosis. Tn the full grown fungus-body nuclear divisions occur with great rapidity, and the fungus passes from the uninucleate to the multinucleate condition. This latter condition seems to be coincident morphologically with the general character of the vegetative thallus of Phycomycetes. However, at this stage the symplast of the host (Kusano, ’07 a,b), on which the fungus relies for its nutrition, is expanded into such an exceedingly thin membrane that its activity must be very nearly nil. Simultaneous with this change the fungus excretes around its body a hyaline membrane which apparently seems to I. For the dimension of nuclei in animal cells consult CARNOY’s paper (’88, p. 270). pap P A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. OF arrest the entrance of nutritive substances from the host into the fungus- body, and indicates perhaps the end of feeding action. Consequently we take this stage as the end of the vegetative, and the beginning of the re- productive period in accordance with HaArPer’s (’99, p. 481) and STEVENS’ (703, p. 406) view. As far as my observations go, the first nuclear division is really mitotic as in S. decipiens, and no amitotic division, such as was found by DANGEARD (790) and confirmed by Rosen (793) in other Synchytriwm, was at all observ- able. In studying the primary mitosis I experienced a similar difficulty as Stevens. The karyokinetic figures are exceedingly small in contrast to the considerable size of the nucleus in the resting condition. Moreover, serial stages relating to the division could not be found out complete in spite of the examination of a great many preparations. So that I shall describe here the successive processes of division up to the metaphase only, from which IT can draw conclusions different from those arrived at by STEVENS in S. decipiens. Tt is somewhat difficult to point out precisely the beginning of the nuclear changes preparatory to mitosis, since the nucleus in the vegetative period is not constant in structure, and there is found no spireme stage preceding the division to indicate the early prophase. However, the critical point, at which the resting stage terminates and division begins, may be practically determined by taking into account certain features in the nucleus, such as its maximal growth, the maximal increase in number of chromatin-granules as well as the secondary nucleoli, and the sudden diminution of these nuclear elements. I will denote therefore this last mentioned condition of the nucleus as an early prophase, corresponding to the spireme stage in the typical karyo- kinesis. As the first step in the diminution of the nuclear elements, we see that the secondary nucleoli begin to disorganize, becoming faintly stained and showing only the achromatic ground substance. They proceed then to fade away in the cavity of the nucleus, setting free the attached chromatin-granules. Simultaneously with this change the chromatin-granules gradually diminish, apparently by dissolution, as may be seen from their becoming smaller in size and less stainable (Figs. 10, 11). When this process proceeds further, 98 S. KUSANO: the nucleus arrives at a stage with exceedingly few visible elements in its cavity and with a hardly recognizable wall. Fig. 12 shows one of the sections of a nucleus at such a stage. We see here only a few chromatin-granules yet remaining and a few secondary nucleoli in indefinite or somewhat globular iorm. ‘The primary nucleolus still retains its original form, but its staining capacity seems to have exceedingly diminished. A similar remarkable diminu- tion of chromatic element was already observed by STEVENS in the correspond- ing stage of S. decipiens. He says, “formerly coarse and lumpy, its globular masses become much more numerous and relatively smaller. They then appear to elongate, the numerous globules being replaced by rod crossed and tangled in inextricable confusion” (p. 410). His globular masses (hia Fig. +) correspond to my secondary nucleoli, as already stated, and the much smaller granules of the next stage (his Fig. 5), to the chromatin-granules proper now set free by the dissolution of the nucleoli to which they had adhered. This I can confirm by my own observation of the same fungus. Further, he mentioned that the chromatin-granules were replaced by the rods of chromatin. It seems to me that a further study is necessary to place this fate of the granules beyond doubt. For, as to the perfcctly similar granules in S. Puerariae there is no doubt that they undergo a gradual dissolution. The nuclear membrane now vanishes, and the large cavity of the nucleus is soon occupied by the surrounding cytoplasm. At this stage the nuclear contents undergo further dissolution. The primary nucleolus is deformed and produces pseudopodia-like processes through which the ground substanee passes out into the cytoplasm. In Fig. 16 is shown a somewhat advanced stage of this process. Here we find numerous rod-shaped granular fibres radiating from the central achromatic mass which represents the residue of ihe primary nucleolus now in the process os further dissolution. Numerous chromatin-granules studding these fibres seem also to dissolve away together in the surrounding cytoplasm. In the next figure (Fig. 17) a still more advanced stage is shown, in which the primary nucleolus has lost its original fourm and been replaced entirely by the fibres. The general feature at this stage may be perhaps comparable to the spireme stage in an ordinary nucleus, the fibres corresponding to the ruclear threads; but as the arrangement of chromatin on the fibres is not so regular as on typical nuclear threads, we A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 99 may regard it rather as a result ef the dissolution of both the chromatie and achromatic substances of the nucleus. A similar change is known in the prophase stage of the vegetative cell of Chara fragilis. According to DEBSKI (798, p. 635), soon after the nuclear membrane disappears, the “Knauel” of the chromosomes takes its: position in the centre of the nuclear cavity and striations appear radiating from it in all directions, often intersecting one another (see his Fig. 9). He thinks that they form the spindle (p. 642). In S. Pucrariae I can not find any genetic connection between these fibres and the spindle. The radiating fibres afterwards appear as granular striations, and a few chromatin-granules left at the focus of the striations enter into the composition of the chromosomes (Fig. 18). While the chromosomes are being built up, the radial striations become fainter and fainter, and acquire a more granular character (Fig. 19). In Fig. 20, in which the spindle is already formed, the striations have dis- appeared almost entirely and are replaced distinctly by a dense granular cytoplasmic mass. The origin of the spindle could not be clearly made out, but it seems certain that the achromatic residue of the ground substance of the primary nucleolus (Fig. 18) is concerned in the formation of the spindle-fibres. The spindle is at first broadly oblong with rounded poles (Figs. 20, 22-25). The fibres are very fine but distinct, though at first it appears some- what gelatinous. At the metaphase the spindle becomes pointed at the poles (Fig. 21). The chromosomes, apparently five in number, are generally spherical (Figs. 22, 25). They often assume during splitting into daughter- chromosomes an oblong form (Fig. 23). I have not been able to obtain further stages of karyokinesis, and so the details of the reconstruction of the daughter-nuclei is still unknown. This gap, however, may be filled by the serial stages easily observable in the secondary nuclei, with which the main features of the desired stages in the primary nucleus probably agree. The nucleolus does not always disappear in later stages of the primary mitosis, and its residue is found frequently near the spindle (Figs. 19, 22). In the metaphase the spindle is surrounded by a halo of granular mass (Fig. 25). 3. Membrane-forming body. The process of membrane-formation in the daught«r-nuclei is certainly a most striking feature in the present study, unlike anything known in general cytology. Although in the foregoing paragraph I mentioned the aster as behaving like the centrosome because of its appearance, there is no doubt that in its function or its relation to nuclear division it does not show any resemblance to the typical centrosome. As is well known, the centrosome is a permanent organ during certain ceil- generations, transmitted by division from nucleus to nucleus. It is, as a rule. the centre of activity during karvokinesis, and is especially concerned in the formation of the spindle. Likewise, it is not comparable to the centro- sphere which may also play a similar role. Further, the polar rays appearing at the poles of the spindles in many higher plants seem to he closely related in function to the centrosome (NEMEC, 01), though their occurrence is quits transitory like the aster in question. The nuclei of the ascus of many Ascomycetes are permanently attended by a centrosome, or Harrer’s “central body,” which is transmitted from one nuclear generation to the next. When the ascospore-formation is beginning, the centrosome acts as the centre in forming the “Hautschicht” of the ascospore ; put the observations of several investigators have not brought to light any proof of its activity in the formation of the nuclear membrane. Even accepting CLAUSSEN’s notion that it may be concerned in the formation of the membrane, we can not vet regard this body as exactly identical with the similar body in Synchytrium, since the period of occurrence is quite different in the two cases. Further, CHAMBERLAIN’S centrosphere in Pe/lia, which appears to be associated with the nuclear membrane, is not comparable in structure to the centrosome-like body in Synchytrium. The problematic body under consideration is characterised by its short existence, occurring only at the telophase. As noted above, it can not be distinguished morphologically from the typical centrosome or blepharoplast, but on account of its peculiar function it must be regarded as a body sui generis, and consequently I venture to call it by the name of “karyoder- matoplast”? in distinction from other allied bodies. The origin of the karyodermatoplast is not at present apparent. I am I. xaov0v = nut, déoya==skin, ZAaaTOs = moulded. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 27 eure that it is not a direct derivative of the nucleus; yet the staining reaction renders it probable that the central granules are of nucleolar origin. Making allowance for NEMEC’s view (99a) that there is a genetic connection be- tween the kinoplasmic fibres and the extranuclear body at the poles of the spindle, I am inclined to think that the karyodermatoplast represents an extranuclear nucleolus which may be transformed wholly into the kinoplasmie fibres while forming the nuclear membrane. This notion agrees well with the prevalent view on the function of the intranuclear nucleolus that it con- tributes to the formation of the spindle-fibres. As already stated, much nucleolar substance is contained dissolved in the cytoplasm. The appearance of a dense granular mass preceding the formation of the karyodermatoplast indicates most probably the accumulation of this substance to reconstitute the extranuclear nucleolus. Admitting this view, we may say that the nuclear membrane is, like other nuclear elements, derived from the parent-nucleus. 4. Comparison with the ovum-cells of animals. Broadly speaking, the primary nucleus of Synchytrium has many points of resemblance to the nuclei of the ovum-cells of animals (WrLson, 700); (1) it is comparatively large, (2).it contains a large amount of karyolymph, (3) there occur, though not in exactly the same form, secondary nucleoli, (4) the greater part of the nuclear elements, mostly chromatin, are thrown off into the surrounding protoplasm, etc. Theze similarities are perhaps associated with similar be- haviour of the nuclei during subsequent development. From the physiological point of view the uninucleate stage of the fungus is strictly comparable with the same condition of the ovum-cell: the fungus at this stage requires to store up or elaborate a large amount of nutritive substance for the subsequent rapid increase of the nuclei, a process in which the nucleolus plays an important role. A most remarkable resemblance lies perhaps in the existence of two different components of chromatin, one concerned in heredity and the other perhaps in nutrition. In the nuclei of the ovum-cells they are distinguished by some authors as “idiochromatin’ and “tropochromatin” respectively (LuBoscH, *02). The same designations may in my opinion be applied to the different kinds of chromatin found in Synchytrium. 128 Samu SANO: VI. Cytology of the Host-Cell. Tt is evident that in all intracellular parasites their development is most intimately dependent upon the condition of the host-cell in which they are enclosed. It is therefore advantageous for the parasites not to inflict direct injury on the host-cell, at least during their vegetative period. In this respect NAWASCHIN (99) has already stated that the myxoamoeba of Plasmodiophora Brassicae is to a certain extent in symbiotic relation with the host, in proof of which it is pointed out that the parasite causes an accelerated development of the protoplast that harbours it. A similar relation between the parasite and the host-cell can be found in the case of Synchytrium. When a cell is infected at an early stage of its growth, it contain a larger amount of protoplasm than the unaffected neighbouring cells (Figs. 1-3), and its chloroplasts can accumulate at the same stage starch-granules like those of unaffected cells. While it is believed that Synchytrium, when full grown, exceeds considerably in size the ordinary cells of the host, it may be easily imagined that accompanying the enlargement of the fungus-body the protoplast encasing it must be expanded, unless a corresponding increase in amount takes place, to such an extent as to mechanically interfere with its normal activities and consequently to he disadvantageous for the further development of the fungus. However, in both S. Pwerariae and S. decipiens the further development of the parasits is facilitated by the formation of sym- plasts, as I have stated elsewhere (707b). The formation of symplasts brings about another advantageous effect upon the fungi. The wall of the cells entering into the formation of the symplast is dissolved by the action of the fungi, and a wide lysigenic chamber results, which affords sufficient space for the enlargement of the fungus-body. Maenes (’97, *01, 702), in his studies of Urophlyctis, has reported that such lysigenic chamber is produced in a quite similar manner, but it is not certain from his descriptions whether a symplast is formed at the same time, and it remains still undetermined whether the formation of the lysigenie chamber and symplast is of such a biological importance in Urophlyctis as in Synchytrium. The group of cells of the host undergoing the dissolution is in almost all cases those that owe their origin to the stimulating action of the fungi. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 129 The number of these cells is different in different parts of the host, or in different stages of development of the affected tissue. Generally the stem and both the petiole and veins of the leaf are more liable to produce such abnormal cells than the mesophyll of the leaf. Also the hypertrophy of the host-tissue is more remarkable when infection takes place at a younger than at a later stage. The varying size of the adult fungus-body is perhaps intimately related with this difference. When the fungi are vet at an early stage of growth, the symplast is found as a somewhat thick coating around them. 3ut further growth of the parasite causes the symplast to be much stretched (Fig. 95), and towards the end of its vegetative or growing period, the symplast becomes, as it were, an investing membrane for the parasite (Figs. 61,85). At this stage the activi- ties of the symplast are certainly brought to a standstill, and related to this con- dition there appears at th's time a hyaline membrane around the fungus-body, making the interchange of substances between the symplast and the fungus possibly difficult or quite impossible. The above fact shows that the fungus does not consume the symplast at the end of its vegetative period, and that the disappearance of the symplast, which takes place after the fungus has further advanced in development to form mature sporangia, is most probably due to selfdisorganization. I take here the hyaline membrane appearing between the symplast and the fungus as a mark separating the vegetative from the reproductive period. It is on the strength of this assumption that T maintain that any subsequent change occurring in the symplast after the appearance of the hyaline membrane is not under the direct action of the fungus. According to NAwAscHIN and Toumey, the protoplast of the host- cell they studied is wholly consumed by the parasite. To determine whether selfdisorganization is concerned in this case in the disappearance of the protoplast or not, is practically impossible owing to the absence of any clear mark separating the vegetative from the reproductive period, though I think it likely that selfdisorganization takes place. Noteworthy is the behaviour of the parasite towards the nuclei of the host. It is not that the action is visible in all the nuclei of the tubercle, but it is more or less apparent in the nucleus of the host-cell or nuclei of the symplast. At the beginning these nuclei appear perfectly normal, but the growth of the parasite brings about a pronounced hypertrophy of the nuclei, accompanied frequently hy deformation 130 S. KUSANO: (Figs. 84, 95, 96). However, the internal structure—the relative amounts of linin and chromatin—seems to be unchanged during the above modification, so that I can detect neither a decrease of chromatin, as was observed by NAWASCHIN and TotMEy in their fungus-materials, nor an increase, as was reported by LOWENTHAL in Synchylrium. At the time the symplast is stretched into a thin membrane, its nuclei become strikingly flattened so as to be disc-shape (Figs. 85, 95). They are then often stained uniformly and deeply, showing the process of disorganiza- tion. Sometimes conspicuous vacuolation takes place before they become compressed (Fig. 86): the chromatin is divided into prominent globules, while the network structure appears somewhat indistinctly. The number of nuclei of a symplast is variable according to the number of the composing cells: at times more than 20 nuclei may be easily counted. Frequently some nuclei lie close together, appearing as if they were produced by the amitotic division or fragmentation of a nucleus. However, any evidence for the occurrence of amitosis or even mitosis was not been found, and at present I am of the view that the nuclei of the svmplast are nothing but the nuclei of the cells that have entered into the formation of the symplast. The multiplication of cells around the diseased spot of the host takes place by means of typical mitosis and the occurrence of amitosis is exceeding- ly doubtful. VII. Summary. The chief results of my observations on Synchytrium Puerariae may be summarised as follows. But it must be remarked that nearly the same results have also been obtained in S. decipiens, so far as my observations have gone. 1. The swarm-spore infects not an epidermal cell, but always a sub- epidermal cell containing very little or no chlorophyll, by responding to the chemical stimulus exerted by the latter. 2. The fungus-body in the uninucleate condition resembles in many points, but especially in the structure of the nucleus, the ovum-cell of animals. 3. The youngest nucleus contains a single prominent chromatin-nucleo- Ins as the only visible element within the membrane. Chromatin-granules and linin-threads that appear later are derivatives of it. A CONTRIBUTION TO THE CYTOLOGY OF SYNCEHLYTRIUM. 151 4. Secondary nucleoli are present, and are especially more numerous in the primary nucleus. They are, at least in the primary nucleus, preformed in the interior of the primary nucleolus and successively pass out through ihe cortical layer of the latter. The relative amount of chromatin and plastin contained in them varies according to the periods at which they are derived, but it is always approximately same as that of the primary nucleolus in which the relative amount of chromatin varies according to the period of developinent. 5. During the growth of the nucleus the primary nucleolus becomes more and more vacuolate; and the secondary nucleolus derived from the primary nucleolus in such a condition is also equally vacuolate. However, condensa- tion process may soon take place on the latter, by which it becomes smaller and more compact and loses the vacuolate character. 6. The primary nucleolus is the morphological centre of the nucleus. It gives rise to chroniatin-granules and linin-threads or achromatic substance. Its ontogenetic origin is the daughter-chromosomes together with residue of spindle-fibres. Thus it shows most clearly the continuity of the chromatic and achromatic substances in the successive nuclear generations, so that WaceEr’s statement about the behaviour of the nucleolus is applicable here without any modification. 7. The composition of the primary nucleolus is not constant throughout the successive stages of one nuclear generation, or throughout different nuclear generations. Generally, at the youngest stage or sometimes throughout the whole stage of development at certain secondary nuclear generations, it may be regarded approximately as chromatin-nucleolus. At an advanced stage it is, as a rule, a plastin-chromatin-nucleolus, and previous to nuclear division it becomes a plastin-nucleolus. 8. The chromatic substance, which is at first uniformly distributed in the primary as well as secondary nucleoli, condenses at a later stage at the periphery, and is ultimately set free as granules in the nuclear cavity. 9. During nuclear division the greater part of the chromatin is thrown off in a dissolved form into the surrounding cytoplasm, and the residue goes into the composition of the chromosomes. 10. The karyolymph contains certain soluble albuminous substances easily precipitated as fine granules or globules by corrosive sublimate. 132 S. KUSANO: 11. The prophase stage of mitosis is atypical. No typical spireme is formed, and most of the nuclear elements dissolve away and the final remnants constitute a spindle. 12. In the secondary nuclei there takes place exudation of the nuclear elements, in consequence of which the spindle-figures are formed in various ways. 13. The dissolution-product of the primary nucleus persists as a dense granular mass around the spindle. At a later stage its peripheral portion fades away into the surrounding cytoplasm and then it assumes the form of a halo around the spindle. The halo is not present during the secondary mitosis. 14. At the telophase stage there appears suddenly a centrosome-like body, called here “karyodermatoplast,” which is concerned in the formation of the nuclear membrane. It disappears when the membrane is completely formed. 15. The chromosomes are generally spherical and definitely five in number. 16. Generally, the nucleolus persists without any remarkable decrease in size during the secondary divisions. It then undergoes a gradual dissolu- tion, but does not entirely disappear even at the time of the next division. 17. The primordial sporangium is formed by progressive cleavage-forma- tion, when the cytoplasm of the fungus-body shrinks by throwing off water or oily matter. Otherwise, it is formed by approximately simultaneous formation of partitions among the cytoplasm. 18. The primordia! sporangium contains a few nuclei and no uninucleate protospore is formed previously. 19. The fungus develops at first in a single host-cell; but its sub- sequent considerable enlargement causes the walls of the surrounding cells of the host to dissolve, and a wide lysigenic intercellular chamber and symplast are formed. 20. The symplast may be active so long as the vegetative or feeding period of the furgus lasts. 21. The number of nuclei in the symplast indicates just the number of the cells brought into fusion. They are remarkably deformed and enlarged. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. £33 Postscript. While the manuscript of the present article was lying in its final shape, I received a most interesting paper of STEVENS, entitled “Some Remarkable Nuclear Structure in Synchytrium™ (Annales Mycologici, Bd V, No. 6, 1907, p. 480), in which he gives many remarkable phenomena in the secondary nuclei of Synchytrium decipiens. In the accompanying plate he seems to haye illustrated exhaustively and comprehensively the most important changes he was able to observe, and his interpretations of the numerous problematic phenomena seem to be based mainly on the figures of that plate. The facts noted by him and to be seen from his figures do not invalidate the conclusions drawn by me in S. Pucrariae. Fortunately enough his figures are quite similar to those given by me in 8. Puerariae, so that they strengthen my con- clusions on the latter fungus, and also confirm my anticipations relating to the nuclear phenomena in S. decipiens. Notwithstanding, there are some differences between us, which lead me to make some remarks upon his paper. They do not, of course, touch the value of his preparations, but have reference to his opinions on those points upon which I have also advanced my views. In this connection it must be noted that he remarked previously in setting forth his opinions, “the greatest difficulty in answering these questions 1s the lack of any definite structure to use as a measure of the period of development to know whether a certain view is younger or older than another view” (p. 482). In S. Puerariae, however, I can say that I have succeeded in determining the period of development more accurately by using a larger number of serial stages than given by Stevens. Although it must be admitted that there ave generally specific differences on various points in different objects, I can not abstain from proposing my own interpretations of the several re- markable structures mentioned hy STEVENS, on the basis of the results obtained in S. Puerariae. Confining ourselves for the present to matters of fact, STEVENS’ figures agree perfectly with my expectations. For, I have already expressed my view in my preliminary note (Ota) that the details of nuclear. division in ¥. decipiens would be found almost identical with those in S. Puerariae (p. 121). It is, therefore, very interesting to compare his figures with my own 15 S. KUSANO: of S. Puerariae, to ascertain how far my statement is justified. 1. SrevENS has given in his Fig. 1 a primary nucleus of anomalous structure, entirely devoid of membrane and consisting of the chromatin and a large nucleolus. Admitting that the nuclear structure given in his first paper (05) as normal, it is but natural to take such structure here figured as anomalous. It is, however, questionable whether the structure regarded by STEVENS as normal is really so or not. To judge from my own observations on S. Puerariae and partly on the same species as STEVENS’, the gelatinous swelling of the nuclear membrane regarded by him as a normal occurrence during mitosis is highly questionable. In S. decipiens, so far as observed by me, the nucleus previous to division is provided with a scarcely recogniz- able membrane (Fig. 91); and in S. Puerariae I could observe with great definiteness the disappearance of the membrane and the consequent coming in contact of the surrounding cytoplasm with the central irregular mass of residual nuclear elements (Fig. 16). STEVENS’ figure seems to represent this process of nuclear division, showing a_ stage just previous to that shown in my Fig. 16 in S. Puerariae, or just following that shown in Fig. 91 in NS. decipiens. So far as I know, this is a normal process during mitosis in both species, and consequently I can not agree with him in taking the karyokinetic figures mentioned in his first paper as normal. This view is strengthened by the fact that these figures have many peculiarities of structure not generally observable. 2. In the multinucleate condition of the fungus he mentions some large homogenous nuclear bodies in the protoplasm surrounded each by a clear space with or without a limiting wall (Fig. 2), and also a few small isolated nucleolar bodies (Figs. 8, 11). All these bodies have been observed by me in S. Puerariae. The large nuclear body of STEVENS corresponds to the youngest stage of the daughter-nucleus, those with membrane beng the later stages of those without it. The body itself represents the nucleolus arising from the chromosomes and a residue of the spindle-fibres. Later the nucleolus gives rise to the chromatin as well as the linin, which both appear in the form of a network in the adult nucleus (his Figs. 13-17). As to the isolated nucleolar body, what I have stated in 8. Puerariae is applicable here ; they are the nucleolar residue of the parent-nucleus now going to be dissolved. In the metaphase he says, “no nucleoli are certainly discernible” (p. 483). A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 135 Judging from my figures, however, this point requires reinvestigation, since in my preparations the absence of the nucleoli during karyokinesis, is not very frequent, and in the majority of cases the spindle is attended by one or two prominent nucleoli, persisting through the telophase and afterwards as extranuclear nucleoli for a short time. 3. A centrosome- or blepharoplast-like body was found by STEVENS in the multinucleate state of the fungus. As to its or’gin he state, “whether these asters are isolated or are connected with nuclei lving in other planes, and which are therefore not visible from the present viewpoint, is not certain. From their abundance it seems rather more probable that many of them are independent of any nuclear connection” (p. 481). However, he mentions in other places certain connection of the aster with the nucleus, saying, “the rays seem to shorten until the centre of the aster touches the nuclear membrane” (p. 481), and “the influence of the rays upon the shape of the nuclear wall is apparent” (p. 481). These observations do not appear to have led him to form a definite view on the significance of the aster, but they are enough to show the essential similarity of phenomena in his and my materials, and the presence of what T have called “karyodermatoplast.” The aster in my case appears always close to the daughter-nucleus at the late telophase, among a dense cytoplasmic mass, and is replaced. when the membrane is completed, by a dense cytoplasmic mass again. If we bear in mind the connection of the aster with the nuclear membrane, many characteristic phenomena relating to the aster, mentioned by STEVENS as yet problematic, will be explained most easily. Firstly, the deformation of the nucleus, which he thought to be due to the action of the astral rays, points out that the aster is going to form the membrane. Secondly, the shortening of the rays is an expression of the progress of the membrane- formation, and lastly, a dense cytoplasmic mass in the place of the aster is, nothing but its remnant. His figures become more intelligible, if we arrange them according to the serial stages of this process as follows: Fig. 3, Fig. 4, Figs. 5, 7, Fig. 6, and Fig. 9. In all of these figures the membrane is represented very sharply, and this fact seems to conflict with my conclusion. However, it must be borne in mind that certain features may be liable to be exaggerated in drawing according to the peculiar view of the observer. In my preparations of S. Puerariae the membrane is not yet formed in the 156 S. KUSANO: nucleus at the time the aster begins to appear, and likewise a reexamination of the preparations of S. decipiens will reveal, I believe, the absence of the membrane at the same stage. As to the multiple asters and blepharoplast-like body (his Figs. 11, 12) I can not express any definite view, since such structures were not found in S. Puerariae. "They may possibly be abnormal forms of my karyodermatoplast whose function is never arrested. A large cytoplasmic mass which appears close to an adult nucleus is for me a problematic body (his Figs. 13, 16, 17). Owing to its large size it is not comparable to the remnant of the ordinary aster, though its position and structure appear to suggest a close resemblance to the latter. STEVENS considered it to be the remnant of a nucleolus or its dissolution-product. How- ever, if, as stated above, the remnant of the parent-nucleolus in S. decipiens is represented by the numerous extranuclear nucleoli scattered in the cytoplasm, the problematic mass does not seem to have any connection with the nucleolus. T am inclined to think that it indicates an abnormal remnant of aster. 4. STEVENS figures several ‘clusters of nuclei as abnormal. Similar clusters occur quite frequently in S. Pucrariaec. TI could not follow the fate of these nuclei, but from the fact that no clusters are found in the fungus-body previous to cleayage-formation, I agree with STEVENS’ view that the nuclei of these clusters separate afterwards. Thus, on the whole, his figures are so completely reproduced that I can draw from them the same conclusions as in 8. Puerariae. Iam emboldened to do so, chiefly supported by the fact that there are many points of resemblance in the two fungi, not only in the nuclear phenomena in the uninucleate con- dition, but also the morphological and biological aspects, as made clear in the present article. At any rate it is true that STEVENS’ paper has confirmed the presence of my karvodermatoplast in another species of Synchytrium, or at least it has shown that the aster is by no means of an accidental occurrence, or an artefact, like the similarly appearing structures in the xaryokinetic figures of the higher plants, on which conflicting opinions are still in vogue. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. bys Shortly after the appearance of STEVENS’ paper, a paper of Gricas entitled “On the Cytology of Synchytrium TIT. The Role of the Centrosomes in the Reconstruction of the Nucleus” (The Ohio Naturalist, VIIT, March. 1908, p. 277) was published. His study was undertaken with STEVENS’ material of S. decipiens, aleoholic specimen, paraffine cakes, slides, and notes of observations on the slides—perhaps the same slides from which STEVENS obtained the results just reviewed. It is, therefore, evident that the present study is an embodiment of the reinvestigation of the same subject and partly of the same preparations. as those of STEVENS. Grices’ present article has brought out many characteristic phenomena occurring in the secondary nuclei of S. decipiens more clearly and definitely. In particular. he has confirmed the absence of the nuclear membrane at the telophase, on account of which he has succeeded in demonstrating the connection between the centrosome- like body and the nuclear membrane in quite the same manner as IT have already described briefly in the preliminary note (707) and more fully discussed in the present paper. He has further described other nuclear phenomena, partly confirming and partly opposing STEVENS’ statement. As they are all intimately related to my own study on S. Puerariae I shall add here some remarks on his paper. . From both STEVENS and Griccs’ investigations there is no doubt that the centrosome-like body in S. decipiens presents great irregularities in struc- tnre. Yet judged from the evidence in 8. Pucrariac, T may ask whether the absence of the central granules or granule in the aster is a constant character or not. In S. Puerariae I am of the view that the granules in the focus of the aster is the centre of activity in relation to the formation of the nuclear membrane. The granules become invisible when the membrane-formation has much advanced, while they are constantly present in the earlier stages. Hence it seems probable that the presence or absence of the central granules in the aster is not constant throughout the whole period of its activity. According to Grices the daughter-nuclei at the telophase contain a certain number of separate chromosomes. This is not in agreement with my observations on S. Puerariae nor with the results of Stevens. In S. Puerariae the daughter-chromosomes fuse together already at the anaphase, and when the mass of the fused chromosomes reach the pole it represents 138 S. KUSANO: itself the nucleolus of the daughter-nucleus. The nuclei in StEveNs’ Fig. 2 (07) are represented each by a large globular mass surrounded by a clear space. Compared with the corresponding figures of mine his figures are, T be- lieve. quite correct as showing the telophase stage. Thus Strvrns and I agree in concluding that at an early stage of development the daughter-nucleus has a very simple structure, and about the time the nuclear membrane is com- pletely formed it becomes more complicated in structure, producing linin and chromatin-granules. According to Griccs’ figures we may think that the daughter-nucleus is more complicated in structure at an early stage than at a later. On these points STEVENS and Grices’ observations on the same materials have brought forth discordant results. 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Naturg., Bd. LIX. I, 1893, p- 25- A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 141 McGILL, C. (‘06): Nucleoli during the Oogenesis of the Dragon-fly. Zool. Jahrb., Abt. Anat. u. Ontog., Bd. XXIII, 1906, p. 207. MiTzkEwiTscH, L. (98): Ueber die Kernteilung bei Spirogyra. Flora, Bd. LXXXV, 1898, p. SI. Mott, W. (’93): Observations on Karyokinesis in Spevogyra. Verh. d. Akad, Van Weter- schappen te Amsterdam, Tweede Sectie, Deel I, No. 9, 1893. Moyntcomery, T. H. Jr. (99): Comparative Cytological Studies with special Reference to the Morphology of the Nucleolus. Journ. of Morph., Vol. XV, 1899, p. 267. NAWASCHIN, S. (99): Beobachtungen iiber den feineren Bau und Umwandlungen von Plasmodiophora Brassicae \Nor. im Laufe ihres intracellularen Lebens. Flora, Bd, LXXXVI, 1899, p. 404. Nemec, B. (99a): Ueber den Einfluss niedriger Yemperaturen auf meristematische Gewebe. Sitzber. d. kg]. béhm. Gesell, d. wiss., Math.—natur, CL., Bd. XII, 1899. , (’99b): Zur Physiologie der Kern- und Zellteilung. Bot. Centlbl., Bi. LXXVII, 1899, p. 241. , (o1): Ueber centrosomenahnliche Gebilde in vegetativen Zeilen der Gefiass. pflanzen. Ber. d. deutsch. bot. Gesellsch., Bd. XIX, 1901, p. 301. NicHots, M. L, (08): The Development of th: Pollen of Sarracenia. Bot. Gaz., Vol- XLV, 1908, p. 31. PoIRAULT, G. (05): Sur une Chytridinee parasite du JZuscari comosum. Bull. mensuel de Pzssoc. frangaise pour l’avanc. des Sei. Paris, 1995, p. 325. Rosen, F. (93): Beitrage zur Kenntniss der Pflanzenzellen II. Studien iiber die Kerne und die Membranbildung bei Myxomyceten und Pilzen. Cohn’s Beitrg. z. Biol., d. PA, Bdpavil5 1893; ps 237/- Ryrz, W. (07): Beitrige zur Kenntniss der Gattung Syxchytrium. Centlbl. f, Bakt., Bd. _ XVIII, 1907, p. 635,799. SCHNEIDER, C. (91): Untersuchungen iiber die Zelle. Arb. zool. Inst. Wien, Bd. IX, 1891. ScUROETER, J. (70): Die Pflanzenparasiten aus der Gattung Syxchytrium. Cohw's Beitrg. z. Biolaid) PA Bd. i; 1870; pate Srevens, F. L. and A. C. (’03): Mitosis of the Primary Nucleus in Synchytrium decipiens. Bot. Gaz., Vol. XXXV, 1903, p. 405. STRASBURGER, E. (co): Ueber Reduktionsteilung, Spindelbildung, Centrosomen und Cilienbil- "Sh dner im PHanzenreich. Jena, 1900. A ? , (07): Die Ontogenie der Zelle seit 1875. Progressus rei bot., Bd. I, 1907, p. 1. TIMBERLAKE, H. G, (01): Development and Structure of the Swarmspores of Mydrodic- tyor. Trans. Wisc. Acad., Vol. XIII, 1901, p. 486. Toumey, G, W. (oo): An Inquiry into the Cause and Nature of Crown-Gall. Publ. Univ. Arizona Agric. Exper. Station, Vol. XXXIII, 1900. Wacer, A. H. (’04): On Fertilization in the Saprolegnieae. Ann. of Bot., Vol. XVIII, 1904, p. 544. 142 Se icUSANO: WILLIAMS, L. (04a): Studies in the Dictyotaceae. 1. The Cytology of the Tetrasporangium and the Germinating Tetraspore. Ann, of Bot., Vol. XVIII, 1904, p. 141. , (o4b): ————. 2, The Cytology of the Gametophyte Generation. Idem, p- 183. Witson, E. B. (’00): The Cell in Development and Inheritance. 2nd. edition, 1900. London. WoLFE, J. ('04): Cytological Studies on Memalion. Ann. of Bot., Vol. XVIII, 1904, p. 607. ZIMMERMANN, A. (96): Die Morphologie und Physiologie des pflanzlichen Zellkernes. Jena, 1896. Explanation of Figures. All the figures are drawn with the aid of ABBe’s drawing apparatus using, except Vigs. 61- 66, the apochromatic 2mm. objective of Zeiss mostly with compensating ocular 12 (Figs. 1-20, 84-94) and 18 (Figs. 22-60, 67, 81). F, fixed in FLEMMING’s solution; K, fixed in KeIsEeR’s solution; T, FLEMMING’s triple stain; I1, IEIDENHEIN’s haematoxylin stain; J, Stained with fuchsin iodine- green. Synchytrium Puerariae. Plate VIII. Primary Nucleus. Fig. 1. Youngest fungus-body in a host-cell, partly hidden from view by the nucleus of the host. (F.T.) Fig. 2. A little later stage of the same. The nucleolus is homogeneous and only three chromatin-granules are visible. (I'.T.) Fig. 3. Advanced stage of the same. Chromatin-granules increase in .umber, achromatic substance bacomes visible. Three large globules studding the nucleolus. (F.T.) Figs. 4-12. One of several sections of nucleus. lig. 4. Nucleus somewhat advanced in growth, with many chromatin-granules and globules along the inner surface of the nuclear membrane and on the surface of the nucleolus. (F.T.) Fig. 5. Nucleus more advanced in growth, with two chromatin-globules and a vacuolate secondary nucleolus attached to the vacuolate primary nucleolus. A large amount of achromatic substance is represented as faint globules. (K.H.) Fig. 6, Nearly full grown nucleus, with a much more vacuolate primary nucleolus (dia- A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 143 grammaticaily shown) and a few secondary nucleoli, and producing a large amount of chro- matic and achromatic substance. (K.H.) Fig. 7. Full grown nucleus with numerous secondary nucleoli carrying chromatin-granules on their surface, Fine chromatin-granules stud the surface of the primary nucleolus whose ground substance has nearly lost affinity for stains. (.H.) Fig, 8. Surface view of the membrane of the nucleus at the sam2 stage. Fine granular substances arranged close together. Immediatzly bencath the membrane are represented nume- rous secondary nucleoli. (I°I1.) Fig. 9. A portion of a full grown nucleus with exceedingly large secondary nucleoli emit- ting large globules of chromatin. ‘The primary nucleolus is nearly empty of chromatic substance. (F.H.) Fig. to. First process of dissolution of secondary nucleoli and chromatin-granules. (F.H.) Fig. 11. Somewhat advanced, stage. (F.H.) Fig. 12. Nucleus approaching division. Most of the secondary nucleoli and chromatin- granules have been dissolved, (F.H.) Figs. 13-15. One of several sections of primary nucleoli at different stages. Fig. 13. Peripheral portion of a vacuolate nucleolus at an early stage. A large and numerous small vacuoles are visible among the deeply staining ground substance, and a large vacuolate secondary nucleolus is now passing out from the primary nucleolus, (KX.H.) Fig. 14. Highly vacuolate nucleolus at Jater stage, poor in chromatin. (I.H.) Fig. 15. Nucleolus at still later stage. Chromatin condenses on its peripheral portion, assuming a complete ring form in section. (F.I1.) Figs, 16-20, Prophase stage of the first mitotic division. Fig. 16. Primary nucleolus in dissolution, producing pseudopodia-like striations. (K.H.) Fig, 17. Striations completely replacing the nucleolus. (K. 1H.) Fig. 18. Dissolved form of nuclear elements represented as radial striations with a small quantity of chromatic and achromatic substances in the focus. (K.I1.) Fig. 19. Striations becoming fainter, and chromosomes and residue of the nucleolus be- coming differentiated. (IX.I1.) Fig. 20, Striations being transformed into granular mass and spindle completed. The spindle is heavily strained and chromosomes are indistinct. (k.T.) Fig. 21. Dissolution-product of nucleolar elements distinctly separated by large vacuoles from the surrounding cytoplasm. (IF.H.) x 920. Fig. 22. The end of prophase stage with chromosomes at equatorial plane and a residue of nucleolus. Striations are entirely transformed into granular mass, (K.I1.) Fig. 23. Early stage of metaphase with broad spindle. (K.T.) Fig. 24. The same stage with narrow spindle, (K.T.) Fig. 25. Somewhat later stage of the same, some of the chromosomes splitted and the dissolution-product of nuclear elements diminished in amount forming a halo around the spindle. (K.T.) 144 S. KUSANO: Plate IX. Secondary Nucleus. Fig. 26. Earliest stage of resting nuclei with very few chromatin. a, with two nucleoli 4, with prominent linin, (F.H.) Fig. 27. More advanced stage of the same with much increased chromatin. @, with prominent linin reticulum; 4, c, with two nucleoli; d, e, with secondary uncleoli, (F.H.) Fig. 28. First stage of dissolution of nucleoli. (F.H.) Fig. 29. Somewhat advanced stage. (F.H.) Fig. 30. Disappearance of nuclear membrane and emission of the nuclear elements into the surrounding cytoplasm in the same manner as shown in Figs. 16, 17. (F°.—.) Figs. 31-37. Other modes of development of nuclei. (K.H.) Fig, 31. Earliest stage of resting nuclei, with heavily stained globular mass budding out from the nucleolus. Fig. 32. Emission of chromatin-granules from nucleoli preparatory to division. a, with achromatic substance arranged diffusely; 4, with achromatic substance condensing in the central portion of the nuclear cavity; c, with achromatic substance about to form spindle, Fig. 33. Chromatin-granules condensed on the peripheral portion of nucleolus. Fig. 34. Direct transformation of nucleolus intc a number of chromatin-granules. a, 4, first step in which the outline of nucleolus is still visible; c, d, nucleolus becomes indistinct, Fig. 35. Chromatin-granules about to form chromosomes. Fig. 36. Advanced stage with definite number of chromosomes. Fig. 37. Abnormal nucleus approaching division, with exceedingly less chromatin, Fig. 38. Two spindles in the same fungus-body at second division. A few granules lying between them represent the residue of the nucleolus of the primary nucleus, (EE) Vig. 39. A pair of spindles at third division, showing the residue of nucleolus betweea them. In the spindle of the left side some of the chromosomes have been divided into daughter-chromosomes, while in that of the right side five mother chromosomes are distinctly shown in polar view. (F.H.) Fig. 40. Spindle with chromozomes at the equatorial plane. a, side view; 4, polar view; ¢, oblique view. (K.H.) Fig. 41. Early metaphase with elongated chromosomes, perhaps about to split. a, side view; 4, oblique view. (K.H.) Fig. 42. Formation of daughter-chromosomes, a, side view ; 4, polar view. (K.H.) Fig. 43. Metakinesis accomplished. a, side view; 4, oblique view. (IX.H.) Fig. 44. Chromosomes appearing thread— or rod—like, perhaps at the beginning ol metakinesis. @, side view; 4, polar view. (K.H.) Fig. 45. Heteromorphic chromosomes at metakinesis. (K.H.) Figs. 46-51. Anaphase stage. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 145 Fig. 46. Early anaphase with daughter-chromosomes going to fuse together. (K.H.) Fig. 47. A little later stage. (K.I1.) Fig. 48 Spindle constricted at the middle before the daughter-chromosomes reach the poles. (K.H.) Fig. 49. Elongated spindle with rounded poles. (K.H.) Fig. 50. Elongated spindle going to break at the median portion. (F.T.) Fig. 51. The same. (K.H.) Figs. 52-55. Telophase stage. Fig. 52. Early stage with large nucleolar residue and spindle-fibre about to be drawn in the daughter-chromosomes. (K.H.) Fig. 53. Development of hyaline space around daughter-chromosomes. (K.H.) Fig. 54. Chromosome-mass becoming globular, with remnant of spindle-fibres attached to it. (K.H.) Fig. 55. Growth cf chromosome-mass and hyaline space around it. The chromosome-mass has now become the nucleolus of the daughter-nucleus, and the residue of parent-nucleolus diminishes in size. (K.H.) Figs. 56-60. Formation of nuclear membrane. Fig. 56. Karyodermatoplast appearing near daughter-nucleolus. @, with numerous granules in the focus of rays, and nucleolus constricted and deformed; 4, with two granules in the focus, and a uniformly stained nucleolus; c, with numerous granules in the focus, and a nucleolus. (F.H.) Fig. 57. The same stage with a single granule in the focus. a@, with nucleolus having chromatic and achromatic substanses distinctly separated; 4, with chromatin-grobules condensed on the surface of the nucleolus. (F.H.) Fig. 58. Astral rays beginning to form the membrane around the hyaline space of daughter-nucleus. a, with nucleolus producing a single chromatin-granule; 4, with nucleolus before production of chromatin-granules. (F.T.) Fig. 59. A stage with membrane nearly complete. The rays become shorter and fainter, and the granules in the focus becomes indistinct. (¥.H.) Fig. 60. Daughter-nucleus completely reconstructed, with residue of karyodermatoplast near it as dense plasmic mass. (F.T.) Plate X. Sporangia, swarm-spores, etc. Figs. 61-66. Sporangium-formation. (F.H.) x 300. Fig. 61. Multinucleate fungus-body just before cleavage, with excretion-product between the cortex and protoplast. Fig. 62. Formation of radial cleavage by which the protoplast is divided into numerous pyramidal masses. 146 S. KUSANO: Fig. 63. Further stage of cleavage formation in which the protoplast is divided into irregular masses. Fig. 64. More advanced stage of the samie. Fig. 65. Formation of primordial sporangia by nearly simultaneous partition of whole protoplasmic mass. Fig. 66. Progressive formation of primordial sporangia. Fi Fig. 67. A part of the fungus body just before the appearance of cleavage. (F.T.) gs. 67-79. Development of sporangia and formation of swarm-spores, Fig. 68. The same at the beginning of cleavage-formation. The nuclei are at younger stage than before. (F.T.) Fig. 69. Primordial sporangium just furmed (compare Fig. 65). (F.H.) Fig. 70. Later stage of the same. (F.].) Fig. 71. ‘The same with nuclei at a more advanced stage. (K.J.) Figs. 7273. The same having much more chromatin-granules, (K.J.) Fig. 74. Nuclei at early prophase. (F.J.) Fig. 75. Nuciei in which the membrane has disappeared and nucleolus become smaller while chromatin-granules are more numerous. (F.].) Fig. 76. Nuclei at metaphases. In Some nuclei duaghter-chromosomes are not yet formed. (F.J.) Fig. 77. Multinucleate sporangium with sporangium-wall completed and mother-nuclei of swarm-spores. (K.J.) Fig. 78. Mature siorangium with nuclei of swarm-spores. (F.H.) Fig. 79. Formation of swarm-spores in the sporangium in water, (K.J.) Fig. 80. Adult swarm-spores liberated from sporangium. a, with oil drops blackened by osmic acid; 4, with the drops bleached. (F.T.) . Fig. 8. Swarm-spores at rest. (F.T.) Fig. 82. Aggregated nuclei at resting stage in a multinucleate fungus-body. (K.H.) X 920. Fig. 83. Anaphase of aggregated nuclei. (k.H.) x920. Fig. 84. Nuclei of symplast and surrourding host-cell. The fungus is yet at uninucleate stage. (F.T.) Fig. 85. Compressed nucleus of symplast at the stage the fungus is about to form cleavage. (F.T.) Fig. 86. Vacuolated nucleus of symplast approaching disorganization, (F.T.) Synchytlrium decipiens. Plate XI. Fig. 87. Young fungus-body with nucleolus emitting secondary nucleoli, (F.T.) Figs, 88-91. One of several sections of nuclei. A CONTRIBUTION TO THE CYTOLOGY OF SYNCHYTRIUM. 147 Fig. 88. A part of nucleus showing primary nucleolus much vacuolated and with numerous secondary nucleoli, (F.T.) Fig. 89, Full grown nucleus with numerous secondary nucleoli and chromatin-granules along the nuclear membrane. The primary nucleolus is shown diagrammatically. (F.T.) Fig. 90. Dissolution of secondary nucleoli leaving behind chromatin-granules freely. (E-T) Fiz. 91. Nucleus approaching to division. Secondary nucleoli and most of the chromatin- granules disintegrate leaving in this section only four chromatin-granules and a single secondary nucleolus still attached to the primary nucleolus. Somewhat fibrous achromatic substance is perhaps the dissolution-product of the secondary nucleoli. (F.T.) Fig. 92. A highly vacuolate primary nucleolus with a large vacuole near its peripheral layer. (F.T.) Fig. 93. Primary and irregular secondary nucleoli. Two large vacuolate secondary nucleoli have just passed out at the opposite side of the primary nucleolus leaving clefts behind. (F.T.) Fig. 94. Surface view of nuclear membrane. (F.T.) Fig. 95. Nucleus of the symplast and of the cell of the host surrounding the lysigenic hamber. The fungus is at this stage uninucleated. (F.T.) Fig. 96. Hypertrophied and much deformed nucleus of the symplast. (F.T.) Fig. 97. Section of a tubercle on the vein of a leaf, showing the fungus developing jast below the stomata. The intercellular space under the stomata is compressed. x400. Fig, 98. Another section of the same. X 400. eae. = Me le 7, A HERE INSERT ‘OLDOUT HERE S. KUSANO DEL. Bull. Agric. Coll. Vol. VIII. PEN ia 37. ‘ « 3 Plate XI. 5S. = A 1s SS 89. : is ms . 3 %e, oa eX 4 ry 6 « x, = } ‘ oY ie Oem go. ) Ff $ @ . 4) 3 ’ > e toe F: be £ 8 g i oat, e-@ “oF opt . Lo o*e ® be ? a ae to” | Gd sas ° “RS s HE lo ° | ; 2 yy ae: ery 7 > a ™ ys 5 \ e ¢ \ “e \ ; . \ 5. . \ : if a : ae. ws ae ® ! . i : ; ‘ 7 NS a3 aE ~~ ‘ : 7? 2. a e's of @.. e. *: 4 &- gh, “ we! aS Png S A of See f > eg, & See na e: os pine. - © Se Oy YC 25 ‘y= & ee ieee ie Se & , a ae beh » va YS met hj Print. by K. Ogawa. Description of a New Species of the Genus Latirostrum, with Remarks on the Generic Character and the Significance of its Long Palpi. BY T. Miyake. With one Figure in the Text. The genus Latirostrum, first erected hy Sir, G. F. Hampson and described in his ‘Fauna of British India, Moths, vol. III, p. 68 (1895), has a peculiar and interesting character in having very long labial palpi. The only species is HAampson’s genotype Latirostrum bisacutum of the Himalayas, des- eribed in the work just cited; and no species has, so far as I know, been added since. Recently Baron N. Taxacurno of the Imperial Agricultural Experiment Station at Nishigahara brought me a moth for identification. On a thorough examination it has turned out to be a new second species of the present genus, and the first Japanese species to be described. I give it the name Latirostrum japonicum (Japanese name: Tengu-atsuba). Latirostrum japonicum noy. spec. oo Ochraceous brown. Head traversed by a median black line extending along the dorsal edges of palpi to their extremity; antennae fuscous-brown ; tegulae edged with bluish-black; inner margins of patagia and tops of meso- and metathorax irrorated with bluish-black; legs and under-side of body pale ochraceous; abdomen except basal segment suffused with fuscous; extremity with tuft of fuscous black hairs. Fore-wing with a bluish-black patch at base; a dentate subbasal purplish- biack line angled outwards on subcostal and inwards on median nervure; a 150 DESCRIPTION OF A NEW SPECIES OF THE GENUS LATIROSTRUM. — ‘ 1 ee on 2 a ae = _ Pg al. Pa oe F 2 : Ze \ Nokes 4 Ste: i 4 Latirostrum japonicum n. St fl. like-wise coloured antemedial line angled inwards on subcostal and outwards on median nervure; both lines somewhat ill-defined towards costa; a small very prominent purplish-black spot in cell between the above-mentioned lines ; a very small rather indistinct spot at end of cell; a rather indistinct but posteriorly well-defined purplish-black postmedial line highly excurved beyond cell; a well-defined almost straight subterminal line edged internally with a narrow silvery streak, which is shaded internally and posteriorly with a fuscous-brown line; outer margin defined with purplish-black; inner margin suffused with black along vein 1. Hind-wing suffused with fuscous along outer margin, with the cilia yellow. Under-side paler; fore-wing with a curved brownish postmedial line, which is outwardly irrorated with brown: a small discoidal spot; hind-wing with a brownish rather straight postmedial line, externally with slight brown suffusion; an indistinct subterminal series of brownish spot; a brown spot at the end of cell. Expanse of wing 42 mm; length of body 19 mm; length of palpi 9 mm. A single male specimen captured by Baron TaxacutHo, on Hikosan, Kiushiu, July, 1906. This species is quite different from the LZ. bisacutum of Hampson and nothing need be said on this point. But the two species are to a certain extent allied, as the two spots in and at the end of cell, the antemedial dentate line and the post-medial and subterminal lines are situated topographically alike in both. DESCRIPTION OF A NEW SPECIES OF THE GENUS LATIROSTRUM. 151i I have slightly modified the generic characters as given by Hampson; namely vein 6 of the fore-wing is not obsolete but distinct like the other veins, while Hampson says vein 5 is “almost obsolete.” In his figure of the vena- tion, vein 8 terminates at the margin before the apex, i.e. on the outer margin, whereas in the present species, it terminates beyond the apex, i.e. on the costal margin (see the figure). Baron TAKACHITHO captured the moth in a forest on Mt. Hikosan, one of the highest mountains in Kiushiu. He says that the moth was resting on the leaf of a certain tree, with its long palpi extended forwards so as to imitate a spine in a very perfect mauner, and he supposes that when it settles on a branch of a tree it may pass unobserved even by keen eyes, showing us the significance of the long palpi of this species. November, 1908. A Revision of the Arctianae of Japan. BY T. Miyake, With six Figures in the Text. The ArcTrAN&:, one of the subfamilies of AncTrap2, include some insects injurious both to our farm crops and forests, especially to the mulberry-tree. Indeed the latter harbours seven species of the present subfamilys these and other injurious species are as follows :— NAME OF SPECIES. NAME OF VEGETABLE AFFECTED. Diacrisia lubricipeda. Malberry-, cherry-tree, &c. Diacrisia bifasciata. Mulberry-tree. Diacrisia obliqua. Mulberry-tree. Diacrisia subcarnea. Mulberry-tree. Diacrisia flammeola. Persimmon-tree. Diacrisia imparilis. Malberry-, peach-, pear-, plum-, cherry-, apple- tree, and many others. Diacrisia infernalis. Mulberry-, peach-, pear-, plum-, cherry-, apple- tree; Quercus serrata, Q. glandulifera; «ce. Amsacta lactinea. Maize, soja bean, &e. Arctia caja. Hemp, rape, mulberry-tree ; Ribes grossulariot- des; and many others. Camptoloma interioratum. Quercus serrata; Q. glandulifera. Japanese Arctianae have been studied, like other groups of Lepidoptera, 7 Z =. ss 2 ea: ee by several foreign entomologists such as BUTLER," LEECH,” PRYER,*? HAMPSON, 1. Ann. Mag. Nat. Hist., (4) XX (1879); Cist. Ent., ili (1885); Trans. Ent. Soc. Lond., 1881; Ill. Typ. Lep. Het., ii, iii (1878-9). 2. Proc. Zool. Soc. Lond., 1888; Trans, Ent. Soc. Lond., 1899. 3. ‘Trans. Asiat. Soc. Jap., vol. XX (1885). 4. Cat. Lep, Phal., iii, (1901). 154 T. MIYAKE: and many others, and I myself have also published a systematic review of the group (Téga-akwa ni kwansuru kenkyu hikoku) in the ‘“Extra-reports from the Imperial Central Agr‘cultural Experiment Station No. 22 (1906), and there is but little room for anything in the way of its systematic. However since then a few species have been discovered, some of which I consider to be rew to science and some to be new to Japan. I therefore propose to describe them in this paper and I may also improve this opportunity by adding a few remarks on some other species and describing the larvae of some. Of this subfamily I recognize 32 species, of which only 8 species are peculiar to Japan. Of the 8 species 5 are found in Japan proper, and the other three are limited to Formosa. The other species are either palaearctic or oriental or both palaearctic and oriental. They are divided as follows :— Palaecaretic.:;- <2: geese tl ee Oriental 22) co teIs- ween tues 7 (24 Common to both regions... ..- ... .. 93 10 of our species are common with Corea, 17 with China, 10 with Amur- land, 1 with Siberia, 9 with Europe, 9 with India, and 4 wth the Malay Peninsula and the adjacent islands. The distribution among the main Japanese islands and the relationship tu other regions may be summarised as follows :— Palaearchic 2.) .--- (es Species occuring in North Japan (Hokkaido)} Oriental ... ... ... 1711 Common to both regions 3 Palaearctic... ... 14 Species occuring in Central Japan (Honto)...)Oriental ... ... ... 2 Common to both regions 3 Common to both regions 1 Palaesretiessc, <2: “sees Species occuring in Formosa (ine. Riukiu)...{Oriental ... 2... 5 Palacarctie!...0 ’s-2ie ee Species occuring in South Japan (Kiushu)...?Oriental ... ... ... 2710 Common to both regions 2 A REVISION OF THE ARCTIANAE OF JAPAN. 155 In order to bring out a few more details, I have drawn up the following table showing the distribution of the species in the principal parts of our Empire, Hokkaido, Honto, Kiushiu and Formosa, as well as in other countries or regions which are more or less related faunistically to ours. In preparing this table I have relied on my own knowledge so far as Japan was concerned, but for foreign countries I have mostly followed the statements of such authori- ties as Hampson, BUTLER, LerEcH, Kirpy, Etwes, STAvpINGER &c. Within the limits of Japan my experience enables me to tell the occurrence of the species with great accuracy, although it is not uncommon that a species known to occur in a limited district may be found in a quite detached place. The classification adopted in this work is most commonly followed to one of Hawpson, as set forth in his “Catalogue of Lepidoptera Phalaenae vol. PEE (1901) -” For convenience, I have included the genus Camptoloma, mentioned in his “Fauna of British India, Moths, vol. II (1894)” but omitted in his catalogue, just referred, as a genus probably to be referred to another family. 156 TMIYAKE: JAPAN Z a> a = | & ) ces < Aa See > | 5 |o/f@ 32/845 Sie | = | O ) Olea) a ie 1. LPhragmatobia fuliginosa. * eZ % * 2. Diacrisia nivea. * c3 * * * * 3h pst punctaria. * 3 - * zs Az DD): lubricipeda. * ‘8 = “3 * x * Fa, BE Lewitst. * * * tee Jap bifasciata. x * 722: tnaegualts. * eS S22: sertatopunctata. * ae a: * oF Hh Vee obligua. * * me * * * 10; . 2: metalkana. * * * x = 1650 2: sannio. * * * * yi. JOR flammeola, * * | 7 18:2 22 Moltrechti. es 192 2D: miparilts. - * x 20.” PD. anfernalis. * * Dis. DP caesavea, * * * x x 22. Amsacta dactinea. * * = 23. Creatonotus transiens. * 24. Creatonotus gangis. - * 25. Creatonotus Koni. * 26. Pericallia picta. Riu- * Altai] * kiu 27. Parasemia plantaginis. % ig 28. 6S oF sig ‘black subterminal spots below costa. Hind-wing scarlet, an antemedial black spot below median nervure, and a small spot on vein 1. Underside of wings scarlet; fore-wing with a black spot near the base and in the middle of cell; a spot below origin of vein 2 and discoidal Junule; hind-wing immaculate. 164 7 MEYAKE: 2. More fulvous yellow; underside of wings with more pronounced’ black spots; fore-wing with subterminal black spots; hind-wing spotted as above, only the conjoined subterminal spots absent. Expanse 37-39 mm. Two males captured by the author at Mionoseki, Shimane-ken, on 28th August 1906, and a female specimen in the collection of the Agricultural College, obtained at Zeze, Shiga-ken. This svecies is allied to D. nebulosa and D. amurensis. But it differs from the former by the absence of the black suffusion of the inner half of the fore-wing, and from the latter by the coloration of the fore-wing and abdomen and the biserrate structure of the antennae of the male. Besides,. this species is smaller than either of the two species. 14. Diacrisia nebulosa Buti. (Benishita-hitori). Rhyparioides nebulosa Butl., Ann. Mag. Nat. Hist., (4) XX, p. 396 (1877) ; Tl. Typ. Lep-eitet, II, p. 5, pl. XXL fig: 2 (1aiaye Leech, Trans. Ent. Soc. Lond., 1899, p. 156. Rhyparioides simplicior Butl., Trans. Ent. Soc. Lond., 1881, p. 6. Rhyparioides rubescens (part) Leech, Proc. Zool. Soc. Lond., 1888, p.. 616. Diacrisia nebulosa Hampson, Cat. Lep. Phal., III, p. 316 (1901). Rather uncommon in Honto but common in Hokkaido; I have not yet captured the species in Tokyo, where FENTON is said to have obtained some examples. The dark suffusion of the fore-wing is very variable, in some very obscure as in D. amurensis, and In some very strong so that the fore-wing is almost entirely dark-coloured. Abdomen ‘is without exception coloured! with crimson. 15. Diacrisia metalkana Led. (No-benishita-hitori). Nemeophila metalkana Led., “Wien. Mon., V, p. 162, pl. III, fig. 12 (1861)”; Leech, Proc. Zool. Soc. Lond., 1888, p. 616. Chelonia flavida Brem., Lep. Ost-Sib., p. 39, pl. IV, fig. 4 (1864). Rhyparioides metalkana Leech, Trans. Ent. Soc. Lond., 1899, p. 155. Diacrisia metalkana Hampson, Cat. Lep. Phal., IIT, p. 299 (1901). A REVISION OF THE ARCTIANAE OF JAPAN, 165 Parasemia metalkana Wirby, Butt. Moths Europ., p. 109, pl. 23, fig. 16 (1903). Very rare. I have only one male specimen obtained by Mr, Ocuma in Tokyo. The specimen seem to have no remarkable difference from the Euro- pean form. 16. Diacrisia sannio I.. (Mon-heriaka-hitori). . ? Bombyx sannio L., “Syst. Nat., I, p. 506 (1758)/ Bombyz russula l., “Syst. Nat., I, p. 510 (1758).” Diacrisia irene Butl., Trans. Ent. Soc. Lond., 1881, p. 6; Leech, Trans. Ent. Soe. Lond., 1899, p. 157. Diacrisia russula Leech, Proc. Zool. Soc. Lond., 1888, p. 615; Trans. Ent. Soc. Lond., 1899, p. 156. Diacrisia sannio Hampson, Cat. Lep. Phal., III, p. 209 (1901). Parasemia sannio Wirby, Butt. Moth. Europ., p. 109, pl. 23, fig. 15ab (1903). Uncommon. I have two females from Suwa, Shinano and a male from Hiroshima. 17. Diacrisia fiammeola Moore. (.ika-hitori). Alpenus flammeolus Moore, Ann. Mag. Nat. Hist., (4) XX, p. 89 (1877) ; Leech, Proc. Zool. Soc. Lond., 1888, p. 617. -Spilosoma flammeolus Leech, Trans. Ent. Soc. Lond., 1899, p. 154. Diacrisia flammeola Hampson, Cat. Lep. Phal., II, p. 301, pl. XLV, fig. 10 (1901). T have a male specimen, prebably captured in Honto, from Mr. Nawa, nd three males from Mr. YANO obtained in Kiushiu. One of the specimens Fig. 3. Diacrisia flammeola Moore. J. +-.* before me has, as is figured, a series of postmedial spots not mentioned by Moore or Hampson. Mr. YANO says that the larvae feed on persimmon-tree and therefore they are more or less injurious to the plant. 166 T MIYAKE: 18. Diacrisia Moltrechti noy. sp. (Chairo-hitori). Brownish ochraceous; head and thorax ochraceous; palpi brownish black;. antennae bipectinate, black; legs brownish, with the femora ochraceous above ;. Fig. 4. Diacrisia Moltrechti n. sp. <7. -|-- abdomen bright ochraceous yellow, the basal joints of which are paler on the ventral side, with dorsal, lateral and sublateral series of black spots. Fore-wing brownish ochraceous suffused with black; a medial series of six black points from costa to inner margin, slightly angled outwards at the median nervure. A postmedial series of five spots, of which two are ex- curved from below costa to vein 3, situated in the end of discoidal cell, then incurved to meet the above-mentioned medial series above vein 1; of the five spots, one, situated between vein 2 and 3, is oblique and somewhat elongated ; another small spot between vein 5 and 6 just beyond the discoidal cell. Hind-wing ochraceous orange, with two discoidal spots and one spot just beyond the discocellulars ; an indistinct patch at vein 1 near the base. Under surface ochraceous without blackish suffusion, similarly marked as above. Expanse 32 mm. ‘ A male specimen in the collection of Mr. Kon of Vladivostok, captured by Dr. Motrrecut on Mt. Arisan in Formosa, 1908. This species is to a certain extent allied to Diacrisia flammeola Moore, D. biseriata Moore, D. flavens Moore and D. eugraphica Walk., but can readily b2 distinguished on many points and is doubtless a distinct species. 19. Diacrisia imparilis Butl. (Kwwa-gomadara-hitori). Spiiarctia imparilis Butl., Ann. Mag. Nat. Hist., (4) XX, p. 394 (1877) ; Ill. Typ. Lep. Het., II, p. 4, pl. XXII, fig. 4 (1878); Ann. Mag: Nat. Hist., p: (5) IV, p. 351 (1879) ; Leech, Proc. Zool. Sec. Lond., 1888, p. 620. Spilosoma imparilis Leech, Trans. Ent. Soc. Lond., 1899, p. 153. Diacrisia imparilis Hampson, Cat. Lep. Phal., III, p. 308 (1901). A REVISION OF THE ARCTIANAE OF JAPAN. 167 Yery common in Honto; I have also received a series of specimens from Hokkaido. The larvae are very common on various plants in Tokyo. Larva. Purplish fuscous, with hairs of greyish white and greyish black; head and legs greyish fuscous; a dorsal and subdorsal series of greyish yellow spots; tubercles mostly ochraceous brown, some of 6-12 somites metallic blue; prothoracic shield metallic blue. Food-plants: mulberry-, peach-, pear-, plum-, cherry-, apple-tree and many others. 20. Diacrisia infernalis Butl. (Kurohane-hitori). Thanatarctia infernalis Butl., Ann. Mag. Nat. Hist., (4) XX, p. 395 (1877); UN Type tepaeeter., LIT, p. 7, pl. XLII, fig, 9 (1879); Leech, Proce. Zool. Soc. Lond., 1888, p. 617; Trans. Ent. Soc. Lond., 1899, pa L60: Diacrisia infernalis Hampson, Cat. Lep. Phal., IIT, p. 512 (1901). Not very rare in Hokkaido and Honto; I have received some specimens captured in Tokyo. Larva.. Purplish fuscous with mixed hairs of whitish and blackish; head ochraceous brown; legs brownish; a yellowish dorsal line with some indistinct irregular lateral lines; tubercles of dosal half metallic blue; lateral ones ochraceous brown. Food-plants: mulberry-, peach-, pear-, cherry-, apple-tree ; Quercus serrata; Q. glandulifera; &e. 21. Diacrisia caesarea Geoze. (Kibara-hitori). Bombyx caesarea Geoze, “Ent. Beytr., III, (8) p. 63 (ATS )e” Bombyx luctifera Esq., “Schmett., ITT, p. 222, pl. XLUI, figs. 1-5 (1784).” Atolmis japonica Walk., “Cat Lep. Het. Suppl., I, p. 223 (1864).” Spilosoma luctifera Leech, Proc. Zool. Soc. Lond., 1888, p. 618. Estigmene moerens Butl., “Cist. Eut., III, p. 114 (1885) .” Arctinia caesaria Kirby, “Cat. Lep. Het., I, p. 276 (1892)”; Leech. Trans. Ent. Soc. Lond., 1899, p. 160. Diacrisia caesarea Hampson, Cat. Lep. Phal., III, p. 313 (1901). Phragmatobia cacsarea Kirby, Butt. Moth. Europ., p. 113, pl. 29, fig. 7 (1903). 158 T. MIYAKE: Very rare. I obtained a male specimen at Sasago (Yamanashi-ken) by the Jamp in May 1906. Genus Amsacta Walk. Hampson, Cat. Lep. Phal., IH, p. 322 (1901). 22. Amsacta lactinea Cram. (Kyd-jord). Bombyx (Aloa, Phalaena) lactinea Cram., “Pap. Exat., II, p. 58, pl. CXXXITI, fig. 0 (1777)2 Bonbyx sanguinolenta Fabr., “Ent. Syst., II, p. 473 (1793).” Aloa lactinea Walk., “Cat. Lep. Het., II], p. 702 (1855)”; Leech, Proce. Zool. Soc. Lond., 1888, p. 620. Rhedogastria lactinea Leech, Trans. Ent. Soc. Lond., 1889, p. 124. Creatonotus lactineus Hampson, Fauna Brit. Ind., Moths, II, p. 27 (1894): Leech, Trans. Ent. Soc. Lond., 1899, p. 163. Amscta lactinea Hampson, Cat. Lep. Phal., III, .p 328 (1901). Commen in Honto. Larva. Head brownish black; tubercles on subdorsal, supra- and sub- spilacular lines, with tufts of longer or shorter hairs of black and reddish brown; skin and legs brownish black. Food-plants: maize, soja bean, &c.—Prof. Sasaki. Genus Creatonotus Hiibn. Hampson, Cat. Lep. Phal., II, p. 331 (1901). 23. Creatonotus transiens Walk. (Hai-iro-hitori). Spilosoma transiens Walk., “Cat. Lep. Het., III, p. 675 (1855).” Amphissa vacillans Walk., “Cat. Lep. Het., ITI, p. 675 (1855).” Aloa isabellina Walk., “Cat. Lep. Het., III, p. 705 (1855).” Phissama vacillians Butl., Il. Typ. Lep. Het., III, p. 5, pl. XCII, fig. 4 (1879) ; Hampson, Fauna Brit. Ind., Moths, II, p. 29 (1894) ; Leech, Trans. Ent. Soc. Lond., 1899, p. 164. Creatonotus transiens Hampson, Cat. Lep. Phal., III, p. 334 (1901). There is a series of specimens in the collection of the Science College from Riukiu. A REVISION OF THE ARCTIANAE OF JAPAN. 169 24. Creatonotus gangis Linn. (Murosuji-hitori). Phalaena gangis Linn., “Amoen. Acad., VI, p. 410 (1764).” Phalaena (Noctua) interrupta Linn., Syst. Nat. I, (2), p. 840 (1767). Bombyx francisca Fabr., “Mant. Ins., IT, p. 151 (1787).” Cretatonotus continuatus Moore. Ann. Mag, Nat. Hist., (4) XX, p. 344 Sathya: Creatonotus interruplus Hampson, Fauna Brit. Ind., Moths, IJ, p. 26 (1894) ; Leech, Trans. Ent. Soc. Lond., 1899, p. 163. Creatonotus gangis Hampson, Cat. Lep. Phal., III, p. 333 (1901). A male specimen in the collection of Mr. KrNosurra from Formosa. 25. Creatonotus Koni nov. sp. (Arisan-hitori). Head and thorax ochraceous white, very slightly tinged with pink; palpi black; legs pale brown, the femora orange above, white below; abdomen orange above, the extremity of anal tuft and ventral surface white; dorsal, lateral and sublateral series of small black spots. Fore-wing ochraceous white with : =. =< -. > mul 2 _ WAS * a ee eee . > a 1 i rs — "i Fig. 5. Creatonotus Koni n. sp. 9. +-. s . ~ Lie ~ + ®, 8 Cp a® -$ oy slight pinkinsh tinge; small black points in and beyond upper and lower angles of discoidal cell; a subterminal series of five small black spots from above vein 5 to vein 1. Hind-wing slightly tinged with ochraceous brown, with three rather elongate black subterminal spots. Expanse 58 mm. A single female specimen in the collection of Mr. Kon captured on Mt. Arisan in Formosa by Dr. Morrrecn. Mr. Kon was very kind to show and lend me the specimen for study and I have a great pleasure in naming the specific name after him. Genus Pericallia Hiibn. Hampson, Cat. Lep. Phal., III, p. 350 (1901). 170 T. MIYAKE: 26. Pericallia picta Walk. (Akasuji-hitori). Deiopeia picta Walk., “Cat. Lep. Het., XXXI, 263 (1864).” Tatargina formosa Butl., Trans. Ent. Soc. Lond., 187%, p. 366; Ill. Typ. Lep. Het., III, p. 8, pl. MGMT, fig. 1 (1879). Tatargina picta Hampson, Fauna Brit. Ind., Moths, II, p. 54 (1894). Pericallia picta Hampson, Cat. Lep. Phal., IIT, p. 353 (1901). Some specimens in the collection of the Sapporo Agricultural College from Riukiu. Genus Parasemia Hiibn. Hampson, Cat. Lep. Phal., IIT, p. 458 (1901) . 27. Parasemia plantaginis L. (//ime-kishita-hitori). Bombyz plantagins L., “Syst. Nat., I, p. 501 (1758).” Bombyz alpicola Scop., “Ent. Carn., p. 205 (1763).” Bombyx hospita Den. and Schiff., “Wien. Verz., p. 310 (1776).” Bombyx matronalis Freyer, “Neu. Beitr.. V, p. 37, pl. 405 (1843).” Chelonia caucasica Herr.-Schaff. “Schmett. Eur., IJ, p. 147, figs. 42-44 (1845).” Nemeophila petrosa Walk., “Cat. Lep. Het., ITI, 626 (1855).” Lithosia nicticans Mén., Schrenck’s Reise. Amur., Lep., p. 50, pl. 4, fig. 4 (1859). Platarctia modesta Pack., “Proc. Ent. Soc. Philad., III, p. 113 (1864).” Platarctia scudderti Pack., “Proe. Ent. Soc. Philad., III, p. 113 (1864).” Eupsychoma geometrica Grote, “Proc. Ent. Soc. Philad., IV, p. 318, pl. 2, fir. 1 (1865).” Nemeophila caespitis Grote and Rob., “Trans. Am. Ent. Soc., I, p. 337, pl. 6, fig..43 (1868) .” Nemeophila cicherii Grote and Rob., Trans. Am. Ent. Soc., I, p. 338, pl. 6, fig. 44 (1868) .” Nemeophila macromera Butl., Trans. Ent. Soc. Lond., 1881, p. 5. Nemeophila macromera vay. leucomera Butl., Trans. Ent. Soc., 1881, p. 5. Nemerophila macromera var. melanomera Butl., Trans. Ent. Soc. Lond., 1881,-p. dS. A REVISION OF THE ARCTIANAE OF JAPAN, ~ 17} Nemeophila geddesi Neum., “Papilio, IIT, p. 137 (1884).” Nemeophila selwynii H. Edw., “Can. Ent., XVII, p. 65 (1885).” Nemeophila plantaginis Leech, Trans. Ent. Soc. Lond., 1899, p. 157. Parasemia plantaginis Hampson, Cat. Lep. Phal., III, p. 458 (1901). The Japanese forms included by Butter under the names of Nemeophila macromera, N. leucomera and N. melanomera are undoubtedly to be considered as varieties of P. plantaginis. The characteristics mentioned by BuTLER, “the white spot in the discodal cell apparantly never touching the costal margin, the subapical sigmoidal stripe not united to the A-shaped marking,” are not constant in our species. As may be seen from the figures, in the form re- presented by fig. b, the above mentioned points are exactly as in the European form, whereas in the form represented by fig. a, the white spot in the discoidal cell touches the costa! margin and in the form or fig. ¢ the 4-shaped marking is united to the subapical sigmoidal stripe. The figure a belongs to the form leucomera and c¢ to macromera, and I have not yet received any example of ielanomera. Fig. b, which is, as Fig. 6. Parasemia plantaginis. +. a. Form leucomera. f. b. Enropean from. ©. c. Form macoromera of. stated above, exactly similar to the European form, is a female in which the hind-wing is orange coloured. Two males and a female were kindly sent to me by Mr. M. Suzvxtr of Kyoto, which were captured on Mt. Asama in the summer of 1907. Genus Arctia Schrank. Hampson, Cat. Lep. Phal., III, p. 463 (1901). 28. Arctia caja L. (Hitori-ga). Bombyx caja L., “Syst. Nat., I, p. 500 (1758).” Phalaena erinacea Retz., “Gen. Spec. Ins., p. 36 (1783).” Euprepiu phaeosoma Butl., Ann. Mag. Nat. Hist., (4) XX, p. 395 (1877) ; 172 T. MIYAKE: ll. Typ. Lep: Het., [Eye pl. XLIL fig. 10 (1879). Luprepia phacosoma var. auripennis Butl., Trans. Ent. Soc. Lond., 1881, Dane Hypercompa phaeosoma Wirby, Trans. Ent. Soc. Lond., 1881, p. 259. Buprepia caja Leech, Proe. Zool, Soc. Lond., 1888, p. 617. Arctia orientalis Moore, Ann. Mag. Nat. Hist., (5) I, p. 230 (1878) ; Hampson, Fauna Brit. Ind., Moths, I, p. 16 (1892). Arctia caia Hampson, Cat. Lep. Phal., IJ, p. 463 (1901) ; Leech, Trans. Ent. Soc. Lond., 1899, pe 159. Rather rare in Honto but very abundant in Hokkaido. The whitish markings of fore-wing are extremely variable. The ground colour and black spots of the hind-wing also variable, the former being usually scarlet, but often orange and rarely yellow. The moths often fly about in the day-time. Larva. Head black with reddish-brown spot at sides; body black; each body-segment with two deep-black tubercles on’ subdorsal line, one on supra-, subspiracular and basal lines; tubereles on subdorsal and subspiracular lines thickly covered with longer or shorter light greyish yellow hairs; tubercles on subspiracular and basal lines with short reddish brown hairs; thoracic legs biack; abdominal legs dark brown. Food-plants: hemp, rape, mulberry-tree. Ribes grossulartoides—Prof. Sasakt. Genus Utethesia Hiibn. Hampson, Cat. Lep. Phal., IIT, p. 480. 29. Utethesia pulchella L. (Peni-gomadara-ltori). Linea pulchella L., “Syst. Nats I, 2, p. 884 (1767).” Noctua pulchra Den. and Schiff., “Wien. Verz., p. 68 (1776).” Geometra lotriz Cram., “Pap. Exot., II, pl. 109, E, F (1779).” Utethesia pulchella Kirby, “Cat. Lep. Het., I, p. 346 (1892)”; Hampson, Cat. Lep. Phal., IIT, p. 483 (1901). Leiopeia pulchella Hampson, Fauna Brit. Ind., Moths, II, p. 55 (1894) ; Leech, Trans: Ent. Soc. Lond., 1899, p. 170. Described from Hokkaido, Kiushiu and Riukiu. JI have received some spec.mens from Formosa. There is also a series of specimens in the collection of the Agricultural College. A REVISION OF THE ARCTIANAE OF JAPAN. - Wes Yenus Rhodogastria Hiibn. Haareson, Cat. Lep. Phal., Til, p. 498 (1901). 90, Rhodogastria astreas Drury. (Tsumaguro-sukashi-h itori). Glaucopis (Sphinaj astreas Drury, “Ins. IT. pl. XXVII, fig. 4 (1773).” Sphina melanthus Cram., Pap. Exot., II, pl. 286, B (1780). Rhodogastria astraca Moore, “Tep. Ceyl., I, p. 76, pl. CVIII, figs. 1, 1a (1882).” Noctua eugenia Fabr., “Syst. Bnt., 3, UL, ps 19. G72) Chelonia madagascariensis Boisd., “Delegorgue, Voy. Afr. Autr., iD eas 598 (1847).” Amerila rhodopa Walk., “Cat. Lep. Het., XXXI, p. 305 (1864).” Creatsnotus communis Walk., “Cat. Lep. Het., XXX, p. 283 (1864).” Amerila vitrea Plotz, “Stett. Ent. Feit. MEN, p. 62 (lean). Rhodogastria astraca Moore, “ep. Ceyl., H, p. "6, pl. CVIII, figs. L 1a (1882).” Amerila bauri Moschl., “Verh. Zool.-bot. Ges. Wien, XXXIII, p. 289, pl. 16, fig. 2 (1884) .” Pelochyta astrea Hampson, Fauna Brit. Ind., Moths, II, p. 38 (1894) 5 Leech, Trans. Ent. Soc. Lond., 1899, p. 167. Rhodogastria astreas Hampson, Cat. Lep. Phal., III, p. 505 (1901). I have not seen any specimen of this species nor have I been informed >t its occurrence by any of our correspondents. LEECH and Hampson have described the species from Formosa. Genus Camptoloma Feld. Hanpson, Fauna Brit. Ind., Moths, II, p. 31 (1894). 24, Camptoloma interioratum Walk. (Sarasa-litorv). Numenes interiorata Walk., “Cat. Lep. Het., Suppl., I, p. 290 (1864); Leech,.Proe. Zool. Soe. Lond., 1888, p. 61. Camptoloma interioratum Kirby, “Cat. Lep. Het., I, p. 359 (1892)”; Hampson, Fauna Brit. Ind., Moths, 1, p. 31 (1894) ; Leech, Trans. Ent. Soc. Lond., 1899, p. 1654. Very common in Honto. The moth emits a peculiar sound by its body segments. i174 T. MIYAKE: Larva. Body greyish yellow except head, the dosal shield of first body segment, legs and last segment, which are blackish in colour; ventral side of body orange; six brownish black longitudinal streaks between dorsal and ventral lines; six brownish spots with white hairs, from dorsal to basal line in each segment. Food-plants: Quercus serrata, (). glandulifera—Prof. Sasaki. Genus Nicaea Moore. Lep. Atk., p. 11 (1879) ; Hampson, Cat. Lep. Phal., III, p. 218 (1901). 32. Nicaea (?) formosana Miyake. (Kiboshi-hitori). Nicaea (?) formosana Miyake, Ann. Zool. Jap., vol. VI, part 2, p. 8 (1907). A female specimen captured at Jukirin, Formosa, in the collection of dhe Science College. Jan. 1909. eS eee eS re = 2 “ y ty y S Tokyo Daigaku. Ndgakubu 19 Bulletin Biological & Medical Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY 331 ef se * Se” x a ates Sof, i