\\ AS . WS . — \\ ‘ WV C POG Ae vue i, a fi ey ae Aven ed — —— 5 ! i 5 eioz snouosied ay} 2] ©} payedIp » { [ duly joy st »C SB UMOUY AjuoOUOD yeu soIWIeU ke Noul2s09 +«snjelop “eWeug jeio> 10 uinb yizpy se uMmouy AjuouIUIOD ayeU nouosiod e “SMIAINY sdejq SI JJo] 0} payalIp peosy ay} YM ‘oyeus aypplur 9y fF ayeug jyeio>y) ueiouog se uMOoUy ATUOULIOS yeu snouosiod e “SnyyUeXxAING sdelq SI ys 0} pe Ip pesy 24} YUM ‘ayeus yaddn qu Pai Fw ft yo gi zr SNAKE VENOMS, AN INVESTIGATION OF VENOMOUS SNAKES WITH SPECIAL REFERENCE TO THE PHENOMENA OF THEIR VENOMS BY HIDEYO NOGUCHI, M.D., M.Sc. »? 8 Phi ow INS UTUTION "rarer WASHINGTON, D. C. PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON 1909 CARNEGIE INSTITUTION OF WASHINGTON PuBiicaTion No. 111 : is ix . Fibe| ris hAby wis > ras ta aT? eM kee) aay tr, A! bie PREPACE.. My interest in an opportunity to study the subject of snake venom I owe to certain peculiar and fortunate circumstances. After my graduation in medicine, I was for several years connected with the Institute for Infectious Diseases, in Tokio, where I came under the instruction of Professor Kitasato. In the autumn of the year 1900 I became assistant in pathology at the Uni- versity of Pennsylvania, where I remained until Professor Flexner resigned his post to assume the directorship of the laboratories of the Rockefeller Institute. It was soon after my arrival in Philadelphia that Dr. S. Weir Mitchell expressed his great desire that the scientific study of snake venom should be resumed and prosecuted along the lines of the new biological conceptions of toxication and immunity, which had become at that time so promising a field of pathological investigation. I had, therefore, the good fortune thus early to become associated in carrying out the studies (which extended over several years), relating to snake venom, which were issued from the pathological laboratory of the University of Pennsylvania. The expenditure involved in the execution of the researches of snake venom was met first by Dr. Mitchell himself, and later, chiefly through his recommendations, by means granted from the Bache Fund of the National Academy of Sciences and by specific grants from the Carnegie Institution of Washington. During the several years of my connection with the University of Penn- sylvania, I was the recipient of many courtesies from the other members of the staff of the Pathological Department, and from Provost Harrison, Dr. John Marshall, the professor of chemistry, and many others, to whom I wish to express my appreciation. In the interval between my leaving the Uni- versity of Pennsylvania and resuming my connection with Professor Flexner at the Rockefeller Institute, I spent a year of study on snake venom at the Statens Serum Institute, in Copenhagen. The expenses incurred were de- frayed by the Carnegie Institution of Washington. For the opportunity to continue my work in the Serum Institute I am indebted to Dr. Madsen, who has also placed me under many obligations by his constant aid and kindness. I am also indebted for many courtesies to Professor Salomonsen and to Dr. Walbum of the Institute. The present monograph on snake venom was projected a number of years ago, and at first it was intended that it should be devoted to a collection of the studies on venom in which I was more or less directly concerned. The ll iv VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Carnegie Institution generously undertook to defray the expenses connected with the preparation and the publication of the monograph. However, as the work of preparation progressed, it seemed more desirable to present a fuller and more balanced exposition of the subject of venom than was at first proposed. No single work in the English language exists at this time which treats of the facts of zoological, anatomical, physiological, and pathological features of venomous snakes, with particular reference to the properties of their venoms. During the interval of the preparation of this monograph there appeared in French the excellent work on snake venom by Professor Calmette, which covers a part of the ground gone over in my monograph. I have availed myself of the opportunity offered by Professor Calmette’s book to complete and improve my own. In this connection I desire to thank Professor Calmette for his generosity in supplying me with cobra venom on several occasions, and Dr. George Lamb also for liberal gifts of cobra and daboia venom. I am also indebted to Director William T. Hornaday, Dr. Raymond L. Ditmars, and Mr. E. R. Sanborn of the New York Zoological Park for permission to consult their collection of skulls and photographs of snakes and to reproduce certain of the specimens. Finally, it is my very pleasant duty to acknowledge the great advantages which I have gained from the long connection with Dr. Flexner, and the many acts of friendship which he has shown me. HipEYO NOoGUCHI. ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH. New York, November, 1908. CONTENTS. Page. RREEACE apt hay shokel ate seas onctchotelteteh car aratote ch ckeveies oto er oat ok Ne aRE a rate shebetoletera: ts, o ere echie erties iii RPMI U Rte a een nah ili d Ae ata on stele a tin icles SARA sae oe ea + Cvle ge eieieae xi I. Systematic Position of Venomous Sridkes a eine recievers stale sare sate sere = I-3 hie Miorplolopy/ of Wenomous snakes’ \cmm ei tetera’ > Sous omisiete eclersieliare « diss, 4-45 verano Ma ONC Caee percep) ee tev oda el tar eles neta ke lotr ohare are evel che tet sh atet= che s/t avaxe 4 WRIHOPI VIM. Le ee eda wee a kt ene aia este Deiat b's ae ee pare c'e eB en thse 4 Subramily, Dispsadomoiphine .: ../c% hs dilewete sek Cares 6 Sete es ie ese 4 SOUL FLAC RILOC ORES 66.5000 apie ces eee halelv nwa Cera peida viawee 10 Stbiamuly Homalopsinve: 2 %)c 4 ae oe sa de ce ss cen Geen omeries 10 POLENOP Uy mliaeeny-seuesessreteiouei sv ckousvouste miensnegatery el tenets ho ett’ ain) chal aoetenele peer a= II SS Unf) RABUN Lyrae Lobe Et casey ah ete ol eto) ce vel sce Nr) eyo aloy ofa leke oe lal etaee lela) oy -1)« II Siilatamally) Eiger piunge: sua deseo tie naa) Sa esl de eos eelalpalas «= 26 amily sViperidcowteveir reticle) iepstayctaa« afsunlsicraiaiai oreraie a auotst farscei nore eee ors 29 SoU endl y, W gpebdeieesr eS 2. cha We He sats riale's Seabees apeitst ols init ae Os aie 30 Subfamily Crotalin ces s..< ie = ota etetate ieee er erereean a ete ed ob eat elereya ts ons 35 lit Phylogeny, of Venomous, SHAKES = rjciiste a: <> sleriateitlt es -leriee ee einai aa «le 2) 40-51 IV. Geographical Distribution of Venomous Snakes ...........0-eeee eee eres 52-57 Vin Polson Apparatusioty Venomous) SNAKES |e o/je <5) \sjo.siieiaelaolste «tare Cletus 4. ie 58-69 NE RNeaD PS eyaconel =F 71 Ifo tality: (Cased siocat eons jaja cher ook sedated os clet en nels «eso recerehsys vei fatek ayels s\n eer 75 VII. Physical and Chemical Properties of Snake Venom...............-.+-5-- 77-93 Chemical’Nature of Snake Venom) <2: ccgyeajepe sloisielaicilsialsvesetouersceisisis e's = 79 VIII. Effects of Various Physical and Chemical Agents upon Snake Venom .... 94-102 ECtSHOlme My Sic Al PAG eI tse. cleseyore trade cioeta/ eRe bel lsfealell aly etclaiapaieia\« <= ore 94 IDfiectssOb vVanious, Chemicals) = cinco a:ccucrersaabalcroe is orepeisinieie: Sietepniie=|=1s, «6 ni) 96 FX. Effects of Ferments upon Snake Venom... 20.2.5. ..eecsscnseesennce cas 103-105 &. Symptoms’ of Venom Poisoning I maM ......60 02.2 ese cece sence ness ens 106-112 Aes VAMETIC Ce aie tree tare Penta ee pats tne gre re eats 4 whch «saiaqeier terete Action of Snake Venom upon the Spleen............00.ecsseeeceees Acton of Snake’ Venom uponithe Heart. :.00. 2... 2.eroes-os eer Action of Snake Venom upon the Muscles ............2eeeeeeeeees SIX, -Ferments in Snake: Venom’. .)jocenuee © owen Penne Pee ee oe Proteolytic Action of Snake Venom atts 0. oan ace cote emer: Diastatic ‘Actions of Snake Venom) 27002) 0. .ocioe.. che sire cole aot sas os Lapolytic: Action of Snake Venom) (2) 42%, (0s. ahec cnet ctme ces serene XX. Antibactericidal Properties’ of Snake Venom. .. 2.0... +. 02000 neaeegn Ge © AL. "Loxicityof the issues of Venomousisnakes. ....c...saddcenias orm. en! XXII. Effects of Snake Venom on Mucous, Conjunctival, and Serous Membranes and Alimentary, Warict. act ith 3 Seca wae aetra ne vere OCT. | Amtiicial Imvatnization: 0 sicc< seis nas ae ee a Active Immunity — Prophylactic Ineculation .¢20..c\.sceis2+s0h+<4 020% Passive Imamunity ——Antivenins 2 /.5.2h)5 ales. oalvo.aidsrce iol eee Lass See XXIV. Specificity and Therapeutic Values of Antivenins ..............0.-.00005 speciacity of Antivenins asa a hole i. tne \ hla eaves Meteitte nae Specificity of Antivenins due to differences in the characteristic toxic principles of the venom of each species ............+.- Specificity of Antivenins due to differences in individual cytotropic toxins of the venoms, of ‘different: Species). t. uavga ok taee an as Crotalus adamanieus Antivenin ; soca oo cea vnl saseeend ooaeeiads «40% Crotalus terrifieus Autivenin 0 522, cis see oo ee Ghe a ee ee eee Ancistrodon piscivorus Antivenin (25.0% i .esess «sociated ees Lachesis lanceolatus) Autivenin’ 2.5 52.) 26d eek Sa ae ec oe es Lachesis flavovirulis Antivenin o25 cic 6 64a sons eee mea ee Calmette’s Antiveni. 2.2 ees asa ee eine A ok cake rae CE Therapeutic: Value’ of Autiventns, 6-5 a, 0e0e tack cea Ge ie XXV. Interactions between Venom and Antivenin ............0..ececeeeeeeees Establishment of the Chemical Nature of Venom-antivenin Reaction .... Regeneration of Venom and Antivenin from their Neutral Combination .... 199 206-209 CONTENTS vii Page. The Ehrlich-Madsen partial Saturation Phenomenon in Venom-antivenin PACs hes vin bey a Beth whose a wee Sa x She PW eeu o\ moa 250 CRAMIUS-VEDIOT PAUIVOMEN). ya.e sien /a vais vie ea n's 08 we ney a viel wl Was mm AeA 257 Wrater-moceasin-venOni ANEIVEME 75/6 <(6 <2)<)e1«) 5202) a)eforsleier« cleleye em sraiate sere 259 ROVE... Precipitin-reaction with) Smake Venom . ... ..esies csc ee wate e ees cee sah ge 261-263 PN Medrem Neat tarellen ra ra TTA DEG ney erate Were getel or oisict Fel sshelopetslaiste a ataldnop /otata el le\tetaredn’e rile = ars) ar Ucar 264-269 CEES Obs Ven Ol: UPOOMO MARES ili. telela eters etetel te ietats ofaiai stata ie et axeiaietanetela 264 Explanation of the Mechanism of Natural Immunity ................. 265 Natural Immunity of Certain Animals from Snake Venom.............. 268 XXVIII. Effects of Snake Venom upon the Blood of Cold-blooded Animals and upon the Nerve Cells, Nerve Fibers, Ova, and Spermatozoa. ..270-284 Action of Snake Venom upon Cold-blooded Animals ................. 271 Effects of Snake Venom upon the Blood Corpuscles of Cold-blooded Ani- TMA S Ses pero Mae kat Naaman AST «tailor oleh arwiwtel catatnans ater ats 282 Effects of Snake Venom on the Nerve Tissues, Ova, and Spermatozoa .. 284 XXIX. Effects of Snake Venom upon Plants and the Process of Germination of SGOdSU ayes care Hass oso ecehtiaea rent ote ae swioha attr sayy (ely bis tee is cine oie 285-286 ROO eh reatmentrOr SMA Ke mE Iker 7s) shares claro teraletceete ote eieherat atete arava pera) afer okalor= Bis 1K 287-296 Non-specific Treatment. Immediate Ligature and Dissection ......... 287 Wocale Pmeatenents: epee sista! wile at steenrs soil snevorasrelckeroletal ojetalietcapelo svensitcetareuPotele = 288 Pofassiimebs erie ant ele wane iaonene acta) telat alot tet elon iste) a) Siete stele siaiet= eters 288 Chloride of Gold, Hypochlorites of Alkalies and Chloride of Calcium....... 291 General Wrcareamieneatton: 2s, Jed vin Se ays eat hein eis wi nierge an, A, B, and C, Skull of Homalopsis buccata. D and E, Skull of Distira stokesii. (Drawings from Boulenger’s Book.) MORPHOLOGY OF VENOMOUS SNAKES 11 Homalopsis buccata. Scales in 37 to 47 rows. Ventrals r60 to 171; anal divided; subcaudals 70 to go. Total length about 3 feet. Bengal (?), Burma, Indo-China, Malay Penin- sula, Sumatra, Borneo, Java. Genus CERBERUS Cuvier. Cerberus rhynchops and 2 other species. Genus HYPSIRHINA Wagler. Hypsirhina plumbea and 14 other species. Genus HIPISTES Gray. Hipistes hydrinus. Head covered with small scales, scales of body smooth, excepting the very nar- row ventrals, which have double keels. Body laterally compressed, resembling in general appearance the Hydrophine. It is piscivorous and swims far out into the sea. It inhabits Siam. Family COLUBRID Boulenger. PROTEROGLYPHA. Corresponds to Cope’s two superfamilies Proteroglypha and Platycerca and Stejneger’s Elapidz. This family contains all snakes with a permanently erect grooved poison fang in the anterior portion of the horizontal maxillary bone. Boulenger and Stejneger divide the members of this family into subfamilies Elapinee and Hydrophine.' As a rule, smaller, solid teeth are carried by the maxilla behind the grooved fangs. Shape of pupil variable, some being round, some vertical. Elaps and Acanthophis have vertical pupils, while the famous Cobra has a round one. Although their poison apparatus is inferior to that of Viperide in Boulenger’s term or Solenoglypha of the usual nomenclature, the Elapine snakes are the deadliest and often the most dangerous of all snakes.2 Their general appearance is not essentially different from that of most harmless colubrine snakes and the presence of the fang is the only reliable difference in these species. Most Proteroglypha are viviparous, but the famous cobra de capello and certain marine snakes are oviparous. Some are terrestrial, others marine. The Elapine snakes are tropical. The whole Australian, Paleotropical and Neotropical regions, with exception of Madagascar and New Zealand, are inhabited by them. They extend northward into the warmer parts of North Africa, and range over a great part of the Palearctic subregion, being found in North Africa and southwestern Asia. They also inhabit the southeastern Asiatic islands and mainland. Subfamily ELAPINZ Boulenger. All are terrestrial, and the general feature is that of the harmless colubrine snakes. The comparatively small eye with vertical pupil, frequent absence of loreal, and indistinctness in width of head and body are often of differential value in determin- ing the species, but the presence of the grooved fangs is the last and reliable criterion. The most remarkable feature of some of the Elapine snakes is that they can dilate certain cervical ribs, assuming a hood-like or fan-formed shape when the snakes are excited. This, together with the conditions of median dorsal scale rows and subcaudals, serves to divide them into several genera. According to Cope the presence or absence of a postfrontal bone draws the line between Naja and Elaps.* They live on small vertebrates: lizards, birds, rats, frogs, snakes, and occasionally 1 Hydrine Stejneger. * The most dreaded species are Naja, Ophiphagus, and Bungarus in India, and Acanthophis and Pseu- dechis in Australia. They all possess very powerful venom and have courage and are often rapid im action. The Ophiphagus elaps of India reaches 12 feet and is the longest venomous snake. 8 Cope gave them the rank of families, Najide and Elapide. 12 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS fishes. ‘Their fondness for rats makes some of them extremely dangerous, as they often invade the human abode for their prey. ‘There are about 150 species, grouped into a large number of genera. Genus NAJA Laurenti. Naja Laurenti, Synops. Rept., p. 90, 1768. The pair of large and grooved poison fangs are separated by an interspace from one to three small, faintly grooved teeth near the posterior end of the maxillary bone. Pupil round. Head but slightly distinct from neck. Neck covered with more numerous scales, and can be expanded into a hood by the spreading and forwarding motion of the ribs. Scales are smooth and pitless; vertebral row not enlarged. Anal entire, subcaudal divided. No loreal. Postfrontal bone present. Naja tripudians. (Plate 2, c; plate 3, A, B, Cc; plate 4, A, B.) This dangerous tropical snake is known as the Cobra di capello aad is much dreaded on account of its powerful venom and its audacity. Oviparous, producing about twenty elliptical soft-shelled eggs of the size of a pigeon’s egg. Coloration varies much. ‘The typical form is yellowish to dark brown with a black-and-white spectacle-like mark on dorsal side of hood and a black-and-white spot on each side of the corresponding under surface. Other specimens are uniform pale brown to blackish-gray, without the spectacle pattern on the hood. Length usually 4 to 5 feet, or even over 6 feet, the tail 9 inches or more. It resorts to places affording easy retirement, such as heaps of stones, stacks of wood, ruins, and deserted hills of termites, and loves the neighborhood of streams. It visits inhab- ited houses to catch rats. The range of habitation is very wide, from Trans-Caspia to China, including all southern Asia, and to Formosa and the Philippines. Many observations show that cobras live in pairs, but otherwise do not take much notice of each other or of other kinds of snakes. They avoid the hot sunshine and hunt late in the after- noon or evening. ‘There are many varieties of this species, of which several will be described below. Naja tripudians typica: A spectacle-shaped white imprint on the most dilated portion of the dorsal surface of the hood; ventral side transversely banded with one or more dark lines. India, Bengal, Ceylon. Naja tripudians ceca: Uniform pale brown to deep gray color, without pattern on the hood, and one or more dark bands across the anterior ventral surface. ‘Trans- Caspian region, India, Bengal, Java. Naja tripudians fasciata: Brown, black, or olive color, with more or less light transverse bands; a white spectacle bordered with black is on the neck, and a black spot on each side, above the mark. Bengal, India, Cochin China, southern China, Hainan, Cambodia, Siam, Malay Peninsula. Naja tripudians sputatrix: Black or deep brown with a yellow white-rimmed spectacle on the side of the head and neck. Southern China, Burma, Malay Pen- insula, Chusan Islands, Sumatra, Formosa, Indo-China. Naja tripudians leucodira: Brown or black without mark on the hood. Naja tripudians miolepis: Brown or black; around the head and neck yellowish; no mark on the head. Sarawak, Labuan, Borneo. Naja samarensis. Black above, sometimes yellowish-black; pale brown to yellow on ventral sur- face; neck black. Grows to 3 feet. Philippine Islands. Naja bungarus s. Ophiophagus elaps s. Hamadryas. ‘“Sunkerchor” or Skull-breaker.” (Plate 2, B; plate 5, A, B.) This is the king cobra or snake-eating snake, or hamadryad. It has a well- expanding hood. Coloration variable, from yellowish to black, with or without an olive gloss. More or less distinct dark bands or rings may be seen in some MORPHOLOGY OF VENOMOUS SNAKES 13 specimens, while others are olive above with black-edged scales, and still others are very dark above and beneath. The distinctive, specific character is the small number of scales. It has only 15 rows on the middle of the body and 1g to ar on the dilatable neck, where in the typical tripudians 29 to 35 rows are found. It reaches the length of even r5 feet, and its venom is very powerful. It is said to kill an elephant within 3 hours by one bite and is liable to attack man. Per- haps this is the most dangerous snake of India. It lives exclusively on other snakes. It haunts the rivers and streams, lives in forests and jungles, and is a very agile climber. Naja haje. (Plate 5, c.) The common hooded cobra of Africa, the “Aspis,” so called on account of its shield or hood. Spectacle-marks on the neck absent or indistinct. General color varies brown to dark brown or blackish above, with or without spots of brown or yellow; below yellow, dark brown, or blackish ; head blackish; the neck with black or brown band on the uniform dark olive or yellow ground-color. It inhabits the border of the Sahara, Egypt, southern Palestine, east Africa to Mozambique. It is very common in central Africa and along the basin of the Nile and the Soudan. In Egypt it is often seen near ruins, under heaps of stones or among bushes. When chased it stops to defend itself. It lives in captivity for 6 or 8 months, but remains wild and vicious. It may reach a length of 6 feet or more. Naja flava. Very similar to the foregoing species. Has a dilatable neck, which is surrounded by a black band in some specimens. Color very variable, uniformly yellowish, reddish, brownish, or blackish, with a light spectacle-mark. Averages about 5 feet in length. South Africa. Naja melanoleuca. Sides of head brown or whitish, labial plates bordered black posteriorly. Reaches nearly 8 feet in length. Tropical Africa. Naja nigricollis. Coloration variable, with a black transversal band under surface of neck. Sene- gambia, upper Egypt to Angola and Transvaal. Naja anchietz. Black or brown above; muzzle yellowish; abdomen yellow or light brown, with or without a black band across under surface of neck. Scales on the neck or body 15 to 17 rows. Grows to 6 feet. Angola. Naja goldii. Eyes large; 15 rows of scales on neck and body. Color uniformly black or with transversal series of small whitish marks; abdominal surface white anteriorly, black posteriorly; subcaudal scales black. Grows to 5 or 6 feet. Lower Niger. Genus SEPEDON Merrem. Maxillary bone more prolonged than palatal bone, and carries one pair of enor- mous fangs, but no other teeth, differing thus from N aja. Neck dilatable. Head not distinct from neck. Eyes moderate in size with round pupil. Body cylindri- cal; scales keeled, in 19 rows. Sepedon hzmachates Merrem. “ Spy-slange” or “ spitting snake.” Known in South Africa as Ringhals or banded neck. This is another hooded snake of Africa. The general description of the genus applies to this species. General color bluish-black with many narrow undulatory and zigzag crossbands of yellow or yellowish-white color; under side of neck black or deep red with one or two white bands around lower portion of neck ; belly grayish. This snake is very well known because of its peculiar habit of spitting the venom to a distance 14 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS of more than 3 feet. This was primarily stated by the African natives, who believe that if the venom gets into the eye the sight will be lost. This seems to be partly corroborated by subsequent observations of some European travelers and settlers. It seems quite conceivable that if the projected venom gets into the eye there will be severe conjunctivitis. Calmette states, however, that he never observed such faculty in Sepedon during its captivity in his laboratory. Boers call it Spy-slange or spitting snake, meaning that it spits out its saliva when excited. Genus BUNGARUS Daudin. Bungarus Daudin, Bull. Soc. Philom. Paris, 1803, 187. The two large, grooved poison fangs are followed by one or two smaller teeth. Eyes small, with round or vertical pupils. Head not distinct from neck. Dorsal ridge prominent and covered with a row of much-enlarged scales. Scales in 13 to 17 rows. Body moderately thick, with comparatively short tail. Subcaudal in one or two series. Bungarus is closely allied to Naja, but has an undilatable neck. About half a dozen species inhabit southeastern Asia. Bungarus candidus!s. ceruleus. (Plate 3, D, E; plate 6, A.) Bungarus candidus var. ceruleus, or often B. ceruleus so called, is one of the most dreaded venomous snakes of the whole of India and adjacent regions. It is known as common krait; has a habit of invading houses in search of rats. It is comparatively small in size, reaching about 3 feet; lives on lizards, rats, and young snakes. It is known to penetrate to a veranda, bathroom, or even a pillow, but usually hides in old trees or walls. Scales smooth, dark brown or bluish-black with narrow crossbars or white specks, or alternately barred brown and yellow; under part uniform white. Krait is the next most dangerous snake to the cobra. Bungarus fasciatus. (Plate 6, B.) General color bright yellow, alternating with blackish bands. About 5 feet long. Fangs comparatively small. Known in northwestern India under the name Koclia-krait, it inhabits Bengal, Coromandel, and Burma. A bite of this species, also called the banded krait, is fatal to a dog within a few hours. Other species of Bungarus are multicinctus,’? which reaches 3 to 4 feet in length and inhabits South China, lower Burma and Formosa; ceylonicus, the Ceylon krait, and lividus, which has less-pronounced median dorsal scales and inhabits Assam. Genus HEMIBUNGARUS Peters. Hemibungarus Peters, Mon. Ber. Berliner Akad. Wiss., 1862, 637. In contradistinction to the genus Callophis, this one has several small solid teeth behind the poison fang. The pupil is round. Head and neck less distinct. Body rather slender and cylindrical. The poison extends sometimes down to the ab- dominal cavity. Scales in 15 rows. ‘Tail short. Subcaudals in two rows. Hemibungarus colligaster. Head purplish with black crossbars separated with narrow white bands; ab- dominal surface and tip of tail red; nose yellow, with black band around the upper lip near eyes. Grows to a length less than 2 feet. Philippine Islands. Hemibungarus collaris. Black on back; black and red bands on belly; yellowish collar on back part of head. Philippine Islands. iBoulenger divided Bungarus candidus into two varieties, ceruleus and multicinctus. Stejneger is inclined to consider Bungarus candidus s. ceruleus as not so closely allied as to be ranked as subspecies, but deserving full specific rank as Bungarus multicinctus — not as Bungarus ceruleus multicinctus. 2 Boulenger’s subspecies of Bungarus candidus, and corresponds to Bungarus semifasciatus Giinther, not of Boie, 1827. MORPHOLOGY OF VENOMOUS SNAKES 15 Hemibungarus nigrescens. Scales in 13 rows. Inhabits western India, Bombay to Travancore. Hemibungarus japonicus Giinther. ‘ Hai.” Callophis japonicus Giinther, Ann. Mag. Nat. Hist., 1868, 428. Hemibungarus japonicus Boulenger, Cat. Snakes, Brit. Mus., III, 1896, 396. Scales in 13 rows. Red above, with one to five black bands crossed by other black bands with yellow margin; muzzle and chin black; ventral surface yellowish with black specks and black transverse bands. Oshima in Riu Kiu Islands. Hemibungarus beettgeri. ‘‘ Hai.” Callophis bettgeri Fritz, Zool. Jahrb., Sept., 1895, VII, 861. See also Kat. Schl. Mus. Senckenberg, H Suateaterns japonicus Boulenger, Cat. Snakes, Brit. Mus., 1896, III, 395. Stejneger, Herpetology of Japan, 1907, 389. Almost identical with the preceding species except in coloration. Color above iridescent blackish blue with four longitudinal light bands, being reddish in the two median and white in the two outer ones. Across these longitudinal lines are about 14 transverse, irregular bands of bluish-black edged with white. These black crossbands are carried across the belly on a single ventral; under side whitish with numerous large and irregular blackish-blue blotches. Okinawa Island in Riu Kiu Islands. Genus CALLOPHIS Giinther. (Plate 6, D.) Callophis Gray, Ind. Zool., II (C. fig.). In many respects this genus resembles Sepedon. The maxillary bone is longer than the palatine, and carries a pair of very strong poison fangs, without any other teeth. Eyes small, with a round pupil. Body cylindrical and slender. Neck not dilatable. The scales are smooth instead of being keeled as in Sepedon, imbri- cated in 13 rows. The head is small and not distinct from the neck. Length not more than 2 feet. All the members of Callophis are elegantly and variedly colored, hence are called by this generic name, which means “beautiful snake.” They live exclusively on other snakes belonging to Calamaride, and do not inhabit any region where. no calamarine snakes are to be found; for example, Ceylon. They are essentially terrestrial and hide around old logs or trunks of trees; they are slow and lazy, being more of a nocturnal nature. While their venom is by no means weak, no fatal accident from the bite of the snakes of this genus has been recorded. Callophis gracilis. Red or pale brown, with three longitudinal black lines indented with black and brown specks, the lateral marks alternating with the vertebral ones; black and yellow bands along under side of abdomen and tail. Total length about 2 feet. Malay Peninsula and Sumatra. Callophis trimaculatus. Head and muzzle black with a yellow speck on each side of occiput; belly uni- formly red. Total length about a foot. Burma and India. Callophis maculiceps. Head and muzzle black with two yellow bands on each side; belly red. Total length about 1.5 feet. Inhabits Burma, Cochin China, Malay Peninsula. Callophis macclellandii. Elaps macclellandii Reinhardt, Calcutta Journ. Nat. Hist., IV, 532. Giinther and Boulenger grouped it in Callophis. Head and neck black with a yellow crossband in rear of eyes; back reddish- brown with regular and equidistant black rings; belly yellow with bands and quadrangular marks of black. Total length about 1.5 to 2 feet. Inhabits Nepal, Assam, Burma, southern China. 16 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Callophis bibronii. Head black anteriorly, reddish posteriorly; body purplish-brown to the tail, with transverse irregular bands. Grows to 2 feet. Found by Bedhome at the altitude of 3,000 feet in Malabar. Callophis univirgatus Giinther. Callophis univirgatus Gimther, Proc. Zool. Soc. London, 1859, 83. Callophis macclellandii var. univirgatus Boulenger, Cat. Snakes, Brit. Mus., III, 1896, 399. Coloration alone separates this from the macclellandit, it having a narrow black line down middle of back, and no regular crossbars. It inhabits Sikkim and Nepal, Assam, Burma, southwestern China, Formosa. In the province of Fokien it is found at an altitude of 3,000 to 4,000 feet. Genus DOLIOPHIS Girard. Adeniophis Meyer. Doliophis differs from Callophis chiefly on account of the presence of an enor- mously developed poison gland in the former. It is not restricted to the head, but extends along the anterior third of the body, gradually thickening and terminating in front of the heart with club-shaped ends. Owing to the extension of these glands, which can be felt through the skin as thickenings at end of first third of body, the heart has been shifted farther back than in any other snake. Doliophis bivirgatus. Color purplish-red or black above, red on head, tail, and belly. ‘Total length 4 to 5 feet. Burma, Cochin China, Malay Peninsula, Sumatra, Java, and Borneo. Doliophis intestinalis. Back brown or black with longitudinal rings of lighter or darker shade. Tail red; belly red with intersections of black crossbars. ‘Total length about 2 feet. Burma, Malay Peninsula, Sumatra, Java, Borneo. Doliophis bilineatus. Back black with two white bands along entire length of body; muzzle white; the belly striated with white and black bands; tail orange. Total length a little over 2 feet. Philippine Islands. Doliophis philippinus. The back carries two longitudinal brown bands, which are intersected by the transverse black bars of the belly; the interspaces of the bands are again annulated with yellow and red; head brownish with small yellow specks. Total length about 18 inches. Philippine Islands. Genus BOULENGERINA Dollo. Cacophis Giinther. The maxillary bone has a length equal to that of the palatine; carries a pair of comparatively large fangs, then a series of three or four small teeth behind the fangs. Eyes small with round pupil. Body cylindrical; scales soft, in 21 rows; the ventral scales are rounded off. Moderate tail. Subcaudal double. It is a small snake, about 7 inches long. The head and neck indistinct in width. Boulengerina stormsi. The only known species of this genus is of brown color with black rings on neck, black tail, the belly anteriorly white, posteriorly brown. Inhabits the region around Lake Tanganyika. Genus ELAPECHIS Boulenger. The length of the maxillary and palatine bones is equal. A pair of enormous poison fangs is followed by two or four small teeth. Head and neck indistinct. Eyes small with round pupil. Body cylindrical. Scales soft, oblique, and in 13 to 15 rows; ventrals rounded off. ‘Tail very short. Subcaudals in two rows. Noguchi Plate 2 Cc A. Skulls of a Harmless Snake and of a Boa Constrictor B. Skull of Naja bungarus. From Photograph. C. Skull and Skeleton of Naja tripu lians (From Photograph ) Noguchi Plate 3 ‘« y _ ——s — ~—s a > Pon ‘ == — bi . = j ANY MENS S OED A, B, and C, Skull of Naja tripudians. D and E, Skull of Bungarus candidus. (Drawings from Boulenger’s Book.) : he whe erat pei hire) A Noguchi Plate 4 B A and B. Photographs of Naja tripudians, from Live Specimen. Noguchi A and B. Photographs of Head of Naja bungarus (King Cobra), from Dead Specimen. C. Photograph of Naja haje (Egyptian Cobra), from Live Specimen. Plate 5 MORPHOLOGY OF VENOMOUS SNAKES 17 Elapechis guentheri. Color whitish or gray above, with black crossbars; belly whitish or brownish, or gray. ‘Total length about 1.5 feet. Gaboon, Congo, Angola, central Africa. Elapechis niger. Whole body black. Not over 1.5 feet in length. Inhabits Zanzibar. Elapechis hessii. Color gray with black crossbars; belly white. Total length about 5 inches. Congo. Elapechis decosteri. Color dark gray; each scale has a black margin; belly white. Total length about 1 foot. Delagoa Bay. Elapechis sundeyvallii. Reddish color with yellow transverse bands; scales bordered with brownish red margin; upper lip and belly yellow. Total length 1.5 to 2 feet. Elapechis boulengeri. Back black with tiny white crossbands; head white, belly grayish-black. Total length only 5 to 6 inches. Inhabits Zambesia. Genus ASPIDELAPS Fitzinger. The maxillary bone is farther advanced than the palatine bone, as in Sepedon, with a pair of very large poison fangs, besides which there are no teeth on maxilla. Head slightly distinct from neck. Moderate-sized eyes with round or vertical pupil. Body cylindrical. Scales oblique and keeled in 19 to 23 rows. Ventrals rounded off. Tail short and obtuse. Subcaudals in two rows. Aspidelaps lubricus. Orange or red with black rings; top of head sometimes completely black. Total length about 2 feet. Cape and Namaqualand. Aspidelaps scutatus. Pale gray with black specks or crossbars; head has a black curved (~) mark; black collar around neck; belly whitish. Total length about 1.5 to 2 feet. N atal, Delagoa, Mozambique. Genus WALTERINNESIA Lataste. The maxillary bone surpasses the palatine bone in length. It carries a pair of very powerful poison fangs, but no other maxillary teeth. Head and neck distinct. Eyes very small, with a round pupil. Body cylindrical. Scales somewhat keeled and in 23 rows; the subcaudals in two series. “The tail is short. Walterinnesia zxgyptia. Dorsal side blackish-brown; belly light in degree. This snake may grow 3.5 to 4 feet long. Egypt. Genus DENDRASPIS Schlegel. The maxillary bone is curved at the base, and carries a pair of strong poison fangs. No other maxillary teeth. A long terminal tooth on each mandible. Head narrow, elongate; eyes moderate in size, with round pupils. Body slightly com- pressed; scales narrow, and very oblique, in 1 3 to 25 rows. Tail long; subcaudals in two rows. Dendraspis viridis. Color uniform olive green; head black; lip yellow; belly and tail yellow, with scales and plates bordered black. Total length about 6 feet. Western Africa, Senegal, St.Thomas Island. 18 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Dendraspis jamesonii. Same coloration, with 15 to 19 rows of scales; tail sometimes black. ‘Total length somewhat over 6 feet. Western Africa, Guinea to Angola, central Africa. Dendraspis angusticeps. Color uniform green, olive, or blackish; belly yellowish or light green. Total length 6 to 7 feet. Western Africa south of Congo; central Africa; eastern Africa; Transvaal; Natal. Dendraspis antinorii. Color olive above, yellowish beneath. ‘Total length 8 to g feet. Abyssinia. Genus OGMODON Peters. The maxillary bone, which is more prolonged than the palatine bone, carries, beside the poison fangs, 6 to 7 grooved reserve teeth. Eyes very small, head not distinct from body. Body cylindrical with smooth scales in 17 rows. ‘Tail short. Subcaudals in two series. Ogmodon vitianus. Pointed snout. Color dark brown, lighter on sides; belly white or slightly spotted with black; tail black. Total length about 1.5 feet. Fiji Islands. Genus GLYPHODON Giinther. (Plate 7, A, B, C.) The general character like Ogmodon. Snout rounded off. The poison fangs are followed by small posterior maxillary teeth with an interspace between them. Head and eyes small; pupil round or slightly vertically elliptical. Body cylindrical, the scales in 17 rows. ‘Tail short, subcaudal in two series. Glyphodon tristis. Color dark brown above, occiput yellowish or pale brownish-red, belly yellow. Total length about 3 feet. Northeast of Australia, southeast of New Guinea. Genus PSEUDELAPS Duméril and Bibron. Maxillary bone is longer than palatine bone and carries a pair of large poison fangs, which, after an interspace, are followed by 8 to 12 small teeth towards rear. Anterior mandibular teeth almost as large as the poison fangs. Head and neck not very distinct. Eyes small, vertical pupil. Body cylindrical. Scales smooth, in 15 to 17 rows. ‘Tail moderate or short, subcaudals in two rows. Pseudelaps muelleri. Color brown with a light vertebral line; the clear band of each side of head crosses over the eyes; belly yellowish or coral-red, with or without black specks. Total length about 1.5 feet. Malay Archipelago (Dutch), New Guinea, New Britain. Pseudelaps squamulosus. Color brown with yellowish band around snout and between the eyes; belly whitish with black specks, which join and form blackish line on each side. Total length about 1.5 feet. New South Wales. Pseudelaps krefftii. Color dark brown with a longitudinal line on each scale; yellow transverse band on occiput, passing over to another yellow band around muzzle; belly whitish anteriorly and black in posterior portion. ‘Total length about 8 inches. Queens- land. Pseudelaps harriettz. Color brown with a longitudinal black line on each scale. ‘Total length about 1.5 feet. Queensland. es MORPHOLOGY OF VENOMOUS SNAKES 19 F Pseudelaps diadema. Color pale brown with a brown net on each scale and a yellow transverse band on occiput; belly uniformly white. Total length about 2 feet. North, East, and West Australia. Pseudelaps warro. Same characteristics as diadema, but with large black collar around neck; top of head black, but not so black as collar. Port Curtis, Queensland. Pseudelaps sutherlandii. Same characteristics as above, except certain color variations. This also has a spectacle-shaped collar on neck. Norman River, Queensland. Genus DIEMENIA Gray. The maxillary bone surpasses the palatine bone and carries a very well-developed pair of poison fangs, behind which are 7 to 15 smaller teeth with wide interspaces between them and the fangs. The anterior mandibular teeth are elongated and present the appearance of poison fangs. Head and neck slightly distinct. Eyes quite large with round pupil. Body cylindrical with 15 to 19 rows of scales on back. Tail is moderate, subcaudals usually in two rows. Color very variable, yellowish, olive, brownish-red, and brown. Medium length about 3.5 to 5 feet. Southeast of New Guinea and Australia. The following species of Diemenia exhibit the characteristics noted: psammophis, torquata, and olivacea have 15 rows of scales; modesta has 19 rows, as have also textilis, the “‘Brown”’ snake, and nuchalis. Genus PSEUDECHIS Wagler. The maxillary bone surpasses the palatine bone markedly, and carries a large pair of poison fangs, followed by two to five smaller solid teeth in the rear. Anterior mandibular teeth long. Head more or less distinct from neck. Eyes rather small, with round pupil. Body cylindrical. Scales smooth, in 17 to 23 rows, having a few more rows on the neck, though the latter is not, or only slightly, dilated. Tail moderate, the subcaudals partly in double, partly in single series. Total length about 6 feet or even longer. Australia and New Guinea. Eight species in this genus. Pseudechis porphyriacus. (Plate 8, A.) Back black with anterior row of red scales; belly reddish with black edges. Pseudechis cupreus. Color copper above, brown or orange beneath; all the plates and scales black- rimmed. Pseudechis ferox. Muzzle greatly rounded off. Scales on body in 25 rows. Dorsal color black, yellowish on under side. Besides the above named, the following species of Pseudechis have the color characteristics noted: P. australis, back pale brown; belly yellowish. P. darwiniensis, brownish-red; head pale brown; belly light yellow. P. papuanus, uniformly black, but chin white. P. scutellatus, dark brown above; muzzle pale brown or yellowish; belly yellow. P. microlepidotus, dark brown above; belly grayish-yellow; head blackish. Genus DENISONIA Krefft. Maxillary bone projecting beyond palatine bone, with a pair of large poison fangs followed by 3 to 5 smaller teeth. Anterior mandibular teeth pretty well 20 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS developed. Head distinct from neck. Small eyes, usually with round pupil; some pupils vertical. Body cylindrical. Scales smooth, in 15 to 19 rows. Tail moderate or short. Subcaudals in one single row except one species. Boulenger enumerates 21 species of Denisonia. Denisonia superba. Brown or salmon color, belly yellow or olive-gray. Length about 3.5 feet. New South Wales, central Australia, Tasmania. Denisonia coronata. Olive color with black band on each side of head; belly yellow or pale olive. 1.5 feet long. Western Australia, New South Wales. Denisonia coronoides. Color brown; yellow lip; belly olive-gray or salmon. Length about 1.5 feet. southern Australia; Tasmania. Denisonia muelleri. Color brownish-gray; lip and chin yellow; belly gray. Length about a foot. Queensland. Denisonia frenata. Scales in 19 rows. Color olive-brown; upper lip yellow; belly white. Length about 1.5 feet. Lake Elphinstone and Queensland. Denisonia ramsayi. Color olive-green; belly yellow; subcaudals black. Length about a foot. New South Wales. Denisonia signata. Color greenish-brown; head brown; belly gray or white. Length 2 to 2.5 feet. Queensland and New South Wales. Denisonia dzemelii. Color olive; head more intense; belly yellow. Length about 1.5 feet. Queens- land. Denisonia suta. Pale olive color; head deeper brown; belly yellow. Length about 7 inches. Middle Australia. Denisonia frontalis. Light brown color; black vertebral line; belly pearl-white, with bronze-colored median band. Length about 1.5 feet. New South Wales. Denisonia flagellum. Color pale brown; occiput and neck black; belly white. Length about 1.5 feet. Victoria. Denisonia maculata. Color brownish-gray or brown; head covered with large dark olive-green or brown speckles; belly grayish. Length about 1.5 feet. Queensland. Denisonia punctata. Pale brown color; head and neck orange; lower lip and belly yellow. Length about a foot. Northwestern Australia. Denisonia gouldii. Color brownish-yellow; neck white; head covered with large greenish-blue speckles from nose to neck; lower lip and belly yellow. Length 1.5 feet. Southwestern Australia. MORPHOLOGY OF VENOMOUS SNAKES 21 Denisonia nigrescens. Color deep olive; head black; belly yellow. Length about 1.5 to 2 feet. New South Wales and Queensland. Denisonia nigrostriata. Yellow color with black stripes; head dark brown; upper lip and belly yellow. Length about 1.25 feet. Queensland. Denisonia carpentariz. Brown color; upper lip and belly yellowish-white. Length 8 inches. North Queensland. Denisonia pallidiceps. Color deep brown-olive; head usually very light; belly yellow. Length about 2 feet. North Australia. Denisonia melanura. Dark brown color; head and side of body red; tail black; belly yellow. Length about 3.25 feet. Solomon Islands. Denisonia par. Color brownish-red in large bands over body, with white intervals; head brown; belly white; tail with red bands. Length about 2.5 feet. Solomon Islands, Straits of Bougainville, and neighboring islands. Denisonia woodfordii. Color light brown, with reticular pattern; head dark brown; belly white. Length about 2.25 feet. New Georgia, Solomon Islands. Genus MICROPECHIS Boulenger. Maxillary bone extending forward as far as the palatine, with a pair of large poison fangs followed by three small solid teeth; anterior mandibular teeth longer. Head distinct from neck; eyes very small and with round pupil. Body cylin- drical; smooth scales, in 15 to 17 rows. ‘Tail short; subcaudals in two rows. Micropechis ikaheka. Color yellow and black on transverse bars; head and tail black; belly yellow. Length 4.5 to 5 feet. New Guinea. Micropechis elapoides. Color cream, with 22 black bands which are larger than the white interspaces which separate the former. Length about 2.5 feet. Florida Islands, group of Solomon Islands. Genus HOPLOCEPHALUS Cuvier. Same characters as Micropechis. The scales in 21 rows. Ventrals angulate and notched laterally. Tail moderate, subcaudals in one row. Hoplocephalus bungaroides s. variegatus. Color black above with yellow speckles forming irregular crossbands on body; upper lip yellow; belly whitish-yellow, and yellower on sides. Length about 6 feet. New South Wales. Hoplocephalus bitorquatus. Ventral scales very angular. Color olive-green; head pale olive with bright yellowish occipital speckle, and two black spots and some smaller spots between and around the eyes; belly olive-gray or brown. Length about 1.75 feet. Queens- land, New South Wales. Hoplocephalus stephensii. Alternative transverse bars of white and black, the black twice larger than white. Head dark, a yellowish w-spot on neck. Length 2.5 feet. Port Macquarie, New South Wales. 22 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Genus TROPIDECHIS Giinther. Same characters as Hoplocephalus and Micropechis. Scales of trunk more keeled in 23 rows. Tail moderate; subcaudals in one row. Tropidechis carinata. Dark olive with darker transverse bands; belly more or less olive-green or yel- low. Length about 2.5 feet. New South Wales, Queensland. Genus NOTECHIS Boulenger. (Plate 8, B.) Maxillary extending forward as far as the palatine, with a pair of large, grooved poisoned fangs followed by 4 or 5 small, feebly grooved teeth; mandibular teeth, anterior longest and feebly grooved. Head distinct from neck, with distinct canthus rostralis; eye rather small, with round pupil; nasal entire; no loreal. Body cylindrical, but scales of trunk are smooth and oblique, in 15 to 19 rows. Lateral scales shorter than dorsal. Tail moderate, subcaudals in single row. Notechis scutatus s. Hoplocephalus curtus. This famous snake, known as “ Tiger snake,” has dark-olive color; belly yellow or olive; scales have often dark rim, in 15 to 19 rows, which are smooth. The olive color of the body is often crossed with dark bands. Genus RHINHOPLOCEPHALUS F. Miiller. Dentition same as Hopflocephalus. Head and neck little distinct. Eyes small with round pupil. No internasals. Body cylindrical, rigid, and smooth scales in 15 rows. ‘Tail is short, subcaudal in one single row. Rhinhoplocephalus bicolor. Olive-gray color above, whitish-yellow on belly; white tongue. Length about 1.25 feet. Australia. Genus BRACHYASPIS Boulenger. Same characteristics as above, but head distinct from neck; eyes small and have a vertical pupil. Body stout and cylindrical. Scales smooth, slightly oblique, in 19 rows. ‘Tail short, subcaudals in one row. Brachyaspis curta. Uniform brown-olive color, with yellowish belly. Length about 1.5 feet. West- ern Australia. Genus ACANTHOPHIS Daudin. (Plate 8, c.) The “death adder” has a maxillary bone equaling the palatine in length, and the former carries a pair of large poison fangs, followed by a series of two or three small teeth in the rear. The anterior mandibular teeth are so elongated as to appear like the fangs. Head distinct from neck. Eyes small with vertical pupil. Body short and thick, covered with 21 to 23 rows of keeled scales. Anterior caudals in one and the posterior in two rows. Tail peculiar in form, being laterally com- pressed, with a thin, horny, terminal spine. This snake is viviparous. Acanthophis antarcticus. This is the real “death adder,” the type of this genus. The colors of the upper parts are a mixture of brown, reddish, and yellow, often spotted with black or brown. End of tail yellow, reddish-brown, or black. Length under 3 feet. Moluccas, New Guinea, Australia. Genus ELAPOGNATHUS Boulenger. The maxillary bone surpasses the palatine bone, with a pair of fairly developed poison fangs, but no other teeth; mandibular teeth of equal length. Eyes moder- ate, the pupil round. Body cylindrical and covered with 15 rows of smooth scales. Tail moderate, the subcaudal in one row. ta ae Wiha, ‘x Noguchi , Plate 6 A, Bungarus candidus (Common Krait). B, Bungarus fasciatus (Banded Krait). C, Elapsfulvius (Coral Snake). D, Callophis. (A, B, and D, Photographic Reproductions from Fayrer’s Thanatophidia; C, Photo- graph from Live Specimen.) Plate 7 Noguchi A, B, and C, Skull of Glyphodon tristis. D, E, and F, Skull of Elaps marcgravi. (Drawings from Boulenger’s Book.) Noguchi Plate 8 A, Pseudechis porphyriacus. B, Notechis scutatus. C, Acanthophis. (Photographic Reproductions from Krefft’s Book.) ‘de oo ‘ 7 ‘ tle } yah. ie ae ‘ Y AOATaSS Tui hades } Ag : 7 ah Pay Ne ae ie ; Tele ; i We } mY i ue tao oe s ee, ; 5 ; ‘ lis ; ot } ath un Hy ns fi ag, ys ; : , L b ¥, : " it le Pane ey way ra A Ph aa Went se ahs y i hh ina . ‘ : 4 ve : ey eae ey th i Ai’ ‘ ONC n bs fi The was al wn) Ce GED hy a 1a 4 Fi i) be Jte> > ; ie _ ps ' c 7 wer ath NY ' J } 1 j 7 1 Ne ! y ; i i ; } ; - A i og A? Vy ' rte Bi ' ; } Vv) ok j ‘EWA Fan! A vives. ; t : ; ihe i : 7 J ' | a jet, ; | Aa i Ie My “ Ae is ‘ i I Ves vi? : : Niiset van! ‘i 7 cf 7, ; h Maye rs ’ i are in’ iF 7 neat i aan, - 1 1 wil a / 1 t i , re q ; , iid ni 3 nut ‘ uN) . } i t ’ : ' 1 ny iv ae: i i , we ; : ui » Aa 7 o a j i , f ; : j 7 At I ‘ 1h é ay X 7 : / 7 i) ! : m ¢ i | RL . 4 " { fy : i) oil : n} ; , Ved ! : é Al ; Dis Oe : i ; Ve i Smee i AA if 1 Ms ui iy Naa 14 ; ¥ i \ Meh cab Tie . Bor p 4 vel < ee) ; , a4 t in) i a Dey Al ih’ ie i J , i é a) a i: ip id ne ; ; aN Aes anny tp At os f : bi ; 4) 4 ; F ; . ‘ ve : hy ve i i i ian Re be ia at , ; a ay eh utes pe i ae ‘i ye mol me Riis, Bhi ty saree ot yn Me at ane MORPHOLOGY OF VENOMOUS SNAKES 23 Elapognathus minor. Dark olive color with a black occipital spot in young specimens; belly yellow or greenish-gray. Length about 1.5 feet. Southwestern parts of Australia. Genus RHYNCHELAPS Jan. Maxillary bone surpasses palatine, with a pair of poison fangs of medium dimen- sion, followed by a wide interspace as far as the two small teeth at extremity of bone. Anterior mandibular teeth longer than posterior. Head small and indis- tinct from neck. Eyes small with vertical pupil. The short, cylindrical body has 15 to 17 rows of smooth scales. Tail very short. Subcaudals in two rows. Rhynchelaps bertholdi. Yellow, with 19 to 4o ordinary black rings narrower than interspaces. The head is more of a brown color and has one large black spot. Rhynchelaps australis. Color red above with irregular transverse bars consisting of black-rimmed yel- low scales; belly white. Length about a foot. Queensland. Rhynchelaps semifasciatus. Color yellow above with brown crossbands; large brown speckle on head; belly white. Length about a foot. Western Australia. Rhynchelaps fasciolatus. Color red above with numerous black-brown crossbands; large brownish-black speckles on head. Length about a foot. Western Australia. Genus FURINA Duméril and Bibron. The maxillary bone surpasses the palatine, carries a pair of medium-sized poison fangs and one or two small teeth near posterior end of bone. Mandibular teeth almost equal in length. Head is small and indistinct from neck. Eyes very small with a round pupil. Cylindrical body, covered with 15 rows of smooth scales. Tail very short; subcaudals in two rows. Furina calonota. Color yellow, with black vertebral ray; black bar crosses extremity of muzzle; a large black spot covers top of head; belly white. Length about 7 inches. West- ern Australia. Furina bimaculata. Color yellow, with large black spots on nose, on middle of head, and the occiput; belly white. Length about 1.25 feet. Western Australia. Furina occipitalis. Black and white bands all over body, narrower on belly; head black with wide white band on occiput and narrower white band on muzzle; nose black. Length about 2 feet. Australia. Genus ELAPS Schneider.! (Frontispiece; Plate 7, D, E, F.) The maxillary bone is rather short and protrudes beyond the palatine, carrying a pair of large poison fangs; only a few or no pterygoid teeth; mandibular teeth of equal length. No postfrontal bone; the prefrontals unite on the median line. Head small, not distinct from neck. Eyes small with vertical pupil, which is ellipti- cal or semi-elliptical. Body cylindrical, with 15 rows of smooth scales. Sub- caudal scales partly in one, partly in two, or throughout in two rows. The rather elongate body, short tail, and small eyes render it difficult to discriminate these from the calamarine snakes without examining the dentition. The scutellation 1This group of snakes holds family rank in Cope’s classification, Elapide. 24 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS of the head is exactly that of Tantilla. Coloration bright, consisting of red and black, with some yellow, arranged in rings or parts of rings. The red is generally the ground-color, and the black rings are either single or in sets of three. The latter may be much narrower than ground-color, or may be so wide as to reduce it to very small proportions (Hlaps semipartitus). Epidermis beautifully iridescent, especially in the black spaces. The colors are much like those of the mineral labradorites, and are probably due to the same physical cause, namely, a micro- scopic lamination of the surface (Cope). On direct antero-posterior view the color is peacock purple; on transverse view it passes from brassy yellow through brassy green to maroon and brown. The colors do not appear if the scales are wet. As to the dangerous character of the coral snakes, all suspicion of doubt has been removed by the many fatalities arising from careless handling of these snakes. The alleged non-dangerousness or even innocuousness of the coral snakes has its origin in the mistaken identity of the snake, as their general appearances are hardly distinguishable from many truly non-poisonous American snakes — for example, Ophibolus (‘scarlet king snake,” O. doliatus; “red king snake,” O. coccineus; O. annulatus, “ringed king snake,” etc.), and Osceola elapsoidea; the ‘scarlet snake,” Cemophora coccinea; Rhinochilus lecontei. One fundamental difference in the color-arrangement seen in the species of H/aps within the United States boundary and that in Ophibolus, Osceola, and Cemophora, is that in our Elaps black rings are bordered on each margin by a yellowish ring, while in the others the yellow rings are bordered on each side by a black ring. Red — yellow — black — yellow — red (Coral snake). Red — black — yellow — black — red (Ophibolus and the like). Elaps fulvius.1 (Plate 6, c.) This species is known as the “harlequin snake.” Color above, red; yellow and black rings; tail yellow and black rings; nose black. Length 3.25 feet. Eastern parts of Southern States of North America, boundary of Ohio and of Missouri down to Rio Grande, Mexico, Central America. ~Elaps euryxanthus. This is known as the “Sonora coral snake.” Color red with 11 yellow-rimmed black rings. Length about 1.25 feet. Arizona and Colorado, northwestern parts of Mexico; in Arizona even at an altitude of 5,500 feet. Elaps marcgravii. Six to ro black rings, the middle ones being larger. Muzzle yellow, nose black; occipital black. Length about 3.5 feet. Tropical South America. Elaps heterochilus. Like E. marcgravii. Length 1.6 to 2 feet. Brazil. Elaps surinamensis. Seven or 8 series of tricolored rings. Length 2 to 3 feet, but can attain a length of 6 feet. Venezuela, Guiana, northern part of Brazil, northeast of Peru. Elaps gravenhorstii. Seven series of tricolored rings. Length under 2 feet. Brazil. Elaps langsdorfii. Dark-brown color with 63 cross series of cream spots, each occupying one scale; belly yellow with red crossband. Length about 1 foot. Upper Amazon. 1 An account of the dangerous effect of the bites of this species was given by Einar Loennberg, Proc. U.S. Nat. Mus., 1894, XVII, 334. See also Cope, 1898, 1123. The habits of the coral snake are admirably described by Ditmars, The Reptile Book, 1907, 397. MORPHOLOGY OF VENOMOUS SNAKES 25 Elaps buckleyi. Orange color with yellow spots; head black; temple yellow. Length about 1.6 feet. North Brazil and East Ecuador. Elaps anomalus. Fifty-five black rings separated by narrow yellow bands; belly reddish; tail yel- low or red with four black rings. Length about a foot. Colombia. Elaps heterozonus. Red or brown color with 17 to 25 black rings which are narrower than inter- mediary spaces; the black band on the head transverses the eyes. Length about 3.6 feet. Eastern Ecuador; eastern Peru; Bolivia. Elaps elegans. The black rings of the tricolor series are separated by yellow spaces; 12 to 17 series; head black with yellow spots. Length about 2.5 feet. Mexico and Guate- mala. Elaps annellatus. Black color with 41 to 49 white rings on body, including 4 to 7 on tail; white ring on head. Length about 1.6 feet. Eastern Peru. Elaps decoratus. Fifteen to 16 series of the three black rings on red; the head yellow with black crossband over eyes. Length about 2 feet. Brazil. Elaps dumerilii. Fight to 9 series of black rings on red and yellow; head black with one yellow crossband over occiput. Elaps corallinus. The “serpent corail.” The black band is separated by a red space with yellow edge; head bluish-black, and a blue line from rear of eye to lower jaw; tail white. Length about 2.6 feet. Tropical South America; St. Thomas, St. Vincent, Martinique. Elaps hemprichii. Black with red and yellow rings, a large ring between two narrow ones; occiput, upper lip, and temple yellow. Length about 2.5 feet. Guiana, Colombia, and Peru. Elaps tschudii. Black rings with larger spaces in series; the interspaces red and yellow; muzzle black. Length about 1.5 feet. Peru. Elaps dissoleucus. _ Same coloration. Length 3.25 feet. Venezuela. Elaps psyches. Alternative rings of black and brown with 48 to 52 narrow yellow rings; head black with yellow spots. Length about 1.6 feet. Guiana. Elaps spixii. Twenty to 38 black rings of the tricolor series; an occipital collar of black is followed by a spacious red band. Length about 5 feet. Venezuela, North Brazil. Elaps frontalis. Coloration regular; red, yellow, black series; head spotted yellow on black. Length about 5 feet. Central Brazil, Paraguay, Uruguay, and Argentine Republic. Elaps lemniscatus. Eleven to 14 series of black on red and yellow ground; head yellow, nose black; a white band on middle of head crosses the eyes. Length 3.5 feet. Guiana and Brazil. 26 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Elaps filiformis. Almost the same as Elaps lemniscatus, this species is characterized by the black muzzle and black band over the yellow head. Length about 2 feet. Amazon and Colombia. Elaps mipartitus. Black with 40 to 60 narrow white rings; head black between the eyes, the rest yellow. Length about 2 feet. Central America, tropical South America. Elaps fraseri. Black with 75 narrow white rings. Length about 2.5 to 3 feet. Ecuador. Elaps mentalis. Body black with 58 to 70 narrow white rings; tail annulated black and yellow. Length about 1.6 feet. Colombia and Ecuador. Elaps ancoralis. Sixteen black rings of series; the middle ring of a series is larger; black spots; the head has an anchor pattern. Length 2.5 to 3 feet. Ecuador. Subfamily HYDROPHIINZ Boulenger. Hydrine Stejneger. In adopting the aquatic or marine life these sea snakes have undergone con- siderable morphological changes. The most remarkable features are the highly compressed, oar-blade-like tail and the narrowly tapered upper part of body, which is in some cases strongly compressed laterally throughout the entire length. As a rule the posterior part of the body is enormously enlarged and presents rather a characteristic appearance in contrast with the tiny anterior part. Head not wider than neck. Eyes small with round pupils. The nostrils are situated on top of the snout and are provided with valves. The tail is prehensile and can keep the body afloat by seizing a polyp. The scales of the body are polygonal and are juxtaposed with short keels or knobs or spines, sometimes in pairs, and totally unlike the scales of other snakes. The ventrals are so reduced in width that they can hardly be recognized as such. The above-mentioned features are typical of the absolutely marine snakes, but in some species still partially terrestrial the ventrals are broader and keeled, and the snakes live near the shores, and occasionally or temporarily climb among the rocks or go ashore considerable distances from the landing-places. The tropical marine snakes can not crawl easily on the land, although agile in water. They survive in an aquarium only two or three days, mostly dying shortly after captivity. Owing to the large capacity of the lung they can dive very deep. The Hydrophiine snakes are proteroglyphous and their poison is extremely power- ful. They live on fish and are viviparous. The geographical distribution of the sea snakes is not easily established, owing to the absence of definite boundaries and to the occasional conveyance from the native to a foreign place by currents. In general it may be stated that outside of the Atlantic Ocean all the tropical and subtropical seas are inhabited by the repre- sentatives of Hydrophiine. About 50 species are known. Genus DISTIRA Lacépéde. (Plate 1, D, E.) Poison fangs are large and followed by 4 to 10 small grooved teeth. Head is larger than Hydrophis. Body more or less flattened. Scales imbricated on ante- rior part of body, less distinct on ventral side, always small. The catalogue of the British Museum enumerates 18 species distributed in the Indian and Pacific Oceans, including from the Persian Gulf to Japan and around New Caledonia. Distira ornata. Uniform dorsal color gray; belly whitish. Length 3 to 4 feet. Persian Gulf, India, Ceylon, and Malay Archipelago and northern part of Australia. MORPHOLOGY OF VENOMOUS SNAKES 27 Distira subcincta. Forty-one dark bands, separated by spaces the width of the bands. The bands stop in the middle of the side of the body; series of small black spots on sides. Length 3.2 feet. Indian Ocean. Distira cyanocincta. (Plate 9, A.) Greenish-olive with quite large black or dark rings on back; the belly has a black longitudinal band. Length 5 feet. Persian Gulf, India, coasts of China and Japan, Papuasia. Distira jerdonii. Olive above with black rings; between the rings black spots. Length 3 feet. Gulf of Bengal, Strait of Malacca, and Borneo. Genus ACALYPTUS Duméril and Bibron. The maxillary bone is longer than ectopterygoids, the head covered with scales behind. Body elongated. Western tropical Pacific Ocean. Acalyptus peronii. Gray or pale olive above, with white belly and dark bands. Length 3 feet. Hong Kong, western Pacific tropical zone. Genus HYDROPHIS Daudin. (Plate 10, A, B.) Maxillary longer than the ectopterygoid, not extending forward as far as the palatine; poison fangs large, followed by a series of 7 to 18 solid teeth. Head small; nostrils superior, pierced in a single nasal shield, which is in contact with its fellow; head-shields large; preocular present; loreal usually absent. Body very long, often very slender anteriorly; scales on the anterior part of the body, imbri- cate; ventrals more or less distinct; very small. Indian and Pacific Oceans, from the Persian Gulf to Southern China and north- ern Australia. Hydrophis obscurus s. stricticollis. Dark olive-green with yellow bars which form rings in the compressed posterior part of body; yellow spots on muzzle and yellow bars on each side of head. Length about 3 feet. Gulf of Bengal; Malay Archipelago. Hydrophis spiralis. Olive above, yellowish beneath, with black rings; head black, with a horseshoe- shaped yellow spot whose convexity rests upon prefrontal scales. Length 1.5 feet. Hydrophis czrulescens. Color gray with coarse black bands, forming complete rings or interrupted annules on ventral surface; head uniformly black. Length about 2 feet. Coast of Bombay, Gulf of Bengal, Strait of Malacca. Hydrophis nigrocinctus. Pale olive above, yellowish on belly, with black rings which are larger beneath. Length 3 feet. Gulf of Bengal, Strait of Malacca. Hydrophis elegans. Pale yellow above, with black rhomboid cross-speckles separated by a black spot; black spots on belly; head covered with black, crossed by a light line from nose and eyes. Length about 2.5 feet. From Malay Islands to northern Australia. Hydrophis gracilis. Olive or bluish back with clear crossbands anteriorly, and rhomboid speckles reaching the belly or interrupted on the sides. Length 3 feet. Persian coasts, India, Ceylon, Malay Archipelago. 28 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Hydrophis cantoris. Dark olive body, with yellowish bands above; posterior part of body olive above, yellow on sides; tail with vertical olive bands; blackish line throughout wholelength of belly. Length about 3.5 feet. Gulf of Bengal. Hydrophis fasciatus. Head and neck black; yellow crossbands on neck, body pale with black rings, which are wider near the back. Length 3.2 feet. Coasts of India, Cochin China, and Malay Archipelago. Hydrophis leptodira. Black with yellow streaks on neck, yellow rings on body. The streaks and rings number 77. Length about 1.6 feet. Delta of Ganges. Genus ENHYDRIWA Gray. Two vigorous poison fangs are accompanied by 4 solid teeth. Body moder- ately compressed. Scales imbricated and distinct, but very small on belly. Color olive or gray with black crossbands; sides of belly white. From the Persian Gulf to New Guinea. Enhydrina valakadien s. bengalensis. (Plate 9, B.) Length about 3.5 feet. The male has stronger scales, keeled or tubercular. Gulf of Persia, coasts of India, of Cochin China, Malay Archipelago and Papuasia. Genus HYDRELAPS Boulenger. Muzzle short, scales of head large. Poison fangs are followed by 6 rear teeth. Body slightly compressed. Scales imbricated; ventral scales small, but well marked. North coast of Australia. Hydrelaps darwiniensis. Black and yellowish white rings, narrower near the belly; head dark olive with black spots. Length 1.5 feet. Northern Australia. Genus HYDRUS Schneider. The maxillary bone of this genus is longer than the ectopterygoid and situated somewhat behind the palatine bone. The poison fangs are cannulated and rather short, followed by 7 or 8 solid teeth with a wide interspace between the fangs and the latter. Snout long. Body rather short; scales hexagonal or squarish, juxta- posed; no distinct ventrals. Indian and Pacific Oceans. Hydrus platurus s. Pelamis bicolor Daudin. (Plate 9, c; plate rz, A, B, C.) Anguis platurus Linneus, Syst. Nat., 12 ed., I, 39r. Hydrophis platurus Latreille, Nat. Hist. Rept., IV, 1802, 197. Hydrus bicolor Schneider, Hist. Amph., I, 242. Color black and yellow, with crossbands bordered with black. Length 2.5 feet. Indian Ocean, tropical and subtropical ‘Pacific. Genus THALASSOPHIS Schmidt. Poison fangs followed by 5 small teeth. Snout short. Body rather elongate. Scales hexagonal and juxtaposed, ventral scales indistinct. Coast of Java. Thalassophis anomalus. Body with black bands, which are wider near back. Length about 3 feet. Java. Genus ENHYDRIS Merrem. Not Enhydris Latreille = Hypsirhina Wagler. Enhydris Merrem = Lapemis Gray, Ill. Ind. Zool II. Lapemis hardwickii, type, Stejneger. Two poison fangs and 2 to 4 slightly grooved small teeth; body short and thick; the scales, which are hexagonal or squarish, have almost completely dis- appeared from the belly. Coast of India to the Chinese Sea and New Guinea. peta a i eee Es MORPHOLOGY OF VENOMOUS SNAKES 29 Enhydris‘curtus. (Plate!1o, C.) Dark crossbands, broader at middle of body; tail black. Length 2.5 feet. Coasts of India and Ceylon. Another species of this genus is Eniydris hardwickii Gray. Genus PLATURUS Latreille. Laticauda Laurenti, Syn. Rept., 109. Two large poison fangs and one or two small solid teeth near end of maxilla. Body very fringed. Scales united and imbricated on body, large on belly and tail. Four species along the eastern parts of Indian Ocean and west Pacific. Platurus laticaudatus s. fischeri. (Plate 10, D.) Olive with yellow belly; 29 to 48 black rings. Length 3.5 feet. Platurus colubrinus. (Plate 11, D, E.) Olive color with 28 to 54 black rings. Length 3.5 to 4 feet. Platurus muelleri. Sixty-two rings. Only in subtropical central Pacific to New Hebrides and Tasmania. Genus AIPYSURUS Lacépéde. Aipysurus Lacépéde = Emydocephalus Krefit, type annulatus, Proc. Z. S. London, 1869, 321. Maxillary slightly longer than pterygoids; the poison fangs and 8 to to hollow teeth are separated by a short interval; the anterior mandibular teeth slightly longer. Body moderate, scales imbricated, ventrals large, and keeled at middle. Aipysurus australis. Brown or cream color, with brown spots forming more or less distinct crossbars. Length about 3.5 feet. New Guinea and Australia. The species: Aipysurus eydouxii, annulatus, and levis‘ may be found along the coasts of Singapore, of Java, of the Philippines, and of Loyalty Islands. Family VIPERIDZ Boulenger.? The maxillaries are very short, movably attached to the prefrontals and ectoptery- goids, so that they can be erected together with the large poison fangs, which with the reserve teeth are the only maxillary teeth. The prefrontals are not in contact with the nasals. The squamosals are very loosely attached. The poison fangs are perforated, having a wide hole on the anterior side of the base, in connection with the large venom gland; the hole leads into a canal which opens gradually as a semi- canal on the anterior surface of the distal third or quarter of the fang. As usual in poisonous snakes, several reserve fangs are stored away behind the acting fang. When the latter is broken off or has served its time it is cast off at the base, and the next reserve tooth takes its place. The supply of reserve fangs is indefinite, half-developed teeth down to mere germs constantly growing. All of the Viperide are very poisonous, and all of them, except the African Atractaspis, are viviparous. They include the terrestrial, arboreal, semiaquatic, and burrowing types. The family is found in every country except Madagascar and Australia. 1 This is Emydocephalus annulatus Krefit. Stejneger describes Emydocephalus ijime, which Boulenger thinks to be identical with Krefit’s Emydocephalus annulatus. See Stejneger, Herpetology of apan, 1907. f a Includes Gobrida and Crotalidz of Stejneger and Atractaspidide, Causide, Viperide, and Crotalide of Cope. 30 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Subfamily VIPERINZ Boulenger. Stejneger’s Cobridz. Cope’s Atractaspidide, Causide, and Viperide together. There is no fossa in the external side of the maxillary bone, between the eye and the nose, as in Crotaline. The vipers are entirely restricted to the Old World, ranging over the whole of Europe, Africa, and Asia, except Madagascar. Their northern extension is limited only by the permanently frozen condition of the ground. Nine genera with about 4o species are known. The head and neck are distinct, covered with small scales, with or without frontal plates and parietals. Eyes small with vertical pupils. Body cylindrical. The scales keeled, with apical fossa, in from 1g to 31 rows. ‘Tail short, subcaudals in two rows. Genus VIPERA Laurenti. Vipera berus s. Pelias berus. (Plate 12, A.) This is the common European viper. The upper jaw not turned at the end; scales of body form 21 rows. ‘The coloration is very variable. Usually the gray, yellowish-olive, brown or red ground-color is set off by a dark zigzag band along the back. The belly is gray, brown, or black, or speckled. The end of the tail is usually yellow or red. Some males are black, through extension of the black mark- ing or through darkening of the ground-color. Males usually are darker and deeper in black markings, with the ground-color lighter, and are mostly somewhat smaller than the females. The viper prefers heaths, moors, and mixed woods with sunny slopes. Brambles, clumps of nettles, hedges, the edge of small copses, and heaps of stones, are favorite places of retreat, affording shelters, and being also the resorts of mice, which form its chief sustenance. At harvest time it is often found in cornfields, and it frequently hides in the sheaves of grain. Vipers are fond of basking on certain spots: on the top of stones, the stump of a tree or a strip of sand; a shower of rain or even passing clouds driving them back into their holes. They are nocturnal and a fire attracts them. ‘They can not climb, and, if they can avoid it, do not go into water. They hibernate for about six months, and a number in the same place. This species reaches a length of about 2 feet. It has a wide range, from the British Isles to Saghalien Island and from Caithness to the north of Spain, and the intermediary countries and districts. It ascends the Alps to an altitude of 6,000 feet. In captivity it refuses to eat. Its food consists of small birds, frogs, lizards, and sometimes fishes. Its bite is sometimes fatal to human beings, and especially to children, but is not often so. Vipera aspis. The asp of the Mediterranean is a more southern and western European viper. The muzzle is slightly turned upwards. The top of the head is usually covered with small scales. The coloration is very variable: gray, yellowish, brown, or red above with zigzag band, usually a U mark on the occiput, and longitudinal black bands from back of eyes; belly yellow, white, gray, or black, with a somewhat lighter space. Length 2 to 2.5 feet. France, southern parts of England, Pyrenees, Alsace-Lorraine, Black Forest, Switzerland, Italy and Sicily, Tyrol. Vipera ursinii. Color yellow or pale brown above, gray or dark brown on the sides, sometimes uniform brown, with more or less regular oval, elliptical, or rhomboid speckles; white stripe along vertebral column; two or three longitudinal series of speckles, dark brown or black on sides; chin and throat yellow; belly black with grayish or white cross series of spots. No sexual difference in coloration. Length about 1.3 to 1.6 feet. Southeastern parts of France, Italy, Istria, Bosnian mountains, plains of lower Austria, Hungary. MORPHOLOGY OF VENOMOUS SNAKES 31 Vipera latastii. Gray or brown above, with a longitudinal zigzag band, usually speckled with white; head with or without speckle on top; black stripe behind eye; belly gray, spotted with black and white; tail yellow with yellow eee Length about 1.6 to 1.8 feet. Spain and Portugal, North Africa. Vipera ammodytes. The upper jaw turns up into a horny appendix. Color gray, brown, or reddish above, with a zigzag dorsal band, ordinarily spotted with white; black stripe from rear of eye; belly gray or violet; end of tail yellow, orange, or coral red. Length about 1.6 to 1.8 feet. Tyrol, Carinthia, Styria, Hungary, Danubian districts, Turkey. Not farther than 48° north latitude. North Africa. Vipera russellii. (Plate r2, D.) Known best as the ‘“‘ Daboia’’; another synonym is Vipera elegans. The scales form about 30 rows on back. Top of head covered with small, imbricating, usually keeled scales. General color pale brown above, with 3 longitudinal series of black, light-edged rings, which sometimes encircle reddish spots; belly yellowish-white, uniform, or with small crescent-shaped black spots. Length up to 5 feet.! Hin- dustan Peninsula, Bombay, Bengal, Ceylon, Burma, and Siam. The species ascends the Himalayas to an altitude of 5,000 feet. Its food consists of small verte- brates, frogs, mice, rats, and birds. It invades the inhabited house to hunt the rat. Its poison is extremely powerful. Vipera superciliaris. Snout round. Body is covered with 27 rows of strongly keeled scales. Colora- tion pale reddish-brown or orange, with blackish crossbands which are intersected by a yellowish longitudinal band on each side; belly white with black speckles. Length 1.8 to 2.5 feet. Mozambique coasts. Vipera lebetina. Upper jaw obtuse and rounded off with marked prominence. Coloration very variable, gray or pale brown above, with a series of large dark speckles; a large — of brown is found on the top of the head; belly is whitish and dotted with brownish-gray; end of tail yellow. Length 3 feet, but the female may attain 3.5 feet. Cyprus, Galicia, Asia Minor, Persia, Beluchistan, Morocco, and northern India. Vipera renardii. Resembles Vipera berus, but with the upper jaw more pointed. ‘The coloration is like Vipera ursinii of Europe, with slight variation. Length about 2 feet. Cen- tral Asia, Turkestan. Vipera raddii. Coloration pale brown or gray, with a series of small reddish dots in pairs along the back; A on head and black band behind the eye; belly yellow, punctulated with black and white. Length 3.5 feet. Armenia. Genus CAUSUS Wagler. Head distinct from neck; eyes moderate, round pupils. Body cylindrical, scales smooth or keeled, oblique on the sides; ventrals rounded off. Tail short, subcaudals in one or two rows. Four species in this genus. Causus rhombeatus. Snout obtuse, slightly prominent; the scales in 17 to 21 rows. Coloration olive or brown, often with a series of v-shaped brownish speckles, which are rimmed t According to Boulenger it can attain a length of over 6 feet. 32 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS with a white edge, and a large A-shaped mark on the back of head; lips bordered black; belly yellowish or gray. Length 2 to 2.5 feet. Tropical and central Africa, Gambia, Cape. Causus resimus. Snout prominent and more or less turned upward. Color olive-gray above, uniformly white on belly. Length 1.5 feet. Central and eastern Africa, Angola. Causus defilipii. Snout prominent, more or less turned upward. Color gray or pale brown above, with a series of v-shaped, brownish-black, rhomboid speckles; large A-shaped speckle on rear of head; dark oblique line from rear of eye; belly yellowish. Length about 1.2 feet. Central and eastern Africa, Transvaal. Causus lichtensteinii. Snout obtuse; scales in 15 rows, the subcaudals in single row. Color dark gray with indistinct marks. Western Africa, Gold Coast, Congo. Genus BITIS Gray. Head very distinct from neck, covered with imbricate small scales; eyes rather small, with vertical pupils. Nostrils directed upward or upward and outward, pierced in a single or divided nasal, with a deep pit or pocket above, closed by a valvular, crescentic supranasal. The postfrontal bone is much larger than that in Vipera. Scales keeled, in 29 to 41 rows; subcaudals in two rows. ‘Tail very short. Bitis arietans. (Plate 13, D, E; plate 14, C.) The “puff adder” has the nostrils on the upper surface of snout. Body very thick, tail short. Head large and triangular. Color yellow or orange with chevron- shaped, large, oblique, black crossbars and an oblique band from rear of eye; belly either all yellow or speckled with small black points. It is hard to see this viper when it is lying on sand, or stony ground; the under parts are sometimes whitish. It is very slow, and trusts to not being discovered when lying in the dry grass; when approached it inflates the body and hisses loudly with a puffing sound, watching the enemy with raised and characteristically bent head and neck; but it bites only when actually touched or attacked. The effect of the bite is very dan- gerous.” Its prey consists chiefly of small mammals, which it hunts during the night. Length about 4 to 5 feet. The whole of Africa, excepting northern coasts, from southern Morocco, Kordofan, and Somaliland to the Cape of Good Hope, and southern Arabia. Bitis peringueyi. Color olive-gray, with 3 longitudinal series of blackish or gray speckles. Length about 1 foot. Angola and Damaraland. Bitis atropos. Color brown or brownish-gray, with 4 longitudinal dark series of black and white dots; belly gray or brown. Length up to 1.5 feet. Cape of Good Hope. Bitis inornata. Eyes smaller than in atropos. Length up to 1.5 feet. Cape of Good Hope. Bitis cornuta. Nostrils high and outward. Head covered with small imbricated scales, strongly keeled; 2 to 3 scales alongside the eyes become so elevated as to appear like horns at upper inner corner of eye on each side. Color gray or brownish-red with black 1 Echidna. 2 The natives of southern Africa claim that this viper can jump so high as to reach a man on horse- back. The Hottentots hunt it to get its head, which is used by them to prepare a poison for arrows, by mixing the pulp of the crushed head and a certain plant juice. Noguchi Plate 9 A, Distira cyanocincta. B, Enhydrina valakadien. C, Hydrus platurus. (Photographic Reproductions from Fayrer’s Thanatophidia.) 4 « : a slit i On a a 7 vk Baa e Pa a a Plate 10 Noguchi atus. D, Platurus laticaud Thanatophidia.) C, Enhydris curtus. Two species of Hydrophis. I A and roductions from Fayrer’s \ep 2 graphic | (Photo Noguchi Plate 1] LQ ? : oS ee ‘ d » A A, B, and C, Skull of Hydrus platurus. D and E, Skull of Platurus colubrinus, (Drawings from Boulenger’s Book. Daron ; J ely er Siete ph Noguchi Plate 12 A, Vipera berus. B, Cerastes cornutus. C, Cerastes vipera. D, Vipera russellii. (Photographs from Live Specimens.) MORPHOLOGY OF VENOMOUS SNAKES 33 spots rimmed with white in 3 or 4 longitudinal series; belly yellow or brown, uni- form or speckled. Length about 2 feet. Cape, Namaqualand, Damaraland. Bitis caudalis. The prominent keeled scales near the eyes take the form of a single horn. Color reddish or grayish-red, with 2 brownish series of spots; belly yellow, uniform or black-spotted on sides. Length about 1.5 feet. South Africa, Angola to Nama- qualand. Bitis gabonica. ‘‘ Rhinoceros Viper,” or “‘ Gabonian Viper.” Nostrils directed upward and outward. Between the eyes the inter-supranasal scales are so prolonged as to form a pair of horns, which are erect and triangular or sometimes even of a tricuspid form. This viper may grow about 4 feet long and is vicious. Color brown, with a single longitudinal vertebral series of black speckles; belly yellow with many small brown or black dots. Head very large and triangular. Body very stout with a small tail ending abruptly in a point. Nocturnal, and dur- ing the day appears to be very lazy and inactive. Its venom is very powerful, but it bites only when molested. Bitis nasicornis. The nostrils directed upward and outward. Supranasals form two triangular, erect horns of 3 or 4 keeled scales. Between the horns are small scales separating them. Color purple or red-brown above, with pale-olive or dark-brown speckles; the vertebral series is of brown spots rimmed white and each speckle assumes rhomboid shape; belly olive with black or yellow spots. Length about 4 feet. Western Africa, from Liberia to the Gaboon. Genus PSEUDOCERASTES Boulenger. This genus is represented by only one species, P. persicus of Persia. Pseudocerastes persicus. Head very distinct from neck, covered with small imbricate scales. Eyes small, vertical pupils. Upper jaw very short, and rounded off. Coloration gray or brown, with four series of large black speckles; the head carries two longitudinal black streaks behind the eyes; belly is whitish, spotted with black. Length 3 feet. Genus CERASTES Wagler. Head very distinct from neck, covered with scales, small, juxtaposed, and lightly imbricate. Eyes small, vertical pupils. Body cylindrical with imbricate scales with apical pits, in 23 to 25 rows. Tail short; subcaudals in two rows. Cerastes cornutus.! “‘ Horned Viper.” (Plate 12, B.) Upper jaw short and large. Two erect horns above the eyes. Color brown- yellowish or gray, with or without 4 to 6 series of brown speckles. Black bands run obliquely behind the eyes; belly white; end of tail black or white. Length 2.5 feet. ‘North of Sahara, Egypt, Nubia, Arabia, and central Palestine; Asiatic side of Suez Canal. Cerastes vipera. (Plate r2, Cc.) Snout very short and broad. No “horns.” Color yellowish, pale brown or reddish, with or without black speckles; end of tail often black above and white beneath. Length 1 foot. North of Sahara, from Algeria to Egypt. Genus ECHIS Merrem. Head very distinct from neck, covered with small imbricate scales; eye mod- erate, with vertical pupil; nostrils directed upward and outward, in a single or 1 The ‘‘ Horned Viper,” or C. vipera, is supposed to be the species which has become famous through the suicide of Cleopatra. In the daytime the horned viper is invisible, being buried in the sand with only the eyes, nostrils, and horns appearing above the surface. ‘This attracts small birds, which mistake the horns for insects and, approaching the viper, are themselves caught. 34 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS divided nasal. Body cylindrical; scales keeled, with apical pits, in 27 to 37 rows, dorsal scales forming straight longitudinal series; ventrals rounded. ‘Tail short; subcaudals single. Africa, north of the Equator, southern Asia. Echis carinatus. (Plate 14, B.) ‘“‘Phoorsa’’?* is characterized with a single row of subcaudal scales. It is fero- cious and aggressive, ready to strike at any time. The body is of more or less dark grayish color and is patterned with stripes and with speckles and dots of blackish- brown; the back is marked with long whitish-yellow undulatory lines, on each side of the vertebral line; the head has y-shaped speckles. The scales produce a peculiar noise, when the snake rubs them together by folding the body. Length reaches 2 feet. India, Persia, Beluchistan, Arabia, Palestine, and Africa, north of Equator. Echis coloratus. The scales on the muzzle are convex and smooth or slightly keeled. No Y mark on head. Length 2.5 feet. Arabia, Socotra, Palestine. Genus ATHERIS Cope. Head very distinct from neck, covered with imbricate scales. Eyes large, vertical pupils. Body slightly compressed. Scales keeled, with apical fossa. Tail moderate and prehensile, subcaudals in one row. Arboreal habits. Tropical Africa. Atheris chlorechis. Color greenish with small yellow dots, end of tail yellow. Length about 1.5 feet. Western Africa, from Liberia to Ogowe. Atheris squamiger. Color olive, with yellow, narrow, more or less regular Y bands, or with green spots; belly dark olive with black spots. Length about 1.8 feet. Western Africa, from Cameroon to Angola. Atheris ceratophorus. Many erectile superciliary scales. Body highly keeled. Dark olive color, with cross-shaped black spots; belly pale olive. Length only 7 inches. Western Africa. Genus ATRACTASPIS Smith.? (Plate 13, A, B, C.) Enormously developed poison fangs. A few teeth on palatine, but none on pterygoid bones characterize this genus. The mandible edentulous, with 2 or 3 small teeth in middle of dentary bone. Head small and indistinct from neck. Eyes minute, round pupil. No postfrontal bone. Body cylindrical; scales smooth, in 17 to 37 rows. Ventrals rounded off. Tail short, subcaudals in one or two rows. General coloration brown, brownish-black, and black. This genus is remarkable as presenting the most extreme specialization in the viperine direction, the poison fangs being as large in proportion as in any other form and the solid teeth on the palate and mandible, which are much reduced in number in many of the Crota- lines, having almost disappeared. The habitats of the several species of Atractaspis and the variations in length are shown in the following list: A. hildebrandii, length 1.5 feet; eastern Africa. A. congica, length 1.5 feet; Congo, Angola. A. irregularis, length 1.66 feet; central and western Africa from ~Gold Coast to Congo. A. corpulenta, western Africa, from Liberia to Gaboon. A. rostrata, length 2 feet; central and eastern Africa. 1‘*Phoorsa” of the Hindoos; Efa or ‘‘Pyramid viper” of the Egyptians. 2'This genus is given family rank in Cope’s system as Atractaspidide. MORPHOLOGY OF VENOMOUS SNAKES 35 . bibronii, length 2 feet; east of Cape Colony, Natal, Namaqualand, Angola. . aterrima, length about 2 feet; central and western Africa. . dahomeyensis, length about 1.5 feet; Dahomey. . micropholis, length about 1.2 feet; Cape Verde. . leucomelas, length about 2 feet; Somaliland. . microlepidota, length about 1.75 feet; western and central Africa. BRR Rw DB Subfamily CROTALINZ Boulenger. The essential difference of this subfamily from the Viperine is the presence of a deep cavity or pit between the eye and the nose, lodged in the hollowed-out maxil- lary bone. This pit is lined with a modified continuation of the epidermis, and is amply supplied with branches from the trigeminal nerve. It is undoubtedly sensory, but we do not know its function.t All snakes belonging to this group are called “ Pit-vipers.” The maxillary bone, into the lower end of which the large hollow fang is immoy- ably fastened like a knife in a handle, is extremely shortened and higher than long, so as to appear to be in a vertical position. On the other side of this bone there is the deep cavity which separates two articular surfaces. The upper surface of the maxillary forms with the corresponding concave face of the prefrontal (lachrymal) bone, which projects from and articulates with the frontal bone, a hinge-like joint allowing considerable freedom of motion. The lower articular surface receives the flattened anterior end of the external pterygoid bone (transversum). If the ectopterygoid be moved forward or backward, the maxillary hinges on the pretrontal, and if the same is pushed forward the fang is erected. The Crotaline are divided into two groups according to whether the eke has the ‘“‘rattle” or not. The rattleless snakes fall into two genera, Ancistrodon and Lachesis; the rattlesnakes are divided into Sistrurus and Crotalus. About 60 species are known. The rattlesnakes are restricted to America, but the rattleless Crotaline snakes are found in North and South America and the southeastern half of Asia. No pit- viper is found in Africa, Australia, the southwestern corner of Asia, and Europe, except one species which enters the extreme southeastern corner. Genus ANCISTRODON Beauvois. Originally transliterated as Agkistrodon, dyx.oTpov, hook, 65wv, tooth. Without a rattle. Sensory pits between nose and eye. Surface of head covered with g large shields, sometimes broken into small scales. Body cylindrical, covered with smooth or keeled scales which have special fossa. ‘Tail moderate or short, subcaudal scales in one or two rows. From northern borders of the Caspian Sea, throughout most of the Asiatic mainland and in North and Central America. In the Asiatic mainland to Himalaya in the south and to Lake Baikal in the north. Japan (Formosa and adjacent islands) has some representatives. Ancistrodon piscivorus Lacépéde. (Plate 15, B; plate 16, A.) Trigonocephalus piscivorus Holbrook. Crotalus piscivorus Lacépéde. Cenchris piscivorus Gray. Toxiophis piscivorus Baird and Girard. Coluber aquaticus Shaw. The water-moccasin or ‘“‘cotton mouth” has a round muzzle. Scales of body keeled, in 25 rows, subcaudals in one row, ending in two rows towards the point. General color dark chestnut-brown, with darker markings. Head above purplish- black. On each side a series of 20 or 30 narrow, vertical, purplish-black bars 1 or 1 A good anatomical account by West, Trans. Linn. Soc., XXVIII. 36 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS 2 scales wide. Of these, sometimes two contiguous to each other form a space darker than the ground-color. Sometimes corresponding bars, from opposite sides, unite and form a ring encircling the body. Sometimes there is a lighter shade bor- dering the dark bars. Beneath, black, blotched with yellowish-white. No loreal plate. The water-moccasin is a water-snake and is found about damp, swampy places or in water, but never far from it. In summer, numbers of these snakes are seen resting on the low branches of such trees as overhang the water, and they plunge into the water at the slightest alarm. ‘They are also found in the ditches of rice fields, especially on the dry bank, where they bask in the sun. It is said that the water-moccasin attacks everything that comes within his reach, erecting his head and opening his mouth for some seconds before he bites, which habits are not observed with other pit-vipers. This snake is very much dreaded, although records of fatal issues are not numerous. Unlike the other pit-vipers the water- moccasin is easily kept in captivity, as it takes food in the cage. Effelds once had a pair so tame as to take fish, cold or warm-blooded animals, or even raw meat, from the forceps in the hand of the keeper. It is said that the bite of the water- moccasin, curiously enough, is more dangerous to other venomous snakes than to itself, and that this snake is very fierce towards other snakes. Length 4 to 5 feet. Eastern states of North America, including North Carolina, Indiana, Florida, and ‘Texas. Ancistrodon contortrix. (Plate 15, c; plate 16, B.) The ‘‘copperhead” is more slender than the water-moccasin. A distinct loreal plate is present. Above light hazel-brown, rather brighter on top of head, and everywhere minutely marked with fine dark points. On each side is a series of 15 to 26 darker chestnut-colored blotches resting on the abdominal scutelle, and suddenly contracting about the middle of the side so as somewhat to resemble an inverted y. These blotches extend to the vertebral line, where they may be trun- cated or end in a rounded apex. Generally those of the opposite sides alternate with each other, but frequently they are confluent and form continuous bands. Color beneath dull yellowish, with a series of distinct large, dark blotches, 35 to 45 in number, on each side; chin and throat unspotted; side of head cream color; labials yellowish-white. Length about 3 feet. The copperhead is called “upland moccasin,” “chunk head,” “pilot snake,” or “deaf adder,” according to different localities, and is much dreaded on account of the absence of warning before the bite and of its aggressive nature. However, only very few cases of fatal poisoning have been recorded. ‘These snakes are easily kept and fed in captivity. Like all Crotaline snakes the copperhead produces living young, 7 or g in number. North America. From central Massachusetts to Texas, and from Florida to Kansas, including Illinois, Louisiana, Indiana, and Mississippi. Michigan, Wis- consin, and Nebraska are said not to be inhabited by this species. Ancistrodon bilineatus. Snout pointed; scales less carinated. Subcaudals single in front, divided in posterior. ‘This ‘‘ Mexican moccasin”’ has a general coloration similar to but deeper than that of the other two species, of which the copperhead is the fairest and most vivid. Length about 3 feet. Mexico, Guatemala, and Honduras. Ancistrodon halys. Snout somewhat upwardly turned, with a rounded end. Coloration yellowish- gray, red, or pale brown above, with dark crossbars; belly whitish, more or less marked with gray or brown. Length 1.5 feet. Border of Caspian Sea, Ural moun- tains, and Yenisei Highland, Turkestan. Noguchi Plate 13 A, B, and C, Skull of Atractaspis atterima. D and E, Skull of Bitis arietans. | F, Skull of Crotalus horridus. (Drawings from Boulenger’s Book.) Noguchi Plate 14 A, Ancistrodon hypnale. B, Echis carinatus. C, Bitis arietans. (Photographic Reproductions: A and B, from Fayrer’s Thanatophidia; C, from Live Specimen.) hans? ie ) pee eh . sai a i oy Wy i ‘ Th ean fu , * 1 i { ‘ate ‘ hr A : ; oe 1 o ar - ” pipe Te vo Dae ' r nt j { s i Salta f t Pa af f : i \ * 4 F : | » es | Noguchi Plate 15 EUS : n > SY hi Photographs of: A, Skull of Lachesis mutus; B, Skull of Ancistrodon piscivorus ; C, Skull of Ancistrodon contortrix; D, Skull of Crotalus adamanteus ; E, Rattler of Crotalus adamanteus. Plate 16 A, Ancistrodon piscivorus. B, Ancistrodon contortnx. (Photographs from Live Specimens.) MORPHOLOGY OF VENOMOUS SNAKES 37 Ancistrodon intermedius. Ancistrodon blomhoffii intermedius Stejneger. About same as halys except that the snout is not turned at the end. Length about 2.5 feet. Central Asia, eastern Siberia, Mongolia, and Japan. Ancistrodon blomhoffii.! Resembles the /alys, but not turned up on end of snout. Coloration variable: gray, brown, or red above, with large black-rimmed blotches in pairs; black bands with red margin; belly yellow or reddish, more or less spotted black. Length about 2.5 feet. Japan, Mongolia, eastern Siberia, central Asia. Ancistrodon blomhoffii brevicaudus 2 Stejneger. Much like Ancistrodon blomhoffit of Japan, but is distinguished from it by ab- sence of white spot at anterior angle of crescentic lower postocular, and by the fewer number of subcaudals. Length about 2 feet. Eastern China, Korea, and Formosa. Ancistrodon himalayanus. Snout hard, markedly turned upwards. Color brown with black crossbars or points; a light-margined black band from eye to the angle of mouth; belly dark brown or more or less whitish. This snake lives chiefly on mice. Length about 2 feet. Himalayas up to 10,000 feet, especially in the northwest. Ancistrodon acutus. The snout is so prolonged as to protrude. Length about 5 feet. Upper Yang- tse, China. Ancistrodon rhodostoma. Snout pointed, sometimes directed upwardly. Length about 3 feet. Java. Ancistrodon hypnale. (Plate 14, A.) Snout more or less curved, with a hard-pointed end. Scales in 17 (most of the Ancistrodon species 21) rows. Length about 2 feet. It is known as “carawalla” in Ceylon, where it is dreaded. Ceylon, western coasts of India to Bombay. Genus LACHESIS Daudin.? (Plate r5, A.) Without a rattle, but the tail has a series of 10 to 12 rows of spinous scales sharp- ened at end. Head covered with plates or small smooth or carinated scales, with or without special fossa. Maxillary bone much reduced in dimensions, but ectoptery- goid is well developed. The absence of the regular shields on top of head makes it easy to differentiate this genus from Ancistrodon. Many species of Lachesis of Asiatic origin are called Trimeresurus. Stejneger points out that it has not been conclusively demonstrated that they are generically identical with the numer- ous American pit-vipers of a similar head scutellation, which are usually known as Trigonocephalus or Bothrops. The South American Lachesis is_ sufficiently characterized by the peculiar scutellation of the tail. The American representatives of Lachesis are as follows: Lachesis lanceolatus. Known as Fer de lance of Martinique, “Jararacussu” of Brazil, and Bothrops lanceolatus of various authors. Snout obtuse, slightly elevated; scales of top of head are small and imbricated, more or less strongly carinated. Subcaudal in two rows. Coloration very variable: gray, brown, yellow, olive, or reddish, uniform or 1 oY According to Stejneger this species is restricted to Japan, including the small southern islands. 2 Ancistrodon blomhoffii affinis Gray was described, but its locality is uncertain; it is supposed to come from Yaeyama Island of Riu Kiu, Japan. 3 Bothrops, Trigonocephalus, and Trimeresurus are synonyms for this genus, 38 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS with more or less distinct dark crossbands or speckles; black band from eye to corner of mouth; belly uniformly yellowish or spotted brown. Length about 6 feet. This is one of the most dreaded venomous reptiles in America, and estab- lishes itself everywhere — in swamps, plantations, forests, in the plains and in the hills. During the daytime it hides quietly, but is ready to cause death at any moment. Its motion is exceedingly rapid. It swims easily. The bite of the Fer de lance is extremely dangerous and causes numbers of deaths in Martinique, especially among the sugar-planters, or coffee-cultivators. Tropical America; Mexico, Martinique, St. Lucia, from Bequia Isle towards St. Vincent, Venezuela, Guiana, Rio de Janeiro. Lachesis mutus. “Bushmaster.” (Plate 17, A.) The “Surucucu” of the Brazilians is another large venomous snake of tropical America. Color yellow or rose on back with a series of rhomboid brown speckles or indented black speckles; a black band extends from eyes to corner of mouth. Length about 6 feet. Central America and the tropical zone of South America. Lachesis atrox. Laboria is another name for this snake. Resembles the Fer de lance, but the body is stouter, the head larger, with more vigorous poison fangs. Length of the fang may reach 3 inch. Color brown with crossbands or triangular speckles. Dark band from eye to corner of mouth. Belly whitish-yellow with brown spots. Length about 3.5 feet. Central America; from Peru to the north of Brazil. Lachesis pulcher. Color olive with brownish crossbands with white rim. Belly covered with fine confluent brown speckles. Length 2 feet. The Andes of Ecuador. Lachesis microphthalmus. Snout short and rounded, eyes very small. Length 2 feet. Peru and Ecuador. Lachesis pictus. Snout obliquely truncated. Length 1 foot. Peru. Lachesis alternatus. Head narrow and elongated. Length 3.5 feet. Southern Brazil, Paraguay, Uruguay, Argentine. ~ Lachesis neuwiedii. The Bothrops urutu of the Brazilians has obtuse snout. Scales highly carinated, as in the foregoing species. Color yellowish or pale brown with brown speckles rimmed black; the dorsal speckles form a single or double series alternately; belly more or less powdered with brown. Length 2.5 feet. Brazil, Paraguay, Argentine. Lachesis ammodytoides. Turned-up snout. Color brownish with large white-rimmed black speckles, or zigzag alternative crossbands; belly yellowish with brown speckles. Length 1.6 feet. Northeast of Chile and Argentine. Lachesis xanthogrammus. Length about 5 feet. Long snout. Ecuador and the Andes of Colombia. Lachesis castelnaudii. Length 3.5 feet. Long snout. Brazil, Ecuador, eastern Peru. Lachesis nummifer. Snout large and rounded; subcaudals mostly in one row. Length 2.8 feet. Mexico and Central America. Lachesis godmani. Snout large and rounded; subcaudals in one row. Length 2 feet. Guatemala. MORPHOLOGY OF VENOMOUS SNAKES 39 Lachesis lansbergii. Snout turned up somewhat as in Vipera aspis. Subcaudals in one row. Length 2 feet. Southern Mexico, Colombia, Venezuela, Brazil. Lachesis brachystoma. Snout turned up. Length 1.5 feet. Southern Mexico and Central America. Lachesis bilineatus. Snout rounded; subcaudals for most part in two rows. Tail prehensile. Color greenish, in contrast to most of the enumerated species, which are mostly brownish, grayish, or dark yellowish, with or without black speckles; belly white; end of tail red. Length 2.5 feet. Brazil, Bolivia, Peru, Ecuador. Lachesis undulatus. Snout short and round. Color olive or brown, sometimes with black speckles. Tail prehensile. Length about 2 feet. Mexico. Lachesis lateralis. Snout rounded, subcaudals in one row. ‘Tail prehensile. Color greenish with yellow line on each side. Length 1.6 feet. Costa Rica. Lachesis bicolor. Much like foregoing. Green with yellow belly. Length 1.2 feet. Guatemala. Lachesis schlegelii. Subcaudals in one row. Tail prehensile. Coloration very variable: Green is predominant, with speckles or bands of black, rose, or red; belly yellow, with green or red; end of tail red. Length 2 feet. Central America, Colombia, Ecuador. Lachesis nigroviridis. Subcaudals in one row. Tail prehensile. Color greenish or olive with black speckles; belly yellow; head black. Length 1.6 feet. Costa Rica. Lachesis aurifer. Snout short and rounded; subcaudal in one row. ‘Tail prehensile. Color green with yellow speckles; black band on temple. Belly greenish-yellow. Length about 2.5 feet. Guatemala. The Asiatic representatives of Lachesis are characterized by the shorter tail, which is often prehensile and enables the snake to climb or grasp the trees during the hunt for its prey. The subcaudals are in two rows. On account of the more general characteristics peculiar to this group, these snakes, though undoubtedly belonging to the common genus Lachesis, are divided into two main groups. As was mentioned previously, the term T'rimeresurus is used by Stejneger in lieu of Lachesis, because the character of the tail is considered by him to be sufficient reason to group the Asiatic pit-vipers under the former designation. A. The first supralabial scale is in contact with its neighbor. I. Scales in 21 to 25 (seldom 27) rows; 129 to 158 ventrals; 21 to 27 subcaudals; 5 to 9 series of scales between supraocular plates. Tail non-prehensile. Lachesis monticola. Snout obtuse. Color brown or yellow above, pale yellow or brown on sides, with a brown temporal band; belly white, with brown spots. Length 2.5 feet. Tibet, Himalaya (up to 7,500 feet), Burma, Malay Peninsula, Singapore, Sumatra. Lachesis okinavensis. Trimeresurus okinavensis. End of snout pointed and raised. Color brown above with dark crossbands and a light temporal band; belly brown with black spots, especially along side of body. Length about 1.25 feet. Okinawa, Riu -Kiu Islands, Japan, 40 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Lachesis strigatus. Length 1.5 feet. Vicinity of Bombay, Deccan, Anamallays, and Nilgherries. II. Scales in 27 to 37 rows; 174 to 231 ventrals; 54 to 90 sub- caudals; tail non-prehensile. Lachesis flavoviridis. ‘‘Habu.” (Plate 18, A, B.) Trimeresurus riukiuanus Hilgendorff. Trimeresurus flavoviridis Boulenger; later, Lachesis flavoviridis Boulenger. Bothrops flavoviridis Hallowell. Scales in 33 to 37 rows; 222 to 231 ventrals; 75 to 90 pairs subcaudals; 8 to 9 supralabials. Coloration pale brown or yellowish-green on the back with black marblings; the head has the longitudinal black streaks disposed symmetrically; belly yellow or greenish with darker speckles. Length about 4 feet. This snake is much dreaded by the natives and occasions many fatalities every year. Oshima and Okinawa subgroups of the Riu Kiu Islands, Japan. Lachesis cantoris. Color pale brown to greenish with small black speckles; side whitish; belly white or greenish. Length about 3 feet. Andaman and Nicobar Islands. IIT. Scales in 21 to 27 rows; 160 to 218 ventrals; 54 to g2 sub- caudals; the tail is slightly or not at all prehensile. Lachesis jerdonii. General coloration greenish-yellow with some black markings. Length about 3 feet. Habitat, Assam, Tibet, Yang-tse. Lachesis mucrosquamatus. Trimeresurus mucrosquamatus Giinther. Trigonocephalus mucrosquamatus Cantor. Not Lachesis mucrosquamatus Wall. General coloration brownish or gray, with blackish speckles. Length about 3.25 feet. Formosa. Lachesis luteus. Trimeresurus elegans Gray. Very much like the foregoing species, but this has somewhat keeled temporal scales. It lives in the crevices of stone walls, or in hollow trees, etc. It is more sluggish than the L. flavoviridis. Length about 3 feet. Yaeyama Islands, Riu Kiu, Japan. Lachesis purpureomaculatus. Color, back purplish-black, sometimes variegated with pale green; sides pale green; belly olive or whitish-green, uniform or with black spots. Some specimens are completely green. Length about 3 feet. Himalaya, Burma, Malay Peninsula, Andaman and Nicobar Islands, Pinang, Sumatra, India. IV. Scales in 21 (occasionally 19 or 23) rows; 7 to 13 series of scales between supraoculars; tail more or less prehensile. Lachesis gramineus. “Green pit-viper.”’ (Plate 17, Cc.) Trimeresurus gramineus. Snout more or less prominent. Color bright green, seldom olive or yellowish. with deeper crossbands; end of tail yellow or red; belly green, yellow, or white. Length about 2.8 feet. Southeastern Asia, Darjeeling, Himalaya, delta of Ganges, Siam, South China, Hong Kong, Formosa, Java, Sumatra, Timor. Lachesis flavomaculatus. (Plate r7, B.) Snout is prominent and obliquely truncated. Color bright green or olive, some- times with reddish-brown bars; belly green, olive, or yellowish-green; end of tail usually red. Length about 3.2 feet. The Philippine Islands. Noguchi Plate 17 A, Lachesis mutus. B, Lachesis flavomaculatus. C, Lachesis gramineus. D, Lachesis anamallensis. (Photographic Reproductions: A, from Live Specimen; B, C, and D, from Fayrer’s Thanatophidia.) Plate 18 B Lachesis flavoviridis. Two views are given to show more completely the characteristics of this“ snake. Noguchi Plate 19 A, Sistrurus catenatus. B, Crotalus adamanteus. C, Crotalus horridus. (Photographs from Live Specimens. MORPHOLOGY OF VENOMOUS SNAKES 41 Lachesis sumatranus. Color bright green with or without black crossbands; yellowish band on each side; belly yellow or green with or without black speckles; end of tail red. Length about 3.5 feet. Singapore, Sumatra, Borneo, Palawan. Lachesis anamallensis. (Plate 17, D.) Color green, olive, yellow or reddish-brown; black temporal band; belly pale green; tail usually black or yellow. Length 2.5 feet. Anamallay and Nilgherries, southern India. Lachesis trigonocephalus. Coloration similar to preceding. Length about 2.5 feet. Ceylon. Lachesis macrolepis. Color bright green or olive. Belly pale green. Length about 2 feet. Southern parts of India. B. First infralabial plate is divided; the separated portion forms a pair of small supplementary dental plates; 144 to 176 ventrals; 38 to 57 subcaudals; tail prehensile. Lachesis puniceus. Scales in 21 to 23 rows. General coloration gray, brown, or red; belly spotted brownish; end of tail red. Length 2 feet. Java, Borneo. Lachesis borneensis. Length about 2.2 feet. Borneo, Sumatra. C. Scales in 19 to 27 rows; ventrals 127 to 156, subcaudals 45 to 55. Tail prehensile. Lachesis wagleri. Color green with lighter or deeper black and yellow speckles. Length about 3 feet. Malay Archipelago and Peninsula. Genus SISTRURUS Garman. Head very distinct from neck, covered with 9 large symmetric plates.'| Eyes rather small, with vertical pupils. Body cylindrical; scales carinated, apical fossa. Tail short, terminated by a horny appendix which produces a peculiar sound (the rattle); subcaudals for the most part in one row. Sistrurus miliarius. ‘‘Southern Pigmy Rattlesnake.” A very small species with stout body and distinct, flattened head. Tail thin, minute rattle. Dark ashy gray, with a series of large, black blotches on the back, these irregularly rounded and separated by reddish spaces; on the sides are several series of black spots, smaller and less distinct than those on the back; tail un- usually reddish; belly white, thickly marbled with black spots or blotches. Length about 1.8 feet. Florida, central North Carolina, coastal region along the Gulf of Mexico to Texas. Along the Mississippi valley to Arkansas and central Oklahoma. Sistrurus catenatus. ‘‘ Massasauga.” (Plate 19, A.) Large and comparatively stouter snake than S. miliarius. Tail shorter, rattle better developed. Ground-color grayish-brown, with a series of large, rich-brown blotches on the back, these faintly bordered with white; tail ringed with dark brown; belly dull gray marbled with black or entirely black; throat paler. The maximum number of joints of the rattles examined by Ditmars was eight. Length about 2.5 feet, but reaches 3.5 feet. 1 Crotalus is covered with granular scales. 42 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS It is a snake of swampy localities, and eats cold-blooded animals, unlike Crotalus; in this and in the presence of the large shields on the head the pigmy rattlesnake resembles the copperhead or water-moccasin. It can be kept in captivity, as it readily becomes tame and takes food. Sistrurus catenatus var. edwardii. Coloration much paler and more yellowish than the typical form. Habitat, from Ohio to Nebraska, Minnesota, Wisconsin, and Michigan, and even some districts in Canada and south to Mexican borders. The Edwards massasauga extends farther south than the regular massasauga. Sistrurus ravus. General coloration yellowish-brown with a dorsal series of dark-brownish blotches, and a series of crossbars; belly yellowish with brown spots. Length about 7 inches. Vera Cruz, Mexico. Genus CROTALUS Linnzus. (Plate 15, D, E,) — —— ee 3 This genus is provided with the rattle as in the Sistrurus, but may be distinguished from the latter by the scutellation of the head; this being small and granular. A few varieties may be covered with some enlarged shields in the front of the eye. The genus comprises 15 distinct species and several varieties; 11 of these species and 2 varieties are found in the United States. The rattle consists of a number of thin, but dry and more or less elastic, hollow, horny cones of successive sizes. These horny cones have a stricture in the middle and are divided into an upper smaller cone and a lower larger cone. The base of the lower cone is inwardly turned and its diameter is smaller than that of the smaller upper section of the next proximal cone, which is constructed in a similar manner. Thus the upper globular portion of the horny cone serves to hold the next cone by means of the inwardly turned edge of the latter. Of course the diameter of the base opening of the second cone is longer than that of the strangulated part of the first cone, but shorter than that of the upper globular part of the latter. The articulation of the rattle-cones is very loose and a slight shaking is sufficient to cause the well-known sound. The number of the cones or “buttons” does not agree with the number of years the snake has lived, as is commonly believed, but increases irregularly irre- spective of the age of the snake; it seldom surpasses 18 to 20 cones. Rattlesnakes live in all kinds of ground, but prefer rocky regions, where they have abundant places of concealment. They are not of quick movement and do not bite quickly unless trodden upon or attacked. It is hard to induce them to take food when in captivity. I. A CHaIn oF LARGE, DARK, PALE-BORDERED RHOMBS OR “ DIAMONDS.’’! (2) Diamond markings closed on sides: Dark olive; rhombs with yellow borders. (Southeastern United States) . Crotalus adamanteus+ Grayish; rhombs with whitish borders. (Texas to southern California) .... Crotalus atrox. Reddish; rhombs with whitish borders. (Southern and lower California) Crotalus atrox var. ruber. Dull white or pinkish, with very obscure, rhomb-like markings. (Southern Calitornia, lower California, and(Arizoma)) (.jhc/.6 <0. «ices Bix Alaa > Crotalus mitchelli. (0) Diamond markings narrowly open at sides and continued downward as narrow bands: Yellow or greenish. ‘Two paler blotches within each rhomb. (Arizona, New Mexico;vand! Mexico)ih5 Br acevoiat cee conte iat ales eies eine oe oa Crotalus molossus. 1 The South and Central American species are not in this key. MORPHOLOGY OF VENOMOUS SNAKES 43 II. A Row or ROUNDED, DARK-BORDERED BLOTCHES, WELL SEPARATED. (2) No horn over the eye: A pale band, one scale wide, in front of eye. (Central United States, Canada LONE CEICO) ete a ter itnthe ots eietelaraie al sl> oloiis akei eve) sel sie /elelarsietolelel s\el= ..- Crotalus confluentus. A pale band, two scales wide, in front of eye. (Extreme western United SCALES) Meee rey sree: ora stabs) wacactelt elo hat hel otarnlshetadatetoreloiegetal ole) chet ei sete (or siei(el=.0) sie! Crotalus oregonus. Two rows of blotches on anterior part, fusing into a single row in rear of body. (Arizonavamd IWlexico) iets ce cre soreiete clots ehevetareleale cine lofe e elerel« cleleloe- ete Crotalus pricet. (6) A horn over each eye: Yellowish; square, dull blotches on back and black spots on sides. (Deserts Ob Arizona NE VAG Asta Calif OxMId) ey merieloe ste eres clolele eo ole eletal ats lavas Crotalus cerastes. III. MARKINGS IN THE Form OF DARK, TRANSVERSE BANDS. (a) Bands angular: Bands regular in the rear — sometimes broken into three blotches — the central the largest. (Eastern United States, Vermont to Florida; westward to the POLIS) Ree efarmeree eccrine cues eace ei sintcray ote ce Grctatchahanere oro) stsyetere) cleaners Crotalus horridus . (6) Bands even: Yellowish or gray; three series of blotches on anterior portion of body. On latter two-thirds of body bands closely situated. (Desert mountains of southern Galitonias ArizomasINEVAGd) i: ae acces cies cvs volo a sis Stee aioe Crotalus tigris. Greenish; narrow and regular black bands at a considerable distance apart. (Region of the Mexican boundary, from western Texas to western PATIO) cere Nate, isae Poca ce erale STOTT aR IS ca cesT over vs wiles kone sie erro the Crotalus lepidus. Crotalus adamanteus Beauvois. ‘‘ Diamond-back Rattlesnake.’’ (Plate 10, B.) Crotalus durissus Linneus. This is the largest species of the whole family and grows over 6 feet and even up to 8 feet in some specimens. Body stout and heavy. Head broad, flat, and dis- tinct from the neck. Scales in 25 to 29 rows, the dorsals highly carinated; 169 to 18r ventrals; 24 to 32 subcaudals. The poison fangs are of highest efficiency both in structure and in dimension. Coloration olive or grayish-green, with a chain of large, diamond markings of a darker hue, these with bright yellow borders about the width of a single scale; toward the tail they become obscure and fuse into crossbands; the tail on top is olive, ringed with black; belly dull yellow. Southeastern United States, from North Carolina to Florida and along the mouth of the Mississippi. This reptile is said to be very bold and alert. A diamond rattler seldom glides for cover, if disturbed. Pine swamps and hummock lands are its favorite haunts. It is mostly of a nocturnal nature and hunts its prey after twilight. Wild rabbits, rats, birds, and the like constitute its food. It swims well, but seldom climbs trees. Crotalus horridus Linneus. “ Banded Rattlesnake” or ‘‘ Timber Rattlesnake.” (Plate 13,F; plate 19, C. The general scutellation is similar to the Crotalus adamanteus. The most familiar coloration is that of a sulphur-yellow ground-color, with wide, dark-brown or black crossbands, these usually wavy or sharply pointed in the rear; tail black. Another common phase is olive. On the anterior portion of the body are three series of dark blotches, margined with yellow; these fuse into wavy, yellow-edged crossbands on posterior two-thirds of body; belly uniformly yellow or spotted with black on yellow. Length about 3.5 to 4 feet. Central Vermont to the northern portion of Florida, thence westward to Iowa, Kansas, Indian Territory, eastern Texas. Abundant in the coastal regions of the Atlantic and the Gulf (variety cane-brake rattlesnake). The mountains of south- ern New York, Massachusetts, and eastern Pennsylvania. 44 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS This species lives in mountainous regions where they find many ledges, cleft with many fissures and large shelving rocks. During the hibernating season they gather together, coiling closely, and thus pass the winter. In the spring, during the mating season, they linger on the main ledge in large numbers, but finally scatter to the timber for the warm months. ‘This is the most mild-tempered species and becomes so docile that it can be handled like the harmless snakes. Crotalus atrox Baird and Girard. ‘‘ Western Diamond Rattlesnake.” Next to the diamond-back rattlesnake of the Southeastern States, this species is the largest of the genus. It attains a length of 7 feet. Color-pattern similar to the preceding species, except the tail, which is white with jet-black rings. The ground-color may be yellowish-gray, pale bluish-gray, or pinkish, according to the localities. General color-pattern much duller than the eastern species. Some specimens from the desert region of Arizona are of distinct pinkish ground-color with vividly white-rimmed rhombs. Tail chalky white with jet-black rings. It mainly inhabits the sub-arid and desert regions of Texas and southwestern United States. Common in central and western Texas, southern New Mexico, Arizona, and southern California. In Mexico the coloration and scutellation of the head differs from the original type. This is one of the wildest and most vicious species. Crotalus atrox var. scutulatus. ‘“‘ Mountain Diamond Rattlesnake.” Crotalus scutulatus Boulenger. Irregular plates cover anterior portion of head, resembling Crotalus oregonus. It is, however, maintained to be a subspecies of Crotalus atrox, which in the Mexican Tablelands has undergone variation in some of the typical forms. Length about 3 feet. Arizona, New Mexico, Texas, northern part of Mexico. Crotalus atrox var. ruber Cope. ‘‘ Red Diamond Rattlesnake.” Differs from the typical atvox in having a more reddish hue. Length about 4.5 feet, but can grow up to 5 feet. Arid regions of California, and the peninsula of lower California; southwestern Arizona. Crotalus confluentus Say. ‘Prairie Rattlesnake.” Body not so stout as most rattlesnakes, and the snake seldom reaches a length of 6 feet. Greenish-yellow, or olive, with a row of large, round, and well-separated blotches of brown upon the back. ‘Toward the tail the blotches fade into obscure bands. The head marking differs from the Pacific rattlesnake. In the prairie rattlesnake a dark band starts from beneath center of eye to angle of mouth, while the Pacific rattlesnake has the dark band commencing behind center of eye. The dark band is bordered with yellow strips — the front strip being narrow, the width of one scale row, whereas the pale strip of the Pacific species is of the width of two scale rows. This species is rather vicious and irritable, but becomes very tame in captivity. In the prairie this snake often prowls.into the burrows of prairie dogs, but instinc- tively it seeks the deserted burrows. Sometimes the snake may be hunting in the burrows to devour some of the young. Western parts of North America, from British Columbia (46° north latitude) to the southern parts of California, Dakota, Nebraska, Kansas, southwestern Texas, northern Mexico. Crotalus oregonus Holbrook. “‘ Pacific Rattlesnake.” Crotalus lucifer Baird and Girard. Rather smaller than the prairie rattlesnake, but resembles it in most other char- acters except head markings, of which mention has been made above. Habits similar to the Prairie rattlesnake. Length under 4 feet. The Pacific region, from southern British Columbia to southern California; also in Idaho, Nevada, and Utah. It occurs in altitudes as high as 11,000 feet. MORPHOLOGY OF VENOMOUS SNAKES 45 Crotalus tigris Kennicott. ‘Tiger Rattlesnake.” This snake seldom attains more than 3.5 feet. The general features not essen- tially different from prairie and Pacific rattlesnakes. Yellowish-gray, with a series of small and not very distinct blotches on back and on each side, for the anterior third of body; on the latter two-thirds, these blotches fuse into regular crossbands, producing a strongly barred effect. It becomes docile in captivity. Desert moun- tains of Arizona, Nevada, and southern California. Crotalus molossus Baird and Girard. ‘“ Black-tailed Rattlesnake.” Head large and quite blunt at snout. On the upper part of snout are 3 pairs of enlarged scales. Body fairly stout and attains a length of 3.5 to 5 feet. The uniform jet-black tail distinguishes this snake from the other southwestern species. Its range is not farther north than the central portion of Arizona. Its range into Mexico is not definitely known. Crotalus cerastes Hallowell. ‘‘ Horned Rattlesnake” or ‘‘Side Winder.” One of the smallest species of Crotalus. It has a horn-like projection over each eye. Body stout, with strongly keeled scales. Pale brown, yellow, or pinkish, with a series of dull blotches, generally separated by white interspaces; irregular rows of small black or brown spots on sides; several black bars on tail. Maximum length about 2.5 feet. Desert areas of Arizona, southern Nevada, southwestern Utah, and eastern California. Crotalus lepidus Kennicott. ‘‘ Green Rattlesnake.” The smallest species of the genus. Body quite slender. Greenish-gray or rich, dark green above, crossed at wide intervals by narrow jet-black bands; the bands are usually bordered with pale greenish-yellow; belly pinkish, or yellowish-white Just behind the head is a black blotch forked in the front. Length about 2 feet. This species inhabits mountainous areas, on both sides of the Mexican boundary. Crotalus pricei Van Denburgh. Rare and very small, resembling Sistrurus catenatus in general appearance. Length about 2 feet. Southern Arizona to northern part of Mexico. Crotalus triseriatus. Length about 2 feet. Mexico. Crotalus polystictus. Length about 2 feet. Tableland of Mexico. Crotalus mitchelli Cope. ‘‘ White Rattlesnake.” The head squamation differs from the other rattlesnakes. The large anterior nasal plate is separated from the rostral plate by small scales. With other species of Crotalus the anterior nasal shield is in contact with the rostral plate. Grayish- yellow or pinkish, the body profusely sprinkled with brown dots; upon the back these dots assume the form of a series of blotches, which imparts much the same effect as the pattern of the western diamond rattlesnake. A bright-red specimen has been taken in Canyon Prieto of Arizona and was named Crotalus pyrrha by Cope, but no more of the same variety have since been encountered. Length about 3.5 feet. Desert mountains of lower California, southern California, southern Arizona, and extreme northeastern Mexico. Crotalus terrificus. ‘‘ Dog-faced Rattlesnake.” Length 4 to 4.5 feet. Northern part of Brazil and northern Argentine. CHAPTER IIL. PHYLOGENY OF VENOMOUS SNAKES. The distinction between the non-poisonous and poisonous snakes is the presence in the venomous species of a special poison apparatus, consisting of a poison gland and poison fangs —teeth especially adapted for injecting the venom into the tissue of the victim. The poison gland is in communica- tion with the poison fang through an excretory duct which originates in the former. The poison fang is distinguished from ordinary maxillary teeth by the presence of a longitudinal groove or a canal running along the axis of the fang on the frontal side. The canal ends in a slit-like orifice on the frontal surface near the apex. A mere presence of the poison gland alone does not, however, entitle a snake to the rank of Serpentes venenata, but it requires the simultaneous presence of the poison fang and the poison gland. The composite and complex nature of the poisonous apparatus creates conditions which directly or indirectly influence the degree of danger from a bite of a poisonous snake. ‘The efficiency of a venomous bite depends to a consider- able extent upon the size and position of the poison fang, the nature and amount of the venom, and also the habit of the snake. These factors decide the degree of danger of the poisonous snakes and render it convenient to group them into dangerous and non-dangerous species. The evidence that the poison apparatus is the result of progressive evolu- tion is made clear in the morphology of the maxillary teeth alone. In the innocuous species the maxillary teeth are small, uniform, and solid, and are designated Aglyphodont. ‘The next group of species has one or more grooved teeth in the rear of the maxilla, but no grooves in the anterior teeth. This group has received the name of Opisthoglyph, and must be considered as the primitive form of the venomous snake. The next step of advancement is shown in certain species in which the anterior maxillary tooth or teeth are grooved in varying degree. The groove may be so deep that the edges pro- trude forward to fuse together and inclose a complete canal. In such case the junction line of the edges is easily recognizable. Behind the grooved or canaliculate teeth there may be some small solid teeth in some species. This group is known under the name of Proteroglyph. Here the grooved teeth attain a larger size than the rest of the maxillary teeth. The last and highest stage of evolution of the poison fang is, however, seen in the group called Solenoglyph, in which all maxillary teeth embrace a longitudinal tubular duct. Only in the newborn specimens a faint fusion mark along the frontal median line of the fang is visible, while no trace persists after ma- turity. The size of the fang reaches large proportions, and the fang is erectile through the movable joint of the maxillary bone with the prefrontal bones. A systematic inquiry into the relation between the poison fang and poison gland in their evolutional chronology is not without certain bearing on the question of the origin of poisonous snakes, and on the evolutional relation of 46 PHYLOGENY OF VENOMOUS SNAKES 47 the poisonous to the non-poisonous species. The question may be raised as to whether the poison gland and fang came into existence at the same time, or whether one antedates the other. If not of simultaneous appearance, which of the two came first? Through elaborate work of various investiga- tors it was made clear that the poison gland does exist in the group of snakes which possesses no grooved or perforated teeth, and the presence of the grooved tooth is always associated with a much more developed venom gland than the rudimentary form of the aglyphous species. The poison gland of the snake is a modified form of the glandula labialis superior and in all probability is equivalent to, if not identical with, the parotid of the mammalia. In the Urodelia and the Anura the oral cavity is provided with the mucus- secreting glands which are located in the internasal and lingual regions, and also in the pharyngeal area in the latter order. In the Chelonia the medial and lateral palatine glands take the place of the internasal, and the sub- lingual appears anew. In the Crocodilia no sublingual gland is present. First in the Sauria, comprising both the Lacertia and the Ophidia, the glan- dula labialis, superior and inferior, are added to the sets already mentioned. For the Ophidia no palatine gland is present, but there is sometimes the poison gland. Phylogenetically considered, the salivary glands appear first in the Amphibia, in which the mucus-secreting glands are the only kind. In the Pisces no salivary gland is found. The majority of the Reptilia possess the mucous gland with a fairly high, clear cylindrical epithelium, which contains more or less granules in the protoplasm (Wiedersheim, 1886). In this class, however, not only the number of glands, but also their shape, arrangement and structure, become more abundant and variable, with acquisition of certain new functions at the same time. In certain Sauria, for example, the Lacertia and Anguis fragilis, a serous and a mucous gland are formed in the area of the sublingual gland, a fact which shows that the con- dition is gradually approaching the mammalian class. The poison gland of snakes has occupied the minds of many great anato- mists for solution of its origin and its relation to the salivary glands, both in the same order and in the orders next to it. Comparative studies of Carus (1834), Tiedemann (1810), Meckel (1829), Stannius (1846), Ellenberger and Hoffmeister (1881), and others demonstrated that in the mouth of birds of prey there are at least four to five sets of glands, corresponding to the sub- maxillary, sublingual, lingual, parotid, and a group of glands (follicle) near the side of and behind the posterior nostrils, which, together with a large group around the Eustachian tube, was held for the tonsils by Rapp (1843). Rapp’s interpretation was shown to be incorrect by the work of Kahlbaum (1854), Leydig (1857), Stéhr (1884), Gadow (1891). The peculiarity of the avian mouth is in the presence of a ‘pair of glands over the maxillary joint. Carus (1834) was not able to decide whether it is to be considered an analogue of the parotid of the mammalians or the poison gland of snakes. Ranvier (1884, 1887) held that the cells of the gland erroneously called the submaxil- laris are mucus-secreting, which is not the case with the gland of the labial commissure. Battelli and Giacomini (1889) described two types of cells, 48 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS a typical mucus-epithelium and a granulated cell, but considered them to be identical only in different stages of function. They considered the gland in the corner of the mouth not identical with the parotis of the mammalian, and named it glandula angularis oris. Cholodokowsky (1892, 1893) declared that the salivary glands of the birds are purely mucus-secreting. Wieders- heim (1898) hesitated to conclude the identity of the gland of the mouth corner of birds with the posterior supralabial gland or the poison gland of snakes, while Gadow (1879) recorded the occurrence of the parotids in various species of birds. As mentioned before, snakes possess the supra- labial glands throughout all families. It was also remarked that only certain snakes have well-developed poison glands. The question may be asked whether the poison gland may be phylogenetically related to the parotid of the mammalia or the glandula angularis oris of the birds. What relation has it to the supralabial glands in general? The situation and the serous character of the secretion and the single excretory duct of the poison gland suggest strongly a possible homology with the mammalian parotid. The histological and the anatomical examinations of the supralabial glands of many innocuous snakes brought out certain interesting facts which seem to help to trace back the origin of the poison gland. The accounts of the anatomical studies of various glands of the oral cavity by the early investigators are mostly macro- scopic. Meckel (1826) stated that the non-poisonous snakes possess far larger salivary glands than the poisonous ones. A similar finding is in Stannius’s work (1846). The supralabial gland (glandula labialis supe- rior, Leydig; glandula maxillaris superior, Oppel) has been exhaustively examined by Tiedemann (1813), Meckel (1826), Cuvier (1810), Cloquet (1821), Dugés (1827), Duvernoy (1832), Schlegel (1837), and Leydig (1873). Leydig made the important discovery that the glandula labialis superior of non-poisonous snakes is composed of two distinct parts. The anterior part is reddish-gray in color and consists of many minute glandular grains, with numerous excretory ducts. The posterior part appears yellowish-white and is of coarser glandular grains. It possesses only one excretory duct and is homologized with the poison gland of the venomous species. Reichel (1882) demonstrated an exactly similar condition of the gland with Tropidonotus natrix, one of the harmless, solid-toothed colubrine snakes. According to Leydig (1873) the poison gland is not a proper gland, but a modification of one of the lobes of the supralabial gland, and may be present in non-poisonous snakes. It may be here stated that, prior to Leydig, Meckel (1826) announced that the poison gland originates in an enlargement and development of the yellowish portion of the supralabial gland. As early as 1833 Duvernoy demonstrated the occurrence of the poisonous gland in many species hitherto considered non-poisonous, and declared thereby that the simultaneous presence of the poison fang is one of the essential characteristics of the poisonous snakes. Phisalix and Bertrand (1894) look upon the toxic property of the adder blood as deriving from the supralabial gland through the internal secretion, and the natural immunity of the same animal against the viperine venom as the result of constant immunization by the same prin- PHYLOGENY OF VENOMOUS SNAKES 49 ciple as echidnin. Jourdain (1894) found that Tropidonotus viperinus, Elaphis esculapti, Coronella levis and Rinachis scalaris enjoy the same immunity against the viper’s venom as T'ropidonotus torquatus. He held it certain that these snakes have the venom-producing apparatus and contain its product in the blood. Jourdain ventured a still more far-reaching gen- eralization, that every snake is provided with the venogenous gland. While the experimental data concerning the toxic property of the posterior yellowish portion, alleged to be homologous with the real poison gland by Leydig, are strikingly meager, a mere anatomical investigation into the extent in which that particular portion of the supralabial gland comes into existence among the non-poisonous species seems. to warrant enough interest to be briefly dwelt upon in this place. Several species of the Aglyphous snakes have been studied by some investigators. Of the subfamily of Natricinz (or Colubrinz) some species of the genus Naérix (or Tropidonotus), of the sub- family Coronelline, some species of the genus Elaphe (s. Coluber), Ptyas (s. Coryphodon) and Herpetodryas,* and of the subfamily Rhachiodontine, the genus Daspeltis have been studied. Tropidonotus natrix s. Natrix torquatus Fleming: the glandula labialis superior consists of a grayish and a yellowish portion. The glandular grains are made of aggregates of tubules. The cellular elements of the yellowish portion are filled up with granules, and appear like the rennet cells. The epithelium of the excretory duct is a high, clear cylinder cell. The entire yellowish part is provided with one single duct, while the anterior parts have many small ducts opening near the teeth. Leydig (1873) drew an analogy between the yellow portion and the parotid of the mammalia. The posterior part of the yellowish portion is described by Leydig as having the dark, granulated epithelium. Reichel described a similar character of the cell and added that the nucleus is situated basally. The examination of the grayish part reveals many tubules with clear cylindrical cells, which show transverse striation when treated with osmic acid. The reddish- grayish portion contains transparent, non-granulated cells, which are cylin- drical and have basal nuclei. Sometimes there are smaller, highly granulated cells with nuclei dislodged. These are usually met with in the edge of the acini. Some alveoli may consist entirely of this type, others chiefly of the first-named kind, and still others of a mixture of the two types. Reichel con- sidered these varying types of the epithelia as the representatives of different stages of cellular activity, the first type being the resting state. Tropidonotus subminiatus Reinwardt: Niemann (1892) has described the supralabial and the yellowish gland separately. He found four smaller excretory ducts in the former and one larger for the latter. The yellowish gland is surrounded by a strongly developed connective tissue capsule, which has a circular cleft to allow the entrance of the blood-vessels. The long-oval tubules of the gland are surrounded by a thin, delicate connective tissue mem- brane. “The glandula labialis superior is wrapped up in a layer of strongly developed connective tissue, within which a lymphatic space is also present. Pee a ee eee Ba a ee 1Probably synonymous with Liopeltis Fitzinger. 50 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Tropidonotus tesselatus Laurenti: In the glandula labialis superior, which is somewhat smaller and narrower than that of the natrix, there is present the yellowish portion with large follicles. Coronella levis Merrem: This species possesses the glandula labialis superior, consisting of one grayish and one yellowish portion. Coluber viridiflavus var. carbonarius Schreiber, or Coluber flavescens s. esculapii: ‘The Aisculapius snake has a less-developed supralabial gland, which has, however, two distinct portions, one grayish and one yellowish portion. ‘The cells forming the tubules of the yellowish portion are highly granulated. ‘The only duct is lined with high, clear, cylindrical cells greatly differing from those present in the glandular tubules. The rest of the gland, composed of smaller acini, has clear, epithelial cells. Leydig (1873). Elaphis virgatus Schlegel: In the rear part of the supralabial gland is formed the yellowish portion, which is distinguished, by its firmer con- sistency, from the glandula labialis superior proper. Three excretory ducts are provided for the latter, but only one for the yellowish portion. The structure of the yellowish part is briefly stated here. This portion (yellow) is surrounded by a scantily developed connective tissue, beneath which lies the lymphatic space with small, irregular, compressed meshes, from above to below, which contain some lymphatic cells. The gland consists of small, mostly tubular spaces, which, while rather narrow, are quite regu- larly built and often present a flattened form. Each of these tubules is inclosed in an extremely delicate, soft connective-tissue layer. The interior of the tubule is lined with epithelia of cubic shape. The connective-tissue capsule of the glandula labialis superior is weaker than that of the yellowish portion. Ptyas korros Schlegel s. Coryphodon korros Jan: In this species the glan- dula labialis superior consists of a grayish and a rear yellowish portion, but the transverse sections of the two are described as similar. Herpetodryas carinatus Boie: No yellowish part is found in this species, although the glandula labialis superior is regularly developed and surrounded by a poorly developed layer of connective tissue in which a lymphatic space is noticeable. In the place of the yellowish portion, which is missing even in a rudimentary form, the glandula membran. nictitant. is enormously developed. Liophis merremii Wiedersheim: ‘This is another example in which no yellowish part is observed in the rear of the glandula labialis superior. Daspeltis scaber Wagler: Kathariner (1898) described in this species, besides other sets of oral glands, an independent poison gland, which, accord- ing to his judgment based upon the anatomical characteristics, can not be considered as a specialized part of the supralabial gland. This gland is a tubular, branched, and compact gland by itself and has one single central duct, which opens at the corner of the upper jaw in a pocket of the mucous membrane of a definite tooth. On the other hand, the glandula labialis superior is composed of numerous alveolar glands which secrete their product into the groove between the maxilla and the upper lip through PHYLOGENY OF VENOMOUS SNAKES 51 their respective ducti excretores. Among these different Aglyphous species only two seem to lack the rudimentary venom gland or the yellowish portion of the glandula labialis superior. One of the most natural outcomes of these morphological investigations would lead to an investigation whether the parotid secretion of these solid- toothed snakes is really toxic when it is introduced directly into the blood circulation of different animals, which are susceptible to the action of the parotic secretion (venom) of some poisonous species. ‘This interesting prob- lem attracted the attention of Bertrand and Phisalix (1894), who discovered that the salivary glands of two European species of Tropidonotus secrete a fluid which acts fatally upon guinea-pigs when injected into them. Still later Alcock and Rogers (1902) examined the poisonous property of the watery extract of the parotid of freshly decapitated specimens of Zam- enis mucosus upon rats and mice and of the watery and saline extracts of the parotid and the Harderian glands of Tropidonotus piscator upon the last-named animal. The injection was made subcutaneously. The parotid extract of the Aglyphous snakes used was a viscid mucin, quite different from the thin, opalescent fluid derived from the Opisthoglypha. The violent con- vulsions that followed the injection of Zamenis extract presented a marked contrast to the characteristic dyspnoeic convulsions caused by the Opistho- glyphous snakes, although a sufficient dose killed these small mammalia. The extract of the Harderian gland was without toxic effect. The poisonous property of the parotid gland, as well as its secretion, of certain harmless, non-groove-toothed snakes, has been established beyond doubt, but there is still much to be done about this problem, especially in regard to the identity of the poisonous principles of these rudimentary forms of the venom glands. A similar investigation of a greater number of non- poisonous snakes and even of the mammalia is highly desirous. The passing of snakes from the non-poisonous into the poisonous kinds is a gradual process and is associated with a definite morphological and functional modification of the parts directly concerned. This modification is an acquisition of the poisonous apparatus by grades, namely, the speciali- zation of the supralabial gland into a venomous one, and then the canali- zation of the maxillary teeth so as to enable them to conduct the venom. These changes are also accompanied by an ascending perfection of these and other accessory apparati. Thus, in one group of snakes there is neither the venom gland nor the poison fang. Ina second there is the venom gland of a rudimentary stage, but no venom-conducting tooth. In a third the venom gland attains larger dimensions, but the fang is still primitive, being moderate in size, shallow in groove, and situated inconveniently in the rear. In a fourth group the conditions are more favorable, as the venom gland is better developed, the fang is longer and has a deeper groove or a canal, and its position is in the anterior of the maxilla. The fifth and last group has a well-developed gland and one or more large and strong fangs. The fang is tubular and situated in the anterior part of the peculiarly short space of the erectile maxillary bone. CHAPTER IV. GEOGRAPHICAL DISTRIBUTION OF VENOMOUS SNAKES. New Zealand is entirely free from snakes. Australia and its adjacent islands are free from the Viperidee—containing neither the Viperine nor the Crotal- ine. ‘The American continents are noted for the absence of the true viperine forms, whereas the Crotalinz, another representative of the Viperide family, is fully established throughout temperate and tropical America. ‘The famous rattlesnakes are the most specialized of all the venomous snakes and are exclusively confined to the New World. The total absence of the poisonous Colubridz from Europe is another remarkable geographical feature, and it is highly interesting to note that the only venomous snakes here belong to the Viperine, of which but one genus is represented. In Africa the repre- sentatives of the Viperinee are most numerous, but there are none of the Crotaline; of the Colubride no Hydrophiinz are found, while the Elapinz are fairly numerous. Asia contains almost every genus of poisonous serpents, except Croéalus. The Elapinz are well represented by Naja, Bungarus, Hemibungarus, Cal- lophis, and Doliophis, while the Hydrophiine are most abundant along the coasts of the tropical regions of Asia. ‘The true vipers are not unknown here, as the Viperinz are represented by four genera. The crotalines are abundantly represented in Asia, as far as its western neighboring continent Africa, both by Ancistrodon and by Lachesis, better known as Trimeresurus, but, as was stated above, no Crotalus or rattlesnake. Thus Asia may be looked upon as a region where the evolutional balance of various venomous snakes is comparatively well preserved. One of the most extraordinary facts is that Australia is an exclusive home of venomous Colubride, of which no less than 16 elapine and g marine genera are enumerated; but, as pointed out previously, there is no representa- tive here of the Viperide. Returning to the American continents, the conditions are found to be quite contrary. Here peculiar relations exist between the Crotaline and the Colubride —both Elapinee and Hydrophiine on one hand, and Crotalinz and Viperinz on the other. The prevailing venomous snakes of America belong chiefly to Crotaline, and the colubrine and the viperine snakes are thrown into the background. Especially no true vipers exist on this continent. Of the colubrine snakes only one genus is represented, Hlaps,! which, although it includes more than 20 species, is in a state of more or less general degra- dation, as may be judged from its diminutive size and its tendency to burrow. 1 Another genus was described, Micropechis, with only one species, elapoides. 52 GEOGRAPHICAL DISTRIBUTION OF VENOMOUS SNAKES 53 The predominance of the crotaline snakes is most remarkable. While the genus Ancistrodon is less numerously represented than in Asia, Lachesis is much more in evidence. Moreover, two new genera have made their appear- ance on the American continent, namely, Crotalus and Sistrurus. Both are characterized by the presence of the “rattles” at the end of the tail. Of 8 different genera, 4 (Vipera, Echis, Pseudocerastes, and Cerastes) are found in Asia and Africa in common, and Vipera also in Europe, but the rest are characteristic of each continent. It does not follow, however, that these same genera occurring in different continents are represented by the same species. On the contrary, the species of a genus vary according to the prosper- ity enjoyed by the genus on the particular continent. The members of the genus Vipera have different species-characteristics, depending upon whether they inhabit Africa, Asia, or Europe. Of 4 genera of the subfamily Crotaline, Ancistrodon and Lachesis inhabit both Asia and America, but the constituent species of these two genera differ widely according to the continent to which they belong. It is also seen that of 28 genera of the subfamily Elapine, only the Naja is met both in Africa and in Asia, and of that genus there is no species common to both continents. It is noteworthy that even the marine snakes, whose pelagic nature would render almost any artificial geographical boundaries of ocean insignificant, seem to have more or less restricted habitats. Thus, some genera prefer to swim about the coasts of tropical Asia, and especially along the Indian and Malayan coasts and Archipelago, while still others seem to be confined near Sydney. In general, however, the habitat of the marine snakes is highly uncertain and reports of the capture of certain species from unexpected parts of the globe add difficulties to this particular point. EUROPE AMERICA Crotalinae** 4 genera, 43 species AFRICA Elapinae | RR 2 genera, 28 species Viperinae 7 genera, 32 species Elapinae AUSTRALIA 7 genera, 21 species Elapinae 15 genera, 61 species * Comprises only Ancistrodon, and Lachesis, but no “ rattlesnakes.” ** Comprises Ancistrodon, Lachesis, Sistrurus and Crotalus. *#* 27 species out of this number belong to genus Elaps. Hydrophinae 9 genera, 10 specles 54 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS List showing the Geographical Distribution of Venomous Snakes. ASIA. COLUBRID &. OPISTHOGLYPHA. Dipsadomorphine. Dipsadomorphus trigonatus. cyaneus. krepelini. Coelopeltis monspessulana. Psammodynastes pulverulentus. Elachistodontine. Elachistodon westermanni. Homalopsine. Homalopsis buccata. Cerberus rhynchops s. Hurria rhyn- chops. Hypsirhina plumbea. PROTEROGLYPHA. Elapine. Naja tripudians compris- ing varieties N. tripudi- ans typica, N. tripudians ceca, N. _ tripudians fasciata, N. tripudians | Burma, sputatrix, N. tripudians | China, leucodira, N. tripudians | Dutch | J plumbea_ s._ Enhydris India, Ceylon, miolepis. India, Naja samarensis. Philip- Naja bungarus s. Ophio- | pines. phagus elaps s. Hama- dryas (king cobra). India, Ceylon, Burma, Indo- China, southern Bungarus fasciatus (banded krait). Bungarus candidus var. ceruleus and multicinc- tus (common krait). B : China, ungarus ceylonicus. ma lividus. India, Borneo. Hemibungarus colligaster. Southeast- Haase: ern Asia, oe India nigrescens. 2 japonicus. | Janet, beettgeri. as J pines. Callophis gracilis. Burma, trimaculatus. Indo- maculiceps. China, macclellandii. Formosa, bibronii. southern univirgatus. China. Doliophis bivirgatus. ) Indo- intestinalis. | China, bilineatus. Malay Pe- philippinus. | ninsula. Hydrophiine. Gulf of Persia, Distira ornata. ' Indian Ocean, subcincta. Gulf of Bengal, cyanocincta. | Strait of Malacca, jerdonii. Sea of China, ASIA. — (Continued.) COLUBRID. PROTEROGLYPHA. Hydrophiine. Philippines, Acalyptus peronii. } Malay Archipelago. Hydrophis obscurus. ) spiralis. ceerulescens. nigrocinctus. elegans. gracilis. cantoris. fasciatus. leptodira. Tropical Enhydrina valakadien s.| and sub- bengalensis. tropical Hydrus platurus s. Pela-! parts of mis bicolor. Indian and Thalassophis anomalus. Pacific Enhydris curtus. Oceans. hardwickii. Platurus laticaudatus s. fischeri. colubrinus. muelleri. Hydrelaps darwiniensis. Aipysurus eydouxii. annulatus. levis. a Turkestan PROTEROGLYPHA (SOLENO- Ural es : oo . LYPHA). Siberia, Viperine. Cancaaie Vipera berus s. Pelias Dares ? oe Armenia, renardn. western ae China, India eee Ceylon , russellii s. V. ele- : 4 ganss. Daboia. ee Pseudocerastes persicus..... Persia. Cerastes cornutus....Arabia, Palestine. Arabia, Echis carinatus s. | Persia, India, Phoorsa. Beluchistan, Afghanistan. Crotaline. Trans- Ancistrodon halys. re intermedius. ‘an blomhofhi. Him ae Ancistrodon blomhoffi bre- leepuie vicaudus. ee himalayanus. China acutus. Pormiosa rhodostoma. : hypnale LES | Ceylon, Java. GEOGRAPHICAL DISTRIBUTION OF VENOMOUS SNAKES 55 List showing the Geographical Distribution of Venomous Snakes. — (Continued.) ASIA. — (Continued.) VIPERID. PROTEROGLYPHA (SOLENOGLYPHA). Crotaline. Lachesis s. Trimeresurus flavoviridis. riukiuanus. okinavensis. monticola. strigatus. cantoris. Southeast- jerdonii. ern Asia, mucrosquamatus. southern luteus. China, India, purpureomacula- Tin. bee China, gramineus. Riggcnoe flavomaculatus. Java, z sumatranus. Sumatra. anamallensis. trigonocephalus. macrolepis puniceus. borneensis. wagleri. J AFRICA. COLUBRID. OPISTHOGLYPHA. Dipsadomor phine. Macroprotodon cucullatus. Ccelopeltis monspessulana. moilensis. PROTEROGLYPHA. Elapine. Naja haje. | Egypt, western flava. and eastern melanoleuca. Africa, nigricollis. | {| Morocco, anchiete. | | Congo, goldii. Angola. ) Southern : Africa, Sepidon hzmachates. Cape of Good Hope. : . Central Boulengerina stormsi. } Aiea: Elapechis guentheri. a Southern Thiastert | and central sundevallii. | mirica. boulengeri. } Eastern and Aspidelaps lubricus. southern scutatus. Africa, Mozambique. Walterinnesia egyptia....... Egypt. AFRICA. — (Continued.) COLUBRID. PROTEROGLYPHA. Elapine. Dendraspis viridis. jamesonii. angusticeps. antinorii. VIPERID. Southern and central Africa, Angola, Great Lakes, Congo, Transvaal. PROTEROGLYPHA (SOLENOGLYPHA). Viperine. Causus rhombeatus. resimus. defilipii. lichtensteinii. Vipera latastii. ammodytes. lebetina. superciliaris. Bitis arietans. peringueyi. atropos. inornata. cornuta. caudalis. gabonica (Rhino- ceros viper). nasicornis. Cerastes cornutus. vipera. Echis carinatus (Efa). West Africa, Gambia, Great Lakes Congo, Angola, Transvaal. Morocco, Tunisia, Algeria, Egypt, _ Mozambique. Zanzibar, Zam besia, Transvaal, Cape, Congo, Gaboon, Benguela, Angola, Senegal, Niger. Eastern Africa, Sahara. Eastern Africa, Tchad, Egypt, Soudan, coloratus. Somaliland, Socotra. Tropical Atheris chlorechis. Africa, Lagos, squamiger. Dahomey, ceratophorus. | Cameroon, Gaboon, Congo. ) Tropical Atractaspis hildebrandii. ee south a rica, congica. — eso irregularis. A 2 corpulenta. arch 7 chad, rostrata. Eahaa bibronii. Dy bnuiey aterrima. Gold ? dahomeyensis. Cicast micropholis. Ganvib leucomelas. se microlepidota. a and, Natal, Cape. 56 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS List showing the Geographical Distribution of Venomous Snakes. — (Continued.) AUSTRALIA AND NEIGHBORING TERRI- AUSTRALIA AND NEIGHBORING TERRI- TORIES. TORIES. — (Continued.) snake). spessulana. | COLUBRID. COLUBRID. PROTEROGLYPHA. PROTEROGLYPHA. Elapine. PS re Elapine. Ogmodon vitianus.. . Fiji Islands. Brachyaspis curta.......... Australia. eRe East Australia, : Glyphodon tristis. a ice: Reaaiiapaie mentee ee Pseudelaps muelleri. ticus (death adder). Papuasia. squamulosus. ‘ eee Australia, Elapognathus minor........ Australia. ae Molucca, Rhynchelaps bertholdi. iadema. Papuasia. australis. : warro. semifasciatus. a sutherlandii. fasciolatus. Diemenia psammophis. Furina calonota. eoxgualee d bimaculata. Australia. olivacea. Australia, occipitalis. modesta. t New textilis (brown | Guinea. Hydrophiine. snake). ea lataeu } Equatorial and sub- nuchalis. J Eas tropical Pacific. Pseudechis porphyriacus | aks cupreus. Thalassophis anomalus. ae australis. ent eal: ew darwiniensis. at te Guinea. aa Guinea. Celebes, scutellatus. Hydrelaps darwiniensis. + Trimor, microlepidotus. Australia. Teniecns roe } Tasmania, enisonia superba. Hydrophis elegans. +} New Caledonia, cocoa New Hebrides. ones eS Distira ornata. Par e9 cyanocincta. ee Enhydris curtus. aan Enhydrina valakadien s. ee = bengalensis. a a Aipysurus australis. frantalis, eae Platurus muelleri. ieee Islands, EUROPE. maculata. ° punctata Tasmania. | VIPERIDZ. gouldii i PROTEROGLYPHA (SOLENOGLYPHA). nigrescens. Viperine. F Ital Carpe are Austria-Hun ary pallidiceps. Vipera berus s. Pel- Gian aa 1 melanura. ias berus. : ys par cesta gium, Sweden, woodfordii. aspis. ees Micropechis ikaheka. New Guinea, latastii. Portugal, Bosnia, elapoides. {| Solomon Isds. ammodytes: Herzegovina, Hoplocephalus bungaroi- southern Russia, des or variegatus (blood- Turkey, Greece. headed snake). Australia. CoLuBRID#. Hoplocephalus bitorquatus OPISTHOGLYPHA. Hoplocephalus stephensii. Dipsadomor phine. Tropidechis carinata........ Australia. Coasts of Notechis scutatus s. Hop- } Neel Chih | Mediterranean locephalus curtus (tiger oo os ee. a talons Spats asmania. France, Italy (only in Liguri), GEOGRAPHICAL DISTRIBUTION OF VENOMOUS SNAKES 57 List showing the Geographical Distribution of Venomous Snakes. — (Continued.) AMERICA. COLUBRID. OPISTHOGLYPHA. Dipsadomor phine. Trimorphodon lyrophanes. upsilon. vilkinsonii. lambda. tau. collaris. Sibon septentrionale. yucatanense. annulatum. personatum. rhombiferum. frenatum. nigrofasciatum. pacificum. Scolecophis zemulus. atrocinctus. Tantilla coronata. eiseni. nigriceps. gracilis. Manolepis putnamii. Conophis. Coniophanes imperialis. imperialis imperialis. lateritius. PROTEROGLYPHA. Elapine. Elaps fulvius (harlequin } snake). euryxanthus. marcgravii. heterochilus. surinamensis. gravenhorstii. langsdorfii. buckleyi. anomalus. heterozonus. elegans. annellatus. decoratus. dumerilii. corallinus. hemprichii. tschudii. dissoleucus. psyches. spixii. frontalis. lemniscatus. filiformis. mipartitus. fraseri. mentalis. ancoralis. Micropechis elapoides. Central America, Bolivia, + Ecuador, Peru, Colombia, Brazil. AMERICA. — (Continued.) VIPERID. PROTEROGLYPHA (SOLENOGLYPHA). Crotaline. Ancistrodon piscivorus (water-moccasin or cot- ton-mouth). Ancistrodon bilineatus. Ancistrodon contortrix (copperhead). Lachesis mutus (bushmas- ter). Lachesis s. Bothrops lan- ceolatus (Fer de lance or jararacussu). Lachesis atrox (Labaria). pulcher. microphthalmus. pictus. alternatus. neuwiedii (Both- rops urutu). ammodytoides. xanthogrammus. castelnaudii. nummifer. godmani. lansbergii. Lachesis brachystoma. bilineatus. undulatus. bicolor. schlegelii. nigroviridis. aurifer. Sistrurus miliarius. catenatus (Mas- sasauga). ravus. Crotalus adamanteus s. durissus (diamond-back rattlesnake). Crotalus horridus (banded rattlesnake). Crotalus confluentus (prai- rie rattlesnake). Crotalus atrox var. scutu- latus. Crotalus atrox var. ruber. oregonus. tigris. molossus. cerastes. lepidus. pricei. triseriatus. polystictus. terrificus (dogface rattlesnake). mitchelli (white rattlesnake). Eastern America, Florida, Texas, Mexico, Guate- mala. Central and South America, Marti- nique, St. Lucia. Central and southern America, Martinique, St. Lucia. Eastern ) America, east of Rocky Moun- tains, Mexico. Southern Canada, British Columbia, Central America, Guiana, Venezuela, Brazil, Uruguay, North of Argentina. J CHAPTER V. POISON APPARATUS OF VENOMOUS SNAKES. Venomous snakes in the act of biting inject a poisonous fluid into the object bitten. This fluid is generally known as “venom” and is the product of highly specialized, well-developed glands which show certain phylogenetic relations to supralabial glands and correspond with the mouth-angle glands of birds and the parotid glands of mammals. The fluid is injected into the victim by means of a series of specialized teeth of the maxilla, which differ from ordinary teeth by the presence of a groove throughout the entire front surface of the teeth or of a complete canal from the base to the apex. These specialized teeth, called “‘venom fangs,’ are larger than the rest, and are in communication with the venom glands by means of the common ducts of the latter. ‘The end of the excretory ducts does not enter the basal opening of the groove or canal of the poison fang, but opens quite close to the latter. The flowing out of the venom is, however, well prevented by a sheath, which is merely a prolongation or fold of the mucous membrane. Naturally the inoculation itself is accomplished by the complicated motion of numerous muscles, which open the mouth, erect the fangs, and close the mouth. Simul- taneous compression of the venom glands by a certain muscular envelopment forces out the glandular secretion through the common duct into the venom- conducting fangs, which are in the meantime inserted into the victim. POISON FANGS. The specialized teeth adapted to conduct the secretion of the venom glands into the interior of the tissue of the victim have certain general features com- mon to widely distant families of venomous serpents. They are provided with either a groove or a canal, and are larger in dimension than the rest of the maxillary, palatine, and mandibular teeth. They are situated on the max- illary bones, to which they are firmly ankylosed. The poison fangs are cres- cent-shaped, with one square, wide end on the base. When in connection with the maxillary bone and ectopterygoid bone (transversum) they resemble a sickle. The base of the fang is, comparatively, very broad and the apex is extremely sharp. In all proteroglyphous snakes the number of active fangs is usually two, arranged side by side. Behind the inner fang are several reserve fangs in developing order, which take the place of the active fang when it is damaged or shed. In one set of proteroglypha the fangs are front- ally grooved longitudinally, while in the other set the groove is completely closed into a hollow tube which again opens as a slit near the frontal side of the apex. This latter set is often designated solenoglypha or tubular-fanged. The furrow of the groove is of varying depth, according to the species of the snake. 58 Noguchi Plate 20 ~~ lao D : Venom gland of Crotalus adamanteus. A. Longitudinal section. X 9. B. Cross-section, showing Tubular Raminous Structure. (Note abundant muscle-fibers running into the interacinous spaces.) X 100. C. Cross-section. X 9. D. Portion of Tubular Acinus, showing Columnar Epithelial Cells with Nuclei near Base of Cell Body. X 1000. ie Re y “yc | ‘ POISON APPARATUS OF VENOMOUS SNAKES 59 In the opisthoglyphous snakes the fangs are short and have usually much shallower furrows than the proteroglypha. The fangs differ greatly in their magnitude according to the snakes. The viperine and crotaline snakes pos- sess the best-developed and longest fangs, while the elapine and hydrophine families have much shorter ones. A fang an inch long is not uncommon in Crotalus and Lachesis. It is hardly necessary to mention that the poison fangs are provided with a regular pulp-cavity which occupies a space just behind the groove or canal separated with a thin septum of dentine and enamel. The relation of the poison duct to the fang has often been misinterpreted. Niemann! even reproduced a picture in which the poison duct was shown to enter directly into the base-opening of the canal of the fang. But Mitchell as early as 1860 described the real method by which the poison duct communi- cates with the groove of the poison fang. It is by means of the cavity sur- rounding the base of the tooth and inclosed by the mucous membrane folds which constitute the proximal portion of the vagina dentis. In Crotalus Mitchell discovered the presence of certain muscular fibrillze in the mucous- membrane sheath, which apparently serves as a sphincter. ‘This arrangement has the advantage that in the replacement of the fang the connection will not in any way be affected, in spite of the change of the position of a new fang. The sphincter which has been found by Mitchell near the termination of the duct in the Crotaline appears to be absent from the proteroglyphous Colubrinz, while in Hydrophiine a non-striated muscle is present near the base of the fang. POISON GLANDS. (Plate 20, A, B, C, D.) Glandula venenata were not definitely discovered until a correct account was given by Fontana. Schlegel (1828) found in many snakes with furrowed posterior teeth a large gland which opens its duct only at the base of this tooth. Duvernoy (1832) then found that a similar gland exists also in numerous sus- pected snakes. The poison gland of fangless snakes is not exactly equal to the fully developed venom gland of fang-possessing reptiles, but is a mixed gland, consisting of anterior grayish-red portion and posterior grayish-white portion. The latter is provided with only one duct. After the careful studies of Rudolphi, Meckel, and Leydig (1873) it became clear that the posterior portion is of the nature of a serous gland, while the anterior portion is that of a mucous gland. It is noteworthy that a serous gland comes for the first time into existence in these snakes, but not in any class in the inferior evolu- tional order, which, as in Amphibia, is provided with a mucous gland. In the Mammalia the existence of the serous gland becomes universal. In the majority of venomous snakes the poison gland occupies a space behind the eye and stretches backwards in length according to the size of the eee 1 Niemann, Arch. f. Naturgeschichte, 1892, I. 60 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS gland. Its anatomical position corresponds with that of the parotid gland of the Mammalia, to which its similarity is still further strengthened by the serous character of the secretion. A certain digestive function of the poison gland has been described and is alleged to be essential to digest the prey. The venom gland like the parotid gland has only one excretory duct. Allu- sion has been made to the fact that the secretion of the poison gland has a double importance to the reptile —to assist digestion and to capture prey. The dimension! of the poison glands is generally in proportion to the size of the snakes, although some exceptions are observed in certain species. Of the Crotalidz with the length of about 5 feet the gland attains the size of an almond. ‘The cobra is provided with a somewhat larger gland. The Euro- pean vipers have much smaller glands, as their size is not very great. It is a curious fact that the genus Doliophis, one of the venomous colubrine genera, is characterized by the possession of a very large, elongated poison gland which extends down one-third of the entire length of the body. It ends in a club form in front of the heart, shifting the latter to the right. Especially Doliophis ? intestinalis and Doliophis bivirigatus are noted for their enormous glands. In the visceral region the glands are in one mass and separate from one another near the head in order to supply the poison fang on each side. Similar glands are described by Meyer in Doliophis philippinus, Doliophis nigroteniatus, and Doliophis flaviceps. The gland is surrounded by striated muscle fibers which run parallel to its longitudinal axis. According to Meyer and Hoffmann (1890) another duct opens near the exit from a second large gland which lies behind the eye. In Causus rhombeatus Reinhardt discovered a poison gland disproportionately large. In a specimen which measured 18 inches the gland with the duct reached 3 inches. The gland runs down along each side and lies on the ribs and muscles, and is provided with a muscle attached to it. The poison gland is for the most part a serous gland. A considerable variation is noted in the structure of poison glands throughout Ophidia. In proteroglyphous colubrine snakes the alveoli of the gland are much larger and have a lining epithelium of short columnar cells inclosing a capacious lumen in which secretion is stored. The supporting framework of interalveolar 1S. Weir Mitchell gives the following measurements for the Crotalus adamanteus kept two to eight weeks in captivity: No. Weight. Length. Meee oe 1 lb. 6 oz. ft. x inch 7} grains 2 Meyer, the discoverer of this visceral poison gland, used the term Calliophis or Callophis, but Wil- helm Peters (1871) devised a special generic name Adeniophis for this particular group. Bou- lenger again employed the third name Doliophis in lieu of Peters’s Adeniophis. POISON APPARATUS OF VENOMOUS SNAKES 61 connective tissue,varies in amount in different species, but in all cases it is developed to a greater extent in the center of the gland in the region of the forwardly converging ducts. ‘The poison duct, which is longitudinally folded for the greater part of its course, has opening into it, throughout its length, a series of small glands completely surrounding it. These minute lobules are mucous glands and are difficult to stain, and the alveoli and cells have a different structure from the rest of the gland. In Hydrophiine the inter- alveolar connective tissue is extensively developed, most noticeably in Enhy- drina hardwickii. Platurus fasciatus is conspicuous for the small size of the external alveoli, especially at the posterior end of the gland. The duct of this group is remarkable for the convoluted course its terminal portion takes. Small lobules are found arranged as in the other Proteroglyphous Colubrinz. In Platurus fasciatus these glands are reduced almost to single alveoli with a lining epithelium like that of the poison gland itself. But in Distira cyano- cincta and Hydrus platurus they are more markedly developed, a few of them in the latter half of the course of the duct becoming mucus-secreting. Towards the termination of the duct the cells of its own lining epithelium also become mucus-secreting. ‘This has been shown to be common in the duct of the parotid and labial glands of the opisthoglyphous Colubrinz, and it forms a pavement layer in the Crotaline. Thus the Ophidia are the only animals in which a considerable admixture of mucus is present in the parotid secretion, this mucus being derived in all cases from some of the cells of the duct and sometimes from special accessory mucous alveoli. The presence of mucous alveoli in the parotid gland and the conspicuous admixture of mucus in the parotid secretion, more especially of elapine Colubrinz, may perhaps present an analogy to the condition in the submaxillary glands of many mammalia. They are all restricted closely to the exit of the duct.’ Johannes Miiller (1830) was, however, the first to recognize the tubular structure of the poison gland and the spongy nature of the inner wall of the tubules. He states also that the glandular tubules stretch continuously from the exit duct to the surface of the organ. According to him the structure of the poison gland of Naja haje is as follows: The connective-tissue capsule of the gland consists of a single layer, but not of double, serous space embrac- ing covers, as is the case of Vipera, while a wide, rather indefinite lymphatic space is present between the poison gland and the upper wall of the oral cavity beneath. Emery (1875) distinguishes two parts in the poison gland of Naja haje. The posterior part is considered as the poison gland proper, the anterior part as belonging to the mucous system, in which all supralabial glands are to be enumerated. In the posterior part the cells near the central zone are cylindrical, while those lining the peripheral zone are flattened epithelia. In the anterior part there are also cylindrical epithelia, but they are somewhat larger and have a much clearer nucleus — not many granular particles*around the nucleus — which is always easily seen. In fact, these : 1 West. Jour. Linn. Soc., 1898, XXVI, 517. 62 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS epithelia are identical with those of the supralabial glands, and the anterior part of the poison gland is called the accessory mucous gland. This assump- tion is supported by the observations that in the epithelia of the anterior part the nucleus is driven near the base of the epithelia, which are spherically swollen. Such appearance is never manifest in the posterior part treated by the same technique. The epithelia of the duct are cylindrical, yet their contents are not so clear and homogeneous, and stain by carmine solution. The arrangement and ramifications of the glandular tubules are described by Emery and are shown to be more complicated in the posterior than in the anterior part. Emery maintains the presence of ramification of the tubules in the posterior glandular part. In the anterior part there are glandular lobes, whose orifices open into the central secretory way in from five to six regular series. No lobes are found in the posterior part. From behind, a protrusion into the lumen of the secretory way is projected. This protrusion contains a collecting canal from the posterior glandular part and represents the continuation of their central substance. The structure of the poison gland of Vipera berus, Vipera aspis, and vari- ous European vipers has been well studied by Fontana, Rudolphi, Meckel, Joh. Miiller, Brandt, de Betta, A. B. Meyer, and Leydig. The poison gland lies in a fascia-like, pocket-like, extension of ligamen- tum zygomaticum. The main muscular coat of the gland is furnished by M. masseter, and certain of the fibers are also sent from M. temporalis. The tough, fibrous capsule divides the gland into many main lobes by send- ing several lamellar foldings within the glandular parenchyma. The tough cover of the poison gland consists of a firm connective tissue which resembles in its texture the ground structure of leather. Beneath this firm capsule the tissue becomes much looser and forms within it a lymphatic space. The con- nective-tissue framework of the gland retains the same soft, loose character described above, and the gland itself represents a tubular construction. The poison gland has a triangular form, the front angle of which is drawn into a secretory duct. The gland lies in a firm connective-tissue sheath and through this it is surrounded by muscles from almost all sides. It is so arranged that in every act of biting a compression of the gland, and the sub- sequent evacuation of the secretion from the poison fang, are brought about. The secretory duct consists of firm, thickly woven, circularly arranged, con- nective tissue, which contains no muscle fiber. The tough sheath of the poison gland transforms in the interior into a wide-meshed frame, which incloses numerous lymphatic spaces. Beyond this network numerous blades radiate internally and hold many glandular tubules together in groups so as finally to result in so-called granules. According to the degree of fulness of the secretion, the lumen of the tubules may be quite wide, or the walls may be in contact with each other. The epithelium of the poison gland is low and cylindrical. The protoplasma is more or less strongly granulated, and some coarse granules are seen. The nucleus lies somewhat apart from the base, but never near the apex. In the POISON APPARATUS OF VENOMOUS SNAKES 63 fresh gland the tubules are sometimes filled with the secretion (venom), which appears as clear, transparent particles. The lymphatic space of the secretory duct gradually disappears near the end. Niemann (1892) and Lindemann (1899) observed that the tubular arrangement of the poison gland of Vipera berus is of irregular nature and to be considered as a second- arily modified tubular gland. While Leydig and Reichel described the epithelia of the poison gland as low and cylindrical, Niemann and Lindemann found them to be cubic. Yet the study of the latter authors revealed the cause of this difference. ‘They found that in the epithelium immediately after the bite the nuclei are dark, stainable, not larger than half the diameter of the basis of cell, and stand slightly apart from the base. The granulation is gradually increased toward the upper part and thickest near the free edge of the epithelium. In a snake which has been kept a long time in captivity and has not bitten for some time the granulation is much lighter and more regularly and evenly distributed throughout the cell-body. The approximate size of the cell remains, how- ever, unchanged. THE PROCESS OF SECRETION OF VENOM. The process of secretion is comparable to that of salivary secretion. In the protoplasma of the epithelium homogeneous droplets appear and render the protoplasma more transparent. Immediately after secretion of the venom the periphery of cells becomes more darkly granulated.* According to a later study of Launoy ? (1903) the process of venom secre- tion can be divided into two phases: (1) a phase of nuclear elaboration, (2) a phase of cytoplasmic elaboration. Besides the passive exchanges which take place between nucleus and cyto- plasma, the nuclear sphere participates very actively in the secretive process. This participation of nucleus is evident from the following reactions: (1) By the difference in the stainability of the chromatin grains. (2) By emission of equal-sized, spherical, well-defined grains, with tincto- rial reaction of the internuclear differentiated chromatin, into cytoplasma. (3) By the exosmosis of the dissolved nuclear substance, which appears in the meanwhile in ergastroplasmic form. These formations constitute, on the one hand, the grains of “‘venogene;”’ on the other, the ‘‘ergastroplasmic venogene.”’ In the venom cell of Vipera aspis and the serous cell of the parotid of Tropidonotus natrix is produced chiefly in a granular form. After reaching to the perinuclear cytoplasma the venogene grains and ergastroplasmic venogene will at once disappear, if it happens to be during the period of cellular excitement; or will remain for some time, if it is at a period when the cell is saturated with the product. During the cytoplasmic activity the venogene grains and ergastroplasmic venogene disappear. 1 Lindemann. 2 Launoy, Thése de doctorat és sciences, Paris, 1903, No. 1138, série A, 454. 64 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS The nuclear and the cytoplasmic elaborations constitute two distinct cytes of secretion. The nuclear elaboration is to furnish the cytoplasma with necessary material for the proper secretory work, while the cytoplasmic cyte is an elaboration, in which not only the basal protoplasma takes part, but also throughout the entire cell, especially the perinuclear cytoplasma. The venogene grain can be distinguished from the venom grain by its affinity to Unna’s blue, safranin, and fuchsin. The venom grain is eosino- philic. It is never secreted in granular form, but always after the intracellu- lar dissolution. In the lumen of the glandular tubules no venogene grain is to be found. DYNAMICS OF THE FUNCTION OF POISON APPARATUS. (Plates 21 and 22.) The bones which are concerned with the insertion of the poison fang into the victim can be divided into direct and indirect. The direct bone is the supramaxillary, to which the fang firmly ankyloses on the alveolar socket. The supramaxilla is a sort of triangular body and has four facets. The under surface presents a somewhat dull triangle, or rather a pentagonalum, with its apex directed forwards. Here near the apex two sockets are found, in which two acting fangs are usually implanted. The anterior external facet has an irregular surface, and in the crotaline snakes this is eclipsed at the posterior external portion by a spherical excavation —a fossa characteristic of the “pit” vipers. The anterior internal surface is very smooth and oblong. The posterior surface is defective by the presence of a spherical depression, which at the same time excavates the upper half of the anterior external facet. In the posterior surface, just above the fossa, is a smooth articulat- ing surface, which connects this bone with the corresponding articulating surface of the prefrontal (lachrymal) bone. Near the internal edge of the posterior surface is a small, deep furrow, at the bottom of which are two holes, one communicating with the spherical exca- vation-pit and the other with the bottom of the alveolar sockets of fangs on the under facet. The border where the under and posterior facets meet forms an- other articulating line —a narrow, straight line —and is connected with the thin, extended end of the ectopterygoid bone (transversum) by a strong hori- zontal ligament. ‘This maxillo-ectopterygoid articulation is freely mobile and is one of the most important factors in erecting the fang. The maxillo- prefrontal articulation is also of such nature that the maxillary bone is easily rotated and erected. The denomination of different surfaces of the maxillary bone is naturally variable with the positions to which this bone may event- ually change, but by the under “‘surface’’ is always meant the facet where the dental alveolar sockets are present. This “under surface’? becomes the ‘posterior’? when the fangs are horizontally folded with their points directed backwards, and the “posterior surface”’ will then turn to the “upper.” 1A minute description of the osteology, myology, and the physiological mechanism of the bite of the Crotalus is given by S. Weir Mitchell, Researches upon the venom of the rattlesnake, Smith- sonian Contributions to Knowledge, 1861, Washington, D. C. Lite DOR 7) ve 7 Sa ie - ry DESCRIPTION OF PLATE 21. BONES OF CROTALUS ADAMANTEUS A. Ventral view of skull. Other bones, right side seen from inside. Premaxillary bone. Prefrontal, or lacrymal, bone. Frontal bone. Post-orbital process. Posterior region of parietal bone. Crest of sphenoid bone. Squamosal bone. Articulating surface of supermaxillary bone (with prefrontal bone, 2). Foramen for entrance of a large branch of trigeminous (into the pit). Supermaxillary bone. Palatine bone. Poison fang. Internal pterygoid bone. External pterygoid bone (so-called transversum). Quadrate bone. Mandibular bone. B. Dorsal view of skull. Other bones, right side, seen from outside. Bis 32. 17. Occipital bone. 18. 19. 20. 2i. 22 23% 24. 25: 20. 27. 28. 29. 30. Articulating surface of temporal bone (with squamosal bone, 24). Parietal bone. Frontal bone. Premaxillary bone. Prefrontal, or lacrymal, bone. Post-orbital process. Squamosal bone. Quadrate bone. Mandibular bone. Internal pterygoid bone. External pterygoid bone (or the transversum). Palatine bone. The pit of the supermaxillary bone (the foramen for the nerves is seen in t2 uw» er position of the pit). Supermaxillary bone. Poison fang. Noguchi Plate 21 B Bones of Crotalus adamanteus POISON APPARATUS OF VENOMOUS SNAKES 65 Among the bones indirectly concerned the ectopterygoid bone and the pre- frontal bone are the most important. ‘The ectopterygoid is connected with the maxilla by a broad, strong horizontal ligament at the lower border of the posterior surface of the maxilla, and at its hind end is attached firmly to the upper surface of the endopterygoidal bone near the middle part of the latter. The prefrontal bone is connected with the articulating, small, ovoid surface of the upper corner of the posterior surface of the maxilla. This joint is mobile. The articulation of the prefrontal with the frontal bone admits a certain amount of movement. Thus there are two points on the posterior surface of the maxilla, one at the upper and one at the lower end. Should the ecto- pterygoid be brought forward by certain muscular movements, it would necessarily result in rotating the maxilla at the maxillo-prefrontal joint and force the maxilla with its fangs to project in a forward direction; hence the erection of the fangs. The endopterygoid bone is two-thirds anterior straight and one-third pos- terior slightly turned upwards. In the anterior end it is jointed with the palatal bone, which is short and vertically flattened. The posterior end is loosely connected with the mandibulo-quadrate joint. The ectopterygoid is, as stated above, firmly ankylosed at the upper surface of the middle way of the endopterygoid. The latter has several teeth (solid) under the surface. Just behind the prefrontal bone (paired) is the frontal bone (paired), and behind the latter the parietal bone (unpaired, but fused at the median line). The frontal bone is slightly depressed in the upper surface (practically a flat roof) and of square shape. On the internal margin it joints with the cor- responding part of the other frontal; in the frontal edge it is free, but forms a posterior wall of the nasal cavity. Below it has a large foramen for the olfactory nerve. The premaxillary bone is connected with the median frontal fusion line of two frontal bones by a tiny projection. The lateral edge of the frontal is a slight curve which forms the upper edge of the orbit. Here the bone is very thin and shows the tendency of a vault underneath. At the posterior edge the frontal is jointed with the parietal bone. The parietal bone has a lateral process on each side, near the articulation with the frontal—the posterior orbital process—which forms the posterior upper edge of the orbit. The process leads inwardly to a transverse crest. On the under surface, near the median line, on each side, there is an oval foramen which communicates with the cranial cavity, and which is the optic-nerve path. The parietal bone occupies the largest dimension of the cranium and incloses in it an irregular, oblong cavity for the reception of cerebral and cerebellar contents. On the upper surface it is flat, but on the under surface the median line develops into a sharp, triangular crest enormously elongated, especially toward the posterior part (sphenoidal crest), into a projection. The tem- poral bones seem to be completely fused with the posterior part of the parietal, and three holes (two larger and one small; the two large ones become one inside) are found near the posterior edge (lateral and inferior surface) of the parietal. Just above them, namely, on the posterior upper surface, 66 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS the squamosal bone is attached with the loose ligament. ‘The squamosal — perhaps called mastoid bone by some authors —is a flat, short oblong bone, which attaches to the parietal (or temporal) region with the entire under surface. At its posterior end it joints with the quadrate bone, which, in turn, joints with the condyle process of the mandible. ‘The quadrate is often called zygoma. It may be stated that the lower maxillary bone of snakes consists of two symmetrical halves, connected by a very strong ligamental band, and that it is capable of a large amount of expansion. The occipital bone can not be distinctly marked out, and this perhaps completely fused with the parietal and temporal bones. Before entering on the description of the muscles which are concerned in the mechanism of the erection of fangs, biting, and ejaculation of venom, it must be stated here that the venom gland, in the majority of poisonous snakes, occupies the space immediately behind the postorbital process (hence, the posterior edge of the orbit) and the entire lateral region alongside the parietal bone on each side. The erection of the fang is effected by the contraction of M. sphenoptery- goid,' which originates in the crest at the base of cranium and, running backwards and outwards, is inserted fan-like upon the pterygoid plate, which is movable. The retraction of the fang is effected by the contraction of M. pterygoid externus,” which arises from the tough aponeurosis covering the zygomatico- mandibular articulation (or quadrato-mandibular) of the lower jaw, and, running forward below the venom gland, is inserted tendinously into an apophysis of the upper maxillary bone exteriorly to the maxillo-ectopterygoid articulation. While passing below and inside the poison gland M. pterygoid externus sends a strong layer of white fascial tissue out upon the capsule of the gland. Some of its lower fibers are finally inserted directly into the two lips or edges of mucous membrane of the fang. Another muscle, M. spheno-palatinus,’ originates from the raphe of the base of the skull, above the sphenopterygoid and thus nearer the skull, and, run- ning diagonally outwards and backwards, inserts along the inside of the palatal bone. As its fibers cross those of the sphenopterygoid, it has an opposite effect to the latter, and thus assists the depressing action of the pterygoid externus upon the fang. The opening of the mouth is effected by muscles, such as M. costo- mandibular and M. vertebro-mandibular, with the help of a muscular layer analogous to M. platysma myoides. The articulation of the jaw is fixed by the double action of the digastricus 4 and cervical angular muscles. The closing of the mouth is effected by the temporal muscles. The most 1M. pterygo-sphenoid posterior. 2M. transverso-maxillo-pterygo-mandibularis. ‘This muscle aids in fastening the fang upon the prey attempting to flee. 3M. pterygo-sphenoid anterior. 4M. digastricus is held responsible by many writers for opening the mouth and lowering the mandible. DESCRIPTION OF PLATE 22. A. Right side, seen from inside. 1. Spheno-palatine muscle. Spheno-pterygoid muscle. Sphenoid crest. BW bp dibular portion. wn dibular portion. Supermaxillary bone. Mandibular bone. Poison fang. Palatine bone. © 31 10. Internal pterygoid bone. 11. External pterygoid bone. 12. Parietal bone. 13. Squamosal bone. 14. Quadrate bone. 15. Premaxillary bone. 16. Prefrontal bone. 17. Post-orbital process. B. Right side, seen from outside. Posterior portion. dibular portion. nN ABwWNH dibular portion. 7. Supermaxillary bone. 8. Mandibular bone. Ome nits 10. Fang. 11. Internal pterygoid bone. 12. Palatine bone. 13. Parietal bone. 14. Post-orbital process. 15. Prefrontal bone. 16. - Premaxillary bone. 17. Quadrate bone. 18. Squamosal bone. C. Head of water mocassin (Ancistrodon piscivorus) seen from above. Skin removed except near snout. 1. Poison gland. 2 Ending of the anterior temporal muscle in capsule of poison gland. 3. Mandibular portion of anterior temporal muscle. 4. Attachment of the middle tem- poral muscle to the tempo- parietal region of cranium. Posterior temporal muscle. Internal suspensory ligament of oe poison gland. Posterior ligament. Digastric muscle. Poison fang. Pits woe tO aco! : » Anterior temporal muscle, man- Posterior temporal muscle, man- External pterygoid muscle. Spheno-palatine muscle. Spheno-pterygoid muscle. Posterior temporal muscle, man- Anterior temporal muscle, man- External pterygoid muscle. 12. mas I4. Io. If. 12. 133 T4. TGs 16. E. Right Tes Ww > Om 10. Ii. Ls Te T4. 15. Shield scales. Eye. Cervical muscles. D. Right side, seen from outside. Poison gland. Duct of poison gland. Internal suspensory ligament of gland. Posterior suspensory ligament. Anterior temporal muscle. Insertion of anterior temporal muscle around the poison gland overlapping from behind and ending in the capsule. Posterior temporal muscle, man- dibular portion. Posterior temporal muscle, cra- nial portion. . Middle temporal muscle, at- tachment to the parietal bone. The fibers end in a fan-like, spreading layer over anterior temporal muscle. External pterygoid muscle. Origin of the external ptery- goid muscle. Spheno-palatine muscle. Digastric muscle. Supermaxillary bone. Internal pterygoid bone. Mandibular bone. side, seen from inside. Poison gland. The duct of poison gland. Mandibular portion of the an- terior temporal muscle. Upper portion of the anterior temporal muscle enveloping posterior region of gland. Cut edge of the middle tem- poral muscle. Middle temporal muscle; the upper torn edge is detached from the parietal bone to which it has been inserted. The lower cut edge is originally continuous with the fibers shown in the opposite cut edge through a short piece of the removed portion. Posterior temporal muscle. Upper portion of the posterior temporal muscle. External pterygoid muscle. Its anterior portion. Spheno-pterygoid muscle. Spheno-palatine muscle. Digastic muscle. internal suspensory ligament. A portion of posterior ligament of poison fang. Plate 22 Musculature of the Poison Apparatus, head of Ancistrodon piscivorus. POISON APPARATUS OF VENOMOUS SNAKES 67 important is the anterior temporal,’ which arises from behind the orbit and from the upper two-thirds of the firm fascia of the poison gland. Its fibers run backwards over the gland and descend between it and the middle tem- poral muscle. In this course the fibers lie posteriorly to the suspensory ligament, and the outer ones, as they fold about the anterior end of the gland, lie in contact with the prolongation of the external lateral articular ligament upon the glandular body. Finally, the muscle winds around the commissure of the lips, and is inserted into the mandible some distance in front of the angle of the lips. ‘The middle and posterior temporal muscles arise chiefly from the temporal fossa and are inserted, one behind the other, into the lower jaw. Their fibers descend nearly vertically and their obvious func- tion is to close the jaw. ‘The function of the anterior temporal muscle is apparently twofold — to exert the pressure upon the poison gland and to aid in shutting the mouth in the meanwhile. The poison gland of Crotalus occupies the side of the head, behind the eye and beneath the anterior temporal muscle. Its posterior end extends three or four lines beyond the commissure of the lips, while the anterior extremity reaches below and just behind the eye. Thus situated, the gland is in rela- tion with the bony surface behind the eye, with the middle temporal muscle, with nerves which emerge under the suspensory ligament, and with the anterior temporal muscle above and behind where that muscle descends to its insertion. Beneath, the gland is in contact with the external pterygoid muscle, with whose aponeurosis it has peculiar relations. The portion of the gland below the anterior temporal muscle and above the line of the lip is in direct contact with the skin, which is here loosely connected with the areolar tissue. The ligaments of the poison gland are firmly connected with the tough, fibrous capsule of the gland, and are really in continuation of the latter. Anteriorly the ligament gradually tapers thin and runs forwards with the duct, constituting a part of its thickness. Posteriorly there is one ligament which attaches firmly upon the fascia of the temporal muscles. Another strong ligament is found to extend from the capsule of the gland to the bony surface beneath the gland. A third attachment of the gland is by means of a fascia which forms a strong expansion upon the external pterygoid muscle and then runs off laterally, to be inserted in the outer capsule of the gland. THE BITE. The mechanism of the bite has been studied most exactly and exhaustively by Weir Mitchell, who made observations on the rattlesnakes. The snake prepares itself for striking by raising the head a little above the rest of the body, but not, usually, more than 3 or 4 inches, even in large snakes. The neck and upper end of the trunk are not thrown into complete circle, but lie in two or three abrupt curves across the mass of the coiled body. The 1M. temporalis anterior of Mitchell may be identical with M. masseter of authors, and his middle temporal muscle with M. temporalis anterior of authors. 68 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS next phase is the forward cast of the body, which is effected by a sudden contraction of the muscles which lie upon the convexity of the bendings formed by the upper part of the snake, so as to abruptly straighten the body and thrust it in a direct line. The projectile range does not exceed a third of its length. The snake can cast itself in every direction — forwards, down- wards, or almost directly upwards. At the instant, and while in motion, the jaws are separated widely, and, in order to bring the points of the backwardly curved fangs into a favorable position to penetrate the opposing flesh, this is done to such an extent that an observer standing above the snake can see the white mucous membrane of the mouth as the blow is given. The for- ward thrust of the body and the opening of the mouth are instantaneously accompanied by the contraction of the spheno-pterygoids of the pterygoid plates, which, through their articulation with the maxilla, bring the fangs to the erect position. The mere act of opening the mouth is not necessarily associated with the erection of the weapon, but, on the contrary, even when the mouth is widely opened, the snake has the most perfect control over the movement of the fang, raising or depressing it at will. At the same moment the cloak-like vagina-dentis is thrust off from the convexity of the fang and is gathered in loose folds at its base. When the erected fangs penetrate the flesh a second series of muscular movements follows. The contraction of the spheno-pterygoid is relaxed and is immediately succeeded by contraction of the pterygoid externus and spheno-palatine. The latter movement, due to the insertion of the posterior apophysis of the maxillary bone and the inside of the palatal bone, respectively, draws the point of the fang violently backwards, so as to drive it more deeply into the flesh. At this instant occurs a third series of motions which result in the further deepening of the wound and the injection of the venom. The lower jaw is closed upon the bitten part or member. The closure is effected by the pos- terior, middle, and anterior temporal muscles. The first two tend simply to shut the mouth, but the anterior temporal is so folded about the poison gland that while it draws up the lower jaw, it simultaneously compresses two-thirds of the body of the gland. This force is applied in such manner as to squeeze the venom out of the upper and posterior parts of the gland and drive it forward into the duct. The middle temporal muscle descends from its attachment at the temporal fossa to its thin fan-like insertion over the external surface of the anterior and posterior temporal muscles, passing downwards in a slightly oblique anterior direction along the inner surface of the posterior one-third of the poison gland, and crossing, in part, that portion of the anterior temporal which is wrapping up the anterior two- thirds of the gland by a curved course which it takes from the front to the rear, where it ends in the capsule of the gland. Thus, the contraction of this muscle exerts also an important rédle in compressing the gland. The anterior lower part of the gland and a portion of the duct is subjected to the pressure at the same instant, owing to the flat tendinous insertion of a part of the external pterygoid upon the parts in question. POISON APPARATUS OF VENOMOUS SNAKES 69 The whoie process — deepening the wound, fixing the prey, and injecting the venom —is the work of an instant, and the next effort of the snake is to disentangle itself from the victim. This step is effected by relaxing the muscles of the neck, so as to leave the head passive, while the continued trac- tion of the muscles of the body pulls upon it and withdraws the fang. ‘The elastic mucus sheath glides over the fang, and the pterygoid externus, again acting, depresses the latter, the snake resuming its posture of defense. It is not uncommon that in a bite but one fang takes effect. Again, it has often been observed that when the snake in captivity is allowed to bite upon the inner edge of a cup it often uses only one fang. Or, the fangs are used alternately with intervals. It may happen that when the object stands too near the snake the latter miscalculates the distance and the fangs are not in the erect position, hence no penetration. In a contrary instance, the object may be beyond reach of the snake and the biting movements may be per- formed before the object is struck. In this case the venom is sometimes pro- jected several feet. The traction of the anterior temporal muscle is associated with the com- pression of the poison gland, and it becomes rather questionable how in a pacific mood the snake prevents the flow of the venom when it uses this muscle for other than the biting purpose. According to Weir Mitchell this is prevented by two means: the most effectual is the sphincter around the duct, and the other is the mechanical pressure upon the duct while it runs over the frontal angle of the maxillary bone just before it reaches the base of the fang. This mechanical compression is instantaneously removed as soon as the fang is erected. CHAPTER VI. TOXIC SECRETIONS OF VENOMOUS SNAKES. AMOUNTS OF VENOM SECRETED. The quantity of venom yielded at one time by a snake, either at a single bite or by squeezing the poison glands, is very variable. It increases, how- ever, in general, in proportion to the size of the snake, although this rule can not be applied to the marine snakes, which secrete an amount that is small in comparison to their size. The continuous failing of health in cap- tivity causes a decrease of venom, both in quantity and in toxicity; especially is this the case where the snakes refuse to take nourishment and are sub- jected to repeated extractions of venom. While it is very important to deter- mine the exact amounts of venom yielded at a single bite, the data bearing upon this subject are rather unsatisfactory. Weir Mitchell has noted the difficulty of extracting venom from the rattle- snakes when the latter are resisting forcible manipulation. He has found that more venom can be extracted when the snakes are put under the effect of an anesthetic, thus relaxing the muscular resistance. Lamb’s careful study of the Indian cobra showed that the amount of venom thrown out vol- untarily by a snake is larger than that obtained through forcible compression. Calmette gives the detailed results of his experiments on two specimens of Naja haje, about 5.5 feet long. During 120 days in his laboratory one was subjected to 5 and the other to 6 extractions. In the fresh state the total amount of the venom for the first specimen was 0.581 gm. (per 5 extractions) which weighed 0.174 gm. in the dried form. The second snake afforded (on 6 extractions) 0.684 gm. fresh venom, which weighed 0.202 gm. on dry- ing. Thus the average single amount of venom to be ejaculated by the adult 0.581 + 0.684 0.174 + 0:202 5 + 6 oe = 0.0331 gm. in the dried form. He also found that 1 gm. of the fresh venom weighed 0.336 gm. upon complete desiccation over calcium chloride. With Naja tripudians, Cunningham estimated the amount of a single bite at 0.254 gm. (dried); Lamb at 0.231 gm. (extracted with fingers, dried) ; 0.373 gm. (by voluntary bite, dried); and Rogers at 0.249 gm. (dried). With Pseudechis porphyriacus, MacGarvie Smith gives the amounts of single bite at 0.1 gm. to 0.16 gm. in the fresh state and 0.024 gm. to 0.046 gm. in the dried form. With Notechis scutatus s. Hoplocephalus curtus the same author gives the amount of venom produced at a single bite at 0.065 gm. to 0.15 gm. liquid and 0.017 gm. to 0.055 gm. dried. The dried venom weighed from 9g to 38 per cent of the fresh venom. Daboia russellii yields on an average 0.15 to 0.25 gm. weighed dry (Lamb). 70 Naja haje is = 0.115 gm. in the fresh state and TOXIC SECRETIONS OF VENOMOUS SNAKES 71 With Lachesis lanceolatus (fer de lance) Calmette gives 0.320 gm. in the fresh and 0.127 gm. in the dried form for a single extraction from both glands. With Crotalus adamanteus,' Flexner and Noguchi put the figures at between 0.179 gm. and 0.309 gm. in the dried form for a single extractable amount from both glands. With Crotalus confluentus, Calmette gives 0.370 gm. in liquid and 0.105 gm. in dried form from a single bite. With Ancistrodon piscivorus, Flexner and Noguchi estimated 0.125 gm. to 0.18 gm. dried as a single extractable dose. With Ancistrodon contortrix the same authors found the average to be 0.03 gm. to 0.06 gm. dried. With two large Egyptian Cerastes specimens Calmette derived 0.125 gm. (0.027 gm. in dry) and 0.085 gm. (0.019 gm. in dry), respectively. With Enhydrina, Rogers could extract only 0.0023 gm. to 0.0094 gm. (as dried form) from both glands of the adult specimens. The loss in weight of venom upon drying has been estimated by various authors. Weir Mitchell and Reichert found it to lie between 25.15 per cent to 27.42 per cent with Crotalus adamanteus, C. atrox, and Ancistrodon piscivorus. Calmette places it at 62 to 80 per cent. C. J. Martin found the loss in weight after drying the Australian venom to be 33 per cent, while Flexner and Noguchi found the solid portion of venom (Crotalus and An- cistrodon) to range from so to 7o per cent of the total weight. TOXICITY OF SNAKE VENOM. Irrespective of the modes of action by which the fatal issue of the venom poisoning is brought about, it is possible to determine the approximate mini- mal lethal dose of each snake venom for a given species of animals by intro- ducing the venom directly into the system of that animal. It may be stated that certain venoms act more powerfully when introduced directly into the blood circulation, while others do not appreciably differ in their final out- come, whether they are given subcutaneously or intravenously. Thus, a much smaller quantity of the venom of Crotalus is effective when injected intravenously than when injected intraperitoneally or subcutaneously, while but little difference is shown with the venom of Cobra in these respects. It is superfluous to emphasize the fact that venoms from different species of snakes differ slightly or widely in their constitution, which is always multiple in nature, and also in their physiological effects. A powerful venom means one which kills or injures the victim by a smaller amount than a weak venom. The cause of these qualitative differences will be treated later im extenso. The susceptibility of animals to venom is also very variable. Speaking in general, warm-blooded animals are more sensitive to the action of venom than cold-blooded animals. There exists among various species of animals a constant susceptibility peculiar to each group. In regard to the toxicity of venom we find certain constancy of strength in 1 Weir Mitchell (1861) states the following details: Crotalus durissus, No. 1, 18 in., 9} 0z.; venom ri drops. No. 2, 25 in., 18 oz.; 19 drops. No. 3, 49} in., 34 lbs.; 29 drops. 72 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS each species. However, certain exceptions must be expected, as a strict uniformity does not rule out biological phenomena. For these reasons the figures given by different investigators do not quite agree, yet they agree sufficiently to furnish us with the approximate toxicity of each venom. The minimum lethal doses of the venoms of various species of snake are given in table 1. The figures indicate the quantities of dried venom in gram per kilogram of the body-weight of the animals. In order to make compari- son of the quantities given by different authors in different standards pos- sible, these are reduced to the uniform standard and unit, unless special statement is made to the contrary. Certain remarkable facts will be noticed by scrutinizing the effects of minimum lethal doses of various venoms upon different classes of animals by different modes of administration. ‘The very high toxicity of the marine- snake venoms, as compared with that exhibited by most of the elapine-snake TABLE I. . Animal experi- Nature of Amount of venom . Species, etc. mented Teor injection. administered. Authority. Colubridz: Elapine: Naja tripudians ..... Rabbit. Intravenous .. | 0.00025 to 0.0005 | Calmette. Dor eee err .. do. =1d0; 5) |O:000g5 Lamb. WON eerie an SCO nied brunt Gowers 0.000245 Fraser. OMe cyereei-t Che ioag |Mstaloa do. .... | 0.0007 Elliot. WD Ora areeosenstgs: Guinea- pig Subcutaneous 0.0004 Calmette.! DOr tesa seks . do. do. .... | 0.0005 Noguchi. DOs ee vereya crest. By koe eer ol ements do; =...) || o:coon8 Fraser. DOS e craves RAR yes. . Hydrochloric acid (0.4 | Very soluble.......... Very soluble sree Do. per cent). Metaphosph. acid..... Insolublencnucae ns sce Imsolubles ie nreeeee eee | Insoluble; yellowish tint. Orxthophosphiadcid) 2-1: Soluble an cscs ees Very soluble .......... Very soluble. Sod. metaphosphate ...} Insoluble............. nisolablestrcs mice ae Do. Sod. orthophosphate...| Very soluble.......... Iessisoluble. ..2.452 sis). Still less soluble. Pot. ‘sulphatess...2) 5. GCOSs Hitac Spacers Tesoluble 73: ec. ss: Insoluble. Calexchiorides jon: Ossett prde meee essisolubler amass oer Less soluble. Acetic acid (5 per cent). : Soluble wimecyskeseiecior Very soluble. Ncetic-acids (Glacial) ih "e sem GOsne saree teres GOP ievats She aerehers Do. Finally, the venom peptone was prepared by dialysis. Elimination of the coagulable proteins by boiling was unsatisfactory, because it never gave PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 81 clear filtrate, and if the boiling was too prolonged the peptone broke down and gave fine coagula. The following reactions were obtained with this fraction: TABLE 4. No immediate coagulation at a tempera- Mercuric chloride, decided precipitate. ture of 100° C. Absolute alcohol, precipitate, redissolved by the Full reactions with the protein color tests. addition of water. No precipitate with weak or strong nitric Mercuric nitrate, decided precipitate. acid. Pot. hydrate, precipitated by saturation. Ferric chloride, no precipitate. Pot. ferrocyanide in presence of weak acetic acid, Cupric sulphate, no precipitate. a precipitate. Moccasin peptone resembles the above (crotalus) closely. On the other hand, Mitchell and Reichert met some remarkable properties of a similar preparation obtained from cobra venom. It was not precipitated by mercuric chloride or absolute alcohol. In watery solution all venom peptones pro- duced fine coagula when boiled for a few minutes. After filtration, the filtrate again gave similar fine coagula on boiling, and so the process of boiling, filtering, and reboiling the filtrate went on repeatedly, yet a clear filtrate could not be obtained even after one hour’s repetition. Mitchell and Reichert considered this peculiarity to be a decomposition phenomenon of a protein as a result of violent physical force. The cobra peptone reacted positively to the xantho-proteic, Millon and biuret. There was no precipitate by strong nitric, hydrochloric, or acetic acids, but precipitation resulted by saturated sodic chloride, tannic acid, and basic-lead acetate. Reverting to the identity of the fractions of coagulable proteins obtained from the different venoms by the same methods, Mitchell and Reichert give comparative lists of their solubility and heat-coagulability. Salt-free sus- pension of the water-venom globulins of Crotalus terrificus and Cobra, and the copper-venom globulin and dialysis-venom globulin of Crotalus terrificus are recorded as coagulable by heat, while no coagulation or even clearing up is observed by them with the three globulin fractions of Ancistrodon piscivorus. Except that the water-venom globulin of this latter snake is insoluble in acetic acid (s per cent or glacial), no remarkable differences are noticeable, and again three fractions behave alike to a certain extent. Mitchell and Reichert give the following composition of the venom proteins in two crotaline and one elapine snakes: TABLE 5. Crotalus adamanteus: 0.5 gm. dried venom water-venom globulin 0.0495 gm. copper-venom globulin 0.0375 gm. dialysis-venom globulin 0.0360 gm. 0.1230 gm. =globulins. 0.3770 gm. =peptone (estimated). Ancistrodon piscivorus: 0.3364 gm. dried venom water-venom globulin 0.0034 gm. copper-venom globulin 0.0182 gm. dialysis-venom globulin 0.0047 gm. 0.0263 gm.=globulins 0.3101 gm.=peptone (estimated). Cobra: o.2 gm. dried venom water globulin 0.0035 : peptone 0.1965 (estimated). 82 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Thus in Crotalus adamanteus 24.6 per cent, in Ancistrodon piscivorus 7.8 per cent, and in Cobra only 1.75 per cent of globulins were found present. In 1886 Norris Wolfenden published some interesting studies upon the constitution of the venom of Naja tripudians and that of Daboia russellii. He first denied the presence of Blyth’s cobric acid, which he affirmed to be nothing but calcium sulphate crystals. The bacterial as well as Gautier’s alkaloid theory has been completely discarded, the presence of alkaloidal principles in crotalus venom being disproven before this by the Mitchell- Gibbs experiments. Wolfenden first established that the toxic power of cobra venom resides in the protein constituents of the secretion, and is lost when the proteins are removed, and diminished as these are diminished. He carefully sets up the possibility that the venomous body is something so intimately linked to the proteins that it varies in intensity with the amount of these bodies present and is precipitated, coagulated, or destroyed by all such means as those which precipitate, coagulate, or destroy proteins. By employ- ing the magnesium sulphate precipitation he succeeded in separating three distinct proteins from cobra venom.! Globulin: This was fractionated by saturating the venom solution with MgsO,. It coagulates at 68° to 75° C. when in solution in water. It is not, however, a pure globulin, but a mixture of acid albumin and globulin. Wolf- enden states that the amount of acid albumin is very small and can be coagu- lated, when the solution minus the coagulated globulin is heated again with MgsO,. A precipitate is yielded by acid albumin upon the addition of acetic acid and ferrocyanide of potassium. The prolonged dialysis of the 1 Armstrong (quoted by Fayrer in Proc. Roy. Soc. Lond., 1884, 156) has made, in 1873, the following analysis of cobra venom. Crude poison. edt Uk Ngobolle Albumin for comparison. _ «| Percent. | Percent. | Percent. | Percent. | Percent. C. ..... 43-56 45-76 43-04 53-5 53-5 ING ees 40.30 14.30 12.45 15.7 15.5 PS ae Aaya 6.60 7.0 7.1 7-0 eee See 2.5 Rel! cane: 1.6 Oreo. aie ets Sera seta 22.0 Na ee shred ee Rae aa ats 0.4 Pedler held it possible that the poison is a mixture of albuminous principles with some specific poisons. (Report of Commission on Indian and Australian Snake Poisoning, Calcutta, 1874.) His ele- mentary analysis gives the following figures: C 52.87 per cent, H 7.1 per cent, N 17.58 per cent. Liquid venom contained 27.74 per cent of solid matter and 6.68 per cent ash. Sir Joseph Fayrer ane saa Brunton compared the action of cobra venom to the alkaloid conin. (Loc. cit., idem. Lacerda at first considered the venom to be an organized ferment like bacteria, but later modified this view only to adopt another enzyme theory in which venom is thought to be analogous to the digestive ferment of the pancreas. His discovery that potassium permanganate stops the action of venom came from the idea that oxidation destroys the ferment. Winter Blyth described a cobric acid as the sole toxic principle of cobra venom. It is not a protein, but microscopic needles crystallized out from the alcoholic filtrate of the venom by means of pre- liminary precipitation with acetate of lead, and then the removal of the lead with H2S and sub- sequent evaporation in vacuo. Another method of Blyth is to shake up the alcoholic filtrate with ether, removing the ether, evaporating it off, dissolving in water, passing through a wet filter, and finally evaporating down. (The Analyst, 1877, I, 206.) PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 83 globulin-magnesium precipitate allowed a small trace of albuminous body to diffuse outside the dialyzer, and the latter gave the precipitate with acetic acid and ferrocyanide of potassium, indicating that this was an acid albumin. Judging from this he thought the dialyzable protein of venom could not be a peptone, but an acid albumin. Serum albumin: A very small quantity precipitable with Na,SO, from the magnesium filtrate of venom was obtained, but precipitation required many hours shaking. It coagulated in redissolved condition at between 70° and 80° C. Syntonin: Some fraction was obtained from the filtrate of an aqueous solution of venom previously heated to 98° C. for 10 to 15 minutes by means of saturated MgSO,, and still more completely by boiling the filtrate with solid MgSO, in saturation. Usually this frees the venom solution of any protein matter, but there may be some exceptions, which Wolfenden, in that case, thought due to the presence of peptone. He found that the filtrate of the boiled venom solution (in water) is acid and forms coagula by neutralization. Peptone: Wolfenden employed the method devised by Hofmeister for testing peptone: (1) Cobra venom which had been precipitated by satura- tion with MgSO, was treated according to that method.* On addition of the acid phosphotungstate of soda there was produced a slight opalescence. (2) An alcoholic extract of cobra venom was treated with the above method and gave similar opalescence with phosphotungstate of soda. The extract gave undoubted protein reaction. (3) The dialysates (mixed) of cobra venom, after 3 days’ dialysis, gave no acid-albumin reaction, but yielded precipitation with Hofmeister’s method. Biuret was negative. (4) A portion of cobra venom which was once treated with MgSO, and Na,SO, gave a slight turbidity with the acid phosphotungstate of soda. From these experi- ments Wolfenden concludes that there are three proteins present constantly in cobra venom, namely, globulin, serum albumin, and syntonin and prob- ably, in traces only, a fourth, peptone. Similar experimental studies have been extended by Wolfenden to the venom of Daboia russellii. In this series for the separation of globulin he employed precipitation by MgSO,, NaCl, and (NHy).SO,, CO,, and dialysis. He found the globulin to coagulate at 75° C. The presence of serum albumin was made apparent by precipitation of the magnesium filtrate by Na,SO, and the occurrence in the solution of the soda precipitate of an opalescence on boiling, within the range of coagulation temperature of serum albumin — BOF t0:.807.G. 1 Hofmeister’s method is as follows: Treat a solution of albumin with saturated solution of sodic acetate and then add ferric chloride, until of a blood-red color. At this point the addition of the iron is stopped. Neutralize it with sodium hydrate up to a slight acid reaction. Then boil the fluid for a few minutes, and filter. The colorless filtrate must not give precipitate with ace tic acid and ferrocyanide. Make the filtrate acid with acetic acid and test with an acid solution of phosphotungstate of soda (acidified with acetic acid). The ratio of the filtrate and the reagent is 4:1. If any peptone is present there is a flaky precipitate, or a turbidity, on standing for a few minutes. All the albumins except peptone are removed by the ferric acetate. This method is about 50 times more accurate than that of biuret, which can detect only in I= 2000. 84 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Albumose and Syntonin: Slow-diffusible proteins are still found in the fil- trate of the venom solution heated to 95° C. Wolfenden believes that they belong either to acid albumin or albumoses, but not to peptone, because they can be removed by ferric acetate. This author expresses the opinion that the substances called peptones by Mitchell and Reichert belong to albu- moses. In 1892 Kanthack’ made a thorough examination of the venom of Naja tripudians atrox. He brought out evidences that the chief active principle of cobra venom is albumose; and he gives the following summing up: (a) The fresh protein precipitate, whether obtained by the addition of absolute alcohol or ammonium sulphate, is amorphous and white, but when dried slowly it forms a translucent colorless mass. (5) Its solution is colorless or slightly opalescent, and neutral or alkaline to litmus paper. (c) It is very soluble in water, quite insoluble in alcohol. (d) It gives a brilliant biuret reaction with caustic potash and a trace of cupric sulphate. (e) Nitric acid gives a precipitate, soluble on heating and coming down on cool- ing if the solution is of sufficient concentration. With dilute solution a trace of sodium chloride must be added to cause precipitation. (f) Picric acid causes a precipitate, dissolved on heating, but appearing or form- ing again on cooling. (g) Boiling produces no physical changes. (h) Saturation with ammonium sulphate gives a precipitate. Letting the fluid stand for 48 hours and filtering off the precipitate, the clear filtrate gives no biuret reaction, while a solution of the white precipitate reacts beautifully. On adding acetic acid to the filtrate no precipitate is obtained. (7) Saturation with sodium chloride causes a precipitate. The filtrate gives no biuret reaction, nor a precipitate on the addition of acetic or nitric acid. (j) The solution gives the ordinary protein reactions, the color with Millon’s reagent being less bright, and more of a pinkish-yellowish hue. From these reactions Kanthack concludes that the substance in question is an albumose, and that only one, a primary albumose, is present. The presence of an alkaloid is denied. According to Kanthack, cobra venom does not contain any appreciable amount of globulin. The substance described by Weir Mitchell and Reichert as globulin is, according to this author, certain derivatives of the proto- albumose of the same venom. 1A. A. Kanthack. The nature of cobra poison. Jour. of Physiology, 1892, XIII, 272. In 1892 Martin published a method for separating the albumoses from snake venom. It is as follows: The poison is slightly diluted with sterilized water, and then thrown into a large excess of alcohol, and allowed to stand for a week. The alcohol is separated off and the white precipitate washed with absolute alcohol, dissolved in sterilized distilled water, and once more precipitated by alcohol. The resulting precipitate is allowed to stand under alcohol for a week, then washed with alcohol and dissolved in water, to which a little thymol is added to _prevent putrefaction. ‘The solution is found to contain no other proteins than albumose. | Hawkins’ method for separating albumose employed by Wolfenden. ‘The solution of cobra venom is saturated with ammonium sulphate and the mixture allowed to stand for several days. The white precipitate is dissolved in water and dialyzed until all trace of the salt has disappeared. The solution is then concentrated by dialysis against absolute alcohol, and subsequently poured into a large excess of alcohol and treated as in Martin’s method. (Brit. Med. Journ., 1890, July 12.) PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 85 By repeated precipitation by absolute alcohol and redissolution of the precipitate in water, thus excluding the possibility of admixture of globulin, he obtained the protein substance which corresponds to the proto-albumose derived from digestion cleavage. Now, he found that if the solution of the albumose be subjected to the action of higher temperature or the sunlight, there appears in the originally clear solution some precipitate, which is in- soluble in distilled water, but soluble in 0.75 per cent sodic chloride solution, from the latter heating, or saturation with NaCl again throwsit down. Accord- ing to Kanthack this precipitate is a hetero-albumose. The mere dialysis did not uniformly produce precipitate from a solution of native venom, but it did‘ occasionally. In this case he thinks it to be a hetero-albumose. At times the precipitate derived either from the diffusible proteid of the venom or from the alcohol-treated albumose is partly insoluble in 0.75 per cent sodic chloride solution in contradistinction to a hetero-albumose. This was thought by Kanthack to be a dysalbumose. Prolonged heating of the proto- albumose yields a hetero-albumose and a dysalbumose, which are harmless. It was found that the solution of cobra proto-albumose exposed to the tem- perature for 12 hours no longer gives the biuret reaction and is entirely innocu- ous (and cloudy from the precipitate). The loss of toxic action by heating the albumose is ascribed to the decom- position of proto-albumose into hetero-albumose and dysalbumose. C. J. Martin and MacGarvie Smith separated the albumoses of Pseudechis s. Notechis porphyriacus of Australia from the coagulable proteins by filter- ing the venom solution previously heated to coagulation. The filtrate was then precipitated with a saturated magnesium sulphate (shaken a couple of hours). The precipitate was collected on the filter and washed with the saturated solution of MgSO,. The filtrate was then dialyzed in running distilled water for 24 hours, then concentrated by dialyzing it in absolute alcohol. This last condensed fluid contained certain proteins in solution, and these were found to be a mixture of hetero-albumoses and proto-albumoses with a little of peptones. Sometimes the peptones were absent. The separation of hetero-albumoses and proto-albumoses was effected by Neumeister’s method. The proteins were precipitated with 5 per cent CuSO, (a few drops), then the deposit was collected and washed with MgSO,, put into distilled water, and then dialyzed during three days. The proteins are thrown down abundantly, and are centrifugalized, clear fluid being pipetted off and then dialyzed in absolute alcohol. Then the residue is finally desic- cated at 40° C. Here the separation is accomplished simply by extracting the dried mass with distilled water, which takes up only proto-albumose, but not hetero-albumose, which is insoluble. The coagulable proteins of this venom consist of albumin and globulin. Thus the poisonous principles of snake venom have been classed with the toxalbumins, among which several plant poisons, like ricin or abrin, are also enumerated. 1Kiihne never found formation of hetero-albumose out of proto-albumose on dialysis. 86 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Starting from the biological observations made by Flexner and Noguchi, Kyes, under the direction of Ehrlich and partly in association with Sachs, finally made a very important discovery on the hemolytic constituent of snake venom. Flexner and Noguchi found that venom becomes hemolytic when there is at the same time the serum of a susceptible species, and thought it to be regular complements of blood serum which activate venom. Calmette discovered that the heated serum contains more activating principles, and all serum becomes, no matter whether originally venom-activating or not, com- plementary for venomon boiling. From these observations Kyes was led to discover the nature of the venom-activating principles of heated serum. It was found to be lecithin. Later Kyes obtained a definite compound of venom and lecithin, which is hemolytic by itself. This is called lecithid of venom. His original method of preparing venom-lecithid is given below: 4o c.c. of at per cent solution of cobra venom in 0.85 per cent NaCl solution are mixed with 20 c.c. of a 20 per cent chloroform solution of lecithin in a flask of about 100 ¢.c. capacity and then shaken in an apparatus for this purpose for about 2 hours. Then the whole mixture is centrifugalized for 45 minutes — 3,600 revolutions per minute. When the process is successful the watery portion of venom separates sharply from the clear chloroform portion below with a compact, whitish, narrow layer between the two portions. The clear chloroform portion has about 19 c.c. as a rule, and can be easily separated by fine pipette. Then mixing it with 5 volumes of ether, there appears a precipitate which is the lecithid. The ee lecithin remains in solution in ether. As a rule Kyes washed the precipitate with original volume of ether ro to 20 times repeatedly, in order to remove all trace of lecithin from the lecithid. The lecithid can be preserved a long time under ether, without change, or it can be dried carefully, but in the latter case its solubility is observed to undergo change, although its hemolytic activity remains intact. From 1 gram of dried cobra venom about 5 grams of dried lecithid has been prepared. A very important finding is that after the preparation of lecithid, the watery portion lost its hemolytic property almost completely, while the neurotoxic principle still remained undiminished in the solution. The cobra lecithid is found to be hemolytic, but not fatal to animals. When injected into a mouse in a quantity which could dissolve 200 c.c. of the blood in vitro it produced only an infiltration of the site of injection. In a rabbit ro c.c. of a 1 per cent solution of lecithid caused an extensive infiltration when injected subcutaneously. The properties of cobra lecithid: The primary product, which is obtained by repeated washing in ether and compressed between filter papers, is insolu- ble in acetone and ether, but soluble in chloroform, cold alcohol, and warm toluol. From solution in chloroform and alcohol it is precipitated by ether and acetone. Lecithid which still contains some ether, or lecithid rapidly freed from ether by air-current, dissolves in water without cloudiness, and presents a clear, slightly yellowish solution. Its solubility in various lipoid PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 87 solvents distinguishes lecithid from cobra venom on one hand and lecithin on the other. The curious phenomenon was observed that when a watery solution of the primary lecithid is allowed to stand in the room temperature the solution gradually becomes turbid and separates out whitish precipitate which settles to the bottom. The whitish deposit consists of crystalline, translucent, highly refractory bodies of microscopic dimensions. This modified product is called secondary lecithid. It is almost insoluble in cold water, soluble in warm alcohol, and reappears on cooling. Its solubility in organic solvents is the same as the primary lecithid. The lecithid requires no incubation time for attacking the blood corpuscles. It is thermostabile, while the native cobra venom becomes inactive when heated to 100° C. for 30 minutes. No biuret reaction is given by the lecithid. The elementary analysis of pure lecithid of cobra venom has been made by different chemists." Willstatter and Liidecke give the following figures: One estimation N = 2.73 per cent P 76 per cent. Other estimation N = 2.8 per cent P 5: 6.03 per cent. The result of von Braun is as follows: N = 2.84 per cent P = 5.56 per cent. H = 10.92 per cent C = 59.07 per cent. The result obtained by H. Weil is as follows: N = 6.35 per cent P = 3.16 per cent S = 7.66 per cent. H = 9.48 per cent C = 56.26 per cent Ash = 9.29 per cent. In this connection Kyes referred to the fact that the percentages of nitrogen and phosphorus of the lecithid closely approach those of monostearyl lecithin and monopalmitin lecithin, which, according to Willstatter and Liidecke, contains N = 2.74 per cent, P = 6.06 per cent in the latter, and N = 2.59 per cent, P = 5.73 per cent in the former. The lecithids were prepared also from the following venoms: (t) Lachesis s. Bothrops lanceolatus. (5) Bungarus fasciatus. (2) Daboia russellii. (6) Lachesis s. Trimeresurus riukiuanus. (3) Naja haje. (7) L. s. Trimeresurus anamallensis. (4) Bungarus ceruleus (krait). (8) Crotalus adamanteus. Kyes met some irregular results in preparing venom lecithid, and finally found that the acidity which develops and gradually increases during the shaking of the lecithin-chloroform solution and cobra-venom solution was the cause. If the acidity of the mixture be removed by appropriate quanti- ties of alkali, the formation of lecithid goes on again until the acidity reaches the inhibiting degree for the process. After completing this process the alkali is removed from the mixture by means of hydrochloric acid. Kyes studied the products which arise from the mixture of an insufficient 1 Kyes. Ueber die Lecithide des Schlangengiftes. Biochem. Zeitschrift, 1907, IV, 99- 88 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS lecithin and sufficient cobra venom, shaken as usual. He obtained four preparations: Substance I. A spontaneous precipitate formed in the watery layer when the latter is separated from chloroform (lecithin solution) by means of 1 volume ether and two-fifths volume of alcohol. It gives a biuret reaction and many other protein reactions — precipitation by potassic ferrocyanide, acetic acid, nitric acid, tannic acid, picric acid, and alcoholic cadmium chloride solution. Hzemolytically very weak (one-tenth of the original venom), but can be complemented with lecithin. Substance II. Obtained by first precipitating the watery portion with 5 per cent phenol, then the oily matter which separates out thereby is again precipitated with alcohol. The precipitate has the property of becoming ro times more hemo- lytic than the native venom when combined with enough lecithin, but otherwise is almost inactive. Substance III. Obtained by precipitating the filtrate (of the alcohol-soluble oily fraction, after separation of substance II) with ether. This is gelatinous and water-soluble, has about the same strength as the native venom. Requires lecithin to be active. Substance IV. Precipitate obtained by treating the filtrate (the soluble portion from which oily matter was removed by phenol) with alcohol. These bodies are called by Kyes incomplete lecithids, which differ from the complete lecithid in their insolubility in alcohol. Of these four, sub- stance II is neutralizable by antivenin, while some of the incomplete lecithids remain unaffected. Complete lecithid is not reactive to antivenin, but is said to be able to produce anti-serum, which neutralizes lecithid and native venom equally. Snake venoms are not the only class of bodies which produce hemolysis in the presence of lecithin. Pascucci! has shown that ricin becomes hemolytic when mixed with leci- thin. Landsteiner and Jagic? discovered that the colloidal silicic acid, which like ricin is usually only agglutinating, becomes upon the addition of lecithin highly hemolytic. Landsteiner and Jagi¢ explain this phenom- enon as due to the adsorption of lecithin to the corpuscles previously or simultaneously impregnated with the colloidal silicic acid. Naturally they found that lecithin is by itself more or less hemolytic even without the inter- mediation of this colloid. Later, Landsteiner and Jagic * also found that the shaking of the mixture of aqueous solution of colloidal hydrate of iron and choloroform solution of lecithin gives rise to a new compound of these two bodies precipitable from chloroform by a large quantity of ether. This relation is exactly what takes place when venom and lecithin are shaken under the same conditions. Reiss ‘ states that chloroform solution of lecithin can take up lac and trypsin from aqueous solutions, but these ferments, which apparently had gone over 1 Pascucci. Ueber die Wirkung des Ricins auf Lecithin. Hofm. Beitr. zur chem. Physiol. u. Pathol., 1906, VII, 457. i 2 Landsteiner and Jagic. Ueber Analogien der Wirkung kolloidaler Kieselsiure mit den Reaktionen der Immunk6rper und verwandter Stoffe. Wien. klin. Woch., 1904, XVII, 63. Landsteiner and Jagic. Ueber Reaktionen anorganischer Kolloide und Immunké6rperreaktionen. Miinch. med. Woch., 1904, LI, 1185. Michaelis and Ehrenreich. Die Adsorptionsanalyse _ der Fermente. Biochem. Zeitschr., 1908, X, 283. ‘Reiss. Eine Beziehung des Lecithins zu Fermenten. Berl. klin. Woch., 1904, XLI, 1185. PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 89 to lecithin chloroform, were not precipitated out by adding a large quantity of ether. Michaelis and Rona! add further knowledge as to the mechanism of lecithid formation. These authors found that chloroform solution of mastix when shaken with aqueous solution of rennet takes up a certain part of this enzyme as chloroform-alcohol solution forms and can equally be precipitated out by means of ether, an exact parallel phenomenon to the process of lecithid prepa- ration of Kyes. — Landsteiner and Jagic and Michaelis and Rona are inclined to regard the phenomenon as of merely physical nature, namely, colloidal reaction. Kyes, however, does not consider the physical explanation of Michaelis and Rona of the mastix-rennet phenomenon applicable to the formation of venom lecithid, inasmuch as there is a very great and important difference between these two sets of superficially analogous processes. Venom lecithid does not contain any trace of the original materials from which it has been derived. No evidence of free venom-hemolytic amboceptors or of native lecithin can be brought out. Its chemical and physical properties are quite different from the original materials. On the other hand, the precipitate of mastix-rennet shows at least that the ferment is intact in all its original charac- teristics. Here it has been a mere physical process, not comparable with the venom-lecithid formation. According to von Dungern and Coca® a very large quantity of oleic acid is separated out during the preparation of cobra lecithid. 11 c.c. of 20 per cent lecithin solution in chloroform plus 22 c.c. of 1 per cent cobra venom solution in 0.8 per cent NaCl solution were shaken 2 hours; 5 times volume of ether; precipitation of lecithid. From 2 gm. of lecithin about 1.206 gm. lecithid were obtained. From to c.c. chloroform solution 0.5642 gm. of pure acid oil went into ether. Its acidity agreed with that of oleic acid. 0.5079 gm. of this oil was dissolved in absolute alcohol and then determination of acidity was made. It required 16.5 c.c. of N/1o NaOH, whereas 0.5079 gm. of pure oleic acid took 17.6 c.c. N/to NaOH. Baeyer’s double bond test with permanganate was positive; lead salt perfectly soluble in ether. In the separated saline solu- tion there was still cobra venom, which, when used in 5 times the original, dissolved the blood in the presence of 0.5 c.c. of 0.05 per cent lecithin just as rapidly as the original 1 per cent venom solution. The hemolytic activity of the lecithid was: 0.00002 gm. dissolved 1 c.c. of 5 per cent suspension of ox corpuscles, but 0.coce1 gm. did not act. A preparation of lecithin purchased from E. Merck they found to contain a large amount of acid (5.5 c.c. N/1o NaOH to1 gm. of lecithin in alcohol, phenolphthalein indicator). With this lecithin hemolysin was also formed. But the hemolysin did not precipitate until 40 parts N/ 10 Na,COs were added 1 Michaelis and Rona. Ueber die Léslichkeitsverhiltnisse von Albumosen und Fermenten mit Hin- sicht auf ihre Beziehungen zu Lecithin und Mastix. Biochem. Zeitschr., 1907, IV, 11. 2Kyes. Bemerkung iiber die Lecithidbildung. Biochem. Zeitschr., 1908, VIII, 42. 3 y. Dungern and Coca. Ueber Hamolyse durch Schlangengifte. Minch. med. Woch., 1907, LIV, 2317. 90 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS to 1 per cent venom solution in saline and then separated with ether and chloroform. (Drawings by Noguchi.) Noguchi Plate 24 Cc A. Medulla oblongata of Rana catesbiana (4 hours in physiol. salt sol.). 500. B. The same, kept 2 hours in Cobra venom solution. X 500. C. The same, kept 4 hours in Moccasin venom solution. X 500. (Photomicrographs.) Nerve Fibers of Rana catesbiana. A. Normal (low power). B. Normal (high power). C. Acted on by Cobra venom for 2 hours (low power). D. Acted on by Cobra venom for 2 hours (high power). (Photomicrographs.) NEUROTOXINS OF SNAKE VENOM 15! employed were those contained in the precesophageal ganglia. The ganglia having been excised from the living animal, they were carefully and minutely teased in sea water, and then brought under the influence of venom also dissolved in sea water. Observations were made under the microscope at inter- vals up to 24 hours. Control preparations of the cells suspended in sea water, were examined at corresponding intervals. The temperature was that of the room during the summer months. A brief summary of facts follows. CELLS OF SYCOTYPUS. (Plates 23, 24, 25.) The controls show two kinds of nerve cells: (a) pigmented and (b) non- pigmented. Between the cells and originating from them numerous fine, non-medullated fibrils occur. The cells are large, reaching 4o », and are readily observed in unstained preparations. They are oval or elliptical in form, and granular. The pigmented cells contain many highly refractive yellow granules of varying size, which more or less completely obscure the nuclei. The pigment is abundant and granular; there is no diffuse pigment. The non-pigmented cells may equal the first in size and also in number of granules. These granules are markedly uniform in size, round in form, and give a dark grayish hue to the protoplasma, in which they occur in such quan- tity as to obscure the nuclei. The unpigmented cells are far less numerous than the previous ones. While the first exist in groups, the second tend to appear singly or in small clumps only. The two kinds of cells show marked differences of resistance to venom in solution. In simple sea water, if protected from evaporation, the nerve cells undergo no appreciable change within 24 hours. After that the cells suffer changes in distinctness of outline, but show no evidence of disintegration for some time. One per cent cobra venom: Five minutes: The cells are swollen and rendered less distinctly visible. Twenty minutes: Swelling increased; nerve fiber coarsely granular; yellow pigment granules beginning to undergo solution. Sixty minutes: Protoplasma of large pigmented cells very indistinct; pig- mented cells disappearing. Twenty-four hours: Almost complete dissolu- tion of the tissue. Here and there some of the large yellow pigment granules have coalesced into globules averaging in size the nucleus of a cell. One per cent water-moccasin venom: This venom is distinguished in effect from cobra only by its somewhat weaker and slower action. At the end of 24 hours the cell bodies have been dissolved and the pigment liberated, but the latter may still retain the general form of the cell from which it came. Occasionally the granules may be seen to have coalesced, as in the case of cobra-venom solution. The nerve fibers, which are even more resistant to cobra venom than the cell protoplasma, finally vanish entirely. One per cent crotalus venom: This venom is much weaker in action than the other two. Even after contact for 24 hours many of the cells still keep their outlines, and,the pigment and many fibrils are still preserved and easily visible. 152 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS The experiments here given confirm previous observations respecting the nerve cells of the mussels and giant sea-clam. The nerve cells of these ani- mals are smaller than those of the periwinkle, and are pigmented; but they are somewhat less satisfactory for study than those of the latter animal. On the other hand, they are somewhat less resistant than the cells of the peri- winkle, as is shown by their more rapid disintegration in the controls and correspondingly more rapid neurolysis in venom. B. HISTOLOGICAL CHANGES OF THE NERVOUS SYSTEM PRODUCED BY SNAKE VENOM. Our knowledge of histological changes produced by various snake venoms upon the peripheral and central nervous system was quite defective until about 1900. Many valuable contributions have since been added and to-day we are in a position to corroborate physiological findings by demonstrating gross and minute histological lesions which are accountable for the functional disturbances of the nervous system under the effects of various venoms. Ewing * found the changes occasioned in the ganglion cells of a rabbit by moccasin venom to be somewhat specific and of extreme grade. Nissl stain showed a general disintegration of the chromatic substance. The outlines of the Nissl bodies were completely obscured; the substance had been de- posited in a finely granular form all over the cell body and even in the peri- cellular lymph space. In the majority of the large stichochromes neither formed bodies nor reticulum could be distinguished. It was evident that the lesions went much deeper than the chromatic substance, affecting the under- lying cyto-reticulum, which was granular, disintegrated, and in places com- pletely destroyed. The nuclei were very opaque and the nucleoli often swollen and subdivided. The dendrites were often irregular, shrunken, or detached. These changes constitute a true acute degeneration of the cell, in contradistinction to the simple disturbances of chromatic substance, which may be entirely physiological. Bailey published his result in the same place. According to him most of the cells of the anterior horn of the spinal gray matter were normal, but a small number presented those modifications in their chromatic elements which probably evidence the early stages of acute degeneration, 7. e., an in- crease in the granularity of the chromophilic bodies and a fraying out at their edges, with some distinct loss in chromatic substance. The cyto-reticulum is normal. The nucleus may be normal, or there may be an intensification of the surrounding membrane and a thickening of the strands of the nucleo- reticulum. A few cells are found in which there is much greater loss of chromatin, the cell bodies appearing extremely pale and no distinct chromophilic bodies being present. 1Tn ie Langmann’s article ‘‘Poisonous snakes and snake poisons.’’ The Medical Record, 1g00, ept. 15. NEUROTOXINS OF SNAKE VENOM 153 Kelvington * studied the central nervous system of rabbits killed by sub- cutaneously injecting varying doses of the venom of Notechis scutatus, the Australian tiger snake. The animals died within from 20 minutes to 36 hours, according to the amounts of the venom given. Death resulted from the paralysis of respiration. There were no signs of inflammation present. Except in the animal which died in 20 minutes after the administration of the poison, the nerve cells exhibited signs of degeneration, which were most marked in those animals which lived the longest (and which received the smaller doses). The changes consisted in a breaking up of the Nissl granules into a fine, dust-like deposit, which was scattered through the cell. This breaking-up takes place apparently in several stages, smaller masses being formed which subsequently subdivide. The disintegration of chromatic material takes place either throughout the cell or unequally. A dust-like deposit can be traced in the dendrites. The most extreme changes are seen in cells which appear as shadows containing a few fully staining particles. Swelling of the cell-body is not a noticeable feature, and though the nucleus loses its dis- tinctness in outline, it still, as a rule, remains in the center of the cell. The nucleolus is nearly always present. The position of maximum intensity of the lesions was in the cells about the central canal of the cord, viz, those at the inner side of the bases of the anterior and posterior horns, and especially the small cells in the gray commissure. In conclusion Kelvington made the following points as to the changes observed in nerve cells after poisoning with notechis venom: (1) Chromatolysis, the Nissl granules breaking up into dust. Ultimately all stainable substance disappears. (2) The staining never becomes diffuse. (3) No swelling of the cell occurs. (4) The outline of the nucleus is lost, but it retains its central position as a rule. In the very worst cells the nucleus disappears. The nucleolus is usually distinct, though it sometimes appears loose in the cell. (5) The changes are very unequal in different cells. (6) The cells around the central canal of the cord show the earliest and most advanced degeneration. (7) With rapidly fatal doses no microscopic changes occur. Its degree is dependent on the time the animal survives. (8) Inflammatory and vascular changes are absent. Since 1904 Lamb and Hunter? have been studying the histological lesions of the nervous system caused by various venoms of Indian snakes. The 1 Kelvington. A preliminary communication on the changes in nerve cells after poisoning with the venom of the Australian tiger snake (Hoplocephalus curtus). Jour. of Physiol., r902, XXVIII, 426. 2 The Lancet, 1904, I, 20. Op. cit., 1904, II, 518. Op. cit., 1904, II, 1146; Venom of Bungarus fasciatus (banded krait). Op. cit., 1905, II, 886. Op. cit., 1906, II, 1231. Action of venoms of different species of poisonous snakes on the nervous system. Op. cit., 1907, II, 1017; Venom of Enhydrina valakadien. 154. VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS venoms of four different colubrine snakes and one viperine snake had been carefully studied previously. They experimented chiefly on monkeys, usually giving the venom subcutaneously, in order to obtain a longer action of the poison on the nervous system in general. These two careful investigators found definite signs of chromatolysis at varying stages to be constantly present in the central nervous system of the monkeys injected with these four colubrine venoms — namely, Cobra, Bun- garus fasciatus, Bungarus ceruleus, and Enhydrina valakadien, but not in the case of daboia-venom toxication. In the case of the venom of Naja tripudians definite histological lesions are demonstrable only when the animal lives longer than two or three hours after the injection of the venom. On the other hand, the venom of Enhydrina may produce within go minutes just as pronounced chromatolytic degeneration as in the cases where death does not result until several hours after the injection. The effects of the venom of Bungarus fasciatus are still somewhat different, inasmuch as an incub- ation period of many hours is required before nervous symptoms appear. Where the symptoms appear within several hours, the animal usually dies within 1 to 3 days, but should the symptoms appear in 2 to 6 days, the animal dies in a week or longer with the nervous and muscular atrophic symptoms. Lamb calls the first group the acute and the last the chronic poisoning. Cobra venom is never known to produce the latter form of toxication, and therefore it agrees with the acute form of poisoning of bungarus venom. The intensity and extent of histological lesions observed in all cases of poisoning by these colubrine venoms appear to depend on the period inter- vening between the time of the injection of venom and the time of death. With the same venom, the longer the interval the more marked are the chro- matolytic lesions. In comparing one venom with another the lesions are more pronounced with the kind which kills the animal after a longer duration of toxication. Besides, a certain qualitative difference in activity seems to modify the result, viz, the venom of Enhydrina is not only rapid in action, but also displays a wider affinity for nerve tissues other than the central ganglia. From their vast materials Lamb and Hunter give a résumé in which all observed lesions are so represented as to enable one to comprehend the processes of chromatolysis and the sphere through which the ganglion cells have to pass under the influence of the neurotropic toxins of snake venom. In the first place, there is a deep and rather diffuse staining of the ganglion cells. In this diffusely stained plasma the Nissl granules are to be seen as deeper-stained bodies, still quite consistent, with rather ill-defined edges. The Nissl bodies next seem to begin to dissolve in the cell plasma, and they suggest the appearance of pieces of metal being acted upon in a strong acid medium. This leads to the next stage, which is still that of diffusely stained cells, but with smaller granules in the plasma. Then the granules and the NEUROTOXINS OF SNAKE VENOM 155 diffuse staining begin to disappear and leave a skeleton cell, with its margin, the reticulum, and nucleus being somewhat deeply stained but very well differentiated. The staining later becomes gradually less intense until we reach the stage in which the cell is little more then a shadow of its former self —the typical ‘‘ghost” cell. Vacuoles next appear in the body of the cell and its margin becomes indented as if little pieces had been snipped out. Then portions of the cell disappear altogether and leave little more than a nucleus with the remains of the cell reticulum attached to it. All this time the nucleus, at least in a large proportion of the cells, seems to be little affected otherwise than is shown by a varying intensity of its staining. It remains central, is only exceptionally found at the periphery of the cell, and though it is sometimes lost to view in the diffuse staining of the earlier stages it is almost always to be seen in the later stages of degeneration. Vacuolation is most seen in the pale (ghost-cell) stage, but it is also to be met with in the more deeply stained cells. The connective tissue elements of the gray matter seem to play a somewhat secondary part in this degenerative process. They may be slightly increased in number around the ganglion cell in its earlier stages of chromatolysis, but this increase is not considerable and it is not till vacuolation comes on and the cell begins to break up that they are seen to cluster definitely around the disappearing cell. During this later stage, and sometimes at an earlier stage, they are found indenting the margins and are sometimes inside the body of the cell. The chromatolytic changes just described appear to be uniformly most advanced in the smaller cortical cells and in the cells of the more central group in the anterior horns of the cord. The larger cortical cells and certain of the cells in the lateral groups of the anterior horns seem to be considerably more resistant to the toxin, for they are slower in showing degeneration, and when it does appear it is hardly ever so extreme as in those other cells. Of the motor nuclei in the pons and medulla the ganglion cells of the third and fifth nuclei were usually affected about equally with those of the cortex and cord. But the seventh, tenth, and twelfth nuclei showed changes less often at a later period, and of a less intensity, than any of the other motor cells in any part of the central nervous system. In addition to the above description they found that the venom of Enhy- drina valakadien, when allowed to act for several hours, produces granular disintegration of myelin and fragmentation of axis-cylinder, thus showing its widespread action on the whole nervous system, not only on ganglion cells, but also on nerve fibers. CHAPTER XV. HAEMORRHAGINS OF SNAKE VENOM. One of the most alarming symptoms of poisoning in the cases of Crotalus or viper bites is the enormous swelling and profuse extravasation of blood around the wound. Usually these local disturbances set in within 30 minutes and increase steadily in intensity and extent up to 24 hours or even a longer period, when half of the entire body may be swollen and almost blackish- purple in color. The bleeding from the wound often persists a long time. In animals, especially in warm-blooded animals, sanguine extravasation and swelling are equally grave, and even local sloughing ensues. Cold-blooded animals seem to be less susceptible to the hemorrhagic toxin of venom. The action of crotalus or water-moccasin venom on the capillary vessels of the omentum or mesentery is very rapid and causes an almost immediate rupture of the endothelial wall of these capillaries, followed by free escape of the blood. This phenomenon can be observed directly under the microscope on the mesentery of frog. If we inject a certain amount of crotalus venom into the peritoneum of animals, the abdominal tension commences to rise in a few minutes and within 30 minutes it is highly distended and becomes difficult to compress. In animals killed with rattlesnake venom after intra- peritoneal administration a multiplicity of hemorrhages appears almost constantly, extending over all the serous membranes, the surface of the visceral organs, the diaphragm, the abdominal muscle-layers, the pericardium, the pleural surfaces, etc. The peritoneal cavity (and pleural cavity in less degree) are filled with bloody exudate. In certain marine animals occasionally intracranial hemorrhages and haemorrhages from the gills are observed. Certain crotaline venoms, such as lachesis venoms, produce severe hemorrhage in the alimentary tract when administered through the mouth or rectum. In the pigeon the pectoral muscles which receive crotalus venom become thoroughly soaked with the blood and are accompanied by a marked softening. Now the question arises as to how such extensive and rapid extravasation of the blood is produced. Weir Mitchell and Reichert have rightly pointed out that the hemorrhages are produced by the venom proteids resembling in their physical and chemical reactions the substances classified under the general name globulin. Thus these authors prepared at least two varieties of globulin, by dialysis precipitation and by copper sulphate precipitation. Weir Mitchell demonstrated long ago that the hemorrhagic principles of crotalus venom are non-dialyzable, are destroyed at 75° or near 80° C., are precipitable but not destroyed by alcoholic treatment, are easily destroyed by weak acids but not by weak alkalies, and finally are destroyed in the ali- mentary canal by the action of gastric or pancreatic ferments. The dura- 156 NOGUCHI A. Haemorthages from Capillary and Small Vessels of the mesenterial membrane of Rabbit, under the influence of the venom of Crotalus adamanteus. B. Hatmorrhagic and Softening Effects of the Crotalus adamanteus upon M. pectoralis of Pigeon. (After Mitchell and Reichert.) PLATE 26 HA MORRHAGINS OF SNAKE VENOM 157 bility of the hemorrhagic activity in glycerin or in a dried state has also been shown by this investigator. It is noteworthy that the hemorrhagic activity of various venoms is parallel with the amount of globulin-like bodies in these venoms. One can distinguish two distinct local effects produced by various venoms, one hemorrhagic and the other oedematous. Usually these two effects are - present simultaneously and are likely to be confused, but a closer analysis seems to have revealed their independence. Thus Weir Mitchell and Reichert found that the hemorrhagic effects are the action of the globulins of venom, but the oedematous effects are produced by the dialyzable, peptone-like, proteid fractions. Cobra venom, which is very rich in the peptone-like pro- teins, causes a marked cedema of the locality of the bite or of the injection of venom, without the characteristic hemorrhage in the same degree as in the case of viperine or crotaline poisoning. (Plate 26.) That the venom of vipers is much the same in its general effects as the crotaline venom is recognized by all investigators from the time of Fontana, Mitchell, Fayrer, and Brunton, and for the purpose of avoiding severe local reactions during immunization of animals Calmette employed various chemical and physical agents to eliminate this principle. The clearer and more defi- nite analysis of the hemorrhagic principles of snake venom was, however, demonstrated by Flexner and Noguchi by their biological methods. The venom of Crotalus adamanteus is richest in the hemorrhagic content, and the removal of this principle by means of heating to 75° C. for 30 minutes deprives this venom of nearly go per cent of its toxicity, that is to say, in order to kill an animal with the heated, hemorrhagin-free venom 10 to 20 times the minimal lethal dose of the original, unheated venom is required, and the cause of death in the case of the heated crotalus venom is chiefly due to the presence of the thermostabile toxic principle of neurotropic nature. Again, the removal of the neurotoxic principle by means of brain emulsion, which is capable of fixing the neurotropic principle of various venoms, does not materially alter the hemorrhagic content, and at the same time no diminu- tion in the general toxicity of the crotalus venom takes place. Antivenin which is able to neutralize a large amount of neurotoxins, hemolysins, and hemagglutinins, but not hemorrhagins, is only effective against the fatal effects of certain neurotropic venoms, such as cobra or some ancistrodon venoms, but proves to be totally ineffective against the fatal action of crotalus venom. Inversely, it was found that the antivenin which is strongly anti- hemorrhagic, but not antineurotoxic, is without any protective action against the neurotropic venoms, while effective against hemorrhagic venom. Before the studies of Flexner and Noguchi there was no distinct demarca- tion drawn between the hemolytic and hemorrhagic processes, and they were described in confusion. But these investigators, by various means, soon cleared away the doubts surrounding the case. On one occasion they found that the antiserum from animals immunized with crotalus serum is markedly antihemolytic against crotalus venom, but that having no anti-hemorrhagic 158 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS property it failed to counteract the fatal effects of the venom in vivo. In another instance they found that crotalus venom solution remained almost unaltered in its general toxicity after being kept at 70° C. for a period of 8 weeks, while that of Cobra had undergone a deterioration down to one- tenth of the original activity. The hemolysins of the crotalus as well as the cobra venom had also undergone a considerable diminution during this period. In testing the hemorrhagic activity of crotalus venom, it was quickly found that the content in hemorrhagin was undiminished. In other words, the hemorrhagins of crotalus venom are remarkably stable in this particular respect, although against high temperature, acids, and other chemical pro- cesses, such as oxidation, they are more sensitive to inactive modification. Here the persistence of the hemorrhagins and the general toxicity in con- tradistinction to the other toxic principles is clearly demonstrated. Flexner and Noguchi also demonstrated that the hemorrhagins of crotalus venom constitute the chief toxic constituents of this venom, by showing the difference which arises from the different modes of introduction into the body. With the neurotropic venoms it matters but little in the final issue whether they are injected into the blood circulation, into the muscular sub- stances, into the serous cavities, or under the skin. Usually death follows more quickly when the venom reaches the central nervous system according to the mode of administration, while the minimal lethal dose remains the same or not very different. On the other hand, if the crotalus venom is injected directly into the brain substance or into the cranial cavity, death is brought about by a small fraction of the minimal lethal dose that is esti- mated by the subcutaneous administration of the venom. Taking guinea-pigs of 4oo grams of body-weight, o.cor gm. of crotalus venom, given subcutane- ously, kills in about 3 hours, while a smaller dose than this produces extensive swelling, hemorrhage, and sloughing, but not death. With the same sample of the venom, 0.00005 gm. suffices to kill the animals in about 3 hours when injected into the brain. Thus the direct application of crotalus venom to the brain is about 20 times more poisonous than that administered under the skin. The above experiments clearly point out the difference in the modes of producing fatal effects by cobra venom on one hand and by crotalus venom on the other. Flexner and Noguchi explain this difference on the ground that the neurotoxin, the chief toxic principle of cobra venom, has a specific affinity to the nervous tissue, namely, the ganglion cells of certain parts of the central nervous system, and is not much absorbed or fixed by the other tissues; hence its final effects are nearly the same, irrespective of the mode of injection, although more time is required for the subcutaneous injections than for the intravenous or intracranial administrations. On the other hand, the hemorrhagin, the chief toxic principle of crotalus venom, has a specific affinity to the endothelial cells composing the wall of the blood and lymph vessels, and when it is introduced at a remote part from the vitally important organ, namely, the brain, it has to travel to the latter in order to produce fatal effects, but as the entire system of the living body is sur- HA MORRHAGINS OF SNAKE VENOM 159 rounded and penetrated by a rich supply of blood and lymph vessels the hemorrhagin seldom invades the central nervous system in a seriously large quantity. Judging from the symptoms only, we readily comprehend that the hemorrhagin is most energetically absorbed at the spot nearest the place of venom injection. It is only when the venom enters the blood circulation that danger to life is more apparent; otherwise one will find that hemorrhage will gradually extend wider and wider, but with gradual diminution in its severity, to the remote parts of the body. Naturally a certain portion of the venom necessarily enters the circulation and finally reaches the vitally important region of the brain, and death may follow even the subcutaneous injections, but only after the application of a comparatively large dose. It is certainly not denied that venom hemorrhagin may have double func- tions and that hemorrhagic effects are only one of the two or more properties it possesses; it may attack certain constituents of the central nervous system as well, but, if it has such action at all, it must be altogether different from that of other neurotropic toxins of venom in general, because its symptoms are entirely different. Moreover, the hemorrhagic and neurotoxic effects — assuming their existence — are produced by the same fraction and disappear at the same time; these are inseparable by our present methods. In this connection reference may be made to ricin. As is well known, this phytotoxin possesses three functions, hemagglutinative, hemorrhagic, and neurotoxic. By pepsin digestion we can destroy agglutinating property, but hemorrhagic and neurotoxic effects still persist. At present we can not separate the two effects as the work of two distinct substances. Antiricin can neutralize all three properties. It may be that these effects are due to the actions of their correspondingly active principles, and the neutralization by the antiricin is due to the presence of three distinct anti-bodies in the latter. On the other hand, as the antiserum produced with the non-agglutinating digested ricin is able to neutralize the agglutinating function of unmodified ricin, it is not at all improbable that the toxic molecule of the ricin has three different toxophore groups and one common haptophore group; hence the neutralization by antiricin is to combine with and render the common hap- tophore group inactive. We therefore may consider this point still open to investigation. At all events, Flexner and Noguchi’s view, ascribing the chief toxic principle of crotalus venom to hemorrhagin, must remain unaffected. Flexner and Noguchi made a quantitative determination of hemorrhagin in various venoms. For this purpose intraperitoneal injections of venom were employed in guinea-pigs. The lethal dose having been left out of considera- tion and the animals which survived having been etherized 30 minutes after the inoculation, the existence and degree of hemorrhage were noted. If, as a standard, 1 mg. of cobra venom is taken as representing 10 minimal hemor- rhagic doses, the same quantities of water-moccasin and copperhead venom would contain 100 minimal hemorrhagic doses and of crotalus venom 1,000 minimal hemorrhagic doses. If the quantity of venom necessary to cause death in a guinea-pig, weighing 300 grams, in 4 hours is injected, there will be 160 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS required respectively, 0.coor gm. of cobra, equaling 1 minimal hemorrhagic dose; 0.2 mg. water-moccasin, equaling 20 minimal hemorrhagic doses; 0.6 mg. copperhead, equaling 60 minimal hemorrhagic doses; and 1 mg. rattlesnake venom, equaling 1,000 minimal hemorrhagic doses. The above estimate would amply justify the view of Flexner and Noguchi that the chief toxic constituent of crotalus venom resides in the hemorrhagin. Morgenroth has also shown that the hemorrhagin of,crotalus venom is extremely sensitive to the influence of acids. This investigator found that the inactivation of hemorrhagin can be brought about by a very weak dilu- tion of hydrochloric acid and its action is almost instantaneous. Even if the acid is injected after the venom, provided the venom was promptly followed by the acid injection, no hemorrhage is produced in the peritoneum of guinea-pigs and consequently no fatality results, even from a large quantity of the venom. I was able to confirm his observation to a large extent. Flexner and Noguchi had shown, prior to Morgenroth, that the hzmor- rhagin is very sensitive to acid treatment. They utilize this mode of modifi- cation of hemorrhagin for immunization, the disagreeable local effects having been easily eliminated without impairing the property of venom to produce anti- hemorrhagin in the immunized animals. They considered this phenomenon as an example of toxoid formation of hemorrhagin in Ehrlich’s sense. It is very interesting also to notice that a weak solution of trichloride of iodine produces a similar modification of the hemorrhagin and can be used for an easy accomplishment of crotalus immunization. Flexner and Noguchi have not made investigations as to the comparative merits of the unmodified and modified venoms in producing antivenins, but, at all events, as the degree of immunity reaches a certain point, the unmodified venom may gradually be substituted, should the modified venom prove in any respect inferior to the unmodified in producing strong antivenin. Lachesis flavoviridis s. Trimeresurus riukiuanus contains chiefly hemor- rhagin, while hemolysin, agglutinin, and neurotoxin are present only in trifling quantities. According to Ishizaka, the hemorrhagin of this venom becomes inactive when shaken with chloroform, or acted upon by hydrogen sulphite, ferric chloride, and acetic or hydrochloric acid. The removal of hemorrhagin by heating to 73° C. diminished its toxicity to one twenty-seventh, while chloroform treatment diminished it to one- seventeenth of its original strength. On the other hand, hemolytic and neuro- toxic effects are not reduced by these treatments. Ishizaka also found, as Flexner and Noguchi did, that tryptic digestion of the venom completely destroys its toxicity. The modified hemorrhagin (chloroform, SH,) of this venom was capable of producing anti-hemorrhagin in the animals by repeated injections—this confirming the toxoid formation of hemorrhagin first described by Flexner and Noguchi. The same author made an attempt to remove hemorrhagin by means of endothelial cells of the aorta and richly vasculated organs, but no absorption was observed. Noguchi Plate 27 Z ‘ssh 2 hi % gee c A. Hemorrhage caused by venom of Crotalus adamanteus on the mesentery of Guinea-pig Showing one large and two smaller ruptures of Capillary Vessel. Picture shows be- ginning of hemorrhage. X 1000. B. Hemorrhage caused by venom of Crotalus adamanteus on the mesentery of Rabbit. Showing defect of Capillary Vessel wall caused by disappearance of an Endothelial Cell. C. Hemorrhage in mesentery caused by Crotalus Venom in Rabbit. X 200. HA MORRHAGINS OF SNAKE VENOM 161 HISTOLOGICAL CHANGES CAUSED BY VENOM HA2MORRHAGINS. In endeavoring to discover the precise mode of the action of heemorrhagin Flexner and Noguchi have resorted to the mesentery of guinea-pigs and rabbits. The venom (Crotalus adamanteus) was injected into the peritoneal cavity, or, following Weir Mitchell’s method, a minute particle of the dried venom was placed on the exposed mesentery. The areas of hemorrhage, when in thin, transparent membrane, were spread over small bottle-tops, carefully fastened with fine silk, excised, and hardened, usually in Zenker’s fluid. The staining was done in hematoxylin and eosin. The preparations are transparent and perfectly adapted for the study of the vascular walls. Places showing the smaller, incipient extravasations are suitable for close scrutiny. The changes in the vascular walls associated with hemorrhages are clear and unmistakable. The extravasations take place, not by diapedesis, but through actual rents in the walls. The explanation of the rents is of much interest. That they are not simple ruptures seems to be proved by the dis- appearance, as if through solution, of the parts of the wall at the point of the escape of corpuscles. The solution of continuity is one-sided, and, in some cases, is attended by a displacement of the adjacent endothelial cells, which are . pushed outward, away from the vessel, by the force of the escaping blood. Other phenomena have been noted. Among those of interest is the occur- rence of stasis in vessels, attended, usually by hemorrhage. That the condi- tion is stasis, with the disappearance of the cell-contours, and not agglutination of corpuscles, is shown by the separate state of the red cells beside the vessels in the rare cases of associated stasis and extravasations. (Plate 27, A, B, C.) Giant cells occur in the course of the vessels in which venom changes are going on. They are fusion giant cells arising from intravascular leucocytes. From their size they must almost, or completely, block smaller veins in which they form. The escape of corpuscles is not limited to the red cells. White cells also pass out. Where the latter are noticeable, they are in far greater relative proportion than in the circulating blood. The manner of their escape, namely, by emigration, is easily followed. But especially interesting is the fact that while, in some areas of specimens, the polynuclear cells predominate, in others the mononuclear are chiefly met with. The escape of corpuscles by dissolution of the walls of the vessels is limited to capillaries and small veins. When acted upon by venom both show irregu- lar bulging of the walls, with which enlargements extravasation is often con- nected. It is probable that the points of contact with venom, and of injury of the vascular coat, are many, but only in a part of these does the vessel give way entirely. In conclusion, Flexner and Noguchi made the following statement: ‘‘ We look upon hemorrhagin, therefore, in the light of a cytolysin for endothelial cells of blood vessels, the destruction of which is the direct cause of the escape of blood’ into the surrounding structures.” CHAPTER XVI. VENOM HAEMOLYSIS AND VENOM AGGLUTINATION. THE EFFECTS IN VIVO AND IN VITRO. Fontana, who observed the loss of coagulability of the blood in cases of death from viper poisoning and the anticoagulating effect of that venom upon the shed blood in vitro, failed to discover any alteration of the corpuscles. Weir Mitchell, who noticed similar effects of crotalus venom in cold-blooded as well as warm-blooded animals, saw no perceptible changes in the cellular elements of the blood, either examined immediately after death or when the blood and venom were mixed im vitro, at least not within any brief period of time, as half an hour. He emphasizes, however, that it is a question open to further study whether or not this direct contact would affect them after a longer time. In his subsequent investigations, once with Reichert and again with Stewart, Mitchell found that the venom of Crotalus destroys blood corpuscles after a long contact in a zone of suitable concentrations of the venom. ‘This observation is very important, as he shows there that a too strong solution of venom again fails to bring about destruction of the corpuscles in vitro, and this phenomenon received confirmation by many later investi- gators with the venoms of other species of snakes, for example, cobra venom. From the blood of guinea-pig, rendered less coagulable or incoagulable by the crotalus venom, Mitchell obtained beautiful crystals of hemoglobin which in no way differed from those prepared from the blood of the normal guinea-pig. Weir Mitchell and Reichert (1886) described a peculiar effect of crotalus venom upon the shape of red corpuscles. Under the influence of the venom the erythrocytes first lose their biconcavity and become spherical, without parting with their pigment. They also exhibit great adhesiveness, arranging themselves into aggregations of various sizes and shapes. The corpuscles comprising these groups sometimes appear to fuse so that their outline can not be determined. (This phenomenon was later confirmed by Flexner and Noguchi, who called it venom-agglutination.) This remarkable condition passes away after a short duration, the corpuscles appearing again in separate spherical form. The amceboid movements of leucocytes are seen to be quickly suspended in the venom solution. Fayrer and Brunton (1874) failed to discover any definite alteration of the blood corpuscles when death resulted from cobra venom, except less inclination to form rouleaux and more marked crenation of the corpuscles. Lacerda (1854), working with a species of Lachesis, probably Lachesis urutsu, mentions crenation of the red corpuscles in chronic poisoning. Some 162 VENOM HASMOLYSIS AND VENOM AGGLUTINATION 163 are elongated, deformed, or broken up; others present shining points and then break up into minute fragments. Direct contact of the blood with the venom in vitro produces adhesion of the corpuscles, which then lose their normal forms. Ina few minutes the dissolution is complete. Feoktistow (1888), who worked with the venoms of Crotalus and Vipera berus, found that 2 per cent solution of these venoms produced dissolution of the corpuscles after 18 to 24 hours. Ragotzi (1890) states that the blood corpuscles of mammals injected with cobra venom or directly mixed with it become convex and lose their tendency to form rouleaux. The corpuscles are dissolved after some hours. When a sample of frog’s blood is mixed with the venom the corpuscles at once become pale and the stroma invisible. The nuclei remain for some time. The laked blood still shows the oxyhemoglobin absorption band, but gradually disappears without losing its clear, bright color. C. J. Martin (1893 and 1896) made observations on the effects of the venom of Pseudechis porphyriacus upon the blood corpuscles of various animals. The blood of frog was mixed with an equal volume of a 0.7 per cent solution of NaCl containing 0.1 per cent of venom. The mixture was observed under the microscope. Within a few minutes a disintegration of the corpuscles occurred. The disappearance of the erythrocytes was so complete that at the end of 15 minutes there was nothing except the slight coloration of the field to distinguish the preparation from one of lymph. The action on the “white cells was much slower. At the end of 15 minutes there was no change, but no amceboid movements occurred. Soon nuclei became clearer and then intensely granular, swollen, and finally disappeared. During this time controls were still actively motile. The corpuscles of pigeon were slightly more resistant. The blood corpuscles of different mammals present remarkable diversity in their power of resisting the destructive effects of the venom, and those of the dog were more sensitive than those of any other animal. With this blood 0.00001 gm. of the venom was just sufficient to destroy roo c.c. of blood, either in the body or in vitro. The corpuscles of rabbits, cats, guinea-pigs, and white rats, and especially those of man, were much less sensitive to the destructive effect of venom. Martin observed that in dogs hemoglobin is easily crystallized out from the laked corpuscles, both in vivo and in vitro. The urine nearly always contains such crystals, and in animals which died with suppression of urine two or three days after the injection of the poison, microscopical examination of the kidneys has shown the tubules to be com- pletely blocked with hemoglobin crystals. Hemoglobinuria is a frequent symptom with animals other than dogs, except with minimal or subminimal doses. On examining the blood of animals, immediately subsequent to death from the injection of pseudechis poison, Martin found, except in those cases which succumbed within a few hours after the injection, increase in the number of leuco¢ytes and occasional gathering together of these cells in grape-like 164 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS masses. In dogs injected with pseudechis venom the number of erythrocytes quickly commences to diminish, in some cases to less than half of the original within a few hours. Leucocytopcenia is also striking, as in some instances the number of leucocytes diminished to an almost complete disappearance from the circulation. But after from 30 minutes to 5 hours they reappear almost as numerously as at first. Leucocytopcenia occurs to a trifling extent when the venom is subcutaneously injected. After a few days leucocytosis reaches its height, often 4 or 5 times the normal proportion. The bile contained hemoglobin, as Ragotzi found also in the case of cobra- venom injection. Cunningham demonstrated in several experiments that if the blood of an animal which has received a large dose of cobra venom, either intravenously or subcutaneously, be withdrawn from the body after death and allowed to clot, the serum which exudates from the clot is of a red color; also that, if a fowl has been employed, many free nuclei of the red cells are found on examining the blood at death; there has been, in fact, a considerable solution of the bodies of the red cells. Further, he has shown that if the dose of venom injected into a fowl is very large, namely, 0.1 too.3 gm., the blood at death contains a great abundance of free nuclei, and the remaining red cells appear deformed; and if the specimen be allowed to stand, at the end of a few hours complete destruction will have taken place. In 1898 Weir Mitchell and Stewart ' described the injurious effects exerted by the crotalus venom upon the erythrocytes and leucocytes of the blood of rabbits, snakes, monkeys, and man when mixed outside of the body. The observations were made under the microscope with sealed slide-prepara- tions. Hemolysis was observed with venom-blood mixtures if the venom is not too concentrated, but not with the blood containing an equal quantity of the fresh venom. Lamb (1903) confirmed the experiments of Cunningham on the destructive action of cobra venom im vivo upon the blood corpuscles of various animals. He also states the instances in case of man where cell-free sanguineous exudate was found around the point of snake bite. In a donkey receiving slightly over the minimal lethal dose of cobra venom he observed a blood-stained mucous discharge from the rectum. With daboia venom Lamb made a series of experiments to determine the effects of intravenous and subcutaneous injections of this venom into monkeys, rabbits, pigeons, horses, and donkeys, and obtained results which clearly demonstrate the destructive action of venom upon the corpuscles in vivo. Ante-mortem and post-mortem examinations of the condition of the blood of the venomized animals showed that the serum separated from the loose clots drawn a little before and after death were always stained with free hemoglobin. If cedema existed around the site of injection of the venom it was red color, but contained no red corpuscles. The urine was dark brown. 1 Weir Mitchell and Stewart. A contribution to the study of the action of the venom of Crotalus ada- manteus upon the blood. Trans. Coll. Physicians of Philadelphia, 1897. 3d series, XIX, 105. VENOM HAMOLYSIS AND VENOM AGGLUTINATION 165 In the investigations cited in the foregoing pages the primary object was to determine whether snake venoms act destructively upon the cellular ele- ments of the blood, either imira corpore or extra corpore. Although their modes of determining the injurious effects of venom upon the blood corpuscles did not allow earlier investigators to work it out in a quantitative way, enough data were accumulated to conclude the presence of more or less destructive agents in various venoms thus tested. Hmolysis and also agglutination in certain cases have been clearly demonstrated. In the meanwhile the era of antitoxin and immunity was making rapid progress along all sides of pathol- ogy, resulting coincidentally in the preparations of antivenomous serums, first by Calmette, then by Fraser, Lamb, McFarland, Flexner and Noguchi, Brazil, Ishizaka, and Kitashima; and it was natural that antivenomous serum should be drawn into the study of the hemolytic property of snake venom with brilliant results. As I will give attention to the immunity questions in later chapters I will not discuss at length the relation between antivenin and venom under the pres- ent head, but it is necessary to recognize here the great influence, direct or in- direct, exerted by the Ehrlich school on the study of venom hemolysis. The accurate techniques for determining the strength of toxin and antitoxin in general had been established by Ehrlich and his pupils, and we were thus enabled to distinguish the specific affinity of a given antitoxin to a group of toxins which otherwise would not have been discriminated. Take snake venom, for example: the venoms of different species dissolve the blood cor- puscles of a certain animal in a similar fashion, and it would be impossible to distinguish the hemolytic principle of one venom from that contained in another kind, unless a specific antivenin is used to differentiate them. The same holds good for the toxic constituents other than hemolysin contained in different snake venoms. No wonder, therefore, that pre-antitoxin investiga- tors were often led to regard the active principles for various sets of cells and tissues as identical in any kind of snake venom. Even after the introduction of antivenin in the study of venom, earlier investigations not unfrequently failed to reveal specificity, and it is evident that this was, to a great extent, due to the lack of pure antitoxins, and also, to a small extent, due to the par- tial specificity which easily might have evaded differentiation by a less strict quantitative technique of standardization. The test-tube experimentation with hemotoxic substances was employed first by Ehrlich in his famous studies on ricin and abrin and their antiserums. Of hemolytic substances, Madsen, under Ehrlich, introduced a colorimetric measurement for determining the quantity of liberated hemoglobin in the fluid containing red corpuscles and tetanolysin, thus enabling observation of the intensity of destruction of the corpuscles by the lysin. The calorimetric estimation of the hemolytic strength of snake venom was first employed by Myers and Stephens‘ in their study on cobra venom. 1 Stephens fad Myers. ‘Test-tube reactions between cobra poison and its antitoxin. Brit. Med. Jour., 1898, I, 620. 166 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS These two investigators made the experiments: (1) With a fixed volume of blood mixed with a fixed volume of cobra poison of known strength in a hemocytometer pipette, and the corpuscles in a given field counted from time to time. (2) Mixing known volumes of blood and poison solutions in small test-tubes and observing in which series of tubes hemoglobin was dissolved. Care was taken to have solutions isotonic or slightly hypertonic. It was found that cobra venom has marked hemolytic powers upon bloods of various kinds of animals as well as man, and that these powers could be checked in the test-tube by the addition of the antivenomous serum. Antivenomous serum, alone, has the power to inhibit the haemolysis. All other sera tried were without effect. ‘They also found that relationship held good for mul- tiples of these numbers. The proportions varied with different bloods. In endeavoring to determine whether there might be some relationship between the neutral point im vitro and the protective power of the antivenin in corpore, they found that, for a guinea-pig weighing 250 to 300 gm., 0.0001 gm. of cobra venom was the minimal lethal dose, death ensuing in 5 to 8 hours. ‘Che haemolytic action of this poison was neutralized by o.1 c.c. of isotonic antivenin, and such a mixture they found was never fatal to animals, but if the hemolytic action was completely neutralized the guinea-pig might or might not die. When, however, larger quantities of the venom, completely neutralized as regards haemolysis, were used, they were found to be rapidly fatal on injection, 5 out of 6 animals dying. ‘Thus the investigators conclude that there is no positive connection between the neutralization and the hemo- lytic and toxic actions of the venom. ‘Their results are summarized thus: (1) Cobra poison is strongly haemolytic in vitro. (2) This action is neutralized by antivenomous serum, and the action of the latter is specific. (3) For certain doses (o.ocor gm.) the measure of this neutralization i vitro is a neutralization in cor pore for guinea-pigs. (4) This neutralization is chemical, not cellular or vital. In a subsequent communication’ on the same subject, Myers and Stephens state that poison solutions containing from 0.002 to 0.0075 gm. in I C.C. hemolyze blood not at all; at other times, less completely than weaker solutions do. Dog’s blood is found to be exceedingly sensitive to the poison, haemolysis in this animal occurring in very dilute solution —for instance, in the strength of 0.5 C.C. = 0.000009 gm. Further, with regard to dog’s blood (and the same holds good for frog) it was observed that the haemolysis was often complete in less than an hour in solutions of various strengths, while in the corresponding tubes for guinea-pig and man hemolysis was not apparent for three to four hours, though eventually complete. In continuation of the investigations which he commenced with Myers, Stephens* made another valuable contribution to our knowledge as to the 1 Stephens and Myers. The action of cobra poison on the blood. A contribution to the study of passive immunity. Jour. of Pathol. and Bacteriol., 1898, V, 279. 2Stephens. On the hemolytic action of snake toxins and toxic sera. Jour. of Pathology and Bac- teriology, 1900, VI, 273. VENOM HASMOLYSIS AND VENOM AGGLUTINATION 167 biological nature of haemolytic and toxic constituents of snake venoms and certain toxic serums. In this article Stephens confirmed the peculiar anti- hemolytic property of cobra venom when employed in a strong concentra- tion, namely, more than 0,cooor gm. in 1 c.c. He failed to find the reason for this non-occurrence of haemolytic phenomenon in a concentrated venom solution, but at the same time he brought out another puzzling phenomenon with cobra hemolysis — that the addition of a large quantity of horse serum to such mixture (large quantity of the venom and the blood) induces a rapid and complete hemolysis. At that time this was entirely inexplicable, although we to-day understand it and I will later return to this phenomenon. In testing the neutralizing power of Calmette’s antivenin against the hamo- lytic principles contained in various snake venoms, Stephens found that it had a very slight neutralizing action on the hamolysins of the venoms of Daboia, Crotalus terrificus, and Pseudechis porphyriacus. Against cobra venom the antivenin neutralized the lytic action of 0.00045 gm., or about 4 minimal lethal doses, while the same quantity could neutralize only 0.00003 gm. of daboia venom, a quantity far less than the lethal dose. Stephens states that normal or antivenomous serums of horse sometimes produce quite marked and progressive haemolysis and sometimes an opposite effect. Thus, when antivenin was introduced in a dose insufficient to neutralize cobra venom completely, a much prompter hemolysis may occur than in the saline venom solution. Here he expresses his uncertainty as to the identity of the hamo- lytic principles existing in different venoms. Stephens’s observations on the hemolytic and toxic effects of the serums of Tropidonotus natrix, python, frog, and eel, as compared with those of venoms, are very interesting. With the tropidonotus serum he found a cer- tain antihemolytic action of antivenin, while normal horse serum had none. He found that a rabbit immunized against the tropidonotus serum to quite a high degree succumbed quickly to a single lethal dose of the serum of an Australian python, and concluded that the haemolytic as well as toxic constitu- ents of these two serums can not be identical. While the serum of horse had marked promoting effect upon the cobra as well as daboia venom hemolysis, an inhibitory effect was found in the serums of the snake. Stephens filtered a 0.25 per cent solution of cobra venom through a gelatinized bougie, which had previously been used to filter bullock serum, until the filtrate gave no albumin reaction. By repeated filtrations under high pressure, the filtrate was tested each time for its haemolytic and toxic actions. The filtrate was still active after the third passage, but no more after the fourth. A trace of biuret reaction was given with this last filtrate. In 1900 Walter Myers contributed another very exhaustive study on the nature of toxic constituents of cobra venom. From his own experiments, as well as from those of many of his predecessors, notably those of Weir Mitchell and Reichert, he concluded a priori that in the venom of cobra di capello there are present at least two poisonous substances. One of these is ham- olytic, and the other causes death, probably by its action on the respiratory 168 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS center in the medulla oblongata. Myers called the first cobralysin and the second cobranervin. ‘The reasons for their independent existence were first furnished by the experiments of Mitchell and Reichert,! who found that the hemolytic principles are precipitated and destroyed by heat before the nerve poison is affected. The second reason is deduced from the fact that the fatally acting principles are set free in a considerable amount when multiple doses of cobralysin are completely neutralized by antivenin. It is only for the minimal lethal dose, or a little over (using guinea-pigs as test animals), that the neutralizations of the two effects run hand in hand. The third reason is that the susceptibility, im vitro, of the red corpuscles of various animals bears no relation to the susceptibility of those animals to subcutaneous inoculation of the venom. In this series of study Myers adopted Ehrlich’s* famous fractionated satura- tion method‘ for diphtheria toxin-antitoxin to analyze the combining prop- erty of cobralysin for anticobralysin. The selection of suitable blood for estimating the haemolytic power of cobra was by no means an easy task, for the corpuscles of the most susceptible animals (namely, guinea-pig and dog) were often hemolyzed by the horse serum alone, rendering the determina- tion of the true action of the venom extremely difficult. The corpuscles of rabbit were more resistant than these two specimens, but much more venom was necessary to obtain enough reaction, and this again made it inconvenient, as the antihemolytic power of the antivenin he had in hand was so weak that it required 1 to 2 c.c. to counteract the hemolytic effect produced by 0.001 gm. Myers finally came to use human blood, which, when suspended in 1 c.c. of isotonic (0.8 per cent) saline solution, was hemolyzed by 0.000003 to 0.000005 gm. in 2 hours at 15°C. It was found that the hemolytic action of o.cor gm. of dried venom was neutralized by 1.3 c.c. of the antivenin. Theoretically the addition of one-thirteenth of this amount of the serum 0.001 0.001 should neutralize =;- gm., and the second fraction again another “3 gm. and so forth. But, in reality this was found not to be the case. The first frac- tion, instead of neutralizing the theoretical portion of the venom on gm., neutralized o.cor X 0.8, leaving only o.cor X 0.2 gm. still in the fluid unneutralized. Speaking of the hemolytic units, o.cor gm. contained 2,000 units, but after the addition of 0.1 c.c. of the antivenin only 4oo units were found to be present in the mixture, whereas it should, theoretically, neutralize only a“ = 153 units by this fraction. The addition of 0.2 c.c. of the anti- venin left 200 units, 0.4 c.c. 125 units, 0.6 c.c. 58.8 units, 0.8 c.c. 20 units, etc. Finally 1.3 c.c. left a dose less than 1 unit of cobralysin. These paradoxical phenomena, first seen by Ehrlich and then by Madsen with other toxins, led Myers to assume that cobralysin consists of a set of substances whose toxic effect can not be observed, but have an antitoxin- 1 Weir Mitchell and Reichert. Smithsonian Contrib. Knowl., Washington, 1886. 2 Stephens and Myers. Jour. of Pathol. and Bacteriol., 1898, V, 279. 8 Ehrlich. Die Wertbestimmung des Diphtherieheilserums. Klin. Jahrb., 1897, VII, 299; and Ueber die Constitution des Diphtheriegiftes. Deut. med. Wochenschr., 1898, XXXVIII, 597- 4 Madsen. La constitution du poison diphthérique. Ann. Inst. Pasteur, 1899, XIII, 568. VENOM H#®MOLYSIS AND VENOM AGGLUTINATION 169 combining property of varying affinity. With this assumption Myers explained these results, and thought that cobra venom contains, besides real intact hemolysin, a number of toxoids, which are still able to unite with the anti- toxin, although their hemotoxophore side-chains are inert. In some instances he found that the first fraction of the serum neutralized somewhat less than the second. In these cases the presence of prototoxoids was assumed. In fact, according to this hypothesis, the neutralization of a given amount of cobralysin means the neutralization of toxin and toxoids (prototoxoid and deutotoxoid, but not the least reactive epitoxoid) ; and the amount of any one fraction of the antivenin to combine is always the same (only differing in the toxic expression), which is due to the toxin alone, but not to the toxically inert, antivenin-combining toxoids. At last, Myers tried to determine the reason for the fact that in a venom there can be present toxin and toxoids. He found that when a solution of cobra venom is placed at room temperature (still quicker at 37°C.) its hemolytic power becomes rapidly reduced. This reduction in hemolytic power was not followed by a parallel loss of its com- bining power with antivenin. In other words, hemolytic toxoids were formed in such solutions. THE NEW ERA OF THE STUDY OF VENOM HAMOLYSIS. Definite proofs of the existence of hemolytic and hemagglutinative sub- stances in different venoms in varying proportions have thus been amply brought about both in corpore and in vitro. In the last few years prior to 1900, the investigations assumed a character of quantitative estimation of the hemolytic principles in various venoms and suggested their specificity as well as their insignificance in the lethal properties of these venoms. The estimate made of their action upon the blood corpuscles was, however, not understood at all. It was Flexner and Noguchi who for the first time found that the active principles of the venom require a second substance to mani- fest their solvent function upon the blood corpuscles, and this marks the opening of the new era of study of the hematoxic actions of the venom. In 1902 these investigators discovered that certain venoms do not dissolve the blood corpuscles from which every trace of serum has previously been removed by repeated washings in an isotonic saline solution. But if the separated serum is restored to each of the several kinds of blood corpuscles treated with venom, lysis takes place. They found that the substance or substances of those serums capable of activating the hemotropic principles of venom have the properties of serum complements, inasmuch as the elective removal of the serum-amboceptors for each kind of the washed corpuscles by absorption in the cold (0° C.) did not impair their activating property, and heating the serum to 56° C. for 30 minutes deprived some of the serums of this activating action. Neither did solution of the venomized corpuscles take place at o° C. The abolition of the activating property by heating to 56° C. was observed with the serums of rabbits, guinea-pigs, and Necturus, but seldom with that of dogs. The velocity of the haemolytic process was 170 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS found to be extremely variable, according to the kind of complement em- ployed. Thus dog’s complements produced very rapid solution of the venom- ized corpuscles, while with those of guinea-pig and rabbit lysis proceeded slowly, taking many hours to complete the reaction. In a second series of experiments Flexner and Noguchi showed that the blood corpuscles of sus- ceptible blood take up the hemolytic principle of venom by absorption. The supernatant fluid obtained by separating the corpuscles from the venom solution by centrifugalization becomes far less active upon the same kind of cells, but is powerfully active upon the other kinds, to which it gives in turn, by absorption, the hemolytic bodies electively if each is treated in succession. From these observations Flexner and Noguchi drew an analogy between the mechanisms of venom hemolysis and serum hemolysis: whereas the inter- mediary bodies in the first are present in venom itself, in the second both the intermediary bodies and the suitable complements are present in the same serum. In Ehrlich’s terminology venom contains amboceptors and the serum contains complements. (Plate 28.) Flexner and Noguchi next mentioned that venom, especially that of Crotalus adamanteus, Ancistrodon piscivorus, and Ancistrodon contortrix, produces agglutination of the washed corpuscles. With the defibrinated blood or the washed corpuscles mixed with suitable activating serum, there is only a momentary agglutination or none, as the cells become quickly dissolved. The more resistant the corpuscles and the stronger the venom solution, the more pronounced and lasting is the phenomenon of agglutination. Venom agglutinins are distinct from venom hemolysins. The corpuscles agglu- tinated by ricin are readily dissolved by venom hemolysins, unless there is avery prolonged action of the former, with more or less delay in liberating hemoglobin from the firmly conglomerated stroma. The same authors studied the effects of venom upon leucocytes in vitro. In studying the changes taking place in the leucocytes under the influence of venom a warm stage (37° C.) was used, the edges of the cover-glasses having first been sealed with vaseline. The leucocytes were obtained from the pleural or peritoneal cavity by injection of chemotoxic substances. The venom solutions varied from 10 per cent to 0.002 per cent in isotonic saline solution. With cobra venom almost no effect was observed in a solution of 0.002 per cent, whereas 0.002 per cent of rattlesnake venom and 0.005 per cent of moccasin venom caused definite changes. Only the granular cells showed motility. Weak active solutions are with- out immediate effect on motion, but begin to manifest an inhibiting action after about an hour, the controls being still motile at the end of 2 hours or longer. After the motility ceases, the cells in general, except the lymphocytes, show increased granulation due to the appearance of coarser and more numer- ous granules in the protoplasma, the nuclei coincidently becoming more distinct. After 6 hours the majority of the largest granular cells have already disintegrated, the nuclei having been liberated. After 24 hours most of the NOGUCHI PLATE 28 Siete ee ee =e Om eg i_< = 7 J ae Naaual: 24 Gan in normal saline at 37° C. vv waa C. Crotalus venom, 0.5 per cent; 6 hours at 37° C. Effect on White and Red Corpuscles of Rabbit. (Drawings by Noguchi.) >. NOGUCHI PLATE 29 ee: Be es eg! a ee cent; 24 hours at 37° C. A. Crotalus venom, 0.5 per FCO 5% acct 0. ¢ os *o x 0 ve fe ve ang ,% ats tad 2 “a e - ” Sie" de ‘ ; mn ees os A ee | he ne °° e4 e, = i : . 4 ae 5 : > : Me Bo C. Cobra venom, 0.5 per cent; 20 minutes at 37° C. Effect on White and Red Corpuscles of Rabbit. (Drawings by Noguchi.) ' VENOM H#MOLYSIS AND VENOM AGGLUTINATION 171 medium-sized granular cells have suffered disintegration, while lymphocytes show but slight and inconspicuous changes. Stronger solutions, varying from 0.2 per cent to ro per cent, cause instantaneous cessation of motility and rapid agglutination without distinction of variety of cells. Within 5 to 3o minutes thereafter dissolution sets in, affecting first the largest, then the medium-sized cells, and finally the small lymphocytes. There are variations in the activities of the several venoms and in the com- pleteness of solution of the cells. Rattlesnake venom is far less active than that of the Cobra. Thus in 2 per cent solutions cobra venom causes complete solution in 30 minutes, while that of the rattlesnake requires 2 hours to bring about the same result. (Plate 29.) The effects upon the washed leucocytes differ from those described in that venom solutions cause agglutination, but with the production of only very little lysis. The next question to decide was whether the hemolysins (erythrocytolysins) are identical with the leucocytolysins. The supernatant fluid free from the erythrocytolysins, as obtained by the usual absorption of copperhead venom with the washed corpuscles of rabbits, was allowed to act upon the leucocytes of the same animal. There was no agglutination, but a complete solution of the cells took place within 30 minutes. On the other hand, the parallel experiment in which venom solution was treated with washed leucocytes yielded a fluid still active for defibrinated blood. From these observations they concluded that — (rt) Venom contains principles which are agglutinating and dissolving for leucocytes. (2) The agglutinating principles may be identical for both white and red cells. (3) The dissolving principles for leucocytes are distinct from those for erythrocytes. : (4) In order that solution of venomized corpuscles shall occur a comple- ment-containing fluid is required. (5) The several varieties of white cells of rabbit blood show different susceptibilities to the action of venom. Calmette made a very important observation in regard to the mechanism of hemolysis caused by venom. He confirmed the finding of Flexner and Noguchi that venom requires a second substance or substances contained in blood serum in order to accomplish its dissolving action upon the corpuscles. Moreover, he found that the substance or substances of serum concerned in venom hemolysis differ from ordinary serum alexines in that they do not lose their activating property at 62°C. At this temperature there may be some diminution of this power, but if heated to 100° C. the venom-activating action of all serums becomes much more powerful than the unheated serums. Closely following the work of Flexner and Noguchi on one hand, and that of Calmette on the other, Preston Kyes, under the direction of Ehrlich, made 172 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS a brilliant contribution to our knowledge of the nature of venom hemolysis. As will be seen below, Kyes, in his first article, confirmed the observations of Flexner and Noguchi as well as those of Calmette and explained at the same time the apparent discrepancies existing between the results obtained by these investigators. He found that there are two kinds of blood corpuscles, according to their susceptibility to the hemolytic action of snake venom — cobra venom being chiefly employed: (1) the corpuscles which undergo hemolysis by venom without a second substance; (2) the corpuscles which become hemolyzed only when auxiliary substances (complements, etc.) are present at the same time. The corpuscles of guinea-pigs, dogs, and man were found to be most susceptible, and those of rabbits and horses quite resistant, while those of the ox, sheep, and goat were completely refractory to the cobralysin. 1 c.c. of 5 per cent suspension of the first-named corpuscles is hamolyzed by only 0.25 c.c. of o.or per cent venom solution, while those of the horse are dissolved by 1 c.c. of 0.1 per cent concentration, requiring nearly 40 times more venom than in the case of corpuscles of dogs or guinea- pigs. Kyes found that the insusceptible kinds of corpuscles can be dissolved by venom if certain suitable fresh serums be introduced. ‘Thus he gives the following combinations as corresponding with the examples of amboceptor- complement-like phenomenon of venom hemolysis: Ox corpuscles, guinea- pig serum; sheep corpuscles, guinea-pig serum; rabbit corpuscles, guinea-pig serum; horse corpuscles, ox serum. ‘The complementing property of guinea- pig serum is seen to disappear by heating to 56° C. for 30 minutes. He was also able to confirm that the cobra amboceptors are absorbed by sheep cor- puscles at o° C., even in the presence of guinea-pig serum. After having confirmed the facts described by Flexner and Noguchi in cer tain particular instances, Kyes now proceeded to clear up the phenomenon why certain kinds of corpuscles are attacked by venom directly. It was found that the susceptible corpuscles contain in their constituents certain substances capable of activating cobra venom. ‘This group of ‘‘activators”’ was called endocomplement and found to be thermolabile. From the ox corpuscles he was able to obtain active endocomplement, notwithstanding these cor- puscles are entirely insusceptible to the cobralysin in their integrity. This phenomenon was explained by assuming that the endocomplement of this kind of corpuscles exists in an unavailable state, but becomes accessible after their disintegration, through which process endocomplement was prepared. Returning to Calmette’s phenomenon, namely, the acquisition of venom- complementing property of various serums, irrespective of whether one was incapable of activating venom in its fresh state or not, after heating to a tem- perature above 62° C., Kyes found that heating all kinds of blood serums to too° C. invariably renders them activating for cobra venom, and, indeed, more active in this respect than in an unheated native state. Finally Kyes discovered that the thermostabile venom activator of blood serum can be VENOM H#®MOLYSIS AND VENOM AGGLUTINATION Ls extracted with alcohol, and that of the substances extracted with alcohol lecithin alone possessed the property of activating cobra venom in the same manner as aheated serum. ‘The hemolysis produced by the combined action of cobra venom and lecithin differed from that caused by the combination of cobra venom and certain suitable blood serum (complement) in the former’s rapid completion, and also its occurrence even at 0° C. Kyes and Sachs went deeper into the search for the closer mechanism of venom hemolysis produced by certain fresh serum on one hand and lecithin on the other. In order to decide whether the venom-activating property of the fresh serum of guinea-pigs is different from lecithin of that serum, they first employed the fresh serum of guinea-pig to see if in small quantities unable to activate the venom this serum still inhibits the venom-activating action of lecithin. It was found that this serum exerts a powerful anti-lecithin action in a dilution incapable of activating venom, showing that its anti-lecithin component exceeds in amount the venom activator contained in it. In still another experiment the serum of a rabbit immunized against guinea-pig complement was employed. This serum became highly antilecithinic after heating to 56°C. This lecithin-inhibitory property was saturated with the addition of lecithin, and then its action upon the venom-activating property of fresh serum of guinea-pig was tested. The result shows that this mixture inhibited the latter’s venom activation in a very small amount. A third differentiation was made by digesting the fresh serum of guinea-pig with papain, which destroyed the activating power of the former, although it had no effect upon the venom-activating action of lecithin. A fourth differentia- tion was found in the elective inhibitory action of cholesterin upon the activat- ing property of lecithin, whereas venom-complement hemolysis is seen to remain unaffected. Taking up once more the question of the nature of the corpuscular venom activator —their original endocomplement —Kyes and Sachs next sought the seat of the activator in the cells. They quickly found that the stroma is activating, but not the soluble laked fluid of the red corpuscles. By alcoholic extraction the activator was obtained in proteid-free state, and behaved in the same manner as lecithin. Again, as to the activating property of the washed stroma, they state that it corresponds with that of lecithin, as it is not inactivated at 62°C., is quite active at o° C., but inhibited by cholesterin. Thus, no endocomplement longer exists. The thermolability of the aqueous solution of disintegrated red corpuscles was found to be due to the simultaneous presence of hemoglobin, which at 62° C. combines lecithin and renders it inactive in regard to venom hemoly- sis. In a series of experiments they showed that lecithin combines with hemoglobin when heated together in solution to 62° C. Kyes and Sachs proceeded further to ascertain the degree of susceptibility of the blood corpuscles of different species of animals to cobra venom, either with the addition of a sufficient amount of lecithin, or in their native state. 174 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS The preparations of lecithins which they employed for venom activation were only slightly hemolytic, and it required nearly 200 times the amount necessary for the activation of cobra venom. A preparation of kephalin was also capable of venom activation, while sinapin was devoid of this property. The following table states the results in succinct form: TABLE 7. With 0.2 c.c. 0.025 per ents cent lecithin solution. No lecithin. I C.c. 5 per cent suspension. ©.00000005 gm. 0.000025 gm. ©.000000I gm. Insusceptible. 0.00000025 gm. 0.0005 gm. ©.0000005 gm. 0.000025 gm. Insusceptible. 0.000025 gm. 0.001 gm. 0.00025 gm. 0.00001 gm. In closing, Kyes and Sachs state that the addition of a small amount of hydrochloric acid to cobra venom increased the resistance of the latter’s hemolytic substance to the temperature of 100°C. The concentration of this acid was one-eighteenth normal, to prevent quick heat-destruction of the venom, in which almost no diminution of lytic power took place in 30 minutes’ boiling, although it was entirely inactive after 2 hours’ heating. Kyes finally succeeded in preparing a new compound which possessed considerable hemolytic activity from the mixture of an aqueous solution of snake venom and chloroform solution of lecithin. The process of pre- paration has been described on page 86. This new compound is the venom lecithid and was obtained in a microcrystalline form. Usually it quickly separates out from the aqueous solution and becomes amorphous. It differs in physico-chemical properties from the native venom by its solu- bility in various organic-fat solvents and in its enormous resistance to high temperature. Its hemolytic action is almost instantaneous and uninfluenced by low temperature (0° C.). It is almost non-fatal, although if given sub- cutaneously in a very large quantity infiltration takes place. It differs from lecithin in its insolubility in ether. Cholesterin has inhibitory action upon the hemolytic action upon venom lecithid. It is important, after the shaking of the venom solution with chloro- form solution of lecithin, that the aqueous portion of the mixture does not contain any further hemolytic substance, while the neurotoxic principle remains still in solution. Besides cobra venom, the venoms of Lachesis lanceolatus, Lachesis anamal- ensis, Lachesis flavoviridis, Crotalus adamanteus, Vipera russellii, Bungarus céruleus, Bungarus fasciatus, and Naja haje were found to form the lecithids. Flexner and Noguchi * kept up their studies on snake venom and found in the meanwhile that the venoms freshly squeezed out of the poison glands of 1 Flexner and Noguchi. The constitution of snake venom and snake sera. Jour. of Path. and Bac- teriol., 1903, VIII, 379. VENOM HAMOLYSIS AND VENOM AGGLUTINATION 175 various crotaline snakes, Crotalus adamanteus, C. terrificus, C. confluentus, Ancistrodon contortrix, and A. piscivorus, do not contain complements and are inactive without the help of second substances of certain blood serums. They found that even repeated washings fail, with some samples, to prevent subsequent dissolution when mixed with cobra venom. With the corpuscles of dogs the removal of serum had the least influence on the retardation of hemolysis. In explaining this phenomenon they assumed the presence of intracellular complements in these corpuscles. In fact, Flexner and Noguchi succeeded in extracting intracellular complements from the dog’s corpuscles by suspending them in the heated serum over night. If the heated serum is separated next day from the corpuscles by centrifugalization and then tested for its hemolytic property on washed corpuscles of guinea-pig, there will be more or less marked hemolysis. The controls with the heated serum of dog or the saline supernatant of the washed corpuscles of dog do not cause any hemolysis. When the washed corpuscles of dog are repeatedly digested in the heated serum of dog (suspension and washing in several successions) the susceptibility of the digested corpuscles to the hemolytic action of cobra venom is seen to be far less than that of the corpuscles simply washed re- peatedly in isotonic saline solution. This phenomenon is of a dual nature, due sometimes to the removal of intracellular venom activators and then to absorption by the cells of the anticomplementary principle’ which has developed in the heated serum of dog. Flexner and Noguchi then studied the nature of venom-intermediary bodies,” especially in regard to their cytophilic and complementophilic groups. The remarkable feature of venom-intermediary bodies is that they are active with various kinds of foreign blood serums. Those who have worked with normal and immune serum hemolysins must recognize that the complemento- philic groups of the amboceptors of these serums are better fitted with the native complements, hence more active in that combination. In other words, as shown by Ehrlich, Morgenroth, Sachs, Neisser, and Doering, the native serum contains more suitable complements for the homologous amboceptors than the foreign serums. Now, why are venom amboceptors active in the presence of foreign serums? What will be the result when we furnish them with their homologous complements? What will be the relation of venom amboceptors to the native and foreign serum complements? In this series the venoms employed were fresh and the complements were obtained by allowing the given kind of corpuscles to absorb all specific serum amboceptors for them by the cold method. The complements employed were from the serums of Crotalus adamanteus, Ancistrodon piscivorus, and Pityophis cateniferis. Each complement was tested for its activating value for the hemolytic amboceptors of cobra, moccasin, copperhead, and rattle- snake venoms. The corpuscles subjected to the action of the combination of snake complement and venom in variable orders were those of the dog and 1 Noguchi. On the thermostabile anticomplementary constituents of the blood. Jour. of Exper. Medicine, 1906, VIII, 726. 2 Intermediary body = Ehrlich’s amboceptor and Bordet’s substance sensibilisatrice. 176 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS guinea-pig, and had been freed from their native complements by washing. The results obtained are closely in parallel. All four of the venoms become active upon the addition of a suitable quantity of crotalus complement. The hemolytic process is, however, very much slower than in the cases where the serums homologous to the corpuscles are added. It was found, again, that the velocity of the hemolytic reaction is increased by using larger quantities of the venom, in combination with the crotalus complement. From this fact Flexner and Noguchi considered it possible to divide the intermediary bodies of venom into two groups, namely, the isocomplementophilic and the heterocomplementophilic. It appears to be remarkable how far the number or amount of the heterocomplementophilic amboceptors exceed the isocom- plementophilic in all venoms. From this it at once becomes evident why venom in general is so powerfully destructive when introduced in a heterolo- gous system, while it has almost no effect upon the homologous blood. The behavior of the complement of Pityophis cateniferis is quite different from the crotalus complement. With none of the venoms here employed did it produce a complete hemolysis even after a very long period of action. As the controls underwent more or less complete hemolysis after some hours, especially with cobra venom, the addition of the pine-snake complement is seen to have acted antihemolytically, at least to a certain extent. There is an indication that the complement of this snake is far less suitable in activating the venom lysins than that of Crotalus adamanteus. In a subsequent series of experiments they found that the crotalus complement can promptly reacti- vate the crotalus-serum amboceptors (the serum heated to 56° C.), but only partially those of the pityophis blood, and vice versa. According to the activability of the venoms by snake complements on one hand, and by heterogeneous complements on the other, Flexner and Noguchi have shown the relative amounts of the isocomplementophilic and hetero- complementophilic intermediary bodies in crotalus, moccasin, copperhead, and cobra venoms. In the case of the corpuscles of guinea-pigs, crotalus venom contained about 7 times as much of the isocomplementophilic as of the heterocomplementophilic intermediary bodies. For the corpuscles of dogs, on the other hand, the hetero-body is present in excess in about the proportion of 12 to 1. With the other venoms the heterocomplementophilic bodies are found to exist in excess in about 60 to roo times. That these figures would differ were the native complements available in each case is indicated by the distinctly weaker action of pine-snake serum upon crotalus venom as compared with its own serum. Flexner and Noguchi then took up the question of the haptophore groups of the venom-intermediary and serum-intermediary bodies. First the cor- puscles of the dog, guinea-pig, and pigeon were subjected to the action of the crotalus serum previously heated to 56° C. for 30 minutes in order to abate the action of the complement. After contact for a few hours the corpuscles were separated by centrifugalization and washed in 0.85 per cent salt solution. These corpuscles had fixed the intermediary bodies to themselves, as they VENOM HMOLYSIS AND VENOM AGGLUTINATION i7T underwent hemolysis upon introduction of the crotalus complement, but scarcely at all when heterogeneous complements were substituted, which shows that the serum-intermediary bodies in this case required for their lytic function the homologous complement. In other words, the crotalus serum- intermediary bodies are isocomplementophilic. Now, what became of the susceptibility of these serumized cells to the action of venom-intermediary bodies? Are they still able to find the venom-inter- mediary bodies and produce hemolysis when a heterogeneous complement is introduced at the same time? Of course, the crotalus complement can not be employed in this test, for the serum amboceptors will grasp the homologous complement and bring about the hemolysis. The serumized corpuscles have proved to be very resistant to the combined addition of crotalus venom and the serum of guinea-pigs (in the case of corpuscles of the guinea-pigs) or that of dogs (in the case of corpuscles of dogs). In fact, almost no hemolysis commenced even after many hours’ contact, whereas complete hemolysis proceeded in the normal manner and rapidity with the corpuscles which had not been previously digested in the inactivated crotalus serum. It would appear from this that the receptors of the corpuscles had been saturated with the serum-intermediary bodies of the crotalus serum and became afterwards unable to fix the venom-intermediary bodies; hence there was no hemolysis in the presence of a heterogeneous complement, which, as already mentioned, is capable of activating the latter set, but not the former set, of the hemolytic amboceptors. It tends to show that the haptophore groups of the crotalus serum-intermediary bodies and the crotalus venom-intermediary bodies are identical and saturate the same set of receptors in the blood corpuscles. If in the foregoing experiment we replace the crotalus venom with water- moccasin or cobra venoms, the inhibition exerted by digesting the corpuscles in the inactivated crotalus serum becomes far less pronounced than is the case with the first venom. What is the reason for these differences? It seems most probable that the hemolytic intermediary bodies in these last two venoms differ from the crotalus intermediary bodies in their structure of haptophore groups and unite with quite other sets of receptors of these corpuscles. Hence the addition of heterocomplementophilic intermediary bodies of cobra or moccasin venoms brings about hemolysis, although some delay is also noticed here. A reversion of experimental orders of the above led Flexner and Noguchi to exactly the same conviction, but through another path. The washed corpuscles of dog or guinea-pig can be easily venomized with crotalus or moccasin venom without dissolving them, at least during several hours. Now, after venomization, still better if the venom be left in the mixture, the corpuscles are subjected to the action of the fresh serums of crotalus or pine snake. What actually happens there is that a previous treatment of these corpuscles with these venoms in strong concentrations, or the simultaneous presence of these venoms in large enough doses, interferes with the hemolytic action of;snake venoms. 178 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS If a fairly large amount of the fresh snake serum is added to the washed corpuscles alone, complete hemolysis will result in a few minutes. But if the same quantity of the serum is added to the venomized corpuscles hemolysis is always imperfect or complete only after many hours’ contact, this especially being more pronounced (inhibited) when moccasin venom is employed. Why is it? It is because the preoccupancy of the receptors of the blood corpuscles by venom-intermediary bodies means the possession of the majority of the receptors by isocomplementophilic intermediary bodies in the case of crotalus venom, but by heterocomplementophilic intermediary bodies in the case of moccasin venom; hence hemolysis may be complete in the first instance in the presence of the homologous complement, but only little in the second instance on account of the non-availability of the venom- intermediary bodies, in this case of the crotalus complement. Even if the moccasin complement be used, hemolysis proceeds slowly. Again, if, instead of homologous serums, some quick-activating heterogeneous serums should be employed, the venomized corpuscles undergo hemolysis, the promptness of action being proportionate to the amount of venom, provided a certain limit is not passed beyond which another phenomenon complicates the process of the antihzmolytic action of venom. Flexner and Noguchi finally resorted to some other methods to identify the haptophore groups of venom-intermediary bodies and serum-intermediary bodies. They found that the anti-serum for crotalus serum was quite anti- hemolytic against the crotalus hemolysin, but less so against moccasin or cobra venoms. Antivenin prepared by Calmette was quite antihzmolytic against crotalus serum. ‘The reason why this antivenin was effective in neu- tralizing the hemolytic action of crotalus serum, as well as crotalus venom, may find its explanation in the fact that Calmette immunized his horses with a mixture of several venoms, of which crotalus venom was a component. Venom hemolysis was found to proceed uninfluenced by the presence of cholesterin. This fact was later confirmed by Kyes and Sachs, who in case of venom-lecithin hemolysis found also a marked protection displayed by this substance. Venom-agglutination occurs with the corpuscles hardened in to per cent formalin in Hayem’s solution, but no hemolysis. The approximate units of hemolytic activity of various venoms upon different kinds of defibrinated blood in vitro are given by Flexner and Noguchi in the following table, which also shows the coincidental agglutinative value: TABLE 8. Minimal hemolytic dose in 1 mg __ Minimal . ; Agglutinative dose in 1 mg. Venom. Guinea-pig. ; Dog. Guinea-pig. Cobrak.iqss.neee see 378 4 20 Water-moccasin 200 230 250 Copperhead ...:.... 100 125 400 Rattlesnake? ja4 8 5 12 VENOM HMOLYSIS AND VENOM AGGLUTINATION 179 The toxoid formation of hemolytic principles, first observed by Myerswith the cobra venom, has been confirmed by Flexner and Noguchi with the Ameri- can venoms. They did not find such rapid deterioration as was mentioned by Myers. They found that hydrochloric acid in the concentration of 2 to 3 per cent caused only a slight deterioration of heemolysins after 48 hours. Lamb (1903) estimated the hemolytic value of cobra venom for a number of the bloods of different animals by using the calorimetric measurement in small test-tubes containing a known quantity and the red corpuscles in question. He brought out no specially new point as to its behavior upon the red cells, but he has shown that 1 per cent solution of cobra venom resists the heating to 73°C. much better than o.1 per cent solution of the same venom. With unheated venom 0.005 c.c. of the blood suspended in 0.5 c.c. isotonic salt solution was dissolved by 0.015 mg. ‘lhe heating of 1 per cent solution of the venom raised this minimal complete hemolytic dose to 0.0312 mg., while that of o.1 per cent solution was raised to 0.25 mg. Now, coming to the hemolytic property of the daboia venom, Lamb found a somewhat singular phenomenon, namely, that this venom, while a powerful destroyer of the red corpuscles im vivo, did not readily dissolve the blood corpuscles when the cells were directly mixed with the venom in a saline solution in vitro. This fact has also been observed by Cunningham and emphasized especially by Stephens and Myers. Lamb found that in order to start hemolysis of any kind of blood it was necessary to have the toxicity of the saline medium somewhat below the isotoxicity, so as to produce a par- tial destruction of the corpuscles before the venom is introduced, recollecting also that Stephens and Myers noticed that the addition of the normal horse serum accelerated the hemolysis with this venom. Weir Mitchell’s phenomenon, namely, non-hemolysis by too strong a con- centration of venom, has also been observed with the daboia venom in vitro. As to the resistance of the hemolytic principle of the daboia venom, Lamb finds that it differs from that of the cobra venom, as the former loses its activ- ity completely when heated to 73° C. in a o.1 per cent solution, whereas only a certain diminution of power takes place in the latter venom. Another point of difference between these two venoms is the far more powerful hemolytic action im vivo possessed by the daboia venom. Noc, working with a large variety of snake venoms, established the fact that the phylogenetic relation between the main families and genera of poi- sonous snakes is not merely an anatomical and morphological factor, but also a biological factor, for he found that the activity of the hemolytic prin- ciples of their venoms obeys the linear course and rank to which each snake is assigned in the natural system. Noc’s experimental data bearing on this point are simple. He determined the length of time required by 1 mg. of each venom to hemolyze completely 1 c.c. of 5 per cent suspension of the washed corpuscles of horse in 0.9 per cent salt solution in the presence of 0.2 c.c. of the normal horse serum heated previously to 58°C. It was neces- sary to add this amount of the heated horse serum to obtain hemolysis, as 180 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS none of the venoms employed could dissolve the washed corpuscles of horse in a serum-free saline medium. His results are shown in table 9, 0.1 mg. being administered in each case. TABLE 9g. Complete Complete ": haseolyeis in— Name. kemalens in— Colubride: Viperidz — Continued. Cobra goss site tatsgo ate 5 mins. Crotalinze — Srey ere eye ere eile ore 10 Lachesis flavoviridis......| 35 mins Nap aymOin es acai: cits ie cs -tonenee 20 Ancistrodon piscivorus....| 40 Notechis scutatus .......... 40 Ancistrodon contortrix ....| 60 Wiperidsest 1 Py ia oe on M Tararactissulcces aa en ra. cites 2 hours Nipering—<—9 9 Oe et Sol Pei ifaramacan sevdepam crete. atte 24 Mipera *berusy ize seisickas «,» Gor on ii erat te cere ei entenrabeiare 3 Vipera) russelliic ())22 11-5 30 ~~ «i | «+ Bachesislanceolatusi-..--: 3 In testing the antiheemolytic power of an antitoxin obtained by immunizing an animal against the venoms of Cobra and Bothrops, Noc found that it neu- tralized the hemolytic action of 1 mg. of the venoms of Cobra, Bothrops, Urutu, Bungarus, Jararaca, Naja noir, and Vipera berus, but failed to do so against that of Trimeresurus of Japan (Lachesis flavoviridis). Here he makes the statement that the antihemolytic power in vitro of a given sample of antivenin is the measure of its antitoxic power im vivo against the same venom. Notwithstanding Noc’s approximate statement in regard to the stronger hemolytic activities with the elapine venoms, it does not follow that all colubrine snakes are included in this general rule. On the contrary, it is found by Rogers that another large subfamily of Colubride, the Hydrophiine, contains no appreciable hemolytic principles in their powerful venoms. Rogers' studied the hemolytic action of Enhydrina, Distira cyanocincta, and Distira cantoris upon the bloods of man and pigeon. When these venoms, especially that of Enhydrina, are mixed with the suspension of the blood in the ratio of over 1 to 1,000, complete hemolysis occurs after 24 hours’ contact. Cobra venom has about 100 times more hemolytic power. When the toxicity of these two venoms is compared the enhydrina venom surpasses the cobra venom by ro times (1 minimal lethal dose for pigeon = 0.00005 gm. for Eny- drina and 0.0005 for Cobra). In still another form of presentation 200 mini- mal lethal doses of enhydrina venom can dissolve only 3qyq part of the blood of the bird, excluding the possible réle in the fatal issue played by the hemo- lytic poison in the enhydrina toxication. Kyes? (1904) took up the question why none of the kinds of bloods of dif- ferent species of animals is attacked by the hemolytic principles of snake venom, in spite of the presence of a nearly equal amount of lecithin in all bloods. Kyes advanced the theory that lecithin in the blood corpuscles of various species does not exist in the same manner, and in some kinds it is ‘Leonard Rogers. On the physiological action of the poison of the Hydrophidz. Proc. Roy. Soc., 1903, LXXI, 48r. Lecithin und Schlangengifte. 2 Kyes, Zeitschr. f. physiolog. Chemie, 1904, XLI, 273. ° VENOM H4MOLYSIS AND VENOM AGGLUTINATION 181 easily grasped by the venom, while in others it is not at all available for venom activation. ‘Testing the antihemolytic power of Calmette’s antivenin, he found that it neutralizes the action of the venoms of Bungarus ceruleus, Bungarus fasciatus, Naja haje, and Naja tripudians, but not at all that of the venoms of Lachesis lanceolatus, Lachesis flavoviridis, Crotalus, and Vipera russellit. In the absence of serum or lecithin none of the venoms hemolyzed the bloods of sheep, ox, or rabbit, except that the last kind was dissolved by Naja tripudians. The blood of man was not attacked by the venoms of Lachesis lanceolatus, Lachesis anamalensis, and Crotalus, but by those of the daboia, habu, kraits, and cobras. This article does not contain any sub- stantial evidence that lecithin exists variously in different kinds of bloods. In 1904 Noguchi’ pointed out that the hemolytic principles contained in the venoms of cobra, rattlesnake, water-moccasin, and daboia are not identical, so far as their affinities to specific antivenins are concerned. Although not in a strict sense, the antivenin derived from an animal immunized against a given venom shows a specific affinity to that venom. In these experiments he employed the antivenin specific for cobra, crotalus, moccasin, and daboia. Lamb ” (1905) took up the question of the mechanism of venom hemolysis and contributed many interesting facts. He employed ten different kinds of venoms, comprising Naja tripudians, Naja bungarus, Bungarus ceruleus, Bungarus fasciatus, Notechis scutatus, and Enhydrina valakadien of the Colubride, and Vipera russellii, Echis carinata, Lachesis gramineus, and Crotalus adamanteus of the Viperide. From the action of these venoms upon the washed blood corpuscles of dog, Lamb divides the venoms into two groups according as they have or have not hemolytic action on these cells without the addition of a free activating agent —lecithin or serum. The first group contains cobra venom and daboia venom, which even in small amount have a complete hemolyzing effect; the venoms of Bungarus ceruleus and Echis carinata, which have also a complete hemolyzing action when used in large quantity, and the venom of Notechis scutatus, which has only a slight effect even in comparatively large amount. Group 2, namely, those venoms which have no hemolytic action on the washed cells of dogs, consists of the poisons of Naja bungarus, Bungarus fasciatus, Enhydrina valakadien, Lache- sis gramineus, and Crotalus adamanteus. Testing their hemolytic value in the presence of the serum of dog or an adequate amount of lecithin developed that, except the venom of Enhydrina valakadien, all these venoms became nearly equally hemolytic, notwithstanding slight variations may be noticed. 0.00001 gm. was about the average dose which could completely dissolve I c.c. of 5 per cent suspension of the washed dog corpuscles (in 0.85 per cent salt solution) in 1 hour at 37° C. and over night in the ice-chest, when 0.5 c.c. of twofold dilution of dog’s serum was added simultaneously. The enhy- drina venom never completed hemolysis even in a dose of 5 mg. in the same 1 Noguchi. Expériences thérapeutiques avec les antivenins (Crotalus adamanteus et Ancistrodon piscivorus). 1904. ?Lamb. Snake venoms in relation to hemolysis. Sc. Mem. Off. Med. and San. Dept. Governm. India, 1905, new series, No. 17. 182 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS quantity of the blood. From these results Lamb explains why, without addi- tion of suitable serum, hemolysis was obtained by Kyes and Sachs, who chiefly worked with cobra venom of group 1, and not obtained by Flexner and Noguchi, who worked with the venoms belonging to group 2. With the same experimental arrangements as the above, but using 0.2 c.c. of o.1 per cent lecithin saline suspension as the activator, Lamb demonstrated the variability of the affinities possessed by different venoms toward lecithin. He found that the venoms of Echis carinata, Bungarus fasciatus, and Bungarus ceruleus become active on the addition of lecithin in the same degree as on the addition of dog’s serum, but lecithin has but slight activating effect upon the venoms of Naja bungarus, Enhydrina valakadien, and Crotalus adaman- teus. In this connection it is important to remember that the venoms of Naja bungarus and Crotalus adamanteus are equally or even more hemolytic when, instead of lecithin, dog’s serum is employed as the activator. "This phenome- non offers some difficulty in generalizing Kyes’s hypothesis that lecithin is solely responsible for the venom-activating property of a blood serum, because pure lecithin is not equivalent, in activating these particular venoms, to a suitable serum. The washed corpuscles of ox and goat bloods are entirely resistant to all venoms here employed, but become hemolyzed upon the addition of lecithin with the venoms belonging to group 1. On the other hand, lecithin does not appreciably accelerate or activate those venoms falling in group 2. ‘These differences were explained by Lamb as though different venoms have varying affinities to lecithin. The next question determined by Lamb relates to the fixability of venom- intermediary bodies by the blood corpuscles. He tried to impregnate the washed corpuscles of certain suitable kinds of bloods with varying amounts of venoms, say I, 2, 4, 6, 8, 10, and 20 minimal complete hemolyzing doses. The tests whether the corpuscles absorbed any portion of the venom from the medium after a period of contact were made by examining the fluid separated from the cells (by centrifugalization) for the remaining activity on a fresh lot of the same kind of blood corpuscles with the simultaneous addition of either homologous serum or lecithin, and also by resuspending the sedimented cor- puscles in a fresh volume of saline solution and the subsequent introduction of suitable venom activators. In no instance could Lamb demonstrate the fixation of venom by the blood corpuscles. These results, which apparently contradict those obtained by Flexner and Noguchi, are not, in reality, com- parable in these instances, as the experimental arrangements in both cases differ so far as the réles of complements in such mixtures are concerned. As Lamb clearly pointed out, Flexner and Noguchi used the defibrinated blood, whereas Lamb employed the washed cells. There are instances where no fixation of intermediary bodies takes place, unless there is present at the same time suitable complements or complementoids.’ 1 Bordet and Gay. Sur les relations des sensibilisatrices avec l’alexine. Ann. Inst. Pasteur, 1906, XX, 467. VENOM HAMOLYSIS AND VENOM AGGLUTINATION 183 Lamb also found the diminution of hemolysis with much larger doses of venom. In the meanwhile Noguchi was still pursuing his study on venom hemo- lysis and tried to clear up the mechanism of lysis produced by venom in the presence of certain thermostabile chemical substances other than that (lecithin) discovered by Kyes. Noguchi thinks that the hemolysis caused by venom can in many instances be due to the sensitizing effects of venom on the hemolytic action of certain substances which are already active by themselves. In other words, venom inflicts upon the blood cells certain injury and renders them more vulnerable to the solvent action of these substances. Thus in the case of lecithin, a typical venom activator, Noguchi points out that this substance is quite hemolytic by itself. The hemolytic power of lecithin is, however, about one-twentieth of what it is when used in the presence of venom. ‘This proportion is not the one which is estimated at the beginning of experiments, but is obtained at the time when the reaction is almost com- pleted. As was shown somewhere else, the velocity of reaction of different chemicals is extremely variable according to the nature of the substance, but the final sum of reaction is not parallel to the velocity of reaction. Now let us take lecithin. This substance does not start to act until many hours after the addition to the blood suspension, but gradually reveals its lytic property in about 3 or 4 hours, and continues to be active until 18 to 24 hours. On the other hand, the venom lecithin hemolysis does not require a latent period, but completes the reaction within half an hour or so. If a com- parison of the hemolytic strength of venom lecithin and lecithin alone be taken within the first 5 minutes the proportion would be no lysis with lecithin against the very powerful rapid lysis of venom-lecithin mixture. Ti an-esti; mate be made after 1 or 2 hours it would be 1 to 200-300 or more in favor of the latter. However, this proportion slides gradually to the advantage of lecithin, as the time requisite for its completing reaction gains, until the ratio is about 1 to 20 after 24 hours. There is no question as to the formation of a definite new haemolytic com- pound (lecithid) in the mixture of venom and lecithin, together with a reduced incubation period of hemolysis, but it is amazing to see how rapidly this reaction takes place. If a sufficiently large amount of lecithin be added, hemolysis occurs instantaneously, while an insufficient amount delays the process very markedly, notwithstanding the comparatively large amount of venom. The rapidity with which lecithin hemolyzes the blood cells with the aid of venom (cobra) is almost without parallel in the enzymic process — perhaps with the exception of lipase upon neutral fats. Another example of a rapid completion of hemolytic process is furnished by sodium oleate. This sub- stance is able to heemolyze about the same amount or slightly more of the blood corpuscles as lecithin or oleic acid, but its reaction is all over within an hour. In this body we see that the zymotoxic (or toxophore) group of the compound is set to a rapid action through the presence of sodium. 184 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Having found that a certain constant proportion exists between the inherent hemolytic power of lecithin and the hemolytic power augmented through the addition of venom, Noguchi undertook a series of studies in which he sought to compare the native hemolytic power and the hemolytic power with venom of various groups of substances, in order to ascertain whether certain chemical bodies are or are not able to perform the function of venom activa- tion. The chemicals subjected to this test are numerous and comprise normal fatty acids from formic up to ceratinic, unsaturated mono-carbonic acids con- taining ihe higher members, aliphatic acids, unsaturated dicarbonic acids, mineral acids, sodium salts of fatty and acrylic acids, neutral fats, lecithin, neurin, cholin, and some saturated and unsaturated alcohols. From his experiments Noguchi discovered that the compounds which contain in their molecules a double bond only can become many times more hemolytic than their native activities upon the addition of venom. Normal fatty, aliphatic, and mineral acids do not act stronger in the venomized corpuscular suspension than without the venom, whereas unsaturated organic acids, especially oleic acid, have their action increased by nearly 10 to 20 times their inherent strengths. This does not mean that each acid of this particular group has an equal hemolytic power — which is far from being the case —but simply means that the proportion or ratio is sufficiently constant to rank them with lecithin or kephalin as venom activators. In reality triolein and oleic acid were the only substances worthy of comparison with lecithin in hemolytic activity, the rest, although maintaining the con- stant ratio, being far inferior in this respect. Again coming to the mechanism of venom activation by these simpler fatty compounds — simpler when compared with lecithin —it was noticed that their velocity of reaction was but little affected with the venom, in contradis- tinction to that with lecithin. It appears from this that the activation caused by these chemicals is essentially different from that brought about by lecithin; nevertheless they are venom activators in a certain sense. Noguchi further studied the quantitative relation of the requirement of venom and venom activators to produce a definite degree of hemolysis. Using a uniform amount of venom and variable amounts of activator, he found that the requirement for producing an equal degree of hemolysis is in proportion to the square root of the amount of venom present in the mixture. In reversing the condition, namely, using a uniform amount of activator and variable amounts of venom, it was found that an increase in activator per- mits of a reduction in venom approximately in proportion to the square of the amount of activator employed. The antihemolytic property of cholesterin against that form of hemolysis where venom and lecithin are concerned has been found by Kyes. Accord- ing to Noguchi the action of cholesterin is directed to lecithin, but not to venom. Noguchi prepared a series of heemolyzing mixtures in which varying amounts of lecithin and venom were so combined as to result in an equivalent hemolyzer. Cholesterin was added to such mixtures in sufficient amount to VENOM H#MOLYSIS AND VENOM AGGLUTINATION 185 prevent hemolysis. To antagonize one complete hemolyzing mixture the amount of cholesterin was found to vary according to the amount of lecithin which such a mixture contains. The greater the amount of lecithin, the more cholesterin was required to inhibit hemolysis. No quantitative rela- tion exists between the amount of venom and that of cholesterin.' Noguchi also demonstrates that the addition of methyl alcohol beyond a certain amount (or concentration) finally nullifies the antihemolytic property of cholesterin, which may indicate that the increase of solubility of cholesterin diminishes its antagonistic action against the mixture of venom and lecithin. MECHANISM OF VENOM HAMOLYSIS. Goebel? discovered that the corpuscles, which are completely refractory to the hemolytic action of cobra venom in isotonic sodium chloride solution, are promptly dissolved when suspended in isotonic saccharose solution instead of the saline medium. Pascucci * investigated the action of various hemolysins, saponin, solanin, tetanolysin, and cobra venom, upon the artificial membrane of lecithin- cholesterin mixture. It was found that these hemolysins produced per- meability of the membrane for hemoglobin solution. This alteration was most pronounced when the percentage of cholesterin in the mixture was least. The higher the percentage of cholesterin the less permeability resulted as the effect of these substances. Morgenroth * confirmed the finding of Kyes and Sachs that the hemolytic principle of cobra venom is much more resistant to the deteriorating effect of boiling when heated together with a small quantity of hydrochloric acid. He also found that the hemolysin (as well as neurotoxin) is so modified by this acid that the specific antitoxin now becomes unable to combine with this principle in the acidified medium. Even the neutral mixture of the lysin and antitoxin is split up by the acid into its original components. If the action of the acid has not continued too long the removal of the acid restores to the lysin its original affinity towards antitoxin. Morgenroth and Pane ® followed up the study of the effects of hydrochloric acid upon the hemolysin of cobra venom. They observed that when cobra venom in an acid medium containing about N/ 20 HClis heated for a long time and its hemolytic power is tested immediately after cooling and neutraliza- 1 Recently Morgenroth made a statement that this experiment was a mistake, on the grounds that cholesterin antagonizes the hemolytic action of venom lecithid, the ready hemolysin. To my mind such a criticism is invalid. First of all we must admit that the resultant lecithid is in proportion to the amount of lecithin acted upon by venom. The amoumt of lecithin is the source of lecithid to be formed by the action of venom; hence, if the amount of venom present is sufficient, the quantity of the lecithid produced is directly proportional to lecithin previously present. in other words, the experiment has shown that the inhibitory action of cholesterin is directed against the activated lysin. * Goebel. Contribution a I’étude de l’agglutination par le venin de cobra. C.r. de la Soc. Biol., 1905. Contribution 4 l’étude de l’hémolyse par le venin de cobra. Loc. cit., 1905. 8 Pascucci. Die Zusammensetzung des Blutscheibenstromas und die Hamolyse. II Mittheil. Die Wirkung von Blutgiften auf Membranen aus Lecithin und Cholesterin. Hofm. Beitr. zur chem. Physiol. und Pathol., 1905, VI, 552. 4 Morgenroth. Ueber die Wiedergewinnung von Toxin aus seiner Antitoxinverbindung. Berl. klin. Woch., 1905, XLII, 1550. ® Morgenroth and Pane. Ueber Beobachtungen reversibler Veranderungen an Toxinen. Biochemische Zeitschrift 1906, I, 354. 186 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS tion, there is always some reduction in this power. But if the neutralized venom solution be left in the room temperature for a few days and its hemo- lytic strength be again measured, it will be found that the venom solution had become once again as strongly hemolytic as its original native venom solution. On the other hand, the hemolytic power of cobra venom heated without HCl remains at its reduced value without regaining its lost strength during the same length of time. Even after a lapse of only 4 hours the return of the hemolytic strength was often seen to be very complete. Faust ' tested his ophiotoxin for the hemolytic property and found that this substance has quite marked action upon the washed or unwashed corpuscles of ox, pig, horse, and sheep. Heating to go°® C. for 15 minutes did not destroy its hemolytic power. Ishizaka? studied the hamatoxic action of the venom of Lachesis flavo- viridis and found that the blood of dog is more susceptible to the hemolytic action than that of cat, rabbit, ox, or rat. 0.05 c.c. of dog’s blood promptly dissolved by 0.000002 gm. of the venom, while 0.co00002 gm. caused only a trace of lysis. Cat’s blood is much agglutinated, but only slightly dis- solved even in a comparatively large quantity of the venom, while 20 mg. of the same failed to hamolyze any part of the blood of mouse, ox, or rabbit. In these cases more or less agglutination took place. In support of the obser- vations of Flexner and Noguchi, he found with this venom that thorough washing of the corpuscles retards or diminishes hemolysis to a great extent. With the most susceptible kind of blood, namely, that of dog, Ishizaka found that after 3 to 6 washings the corpuscles become completely insus- ceptible to the smaller quantities of the venom. He found that lecithin can activate this venom very easily. Cholesterin is found to inhibit hemolysis caused by the habu venom, but not by combining directly with the latter; this latter fact has been shown by Noguchi in his former studies. In 1907 Noguchi attempted once more to clear up the questions concerning the susceptibility of the corpuscles of certain kinds of bloods and the non- susceptibility of those of some other kinds to the hemolytic action of venom. It appeared to him still quite uncertain why one set of the blood serums is able to activate venom, while the other is not. As to the nature of the thermo- stabile venom activators of the blood serum, Noguchi agrees with Kyes that it is chiefly lecithin which is capable of venom activation. But some serum contains, besides lecithin, certain thermolabile activators, whose action dis- appears when heated to 56° C. for half an hour. ‘There are numerous serums which are devoid of venom-activating property in the fresh state, but these all become invariably activating when heated to the temperature near or above the coagulation point. In these instances there is no doubt that leci- thin is liberated by heat and activates venom freely. This, however, does not explain the venom-activating property of the fresh serums; it only sug- gests that lecithin may exist in one set of serums in a state to be acted upon 1Faust. Ueber das Ophiotoxin aus dem Gift der ostindischen Brillenschlange. Leipzig, 1907, 19. 2 Ishizaka. Studien tiber das Habuschlangengift. Zeitschr. f. experim. Pathol. u. Therapie, 1907, IV, 88. VENOM HZMOLYSIS AND VENOM AGGLUTINATION 187 by venom, while not in the other set. This theory does not readily explain the activating property of a serum which becomes inactive at 56° C., because if it were lecithin which activates venom, heating would not suppress its activ- ity, as it is found by Noguchi that the acquired activating property of a non- activating serum through the addition of an adequate quantity of available lecithin is not to be suppressed by a subsequent heating to that temperature. Lecithin is also characterized by its prompt activation, no matter how it is introduced into a serum. As to the quantities of lecithin existing in various kinds of the activating as well as non-activating serums, there are no great differences among them. The same difficulty is encountered in explaining the susceptibility and non-susceptibility of the blood corpuscles of different species of animals. They all contain lecithin in about the same quantities, but venom can not attack them with equal readiness. In the case of dog’s corpuscles there is undoubtedly an indication that lecithin is concerned in venom hemolysis. Again, in the case of serum of that animal the venom- activating property of the fresh serum appears to be caused, at least in part, by the presence of available lecithin. On the other hand, the thermolabile activators contained in the serums of guinea-pig, horse, cat, pig, rabbit, pigeon, hen, goose, and man are distinguished from lecithin by their slow activation. Besides, activation of venom due to lecithin can not be prevented by the addi- tion of calcium chloride, while activation caused by substances which are not of the nature of lecithin is easily removed by this salt. Ether can not remove lecithin from the mixture with serum; hence the activating property of the serum which contains available lecithin remains undiminished after ethereal extraction. On the other hand, the activators of the second group of serums go over to ether and render the extracted serums no longer activating. Meanwhile the substances extracted with ether can, when transferred to non-activating serum, confer the activating property upon the latter in a marked way. According to Noguchi the second group of activators consist mainly of non- phosphorized fats, fatty acids, and their soluble salts; while in serum these bodies are not hemolytic, but become quite hemolytic when venom is intro- duced. Their mode of activation therefore differs essentially from that caused by combination of venom and lecithin. The activating property of these bodies disappears when mixed with calcium chloride or heated to 56° C. in the presence of serum components. On mixing non-activating serum with oleic acid or soluble compounds of oleic acid, the former acquires activating power very similar to that possessed by the fresh serums of the second group. In regard to the variable susceptibility of the washed corpuscles of various kinds of bloods, Noguchi found an existing relation similar to that of activa- bility of the different kinds of blood serums. The water-laked solution or the stroma of the corpuscles of the non-susceptible kind does not contain venom activators, while the reverse is true of the susceptible variety. Of the cor- puscular activators there are again two groups — one that of lecithin and the non-phosphorized lipoids. The corpuscles of dog contain both sets, but 188 | VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS most bloods contain mainly the second group. Ethereal extraction removes from the latter the greater part of the activators, which, when introduced into the non-susceptible variety of corpuscles, make the latter susceptible to the hemolytic effect of venom. Calcium chloride removes the activating property very effectively, but not that of the first group (dog). In accordance with this finding the defibrinated bloods of the second group are very well protected by the addition of calcium chloride, but this salt fails to protect the blood of the first group. Even the washed corpuscles of the latter group may finally be hemolyzed by venom in spite of the presence of calcium chloride, demon- strating that lecithin forms at least a part of the activators in these cells. It may be recalled at this place that both from the non-activating serums and from the non-susceptible corpuscles a certain quantity of lecithin is easily extractable with hot alcohol. It has repeatedly been stated that every serum becomes venom-activating when heated to coagulation and that it matters not whether the serum was originally activating or not. The activating property developing after heat- ing is uninfluenced by calcium chloride and its action is very rapid. ‘There can be no doubt that it is due to the liberation of lecithin by heat. Noguchi found, however, that non-activating serum is here again quite inferior to activating serum in its activating power after heating. In both instances ethereal extraction fails to remove the activator from the clear filtrate which alone is activating. It appears that lecithin exists as a non-coagulable pro- tein compound, comparable to Chabrie’s albumin. Now, taking up the question why lecithin is available for venom activation in dog’s serum and not in ox’s serum, Noguchi was able to show that lecithin as existing in paired state with serum albumin and serum globulins is entirely inactive in regard to venom activation. It is the same with non-activating or activating serums. But in the activating variety (dog) there exists in the serum a certain protein compound of lecithin capable of venom activation, but not in the non-activating serum. ‘This compound remains in solution when serum globulins are precipitated by dialysis, but can be precipitated by half-saturation with ammonium sulphate. It is perfectly soluble in water, and is not coagulable in neutral alkaline salts solutions upon boiling. Cal- cium chloride has no inhibiting influence on its venom-activating property and warm alcohol extraction of this protein yields much lecithin, but not with ether. In this place it may be remarked that boiling of fractionated albumin and globulins of ox serum did not produce any striking amount of venom activator. No study has been made to find out why the whole serum pro- duces and the fractionated proteins do not produce venom-activating lecithin compounds on boiling, but the removal of certain salts and lipoids through fractionation may in part account for the difference. Noguchi is inclined to consider the activation of venom by certain fatty substances, at least in part, as a sort of cumulative action of venom and these chemicals. ‘That venom inflicts upon the washed corpuscles a rather marked injury and renders the latter considerably more subject to all kinds of destruc- VENOM H#MOLYSIS AND VENOM AGGLUTINATION 189 tive effects of a physical nature has already been shown. But it is not at all improbable that these fatty substances have acted as mordants and enabled the interaction of venom and lecithin compounds of proteins to take place, and hence hemolysis. At the same time we must not forget that most normal fatty acids can not take the place of acrylic acids as venom activators; that the inherent hemolytic powers of higher members of the latter acids are nearly ro times greater than any other acid, organic or mineral; that lower alcohols, which have quite large lecithin-solving property with but little hemolytic power, fail to accelerate venom hemolysis to any marked degree; and finally that mineral as well as organic bases favor slightly if at all the attack of venom on the blood corpuscles. There is one thing which seems to be necessary for the venom-activating property of these acrylic acids and their compounds —that is, the powerful hemolytic quality. It is still undetermined just how far the lipoid solvent property of these acids and soaps is concerned in their inherent hemolytic property and also their venom-activating property. The alterations of physical factors, such as the diminution of the osmotic tension of the fluidal media through the introduction of these particular chemical bodies, may play certain important parts in regard to the bursting of the delicate lipoid-like membrane of the blood corpuscles. Gengou ! found that sodium citrate when used in 2.1 per cent solution can stop the hemolysis caused by cobra venom and suitable blood serum. This antihemolytic property is, however, removed by adding sufficient calcium chloride to such mixture. The introduction of the latter salt will cause the formation of calcium citrate and sodium chloride, thus removing the inhibit- ing effect of the soluble citrate from the mixture. Gengou used just enough calcium chloride to remove the citrate soda and did not notice the antihemo- lytic property of the lime salt. He employed the laked solution of the washed corpuscles of guinea-pig’s blood as a venom activator and obtained a similar result in regard to the antihzemolytic action of sodium citrate. TABLE Io. Mixture = (1) Washed corpuscles of ox, rdrop. (2) 0.4 per cent cobra-venom solu- tion, 0.2 c.c. (3) Fresh guinea-pig serum in doses indicated in the table. (4) Solu- tions of respective chemicals in doses to make up the total quantity of the resulting fluid, 1 c.c. per tube. Hemolysis produced. Calcium chloride o.8 per cent solution. Slight in 24 hours None in 24 hours None in 24 hours None in 24 hours None in 24 hours Sodium citrate 2 per cent solution. Slight in 24 hours Trace in 24 hours None in 24 hours None in 24 hours None in 24 hours Sodium chloride 0.9 per cent solution. Complete in 20 min. Complete in 20 min. Complete in 40 min. Complete in 20 min. None. Noguchi compared the antihemolytic powers of sodium citrate and calcium chloride with cobra venom and was able to confirm Gengou’s observations. 1Gengou. Compt. rend. de la Soc. de Biol., 1907, LXII, 409. 190 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Both sodium citrate and calcium chloride are very effective antihemolytic agents against venom. Substituting guinea-pigs’ cells for ox corpuscles, the result was the same. In the next series of experiments the concentrations of these two chemicals were gradually reduced, whereas 0.9 per cent solution of sodium chloride was used as diluent. TABLE It. Mixture =Washed corpuscles of guinea-pig, 1 drop. o.4 per cent cobra-venom solution, 0.2 c.c. Fresh guinea-pig serum, 0.35 c.c. Amounts of . aaiesrclvic Hemolysis produced. salt solutions added to the mixture, making Calcium chloride, Sodium citrate, the total 1.5 c.c. 0.8 per cent solution. 2 per cent solution. None. Complete in 2 hours. Complete in 60 min. Complete in 40 min. Complete in 25 min. Complete in 20 min. Complete in 4 hours..| Complete in 15 min. Complete in 2 hours..| Complete in 15 min. Complete in 30 min...| Complete in 15 min. Complete in 10 min...| Complete in 10 min. It is noteworthy that calcium chloride has a far stronger antihemolytic power than sodium citrate and seems to have no relation to the increase of tonicity of the mixture. On the other hand, the concentration of sodium citrate is quite above its isotonicity in effecting the antihemolytic action of this salt, which is isotonic at a concentration of about 1.76 per cent in the case of guinea-pig corpuscles. 0.8 per cent solution of calcium chloride is here isotonic. In a third series of experiments the comparative antihemolytic powers of these two chemicals were tested against different venom activators in the presence of cobra venom. TABLE 12. Washed corpuscles of ox, 1 drop. 0.4 per cent cobra-venom solution, o.2 c.c. Hemolysis produced. Activators 0.2 c.c. each. Calcium chloride 0.8 per | Sodium citrate 2 per cent | Sodium chloride 0.9 per cent cent solution 1 c.c. solution r c.c. solution 1 c.c. N/ 1000 lecithin Complete and prompt | Complete, but with Complete and prompt. delay. I per cent ovovitellin. .. d Do. Heated dog serum .... Do. Fresh guinea-pig serum | None ig Complete, but slowly. N/tooo sod. oleate .... Complete and fairly quick. N/tooo neurin oleate . . Do. N/1o00o ammon. oleate . Do. N/ tooo oleic acid Do. No activator None. VENOM HEMOLYSIS AND VENOM AGGLUTINATION 191 From the above table it appears that the antihemolytic property of sodium citrate is not directed against the same set of activators as that of calcium chloride. Sodium citrate has a certain inhibiting influence upon hemolysis caused by venom and lecithin, but almost none against the combination hemolysis of fatty substances and venom. This relation is exactly the reverse with calcium chloride. Teruuchi* found that cobra hemolysin and its antitoxin are destroyed by dog’s pancreatic juice, activated with the intestinal juice. Cobra lecithid is not affected by this treatment. Through digestion of the neutral mixture of cobralysin and antitoxin by pancreatic juice a portion of the lysin can be restituted. On the other hand, the combination of the mixture of cobralysin and antitoxin with lecithin before digestion seems to prevent the liberation of active lysin under the influence of the pancreatic juice. Von Dungern and Coca studied the constitution of cobra hemolysins and found that the washed corpuscles of ox, which are completely insusceptible to the venom, can be dissolved by adding either fresh guinea-pig serum or lecithin. This was first discovered by Flexner and Noguchi and Kyes and Sachs. Von Dungern and Coca investigated whether the cobralysin is ab- sorbed by these corpuscles or not. By allowing the washed ox corpuscles to remain in contact with cobra venom for some hours they found that the cells absorbed a certain portion of cobralysin from the fluid in which they had been suspended. The evidence of absorption was brought out by washing the corpuscles with saline solution, freeing them from cobralysin, and then examining the corpuscles for susceptibility to serum complements and leci- thin. If the corpuscles were laden with venom amboceptors, or sensitized with venom sensibilisatrice, hemolysis would occur on adding complements or lecithin. In fact, they discovered that the venomized corpuscles are easily dissolved by adding guinea-pig complements, but not lecithin. They exam- ined the venom solution, after separation of the corpuscles, for its haemolytic property, and again demonstrated that it retained all its hemolysin con- tent by adding lecithin, but lost all its complement-activable hemolysin. In other words, venom contains two different types of hemolysins, one resem- bling typical serum amboceptor, the other quite different from that class of hemolysins. ‘The latter is active in the presence of lecithin, but not of serum complements, and is not absorbed by the ox corpuscles. Not knowing the discovery of Noguchi, von Dungern and Coca, independ- dently of him, found that calcium chloride, barium chloride, and magnesium chloride can prevent hemolysis caused by venom — especially that calcium chloride is of much greater power than the other two. Like Noguchi, they also observed that their antihemolytic properties are due to suppression of the activating property of serum complements, without preventing the ambo- ceptors from being absorbed by the corpuscles. Only a slight inhibition can 1Teruuchi. Die Wirkung des Pankreassaftes auf das Hamolysin des Cobragiftes und seine Verbin, Se mit dem Antitoxin und Lecithin. Hoppe-Seyler’s Zeitschr. f. physiol. Chem., 1907- » 478. 5 192 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS be obtained by these salts against venom-lecithin hemolysis. Peculiarly enough, barium chloride exceeds the other in its antagonistic action against this form of hemolysis. Von Dungern and Coca incidentally refer to the regeneration of the com- plement once inactivated by barium chloride by means of adding sodium sulphate. This confirms the observations of Noguchi, who studied the same phenomenon with numerous complements and chemicals. As to the cause of certain inhibitory influence exerted by barium chloride against cobra venom in the presence of lecithin, these investigators ascribe it to the interference of this salt on the formation of lecithid, because the prepared lecithid is as hemolytic in the barium solution as in the sodium-chloride solution. On the other hand, the prepared lecithid shows less strength in sugar solution (9.35 per cent) than in the saline solution (0.8 per cent). Formation of cobra lecithid seems to be much easier in the sugar solution than in the saline solution, because certain kinds of blood are easily hemolyzed in the former, but not at all in the latter medium (Goebel). This phenomenon is interesting, as it presents a reverse relation to the hemolysis caused by the serum ambo- ceptor and complement. Von Dungern and Coca studied the nature of cobra lecithid in regard to its bearing on immunity. They prepared lecithids in the usual way, originally given by Kyes. The results of their experiments do not favor the toxin-like view of lecithid. They immunized rabbit with lecithid and obtained anti- hemolytic serum. But the antihemolytic power of the immune serum does not manifest itself until it has been heated to 64° C., because the normal rabbit serum is found to be almost as strongly antilecithidal as the immune serum in the fresh state. This peculiar phenomenon was first observed and described by Kyes in his studies of immunization against lecithid. Kyes’s interpretation was that the normal rabbit serum contains a certain amount of anti-body against lecithid, and its destruction by means of previous heating of the serum to 64° C. is necessary to make the specific antiheemolytic prop- erty of the immune serum appear in a more striking degree. Von Dungern and Coca gave a very different explanation for this phenom- enon on the grounds of their own experiments. They assume that lecithid contains a certain quantity of lecithin-splitting venom-ferment or heemo- lysin and the immunization of animals with such mixture causes appearance of anti-body for this minute quantity of venom lysin in its blood serum. The reason why the heated normal serum becomes inactive and the heated immune serum still more or less active against lecithid is that the former furnishes enough liberated lecithin to be acted upon by the native venom contained in the lecithid, hence more hemolysis than with the fresh normal serum. On the other hand, the immune serum contains, even after heating (64° C.), a specific anti-body to neutralize the native venom of the leci- thid; hence the presence of free lecithin in the heated immune serum is 1 Noguchi. Ueber die chemische Inaktivierung und Regeneration der Komplemente. Biochem. Zeitschr., 1907, VI, 327. VENOM HAMOLYSIS AND VENOM AGGLUTINATION 193 no longer attacked and rendered lytic by the otherwise active native venom of the lecithid. At the same time von Dungern and Coca show that a subminimal hemo- lytic dose of cobra lecithid becomes a complete hemolyzing dose if enough of lecithin be added to the mixture, showing that there is still present in the so-called pure lecithid enough lecithin-splitting venom component. On the other hand, the antihzemolytic powers of the heated normal and immune rabbit serums do not differ if they are tested against the lecithid which had been heated to 100° C. for 3 hours, because here the specific anti-body is not concerned in the reaction. In the case of the heated normal serum the lecithin remains unattacked by the lecithid without the adhering native venom and there will be no more hemolysis than in the case of the fresh serum. Von Dungern and Coca do not consider it necessary to assume that cobra lecithid is a chemical compound of venom and lecithin, but entertain the view that the hemolytic substance is only a split product of lecithin and is contaminated with a minute quantity of native venom component, the latter playing but little part in the hemolytic activity of the whole lecithid. From various preparations of ovolecithin they were able to isolate a highly hemolytic substance with all the physical and biological characteristics of cobra lecithid, except that it did not show evidence of the adherence of native cobra venom, and hence no increase in its lytic power by lecithin addition and no antilysin formation in the animal body. Its hemolytic activity was about half that of cobra lecithid. In a subsequent paper’ von Dungern and Coca attempted to solve the mechanism of venom hemolysis induced by adding oleic acid or sodium oleate in subminimal inherent hemolytic quantities. As already stated, Noguchi, among many other acrylic acids and soaps, found these two chemicals espe- cially suitable for rendering the blood corpuscles hemolyzable by venom. Von Dungern and Coca treated the ox corpuscles with cobra-venom solu- tion for 1 hour (20 c.c. 5 per cent corpuscular suspension and 2 c.c. 1 per cent cobra-venom solution), and then, after separation from the venom, their sus- ceptibility to the hemolytic action of oleic acid and sodium oleate was tested. The result shows that the degree of hemolysis is the same whether the cor- puscles have been venomized or not. Thus they could find no comparison between the hemolytic serum complement and these oleic compounds. Then they tried to ascertain if cobra venom can act directly on these chemicals and render them more active, but this was found not to be the case. On the contrary, oleate soap, if allowed to act long, rather diminished than increased the action of cobra venom when there was no blood or lecithin in the mixture. However, if the soap, venom, and lecithin are allowed to remain in contact for a much longer period, the hemolytic activity of such mixture is rather greater than that of a mixture in which soap is added at the same time as or just before the addition of blood. Oleic acid always exerts an accelerating 1 Von Dungern and Coca. Ueber Hamolyse durch Kombination von Oelsiure oder élsaures Natrium und Kobragift. Miinch. med. Woch., 1908, LV, ros. 194. VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS influence, but is not an activation of this acid by venom. Sodium oleate, which is at first somewhat inhibitory, later accelerates the lecithin-splitting process of the venom, and oleic acid favors this reaction under all conditions. Von Dungern and Coca finally advanced the theory that oleic acid and sodium oleate alter the solubility of cobra-venom components and enable the venom to attack the lecithin of the serum or corpuscles. ANTIHA-MOLYTIC PROPERTIES OF CHOLESTERIN. Abderhalden and Le Count’ made an extensive investigation on the mechanism of the antihemolytic property of cholesterin against venom- lecithin hemolysis. By using various derivative products of cholesterin and also many cleavage and synthetic compounds of proteins they tried to locate the radical upon whose existence the antagonistic property of cholesterin is dependent. ‘They tested a few amino-acids and numerous peptids— dipep- tids, tripeptids, and tetrapeptids, peptone Siegfried, etc., but none of these bodies was able to exert the antihemolytic action. Of cholesterin and its derivates they employed (1) cholesterin from human gall-stone, C,,;HyO; (2) cholesterin from egg-yolk, C,,H,,O; (3) a cholesterin-like body obtained from the radish oil; (4) cholesteryl chloride, C,,H,;Cl; (5) cholesteryl acetate, C,,H,OC,H,O; \(6) cholesteryl benzoate, C,,H,,;C,H;CO,, cholesten, C,,Ha; cholesteron, C,,H,,O (ketone); cholesteron-oxim, C,,H,ON (normal oxim of ketone); oxynitrocholesteryl nitrate, C,,;H,,.N.O,; cholesteronol acetate, C,,H,,0, * C,H;0; cholesteronol formiate, C,,H,,;0 * CHO; cholestandion, C,,H,,O.; cholestenon diacid, C,,H,,O; (ketodicarbonic acid); dimethyl- ester of cholestenon diacid, C,,H,,O; and its sodium salt; chlordicarbonic acid, C,,H,,ClO,; lactone acid, C,,H,,O,. These substances were first tested for their inherent hemolytic powers. They all reacted acid when dissolved in a fluid containing methyl alcohol sufficient to hold them in solution. The acidity was neutralized by adding N/1o NaOH (indicated by phenolphthalein), the latter solution being pre- pared by mixing 1 part of normal NaOH with 9 parts of methyl alcohol, thus preventing precipitation of the cholesterin and the cholesterin derivatives from the diminution of alcohol percentage during neutralization. The amount of NaOH required for neutralization was so small that that alone could not produce any marked hemolysis. Cholesterin from egg-yolk after neutrali- zation became somewhat hemolytic; the alkali added to it was enough to cause hemolysis by itself. The experimental plans were as follows: Cobra venom o.1 c.c. of 0.005 per cent solution uniform. Lecithin 0.1 c.c. of 0.05 per cent solution uniform. 1 c.c. of 5 per cent suspension of the blood corpuscles — horse and goat — in an 8 per cent methyl-alcohol, isotonic, salt solution. Then decreasing amounts of the solutions of cholesterins and various derivatives were added. 1 Abderhalden and Le Count. Die Beziehungen zwischen Cholesterin, Lecithin und Cobragift, Te- tanus-Toxin, Saponin und Solanin. Zeitschr. f. Pathol. u. Therapie, 1905, II, 199. VENOM H#MOLYSIS AND VENOM AGGLUTINATION 195 The concentration of the cholesterins and their derivatives was usually 0.02 per cent. 1 c.c. or less of such solution was added to the above combinations. The results obtained by Abderhalden and Le Count show that choles- terins obtained from gall-stones and egg-yolks displayed marked and about equal antihemolytic powers, being still able to prevent hemolysis in doses of 0.15 c.c. and upwards. A cholesterin-like preparation from the radish oil was without this property. Cholesterin showed a slight inhibition when used in dose of 1 c.c. Cholesteron was entirely inactive in this regard. The chloride, acetate, and benzoate of cholesterin were devoid of antilytic power. Cholesteron-oxim was highly inhibiting, while cholesteron itself was inactive. As to the effect of neutralization of cholesterins with NaOH they found this property almost unaffected, save the hemolysis which attends the larger quantities of the neutralized cholesterins,' perhaps due to the dissociable alkali under the circumstances. The rest of the chemicals tested by them were inactive in regard to the venom-lecithin hemolysis. From these results Abderhalden and Le Count conclude that the free hydroxyl group is indispensable to the antihzmolytic action of these bodies, and that the double bonds may not be entirely indifferent. In 1905 Noguchi made a more exhaustive study of the mechanism of Mitchell’s phenomenon, or the non-hemolyzability of the blood corpuscles in a very concentrated venom solution. Mitchell and Stewart observed that in a mixture of blood and fresh venom, in equal parts, the corpuscles, instead of undergoing hemolysis, were actually preserved from disintegration for a period considerably greater than in the control specimens to which no venom had been added. On the other hand, if the amount of venom employed was less than ro per cent of the mixture, hemolysis occurred in the usual way. They were led by their experiments to regard the once-dried venom as being a less effective preservative than the fresh secretion, and they noted that, of the corpuscles tested, those of the rattlesnake were most perfectly pre- served by crotalus venom. Stephens and Myers, Kyes, Kyes and Sachs, and Lamb encountered the same phenomenon in the course of their studies with cobra, bungarus, and daboiavenom. Flexner and Noguchi also confirmed the protective property of crotalus venom upon rabbit corpuscles. It may now be regarded as well established that when the optimum of the hemolytic action of venom is exceeded, the degree of hemolysis which it is capable of producing diminishes gradually as the dose of the venom increases. Among the natural biological hemolysins, venom alone is known to possess this property, but, in the course of a study of bacteriolysis with certain immune sera of high potency, Neisser and Wechsberg observed an inhibition of bac- tericidal effects when an excess of amboceptors, relative to the complement content, was brought into bacterial suspension. A similar, although probably distinct, phenomenon has been described by Detre and Sellei in their studies of hemolysis caused by bichloride of mer- 1 Against tetanolysin the antilytic power was increased by neutralization of cholesterins, while against saponin and solanin it disappeared after neutralization. 196 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS cury. As has been shown by Madsen and Walbum, acids in excess produce non-hemolyzability of the corpuscles, whereas their weaker concentration causes regular dissolution of the cells. Kyes and Sachs explain this phenomenon by assuming that an excess of venom amboceptors produces “side-tracking”’ of lecithin and renders the latter unable to attack the venomized corpuscles. In other words, the hemo- lytic amboceptors of the venom are responsible for this phenomenon when used in great excess. They did not, however, produce the evidence that the venom from which all coagulable constituents have been eliminated by a brief boiling, which is insufficient to diminish its hemolytic power, is still capable of producing this protective phenomenon. Noguchi analyzed this phenomenon in quite a different manner and reached an entirely different conclusion. He treated the washed corpuscles of horse with varying strengths of different venoms, ranging from a superhemolytic (or non-hemolyzable) dose to an optimum hemolytic dose (this latter con- centration is only hemolytic in the presence of suitable venom activators). When venom is present in more than 5 per cent concentration the hemolysis of the defibrinated blood of horse is retarded for nearly 12 hours at 20° C., causing no lysis within the first 6 hours. But as long as there is a trace of serum constituents in the mixture complete dissolution can not be prevented by any practicable concentration of venom. An equal mixture of the defibrin- ated blood and 2.5 per cent cobra-venom solution will undergo hemolysis in I or 2 hours. It has already been stated that the thoroughly washed corpuscles of horse are not hemolyzed by venom, no matter what the degree of concentration. If cobra venom in strengths above 4 per cent be mixed with a corpuscular suspension of 5 per cent, no change may take place in the mixture in many weeks; while with a quantity of venom as small as o.1 per cent the cells will disintegrate rather more quickly than in control tubes which are not entirely sterile. Corpuscles which had been brought into contact with the stronger solutions of venom were tested for resistance to salt solutions of varying toxicity. These tests disclosed the unexpected fact that corpuscles thus highly venomized, and in the presence of an excess of venom, are not hemolyzable even by water. At the same time their susceptibility to heat is changed. It has been found that the control tubes of blood corpuscles alone are hemolyzed completely in from 175 to 180 minutes when kept at the constant temperature of 53°C. In the presence of venom of a concentration not exceeding 1 per cent, complete hemolysis will take place at this temperature in from 5 to 15 minutes; with a concentration of venom as low as 0.01 per cent 30 minutes will be required; with a concentration of 10 per cent there is no perceptible change in the corpuscles for the first 20 minutes, after which laking com- mences. This last laking is not, however, typical. A bright haemoglobin color does not appear in the fluid, but the cells undergo disintegration, the color of the mixture becomes coffee-like, and in about an hour a turbid pre- VENOM HZMOLYSIS AND VENOM AGGLUTINATION 197 cipitate of cell particles and venom granules can be discerned under the microscope. Strong solutions of venom are capable of protecting the corpuscles from destruction by water, but venom solutions below 2 per cent in strength render the cells more sensitive to salt solutions, as measured by the toxicity of the fluid for the corpuscles; and as the strength of the venom descends from this limit the susceptibility, as measured by the degree of toxicity, diminishes. The next step of inquiry into the mechanism of this phenomenon was taken by Noguchi, who first determined whether the hemolytic amboceptors of venom have any relation to it. The washed corpuscles of horse were mixed with ro per cent and 2 per cent solutions of cobra venom and acted upon by the latter for two hours. After this period of contact an excess of horse serum or lecithin was introduced into the mixtures. No hemolysis took place in the mixture containing ro per cent venom, while complete hemolysis occurred in the mixture with 2 per cent venom in it. An activator deviation does not exist here. Another way of demonstrating the non-participation of hamolysins in this protective phenomenon was brought out by heating the venom to 95° C. and 100° C. for a brief period. The venom which had been heated to gs° C. was a milky fluid with fine precipitate, but it still had both the protective and hemolytic properties. By separating the coagula by filtration the protective body remains on the filter, while the entire content of hemolysin reappears in the clear filtrate. When the venom is heated to 100° C. for 5 minutes it becomes non-protective, but the heated solution contains the greater portion of the hemolytic principle. Heating to 135° C. destroys both the hemolytic and protective properties in foto. That the hemolytic filtrate of the heated poison exerts a markedly injurious effect on the integrity of washed corpuscles of horse —even in a concentration of 10 to 20 per cent of heated venom — is also shown by the reduction of their resistance to toxicity. Until a specially injurious agent, which predisposes corpuscles to laking by physical agents, is discovered in venom, Noguchi is inclined to believe that the injurious action of the filtrate is due to venom hemolysin. Another interesting fact was brought out in regard to the venom-protection phenomenon. The serum of rattlesnake is highly agglutinative and hemo- lytic for corpuscles of horse, and yet it does not protect them in any degree against injurious physical agents. The corpuscles of horse, after a contact of 12 hours with the inactivated rattlesnake serum in excess, were heemolyzed by salt solution of 0.45 per cent strength. The protection afforded by the strong concentration of venom disappears when the corpuscles are washed in salt solution and freed from the venom. Such corpuscles show a greater diminution of their physical resistance than those treated with a weaker venom solution. It is very singular that the protection is closely associated with the presence of the venom. The venomized corpuscles, which are non-hemolyzable even in water, are readily hamolyzed by weak solutions of acid or alkali, and in these cases the 198 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS venomized cells succumb to the latter effects much more easily than the nor- mal corpuscles. Saponin, on the other hand, fails to hamolyze the venom- protected corpuscles, unless the venom has previously been removed, in which case laking is more prompt in the treated cells than in the untreated cells. The venom renders the corpuscles unhemolyzable in about 20 minutes. Noguchi finally proceeded to find out the nature of this protective phe- nomenon. It was ascertained that the aqueous solution of horse corpuscles does form minute precipitates when mixed with a strong solution of venom. The stroma separated from the water-laked corpuscular solution did not give any appreciable quantity of precipitation. By using an aqueous solution of pure hemoglobin he discovered that venom produces precipitation with this solution. But in a saline solution at 0.9 per cent the precipitate was of coarser and scantier nature. The weak solutions of acid or alkali promptly dissolve the venom-hemoglobin precipitate. This seems to explain why acid and alkali hemolyze the venom-protected corpuscles, while water does not. Globin obtained from the horse hemoglobin was found to be readily precipitated out by venom from its aqueous, but not saline, solution. Further, it was found that not every hemoglobin solution is precipitable by venom. ‘The intracorpuscular contents of horse, rat, and rabbit bloods gave abundant precipitate, while none was obtained with those of dog and guinea- pig bloods. The solutions of pure hemoglobin and globin of dog’s blood corpuscles did not give precipitation with venom. It may be mentioned here that the corpuscles of dog and guinea-pig are never protected by any high strength of venom, while those of horse, rabbit, and rat become entirely non-hemolyzable when mixed with strong venom solution. Noguchi adds that when the serum globulin of horse serum — obtained by dialysis —is suspended in water or weak saline solution, the venom quickly brings down the suspended particles, which, left to themselves, subside very slowly, if at all. Of globulins, pseudoglobulin gave the most abundant pre- cipitation with venom. The venom-protected corpuscles resist the hemolytic action of tetanolysin and the destruction by fluorescent aniline dyes when exposed to the sun’s rays. CHAPTER XVII. CYTOLYSINS IN SNAKE VENOM. The death-dealing neurotoxins and the hemorrhagins are most important of all cytolysins contained in snake venom, and the best-studied are the heemo- lysins, including the erythrocytolysins and leucocytolysins. Full descriptions of these three groups of cytolysins have already been given elsewhere under separate headings and I shall not repeat them in the present section. Apart from the neurolysins, hemorrhagins, and hemolysins several other cytolysins have been demonstrated in snake venom by Flexner and Noguchi,’ and somewhat later by Calmette and Noc.? The results of experiments obtained by Flexner and Noguchi are given in table 13. The venoms employed were those of Cobra, Ancistrodon piscivorus, Crotalus adamanteus, Daboia russellii, and Lachesis flavoviridis. The animal cells came from a wide range of animals, including warm- blooded and cold-blooded species. The former, limited to certain species of mammalia, had served for the study of the effects of venom upon the cells of the liver, kidney, and testes; the latter for that upon the spermatozoa, ova, and nerve cells. The cells were obtained by preparing emulsions of the solid organs and by suspending the expressed spermatozoa or separated ova in appropriate fluids. In the case of warm-blooded animals 0.85 per cent saline solution, and in the case of the marine animals fresh sea-water, were employed. The strength of the emulsions of the cells was approximately 5 per cent of the organs used. The venom was dissolved in 0.85 per cent saline solution or in sea-water. The temperature to which mixtures of emulsion and venom were exposed were those of the room or thermostat (37° C.), depending, usually, on the origin of the cells. Observations of the effects were made (1) in test-tubes with the naked eye and (2) by means of microscopical examination. EFFECT OF VENOM ON CELLS OF WARM-BLOODED ANIMALS.’ The animals used in these experiments were dog, guinea-pig, rabbit, rat, and sheep. The venoms employed were daboia and crotalus. The experi- ments given would seem to prove conclusively that venom contains solvents for the parenchymatous cells of several animals, and that considerable dif- ferences in activity in this respect occur, according to the source of the venom. Flexner and Noguchi have also tested other venoms, for example, from the cobra, water-moccasin, and habu, and have found them to possess similar cytolytic properties; but their experiments show that daboia venom contains the most and crotalus venom the least active solvents, the other venoms arranging themselves in the order: water-moccasin, cobra. (Table 13.) 1 Flexner and Noguchi. On the plurality of cytolysins in snake venom. 2Noc. Sur polars propriétés physiologiques de différents venins de serpents. Ann. Inst. Pasteur, 1904, » 387. : 3 Rabbits’ s atozoa are seen to stop their motion under the influence of crotalus venom. (Weir Mitchell and Reichert.) 199 200 TABLE T3> [Experiment I: Dog’s Organs.] Gelb and Control. I per cent daboia venom. I per cent crotalus venom. Liver, Contains separated and | Marked agglutination and clearing of fluid; | Slight agglutination; no solution 3 hours. united liver cells; few complete disappearance of cell outlines of cells, but only swelling with free granules and cre- and forms. Only ground substance con- indistinctness of cell outlines. nated red corpuscles. taining numerous refractive granules re- Nuclei invisible. mains. No nuclei visible. Kidney, | Cells much broken; few | Marked agglutination and clarification of | Slight agglutination; granules 3 hours. complete cells; many emulsion; no complete cells visible; ground partly dissolved; individual free nuclei and gran- substance granular. Glomerular blood cells visible; glomerular blood ules. Some complete dissolved and cells cleared; nuclei of capil- dissolved; capillary walls and tubules and glomeruli. laries distinct. nuclei distinct. Testis, | Testicular cells and sper- | Marked agglutination; complete disappear- | Slight agglutination; complete 3 hours. matozoa_ well pre- ance of cell-bodies; occasional sperma- disintegration of testicular served; few free gran- tozoa and refractive granules remain. cells; large numbers of free ules. Agglutination masses show no preserved nuclei visible; spermatozoa cells. little altered. [Experiment II: Guinea-pig’s Organs.] Liver, Cells chiefly in smaller | Marked agglutination; clearing of fluid; cell }| Slight agglutination. Only swell- 3 hours. and larger groups; nu- protoplasm clear; nuclei distinct. Ground ing of cells; otherwise un- clei obscured; many substance in which cell outlines are indis- altered. granules and crenated tinctly visible show granules and fat. red corpuscles. Kidney, | Cells much broken up; | Marked agglutination and clearing. Nearly | Slight agglutination; swelling of 3 hours. many free granules and complete disappearance of cells and gran- cell protoplasm and imper- nuclei; some ‘red cor- ules; indistinct free nuclei; glomerular fect solution of granules; nu- puscles and glomeruli. blood, capillary walls and nuclei distinct. clei indistinct. Testis, Testicular cells perfectly | Marked agglutination; emulsion cleared. | Very slight agglutination; cells 3 hours. preserved; numerous Loss of cell outlines and nuclei. Sperma- have lost refraction; are finely preserved spermatozoa; tozoa little altered, but entangled in clear granular and present eroded very few free granules. ground substance. appearance; spermatozoa swollen; many free granules. [Experiment III: Rabbit’s Organs.] Liver, Very few free cells; many | Marked agglutination and clearing; cell | Slight agglutination; cells co- 3 hours. small groups of cells; clumps show very indistinct outlines; nu- alesced, but outlines can still many red corpuscles. merous granules; occasional visible nuclei be made out; no solution of in cells. granules; nuclei invisible. Kidney, | Many complete cells and | Marked agglutination; almost complete dis- | Moderate agglutination; al- 3 hours. tubules; numerous free appearance of all cells; few granules re- most no solution, but gran- granules and red cor- main; nuclei invisible. Whole tubules ules remain. Cells swollen puscles. very indistinct. and granular; nuclei more or less visible. [Experiment IV: Rat’s Organs.] Liver, Cells in small groups; | Agglutination; emulsion clear; cells coa- | Slight agglutination; swelling of 3 hours. many disintegrated lesced; outlines gone; granules largely dis- cells; few outlines remain; cells; free granules, nu- solved; nuclei indistinct, but in part free. granules remain; nuclei in- clei and red corpuscles. visible. Kidney, | Individual cells and al- | Marked agglutination; almost complete dis- | Slight agglutination; cells swol- 3 hours. most complete tubules; appearance of cells. Complete tubules len, but no solution; gran- few free granules and show indistinctness of outlines and solu- ules remain; cells appear red corpuscles. tion of cell granules. Glomeruli cleared. eroded. Testis, Cells well preserved; nu- | Marked agglutination; almost complete dis- | Marked agglutination; cells 3 hours. merous spermatozoa; appearance of cells; ground substance granular and eroded, but no few free granules. contains large numbers of twisted sper- solution; nuclei indistinct; matozoa. spermatozoa preserved. [Experiment V: Sheep’s Organs.] Liver, Cells in separate state and | No agglutination; cells swollen and granules | Almost unchanged. 3 hours. small groups; outlines partially dissolved; nuclei more visible; sharp and _ distinct; free granules partially dissolved. granules numerous; nu- P clei usually invisible. Kidney, | Imperfectly separated; | Slight agglutination; almost complete solu- | Partial solution of cells; many 3 hours. cells much broken; tion of cells, leaving only granules and granules and free nuclei re- VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS some free granules and nuclei; some glomeruli. occasional nuclei; glomeruli cleared; out- lines of capillaries and nuclei visible. main. Glomeruli partially cleared. CYTOLYSINS IN SNAKE VENOM 201 EFFECT OF VENOM ON CELLS OF COLD-BLOODED ANIMALS.! For the purpose of this study Flexner and Noguchi employed three differ- ent kinds of cells: (a) nerve cells; (6) spermatozoa; (c) ova. Nerve cells: In regard to the neurolysis by venom the nerve cells contained in the pre-cesophageal ganglia of Sycotypus canaliculatus, Modiola modiolus, and Mactra solidissima were employed and seen to undergo rapid disintegra- tion under the influence of venom. For the details I refer to the separate heading ‘‘venom neurolysis in vitro.” Sperm cells: For the study of venom spermatolysis, the spermatozoa of several different orders of animals —the reptilia, arthropoda, vermes, pisces, and echinodermata —were employed. The method of study consisted in suspending the spermatozoa in sea-water or normal saline solution (0.85 per cent), depending upon the nature of the animal. To the uniform milky sus- pensions the venom in 1 per cent solution was added. The effects were noted im vitro by the naked eye and under the microscope. Below are two typical experiments given to avoid detailed descriptions for each species: TABLE 14. [Experiment I: Spermatozoa of Chrysemys picta (Painted Turtle).] Control. Cobra. Moccasin. Crotalus. Spermatozoa ac- | The milky hue becoming | Suspension becoming clearer, | No change; active tive; normal lighter; motility lost; no motility; swelling of motility. appearance; 30 many partially dis- middle piece especially | minutes. solved. marked. Like control; 2 | Fluid clearing and al- | Moderate agglutination; con- | No marked change hours. most without deposit. siderable deposit and much | to naked eye. Mo- Microscopically, frag- clearing of the suspension. tility present in ments only visible. Many of the cells dissolved. some individuals. No motility; ten- | Fluid almost completely | Clear fluid, but whitish; ag- | Motility absent; de- dency of cells cleared. All cells glutinated deposit in which posit of cells; no to sink to bot- practically dissolved. many of the cells remain in agglutination. tom of test- a swollen condition. tube; 4 hours. [Experiment II: Spermatozoa of Tautogolabrus adspersus (Cunner).] Very active mo- | Motility gone in 5 min- | Clearing in 30 minutes owing | Slight agglutination; tility; 30 min- utes; beginning in 10 to marked agglutination; | motility much re- utes. minutes to clear; no deposit forming. duced. agglutination. ne Very active mo- | Solution complete. Deposit undergoing solution. | Very slight motility tility; 1 hour. | remains. : Very active mo- | Solution complete. Only débris remains. Motility gone; little tility; 2 hours. | if any solution. These two experiments will suffice to show the rapid action of cobra and the weaker effect of water-moccasin and crotalus venom in causing sperma- tolysis. ‘The effect of crotalus venom is, indeed, but slightly injurious, pro- ducing, as it does, agglutination, but almost no solution of the cells. 1 Mitchell and Reichert observed that the crotalus venom causes the cilia of pharyngeal epithelia to cease their motions, but those of the tunic of oysters remained unaffected. * 202 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Other kinds of spermatozoa which are acted upon in much the same manner as the two examples given are those of the following orders and species: Pisces: Fundulus heteroclitus (minnow), Cyprinus carpio (German carp.) Arthropoda: Homarus americanus (lobster), Eupagurus longicarpus (small hermit-crab), Eupagurus pollicaris (large hermit-crab), Limulus poly- phemus (horse-shoe crab), Libinia (spider-crab). Vermes: Cirratulus grandis, Lepidonotus squamatus (scale-worm), Lum- briconereis opalina, Nereis virens (clam-worm). Echinodermata: Asteria vulgaris (star-fish), Arbacia punciulata (purple sea-urchin). The following kinds of spermatozoa are found to be wholly refractory to the effect of venom, even cobra: Phascolosoma among the vermes, and Pentacta frondosa (sea-cucumber) among the echinodermata. Egg cells: For the study of ovolysis the egg cells of several orders of cold- blooded animals were employed: pisces, arthropoda, vermes, and echinoder- mata. Not all of these cells are affected equally, and some are not acted on at all. In some instances pigmented ova, under the influence of venom, give up their pigment, which is diffused along with other interior contents into the surrounding medium, tinting this so as to suggest the liberation of heemo- globin from red blood-corpuscles by venom and other heemolytic substances. Table 15 illustrates the changes in unfertilized eggs brought about by venom. The experiments prove the susceptibility to venom cytolysis of certain ova of cold-blooded animals. Of the ova tested, those of Limulus and Nereis failed to show distinct changes leading to more or less complete dissolution. Among other susceptible ova are those of Phascolosoma and Cirratulus, with which may be mentioned the fact of the insusceptibility of sperm cells of the former worm under the same conditions of venom treatment. (Plates 30 and 31.) TABLE 15. [Experiment I: Ova of Asteria (Star-fish).] Time of 2 per cent moccasin 2 per cent crotalus observation. Control in sea-water. I per cent cobra venom. erat ae 30 mins. Pale, pinkish-yel- | Slight agglutination; con- | Marked aggluti- | Slight agglutina- low round cells; tents dissolved; first nation; solu- tion only. germinal ves- deuteroplasm and then tion of cells be- icle visible. germinal vesicle. gun. 3 hours. No change. Only empty membranes | Destruction more | No solution. and fragments left. advanced, but less than cobra. 6 hours. No change. Only empty membranes | Many cells have | No solution. and fragments left. lost contents. [Experiment II: Arbacia (Sea-urchin).] 30 mins. Round, semi- | Marked agglutination; | Marked aggluti- | Moderate ageluti- opaque cells of cells swollen and pig- nation; _pig- nation only. faintly purple ments liberated. ment partially color. Nucleus liberated. visible. 3 hours. No change. More advanced solution. | More advanced | Pigment paler; solution. eggs swollen. 6 hours. No change. All cells have yielded their | Somewhat less | No change. contents; membranes advanced than visible. cobra. NOGUCHI PLATE 30 4 Rake: oy ‘ 2 & } 5 | > C en, ua opcesia atau ra x - Loe Lay, os ‘ 1 aie ; [fe Sy E . ; (ez “Ny E ‘ ° 4 wk ne 6 4 | ; 0 ! ’ 5S 1 to 4. Eggs of Arbacia punctulata. 1, Normal. 2 to 4, Different stages of Ovolysis caused by | per cent Cobra venom in sea-water. 5 to 8. Eggs of Lepidonatus squamatus. 5, Normal. 6 and 7, Different stages of Ovolyais caused by | per cent Cobra and 2 per cent Crotalus venom, showing no real Ovolysis, but peculiar Retraction of Deutoplasm. » 9to 11. Pluteus stage of Arbacia punctulata. 9, Normal, active pluteus. 10, Plu- teus under the effect of 2 per cent Cobra venom, 11, Disintegration under the effect of same venom. (Drawings by Noguchi.) ee \ wei a - ‘| a ie mi re i i Vy , } t x ‘ \ . \ hi Tae i ‘ 5 . SUNe . f i” To . : : / NOGUCHI PLATE 31 Ovolysis—Eggs of Asteria vulgaris. 1, Normal unfertilized egg. 2, Action of | per cent Cobra or 2 per cent Moccasin venom. » 3, Result of complete Ovolysis by these venoms in 6 hours. —_ 4, Action of 2 re cent ° Crotalus venom in 6 hours. 5, Normal egg with polar body formation. 6, lysis by venom. 7 to 15, Ovolytic effects of venom upon fertilized eggs. (Drawings by Noguchi.) [ fo a ‘) i f 7 mot ae a CYTOLYSINS IN SNAKE VENOM 203 TABLE 15.— Continued. [Experiment III: Lepidonatus squamatus (Scale-worm).] Time of 18 2 per cent moccasin 2 per cent crotalus = in water. I nt cobra m. at en. Control in sea per ce veno cared. Large, transpar- | Very marked agglutina- | Like cobra. Moderate aggluti- ent cells with tion; membrane intact; nation; no other distinct mem- vesicle granular; nuclei change. brane; germi- distinct. nal vesicle, etc. No change. Little change. Little change. Moderate aggluti- nation; no other change. No change. Cell membranes unrup- | Little change. Germinal vesicles tured; germinal vesicles granular; no fragmented. other change. [Experiment IV: Fundulus heteroclitus (Minnow).] 30 mins. Greenish - blue- | Pigment quickly dis- | Similar to cobra | Unchanged. opaque cells solved; germinal vesicle venom. with irregular much swollen or frag- contours; ger- mented. minal vesicle covered with pigment and obscured. No change. Many cells have ruptured | Similar to cobra | Swelling of cells. and emptied contents. venom. No change. All membranes emptied. Few membranes | Partial disintegra- with contents. tion. Flexner and Noguchi next determined the action of venom upon the fer- tilized ova of Fundulus, Arbacia, and Asteria. Observations upon these cells were conducted under two sets of conditions: (a) fertilization was attempted in venom solutions, and (b) venom in solution was added to the fertilized eggs at different periods of segmentation. The following facts appeared: Fundulus ova: (1) Venom in strong®solution (5“per cent cobra) dissolves the-fertilized ova, but in weaker solution only delays segmentation. (2) After fertilization and beginning segmentation weak solutions of venom do not prevent further segmentation. (3) Brief treatment with weak venom solutions of the segmenting ova up to the time of the morula stage causes only delayed development. (4) Brief treatment of the embryos with weak solutions at about the period of formation of the brain and optic vesicles (36 hours) causes malforma- tion and delays the hatching. (5) More advanced embryos are more resistant, but finally may succumb to venom poisoning. Arbacia and Asteria ova: (z) Strong solutions (5 per cent cobra) of venom prevent fertilization, but weak solutions permit its occurrence. (2) Strong solutions of venom delay segmentation, while weak solutions cause imperfect and irregular segmentation. (3) Brief treatment with weak venom solution accelerates the blastula forma- tion, while the plutei are killed by strong solutions of venom. 904. VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Flexner and Noguchi found that the cytolytic principles of snake venom are still active after moist heating to 85° C. for 30 minutes and are not de- stroyed by moist heat at a temperature of 100° C. maintained for 15 minutes. Dry heating for 2 hours at 140° C. suffices to diminish, but not to abolish, the activity of daboia venom. The mechanism of venom cytolysis was also studied. They found that previous heating of susceptible somatic cells and nerve cells to 55° C. for 30 minutes renders the cells almost insusceptible to the solvent action of venom, although agglutination and some granulation of the cells may occur. Addition of fresh serum or fresh body fluid to the mixture of the heated cells and venom causes a certain amount of dissolution and disintegration, which are never so strong as in the case of unheated cells. Repeated washings of the fresh cells in sea-water or saline solution do not suffice to prevent or even delay considerable solution of the cells by venom. They also pointed out that the cytolysis can not be due to the action of proteolytic ferment of venom, for the latter directly attacks the gelatin and is completely destroyed by 80° C. In an extensive series of experiments these investigators were able to show that venom contains a number of cytolysins, each having a special preference for one given group of cells. They also consider that cytolysins of one group attack their corresponding cells with varying severity according to the source of the cells, which fact they regard as due to the differences in the receptor apparatus of similar cells in different species of animals. Since the appearance of the above work of Flexner and Noguchi, the important discovery by Kyes of the interaction between venom and lecithin has appeared and makes it probable that the mechanism of venom cytolysis, especially the dissolution of egg cells, may have some resemblance to the venom-lecithin hemolysis. As is shown by Jacques Loeb and others, the membrane of ova seems to be of a lipoidal nature and the deutoplasma contains a considerable amount of lecithin. There may be, at least in part, certain relation between the lecithinophilic property and the ovolytic processes in this case. Loeb and others have shown that artificial parthenogenesis can be accomplished by the combined effects of fat-solvent or fatty acids and changes in the toxicity of the medium in which the ova are placed — in the presence of oxygen. I have made an attempt to produce a similar phenome- non by substituting fat-solvents with weak venom solutions, but so far with- out definite result. It is significant that Flexner and Noguchi observed the accelerating influence of weak venom solution upon blastula formation. In this connection the interesting observations of Féré* on the influence of venom upon the evolution of chicken embryos must not be overlooked. This biologist introduced the venom of viper (in dose of 0.00005 gm. per egg) into the egg-white and found that 83 out of too toxicated eggs presented vari- ous anomalies of development when opened and examined 72 hours after the inoculation. 1Féré. Evolution de ’embryon de poule. Influence de l’introduction du venin dans l’albumen de Poeuf de poule. C. R. de la Soc. Biol., 1896. IIme Série, 8. CYTOLYSINS IN SNAKE VENOM 205 CYTOLYTIC ACTION OF SNAKE VENOM ON MICRO-ORGANISMS. Flexner and Noguchi’ first stated that snake venom produces on B. anthra- cis, B. coli, and B. typhi rapid involutions, degeneration, and plasmolysis when it is mixed with nutrient media. Cobra venom was the strongest and crotalus venom the feeblest, while ancistrodon and daboia venoms were intermediate. (Plate 32.) Somewhat later Calmette and Noc? found that 1 per cent cobra venom quickly dissolves Vibrio cholera and asporogenous strain or young culture of B. anthracis. Staphylococcus aureus, B. diphtherig, and young B. subtilis were equally affected, while B. pestis, B. coli, and B. typhi were more resistant; and B. pyocyaneus, and B. prodigiosus were almost unaffected. B. tuberculosus proved totally insusceptible. The removal of the bacteriolytic substance for one kind means the same for the rest, showing the non-specific nature of this particular principle of venom. Calmette’s antivenin effectively stops the bacteriolytic action of cobra venom. ‘The reappearance of this property out of the neutral mixture of venom and antivenin does not occur when heated to 80° C. The bacterio- lytic property disappears when heated to above 8 5° C. for 30 minutes; hence it is not due to the proteolytic property of venom, which disappears at 80° C. Trypanosomes are also dissolved by 1 per cent cobra venom in 30 minutes. 1 Flexner and Noguchi. Snake venom in relation to hemolysis, bacteriolysis, and toxicity. Jour. xp. Med., 1902, VI, 294. Foot-note. ?Noc. Ann. Inst. Pasteur, 1905, XIX, 200. ’Calmette. Les venins. 1907, Paris. Goebel. Ann. Soc. Med. de Gand, 1905. CHAPTER XVIIL. HISTOLOGICAL CHANGES PRODUCED BY SNAKE VENOM ON VARIOUS ORGANS AND. TISSUES. Before presenting the facts derived from the elaborate and extensive studies of various investigators concerning the histological alterations produced by snake venom, a general review of the nature of these changes may be per- mitted at this place. As we shall presently see, the histological changes, which were demon- strable with the practicable methods of our past and present histological status, fall into two great groups. ‘The first group is the fatty degeneration of the protoplasma of the diverse kinds of cells, and the other comprises the changes designated necrosis. The question quickly arises whether both are the action of the same principles or the actions of specific agents for each group of alteration. In view of the recent development in the biochemical investigations of the active principles contained in snake venom and other similar cellular toxins, it appears that the latter hypothesis conforms to the observed facts. The lipolytic properties of snake venom— especially the libera- tion of fatty acids from phosphorized and non-phosphorized fats by certain ferment-like principles of venom — render it probable that fatty degeneration is the result of the cytolysis of such agents. It is partly due to the Ehrlich- Kyes phenomenon (or formation of lecithids and liberation of free fatty acids) and to the Neuberg-Rosenberg phenomenon (or the splitting of neutral fats), both being liable to take place in media as rich in lecithin and fats as the cell protoplasma. Considering the potency which a minute quantity of venom circulating in the venomized body possesses, it is again reasonable that in the production of fatty degeneration lecithin-splitting plays the foremost part. The necrotic changes I consider due to the specific phenomena peculiar to each group of somatic as well as nervous tissues, and brought about by the action of specific cytolysins in the sense of Flexner and Noguchi, namely: there exists between the cells and the active principles a special affinity, if not specific. ‘The relation between fatty degeneration and the necrotic altera- tions of these cells is not quite clear, inasmuch as various fatty and lipoid substances display marked destructive effects on various cellular elements. It may be true, at least in part, that the necrotic processes are caused second- arily by the primary fatty degeneration. On the other hand, there are many evidences that point in the opposite direction. Fatty degeneration may be entirely absent from the cells showing marked disorganization of their con- stituents. Equally we may assume that fatty degeneration may be produced secondarily by the cessation of the normal oxidating function of the cells, through specific toxins of venom. In deciding this point the results which I 206 Noguchi re & 7 oe : va 5 . pe RSS \ a ee NY ee ay — A \ ‘ \ 4 a Peepers \ \ =H 7 =. t ~ \ Papen ; f = Mh i = I / ¢ 2 SSS: ris i 7 Roe Ee , ‘ shames 2 Sas NN ‘ 7 2, f So 5 ee PRIS ie a Se af me A\m~ r / .. fi Ziv. i . / > rrr x % \ Sy ue ge ae Bn Sy ‘ ‘ Me, / eo . = wim ae ‘ = f gee a a Soar Pte it a ae ¢ o=Sia— Se i = ; \ iy SN ie P i SS oe . KG Z ; “i é phe H \ e 4 ' i 3 ~ ~ «4 =a Fi ‘ } “ee ! \ i 1 A ' ~~ Oy x I i ~*~, > } Na. I a Ne Finn, f { XQ = é . =~. = j A pee) 4 / r ‘ sf - ne 5 ¥ \ Hf b>: / N + % / { 7 o 2 é 2 \ os © h<« -" g « ¢ - e i ‘ ; ‘ we s . * - oy 4) . 4 — ee ae “es -- \e s ‘ ie ‘ be a \ So “e \ : x Plate 32 Cc D A, Young Agar Culture of B. anthracis. B, C, and D, Young Cultures of B. anthracis on Venomized Agars. (Drawings by Noguchi.) A eae we i i i vi pont ¥ i ara kine Hea BA mew a Me La a ci ny HISTOLOGICAL CHANGES PRODUCED BY SNAKE VENOM 207 obtained through studies on the effects of various venoms upon the excised tissues in vitro have a certain weight. In this case the vital processes have no parts. Our present knowledge of the pathological histology of venom toxication is derived from the investigations of S. Weir Mitchell and Reichert,! Hindale,’ Karlinski,? C. J. Martin,! Nowak,®° Ewing and Bailey,’ Kelvington,’?’ Lamb and Hunter,’ Hovius,® Zeliony,’® Flexner and Noguchi," Jousset ” and that of the writer added in the present work. The histological changes produced by the neurotoxins and hemorrhagins, owing to their direct importance in the fatal effects of the venoms they contain, have been dealt with in detail in the separate sections, and these will not be repeated here. | The solvent actions of snake venom upon various sets of cells in the fresh state must properly fall under the present heading and the phenomena can be compared with the changes demonstrated in the sections as stained specimens, but the writer has given the former a special space, on account of the unique manner of study by which Flexner and Noguchi worked. ACTION OF SNAKE VENOM UPON THE LIVER. This organ is especially susceptible to the action of venom. In the case of rapid death the protoplasma of hepatic cells is turbid and granular, and the granules take stains well along the periphery, but not in the interior of the cell. In the case of slower death, namely, prolonged toxication with venom, the protoplasma heaps up to certain parts of the cell and forms vacuoles of indefinite contour. One part of the protoplasma is necrotized and destroyed. Here the nuclei undergo certain alterations. Their con- tour is well marked and sharp, but the chromatin in the interior presents granular fragmentation, and the entire nucleus takes basic dyes faintly, due to the diffusion of dissolved chromatin substance into the nuclear fluid. When the protoplasma of hepatic cells undergoes further changes its chromatin mass of nuclei diminishes and gradually loses the property to take up stains, and finally the entire cell contains a small quantity of granules without the nucleus. 1 Weir Mitchell and Reichert. 1886, Washington. 2Hindale. Medical News, 1884, XLIV, 454. ; 3 Karlinski. Zur Pathologie des Schlangenbisses. Fortschritt der Medicin, 1890, VIII, 617. 4C. J. Martin. On the physiological action of the venom of the Australian black snake (Pseudechis porphyriacus). Proc. Roy. Soc. of N. S. Wales, 1895. ®’ Nowak. Etude expérimentale des altérations histologiques produites dans l’organisme par les venins des serpents venimeux et des scorpions. Ann. Inst. Pasteur, 1898, XII, 369. * Ewing and Bailey. Appendix to Gustav Langmann’s ‘Poisonous snakes and snake poison.” Med. Record, 1900, LVIII, gor. 7 Kelvington. preliminary communication on the changes in nerve cells after poisoning with the ss of the Australian tiger snake (Hoplocephalus curtus). Jour. of Physiol., 1902, XXVIII, 2 426. 8 Lamb and Hunter. See under “ Neurotoxins.” ® Vailant Hovius. Thése Bordeaux, 1902. 10 Zeliony. Path.-histolog. Verainderungen der querstreiften Muskeln an der Infektionsstelle des Schlangengiftes. Virchow’s Arch. f. pots Anat. und Physiol., t905, CLXXIX, 36. 1 Flexner and Noguchi. The constitution of snake venom and snake sera. Jour. of Path. and Bac- teriol., 1908, VIII, 379. 14 Jousset. Lésions produites par les venins de serpents. Art. Med., Paris, 1899, LXXXVII, 358. 208 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS In certain cases there is extensive lesion of fatty degeneration and in parts numerous small foci of total destruction of hepatic tissue. The liver of the dog is very sensitive and the microscopic structure of parenchyma may some- times be completely destroyed. ‘There is no lobular disposition to be further distinguished; the trabecules are broken up and only confused agglomeration of the cells in the extravasated blood can be made out. With the animals which lived a long time after venomization there are also some changes in the biliary tract. The epithelial cells are subjected to fatty degeneration. In dog and other small mammals there are infiltrations of mononuclear cells between the epithelia of the biliary canalicules. The latter may sometimes be tumefied, swollen, or vacuolated. Thus the histo- logical changes of the liver are fatty degeneration, necrosis, and the infiltration of lymphocytes in the biliary tract. ACTION OF SNAKE VENOM UPON THE KIDNEY. This organ is also very susceptible to the action of venom. The glomerula are affected in their three parts. The blood-vessels in the cortex are static and their walls are sometimes ruptured, causing the escape of blood into the capsular cavity. The capsular cavity is filled up with granular exudate. The epithelial coat of Bowmann’s capsule is bulged and the nucleus stains badly. In the tubule contorts the cellular lesions present a great analogy to those of the liver. There are granulations and vacuolation, and the nucleus be- comes diffuse. Their lumen is filled with the necrotic cell débris. Similar obliterations occur in the Henlé loops. In the straight tubes and collecting tubes the epithelia are sometimes detached in blocks. Some of these canals are obliterated with granular cylinders or by the swelling of the epithelial cells. The vessels in the renal parenchyma are always distended and sometimes ruptured, resulting in small foci of interstitial hemorrhage. It is not un- common for the extravasated blood to destroy the parenchyma. In case of pseudechis poisoning there are frequently radial hemorrhages in the cortex, acute necrosis of epithelium lining the convoluted tubes. The hemoglobin from the disintegrated corpuscles exhibits an abnormal tendency to crystallize, and this sometimes happens in the tubules of the kidney to such an extent as to block the greater number. ACTION OF SNAKE VENOM UPON THE LUNGS. In the lungs venom produces numerous small impacts, around which are seen capillaries much dilated and the pulmonary vesicules are rendered very small. ACTION OF SNAKE VENOM UPON THE SPLEEN. Nowak observed only a slight degree of fatty degeneration in the spleen in those cases where the lesions in the liver and kidney were very advanced and extensive. Noguchi Plate 33 my SrneRensusime aa 6 |< EG cer stoi Se Bee Se Sse ete eters? an Yer ete - —) +0.4 per cent MnSOs 2 cc........... Castor oil ro c.c. (acidity =4.1 c.c.) +1.5 per cent cobra venom 2 c.c. +0.4 per cent MnSQOs 5 c.c........... Castor oil 5 c.c. (acidity = 2.05 c.c.) +1.5 per cent crotalus venom 2 c.c. (acidity = 1.4 c.c.) +0.4 per cent MnSQOz 2 c.c........... Castor oil to c.c. (acidity =4.1 c.c.) +1.5 per cent crotalus venom 2 c.c. +-0.4 per cent MnSO.z 5 cc........-.- From table 17 the participation of venom lipase in typical venom-hemolysis seems to be excluded. According to the same authors, ricin, and crotin in much less degree, possess a similar fat-splitting property, somewhat stronger even than crotalus venom. Bee venom also seems to have a certain lipolytic power. It is most desirable to investigate the thermal and chemical resistances of the lipolytic ferments of snake venom and other toxins in order to elucidate their importance as factors in the general toxicity of these substances. CHAPTER XX. ANTIBACTERICIDAL PROPERTIES OF SNAKE VENOM. S. Weir Mitchell has repeatedly pointed out the danger of a secondary bacterial infection in the subject which survives the primary fatal effects of snake venom, and also of the rapid decomposition of cadavers dead of venom toxication. He perceived that there must be a definite alteration produced by venom in the preservative properties of the tissues, but did not establish this point on an experimental basis. In 1893 William H. Welch, together with C. B. Ewing,' finally discovered that rattlesnake venom has the property of annihilating the bactericidal power of the blood. A rabbit was fatally poisoned with venom and just after its death the blood was collected from the large veins. Control blood was obtained from a healthy rabbit. The bactericidal power was tested by introducing into the serums obtained from those bloods, cultures of the anthrax bacillus and of cholera bacillus. It was found that while normal serum destroyed thousands of the respective bacilli, the venomized serum had lost this power. It is supposed that the rapid decomposition of the bodies of those who die of snake poisoning, as well as the extensive suppuration from which they suffer, may depend upon this cause. An exhaustive study of the same phenomena was next made by Flexner and Noguchi,? and the results are briefly mentioned below. The animals employed were the dog, rabbit, and Necturus; the venoms belonged to the cobra, moccasin, copperhead, and rattlesnake, and the bacteria were B. typhi, B. coli, and B. anthracis. The method consisted in — (1) Introducing venom into the animal and drawing the blood from the femoral artery into sterile Nuttall bulbs. (2) Permitting the blood from the normal animal to enter Nuttall bulbs in which the venom solution was contained. (3) Admixture of the venom in sterile solution (heated for 4 days to 56° to 60° C., 30 minutes each time) with separated serum. The bactericidal effects of the normal serums were first established. Rabbit serum is highly destructive for B. typhi and B. anthracis and least for B. coli. Dog serum is highly destructive for B. typhi. Necturus serum is also very destructive for B. typhi and B. coli, but without marked effects on B. anthracis. (x) Serum venomized in vivo: Cobra venom was most active. Blood from rabbits which had received 0.01 gm., taken 57 minutes after injection — 1 Welch and Ewing. The action of rattlesnake venom upon the bactericidal properties of the blood. Trans. of the First Pan-American Medical Congress, Washington, 1893, I, 354. 2 Flexner and Noguchi. Snake venom in relation to hemolysis, bacteriolysis, and toxicity. Jour. of Exper. Medicine, 1902, VI, 277. 215 216 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS moribund state —showed great loss of bactericidal properties. The three kinds of bacteria multiplied in this serum very freely, while all were com- pletely destroyed except (in a few experiments) B. coli, which showed con- siderable diminution until after 6 hours, when increase began. In another series, 0.03 gm. of crotalus venom was injected into the blood of animals, taken after 45 minutes; full serum was used for inoculation of bacteria; practically the same result as in the previous series was obtained. (2) Blood mixed with venom in vitro: In this series rabbits only were employed. The venom solutions were placed in Nuttall bulbs and the blood from the femoral artery was permitted to stream into them. In each experi- ment 0.006 gm. of venom were mixed with 20 to 30 c.c. of the blood. Coagu- lation was very slow or completely inhibited, and the serum was obtained when necessary by centrifugalization. It invariably contained hemoglobin. The results all show that the bactericidal power of the venomized blood is completely abolished. This series of experiments may be open to the criticism that the increased nutritive value of the serum because of the hemoglobin present may have been the cause of the effects noted; asa control, therefore, peptone was added to the serum in the proportion of 0.006 gm. of peptone to 20 c.c. of serum. From this experiment it follows that improve- ment in nutritive value reduced bactericidal effect, but in far less amount than is noted in the parallel case of venom. That the nutritive change is unimportant is shown by the first series of experiments, in which the poison- ing was done in vivo, and also by the following experiments in which venom was added directly to the separated serum. (3) Direct addition of venom to the serum in vitro: In this series serums of normal rabbits and dogs were employed. To 1 c.c. of rabbit serum 0.001 gm. of crotalus venom, and to 1 c.c. of dog serum 0.006 gm. copper- head venom were added. The results were the abolition of the germicidal powers of these serums. In order to determine the least quantity of venom required to remove the bactericidal properties of the serum varying quan- tities of copperhead venom were employed. Dog serum was chosen with B. typhosus. In each case 1 c.c. of serum was used. Table 18 gives one of these experiments, and the number of bacteria grown on one plate is shown. TABLE 18. [Experiment XXXII (a): 1 c.c. dog serum and varying amounts of copperhead venom.] Amount of copperhead venom employed. Time of contact before plating out. 0.0005 gm 0.0002 gm 0.0001 gm. 0.00005 gm 0.00002 gm. Immediately. .] 5,970 3,070 4,290 4,940 3350 r hourse a eS B o 30 o.1 83-5 82.5 oO. 78.2 79-5 0.15 71.3 74 80 0.2 56.6 59-5 0.2 64.5 66 0.3 39-8 40.5 0.25 56.2 57 - 21.6 0.3 44.4 48.5 70 5-3 0.4 33-3 32 0.5 15-9 15 i 354 60 50 40 30 0 0.1 0.2 0.3 0.4 0.5 0.6 o Neutralization with antivenin I. p Neutralization with antivenin II. Fic. 14. — tela i antilysin. Horse blood with lecithin. WATER-MOCCASIN-VENOM ANTIVENIN. With this venom the partial neutralization with its specific antivenin gave a singular result. When 2 c.c. of the antivenin were added to 10 minimal lethal doses of the venom (0.012 gm.) the mixture contained about 6 minimal lethal doses, but with 8, 10, 20, and 40 c.c. there was no complete neutralization. Madsen and Noguchi left this phenomenon to a later investigation, not having enough material for further work at that time. The neutralization of the hemolytic component of water-moccasin venom with the specific antivenin gave the following results: 8 c.c. 5 per cent suspen- sion of dog blood in o.g per cent NaCl were used for the test reaction. 260 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS “‘qcalc.”’ were calculated on the hypothesis that 1c.c. of antivenin is equiva- lent to 1.2 c.c. of the venom solution and that K = o. In this case the devia- tion of the calculated values with the increase of antivenin is much greater than in the cases of crotalus and cobralysins. From these observations Madsen and Noguchi point out that the neutrali- zation of the toxic principles of cobra, moccasin, and crotalus venoms by their specific antivenin shows analogy with the results obtained by Madsen, Arrhenius, and Walbum with many other toxins and antitoxins, and can be interpreted according to the view held by Arrhenius and Madsen. With the hemolytic components of these venoms the neutralization shows almost a straight line and there is only slight dissociation. The results obtained by Kyes' regarding the neutralization of cobralysin by antilysin show also that the reaction between these two bodies is very strong and gives a straight line. Criticizing the results obtained by Myers and by Flexner and Noguchi, who found a marked curve indicative either of TABLE 38. I C.C. 0.05 per cent solution of moccasin venom + 7 c.c. of antivenin + 1-7 c.c. of r per cent NaCl solution. nN q obs. qcalc. ° 100 100 0.05 93 94 0.1 87 88 0.15 82 82 0.2 in 76 0.25 7° 7° 0.3 63 64 0.4 53 22 0.5 42 40 0.6 26 28 0.7 17 16 0.8 10.5 4 I 2 ° Fic. 15. — Ancistrodonolysin-antilysin. Dog’s blood. the presence of toxoids in Ehrlich’s sense or of the strong dissociation in Arrhenius and Madsen, Kyes points out the importance of the presence of sufficient amount of activators (lecithin) in order to obtain the real expression of the reaction. With a defective supply of venom activator it would be natural to obtain a false result. Kyes proves his claim by a very interesting series of experiments in which the amount of activator was used in variable proportions. In the case of sufficient supply of activator he obtained a straight, and in the defective supply of activator a curved, neutralization line. 1Kyes. Cobragift und Antitoxin. Berl. klin. Woch., 1904, XLI, 494. CHAPTER XXVI. PRECIPITIN-REACTION WITH SNAKE VENOM. That a comparatively specific precipitating substance is developed in the serum of animals through repeated injections of the serums of alien species or various other proteid substances was first shown by Bordet and then carefully worked out in detail by Nuttall, Uhlenhuth, Wassermann and Schiitze, Myers, Linossier and Lemoine, and others. The precipitation reac- tion has been shown to be highly specific, but not absolutely so. Besides the specific precipitins for numerous kinds of serums of different species, including human serum, precipitins for such proteids as casein of milk, the albumins occurring pathologically in the urine, crystallized egg albumin, pure serum globulin from sheep and from bullock, Witte’s peptone, and muscle have also been produced by various investigators. Nuttall observed that the reaction is more intense when the host animal and the animal furnishing the serum for injection are widely distant. Myers found that precipitin prepared with egg albumin does not precipitate other proteids, such as the serum globulins obtained from the sheep or bullock, and Witte’s peptone. Uhlenhuth and Wassermann and Schiitze have worked out the specific nature of the precipitin for human blood and applied this reaction to identify the source of a given unknown blood for the medico- legal purpose. Quite recently this reaction has been recommended by Wassermann to detect the adulteration of sausages with certain meats legally prohibited from sale as human diet. According to Noguchi precipitins can be produced even in certain invertebrate animals, such as crustacea. In 1902 Lamb! first studied the precipitin formation with snake venom and employed this reaction to distinguish between the proteids of different snake venoms. He prepared an immune serum in rabbit with pure cobra venom ‘and obtained a markedly precipitating serum when mixed with the homologous venom (cobra). He tested its precipitating property, 3 or 2 parts of the serum being mixed with 1 part of venom solution, varying in strength from o.1 per cent to 0.0001 per cent, and observed the amount of pre- cipitate formed after 18 to 24 hours. 1 per cent venom solution was not suitable for the test, as this concentration produced more or less precipitate even with normal rabbit serum. Among the results obtained by Lamb special interest is attached to the facts that daboia venom behaved in almost every respect the same as cobra venom: precipitate was formed practically in the same quantity. Secondly, the heated venom solutions (75° C.) of these two venoms — heat-coagulated proteids being removed by filtration — gave just as much precipitate as the snake poisons. Lancet, 1902, II, 431. 261 262 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS unheated venom solutions did. Heating of the immune serum to 55°C. for 30 minutes did not affect its precipitating quality and quantity. The reaction appeared with the dilutions down to o.cor per cent, but not with 0.0001 per cent. The precipitin tests made with the venoms of Bungarus fasciatus, Notechis scutatus, and Echis carinata were all negative, namely, no precipitate was formed with these venoms. The above experiments of Lamb clearly point out that the proteids of two entirely different snake venoms, both from the physiological and zoological standpoints, can eventually be identical; hence these proteids can have no relation to the real toxic components of these venoms. While a venom of a colubrine snake (cobra) and that of a viperine snake (daboia) may have similar proteids, it does not follow that all viperine venoms are similar in this regard. On the other hand, Lamb found that the venoms of two colubrine snakes (Bungarus fasciatus and Notechis scutatus) did not contain any pro- teids which could react with the cobra anti-serum. Echis carinaia, a viper, also has no corresponding proteids with those existing in the venoms of cobra and daboia. In a subsequent communication Lamb!’ extended the precipitin test to various venoms representing practically the entire class of Ophidia. The immune serum used as the precipitin was prepared in rabbit by repeated injections of pure unheated cobra venom. With this cobra anti-serum he obtained the following results: COLUBRID: COLUBRID: Elapine : : Hydrophine : Naja tripudians = + Enhydrina valakadien bungarus ° Bungarus ceruleus o fasciatus o Notechis scutatus 0 VIPERIDE: VIPERIDE : Viperine : Crotaline : Vipera russellii + Lachesis gramineus Echis carinata oo Crotalus adamanteus o + = strong reaction. X = weak reaction. — = no reaction. From the foregoing table it becomes evident that the precipitin-reaction bears no relation to the zoological classification of snake, but is of the most irregular nature. Neither does it seem to have any relation to the physiologi- cal or toxicological constitution of the snake venom. Lamb also brought out the fact that the antitoxic value of antivenin has no relation to the precipitin content of the serum. Three antivenins of high strength have been found to possess almost no precipitating property. He sought the reason for this non-occurrence of precipitation in the species of the animals, holding that asses and horses are unsuitable for the precipitin production. 1Lamb. On the precipitin of cobra venom. Lancet, 1904, II, 916. PRECIPITIN-REACTION WITH SNAKE VENOM 263 In the meanwhile, Flexner and Noguchi! were engaged with the study of precipitin formation with different venoms. They made the following statement regarding this particular phenomenon. Precipitins are formed from venom along with or independent of the immunizing principles for venom. ‘There is no relation between the degree of protection afforded by and the amount of precipitin present in the immune serum. Precipitins may arise in treated animals even when the modified venoms are incapable of provoking the production of immunizing substances. Precipitins for different venoms —crotalus, cobra, and daboia —are highly, although not absolutely, specific. Below is a concise statement of their work on the venom precipitins. TABLE 39. Precipitation. protectiveness. Daboi aboia Crotalus venom.| Cobra venom. Verioni Crotalus I Feebly Moderate None None Do. 2 Feebly Copious None None Do. e Feebly Slight None None Do. 4 Strongly Moderate None None Do. 5 Not at all Copious None None Do. 6 Strongly Copious None None Cobra 7 Not at all None Moderate None Daboia 8 Rabbit Not at all None None Slight The immune serums 1, 2, 3, 4 were prepared with the venom modi- fied with weak hydrochloric acid; 5 with hydrochloric acid and pepsin; 6, 7, 8 with weak solution of trichloride of iodine. The test was made in test- tubes, each containing o.s5 c.c. of the immune serum and o.5 c.c. of 0.5 per cent venom solution. Ishizaka? found that while the pure antivenin of Lachesis flavoviridis produces a copious precipitate with that venom, it produces only a slight precipitate or none at all with the venom of viper. 1 Flexner and Noguchi. Production and properties of anticrotalus venin. Jour. of Med. Research, 1904, n.s., VI, 363. ’ 2 Ishizaka. Studien iiber Habuschlangengift. Zeitschr. f. exper. Path. u. Therapie, 1907, IV, 88. CHAPTER XXVII. NATURAL IMMUNITY. EFFECTS OF VENOM UPON SNAKES. The experimental data bearing on this question are more or less conflicting, but one fact has been established beyond any doubt, namely, that venomous snakes are not absolutely immune to the action of their own and alien venoms, though their susceptibility — subject to more or less fluctuation — is far less than that of the majority of innocuous snakes and saurians. The deter- minations of the effects of venom were carried out in two ways: one allowing the snake to bite either itself or other snakes; the other (which is more accurate and reliable) injecting the venom into the subject on which its effects were to be tested. Fontana‘ in his biting experiments failed to produce death on vipers by the bite of the same species. Claude Bernard? has, however, found that vipers succumb to the bite of vipers within 3 days. In 1861 Weir Mitchell made a series of experiments as to the effect of crotalus bite on the same species. His experiments are somewhat unique in their arrangement, as he tested the action of this venom upon the same snake from which it was taken, or by letting a snake bite itself at a spot denuded of its skin. In the biting method he obtained 3 positive results out of 4 experi- ments. In two of these cases death occurred in 1o days, and in another it took place in 14 days. By the injection method all three rattlers succumbed to their own venom. ‘The first, receiving 10 drops of its fresh venom, died in 36 hours; the second, receiving 8 drops, died in 67 hours; the last was killed in 7 days with 7 drops of its venom injected. The autopsies showed softening of the sites of the bite or injection, but not much alteration could be ob- served in the internal organs. Mitchell quotes the self-biting experiments made by Burnett on Crotalus, in which death usually followed the bite in a few minutes! Russell, Fayrer,? and Waddell,‘ working on the Indian venomous snakes, have obtained more negative results. In the majority of cases the snakes remained almost unaffected, or at least survived a large quantity of their own or alien venoms introduced either by the biting or by the injection methods. There are, however, a few instances where a venomous snake was killed by another species of venomous snake within a few days after the bite. Fayrer studied Cobra, Bungarus, Echis, and Daboia in this regard. Waddell, like Russell, obtained negative results. ‘Fontana. Abhandlung iiber das Viperngift, 1787, Berlin. ? Claude Bernard. Lecons sur l’effet des substances toxiques, 1857. 3 Fayrer. The Thanatophidia of India. 1874. ‘Waddell. Are venomous snakes antitoxic? An inquiry into the effect ‘of serpents’ venom upon the serpents themselves. Sci. Mem. Off. Arm. India, 1889, IV, 47. 264 NATURAL IMMUNITY 265 The action of snake venom, especially that of Cobra, is quite powerful upon different species of non-poisonous snakes. The bite of Cobra was fatal within 30 minutes to several hours to the following snakes: Passerita mycteri- zans (green whip-snake), Tropidonotus quincunciatus (grass-snake), Den- drophis picta (tree-snake), and Dryophis (green tree-snake). Ptyas mucosus is much more resistant to cobra venom and often escapes death from several successive bites. If death occurs it usually comes over 24 hours after the infliction of the venomous bite. EXPLANATION OF THE MECHANISM OF NATURAL IMMUNITY. On what does the relatively high natural immunity of venomous snakes depend? Why are the venomous species of snakes more resistant to venom than their innocuous congeners, and why do the latter possess a greater resist- ance than various mammals and birds? Bearing on these interesting ques- tions numerous experiments were performed. The work of Leydig, Phisalix and Bertrand, Jourdain, and other anato- mists and physiologists established in the non-venomous snakes the existence of poison-secreting glands and made the differences in the non-venomous and venomous snakes appear as a matter of grade in the evolutional phase. (See Phylogeny of snakes.) The physiological analogies between the venomous and innocuous species have been shown by the toxic properties of the parotid glands of the latter to be somewhat comparable to the poisonous action of venom (Alcock and Rogers). Again, the poisonous properties of the blood serum of various poisonous snakes, as well as those of the innocuous kinds, came to light, and it was shown that these serums are rather powerfully poisonous, being strongest in the serum of the snake with the most active venom. These facts have formed the basis on which Phisalix and Bertrand built their theory that the non-susceptibility of snakes, especially of the venomous species, to venom is due to the constant internal secretion of venom. In 1893 Phisalix and Bertrand’ studied the relation of the poisonous properties of the blood of viper and its venom, and concluded that they are identical in their physiological actions. The source of the toxic principle in the blood was sought in the constant absorption of the venom. Calmette ? found that the blood of Cobra is highly toxic for the rabbit. The fact that certain non-venomous snakes sometimes enjoy a compara- tively high immunity to venom demands explanation. Phisalix and Bertrand® investigated this point carefully and have shown that while adders have no venom apparatus by which it is possible to produce a poisonous wound, the secretion of the parotid glands resembles venom in the effects it produces when artificially introduced into animals. Extracts of the various organs 1 Phisalix and Bertrand. ‘Toxicité du sang de la vipére. C. R. Soc. Biol., 1893, 10 ser., V, 997; and C. R. Ac. Sci., 1893, CXVII, 1099. 2Calmette. Sur la toxicité du sang de cobra capel. C.R. Soc. Biol., 1894, ro ser., I, rr. 8 Phisalix and Bertrand. Sur la présence de glandes venimeuses chez les couleuvres, et la toxicité du sang de ces animaux. C. R. Soc. Biol., 1894, ro ser., I, 8. C. R. Acad. Sci., 1894, CXIII, 7. 266 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS were used experimentally, but only those of the major maxillary glands were found to possess the poisonous action described. The blood of these snakes was also found to be poisonous, and from it a precipitate was secured which behaves exactly like the active principle secured from the blood of vipers. As a logical consequence it should follow that the removal of the poison gland must also remove the toxic properties of the blood of venomous snakes. An experiment of this sort is by no means easy to perform. Phisalix and Bertrand ' have, however, carried out this experiment on 46 vipers. The poison glands were removed from one-half of them. After 168 days from the ablation of the glands the blood was taken from the heart under the influence of chloroform and tested for its toxicity. All guinea-pigs survived, while the blood from the control vipers was highly poisonous and killed the animals. The same authors? went further in this inquiry and finally found that the infection of the blood of viper or adder heated to 68° causes no symptoms in guinea-pigs, but confers upon the latter an increase in resistance to the toxic effect of viper’s venom. This phenomenon was considered by them to be analogous to the protection conferred by the injection of the attenuated viper’s venom. Calmette * found that the bloods of Cobra, Naja tripudians and Naja haje, Crotalus, and Cerastes are highly toxic upon guinea-pigs, but instead of accept- ing the hypothesis of Phisalix and Bertrand he considers the active principles of the blood and venom of venomous snakes to be different. The toxicity of the blood disappears on heating to 68° C., while the venom retains its activity at this temperature. The repeated injection of the sublethal quanti- ties of snake blood produces an immunity not only against the same blood but also against the venom. From this Calmette infers that the toxicity of the blood is due to the presence of a forerunner of venom from which the latter is produced through the process of secretion of venomous glands. The later study of Flexner and Noguchi‘ shows that the blood serums of Crotalus and Ancistrodon are highly toxic upon guinea-pigs, while that of the innocuous pine snake (Pityophis catenifers) is less so. Taking up the hemolysis as the test reaction these authors found that the antiserums of Crotalus and Pityophis serums have neutralizing properties upon the hemo- lysins contained in these serums as well as in the case of the venoms of Cobra, Ancistrodon, and Crotalus. They have, however, noticed that the antihemolytic actions of these immune serums are quite specific and display more protection against the serums with which they were produced. ‘The neutralization of toxicity of these two snake serums (Crotalus and Pityophis) by their respective antiserums was also found to be highly specific. With- out definite conclusion as to whether the venom is the cause of the toxicity 1 Phisalix and Bertrand. Sur les effets de ablation des glandes & venin chez la vipére. C.R. Soc. Biol., 1894, ro ser., I, 747. C.R. Acad. Sci., 1894, CXIX, g19. 2 Phisalix and Bertrand. Sur l’emploi du sang de vipére et de couleuvre comme substances antiveni- meuses. C. R. Soc. Biol., 1895, ro ser., II, 751. C. R. Acad. Sci., 1895, CXXI, 754. 3 Calmette. Le venin des serpents. 1896, Paris. 4 Flexner and Noguchi. Constitution of snake venom and snake sera. Jour. of Path. and Bacteriol. 1903, VIII, 379. NATURAL IMMUNITY 267 of the blood or the toxic elements of the blood are the source of venom, Flex- ner and Noguchi distinguished the differences in these two sets of active principles by their capability to unite with or to be activated by their homolo- gous and heterogeneous complements. According to these authors venom lysins are capable of being activated by isocomplements as well as hetero- complements, while the amboceptors of the snake serums are active only in the presence of their own complements. ‘This explains why the snake serum loses its toxicity when heated to 56° C. or above; here the inactivation is due to the disappearance of suitable activators —isocomplements in this case. At present our knowledge concerning the venom activators is so enlarged that the so-called heterocomplements, in Flexner and Noguchi’s sense, com- prise lecithin, certain fatty substances, and also serum complements. It becomes probable that Calmette’s view on the relation of blood toxicity and venom toxicity was a proper one. Stephens and Noc found that Calmette’s antivenin neutralizes the heemo- lytic principle of snake serums, especially that of Cobra. Theoretically considered, natural immunity must be regarded as the ex- pression of combination of many factors. It is seldom that the blood of an animal refractory to the effect of a toxin contains a definite anti-substance comparable to the product of artificial immunization known as immune body or antitoxin. In the cases of poisons, such as saponin and other glucosids, the blood may contain a definite substance (like cholesterin) capable of direct neutralization, but in other cases—such as certain alkaloids— toleration through the repeated introduction of these bodies into an animal body may be attained without inducing the formation of any definite anti-body. In still other cases, normal serums often contain substances similar to real antitoxins, as in horse serum for tetanus toxin. Again to-day we find very instructive instances of another set of phenomena pointing to the cellular and vital processes of immunity, either natural or acquired — namely, the rdles of phagocytosis advocated so long by Metchni- koff and his collaborators. In this instance the substances called opsonins by Wright and cytotropic substances by Neufeld must be regarded as the cause of natural as well as acquired immunity. Still other examples of natural immunity are the cases of chicken against tetanus, and hen and tortoise against abrin. There are no antitoxic prop- erties in these bloods. This may be due to the absence of suitable receptors in the sense of Ehrlich’s side-chain theory. In considering the nature of natural immunity in snakes against their own and alien venoms, we must take the above factors into account before we can reach a conclusion. I am inclined to think that the phenomenon is the expression of not one single circumstance, but of several circumstances here enumerated. The lack of suitable receptors especially seems to be playing a dominant part. 1Stephens. On the hemolytic action of snake toxins and snake sera. Jour. of Path. and Bacteriol., 1900, VI, 273. 268 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS NATURAL IMMUNITY OF CERTAIN ANIMALS FROM SNAKE VENOM. It has been a popular belief that certain warm-blooded animals are im- mune to the bites of poisonous snakes. Of the mammals the mongoose (Herpestes ichneumon), hedgehog (Hrinaceus europeus) and hog (Sus); and of the birds the Ajaja, Cancroma, Botaurus, and Mycteria, known under the names of culebrero and guacabo in Colombia, are alleged to resist the effects of venom. Three experiments made by Fayrer on the mongoose show that this animal is not immune to the venom of Cobra, but is less sensitive than other warm- blooded animals. ‘The quick movements of this animal and its thick hair seem to protect it from being bitten by the Cobra. One of Fayrer’s descrip- tions of the experiments is quoted below: A mongoose and a full-sized cobra were put into a large wire cage at 1 p.m. The snake struck at the mongoose and they grappled each other frequently, and apparently the mongoose must have been bitten, as the snake held on to it about the neck or head. At 12 15™ p.m. there was no effect on the mongoose; both it and the snake were much excited and angry, the snake hissing violently. 2h 3o™ p.m. no effect on the mongoose. The snake is bitten about the neck, and shows the bleeding wounds. 2h 51™ p.m. They occasionally attack each other, but the mongoose jumps over the snake and tries to avoid it. Next day at noon both were well; the snake frequently struck at the mongoose, but did not appear to injure it; both seemed very savage, but the mongoose would not bite the snake; he jumped over it. There had been two cobras in the cage with the mongoose during the night, both equally fierce and striking at each other and the mongoose; but the latter was not poisoned. He was rather severely scratched on the head by the cobra. But on being bitten in the thigh by the same cobra, when both were taken out of the cage the mongoose succumbed to the poison and died very soon. According to Calmette ' the blood serum of the mongoose is devoid of any antivenomous properties against the venom of Cobra, although death may be slightly delayed. He injected into three normal mongooses the doses of cobra venom respectively corresponding to 4,6, and 8 minimal lethal doses for the rabbit. The first showed no symptoms; the second was ill for two days, but recovered; the third mongoose died in 12 hours. Thus the immunity of the mongoose is relative only. Flexner and Noguchi? found that the defibrinated blood of the mongoose from Jamaica was not hemolyzed by crotalus and ancistrodon venoms in the concentrations from 20 per cent to o.o5 per cent. The addition of an adequate amount of the crotalus serum to not too high concentrations of these venoms produced a rapid dissolution of the mongoose corpuscles, while the concentrations above 0.5 per cent of the venoms again progressively dimin- ished the amount of hemolysis in the presence of a uniform quantity of the 1Calmette. Les venins. 1907, Paris. 2 Flexner and Noguchi. Constitution of snake venom and snake sera. Jour. of Path. and Bacteriol., 1903, VIII, 379. NATURAL IMMUNITY 269 crotalus serum. Unlike the venom, the crotalus serum is highly hemolytic upon the mongoose corpuscles. Lewin’ and Phisalix and Bertrand? made numerous experiments on the immunity of the hedgehog against the venom of Vipera berus. It is known that the hedgehog chases the viper and eats it very eagerly. The spinous coat of the animal protects it from being bitten by the snake and the animal is very skilful in catching the reptile at vulnerable parts of the body. Should the snake succeed in biting the hedgehog, the latter seldom dies. The inoculation of about 40 minimal lethal doses for guinea-pig of this venom into the hedgehog is required to kill. Whether or not the resistance of this animal is due to the presence of an antitoxic substance in the blood has been studied by Phisalix and Bertrand, who state that the serum of this animal heated to 58°C. in order to deprive it of its inherent toxicity for the guinea- pig can, when mixed with 2 minimal lethal doses of the viper poison in quan- tities of 8 c.c. of the serum, protect the guinea-pig from the fatal effects of venom. In regard to the natural immunity of hogs, which is popularly credited in the Mississippi Valley, it is still unconfirmed experimentally. Calmette ® found the pig to resist more cobra venom than dogs (speaking proportionally), but its serum contained no antivenomous properties. Along the valleys of the lower Mississippi it is said that the hog swallows young crotalus with evident liking. Concerning the grallics of Colombia, the culebrero and guacabo, nothing definite is known, except that these birds hunt young snakes for their prey. Probably they are relatively immune to the bite of poisonous snakes, partly because they understand how to kill the snake, and partly, perhaps, because they are less sensitive to the effect of the venom itself. 1 Lewin. Beitr. z. Lehre von der natiirl, Immunitit. Deutsch. med. Woch., 1898, XXIV, 629. ? Phisalix and Bertrand. Recherches sur ’immunité du hérisson contre le venin de vipere. C. R. Soc. Biol., 1895, 10 ser. II, 639. Ibid. Bull. de Muséum d’Histoire natur., 1895, I, 294; op. cit., ET, x00. 8Calmette. Les venins, 1907, Paris. CHAPTER XXVIII. EFFECTS OF SNAKE VENOM UPON THE BLOOD OF COLD- BLOODED ANIMALS, AND UPON THE NERVE CELLS, NERVE FIBERS, OVA, AND SPERMATOZOA. Fontana,’ as early as 1787, performed experiments on the effects of the venom of viper on vipers, two innocuous snakes, one salamander, turtles, and leeches, and found that none of these cold-blooded animals die of viper poisoning. On the other hand, he found certain fish and frogs to be sus- ceptible to the viper’s venom, death following the bite after a much longer time than in the cases of warm-blooded animals. In 1860-1861 S. Weir Mitchell? made a careful study of the effects of crotalus venom upon frogs, and observed two kinds of venom poisoning — an acute or rapid and a chronic or slow poisoning. He pointed out the rela- tively greater resistance of frogs to the venom. For physiological and pharmacological experiments with various kinds of venom on isolated organs, the frog has been nearly always employed by in- vestigators, and it would be superfluous to give any further description of the effects which venom produces on this animal. Brunton and Fayrer * also made a few experiments on cold-blooded animals. Two fishes, Ophiocephalus marulius and a carp, succumbed to the cobra venom. Cobra venom seems to destroy the irritability of snails. According to Rogers, the venom of marine snakes, as compared with that of land snakes, acts more powerfully upon marine animals than upon the warm-blooded animals, although the absolute susceptibility of these two orders of animals to the first venom is greater in the case of the warm-blooded animals. A more systematic study of the effects of snake venom on cold-blooded animals has been recently made by Noguchi. The results obtained by him were first issued as Publication No. 12 of the Carnegie Institution of Wash- ington, and are quoted in the following pages (271-280). 1Fontana. Abhandlung iiber das Viperngift. 1787, Berlin. 2 Weir Mitchell. Physiology and toxicology of the venom of the rattlesnake. Smithsonian Contr. to Knowledge, 1861, Washington. 8 Brunton and Fayrer. On the nature and physiological action Bh the poison of Naja tripudians and other Indian venomous snakes. Proc. Roy. Soc., 1874, 6 270 THE ACTION OF SNAKE VENOM UPON COLD-BLOODED ANIMALS 271 THE ACTION OF SNAKE VENOM UPON COLD-BLOODED ANIMALS. Since the writings-of Fontana, Weir Mitchell alone seems to have concerned himself with the study of the action of snake venom upon cold-blooded animals. Having studied and described the action of rattlesnake venom upon frogs and upon Crotalus itself, he intended, as appears from a paragraph in his earlier paper on venom, to extend his observations to a wider class of animals. Thus he writes: “Tt was my intention to examine, in the next place, the effects of the venom upon leeches, fish, eels, and crustacean animals, but for some reasons, which it is needless to relate, 1 was obliged to postpone these observations until some future occasion.”’! The following orders of animals were tested against venom: Reptilia, Amphibia, Pisces, Insecta, Crustacea, Vermes, Mollusca, Echinodermata. Several kinds of venom were employed: cobra, water-moccasin, and rattlesnake. All had been previously dried, and hence they were dissolved, before injection, in sterile sea-water or normal saline solution, according as they were to be introduced into fresh or salt water animals. The mode of injection varied with the animal species employed: in higher forms the peritoneum was selected, in lower forms the body cavities or water vascular system. Some of the vermes gave unsatisfactory results in respect to the dosage because of strong muscular contraction produced by the needle puncture and the presence of septa throughout the body. It was almost impossible to calculate the exact amount of venom introduced into these animals. Each experiment was accompanied by at least two control animals maintained under precisely the same external conditions. In every case in which the cause of death was doubtful the experiment was repeated. In general, it may be stated that the animals used in the experiments stood the necessary handling and cap- tivity without serious drawbacks. But in a few instances the degree of sensitive- ness to these procedures was found to be very great. ‘Thus, in the case of several kinds of small fish, e.g., pollack, silver-side, pipe-fish, this sensitiveness was so great that they did not survive beyond 24 hours in captivity. Animals surviving the injections were, as a rule, killed at the end of the experiment and examined for local and general lesions. The results of the study are given in tabulated form. In reviewing the tables, one is impressed with the wide degree of susceptibility to snake venom exhibited by cold-blooded animals. On analyzing the effects produced, it becomes quickly evident that cobra venom exerts little if any local action, although it is the most toxic of all venoms employed. Crotalus venom, on the other hand, while exhibit- ing the least general toxicity, displays the greatest local action. Water-moccasin venom occupies an intermediate position in this regard. The chief local effect produced by rattlesnake and water-moccasin venoms is the escape of red blood corpuscles from the vessel; only rarely is macroscopic necro- sis of tissue visible. This production of hemorrhage is, however, not restricted to the site of injection of the venom, but in some animals generalized hemorrhages also take place. ‘This latter effect was noticed chiefly in fishes, from which the blood may escape in such large quantity from the gills as to color the sea-water. In other instances, hemorrhages into the skin occur, and I have noticed during life, in the dog-fish poisoned by crotalus venom, the occurrence of intracranial hamor- rhage. Only one species of fish — the puffer —was wholly insusceptible to the locally irritating principles of venom; it succumbed, however, to the general toxic effects of all the venoms. It would appear as if the chief toxic effects of crotalus and moccasin venoms are the outcome of their local action, and yet the general toxic constituents which 1 Researches upon the venom of rattlesnake, with an investigation on the anatomy and physiology of the organs concerned. Smithsonian Miscellaneous Collections, vol. XII, Washington, 186r. 272 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS they contain can not be without marked action in some cases. These venoms may, therefore, cause death either through a destructive local action or through the operation of the neurotoxin upon the central nervous system. In the case of cobra venom, the toxic action must be ascribed to neurotoxin. There the local effects are almost nil, while the respiratory disturbances are very apparent. ‘The poisoned animals suffer from dyspnoea and from motor paralysis. Among fishes cobra venom causes rapid loss of equilibrium, so that the venomized animal swims with a rotary motion until it becomes too weak to struggle further. Crotalus and moccasin venoms cause far less disturbance of equilibration, while, on the other hand, their action at the beginning is likely to be irritative; the animal dashes about furiously without exhibiting evidence of a marked loss of balance. Speaking generally, cobra venom is most toxic and crotalus venom least toxic for cold-blooded animals. Moreover, this rule applies to the different classes as well as to the various species of animals employed. In other words, cold-blooded animals are more highly susceptible to the toxic action of neurotoxin than to that of hemorrhagin.' Crotalus venom is effective chiefly in those instances in which the local lesions are marked; while in instances in which it acts independently of the local lesions a far larger dose, in keeping with its small proportional content of neurotoxin, is required to produce fatal results. Snakes and frogs succumb easily to cobra venom, but they are relatively insus- ceptible to crotalus and moccasin venoms. ‘They would seem to be entirely resist- ant to the action of hemorrhagin. Turtles are more susceptible to all venoms than the foregoing animals, and fishes exceed turtles in this respect. The grass- hopper succumbs only to large doses of venom. Among the crustaceans the horse- shoe crab is almost insusceptible, and other species of crabs are only moderately susceptible to venom poisoning. The lobster is only moderately resistant. Excepting the earthworm, all the worms with which I experimented showed a low degree of susceptibility. While the first will die zm toto if injected with venom, the others show at times general effects, but they suffer only partial necrosis, from which they finally recover. After separation of the dead parts the worms seem to have been entirely restored. On the injection of Phascolosoma with an enormous dose of venom I have seen the muscular contractibility of the injected part disappear for a period of a week or longer, but in the end it was recovered. If necrosis occurred a slough was formed and was finally cast off. Upon echinodermata venoms produce little effect. The sea-urchin succumbed to all the venoms, while star-fish and sea-cucumbers were not perceptibly affected. The general toxicity of venoms upon the adult organism, as compared to their special effects which are produced upon the embryological elements” of the same species, is of considerable interest. ‘The ova or spermatozoa of some vermes and echinodermata are easily dissolved or fragmented by venoms, while the adults of cor- responding species are almost entirely insusceptible tothem. On the other hand, the reverse is possible. ‘Thus the eggs of Fundulus —a fish — are comparatively insus- ceptible to venoms, as they can be fertilized in sea-water containing rather a large amount of venoms and development of the fertilized ova progresses in the normal way, but the adults are found to be highly susceptible to the same kind of venoms. A close examination as to the relation existing between the general toxicity and the hzematoxic power * of venoms upon cold-blooded animals adds further interest- ing as well as important facts to the understanding of the nature of the action of snake venom in vivo. 1 Flexner and Noguchi. The constitution of snake venom and snake sera. Journal of Pathology and Bacteriology, 1903, VIII, 396. 2 Flexner and Noguchi. On the plurality of cytolysins in snake venom. Univ. of Penna. Medical Bulletin, 1903, July-August. 3 Noguchi. The effects of venom upon the blood of cold-blooded animals. Univ. of Penna. 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Coe Ba SS ee eS SS 282 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS THE EFFECTS OF VENOM UPON THE BLOOD CORPUSCLES OF COLD- BLOODED ANIMALS. The following article is reprinted from the University of Pennsylvania Medical Bulletin, July-August, 1903: In the course of experiments upon hemolysis carried out during the past sum- mer at the Marine Biological Laboratory, Woods Holl, I took advantage of the opportunity to study the agglutinative and lytic action of venom upon the blood corpuscles of a wide series of cold-blooded animals. The study is of sufficient interest, I think, to warrant presenting the results in a tabulated form. (See table 41.) The blood was obtained from animals belonging to the classes of reptilia, am- phibia, pisces, insecta, crustacea, vermes, mollusca, and echinodermata. The dried venoms were dissolved either in 0.9 per cent or 2 per cent saline solu- tion immediately before conducting the experiments; the venoms employed were cobra, water-moccasin, and rattlesnake; the blood was used in 5 per cent suspension; the temperature was that of the room, and varied between 20° and 30° C. In order to determine the minimum hemolytic, leucolytic, or agglutinative quantity (dose) of venom, the reactions were noted at a fixed interval. Thus, for hemolysis and leucolysis, after 12 hours; for agglutination, after 4 hours of contact. While solution of the erythrocytes can be readily observed in test-tube reactions, leucolysis can be determined only by direct microscopic examination. In general, there is no difficulty in distinguishing leucolysis and leuco-agglutination; but in some instances the act of defibrination causes considerable alteration of the white corpuscles, and these cells exhibit a tendency to undergo spontaneous agglomera- tion. When the result was mistakable it was indicated by the use of the term “doubtful.” The action of venom upon washed corpuscles was also studied, and it was deter- mined that hemolysis occurred not at all with water-moccasin and rattlesnake venom, while with the cobra venom a delayed solution would set in. This result with cobra venom recalled the similar one which Professor Flexner’ and I had met with in our studies of cobra-venom hemolysis in warm-blooded animals, and is now sufficiently explained on the basis of the existence of intracorpuscular com- plements as observed by us and by Kyes,’ and of Kyes’s important researches on lecithin in relation to its action as complement to cobra-venom amboceptor. The heat liability of venom agglutinins for the blood cells of cold-blooded ani- mals was found to vary between 68° C. and 72° C. for an exposure of 30 minutes. The temperature of too? C. maintained for 30 minutes abolishes largely the hamo- lytic power of venom over these corpuscles. Crotalus venom proved most susceptible, as its activity is greatly reduced at go° C. in 30 minutes. 1 Flexner and Noguchi. The constitution of snake venom and snake sera. Univ. of Penna. Medical Bulletin, 1902, XV, 345; Journal of Pathology and Bacteriology, 1903, VIII, 370. 2Kyes. Ueber die Wirkungsweise des Cobragiftes. Berliner klin. Wochenschrift, 1902, 886, 918. Kyes and Sachs: Zur Kenntniss der Cobragift activirenden Substanzen. Berliner klin. Wochen- schrift, 1903, XLI, 21, 57, 82. THE ACTION OF SNAKE VENOM UPON COLD-BLOODED ANIMALS 283 TABLE 41. —_——_——__---—————————————————————— eee, Caled venom. Water-moccasin} | Crotalus ada- venom, manteus venom. Name. Mini- | M2) ini- | Mini- | Mini- | Mini! Remarks. mum lu- mum mum mum agglu- hemo- oot hemo- |aggluti-| hemo- mt lytic Ato lytic | native | lytic ae dose. dose. | dose: dose. | dose. anes -cteey p)PkCteyl| ek cts ES Cra |p cts .cts Bascanium constrictor (black snake) ..... 0.005 | 0.1 | 0.1 0.05 | o ° Typical Cyclophis,vernalis (green snake) ......... 0.01 O.I | 0.2 0.02 | o o_|laking of Aromochelys odorata (musk,turtle) ....... 0.005 | 0.2 | 0.2 0.01 | o I erythro- Chelopus guttatus (speckled turtle) ....... 0.005 | 0.2 | o.1 0.002 | o 0.5 cytes Chrysemys picta (painted turtle) ......... 0.002 | 0.1 | 0.05 0.005 | 0 0.5 Do. Chelydra serpentina (snapping turtle) ..... 0.002 | 0.2 | 0.02 0.01 | 0 0.5 Do. Emys meleagris (Blanding’s tortoise)...... 0.005 | 0.1 | 0.2 0.05 | 0 0.5 Do. Rama span (leopardsinagiy a: acim shee saat 0.02 0.2 | 0.5 0.02 | 0.4 I Do. Rana, catesbiana (bullfrog) ............. 0.1 0.5) | 0.85 o.or | o ° Do. Acanthus!sprr(ScUlpin) 7 ay.6 sis nates ere 0.005 | 0.1 | 0.02 O.1 0.05 | 0.5 Do. Amphiuma means (Congo eel) ........... 0.005 | 0.2 | 0.02 0.1 ° I Do. Anguilla De (Cel) eta stesco cs wet 0.005 | 0.05 | o.or 0.02 | 0.1 2 Do. Apeltes quadracus (stickleback) ......... 0.01 0.2 | 0.02 0.2 0.05 | o Do. Brevoortia tyrannus (menhaden) ......... 0.005 | 0.5 | 0.005 | o.1 0.02 | 0.4 Do. Clupea harengus (herring)............... 0.02 0.5 | 0.01 0.05 | 0.02 | 0.5 Do. Cynoscion regalis (squeteague) .......... 0.05 I 0.08 0.1 ° ° Do. Fundulus heteroclitus (common minnow). .| 0.05 ° 0.2 0.5 0.4 I Do. Microgadus tomcod (tom-cod) ........... 0.001 | 0 2 ° O.1 ° Do. Morone americanus (white perch) ........ I ° ° ° ° ° Do. Murenoides gunnella (butter-fish) ....... @:002, | 0.1 | 61005 || o:or | /o.01 | 0 Do. Mustelus canis (smooth dog-fish) ........ 0.01 0.2 | 0.005 | o.1 ° I Do. Carcharinus littoralis (sand shark) ....... 0.5 I 0.8 I ° ° Do. @psanus tau! (toad=fish) 95.5 52055 lets. 0.05 ° ° ° ° ° Do. Osmerus;mordax (SMELL); = c.5sccieneciee = 0.02 0.5 | 0.2 0.2 0.4 ° Do. Paralichthys dentatus (summer flounder) ..] 0.002 | 0.2 | 0.008 | o.r 0.02 | 2 Do. Pleuronectus americanus (flat-fish) ....... 0.001 | 0.2 | 0.02 0.05 | 0 I Do. Prionotus strigatus (red sea-robin) ...... 0.001 | 0.2 | 0.002 | 0.02 ° 2 Do. Pterophryne histrio (Summer skate) ...... 0.02 0.4 | 0.002 | 0.2 0.4 ° Do. Raja ocellata (winter skate) ............. 0.01 0.5 | 0.02 O.I ° I Do. Raja levis (barn-door skate)............. 0.0001 | 0.5 | 0.05 0.2 0.5 I Do. Pomatomus saltatrix (blue-fish) .......... 0.002 | 0.5 | 0.004 | 0.2 0.02 | o Do. Nara sarda (ORO)... ccc ss ses fa met 0.01 I 0.5 0.5 ° I Do. Scomber scombrus (mackerel) ........... 0.02 I 0.6 0.5 ° ° Do. Siphostoma fuscum (pipe-fish) ........... 0.02 I 0.1 0.5 0.2 ° Do. Spheroides maculatus (puffer) ........... ° 4 ° 0.2 ° ° Do. Stenotomus chrysops (scup).............. 0.2 0.5 | 0.001 | o.1 0.01 | 1 Do. Tautogolabrus adspersus (cunner) ....... 0.05 I 0.002 | 0.2 0.02 | 2 Do. PautopaOnitis (CAUtOe)) 2.2% soc sca .eate ore 0.05 I 0.2 0.1 0.5 ° Do. CentropristesiStilatusyiisies. cisjeecvoaeiete caer 0.08 2 0.5 0.3 I 2 Do. Pollachius virens (pollock) .............. 0.2 O.5° (0:5 0.2 0.5 2 Do. Mugil cephalus (mullet)................. 0.001 | 2 0.0005 | 0.2 0.02 | o Do. Menidia notata (silverside) .............. 0.02 ° 0.05 2 0.2 ° Do. Acridium americanus (grasshopper) ...... 0.05 2 2 I 5 ° No ery- Carcinus granulatus (green crab) ........ 0.05 I O.1 2 0.5 ° throcy- Eupagurus longicarpus (small hermit crab) | 0.05 I o.1 I 4 ° tes; leu- Eupagurus pollicaris (large hermit crab) ..| 0.05 I 0.1 I 4 ° colysis Homarus americanus (lobster) ........... 0.4 2 0.3 0.2 0.6 3 only. Libinia canaliculata (spider crab) ........ 0.2 I 0.8 0.5 2 ° Do. Limulus polyphemus (horseshoe crab) ....] 0.02 I 0.02 0.02 | o.% 2 Do. Platonychus ocellatus (lady crab) ........ o.T ? 0.4 ip ° ° Do. Lumbricus terrestris (earth worm) ....... 10 ° ° ° ° ° Do. ATNPDIEEILe OLNAtA 1c neice wacins en nlew te = 5 ° Io ° ° ° Do. @inratwlasl praise site er.cts tony oahe eoicterss 0.04 ° 0.5 ° I ° Do. Mepidonotisisquamatus. 0.6. 5.- 6. ss see 6 ° 10 ° ° ° Do. Nereis virens (clam worm) .............. 5 o {10 ° ° ° Do. Phasolosomalgouldie 2... cewne« waists sess = 5 ° ° ° ) ° Do. Ensatella americana (razor clam) ........ O.1 I 5 o.1 ° ° Do. Walipospeality is 20 acr-cys: ssh sis aaacsoreecea 0.05 2 O.I 0.5 0.5 ° Do. Mactra solidissima (sea clam) ........... 0.5 I 0.3 0.2 ° ° Do. Modiola modiolus (mussel) ............. 0.5 rt 3 2 ° ° Do. Sycotypus canaliculatus (whelk, periwinkle) | 0.1 I 0.5 0.5 5 ° Do. Asteria vulgaris (common star-fish) ....... 2 ° 5 2 ° ° Do. Arbacia punctulata (purple sea-urchin) ...| 0.005 | 2 0.02 I 0.5 ° Do. Pentacta frondosa (northern sea-cucumber) | 2 P 5 ? ° ° Do. 284 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS SUMMARY. 1. The lytic principles of venom for blood corpuscles are active over a wider group of animals than the agglutinative principles. 2. The more distant, as a rule, the animal groups are from the vertebrates the less the susceptibility of their blood corpuscles to venom lysins and agglu- tinins. 3. In one instance only—that of Sphenoides maculatus—did the blood corpuscles prove wholly insusceptible to the action of venom. ‘This animal is, however, susceptible to the toxic action of venom, although crotalus venom produces death without causing hemorrhage. According, therefore, to the view of the constitution of venom held by Professor Flexner and myself, this animal is subject chiefly to the action of the neurotoxic constituent of venom. 4. Cobra venom contains the largest and crotalus venom the smallest num- ber of hemolytic units, while moccasin venom contains the largest number of agglutinative units for these bloods. 5. The mechanism of venom lysis in these animals is identical with that in warm-blooded animals. Complements are therefore present in all verte- brates and many, at least, invertebrate species. 6. The heat liability of the venom agglutinins and hemolysins for cold- blooded animals agrees closely with that for warm-blooded animals. EFFECTS OF SNAKE VENOMS ON THE NERVE TISSUES, OVA, AND SPERMATOZOA. The marked cytolytic properties of various snake venoms upon the nerve tissues, ova, and spermatozoa have already been described in detail under the heading cytolysins in snake venom and neurolysis im vitro, and I shall not repeat at this place. It suffices to say that the individual groups of these cells are affected by various venoms in various manners, depending upon the source of the materials. CHAPTER XXIX. EFFECTS OF SNAKE VENOM UPON PLANTS AND THE PROCESS OF GERMINATION OF SEEDS. Under the heading “Cytolytic action of snake venom upon micro-organ- isms” the energetic destructive action of various venoms upon unicellular plants (bacteria) has already been described. Now it is of some interest to find out whether venom has any influence on the vital processes of multi- cellular plants. The literature on this subject is rather meager and there are only a few experiments to be referred to here. In 1854 B. J. Gilman ' inoculated several small but vigorous and perfectly healthy vegetables with the point of a lancet well charged with venom. The next day they were withered and dead. No control was made, nor were the size of the plants and the amount of venom employed stated. In the same year Salisbury? experimented with the venom of Crotalus adamanteus upon four young shoots of the lilac (Syringa vulgaris), a small horse-chestnut of one year’s growth (Esculus hippocastanum), a corn plant (Zea mays), a sunflower plant (Helianthus annuus), and a wild cucumber vine. Without testing the toxicity of the venom on animals, he introduced the venom into the plant, just beneath the inner bark, with the aid of the point of a pen-knife. The quantity of venom was that which adhered to the point of the instrument. No visible effect from the poison was perceptible until about 6 hours after it had been inoculated. At this time, the leaves above the wound, in each case, began to wilt. The bark in the vicinity of the incision exhibited scarcely a perceptible change. 96 hours after the operations nearly all the leaf-blades in each of the plants, above the wounded part, were wilted and apparently quite dead. On the fifth day the petioles and bark above the incisions began to lose their freshness, and on the sixth day they were considerably withered. On the tenth day they began to show slight signs of recovery. On the fifteenth day new but sickly-appearing leaves began to show themselves on the lilacs, and the other plants began to show slight signs of recovery in the same way. Neither of the plants was entirely deprived of life. The edges and apices of the leaves were the parts first attacked. There was no effect on the leaves below the point of inoculation, and those on the side upon which the venom was inserted were the first to suffer. 1 Quoted by Mitchell. 2 J. H. Salisbury. Influence of the poison of the northern rattlesnake (Crotalus durissus) on plants. Jour. of Med., 1854, XIII, 337- 285 286 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOM Weir Mitchell! made a similar experiment in 1859 and obtained results which tend to show that the effects observed by Salisbury were caused by the mechanical injuries from the insertion of the instrument. He experimented first with active crotalus venom on four young shoots of Tradescantia, a very succulent and tender trailing plant. Each of the shoots was split half-way through, and about a third of a grain of dry, pulverized venom was dropped into the opening, which was then allowed to close on the poison. Next the plants were well watered and a drop or two allowed to fall on the line of incision. Four controls were made without venom. During a week no result was obtained. After that period two of the unvenomed shoots and one of the poisoned became sickly and gradually lost most of their leaves within the ensuing fortnight. In his second series of experiments Mitchell employed (1) a young shoot of common bean; (2) a long flower or budding flower-stalk of medicinal colchicum, C. autumnale; (3) three branches of geranium, growing on a large and healthy plant; (4) a small succulent garden weed; (5) a young dahlia. The venom was freshly collected and had been tested for its potency on animals. The mode of introducing the venom varied according to the kind of plant, but it was accurate and reliable, the quantity of venom being one or two drops. Unfortunately some of the plants had no controls. The period of observation was three weeks, but neither in the bean, colchicum, or geranium did the leaves die or the plants suffer in any way. Mitchell, how- ever, reserved any definite conclusion as to the effect of venom on higher plants in general. One of the most interesting experiments is the inhibiting influence of cro- talus venom upon germination of seeds of certain plants. Mitchell placed a number of the seeds of canary and mignonette in venom solution (1 or 0.5 drop of fresh venom to 8 drops of water) and in plain water. None of the seeds in the venom solution germinated, while germination took place in plain water under otherwise similar circumstances. C. Darwin? observed that 0.015 gm. of cobra venom dissolved in 8 c.c. of water acted powerfully on Drosera. A minute drop on a small pin’s head acted energetically on several glands, more powerfully than the fresh poison from a viper’s fang. ‘Three leaves were immersed in 90 minims of the solu- tion; the tentacles soon became inflated and the glands quite white, as if they had been placed in boiling water. After 8 hours’ immersion they were taken out and placed in a fresh lot of water, and after about 48 hours re-expanded, showing that they were by no means dead. 1 Weir Mitchell. Smithsonian Contr. to Knowledge, 1861, Washington, D. C. 2 Quoted by Brunton and Fayrer. Proc. Roy. Soc., 1875, 273. CHAPTER XXX. THE TREATMENT OF SNAKE BITE. NON-SPECIFIC TREATMENT —IMMEDIATE LIGATURE AND DISSECTION. In order to prevent the absorption of the venom a ligature should imme- diately be placed on the limb above the point bitten. It must be applied where there is only one bone and not on the forearm or lower leg, and must be tight. A stout India-rubber band is very suitable for this purpose, but in ordinary circumstances only part of the clothing would be available and answer quite well, a stick being passed under the ligature and twisted. The value of the ligature differs according to the nature of the venom. Should the venom contain fibrin ferment, as in Daboia, Echis carinata, Not- echis scutatus, and Pseudechis porphyriacus, the benefit of the ligature is very great. In these cases the ligature prevents the absorption of the venom and brings about intravascular thrombosis throughout the peripheral vessels into which the venom enters. There is then no further absorption of venom into the circulation, and upon the removal of the ligature no general venom- toxication follows. Martin established this interesting and important fact upon animals. On the other hand, the ligature has no more advantage in most colubrine venom-poisoning than to prevent the absorption of the venom mechanically. As soon as the ligature is removed the usual venom poisoning sets in, and the ligature can never be left for longer than 30 minutes without danger, lest the entire limb undergo gangrenous mortification. In that case, the destruction of the venom deposited locally must promptly be commenced by means of certain chemical agents, such as potassium permanganate, chlorides of gold and calcium, and others. There is another way of preventing the absorption of the venom, that is, dissection of the bitten locality. Wall recommends careful and deep dissec- tion with the knife of all parts likely to contain the poison. The dissection must be free in all directions, especially so in the direction of the lymphatics and venous return. In the case of the fingers, hand, and such parts it should be carried clear down to the bone. After this free and careful dissection the wound should be freely washed out with a strong solution of potash permanganate. Martin and Lamb call attention to the danger which would arise from the injection of chloride of gold or chloride calcium, as these solutions are liable to attack the least resistant tissues instead of following the venom, and would produce a nasty slough. These authors believe that the washing of the - punctures with any reagent is futile and that the application of any destruc- tive agent to an incision through wounds is almost useless, because the exact site of the venom deposited is extremely difficult to strike in this way. To suck the wounds is absolutely useless. 287 988 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS LOCAL TREATMENT. Local treatment of the place of the snake bite is of great importance and should never be neglected, no matter whether the therapeutic agent be a non- specific or a specific. The retardation of the venom by means of a timely ligature above the point of the wound is first to be resorted to, and imme- diately afterwards the fang punctures must be freely and widely incised in order to facilitate the closer contact of a chosen reagent with the venom in situ. Meanwhile, the solution of the antidotal reagent is to be injected intra- muscularly in such manner that the solution will besiege the venom in the spot, and any attempt of the latter to escape and be absorbed into the system will necessarily meet with the destructive reagent. Of course certain re- agents — non-specific as they are—are much more promptly diffusible than the venom and have a better chance to reach the venom even when the former are injected somewhat distantly from the wound. Notwithstanding numerous experiments, we are to-day in a position to choose for the local treatment only certain reagents which are comparatively less destructive to the tissues than others. In the following I describe some whose usefulness has been fairly established. The chemicals which destroy venom are numer- ous, but most of them can not be used for local treatment on account of their injurious action on the tissues themselves. As to the local treatment with specific agents, namely, the specific antivenins, it must be remembered that certain snake venoms, such as the viperine and crotaline, are enormously destructive to the local tissues, and it is but rational to inject the antivenin at and around the point of the wound. This does not mean that the intravenous injection of the antivenin can be neglected; on the contrary, the injections should be made both intravenously as well as locally. Even in the case of colubrine poisoning local treatment with the antivenin must be resorted to, especially when the case comes under treatment soon after the incident. Naturally here the intravenous injection deserves the primary attention. It would be of great value to use non-specific and specific agents in com- bination, should the patient come early under observation. In such case the non-specific chemicals must be applied closer to the bitten place and the antivenin should be applied somewhat distantly from the point of the injec- tion of the solution of the non-specifics, in order to avoid the destructive action of the chemical upon the antivenin. Here it is understood that the antivenin is also to be given intravenously. POTASSIUM PERMANGANATE. Early in 1860 S. Weir Mitchell made thorough studies on the effect of various chemicals upon the toxic properties of rattlesnake venom and found numerous agents capable of depriving the venom of its fatal activity. The first experiments on the use of permanganate of potash as an antidote were, however, made by Fayrer! in 1869, both by local application and by intra- venous injections, but without satisfactory results. 1 Fayrer. The Thanatophidia of India, 1872, p. 95. London. TREATMENT OF SNAKE BITE 289 Winter Blyth’ showed that cobra venom becomes innocuous when it is mixed with potassium permanganate in vitro. Couty and Lacerda,’ in 1881, made a number of experiments upon the effect of permanganate of potash on snake venom (Lachesis) and found that this substance not only destroyed the lethal action of the venom when mixed with it 7” vitro, but also preserved life when a 1 per cent solution was injected into the tissues close to the place where the venom had been previously in- jected, and also where venom and antidote were injected directly into the vein. Vincent Richards, also in 1881, similarly showed that the cobra venom is destroyed by permanganate of potash im vitro, so that death does not follow the injection of the mixture. But after the development of the poisoning symptoms no beneficial effect was to be had from the injection of this chemical. In 1902 Brunton devised an instrument by which the bitten person him- self can at once apply potassium permanganate to the place of snake bite. The instrument consists of two principal parts, one for opening the wound by incision and the other for holding a quantity of crystals of potassium permanganate. The first is a fine steel lancet and the latter is a hollow excavation in the opposite end of the wooden handle, to which the lancet is also fastened at the other end. Each of these main parts of the instrument _ is covered with a wooden cap. In an emergency the limb on which the bite occurred must be ligated with a tight bandage and the puncture of the fangs must be at once opened by free incision, when the crystals of the permanganate are to be applied —a few drops of saliva for facilitating its solution may be used —to the wound, and rubbed in it thoroughly. Experimental Merit of the Treatment: Leonard Rogers made a series of very instructive and thorough experi- ments to determine if potassium permanganate can nullify the toxic effects of various kinds of snake venom on certain animals and thus prevent death. A ligature was simultaneously applied to the bitten limb. Rogers sum- marizes his results as follows: The venoms tested were these of Cobra, Daboia russellii, Crotalus terrificus (the pit viper), African puff adder, Bungarus 1 Winter Blyth. The poison of cobra. The Analyst, 1877, 204, 2 Couty and Lacerda. C. R. Acad. Sci., 1881, XCII, 465. Also, Lacerda. C. R. Acad. Sci., 1882, XCIII, 466: O veneno ophidico e seus antidotos. Rio de Janeiro, 1881. 3 Brunton, Fayrer, and Rogers. Experiments on a method of preventing death from snake bite capable of,common and easy practical application. Proc, Roy. Soc., London, 1904, LXXIII, 323. 290 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS fasciatus, and Enhydrina valakadien. In the case of each 1o or more lethal doses were neutralized by very small quantities of permanganate in solution (ro per cent), and in most of them 20 lethal doses were readily rendered harmless. This salt will neutralize about its own weight of the venom and is effective against every class of snake venom. After the injection of the venom the ligature is to be applied to the limb for 30 seconds to 10 minutes, the release occurring in from 2 to 3.5 minutes. The results with rabbits were very encouraging, but better results were obtained with the experiments on cats. Practical Merit of the Treaiment : The practical value of the potassium permanganate treatment on human subjects may be very great. Rogers * has already reported 17 cases of snake bite in India in which this local treatment has been resorted to. The results recorded by him show that only 2 out of 17 cases ended fatally. In these cases nothing definite about the quantities of venom injected by the snakes could be learned, hence no conclusion can be drawn as to the absolute efficacy of potassium permanganate. It seems, however, that this salt has done much in averting death, as evidenced by the small mortality of cases thus treated in comparison with the deaths usually following the bites of these deadly Indian serpents where this treatment is not used. The table given by Rogers presents many interesting facts and it is given here for those interested in scrutinizing various points concerning the cases of snake bite in human subjects (table 42). TABLE 42. Sex Age Snake Fang | Site of bit Aneel) orimelhs “ : ; pa e. eaten ime bitten. Result. Male Ad. Daboia 1 ?6 Arm At once Day Recovered. Male 30 Cobra 1 2 Foot 30 mins. Evening Do. Female | 40 Cobra ! is Forearm 11 hrs. Midnight Died. Female | Ad. Daboia 1 2 Foot 30 mins. Morning Recovered. Male Ad. Daboia 1 2 Toe 45 mins. 9 P.M. Do. Male Ad. Daboia ! wa ? At once Day Do. Female Ad. Daboia 1 2 Foot 4 hrs. in Do. Male Ad Daboia ! 2 Foot 1 hr. Afternoon Do. Female 35 Cobra (seen) 2 2 Finger 30 mins. Io A.M. Do. Female Ch: p32 I Finger At once Noon Do. Male Ad. ras 2 Foot Soon 10 P.M. Do. Female | Ad. P2 a ? 9 hrs. 3 A.M. Died. 1 Killed and identified. 2 Snake not killed. Several cases of snake bites have lately been successfully treated by the same method, but I deem it superfluous to record them at this place. 1 Rogers. Five cases of snake bite successfully treated by the local application of permanganate of potash. Indian Med. Gazette, 1905, XL, 41. Twelve cases of snake bite treated by incision and application of permanganate of potash with ten recoveries. Indian Med. Gazette, 1905, XL, 369. TREATMENT OF SNAKE BITE 291 CHLORIDE OF GOLD, HYPOCHLORITES OF ALKALIES AND CHLORIDE OF CALCIUM. As energetic destroyers of snake venom in loco, chloride of gold, hypo- chlorites of alkalies, and chloride of calcium have been recommended by Calmette ! for local treatment of snake bites. If promptly injected 1 per cent solution of chloride of gold and hypochlorites of alkalies can destroy the activity of various snake venoms and save the animals from death. These reagents have the advantage over many other venom-destroying chemicals of being less caustic on the tissues into which they are injected.” Chloride of calcium, freshly dissolved in a ratio of 2 gm. per 100 c.c. of water, and having the titration of go c.c. of gaseous chlorine per 100 gm., is most highly recommended by Calmette. Owing to the easy diffusibility of chlorine gas to a considerable distance from the spot of injection the venom is quickly destroyed by the lime solution, even after absorption commences. The simultaneous application of an elastic ligature is also recommended. Certain acids seem to have more or less pronounced destructive action upon snake venom. Kaufmann* recommends the local application of chromic acid — in 1 per cent solution — for the purpose of postponing the lethal effect of venom. The local irritating properties are completely destroyed by this reagent, but not the toxic properties. The early work of Weir Mitchell also indicates the destructive action of certain acids upon the hemorrhagic principles of crotalus venom, although he did not recommend the acid as a practical means of combating the effects of the venom. Recently Morgenroth found that guinea-pigs which had received some lethal doses of crotalus venom into the peritoneum can be saved from death by prompt injection of dilute hydrochloric acid. I was able to confirm this phenomenon. But how much benefit can be derived from the acid treatment in the subcutaneous venom-poisoning remains to be seen. GENERAL MEDICAMENTATION. Fayrer and Brunton recommended the administration of strychnine as a means of prolonging the life of the bitten person. This notion is derived from their experiments on the beneficial effect of artificial respiration on snake poisoning, when strychnine, as a cardiac and respiratory stimulant, was thus introduced. Feoktistow as well as Aron failed to discover any curative in- fluence either by the artificial respiration or the injection of strychnine. Aron also tested the effect of atropin and caffein without obtaining any beneficial result. Feoktistow thinks that the use of strychnine and caffein should be forbidden because of the danger of increasing hemorrhage through the rise 1Calmette. Contribution a l’étude du venin des serpents. Ann. Institut Pasteur, 1894, VIII, 275; also, Les venins. Paris, 1907. 2 Martin and Lamb state the danger of slough from the injections of these chemicals. 3 Kaufmann. Sur le venin de vipére. Bull. Soc. Centr. de Méd. vet. Par., 1889, n. s., VI, 187. C. R. Soc. Biol., 1894, 10 ser., I, 113. 292 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS of blood pressure. In America some cases were reported where strychnine and brandy had been given with success, although no therapeutic value of this substance could be confirmed in these cases. According to Elliot, strychnine has no stimulating action upon the cases where the patients are in an aphasic state. Raston Huxtable * collected 426 cases of snake bite, of which 113 were treated with strychnine, with 15 cases of death, a mortality of 18.2 per cent, while 313 without strychnine resulted in 13 cases of death, the mortality being only 2.4 per cent. Although Fontana rejected it as quite worthless, ammonia enjoyed much reputation as an antidote against snake bite for many years without, how- ever, any definite experimental verification. The strongest advocate of ammonia as an antidote against venom poisoning was Halford, who recom- mended the injection of 10 to 4o drops (diluted with 2 to 3 parts of water) of ammonia into the vein in all cases of snake bite. Yet the injection of ammonia is by no means harmless, but is often followed by serious complications, such as phlebitis, perivascular necrosis, etc. Experimentally ammonia is entirely powerless to delay the usual course of toxication with venom. There may still be certain physicians who adhere to this worthless traditional am- monia treatment, but its practice should be discontinued. Alcohol, in the forms of wine, whisky, and brandy, has been freely admin- istered by physicians, perhaps partly encouraged by the popular belief ascrib- ing certain cases of recoveries of snake bite to their use, although there is no solid foundation whatever for this notion. Certain investigators recommended the use of alcoholic beverages with the supposition that the absorbed venom is partly secreted from the stomach and this can be precipitated by the alcohol before its reabsorption. But, as we all now know from experiment, alcohol precipitates but does not impair the toxic properties of the venom, hence alcohol administered per os can have no value as an antidote. Indeed, it was shown long ago by Mitchell and Reichert that in animal experimenta- tion, at least, alcohol has a distinctly injurious influence and quickens death from venom-toxication. It is, therefore, not advisable to prescribe whisky or brandy in the cases of snake poisoning. It is certainly harmful to give alcohol in any excessive quantity. Certain diaphoretics, e.g., Folia jaborandi and philocaspin, have also been recommended with the view of eliminating venom, but in reality such would be of no avail. In some cases, where alcohol was given, stomach-irrigation was practised with the hope of removing the precipitated venom. In the case of violent excitement, potassium bromide, morphia, and other narcotics have often been used. Rogers recommends the administration of adrenalin chloride in the case of bites from Daboia and those snakes the poisons of which have a marked paralytic action on the vasomotor center. 1 Huxtable. Transaction of Third Intercolonial Congress, 1892, 152. TREATMENT OF SNAKE BITE 293 CERTAIN ALLEGED ANTIDOTES FOR SNAKE POISONING. It would be purposeless to enumerate at this place all snake remedies, popularly credited as such, and I shall describe only a limited number of the antidotes most frequently referred to. None of these are warranted as to their antidotal value. The best known, guaco or huaco, also known as herba de cobra or yerba capitana, is a syntherea with strong aromatic perfume and is found in Colom- bia and other parts of tropical South America. Its proper name is Mikania guaco Humb. et Bonnpl. Its leaves are well decocted and administered internally as well as locally. It is also inoculated for prophylactic purposes against snake bite. Chambers failed to obtain any protective action of guaco against the venom of Vipera arietans on rabbits. Another reputed plant remedy is Simaba cedron, the nuts of which are also believed by the South American natives to be antidotal. In the West Indies, the roots of Dorstenia contrayerva and Chicocecca anguifuga are also reputed to be valuable as antidotes. In North America the roots of Aristolochia serpentaria and Polygala senega or Euphorbia prostrata, the swallow-root of Arizona, are often used internally and externally in the case of rattlesnake bite. Among the East Indian vegetables the roots of Ophiorohiza mungos and many varieties of Aristolochia, and the wood of Sérychnos colubrina and Ophioxylon, are the best known. Olive oil and sugar-cane juice are also employed. Ruta graveolens and Dictamnus albus are popularly supposed in Europe to be antidotal, but in reality are not at all so when experimentally tested. Various ethereal oils, namely the essences of camilla, peppermint, thyamin, and baldria, are equally inactive. Among certain composite antidotes Bibron’s antidote and Tanjora pills may be mentioned. The former consists of potassium iodide, mercuric chloride, and bromine water, while the latter contains chiefly arsenic acid. Mitchell found Bibron’s antidote worthless, while Fayrer did not discover any efficacy in the Tanjora pills. Psychic treatment is also in practice among the East Indians (though they believe it a real antidote), in the form of snake stones. The snake stone is obtained from the stomach (?) of cobra and is the concrement known as bezoare. The round concretion of cinerated acorn and a dark achatstone are also among the Indian snake stones. These are applied locally to the place of the bite. The cinerated acorn or achatstone may absorb some of the venom, but never any considerable amount. Thus, the snake stones can have no real curative value except certain psychic effect upon the super- stitious natives. The alleged cases of successful treatment of snake bite with snake stones must have been cases which would have recovered without the stone treatment. 294 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS SPECIFIC TREATMENT. The most rational treatment of snake poisoning is by specific anti- venins. That the action of antivenin is highly specific has been related under the chapters concerning antivenin. The therapeutic value of anti- venin is also discussed at length, and we may duly hope that in the near future specific antivenins of higher potency for the most deadly venoms will be produced and placed in the hands of practitioners in the countries where accidents and death from venomous serpents are frequent. Up to the present time a polyvalent antivenin is not sufficiently strong to be of much practical use, and it would be necessary to employ various monovalent specific antivenins for the cases indicated. There are, at the least, several available antivenins for practical purposes, as follows: Cobra antivenin, Daboia antivenin (issued from the Pasteur Institute, India), No- techis antivenin (Tidswell, Australia), Lachesis antivenin (Brazil,) Crotalus antivenin and Moccasin antivenin (Rockefeller Institute, New York). A seventh is another Lachesis antivenin (Trimeresurus antivenin) and may be gotten from the Institute for Infectious Diseases, Tokio. Besides these monovalent antivenins there is a polyvalent antivenin pre- pared by Calmette at the Pasteur Institute, Lille, although its action is more pronounced against cobra venom than the other venoms. As Martin and Lamb rightly pointed out, the antitoxic powers of all these antivenins are still feeble and are far from being satisfactory for practical purposes. But, asI have stated elsewhere, the amounts of venom introduced into the body by the bite are extremely variable, and death may occur in certain instances as the result of a small excess over the quantity which by itself is insufficient to be fatal. Here antivenins are of immense service in averting death by neutralizing that excess. The excess may again be very variable, but there must be a range of doses by which the antivenins, however feeble they may be, can prevent the venom from reaching the vital organs of the organism. Remembering that the antivenins are the only agents that can neutralize the venom after the absorption of the latter into the general system, we are amply justified in injecting as large a dose of antivenin as practicable in every case of snake bite and, indeed, no practitioner would be justified in hesitating to use this specific agent freely simply because of the comparatively low antitoxic value which our present preparations of anti- venins possess. To my mind there can be no room for the slightest doubt as to the great service that antivenin has rendered towards saving the unfortunate victims of snake poisoning from certain death. In many instances the cases of recovery might have been due to the shortage of amount of venom introduced, but equally as many cases must have been due to the removal of the excess of the venom by the use of antivenin. The cases of death in spite of the injection of antivenin are the instances where the excess of the venom has been more than the employed dose of antivenin could neutralize. TREATMENT OF SNAKE BITE 295 For these obvious reasons I earnestly caution practitioners not to view the future of the antivenin therapy of snake bite in a gloomy light. All thera- peutic experiments on animals point to the high curative property of anti- venins, and this is especially marked in the cases of crotalus antivenin and ancistrodon antivenin. Enough has been said to uphold the practical value of antivenins, and it now remains to give the rules for administering these specific antidotes: (1) The injection should be made as soon after the bite as possible. (2) The injections should be made both intravenously and locally. In the latter the intramuscular as well as subcutaneous injections should be made somewhat distant from the incised wounds of the fang punctures. Here 1 per cent solution of potassium permanganate should be injected at and around the point of the bite, and the antivenin introduced somewhat remote from the chemical. (3) With the present preparations of antivenins a quantity, at least, of 100 c.c. should be injected into the vein and also as much into the bitten limb or parts of the body as will be absorbed by the tissues. ‘The injections there may be made not at one spot, but at several places surrounding the entire circumference, for example, of the limb involved. Where specific antivenins are employed general medicamentation becomes entirely superfluous and any excessive use of alcohol is decidedly objectionable. A decidedly favorable report has recently been made by Kitashima on the antivenin treatment of Habu poisoning in man. Antihabu serum has been tried upon a number of cases in Anami, Oshima, and Riu kiu since 1905. In most cases the serum has been gratuitously distributed among the practitioners of the islands. The neutralizing power of the antivenin was such as Io C.C. of it will render 0.1 gram! (dry weight) of the habu venom completely inactive when mixed in vitro and allowed to act during 30 minutes at 37°C. At present 40 c.c. of this antivenin are put in a bottle as a curative dose for one case. In achronic case, twice or more is used. The injection is made near the bite, which is incised slightly, washed and dressed in the usual manner. 115 cases were treated with the antivenin, of whom 5 died, making a death rate of 4.2 per cent. One patient was brought in 3 hours after the bite and died in a few minutes after receiving the antivenin. Kitashima states that if the antivenin is given immediately after the bite, swelling only makes its appearance, with only the slightest phenomena of toxication. In ordinary cases general symptoms, such as vomiting, colic, and pain soon disappear. Pain at the site of the bite also decreases in 2 to 3 hours and the swelling subsides after serum treatment. In the future when r c.c. of antivenin may become so active as to neutralize, for example, 0.or gm. of venom or even more, the incision or dissection may be abandoned and the non-specific venom-destroyers, such as permanganate of potash and certain chlorides, may also become superfluous. Aces en UNE RNS Bee seeen mals Nope ude eer ee 1 A habu‘discharges, under natural circumstances, 0.3 to 0.5 C.c. of venom, equaling about 0.1 gram of the dried venom. 296 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS Of course, these measures — incision, dissection, and chemical destruction of venom — are chiefly for instant use and are easily practised by the person bitten, so that their value remains almost unaffected by the improvement of the antivenins. In conclusion, it may once more be emphasized that the ligature must always be placed immediately after the bite. BIBLIOGRAPHY. Abderhalden (E.) and Le Count (E.R.) 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Zeitschr. f. wiss. Zool., 1876, X XVII, 1-50. Wilson cw. HS) ithe physiological action of scorpion venom. Proc. Physiol. Soc. Lond., 1904, p. Xlviii. Wilson (W. H.) The immunity of certain desert mammals to scorpion venom. Proc. Physiol. Soc. Lond., 1904, p. I-lii. On the venom of scorpions. Rec. Egypt. Gov. Sch. Med., Cairo, 1904, II, 7-44. Wolfenden (R. N.) On the nature and action of the venoms of poisonous snakes. Journal of Physiology, 1886, VII, 327-364. Wolff. Folgen von Schlangenbissen. Arch. f. med. Erfahr., Berlin, 1821, II, 42-44. Woods (F. H.) Five cases of snake bite. N. S. W. M. Gaz., Sydney, 1873-4, IV, 129-132. Woodward (B.) Jodine as a remedy in rattle- snake bite. Northwest. M. and S. J., Chi- cago, 1856, XIII, 61. INDEX. PAGE. Absorption of venom from serous and mu- cous membranes .....-.-+++-+-eer'= 221, 222 PROB G EU io 515. cyes ein i> alee nye ayn eye nce a ah nee 27 PETONI.. 0. eee e eres cece e eee 27 Acanthophis ........+.eseeeerensecerces 22 ANTALCHICUS caso © seein ieee 22 Acetone, effect on venom.......----+-+-+++> 102 Acids, effects on snake venom ......-.++--+ 98 Acrylic acids.......---s2eseeeeeserees 189, 184 Adrenalin, in treatment of snake bite .....- 292 Africa, venomous snakes of.......---+++++ 53 Ry PHA) < odeciaye: crayon eae ese sine sens a irieees I Aglyphodonta.......----2+eeeeereee eters 1,46 RIPYSUEUS «api nes cs ieee eosin eet 29 AUISTEAMS (2s ne csveialn lm with ene v suatne 29 Albumoses of venom .....-.---++++2++:> 84, 85 Alcohol, alleged value in treatment of snake ifeercccer sia Heotvise fos eo earoeaee 292 effects on snake venom ......----- 79 Alimentary tract, effects of snake venom on. 220-222 PAA ato a easinaleaueye epson a saniauaes 5 Alum, effects on snake venom......------- 98 Amblycephalide, definition of .....-.-.--- 2 Amblyodipsas.......---+2ss+erscrteesres 10 Ammonia, alleged value in treatment of snake Bite ayn ote ee rete Gieheteae 292 effects on snake venom ......-. 97 Ammonium, carbonate of, effects on snake VANOIINNE TR ein se cy nies errs IOI effect on snake venom........- 100 Amphibia, effects of snake venom upon. .... 274 mucous glands of oral cavity .-- 47 Amplorhinus ........-----s+ssseerteeees 6 PAGE. Anticoagulating property, negative phase Off ecb: sie 134 Notechis ...... 141 Pseudechis .... 141 snake venoms. . 133, 139-142 Antihzmolytic properties of cholesterin. .174, 178, 184, 185, 186, 194 Antihemolytic properties of cholesterin de- MUVALLVES © enc a eis eheicdsie = ie era 194 Antihemolytic properties of excessive amount Of “Venom. ... 55. -«- 1 179, 195-198 IAntriCin pac eee ents Genes ee aheeet = 159 Antivenin of Ancistrodon piscivorus ......-- 240 Calmette’s 2 e.stis.c acckle > ees enateieny 241 Cobran a cen ot on aries 239, 258 Crotalus adamanteus ........-- 239 Grotalus terevicus 3 oiecsees oe 240 @rotahisvenomi. 2 2. alee Gree 257 Da Ola ee Sle cteia «20 cheaper 238 Lachesis flavoviridis ........--- 241 Lachesis lanceolatus ......-..--- 240 Notechis scutatus .......-...--- 238 water-moccasin venom .......-- 259 and venom regenerated from their neutral combination.........-- 248 IATEVORUINS) fet cos cts Ciera aintebakel tay sietatalle 225-241 data as to the effective dosage Be ids ah ae alate taitesen 244 o difficulty of obtaining strong.... 245 duration of effects after injection 231 effect ob alcoholh. <=. 36 specificity of ........-.---- 234-245 amount of venom therapeutic value of.......-. 241-245 secreted 35 | Asia, venomous SNAKESOL o-0 ec cels eee = 53 amount of venom se- INSTR cro th creleul steak + eiekehtinoi oie) sietpie = Fo eps I Crete eee 4x | Aspidelaps ...--.+.0+sseesereecseneneces 17 effect of venom upon WLICUSH os naan aet me Ses 17 the nervous system 124 SCUTALUS tc ootreke Mareen sieisteiet ste iret 17 VenomlOf .....:5c:- ae Ns PIS tal Stee ya dm mmineptiage So > wwe = RASS 34 poisoning, experimental ....... 115 ceratophorus .....-.--+++++++ee> 34 Phodostoma .2..2.5- +. ese ss 3H Ghlorechis 1.25406 See arin amine 34 Angiostomata ......---+2+sseerse esters I squamiger .....-++-eeseeeeereeee 34 Anguis fragilis, glands of ........--------- ey) | AATACUASPIS <5. os 2 += 5 Coes eae 34 Animals, natural immunity from snake aterrima) | «+a sxlas'aicid is a= eres 35 VETO oorva scare oop Sgemieds Moyen sare aero spkene oer ek 268 BiDEOU fe ois sscasigrnus = siete ey as 35 Antibactericidal properties of snake venom. . EANPICK:. 3.44 aed oar cssome aoe 34 215-218 corpulenta ......--+-+++++++: 34 Anticoagulating property, effect of anti- dahomeyensis .....-.+---++++> 35 venins on. . 238-239 hildebrandii .......-.-+++++++5 34 effect of heat upon 141 irregularis .....--+-+-+++++++00> 34 of Daboia venom. 141 leucomelas ......--+-++++2e0e> 35 . Echis: 502.03 141 microlepidota ..-.--+++++-+5+ 35 307 308 INDEX PAGE PAGE Atractaspis micropholisiys 26 ered) sia. © ani) Cavisiismes eer wip Saleen hehe aie wacke gl pioreietea Sena 31 NOSHALA «-- -, sopegiec! Ate xeNrus eee 34 Ge tiipni ote. ete ese Let eter 32 Australia, exclusive home of Colubride..... 52 lichtensteinid -... 1-60. e.ee seen ees 32 free from Viperide hs A tee Re ae 52 TESUITUUTS cs ity eset ROS OL ano eae 32 rhombestus! seacoast eee 31 Bacillustamthracis ieyyr-\evscwy sitet ak 205, 215 poison glands of........ 60 COW eekitsss castes ce 208 wane, a nOsom nln Cellsiofisy Coty pUS mel ier: elit yiener ee 151 diphtheria werkt) hcheclgeioies eens BOSH mC enastes | wallaciweer cleat errr Ee ne eee 33 PYOCVANCUS sine eniboaniaied Soe 205 amount of venom secreted......... aI SuloiiISSonsscoscsssb sods sae sdse 205 COrNULUS .-- +--+ eee eee ee eee eee 33 TUDETCULOSUSIRa ri. acioeus mclaren i 205 Vipera) to) Sos cia jose oem eee 33 Eyes Ce tee ere ere eee 205, 20s | Cerberus: coo? etricmns sae cise) ete mee II Barium chloride, antihemolytic property of. 191 | Chameetortus.................... eee 5 Bengal, mortality from snake bites in ...... 75 | Chelonia, mucous glands of oral cavity..... 47 Bibron’s antidote, used for snake bite...... 293 | Chemical effects of snake venom .......... 96 Bile, antivenomous properties of....... 103-104 nature of snake venom.......... 79 Bite of Australian species of snakes........ 110 the venom-antivenin re- snake, mechanism of ............. 67-69 ACH ON 2) Fewer 246 PETS een chee ein eeaeon) rece sath ts eee Sele oe 32 properties of snake venom ...... 7 -O3) AIECAM See rare eae ore ea Ee 32 | Chicken egg, effect of venom on evolution of mortality caused by ......... 76 embryo nies Nae noe eee 204 AEE ODOSI eer ta ae ere eae aeons 32 | Chicocecca anguifuga, used for snake bite .. 293 Cad alts ais ie ngeyen cramls wan tayeeustne re sas eer 33 | Chloride of calcium, experiment with....... 220 COEMUTA: rs Poiarc vests caste cuss vee pele roeerene 32 gold, experiment with.........-. 226 BADONICA 2. as e-eteuete coxa eieters oye ee es 33 water, effects on snake venom TNO TEA ALA ivenetretoh atc detoncts te tenen sie one eekeetoeats ia 32 99, 101 MASICOLMIS' sis wiesv fica nraeutebser a ayer ie it 33 | Chlorine water, effects on snake venom . .98, ror DEVIN PUI Aan ee Okra 32) | Chloroform ettect on venom) .\4-0 94.) ee 102 Blood corpuscles, protective property of Cholesterin 27's 27. cicen heey 149, 174 VENOM OME cio 4 ye sen actEs aks oie 195-198 antihemolytic properties of . .194-198 Blood pressure, in venom toxication.. ..... 125 effects on venom hemolysis.... . 174, Bode idelimition Ole es seems eis Sess 782 178, 184, 185, 186 [BOISE NL eek ent a eamoe eee cuits ae | ¢@holing, oc csneh aso eee ia ae aoe 184 iBothropsi(@achesis)mies aes e one oe Sens ay) ||) Chromatolysis) 1.01 ies > eee 154 Boulenger on systematic position of venom- Chrysopelea: i: acces eater ce oe ee eee 10 OUSESUAISES emer criny Tora teehee erento ets 2-3 | Coagulability of blood,increased and dimin- Boulengennavs: se eee ee ee ee 16 ISHE! Bay Me ie es oi ees Tes en ae ee ma) Boulengenmatstormsie-- eee see eee 16 | Coagulating property, counteracted by anti- Bra Ghivasplsier pretense ae aero orerom eas chtoe de 22 coapulating *propertyecn...c ea 142 CUTER ae eile: eictak tice ae 22 | Coagulating property, effect of antivenoms on IBrachy opis see aac seamen tian eae 10 238, 239 San yearn ete emer eee ee meets c esa eke 292 | Coagulating property of Daboia venom, effect Bromine, effects on snake venom .......... 98 Ohicobra venom on -aee. ae ee ee I4I, 142 water, effects on snake venom .... ror | Coagulating property of venoms, comparative BUNSATIS Mera Penis ei es ee ahaa ee 14 Strengths Of) 2252 nE ms sce eee 138 ceruleus, effect of venom of, upon Coagulation, intravascular...............- 133 nervous system ...... 129 Cobra, effect of venom of, upon the nervous effects upon coagulabil- SYStETO eye Getic henner 125 ity of the blood ...... 137 Chemicalyamalysisvens a. ae ater 87 mortality caused by..... 75 digestibility by trypsin............. 105 poisoning in man...... IIo lecithid’::< 251 Boy = Gene ES eke 86 candidus’s: ceruleus.. 07). -..55- 14 Poisoning nian. Scie soe ee 110 PASCLALUSH My cst anc tare iene yarns epee 14 preparation properties............ 86-87 effects of venom of, upon Venomiantiventnis. sere seers 258 nervous system....... r299 | "Cobralysin® .3..¢ 2565252 sce ae eee 168 mortality caused by 2-1. arses 75 absorbability by blood corpuscles 191 poisoning, experimental ......... TES || \Cobnade: ac \.\aces ae oe eee Ge Oe ee 30 Coclopeltis. 25.53. see hee oe oe oe Caffein, effect on venom toxication....... "291 lacertina, venomous nature of bite Calanelapswe ses Hct ane ee eaten 10 OR ES ae err pa tee be 122 Calcium chloride, antihemolytic property of. PT OUMEMSISK yay hes onetels css ie veel 7 188-190 monspessulana) sacacrasc)- «eo eflectiona. eile 134, 135, 136 venomous nature of Calliophis tac ecwseoen neyo k a cede eck 15 pitevoiae rk a 122 Callophisi ere en scnia ae cicinas Seis eee 15 | Cold-blooded animals, effect of snake venom bibroniiin. cis), .iwecmeters sotreee ore 10 pons sage cer 270-284 STAG es ure eee eee cae ne eee 15 effect of snake venom macclellandis ass ae eet 15 on cells of ..... 201-204 maculiceps! “Racuise ones eee qs 9) Cold, eftect-on snake venontas ecient 95 traimaculatusy os seers were ace 5 ay Coluber ces culappiih eaeenseye pete eres ie 50 WGLVIESABUS yoy tee twee nie esses 16 HavescEnSixty: csc cm chin meet etectets 50 Calmette’s antivenin ............ 150, 167, 241 viridiflavus var. carbonarius...... 5° Carbolic acid, effect on snake venom. ...99, 100 Colubridae 3 5s oa at anette 3545 LE Catodontans) et sect sees ee I GehnitiOMOla sry ese eion\e eee 2 Caustic alkalies, effects on snake venon..... 97 Colubring 22752 Se aaron 3. CWansidse ts. ho ke do aT) he ae toe ie es Colubroideats 2. ne co < esac sclera ole eels rg INDEX 309 4 : 4 e PAGE. PAGE. Complement, chemical inactivation and re- Daboia, anticoagulating properties of venom of 141 MCUVAUONOL 2 acc caren ors 194 POWOUINS IW May. ce s+. 108, 135 destruction of, by venom...... 218 russellii, effects of, upon the coagu- disappearance of activating lability of the blood. ..... 137 _ property by heating........ 172 amount ofvenom secreted . 70 : in venom hemolysis ..170, 171, 175 mortality caused by...... 75 Coniophanes. AN Ba eae eter reenter G | Daspelis:ecaber. 2. cone. eet eae 5° ATEPETIALI Se occ ital ately ese aee Os | Deneraspisy 7 sictr'd omic sige tae on marr eras 17 imperialis imperialis .......... Io AURUSHICEDS, cere eee ee 18 imperialis proterops........... 10 SHENOY Sei a. coe ea ce 18 IRTeritinishsers he tceoe ee Re. 10 JAMMCSONI-. ccs | ee eee ie oe 18 Conjunctival membranes, effects of snake WILIGUS sicetl clon, wince name eee make 17 UROL OU anger peters tele rad eee oe 220= 2226 | SPIORISONIA ws tee cats Nove tie re. cee iol ets 19 MOSRATSTOINIS Acre meee «Mestalla (ach rar bees aoe Carpentarie’ = rset yee see 21 Cope on systematic position of venomous COLONSIAy cs vascredon ea ne 20 RES) GY i ae eee nay hee este Se Pn Seto a COMOHOIUES 2. has sine eee 20 Copper-venom globulin .................. 80 Mapelleny sc cei. ha coe ee ee 20 ASOT N CAL OREHEK Yemen ouch.) oss Seas 124, 130 Prenat ect ee otee eeees 20 SS OTOME Leila viSh mais. oe ik he eee Aaa rie as 49, 50 NLL on ASS ee and orate eer eee 20 Sorypnodon KOREGS! Moma weanc ean ee: 50 miaen lata tence eee 20 Crocodilia, mucous glands of oral cavity.... 47 NTIGIQRMTUITA? cas ae cr ce ae ee ata 21 MBE OEAL ee ey pe ened ts ns Chien ae heer. cece 3 TAVICLERE po! ita ya'c's osc eet mee eR ie 20 Remo nalan ae fo) ek Rm Ne Boras See y si wren 35 TIPTESCENIS'. 0,-1.0 ae heii wets 21 effect of venom upon nervous sys- TI PTOR IAEA Ty ¢ het tyed oe Cease oe 2 CERN segs ep creer ye are aa foate emia 124 Dallicicepsy:.%.% 4 nec. eens res 21 Wropalusi res. wiasisa cruth caharpe tices emits 6 5 42 Dale oct ee «coh atten 21 AGADIADLEUS cs, c. 38 AUNT aeT S13 ces tea Rene tas) roy 2 hich Se east chew Oral) Mla elie aeldlen reeks Salen. teers Sete eraiauriars 102 Elysiids, (definition! Of: 5......000 w+ > en one 2 Asem predn ase 5 ani, ciestene toto aol ible» AWA ees sees Immunity, active, against venom ....... 223-225 | Lecithid, alleged presence in ordinary lecithin RIALS ership oa eiers2, 6 264-269 preparation .......-..-+.++0-5 89 PABELVCo hs ae cho tacetiota oven wine Giers 225-241 efiect of heating on .......-... 192, 193 produced by feeding........... 228 hemolytic properties CEM ee nae 174 Immunization against venom, principles of.. 226 in relation to immunity ......-.-. 192 ALICIA Ps che valet sss «aden sates 223-232 physico-chemical phenomena re- Dyleedingeen aan. teens since 228 sembling.........-. cdiveysves 88-89 Inoculation, prophylactic, against venom.... 223 preparation and properties of . . .86-87 Insects, effects of snake venom upon....... 278 AGKUCIEY OKs Amie oo Wine) sis eae Nisie ieee 174 312 INDEX PAGE. PAGE Lecithin, activating property for hemolysin..173, | Najasamarensis .......................- 12 183, I9I tripudians 2. ou. Seieeese ee eer eee 12 antagonistic property of cholesterin amount of venom secreted. 70 OU eh eee eee 178, 184-185 effect of venom of, upon the hemolytic action on blood cor- nervous system..... 124, 126 USCLES Ne anca shee ene eh eerie 183 effects of, upon the coagula- protein compound of ............ 188 bility of the blood ...... 137 Leech extract, effect on venomized plasma.. 136 mortality caused by....... 75 Ieewcocytolysin cece cee oe hort EVE Nada! (5. curs oe coed eine eee 3 Ligature, as a means of preventing absorp- Narcotics in treatment of snake bite........ 292 TOM OL VENOM eee eae es he he are 287) | Natrix torguatuss ee try cic mny een ere 49 Isto phis geen. 3) hs are yey tole Seles hale 50 | Natural immunity from snake venom... . 264-269 WeIpAase, Wms VENOM’ Slay ue seen ciate tet 213 | Nerve cells, changes produced by Notechis WET OLdS ee cee ehmcka cys iedsereichaas oF eesrareiser 187 VMOU GH eyesore ee Get ane 153 Lipolytic ferment in snake venom ......... 213 effects of snake venom upon. 201, 270 Liver, histological changes caused by venom. 207 Lungs, histological changes caused by venom 208 Lev COUryaSs «pclae ae oes meen aye 5 by Gowers anmamdadacaso ve bch olbm Geet 6 Macrelaps) sare ce een cei cetera seal 10 IMacroprotodontenr ites eee tome nt 7 cucullatus® Gewese ee chee 7 Macrostomataee ser one eae a ne toe I Magnesium chloride, antihemolytic property Ole Bante eter cies oman crete ae rer eicens 1Ql Malpolon insignitus, venomous nature of bite OD eR eins sec tae araincee a eee tae 122 Mammalia, mucous glands of oral cavity ... 47 Mam Ole DIS mace amin cree hetetats a temgets era et 9 PULMAMTVEY yep eae aerate 9 IMiassasatga, =) stot j.28 cick setae eee eo eee erecta 10 Oxyrhopusy. t20 cme aecr ee ae erre 6 Pancreatic digestion, on venom neurotoxin, hemolysin, hemorrhagin ............... 105 Pancreatin, effect on venom..........-....- 104 Bapalmcss cures tac en cele etek octine een e sien 105 Paraglobulingss2c. ce: -. lec cui hele ean ero 135 Peliasibenus! 3225955 os Gece ema ecreeer 30 Pepsin, effect on various toxic constituents OL VEHOMSes weet ee ees 105 efeCEON VENOM.) soe eee 105 Peptic digestion: 2-year oe aor a LOS Peptone® Veer hee ects eke meer ettoeherens 83 Permanganate of potash, effects on snake VETOMD ois era nie ee ta ee ae cision nae IoL Permanganate of potassium, effects on snake VETIOMI ese Greece seater son eae arene 100 Peropoda’ wsijiss se eaeer scene Bae e eh eheiereasis I INDEX PAGE. Peroxide of hydrogen, effects on snake MES PLCS ED getaten stat ies 0 dear e's jai(a. « ay sss) Maeenann 98, 101 Philodryas ..... Rei ae cockewaaterepres cues His. Phosphoric acid, effects on snake venom.... ror Photodynamic substances, effects of..... eee PPRTEMIC NETVE 6 uo. cece eens © 128, 129, 131, 132 Phylogeny of venomous snakes............ 46-51 Physical properties of snake venom....... 77-93 Pisces, mucous glands of oral cavity........ 47 Plants, effects of snake venom upon... .285, 286 laStrIaCHETAte slave eA esc ees one cela 135, 037 ORB ALC yarn inlays crete eee 136, 137 PASI ELIS ee A pce nha Siars) ices fakes SOR ance 29 BON IDTID US: eyes foe ois -nasahovslnlsistetene 29 fasciatus, poison glands of........ 61 laticaudatus s. fischeri........... 29 PIMISH OR Ee reps cya aiatnce ara eiat fsvatss (els 29 ER ALV.COECA yoo sia so omrcig keener assis hu veiakepoet tats 3 Poison apparatus of snakes ....... 46-51, 58-69 dynamics of function of. .64-67 TCs sxtings Siero teapatnets, eyNteim ots SiS: havea ee 59 ERIN ES oases wperrn) skase ee peel. ohe/ Ee Re eke 58-59 lAMCS end te ch. torie a areca eR sae 59-63 PODER ON eae tk aude ncpis, cneaneatrai tierce eicce os 10 Polygala senega, used for snake bite........ 293 Post-mortem examination after snake-poison- AI Ete re ea cicc erate SR essai ure hors 68 Smears Ir2 Potassic bichromate, effects on snake venom 98 carbonate, effects on snake venom . 97 iodide, effects on snake venom..... 98 permanganate, effects on snake SUT OM ne sery aie ney aleeeenbre siclc/s\s eAs.custe 98 Potassium permanganate used for snake bite 287, 290 Precipitability of various venom constituents. 81 Precipitin-reaction with snake venom. . . . 261-263 Preservation, effect on snake venom........ 94 Proteolytic ferment, in venom......... 204, 211 FEOLEEO RYN eres 2s Fare sont sei ae or ast I, Lr, 46 PSROEOUML OED fo1e os suesor ea tate pater sles cite e 8c 2 137 IBSATNMMOCLYNASLES” reyaicsu cele « tire wheisin sie! 's1 18 CAEN pelea ie). tie borosaokersiatate 19 PARCEL peers setslcaeictaneys New «ofa /o7e- 18 ICME LADD Sevag cre nis arose piauarece onda 18 PRISER ETA ech conch Senrey cot bickotie ce Urals 18 SOU AMTMLOS US cca ve risiers) esa as 18 SUCHET AINE a ac sage 2/>) le 29 = 4 22 COMO erst hie ao a teers 22 NTUDOSTONIAN cs vice, Perais's - 288 MIGCOADISINI OF 515045 «ne AsO 67-69 treatment. Of <0... 06001 < sna 287-296 314 INDEX PAGE. PAGE. Snake bite poisoning, certain alleged anti- Tarbophis vivax, venomous nature of bite of. 123 GOLESIOR ae gee ee cores 2093" |) dihalassophisn: 7c. s5,a cack oe eee 28 stones, used for snake bite.......... 293 anomalisiys)5) isch! cers 28 venom, action upon heart .......... 2009, || dihamnodynastesiey aria ees 6 eid neyaarrorisee: 208 ||| Juhanatophidiay cn) rs aerate oo eee I Livery arrestee 207°. )|\ AhelotOrnish.s rasa Ae aa al ee 10 anal See ee eee 208 | Therapeutic values of antivenins ....... 233-245 TMUSCLES eke 200: || Tbhronibogen Hi aalttey. peer ces eee eos 137 SPlee ieee tee 208 | Thrombosis, intravascular............. 135, 144 amount secretedi a sees Ho. Momadonn,. gacean sewers ae eee ee 6 antibactericidal properties of MOrtricin aly tps con ae ee On eee I gre —on6)\|) Oxalipumalias) eysyed-ie eral ey clare ie erie 85 cytolysinsiinw er ene eau: 199-205 | Toxic secretions of venomous snakes...... 70-76 descriptionioine sass ee. 79-80 | Toxicity of snake venom...............:. 71-76 diastatic actions of ......... 213 tissues of venomous snakes...... 219 effect of, on cells of cold- Moxoidithzemolysinye epee |e ee ae ee 179 blooded animals ...... 201-204 HeMOLMAgIN as dante ones 169 effect of, on cells of warm- Tragops prasinus, venomous nature of bite blooded animals ....... 199-200 OB etch jardin ase ee eae eee 123 effect of, on coagulability of ‘Treatment of snake bite with antivenins. 294-296 blood ise nance See cee 133 | Trichloride of iodide, effects on snake venom 99 effect on nervous system .124~132 siniponocephaltisia sets ty eee 37 PEGE SUESMIC a Ae eae ZTO-en4 ||) AlnimMereSUbUS! pe curiers eaten ee eee 37 nzemolysins Ots-y.cne ee ees 145 riukiuanus, effect of venom of, histological changes produced upon the nervous system.... 125 on various organs and tissues riukiuanus, mortality caused by 75 200-200) ||, Lenimerorhints) yy. .o.5 seer (eee eee lipolytic action of .......... 203) ||| eirimorphodontes aes ee oie NEULOLOXIMS (Obs yeys eres 143-155 biscutatus, venomous nature physical and chemical prop- Of IbiteiOhe aeaerc ears 122 EXLESOleaee tet see ys 77-93 CONATISGushiee eon k oer 8 precipitin-reaction with . . 261-263 lamabday ccs joie Napeisece 8 proteolytic action of ........ 211 lyrophanesies wre eae 8 LOMICIRVMOL.. Paneer cy 71-76 PAU uae oom Siti ses, aro, eae tS 8 Snakes, effects of snake venom upon....... 264 Upsilones ove ae Coe 8 Sodium, carbonate of, effects on snake venom ror Ville SOM este ean eee 8 CURT ALC erp en ah Roe eh vem tain aie £35,030, |) JUTOpIdechiswaac cues ean ee eee 22 antihemolytic property. .189, 190 Cavite Me Cos eee eR 22 fluoride, effect on venomized plasma 136 | Tropidonotus natrix................... 48, 49 OlEAT EE Be repsins Se vrini cere eee 183, 193 PISCALOR Fees a.20.0aN tara 51 Solenogly phan stn cvcee cee ee i EL, 46 Subminiatusip cick. ae 49 Solubility of various protein constituents of tesselatuspe tyr ns teicher 50 WVEDOMDG., sev. orev kote aug Mats iarsnrePaere a seve ee 80 LOEGUALUS) fe pie< io. she Ses sic serene 49 Specificity of antivenins ... css.) 24. .: 233-245 VIPELINIUIS evict. ey otras erener 49 Spermatozoa, effects of snake venom upon..201— | Trypanurgos ..................0.eee000- 6 202, 270-284 | Trypanosomes, effects of venom .......... 205 Spinalcord, paralysis, in venomitoxication'sem27, ||) dinypsiny sa ein ate eee ee 105 130; 190, ca2) ||) Dyphlopide, detinitiontof -—) ..a0-meaeeeee 2 Spleen, histological change in, caused by VENOM Sa shee as ae eee en ee 208 | Urodelia, mucous glands of oral cavity...... 47 Staphylococcus;aureus) 4-0 14.6205 e eee 205) |) Uropeltide detinitionlot ss eae a reere 2 DLCHOP MIS Hake ree ee Ce eee ae CO iF STEMOEIN aye ee, pee et whe ath ane eee HOV) VaRUSt ere vat ulsiseeee en 125, 128, 029, 130 Strychnine, value of, as an antidote to venom Viasomotor ‘center? 3.00509 se ae 124, 125, 132 LORICA TI OTE A preter ch thee Roe ote eee 200292) |) VEHOMsactivators.- roe ae eee 183-189 Strychnos colubrina, used for snake bite.... 293 agglutination. s+. 42 e 162-198, 282-284 Sugar-cane, used for snake bite ........... 293 albumin) ee es an ae ne 82 Sulphuric acid, effects on snake venom. . .98, ror and antivenin regenerated from their Sun, effects upon neurotoxin of snake venom. 144 neutraliicombination,... 2. «s/t = 248 Supervenomization of blood corpuscles ..... 195 cytolysis, mechanism of ............ 204 Swallow-root, used for snake bite.......... 293 TEFIMENtS race semen olor eG 210-214 SYCOLY MUS celISioOhi tu Rani sere i ieee aa 151 roles of, in venom toxication 210 Symptoms, venom poisoning in man... .. 106-112 gidbulinss;AAee aero Cre ace 80, 82 SYDLOMM renee ae ater er star 83, 84 haemolysis, amboceptor nature of .... 170 Systematic position of venomous snakes. .... 13 effect of/antivenin==.--4.- 168 injurious effects on blood HRA CHYRIEDIS at: G:c ek ae ene esc 6 COLPUSCIES 7. eae 189 Tanjora pills, used for snake bite.......... 293 protective property of Tannic acid, effects on snake venom....... 98 AGIdS Ons. nae aero 185 Wearrtillay p.t272-2% hitsks eho tess meso ee eae ae 9 toxoid formation of ...... 169 coronata: )sofsst/aety ee ete ear 9 hemolysis); Ge on eeeoeeee 162-198 EISGTT Ay ate ee ee rae epee eel 9 among cold-blooded ani- PTACISN 2 hice mies eee eee tar 9 MALS ray. wera eck ats 282, 284 HIGTICEPS) seen eee Te eRe 9 in saccharose solution.... 185 (aphrometopon: oui sce oe aoe aac 6 mechanism of........ 185-194 Warbophis eh csc aerate eran eerie 6 new era of study of...... 169 INDEX PAGE. VENOM MANASC ote crete wat cme tee a 211, 213 HE CIENT Swrectchs crt eicione cae tee 146 EASE metro tare uae erie eer era ea 213 NEUROSIS AM WET” Fi. se a aan os 150 TSE DEOUICS Decree avs Shur atta si eeeiiey, dass 81, 83 poisoning in animals, experimental . . 113-123 man, symptoms of... 106-112 process of secretion of ............ 63-64 proteolytic, anticoagulating property OLeM ices oo Le 212 PETIMENE se ois leas aie © 211 properties of .......; 211-212 Venomous snakes, geographical distribution Olpete See ea rene Cea of American continents . 52 phylogeny of.......... 46-51 toxic secretions of ..... 70-76 systematic position of... 1-3 toxicity of tissues of..... 219 WMenoms, Secretion! 20 6 o5.saceccs arenes 63 Wir OeCh@lercess store viva auhcts isle suerte aaiaistec 205 NEN AN eerie cos Pans AN olay chen ana ae ala\t 30 BMAMOMVEES ayia rls s wees le rene 31 effect of venom of, upon nervous system....... 126 effects of, upon coagu- lability of blood...... 133 Nitnenitaspiswaja tachi amos tn aicicham acct \, Sieie aire 30 berus, effect of venom of, upon the HEGVOUS! SYSTEM 6 ceca 126 effects of, upon the coagula- bility of the blood ......... 133 mortality caused by ........ 76 POSOnINE in man fs 2-5) <1 109 > PAGE Vipera berus,s. Pelias berus ............. 30 NBUSTNNsihariatie tah athe ate © «aioe ers 31 eben ge ole csc sis .c0 ee te eae oe 31 poisoning, experimental............ 116 ELAN, sh tres cite semis ae nice al ote alae aca ar PETIT Lie aerate sie aior as tate natal ee 31 MUBSELUD s/s enki caer cc aininis, 2 os meas 31 mortality caused by ........ 76 sp., poison glands of. ....0..-.+4%.- 61-63 Supercilianign |... Wi.) iets tee ale aul 31 PICS. Adare ir eainuth tn tote sees 30 WHET Ca Be ies. 51.0 ais apslat ecciel ais an eet e 3, 29 definition ors,.,.. a.s sos, -ian tee 2 MIDETINGE aes tear wale & oa aware oes ae 30 effect of venom of, upon nervous SYSCCON Sep nae nae shoe ate 126 Woaltertnmesta 22.013. soe sich nce ein oieiote 17 EDV DUAN es aicus sactoictsrectieree s 17 Warm-blooded animals, effect of snake venom OWCOMS/ ON crm cae rn nas een eaters 199-200 Water-moccasin venom antivenin ......... 259 Water venom globulin) 25 0.7.2 1