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Spring 1969
VARIABILITY IN ACANTHOCEPHALUS BUFONIS FOUND IN AMPHIBIANS FROM TAIWAN
FRANCELLA ALICE WOODS
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Recommended Citation WOODS, FRANCELLA ALICE, "VARIABILITY IN ACANTHOCEPHALUS BUFONIS FOUND IN AMPHIBIANS FROM TAIWAN" (1969). Doctoral Dissertations. 900. https://scholars.unh.edu/dissertation/900
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WOODS, Francella Alice, 1927- VARIABILITY IN ACANTHOCEPHALUS BUFONIS FOUND IN AMPHIBIANS FROM TAIWAN.
University of New Hampshire, Ph.D., 1969 Zoology
University Microfilms, Inc., Ann Arbor, Michigan VARIABILITY IN ACANTHOCEPHALUS BUFONIS FOUND
IN
AMPHIBIANS FROM TAIWAN * auCe FRANCELLA WOODS
B. S ., U n iv e rsity of Nev Hampshire 1951
M, S,, University of Nev Hampshire 1952
A THESIS
Submitted to the University of Nev Hampshire
in Partial Fulfillment of
The Requirements for the Degree of
Dootor of Philosophy
Crraduate Sohool
Department of Zoology
May, 1969 This thesis has been examined and approved
m & L , R . U r r l J L . OhJMLu)^ U~
^H/d$X Deep appreciation Is expressed to Professor Wilbur Bullock for his generous use of time and patienoe in guiding me in the pursuit of this problem. Dr, Bullock also made it possible for me to secure * Government-sponsored research grant under Navy Contract 168-3762 as well as selecting me for a project assistant for National Science Re search Grant GB-6408 "The Morphology and Taxonomy o f the Acantho- oephala, 11 For this assistance I would like to acknowledge my thanks, I am indebted to Dr. Robert Kuntz for supplying materials and for help in securing the above Navy contract. I would like to express thanks to Dr, John Cross and the Parasitology staff of NAMRU 2, Taipei, Taiwan, for the use of the facilities, as well as help and encouragement while working on this p ro jec t, I would like to express appreciation to Mr, F, F, Mlddleswart of Wilmington, Delaware, for his assistance and advice in the statistical study and for a ll the computer analysis done at Dupont, Finally, I would like to say thank you to my brother-in-law, Mr. Robert Costantino, for his assistance in the preparation of illustra tio n s . 1 TABLE OF CONTENTS LIST OF TABLES...... v LIST OF ILLUSTRATIONS...... v i I . INTRODUCTION...... 1 I I . LITERATURE REVIEW...... 3 1. H isto ry ...... 3 2. The genus Aoanthocephalus ...... 7 3. Morphology...... 8 h, Aoanthocephala found in amphibians ...... 11 5. Aoanthocephala found in amphibians from the Far East.. ih I H . MATERIALS AND METHODS...... 21 1. Definitive hosts,...... 21 2. Zoogeography...... 25 3. Preparation of speaimens ...... 26 Jf. Morphological study ...... 27 5. A study in variation...... 27 IV. OBSERVATIONS AND RESULTS...... 29 1. Body sise - length and width...... 29 Comparison of males and females - length.,,, 29 Length of specimens from one location - Tounan,... 32 Observations on length ...... 3h Width ...... 36 D iscussion ...... 37 2. Proboscis i length and width ...... h i Range and mean from one loo at ion - Tounan ...... h i Comparison of three hosts t B, melanostiotus. B, bankorensis. and Rana...... h3 D iscussion o f proboscis s i s e ...... h 6 3. Variation in the maximum sise of the proboscis hooks., h7 Sexual dimorphism ...... h7 Host-associated variation ...... h7 Location differences ...... 51 Discussion of maximum hook sise ...... 51 i i 4. Hook arrangement...... 53 Variation in the number of longitudinal rows of hooks...... 53 Variation in the number of hooks per longitudinal row ...... 58 Combination of the number of longitudinal rows and the number of hooks per row ...... 61 Discussion on hook arrangement...... 62 5. The lem nisei ...... 64 Size of the lemnisei compared to the size of the proboscis receptacle ...... 64 Size of the lemnisei in comparison to each other,... 69 Discussion of the lemnisei ...... 70 6. Length of the proboscis receptacle...... 72 7. Variation in cement glands...... 72 Number of cement glands ...... 72 Form and shape ...... 75 Discussion of cement glands ...... 78 8. Variation in the testes ...... 80 Range and mean fo r th e a n te rio r and p o ste rio r t e s t e s . , , , ...... 80 Discussion of the testes ...... 81 9. Variation in the eggs ...... 81 S tru c tu re ...... 81 Size comparison ...... 86 Discussion of the eggs...... 86 10, The female genitalia...... 93 V, DISCRIMINANT ANALYSIS OF MORPHOLOGICAL VARIATION FOUND IN ADULT ACANTHOCEFHALANS FROM AMPHIBIANS ON TAIWAN...... 94 1. Discussion ...... 95 2. The Coefficient of Variation, ...... 96 3. The Coefficient of Difference ...... 99 4. The Nt” test ...... 101 5. Pearson's Correlation Coefficient ...... 105 6. Point Bl-serlal Correlation Coefficient, ...... 108 7. A Nested Analysis of Variance ...... 109 8. Other statistical techniques used ...... I l l Two-Variable Scatter Plots ...... I l l Cluster Analysis ...... 112 i l l M u ltl-v ariate Cla s sific a tio n Technique...... 112 P rin c ip a l Component An aly sis...... 113 VI. CONCLUSIONS...... 115 LITERATURE CITED...... 117 APPENDICES...... 123 iv LIST OF TABLES Number Page I, Comparison of body length by classes from various hosts ...... 30 II, Comparison of bod$r sise ,...... 31 III, The range and mean of body length for Tounan specimens - grouped by Individual hosts ...... 33 IV, Length and width of the proboscis of parasites from one location ...... * ...... 41 V, The range and mean of the proboscis length of female specimens from Tounan, grouped by host ...... 42 VI, Comparison by host of the proboscis length ...... 44 VII, Comparison by host of the proboscis width ...... 4-5 VIII, Sexual dimorphism demonstrated in the maximum sise of proboscis hooks of specimens from Bufo...... 48 II, Host-associated variation of the maximum hook sise ...... 50 X, Length of the proboscis receptacle,,.,...... 72 XI, Variation in the number of cement glands ...... 75 X II, Range and mean fo r th e a n te rio r and p o ste rio r te s te s 80 XIII, Size comparison of the anterior and posterior testes 81 XIV, Ranges in egg and embryo sise according to the several host groups...... 87 XV, Comparison of egg sise for all amphibian hosts...... 91 LIST OF ILLUSTRATIONS Number Page I, Map of Taiwan, ...... 2k II, Graphic arrangement of the number of longitudinal rows of hooka arranged' by the several host groups - male ...... 5k III, Graphic arrangement of the number of longitudinal rows of hooks arranged by the several host groups - female 55 IV, Graphic arrangement of the hooks per longitudinal row arranged by th e sev eral h o st groups - m a le ,,,...... 59 V, Graphic arrangement of the hooks per longitudinal row arranged by the several host groups - fem ale,,,,,,,,,,..,, 60 VI, Plate # 1. Size of the lemnlsoi compared to the sise of the proboscis receptacle ...... 65 VII, Plate # 2. Atypical lemnisei...... 67 VIII, Plate # 3, Variation in the number of cement glands.,,,,, 73 IX, Plate # k. Variation in the form and arrangement of the cement glands ...... 76 Z. Drawing # 1, Drawing of an egg from Bufo melanostictus... 82 XI. Drawing # 2, Drawing of an egg from Rana longlorus.. 63 XII, Drawing # 3. Drawing of an egg from Bufo bankorensia 8k vi ABSTRACT The genus Acanthocephalus i s found in amphibians of Taiwan, The type of host, the number of parasites harbored by a given host, the collecting time as well as the place collected are all factors which demonstrably contributed to morphological variation. Statistical methods were used involving computer analysis, which demonstrated that there was only one species in this series of specimens. The inherent variability of the cement glands and lemnisei was clearly shown. The adaptive character of the egg was seen in Bufo and Rana, where polar elongations were present in both or lacking in both genera. The species of Acanthocephalus f ound in Amphibians from Taiwan and other places in the Par East is probably Aj_ bufonis. The amount of variation found to be present in the morphological characters of A^ bufonis is tremendous. SECTION I INTRODUCTION The Aoanthocephala are a group of endoparasites that occupy an uncertain phylogenetic and systematic position. Adults of all known species are found as parasites In the intestine of vertebrates. Typically, the Intermediate host is an arthropod. The genus Acanthocephalus is found in the amphibian parasite fauna of North and South America, Australia, Madagascar, Europe, Russia, and Asia, There have been six species of Acanthocephalus reported from amphibians of the Far East. They arei A. bufonis Shipley, 1903» A, lu cidus Van Cleave, 1925» A, a rta tu s Van C leave, 1925 1 A. sin e n sis Van Cleave, 1937, A, nanus Van Cleave, 1925, and A, elongatus Van Cleave, 1925. One of the purposes of this study was to survey the amphibians from Taiwan and determine what aoanthocephalan parasites they harbored, A study of variation was undertaken to contrast and compare the morphological characters of a large number of apeolinens from various hosts, different collecting points, and varying times of collecting. As far as reports in the literature permitted, a comparison with other species of Acanthocephalus from amphibians around the world was made. Sophisticated statistical techniques, using the computer, were em ployed to discover the actual quantity of variation present and to decide if these differences were significant or merely due to ohanoe alone. Previously, some species have been set up on the basis of one or two specimens or because of the presence of scorn slight difference in one 1 . morphological feature, A study of differences in the sise, shape, and number was made of the cement glands and lemnisei, as these two charac ters seem to differ Inherently. Other characters are adaptive and vary according to the environment. The present study was undertaken to throw light on the wide area of variation that occurs in the genus Aoantho- cephalus. It is hoped that this work will aid in an understanding of some of the problems of speciation in this extremely variable genus. 3 SECTION I I . LITERATURE REVIEW H isto ry Acanthocephalan worsts were observed by early workers, probably by Redi In 1684 and by Von Leeuwenhouk in 1695 *&d by ,other workers about the begin ning of the 16th century (Tamagutl l?63t Hyman 1951). Koelreuther (1771) proposed the name Acanthocephalus for a spiny-headed worst isolated from the Intestine of a fish. Zoega and Mttller (1776) used the name Eohinorhynohua for the same organism. Up until 1891 all speoies were described as belong ing to the latter genus. The first mention of this genus in the Linnean system was in the 13th edition of the Systems Naturae. The generis name used was Eohinorhynohua and they were included in a listing of roundworms and flatworms with a similar habitat. Pallas (1781) called the fam iliar thorny-headed worm of the pig Taenia hirudinaoea. Rudolphl (1808) established the order Aoanthocephala, He derived the name from two Greek words i "akantho", which means spiny, and "kephalo", which means pertaining to the head. Only one genus, Eohinorhynohua. was recog nised which had the characteristics of the order. In 1619 he assigned the following characteristics to itt "Corpus teretinaculum, ultrlculore, elas- tloum. Proboscis aeriatum unoinata," Westrumb (1821) briefly reviewed the earlier observations made on Aoanthoaephala. His papers were in Latin and he listed 90 speoies of Echine- rhynchua. Dlesing in 1851 concurred with Rudlophi's classification in the accept ance of only one genus, but he considered the order Aoanthocephala to be a tribe which ho lnoluded In the sub-order Aproote, along with the gregarlnes. In 1876 the family Eohinorhynchldae was ereoted by Cobbold to aooom- modate the single genus Echinorhynohus. The characteristics were not defined, Leuekart ( 1876) used the family name Acanthoaephalidas, His works and that of his students provided much of the basic materials which were used in later monographs by Kaiser (1891-3) and by Hamann (1891-5), Van Cleave (19i+8) gave Hamann the distinction of being the founder of modem taxonomic grouping of speoies in the Aoanthocephala, Hamann attempted to divide the group into three fsmiles 1 Echlnorhynchidae (genus Echinorhynohus). Giganto- rhynehldae (genus Gigantorhynohus) , and Neorhynohidae (genus Neorhynchua ) 1 based upon fundamental differences in bodily structure. This is one of the earliest works on Aoanthocephala dealing with the development, history, and anatomy, as well as containing the best early descriptions. Van Cleave (19^8) states, "All modem taxonomy at the level of genera and families of Aoanthocephala is but an expansion of his morphological analysis," The families roughly correspond to the three orders of the present day. Max Lithe (1911), in his significant historical treatment of Acantho cephalan nomenclature, made a critical review of all the earlier specific descriptions. The position of the Aoanthocephala among the animal phyla has been uncertain. The Aoanthocephala show remarkable uniformity in general anatomy, life history, and habits, and yet, at the same time they are widely diver gent from other groups of worms. Considered from an historical standpoint, their relationship to other phyla is interesting. They have been placed with the platyhelminths by some and with the nematodes by others. In their development, and in their relationship within the host, they are closely parallel to the oestodes. They are divorced from the flatworms in the structure of the oocyte and the mode of segmentation of the egg. The number of nuclei is constant in a given speoies. After reaching the final host there is no farther cell division, except in the germ cells, even though the body may grow to hundreds of times its original sise. Today it is acknowledged to be a unique, separate phylum. When one considers the classification of the phylum, Anton Meyer's monograph (1932-3) is extremely noteworthy. It is a comprehensive, elaborate, highly consistent work based upon a wide understanding of comparative morphology and biological data. He recognised 12 families with 58 genera and somewhat more than 300 species. He included two o rd ers 1 Arohlaoanthocephala and Palaeaoanthocephala, Later Van Cleave (1938) added another order, Eoaoanthocephala. Petrotsohenko (1958, 1958) published a two-volume work, in Russian, which contains a large section on general discussion and his descrip tions of all orders, families, and speoies. It also contains many keys, Golvan published his firs t monograph in 19581 his works are also to be found in the current literature, Golvan and Houin (1963) in their revision of the Palaeaoanthocephala statet J, G. Baer, dans le cadre obligatoirement etroit d'un traite de Zoologie. a fait one exoellente synthese de la Systematlque des Aoanthocephales (1961), Les grandes divisions de l'Embranchement, crises par Meyer (1931) puis par Van Cleave (1936) demeurent, II r e s ts 1 sav o ir 1 quel niveau 11 faut placer les trols ooupures, k ce propos Baer (1961) eoritt "Les oaracteres des classes et des ordrea ont e te rdsumrfs p ar Van Cleave (1953) dans un d e rn ie r t r a vail paru aprss sa mort, , Golvan (1959) supprlme la olasse des Metacanthooephales pour elever les deux ordres qu'elle renfermait au rang de classes^diptinots. Toutefols, les bases sur lesquelles les classes ont ete erigees nous paraissent jtrop Itroites pour slparer deux groupes hlerarchlques ^ussl sieves et^p'est pour oette raison que nous adoptsrons la premiere suggestion emise jpar Van Cleave et les considers rons oomme des ordres. Par consequent les classes admlses par Golvan deviendrcnt des ordres. La olasslfioa- tion proposes par Petrotsehehko (1956 et 1958) repose sur des premisses a priori non Just If lees, a s avoir quo les Aoanthooephales seraient dies parasites hautement specif Iques, et par consequent, qua ohaque groups d'hotes renfeme des espeoes et des genres qul lnl sont propres. Sur oette base, 1*auteur est amene a oanslderer ipso faoto que les formes hebergles par des Polssons represeptent les plus primitives ou arohalques et nous avons dlja signals plus haut toutss les difficultes qul surgissent dans oe case. Nous aooepterons done la suggestion de Baer et divlserens areo lu l 1*Embranohement en tro ls ordres i Goao anthooephala Van Cleave 193&* Palaeaoanthoeephala Meyer 1931, et Arohiaoanthooephala Meyer 1931* Tamsgutl (19&3) edded to h is o th er volumes on trem atodes and oea- todes a volume on Aoanthocephala which contains excellent keys but his system of classification is very different from Golvan* s. He has a kind of arbitrary taxonomy, much of which he adapted from Petrotschenko. He divides the group Intoi 1) Eehlnorhynohldea, 2) Glganthorhynehldea, 3) Neoechlnorhynehidea, 4) Apororhynchldea, He does this according to the law of priority. From an ecological standpoint, the definitive hosts can be divided by habitat in a manner roughly equivalent to the taxonomic divisions. The Palaeaoanthooephala have mostly aquatic hosts as they Inhabit fishes, aquatic birds, and mammals. The Eoacanthooephala also have aquatic hosts but they are found In fishes with but a few species located In turtles and amphibians. Terrestrial vertebrates harbor worms belonging to the Arohiao anthooephala. All Aoanthocephala have an arthropod intermediate host. Some may have a paratenlo host. Manter (1967) reports the utilisation of auxili* ary or paratenlo hosts in a few trematodes of fishes but it Is more widely developed in the Aoanthocephala. The definitive host can be found In nearly all the known classes of vertebrates. The Genas Acanthocephalua Van Cleave (1919) gives the following generic diagnosis of Aoantho- oephalus. K oelreuther 1771• Aeanthoeephala of small to medium sise, paras it lo as adults In the alimentary oanal of fishes and amphibians. Subcut inula and lemnisei provided with numerous small nuclei. Proboscis ovate or a short cylinder, Body proper and neck spineless. Proboscis-receptacle a two-walled muscular sac Inserted at the Junction of proboscis and neck. Meyer (1933) g*?e a description and definition of the genus Acan thocephalus. K oelreuther 1771» Ltthe 1911. KBrper kleln bis von m ittlerer GrOsse, nahezu sylindrisch, mit zahlreiohen kellnen Kemfragmenten In der Haut. Proboscis m&ssig lang, kurz zyllndrisoh bis el- oder keulenffirmig, mit nicht sehr zahlreoihen Haken, dlase von der Spitze naoh der Mitte zu an GrSsse zunehmend, naoh der Basis hin welder erheblioh kleiner werdend. Das Ganglion 1st lm Probosols-Reoeptaeulum hinten gelegen, Zement- drdsen o, tells neben- tells hlnterelnander, mehr oder wenlger in 3 Paaren, birnfhrmig, mit scharf abgesetzten Ausftthrungsglngen. Uterus ms 1st verhttltnismlssig kurz (kttrzer als lm Genus Eohlno- rhynohus). Parasitisoh In Fisohen, marin und lm Sfisswasser, - Verbreitungi Buropa, nfirdliohe Meere und Mittelmeer, Nord-Amerlka, China, Japan, Brasillen (?), sttdlioher Atlantlk, (17 speoies.) Tamagutl (1963) followed the classification of Petrotschenko (1956) 111 dividing this genus an the basis of the host being aquatic or terres trial t the embryonic larva of the former being spinose at eaoh extremity alone and having the middle eggs shell with polar prolongations, Aoanthocephalans that are parasitlo mainly In terrestrial forms and which lack polar elongations of the middle shell he plaoed In another class, the Glgantorhynohlnea In the new genus Pseudoaoanthooephalua. There axe four speoies lnoluded In this genus, according to Golvan, all parasitic In terrestrial amphibians, Golvan (i960) reviews the classification given by Petroehenko of the genus, Pseudoaoanthooeiahalus, He lists k spec les i P. bufonis (Shipley 1903) Petrotschenko 195k, £• bufonisola (Kostylew 19kl) Petrotschenko 1956, P. Caucasians (Petrotsehenko 1953) Petrotschenko 1956, and P, elongates (Van Cleave 1937) Petrotschenko 1956, These are all parasitic of terrestrial amphibians. The embryos are oral, possessing three membranes, the middle of which is thick and brilliant bat polar elongations are lacking. The embryo is smooth (?) and completely covered with minute spines. Golvan*s remarks concerning the splitting of the genus Ac anthoc ephalua are as followsi La creation de ce dernier genre nous par ait, reposer sur des caraetdrps trop peu Import ants pour que nous en aeaaptions la valldite, En effect, en oe qui concerns 1 'anatomje des embryons, Petrotschenko dlt, dans sen texte, qu'il n'a pa etudler que ceux d'une seule des quatre esp&ces qu 'il classe dans sen genre Pseudo- acanthocephalus et que les trois autres ont "probablement" des embryons prdsentant les memes oaracteres, Le second argument que donne 1*auteur russe pour justifier la creation de son genre est encore beaucoup plus discutable, Selon lui, les differences Ithologiques et ecologiques entre Anoures "terrestres" et Anoures "aquatiques"^ sont telles que ces deux groupes sont parasites par des Ac anthoc eph ales qui appartiennent non seulement a des genres, s mals k des olasses diffdrents (les Acanthocephalus appartiennent a la sous-olasse des Echinorfavnchinea Petrotschenko 1956 et les Pseudoacanthocephalus ~k cello des Gigantorhynchlnea Petrotschenko 1956). Si nous aocordons une tres grande valour taxonomlque aux arguments d'ordre biologlque, dans ce cas precis, nous ne pouvons aooepter 1*opinion de Petrotschenko. En effect, nous savons que tons les Anoures sont aquatlques, non seulement pendant leur vie larvaire mals aussi pendant la perlode annuelle de reproduction 1 que leur regime alimentaire, mdme pour ceux qul, comme Rana esoulenta. ne qulttent jamais le bord des eaux, repose presque exclusivement sur la capture d'Arthropodes terrestres. Nous pro- posons done, jusqu'a ce que nous ayons des renseignements plus preols, de conserver a ces quatre espeoes la place qu'elles oocupaient dans le genre Acanthocephalus Koelreuther 1771* Morphology The thorny-headed worms form a unique group by reason of their structure and extreme parasitic habits. The very name Aoanthocephala reflects the distinotiveness of the proboscis which serves as an organ of attachment. Distinctive features of this phylum are complete absence of a digestive tract, a more or less flattened body, a sheathed, retractile proboscis armed with rows of recurved hooks, and the sexes are separate. The life oycle of the Aoanthocephala involves an intermediate host which is an arthropod. The parasites of aquatic animals probably have crustaceans or larval Insects as intermediate hosts and those of terrestrial animals have various inseots. The body Is divided into two parts i the presona, made up of the proboscis, the neck, the proboscis receptacle, the proboscis ganglion, and the lemnisei, and the trank which makes up the remaining, much larger portion of the body. The trank has a central fluid-filled cavity in which the reproductive organs are situated. The surface of the proboscis is covered with rows of recurved hooks which are usually similar in sise and shape. The proboscis, in most species, is retractile into a proboscis sac or receptacle by being turned inside out, In fact, the whole presona is retractile, without inversion, into the fore part of the trunk by means of special retractor muscles Inserted on the trunk wall. The proboscis has distinctive value as a functional feature (Van Cleave, 1952), Mechanically, this is a most ingenious device j the principle under which it operates remains remark ably uniform throughout the phylum. The taxonomy of this group is based, to a large degree, on the proboscis armature. The hooks are fairly delicate with fine slender spines either lying parallel to or projecting At a slight angle from the surface, Not all hooks are of identical form and sise. They may be of even size over th e proboscis b u t commonly those a t th e base are smaller* The hooks may be largest at the summit and decrease gradually toward the base or often are largest at the middle region, decreasing toward the ends. The nook is variable In development but it is invariably devoid of spines. It is separated from the trunk by a o ire alar ring posteriorly and extends anteriorly to the last row of hooks. The probosols receptacle is a heavy-walled muscular sac. The fibers of each muscle layer run in a clockwise spiral around the recep tacle making an angle of about 45° to its longitudinal axis. The pro boscis and receptacle enclose a fluid-filled cavity. It is fastened in a circle to the inner surface of the proboscis wall and hangs into the pseudocoel. Within the proboscis receptacle is a cephalic ganglion or "b rain ". The lemnisei are two fingerlike projections located an either side of the proboscis sac. They originate from the inner layers of the wall of the neck. They contain numerous fluid-filled spaces which communi cate with those in the proboscis wall, Hammond (1966) notes that they are completely invested by the so-called "neck retractor muscles". He also demonstrates that fluid flows from the lemnisei to the proboscis wall only after the probosols has become evaglnated. The lemnisei can not, therefore, aid in the evaglnation of the proboscis, ( 1966b) Crompton and Lee (1965) thought the lemiscl were involved in the absorption of fat. The passage of Scharlaoh R from the intestine of the host to the lemnisei suggests that these organs are more than a mere hydraulic reservoir. This was demonstrated by Hammond (1966) in his work with A. ranae. The lacunar system consists of median or lateral longitudinal main vessels and numerous transverse, often anastomosing, branches. There are no definite walls, A more or less cons pic lous network forms in the trunk while the lacunar system of the probosols is made up of irregular. interconnected fluid-filled spaces and does not have a definite pattern of channels. The gonad of the female is not penaanent for very early in adult life it becomes fragmented into numerous masses of oells which produce eggs. The ligament sacs or the body cavity becomes filled with rounded masses which are called "egg masses" or ovarian balls. When the egg masses break up, the body of the female becomes filled with developing eggs, each of which is surrounded by & series of shells or membranes. They are actually embryos as fertilization has already taken place and cleavage started. The reproductive ducts of the female consist of a uterine bell, uterine duct, uterus proper and the vagina. The funnel- shaped uterine bell has at its base a group of oells commonly called the selective apparatus as well as a pair of dorsal pouches which appear to regulate which eggs are passed on into the uterus and which are returned to the body cavity via ventral openings in the uterine bell. The vagina is funnel-shaped j its greater posterior portion is surrounded by a mus- oular sphincter. The male genitalia includes two testes, cement glands which vary in number and shape in different groups, as well as accessory structures. There are ducts for the storage of sperm and cement. There is also a complicated eversible bursa or pouch for copulation, surrounding the cirus, along with a " tJaefftigan's pouch, Ac anthooephala Found in Amphibians Worth and South America Aoanthooephalua ao u tu lu s. Van Cleave, 1931» found in T ritu ru s viridesoens in the U.S.A. i A, lfltsl. Hamaan, 1891. Syn. Eohinorhynohua lu tsi. Hamann found this in Bufo auga in Brasil and Cordero found it in P&ludloola darwlni. Bufo arenarum and Hyla radd lam In Uruguayi A, ranae (Sohrank, 1?68) Luhe, 1911 Syn. Eohinorfaynchus haeruoa (P all,, 1788) Rod., 1802, found In DiemictylUB ylrldeaoena In U.S.A. 1 A. van~oleave! Hughes e t Moore, 1943 In Burycea tra e re n s ls from Oklahoma, A u stralia A, hylae In Centropus phaslanus. Podargus strlgoldes In Australia, Madagasoar A. nadagasoarlens Is Golvan, 19&5 found In Madagaaoar In Rhaoophorua sp. This is a tree frog, Afghanistan A, kabulensla Datta et Soota, 195& In Rana sp. at Kabul, Afghanistan, Europe A. anthurla (Du j, , 1845) Luhe, 1911 in Molge cristate. M. vulgaris. Triton punetatus. also In Emys orbicularis 1 A. faleatus (Froel,, 1789) Luhe, 1911* Syn, Echlnorfaraohus haeruoa. found In Salamandra atra and Tropldonotus natrlx In Europe 1 A, ranae (Sohrank, 1788) Luhe, 1911 • found In Rana. Bufo. Bomblnator. Hyla. Trlturua. Salamandra In Europe, Russia A, bufonioola K ostylev, 1941 in Bufo v u lg a ris and B, bufoi A, bufonis Shipley, 1903, Syn. A, sinensis Tan Cleave, 1937 in Bufo vlrldia (Petroohenko, 1953) lists two other hosts 1 B. m elanostictus and B, penangensls but does not say that these were found in Russia.) A. eau- oaslous Petrotschenko, 1953 in Rana maerooneals. and Bufo v lrid lsi A, ranae (Sohrank 1788) Luhe, 1911 found in Tropldonotus natrlx. Bcmbina bombina. Rana esoulenta. Rana ridlbunda. R. tenporarls while occurrence in the green toads (Bufo vlrldia) as reported by Bykhovskii in 1935 is doubtful, 1 3 . Asia. A, bufonis Shipley, 1903 in B. melanostictus. B. penangensls. B. virdla in China and Indochina, also in R. tlgrina. and B. as per in Japan) A, elongatus Van Cleave, 1937 in B. formosus from China, A. luoidus Van Cleave, 1925, Syn, A, artatus Van Cleave, 1925, Harada 1935, found in R. japonica. R, tem poraria. R. o rn a tiv o n tris . R, nigrcm aoulata. R, tagoi. B, vulgaris iaponlcns. Megalobatrachus .japenious. Polrpedates buargeri in Japan) A, sinesnis Van Cleave, 1937 in R, nigroanaoulta. B, formo3tta in China) A, tigrlnae Shipley 1903 in R, tlgrina from Siam) A, nanus Van Cleave, 1925, in Diemictvlus pyrrhogaster Rana rugosa in Japan and Indochina and in Megalobatraoh.ua .japonicas in Japan, There have been some references in the literature to Acantho cephalus from amphibians but the speoies has not been stated) e.g., Benett and Humes, 1938) Dinnich, 1926) Pearse, 1932 from Japanese sala manders ) and Rankin, 1937 mentions Acanthocephalus sp. from North Caro lina salamanders. Van Cleave (1931) lists records of Ward, La Rue and Leidy which show no Aoanthocephala in 300 amphibians examined from Illinois, Michigan, Kansas, Nebraska, Ohio, Pennsylvania, Oklahoma, and M issouri, Niokol (19^7) lists Fessisentis neoturorum as the first member of that genus reported from salamanders. He also reports finding A. acutulus in Plethodon glutinosus. Reports of accidental infections are also recorded, Brandt (1938) quotes Sandground (1926) as reporting Moniliformis from B. Marinas in Honduras. Rankin (1937) reported a single specimen of Pcwphortanohus bolbicclli found in the newt, Triturus virldescens. from Leverett, Mass, Golvan lis ts Bufo as harboring Maoranthorfaypchua. Juvenile forms, the adult stages of which parasitise birds, use amphibians to serve as an intermediate link In the life cycle. They probably serve as reservoir hosts as well. The genus reported from around the world In a variety of Amphibians is Centrorhynchus (Van Cleave, 1915* Brant, 1936* Petrochenko, 1953* Yamaguti, 1963), Coryno- soma has also been utilised by larval forms (Muhling, 1898; Van Cleave, 1915* Petrochenko, 1953). Petroehechenko also mentions two other genera, Arhythmorhvnchus and Luheria as being host to larval forms, Golvan (I960) adds two more names 1 Porrorchinae and Sphaarechinorhvnohlnae. There are also records of undesignated encysted larval forms from newts (Pearse, 1932); and Van Cleave (1036) notes encysted post-larval forms in both amphibians and reptiles from China, Aoanthocephala Found in Amphibians Fran the Far East There have been six species of Acanthocephalus reported from amphibians of the Far East, These have been the subject of much dis cussion and no little confusion. They are: A, bufonis Shipley, 1903* A. luoidus Van Cleave, 1925* A. a rta tu s Van Cleave, 1925* A. sin e n sis Van Cleave, 1937. A, nanus Van Cleave, 1925* and A. elongatus Van Cleave, 1925. A. bufonis (Shipley, 1903) Southwell and MacFie, 1925. This speoies was described by Shipley (1903) under the genus name of Eohino- rhynohus. He stated that the body was curved, the female was 15 mm long, the male 5 mm long. The proboscis was ovoid with hooks In lJf-l6 rows, Southwell and MacFie (1925) redesoribed the animal in order to give greater aoouraoy. Meyer (1932) gave the following description! The length of the female 20-25mm, the male 9mm while the width varied between 1.5 - 1.8ma; the greatest width btlng near the anterior end of the body. The probosols hooks in 18 - 24- rows of 6 - 8 eaoh while the probosols cy lin d er measures 0 .5 - 0.6 x 0,3fem. The length of th e middle hooks was 80 - 90 microns t the roots are similar to A, ranae being strongly genloulated. The receptacle about 1 mm by 0,4 mm vide with a ganglion at Its base. The lemnlsai about double the length of the probosols receptacle. The testes* measuring 0.6 x 0,3 mm, lie at the beginning of the posterior half of the body* one behind the other. Joyeux and Baer (1935) described this parasite and compared it to other speoies, In 195** Yamaguti, on the basis of 1** males and 15 females* redescribed it, Petrotsohenko (19 5*>) ascribed it to the new genus Pseudoae an tho se phalus (Giganthorhynchides). Golvan (I960) again placed it in Ac anthocephalus. Shipley Indio ate s its host as Bufo melanostlotus and B, penangensls from Hong Kong, Meyer (1932) sta te s th a t A, bufonis was isolated from Bufo sp, and a turtle, Petrotsohenko includes the host B, v ir id is . A, lueldus Van Cleave. 1925. Van Cleave described the new speoiesi Males about 4 mm in length* females 12-17mm. Proboscis of type male 0.35 x 0,3mm, L ongitudinal rows of hooks numbered 12 to 16 w ith four hooks In eaoh rovi those at the tip slightly smaller and shorter (70 microns) than those on the remainder of the proboscis (82 to 88 mlorons), The lemnlsol but slightly longer than the probosols receptacle. Embryos from the body cavity of gravid females 9** to 106 microns long and 24 to 26 microns in diameter. Type host, Bans .japonica found in the Province Shlmosa* Japan. Walton (19**2) lis ts the following hosts t Megalobatrachus japonlcus. Polypodates buereeri. Bans .japanloa. Rana nigromoulata. Rana tagol, Rana temporla ornatlventris. Ri b i tigarlna. Yamagutl (1954) reports Bufo vulgaris japontcus as a host, A, artatua Van Cleave 1925. Van Cleave described this as a new species with the following characteristics t Body of mature Individuals about 10 or 11 mm long. The proboscis at least partly obsoured in all specimens available, length not obtainable, diameter about 0,27 mm, form similar to that shown for A, luoldus. Armed with about 14 to 16 longi tudinal rows of four or more hooks each} those near the base of proboscis about 90 microns long, near the middle about 100 microns long. Embryos 69 - 83 microns long and 18-24 microns In diameter. Type host Rana japonic a from Province Shlnano In Japan, Van Cleave had six individuals all of which had the proboscis retracted or Inverted, Re stated that the species seemed to be closely allied to A, luoldus but he differentiated them on the specific differ ence in the size of the embryos. Other hosts listed for this parasite are R, temporaria. R, nlgramaoulta. R. tagoi. Megalobatraohus japonicus. and Polypedatea buergeri, Harada (1935) adds a note about the size of hooks in A, luoldus which become progressively larger from anterior to posterior. The female hooks vary from 95 to 125 microns and the male hooks from 70 - 90 microns. He mentions that the two species A. luoldus and A, artatua from Japan are not very clearly distinguished on the basis of proboscis hooks as they vary greatly in number, position, size, and even according to sex. He found that the eggs of his specimens, in the preserved state, were 80- 100 microns In length and 26 microns wide. Thus the gap between the two species described Ly Van Cleave was filled. On the basis of his own specimens Harada (1935) reduced A, artatua to synonymy with A, lu o ld u s. Tamaguti (195*0 adds details of measurements of various parts, but finally agrees with Harada and reduces this parasite to synonymy with A. lu o ld u s. A. sinensis. Van Clears 1937. This species is frost Changsha in central China with other localities In the sane general area mentioned. The hosts are Rana nigromaoulata and Bufo formosus. Van Clears used **5 Individuals In his study of this speolss. He desorlbed the new species as haring females with a length of 10 - 15 mm while the males were 5 - 8 mm In length. The proboscis was armed with 15 - 19 longitudinal rows of 4 - 6 hooks each. The hooks showed great Individual variation with those of the female tending toward greater siae than those of the male. Hooks on the anterior region of the proboscis of males ranged from 53-9** microns, of females 52 - 115 micronst on the mid-region of the proboscis of males 66 - 103 microns, of females 79 * 115 micronsi on the basal region males ranged from 3 3 -9 8 microns while females varied from 53 - 98 microns. Embryos were reported as 45 - 60 x 12 - 15 m icrons. Van Cleave differentiates this species on the basis of the sise of the embryos which are distinctly smaller. The nearest species, A, artatus. has embryos measuring 69 - 83 x 18 - 24 microns. He also uses geographical locations pointing out that the latter have only been reported from Japan, All other species reported from the Orient have larger embryos. However, the fish parasites. A, acerbus and A. aouleatus. fa ll within the embryo sise range but their number of longitudinal rows of proboscis hooks is constantly less than A, sinensis. A. nanus Van Cleave. 1925. Van Cleave desoribeo this parasite of salamanders aai Hales usually about 5 long with a maximum diameter of 0.6 - 0,8 mntf females 8 - 11 x 0,7 - 0,9 mm. Proboscis cylindrical, usually O.h x 0,18 mm. Armed with 10-12 longitudinal rows of 6 to 8 hooks each. Hooks at base of proboscis h? - 53 microns, at the tip hi - h7 microns, remainder 53 - 59 microns. The proboscis receptacle 0.18 mm long, Lemnisoi about one and a half times the length of the receptacle. Type has a proboscis 0.35 x 0.18 mm. Embryos within the body cavity of gravid female 77 - 8h microns long by 9 - 12 microns in diameter. The type host is Deimiotylus pyrrhogaster from Province 0ml, in Japan, More than 20 specimens used in the study were in a poor state of preservation. Other hosts Inoludei Melalobatraohua .japonic us. Rana rug os a. and Trlturus pyrrhogaster. Walton (19h2) lists Asellua aquaticus. an iso pod, as the Intermediate host, Yamaguti (1935) states that the proboscis sheath is constantly longer than the proboscis and conoludes that Van Cleave's measurement of 0,18 mm to be obviously erroneous. He also disagrees with the embryo measurement as his specimens would seem to be larger, A* wlongatua Van Cleave 1925. Van Cleave's original description is based on one specimen which was an immature female taken from Bufo formoaus in China, The most diagnostic characteristic of this species is the very slender* much elongated proboscis. The European species A, anthuris has a similar number of hooks but they are larger in sise while the proboscis itself is much smaller. Also in comparison with A. gotoi from Japanese fish there is a comparable hook count but the proboscis of A. elongates is considerably larger and more filiform. Jpyeux and Baer (1935) found A,_ artatus in Indoohina and they stated that it appeared to them that A^ artatus vas very olose to A^ bufonis. even identieal with it. They also noted that this species was very olose to the European species, A^ ranae. from which it conic, be dis tinguished by the sixe of the lemnisoi and the consistently larger eggs. A. bufonis cement glands are elongated (clavate or club-shaped), while those of A. ranae were globular or rounded. However, one can also find elongated cement glands in three other species very similar to Aj_ bufonis (A. artatus. A. luoldus. and nanus). all of which are extremely difficult to separate. Southwell and MaoFie (1925) sttte that the egg sise of their specimens is in agreement with A. bufonis in Shipley's description. They also add that it differs from A. ranae in the greater length of the lemnisoi and in the greater breadth of the egg. Regarding the olose morphological sim ilarity of these Far Eastern species. Joyeux and Baer (1935) have a most intriguing final paragraph from which I shall quote i Quoiqui'il en soit, ces parasites des Anbures d'Estreme-Orlent sont tr^s voislns les uns des autres. L* etude d'une grande quantite de materiel pourra settle decider s 'il s^aglt la de variations d'une memo forms, ou de trois formes differentes^ Nous avon^ reunis dans le tableau ei-dessus les prinoipaux caraoteres des especes du genrer Aoanthocephalus hebergees par les Batraoiens d * Extreme-0rient, Harada (1935) brings out the fact that Yamaguti*s description of A. artatus from R^. .japonica (1935) corresponds to the description for A. lu o ld u s. Thus, from the literature A^ lucidus and A. artatus are considered to be one and the same, A^ sinensis has been reduced to synonomy with A. bufonis (Harada, 19351 Yamaguti, 1935)» although in his 19&3 edition Yamaguti keeps A^ sinensis as a unique species. A^ nanus as a parasite of salamanders, w ill not be Included In a detailed study. This leaves one land form and one aquatic form to be considered! A. bufonis and A, lu cldus. SECTION H I MATERIALS AND METHODS Definitive Hosts All of the definitive hosts collected belong to the order Anura or Salientia, Four fsullies of six genera and 2$ species have been reported from Taiwan (Chen, 1950), Hosts involved in this study represent the families Bufonldae, Hylidae, and Ranidae, In the family Bufonidae, there is only one genus, Bufo. which exists on Taiwan. Bufo melanostiotus Schneider i s commonly known as th e speotacled toad, Chen l i s t s i t as being found in Taipei, Hsinohu, Chiayi, Tainan, Hengohu, Yilan, Huallen, Pingtung, and Taitung. The following loo all ties may be addedt Changhua, Hsilo, Grass Mountain, Kaohsiung, Toxin an, and Taihsi, This would make the distribution of the species island-wide. The other species of Bufo listed by Chen is B. bankorensls Barbour, which has been reported from Pingtung, Huallen, Taichung, and All Mountain. In the central area, Sun Moon Lake and Taitung may be added to the lis t of places where B. bank orensls is found. The family Hylidae contains only one genus and one species on Taiwan, Hvla aMwwaia. These are small, beautiful green Chinese tree toads. They were collected in Taipei and Grass Mountain * but contained only larval aoanthooephalans. The family Ranidae which contains the genus Rana has 20 species feported from Taiwan, Hosts in clude i R. U m aaiw H a Wieokann, commonly c a lle d th e ric e fro g o r the pond fro g , and R. tle r in a (var, nantherlna) F ils Inge r, known as the f ie ld - chicken, which many people enjoy eating, are both found island-wide. They can be bought at any market and are often sold doer-to-door. R, lcngicrus Stejneger has been reported from Taipei, Tilan, Taoyuan, Hsinchu, and Chiayi, Yung-foh-lee and Graas Mountain nay now be In cluded in the range of R. latouohi. R. swinhoana Boulanger was oolleoted in Huallen Helen, R, guentheri Boulanger was ploked up at Tung-foh-Lee and R. sauterl Boulanger gathered fro® ill Shan were very few in num- berj only one or two per host species. The parasites contained therein were also few in nuaber and unfortunately In extremely poor condition*. R. saute rl had been oolleoted previously at Kuan-to a-ling, whioh has an elevation around 2,000 feet. Rhacophoroua robustus Boulanger was col lected in Huallen, but the hosts were mixed with R, llmnoreharls. There were only two parasites present, but to whioh host they belong is not known. There is one Immature female and one mature female which has eggs which have spines, polar elongations, and pronounced hooklets. Dr. Robert Kunts, while serving with the United States Navy and stationed at NJkMRU 2 in Taipei, Taiwan, examined a wide variety of Taiwanese fauna for parasites. The aoanthooephalans which were Isolated from amphibians were sent to Dr, Wilbur L, Bullock, University of New Hampshire, Dr. A. James Haley, University of Maryland, and Dr. David Llnoloome, Howard University. They in turn gave their material to me. These specimens, together with those oolleoted personally over a period of four years is the basis of this study. The total collecting period was between 1957 and 1967. In records of previous collections, the small intestine was desig nated as the location of the parasite. In a few oases only, were the parasites found in the large intestine. For the most part, the parasites were recovered from the anterior region of the intestine except in oases of heavy infection, when the parasites were found all along the digestive tr a c t. 23. Larval fonts of Aeanthooephala were found In Bufo on tiro occasionsj onee in the small intestine and once in the mesenteries, A oyst from the body cavity of Bj_ bankorensis was recovered from a host from Sun Moon Lake, By far the majority of encysted forms were found in Rana. The locations were quite varied, e.g., the surface of the small intes tine, within; the small intestine, in the body cavity and in the body wall, Hyla housed only larval forms which were located in the body cav ity . As far as possible a study of the parasite number per host was made, Toads had a much higher incidence of infeation, with some harbor ing well over 100 specimens. In some areas the toads were startingly clear of any kind of intestinal helminth while at other times they were very heavily infected. The frogs had a low incidence of acanthocephalan parasites but had a comparatively large number of trematodes, nematodes, and Snarganum. The frog, at all times,was host to some type of intes tinal worm. Out of 362 frogs dissected, 21 specimens produoed 98 Aeanthooephala. One host held 1? Aeanthooephala, while many contained only one. This is pointed out by other investigators, Lees (1962) pointed out that frogs were more likely to be parasitised by nematodes than aoanthooephalans in Great Brltian. Over a four-year period, he examined 2feL3 frogs and found that h37 harbored A^ ranae. Dogiel et al, (1958) observed that ranae ooourred only rarely in the absence of a trematode infection. It appears that the distribution of Aj_ ranae in Europe and Russia is looal, but where it is found it may be abundant (Goto and Hewer, 19^9). Hosts in mountain areas seemed to have fewer ao an thoa e phalans, but the frogs were heavily infected with everything e ls e . 2 4 . MAP OF TAIWAN Keelung Taipei Talehun Huallen Pingtung Tsoying i 9 40 m iles Zoogeography The nap shows clearly the wide collecting area. For convenience, the Island la divided Into north, central, and southern sections, The northern areas include Taipei and suburbs, Keelung, and Grass Mountain, the oentral section covers Yilan, Huallen, All Shan, Sun Moon Lake, Wushlh, Chunghua, Tounan, sad Talhsi. The southern region contains Tsoying, Kaohsiung, and Pingtung, Taiwan is an island lying close to the east coast of continental Asia, The Taiwan Straits separate Taiwan from the mainland of China. The Island is small and very mountainous with Mount Morrison reaching an elevation of 13,103 feet. The topography Is extremely varied for such a little place. The Tropic of Cancer crosses the island Just below the mid point, passing through the southern part of the city of Chiayi. Life cones range from sub-tropical at sea level up to aub-artio cones of the snow-capped mountains of central Taiwan, With its extensive Irrigation system to provide water for rice, water is literally everywhere, Taiwan oeuld well be called an * Amphibian Paradise", In southern Taiwan, where there are three rice crops a year, water is really In abundance. The dream of every Taiwanese woman. In fact it is something of a status symbol, is to have a home with a fish pond overshadowed by a weeping willow tree. This was the environment from which the Tounan toads were collected. Often along the road one can see a sum with a long rod, connected to a battery, walking along the irrigation ditches or between the rice paddles, catching frogs by stunning them with electricity. Sanitation in some places leaves much to be desired and In places which lacked 26* running water the parasite fauna of the amphibians was much higher. There are seasonal changes which are difficult to delineate because of the variation of the environmental conditions, type of host* the life oycle of the parasite, etc. The plains do not have great changes in temperature whloh probably contributes to a continuing infection of amphibisns. One generation might overlap with another generation. During the spring In northern Taiwan, the noise of the peepers is almost deafening. The breeding season is in April and tadpoles could be picked up almost anywhere. Preparation of Specimens The wonts were carefully removed from the amphibian hosts by dividing the alimentary tract into sections, and by placing each part In a separate petri dish. The sections were split longitudinally under a dissecting microscope and carefully examined for worms. The acantho- oephalans found were placed In distilled water to relax them and to bring about the evaglnation of the proboscis. Sometimes this Involved leaving them In the refrigerator overnight. The specimens were then fixed In AFA (alcohol-formalin-acetie acid) after which they were stored In 70$ ethyl alcohol with glycerine (5$)* Each individual specimen was pricked to Insure penetration of the stains and other reagents. Some of those specimens given to the author had already been stained with iron hematoxylin, carmine, etc,, but generally Grenaoher's borax-carmine has been used. For the staining procedure, see Appendlx.23. In one instance, some of the female worms were opened Immediately with the pseudoooelomlo contents of the worms smeared directly on a clean slide with saline added and photographs taken of the eggs, A few of these slides were preserved In inseet media. Morphological Study Ratio; of males to females. The specimens taken fram Bufo num bered 1,072 of which 563 were females and 509 were males, Rana harbored a total of 277 Individuals, 153 of which were females and 124 males. Of th e p a ra site s in Bufo. 52$ were fem ales, w hile those In Rana showed a slightly higher percentile, at 55. Characteristics measured. The following characteristics were analysed 1 1) body sise, including length and width, 2) the proboscis, which encompasses the size, length, and width and also a study of the armature Including number, arrangement, and size of the hooks, 3) the proboscis receptacle, which included a comparison in size with the lemnisoi, 4) the lemnisoi - a study of the sise relative to eaoh other was made, 5) the eggs, 6) the cement glands and testes, and 7) th e length of the genitalia, All hooks counts Involved at least two complete counts of the rows of hooks around the proboscis. The only specimens included were those Individuals on which the count was clear. Measurements were made on hooks that could be studied In full profile viewj these measurements Included those In the basal, middle, and anterior portions of the proboscis but only the msariMOTi measurements were Included In the sta tistica l study. Mature embryos were measured In the body cavity of the stained mounts. A Study in Variation Because of the tremendous amount of variation present for eaoh morphological characteristic studied, a statistical analysis of variation was undertaken. On* location with one Individual host was used as a base line In this study of variability. Because of the large number of parasites present in this one host animal, this mas possible, This was farther developed to use similar hosts bat keeping the location the same. These were compared and contrasted against those from like hosts bat from different locations. Finally, other hosts as well as other loca tions were used. As far as collected material permitted, there are some observations as to variations which accompanied locales and time of year c o llec ted . Each characteristic was studied individuallyj histograms, graphs, and charts were utilised to demonstrate areas of difference and similar ity, Finally a discriminant analysis of morphological variation was carried out. The mean, variance, and standard deviation were machine- computed for all individuals and for the several characteristics men tioned above. Other statistics computed were Coefficient of Variation, Coefficient of Difference, Pearson's Correlation Coefficient, and Point Bi-serial Correlation where applicable, A vested Analysis of Variance was calculated for several sets of data, and ”t" tests were made to evaluate possible sim ilarities. Four other statistical techniques were employed in an attempt to establish measurement groupings that aould be inferred to represent different species. These techniques we ret Two- Variable Scatter H ots, Cluster Analysis, Multi-variate Classification Technique, and Principal Component Analysis. Hr, F, F, Hlddleswart helped In the programming and ran the raw data through the computer at Dupont in Wilmington, Delaware, He also gave valuable aid in the analysis of the data sheets. SECTION IV OBSERVATIONS AND RESULTS Body Size - Length and Width Comparison of males and females - length The male specimens of Aeanthooephala had a much more limited size range (2.96 - 11.1 mm) than the females, They also shoved a greater tendency to group around one mode, which in this oase was 5*0 to 5*9 mm. The concentration in this made was 32$ of the total. In con trast, the range of the females was much broader ( 4,08 - 22,8 mm), while the highest area of concentration was found In the 10 mm bracket, which accounted for 16$ of the to tal number. The 8 mm and 9 me classes were quite olose to the 10 mm group, with each containing approximately lh$ of the total. These variances reflect the sexual dimorphism of the species. (See Table II, page 31.) When comparing the males recovered from Bufo melanostiotus with those from B, bankorensis. 33$ of the former were grouped in the 5 nm column, while the latter had 20,6$ in the 9 mm column. Hales recovered from Rana were concentrated in both the h (28$) and $ mm (27$) groups. Males from Rana tended to be smaller, while those from B, bankorensis. while fewer in number, tended to be the largest. Table 1. Comparison of body length by classes from various hosts. Male and female. Size in mm length Male Hoat # 2 3 4 5 6 7 8 9 10 11 £, aelanoatictoiB 316 6 49 108 70 53 20 7 3 B. bankorensis 34 2 2 6 6 7 5 3 2 1 Rana 96 1 9 28 27 20 8 3 Total 446 1 17 79 141 96 68 28 10 5 1 Feaale Host # 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 B. melanostictua 337 1 4 18 22 53 49 53 34 18 12 12 12 4 5 1 2 1 2 1 C'v i—i r\ B» bankorensis 23 1 1 2 4 3 1 2 1 1 Rana 96 2 4 8 5 11 15 17 15 12 7 1 1 T otal 458 2 6 26 27 65 66 73 *8 33 24 15 14 7 5 2 3 1 2 1 Table II, Comparison of Body Size* £ Average Range LENGTH Females B. melanostletns 336 11.03 4. 92-22,8 B. bankorensis 22 15.06 13.53-19.38 Rana 92 10.OM’ 4.08-16,72 Hales B, a elan o stlo tn s 316 6.3 3.1*4 - 10.6 B, bankorensis 37 6.82 8.13-11.1 Rana 96 5 .214- 2.96-8.133 WIDTH Females B, m elanostletns 336 1.12 0.48-2.2 B. bankorensis 25 1.25 0,84-2.1 Rana 89 1.03 0,44—1,8 Hales B. melanostletns 316 0.89 0.44-1.5 B, bankorensis 33 1.1 0.64-1.64 Rima 94- 0.85 0.40-1.4 * All measurements in mm. Hales recovered from B. bankorensis had a mooh narrower range, in fast less than half that of the parasites reoovered from the other two host groups. The fem ales reoovered from B. m elanostiotus showed the widest range covering 17.88 mmt those from Rana had a range of 12,64 mm, while the females from B. bankorensis had the narrowest range of 5.8 mm. Length of specimens from one location - Tounan The fem ales, when a l l recovered from one sin g le h o st, showed a slightly more limited range as compared to that of the pooled specimens from the same location. This difference was more pronounced among the females than the males. The largest parasites were recovered from those hosts which housed fewer total parasites. One can see a tendency for a higher mean sise when fewer parasites are found, (See diagram, page 33 and Appendix 11I I ,) All of these toads were caught in one collecting trip and later sacrificed at the same time. Of the worms removed from one individual host (host number 1), 24# were 8 mm in length, while 25# were 10 mm long. They ranged in sise from 5*85 - 17.18 mm. In the pooled collection of females (host numbers 3 - 8), 14# measured 9 mm, 15$ were 10 mm, and 17# measured 11 mm. The sise range was from 7.80 - 22,65 mm. The largest me was from a host which contained a total of 9 worms. The smallest one was from a host which housed 43 parasites. S ix ty -th ree male specimens removed from one sin g le h o st showed a 5 mm mode which contained slightly over one-half of the total number of 3 3 . TABLE I I I Range and mean of body length of Tounan specimens grouped by Individual hosts. Female 25 20 15 10 Host #13^5 6 7 8 3-8 Parasite# (102) (3) (5) (3) (23) (16) (if 9) (if9) Male 15 10 ______I Host # X 5 6 7 8 3-8 P a ra site # (102) effect, Crompton and Harrison, 1965, observed the effect of large num bers of Polymerphus minutes in wild fowl. The females did not tend to concentrate in any one sise group but spread out over the whole range. When a ll the specimens from Tounan were combined, $ of the males were concentrated in one mode (5 mm). The females were most numerous around th e 10 mm mode. There would appear to be a larger concentration around one mode the larger the number of speoimens oolleoted. This is very apparent among the males. As both the time of collection, the place of collection, and the host species were the same, the differences demonstrated should be attributable to the number present within the host. Another factor could be the length of time over which infection was acquired. Combining all the specimens collected from Tounan would tend to eliminate the effects of overcrowding found in the one host which contained such an overload of parasites. Observations on the length (measurements are given in Appendices 7-11) 1, Males tended to oonoentrate in greater numbers around a single norm whereas females showed a greater tendency to spread out over a larger area. If all the females had been mature gravid females, there would probably exist this state general concentration around one norm. 2. Hales obtained from Rana tended to be smaller than those from Bufo. The range among these males was also much narrower. The more speoimens there were, the wider the ranges tended to be, 3. Female specimens from Rana showed a tendency for greater con centration In one mode, than did those obtained from B, melanostletns. h, Hales oolleoted from B, melanostletns In Tounan were medium in siz e . 5. In the specimens obtained from one location, the females showed a larger percentage concentration in one size mode* this reflects greater uniformity as the variables are minimized, 6. The females found in Rana showed a narrower range. R. Hmnorohis from the north possessed females of a greater size range than did speoimens from the south. The females from the south showed a greater tendency to cluster around one mean, which was larger than the majority of those from the north, 7, The largest females were found In hosts with fewer worms t a t least there was a trend In this direction. When the numbers of parasites present were more nearly equal, the range In size was comparatively closer. This can be demonstrated less spectacularly In the males than In the females, 8, Females from Grass Mountain were on the average larger} their range startllngly higher, 9* In both male and female speoimens, those from B, bankorensis were on the average larger than those from either B, melanostlctus or Rana. 10, For the most part, southern forms tended to be smaller, 3 6 . 11. Parasites from R. llmnorchls had a range In both sexes which was greater than that for any of the other of species of Rana. covering 11,8^ mi In the females and h.97 in the males, R, longlorns females had a range of 6,20 mm with only two specimens, while the males from the same species had only a 0.^4 mm range. Ten male speoimens from R, latoochl had a range of 2,52 mm, while the females from the same species had a range of “m* 12. Of all the specimens collected from Rana. those from R, limnoroharls housed the largest females and the smallest males, 13. A Coefficient of Difference of ,029 was found when 10 specimens from R, latouohi were compared with 63 speoimens from R, limnoroharls. The C,D, of h parasites from R, tigrina and 75 from R, Hgmoroharls was , 065. This reaches th e 73$ of joint non-overlap for subspecies. This demonstrates a within group variation In parasite size. Width (Measurements are given in Appendices 13*15) The w idth showed a g re a te r uniform ity among the d iffe re n t groups, A lack of sim ilarity was readily evident when Immature females were present, The worms from Tounan contained many immature forms, even though the total range was wide. The males from one single host in Tounan were equal In width to those from the pooled collection from the same place, Width - oemnarlson by species'11 # females * stales B. m elanostletns Tounan #1 102 .8 -1 .4 63 .69-1.35 Tounan 3 -8 66 .6-1.35 49 .69-1.38 North 181 .4 -2 ,2 181 .4-1.48 South 1 1.2 5 .7 6 -. 91 T otal .4-2,2 .48-2,2 B. bankorensis 25 ,844-2.1 33 .64-1.64 Bans .4 -1 .8 .4 -1 .7 ♦all measurements In mm The specimens from B, melenestletus mere slightly wider than those from Ryia. The Individuals from the north were for the most part wider. The pooled collection of speoimens interestingly enough started at the narrower end of the range, where there were fewer individuals present and conversely the range leaned toward the right with more speoimens that were wider, A large percentage was found on the wider end. There was a tendency to group around a single norm In the pooled collection of speoimens from Tounan, The w idth did n o t show th e range of v a ria tio n that was found In the length. When there was a larger number of speoi mens present, there was a smoother distribution. The range In widths of specimens from B. bankorensis was smaller than the ranges of widths from the other two hostsj that is, the minimum width of these speoimens was larger and the maximum width smaller than the corresponding maxima and minima from th e o th er h o sts. D iscussion It Is generally acknowledged that sise is not a useful specific criterion. To the casual observer, the degree of body contraction causes a tremendous sise differential, even In one individual specimen, Haley (1962) reviewed some factors In sise difference Including such artificial 38. factors as bias in the selection of speoimens, the size of the sarnie, and the processing and staining techniques. The size within a single host species may be influenced by the host - its age and diet. Dogiel e t a l , , 1961, showed the e ff e c t of age and the number o f p a ra site s in a given fish population. The number of worms present, overcrowding, and even previous exposure to the parasite, Immunity or the presence of another type of parasite, and competition are all factors which may cause variation. Using one location as a reference point reduces environmental variation. Specimens from one individual host face less variation due to Internal environmental ohange, but conversely they are affected by overcrowding, competition for attachment sites, and available food. Actually the smallest males from Tounan were found in a toad, B, melanoatlotus. which harbored 43 parasites, of mhioh 16 were males. The overall range for these parasites was 4.02-5.76 mm. The lower lim it of this size range contained a specimen smaller than the smallest one (4,38 mm) obtained from the one host which harbored 63 male worms. The smallest female observed was not recovered from a host in Tounan, but it was a 3.44 mm speolmen found in B, melanostiotus from the north. In the pooled collection from Tounan, the smallest male was 4,02 mm. The smallest male from host #1 measured 5.85 mm. The other end of the range showed the opposite to be true, with the speoimens from one single host measuring 17. l6 mm, and worms from the pooled collection measured 22.65 mm. One speolmen from the north outdistanced this with 22.8 mm. Thus specimens from B. melanostiotus from Tounan were well within the range of wonts obtained from B, melanostiotus from other places. Thus similar environmental conditions out dotm the total range In both 8exesa By pooling all the spoolmens from Tounan# a fair picture of overall range In size Is obtained with the environmental variations out to a minimum. Among the female aoanthooephalans from Far Eastern amphibians recorded in the literature, the smallest worm recorded was A, elongatua. which measured 4.1 mm In length (this was an Immature specimen) j the largest speolmen was A, bufowi«. which was 25 mm. The narrowest parasite was A. nanus which measured 0.7 mm, while A. luoldus had a length of 2.0 mm. The smallest female speoimens observed In the present study were obtained from Rana. measuring 4,08 mm., and the largest from B. melanos- tlotus, measuring 22.8 mm. Rana harbored the narrowest worn whioh was 0,44 mm wide, while the largest came from B, melanostiotus with a 2,2 mm w idth. When reviewing Aoanthooephalans from other parts of the world, the smallest males were A, faloatus. whioh measure 2.02 mm, while the largest, A, ranae. measured 12 mm. A, Aoutulua. was the narrowest, measuring 0.4 mm, while A, bufonis was the widest at 1.5 mm (from Russia). The overall number of measurements for width found In the literature were rather limited. Among the females the smallest was A. faloatus at 2.44 mm long and the longest was A. ranae at 60 mm. The width varied from 0,46 mm in A* f*l°atus to 1,9 mm for both A, kabnlensis and A, oauoasious. I t would seem that the greater the number of speoimens stfcdied, the wider is the observed range. This Is especially noticeable among the females. The more speoimens observed, the more ohanae there is of having the sample more realistically refleot the size of the whole population. Van Cleave (1952) noted a correlation between the size of the fish host and the type of parasite present In the larger suckers which feed primarily on snails and not on ostraoods, Grabda-Kasubska (1962) noted that the slso of the female was greatly influenced by the species of th e host. Studies have been made on hosts which harbor multiple infections of parasites* e.g ., Acanthocephalan and Cestodes (Holmes* 1961*, Dunn, 1963* Chubb et al, 196*0, Crowding effects are reflected by the smaller sise of the parasite, A correlation between the sise of the parasite and the host can be observed by a review of the literature, Male specimens of A, lucidus obtained from Rana vary from - 16.8 mmi from Bufo. which harbored A, bufonis. the length varied from ~ 9 mm* while those from other amphibians* e.g.* A, faloatus. varied from 2.12 - 6,8 mm. The females from Rana showed A, kabulensis as the sm a llest, measuring 6,17 mm, and the largest was A, ranae at 60 mm. A, bufonis from Bufo measured ^.1 - 26 mm, while other amphibians contained A, faloatus with both the smallest (2,*4- mm) and the largest (13 mm) speoimens. The body length of speoimens from Urodeles are generally smaller in both sexes than those Isolated from Anura; their ranges are also much narrower. In fact, the largest female specimen of any s pea les was one of A, faloatus. which only measured 13 mm for body length. Although the lines of demarcation are somewhat overlapping* the terrestrial Anura tend to be smaller than aquatic Anura, Females of A. bufonis. however, run counter to this observation among speoimens fro* the Far East where they have twice as large marl mum measurements than do any other species from toads. A, ranae is well known for its large sise. The exact opposite is true for speoimens from Taiwan, where the parasites from frogs tended to be smaller and narrower than specimens obtained from toads. Goto and Never (1949) concluded that A, ranae did not reach the same large size In B. bufo as It did in R, temporaria. It is obvious that size variation is definitely associated not only with the maturity of the worm but also with environmental conditions, location, numbers present, and the host. Perhaps it would be well to agree with Moore (1962) in his suggestion that size can only be con sidered in a general way* Proboscis Length and Width* Range and mean from one location - Tounan The specimens from number one were a l l obtained from one h o st j those numbered 3 - 8 include five different toads all belonging to the same species. Detailed records of proboscis length and width measure ments are in Appendices 16 - 19. Table IV. Length and Width of the Proboscis of Parasites from One Location Length of the Proboscis Female .300 .400 .500 Mean Male .300 .400 . 500 Mean Range #1 (101) 9 73 19 7*57 .330-. 597 1537 31 22 7155 .309-.494 $ 9 72 19 58 42 3-8 (70) 6 42 22 .470 . 340-.587 (49) 22 27 . 390 . 309-.450 $ 9 60 31 45 55 1-8 (171)15 115 41 .462 . 330-.597 (L02) 53 49 . 389 .309-.494 % 9 6? 24 52 48 Width of the Proboscis Female .200 . 300 .400 Mean Range Male .100 . 200 . 300 Mean Range fl (101) 39 63” 2 ."3 1 2 ”24777433 (53) 1 4^ 3~ .252 .196-7340 $ 39 59 2 3 92 6 3-8 (70) 30 39 1 .314 . 206-.546 (49) 2 42 5 .258 . 196-.360 $ 43 56 1 4 86 10 1-8(171) 69 99 2 . 312, 206- . 546 (102) 3 91 8 .255 .196-. 360 % 40 59 1 3 89 8 *A 11 measurements in mm, except when otherwise stated. TABLE V The range and mean of the probosois length of female specimens from Tounan, grouped by host. ,600 ,500 ,1*00 .300 .200 Hostj # females fr - 5 #5-3 #8-16 #7-17 #6-17 # 1 - 1 0 1 Total # present (?) (7) (31) (36) (1+3) (176) The probosois measurements for worms from host number one contained the longest as veil as the shortest examples In both sexes. This did not hold for the width, whloh shoved the opposite effect. It naturally follows that in the pooled collection, the individuals would tend to be larger and the arithmetic mean would thus be larger. The range in fe males was greater than that found in stales. Females showed a greater concentration around one mean specifically in the individuals from one single host. The males showed a difference in the two groups which averaged out to make for an equal amount in the .300 and ,h00 mm groups. The greatest uniformity In width was seen among the males in the ,200 mm bracket, which contained 92$ of the total, Among the pooled collection, 86$ fell in this group. The females tended to be larger with 59$ of the specimens from host number one in the ,300 mm class and 39$ in the .200 mm group, (See table above on the length of the probosois.) The pooled collection showed the same general tendency, w ith 5^$ in th e ,300 mm bracket and kj$ in the ,200 mm division. The females thus demonstrated a greater concentration around one norm, while the males showed an even greater consistency in width when taken from one location. The hosts containing the greater number of specimens tended to have a greater range in some eases, but a lower mean. This tendency can be noted from the females collected from one looatlon. Comparison of three hosts* B, melanostlotus. B, bankorenala. and Sana There was a higher concentration around one elass in the males than in the females, This was true for all three host groups. Parasites from B. bankorensis had the narrowest length range in both sexes, while spec lmensfran Rana had the widest range. It is noteworthy that the highest concentration of nales ocourred in the ,300 mm class, except for specimens from the pooled collection from Tounon, where the largest per centage was found In the .400 mm class. When all the specimens from Tounan were combined, however, they were also in the .300 mm mode. The mode for all the females was in the ,400 mm class. The w idth o f the probosois among th e females showed the modal c la ss to be ,300 mm. The mode for the males was in the ,200 mm group, with an even greater concentration of specimens manifested. In both sexes, the specimens from Tounan had the widest probosois range while Rana had the narrowest range for this characteristic. Table VI, Comparison by Host of the Proboscis Length Females .200 .300 .400 .600 Mean Range B, m elanoatiotus (79) 26 36 16 1 .441 .323-,616 33 45 20 1 B, bankorensls (14) 4 9 1 .432 .346-.505 28,6 64.3 7.1 Tounan 15 115 41 1 ,462 .330-.597 * 9 67 24 1 Rana 1 10 32 8 1 .438 .262-,616 i 2 20 62 2 Males B, m elanostictus " (100) 8 73 19 .397 ,231-,462 $ 8 73 19 B. Bankorensls (18) 12 6 ,376 .308-.424 % 67 33 Tounan (102) 53 49 .389 .309-.494 $ 52 48 Rana (33) 23 9 1 .370 .300-.515 70 27 3 4 5 TABLE V II. Comparison by host of the probosois width. Female .100 .200 .300 .400 ,500 Mean Range B. itelanost ictus ...... T 7 '« " ' 2 28 45 4 .312 .169-462 * 3 35 57 5 B. bankorensls (20) 4 14 2 .326 , 231-,474 i 20 70 10 Tounan (171) 69 99 2 1 .312 . 206-,546 $ 40 58 1.2 .8 Rana (52) 1 14 30 1 .304 . 162-.408 — i 2 27 69 2 Hale .100 .200 .300 .400 Mean Range B. m elanostictus — nwj— 5 77 18 .289 .162-3.46 7> 5 77 18 B. bankorensls & ? r 15 11 3 .295 ,200-,412 t 52 38 10 Tonnan (102) 3 91 8 .255 . 196-.360 i 3 89 8 Rana 23 10 .277 .216-.330 ¥ * > 70 30 Discussion - Proboscis Slaa Sexual dimorphism in the slaa of the probosois has been noted by Ward and Nelson (1967) In their work with Moniliformis. The males were aignifioantly smaller than the females* although the mode for both sexes was the same, Bullook (1962) reported that the probosois size in A, .lacksoni showed sexual dimorphism, Host-related differences can be noted in the average values of the probosois length* where Tounan females had the largest average aize (.462) and also the largest man in the males (,389). In oontrast* the width of the probosois of males from Tounan had the lowest average (.255)* while males from B, bankorensls had the widest (, 30*0, The females from B, bankorensls were also the widest (. 30*+), Overcrowding is demonstrated in both length and width for the pooled collection of parasites from Tounan* which were on the average bigger than those obtained from one individual host. The largest percent concentra tion among all the males in width was found in Tounan with 89$ of 102 worms in the ,200 mm braoket. Sexual dimorphism was observed in the present study in both the length and width of the probosois. The males were 0,3 mm* while the females were 0.*+ mm. A, luoidua shows this size differential* where the females are 0.*+-0.6 x 2,5-0.*+8* and the males are 0,3-0,5 x 0,15-0,32. Yamaguti (1953) reported a aize variation in A, bufonls among the fe males as 0,28-0.70 x 0,22-0,*+3* and the males as 0,35-0.*+3 x 0,25-0,*+7. Petroohenko (1953) records the proboscis dimensions for the same para site asi males 0,63 x 0,28-0.33 and the females as 0,6*+ x 0.*+l, thus limiting the range and practically eliminating any sexual dimorphism in the length. A survey of aoanthocephalans from amphibians In other areas o f the world showed th e same d ifferen e e between the sexes. Specimens from B. aelanoatlotus oolleoted In the Taipei area, out side of Namru 2, had a higher mean for the probosois length in both sexes, Conversely, the width was narrower for both sexes from the same group. Females oolleoted on Grass Mountain had a probosois length shorter in the females but wider in the males. Among the females from R, llmnorohls. the southern forms had the largest mean (.****-1). This was also true for the probosois width (.3^9), Thus location differences contribute to variation. Variation in the Maximum Size of the Probosois Hooks Sexual Dimorphism A collation of the maximum measurements of parasites harbored in toads when pooled readily demonstrates sexual dimorphism. In the graph, the total number of Individuals was plotted against the maximum size in microns. There were 122 males ranging in size from 65-7** microns, while there were 106 females exhibiting a range 10 microns higher. Host-Associated Variation Bufo oolleoted from one location. Tounan. The males oolleoted from one single host in Tounan had the largest concentration of maximum hook size in the 75 - 79 bracket, while the female worms from the pooled collection had the largest number in the 70-7**' class. However, the overall average was greater for specimens from the pooled collection (75 mlorons), while the male worms from the single host wore slightly smaller (73#6 microns). 4 8 . TABLE VHI Sexual dimorphism demonstrated in the maximum size of probosois hooks of specimens from Bufo. 60 I 50 40 30 20 10 I III 50-54 55-59 60-64 65-69 70-74 75-79 80-84 85-59 90-94 95-99 100-104 105 Length o f Rooks in Microns o f Bufo specimens. Female Male Among the females, both groups had the highest concentration In the 80 - 8b bracket, However, the female worms from number one averaged a slightly larger slse (85 microns) than did the Individuals from the pooled collection, whioh had a mean of 83.6 microns. The pooled collection of males had both the smallest and largest hooks. The largest hooks among the females came from host number one. Rana limnooharis. When specimens from R. llmnocharis collected from the north and oentral areas were compared with a pooled collection of specimens recovered from a number of different species of Rana from the same general areas, the minimum hook slse of the latter tended to be greater, the former tended to lack worms which had smaller maximum slse hooks In both males and females. The males obtained from R. limnooharis from the northern area showed the highest concentration In the 70 - 7** micron class, while those from the south were In the 75 79 micron group. The females, on the other hand, from the oentral area showed the highest category to be 70 - ?4 micron, while those from the north were grouped In the 7 5 - 7 9 micron mode. Thus, females from the north tended to be larger, whereas males from the south tended to have larger maximum hook slse. When all the specimens from the pooled collection were ob served, the southern groups had a smaller maximum hook slse In the males. The pooled females from the north, however, had a muoh shorter range. This pooled group had a number of different Rana speoles serving as hosts, so it is to be expected that a greater variation would be present. Several host species compared. It Is noteworthy that the specimens from B, bankorensls had the largest maximum hook: slse for both sexes. Female worms from Rana had an average maximum hook slse of 81.4 microns, TABLE IX. Host associated variation of maximum hook size. (I'easurements in microns) 11 Host Sex 50- 55- 60- 65- 70- 75- 80- 85- 90- 95- 100- 105- Average Range 54 59 64 69 74 79 84 89 94 99 104 — #1 o’ 51 1 2 10 9 14 13 2 73-6 62-87 10 Op 2 4 20 18 27 13 4 ? 37 4 10 14 31 13 12 2 1 85 69-101 100# 5 11 16 36 15 14 2 1 CO n 1 48 1 1 1 8 13 9 11 3 1 75 51-92 100# 2 2 2 16 26 18 22 6 2 6 8 14 83 .6 69-87 r 63 3 13 19 100# 5 10 21 30 13 22 B. bankorensis o* 26 2 5 6 3 3 2 5 73 61-94 100# 8 15 23 12 12 8 15 16 1 0 2 2 4 86 61-114 9T 3 5 100# 6 13 19 31 13 19 B. melanostictus 82 2 10 9 32 18 6 5 74 52-96 100# 2 12 11 39 22 7 6 65-110 9 48 3 10 15 2 12 2 4 82 100# 6 21 31 4 25 4 8 Rana 47 2 13 10 10 11 1 73 60-89 100# 4 28 21 21 24 2 Q 47 1 1 10 10 10 10 3 1 1 81.4 61-120 T 100# 2 2 21 21 21 21 4 2 2 8 yet they had the largest overall range in hook slse. When analysing the females i Tounan, parasites from one individual host, had 36$ that were in the 80-84- bracket, while the pooled oolleotion from the same area had 30$ in th e same mode. Hale speoimens from B, bankorensls had 23$ In the 70 - 74 elass bat also had 15$ in the 90 - 9^ group. The female worms from B, bankorensls had equivalent amounts (19$) in two separate classes i the 75 - 79 class and the 110 - 115 group. Location Differences In a study of B. melanostiotus from the northern area, the data from three collecting sites were selected. Namru 2 was used as a base and guideline for the north» a good deal of uniformity was evident in both males and females as far as the maximum hook else was concerned, with 50$ or more of the speoimens consolidated In one group. The second site covered a number of locations in and around Taipei which were oombinod. The range among the males was more scattered with a bias toward a smaller maximum hook slse in both saxes. The third location was Grass Mountain, which is approximately 1,000 feet higher and 10 degrees oooler than Taipei, Here there was demonstrated a definite shift towards a larger hook slse for both sexes. This is noticeable, especially in the males, whose hook measurements commenced at 70 microns in contrast with 50 microns for Taipei and environs. The presence, among the females, of hook slse of over 100 microns makes this location difference very striking, D isoussion - Maximum Hook S ite Nelson and Ward (1967) *&d Bullock (1963) both reported sexual di morphism in the length of the longest hooks for M awilifwia and A. jacksani. respectively, This is also apparent In the present study. References in the literature give a vide range in hook slse for A. luoidus and A, b u fo n ls. the l a t t e r range being 60 - 121 m icrons, and the range of the former 60 - 155 microns, Fetroohenko gave 70 - microns for male hooks and gave the female range as somewhat larger for A. bufonls. The range of A, nanus is much lower at 44 - 69 microns. Aside from those already mentioned, no other Acanthooaphalus from am phibians has bean reported with hooks larger than 90 microns j however, this figure was reached by A. kabulensis. which has a range of 70 - 90 microns. The other species are all smaller. Many times maximum hook size is not to be found in the literature. Two Far Eastern species, A. bufonls and A. luoidus, have the greatest range in maximum hook size - more than any other Aoanthooaphelus from amphibians. Host-related differences in hook size have been reported by Grabda- Kazubska (1962) in her work with A. falcatua. This difference is seen in specimens obtained from B. bankorensls. which had the largest hook averages in males (78)* as well as in females (86), On the opposite end, specimens from Rana had the smallest average for the maximum hook size among the males (73) as well as for the females (81,h). Geographic distribution was not found to affeet the size of the hooks in Bullock's study of A. jaoksoni, but he mentioned that a study of Illiosentis furoatus showed a larger size of hooks in speoimens obtained from Woods Hole in contrast to speoimens from Texas and Loui siana. This location difference may be demonstrated by specimens ob tain ed from R. H m m Iu iHii frost the north, which tended to have a larger hook size (75 * 79) among the females, whereas this sex from the south tended to have a smaller maximum hook size (70 - 7*0, On the contrary, males from the south tended to be larger (75 - 79)» while males from the north tended to be smaller (70 - 74). Combining several locations In and around Taipei, the range beoame much more scattered with a bias toward a smaller maximum hook slse in both sexes when more looations were contrasted to one location, Namru 2, Speoimens from Grass Mountain exhibited an even more dramatic change with a tendency toward larger hooks, even to going over 100 microns among the females. This was also shown in relation to B. bankorensls specimens from All Shan, where the ranges were even higher for both sexes. The basal hooks in some specimens were a ll smaller than the rest of the hooks. In other parasites, the basal hooks in some rows were much sh o rte r than the b asal hooks in the row adjoining . Van Cleave (1915) notes a like factor in his study North American specimens of A, ranae. Hook Arrangement V ariatio n in th e Number of L ongitudinal Rows of Hooks The range. The number of longitudinal rows of hooks among the females ranged from 1*4- to 24 rows and 13 to 21 rows in the males. The range covered by individuals secured from one single host was 16 - 23 for the females and 16 - 21 for the males. This range was extended by one, to 15, on the lower side when the speoimens from the pooled collec tion were studied for the males. The overall pooling of the specimens from one location did not extend the range for the females, B. melanoa- tlotus contained one specimen with a oount of 24 rows of longitudinal hooks, which was the largest number of rows found among a ll the females. One female speoimen from Rana had a oount of 14 rows of hooks. The speoimens from Rana had a range which started at a lower point but did & Graphic arrangement of the number of longitudinal rows of hooks arranged by the several host groups - female. B. melanostictus #1 . melanostictus #3-- 3_._ melanostictus (all others) Rana______B. bankorensis 20 10 15 15 1? IB 19 2p 21 22 23 2k n Graphic arrangement of the number of longitudinal rows of hooks in arranged by the several host groups - male. B. melanostictus #1 B. melanostictus #3-8 B. melanostictus (all others) Rana B. bankorensis hO 30 20 1 10 13 14 15 16 17 18 19 20 21 not go as high. Whan all tha spaoloans were considered togethar, tha largest ooncantratlon of Individuals among tha famalaa mas 19 or 20 and for tha malas It was 18, with 19 naxt abundant. (Saa Chart 9 or Appendix 20.) Host-associated variability. The worms oolleoted from toads in Tounan has a marked consistency with an almost equal representation between 19 and 20 rows of longitudinal rows of hooks among the females. The range of the males was narrower. The females recovered from one single host showed tha greatest conoentration around one mean, B, m elanostictus showed a wider spread over 18, 19, and 20 rows of lon g i tudinal rows of hooks. This host had more looations represented, as well as a larger number of speoimens. In fact, there was a rather ob vious low concentration over a wide range evident here. The males demonstrated a similar wideness of spread, covering 17, 18, and 19 rows. Rana showed no great concentration in any one groupi among the females 19 rows had the highest oount, followed closely by 18. The actual sweep of the individuals from Rana. among the males, was quite close to that encompassed by worms from Tounan, It would seem that different hosts do not drastically influence the number of longitudinal rows of hooks. Sexual dimorphism was quite apparent, except in Rana. Female worms from Rana showed a higher ooncentratlon at 19 rows, and the males at 18, although many males a lso had 19 rows. Location differences. B. melanostictus from three locations in the north showed that the Individuals from one location (Hamru 2) exhibited an inclination to be unlmodal. In this case both sexes had 18 longitu dinal rows of hooks. The worms from Taipei and suburbs exhibited a 15 - 2k row oount among the females and a sweep of 13 to 20 in the males. 5 7 The so ope of this range was made nore vivid because of lack of concen tration at any one norm. The speoimens from Grass Mountain evidenced sexual dimorphism, with a range among the females of 16 to 22 and for the males 16 to 19. An increase in the number of locations which were pooled evidenoed a marked increase in overall diversification. FEMALES MALES L ongitudinal row L ongitudinal row Location of hooks hooks Kamru 2 T aipei 19 (253C) ^ Grass Mountain (6) 20 (32*) ^ (11) 16 (2m % 17 (33*) T otal (53) 18 (3*#), 20 (21# ) ( 93) 1? (2656) , 18 (2756) When considering parasites from Rana from a ll three general areas, north, south, and oentral, there seemed to be an inclination toward greater amounts of variation and less concentrating around one class. The number of rows of hooks from both sexes from the north were uni- nominal, The oentral area encompassed a broader sweep, perhaps influ enced by the multlfariousness of the collection sites, with a tendency toward fewer rows of hooks. Specimens from the south leaned toward more longitudinal rows of hooks, at least among the females. Sexual dimorphism was almost lacking. FEMALES MALES Location # of rows of hooka hooka North 18 (27*), 19 (27*) C en tral 17-19 (each 18*) South 18 (4055), 21 (4055) T o tal 18 (21#), 1? (20)5), 20 (ldj5) Speoimens from Grass Mountain showed a tendency for fewer hooks per row as did the worms from All Shan which were recovered from B, banko- re n s is . Host differences do not apparently influence the number of hooks p er row. Sob* irregularities were noted such as hooka being completely miss ing, and one speoimen had two hooks which ware turned around with the thorns directed anteriorly. Variation in the Number of Hooka per Longitudinal Row Range. The number of hooks per row for the females varied from h to 7. The males varied from h/5 to 7* There was only one female speoi men recovered from B. bankorensls which had a count of h hooks per row. The speoimens from Tounan covered the whole sweep in both sexes. The males were marked by a greater uniformity as demonstrated by more con solidation around one norm. When considered as a unit, the preponderance of 6 hooks per row was evidenced among the females, while the dominant number among the males was 5 with 5/6 close behind. Host-related variability. Among the total speoimens oolleoted from Tounan, $ of the females had 6 hooks per row, while k6$ of the males had 5 hooks per row. B, melanostictus parasites had the greatest numbers clustering in the 6-hook mode among the females and showed a spread among the males between 5* 5/6, and 6 hooks per row. Female parasites from Rana showed a 50$ concentration at 6 hooks per row, and the males had kZ$ a t 5r w ith 5/6 a clo se runner-up. The hooks per row in the female specimens from Tounan, B. melanostictus. and Rana are almost identical - all showing the greatest concentration in 6 hooks per row. It would appear that the number of hooks per row was little influenced by different hosts, as shown in greater ooneentration around one class, but this might simply be a reflection of less total variables. Location differences. B, melanostictus frost three different loca tions in the north showed the greatest lack of uniformity in the 5 9 . Graphic arrangement of hooks per longitudinal row arranged by the several host groups - males. 70 l\ f \ I \ I \ 60 I \ I \ I \ I \ 50 40 30 20 10 .* 5/6 6 6/7 7 hooks per longitudinal row B. ao lan o stio tn a *1 B. a elan o stlg tn s r3 -o B. gelanoatlotus (except Tounan) ‘Rana ~B. bankorensls 60 t«* t v * < £ FKMAIES MALES Location f Hooks/Rows # ______Hooks/Rows______Namru 2 7W $ (34*) J m 5 (2^%), 5,6 (W), 6 (34*) Taipei (34) 5,6 (4o*), 6 (34*) (53) 5 (30*), 5,6 (28* ), 6 (26*) Grass (6) 4,5 (1/3), 5 U/3), (11) 4,5 (2?*), 5 (1036), 5,6 (45*) Mountain 6 (1/3) Total (52) 5,6 (34*), 6 (42*) (91) 5 (32*), 5.6 (32*), 6 (26*) individuals from tho Taipei area. The speoimens from the mountain shooed a tendeney for fewer hooks per row. This same tendency was seen In specimens from B, bankorensls from high elevations, Rana from three general areas. Speoimens from the northern area tended toward fewer hooks per row among the females. Among the speoimens from the north, the largest concentration in both males and females was 5/6 hooks per row. In the oentral area 6 hooks per row predominated and 5 was the number found 53* of the time among the males, FEMALES MALES Location # Hooka per Row # Hooks per Row North 7227 5 <32*), 5.6 (41*), 6 (2?*) 7W 5 (31*), 5,6 (50*), 6 (tjjb) Central (22) 5,6 (14*), 6 (72*) (17) 5 (5336), 5,6 (24*) South ( 2) 5,6 (50*), 6 (50*) ( 3) 5,6 ( 6?*) Total (46) 5,6 (29*), 6 (50*) (36) 5 (42*), 5,6 (39*) Combination of the Number of L ongitudinal Rows of Hooks and the Number of Hooks per Row Range and mean. When a ll the females were considered together, the bracket containing the most individuals was 19*6, with the next highest group 18i5/6» The males were also consolidated In two classes - 18*5/6 and 18 15. When speoimens from the different hosts were considered se p a ra te ly , the same uniform ity h eld . Various host speoies. The speoimens oolleoted from the pooled col lection in Tounan reveal the largest oonoentratlon of females at 19*6, as do the worms o o lleo ted from Rana. Speoimens from B, m elanostictus contained a larger number In the I 816 class. Worms from Rana oovered I6t5 to 2016, The malms from Tounan were in th e I8 i5 group, while parasites from B, melanoattctus were 17i5/6. The range of male worms from Rana was l6tk/5 to 18:5/6, Females from B. bankorensls ranged from 18:5/6 to 20*6, while the male range was from 17»^/5 to 20*6, Time of c o lle c tio n . During th e w inter months, p a ra site s from both Rana and Bufo had hook oounts which were essentially the same, Males from Rana were 18:5, while females were 19(6, Males from Bufo were 18:5 and females were 18,19, both with 5/6 hooks per row. During the spring months, worms from Bufo tended to turn up with more In the 17 oount. During th e summer, 18 rows were predominant in speoimens from Bufo. with 19 for worms from Rana. The fa ll months had fewer hooks from parasites from toads and a wide range was found In the parasites from the frogs. Actually there was not enough material to study the influence of the season, as there were other factors involved. The differences demonstrated were probably not so much due to the time of eollection as to physiological factors connected with temperature and development of the worms. Hook Arrangement - Discussion Some groups among the Acanthooaphala completely lack any evidences of variation in hook pattern, Lflhe (1912) was among the first to call attention to irregularities in pattern. Ward and Nelson (1967) reported from M«n^ n-farmta that females had more rows of proboscis hooks than males, but that this was not highly significant. The row range of Moniliformis varied between 11 - lh, with 12 the mode for both sexes, but the stales had 70,2$ with 12 rows, while the females had only 57. % w ith th e same number of rows, Variation in the number of longitudinal number of rows in the genua Aoanthooephalua is almost axiomatic, Van Cleave (19W) refers to this phenomena as being normal in members of th is genus. Most of the data recorded in the literature for the number of rows of hooks is given in one range covering both sexes, e.g., A, nanus has 10 - 14 rows, A, luoidus has 12 - 17 rows, and A. bufonia 1*4- - 20 rows, Petroehenko (1953) would lim it the number of rows of hooks for A, bufonls to 20, Grabda-Kasubska (1962), in her work with A, faloatus. reported that males had 11 - 15 (13) rows, and the females had 12 - 1? (1*0, thus showing sexual dimorphism. The females in this study had a range of 1** - 24 rows, with the m ajo rity of speoimens having e ith e r 19 o r 20 rows. The male range was from 13 - 21, with the largest number of worms having 18 or 19 rows. This difference between the sexes is quite apparent in speoimens from both Bufo hosts, but is looking in specimens from Rana. Differences in the host species did not seem to greatly influence the number of rows of hooks, Bullock reported that New Hampshire speoimens of A, jacksonl had, in contrast to Massachusetts speoimens, more hooks. Geographical influ ence can be demonstrated by speoimens from B, melanostictus from the north, Speoimens from Namru 2 exhibited a tendency to be unlmodal with h0$ of the females with 18 rows and 5&f> of th e females had the same num ber of rows. Specimens from the Taipei area had a wide range in the females (15 - 2*0 and the males (13 - 20), without any concentration in one group. Worms from Grass Mountain showed a definite sexual dimorphism with the males tending to have fewer rows of hooks than the rest of the northern speoimens. An increase in the number of loeatiens pooled evidenoed a marked increase in overall diversification. The various speoies of Acanthooephaiaa found in amphibians show a vide amplitude of overlap in hook counts from widely scattered looatlons. The females had a range of - 7, but the majority of specimens had 6 hooks per row. The males' range was *t/5 to 7, with the largest con centration of speoimens having either 5 or 5/6 , Sexual dimorphism in the number of hooks per row has been reported by Ward (1967)* Bullock (1963 ), and Grabda-Kasubska (1962), A. luoidus has a hook per row oount of k - 5 and occasionally 3, while A, bufonia had a range of 5 - 8. Petrochenko limits this to 6 - 7 hooks per row. Hooks po inting in random d ire c tio n s w ithout reference to the o rien tation of the normal hooks perhaps indicates that the hook pattern is subject to individual modification. The Lemnisoi Sise of the Lemnisoi Compared to the Slse of the Probosois Receptacle The lemnisoi manifested a prodigious amount of variation. (See Plate #1, page66 ). At times they were shorter than the proboscis receptacle, and in other speoimens they were more than double the length of the proboscis sac. In one instance, the lemnisoi measured almost four times longer than the proboscis sheath. Actually, they could measure anywhere between the two extremes just mentioned. The majority of the speoimens measured had lemnisoi which were slightly longer or up to one- half again as long as the receptacle, Bana and Bufo were very similar in this respect, Rana exhibited a greater uniformity in having more lemnisoi which were nearer to the slse of the probosois sae, Lemnisoi sise varia tion was greater in individuals from Bufo than in speoimens from Rana. (See Appendix 21,) 65. P la te # 1 Slse of the lemnisoi compared to the slse of the probosois receptacle All from one individual host animal // I \ 67. P late # 2 Atypical lemnisei: (1) lobed, (2) ^ lemnisei all of almost equal size. (3) one long and two short lemnisei. (4) two small lemnisei. (5) four lemniseii 2 large. 2 small. rfM i' Atypical lemnisei were seen in two specimens which had 2 largo and 2 small lemnisol, At least one vonn had one large and one small lemnisol. Some specimens ended up with three lemnisei with lengths almost equiva lent. There was even one worm with a bulb formed on one of the lemnisei. There were many specimens that were lobed basally. (See Plate #2, page 6?t Size of the Lemnisei in Comparison to Each Other When the two lemnisei were compared in size to each other, the males from Tounan showed the greatest distribution range i however, the females had the highest number of Individuals with lemnisei of equal size. Actually, the two lemnisol were quite near to each other in length, as is shown by th e f a c t th a t 5^f> of the females and 60$ of the males came with in the .90 - .99 group. (The nearer the figure is to 1.0, the nearer the lemnisei axe in length. This figure was obtained by dividing the smaller lemnisol by the larger. Details are given in Appendix 22.) Males from the pooled collection from Tounan showed a wide range of variation, but the range of the females was even greater. The males showed an even greater conformity to equivalence in size, actually about 70$. The greatest difference among all the females was found in the pooled collection from Tounan. B, melanostiotus showed a bias toward near equal size, the distribution being quite similar to the specimens from Tounan. The range was not as great. Among the males from Rana. there was the greatest concentration of equal length lemnisei. The parasites from Hana tended to demonstrate a greater difference between the length of the two lemnisei. The females showed the greatest range from B, melanostiotus. while the greatest range in the males was from Ran*. Of a ll the specimens examined, 11$ had lemnisei of equal length. It la Interesting that the greatest range in size oame from those specimens from one geographical location. It mould seem that the place does not greatly affect the length of the lemnisol. Lemnisol - Discussion As early as 1925* Southwell and Macfis reported that even in mature worms, the length of the lemnisol appeared to vary within rather wide lim its. They assumed this variation to be present In growing forms. They also suggested that the length varied with the age. Besides, the lemnisei length relative to the total length of the body showed variation correlated with the state of contraction of the worms. They listed the lemnisol among the somewhat unsatisfactory characteristics upon which to base classification, Harada (1935) considered the lemnisol to be an unreliable character i s t i c , Grabda-Kazubska (1962) in her work in Poland stated that the shorter lemnisol were only met with In very young specimens of A, ranae. She also reasoned that weakly formed lemnisol probably accounted for the absenoe of a neok in A, falcatus. Grabda added that A, faloatus had lemnisol normally shorter than the proboscis receptacle which were strongly fla t tened and adhered olosely to the receptacle, while those of A, ranae, for the most part, were longer than the proboscis receptacle and were projected away from i t . Bull oak (1963) made a study of variability In A, Jackson! indicating the presenoe of variability of the lemnisol, not in total size, but in the presenoe of lobes, the presenoe of irregular crenulated margins, and reductions in size and pointed out that in at least one specimen there was only one lemnisol, Hammond ( 1966), in his research on the function of the lemnisei, demonstrated that the fluid flows from the lemnisol to the proboscis wall only after the proboscis has become evaginated, This eliminates the possibility that the lemnisol aid in the evaglnatlon of the probos cis, He referred to Graybill (1902) as having reached a similar conclu sion, The passage of dye from the Intestine of the host to the lemnisei suggests that these organs are more than mere hydraulic reservoirs. Thus the state of evaglnatlon of the proboscis does not affect the size of the lemnisei. Schmidt and Kuntz (1966) considered the character of the lemnisei to be useful for toxemic purposes if oare Is taken in selecting specimens that are mature and typical. They also noted that the lengths of the lemnisol relative to each other and to the proboscis sheath are especial ly useful. When thinking of the large amount of variation present in amphibian lemnisei, one could ponder this statement. On the basis of difference of the length of the lemnisei compared to the proboscis sheath, Schmidt and Kuntz established a new species Plasiorfaynchus taiwanensis. Travasses (1920) recognized a genus Lueheia. characterised by the presenoe of k to 6 lem nisol, Werby (1938) erected a whole new genus, Furcata. on the basis of forked lemnisol. Van Cleave (19^2) presented a good review of a typical lemniscus, He mentioned the very common disparity in size of the two lemnisei and other abnormalities. 72. Length of the Proboscis Receptacle Specimens from Ran*, whether male or female, had the largest average size for the probosois receptacle. Those taken from one location were on the average smaller than those obtained from a variety of locations. Sexual dimorphism was evident. Table X, Length of the Proboscis Receptacle Source Female Average Range Male Average Range #1 101 562 361-937 57 4?6 319-639 3-8 65 531 227-876 50 /+6? 299-659 Bj. m elanostiotus 685 323- 2 ,0 2 48 669 231- 1 ,1 8 B. bankorensis 18 710 381-880 35 626 412-94? Rana 90 740 462-1.09 96 621 464-1,06 The la rg e s t speoimen from B. m elanostiotus was secured from Grass Mt, The probosois receptacle is a muscular organ which can easily con tract and expand. The amount of contraction is influenced by the fixing process. This is why specimens from one lot w ill tend to be alike as far as the length of this organ is concerned, but specimens fixed the very next day from a similar host may have a receptacle which differs widely. V ariatio n in Cement Glands Number of Cement Glands The normal number of cement glands for the genus Acanthooephalus is six. From the chart it is obvious that 14^6 of the males from B^ melanos- tictus and 22$ of the worms from Rana demonstrated deviations from this norm. The total count of oement glands varied from 4 up to 12, The specimens collected from one Individual host showed a very wide range of variation, even more so than the specimens from the pooled P la te # 3 Variation in the number of oement glands (1) Twelve oement glands, 2 groups obvious, host Rana (2) Eight oement glands (3) Eleven oement glands (*0 Twelve oement glands (5) Three oement glands (6) Seven oement glands Numbers 2 - 6 a l l from B, m elanostiotus collection from the same loaation. Hales from B, celanoatiotug showed the greatest consistency, with 92# of the total having 6 glands. The range for specimens from Rana was wider, with a resultant lack of con centration in the noxmal number and with the number of oement glands going up to 12, (See Plate 3.) Table XI. Variation in the Number of Cement Glands Source £ 4 1 6 1 8 2 12 #1 64 2 51 8 1 l 1 # 3 80 12* 1* l* 1* 3 - 8 52 1 5 43 3 # 2 10 82 6 B. m elanostictus 80 k Ik 1 1 * ...... 5 92* 1* i* B. bankorensls k5 1 1 40 2 1 * 2 2 89 5 2 Rana 83 7 k 65 6 1 £ 9 5 78 7 1 T o tal 196 1 11 168 12 2 i 1 B. melanostictus * 5 85* 6 1 * * There was more variation, in fewer specimens, from those collected a t Namru 2 than for those from many stir rounding areas where the variation was practically nil. Specimens collected from B. melanostictus in the central area showed no variation. Specimens collected from R. Ilmnor- o h aris in th e north showed a lack of v a ria tio n w ith only 6# w ith 5 oement glands, w hile th e same species from th e n o rth showed those w ith seven cement glands to be 17# of the to ta l. Form and Shape The oement gland shape manifestly varied from elongate, bunched, grouped with a 2,2,2 combination or with a 4,2, and seme groups defy 7 6 * P lato # 4 Variation in the form and arrangement of the oement glands All from one location j 1-4 all from one host animal Q 0 0 O description. As for the form, they could be called stubby, slender, plump, tubular, pear-shaped, compact, all of equal length, etc., depend ing on which specimen i s being observed a t the moment. P ra c tic a lly any descriptive term found in the literature about the oement glands could be used to describe some specimen in this group. (See Plate #4,) Seoause of the vide variation, the specimens collected from one individual host have been used for comparison to show this acute variety found in the shape. Cement Glands - Discussion Ltthe (1911)» Meyer (1932-33), Van Cleave (1936, 19W, and 19^9), and Golvan (I960 and 1962) have all used the shape, number, and structure of the cement glands in setting up taxonomic differences, Thap&r (1927), however, considered the number and shape of cement glands to be highly variable. As early as 1925, Southwell and Maofie were questioning the relia b ility of the cement glands in diagnostic work because of the differences in appearance. They mentioned this particularly for Immature worms, but they went on to point out the fact that anyone who has worked with this characteristic readily acknowledges, that is, that in mature forms it was frequently extremely difficult to determine the exact number of oement glands. They noted that the shape as well as the arrangement of the oe ment glands was by no means constant. Even differences of considerable degree were not taken to be significant. Within the genus Ananthooenhalus. Meyer (1932) described a specimen of A, lu tsi (Hanuum, 1891) as having four oement glands, Hughes and Moore (19^3) reported the presence of 7 oement glands In A. vanoleavei. Bullock (1962) reported oement gland variability In A. iaekaoni and shoved th a t 36,9/6 of 217 males had other than six oement glands. Undue emphasis on the variability in the number and shape of the oement glands may cause one to forget that glands of the Palaeaoantho- oaphela are always multinueleate. Van Cleave (19^9) has differentiated, on the basis of the morphology of the oement glands, three basic oyto- logical kinds of oement glands within the phylum which correspond to the main divisions of the Ao anthoc ephela. Petrochenko (1953) used the disposition of the oement glands as one of the criteria for differentiation between A, bufonis and A, oauojsious. The oement glands of the former species take the form of a bouquet, while the glands of the latter species arise in successive pairs over a considerably longer part of the body. Variations have not been noted especially in amphibian aoantho- cephala from the Far East, or elsewhere, except to note the variety of language used to describe the shape. The following terminology has been used to describe the glands 1 ’’the form of a bouquet", "round - lying compactly", "oval", "small-pyrlform”, "funnel-shaped", "arranged in three pairs like a rosette", "sloping rows of three", "elongate", "slender", etc, Datta and Soota (195*0 observed that A, kabulensis practically agreed with A, opsarlchthvdla. Tsmaguti, 1935* with respect to the body shape, the number of longitudinal rows of hooks, and the number of rows of hooks in each row, but differed from it in the arrangement of the prostate glands. This gives cement glands specific diagnostic value in the dif ferentiation of these two species. On many slides it was impossible to ascertain aoourately the exact number of cement glandst if they could have a ll been counted, perhaps the number variation pictured herein would have been different. Variation In the Teates Range and Means f o r the I n te r io r and P o ste rio r Testes The specimens from Toonan shoved the smallest average size, the ones from one single host being smaller In average slse than those from the pooled collection from the same area. Males from Rana tended toward a larger size In both the anterior and posterior testes among Individual specimens. Same of the males from R. longurls and R, latonohi had the largest testes. In some cases, one testis was larger If the other one had started to disintegrate. Table XU, Range and Mean for Anterior and Posterior Testes Average Average Source t Anterior Posterior Ranee - Anterior Ranee - Posterior H 64 511x335 499x342 265-700x175-4^3 381-670x247-484 3 - 8 53 522x347 505*347 360-690x216*53 350-693x216-453 1 - 8 117 515x340 501x3** 268-700x175-453 350-693x216-453 B. m elanostictus 160 550x396 535x392 384-639x299-539 408-616x288-515 B. bankorensis 38 620a*35 605x^25 414-906x350-556 371-989x309-546 Rana 92 552*386 529x395 239-809x227-539 231-924x192-562 In the majority of cases, the anterior testes was the largest. In a muoh smaller percentage, the two were equal In length, Monorchids existed in some instances up to 8$ of the total number, but the average for males from B, melanostictus was four per eent, while males from Rana having only one testis amounted to The fact that one of the testis appeared to disintegrate has been mentioned in the literature. This was found quite often In this study. Table X IH , Sise Comparison of the A nterior and Posterior Testes A nterior P o ste rio r Source f la rg e r la rg e r Equal Only one *1 ...... 6b 37 (Stf) 2b (37& ) 1 < i» 2 ( 1#) 3 - 8 53 28 (71#) 13 (2Sg) 2 M) B. m elanostictus lb 8 93 (6336) 3b (2336) 12 (8*) 9 (636) B. bankorensis 37 23 (569 6) 12 (2936) 3 ( # ) 3 (836) T otal - B, melanostictus 25*f 158 (62?6) 71 (2836) 16 m 11 (336) Rana 81 57 (7036) 18 ( 2296) » (318) 2 (396) Testes - Discussion According to the literature, the testes are generally located in the posterior third of the mature worm, although there have been a few in stances where they were said to be located in the posterior half of the worn. When only one testis was present, this did not necessarily cause the remaining one to be enlarged. In a few oases this was true, but In the majority it was not so. This enlargement of the testis In monorohids has been reported by Bullock (1962), He also pointed out that In 208 specimens of A, .laokscnl. Ilf had only 1 testis, Haley (1962) concluded that the finding of testes In unusual locations showed interhost varia tion but that the number of testes did not. Variation in Eggs S tru ctu re The eggs In specimens from a ll hosts had four membranes. These membranes were re a d ily d istin g u ish ed when liv in g m ateria l from B, melanostictua was examined, but in stained specimens they are difficult to differentiate. This was noted by West (196b) in his study of aeanthor membranes. The o u ter membrane was a v ery th in covering which was tra n s parent and which showed some variation from egg to egg. In some stained 8 2 . Aoanthocephalus bafonls O uter membrane fibrillar coat fertilization membrane - in n er membrane Hosti Bnfo melanoatlctua (fresh eggs In saline - preserved in insect media) SOm . $ ot Rn longlorna Rana Hosts sane pemn n te oy a t ) ity cav body the in speeimen ed (stain awhopals bttfonis Aaawthnoepfaalus ooat r e l l i r b i f membrane n o i t a s i l i t r e f ue membrane Outer aoanthor nne membrane er n in Ao anthocephaias bufonis outer membrane -fibrillar ooat - f e r tilis a tio n membrane -aeanthor -Inner membrane Host l Bufe banknrenais (stained specimen in the body oavity) eggs, this area was vary large relative to the rest of the egg size, In eggs from th e u te ru s , th is membrane was sm aller. In many stain ed speci mens, th is membrane was not o le a r. The f i b r i l l a r eo at was v ery obvious and oould be seen clearly at the end of the shell, since there was a greater concentration of the fibers In this area. The fertilization membrane was most prominent, and it was very distinct in all specimens. The polar elongations varied somewhat, one end appearing more pronounced than the other. It would seem as though this end were more like a knob. The innerm ost membrane adhered c lo se ly to th e ao au th o r's surface and was quite difficult to distinguish, (See drawings 1 & 2) The embryos had prominent hooks and spines covering the complete body of the embryo, with a definite concentration of nuclear material almost in the center of the aoauthor. Some specimens had very prominent polar elongations and the eggs appeared longer and thinner. One specimen from R. latouchl from Yong- foh-lee had an extremely large outer membrane which was not a smooth oval but which came out at the poles like a fan, (See drawings,) The fib rilla r coat was very pronounced and the polar elongations were long and drawn out. The embryo appeared longer and thinner, although in actual measurement It was not. It also looked as If it had two eyes which were actually pronounced embryonic booklets. Specimens from R. llmnocharls from Keelung had eggs with practically no polar elongations. Eggs from the body cavity in many oases were immature. Descriptions and measurements were made only on those eggs which appeared to be mature. The basis for discrimination was the presence of polar elongations and the ring of hooks on the embryo. Eggs within the uterus were usually mature, but It was not always possible to find specimens in such a p o sitio n . Ranges in egg and embryo sise aoaording to several hosts, (sise in miorons) Average Average Range Range Egg —Embryo______Egg______Embryo Source #1 71 x 19 * 8 x 12 67-76 x 16-21 **-53 x 9-16 3-8 78 x 20 5 * x 13 67-92 x l*-23 * 2-58 x 9-25 T otal - Toonan 73.8x19,* *9.2x13 67-92 x l*-23 37-58 x 9-25 B.melanos- tio tu s ? 8,*x 2 0 ,6 * 0 , 7x12.* 71-82 x 16-25 39-51 x 9-18 B, bankorensls A ll Shan 109 x 22 60 x 13 101-117 x 18-25 55-6* x 9-16 B, bankorensls others 7* x 18,6 * 3 .8 x 12,3 67-80 x 15-23 *1-53 x 9-1* ’ T otal - B. bankorensls 87 x 19.6 50 x 12.5 67-117 xl5-25 * 1 - 6* x 9-16 Rana speoles 70 x 1? ** x 13 62-85x16-23 37-51 x 9-16 R. tig r in a 66.9 x 19 *1 x 12,5 65-68 x 16-23 37-** x 11-1* R. longiorus 69.2 x 1 7 .8 39.5 x 1 2 .2 6*-79 x 16-21 3*^*5 x 9-1* R. llm nooharls 73.7 x 18.* * 3 .7 x 10 71-80 x 16-25 *1-48 x 9-1* R. latouohl 75.5 x 20 * 3 .8 x 13 7*-79 x 18-26 * l -*6 x 12- 1* Rana t o ta l 72 x 19 ** x 13 62-85 x l *-26 3*-51 0 9-16 Size Comparison Examination of eggs on the basis of different hosts shoved no clear- out lines of demarcation in size, except for those from B. bankorensls from All Shan. (See next page.) On the average, specimens obtained from Rana tended to be smaller. Differences in the size and shape of the polar elongations mere difficult to determine, especially in the oases where they were small and appeared to be lacking. This could have been due to immaturity of the speoimen. Some individuals had polar elongations with a distinct conical appearance at the tips. Hooklets were observed in all eggs studied, and spines covered the body of the acanthor. Ranges in Egg Size (Measurements in Microns) Egg Embryo 8. melanostictus 71-82 x 16-25 39-51 x 9-18 B, bankorensls 67-117 x 15-25 hl- 6h x 9-16 All Shan 101-117 x 18-25 55-6** x 9-16 Hot All Shan 67-80 x 15-23 M-53 x 9-1^ Rana 62-65 x lh-26 3*4-51 x 9-16 The egg range from B, melanostictus was slightly wider than that of eggs from Rana. Specimens from Rana had smaller individuals and lacked larger ones. It was noted that as the eggs became more mature, it did not necessarily follow that they beaame wider. In fact, the oontrary was often demonstrated by the fact that the more mature the eggs were, the thinner and more spindle-shaped or oval-shaped they appeared to be. Discussion - Eggs The egg has been considered by some to be the characteristic that is least affected by environmental extremes, as it is influenced by neither the number of parasites per host nor by the species of the host (Baer, 1951)• Joyeux and Baer (1935) considered the egg along with the hooks the only two reliable morphological characteristics of the genus Acanthocephalus. Petroohenko (1956) and Yamaguti (1963) placed suah importance on the egg structure that they have established whole new taxonomic cate gories to accomodate these ideas in the classification of those parasites from amphibians. The characteristics which they consider to be decisive are the polar elongations of the middle shell of the egg, the hooklets of the embryo, and the spines on the body surface of the acanthor. Be cause of this concept, A, bufonis has been placed in a new genus Pseudo- acanthooephalus. which is a different family (Pseudoacanthocephalidae of the Gigantorhychidea), while other species of Acanthocephalus were re tained in the Echinorhynchldea, This is crossing class lines. This new genus was erected to accomodate embryos without polar elongations, but which do have embryonic hooks and the acanthor is spined, These are all parasites of terrestrial amphibians, Golvan (I960) included four speoies in th is genus. Another example of th is same type o f reasoning i s when Petroohenko and Yamaguti placed the genus Prosthorhynchus along with Porrorchis and Pseudoporrorohia in the Prosthorhynchidea of Petroohenko (1956), which is included in the Glganthorhynehldea, The genus Flaglo- rhynchus is retained in the Plagiorhynohldae, a family in the Eohino- rhynohidea, Schmidt and Kunts (1966) observed that the host habitat is not an invariable character and that the type of egg is not (with or without polar elongations) strictly aquatic or terrestrial. They concluded that the differences between the two speoies are not significant even at the generic level. They proposed synonymy of Plagiorhynchus and Proatho- rhypohus, with the former having priority. FilicoUidae was also placed In the Glgantorhynchidea on the basis of the lack of polar elongations, in spite of marked oloseness in other characteristics to Polymorphidae. This was later ohanged by Yamaguti (19^3). Schmidt and Kunts (19&7) statedj "In our opinion the worms in question are clearly within the definition of Polroorphus. The presence or absence of polar swellings cannot alone be regarded as generic dif ferences in this order,M In the genus Corynosoma there are also species with and species without polar swellings of the middle shell. Formerly Yamaguti (1935* 1939) described A, bufonis as having polar elongations, Petroohenko (1953) reported A. caucasicus from both Bufo and Rana and said that the eggs formed protrusions at the poles. In the same paper, he described A, bufonis as having ellipsoidal eggs and that the intermediate shell formed no polar protrusions or folds, although other acanthooephalan species at the same stage displayed such bulges on the second egg envelope, at least incipiently, at a similar stage of m aturity, Grabda-Kasubska (1962) observed in her work with A. falcatus that eggs from young females had shorter polar elongations. She also used the sise and form of the eggs to distinguish A. falcatus and A. ranae. In A, falcatus the eggs were oval with short terminal extensions | in con trast, those of A, ranae were spindle-shaped with long terminal extensions, Golvan and Deltour (196*0 discussed the problem of spines found on embryos and discussed their Implication in systematise, Golvan (1965) reported for A, madaaascarlensis, which had for a host Rhacouhorus sp, (a tree frog), that the embryo had polar elongations and also booklets as well as spines on the acanthor surface. Re suggested that spination of the embryo was a characteristic with merit but was not of sufficient importance to make it of taxonomic value at the class le v e l. Work by Grabda-Kazubska (196*0 added some In te re stin g arguments to this line of reasoning. She concluded her study on the armature of embryos of acanthocephalans by statingt The present investigations indicate that the types of embryo struc ture distinguished by Petrocenko 1956* 1958 cannot be aooepted,,, i,e ,, the transfer of certain species of the genus Acanthocephalus to a new genus Pseudoacanthooephalus (due to cutloular spines on the embryo surface) and placing th elatter in the subclass Gigan- torhynchinea, or the transfer of Centrorhynoh.ua and FilicoT n « to this subclass are not Justified, A cursory review of the literature reveals that Aoanthooephalus from aquatic hosts tend to have larger eggst A, ranae 110 - 130, A, cauoasious 94 - 10*+, A. kabulensis 90 - 100, A, lucldus 93 - 114, while those from terrestrial hosts have eggs which tend to be smaller in sise, e.g,, A. bufonloola 64 - 71, A, bufonis 75 - 84, A, madagasoariensis which has a maximum size of 75* Contrary to expectations, the embryos from aquatic hosts (Rana) had a lower range (62- 83) than did the eggs from the terrestrial host (B, melanostictus). which had a range of 67 - 92, while specimens of B, banko- rensis ranged from 71 - 113? which was by far the widest range. Breaking down this range, hosts captured on the plains contained specimens with an egg range of 71 - 76, while specimens from All Shan had an egg range of 101 - 113* This could have been due to the difference in geographic lo ca tio n . If Van Cleave's egg measurements for A, artatus of 60 - 83 x 18 - 24 are considered, this would Increase the range of A. lucldus to 60 - 114 x 18-32, His whole differentiation pf this spedles, A. artatus, was based on the specific difference in embryo size, as all the specimens he studied had the proboscis Inverted. TABLE XV. Comparison of egg else lo r a ll amphibian host a. (Measurements in miorons) Urodels Location Egg Embryo Af an th u rls Europe (Franoe and Italy) 90-100 falo a tu s Europe (Alps) 74-87 x 71-21 3 8 -4 7 Af aoutulus North Carolina Not observed A, nanus Japan 77-100 x 9-20 42-57 x 9 -1 1 Anura-aquatio At ranae Europe 110- 130x 13-16 A. oauoasiaus Russia 9*4-104x23 £ kaSalersis Afghanistan 90- 110x 50-80 68 x 16 A. tlgrlnae Siam 80 x 20 A. luoldus Japan 9 3 « 1 1 4 x 24-32* 4 1 -8 3 x 13-24 Anura - terrestrial At lu te B ra sil A. bufonicola Russia 64-71 x 23-32 A. bufonis Russia 74 x 29** At bufonis Far E ast 7 5 -8 4 x 23-27 A. madagasaarlen- s is Madagascar 75 x 23*^* 3 5 * 12 * This does not include VanC leave's measurements of A. artatus of 60-83 x 18-24. ** Not f u lly mature *** Maximum sise Yamaguti (1963) retained the species A, sinensis Van Cleave, 1937* even though this speoies vas synonymized with P. bufonla (*Aoanthooopha- lus b,) (Yamaguti, 1954). Ha doss this because the eggs are distinctly smaller (46 - 80 x 18 - 15) than those of the nearest speoies, as well as the difference in the polar elongations of the middle shell. He lists one aquatic and one terrestrial host. At first I supposed rather naively that the presence of polar elongations and the more elongate, spindle shape of the eggs from aquatic hosts would account for the difference in egg size. However, many eggs which appeared longer, due to the pronounced polar elonga tions, by actual measurement were not. There are several alternativest 1) On the basis of this egg range, the present specimens could be included with A, lucldus. 2) On the basis of egg size difference, the lowland specimens could be considered as A, bufonis. regardless of the host species or the habitat of the hosti and the parasites from All Shan Identified as A. lucldus. even though the host is terrestrial, (One wonders at the advisibllity of this when one realises that the eggs from Tounan had a range of 6? - 92.) 3) Be cause of the overall spread of the sise range and the mix-up of polar elongations in both Bufo and Rana (the largest eggs had polar elonga tions, but theyiwere found in Bufo). they could all be considered to be A. bufonis. It would seem that the presence or absence of polar elongations of the middle shell is not strictly aquatic or terrestrial, Schmidt and Hunts (1966) did not consider polar elongations to be significant, even at the generic level. This study supports the conclusion that this characteristic is not significant, even at the specific level. Female Genitalia Thera la not orach variation in the size of the female genitalia when oompared with total body size. The genitalia length was leas than .15 of the total body length in 97$ of the apeolmens. Length of Female Genitalia Compared to Body Sise £L 2-8 B. m elanostiotus B. bankorenaia Rana . 1 6 -,20 1 2$ .11-,15 29 9 9 1 11 32$ 21$ 11$ 8$ 21$ . 06- 1 .0 61 32 30 11 38 68$ lh$ 71$ 8h$ 73$ .01-0.5 2 2 1 3 9$ 9$ 8$ 6$ The average size was from 0 ,8 8 3 - 0,968 mm. The range waa from 0,*39 - 1,675 am. The la r g e s t average measurement was from B, bankoren- sis. The ones from Tounan were generally amaller. # Average Range #1 W .939 • 7*2-1,082 3-8 66 .968 .731-1.082 B. m elaneatlo tu s 50 .883 .*39-1.369 B. bankorensla 1* .976 .8*7-1.236 Rana 57 .908 ,65*-1.15* The malea ahoned orach more variation In the genitalia length, mainly beoauae the meaaurement of this eharaoterlatlo was affected by the bursa, which may or may not have been fully extended. Discriminant Analysis of Morphological Variation Pound in Adult Aeanthocephalans frost Amphibians on Taiwan Variation Is ubiquitous when a comparative morphological study is made which involves a large number of specimens from various sources. Analyzing variation proves difficult because of variant characters which are infrequently expressed. Integrations between extreme morphological differences can usually be found if the sample population is large enough to adequately represent the actual population. The mean, variance, and standard deviation were machine-computed for all individuals and characteristics studied. Other statistics com puted were Coefficient of Variation, Coefficient of Difference, Pearson's Correlation Coefficient, and Point Bi-serial Correlation Coefficient, when applicable, A nested Analysis of Variance was calculated for several sets of data, and "t" tests made to evaluate possible similari ties. Four other statistical techniques were employedt Two-Variable Scatter Plots, Cluster Analysis, Multl-varlate Classification Techniques, and P rin c ip a l Component A nalysis, The data analyzed were divided into three basic groups. The first group consisted of data from worms collected from one location - Tounan. This group in turn was subdivided to include a comparison of worms col lected from one host Individual with a pooled collection of parasites from the same host speoies, Bufo melanostlotua. The second set of data was made up of specimens from B. melanostictus from varying locations which excluded Tounan, The third set of data covered all worms collected from Rana. regardless of the host speoies or the collection data. The following characteristics were observed in both sexes t total length, maximum width, probosais length, hook number, size of largest hooks, sise of ths lemnisei, and tho longth of the probosols receptacle; In the male; the length and width of the testes and the number of cement glands; ln,£he female; the length and width of both egg and embryo, as well as the length of the genitalia, D iscussion Speolation work involves population analysis and a study of quan titative characteristics. Statistical methods are indispensable in making accurate quantitative descriptions of populations and they also help to delineate in such a way as to facilitate comparison. Feller (19^9) felt that the major abuse of statistics has been overemphasis on the role of statistics in evaluating results. However, in any experiment, data are bound to be accumulated, and the evaluation and Interpretation of said data are necessary. This would be a simple task if experimental determinations were infallibly ac our ate and perfectly reproducable, and if the differences between groups being compared were obvious on inspec tion, Observations are useless unless they are interpreted. Statistical analysis, as such, does not constitute evaluation or interpretation of data - it is only an aid. Appropriate use of formal statistical methods often can increase experimental efficiency by increasing the amount of reliable information gained and by decreasing the amount of effort ex pended, A more precise description of data can be obtained through the use of various statistical methods. Host properties vary continuously so that clear out separation into classes may be somewhat arbitrary, since ths dividing lines are often ill-defined and their exact positions a matter of opinion, Moore (1962) stated that the important thing is the quality of the difference, and if the difference is large enough to justify classification of the popula tion into different groups or categories! i.e ., the interpretation of the d ata. The C o e ffic ie n t of V ariation The Coefficient of Variation is a measure of relative variability used to compare dispersion and variability of items of different sizes. This measure of variation is obtained by dividing the Standard Devia tion by the Mean, Sinoe this quotient is usually a very small number, it is multiplied by 100 and expressed as a percentage. The Coefficient of Variation (C.V.) is divorced from any unit of measure. It has been considered by many biologists as a very good measure of relative dis persion and has been called by Simpson, Roe, and Lewontin (I960) "by far the most useful measure of variability that has yet been proposed." The C.V, represents the percentage of the mean which is contained In the standard deviation, When this number is small, this simply says that the variation between the individual items in the population sam pled is small relative to the mean. When one compares two or more popu lations and the C. V, of one is significantly smaller than the C.V. of the other, then one concludes that the Individuals in the first popula tion are less variable than those in the second population relative to their respective means, Mayr, Linsley, and Uslnger (1953) regarded the C.V, as a sensitive indicator of the homogeneity of samples. They also asserted that this measure is particularly useful when comparable samples of the same species from different localities are investigated or when the variability of different variates is compared. Tables of C.V, *s for the several aharaoterlstlos and host groups are in Appendix I . A b r ie f summary of s ig n ific a n t fin d in g s follows* The length of the male specimens from Rana and B. melanostlctus from a variety of locations showed a higher C.V* than the length of stale specimens from B, melanostlctus from Tounan, In both of the former groups* the number of locations and the number of Individual hosts were quite large. Among the female specimens* the C.V. of lengths of worms from Rana and B, melanostlctus were between that of the two groups from Tounan. The same holds true in general when comparing the C.V, of widths and of maximum hook e ls e . When comparing the number of longitudinal rows of hooks* the male specimens from Tounan had a slightly higher C.V, than did the worms from B, melanostlctus. while male parasites from Rana had a comparable C.V. to that of the male specimens obtained from one host individual in Tounan. In the females, the worms from B, melanostlctus had the largest C.V, * while the female parasites from Rana had a C.V. equivalent to that of the pooled collection from Tounan, The respective C.V.'s for the three characteristics - hooks per longitudinal row* proboscis width* and proboscis receptacle length - were comparable between the various host groups for both male and female worms, except that the C.V, of the proboaals receptacle length of female worms from Tounan was the largest of all groups. The length of the larger lemnlsci showed male worms from B, melanos tlctus with almost twice the C.V, as male specimens from Tounan* while males from Rana also had a substantially larger C.V, than did the male specimens from Tounan. The C.V.*s for this characteristic for all female groups were about the same. The C.V.'a for the length of the anterior testis varied between groups. The values from specimens from B. nelanostiotus and Rana were muoh higher than that of the male worms from Tounan. The posterior testis did not exhibit this marked difference. The C.V. *s for genitalia length varied considerably between host groups, ranging from 29.1$ for worms from B. melanostlctus.through 16.0# for parasites from Rana. down to 11.0# for the combined Tounan hosts. The C.V,*s for egg length varied little between groups, ranging from 8.5# for worms from Rana through 7.6# for specimens from B. melanostlctus. to 6,5# for parasites from the combined hosts from Tounan. In contrast, the C.V, for embryo length of parasites from Tounan was twice that for parasites from the other two host groups. Female worms from Rana revealed a higher C.V, in egg length, while female parasites from Tounan had the highest C.V. for the length of the embryo. D iscussion The basic implication herein is that dispersion relative to the mean is a measure of biological variability. This is only a broad rule, mainly empirical in foundation and always open to exception. The idea of the C.V, as a statistic to measure the scatter is convenient when the variates are homologous. However, Simpson (I960) stated that if the variates are not homologous, experience suggests that comparison using C.V, *s are s till generally valid if they are all linear measurements of anatomical elements. There are some difficulties connected with using this statistic. One cannot te ll too muoh about the C.V. by itself when comparing two populations, for if the means were quite different and the standard deviations of each were In proportion to the respective means, one would have nearly equal C.V. 's, but radically different population characteristics. Since the distribution function of the C.V, is com plex, one does not have a readily understood way of assessing significant differences. Inferences to be dr aim from the C.V, measurements may be summarized as follows! 1. The C.V, 's for parasites from B, melanostlctus and Rana are more alike for the several characteristics and generally larger than those for the respective C.V.'s from the Tounan hosts, 2, The greater variability in the worms from B. melanostlctus and Rana compared to that from the Tounan worms may be attributed to either a real difference in species or the effect of the varied locations of the specimens from B, melanostlctus and Rana. The C.V, data do not offer conclusive evidence to choose between species differences or location differences. The Coefficient of Difference (C.D.) This statistic is simply a difference of the means of two samples divided by the sum of the standard deviations of the two samples. It assumes that both samples are taken from normal populations and is used in connection with a C.D. table (See Appendix III) to estimate the joint non-overlap percent of the two populations. The C.D, has been called by some biologists "a rough approximation", and some statisticans olasslfy it as a "quick and dirty" test. As seen from the list of calculated C.D,'s (Appendix III), the only characteristic whioh showed any significance according to the C.D. tables, Just mentioned, me that for the proboscis receptacle length, which had C,D,'s of 0.8oh, 0,7*t>5, and 0,66, whioh represent Joint nonoverlap values of 80$, 73$» and 73ft respectively. This characteristic is in itself highly variable due to an inherent high experimental error. It should be noted that this characteristic had an extremely low C.D, of 0,13 0,136 for males and females when the two groups from Tounan were compared, where under the applicable experimental conditions, one would expect a low experimental error (See Appendix IV.) The worm length of female worms had a C.D, of 0,83 when a ll the female specimens from Tounan were grouped and compared with a ll the female parasites from Rana. This indicates a Joint non overlap of 80$, which is not highly significant for this statistio. The C.D, readings from Tounan were so low in every characteristic that the test was indiscriminate, except as an indication that when the host species are the same and the location is the same, the variation is extremely small among the samples. None of the C.D, values were as high as 1,28 or the 90$ level of Joint nonoverlap which Mayr (1953) sot up as the subspecies level, Disoussion Mayr, Linsley, and Usingsr (1953) pointed out that there is no general agreement as to how two populations can be recognized as two different speoies. The most frequently proposed convention Is the so- called "75 percent rule" to delineate subspecies, Mettrlek (1963) Indicated that parasitologists accept a level of speeiation whioh is comparable with the sub-specific levels proposed for other groups. He would prefer that a C.D, of 1,96, or higher, rather than 1,28, be used to Indicate a specific difference. He added a note of caution that with large population samples an erven higher C.D. is in order and placed the figure at 2,18, There are obvious difficulties in trying to distinguish two popula tions on the basis of one characteristic. The use of multiple-character analysis is preferable, Scott (I960) came to this conclusion in his work with trematedes. In the present study, the C.D. values were so small that they did not indicate the presence of any sub-species. The " t M T est This test was used to test whether or not the observed means of the sample could be considered as being derived from a common population of such values, i.e ., if the specimens in question were similar enough in characteristics to be considered as belonging to one species. When significance is used herein, it is a statement of the probability that an observed difference is a true difference, and not merely a result of chance. It is admitted that a statistical difference does not establish the existence of true differences, but merely establishes that such a result is unlikely to have occurred by ohanoe alone. Probability levels of 0,10, 0.05, 0.01, and 0,001 have been used. Observed differences whose estimated probability of ohanoe occurrence correspond to (or are less than) those levels are designated as being significant at the 10 per oent, 5 P*r cent, 1 per cent, or 0.1 per cent levels, respectively, A tentative hypothesis was set up saying that the worms obtained from one host speoies represented one population or one speoies of para site, regardless of the location from which they were drawn, If the observed results were Improbable of occurrence, the hypothesis vas re jected and the alternate hypothesis that the differences truly existed vas accepted. Homogeneity of the sample is indicated when the hypothesis is not rejected. First of all, one location vas used, comparing those individual worms secured from one individual host with a pooled collection from several similar hosts. Sexes were compared separately and then com pared to each other to demonstrate sexual dimorphism. The first host had 57 males present, which were oompared to the pooled group of 61 males from the same lo c a tio n . The d ifferen c e s between the means of the number of rows of hooks vas significant at the 1# level, while the differences between the means of the size of the larger lemnisci vas significant only at the 10$ level. The other twelve characteristics showed no significance. This general homogeneity indi cates th a t these worms came from one population. The 98 male specimens colleeted from B, melanostlctus from various locations were compared with the 75 male specimens from Rana. The specimens from Rana represented a wide variety of hosts as well as lo c a tio n s. The Mt" t e s t r e s u lts showed more s ig n ific a n t d iffe re n c e s than did the males from one location, but not enough to infer that real speoies differences were present. The number of rows of hooks and the proboscis receptacle length were significantly different at the 10$ level; the number of hooks per row and the proboscis width were signifi cantly different at the $f> level; and the size of the larger lemnisci vas significantly different at the 1$ level. When all the male parasites from Tounan were pooled together and oompared with B, melanostiotus from a ll other locations, the character istic shewing significant difference vas in the probosois receptacle length, which had & much greater than 0,1# significance. The body width, the proboscis length, and the size of the lemnisci also showed significant differences at slightly more than the 0,1# level, The length, but not the width, of the testes was significantly different, as was the number of rows of hooks and the number of hooks per row, all at th e 5# sig n ifican ce le v e l. The same general tren d was evidenced when a l l th e male p a ra site s from Tounan were pooled and compared to the male parasites from Rana. When the female parasites from one individual host were compared with the pooled collection from Tounan, significant differences were almost nil. The proboscis receptacle length was signficantly different at the 10# level, and the length of the worm was significantly different at slightly greater than the 1# level. This would indicate a lack of heterogeniety in the two samples. From the comparison of 55 female specimens from B, melanostlctus and 58 female specimens from Rana. characteristics which were signifi cantly different at the 10# level were the proboscis receptacle length, the length of the larger lemnisci, and the genitalia length. The worm length and width, as well as the length of the smaller lemnisci, were significantly different at the 1# level. The combination of all the female parasites from Tounan into one group revealed, when compared to a ll the worms from B. melanostictua. much less variation than did the males In a comparable test, A greater than 1# significance was exhibited in the differences between the means of the length of the proboscis receptacle, the size of the lemnisci, and the total length of the worm. When the female parasites from Rana were compared with the total number of female parasites from Tounan, the differences were of little significance. The only characteristic whioh showed extreme sig n ifican ce was th e proboscis recep tacle length. Sexual Dimorphism Specimens secured from one individual host, when oompared according to sexes via the "t" test, showed significance in every characteristic studied, (See Appendix V.) The body length showed by far the greatest significance with a muoh lass than . 1$ ohanoe of coming from the same population, Next in significance was the proboscis length and width, followed olosely by the number of rows of hooks, the maximum size of the hooks, and the number of hooks per row. The size of the two lemnisoi showed about the same amount of sig n ific a n c e . The w idth did n o t vary as muoh as soma of the other characteristics, while the proboscis reoeptaole len g th showed the l e a s t amount of d iffe re n c e w ith a sig n ifican ce le v e l of 156. The pooled specimens from Tounan were significant at muoh more than . 1# in all the characteristics. The length and width of the worms, as well as the proboscis length and width, were more highly significant than the same c h a ra c te ris tic s when observed from one in d iv id u al h o st. There was also a much greater significance in the size of both lemnisci. The comparison of males and females from B. melanoatlctus from all locations, excluding Tounan, showed significance at more than the ,1 f> level for a ll characteristic s except for the number of hooks per row, which showed sig n ific an c e a t th e 5$ le v e l. The p a ra s ite s from Rana exh ib ited sexual dimorphism a t g re a te r than the ,1$ level in all characteristics except twot the length of the pro boscis reoeptaole, and the length of the smaller lemnisoi, both of whioh were significant at greater than the Vf> level and almost reached the .1 $ le v e l. log. D iscu ssio n Tho bost estimate of the actual difference is provided by compari son of the means. However, means of samples are only estimates of true population means and are subject to error. The mere establishment that means are not statistically significant ly different is not neaessarily of prime Importance. Statistical signi- flanoe often may not oonnote experimental significance or importance. One does have an estimate of the magnitude of the differenoe and some measure of the uncertainty of that estimate. Differences in eggs from the Tounan specimens was probably due to the low number of samples. By far the majority of the female specimens lacked any mature eggs, Any variation found in the length of the para sites from Tounan was undoubtedly due to overcrowding. Technique factors are responsible for the differences recorded in the proboscis reoeptaole length. In spite of similarity of fixing conditions and technique in volved in the specimens recovered from one host in Tounan, the length of the proboscis reoeptaole still showed significance at the 1$ level. It is of interest that the parasites from one individual exhibited a greater amount of sexual dimorphism than did the pooled collection from the same place, Pearson Correlation Coefficient The correlation coefficient is used to test the significance of the sise relationships between two variables. The limiting values are plus one and minus one. Positive values indicate that ths two variables tend to vary in the same direotion (increase or decrease), and negative values indicate that the relative variation is in the opposite direction. If the spec linens front one host exhibit a different correlation pattern than the data front another set of specimens, then one night conclude that the speoies represented by the second set of data vas different front the first species. This test vas ran only on parasites front Tounan, which were sepa rated into tvo groups. Males and females ware treated separately. The 96 male parasites, obtained front one host in Tounan, shotted correlation in the worm length with the width, hook size, and the pro boscis length and width. There was a non-significant negative correla tio n in the number of rows of hooks. The width of th e worms showed correlation with the maximum hook size and the proboscis length and width. Non-significant negative correlation was seen in the number of rows of hooks. The maximum hook size showed no signfleant correlation with other characteristics, A negative correlation was seen in a number of characteristics. The number of rows of hooks revealed no significant correlation. The number of hooks per row did show a significant corre la tio n w ith the proboscis len g th . The proboscis len g th showed c o rre la tion with both the proboscis width and the length of the proboscis receptacle. Neither the proboscis width nor the length of the proboscis receptacle showed significant correlation with other characteristics. The greatest amount of correlation is seen in the length of the larger lemnisci and the smaller lemnisci. The lemnisoi do not show correlation with any other characteristics. The pooled collection from Tounan had fewer females In the sample, so th e c o rre la tio n number should read hig h er. The len g th showed c o rre la tion with the width, the maximum hook size, the proboscis length, and the siz e of the lem nisoi. The w idth showed c o rre la tio n w ith the maximum hook size and the size of the lemnisoi. The maximum hook size showed correlation with the proboscis length. The proboscis length and width shoved a definite correlation, as did the size of the lemnisci relative to each other. The males from Toonan shoved the same general c o rre la tio n p a tte rn as did the females. The von width correlated with the maximum hook sise and the proboscis length. The won length shoved correlation with the won width, the maximum hook sise, and the length and width of the pro boscis, The lemnisoi shoved a definite correlation to each other. It is interesting to note that the testes showed high correlation between each length and width, bat no correlation between the anterior and pos terior testes in either length or width. The pooled collection of males from Tounan showed the width cor relating with even more characteristics than the females from the same location by adding the sise of the lemnisci and the sise of the testes. The maxi mam hook sise shoved no c o rre la tio n . The proboscis len g th showed no correlation with the width. The width of both testes shoved correla tio n . From the above, it would appear that there is definite evidence to support the existence of correlation among the different characteristics of th e aoanthooephalan worms. The correlation coefficients did not appear to vary much from the various host speoies. That the correlation pattern was the same is ob vious, but this does not necessarily prove that the speoies represented by the different sets of data are the same. Point Bi-serial This is a diohotomous test which shows the presence of overlap. It also discriminates by showing where there is high correlation. The oloser the calculated value is to one. the less overlap there is present, When a point bi-serial test was run comparing a ll the males from Tounan with all other males from B, melanostlctus. the coefficient of correlation showed overlap in every characteristic. This was also true for males from Tounan when oompared with males from Rana. (See Appendix VI.) The females showed the same lack of correlation as the males, in dicating that there was an overlap present between all the male groups studied and also between all the female groups observed. D iscussion Statistical correlation relates specifically to how two variables behave relative one to the other and is independent of the values of the mean and standard deviation of the variables in question. It was hoped that the correlation coefficients might give some indication of a novel way to distinguish species. For example, if there was a certain pattern of correlation between measurement characteristics of me suspeoted species and the next set from another host exhibited a distinctly different pattern of correlation between the same character istics, then one might conclude that the speoies represented by the second set of data was different from the first species, This did not occur. The correlation within the groups proved to be of real interest. The correlation between the length and width was not strange, and a like correlation between the proboscis length and width was to be expected. But the correlation of the total von length with the proboscis length was surprising, to a ay the least. It is also noteworthy that the number of hooks per longitudinal row did show a significant correlation with the length of the proboscis, Another unexpected correlation which runs counter to previous assumptions was that between maximum hook sise and proboscis length. This indicates that the hook sise increases as the worm becomes larger, BayUs (1927) observed that in Polyaoanthorhynohus "the number and sise of the hooks also appear to increase with the age of the specimen,M Van Cleave (19^1) noted that such conditions are extremely rare in the Acanthocephala and deserve critical study. The characteristics which showed the highest correlation were the sise of the lemnisoi. It was Interesting that the testes showed high correlation with their own respective length vs, width, but revealed no significant correlation to each other. Because the correlation pattern was the same between the two sets of data studied, this does not necessarily prove that the species represented by the two sets of data are the same, but yields no data to support an argument that a difference exists. Nested Analysis of Varlanee on the Pooled Group from Tounan The nested analyses of variance for pooled specimens from Tounan for both sox groups were computed to show variances between hosts and between specimens within hosts. Significant differences at the indica ted levels were found to exist between host means for the following characteristicsi Hales Females f;h«»«at-eriatle Level ofSignificance Significance Level Level of of Sl^nlfj Significance Body length Body w idth , ijt ,i The pattern of differences between individual host means for the several characteristics is such that one cannot infer that true species differences exist between hosts. I t was found that the variance component, with but one exception, due to specimens within hosts, was larger than the variance component between hosts for all characteristics in both sex groups. In most oases, the ratio between these components was 3 or h to 1, or greater. This high variance at the specimen level may Indicate either substantial variability between specimens from a single host or large experimental error, or both. Variance Components MALES Between Between Characteristic Hosts Specimens Body length .933457 .494316 Body w idth ,016965 .016279 Maximum hook 20.822582 39.671056 Rows of hooks -.157408 2.320059 Hooks/row .031018 ,144499 Probosois length 803.640920 2226.174000 Proboscis width -89.874530 1283.573400 Probosois reoeptaole 2225.576900 6706.628700 Larger lemnisci 9183.113700 12677.525900 Smaller lemnisci 11083.341200 19127.865000 Anterior testis - L 550.375240 4740.729900 Anterior testis - W 785.603660 2888,618800 Posterior testis - L 2205.482400 4456.870500 Posterior testis - W 1279.702300 2812,282600 FEMALES Body len g th 3.510951 6.312671 Body width .025859 .028922 Maximum hook 3.430039 42,188885 Rows of hooks ,488054 1.576875 Hooks/row .089313 .135434 Probosois length -73.962768 3018.545600 Probosois width 154.067140 2476,364900 Proboscis reoeptaole 4674.556400 14011.059300 Larger lemnisoi 12328,296000 37743.757000 Smaller lemnisoi 10248,718700 35105.485000 Genitalia length 306.432200 14096.431000 Other Statistical Techniques Used Four other statistical techniques were employed in an attempt to establish measurement groupings that could be inferred to represent different speoies. These techniques were as follows, Two-Variable Scatter Plots Various pairs of measured characteristics thought to be significant in differentiating between species were uied to form scatter plots. Data were from 322 specimens from many hosts. Other than the known sexual dimorphism, no groupings were evident on these plots, Henoe no inferences could be made of the presence of more than one species through the use of this method. Cluster Analysis This is a mathematical technique for examining the structure of multi-variate data to determine if the data represented a» points in a k - dimensional space (where k is the number of characteristics measured in each specimen) fall into natural Clusters. In this analysis, nine characteristics from 107 male specimens from a variety of hosts and lo cations were used. The computer used was a Univac 1108, having a sophisticated program for cluster analysis. No statistically valid clustering was apparent, thus suggesting that no species differences could be inferred by this technique, Multi-variate Classification Technique A sophisticated, as yet unpublished, computer technique made avail able through private communication with its developer, D, W. Marquardt, was utilised. This technique, whioh requires a large-scale computer for its implementation, has been successfully tested in multivariate botanical taxonomic analyses. It operates as follows, where species differences are suspected due to differences in one or more of the measured charac teristics t First, the specimens are divided into the suspected different' groups. Then about half the specimens from each group are chosen at random, and for each suspected group the parameters for from k to 7 likely multi variate probability density functions are computed - for example, the Normal Distribution, two or three forms of Pearson Type 2, and two or more forma of other appropriate Pearson type functions. These probability density functions thus formed are known as the classification funotlons. All the data are now substituted, one specimen at a time, into these density funotlons for each group, and each specimen is assigned as being * member of that group for whioh it had the greatest probability value. If this selection prooedure for any one of the probability functions classifies the specimens into the suspected groups in a consistent man ner (greater than 93$ of the time), then one oan conclude that the sus pected groupings are valid, and in taxonomic studies one oan suspect that each group represents a species if there are no other known factors which would cause the grouping. In the analysis made, the specimens were divided into the following suspected groups t Group 1 Tounan. B. melanostlctus single host male 1 Group 2 Tounan. B. melanostlctus single host female 3 Group 3 Various locations B. melanostlctus Various hosts male 5 Group kr Various locations B. melanostlctus Various hosts female 6 Group 5 Various locations Rana Various hosts male 2 Group 6 Various locations Rana Various hosts female 7 322 specimens were used, with 3 Pearson Type 2, 2 Pearson Type 7, and the Normal Distribution function as the classification functions. In no grouping was the classification oorreot as high as 70$ of the time for any of the six distribution funotlons used, Fran these results, one in fers that if different species were present, they could not be identified with the above groups, Since the Cluster Analysis involving only male specimens from B. melanostlctus from various loo at ions and hosts showed no significant clustering, no further attempt was made to regroup the specimens according to other oriteria and reapply the multivariate clas sification technique to such regroupings. P rin c ip a l Component Analysis Often in multivariate analysis where there is correlation between the variables, an examination of the eigen values and eigenvectors of the correlation matrix shads light on the structure of the data. These statistics vara calculated for eaoh of ths six groupings listed In the previous section. It was found that although there were nine eigenvalues one dominated eaoh group and the three largest accounted for over 99$ of the sum of all eigenvalues, The original sets of nine characteristics for eaoh specimen were therefore transformed into sets of three values per specimen, corresponding to the eigenvectors related to the three largest eigenvalues. Scatter plots were made of all points by taking the three transformed values in pairs. The sexual dimorphism vas most evident in these plots, but no other groupings were apparent. Here again no speoies differences could be inferred, D iscussion From the results of the use of these four powerful techniques for establishing differences in multivariate domains, no conclusive evidence of species differences oan be inferred. However, the known sexual dimor phism was clearly established, thus giving validity to the methods and supporting the inference that other groupings were not evident, i.e., no species difference could be inferred unless the speoies are completely confounded with the sexes. SECTION V I CONCLUSIONS The problem of separating A, bufonis and A, luoldus an the basis of information found In the literature Is extremely difficult. As early as 1936, Joyeux and Baer felt that A. artatns In Indoohlna vas very close to A, bufonis. even ldentloal to It. The two species are similar in body size, number of longitudinal rows of hooks, maximum hook size, and probosois size. They differ in the size of the embryo (75-8^ miorons oompared to 93 - 114 microns) and in the range of the number of hooks per row, whioh is greater in A, bufonis (5-8 oompared to 4 - 5). The size discrepancy in the hook size is eliminated if the sizes recorded by Van Cleave are included. Up until the present time A, luoldus has been reported from China, Hong Kong, Slam, and Russia, In the past, with some exceptions, aquatic amphibians have harbored A, lucldus. while terrestrial amphibians have been host to A, bufonis. The overlapping ranges of almost all the characters of the two speoies is very evident. The statistical study, especially in the multivariate tests, bears out to a marked degree the conclusion that only one species is represented by this series of speolmens, in spite of the wide range of variation. A, bufonis resembles A, ranae. the common member of the genus from a variety of European amphibians. The variability present in this species is well knasm, A, bufonis differs from A, ranae in the embryo size. The range of A. ranae is 110 to 130 microns, while the range for A. bufonis as recorded in the literature is 74 - 84 miorons. In the present study, the range in one host animal was from 7 1 -9 2 miorons. It may eventually be possible to minimise the differences between A, bufonis and A, ranae and consider the Asian worms as a subspeoies of A. ranae. Variation in different characters is demonstrably influenced by the type of host, the number of parasites present In a given host, the location from which collected - even the number of locations in a given general area - as well as the time of collection. Geographical influence is seen in the variation in egg size from Ali Shan. The extent of this variation remains an open question which w ill only be solved when there are more specimens available for a de tailed study. Other oharaoters showed the same tendency for overlapping in the specimens from Ali Shan. The correlation of maximum hook size and the size of the worm was unexpected and contrary to previous assumptions. Egg morphology,i.e., the presence or absence of polar elongations, is not a good characteristic to subdivide even on the species level. Adaptive characters are subject to environmental changei this was demon strated in the eggs from frogs and toads. The tremendous amount of variation present in the size, shape, and number of both the lemnisoi and the cement glands would seem to Indicate that differences in these characters are not valid criteria for estab lis h in g new sp ecies. One is le ft almost overwhelmed with the sweep of variation present in the morphological characters of A^ bufonis. 1 1 7 . LITERATURE CITED. Baer, J. 6. 1951 Eoology of animal parasites, Univ. of 111, , Ur- bana, Illinois, 224 pp. Baylla, H. A. 1927 Soma parasitic worms from Arapaima gigas (Teleo- staan fish) with a dascription of Fhllometra senticosa n. sp. (Fllarloldas), Parasitology 19»35-47, Brandt, B, B. 1936 Parasites of certain North Carolina Salientia, Ecol, Honog, 6t491-532, Bullock, W, L, 1962 A new species of Aoanthooaphalus from New England fish, with observations on variability, J. Parasit, 48 1 442-451. 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Mlor, Soo. 6l i 206-210. ______1948 Expanding horisons in the recognition of a phylum, J, Parasitol, 3411-20, ______1949 Morphological and phylogenetic Interpretations of the eenent glands in the Aoanthooephala. J. Morph, 84 i427-457. Van Cleave, H. J. 1952 Spec i at ion and formation of gonora in Aoanthoeephala. Sys. Zool, li72-83, Walton, A, C, 19^2 Aoanthooephala as parasites of Amphibia 4, Contributions from the Biological Laboratories of Choi College Ho. 79. Ward, H. L. and D, R. Nelson 1967 Aoanthoaephala of the genus Moniliformis from the rodents of Egypt with a description of a new speoies from th e Egyptian spiny mouse (Acomya cahlrinus.) J, Parasitol. 53*150-156. Werby, Helen 1938 A new genus of Aoanthoeephala with forked lemnisei, Reans, Amer. Micr, Soc. 57*204-212, West, A. J, 1964 The acauthor membranes of two species of Aoantho oephala, J. Parasitol. 50*731-73*+. Westromb, A. H. L. 1821 The helminthbus aoanthocephalis. Hanoverae, Yamaguti, S, 1935 Studies on the helminth fauna of Japan. Ft. 8 , Aoanthooephala I, Japan 5 . Zool. 6 i 247-278, ______1939 Studies on the helminth fauna of Japan. Part 29, Aoanthooephala XI. Jap, J, Zool, 81317-351. 195*+ Parasitic worms from Celebes. Part 8. Aoantho oephala. Acta Medio a Okayama 81406-413. ______1963 Systems . Helminthum. Vol. V, Aoanthooephala, Intersoienoe New York, 423 pp. 12-3, rbsi width Proboscis rbsi Recep Proboscis Maximum Width rbsi length Proboscis Maximum size hook ogtdnl rows Longitudinal si - 1 - estis T tl Length otal T agr e ic 6118.7 16.1 nisci lem Larger si - 1 - estis T ok pr row per Hooks mle lmnisci lem Smaller si - 2 - estis T 2 - estis T it 18.1 width width f hooks of length length acl length le c ta H J 6 +J •H * 5 3 5 •** i « H « ^ Vi ^ o o ^ g£ a 5pi 5 a £ ;g £ £ H j H rj O H -H H i j H fj fi o s ■ B pedx 1 Appendix Table of c o e ffic ie n t of V ariation fo r r fo ariation V of t n ie ffic e o c of Table 12# Maximumwidth aiu ho size Maximum hook rbsi recep Proboscis width Proboscis length Proboscis Longitudinal mro width Embryo length Embryo ntla length enitalia G length otal T agr e ic 15.2 nisci lem aller Sm nisci lem Larger g width Egg length Egg ok pr row per Hooks os f hooks of rows acl length le c ta V C Vi O H a, o H H « O -H •H 8 P o ■P 10.8 13.3 22.9 10.7 19.^ 824.6 18 # **“ s o C m (# a _ c H _ C -P * C O H O © O H fct .4 6 7.9 . 7.2 8.7 4.1 9.6 9.6 9.8 b « •b I 016.1 20 16.2 11.6 26.3 23.4 5424.1 25.4 11.7 17.3 12.5 . ■ * rt S t -r U « 8 8 « m o CS a O Vi © . 8.0 8.2 Females . 7.9 7.9 7.2 18.8 11 20.8 13.3 11.2 < 23.4 21.7 10.7 15 8 P <8 U 6>.8 6.5 vt C ml 14.9 18.2 15.2 25.6 21.1 23.9 29.1 23.3 51 - 20.2 14 8.3 9.2 2.'5 7.6 5.3 11.3 21.7 12.1 22.1 19.8 15.8 16 23 14.2 23.4 8.5 . f te eea caatr and characters several the r fo 7.2 . hot rus or eae specimens. female r fo groups ost h 7.5 9.6 5.6 pedx 2 Appendix Table of c o e ffic ie n t of V ariation ariation V of t n ie ffic e o c of Table Percentage of non overlap of partially overlapping curve a associated with stated values of the coefficient of dlfferenoe (C,D.) Appendix 3 Values C.D, Joint nonoverlap percent Below the level of conditional 0.675 75 subspeoifio distinctness 0.84 80 0.915 82 0.995 84 1.04 85 1.08 86 1.13 87 1.175 88 1.23 89 Conventional level of subspeoifio difference 1.28 90 Above the level of conventional subspeoifio difference 1.3* 91 1.405 92 1.48 93 1.555 94 1.645 95 1.75 96 Methods and Prlnoiples of Systematic Zoology, page 146, Mayr, Llniley, and Usinger, 1953* Table of Coefficient of Difference for several characters for both sexes. $ H Worm length 0.056 0.22 0.04? 0 .2 4 0.16 0.83 Worm width 0.007 0.026 0.28 0.104 0.31 0.22 Maximum size of hooks 0.071 0.03^ 0.06 0.10 0.137 0.77 Longitudinal rows of hooks 0.33 0.104 0 .1 4 0.21 0.039 0.18 Hooks per row 0.0017 0.07 0.1 74 0.11 0.059 0.14 Proboscis length 0.0014 0.088 0.298 0 .1 7 0.50 0.17 Proboscis width 0.11 0.047 0.145 0.16 0.35 0.025 Proboscis Recep tacle length 0.13 0.136 0.804 0.7^5 0.66 0.53 Larger Lemnisci 0 .1 9 0 .0 0 0 8 0 .4 9 0.50 0.257 0.17 Appendix 'JH Smaller Lemnisci 0.12 0.039 0 .4 1 0 .5 4 0.35 0.198 Testes - 1 length 0.018 0.0298 0.09 Testes - 1 width 0 .0 5 0 .41 o'. 27 Testes - 2 length 0.091 0„l6 0.22 T estes - 2 width 0 ,0 3 0 .4 4 0.39 Genitalia length 0.039 0.73 0.247 Egg length 0.105 0.5 6 Egg width 0.23 0.62 Embxyo length e 0.004 O.65 n o . ID Embryo width 0.044 1 to B r-l o o o m a H S <0 H e t* m The calculated "T’s" for males and .females from various hosts (levels of significance as stated*) Appendix 5 Total length 12.813 14.937 14. 611 1A,706 Maximum width 3.888 5.807 7.138 **.655 Maximum hook size 5.826 5-745 5.217 4.854 Rows of hooks 6.410 4.401 4.814 3.635 Hooks per row 5.528 5-351 1.996 4.545 Proboscis length 8.305 3.334 8.704 5.057 Proboscis width 7.134 10.566 6.658 4.114 Proboscis receptacle length 3.158 4.264 4.017 3.146 Larger lemnisci 4.344 9.409 3.402 3.951 Smaller lemnisci 4.373 3.610 4.805 3.279 i o W in 0 w * T - 10 = 1.66 T - 05 = 1.98 T - 01 = 2.62 T - 001 = 3.37 Table of Point 3-Serial test results for different host groups. Appendix 6 Tounan males vs. All Tounan males All Tounan, females All Tounan females Location B. melanostictus vs. Rana males vs. B. melanostictus vs. Rana Worm length .0501 .1539 .2235 .0695 Worm width .2484 .2905 .0972 .1857 Maximum hook size .0604 .1247 .0 94-5 .0707 # of longitudi nal rows of hooks.1562 .0225 .1710 .1645 # of hooks per row .1722 .0562 .0960 .1051 Proboscis length .2809 .1212 .1514 .13 66 Proboscis width .1441 • 3731 .0335 .0253 Proboscis receptacle length .6236 .5494 .5453 .4307 Longer Lemnisci . 4268 .2003 .4222 .1557 Smaller Lemnisci .3379 .3247 • 3375 .1629 Testes 1 - length .0320 .1874 Testes 1 - width .3339 .24-90 Testes 2 - length .1751 .2035 Testes 2 - width .3217 .3360 Genitalia - length .0682 .2177 Egg length .4789 Egg width .1531 Ehbryo length .3336 Embryo width .0803 Summary Table for length of Taiwan specimens. Appendix 7 Length of worm in mm. # 3 4 5 6 7 8 9 10 11 12 13 1 ^ 1 5 16 17 - 18_ 19_ 20 21 22 SfiDgft O »f\ GO V"\ r-4 CM #1 ? 102 1 1 6 13 6 1 2 1 5. 850- 17.160 e> 63 8 32 15 4.380-10.230 #3 1 1 1 11.910-18.36 cf 5 1 1 2 1 6. 690- 9.120 #4 ¥ 5 2 1 1 1 11.37-22.65 & 3 1 2 6. 270- 8.610 #5 2 1 15-54-16.05 o»? ? 1 1 7.20 #6 ¥ 23 3 2 3 6 2 4 2 1 6 .2 4 -1 6 .8 9 6) 12 6 6 4 .0 2 -5 .7 6 #7 ? 16 1 2 4 3 5 1 7 .8 0 -1 3 .0 CTIB 10 6 2 5 .4 0 -7 .9 0 #8 $ 16 1 2 1 2 3 1 2 1 2 1 7.4 1-1 8.39 oUO 3 6 1 5 .4 3 -7 .2 9 # > 8 ? 66 3 4 5 9 10 11 8 2 3 4 '-4 2 1 7.80-22.65 o?49 6 16 6 7 10 2 4 .0 2 -9 .1 2 #1-8 n 6 9 1 4 12 29 23 35* 17 n 8 4 6 4 1 2 1 5.85-22.65 0*112 14 48 23 12 11 3 1 4 .0 2 -10 .23 Summary Table for length of Bufo specimens• Appendix 8 (Measurements in mm) HsaL *--2 J5 fi Z 2___9 10 11 12 IB 14 IS 1 6 17 18 19 20 21 22 #1-8 168 ? 1 4 12 29 23 35 17 21 8 4 6 4. 1 2 5.85-22.65 112 o’ 14 48 23 12 11 3 1 4.02-10.23 B .aalan o a- tie tu * 169 ? 1 3 14 10 24 26 18 17 13 10 8 6 8 3 3 1 4.92-2.28 North 199 ^ 5 34 58 47 40 9 4 2 3.440-10.6 B .m alanoa- tic ta a 1 9 1 12.52 South 5 1 1 2 1 3.960-8.76 B .m alanoa- tic tu a 170 9 1 3 14 10 2426 18 17 14 10 8 6 8 3 3 1 4.92-22.8 No.&So. 204 .6 35 60 47 41 9 4 2 3.44-10.6 A ll fi*. ■ alanoa- 338 9 1. 4 18 22 53 49 53 34 18 12 12 12 4 5 1 2 4.92-22.8 tlc tiia 316 * 6 49 108 70 53 20 7 3 3.44-10.6 g . hanko- XfiQfilflL 6 ? 1 1 1 1 2 5*569-13*4 North 8 h* Summary Table for lengths of different species of Rana Appendix 9 * 2 3 4 5 9 7 9 9 10 U 12 n 14 i s 19 Range fiaai «p 1 4 ? 2 1 1 2 8 8.3 4-13.80 9 jgtsufitii 21 1 4 .0 8 -9 .1»8 10* 1 2 3 2 1 1 2.96-5.J»8 R. tlg rin a 4? 2 1 1 l l . 36- i 3.38 5** 5 6 .^ 8 —6.80 gttentbfld 1? 1 15.32 2 * 1 1 5 .7 6 -7 .0 1 4 8 4 9 13 16 11 3 5 1 4 .8 8 -1 6 .7 2 6 3* 7 21 21 10 3 2 3 .1 6 -8 ,1 3 T o ta l Rana 98? 2 4 8 5 11 15 17 15 12 7 l 1 4 .0 8 -16 .72 9 6 * 1 9 28 27 20 8 3 2 .9 6 -8 .1 3 132. Table of seasonal variation found in body length of B. n elen o stietu s Appendix 10 Males Females January end February Place # Average Range # Average Range T aipei 12 6.44 **.50-7.25 8 13.41 8,16-22.80 Tsoylng 8 5.13 4.32-6,48 3 10,48 8.19-11,70 May and June T aipei 9 7.44 6.06-924 11 13.50 8.20-19,36 T aipei 14 **.98 3.52-6.80 19 8.16 5.76-12.00 Namru 21 6.96 5.72-9.99 20 10.69 6.56-16.72 Namru 21* 5.45 4.28-7.52 12 10.06 9.20-13.68 Tounan 112 6.22 4.38-12.51 168 10.90 5.85-22.65 Tsoylng 20 5.12 3.76-5.92 19 8.16 5.76-12.00 July and August T aipei 2 5.31 5.16-5.46 T aipei 1* 5.6? 3.44-7.84 33 11,42 7,760-17.08 T aipei 1 12.52 T aipei 5 6.11 5.68-7.84 8 11.17 6.92-18.80 Namru 31 6,29 4,12-8.60 21 11.36 5. 00- 16.6 Grass Mt, 7 7.3** 3.52-10.60 11 15.49 12,22-20.1< Tung Foh 3 5.93 5.2-6.96 5 10.12 5.75-13.40 Sun Moon Lake 6 7.98 6.99-10.41 1 13.80 November and December T aipei 20 5.12 3.76-5.92 16 8.32 5. 20-11.52 Table of seasonal variation found in Rana In the North, Appendix 11 Males Females Location # Average Range # Average Range J anuary-February Tung-Foh-Lee 1 if. 68 T aipei 10 5.09 if ,2if—6 20 1 16.72 May and June Taipei 2 6.7*1- 6,if8-6 80 19 8 ,lif 5.28-10.88 Namru - 2 7 5.3^ if. 80-6, 2 Grass Mountain 2 5.00 if. 88-5 12 2 10,18 7.08-13.28 Eeelung 3 8.77 7.60-9, 6if Eeelung 10 if, 61 3.16-5 76 12 9.12 5.76-11.36 N ovember-Deoember T aipei 8 if. 10 2.96-5 if8 1 9,if80 Tung-Foh-Lee 1 7.68 Eeelung 5 if. 32 2 11,22 9.32-13.12 Table of seasonal variation found In Rana fro* the South. Appendix 12 Hales Females Location # Average Range f Average Range January and February Sun Moon Lake 7 6.69 5.12-8.13 3 10.48 9.20-12.76 Sun Moon Lake 5 5.72 4,88-6.24 1 4,08 Sun Moon Lake 1 6,32 2 10,14 9.68-10,60 Sun Moon Lake 8 4.17 3.44-4.60 8 7.90 6.24-11.20 May and June Wu-Shih 1 7.96 Tounan 9 7.57 4.83-7.96 12 11,9 9.45-12.90 July and August Tounan Tsoylng 3 6.63 6.52-6.80 2 12.37 11.36-13.38 Suaaary Table of widths of Taiwan specimens. Appendix 13 Width in mm. # .20 .10 .40 -50 .60 .70 .80 -90 1.6 1.1 1.2 1.3 1.4 1.5 1.6 1-7 1.8 fantt. #1 102? 7 14 29 13 21 11 7 .840-1.44 63 c? 1 4 16 18 9 10 2 3 .690-1.35 #3 3 ? 1 1 1 1.14-1.53 5^ 2 2 1 1 .90-1.2 #4 5? 1 1 1 1 1.08-1.83 3 #5 3 ? T 1.32-1.35 i 2 3 ? 1 1 13 2 3 2 1 .69-1.38 12 ^ 3 3 3 3 . 69-.9 9 #7 16? 3 3 5 5 .90-1.23 lfltf- 5 9 1 3 .87-1.14 #8 169 1 4 3 4 1 1 1 1 .90-1.68 10 # 2 1 3 2 2 .90-1.38 #3-8 66? .1 1 17 10 13 11 6 2 2 1 1 1 .69-1.68 4 3 3 8 18 2 8 4 3 .69-1.38 %)ZX Summary Table of Widths of BHla specimens. Appendix 14 Width in non. t ___ ^ 2 _ .W *3) .6 0 .70 .0 0 .90 1.0 1.1 1*2. -U it 1 .6 1 .Z 1, •0 1 .9 2*1 2.2 fiangft # 1 - 8 1 6 8? 1 1 7 31 39 2 6 32 17 9 2 1 1 1 .69-1-68 1 2 2 o’ 4 7 24 36 1 1 1 8 6 6 .6 8 -1 .3 8 tic tu a North 1 8 1 ? 2 2 14 14 29 19 31 17 21 10 11 3 4 2 1 1 .4 8 -2 .2 2 0 0 4 6 24 14 6 2 35 4 0 7 4 1 3 .4 4 -1 .4 8 B .eel anna. tle tu a 1 ? 1 1 .2 South 1 3 1 .7 6 -.9 1 B.melan na. tic tu a 1 8 2? 2 2 4 14 29 19 31 17 22 10 11 3 4 2 1 1 .4 8 -2 .2 No.&So. 205 tf 4 6 25 14 65 36 hO 7 4 1 3 .4 4 -1 .4 8 T otal £ . melanoa- 3 5 0 ? 2 2 15 15 36 50 70 43 54 27 20 5 5 1 3 1 1 .4 8 -2 .2 tlc tu a 317 & 4 6 29 2 1 89 72 51 25 10 7 .4 4 -1 .4 8 fiibanto- 3 renaia 8 t 3 3 1 1 .8 4 -1 .7 6 North 8 d> 1 1 1 2 2 1 .6 8 -1 .6 4 B.hanko- renaia 1 2 ? 4 1 3 2 1 1 .0 4 -2 .8 Central 19 o* 1 1 6 2 2 2 3 1 l .6 4 -1 .5 B.banko- ranaia 2 0 ? 3 7 1 3 2 1 1 1 .8 4 -2 .1 No.&So. 27 d> 2 1 7 2 1 2 4 3 3 1 1 .6 4 -1 .6 4 B.bankoe renaia 5 ? 2 1 2 1 .2 -1 .5 A liahan renaia 2 5 ? 3 7 1 5 3 1 2 1 1 .8 4 -2 .1 T otal 3 3 c? 2 1 7 2 3 4 6 3 3 1 1 .6 4 -1 .6 4 Suxaaiy Table of widths of species of Rana Appendix 15 #_ ^20 .30 .40 ,50 .60 .70 .80 .90 1.0 1.1 1.2 1.3 1 .4 1.5 1.6 1.7 1-8 fiangft fiADft. *P* 1 4? 2 3 6 2 1 .81-1.26 10 ^ 15 12 1 .75-1.38 &• landfiEBA 2? 1 1 .80-1.8 3tf> 1 1 l . 60- 1.8 R. latouche 2 ? 1 1 .90-1.64 10 d* 2 2 2 2 1 1 .60-1.6 &. tiorina 4? 12 1 .76-1.24 5 c? 1112 .72-1.2 &• gaanthari 1 ? 1 1.6 2 c*1 11 1.04-1.2 flawtert 3* 111 .47-78 &• iimQfiharia 7 5 ? 1144 13 8 17 9926 1 " .44-1.48 66 1 3 11 16 12 9 11 1 2 .40-1.4 T otal 98? 1 1 5 4 16 1226 11 1 1 2 6 2 .40-1.4 96 & 2 5 15 19 21 10 17 1 3 1 2 1 .4 0 fi/4 Table of Proboscis length of specimens arranged by geographic location. Appendix 16 Female Hale # .20 .30 .40 .50 .60 Nean Range .4at .20 .30 .40 .50 Mean Range B.melanos- tictus Namru 2 16 4 10 2 .439 .346-539 23 20 3 • 358 .308-439 Taipei 53 16 24 12 1 .441 .323-.616 64 6 45 13 .365 .254-.447 Grass Ht. 8 5 1 2 .414 .346— 516 12 1 8 3 .358 .231— 462 Total- North 77 25 35 16 1 .438 .323-.616 99 7 73 19 .363 462 Central 2 1 1 .378 .331-.424 1 1 .254 North Ft Central 78 26 36 16 1 .323-.616 100 8 73 11 .231— 462 Tounan 171 15 115 41 .462 • 330-597 102 53 49 .403 .309-.494 Central & South 248 41 151 57 2 .323-616 201 7 126 68 .231-494 3. banko- rensis AliShan 4 4 .412 .402-.422 North & South" 14 4 9 1 .432 •346-.505 14 12 2 .368 .308-424 Total 14 4 9 1 .432 .346-505 18 12 6 .376 .308-.424 Rana lin- nocharis North 23 7 13 3 .426 .316-516 9 7 2 .360 .300-.424 Rana-other 4 2 1 1 .418 .362-539 5 4 1 .356 .316-354 Total - North 27 9 14 4 .424 .316-539 14 11 3 .359 .300-.424 Total - 0 Central 24 2 7 11 4 .438 .252-616 y 7 2 .357 .308-.462 Rana South 2 1 1 .466 .421-.500 3 2 1 .378 .347 -.416 Rana-lin- norcharis 31 o 19 14 1 .428 .308-.6l6 18 14 4 .359 .300— 462 All Rana Central F: n South 26 3 c 11 h .440 .262-616 12 7 4 1 .363 .308— 462 1 5 9 . Table of Probosols Length arranged by epeoles. Female Appendix 17 1 * .2 0 /. 30 .40 •50/.60 1 Range #1 108 9 73 19 .*57 .330-.597 3-8 70 6 42 22 .470 .3*0 -.587 1-8 171 15 115 41 .462 .330-.597 B. melanos- tio tu s 79 26 36 16/1 .441 .326-.616 B. banka- ren aia 14 4 9/1 .*32 .346-.505 R. lin n o - o h a ris 30 7 19 3/1 .*33 .316-.616 R. 1 one- o ra s 3 1 1 1 .*33 . 362-.539 R. tlgrina 2 1 1 .466 .431-.500 R. lato u o h i 2 1/1 .312 .262-.362 Rana ap. 15 1 11 3 .462 .381-.556 Total Rana 52 1/10 32 8/1 .*38 . 262-.616 Hale # .20/.30 .40 , 50/.60 Average Range #1 53 31 22 .388 .309-.*9fc 3-8 *9 22 27 .390 .309-.*50 1-8 102 53 49 .389 .309-.49** B. nelanoa- tio tu s loo 8/73 19 .397 ,231-.462 B. banka- ren aia 48 12 6 .376 ,308-,424 R. lim no- oharla 17 13 4 .353 .300-.462 R. lone- e w 2 2 .350 .3*6-.35* R. tierlna 5 4 1 .35* .316-.*16 R. latouohl 1 1 .*39 ftana sp. 8 4 3 1 .397 ^309-.515 Total Rana 33 23 9 1 .370 .300-.515 Table of Proboscis width of specimens arranged by geographic location. ‘ppendix 18 Females Kales a # .10 .20 .30 .tt> .50 Kean Range IT .10 .20 • 30 .1+0 Kean Range B. melanos- tic tu s Namru 2 16 3 11 2 .3^5 .231-. 1*62 23 18 5 .279 .231-. 31+6 Taipei 53 1 20 31 1 .304 .169-.385 63 1+ 50 9 .261 .177-.3b6 Grass Mt, 8 b 3 1 , 32b .228-. 1+08 12 1 7 b .269 .162-. 31+6 North 77 1 27 b5 4 .315 .169-. 1+62 98 5 75 18 .266 .162-.31+6 Central 2 1 1 .228 .185-.270 1 1 .251+ Tounan 171 69 99 2 1 .312 .206-.5b6 102 3 91 8 .255 .196-.360 Total 3 ^ 3 99 112 6 1 .169-.5b6 210 10 181 5b 2 .162-.360 B. banko- rensis AliShan b 3 1 .376 .360-. 1+12 North ?: South 20 . b 11* 2 .1*32 .231-. 1+71+ 23 15 8 .280 .200-.319 Total 20 b lb 2 .lt32 .231-. 1+71+ 27 15 11 1 .295 .200-. 1+12 R. limno- charis North 23 3 20 .319 .262-. 31+6 9 7 2 .282 .239-.323 Rana-other 3 1 .281 .251+-. 31*6 5 2 3 .288 .238-.308 Total-North 2 7 6 21 -313 .251*-. 31+6 11+ 0s 5 .281+ .238-323 Total-Central 2b 1 10 13 .296 .231-.361 9 7 2 .273 .231-. 31+6 Rana-South 2 1 1 .369 .331-008 3 3 .296 .277-.293 Rana-1dm- norcharis 31 8 23 .311 . 2 3 1 - 3 ^ 18 lb 1+ .277 .231-. 31+6 Rana-total 26 1 11 11* .231-. 1+08 12 '10 2 .279 .231-.293 Table of Proboscis length arranged by species. Appendix 19 Female Hale # # ..20/^0 *40 - #1 108 9 73 19 .457 •330-.59? 53 31 22 .388 .309-.494 #3-8 70 6 42 22 .470 . 340-.587 49 22 27 .390 .309»*450 #1-8 171 15 115 41 ,1*62 /330-.597 102 53 49 ,389 .309-.494 79 26 36 16/1 .441 . 326—.616 100 8/73 19 .387 .231-.462 14 4 9/1 .432 .3*t6-.505 48 12 6 .376 .308-.424 R.limgfiharia 30 7 19 3/1 .433 •316-.616 17 13 4 .359 .300-.462 3 1 1 1 .433 . 362-.539 2 2 .350 .346-.354 R- tim pina 2 1 1 .466 .431-.500 5 4 1 .354 .316-.416 2 1/1 .312 .262-.362 1 1 .437 fiaai 8p. 15 1 11 3 .462 .381-.556 8 4 3 1 .397 .309-.515 T otal Rana 52 1/10 32 8/1 .438 .262-.616 33 23 9 1 .370 .300-.515 Table of host associated Variation in hook count Appendix 20 # of longitudinal rows of hooks Hooks per row t 1 3 1 4 1 5 1 6 17 IB 19 20 21 22 ZLJ& t 4 4/5 5 5/6 6 6/7 7 #1 a 92 1 1 9 32 25 17 5 90 16 19 43 5 2 5 16 15 7 6 52 1 25 18 7 3-8 g. 71 2 7 10 14 20 13 5 72 1 7 22 36 4 5? 48 4 5 11 11 9 7 1 48 21 16 10 1 1-8 o 163 1 2 8 19 46 4-5 30 10 162 1 23 41 79 < c?100 4 7 16 27 25 14 7 100 1 46 34- 17 ] £ . gg]g|^ o stic tu a 60 2 6 16 13 14 5 3 1 59 2 5 19 27 5 118 1 l 4 IS 26 32 25 8 3 84 5 27 25 24 3 Saba $ 50 1 4 7 12 10 9 7 46 9 13 23 1 <$l18 3 7 3 16 14 3 2 34- 2 15 14 4 1 B.banko- renaia g 20 1 4 5 6 2 1 14 1 1 2 6 4 o*36 2 3 12 7 5 7 16 2 l 11 2 T otal ?300 1 1 8 23 56 78 71 4-5 13 288 3 41 83 136 15 10 0*319 2 1 13 41 53 94- 72 32 12 270 1 13 107 96 48 5 1 21? 21? T Size of lemnlsci In comparison to the proboscis receptacle. Appendix 21 #1 3-8 B. banko- B. melanoa- Rana rensis tiotus Shorter than reoeptaole 5 11 6 9 3 3 8 27 5 24 Equal 1,0 U 4 6 2 3 6 7 22 16 26 Longer 1.1-1,5 72 56 26 44 5 26 104 136 68 70 1 .6 -2 .0 61 38 39 31 7 21 21 41 12 21 2 ,1 -2 ,5 37 18 10 1 12 3 2 3 5 2.6-4.75 1 11 1 (Approaoh 1.0 , lemnisci and proboscis reoeptacle equal length. Under 1.0 lemnisoi smaller than proboscis reoeptaole. Over 1.0 lemnisoi longer than proboscis reoeptaole.) 1 4 4 , Size of lemnisoi compared to each other. Appendix 22 #1 3-8 B. banko- B. aelanos- Rena rensis tictua ,10-,19 1 .20-.29 2 .30-.39 1 .40-.49 1 1 2 .50-.59 2 1 1 2 1 .60-69 1 1 3 2 1 1 .70-.79 7 5 4 2 2 6 4 .80-,89 30 12 17 10 3 13 12 13 13 17 .90-.99 58 32 26 4-5 2 13 18 18 23 26 1.0 8 3 1 4 7 11 5 20 STAINING PROCEDURE Appendix 23 After m m hare been fixed In ale ohol-f oraalin- ac e t ie aold mixture or other fixative for a few hours or overnight the speoinens may be transferred to 70$ ethyl alcohol with 3$ glycerin added. Each specimen must be cate fully pricked through the body wall in several places. Staining then proceeds as followst 1. In the morning (i.e. before 9 A.M.) replace alcohol with undiluted Grenaoher’s borax carmine, 2. In the late afternoon carefully add one drop of concentrated HC1 for eaoh 5 ®1 of stain. Quickly mix by inverting vial several times. 3. The next day (preferably in the morning) replace the stain w ith 1$ HC1 in 70 $ alcohol and destain until a light pink oolor has been obtained. During the destainlng prooess re place aold aloohol frequently, 4. After destainlng is completed, transfer to 8 % aloohol for 18-24’ hours. Be sure to change the 8336 aloohol several times during this period to remove excess aold, 5. Transfer to 95$ aloohol for 6-18 hours 6. Transfer to lOOJf alcohol for 6-18 hours 7. Transfer to 100$ aloohol terpineol 23$ terpineol for 6-18 hours 5056 terpineol for 6-18 hours 73$ terpineol for 6-18 hours 8. Transfer to pure terpineol for 18-24 hours 9. Mount worms in thick perm cunt and apply ooverslips. •BIOGRAPHICAL DATA Name In Falla Franeella Alice Woods Data of Birtht December 17, 1927 Plaoa of Birthi Union City, New Jersey Secondary Education: Austin Cate Academy, Center Strafford, New Hampshire Collegiate Institutions Attended: Dates Degree Providence Bible Institute 191* 5-191*6 University of New Hampshire 1948-1951 b ; S. University of New Hampshire 1951-1952 M, S, University of New Hampshire 1963-1969 Ph. D. Positions Held: Science Teaoher, Morrison Academy 1960-1962 Taichung, Taiwan Graduate Assistant in Biology 1963-1964. University of New Hampshire Assistant Professor of Biology 1964-1968 C hrist's College, Taiwan (5) (3) (23) (16) (if9) (if9) specimens (51$) * The overall range was slightly larger than that of the 49 pooled speoimens from Tounan mentioned above. Among the pooled speci mens 33# mere found In th e 5 nm node. The pooled forms had a g re a te r concentration In the larger measurement groupst e.g ., 20$ mere 8 mm, whereas in the parasites from host #1 there was only slightly over one percent at this length, When fewer parasites were present, there was a comparable increase in sise, which I believe demonstrates the crowding