This dissertation has been microfilmed exactly aa received 70-6746
CHRISTIAN, Frederick Adetokunbo, 1939- LIFE HISTORY STUDIES OF SPINITECTUS MICROSPINOSUS SP. N (NEMATODA:RHABDOCHONIDAE) WITH EMENDATION OF THE GENUS SPINITECTUS FOURMENT, 1883.
The Ohio State University, Ph.D., 1969 Zoology
University Microfilms, Inc., Ann Arbor, Michigan LIFE HISTORY STUDIES OF SPINITECTUS MICROSF1NOSUS SP. N
(NEMATODA:RHABDOCHONI DAE) WITH EMENDATION OF
THE GENUS SPINITECTUS FOURMENT, 1883
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Frederick Adetokunbo Christian, B.Sc., M.Sc.
#*»*#**
The Ohio State University 1969
Approved by ACKNOWLEDGMENTS
The author wtshes to express his sincere thanks and appreciation to all the people whose help, kindness, cooperation and suggestions have made these studies possible.
I am Indeed very grateful to my academic adviser. Professor
J. L. Crltes for his helpful suggestions, patient assistance, unre lenting encouragement, and constructive criticism throughout the course of these studies. I also thank the reading committee,
Professors Carl R. Reese and L. S. Putnam.
Words will be inadequate to express my sincere appreciation to Mr. and Mrs. R. Seeds and family who spent their time, money, and energy to assist me in the col lection of the fish, LepomIs macrochIrus. from their privately-owned pond in North Ridge Township,
Fairfield Co., Ohio.
I wish to thank Messrs. Rennie and Ronnie Fowler for their assistance In the collection of the Asellus Intermedlus. I also wish to thank Messrs. R. Burnard, D. Radabaugh, A. White, and J.
Zapotosky for taking the photographs of the nematodes, and Mr.
Lawrence Negulesco for his ever ready help in the collection of the fishes.
I wish to thank the Academic Faculty of Zoology, College of
Biological Sciences, The Ohio State University, for giving me the opportunity to study here and for providing the facilities that made
these studies possible. t| VITA
July 8, 1939 ...... Born - Lagos, Nigeria
December, 1962 ...... B.Sc., Allen and Benedict College, Columbia, S. C.
1963 - 1965 ...... Graduate Teaching Assistant and Associate, Biology Department, Wayne State University, Detroit, Michigan
1965 ...... M.Sc., Wayne State University, Detroit, Michigan
1965 - 1969...... Teaching Associate, Academic Faculty of Zoology, College of Biological Sciences, The Ohio State University, Columbus, Ohio
PUBLICATIONS
"Lanqeronla parva sp. n (TrematodatLecithodendrlIdae) from Rana pip lens with a Revision of the Genus Lanqeronia Caballero y et Bravo-Hollls, 1949." (Manuscript in Preparation)
FIELDS OF STUDY
Major Field: Zoology
Studies in Invertebrate Zoology. Professor J. L. Crites
Studies In Parasitology. Professor J. L. Crites
Studies in Veterinary Parasitology. Professor F. Koutz
Studies in Acarology. Professor G. W. Wharton
Studies In Protozoology. Professor J. P. Kreler
Studies in Medical Entomology. Professor C. Venard
Ml TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... H
VITA ...... HI
LIST OF T A B L E S ...... vl
INTRODUCTION ...... I
HISTORICAL BACKGROUND...... 4
History of the genus Spin!tectus ...... 4 General Patterns and Life rflstories of Some Spiruroid Nematodes ...... 13
MATERIALS AND M E T H O D S ...... 16
Examination of the Fish for Nematodes and Preparation of Nematodes for Study ...... 17 Arthropods Used in the Trial Feeding Experiments ...... 19 Collection and Laboratory Rearing of Asellus Intermedius . 19 Definitive H o s t ...... 20
GENERAL INFORMATION ...... 22
Information on Egg Types of Spiruroid Nematodes ...... 22 The Larval Stages (=Juveniles or Postembryonlc Stages) . . 23
THE LIFE HISTORY STUDIES OF SPINITECTUS SP...... 25
Reproductive Habits ...... 25 Feeding and Infection Experiments ...... 26 Description of the Eggs and Developmental Stages in the Life History S t u d i e s ...... 29 Description of the Fully Mature Adults of Spinltectus sp. 37 Remarks on the Life H i s t o r y ...... 43 Summary of the Life History Studies ...... 48
SYSTEMATIC REVIEW OF THE GENUS SPINITECTUS ...... 53
lv TABLE OF CONTENTS (CONT.)
Page
EMENDATION OF THE GENUS SPINITECTUS AND THE FAMILY RHABDOCHONI DA E ...... 58
DESCRIPTION OF NEW S P E C I E S ...... 60
M a l e ...... 61 F e m a l e ...... 62 Discussion...... 63
SUMMARY ...... 67
APPENDIX Plates and Explanation of Plates ...... 71
REFERENCES C I T E D ...... 90
v LIST OF TABLES
Table Page
1. Species, Hosts and Geographical Distribution of the Genus Splnltectus...... 9
2. Number of Species of Spln|tectus According to Countries . 12
3. Measurements of Splnltectus sp...... 42
4. Outline of Life Cycle of Spin I tectus sp...... 52
5. Comparison of Species of Splnltectus reported from Freshwater Fishes of North America ...... 66
vl INTRODUCTION
This study was undertaken by the author at the suggestion of his
Academic Adviser, Dr. J. L. Crites who observed the present Investiga tor's Interest In attempting to survey the helminth parasites of certain freshwater fishes In Ohio. Examination of many centrachld fishes from different counties In Ohio revealed the presence of some species of spiruroid nematodes belonging to the genus SpInltectus In the intestines of these fishes.
A comprehensive review of the world literature shows that the works concerning genus Spin!tectus are predominantly taxonomlv. and ecological In nature except for a pioneering study by Gustafson
(1939) on the life cycle of SpInItectus gracIlls. This, In Itself, is a very short note published as an abstract and details of this study are lacking. This study by Gustafson (1939), although meagre, is the only available Information on the life cycle of any of the described species of Spin!tectus. So, at the suggestion of the present
Investigator's academic adviser, the present study was undertaken to elucidate the details of the life cycle of another species of the genus Spin Itectus.
The genus Splnltectus erected by Fourment in 1883, belongs to the superfamfly Splruroldea, a group of parasitic nematodes that are known to require In their life cycles an arthropod Intermediate host
I 2 and a vertebrate definitive host. Two species have been described
for the genus Spin Itectus from North American freshwater fishes.
These are S. gracilis Ward and Magath, 1917, and S. Carolina Hoi I,
1928. Anthony (1963) mentioned another four species but did not name or describe them. The two described species, S. gracilis and S^. Caro
lina, have been reported from a large number of genera of North Ameri can freshwater fishes from many different localities In the United
States and one specimen of S^. carol Ina has been reported from an
amphibian host, Trlturus vlridescens by Hoi 1 In 1928.
Review of world lit* “ature shows that 29 species have been
described and named for the genus Spin!tectus. Of these only 26 are
now considered valid. As mentioned above, no details of complete life
cycle are known for any of these species. Gustafson (1939) found that mayfly larvae of the genera Hexagenea, Heptagenea, and Streptonoura may serve as Intermediate hosts of S_. gracl 11s, and he utilized the green sunflsh, Lepomls cyanellus as a definitive host.
Yamagutl and Ntslmura (1944) found what they believed to be a
specimen of the larva of Splnl tectus gig I Fuljlta, 1927 fr. a crus
tacean host, Caridina denticulata.
The above constitutes the only available Information concerning
the life history studies of any of the known valid species of the
genus Splnltectus. Therefore, the present study was undertaken In an
effort to work out the details of the life history of S_. caroIina, the
second described species from the North American freshwater fishes.
In the course of this study, a l'~ge number of Bluegllls, Lepomls 3 macrochIrus, from different localities In Ohio were examined. A hitherto undescribed species of the genus Spin Itectus was encountered
In the Intestine of these fishes and the author directed his efforts to working out the details of the life history of the new species encountered.
The objectives of the present life history study therefore were: to find out what arthropod Intermediate hosts might be Involved in the life cycle; to describe the mode of Infestation by the nematode to both the Intermediate and definitive hosts; to describe the growth and development of the nematode In both hosts; to find out the length of time from one juvenile stage to the next, and egg to egg cycle; to study the host-paraslte relationship.
The proper systematic position of the genus Spin Itectus has been a matter of differences of opinion. So, the present systematic position of this genus will be reviewed and the genus emended in the
light of what is known concerning the described species of the genus.
Emendation of this genus will also necessitate emendation of the family to which it is now assigned.
The new species, Splnltectus sp., the life cycle of which is worked out herein, will be described, compared with and contrasted to the other described species, gra d 11s and S^. carol ina from the freshwater fishes of North America. HISTORICAL BACKGROUND
History of the Genus Spin Itectus.
Fourment (1883) established the genus SpInltectus with Its type species S. ovlfI age 11 Is for some spiruroid nematodes he recovered from the Intestine of marine mackerel fish, Merlangus vulgaris. The type species S. ovlfI age I I Is was described from female specimens only when
Fourment (1883) erected the genus and Its type species. Fourment, In
1884, gave a more detailed morphological description of the female specimens and eggs of S. ovlfI age Ills. This Is the only species of
Spinitectus known to have been described with eggs having polar filaments. Schuurmans-Stekhoven (1935) also recovered female speci mens of S. ovlfI age 11 is from Gardus meriangus on the French coast of the North Sea.
Rahman (1964) recovered both male and female specimens of ovlfI age I I Is from the Intestine of marine whiting, Merlangus vulgaris
(= Gardus merlangus) collected off the west coast of Scotland, He gave a detailed morphological description of the male specimens and compared them with the males of other described species of the genus,
S^. Inermis and cr I status, both of which were also found In sea-fish of Europe and America.
Rudolph, In 1809, described and named some spiruroid nematodes he recovered from eels, AngulI la vulgaris, as Liorhynchus dentlculatus but these nematodes had been named Soezla Inermis by Zeder In 1800. It was later discovered that G. Inermis actually belonged to the genus Spin!tectus, hence Goezla becomes a synonym of Splnltectus In part and according to Schneider, Stiles, Hassall, and Rail I let in
"Yorke and Maplestone's Nematode Parasites of Vertebrates, 1926", the genus Llorhynchus Rud., 1801, has for Its type an undeterminable species L. truncatus (Rud., 1873) and should therefore be abandoned.
So both Goezla and Llorhynchus become synonyms of Splnltectus In part and both Llorhynchus dentlculatus Rd., 1809, and Goezla Inermis Zeder,
1800, become Splnltectus Inermis (Zeder, 1800). Neveu-Lemalre (1927) redescribed S. Inermls in more detail.
Llnstow (1878) described a species as FIlarla echlnata from
Alburnus lucldus and AngulI la vuIgaris. This species undoubtedly belongs to the genus Splnltectus as RalI I let and Henry (1915) thought and rightly so. According to Morlshlta (1926) this species Sp1n1tec tus echlnatus (Llnstow, 1878) seems to be synonymous with S^ oviflag- el Us The species has also been referred to as a larval form of $_.
Inermis by York© and Maptestone, but It Is now considered as a valid species of Splnltectus as determined by RalI I let and Henry (1915).
Stewart, In 1914, described a single male specimen of spiruroid nematode he called SpIroptera dentlculata var. ml nor from WalI ago attu
In India, but this was subsequently established as a distinct and valId species of Splnltectus designated as Spin Itectus minor by Bay I is
In 1936 and was redescrlbed by All In 1956. RalI I let and Henry In 1915 renaned FIlarla serrata of Linton, 1901 as Splnltectus crI status.
Ward and Magath In 1917 described and named Splnltectus gracilis 6
from some North American freshwater fishes and Gustafson (1939) did
a pioneering study on the life cycle of this species; S. gracilis was
redescribed In detail by Mueller and Van Cleave In 1932. The first
described species of SpInitectus from an amphibian host was S, ranae
described by Morlshlta in 1926 from the stomach of frogs Rana
nlgromaculata In Japan. This species was also redescribed by YamagutI
In 1941(a).
§P?P f tecuts gig I was described from fish In Japan by Fuljlta
(1927) and was later redescribed by YamagutI In 1941(b). Yamaguti and Nislmura (1944a) stated that they found what they believed to be
a juvenile of S_. gigl In a crustacean host Carldina denticulata.
Spin Itectus asper was described from Prochllodus scrofa In BraziI by
Travassos, Artfgas, and Pereira (1928) and they also described yorkeI from Plmelodella laterlstrlga In 1928 from BraziI. Hoi I
(1928) described carol Ina from some freshwater fishes In North
America; he also recovered one specimen of this species from an amphibian host, Trlturus vlrldescens, a salamander. Mueller and
Van Cleave (1932) redescrfbed thTs species in detail In their mag nificent work on the parasites of Lake Oneida In New York.
Bay I Is (1929) described guntheri from an unknown fish host
from a depth of 1000 meters off the coast of the southwestern part of Africa, but Campana-Rouget (1955), while working In Lakes Albert and Edwards In Africa, recovered nematodes very similar to or Identical to what was described by Baylts In 1929 as S^. guntheri and he reassigned this species to the genus Metabronema. Since Bay I Is1 specimens of S^. 7
guntheri are not available and can only be Identified by his descrip
tion, further work remains to be done to either validate this species
or justify Its removal and reassignment to the genus Metabronema as
proposed by Campana-Rouget (1955).
In India, Verma and Agarwala (1932) described S_. Indicus from
SIlurold fishes, PseudotropI us qarua and EutropIIchthys vacha. In
Brazil, Vaz and Pereira In 1934 described Sptnltecuts rudolphlherlnqi
from Plmelodella laterlstriga and Salmlnus hi lari I; and In the follow
ing year, 1935, Yamaguti described another species, mogurndae, from
Mogurnda obscura In Japan. Moorthy (1938), working in India, added
S. cortl from Ophlocephalus qachua to the 11st from Indla; and thIs
species was later redescribed by All In 1956.
Johnston and Mawson (1940) described three species of Splnltectus
from Australia; these are (I) S.. plectrop11tes (based on females only)
from P lectrop I i tes omblquus, (2) S^. percalates (based on males only)
f rort' Perea I ates colonorum and (3) S_. bancroft I from Mogurnda adspersus.
Accorolng to Khera (1956), S^. perca I aie * Is a synonym of £. p lectrop-
11tes.
Karve and Natk (1951) added three new species from India. These
species are (I) j5. notopterl from teleost fish Notop terus notop- terus. (2) S_. mastacembe 11 from the stomach of Mastacembelus armatus
and Notopterus notopterus, and (3) S . ne1111 from Barbus nelI 11.
Khera, In 1956, described another species, Spin Itectus major, from
Mastacembelus armatus In India. 8
Alf (1956), while studying the nematode parasites of fishes and birds of Hyderabad State In India for his doctoral dissertation, added four more species of Splnltectus to the list. All also redescrlbed those species previously but poorly or Inadequately described and provided a key to the species described up to that date. The new specios which All added are: (I) S_. armatus from Mystus tengara,
(2) S . longlpapi I latus from Rlta hastata, (3) S^. singhI from Masta- cembeI I us armatus, and (4) £. thapari from Notopterus.
Chakravarty, Sain, and Majumdar, in 1961, described Sp!nitectus benga lens I s frc;.i ine stomach of Notopterus notopterus In Calcutta,
India. Sahay and Prasad (1965) described Spin!tectus komlyal from
Eutropichthys vacha at Patna in the State of Bihar in India. They expanded on Ali's (1956) key to include additional species described up to 1965.but, unfortunately, some species were not Included. 9
Table t. Species, Hosts and Geographical Distribution of the
Genus Splnltectus.
Splnltectus spp. Hosts Loca1111es
S. ovlflaqellls Fourment 1883 Merlanqus vulgaris Not given - 5~. ov I f I age I I i s Rahman 1964 MerIanqus vulgaris Off the west coast of Scot land
2. S_. Inermis (Zeder, 1800) Eel, AnquII I a Baltic, vulgaris S. Europe
3. S^. echinatus (Llnstow. I878a ) Alburnus llcidus Not given
4. S. minor Stewart. 1914; WaI I ago attu and Lucknow, India Bay I Is, 1939 Molva molva
SL crlstatus Ra i111et Phycls tenuIs and Off Nantucket and Henry, 1915 Molva molva and Porcupine Bank
qracl11s Ward & Magath, Many genera and United States 1917; Mueller 4 Van Cleave, species of of America 1932 freshwater fIshes
7. S. ranae Morlshita, 1926; Rana nlqromacu- Japan Yamaguti, 1941
8. S. gfgl FullIta, 1927; Pelteobagrus Japan Yamaguti, 1935 nud I cepis
9. £. yorkeI Travassos, Artlgas, Pime I ode I I a BraziI and Pereira, 1928 I atarisfFlga
10. S. asper Travassos, Artlgas, Prochllodus Brazi I and Pereira, 1928 scr°fa
II. S^. carol Ina Hoi 1, 1928; Many genera and U.S.A. MueIler & Van Cleave, 1932 species of freshwater fishes 10
Table I. (Continued)
Splni tectus spp. Hosts LocalItles
l2* i* guntherib Bay 1 is, 1929 Unknown fish 1000 meters depth off coast of S.W. Africa
13 . S. indlcus Verma and Eutroplchthys India Aga rwa1, 1932 vacha and Pseudotroplus garua
14 . S. rudolphlheringl Vaz Pime lode 11 a BraziI and Pereira, T934 laterlstriga and Salminus hi Iar"i i
15. S. mogurundae Yamaguti, 1935 Mogurnda obscura Japan
16 . S. cortl Moorthy, 1938; Ophlocephalus India All, 1956 gschua
17 . plectrop11tes Johnston Plectrop11tes Aus+raI la and Mawson” T940 amblguus
18. S. percaIatesc Johnston Percalates Austral la and Mawson, !940 colonorum
19 . $_. bancroftel Johnston Mogurnda adspersus Australia and Mawson, 1940
20. S. notopterl Karve and Notopterus India Nalk, 1951 notopterus
21. S. mastacembel1 Karve Mastacembelus India and Nalk, n?5l armatus and NoTopterus notopterus
22. S. net 111 Karve and Barbus nel111 India Nalk, 1951 Table I. (Continued)
Spinttectus spp. Hosts Localities
23. S. major Khera, 1954 Mastacembe1 us India armatus
24. S. armatus All, 1956. Mystus tengara Hyderabad Deccan, India
25. S. longipapi1latus Ali, 1956 Rita hastata Hyderabad Deccan, India
26. S. singhi Ali, 1956 Mastacembelus Hyderabad armatus Deccan, India
27. S. thapart All, 1956 Notopterus Hyderabad notopterus Deccan, India
28. S. bengalensis Chakravarty Notopterus Calcutta, et a 1, 1961 notopterus India
29. S. komiyai Sahay and Eutorplchthys Patna, In Stc Prasad, 1965 vacha of Bihar, India
30. S. sp. Christian, 1969 Lepomls macro- Ohio, U.S.A. chi rus and Lepomi s cyanelI us
a echlnatus is considered juvenile stage or form of S. inermis by Yorke and Maplestone and as a synonym of S. ovlflageliis Fy Morishlfa (1926). “
k 1L* 9unther t Bay I Is, (929, has been transferred to the genus Habronema by Campana-Rouget (1955), hence is no longer a Spinitectus sp.
c According to Khera (1954), ji. percalates is a synonym of S.* p lectrop I jtes. 12
Table 2. Number of Species of SpIni tectus According to Countries.
13 species have been described from India
4 species (this report inclusive) have been described from U.S.A.
3 species have been reported from Australia
3 " " " " " Brazil
3 ” " " " " Japan
I " has " " ” Scotland
I " " " fl M Southern Europe
I " " " " ” S.W. Africa
I " ” " " with no locality given 13
Generaf Patterns and Life Histories of Some Spiruroid Nematodes
The suporfamtly Spiruroidea Rail I let and Henry, 1915, contains a number of parasitic nematodes of alimentary canal, respiratory system, orbital, nasal or oral cavities of verTebrates. In all of the known life histories of spiruroid nematodes, arthropod Intermediate hosts are involved.
Eggs are embryonated at the time of ovlposition. The embryonated eggs are ingested by the arthropod Intermediate hosts in which the first stage juveniles hatch and further development of the Juvenile worms to infective stage occurs. The vertebrate definitive hosts become Infected by ingesting the arthropod intermediate hosts harbor ing the ensheathed third stage juveniles. The third stage Juveniles are released in the vertebrate host’s intestine where they remain or migrate to specific sites In the body and later undergo ecdyses and develop to adults.
Brumpt (1931) demonstrated that Blatella germanfca and nymphs of Rhyparobia madera act as the Intermediate hosts for Protospirura bonnet, a spiruroid nematode of rats. He reported that the juvenile worms were found in the digestive tracts of these insect Intermediate hosts; and that these juvenlle worms later burrowed through the intes tinal watt into the haemocoel of the Insects. In the haemocoel they move around, become encapsulated and undergo ecdysls to become third stage juveniles. He then fed the Infected Insects to laboratory- reared rats In which the Juvenile worms ecdysed and grew to adults. 14
Burnett et at. (1957) reported In the case of Thelazla cat 1fornl- ensls a sptrurotd nematode found In the conjunctiva of dogs, deer, cats, and sheep, that muscotd dtptera Fannla canlcularls and Fannta banjamlnt serve as Intermediate hosts. Natural Iy-Infected Fannla spp. with ensheathed juvoniles of these worms were found. Experimentally and naturally Infected Fannla spp. were fed to uninfested dogs, deer, cats, and sheep, and the adult worms were recovered In the eyes of these vertebrate definitive hosts some days after. Petri (1953) showed that german cockroaches, Blatella germanlca, serve as the
Intermediate hosts or vector of Physaloptera rara. He also success fully Infected flour bettles TrIboll urn confusum In which he recovered the third stage juveniles. These Infected roaches and beetles were fed to uninfected cats and dogs In which the juvenile worms developed to adults.
Schell (1952) reported the juveniles of Physaloptera hisplda developed to the Infective stage In german cockroach (Blate I I a germanlca) and that the normal definitive host for this spiruroid nematode parasite Is the cotton rat (Sigmodon hlspldus); and he reported that there were tissue reactions of the Intermediate host to the para site, He also showed that the activities of the parasites cause local destruction of epithelial cells of either the colon or rectum of the cotton rats.
Gustafson (1939) did a pioneering work on the life cycle of
Splnltectus gracilis. He found that the mayfly larvae of the genera
Hexaqenea, Heptagenea, and Streptonoura Ingested the embryonated eggs 15
Spin}tectus gracilis and the juvenile worms were found In the haemocoels of these Insect larvae, showing specific characteristics within 8 days. He fed the Infected mayfly larvae containing 11 —day— otd juvenile worms to the green sunflsh Lepomls cyanellus and recovered young adult nematodes of graclIlls from the Intestines of the fish three days later MATERIALS AND METHODS
Freshwater fishes: Blue gills, Lepomls macrochtrus; green sun- fish, Lepomls cyanellus; white crappfes, Pomoxls annularis; black crapples, Pomoxls nlgromaculatus; small mouth bass, Mlcropterus dolo- roleul; largemouth bass. Mi cropterus salmoldes; pumpkinseed, Lepomls glbosus; madtom stonecat, Noturus flavus; yellow bullhead, I eta Iurus natal Is; black bullhead, Ictalutus me I as; and creekchubs, SemoteI us atromaculatus were collected by lines and seining from different local ponds, streams, and rivers In Franklin, Perry, and Fairfield counties
In Ohio.
Fishes were transported to the laboratory In coolers and fish- buckets and were held In fish tanks until ready for examination for the helminth parasites.
Examination of these fishes revealed the presence of many species of trematodes, cestodes, acanthocephala, and nematodes. Among the nematodes recovered are four species of Splnltectus. two of which had been already described and the other two are as yet undescribed.
One of these undescribed species Is found In targe numbers In the
Intestines of the bluegllls, Lepomls macrochlrus, collected from a privately-owned local pond belonging to Mr. and Mrs. Robert Seeds In
Snorttn Place, Snortln Ridge, Fairfield County, Madison Township, Ohio.
The pond Is thickly populated with blue-gltls, Lepomls macro chlrus, which Is the only species of fish present. There Is no
16 17 available Information as to the origin of this pond or the Introduction of the blue gills, lepomls macrochirus, Into It. Every one of the fish examined harbored 6-10 specimens of Spin I foetus sp. In Its
Intestine.
Examination of the Fish Host for Nematode and Preparation of Nematodes for Study
Mature males and females of Spin Itectus sp. were recovered from the intestines of the blue gills, Lepomls macrochirus. Gravid females and mature males are left In Ringer's "cold" solution overnight In small petrl dishes in which the gravid females oviposited. Reproductive habits and sexual behavior of these worms were observed by examining the males and females In the petrl dishes under a dissecting scope.
Eggs were also recovered In this way.
Sometimes, hundreds of eggs already laid while the worms were In the fish Intestine were found adhering to the intestinal mucus and contents, and sometimes eggs were separated from the fish faeces with the aid of dissecting microscope. So, eggs were obtained In two ways, either by allowing the gravid female worms to oviposit In small petrl dishes containing Ringer's "cold" solution or by microscopically separating of eggs from the Intestinal mucus and contents.
The embryonated eggs obtained as described above were used In the feeding or Infection experiments to determine the arthropod Inter mediate hosts Involved In the life cycle of this species of Splnltectus.
Worms were studied alive under cover slips In Ringer's "cold" solution as well as In the small petrl dishes for observation on 18 sexual behavior, morphology, formation of egg membranes, egg production, and egg laying. Adults and juvenile worms were relaxed In Ringer's
"cold" solution and fixed In warm 75^ alcohol or A.F.A. fixing reagent.
They were then gradually cleared In 1056 solution of g lycert n-alcohol which was prepared by mixing 90 cc of 15% ETOH with 10 cc of glycerin.
The specimens were then placed In a desiccator until the alcohol portion of the solution had evaporated and glycerin penetrated the worm, thus clearing the worm, leaving It suspended In the pure glycerin.
The entire tall of the male worm was cut off with a sharp razor blade while the worm was still alive and rolled to a ventral view under a cover slip. The caudal alae were spread out and the pap 1 Ma r arrangements were exposed. The entire tall was then fixed with A.F.A. fixing reagent under a cover slip and gradually cleared In 1056 glycertn-ETOH and mounted In pure glycerin for microscopic studies of the caudal alae; number, arrangement, and structure of genital papillae; and cuticular ridges.
En-face view mounts were also prepared by cutting off the lips of cleared nematodes with a sharp razor blade and mounting them In glycerln-jelly. These were later studied under the microscope for de fa11ed structures.
Cross sections of the fish Intestines containing the fourth and fifth stage juvenile worms and the adult worms were made. These
Intestines were fixed In A.F.A, fixing reagent; dehydrated through ascending alcohol series of 5056, 70J6, 8556, 95J6, and I00?6. They were cleared In xylene and Imbedded In paraffin and cross-sectioned at 19
10 and 7 microns and stained with Ehrllch's acid haematoxylln and eosin, and mounted in Balsam. Drawings were made with the aid of a microprojector, light compound microscope, and camera lucida.
Arthropods Used In the Trial Feeding Experiments
Aquatic arthropods were utilized in trial feeding experiments to determine what arthropod intermediate hosts are Involved In the life cycle of Spinitectus. The aquatic arthropods used were: aquatic insect nymphs of Calopteryx spp., aquatic insect nymphs of
Dldymops transversa, aquatic Isopods, AseI I us t ntermed1 us; amphlpods,
Hyalella Knickerbocker!, and cladocerans, Daphnla spp. Of all of these arthropods, the aquatic Isopods, Asellus Intermedlus, were found to ingest the eggs and became infected. So, efforts were directed to the rearing a population of helminth-free Isopods, AseIius
Intermedlus, in the laboratory for the life cycle studies.
Collection and Laboratory Rearing of Asellus intermedlus
Aquatic Isopods, Asellus intermed1 us, were collected along the banks of Olentangy River and ditches adjoining the river In the
Worthington area in Franklin County, Ohio. The area of collection was restricted to a small ditch completely cut off from the river and
It Is only fed with rain water or whenever the water level In the river becomes high. The ditch is always full of water, aquatic vegetation, rotting logs, and has a muddy bottom. The ditch Is thickly populated 20 with Asellus Intermedlus and many other aquatic arthropods. AseI I us
Intermedlus were collected with wire mesh dip nets and by hand picking and washing them off from stones, rotting logs, and submerged aquatic vegetation, A large number of the Isopods collected from this area were dissected for helminth parasites, but they were all negative for juvenile nematodes or trematode and cestode larvae of any other helminth parasites. The rest of these Isopods were therefore considered good to start with In the laboratory-rearing of Isopods for the Infection experiments.
The Isopods were maintained In the laboratory In open enameled trays (45 cm by 30 cm by 9 cm) and finger bowls (12 cm by 6 cm).
The tsopods were regularly supplied with filamentous algae and dried grassy weeds and leaves. It was observed that these Isopods were able to survive, grow and reproduce prodigiously In the laboratory at room temperature ranging from 16-32°C. The nymphs hatch In the marsupia of the mothers, grow and are later released. Upon release, the young Isopods were Immediately separated from the parents and were placed In separate finger bowls In which they molt, grow, and finally reach adulthood. These and their succeeding generations were used for the feeding Infection experiments.
Definitive Host
Helmlnth-free l-year- and 2-year-old blue gills, Lepomls macro- chlrus, were obtained from the State Fish Hatchery In Newtown, southern
Ohio. These fish were hatched from the eggs and raised In the labora tory. The flngerllngs were also brought to laboratory and raised to 21 adults. These fish were used In the feeding Infection experiments
In the life history studies of Splnitectus sp.
Blue gills, Lepomls macrochirus, were caught from the pond at
Snortin Ridge throughout the year and every fish was found to harbor
Splnitectus sp., both gravid female adults and mature males through out the year.
Other materials and methods employed In this Investigation will
be explained In their appropriate sections. GENERAL INFORMATION
Information on Egg Types of Splrurold Nematodes
Little can be said concerning the general characteristics of the eggs of splrurold nematodes, yet much Is known about the eggs of many genera and species of this group. The eggs usually have smooth,
transparent thick shells and are embryonated at the time of ovfposltfon.
The egg envelope Is composed of three typical membranes; the protein
coat, the chltlnous true shell; and the vitelline membrane. The general shape Is that of a regular ellipse, but various specialized
shapes exist. Terminal filaments, opercula, and mammilatlons have
been reported for the eggs found In this superfamily of nematodes.
The eggs of the following genera of the splrurold nematodes have
been reported to have terminal filaments; Tetrameres, Cystldlcola,
Metabronema, Ascarophts, Rhabdochona, and Splnitectus. It has been
found that there is no uniformity In possession of polar filaments by
the eggs of the genera that possess this characteristic. Only one species of Splnitectus, S_, ovlf I age 11 is, has eggs with polar filaments.
Operculatton Is a fairly common phenomenon in the eggs of splrurold
nematodes (Chandler, 1919). Metabronema magnum has been found to
possess button-like opercula. Chandler (1919) and Baylls (1931) have
observed the eggs of Hedrurls slredonls and Hedrurls splnlgera,
respectively, to have opercula, but no operculatlon of any sort has 23 been reported for the egg of any of the described species of
Splnttectus.
The Larval Stages (-Juveniles or Postembryonlc Stages)
The term ecdysls has been adopted to describe the type of molting that occurs In nematodes. Nematologlsts have found that In both free- living and parasitic nematodes development Is well accomplished at the time of hatching from the egg membranes, except for size and reproduc tive system. In nematodes to which Splnitectus sp. belongs, ecdysls
Is characterized by shedding of entire cuticle, Including the lining of the labial region, pharynx, rectum, and vagina.
Most authors have referred, and still do, to the postembryonlc development or preadult stages of nematodes as "larvae”. Hyman (1951) was of the opinion that the term "larvae" Is Inappropriate and sug gested the term "juveniles" to describe the postembryonlc stages In the development of life cycle of nematodes. Hyman thought this term
"juvenile" Is more appropriate and more descriptive since metamorphosis
In Insects Is not comparable to the postembryonlc development in nema todes. Therefore, In agreement with Hyman (1951) the term "Juveniles"
Is here used to describe the postembryonlc stages or preadult stages
In the life history of Splnitectus sp.
As in other nematodes life cycles, four ecdyses are observed In the life cycle of Splnitectus. Periods of growth are separated by these.ecdyses. Each of these ecdyses Is characterized by shedding of entire cuticle, Including lining of the stoma, esophagus, rectum, and, 24
In the fourth ecdysls, of vagina and cloaca. As the result of fourth ecdysls, fully developed young adult nematodes emerge. They a^e matured except for the production of eggs or spermatozoa. Growth Is observed to continue for some time after the fourth ecdysls In the case of Splnitectus sp. under study. Gametes are produced later after fourth ecdysls. THE LIFE HISTORY STUDIES OF SPINITECTUS SP.
Reproductive Habits
This species Is dioecious and there is a marked sexual dimorphism.
The adult females are always larger In size, width, length, and thick ness than the adult males.
The adult males are provided with two unequal and dissimilar spicules, narrow caudal alae, genital papillae. The genital papillae have been found to function as tactile organs during copulation In nematodes. Like some other adult male nematodes that have caudal alae, the males of Splnitectus sp. use their caudal alae as a rein forcement to the tail in clasping the females during copulation.
The male Inseminates the female by Inserting Its spicules through the vulva Into the vagina of the female, thus, the spicules are used In sperm transfer.
The eggs are produced In the ovaries, and fertilized In the ovi ducts, then a vital line membrane Is formed around the fertilized egg.
When the eggs get to the uteri, a very thick layer of shell Is formed around them, and they are later surrounded by a third thin layer of external protein coat, believed to be secreted by the uterine wall.
Not all three layers of membranes are present In all nematode eggs, but the three layers are present and are easily observable In the eggs of this species of Splnitectus.
25 26
Eggs containing embryo log leal stages can be seen In the ovi ducts, uteri, and vagina of the gravid female Individual nematodes, both living and in fixed specimens. The egg contains a colled embryo
Inside at the time of Its ovlposltion.
Each gravid female may lay a large number of eggs ranging from
200 to 500 In a period of 24 hours when placed In Ringer's "cold" solution. The gravid females normally lay their eggs in the fish
Intestines and these eggs are found adhered to the Intestinal mucus and contents of the fish. The eggs are passed out In the fish faeces as revealed by fecal examination of the fish definitive host,
LepomIs macrochlrus.
Feeding and Infection Experiments
Series of finger bowls were half filled with Ringer's "cold" solution. Approximately 200 embryonated eggs of Splnitectus sp. were placed In each of these finger bowls, and four uninfected Isopods,
Asellus Intermedlus, were added to each bowl. The isopods Immediately began to ingest the embryonated eggs of the worm with Incredible rapidity.
Another group of Isopods, Asellus Intermedlus, was placed In finger bowls half filled with Ringer's "cold" solution but no embry onated eggs were added. These served as control group.
The Isopods in each group, controls and experiments, were dis sected at Intervals of 3 hrs. each. Ten to 12 hours after ingestion of embryonated eggs of Splnitectus sp., the first stage Juvenile worms 27 were observed inside the Intestine of the Isopods, Asellus intermedI us.
The control groups were negative for the first stage Juveniles In their Intestine or any organ.
From then on, examinations of the isopods were done dally, that
Is, from day to day examination. Dally examination by dissection of these Infected isopods, showed that the first stage Juveniles persist
In the Intestines of the Infected Isopods fir 2-3 days with the first ecdysls occurring on the third day after the hatching of the first stage juveniles. The second stage juveniles emerged as the result of first ecdysls. The second stage juveniles penetrate the wall of the intestine and burrow Into the haemocoel of the Isopod, The second ecdysls occurs In the haemocoel of isopods on the fifth day; and the third stage juveniles emerge and later become encapsulated In a very thin membranous cyst. Third stage juveniles become Infective to the fish nine days later.
The infected Isopods containing 9 to 12-day-old 3rd stage juve nile nematodes were then fed to nematode-free I and 2-yoar-old blue gilts. The infected fish were examined by dally dissections. The third stage juvenile worms continue to grow and develop further within the Intestinal mucus and crevices of villi of the fish Intestine.
Third ecdysls occurred the 4th to 5th day after the ingestion of the Isopods containing the 3rd stage Juveniles. Fourth stage Juve niles emerge as the result of the third ecdysls In the Intestine of the fish host. Most of these 4th stage Juvenile worms were always found with thetr anterior ends burled within the Intestinal mucosa 28 and submucosa of the fish and sometimes some were found within the mucus In the fish Intestine.
Fourth ecdysls occurred 21 days after the Ingestion of the
Infected Isopods by the fish. This ecdysls occurred within the cast
Integument of the fourth stage Juvenile, Fifth stage juvenile worms emerge as the result of the fourth ecdysls and young adults were observed In the Intestine of the fish host 21-27 days after Ingestion of the Infected Isopods. All of the control Isopods and fish were negative for any juvenile stage of the worm or the adult worms.
Experiments were performed on the eggs to check the effect of
Intestinal Juice of Isopods on these eggs. Approximately twenty eggs were placed on a depression slide and Intestinal Juice of Isopods added and covered with a cover slip sealed with vaseline. Another slide was prepared with 20 eggs but with a drop of water added. First stage Juveniles hatch from the eggs In Intestinal juice but first
Stage Juveniles failed to hatch from the egg In a drop of tap water.
The possibility of auto-infection was attempted by feeding embryonated eggs to the uninfected fish. Ten fishes, Lepomls macro- chlrus, were fed with approximately 200 embryonated eggs of SpInI — tectus sp. each and ten other fish were not fed with eggs of the worm at all but were used as controls. The fish from each group, the experiment and control, were examined by dissection of one fish per group at intervals of 24 hours for the Juveniles. In every case, the fish were found to be negative for any Juvenile stage In the life cycle of Splnitectus sp. So, the author concluded that auto-infection 29 does not occur in this case and that the life cycle of Splnitectus sp. Is not a dIrect 11fe eye Ie.
Description of the Eggs, and Developmental Stages In the Life History Studies (AlI measurements are In millimeters)
Eggs. Eggs In various stages of development are observed inside the ovaries, oviducts, uteri, and vagina. Eggs Inside the uteri and vagina and sometimes within the ovejector are thick-shelled and each contains a colled embryo. Each oviposited egg Is regularly elliptical
In shape (Figs. 4, 5) smooth in out 11 no and contains a colled sausage shaped embryo. The eggs measure approximately 0.039 long by 0.024 wide; the true shell Is approximately 0.005 thick. The egg Is non- operculate. (Figs. 1-6)
Hatching of the first stage juveniles. Development to the vermi form first stage juveniles occurs only in the swallowed eggs inside the Intestine of the isopod; and this development stage does not occur while the egg Is Inside the gravid female worm.
The eggs recovered from one- to few-day-old faeces of fish are found to contain embryos that move very little Inside these egg membranes but the embryos Inside the eggs Just laid do not move at all. This the present author suggests to be due to some extrinsic or external factors that are necessary for this further development into this subvermiform and moving stage.
The eggs placed in the Ringer's "cold" and "warm" solutions and different buffer solutions for several days up to one week never give 30 rise to the hatching of first stage Juveniles, but the embryos Inside did move a little. When the embryonated eggs are swallowed by the aquatic Isopods, Asellus Intermedlus, the first stage Juveniles hatch from the egg membranes Inside the Intestines of the Isopods within 10 to 12 hrs after the Ingestion of the embryonated eggs by the Ase11 us
Intermedlus. Factors controlling the hatching of the first stage Juve niles are not worked out but the present author Is of the opinion that these may be endogenous to the chemical substances present in the digestive tract of AseI I us 1ntermed1 us In addition to the factor of
Incubation Inside these Isopods.
The point of exit seems to be identical in form and position for all of the first stage juveniles. There is no splitting or rupturing of the egg shell. It seems there Is a common weak point digested away either by the juvenile worm Inside or by the Intestinal enzyme system of the Isopods. The emerging first stage Juvenile wriggles out of the egg In a jerky, twisting, and convulsive manner with its anterior end coming out first. After exit or hatching of the first stage
Juvenile, an opening Is left behind on the empty egg membranes, remi niscent of an operculated egg (Fig. 6).
The Identifiable structures of the first stage Juvenile Include the cephalic end with a mouth silt, followed by an area of globules which seem to be the forerunner of the digestive system; this area extends to the posterior end of the Juvenile worm. The body tapers gradually to a posterior end with a tall. No circlets of spines were observed at this stage. The digestive system terminates In an anal slit near the posterior end of the Juvenile worm (Fig. 7). 31
The first stage juvenile worms vary In length; they measure 0.18 to 0.21. They wander around with convulsive, Jerky motions Inside the Intestine of the Asellus Intermedlus and remain there for 2-3 days and finally become quiescent and undergo the first ecdysls to become the second stage juvenlle on the 3rd day.
Second stage Juveniles. (Figures 8,9) By the fourth day after
Ingestion of the embryonated eggs by the Isopod, the second stage
juveniles have penetrated the Intestinal wall of the isopods and got
Into the haemocoel of the Isopods. They move around freely in the
Isopod’s haemocoel. They measure 0.41 to 0.64 In length. At this
stage, two portions of the oesophagus are becoming apparent; the nerve
ring Is formed; anus and cloacal region are quite observable; and the oral capsule Is becoming visible.
The second ecdysls occurs on the 7tti day after swallowing of the embryonated eggs and hatching of the first stage Juvenile worms. The
second ecdysls occurs while the second stage juvenile worms were In
the haemocoel of the isopod, giving rise to the third stage juvenile
worms (Figure 10).
Third stage Juvenile. (Figures 11,12)The Juveniles at this stage
are encapsulated In delicately thin, transparent cyst wall In which each juvenile worm colls up and moves very readily. The Juveniles measure 0.891 to 0.99 In length. The nerve ring Is prominent.
The genital prlmordlum Is readily visible and Its cells have
begun to proliferate posteriorly and anteriorly from the middle of the
length of the Juvenile worm. These later form a longitudinal row of 32 cells. The characteristic splrurold type of esophagus with muscular and glandular portions has become evident but cutfcular circlets of spines characteristic of the genus Splnitectus are not yet formed at this stage. The esophagus lengthens, measuring 291 to 333 microns; the muscular and glandular portions together are about l/3rd of the whole body length of the juvenile worm; and the Intestine, which Is also prominent, constitutes about 1/2 the length of the worm.
The oral capsule becomes readily visible. The Juvenile worm tapers posteriorly from the anal region Into a slenderly pointed end with a small short knob-like projection at the tall end. This stage constitutes the infective stage to the fish definitive host that may
Ingest the Infected isopods. Further development and growth of the third stage juveniles continues in the intestine of the fish. When the fish, lepomls macrochlrus, ingests Infected Isopods, Ase11 us intermedlus, containing the encapsulated third stage Juveniles, these juveniles are released in the intestine of the fish. They are found within the intestinal mucus of the fish some hours later. Some of these are found In crevices of the intestinal villi and some had already begun to penetrate the Intestinal mucosa. One day later, these third stage Juveniles are found with their anterior ends buried within the mucosa of the fish and grow in length.
Toward the end of this stage, the patterns of reproductive sys tem are well set. Male can be differentiated from the female Juveniles, but both sexes develop at about the same rate and no significant dif ferences in length and width of these Juveniles. Very late In this 33 stage the short spicule of the male becomes evident. Rudiments of the female vagina and uteri are distinguishable under 440 diameters magnification. The characteristic circlets of spines are beginning to develop toward the end of this stage. These are readily discern
ible on the anterior I/3rd portion of the body.
Third ecdysls occurs 5 days after the Ingestion of the Infected
Isopods by the fish, Lepomis macrochlrus. The fourth stage Juveniles emerge.
Fourth stage Juveniles. (Figs. 14-16,l8-f?3) The juvenile worms get out of the ecdysed skin to wander around In the Intestine of the fish for one to two days. They later become attached to the Intestinal wall of the fish with their anterior l/3rd of the body burled within the
Intestinal wall of the fish. This Is a period of attachment and de tachment by the juvenile worms to and from the Intestinal wall of the fish. This stage is also characterized by rapid growth In size and of organ systems especially the reproductive system. Much differ entiation In the gonadal development begins to occur; for example, male and fema'e reproductive systems are well established so that It
Is easy to tell the sexes apart. Sexual characteristics, such as the vagina, amphidelphlc uteri, oviducts, and ovaries are now well devel oped and easily recognizable in the females. Testis and two spicules
In the males are now prominently observable, but the short spicule Is better developed.
The circlets of spines characteristic of the genus Splnitectus are now very prominent. Early fourth stage Juveniles are characterized 34
by same rate of growth In size of both sexes, but fater In the stage, there is a difference In the rate of growth; the female grows faster
in length and width than the male. Oral capsule Is now well defined.-
The muscular and glandular portions of the oesophagus become wider and bigger In size and there Is a well-defined oesophago-Intest Inal valve between the posterior end of the esophagus and the anterior end of the
Intestine. The intestine becomes longer than it Is In the third stage
Juveniles; it Is composed of a unicellular layer of tissue and termin ates In a cloaca or rectum, which Is characterized by chltlnous wall and opens to the outside through an anus. The posterior end tapers
Into a short spike I ike knob at the caudal tip of the female worm and a short pointed spike in the male.
Female fourth stage Juvenile. (Figure 15) The circlets of spines are formed at the time of third molt Inside the shed cuticle of the third stage juvenile. Cephalic region measures about 66 microns and the first circlet of spines appears at about 66 microns from the anterior tip of the worm. The growth In length of the whole worm
reaches about 4.25 to 5.20.
The head Is provided with two Indistinct lips and six papillae; a straight-sided chltlnous oral capsule that leads Into a bipartite oesophagus. The muscular esophagus measures 240 microns to 300 microns; the glandular esophagus measures about 1.30 to 2.00. There
Is a well-marked esophago-Intestinal valve at the Junction of the esophagus with Intestine at a short distance anterior to the vulva.
The Intestine measures about 2.30 to 2.60 and terminates in a rectum 35 which Is readily visible ventralfy and laterally. The rectum opens to the outside through an anus situated at about 96 to 99 microns from the tip of the tall. The tall narrows posteriorly measuring 96 to 99 microns In length and terminating In a splke-llke knob measuring about 5.0 microns In length.
The vulva Is not yet opened to the outside; It appears to be sealed off or walled off by the cuticle of the worm. The presumed position of the vulva Is about 1.90 to 2.0 from the posterior extremity and about 2.60 to 3.5 from the anterior tip of the worm. The ovejector
Is well developed and is fairly large; it leads Into the fairly mus cular vagina that extends a short distance to form amphidelphic uteri, one uterus going anteriorly and colling around the anterior end of the
Intestine while the other uterus extends posteriorly slde-by-slde with the Intestine, then coils around It and reflexed back anteriorly to join the ovary. It Is difficult to distinguish the oviduct from the uterus at this stage. The excretory pore Is located between the 7th and 8th circlets of spines. The nerve ring Is formed at the level of the 4th circlet of spines.
Male fourth stage Juvenile. ( Figure 16). Similar to the females in anatomical architecture except In length of the body and the reproductive system. Males measure about 4.25 to 4.60 In length.
The nerve ring is visible at level of the fourth circlet of spines.
Excretory pore Is located between the 7th and 8th circlets. Muscular esophagus measures about 200 to 240 microns In length and glandular esophagus Is about 1.30 to 1.50 long. The Intestine Is about 2.20 to 36
2.50 long. Tall measures 105 microns to 110 microns In length.
There are about 56 to 72 very fine spines per visible circlet. The
circlets are farther apart anteriorly but become closer progressively
backward until very close together at about 15th to 18th circlets.
The testis Is a slender longitudinal tubelike structure situated
about 1.84 to 2.20 from anterior tip. It goes anteriorly a short
distance, then reflexes at about 0.2 to 0.25 posterior to esophago-
Intestlnal junction, colls around the Intestine making about 4 to 5
loops, then goes posteriorly and joins with the vas deferens. The
vas deferens Joins to the cloaca. The long spicule Is about 0.198
to 0.20 long and the short spicule measures about 0.06 to 0.08 long.
The cloaca opens to the outside through the anus. In the late fourth
stage male, four preanal and six postanal genital papillae are
readily discernible.
Fifth stage Juveniles or young adults. ( Figure 25). The
ecdysls of the fourth stage juveniles both males and females occurs
the 21st to 24th day after Ingestion of infested Isopods by the fish.
This results In the 5th stage juveniles or young adult worms.
Prior to the 4th ecdysls, the fourth stage juveniles penetrate
the fishes' Intestinal mucosa and submucosa until the anterior I/5th of the body Is burled In the tissue. Thus, the fourth ecdysls occurs
while the worm is still surrounded by the shed cuticle of the fourth
stage juvenile and a rapid growth period begins In length and width at
this stage. After the ecdysls, the fifth stage Juvenile gets out of
the shed cuticle of the fourth stage Juvenile and moves around In the 37
fish InfestIne for some days. It reattaches to the Intestinal mucosa and submucosa with its anterior end burled In these tissues (Figures
23,24). The rate of growth In length and width becomes greater In the
females than In the males. More differentiation occurs In the repro ductive system. In the male, two spicules, a short one and a long one, are now clearly defined. The cutlcular ridges, which are arranged In
two parallel longitudinal rows anterior to the anus, are present In the male. Caudal alae and genital papillae are also present In the males at this stage. All of these structures are absent in the
females. In the female, the vulva Is near the middle of the body.
The ovejector is well developed and a muscular vagina approaches It posteriorly. The female gonoduct is amphtdelphic with one branch going anteriorly and the other branch going posteriorly, each termin ating in an oviduct joining an ovary respectively. The tall ends In a very short spike-like projection.
Description of the Fully Mature Adults of Splnitectus sp.
In living condition, these worms are often found attached to the
Intestinal mucosa and sometimes found coiled around the outside of
Intestinal contents or attached to the mucus substances In the Intestine.
The females are larger In size than the males. The anterior end of the worm is somewhat conical In both sexes and Is devoid of spines. The tall, In the female, terminates In a very short splkellke projection which Is obscured In the males by the caudal alae.
The cuticle bears circlets of spines characteristic of the genus 38
Splnitectus. The spines In each circlet are not continuous around the circumference of the worm, but are divided Into dorsal, ventral, and two lateral groups or quadrants. Sometimes these four groups of spines are out of tine at the points of their meeting so that slight faults are formed and sometimes there Is a definite discontinuity marked by an Interval without spines. The cutlcular spines are very small In
size, fine and numerous, each circlet bears 56 to 72 spines. The
first 3 anterior circlets are farthest apart, and the next 5 to I I circlets are less farther apart than the first 3 anterior circlets; the next 12 to 22 circlets are set apart approximately 1/2 the dis
tance of the 4th to llth circlets. The change In spacing of the cir clets of spines is not accompanied by diminution In size of the spines
In the first 1-12 circlets until 13th circlet posteriorly when spacing
Is accompanied by diminution in size of the spines. It becomes Increas
ingly difficult to see the spines from the level of the Junction of the
two esophagi posteriorly.
The mouth is terminal and guarded by two small lateral lips.
The en-face view studies show the presence of six labial papillae
t Figure 26). The mouth leads Into an oral capsule which Is straight,
thin-walled, longitudinal, cutlcularlzed tube terminating posteriorly at the level of first circlet of spines where It joins with the anterior end of the esophagus.
The esophagus consists of two parts, a short muscular portion
and a long glandular portion. The anterior portion Is muscular,
shorter in length, and smaller In width than the posterior glandular 39 portion. The muscular portion Is surrounded by the nerve ring at the level of the 4th circlet of spines. The junction between the muscular and glandular portions of esophagus Is marked by the levels of the 17th and 18th circlets of spines In males, 19th to 22nd circlets In the females. The glandular portion of the esophagus Is long and It Is about eight times the length of muscular portion In the females and about six times as long In the males. The esophagus Is joined pos teriorly to the Intestine by a distinct esophago-Intestlnal valve.
The Intestine Is a single cell-layered, longitudinal tube wider anteriorly and narrower posteriorly and joins with the rectum by a recto-Intestinal valve. The rectum opens to the outside through an anus.
The excretory system Is renette cells which open to the outside between circlets 7th and 8th In the males; 9th and IOth In the females.
Female. (Figures 25-29). The females vary In length 16.0 to
20.0 long, 0.179 to 0.182 wide at level of vulva. Head region measures
0.099 to 0.10 In length. The tall Is conical and tapers to a spike)Ike posterior extremity; It measures 0.146 to 0.165 In length.
The vulva Is non-sal lent, situated slightly posterior to middle of the body and measures 8.5 to 10.2 from anterior tip, 8.2 to 9.7 from posterior tip. The muscular ovejector Is well developed and approaches the vulva posteriorly; It lies between the vagina and the vulva. The vagina Is strongly muscular and approaches the ovejector from posterior. It measures 0.35 to 0.38 in length and terminates In didelphfc amphidelphfc uteri with one branch going anteriorly, the 40 other branch turns and passes posteriorly. In fully gravid female
Individuals, the uteri are engorged with eggs. Uteri terminate In oviducts. The oviducts are long and extensively colled beside the
Intestine In their courses. The anteriorly directed duct extends to and colls around the posterior half of glandular portion of esophagus and then connects with the ovary. Posteriorly directed duct Is connec ted to the ovary which reflexes about 0.106 anterior to the anus, then turns anteriorly to terminate about 0.226 anterior to the anus.
Eggs are elliptical in shape, smooth In outline and are thickly shelled. Each contains a coiled embryo at the time of ovlposltlon and measures 0.039 long by 0.024 wide.
Male. (Figures 30-32) The males are similar to the females In anatomy of the anterior region but are smaller In size than the females. The males are shorter In length and are more slender than the females. They measure 7.6 to 8.2 In length. The head region Is about 0.09 long. The tall colls two complete turns. When straightened out and viewed ventrally, the tall Is provided with two lateral, narrow caudal alae, 0.02 wide, on each side of the tall. The caudal alae extend from a level Just anterior to the level of anus to the posterior extremity of the tall. In life, eleven pairs of caudal genMal papillae are ot srved with the last pair of the postanal papillae being very sm ! and are not easily located In the fixed male specimens. There e four preanal papillae and seven postanal papillae. All of these papillae are borne on stalks, that Is, they are stalked or pedunculate except the last pair of the postanal papillae (Figure 32). 41
The reflexed test Is Is In form of a narrow tube that extends anteriorly, then reflexes at a distance of about 1.32 posterior to the esophago-intestinaI Junction, It turns posteriorly and Joins the vas deferens. Two spicules are present. These are unequal and dis similar. One Is short, thick, and arcuate, and measures 0.086 to
0.094 In length, Its posterior tip is pointed and provided with very fine, ventrally curved barb. The other spicule Is long and slim; its anterior l/3rd Is thick and tubular but Its posterior 2/3rds is slightly compressed and rounded into a trough-like shape. It tapers posteriorly to a fine point. The long spicule measures 0.296 to
0.312 long.
Two parallel, longitudinally arranged rows of cuticular ridges are present starting about 0.039 to 0.040 anterior to anus, and extend anteriorly beyond level of anterior end of long spicule, and anterior to the level of the first pair of preanal papillae
(Figure 31). The author is of the opinion that the number of these cuticular ridges in a row and the number of rows may be of taxonomic
Importance on the species level. 42
Table 3. Measurements of Splnitectus sp. (A11 measurements In ml 11Imeters)
Females Males
Length 16 - 20 7.6 - 8.2
Width 0.168 - 0.179 0.09 - 0.10
Length of oral capsule 0.099 - 0,10 0.09 - 0.094
Width of oral capsule 0.005 0.005
Esophagus: Muscular: Length 0.340 - 0.363 0.297 - 0.312 Width 0.031 - 0.034 0.20 - 0.027 Glandular: Length 2.950 - 2.970 1.914 - 2.030 Width 0.098 - 0.109 0.066 - 0.070
Distance of vulva from: Anterior tip 8.5 - 10.2 --- Posterior tip 8.2 - 9.7
Length of tal1 0.165 0.140 Length of caudal alae 0.296 Width of caudal alae 0.02 Length of spicules: Short spicule 0.086 - 0.094 Long spicule 0.296 - 0.312
Number of cauda1/gen 1ta1 papillae: Preana1 --- 4 Postanal 7
Length of vagina 0.35 - 0.38 Size of ova (eggs) 0.039 x 0.024 Position 'of the nerve ring 4th circlet 4th circlet Number of spines per circlet 56 - 72 56 - 72 Length of spine 0.005 0.005 Number of rows of cuticular ridges 2 Number of cuticular ridges In a row 54 - 56 Position or location of excretory pore between 9th A 7th & 8th 10th circlet Distance of excretory pore from anterior tip 0.312 0.289 43
Remarks on the Life History
The experimental results In the life history studies of SpInI - tectus sp. demonstrate the pattern of a splrurold nematode life cycle whereby an arthropod Intermediate host Is Involved In the life cycle.
An aquatic isopod, Asellus Intermedlus, serves as the arthropod Inter mediate host of the SpInitectus sp.
The egg of Splnitectus sp. contains a coiled embryo at the time of oviposltlon. Experimental evidence shows that the first stage
Juvenile never hatch from the egg left In Ringer's "cold" or Ringer's
"warm" and different buffer solutions for seven days. The eggs must be Ingested by the arthropod Intermediate host which. In this case,
Is Asellus Intermedlus before the hatching of the first stage juvenile can occur.
When the embryonated eggs are Ingested by Asellus Intermedlus, the first stage Juveniles hatch !n the Intestine of these Isopods.
Factors affecting the hatching of the first stage Juveniles In the intestine of the Isopods were not studied. The present author Is of the opinion that the factor of Incubation In the Isopod and chemical substances or the enzyme system of the Isopod may be responsible for the hatching of the first stage juveniles.
It was observed that when eggs are placed In the Intestinal Juice of AseII us IntermedI us, the first stage Juveniles hatch from the egg membranes.
Detailed microscopic studies of the eggs of Splnitectus sp. revealed no presence of any observable operculum. Each of the first 44 stage juveniles was observed to emerge from the egg at a fixed point of exit at one end of the egg membranes. There Is no cracking or splitting of egg shell for the juvenile worm to hatch out of the egg membranes. The point of exit can be clearly seen mIcroscopIca11y on the empty shell after the emergence of the first stage juvenile worm as a small but almost round space with smooth edge (Figure 6). The juvenile worm gets itself out of the shell by a lashing motion and the empty shell is left behind as If It has an operculum.
Since the eggs are passed out with faeces of the fish, they are subjected to external environmental factors that will affect their survival.
Since the adult worms, both gravid female and mature male worms, are recovered from the Intestines of the fishes, Lepomls macrochlrus and Lepomls cyanellus, during the winter, It is believed that the worm overwinters as adult In the fish Intestine.
Feeding experiments showed that four ecdyses occur In the life cycle of Splnltectus sp. The first ecdysls occurs In the Intestine of Asellus intermedlus three days after hatching of the first stage
Juvenile from the egg membranes. The second stage juveniles emerge and later burrow through the Isopod's intestinal wall and get Into the haemocoel of the Isopod. The second ecdysls occurs In the isopods' haemocoel the 7th day after hatching of the first stage juveniles.
This second ecdysls results In emergence of the third stage juveniles which become encapsulated In the tissue surrounding the haemocoel of the Isopods. Third ecdysls does not occur until the Infected Isopod 45 containing third stage Juveniles Is Ingested by fish, Lepomls macro- chlrus. If the Infected Isopod Is not Ingested by the fish, the third stage Juveniles remain encapsulated In the tissue around the haemocoel and no ecdysls occurs.
When the Infected Isopods containing third stage juvenile worms are Ingested by the fish, Lepomls macrochlrus, experiments show that the 3rd stage Juveniles are released In the fish Intestine. Further growth and development of third stage juvenile worms continue In the fish Intestine. Third ecdysis occurs In the Intestine of the fish between 4th and 5th day after Ingestion of the Infected Isopods. The fourth stage juveniles emerge and wander around In the Intestine for
2 to 3 days to later become attached to the Intestinal wall of the fish for 2-4 days and later become detached. Actually this Is a period of attachment and detachment of the fourth stage juvenile worms to and from the Intestinal wall of the fish. The fourth stage Is also a period of rapid growth and development of organ system, size in length and width of the juvenile worms. Reproductive system continues to become better differentiated.
The fourth ecdysls also occurs In the ftshTs Intestine between
21 to 24 days after Ingestion of the Infected isopods. The fifth stage
Juveniles or young adult emerge as the result of the fourth ecdysls.
These fifth stage Juveniles are morphologically Identical with the adult worms except In length and lack of eggs In the female fifth stage Juveniles. Growth and development continues In the fifth stage until maturity Is attained. Egg to egg cycle requires 45 to 50 days. 46
In order to Investigate the possibility of autoinfection or direct life cycle In which no intermediate host is involved, 150 to 200 embryonated eggs of Splnltectus sp. were force-fed to each of ten young blue gills, Lepomts macrochfrus. Another ten fish were used as control without being fed with the embryonated eggs. These fishes were dissected and examined for the first, second, or third stage
Juvenile worm at intervals of 24 hours for ten days. The results were negative in ail cases. The author therefore concluded that the life cycle of Spinitectus sp. Is not direct. In the experimented fishes, empty egg shells were found fn few cases In the intestine of two fishes but no juvenile stages of the worm nor adult stage were found.
The control group fish, not fed with the embryonated eggs, also showed negative results (Figure 19).
Pathological conditions do occur to the l-year old fish definitive host during the fourth stage of the juvenile worms. This stage, as mentioned before, Is a period of much attachment and detachment of the
Juvenile worms to and from the Intestinal wall of the fish; It Is also a period of rapid growth and development of the Juvenile worms. These
Juvenile worms are found with their anterior l/5th of body burled In the intestinal mucosa and submucosa of the fish. A lot of this burrow ing In and out by the Juvenile worm occurs during the fourth stage.
This situation causes pathological conditions In the Intestine of the fish. During this period, some of the young fish died from worm burden and the worm activities of burrowing In and out of the intestinal mucosa and submucosa of the fish. 47
Studies of the cross sections of the fishes' Intestines contain ing 4th and 5th stage juveniles (Figures 18-24)showed that definite pathological conditions do exist In the fish Intestine due to the attachment and detachment of these 4th and 5th stage Juvenlle worms.
The adult worms are also found to burrow Into the Intestinal mucosa and submucosa of the fish, but most of the time they are found
In the Intestinal mucus and contents.
Some naturally Infested yearling fish were found to be heavily
Infected with 4th stage juveniles and these fish were In poor health.
Most of these fish died. Fish with light infections of not more than ten worms were found to survive the Infestation and the worms develop to adults.
Gustafson (1939) reported In his brief note on the life cycle of
Splnitectus gracilis that the life cycle took approximately 15 days
In the mayfly larvae and the green sun fish, lepomls cyanelI us. He did not give the details of stage by stage development.
Gustafson's (1939) report is so meagre that It does not offer much In the way of analytical comparison between the life cycles of these two species, S.. gracl 11s and Splnitectus sp. Both species may be found In the green sun fish, Lepomis cyanelI us. but the present author has not been able to find S.. gracl 11 s In the blue gills,
Lepomls macrochlrus. The species reported here, Splnitectus sp. has been found predominantly In the blue gills, Lepomls macrochlrus. and to a lesser extent in the green sun fish, Lepomls cyanellus. 48
Gustafson (1939) utilized mayfly larva (nymphs) as the Intermediate host of S_. graclI Is, but aquatic Isopods, Asellus Intermedlus serve, under laboratory conditions, as the arthropod Intermediate host for the new species of Spfnitoctus In-this study.
Length of time required for completion of fife cycle of S. gracl11s
Is shorter than reported for the Splnitectus sp. under study.
The discovery of Asellus Intermedlus as the probable arthropod
Intermediate host (experimentally) for Splnitectus sp. neatly fits Into the ecology Involved In the life cycle of this nematode In two ways:
(a) Asellus Intermedlus are found In large numbers In ponds, streams, rivers, and lakes In southern Ohio.
(b) The dietary habits and behavior of Asellus Intermedlus also play significant roles In the food web. These Isopods, AselI us
Intermedlus, are detritus feeders, feeding on the rotting aquatic vege tation at the bottom of the water; here they may Ingest nematode eggs that are found with detritus. Experimental evidence supports this dietary behavior because feeding experiments show that Asellus Inter med I us readily Ingest the embryonated eggs of Splnitectus sp. when exposed to them.
Summary of the Life History Studies (See Table 4)
I. Splnitectus sp. Is a splrurold nematode parasitic as an adult In
the Intestine of blue gills, Lepomls macrochlrus, and green sun
fish, Lepomls cyanelI us. The gravid females on the genus Splnitec
tus oviposit their eggs In the Intestines of the fish definitive
hosts. 49
2. The eggs measure 0.039 mm long and 0.024 mm wide. They are pro
vided with thick and transparent shell membranes; and each egg
bears a colled embryo at the time of oviposit Ion. The eggs are
non-oporcu!ate.
3. The eggs are passed out In the faeces of the fish, Lepomls
macrochlrus. Further development of the colled embryo to Infec
tive first stage juvenile for the Isopod occurs Inside the egg
shell with favorable environmental conditions.
4. When the eggs are swallowed by the aquatic Isopods, Asellus
Intermedlus, the first stage Juveniles hatch out of the egg
membranes In the Intestine of the Isopod within 10 to 12 hours.
These first stage juveniles develop In the Intestine of the Isopod
and undergo the first ecdysls to become second stage Juveniles
about 2 to 3 days later.
5. The second stage juveniles burrow through the Intestinal wall
and get Into the haemocoel of the isopods. These second stage
Juveniles wander around In the haemocoel of the tsopod for some
days. They later undergo second ecdysis to become the third
stage Juvenile worms In the haemocoel of the isopod on the 7th
day after the ingestion of the embryonated eggs of the Splnitectus
sp. by AseI I us Intermed I us. These third stage juveniles become
encapsulated within delicate, thin, transparent membrane In the
tissues around the haemocoel of the Isopods. Further anatomical
development occurs and the third stage Juvenile worms become
Infective to the fish definitive host, Lepomls macrochlrus. 50
6. The third stage Juveniles are released Into the Intestine of the
fish. These encapsulated Juveniles excyst and wander around In
the Intestine and burrow their anterior ends In the Intestinal
mucosa. Further development of the Juvenile worms occurs. Third
ecdysls which results In fourth stage Juvenile worms occurs 4 to
5 days after Ingestion of Infected Isopod by the fish.
7. After the third ecdysls, the fourth stage Juveniles may be found
In the Intestinal mucus of the fish. Some days later, they are
found attached to the Intestinal wall of the fish with their
anterior l/5th of the body burled within the Intestinal mucosa
and submucosa of the fish. Further growth and development occur
rapidly. This Is a period of attachment and detachment to and
from the Intestinal wall of the fish. Sexual differentiation Is
well marked at this stage. The characteristic circlets of spines
are well developed at this stage. Fourth ecdysls that results In
fifth stage Juveniles occurs 21 to 24 days after the Ingestions of
Infected Isopods by the fish.
8. Pathological conditions were observed In the Intestines of the
fish harboring the fourth stage Juveniles due to the burrowing In
and out by these fourth stage Juveniles Into the Intestinal wall
of the fish.
9. The fifth stage Juveniles of Splnitectus sp. emerged from the
shed cuticle of the fourth stage Juveniles as the result of the
fourth ecdysls. At first these Juveniles are found In the Intest
inal mucus of the fish but later on they are found attached to the 51
Intestinal mucosa. This attachment lasts but a few days. They
become detached again. Growth and development continue. Repro
ductive systems and other organ systems are fully developed at
this stage but growth in size continues. Later In this stage,
the males develop caudal alae and the females begin to produce
eggs. Copulation occurs. Eggs containing colled embryos are
seen In the uteri of female worms 45 to 50 days after ingestion of
Infected isopods by the fish.
10. Egg to egg cycle of Splnitectus sp. lasts about 45 to 50 days
under laboratory conditions.
11. The life cycle of Splnitectus sp. Is Indirect In that an aquatic
Isopod, Ase11 us IntermedI us, acts as the Intermediate host.
Experimental evidence ruled out the possibility of autolnfectlon. 52
Table 4. Outline of Life Cycle. (J^Juvenlles; E=ecdysls; sub script numbers represent stages)
- 1 8 - 2 1 p J formed In egg shell l hatches In gut of Isopod
E Intestine of fsopod 1
41 - 64 y J Intestine and haemocoel of Isopod 2
7 days after hatching E haemocoel of Isopod 2
89 - 99 p J ) 3 )f a ) Tremendous 4-5 days after Ingestion E intestine of fish ) growth 3 of Isopod by fish ) (In size) (11-12 days after ) hatch Ing) )
Male 4.2 - 4.6 mm ) k Female 5.2 - 7.25 mm ) ) Tremendous 21-24 days after Intestine of fish ) growth H Ingestion K ) (In size) 28-29 days after ) hatch!ng ) (young adult) J5
45-50 days Adult intestine of fish Male 7,6 - 8.2 mm Female 16 - 20 mm
8 Greatest growth occurs, In size of cells, at last two ecdyses (actually not an increase In number of cells). SYSTEMATIC REVIEW OF THE GENUS SPjNITECTUS FOURMENT, 1883
The genus Splnitectus was established by Fourment In 1883 with
S. ovlfI age 11 Is as the type species of the genus. The systematic position of the genus Splnitectus with reference to the family and subfamily to which It belongs has been a matter for differences of opinion. Fourment in 1883 did not assign this genus to any family or subfamily. Baylls and Daubney (1926) placed the genus as an appendix to their newly created subfamily Thelazlinae under the family Spirurldae. Yorke and Maplestone (1926) Included the genus
Splnitectus In the subfamily RIctulari 1nae Hall, 1913 under the family Rictularlidae Rail I let, 1916.
Travassos, Artlgas, and Pereira (1928) created a new subfamily
Rhabdochontnae to which they transferred the genera Rhabdochona
RalI Met, 1916; Splnitectus Fourment, 1883; and Cystidicola Fischer,
1798 under the family Spirurldae. According to All (1956) these authors apparently have recognized the fundamental similarity of structure In these three genera besides their being parasitic In fish hosts.
Mueller and Van Cleave (1932) believed that Splnitectus, Cystl- dlcololdes, Cystidicola, and Rhabdochona belong In the family Splru- rldae and regarded Thellzildae as a separate family meaning that the above mentioned genera do not belong to the family Thelazffdae. They
53 54 further assigned these genera to subfamily Splrurlnae based chiefly upon negative characters such as their lack of specialized lips and vestibular structures.
Chitwood and Wehr (1932) placed both Cys+tdocola and Splnitectus under family Spirurldae rather than Thelazildae as done by others, but he further separated Splnitectus as an appendix to the family
Spirurldae, but Mueller and Van Cleave (1932) doubted the soundness of such an action.
Skrjabln (1946) Included all Splrurold nematode parasites of fish
In his newly created family RhabdonchonIdae with three subfamilies:
Rhabdochonfnae, CystfdlcolInae, and Spfnltectlnae, thus removing
Rhabdochona, Cystidicola, and Splnltectus from the family Spirurldae because they all differ from the type genus Splrura. Skrjabin (1946) emphasizes the similarity of their host group and their structural slmilari ties.
Khera (1954) while agreeing with Travassos et a_l_. (1928), dis agreed with SkrjabIn's (1946) classification on the grounds that the three subfamilies created by Skrjabln are unfortunately too widely different to be grouped In one family and that their affinity being only In the similarity of the host group. He also questioned Skrjabin's family diagnosis of RhabdochonIdae: thin delicate body, mouth opening
Into a funnel-shaped oral cavity, a clearcut double oesophagus, unequal and dissimilar spicules, and numerous post-cloacal papillae. These characteristics, according to Khera (1954), are too common and are met with also In many other splrurold nematodes; and besides, equal 55
spicules have been reported in a species of Splnitectus, I.e., S.
Indlcus Verma and Agarwala, 1932. Furthermore, continued Khera, the
Subfamily Rhabdochoninae has been characterized by the possession of
smooth cuticle and eggs without polar filaments, but Thapar (1950) had
described two species of Rhabdochona, R. kashmlrensls and R. hospetl
with eggs possessing long polar filaments and small threads, respec
tively, and that other splrurold nematodes of fishes are not accounted
for in Skjabln's classification. So, Khera (1954) suggested that It
Is better to follow the older classification of Spirurldae as Indicated
by Travassos et a I. (1928). This author does not agree with Shera
In Including Splnitectus in the subfamily RhabdochonInae of Travassos et aJL* (1928) for the reasons that will be given later.
This author is of the opinion that Skrjabin’s classification Is a step toward the settlement of disputes that have accompanied the
classification of these groups, especially the genus Splnitectus.
All (1956) did not totally agree with Skrjabin’s scheme of
classification but followed Travassos et aj_. (1928) In their use of the subfamily RhabdochonInae to accommodate the related forms. He was not In favor of emphasizing the host relationship so far as to
raise the subfamily RhabdochonInae to the family rank as did Skrjabln
(1946). All (1956) was of the opinion that the three subfamilies created by Skrjabln (I946)stould be included In the family Thelazlldae
and not In the family RhabdochonIdae.
All (1956), as well as some authors, are In total agreement In
shifting of the genus Splnitectus from the family RictularlIdae In 56 which Yorko and Maples+one Cl 926) put It to the family The Iaz11dae.
The reason for this action Is that the stoma of Splnitectus differs radically from that of RIctularla, the type genus of the family
RtctularlIdae. All (1956) Included the subfamily Splnltectlnae In the family TheIaz11dae because the stoma of the genus Splnitectus closely resembles the forms Included In the family Thelaztldae.
Sahay and Prasad (1965) agreed with All (1956) on the classification of the genus Splnitectus.
The discrepancies that had accompanied the taxonomic position of the genus Splnitectus and Its related forms, are due In part to the lack of knowledge as to the true nature of these groups and to the fact that little Is known concerning the life cycles of the groups. As more and more Is known concerning these groups, their true natural relationships become apparent. So, Yamagut! (1958) In
Systema Helminthum Vol. Ill revamped the systematlcs of the whole group of Splrurold nematodes parasitic In various classes of verte brates. This classification was broad and Inclusive In scope. View points of previous authors were well taken. The family Thelaztldae was upheld and was not reduced to subfamily, as Indicated by All
(1956); and thus, dealt with properly. It was the belief of Mueller and Van Cleave (1932) that the genus Splnitectus and related genera do not belong to the family Thelazlldae, but rather Thelazlldae was considered by these authors as an entirely separate family.
Those genera that were unaccounted for In Skrjabin’s classification were considered and properly dealt with In YamagutI’s classification. 57
Not only morphological characteristics were employed In this scheme, both definitive and Intermediate hosts were considered when Known.
The author agress with Skrjabln (1946) and Yamagut! CI958) In
Including the genus Splnitectus and related genera In the family
RhabdochonIdae and the genus Splnitectus under subfamily Splnltectlnae.
The present author Is of the opinion that the genus Splnltectus and the family RhabdochonIdae should be emended In the light of what
Is known concerning the genus. Though the author realizes that, with more work and Information, YamagutI's Cl958) classification for these groups may. In time, be changed, yet It seems to him as the most reasonable classification yet proposed for these groups of Splrurold nematodes. So, the classification of the genus Splnitectus should read as fo11ows:
PhyI urn Nematoda Class Secernentea Order Splrurlda Suborder Splrurlna Superfamlly Splruroldea Family RhabdochonIdae Subfamily Splnltectlnae Genus Splnitectus EMENDATION OF THE GENUS SPINITECTUS AND THE
FAMILY RHABDOCHONI DAE
Since males of one species, S. Indfcus Verma and Agarwal, 1932, had been reported to possess equal and similar spicules, and the females with vulva located In anterior half of body, It Is the opinion of this author that the generic diagnosis of the genus Splnitectus should be emended to accommodate S_. Indicus. All (1956) also reported that the males of S. long I pap i11atus have similar but unequal spicules. So, the spicules which are important taxonomic characters in this genus may or may not be equal, similar, or dissimilar.
(Note: Words In capital letters Indicate emendations)
Splnitectus Fourments, 1883
Syn. Goezia Zeder, 1800, partim
Cochi us Zeder, 1803, partim
Liorhynchus Rud., 1801, partim
Generic diagnosis: RhabdochonIdae, Splnitectinae: cuticle provided with a series of transverse rings, to the posterior edge of which are attached backward Iy directed spines diminishing In size and number posteriorly. Mouth with Indistinct lips; buccal cavity cylindrical or funnel-shaped; esophagus consisting of two parts, muscular and glandular. Male: tall spirally colled; CAUDAL ALAE NARROW OR FAIRLY
58 59
WIDE, sometimes with denticulate crests in front of cloaca; preanal and postanal pap I Mae-present CIO to 15 pairs In all; SPICULES MAY
OR MAY NOT BE. EQUAL OR SIMILAR. Female: vulva In ANTERIOR HALF OF
BODY or in middle, or posterior region of body; oviparous; eggs small, ellipsoidal, thick-shelled, sometimes with polar plugs bearing long filaments. Parasitic in stomach and intestine of fishes and frogs.
Modified and Emended from YamaguTl (1958)
Family RhabdochonIdae Skrjabln, 1946
Famlly diagnosis: Spiruridea: cuticle with or without ornamentations.
Mouth with or without lips. Buccal capsule funnel-shaped or cylindri cal, may or may not be provided with longitudinal thickenings or teeth.
Esophagus consisting of two portions. Male: posterior extremity may be rolled up ventrally or spirally cofled. CAUDAL ALAE NARROW OR
FAIRLY WIDE, sometimes with denticulate ridges in precloacal region.
Caudal PAPILLAE SESSILE OR PEDUNCULATE, usually not numerous. SPICULES
MAY OR MAY NOT BE EQUAL OR SIMILAR. Female: Vulva in anterior or posterior half of body. Oviparous. Parasitic in stomach and Intestine of fishes and amphibians.
Emended from Yamagutl (1958) DESCRIPTION OF NEW SPECIES
Splnl tectus mlcrosplnosus sp. n
(Figs. 25-32)
Description: (Based on 10 mature males and 10 mature females. All
measurements are In millimeters unless otherwise
Indicated)
Body filiform, slightly attenuated at extremities; anterior bluntly conical; posterior end pointed. Cuticle provided with series of circlets of backwardly projecting spines. Each circlet bearing 56 to 72 spines; each spine minute, fine, 0.005 long. Head region devoid of spines, bluntly, conical anteriorly, demarcated posteriorly at level of first circlet of spines. Distances between circlets I, 2, 3 greater than those for circlets 4 through 12; distances between cir clets 13 through 22, half those of circlets 4 through 12. Circlets becoming very close together with diminution In spine size at level of junction of two portions of esophagus. Mouth terminal, guarded by two small, lateral. Indistinct lips. Oral capsule, cylindrical, straight sided cutlcularlzed tube, terminating at level of first cir clet of spines. Esophagus, starting at level of first spinous circlet.
In two parts; anterior muscular portion, being narrower and shorter than posterior, glandular portion. Nerve ring surrounds muscular esophagus at level of 4th spinous circlet. Posterior end of glandular esophagus
60 4 61
communicates with anterior end of intestine through distinct esophago-
Intestinal valve. Intestine, longitudinal tube, wider anteriorly,
narrower posteriorly, Joining rectum through Inconspicuous rectal-
Intestlnal valve. Rectum opening outside through anus. Tall, slenderly
tapering posteriorly.
Male: Length 7.6 to 8.2. Maximum width 0.09 to 0.10. Head region,
0.094 to 0.099 long; 0.032 to 0.039 wide at level of first spinous
circlet. Oral capsule 0.094 to 0.098 long; 0.005 wide. Muscular esophagus 0.297 to 0.312 long, 0.02 to 0.03 wide; glandular esophagus,
1.914 to 2.03 long, 0.066 to 0.07 wide. Nerve ring 0.160 to 0.164
from anterior tip of body. Junction between muscular and glandular esophagus, marked at level of 17th and 18th circlets of spines.
Excretory pore, between 7th and 8th circlets, about 0.286 to 0.289
from anterior tip. Testis single, reflexed, extends anteriorly to
1.32 of esophago-intestinal valve and turns posteriorly to Join vas deferens. Two spicules, unequal, dissimilar, channeled, anterior ends attached to retractor muscles. Short, spicule; thick, arcuate,
0.086 to 0.094 long, psoterlor end pointed, provided with fine ventrally curved barb. Long spicule: 0.296 to 0.312 In length,
anterior l/3rd thicker, tubular; posterior 2/3rds slightly compressed
rounded Into trough shape posteriorly, tapers to fine posterior end.
No gubernaculum. Anus, 0.130 to 0.133 from posterior end of tall.
Tall, somewhat rounded tip end, 0.130 to 0.140 long, thrown Into two complete or Incomplete turns. Narrow, ventral caudal alae, 0.02 wide on either side of tall, and 0.296 long starting from at about 62
0.03 anterior to first pair of preanal papillae and 0.156 anterior to anus, extending to posterior tip of tall. II pairs of caudal papillae present; 4 pairs preanal, pedunculate; 7 pairs postanal of which 6 pairs are pedunculate, one pair small, sessile. Two parallel, longitudinal rows of cutlcular ridges present, starting about 0.039 to 0.040 anterior to anus, extending anteriorly beyond anterior level of re tracted long spicule.
FemaIe: Vary In lengti, 16 to 20. Width at level of vulva, 0.168 to
0.179. Head region marked off at level of first circlet of spines,
0.099 to 0.109 long, 0.06 wide. Oral capsule, 0,09 to 0.107 long,
0.005 wide. Muscular esophagus, length, 0.34 to 0.363; width, 0.031 to 0.034. Glandular esophagus, length, 2.95 to 2.97; width, 0.098 to 0.109. Nerve ring, about 0.168 to 0.172 from anterior tip.
Excretory pore between 9th and 10th circlets cf spines, about 0.312 from anterior tip. Muscular esophagus, 0.34 to 0.363 long, 0.031 to
0.034 wide; glandular esophagus, length, 2.95 to 2.97; width, 0.098 to 0.109. Vulva, nonsallent, 8.5 to 10.2 from anterior tip; 8.2 to
9.7 from posterior tip of tall. Ovejector present, between vulva and vagina. Vagina, strongly muscular, 0.35 to 0.38 long, directed posteriorly, terminating in amphtdelphlc, opposed uteri. Uteri, direc ted anteriorly and posteriorly, respectively, filling almost entire width of nematode. Posterior ovary, reflexed, about 0.106 anterior to anus. Uteri and vagina, engorged with eggs. Eggs, elliptical, with thick, transparent shell, without polar filaments, containing colled embryo at ovlposltlon, measure 0.039 long, 0.024 wide. Anus 63
0.158 to 0.(65 from posterior tip of tall. Tail, slenderly tapers posteriorly, measures 0 , 146 to 0.165 with short, splkellke projection,
0.005 long.
Host: Lepomls macrochlrus
Site of Infection: Intestine
Locality: Snortln Ridge, Madison Township, Fairfield Co., Ohio
Specimens: Laboratory of parasitology Faculty of Zoology The Ohio State University Columbus, Ohio 43210
Discussion
Two species of Splnitectus have been described from freshwater fishes of North America. These are Splnitectus gracilis Ward and
Magath, 1917, and S_. carol ina Ho I 1, 1928. Both species were redes cribed by Mueller and Van Cleave (1932) and they suggested that the shape size, form, and ratios of anatomical structures should be employed in separating species belonging to this genus. Mueller and
Van Cleave employed this idea to separate these two species reported from freshwater fishes of North America. All (1956) followed
Mueller and Van Cleave's (1932) suggestions and constructed a key which, though unwieldy. Is useful in separating most of the species reported for the genus. Sahay and Prasad (1965) expanded on All's
(1956) key but unfortunately some species were left out. So, sug gestions of Mueller and Van Cleave (1932) will be followed in separating the species herein reported, from the other two species, 64
L* graclI Is and S. carol 1na, reported from freshwater fishes of
North America.
S. mlcrosplnosus differs from S. gracl11s In the form and size of the two spicules In the males. The long spicule In graclI Is
Is tubular throughout Its length and measures 0.6 mm long while In
mlcrosplnosus It Is tubular l/3rd of Its length and laterally, slightly compressed for the remaining 2/3rds of Its length and measures 0.150 mm while that of S^. mlcrosplnosus Is arcuate and measures
0.086 to 0.094 long. In respect to spicules, S_. mlcrosplnosus super- ficlally resembles S_. carol I na but differs from It In size and form of
long spicule which Is tubular 1/2 Its whole length and slightly com pressed the other 1/2, and measures 0.270 mm In S. carol 1na. There Is also a difference In size of the short spicule which Is 0.070 mm and provided with a large ventral barb In S^. carol I na but 0.086 to 0.094 mm and provided with a fine ventral barb in S^. mlcrosplnosus.
The vagina approaches the vulva anteriorly In 5_. gracl11s but posteriorly In S. mlcrosplnosus, thus sp. Is similar to S^. carol Ina
In this respect but differs In size; vagina measures 0.280 mm In S. carol Ina but 0.330 to 0.380 mm In S. mlcrosplnosus. Esophagus extends far In front of 1st circlet In S. gracl11s but terminates at level of or Just anterior to the 1st circlet In £. mlcrosplnosus, while at level or behind of second or third circlet In S_. carolIna.
Ratio of esophagus to total body length In females differs In the three species: In S^. gracl I Is It Is 1:9 or 1:10; In S_. carol Ina,
1:3 or 1:4; In S^. mlcrosplnosus, 1:5 or 1:6. S_. mlcrosplnosus a I so 65 differs from S. graclI Is and S. carolIna In position of vulva: pre
vulva to postvulva length ratio In S. gracl I 1 s Is 3:1; In S_. carol I na
It Is 4:3; In S. mlcrosplnosus It Is approximately I:I or 1.2:1.
mlcrosplnosus differs from S. gracl I Is In the arrangement of genital papillae which are doubly paired but unevenly set apart In
S. mlcrosplnosus; In S^. carol I na the par Is are evenly set apart.
S. mlcrosplnosus differs from S. carol Ina In number of postanal papillae which Is 5 In S^. carol I na and 6 In S. mlcrosplnosus. Seven pairs of postanal paplllae were observed in _S. mlcrosplnosus when examined a IIve.
S^. mlcrospl nosus also differs from both S_. gracl 11 s and S_. carol I na
In being the largest of the three In the size of the females which are
twice as large as their males. S_. mlcrosplnosus also has the largest
number of spines per visible circlet, being 56-72 for S^. mlcrosplnosus
and 25-35 for S^. carol I na, and 35-50 for S^. gracl I Is.
Because of the differences given above, S^. mlcrospl nosus Is con
sidered a new species of the genus Splnltectus and the third to be
described from freshwater fishes In North America. Table 5. Comparison of Species of SpInItectus reported from Freshwater Fishes of North America
S. graclI Is carol 1na S.______
1. Length, mature females 10mm to 15mm 7mm to 8mm 16mm to 20rnrt 2. Length, mature males 8mm to 10mm Slightly sm. than fern. 7.6mm to 8.2mm 3. Length of oral capsule 0.025mm 0.10mm 0.09 to 0.107mm 4. Vagina approach vulva from Anterior Posterior Posterior 5. Length of vagina 0.80mm 0.28mm 0.33mm to 0.38mm 6. Ratio of esophagus to body length 1:9 or 1:10 1:3 or 1:4 1:5 or I:6 7. Ratio of mus. to glan. esophagus 1:3 1:5 or I:6 I:6 male - 1:8 female 8. Long spicule length Tubular throughout Distal 1/2 com Distal 2/3 guttershaped, Its length 0.6mm pressed, 0.27mm anterior 1/3 tubular, 0.296 to 0.312mm 9. Short spicule length Bent twice, 0.15mm Arcuate, 0.07mm Boat shaped, 0.086 to 0.094mm 10, Number of spines In each of Long, 35-50 spines Spines very long and Spines very fine 0.005 anterior circlet, per clrclet sharp (25-35) long, 56-72 per cl re. 11. Position of nerve ring Usually bet. 1st and Between 4th and 3rd and 4th circlets 2nd circlets, may be 5th cl relets posterior to 3rd 12. Number of rows of cutlcutar Series of these Series of parallei 2 rows (54-56 In a row) longitudinal rows 13. Number of preanal papillae 4 pairs 4 pairs 4 pairs 14. Number of postanal papillae 6 pairs 5 pairs 7 parls 15. Position of excretory pore Not described Not described Bet.7th & 8th circlet, mates; 9th & I0th,fem. 16. Position of vulva-ratlo of pre 3:1 4:3 1.2:1 or 1:1 vulva and postvulva region 17. Alae Falrly wide Narrow Narrow 18. Size of eggs 0.040mm x 0.024 0.036mm long x 0.023 0.039mm x 0.024mm 19. Lips without No definite 2 Indistinct 20. Head length Not given 0.08mm 0.099 to 0.107mm
o\ O* SUMMARY
1. Splnitectus mlcrosplnosus, a new species of splrurold nematode Is
described from the Intestine of freshwater fish, Lepomls macro
chlrus, In Ohio. It Is compared with and contrasted to the other
two described species of the genus, S_. gracl11s Ward and Magath,
1917, and S^. carol Ina Hoi 1, 1928, from freshwater fishes In North
America.
2. The life cycle was worked for the new species S^. mlcrosplnosus.
An aquatic isopod, Asellus intermedlus was found to serve as the
arthropod Intermediate host of £. mlcrosplnosus In the laboratory.
3. Eggs of S_. mlcrosplnosus have thick, transparent shell, without
polar filaments. Each egg contalnes a coiled embryo at the time
of ovlposltion. These eggs are laid In the Intestine of the fish
and are voided In the fish faeces.
4. Further development of the coiled embryo In the egg membranes
occurs when reached favorable external environment.
5. Asellus intermedlus becomes infected by Ingesting eggs of
Splnitectus mlcrosplnosus that contain colled embryos. First
stage juvenile worms hatch from the egg membranes In the intestine
of the Isopod within 10 to 12 hrs. Four ecdyses occur In the
life cycle of Splnitectus mlcrosplnosus.
67 68
6. The first stage Juvenile worms develop In the Intestine of the
Isopod and undergo the first ecdysls to become second stage
Juveniles about 2 to 3 days later.
7. Second stage Juvenile worms penetrate the Intestinal wall of
the lsopods and get Into the haemocoel. In which they wander
around, develop and undergo second ecdysls on the seventh day
to become the third stage juvenile worms.
8. The third stage juveniles grow and develop. The genital prlmodlum
develops and Its cells begin to proliferate. The digestive
system Is well developed. The third stage juveniles then become
encapsulated within the tissues around the haemocoel of the
Isopod.
9. The fish, lepomls macrochlrus, becomes Infected by Ingesting
Infested lsopods, Ase11 us 1ntermedI us, which harbor the encapsu
lated third stage juvenile worms.
10. The third stage juvnfles are released In the Intestine of the
fish. These juveniles get Into the crevices of Intestinal villi
and Into the Intestinal mucus of the fish. They grow, develop
further, and later penetrate the Intestinal mucosa. Spines begin
to show development.
11. The third ecdysls occurs 4 to 5 days after Ingestion of the
Infected AseI I us Intermed1 us, giving rise to the fourth stage
JuvenIles.
12. Fourth stage juveniles are characterlzed by sexual differenti
ation, rapid growth, development, and establishment of the
characteristic circlets of spines of the genus Splnitectus. 69
The reproductive system, and excretory system are we I I developed
late in this stage, but the reproductive system of the female
worms still lacks a vulvalar opening, and the spicules of the
males are not fully developed. This fourth stage Is also charac
terlzed by attachment and detachment of the Juvenile worms to
and from the Intestinal wall of the fish.
13. A study of the cross sections of the Intestine of the Infected
fish with fourth stage Juvenile shows that pathological con
ditions In the Intestines of the fish do exist during the worm's
fourth stage. These conditions are due to the burrowing In and
out of the fish Intestinal mucosa and submucosa by the fourth
stage Juvenlles.
14. The fourth ecdysis occurs 21 to 24 days after the Ingestion of
Infected Isopods. The fifth stage juveniles emerge from the
Integument of the fourth stage juveniles. Male spicules, caudal
alae, and genital papillae are now well developed but the
spicules continue to grow In length. The female reproductive
system now opens to the outside of the nematode through the
vulva. Copulation occurs later on In this stage. Eggs were
seen In the uteri and vagina of the females 45 to 50 days after.
Egg to egg cycle lasts about 45 to 50 days under laboratory
conditions.
15. Based on additional Information available from the newly des
cribed species, the author emended the genus Splnltectus and 70
the family Rhabdochonldae to which It Is assigned.
16. A table of geographical distribution, and hosts of the known
described species of the genus Is presented. A table of the
comparison between the species of the genus, reported from
North American freshwater fishes, Is presented. APPEND! X 72
EXPLANATION OF PLATE I
(All scales represent 0.01 millimeter. Figures I to 4 represent embryologleal stages Inside the eggs as found In the uterus. Fig ure 5 represents oviposited egg. Figure 6 represents empty egg membranes)
Fig. I Morula stage
Fig. 2 Subvermlform stage
Fig. 3 Tadpole stage or late subvermlform stage
Fig. 4 Vermiform stage
Fig. 5 Egg containing first stage juvenile worm.
Fig. 6 Empty egg membranes from which first stage Juvenile hatched I 31Vld ZL 74
EXPLANATION OF PLATE II
(AM measurements In millimeters)
Fig. 7 Early first stage Juvenile
Fig. 8 First stage Juvenile, ecdyslng
Fig. 9 Second stage juvenile, four days after Infection
Fig. 10 Second stage Juvenile, ecdyslng
Fig. II Third stage juvenile, eight days after Infection
Fig. 12 Third stage juvenile, encapsulated 75 PLATE II
O 05
O oS
H EXPLANATION OF PLATE III
(AN measurements In millimeters)
Fig. 13 Late third stage juvenile In the fish
Fig. 14 Third stage Juvenile, ecdyslng to become fourth stage Juvenile
Fig. 15 Fourth stage juvenile, female
Fig. 16 Fourth stage juvenile, male 77 PLATE III
10 78
EXPLANATION OF PLATE IV
Fig. I Cross-sect Ion of intestine of uninfected fish, one of the control group (under magnification of 440 diameters)
Fig. f Cross-section of Intestine of infected fish with 3rd stage JuvenIles PLATE IV 80
EXPLANATION OF PLATE V
Fig. I Cross-section of Intestine of an Infected fish with fourth stage juvenile showing the worm In the mucosa
Fig. 20 Cross-section of Intestine of Infected fish showing the cross-section of fourth stage Juvenile worm In the submucosa PLATE V
4 r
f; , ft.
\ ° i 82
EXPLANATION OF PLATE VI
Fig. 21 Cross-section of the Intestine of Infected fish with the fourth stage juvenile on the columnar epithelial cells of the Intestinal villi
Fig. 22 Cross-section of the Intestine of fish Infected with late fourth stage Juvenile, showing the worms In the submucosa
84
EXPLANATION OF PLATE VII
Fig. 23 Cross section of Intestine of fish Infected with fourth stage juveniles
Fig. 24 Cross-section of Intestine of fish infected with early fifth stage juveniles showing the anterior l/5th of worm in the submucosa of Intestinal wall of the fish PLA TE VII
23 86
EXPLANATION OF PLATE VIII
Fig. 25 Anterior end of adult female worm lateral view
Fig. 26 Lips, en-face view
Fig. 27 Vulvar region, lateral view, showing vulva ovejector, vagina, and parts of uteri gorged with eggs containing colied embryos
Fig. 28 Posterior region of adult female showing the ovary, anus, and tail end
Fig. 29 Female reproductive system, dissection 87 P LA T E VIII 88
EXPLANATION OF PLATE IX
Fig, 30 Posterior half of adult male worm showing portion of digestive system, reproductive system and colled talI.
Fig. 31 Spicules of the males
Fig. 32 Ventral view of the posterior region of adult male showing the arrangement of genital papillae, spicules and caudal alae PLATE REFERENCES CITED
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