STUDIES ON THE LIFE HISTORY OF RICTULARIA COLORADENSIS HALL, 1916 (NEMATODA: THELAZIIDAE), A PARASITE OF PHtoMYSCUS LEUCOPPS NOVEBORACENSIS (FISCHER)

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio S tate U niversity

Bbr VERNON HARVEY OSWALD, B. S ., 11. A.

*****

The Ohio State University 1956

Approved by:

f Department of Zoology and [ y Entomology ACKNOWLEDGMENTS

This study was accomplished through the direct and indirect assistance of many of the faculty members, fellow students, and technical personnel in the Department of Zoology and Entomology of The Ohio State University. The author wishes to thank Dr. Josef N. Knull and Mr. Richard D. Alexander of the Department of Zoology and Entomology for identifying ground beetles and field crickets, respec­ tively, which were used in th is study. Dr. Edward Thomas of the Ohio State Museum, Columbus, kindly identified wood roaches and camel crickets for the author. Special thanks also go to Mr. Donal (3. Myer for help in collecting material and to Mr. John L. Crites who was concurrently working on the life history of Cruzla americana and who offered many suggestions on techniques which were used by the author. Dr. William H. Coil, a Muellhaupt Scholar in the Depart­ ment, took an active part in collecting material and is also respon­ sible for the photomicrographs included in this work. Lastly, the writer wishes to acknowledge the many helpful suggestions and c riti­ cisms given hy his adviser, Dr. Joseph N. Miller of the Department of Zoology and Entosology.

- i i TABLE OF CONTENTS

INTRODUCTION ...... 1 HISTORICAL REVIEW...... 3 MATERIALS AND METHODS ...... 8 CLASSIFICATION...... 13 OBSERVATIONS AND RESULTS Occurrence of Rlctularia coloradensls in Naturally Infected Mice ...... 16 Experimental Infection of Arthropods...... 17 Natural Intermediate H osts...... 22 Development in the Intermediate H ost...... 2U Pathology in the Intermediate H ost...... 31 Infectivity of the Larval Stages ...... 33 Development in the Laboratory Mouse ...... 35 Pathology in the Definitive H ost...... UU Experimental Infection of other Mammalian Hosts hL Description of the Adult ...... U7 DISCUSSION General Aspects of the Life Cycle ...... 51 Taxonomic Considerations...... 55 S1WMARY...... 68 LITERATURE CITED ...... 71 PLATES ...... 76 AUTOBIOGRAPHY ...... 90

- i i i - LIST OF TABLES

TABLE 1. Incidence of Rictularia coloradensls in Peromyscus leucopus novcboracensls ...... 16 TABLE 2. Experimental infection of arthropods with Rictularia coloradensls following the feeding of infective eggs...... 19 TABLE 3. Insects examined for natural infections of Rictularia larvae ...... 22 TABLE U. Worms recovered from experimentally Infected

laboratory mice ...... 36 TABLE 5. Comparison of the North American species of R i c t u l a r i a ...... 61

- iv - LIST OF ILLUSTRATIONS

TEXT FIGURE 1. Average growth rate of R^ coloradensls In laboratory m ice ...... 39 TEXT FIGURE 2. Effect of differential growth rates of the esophagus and the prevulvar and postvulvar portions of the body on the relative location of the vulva of R. coloradensls ...... U2 TEXT FIGURE 3» Diagrammatic representation of the develop­ ment of R^ coloradensls in experimental h o sts...... 52 TEXT FIGURE U. V ariation in the to ta l number of p airs of cuticular processes in male IL coloradensls ...... 57 TEXT FIGURE 5« Variation in the total number of pairs of cuticular processes in female FL ooloradensls ...... 57 TEXT FIGURE 6, V ariation in the number of p a irs of prevulvar cuticular processes In female [L coloradensls ...... 57 TEXT FIGURE 7. Distribution of R^ coloradensls and related North American species of R ictularia...... 65 PLATE 1, FIGS, 1-6, The egg and the first and second larval stages of R* coloradensls ...... * ...... 77 PLATE 2, FIGS, 7 -13» The third larval stages of R« coloradensls ...... 79 PLATE 3, FIGS, lh-21. The adults of R. coloradensls ...... 81

PLATE Li, FIGS. 22-23. Encysted larvae of IL coloradensls...... 83 PLATE 5» FIGS. 2h-25. Encysted larvae of R. coloradensls...... 85 PLATE 6, FIGS. 26-27. An immature female and the comb of

a mature, female R^ coloradensls...... 87 PLATE 7» FIGS. 23-29. Spine of an adult, female IL colora-

densis and the combs of Rictularia sp. from a wood rat .....89 - v - INTRODUCTION

In the following pages, various aspects in the life history of the sp iruroid roundworm, R ictu laria coloradensls H all, 1916, w ill be elucidated. This nematode, originally described from a chipmunk, has been reported from several species and subspecies of white-footed ndce, and it is with material from the latter host that this work is concerned. This study was suggested while the writer was collecting para­ sites during work on his master's degree. Rhododendron Hollow, located in the extreme northwest corner of Hocking County, Ohio, was found to have a large population of white-footed mice, Peroaorscus leucopus noveboracensls, and the incidence of infection of this host with R^ coloradensls was very high. In any life history study, an abundance of both host and para­ site material is highly desirable, if not a necessity, for the suc- cussful completion of the work. In this regard, the situation in Rhododendron Hollow was very favorable. The problem also seemed to lend itself well to a laboratory study. In all spiruroid life cycles, an intermediate host, usually an insect, is required. Cockroaches, which are readily reared in the laboratory, are quite frequently suit­ able as experimental Intermediate hosts for members of this group. It was also anticipated that laboratory mice might serve as experimental definitive hosts for this parasite. This study is of special interest since only one fragmentary life cycle is known in the subfamily to which this parasite belongs. There is also some question regarding the specific status of a number of species in the genus Rictularia, and this work, together with studies on the life cycles of other species in the genus, may eventually solve these problems. HISTORICAL REVIEW

Rictularia coloradensls was described by Hall (1916) from one male and an incomplete female specimen obtained from a chipmunk, Eutamlag quadrlvittatus, collected in Pagosa Springs, Colorado. Harkema (1936) rep o rted R^ co lo rad en sls in Peromyscus 1. leucopus from North Carolina, but only female specimens were found. According to Tiner (19U8a), Harkema*s specimens were identified by Dr. B. G. Chitwood. Rankin (19U5) reported IU coloradensls in Eutamias amoenus, Peromyscus maniculatus, and Microtus longicaudus from Northrup Canyon, Washington. Tiner (19U8a) redescribed coloradensls from male and female specimens taken from Peromyscus maniculatus bairdii collected in Wisconsin, a male specimen from P^ leucopus novebora- censis collected at Bowie, Maryland, and Harkema*s North Carolina specimens. The genus R ic tu la ria was erected by F ro e lic h (1802) fo r R. cristata from a dormouse, Muscardinus avellanarius. A large number of species have now been described for this genus; in a review, Dollfus and Desportes (19h5) list a total of thirty-two. In addition to the species listed by these authors, the following have been named: R. nycticebl (MBnnlg, 1920); R._ oligopectinea Wu and Hu, 1938; R» onychomis C uckler, 1939; R^ ondatrae Chandler, 19U1; R^ nana C aballero, 19b3; R^ dlpodonds T iner, 19U8; R^ m icro ti McPherson and T iner, 1952; and R^ baicalensls Spasski, Rishikov, and Sudarlkov, 1952. Tiner (19b8a) reduced R^ ha 111 Sandground, 1935 in to synonorqy w ith R. c l t e l l i McLeod, 1933» end he considered R^ nana C aballero, 19^3 * synonym of R^ macdonaldi (Dobson, 1880). Cuckler (1939)» in present-

- 3 - - li - ing a key to the known species in the genus, states that males are unknown for more than half of the species, and that many of the species are inadequately described. Therefore, the validity of a number of species is in doubt. At the present time, however, the genus contains thirty-eight named species when both valid species and species inquirendae are considered. Species of Rictularia have been reported from insectivores, bats, marsupials, rodents, carnivores, and primates. disparllis Irwin- Smith, 1922 from a lieard has been placed in a separate genus, P s e u d o ric tu la rla , by D o llfu s and D esportes (19L5)» Members o f th e genus have been reported from every continent with the exception of Australia, and two species have been reported from the Philippines. The following lis t of species has been reported from North Americas R. coloradensls Hall, 1916 Type hosts Eutamias quadrivittatus (Say) - chipmunk Other hosts: Eutamias amoenus iEicrotus longicaudus (Merriam) - vole Peromyscus 1. leucopus (Rafinesque) - white- footed mouse P* leucopus noveboracensis (Fischer) FT m aniculatus b a ir d ii IHoy and K ennicott) R eferencess H all (1916), Harkema (1936). Rankin (19U5)t W ilson (19U5>), T iner (19U9a), McPherson and Tiner (195?) Rj_ splendida Hall, 1913 Type hosts Canis nebracensls Merriam - coyote Referencess Hall (1913) R. scalopis Goodrich, 1932 Type hosts Scalopis aquaticus (Linn.) - mole Referencess Goodrich (1932), Tiner (19h8a) c i t e l l l McLeod, 1933 Synonym: R. ha111 Sandground, 1935 Type hoats: Cltellus frank 11 ni (Sabine) - apemophlle C. t. trldeclmllneatus (Mitchill) Other hosts: Tamias striatus lysteri (Richardson) - chipmunk t. siriatus grTseus ^Mearns) T7 slrlatus ssp. Sciurus carolinensis leucotis (dapper) - gray s q u irre l References: McLeod (1933), Sandground (1935), Tiner (191:8a)» Rausch and Tiner (191:3), Chitwood, M. B. (1952) onychomis Cuckler, 1939 Type h o st: Onychomys leu co g aster (Weid) - grasshopper mouse Other hosts: Peronyscus leucopus ssp. (?) - white-footed mouse References: Cuckler (1939)* Reiber and Byrd (19U2), Tiner (I9h8a), Chandler and Melvin (1951) ondatrae Chandler, 19U1 Type host: "Muskrat" Other hosts: Ondatra rivalica (Bangs) - muskrat slgmodon hlspldus ssp. - cotton rat Oryzomys p. palustris (Harlan) - rice rat References: Chandler (I9hl)» Penn (19L2), Tiner (191:8a), Melvin and Chandler (1950) macdonaldi (Dobson, 1880) Synonym: R. nana C aballero, 19U3 Type h o st: Megaderma fro n s S t.-H il. - A frican b at Other hoats: Balantlopterix ochoterenai - Mexican bat tlyotis luclfugus (LeConte) - N. American bat References: Jaegerskioeld (1909), Caballero (191:3), Tiner ( l 9U8a) dipodomis Tiner, 191:8 Type host: Dlpodomys sp., probably merrlami - kangaroo rat Other hosts: D. morroensis (Merriam) B7 panamlntlnus mohavensls - 6 - References* Tiner (19l*8b), Read and Ulllemann (1953) R. m icroti McPherson and T iner, 1952 Hostsi Mlcrotus oeconomas lnnoitus - vole M. ndurus panekl References* McPherson and Tiner (1952), Rausch (1952) Rictularia sp. Hosts* Sciurus niger ruflventer (Qeoffroy) - fox squirrel S. c. carollnensls omelin - gray squirrel TairJasciurus Jm3sonlcus ssp. - red squirrel References* lCats (1938)» Oraham and Uhrlch (191*3), Rausch and Tiner (191*9), Tiner (19l*8a) Rictularia sp. Host* Neotoma m aglater - wood r a t References* Tiner (19l*8a), McPherson and Tiner (1952) All North American species of Rictularia appear to be exclusively nearctic with the exception of R^ macdonaldl which was originally described from an African bat. Hall (1913) erected the subfamily Rictulariinae for the genus Rictularia. He interpreted the cuticular thickenings in the cloacal region of some males as an open bursa, and therefore he placed the subfamily in the family Metastrongylidae Leiper, 1908, which is included in the superfamily Strongyloidea Weinland, 1858. Railliet (1916) recognised the spiruroid affinities of Rictularia, and he raised Hall's subfamily to family rank and placed it in the super- family Splruroldea Railliet and Henry, 1915. Baylis (1928) considered Rictularia to belong in the family Spiruridae Oerley, 1885, subfamily Thelazlinae Baylis and Daubney, 1926. On the basis of cephalic char­ acters, Chitwood and Wehr (1931*) placed the subfamily Rictulariinae Hall, 1913, together with four other subfamilies, in the family Thelaiildae Railliet, 1916. In their review of the genus, Dollfhs - 7 - and Desportes (19b5) follow the classification proposed by Chitwood and Wehr. They place four genera in the subfamily Rictulariinae: Rictularia Froelich, 1602 (type genus); Pneumonema Johnston, 1916;

Echinonema Linstow, 8 I 9 8; and R ictu lario id es H all, 1916. Chabaud (1951j) attested to formulate a classification of the order Spirurida on the basis of biological characters. He concluded that, in most cases, the classification of Chitwood and Vehr is admirably in accord with the known biological characters. Up to the present time, only one partial account of a life cycle in the genus Rictularia has been published. Witenberg (1928) obtained R. cahirensis Jaegerskioeld, 190b experimentally by feeding young dogs the viscera of reptiles which contained nematode cysts in the serous membranes. Two molts occurred in the intestine of the dog, giving r is e to ad u lt worms. Witenberg considered the r e p tile s to be probable second intermediate hosts; the larvae were postulated to develop initially in coprophagous insects which were subsequently ingested by the reptiles. Unfortunately, Witenberg's contribution is in the form of a note, and no experimental data nor drawings are presented. No life cycles are known for related genera in the subfamily Rictulariinae. MATERIALS AND METHODS

White-footed mi ce were collected with conventional mouse traps baited with either bacon or peanut butter. Traps were placed at holes, under rock ledges, or at the bases of trees where signs of activity were noted. Some mice were also captured in live traps fashioned from No. 2 tin cans. A lid was hinged to one end of the can, and a regular mouse trap was wired to the inside. A wire connecting the hinged lid to the spring of the trap caused the lid to snap shut when the treadle of the trap was disturbed. A few dog chow checkers were placed in the can, and the treadle of the trap was baited with bacon. Some of the mice caught in this manner were used as sources of nematode eggs, and others were used in feeding experiments. Live Peromyscus, together with laboratory mice and rats used for feeding experiments, were kept in the basement of the Botany and Zoology Greenhouse. All animals were fed Purina Dog Chow Checkers. The mice were marked for identification purposes by cutting slits in the ears according to a predetermined code. Fecal examinations were made by d ir e c t smears. German roaches for infection experiments were reared in wide- mouth Jars of about two quart capacity. Shredded paper toweling was placed in the bottom of the Jar, and the top was greased with vase­ line and covered with cheese cloth to prevent the roaches from escap­ ing. The roaches were fed a diet of ground dog chow checkers, powder­ ed yeast, and Starlac. Vater was provided in a vial stoppered with cotton. American, oriental, and brown-banded roaches were obtained from cultures maintained in the Entomology Department, and meal worms

- 8 - were obtained from a culture maintained in the General Zoology Store­ room. Wood roaches, camel crickets, sow bugs, and ground beetles for infection experiments were collected at the University Woodlot. Field crickets were collected on the premises of the Hudson Bait Company of Columbus, Ohio. Wood roaches and field crickets were maintained in the laboratory in culture Jars similar to those used for German roaches. A Petri plate containing a cotton pad saturated with water was placed in the culture jars containing camel crickets to maintain a moist condition. Sow bugs and ground beetles were kept in four-ounce Jars with a moist cotton pad. Eggs of Rictularia were obtained from the feces of Infected Peromyscus which were kept in a cage with a wire-mesh bottom. The feces were suspended in tap water and centrifuged repeatedly until the supernatant fluid was clear. The moist, fecal residue, which contained the eggs, was placed in a vial and stored in a refrigerator. Some eggs were also obtained by race rating r&ture female worms in tap w ater. Four-ounce jars were used for infection experiments with insects. One to two dozen insects were aspirated into a clean Jar and kept without food or water for one or two days. Small pieces of ground beef were covered with the egg-feces mixture and placed in the jar with the insects. In cases where the rate of development of the larvae in the insect host was being studied, the contaminated food was removed at the end of three hours, and clean food and water were added to the container. In cases where the rate of development was not being studied critically, the insects were exposed to the eggs for one or two days to build up a larger infection. When the Insects were examined for larvae, they were decapitated - 10 - and placed In a watch glass containing Ringer's cold solution. One or two terminal body segments were separated from the rest of the body with dissecting needles, after which the entire digestive tract could usually be pulled from the body of the insect. The digestive tract was macerated in a drop of Ringer's solution on a slide and examined for larvae. Mice and rats were fed infective larvae by stomach tube. Mice were tubed with an 18 ga. hypodermic needle about one and one-half inches long which had the point removed to form a blunt tube. The tube was used with a 1 ml tuberculin springe. Encysted larvae were removed from the intermediate host, placed in tap water, and allowed to escape from the cyst membranes before they were fed. This allowed the number of larvae which were pulled into the syringe to be deter­ mined more accurately, and it also prevented the cysts from adhering to the inside of the stomach tube. About 0.175 ml of water was drawn

Into the syringe after which 1 £ to 20 larvae were pulled into the tube with an additional 0.02£ ml of water. The mouse was grasped by the scruff of the neck with the thumb and index finger of the left hand, and the ta il was held against the palm of the hand with the fourth and fifth fingers. The stomach tube was then inserted its full length, and the contents of the syringe were slowly discharged. The tube had to be inserted with care to avoid damaging the esophagus.

Laboratory rats were tubed with a No. 8 French rubber catheter. The catheter and syringe were filled with water, and all air bubbles were eliminated. Fifteen to twenty larvae were then drawn into the end of the catheter with a small amount of water, and the catheter was lubricated on the outside with glycerine. The rat was grasped behind the head in the palm of the left hand with the thumb and index finger encircling the body Just anterior to the front lege. A wooden tongue depressor with a hole large enough to admit the catheter was inserted into the mouth behind the incisors* With this wooden gag in place, the rat could neither open nor close its mouth, and the stomach tube could be inserted through the hole in the gag into the esophagus and stomach. In examining the anim als, the small in te s tin e was removed and placed in a Petri dish with a small amount of tap water or Ringer*a solution. The intestine was opened with a scissors under a dissecting microscope, and the nematodes were removed with a dissecting needle into a dish of clean water or Ringer's solution. When very small developmental stages were expected, the mucosa of the Intestine was scraped with a scalpel, and the scrapings were then examined under a dissecting microscope. The smaller larval stages were usually studied alive, but occa­ sionally they were fixed by running alcohol-formalin-acetic acid solution (Lavdovsky's formula) under the coverslip. The larvae could be studied alive more easily if they were first stunned by placing them in an oven at 60° C. for three to five minutes. The larger larval stages and adult worms were fixed in75% alcohol or AFA heated to 70° C. Specimens were cleared in a stendor dish of glycerine alcohol (1 part glycerine in 9 parts 75% alcohol). The dish was covered with lens paper or a larger inverted container to exclude dust, and the alcohol was allowed to evaporate gradually. Temporary slides were made by mounting the specimens in glycerine with the coverslip sealed with paraffin. Some of the specimens were stained by adding a very small amount of Semichon1s stain to the glycerine alcohol. Material to be sectioned was fixed in Bouin's or AFA and handled according to standard technics. Sections were stained with Ehrlich's glycerine alum hematoxylin and counterstained with eosin. En face preparations were made according to the technique out~ lined by Buhrer (19U9). The worm was imbedded in a drop of glycerine Jelly on a slide. After the glycerine Jelly had solidified, the slide was placed under the high power of a dissecting microscope60 X), ( and the worm was decapitated Just behind the buccal capsule with a small cataract knife. The slide was heated gently to remelt the glycerine Jelly, and a small coverslip was applied. The coverslip was manipulated with a dissecting needle until an en face view was obtained, and it was held in position until the glycerine jelly hardened* Drawings and some measurements were made with the aid of either a camera lucida or a microprojector. CLASSIFICATION

The economic importance of nematode parasites of plants and animals is attracting an increasing number of workers, and the field of nematology is rapidly becoming a separate specialty. A large number of new species is being described annually, and, as in all groups which are undergoing extensive study, the classification is being expanded to indicate more closely the natural relationships of the members in the group. It has been customary in the past to consider the nematodes as a class, together with the Acanthocephala and Nematomorpha (Gordiacea), in the phylum Nemathelminthes. Chitwood and Chitwood (1950) proposed that the class Nematoda be given the rank of a phylum. Such a major change is not accepted readily by most zoologists. In her volume on nematodes and related invertebrates, fyman (1951) separated the Acanthocephala into a separate phylum. However, she considered the Nematoda as a class in the phylum Aschelminthes (Nemathelminthes) in which she also placed the classes Rotifera, Gastrotricha, Kinorhyncha, Priapulida, and Nematomorpha. The following classification of Rictularia coloradensls is taken from the f i r s t edition of "An Introduction to Nematology" pub­ lished by Chitwood and Chitwood in 1937 in which the nematodes are considered as a class in the phylum Nemathelminthes. The superfamily, family, and subfamily diagnoses are from Chitwood and Wehr (193U), and the generic diagnosis is from Hall (1916).

- 13 - - Ik - Phylum Nemathelminthes (Aschelminthes) Class Nematoda Subclass Phasmidia Chitwood and Chitwood, 1932 Order Spirurida Chitwood, 1933 Suborder Splrurina (Railliet and Henry* 1915) Superfamily Spiruroidea R ailliet and Henry, 1915 Plagnosis* Oral opening oval, circular, hexagonal, or dorsoventrally elongated, and occasionally bordered by teeth. Papillae of external circle consisting of four (laterodorsals and lateroventrals) or eight (dorsodorsals, laterodorsals, ventroventrals, and lateroventrals), the dorsodorsals and ventroventrals, in some cases, showing a marked tendency to disappear or to fuse partially or completely with the laterodorsals and lateroventrals, form­ ing double or duplex papillae; ventrolaterals absent, so far as known. Amphids lateral, pore-like, not externally modified; never posterior to labial region. True lips reduced or absent; pseudolabia usually and interlabia some­ times p resent. Stoma usually w ell developed, prostom often everted. Esophagus usually differentiated into an anterior short, narrow, muscular part and a posterior long, wide, glandular part, or into an anterior muscular clavate part and a posterior glandular clavate part, the two divisions approximately equal in length, or not distinctly differenti­ ated into two parts, the esophagus showing either a cylin­ drical or clavate form. Spicules usually unequal and dissimilar, sometimes equal or subequal and similar. Qubernaculum present or absent. Caudal alae present or absent. Subventral excretory cells absent, excretory system inverted U-shaped. Family Thelazildae R ailliet, 1916 Diagnosis* Spiruroidea. Oral opening oval or hexagonal, dorsoventrally or laterally elongated. Papillae of external circle four or eight in number, not approaching in pairs, sometimes with dorsodorsals and ventroventrals internal to laterodorsals and lateroventrals; papillae of internal circle reduced, rarely rudimentary. Pseudolabia usually absent. Cordons or other cephalic ornamentation absent. Stoma usually well developed, prostom not everted (except in Physocephalus), telorhabdions inconspicuous. Cuticular spines present or absent. Caudal alae present or absent. Spicules equal or unequal and sometimes dissimilar. Quber­ naculum present or absent. Vulva usually pre-equatorial, rarely equatorial or post-equatorial. Oviparous or vivi­ parous. - 15 - Subfamily Rictulariinae Hall, 1913 emend. Chitwood and Wehr, 193h Diagnosis: Thelazlldae. Oral opening usually shifted dorsad, degree of shift varying with the species. Lips and pseudolabia absent. Papillae of internal circle distinct with termini pore-like, dorsodorsal and ventro- ventral papillae of external circle small, situated near the laterodorsal and lateroventral papillae, the latter large; amphids and internolateral papillae, usually ventrolateral in position. Cuticle armed with hooks or spines; cuticular bosses absent. Caudal alae present or absent. Spicules equal or unequal. Qubernaculum present. Vulva pre-equatorial or equatorial. Oviparous. Type genus Rictularia Froelich, 1802. Qenus Rictularia Froelich, 1802 Diagnosis: Rictulariinae. ftjccal capsule well developed and narrow, with its aperture more or less distinctly dorsal and surrounded by a circlet of denticles, and with its base armed with teeth and spines. Along practically the entire ventral surface of each side there are two rows of cuticular combs or spines. Male with or without bursa; the bursa when present is always small and always remains open. Two small, equal or unequal spicules. Vulva anterior, near the posterior end of the esophagus. Oviparous, the egg containing a well-developed embryo when oviposited. Parasitic in the small intestine as a rule. Species R. coloradensls Hall, 1916

Diagnosis: See page U8. OBSERVATIONS AND RESULTS

Occurrence of Rictularia coloradensls In Naturally Infected Mice

A total of eighty-two white-footed mice, Peromyscus leucopus noveboracensiSf has been examined for parasites since 1950. Thirty of these mice ( 3 6 .6 per cent) were parasitised with Rictularia colora­ densls. The mice were collected from three different locations: 1.) Rhododendron Hollow, Hocking County, Ohio, 2.) the Ohio State Uni­ versity Woodlot, and 3.) the wooded area bordering the Olentangy River north of the Ohio State University Campus. The incidence of Rictu­ laria in these areas is given in Table 1. The per cent infections given in the table may not be entirely accurate since the young nema­ todes are very small, and some may have been overlooked in earlier examinations.

TABLE 1, Incidence of Rictularia coloradensls in Peromyscus leucopus noveboracensls.

No. Infected / No. Examined Total Location ------■ Per Cent Nora Apr. Uay June July Aug. Sept. Oct. Infection Burden^ M BBaanBM BBaaBBssBaB^aBsnK xanaanaBaK Rhododendron Hollow 11/16 7/8 — — 5/7 3/3 — 76.5 1-10 (Av. 3 ) U niversity Woodlot 0/1 — 1/2 1/6 — — 2/lh 17. h 1-7 (Av. 2.8) Olentangy River 0/15 0/6 — — 0/h — - - 0 ----

1 Only adult worms are tabulated. - 16 - - 17 -

Rictularia was not found in mice collected from the swamp forest bordering the Olentangy River. In Rhododendron Hollow, on the other hand, about three-fourths of the mice were infected* Rictularia is endemic in the University Woodlot, but the incidence of infection is low (17.U per cent). The worm burden ranged from one to ten with an average of three adult worms per mouse. Two white-footed mice collected at Rhodo­ dendron Hollow also harbored ten and fifteen Rictularia larvae, respectively. Out of a total of eighty-nine adult worms from natur­ ally infected Peromyscus, only one male was found. The low worm burden and the male-female ratio will be discussed later in the section dealing with the development of R^ coloradensis in the definitive host.

Experimental Infection of Arthropods

An intermediate host is utilised in all life cycles which are known for spiruroid nematodes. This host is usually an insect, but sow bugs, copepods, and other crustaceans are used by some species. Eleven species of insects and one species of isopod were fed eggs of R. coloradensis to determine the range of intermediate hosts in which this parasite could develop, and also to determine, if possible, the species which might act as natural intermediate hosts. Some of the experimental hosts were obtained from cultures reared in the laboratory, and others were collected in the field. The following species were usedi - 18 - Species reared in the laboratory: hlattella germanica (Linn.) - German roach Blatta orientails Linn. - oriental roach Periplaneta amerlcana (linn.) - American roach Supella supellectillum Serv. - brown-banded roach Parcoblatta pensylvanica (DeGeer) - wood roach Tenebrio molltor Linn. - meal worm Species collected in the field; Trachelipus sp. - sow bug Parcoblatta pensylvanlca (DeGeer) - wood roach P. virgin!ca (Brunner) - wood roach Acheta assimilis Fabricius - field cricket Ceuthophilus sp. - camel cricket Dicaelus sculpt!lis Say - ground beetle Chlaenlus sp. - ground beetle A preliminary feeding experiment with wood roaches indicated that the larvae developed to the infective third stage within ten to twelve days. Therefore, the insects and sow bugs were examined for nematode larvae twelve days following exposure to eggs. The results of the dissections are given in Table 2. Feces of the sow bugs were examined on the day following exposure to eggs, and many dead larvae and several unhatched eggs were found. The dead larvae measured 326 microns long andlh microns in diameter, and they resembled in all details the larvae of Rictularia Just after hatching. Although the eggs hatch readily, sow bugs apparently do not offer an internal environment suitable for continuing develop­ ment, since no larvae were found when the sow bugs were examined at the end of twelve days. - 19 - TA^LE 2. Experimental Infection of arthropods with Rictularia coloradensls following the feeding of infective eggs.

Species Stage of Number Infected Number Host Number Examined of Cysts

Crustacea Isopoda Trachelipus sp. Adult 0/16 0 Cnsecta Orthoptera B lattidae Parcoblatta pensylvanica Adult Nymph IS IS Parcoblatta virglnica Adult Nymph IS Blattella germanica Adult Nymph IS IS Blatta orientalis Nymph 3/12 1- 7( 0 ) Periplaneta americana Nymph h/10 1-9 Supella supellectilium Nymph 7/9 1 -2 G ryllidae Acheta assimilis Adult (a) (a) Cryllacrididae Ceuihophilus sp. Adult 2/3 2-20 Coleoptera Tenebrionidae Tenebrio molitor Larva i / 6 lid) Adult 0/3 0 Carabidae Dicaelus sculptulis Adult 1 /2 3 (c) Chlaenius sp. Adult 2/3 8- 1 0 (c)

(a) An undetermined number of specimens was used to rear larvae for feeding experiments. The per cent infection was high, and usually more than twenty cysts were found in each in se ct. (b) This host was used to study the developmental stages in the inter­ mediate host. The per cent in fectio n was high, but the number of cysts was never large. (c) Cysts pigmented and containing dead or dying larvae. (d) Larva dead but cyst not pigmented. - 20 - Soae degree of larval development took place in each species of Insect exposed to eggs. Cysts containing larvae were found on the hindgut and/or free in the body cavity. In the American and oriental roaches, cysts were located on the hindgut just posterior to the Malpighian tubules. In these two species, however, the cysts contained a reddish-brown pigment, and the larvae appeared to be dead or dying. In heavily pigmented cysts, the larvae had disin­ tegrated, but in lightly pigmented cysts, the larvae appeared to have developed to the third stage. In German and brown-banded roaches, the larvae developed to the infective third stage in cysts which remained attached to the hindgut posterior to the Malpighian tubules. There were no indications of pigmentation in the cysts which developed in these two species. In wood roaches and field crickets, cysts containing larvae in the third stage of development were found both free in the body cavity and attached to the hindgut. In the camel crickets, all the cysts were free in the body cavity at the time of dissection. A single cyst was found in the body cavity of a meal worm larva. This cyst contained a dead larva which had undergone some development but had not reached the Infective stage. In one specimen of the ground beetle Dicaelus sculptulis, three small pigmented cysts were found in the body cavity. The larvae in these cysts were completely d isin teg rated . In Chlaenius s p ., a number of pigmented cysts were found attached to the hindgut and also free in the body cavity. All the larvae in these cysts were dead, but when the hindgut of one specimen was teased apart, six viable larvae were released. The cysts containing these larvae were not visible on the surface of the hindgut, - 21 - and the defensive mechanism of the host tissu e apparently had not reached the stage where pigmentation and death of the larva occurs* Since there was the possibility that the insects collected in the field were already infected with some spiruroid roundworm other than Rlctularia, feeding experiments ware conducted to confirm the identity of the larvae. Larvae from Parcoblatta virginlca, Ceuthophllus sp., and Acheta assim ilis (approximately twenty from each source) and the six viable larvae which were recovered from the intestinal epithelium of Chlaenius were fed to uninfected white mice. After eight days, three immature female and two immature male speci­ mens of Rictularla were recovered from the small intestine of the mouse fed larvae from Parcoblatta j one immature female was recovered from the mouse fed larvae from Ceuthophllusj and two immature females were obtained from the mouse fed larvae from Acheta. The mouse fed the larvae from Chlaenius was negative. However, the small number of larvae fed in the latter case may account for the failure to establish an infection. It is also possible that larval development had been inhibited in this host, and that the larvae had not reached an infective age. All worms recovered from the mice were easily identi­ fied as R ictu larla by the two rows of cu ticu lar combs and spines. The data obtained from these laboratory studies indicate that, although some development will probably take place in a large number of different species of Insects, there is a definite host reaction in some species which results eventually in the destruction of the parasite. The resulting host specificity appears to be on a species level, since in the Orthopterw, development without adverse host reaction occurs in field crickets (Qryllidae), camel crickets (Qryll- acrldidae), and wood roaches, German roaches, and brown-banded roaches - 22 - (Blattldae). Within this last family, however, tissue reaction in the American and oriental roaches results in the death of the larvae. Although the number of species of insects used in these labor­ atory studies was small, the results indicated that wood roaches, camel crickets, or field crickets might act as the natural inter­ mediate hosts for R^ coloradensis. German and brown-banded roaches are domestic species, and they would not be expected to act as hosts under natural conditions.

Natural Intermediate Hosts

In view of the results obtained from the feeding experiments, a number of Orthoptera were collected from Rhododendron Hollow and the University Woodlot and were examined for encysted larvae of Rlctularia. The results obtained from this survey are given in Table 3.

TABLE 3. Insects examined for natural infections of Rlctularia larvae.

No. Infected / No. Examined______No. Insect University Woods Rhododendron Hollow Cysts

Camel crickets 0/5 16/32 1-31 (Av. 9.7) Wood roaches l/ti3 0/2 1 Field crickets O/l 0/1 0

In Rhododendron Hollow, fifty per cent of the camel crickets harbored cysts containing the infective third-stage larvae of Rictularla. These camel crickets were identified as Ceuthophllus g. g racilip es (Haldeman) by Dr. Edward Thomas of the Ohio S tate - 23 - Museum. They undoubtedly serve as the primary Intermediate host of R. coloradensis at Rhododendron Hollow. One cyst containing a single larva was found in a wood roach, Parcoblatta virgin!ca, collected at the University Woodlot. This larva agreed with the larvae of R. coloradensis from other insect hosts, but its identity was not posi­ tiv e ly confirmed by feeding. However, since i t was found th a t larvae develop readily in wood roaches in the laboratory, it seems probable that Parcoblatta spp. also serve as natural intermediate hosts. Ecological evidences also point to camel crickets and wood roaches as natural intermediate hosts. Rhododendron Hollow is a deep gorge with precipitous, heavily-wooded sides which are characterized by numerous outcroppings of Blackhand conglomerate sandstone. There are abundant signs of the activity of white-footed mice around these outcroppings. These mice are known to feed not only on nuts and seeds but also on in se c ts. A large number of camel crick ets were collected one evening on and around the outcroppings, which would suggest th a t camel crickets could easily comprise part of the diet of the mice in this area. The population of wood roaches is small in Rhododendron Hollow (only two were found during several diligent searches of the area). In the University Woodlot, on the other hand, wood roaches are abundant under fallen logs and pieces of bark. Since white-footed mice also make use of these same ecological niches, it would seem likely that wood roaches, possibly together with camel crickets which were found occasionally in these same habitats, serve as the inter­ mediate hosts for R. coloradensis in this latter area. - 21* - Development in the Intermediate Host

In the general spiruroid life cycle, the eggs, containing first- stage larvae, hatch in the intestine of the intermediate host, wMch, in all known cases, is an arthropod. These larvae usually enter specific organs or tissues where they are surrounded by a cyst of host tissue. In some cases, however, they develop in the body cavity of the host without becoming encysted. The developing larvae undergo two molts in the intermediate host, the second molt giving rise to infec­ tive third-stage larvae. No further development occurs until such infective larvae are Ingested by s suitable definitive host. In the development of coloradensis in the intermediate host, the larvae become encysted and follow the general rule of two molts. The descriptions of the stages in the life cycle which follow are based on the development of the larval stages in the German roach at room temperatures which varied between 72° and 8o° F. The egg. The egg is fully embryonated when laid, and it is surrounded by the three membranes characteristic of nematode ova (Plate 1, Fig. 1). The outer or albuminous coat is ovoid, £1.6 to ££.0 microns long (Av. £3«M and 39.1* to microns wide (Av. b0.6), and it is about 2 microns thick. The albuminous coat has about the same refractive index as water and is very difficult to see in eggs which have been laid in water. If a drop of India ink is added to such a preparation, the albuminous coat is easily visible as a clear area between the colloidal particles of pigment in the surrounding water and the chitinous shell of the egg. In eggs recovered from the feces of the definitive host, small particles of debris adhere to the albuminous coating, delim iting i t s o u tlin e. However, the albuminous coat is sometimes missing in eggs found in fecal specimens. The - 25 - chitinous or true egg shell is U7*6 to 50.2 microns long (Av. 1*6.9) and 3h*7 to 39*U microns vide (Av. 36.2), and i t is 1.9 to 2 microns in thickness. The inner or vitelline membrane is a thin, refractive layer closely applied to the chitinous shell. The first-stage larva is extremely coiled and flexed, and it takes up the entire space within the egg membranes. Such larvae in fully embryonated eggs, i. e., those found in fecal specimens or those laid in water, appear to be in a state of diapause, and they were never seen to undergo any type of movement within the egg. Eggs in the uterine horns some distance from the vulva contain younger, developing larvae which exhibited sluggish movements. The excretory vesicle, a hyaline vesicle three to four microns in diameter, is located about 112 microns from the anterior end of the larva and is very prominent in unhatched eggs (Plate 1, Pig. 1). The vesicle was observed to contract suddenly every one to three minutes to about one-third its original diameter and then slowly return to its original size. Attempts to hatch eggs in vitro were unsuccessful. Eggs failed to hatch after being kept in either tap water or Ringer's cold solu­ tio n fo r several weeks a t room tem perature. Eggs placed in a r t i f i c i a l gastric juice (0.5*3 pepsin in 0.2£ HC1) and in macerated roach intes­ tine also failed to hatch. The first-stage larva. The eggs hatch in the midgut of the German roach, and first-stage larvae were found in this location with­ in three hours after the roach was exposed to eggs. Eggs recovered from the foregut were always unhatched, and the larvae within the eggs were non-mo ti l e . The mechanism of hatching was not determined. The shells of hatched eggs have one end pushed outward and ruptured, but - 26 - both the albuminous and chitinous layers appear to be Intact except at the ruptured end. This would suggest that hatching is not a result of the digestion of the egg membranes but is the result of a pressure applied from within the egg, probably by the larva itself. First-stage larvae were found in the hindgut of the roach twenty- four hours after exposure to eggs. These larvae invade the epithelium of the hindgut, and further development takes place within a cyst formed by host tissues. The reaction of the host tissue to the parasite will be discussed in a later section. The larvae, soon after hatching, are 306 to 326 microns long and 12.5 to 15.6 microns wide. They undergo little development up to the end of the first day (Plate 1, Fig. 2). The head bears a small, lateral, refractive protuberance on each side. Such protuberances may correspond to the teeth or spines reported for other first-stage spiruroid larvae. The excretory vesicle is located ventrally about 110 to 115 microns from the anterior end. This structure is associ­ ated with a large nucleus, containing a nucleolus, which probably belongs to the renette cell} however, the cell membrane of this cell was not visible. Neither the esophagus nor the intestine are differ­ entiated by the end of the first day, although the anterior end of the intestine is vaguely indicated by an accumulation of cellular material. The body of the larva is filled with ill-defined, granular cells. A large nucleus, probably that of the anlage of the genital primordium, is located about two-thirds of the distance from the head to the tip of the tail. The intestine is the first structure to develop. By the end of the second day of development, a lumen is present from the anterior end of the intestine to a point Just posterior to the genital - 27 - primordium, which is now a distinct cell. The intestinal lumen continues to develop posteriad in succeedingdays. The esophagus develops from its posterior end anteriad, and differentiation is apparent for the first time in the posterior half of the esophagus at the end of the third day (Plate 1, Pig, 3)• The lumen of the rectum is vaguely indicated at the end of the fourth day of develop­ ment. At the end of the fifth day of development, the cuticle of the head and tail region is pulled away from the body of the larva, indicating the approach of the first molt (Plate 1, Fig, b). However, the first molt does not actually occur until the seventh or eighth day after exposure to infective eggs. Larvae in the pre-molting stage are 32U to 355 microns long and about 25 microns in diameter. In the darly pre-molting stage, the anterior third of the esophagus is not clearly differentiated, but the posterior two-thirds is well developed and somewhat bulbous at the posterior end. The esophagus is fully developed just prior to the first molt, and is about 135 microns long. The walls of the intestine, especially in the anterior portion, contain many refractive globules of varying size. The intestinal lumen is developed as far as the rectum but does not connect with it. The excretory pore is located about 100 microns from the anterior end. The renette cell is ovoid, about 25 microns long, and displaces the esophagus dorsad. It is connected to the excretory pore by the excretory vesicle. The genital primordium is a compact, ovoid mass containing 6 to 8 nuclei. It is located about 215 microns from the anterior end. The tail is bO to 52 microns long and rounded at the tip. The nerve ring is distinctly developed in the advanced pre- molting stage and is located about 60 microns from the anterior end. - 28 - The second-stage larva. Second-stage larvae (Plate 1, Fig. 5) were first found at the end of the seventh day of development, at which time the cuticle was already pulled slightly from the tip of the tail, indicating the approach of the second molt. The cuticle begins to pull away from the head region by the end of the eighth day, and the buccal capsule of the third-stage larva begins to form at the end of the ninth day (Plate 1, Fig. 6). Second-stage larvae are 1x22 to bS9 microns long and 30 to 36 microns wide. The mouth opens into a shallow buccal capsule which is about 3 microns deep. The esophagus starts 11 to 13 microns from the anterior end and is 112 to 13b microns long. The esophago-intestlnal valve is not developed. The nerve ring is located 62 to 6$ microns from the anterior end. The excretory pore is on a slight prominence 87 to 93 microns from the anterior end. The excretory vesicle of the first-stage larva is replaced by an excretory duct which leads into the renette cell. In late second-stage larvae, the longitudinal excretory ducts are visible, passing from the renette cell into the lateral cords. The genital primordium is slightly elongate, 3b to 37 microns long, and located2$0 to 280 microns from the anterior end. The intestino-rectal junction is not completely developed. The tail is b2 to 56 microns long. The cuticle of the second larval stage is rounded at the tip of the tail, but, in all specimens observed, the pointed tail which is characteristic of the third-stage larva was apparent under the cuticle. The third-stage larva. The second molt occurs during the twelfth or thirteenth day of development and gives rise to the third larval stage (Plate 2, Fig. 7)* The third-stage larva, which is the infec­ tive stage, is structurally complete after the second molt. The only - 29 - additional development in the intermediate host appears to be a slight Increase in overall size and in the length of the genital primordium. Fully grown larvae are 506 to 593 microns long with a maximum width at the posterior end of the esophagus of 56 to 61 microns. The cuticle is 5 to 8 microns thick and is transversely striated. The cuticle is also thickened laterally, forming longitudinal ridges which extend from the head to the tail. The cervical papillae are located dorsal to the longitudinal ridges at a distance of 77 to 85 microns from the anterior end. The buccal capsule is 11 to 1U microns deep and is surrounded anteriorly by a circlet of fine denticles (Plate 2, Fig. 8). The latero-dorsal and latero-ventral papillae of the external circle are present, but the presence of the dorso-dorsal and ventro-ventral papillae of the external circle could not be veri­ fied in lateral view. The esophagus terminates In an esophago- intestinal valve 191 to 213 microns from the anterior end. The nerve ring is located 52 to 55 microns from the anterior end, and the excretory pore at 93 to 110 microns from the anterior end. The intestino-rectal Junction is complete, and the tail is pointed, U9 to 57 microns long. The genital primordium is rod-shaped, liO to 73 microns long, and located slightly posterior to the mid-point of the body. First and second-stage larvae die soon after being placed in tap water. Third-stage larvae, on the other hand, are very resistant, and some larvae were still viable, i. e., exhibited motility, after being kept in tap water at C. for 100 days. Such larvae were not infec­ tive, however, when fed to laboratory mice. - 30 - Additional observations on larval development. The effect of temperature on larval development was not determined accurately since all feeding experiments were conducted at room temperature. However, there Is reason to believe that temperature is an important factor in determining the rate of larval development in the intermediate host. The first molt occurred during the seventh day in German roaches kept at 72° to 80° F. In one series of German roaches infected during August, however, the first molt occurred as early as the fifth day of development. The time of the second molt was not determined in this series, but it would probably also occur relatively sooner than the data mentioned above would indicate. Unfortunately, a record of the range of temperatvres in the laboratory was not obtained during the time th is se rie s was in progress. However, data from the Heather Bureau Station at Port Columbus gave an average minimum temperature of 68.3° F. and an average maximum temperature of 92.5° F. during the time that this series was in progress, which would seem to indicate that higher temperatures may be responsible for the increased rate of development. In brown-banded roaches, the first molt occurred during the seventh day of development, and the temperature during this time ranged from an average minimum of 69.9° f. to an average maximum of 93*9° F. at the Heather Bureau Station. These average temperature extremes are comparable to those reported above, but the first molt was apparently delayed two days in this species. This would indicate that not only temperature but also the species of intermediate host utilized can influence the rate of larval development. The first molt occurred during the sixth day of development in field crickets Infected late in September. Since the buildings were being heated artificially at this time, the temperature probably ranged between 70° and 80° F. These data leave much to be desired, but it seems probable that the first molt will occur during the fifth to eighth day and the second molt during the tenth to thirteenth day of larval development, except under extreme environmental conditions. The exact influence of temperature and of the species of intermediate host on the rate of larval development necessitate experimental feedings performed at constant temperatures.

Pathology in the Intermediate Host

After the eggs of R^ coloradensis hatch in the midgut of the intermediate host, the first-stage larvae enter the epithelium of the hindgut where, after two molts, the third-stage larvae develop. The presence of the larva in the epithelium results in tissue damage and the formation of a cyst of host tissue around the developing p a ra site . Schell (1952b) described the tissue reactions of the Qerman roach to developing larvae of Physaloptera hispida. The pathological changes caused by the larvae of Rj_ coloradensis appear to be very similar to those described for P^ hispida. First-stage larvae of R^ coloradensis were found in the epi­ thelium of the hindgut twenty-four hours after Qerman roaches were exposed to eggs (Plate li, Fig. 22). A section through a cyst, con­ taining a third-stage larva after twelve days of development in the Qerman roach, is shown in Plate U, Fig. 23. The larva is surrounded by a zone of diffuse cytoplasm containing hypertrophied nuclei of the - 32 - intestinal epithelium. In this cyst, the zone of diffuse cytoplasm is loosely surroundedby connective tis s u e c e lls . However, th e initiation of fibrosis does not appear to be a strict function of tim e, since another cy st in the same roach (P la te 5, Pig. 2U) shows no indications of infiltration of connective tissue cells. Cysts containing third-stage larvae were first visible protruding from the surface of the hindgut on the fourteenth day of development. Cysts may become pedunculate in the German roach, but they were never found free in the body cavity. In field crickets, wood roaches, and camel crickets, the larvae invade the hindgut, but the developing cysts break away from the surface of the gut and are found free in the body cavity. In field crickets, cysts were first foudd free in the body cavity ten days a f te r exposure to eggs. Even a f te r tw enty-three days, however, some cysts were still attached to the hindgut. A section through a cyst in the epithelium of the hindgut of a field cricket twelve days after infection is shown in Plate 5, Figure 25* There appears to be a relatively larger zone of diffuse cytoplasm than was found in the German roach. Cysts recovered from the body cavity of field crickets, camel crickets, and wood roaches appear to be similar. The cyst (Plate2, Fig. 9) is surrounded externally by a thin, anucleate membrane. Internally it contains diffuse cytoplasm with many hypertrophied nuclei. The diffuse cytoplasm is surrounded externally by a thin, elastic membrane, and the cavity containing the larva is lined with a similar membrane. There is a thin, hyaline space between the ex tern al w all of th e cy st and th e membrane surrounding th e d iffu se cytoplasm. Each cyst may contain from one to as many as twelve larvae. In compound cysts, there may be up to three larvae within a single cavity within the cyst, and there may be several cavities containing larvae. In C euthophllus s p ., compound c y sts up to 1*80 microns in diam eter were found. As was mentioned in an earlier section, cysts which developed in American roaches, oriental roaches, and ground beetles contained a reddish-brown pigment, and the larvae within these cysts were dead or dying. This phenomenon has been observed by other authors, and the data and opinions which have been advanced are reviewed by Schell (1952b). The process of pigmentation has been commonly referred to as "chitinisation," but Schell was unable to get a positive test for chitin. It is his opinion that "...pigmentation represents a more effective defensive mechanism than encystment, as the latter merely walls off the parasite while pigmentation is associated with the death of the parasite."

Infactivity of the Larval Stages

The larvae of spiruroid roundworms are usually considered to become infective when they have developed to the third stage; however, Schell (1952a) found that second-stage, as well as third-stage, larvae of Physaloptera hispida were infective. When second-stage larvae were fed to cotton rats, the second molt, which would normally occur on the twenty-fourth to twenty-seventh day in the intermediate host, was delayed about thirteen days. The first-stage larvae of hispida were not infective. The stage in which the larvae of coloradensis become infective was determined experimentally by feeding larvae of varying ages to - 31* - laboratory mice. Since the first and second larval stages are too small to be handled conveniently when removed from their cysts* each mouse was fed the Intestines of three Infected Oerman roaches by means of a stomach tube. Larvae which had undergone 1* 3* St 8 * 10* 12, lb, and 16 days of development in the Oerman roach at 72° to 00° F. were fed* and the mice were examined five days following the last feeding. Idee fed 1* 3* St and 8 day larvae were negative. The mice fed 10* 12* lb* and 16 day larvae harbored 2* 1* 2* and 2 immature* adult Rictularla respectively. These data were confirmed by feeding larvae 6, 7* 8* 9* 10, 11* and 12 days old. Each mouse was examined five days after being fed. The mouse fed larvae 10 days old harbored one immature* adult Rictularla * and the mouse fed larvae 12 days old harbored one third-stage larva which was about ready to molt into the adult stage. The remaining mice were negative. These data indicate that the larvae of FU_ coloradensis become infective at least as early as the tenth day of development under the laboratory conditions stated above. Since the first molt occurs during the seventh or eighth day and the second molt during the twelfth or thirteenth day of development in the Oerman roach at 72° to 80° F., it would appear that the larvae become infective about two days prior to the second molt. The characters of the third-stage larva appear to be well developed by the end of the tenth day* and it would seem to be a matter of definition whether ten day larvae are designated as second-stage or third-stage. However, the ecdysis or shedding of the larval cuticle was used in this research as the cri­ terion for separating the larval stages. From this point of view* therefore, the larvae would be designated as infective in the late second stage. - 35 - Development In the Laboratory House

Webster's strain of white Swiss mice, Mus musculus,^ were used as the experimental definitive host to study the development of R. coloradensis from the infective third-stage larva to the adult. The mice were fed approximately fifteen infective larvae each, and a series of developmental stages was obtained by examining the mice at varying intervals as *iven in Table ij. Out of a total of twenty-five mice used in this series, worms were recovered from nineteen (76 per c e n t). larvae were found in both the stomach and the duodenum at the end of three hours. In all mice examined after three hours, the worms were found usually in the upper third and occasionally in the middle third of the small intestine. Growth of the third-stage larva, which is in a state of diapause in the intermediate host, is resumed soon after entering the defini­ tive host. Sexes can be distinguished by the end of the first day of development in the mouse; the cells in the center of the genital primordium of the female be^in to become vacuolated, while the genital primordium of the male remains a compact, rod-shaped mass. The buccal capsule becomes sclero tized and c h a ra c te ristic of the ad u lt stage during the third day of development. The cuticle of the third-stage larva begins to loosen during the fourth day of development, indicating the approach of the third molt. Females in the third pre-molting stage after four days in the mouse are 850 to 1038 microns long and 50 to 62 microns wide. The buccal capsule (Plate 2, Fig. 10) is 9 to 10 microns in outside

1 Obtained from Taconic Farms, In c ., Germantown, New York. - 36 -

TABLE U. Worms recovered from experimentally infected laboratory mice.

Worms Recovered Interval after Infection Male Female Total

3 hours _ • k 18 hours - - 3 1 day - - 7 2 days - - 2 3 days - - 5 U days (Material from two mice) - - 11* 5 days (Material from two adce) 2 7 9b 10 days 1 2 3 15 days 3 2 5 20 days 0 3 3 25 days 0 2 30 days (Material from two mice) 2 3 5 35 days 2 5 7 US days 0 3 3 55 days 0 1 1 65 days 0 1 1

* Worms shorn evidence of approaching molt. ® Four females and one male molted; others show evidence of a molt. - 37 - diametert with Its base located 1U to 16 microns from the anterior end of the worm. The anterior end of the buccal capsule Is surrounded with denticles, and the base is provided with three triangular jaws which project anteriorly into the mouth. One Jaw is located dorsally and two are located ventro-laterally. The esophagus is cylindrical, 223 to 286 microns long. There is a prominent valve at the Junction of the esophagus and intestine. The nerve ring is located 81* to 97 microns from the anterior end, and the excretory pore is located ventrally, 132 to 139 microns from the anterior end. The vulva appears as an opening in the ventral body wall 506 to 580 microns from the anterior end (Plate 2, Fig. 11). The vulvar opening is con­ nected with the genital primordium by several enlarged cells. The genital primordium itself extends both anteriorly and posteriorly from the vulva, and it is 251* to 277 microns long. There is a group of highly vacuolated cells in the center of the primordium which appears yellow in living worms. One dorsal and two ventro-lateral cells sur­ round the rectum at its junction with the intestine (Plate 2, Fig. 12). The ta il is pointed and 62 to 61* microns long.

Males after four days of development in the mouse are638 to 989 microns long and 1*5 to 62 microns wide. The buccal capsule is 9 to 10 microns in diameter, and the base is located 12 to 11* microns from the anterior end. Structurally, the buccal capsule of the male resembles that of the female. The esophagus is 2U9 to 268 microns long, and the nerve ring is located 85 to 9b microns from the anterior end. The excretory pore is 127 to 132 microns from the anterior end. The genital primordium of the male extends along the ventral region from about the middle of the larva to the posterior end of the intestine (Plate 2, Fig. 13). The anterior end of the genital primordium is - 38 - flexed posteriad for a short distance. The spicules of the male are beginning to develop (Plate 2, Fig. 13). The tail is 62 to 65 microns long. The third molt occurs in laboratory mice during the fifth or early part of the sixth day aftet* infection. The genital papillae were visible under the old cuticle in males which had not yet molted by the end of the fifth day. The spines and combs characteristic of the adult stage were also visible under the old cuticle of the third- stage larva in both male and female specimens which had not molted by the end of the fifth day. The age of the third-stage larva at the time of ingestion appar­ ently has little effect on the rate of development in the definitive host. In the infactivity study, larvae ten days old molted as early as the fifth day in the mouse. On the other hand, larvae which were obtained after thirty-five days of development in a wood roach did not molt sooner than the fifth day when fed to laboratory mice. The third molt gives rise to worms with all the structural characters of the adult stage (Plate 6, Fig. 26). Further develop­ ment appears to be confined to growth and to the maturation of the genital systems. No additional molts were documented in the defini­ tiv e host. The average growth rate of coloradensis in the laboratory mouse is plotted in Text Figure 1. Unfortunately, the average lengths at the various time Intervals were determined from small samples (see Table U). In addition, variations in size which might be caused by physiological differences in individual mice are not averaged out, since moat of the time intervals are represented by material from a single mouse. E FGR 1 Aeae rwh ae f * clrdni i te aoaoy aonae. laboratory the in R*. coloradenaia of rate growth Average FIGURETEH 1. LENGTH IN MM. 30 0 2 25 10 15 ------

10

| i | j •

FEMALE

“ < _ _ _ _

< i _

1 i i UJ ------20 ------

' 25 ______IE N DAYS IN TIME < 30

36 > ___ 45 i | ; j ; 50 i i

55 Y

J . 0 6 T i i j

65 - UO - At the time of the third molt (five days after infection of the mouse), both males and females are about 1 to 1.25 nm long* At the end of twenty-five days, females were between U.3 and 6.1 on long. There is a rapid increase in length between the twenty-fifth and thirty-fifth day, and females recovered at the end of thirty-five days were between 16.2 and 20.9 mra long. Fertilized eggs, which were in early cleavage stages, were first observed in the uteri of females recovered after thirty days of development. At the end of thirty-five days, some of the uterine eggs contained larvae.A single female

2 3 .7 mm long which was recovered after fifty-five days of development contained eggs in the ovejector which would indicate that it had started to lay eggs. Growth continues for some time after the female reaches maturity, i. e., has eggs in the uterine horns, and specimens up to U2.5 nn long have been recovered from natural infections in Peromyscus. Males are relatively small, and they mature much sooner than females. The largest male specimen was recovered at the end of thirty days in a laboratory mouse and measuredU.6 mm long. However, males appeared to be sexually mature as soon as ten days after infection, since such specimens had spermatozoa in their seminal vesicles. The vulva of the female is located slightly posterior to the mid­ point of the body during the fourth day of development in the mouse. In mature females, on the other hand, the vulva is in the anterior part of the body, usually slightly anterior to the end of the esopha­ gus. In all adult female specimens, however, there are between twenty- nine and thirty-two prevulvar cuticular processes, regardless of the age of the worm. The difference in the location of the vulva in young - u i - and mature females Is caused ty a differential growth rate of the esophagus, prevulvar, and postvulvar regions of the body. The effect of the differential growth rates of the esophagus and the prevulvar and postvulvar portions of the body on the relative loca­ tion of the vulva of coloradensis is shown graphically in Text Figure 2. The values plotted in this graph are ratios; the relative location of the esophagus was obtained by dividing the average length of the esophagus by the average length of the worm, and, in the case of the vulva, the average prevulvar length was divided by the average length of the worm. Thus a value of 0.$ designates the mid-point of the worm, while a value of 1.0 indicates the tip of the tail, and a value of sero indicates the tip of the head. The relatively rapid growth of the postvulvar region in comparison to the prevulvar region causes the vulva to become located in the anterior end of the body. The esophagus grows at a faster rate than the prevulvar portion of the body wall, and the posterior end of the esophagus gradually approaches and eventually becomes located slightly posterior to the vulva. In none of the specimens obtained from laboratory mice did the vulva lie in a position anterior to the posterior end of the esophagus. There was some variation in the relative rates of growth, however, and in one specimen only 13.7 mm long after forty-five days of develop­ ment, the esophagus and vulva were on the same level. In Text Figure 2, time interval "x" represents specimens of unknown age from natural infections in which the vulva is located anterior to the posterior end of the esophagus. In specimens 22 to 23 mm long from naturally in­ fected Peromyscus, the vulva was slightly anterior to or at the level of the posterior end of the esophagus. VULVA ESOPHAGUS z o o o -J 0.5 UJ > H < _l Ui cr

2 0 30 40 46 50 55 60 6525 TIME IN DAYS SPECIMENS Of UNKNOWN AOE FROM NATURALLY INFECTED PER0MY8CU8.

TEXT FIGURE 2. Effect of differential growth rates of the esophagus and the prevulvar and postvulvar

portions of the body on the relative location of the vulva of IL. coloradensis. - U3 - Three times as many females as males were obtained from the experimental mice (Table U). A paucity of males appears to be charac­ teristic of the genus Rlctularia. A partial explanation, which appears to have some basis in the data obtained, might be that the males leave the body of the host soon after copulation with the females. Females mature, i. e., have fertilised eggs in the uteri, approximately thirty days after infection of the mouse, which would indicate that copulation had already taken place. Males were not encountered in mice examined on or after the forty-fifth day (Table U), which may indicate that the males leave the body of the host during the interval between the thirty-fifth and forty-fifth day. Males are known to leave the body of the host after copulation in such roundworms as the human pinworm, Enteroblus vermicularis, and the pork nematode, Trichinella spiralis. A small worm burden was characteristic of natural infections in Peromyscus, and usually only one to three mature females were present in each mouse. In heavier infections of up to ten female worms, the individuals appeared to be relatively young (approximately 20 mm or less). Laboratory mice examined fifty-five and sixty-five days after infection harbored only a single female each, which might be indicative of a decrease in the number of worms as the age of the infection in­ creases. Since the experimental mice were fed only about fifteen larvae each, a high worm burden would not be expected. On the other hand, camel crickets at Rhododendron Hollow frequently harbored over fifty third-stage larvae each. Twenty-six infected Peromyscus from this locality were examined, but no more than ten adult Rictularla were found in each host. - Uh - These data would suggest some mechanism which re s u lts in the elim ination of a l l except a few worms. Although the nature of th is mechanism i s not known* i t would not appear to be an immune reaction of the host* since super-infections were occasionally found In Pero­ myscus. In one mouse* fo r example* four mature females* one immature female* and ten larvae were found* representing infections from at le a s t three separate feedings. I f the host had developed an immunity* it would have been resistant to subsequent infections.

Pathology in the Definitive Host

A thorough study of the pathology in the definitive host was not accomplished because of a lack of material suitable for section­ ing. However* no gross pathological changes* e. g.* hemorrhagic areas or ulceration* were observed in infected mice. Mature worms occupied the lumen of the small intestine* but they were never found to be firmly attached to the mucosa of the intestine as is sometimes the case with other nematodes. Mature female worms are a translucent* salmon-red color. However* this does not seem to be caused by ingested blood* since the color is not confined to the intestine of the worm. Because of the low worm burden found in the mice* and also because of the lack of gross pathological changes* it would appear that adult specimens of coloradensis cause little damage to their host. Experimental Infection of other Mammalian Hosts

Additional observations were made on the development of R*_ colora­ densis in white rats (Rattus norveglcus), captive white-footed mice (Peromyscus leucopus noveboracwnsis)* and a meadow mouse (Microtus p. - 1*5 - pennsylvanlcua). The meadow mouse was obtained from a window w ell at the Kenny Road laboratory of Battelle Memorial Institute. The specimens of Peromyscus were collected in live traps from the wooded area along t.he Olentangy River just north of the Lane Avenue Bridge. The examination of a number of w hite-footed mice from th is location was negative for Rlctularia, and, in addition, the feces of the hosts were examined before they were infected in the laboratory and were found to be negative for spiruroid eggs. Any infection established in these animals could, therefore, be attributed to the experimental feedings. Two white rats were fed fifteen infective larvae each. One of these was negative at the end of five days. The other rat was examined at the end of the sixth day, and six larvae were recovered from the duodenum. These larvae were only 6U3 to 710 microns long, and thqy showed no evidence of advanced development or of molting. Two addi­ tional rats were fed twenty larvae each, and a laboratory mouse was fed fourteen of the larvae from the same source. The mouse and one r a t were examined a t the end of fiv e days. Four young adults and two larvae in the process of molting were recovered from the mouse. Only two larvae were recovered from the rat, and they were retarded in growth and development. The second rat in this series was examined at the end of twenty days and was negative. Since IL coloradensis has been reported from chipmunks (family Sciuridae), white-footed mice (family Cricetidac, subfamily Cricetlnae), and a vole (family Cricetldae, subfamily Microtinae), it would appear that the host specificity of R^ coloradensis is fairly broad. The laboratory mice used for experimental hosts in this study add another family of rodents (Muridae) in which this parasite can develop. - U6 - Although laboratory mica and rats belong to the same family and were fed the same d ie t, th e larvae appeared to be unable to grow and develop in the rat. Whether coloradensis can develop in a given host would appear, therefore, to be under the control of delicately balanced factors. Whether the factors involved are of a chemical or physical nature is unknown. A total of nine specimens of Peromyscus were fed approximately fifteen larvae each. One mouse was examined at the end of three days, and nine female and four male third-stage larvae were recovered. These larvae were in an advanced pre-molting condition. Another mouse was examined at the end of four days, and one male and one female in the adult stage were recovered. Two female worms were recovered at the end of fourteen days, but one w hite-footed mouse examined a t twenty- eight days, one at thirty days, and four at thirty-four days were negative. The meadow mouse was fed about twelve larvae, but it was negative when examined at the end of forty-three days. The third molt appears to occur one day earlier in Peromyscus than in laboratory mice. After molting, however, the worms do not appear to be able to continue their development to maturity under laboratory conditions. Since the worms molt one day earlier in Pero- myscus, their development at fourteen days should be comparable to the development at fifteen days in laboratory mice, if one assumes the same ra te of development in both hosts. However, th ere appeared to be a significant difference in the length of the worms (2.8 to 3*0 me after fourteen days of development in Peromyscus as compared to 3*5 to U.6 mm after fifteen days of development in white mice). - U7 - The causes underlying the failure of coloradensls to grow to maturity in Peromyscus ugder laboratory conditions are unknown. The change in diet or the stress caused by being kept in captivity may be sufficient to cause a change in the internal environment of the captive white-footed mice which is beyond the range of tolerance of the worms. Chabaud (19f>U) mentions th a t the data obtained from the experimental infection of captive animals are often deceiving. It has been found that captive animals under the influence of changes in their environment, and probably also under the influence of more complex factors, very frequently free themselves spontaneously of intestinal parasites. He concludes, therefore, that it is not sur­ prising that experimental infections in captive animals, even when performed under the most favorable conditions, very frequently result in failure.

Description of the Adult

The following description of the adult stage is based on material from naturally infected Peromyscus and also on specimens obtained from experimentally infected mice. Since only one male was found in a natural infection, the description of the male is based almost entirely on material from laboratory Infections, measurements are restricted to nature individuals; in the case of the female, this is interpreted to be those specimens which contain eggs in the uterus. Specimens of all ages were used for observations on the number of cutieular processes. - 1*8 - R ictu larla coloradensia H all, 1916 (Plate 3» Pigs, lli—21* Plate 6, Fig. 27; and Plate 7» Fig. 28) General. Oral opening anterior, with well developed and heavily sclerotized buccal capsule. Anterior margin of buccal capsule sur­ rounded with denticles and base provided with a tri-radiate opening surrounded by a dorsal and two ventro-lateral jaw-like projections with serrate edges. Six feebly-developed lips surround oral opening. Six papillae in inner circle, two amphids and eight papillae in outer circle (Plate 3» Fig. 16). Cuticle characterized by two longitudinal rows of cuticular processes located ventro-laterally. Female. Mature living specimens salmon-red in color. Length 13.7 to U2#5 mm; width 33b to 732 microns. Buccal capsule 80 to HO microns deep and 58 to 91 microns in outside diameter at base; sur­ rounded anteriorly by 16 to 20 denticles (en face observations on 7 specimens). Cervical papillae laterally located at equal or subequal levels, U50 to 578 microns from anterior end at level of 7th or between 7th and 8th comb. Excretory pore ventral, U62 to 516 microns from anterior end between 6th and 7th comb. Esophagus cylindrical, 2.1 to 5.L mm long, terminating in a prominent esophago-intestinal valve. Nerve ring, surrounding esophagus between bth and 5th or at level of 5th comb, 292 to 1*09 microns from anterior end. Vulva 2.3 to U*8 mm from anterior end, usually anterior to end of esophagus, but occasion­ ally on a level with or slightly behind esophagus, especially in small­ er specimens. Vulva opens into a muscular ovejector 136 to 512 microns long. Ovejector followed by a short vagina which branches, giving rise to a didelphic uterus. Uterine horns extend posteriad either to level of anus or, in some specimens, to a point about 500 microns in front of anus. Uterine horns then flex anteriorly and terminate in - 1*9 - ovaries slightly posterior to vulva. Entire female tract highly con­ voluted, filling most of body cavity from vulva to anus. Cuticular processes 63 to 69 pairs of which 29 to 32 pairs are prevulvar. Anterior cuticular processes are combs (Plate 6, Fig. 27) and poste­ rior processes are spines (Plate 7, Fig. 28); transition from combs to spines takes place in vulvar region, with 1 to 3 spines located anterior to vulva. Maximum length of combs (anterior apex to project­ ing tip) 69 to 97 microns; maximum length of spines 72 to 86 microns. Last one or two spines may be vestigial* in which case the last spine is located on a level with the anus; in specimens in which rudimentary spines could not be found, last spine 0*7 to 1.0 mm in front of anus. T ail 3U6 to 1*28 microns long. Eggs embryonated when laid, 30*6 to 32.0 by 39.8 to 1*3.6 microns in size in fixed material. Male. Length 2.1 to 1*.6 nun; maximum width, attained in posterior part of body, 11*0 to 252 microns. Two rows of combs extending from Just behind the buccal cppsule to a point 212 to microns anterior to cloaca; combs numbering U2 to 1*1* pairs, last pair frequently ves­ tigial; maximum length of combs, 67 to 109 microns. Buccal capsule, resembling that of female, 28 to i*i* microns deep and 25 to 3l* microns in diameter; surrounded anteriorly by a circlet of 18 denticles (en face observations on two specimens). Nerve ring between l*th and 5th p a ir of combs, 125 to 172 microns from a n te rio r end. Cervical p ap illae at level of 7th pair of combs, 187 to 21*9 microns from anterior end. Esophagus 0.5 to 1.0 mm long, cylindrical, slightly wider in posterior two-thirds than anterior third; esophagus terminated with an esophago- intestinal valve between 16th and 21st pair of combs. Excretory pore at about level of 7th pair of combs, 161* to 266 microns from anterior end. Cloacal opening 102 to 170 microns from tip of t a i l . Male - so - system single, extending from cloaca to about two-thirds of distance between cloaca and esophagus; terminal part of testis flexed posterlad for a short distance. Spicules equal, subequal, or unequal; right spicule, measured along curvature from origin to tip, 156 to 30U microns; left spicule 210 to 281 microns long. Oubemaculum present. Fans usually absent; one specimen with one fan (Plate 3* Fig. 21) obtained froma natural infection, and one specimen with two and one with three fans (Plate 3» Fig. 20) obtained from experimental feedings. Ten pairs of caudal papillae present (Plate 3* Figs. 19-21); two pairs anterior to cloaca, one pair at level of cloaca, and three pairs posterior to cloaca, with a fourth pair more laterally placed; these followed by three pairs of simple papillae, located close to ventral median line; last pair small and often difficult to see. A single, unpaired p ap illa located on an terio r lip of cloacal opening. Fhasmids open laterally at tip of tail. Hosts: Peromyscus leucopus noveboracensls Hus muscuius (experimental) Tissue s ite : Upper tw o-thirds of small in te stin e . Localities: Natural infections found in Franklin and Flocking Counties, Ohio. Life cycle: Larval stages found in Ceuthophilus g. gracllipes and Parcoblatta virgin!ca. DISCUSSION general Aspects of the Life Cycle

It is generally accepted that there are five stages in the devel­ opmental history of nematodes. The first four stages are larval stages, and each is terminated by a molt. The fourth molt* the molt of the fourth-stage larva, gives rise to the adult, sexual stage. Molting consists essentially of the formation of a new cuticle under the old cuticle} the latter is finally shed or, in some cases, retain­ ed as a sheath surrounding the developing larva. Each molt is usually accompanied by changes in the morphology of the buccal capsule, ta il, or some other structure covered or lined with cuticle. Growth per se does not appear to be the primary stimulus initiating the molt since, in the adult stage, nematodes may increase markedly in sise without molting. In spiruroid nematodes, the first two molts usually occur in the intermediate host, the second molt giving rise to the infective third- stage larva. Two additional molts occur in the definitive host, and the last or fourth molt gives rise to the adult stage. A diagrammatic representation of the development of coloradensia under laboratory conditions is shown in Text Figure 3. Temperature and the species of insect utilised as the intermediate host appear to have some effect on the rate of development. It was also shown that the third molt occurs one day sooner in the natural definitive host, Peromyscus leucopus, than in the laboratory mouse. Therefore, slight deviations from the time intervals given in Text Figure 3 c*n be expected under different environmental conditions and when different hosts are utilised.

- 5 1 - DEVELOPMENT IN GERMAN ROACH, DEVELOPMENT IN LABORATORY BLATTELLA GERMANIC A. AT 72*-8Q*F MOUSE, MUS MUSCULUS I S 2 a Sfl e bJ CM g _o _o _o H -8 -ai - -

-

FRT MOLT FIRST « i I SECOND MOLTI t NETO O ECSE, NETV LARVAE INFECTIVE ENCYSTED, OF INGESTION EGGS HATCH} LARVAE HINDGUT OF EPITHELIUM ENTER INGESTION EMBRYONATED OF EGGS LARVAE MOUSE INFECTIVE TO BECOME MALES MATURE (APPROX. TIME) MATURE (APPROX. MALES HR MOLT THIRO E0G8 EMBRONATED OF DEPOSITION EAE MTR (PRX TIME) MATURE (APPROX. FEMALES ENCYSTMENT APO. TIME) (APPROX. 2 - 52 -

TEXT FIGURE 3* Diagrammatic representation of the development of R*. coloradenais in experimental hosts - 53 - The development of IU coloradenals apparently forms an exception to the general rule of four molts in the Nematoda. The normal two molts occur in the intermediate host, but in the definitive host, the third-stage larva apparently gives rise directly to the adult without the interpolation of a fourth-stage larva. Third-stage larvae after four days of development in the laboratory mouse appear to correspond developmentally to fourth-stage larvae described for other worms. Sexes can be distinguished readily at the end of the fourth day, and the vulva of the female and spicules of the male are present, larvae were studied carefully from three hours after infection of the mouse up to the time of molting during the fifth day, and there were abso­ lutely no indications of a molt between these times. Therefore, the molt which gives rise to the adult stage during the fifth day must be the third molt. There is apparently only one well documented account a of nema­ tode life cycle with less than four molts, Chabaud (195U) described a shortened life history for Spirura rytipleurites seurati. The third-stage larva of this worm gave rise directly to the adult stage, and in this respect the development seems to be similar to that of R. coloradenals. Instead cf becoming passive after the second molt, however, the third-stage larva of rytipleurites was able to con­ tinue its development to an advanced third-stage larva in the inter­ mediate host. Chabaud found that this advanced development in the insect host was not mandatory. Similar development would take place in the definitive host when young third-stage larvae were fed, and this development terminated in a molt which gave rise directly to the adult stage. It is noteworthy that both IU coloradensis and pieurites are members of the superfamily Spiruroidea, although the - 51* - former ia a member of the Thelazlldae and the latter of the Spiruridac. Further studies of other members of the Spiruroidea may reveal other exceptions to the general rule of four molts. Moorthy (1938) In his study on the life history of Camallanus sweetl stated,"...only a single specimen of Camallanus larva under- going(?) the fourth molt was obtained. Judging from the structure of the old stoma which was just being cast off and of the new one just being formed, it was thought at first to be undergoing the third molt. However, the structure of the genital primordium and already formed vulva suggested further development than the third stage, and it is described as undergoing the fourth molt." The description of this larva suggests the developmental condition of late third-stage larvae of R^ coloradenals in regard to the already formed vulva. It is con­ ceivable that lloorthy's initial interpretation was correct, and that the larva was actually undergoing the third molt. If this were true, the development of sweeti would be similar to that of R^ colora- densis in regard to the number of molts. Vfitenberg (1928) stated that the larvae of Rj_ cahirensis, a para­ site of dogs, underwent two molts in the intestine of the definitive host which would suggest a normal development with four molts. Since he presented no data or drawings, it is impossible to compare his developmental stages with those of R^ coloradenals. The life cycle of R. cahirensis also differs from that of R. coloradenals in that the infective larvae were found encysted in the mesenteries of reptiles. Witenberg considered the reptiles to be second intermediate hosts, and he postulated that the larvae developed initially in insects. It was not established whether the reptiles were true second intermediate hosts or whether they served only as transport hosts. Physocephalus - 5 5 - sexalatua, the swine stomach worm, can utilise vertebrates as trans­ port hosts, but the vertebrate is not considered a true second inter­ mediate host since swine can become infected by ingesting third-stage larvae in the insect intermediate host (Seurat, 1913} Alicata, 1935)* Hall (1913) distinguished two groups of species in the genus Rictularla. Zn one group of species, which parasitise carnivore^ the transition from combs to spines in the female is very gradual and occurs posterior to the vulva. The combs extend to, or almost to, the cloaca in the males in this group. The other group of species para­ sitise rodents, bats, and insectivores. In this group, the transition from combs to spines is abrupt and takes place in the region of the vulva} th e males in th is group have the combs ending somewhat a n te rio r to the cloaca. It seems likely that vertebrate transport hosts are utilised by the group of species which are parasitic in carnivores, and that rodents, bats, and insectivores become infected directly by ingesting insect intermediate hosts containing encysted third-stage larv ae.

Taxonomic C onsiderations The genus Rictularla poses several distinct difficulties for the taxonomists 1.) The number of specimens available for study is usually small. 2.) Hales are encountered relatively infrequently and are un­ known for some species. Consequently, the differentiation of species has been based mainly on the characters of the female. 3*) Morpho­ logical evidences of specietion are based mainly on variable characters. The range of variation in a given species is usually qpite inadequately known because of the sm all number of specimens av ailab le fo r study. - 56 - In this study, it has been possible to examine a larger number of specimens than is usually available to the taxonomist. The speci­ mens from experimentally infected mice originated from eggs of pro­ bably less than six mature, female worms, and specimens from natural infections were collected within a very limited geographical area. Within these limited populations, however, it has been possible to demonstrate considerable variation in taxonomic characters. The redescription of Rj_ coloradensis given by Tiner (19h8a) is adequate for the most part, and there is no doubt of the conspeci- ficity of the material available to the author and that studied by Tiner. However, the range of variation of several diagnostic charac­ ters must be extended. Tiner lists ill to k2 pairs of combs in the male and 60 to 6k

pairs of combs and spines in the female, 29 to31 pairs of which were prevulvar. The males observed during the course of the present study had U2 to UU pairs of combs, and the females had 63 to 69 pairs of combs and sp in e s, o f which 29 to 32 p a irs were p rev u lv ar. The f r e ­ quency in occurrence of these variants is shown in Text Figures U, 5» and 6. Most of the males had U3 pairs of combs, and the females

usually had 6U to 6? pairs of combs and spines, with 30 o r 31 p a irs located anterior to the vulva. One or two of the terminal pairs of spines may be vestigial in the female, and the last pair of combs is frequently vestigial in the male. Tiner does not mention the presence of vestigial processes, which probably explains his lower ranges in the number of cuticular processes. The location of the vulva in relation to the posterior end of the esophagus has probably been given greater significance than this char­ acter warrants. It has been shown earlier that the location of the - 57 -

10

>- o X h i 53 a hi OE It.

42 43 44 PAIRS OF PROCESSES TEXT FIGURE lu Variation in the total number of pairs of cuticular processes in male R*_ coloradensis.

10 T

SEZE S3 64 65 44 67 68 69 PAIRS OF PROCESSES TEXT FIGURE •j. Variation in the total number of pairs of cuticular processes in female R. coloradensis.

20

> 0 I 10 f a1 oehi II

ja29 SO ils 31 32 PAIRS OF PROCESSES TEXT FIGURE 6. Variation in the number of pairs of prevulvar cuti­ cular processes in female R^ coloradensis. - 5 8 - vulva is a phenomenon of growth, and that the vulva gradually shifts from an equatorial position, which is posterior to the posterior end of the esophagus, to an an te rio r po sitio n somewhat in fro n t of the posterior end of the esophagus. Although the vulva in mature speci­ mens of R. coloradensis is usually anterior to the posterior tip of the esophagus, slight variations in the location of this structure are of no taxonomic value. According to Tiner, the eggs of FU coloradensis are 32 by J*5 microns in size. In the present study, the size of the eggs in fixed specimens (3 0 .6 to 3 2 .0 by 39.8 to 1*3*6 microns) was almost identical to that given by Tiner. These measurements do not include the albu­ minous coat which is difficult to see in fixed eggs. Fixation appears to cause some shrinkage, since the size of the chitinous shell in liv in g eggs ranged from 3U.7 to 39.1* by 1*7.6 to 5 0 .2 microns, Tiner (19l*8a) and UcPherson and Tiner (19$2) consider the maximum length of the combs of female Rictularla to be a valuable taxonomic character. The maximum comb length in the female specimens available to Tiner was 96 to 122 microns. The maximum length of the combs in the specimens studied by the author appeared to be smaller (65 to 97 m icrons). In the original description of coloradensis given by Hall (1916), the male did not possess mid-ventral fans which are present in the males of many other species of Rictularla. Tiner (19l*3a) found three fans anterior to the cloaca in the males which he studied. UcPherson and Tiner (1952) subsequently noted a male from Peromyscus leucopus which was without fans. Host of the males in this study were without fans, but one male with one fan, one with two fans, and one with three fans were observed. Fans are known to vary in number in - 59 - other species in the genus Rictularla, and they are apparently of little taxonomic significance. The length of the spicules, normally considered to be a reliable taxonomic character, is known to be variable in JU coloradensis. In the male described originally by Hall (1916), the right spicule was llt5 microns long, and the left was 180 microns long. Zn liner's redescription, the right spicule ranged from 50 to 53 microns long and the left from 92 to 99 microns long. McPherson and Tiner (1952) mention two males of coloradensis, one with the spicules equal, each 238 microns long, and the other with the right spicule 39 microns long and the left spicule 88 microns long. In the present study, the spicules of mature males were either equal, subequal, or unequal; the right spicule ranged from 156 to 3OI4 microns and the left from 210 to 281 microns in length. In males with unequal spicules, the right spicule was usually sh o rter than the l e f t . However, one male obtained after thirty days of development in a laboratory mouse had the right spicule 30 U microns long and the left spicule 281 microns long. The genital papillae were constant in number and location in all male specimens which were studied, and they agree with the specimens studied by Tiner. Unfortunately, the pattern and the number of papil­ lae are shared by several other North American species of Rictularia (see Table 5)» and therefore, this character is of limited value for separating species. The number of o ral d en ticles counted in female coloradensis ranged from 16 to 20, which overlaps and somewhat increases the range of 15 to 17 given by Tiner. Tiner found 12 denticles in the males, while 18 were present in two males which were examined en face by the writer. The denticles can be counted accurately only when specimens - 6 0 - *re examined en face, and the range of variation of this character has not been determined for most North American species in the genus. The North American species in the genus Rictularia are compared in Table £. The species may be divided into two groups on the basis of the oral opening. In one group, the oral opening is transverse and located in a distinctly dorsal position} in the other group, the oral opening is circular and located anteriorly. The oral opening is dorsal in Ri splendida, R. citelli, R. scalopis, and nacdonaldl which d iffe re n tia te s them from IU coloradensis and the other North American species of Rictularia in which the oral openings are anterior (ter­ m inal). R. ondatrae possesses a la rg e r number of cu tic u lar processes in both the male and female than is found in Rj_ coloradensis. Although the number of caudal papillae appear to be the same in the males of both species, the pair found on the level of the cloaca in IL colora­ densis appears to be shifted slightly posteriad in ondatrae accord­ ing to a reconstruction of this species made by Tiner (19hSa). R. dipodomis differs from Rj_ coloradensis by having slightly fewer cuticular processes in the male and slightly more in the female. The female, however, has about ten more pairs of prevulvar processes than are present in IL coloradensis. In the male, there is one more pair of precloacal papillae and one less pair of postcloacal papillae than found in R. coloradensis. In a redescription of R^ dipodomis, Read and Millemann (1953) do not mention or illustrate a papilla on the anterior lip of the cloaca, and they state that there are "at least" two pairs of simple papillae at the tip of the tail. These simple papillae are often difficult to see in lateral views of fixed TABLE 5. Comparison of the North Aaerlcan species of Rictularia.

Position Length Nuaber of Ifcirs of Coabs Nuaber of Nuaber A ipillae Species of (sa) and Spines D enticles of of , . Mouth Male Feaale Male feaale ft*e vulvar Male Feaale Fans I f c le ^ splendida Dorsal 5 8-10 108-109 136-138 55 15-20 7 8 7 e l ta l l l Dorsal 4-14 47-70 37-38 30-38 (b) 25-28 ? 7 4 ,5 ,6 2-(l)-l-2-l-3 Dorsal 7 54 7 7 287 7 7 7 7

MfldfiDlldl Dorsal 3 15-29 43 70-73 30-32 7 7 3,4,5 2-(l)-l-2-l-3 ondatras Anterior 7 25-30 52 73-75 3 2 7 18 1 2-(l)-0-4-l-3 dinodoaia A nterior 4-5 18-29 38-40 71-74 40-41 7 18 3 3-(7)-l-2-l-27 n n ^ h n ^ g lc ) A ntarior 7 24-42 7 56-60 32 7 26 7 7 a ic ro tl Anterior 5-10 11-28 45 64-66 32-33 26 24-25 0,1 coloradanaia A nterior 3 16-29 41-42 60-64 29^31 12 15-17 3 2-(l)-l-3-l-3 (Tiner, 1948s) color* A nterior 2-5 14-43 42-^4 63-69 29-32 18 16-20 0,1,2,3 2-(l)-l-3-l-3 (This study) s Explanation of foraolav 1st nuaber * pairs of pre-cloacal papillae) 2nd ncaber * presence or absence of unpaired papilla on anterior lip of cloaca) 3rd nuaber * pairs of papillae on level of cloaca) 4th nuaber * pairs of post-cloacal papillae near aedian line) 5th nuaber * pairs of post-doacal papillae near lateral line) 6th nuaber * pairs of slapis, terainal papillae, b All spines nay not have been counted, c Male unknown. - 62 - specimens, and it seems possible that the third pair is present in R. dipodomis but was not seen by these authors. R. microti appears to be quite similar to IU coloradensis. The ranges in the number of cuticular processes overlap or are contiguous in both species, and the papillae of the male are similar in number and location* The number of denticles in both male and female speci­ mens is greater than has been found in R. coloradensis, and in this respect IL m icroti seems to be more sim ilar to Rj_ onychoads. The maximum length of the combs in the female ranges from 131 to 1U5 microns, and thus exceeds the range of 65 to 122 microns which has been reported for R^ coloradensis. The male of R^ onychomis is unknown. The female d iffe rs from R. coloradensis by having a slightly smaller number of cuticular pro­ cesses, a la rg e r number of o ral d en ticles, and la rg e r combs (2lj0 microns). Reiber and Bjyrd (19U2) found "R^ onychomls" in Peromyscus leucopus from Tennessee; however, Tiner (19li8a) considers their mate­ rial to be conspecific with R^ coloradensis from Peromyscus spp. in other localities. Chandler and Kelvin (1951) also reported R^ ony­ chomis from leucopus collected in Pennsylvania. They state that the number of te e th bordering the stoma number 20 instead of 26. Since this is in the range of denticles reported for R^ coloradensis, it would appear highly probable that they were dealing with the latter species. McPherson and Tiner (1952) believe that "...several additional species which are closely related to R^ coloradensis occur in North America, and th a t maximum comb lengths, number of combs, and number of denticles are some of the characteristics that can be used to separate them in to h o st-sp ecific groups." Tiner (19U8a) mentions - 63 - RlctnTarla sp. with a maximum comb length of 11*8 to 2 1 1 microns which parasitises various species of tree squirrels, and Rictularia sp. with a maximum comb length of ll*B to 160 microns which is a parasite of wood ra ts . The writer was able to examine a single female specimen of Rictu­ laria which was collected by Kr. Donal Hyer from a wood rat, Neotoaa pennsylvanica, at Carter Caves, Kentucky. This specimen was similar to coloradensis in most respects. There was a total of 61* pairs of combs and spines with 31 pairs located anterior to the vulva. The transition from combs to spines was very abrupt, and two pairs of spines were anterior to the vulva. The combs were prominent (Plate 7t Pig. 29)# with a maximum length of 120 microns* There appeared to be about 18 denticles (determined from lateral view). All these charac­ ters fall within the ranges which have been reported for Rj_ colora- densis. The maximum length of the combs, 120 microns, is greater than the range of 65 to 97 microns found by the writer in his material from Peromyscusi but it is in the range of 96 to 122 microns given by Tiner for R^ coloradensis from Peromyscus spp. On the other hand, the maxi­ mum comb length is less than ll*8 to 160 microns which Tiner reported for his specimens from wood rats. This may be an indication of the variation in maximum comb lengths which can be expected when specimens from various geographical locations are studied. Although the writer does not wish to give a definite opinion after studying only one speci­ men, i t seems probable th a t the R ictu laria p a ra s itisin g wood ra ts is actually coloradensis. Spasski e t a l. (1952) described R. b alcalen sis from Itus muscuius and Apodemus agrarius collected at Lake Baikal in the Soviet Union. In an abstract of this paper, it was stated that R. baicalensis - 6U - resembles R^ coloradensis In that the mouth opens terminally, and they both have the same number of combs. In th is new species, the spicules were equal, and the eggs measured 29 to 33 by 37 to U2 microns. Appar­ ently these authors compared their material with the original descrip­ tion of R^ coloradensis given by Hall (1916) in which the spicules were equal, and the eggs were said to measure 22 by 3d microns. The egg size of R. baicalensis is identical with the measurements obtained in the present study, and it has been shown that the spicules cannot be used as reliable taxonomic characters. Further comparison of R. baicalensis with R^ coloradensis may show that they are identical. The fact that R^ coloradensis can mature experimentally in Mus muscuius strengthens this supposition. There is no doubt that R^ coloradensis, R. baicalensis, R. microti, R. onychomis, and Rictularla spp. from tree squirrels and wood rats comprise a group of closely related species. The known geographical distribution of the North American species in this group is illustrated in Text Figure 7. R^ coloradensis appears to have a fairly continuous distribution across the northern two-thirds of the United States, although it has not yet been reported from the Oreat Plains. The dis­ tributions of Rictularia sp. from tree squirrels and Rictularla sp. from wood rats are continuous with or overlap the distribution of R. coloradensis. R. onychomis is known definitely only from the type lo c a lity in Nebraska, and R^ m icroti is known only from S t. Lawrence Island in the Bering Sea and the Brooks Mountain Range in Alaska. R. baicalensis, which is not shown on the map, is reported from Lake Baikal in the Soviet Union. The land bridge which at one time connec­ ted Asia with North America could have served as a migration route for hosts which harbored a common ancestor of the species in this group, M TEXT FI"URji 7. Distribution of coloradensis and related North

American species of Rictularla. - 66 - and this may explain the presence of a species closely related to R. coloradensis in the Soviet Union. R^ microti and Rj_ onychomis may possibly represent speciation from a "coloradensis type" under iso­ lated contitions. R. baicalensis was reported from murine rodents, microti from microtine rodents, onychomis from cricetine rodents, and Rictularia spp. from sciurid and cricetine rodents. Since coloradensis has been reported from sciurid, microtine, cricetine, and experimentally, from murine rodents, it would appear that host distribution per se is not sufficient grounds for separating species in this group. At the present time, slight variations in the number of cuticular pro­ cesses, oral denticles, and the maximum length of the combs appear to be the only means of separating these species. These characters do not appear to be very reliable because of the overlapping of the ranges of v a ria tio n . In the foregoing discussion, an attempt was made to show that R. coloradensis, R. baicalensis, R. onychomis, R. microti, Rictularia sp. from tree squirrels, and Rictularia sp. from wood rats are so closely related that they way actually represent a single, variable species. From the standpoint of host distribution, R^ coloradensis would appear to be th e b asic sp ecies, and th e o th e r members o f th is group may represent only slight variations from the typical R^ colora­ densis. On the other hand, genetic shifts in isolated populations may have progressed to the point where separate species have evolved. Whether maximum comb lengths and other slight morphological variations are adequate indicators of speciation has not as yet been established conclusively. It is suggested that additional studies of life cycles in this group may eventually solve these problems. Laboratory studies - 67 - of this sort should not be difficult since experimental hosts are readily available in most laboratories, and considerable data could be obtained from the eggs of even one mature female worm. SUMMARY

Rictularia coloradensis was found in the small intestine of 76.5 per cent of the white-footed mice, Peronyscos leucopus noveboracensis, collected at Rhododendron Hollow in Hocking County, Ohio, and in.U 17 per cent of the white-footed mice collected in The Ohio State Uni­ versity Woodlot. Infected mice harbored from one to ten adult worms, but only one male was found in a natural infection. The larvae of R^ coloradensis were found to develop experimentally in a number of insect hosts. The larvae developed normally to the infective third stage in Herman roaches, brown-banded roaches, wood roaches (Parcoblatta spp.), field crickets (Acheta assim ilis), and camel crickets (Ceuthophilus sp.). However, in oriental roaches, American roaches, ground beetles (Dicaelus sculptilis and Chlaenius sp.), and meal worms (Tenebrio molitor), the larvae underwent some development, but on or before the twelfth day of infection, the cysts became pigmented, and the larvae were dead or dying. Eggs hatched in the sow bug, Trachelipus sp., but the larvae failed to develop. Larvae of R^ coloradensis were found under natural conditions in camel crickets, Ceuthophilus g. gracilipes, at Rhododendron {follow, and one larva was found in a wood roach* Parcoblatta virginica, at the University Woodlot. The eggs of R^ coloradensis are fully embryonated when laid. After being ingested by the intermediate host, they hatch in the midgut. First-stage larvae were found in the cells of the epithelium of the hindgut twenty-four hours after exposure to eggs. The larvae develop to the infective stage within a cyst formed by the tissue of the host

- 68 - - 69 - gut. In German roaches kept at room temperature (72° to 80° F.), the first molt occurs during the seventh or eighth day of development and gives rise to the second-stage larva. The second molt occurs during the twelfth or thirteenth day of development and gives rise to the third-stage larva. In German and brown-banded roaches, the cysts con­ taining the third-stage larvae remain attached to the wall of the hind- gut, In wood roaches, field crickets, and camel crickets, the cysts are sloughed from the hindgut into the body cavity of the host. Cysts were found free in the body cavity of field crickets as early as the tenth day of infection. Larvae become infective to the definitive host after ten days of development in the German roach at 72° to 80° F. Development in the definitive host forms an exception to the rule of four molts. The third-stage larva molts, giving rise directly to the adult stage with­ out the interpolation of a fourth-stage larva. This molt occurs during the fifth or sixth day following the infection of laboratory mice. Males become sexually mature about ten days after infection of the mouse. Females mature later, on about the thirtieth day after infection. The females begin to deposit eggs after about fifty-five days of devel­ opment in the laboratory mouse. In Peromyscus, the third molt, which gives rise to the adult, occurs about one day sooner (during the fourth day) than in the labor­ atory mouse. However, worms appeared to be unable to develop to maturity in captive white-footed mice kept under laboratory conditions. Two female R ictu laria were found in one w hite-footed mouse examined fourteen days after infection, but all mice examined after fourteen days were negative. An attempt to infect a meadow mouse (Microtus p. pennsylvanicus) was unsuccessful. Third-stage larvae were recovered - 7 0 - from T'hite rats up to six days after infection, but they failed to show evidence of advanced development. Variation in the taxonomic characters of adult coloradensis is documented and discussed, and th is species is compared with the other North American species in the genus. LITERATURE CITED

Alicata, J. E, 1935. Early developmental stagea of nematodes occurring in swine. U. S. Dept. Agric. Tech. Bull. No. Ii89> 96 pp.

Raylis, H. A. 1928. On a collection of nematodes from Nigerian

mammals (chiefly rodents). Parasit. 20: 280-30U. Buhrer, E. M. 19U9. Technique for the beheading and en face exami­ nation of nematodes and similar animal types. Proc. Helminth. Soc. Wash. 16: 3-6. Caballero y C., E. 19b3* Nematodos de los murcielagos de Mexico. IV. Descripcion de una nueva especie del genero Rictularia y breves consideraciones sobre la sistematica de las especies comprendidas en este genero. An. Inst. Biol. Mex. Ill* U31-L38. Chabaud, A. 0. 195lw Sur le cycle evolutif des spirurides et de nematodes ayant une biologie comparable. Valeur systematique des caracteres biologiques. Ann. Parasit. Hum. et Comp. 29* U2-

88, 206-2li9, 358-1j25.

Chandler, A, C. 19Jj1. Helminths of muskrats in southeast Texas. J. Parasit. 27* 175-191. Chandler, A. C. and Melvin, D. M. 1951* A new cestode, Oochoristica pennsylvanica, and some new or rare helminth host records from Pennsylvania mammals. J. Parasit. 37* 106-109. Chitwood, B. G. and Chitwood, M. B. 1937* "An Introduction to Nematology." Sect. 1, Part 1. Monumental Printing Co., Balti­ more. 53 pp. Chitwood, 8. G. and Chitwood, M. B. 1950. "An Introduction to Nematology.* Sect. I. Revised Ed. Monumental Printing Co., Baltimore. 213 pp. Chitwood, B. 0. and Wehr, E. E. 193U. The value of cephalic struc­ tures as characters in nematode classification, with special reference to the superfamily Spiruroidea. Zeit. f. Parasitenk. 7* 273-335. Chitwood, M. B. 1952. Some observations on Rictularia halli Sand- ground, 1935 (Nematoda). Proc. Helminth. Soc. Wash. 19* 121-123. Cuckler, A. C. 1939* R ictu laria onychomjs n. sp. (Nematoda* Thelasiidae) from the grasshopper mouse, Onychoaprs leucogaster (Weid). J. P arasit. 25* 1*31-1*35. Dollfus, R. P. and Desportes, C. 191*5. Sur le genre Rictularia Proelich, 1802 (Nematodes Spiruroidea). Ann. Parasit. Hum. et

Comp. 20* 6-3U, 208. Proelich, J. A. von. 1802. Beitrige zur Naturgeschichte der Einge- weidewflrmer. Naturforscher 29* 5* Ooodrich, A. L. 1932. Rictularia scalopis sp. nov., a nematode from the mole, Scalopua aquaticus (Linn.). Trans. Amer. Micros. Soc. 5 1* 216- 218. Oraham, E. and Uhrich, J. 19U3* Animal parasites of the fox squirrel, Sciurus niger rufiventer in southeast Kansas. J. Parasit. 29* 159-160. Hall, M. C. 1913. A new nematode, Rictularia splendida, from the coyote, with notes on other cqyote parasites. Proc. U, S. Nat. Mus. 1*6* 73-81*. Hall, M. C. 1916. Nematode parasites of mammals of the orders Rodentia, Lagomorpha, and t(yracoidea. Proc. U. S. Nat. Mus. 50* 168-175. Harkema, R. 1936. The p arasites of some North Carolina rodenta.

Ecol. Monogr. 6t 153- 2 3 2. Ityman, L. H. 1951* "The Invertebrates: Acanthocephala, Aschelndnthes, and Entoprocta. The pseudocoelomate Bilateria." Vol. III. McGraw-Hill Book Co., Inc., New York. 572 pp. Irwin-Soith, V. A. 1922. A new nematode parasite of a Heard. Proc. Linn. Soc. N, S. Wales. 1*7* 311-318. Jaegerskioeld, L. A. 1909. Nematoden aus Aegypten und dem Sudan (eingesammelt von der Schwedischer Zoologischen Expedition). Results of the Swedish Zoological Expedition to Egypt and the White Nile, 1901, under the direction of L. A. Jaegerskioeld. Part 3» No. 25, pp. 1—1*5- K. W. Appelberg, Uppsala. Kate, J. S. 1936. A survey of the parasites found in and on the fox squirrel (Sclurus niger ruflventer Qeoffroy), and in the southern gray squirrel (Sclurus c^ carolinensls Qmelin) in Ohio. Unpublished Master's Thesis, The Ohio State University. McLeod, J. A. 1933. A parasitological survey of the genus Citellus in Manitoba. Canad. J. Res. 9* 108-127* McPherson, S. E. and Tiner, J. D. 1952. A new nematode (Rictularia microti) from a vole on St. Lawrence Island, Alaska. Nat. Hist. Misc., Chicago Acad. S c i., No* 108, 7 pp. Melvin, D. M. and Chandler, A. C. 1950. New helminth records from the cotton r a t, Sigmodon hispidus, including a new species, Strongyloides sigaodontis. J. Parasit. 36: 505-510. MBnnig, H. 0. 1920* Filaria nycticebl, eine neue Filarla aus dem Nyctlcebus. Centralbl. Bakt. Jena Abt. I Orig. 85* 216-221. Moorthy, V. N. 1938. Observations on the life history of Camallanus sw eeti. J. P a ra sit. 2U: 323-3U2. - 71* - Penn, 0. H. 191*2. Parasitological surrey of Louisiana muskrats. J. P a ra sit. 28* 31*8-31*9. Rankin, J. S. 191*5. Ecology of the helminth parasites of snail mammals collected from Northrup Canyon, Upper Qrand Coulee, Washington. The Ifurrelet. 26* 11-11*. Rausch, R. 1952. Studies on the helminth fauna of Alaska. XI. Helminth parasites of microtine rodents - taxonomic consider­

ations. J. Parasit. 3 8* 1*15-1*1*1*. Rausch, R. and Tiner, J. D. 191*8. Studies on the parasitic helminths of the North Central States. I. Helminths of Sciuridae. Amer. Midi. Nat. 39* 728-71*7. Read, C. P. and Uillemann, R. E. 1953* Helminth parasites in Kan­ garoo rats. Univ. Calif. Publ. Zool. 59* 61-80. Reiber, R. J. and Byrrd, E. E. 191*2. Some nematodes from mannals of Reelfoot Lake in Tennessee. J. Tenn. Acad. Sci. 17* 78-89. Sandground, J. H. 1935* Spirura n&chiganensis n. sp. and Rictularia halli n. sp., two new parasitic nematodes from Butamias striatus lysteri (Richardson). Trans. Amer. Micros. Soc. 51** 155*166. Schell, S. C. 1952a. Studies on the life cycle of Phyealoptera hispida Schell (Nematoda* Spiruroidea), a p arasite of the cotton

r a t (Sigmodon hispidus l i t t o r a l i s Chapman). J. P a ra sit.3 8 *

1*62 - 1*72 . Schell, S. C. 1952b. Tissue reaction of Blatella germanica L. to the developing larva of Phyealoptera hispida Schell, 1950 (Nema­ toda* Spiruroidea). Trans. Amer. Micros. Soc. 71* 293*302. Seurat, L. 0. 1913. Sur 1*evolution du Physocephalus sexalatus (Molin). Compt. rend. Soc. Biol. 79* 517-520. - 75 - Spasskiy A. A.,RichikoVy K. M. and Sudarikov, V. E. 195?* [The helminth fatina of wild mammals in the region of Lake Baikal^ Trudi Gelmintologicheskai Laboratorii. Akademii Nauk S. S. S. R. 6* 85-113 (In Russian) T inerf J. D. 19U8a. Observations on the R ictu laria (Nematodat Thelaiiidae) of North America. Trans. Amer. Micros. Soc. 67* 192- 200 . Tinery J. D. 19U8b. Rictularia dipodomis n. sp. (Nematodat Thela- slidae) from the kangaroo rat Dipodomys sp. J. Parasit. 3l*t 332-335. Wilsony L. W. 19U5* Parasites collected from the wood mouse in West V irginia. J. Mammal. 26: 200. Witenbergy 0. 1928. Reptilian als Zwischenwirte parasitischer Wttrmer ▼on Katie und Hund. Tier&rstliche Rundschau.3ht Nr. 32y p. 603. Why H. W. and Huy Y. T. 1939. Some parasitic nematodes from Hainan. Proc. Sixth Pacific Sd. Congr. I*t 253. - 7 6 -

EXPLANATION OF PLATE 1

(Figures 2 to 6 are lateral views) Figure 1 Embryonated egg. Figure 2 First-stage larva, 1 day. Figure 3 First-stage larva, 3 days. Figure h First-stage larva at the beginning of the first molt, 5 days. Figure £ Second-stage larva, 7 days. Figure 6 Anterior end of second-stage larva showing the beginning of the second molt, 9 days. PLATE I - 78 -

EXPLANATION OF PLATE 2

(Figures 7* 8* 10 to 13 are lateral views) Figure 7. Third-stage larva, 35 days in rood roach, Parcoblatta v irg in lc a . Figure 8. Anterior end of third-stage larva. Figure y. Compound cyst from the body cavity of a wood roach, Parcoblatta virginlca. Two third-stage larvae have escaped from the cavity to the right. F igure 10. Anterior end of third-stage larva,k days in laboratory mouse. Figure 11. Genital primordium of female third-stage larva, L days in laboratory mouse. The vulva is beginning to form. Figure 12. Tail of female third-stage larva, it days in laboratory mouse. Figure 13. Posterior half of male third-stage larva, h days in laboratory mouse. The genital primordium is greatly elongated, and the spicules are forming. - 79 - PLATE 2 - 80 -

EXPLANATION OF FLATE 3

(Figures lli, 1$, 17 to 21 are lateral views) I'igure 1U Figure 15 Figure 16 Figure 17 Figure 18, Figure 19 Figure 20 Peromyscua. Figure 21. Tail of male, naturally infected Peromyscus. - 81 -

PLATE 3 - 82 -

EXPLANATION OF PLATE U

Figure 22 . Larva in epithelium of hindgut of German roach,2h hours. Figure 23 . Section through cyst in epithelium of hindgut of German roach, 12 days. This cyst illustrates the initial stages of fibrosis. Value of Scales Fig. 22. 100 microns. Fig. 23. 100 microns. - 83 -

PLATE 4

23 - 81* -

EXPLANATION OF PLATE 5

Figure 2h. Section through a cyst in epithelium of German roach, 12 days. There are no indications of fibrosis in th is cy st. Figure 25 Section through a cyst in epithelium of hindgut of field cricket, Acheta asslmllis, 12 days. Value of Scales Fig. 2U. 100 microns. Fig. 25. 100 microns. - 35 -

PLATE 5

24

25 - 86 -

EXPLANATION OF PLATE 6

Figure 26. Adult female from small intestine of laboratory mouse, 8 days. Figure 27. lateral view of combs of adult female from laboratory mouse, 35 days.

Abbreviations

E - p o ste rio r end of esophagus. V - vulva. Value of Scales Fig. 26. 500 microns. Fig. 27. 100 microns. - 87 -

PLATE 6

27 - 88 -

EXPLANATION OF PLATE 7

Figure 28 * Lateral view of spine of adult female from labor­ atory mousef 35 days. Figure 29 . Ventro-lateral view of combs of adult female Rictularia sp. from a wood rat, Neotoma magister.

Value of Scales Fig. 28. 100 microns. Fig. 29. 500 microns. - 89 -

PLATE 7

29 AUTOBIOGRAPHY

I, Vernon Harvey Oswald, was born at Hartville, Ohio, on Hay 11, 1925* I received ray secondary school education at Hartville High School. Both qy undergraduate and graduate training were received a t The Ohio S tate U niversity. I received the degree Bachelor of Science in 19b9 and the degree Master of Arts in 1951* I held the position of Graduate Assistant in the Department of Zoology and Entomology from 19U9 to 1951* I have been employed at Battelle Memorial Institute, Columbus, Ohio, as Principal Biologist since August, 1951* I obtained a leave of absence from Battelle during the school year 195^-55, during which time I served as a Graduate Assistant in the Department of Zoology and Entomology while completing ray residency for the degree Doctor of Philosophy at The Ohio State University.