LIFE CYCLE STUDIES OF PHYSALOPTERA HISPIDA N. SP. (NEMATODA: SPIRURATA) FROM THE COTTON RAT, SIGMODON HISPIDUS LITTORALIS, CHAPMAN

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U N I V E R S I T Y O F ILLINOIS THE GRADUATE COLLEGE

OCTOBER 1 0 ,

I?fo

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY STEWART CLAUDE SCHELL

SUPERVISION BY.

L I F E CYCLE STUDIES OF PHYSALQPTERA HISPIDA N . S P . (NEMATODA:SPIRURATA) FROM THE COTTON RAT, SIGMODON HISPIDUS LITTORALIS CHAPMAN

F.MTTTT.PT>

BE ACCEPTED* AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE DEGREE OF_

DOCTOR OF PHILOSOPHY IN ZOOLOGY

/^^^^^^t^a^XZ^r-^^

J

Head of Department

Recommendation concurred inf

C7?,C7^W$

Committee on

QycSUva&^c * Subject to successful final examination in the case of the doctorate, f Required for doctor's degree but not for master's.

6M—12-48—40199K

Final Examination!

LIFE CYCLE STUDIES OF PHYSALOPTERA HISPIDA n. sp. (NEMATODA: SPIRURATA) FROM THE COTTON RAT, SIGMODON HISPIDUS LITTORALIS CHAPMAN

BY

STEWART CLAUDE SCHELL B.S., Kansas State College, 1939 M.S., North Carolina State College, 1941

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE GRADUATE COLLEGE OF THE UNIVERSITY

OF ILLINOIS, 1049

URBANA. ILLINOIS

COPYRIGHTED by STEWART CLAUDE SCHELL 1950

ACKNOWLEDGMENTS The work was done in the Zoological Laboratories of the University of Illinois under the direction of Doctor Lyell J. Thomas.

The writer expresses his sincere thanks to Doctor Thomas

for his assistance and continued Interest in the work.

My grati-

tude is extended to my wife, Dorotnydean Vlets Schell, who helped in many ways including the typing of the manuscript.

The author

is also Indebted to Doctor Donald Hoffmeister, Department of Zoology, University of Illinois, who supplied technical information concerning the mammalian hosts, and also to Doctor Milton Sanderson, Illinois Natural History Survey, for the identification of some Arthropod hosts.

The writer is very grateful for the

assistance given by Doctor C. 0. Morrill of the Division of Veterinary pathology for technical assistance concerning the involved pathology.

TABLE OF CONTENTS Page I.

INTRODUCTION

1

II.

REVIEW OF LITERATURE

1

III.

MATERIAL AND METHODS

3

IV. DESCRIPTION OF THE ADULT V. VI.

SPECIAL FEATURES OF THE INTERNAL ANATOMY OF THE ADULT

5 9

EGG FEEDING EXPERIMENTS

11

DEVELOPMENT IN THE INTERMEDIATE HOST..

13

VIII.

DEVELOPMENT IN THE DEFINITIVE HOST

18$

IX.

INFECTION WITH SECOND STAGE LARVAE

20

INFECTION OF HOSTS OTHER THAN THE COTTON RAT

21

SUPERINFECTION

22

PATHOLOGY IN THE INTERMEDIATE HOST

23

PATHOLOGY IN THE DEFINITIVE HOST

31

SUMMARY

33

REFERENCES

35

VITA

3g

VII.

X. XI. XII. XIII. XIV. XV. XVI.

1 I.

INTRODUCTION

In the fall of 19*1-7 two cotton rats (Slgmodon hispidus llttoralls Chapman) were sent to Doctor L. J. Thomas by the Hegner Zoological Research Supply Company of Sarasota, Florida.

One of

the animals had died enroute and was promptly examined upon arrival in the laboratory.

The stomach contained a number of large nema-

todes which were later identified as a species of Physaloptera. Later the other cotton rat was autopsied and found to contain the same parasite.

Since no complete life cycle had ever been estab-

lished for nematodes of the family Physalopteridae it was thought advisable to carry out studies in this direction.

The following

account represents the combined results of this work.

Although a

considerable amount of information has been accumulated, some new problems have arisen in the course of the work and will require further investigation. II.

REVIEW OF LITERATURE

Although no complete life cycle is known to date for any member of the nematode genus Physaloptera, some fragments of information have been contributed by several workers. Cram (1931) rather cautiously identified encysted larvae found in the breast and leg muscles of bob-white quail and ruffed grouse in Minnesota as belonging to a species of Physaloptera.

Cram

stated, "the most probable explanation in cases such as these is that the young parasites found their way into the wrong host, due to the fact that that host had ingested arthropods serving as intermediate host of the parasite; however, there is a possibility that partial development in some such host as the bird is a necessary step in the life cycle." There was a clear indication here that a second intermediate host might be required to complete the cycle.

2 Up to this time no one had experimentally infected any intermediate host with any species of physaloptera.

There was therefore the

possibility of a misidentiflcation of the larvae. Boughton (1937) apparently reports upon the same parasites that had been studied previously by Cram. infection of 3.25$ in ruffed grouse.

Boughton found a cyst

The larvae were 2.-3. mm.

long and were encysted near the surface of the breast and leg muscles.

The cysts were yellowish brown and measured about 2 mm.

in length. Alicata (1937) was able to infect Blatella germanica by feeding embryonated eggs of Physaloptera turglda Rudolph! 1319 from the opossum (Didelphi8 vlrglnlana).

He gave a brief description of the

egg and larval stages and attempted to experimentally infect a dog, cat, rabbit, guinea pig, rat and a chick with encysted third stage larvae.

An autopsy was performed on these animals one month later

but no developing or mature P. turgida were found.

Third stage

Physaloptera larvae were recovered from washings of the stomach of the cat and rabbit.

Encysted third stage larvae were found in the

stomach wall of the rat.

There was no trace of Physaloptera in

the dog, guinea pig or the chick. Hobmaler (194-1) Infected Blatella germanica by feeding eggs of Physaloptera maxillarls which has been reported as a parasite of various skunks, badger, mink and raccoon.

He gave a short account

of the first and third stage larvae; but apparently did not encounter the second stage larva.

Hobmaier fed infected cockroaches

to cats, dogs, and guinea pigs.

These animals were examined after

a six weeks interval but no parasites were found.

3 III. MATERIAL AND METHODS Live trapped cotton rats (Sigmodon hlspidus llttoralls Chapman) were obtained from the Hegner Zoological Research Supply Company, Sarasota, Florida. Upon arrival in the laboratory mature male and female rats were paired off in cages. When females were observed to be in an advanced stage of pregnancy cotton batting was placed in the cages for nesting material. The young rats were weaned at lg-20 days, and placed In clean cages in a separate room to avoid accidental worm infections. The gestation period for 9 litters varied from 21-27 days, with from 3-7 rats per litter. The newborn rats are quite active and covered with hair. The eyes open 20-24- hours after parturition. Males and females were usually compatible and the males were left in the cages with the females and young.

In only one instance was the litter destroyed

by the female. The rats were fed a dally diet of laboratory rat chow which was supplemented with semi-weekly feedings of a vegetable such as lettuce, swiss chard, carrots or tomatoes. Yellow whole kernel corn and a few grams of horse meat was fed once a week. Cotton rats are wild excitable animals and show little tendency toward taming. They are not vicious but will bite when handled. Arthropod hosts used for infection experiments were either laboratory reared or collected in protected habitats where natural infections were not likely to occur. Blatella germanica and Periplaneta americana were reared in quart Jars containing a one inch layer of wood shavings and were fed rat biscuit and lettuce, parcoblatta pennsylvanica were reared in Jars of autoclaved leaf mold which was kept sufficiently moist

4to support the growth of some fungi. Woodroach eggs required considerable moisture for hatching.

The young woodroaches promptly

devoured the adults upon hatching. Pillbugs (Armadlllum vulgare) were collected from under the benches In the Natural History Survey Greenhouses. The earth-boring dung-beetles (Geotrupes sp.) were collected from beneath piles of fresh horse manure and kept outdoors in deep boxes containing equal quantities of horse manure and soil. Eggs, larvae and adults of three species of Aphodlus were collected from swine droppings which were placed in gallon Jars and kept outdoors until needed. A number of Tenebrlo larvae were obtained from the Entomology Department.

These had originally been field-collected specimens.

Larvae and adults of Tribollum sp. were reared in boxes of oatmeal. Attempts to rear crickets and grasshoppers from eggs were generally unsuccessful. For studies of the pathology in the intermediate and definitive host, tissues were fixed overnight in Bouin's fixative, embedded in paraffin, sectioned and stained in Harris' Haematoxylln with a counterstain of Eosin or Orange "G". The nematode larval stages were successfully fixed in hot Travassos1 fixative, cleared in glycerine and then mounted in glycerine jelly. In order to follow the development and migration of the parasite in the intermediate host (Blatella germanica) the head of the cockroach was first pinched off with sharp-pointed forceps. The entire alimentary tract was then pulled out through the posterior end and immediately placed on a clean glass slide in a fresh drop

5 of physiological saline.

The crop, ventriculus, and proctodaeum

were then carefully separated and each part placed on a slide in a separate drop of saline.

Each part of the alimentary tract as

well as the body cavity was carefully teased apart with fine dissecting needles and examined under a cover slip with a compound microscope. The Sudan IV technique for the demonstration of fat was carried out as follows:

Solutions for staining were mixed according

to the method of Kay and Whitehead (1935).

Pieces of cockroach

colons were flattened on a microscope slide under a coverslip and fixed with 10# formalin.

These tissues were stained in the Sudan

IV solution for 30 minutes and then rinsed in distilled water, cleared in glycerine and finally mounted in glycerine Jelly. The test for chitin was the modified van Wlsselingh test as outlined by Campbell (1929).

It consists essentially of treating

the tissue with concentrated potassium hydroxide (slightly under saturation to avoid crystallization) for £-1 hour at 130°-l4o° C. Any material that does not dissolve is then hydrated gradually through 95$; 70$; 35$ ethyl alcohol; rinsed in distilled water and then treated with 1 drop of a 0.2$ iodine solution in potassium iodide.

To this, is added 1 drop of l.# sulphuric acid.

alkali treatment changes chitin to chitosan.

The hot

The acid-iodine causes

the production of a rose violet color if chitosan Is present.

Com-

plete dissolution in hot alkali is a negative test for chitin. IV.

DESCRIPTION OF THE ADULT

Nematodes of the genus Physaloptera have two large triangular lateral lips, each with two external papillae, one externolaterai

6 tooth, and three internal teeth; no denticles on the margins of the lip; cuticle may or may not be reflected over the lips to form a collarette; cervical papillae behind the nerve ring; esophagus consists of an anterior muscular and a posterior glandular portion. Female with the vulva anterior to the middle of the body.

Male

with caudal alae meeting ventrally in front of the anus; with at least four pairs of stalked papillae.

Uterus di-, tri-, tetra-, or

polydelphys. Physaloptera hlspida n. sp. Robust worms with fine cutlcular striatlons; females pink with tiny scattered flecks of brown pigment; male white and smaller than the female.

Two triangular lateral lips at the anterior end, each

provided with one lateral amphid, one large externolateral tooth, three internolateral teeth, one subdorsal papilla and one subventral papilla; the papillae are 12.2 yu in diameter; collarette absent. Male: O.56-O.73

mm

Length 30-4-2 mm.; width 0»9-l»4- mm.

Muscular esophagus

» long; glandular esophagus 3•7-4-.5 mm. long; entire

esophagus ranging from 4-.2-5.3 Tam" l*1 length.

Cervical papillae

opposite to slightly asymmetrical and 7i3.-i06l.yU from the anterior end; excretory pore g78>.-llS>9. p. from anterior end; lip-nerve ring distance 54-9.-695. yu.

Caudal end of male flexed, (Fig. 30) blunt

pointed; with caudal alae; ventrally there are four pairs of evenly spaced pedunculate papillae, the two middle pairs the longer; three seseile preanal papillae and five pairs of sessile post anal papillae; the first two pairs are on the posterior margin of the anus; the third pair moderately to widely asymmetrical with the left papilla the more anterior; the fourth pair also asymmetrical with

7 the left anterior to the right; the last pair opposite and close together.

The spicules are unequal in size, the right one (Fig. 24-)

the longer of the two, lanceolate and uniformly tapering toward the tip and measuring 390.-54-9. ja; the left spicule (Fig. 22) bladeshaped and measuring 34-1.-4-77• /*• Female:

Length 53.-64-. mm. long, 1.9-2.0 mm. in width; uterus

dipelphys (Morgan's 2-B type); anterior muscular esophagus 0.64-0.69 mm. long; glandular esophagus 4-.4—6.3 mm. in length; the entire esophagus measures 5.0-7*0 mm.

Cervical papillae opposite to

slightly asymmetrical, and 677.-917. /1 from the anterior end; excretory pore 34-1.-IO79. ju from the anterior end; lip-nerve ring distance 710.-74-9. p.; vulva 11.-16. mm. from the anterior end; posterior end variable in shape depending upon condition of cuticular prepuce.

When prepuce Is fully extended the phasmids lie half-

way between anus and tip of tall. Eggs measure 24-.-30. ja by 4-0.-52. ja. Host:

Slgmodon hlspldus llttoralls Chapman.

Location:

Pyloric region of stomach.

Locality:

Sarasota, Florida

Types:

U. S. Nat. Mus.

Paratypes:

Helminth.

U. S. Nat. Mus.

Collection #

Helminth.

.

Collection #

.

Described from a large series of specimens of both sexes. Remarks:

Other species of Physaloptera reported as parasites

of the cotton rat are p. murls-braslliensls Die sing, 1S61 and P. bisplculata Vaz and Perelra, 1935«

Morgan (194-3) placed p.

blsploulata as a synonym of P. getula Seurat, 1917.

There is some

doubt in the writer's mind as to the advisability of this.

p.

g bisplculata was originally described from speolraens found in the stomach of Neotomys squamlpes, a wild rat In Brazil. tion is reasonably complete.

The descrip-

P. getula was described as a parasite

of Mus rattus, a common rat of Morocco, Africa.

Seurat»s 1917

description is rather vague and without illustrations.

In 1937

Seurat again described p. getula, this time from the stomach of Merlones shawl Rozet from Africa (Zemmora, Oranie).

This descrip-

tion still did not reveal the exact arrangement of the male ventral papillae.

In view of these circumstances It is questionable as to

whether there is a case in favor of synonymy.

Morgan did not have

access to types of P. getula; however he did see paratypes of P. bisplculata.

The writer seriously doubts whether P. getula should

even be regarded as a parasite of the cotton rat. Differences between the four species are given below in Table I. TABLE I Length Length P o s i t i o n Right Left •£ mm. o^mm. of Vulva S p i c u l e S p i c u l e *P. murisbraslllensls

34-4-3

22-2S

Ant.

400/a

400 >u

Ortlepp 1922

P. murls"brasTTTensi s

43-53

23-33

6.-6J mm. 523 ja

54-0 ja

Morgan 194-2

*Z' b i s p l c u l a t a

27-55

25

Ant.

400 ja

4-60/1

Vaz & P e r e i r a 1935

P. bisplculata

3&-55

22-30

10.-16.

34-7-4-75 4O0-4S6 Morgan 194-2

13.5

16.3

31

20.6 30-4-2

*£• gQtiila P. getula

£• M s P l d a 53-64(» from Morgan 194-3)

350^

4-SOyW

Seurat 1917

6.

4-50yu

4-501/"

Seurat 1937

11.-16.

390-54-9 3^1-^77 S c h e l l 1949

9 All species have the 2-B type of uterus except p. murlsbrasiliensls which has a 2-A type.

In addition,the third and fourth

pairs of sessile postanal papillae of the male are opposite in P. bisplculata and consistently asymmetrical in p. hispida. V.

SPECIAL FEATURES OF THE INTERNAL ANATOMY OF THE ADULT

It occurred to the writer that certain features of the internal anatomy of Physaloptera hispida are of special interest.

The eso-

phagus, as in other Splruroldea is composed of an anterior muscular portion and a posterior glandular esophagus.

Figure 4-2 is a cross

section through the muscular portion at about the level of the nerve ring.

The muscle fibers have a distinct radial arrangement with

nuclei scattered through the full length of the muscular portion. The amphids are lateral in position between the esophagus and the lateral chords. the esophagus.

Some nerve cells can be seen distributed around These are dark-straining cells in sections treated

with Harris' Haemotaxylin. Sections through the glandular esophagus at the level of the excretory pore show the terminal excretory duct (Fig. 3 2 ) . At this same level the duct from the dorsal esophageal gland enters the lumen of the esophagus.

The ducts of the two subventral glands

enter the esophageal lumen 20-90 ja posterior to the dorsal duct (Fig. 23). The glandular esophagus is composed of a rather loose tissue and scattered muscle fibers.

The nuclei are small and are

scattered evenly along the full length of the glandular esophagus in all sectors.

The lumen is triradlate with a cuticular lining.

Hsu (1933) described the glandular esophagus of Physaloptera clausa. In this species the gland ducts are separated by a distance of 4-0 AX.

10 Hsu also noted that the nuclei were distributed along the full length of the glandular esophagus, but seemed to be concentrated more toward the posterior end. The nuclei of the lateral chords are small and numerous and distributed evenly throughout the chords.

The longitudinal muscles

of the body wall are polymyarlan and coelomyarian with prominent flat fibers extending across the base and some distance up the sides of each muscle cell. The excretory system is of the Inverted U-type (Fig. 20) with the longitudinal canals directed posteriorly along the median surface of each lateral chord.

These main canals merge across the

ventral surface of the glandular esophagus to form a transverse canal.

At this Junction there is a slightly enlarged portion which

in turn communicates with a short terminal excretory duct which empties through the ventral excretory pore. The ovljector (Fig. 22), is composed of an anterior muscular vagina through which eggs pass in single file (Fig. 4-3), and a posterior egg chamber provided with a thin layer of circular muscle fibers (Fig. 4-1). Figure 4-4- shows the vagina in cross section with its heavy outer layer of circular muscle fibers and thinner layer of radially arranged longitudinal muscle fibers. branches are directed posteriorly.

The two uterine

Only embryonated eggs are

ejected. A median sagittal section through the posterior end of a male specimen is shown (Fig. 3 1 ) demonstrating the arrangement of the terminal ducts as they enter the cloaca. ventral to the intestine.

The testis Is single and

The writer has frequently observed males

In which the cloaca, ejaculatory duct

and the terminal portion of

11 the intestine were packed with embryonated ova. ently continues ejecting eggs during copulation.

The female apparFully embryonated

eggs can be found approximately one third of the way up the uterus. The seminal receptacle is a prominent oblong structure located at the Junction of ovary and uterus. VI.

EGG FEEDING EXPERIMENTS

Although the writer learned very early in the course of the investigation that Blatella germanica would serve satisfactorily as an intermediate host for Physaloptera hispida, an attempt was made to infect other arthropods in the hope of finding one that would be more likely to occur in the natural habitat of Slgmodon hlspldus. Cotton rats will eat insects readily.

In a few instances infected

cockroaches were folded into pieces of horse meat which were then dropped into the cages.

Several times the rats were known to pick

out the cockroach and eat it first and then eat the meat.

They

will also eat isopods and beetles. The results of the egg feeding experiments are recorded in the following table: TABLE II Arthropod Armadlllum vulgare (Plllbug)

No• Examined

Result

31

Neg.

Many

Pos.

Perlplaneta amerlcana

35

Neg.

Parcoblatta pennsylvanica

30

Neg.

Aphodius flmetarlus

12

Neg.

Aphodius dlstlnctus

23

Neg.

Blatella germanica

(continued)

12 TABLE II Arthropod

(continued) No. Examined

Result

Aphodius femoral is

21

Neg.

Geotrupes sp.

24-

Neg.

Tenebrlo sp. (larvae)

12

Neg.

» Tripoli urn sp. (larvae)

25

Neg.

»»Trlbolium sp. (larvae)

14-

Neg.

4-0-50

Neg.

Trlbollum sp. (adult) *

Examined as larvae.

**

Examined as adults.

Embryonated eggs were added to pulverized rat biscuit in shell vials (ri11 x 3") a nd fed to the cockroaches, woodroaches, and mealbeetles.

The Isopods and dung-beetles were given a mixture of

embryonated eggs and pulverized rat feces in 2" stender dishes. An examination of specimens of Perlplaneta amerlcana and parcoblatta pennsylvanica 24-4-2 hours after feeding eggs revealed that many of the eggs had been ingested and some had hatched but all of the larvae were dead. Both larvae and eggs were passed in the feces. Some of these roaches had been given repeated feedings of eggs in order to Insure an infection, but to no avail. The earth-boring dung-beetles (Geotrupes sp.) and the isopods consumed huge quantities of eggs, but none were ever known to hatch in the Insect. All specimens examined 25 days later were negative for Physaloptera. A very high incidence of Infection can be established in Blatella germanica. Usually they were given access to eggs for a

13 period of 4-2 hours after which the insects were removed to a clean cage and given fresh food and water. In such feeding experiments the nymphal stages become Infected more readily than the adult cockroaches.

This may simply be due to the fact that Immature roaches

consume more food than the adults because in some instances when the roaches had access to the eggs for more than 2 hours the adults obtained a fairly high Infection.

Specimens of B. germanica may

carry as many as 3°~35 encysted larvae. In such cases the entire wall of the colon is a mass of cysts. VII. DEVELOPMENT IN THE INTERMEDIATE HOST Embryonated eggs, when Ingested by Blatella germanica. pass unaltered through the crop, proventriculus and ventrlcuius to the colon and rectum where they hatch. Numerous empty egg shells and newly-hatched larvae can be found in the colon and rectum but never in any other portion of the alimentary tract. The newly-hatched larva shows very little Internal structure and at the time of hatching measures 164-123/1 in length by 9.7-10.9/1 in width. There is a prominent tooth at the anterior end. The larva penetrates the peritrophlc membrane of the host and invades the colon epithelium and causes considerable local destruction of the epithelial cells. By the fourth day the larva develops a prominent renette cell which opens ventrally through a pore in the anterior third of the body. At this time the esophagus and intestine are visible; however, the posterior part of the larva is still an undifferentiated mass of cells. By the seventh day (Fig. 17) the genital primordium is evident as a single cell about midway of the Intestine. From the tenth to the eleventh day the coiled larva can be observed in

Ik the colon wall in specimens pressed flat under a cover slip.

The

area of the colon containing a larva appears to be more translucent than non-parasitized areas, due to the partial destruction of the epithelium by the parasite.

By the thirteenth day (Fig. 9) the

larva has Increased to a length of 311. p. and a width of 25. >u. The excretory pore is 79 /* from the anterior end and the esophagus is ll6 /1 long. nucleus.

The rectal glands are prominent and have a large

The cuticle now loosens in preparation for the first molt

which occurs during the l4-17th day.

Just previous to the molt the

features of the second stage larva can be seen through the loosened cuticle.

The tooth is lost with the old cuticle.

The second stage larva (Fig. 1) grows rapidly.

The esophagus

becomes more cylindrical; the posterior end is no longer bulbous. The renette cell becomes more compact and develops a fine long terminal duct.

Between the esophagus and intestine four distinct

cells are visible.

These are exclusively structures of the second

stage larva and might possibly contribute to the formation of a valve between esophagus and intestine.

The second stage larva has

two distinct lips bearing no armature of any kind, but each lip bears laterally a Y-shaped suture which is definitely a part of cuticle.

Just behind the lips are several large gland-like cells,

the nature of which is unknown. made up of rather large cells.

The intestine is a straight tube The rectal glands tend to become

more compact as the larva develops. At 16-12 days the genital prlmordlum is composed of 2-12 cells, but sexual differentiation is still not possible.

At 20 days the cysts can be dissected from the

colon wall as separate spherical membranous structures although at

15 this early stage the cyst membrane is delicate and may rupture easily. The second molt occurs between 24-27 days at a temperature of 22° C.

The loosened cuticle (Figs. 6 and 11) is especially evident

at the anterior and posterior ends of the larva and around the excretory pore.

The features of the third stage larva are clearly

outlined beneath the old cuticle. process of undergoing a molt.

One larva was observed in the

The larva was flexed ventrally, the

cuticle was ruptured all around the larva about midway between the anterior and posterior ends.

With repeated flexing movements the

cuticle was gradually worked off of the anterior and posterior ends* The Y-shaped sutures on the molted cuticle remained intact.

The

lining of the cloaca was a part of the molted cuticle. The third stage larva (Figs. 2 and 5) has two triangular lips, each with a prominent lateral tooth, and one subdorsal and one subventral oral papilla. strated.

The presence of amphids could not be demon-

The esophagus is now composed of an anterior muscular

portion, surrounded by the nerve ring, and a long posterior glandular region.

The renette cell is more compact, its terminal duct

looping back so that the excretory pore is on approximately the same level with the posterior tip of the renette cell.

The intestine

grows more rapidly than the rest of the larva and is thrown into folds (Fig. 16). These folds are no longer present after 10-12 days in the definitive host.

The extra length appears to be utilized

in the rapid Initial growth in the final host. tion is now possible.

Sexual differentia-

The female genital prlmordlum (Fig. 13) is

a simple sac-shaped structure attached by. one end to the ventral

16 body vra.ll.

I t tends to be located farther forward than the male

prlmordlum (Fig. 12) which i s composed of two knob-like portions without apparent union to the body wall.

Later there i s evidence

of attachment. Alicata (1937) noticed a difference in size between the male and female t h i r d stage larvae of Physaloptera turglda.

Measure-

ments of male and female larvae of P. hispida do not show consistent differences. The r e c t a l glands are more compact with prominent n u c l e i . Several r e c t a l and cloacal muscles are present.

The t h i r d stage

larva i s infective for the cotton r a t at any time following the second molt; but i t does not complete i t s growth u n t i l the end of 30-35 days.

The f u l l y developed third stage larva i s 1.04-1.2 mm.

long by 79.-101. ja wide.

The muscular esophagus i s 103.-122/1 and

the glandular esophagus i s 402.-4-29. p. i n length; the excretory pore i s 14-3.-152 p. and the nerve r i n g I s 97. p. from the anterior end. The male g e n i t a l prlmordlum i s located 4-24-.-567. p. from the posteri o r end; the female prlmordlum ranges from 504.-621. p. from the p o s t e r i o r end of the l a r v a . The cyst increases in diameter from 195. p at 14 days to 4-20. yu in the case of the fully developed t h i r d stage l a r v a . infections one cyst may contain several larvae. bulge s l i g h t l y from the colon wall.

In heavy

The younger cysts

As development progresses the

cysts protrude more and more, some of them eventually become pedunculate, being attached only by a thin, s t a l k .

Upon exactly four

occasions the writer found detached cysts entangled in the malpighlai tubules of the cockroach.

In a l l such cases the o r i g i n a l point of

17 attachment was evident.

In addition, detached cysts always contain

cellular elements which serve as a landmark.

(Cf. Hypertrophled

nuclei under pathology In the Intermediate Host.) Alicata (1937) working with Physaloptera turgida, examined several cockroaches 14- days after feeding eggs and found numerous first and second stage larvae. "free in the body cavity."

He stated that these larvae were,

Twenty-six days later he dissected two

more cockroaches and found third stage larvae.

These larvae were

encysted in tissues surrounding the body cavity. The writer has had the opportunity to make an Intensive comparative study of the development of both Physaloptera turgida and £• hispida in Blatella germanica.

The results are contradictory to

those of Alicata. After making a critical study of numerous dissections and sectioned material the writer has arrived at the following conclusions: 1.

The larva never completely penetrates the alimentary tract of the cockroach and therefore never gains access to the haemocoel.

2.

The entire larval development takes place within the wall of either the colon or the rectum.

3«

A study of sections revealed that encystment is a gradual complex process, beginning at the time the larva first enters the epithelial cells and that cyst formation involves pronounced cytological and histological changes, all of which contribute to a definite pathology.

4-. Encystment has never been observed in any part of the

12 a l i m e n t a r y t r a c t o t h e r than t h e colon and rectum; n e i t h e r do c y s t s occur on any p o r t i o n of t h e body w a l l . 5«

The manner of development of P . h i s p i d a and p_. t u r g i d a l n

B l a t e l l a germanica was found to be i d e n t i c a l . VIII.

DEVELOPMENT IN THE DEFINITIVE HOST

E n t i r e i n f e c t e d cockroach colons were folded i n t o p i e c e s of horse meat and fed t o young l a b o r a t o r y - r e a r e d c o t t o n r a t s .

The

f l o o r of each cage was l a t e r examined for t r a c e s of meat i n o r d e r to determine whether t h e c y s t s had been e a t e n .

The f e c e s of t h e

r a t s were examined a t i n t e r v a l s for the presence of P h y s a l o p t e r a eggs.

The time for development i n the d e f i n i t i v e h o s t v a r i e s from

73-102 d a y s .

The r e s u l t s of t h e s e experiments a r e given i n Table

III. TABLE I I I Date Infected

No. l a r v a e Fed

*52

11/23 A 2

k

1

2

2/4-/49

73

4-6

9/26/4-2

15

3

3

1/3/4-9

99

4-7

9/27/4-2

15

3

3

1/7A9

102

21

3

0

2/2/4-9

7^

4-/4-/4-9

22

51

11/12-20/4-2

Parasites Recovered

Feces Positive

No. days for Development

Rat No.

4-9

1/12/4-9

Ik

2

1

4-2

11/4-5/4-2

32

13

10

1/31A9

27

*55

1/20/49

12

1

l

4722/4-9

92

f&inn, 29

W * 5/29/43

22

3

2

V26/4-9

25

9

6

3

2/24-/4-2

$1

* Rats #52 and 55 were also used in superinfection experiments.

19 Another series of rats were utilized for the study of the development of the larval stages.

These rats were killed and exam-

ined at various intervals of time. The parasites undergo all of their development attached to the wall of the pyloric region of the stomach.

The larvae feed singly during the first 30-4-0 days,

causing scattered inflamed areas. After this period they tend to congregate and feed in a compact group.

The mucosa is destroyed

and continued feeding eventually results in the formation of a chronic ulcer. The larvae grow rapidly during the first 20-25 days. Following this period the growth rate retards somewhat. TABLE IV r

Length

Width

5-7

4-6 mm.

10-14-

6-10 mm.

91.-173. M 109.-210. P

16-12

9-11 mm.

219.-256. M

22

9-12 mm.

219—292. M

25-22

14-12 mm.

274-.-366. P

54- female — male

29-30 mm. 12-21 mm.

0 . 7 mm. 0 . 5 mm.

57 female — male

^0-4-7 mm. 27-29 mm.

1*3 mm. 0*7 mm.

e of l a r v a i n days

The male and female genital primordla after 16-12 days in the rat are shown in Figs. 21 and 29. The writer was unable to detect the number of molts that occur in the definitive host. After the 60th day the males and females are frequently found in copulation. The parasites continue growing for some time after reaching sexual maturity.

20 IX. INFECTION WITH SECOND STAGE LARVAE Two young cotton rats were given 30 and 32 cysts respectively, containing only second stage larvae 19 and 20 days old. After an interval of 101 days the rats were killed and examined.

Both rats

were positive; the parasites in each were feeding in a compact group and had caused the formation of small ulcers 5.-6. mm. in diameter. The larvae were uniform in size in both rats, and the sexes were approximately equal. They had attained about half of their normal growth for the period of time involved.

Some pairs were

copulating; however, there were no eggs in the uterus. TABLE V Rat #50

Rat #54-

101

101

No. of second stage larvae fed:

32

30

No. of specimens at autopsy:

26

2

Time in days:

From these results it appears that the cotton rat might obtain an Infection by ingesting second stage larvae. The possibility of a molt in the egg was taken into consideration, and a special effort was made to detect such a molt. Normal hatching of eggs was not observed; however, recently hatched larvae and empty egg shells have frequently been seen in the contents of dissected colons. In addition, numerous embryonated eggs have been subjected to pressure under a coversllp in order to crack the egg shell and force the larva out of the egg. In no instance was there any indication of a molted or of a loosened cuticle or sheath on these larvae. The interior of the egg was completely empty. Two larval molts had already been observed.

21 The method of development of these second stage larvae in the definitive host will require further study.

There is the possi-

bility of the larva excysting and attaching itself to the stomach wall for feeding, with continued development.

The larvae in this

case would go through the second molt in the excysted conditions. A second possible mode of development would consist of excystation after ingestion followed by re-encystment in the stomach wall of the rat, undergoing the second molt in this cyst and then excysting again for final development. the developmental period.

Whatever the procedure, it prolongs

Alicata (1937) found that third stage

larvae of Physaloptera turgida could reencyst In the stomach wall of an abnormal (rat) host. X.

INFECTION OF HOSTS OTHER THAN THE COTTON RAT

Encysted larvae of Physaloptera hispida were fed to two peromyscus sp.; two rice rats (Oryzomys palustris natator Chapman), two albino laboratory rats and two brown Norway rats (Rattus norveglcus). bino rats.

All of these animals were live-trapped except the alThe rice rats were from the vicinity of Sarasota, Florida

and had been in our animal house for 12 months previous to the time of the experiment.

The Peromyscus and Norway rats were from the

vicinity of Champa!gn-Urbana, Illinois.

The feces of all of the

above animals had been examined several times for helminth infections prior to the feeding experiment and all fecal samples were negative. o i

One specimen of Peromyscus had been fed embryonated eggs

Ascaris columnaris as part of another experiment.

encysts in the viscera of Peromyscus.

This parasite

22 At autopsy the Peromyscus and Qryzomys were negative for Physaloptera.

The wall of the descending colon of one rice rat

contained an encysted Infective larva of protosplrura sp. Albino rat #4-5 was examined 54- days after infection.

The stomach contained

11 Immature specimens of P. hispida. Albino rat #54- was examined 35 days after infection. autopsy.

The feces had been positive on the day of

The stomach of this rat contained five mature specimens

of P. hispida. The two Norway rats were examined 25 days after Infection. The stomach of one rat contained two developing larvae; the other contained ten larvae.

The larvae seemed to be developing at a

normal rate.

No. enc ysted larvae fed

Hosts

Parasite at autopsy

No. days

Peromyscus sp. #1

39

Neg.

3^ (Died)

Peromyscus sp. #2

Neg.

5*

Oryzomys palustrls "A"

35-37 4-0

Neg.

93

Oryzomys palustrls "B"

39

Neg.

Albino Lab. Rat #54-

22

3^2?

93 25

Albino Lab. Rat #4-5

25

2c** 3 ?

5^

Rattus norvegicus #32

9

2 (larvae)

25

Rattus norvegicus #39

12

10 (larvae)

25

XI.

SUPERINFECTION

Two cotton rats were given several feedings of cysts at intervals in order to determine whether superinfections were possible.

23 TABLE VII

Rat #52

Rat #55

Feeding Date

No. Cysts

Nov. 23, 19^2 Jan. 20, 194-9 Apr. 5, 1949

10 12

Jan. 20, 1949 Mar. 9, 1949 Apr. 1, 194-9

12 10 15

4-

parasites Recovered

Autopsy Date

2 4-

1 (mature) 4-23-49 1 (immature) (14-) (12 days old)

1

1 (mature) 4-23-4-9 (4-) (4-5 days old) (12) (23 days old)

There is an indication from the above data that an infection may build up gradually in the cotton rat. The stomachs of several live-trapped cotton rats contained physalopt era hispida which could be sorted into two different size groups. The largest number of specimens ever found were 23, a n of which were mature, from a live-trapped cotton rat. Morgan (194-lb) records finding up to 32 specimens per rat stomach. XII, PATHOLOGY IN THE INTERMEDIATE HOST Arthropods serve as intermediate hosts to a long list of helminth parasites; however, very little is known concerning the pathology initiated in the arthropods by these parasites. A considerable amount of literature has been published dealing with host tissue reactions to fungous, bacterial and protozoan infections. Chen, (1934-) gives a fine review of most of this literature. To the writer's knowledge the first work on host response to a helminth infection in an arthropod was that of Hollands (1920) who gives an account of the reactions of the tissues of Dytiscus marglnails L. to an infection by a larval Distome. The Distomes were found in pedunculate cysts on the wall of the ventrlculus (intestln moyen) and also on the wall of the proctodaeum. The

24larvae were enclosed in a cyst membrane around which was a layer of muscle fibers and a mass of Insect blood cells.

Enclosing all

of this was a thin pericystic membrane which Hollande believed to be an extension of the peritoneal membrane.

He noted that some fat

body cells tended to undergo amitotic division and that some cells developed 2-4- nuclei.

In a few fat body cells the nuclei became

hypertrophled and rich in chromatin.

All of these changes he attri-

buted to mechanical irritations and the release of toxins by the parasite.

Hollande*s observations were made upon a single male

specimen of Dytiscus marglnails captured outdoors.

This provided

a rather statio picture of what might have really happened during the development of the parasite. Chen (1934-) studied the reactions of Ctenocephalldes fells to Dlpylldium canlnum.

The cysticercold of this tapeworm undergoes

its development in the haemocoel of the larva, pupa and adult of the cat and the dog flea.

Some of these cysticercoids are encap-

sulated and destroyed by the haemocytes of the flea.

Chen noted

that all types of haemocytes enter into the reaction; however, the reaction of the haemocyte was slow at first.

No appreciable respons^

was noticed until after the fifth day of Infection.

Not all of the

cysticercoids were encapsulated, but those that were encapsulated, soon died and were transformed into a mass of yellow pigment.

In

the capsule the haemocytes did not form a syncytium, neither was there any evidence of giant cell formation.

Each haemocyte main-

tained its integrity and individual cells could be made out easily in sections.

The yellow pigment which later turned brown was dis-

posed of through the epithelium of the ventriculus into the lumen of the intestine.

25 The eggs of Physaloptera hispida hatch In the colon of the cockroach and within the first 24- hours the larva Invades the epithelium of the colon wall. Just entered the epithelium. round or oval nuclei.

Figure 36 shows a larva that has The epithelial cells are columnar with

The epithelium rests upon an outer layer of

circular muscle fibers which are covered by a thin muscle sheath. The surface of the epithelium next to the lumen of the colon is covered by a cuticle. The growth and activity of the larva causes mechanical injury and general disruption of the epithelial cells.

Fig. 34 shows a

larva in section, four days after invasion of the epithelium.

The

portion of the epithelium adjacent to the larva becomes completely disorganized by the sixth day to such an extent that two separate zones are evident,

A thin layer of epithelium persists next to the

lumen of the colon.

Between this inner layer and the muscle layer

there develops a layer containing a mass of damaged epithelial cells.

By the 7th day (Fig. 3^) the epithelial nuclei adjacent to

the larva show signs of hypertrophy.

This nuclear hypertrophy is a

constant feature of parasitized epithelium.

The round nuclei of

normal colon epithelium measure 9.-12. p In diameter while oval nuclei measure 9.-12. p by 14-.-12. p.

Hypertrophled nuclei, on the

other hand, may attain dimensions of 12.-22. p. in the oase of round nuclei and 14-.-16. p by 23.-25. p for oval nuclei.

Eventually all

of the nuclei in the parasitized area become hypertrophled.

The

nuclei in the epithelium next to the colon lumen retain their normal size (Fig. 4-0). There Is no indication that hypertrophled nuclei ever undergo

26 division; instead they show definite signs of deterioration as the parasite develops.

On several occasions second stage larvae were

seen tearing nuclei to pieces.

The hypertrophled nuclei plus the

remaining diffuse cytoplasm are finally enclosed within the cyst membrane and it is possible that this material is utilized as food by the larva which undergoes approximately two-thirds of its development within a cyst membrane.

By the time the completely

developed third stage larva is produced (30-35 days), the interior of the cyst is completely devoid of hypertrophled nuclei and cytoplasm (Fig. 39). The nuclei of normal colon epithelium divide mitotically. Mitotic figures have been observed on a number of sections. Regeneration cells are not present in the cockroach colon.

Such cells

have been reported as being present in crypts in the wall of the ventrlculus.

According to Snodgrass (1935) when regenerative cells

are present they divide mitotically to produce new epithelium as it is needed, while the epithelial cells have a secretory function and usually do not divide. Alicata (1935) illustrates "rounded bodies" within the cysts of Ascarops strongyllna.

He called these structures "fat cells."

The writer considered the possibility of the hypertrophled nuclei in cysts of P. turgida and p. hispida being the same type of structures; however, there is no blackening with osmic acid treatment; neither would they stain with Sudan IV.

Both of these techniques

were checked with cockroach fat body with positive results. A study of serial sections, especially of the earlier stages of larval development in the colon epithelium, furnished conclusive evidence

27 that these structures could be nothing other than hypertrophled nuclei of colon epithelial cells enclosed by the cyst membrane.

The

development of P. hisplda and p. turgida was found to be Identical. Marked activity is noted during the 13-l2th day.

Sections of

infected colon taken during this period exhibit what appears to be a heavy infiltration of host blood cells. this infiltration in progress.

Figures 37 and 4-0 show

The insect haemocytes accumulate

and pile up against the muscle layer.

In some sections the muscle

fibers seem to be literally forced apart by the infiltrating haemocyte. cyte.

There is no sign of nuclear division among the haemo-

They infiltrate and become distributed around the parasitized

area, enclosing it and separating it completely from the rest of the epithelium.

The haemocytes flatten and become densely packed

to form a cyst membrane.

By the 20th day the cyst can be dissected

out of the colon as a separate spherical structure containing the developing larva and the hypertrophled nuclei; however at this time the cyst membrane is quite delicate and may rupture easily. Toto mounts of cysts, twenty days old stained with haematoxylin show numerous nuclei in the cyst membrane.

Later (35-4^0 days) these

nuclei can no longer be demonstrated in this manner and the membrane resembles other enucleate connective tissue membranes of insects. The extensive work of Lazarenko (1925) showed a direct relationship between the blood cells of larvae of Oryctes naslcornis L. and the formation of connective tissue membranes.

He Introduced

particles of celloldln into the haemocoel of the insect and studied the reaction of the blood cells to the presence of these foreign bodies.

The blood cells accumulated around the celloldln, flattened

22 against its surface and formed a syncytium.

Eventually their

nuclei were lost, resulting in complete encapsulation by a connective tissue membrane.

There was never any indication of cell divi-

sion among the blood cells. The volume of the cyst when first formed is many times larger than the larva occupying it; however by the time the larva has completed its development it fills the cyst.

The cyst also in-

creases in size. All of the second and third stage development occurs within the cyst membrane.

The presence of the parasite

does not appear to have any disastrous effects upon the host.

Cock-

roaches carrying extremely heavy Infections, in which the colon wall was literally a mass of cysts, seem to thrive as well as uninfected ones.

Very little is known of the function of the Insect

colon, but whatever It is it seems not to be impaired by the presence of large numbers of encysted parasites. Hollande (1920) found that fat body cells of Dytlscus marginalis were affected by the presence of the Dlstome parasite encysted in the haemacoel.

Some fat body cells became polynucleate,

the nuclei became hypertrophied and rich in chromatin. body cells divided amitotically.

Some fat

The writer examined the fat body

of infected and uninfected cockroaches.

Parasitism seemed to have

no effect upon these cells. Occasionally a cyst was found in Blatella germanica containing a yellowish brown pigment along with a dead or dying larva.

Alicata

(1937) found similar pigmented cysts in B. germanica infected xvlth Physaloptera turgida.

He refers to the deposit as a "chitinous-llke

substance" and regards the phenomenon as a defense reaction on the

29 part of the host. He also refers to a similar condition associated with encysted larvae of Gongylonema pulohrum.

Hobmaier (194-1) In

his studies of Physaloptera maxillarls observed cysts having a golden brown color, with or without destruction of the enclosed larva. Brug (1932) summarizes data concerning the chitlnizatlon of various bacterial, protozoan and worm parasites of mosquitoes.

It

is difficult to determine whether all of his examples are the result of the same type of reaction.

Brug points out that if chiti-

nous encapsulation is a defense reaction it is a poor one.

As an

example he cites Manson-Bahr (1912) who figures one fllaria enclosed In chitin adjacent to numerous apparently healthy filarla larvae. The writer found a similar condition in the case of Physaloptera hisplda and p. turgida in Blatella germanlca.

Pigmented cysts are

rare in this host and may occur in the same insect along with many normal encysted larvae.

Brug indicates that due to the fact that

some larvae are chltinlzed and some are not, there are two kinds of parasites, dead ones and live ones, and that, chitlnizatlon prevents resorption of the dead parasite bodies by the host. Apparently this conception must have originated through the belief that chitlnizatlon means complete envelopment in an extra membrane, insulating it or sealing it off from the host body. The writer found that some pigmented cysts in Blatella germanica contain larvae that are still alive but showing signs of deterioration. larva.

Pigmentation can definitely precede the death of the

In perlplaneta amerlcana and Tripoli urn sp., in which

Physaloptera hispida will not develop, a few pigmented cysts were

I

_ _ — the only ones ever found.

=

30

Such pigmented cysts contained the re-

mains of larvae that had died in the early stages of development. Several of these cysts were carefully dissected.

Most of the brown

pigment is concentrated toward the center of the cyst.

The larva

is enveloped in a yellow encrustation which has imprinted in it the fine transverse strlations of the cuticle of the larva.

The

general picture Is that of a larva unable to undergo a normal molt and free itself of the old cuticle.

The writer is of the opinion

that this is not a defense reaction but only an event that occurs In connection with unhealthy or dead larvae. Chen (1934) in connection with his work with the cysticercoids of Dlpylldlum canlnum in the larvae of the cat flea, describes a yellow pigment deposit in encapsulated cysticercoids.

This yellow

pigment later turned brownish-yellow and was extruded through the epithelium of the ventrlculus Into the lumen of this organ.

Not

all cysticercoids were encapsulated, but when this did occur the cysticercoid was always destroyed, and pigment was produced and extruded.

He explained the pigment formation as the result of a

reaction between an enzyme, tyrosinase, in the haemocytes of insects and a chromogen in the plasma.

Any plasma adhering to the

encapsulated cysticercoid will have its chromogen slowly oxidized

§ I

by the tyrosinase from the surrounding haemocytes. The glib usage of the term chitin and chitlnizatlon in all of these reports is misleading.

In no case is there any mention of a

specific test for chitin having been made upon any of the material. The writer tested pigmented cysts for the presence of chitin, using j a modified van Wlsselingh test for chitin as outlined by Campbell

B

31 (1929).

The test was repeated several times upon excised cysts and

upon cysts left in_ situ in the colon wall.

Excised cysts were given

the hot concentrated alkali treatment in one inch watch glasses which were placed upon the stage of a binocular dissecting microscope so that the samples could be observed. cysts dissolved completely.

Individual pigmented

The pigment disappeared first, then

the cyst membrane and finally the larva dissolved.

Entire colons

containing pigmented cysts were given similar treatment.

The only

structure remaining undissolved was the peritrophlc membrane which gives a definite positive color test for chitin.

It can be con-

cluded that pigmented cysts containing larvae of Physaloptera hispida or P. turgida contain no chitin.

Campbell calls attention to

the fact that chitlnizatlon Is not related to pigmentation or hardness. XIII.

PATHOLOGY IN THE DEFINITIVE HOST

During the first 4—5 weeks of development in the definitive host the larvae are scattered and attach and feed separately on the wall of the pyloric region of the stomach.

After this initial

period the larvae tend to be more gregarious and are found feeding in compact groups.

Usually, all of the worms will be found feeding

(Fig. 26) in a single group. As a result of their feeding activities the mucosa is penetrated and the lips become imbedded (Fig. 4-2) in the submucosa.

persistent concentrated feeding eventually re-

sults in the formation of a chronic ulcer which may vary from 3. to 10. mm. in diameter.

On the outer surface of the stomach the

ulcerated area is hard and has a whitish appearance.

On the inner

32 surface the ulcer is circular in outline with a raised margin and a hollowed-out inflamed central area in which the parasites are attached. Sections of an ulcer (Fig. 4-7), show that the mucosa is completely destroyed in the central feeding area.

There is a tremen-

dous infiltration of polymorphonuclear leucocytes. ulcers stab cells are abundant. at the base of the ulcer.

In the larger

Some lymphocytes may be present

The inner muscularis is penetrated but

the outer muscle layers always remain intact.

Granulation is evi-

dent, with numerous fibroblast tracts running at right angles to the surface of the ulcer.

Vascular endothelium, forming a new

capillary bed, runs parallel with the fibroblast tracts.

In the

deeper areas the fibroblasts run parallel with the surface of the ulcer.

There is a very marked fibrosis at the base of the ulcer

resulting in a walling off or attempt at encapsulation.

This is

apparently responsible for the induration as evidenced on the outer surface of the stomach wall.

The margins of the ulcer (Fig. 4-7)

exhibit a marked hyperplasia which becomes more pronounced in older ulcers. Infected cotton rats exhibit no noticeable external symptoms. An attempt was made to determine whether a leucocytosis was evident in the circulating blood, but the results were so variable as to make the venture impractical. Seurat (1937) describes and Illustrates a similar ulcer, which he referred to as a "cupule", caused by physaloptera getula Seurat 1917 from the stomach of a rodent, Merlpnes shawl Rozel from N. Africa.

Yokogawa (1922) gives a brief description of a "tumor"

caused by Physaloptera formosana.

33 Hoeppll and Feng 1931 studied the effects of secretions of the esophageal glands of p. olausa Rudolphi 1219 from the hedgehog upon bacteria and upon the tissues of various rodents.

These workers

were unable to demonstrate any bactericidal effects of glandular secretions against Staphylococcus aureus and Bacillus coll in hanging drop preparations.

Subcutaneous and intracutaneous injec-

tions of sterile emulsions of the esophagus into rabbits, hamsters and guinea pigs produced edema, hyperanemia and infiltration with polymorphonuclear leucocytes, but there was no liquefaction of tissues. XIV.

SUMMARY

Physaloptera hisplda is a new species of Physaloptera found in the stomach of the cotton rat (Sigmodon hispidus llttoralls Chapman). A complete life cycle is established for the parasite with Blatella germanica serving as the intermediate host. Attempts to infect other arthropod hosts were unsuccessful. In the intermediate host the larva becomes encysted in the wall of the colon or the rectum and there undergoes two molts during its development.

A completely developed third stage larva is pro-

duced in 30-35 days.

The parasite attains sexual maturity in 73-

102 days in the definitive host.

Cotton rats were successfully

Infected by feeding cysts containing second stage larvae.

P. his-

pida will develop in the albino laboratory rat and the brown Norway rat (Rattus norvegicus), but will not develop in Peromyscus sp. or in the rice rat (Oryzomys palustrls).

There is some evidence to

show that superinfections may be possible.

3^ In the intermediate host the larva undergoes all of its development within the epithelium of the colon or the rectum, causing considerable local disruption and Injury to the epithelial cells. Epithelial nuclei adjacent to the larva become hypertrophled. Haemocytes accumulate next to the muscle layer of the colon and rectum and large numbers of haemocytes Infiltrate and surround the developing parasite and the damaged epithelial cells.

The haemo-

cytes flatten out, form a syncytium and gradually produce an enucleate connective tissue cyst membrane. In the definitive host the parasites cause the development of a chronic ulcer as a result of their feeding activities.

Ulcers

in section exhibit evidence of leucocyte infiltration, marked fibrosis, Induration

and hyperplasia of the stomach epithelium.

35 XV.

REFERENCES

Alicata, J. E-, 1935* Early Developmental stages of Nematodes Occurring in Swine. U.S.D.A. Tech. Bull. #4-29:1-97. , 1937* Larval Development of the Spirurld Nematode Physaloptera turgida in the Cockroach, Blatella germanica L. papers on Helminthology published in commemoration of the 30th Year Jubileum of the Scientific, Educational and Social Activities of K. J. Skrjabln.pp. 11-14-. Boughton, R.V., 1937* Endoparasitlc Infestations in Grouse, Their Pathogenicity and Correlation with Meteoro-Topographical Condition. Univ. Minn. Agr. Exper. Sta. Tech. Bull. #121: 1-50. Brug, S. L., 1932. Chitlnizatlon of parasites in Mosquitoes. Entom. Res. 23(2);229-231.

Bull.

Campbell, F. L., 1929* The Detection and Estimation of Insect Chitin; and the Irreiatlon of "Chitlnizatlon" to Hardness and Pigmentation of the Cuticula of the American Cockroach (Perlplaneta amerlcana) Ann. Entom. Soc. Amer. 22:401-4-26. Chen, H. T., 1934-. Reactions of Ctenocephalides fells to Dipylidl urn caninum. Zeitschrlft f. Parasitenkunde 6:603-637. Chitwood, B. G. and E. E. Wehr, 1934-. The Value of Cephalic Structures as Characters in Nematode Classification, with Special Reference to the Superfamily Splruroldea. Zeitschrlft f. parasitenkunde 7:273-335. Cram, E. B., 1931- Recent Findings in Connection with Parasites of Game Birds. Trans. Amer. Game Conferences 12:24-3-24-7. Diesing, K. M., l26l. Revision der Nematoden Sitzunsb. K. Akad. Wissensch. Wien, Math. Naturw. CI. (1260) 42(22):595-736. Haber, V. R., 1926. I. The Blood of Insects; with Special Reference to that of the Common Household German or Croton Cockroach, (Blatella germanica). Bull. Brooklyn Entom. Soc. 21:61-100. Hobmaler, M., 194-1. Extramammalian Phase of Physaloptera maxi llarl s Molin 1260. J. Parasitol. 27(3):233-235. Hoeppll, R. and L. C Feng, 1931. On the Action of Esophageal Glands of parasitic Nematodes. Nat. Med. J. China 17: 529-592. Hollande, A. C., 1920. Reactions des Tissue du Dytlscus marglnails L. au Contact de Larves de Distome Enkystees et fixees aux parols du Tube Digestif de I'insecte. Archives de Zoologle Expr. et Gen. 59:54-3-563.

36 Howell, A. H-, 1943. Two New Cotton Rats from Florida, proc. Biol. Soc. wash. 56:73-76. Hsu, H. P., 1933. A study of the Esophageal Glands of Some Species of Splruroldea and Fllarioldea. Zeitschrlft f. parasitenkunde 6(3):277-227. Kay, W. W. and R. Whitehead, 1935* The Staining of Fat with Sudan IV. J. Path, and Bact. 4-1(2) :303~3o4-. Lazarenko, T., 1925. Beltrage zur verglelchenden Hlstologie des Blutes und des Bindegewebes. II Die morphologlsches Bedeutung der Blut-und Bindegewebeelemente der Insekten. Zeltschr. f. Mikr. Anat. Forsch. 3:4-09-499. Manson-Bahr, p. H., 1912. Filarlasle and Elephantiasis in Fiji. Witherby and Co., London. 192 pp. Meyer, B. J. and R. K. Meyer, 194-4-. Growth and Reproduction of the Cotton Rat, Sigmodon hispidus hispidus, under Laboratory Conditions, J. Mammal. 25:107-129. Morgan,

B. B., 19.4-1 a. A Summary of the Physalopt erinae of North America, proc. Helm. Soc. Wash. 2(l):22-30. , 194l b. The Physalopterinae (Nematodes) of North American Vertebrates. Unpublished Doctoral Dissertation, Univ. of Wisconsin.

, 194-3*

The Physaloptera (Nematoda) of Rodents, Wasmann

Collector 5 ( 3 ) : 9 9 - 1 0 6 . , 1946. H o s t - P a r a s i t e R e l a t i o n s h i p s and Geographical D i s t r i b u t i o n of the P h y s a l o p t e r i n a e (Nematoda), Trans. Wisconsin Acad. S c , A r t s , and L e t t e r s , 32:273-292. O r t l e p p , R. J . , 1922. The Nematode Genus p h y s a l o p t e r a Rudolphi. P r o c . Zool. Soc. London, 1922 ( 2 ) : 9 9 9 - l l 0 7 . Seurat, L. G., 1917* P h y s a l o p t e r e s des Mammiferes du Nord-Africaln. Comp. Rend. Seances e t Mem. Soc. B i o l . 20:210-212. _, 1919« Contributions Nouvelles a 1 ' e t u d e des Formes L a r v a l r e s des Nematodes P a r a s i t e s Heteroxenes. B u l l . B i o l . F r . e t B e l g . , P a r . et Lond. 52(4-):34-4-372. , 1937. Sur Quelques Nematodes de l ' e s t o m a c des Murides et l e s Reactions q u ' l l s Provoquent. B u l l . Soc. Nat. Afr. Nord. 22:4-22-4-31. Snodgrass, R. E., 1935* P r i n c i p l e s of I n s e c t Morphology, H i l l Book Co., N. Y. and London, 667 p p .

McGraw-

37 Svlhla, A., 1929. Life History Notes on Sigmodon hispidus hispidus. J. Mammal. 10(3):216-220. 10(4-) 5352-353. Vaz, Z. and C. Pereira, 1935« Some New Brazilian Nematodes. Amer. Micro. Soc* 54-(l):36-4-0.

Trans.

Wigglesworth, V. B., 1937- Wound Healing in an Insect, Rhodnius prollxus (Hemiptera). J. Exper. Biol. l4-:364-32T7 , 1939. The principles of Insect Physiology. Co., London 4-34- pp.

Methuen and

Yokogawa, S., 1922. On a New Species of Physaloptera (p. formosana) and the Tumor caused by this parasite. Trans. Jap. path. Soc. 12:201-202. Yorke, W. and p. A. Maple stone, 1926. The Nematode Parasites of Vertebrates, P. Blakiston's Son and Co., Phila., penna.

536 pp.

Vita Stewart Claude Schell was born February 4-, 1912 i n Reading, Pennsylvania.

He a t t e n d e d the p u b l i c schools of West Lawn,

Pennsylvania, and i n June, 1930 was graduated from t h e Wilson High School of West Lawn, Pennsylvania. He e n t e r e d Kansas State College i n September, 1935 and pursueda four year course i n general s c i e n c e .

He was graduated i n June, 1939

r e c e i v i n g a Bachelor of Science degree i n Entomology.

He entered

North Carolina S t a t e College i n September, 1939 as a h a l f - t i m e teaching fellow i n Zoology.

In June, 194-1 he received the degree

of Master of Science i n Zoology and Entomology.

His M a s t e r ' s t h e s i s

d e a l t with a study of the biology of a p a r a s i t e i n the eggs of the Squash-bug (Cf. P u b l i c a t i o n s ) . He married Dorotnydean Vlets of Lawrence, Kansas on December l 4 , 194-1.

He remained a t the North Carolina S t a t e College u n t i l

A p r i l , 1942 a t which time he accepted a p o s i t i o n with t h e United S t a t e s Public Health Service working on s t a t e - w i d e malaria c o n t r o l projects.

In A p r i l , 1943 he was commissioned a s A s s i s t a n t Sani-

t a r i a n Reserve i n t h e United S t a t e s Public Health Service and served with t h i s o r g a n i z a t i o n i n connection with malaria and endemic typhus c o n t r o l a c t i v i t i e s u n t i l August, 1946.

The summers of 194-6 (on

leave) and 194-7 were spent a t the University of Michigan, B i o l o g i c a l S t a t i o n , where he was engaged i n r e s e a r c h i n helminthology.

He

entered the U n i v e r s i t y of I l l i n o i s i n the f a l l of 194-6 and began graduate xrork toward a degree of Doctor of Philosophy i n the Department of Zoology.

He i s a member of Sigma XI.

Publications: Schell, Stewart C., 1943- The Biology cf Hadronotus ajax Gir., a parasite in the eggs of the Squash-bug (Anasa trlstls DeGeer.) Ann. Entom. Soc. Amer., 36:625-635.

FIGURE LEGEND All figures are reproductions of camera lucida drawings except figures 19 and 26 which are sketches, and 46, 4-7, and 42 which are photomicrographs. The projected scales are as follows: Figs. Figs. Figs. Figs. Figs.

1, 2,3, 4-, 5 6, 7, 2, q, 11, 12, 13, 14-, 17 = .05 mm. 10, 16, 22, 24-, 25, 42, 4-3 = 0.1 mm. 15, 30, 31 = 0.6 mm. 12, 20, 33 = 0.3 mm. 21, 23, 27, 29, 32 = 0.15 mm. Fig. 22 = 1.0 mm.

Figs. 34, 35, 36, 37, 32, 39, 40, 4-1, 44, 4-5 = 0.3 mm. Fig.

1.

Fig.

2.

2.

Posterior end of third stage larva (24- days).

Fig.

3-

Larva forced out of egg by pressure on coverslip.

Fig.

Ik

Fig.

5. 6.

Anterior end of third stage larva, lateral view (24- days).

Embryonated egg as ejected by female.

Fig.

72.

Fig.

9.

Fig. 10.

First stage larva preparing for molt (13 days). Encysted second stage larva with hypertrophled nuclei and cytoplasm inside of cyst.

Fig. 1 1 .

Larva preparing for second molt, posterior end (24- days).

Fig. 12.

Male genital prlmordlum, third stage larva.

F i g . 13.

Female genital prlmordlum, third stage larva.

F i g . 14.

Embryonated egg from rat feces, with debris adhering to the shell.

F i g . 15.

Colon of Blatella germanica bearing cysts.

F i g . 16.

Encysted third stage larva, completely developed (31 days).

F i g . 17.

First stage larva (7 days).

Fig. Fig.

1.

Second stage larva, lateral vlevr (16 days).

Ventral view of lips of third stage larva.

Larva preparing for second molt, anterior end (24 days).

Empty egg shell from cockroach colon.

Fig. 12. Anterior end of adult male, ventral view. Fig. 19.

Male and female in copulation.

Fig. 20.

Internal view of excretory canals.

Fig. 21.

Male genital prlmordlum after 16-12 days in the definitive host.

Fig. 22.

Left spicule.

Fig. 23.

Cross section of adult, showing glandular esophagus at level of subventral ducts.

Fig. 24-. Right spicule. Fig. 25.

En face view of adult female.

Fig. 26.

Group of adult worms feeding in ulcer.

Fig. 27.

Posterior tip of adult female, ventral vlex/, showing openings of phasmids.

Fig. 22.

Ovljector of female.

Fig. 29.

Female genital prlmordlum after 16-12 days in the definitive host.

Fig. 3°. Posterior tip of adult male, ventral view, showing arrangement of papillae. Fig. 31.

Median sagittal section of male showing ventral ejaculatory duct, intestine and cloaca.

Fig. 32.

Cross section of adult at level of terminal excretory duct, and dorsal gland duct.

Fig. 33*

Anterior end of adult female, lateral view.

Fig. 34.

Section of parasitized colon of Blatella germanica (4- days).

Fig. 35"

Section of unparasitized colon, showing normal epithelium.

Fig. 36.

Section of colon epithelium showing larva that has just entered (24 hours).

Fig. 37-

Haemocyte accumulation and infiltration (14 days).

Fig. 32.

Colon epithelium, showing initial hypertrophy of epithelial nuclei (7 days).

Fig. 39.

Section through cyst, showing completion of infiltration and flattening of haemocytes (35 days).

Fig. 4-0.

Haemocyte accumulation and early infiltration (14- days

Fig. 4-1.

Sector of wall of egg chamber, cross section.

Fig. 4-2.

Muscular esophagus of adult, showing amphids and some nerve cell nuclei, cross section.

Fig. 4-3.

Muscular vagina, side view.

Fig. 4-4-.

Muscular vagina, cross section.

Fig. 4-5.

Section through cyst (4-5 d a y s ) .

F i g . 46.

Photomicrograph of tvro c y s t s i n cockroach colon (35 davs) x 105. ' .

F i g . 4-7.

Photomicrograph of s e c t i o n of chronic u l c e r ,

Fig. 4-2.

Photomicrograph of s e c t i o n of u l c e r showing l i p s of p a r a s i t e embedded i n submucosa. x 3 5 .

x 16.