Delafield's Hematoxylin Research Articles

A Survey of Helminths of the Muskrat, Ondatra z. zibethica Miller, 1912, in Portage County, Ohio

From 130 muskrats collected at 13 sites in Portage County, Ohio, 10 species of trematodes, two species of cestodes, five species of nematodes, and one genus of acanthocephalans were identified. The finding of Corynosoma sp. represents the first record of this acanthocephalan genus from North American muskrats. The recovery of the nematode Longistriata dalrymplei Dikmans, 1935 and of the trematode Mediogonimus ovilacus Woodhead and Malewitz, 1936 constitute second reports of these species. A table summarizing the published literature (1951-66) on helminths from North American muskrats is included. Numerous helminthological surveys concerning the muskrat Ondatra z. zibethica Miller, 1912 have been made in North America. Several reports have summarized the literature up to 1951 (Meyer and Reilly, 1950; Knight, 1951). As an aid to individuals currently conducting similar research, Table I lists the literature in North America from 1951 through 1966. Prior to this report, the only published survey of helminths of Ohio muskrats was that by Rausch (1946). His investigation covered the period from March 1943 to March 1945. Seventy muskrats were examined from seven scattered Ohio localities. Four species of trematodes (Echinostomum coalitum Barker and Beaver, 1915; Notocotylus quinqtieserialis Barker and Beaver, 1915; Wardius zibethicus Barker and Beaver, 1915; and Plagiorchis proximus Barker and Beaver, 1915), one species of cestode (Taenia taeniaeformis Batsch, 1786), two species of nematodes (Capillaria ransomia Barker and Noyes, 1915 and Trichuris opaca Barker and Noyes, 1915), and one genus of cestodes (Hymenolepis) were identified. The objective of our investigation was to determine if these same species, as well as others, parasitized muskrats in Portage County, Ohio. MATERIALS AND METHODS During the 1963 and 1964 Ohio trapping seasons, 13 collection sites were established in Portage County. These included portions of eight lakes and ponds, the Mahoning and Cuyahoga rivers and three swampy-creek areas. Fur dealers and trapReceived for publication 17 February 1967. * From a thesis submitted to the Graduate School of Kent State University in partial fulfillment of the requirements for the degree of Master of Arts. pers were the primary sources of skinned carcasses, but 30 animals were trapped by one of us (Beckett). Ten muskrats from each collection site were data-tagged, frozen, and stored at -20 C until ready for study. All of the worms recovered were fixed in A. F. A. Delafield's hematoxylin and Mayer's paracarmine were used to stain cestodes and trematodes. Fastgreen in 95% ethyl alcohol was used to counterstain the ventral glands of monostomes and the cephalic spines of members of the Echinostomatidae. Acanthocephalans were stained with Delafield's-Ehrlich's hematoxylin. Nematodes were cleared in glycerine and mounted in glycerine jelly. RESULTS AND DISCUSSION A total of 130 muskrats, comprising 68 males and 62 females, were examined from the 13 collection sites. Ten species of trematodes, two species of cestodes, five species of nematodes, and one genus of acanthocephalans were identified (Table II). Only those helminths identified during this survey and reported infrequently from North American muskrats will be discussed.

Timing of Ovulation and Implantation in the Cottontail Rabbit

Timing of Ovulation and Implantation in the Cottontail Rabbit

Studies on the Development of Endosperm in Rice : 3. Observations on the cell division.

Extending over the 6th to 8th day after anthesis, ripening rice caryopses were collected every two hours. Samples thus collected were fixed with FAA, and then cross sections obtained from the middle portion of the endosperm were stained with Delafield's Haematoxylin. Cell division occurred mainly in the early morning (from 2 a. m. to 8 a. m., accounting for 50-72% of the all samples), and after passing through the rest stage in the day-time (from 10a. m. to 4 p. m. ), it reoccurred in the evening. It was observed that from evening to midnight, the time for cell division and resting stage were repeated alternately (Tables 1 and 2). The periodicity of cell division had connection with the day-night rhythm, while it was thought that the pattern of the division cycles might differ according to age of the tissue. Average time required per cell division was 45-55 minutes. Resting time in early morning was very short, while that in the day-time was very long. It became evident that the section of the cell division was not confined to the outermost cell layer of the endosperm. Namely, 86% of the total cell division was observed in the outermost cell layer of the tissue, and 14% in the 2nd and interior cell layers. Basing on the observations, the process of the tissue formation shown in the Report 1 on this investigation has come to be revised, because in the case of Report 1, the tissue formation process had been deduced from the hypothesis that the cell division occurred only on the outermost layer. In the case of division of the cells found in the interior portion of tissue, the features have been found in that the cells were divided into two caused by the nuclear division which occurred after the enlargement of size of cells has reached twice as large as the surrounding ones.

Redescription of Prosthopycoides lynchi (Ingles, 1936) gen. et comb. n. (Trematoda: Lecithodendriidae)

The trematode Brachycoelium lynchi Ingles, 1936, is redescribed and transferred to Prosthopycoides gen. n. This genus differs from Lecithodendrium by having preacetabular testes and prececal vitellaria; from Prosthodendrium by having a pseudocirrus, anterior uterine loops and no genital sinus; and from Pycnoporus by having prececal vitellaria, shorter esophagus, and oval shape. This species is the only member of the subfamily Lecithodendriinae found in amphibians. Ingles (1936) discovered a fluke in the redlegged frog in California and named it Brachycoelium lynchi. Manter (1938) suggested that this worm probably belonged in the genus Lecithodendrium Looss, 1896, and Rankin (1938) followed that suggestion by transferring the species to Lecithodendrium on the basis of the original description and figure. Cheng (1958) studied the type and paratypes deposited in the U. S. National Museum by Ingles and concluded that Rankin (1938) was correct in removing this fluke from Brachycoelium but doubted that Lecithodendrium was the correct genus. Yamaguti (1958) retained this species in Brachycoelium; however, he noted Rankin's proposed change to Lecithodendrium. Dubois (1960, 1961a, b) did not mention this fluke in his revision of the lecithodendriids. After comparing my own specimens with those described by Ingles, I am convinced that this species should be redescribed and a new genus erected to accommodate it. The trematode was found only in Rana aurora although 13 other species of frogs and salamanders were examined in Oregon. Specimens were fixed in Lavdowsky's AFA or Gilson's fluid under slight cover slip pressure and stained with Delafield's hematoxylin. Drawings were made using a Wild drawing apparatus. Measurements were taken from eight egg-bearing adult specimens from two hosts, and are in microns. The average measurement is followed by the range in parentheses. The Received for publication 16 February 1966. * Based in part on a thesis submitted to the Graduate School, Oregon State University in partial fulfillment of requirements for the Doctor of Philosophy degree. t Present address: Dept. of Zoology, Seattle Pacific College, Seattle, Washington 98119. long axis or anterior-posterior measurement is followed by the width measurement. Prosthopycoides gen. n. Diagnosis Lecithodendriidae-Lecithodendriinae. Small, spinous, slightly flattened distomes. Suckers subequal; oral sucker subterminal, mouth subterminal with funnel-shaped opening leading to short prepharynx. Pharynx globular, smaller than oral sucker, surrounded by small peripharyngeal glands. Esophagus about as long as pharynx; ceca very short, widespread. Acetabulum muscular, in posterior half of body. Testes entire, opposite, preacetabular. Pseudocirrus sac intertesticular, curved with distal end turning posteriorly to join genital atrium; genital pore median, ventral, directly anterior to acetabulum; seminal vesicle large, coiled; ejaculatory duct, surrounded by prostatic cell mass, leads from seminal vesicle to form terminal, eversible pseudocirrus (as defined by Pande, 1935 and Dubois, 1960). Ovary entire, intertesticular, dextral or sinistral. Seminal receptacle extending posteriorly from ovary; Laurer's canal arises from seminal receptacle; Mehlis' gland present. Vitelline follicles in a grape-like cluster on each side, posterolateral to oral sucker, anterior to ceca; vitelline duct extends from each cluster to vitelline reservoir between ovary and seminal receptacle. Uterus extensive posterior to acetabulum, anterolateral loops extend to vitellaria. Eggs small, operculate. Excretory pore median; bladder V-shaped, with widespread crura reaching acetabular level. Intestinal parasites of amphibians. Type-species: P. lynchi (Ingles, 1936). Type-host: Rana aurora, red-legged frog. Prosthopycoides lynchi (Ingles, 1936) gen. et comb. n. (Figs. 1-4) Syn. Brachycoelium lynchi Ingles, 1936 Lecithodendrium lynchi (Ingles, 1936) Rankin, 1938

Caudouterina rhyacotritoni gen. et sp. n. (Trematoda: Digenea) from the Olympic Salamander

A new genus and species of allocreadioid trematode, Caudouterina rhyacotritoni, from the intestine of Rhyacotriton olympicus Gage (salamander) collected in western Oregon is described. The host salamander, Rhyacotriton olympicus Gage, was collected under rocks in and along fast-moving mountain streams in the Oregon Coast Range Mountains. Only those salamanders collected from streams descending the east slope of Marys Peak, Benton County, Oregon, harbored this fluke. Eighteen of 21 R. olympicus harbored from one to six (avg 2.3) flukes. Five other Olympic salamanders collected near Hebo, Oregon, 60 miles northwest of Marys Peak, were not infected. The flukes were removed from the intestine and placed in amphibian Ringer's solution, observed alive and later fixed in AFA (Lavdowsky's solution) and stained with Delafield's hematoxylin. Frozen frontal sections were made on a cryostat and stained with Delafield's hematoxylin. All drawings were made with the aid of a Wild drawing apparatus. Measurements are in microns unless otherwise stated. The average measurement appears first with minimum and maximum in parentheses. Caudouterina gen. n.

The Intestinal Phase of an Experimental Infection of Chicks with Trichinella spiralis

Quantitative studies of Trichinella spiralis infections in other than mammalian hosts are rare. The intestinal phase of T. spiralis infections in 8-week-old mice and 1-day-old chicks were compared. Recovery of 1% to 4% of the initial infection was common from the chicks compared to 20% to 30% from mice. Primary location of the adult worms was in the ceca of the chicks rather than in the small intestine and gravid females were recovered from this location during the 47-day duration of the experiment. No gravid females were recovered from the mice after the 14th day. There was no statistically significant difference in the size of the adult worms recovered from the two hosts. Augustine (1933) and Matoff (1937) showed that when chickens and pigeons are experimentally infected with Trichinella spiralis some adults develop and copulate in the gut. They reported the production of embryos and limited recovery of larvae from the muscles of birds. Both authors cite the gizzard as preventing large numbers of adult trichinella from developing in the gut. While other factors impede the establishment of larvae in the muscle, a complete explanation of resistance in birds is not available. Quantitative reports regarding growth and development of T. spiralis in the avian host are lacking. The purpose of this paper is to describe the location, development, and longevity of the adult worms in the bird intestine. MATERIALS AND METHODS All larvae were obtained from infected stocks of white mice maintained in the laboratory. No larvae less than 6 weeks old were used for infection. Since infection in mice has been studied thoroughly by numerous investigators, mice were used for comparison. The mice for this study were maintained as an inbred colony in the laboratory. All mice were 8 weeks old at the time of the initial infection. Laboratory chow and water were available to them at all times. All chicks were 1 day old on the day of infection. They were cockerels of a lightweight white leghorn breed. Commercial chick starter and water were constantly available except for 1 day prior to killing. Larvae were obtained by artificial digestion Received for publication 12 January 1966. *A portion of a dissertation submitted by the author in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Zoology in the Graduate College of the State University of Iowa. Present address: Westmar College, Le Mars, Iowa. using a 1% solution of pepsin with 0.5% hydrochloric acid. A blender was used to prepare the mouse carcass for digestion. Infective doses were counted by the dilution method of McCoy (1931). Actual counts of such doses were shown to be consistently within a deviation of 10% from the desired dosage. The mice were etherized and inoculated orally by placing the desired number of worms into the sto ach with a glass pipette. The chicks were infected similarly but the worms were placed in the crop without anesthesia. The same procedure for recovery of adult T. spiralis was used for both the chicks and mice. The animal was deprived of food the night before it was killed. The intestinal tract was removed, from stomach or gizzard to the anus. The ceca, large intestine, and four equal lengths of the entire small intestine were each placed in a separate dish of 0.85% saline. Each portion of the intestinal tract was opened lengthwise and the mucosa scraped from the muscularis. The muscularis and fragmented mucosa remained in saline for at least 1 hr. Then the large pieces of intestine were shaken vigorously, washed several times, and discarded. The remaining sediment was washed by adding saline, allowing a period of time for settling, and decanting the upper portion of fluid. After repeated washing, the fluid became clear; it was discarded and the remaining sediment which contained the larvae was spread in rows on a glass slide for counting. For preparation of whole mounts, adult trichinella were fixed in hot alcohol-formol-acetic solution, transferred to glycerin-alcohol, and mounted in glycerin jelly. All measurements of adult worms were made from these permanent mounts. Penetration of the gut wall was studied by fixing portioiis of the gut in Bouin's solution, imbedding in paraffin, and sectioning at 12 Y. Delafield's hematoxylin and eosin were used for staining. All statistical comparisons were made using Student's t distribution. EXPERIMENTS AND RESULTS A preliminary experiment was conducted with 1-day-old chicks to determine the LD50 of the trichinella larvae for chickens. Three

Aging White-Tailed Deer by Annuli in the Cementum of the First Incisor

Histological preparations of the primary incisors of white-tailed deer (Odocoileus virginianus) revealed the presence of layers in the cementum corresponding to yearly growth. The method was confirmed by using known-age material from Michigan. Since regional variation and inconsistency have been shown to occur in the use of the tooth-wear method of aging the white-tailed deer (Ryel et al. 1961, Gilbert 1964), it is necessary that another more general and more accurate criterion be found. Recent progress on studies of the annual increment in body structures of many species of mammals suggested the approach used in this study. Annual rings in either the dentine or cementum have been described for other big game species: for moose (Alces alces) by Sergeant and Pimlott (1959), for caribou (Rangifer tarandus) by McEwan (1963), for the mule deer (Odocoileus hemonius) by Low and Cowan (1963), and for the white-tailed deer by Ransom (1966). Known-age material in this study was made available through the Michigan Department of Conservation; the cooperation of Dr. L. Fay and L. Verme is much appreciated. I am grateful to Dr. D. Dodds and the Biology staff at Acadia University, who directed the study through its early stages, and to A. T. Cringan and Dr. S. Dixon of the Zoology Department of the University of Guelph, who supplied the needed facilities for the completion of the study. MATERIALS AND METHODS When the teeth from wild specimens of white-tailed deer were ground, examination indicated the presence of annuli in both the dentine and cementum of the incisiform and molariform teeth. Owing to the greater ease of removing and decalcifying the primary incisor (Ii), this tooth was used for the perfection of a histological technique. McEwan's (1963:111-112) procedure formed the basis of this technique, and it was finally refined to the following: (1) decalcification of the incisor in 30 percent formic acid for 96 hours (or longer if necessary), (2) immersion in Cellusolve for 24 hours, (3) three paraffin (melting point, 54-56 C) baths of 3, 2, and 2 hours each, (4) longitudinal sectioning of the tooth at a thickness of 10 microns by means of a microtome, (5) standard staining technique using Delafield's hematoxylin with a maximum staining time of /2 hour. Microscopic examination of sections mounted in balsam by means of transmitted light proved entirely satisfactory. The technique was then tested for veracity against a sample of known-age whitetailed deer teeth (1%/-11 years) from MichiTable 1. Data on 10 known-age deer specimens from Michigan from whose primary incisors the aging technique was confirmed. LIVING DATE AGE AT CONDISEX OF DEATH TIONS DEATH (IN YEARS) Experimental F 4/19/58 1% Experimental F 10/31/60 2? Experimental F 3/16/60 5% Natural F 3/23/60 6% Experimental M 12/ 6/63 7? Natural F 2/23/60 8% Natural F 11/16/59 91/2 Natural F 4/12/53 11 Natural F 1/ 4/65 3% Natural M 10/29/63 4?

Electron microscopic evidence for the systemic invasion of an insect host by a plant pathogenic virus

Ultrathin sections were prepared of viruliferous Agallia constricta Van Duzee, a leafhopper vector and host of the plant pathogenic wound-tumor virus (WTV), in an attempt to localize the virus within tissues and cells of various anthropod organs. Earlier infectivity and serological tests had revealed the presence of virus in some insect organs and tissues, without permitting precise localization. In the present study, excised fat body tissues, gut, Malpighian tubules, and salivary glands, as well as whole abdomens, were fixed and embedded in methacrylate or in epoxy resin. Alternating thick and ultrathin sections were made with a Porter-Blum microtome and stained with uranyl acetate and lead. For light microscopy, the thick sections were stained by Delafield's hematoxylin and eosin. Thin sections, dried on carbon-coated collodion or Formvar film, were examined by a Siemens Elmiskop I. Largest accumulations of WTV, often in the form of microcrystals, were found in the fat body tissues; crystalline arrangements were also seen in muscle cells. Virus clusters were observed in defined cytoplasmic structures within the Malpighian tubules, mycetome, trachea, and epidermis. Only one or two lobes of the salivary gland contained scattered virus particles not associated with defined cytoplasmic structures. The findings point to the fat body as the most likely site of virus multiplication. The occurrence of virus accumulations in almost all organs of A. constricta is interpreted as direct evidence for systemic invasion of this arthropod host. Lack of a sizable accumulation of WTV in the salivary gland may explain the erratic and poor transmission record of the insect vector. This finding is in contrast with findings made with the very efficient vector of rice dwarf virus, in which the lobes of the salivary gland contain large masses of virus particles.

Plagitura intermedia sp. n. (Trematoda : Plagiorchiidae) from the Common Newt, Notophthalmus viridescens viridescens

Plagitura intermedia sp. n., a new plagiorchiid trematode, is described from the common newt, Notophthalmus v. viridescens, collected in Bear Lake, Shawnee State Park, Ohio. In general arrangement of the gonads, P. intermedia is similar to P. parva, the smaller of the two previously named species, but three to four times larger in size. Immature specimens are reported which are larger than the maximum size reported for either P. parva or P. salamandra. Cuticular spines extend posteriorly for three-quarters of the body length, rather than one-third as in the other species. Many trematodes of the genus Plagitura were taken from the small intestines of more than 100 adult Notophthalmus viridescens viridescens collected in Bear Lake, Shawnee State Park, Ohio; none were found in 25 larval newts, or in seven red efts. Some specimens were considerably larger than the maximum size of the two described species, P. parva Stunkard, 1933 and P. salamandra Holl, 1928, both of which were also identified from the same locality. The large worms are three to four times as long as P. parva, but are similar to that species in general arrangement of the gonads. Worms were studied alive in amphibian Ringer's solution, and also as permanent whole mounts. Worms were fixed, under light pressure, in Bouin's fixative, and stained in Semichon's acetic carmine, Ehrlich's hematoxylin, Delafield's hematoxylin, or Gower's carmine. Plagitura intermedia sp. n.

Eimeria sceloporis and Eimeria molochis spp. n. from Lizards

Two new species of coccidia are described: Eimeria sceloporis sp. n. in the small intestine of lizards (Sceloporus spp.) from southern California and Mexico; and Eimeria molochis sp. n. in the Australian horned toad, Moloch horridus, from the Queensland desert, probably also an intestinal parasite. The oocyst of E. sceloporis is broadly ellipsoid, usually 27 by 22 au, index 1.23, containing four oval sporocysts 8 by 7 i, index 1.16; a temporary oocyst residuum is formed; sporulation time is 8 hr, range 8 hr. The oocyst of E. molochis is also broadly ellipsoid, usually 34 by 26 u, index 1.31, containing four oval sporocysts (with temporary Stieda body) 10 by 7.5 u, index 1.33; no oocyst residuum formed; sporulation time is 8 hr, range 9 hr. The known species of Eimeria from lizards are compared in a table. Among nearly 600 Eimeria spp. which have been recorded (Pellerdy, 1963) 20 are mentioned from lizards, of which 17 have been described as distinct species. Most of these were reviewed, but incompletely compared, by Arcay de Peraza (1963) in a table which included no data on oocysts other than gross dimensions and shapes. We therefore present an extended table to supply additional information (Table I). Some reports of coccidia in reptiles suggest a low incidence of infection (Moskowitz, 1951; Bovee, 1962). However, eimerians and other sporozoons have usually been found in lizards when sought. A survey of parasites and inquilines in reptiles revealed two eimerians which we consider to be new species: Eimeria sceloporis sp. n. in Sceloporus clarki boulangeri from Mexico and in S. occidentalis biseriatus and S. magister from southern California; and Eimeria molochis sp. n. in the horned toad, Moloch horridus, from the Queensland desert of Australia. MATERIALS AND METHODS Fresh feces from a juvenile Sceloporus clarki were dispersed in amphibian Ringer's solution, and abundant unsporulated oocysts of the coccidian species were found (about 15 per 43 X microscopic Received for publication 17 June 1964. * Supported by USPHS Training Grant No. 2E-70 and USPHS Grant No. 6462. 85 field) so that flotation and concentration techniques were superfluous. Three other S. clarki, an adult S. magister, and several juvenile S. occidentalis, were found infected with the same, but no other coccidian species. Two Moloch horridus were found infected with a different eimerian species, and no other coccidian species. Fresh oocysts placed in 3% K2Cr207 were pipetted at intervals onto clean slides and observed microscopically to determine duration of sporulation. One hundred cysts were measured at each stage of development, the mean derived, and most frequent measurements noted, as recommended by Doran and Jahn (1952). Observations were made with bright-field, variable phase contrast, and interferometric microscopy at 100 X to 1,000 X magnifications. Measurements were made at 400 X and 1,000 X with a calibrated ocular micrometer. Room temperature was kept at or near 25 C during the study of the duration of sporulation. The endogenous part of the life cycle was studied from stained serial sections of tissue (Delafield's iron hematoxylin with eosin or fast green counterstain) for E. sceloporis from Sceloporus clarki and S. magister. The viscera of S. occidentalis and of Moloch horridus were inadvertently discarded by a laboratory technician. OBSERVATIONS I. Eimeria sceloporis A. Endogenous development 1. Locale of infection: In the juvenile S. clarki only a short part of the small intestine just posterior to the duodenum was infected, but there a great majority of the villi was parasitized. Tips of villi were seldom invaded, but the sides of villi were heavily parasitized, This content downloaded from 157.55.39.211 on Mon, 08 Aug 2016 06:20:39 UTC All use subject to http://about.jstor.org/terms 86 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 1, FEBRUARY 1965 TABLE I. A comparison of the various species of Eimeria from lizards. Species Host Site of infection Oocyst Zygote E. boveroi Carini and Pinto, 1926 E. knowelsi Bhatia, 1936 E. raillieti Leger, 1899 Lainson, 1959 E. mabuia Carini, 1938 E. sceloporis sp. n. Bovee and Telford, 1965 E. gekkonis Tanabe, 1928 E. intermedia Ruiz, 1959 E. stenocerci (tropiduri) Carini, 1940 E. molochis sp. n. Bovee and Telford, 1965 E. minetti Ray et al., 1942 E. acanthodactyli Phisalix, 1930 E. scinci Phisalix, 1923 E. colonorum Prasad, 1960 E. cystis-fellae var. americana Amrein, 1952 E. mirabilis Yakimoff, 1936 E. flaviviridis var. americana A. de Peraza, 1963 E. rochalimai Carini and Pinto, 1926 E. agamae Laveran and Pettit, 1910 E. koidzumii Matubayasi, 1941 E. flaviviridis Setna and Bana, 1935 E. hemidactyli Knowles and Das Gupta, 1935 E. sp. Brygoo, 1963 Hemidactylus mabui Ameiva ameiva Hemidactylus flaviviridis

Callus Formation and Isolation of Single Cells from Rice Seedlings

1. This experiment was designed to elucidate the course and the rate of callus production in rice seedlings and to study the single cells isolated from the callus under sterile conditions. Nutrition used here contained 10 μM 2, 4-D, 10 μM NAA, 10 μM kinetin, 5g/1 yeast extract and the basal medium of the modified Murashige and Skoog's (Table 1). 2. Vigorous callus formation was not induced by NAA but 2, 4-D (Fig. 1). Nutritional requirements for the development of callus resulted in the use of yeast extract (Table 2). When the small pieces excised from rice callus were subcultured or rice seeds were cultured for prolonged periods, they developed a huge callus producing a lot of roots with root hairs (Figs. 3 and 4). The fresh weight of rice seedlings with callus increased about 20 fold in 50 days; namely, a seed of 30 mg developed to a lump of callus of 600 mg or more during the incubation period (Fig. 15). From detailed observations on the process of callus formation, it was ascertained that the callus is induced not only from the scutellum or coleoriza but also from the basal regions of the coleoptiles, leaf tissues and root apicles (Figs 5 to 12). 3. Many single cells were freely separated from the callus in suspension culture by rotating the tubes in which the callus and the medium were contained at 1 rpm. The cells were stained with Delafield's hematoxylin, neutral red or methylene blue. The size and form of the cells were observed microscopically. The length and the width of the single cells (means of 100 cells) were 41.1 and 18.8μ, respectively (Table 3). The single cells were of various forms; round, rectangular, crooked and very long. Protuberances similar to budding in unicellular organisms were observed in the single cells. Many pairs of divided cells suggested that longitudinal division occured in the single cells (Figs 13 and 14). 4. The callus and the single cells separated from rice seedlings will provide a system that is of interest in genetical and cell physiological studies on rice plants.

Raillietina (Raillietina) loeweni sp. n. (Cestoda: Davaineidae) from the Hare in Kansas, with Notes on Raillietina of North American Mammals

Raillietina (Raillietina) loeweni sp. n. is described from the black-tailed jackrabbit (Lepus californicus melanotis Mearns) from western Kansas. A key is given for the species of Raillietina of North American mammals. Subfamily and generic emendations are made to include R. loeweni. Data are given on the seasonal and monthly prevalence of this cestode in the host population in the study area. Surveys of the parasitic helminths of hares (Lepus spp.) and rabbits (Sylvilagus spp.) in the United States and Canada have revealed Raillietina infections as follows: R. salmoni (Stiles, 1896) from rabbits in Texas (Price, 1928); Raillietina sp. from Lepus alleni from Arizona (Vorhies and Taylor, 1933); R. retractilis (Stiles, 1896) from Sylvilagus spp. from Wyoming (Honess, 1935); R. salmoni from cottontail rabbits in Alabama (Moore, 1939); R. retractilis from cottontail rabbits in California (Herman and Jankiewicz, 1943); R. retractilis from S. nuttalli grangeri, S. auduboni baileyi, S. aud. arizonae, and L. townsendi campanius; R. salmoni from S. aud. arizonae, S. floridanus, and L. cal. melanotis in Wyoming (Honess and Winter, 1956); R. retractilis from L. cal. deserticola in Utah (Grundmann, 1958); R. retractilis from L. californicus in California (Lechleitner, 1959); and R. salmoni (later Received for publication 20 September 1963. * This study was supported in part by grant No. E-1676, National Institutes of Health; by Kansas State University, Agricultural Experiment Station Project No. A-488; and by the Kansas Forestry Fish and Game Commission. Contribution No. 326, Department of Zoology, Kansas State University, Manhattan, and Zoology Series No. 357. t A portion of a dissertation presented by Monroe H. Bartel in partial fulfillment of the requirements for the degree Doctor of Philosophy in Parasitology at Kansas State University, Manhattan. Present address: Department of Natural Sciences, Moorhead State College, Moorhead, Minnesota. identified by senior author as R. loeweni sp. n.) from L. cal. melanotis in Kansas (Lyons, Hansen, and Tiemeier, 1960; Bartel, 1960). A helminth parasite survey was conducted on the black-tailed jackrabbit, Lepus californicus melanotis Meams, from January 1960, through November 1961, and June through October exclusive of September 1962, near Lakin in Kearny County, Kansas. One hundred ninety hares (70.4%) of 270 examined harbored Raillietina (R.) loeweni sp. n. Inference of the monthly infection rates of the host population by the cestode was based on the infections of ten hares collected each month. The sample of ten hares, showing no shot damage to the intestinal tract, was taken from larger monthly collections of 11 to 37 hares collected within an 11-mile radius of Lakin, Kansas, north of the Arkansas River. The hares were classified as adult or juvenile on the basis of body size, condition of reproductive organs, and the degree of epiphyseal closure of the humeri according to the method of Plenert (1962). The cestode is named in honor of Dr. S. L. Loewen, parasitologist, Tabor College, Hillsboro, Kansas. The description is based on eight gravid cestodes fixed in Ward's fixative and stained in Delafield's hematoxylin. Additional specimens were observed fresh or cleared in lactophenol. Egg pouches, teased from fixed gravid proglottids, were measured and counted. The size of eggs and oncospheres was determined from eggs teased from living proglottids. Living oncospheres were stained with 1 : 10,000 neutral red or 1: 5,000 brilliant

Brugia beaveri sp. n. (Nematoda: Filarioidea) from the Raccoon (Procyon lotor) in Louisiana

Brugia beaveri sp. n., from the lymph nodes, skin, and carcass of the raccoon (Procyon lotor) in Louisiana is described. This is the first Brugia species to be reported from mammals in North America. It differs from the six other described species of the genus generally in the smaller body size of the female, the size and structure of the spicules, and the larger microfilaria which has a distinctively long cephalic space. A sheathed microfilaria of the Brugia type was first discovered in the United States in 1953 in a thick blood film from a raccoon (Procyon lotor) from Lafitte, Louisiana (Beaver, personal communication). A similar microfilaria was found in the blood of a bobcat (Lynx rufus) in Florida in 1960 (Orihel, personal communication). In neither case was the adult worm recovered. In February 1963, sheathed microfilariae were again found in raccoons in Louisiana. The adult worm was subsequently recovered and is here described as a new species of the genus Brugia Buckley, 1960. It is the first species of this genus to be described from the United States, the second reported from the Western Hemisphere (Orihel, 1963). MATERIALS AND METHODS Seven raccoons were collected from an area near Rosedale, Louisiana, approximately 90 miles northwest of New Orleans. As six of the animals had been captured alive, their blood could be examined repeatedly before they were necropsied. Microfilariae in blood samples were fixed in 2% formalin. A dilute Azur II solution was added to formalin-fixed microfilariae to bring out morphologic details. Microfilariae were also studied in thick and thin blood films. Thick films were air dried, dehemoglobinized in 0.85% saline, fixed in hot (60 C) 70% alcohol, and stained with dilute Delafield's hematoxylin. Thin blood films were Received for publication 8 August 1963. * This study was supported in part by grants 2E-2 and AI-04919-01 from the National Institutes of Health, Public Health Service. fixed in methyl alcohol and stained 1 hr with Giemsa (2% in pH 7.0 buffered water). Adult worms were recovered by soaking the skin, carcass, and lymphatic tissues in warm 0.85% saline solution as described by Dunn (1931). In the examination of one raccoon in which seven worms were recovered the soaking fluid was, in addition, filtered through coarse filter paper; the material on the surface of the filter paper was then washed into a large petri dish and examined with a dissecting microscope. Adult worms were fixed in glacial acetic acid (Berland, 1961), transferred to 70% alcohol containing 5% glycerine, and then gradually brought into pure glycerine, the medium in which they were studied. All drawings were made with the aid of a camera lucida. Brugia beaveri sp. n. Brugia microfilariae were found in five of the seven raccoons examined. Microfilariae of a Dipetalonema sp. were found in six and those of Dirofilaria tenuis in all of the animals. Adult worms of the new Brugia species were recovered from only two. However, one of the seven raccoons was dead when brought into the laboratory and, although it had Brugia microfilariae in the blood, it was not examined for adult worms. From one raccoon a single male was recovered from the saline in which the skin had soaked. From the other, two complete males, a broken male, three complete females, and a broken female were recovered. One male was recovered from an axillary lymph node and another from a popliteal node; all other specimens were found in the saline in which the skin and carcass had been immersed.

ONTOGENY AND DIFFERENTIATION OF SCLEREIDS IN RAUWOLFIA

ONTOGENY AND DIFFERENTIATION OF SCLEREIDS IN RAUWOLFIA

Early Development of the Secondary Bronchi in the House Sparrow Passer domesticus (Linnaeus)

This study presents the origin, development and relationships of the secondary bronchi in the embryo of the House Sparrow, Passer domesticus (Linnaeus). Serial transverse, frontal or sagittal sections at six or eight microns were made of one or more embryos, fixed in Bouin's or Lavdowsky's fluid, cmbedded in paraffin and stained with Delafield's Hematoxylin, for each half-day interval from two through nine days incubation. Photomicrographs, camera lucida tracings, direct projections were used to make diagrammatic reconstructions at regular intervals of incubation. The time and point of origin and general destination of each secondarv bronchus is described. Three groups of secondary bronchi occur in this species. The five mesiobronchi arise on the dorsal, dorsomesial or dorsolateral surface of the mesobronchus and occupy the anteromesial and mesial regions of the lung. The nine dorsobronchi arise on the dorsal and dorsolateral surface of the mesobronchus and occupy the anterodorsal, dorsal and dorsolateral regions of the lung. The nine lateroventrobronchi arise on the lateroventral and lateral surfaces of the mesobronchus and occupy the ventral and lateroventral portions of the lung. Detailed description is given for the development of the mesobronchus, secondary bronchi and tertiary bronchi concerned with the origin of the individual airsacs. Primary and secondary ostia of the intermediate and anterior abdominal airsacs are described. Migration of origins or mouths of secondary or tertiary bronchi from the lung to the airsac side of an airsac ostium are given for the intermediate, anterior abdominal and posterior abdominal airsacs. The individual secondary bronchi are compared with those described in other species. The time of origin of the secondary bronchi in the House Sparrow are compared with those of the chicken; these structures appear earlier in the House Sparrow than in the chicken.

Micro-Anatomy of the Proventriculus of the Common Rat Flea Xenopsylla cheopis (Rothschild)

Living specimens were obtained from an extremely flourishing culture maintained by adopting Leeson's method (1932), and the insects were anaesthetized and dissected in physiologically normal saline for observing living movements of the proventricular walls. Proventriculi were also teased out in normal saline for studying the spines. Mounting of freshly teased out spines in the DeFaure's fluid tinted light pink with borax carmine yielded excellent results. Different fixatives were tried but Carnoy II was found to be most satisfactory. Double embedding (celloidin and paraffin wax) method was employed for cutting serial sections. Delafield's haematoxylin and eosin were mostly used for staining. The blow and roll technique devised by the author and described below, proved to be of a great help for the study of the entire surface anatomy of the organ, including the arrangement, pattern and distribution of the spine bases, and for calculating the total number of proventricular spines in both the sexes. Freshly dissected out proventriculi were allowed to float freely in normal saline contained

Effects of nitrogen, phosphorus and potassium on the structure and physiology of the vegetative shoot apex in the rice plant

The effects of nitrogen, phosphorus and potassium on the structure of the vegetative shoot apices in rice plants were investigated cyto-histologicaiiy, using the variet of paddy rice Aichi-Asahi In the first experimcnt (1956, Soil culture), three plots were provided according to the quality of nitrogen, phosphorus and potassium supplied, and in the second experiment (1957, Sand culture), seven plots (1. N, 2. P, 3. K, 4. NP, 5. NK, 6.PK, 7. NPK) were providee, using the Dr. KASUGAI's complete culture solution for rice plants as the standard solution. Dissected stem tips of the young plants of about middle stage of vegetative growth were fixed in modified Navashin's fluid or F. A. A. and embedded in paraffin wax and serial sections cut at about 7μ were stained with the Delafield's hematoxylin. The results obtained in the experiments are as follows : (1) The structure of the vegetave shoot apices in rice plants shows several cyto-histological changes according to different conditions of mineral nutrients. (2) In the plots of high nitrogen supply or nitrogen only, the number of the tunica layers are increased and 2 or 3, sometimes 4 layered tunica is observed. the anticlinal division in the peripheral region of the apices being promoted. (3) On the contrary, in the plots of hlgh phosphorus supply or phosphorus only, the stratification in the tunica is distutbed and thc number of the tunica layers is decreased to 2 or 1 layer, accampanied by the increase of the all direction division in the corpus. (4) Potassium seems to have an effect to establish the topographical differentiation in a apex distinctly and to restrain the one-sided changes in the number of tunica layers by nitrogen or phosphorus supplied. Refering to the writer's observations on the developmental changes in the structural pattern of thc vegetative shoot apices in rice plants, it is considered that high nitrogen supply tends to strengthen the vegetative characteristics in the apices, and the other way, high phosphorus supply tends to diminish the vegetative features in them.

The Reproductive Cycle of the Field Mouse, Microtus californicus

The cricetid rodent Microtus has been studied extensively in recent years due to its widespread geographic distribution and to the cyclic abundance shown by most of its species. Reports are available on certain aspects of the reproduction of Microtus californicus (Hatfield, 1935), M. agrestis (Brambell and Hall, 1939), M. pennsylvanicus (Hamilton, 1941), and M. guentheri (Bodenheimer, 1949). With these exceptions, the remaining work is primarily concerned with the population dynamics of Microtus . The present report, based on work in the field and in the laboratory, describes some of the anatomical and physiological features of the reproductive cycle of females of Microtus californiens and emphasizes certain aspects in which M. californicus differs from other microtines or from other rodents. The present study is based on dissection of 581 female Microtus californicus collected over a 21- month period from 1951 to 1953. Most of these animals were trapped in grassland adjacent to San Francisco Bay, near San Rafael, Marin County, California. Approximately 50 were kept in captivity. Appropriate reproductive tracts were preserved in Bouin's fluid, paraffin embedded by the dioxane method, sectioned at 10 micra, and stained with Delafield's hematoxylin and eosin. A summary of the field data may help in placing the laboratory findings in proper perspective. Microtus californicus is polyestrous, breeding throughout the year. The major breeding season is in the late winter and spring months with a minor period of reproductive activity in the fall. During the rest of the year sporadic pregnancies occur. The mean litter size is 4.2 with a range from 1 to 9 embryos. However, there is considerable seasonal variation in litter size depending upon the age of the females. Pregnancy lasts 21 days (Hatfield, 1935), and the young can be weaned …

A New Species of Benedenia (Trematoda: Monogenea) from Girella nigricans, the Opaleye

Judging from the color and condition of the preserved material it seems probable that the worms were fixed under slight pressure in Bouin's fluid. The following description is based on a large series of whole mounts stained with alum cochineal, hematein or Delafield's hematoxylin, cleared in deacidified beechwood creosote and mounted in piccolyte. Unstained whole worms mounted in Diaphane were also employed. All measurements were made by the use of a calibrated ocular micrometer and are given in millimeters. In citing the measurements the mean is given, followed by the minimum and maximum in parentheses. The number of measurements used in deriving the mean is usually the same as the number of specimens measured, but where different the number employed appears in parentheses before the measurements. All measurements of curved structures are across the lines subtending the greatest arcs described by those structures. Drawings were made with the aid of the camera lucida.

Trematodes Parasitic in the Salientia in the Vicinity of Ann Arbor, Michigan

Materials and methods.-The 102 hosts used in this study were collected during April through September, 1951, from four stations in the vicinity of Ann Arbor, viz., ponds along Zeeb Road, Whitmore Lake, Flemming Creek, and the Huron River. Most of the amphibians were killed within 48 hours after capture. The worms were fixed with hot A.F.A. Borax carmine, Mayer's paracarmine, and Delafield's hematoxylin were used as stains.