Proceedings of the Helminthological Society of Washington 36(1) 1969

Volume 36 January 1969 /Nurrtbef V

PROCEEDINGS

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;ABRAM, JAMES B. , Some Gastrointestinal' Helminths of Ondatra zihethicus , . ,; .Linnaeus, tlie Muskrat in Maryland -,...------:_-..--..--.--.._..-A----->------l-- ---.---.rr ,93 -CABLE, ;R. MJ, AND HADAR ISSEROFF^ VA Protandrotis Uaplopqrid Cercaria, r , Probably Jthe Larva of SaccocQelid.ides sogandanqsi Lumsden, 1963 1..^. COI.GLAZIER, M. L., K. C. KATES/kNb F, D. ENZIE. \. Activity of , ,'V-Tetfainisole, Thiabendazble, and Purified Fin^ Particle Thenothiazine, .-.':Against Experimental/Infections of Haernojichus contorUis--JanA Triclio- stroiigylus Speeies in Sheep ,Liivir— .:-—1^ l-.,r.__...-.i-.J.«__:.: -4^212 68 CROLL, N./A^ ANDLTD, R. \ViGLjERcmo. Osmpregulatioh arid the Uptilce pf \;:; > ^/. Ions in a Marine Nematode ..^^.^.: ....::..„-.-../...... ^^.,^...^..'r'r-l.^.....L~^.'.. •;"-!' DORAN, DAVID J., AND JO&N M.^VEiTteRriiNG. Infectiyity of Two Species of ' • r J Poultry Coccidia After Freezing, arid Storage in. Liquid JNfitrogen Vapor\:l._- r30 DORAN;) DAVID ,J.y AND JOHN M. VErrteRirNTG. Influence of Storage Period;on; /' Excystation; and i Development in Cell Culture bf Sporozoites of Eimeria '...-' ••£ - fneleOfgrimitis Tyzzeir, 1929^. -.^--^—^.. .--l.u--.-Z2 ^_ii.r.—....^..^.-:_^lL-. 'f /,,;- -X;K ;/; "';"^ vv ;;'K r- - / -'(Continued on BackCover),' - J- '.: .^,;/ \ ... .r -''/:> '•#•

' ' ".' ''S ' •' ' ' -~ ' ' -J' '' / ' ' • . ' '~~L.' ' ' "'• -~~' "I •-•' -'""' •' ''I *'_- .'' -' "• ^ '- / i -;'.'.;"r'••••;• THE SOCIETY meets once a mpriUi from October dirough May for the presentation, and T ^/ •;' discussion'pf papers in .any \and all branches of parasitology or "related .sciences. All .interested f /_r persons are invited to attend. ;J ' //' "/' . ' •.""'-"-,'; / :\, x ; ''Persons interested^in inembership in die HelrriintliQlogical Society of.;Was3bin"gton.i may obtain Vir vapplicatibn blanks from the Gorresponding gecretalry-Treasurer^ Miss Edna ,/M. Bulirer, Belts-- /, N '; ville Parasitplogidal Laboratory, Agricultural Research Center, Beltsville, Maryland, 20705.. A i ^ year's subscription to,, die Proceedings is^included in die1 annual dues ($6:00).v ;"/ --/

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VOLUME 36 JANUARY 1969 NUMBER 1

Osmoregulation and the Uptake of Ions in a Marine Nematode

N. A. CROLL1 AND D. R. VlGLIERCHIO Department of Nematology, University of California, Davis, California

Past studies have demonstrated that if cer- conditions the worms remained in an appar- tain animal-parasitic, plant-parasitic and free- ently good state for up to 6 weeks. All experi- living nematodes are placed in hypertonic ments were conducted on adult worms from solutions they lose water causing a reduction this supply at 25 C. in volume (Stephenson, 1942; Osche, 1952; Through water transfer nematodes become Lee, 1960; Wallace and Greet, 1964; Anya, greatly reduced in size and distended in hyper- 1966; and Myers, 1966). In most cases osmo- tonic and hypotonic solutions respectively. The regulation has then occurred enabling the nematode response to osmotic solutions and nematodes to regain their original size. Sim- osmoregulation was measurable by changes in ilarly when placed in hypotonic media there its physical dimensions. The distance from the is an increase in volume and sometimes a re- oesophago—intestinal junction to the anterior turn to normal body size (Stephenson, 1942; tip was found to be a convenient sensitive Anya, 1966; and Myers, 1966). This com- parameter. In preliminary experimentation it munication complements these observations by was found that this measurement was in direct characterizing osmoregulatioii and uptake of proportion to changes in width and overall ions in the marine nematode—Deontostoma length. (= Thoracostoma) calif ornicum. Each worm was placed in a perspex groove Reviewing the data on osmoregulatory mech- containing seawater, held by a coverglass and anisms in invertebrates, Beadle (1957) was measured with an ocular micrometer. The able to say that "There is no convincing evi- nematodes then were placed in test solution dence as yet that osmotic gradients between in 2 cm diameter glass cells in covered Petri body fluid and external medium are due in dishes. A little tap water was placed in each any degree to the active transport of water." Petri dish to ensure a high humidity while It is, therefore, with the movements of ions allowing free gaseous exchange. The nema- that osmoregulation is achieved and the ability todes became inactive or less active during to osmoregulate will depend on the ability to the experiments. take up and lose ions. Frequently, exosmosis proceeded until con- Experimental Methods and Materials tortion and crumpling of the worms occurred and at these extremities of water loss, volume Deontostoma calif ornicum was collected changes were not truly reflected by length from the holdfasts of Laminaria digitata and measurements. These excessive losses were Egregria laevigata at Dillon Beach, California, at extreme low water spring tide. D. califor- accommodated within 15 min and the usual nicum was removed from the holdfasts and body shape was recovered. The first reliable stored in seawater on gauze at 5 C as this was measurement of water loss (ME) was on the found to reduce "clumping." The seawater was recovery curve R which approximated to changed at weekly intervals, and under these linearity in all but the two cases described below. T was the time taken to regain original

1 Fulbright Scholar, permanent address: Department of (seawater) size (Fig. 1). The rate of recovery Zoology and Applied Entomology, Imperial College, Uni- versity of London. was expressed as ME/T or tan «°.

Table 1. Osmotic regulation of Deontostoma californicum following immersion in various hypertonic media. PERCENTAGE CHANGE IN LENGTH Compound Regulation-;- Compound Regulation'!'

NaCl + MgCl2 + NaBr* + CaCl2 + Nal + KMnO, O NaNO2* -)- Oxamic acid o (partial) (COOH-CO-NH2) Figure 1. Schematic diagram of the measure- NaNO., + Oxamide o ments made of the osmoregulatory process in NH2-CO-CO-NH2) Deontostoma californicum, following immersion in Na2SO4 — Formamide o hypertonic media. ME, maximum recorded exos- (CHO-NHg) mosis; T, recovery time; R, recovery line; tan a, Na^O., Dimethyl oxalate o rate of recovery. (CH.,-CO-CO-CH3) Na2MoO4 — Ribose NaWO, — Xylose Osmoregulation in hypotonic and KC1 + Sorbitol : hypertonic solutions of NaCl KBr -)- Inositol — Using distilled water, 0.4, 0.6, 0.8, and 1.0 M Kl -f- Galactose solutions of NaCl, the length changes of D. (slight) californicum were measured with respect to K1O, O Glucose time. The isotonicity of the worm was thus K3Fe(CN)(. — Fructose - approximated as well as its ability to recover K1Fe(CN)(. — • Trehalose its seawater dimensions. NH(C1 + Lactose — RESULTS: In both hypertonic and hypotonic LiCl + Maltose solutions there were immediate changes in LiNO., + Sucrose (slight) length (Fig. 2). D. californicum was unable SrCl2 to osmoregulate in hypotonic solutions (Fig. SrBr2 +

2) even after 24 hr, but recovered its normal Srls length and mobility in seawater. In hypertonic + ::: Nonlinear recovery. solutions recovery occurred at an essentially t +, Recovery to original length; —, No recovery but constant rate until the original length had been normal on return to seawater; Q, No recovery, dead on reached; R was treated as being linear. There return to seawater. frequently occurred an "over-osmoregulation" giving a length larger (usually 5-10%) than ing overcompensated, a subsequent return to the original seawater length (Figs. 2, 4). Hav- normal size was never observed. Following osmoregulation in a hypertonic solution and transfer to distilled water, there was an immediate and gross increase in size which usually led to bursting of the worm. This degree of > increase did not occur in changes from seawater to distilled water. Osmoregulation in other solutions A comparison was made of the ability of D. californicum to return to original seawater length following immersion in hypertonic solutions of selected electrolytes and nonelectrolytes. Figure 2. Percentage length changes of D. cali- RESULTS: The ability or inability of the fornicum with respect to time in NaCl solutions of nematodes to recover normal length in the varying molarity. hypertonic media, together with the return to

SECOND SALT

Figure 3. Percentage length changes of D. californicum with respect to time to hypertonic solutions of NaBr and NaNO2, showing nonlinear recovery.

, _ .50M NaCl / normal activity in seawater following the treat- / ,- .75M NaCl ment, is tabulated (Table I). D. californicum returned to its original size, or increased slightly in most electrolytes. The recovery rates (tan «) differed, while the recovery line R approximated to linearity except in the cases of NaBr and NaNO2 (Fig. 3). Very slight recovery occurred in sucrose and galactose and none was observed in any of Figure 4. Schematic diagram showing length the other sugars. changes resulting from immersion in a second solu- Oxamic acid, oxamide, formamide, dimethyl tion following regulation in the first. A. Transfer oxalate, and potassium periodate were used to from 1 M solution of first electrolyte to 1 M solu- investigate the significance of spatial configura- tion of a second electrolyte. B. Transfer from 1 M tion and charge in the regulatory mechanism. NaCl to 0.5 M or 0.75 M NaCl. (Arrow indicates Their low solubility, together with their appar- the point of transfer.) ently high toxicity, made reliable conclusions impossible. 0.75 M NaCl and any changes in length were In seawater those individuals that had osmo- measured. regulated, expanded in size and frequently RESULTS: Every change to a molar solution burst; all of the others (except the oxalic acid of the second salt, following regulation in the derivatives, formamide and potassium perio- first, resulted in an additional increase in size date) regained their normal size and mobility. (Fig. 4A), though'the increase "was subject to Vigorous activity occurred for up to 30 min some individual variation. The following pairs following transfer to sodium thiosulphate. of 1 molar successive changes were made: LiN03 -^ NaCl, NaNO3 -» NaCl, The influence of successive immersions NaCl -* LiNO3, NaCl -> NaNO3. in different hypertonic solutions on osmoregrilation D. californicum. that had osmoregulated in 1 M NaCl also increased further when placed After D. californicum had regulated in a 1 in 0.5 M or 0.75 M NaCl, the increase being molar solution of an electrolyte, it was im- greater with the more dilute solution (Fig. 4B). mersed in another molar solution of a second salt. Salts were selected from those known to The influence of sulphate on the allow regulation (Table 1) in order to show regulatory mechanism any cationic or anionic specificity of the regu- Since D. californicum was unable to regulatory mechanism. late in sulphate (Fig. 5), thiosulphate, molyb- Nematodes that had osmoregulated in 1 M date, and tungstate (Table 1), recovery was NaCl were also transferred to 0.5 M NaCl and estimated in various volume to volume hyper-

v/v N.,SO./H»C1

0/100 55'

Figure 5. Percentage length changes of D. californicum with respect to time in hypertonic solutions of the sulphate and nitrate salts of lithium and sodium. tonic mixtures of 1 M Na2SO4 and 1 M NaCl. The rate of regulation varied with different Figure 6. Percentage length changes of D. californicum with respect to time in various volume/ v/v mixtures of Cl~ and SO4= (Fig. 6). In 100% and 75% sulphate solutions there was volume mixtures of 1 M NaJSO.t and NaCl; showing the dependence of angle « (Fig. 1) or the Na2SO4/ no recovery from ME (« = 0°). At 50:50 NaCl ratio. mixtures there was slight osmoregulation, and the rate of osmoregulation increased with greater dilutions of sulphate. From 1% to 50% were found for worms exosmosed only (X), SO4= the rate of recovery against dilution plot this apparently being due to adsorption or some followed closely an exponential relation. other form of binding or trapping by the in- The inhibitory effect of high SO.t" concen- tegument. The increases in total cation follow- trations was temporary and the worms re- ing regulation in 1 M NaCl and MgCl2 approxi- covered normal size and motility on being mated to the calculated value for a volume of transferred to seawater. 1 M solution equivalent to the volume of solution taken up in the nematodes (Table 2, Flame photometric determination of ions volume of free water R-X). Insufficient K+ To establish the uptake of ions during osmo- ions entered to account for the total required regulation, groups of 10-12 D. californicum increment in osmotic pressure needed to attain were weighed (wet wt), dried to constant isotonicity in 1 M KCl. weight for 3 hr at 100 C and ashed at 600 C If the ionic concentration of the water lost for 8 hr (ash wt) following treatment. In on drying of D. californicum (wet wt-dry wt addition to controls taken directly from sea- 100 C) is assumed equivalent to seawater for water, other groups were exosmosed (X) for purposes of comparison (it is not, of course, 3 min in 1 M solutions of NaCl, KCl, and because of the hydration of tissues and organic MgClo, respectively. The other treatment (R) solutes, etc.), it appears that Mg++ and Na+ was exosmosis and recovery in 1 M NaCl, KCl, are present within the animal in concentra- and MgClo prior to weighing and ashing. The tions of the same order present in seawater. concentration of Na, K, and Mg were then Potassium ions, however, are present in much estimated with a flame photometer. higher concentrations. RESULTS: Flame photometric measurements of ionic concentrations of K1, Na+, and Mg++ Localization of the regulatory mechanism following regulation (R) in .1 M KCl, NaCl, Following regulation in hypertonic solutions, and MgClo, respectively, showed increases in D. californicum increased greatly in size and all cases. Increases in ionic concentrations also often burst when transferred to distilled water.

Table 2. Distribution of ions in Deontostoma calif ornicum, exosmosed only (X), osmoregulated (R), and from seawater controls (C).

M s •1 a c c •Sx .2 ^ ;02) 2S ms^ *y. wa t

M a f •^£ o .« rt O ^ /i g /tg/m l

C bo c 0 C'+3 equival e I1' o C 3 ° .« -^ '^.2^^ i n I 1j tissu e £ to wate r tio n :io n reas e ieawate r ll a ff, la « S<^ 1 la Q £^ 13 OS U" O ^ £ g U.S'o Potassium 9.72 1.3 4.0 50.80 0.495 K+0 22.9 37.5 2.73 18.9 K+x 23.4 38.6 8.62 18.9 K>K 23.6 38.5 11.30 22.3 Sodium 457.00 0.4 17.0 9.20 4.200 Na+c 22.9 37,5 44.10 18.9 Na+x 23.1 37.9 60.70 14.0 Na+jt 23.8 38.9 78.30 14.7 Magnesium 53.40 1.5 22.2 36.48 1.950 Mg++c 22.9 37.5 6,37 18.9 Mg++x 22.8 38.0 16.50 11.0 Mg++R 23.8 38.2 40.40 14.9

Ligaturing experiments were conducted to iso- RESULTS: Crystal violet (Gentian violet), late those regions of the body important in eosin, and methylene blue entered best and the osmoregulatory mechanism and in the colored the entire body contents. No canals uptake of ions. Using hypertonic 1 M NaCl, were found and no pores were visible for water ligatures were tied with 7.0 Ethicon braided passage in or out of living worms. silk. RESULTS: When ligatured in the middle of The longitudinal movement of the body while still in seawater and placed in water in D. californicum 1 M NaCl, both anterior and posterior halves Worms from seawater were placed across a of the body decreased in size. Both halves vaseline barrier separating distilled water and became grossly distended on being returned 1 M NaCl. This design contrasted with liga- to distilled water. When ligatured behind the turing as the environment was separated but stoma and anterior to the anus, the nematodes the movement of ions and water in the worm became smaller in hypertonic media and fol- was free and continuous through its whole lowing osmoregulation burst when returned to length. distilled water. The two halves of the worm behaved inde- Localization of the area of water uptake pendently with respect to osmotic change. The ligaturing experiments indicated that Although the stains entered the portion of the both osmoregulation and the uptake of water worm in distilled water, there was no indica- occurred through the body wall. After osmo- tion of the dye moving longitudinally toward regulation for 30 min in 1 M NaCl (incomplete that part of the worm in 1 M NaCl. This may recovery), D. calif ornicum was placed in dilute have been due to adsorption of the dyes onto vital stains to determine whether the entry membranes preventing its free passage. Never- of water (and with it the stain) was in any theless, an appreciable longitudinal move- pores or canals. Cotton blue, eosin, congo red, ment of water could not be detected in our janus green B, crystal violet, and methylene techniques. blue were all used. The reverse process was also examined where stained worms were Discussion exosmosed to see any pores or canals from The environment of all the animal parasitic which the dyes diffused out. nematodes examined to date are hypertonic to

Copyright © 2011, The Helminthological Society of Washington PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY the body fluids of the nematodes (Pannikar and Sproston, 1941; Lee, 1960; Rogers, 1962; Anya, 1966). It has been consistently demonstrated that these forms are able to osmoregulate well in hypertonic media. They are, however, less able or unable to regulate in hypotonic media. In contrast the free-living Rhabditis terrestm in culture (Stephenson, 1942) and other Rhab- ditis species (Osche, 1952) and Panagrellus redivivus in water (Myers, 1966) inhabit hypotonic habitats and regulate well in hypotonic media. Isotonicity for these is about 0.15 M NaCl. Because of the apparent adaptation of osmoregulation to habitat, it has been Figure 7. The relationship between molecular suggested that marine nematodes would be weight and the rate of recovery (tan a); — joins unable to regulate their osmotic pressure common cations, joins common anions, = joins (Rogers, 1962; von Brand, I960; Lee, 1965). the chlorides of divalent cations. Isotonicity of D. californicum. is, however, 0.6 M NaCl, or about four times greater than It may be seen from Figure 7 that the dis- for any nematode known. Many marine inver- tribution of salts in a molecular weight against tebrates have poor powers of osmoregulation tan a (rate of osmoregulation) plot is not but D. californicum maintains an osmotic equi- random. When using the chloride, bromide, librium in hypertonic media—a feature it and iodide salts of Sr++, Na+, and K+, iodides shares with some animal parasitic forms. allowed the fastest rates of recovery, chlorides These observations suggest that D. califor- the slowest, and both SrBr2 and KBr were nicum, is partially adapted to intertidal life and intermediary. The position of NaBr (Figs. 3, 7) can tolerate exposure and higher salinity. This cannot be explained, but von Brand (1943) nematode may be poikilosmotic as suggested showed it to be toxic to larval Eustrongylides by Osche (1952) for free-living soil forms and ignotus. In a M wt X tan « plot (Fig. 7) the Rogers (1962) for some animal parasitic halogen salts of each cation had slopes of the nematodes. same order. Approximately parallel lines join Beadle (1957) emphasized the importance respectively: Nal and KI, NaBr and KBr, and of ionic exchange arid uptake in the regulation the chlorides of Li+, NH4+, Na+, and K+. of osmotic pressure and water content. Hobson The anionic and cationic sequence in de- et al. (1952a, 1952b) showed that the body creasing order of preference in the regulatory fluids of Ascaris lumbricoides changed in ionic mechanism is: composition following incubation in diluted seawater. Myers (1966) reported evidence of Anions I —» Br~ —» Cl~ ionic absorption in the osmoregulation of Pana- Cations K< -> Na1 -» NH4+ —» Li+ grellus redivivus and Aphelenchus avenae. The Stoke's Law radii and ionic mobilities of Regulation of D. californicum relates to these Sr++ and Ca++ are practically identical and findings in other nematodes, taking up ions in Mg++ is very similar (Table 3), corresponding hypertonic solutions until osmotic equilibrium to the rates of regulation in the chlorides of is reached with the environment. Bursting of these ions (Fig. 7). The ionic mobilities and the nematode upon re-entry in distilled water, Stoke's Law radii of K+, Na+, and Cl+ also form together \vith the flame photometric demon- a series reflecting the rates of regulation of D. stration of an increased concentration of ions californicum (Fig. 7). Having the lowest M following regulation, supported the theory of wt, Li+ has the highest charge density, greatest ionic uptake. There was no suggestion of an hydration, lowest molecular mobility and, outward movement of ions either in hypotonic therefore the highest Stoke's Law radius. media or following over-osmoregulation in hy- Of the anionic halogens the increasing order pertonic solutions (Figs. 2, 4B). of preference for regulation is Ch —> Br~ —> L

Table 3. Ionic mobilities (\°) Stoke's Law Radii, and energy of hydration (~A HK cal mole"1) of ions at 25 C.

-AHK-Kcal Ion X° Stokes' r mole-1 K+ 73.50 1.25 - 83.37 Na+ 50.10 1.84 -103.55 Figure 8. Schematic model illustrating a possible mechanism whereby polarizability of an anion Li+ 38.60 2.39 -129.67 facilitates its passage through a charged membrane Mg++ 53.0 3.47 -473.29 of the body integument. A, anion approaching Ca++ 59.5 3.10 -394.50 charged membrane; B and C show7 polar reversibil- Sr++ 59.4 3.10 -359.22 ity on passage through the "pore"; D, completion I- 76.8 1.20 - 61.8 of entry. Br- 78.14 1.18 — 72.2 ci- 76.35 1.21 - 80.3 SO4= 2.30 function of the square; there is a nonlinear Fe(CN)03- 2.76 increase, therefore, in the area for ionic uptake. Fe(CN),,''- 3.35 The resultant of the decreasing rate of diffusion and increasing the surface area tends to give Data after Noyes (1962) and Robinson and Stokes (1959). a linear recovery line. It is suggested that the entry of the cation (Fig. 7). These anions have low hydration, may be essentially dependent on differential and Stoke's Law radii and their ionic mobility ionic mobility, reflecting hydrated diameters are essentially the same (Table 3). The series and modified by the anion depending on its Cl~ —> Br~ —» I~ does, however, manifest de- polarizability. Their entry depends on a living creasing charge density and increasing polar- system possibly charged and has the char- izability. This significance of this polarizability acteristics of a one-way selective diffusion, appears to be correlated with increasing ease there being no evidence for the outward of recovery. A model scheme for a possible movement of ions. mechanism by which an anion could pass In attempting to explain the inability of through a "pore" in the membrane is illustrated SO4= and other Group VI anions to enter, it (Fig. 8). was not possible to distinguish between the The results suggest that in hypertonic solu- molecular structure or the multiple charge. tions of single salts, ions pass in by selective Monovalent anions of like spatial configura- diffusion until an equilibrium is reached for tion; C1O4~, IO4~, MnO4~ and unlike configura- that salt. The "distention" over normal size in tion, oxalic acid derivatives, as well as poly- a regulated animal may be explained in part valent anions Fe(CN)G4^, Fe(CN)G3- were by the osmotic effect of normal residual soluble either toxic or insufficiently soluble for testing. components, over and above that due to the The penetration of extremely dilute solu- electrolyte of hypertonicity. This is consistent tions of eosin, methylene blue, and gentian with the two-step distention of an animal violet, would suggest that there is transfer of treated successively in two different salts. In diverse ions and molecules through the integu- a second hypertonic salt, following equilibrium ment, but that it is of an order several times in the first, there is a second equilibrium giving removed from that involved in osmotic effects. a greater resultant osmotic pressure, and fur- The inability to osmoregulate in hypertonic ther increase in size. sugar solutions may rest on the very limited The recovery line R (Fig. 1) is approxi- ability of nonelectrolytes to cross the integu- mately linear; the explanation for this may ment (Mueller, 1929). The inability of Group be found by the interaction of two opposed VI anions to enter is consistent with the results factors. The entry of ions by physical means of Tweedie and Segel (1967) and Yamamoto would tend to be greatest at the onset of ionic- and Segel (1966) on Penicillium chrysogenum entry ME (Fig. 1) and gradually become re- and Aspergillus sp. Krogh (1939) found that duced. As ions enter and the worms dimen- SO4= did not enter marine invertebrates well; sions increase, its surface area increases as a and Robertson (1957) found that SO4= in

Copyright © 2011, The Helminthological Society of Washington PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY many marine invertebrates was usually below which have at some time been implicated in the equilibrium value for seawater. The inhibi- nematode osmoregulation. The gut was shown tory effect is probably of little importance in not to play an essential role in ionic regulation, the ocean, where SO4~ ion in the Dillon Beach although it probably contributes in the natural locality are present in 2.64 parts per thousand habitat. at a salinity of 3.48% (Hedgpeth, 1964). There is good evidence that in hookworm Nematode recovery can be visualized as in- larvae osmoregulation is restricted to the so- volving several phases: (a) Passage of anions called excretory system as it is in flatworms and cations through a selectively permeable (Weinstein, 1952 and 1960). This difference membrane. The effect between the activity of is consistent with the broad postulate in biology the monovalent cation series and the mono- that marine organisms, which are usually iso- valent anion series may be in part a reflection tonic with their surroundings, undergo ionic of manner of hydration. Since the water mole- exchange and water loss over their entire sur- cules around a cation extend their protons face. While with migration onto land or fresh- outward so that ions having a wide range of water or another nonisotonic environment, sizes may assume configurations more adapt- comes a reduction in the surface area of such able to the surrounding water structure while membranes and a localization of osmotically anions require the hydrogen of water molecules active surfaces. to be directed inward and as such, the configurations of the hydration spheres may be Summary more dependent on the central ion size, (b) Deontostoma californicum was able to re- The formation of an osmotically active body cover its osmotic pressure in hypertonic NaCl solution, made up in part by imbibition of but not in hypotonic solutions. Osmoregulation salts, increased solubilization of organic sub- was not possible in hypertonic nonelectrolytes stances and an increase in bound water from and in SO4=, and other Group VI anions. In increased tissue hydration such as may be the other electrolytes used, the rate of recovery induced by or reflected in cation mobility. varied with ionic mobility and energy of In view of experimental observation it is hydration in cationic species with the degree tempting to think that monovalent anions, de- of polarizability in anionic species. A hypoth- pending upon their polarizabilities, control the esis is presented that relates the chemical and passage of salts across the semipermeable membrane. The cations, however, are pri- physical characteristics of ions and the proper- marily involved in solubilization of organic ties of the integumental membrane to the components and enhanced hydration of tissues mechanism of ionic and osmotic regulation. manifested in increased rates of recovery of Using ligaturing experiments and aqueous body lengths, according to the series of in- dyes it was concluded that the uptake of ions creased ionic mobilities of monovalent cations. and entry of water occurred over the entire The accumulation of K+ and possibly the body surface. Finally, our results have been other salts suggested in the flame photometric compared to observations on other free-living analyses, may be explained by the observed and parasitic nematodes. unidirectional movement inward of salts at high tide and the partial loss of water through Literature Cited drying at low tide over long periods. Anya, A. O. 1966. Investigation on osmotic If this were the case then it would be neces- regulation in the parasitic nematode Aspi- sary to postulate an equally slow elimination of culuris tetraptera Schulz. Parasitology 56: other cations Na+, Mg++ by metabolic means 583-588. eg. excretion, loss through the gut, so that the Beadle, L. C. 1957. Comparative Physiology: balance with seawater was maintained. Osmotic and ionic regulation in aquatic animals. Ann. Rev. of Physiol. 20: 324-358. The ligaturing experiments suggested that Hedgpeth, J. W. 1964. Introduction to Sea- the uptake of ions occurred through the integu- shore Life of the San Francisco and Bay ment just under the cuticle. It was also found Region and the Coast of Northern Califor- that the process did not depend on localized nia. University California Press, Berkeley. receptors, amphids or caudal glands all of Hobson, A. D., W. Stephenson, and L. C.

Beadle. 1952a. Studies on the Physiology Robinson, R. A., and R. H. Stokes. 1959. of Ascaris lumbricoides. I. The relation of Electrolyte Solutions. The Measurement and the total osmotic pressure, conductivity and Interpretation of Conductance, Chemical chloride content of the body fluid to that of Potential and Diffusion in Solutions of Sim- the total environment. J. Exp. Biol. 29: 1-21. ple Electrolytes. Second Edition, Butter- Hobson, A. D., W. Stephenson, and A. Aden. worths, London. 1952b. Studies on the Physiology of Ascaris Rogers, W. P. 1962. The Nature of Para- lumbricoides. II. The inorganic composition sitism. Academic Press, New York. of the body fluid in relation to that of the Stephenson, W. 1942. The effect of variations external environment. J. Exp. Biol. 29: 22- in osmotic pressure upon a freeliving soil 29. nematode. Parasitology 34: 253—265. Krogh, H. 1939. Osmotic Regulation in Aqua- Tweedie, J. W., and I. H. Segel. 1967. Spec- tic Animals. Cambridge University Press, ificity of Group VI anion transport in London. filamentous fungi. Bacteriological Proceed- Lee, D. L. 1960. The effect of changes in the ings, p. 29. osmotic pressure upon Hammerschmidtiella diesingi (Hammerschmidt, 1938) with ref- Von Brand, T. 1943. Physiological observa- erence to the survival of the nematodes during tions upon a larval Eustrongylides. IV. In- moulting of the cockroach. Parasitology 50: fluence of temperature, pH and inorganic 241-6. ions upon oxygen consumption. Biol. Bull., . 1965. The Physiology of Nematodes. 84: 148-156. Oliver and Boyd, London. . 1960. Influence of pli, ions, and Mueller, J. F. 1929. Studies on the micro- osmotic pressure on life processes in Sasser scopical anatomy and physiology of Ascaris and Jenkins. Nematology. University of lumbricoides and Ascaris megalocephala. Z. North Carolina Press, Chapel Hill. Zellforsch 8: 361-403. Wallace, H. R., and D. N. Greet. 1964. Myers, R. F. 1966. Osmoregulation in Pana- Observations on the taxonomy and biology grellus redivivus and Aphelenchus avenue. of Tijlenchorhtjnchus macrurus (Goodey, Nematologica 12: 579-586. 1932) Filipjev, 1936 and T. icarus sp. nov. Noyes, R. M. 1962. Thermodynamics of ion Parasitology 54: 129-144. hydration as a measure of effective dielec- Weinstein, P. P. 1952. Regulation of water tric properties of water. J. Am. Chem. Soc. balance as a function of the excretory system 84: 513-522. of filariform larvae of Nippostrongijlus muris Osche, G. 1952. Die Bedeutung der Osmoreg- and Ancylostoma caninum. Exptl. Parasit. ulation und des Winkverhaltens fur freile- 1: 363-376. bende Nematoden. Z. Morph. w. Oekol. . 1960. Excretory mechanisms and ex- Tiere 41: 54-77. cretory products of nematodes: an appraisal. Pannikar, N. K., and N. G. Sproston. 1941. In: Host Influence on Parasite Physiology. Osmotic relations of some metazoan para- Ed. Stauber. Rutgers University Press, New sites. Parasitology 33: 214-223. Jersey. Robertson, J. D. 1957. Osmotic and ionic Yamamoto, L. A., and I. H. Segel. 1966. regulation in aquatic invertebrates. Recent The Inorganic Sulfate Transport System of Advances in Invertebrate Physiology. Uni- Penicillin chn/sogenum. Arch. Biochem. Bio- versity Oregon, Eugene. phys. 114: 523-538.

Fine Structure of the Somatic Muscles of the Free-Living Marine Nematode Deontostoma californicum Steiner and Albin, 1933 (Leptosomatidae)

W. DUANE HOPE1 Department of Invertebrate Zoology, Museum of Natural History, Washington, D. C.

The somatic musculature of nematodes is, rum by Hinz (1963) it was demonstrated for in general, comprised of a single layer of longi- the first time that the "contractile fibers" or tudinally oriented, spindle-shaped muscle fi- "contractile ridges" of light microscope observa- bers. Each fiber has a noncontractile region, tions, are comprised of myofilaments. Hinz which gives rise to a "neuromuscular" also mentioned the presence of invaginations process. The contractile region of the fiber of the sarcolemma which we would now recog- is applied to the hypodermis of the body-wall nize to be "T-tubules." But a "T-system," and encloses an array of numerous ribbonlike which is now a well-known structure of verte- bands or "fibers" (Chitwood and Chitwood, brate muscle, was first clearly demonstrated in 1950). Schneider (1860) observed that these nematode muscles by Rosenbluth (1963; ribbons are perpendicular only to that portion 1965a) and Reger (1964) in studies of A. of cell membrane adjacent to the hypodermis himbricoides. They also showed that cisternae in some nematodes, whereas in others they of the sarcoplasmic reticulum are closely asso- may be perpendicular to the membrane at the ciated with the T-tubules forming both dyads base and sides of the cell as well. The former and triads, and that both thick and thin myo- he designated as platymyarian and the latter filaments are present. coelomyarian muscle. Chitwood and Chitwood The arrangement of myofilaments in the (1950) have since described cylindrical muscle type of muscle that occurs in nematodes differs cells in which the ribbonlike bands are per- from that in vertebrate cross-striated muscle. pendicular to the cell-membrane all around the Our understanding of this arrangement began cell, enclosing a central core of sarcoplasm, and with early studies by Kawaguti and Ikemoto which they termed circomyarian. (1958c) on the muscle of Ascaris and on sim- As others have indicated (Hanson and Lowy, ilar muscle occurring in annelids (1957b; 1960), our understanding of the structure of 1958a, cl), molluscs (1957a; 1961) and a the contractile bands and other features of sea squirt (1958b). Their studies revealed nematode muscle has not been advanced sig- that these muscles have in common an oblique nificantly in recent years by light microscope longitudinal striation and a repeating pattern observations. But, recent electron microscope of transverse bands of thick and thin fila- studies have contributed substantially to an ments. Rosenbluth (1963; 1965a) and Ike- understanding of the fine structure of nema- moto (1963) showed that these features were tode muscle. In a study of the coelomyarian due to a uniform arrangement of longitudinally muscles of Ascaris himbricoides by Kawaguti staggered myofilaments in muscle of Ascaris and Ikemoto (1958c) and of Parascaris equo- and an oligochaete, respectively. While these and other studies (Auber-Thomay, 1964; 1 This study was conducted in the Department of Para- sitoloRy, University of Toronto, Toronto, Canada and sup- Jamuar, 1966; Lee and Miller, 1967; Watson, ported by NRC grant A 3757 from the National Research Council of Canada. 1965a; Wright, 1964) have revealed that the

Figure 1. Transverse section of muscle fibers showing noncontractile region (S) bearing the nucleus (N), and contractile regions (C) with horizontal basophilic (A) and nonstained zones (I) and vertical nonstaiiied zones (V). Polyethylene glycol. Haematoxylin. X 2,150. Figure 2. Transverse section. Dark stained material around the nucleus (N) is glycogen (G); that between cells is the glyco-protein of the sarcolemma (M). Paraffin. PAS. X 2,150.

arrangement of myofilaments is basically the water and then placed in a Bureau of Plant same in most nematode muscle, they have also Industry Dish filled with a 2.5% aqueous solu- disclosed deviations in the extent of develop- tion of polyethylene glycol 400. The dish was ment and form of the "electron dense material" covered with a cover glass to extend evapora- and sarcoplasmic reticulum, and in the pres- tion of the water over a period of about one ence or absence of a T-system. week, in much the same way that nematodes The above studies constitute a significant are conventionally dehydrated in glycerine. advance in our understanding of nematode This procedure, in lieu of passing the nema- muscle, but further studies are desirable to todes through a graded polyethylene glycol understand not only the structural relationship 400 series, eliminates collapsing of the body between platymyarian, coelomyarian, and cir- and minimizes distortion of tissues. After com- comyarian muscle of nematodes, and between pleting the embedding as recommended by nematode muscle and obliquely striated and Riopel and Spurr, the nematodes were sec- double obliquely striated muscle of other in- tioned at 5 /JL on a rotary microtome and stained vertebrates, but the significance of variations for 5 minutes in 0.1% aqueous hematoxylin at of the sarcoplasmic reticulum and "electron pH 2.4 by the method of Craig and Wilson dense material." In view of this, light and (1937). electron microscopy have been employed in a Fasted and nonfasted specimens to be ex- study of coelomyarian muscle of the free-living amined for the presence of glycogen were fixed marine nematode, Deontostoma californicum in Rossman's picric acid-alcohol—formalin fixa- Steiner and Albin, 1933, and the observations tive (Gray, 1954), embedded in paraffin, sec- are compared with those from previous studies tioned at 10 fj., and stained by the periodic on nematodes and higher metazoans. acid-Schiff method. Control sections were treated with ptyalin. Materials and Methods ELECTRON MICROSCOPY: Whole specimens Specimens were collected from sediment re- were fixed at room temperature for 20 min- moved from holdfasts of the alga Egregia sp. utes in a mixture of 1 volume of Acrolein and from intertidal rocks at Dillon Beach, Marin 9 volumes of M/10 solution of Sorensen's phos- County, California. All nematodes were re- phate buffer adjusted to pH 7.4. tained in sea water at 15 C until fixed; some They were then rinsed in the buffer and cut were fixed within 24 hours while others were into pieces approximately 2 mm long in 1% kept in sea water at 15 C for up to one month. OsO4 in veronal acetate buffer (pH 7.4) con- LIGHT MICROSCOPY: Specimens used for taining sucrose (Palades sucrose fixative) and general histological examination were fixed post-fixed in the same solution for 1 hour. whole for 24 hours in 10% formalin in sea After dehydration in cold ethanol, the tis- water. To avoid distortions that usually occur sues were embedded by the method of Wright during paraffin embedding, the specimens were and Jones (1965) in a mixture of 9 volumes embedded in a 39 to 1 mixture of polyethylene of Maraglas 655, 1 volume of Cardolite Nc glycol 1540 and 4000, respectively, by the 513, and 0.25 volumes of DMP-30 (Freeman following modification of the method of Riopel and Spurlock, 1962). and Spurr (1962). Fixed specimens were Sections were cut on LKB and Porter Blum washed for approximately 30 minutes in tap ultramicrotomes, mounted on Formvar or

Figure 3. Parasagittal section of a muscle fiber. Basophilic zones (A). Nonstained zones (I). Spindle- shaped profiles (Z). Polyethylene glycol. Haematoxylin. X 2,800. Figure 4. Electron micrograph of glycogen particles in perinuclear sarcoplasm of nonfasted specimen. Lead hydroxide. X 91,000. Figure 5. Electron micrograph of cross-sectioned contractile region showing various profiles of thick zone (A and I Bands) material. Note the presence of glycogen particles (G) in the Z planes (ZP). Lead hydroxide. X 33,800.

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Formvar and carbon coated grids, stained with the nonstained zones, thus resembling similar Watson's (1958) saturated lead hydroxide or structures appearing in longitudinal and ob- with permanganate and lead citrate (B. L. lique sections of Ascaris (see Rosenbluth, Soloff, personal communication), and exam- 1965a). ined with a RCA EMU-3E (operated at 50 kv) The entire fiber is enclosed in a PAS posi- or Zeiss EM 9 electron microscopes. tive sarcolemma (Fig. 2) which remains reac- tive after ptyalin digestion. Observations ELECTRON MICROSCOPY: The sarcoplasm of LIGHT MICROSCOPY: The body wall muscu- the noncontractile region of muscles in non- lature of D. califo-rnicum consists of a single fasted specimens contains a high density of layer of longitudinally oriented, spindle-shaped lead-stained particles 100—230 A in diameter muscle fibers. In cross-section, an average of (Fig. 4). These are surmised to be particles 24 fibers appear in each midbody quadrant. of the beta form of glycogen because of their The more obvious features of each fiber in- size, form, and staining properties, and also clude the enlarged, noncontractile region, their distribution is coincident with the PAS which extends into the pseudocoelom, and a positive, ptyalin-digestable substance observed relatively slender contractile region, the base in light microscope preparations. Glycogen of which is adjacent to the hypodermis (Fig. 1). particles may also occur in the contractile por- The noncontractile region contains a nucleus tion of fibers (Fig. 5). Alpha particles oc- and substantial quantities of PAS positive ma- curred infrequently in all sections examined, terial (Fig. 2), which is removed by ptyalin and some specimens maintained in sea water digestion. From these observations, and those for as little as one week without food, con- made with the electron microscope (see be- tained little glycogen in either the noncontrac- low), it is concluded that this material is tile or contractile regions (Fig. 6). glycogen. Mitochondria occur in the noncontractile The contractile portion of the fiber, when region and occasionally were observed subja- viewed in cross-section, appears to be com- cent to the cell membrane in the contractile prised of basophilic zones which usually pass region as well. Slender strands of presumably uninterrupted from one side of the fiber to the noncontractile filaments and vesicles bound by other. These zones are separated from one smooth membranes are also evident in non- another by nonstained zones that also pass contractile regions and more readily observed from one side of the fiber to the other (Fig. 1). in specimens depleted of glycogen. Golgi zones Sometimes, however, nonstained zones occur were not observed. in or near the sagittal2 plane of the muscle Low magnification electron micrographs of fibers as well, extending varying distances cross-sectioned muscle fibers reveal, in the from the perinuclear sarcoplasm toward the contractile region, an arrangement of wide base of the fiber and separating the stained transverse zones containing small circular pro- zones of this region into right and left halves files of cross-sectioned filaments and, alternat- (Fig. 1). The vertically oriented nonstained ing with the above zones, much narrower zones zones resemble the sarcoplasmic core of Ascaris containing bars of electron dense material (Fig. muscle, but differ in being narrower, not ex- 6). This alternating pattern superficially re- tending as far toward the base of the fiber, and sembles the stained and nonstained zones of in appearing less frequently. light microscope observations (Fig. 1), but the It is evident from parasagittal sections of difference between the widths of the stained the fiber, that the basophilic and nonstained and nonstained zones is not nearly so great as zones described above are ribbonlike and ex- that between the wide and narrow zones of tend over much of the length of the fiber (Fig. electron microscope observations. Commonly, 3). In the same sections, small spindle-shaped the wide zones extend uninterrupted from one profiles are spaced at rather regular intervals in side of the fiber to the other, each appearing in cross-section as a broad rectangle with its 2 Sagittal (para-) is used here to indicate longitudinal longer sides parallel to the base of the fiber. sections whose plane extends from the base of the fiber to the apex; frontal sections are also longitudinal with the Occasionally, in the apical region of the fiber, plane extending between the right and left sides of the fiber. each wide zone may be intercepted by vertical

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 15 m . •:'/•'•'. 'it'- ' -'• .*. - * -^feir'x^i^.t^i^V'-. »'**-. >•*."','" *:^^*''' ':-'"'• '"•

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Figure 6. Electron micrograph of cross-sectioned muscle showing wide zones (W) and narrow zones (ZP) with bars of electron dense material (Z). Note absence of glycogen in sarcoplasm at upper left and vertical Z plant at right (V). Permanganate and lead citrate. X 25,500.

Copyright © 2011, The Helminthological Society of Washington 16 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY narrow zones located near the sagittal plane of was not observed, nor did there appear to be the fiber (Fig. 6) resembling the vertical non- a regular arrangement of thin filaments around stained zones from light microscope observa- the thick. Thin filaments extend to the margin tions. When separated in this manner, the of the narrow zones and here they frequently wide zones on one side of the fiber are usually appear to be segregated in small clusters (Fig. at the same level as wide zones of the opposite 7) much as has been found in the case of side; less frequently are wide zones opposite Ascaris lumbricoides (see Rosenbluth, 1965a). narrow zones. Sometimes, several irregularly From parasagittal sections it is evident that oriented narrow zones divide wide zones into the wide zones extend uninterrupted for con- profiles of various shapes, including circles siderable distances through the fibers in a (Fig. 5) and triangles, immediately basal to longitudinal direction, slightly oblique to the the nuclear region and at the base of the cell longitudinal axis of the fiber. The bands of as well. wide zones are not nearly as distinct in these Examination of cross-sections at higher mag- sections as in those that are transverse, but nifications shows that the wide zones are com- it is at least possible to identify the A bands posed of two sizes of myofilaments, the thicker (Fig. 8). with a maximum diameter of about 200 A, and The narrow zones are characterized at low the thin about 60 A. These filaments are dis- magnifications by bars of electron dense ma- tributed so as to form five transverse bands in terial, the extent of which varies from almost each thick zone. That is, there are two outer none in some narrow zones to uninterrupted bands of thin filaments (Fig. 7, i); medial to bars passing from one side of a fiber to the these are two comprised of both thick and thin other. Most frequently, however, the dense filaments (Fig. 7, a); and in the median plane material appears one to several times for of each thick zone, a single band of thick fila- short distances in cross-sections of narrow ments only (Fig. 7, h). This pattern is char- zones and often appears closely applied to, or acteristic of obliquely striated muscle (Ike- in some way fused with, the plasma membrane moto, 1963; Rosenbluth, 1963; 1965a) and at the sides of the fiber (Fig. 6). the bands are comparable to the I, A, and H In longitudinal sections, the electron dense bands, respectively, of vertebrate striated mus- material is apparent as a series of spindle- cle. I bands occur on both sides of thin zone shaped profiles regularly spaced in a longi- material regardless of the orientation of the tudinal plane that is slightly oblique to the latter. The filaments of the I bands do not longitudinal axis of the fiber. Their periodicity stain with hematoxylin as employed in this is about 2.0 ^ which agrees very closely with study, and so the nonstained zones described comparable measurements made from photo- above correspond to the I bands and thin zones micrographs. A narrow strand, presumably a as well, which they flank. Thus, the nonstained bundle of thin filaments, appears to extend zones are not wholly comparable to the thin from each end of each spindle, and merge with zones of electron microscope observations. the filaments of the I band (Fig. 8), as has Thick filaments closest to the Z bands are been observed in the case of Nippostrongyhis slightly smaller in diameter than those of the muscle (Jamuar, 1966). Consequently, the H band and, therefore, the thick filaments are filaments from one end of a spindle pass into assumed to be slightly tapered as in Ascaris the I band below, and those from the opposite (see Rosenbluth, 1965a). Cross-linking be- end pass into the I band above the spindle. tween thick and thin filaments in the A bands This apparent continuity between electron

Figure 7. Cross-sectioned muscle showing thick (T) and thin (t) filaments arranged into I, A, and H bands. Note clusters of thin filaments (t') at the margin of the Z plane (ZP). Permanganate and lead citrate. X 81,900. Figure 8. Electron micrograph of a parasagittal section showing the thick filaments (T) of the wide zones (A bands) and what are interpreted to be clusters of thin filaments (t') continuous with cross- sectioned profiles of Z bars (Z). Permanganate and and lead citrate. X 34,400.

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dense material and I band filaments suggests Furthermore, tubules and vesicles associated that at least a portion of the electron dense with the Z plane proper have a similar electron material is a Z component comparable to the translucent medium within them. Z band material of vertebrate striated muscle. Fingerlike projections of the vesicles may For this reason, and for reasons given in the extend into the I band (Figs. 9, 10) where, in discussion, the bars of electron dense material cross-section, they appear as small circular will be referred to here as Z bars, and the profiles (Fig. 12). Therefore, the sarcoplasmic oblique plane bearing them, the Z plane. reticulum is not entirely restricted to the Z Frontal sections of muscle fibers disclose that plane. In no instance was it possible to iden- the Z planes also bear fibrils oriented at right tify dyads or triads within the Z plane, al- angles to the myofilaments. In some instances though vesicles (diads) occur subjacent to the it appears that the fibrils are adjacent to, but lateral sarcolemma (Figs. 13, 14). The long not enmeshed with the Z bars (Fig. 9), while axis of the vesicles, measured from cross-sec- in other cases these components do appear to tions of the fiber, has an average length of be joined with each other (Fig. 10). It is also 0.34 fji and the short axis, 0.12 ^. The average possible, however, that the latter may be an distance separating the plasma membrane from illusion attributable to superposition. the membrane of the vesicle is 120 A. In some Sarcoplasmic reticulum in the form of nar- instances the lumen of the vesicle is completely row tubules and dilated vesicles also occurs filled with a rather electron dense, granular consistently in Z planes. The tubules are usu- material (Fig. 14), while in others it may have ally closely applied to the surface of the Z bars relatively little (Fig. 13). The association be- so as to separate the latter from the I bands; tween the vesicles and the plasmalemma is in fact, wherever a Z bar is present in cross- here regarded as a diad and is in accord with sections, at least one and usually both of its similar views stated by Rosenbluth (1968) surfaces bear sarcoplasmic reticulum (Figs. regarding a comparable structural arrangement 11 and 12). Tubules of sarcoplasmic reticulum in Ghjcera muscle. do not alternate with Z bars in the Z plane as A T-system was not observed in this muscle, they do in Ghjcera (see Rosenbluth, 1968). but two types of shallow invaginations of the Seldom is the lumen of the tubules more sarcolemma do occur and both have asym- than 400 A wide and often it is so narrow that metrical unit membranes. The first type of a lumen is hardly perceptible (Figs. 11, 12). invagination (Fig. 15) more closely resembles Vesicles, on the other hand, commonly have a the T-tubules of Ascaris (Reger, 1964; Rosen- diameter approximately equal to the width of bluth, 1965a; and Watson, 1965b) in that the narrow zone (Figs. 12, 13). In the Z plane they have been observed only at the level of they occur most frequently in those areas de- the Z planes and have a diameter comparable void of Z bars. Continuity between the tubules to that of Ascaris T-tubules. They differ, how- and vesicles is clearly evident in numerous ever, in being very infrequent, shallow, and instances (Fig. 12), and tubules from both sarcoplasmic reticulum has never been ob- surfaces of the dense material may have con- served near them as it occurs in diad and triad tinuity with a common vesicle (Fig. 12). Al- formations of Ascaris. The second occurs in though it was not possible to resolve the unit both the noncontractile and contractile regions membrane structure of the sarcoplasmic retic- of the fibers. It is characteristically a very ulum, or to measure accurately its membrane narrow infolding, the opposing membranes width, membranes enclosing vesicles do not separated by a uniform distance of 80 A. The appear to differ from those enclosing tubules. infoldings may be rather extensive, occasionally

Figures 9 and 10. Electron micrographs of frontal sections demonstrating portions of Z bars (Z) and thin filaments (t) which become continuous (f) with them. Filaments (F) presumed to be of the cyto-skeletal system, traverse the Z plane at nearly right angles to the thin actin filaments. Note tubules and vesicles of sarcoplasmic reticulum (SR) extending into the I bands. Permanganate and lead citrate. Figure 9, X 80,800. Figure 10, X 81,700.

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Copyright © 2011, The Helminthological Society of Washington 20 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY appear to ramify, and always remain close to other tissues including the gut which gave the cell surface (Fig. 16). They have never the next most intense reaction. Large deposits been, observed to extend between bands of of glycogen have been found in A. lumbricoides filaments, but may pass between filaments and Dirofilaria immitis (Rosenbluth, 1965b; and subsarcolemmal cisternae, although in this Lee and Miller, 1967). Rosenbluth has pointed instance, the distance between the membranes out that the noncontractile region of the muscle of the subsarcolemmal cisternae and the in- may serve as a glycogen storage depot which foldings may be less than that between the may sustain the parasite during periods in former and the cell surface. which its host is fasting. This is very likely The sarcolemma of Deontostoma muscle the case, but because of comparable deposits consists of a cell membrane 100 A wide and of glycogen occurring in D. californicum, a an extracellular coating 1.5 ^ wide. It is likely free-living species, it is apparent that storage that this coating is responsible for the PAS of glycogen in the muscle belly is not strictly positive staining both before and after ptyalin an adaptation to meet the vicissitudes of digestion, and is a form of glycocalyx which parasitism. invests cells of many types (Fawcett, 1966). CONTRACTILE REGION: Differences between Unlike Ascaris, however, it is not comprised Deontostoma muscle and the muscle of other of bands or lamellae. nematodes are to be found among components Wright (1966) reported the occurrence of of the Z plane, namely the electron dense ma- cytoplasmic bridges between muscles of Deon- terial or Z bars and the sarcoplasmic reticulum. tostoma californicum which were again ob- This electron dense material may extend served in this study. uninterrupted for considerable distance across the width of a fiber or may appear intermit- Discussion tently in the same Z plane. In photomicro- Light and electron microscope studies of graphs and electron micrographs of parasag- D. californicum reveal that its muscles have ittal sections of Deontostoma muscle, it can the usual noncontractile portion which is lobe seen that the obliquely regimented pro- cated along the pseudocoelomic side of the files of the dense material occur with regu- fibers and a more slender contractile region larity that suggests they may be an integral adjacent to the hypodermis. part of the staggered bands of myofila- NONCONTRACTILE REGION: No attempt has ments. Indeed, it can be seen at higher mag- been made to study this region of the muscle nifications of the same sections (Fig. 8, Z) in detail, but it is worth noting that it con- that each profile is tapered and appears to tains the nucleus, mitochondria, noncontractile be continuous with thin filaments of adjacent fibrils, sarcoplasmic reticulum, and glycogen I bands. For these reasons it is suggested that particles as has been reported for other species at least a part of the electron dense material (Wright, 1964; Reger, 1964; Rosenbluth, of this muscle is Z material; comparable to the 1965b; Lee and Miller, 1967). The abun- constituents of Z bands in vertebrate muscle. dance of glycogen is particularly impressive as A second component in the Z bars is evidenced the intensity of the PAS reaction was much by frontal sections in which filaments (Figs. greater in somatic muscle than in any of the 9 and 10, F), presumably of the cyto-skeletal

Figures 11 and 12. Electron micrographs of cross-sectioned muscle cell showing Z bars (Z) and tubules and vesicles of sarcoplasmic reticulum (SR). Note continuity between tubules and vesicles. Lead hydroxide. X 76,340. Figure 13. Electron micrograph of cross-sectioned muscle fibers demonstrating an enlarged vesicle of sarcoplasmic reticulum (SR) closely applied to the plasma membrane (P) forming a diad at the level of the Z plane (ZP). The vesicle is partially filled with granular material. Lead hydroxide. X 97,400. Figure 14. Same as Figure 13, but the diad is at the level of an A band with a tubule directed into the Z plane. The vesicle is filled with granular material. Permanganate and lead citrate. X 156,000.

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Copyright © 2011, The Helminthological Society of Washington 22 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY system, are observed traversing the Z plane appears that there are aggregates of thin fila- at right angles to the longitudinal axis of the ments in the Z plane (Lee, 1966) and that fiber. These filaments were observed infre- clearly these are bundles of thin filaments con- quently which suggests that they either occur tinuous with the Z component (Jamuar, 1966). infrequently or that, if they are a common By this interpretation, the structure in Ascaris, component of Z bars, they are fused or en- designated by Rosenbluth as "Z bundles" and meshed with the Z component and, therefore, "ovoid dense bodies," may in reality be aggre- difficult to identify. gates of converging or diverging thin fila- Z bars, or their counterpart have not been ments and ovoid Z bodies, respectively. described in detail for ncmatodes except in From their study on DirofiJaria immitis the case of Ascaris. According to Rosenbluth muscle, Lee and Miller (1967) concluded (1965a) the dense bands (Z planes) in Ascaris that what had been termed "supporting contain "bundles of fibrils" (presumably of the fibers" in the myofibrillar and amyofibrillar cyto-skeletal system) which appear in cross- area of nematode muscle, are identical with section as tightly packed ovoid "dense bodies" the Z bundles of Rosenbluth (1965a). But and a second component, which he designates evidence of fibrils traversing the Z plane at as "Z bundles," comprised of "small, sheaflike right angles to the myofilaments in Deonto- aggregates" of the thin filaments in which the stoma muscle, supports earlier concepts of a latter "appear to be linked together." These system of cytoskeletal fibrils separate and dis- bundles occur not only at the edges of the I tinct from the contractile filaments. zones but within it as well. He further states Whereas in Ascaris the Z junctures are in that "thin filaments sometimes seem to join the the form of an ovoid dense body, and barlike dense bodies" and later (1967), without fur- in Deontostoma, they apparently have the form ther comment, states that the "thin filaments of dense thickenings subjacent to the plasma insert into opposite ends of the dense bodies." membrane in Capillaria hepatica muscle stud- From these comments it is not entirely clear ied by Wright (1964). The distribution of whether in Ascaris muscle there are Z bundles these dense areas along the base and sides of in addition to dense bodies, or whether the the fibers suggests that it is coelomyarian, but dense bodies themselves are the Z junctures highly specialized, and possibly degenerate. between "sarcomeres." The fact that the Z juncture is plaquelike and What are interpreted to be aggregates of limited to the periphery of the cell, not extend- thin filaments occur in the Z plane of Deonto- ing across the fiber as a series of dense bodies stoma muscle (Fig. 7, t'), as in Ascaris. How- or a bar, may explain the absence of distinct ever, it is suggested that these are not Z transverse bands of thick and thin filaments.3 bundles, but are aggregates of thin filaments The Z junctures in Nippostrongylus brasil- grouped together as they converge upon (or emerge from) the Z bars. A similar situation 3 Wright (1964) reported myofilaments of but one diameter, but disclosed in personal communication that he may exist in Nippostrongylus muscle, since it has since found thin filaments as well.

Figure 15. Cross-section of a fiber demonstrating one of the few observed examples of relatively broad invaginations of the sarcolemma located only at the level of the Z plane. Permanganate and lead citrate. X 131,200. Figure 16. Cross-section of a fiber with examples of the numerous narrow invaginations of the sarcolemma that occurred in contractile and noncontractile regions of the fiber. Permanganate and lead citrate. X 98,200. Figure 17. Composite illustration of the Z plane of Deontostoma muscle. Electron dense Z material presumably with incorporated filaments of cytoskeletal system (Z); vesicles and tubules of sarcoplasmic reticulum (SR). Figure 18. Composite illustration of the Z plane of Ascaris muscle based on the authors interpretation of electron micrographs published by Rosenbluth (1965A; 1967). T-tubule (T); electron dense Z material with fibrils of cytoskeletal system (Z); sarcoplasmic reticulum (SR); and filaments of the cytoskeletal system (F).

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Copyright © 2011, The Helminthological Society of Washington 24 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY ien-sis muscle are also of interest, not because other, is that the former appears to have a of the shape of the juncture itself, which ap- component of cytoskeletal fibrils which have pears to be rodlike, but in that they are not been observed in the latter. Otherwise, the oriented at right angles to the plasma mem- Z structures seem to be quite comparable in brane at the base of the fiber instead of at the obliquely and double obliquely striated muscle, sides and are, therefore, platymyarian. The and it seems they are also comparable to the significance of this is discussed below with Z bands of vertebrate cross-striated muscle. regard to oblique striation. SARCOPLASMIC RETICULUM: The sarcoplas- The above variations in the Z component of mic reticulum of the Z plane of Deontostoma nematode muscle, with the possible exception muscle is extensively developed while a T- of C. hepatica, can also be found among the system is lacking or poorly developed. In obliquely striated and double obliquely striated Ascaris, on the other hand, the sarcoplasmic muscle of annelids and molluscs. The Z com- reticulum is not as extensive and a T-system ponent appears to have the general shape of is well developed. These differences are mani- an ovoid structure in the heart muscle (J-gran- fest in the basic arrangement of Z plane com- ules; Kawaguti, 1963), and mantle muscle of ponents. Transverse sections of Deontostoma cuttlefish (J-particles; Kawaguti and Ikemoto, muscle fiber disclose that, when all components 1957a), and is more rocllike in leeches (J-rods, of the Z plane co-exist, the electron dense compact sarcotubules and cross filaments; material is medial in the Z plane and the nar- Kawaguti and Ikemoto, 1958a; Pucci and row membrane bound cisternae are peripheral Afzelius, 1962; Rohlich, 1962), polychaetes to it. Diad or triadlike formations were never (J-rods; Z lines; Kawaguti and Ikemoto, 1958d; observed in the Z plane (Fig. 17). In Ascaris, Rosenbluth, 1968) and oligochaetes (J-rods; by contrast, the medial region of the plane is Ikemoto, 1963). But in all instances these occupied by a relatively broad membrane- structures are located in what is comparable bound structure (T-tubule) with the dense to the Z plane of Deontostoma muscle, and, in material and narrow cisternae of sarcoplasmic many instances, there is evidence that thin reticulum (which are the lateral elements of filaments are continuous with them (Kawaguti the diads and triads) displaced to the periph- and Ikemoto, 1957a, b; 1958a, b, c; Rohlich, ery of the Z plane (Fig. 18). Commonly, rela- 1962; Rosenbluth, 1968; Ikemoto, 1963). tively broad vesicles limited by membranes At present it seems that the greatest differ- (which are sarcoplasmic reticulum) may oc- ence between Z structures of nematode muscle, cupy the entire width of the Z plane in Deon- on the one hand, and those of the obliquely tostoma muscle, superficially resembling the striated muscles of higher metazoans on the T-tubules of Ascaris. However, when this

Figure 19. Cut-away diagram of platymyarian muscle fiber depicting the patterns of oblique I and A bands (H bands not shown) and Z planes. Based on the authors interpretation of electron micrographs published by Lee (1966) and Jamuar (1966). Figure 20. Cut-away diagram of a coelomyarian muscle cell showing the pattern of double oblique striation (partial helix) that would result if the platymyarian arrangement of oblique bands were extended onto opposing sides of the fiber. Based on the authors interpretation of electron micrographs published by Rosenbluth (1965A; 1967). Figure 21. Cut-away diagram of circomyarian muscle fiber showing double oblique striation that would result if the platymyarian arrangement of oblique striations were extended around the entire periphery of the fiber forming complete helices. Inferred from authors interpretation of data by Chitivood (1951), Lee (1966), Jamuar (1966), and Rosenbluth (1965A; 1967). Figure 22. Enlarged portion of comparable contractile region bracketed on each of the cut-away diagrams. The same sequence of H, A, and I band and Z planes occurs in all obliquely and double obliquely striated muscle. Figure 23. Cut-away diagram of Deontostoma muscle showing the probable arrangement of bands and Z planes inclined in the same direction and at the same angle in both right and left sides of the fiber.

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situation exists, electron dense material and therefore, move rather infrequently with slow membrane-bound vesicles are not peripheral cycles of contraction and relaxation. to it. That these dilated vesicles are com- A system of sarcoplasmic reticulum similar ponents of the sarcoplasmic reticulum is fur- to that in Deontostoma muscle is more common ther evidenced by their continuity with the in annelid muscle, having been reported in the much narrower cisternae. Continuity between case of Glycera muscle (Rosenbluth, 1968), the sarcolemma and either the vesicles or Eisenia muscle (Ikemoto, 1963), Lumbricus cisternae was never observed and, since the muscle (Heumann and Zebe, 1967), and leech plasma membrane of the fiber has asymmet- muscle (Pucci and Afzelius, 1962). However, rical unit membrane structure, while unit mem- in these annelids the tubules of sarcoplasmic brane structure of the sarcoplasmic reticulum reticulum are not in direct association with the could not be resolved, it seems unlikely that Z bar as they are in Deontostoma, but occur in any of the membrane-bound vesicles within the Z plane midway between each pair of Z the Z plane are derived from the sarcolemma. bars. This differenece may have some rele- Rather, it is interpreted that the membrane- vance to speed of contraction and relaxation. bound structures within the Z plane and sub- OBLIQUE AND DOUBLE OBLIQUE STRIATION : All jacent to the sarcolemma are components of muscles with longitudinally staggered bundles the sarcoplasmic reticulum and the vesicles of myofilaments (obliquely striated) appear to lying adjacent to the sarcolemma, situated have similar organization and structure whether either above or below Z planes, are associated from representatives of the Nemata, Annel- with the sarcolemma so as to form diads. ida, or Mollusca. However, there are repre- Sarcoplasmic reticulum in the form described sentatives of the latter two taxa for which for Deontostoma has not been previously de- double oblique striation has been clearly dem- scribed for other nematodes, but it appears onstrated, namely the clamworm (Kawaguti that the "membrane units" described for Ca- and Ikemoto, 1958d), tellinid clam (Kawaguti pillarla by Wright (1964) may also be sub- and Ikemoto, 1961) and the annelid Myxicola sarcolemmal cisternae or diads without in- sp. (Hanson and Lowy, 1960). In these wardly directed tubules. Nippostrongylus examples, bands of myofilaments occur on brasiliensis muscle may also be similar to that opposing sides of the cell and the bands on of Deontostoma with respect to the absence one side are oblique in a direction opposite to of a T-system and greater development of those of the other side. sarcoplasmic reticulum, while Euchromadora Patterns of double oblique striation have not vulgaris and Dirofilaria immitis seem to more been reported for nematode muscle, but in con- closely resemble Ascaris muscle in this regard. sidering the following information from light The function(s) of sarcoplasmic reticulum and electron microscope studies, it seems quite is, as yet, not well understood, but one pos- possible that patterns of double oblique stria- tulate, which has been reviewed by Porter tion may occur in at least certain types of (1961), states that it may be involved in nematode muscle. From light microscopy it is muscle relaxation, by uptake of calcium, by known that "contractile ridges" (bands of myo- production of a "relaxing factor," or both. filaments) are only at the base in platymyarian With regard to this theory, it is presumed that (Fig. 19), at the base and sides in coelo- hypertrophy of the sarcoplasmic reticulum is myarian (Fig. 20) or around the entire periph- directly related to rapid cycles of contraction ery enclosing the medial sarcoplasm in cir- and relaxation. If this theory proves to be comyarian muscle cells (Fig. 21). Further, correct, it may explain the relatively greater platymyarian muscle cells tend to be flattened, abundance of sarcoplasmic reticulum in Deon- that is as wide, if not wider, than tall (Fig. tostoma, which is a free-living nematode that 19); those that are coelomyarfan are usually presumably moves about in search of food by taller than wide with an enlarged perinuclear means of frequent cycles of muscle contraction region at the apex and a narrower base con- and relaxation, and less sarcoplasmic reticulum taining the myofilaments (Fig. 20); and in adult Ascaris, which retains itself in the circomyarian fibers are cylindrical (Fig. 21). lumen of the gut of its host by bracing against Many nematode muscle cells tend to have an the gut wall (Makidono, 1956), and may, organization and shape intermediate to those

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 27 assigned to these categories. From the illus- It is of interest to note further, however, that trations in Chitwood and Chitwood (1950) because the striations are inclined in opposite it appears that the somatic muscles of Ethmo- directions on opposite sides of the fiber in laimiis revaliensis, are very low coelomyarian to the above-suggested situations, each of the platymyarian while those of Theristus setosus bands on one side, in cross section, cannot are higher coelomyarian tending toward circo- always be on the same level with a compa- myarian. Some muscle fibers, such as those of rable band of the opposite side. In other words Ascaris, are coelomyarian in the midregion and the bands of one side will be out of register circomyarian toward their ends. Furthermore, with those of the other. An example of this, according to Chitwood and Chitwood (1950), and in which double oblique striation has been Martini has shown that some nematodes are demonstrated as well, is to be found in the platymyarian as first stage juveniles, and later adductor muscle of the tellinid clam (Kawaguti develop coelomyarian muscle. The above ob- and Ikemoto, 1961). servations suggest a similar structural relation- From electron micrographs of cross sections ship between the muscles of these categories. of Ascaris muscle (Rosenbluth, 1965a) it ap- From electron microscope observations of pears that the bands of one side are out of cross-sectioned fibers it is known that the same register with those of the other which sug- sequence of H, A, and I bands and Z planes gests the possibility of double oblique striation occur in coelomyarian muscle (Rosenbluth, in the muscles of this species. However, this 1963, 1965a; Reger, 1964; Watson, 1965a; is contrary to the situation in Deontostoma, and Lee and Miller, 1967) as well as platy- where in cross section the majority of the myarian muscle of Nippostrongylus brasiliensis bands on one side of the fiber are precisely on (see Jamuar, 1966) and that all bands and Z the same level with a comparable band of the planes are at right angles to the cell membrane other side giving the appearance of continuity (Fig. 22). Therefore, the longitudinal stria- the full width of the fiber (Figs. 1, 2, 23). A tions of platymyarian muscle, if viewed from similar arrangement exists in Glycera muscle the base or other appropriate angle, would (Rosenbluth, 1968). This "in register" con- appear to have slightly oblique orientation dition is here interpreted to indicate that the from the right side of the fiber to the left or bands are not only inclined at the same angle, vice-versa (Fig. 19). If exactly the same pat- but in the same direction and each myofila- tern of striations were formed adjacent to a ment band would give the appearance of a cell membrane that was not broad at its base, lamella that spans the full width of the fiber but curved or folded so as to appear U-shaped and extends obliquely from the apex to the in cross section with continuous bands occur- base of the fiber (Fig. 23). Even where ring on both sides of the fiber as \vell as at separated by a sagittal Z plane, the bands on the narrower base (coelomyarian), then the one side are usually "in register" and parallel same pattern of striations would pass from with those of the opposite side. Therefore, near the apex to the base on one side, continue even under favorable conditions, double across the base, and upward toward the apex oblique striation most likely could not be of the opposite side (Fig. 20). If these as- demonstrated in Deontostoma muscle. sumptions are correct, then I, A, and H bands What significance these considerations have and Z planes would form partial helices in cannot be fully appreciated at this time, but coelomyarian muscle and if the same pattern differences may be relevant to the function of were to occur in circomyarian muscle, com- nematode muscle and the relationships of plete helices would be formed (Fig. 21). If nematode taxa. Perhaps functional implica- fibers of either of these two types were ob- tions will not be as readily apparent and may served in longitudinal optical section with be appreciated only as more is learned regard- sufficient depth of focus to simultaneously ob- ing the mechanics of nematode muscle con- serve the bands and Z plane on both sides of traction. But, on the other hand, if subsequent the fiber superimposed on one another, it investigations disclose that somatic muscles would have the appearance of double oblique now collectively designated as coelomyarian striation (Figs. 20, 21) comparable to that of prove to be a composite of two types, one annelids and molluscs. helical or double obliquely striated and the

Copyright © 2011, The Helminthological Society of Washington 28 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY other bilaterally parallel, and if all members ing this study, and especially to Dr. Wright of the order Enoplida (to which Deontostoma for his invaluable guidance. I also wish to belongs) have muscle of the former type and thank Dr. Rosenbluth for his helpful review of those of Ascarida (to which Ascaris belongs) the manuscript and the very capable Mrs. the latter, then these differences will give fur- Carolyn Cast for preparing the illustrations. ther justification for the wide separation between these two taxa in the taxonomic hier- Literature Cited archy of nematodes and may provide further Auber-Thomay, Michele. 1964. Structure et clues to phylogenetic relationship within the innervation cles cellules musculaires de nema- Nemata. todes. Microscopie 3(1): 105-113. Chitwood, B. G., and Maybelle B. Chitwood. Summary 1950. An Introduction to Nematology. Sec- tion I, Anatomy. Monumental Printing Co., The structure of somatic muscles of Deonto- Baltimore. 213 pp. stoma californicum is described from light and Craig, B., and C. Wilson. 1937. The use of electron microscope observations. The non- buffered solutions in staining: theory and contractile region bears essentially the same practice. Stain. Technol. 12(3): 99-109. organelles as described for previously studied Fawcett, D. W. 1966. An Atlas of Fine nematodes and contains considerable quanti- Structure. The Cell, Its Organelles and In- clusions. W. B. Saunders, Philadelphia and ties of glycogen. London. 448 pp. The basic organization of the contractile Freeman, J. A., and B. O. Spurlock. 1962. region is that of obliquely striated muscle with A new epoxy embedment for electron micros- characteristic repeated patterns of H, A, and I copy. J. Cell Biol. 13: 437-443. bands and Z components and resembles the Gray, P. 1954. The Microtomists' Formulary obliquely striated muscles of other nematodes, and Guide. Blakiston Co., Inc., New York. annelids, and molluscs. Deontostoma muscle 794 pp. differs from other nematode muscle in that its Hanson, Jean, and Jean Lowy. 1960. Struc- Z components appear barlike, and a well- ture and function of the contractile apparatus in the muscles of invertebrate animals. In developed T-system is absent. Furthermore, it Bourne, G. H. The Structure and Function has a well-developed system of sarcoplasmic of Muscle I. Structure. Academic Press, reticulum consisting of tubules and vesicles in New York and London. 472 pp. the Z plane which are continuous with diads. Hinz, E. 1963. Elektronenmikroskopische Un- Functional implications of the differences in tersuchungen an Parascaris equorum (Integ- the extent of the development of sarcoplasmic ument, Isolations-gewebe, Musculature und reticulum are briefly reviewed. Nerven). Protoplasma 56(2): 202-241. In Deontostoma muscle the continuity of Z Heumann, H. G., and E. Zebe. 1967. tJber components and myofilament bands from one Feinbau und Funktionsweise der Fasern aus dem Hautmuskelschlauch cles Regenwurms, side of the fiber to the other suggests that each Lumbriciis terrestris L. Z. Zellforsch. 78: band forms lamellae which span the full width 131-150. of the fiber and extend obliquely from the Ikemoto, N. 1963. Further studies in electron apex to the base of the fiber. Therefore, the microscopic structures of the oblique-striated organization of Deontostoma muscle is such muscle of the earthworm, Eisenia foetida. that it could not display double oblique stria- Biol. J. Okayama Univ. 9(3-4): 81-126. tions possibly present in circomyarian and Jarnuar, M. P. 1966. Electron microscope studies on the body wall of the nematode some coelomyarian nematode muscles. It is Nippostrongylus brasiliensis. J. Parasit. 52 suggested that these differences may have (2): 209-232. functional and/or phylogenetic implications. Kawaguti, S. 1963. Electron microscopy on the heart muscle of the cuttlefish. Biol. J. Acknowledgments Okayama Univ. 9(1-2): 27-40. Kawaguti, S., and N. Ikemoto. 1957a. Elec- The author wishes to express sincere grati- tron microscopy of the smooth muscle of a tude to Dr. A. M. Fallis and Dr. K. A. Wright, cuttlefish, Sepia esculenta. Biol. J. Okayama Department of Parasitology, University of Univ. 3(3): 196-208. Toronto, for the hospitality they gave me dur- . 1957b. Electron microscopy on the

smooth muscle from the body wall of the Rohlich, P. 1962. The fine structure of the earthworms, Pheretima communissima and muscle fiber of the leech Hirudo rnedicinalis. Eisenia foetida. Biol. T. Okayama Univ. 3 J. Ultrast. Res. 7(5-6): 399-408. (4): 223-238. Rosertbluth, J. 1963. Fine structure of body —. 1958a. Electron microscopy on the muscle cells and neuromuscular junctions in smooth muscle of the leech, Hirudo nipponia. Ascaris lumbricoides. J. Cell. Biol. 19(2): Biol. J. Okayama Univ. 4(1-2): 79-91. 82A. . 1958h. Electron microscopy of the . 1965a. Ultrastructural organization of heart muscle from the sea squirt, Ciona obliquely striated muscle fibers of Ascaris intestinalis. Biol. J. Okayama Univ. 4(1-2): lumbricoides. J. Cell Biol. 25(3): 495-515. 93-101. . 1965b. Ultrastructure of somatic mus- . 1958c. Electron microscopy on the cle cells in Ascaris lumbricoides II. Inter- smooth muscle of the roundworm, Ascaris muscular junctions, neuromuscular junctions, lumbricoides. Biol. J. Okayama Univ. 4(3— and glycogen stores. J. Cell Biol. 26(2): 4): 177-190. 579-591. . 1958d. Electron microscopy of the . 1967. Obiquely striated muscle. III. smooth muscle of the clamworm, Nereis Contraction mechanism of Ascaris body japonica. Biol. J. Okayama Univ. 4(3-4): muscle. J. Cell Biol. 34(1): 15-33. 207-216. 1968. Obliquely striated muscle. IV. 1961. Electron microscopy on the ad- Sarcoplasmic reticulum, contractile appara- ductor muscle of a tellin, Fabulina nitidula. tus, and endomysium of the body muscle of Biol. J. Okayama Univ. 7(1-2): 17-29. a polychaete, Glycera, in relation to its speed. Lee, Chin-Chiu, and J. H. Miller. 1967. J. Cell Biol. 36(1): 245-259. Fine structure of Dirofilaria immitis body- Schneider, A. 1860. Ueber die Muskeln und wall musculature. Exper. Parasit. 20: 334- Nerven der Nematoden. Arch. Anat, Phys- 344. iol. u. Wiss. Med. 224-242. Lee, D. L. 1966. An electron microscope study Watson, B. D. 1965a. The fine structure of of the body-wall of the third stage larva of the body-wall in a free-living nematode, Nippostrongylus brasiliensis. Parasitology 56: Euchromadora vulgaris. Quart. J. Microsc. 127-135. Sci. 106(1): 75-81. Makidono, J. 1956. Observations on Ascaris . 1965b. The fine structure of the body- during fluoroscopy. Amer. J. Trop. Med. wall and the growth of the cuticle in the Hyg. 5: 699-702. adult nematode Ascaris lumbricoides. Quart. Porter, K. R. 1961. The Sarcoplasmic Retic- J. Microsc. Sci. 106(1): 83-91. ulum: Its recent history and present status. Watson, M. L. 1958. Staining of tissue sec- J. Biophys. and Biochem. Cytol. 10(4/2): tions for electron microscopy with heavy 219-226. metals. II. Applications of solutions con- Pucci, Ida, and B. A. Afzelius. 1962. An taining lead and barium. J. Biophys. Bio- electron microscope study of sarcotubules chem. Cytol. 4: 727-730. and related structures in the leech muscle. Wright, K. A. 1964. The fine structure of the J. Ultrast. Res. 7(3/4): 210-224. somatic muscle cells of the nematode Capil- Reger, J. F. 1964. The fine structure of the laria hepatica (Bancroft, 1893). Can. J. fibrillar network and sarcoplasmic reticulum Zool. 42: 483-490. in smooth muscle cells of Ascaris lumbri- . 1966. Cytoplasmic bridges and mus- coides (var. suum). J. Ultrast. Res. 10: cle systems in some polymyarian nematodes. 48-57. Can. J. Zool. 44: 329-340. Riopel, J. L., and A. R. Spurr. 1962. Carbo- Wright, K. A., and N. O. Jones. 1965. Some wax for embedding and serial sectioning of techniques for the orientation and embedding botanical material. Stain Tech. 37(6): 357- of nematodes for electron microscopy. Nema- 362. tologica 11: 125-130.

Infectivity of Two Species of Poultry Coccidia After Freezing and Storage in Liquid Nitrogen Vapor

DAVID J. DORAN AND JOHN M. VETTERLING Beltsville Parasitological Laboratory, Animal Disease and Parasite Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland

Kouwenhoven (1967) reported that sporo- sterile distilled water for 24 hours before freez- zoites of Eimeria tenella (Railliet and Lucet, ing or excysting sporozoites. Sporocysts were 1891) Fantham, 1909, frozen within sporo- treated with excystation fluid as previously cysts released from oocysts, produced infection described (Doran and Vetterling, 1967), until in chickens 24 hours after storage at —79 C. more than 85% of the sporozoites had excysted. He also found that sporozoites within intact Oocysts, sporocysts, and sporozoites of E. oocysts were noninfective after the same inter- tenella were placed in media consisting of 86% val at -79 and -196 C. Doran and Vetterling Earle's (Earle, 1943) balanced salt solution, (1968) stored E. tenella and E. meleagrimitis 5% lactalbumin hydrolysate (2.5% solution in Tyzzer, 1929 sporozoites, excysted and within Earle's B.S.S.), 2% calf serum, and 7% di- released sporocysts, in liquid nitrogen vapor methyl sulfoxide (DMSO). Those of E. for one month. After either manner in which meleagrimiti-s were placed in media containing sporozoites were stored, they found that ma- 88% Medium 199 (Morgan et al., 1950) in ture schizonts developed in cell culture. They Hank's B.S.S. (Hanks and Wallace, 1949), 5% also found that frozen excysted sporozoites chicken serum, and 7% DMSO. The media inoculated into birds produced oocysts and were at 37-43 C when parasites were added. lesions similar to those produced in birds inoc- Suspensions were quickly dispensed in 5-ml ulated with freshly excysted sporozoites, amounts into 10-ml round-bottom screw-top whereas sporozoites within frozen sporocysts tubes using a Cornwall automatic pipette. produced less intense infections than in birds The tubes were then immediately placed in a fed freshly released sporocysts. liquid nitrogen freezer (Canal Industrial Corp., This report pertains to the infectivity of Bethesda, Md.; Model 2) and frozen to -80 C excysted sporozoites, sporozoites within re- at the rate of 1 degree/min. After the tem- leased sporocysts, and sporozoites within in- perature had dropped to that of the vapor, tact oocysts of E. meleagrimitis and E. tenella the tubes were placed in a cylindrical storage after storage in liquid nitrogen vapor for inter- tank (Lansdell Cryogenics, Hyattsville, Md.; vals up to 4 months. Type LR-40) about 4 to 5 inches above liquid nitrogen. Each tube contained 2.5 to 3.5 Materials and Methods million excysted sporozoites, released sporo- Oocysts were collected from droppings of cysts, or intact oocysts. chicks or turkey poults during the first 72 hr One tube of oocysts, sporocysts, and sporo- of the patent period. The methods for recovery zoites of each species was removed from stor- and sporulation were the same as previously age at 1 week, 1 month, 3 months, and 4 described (Vetterling, 1969). All oocysts months, respectively. After their contents were used were from single suspensions that had quickly thawed by vigorous agitation in warm been made bacteria-free by treatment with water at 43 C, the number of oocysts, sporo- undiluted Clorox (Jackson, 1964). They were cysts, or sporozoites per respective tube was stored in Ringer's solution at 3 to 6 C and were determined. Groups of 3-week-okl chicks and either 35 days or 2.5 months old when ground turkey poults, arranged by weight into lots, oocysts (sporocysts) and sporozoites or oocysts, were inoculated with either the frozen para- respectively, were frozen. sites or fresh oocysts. Uninoculated groups Sporocysts were released from oocysts by were included to compare weight gains. Sporo- grinding with a tissue grinder (Doran and zoites (200,000) were inoculated by syringe Vetterling, 1969). They were placed in into either the duodenum (E. meleagrimitis)

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUiME 36, NUMBER 1, JANUARY 1969 31 or cecum (E. tenella); oocysts (25,000) and Results sporocysts (100,000) were fed by gavage. Oocysts of both species appeared in excel- Chicks receiving E. tenella were maintained lent condition when examined immediately until 8 days after inoculation. The birds were after thawing. They were similar to fresh then weighed and necropsied. After the cecal oocysts in all respects. However, they did lesions were evaluated, the ceca and contents not produce infection when fed to their natural were mixed with potassium dichromate and host. homogenized in a Waring blender. Droppings The membranes of many frozen sporocysts from turkeys that received E. meleagrimitis were either slightly indented or collapsed on were collected between 116 and 192 hr after one side. A few empty sporocysts were also inoculation. They were placed in potassium found. It was impossible to determine whether dichromate and stirred for several hours. At sporozoites within the sporocysts were alive. 192 hr the surviving poults were weighed, In their dormant condition, they appeared necropsied, and their duodenal lesions eval- morphologically similar to those in fresh uated. After the cecal homogenate and fecal oocysts. When fed to their hosts, both species suspension had been diluted with tap water, produced infection but less severe than that oocyst counts were made from samples with of their controls as judged by the AI (Table 1). the aid of a Levy counting chamber with Excysted sporozoites of both species ap- Fuchs-Rosenthal ruling. The total number of peared morphologically similar to freshly ex- oocysts recovered from each group was cal- cysted sporozoites; however, because of their culated, and this was divided by the number typically sluggish motility, it was impossible of surviving birds in the group to give an esti- to tell whether they were alive. Counts indi- mate of the relative oocyst production per cated a loss of less than 5% per vial. When surviving bird. sporozoites of both species were injected into The anticoccidial index (AI), as proposed their natural host, infections equal to or greater by the Merck technical staff (Merck and Co., than those of the controls were produced. The 1960), was calculated for each group. This AI's calculated for E. tenella infections were appeared to be the best available method for nearly identical, while those of the frozen E. comparing the results obtained with fresh and meleagrimitis infected principals were lower frozen parasites. With this method, the growth and survival of the uninfected control (UC) than those for the control groups. birds are the standards for comparison, and Discussion this group is assigned an index of 200. Lesions of the infected birds are scored on the basis The infections produced in birds with frozen of 0 = normal; 1 = detectable; 2 = moderate; sporocysts were entirely due to sporozoites and 4 = maximal. A lesion value of 2.5 is within the sporocysts and not to free sporo- arbitrarily assigned to each bird that dies of zoites released by grinding. A small number coccidiosis during the experiment (McLoughlin (~3%) were released, but these were killed and Chute, 1966). Oocyst production is scored during the 24-hour storage before freezing. by the following schedule: We previously found that excysted sporozoites Oocysts Oocyst will not survive 1 hour in water only. Success recovered/surviving index with sporocysts and not oocysts was probably bird X 10° score due to differences in permeability of the oocyst 0 and sporocyst membranes to the freezing 0.1-1.0 1 protectant. 2-5 10 After 4 months storage, frozen sporozoites 6-10 20 of both species produced infections of equal 40 or greater intensity than those produced by a The AI is calculated according to the equa- similar number of sporozoites in the dosages tion AI = (% survival + % UC weight gain) of fresh oocysts. However, it is doubtful - (mean lesion score X 10 + oocyst index whether this really means that the 200,000 score). frozen sporozoites inoculated into the duo-

Table 1. Infection of turkey poults and chicks with frozen and nonfrozen Eimeria meleagrimitis and E. tenella.

Mean wt grain/ surviving bird Mean Oocysts recovered/ Birds surviving/ lesion surviving bird Anticoccidial birds started Grams %UC* score X 10" index Eimeria meleagrimitis Uninoculated control 10/10 128 100 0 0 200 Fresh oocysts 4/10 85 66 2.0 31.0 46 Frozen sporocysts 9/10 125 98 1.0 31.0 138

Uninoculated control 9/9 121 100 0 0 200 Fresh oocysts 5/9 90 74 1.5 24.5 75 Frozen sporozoites 3/10 41 34 2.1 38.5 4 Eimeria tenella Uninoculated control 10/10 173 100 0 0 200 Fresh oocysts 10/10 93 54 2.2 4.5 122 Frozen sporocysts 10/10 173 100 0.1 0.2 198

Uninoculated control 10/10 130 100 0 0 200 Fresh oocysts 10/10 133 100 1.4 9.9 165 Frozen sporozoites 10/10 153 100 1.5 7.5 167 Uninoculated control.

denum or cecum were all alive and just as Summary potentially infective or viable as 200,000 within Oocysts, sporocysts released from oocysts, 50,000 oocysts given orally. When a dosage and excysted sporozoites of Eimeria melea- of oocysts is given orally, a lot of the sporo- grimttis and E. tenella. were placed in media zoites are lost—some because of oocysts fail- containing 7% dimethyl sulfoxide, frozen to ing to rupture in the gizzard, some due to -80 C at the rate of 1 degree/min, and stored failure to excyst, and others, after excysting, above liquid nitrogen for intervals up to 4 passing out in the feces. Perhaps similar re- months. sults were obtained with fresh and frozen Oocysts produced no infection when fed to sporozoites because a loss from the control turkey poults or chicks. After 3 months, sporo- equalized a loss in the countable frozen cysts of both species produced infections, but inoculum. less than those produced by fresh oocysts. There should be no further deterioration or However, inoculation of sporozoites that had loss of viability during storage for years at been stored for 4 months into the duodenum the temperature of liquid nitrogen vapor (Le- or cecum of their natural host resulted in infec- vine and Andersen, 1966). Freeze-storage of tions comparable to those caused by fresh coccidia (excysted sporozoites or released oocysts. sporocysts) by the method described herein should save workers a considerable amount Literature Cited of the time, effort, and expense involved in Doran, D. J., and J. M. Vetterling. 1967. maintaining cultures by frequent serial passage Cultivation of the turkey coccidium, Eimeria through the host. It also makes possible (1) meleagrimitis Tyzzer, 1929 in mammalian the retention of pathogenic and drug-resistant kidney cell cultures. Proc. Helm. Soc. Wash. strains without genetic alteration and (2) the 34: 59-65. establishment of a "bank" from which workers , and . 1968. Preservation of could obtain coccidia for experimental pur- coccidial sporozoites by freezing. Nature 217: 1262. poses. Since the morphology of intact oocysts , and — . 1969. Survival and was not altered by freezing (Kouwenhoven, development of Eimeria meleagrimitis Tyzzer. 1967; present study), it is possible that a type 1929 in bovine kidney and turkey intestine specimen collection could also be established. cell cultures. J. Protozool. (In press.)

Earle, W. R. 1943. Production of malignancy Frozen storage of Tritrichomonas foetus for in vitro. IV. The mouse fibroblast cultures 5.6 years. J. Protozool. 13: 199-202. and changes seen in living cells. J. Am. McLoughlin, D. K., and M. B. Chute. 1966. Cancer Institute 4: 165-212. Preliminary laboratory trials with novasat in Hanks, J. H., and R. E. Wallace. 1949. Re- Eimeria tenella infections. Avian Diseases lation of oxygen and temperature in the 10: 410-12. preservation of tissues by refrigeration. Proc. Merck and Co. 1960. Technical Bulletin, Am- Soc. Exp. Biol. Med. 71: 196-200. prolium, 56 pp. Jackson, A. R. B. 1964. The isolation of Morgan, J. F., H. J. Morton, and R. C. viable coccidial sporozoites. Parasitology 54: Parker. 1950. Nutrition of animal cells 87-93. in tissue. 1. Initial studies on a synthetic Kouwenhoven, B. 1967. The possibility of medium. Proc. Soc. Exp. Biol. Med. 73: 1. low-temperature freezing of Eimeria tenella Vetterling, J. M. 1969. Continuous-flow sporocysts. Tijdschr. Diergeneesk. 92(24): differential density flotation of coccidia 1639-42. oocysts with a comparison to other methods. Levine, N. D., and F. L. Andersen. 1966. J. Parasit. (In press.)

Influence of Storage Period on Excystation and Development in Cell Culture of Sporozoites of Eimeria meleagrimitis Tyzzer, 1929

DAVID J. DOR AN AND JOHN M. VETTERLING Beltsville Parasitological Laboratory, Animal Disease and Parasite Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland 20705

Prolonged storage of oocysts results in loss Materials and Methods of infectivity. There are no reports available OOCYSTS: All oocysts were of a single strain that indicate whether the decrease in infec- isolated from a single oocyst. They were col- tivity is due to one or more of the following: lected on the third day of the patent period, (1) death of sporozoites within sporocysts and the method for their recovery and sporula- during storage; (2) inability of the sporozoite tion was as previously described (Vetterling, to excyst because of nonremoval of the sporo- 1969). After they had been rendered free cyst plug or Stieda body; or (3) inability to of bacteria by treatment with undiluted Clorox survive and develop through the first asexual (Jackson, 1964), they were stored in Ringer's generation. In previous work on cultivation (Doran and solution at 3 to 6 C until used. Vetterling, 1967a, b, 1969), oocysts less CELL CULTURES: Cell cultures were of bo- than 18 weeks old were used. In order to have vine embryonic kidney (cell line; 20th pas- such cultures available, it was necessary to sage). They were on 10- by 35-mm cover propagate oocysts several times. Consequently, glasses in 16- by 150-mm Leighton tubes. Only many bacteria-free cultures of different ages cultures that were confluent and did not con- were accumulated. In an attempt to determine tain cell aggregates were used. The growth some of the reasons for loss of infectivity or and maintenance media were the same as decreased oocyst production in the host, we before (Doran and Vetterling, in press). excysted sporozoites from these cultures and EXCYSTATION: Five hundred thousand sporo- inoculated them into cell cultures. This re- cysts, released from oocysts of each age by port concerns Eimeria meleagrimitis cultures grinding (Doran and Vetterling, 1969), that were between 5 and 60 weeks old. were treated with excystation fluid (Doran

Table 1. Survival and development of Eitneria meleagrimitis.

Per cent 48 hr 72 hr decrease* c , . i c u; „„» _, . „ „ c no. no. ( weeks ) zoites zoites I M % zoites I M % 48 hr 72 hr 1 4 5 3,242 2,525 100 26 4.8 1,735 22 32 3.0 18.2 44.8 2 27 2,900 2,101 64 45 4.9 1,803 19 59 4.1 23.8 35.1 1 53 920 192 4 1 2.5 151 1 0 0.7 78.6 83.5

2 4 12 2,911 2,411 89 49 5.4 1,721 19 39 3.3 12.4 38.9 3 29 2,875 2,120 76 72 6.5 1,523 29 78 6.6 21.1 43.3 2 34 3,146 2,477 92 18 4.2 2,311 27 82 4.5 17.8 23.0 1 60 751 201 2 0 1.0 82 0 2 2.4 73.0 88.8 * Total number of parasites. t I, immature; M, mature (with merozoites). and Vetterling, 1967a) for 20 minutes. Counts the total number of organisms found by exam- were then made of the number of empty sporo- ining every 4th "row" across the length of cysts, sporocysts containing one and two sporo- each of four glasses. Variation among the four zoites, and free sporozoites. Percentage ex- cultures ranged from 11 to 31 per cent. cystation (PE) was calculated by two methods: Results (number of number of sporo- empty sporo- -j- cysts with one Similar results were obtained in cell culture cysts X 2) sporozoite Method A PE — X 100. with sporozoites from oocysts 5 to 34 weeks total number of sporocysts X 2 old (Table 1). There was little difference in: number of free sporozoites (1) numbers of sporozoites found within cells Method B PE = X 100. total number of sporozoites at 5 hr; (2) percentage of developmental (free and within sporocysts) stages at 48 hrs; and (3) percentage decrease INOCULATION AND MAINTENANCE OF CELL in the total numbers of parasites after 5 hr. CULTURES: Sporozoites were concentrated and However, the results with oocysts 53 and 60 resuspended in 20 ml of inoculation medium weeks old were decidedly different. consisting of Medium 199 in Hank's (Hanks The smaller number of sporozoites from and Wallace, 1949) balanced salt solution + oocysts 53 and 60 weeks old in cells at 5 hr 3% calf serum. After adjusting the pH to 7.0— is mostly due to death of sporozoites before 7.2 and thoroughly agitating the suspension, inoculation of cell cultures. There was little 1.2 ml was immediately pipetted into each of difference between oocysts of all ages when 10 cell cultures. the percentage excystation was calculated by At 3.5 hr the sporozoite-containing medium Method A (Fig. 1). When Method B was was removed. The cells were washed once used, however, only about 40% excystation with warm inoculation medium and 5 ml of was obtained for the 53- and 60-week oocysts. maintenance medium (pH 7.2-7.4) was added. This finding indicates that many sporozoites Changes of medium were made when neces- either died before or shortly after excystation. sary in order to maintain the pH at 7.0—7.4. At least half of the sporozoites inoculated into Cultures were kept in an incubator adjusted these cultures were dead at the time of inocula- to alternating intervals of approximately 12 hr tion. Many sporozoites from the 60-week cul- at 40.6 and 43 C. tures were observed to break up while ejnerg- FIXING, STAINING, AND COUNTING: At 5, 48, ing from the sporocyst. and 72 hr, cover glasses were removed from four tubes in each set of inoculated cultures. Discussion They were fixed and stained as previously Data indicate that decreased infectivity of mentioned (Doran and Vetterling, 1969). older oocyst cultures is not due to the inability Counts were made at 645 X. They represent of sporozoites to excyst because of nonremoval

100 -i Exp. Exp. 2 many sporozoites are also unable to survive within cells and develop through the first asexual generation. 80. Summary Sporozoites, excysted from sporocysts released from Eimeria meleagrimitis oocysts of 60, different ages, were inoculated into cell cultures of bovine embryonic kidney (cell line; 20th passage). Data obtained indicate that o p 40 decreased infectivity of older oocyst cultures is due to: (1) death of sporozoites within sporozoites during storage; and (2) inability of sporozoites to survive and develop after 20 entering cells. Literature Cited Doran, D. J. 1966. Pancreatic enzymes ini- 26 53 12 29 34 60 tiating excystation of Eimeria acervulina AGE OF OOCYSTS (WKS.) sporozoites. Proc. Helm. Soc. Wash. 33: Figure 1. Excystation of sporozoites from oocysts 42-43. of different ages. A - Method A; B = Method B. , and J. M. Vetterling. 1967a. Cultiva- tion of the turkey coccidium, Eimeria meleagrimitis Tyzzer, 1929 in mammalian kidney of the sporocyst plug. When calculated on the cell cultures. Proc. Helm. Soc. Wash. 34: basis of empty sporozoites (Method A), the 59-65. , and . 1967b. Comparative cul- percentage excystation of sporozoites from all tivation of poultry coccidia in mammalian cultures was similar. kidney cell cultures. T- Protozool. 14: 657- The low percentage excystation from the 662. older cultures when calculated on the basis of , and . 1969. Survival and surviving sporozoites (Method B) and the development of Eimeria meleagrimitis Tyz- presence of degenerating sporozoites emerging zer, 1929 in turkey intestine and bovine from sporozoites indicate that many sporozoites kidney cell cultures. J. Protozool. (In press.) in older oocysts are either dead within the Hanks, J. H., and R. E. Wallace. 1949. Re- sporocyst or die shortly after excysting. The lation of oxygen and temperature in the breaking up of sporozoites while emerging from preservation of tissues by refrigeration. Proc. sporocysts suggests that excystation of sporo- Soc. Exp. Biol. & Med. 71: 196-200. Jackson, A. R. B. 1964. The isolation of zoites depends on external factors (trypsin, viable coccidial sporozoites. Parasitol. 54: bile) and is probably not due to the secretion 87-93. of any enzyme by the sporozoite as previously Vetterling, J. M. 1969. Continuous-flow thought (Doran, 1966). differential density flotation of coccidia The lower percentages of development and oocysts with a comparison to other methods. percentages of survival after 5 hr suggest that J. Parasit. (In press.)

Megodontolaimus New Genus (Nematoda: Chromadoridae), with a Description of Two New Species

R. W. TIMM Visiting Professor, Department of Nematology, University of California, Davis

Two new species of a new genus of marine toward the posterior (e.g. H. steineri Wieser, nematodes of the family Chromadoridae, sub- 1954), but may occasionally be as prominent family Hypodontolaiminae, were found in the as in Megodontolaimus (e.g. H. inaequalis and Bay of Bengal. The smaller species has six H. buetschlii in the illustrations of Bresslau longitudinal alae and a single esophageal bulb, and Schuurmans Stekhoven, 1940, plate 9, while the larger species has two lateral alae and Figs. 44 and 45). Hypodontolaimus has four a double esophageal bulb. Although the two cephalic setae (may have six setose labial species differ in these prominent characters, papillae), whereas Megodontolaimus has ten; they are alike in stomatal structure and so in the latter the punctation is larger and more have been kept in the same genus. distinct, while the body is paler in color and the excretory cell is much less distinct. Megodontolaimus new genus Megodontolaimus coxbazari sp. n. DIAGNOSIS: Chromadoridae, Hypodontolaim- inae. Four long and six short cephalic setae. (Fig. 1 A; Fig. 2 A-B) Cuticle with prominent punctation; two or six FEMALE (n = 20): Length = 0.58-0.78 alae present, with fine cross bars. Prominent mm; maximum body diameter = 22-30 p.; pharyngeal bulb surrounding stoma, asymmet- esophagus = 99-125 p.; esophagus-vulva = rical, much larger on dorsal side. Long ven- 0.21-0.30 mm; vulva-anus = 0.19-0.29 mm; tral tooth, turning to left side, with tip hooked tail = 69-80 p.. inwardly and dorsally. Dorsal tooth two- MALE (n = 13): L = 0.61-0.74 mm; mbd pronged, with cresentic thickening extending = 18-22 p.; e = 99-118 p, e-a = 0.44-0.56 along dorsal wall of pharyngeal bulb. Trans- mm; tail = 58-77 p.. verse elliptical amphids. Ovaries amphidel- HOLOTYPE MALE: L = 0.69 mm; mbd = phic, with flexures.; single outstretched testis. 19 fr e = 109 p.; e-a = 0.51 mm; t = 67 /*. Spicules cephalate; gubemaculum large, par- ALLOTYPE FEMALE: L = 0.75 mm; mbd = allel, with lateral sleeves. Preanal midventral 26 p.; e = 115 ^ e-v = 0.28 mm; ovi = 0.19 setae present. Tails conical, with pointed tip. mm; ov2 = 0.13 mm; v-a = 0.28 mm; t = 80 p.. TYPE SPECIES: Megodontolaimus coxbazari DESCRIPTION: Body outstretched in death, sp. n. with tail loosely curved to ventral side. Cutic- REMARKS: Megodontolaimus is most closely ular punctations elliptical, larger at anterior of related morphologically to that group of Hypo- body and bordering the alae. Annules inclined dontolaimus with a prominent pharyngeal bulb forward in cervical region and backward from in which the somatic setae are abundant and opposite tip of anterior ovary. Lateral alae at least as long as the body diameter. The extending full length of body from amphids to most obvious distinction between the two tail tip, crossed by thin transverse bands. Head genera is the long thin crescentic apophysis on broadly rounded; head diameter 16—19 p.; 12 the dorsal side of the pharyngeal bulb in Mego- fine cheilorhabdions surrounding mouth open- dontolaimus, together with the two-pronged ing. Ten fine cephalic setae, the four subdorsal tooth. In all Hijpodontolaimus species median longer setae 15—18 p., the six shorter the large tooth is said to be dorsal (cf. Wieser, setae 6-7 p. long. Six setiform labial papillae. 1954, p. 75), whereas in Megodantolaimus it Somatic setae along full length of body, but is basically ventral (Fig. 1A, B; Fig. 2A). The not grouped in sets at same body level; somatic pronounced dorsal swelling of the pharyngeal setae up to 20 p. long. Amphids at extreme bulb may or may not be present in Hijpo- anterior, transversely elliptical (appear to be dontolaimus; if present, it is usually tapering flattened-spiral in one specimen). Large pha-

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Figure 1. A. Megodontolaimus coxbazari, female head. Megodontolaimus sonadiae: B. Male head. C. En face view. D. Optical section at level of ventral tooth tip. E. Optical section just behind ventral tooth tip. ryngeal bulb, 24-29 ^ long in female, 24-26 p truding into cheilostome; long thin crescentic in male, much more expanded dorsally; con- dorsal apophysis along outer margin of pharyn- spicuous oblique pharyngeal muscles. Large geal bulb. Large double esophageal bulb, 32— ventral tooth, appearing obliquely lateral when 42 ju, long in female, 21-32 ^ in male, with viewed from side; tip turned in toward center external indentation into two parts and inter- of stoma. Dorsal tooth two-pronged at tip, pro- nal division. Cardia inconspicuous. Intestine

Figure 2. Megodontolaimus coxbazari. A. Female esophageal region, ventral view. B. Male tail. Megodontolaimus sonadiae. C. Entire female. D. Male tail.

thin-walled, with very scattered pale yellowish spicules. Three fine midventral setae anterior globules. Amphidelphic ovaries, with flexure to anus, equally spaced. Tails in both sexes up to one-half the distance to vulva; vulva at conical, tapering to pointed tip; tail 4-4.6 anal 51-56% of body length; spherical sperm in body diameters long in female, 3.4—4.8 in male; uterus, 2 ^ in diameter, with dense refringent three caudal glands opening by a simple slit. matter in form of a crescent. Testis conspic- TYPE HABITAT AND LOCALITY: Fine slit uous, one-half body diameter in width for most above sand, tidal pool, Cox's Bazar, East of its length. Spicules slightly arcuate, 21- Pakistan. 22 p, long across arc, with narrow pointed tips. HOLOTYPE MALE: Collected by R. W. Timm, Gubemaculum broad, thickly cuticularized, 22 December 1964; Slide 1080, University of 20-24 p. long, with thin lateral sleeve around California Nematode Collection, Davis.

ALLOTYPE FEMALE: Same data as holotype; elliptical. Pharyngeal bulb 23-32 p, long in Slide 1081, UCNC, Davis. female, 19-22 p. in male, expanded dorsally. PARATYPES: Slide CB 53 sent to B. E. Teeth as in previous species. Single esophageal Hopper; Slides CB 54-61 in collection at Notre bulb, 27-33 p. long in female, 16-22 p, in male. Dame College, Dacca, East Pakistan. Inconspicuous excretory cell a short distance DIAGNOSIS: This species is distinguished posterior to esophageal bulb; inconspicuous from the following species by its greater excretory pore between bulb and nerve ring. length, double esophageal bulb and the pres- Yellowish refringent circular to elliptical intes- ence of only two alae. tinal inclusions. Amphidelphic ovaries, with flexure up to one-half or more of distance to Megodontolaimus sonadiae sp. n. vulva; ova 42 by 22 p.; vulva at 52-56% of (Fig. 1 B; Fig. 2 C-D) body length. Spicules highly arcuate, 28—31 p. FEMALE (n = 20): L = 0.42-0.65 mm; long across arc. Gubernaculum 18—19 p, long, mbd = 22-35 p, e = 80-118 ^ e-v = 0.13- with lateral sleeve around spicules. Eight mid- 0.23 mm; v-a = 0.14-0.23 mm; t = 48-75 p.. ventral short, thick, flabby setae anterior to MALE (n = 20): L = 0.41-0.53 mm; mbd anus, not equally spaced. Tails in both sexes = 22-35 p; e = 73-102 p, e-a = 0.27-0.37 conical, tapering to acute tip; tail 2.2—4.4 anal mm; t = 54-70 /A. body diameters long in female, 2.3-3.5 in male; HOLOTYPE MALE: L = 0.5 mm; mbd = 29 three caudal glands opening by a simple slit. p; e = 93 p.; e-a = 0.34 mm; t = 68 /*. TYPE HABITAT AND LOCALITY: Mud of inter- ALLOTYPE FEMALE: L = 0.5 mm; mbd = tidal zone, north side of Sonadia Island, Bay 29 p,; e = 96 p.- e-v = 0.17 mm; ov1 = 112 p.; of Bengal, East Pakistan. Other locality: St. ov2 = 128 p.; v-a = 0.18 mm; t = 51 p. Martin's Island, Bay of Bengal, East Pakistan. DESCRIPTION: Body assuming a spiral form HOLOTYPE MALE: Collected by R. W. Timm, or loose curve in death. Cuticle bearing con- 22 December 1964; Slide 1082, UCNC, Davis. spicuous rounded punctations, larger at an- ALLOTYPE FEMALE: Same data as holotype; terior and posterior of body and wider apart Slide 1083, UCNC, Davis. than in previous species. Annules inclined PARATYPES: Slides 1084 and 1085, UCNC, forward in cervical region and backward from Davis; Slide So 51 sent to B. E. Hopper; Slides opposite anterior of ovary. One pair lateral So 3, 20, 33, 35, 40, 52, 53 in collection at alae extending from midesophagus to midtail; Notre Dame College, Dacca, East Pakistan. two pairs of submedian alae in region from esophagus to anus; all alae with fine cross bars. Literature Cited Head broadly rounded; head diameter 16-19 Bresslau, E., and J. H. Schuurmans Stekhoven. p.; 12 fine cheilorhabdions surrounding mouth 1940. Marine freilebende Nematoda aus der opening (Fig. 1C). Ten fine cephalic setae, Nordsee. Mus. Roy. Hist. Nat. Belg., 1-74 + 12 plates. the longer 8-13 p. long, the shorter 3 p, (may Wieser, W. 1954. Free-living marine nema- be setiform labial papillae). Somatic setae todes. II. Chromacloroidea. Chile Reports extending full length of body, up to 13 p, long. 17. Lunds Univ. Arsskr., N.F. Avd. 2, 50: Amphids at extreme anterior, transversely 1-148.

Galvanotaxis of Pelodera strongyloides (Nematoda: Rhabditidae)

FRED H. Department of Biology, University of Louisville, Louisville, Kentucky

Numerous experiments concerning the re- from the electrode to the Ringer's one arm of sponses of various organisms in an electrical a U-shaped piece of glass tubing filled with field have been carried out within the past KC1 agar was inserted through the other hole sixty years. Some examples listed by Warden into the HgCl, KC1 solution, and the other arm et al. (1940) of the many different organisms of the agar bridge was immersed in the Ring- that have exhibited a galvanotactic response er's solution in the dish. The agar bridge also are certain protozoans, bacteria, slime molds, functioned in preventing excessive polarization. flatworms, annelids, molluscs, insects, crusta- Direct current was produced by a DC power ceans, and echinoderms. supply equipped with filters to reduce the However, as far as it could be determined ripple. An ammeter was placed in the circuit from the literature, there are only two reports so that total amperage could be read at any of previous work of this nature with nematodes time and current densities calculated. Decade (Caveness and Panzer, 1960; Ronald, 1963); resistance boxes were also used when small hence, the following experiment was under- amperages were desired. taken to determine whether a galvanotactic The orientation of the worms was observed response could be elicited from Pelodera with a dissecting microscope. strongyloides. Since it was impossible to determine the exact values for the current passing through Materials and Methods the worms, the responses are thus described in All the stages of P. strongyloides used in terms of the total amperage and/or current these experiments were cultured in Petri dishes density at a particular moment. on a blood fibrin nutrient agar medium that For controls, worms were placed in the had been inoculated with the bacterium Pseu- glass dish filled with Ringer's solution and domonas fluorescens. observed for 2 hours in the absence of an The dauer larvae were tested as one group, electrical current. and the other larval stages with the adults constituted the other group. For each series Results of the experiment, approximately 200 washed DAUER LARVAE: It was found that at a total worms were placed centrally by means of a amperage of 3 ^a (current density of 0.00064 pipette on the bottom of a glass dish measuring //,a/mm2) with a resistance of about 2 meg- 12.2 by 3.8 by 3.3 cm that had been filled ohms, all the dauer larvae showed a definite with Ringer's solution. threshold response by moving toward the Two calomel electrodes were constructed for anode. In all the experiments the worms first use in the experiments. Each electrode con- moved like one integrated mass toward the sisted of a 10 by 4 cm shell vial in which were observer (perpendicular to the lines of force) placed about 15 ml mercury and 50 ml of a for about 10 minutes and then in mass toward saturated solution of HgCl and KC1. Each the anode. Gradual increase of the total am- vial was fitted with a two-hole cork stopper. perage to 100 p.0. had no effect upon the rate Through one hole was inserted a piece of glass of movement of worms. With undulating and thrashing movements, the dauer larvae traveled tubing in which was fused a piece of platinum the 6 cm in approximately 2 hours eventually wire, one end of which passed into the mer- aggregating near the anode. The experiment cury and the other end extended a short dis- was repeated several times with similar results. tance above the cork. To conduct the current The presence of the electrical field appeared to affect all the worms simultaneously and 1 This work represents a portion of a thesis submitted to the Graduate School, University of Illinois, in partial ful- with equal intensity. This showed that gal- fillment of the requirements for the degree, Doctor of Philosophy. vanotaxis was not due to electrolytic products,

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 41 in which case those worms nearest one or the ment was noticeably absent in the response of other electrode would exhibit galvanotaxis first the nondauer larvae. and then the other worms in succession. The cessation of movement toward the Passing current through the solution before anode at 70 /j.a by the nondauer larvae may introducing the worms had no effect upon indicate some inhibitory effect at this amper- orientation or movement. There was no differ- age level. ence in the amperage level at which the thresh- The results of this work differ from those old response occurred or in the rate of migra- of previous investigators. Caveness and Panzer tion of the worms toward the anode when the (1960) working with adult Panagrellus redi- direction of current flow was reversed. viviis and a number of other nematode genera The control worms which were not sub- in a field of direct current, reported a galvano- jected to any current showed random move- tactic movement of these worms to the cathode ments. on an agar substrate. These investigators re- ADULTS AND OTHER LARVAL STAGES: The corded for P. rediviviis a threshold response at adult worms showed no galvanotactic response 0.02 ma below which movement was not sig- within a total amperage range of from 1 ^a nificantly different from the random move- to 10 ma. Throughout the experiments these ment of the controls. On a soil substrate, P. worms remained within the visual field of the rediviviis was reported to have migrated also dissecting microscope and distinctly showed to the cathode, but in numbers less significant very slow random movements, there being no than those which migrated to this electrode on indication of movement toward either of the agar. electrodes. Using distilled water and dilute saline solu- In regard to the nondauer larvae, there was tions as media, Ronald (1963) was unable to no change in the random movements until a obtain any directional movement with larvae total amperage of 32 ^a was reached, at which of Terranova decipiens in continuous direct level the young larvae appeared to orientate current, in intermittent direct current or by very weakly toward the anode and extremely reversing the polarity. He did record, how- slow, undulating movements toward this ever, a condition of statis with both polarity electrode then ensued. A slow constant rate reversal and alternating current. of movement continued until a total amperage Insufficient knowledge of the physiology and of 70 p.a. was reached at which point no further biochemistry of P. strongyloides, especially un- movement was evident. A current increase to der these experimental conditions, precludes 200 p,a. did not induce any further movement; any accurate interpretation of the responses however, after decreasing the amperage below obtained in these experiments. However, one 70 fj.a., a resumption of movement occurred, broad explanation for such responses as pro- and the worms eventually aggregated near the posed by Hyman and Bellamy (1922) en- anode. deavors to relate metabolic gradients to elec- The controls for these stages showed random trical gradients. These investigators suggested movements. that differences of potential in organisms, particularly those permanent differences that Discussion occur along the main axis of animals, are due The fact that the larval nematodes showed to differences in metabolic rate at different a constant rate of movement throughout the regions, the region of highest metabolic rate amperage ranges indicates that the anodal being the most negative in the external circuit response should not be attributed to electro- and most positive in the internal circuit. They further suggested that internal potential differ- phoresis. Had the response been electro- ences may account for the galvanotaxis of phoretic, a greater rate of movement would many animals so that in a direct current, ani- have resulted with an increase in current. mals which are electro-positive internally be- It cannot be stated with certainty why the come oriented toward the cathode, while those dauer larvae first moved perpendicular to the which are electro-negative, become directed lines of force before orienting and migrating toward the anode. These workers contend that toward the anode. Such perpendicular move- such bioelectric currents are chemical in origin

Copyright © 2011, The Helminthological Society of Washington 42 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY and probably arise through differences in the Summary rates of oxidation-reduction processes in differ- The galvanotactic response of the larval ent regions of the animal. stages of the nematode Pelodera strongyloides As a matter of speculation based on the is reported. Ringer's solution was employed aforementioned considerations, the tactic reas the medium in which the larvae migrated sponse of the larvae of P. strongyloides may to the anode. A threshold response of 3 ^.a be attributed in part to a rate of metabolism was recorded for the dauer larvae and one of greater than that which occurs in the adult. 32 ^u,a for the nondauer larvae. The adult Oxidation-reduction processes, occurring at a worms showed no galvanotactic response within greater rate in the larvae, could be associated a total amperage range of from 1 ^a to 10 ma. with, among other things, growth of the orga- The results of two previous experiments con- nism, tissue differentiation and development of the reproductive system and would produce cerning the galvanotaxis of nematodes are larger amounts of organic salts and other sub- briefly reviewed, and an explanation, entirely stances. In an electrical field, the cations of speculative, is offered as to why the nematodes these organic salts would be attracted to the in the present experiments migrated to the cathode, and by nature of their structural anode. simplicity and small size, would be able to Literature Cited pass through membrane barriers fairly readily. On the other hand, most of the corresponding Caveness, F. E., and J. D. Panzer. 1960. organic anions, primarily because of their Nemic galvanotaxis. Proc. Helm. Soc. Wash. 27: 73-74. larger size, would be unable to pass through Hyman, L. H., and A. W. Bellamy. 1922. the organelle and cellular membranes to the Studies on the correlation between metabolic anode. Such a condition may therefore result gradients, electrical gradients and galvano- in the tactic response of the entire organism taxis. I. Biol. Bull. 43: 313-347. toward the anode as occurred in the case of Ronald, K. 1963. The effects of physical the larvae of P. strongyloides. stimuli on the larval stage of Terranova Further investigations in the area of galvano- decipiens. III. Electromagnetic spectrum and galvanotaxis. Can. J. Zool. 41: 197-217. taxis are obviously necessary and may even- Warden, C., T. Jenkins, and L. Warner. 1940. tually reveal that in some cases such responses Comparative psychology, Vol. 2: Plants and are an active behavioral process, while in other invertebrates. 1070 pp. Ronald Press, New cases, the responses are of a passive nature. York.

IN MEMORIAM

Charles G. Durbin 1919-1969 Member since 1949 Recording Secretary 1953 Vice President 1960 President 1964

New Species of Tylenchs Associated with Bark Beetles in New Mexico and Colorado

CALVIN L.

Members of the superfamily Tylenchoidea lectecl from the galls of chestnut oak, decaying are among the most important nematode para- fungus (Pleurota sp.), and under the dead sites and associates of bark beetles and other bark of cottonwood. Original specimens of insects throughout the United States. In the Deladenus obesus were taken from the frass opinion of the writer and other workers in the of bark beetles under the bark of dead white field, many of the free-living or saprophagous fir Abies concolor Lindl. It appears that both forms associated with insects evolve into true species were collected from normal insect parasites. At times, the sexual free-living stages habitats. of nematode parasites of insects exhibit striking As research intensifies on the relation of resemblances to soil-inhabiting or associated nematodes to insects and their ecology, it can forms. Numerous species of internal parasites be reasonably expected that more and more at some period in their taxonomical evolution examples of the similarity of parasites to free- have been placed in genera which are known living nematodes will arise, and the role of to have only a free-living existence. Ruhm the associated forms will assume increasing 1956 placed a parasite of Pissodes pini in the importance. genus Stictyhis because of the resemblance of Six new species of tylenchs were collected the sexual forms of the parasite to the sexual from trees infested with bark beetles; they forms of that genus. The parasite had been were either associated with the various stages previously included in the genera Tylenchus, of the insect or were residing in a habitat that Allantoncma, and Parasitijlenchus. Recently had been abandoned by the beetles. All type the same parasite was placed in the genus specimens are in the nematode collection of Spfaaerutariopsis Nickle 1963. The genus the Rocky Mountain Forest and Range Experi- Stictyhis Thorne 1941 includes species that ment Station at Albuquerque, New Mexico. are known only to be free-living, although their habitat lends credence to Ruhm's desig- Neoditylenchus puniwopus sp. n. nation. The writer has been unable to asso- (Fig. 1 A-C) ciate known forms of Stictyhis with a parasitic- FEMALE (N = 3): L = 0.93-1.17 mm; a = counterpart, but closely related genera have 34.0-44.0; b = 6.2-7.4; c = 31.1-35.2; V = been collected from insect habitats. 90%. More recently, Bedding (1967) in his studies MALE (N = 3): L = 0.59-0.71 mm; a = on nematode parasites of the horntail genus 39.2-51.3; b = 4.5-5.3; c = 23.5-30.8. Sirex noted the remarkable similarity of the FEMALE: Figure IB. Body slender. Cuticle sexual forms of the parasite to the genus nearly smooth, transverse striae faintly visible in younger specimens. Head continuous with Deladenus. The writer, working with the same body contour. Lip region twice as wide as parasite at Rotorua, New Zealand, confirmed deep. Stylet relatively slender with prominent the resemblance. The genus Deladenus Thorne basal knobs over one-third longer than width 1941, at the time of erection, included two of head. Esophagus as in Figure IB, meta- species, Deladenus diirus and Deladenus corpus spindle-shaped with large central val- obesus. Deladenus durus was originally de- vular plates and ending in a well-developed scribed in the genus Tylenchus and was col- terminal bulb. Nerve ring a body width pos-

1 Rocky Mountain Forest and Range Experiment Station, terior to metacorpus. Excretory pore adjacent Forest Service, U. S. Department of Agriculture, with central headquarters maintained at Fort Collins in coopera- to nerve ring. Hemizonid posterior to excretion with Colorado State University; author is located at tory pore. Ovary single, outstretched, quadri- Albuquerque in cooperation with the University of New Mexico. columella two body widths in length. Post- The writer wishes to thank Mr. Gerald Thorne for his suggestions on the preparation of the manuscript. uterine branch a body width in length. Lips

Figure 1. Neoditylenchus puniwopus sp. n. A, Head; B, Female; C, Male tail.

Figure 2. Neoditylenchus yasinskii sp. n. A, Female; B, Male tail.

Copyright © 2011, The Helminthological Society of Washington 46 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY of vulva slightly protruding in some specimens, of tail, and gubernaculum. Stylet knobs con- transverse. Anal opening barely discernible siderably more prominent than pinophilus. under oil immersion objective. Terminus Also related to Neoditylenchus panurgus broadly rounded. (Ruhm, 1956) Meyl 1961—differing from that MALE: With anterior body characters of species in absence of a lateral field. female. Testes single, outstretched. Spicules TYPE LOCALITY: Associated with Dendroc- paired tylenchoid, gubernaculum as in Figure tonus ohesus (Mannerheim) in Engelmann 1C, one-fourth the length of the spicules. spruce Picea engelmanni Parry. Bursa enveloping terminus which is subacute. DIAGNOSIS: Closely related to Neoditylen- Sychnotylenchus scolyti sp. n. chus ahieticolus (Ruhm, 1956) Goodey 1963, (Fig. 3 A-D) differs in body length and width, in its longer FEMALE (N = 3): L = 0.71-0.87 mm; a = and more slender stylet and in the nearly 30.6-33.8; b = 5.5-6.2; c = ?; V = 89-90%. smooth cuticle. MALE (N = 3): L = 0.70-0.72 mm; a = TYPE LOCALITY: Santa Fe National Forest 52-54; b = 5.2-5.4; c = 31-35. near Pecos, New Mexico. FEMALE: Figure 3A. Body narrowest at TYPE HABITAT: Associated with the Doug- anterior end, widest immediately anterior to las-fir beetle Dendroctonus pseudotsugae Hop- vulva. Cuticle almost smooth, transverse stria- kins, in Douglas-fir. Pseudotsugae menziesii tions very faint. Head slightly set off. Lip var. glauca (Mirb.) Sudw. region more than twice as wide as deep, lateral lips narrower than other four and protruding Neoditylenchus yasinskii sp. n. beyond body contour. Figure 3B. Stylet stout (Fig.2A-B) without basal knobs, slightly longer than width FEMALE (N = 3): L = 1.25-1.28 mm; a = of head, Figure 3C. Metacorpus of esophagus 32.60-38.60; b = 8.54-8.62; c = 20.77-22.82; with prominent valvular apparatus, spindle- V = 89%. shaped, muscular. Isthmus slender, ending in MALE (N = 1): L = 0.94 mm; a = 37.16; a prominent elongate terminal bulb. Nerve b = 6.49; c = 18.8. ring one-half body width posterior to median FEMALE: Figure 2A. Body nearly straight bulb. Excretory pore one body width anterior when relaxed. Narrow at anterior end, widen- to median bulb. Hemizonid about opposite ing rapidly near the vulva. Cuticle with mod- anterior end of basal bulb. Ovary outstretched, erately fine transverse striations. Lip region relatively short. Quadricolumella prominent, twice as wide as deep. Head continuous with approximately one-seventh ovary length. Pos- contour of body. Stylet with distinct basal terior uterine branch 1% body widths long. knobs, one-third longer than the width of the Vulva transverse, lips protuberant. Anal open- head. Esophagus typical of genus. Nerve ring ing not discernible. Terminus broadly rounded. two body widths posterior to metacorpus. Ex- cretory pore three-fourths of a body width pos- MALE: With head and esophageal char- terior to nerve ring. Hemizonid immediately acters of female. Testes outstretched. Spicules posterior to excretory pore. Ovary single, out- paired, tylenchoid. Gubernaculum one-third stretched on some specimens, reaching nearly length of spicules, Figure 3D. Bursa envelop- to basal bulb of esophagus. Postuterine branch ing tail. Terminus subacute. one body width in length. Lips of the vulva DIAGNOSIS: Differs from Sychnotylenchus protuberant. Vulva transverse. Anal opening iilmi Ruhm 1956 in stylet length, absence of discernible only under oil immersion. Terminus basal knobs, form of metacorpus, and absence subacute. of a discernible anal opening. Sychnotylenchus MALE: Spicules tylenchoid. Gubernaculum scolyti is in general a smaller species than as in Figure 2B. Bursa enveloping tail, ter- S. ulmi. minus narrowly rounded. TYPE LOCALITY: Ft. Collins, Colorado. DIAGNOSIS: Closely related to 'Neoditylen- TYPE HABITAT: Associated with Scolytus chus pinophilus (Thome) Goodey 1963. Dif- multistriatus Marsh, in American elm, Ulmus fers from that species in shorter length, shape americana L.

Figure 3. Sychnotylenchus scolyti sp. n. A, Female; B, Face view; C, Head; D, Male tail.

Figure 4. Sychnotylenchus phloeosini sp. n. A, Head and neck; B, Head; C, Face view; D, Female midbody; E, Male tail; F, Female tail.

Sychnotylenchus phloeosini sp. n. FEMALE: Body stout, narrow at anterior (Fig. 4 A—F) end, becoming widest immediately preceding FEMALE (N = 3): L = 0.96-1.05 mm; a = vulva. Cuticle with faint longitudinal and 21-24; b = 9.3-9.5; c = ?; V = 92.6-93.6%. transverse striations, the striations most dis- MALE (N = 3): L = 0.66-0.89 mm; a = cernible in neck region, becoming very faint 29-38; b — 5.5-7.8; c = 38-39. at midbody in mature specimens. Head slightly

Figure 5. Aglenchus exiguus sp. n. A, Female; B, Head; C, Male tail; D, Tail showing lateral field; E, Head and neck showing lateral field.

Copyright © 2011, The Helminthological Society of Washington 50 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY set off, flattened, lip region twice as wide as terminal bulb of esophagus. Nerve ring at deep, the lateral lips much narrower than other middle of isthmus. Ovary single, outstretched, four, protruding beyond body contour in face very short in mature specimens; postuterine view, Figure 4C. Stylet moderately slender branch short, one-half body width in length. with basal thickenings, Figure 4B. Procorpus Vulva transverse. Lips covered by vulval flap. of esophagus stout, narrowing into a prominent Anal opening not discernible. Tail elongate, ovular median bulb in which the valvular ap- terminus acute. paratus is quite distinct. Terminal bulb as in MALE: With general body characters of Figure 4A. Nerve ring at the middle of the female. Testis single, outstretched. Spicules isthmus. Excretory pore slightly posterior to typically tylenchoid. Gubernaculum and bursa median bulb. Hemizonid not discernible in as in Figure 5C. Tail elongate, terminus acute. specimens examined. Amphidelphic, the ovary DIAGNOSIS: Closely related to Aglenchus reaching to the median bulb in mature speci- bryophilus (Steiner, 1914 )Meyl 1961, varies mens, posterior uterine branch as in Figure 4F. in the shape of the spicules and gubernaculum. Lips of the vulva protuberant. Vulva nearly Female without discernible anal opening. transverse, anal opening not discernible. Ter- TYPE LOCALITY: Mt. Taylor, New Mexico. minus broadly rounded as in Figure 4F. TYPE HABITAT: Taken from base of Engel- MALE: Body relatively slender. Head and mann spruce Picea engelmanni Parry infested lips as in female. Testes outstretched, reaching with the Engelmann spruce beetle Dendroc- nearly to terminal bulb. Spicules paired, tylen- tonus obesits (Mannerheim). choid. Gubernaculum one-third to one-half length of spicules. Figure 4E. Bursa envelop- Neotylenchus nitidus sp. n.- ing tail. Terminus subacute. Figure 4E. (Fig. 6) DIAGNOSIS: Closely related to Sychnotylen- FEMALE (N = 4): L = 0.79-0.86 mm; a = chus intricati Ruhm 1956. Varies from that 31.6-37.3; b = 5.7-6.8; c = 11.1-13.3; V = species in placement of excretory pore and 83%. nerve ring, absence of a lateral field, and dis- MALE: Unknown. cernible anal opening. FEMALE: Figure 6. Body extended when TYPE LOCALITY: Bandelier National Mon- relaxed. Cuticle with moderately fine trans- nument, New Mexico. verse striations. Lip region hardly set off, TYPE HABITAT: Associated with Phloeo.sinu.s continuous with body contour, twice as wide sp. in Juniperus utahensis (Engelm.) Lemm. as deep. Stylet moderately coarse with prominent basal knobs. Corpus of the esophagus Aglenchus exignus sp. n. cylindrical, somewhat widened at the center (Fig. 5 A-E) of its length, then narrowing as it passes FEMALE (N = 2): L = 0.31 mm; a = 19.7; through the nerve ring, ending in a prominent b = 4.6-4.9; c = ?; V = 64%. basal bulb. Excretory pore immediately pos- MALE (N = 2): L = 0.31-0.34 mm; a = terior to hemizonid, both approximately one- 24.3-25; b = 4.6; c = 3.7. half body width anterior to the junction of the FEMALE: Figure 5. Body slender ending in esophagus and gut. Ovary single, outstretched, an elongate tail. Cuticle coarsely annulated, quadricolumella occupying one-eighth of its the annules widest at midbody and interrupted total length. Posterior uterine branch well- by very prominent lateral fields formed by two developed, approximately one body width in bright lines, Figure 5C, D, E. Lateral fields length, vulva transverse. Anal opening only extending from two body widths posterior to faintly discernible in some specimens. Ter- anterior end to eight body widths anterior to minus acute. terminus. Head slightly offset, Figure 5B. DIAGNOSIS: Closely related to Neotylenchus Lips only faintly discernible in lateral view. acutus Thorne 1941, varies from that species Stylet slender with prominent basal knobs, in the prominence of the knobs on the stylet, Figure 5B. Esophagus with valvate metacorpus, the procorpus and metacorpus equal 2 Nickle (1968) has made Neotylenchus a synonym of Hexattjlus. It is the opinion of the writer that Ncotylenchus in length to isthmus and terminal bulb. Ex- is a valid genus. The character of the junction of the esophagus and intestine in members of the group is of cretory pore prominent, adjacent to center of sufficient variation to justify generic rank.

Figure 6. Neotylenchus nitidus sp. n. Female.

in the character of the junction of the esoph- Siisswassern nematoden (Fadenwiirmer) In. agus and gut, and in the presence of a pos- Die Tierwelt Mitteleuropus (Bohmer, Ehr- terior uterine branch. It also varies in the man and Ulmer) Quelle and Meyer, Leipzig, absence of a lateral field. pp. 1-273, 54 pis., Figs. 1-889. Nickle, W. R. 1963. Bovienema (Nematoda: TYPE LOCALITY: Red Feather Lakes Colo- Allantonematidae), A New Genus Parasitiz- rado, Roosevelt N.F. ing Bark Beetles of the Genus Pityogenes TYPE HABITAT: Abandoned galleries of Bedel with Notes on Other Endoparasitic Dendroctonus obesus (Mannerheim) in Engel- Nematodes of Scolytids. Proc. Helm. Soc. mann spruce, Picea engelmanni Parry. Wash. 30(2): 256-262. Nickle, W. R. 1968. Observations on Hexa- Literature Cited tijhis viviparus and Neotylenchus abulbosus (Neotylenchidae: Nematoda). Proc. Helm. Bedding, R. A. 1967. Parasitic and Free- Soc. Wash. 35: 154-160. living Cycles in the Entomophagous Nema- Ruhm, W. 1956. Die Nematoden der Ipiden. todes of the Genus Deladenus. Nature Vol. Parasitologische Schriftenreihe Jena, No. 6: 214: 174-175. 1-437, Figs. 1-145. Goodey, T. 1963. Soil and Freshwater Nema- Thorne, G. 1941. Some Nematodes of the todes. Methuen and Co. Ltd. London, 544 Family Tylenchidae which do not Possess a pp., illus. Vulvular Median Esophageal Bulb. Great Meyl, A. 1961. Die freilebenden erd und Basin Naturalist 2(2): 37-85.

The Proposed Synonymy of the Monogenean Genera Cleidodiscus Mueller, 1934 and Urocleidus Mueller, 1934, with the Proposal of Cleidodiscus bychowskyi sp. n.1

C. E. PRICE2 AND A. MURA3

A total of 43 reports of monogenetic trem- each other by a single character: the accessory atodes parasitizing North American catfishes piece is basally articulated to the cirrus in (family Ictaluridae) has been recorded. In Cleidodiscus whereas these structures exist all cases, the parasite recovered was either separately in Urocleidus. Price (op. cit.) de- Cleidodiscus floridanus Mueller, 1936 or C. clined to amalgamate the two genera. After pricei Mueller, 1936. In light of the numerous additional consideration, the authors feel that reports of these two monogeneans from the this single trait is not of generic magnitude. gills of several species of Ictaluridae, it ap- We consider these genera identical and recom- pears that close parasite—host relationships exist mend that species presently included under between Cleidodiscus parasites and their icta- Urocleidus be transferred to Cleidodiscus. lurid hosts. The present study includes a description of Cleidodiscus bychowskyi, the third Materials and Methods species of Monogenea to be recovered from a Host specimens utilized in this study were member of this catfish family. donated by Mr. W. L. McCann of Alexandria, In a comparative study of Cleidodiscus and Louisiana, to whom the authors wish to express Urocleidus, Price (1967) expressed the opinion their thanks. that these genera were very nearly identical Hosts were received frozen. After thawing, in morphology. They could be separated from gills were removed and preserved in 85% iso- propyl alcohol. From this point, gills and 1 Study supported jointly by the Departments of Biology of Augusta College and Millersville State College and by recovered parasites were treated as prescribed a Grant-in-Aid of Research from the Society of Sigma Xi. '- Department of Biology, Millersville State College, by Price (1966). Anatomical terminology em- Millersville, Pennsylvania 17551. ployed is that recommeneded by Price and 3 Department of Biology, Augusta College, Augusta, Georgia 30904. Arai (1967) for monopisthocotylean Mono-

Figures 1-10. Camera lucida illustrations of Cleidodiscus bychowskyi sp. n. 1, whole mount (ventral view); 2, detail of anterior region, showing nature of head organs, cephalic glands, and eyespots with associated lenses; 3, ventral anchor; 4, dorsal anchor; 5, ventral bar; 6, dorsal bar; 7, 8, hooks, showing two of the many possible relative positions of the associated domus; 9, cirrus; 10, accessory piece. All figures except Figure 1 drawn to same scale.

genea and by Hargis (1958). Measurements Length of ventral bar 56 (48-63); length of were made according to the recommendations dorsal bar 60 (53-68). of Mizelle and Klucka (1953). Hooks 14 in number (7 pairs), all similar Appropriate measurements and illustrations in shape and size; hook shape somewhat were made with the aid of a calibrated filar atypical for Cleidodiscus (Figs. 7, 8). Five micrometer ocular and a camera lucida, respec- pairs of hooks located on ventral aspect of tively. Average measurements are given first, haptor, remaining two pairs on dorsal aspect. followed by minimal and maximal values en- Each hook composed of: (1) a solid elongate closed in parentheses. All measurements are base joined to (2) a much narrower shaft, and expressed in microns. (3) a sickle-shaped termination provided with We found that a tincture of Poinceau S a decurved opposable piece. Each hook pro- served as an excellent stain for differentiating vided with a domus. All hooks between 14 head organs and cephalic glands. and 17 in length. Copulatory complex composed of a cirrus Cleidodiscus bychowskyi sp. n. and a basally articulated accessory piece (Figs. HOST AND LOCALITY: Ictalurus punctatus 9, 10). Cirrus equipped with an elongate, (Rafinesque), the channel catfish; Calcasieu heavily sclerotized base. Cirrus tube tapers River, near Lake Charles, Louisiana (Calcasieu gradually to a point; length of cirrus in situ Parish). 80 (73-88), estimated total length 110 (95- 129). Accessory piece in form of a slightly NUMBER OF SPECIMENS STUDIED: Ten. curved sclerotized bar with a flared termina- TYPES: Holotype USNM Helm. Coll., No. tion and provided with a deep groove on its 71238. Paratypes in authors' collections. inner surface; length 34 (28-37). Prostatic DESCRIPTION: A dactylogyrid of moderate reservoir single, filled with a yellowish gran- size, provided with a thin cuticle. Anterior ulated material and emptying into cirrus base two-thirds of body proper fusiform, tapering by a fine duct. Vagina, when visible, opens uniformly toward peduncle; length 793 (705- ventrally near right body margin; vaginal 878), greatest width of body 162 (144 to duct opens into a thin-walled seminal recep- 209). Cephalic lobes moderately developed. tacle. Ovary somewhat elongate longitu- Peduncle and haptor variable in shape, de- dinally, larger than the subspherical, post- pending upon state of contraction, haptor ovarian testis. Vitellaria composed of dark usually well set off from body proper (whole granules of essentially uniform size, the vitel- mount: Fig. 1). Two pairs of eyespots, mem- laria forming two broad bands laterally. In- bers of posterior pair larger (Figs. 1, 2). testinal limbs confluent posteriorly. Pharynx prominent, very muscular, and subspherical in ventral view. Four pairs of gland- Discussion ular head organs, connected to the multilobed C. bychowskyi does not appear to closely cephalic glands by a common duct (Fig. 2). resemble any known form presently ascribed to Two pairs of anchors, one pair in ventral either Cleidodiscus or Urocleidits. It would portion of haptor, other pair in dorsal. All serve no apparent useful purpose to designate anchors similar in both shape and size (Figs. species possessing bars and/or anchors resem- 3, 4). Each composed of: (1) a solid base bling those of the present new species, as a provided with a prominent superficial and a great degree of homogeneity of these struc- blunt deep root, (2) a solid shaft, and (3) a tures exists among most of the included forms. solid point which meets shaft at a definite The accessory piece of C. bychowskyi is unique angle. Anchor wings present. Ventral anchor among members of either genus. The hooks length 45 (4.1-49), width of base 23 (21-25); of the present form are atypical for both dorsal anchor length 47 (45-51), width of Cleidodiscus and Urocleidus. base 22 (18-25). Bases of each anchor joined DERIVATION OF SPECIES NAME: This parasite by a simple haptoral bar, the bars similar in is named in honor of Dr. Boris E. Bychowsky, size but dissimilar in shape (Figs. 5, 6). Director of the Zoological Institute of the

USSR and author of the most complete account Literature Cited of the Monogenea thus far published (By- Bychowsky, B. E. 1957. Monogenetic trema- chowsky, 1957). Dr. Bychowsky (personal todes—their systematics and phylogeny. communication) has been a great source of (Originally in Russian. American translation both information and encouragement to the edited by W. J. Hargis, Jr.) Graphic Arts Press. Washington, D. C. 506 p. senior author. Hargis, W. J., Jr. 1958. A revised, annotated Summary list of terms useful for morphological studies of monogenetic trematodes. (Mimeographed The very similar morphological features of at Virginia Fisheries Laboratory, Gloucester the genera Cleidodiscus Mueller, 1934 and Point, Virginia.) 12 p. Mizelle, J. D., and A. R. Klucka. 1953. Urocleidus Mueller, 1934 have been compara- Studies on monogenetic trematodes. XIV. tively studied. It is proposed that these genera Dactylogyridae from Wisconsin fishes. Amer. are similar to the extent that they are con- Midi. Nat. 49(3): 720-733. sidered to compose the single genus Cleidodis- Price, C. E. 1966. Urocleidus cavanaughi, a cus. It is recommended that species now in- new monogenetic trematode from the gills cluded under Urocleidus be transferred to of the keyhole cichlid, Aequidens maroni (Steindachner). Bull. Georgia Acad. Sci. Cleidodiscus. 24(3): 117-120. A new species of the North American Mono- . 1967. Notes on the trematode genera genea, Cleidodiscus bychowskyi, recovered Cleidodiscus and Urocleidus. Quart. J. Flor- from the gills of channel catfishes [letalums ida Acad. Sci. 30(1): 61-67. -, and H. P. Arai. 1967. A proposed punctatus (Rafinesque) ] captured in Louisi- system of anatomy for freshwater Mono- ana, is described. genea. Canadian J. Zool. 45(6): 1283-1286.

Studies on Haplobothrium bistrobilae sp. nov. (Cestoda: Pseudophyllidea) from Amia calva L.

G. PREMVATI1 Department of Biology, Florida Agricultural and Mechanical University, Tallahassee, Florida

The genus Haplobothrium Cooper, 1914 of The cestodes were flattened in hot water the family Haplobothriidae Meggitt, 1924 has and fixed in 10% formalin. The worms were so far only one species, H. globulijorme stained for whole mounts with either cochineal Cooper, 1914, described from only one host, or Semichon's carmine or aceto alum carmine Amia. calva L., 1766. An examination of the or hematoxylin. For sections, worms were same fish from Florida fresh waters revealed fixed in hot Bouin's fluid and stained in hema- the presence of a new species of Haploboth- toxylin and eosin. Serial frontal and cross sec- rium. Ten bowfins were examined and all tions were prepared from the region of primary were found to be infected with this cestode. scolex, pseudoscolex, immature strobila, and Four were male fish and had very light infec- mature strobila. Osmoregulatory system was tion: each fish harboring not more than three studied and photographed in living specimens. to four immature worms: one to two with pri- All measurements are in microns unless mary scolices and two to three with pseudo- otherwise indicated. scolices. In the remaining six female fish, the Haplobothrium bistrobilae sp. nov. number of worms varied from 10-18 with pri- (Figs. 1-15) mary scolices and 55 to 65 with pseudoscolices. HOST: Amia calva L., 1766. LOCALITY: Lake Mnnson, Leon County, 1 Present address: Department of Zoology, University of Lucknow, Lucknow, India. Florida.

LOCATION: First half of intestine. ments. Terminal proglottid conical in shape. HOLOTYPE and nine paratypes in USNM Testes rounded, medullary in position and Helm. Coll. Nos. 71209 and 71210. measure 80-100 by 45-50; number of testes DESCRIPTION: Specimens very active and in immature proglottids varies from 12—14 on show considerable relaxation and contraction, each side, arranged in two rows, i.e., total occurring in two phases: primary strobila and number 24-28. This number becomes double secondary strobila. Primary strobila with true after about five or six proglottids, and 24-28 scolex, followed by a growth zone (neck testes lie in double alternating rows on each region), 10—25 mm in length with only ex- side. Total number of testes not seen to exceed ternal segmentation, followed by a long chain 56 in any proglottid. Vasa efferentia on either of narrow segments. Each segment subdivides side join and form vas deferens, submedian in anteriorly into three to eight segments of sec- position and leads to a globular external ondary strobila. Anterior segments of sec- seminal vesicle, followed by a cirrus sac en- ondary strobila, when complete, develop four closing internal seminal vesicle, ejaculatory auricular lobes: one dorsal, one ventral, and duct and cirrus. Cirrus sac measures 250 in two lateral. Original segments with a number diameter; cirrus short, spiny, cylindrical, mus- of anterior proglottids break off from primary cular and eversible, opening at level of external strobila and develop into secondary strobila. seminal vesicle. Thus primary strobila is simply a dividing Ovary measures 200—250 by 240, median, strobila which gives rise to secondary strobila. and lies at posterior portion of proglottid. Genital organs never seen to differentiate in Lobes of ovary connected by isthmus; oviduct proglottids of primary strobila. arises from isthmus, runs anteriorly for a short Primary strobila has a typical trypanorhyn- distance, and joins common vitelline duct. chan scolex, with four protrusible and eversible Vitellaria follicular, medullary, and extend proboscides. Each proboscis has a proximal from anterior margin to posterior margin of spiny base or stump which can also be pro- proglottid on two lateral sides. Follicles mea- truded or retracted, and a distal nonspiny sure 40—60 by 40. Two anterior and one elongated tentacle. Proboscides enclosed in posterior vitelline ducts join in region of ovary invaginated saclike structures. Cross sections and form a thick short vitelline duct, running show each proboscis enclosed within a mus- transversely on either side. Two transverse cular sac, and surrounded by a cuticular layer, vitelline ducts join in space between two circular layer and longitudinal layer. limbs of ovary and form a very short vitelline Secondary strobila between 85-100 mm in reservoir. From this, the common vitelline length. Anteriormost segment becomes modi- duct runs posteriorly to join oviduct just before fied into a pseudoscolex, with spines develop- ootype, which is surrounded by Mehlis' gland. ing on its anterior margin. Segmentation com- Uterus runs anteriorly as a coiled tube for a plete and begins immediately posterior to short distance, and then enlarges to form a pseudoscolex. Anterior segments broader than uterine sac, with a permanent uterine pore. long, with distinct four auricular lobes, and Vagina opens on ventral aspect by a circular then gradually become longer than broad, and pore just posterior to cirrus opening. Vagina appear beaded to naked eye. Reproductive enters female genital space and enlarges to organs appear after about 20—45 segments. form seminal receptacle. Eggs measure 59-60 With growth of genitalia, internal segmentation by 25. starts to disappear first, and ultimately external Osmoregulatory system may be studied in segmentation also not visible in gravid seg- three parts: (1) in primary scolex and neck

Figures 1-6. Haplobothrium bistrobilae sp. nov. 1. Camera lucida sketch of primary scolex of living specimen to show the proboscides and osmoregulatory system. 2. Pseudoscolex of secondary strobila with immature proglottids. 3. Two mature proglottids: the first showing vitellaria and the other position of testes. 4. Enlarged view of female genitalia. 5. Cross section from the region of ovary of a mature proglottid. 6. Cross section from the region of external seminal vesicle of a mature proglottid.

Copyright © 2011, The Helminthological Society of Washington 58 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY region; (2) in immature strobila; and (3) in In last proglottid, two ventral canals seen to mature and gravid strobila. open separately by excretory pores a small dis- Osmoregulatory canals in primary scolex tance from posterior end. Two dorsal canals with two pairs, a ventral and a dorsal canal form two excretory vesicles at posteriormost on either side. Ventral canals with sinous portion of proglottid (Fig. 11). No connection course, run on outside and on reaching basal between ventral canal and dorsal canal of spiny portion of proboscides, curve inwardly; either side in any proglottid. But when pro- two dorsal canals also sinuous and run inner glottids break up and anterior end forms a to ventral canals, and as they reach anterior pseudoscolex of secondary strobila, two ven- half of proboscides, they are connected with a tral canals connect with single median dorsal transverse canal. Three small commissures join canal without forming any ring canal (Fig. 2). ventral canal to dorsal canal on either side of Flame cells numerous in each proglottid, in anterior half of scolex. Thereafter dorsal canal groups of four and as many as twenty could runs anteriorly and seen to be confluent with be counted on either side of one proglottid. ventral canal (Fig. 1). In neck region, two ventral canals run as two separate canals, one Discussion on either side. Dorsal canal single and median, Gen vis Haplobothrium Cooper, 1914, family and bifurcates into two canals after forming Haplobothriidae Meggitt, 1924, seems to be one or few islets (Figs. 10, 15). confined to only one host, Amia calva L. which In immature strobila, only three longitudinal is now found only in North America. H. canals run along vertical axis of body, one globuliforme Cooper, 1914, the only species of ventral on either side of body, and a single this genus, is so far reported mostly from median dorsal canal (Fig. 13). Dorsal canal northern parts of the United States and from slightly thicker at places, otherwise all three Mississippi in the South. This is the first record canals practically of uniform width and have of this genus from Florida. sinuous course. The morphology and systematic position of In mature strobila, two ventral canals run H. globuliforme was described in detail by longitudinally in a sinuous course, one on Cooper (1914, 1919). Essex (1929), Thomas either side of body. Course of median canal (1930), and Meinkoth (1947) described its rather unique. It runs both as a single canal life cycle and showed that it is similar to that and as two bifurcated canals. When present of pseudophyllideans, involving the develop- as two dorsal canals, they run on either side of ment of procercoid and pleurocercoid, and that genital organs, and may unite either in same the characteristic trypanorhynchan scolex de- proglottid or may run as two separate canals velops later in the pleurocercoid stage. for a couple of proglottids and then join (Fig. Haplobothrium bistrobilae sp. nov. differs 14), thus showing no regularity in bifurcation from H. globuliforme in the following: (1) or union of dorsal canals in mature strobila. The total number of testes is only 48-56:24-28

Figures 7-15. Haplobothrium bistrobilae sp. nov. (Photomicrographs). 7. Living primary scolex showing completely retracted proboscides. 8. Living primary scolex showing protruded basal spiny portion and retracted tentacles of the proboscides. 9. Frontal section of primary scolex. 10. Osmoregulatory system in neck region of living specimen. Primary strobila, showing bifurcation of median dorsal canal. 11. Pos- teriormost proglottid in living specimen showing excretory pores of ventral canals and two excretory vesicles of dorsal canals. (Note: Figure retouched.) 12. Cross section of primary scolex showing proboscides. 13. Osmoregulatory system in living immature proglottids, showing two ventral canals and single median dorsal canal. 14. Osmoregulatory system in living mature proglottids, showing two ventral canals and union and bifurcation of dorsal canal. 15. A portion of dorsal canal of neck region of living primary strobila to show islets before bifurcation into two dorsal canals. Abbreviations used: B, basal spiny portion of proboscis; C, cirrus sac; DC, dorsal canal; G, male genital pore; L, auricular lobe; M, Mehlis' gland; O, ovary; P, proboscis; PS, pseudoscolex; R, seminal receptacle; S, external seminal vesicle; T, testis; U, uterus; UP, uterine pore; V, vitellaria; VA, vagina; VC, ventral canal; VD, vitelline duct; VP, vaginal pore.

...'•• vr.;;a*«s, • •v-.-r/ry-si

Copyright © 2011, The Helminthological Society of Washington 60 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY on each side of proglottid arranged in two Haplobothrium bistrobilae sp. nov. differs alternate rows. In H. globuliforme, the num- from the only other species, H. globuliforme ber of testes is 80:40 on each side. (2) Uterine Cooper, 1914, in the number of testes, pres- pore persists even in gravid proglottids, but ence of permanent uterine pore, size of eggs, ceases to exist in those of H. globuliforme. in having completely retractable proboscides, (3) The proboscides are completely retract- and in not having a single continuous dorsal able, while in H. globuliforme the stump or canal. the base remains permanently protruded. (4) The disposition of vitelline ducts differs from Acknowledgments that of H. globuliforme. (5) The eggs are The author wishes to thank Professor Robert smaller as compared to those of H. globuli- B. Short of the Florida State University for forme. (6) The course of dorsal canal ("me- the use of his personal library; Dr. N. A. dian vessel" by Cooper) does not remain single Meinkoth, Department of Biology, Swarthmore as in H. globuliforme but becomes double at College, Swarthmore, Pennsylvania, for the places in mature and gravid proglottids. The generous supply of slides and fixed material terminal proglottid also shows two distinct of Haplobothrium globuliforme for purposes of vesicles of dorsal canals at posteriormost end. comparison, and Dr. W. W. Becklund of the The two openings of ventral canals are seen Beltsville Parasitological Labortaory for pro- to lie separately on the lateral sides of the viding type and paratype slides of H. globuli- last proglottid. forme from USNM collection. Summary Literature Cited Haplobothrium bistrobilae sp. nov., the sec- Cooper, A. R. 1914. A new cestode from ond species of the genus Haplobothrium Amia Calva L. Trans. Roy. Canad. Inst. 10: Cooper, 1914, family Haplobothriidae Meggitt, 81-119. 1924, is described from Amia calva L. from . 1914. On the systematic position of Florida fresh waters. It is characterized by Haplohothrium globuliforme Cooper. Trans. having 48-56 testes (24-28 on each side); Roy. Soc. Canad. 8: 1-5. 1919. North American pseudophyllid- permanent uterine pore; completely retract- ean cestodes from fishes. Illinois Biol. able proboscides with spiny base and nonspiny Monogr. 4: 288-541. distal tentacles, and a unique course of dorsal Essex, H. E. 1929. The life cycle of Haplo- osmoregulatory canal. Photomicrographs of bothrium globuliforme Cooper, 1914. Science the osmoregulatory system studied in living 69(1800): 677-678. worms show that the two ventral canals have Meinkoth, N. A. 1947. Notes on the life a sinuous course throughout the primary and cycle and taxonomic position of Haplo- secondary strobila. The dorsal canal is single bothrium globuliforme Cooper, 1914, a tape- in immature proglottids but bifurcates at places worm from Amia calva L. Trans. Amer. Micr. Soc. 65: 256-261. in mature and gravid proglottids of secondary Thomas, L. J. 1930. Notes on the life cycle strobila. The terminal conical proglottid shows of Haplobothrium globuliforme Cooper, a two distinct vesicles of dorsal canals and two tapeworm of Amia calva L. T. Parasit. 16: excretory pores of ventral canals. 140-145.

Reesimermis nielseni gen. et sp. n. (Nematoda: Mermithidae) Parasitizing Mosquitoes in Wyoming

YUAN-HWANG TSAI1 AND ALBERT W. GRUNDMANN Tropical Disease Research Center, St. Clare's Hospital, New York City, New York, and Department of Environmental Biology, University of Utah, Salt Lake City, Utah

There has been a renewed interest in the Gastromermis was reported by Cox (1966) parasites of mosquitoes as the search for orga- from Anopheles funestus Giles in west Africa. nisms that could prove valuable in biological Welch (I960) reviewed the status of re- control is pursued. As a result of such a study ported mosquito-inhabiting mermithids from in northern Utah and southern Wyoming re- North America and proposed referring Para- gions, a species of mermithid nematode was mermis canadensis to species inqiiirendae be- found inhabiting six mosquito species at a cause it was indicated that the description had site near Lone Tree, Uinta County, Wyoming. been based on immature specimens. He fur- Following an extensive study of the life his- ther stated that a definite status for Agamo- tory, pathogenesis in mosquitoes, and mor- mermis culicis would be unwise at this time phology of this organism, a new genus and since the female was unknown and thus the species name is proposed. generic diagnosis insufficient. Reviewing the Mermithid nematodes have been reported world status of mermithids inhabiting mos- frequently from both larvae and adults of mosquitoes, three species belonging to three genera quitoes in North America and elsewhere. Jen- currently are determined valid, while four may kins (1964) listed four defined species, Lim- be determined only to genus. nomermis aquatilis Dujardin from larval Anoph- The systematics of the Family Mermithidae elines in France, Agamomermi-s culicis Stiles Braun 1883 is difficult due to the scattered from adult Aedes sollicitans (Walker) from the literature and observations on immature forms. United States, Paramermis canadensis Sterner Best references are Filipjev (1934), Filipjev from Aedes vexans (Meigen) and A. strictus and Schuurmans (1941), and the work of a in British Columbia, Canada, and a Mermis number of Russian workers that were nicely sp. from larval Aedes aegijpti in Africa reviewed by Welch (1965). (Gendre 1909). Other records of Mermis sp. In North America, Johnson (1963) erected are: Muspratt (1945) in larval and adult the genus Octomyomermis from Chironomids. Anopheles, lyengar (1927) in India and Poinar (1964) established Orthomermis, also Walandouw (1934) in Sumatra. At least from Chironomids. Welch (196()a, 1962b, fifteen additional occurrences in mosquitoes 1963a, 1964, 1965) has described species from have been reported in North American liter- six genera in Simulid larvae. ature. Other mosquito-inhabiting species that have General Description been described are Hydromermis churchillensis Genus Reesimermis, gen. n. Welch 1960 from larval Aedes communis (De Reesimermis: Mature adult worms elongate, Geer) from Manitoba, Canada. This species cylindrical, with obtuse ends in both males was also reported from Canada by Jenkins and and females. Cephalic papillae consisting of a West (1954) from A. communis, A. impiger crown of six in a hexagonal position, which (Walker), A. nigripes (Zetterstedt), and A. may represent two lateral and four submedial pionips Dyar. Smith (1961) reported what is cephalic nerve endings. Eight longitudinal assumed to be this same species from larval hypodermal chords in dorsal, ventral, lateral, A. pullatus in Colorado. Welch (1964) de- and submedial positions (Fig. 10). Spicules scribed Romanomermis iyengari from Anoph- paired, slightly curved, medium-sized, fused eles subpictiis Grassi in India. A species of posteriorly for more than half of their length (Fig. 8). Vagina straight, of medium length, 1 Present address: Microbiological Department, Bristol Laboratories, Syracuse, New York 13201. muscular and pear-shaped. Cuticle smooth, no

Copyright © 2011, The Helminthological Society of Washington 62 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY criss-cross striae on the middle cuticle layer. authors, it is not possible to accurately state Mouth opening terminal. Postparasitic juve- that Octomyomermis should be synonomizecl niles with tail appendage. Aquatic habitat. with Romanomermis. Other genera possessing paired spicules, pear-shaped vagina, and eight Diagnosis longitudinal chords are Allomermis and Ortho- Reesimermis is the second genus to be de- mermis, but these differ from each other and scribed in the family Mermithidae possessing from Hydromermis in the arrangement of two spicules that are fused posteriorly for more cephalic papillae. The following key separates than half their length. Four established genera these genera from Reesimermis: are found that possess eight longitudinal 1. Four cephalic papillae in one plane; chords, and Reesimermis differs from each of two spicules completely separated; these in several distinct anatomical features. criss-cross striae on the cuticle; The genus Hydromermis, which contains H. mouth opening ventral Allomermis churchillensis and H. contorta, differs from Different arrangement and number of Reesimermis in the shape of the vagina which cephalic papillae; two spicules sep- is S-shaped in Hydromermis but straight and arated or fused posteriorly for more pear-shaped in Reesimermis, and in the mor- than half of their length; no criss- phology of the spicules of which there are two cross striae on the cuticle; mouth fused for half of their length in the new genus opening terminal 2 and described as single in Hydromermis. How- 2. Two lateral cepalic papillae in one ever, H. churchillensis and H. contorta possess plane with six papillae in ring un- two spicules fused for more than half their derneath; two spicules separated .... length in contradiction to the generic charac- Orthomermis ters but have the S-shaped vaginal characteris- Six cephalic papillae in one plane; two tics of Hydromermis. These two species may spicules separated or fused more be considered as intermediate evolutionary than half of their length 3 forms occurring between the separate, paired 3. Paired spicules separated spicules of Isomermis Comaii 1953 and the Romanomermis (Octomyomermis) single spicule of Hydromermis. Both genera Paired spicules fused for more than are similar in other characteristics. half of their length Reesimermis The genus most closely related to the new genus is Romanomermis which differs pri- Reesimermis nielseni sp. nov. marily in having two completely separate GENERAL: All following measurements are spicules. Both genera inhabit mosquitoes. An- in millimeters unless otherwise noted. Worm other genus appearing in the literature that long and slender with smooth cuticle. Criss- should also be considered is Octomyomermis, cross fibers in middle cuticular layers absent. but when the published description was com- Cuticle thin, 5.5—7.7 /JL in adults, and 2—3 /j. in pared with that of Romanomermis, the two postparasitic larvae. Head convex, rounded seemed to be almost identical. Since no speci- with six cephalic papillae consisting of four mens of either group were examined by the submedian and two lateral papillae arranged

Figures 1-10. 1. Lateral view of postparasitic male juvenile tail. T, trophosome; CP, caudal appendage (X 100). 2. Lateral view of postparasitic female juvenile tail (X 100). 3. Lateral view of female postparasitic juvenile vagina. GP, genital primordium (X 150). 4. Lateral view of vagina (mature female worm). O, ova; U, uterus; V, vulva; Va, vagina (X 200). 5. Face view of mature worm head. CP, cephalic papillae; M, mouth opening (X 400). 6. Dorsal view of mature female head. CP, cephalic papillae; A, amphid (X 500). 7. Lateral view of mature female tail. T, trophosome (X 100). 8. Lateral view of mature male tail. Sp, spicule; Gp, genital papillae (X 100). 9. Ventral view of mature male tail. Sp, spicule; Gp, genital papillae (X 100). 10. Cross section in anterior region of female. D, dorsal hypodermal chord; L, lateral hypodermal chord; R, reproductive organ; S, submedial hypodermal chord; T, trophosome; V, ventral hypodermal chord (X 400).

Copyright © 2011, The Helminthological Society of Washington 64 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY hexagonally in one plane and in a slight de- papillae 0.050 (0.048-0.053); width at middle pression of cuticle (Fig. 5). Mouth terminal body 0.144 (0.120-0.160); and width at anus in slight depression of cuticle. Small neck 0.116 (0.096-0.130). Tail length 0.157 constriction visible behind papillar depression. (0.140-0.175). Amphids same as in female. Amphids located behind cephalic papillae Paired spicules, fused posteriorly for 63-68% pouch-shaped, pores small, situated about of their length, 250 (205-310) p. in length. 2-4 p, behind lateral papillae. Ligament con- Individual bases of the separated proximal necting amphids absent. Oesophagus folded, ends 22-26 p.. Width of single separate spic- diameter 4.5-5.5 p. (average 5 /*) near mouth ule 4.4 p, at midpoint, and that of the middle opening, and 4 ^ along main tract; extends fused part 6.5 p,; gently curved, tapering to over three-fourths of body length. Nerve ring sharp pin-point. Testes two parallel tubes 265-290 p. from mouth. Excretory like glands running to middle of body possessing some 0.66—0.85 mm from mouth opening. Sticho- flagellated sperm. Three rows of pregenital cytes beside oesophagus numerous and gland- papillae. Median row with 19—22 papillae, ular-shaped. Lateral hypodermal chords with lateral row with 16-19 papillae, postgenital three cells (a few with paired cells). Male papillae irregular, clustered and tiny. The two and female worms have obtuse terminus and lateral papillar rows converge close to median are slightly curved ventrally. Free-living infec- row at anal area (Figs. 8, 9). tive juveniles, postparasitic and parasitic juveniles retaining needle-shaped caudal ap- iii. Eggs pendage. Flattened ellipsoid shaped, 84 by 74 (80-90 by 70-80) p., with cross-section 74 by 68 (70- i. Female 80 by 63-70) p.. Egg shell thin, transparent Seventeen specimens. Body length 15.10 and sticky outside, with very thin vitelline (11.0—21.2), stout, blunt at posterior end, membrane beneath the shell (visible only in tapering at anterior end. Greatest width of preserved specimens). Eggs unembryonated head at level of papillae 0.060 (0.055-0.065); with one egg cell when deposited. Size of eggs at vulva 0.188 (0.160-0.225); and at tail in ovaries smaller and irregular in shape, measured at the terminus of trophosome 0.141 crowded side by side. (0.125-0.195). Arnphid 13 by 5 p.. Distance from anterior ovary to mouth opening 0.84 iv. Preparasitic juveniles (0.32-1.33), that of posterior ovary to base Body length 0.56-1.20; width at head region of tail 0,57 (0.43-0.69). 6-7 p., width at the middle of body trunk 15- Vulva, 45.7% (41.1-53.1%) of body length, 17 p,. Onchostylet sharp and thin, about 12-14 a transverse opening slit (0.049) in ventral p, in length. Numerous granular cells in body. view, straightly located in lateral view, lead- Head rounded, tail slender, long, whiplike. ing to muscular pear-shaped vagina that is v. Parasitic juveniles straight thick-walled tunnel, 0.112 (0.095- Body length and width vary with state of 0.130) in length, connected to opposed amphi- development. In 28 specimens measured, delphic musculated uteri, 0.070 in width and length 0.88-13.4; greatest width 0.034-0.125. 0.190 (0.180-0.198) in length. Head rounded, tail long, slender. Rate of anterior ovary to body length 41.8% vi. Postparasitic juveniles (37.4—46.2%) and that of posterior ovary to length is 48.7% (42.3-53.9%), so v = Body length 7.4-25.5; greatest width 0.103- 0.190. Ratio of head to body width 0.31-0.44. 41-845.748-3. Length of posterior end of trophosome to base of tail 0.150 (0.100- Anterior trophosome arising 0.370 (0.310- 0.170), that of anterior end of trophosome to 0.420) from head end, posterior trophosome degenerate mouth opening 0.420; no anal arising 0.085—0.200 from base of caudal ap- opening (Figs. 4, 6, and 7). pendage. Caudal appendages 0.150—0.190 long. Sexes are distinguishable (Figs. 1, 2). ii. Male SPECIMENS DEPOSITED: Holotype—10 fe- Eleven specimens. Body length 10.2 (6.2- male adults and 10 male adults in the Uni- 14.10). Head width measured at level of versity of Utah Parasitological Collection.

Table 1. Seasonal fluctuation in female-male ratio (FMR) for postparasitic juvenile populations of Reesimermis nielseni.

Female Male No of collected worms No. Per cent No. Per cent FMR* 21 May 1966 142 32 22.53 110 77.47 0.29 12 June 1966 611 327 53.50 284 46.50 1.15 19 June 1966 277 105 37.90 172 62.10 0.61 28 June 1966 61 29 47.54 32 52.46 0.91 Total worms examined 1,091 493 45.20 598 55.80 0.824 * FMR is obtained by dividing number of females by number of males

TYPE HOST: Aedes communis (De Geer). the prefemale stouter at the base and longer ADDITIONAL HOSTS: Aedes cinereus Meigen, (170-190 /A) . Also the distance from the end A. fitchii (Felt and Young), A. increpitus Dyar, of the terminal trophosome to base of the A. pullatits (Coquillet), and Culiseta impatiens caudal appendage in the premale is much (Walker). greater (170-200 ^ Fig. 7) than in the pre- LOCATION: Haemocoel of host larvae, occa- female (85-130 ^ Fig. 8). The morphology sionally in the pupae and adults of Aedes of the caudal end is thus a useful character increpitus and A. pullatus. that can be observed under the low power of a LOCALITY: Lone Tree, Uinta County, Wyo- dissecting microscope. ming. Elevation 8,000 feet. Collected May- The primordium of the vagina, located near June 1965-66. the middle of the body (Fig. 3) has been previously used to separate premale and pre- Morphological differences between female worms. It is recognizable as a small the male and female slit appearing beneath the juvenile cuticle that postparasitic juveniles does not open to the outside at 46.5% of the The chief morphological differences be- distance from the anterior end. This feature tween premale and prefemale postparasitic is difficult to recognize in active specimens. juveniles obtained from mosquito larvae have Size differences between premale and pre- been briefly described by Welch (1960) as female worms can be used for rapid identifica- being the larger size of the female and pres- tions under some conditions, but should be ence of the genital primordium of the vagina applied in conjunction with the previous in the middle of the female body. Knowledge methods. Postparasitic juvenile females are concerning the differential diagnosis of the longer and stouter, averaging 25 mm in length male and female free-living postparasitic stage while juvenile males are shorter and thinner is inadequate because in cases of superinfec- (average 15 mm in length). Sometimes two tion, male and female worms are often hard to prominent incisions holding big excretorylike separate on the basis of size, and in cases of gland cells are visible on the ventral side, just single or double infections, the males are quite under the stichosome about one-tenth to one- large. The presence of the genital primordium twelfth distance from the anterior end. These of the vagina is difficult to observe in pre- are far more prominent in premales. served material and may be very difficult to detect in live specimens. Therefore, an at- Female—male ratio (FMR) in tempt was made to distinguish the sexes of postparasitic juveniles the postparasitic juveniles obtained following Wide fluctuation appeared in sex ratios emergence from the bodies of mosquito larvae when postparasitic worms were sexed at dif- by means of the morphology of the caudal ap- ferent times during the season (Table 1). pendages and primordium of the vagina. Males made up 77.4% of the population on The caudal end of the postparasitic larvae 21 May 1966, dropped to 46.5% on 12 June, was found to be characteristically different in increased to 62.1% 19 June, and 52.46% on males and females, with that of the premale 28 June. The fluctuations correlated well with being thin and short (150-160 ^ and that of the size of worm burdens per host with the

Copyright © 2011, The Helminthological Society of Washington PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY worms being predominately males when the develop. Approximately 2 weeks later the worm burden was greater than four and almost larvae emerge from the eggs and use their exclusively female when less than four were long, slender tails for swimming in search of a present. This phenomenon has been described host. The larvae can exist for about 4 days previously by a number of workers (Christie, under April and May temperatures and readily 1929; Johnson, 1955; Couturier, 1963; and attach by their onchostylets to the mosquito Parent!, 1965) who pointed out that sex de- larvae that appear at this time. Penetration termination in mermithids may be environ- into the host body cavity takes several min- mentally controlled with crowding being the utes after which the larvae gradually become primary factor. Another factor was introduced motionless. by Strelkov (1964) who showed that sex of The parasitic juveniles grow rapidly and are the parasite corresponds to that of the host in late stage juveniles in 10-15 days. Emergence Filipjevimermis singularis. No attempt was from the host occurs soon after this stage of made to resolve the latter in this study. development is reached. The worms emerge Female-male ratios were obtained both by by pushing the anterior end through the con- examination of free-living postparasitic juve- junctivum between the segments of the thorax, niles that had emerged spontaneously from abdomen and neck. Complete emergence takes hosts in the laboratory and through dissection about 1 minute and is fatal to the host. The of infected hosts. FMR were similar in both free-living postparasitic juveniles burrow into phases done on the same days, indicating that the bottom soil where they become motionless it would be possible to predict population for 2-4 weeks. Molting occurs at the end of changes through examination of free-living this period with the males preceding the fe- individuals in soil samples from the bottom males. Development and sexual differentia- of ponds, and thus to provide data of value tion are slow and it is usually 1—2 months to potential biological control of mosquitoes. before copulation occurs. Another 10-30 days FMR ratios are calculated by dividing the elapse before egg production occurs and the number of females by the number of males, number of eggs produced depends upon the with a low FMR being less than one (Table 1). female size and the size of the trophosome which was determined by the conditions of Life history parasitism depending mainly on the number The life history of R. nielseni is similar to of larvae that inhabit a single host. A large that described by Welch (1960a) for Hydro- female can produce as many as a thousand mermis churchillensis, a parasite of Aedes eggs. After oviposition, the female dies in communis in Canada. 5—6 days, having exhausted the food stored Unembryonated eggs are deposited in the in the trophosome. Males survive longer than bottom soil of shallow pools. Observations the females since they require little stored showed embryonation to be evident in 5-10 material for sperm production. days and the eggs contained fully developed Reesimermis nielseni has one generation per larvae seven days later. The larvae do not year, a condition well adapted to the life hatch upon completion of development under cycles of its hosts that are "single brooded" cold dry field conditions* but remain dormant mountain Aedes species confined to the higher until favorable conditions are present. The altitudes. eggs are vulnerable to desiccation so that dry- Summary ing is immediately fatal if the duration is more than a few seconds, but eggs appear to be All life stages of Reesimermis nielseni, gen. resistant to freezing winter temperatures in et sp. n., a parasite of mosquitoes from Lone soil. Tree, Wyoming, are described. A key to the In the type locality, snow is present from closely allied genera of Mermithidae is in- October to April when melting snow water cluded. The type host is the larva of Aedes forms the pools in which the mosquito hosts communis De Geer but the species was also found parasitizing Aedes cinereus Meigen, A. * The eggs, upon completion of development, could fitchii (Felt and Young), A. increpitus Dyar, hatch after 1.5-2 months in soil water at 18-20 C in the laboratory. A. pullatus (Coquillett), and Culiseta im-

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 67 patiens (Walker). A method of sexing post- Muspratt, J. 1945. Observation on the larvae parasitic juveniles is described as well as the of tree hole breeding Culicini (Diptera: seasonal fluctuation obtained in the female- Culicidae) and two of their parasites. J. male sex ratios during May and June of 1966. Entomol. Soc. S. Africa 8: 13-20. The life history is included and shows one Parenti, U. 1965. Male and female influence generation per year. of adult individuals on undifferentiated larvae of the parasitic nematode Paramermis con- Literature Cited torta. Nature 207(5001): 1105-1106. Poinar, G. O. 1964. A new nematode, Ortho- Christie, J. R. 1929. Some observations on sex mermis oedobranchus gen. nov. (Mermith- in Mermithidae. J. Exp. Zool. 53: 59-76. idae), parasitizing Smittia larvae (Chironom- Couturier, A. 1963. Recherches sur des Mer- idae) in England. Nematologica 10: 501— mithidae, nematodes parasites du henneton 506. commun (Melolontha melolontha L.). Ann. Smith, M. E. 1961. Further records of mer- Epiphities 14: 203-267. mithid parasites of mosquito larvae. Mosq. Coz, J. 1966. Contribution a 1'etude du para- News, 21: 344-345. sitisme des adultes d' Anopheles funestus par Strelkov, A. 1964. Biology of Filipjevimermis Gastromermis sp. (Mermithidae). WHO/ singularis sp. nov. found in the Rybinski EBL/66.76, pp. 1-11. Filipjev, I. N. 1934. The classification of the Reservoir. Vestnik Leningr. Univ. No. 3, free-living nematodes and their relation to Ser. Biol. 19(1): 55-69. the parasitic nematodes. Smithsonian Misc. Walandouw, E. K. 1934. Nematoden als bes- Coll. 89(6): 1-63. trijders von anopheles larven. Geneesk Tijd- , and J. H. Schuurmans Stekhoven, Jr. schr. Ned.-Ind. 74(19): 1219-1224. 1941. A manual of agricultural helminthol- Welch, H. E. 1960a. Hydromermis churchil- ogy (E. J. Brill, Leiden, Netherlands.) 878 pp. lensis n. sp. (Nematoda: Mermithidae) a Gendre, E. 1909. Sur des larves de Mermis parasite of Aedes communis (Deg.) from parasites des larves du Stegomyia faciata. Churchill, Manitoba, with observations on its Bull. Soc. Path. Exct. 2: 106-108. incidence and bionomics. Can. J. Zool. 38 lyengar, M. O. T. 1927. Parasitic nematodes (3): 465-474. of Anopheles in Bengal. Far Eastern Assoc. . 1960b. Notes on the identities of mer- Trop. Med., Trans. 7th Congr. Brit. India. mithid parasites of North American mos- 3: 128-133. quitoes, and a redescription of Agamomermis Jenkins, D. W. 1964. Pathogens, parasites and culicis Stiles, 1903. Proc. Helmin. Soc. predators of medically important arthropods. Wash. 27(2): 203-206. Wld. Hlth. Org. Organisation Mondiale de . 1962. New species of Gastromermis, la Sante, 150 pp. Isomermis, and Mesomermis from black fly , and A. S. West. 1954. Mermithid larvae. Ann. Ent. Soc. Am. 55: 535-542. nematode parasites in mosquitoes. Mosq. 1963. Amphimermis bogonae sp. nov. News, 14: 138-143. and Hexamermis cavicola sp. nov. from the Johnson, A. A. 1963. Octomyomermis itascen- Australian bogong moth, Agrotis infusa sis gen. et sp. nov., a parasite of Chironomus (Boisd.) with a review of the genus Amphi- plumosus (L.). Trans. Am. Micr. Soc. 82: mermis Kaburaki and Imamura 1932. (Nema- 237-241. toda: Mermithidae). Parasitology 53: 55-62. . 1965. Hydromermis itascensis sp. n. (Nematoda: Mermithidae), a parasite of . 1964. Romanomerrnis iyengari n. sp., Glyptotendipes labiferus (Say) (Diptera: Pilot Register of Zool. Card No. 4. Chironomidae) and the taxonomic status of . 1965. Entomophilic nematodes. Ann. Gastromermis. J. Parasit. 51: 53-56. Rev. Ent. 10: 275-302.

Anthelmintic Activity of Tetramisole, Thiabendazole, and Purified Fine Particle Phenothiazine Against Experimental Infections of Haemonchus contortus and Trichostrongylus Species in Sheep

M. L. COLGLAZIER, K. C. KATES, AND F. D. ENZIE Beltsville Parasitological Laboratory, Animal Disease and Parasite Research Division, ARS, USDA, Beltsville, Maryland 20705

The efficacy of tetramisole (2,3,5,6-tetra- were fed a balanced, pelleted ration in ade- hydro-6-phenyl-irnidazo [2,1-b] thiazole hy- quate quantity to maintain moderate but not drochloride), a broad spectrum anthelmintic maximum growth. for man and animals, was first reported by ORIGIN AND ENUMERATION OF INFECTIVE Thienpont et al. (1966). Rohrbacher et al. LARVAE: An isolate of H. contortus, designated (1967) and Bullock et al. (1968) reported AH-2, was established in 1966 from the sheep that the original optically inactive cW-tetra- flock at the Animal Husbandry Division, Agri- misole had been separated into optically active cultural Research Service, Beltsville, Maryland. d and / compounds and that anthelmintic ac- Infective larvae used in this trial were obtained tivity was ascribable to /-tetramisole. In pre- from standard cultures of feces from a stock liminary trials, /-tetramisole had about the same lamb infected with this isolate. anthelmintic activity as the dl compound when Infective larvae of Trichostrongylus spp. given at about half the dose rate. Published were of the KH isolate (Kates and Thompson, data on the activity of tetramisole in sheep and 1967), which contained, on the basis of pre- goats pertain to d/-tetramisole; see Corn well vious sheep necropsies, about 28 per cent et al. (1967), Fitzsimmons (1966), Forsyth T. axei and 72 per cent T. colubriformis. (1966), Gibson (1966), Guilhon (1966), Prior to larval inoculation of the lambs, Lyons et al. (1968), Pankhurst and Sutton infective larvae in the two suspensions (one of (1966), Reinecke (1966), Ross (1966), Shone H. contortus and one of Trichostrongylus spp.) and Philip (1967), Thomas and Bainbridge were quantitated as follows: After appropriate (1967), and Walley (1966). Because of the dilution, the larvae in ten 1-ml samples from marked promise shown by tetramisole as an each suspension were counted in Scott count- anthelmintic for sheep, a trial was conducted ing chambers; undiluted formalin was added to compare the activity of the dl and / com- to immobilize the larvae. The per cent stan- pounds with that of thiabendazole and pheno- dard error (SE) of the larval counts of the H. thiazine against experimental infections of contortus suspension was 1.87 and of the Haemonchus contortus, Trichostrongylus axei, Trichostrongylus spp. was 1.55. and Trichostrongylus colubriformis in sheep. LARVAL DOSE PREPARATION AND INOCULA- TION OF LAMBS: Each larval dose per lamb Materials and Methods contained 5,000 ± 93 SE H. contortus and EXPERIMENTAL ANIMALS: Polled Dorset 44,400 ± 688 SE Trichostrongylus spp. (95% lambs (18 wethers and 2 females) used in this confidence interval = SE X 2.26). Each dose trial were 10-10.5 months old, weighed 21-32 was concentrated by centrifugation and pi- kg (avg 27.7 kg), and were raised parasite- petted onto filter paper. The latter was then free except for insignificant infections of inserted into a gelatin capsule and adminis- Strongyloides papillosus and coccidia. The tered by balling gun. lambs were allocated to five groups of four ANTHELMINTICS EMPLOYED, THEIR ADMIN- lambs, each group having comparable mean ISTRATION, AND DOSAGES: (1) Tetramisole: weights (Table 1). Each group was held in chemically pure / and dl compounds; Amer- separate, concrete-floored pens which were ican Cyanamid Co., Princeton, N. J. (2) Thia- thoroughly cleaned every 2 days. The lambs bendazole: Thibenzole; Merck & Co., Railway,

Table 1. Activity of anthelmintics against H. contortus based upon necropsy worm counts.

No. of lambs1 Dose rate Adult 4th-stage larvae Total worms Per cent efficacy Drug ( Avg wt, kg ) mg/kg Avg (range) Avg (range) Avg (range) 5th-stage 4th-stage- Z-Tetramisole 4 (28.8) 8 0 5 5 100 99 (0-20) (0-20) dZ-Tetramisole 4 (26.8) 15 0 0 0 100 100 Thiabendazole 4 (27.5) 50 77 1,470 1,547 65 3 (0-140) (640-2,380) (740-2,449) Phenothiazine 4 (27.6) 550 <1 1,390 1,391 99 8 purified, 2—3 /JL (0-3) (160-3,120) (163-3,120) Controls 4 (27.7) — 226 1,520 1,746 (143-393) (920-2,040) (1,063-2,293)

1 All males (wethers), except one female each in dZ-tetramisole and thiabcndazolc groups. 2 See text.

N. J. (3) Phenothiazine: 2-3 p. average par- Efficacy calculations were made by the ticle diameter; purified grade (approximate standard method for the controlled anthel- purity 99.9%); wetting agent 1% lecithin; mintic test. Atomic Basic Chemical Co., Pittsburgh, Pa. SUPPLEMENTAL LAMB INFECTIONS: Because All the anthelmintics were prepared as substantial numbers of apparently inhibited aqueous drenches and administered with a 4th-stage PI. contortus were recovered 35 days dose syringe on the 28th day postinfection. postinfection from lambs in the control and The two commercial products were given at two treatment groups (Table 1), four more commonly used dose levels, thiabendazole at lambs were employed to determine the inhibi- 50 mg/kg and phenothiazine at 550 mg/kg. tory influence, if any, of simultaneous infec- d£-Tetramisole and /-tetramisole were given at tion of Trichostrongylus spp. on the develop- 15 and 8 mg/kg, respectively. ment of H. contortus. Two lambs were EXPERIMENTAL PROCEDURES: Data on weight inoculated with similar numbers of larvae of gains, packed red-cell volume (micro-hemato- H. contortus and Trichostrongylus spp. (5,000 crit), and worm eggs per gram of feces were and 44,200, respectively) of the same isolates obtained weekly on all lambs. that were used in the anthelmintic trial, and All lambs were killed 1 week after treat- each of two lambs was inoculated with 5,000 ment. The parasites remaining in the treated H. contortus larvae only (Table 4). Subse- and control lambs were recovered and counted quently, these lambs were penned, fed, ne- by the following procedures: The contents of cropsied, and their parasites recovered and each abomasum and small intestine were re- counted in the same manner as for the lambs covered and stored in a large container to in the anthelmintic trial. which undiluted formalin was added. Each organ was then separately subjected to arti- Results ficial peptic digestion overnight at 37 C. The This trial was planned to obtain compara- next day the contents and digests of each tive data on the activity of certain anthel- organ from each lamb were combined, and the mintics against adult populations of three im- final preparations made up to 2,000 ml for portant nematode parasites of sheep. Conse- sampling. From each preparation, five 20-ml quently, the lambs were treated 28 days post- samples were taken for calculating the numbers infection when practically all worms ordinarily of Trichostrongylus spp. and larval parasites should have become adults. Surprisingly, how- in each organ. After the samples were removed ever, the majority of the H. contortus re- from the total abomasal material, the remainder covered at necropsy from the control lambs was washed through screens and the adult //. and from two of the four treatment groups contortus recovered. All adult H. contortus (thiabendazole and phenothiazine) were 4th- were counted, but total numbers of the small stage larvae (Table 1). Therefore, for this nematodes were calculated from the sample and other reasons discussed later, we believe counts. that considerable numbers of inhibited 4th-

Table 2. Activity of anthelmintics against Trichostrongyhis spp. based upon necropsy worm counts.

Per cent efficacy T. axei- T. colubriformis No. of Dose rate Both species T. axei T. colubri- Drug lambs1 mg/kg Avg (range) Avg (range) Avg (range) formis Z-Tetramisole 4 8 65 0 65 98 100 (0-180) (0-180) rfZ-Tetramisole 4 15 0 0 0 100 100 Thiabendazole 4 50 0 0 0 100 100 Phenothiazine 4 550 30 655 685 99 94 purified, 2-3 /J. (0-60) (140-1,260) (200-1,300) Controls 4 3,675 10,975 14,470 (2,660-4,400) (7,060-13,900) (9,720-18,300)

1 Same lambs inoculated simultaneously with II. contortus (Table 1). 2 All Trichostrongijlus spp. recovered were adults. stage II. contortus were present in lambs of tention of adult H. contortus after treatment. all 5 groups when the anthelmintics were No eggs were seen in the posttreatment fecal administered. If this assumption is correct, samples from lambs treated with tetramisole. data were obtained on the activity of the drugs The drugs were well tolerated in all trials. against 4th-stage //. contortus as well as adults. Average pretreatment egg counts in the The data on the activity of the anthelmintics five groups of lambs were quite uniform, vary- against adult and 4th-stage H. contortus are ing only from 2,445 EPG in the group given summarized in Table 1. Both dl- and Z-tetrami- phenothiazine to 3,563 EPG in the control sole were 100% effective against adult H. con- group. This uniformity in the pretreatment tortus and almost as effective against 4th-stage egg counts indicated that reasonably uniform larvae; only one 4th-stage larva was found in adult infections were established initially in 20 abomasal samples from the four lambs the lambs. However, an average of 226 adult given Z-tetramisole. Phenothiazine was 99% H. contortus (55% females) were present in effective against adult H. contortus, but was the control lambs at necropsy, whereas the ineffective against 4th-stage larvae. Thiaben- same lambs had an average of 14,470 adult dazole was only 65% effective against adult Trichostrongijlus spp. (54% females) (Tables H. contortus and almost totally ineffective 1, 2). Therefore, substantial numbers of the against 4th-stage larvae. eggs counted pretreatment were probably Data on activity of the anthelmintics against those of Trichostrongylus spp. No attempt was T. axei and T. colubriformis are summarized made to differentiate the eggs of H. contortus in Table 2. All were very effective (94- and Trichostrongijlus spp. because quantita- 100%) against adults of both species. tive differentiation of T. axei and H. contortus All anthelmintics reduced the parasite egg eggs in mixed infections is unreliable. counts 92-100% (Table 3). Thiabendazole The reduction in average hematocrit values caused the least reduction because of the re- over the 4 weeks from infection to treatment

Table 3. Activity of anthelmintics against H. contortus and Trichostrongyhis spp. based upon combined species fecal egg counts.

Avg EPG EPG No. of Dose rate Pretreatment Post-treatment reduction Drug lambs1 mg/kff 4th week at necropsy per cent Z-Tetramisole 4 8 3,550 0 100 dZ-Tctramisole 4 15 2,508 0 100 Thiabendazole 4 50 2,825 205 92 Phenothiazine purified, 2—3 fi 4 550 2,445 30 98 Controls 4 - 3,563 2,645 25

1 Saine lambs for which necropsy worm counts are summarized in Tables 1 and 2.

Table 4. Supplemental lamb infections. Necropsy worm counts 35 days postinfection of nonmedicated lambs infected (a) simultaneously with H. contortus and Trichostrongylus spp. and (b) with H. contortus only.

H . contortus Avg ( range ) No. of Age Larvae inoculated Adults 4th-stage T. axei- T. colubriformis- T. a. + T. c. lambs1 mo. per lamb larvae Avg (range) Avg (range) Avg (range) (a) 2 ,3.5 5,000 H. contortus 1,664 20 4,530 19,850 24,380 44,200 Tricliostrongi/his (806-2,522) (0-40) (4,300-4,760) (16,500-23,200) (21,260-27,500) (b) 2 11 5,000 H. contortus 1,240 0 (998-1,482)

1 All males (wethers) except for one female in (a); weight range 17—25 kg. " All in the adult stage. varied only from 5-6% per group, which sup- Discussion ports our conclusion that average infections Our results confirm those of previously cited per group at the time of treatment were authors that rf/-tetramisole given orally at 15 comparable. nig/kg is very effective in removing adult Our conclusion that the 4th-stage H. con- H. contortus, T. axei, and T. colubriformis tortus larvae recovered at necropsy were from lambs, and that /-tetramisole at about derived from the original inocula is supported half the dose rate (8 mg/kg) of the dl com- by the fact that similar percentages of pound is equally effective. If it is assumed H. contortus (34.9%—larvae + adults) and that at the time of treatment the two groups of Trichostrongylus spp. (32.6%—adults) of the lambs treated with tetramisole compounds as original inocula were recovered at necropsy well as the control group had comparable from the control lambs. numbers of inhibited 4th-stage H. contortus, There appears to be no obvious explanation which seems likely, then both tetramisoles were for the presence of large numbers of 4th-stage highly effective against these larvae. Gibson H. contortus in the control and some treatment (1966) reported 100% efficacy of 15 mg/kg groups at the termination of this trial. The of rfZ-tetramisole against all larval stages of results of the small scale test (Table 4) con- H. contortus in lambs, and Lyons et al. (1968) ducted to determine if simultaneous Tricho- reported that the dl compound at the same strongylus spp. infections had a reproducible dose rate was 97% effective against 4th and inhibitory effect on the development of H. 87% against early 5th-stages of H. contortus. contortus were essentially negative. Substan- Also, Shone and Phillip (1967) reported that tial numbers of adult H. contortus (avg 1,664), at 15 mg/kg d/-tetramisole was more than and very small numbers of 4th-stage larvae 99% effective against 4th-stage H. contortus, (avg 20), were recovered at necropsy from but was only 72.5% effective against parasitic the two lambs inoculated with all three species 3rd-stage. Furthermore, Gibson (1966) re- of nematodes. Also, substantial numbers of ported that, at 15 mg/kg, dZ-tetramisole was adult worms (avg 1,240) and no 4th-stage highly effective against immature T. colubri- larvae were recovered from the two lambs formis in sheep. Data are rapidly accumulat- inoculated with comparable numbers of H. ing to show, therefore, that the two tetramisoles contortus only. It is also noteworthy that the at appropriate dose levels are highly effective two lambs with mixed infections harbored against both adult and larval stages of H. larger numbers of adult Trichostrongylus spp. contortus and Trichostrongylus spp. in sheep. (avg 24,380 = 55% of inocula) than the con- We obtained relatively poor action with trol lambs in the anthelmintic trial (avg 14,470 thiabendazole against adult H. contortus (AH- = 32% of inocula). The results of this supple- 2). These findings were not especially sur- mentary test indicate, therefore, that the con- prising, however, because certain unpublished current Trichostrongylus spp. infection was not observations involving the source flock also a major cause of the inhibited development of suggested that the sheep may harbor H. con- H. contortus in the anthelmintic trial. tortus that is somewhat resistant to thiabenda-

Copyright © 2011, The Helminthological Society of Washington 72 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY zole. The fact that we obtained with the AH-2 destroyed immature H. contortus 10 and 15 isolate an efficacy of only 65% against adult days old. Later, Southcott (1963) reported H. contortus, and apparently no activity against that phenothiazine in similar doses was less inhibited 4th-stage larvae, tends to support the effective against H. contortus 12 hr to 7 days earlier observations, and indicates that the old than against parasites 10—28 days old. AH-2 isolate may be resistant to thiabendazole A unique finding in this trial was the re- at the 50 mg/kg level. Further studies are in covery of substantial numbers of 4th-stage progress to compare more precisely the efficacy H. contortus from lambs of the control, pheno- of thiabendazole against this and other isolates thiazine, and thiabendazole groups. These of H. contortus. Most reports (see Drudge 4th-stage larvae were 1.5—2.5 mm long, and et al., 1964; Gibson, 1965) indicate that thia- were equivalent in size and development to bendazole at 50 mg/kg is at least 90% effec- 4th-stage larvae 4—6 days old, as described by tive against adult H. contortus in sheep. In- Veglia (1915). For reasons given below, we deed, Gibson (1965) recommends this regimen believe these larvae developed from the orig- as the treatment of choice. Conway (1964), inal inocula, were inhibited in their develop- on the other hand, reported poor results at ment by some factor(s) not now apparent, and this dose level, but improved efficacy at 80 were present in all groups of lambs at the time mg/kg. At the dose rate of 44 mg/kg, Drudge of treatment 28 days postinfection. Therefore, et al. (1964) administered six successive efficacy calculations are given in Table 1 also drenches at 4-week intervals to naturally in- for activity of the anthelmintics against 4th- fected sheep on pasture with notable lack of stage H. contortus. On this basis, only the two success in controlling H. contortus. Although tetramisoles showed action against these in- fewer H, contortus were recovered at necropsy hibited larvae. from the principals than from the controls, the We cannot be certain the 4th-stage H. con- residual worm burdens in the treated animals tortus recovered from the trial lambs came were almost pure infections of H. contortus. from the original inocula and were inhibited in They concluded that this treatment program their development, but the following reasons apparently selected, from the original popula- support this thesis: (1) The animal pens were tion of H. contortus, a strain resistant to the cleaned regularly. (2) The experiment was drug at this dose level. Attention should be conducted in midwinter and the animal quar- called to the reports of Gibson (1964), Gordon ters were too cold for normal development of (1964), Reinecke et al. (1962), and Southcott infective larvae in the pens. (3) No parasitic (1963) that thiabendazole at 50 mg/kg body larvae of Trichostrongylus spp. were recovered weight is effective against immature H. con- from any of the 20 lambs on trial, which tortus. However, only Reinecke et al., exam- strongly supports our contention that reinfec- ined digests for the immature stages. tion did not occur from pen contamination. Purified, 2—3 ^ phenothiazine given at 550 (4) The numbers of adult H. contortus were mg/kg to a group of four lambs removed 99% low and the numbers of adult Trichostrongylus of adult H. contortus (AH-2), but apparently spp. were high in the lambs; therefore, if had no effect on inhibited 4th-stage larvae. reinfection had occurred in the pens, substan- These results may be compared with those tial numbers of immature Trichostrongylus reported by Colglazier et al. (1967) for three spp. would have been recovered along with other phenothiazine products given at the same immature H. contortus, which was not the dose rate to lambs experimentally infected with case. (5) The total numbers of H. contortus another isolate (AH) of H. contortus from the and Trichostrongyhis spp. recovered at ne- same sheep flock. They reported efficacies cropsy from the controls in both instances were against adult parasites of 12% for N.F. 10 ^ about one-third of the original inocula. phenothiazine, 40% for N.F. 7 ^ phenothiazine, and 75% for purified 6.9 p. phenothiazine. Summary There is a lack of data on the effect of pheno- In a controlled test with experimental infec- thiazine against immature H. contortus. How- tions of Haemonchus contortus, Trichostron- ever, Gordon (1940) reported that pheno- gylus axei, and Trichostrongylus coluhriformis thiazine in doses of 600 mg/kg of body weight in lambs, /-tetramisole (8 mg/kg), df-tetrami-

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 73 sole (15 mg/kg), thiabendazole (50 mg/kg), the controlled test. Parasitology 54: 545- and purified, 2-3 /j, phenothiazine (550 mg/ 550. kg) were all highly effective (94-100%) . 1965. Veterinary anthelmintic medi- against adult parasites of all species, except cation. Tech. Com. No. 33, Commonwl. Agric. Rur., Rucks, England, 206 pp. for thiabendazole which was only 65% effec- 1966. An evaluation of the anthelmin- tive against adult //. contortus. The poor tic tetramisole using the improved control results obtained with thiabendazole suggest test. Vet. Rec. 79: 601-602. that the H. contortus isolate (AH-2) used in Gordon, H. McL. 1940. The anthelmintic effi- this work may be resistant to the drug at the ciency of phenothiazine against immature 50 mg/kg level. Substantial numbers of ap- Haemonchus contortus. J. Council Sci. Ind. parently inhibited 4th-stage H. contortus were Research 13: 245-246. recovered from all lambs except those treated . 1964. Studies of anthelmintics for with tetramisole. sheep: Thiabendazole. Austral. Vet. J. 40: 9-18. Acknowledgmen t Guilhon, J. 1966. Action d'un derive de 1'Imi- dazo-thiazole (Tetramisole) sur les Strongy- The authors gratefully acknowledge the lides gastro-intestinaux des ovines. Rull. technical assistance of R. H. Burtner. Acad. Vet. 39: 255-264. Kates, K. C., and D. E. Thompson. 1967. Literature Cited Activity of three anthelmintics against mixed infections of two Trichostrongylus spp. in Bullock, M. W., J. J. Hand, and E. Waletzky. gerbils, sheep, and goats. Proc. Helmin. Soc. 1968. Resolution and racemization of dl- Wash. 34: 228-236. tetramisole, dl-6-phenyl-2,3,5,6-tetrahydroim- Lyons, E. T., J. H. Drudge, and S. C. Tolliver. idazo-[2,l-b] thiazole. J. Med. Chem. 11: 1968. Activity of tetramisole against gastro- 169-171. intestinal helminths in a controlled test in Colglazier, M. L., F. D. Enzie, and R. P. Leh- lambs. Am. J. Vet. Res. 29: 2157-2162. mann. 1967. Strain variation in the re- Pankhurst, J. W., and D. O. Sutton. 1966. sponse of Haemonchus contortus in sheep and Field trials with a new anthelmintic in lambs. goats given phenothiazine: Efficacy of N.F. Vet. Rec. 79: 166-170. and purified products on pure parasitic infec- Reinecke, R. K. 1966. A larval anthelmintic tions. Am. J. Vet. Res. 28: 1711-1722. test. J. So. Afr. Vet. Med. Assn. 37: 29-33. Conway, D. P. 1964. Variance in the effec- , A. J. Sniders, and I. G. Horak. 1962. tiveness of thiabendazole against Haemon- A modification of standard procedures for chus contortus in sheep. Am. J. Vet. Res. evaluating the relative efficacy of anthel- 25: 844-845. mintics. Onclerstepoort J. Vet. Res. 29: Cornwall, R. L., R. M. Jones, J. Berry, T. 241-257. Jordan, E. A. Mercer, D. A. Pullen, and Rohrbacher, G. H., J. Emro, and E. Walet- C. J. Riley. 1967. Field trials in sheep sky. 1967. A comparison of the anthel- with the anthelmintic pyrantel tartrate. IV. mintic activity of I and c/Z-tetramisole against Comparison of pyrantel tartrate and tetram- nematodes of cattle and sheep by drench. isole in the prevention of parasitic gastro- Prog, and Abst. 42nd Ann. Meet., Am. Soc. enteritis. Vet. Rec. 80: 676-679. Parasitol., p. 44 (also personal communica- Drudge, J. H., J. Szanto, Z. N. Wyant, and G. tion ). Elam. 1964. Field studies on parasite Ross, D. B. 1966. Critical trials with tetrami- control in sheep. Comparison of thiabenda- sole given to lambs experimentally infected zole, ruelene, and phenothiazine. Am. J. Vet. with Haemonchus contortus, Ostertagia cir- Res. 25: 1512-1518. cumcincta, Nematodirus battus, and Tricho- Fitzsimmons, W. M. 1966. The effect of tet- strongylus colubriformis. Vet. Rec. 79: 392- ramisole on the parasitic stages of Tricho- 395. strongylus colubriformis in experimentally in- Shone, D. K., and J. R. Phillip. 1967. An- fected goats. Vet. Rec. 79: 599-600. thelmintic and toxicity studies tetramisole: I. Forsyth, B. A. 1966. Tetramisole: A new Anthelmintic efficacy. J. So. Afr. Vet. Med. anthelmintic for sheep. Austral. Vet. 42: Assn. 38: 165-176. 412-419. Southcott, W. H. 1963. Ovicidal effect of Gibson, T. E. 1964. The evaluation of anthel- thiabendazole and its activity against im- mintics for the removal of gastrointestinal mature helminths of sheep. Austral. Vet. J. nematodes of sheep—an improved form of 39: 452-458.

Thomas, R. J., and M. H. Bainbridge. 1967. spectrum anthelmintic. Nature. 209: 1084- The influence of the anthelmintic tetramisole 1086. on the productivity of fattening lambs. Vet. Veglia, F. 1915. The anatomy and life-history Rec. 80: 266-269. of the Haemonchus contortus (Rud.). Third and fourth Rpts. Dir. Vet. Res., Dept. Agric., Thienpont, D., O. F. J. Vanparijs, A. H. M. Un. So. Afr., pp. 347-500. Raeymaekers, J. Vandenberk, P. J. A. Walley, J. K. 1966. Tetramisole (dl 2,3,5,6- Demoen, F. T. N. Allewijn, R. P. H. tetrahyclro-6-phenyl-imidazo [2,1-b] thiazole Marsboom, C. J. E. Niemegeers, K. H. L. hydrochloride—Nilverm) in the treatment of Schellekens, and P. A. J. Janssen. 1966. gastrointestinal worms and lungworms in Tetramisole (R 8299), a new, potent broad domestic animals. Vet. Rec. 78: 406-416.

A New Trematode Cephalogonimus sireni sp. nov. (Digenea: Cephalogonimidae) from Florida Mud Eel, Siren lacertina

G. PREMVATI1 Department of Biology, Florida Agricultural and Mechanical University, Tallahassee, Florida 32307

In amphibians, a single genus Cephalogon- Description imus Poirier, 1886, of the family Cephalo- gonimidae, has been recorded mostly from Body oval, spinous, small, 1.04—1.44 mm frogs of the genus Rana. From urocleles, only long, and 760-870 wide. Oral sucker sub- one species, C. amphiuma Chandler, 1923, has terminal, 170 in diameter; ventral sucker very been reported from Amphiuma means in large, 340 in diameter; both suckers have three Louisiana and subsequently Manter (1938) to six rows of spines on their margins. Pre- reported the same species from Siren of pharynx thick and muscular; pharynx 125-130 Florida. Two mud eels, Siren lacertina, were by 110, very muscular, globular, resembling examined for helminth infection, and one was a sucker; esophagus absent; intestinal ceca found to be infected with a new trematode of terminate almost at posterior end of body. the genus Cephalogonimus. The worms were Distance between ceca and posterior end of fixed under slight coverglass pressure in hot body 140-150. AFA and stained with Semichon's carmine. Genital pore median and terminal. Testes All measurements are in microns, unless otherwise indicated. 230-270 by 200-230, ovoid to spherical, symmetrically opposite, immediately posterior to Cephalogonimus sireni sp. nov. ventral sucker. Vasa efferentia join in acetab- (Fig. 1) ular region to form the vas deferens. Cirrus HOST: Siren lacertina Linnaeus, 1766. sac preacetabular, elongated; enclosing elon- LOCALITY: Lake Munson, Leon County, gated seminal vesicle, ejaculatory duct, cirrus Florida. and prostatic cells; runs toward left of phar- LOCATION: Latter half of intestine. ynx and then joins uterus to form hermaphro- NUMBER OF WORMS: Five from one host. ditic duct that runs dorsal to oral sucker. SPECIMENS: Holotype and one paratype in Ovary submedian, ovoid to lobed, dorsal and USNM Helm. Coll. Nos. 71236, 71237. to right of ventral sucker, measures 160-170 by 100-110. Laurer's canal and seminal re- 1 Present address: Department of Zoology, University of Lucknow, Lucknow (U.P.), India. ceptacle present. Vitellaria follicular, extends

Oral sucker is larger than ventral sucker in the three genera, Emoleptalea, Paracepha- logonimus, and Oudhia, and in all species except five, of the genus Cephalogonimus. Two of these five species, namely C. com- pactus Stunkarcl, 1924 and C. japonicus Ogata, 1934, have oral sucker equal to ventral sucker. In the remaining three species, the oral sucker is only slightly smaller than the ventral sucker, the sizes being: 240 and 290 in C. lenoiri Poirier, 1886; 220 and 270 in C. vesicaudus Nickerson, 1912; 220-270 and 310-330 in C. manchuricus Oguro, 1941. But in C. sireni, the oral sucker is half the size of ventral sucker and they measure 170 and 340. This ratio of 1:2 in the sizes of the two suckers is not seen in any species of the genus Cephalogonimus. The position of testes in all species of the genus Cephalogonimus is tandem or diagonal. But in C. sireni, the testes are symmetrically opposite. In all species of the genus Cephalogonimus, the shape of the ovary is entire. But ovary is lobed in C. sireni. The position of testes and shape of ovary are two of the major characteristics used to distinguish the two families Prosthogonimidae Figure 1. Cephalogonimus sireni sp. nov. from and Cephalogonimidae: the former has sym- Florida mud eel, Siren lacertina. Holotype: ventral metrically opposite testes and lobed ovary, while the latter has tandem or diagonal testes and entire ovary. Cephalogonimus sireni shows prosthogonimid characters in having sym- from intestinal bifurcation to testicular level. metrically opposite testes and lobed ovary; Two transverse vitelline ducts dorsal to ventral sucker and join to form an elongated vitel- but it has typical cephalogonimid shape of the line reservoir. Whole female genital complex body, elongated cirrus sac, genital pore at lies in acetabular region. Uterus extends pos- anterior extremity on the dorsal side, and an teriorly and then runs anteriorly on left of amphibian host. ventral sucker. Excretory bladder Y-shaped, Thus C. sireni differs from all the present stem of Y being small. Eggs small, numerous, species of the genus Cephalogonimus in the and measure 23—25 by 14-15 (30 measured). following: (1) ventral sucker very large, twice the size of oral sucker; (2) symmetrically Discussion opposite testes, and (3) lobed ovary. It is, The subfamily Cephalogoniminae Looss accordingly, regarded as a new species. (1899), subsequently raised to family Cepha- logonimidae Nicoll (1914), includes four Summary genera: Cephalogonimus Poirier, 1886, Emo- Cephalogonimus sireni sp. nov. (Digenea: leptalea Looss, 1900 (Syn.: Leptalea Looss, Cephalogonimidae) is described from Florida 1899), Paracephalogonimits Skrjabin, 1950 mud eel, Siren lacertina. It is characterized and Oudhia Dayal and Gupta, 1954. by: (1) very large ventral sucker, twice the

Copyright © 2011, The Helminthological Society of Washington 76 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY size of oral sucker; (2) symmetrically opposite Ack no wledgmen t testes, immediately posterior to ventral sucker, Thanks are extended to Professor Robert B. and (3) lobed ovary. In the position of testes Short of the Florida State University, Tallahas- and shape of ovary, C. sireni shows characters see, for the use of his personal library. of the family Prosthogonimiclae; but it has Literature Cited typical cephalogonimid shape of the body, Manter, H. W. 1938. A Collection of Trema- elongated cirrus sac, genital pore at the anterior todes from Florida Amphibia. Trans. Amer. extremity on the dorsal side, and an amphibian Micr. Soc. 57: 26-37. host.

Intestinal Helminths of the Bullfinch, Pyrrhula pyrrhula (L.), in Southern England

I. C. WILLIAMS1 AND I. NEWTON2

The bullfinch, Pyrrhula pyrrhula (L.), is a from the gizzard forward was used for an small passerine bird which is widely distributed analysis of the food (Newton 1967a), while in Europe and northern Asia. Over most of its the rest was stored in 5% formaldehyde and range it favors coniferous or mixed forests, and examined later for helminths. The results in Britain thick deciduous woods and hedge- reported herein thus relate to the duodenum, rows. It lives chiefly on seeds and buds, but small and large intestines, rectum, cloaca, and feeds its young on a mixture of seeds and bursa Fabricii. Birds are classed as juvenile small invertebrates. By eating the buds of from the time they left the nest to the end of fruit trees it has become a serious pest in fruit the postjuvenile moult (3-4 months), first- growing areas of southern England, where it year from then until the first complete moult is resident throughout the year. The ecology a year later (age 15-16 months) and adult and general biology of the bullfinch have re- thereafter. No nestling bullfinches were ceived considerable attention (Wright and examined. Summers 1960; Newton 1964, 1966, 1967a, b); on the other hand, knowledge of the helminth Results (a) Incidence of infection parasites of this bird is confined to a few scattered records and the brief report on Urotocus Intestinal helminths were found in 71 rossittensis (Miihling, 1898) by Newton and (5.6%) of the bullfinches examined; they Williams (1967). The large numbers of bull- comprised three species of trematodes and finches killed annually in pest-control programs two species of cyclophyllidean cestodes (Table in fruit growing areas of southern England 1). No nematodes or Acanthocephala were provided an opportunity to investigate their found. The trematodes were members of the intestinal helminths. family Brachylaimidae, namely Brachylaima Through the kindness of fruit growers, dead arcuata Dujardin, 1845, Leucochloridium bullfinches were received regularly between macrostomum (Rudolphi, 1803) and Urotocus January 1965 and January 1966 from 37 local- rossittensis (Mtihling, 1898), and the cestodes ities in 10 English counties. A total of 1,265 were Anomotaenia borealis (Krabbe, 1869) birds was examined. The gut was removed and Hymenolepis passeris (Gmelin, 1790). from each bird soon after arrival and that part Not more than one species of helminth was found in any infected host and, combining all 1 Department of Zoology, The University, Hull, England. the data, there was no significant difference - Edward Grey Institute for Field Ornithology, Botanic Garden, Oxford, England. in incidence of infection between male (42/

Table 1. The occurrence of five helminth species in Bullfinches, Pyrrhula pyrrhula (L.), from different English counties.

Brachylaima LeucocJiloridiiim Urotocus Anomotaenia Hymenolepis No. of No. of arcuata1' " macrostomum rossittensis borealis- passeris Total local- bull ities in finches No. % No. % No. % No. % No. % No. % County county examined inf. inf. inf. inf. inf. inf. inf. inf. inf. inf. inf. inf. Bedfordshire 1 23 0 0 0 0 1 4 0 0 0 0 1 4 Berkshire 3 131 2 1.5 1 0.8 23 17.6 0 0 0 0 26 20 Buckinghamshire 5 119 3 2.5 0 0 3 2.5 0 0 1 0.8 7 6 Essex 5 207 1 0.5 0 0 2 1 1 0.5 0 0 4 2 Hampshire 1 62 1 1.6 0 0 10 16 0 0 0 0 11 18 Herefordshire 1 75 0 0 0 0 5 7 0 0 2 2.7 7 9 Kent 12 329 4 1.2 0 0 1 0.3 0 0 1 0.3 6 2 Suffolk 1 92 2 2 0 0 4 4 0 0 0 0 6 7 Sussex 6 214 0 0 1 0.5 2 1 0 0 0 0 3 1 Warwickshire 2 13 0 0 0 0 0 0 0 0 0 0 0 0 Total 37 1,265 13 1 2 0.2 51 4 1 0.1 4 0.3 71 5.6

1 New record in the British Isles. - New host record in the bullfinch.

698, 6.0%) and female (29/567, 5.1%) birds. arcuata in birds from Berkshire and Hamp- The most common parasite found was U. rossit- shire are added to the figures for U. rossittensis tensis in 51 birds from 13 of the 37 localities the percentage incidence of infection with age involved and within these areas the propor- becomes 25, 19, and 16, respectively. Only tion of infected birds varied greatly. Berk- one group of juveniles (hatched 1965) was shire and Hampshire provided the greatest examined, but two groups of first-year birds proportion of infected birds (33/193, 17%) (hatched 1964 and 1965) and two groups of and the southeastern counties of Kent, Essex, adults (hatched pre-1964 and pre-1965) were and Sussex the smallest, with under 1% (5/ received. The incidence of trematodes among 750) infected. The second most frequent these five groups from Berkshire and Hamp- helminth found was Brachylaima arcuata in shire was: adult, hatched pre-1964, 3/20 13 birds, six of which were from the central- (15%); first-year, hatched 1964, 7/53 (13%); southern counties (Berkshire, Buckingham- adult, hatched pre-1965, 3/17 (18%); juve- shire, and Hampshire, 6/312, 2%) and five nile, hatched 1965, 6/24 (25%); first-year, from the southeastern counties (Essex, Kent, hatched 1965, 18/79 (23%). Comparison of 5/536, 1%); the remaining two infected birds the two groups of first-year birds suggests were from one locality in Suffolk from which that, within these localities, there may be dif- 92 birds were examined. The distribution of ferences in incidence of infection from year bullfinches infected with L. macrostomum and to year. the two species of cestodes was sporadic but Among the two species of cestode it was it was noticeable that no tapeworms were noticeable that no infection of adult bull- found in any birds from Berkshire and finches was found. Hampshire, although these areas contained bullfinches with the highest incidence of (b) Intensity of infection trematodes. The number of individual parasites per in- Among birds from Berkshire and Hamp- fected host varied considerably (Table 2), and shire, there was an apparent decline in inci- there was a tendency for the intensity to be dence of infection with U. rossittensis with greater in first-year and adult birds than in increasing age (Table 2). Thus, 25% of juveniles, with the exception of U. rossittensis juveniles from these two counties contained in which the reverse was seen. With U. rossit- this species compared with 18% of first-year tensis there was a marked difference in the birds and 14% adults, but these differences mean intensity of infected male (9.4) and were not significant statistically. If the inci- female (2.9) birds; but this was chiefly due dence of L. macrostomum and Brachylaima to two of four juvenile males and three of 25

Table 2. The occurrence of five helminth species in Bullfinches, Pyrrhula pyrrhula (L.), of different age.

Age class of host Juvenile First winter Adult Total and means Number examined 301 770 194 1,265 Urotocus rossittensis a. All localities1 Number infected 9 37 5 51 Per cent infected 3 5 3 4 Mean intensity2 10 7 5 7 Range of intensity 1-42 1-73 3-7 1-73 b. Berks, and Hamps. Number infected 6/24 22/132 5/37 33/193 Per cent infected 25 18 14 17 Lciicochloridium macrostomum Number infected 0 0 2 2 Per cent infected 0 0 1 0.2 Mean intensity - - 15 15 Range of intensity - - 1-29 1-29 Brachyluima arcuata Number infected 1 10 2 13 Per cent infected 0.3 1.3 1 1 Mean intensity 1 1 1.5 1.3 Range of intensity 1 1-2 1-3 1-3 Anomotaenia borealis Number infected 1 0 0 1 Per cent infected 0.3 0 0 0.1 Intensity 2 - - 2 Hymcnolepis passcris Number infected 1 3 0 4 Per cent infected 0.3 0.4 0 0.3 Mean intensity 1 6.3 - 5 Range of intensity 1 1-16 - 1-10 All species Number infected 12 50 9 71 Per cent infected 4 7 5 5.6

1 If data for all areas are combined, as in a, a possible decline in incidence with increase in age of host is masked by the fact that relatively more juveniles than older birds were received from areas of low infection. The data for Berk- shire and Hampshire are, therefore, given separately. - Intensity, being the number of parasites per infected host. first-year males containing exceptionally large are especially rich in lymphoid follicles. The numbers of parasites (42, 28, 39, 73, and 31 effect is to distort and, particularly in a heavy respectively). If these birds are excluded, the infection, destroy the functional division of mean intensity of infected males becomes 3.2 lymphoid follicles into medulla, which pro- compared with the females 2.9. Correspond- duces lymphocytes, and cortex, where these ing adjusted figures for juveniles, first-year mature (Figs. 1, 2). At the same time the birds and adults then become 2.9, 2.9, and extensive vascular and connective tissue net- 5.2 respectively, with an overall mean intensity work, which surrounds and supports the fol- of 3.1 instead of 7 (Table 2). The average licles, is compressed and ultimately destroyed. number of U. rossittensis in infected hosts from Within the bursa U, rossittensis feeds on blood, localities with few birds infected was 1.8, com- lymphoid cells and connective tissue. The pared with 9.6 for Berkshire and Hampshire. breakdown product of hemoglobin, hematin, is especially noticeable in histological sections (c) Pathology of infection and can be recognized microspectroscopically. Of the five helminth species found only For the most part the worms lie with their U. rossittensis was seen to have an appreciable mouth toward the periphery of the bursa as affect on its host. This species is fusiform in though browsing on its inner surface. No shape and may attain 7 mm in length by 0.4 instance of cannibalism by Urotocus was seen. mm in diameter; it lives in the ovoicl, saclike, In addition to damage through pressure and bursa Fabricii where it presses upon the finger- feeding, the cuticular spines of the worm like projections which line this organ and which abrade the surface of the lymphoid tissue.

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Figure 1. Sagittal section of the uiiinfected bursa Fabricii of a juvenile male bullfinch showing numerous lymphoid follicles.

As well as causing damage within the bursa infection occurs during this early period. Al- Fabricii, heavy infections with U. rossittensis though all the nestling's food, consisting mainly greatly distend the bursa producing a "cyst" of caterpillars, flies, spiders, slugs, and snails, which may attain 1 cm in diameter. As the is collected by their parents the adults, appar- bursa lies on the dorsal side of the cloaca, ently, do not eat invertebrates at this time, nor it is possible that a heavily infected and dis- do they predigest it for their young (Newton tended bursa might, through its presence, 1967a); they are, therefore, unlikely to become interfere with the normal functioning of the infected. Yet many helminths were found in organs of the pelvic region. birds of postnestling age. This situation thus provides an unusual opportunity to examine Discussion the longevity of these various parasites in Bullfinches acquire the various helminth natural infections, and results show that parasites reported above by ingesting the re- Brachylaima arcuata can live in bullfinches spective larval stages, present in certain inver- for at least 18 months, L. macrostomum 25 tebrates, with their food. As animal material months, U. rossittensis 30 months, Anomotae- is normally eaten by bullfinches only during nia borealift 2 months and Hymenolepis passeris the first 10 days or so of life, presumably 4 months. As many bullfinches older than

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Figure 2. Sagittal section of the bursa Fabricii of a juvenile male bullfinch heavily infected with Urotocus rossittensis; the peripheral region of the bursa only remains.

those containing the two cestode species were least, that infected intermediate hosts were examined, it is possible that these particular- present but not eaten. parasites can live only a few months in this An interesting comparative study is that of host. This agrees with Kintner's (1938) ob- Sokolova (1959) in Russia on the parasites of servations on the parasites of 197 house spar- the chaffinch, Fringilla coelebs L., which is rows, Passer domesticus (L.), in Indiana, in closely related to the bullfinch. Unlike the which cestodes were found only in birds ob- latter, however, invertebrates predominate in tained before November, with none from No- the diet of the adult chaffinch during the sum- vember to March. Kintner suggested that the mer and the young are reared entirely on cestodes concerned, which included H. pas- animal material (Newton 1967b). From 143 seris, either were short-lived, or that the inter- birds Sokolova obtained four species of trema- mediate hosts were not available or did not todes; two of these, Prosthogonimus ovatus transmit them in winter. Our own observa- and P. cunsatus, inhabited the bursa Fabricii, tions on H. passeris in the bullfinch suggest and the other two, Plagiorchis maculosus and that Kintner's first alternative, that the ces- Leucochloridium sp., the intestine. The cestodes were short-lived, is very likely, although todes found included "Taenia exigua Dujardin there is the possibility, in the present study at 1845" and some unidentified specimens. None

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 81 of these parasites was found in bullfinches. woodland areas receive appreciably greater Sokolova found that trematodes infected proportions of animal food in their diet than mainly nestlings but, in contrast to our re- broods raised in farmland areas, may offer a sults, were rare or absent in fully grown birds. possible explanation of the present observa- The low mean intensity of infection of bull- tions but we have no direct evidence to this finches with Brachylaima arcuata (1.3) is in effect. Furthermore, infection must occur agreement with that reported for the related when the canal between the cloaca and the species B. fuscata in the starling, Sturnus vul- bursa is large enough to admit the young garis L., in northern England (3.4) by Owen parasite, which when infective measures 0.2- and Pemberton (1962), and south Wales (4.12) 0.6 mm in length (Timon-David 1957). As by James and Llewellyn (1967). The latter we were unable to measure the width of the authors also reported that the mean numbers lumen of the canal we do not know at what age of B. fuscata from infected mistle-thrushes, it becomes too narrow to permit entry of the Turdus viscivorus L., at 5, from a song-thrush, infective larva. Only then is the bird free T. ericetorum. Turton, at 9 and from 57 in- from the risk of further infection with this fected redwings, T. musicus L., at 9.8. It is, species of parasite. Of course, some time perhaps, surprising that the intensities should earlier the canal will have become too narrow be fairly low whereas the corresponding inci- to permit the adult 17. rossittensis from leaving dences of infection varied considerably (1% the bursa. Thus in adult birds the worms are of 1,265 bullfinches; 24 of 358 (6.7%) star- effectively encapsulated within the fibro- lings (Owen and Pemberton 1962); 25/122 muscular wall of the atrophied bursa. (21%) starlings (James and Llewellyn 1967); As stated above, exceptionally heavy infec- 2/5 (40%) mistle-thrushes, 1/11 (9%) song- tions were found only in comparatively young thrushes and 57/171 (33%) redwings (James birds and there was also an apparent decline and Llewellyn 1962)). in incidence with increasing age of the bird. It is surprising that neither Owen and Pem- Both these effects might be due either to the berton (1962) nor James and Llewellyn (1967) parasite dying, and presumably being eaten found L. macrostomum. in the 470 starlings and by their neighbors although no instances of 235 passerines of six other species they exam- cannibalism were seen in our sections, or to ined, as these birds have previously been reinfected birds dying in greater proportion than corded as hosts for this parasite. In Poland, uninfected. The role of the bursa appears to however, Joszt (1962) found L. macrostomum be particularly important during the first few in 3 of 190 (1.6%) house sparrows. days of life and in the fowl it is known to pro- Urotocus rossittensis presents an unusual duce both lymphocytes (Ackerman and Knouff case owing to the temporary nature of its 1964) and circulating antibodies (Click 1964) habitat which atrophies with the approach of during this period. Thus, impairment of the sexual maturity and is not normally found in host's defence mechanisms very early in life the adult. Yet we found 2.6% of all adult may lead to death, if not directly from worm bullfinches infected with this species, while infections, perhaps from secondary bacterial the proportion of infected adults from Berk- infections. In addition, it is possible that a shire and Hampshire was much higher at 14%. swollen, heavily infected, bursa may press Undoubtedly infection with U. rossittensis must upon adjacent organs and thereby interfere take place at an age when animal food is eaten, with their function. that is as a nestling. Thus the proportion of In conclusion, we believe that the aquisition animal material in the diet and, also, the inci- of helminth parasites by bullfinches is almost dence of infective larvae in intermediate hosts certainly restricted to the nestling stage, but will influence the incidence and intensity of some species at least remain alive in the host infection in the bird. These two factors may for many months and beyond the stage at operate to produce the differences described which the bird reaches sexual maturity. Of above, in infection of bullfinches from differ- the five helminth species found only one, ent localities. The observations of Newton Urotocus rossittensis which inhabits the bursa (1967a), that bullfinch nestlings reared in Fabricii, does any appreciable damage to the

Copyright © 2011, The Helminthological Society of Washington 82 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY host and might influence nestling and juvenile Click, B. 1964. The Bursa of Fabricius and mortality. the development of Immunologic compe- tence. In The Thymus in Immunobiology (ed. Summary Good, R. A., and A. E. Gabrielson), pp. 343-58. London: Harper and Row. Seventy-one of 1,265 (5.6%) bullfinches, James, B. L., and L. C. Llewellyn. 1967. A Pyrrhula pyrrhula (L.), received from 37 quantitative analysis of helminth infestation localities in 10 counties in southern England in some passerine birds found dead on the between January 1965 and January 1966 were island of Skomer. J. Helminth. 41: 19-44. infected with helminth parasites. These com- Joszt, L. 1962. Helminth parasites of sparrow prised five species, three being trematodes and —Passer domesticus (L.) in the environment of Warszawa. Acta Parasit. Pol. 10: 113-6. two cestodes, namely Brachylaima arcuata Kintner, K. E. 1938. Notes on the cestode Dujardin, 1845, Leucochloridium macro- parasites of English sparrows in Indiana. stomum (Rudolphi, 1803), Urotocus rossit- Parasitology 30: 347-57. ten-sis (Miihling, 1898), Hijmenolepis passeris Newton, I. 1964. Bud-eating by Bullfinches in (Gmelin, 1790) and Anomotaenia borealis relation to the natural food-supply. J. Appl. (Krabbe, 1869). Urotocus rossittensis was the Ecol. 1: 265-79. most common parasite with an incidence of . 1966. The moult of the bullfinch 4%, although in birds received from Berkshire Pyrrhula pyrrhula. Ibis 108: 41-67. and Hampshire the incidence was locally as —-——. 1967a. The feeding ecology of the Bullfinch (Pyrrhula pyrrhula L.) in southern high as 17%; the other four species were com- England. J. Anim. Ecol. 36: 721-44. paratively uncommon. Brachylaima arcuata . 1967b. The adaptive radiation and has not previously been reported from the feeding ecology of some British finches. Ibis British Isles, and neither this species nor 109: 33-98. Anomotaenia borealis have been recorded -, and I. C. Williams. 1967. A trema- before from the bullfinch. tode infection of Bullfinches Pyrrhula pyr- The incidence and intensity of infection of rhula. Ibis 109: 110-3. the five species with respect to age, sex, and Owen, R. W., and R. T. Pemberton. 1962. locality is considered, and the pathology of Helminth infection of the Starling (Sturnm vulgaris L.) in northern England. Proc. Zool. infection of the bursa Fabricii of the bullfinch Soc~ Lond. 139: 557-87. with U. rossittensis is described. It is believed Sokolova, G. A. 1959. [Dynamics of the para- that infection of the bullfinch with helminth site fauna of the Chaffinch (Fringilla coelebs parasites takes place during the nestling stage L.).] Vest, leningr. gos. Univ., 3, Ser. Biol. because only then is animal food included in 1: 83-90. (In Russian.) the bird's diet. Timon-David, J. 1957. Recherches experimen- tales sur le cycle evolutif d'un trematode du Literature Cited genre Urotocus Looss (Digenea, Leuco- chloridiidae). Annls Parasit. Hum. Comp. Ackerman, G. A., and R. A. Knouff. 1964. 32: 219-42. Lymphocytopoietic activity in the Bursa of Wright, E. N., and D. D. B. Summers. 1960. Fabricius. In The Thymus in Immunobiology The biology and economic importance of the (ed. Good, R. A., and A. E. Gabrielson), pp. Bullfinch. Ann. Appl. Biol. 48: 415-8. 123-49. London: Harper and Row.

Euparadistomum cercopitheci sp. n. (Dicrocoeliidae), a Digenetic Trematode from the Talapoin Monkey from Rio Muni1

JACOB H. FISCHTHAL

The specimens of this report were collected wide, anterior extremity round, posterior nearly by Robert W. Cooper, D.V.M.,- Director, NIH truncate, tending toward trigonal shape, 3,180- Primate Colony, Institute of Comparative Biol- 4,385 by 2,630-3,920. Tegument thick, annu- ogy, Zoological Society of San Diego. They lated; papillae and spines absent. Forebody were brought to the author's attention by G. E. 1,300-1,815 long, hindbody 997-1,450 long; Cosgrove, M.D., Biology Division, Oak Ridge forebody-hindbody length ratio 1:0.70-0.87; National Laboratory. Dr. Cooper (personal preoral space 60-115 long. Suckers trans- communication) noted that "The talapoin versely elongate; oral sucker subterminal ven- monkey from which the five trematodes were tral, 675-835 by 760-974; acetabulum 860- collected was received in our colony from Bata, 1,166 by 997-1,304, center at level of anterior Rio Muni (West Africa) on 26 April 1968. 52-57 per cent of body length; sucker length This animal was an adult female. . . . Talapoin ratio 1:1.22-1.39, sucker width ratio 1:1.29- monkeys are extremely common in Rio Muni 1.45. Prepharynx absent; pharynx usually and it is unlikely that the specimen was cap- longitudinally elongate, 205-290 by 200-230, tured more than 25 miles from Bata. ... In a entirely dorsal to or considerably overlapping total of 20 examinations performed on tala- oral sucker; esophagus 70-450 long; cecal poins in Rio Muni, in Cameroun, and here in bifurcation 335—510 preacetabular; ceca wide, San Diego, I have found this particular trem- terminating 220-560 from posterior extremity. atode on only one other occasion. In this case Testes two, situated symmetrically at antero- a single immature specimen was recovered lateral margins of acetabulum in three speci- from the gall bladder of a freshly captured mens, oblique with left testis anteromedian to advanced pregnant female which was pur- acetabulum in two others, usually overlapping chased in Bata from a native on 3 January acetabulum, margins smooth to slightly lobed, 1968." Specimens were fixed, without pres- usually transversely elongate but sometimes sure, in Roundabush solution, stained in Semi- longitudinally elongate, right testis 225-335 chon's acetocarmine or Mayer's carmalum, and by 225-360; left testis 245-300 by 260-365. mounted in permount. Measurements are in Vasa efferentia uniting at cirrus sac. Latter microns. median to submedian, straight to curving slightly, 250-440 by 125-160, lying 75-160 Euparadistomum cercopitheci sp. n. preacetabular, containing saccular, undulating (Figs. 1-4) seminal vesicle, short pars prostatica sur- HOST: Cercopithecus (Miopithecus) tala- rounded by prostate cells, and muscular cirrus. poin Schreber (Cercopithecidae). Genital atrium round, wide, shallow. Genital HABITAT: Gall bladder. pore median, bifurcal to postbifurcal, situated LOCALITY: Vicinity of Bata, Rio Muni; 205-520 postpharyngeal, 205-475 preace- Equatorial Guinea. tabular. SPECIMENS: USNM Helm. Coll. No. 71288 Ovary smooth to slightly lobed, median to (holotype); No. 71289 (paratypes). submedian (left), overlapping posterior part of acetabulum, longitudinally or transversely DIAGNOSIS (based on five mature specimens elongate, 265-375 by 290-335. Mehlis' gland from one host): Body flat, slightly longer than well developed, very compact, round to longitudinally elongate, situated dextral to ovary 1 Contribution from the Department of Biology, State University of New York at Binghamton, Binghamton, New in three specimens, sinistral in two others, York 13901. - Under contract No. PH43-63-56 within the Special usually overlapping ovary dorsally, 140-205 Virus Leukemia Program of the National Cancer Institute, NIH, USPHS, covering studies of the biology and potential by 120—185. Seminal receptacle situated as biomedical research utility of talapoins and other small primates. for Mehlis' gland, large, saccular, longitudinally

Figures 1-4. Euparadistomum cercopithecL 1. Whole mount, holotype, ventral view. 2. Position of testes in two paratypes, ventral view. 3. Terminal genitalia, paratype, ventral view. 4. Annulated tegument, holotype, anterolateral part of body at level of cecal bifurcation. Abbreviations: C, cirrus; CS, cirrus sac; GA, genital atrium; GC, gland cells; GP, genital pore; M, metraterm; PC, prostate cells; PP, pars prostatica; SV, seminal vesicle; U, uterus; VE, vas efferens.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 85 elongate, 300-385 by 195-220. Laurer's canal paraense (Jansen, 1941) Travassos, 1944, extending to dorsal surface near seminal recep- from a didelphid marsupial from Brazil; E. tacle. Vitelline follicles in lateral extracecal buckleyi S. N. Singh, 1958, from a fox (Can- fields, extending from pretesticular, testicular idae) from India. E. cercopitheci sp. n. ap- or acetabular level to level anterior or pos- pears closest to E. paraense, E. buckleyi, and terior to or at cecal ends, anterior and/or pos- E. francolini R. Gupta, 1959 (from a phasianid terior limits of fields in individual specimen bird from India). It differs from them in body may be subequal, one or both fields may be shape, and in possessing an annulated tegu- interrupted at ovarian level or both may be ment. It differs further from E. paraense in uninterrupted. Uterine coils extending to pos- lacking tegumental papillae, and in having terior extremity, overlapping vitellaria ven- smaller eggs and sucker ratios; from E. buck- trally, anteriorly extending to sides of oral leyi in possessing a well developed Mehlis' sucker and lateral body margins; coiling essen- gland; and from E. francolini in having smaller tially as schematically presented by Buckley eggs. and Yeh (1958) in figure 3 of their new The differences cited above for declaring species Euparadistomum heischi. Metraterm E. cercopitheci a new species appear to be shorter than cirrus sac, muscular, anteriormost relatively small in view of experimental studies extent anterior to cirrus sac and genital pore, on host influenced intraspecific morphological surrounded by gland cells. Eggs numerous, variations reported for Opisthioglyphe ranae operculate; shells of younger eggs yellow in (Frolich, 1791) Looss, 1907 (Plagiorchiidae) color, becoming yellow-brown, and finally by Grabda-Kazubska (1967), and for Telor- brown as they progress from proximal to distal chis bonnerensis Waitz, 1960 (Telorchiidae) uterine coils; 20 older eggs measuring 34-41 by Watertor (1967). However, experimental by 22-26. data on morphological variability and on life Excretory bladder Y-shaped, stem post- cycles are not known for any species of ovarian, arms extending to ovarian level; pore Euparadistomum. Additionally, E. cercopitheci terminal. is from Africa, whereas the most closely related species are either from Brazil or India. There- Discussion fore, the author feels that the new species Species of Euparadistomum Tubangui, 1931, designation for the present specimens is have been reported from lizards, birds, and justified. mammals from the Philippine Islands, North Borneo, British Solomon Islands, Malaya, Literature Cited Burma, India, Madagascar, Belgian Congo, Buckley, J. J. C., and L. S. Yeh. 1958. On Kenya, and Brazil. African species are: E. Euparadistomum heisclii n. sp. from the liver varani var. madagascariensis Capron, Deblock of a domestic cat on Pate Island, Kenya, and and Brygoo, 1961, from chamaeleonid lizards a new sub-family Euparadistominae (Dicro- from Madagascar; E. pipistrelli (Sandground, coeliidae). J. Helm. 32: 81-88. 1937) Travassos, 1944, from a vespertelionid Grabda-Kazubska, B. 1967. Morphological variability of adult Opisthioglype ranae (Frolich, bat from the Belgian Congo; E. heischi Buck- 1791) (Trematoda: Plagiorchiidae). Acta ley and Yeh, 1958, from a domestic cat Parasit. Polonica 15: 15-34. (Felidae) from Kenya. Species from mam- Watertor, J. L. 1967. Intraspecific variation malian hosts, in addition to the latter two of adult Telorchis bonnerensis (Trematoda: listed above, are: E. cerivoulae Gogate, 1939, Telorchiidae) in amphibian and reptilian from a vespertelionid bat from Burma; E. hosts. J. Parasit. 53: 962-968.

Survival and Egg Laying of Turtle Blood Flukes (Trematoda: Spirorchiidae) on the Chick Chorioallaiitois1

BERNARD FRIED AND RONALD E. TORNWALL Biology Department, Lafayette College, Easton, Pennsylvania 18042

Turtle blood flukes, tentatively identified on the chorioallantoic membranes of 56 eggs, as Spirorchis elegans Stunkard, 1923 from the and 60 S. elegans were placed on the mem- esophagus, and Spirorchis scripta Stunkard, branes of 53 eggs. 1923 from the head of naturally infected mid- western painted turtles, Chrysemys picta bellei Results (Gray), have been transplanted to the chick The results of the transplant experiments chorioallantois (Fried and Tornwall, 1965), are presented in Table 1. to determine if this site would be suitable for Twenty-eight of the 119 chorioallantois- handling spirorchiid flukes outside of the host. spirorchiids, including the 11 flukes recovered The details are presented herein. live, laid from 1-125 eggs on chorioallantoic membranes. One live fluke recovered 2 days Materials and Methods after transplantation laid about 70 eggs on the Chrysemys picta bellei obtained from a com- chorioallantois, and a second worm recovered mercial dealer (Fried, 1965) were decapitated 3 days postimplantation laid about 125 eggs and the heads cut midsagittally to expose the on the chorioallantois. Fifty chorioallantois- brain and cranial cavity. Spirorchis scripta spirorchiid eggs incubated in pond water at were removed from the surface of the brain room temperature for 10 days failed to and cranial cavity and transferred through embryonate. several changes of sterile Ringer's solution S. elegans and S. scripta removed from tur- (Paul, 1960). A turtle's plastron was severed tles and maintained at room temperature in with bone cutters and the esophagus was re- Ringer's survived no longer than 6 hr and did moved and placed in Ringer's solution. Spiror- not lay eggs. chis elegans dissected from the submucosa and Live parasites were sluggish on the chorioal- periconnective tissue of the esophagus were lantois and did not attach firmly to its surface. transferred through several changes of sterile However, when removed to Ringer's 1-3 Ringer's. days after transplantation, chorioallantois- Fertile white leghorn hens' eggs were incu- flukes were as active as in vivo spirorchiids bated at 37-38 C for 9-13 days. Sterilization placed in Ringer's, and displayed characteristic procedures, methods used to operate on eggs turtle blood fluke undulations. Dead chorioal- and to implant spirorchiids on chorioallantoic lantoic-flukes were either disintegrated and membranes were as previously described for easily lifted from the chorioallantois or were eye flukes (Fried, 1962). Spirorchiids were walled off by the chorioallantois and firmly maintained on chorioallantoic membranes for attached to its surface. 1-3 days postimplantation at 30 ± 1 C and Discussion eggs were examined as previously described (Fried, 1962). The fact that 11 (15%) of the 74 spirorchiids One hundred and nineteen spirorchiids were recovered on the chorioallantois survived for placed on the chorioallantoic membranes of 1—3 days postimplantation at 30 ± 1 C (Table 109 eggs. One to three flukes were used per 1) indicates that chick eggs can provide ade- egg and the membranes were examined 1—5 quate physicochemical conditions for turtle days later. Fifty-nine S. scripta were placed blood flukes for at least 3 days. Since 28 spirorchiids laid eggs on the chorioallantois, 1 Supported in part by a research grant from the Ad- including two specimens that laid an average vanced Studies and Research Fund of Lafayette College and in part by NIH Grants AI-03443 and AI-06835. of 35-40 eggs per day for 2-3 days, turtle

Table 1. Survival of spirorchiids on chorioallantoic membranes at 30 ± 1 C.

No. of No. of No. of days No. of live No. of dead No. of Exper. eggs worms on chorio- worms worms contaminated species used1 used2 allantois recovered3 recovered1 or dead eggs S. scripta 56 59 1-5 5 31 12 S. elegans 53 60 1-5 6 32 10 Total 109 119 1-5 11 63 22

1 Nine to 13 days old. 2 One to 2 worms per egg. 3 One to 3 days postimplantation. 4 On shell or chorioallantois. blood flukes can produce and lay eggs in this Summary site. Spirorchiid eggs from the chorioallantois Turtle blood flukes, tentatively identified as failed to embryonate, possibly because intra- Spirorchis elegans from the esophagus, and specific mating is necessary for normal egg S. scripta from the head of Chrysemys picta production. bellei, have survived on the chick chorioal- A survival rate of 15% indicates that con- lantois at 30 ± 1 C for up to 3 days. Spiror- ditions on the chorioallantois although ade- chiids maintained in Ringer's at room tempera- quate are suboptimal. Physicochemical con- ture survived no longer than 6 hours and did ditions on the chorioallantois differ from those not lay eggs. in normal sites, esophageal submucosal and Eleven of 119 spirorchiids transplanted to periconnective tissue, and cranial cavity and the chorioallantoic membranes of 109 9- to 13- surface of the brain. Spirorchiids in their day-old chick embryos maintained at 30 rt 1 C, were recovered live and laid eggs up to 3 days normal sites are usually surrounded on all sur- after transplantation. One chorioallantoic-fluke faces by host tissue and fluid, whereas the recovered 2 days after transplantation laid ap- chorioallantois makes contact only with the proximately 70 eggs, and a second worm re- ventral surface of the worm. Moreover, spiror- covered 3 days postimplantation laid about chiids have a weak oral sucker, lack an ace- 125 eggs on the chorioallantois. Attempts to tabulum and pharynx and might not be as embryonate these eggs in pond water were capable of obtaining blood and tissue from unsuccessful. the chorioallantois as are trematodes that have a pharynx and well-developed suckers and Acknowledgments have been maintained on the chorioallantoic We wish to thank Mr. Jack Carty for membrane for periods in excess of 3 days, i.e., valuable technical assistance. Philophthalmus hegeneri and Polystomoides sp. (Fried, 1962; 1965). Literature Cited Specimens of Spirorchis scripta obtained Byrd, E. E. 1939. Studies on the blood flukes from the head and Spirorchis elegans from the of the family Spirorchiidae. Part II. Revision esophagus of Chrysemys picta bellei are ref- of the family and description of new species. erable to the descriptions of S. scripta and J. Tenn. Acad. Sci. 14: 116-161. S. elegans by Stunkard (1923), Byrd (1939), Fried, B. 1962. Growth of Philophthalmus sp. and Schroecler and Ulmer (1959). Goodchild (Trematoda) on the chorioallantois of the and Kirk (1960) described the life history of chick. J. Parasit. 48: 545-550. Spirorchis elegans from the heart of Chrysemys . 1965. Transplantation of a monoge- picta picta in Georgia. It is questionable netic trematode, Polystomoides sp., to the chick chorioallantois. J. Parasit. 51: 983-986. whether their S. elegans is the same as the , and R. E. Tornwall. 1965. Survival S. elegans used in this study. The possibility and egg-laying of turtle blood flukes (Trema- also exists that S. scripta and S. elegans from toda: Spirorchiidae) on the chick chorioal- Chrysemys picta bellei are morphological var- lantois. J. Parasit. 51(Suppl.): 46. iants of the same species. Goodchild, C. G., and D. E. Kirk. 1960. The

life history of Spirorchis elegans Stunkard, Host-parasite relationships of Spirorchis ele- 1923 (Trematoda: Spirorchiidae) from the gans Stunkard (Trematoda: Spirorchiidae). painted turtle. J. Parasit. 46: 219-229. Proc. Iowa Acad. Sci. 66: 443-454. Paul, J. 1960. Cell and Tissue Culture. 2nd Stunkard, H. W. 1923. Studies on North ed. Williams & Wilkins Co., Baltimore, Md. American blood flukes. Bull. Amer. Mus. Schroeder, P. J., and M. J. Ulmer. 1959. Nat. Hist. 48: 175-221.

Pathogenicity of the Northern Root-knot Nematode (Meloidogyne hapla) to Potato1

G. D. GRIFFIN AND E. C. JoRGENSON2

The northern root-knot nematode, Meloi- decline in the mobility of Heterodera schachtii dogyne hapla, is considered a serious pest of as soil moisture decreased, which may directly potatoes in the Western United States. We affect root infection. have seen fields where approximately all of The role of host age on root infection by the tubers are either infected or galled by this root-knot nematodes was observed by Christie nematode, making them commercially unde- (1936), who stated that second stage root-knot sirable. We have also observed the occurrence larvae usually enter roots near the tip. This of a deep-seated tuber infection (nematodes accounts for the congregating of nematodes near the center of the potato tuber). We sus- at or near the meristematic root tissue up to 24 pected that age of plant, soil temperature, and hr until the root is penetrated (Godfrey and soil moisture determine the extent of invasion Oliveira, 1932; Peacock, 1959; Loewenberg, and severity of galling of potato roots and Sullivan, and Schuster, 1960). The ability of tubers. Gross (1940) found galling of potato root-knot larvae to penetrate only delicate root tubers by Heterodera marioni (Meloidogyne tissue is evidenced by the galling of only the sp.) less in soil irrigated every 3—5 days at a tip of large tap roots, in some instances in soil temperature of 17-18 C, than under less fumigated soil. frequent irrigations of 7—9 days where soil This study was made to determine (1) the temperature went as high as 26.5 C. Godfrey relationship between soil temperature, soil (1926) reported an increase in root-knot moisture, and the pathogenicity of Meloi- galling of potato plants up to 27 C. Parris dogyne hapla on potato under controlled con- (1948) reported that the higher the soil ditions, and (2) if size or age of roots and moisture, the greater tuber galling by Hetero- tubers affect penetration and galling by root- dera marioni (Meloidogyne sp.). He found knot larvae. larvae near the cambium below swollen lenticels and concluded that nematodes entered Materials and Methods through lenticels of tubers grown in soil with To study the effect of temperature on patho- a high moisture content. Godfrey (1926) genicity of M. hapla Chitwood on potato found that optimum root-knot galling of to- (Solanum tuberosum L.), Russet Burbank mato roots occurred between 50 and 80% potato seed pieces were surface sterilized with field capacity, and Peacock (1957) obtained 1:600 solution of rnethylmercury dicyandi- similar data. Wallace (1956) observed a amicle for 60 sec, suberizecl for 24 hr and

1 Cooperative investigation of Crops Research Division, planted in flats of fumigated soil. After 15 Agricultural Research Service, United States Department days, plants, minus seed pieces, were trans- of Agriculture and the Utah State Agricultural Experiment Station. Journal Paper No. 747. planted in 3.8 liter polyethylene containers of 2 Research Nematologists, Crops Research Division, Agri- cultural Research Service, United States Department of Provo sand (M.H.C. = 17%) and inoculated Agriculture, Crops Research Laboratory, Utah State Uni- versity, Logan, Utah. with 1,000 M. hapla larvae. Plants were grown

Table 1. Effect of temperature on pathogenicity of Meloidogyne hapla to Russet Burbank potatoes.1

Roots Tubers

°c Avg wt. Gall rating2 No. per plant Avg wt. Gall rating2 20 4.16b 2.14d 2.00f 6.24g 0 25 3.48b 4.00e 3.00f 2.85g 0 30 4.51b 3.71e 0 — 35 0.25a 0.67c 0 - Numbers with same letter do not differ significantly at 5% level. 1 1,000 M. hapla larvae per plant. 2 Gall rating: 0, none; 1, very few; 2, slight; 3, moderate; 4, severe; 5, very severe. at temperatures of 20, 25, 30, and 35 C. Each desired level. To be sure the soil moisture was treatment (temperature) was replicated four maintained at the desired level, control plants times, and grown under a 16 hr day. The of comparable size were weighed weekly and experiment was concluded after 60 days, and compensation was made for any increase in plant growth and iiematode pathogenicity plant weight. The study was terminated after determined. 130 days; root and tuber growth and infection The relationship between galling and plant and galling of roots and tubers by M. hapla age was determined by inoculating Russet was determined. Burbank seed pieces, and 3- and 6-week-old The other method, the "field capacity plants (minus seed pieces and rhizomes) with method," was similar except that the soil 1,000 larvae of M. hapla. Plants were grown moisture of all containers was initially adjusted on a greenhouse bench in 15.2 cm clay pots to field capacity. Water was not added to at 22 ± 4 C; treatments consisted of six the containers until soil moisture had dropped inoculated and six control plants. The experi- to or below previously determined soil mois- ment was ended after 140 days, and tuber ture levels of 25, 50, 75, and 100% E.G.; then and root weights, and nematode pathogenicity enough water was added to restore F.C. determined. (Couch, Purdy, and Henderson, 1967). Repli- The effect of tuber size on nematode infec- cates consisted of six inoculated and six con- tion was studied by inoculating plants from trol plants at each soil moisture level. Plants which all but one potato bud or tuber were were grown for 80 days on a greenhouse bench removed. Twenty-five 12-week-old Russet at 22 ± 4 C, and root growth, and root infec- Burbank plants, each labeled as to size of bud tion by M. hapla was recorded. or tuber, were inoculated as previously stated and transplanted into a 15.2 cm clay pot of Results and Discussion Provo sand. Potato buds and tubers ranged Potato plant growth was affected as much from 5—57 mm in diameter. These plants were by temperature as by the pathogenicity of grown for 60 days at 22 ± 4 C, and tuber M. hapla. The least root growth occurred at infection was determined. 35 C, but there were no significant differences Two methods were used to study the effect in root growth of plants from 20-30 C (Table of soil moisture on infection and galling. The 1). However, tubers were formed only at "percent soil moisture method" involved trans- 20 and 25 C, and root galling -was most severe planting 15-day-old plants into 3.8 liter poly- at 25 and 30 C; root galling, like root growth, ethylene containers of Provo sand and inoculat- was adversely affected at 35 C. It appears that ing each plant with 1,000 larvae of M. hapla. a temperature of approximately 25 C is opti- Containers of soil with the moisture levels of mum for maximum expression of root galling 25, 50, 75, 100, and 125% of field capacity on Russet Burbank potato. The slight amount (F.C.) were replicated 6 times per treatment of galling at 20 C agrees with Gross (1940) in a chamber with relative humidity of 80— who found galling of tubers to be less at 17-18 100%, and a soil temperature of 22 ± 2 C. C than at temperatures up to 26.5 C. This Each container was weighed daily, and dis- undoubtedly affects the degree of severity of tilled water added to maintain moisture at the tuber galling from year to year when differ-

Table 2. Effect of age of plant at time of inoculation on root and tuber galling of Russet Burbank potatoes by Meloidogyne hapla.

Age of Avg root Root Avg Per cent Tuber inoculated weight gall tuber Per cent tubers gall plant (g) rating1 weight infected galled rating1 6 weeks — Inoculated 0.78a 2.00c 10.50d 85 85 S.OOf Control 5.1 Ob 21.20e 3 weeks — Inoculated 2.25a 2.22c 8.60d 92 92 3.33f Control 5.56b 20.74e Seed pieces — Inoculated 4.35b 2.29c 6.86d 83 35 3.57f Control 5.33b 7.26d Numbers with same letter do not differ significantly at 5% level. 1 1,000 M. hapla larvae per plant. Tuber and root gall rating: 0, none; 1, very few; 2, slight: 3, moderate; 4, severe; 5, very severe; temperature, 20- 25 C. ences in soil temperatures are noted. However, from 41—57 mm in diameter, which may be we have no data on soil temperatures in potato explained by the greater surface area exposed fields, and can only speculate. to larval invasion. Tuber infection was not affected by differ- The pathogenicity of M. hapla to Russet ences in plant age at time of inoculation. Burbank potato roots and tubers was affected Tubers were readily infected on plants grown by soil moisture. Roots and tubers were galled from inoculated seed pieces, and from 3- and at all moisture levels from 25-100% F.C. with 6-week-old plants (Table 2). However, fewer the "percent soil moisture method" (Table 4). tubers were galled on plants from inoculated Tubers were not galled at 125% F.C., but 74% seed pieces, which apparently was due to the of the tubers were infected. Root galling was late tuber set as shown by the small-sized significantly less at 125% of F.C. than at other tubers when compared with those grown from moisture levels. This was apparently explained 3- and 6-week-old plants. There were signifi- by Shepherd and Wallace (1959), who found cant differences between root growth of con- that the infection of hosts by Heterodera trol and inoculated 3- and 6-week-old plants, schachtU and H. gottingiana was greatest when which showed the pathogenicity of M. hapla most of the soil pores are free of water. This to potato plants. However, there were no may also be attributable to low oxygen and significant differences between inoculated and gaseous exchange in near saturated soil (Van control plants from seed pieces, because more Gundy, Stolzy, Szuszkiewicz, and Rackman, than one root system developed from a single 1962). seed piece. Maximum root growth occurred at 50, 75, Infection was not greatly affected by tuber and 100% F.C., and minimum root growth was size. Larvae were able to penetrate large and at 25 and 125% F.C. small tubers (Table 3). There were, however, The "field capacity method" gave results a greater number of larvae infecting tubers similar to those obtained with the "percent soil moisture method," and resulted in root and tuber infection at 25, 50, 75, and 100% F.C. Table 3. Influence of bud and tuber size of (Table 5). However, tubers were not galled, Russet Burbank potato on infection by Meloi- dogyne hapla. because of the short duration of the study. The greatest root growth occurred at 100%, while Bud and tuber Per cent of Degree 25% of F.C. resulted in the smallest root sys- diameter buds and tubers of ^YniTv)1 infected infection- tem. There \vere no differences in root weights between control and inoculated plants at 25% 5-15 100 2.16 ± 0.42 16-25 100 2.42 ± 0.28 F.C.; roots of these plants were reduced as 26-41 100 2. 65 ±0.36 much or more by low soil moisture as by nema- 41-57 100 3.17 ±0.51 tode pathogenicity. However, at 50, 75, and

1 Size at time of inoculation. 100% F.C. roots of inoculated plants were sig- -Degree of infection: 0, none; 1, light; 2, moderate; 3, heavy; 4, very heavy. nificantly smaller than control plants, although

Table 4. Effect of soil moisture on root and tuber galling of Russet Burbank potatoes by Meloidogyne hapla in controlled moisture chamber of 80-100% relative humidity at 22 — 2 C ("Percent soil moisture method").

Soil moisture Avg Root Per cent Per cent Tuber ( per cent field root wt. gall tubers tubers gall capacity ) (g) rating1 infected galled rating 25 15.80a 4.38e 95 95 4.25g 50 26.32b 4.60e 96 96 4.60gh 75 23.66B S.OOf 100 100 4.67gh 100 29.600 S.OOf 100 100 S.OOh 125 18.34a 3.18d 74 0 0 Numbers with same letter do not differ significantly at 5% level. 1 15-day-old transplants inoculated with 1,000 M. hapla larvae per plant, and grown for 130 days. Root and tuber gall rating: 0, none; 1, very few; 2, slight; 3, moderate; 4, severe; 5, very severe. galling was as severe at 25 as at 50% F.C. mobility (Wallace 1956) and migration of the The point of tuber penetration was appar- nematode to the site of infection. Nematodes ently through the lenticels, because most of were found in the tuber just outside the cam- the nematodes were found immediately below bial layer at all moisture levels, and less than or near the lenticels at 125% F.C. However, 5% of the nematodes were found beneath the there was no affinity for the area below or cambium. There was no deep seated infection near the lenticels at the other soil moisture observed in any of the infected tubers. It is, levels, and an epidermal penetration remains therefore, apparent that deep-seated infection a possibility. Parris (1948) stated that root- of tubers by M. hapla is fortuitous or caused knot larvae enter potato tubers through swollen by factor(s) not considered in this study. lenticels. We found no significant differences Comparing the two methods used for con- between tubers infected at 50 and 75% F.C., trolling soil moisture, the "field capacity although tuber lenticels were swollen only at method" is considered the better method since 75, 100, and 125% F.C. The low soil moistures it more closely compares to field conditions, of 25 and 50% F.C. apparently inhibited len- and is more easily controlled in the greenhouse. ticel swelling, but did not prevent infection Although there were significant differences and galling of tubers by M. hapla. Tubers with in root and tuber infection and galling between swollen lenticels at 100% F.C. were generally the different soil moisture levels, it appears the most heavily galled, which suggests that that soil moisture is not of major importance tubers may be penetrated more readily through in the host—parasite relationship. Over 70% the swollen lenticels. However, the very severe of all tubers were infected at all soil moisture tuber galling at 100% F.C. may be partly due levels; root and tuber galling was severe or to the effect of soil moisture on the nematode very severe at all moisture levels but 125%

Table 5. Effect of soil moisture on root galling of Russet Burbank potatoes by Meloidogyne hapla on greenhouse bench at 22 — 4 C ("Field capacity method").

Soil moisture Avg Root Per cent Per cent (per cent field root wt. gall tuber tubers capacity ) (g) rating1 infected galled 25 Inoculated 4.42a 4.24e 71 0 25 Control 4.86a - — 50 Inoculated 7.86b 4.49e 100 0 50 Control 10.71c . — - 75 Inoculated 8.09b S.OOf 100 0 75 Control 12.29c — - 100 Inoculated 8.31b S.OOf 100 0 100 Control 16.30d — - Numbers with same letter do not differ significantly at 5%. 1 15-day-old transplants inoculated with 1,000 M. hapla larvae per plant and grown foi- clays. l-loot gall rating: 0, none; 1, very few; 2, slight; 3, moderate; 4, severe; 5, veiy severe.

F.C.; nematode infection occurred outside the Virginia Polytecnic Inst., Dept. Plant Path. cambial layer, and very few (less than 5%) Bull. No. 4. 23 pp. were found immediately below the cambial Godfrey, G. H. 1926. Effect of temperature layer. However, root weights of infected plants and moisture on nematode root knot. J. Agric. Res. 33: 223-254. at all soil moisture levels but 25% F.C. were , and Juliette Oliveira. 1932. The significantly less than those of uninfected development of the root-knot nematode in plants. Therefore, we conclude that variation relation to root tissue of pineapple and cow- in soil moisture is important only in the degree pea. Phytopathology 22: 325-348. of pathogenicity, and while it may contribute Gross, A. E. 1940. Controlling root-knot nema- to difference in severity of galling, the presence tode in potatoes in the Klamath Basin by or absence of root and tuber galling, and deep- means of irrigation. Plant Dis. Reptr. Suppl. seated potato tuber infection are conditioned 124: 142. Loewenberg, J. R., T. Sullivan, and M. L. by other factor(s). Schuster. 1960. Gall induction by Meloi- dogyne incognita incognita by surface feed- Summary ing and factors affecting the behavior pattern Root galling of Russet Burbank potatoes by of the second stage larvae. Phytopathology the northern root-knot nematode, Meloidogyne 50: 322. hapla, increased as temperature increased from Parris, G. K. 1948. Influence of soil moisture 20-30 C. Roots were slightly galled at 20 C, on the growth of the potato plant and its and severely galled at 25 and 30 C. There infection by the root-knot nematode. Phy- topathology 38: 480-488. was a very light galling at 35 C, which was Peacock, F. C. 1959. The development of a associated with very little root growth. There technique for studying the host-parasite rela- were no differences in root growth from 20-30 tionship of the root-knot nematode Meloi- C, but tubers were formed only at 20 and 25 C. dogyne incognita under controlled conditions. Tuber infection was not affected by plant Nematologica 4: 43-55. age or tuber size at time of inoculation. Shepherd, A. M., and H. R. Wallace. 1959. A comparison of the rates of emergence and M. hapla infected potato tubers at moisture invasion of beet eelworm, Heterodera schactii levels from 25-125% F.C. in Provo sand. The Schmidt and pea root eelworm Heterodera greater number of larvae infected tubers at gottingiana Liebscher. Nematologica 4: 227- 100% F.C., and the least at 125%. Infection 235. was mainly through the lenticels. Van Gundy, S. D., L. H. Stolzy, T. E. Szuszkie- wicz, and R. L. Rackman. 1962. Influ- Literature Cited ence of oxygen supply on survival of plant- parasitic nematodes in soil. Phytopathology Christie, J. R. 1936. The development of 52: 628-632. root-knot nematode galls. Phytopathology Wallace, H. R. 1956. The emergence of 26: 1-22. larvae from cysts of the beet eelworm, Couch, H. B., L. H. Purdy, arid D. W. Hender- Heterodera schachtii Schmidt in aqueous son. 1967. Application of soil moisture solutions of organic and inorganic substances. principles to the study of plant disease. Ann. Appl. Biol. 44: 274-282.

Some Gastrointestinal Helminths of Ondatra zibethicus Linnaeus, the Muskrat in Maryland1

JAMES B. ABRAM Biology Department, Maryland State College, Princess Anne, Maryland

The muskrat, Ondatra zibethicus, has been for helminth recovery. The aliquot technique an important wildlife entity in Maryland, both involved the use of a dissecting microscope in from the standpoint of adding to the economy examining around 10% of the total volume of and in providing opportunity for scientific the contents of each organ. The sieving investigations. The State has long been recog- method entailed placing a wide strip of fine nized for its large production of pelts, and gauze material over the top of a metallic test muskrat meat is a table delicacy in the area. tube rack and slowly sieving the organ con- Several muskrat studies have been contents through the gauze. The helminths were ducted in the Dorchester County marshes on retained on the gauze and were removed by a the Eastern Shore. The biology and habitats dissecting needle. Routine methods for the of the species have been written of exten- preparation of the helminths followed. This sively, but the aspect of parasitism has been included: staining trematodes with alum neglected. Not since the study of helminths cochineal, borax carmine, Semichon's aceto- of Maryland muskrats by Price (1931), who carmine, and Delafield's hematoxylin. The described four new trematode species, has a nematodes were not stained. After dehydra- similar study been reported. tion in ethyl alcohol, the helminths were The muskrats of the present study were cleared in beechwood creosote or methyl salicy- trapped near Thurmont in Frederick County late and mounted with Canada balsam. and in the Dames Quarter marshes in Somerset County, from November 1966 through No- Results and Discussion vember 1967. The gastrointestinal tracts of the muskrats The collecting areas were ecologically dif- examined yielded five species of trematodes ferent in several respects. The Thurmont and one nematode species, as shown in Table I. study area consisted of five freshwater ponds, Approximately 71% of the Thurmont area located within a 10-mile radius of one another, muskrats and about 51% of those of the Dames near the Catoctin range of the Blue Ridge Quarter area were infected with one or more Mountains. The Dames Quarter marshes, in- of the helminths listed. None of the three fluenced by tides from Tangier Sound and the trematode species recovered from the Thur- Manokin River, are a preferred muskrat habi- mont group was found in the Dames Quarter tat. Their salinity averaged around 11.91^ group; neither trematode collected from the during the study. latter group occurred in the Thurmont group. The nematode, Trichuris opaca Barker and Materials and Methods Noyes, 1915, occurred in both localities. The The muskrats were captured in a Conibear Dames Quarter group had a higher incidence trap which kills the animals by drowning. All of infection with it. taken in the Thurmont area were Ondatra A study of the literature indicated that all zibethicus zibethicus; all captured in the of the helminths recovered in this study are Dames Quarter area were O. z. macrodon. widely distributed in muskrats in this country. Organs of the gastrointestinal tract, stomach All prior reports hereinafter cited pertain to through large intestine, of each muskrat were occurrence in this host. separately examined for helminths. An aliquot T. opaca reportedly occurs in Michigan technique or a sieving method was employed (Ameel, 1944), Ohio (Rausch, 1946; Beckett and Gallicchio, 1967), New York (Edwards, 1 A portion of a thesis submitted in partial fulfillment of 1949), Wisconsin (Tiner, 1950), Colorado the requirements for the degree of Doctor of Philosophy, Oklahoma State University, Stillwater, Oklahoma. (Ball, 1952), Illinois (Gilford, 1954), Oregon

Table 1. Results of survey of the gastrointestinal helminths of 154 Maryland muskrats.

Study area and Location No. animals No. animals Per cent helminth in host examined infected infected Thurmont Echinostoma rec/olutum Small intestine 79 42 53.2 Quinqueserialis quinqueserialis Caecum 79 19 24.0 Wardius zibethicus Caecum 79 26 32.9 Trichuris opaca Caecum 79 7 8.9 Dames Quarter Nudacotijle novica Small intestine 75 27 36.0 Echinochasmus schivartzi Small intestine 75 3 4.0 Trichuris opaca Caecum 75 18 24.0

(Senger and Neiland, 1955), Alaska (Duna- the Dorchester County marshes, occurred in gan, 1957), and Pennsylvania (Anderson and only 4% of the Dames Quarter sample. It also Beaudoin, 1966). occurs in Texas (Chandler, 1941), Louisiana Echinostoma revolutum (Frolich, 1802), (Penn, 1942), and Tennessee (Byrd and which was recovered only from the Thurmont Reiber, 1942). group, had the highest helminth infection rate On the basis of the limited studies reported, (53.1%). This helminth also occurs in Massa- the helminth fauna of muskrats in Maryland chusetts (Rankin, 1946), New York (Edwards, appears to vary from one locality to another. 1949), Illinois (Gilford, 1954), Oregon (Sen- In this particular study, the chief environ- ger and Neiland, 1955; Rider and Macy, mental factor that appears to be the most in- 1947), Pennsylvania (Anderson and Beaudoin, fluential on these parasite differences is the 1966), and Ohio (Beckett and Gallicchio, consistent salinity of the coastal marshes. This 1967). factor limits the ability of the freshwater gas- Wardius zibethicus (Barker and East, 1915), tropods, preferred intermediate hosts for cer- the second most frequently occurring helminth tain trematodes, to persist in the Dames Quar- in the Thurmont muskrats, has been reported ter marshes. in Ohio (Rausch, 1946; Beckett and Gallicchio, While this article does not dwell on the 1967), Illinois (Gilford, 1954), and Penn- influence that ecology plays in controlling the sylvania (Anderson and Beaudoin, 1966). range of helminths, it is clear that differences Quinqueserialis quinqueserialis Barker and in the biological and physical environment play Laughlin, 191.1, which also occurred only in a key role in parasite distribution. Thurmont muskrats, occurs in Tennessee (Har- wood, 1939), New York (Edwards, 1949), Literature Cited Maine (Meyer and Reilly, 1950), Illinois Ameel, D. J. 1944. A life history of Nuda- (Gilford, 1954), Oregon (Senger and Neiland, cotyle novica Barker, 1916 (Trematoda, 1955), Alaska (Dunagan, 1957), Utah (Sen- Notocotylidae). J. Parasit. 30(4): 257-263. ger and Bates, 1957), Pennsylvania (Anderson Anderson, D. R., and R. L. Beaudoin. 1966. and Beaudoin, 1966), and Ohio (Beckett and Host habitat and age as factors in the prev- Gallicchio, 1967). alence of intestinal parasites of the muskrat. Nudacotyle novica Barker, 1916, the most Bull. Wildl. Dis. Assoc. 2: 70-77. frequently occurring helminth in the muskrats Ball, L. 1952. Notes on the helminth para- of the Dames Quarter marshes, occurs in fresh- sites of muskrats from western Colorado. and brackish-water habitats, and in Texas J. Parasit. 38: 83-84. (Chandler, 1941), Louisiana (Penn, 1942), Beckett, J. V., and V. Gallicchio. 1967. A survey of helminths of the muskrat, Ondatra Michigan (Ameel, 1944), Ohio (Rausch, 1946; z. zibethica Miller, 1912 in Portage County, Beckett and Gallicchio, 1967), New York (Ed- Ohio. J. Parasit. 53: 1169-1172. wards, 1949), Maine (Meyer and Reilly, 1950), Byrd, E. E., and R. J. Reiber. 1942. Mam- and Illinois (Gilford, 1954). malian trematodes II. Three flukes from Echinochasmus schwartzi Price, 1931, al- small mammals. J. Tenn. Acad. Sci. 17: though originally described from muskrats of 143-148.

Chandler, A. C. 1941. Helminth parasites of sites of the muskrat. Proc. U. S. Natl. Mus. muskrats in southeast Texas. T- Parasit. 27: 79: 1-13. 175-181. Rankin, J. S. 1946. Helminth parasites of Dunagan, T. T. 1957. Helminth parasites of birds and mammals in western Masschusetts. Alaskan muskrats. Trans. Amer. Micr. Soc. Amer. Midi. Natural. 35: 756-768. 76: 318-320. Rausch, R. L. 1946. The parasites of Ohio Edwards, R. L. 1949. Internal parasites of cen- muskrats. J. Wildl. Mgmt. 10: 70. tral New York muskrats (Ondatra zibethica Rider, C. L., and R. W. Macy. 1947. Pre- zibethica L.). J. Parasit. 35: 547-548. liminary survey of the helminth parasites of Gilford, J. H. 1954. A survey of muskrat muskrats in northwest Oregon, with descrip- helminths in Illinois. J. Parasit. 40: 702-703. tion of Hymenolepis ondatrae n. sp. Trans. Harvvood, P. D. 1939. Notes on Tennessee Amer. Micr. Soc. 66: 176-181. helminths. IV. North American trematodes Senger, C. M., and J. W. Bates. 1957. The of the subfamily Notocotylinae. J. Term. occurrence of Hymenolepis evaginata and H. Acad. Sci. 14: 332-340; 421-437. ondatrae in Utah muskrats. Proc. Helm. Meyer, M. C., and J. R. Reilly. 1950. Para- Soc. Wash. 24: 141-142. sites of muskrats in Maine. Amer. Midi. , and K. A. Neiland. 1955. Helminth Natural. 44: 467-477. parasites of some fur-bearers of Oregon. J. Parasit. 41: 637-638. Penn, G. H. 1942. Parasitological survey of Tiner, J. D. 1950. Two new species of Trich- Louisiana muskrats. J. Parasit. 28: 348-349. uris from North America, with redescrip- Price, E. W. 1931. Four new species of trema- tions of Trichuris opaca and Trichuris leporis tode worms from the muskrat, Ondatra (Nematoda: Asphasmidia). J. Parasit. 36: zibethica, with a key to the trematode para- 350-355.

The Use of Irradiated Third-stage Larvae of Angiostrongylus cantonensis as Antigen to Immunize Albino Rats Against Homologous Infection1

SHERIDAN H. LEE Parasitology Laboratory, Department of Animal Sciences, University of Hawaii, Honolulu, Hawaii 96822

Lim, Ow-Yang and Lie (1965) and Heyne- has a long brain phase 28-day plus, in its life man and Lim (1965) reported that rats which cycle (Mackerras and Sandars, 1955). received one to three sublethal feedings of normal third-stage larvae of Angiostrongylus Materials and Methods cantonensis were able to develop some degree Third-stage larvae of A. cantonensis were of resistance to reinfection as manifested by harvested from laboratory infected giant Afri- the reduced worm burden. Like many other can snails, Achatina fulica, by digesting minced helminthic infections, no sterile immunity was snail tissue in \% pepsin and 1% concentrated obtained (Sprent, 1963). hydrochloric acid solution. Digested snail tis- Jarret et al. (1959) reported success in ob- sue was sieved through a 40-mesh wire screen taining sterile immunity to Dictyocaulus viviparus by using irradiated larvae. This sug- into an Imhoff flask and allowed to settle for gested that a similar result may also be 40 min. After the supernatant liquid was obtained with A. cantonensis since the latter siphoned off, the sediment was baermannized in saline through two 16-ply gauze pads sup- 1 This investigation was supported by Research Grant ported by a 40-mesh copperwire sieve. The NB-04965 from the Institute of Neurological Diseases and Blindness, NIH, USPHS, to Dr. J. E. Alicata. Published larvae were harvested 30 and 50 min later. with the approval of the Director of the Hawaii Agricul- tural Experiment Station as Technical Paper No. 905. Harvested larvae were rebaermannized and

Table 1. Number of worms recovered from the lungs of immunized and control rats of Experiment I, II, and III following a challenge with 400 normal larvae.

Experiment I Experiment II Experiment III

Rat no. Immunized Control Immunized Control Immunized1 Control 1 94 152 6 145 16 (2) 254 2 81 234 10 178 14 (0) 238 3 39 198 2 122 34 (6) 181 4 61 260 0 72 14 (12) 134 5 46 173 0 124 23 (8) 156 6 49 234 9 132 19 (0) 223

1 Number of young adults on brain surface in parenthesis. then washed three times in distilled water. received 200, 40 Kr treated larvae each. The About 10,000 washed larvae were placed in following numbers of worms were recovered 2 cc of distilled water in small screw capped from the lungs of the experimental rats which vials and irradiated with the desired dosage. were challenged with 400 normal larave: 94, The radiation source was a 30,000-curie 81, 39, 61, 46, and 49. The number of worms cobalt-60 irradiator located in the department found in each control rat was 152, 234, 198, of Food Science and Technology of the Uni- 260, 173, and 234 (Table I). versity of Hawaii. When this study began, the EXPERIMENT II. The number of worms output was 4.7 Kr/min at 5 inches. All larvae recovered from the lungs of each experimental irradiated were drawn from a common pool. rat, which had received two feedings of 200, EXPERIMENT I. Twelve rats were used, each 40 Kr treated larvae before challenge, was 6, being given one feeding of 200, 40 Kr treated 10, 2, 0, 0, and 9; whereas, the number of larvae. Six of these rats were killed on the worms found in each control rat was 145, 178, 30th day after feeding; the other six were 122, 72, 124, and 132 (Table I). All the challenged with 400 normal larvae each, 30 worms recovered in both experiments were days after feeding, and killed 42 days after fertile and none of the rats had any worms challenge. in the brain. None of the rats appeared to EXPERIMENT II. Six rats were used, each have suffered any ill effects during the entire was given two feedings of 200, 40 Kr treated period of this experiment. larvae with a 30-day interval between each EXPERIMENT III. The following results feeding. Thirty days after the last feeding, each was challenged with 400 larvae and killed were obtained from rats each of which re- 42 days later. ceived three feedings of 200, 20 Kr treated EXPERIMENT III. In this experiment, each larvae before challenge: Rat 1 had two dead young adults on its brain surface and in its of six rats was given three feedings of 200, lungs were 16 adult worms, 7 males and 9 20 Kr treated larvae with a 15-day interval females, four of the latter being sterile. Rat 2 between each feeding. Thirty days after the had in its lungs 14 adult worms, 5 males and last feeding, each rat was challenged with 400 normal larvae and killed 42 days later. 9 females, five of the latter being sterile. A few first-stage larvae were found in the lung An equal number of control rats were given tissue. Rat 3 had four live young adults and 400 normal larvae to accompany all the above two dead young adults on its brain surface, 7 experiments. All the control rats were of the males and 27 females, in its lungs. Five of same age as the experimental rats. EXPERIMENT IV. Two rats were given 200, the 27 females were dead with only their 20 Kr treated larvae each. One of these rats cuticle and degenerate digestive tract remain- was killed 40 days later and the other was ing. First-stage larvae were present in the killed 62 days later. lung tissue. Rat 4 had three live young adults on its brain surface plus the remains of nine Results dead young adults which appeared as yellow- EXPERIMENT I. One adult female worm was ish green lines. In its lungs were 3 sterile recovered from one of the six rats which males and 11 females, four with eggs and

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 97 two without eggs. Five females were in the It would seem that the protracted stay of process of being absorbed. Rat 5 had seven young adults in the subarachnoid space should partially absorbed young adults plus one live cause the rat to have a stronger immune re- young adult on its brain surface. In its lungs sponse. However, the results of Experiments were 13 females, three without eggs, and 10 II and III do not support this idea, at least males, seven of which were sterile. A few when the quality of immunity is measured in first-stage larvae were present in the lung terms of worm burden after reinfection. Why parenchyma. Rat 6 had no worm on its brain the dosage of 40 Kr makes the third-stage lar- surface. There were 8 males and 11 females vae of A. cantonensis serve as a better antigen in its lungs, seven of which were sterile. No as compared with 20 Kr against homologous first-stage larvae were found in the lung infection remains an unsolved but interesting parenchyma. The number of worms found in question. the control rats for Experiment III was 254, Summary 238, 181, 134, 156, and 223 (Table I). EXPERIMENT IV. Eighteen sterile worms Of six rats which received two feedings of were found in the lungs of the rat killed on 200, 40 Kr irradiated third-stage larvae of the 40th day after being fed 200, 20 Kr treated Angiostrongyhls cantonensis, two became com- larvae. There were no worms in its brain. The pletely refractive and four showed significant second rat which was killed on the 62nd day resistance when challenged with normal larvae. Twenty Kr radiation not only destroyed the after being fed 200, 20 Kr treated larvae had germ cells of A. cantonensis, but also caused six sterile females in its lungs and six young adults on the surface of its brain. Two of the most of the young adults to lose their ability to migrate to the lungs of their definitive host. six young adults were still alive whereas the other four were dead. Acknowledgment The author wishes to acknowledge the tech- Discussion nical assistance of Dr. M. D. Upadhya in Experiment I indicated that a radiation irradiating all the larvae used in this paper. dosage of 40 Kr was quite detrimental to the Literature Cited further development of the third-stage larvae of A. cantonensis. Yet the antigenecity of these Heyneman, D., and B. L. Lim. 1965. Pro- longed survival in rats immunized by a 40 Kr treated larvae was not lost as indicated small number of low-level doses of Angio- by the reduced worm burden of the immunized strongylus cantonensis and challenged with a rats when challenged. This experiment also lethal level of infective larvae. Med. J. indicated that immunity acquired by the ex- Malaya 20(2): 162-163. perimental rats did not depend on the existence Jarrett, W. F. H., F. W. Jennings, W. I. M. of live worms within the rats. Mclntyre, W. Mulligan, N. C. C. Sharp, In Experiment II, the complete absence of and G M. Urquhart. 1959. Immunolog- worms in two of the experimental rats and ical studies on Dictyocaulus viviparus in the relative low number of worms in other calves—double vaccination with irradiated larvae. Am. J. Vet. Res. 20: 522-526. rats of this group suggests that two feedings Lim, B. L., C. K. Ow-Yang, and K. J. Lie. of 200, 40 Kr treated larvae offers the best 1965. Natural infection of Angiostrongylus immunizing procedure. cantonensis in Malaysian rodents and inter- Experiment IV indicated that most likely, mediate hosts, and preliminary observations all the sterile worms recovered from the experi- on acquired resistance. Am. J. Trop. Med. 14(4): 610-617. mental rats of Experiment III were developed Mackerras, M. J., and D. F. Sandars. 1955. from the 20 Kr treated larvae used for im- The life history of the rat lungworm, Angio- munization. It is interesting to note that strongylus cantonensis (Chen) (Nematoda: Experiment IV not only demonstrated the Metastrongylidae). Austr. J. Zool. 3: 1-25. detrimental effect of the dosage of 20 Kr to Sprent, J. F. A. 1963. Parasitism—an introduction to parasitology and immunology for the germ cells of A. cantonensis but also students of biology, veterinary science and revealed that this dosage will prolong the medicine. Williams and Wilkins, Baltimore, brain phase of this worm. 145 pp.

Hypsoperine ottersoni sp. n. (Nemata, Heteroderidae) Infesting Canary Grass, Phalaris arundinacea (L.) Reed in Wisconsin

GERALD THORNE1

Unusual types of nematode-incited galls nemas were directed toward the root terminus. were observed on canary grass in the Wind Eggs usually are deposited in a gelatinous Lake area near Kenosha, Wisconsin by Dr. matrix within the gall tissues, but in some Henry Otterson, Agricultural Chemist and Con- instances they and the matrix are extruded sultant, who submitted specimens for identifi- outside the root. Feeding takes place along cation in 1958, which later were recognized as the cells between the xylem tubes which fre- belonging to the genus Hypsoperine Sledge quently are multiplied and broken apart (Fig. and Golden, 1964. Detailed studies were not IB). Even in extreme instances like this, the begun until 1963 when Arthur Weber under- root cap and terminal cells remain intact, took the project as a thesis problem in grad- enabling continued growth. uate work under the direction of Dr. K. R. Barker, University of Wisconsin, Madison. Hypsoperine ottersoni sp. n.- Validity of the genus Hypsoperine has been Morphology questioned by Dr. M. W. Allen (personal FEMALE (10): 0.27 by 0.45 mm (0.18- communication). It is true that the elevated 0.32 by 0.39-0.52). Variable in form as indi- perineal region sometimes is not evident and cated by these measurements (Fig. 2N—S). the cuticle is not thickened on this species, as Spheroid to elongate pyriform with neck pro- reported by Sledge and Golden (1964) for jecting at different angles (Fig. 2N-S). Vulva H. graminis. However, the physiological effect and anus usually on a slight elevation, but on the host, causing an unusual type of gall sometimes this is not present and the posterior formation with larvae always lying in the root end is similar to that of Meloidogyne. Cuticle with heads toward the apex, is unlike that of 3—6 /j. thick on main part of body, leathery usual Meloidogyne infestations. The hemizonid with extremely fine striae except on head and of H. graminis is reported anterior to the ex- neck where they are easily visible and form cretory pore, while that of H. spartinae is annules variable in width. Lip region un- posterior, according to Rau and Fassuliotis striated, set off by constriction. A face view (1965). (Fig. 2L) shows six well-developed lips, the Infestation always takes place just behind lateral ones being somewhat larger than the the apical cells and does not stop growth of submedian. Amphid apertures slightly oval, the root which generally forms an arcuate, located at apices of lateral lips. Vestibule elongated gall and then proceeds to develop slightly sclerotized, forming a spear-guiding a series of similar galls, each containing from apparatus. Spear 10-12 ^ long with small 1-6 females and in rare instances 15 or 20 may rounded knobs. Dorsal esophageal gland ori- occur. Branch roots are produced on the outer fice almost adjacent to spear base. Anterior periphery of each gall and these in turn are portion of esophagus set off from median bulb infested until a complicated, chainlike mass by constriction. Bulb spheroid with refractive of roots results (Fig. 1A). Occasionally a valve and strong radial muscles. Basal bulb progressive multiple infestation occurs in a a flattened, irregular shaped lobe extending terminal gall with mature egg-producing fe- back over intestine, containing the usual three males at the gall base, while elongated young nuclei which generally are difficult to observe. females are located near the terminus, resulting Isthmus very short. Nerve ring obscure. Ex- in a series of various-aged individuals (Fig. cretory pore far forward, almost opposite spear IB). base (Fig. 2K). Intestine and body contents In all instances observed, the heads of the densely granular, making observations very

1 Professor Emeritus, Departments of Plant Pathology and " First used as a nomen nudum by Weber and Barker Zoology, University of Wisconsin. (1967).

Figure 1. A, Root of Phalaris arundinacea infested with Hypsoperine ottersoni. Note how secondary roots spring from outer contours of the sickle-shaped galls. Upper left and right are females ejecting eggs from the galls as occasionally occurs. B, Unusual terminal gall with progressive infestation. The first larvae entering it are now adult females producing eggs, while those recently entering near the terminus are still in early stages of development. difficult. Ovaries two, elaborately convoluted vae which probably are not ejected. A gelat- (Fig. 2N), at maturity completely filling body inous matrix surrounds eggs deposited either cavity. Vulva transverse. Vagina cylindroid, within the roots or into the soil. muscular. Perineal pattern with simple MALE: (7) 0.9-1.0 mm long, rarely 0.7 mm; rounded striae, usually on a slight terminal a = 34, rarely 40. Body twisted until a true elevation (Fig. 2T). Anal opening visible lateral view of head and tail can be seen only from a lateral view and rarely from ventral by cutting specimens after fixation. Striae (Fig. 3C). Neither spermatheca nor sper- about 2 fj(, apart at midbody, narrower near matozoa observed. head and sometimes absent on tail. Lateral Eggs (15) 45 by 105 ^ (40-50 by 95-115), fields about one-fifth body width, marked by deposited before segmentation by young fe- four incisures, the two median ones sometimes males. Senile individuals may retain from 5- very obscure. Lip region unstriated; labial disc 25 eggs which segment and develop into lar- visible only from a dorsal or Ventral view when

Figure 2. A, B. Anterior and posterior portions of larva X 1,000. C, D. Male head lateral and dorsal views. E, F, G. Lateral and dorsal views of posterior portion of males. H. Specimen with two testes. I, J. Variations in median esophageal bulbs. K, L, M. Head, face view and anterior end of female. N-S. Variations in form of females. T. Perineal pattern. U. Duboscquia sp. parasites.

Figure 3. A, B. Lateral and dorsal views of posterior portion of female. C. Ventral view showing anus, vagina, uterus, and attachment of the two ovaries and saccate rectal glands. the slitlike amphid apertures set it off from width behind bulb. Dorsally the esophagus the lip contour (Fig. 2D). Cephalids obscure, base is short and rounded while ventrally it is visible only from a lateral view, located well extended in a long lobe 5-7 times the body forward of the spear base as in Meloidogyne width. Excretory pore slightly behind nerve (Fig. 2C). Spear 14-16 ^ long with strong, ring. Anal opening very obscure, often un- backward-sloping knobs. Nerve ring one body identifiable. Tail ending in an irregularly width, and excretory pore twice that distance clavate or knobbed terminus. behind median bulb. Lumen of esophagus Holotype female, allotype male and other joining intestine about one body width pos- specimens as indexed under Hypsoperine 1, terior to bulb, the junction being rather abrupt University of Wisconsin nematode collection. as illustrated for the larvae (Fig. 2A). Hemi- TYPE HOST AND LOCALITY: Canary grass, zonid about two annules anterior to excretory Phalaris arundianacea (L.) Reed, Homer pore. Basal esophageal lobes average about Ranch, Wind Lake area, Wisconsin. Found three times as long as body width. Testes wherever canary grass is growing in Racine, 53-65% of body length, usually single, out- Milwaukee, and Waukesha counties and near stretched, rarely reflexed. Males rare; among Waupun in Green Lake County. Many obser- 14, two were observed with double testes (Fig. vations by Dr. Otterson and others have failed 2H). Spicula slightly arcuate 19-23 p, long to reveal it attacks any crop plants. but difficult to measure because of the twisted Hypsoperine ottersoni is distinguished from bodies. Gubernaculum 3 or 4 ^ long, well- H. graminis, Sledge and Golden, 1964 by the thickened, troughlike. Phasmids almost ter- more simple annulation and absence of lateral minal (Fig. 2G). Tail variable in form and lines in the perineal pattern, smaller males 0.9: length (Fig. 2E, F). Sperm ducts packed 1.5 mm, and irregular-clavate terminus of lar- with spheroid spermatozoa indicating that males may be functional although sperms were vae. From H. spartinae Rau and Fassuliotis, not observed in the uteri. 1965, it is recognized by absence of labial LARVAE (10): 0.43-0.50 mm; a = 23-30, annules (two in H. spartinae}, simple lines of b and c measurements not made because the perineal pattern compared to elaborate ar- esophagus base and anal opening could rarely rangement, and presence of male hemizonid be accurately determined. Lateral field marked anterior to excretory pore which is posterior by four fine incisures visible only on favorable in H. spartinae. specimens. Framework of the rounded lip Perineal sections should be mounted in a region practically undeveloped. Vestibule drop of hard glycerin jelly to support the forming a minute sclerotized spear guide. cover glass, otherwise the weight will produce Spear 13—15 p, long with well-developed knobs. folds and wrinkles which destroy the actual Dorsal gland orifice about 4 fjl behind spear. pattern of the striae. Nerve ring adjacent to bulb, practically en- Many larvae and some males were attacked veloping the unusually short isthmus. Esoph- with sporozoans, Duboscquia sp.; in some in- ageal lumen joining intestine about one body stances these were observed in clusters of

Copyright © 2011, The Helminthological Society of Washington 102 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY eight, unlike any previously recorded (Fig. Sledge, E. B., and A. M. Golden. 1964. Hyp- 2U). soperine gramini-s (Nematoda: Heteroderi- dae), a new genus and species of plant Literature Cited parasitic nematode. Proc. Helm. Soc. Wash. 31(1): 83-88. Rau, G. J., and G. Fassuliotis. 1965. Htjpso- Weber, A. J., and K. R. Barker. 1967. Biol- perine spartinae n. sp., a gall-forming nema- ogy of the pseudo root-knot nematode Hijpso- tode on the roots of Smooth Cordgrass. Proc. perine ottersoni. Phytopathology 57(7): Helm. Soc. Wash. 32(2): 159-162. 723-728.

Studies on Freshwater Larval Trematodes. XXI. Two New Species of Macrocercous Cercariae1

PIR NASIR, MARCOS TULIO DIAZ, Luis J. HAMANA SALAZAR, AND S. GUEVARA Laboratorio de Parasitologia, Depto. de Biologia, Escuela de Ciencias, Universidad de Oriente, Cumana, Venezuela

Until now, only bivalve mollusks have been to elucidate life cycles. Even if infected snails reported as the first intermediate hosts of the had been more numerous, only a few cercariae known macrocercous cercariae. This is the emerged at a given time; however, efforts are first record for the involvement of univalves in progress. It may be mentioned here that as well. The cercariae treated in this paper, on one occasion C. yacalicola was escaping as a result of comparative studies discussed from the same specimen of the snail, Pomacea later, proved to be new species and have been glauca, which was also discharging two addi- named, respectively, Cercaria latigazica and tional cercariae, C. cumanensis Nasir, 1965 C. yacalicola. The latter is readily distin- and C. allomacarapanensis Nasir and Diaz, guished by the presence of a cercarial cham- 1968. ber, in the proximal part of its tail, in which The recovery of sporocysts presented an the cercarial body is characteristically enclosed. intriguing situation. Although several snails The former lacks this structure and its tail is were dissected immediately after the appear- frequently thrust out in a peculiar whiplike ance of cercariae, sporocysts were never en- fashion. It is evident from the works of other countered in the usual sites like gills and hepa- authors that various species of tadpoles and topancreas. A careful examination revealed the naiads of insects serve as the second interme- presence of a few, anteroposteriorly elongated, diate hosts for macrocercous cercariae while considerably thick-walled, pigmentecl sporo- urinary bladders of frogs, salamanders, and cysts in the lumen of intestine. fishes are the usual sites of adult infections. All observations have been made on freshly In certain cases like Phyllodistomiim simile, emerged cercariae, except measurements which the precociously encysted cercariae while still are based on specimens killed by irrigating with inside sporocysts when experimentally fed to hot 10% formalin. trout resulted in the development of corre- All measurements are in millimeters. sponding adults (Thomas, 1958). Of course, we are not sure whether these "precocious 1. Cercaria latigazica sp. n. metacercariae" were formed by the penetra- (Figs. 1, 2, 7) tion of cercariae from without. Due partly to HOST: Pomacea glauca (L.). the dearth of material, we have been unable LOCALITY: La Chorrera, near Caripito; Los Yacales, La Llanada de San Juan, near Uni- 1 Supported in part by a grant from Comision de Desar- versidad de Oriente, Cumana, Venezuela. rollo e Investigaciones Cientificas de la Universidad de Oriente, Venezuela. DESCRIPTION: Body without spines, beset

Figures 1, 2, 7. Cercaria latigasica sp. n. 1. Cercaria, note peculiar structure of tail, stylet not shown. 2. Stylet. 7. Sporocyst, note considerably thick wall and pigmented specks. Figures 3-7. Cercaria yacalicola sp. n. 3. Stylet. 4. Body only, note two groups of penetration glands on each side. 5. Cercarial chamber and part of tail. 6. Cercarial chamber along with distal, elongated swollen region. 7. Sporocyst.

Copyright © 2011, The Helminthological Society of Washington 104 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY with 22 rows of setate papillae. Stylet without group of five anterior to ventral sucker; this a basal bulb (Fig. 2). Central parts of suckers latter group may extend laterally around ven- occupied by papillae without setae and irreg- tral sucker. Genital rudiments composed of ularly distributed bristles. Periphery of both two cellular masses, one anterior and other suckers with a row of setate papillae. Ventral posterior to ventral sucker. Excretory vesicle sucker surrounded by a muscular fold. Pharynx tubular, with a swollen posterior region. absent. Esophagus long, not extending to ven- Twelve to 14 transversely elongated glands tral sucker. Intestinal ceca extending to pos- with coarsely granular material on each side terior end of body. Penetration glands small, of vesicle. Main excretory tubes dividing into six on each side along posterior part of esoph- anterior lateral and posterior lateral collecting agus, not extending to ventral sucker. Genital tubes at about equatorial level of preacetabular rudiments represented by two cellular masses, region. Flame cell formula: 2[ (3+3+3) + one anterior and the other posterior to ventral (3+3+3+3+3+3+3)] = 60. Tail without sucker. Excretory vesicle tubular, with a swol- spines or papillae, divided into two regions: len posterior region; eight transversely elon- proximal swollen region or cercarial chamber gated glands with granular contents on either and a distal, narrow, elongated region which side of vesicle. Main excretory ducts extending often presents irregular swellings. Measure- as far anteriorly as about halfway in preacetab- ments of 10 specimens: body of cercaria ex- ular region and after forming a small loop in pressed from cercarial chamber 0.235-0.370 by same region dividing into anterior lateral and 0.112-0.138; oral sucker 0.060-0.066 in diam.; posterior lateral collecting excretory tubules. esophagus 0.060-0.067 long; ventral sucker Flame cell formula: 2[(3+3+3) + (3+3+3 0.072-0.085 in diam.; stylet 0.027-0.033 by +3+3+3+3+3)1 = 66. Tail without spines, 0.007-0.009; cercarial chamber 0.243-0.379 by without cercarial chamber, capable of extend- 0.159-0.300; distal part of tail without cer- ing in a peculiar whiplike fashion. Measure- carial chamber 2.302-2,570 by 0.159-0.300. ments of 10 specimens: body 0.210-0.267 by Sporocysts similar to that of C. latigazica. 0.057-0.090; tail 0.300-0.375 by 0.078-0.114, when extended more than twice as long as its Discussion normal length; oral sucker 0.040-0.044 in For the specific identification only those diam.; esophagus 0.060-0.065 long; ventral characters which are fairly constant or vary sucker 0.043-0.045 in diam.; stylet 0.013- little like the shape and size of stylet, esoph- 0.016 by 0.003-0.005. Sporocysts 0.300-0.817 ageal length, number and arrangement of by 0.206-0.404. penetration glands, number of "cystogenous glands" around excretory vesicle, type of ex- 2. Cercaria yacalicola sp. n. cretory system mesostomate or stenostomate, (Figs. 3-7) number and arrangement of flame cells and HOST: Pomacea glauca (L.). finally presence or absence of cercarial cham- LOCALITY: Los Yacales, La Llanada de San ber have been considered. Unless the relative Juan, near Universidad de Oriente, Cumana, differences in size are of a considerable magni- Venezuela. tude, too much reliance on this character alone DESCRIPTION: Body without spines, covered could be misleading. Some "new species" with setate papillae. Stylet without a basal erected on the basis of minor discrepancies bulb (Fig. 3). Central parts of suckers with should be suppressed. Cercaria eriensis Coil, papillae without setae and irregularly distrib- 1953 and C. lampsilae Coil, 1954 are indis- uted bristles. Periphery of suckers bordered tinguishable in having approximately the same with rows of setate papillae. Pharynx absent. size suckers, esophageal length, intestinal Esophagus long, extending to ventral sucker. length, shape, and extent of excretory vesicle, Intestinal ceca extending only slightly pos- similar number of glands around excretory terior to ventral sucker. Penetration glands vesicle and finally both of the species abound with uniformly granular contents, in two in the same locality, Lake Erie, Ottawa County, groups on each side of body: one group of USA. Furthermore, the molluscan intermediate three posterior to oral sucker and another hosts are the very closely related species,

Lampsilis ventricosa and L. siliquoidea. Ac- size is concerned there is only a difference of cording to Coil, 1954, "C. lampsilae differs 2 /x,; the stylet of C. eriensis is 14—17 ^ in from C. eriensis, its closest relative, by possess- contrast with 16-19 of C. lampsilae. The ar- ing (1) a stylet which is slightly shorter and rangement of papillae on oral sucker is also of a different shape, (2) a different disposition a matter of interpretation. Finally, the cri- of sensory papillae on the oral sucker, (3) a terion of body size is also questionable. Cer- body length which is shorter." In our experi- caria of Gorgodera amplicava Looss, 1899, as ence none of these characters is of substantial described by Krull, 1934, is 4.4-7.5 mm long taxonomic importance. The "differences" be- whereas the same cercaria reported by Good- tween the stylets of C. eriensis and C. lamp- child, 1945, is 12.0-17.4 mm long. It is there- silae, as depicted in diagrams (Coil, .1954; fig. fore suggested that C. lampsilae should be 5—6), are hardly differences at all, because synonymized with C. eriensis. a slight pressure of coverglass will distort the Only those styleted macrocercous cercariae shape of one into that of the other. We have about which sufficient structural details are often encountered this type of distortion while known are compared and these are separated studying xiphidiocercariae. Insofar as stylet by the following key: I. A. Tail with a cercarial chamber II B. Tail without a cercarial chamber III II. Tail with cercarial chamber A. Penetration glands arranged in two groups on each side of body 1. Groups of 5 and 3 on each side —. C. yacalicola sp. n. 2. Groups of 6 and 4 on each side C. raiacauda 3. Groups of 8 and 2 on each side ..,. C. steelmani B. Penetration glands in only one group on each side of body 1. Constant number present a. eleven on each side Phyllodistomum staffordi b. four on each side C. coelocerca c. six on each side la. Number of flame cells known aa. 18 each side C. macrocerca bb. 16 each side C. donecerca cc. 24 each side Phyllodistomum simile Ib. Number of flame cells unknown a'. 10 sensory tubercles around oral sucker aa'. stylet 33—37 p. long Gorgodera amplicava b'. 2 rows of 10 each bb'. stylet 23-25 ^ long Phyllodistomum solidum — C. conica c'. about 40 cc'. stylet 35 ^ long Gorgoderina attenuata 2. Number not constant a. 3 to 4 pairs on each side C. papillostoma b. not arranged in 3 to 4 pairs la. Excretory system stenostomate C. wabashensis 2a. Excretory system not stenostomate C. sphaerocerca III. Tail without cercarial chamber A. Six penetration glands each side C. latigazica sp. n. B. Four penetration glands each side C. eriensis Literature Cited tions on the life history of Gorgodera ampli- Coil, W. H. 1954. Contributions to the life cava Looss, 1899. J. Parasit. 31(Suppl.): cycles of gorgoderid trematodes. Amcr. Midi. 22-23. Natl. 52: 481-500. Krull, W. H. 1934. Studies on the life history Goodchild, C. G. 1945. Additional observa- of a frog bladder fluke, Gorgodera amplicava

Looss, 1899. Pap. Mich. Acad. Sc. Arts & Thomas, J. D. 1958. Studies on the structure, Letters 20: 697-710. life history and ecology of the trematode Rankin, J. S., Jr. 1939. The life cycle of the Phtjllodistomum simile Nybelin, 1926 (Gorgo- frog bladder fluke, Gorgoderina attenuata deridae: Gorgoderinae) from the urinary Stafford, 1902 (Trematoda: Gorgoderidae). bladder of brown trout, Salmo trutta L. Amer. Midi. Natl. 21: 476-488. Proc. Zool. Soc. London 130: 397-435. Schell, S. C. 1967. The life history of Phtjtto- distomum staffordi Pearse, 1924 (Trematoda: Vickers, G. G. 1940. On the anatomy of Cer- Gorgoderidae Looss, 1901). J. Parasit. 53: caria macrocerca from Sphaerium cornenm. 569-576. Quart. J. Micr. Sc. 82: 311-326.

On the Morphology and Ultrastructure of the Esophageal Region of Trichodorus allius Jensen

D. J. RASKI, N. O. JONES, AND D. R. ROGGEN University of California, Davis and Laboratoria voor Morfologie en Systematiek, Gent

The search by electron microscopy for plant- Female and larval nematodes were wet-sieved pathogenic viruses in nematode vectors began from the soil then hand-picked into tap water. with Xiphinema index, the vector of the fan- The nematodes then were transferred to 0.9% leaf strain of arabis mosaic virus. No evidence NaCl and frozen by dry ice in a fume-chamber of the virus was found in X. index but the with 2% OsO4 in distilled water. After being study did yield valuable information on the allowed to return slowly to room temperature ultrastructure of the nematode vector (Wright, they were fumed overnight. This treatment was 1966; Roggen, Raski and Jones, 1966, 1967). followed by 2 minutes immersion in 15% More recently, the search has been directed DM SO (dimethylsulf oxide) in distilled water to Trichodorus allius, a vector of tobacco rattle and 20 minutes in 2% OsO4 in distilled water. virus, in a similar study of nematode-virus re- This procedure was used unless otherwise indi- lationships. One advantage of this virus is the cated in plate text. The nematodes then were rod-shape of its particles which might be easier handled according to Wright and Jones (1965). to detect in animal tissue than the polyhedral Embedded worms were cut by diamond knives particles of fanleaf virus. on an LKB Ultrotome and examined on an A study of ultrathin sections of T. allius has RCA EMU-3G or modified EMU-3E electron not been successful in locating virus particles microscope each having a 50 ^ objective aper- but has been fruitful in developing a better ture and operating mostly at 50 kv, occasionally understanding of the odontostyle and mecha- at 100 kv. nism of feeding and of the ultrastructure of the esophagus. Observations Cuticle Materials and Methods The cuticle of the adult female of T. allius Nematodes used in these studies were col- (Plate 1, A—D; Fig. 1, A) appears to be com- lected originally from soil about the roots of posed of eight layers as follows: sugarbeet (Beta vulgaris) and alfalfa (Medicago L1. A triple-layered, thin (0.025 _«,) osmio- sativa) near Salinas, California. Populations philic membrane. were reared at Davis, California, under green- L2. A thick (0.16 /a) layer, uniform in tex- house conditions feeding on the roots of alfalfa, ture. There may be two sub-layers making up tobacco (Nicotiana tabacum), but principally L2 because in one section (OsO4 fixative plus on sweetpea (Lathyrus odoratus). No males of Pb(OH)2 postsectioning stain for 15 minutes) T. allius were seen in any of these populations. a fine, outer, whitish band and dark inner band

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 107 were observed. These were not consistently or (L1) which is continuous with the outermost certainly set apart in other sections. layer of the body cuticle. Fine cross-striations (cs) (Plate 1, A—C), The opening is set in L2 of the cuticle which approximately 3 ^ apart, were present on most forms a roughly hexagonal shape, each side of the body and extended almost to the termi- about 0.7-0.9 p. long. The six angles of the nus. The striations were formed by an infolding hexagon are directed each between two inner of L1 and L2 and at most created a slight in- labial papillae (IL) and anteriorly these angles dent in the outer surface of L3. extend outward joining a similar layer of tissue L3. A layer, varying in thickness, from outside the papillae. The triradiate oral open- 0.48—1.50 p, with an average of about 0.64 p,. ing also is oriented with each arm directed be- This layer also appeared to have two com- tween two inner labial papillae. Each papilla ponents, different in texture and somewhat in is surrounded by more coarsely granular L3. color, but there was no sharp demarcation About 0.6—0.8 /A posteriad from the anterior between the two. The outer portion (L3a) surface (Plate I, D) is a crease or folding of comprised 74-82% of the total and was coarser- the oral aperture extending parallel with the grained in appearance than the inner portion. anterior surface. This fold is about 0.6 ^ long In cross-section there was only a vague sug- in both directions perpendicular to the longi- gestion of structure as concentric lines but tudinal aperture. The osmiophilic layer lines without regularity. In longitudinal section this fold and is continuous with the lining of (Plate I, C) there were many lines running the oral aperture. longitudinally which appeared to set off sub- Stoma layers, 18—20 in number, whitish with dark borders. The inner 14 or 15 were broader, nar- The first evidence of the stomatal wall (Plate rowing (or doubling) near L2. The inner por- II, B) appears just below the thick anterior tion (L3b) comprised 18-25% of the total of cuticle and is composed of: L3, was darker in color and of finer grain than L1. The osmiophilic layer forms a very ir- L3a. In longitudinal section there were 11 or regular outline of the stomatal lumen and is 12 sublayers similar to those in L3a but dark grossly convoluted or jagged. This lasts only a bands with light borders. short distance and soon becomes more simple, L1. A layer variable in thickness from mini- triradiate with a single bulge at the midpoint mal or nonexistent to 0.68 p., spongy or amor- of each side (Plate II, C). Where the lining is phous in texture. convoluted (Plate II, B) there are some mem- L"~7. The next three layers were granular branelike folds inside the lumen of the stoma. with no apparent structure and about equal in L2. This layer is relatively thick (0.1-0.2 p.), thickness (0.09 p. each). The innermost (L7) at first uniform in texture, and definitely set had an irregular inner surface projecting in off from the adjacent L3 by a dark line. Later rough, lumpy protuberances. a well-defined membrane appears around L2 Ls. Underlying L7 was an irregular and light (Plate II, C) and soon afterwards tissue ap- gray layer the nature of which is unknown. It pears with the membrane. This tissue probably was intimately connected with the epidermis is the most anterior projection of pharyngeal which, on the outer margin, formed an irreg- tissue (ph). ular edge with polyp like protuberances inter- At the same level where the above mem- mingled with Ls. brane first appears there is also the first evidence of the lining of the pharyngeal lumen Oral Aperture (pll) inside L2. The innermost lining of the The most anteriad section shows the oral pll seems to be similar to, if not identical with, opening to be triradiate (each arm about 0.5— L1 and continuous with it. As the pll expands 0.7 /i, long). These arms are almost straight the L2 diminishes to a thin concentric band but slightly posteriorly they become irregular around it, finally becoming an incomplete band with several short side branches, the distal (Plate II, D) and later disappears. branches on two of the arms give an almost The double-layered L3 surrounds L2. Out- forked appearance (Plate II, A). Lining the side of L3 is a membrane which broadens oral opening is the thin, dark osmiophilic layer slightly to a thin line of epidermal tissue. Sur-

Copyright © 2011, The Helminthological Society of Washington 108 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY rounding the epidermal layer is another which tip of the angles of the lumen and one slightly appears to be Ls and it in turn is surrounded larger on each side. by another layer of epidermis. The next change in these bands is their dis- L3 retains its double-layered appearance appearance in the apical area on all three rays (Plate II, C) through level 1 (Fig. 1, A-B) of the lumen accompanied by a lengthening where the pharyngeal tissue appears between of the dorsal wall which is gently arched (Plate it and L2. The pharyngeal tissue continues to IV, A). On the ventral ray of the lumen the expand through succeeding levels further sep- refractive bands divide to form two pairs. arating off L3. As this happens L3 becomes These refractive longitudinal bands persist homogeneous in color and evenly granular. some distance until finally the two pairs on Posteriorly L3 disappears (Plate II, E). This the ventral ray disappear (Plate II, F) and seems to be the inner limit of the external soon afterwards the pairs on the two subdorsal cuticle and probably marks the posterior end rays disappear. of the stoma. This is supported by the fact that Posteriorly the next development is a smooth, molted cuticles of larvae show only a very short grayish thickening of L1 lining the two sides stomatal cuticular lining which is cast off. of the ventral ray of the lumen (Plate III, G). Pharynx These thickenings extend posteriorly through the folds of the guide ring then disappear. The Coincident with the very first appearance of rest of the pll outside the L1 becomes re- the pll there are light colored areas (Plate II, stricted to small narrow areas on both walls C; level 1, Fig. 1, A—B) which seem to be of the ventral ray on each side of the swelling strengthening rods or bands (Pr) running and finally the entire lumen wall becomes a longitudinally. These may serve as muscle at- uniform, gray texture. tachments and are located centrally near the osmiophilic layer. Similar refractive bands soon Odontostyle appear (Plate II, D) at the ends of the tri- The odontostyle is first seen (Plate II, E) radiate lumen. The 3 inner bands are shorter as a simple circular body about 0.2 ^ across, than the distal ones and disappear just before free in the lumen. It has a uniform gray color the stylet tip is seen (Plate II, E). The three with a small central darker dot and a fine, distal bands divide into three each—one at the slightly darker, outer border. Posteriorly it en-

Plate I. Cuticular layers and markings. A, Longitudinal section, 12,000 X; B, Longitudinal section, 38,000 X; C, Cross section, 20,800 X; D, Longitudinal section in stomatal region, (IL) innervations to interne-labial papillae, 43,700 X. Plate II. Cross sections of stoma and pharynx including the odontostyle and its extension. A, Near oral aperture, 14,000 X; B, Stoma, 23,100 X; C, Transition from stoma to pharynx and first evidence of pharyngeal lumen lining (pll), 19,900 X; D, Transition slightly posterior to D, 18,200 X; E, Pharynx with first section of odontostyle (Od), 30,400 X; F, Pharynx posterior to E, 20,000 X. Plate III. Cross sections of stoma and pharynx (cont'd). G, Odontostyle near attachment to dorsal wall, 19,400 X; H, Fusion of odontostyle with dorsal wall and guide ring, 13,400 X; I, Stylet extension posterior to H, 12,500 X; J, Stylet extension posterior to I, 13,800 X; K, Attachment of odontostyle protractor muscles to stylet extension, 22,000 X; L, Transition of stylet extension with wall of pharyngeal lumen, 12,500 X. Plate IV. Esophageal muscles. A, Cross section at collar of (Ap) amphid, (PPm) perpendicular plvaryngcal muscles, (LPm) longitudinal pharyngeal muscles, 19,800 X; B, Cross section near base of stylet extension, (OPm) odontostyle protractor muscles, 23,100 X; C, Cross section of esophagus in narrow region posterior to stylet, (ELm) esophageal lumen muscles, 20,200 X. Plate V. A-B. Longitudinal sections of amphid (P — amphidial pouch; C = collar; -> indicates basal plates of cilia). A, Near base of amphidial pouch, 9,500 X; B, Junction (S) of cilia and nerve (N), 15,300 X; C, Longitudinal section of esophageal gland orifice into lumen of esophagus cut across two rays of lumen (Lu), 30,400 X; D, Longitudinal section between two rays of lumen (Lu), 31,200 X.

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Copyright © 2011, The Helminthological Society of Washington 114 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY larges to about 0.3 ^ across and shows two banded diagonally across the dorsal wall (Plate layers, a darker band (about 0.05 //, wide) as III, L). The lumen, as it courses through the a concentric ring surrounding the solid inner narrow esophagus to the glandular bulb, has core with a central black dot (Plate II, F). the familiar osmiophilic membrane lining it Later the inner core and the outer band ex- -with simple rounded ends on the three radi- pand and the central dot enlarges, becom- ating arms of the lumen. ing more complex and eventually developing an outer membrane surrounding tissue Musculature which includes endoplasmic reticulum and The somatic muscles possess two kinds of mitochondria. elements (myofilaments), fine and large, similar The outer portion of the odontostyle reaches to those in Xiphinema index. These form iden- a maximum thickness of about 0.1 ^ when the tifiable areas in the cells where only large ones total diameter is 0.5 //,. Posteriad this outer occur (near the midline of the cell), or where portion becomes more narrow as the inter- both occur (these areas surround the former). mediate whitish layer expands to 0.8-1.0 p. in Finally the fine elements seem to be free of diameter. At the same time the central core the larger elements in the outer margins of enlarges with a membrane surrounding its tis- each cell. Neither striae nor any regular re- sue. When the outer band has disappeared peating markings were seen in longitudinal (Plate III, G) the odontostyle consists of a sections. circular, uniformly light, cuticular material sur- In the esophageal region there appear to be rounded by a thin, dark line with a maximum six or seven sets of muscles. diameter about 1.1 p, and with an inner tissue 1. LABIAL MUSCLES (LM) (Fig. 1, A). For core about 0.3 ^ in diameter. The cuticular want of a better term these are referred to as lining of the odontostyle is similar in texture labial because they are the first to appear at to the odontostyle extension and continuous the anterior end. There seem to be four (two with it. each subventral and sublateral), quite large, Posteriad the odontostyle begins its attach- extending from the outer stomatal cuticular ment to the dorsal wall and the "guide ring" lining to the body wall and are almost as appears (level 5, Fig. 1, A). The attachment broad as long at their maximum development. begins first toward the left side of the dorsal They each occur laterad to their respective wall (Plate III, H) and is completed in a short dorsal or ventral inner labial papillary inner- distance. Where the odontostyle joins the dor- vation processes. sal wall (Plate III, H) the pi I folds anteriorly It is not certain just where they attach. then again posteriorly, still showing the thick- These are not long muscles and seem to dis- enings of the L1. In the region of the odonto- appear before the stylet tip is found in the style extension posterior to the "guide ring" the buccal cavity. It is possible these represent the lumen of the esophagus (Lu) is small, narrow anterior extremity of the pharyngeal muscles and immediately ventrad to the extension (PPM). (Plate III, I, J). 2. PHARYNGEAL MUSCLES (LPM) LONGITU- In the odontostyle extension the inner core DINAL. Four smaller muscles (LPM) appear at of tissue with membrane moves dorsad (Plate about the same level at which the labial mus- III, I) and finally merges (Plate III, J) with cles disappear. These four are closer in near the nearby tissue (epidermal?) of the pharynx the stomatal region with two each between and the odontostyle extension thus forms a the subventral and subdorsal inner labial pa- U-shape in cross-section with the open end to pilla innervations (Plates II, D; IV, A; Fig. 1, the dorsal side. By the time the core of tissue A). The muscles are narrow and longitudinally is no longer part of the extension, the latter, parallel with but outside the pharyngeal tissue. in cross-section, is flattened ventrally where it The muscles seem to diverge and change loca- is part of the lumen wall and rounded dorsally. tion, one continuing on the left lateral side, As the extension begins to diminish it becomes the others as a single subdorsal and two sub- irregular in dorsal outline and mottled in tex- ventral in location. The left lateral muscle per- ture (Plate III, K). Finally as the last of the sists longest but not quite to the end of the whitish extension tissue disappears it becomes odontostyle proper.

Copyright © 2011, The Helminthological Society of Washington L1"3, layers of cuticle Lm, labial muscles PPm, perpendicular pharyngeal muscles Pr, strengthening rods in pharynx Epi, epidermis LPm, longitudinal pharyngeal muscles Sm, somatic muscles Pll, lining of pharyngeal lumen Copyright Od, odontostyle GR, guide ring OdAt, attachment of

2011, pharynx OdEx, odontostyle

The extension Lu, lumen of pharynx Helminthological OPm, odontostyle protractor muscles Society of Washington

A B Figure 1. Schematic drawing at head region and esophagus of T. allius. 116 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY

3. PHARYNGEAL MUSCLES (PPM) PERPENDIC- gus posterior to the odontostyle and also of the ULAR TO LONGITUDINALS (LPM). A set of three enlarged glandular region, show some muscles muscles are present in the area near the an- radiating outward from the angles of the terior tip of the odontostyle when in retracted lumen. These appear to attach at the angles of position. One is about midventral in position the lumen or on the swellings on the arms of attaching to the ventral apex of the triradiate the lumen (Plate IV, C). Some sections show stoma (Plate IV, A; Fig. 1, A). The other two evidence of attachments of muscles at the ends are subdorsal attaching to the two subdorsal of the rays of the lumen. apices of the stoma. The muscles extend dis- Sensory organs tally and proceed between somatic muscles to the body wall, possibly to the innermost 1. INNER LABIAL PAPILLAE. The six inner layer of the epidermis. Internally the muscles papillae when first seen (Plate II, A) are ar- end at a perpendicular line darkened in color ranged symmetrically about 1 p, from the cen- and adjacent to the basement membrane of ter of the oral aperture. The papillae show a the epidermal tissue surrounding the pharynx. scattered arrangement of microtubular ele- 4. ODONTOSTYLE PROTRACTORS (OPM). These ments. Posteriorly these arrange into four cili- are strongly developed muscles which begin at ary elements in each papilla (Plate II, B—C). a level anterior to the base of the odontostyle. 2. OUTER LABIAL AND CEPHALIC PAPILLAE. Here, there seem to be three, one dorsal and Six outer labial and four (two subdorsal and two subventral, near the cuticular lining of the two subventral) cephalic papillae are present pharyngeal lumen. Nearer the end of the odon- with two or three ciliary elements in each. tostyle these move to the outer edge of this 3. AMPHIDS. These are quite similar to those pharyngeal tissue and appear as four or five found in X. index and are formed by invagina- large, irregularly shaped muscles. At the begin- tions of the cuticle. The inner lining is a con- ning of the odontostyle extension the muscles tinuation of the osmiophilic layer. Adjacent to divide into elements (Plate IV, B), 15 or more the osmiophilic layer and surrounding it was in number almost lining the entire outer limit cuticular material apparently similar to L3 of of the pharyngeal tissue. Some are dark stain- the outer cuticle but also perhaps some rem- ing while others are similar to the muscles nants of L2. anterior to this level. These muscles attach The sensillar (amphidial) pouch is evenly ventrally, two being slightly subventral leaving granular in texture near the aperture but im- a corridor of epidermal tissue directly ventrad mediately posteriad is provided with some ele- to that ray of the triradiate lumen. Each of ments. At first the pouch (P) is irregular in these two becomes bifurcate as they approach outline then assumes a shape roughly rectangu- the outer edge of the pharyngeal tissue. Where lar in outline. The shape then narrows into a the core of the extension emerges, the muscles circular form with a thick ring of cuticle (C) shift toward the center of the pharynx. Here surrounding it (Plate V, A). In the longitudinal mitochondria are large and numerous. Muscle section the amphid has a goblet-shape with a attachments to the extension occur where the concentration of ciliary elements in the narrow sclerotized wall begins to reduce in size and collar (Plate V, A). Posteriorly the amphid then almost cover the outer dorsal wall of that widens again as numerous ciliary elements organ (Plate II, K). with basal plates (arrows) connect (?) with 5. SOMATIC MUSCULATURE (SM) (Fig. 1, A). nerve cells (N) (Plate V, B). Ciliary struc- These muscles begin at about the level where tures are similar to those in X. index, dif- the amphidial pouches enter the body, appear- fering only in number of elements in individual ing first dorsad and ventrad to the pouches. sensory organs. The cuticular structures of the As the pouches move inward leaving a space amphids are molted by larvae and persist as laterad to them, the muscles appear between prominent remnants in the shed cuticles. the pouches and the outer wall. Compared to 4. NERVE RING. First seen at the level near Xiphinema index, the somatic musculature is the posterior end of the odontostyle extension not very high in cross-section. the nerve ring forms a U-shaped arc of nerves 6. ESOPHAGEAL LUMEN MUSCLES (ELM). on the ventral side but not entirely encircling Some sections of the narrow part of the esopha- the esophagus. In the region of the nerve ring

Figure 2. Schematic drawings illustrating feeding mechanism of T. allius. a—c, stylet protraction; d—e, stylet retraction; 1, labial muscles (Lm); 2, perpendicular pharyngeal muscles (PPm); 3, longitudinal pharyngeal muscles (LPm); 4, somatic muscles (Sm); 5, odontostyle protractor muscles (OPm). and posteriorly around the esophagus are many The orifices of the dorsal and two larger nuclei with dense chromatin arranged around subventral glands are provided with minute, the periphery of and also scattered throughout complex structures which appear to serve as the nucleus. collection mechanisms. These measure about ESOPHAGEAL GLANDS. There are five nuclei 2.5 n long, 1 fj, in width and height and are visible in the esophagus of T. allius (Fig. 1, located next to the lumen and each is between B). One is the large dorsal gland nucleus lo- two rays of the lumen. The structure seems to cated in the dorsal sector of the esophagus be a sinus with radiating and branching arms about midway in the posterior bulb or glan- numbering 20 or more. It is completely sur- dular region. Two large subventral gland nuclei rounded by a triple-layered membrane. A sagit- are more posterior in the glandular region. tal section crossing both rays of the lumen Slightly anterior to the dorsal gland nucleus shows a kidney- or oval-shaped outline (Plate are two more subventral nuclei, smaller in size V, C), becoming a common sinus in the center. and less noticeable. Sagittal sections bisecting two rays shows three The dorsal gland extends almost the full or four separated arms of the sinus or 5—10 or length of the dorsal sector but has its orifice more fingerlike radiating arms becoming fan- into the lumen of the esophagus in the anterior shaped and converging to the orifice entering end of the glandular region. In many specimens the esophageal lumen (Plate V, D). the narrow part of the esophagus between the stylet and the posterior bulb forms a distinct Discussion loop or S shape. CUTICLE. The report on X. index (Roggen The two posterior subventral glands connect et al., 1967) concluded that the structure of the with the lumen of the esophagus in the pos- cuticle of nematodes is too variable and too terior bulbar region not far from the nuclei few species have been studied to permit gen- themselves. However, the orifices of the two eralizations. It was not possible to homologize smaller anterior subventral glands were not the layers of the cuticle of X. index with those certainly established. of other nematodes.

Here the same appears to be true and aside and perpendicular pharyngeal muscles results from the outer osmiophilic layer the cuticular in reinvagination of the stoma. components of T. allius show no certain ho- The functioning of the pharyngeal lumen mology with those of X. index. The second appears to be by the classical mechanism found layer in T. allius may be the cortical layer but in most nematodes: opening of the lumen by it does not appear to be at all similar to the muscular action, closing by turgor pressure. second layer of X. index. Eversion of the stoma during feeding may Layer four is interesting in that it varies so possibly account for the fine folds in the inner much in thickness and is very spongy in nature. stomatal lining or osmiophilic layer (Plate II, The cuticle of most Trichodorus species is B). If eversion does take place the cuticular known to be very loose and tends to fold or lining would be stretched and the layers closest wrinkle during movement. In some fixatives to the lumen would be stretched most. A rea- it swells, sometimes ballooning out and forward sonable adaptation would be to provide folds beyond the anterior end of the body. The sec- in the cuticle which would disappear on stretch- tions seen here indicate most of this change ing. The folds would have to be most pro- takes place in L'. This suggests osmotic regula- nounced in the innermost layers and less in the tion may be active in this layer. outer layers of the stoma wall. Furthermore, it FEEDING PROCESS. The feeding process of T. would be expected that thinner membranes allius is accomplished by protraction of the could not withstand as much stress as the stylet to puncture plant cells and to ingest con- thicker ones and should be more folded. The tents of the cells through the triradiate lumen folded osmiophilic layer fits this concept. of the pharynx and esophagus. The structure of the odontostyle being toothlike with a solid Summary tip (Plate II, E) precludes the ingestion of Observations are reported here on the ultra- food through the odontostyle even though it structure of the cuticle, pharynx, musculature, appears to be hollow in sections just prior to sensory organs and esophagus of females of the tip. Anterior to the "guide ring" the stylet Trichodorus allius. The feeding process is also is located inside the triradiate lumen of the discussed. esophagus. Posteriorly the stylet lumen is ventrad to the extension. The muscle structure of Literature Cited the pharynx, as determined by their sections, Roggen, D. R., D. J. Raski, and N. O. Jones. suggests its function in the protraction and re- 1966. Cilia in nematode sensory organs. Sci- traction of the stylet to be as follows (Fig. 2): ence 152: 515-516. (a) rest position with all muscles relaxed; (b) . 1967. Further electron microscopic observations of Xiphinema index. Nemato- by contraction of the somatic muscles, labial logica 13: 1-16. muscles and perpendicular pharyngeal muscles, Wright, K. A. 1965. The histology of the the stoma is everted with a slight protraction oesophageal region of Xiphinema index of the stylet; (c) contraction of the odontostyle Thome and Allen, 1950, as seen with the protractor muscles results in protraction of the electron microscope. Can. J. Zool. 43: 689- stylet and increased turgor pressure in the 700. , and N. O. Jones. 1965. Some tech- pharyngeal wall; (d) upon relaxation of the niques for the orientation and embedding of protractor muscles, turgor pressure effects sty- nematodes for electron microscopy. Nemato- let retraction; (e) relaxation of somatic, labial logica 11: 125-130.

Penarchigetes oklensis gen. et sp. n. and Biacetabulum carpiodi sp. n. (Cestoidea: Caryophyllaeidae) from Catostomid Fish in North America

JOHN S. MACKIEWICZ University of Oklahoma Biological Station, and the State University of New York at Albany

During June and July of 1963—1966 approxi- (Orange Co.) and University Creek, a tributary mately 400 catostomid fish (Cypriniformes: of the Eno. Catostomidae), comprising five genera and nine species from southern and eastern Okla- Order Caryophyllidea Van Beneden homa, were examined for caryophyllid cestodes. (in Olsson 1893) From these collections three new species have Family Caryophyllaeidae Leuckart, 1878 been described (Calentine and Mackiewicz Penarchigetes gen. n. 1966; Mackiewicz 1968b); two other new (Figs. 1-7) species are described in this paper. Reference GENERIC DIAGNOSIS: Caryophyllaeidae. Sco- to the new species in the genus Biacetabulum lex with a pair of median bothria, two pairs Hunter 1927, described below was made in an of lateral loculi, and a small terminal disc. earlier abstract (Mackiewicz 1964). Cirrus joining uterovaginal canal. Ovary H or Cestodes were removed from freshly killed dumbbell shaped. Coils of uterus extending to fish, fixed usually by shaking them vigorously anterior level of cirrus. Preovarian vitellaria in a vial of 5% formalin, stained in Semichon's lateral, sometimes continuous with the post- carmine or Mayer's paracarmine and mounted ovarian vitellaria. External seminal vesicle in permount (Fisher) or Kleermount (Carolina absent. Type species: Penarchigetes oklensis. Biological). Hematoxalin and eosin sections were prepared in the usual manner. Drawings Remarks were made with the aid of a microprojector; In having a single gonopore, lateral vitellaria, measurements are in millimeters unless other- small terminal disc, and uterus not extending wise indicated. anteriorly beyond the cirrus in non-gravid in- Comparative material included: Biacetabu- dividuals, Penarchigetes most closely resembles ulm meridianum Hunter 1929, holotype USNM the caryophyllaeid genus Archigetes Leuckart Helm. Coll. No. 51059 (serial longitudinal sec- 1878. Unlike Archigetes, however, the new tions), and 11 of Hunter's original slides of genus lacks all trace of an external seminal this species (his numbers 687.2 to .6; all are vesicle (Figs. 6—7) which in Archigetes is a serial cross or longitudinal sections) now in the prominent muscularized structure often ap- author's collection; wholemounts or sections of proximating the size of the cirrus sac. Archigetes sieboldi Leuckart 1878, A. brachy- Penarchigetes superficially resembles Para- urus Mrazek 1908, A. cryptobothrius Wisniew- caryophyllaeus Kulakovskaya 1961; however, ski 1928, A. limnodrili (Yamaguti 1934), and since the placement of the inner longitudinal A. iowensis Calentine 1962; and a single whole- muscles is not described nor the presence or mount of Paracaryophyllaeus dubininae Kula- absence of the external seminal vesicle estab- kovskaya 1961. lished, or the number of gonopores mentioned, Since all available examples of B. meridi- adequate comparisons can not be made be- anum consisted of serial sections, attempts were tween the two genera. These important fea- made in August 1966 to recollect this species tures could not be discerned with certainty on from the type locality in North Carolina. Re- the single specimen of Paracaryophyllaeus ob- grettably, only B. infrequens Hunter 1927 was served. Nevertheless, on the basis of an un- found in two of 14 Moxostoma sp. (total length specialized scolex and anterior position of the 225—425) and four of five Erimyzon sucetta uterus of Paracaryophyllaeus as well as a great (Lacepede) (total length 150-225) from the difference in the distribution and hosts of the Eno River near Hillsboro, North Carolina two genera—palearctic Cobitidae for Paracary-

Figures 1-7. Penarchigetcs oklensis gen. et sp. n. from the spotted sucker, unless otherwise specified. 1A. Cross section through anterior part of bothria. IB. Cross section through middle part of bothria illustrating the absence of acetabular musculature. 2. Midsagittal section through gonopore. 3. Immature individual. 4. Gravid individual with cirrus everted. 5. Cross section through midpart of body. 6. Fron- tal section through cirrus and external seminal vesicle of Archigetes iowensis Calentine, 1962. 7. Frontal section through cirrus and vas deferens of P. oklensis illustrating the absence of the external seminal vesicle. Abbreviations: esv, external seminal vesicle; ut, uterus; vd, vas deferens.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 121 ophyllaeus and nearctic Catostomidae for Pen- HABITAT: Intestinal swelling and fore part archigetes, the two genera are regarded as of intestine; weakly attached between mucosal distinct from each other. folds. Clearly it is necessary to erect a new genus TYPE SPECIMENS: Holotype No. 71262 (Coll. to receive this form for which the name Pen 14 July 1965) and Paratypes (4) No. 71263 (pene, L. near) archigetes is proposed. Other of the USNM Helm. Collection; Paratypes (2) genera in the family Caryophyllaeidae first Nos. 1968.7.23.1 and 2 of the British Museum described from North American Catostomidae (Natural History) Helm. Collection. (suckers) are: Glaridacris Cooper 1920, Biace- Supplementary material (six slides) consist- tabulum Hunter 1927, Hijpocanjopliyllaeus ing of immature worms and sections include: Hunter 1927, Hunterella Mackiewicz and Mc- USNM Helm. Coll. No. 71263 and British Crae 1962, Isoglaridacris Mackiewicz 1965, Museum (Natural History) Helm. Coll. Nos. and Promonobothrium Mackiewicz 1968. 1968.7.23.3 and 4. Penarchigetes oklensis sp. n. Remarks SPECIFIC DIAGNOSIS (measurements based on This species, one of the smallest yet de- 11 gravid and three immature specimens, five scribed, offers some interesting morphological sectioned): Gravid individuals 1.2 (0.9—1.7) characteristics. For example, the scolex is long and 0.2 (0.26—0.44) wide at gonopore. expanded and appears to be intermediate Immature individuals from fish 0.9 (0.8—1.0) between the cuneiform-type of Glaridacris long. Length 3.3—5.0 times combined length catostomi Cooper 1920 and the disc-type of of neck and scolex. Scolex 0.34 (0.28-0.39) Glaridacris laruei (Lament 1921) or Archigetes wide. Neck indistinct. Outer longitudinal mus- ioivensis (Figs. 3, 4). In all gravid specimens cles poorly developed. Inner longitudinal mus- the testes contained only a few spermatogonial cles consisting of small fascicles, chiefly in a cells along the testis membrane; this condition dorsal and ventral group that form four large made it difficult to count the testes, particu- fascicles at base of scolex. Testes number 15 larly the most anterior ones. In immature indi- (11—21) in two irregular rows beginning 0.29 viduals, or ones with just a few ova in the (0.25—0.41) from tip of scolex and extending uterus, the uterus does not extend anterior to to a short distance anterior of cirrus sac. Cirrus the cirrus sac; in gravid individuals it does sac round; cirrus eversible. Preovarian vitellaria (Figs. 2—4). With some vitelline follicles dorsal beginning 0.33 (0.26-0.41) from tip of scolex, to the ovary (Fig. 3) Penarchigetes appears to usually continuous with postovarian vitellaria exhibit an intermediate condition between a over dorsal part of ovary. Postovarian vitelline continuous and discontinuous vitelline distribu- field longer than ovary. Ovary dumbbell- tion. These vitelline cells have the vacuolated shaped, compact follicular wings 0.10 (0.06— nucleus as recently described by Mackiewicz 0.15) long. Well developed seminal receptacle (1968a). absent. Osmoregulatory canals diffuse, no spe- Of eight M. melanops ranging in total length cific number in midpart of body. Egg with 277 (143-331), only two were infected with smooth shell, 56 by 40 ^ (one measured in 13 and 14 cestodes, respectively. Some cestodes utero); presence or absence of operculum were unquestionably overlooked because of could not be definitely established. their small size. Biacetabulum banghami Mack- The specific name refers to the state ot iewicz 1968 was the only other helminth as- Oklahoma. sociated with P. oklensis. Two Ictiobus bubalus DEFINITIVE HOST: Spotted sucker, Miny- (Raf.) and one I. ct/prinellus (Val.) were not trema melanops (Raf.) (Cypriniformes, Cato- infected with caryophyllids but one of three stomidae). USA, Oklahoma, Cherokee Co., Cyprinus carpio L. contained Atractohjtocestus Northeastern Outing Club Lake, 22 km NE huronensis Anthony 1958 and Khawia iowensis of Tahlequah on Okla. rt. 10. This lake is an Calentine and Ulmer 1961. ox-bow lake of the nearby Illinois River; it is Penarchigetes oklensis is the fourth caryo- approximately 10 acres in size, has a mean phyllaeid to be reported from the monotypic depth of 1 m and is periodically confluent with spotted sucker, the others are: Monobothrium the river. ulmeri Calentine and Mackiewicz 1966, Bi-

14 15 Figures 8-15. Biacetabulum carpiodi sp. n. from the river carpsucker. 8. Gravid individual. 9. Three immature individuals; note that the acetabular sucker is visible even on the smallest specimen (scale the same as in Fig. 8). 10. Midsagittal sections through the gonopore illustrating an expanding cirrus. 11. Midsagittal section through the scolex showing the basal membrane of the acetabular sucker. 12. Scolex with the acetabular sucker contracted. 13. Outline of an operculate egg. 14. Cross section through midpart of body. 15. Detail of the posterior part of body, ventral view.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 123 acetabulum banghami Mackiewicz 1968, Pro- C. cypriniis (LeSueur). TENNESSEE: Anderson monobothrium minytremi Mackiewicz 1968 Co., Oak Ridge, Clinch River at the Rullrun (Mackiewicz 1968b.) Two others, still under Steam Plant. Carpiodes sp. IOWA: Iowa River, study appear to be Biacetabulum infrequens South Fork Iowa River (coll. R. Calentine). Hunter 1927 and Glaridacris sp. HABITAT: Usually in the intestinal swelling "stomach," occasionally in the anterior part of Biacetabulum carpiodi sp. n. the intestine. Securely attached with scolex (Figs. 8-15) completely imbedded in the mucosa. SPECIFIC DIAGNOSIS (measurements based on TYPE SPECIMENS: Holotype No. 71264 (coll. 23 gravid and 11 immature individuals; 11 10 July 1965) and Paratypes (5) No. 71265 of sectioned): Gravid individuals 5.5 (4—7.5) the USNM Helm. Collection; Paratypes (2) long, 0.47 (0.32-0.70) wide at gonopore. Im- Nos. 1968.7.23.5 and 6 of the British Museum mature cestodes from fish 3.1 (0.9—5) long. (Natural History) Helm. Collection. Length 3.9 to 7.2 times combined length of Supplementary material (8 slides) consist- neck and scolex. Scolex with a pair of well- ing of immature worms and sections include: developed acetabular suckers; no accessory USNM Helm. Coll. No. 71265 and British loculi. Neck distinct 0.31 (0.24-0.46) wide. Museum (Natural History) Helm. Coll. Nos. Outer longitudinal muscles poorly developed. 1968.7.23.7 to 10. Inner longitudinal muscles consisting of large Remarks fascicles irregularly arranged about the cortical parenchyma. Testes begin 1.7 (1.2—2.2) from There are several morphological features tip of scolex and extend posteriorly to the which distinguish this species from all others in cirrus sac. Testes number 69 (59—84) and the genus. The pre- and postovarian vitellarian average 130 (90-210) p in diameter (5 fields are sometimes continuous dorsally over from each of 5 worms). Vas deferens extends the ovary (Fig. 15) similar to that described anteriorly between two rows of testes. Cirrus for Penarchigetes. The testes extend in two sac oval; male gonoduct joins uterovaginal rows on each side of the vas deferens (Figs. canal very close to ventral surface forming an 8, 15). And lastly, the cirrus intercepts the atrium with a large gonopore 50-100 p, in uterovaginal canal very close to the ventral diameter. Preovarian vitellaria in median as surface and with it forms a common atrium well as lateral position, extending 1.2 (0.8— with a large, single gonopore (Fig. 10). Be- 1.4) from tip of scolex to ovary or sometimes cause of this large gonopore, an expanded continuous with postovarian vitellaria. Post- cirrus and female gonopore may give the ap- ovarian vitelline field longer than ovary. Ovary pearance of two gonopores. By its small size shaped like letters H or M; compact follicular and reduced number of testes, B. carpiodi wings 0.3 (0.2-0.5) long. Seminal receptacle most closely resembles B. meridianum and B. present. Fourteen osmoregulatory canals at macrocephalum McCrae 1962 (Table I). How- mid-region of body. Eggs operculate, shell ever, both of these species have few postovarian vitellaria and those present do not smooth, 53 (51-56) ^ long by 32 (28-34) ^ reach the ovary as in B. carpiodi. In addition wide (5 measured in utero); undeveloped to the gross morphological features mentioned when shed. above, B. carpiodi also lacks the well-developed The specific name refers to the host. outer longitudinal muscles of B. meridianum. DEFINITIVE HOSTS: River carpsucker, Car- Incidence and worm burden varied with host piodes carpio (Raf.) (Cypriniformes: Cato- and locality. Five of 70 river carpsuckers (total stomidae). IOWA: Des Moines River (coll. D. length 232-475) from Lake Texoma, Okla- Williams). OKLAHOMA: Marshall Co., Lake homa, contained 1—3 worms per fish whereas Texoma; Payne Co., Lake Carl Blackwell (coll. four of 16 fish (total length 232-414) from R. D. Spall). TENNESSEE: Lake Co., Mississippi the Red River, just below Denison Dam of River at Powell's Ferry opposite Caruthersville, Lake Texoma contained 3—37 parasites. From Missouri, New Madrid Co. TEXAS: Grayson the Clinch River, Oak Ridge, Tennessee, five Co., Red River below Denison Dam of Lake of 38 quillback carpsuckers (total length 212— Texoma (Type locality). Quillback carpsucker, 375) contained 1—2 parasites. In all cases,

Copyright © 2011, The Helminthological Society of Washington 124 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY which represented 91 cestodes, there were no maturing individuals before ova have greatly concurrent infections with other caryophyllids distended the uterus. although Spartoides wardi Hunter 1929 and Whether or not the pre- and postovarian another species of undetermined generic status vitelline fields are continuous may be also an (from Tenn.) occurred in the same host. Fur- important systematic character although at thermore, this species appeared to exhibit a what level is not clear at present. In some marked host-specificity for Carpiodes sp. not genera, for example in Archigetes (except for being found in approximately 275 other cato- A. sieboldi), Atractohjtocestus Anthony 1958 stomid fish representing six genera and 11 (? = Markevitschia Kulakovskaya 1965), Brev- species. iscolex Kulakovskaya 1962, Capingens Hunter The only other Biacetabulum reported from 1927, or Wenyonia Woodland 1923, all of the carpsuckers is that of B. meridianum from 201 vitellaria clearly appear as one continuous C. carpio from Lake Texoma (Timmons, 1951; unit. In others, such as Caryophullaeus Gmelin Self and Timmons, 1955); most infected fish 1790, Glaridacris, and Isoglaridacris, to men- were collected in May. Specimens of these tion a few, the vitellaria are definitely confined Biacetabulum were lost in the mail before they to separate pre- and postovarian regions. Pen- could be re-examined. Except for having testes archigetes and B. carpiodi as well as Caryo- numbering 45-53 (N = 6). Self and Tim- phyllaeides fennica (Schneider 1902) and mons reported that all other characters closely Khawia baltica Szidat 1942 appear to exhibit complied with Hunter's description of B. an intermediate condition. In the absence of a meridianum. In the absence of the original distinct continuity it would appear that this slides it is difficult to appraise the validity of intermediate condition is a variation of the dis- this record; however, all Biacetabulum from continuous distribution. More precise details Lake Texoma Carpiodes were first called B. of the vitelline duct system might aid in the meridianum until critical study indicated that interpretation of this character. a new species was involved. The genus Biacetabulum has been subject Self and Timmons (1955) also reported G. to various interpretations since described by confusa from C. carpio; specimens that could Hunter (1927). Szidat (1937) was the first to be re-examined were redetermined as Sparti- consider some palearctic Archigetes as conge- odes wardi Hunter 1929. neric with Biacetabulum. Calentine (1965), reviewing the complex Archigetes-Biacetabu- Discussion lum question, clearly demonstrated the validity Both of these species offer variations in cer- of the two genera on the basis of life-historv tain generic characters. For example, the posi- and morphological characters. A further analy- tion of the uterus with respect to the cirrus sac sis of this same problem by Kennedy (1965) has long been an important generic criterion resulted in the synonymy of all palearctic in the order Caryophyllidea. In some genera, Biacetabulum under the genus Archigetes. Ex- e.g. Biacetabulum, the uterus clearly twists cept for the Australian species B. tandani and forms loops anterior to the cirrus sac with Johnston and Muirhead 1950, incompletely de- the uterine primordia assuming this position scribed from a single specimen and therefore before gametogenesis commences (Fig. 9). In of questionable status (it has been omitted in others, such as Penarchigetes, Archigetes, or Table I), all other species (six) of Biacetabu- Glaridacris (especially G. laruei), in which the lum are from the nearctic. uterus normally reaches the anterior level of All of these species, including B. carpiodi, the cirrus sac, its position may vary with the can be arranged into distinct groups on the state of maturity of the cestode. That ova can basis of scolex morphology and preovarian extend the uterus beyond its normal position vitellaria distribution (Table I). There can be has recently been forcefully illustrated by Ken- little doubt that Group I constitutes a good nedy (1965) in the genus Archigetes. While species group, although some of the species, this uterus-cirrus sac relationship may be a B. infrequens for example, may represent a continuously variable character, whatever its species complex. The relationship of Groups nature it should be judged in immature and II and III to each other and to Group I is

Table 1. Comparison of the species of Biacetabulum Hunter, 1927.1

Posterior limit of: Acetab- ular vitellaria Testes Preovarian Testes Length Most common Species suckers distribution vitellaria no. ( mm ) host Group I B. infrequens Yes Median and Uterus Cirrus 420-440 16—22 Moxostoma anisurum Hunter, 1927 lateral (Raf.) B. meridianum Yes Median and Vas defereiis External 64-95 5—15 Erirnyzon sucetta Hunter, 1929 lateral seminal ( Lacepede ) vesicle B. giganteum Yes Median and Vas dcferens External 165-215 7-16 Ictiobus bubalus Hunter, 1929 lateral seminal (Raf.) vesicle B. macrocephalum Yes Median and Uterus Cirrus 75-95 4—6.5 Catostomits commersoni McCrae, 1962 lateral ( Lacepede ) B. carpiodi Yes Median and Cirrus Ovary or 59—84 4—7.5 Carpiodes carpio Mackiewicz lateral continuous (Raf.) (this paper) with POV Group II B. biloculoides No Median and Uterus Cirrus 50-90 3.7—11 C. commersoni Mackiewicz and lateral (Lacepede) McCrae, 1965 Group III B. banghami No Lateral mostly, Uterus Ovary 63-91 3.5—13 Minytrema melanopft Mackiewicz, 1968 continuous (Raf.) anteriorly

1 Data for described species from original descriptions, illustrations, or type specimens. much more complex, perhaps involving the deed, it is becoming increasingly clear from introduction of subgenera or possibly of sepa- these southern species as well as others from rate genera, depending upon the systematic different parts of North America, and sup- value given the scolex and the vitelline ported by examples from other parts of the distribution. world, that reliable generic criteria in the Similar problems of generic definition are il- Caryophyllidea have not been, and can not be, lustrated by the palearctic and nearctic Caryo- established until there is a more comprehensive phyllaeus, Monobothrium, and Khawia in view of morphological types and variation in which different scolex types and/or vitellaria the order. distribution patterns occur within the same Acknowledgments genus. Even in Archigetes there are median vitellaria in A. sieboldi but not in the other For assistance in collecting hosts, the coop- species; while in Glaridacris the vitellaria are eration of the Oklahoma Department of Game and Fish is gratefully acknowledged. Apprecia- annularly arranged in G. catostomi but in tion is extended to Dr. J. Teague Self and Dr. lateral rows in the other species. These few C. Riggs, Director of the University of Okla- examples illustrate the need for a critical re- homa Biological Station, for their generous appraisal of generic criteria in an order in cooperation; to Dr. O. Kulakovskaya of the which 60% of the approximately 35 genera are Lvov Museum of Scientific Nature Study, monotypic. In recent collections from catosto- Ukrainian Academy of Sciences, for loan mid fish in Tennessee, Alabama, and North of material; and to Drs. R. Calentine and Carolina, numerous new species were found C. Kennedy for examining preparations of that had different generic characters in various Penarchigetes, combinations with each other, thus posing This study was supported in part by the serious problems of generic redefinition. In- Oklahoma Biological Survey and Grant-in-Aid

No. 20-0160 B from the Research Foundation . 1968a. Vitellogenesis and eggshell for- of the State University of New York. mation in Caryophyllaeuf, laticeps (Pallas) and Canjophyllaeides Fennica (Schneider) Literature Cited (Cestodea: Caryophyllaeidea). Z. f. Para- Calentine, R. L. 1965. The biology and tax- sitenk. 30: 18-32. onomy of Biacetahulum (Cestoda: Caryo- . 1968b. Two new caryophyllaeid ces- phyllaeidae). J. Parasit. 51: 243-248. todes from the spotted sucker, Minytrema , and J. S. Mackiewicz. 1966. Mono- nielanops (Raf.) (Catostomidae). J. Parasit. bothrium ulmeri n. sp. (Cestoda: Caryophyl- 54: 808-813. laeidae) from North American Catostomidae. Self, J. T., and H. F. Timmons. 1955. The Tr. Am. Micr. Soc. 85: 516-520. parasites of the river carpsucker (Carpiodes Hunter, G. W., III. 1927. Notes on the carpio Raf.) in Lake Texoma. Tr. Am. Caryophyllaeidae of North America. J. Para- Micr. Soc. 74: 350-352. sit. 14: 16-26. Szidat, L. 1937. Archigetes R. Leuckart, 1878, Kennedy, C. R. 1965. Taxonomic studies on die progenetische Larve einer fiir Europa Archigetes Leuckart, 1878 (Cestoda: Caryo- neuen Caryophyllaeiden-Gattung Biacetabu- phyllaeidae). Parasitology 55: 439-451. Mackiewicz, J. S. 1964. Variations and host- lum Hunter, 1927. Zool. Anz. 119: 166-172. parasite relationships of caryophyllaeids (Ces- Timmons, H. F. 1951. A study of the hel- todea) from fish of Lake Texoma, Marshall minth parasites of the river carpsucker County, Oklahoma. J. Parasit. 50 (3, sec. 2): (Carpiodes carpio) in Lake Texoma. Master's 31 (abstract). thesis, University of Oklahoma, 49 p.

The Chromosomes of Hunterella nodulosa Mackiewicz and McCrae, 1962 (Cestoidea: Caryophyllidea)

JOHN S. MACKiEwicz1 AND ARTHUR W. JONES Department of Zoology and Entomology, University of Tennessee, Knoxville, Tennessee

Cytological studies on tapeworms have getes, it is impossible to judge the worth of been almost entirely confined to Cyclophyllid- similar cytotaxonomic studies in the monozoic ean cestodes (for reviews see Jones 1945, tapeworms of the order Caryophyllidea. One Walton 1959). Exceptions have been the purpose of the present study, one of a pro- studies of four species of the Pseuclophyllidea jected series, is to learn if cytological informa- by Wolcott (1959), and Wikgren and Gus- tion on the Caryophyllidea will aid in the tafsson (1965), and of Archigetes sp. (order practical problems of taxonomy at the family, Caryophyllidea) by Motomura (1929). Where genus and species levels; another is to learn if comparison between species in the same genus such studies can help in appraising the rela- was made, especially of Hymenolepis by Jones tionship of these unusual tapeworms to the (1945) and Diphyllobothrium by Wikgren and more common strobilate groups, from which Gustafsson (1965), the chromosome numbers they differ greatly in morphology and general and/or karyotypes showed little variation and biology. therefore could be used little as an aid in the Hunterella nodulosa Mackiewicz and Mc- taxonomic analyses of the polyzoic cestodes Crae, 1962 (Cestoidea: Caryophyllidea) of the studied. family Caryophyllaeidae was chosen for de- From the single investigation of Motomura tailed cytological studies for two reasons. First, (1929), who was primarily interested in the in a preliminary survey of 17 species of caryo- embryology rather than the cytology of Archi- phyllids studied by the squashing technique, squashes of H. nodulosa had large numbers of 1 On academic leave from State University of New York at Albany, New York. good metaphase plates with relatively large,

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 127 well stained chromosomes in the testes and acetic acid. A large proportion of the testes vitellaria. Second, H. nodulosa is easily identi- and vitelline follicles were teased out and, fied, widely distributed in North America, and along with the remaining tissues, dissected relatively easy to obtain, occurring as a com- into small fragments; only large pieces of the mon intestinal parasite of the white sucker, integument were discarded. A 22 mm square Catostomus commersoni (Lacepede). It is coverslip (No. 1 or 0) was applied and, after among the best known of North American excess acetic acid had evaporated, the prepara- Caryophyllidea with recent studies on syste- tion was squashed by placing the slide within matics, morphology, and distribution (Mack- one fold of paper towel and applying with the iewicz, I960; McCrae, I960; Mackiewicz and thumb the full weight of the body. The pres- McCrae, 1962) periodicity (Calentine and ence of Newton rings under the coverslip in- Fredrickson, 1965) and larval development dicated a satisfactory squash. The preparation (Calentine, 1965). was made permanent by using the dry ice method of Conger and Fairchild (1953), de- Materials and Methods hydrating in 95% or 100% ethyl alcohol for 1 Parasites were obtained on 27 November min and mounting with diaphane. 1967 from the white sucker, Catostomus com- Dividing cells were most common in the mersoni (Lacepede) (Cypriniformes; Cato- testes but excellent mitotic figures also oc- stomidae) from Jacks Creek, a tributary of the curred in the vitelline follicles, particularly in Smith River (Roanoke drainage), Patrick Co., cestodes in which spermatogenesis was just be- Virginia and from the same host from the Little ginning. Mitoses were rare in the parenchymal River, Walland, Blount Co., Tennessee on 29 cells. March 1968. Living cestodes were dropped Eighteen squash preparations representing into a vial of Carnoy's fixative (6:1:1, ab- a large number of testes and vitelline follicles solute ethyl alcohol, chloroform, glacial acetic from eight cestodes were studied. All illustra- acid) that was placed in ice water and later tions and most of the observations were made stored in a refrigerator until squashes were on squashes from immature worms collected in prepared in May, 1968. No differences in Virginia; the Tennessee collections consisted staining quality were detected in material of mostly gravid worms and were used to sup- stored for different periods. Carnoy fixation plement and verify the other observations. gave good spreads and preservation of the Camera lucida drawings of 16 metaphase plates chromosomes, unlike the results of Wikgren studied under oil immersion (900 and 1,800 (1964) who worked with plerocercoids of D. powers) were prepared at an enlargement of latum. 2,600 X. From 11 of these drawings of meta- Squashes were prepared using the leuco- phase plates (three from testes and eight from basic fuchsin (Feulgen) stain prepared ac- vitelline cells of two worms) an idiogram was cording to Darlington and La Cour (1960). constructed by measuring the chromosomes to Cestodes were removed from the vial of fixa- the nearest 0.5 mm and expressing each chro- tive, hydrated with successive changes (2 hrs mosome or arm as a percent of the total chro- each) of 95%, 50% (ethyl alcohol), and tap mosome length in the complement. In this way water. The whole worm was hydrolyzed in 1 there was an adjustment for the differences in N HC1 at 55 C to 60 C for 9-11 min (10 min chromosomes due to squashing or other factors. was later found to be a good average time) In those cases where either the centromere po- and removed to the Feulgen stain for one hr. sition could not be determined or the chromo- Usually the testes and ovary began to show a some was folded, no measurements were at- staining reaction within the first minute. When tempted; the sample size for each chromosome these organs became deeply stained, (usually measured therefore varied. The position of the within one-half hour), the whole worm was centromere of the small rods could not be ac- placed in 45% acetic acicl for 5-10 min in curately determined but from indirect evidence, order to soften and swell the tissues. A small as discussed under "observations," these chro- part of the body, approximately one-half mm mosomes appeared to be acrocentric and are so long, containing testes and vitellaria was placed indicated on the idiogram. Karyotype analysis on a slide and teased apart in a drop of 45% was based on the above eleven plates plus five

Figures 1-5. Chromosomes of Hunterella nodulosa. Feulgen stained squashes. 1, 2. Metaphase plates from vitelline cells. 3. Diakinesis. Note that more than one chiasma is readily visible on most bivalents. 4. Metaphase I, showing seven bivalents. 5. Metaphase II, Anaphase II, both dyads and monads are visible.

others, only parts of which were suitable for ("J's") (Figs. 6, 7). At mitotic metaphase in purposes of an idiogram, that included two the spermatogonia and vitelline cells the long- from testes and three from vitelline cells of est chromosomes are 6—8 ^, the shortest 2.5-3 three worms (two from Virginia, one from ^ long. As shown by the idiogram (Fig. 6) Tennessee). there are four large "V's," two small "V's," two The photographs were made with a Zeiss large "J's," and three pairs of small rods. The Photomicroscope using phase contrast optics, largest chromosome represents approximately including an apochromatic oil immersion lens 12% of the total chromosome complement and a green filter. (TCL). No satellites or fragments were observed. The location of the centromere of the Observations mitotic metaphase chromosomes could be de- Hunterella nodulosa has 14 chromosomes, termined for all but the six rods, but from diploid (Figs. 1, 2, 7, 8). These include three observations of metaphase I of spermatogenesis pairs of metacentrics, ("V's", two long and one it appears that the centromeres of these rods short pair), three pairs of short acrocentrics are terminal or nearly so. ("rods"), and one pair of long submetacentrics Meiosis was studied only in spermatogenesis

2N = 14 HUuu

Figures 6-8. Chromosomes of Hunterella nodulosa (cont'd). 6. Idiogram; TCL, total chromosome length. 7. Karyotype from a single vitelline cell nucleus. 8. Two metaphase plates from vitelline cells. since no meiotic activity was observed in intra- Motomura designated it as Archigetes appendic- uterine eggs. Spermatogenesis proceeds as ulatus Ratzel. Unfortunately there have been usual with normal sperm being formed after two cestodes with this same binomial, A. ap- two meiotic divisions. Two meiotic or matura- pendiculatus Ratzel 1868 and A. appendicula- tion divisions have also been reported for Tri- tus Mrazek, 1897. It has long been recognized, anophorus lucii (Mull., 1776) (Pseudophyl- however, that while Ratzel's species was in lidea) by Rybicka (1962). At diakinesis struc- fact the larval stage of Caryophyllaeus laticeps ture of the chiasmata was readily observable in Leuckart, A. appendiculatus Mrazek repre- most bivalents (Fig. 3). Chiasma frequency sented a distinct species (Nybelin, 1922). was not worked out but it appears to be 5.0 Since Mrazek's name has been in more com- or 6.0 per bivalent, at least in the case of the mon usage with the binomial it is difficult to longest chromosomes. No attempt was made to understand why Motomura did not use it. The ascertain the number of primary spermatocytes complex case involving these two binomials resulting from a single spermatogonium, details has been thoroughly discussed by Wisniewski best studied in sections. A single sperm could (1930). Kennedy (1965b) in his recent re- easily be seen adjacent to or wrapped around vision of Archigetes does not list Ratzel's spe- the ovum in intrauterine eggs. cies (since it refers to Canjophyllaeus); he places A. appendiculatus Mrazek in synonymy Discussion under A. sieboldi Leuckart, 1878. A further Motomura (1929) was the first to report the complicating factor has been the discovery of chromosome number of a caryophyllidean; another species of Archigetes, A. limnodridi however, there appears to be some question (Yamaguti, 1934), in Limnodrilus sp. and a regarding the identity of the cestode studied. cobitid fish in Japan (Yarnaguti, 1934; Ken-

Copyright © 2011, The Helminthological Society of Washington 130 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY nedy, 1965a). Motomura was most certainly some numbers may have systematic value. It is dealing with Archigetes, because gravid speci- too early to judge if karyotypes show similar mens came from aquatic oligochetes (Limnod- significance. Whether differences in chromo- rilus and Tubifex) yet he did not further de- some numbers between Archigetes sp. and H. scribe or illustrate his species. It is therefore nodulosa have systematic value can not be de- impossible to know whether he had A. sieboldi termined until there are data from more spe- (which is not reported from Japan), A. lim- cies. Studies now in progress indicate that nodrili (originally described from that coun- there is more variation in the chromosome num- try), or another, perhaps undescribed, species bers of caryophyllidean species than any other of Archigetes. group of cestodes yet studied, that the chromo- Adequate comparisons between the present somes are the largest yet described for any work and that of Motomura (1929) are not group of cestodes, and that the chromosomes possible since cytological observations were but can be demonstrated with few technical a minor part of his study. Motomura observed difficulties. a diploid number of 18 and that the chromosomes were of unequal size. From his Figure 2 Acknowledgments of the first maturation division in the ovum, we Thanks are extended to the Departments of measure the largest chromosome to be 3.3 p. Fish and Game of Virginia and Tennessee for long. H. nodulosa, on the other hand, has a permission to collect hosts, to Dr. Etnier for diploid number of 14 and chromosomes of assistance in collecting and to Dr. B. Hochman unequal and larger size (Fig. 8). According for photographic assistance. to Walton (1959) the only other cestode with This study was supported in part by Grant- 14 diploid chromosomes is Liga brasiliensis In-Aid 20—0244 A from the Research Founda- Ransom, 1909 [= Liga punctala (Weinl. 1856) tion of the State University of New York. Weinl. 1857 of Yamaguti (1959) and L. brasiliensis Parona, 1901 of Wardle and Mc- Literature Cited Leod (1952)] a cyclophyllidean of birds Calentine, R. L. 1965. Larval development of (Jones, 1945) but distantly related to four caryophyllaeid cestodes. Iowa Acad. caryophyllideans. Sci. 72: 418-424. More closely related are the Pseudophyllidea, , and L. H. Frederickson. 1965. Perio- an order under which caryophyllideans have dicity of caryophyllaeid cestodes in the often been placed as a family (Hunter, 1930; white sucker, Catostomus commersoni (Lace- Joyeux and Baer, 1961). Working with colchi- pede). Iowa State J. Sci. 39: 243-250. cine-treated pleroceroids Wikgren and Gus- Conger, A., and L. Fairchild. 1953. A quick- tafsson (1965) observed that Diphyllobothrium freeze method for making smear slides per- latum, D. osmeri and D. dendriticum have dip- manent. Stain Tech. 28: 281-283. loid numbers of 18 and that the karyotypes of Darlington, C. D., and L. F. LaCour. 1960. The Handling of Chromosomes. 3rd Ed. somatic cells of all species are apparently iden- George Allen and Unwin Ltd. London. 247 p. tical. The largest chromosome of D. latum is Hunter, G. W., III. 1930. Studies on the 3.5 //, long with seven pairs of metacentrics and Caryophyllaeidae of North America III. Biol. two of submetacentrics making up the comple- Monog. 11: 1-186. ment. In contrast, the longest chromosomes of Jones, A. W. 1945. Studies in cestode cytol- H. nodulosa are 8 /j, long with only three pairs ogy. J. Parasitol. 31: 213-235. of metacentrics in the complement. On cytolog- Joyeux, C. E., and J. G. Baer. 1961. Classe ical grounds the relationship of Diphylloboth- des Cestodaires. In: Traite de Zoologie, T. rium and Hunterella does not appear to be IV fasc. 1: 327-560. close. Kennedy, C. R. 1965a. The life-history of Unlike the experience of Jones (1945) or Archigetes limnodrili (Yamaguti) (Cestoda: Wikgren and Gustafsson (1965) in which little Caryophyllaeidae) and its development in the invertebrate host. Parasitology 55: 427- variation in chromosome number or karyotype 437. was found among the species studied, prelimi- . 1965b. Taxonomic studies on Archi- nary studies on the cytology of cestodes of the getes Leuckart, 1878 (Cestoda: Caryophyl- order Caryophyllidea indicate that chromo- laeidae). Parasit. 55: 439-451.

Mackiewicz, J. S. 1960. Studies on the Caryo- dien Triaenophorus lucii (Miill., 1776). Bull. phyllaeidae (Cestoidea) of Catostomus com- Soc. Neuchatel. Sci. Nat. 85: 177-181. mersoni (Lacepede), with emphasis on those Walton, A. C. 1959. Some parasites and their from fish near Ithaca, New York (Tompkins chromosomes. J. Parasit. 45: 1-20. County), USA. Ph.D. thesis, Cornell Univ. Wardle, R. A., and J. A. McLeod. 1952. The Microfilm No. 61-1439. Univ. Microfilms, Zoology of Tapeworms. Minneapolis. Univ. Inc., Ann Arbor, Mich. Minn. Press. 780 p. , and R. McCrae. 1962. Hunterella Wikgren, B.-J. 1964. Studies on the mitotic nodulosa gen. n., sp. n. (Cestoidea: Caryo- activity in plerocercoids of Diphyllobothrium phyllaeidae) from Catostomus commersoni latum L. (Cestoda). Comm. Biol. Soc. Sci. (Lacepede) (Pisces: Catostomidae) in Fenn. 27: 1-33. North America. J. Parasit. 48: 798-806. , and Margareta Gustafsson. 1965. McCrae, R. 1960. Studies on the Caryophyl- The chromosomes of somatic cells of three laeidae (Cestoda) of the white sucker Diphyllobothrium species, with notes on the Catostomus commersoni (Lacepede) in north- mode of cell division. Acta Acad. Aboensis, ern Colorado. Ph.D. thesis, Colorado State Ser. B. 25: 1-12. Univ., Microfilm No. 60-6797, Univ. Micro- Wisniewski, W. 1930. Das genus Archigetes films, Inc., Ann Arbor, Mich. R. Leuck. Eine Studie zur Anatomic, His- Motomura, I. 1929. On the early develop- togenese, Systematik und Biologic. Mem. de ment of monozoic cestode, Archigetes appen- L'Acad. Pol. des Sci. et des Lettres., Class des Sci. Math, et Nat. Ser. B. II: 1-160. diculatus, including the oogenesis and fer- Wolcott, G. B. 1959. The chromosomes of tilization. Annot. Zool. Japan. 12: 109-129. Dipht/llobothrium ursi. J. Parasit. 45: 378. Nybelin, O. 1922. Anatomish—Systematische Yamaguti, S. 1934. Studies on the helminth Studien iiber Pseudophyllideen. Goteburgs fauna of Japan. Pt. 4. Cestodes of fishes. Kungl. Vetensk. Vitterh. Samh. Handl. Jap. J. Zool. 6: 1-112. Fjarde Puljden 26: 1-228. . 1959. The Cestodes of Vertebrates. Rybicka, Krystina. 1962. Observations sur la Systema Helminthum, Vol. II. New York, spermatogenese d'un cestode pseudophylli- Interscience Pub. 860 p.

A Protandrous Haploporid Cercaria, Probably the Larva of Saccocoelioides sogandaresi Lumsden, 1963

R. M. CABLE AND HADAR ISSEROFF Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907, and Department of Biology, Rice University, Houston, Texas 77005

During the summer of 1967, an unusual trematode. It has a large seminal vesicle filled cercaria was found among larval trematodes with sperms but the testis consists of a largely developing in Amnicola comalensis collected empty sac when the larva is shed by the snail. from a drainage canal near Galveston Bay, That gonad becomes conspicuously developed Texas. It not only is the first freshwater in the cercarial embryo, produces sperms, and cercaria reported for the Haploporidae, a pre- retrogresses before development in the snail is dominantly marine family of trematodes, but complete. In respects other than sexual devel- also is sexually precocious with a degree of opment, the cercaria agrees with the only other protandry apparently unique among larval reported haploporid larva, a marine species trematodes. Many cercariae have well devel- (Cable, 1962). Given a testis like that of the oped genital primordia and a few azygiid lar- cercarial embryo, the body would have fea- vae even contain eggs on escape from the tures of the genus Saccocoelioides which Szidat mollusk. The Texas species lacks eggs but the (1954) erected for freshwater species in body is otherwise equivalent to an adult Argentina.

Infected snails were collected near the pharynx about midway between suckers, 41—58 brackish ponds where Lumsden (1963) found long, 53—62 wide. Prepharynx almost as long sailfin mollies, Mollienesia latipinna, infected as pharynx, esophagus longer; bifurcation near with a new species of Saccocoelioides, S. so- posterior edge of ventral sucker; ceca short, gandaresi. Several dimensions of the haploporicl divergent, inflated. Testis in emerging larva an larva fall within the ranges Lumsden gave for elongate, largely empty sac superimposed on his species. Although the two differ in some excretory bladder; well developed in embryo, other respects, they probably are stages of the smooth at first but becoming slightly indented same species as Sogandares has suggested in a as sperms form. External seminal vesicle filled personal communication. Evidence to that ef- with sperms, most conspicuous feature of hind- fect will be presented after describing the body; beginning as a wide sac near midlevel stages provided by infected snails. of that region, then tapering as it extends an- The entire cercaria was drawn to scale from terolaterally and loops clorsomedially at right measurements of a representative specimen of ventral sucker to turn anteriorly and enter among spontaneously emerging larvae killed in hermaphroditic sac. That sac spherical, over- near-boiling 2% formalin and measured im- lapping ventral sucker anterolaterally; contain- mediately under a freely floating cover glass. ing internal seminal vesicle, prostatic complex Details were added freehand from the study of and metraterm. Ovary small, ovoid, with few living specimens and wholemounts stained with cells; slightly to left, overlapping external Semichon's carmine and counterstained with seminal vesicle posterolaterally. From ovary, indulin. The metacercaria was drawn from a oviduct extends anterolaterally to left, joined living specimen in the same manner, and the first by Laurer's canal, then by common vitel- cercarial embryo by microprojection of a line duct before bending medially to join wholemount. All measurements are given in ootype posterolateral to ventral sucker. Uterus microns; lengths and widths of certain cercarial short, curving to right from ootype, around structures were measured parallel to those di- ventral sucker to cross neck of external seminal mensions of the entire larva. vesicle, enter hermaphroditic sac and join male duct anterior to pars prostatica. Common gono- Description of Stages duct short; genital pore median, near anterior Cercaria (Figs. 2-4) edge of ventral sucker. Seminal receptacle Biocellate, distomate; tail slender, unadorned, absent, amphitypy not observed. Vitelline cells attached subventrally. Larva strongly photo- large, in massive follicles not in two lateral positive, swimming smoothly and almost con- fields but filling parenchyma of hindbody not tinuously before encysting in the open. Body otherwise occupied and compressing incon- linguiform to pyriform, 410—436 long; maxi- spicuous cystogenous glands against subtegu- mum width 175—190, at or somewhat posterior mental muscles. Excretory system stenostomate; to midlevel. Tail 485-510 long, 41-45 wide at primary pores at sides of tail, near base. Blad- slight swelling near base. Tegument of body der elongate sacciform, extending almost to thick, colorless, uneven; with fine spines from ovary; with distinctly thickened wall contain- anterior end almost to eyespot level; par- ing nuclei. From bladder, main tubules extend enchyma yellowish, with much brown pigment to sides of oral sucker, then posteriorly to ven- posterior to eyespots, first clumped, then dis- tral sucker where each receives an anterior and persed except for concentration near caudal posterior secondary tubule. Flame cells num- attachment; eyespots 23 by 20. Oral sucker erous, apparently three groups served by each 61—67 long, 70—76 wide; ventral sucker equa- secondary tubule; five or six flame cells per torial, 69—76 in diameter. Mouth subterminal; group posterior to ventral sucker. Development

Figures 1-5. 1. Young redia drawn freehand to show excretory system of one side. 2. Cercaria, ventral view. 3. Same, showing excretory system on one side of body. 4. Lateral view of cercarial embryo showing precociously developed testis. 5. Metacercaria. Abbreviations: ES, external seminal vesicle; EV, excretory vesicle; GP, genital pore; HS, hermaphroditic sac; IN, intestine; IS, internal seminal vesicle; MG, Mehlis' gland; OT, ootype; OV, ovary; TE, testis; UT, uterus; VI, vitelline follicles.

Copyright © 2011, The Helminthological Society of Washington 134 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY completed in digestive gland of snail after and presumably older specimens; even then, it escape from redia. usually had the appearance of a young testis HOST: Amnicola comalensis Pilsbry and Fer- before the various stages of spermatogenesis riss, 1906. are evident as cell clusters. The cercaria in HABITAT AND,LOCALITY: Drainage canal near question would account for that situation by Galveston Bay, Texas. providing sperms for early egg production be- DEPOSITED SPECIMENS: Nos. 71321-71323 fore the testis redevelops and becomes func- USNM Helm. Coll. tional. Larger adults are about one-fourth longer and considerably wider than the cer- Redia (Fig. 1) carial body but dimensions of the suckers and Elongate, without collar or "feet"; gut short pharynx are essentially the same in both. Dur- and wide in older rediae. Pharynx of young ing growth in the vertebrate, utilization of redia 32 in diameter. Excretory pores lateral, sperms and vitelline cells provided by the just posterior to pharyngeal level. Excretory cercaria could account for the less extensive formula 2[(3) + (3)] = 12 flame cells. seminal vesicle and vitellaria in the adult. Metacercaria (Fig. 5) Meanwhile spination limited to the anterior Cyst symmetrically ovoid, not flattened on end of the cercaria could develop over the one side or adhering to surfaces; with indis- entire body. tinct outer layer and well defined inner one, Morphological differences exclude as the fibrous, 8—11 thick when formed but becoming adult of the present cercaria two other hap- somewhat thicker with age and appearance of loporids known to occur in the region. None space between worm and cyst wall. Cavity of was found in a variety of fishes which we cyst 260-293 long, 200-230 wide. Eyespots seined from the canal where infected snails begin to dissociate soon after encystment. occurred, but Mollienesia latipinna, the definitive host of Saccocoelioides sogandaresi was Discussion not included. Lumsden (personal communica- Although the bladder wall of the cercaria is tion) found in a different snail species a appreciably thickened and contains nuclei, it haploporid cercaria which encysted in the does not appear to be of the epithelial type. open. He fed metacercariae to M. latipinna but Instead, the nuclei seem to be those of the with negative results which he considered in- primary excretory tubules and retained after conclusive because he had found another fusion of the tubules to form the bladder, giv- haploporid adult, Saccacoelium beauforti, in ing it the appearance of being epithelial. A mullet from the same region. It now seems that similar bladder has been seen in bucephalid his cercaria may well be that species and the cercariae (unpublished studies). present one the larva of S. sogandaresi. Features which at first made the cercaria Although haploporids occur mostly in marine seem unlikely to be the larva of Saccocoelioides fishes, Szidat (1954) reported several species sogandaresi were the limited body spination, in Argentine freshwater hosts. He regarded the long prepharynx, extent of the vitellaria, large parasites as relicts of the ancient Tethys Sea external seminal vesicle, and reduction of the which extended from the Mediterranean region testis after its conspicuous development in the to eastern South America and covered the cercarial embryo. The last feature suggested lower watershed of rivers now in that area. In that the adult would be short-lived, grow little a similar manner, former inundations of central if any in the vertebrate host, and have avail- United States could account for the far inland able only the sperms provided by the cercaria. occurrence of a few trematodes in such pre- In contrast, the adult of S. sogandaresi is de- dominantly marine families as the Micro- scribed as having a large testis. However, phallidae, Lepocreacliidae, Miero.scapbj.idae, examination of the paratype of that trematode Cryptogonimidae, and Didymozoidae. The life and several additional specimens provided by history has been elucidated for at least one Dr. R. D. Lumsden showed that worms com- freshwater species in each of those families parable to the cercarial body in size contained except the Microscaphiidae and Didymozoidae, eggs but not what could be recognized as a and recent unpublished studies have revealed testis. That gonad was distinct only in larger the cercariae of Microphallus opacus and two

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 36, NUMBER 1, JANUARY 1969 135 lepocreadiids, Homalometron sp. and Micro- family develop in snails of the genus Cerithium. creadium sp., developing in Amnicola limosa It thus is evident that discussions of host Say in northwestern Indiana. specificity and zoogeography of digenetic It seems significant to the zoogeography of trematodes have unduly neglected molluscan digenetic trematodes that all known central hosts, and not entirely for lack of information and eastern North American freshwater cer- concerning life histories. cariae in the above families as well as the Haploporidae develop in species of Amnicola, Summary and brackish-water larvae in species of Hy- A cercaria developing in Amnicola comalen- drobia. The fact that malacologists disagree as sis from a freshwater canal near Galveston Bay, to whether those genera and Truncatella, a Texas, is essentially an adult with a tail except genus of marine snails, belong in the same for lacking eggs. A conspicuous testis devel- family indicates their close relationship. For- ops in the embryo, fills a large seminal vesicle merly, all were placed in the Rissoidae which with sperms, and retrogresses to a largely now also includes the marine host of the only empty sac before the cercaria emerges from other described haploporid cercaria (Cable, the snail. Encystment is in the open. The 1962). Knowledge of the life histories of Ar- morphology and ecology of the cercaria indi- gentine freshwater haploporids might reveal cate it to be the larva of Saccocoelioides such a close affinity of their molluscan hosts to sogandaresi. The redia, cercaria and meta- marine gastropods. Manter (1957) pointed out cercaria are described. Representation of the that their vertebrate hosts are distantly related Haploporidae and certain other predominantly to those of Mediterranean haploporids, but he marine families by freshwater species is dis- suggested that such euryhaline fishes as mugi- cussed, suggesting that molluscan hosts have lids may have served as ecological "bridges" been neglected as factors in the spread of for trematodes between marine and freshwater trematodes both within and between marine environments. and freshwater environments. Present knowledge of life histories strongly indicates that digenetic trematodes as a group Literature Cited are less specific for their vertebrate than mol- Cable, R. M. 1962. A cercaria of the trema- luscan hosts. Although both are essential to tode family Haploporidae. J. Parasit. 48: those parasites in extending their range, in do- 419-422. ing so they would seem as a rule more adapt- Lumsden, R. D. 1963. Saccocoelioides sogan- able in acquiring new vertebrate than mollus- daresi sp. n., a new haploporid trematode can hosts. Such adaptability within the marine from the sailfin molly, Mollienisia latipinna environment is demonstrated by the crypto- Le Sneur in Texas. J. Parasit. 49: 281-284. gonimid, Siplwdera vinaledwardsii, which para- Manter, H. W. 1957. Host specificity and sitizes shallow-water fishes from Cape Cod to host relationships among the digenetic trema- South America. Throughout that range, the todes of marine fishes. Premier symposium cercaria develops in snails of a single genus sur la specificite parasitaire des parasites de whereas the parasite changes its vertebrate host vertebres. Inst. Zool. Univ. Neuchatel. 185- to fishes of different orders. Similarly, adults 198. Szidat, L. 1954. Trematodos nuevos de peces in the Bivesiculidae parasitize a variety of de aqua dulce de la Republica Argentina y fishes in world-wide tropical and subtropical un intento para aclarar su camcter marine. seas, but all of several known cercariae in that Rev. Inst. Nac. Invest. 3: 1-85.

Lissorchis heterorchis sp. n. (Trematoda: Lissorchiidae) from Catostomus macrocheilus Girard in Oregon

B. BRUCE KRYGIER AND RALPH W. MACY Department of Biology, Portland State College, Portland, Oregon 97207

The only previous record of a species ot and reaching left body margin, 547 (354—708) Lissorchis in the Pacific Northwest appears to long by 121 (83-165) wide; seminal vesicle be that of L. attenuatum which was found by bipartate with anterior part smaller; prostate Bangham and Adams (1954) in Catostomus gland cells present. Genital pore marginal at catostomus and C. macrocheilus in British midlevel of acetabulum; cirrus protrusible, Columbia. Magath (1917) established the spined (Fig. 2). Ovary multilobed with vari- genus Lissorchis which, with the new species able number of lobes immediately anterior to described herein, at present includes eleven anterior testis, 269 (212-342) long by 324 species. (260-401) wide. Vitelline follicles broadly interconnected, fields overlapping ceca and Materials and Methods uterine arms, extending from midlevel of ace- Specimens were removed from the small tabulum to midlevel of posterior testis. Trans- intestine of the host, fixed in Gilson's fluid at verse vitelline ducts meeting at posterior mar- room temperature, under slight cover glass gin of ovary at vitelline reservoir; common duct pressure, stained in Ehrlich's acid hematoxylin, then proceeds anteriorly over ovarian surface and counterstained with 0.02% fast green in where it is joined by short oviduct leading to 95% alcohol. Drawings were made with the aid ootype and beginning of uterus. Laurer's canal of a microprojector and camera lucida. The de- and seminal receptacle not seen. Uterus pro- scription is based upon the six largest speci- ceeds posteriorly along right side almost to end mens. Measurements are in microns unless of body then returns on same side to the level otherwise stated and the average of each is of ovary, then crosses to the left side where it followed by the minimum and maximum in duplicates the same course on the left side of parentheses. the body, terminating in the metraterm which lies under the cirrus sac, and reaching the Lissorchis heterorchis sp. n. common genital pore. Mature uterine eggs, (Figs. 1-3) brownish yellow, averaging 26 long by 16 Diagnosis wide. Body elongate, anterior and posterior ends TYPE HOST: Catostomus macrocheilus Girard. rounded, 3.1 (2.4-4.0) mm long by 0.87 HABITAT: Small intestine. (0.65—1.10) mm wide; cuticular spines ex- TYPE LOCALITY: Multnomah Creek, Mult- tending along body margins from anterior end nomah County, Oregon. to a short distance posterior to posterior testis, INCIDENCE: Seven specimens found in one with few spines on ventral surface. Oral sucker sucker. subterminal, aspinose, 322 (283—389) long by SPECIMENS DEPOSITED: USNM Helm. Coll. 365 (313—454) wide, acetabulum in anterior Holotype No. 71344 Paratype No. 71345. third of body, 374 (307-460) long by 401 (319—513) wide. No prepharynx, esophagus Key to the species of short, ceca of moderate width, extending to Lissorchis Magath, 1917 near posterior end of body. Testes tandem, 1. Vitelline follicles 7—12 on each side .... 2 lobed, posterior to midbody. Anterior testis Vitelline follicles 16 or more on each smaller, 408 (342-472) long by 330 (260- side 3 413) wide; posterior testis 605 (460-779) 2. Ovary deeply trilobed, lobes com- long by 314 (248-389) wide. Cirrus sac pletely separated; length of body oblique, proximal end just posterior to acetabu- posterior to hind testis approxi- lum and distal end at midlevel of acetabulum mately equal to combined length of

Figures 1-3. Lissorchis heterorchis sp. n. 1. Holotype, dorsal view. 2. Detail of genital region. 3. a, b. Outline of testes of two paratypes to show variation.

the three gonads L. simeri (Van L. attenuatiim (Van Cleave Cleave and Mueller, 1932) and Mueller, 1932) Ovary compact, moderately lobed Vitelline fields reaching posterior third but not appearing as three separate of body; testes in posterior half of structures; length of body posterior body to hind testis less than length of the L. polylobatum (Haderlie, 1950) three gonads L. mutabile (Cort, 1918) Results and Discussion 3. Cuticular spines extending to posterior The new species is relatively large with a end of body; postacetabular region broad posterior region. It is the only species of much narrowed and triangular 4 the genus with lobed testes. The posterior Cuticular spines extending no farther testis is much longer than the anterior one and than the middle of the posterior the ovary is deeply lobed. The cirrus sac is testis: postacetabular region not nar- relatively short and its proximal end does not rowed, posterior end more broadly extend much posterior to the acetabulum. The rounded 5 cirrus is spined. 4. Cirrus armed Following Smith (1968), we consider Tri- L. gullaris Self and Campbell, 1956 ganodistomum to be synonymous with Lis- Cirrus not armed sorcliis. Simer (1929) erected a new genus L. fairporti Magath, 1917 Alloplagiorchis for A. garricki but this genus 5. Ovary overlapping both testes, testes also is a synonym of Lissorchis. wider than long, body flask-shaped An examination of paratypes of Lissorchis L. garricki (Simer, 1929) crassicrurum Haderlie and L. polylobatum Ovary not overlapping both testes, Haderlie revealed that the vitelline follicles testes longer than wide, body more are more numerous than shown in the author's elongate 6 drawings; in the former species the vitelline 6. Anterior limits of vitelline fields much fields extend nearly to the inner margins of posterior to acetabulum the ceca, in the latter species the vitelline L. hypentelii (Fishthal, 1942) follicles are overlapping and closely packed. Vitelline fields reaching level of ace- Magath (1917) found that the first and tabulum 7 second intermediate hosts of Lissorchis fair- 7. Acetabulum about twice the size of the porti were the snail Helisoma trivolvis and oral sucker chironomid larvae, respectively. The cercaria L. transluscens (Simer, 1929) possessed both stylet and tail in contrast to Acetabulum less than twice the size the cercaria of L. mutabile which, according of the oral sucker 8 to Wallace (1941), lacked both stylet and 8. Cuticular spines only on the ventral tail. Cercariae of the latter encyst in the corn- body surface and not on body mar- mensal annelid Chaetogaster limnaei in the gins; combined width of ceca equal respiratory chambers of Helisoma trivolvus and to one-third of body width H. campanulata. The tailless cercariae of L. L. crassicrurum (Haderlie, 1953) mutabile encyst after being eaten by the an- Cuticular spines on body margins, not nelid. Thus, there is a correlation between confined to ventral surface; ceca the habitat of the second intermediate host narrower 9 and presence or absence of a tail on the cer- 9. Testes distinctly lobed; posterior testis cariae. Smith (1968) found that L. mutabile about one-third longer than anterior also utilizes freshwater limpets and planarians testes L. heterorchis n. sp. as intermediate hosts. Testes entire; posterior testis not one- No Plelisoma snails are known to occur in third longer than anterior testis 10 the area inhabited by the sucker host of 10. Vitelline fields largely confined to an- Lissorchis heterorchis. More probable snail terior half of body; testes not con- hosts are Flumenicola or Oxytrema, common fined to posterior half of body stream snails of the region.

Summary Literature Cited Seven specimens of a trematode found in Bangham, R. V., and J. R. Adams. 1954. A the intestine of a sucker, Catostomus macro- survey of the parasites of freshwater fishes cheilus Girard, collected in Multnomah Creek from the mainland of British Columbia. J. below Multnomah Falls, Multnomah County, Fish Res. Bd. Canada 11: 673-708. Oregon, were determined to be an imdescribed Magath, T. B. 1917. The morphology and life species of Llssorchis Magath (1917). The new history of a new trematode parasite Lis- sorchis fairporti nov. gen., et nov. spec, from species herein described is characterized es- the buffalo fish, Ictiobus. J. Parasit. 4: 58- pecially by lobed testes unequal in size, a 69. spined cirrus, and numerous, broadly joined Simer, P. H. 1929. Fish trematodes from the vitelline follicles. The genus Triganodistomum lower Tallahatchie River. Amer. Midi. Nat. is considered to be a synonym of Lissorchis. 11: 563-588. A key to the species of Lissorchis is presented. Smith, R. J. 1968. Ancylid snails as first intermediate hosts of Lissorchis mutahile comb. n. Acknowledgments (Trematoda: Lissorchiidae). 1. Parasit. 54: Thanks are expressed to Wanetah D. Bell 283-285. for technical assistance and to W. W. Beck- Wallace, H. E. 1941. Life history and embryol- lund for loan of paratypes. This project was ogy of Triganostomum mutabile (Cort) Lis- supported by National Science Foundation sorchiidae, Trematoda. Trans. Am. Micr. Soc. Grant GB-5384. 60: 309-326.

Survival of Eggs and Larvae in Cysts of the Soybean Cyst Nematode, Heterodera glycines, Ingested by Swine

GROVER C. SMART, JR., AND H. R. TuoMAS1

In Virginia, fields infested with the soybean chiensis Wollenweber, 1923 did not survive cyst nematode, Heterodera glycines Ichinohe, passage through the digestive tract of the 6- 1952, are planted to peanut, Archis hypogaea, week-old pig. Goffart (~1934) fed 3,000 cysts in the rotation system. Although peanut is not of PI. rostochiensis to a rabbit and mixed the a host of the nematode, cysts do adhere to the excrement in sterilized soil planted to potato. pods and hay when this crop is grown in in- Upon examining the potato roots, he found one fested soil (Miller, 1960; Smart and Wright, larva. Ellenby (1944, 1946) reported that 1962). After peanuts are harvested, swine are eggs in cysts of H. rostochiensis survived pas- often allowed to roam the fenced fields to eat sage through the gut of earthworms, and the waste and cull peanuts and peanut vine yielded a greater number of larvae in larval hay. On infested land, they undoubtedly ingest emergence tests than cysts which did not pass nematode cysts. Swine then may be moved to through earthworms. noninfested fields, and the possibility exists of Girard (1887) reported that cysts of H. spreading the nematode to new locations schachtii Schmidt, 1871, which were not dam- through the fecal droppings. aged by the teeth of sheep eating infected Triffit (1929) reported that eggs and larvae sugarbeets, survived passage through the di- in cysts and larvae not in cysts of H. rosto- gestive tract of the sheep. He observed larvae emerging from such cysts and was able to ob- 1 Respectively, Assoeiate Professor of Nematology, De- tain reproduction by adding infested sheep partment of Entomology and Nematology, University of Florida, Gainesville, and Associate Professor of Animal excrement to sand taken from a quarry and Science, Virginia Polytechnic Institute Division of Re- search, Holland, Virginia. The research was conducted in seeding sugarbeet. Chatin (1890) was unable 1962 while the senior author was located at Holland, Va. to recover viable H. schachtii from sheep fed Florida Agricultural Experiment Stations Journal Series No. 3086. infected sugarbeet, some of which were arti-

Copyright © 2011, The Helminthological Society of Washington 140 PROCEEDINGS OF THE HELM1NTHOLOGICAL SOCIETY ficially infested with large numbers of larvae, rectal temperatures were recorded in six other males, and females. He did recover eggs and 13-week-old pigs (three of each sex) on three larvae of parasites of sheep and believed that consecutive days. Temperatures were re- earlier workers had misidentified those as H. corded with the pigs unrestrained and in a schachtii. Willot's (1890) results were iden- standing position. The average rectal tem- tical to those of Chatin. perature of the six pigs was 104.2 F (40.1 C) Kofoid and White (1919) reported that eggs ranging from a low of 103.0 F (39.4 C) in a of a nematode were recovered from the feces female to a high of 105.1 F (40.6 C) also in of military troops. Sandground (1923) showed a female. that the eggs undoubtedly were those of a Six concrete-floored pens were washed root-knot nematode, Ateloidogyne, and con- thoroughly using a high pressure hose (110 ducted experiments showing that root-knot psi pressure at the pump). One animal was nematode eggs passed through the human di- placed in each pen and feed was withheld for gestive system and stated, "They were slightly 40 hr prior to initiating the study; however, brown in color and were in all stages of de- drinking water was supplied ad lib. Once- velopment, with the metameres so sharply each 24 hr for 3 consecutive days each pig defined as to indicate their viability." He ingested 1,000 cysts of H. glycines which further stated that the eggs were "... appar- were mixed with the daily ration of 2 Ib (0.9 ently uninjured." Martin (1968) showed that kg) of a 15.6% crude protein feed. One of Meloidogyne arenaria (Neal, 1889) Chitwood, the pigs (pig 1 in Table 1) ingested cysts from 1949 survived passage through the digestive the Holland, Virginia, area, and the other five tract of a cow and subsequently infested ingested cysts from the Wilmington, North tomato roots. Carolina, area. Control cysts were kept at 4 C. According to Leukel (1924, p. 944), Mar- The feed was the standard growing ration used cinowski (1909) showed that Anguina tritici at the Holland, Va. Station and was of the (Steinbuch, 1799) Filipjev, 1936 in wheat following mixture: 1,598 Ib (726,3 kg) of galls survived passage through the intestines corn, 252 Ib (114.5 kg) of soybean oil meal of the sparrow, goldfinch, pigeon, and mouse, (50% crude protein), 100 Ib (45.4 kg) of but did not survive passage through the tankage, 20 Ib (9.1 kg) of limestone, 10 Ib chicken, gopher, or marmot. However, Da- (4.5 kg) of salt (NaCl), 5 Ib (2,3 kg) of vaine (1857) found that A. tritici in wheat Vitamin Pre-Mix (Charles Pfizer), 0.6 Ib (272 galls did not survive passage through the intes- g) of Terramycin TM50, 100 g of zinc sul- tines of the chicken, pigeon, or sparrow, but phate (ZnSO4), and 12 g of copper sulphate did survive passage through the frog, triton, (CuSO4). salamander, and goldfish. Leukel (1924) Feces were collected initially from each pen found that A. tritici did not survive passage just before the second feeding of cysts. There- through the digestive tract of the horse, cow, after for 4 additional days, feces were col- hog, sheep, or chicken. Wilson (1930) found lected 3 times a day and composited. After that Ditylenchiis dipsaci (Kiihn, 1857) Filip- the third collection the feces were examined jev, 1936 in diseased Phlox did not survive by washing through a 60-mesh and a 325-mesh passage through the intestine of a rabbit. screen. Residues from the 60-mesh screen were Research reported in this paper was con- examined directly for cysts, and residues from ducted to determine whether eggs and larvae the 325-mesh screen were placed in Baermann in cysts of Heterodem glycines would survive funnels for recovery of any larvae that might passage through the digestive tract of swine. have been liberated within the alimentary An abstract has been published (Smart, 1963). canals of the pigs. On the fourth collection day, 50 cysts re- Materials and Methods covered from the feces of each pig were placed Six pigs weighing an average of 63 Ib (28.6 in 4-inch (10.2 cm) pots of autoclaved soil kg) each (60-65 Ib = 27.5-29.5 kg) and with and soybean seed, Glycine max Merr. var. an average age of 13 weeks (80—96 days) were "Lee," planted. All other cysts on all collection used in this study. Three of the pigs were days were isolated and placed in sterilized dis- females and three were males. At a later date, tilled water in 60 mm Petri dishes at the rate

Table 1. Number and percentage of cysts of Heterodera glycines recovered from the feces of each of 6 pigs on 5 consecutive collection days and the number of larvae emerged during incubation of the cysts for 21 days at 22 C. Each pig ingested 3,000 cysts (1,000 on 3 consecutive days).

Pig number and sex, number of cysts recovered, and number of larvae emerged

Day feces I1-Female 2-Female 3 -Female 4 -Male 5-Male 6-Male Control collected Cysts Larvae Cysts Larvae Cysts Larvae Cysts Larvae Cysts Larvae Cysts Larvae Cysts Larvae 1 105 3 15 0 8 0 142 1 53 0 87 0 200 3,596 2 80 0 39 0 47 0 68 0 68 0 133 0 200 3,776 3 133 1 198 0 278 0 190 0 300 0 134 0 200 1,767 4- 185 0 215 0 150 0 127 0 189 0 180 0 136 65 5 28 0 46 0 27 0 78 0 104 1 90 0 100 59 Total 531 513 510 0 605 714 624 0 836 9,263 Percentage of cysts recovered per pig 17.7 17.1 17.0 20.2 20.8

1 Pig 1 ingested cysts from the Holland, Va. area, and all other pigs ingested cysts from the Wilmington, N. C. area. - Fifty cysts from each pig collected this day were placed immediately in 4 inch clay pots of soil seeded with "Lee" soybean.

of 10 cysts per dish and incubated at 22 C. were vacuolated. These larvae were dead; After incubation for 10 days, emerged larvae they remained in a bent position and did not were counted and the cysts were transferred move either autonomously or from tactile to fresh dishes of sterilized distilled water. stimulation. After 11 more days the emerged larvae were No larvae were recovered from the Baer- again counted. The cysts which had been mann funnels containing the debris caught on incubated and all emerged larvae were placed the 325-mesh screen. in 4-inch (10.2 cm) pots of autoclaved soil No soybean cyst nematodes developed on planted to "Lee" soybean. After 60 days, the "Lee" soybean during a 60-day period from soybean plants were removed from the soil. the cysts and six emerged larvae used as inoc- The roots were placed on a 60-mesh screen ulum. From the control cysts, an average of and washed with a spray of water using enough 19 cysts or white females were recovered for pressure to remove cysts from the roots. The each cyst used as inoculum. soil was roiled in a container of water and washed through a 60-mesh screen with the Discussion process repeated twice. The residue was dis- Although a few larvae survived passage tributed in counting trays and any cysts present through the digestive tract of the 13-week-old were counted with the aid of a dissecting mi- pig, they did not appear as "normal," based croscope at 15 X. on the vacuolated appearance of the intestine, as larvae not similarly treated. Balmer and Results Cairns (1963) stated that infectivity dropped A total of 3,497 or 19.4% of the 18,000 sharply in root-knot nematode larvae contain- cysts fed to the six pigs were recovered (Table ing five or more large-sized vacuoles in the 1). Only six larvae emerged from the 3,197 intestinal region. The larvae of H. glycines cysts subjected to larval emergence tests. This which survived passage through the pig, as was an average emergence of 0.002 larvae per well as those which did not survive, had highly cyst. The control cysts yielded 9,263 larvae vacuolated intestines. (The number of vac- for an average emergence of 11 larvae per uoles were not counted since this work was cyst. The six larvae which emerged from cysts done prior to the publication of the work by recovered from the pigs were alive as deter- Balmer and Cairns.) mined by movement both autonomously and Triffit (1929), in seeking an explanation for from tactile stimulation. The intestines of the the death of H. rostochiensis ingested by the larvae, however, were highly vacuolated. The 6-week-old pig, pointed out that the tempera- intestines of larvae manually released from ture in young pigs is higher than in older ones, eggs in the cysts recovered from pigs also but she did not believe body heat to be the

Copyright © 2011, The Helminthological Society of Washington 142 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY decisive factor in death of the nematode. We Heterodera rostochiensis Wollenweber. Ann. recorded the average rectal temperature of pigs Appl. Biol. 31(4): 332-339. 5 weeks old, 13 weeks old, and 24 weeks old 1946. Ecology of the eelworm cyst. Na- with the following average temperatures, re- ture 157(3988): 451-452. spectively: 104.2 F (40.1 C), 104.2 F (40.1 Endo, B. Y. 1962. Responses of Heterodera C), and 102.4 F (39.1 C). Since Endo (1962) glijcines to lethal temperatures. Phytopathol- ogy 52(1): 9. (Abstr.). showed that larvae of H. ghjcines in cysts lived Girard, A. 1887. Sur le developpement des ne- more than 8 hr at 110 F (43.3 C), we do not matodes de la betterave, pendant les annees believe that body heat of the pigs alone was 1885 et 1886, et sur leurs modes de progaga- responsible for the death of most of the larvae tion. Compt. Rend. Acad. Sci. 104, S. of H. ghjcines. Gof'fart, H. 1934. Ueber die Biologic und Be- Sex of the pigs apparently had no effect on kampfung des Kartoffelnematoden (Hetero- survival or death of the larvae, since larvae of dera schachtii Schmidt). Arbeiten Biol. Reichs. H. ghjcines emerged from cysts recovered from Land-u. Forstwirtschaft. 21(1): 73-108. the feces of both males and females. Kofoid, C. A., and A. W. White. 1919. A new Since a very few larvae in cysts of H. nematode infection of man. J. Amer. Medical ghjcines did survive passage through the diges- Assoc. 72: 567-569. Leukel, R. W. 1924. Investigations on the nem- tive tract of swine, it is possible that the nema- atode disease of cereals caused by Tylenchus tode could be disseminated through swine tritici. J. Agr. Research 27(12): 925-955 plus feces. However, based upon the vacuolated 5 plates. appearance of the intestinal region of those Marcinowski, K. 1909. Parasitisch und semi- larvae, and upon the lack of reproduction on a parasitisch an pflanzen lebende nematoden. susceptible host plant, we believe that dis- Arb. k. Biol. Anst. Land. u. Forstw. 7: 1-192. semination of the nematode through the feces Martin, G. C. 1968. Survival and infectivity of is unlikely. eggs and larvae of Meloidogyne arenaria after ingestion by a bovine. Plant Disease Reptr. Summary 52(2): 99-102. Miller, L. I. 1960. Soybean cyst nematode re- Of 18,000 cysts of Heterodera ghjcines in- search in Virginia. Virginia-Carolina Peanut gested by six pigs, 19.4% were recovered from News. 6(1): 9-11. the feces. Six larvae emerged from the cysts Sandground, J. H. 1923. "Oxyuris incognita" incubated for larval emergence. The larvae or Heterodera radicicola? J. Parasit. 10: 92- were alive, but had highly vacuolated intes- 94. tines. When these larvae and all cysts re- Smart, G. C., Jr., and Barbara A. Wright. 1962. covered from the feces were placed in pots of Survival of the cysts of Heterodera glijcines soil containing "Lee" soybean, a known host, adhering to stored sweet-potato, peanut, and no cyst nematodes developed. peanut hay. Virginia J. Sci. (N.S.) 13(4): 219-220. (Abstr.). Literature Cited Smart, G. C., Jr. 1963. Survival of encysted eggs and larvae of the soybean cyst nematode, Balmer, E., and E. J. Cairns. 1963. Relation of Heterodera glijcines, ingested by swine. Phyto- physiological age of root-knot larvae to infec- pathology 53(8): 889. (Abstr.). tivity. Phytopathology 53(6): 621. (Abstr.). Triffit, M. J. 1929. Observations on the inci- Chatin, Joannes. 1890. Le mouton peut-il pro- pager YHeterodera Schachtii? Compt. Rend. dence of Heterodera schachtii at the Ormskirk Soc. Biol. 9th Series, 11(2): 17-18. Potato Testing Station. J. Helminth. 7(2): 93- Davaine, C. 1857. Recherches sur 1'anguillule 98. du ble niele considered au point de vue de Willot. 1890. Destruction cle YHeterodera sch- 1'histoire naturelle et de 1'agriculture. Compt. achtii. Compt. Rend. Acad. Sci. 3,S: 801-803. Rend. Soc. Biol. 3(2): 201-271. Wilson, G. F. 1930. Further investigations on Ellenby, C. 1944. Influence of earthworms on the eelworm disease of Phloxes. J. Royal Hort. larval emergence in the potato-root eelworm, Soc. 55(1): 88-100.

The Intrapulmonary Localization of Angiostrongylus cantonensis in the Rat

THOMAS M. SODEMAN,* WILLIAM A. SODEMAN, JR.,2 AND CHARLES S. RICHARDSS

Angiostrongylus cantonensis was described formaldehyde (40%) and 200 gm of sodium by Chen (1935). Mackerras and Sandars acetate, brought to 5,000 ml with distilled (1955) in a detailed description of its life water. The inflated lungs were fixed in the cycle showed that the adult worm migrates formalin acetate mixture for a minimum of from the rat's subarachnoid space to its lungs 2 days. between the 28th and 31st day of infection. The lungs were separated from the trachea Fertilization takes place in the pulmonary and heart. Each lung was washed for 72 hr arteries and oviposition begins about the 35th in running water into which 5% nitric acid day. was dripped. The entire lung was embedded The purpose of this investigation was to in a solution of 250 mg 80—100 bloom gelatin, study the localization of adult worms, eggs, 40 ml ethylene glycol mono-ethyl ether, 5 ml and larvae in the lungs of experimentally carpyl alcohol, 5 ml 1% ethylmercurithio- infected rats. Because of the difficulty in salicylate and 850 ml distilled water. The maintaining spatial relationships after re- specimen was allowed to stand for 1 hr and moval of organs from an animal, particularly was then transferred to an incubator and kept when making histologic sections from small for 48 hr at 35 C. The entire block was frozen blocks of the whole organ, it was elected to and kept at —20 C until sectioning. Sections section the whole lung. The Gough-Went- were cut at 200 JJL on a Reichert microtome as worth (1960) technique for making thick sec- thawing occurred and they were stored in tap tions (200 ju.) of whole, inflated fixed lungs water. The sections were floated onto glass was used. These sections permitted the local- slides for examination with a dissecting micro- ization of lesions within the matrix of the scope. Several sections were stained with pulmonary parenchyma with little loss of hematoxylin and eosin. the spatial relationships between pathologic Lungs from each group were also embedded changes and normal tissue. in paraffin and sectioned at 5 p. These sections were stained with Giemsa for comparison Methods and Materials with the thick sections. Weanling, Sprague-Dawley rats were in- Results fected with A. cantonensis by feeding of one- half of a Biomphalaria glabrata (= Aiistralorbis The lungs of a rat infected with A. canto- glabratus) snail harboring infective third stage nensis 65 days prior to sacrifice revealed ma- larvae. The rats were divided into six groups. ture worms only within the vascular system. The first group was sacrificed on the 28th They were restricted to the larger pulmonary day of infection and thereafter a new group arteries. The thick sections permitted visualiza- was sacrificed on the 34th, 37th, 39th, 43rd, tion of large portions of the adult worms, most and 65th day of infection. The heart and with ingested blood outlining their digestive lungs were removed en block, avoiding injury tracts, within the mainstem pulmonary arteries to the visceral pleural surfaces. The lungs were and peripherally in the larger branches (Fig. inflated with a formalin acetate mixture 1). There was no evidence that the mature through the trachea by a cannula connected worms penetrated into the pulmonary paren- to a reservoir 4 feet above. The formalin chyma or the bronchial system at 65 days. acetate mixture consisted of 500 ml of liquid Eggs surrounded by fluffy white consolidation were scattered throughout the lung fields.

1 Section on Primate Malaria, Laboratory of Parasite From serial sections it was apparent that egg Chemotherapy, NIAID, P.O. Box 80190, Chamblee, Georgia 30341. lesions were associated with blood vessels. A 2 Liberian Institute of Tropical Medicine, Harbel, Liberia. portion of the left middle lobe was infarcted 3 Laboratory of Parasitic Diseases, NIAID, National In- stitutes of Health, Bethesda, Maryland 20014. (Fig. 2). Serial sections of the lung revealed

Figures 1—4. Thick sections of the left lung from a rat sacrificed 65 days after infection. 1. Angio- strongylus cantonensis fill the main pulmonary artery. Egg lesions are seen as patchy white areas (X 2.5). 2. Infarcation of a portion of the middle lobe (X 2.5). 3. Thrombosis of the artery leading to the infarcted lobe in Figure 2. The proximal portion contains eggs, the distal contains developing larvae (X 200). 4. A thick section of the lung demonstrating eggs embolized to the terminal arteries (X 200).

Figures 5—6. 5. A thin section of the lung from a rat sacrificed at 39 days with an egg in a capillary of an alveolar wall (X 735). 6. A thin section of the lung from a rat sacrificed at 65 days with eggs in advanced stages of development surrounded by organizing tissue. A small artery can be traced to an egg lesion. The proximity of the eggs to alveoli and a bronchus is evident (X 580). thrombosis of the artery to that lobe (Fig. 3). pulmonary arteries. In the early stages of in- The thrombus was composed of two distinct fection, eggs were clearly found only in pul- sections; the older, more distal portion con- monary vessels (Fig. 5). With advancing age tained hatched larvae, the proximal portion, an extensive reaction formed about the eggs developing eggs. Where eggs were seen out- with replacement of blood vessel structure by side of the arterial system, the integrity of the active fibroblastic proliferation and marked vessel was in question because of surrounding leukocytic and monocytic infiltration. As the hemorrhage or an intense inflammation with larvae developed, there was an increased disruption of normal structures. eosinophilic reaction. In many places eggs In many cases, the plane of the section and lying in their connective tissue masses were its thickness permitted the tracing of arterial separated from alveoli or bronchi only by a vessels leading to egg lesions (Fig. 4). In this thin wall of tissue (Fig. 6). After 65 days of figure an egg can be seen occluding a branch infection larvae were seen in the alveoli and of a small artery just distal to its bifurcation surrounded by eosinophils. The patchy white from the main blood-filled lumen. The pattern consolidation in the thick sections was seen to of egg lesions and their association with arteries consist of organized tissue, collections of suggested embolization. The bronchi contained macrophages and polymorphonuclear leuko- no eggs. cytes in the thin sections. Alveolar septa asso- Lungs from rats sacrificed on the 28th, 34th, ciated with intracapillary eggs were thickened and 37th days were normal. Those sacrificed and infiltrated with polymorphonuclear leuko- on the 39th, 43rd, and 65th days postinfection cytes, lymphocytes, and macrophages. In the and processed as 5 /A paraffin sections showed more advanced stages, the large pulmonary histologie changes similar to the thick gelatin arteries were dilated and contained masses of sections. Adult worms were localized in the eggs.

Discussion the pulmonary parenchyma acts as a secondary The results of the thick, as well as thin, site for development when eggs escape from sections indicated that the adult stages were the vascular system. It is likely that the larvae restricted to the pulmonary arteries. Their migrate from the arteries through the orga- size limited them to the larger arteries. No nized tissues directly into the bronchial system evidence in the stages studied, either in the or out into the alveolar sacs. thick gelatin or thin paraffin sections, indicated The use of the thick section technique per- that the adult worm penetrated into the pul- mitted better visualization of eggs within the monary parenchyma. The findings suggested lung substance without loss of orientation. The that oviposition occurs in the blood stream and authors feel this technique deserves considera- that the wide distribution of pulmonary in- tion as an experimental method in studying volvement resulted from embolization of eggs. pulmonary parasitic lesions. This embolization and impaction in blood ves- Summary sels resulted in thrombosis and infarction. That development may progress within the throm- The intrapulmonary localization of Angio- bus was evident in Figure 3 where hatched strongyhis cantonensis in experimentally in- larvae were seen in the distal portion of a fected rats was studied in lungs embedded in thrombus, while in the recently formed prox- gelatin and sectioned at 200 /.i. Adult worms imal portion, eggs were found. There was were found only intravascularly. Egg deposi- nothing to suggest a preferential distribution tion and larval development within lungs were of egg lesions in the lungs. However, because primarily in blood vessels. Eggs were distrib- of the close association of the bronchial and uted by embolization. It is suggested that the vascular systems, many of the egg lesions were preferred site of larval development is intra- in peribronchial locations. vascular rather than in lung parenchyma. When eggs were seen in the pulmonary par- Literature Cited enchyma, they were surrounded by inflamma- Chen, H. T. 1935. Un nouveau nematode pul- tion and organization that obscured their orig- monaire, Pulmonenui cantonensis, N. G., N. inal intravascular position. Furthermore, it was SP. Des rats de Canton. Ann. Parasit. Hum. evident that eggs outside of vessels may have Comp. 13: 312-317. resulted from disruption of vessel walls with Mackerras, M. J., and D. F. Sandars. 1955. hemorrhage or from infarction. Both processes The life history of the rat lung-worm, Angio- were evident in the sections. The extensive strongylus cantonensis (Chen) (Nematoda: organization around the eggs in the arteries Metastrongylidae). Aust. J. Zool. 3: 1-21. Gough, J., and J. E. Wentworth. 1960. Thin extended into the perivascular and frequently sections of entire organs mounted on paper. into the peribronchial tissue. The materials Recent advances in pathology, 7th Ed. examined suggest that the primary site of de- (Edited by C. V. Harrison, M.D.), Little, velopment is within the arterial system and that Brown and Company, pp. 80-86.

BUSINESS OFFICE For the convenience of members and subscribers and to better serve both members and subscribers, the Society on January 1, 1969 established its business office at Allen Press, Inc., in Lawrence, Kansas. All dues, subscriptions, orders for back issues, and any and all correspondence with reference to these items or the receipt of Pro- ceedings should be addressed to: HELMINTHOLOGICAL SOCIETY OF WASHINGTON c/o ALLEN PRESS, INC. 1041 NEW HAMPSHIRE ST. LAWRENCE, KANSAS 66044 U.S.A.

Tylenchus vesiculosus, sp. n. (Nematoda: Tylenchidae) From Soil in Michigan

NATALIE A. KNOBLOCHX AND JOHN A.

Tylenchus vesiculosus sp. n. was recovered FEMALE (HOLOTYPE) : Body filiform slightly from soil samples collected 16 October 1967 arcuate ventrally, tapering more towards pos- from a potato field near East Lansing, Mich- terior than anterior end. Cuticle of body igan. The habitat, a field in which potatoes coarsely striated; annules 1.9 ^ wide at middle had been under cultivation for several years, of body. Lateral field a plain band bordered was organic soil (muck) with two out- by two bright lines one-fourth to one-fifth cropping knolls of sandy mineral soil. T. greatest body width (Fig. 3).Phasmids not vesiculosus, while not widely distributed observed. Lip region conoid, rounded, contin- throughout the field, was found in large num- uous with neck contour; no lip annules ob- bers around the root systems of potato plants served. Spear delicate, 9.75 p long, with well- growing in the sandy soil. Only one male was developed knobs (Fig. 2). Median bulb of recovered from the original soil samples. oesophagus ovate with crescentic valve plates; A ratio of one male for about every 50 fe- the metacorpus obscure in many of the glyc- males was recovered from samples processed in erine specimens. Isthmus slender, surrounded April 1968 but these males were inadvertently by the nerve ring slightly anterior to where destroyed in the laboratory. Since the original the isthmus joins the basal oesophageal bulb. infestation was to be fumigated prior to spring Basal bulb elongate-pyriform; cardia conical, planting, soil was transferred from the original well defined. Excretory pore opens at about habitat to a microplot at the Entomology Re- the middle of the basal bulb (Fig. 1). Vulva search Farm, Michigan State University in an a depressed transverse slit; vagina thick- attempt to preserve the species for future biol- walled, occupying about three-fifths body ogy investigations. width; length of posterior uterine sac about The following description of the species four-fifths vulval body width. Ovary single, is based on preserved specimens. Measure- outstretched anteriorly, wtih a round to oval ments were made on specimens which had shaped spermatheca with an extended pouch- been heat relaxed, fixed in F.A. 4:10, pre- like extension (Fig. 5). Spermatheca and the served and mounted according to the glycerol- extension contained many spermatozoa. Ovary ethanol method of Seinhorst (1959). with oocytes arranged in a single file and extending two-thirds of the distance to oesoph- Tylenchus vesiculosus sp. n. agus (Fig. 4). Anus distinct; tail length about (Figs. 1-7) 9 times anal body width, ending in a rounded tip. HOLOTYPE FEMALE: Length = 0.55 mm; a = 29; b = 6.2; c = 4.7; V = ^66; spear = MALE (ALLOTYPE) : Length = 0.48 mm; 9.75 p.. a = 30; b = 55; c = 4.5; T = 46; spear = 9.7 p. PARATYPE FEMALES (12): Length = 0.52 (0.51-0.55) mm; a = 31.3 (28-34); b = 6.1 Body shape, head and oesophagus same as in female (Figs. 1, 2). (6.0-6.6); c = 4.8 (4.7-4.9); V = 66 (66- Testis single, outstretched anteriorly; sper- 68); spear = 9-11 p. matocytes arranged in single file. Spicules ALLOTYPE MALE: Length = 0.48 mm; a = paired, curved, of usual tylenchoid type. 30; b = 5.5; c = 4.5; T = 46; spear = 9.7 p.. Gubernaculum thickened, troughlike; bursa faintly crenate, rising at a point a little above 1 Research Technician, Entomology Department, Mich- proximal ends of spicula and extending past igan State University, East Lansing, Michigan. - Assistant Professor, Entomology Department, Michigan anus a distance equal to about 1% anal body State University, East Lansing, Michigan. Published with the approval of the Director of the Mich- diameter (Figs. 6, 7). Tail same shape as in igan Agricultural Experiment Station as Journal Article Number 4453. female; about 10 times anal body width.

V-'.-V

Figures 1-7. Tylenchus vesiculosus. 1. Anterior portion of female. 2. Female head region. 3. Lateral field of female. 4. Female reproductive tract showing position of spermatheca. 5. Sketch of spermatheca showing ventral view of attached pouch. 6. Male tail. 7. Posterior portion of male with lateral lines.

DIAGNOSIS: Tylenchus vesiculosus sp. n. can ment of Entomology Collection, Michigan be differentiated from all known species of the State University, East Lansing, Michigan. genus by the spermatheca with an extended TYPE HABITAT: Organic and mineral soil pouch. It is small (0.54 mm) with thick, heav- around roots of potato, Solanum tuberosum L. ily annulated cuticle, plain bandlike lateral field and tail with a distinctly rounded termi- TYPE LOCALITY: A cultivated farm field of nus. It differs from Tylenchus davainei Bastian, potatoes grown in organic and mineral soil 1865, in the straight tail, bandlike lateral field, near East Lansing (elevation 845 feet), unstriated lip region and the spermatheca with Michigan, USA. the pouch. Distinguished from Tylenchus The authors are grateful to Professor Gerald ditissimus Brzeski (1963) by the coarser body Thorne, Department of Plant Pathology, Uni- striation, no annules in the lip region, bandlike versity of Wisconsin, Madison, Wisconsin for lateral field, position of hemizonid, longer his advice during this work and to Richard J. length, possession of post uterine sac and a Snider for his patient counseling in drawing spermatheca with a pouch. The extended techniques. pouch of the spermatheca is known for no other species of the genus Tylenchus. How- Literature Cited ever, a similar pouch is described for Dac- Brezeski, M. W. 1963. Tylenchus ditissimus ti/lotylenchus crassacuticulus Wu (1968). sp. n., a new nematode from Poland (Nema- HOLOTYPE, FEMALE: Collected 16 October toda, Tylenchidae). Bull. Acad. Pol. Sci. 1967 from a potato field in East Lansing, (11): 531-535. Michigan. Slide T-118t deposited with the Seinhorst, J. W. 1959. A rapid method for the USDA Nematology Collection at Beltsville, transfer of nematodes from fixative to anhy- Maryland. drous glycerin. Nematologica 4: 67-69. ALLOTYPE, MALE: Same data as holotype, Wu, L. Y. 1968. Dactylotylenchus crassacuti- slide T-117t. culus, a new genus and a new species (Tylen- PARATYPES, FEMALES: Same data as holo- chinae: Nematoda). Can. Jour. Zool. 46(5): type, 12 slides Tv3-Tvl4 deposited in Depart- 831-834.

NEW JOURNAL The Society of Nematologists announces the publication of a new journal, the JOURNAL OF NEMATOLOGY. Appearing quarterly beginning in January 1969, it is intended as a medium for publication of original research in all phases of nematology. Membership in the Society of Nematologists (annual dues $6.00) includes the Journal of Nematology. Subscriptions are available at $10.00 domestic and $11.50 foreign, per volume. Inquiries should be directed to: Society of Nematologists, Department of Botany and Plant Pathology, Auburn University, Auburn, Alabama 36830; or Dr. Seymour D. van Gundy, Editor, Journal of Nematology, Department of Nematology, University of California, Riverside, California 92502.

Helminths of the Crow, Corvus brachyrhynchos Brehm, 1822, in North Carolina1

LARRY D. HENDRicics,2 REINARD HARKEMA, AND GROVER C. MILLER Department of Zoology, North Carolina State University at Raleigh

There are few comprehensive reports on hematoxylin, Semichon's carmine, or fast green, the helminths of crows in North America. cleared in methyl salicylate, and mounted in Ward (1935) conducted the first extensive Canada balsam. Nematodes were cleared in survey of crows. This work included data lacto-phenol. on the helminths of 50 crows collected in Oklahoma. Morgan and Waller (1941) ex- Results and Discussion amined 112 crows from Wisconsin and Iowa. Twelve species of helminths were recovered Daly (1957a, 1957b) examined 103 southern from 158 crows. These included two trema- crows (Corvus brachyrhynchos paulus Howell) todes, two cestodes, three acanthocephalans, from Virginia. Most references on parasites and five nematodes (Table 1). from the crow are for particular species or on Brachijlecithum americamnn Denton, 1945 surveys of hosts which included crows as part is a common parasite in the liver of birds of of the study. The object of our investigation the families Corvidae and Icteridae and was was to determine the parasitic fauna and the described from specimens obtained from mea- incidence of parasitism of both external and dowlarks, grackels, crows, and blue jays from internal parasites of the crow in North Caro- Georgia, Tennessee, and Texas. It was found lina. The external parasites are reported else- in the liver of 122 of the 158 crows examined. where (Hendricks and Axtell, 1968). It was reported from the yellow throat, Geo- thlypis trichas at Durham, N. C. (Wells and Materials and Methods Hunter, 1960). One hundred and fifty-eight crows, 76 fe- Echinostoma revolutum Froelich, 1802 was males and 82 males, were collected and ex- found in the small intestine of seven crows. amined for parasites between 5 October 1965 Daly (1959) reported this form from the and 22 April 1967. The majority of these southern crow in Virginia. This is the first birds was taken in the spring of 1966 and report from crows in North Carolina. 1967 in conjunction with the annual hunt of Only one specimen of Anomotaenia sp. was the Southeastern Crow Shooters Association in found in the small intestine. It possibly is the vicinity of Henderson, North Carolina. A. constricta (Molin, 1858) which was re- All hosts were data-tagged and sealed in plastic ported from the southern crow in Virginia collecting bags. Whenever possible hosts were (Daly, 1959). Ransom (1909) listed the fish autopsied within a few hours after collection; crow (C. ossifragus Wilson), the raven (C. otherwise they were frozen and stored. corax L.) and the crow as hosts for A. Trematodes were killed and fixed in hot constricta. A.F.A.; acanthocephalans and tapeworms were Variolepis variabilis (Mayhew, 1925) was killed in boiling water and then fixed and found in the small intestine of 34 of the hosts stored in A.F.A.; nematodes were killed and examined and North Carolina is a new locality fixed in hot 70% ethanol and stored in 10% record. It has been reported from the crow glycerine in 70% ethanol. in Illinois (Mayhew, 1925), Wisconsin (Mor- For microscopic study, trematodes, cestodes, gan and Waller, 1941) and Virginia (Daly, and acanthocephalans were stained with Harris' 1959). Mediorhynchiis grandit Van Cleave, 1916 1 From a thesis submitted to the Graduate Faculty of North Carolina State University at Raleigh in partial ful- was found in the small intestine of two crows. fillment of the requirements for the degree of Master of The two specimens recovered were immature Science. This project was supported in part by Grant No. AI-05927, National Institutes of Health. males. This species has been reported from 2 Address: 9th Medical Laboratory, APO San Francisco 96307. the grackle, eastern meadowlark (Van Cleave,

Table 1. Helminths recovered from 158 crows, (Williams, 1929), Oklahoma (Ward, 1935), Corvus brachyrhynchos Brehm. New Jersey (Beaudette and Hudson, 1936), Wisconsin and Iowa (Morgan and Waller, Average number Number of helminths per 1941). This is the first report of this species Parasite infeeted infected host in North Carolina. Trematodes Diplotriaena tricuspis (Fedtschenko, 1874) Brachijlecithum am ericami m 122 14.5 was recovered from an abdominal air sac of Echinostoma revolutum 7 2.8 Cestodes one crow. Probably this is the only adult Anomotaenia sp.* 1 1.0 filarid species reported from crows in North Variolepis variabilis* 34 2.1 America (Beaudette and Hudson, 1936). Acanthocephala Wells and Hunter (1960) reported an imma- Mediorhynchus grandis 2 1.0 Macracanthorhynchus sp.f 2 1.5 ture Diplotriaena sp. from the yellow throat in Plagiorhynchns formosum 1 1.0 Durham, N. C. Nematodes It is interesting to compare this study with Capillaria contorta* 21 1.4 that of Daly (1959) who examined 103 south- Hystrichis sp.*t 12 1.8 Physaloptera sp.*f 2 2.0 ern crows taken in Albemarle County, Vir- Acnaria anthuris* 35 2.4 ginia. There is a distance of 100-150 miles Diplotriaena trictispis* 1 1.0 from the counties in which the majority of * New locality record. the crows in this survey was taken, yet only f New host record. three parasites were common to both studies. These were: the trematode Echinostoma 1916), and the crow (Van Cleave, 1918, 1947; revolutum Froelich, 1802, the cestode Hyme- Daly, 1959). nolepls variabile (Variolepis variabilis of this The infection of two crows with Macra- paper), and the acanthocephalan Mediorhyn- canthorhynchus sp. probably was accidental. chus grandis Van Cleave, 1916. The specimens are immature females and resemble M. hirudinaceus. Summary Plagiorhynchus formosum Van Cleave, 1918 A total of 158 crows harbored the following was found in the small intestine of one crow. helminths: Trematoda: Brachylecithum. amer- It has been reported from the crow in the icanum, Echinostoma revolutum; Cestodes: District of Columbia (Jones, 1928) and the Anomotaenia sp., Variolepis variabilis; Acan- Macgillivray seaside sparrow in North Carolina thocephala: Mediorhynchus grandis, Macra- (Hunter and Quay, 1953). canthorhynchus sp., Plagiorhynchus formosum; Capillaria contorta (Creplin, 1839) was Nematodes: Capillaria contorta, Hystrichis sp., removed from the mucosa or submucosa of Physaloptera sp., Acuaria anthuris, Diplotriaena the esophagus of 21 hosts. Canavan (1931) tricuspis. New locality records are noted for reported this species from a crow in the Anomotaenia sp., V. variabilis, Hystrichis sp., Philadelphia Zoological Garden and Morgan A. anthuris, and D. tricuspis, and new host and Waller (1941) reported it from crows in records for Macracanthorhynchus sp., Hys- Wisconsin and Iowa. This is the first report trichis sp. and Physaloptera sp. of this species from North Carolina. Hystrichis sp. was recovered from the glands Acknowledgments of the proventriculus of 12 of the 158 crows. Sincere appreciation is extended to Dr. The specimens constitute a new species and Helen Ward for her assistance in the spe- it will be described separately. This is the cific identification of the acanthocephalans, first report of a representative of this genus and to Mrs. MayBelle Chitwood for her opin- in North America and the crow is a new host. ions and suggestions on the new species of Two immature female specimens of Phy- Hystrichis. saloptera sp. were recovered from one host. They resemble F. alata Rudolphi, 1819. Literature Cited Acuaria anthuris (Rud. 1819) was found in Beaudette, F. R., and C. B. Hudson. 1936. the crop and gizzard of 35 hosts. This species Parasitism and tuberculosis in a crow. J. Am. was reported from the crow in Nebraska Vet. Med. Assoc. 89: 215-217.

Canavan, W. P. N. 1931. Nematode parasites rhynchos brachyrhynchos Brehm). Bird-Band- of vertebrates in the Philadelphia Zoological ing 12: 16-22. Garden and vicinity. Parasitology 23: 196- Van Cleave, H. J. 1918. The acanthocepliala of 229. North American birds. Trans. Am. Micros. Daly, E. F. 1959. A study of the intestinal hel- Soc. 61: 206-210. minths of the southern crow (Corvns brachy- . 1947. The acanthocephalan genus, Me- rhynchos paulus) in Virginia. Proc. Helm. diorhynchus, its history and a review of the Soc. Wash. 26: 66. species occurring in the United States. J. Hendricks, L. D., and R. Axtell. 1968. Arthro- Parasit. 33: 297-315. pods from passerine birds of North Carolina. J. Elisha Mitchell Sci. Soc. 84: 312-314. Ward, J. W. 1935. Some parasites of crows of Hunter, Wanda S., and T. L. Quay. 1953. An central Oklahoma. (Preliminary report) Proc. ecological study of the helminth fauna of Okla. Acad. Sci. 15: 6.5-66. Macgillivray's seaside sparrow, Ammospiza Wells, M. J., and Wanda S. Hunter. 1960. maritima macgttlivraii (Audubon). Am. Midi. Helminths of the yellow throat, Geothlypis Nat. 50: 407-413. trichae, during migration. J. Parasit. 46: 623. Jones, M. F. 1934. An acanthocephalid, Pla- Williams, O. L. 1929. A critical analysis of the giorhynchus formosum, from the chicken and specific characters of the genus Acuaria, nem- robin. J. Agr. Res. 36: 773-775. atodes of birds, with descriptions of new Morgan, B. B., and E. F. Waller. 1941. Some American species. Univ. Calif. Publs. Zool. parasites of the eastern crow (Corvus brachy- 33: 69-107.

SECOND INTERNATIONAL CONGRESS OF PARASITOLOGY Mayflower Hotel, Washington, D. C. U.S.A. September 6-12, 1970 The Second International Congress of Para- It is proposed to have a series of informal sitology was organized by the American Society discussions in the afternoon sessions. Thus of Parasitologists and is co-sponsored by the free contributions (submitted papers) would Helminthological Society of Washington, Na- be grouped in appropriate sessions. It is fur- tional Academy of Science, Society of Nema- thermore proposed to prepublish these as tologists, Society of Protozoologists, Wildlife longer than average resumes (perhaps 500 Disease Association, and World Federation for words) to serve as the basis for informal dis- the Advancement of Veterinary Parasitology. cussion. Thus the chairman or any participant The program will include any and all aspects might ask for elaboration, clarification, or dis- of human and animal parasitology and plant cussion of any paper or any aspect therein. nematology except the description of new spe- Lantern slide projectors will be available. cies. Parasitology is used in its generally ac- However, it is hoped that free discussion will cepted connotation and hence would not in- not be stifled by the routine reading of a clude bacteriology, virology, mycology, pests series of short papers. or pesticides. This is frankly an experiment in international Plans for the development of the meetings meetings. The concept has been enthusiasti- are progressing well. The ad hoc Organizing cally endorsed in many quarters. We invite Committee and the ad hoc Program Committee comments and suggestions on the concept and have developed the keynote of informality for any and all aspects of the program. The ad free and informal exchange. Every effort is hoc Program Committee welcomes suggestions being made to recapture the spontaneity, en- for presiding officers at these sessions which thusiasm, and stimulation that has character- could number as many as 60 or more. ized some of the few unscheduled and im- Plan to attend the Congress and participate. promptu discussions that have developed GILBERT F. OTTO within some other international meetings. Ac- Secretary General cordingly it is proposed to limit formal presen- Zoology Department tations, with invited papers, to the morning University of Maryland College Park, Maryland 20742

Exposure of Chicks to the Metacercaria of Echinostoma revolutum (Trematoda)1

BERNARD FRIED AND LINDA J. WEAVER Department of Biology, Lafayette College, Easton, Pennsylvania 18042

Although Beaver (1937) has contributed an Results and Discussion extensive monograph on the biology, syste- Twenty-nine (93.6%) of 31 chicks each matics and life history of Echinostoma revo- exposed to 50 ± 10 to 100 ± 20 cysts and lutum (Froelich, 1802) and Senger (1954) autopsied 22—35 days later were infected has studied aspects of growth, development (Table I). Forty (95.2%) of 42 chicks each and survival of this parasite, detailed studies exposed to 50 ± 10 cysts in 3% NaHCO:!, 3- on its infectivity in domestic chicks are lacking. salt Ringer's or distilled water and autopsied During studies on the growth and development 14 days later were infected (Table II). Senger of this fluke in ectopic sites (Fried and Von- (1954) previously demonstrated that the roth, 1968; Fried, Weaver, and Kramer, 1968) domestic chick could serve as a host of E. observations on the infectivity of E. revolutum revolutum and reported that 22 (61%) of 36 metacercariae in the chick were made and are chicks fed metacercariae in water became recorded herein. infected. Materials and Methods Bacha (1964) found that an increased worm burden of Zygocotyle lunata (Diesing, 1836) Helisoma trivolvis (Say) naturally infected in white rats could be obtained if metacer- with E. revolutum larvae were placed in finger cariae were pretreated in NaHCO3, possibly bowls containing pond water with laboratory- because the bicarbonate counteracted the reared Physa sp. snails. Cercariae emerged acidity of the stomach. However, in matched from the H. trivolvis and encysted primarily experiments using NaHCO3 and distilled water in the kidney of the Physa. The number of similar worm burdens of E. revolutum were cysts varied depending upon the size of the obtained (Table II). Moreover, when cysts Physa but ranged from 25—250. Physa were were pretreated in Ringer's the worm burden crushed in pond water from 1-10 days post- increased approximately twofold. Many fac- infection and cysts were transferred to 3-salt tors influence the rate of infectivity of E. Ringer's (Paul, 1960) and teased into clumps revolutum, in chicks including the strain of of approximately 25, 50, or 100 cysts. Exact parasite and host, the emptying time of the numbers of cysts were difficult to count so gut, mechanical and chemical damage of cysts estimates of ± 5, 10, 20 were usually made. in the crop, proventriculus and gizzard and Except in experiments F and G (Table II) further investigations are needed to elucidate where cysts were pretreated in Ringer's or dis- these factors. tilled water and in the cloacal exposure study, In older infections more worms were found cysts were pretreated in 3% NaHCOH for 30— in the rectum than in the ileum. Thus when 60 min and then fed to chicks in 1-3 ml of chicks were autopsied in 2 weeks (Table II) the bicarbonate. 272 worms were in the ileum and 241 in the Day-old chicks were used in all experiments rectum, whereas in hosts autopsied 3—5 weeks except the reinfection studies (Table III). (Table I) postinfection 105 worms were in the Chicks were not given food or water on the ileum and 193 in the rectum. E. revolutum is shortlived in chicks and our records indicate day of exposure or autopsy. They were killed by decapitation and either total worm counts that rarely do infections persist up to 8 weeks. Senger (1954) reported the life span of E. were made or parasites found in the rectum, revolutum in chicks to be about 35 days. Many ceca, and ileum were recorded. worms as they age migrate posteriorly from

1 This work was supported by NIH Grant AI-06835. The their original site and either reestablish or pass authors gratefully acknowledge the technical assistance of Mr. Jack Carty. out of the host.

Table 1. Summary o£ exposure experiments using 50±10-100±20 cysts pretreated in 3% NaHCO». Chicks were autopsied 22-35 days postinfectlon.

No. of No. of No. of Age of Number of worms recovered chicks infected cysts per worm at Per cent Exp. exposed1 chicks chick autopsy Ileum Ceca Rectum Total recovery A 8 8 50±10-100±20 22 7 53 72 132 22.0 B 3 3 50±10 23 0 14 46 60 40.0 C 17 15 50±10 25 58 29 75 162 21.6 D 3 3 100±20 35 40 6 0 46 15.3 Total 31 29 50±10-100±20 22-35 105 102 193 400 22.2

1 All chicks day-old when exposed.

To determine the effects of storage on infec- infection on a primary one 12, 3-day-old chicks tivity, cysts maintained for 7 weeks in 3-salt were each exposed to 100 ± 20 cysts and were Ringer's at 15 C were fed to chicks. Three of reexposed to the same number of metacer- four chicks fed 100 ± 20 cysts and autopsied cariae 13 days later. Twelve additional chicks 7-14 days later were infected with 9, 10, and were used as controls, half of which received 23 worms. Senger (1954) reported that cysts only the primary exposure and the other half stored in 0.4% saline at 5 C for 31 days were the secondary exposure. Chicks were autop- viable. Storage of cysts is important in E. sied 9-10 days after the secondary exposure revolutum studies since many Phijsa that be- (Table III, H, I, J). To determine if chicks come heavily infected with metacercariae die older than 1 month could become reinfected, within 2 weeks postinfection. 14 chicks that were 12 days old at the time of To determine if chicks could become in- a primary exposure to 50 ± 10 cysts were fected with cysts via the cloaca, cysts were reexposed each to 50 ± 10 cysts 31 days later. implanted into the cloacas of four chicks that Chicks were autopsied 10—11 days after the were autopsied 7—10 days later. Two chicks secondary exposure (Table III, K). The re- each received 100 ± 20 cysts pretreated in infection experiments indicate that a primary bicarbonate and two chicks received the same infection of E. revolutum does not protect number of cysts pretreated in Ringer's. None against a secondary infection. Fried (1963) was infected. found that a primary infection of Philo- Cysts occasionally formed ectopically on the phthalmus hegeneri Penner and Fried, 1963, mucus trails of H. trivolvi-s. To determine if did not protect chicks against a secondary these cysts were infective, three chicks were infection and reviewed the literature on re- fed approximately 20, 60, and 175 mucus cysts infection in trematodes. The reinfection ex- and autopsied 7 days later. Only the one chick periments also indicate that chicks older than fed approximately 175 cysts was infected with 2 weeks (Exp. J) can become infected and four worms suggesting that ectopically formed that chicks older than 1 month (Exp. K) can cysts are not as infective as those obtained from become reinfected indicating that an age the kidney of Physa sp. snails. resistance to this worm was not apparent, at To determine the effects of a secondary least within the first month.

Table 2. Summary of exposure experiments using 50±10 cysts and different pretreatment solutions. Chicks were autopsied 14 days postinfection.

No. of No. of Number of worms recovered chicks infected Pretreatment Per cent Exp. exposed1 chicks solution Ileum Ceca Rectum Total recovery E 18 16 3% NaHCO., 104 55 63 222 27.7 F 12 12 3-salt Ringer's 130 73 105 308 51.3 G 12 12 Distilled H2O 38 35 73 146 24.3 Total 42 40 272 163 241 676 32.2

1 All chicks day-old when exposed.

Table 3. Summary of reinfection experiments.

Age of No. of No. of Age of Age of No. of chick at worms No. of worms No. of chick at chick at pos. chicks time of recovered pos. chicks recovered chicks 1° infection 2° infection w 1° autopsy from 1° w 2° from 2° Exp. exposed ( days ) (days) infection ( days ) infection infection infection H 12 3 16 10 25-26 83 9 124 I 6 3 16 3 25-26 24 - - J 6 3 16 - 25-26 - 5 174 K 14 12 43 8 53-54 15 7 132

Chicks in Exp. H received 100 ± cysts in 1° and 2° infections. Chicks in Exp. 1 received 100 ± cysts in 1° infection !only. Chicks in Exp. J received 100 ± cysts in 2° infection only. Chicks in Exp. K received 50 ± cysts in 1° and 2° infections.

Summary Literature Cited Sixty-nine (94.5%) of 73 day-old chicks Bacha, W. J., Jr. 1964. Effect of salt solutions exposed to 50-100 Echinostoma revolution on the establishment of infections of the trem- metacercariae and autopsied 2—5 weeks post- atode Zygocotyle lunata in white rats. J. infection yielded worm burdens of 15.3—51.3%. Parasit. 50: 546-548. Beaver, P. C. 1937. Experimental studies on Forty (95.2%) of 42 day-old chicks exposed to Echinostoma revolutum (Froelich) a fluke metacercariae pretreated in distilled water, 3% from birds and mammals. 111. Biol. Monog. NaHCO.j, 3-salt Ringer's and autopsied 2 15: 3-96. weeks later yielded worm recoveries of 24.3%, Fried, B. 1963. Reinfection of chicks with an 27.7%, and 51.3%, respectively. Cysts stored ocular trematode, Philophthalmus hegeneri in Ringer's at 15 C for 7 weeks were viable. Penner and Fried, 1963. J. Parasit. 49: 981- Cysts formed ectopically on the mucus trails 982. of Helisoma trivolvis snails were not as infec- , and W. Vonroth, Jr. 1968. Trans- plantation of Echinostoma revolutum (Trem- tive as cysts formed within the kidney of atoda) into normal and surgically altered Physa sp. snails. Worms were not recovered chick intestine. Exper. Parasit. 22: 107-111. from cysts implanted into the chick cloaca. -, L. J. Weaver, and M. D. Kramer. 1968. A primary infection of E. revolutum did not Cultivation of Echinostoma revolutum (Trem- protect chicks against a secondary infection. atoda) on the chick chorioallantois. J. Para- Chicks older than 2 weeks became infected sit. 54: 939-941. and chicks older than 1 month became re- Paul, J. 1960. "Cell and Tissue Culture." 2nd Ed., Williams, Baltimore, Md., 312 p. infected indicating that an age resistance to Senger, C. M. 1954. Notes on the growth, de- this worm was not apparent, at least within velopment and survival of two echinostome the first month. trematocles. Exper. Parasit. 3: 491-496.

Academician K. I. Skrjabin — Ninetieth Birthday

On December 7, 1968, Academician K. I. together with his lectures on helminthology Skrjabin celebrated his ninetieth birthday and soon attracted increasing numbers of students. the completion of more than six decades of Over 100 candidates have received doctorates scientific work. Regarded as the outstanding under his guidance, and many more can be helminthologist of the Soviet Union, he has counted as students of his students. Among gained worldwide recognition for his researches the former are a number of corresponding in this field. members of the Academies of Sciences of the Graduating as a veterinary surgeon in 1905 USSR. from the Juriev Veterinary Institute, Dr. Dr. Skrjabin's investigations in the field of Skrjabin first became interested in the study morphology, biology, phylogeny, and syste- of helminths during an assignment in Turkes- matics of helminths, on ecology, epidemiology, tan. He made extensive collections from both and epizootiology of helminthic diseases and domestic and wild animals and later (1912- their control have been reported in over 600 1914), studied them under the guidance of publications. Now internationally known Braun, Fuhrmann, Luhe, Railliet, and Henry. through these publications, his contributions In his master's thesis (1916), based on the to his country's scientific development as an Turkestan collections, he stressed the impor- educator, mentor, and administrator have been tance of establishing helminthology as a sci- even more remarkable. To list only some of the ence in Russia. In the following year, he was honors accorded him in recognition of his ac- named to the first chair of parasitology in complishments, he has received honorary de- Russia, established at the Novocherkassk Vet- grees from Humboldt University (Berlin), and erinary Institute. the Institute of Agriculture (Czechoslovakia). From this vantage point, Dr. Skrjabin gained He is a member of the Veterinary Academy of increasing recognition for his concepts of the France, a member of the Academies of Science significance of helminthic disease in agricul- of Bulgaria, Hungary, Czechoslovakia, and ture and public health. In the period 1920- Poland, and an honorary member of the Amer- 1922 he organized or was instrumental in ican Society of Parasitologists (1932), the founding three centers of helminthological re- Indian Society of Helminthologists, Parasito- search: The Helminthological Department of logical Society of Poland, and the Rumanian the State Experimental Veterinary Institute Parasitological Society. He is a member of which was later named the Ail-Union K. I. three academies in the Soviet Union; Academy Skrjabin Institute of Helminthology in honor of Sciences of the USSR, Academy of Medical of its founder; the Helminthological Depart- Sciences, and the All-Union Academy of Agri- ment of the Moscow Tropical Institute; and cultural Sciences. He is president of the All- a Department of Helminthology at the Moscow Union Society of Helminthologists and founder State University. In addition to the organiza- and Chairman of the Editorial Board of Hel- tion of helminthological institutions in Moscow, minthologia. Among other honors bestowed he was the moving force in establishing the by his government are five Orders of Lenin, study of helminthology in research institutions and on the occasion of his eightieth birthday and universities throughout the Soviet Union. the name of Hero of Socialist Labor. Despite his administrative activities, Dr. The Helminthological Society of Washington Skrjabin found time to continue his morpho- congratulates Academician Skrjabin on his logical and taxonomic studies. Beginning in ninetieth birthday, a milestone in a career that 1919, he organized and directed surveys of has exerted a profound influence on all phases the helminth fauna of man and animals in the of helminthology in the Soviet Union. Soviet Union. Publication of these studies —FRANCIS G. TROMBA

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'"•\a /-*trv '^' '\\- Cairns, {E . J.,Crawley- --, 0Lil ~a R. -' ::: Ehrenford, F. A.-, -f '"-" .-° Golden, AllM.. •.,'."•. ., V. ... ^ Draggan, S. ' ' , '-Ferris, Virginia R. . s ;i-Good, J.vM.,'Jr. , ,•", ; -Ford, B.,R. : •'•;•'- •• "Duxbury, R. "El . r X> \. F. Haley, A. T. y, • : "*. Frandsen,J. "CJ-.-— .... .v .Ericksonj D. G. , Thorson, R. E. ':. . , Hechler, Helen Carol >;.Isensteinr-R. S.v ' • •/Gonzalez, Stella M. - Weinstein, P. P. , - ., ^Herlicb.H. V '•; ." I Rogers; W. A; r .,- Habermann, R. T. '' . ,/'' IqWo . J- /'.,'; , ; Herman, C. 'M-^ ,-.-V Wellborn.'.T. L., Jr. 'Hope, \V- D. ' " Blankespoor, H. D. Hernandez, A. - -V • ^ ^Alaska Kenworthy, F. T.,. ; - ; -1 Ellis, C. J. Hill, C. K . ' ' -iRausch, R. •'-._ "• '; •-•'• : Lincicome, D. R. ( ' .' 'Epstein, A. H. -' . Holbrook, 'A. A. ' / Shults, L. M. > f Martin, L. K. Goss, R. C. .... '-:'•' Huff.C.G. Arizona V ' ' * Oh'vier, Li J. / i Greve1, J. H. . ,'> Humphrey, Judith M. ; ~,f • '• Anderson, G. A. Radke, M. G. - . Hsu, H. F. - ^ ^ achowski, L. A., Jr. , ,""• : Nigh, E.CL., Jr. ^ledington, B. C. >. Norton, D. C./" '',' Jacobs, L. ; . . -• .Reynolds; H. W. N \ Sadun, E. H. f :J iu\mei, M. j. -.>•;,: Tahnes, W. G. - '__ \VVilkes S.N. - Slie, RitaB. , . , Williams, D.:.D. Kates, K. C. + ' Arkansas * /' ft '^ ... Wells, Gladys A./: i. -; ', Ziminermann,' W. J. -- •/• Xriight, R. A. : . ; ~- v - Slack, D. A, ;y '7, Florida -- ~-< \,H , Kansas': ;; /• ..^__',-' Krusberg.'L. R...... ' .' r California ' / Christie, J. R. v, : • Ackert, Ji E., f Lawless, D. K. - ' Allen, M. W. Esser, R. P. ' . ' •Ameel, D. Jj Leek, R. G. >./ ,Ash, L>R. - V Hahnon, C.xL- T '''<-' •" ' Ay tan, Songul V(.-~ Lesser, ,E. ^ • -.. Baker;' N./F.\ .' , . ,.-•'• Kincaid, R. R. " Cml,-W. H7- -;•'/"% Lichtenfels, J. R. V J -,,"''.•. , Brown, R. L. ;L\' v •., O'Bannon, JVH. . i S . Dickerson, O. J. \ J Lotze, J.G. ' ''. /'••)*•• Caveness, F. E. /i:, ,•••' •' > N Overman, Arnegda J.\, V' . .G .:Haiiseh, M. F. ^ '/• Lund, E. E. ,,'• / . .- Dailey,,' M..D. -( '-. . •• \y " /_\V Lunde, 'M. N: v ' ,- , f • Hart, W. H: ' ,.-. . j Short, 'R. B. . '' .Chapman, R: A, ~ • Lwtterrnoser,-G. W. •' Hasbrouck, IS. JR. . Smart, G. C., Jr. ;/ prudge.J. H. v: , Malakatis, G. M. S Heyneman, ©." Tarjan, A. G. .~~~-/ v~ /Eversmeyer, H- "EiS Marchbank, Dorothy 'F. {- Holdeman, QJ L.. , ' Taylor, A. L.' '. > Lyons; E. T. .;•:;. •-.'' . ;McIntosh, A. ' - ' Y" : :Kinsella,J.M. ; .' , Georgia . ; '/Whittaker, F.H.. >; - McLoughliri, D. K. . ' :'. Rrassner,""S. M. Bird, G. W,. ' / : Louisiana ••' • • -• • ' '.'{ MorriS, J. A. .:-. . , Lamberti, vF.' -r > " ^ '• Ciordia, H. > (J'i Acholomi, A. D. ;Murpiy, D. G. " 1 Lear, B. - / , -. Cross, Hi F. x ; • •: ''-. ' Bello, T. R. -- ;• ' i 1 Nickle, ;W. R. ' • -., ' , •• ->-', \, H. W; - Denton, J. F.~ .Besch, E. D. . ' ,W Osborne, H. SV >„.• /-."Loos, C. A. -\. ., ;. .Eni, E.-U. r! '£ )- Birchfield, W.v .- ' • ; ; OtWi, G. F. . :.-.-1 ; : - •'•'•.'• , Maggenti, A. R. • ' :'S • Goodchild, C. G. " __"', Hollis; J. P.* Jr. - ;r Pachecd, Gi . • '•• -.Mahkau, R. ..Heara, R. W., Jr. - ' > Lumsden, R. .D. -?.' i Phillips, B. P. , ^ ' ••f ', Martin', W. E. ::. "'- Kaganj I. G. i , ' / •' Martin', W. J. , 'v r IPipkin, .A; :C.-" -- ' ' ; v . ' , ' McBeth, C. W; Marti, O. G., Jr. Newsom, L. D. Poiley, Judith A. ' y , McGuirei W. G. ~- Minton, N. A- \ '• v ., Overstreet, R. M- . - Poole, J. B. - < ' ,;. Mizelle, :J,D.' . Ruehle, J.;L:/ / ; SogandaresrBernal, F; ;:Powcrs, K. G. ; . '/•' Nahlias, F. M., - - S, -Stewart, T. B. , -;;. " Warren,; ,L. G. / >x ,#rice,:D>L. --' ... :' ' :.~ •"•Noffsiriger, Ella Mae v xSullivan, J. JV"; '" .Jfamaguti, S^ i, . /. 1 vQuigley, Diane '• Paxman, G. A. <• -f f Wehunt, E. J. 5>v,' Maine ' \ \j-'' *'-"• / Bichards 'C.NS., ^. ; /' . Poinar,; G. O.^Jr. ^ / Hawaii '>• f •-. /• v-''r ''' {.,' Meyer, M. C." . '•': . . ., . Robinson.'-Marma M. Alicata,/J. E. 'Maryland -. -, , ' ••fkV\ '-' V Roby, T. O. T '.-.'- .. •Ros^an^R. 'N. , /^ Apt, w. j. / • r , V *. Abram, J. B. : { • v Rogers, S.;H. v - Rothman, A. H. Cheng, :T. C.- i ' . . • r Anastos,^G. , ' ' - • -N'. ' yViRomanowski, R. D. / Seh,-A/K.1% ''I,.''- .'V-' Goto, S. c /..f -Anderson, R. I. (\ \ .•AndrewsV,,- - Rose, J. iS J. . E.: <-. .- : . \, S. A. Lee,S.H.' "..»:. j, Rozeboom, L. E. -^ ' V SiddiquH. ./A. V •;" Rifkin, E. , " - (Bang^F. B. ^_ ' Sa%vyer, T. K-. V ' v Steele, A. E. Idaho , ." , , , '\' •. •/' •£ Beaudoin, R. L. -r:^ Sayre, R. M. , • : '' •;. •\..,;..'Van Gundy, S. D. -, '•» 'i •'"- vDalliinore,1 G.. E. Becklund, W;,W. -> Schiller,. E..L. ,,, '"(" '. ';' '.'.••..; Viglierchio, ;DrR. Illinois - ' Bergquist, E. J^-_ •• -;• Schmunis, G. A- ";••"•' \, C.M. t' Voge, Marietta •'.. . T Amosuy Ov , , • ': -1 vori Brand, 'T. ' /' • ' '-^xr • / i? Ti ' ."'• •-•" ' , Bauman, P. M, •. '• '- "Buhrer, Edna M. ) Scott, J. A., ... - . ' WeiriYnann, ~C. J. ; '. Duuagan, T,\T; . . si_v ;Chitwood,; May B.^-- x \r Dorothy B- > Canal Zone Edwards, D, I. '••- 'Gblglazier, M. L. ^ x Sheffield, H. G. Walton, B..C. v 'Garoiah, G. \, W.-B. -' "..... : Shiroishi, Tsugiye;', . -" . Colorado ' ,TV Huizinga, H- W- _ "< '.'.-.•• Diamond, L. S. ' V Solomon, G. B.'. N /, ••' Olsen, O. W. ' , Kruidenier.^F. 7- 'Doran, D. J. '- i- / . Spindler, L. A. ; tl Schmidt, G. D,' Levine-.-N': D. i Doss, "Mildred A.' V' Sprague, V. ', '•' Stable^, R.,M. . - ,o \, R. B.1 ''. ,Douvres,>;F. W. • iStifewalt, -Margaret A. Connecticut . v \ McCrae, R. C.' , , *- : , Dropkin, V.JH. , Stringfellovv, F. ' . Miller, P. M:; " " •' ' ,.- Qstdiek, J. L. ; ,, '•'- Durbin, C::G. Tarshis, L.JB'. .,'' . ; •^l- Penner, L. R.>r/ / .. Pillai, J. K. , - '-'• /'Edwards, Shirley J. , / j . Terziaiii 'L. A. ' Stoffolano, J. G.', Jr. Schneider, M. D. : IS vEndo, B. Y.. r\' •••'- Traub,^. /•*•£/"'( • • • 'Delaware ': • ^ /-^ , Seitaer, P:iG, •--"• 'i :| Enzie, F, D. A" . TrorribarF.;G. . • < , Fielding, M. J. . ' ) ( Singer, I. , ---•' ' . ?• ' 7,\, 'JMarion M. ,v >' .' ,' 7 Turner, J. H. •'•',, .' .^'/District of Colurnbio -Taylor, D. PS ! ---;•, ^l.iFayer.iR. --'';-., ' :.., Undervvood, P. C; ••-.I . Baisden,(L. A. , r- Thomas, L. J, •A Feldmesseri, J. • Vegprs, Hi H. 1 v Betz, D. O., Jr. •' - .'•' • r Todd, K. S., Jr./K ; Ferguson, M..S.' '• :'x~ '"' )Wehr,E..E-. • \*V.' • •:- , p ;BriggsrN. T. ' ',.v Indiana ' ,'\, R.'J. cFoster; A. O. ' , Williamson, ;F.' S. %.^ , : -'- 1 ; x,i'.^ Buchheit, J. R.' •* Friedman, W: •Wiseman, ;H.-G. /. ;- •• V-'\;Bufke, J. C. ' "\v^: \.'' ' ' Cable, R. M. . f ; • • :' Goldberg, A. / :,;i •,. .:

Copyright © 2011, The Helminthological Society of Washington i r ">')••-, \VOLUME 36 1969 NUMBER .1 C '/./' •/ •'"^••'^.'••y -' *- .,". -if"' '•,;. CONTENTS v .,..., T, '••;^y '.•;•'- ';'.' — •.'/''-•''•' '

FiscHTHAL, JACOB H/ Euparddistomum cercopjthepi sp. n. (Diprbebeliidae), ;af Digenetic t Trematode from the Talapoin /Monkey fjrom 'Rio Muni -;.r_-..î,._.I—;._î±--r-;------•>--% 183' FRIED, BERNARD,-AND RONALD E. TOKSTWALL// Survival arid Egg;Laying( of4T\urtle ,^ >>Blood Flukes (Trernatoda: Spirorphiidae) on $e'jChick Chorioallantois _^.^r-_.:-_.. FRIED, BERNARD, AJSTD LINDA J, WEAVER. —Exposure of Chicks to the Metacercafia of v^ Echinostpma/revolutum'^ Trernatoda) •^^.^.:..^l.:....^:^..^.--.^^..^,...... -.^.-..-.^.,..:... GRIFFIN/ G. D., AND: E. C.' JoRdENSON. Pathbgenipity of ;the Northern RoptTknot V Nematbde/ (Meloidogyne hdjila) to Potato _:-^—__--7—7-t.—,-i „—;::. _k 1*^— HENDRICKS, LAKRY D., REINARD HABKEMA^ AND GROVER C. MILLEB. jHelminths/of the Crow, Cbrvus brachyrJiynchos Brehm, 1822, in Norî Carbh'na —-I'-^ll. i.;.-...J—..„_.. HOPE, W. DUANE. -Fine Structure of the Somatic; Muscles of the Free-Living Marine cyVj . Nematbde peontostpmq.californicum Steiner and Albin, 1933 '.(Leptosomatidae).,_.....' 10 ' Iri .Memoriam .^^.__^= ;i-—--~:-7--"7"f-—---—~l~~~:-l-r:VJ-^_^._L._'Jl'r-.._^—_ --,-.. KN6BLpcH",xNATALjE A., AND JOHN A;^^ KNiEHiM. Tylenchus vesjculosus, sp. n/ (Nema- toda: Tylenchidae) from "Soil, in 'Michigan^-:--l-:-----r_^r----^ —-.:: 147 -KRYGIER, B; BRUÊ, AND RALPH W. MACY, JZisôrchis'heterbrchissptn.j( Trernatoda: i: Lissorchiidae^ from Cytbstomus mactocHeiliis Giriard in Oregon „_„,„.^ -„„__;.:_ 136 LEE, SHERIDAN-H. .'/The lUse of Irradiated ;Third-stage Larvae- of Angiostrongylus ^( (cantonensis as Antigen tblrnmiinize. Albino'Rats Against Homologous Infection'"^:.... MACKIEWICZ, JOHN S. Pjenqrchigeies okhiteis gen. et sp. n. and jBiefeetabulum'carpiodi :' sp. ii. "(Gestoidea: Caryophyllaêidae) ^frorn Gatostomid Fish, in Islortii America,-,..— 119 MACKIEWICZ, JoHN:qS.,: ANIJi AHTHXIB, W.^ JONES, ^fhe Clirbmosomes -of Hunterella / nodulosa Matkiewicz iand McGrae, 1962 (Gestoidea: Carybphyllidea) ...... jr.^...^...^ 126 - .MASSEYy):CALyiN MURA. The Proposed- Synonymy of !vthb Mohogenean^ Genera '. - Cleidodiscus Mueller, 1934 and tlrocleidus Mueller, -1934, with the Proposal of, ^ Cleidoldisci/s .fo^c/ioipsfcVMP^1 n-',.--...^--—-—- .1--...—-,-,-i-.I—^-r-~^-^~-——il-,_-i. !52, RASKj;D.!,,J., N, O. JONES, AND DVTl. ROGGEN. " On the Morphology and Ultrastructure .x ;of the Esophageal Region of Tric)i()dorus allius Jensen ' '....—....V.^-1——.:....:._.....^-. 106 GROVEPI C., JR.,' AND H. RC TH:OÂS. Survival oL Eggs and Larvae in Cysts of the 'Soybean* Cys,t Neniatode, Heterodera gli/cines^lngesied by Swine; 1J...... —..I 139 THOMAS M., ^VILLIAM A. SODEMAN, JR., AND CHARLES S. RICHARDS. The ,- 4 Intrapulmohary Lbcalizatioiivof Angiostrongylus cqntonenisis in the Rat'...... —;.... 143 THORNE, -GERA-Lp- : Hypsoperine ottersoni sp.^-n. (Nernata, . Heterpderidae) Infesting — < Can,ary Grass,'J?halaris "arundinacea (j^.~) Reed in Wisconsin ..-:...—:...^-. .„.!..„:...... 98 TIMM, R. W. ; Megadohtolaimys New Genus (Nematoda: Chromadoridae), 'witii a \ - Description of Two ;New Species'—..:.._-- —..._;—--:------,-—--'-——------— - :36( TROMBA:; FRANCIS G; Academician K. I. Sfcrjabin-—rîhetieth Birthday _.^.....;...... _. 156 • TSAI, YuAN-HwxVNG,-AND ALBERT W. GRTJNDMANN; Reesimermis nielseni gen. et sp. n./ '--''.-'• ^, (Nematoda: M.erniitliidae") /Parasitizing Mosquitoes'/in Wyoming /_!:.._„.-__—_.,..——- .61 WHITTAKER, (FRED H. Galvanotaxi§ o'tPelodizra strongyloides (Netnaloda:' Rhabditidae,)/ \0 [. C., AND I. Nj^VTON.l—tntestmal Helminths of tlie ^Bullfinch, fynliula pyrrjiula (L.), in)Southern England/ ..L.iv— I..;—---^------'---- -^-^.-.,.-1-.——r 1\: V7 6 i.- -.,-• '' -; ' ' ' ':"".... ANNQUNPEMENTS \\_.^:':' :'fcX: Businejss Offic^,—-.—.—-.———_.._;^.-.i-._.-_^:_...—-i.-r-.-.L--.-.::^L 146 New' Journal'- ,C---- —-•- 1T,-^ . ^ -_..ji.—..._.:..l—.-. 149 Second International Gongress of ^Parasitolqgy. -—_.——_- —...... - 152 ,\J rSj/V1 '\ •• -•- X; • '' ^ ...-":'*i^'''1* •• .'*" ,'-v' --.-. -, • , '^1^ :r/!;H .;," lx>-; Date of Publication^SOJaWary/1968 •i(: ALLEN PRESS) INC. .LAWRENCE, KANSAS