Heterodera schachtii responses to exogenous hormones

EXPERIMENTAL PARASITOLOGY 27, 301-309

Heferodera

schachfii

(1970)

Responses to Exogenous

R. N. Johnson1 Department

of Nematology, (Submitted

and

University

David

R. Viglierchio

of California,

for publication,

Hormones

Davis,

6 October

California

95616

1969)

schachtii ReJOHNSON, R. N. AND VIGLIERCHIO, DAVID R. 1970. Heterodera sponses to Exogenous Hormones. Experimental Parasitology 27, 301-309. Infective larvae of H. schachtii, the sugar beet nematode, were treated with aqueous and oil solutions of selected steroids and sesquiterpene derivatives then placed on host plants for development. Among the larvae treated with oil testos’terone solutions, only females were observed to develop with a hypodermal tuberculum in the dorsal sector anterior to the circumesophageal commissure. Among larvae treated with oil solutions of 2,3-cis/trans-6,7-trans farnesyl diethylamine and methyl ether only males were observed to manifest prodigious testicular development together with consequent morphological aberrations. INDEX DESCRIPTORS: Hornones; Sugar beet nematode; Heterodera schachtii; Plants; Development; Teratoma testicular; Hypodermal tubercula; Life history; Nematoda.

This study reports the effect of a number of synthetic and naturally occurring hormones of other animals on the development of the sugar beet nematode, Heterodera schachtii.

Although the life stages of many nematodes are well-known, the available knowledge concerning the physiological processes initiating molting and growth is woefully inadequate. The suggestion that an endocrine system must be involved in these processes is not a new concept and has been predicted by several workers (Sommerville, 1957; Rogers, 1965; Davey and Kan, 1967). Davey and Kan ( 1968), working with Phocanem decipiens, a nematode parasite of vertebrates, have provided experimental evidence indicating the production and release of an unidentified neurosecretory hormone involved in ecdysis. Two hormone-like compounds, cholesterol and farnesol, were tested to determine their effect on the development of TrichineZla spirak larvae ( Meerovitch, 1964). Famesol inhibited the development of the larvae, while cholesterol had no effect on development but did enhance the rate of larval survival.

MATERULS

AND METHODS

The hormones employed belong to two major chemical groups, steroids and terpenoids. The steroids used included an adrenocortical hormone, cortisone acetate; the mammalian sex hormones, estrone, progesterone, and testosterone and a steroid known to have insect-molting hormone activity, ecdysterone. All of the terpenoids employed are known to have juvenile hormone activity on insects and all are farnesyl derivatives: famesinic acid lO,ll-epoxyfamesenic acid ethyl ester, 2,3&/tram-6,7-tram farnesyl diethylamine, and 2,3-cisltrans-6,7-bans farnesyl methyl ether. All of the test hormones were prepared at 0.01 M concentrations in oil except for e&-one, ecdysterone, and cortisone acetate which were used as a fine oil

1 Present address: Department of Biology, Community College of Denver, Denver, Colorado 80220. 301

302

JOHNSON

AND

VIGLIFBCHIO

FIG. 1. H. schachtii females showing the hypodermal tubercula on A. adult female, stage female, C. and D. third-stage females. ht, hypodermal tuberculum; fh, functional separation or initiating furrow separating ht from fh; st, stylet; hc, hypodermal cells.

B. fourthhead; s,

H.

S&U&ii

RESPONSE

TO

HORMONES

303

FIG. 2. The anterior region of A, fourth-stage female and B, adult female showing the structureless internal character of the hypodermal tuberculnm. ht, hypodermal tuberculum; fh, functional head.

suspension. Acetone was used as a solubilizing aid for oil, since these hormones, nearly insoluble in water, are difficult to solubilize directly into oil. The acetone is then removed in vamo. Portions of one group of second-stage larvae from a stock supply were treated with aqueous-saturated hormone solutions. The saturated hormone solutions were prepared by dissolving excess hormone in 0.25 ml of acetone, which was added to 0.5 gm of fine siliceous sand in a BP1 dish, thoroughly mixed, and allowed to evaporate so that the vessel and sand particles would take up a coat of hormone. Water (0.25 ml) was then added and allowed to equilibrate for a minimum of 24 hours before larvae to be treated were added to the mixture and allowed to move through the sand mass and absorb hormone for a 24-hour incubation period. Portions of a second group of larvae from the same stock were pretreated with an 0.01 M mineral oil-hormone solution or fine suspension, if insoluble, for 30 minutes before transfer to a duplicate set of coraqueous-hormone saturated responding

BP1 dishes for 24 hours. To improve efficacy of the oil-hormone treatment excess aqueous surface film coating

the the the

FIG. 3. An adult male H. schachtii having accomplished the molting of its original and normally terminal adult male cuticle. sp, spicules; st, stylet.

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JOHNSON

AND

larvae was allowed to evaporate before immersion in the mineral oil solutions. After the period of oil treatment, the larvae were resuspended in the corresponding saturated aqueous hormone solution before transfer to the corresponding BPA dish for a subsequent incubation period. At the termination of each hormone treatment the larvae were resuspended and separated from the sand. Nine replicates of sugar beet plants (Beta dgu~is, U.S. 75) were inoculated with known numbers of viable nematodes. Additional plants inoculated with untreated larvae and larvae treated with hydrated mineral oil served as controls. Host plants were exposed to the nematode inoculum for 48 hours, after which the plants were removed from pots, the root systems washed in running tap water, transplanted into fresh pots of sand, and watered with Hoagland’s nutrient solution. As a result, the majority of nematodes in the host roots were approximately at the same stage of development. Three repli-

VIGLIERCHIO

cates of each treatment were harvested 5, 9, and 15 days after inoculation. Plant tops were discarded and roots were fixed in formalin-acetic acid-alcohol (FAA) and stained in acid fuchsin-lactophenol, The nematodes dissected from the roots of each replicate of a series were combined. Fortyfive to 50 specimens were randomly selected and placed on slides for microscopic examination. RESULTS

Detectable effects, in terms of morphological deviations, were obtained only with animals treated with three oil-hormone solutions or suspensions. Testosterone was the only steroid showing activity. A number of females developed a hypodermal tuberculum dorsally on the functional head (Fig. lA-B). The presence of the hypodermal tuberculum is evident in the fourth (Fig. 1B) and adult stages (Fig. lA), and its position in all cases was in the dorsal sector of the head region. The initiation of the hypodermal

FIG. 4. Abnormally developing males. A. fourth-stage male developing a large gonadal mass while still encapsulated in the saccate third cuticle. B. adult male developing within molting or molted fourth-stage cuticle, all encapsulated in unbroken third-stage cuticle. g, gonad; sp, spicules.

H. schachtii

RESPONSE TO HORMONES

tuberculum can be seen in the third-stage larvae by the separation of that tissue from the tissue of the functional head (Fig. lCD). From whole-mount specimens the tuberculum appears to be composed of hypodermal tissue only and there is no suggestion of a stylet or lumen in the structure of the fourth or adult stage (Fig. 2A-B) . The other two hormones showing activity were the famesyl diethylamine and farnesyl methyl ether derivatives; both are sesquiterpenoids. Of the two, the diethylamine affected a greater range of morphological deviation. An expression of continued ac-

305

tivity is indicated by males induced to molt once again after reaching the normally terminal adult stage, as manifested by the presence of the molted spicules and stylet tip (Fig. 3). Other males were observed to have undergone other abnormal developments (Fig. 4A). Such males were still encapsulated within the cuticle of the thirdstage larvae, their body shape tended to be saccate, and gonadal development was restricted to a spherical mass of cells filling the central portion of the body. The spicules are fully developed but a molted or molting fourth-stage cuticle is evident ( Fig. 4A-B) .

FIG. 5. Fourth-stage males of H. schachtii within the third-stage cuticle showing the typical gerated gonadal growth. A. Lower magnification showing the third-stage cuticle, c; the gonadal g. B. High magnification of the gonadal mass, g.

exagmass,

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JOHNSON

AND VIGLIFXCHIO

FIG. 6. Adultoid male morphon within the body of a third-stage male H. s&u&ii. A. low-magnificalarva; C. the tion view of the host-morphon complex; B. the anterior region of the third-stage morphon; D. the anterior region of the morphon. st, stylet; mb, median bulb; i syn, intestinal syncytium; a, anus; g, gonad; lu, lumen; sp, spicules; mt c, molted cuticle.

H. schachtii

307

RESPONSE TO HORMONES

nematode body was always solidly filled with tissue. An adultoid morphon within the body of an apparently viable atypical third-stage larva was the most striking morphological result observed in any of the treatments ( Fig. 6). This adultoid morphon is believed to have arisen out of a testicular teratoma. The arguments concerning the origin and development of this abnormal animal that lead to this conclusion have been discussed elsewhere (Johnson and Viglierchio, 1970). Specimens with testicular neoplasms were also observed in nematodes developing from the farnesyl methyl ether treatment. Testosterone, famesyl diethylamine, and farnesyl methyl ether all caused extreme activity of the hypodermis as expressed by the greatly enlarged and densely stained hypodermal cells (Fig. 1D and Fig. 7).

DISCUSSION

FIG. 7. Adult male of H. schachtii showing enlarged and densely staining hypodermal cells. hc, hypodemlal cells; mt c, molted cuticle; sp, spicules.

An additional striking effect was the neoplastic growth of the testis external to the body of either fourth-stage larvae or males having adult secondary sex characteristics. The mass of testicular neoplasm in some cases approximated that of the entire animal to which it was attached (Fig. 5A-B). In all cases specimens manifesting this abnormal form were enclosed within the third-stage cuticle (Fig. 5A) and whenever this cuticle was micrurgically opened to allow the gonad to’ float freely it was found attached to the animal through an opening in the body wall. This did not appear to be an accidental rupture, since the

The physiological response of secondstage H. schachtii larvae to treatments with selected hormones and hormone mimics was expressed morphologically. Alterations in morphological structures could not be of embryogenic origin, since second-stage larvae, not eggs, were treated. For practical considerations it was undesirable to make direct physiological determinations in an initial exploratory investigation. Of the nine compounds tested, three were active: testosterone, famesyl diethylamine, and the farnesyl methyl ether. Testosterone was specific for females and caused the formation of hypodermal tubercula on the neck region in the dorsal sector of the body. Speculation on the genesis of such a structure resulting from a treatment of the second-stage larvae must be based upon a breakdown of a fundamental endogenous control mechanism normally exercised over key cells of the nematode.

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JOHNSON

AND

This particular structure must have had its origin from some cell(s) in the dorsal sector of the region anterior to the circumesophageal commissure, and, since the hypodermis appears to be the only type of tissue comprising the tuberculum, the initiating cells are probably hypodermal. From what is generally known about the morphology of that region of the body, the nuclei of the dorsal cord must be suspected as the key control centers. The dorsal sector of the body is unusual in that the dorsal cord has only one row of nuclei anterior to the circumesophageal commissure and possibly in the tail region, while the ventral and lateral cords have nuclei throughout the length of the body (Chitwood and Chitwood, 1950). These dorsal nuclei may be less determinate than those found in the other body cords. Interference with the endogenous control in one or more of these key cells has released its potentiality for neotonous development of a hypodermal tuberculum. The sex specificity of this reaction may possibly be a consequence of the dimorphism displayed by this species. The stylet and median bulb of third-stage males are smaller and less robust than the comparable structures of the females, which may be indicative of a reduced feeding requirement and cellular activity. Reduced cellular activity, together with the different endocrine balance and possibly other physiological differences would permit the males to escape the consequences of the testosterone treatment observed in the females under the conditions of these experiments. The two active farnesyl derivatives are closely related and displayed similar inductive effects. Under our experimental conditions, the activity was specific for males. Other than the occasional redundant molt of a normal adult male, the male’s susceptibility was localized in the gonadal tissue. Differences in the degree of response could likely be attributed to minor variations in

VIGLIERCHIO

physiological factors within individuals, as well as to the varying amounts of active substance the larvae absorbed during the exposure period. The extracorporeal gonadal growth in males probably arose from the testicular neoplasia developing within the body. Upon filling the body, the continued growth increased the stress until the thin, fragile body wall ruptured. The neoplastic growth of the gonad then extended through the opening into the space enclosed by the third-stage cuticle. Despite the rupture of the body wall to allow the escape of the extracorporeal portion of the gonad, the individual is still protected by the intact third-stage cuticle and it is not surprising to find continued development in these specimens. The principal characteristic of the normal third larval stage appears to be the production of gonadal tissue (testis or ovary), and the treatment appears to have overstimulated or otherwise inactivated the control of this process in the males. The extensive neoplastic growth facilitates an occasional deviation in cellular activity such that nuclear multipotentiality is uncovered or expressed, and the development of an adultoid morphon within the viable atypical thirdstage larvae takes place. ACKNOWLEDGMENTS

We thank Dr. W. E. Scott of the Research Division of Hoffmann-La Roche, Inc. of Nutley, for the supply of lO,ll-epoxyNew Jersey, famesenic acid ethyl ester, 2,3-cis/truns-6,7-t7ans famesyl diethylamine, and methyl ether. Contributions for the completion of this study were also provided by Hoffmann-La Roche, Inc. The interest and discussions of Drs. A. It. Maggenti, R. W. Timm, and Mr. G. A Paxman concerning these experiments are gratefully acknowledged. REFERENCES

K. G., AND KAN, S. P. 1967. An endocrine basis for ecdysis in a parasitic nematode. Nature London 214, 737-738.

DAVEY,

H. schachtii

RESPONSE TO HORMONES

DAVEY, K. G., AND KAN, S. P. 1968. Molting in a parasitic nematode, Phocanemu decipiens. IV. Ecdysis and its control. Can. J. Zool. 46, 893-898. JOHNSON, R. N., AND VIGLIERCHIO, D. R. 1970. Nature London. In press. MEEROVITCH, E. 1964. Studies on the in vitro axenic development of Trichine& spiralis. II. Preliminary experiments on the effects of

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famesol, cholesterol, and an insect extract. Can. J. Zool. 43, 81-85. ROGERS, W. P. 1965. The role of leucine aminopeptidase in the moulting of nematode parasites. Comp. Biochem. Physiol. 14, 311321. SOSIMERVILLE, R. I. 1957. The exsheathing mechanism of nematode infective larvae. Exp. Parasitol. 6, B-30.