Food Uptake by the Insect-parasitic , Sphaerularia bombi ()

GEORGE O. POINAR, JR. and ROBERTA HESS 1

Abstract: The insect-parasitic female of Sphaerularia bombi everts its uterus and associated reproductive structures into the body cavity of its host. This uterine sac then takes over the normal functions of the parasite and leads an independent existence. An examination of this sac shows that the surface of the uterine cells are differentiated into a network of saccular indentations separating off fine cytoplasmic extensions. The folding of adjacent cytoplasmic extensions around portions of the host's hemolymph results in the formation of pinocytotic vacuoles. Intracellular vacuoles are also formed at the base of the saccular indentations. It appears that the first stage of intracellular digestion in S. bombi initially occurs by pinocytosis in the outer surface of the uterine cells. The inner and outer surfaces of the ovary and oviduct are modified into lobelike projections to increase the absorptive surface area, and electron-dense droplets originating in this tissue were also observed in the developing eggs. The hypothesis is presented that soluble nutrients are passed into the ovary-oviduct tissue where, after being reconstituted into droplets, they enter the developing eggs. Key Words: nourishment, parasite, bumblebee.

Little information is available on the uptake hemocoel where they develop to third-stage ef nutrients by . Absorption juveniles (3). Since there is no growth or normally occurs through the intestinal cells, development of the original female, it appeared although simple diffusion of nutrients may also that host nutrients must be absorbed by the occur through the cuticle in -parasitic uterus, which has assumed an independent role. nematodes. However, there has been no The purpose of this study was to determine evidence of phagocytosis, pinocytosis, or how nutrients were absorbed by the developing intracellular digestion in the roundworms (2). parasite and whether special morphological Special adaptations associated with the uptake modifications were present. of nutrients have been reported in some entomogenous nematodes. Riding (5)reported MATERIALS AND METHODS finding microvilli on the body wall of the Mature uterine sacs of Sphaerularia bombi parasitic tylenchid female of a species of (Dufour) were removed from Bombus lueorum Bradynema from the phorid fly, Megaselia L. and Bombus terrestris L. collected during halterata Wood, and the present report May and June, 1971, in Holland. They were discusses still another adaptation for obtaining prefixed for 2 hr in 3% gluteraldehyde, then nourishment in an insect nematode. washed in 0.05 M phosphate buffer and fixed in A special case of parasitism is exhibited by 1% osmium in phosphate buffer (pH 7) for 1 Sphaerularia bombi (Dufour), a parasite of hr. After dehydration in a graded ethanol series, bumblebee queens, since in the host, the uterus the specimens were embedded in Araldite 6005 ~nd associated reproductive structures of the and sectioned with glass knives mounted in a Nematode become everted through the vulva, a Porter-Blum microtome (MT-2). The sections condition referred to as "prolapsed uterus". were stained with a saturated aqueous solution The uterine cells greatly expand, forming a of uranyl acetate followed by lead citrate (4) huge uterine sac containing the ovary, oviduct, and examined with an RCA EMU-3F and sperm, and dwarfing the remainder of the Philips EM-300 electron microscope. For light female body (Fig. 1). microscopy, similar specimens were fixed in 3% The eggs develop normally within the Formalin for 1 week and processed to glycerin uterine sac and are passed out into the host's using alcohol dehydration.

RESULTS Received for publication 6 March 1972. During the development of Sphaerularia 1Division of Entomology, University of California, bombi in the hemocoel of Bombus, the internal Berkeley 94720. The senior author thanks Dr. M. surface of the prolapsed uterus makes contact Oostenbrink and members of the Laboratorium voor Nematologie, Wagcningen, The Netherlands for with the hemolymph of the host. The uterus is providing laboratory space and facilities during the one cell thick, and enlargement apparently course of these studies, and Dr. A. van Kammen, Dr. occurs not by cell division, but by hypertrophy, T. S. Ie and Miss Magda Usmany of the Laboratorium voor Virologic, Wageningen, The Netherlands for aid causing cell nuclei and surrounding cytoplasm in the preparation of specimens. to stand out as projections over the surface of

270 Food Uptake by Sphaerularia: Poinar, Jr., Hess 271 the organ (Fig. 1). The cell borders remain and numerous heterogenous cytosomes. Tile distinct and become interdigitated with cytosomes varied from membrane-bound desmosomes during the enlargement of the vacuoles containing flocculent material in the uterus. apical regions of the cell to basally located In the fully developed uterus, the nucleated electron-dense vacuoles containing membranous portion of the cells with associated cytoplasm inclusions. becomes enlarged and swollen. Each cell The entire surface of the uterus exposed to contains a pleiomorphic nucleus with sparse the host's hemolymph was modified into a chromatin, extensive rough endoplasmic network of saccular indentations separating off" reticulum, elongate mitochondria, lipid droplets fine cytoplasmic extensions (Fig. 2, 3, 4). The

b m

,

,.L

~"~ ~ ~" ~-~'~ 1.5mm

FIG. 1. Mature female of Sphaerularia bombi removed from the body cavity of a queen Bombus sp. (b = body of female, u = enlarged uterine sac). 272 Journal of Nematology, Vol. 4, No. 4, October 1972

FIG. 2. Section of the wall of the uterus (u) and ovary-oviduct (o) ofS. bombi (s = saccular indentations on the surface of the uterus; I = lobe-like extensions on the surface of ovary-oviduct). Food Uptake by Sphaerularia: Poinar, Jr., Hess 273

FIG. 3. A. Outer surface of the uterus showing the saccular indentations (s) surrounded by cytoplasmic extensions (c) and the formation of intracellular vacuoles (iv) containing fiocculent deposit. B. Section cutting through the cytoplasmic extensions parallel to the outer surface of the uterus. 274 Journal of Nematology, Vol. 4, No. 4, October 1972

FIG. 4. A. Outer surface of uterus showing tips of cytoplasmic extensions enclosing portions of host material m "pinocytotic vacuoles" (p.v.). B. Close-up of 3A showing the presence of intracellular vacuoles (i.v.) forming at the base of the saccutar indentations.

i Food Uptake by Sphaerularia: PoiHar, Jr., Hess 275

?

2

~4

J

O ;" • "5 p 4

o

• 'i!,.¸,S

I:IG. 5. A. I.;lectron-dense droplet Id) between the ovary-oviduct •o) and developing egg (e). B. Electron-dense droplets (d) in the tubular indentations of the ovary-oviduct (o) and outer and inner portions of the developing egg (e). 276 Journal of Nematology, Vol. 4, No. 4, October 1972 outer surface of the uterus, as well as the into the host's hemocoel so that its absorptive contents of the saccular indentations, was surface is exposed to the exterior. This surface covered with a fine flocculent deposit that is increased by the bulging shape of the cells ultrastructurally resembled a mucous or and by the differentiation of the cell mucopolysaccaride (Fig. 2, 3, 4) (1). The tips membranes into saccular indentations. of the cytoplasmic extensions were sometimes The flocculent deposit covering the uterine bent towards each other and appeared to be cells may serve as a protective layer to keep the enclosing portions of the flocculent deposit host's blood cells from adhering to the surface into pinocytotic vacuoles (Fig. 4A). of the cells. It could be a residue originating Intracellular vacuoles or vesicles containing from the host's hemolymph, or a secretion flocculent deposit also were formed at the base arising from the uterine cells. However, if the of the saccular indentations, and these deposit were being produced by the uterine structures were seen at various distances from cells, one would expect to find well-developed the surface of the cells (Fig. 3A, 4B). The lower Golgi apparti, since these are generally assumed surface of the uterine cells was ramified by to be responsible for the synthesis and extensive infolding of the cell membrane and concentration of substances like contained cytosomes (Fig. 2). A basal lamina mucopolysaccharides. Yet, these cell organelles (typically found in transporting cells) occurred were very poorly developed. This, plus the along the outer edge of these cells, and a presence of microvillar type projections which flocculent deposit, similar to the one covering are generally accepted as transporting material the apical surface but denser, also lined the in one direction (as in renal cells), makes us lower surface of the cells (1). believe that host material is being incorporated The inner and outer surfaces of the into the uterine cells. ovary-oviduct were modified by projections and The presence of pinocytotic vacuoles in the indentations differing from those on the uterus terminal portions of the cytoplasmic extensions surface. The ovary-oviduct cells contained and intracellular vacuoles at the base of the branched tubular indentations surrounded by saccular indentations strongly suggests that the lobelike extensions of the cytoplasm (Fig. 2, 5). incorporation of nutrients is accomplished by Both surfaces of the oviduct were coated by a pinocytosis. fine deposit 30-45 nm thick. The cytoplasm of the ovary-oviduct cells contained electron-dense These vacuoles and vesicles (earlier referred droplets concentrated near the egg side of the to as cytosomes) become modified in the lower cell (Fig. 5). These droplets appeared to form part of the uterine cell and disappear. within the rough endoplasmic reticulum. The "coated" tubular indentations of the Similar electron-dense droplets were observed outer surface of the ovary-oviduct suggest that in the space between the ovary-oviduct and the absorption occurs in that locality. developing eggs as well as in the outer and inner Electron-dense droplets are formed within the portions of the developing eggs (Fig. 5). They rough endoplasmic reticulum of these cells and were never observed in the uterine cells. are also found in the space between the ovary-oviduct and the eggs as well as in the DISCUSSION layers of the developing eggs. The extruded uterus of Sphaerularia bombi In conclusion, our results indicate that the in the hemocoel of its insect host functions as intracellular digestion of food material by S. an organ for absorbing nutrients from the host's bombi initially occurs by pinocytosis in the hemolymph. The uterus in nematodes outer surface of the uterine cells. We then commonly has several functions. It serves as a suggest that these food vacuoles are digested portion of the reproductive system through and the soluble constituents passed into the which the eggs pass on their way to the ovary-oviduct where they are reconstituted into exterior; parts of it function as a spermatheca, droplets. These droplets move out of the ovarial other portions may secrete the outermost egg tissue and enter the developing eggs, thereby membrane, and the posterior portion may act providing nutrients to the eggs. as a storage organ for eggs. We now show an Experimental evidence will be necessary to additional function-that of absorbing nutrients prove the above hypothesis concerning the from the environment. movement of nutrients into the ovary-oviduct The uterus is everted during its extrusion and developing egg since our evidence is based Food Uptake by Sphaerularia: Poinar, Jr., Hess 277

solely on visual interpretations of the electron Freeman and Co., San Francisco. 154 p. micrographs. 3. POINAR, G. O., JR. and P. A. VAN DER LAAN. 1972. Morphology and life history of Sphaerularia bombi (Dufour) (Nematodea). Nematologica (in press). LITERATURE CITED 4. REYNOLDS, E. S. 1963. The use of lead citrate at 1. FAWCETT, D. W. 1966. An atlas of fine structure, high pH as an election-opaque stain in electron the cell, its organelles and inclusions. W. B. microscopy. J. Cell Biol. 17:208-212. Saunders Co., Philadelphia, Pa. 448 p. 5. RIDING, IRENE L. 1970. Microvilli on the outside 2. LEE, D. L. 1965. The physiology of nematodes. of a nematode. Nature 226:179-180.

Pathogenic Potential of Tylenchorhynchus dubius on Selected Turfgrass 1

C. W. LAUGHLIN and J. M. VARGAS, JR. 2

Abstract: Tylenchorhynchus dubius was observed to feed on 'Toronto' creeping bentg~ass and 'Merion' Kentucky bluegrass, and was a key participant in reducing the vegetative growth of both grass species. The severity of foliar and root macrosymptoms incited by T. dubius to 'Toronto' bentgrass was greater on plants grown at 16 C than on plants maintained at 21, 27 and 32 C. These parasitized plants exhibited a suppression of secondary stolon formation, shortened internodes and premature inflorescence initiation. Initial inoculum densities of 500 and 1000 nematodes/test pot produced similar pathogenic effects on the host. Feeding was primarily on root hairs and epidermal cells immediately behind the meristematic region. No necrotic lesions or other diagnostic symptoms were visible at the feeding sites. Nematodes were not observed inside the roots. Key Words: stunt nematode, temperature, feeding.

INTRODUCTION growing 'Toronto' creeping bentgrass (C-15) turf areas and 'Merion' Kentucky bluegrass In 1954, Troll and Tarjan (9) found lawns in Michigan showed the occurrence of T. Tylenchorhynchus spp. to be the most widely dubius often in populations exceeding 500 distributed phytoparasitic nematode associated nematodes/100 cc of soil (Laughlin and Vargas, with turfgrasses in Rhode Island. They unpublished data). The use of nonfumigant concluded that T. claytoni Steiner and T. nematicides in Michigan resulted in lawns of a dubius (Biitschli) Filipjev were likely to play darker green color and more vigorous foliar major roles in devitalizing bentgrass (Arostis growth than untreated areas within the same palustris Huds.) turf. Taylor et al. (7) recovered lawns (4). These treatments concurrently Tylenchorhynchus spp. from all bentgrass golf reduced the populations of T. dubius associated greens sampled in Illinois. Sumner (6) reported with 'Merion' Kentucky bluegrass turf. To that Tylenchorhynchus spp. appeared to investigate the pathogenic potential of T. contribute to fading-out of bluegrass in in vitro dubius on 'Toronto' creeping bentgrass and studies. Troll and Rohde (8) reported that T. 'Merion' Kentucky bluegrass, the following claytoni was pathogenic to creeping red fescue studies were initiated. (Festuca rubra L.), did not effect Kentucky bluegrass (Poa pratensis L.) and appeared to MATERIALS AND METHODS stimulate shoot growth of annual ryegrass (Lolium multiflorum Lam.). A survey of poorly Pathogenicity Studies. Five cuttings of 'Toronto' creeping bentgrass previously rooted Received for publication 13 March 1972. in distilled water were placed in washed sand, which had been fumigated with methyl 1Contribution from the Michigan Agricultural Experiment Station. Paper No. 5845. bromide, in a 7.6-cm diam plastic pot. 2Assistant Professors, Departments of Entomology, Population densities of 500 and 1000 and Botany and Plant Pathology, respectively, individuals of T. were pipetted directly Michi&an State University, East Lansing, Michigan dubius 48823. onto the roots. Supernatant, which was