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R E S E A R C H N O T E J o u r n a l o f N e m a t o l o g y 2 7 ( 2 ): 2 4 4 -2 4 7 . 1 99 5. T h e S o c i e ty o f N e m a t o l o g i s t s 1 99 5.

A R a p i d a n d S i m p l e M e t h o d f o r S t a i n i n g L i p i d i nF i x e d N e m a t o d e sW M . T E R R E L L S T A M P S A N D M A R C J L I N I T 2

Abstract: A m e t h o d is d e s c r i b e d f o r s t a i n i n g li p i d i n f o u r t h - s t a g e d i s p e r s a l j u v e n i l e n e m a t o d e sf i x e d w i t h f o r m a l - a c e t ic f i x a ti v e (F A 4 : I ) . Bursaphelenchus xylophilus f o u r t h - s t a g e d i s p e r s a l j u v e n i l e sw e r e f i x e d w i t h h o t F A 4 : 1 f o r 2 4 h o u r s , e x c e s s f i x a t iv e w a s r e m o v e d , a n d a s o l u ti o n o f s a t u r a t e d o i lr e d O i n 9 6 e t h a n o l a d d e d a n d a l l o w e d to s it f o r 2 5 m i n u t e s a t 6 0 C . E x c e s s o i l r e d O w a s r e m o v e d ,n e m a t o d e s w e r e w a s h e d t w i ce w i t h 7 0 e t h a n o l , a n d w e r e p r o c e s s e d to p u r e g l y c e r in . L i p i d d r o p l e t sw i t h i n t h e n e m a t o d e s w e r e v i e w e d by l ig h t m i c ro s c o p y a n d a p p e a r e d a s d a r k r e d s p h e r e s o f v a r io u ss iz es . C o m p u t e r i z e d i m a g e a n a ly s is w a s u s e d t o q u a n t i f y l i p i d d r o p l e t a r e a .Key w ords: Bursaphelenchm xflophilus p i n e w i lt , p i n e w o o d n e m a t o d e , o i l r e d O , l i p i d , s ta i n in g .

L i p i d i s e s s e n t i a l f o r n e m a t o d e s u r v i v a la n d is t h e m a j o r s o u r c e o f e n e r g y f o r t h ed a u e r l a r v a e o r i n f ec t iv e s t a g e s o f v ir t ua l lya ll a n i m a l a n d p l a n t -p a r a s it i c n e m a t o d e s( 1, 3) . T h e f a t e o f l ip i d u n d e r v a r i o u s c o n -d i t i o n s a n d t h e r a t e o f i ts u t i li z a t io n a r e o fi m p o r t a n c e i n u n d e r s t a n d i n g n e m a t o d ee c o l o g y , b e h a v i o r , a n d p h y s i o lo g y . E n e r g yu t i l i z a t io n , m e t a b o l i c r a te s , a n d a g i n gp r o g r e s s c a n b e d e t e r m i n e d f r o m t h e li pi dc o n t e n t o f n e m a t o d e s ( 1, 10 ).Bursaphelenchus xy loph i lus ( S t e i n e r a n dB u h r e r , 1 9 3 4 ) N i c k l e , 1 9 7 0 is t h e c a u s a la g e n t o f p i n e w i lt ( 4) . T h e f o u r t h - s t a g e d i s-p e r s a l ju v e n i l e o f B. xylophilus is a n o n f e e d -i n g s t a g e t h a t is m o r p h o l o g i c a l l y a n d p h y s -i o l og i c a ll y d i s t i n c t f r o m o t h e r l if e s ta g e s . I tc o n t a i n s a l a r g e a m o u n t o f l i p id t h a t is u t i-l i z e d a s a n e n e r g y s o u r c e ( 5 , 6 ) . F o u r t h -s t a g e d i s p e r s a l j u v e n i l e s a r e c a r r i e d f r o mi n f e c t e d p i n e s t o n e w h o s ts b y c e r a m b y c i db e e t l e s , M o n o c h a m u s s p p . ( 7 ) . W e a r e i n -t e r e s t e d i n t h e r e l a t i o n s h i p b e t w e e n l i p i dc o n t e n t a n d e x i t b e h a v i o r o f d is p e r s a l j u -v e n i le s f r o m b e e t l e v e c to r s . T h i s p a p e r r e -p o r t s o n a l i p i d - s t a i n i n g t e c h n i q u e ( m o d i -f i c a t i o n o f C r o l l ( 2) a n d S e i n h o r s t (8 )) t h a t

Received for publication 19 September 1994.1 Contribution from the Missouri Agricultural ExperimentStation. Journal Series No. 12,258.Department of Entomology, University of Missouri, Co-lumbia, M O 65211 . Address all correspondence to M. J.Linit.2 4 4

f a c il it a te s q u a n t i f i c a t i o n o f l ip i d c o n t e n t i ns in g le o r a s m a ll n u m b e r o f n e m a t o d e s .T h e t e c h n iq u e w a s m o d i f i e d f r o m t h ew o r k o f C r o ll ( 2) . I n C r o l l' s s t u d y , t h e n o n -f e e d i n g i n f e ct iv e s ta g e o f t h e h o o k w o r mAncylostoma tubaeforme ( Z e d e r ), a v e r t e b r a t ep a r a s i t e , w a s a l i v e i n w a t e r w h e n s t a i n e dw i t h oil r e d O a n d p r o c e s s e d t o g l y c er i n b ya m e t h o d m o d i f i e d f r o m S e i n h o r s t ( 8) .T h e n e m a t o d e s w e r e t h e n a n a l y z e d w i th as c a n n i n g m i c r o d e n s i t o m e t e r a t 5 1 7 n m .O u r m e t h o d i s f o r f i x e d s p e c i m e n s .

M A T E R I A LS A N D M E T H O D SF o u r t h - s t a g e d i s p e r sa l j u v e n i l e s w e r e

o b t ai n e d f r o m n e w ly e m e r g e d a d u l t M o n ochamus carolinensis ( O l iv i er ) b e e t l e s t h a t h a dd e v e l o p e d i n j a c k p i n e, P in u s b a n ks ia n aL a m b . , l o g s i n f e c t e d w i t h B. xylophilus.N e m a t o d e s w e r e c ol le c te d f r o m a d u l t b e e -t l e s u s i n g a m o d i f i e d B a e r m a n n t e c h n i q u e(9 ) a n d w e r e t r a n s f e r r e d t o w e l ls o f a 2 4 -w e l l p l a s t i c t i s s u e c u l t u r e p l a t e ( F a l c o n3 0 4 7 , B e c t o n D i c k e n s o n , L i n c o l n P a rk ,N J ). M o s t o f t h e w a t e r w a s r e m o v e d f r o me a c h w e l l w i t h a P a s t e u r p i p e t t e u n d e r ad i s s e c t i n g m i c r o s c o p e t o p r e v e n t r e m o v a lo f n e m a t o d e s . F o l l o w i n g a s t a n d a r dm e t h o d f o r f i x i n g n e m a t o d e s , 2 - 3 m l o fh o t ( 9 0 C ) f o r m a l - a c e t i c f i x a t i v e 4 : 1 ( F A 4 :1; 1 0 p a r t s f o r m a l i n ( 4 0% f o r m a l d e h y d e ) ,1 p a r t g l ac i a l a ce t i c ac i d , 8 9 p a r t s d i s t i l l edw a t e r ) w a s q u i c k l y a d d e d t o e a c h w e l l ( 9 ) .T h e t i s s u e c u l t u r e p l a t e w a s c o v e r e d , k e p t

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Staining Lipid in Fixed Nematodes: S tamps L in i t 245at room temperature for 24 hours, andthen most of the fixative was removedfrom each well with a Pasteur pipette un-der magnification, as described above.For our staining method, a saturated so-lution of oil red O (no. 0-0625, SigmaChemical Co., St. Louis, MO) in ethanolwas made by adding 1-3 g of oil red Opow der to 100 ml of 96% ethanol (Croll (2)used 70% ethanol) and was stirred for 20minutes. The solution was then filteredthrough a vacuum filter apparatus using aWhatman no. 2 paper filter.Each well received 1.5 ml of the satu-rated oil red O solution, and the plate wascovered and held at 60 C for 25 minutes inan oven (2). Upon removal from the oven,most of the oil red O solution was removedfro m each well with a Pasteur pipet te and 2ml of 70% ethanol was adde d to each well.After sufficient time had elapsed for thenematodes to settle to the bottom of eachwell (about 10 minutes), the excess ethanolwas removed with a Pasteur pipette. Theethanol wash was repea ted a second time(modified from distilled water washes asdescribed in Croll (2)).Instead of adding a 50/50 (V:V) water-glycerin Solution to the wells and allowingthe water to evaporate as described byCroll (2), we processed the nematodes toglycerin in the wells of the tissue cultureplate following a modification of the quickmethod of Seinhorst (8,9). Two ml ofSeinhorst I solution (20 parts 96% etha-nol, 1 part glycerin, 79 parts distilled wa-ter) at room tempe rat ure (21 C) was addedto each well. The uncove red tissue cultureplate was placed in a closed vessel contain-ing a small volume of 96% ethanol. Thisvessel was placed in an oven at 35-40 C for24 hours. The excess liquid in each wellwas removed with a Pasteur pipette, a nd 2ml of a modifi ed Seinhorst II solution(10 parts glycerin, 90 parts 96% e thanol) atroom temperature (21 C) was added. Theuncovered tissue culture plate, not in theclosed vessel, was re tu rned to the oven un-til all ethanol had evaporated, by whichtime the stained nematodes were then inpure glycerin and ready to mount. The

nematodes were mount ed in drops of pureglycerin on standard microscope slides us-ing the wax ring method (9) and viewedwith the use of transmitted light micros-copy.RESULTS ND DISCUSSION

Once the nematodes were in pure glyc-erin, the lipid droplets appeared as darkred spheres of various sizes and were quiteobv ious w hen the nemat odes w er emounted and viewed under light micros-copy (Fig. 1). Th e lipid droplets wi thin thenematodes did not appear to be red or ap-peared to be stained very lightly red whilegoing through the process. This was usu-ally an artifact of processing.Chemical methods of quantifying lipidcontent often require large numbers ofspecimens, and they are impractical forsingle or a few nematodes. The methodpresented here is excellent for examininglipid in a very small numb er of specimensbecause individuals can be tracked u nd er amicroscope th rou gho ut the process. Use ofa 24-well tissue culture plate also allows alarge number of specimens or groups ofspecimens to be processed at one time and,at the e nd of the processing, provides con-venient long-term storage. Specimen in-format ion can be reco rded on the lid. Thistechnique produces preserved specimensthat can be re-examined later. Nematodesalready in glycerin appear unh ar me d aftergoing through part of the process a sec-ond, third, or fourth time.

The intensity and precision of the stain-ing for lipids have allowed us to quantifylipid content in individual nematodes. Im-ages of nematodes unde r transmitted lightmicroscopy were recorded by a video cam-era attached to a video capture board ina personal computer and analyzed withMOCHA (Jandel Scientific, San Raphael,CA), an image analysis program that candistinguish colors and levels of density.Lipid droplet area and whole body areawere quantified, and percentage lipid area(lipid per body) was used as a measure oflipid content for fourth-stage dispersal ju-veniles under various treatment regimes.

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2 4 6 Jo u r n a l o f Nem a to lo g y V o l u m e 2 7 No . 2 Ju n e 1 9 9 5

FIG. 1. Photom icrographs of A an unstained Bursaphelenchusxylophilus ourth-stage dispersal juvenile a ndB an oil red O-stained fourth-stage d ispersal juvenile. Red-stained lipid droplets fill mu ch o f the bod y of thenem atode and app ear black in the photomicrograph. Head region in A and B at up pe r lef t. Scale bar =0.1 ram.

T h e t e c h n i q u e p r e s e n t e d h e r e w o r k sv e r y w e l l f o r s t a i n in g f o u r t h - s t a g e d is -p e r s a l j u v e n i l e s o f th e n e m a t o d e B. xylo-philus a n d s h o u l d w o r k e q u a l ly w e ll w i t h

o t h e r f i x e d n e m a t o d e s p ec ie s t h a t c o n t a i nl ip i d d r o p l e t s . T h e m e t h o d is c o n v e n i e n t ,r a p i d , a n d c o n s i s t e n t i n t h e u n i f o r m i t y a n dd e n s i ty o f s ta i n in g p r o v i d e d .

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Staining Lipid in Fixed Nematodes: S tamps L i n i t 47LITERATURE CITED

1. Cooper, A. F., and S. D. Van Gundy. 1971. Se-nescence, quiescence, and cryptobiosis. Pp. 297-318in B.M. Zuckerman, W. F. Mai, and R.A. Rohde,eds. Plant parasitic nematodes, vol. 2. New York: Ac-ademic Press.2. Croll, N. A. 1972. Energy utilization of infectiveAncylostoma tubaeforme larvae. Parasitology 64:355-368.3. Dropkin, V.H . 1989. Introduc tion to plantnematology. New York: John Wiley.4. Kiyohara, T., and Y. Tokushige. 1971. Inocula-tion experiments of a nematode, Bursaphelenchus sp.,onto pine trees. Journal of the Japanese Forestry So-ciety 53:210-218.5. Kondo, E. 1986. SEM observations on the intra-tracheal existence and cuticle surface of the pinewoodnematode, Bursaphelenchus xylophilus associated withthe cerambycid beetle, M onocham us carolinensis. Ap-plied Entomology and Zoology 21:340-346.

6. Kondo, E., and N. Ishibashi. 1078. Ultrastruc-tural differences between the propagative and dis-persal forms in pine wood nematode, Bursaphelench~lignicolus with reference to the survival. Applied En-tomology and Zoology 13:1-11.7. Linit, M. J. 1988. Nematode-vector relationshipsin the pine wilt disease system. Journal of Nematolo-gy 20:227-235.8. Seinhorst, J. w. 1959. A rapid method for thetransfer of nematodes from fixative to anhydrousglycerin. Nematologica 4:67-69.9. Southey,J. F., ed. 1986. Laboratory methods forwork with plant and soil nematodes. Ministry of Ag-riculture, Fisheries, and Food (Great Britain) Ref.Book 402. London: Her Majesty s Stationery Office.10. Wilson, P. A. G. 1965. Changes in lipid and ni-trogen content of Nippostrongylus brasiliensis infectivelarvae aged at constant temperature. ExperimentalParasitology 16:190-194.