Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae)

Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae),

an associate of bees in the genus Halictus (Hymenoptera : Halictidae)

Robin M. GIBLIN *, Jennifer L. SWAN and Harry K. KAYA Divis ion of Nematology, University of California, Davis, Ca 95616, U S A

SUMMARY

Adults and immature stages of Bursaphelenchus kevini n. sp., an associate of Halictus spp., are described and illustrated. B. kevini n. sp. developed and reproduced on two of seven fungi tested and did best on Moni l in ia fructicola followed by a F u s a r i u m sp. Generation time, from second-stage juvenile (52) to J 2 eclosion of the next generation is eight days on M . fructicola grown on potato dextrose agar a t 250. Biometrics of adults were compared from 14 and 28 day old cultures O € B. kevini n. sp. grown on M . fructicola at 250. Isolates from Halictus from three different localities (Davis, California ; Parma, Idaho ; and Newberg, Oregon) were also compared.

RÉSUMÉ

Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae), associe’ a u x abeilles du genre Halictus ( H y m e - noptera : Halictidae).

Les stades adultes e t immatures de Bursaphelenchus kevini n. sp., associé de Halictus spp., sont décrits e t illustrés. B. kevini n. sp. se développe e t se reproduit sur deux des sept champignons testés, le meilleur étant Motzilinia fructicola suivi par Fusar ium sp. La durée du cycle, depuis le juvénile de second stade (52) jusqu’au 52 de la généra- tion suivante, est de huit jours sur M . fructicola poussant sur PDA à 250. Une comparaison biométrique a été faite entre les adultes provenant d’élevages sur M. fructicola, à 250, aprBs 14 et 28 jours. Des isolats provenant de trois localites différentes (Davis, Californie ; Parma, Idaho et Newberg, Oregon) ont été également comparés.

An unidentified aphelenchoidid was reported €rom the bursa copulatrixes of females and the penises of males of the sweal; bee, Halictus farinosus Smith (Giblin, Kaya 2k Brooks, 1981). Giblin and Kaya (1981) identified this nematode as Iluntaphelen-- choides sp. based on the spicule shape, which was intermediaLe t o the spicule shape of Hurziaphelen- choides Niclde, 1970 and Bursaphelenchus Fuchs, 1937. Subsequently, the genus Nulltaphelerzchoides was synonymized with Bursaphelenchus by Giblin and ICaya (1983a). The nematode in this study and cited above was determined t o be a new species and is described herein as Bursaphelenchus keuini n. sp.

Fungi isolated from the brood cells of H . farinosus, and insect and plant pathogenic fungi were tested as hosts for B. kevirzi n. sp. In addition, studies on the life cycle and generation time of B. keuirzi n. sp. cultured on the fungus, Morzilirzia fructicola (Wint.) Honey, a t 250 were conducted. Comparisons were made of the morphometric variation present in adults

of B. kev in i n. sp. isolated as dauer juveniles ( J I I I s ) from H . farirzosus or H . ligatus Say from three different locations and cultured on M . fructicola al; 250 for 14 and 28 days.

Materials and methods

MEASUREMENTS

Dauer juveniles ( J I I I ) of B. keuini n. sp., isolated from the bursa copulatrix and common oviduct of a heavily infested reproductive female of H. farinosus from Davis, Y010 County, Calilornia on April 25, 1980, were used t o establish a culture on M . fructicola. Type adults of B. keuirzi n. sp. were collected from an eighteen day old culture. Type specimens were heat killed, preserved in formalin-glycerol, fixed in TAF for 24 h, slowly processed into glycerol (Southey, 1970), mounted and measured. Median spicule measurements were taken from a straight line

* Present address : Department of Nematology, University of California, Riverside, Ca 92521, USA.

Revue Nématol . 7 (2) : 177-187 (1984) 177

R.M. Giblin, J.L. Swan di: H.K. K a y a

drawn from the apex of the capitulum to the ,dista1 most point of the lamina. Drawings were done with a camera lucida.

LIFE STAGES

Life stage observations were made with B. kevini n. sp. from Davis grown on one week old cultures of M. fructicola on potato dextrose agar (PDA) for two tao four weeks. B. kevini n. sp. were harvested from cultures, heat killed, stained in 45 % acetic orcein for more than 24 h (Giblin & Kaya, 1983a) and drawn and measured. Eggs of B. kevini n. sp. were also stained, drawn and measured. Dauer juveniles (JIIIs) of B . keuini n. sp. were isolated from the bursa copulatrix and common oviduct of an overwintered reproductive female of H. farinosus from Davis and from old PDA cultures (more than twelve weeks old), and were stained, drawn and measured. Juvenile propagative stages, the dispersa1 stage (dauer juvenile = JIII) and the intermolts are designated as described by Giblin and Kaya (1983a).

LIFE CYCLE

Generation time was determined as follows : B. kevini n. sp. eggs were transferred from a culture into a dish filled with distilled water. Newly-hatched nematodes were collected a t 3-8 11 intervals and ten to twenty nematodes were added to M . fructicola mycelia in each individual tissue culture well as described by Giblin and Kaya (19833). Nematodes were collected from several Wells a t 24 h intervals, until J2s of the next generation were first observed. Al1 nematode cultures and tests were maintained a t 25O.

FUNGAL HOSTS

B. kevini n. sp. stock cultures were maintained on M . fructicola on PDA. Three species of Pusarium were isolated into pure culture from the brood cells of H . farinosus collected in Davis. In addition, M. fructicola, Botrytis cinerea Pers. ex Fries, Beauveria bassiana (Balsamo) Vuillemin and Ascosphaera apis (Maa- sen e s Claussen) Olive & Splitoir were tested as hosts for B. kevini 4. sp. Petri dishes (100mm x 15mm) with ca 15 ml of PDA were inoculated with a small block of fungal mycelia .The'fungi were incubated for one week before inoculation with B . keuini n. sp. Nematodes were extracted from stock cultures and surface sterilized in two steps : ( 1 ) nematodes were placed in 1% water agar containing the fungicide, Aretan@ (methoxyethyl mercury chloride) for 36 h (Moody, Lownsbery & Ahmed, 1973) and ( 2 ) they were then placed in 0.1 g/1 Merthiolate@ (Eli Lilly and

178

Co., Indianapolis, Indiana) as described by Hara, Lindegren and Kaya (1981) for 3 h. Nematodes (240) were pipetted aseptically into three plates of each of the test fungi and incubated for six weeks. Nematodes were extracted for 3 h on a Baermann funnel and counted as described by Giblin and Kaya (1983b).

COMPARISON OF BIOMETRICS OF B. keuini N. SP.

In addition to the cultures of B. kevini n. sp. from Davis, separate cultures were established from JIIIs isolated from the bursa copulatrix and common oviduct of a reproductive female of H . farinosus, collected in Newberg, Yamhill Co., Oregon and from a reproductive female of H. ligatus from outside of Parma, Payette Co., Idaho. An undetermined number of B . keuini n. sp. from an established culture from a given location were inoculated ont0 one week old M . fructicola and grown for 14 and 28 days, respectively. Adults of both sexes of B. keuini n. sp. from a given location and culture time were extracted and measured in temporary water mounts. Comparisons were made between the morphology and biometrics of B. keuini n. sp. from the three different collection sites and from the two different culture times.

Al1 statistical comparisons were made with a one- way analysis of variance (ANOVA) and separation of means was done with Duncan's Multiple Range test.

Cultured adults of B. kevini n. sp. were heat kilied, fixed and prepared for photomicrographs and .for scanning electron microscope (SEM) observations as described by Giblin and Kaya (1983a).

Bursaphelenchus kevini n. sp. * (Fig. 1-5)

MEASUREMENTS

(See Table 1 for Paratypes measurements) Hololype (male) : L = 0.87 mm ; W (width) =

27 pm ; a = 32 ; E (distance from anterior end to junction of esophagus and intestine) = 67 pm ; b = 13.0 ; tail = 46 pm ; c = 18.9 ; spicule = 19 pm ; stylet = 14 pm.

Allotype (female) : L = 0.92 mm ; W = 32 pm ; a = 29 ; E = 78 pm ; b = 11.8 ; tail length = 50 pm ; c = 18.4 ; V = 80% ; stylet = 17 pm.

* Bursaphelenchus keuini n. sp., is named for Kevin Randal Giblin, brother of R.M.G., Who has succeeded in coping with the adversity of a severe handicap.

Revue Nématol. 7 (2) : 177-187 (1984)

Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae), an associate of bees in tne genus Halictus

DESCRIPTION

M a l e : Body cylindrical, annulated, body annules about 0.9 Pm wide. Lip region offset. Lip annules visible with SEM only ; en face view with light microscope with six lips with inner hexaradiate framework; lateral lips narrower than sublaterals. Amphidial apertures pore-like. Cephalic papilla on the outer margin of each sublateral lip. Labial disc demarcated by ridge (seen with SEM only). Six outer labial papillae appear as refractile dots in light microscope, only lateral outer labial papillae visible with SEM. Four sublateral inner labial papillae visible with SEM only. Stylet, two-part ; cone short, shaft wjth basal thickenings. Procorpus ending in well-developed metacorpus. Esophago-intestinal junction about 1.5 nietacorpus valve lengths behind metacorpus. Pos- tcorpus, glandular, dorsally located, 58 pm long. Postcorpus length in other male type specimens 52- 67 pm (58 f 5). Excretory pore a t level of base of metacorpus anterior to nerve ring, in other type specimens, excretory pore located between base of metacorpus and level of esophago-intestinal junction. Hemizonid obscure, when visible, ca 40 pm behind base O€ metacorpus. Gonad outstretched. In some recently molted males gonad reflexed apically. Lat- eral field with four incisures, beginning midway between anterior extremity and start of metacorpus, extending posteriorly about one anal body width above papilla 1 ; then reduced to three incisures t o bursal flap. Tail 2.3 anal body widths long ; in other male types 2.1-3.1 (2.5 & 0.3). Tail veutrally curled ;

Table 1

Cornparisons of the Marphometries.of Three Populations of

in other type specimens extent of curl variable. Tail terminus clawlike in lateral view. Bursal flap sur- rounds tail terminus, terminus variably shaped from ventral view. Nine ventral preanal and postanal papillae : one preanal papilla (P l ) in ventral midline 6 pm above cloaca, one pair preanal subventral papillae (PZ) a t level of P l , one pair of subventral postanal papillae (P3) a t 37 % of tail length from cloaca, position of P3 varied in other male type specimens from 36% to 51 %, another pair of subventral postanal papillae (P4) posterior to P3 and 66% of tail length behind cloaca, one ventral pair of papillae (,P5) a t level of bursal flap, obscure in most specimens. Spicules separate ; rostrum sharply pointed, spicule median length/capitulum length ratio = 1.5-1.7 (1.6) ; lamina complex ; dorsal aspect of blade appears separate from ventral aspect of blade which appears to have transverse bar, end of lamina wide and rounded.

Female : Adult male and female cuticular features and cephalic regions similar. Ovary reflexed apically, in other specimens ovary outstretched. Vulva, transverse slit, vulval lips protrude slightly. Vagina vera cuticularized. Thin, paired, four pronged structures a t junction of uterus and postvulval sac similar to structures in B. seani (Giblin & Kaya, 1983~). Length of postvulval sac ca 4 distance from vulva to anus. Some female specimens, postvulval sac with egg. Lateral field extends to ta11 tip. Anus dome-shaped slit. Tail 2.7-3.4 (3.1 & 0.2) anal body widths long, uniformly conoid with mucron.

11 8

h . 0 . (rsnse) (810-980) (800-950) 880Of50 87ObCt60

Body diea. (um) 27Ct2

L (um)

(29-38) (24-30)

(72-78) (63-75) 74Et2 69%4

Tail (um) (43-53) (46-62) 48'33 53dt6

27Atl 3Zd13 (26-29) (27-36)

E*

b 11.9At0.7 12.7't0.9 (11.1-13.6) (11.8-14.8)

18.3"t0.8 16.7a?1.5 (16.9-19.1) (14.7-18.9Y

V 79"tl (76-81)

-- -_ Stylet (em) 16'tl 14Ctl

(15-17) (14-16)

Spicule (um) -- 20Cf2 _- (18-22)

11 8

(900-1200) (890-1120) 1040BCt90 103Oa270

(42-58) (35-51) 52"*6 4Znbt5

89" 93bCt4 (82-94) (87-102)

60BCt5 67abct8 (50-69) (58-83)

20°t1 24Edt2 (18-23) (22-27)

(10.1-12.8) (9.8-11.9) 11.7'21.0 ll.O'btl.O

17.3ABCt0.7 15.5"bt2.0 (16.3-19.0) (12.3-19.0)

81A'tZ -- (79-86) -_ 18'tl 17nbtl (17-20) (16-18)

26abt2 *- __ 03-28)

11 8 11 8 11 8 11 8 Il 8

900'?210 810c& 1160ABt110 97Oab?100 980CDt160 1070nt140 1220'290 1080't130 100OCn?90 81OCt90 (560-1200) (700-1120) (990-1300) (840-1110) (780-1230) (800-J250) (990-1310) (900-1260) (850-1110) (910-1150)

4BBCt15 35bf8 49ABt4 41abt3 43'?6 40abt8 58'27 47'5 4gBCt9 Xbt6 (24-67) (27-50) (42-55) (38-46) 06-53) (27-49) (50-71) (39-52) (37-63) (29-46)

82'+7 84dt2 95't5 88ed*5 89't5 9ZCt5 10ZA26 9gabt6 9EABt7 10Znt5 (68-91) (82-88) (88-100) (81-96) (83-100) (88-104) (91-113) (93-113) (88-110) (98-113)

(48-73) (58-73) (63-76) J58-71) (55-78) (60-77) (57-78) (58-92) (44-63) (66-91) 5gBCt9 64bct5 6gAt4 63't5 6ZBCt8 70Bbct6 66ABLBt6 738bt10 56't6 74'tB

(16-23) (22-28) (21-25) ' (21-25) (21-26) (24-30) (19-24) (18-25) (13-25) (24-36) 19'22 2SCdt2 24'3 23d?l 23"tl 27bC*3 21CDtZ 23dt2 2lCof3 30nbt4

10.9A8t1.9 10.1bt1.7 12.ZAt1.4 10.9ab%1.0 10.9'Btl.3 11.7abt1.8 12.0Atl.l 11.0°bt1.8 10.3'tl.Z 10.3bt0.9 (8.2-13.2) (8.3-13.5) ..(10.3-14.4) .(9.4-12.2) (9.4-13.1) (8.8-13.9) (9.6-13.4) (8.0-13.3) (8.7-11.6) (8.8-11.5)

. .

(11.7-17.5) (10.0-17.5) (14.4-18.1) (12.1-19.1)(13.7-22.4) (11.8-16.8) (15.6-21.0)(13.2-19.0) (16.2-19.3) (12.3-17.0) 1 d t 1 . 7 13.4~tz.z 16.8~~~tl.l is~'~tz.3 15.8~~*2.4 15.2~~t1.7 18.7~t1.6 15.0~~22.0 le.0~31.0 14.3~~t1.6

78't2 80ABC21 -- 80ABCf2 -_ 82At1 (76-82) -- (78-83) -- -- 79"tl _-

(77-83) -- . (80-84) -- (78-82) __ llBCtZ 16btl 18Btl 178btl l8'tl llabtl 19'fl 18'tl ZO'tl lBn*l (13-19) (14-18) (16-20) (15-18) (17-19) (16-18) (18-21) (17-20) (18-21) (16-19)

27*tl -- 26nbt2 -- 2Sabt2 -- . 24b?2 (26-30) -- (23-28) -- (23-28) -- (21-26)

-- 24bt2 -- -- (21-28) --

*E - distance from anterior end CO ocsophnso-inrasrinnl Junetion.

differenr upper'case lettars and means of mnlca folloved br differenr lwer case letcers (Nemradas w ~ r e culrured onhlondZinio f r u C t i W k o n PDA. Hcnns of femrlca folloued by

are significanrly different (P < 0.01) according CO I Duncan's Multiple ~ n n g c rcsr.)

Revue Nèmaiol. 7 ( 2 ) : 177-187 (19S4) -

~

179

Fig. 1. Bursaphelenchus kevini n. sp. (A-E, SEM photomicrographs ; F-G, photomicrographs). A : Face view ; B : Tai1 region of male (subventral) ; C : Face (lateral) ; D : Female tail (subventral) ; E : Female, vulva (ventral) ; F-G : Male spicules (lateral). AA : amphidial aperture; CP. : cephalie papilla ; P l . : single ventral preanal papilla ; PZ : preanal papilla ; P3-P5 : postanal papillae. (A, C : bar = 1 Pm) ( B , D, E , F , G : bar = 10 Pm).

180 Revue Nèmaiol. 7 (2) : 177-187 (1984)

Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae), an associate of bees in the genus Halictus

TYPE MATERIAL

Holotype (male) : CoIIected April 25, 1980 by Robin M. Giblin. Catalogue No. UCNC 2025, Univer- sity of California, Davis. Paratypes (males and females) : Same data as holotype. Deposited a t Uni- versity of California, Davis ; USDA Nematode Col- lection, Beltsville, Maryland ; Nematology Depart- ment, Rothamsted Experirnental Station, Harpenden Herts., England ; Lab. voor Nematologie, Binne- haven 10, Wageningen,.Netherlands.

TYPE HABITAT

Associated as dauer juveniles ( J I I I ) in the bursa copulatrix and common oviduct of a reproductive female of H. furinosus from Davis, Y010 Co., Cali- fornia.

TYPE CULTURE

Type specimens harvested from eighteen day old type culture on M . fructicola on PDA.

DIAGNOSIS AND RELATIONSHIPS

Based upon spicule morphology, Brtrsaphelerzchus kev in i n. sp. is closest t o B. pol igraphi Fuchs, 1937 and B. talonus (Thorne, 1935) Goodey, 1960. Males of these species have paired spicules that have a median length/capitulum length ratio of 1.5-1.7, a sharply pointed rostrum, and a comples lamina with the dorsal aspect of the blade appearing separate from the ventral aspect. Also, the lamina appears to have a transverse bar and is wide or digitate a t the tip.

Examination of type specimens of B. ialonus from Horse Creek, Utah, 1933 (Slide 9a) revealed tha t the stylet is without basal thiclrenings, the escretory pore is above the level of the nerve ring, at least 2.5 metacorpus valve lengths behind the base of the metacorpus (not originally described (Thorne, 1935)), and a single ventral preanal papilla ( P l ) is present in males (not originally described by Thorne, 1935)). The position of the excretory pore, and the absence of basal thickenings on the stylet in both males and fernales and the blunl; rounded terminus of the fe-

Fig. 2. Bursaphelenchus keuini n. sp. A : Adult cephalic region and esophagus (lateral) ; B-D : Male Tai1 (ventral) ; E : Face view ; F : Female body, posterior region (lateral) ; G : Male Lail (lateral) ; 1-1 : Dauer (JIII) tail (lateral) ; 1 : Dauer (JIII) cephalic region and esophagus (lateral).

Revue Nhna to l . 7 ( 2 ) : 177-187 (1984) 181

R.M. Giblin, J.L. Swan h H.K. K a y a

male tail are characters that differentiate B. talonus from B. keuini n. sp. and B. poligraphi.

B. kevini n. sp. and B. poligraphi males can be separated by tail length ; B. keuini n. sp. m,ales have longer tails (46-92 Pm) than B. poligraphi (25-32 Pm, see Rühm, 1956). Also B. keuini n. sp. males possess nine ventral pre and postanal papillae (see description), whereas according to Rühm (1956), B. poligraphi males possess six papillae. B. keuini n. sp. females possess paired, four pronged structures a t the junction of the uterus and postvulval sac (Figs 2F & 5A), while B. poligraphi females apparently do not. B. keuini n sp. and B. poligraphi females can also be differentiated by the position of vulva 69-72% and 76-84%, respectively. Furthermore, B. keuini n. sp. and B. poligraphi can be differentiated by measurements of dauer juveniles from their insect h0st.s. B. keuini n. sp. dauers are much larger (longer and wider) with much longer tails (L = 730-1180 Pm, W = 23-38 Pm, and tail = 93-113 Pm) than B. poli-

graphi dauers (L = 300-390 pm, W = 13-14 Pm, and tail = 24-32 Pm, see Rühm, 1956). Dauers of B. keuini n. sp. are closely associated with their bee host and are dispersed in the bursa copulatrix and common oviduct of the female (Fig. 5B) or the penis of a male halictid bee whereas dauers of B. polygraphi and probably B. talonus are carried underneath the elytra of bark beetles (Rühm, 1956).

Life cycle of Bursaphelenchus keuini n. sp.

EGG

Ten eggs containing 52 juveniles, just prior t o eclosion, were measured. The length was 83-133 pm (106 & 17) ; W was 37-60 Pm (47 f 8), and the L/W ratio was 2.0-2.5 (2.3 -J= 0.2). The first molt (51-52) occurred within the egg. The shed cuticle of the J I was seen as a small cap on the head of the 52 within the egg (Fig. 3B). Eggs took longer than 24 h to hatch.

Fig. 3. Bursaphelenchus kevini n. sp. Life stages (lateral view). A : 52 ; B : Egg, with 52 enclosed within 51 cuticle ; C : 52 gonad ; D : Male 52-53 midbody and tail ; E : Male 52-53 gonad ; F : Female 52-53 midbody and tail ; G : Female 52-53 gonad ; I-1 : Male 53-54 midbody and tail ; 1 : Male 53-54 gonad ; J : Female 53-54 midbody and tail ; K : Female 53-54 gonad.

182 Revue Nimatol. 7 (2) : 177-187 (1984)

Bursaphelenchus kevini rz. sp. (Aphelenchida : Aphelenchoididae), an associate of bees in the genus Halictus

SECOND-STAGE JUVENILES

Before and after eclosion, the 52 had a blunt tail and a four-celled .genital primordium (Figs 3A-C). The germinal ce11 nuclei were larger and stained lightly compared with the somatic ce11 nuclei, as observed for B. seani (Giblin Sr. Kaya, 1983a). The lip region and stylet of the 52, 53, and 54 were morphologically similar to the adult. The tail shape of the 52 was blunt compared to the uniformly conoid tail of the 53, 54 and adults (Figs 3A, D, F, H, J ; 4A, B). The second molt began about 48 h after eclosion. Just prior to the second molt, the cells of the genital primordium of both sexes began to divide and a spicule primordium became visible in males. The gonad of the late intermolt (52-53) usually consisted of four germinal cells (range = 3-6), seven somatic cells

(range = 6-11), and two cap cells (Figs 3D-G). So- matic ce11 proliferation was posterior in females and anterior in males as observed by Giblin and Kaya (1983a) for B. seani.

THIRD-STAGE JUVENILES

J3s were not observed until 72 h after eclosion. Germinal and somatic cells were dimcult to differentiate with acetic orcein staining during the third- stage. The male gonad of 53s continued to grow anteriorly and cells of the spicule primordium divided several times. The female gonad grew posteriorly a t the beginning of the third-stage. At the end of the third-stage, the cells in the posterior third of the female gonad divided, creating a darkly stained region slightly wider than the rest of the gona,d (Figs 3J-K).

/ I I

I r

l I

F

Fig. 4. Bursaphelenchus keuini n. sp. Life stages (lateral view). A : Male J4-adult midbody and tail ; B : Female J4-adult midbody and tail ; C : Male J2-JI11 ; D : Male J2-JI11 gonad ; E : Female J2-JI11 ; F : Female J2-JI11 gonad.

Revue Némafol. 7 (2)": 177-187 (1984) 183

R.M. Giblin, J.L. Swan & H.K. Kaya 7 ..

This bulge of cells eventually differentiated into the uterus' and postvulval sac. The third molt (53-54) began a t 96 h. Bath the male and female gonads continued to lengthen anteriorly throughout the molt (Figs 3N-10. In some males the gonad began to turn 1800.

FOURTH-STAGE JUVENILES

Fourth stage juveniles were first observed a t 120 h. During the fourth stage the male gonad made a complete 1800 turn, and both ends of the gonad grew rapidly as observed for B. seani by Giblin and Kaya (1983~). The female gonad lengthened slowly. Ven- tral chord nuclei proliferated and enlarged in the vicinity of the bulge. Some of t.he ventral chord nuclei evaginated into the bulge to form the vagina. Before

the late JQadult iatermolt, the vas deferens joined with the rectum to form the cloaca, and the spicule was formed (Fig. 4A). In the late female intermolt the cuticularized vagina became evident and the gonad differentiated into the ovary, oviduct, uterus and postvulval sac. The paired, pronged structures, opposing the vagina at the junction of the uterus and postvulval sac were visible during the late internlolt (Fig. 4B). The gonad continued to elongate in both sexes.

ADULTS

Adults were first observed a t 144 h. Adult males and females increased in size after the molt as indi- cated by comparisons of J$-adult, intermolts (Tab. 2,) with adults (Tab. 1). The life cycle for B. keuirzi from

Fig. 5. (Photomicrographs). A : Female Bursaphelenchus kevini n. sp., four pronged structures at junction of uterusland postvulval sac (ventral) (bar = 10 Pm) ; B : Reproductive organs of Halictus Zigatus infested with dauer juveniles (JIIIs) of B. keuini n. sp. (ventral) (bar = 100 Pm). C O : common oviduct; C S : cuticular structures ; D G : Dufour's gland ; N : nematodes ; OV : ouary ; PS : poison sac ; S : sting.

184 Reuue Nénzatol. 7 ( 2 ) : 177-187 (1984)


52-52 on M . fructicola took eight days a t 250. This is slow compared with B. seani (Giblin & Kaya, 1983a) and B. xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 (= B. lignicolus) which went from 52-52 in less than five days a t 250 (Mamiya, 1975).

DAUER JUVENILES (JIII)

52s of B. kev in i n sp. were observed molting to the JI11 stage in older cultures, as observed for B. seani by Giblin and Kaya (1983a). The gonad of the J2-5111 intermolt which had two germinal cells (range = 2-5) and six somatic cells (range = 5-10) (n = 12) (Figs 4D & F) was very similar to the gonad of the 52-53 intermolt (see life cycle above. 5111s from reproductive female Halictus had two or four germinal cells (range = 2-4) and ten somatic cells (range = 7-10) (n = 11). Gonad length was not significantly different (P > 0.05) between 52-53, 5111s isolated from female bees and 5111s harvested from cultures (Tab. 2 ) . Gonads from these stages were significantly smaller (P < 0.05) than the gonads of the 53-54 and 54-adult stages. 5111s could be sexed like normal 52-53 intermolts by the direction of somatic ce11 proliferation in the gonad and by the presence or absence of a spicule primordium.

The 5111 was morphologically distinct from the 53. The 5111 had a dome-shaped head, the stylet, esophagus and intestine were indistinct (Fig. 2I), and

the tail in both sexes was significantly longer (P < 0.01) (Fig. 2H) than the tail of 52-53, 53-54, and J4- adult intermolts (Tab. 2). In older cultures, the long tail of the 5111 was often bent back upon itself, inside the unshed cuticle of the 52 (Figs 4G & E).

5111s from the reproductive tracts of overwintered adult female Halictus and laboratory cultures were morphologically identical. However, there were significant differences (P < 0.01) between the morphometrics of JIIIs from cultures and from overwintered Halictus (Tab.2). 5111s from the reproductive tracts of overwintered female Hal ic tus were significantly longer (P < 0.01) than cultured 5111s or any of the other intermolt stages (Tab.2). 5111s from cultures were significantly thinner than al1 other stages, including 5111s from overwintered Hal ic tus (P < 0.01) (Tab. 2 ) . The esophagi of JIIIs were significantly longer (P < 0.01) than the esophagi of other stages (Tab. 2) . 5111s from overwintered Hal ic tus have significantly longer tails (P <: 0.01) than JIIIs from culture (Tab. 2).

Giblin and Kaya (1983~) reported for B. searzi tha t there were no significant differences in body or gonad length (P < 0.05) between J2-JI11 and J I I I - J~s from cultures and 5111s from the oviducts of the bee, Anthophora bomboides stanfordiana Cockerell. They also reported that culture produced 5111s of B. seani could be subcultured on fresh M. fructicola mycelia. Conversely, JIIIs of B. kev in i n. sp. from the bursa

Table 2

Cornparisons of the Morphometrics of lntermolts and.3lIls of Dursaphelenchus n. sp.

Nematode Stage 52-53 SC%

52-53 female

JI11 JI11 JI11 JI11 53-54 54 adult 54 adult male

53-54 female

origin Culture Culture male female Clale

Culture Culture Bee female male Culture Culture Culture Culture Bee

fernale Clale

n 10 10 10 10 10 10 10 10 10

L (rm) k . D . 540DE t 28 530E t 28 696B f 63 1374~~ t 55 930A r 105 930A f 102 640’” t 73 590CDE t 85 710’ t 97 720’ f 60 (range) (480-580) (490-580) (620-820) (600-770) (820-1180) (730-1100) (510-710) (460-700) (600-910) (630-830)

Body dism.(pm) 30BC t 4 29’ t 2 22’ t 2 (28-37)

21D f 2 33ABc f 3 3ZBc t 5 30BC 1 5 29’ f 7 3? t 8 3gA f 7 (25-33) (17-25) (17-24) (28-38) (23-38) (22-37) (20-40) (22-48) (27-50)

10

E* 86” f 4 78’ f 6 133AB f 18 111’ t 24 120A t 3 123& t 8 93’ t 7 90’’ f 10 92” t 10 94’ 5 6 (83-95) (67-87) (100-153) (87-150) (116-123) (113-137) (83-103) (73-103) (73-107) (87-107)

Tai1 (pm) 52’’ t 5 4ECD ? 8 84’ t 8 81’ t 4 105A f 8 105A t 10 4ECD t 10 4gCD t 9 46’ t 8 57’ f 7 (43-62) (37-60) (73-93) (73-87) (93-113) (93-113) (33-67) (35-67) (35-57) (47-67)

Canad lgth.(um) 24B f 3 20’ t 4 21B t 3 2ZB t 4 , (19-28) (15-28)

27’ t 7 (17-25) (17-28)

25’ f 6 86’ f 19 76B t 16 296A t 120 351A f 85 (17-40) (13-33) (52-110) (53-103) (140-560) (230-500)

a 18A t 3 (15-24) (16-22) (26-38)

3ZA f 5 (25-43) (24-38) (25-38) (17-23)

21’ f 3 20B t 3 19B t 3 (17-25) (17-27) (15-23)

lgA f 2 33A f 4 28A t 4 29‘4 f 4 21’ t 2

f 10.6’ f 0.9 11.2O t 2.4 8.3E f 0.8 8.3E t 0.5 8.gE t 0.7 8.gE f 1.1 13.6’ t 1.9 12.lBCD 2 1.2 15.EA t 1.8 12.8BC5 1.0 (9.4-12.3) (8.8-14.6) (6.7-9.5) (7.2-8.9) (8.1-10.1) (7.3-11.3) (10.4-16.9) (10.0-13.5) (13.5-18.7) (11.7-15.3)

*E - distance from anterior end to,ocsophago-intestinal junction. overwintered Halictua f a r i n o ~ l ~ f o l l w e d by differant upper case lettirs are significantly different ( P L 0.01) according tn a (Nematades were eultured onMonilinia fructicolaon PDA; nematodes from bees were reCOVRred from the bursa capulatrix and cornmon oviduct of an

Duncan’s Mult-g-

Revue Nèmatol. Y (2) : l Y Y - 1 8 Y (1984) 185

R.M. Giblin, J.L. Swan di. H.K. Kaya

copulatrix and common oviduct of an overwintered Halictus were much larger than JIIIs produced in culture. Also, J I I I s of B. kevini n. sp. from cultures could not be subcultured directly ont0 fresh M . fructicola mycelia (Giblin, unpub. obs.) and probably need some host contact to complete their life cycle. JIIIs of B. keuini n. sp. migrate into the reproductive tracts of adults of Halictus prior to emergence of the bees from their brood cells and overwint>er inside mated female bees from late summer to early spring (Giblin & Kaya, 1983~). B. keuini n. sp. dauer juveniles may sequester compounds from the bursa copulatrix or common oviduct of the worker or the overwintering female of Halictus, but more work is needed to verify this hypothesis.

Fungal hos ts

B. keuini n. sp. did not grow or reproduce on most of the fungi tested, including B. bassiana, A. apis, or B. cinerea, and two species of Fusarium isolated from the brood cells of H . farinosus. B. kevini n. sp. did achieve populations of greater than 110 O00 nematodes/plate within six weeks on M. fructicola and growth and reproduction (populations of greater than 2 O00 nematodes/plate) were recorded on one of the Fusarium spp. isolated from the bee brood cells. These results indicate the B. keoini n. sp. is fastidi- ous when compared with other species of Bursaphe- lenchus, which appear to have relatively wide fungal host ranges (Giblin & Kaya, 1983b).

Comparison of biometr ics of B. keuini n. sp.

Comparisons of biometrjcs of the three different populations and type specimens of B. kevini n. sp. are presented in Table 1. Glycerol processed type specimens from eighteen day old cultures from Davis were significantly smaller (P < 0.01) (female L, and male and female W, E, and tail length) and ratios (a, b, c) were significantly larger (P < 0.01) than specimens collected and measured in water mounts from any of the locations at 14 and 28 days (Tab. 1). Similarly, Stynes and Bird (1980) reported tha t infective larvae of Anguina agrostis (Steinbuch, 1799) Filipjev, 1936, killed and measured in water mounts, were significantly larger than those nematodes processed into glycerol via the slow method. In general, the biometrics for B. keuini n. sp. from a given location and for different culture times were similar, although 28 day old cultures often produced smaller adults than 14 day old cultures (Tab. 2). Ishibashi and Kondo (1977) reported no significant differences in the measurements of B. xylophilus (=B. lignicolus) cultured for different length of time (30-90 days).

Biometric differences among the three populations of B. keuini n. sp. were not as great as the differences between glycerol processed us. water mounted and measured nematodes. However, females from Parma had significantly longer stylets (P < 0.01) than females from Davis and Newberg. Variation in the biometrics among populations of nematodes is common. For instance, Baujard (1981) reported variation in the biometrics of Laimaphelenchus penardi (Steiner, 1914) Filipjev & Sch. Steck., 1941 from different populations. Tarjan and Baeza-A. (1982) did a cluster analysis on the biometrics of two populations of B. poligraphi and concluded that the intraspecies variation was greater for B. poligraphi than the interspecies variataion for some members of Bursaphelenchus. The biometrics presented in this paper should represent much of the variation present in contemporary populations of B. keuini n. sp. How- ever, more variation may be observed when culture times are extended for longer than 28 days and/or when different fungal hosts are used.

ACKNOWLEDGEMENTS

We thank Drs. A. R. Maggenti and R. W. Thorp for reading the manuscript, Dr. A. M. Golden for lending us type specimens of Bursaphelenchus talonus for examination and comparison, and Dr. T. Matsumoto for assistance in the isolation and identification of fungi from Halictus farinosus brood cells.

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Bursaphelenchus kevini n. sp. (Aphelenchida : Aphelenchoididae)