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Host Specificity of Microsporidia (Protista: Microspora) from EuropeanPopulations of Lymantria dispar (Lepidoptera: Lymantriidae)

to Indigenous North American Lepidoptera

L E E L L E N F. S OLTER,* J O S E P H V. MADDOX,* AND MI C HA E L L . MC MANUS †

*Center for E conomic En tomology, I l l inois N atur al H istory Sur vey, 607 E. Peabody D ri ve, Champai gn, I l l i nois 61820; and † USDA Forest

Servi ce, Nort heastern Center for F orest H ealt h Research, 51 M il lpond Road, H amd en, Connecti cut 06514

Received J uly 3, 1996; accepted December 16, 1996

Results of traditional laboratory bioassays may not

accurately represent ecological (field) host specificity

of entomopathogens but, if carefully interpreted, maybe used to predict the ecological host specificity of

pathogens being considered for release as classical

biological control agents. We conducted laboratory

studies designed to evaluate the physiological host

specificity of microsporidia, which are common proto-

zoan pathogens of insects. In these studies, 49 nontar-

get lepidopteran species indigenous to North America

were fed five biotypes of microsporidia that occur in

European populations of L y m a n t r i a d i s p a r but are not

found in North American populations of L . d i sp a r .

These microsporidia, M i c r o sp o r i d i u m sp. from Portu-

gal, M i cr osp or i di u m sp. from Romania, M i cr ospo- r i d i u m sp. from Slovakia, N o sem a l y m a n t r i a e, and

E n d o r et i c u l a t u s sp. from Portugal, are candidates for

release as classical biological control agents into L .

d i s p a r populations in theUnited States. The microspo-

ridia produced a variety of responses in the nontarget

hosts and, based on these responses, the nontarget

hosts were placed in the following categories: (1) no

infection (refractory), (2) atypical infections, and (3)

heavy infections.E n d o r et i c u l a t u s sp. produced patent,

host-like infections in nearly two-thirds of the nontar-

get hosts to which it was fed. Such generalist species

should not be recommended for release. Infectionscomparable to those produced in L . d i s p a r were pro-

duced in 2%of thenontarget hosts fed M i c r o sp o r i d i u m

sp. fromPortugal, 19%of nontarget hosts fed M i c r o sp o -

r i d i u m sp. from Romania, 13% fed spores of M i c r o sp o -

r i d i u m sp. from Slovakia, and 11%of nontarget species

fed N . l y m a n t r i a e. The remaining nontarget species

developed infections that, despite production of ma-

ture spores, were not typical of infection in L . d i s p a r .

We believe it is very unlikely that these atypical infec-

tionswould behorizontally transmitted within nontar-

get insect populations in the United States. r 1997

Academic Press

K EY WORDS:entomopathogen;classical biological control; host range; ecological host specificity; physiologi

cal host specificity; N o sem a l y m a n t r i a e; V a i r i m o r p h a

E n d o r et i c u l a t u s .

INTRODUCTION

A ba sic premise of classical biologica l contr ol is t hath e ta rget pest is brought under some degree of contr ow hile indigenous nonta rget organ isms ar e not a ffectedThe introduced biological control agent must be relatively host specifi c in order to achieve this goal. Am a jordilemma in classical biologica l contr ol programs is th athe ecological host range of an exotic biological controagent can be unequivocally determined only after release of the agent into t he environment. Thereforephysiological host range data produced in laboratorys t u d ie s h a v e b e en t ra d i t ion a l ly u s ed t o p red ict t h eecological host range of an organism. These studies arera re ly d e s ig n e d t o a s s e s s t h e t y p e , q u a l i t y , o r b a s icmecha nisms of host specifi city, yet this specifi c information is needed to predict the ecological host range ofnonindigenous organisms and estimate the likelihoodthat organisms could expand their host range in a newha bita t (Federici a nd Ma ddox, 1996). B a sic know ledgabout host specificity is fundamental, not only to re-s olve s a fet y a n d reg u la t ory iss u es (M a d d ox , 1 992Maddox et a l ., 1992), but also to develop sound ecological and evolutionary hypotheses about organisms usedin biological cont rol progra ms.

S t u d ies h a v e s h ow n t h a t m a n y i ns ect p a t h og en sh a v e a b r oa d h os t r a n g e w h e n u n us ua l m et h od s oexposure a re used under laboratory condit ions, buw hen more realistic methods are used, fewer nonta rgeh os t s a re s u scep t ib le t o in fe ct ion b y t h e p a t h og en(Fisher a nd S a nborn, 1962; U ndeen an d Ma ddox, 1973Fournie et al., 1990; H a jek et al., 1995). C onditions inthe laboratory are often ideal for infection because the

J O U RN A L O F I N V ERT EBRA TE P A TH O LO G Y 69, 135–150 (1997)

A RTI C LE N O . I N964650

135 0022 -2011/97 $25.0Copyright r 1997 by Academic Pres

All rights of reproduction in a ny form reserved


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host receives a ma ximum or optima l dose of the pat ho-gen. Exposures of this kind do not take into accountecological fa ctors such as t hresh old numbers of hosts ornontarget hosts , spatial or temporal overlap betweent h e h os t a n d n on t a rg et p op u la t ion s (On s t a d et a l . ,1990; Onstad, 1993), or the distribution and survival ofinfectious forms in the environment (Kra mer, 1973;Maddox, 1973; J effords et al., 1989). Nevertheless, if

the biology, ecology, a nd ta xonomy for a pat hogen a ndits natural host in the native range are known, physi-ological host specifi city test ing can supply the addi-tional information needed to make predictions regard-ing th e ecologica l host ra nge of the pat hogen w hen it isi nt r od u ced i nt o a n ew h a b it a t (C a t e a n d M a d d ox ,1994).

We applied t hese considera t ions to dat a obta inedfrom host range studies of several isolates of microspo-ridia (Protista) found in European populat ions of thegypsy moth, L y m a n t r i a d i s p a r . Foreign explora tionsan d fi eld studies, primarily in central E urope but also

in Portugal, recovered 11 distinct isolates of microspo-ridia from L . d ispar populations (Weiser, 1964; McMa-nus et al., 1989; Maddox and McManus, unpublishedcorrespondence). The isolat es probably represent a tleast six different species, but sufficient information tocla ssify th e undescribed species is not currently a va il-a ble and w e will refer t o all of th e isolat es as biotypes inthis paper. F ive of t hese biotypes, Microsporid ium sp.,Portugal isolate (MP), Microsporid ium sp. , Romaniaisolate (MR), M icrospori d i um sp., Slovakia isolat e (MS ),N osema l ymant r iae, Czech Republic (NL), an d E ndore- t i c u l a t u s sp., P ortuga l isolate (EP ) are currently being

considered for introduction into the United States asclassica l biological control agents of L . d i s p a r . Tw obiotypes, MP and EP, were experimentally released inisolat ed woodlots to evalua te t heir potentia l to cycle inL. d ispar popula tions. The MP biotype w a s successfulin cycling for at least 3 years (J effords et al., 1989). Nonaturally occurring microsporidia have been recoveredfrom L . di spar populat ions in North America (Ca mpbelland Podgwaite, 1971; Podgwaite, 1981), and host speci-fi c i t y in fo rm a t io n is n o w b e in g o b t a in e d fo r t h e fi v ebiotypes of microsporidia a s a pr erequisit e for consider-a tion of their perma nent int roduction int o North Ameri-

ca n L . d ispar populations.In previous unpublished studies, we examined the

development and progression of infection of the fivemicrosporidian biotypes in L. dispar. This progressionbegins when microsporidian spores, ingested by a host,germinat e in the midgut. Apolar fi lament extrudes an dinjects the internal contents of the spore (the sporo-plasm) into h ost midgut cells . The injection of thesporoplasm into the host midgut cells constitutes aninvasion of the host; by definition, this is an infection.The injected a moeboid form reproduces vegeta tivelya n d in o n e s p e c ie s , E P, fo rm s s p o re s in t h e m id g u t

tissues only (Zwölfer, 1927; Spra gue, 1977). Thesspores are ma tur e, infective, and relat ively resistan t toe nv iron m en t a l con d it ion s ou t s id e t h e h os t a n d a rereferred to a s ‘‘environment a l spores’’ (‘‘MC’’ spores oIwano and Ishihara, 1991). Four of the microsporidianbiotypes produce a distinct primary sporulation stage‘‘prima ry spores’’ (‘‘FC’’ spores of Iw a no a nd Ishiha ra19 91), k n ow n for on ly a few ot h e r m icros porid ia n

species (Avery and Anthony, 1983; Fries et al., 1992Iw a n o a n d Is h ih a ra , 1 9 9 1 ; Iw a n o a n d Ku rt i i , 1 9 9 5 )These spores a re bounded by a very t hin exospore, ha vea short polar fi lament, a nd a re not infective to susceptible hosts per os because they do not survive outsidet h e t is s u e s o f a n in fe c t e d h o s t . Pr im a ry s p o re s a reproduced in th e midgut cells betw een 24 and 60 hr aft elarvae ingest spores. The target t issues are invadeda f t e r t h e s e p rim a ry s p o re s g e rm in a t e in t h e m id g u tt issues. In the target t issues, principally the fat bodya n d s a l iv a ry g la n d s fo r L . d ispar, the environmentas pore s a re form ed ; t h e se b ecom e t h e in ocu lum for

transmission of the microsporidia in the host populat i on . An a t y p ica l p ri ma r y s por u la t i on s t a g e i n t h emidgut t issues could prevent the development of apat ent, horizonta lly tra nsmissible infection in a nonta rget host.

B ecause microsporidia evolve with an d a re a daptedto a specific host or host group, we conjectured thatmicrosporidia n infection in nonta rget hosts is differenfrom infection in t he na tur a l host. This study compar esin fe ct ion s b et w e en n on t a rg e t h os t s a n d t h e n a t u rahost, L . d i spar, and addresses the following questions(1) Can the microsporidium infect the nontarget host?

(2) I f s o, w h a t t i ss ues a r e i nf ect ed ? (3) D oes t h esequence in which tissues become infected differ? (4Are there differences in the intensity of infection? (5Are ma tur e, environmenta l spores produced? (6) Howdoes t he development of the pathogen in nonta rgehosts differ from development in L . d i s p a r ? (7) If a nydifferences occur, how can the information be used topredict th e ecologica l host ra nge of the pa thogen fromthe phy siologica l host r a nge?

MATERIALS AND METHODS

Five biotypes of microsporidia, all of which wererecovered from fi eld popula tions of L . d ispar in Europewere tested for infectivity in forest Lepidoptera nativeto the north eastern U nited St a tes. Most of these nonta rget Lepidoptera a re sympat ric w ith L . dispar.

M icrospor id i an biotypes. Th e m icro sp orid ia w etested in these st udies a re described in Ta ble 1. All ot h e i sol a t es w e r e s t or ed i n l iq u id n it r og en i n ou rlaboratory. A portion of the small subunit rRNA genefrom all fi ve biotypes ha s been sequenced a nd MP, MRM S , a n d N L a p p e a r t o b e c lo s e ly re la t e d ( M . Ba k e rpersonal communicat ion). Spore morphology, differ

136 SOLTER, MADD OX, AND MCMANUS


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e nce s in l ife cy cles , p a t h og en es is, a n d h os t ra n g e ,however, confi rm t ha t t hese biotypes, as w ell a s EP, ar edistinctly different from one another.

Production of spor es. The microsporidia used inthese studies were produced in laboratory-reared L .dispar larva e using the following procedures. Ea chmicrosporidia n biotype wa s removed from stora ge an d100 µl of a spore suspension were sprea d on th e surfa ceof meridic diet (B ell et al., 1981) in 150-ml pla st ic cups.The spore concentrations were predetermined for each

biotype to infect 80–100%of th e lar va e wit hout produc-ing ea rly morta lity. Third insta r la rva e, 10 lar va e/cup,were fed a pproximat ely 106 spores of NL, 107 spores oft h e Microsporid ium biotypes, or 108 spores of EP. Thecu ps w e re h eld in e n viron m en t a l g row t h ch a m b ers(24.5° 6 1°C , 16 hr light /8 hr da rk) for 15 to 20 da ys.Th e la rv a e w e re d iss ect e d b efore p up a t ion a n d t h einfected t issues were harvested and homogenized intap water in a glass t issue grinder. The homogenatew a s fi l t e re d t h ro u g h t ig h t ly w o v e n n y lo n c lo t h in t o15-ml centrifuge tubes and then was centrifuged forapproxima tely 15 min at 3200 rpm. The supernata nt

wa s disca rded a nd t he spores were resuspended in 1–2ml cold ta p w at er. A small a liquot of each suspensionwas diluted and spores were counted with a bacteriacounter. The remaining spore suspension wa s thenmixed w ith a n equa l par t of glycerol, poured int o 1-mlplast ic cryovials , and placed in liquid nitrogen. Foreach set of bioassa ys, a cry ovial of spore suspension wa sremoved from storage and used to produce fresh sporesby infecting L . d i s p a r la rv a e . T h e s p o re s w e re h a r-vested, clea ned a s described a bove, an d th e fresh sporeseither used immediately or frozen in liquid nitrogenwithout glycerol for use within 2 months of production.

Spore concent r ati ons. B a sed on prelimina ry testingfor L . d i sp ar susceptibility to each microsporidianbiotype, two spore concentrations were chosen for testsof nonta rget h ost susceptibility to t he pa thogens. F orall fi ve biotypes, a concentrat ion of 103 spor es/µl w a schosen to test for lower susceptibility or hypersensitiv-ity reactions in the nonta rget species. Lepidopteranmicrosporidia we ha ve tested ra rely cause premat ure

m or t a l it y w h en t h e n a t u ra l h os t is f ed t h is s por econcent ra tion by either spreading 40 µl on th e surfa ceof meridic diet in a 30-ml cup (900 mm 2) or dippingfoliage cut to the sa me approximate surface area in aspore suspension (Ma ddox, 1973; personal observations). A concentr a tion of 105 spores /µl of ea ch of t hes emicrosporidian biotypes produces infections in 80–100% of L . d i s p a r larvae fed spores and is probablyconserva tively high for a fi eld situa tion.

R ea r i n g L . d i s pa r. E g g m a s s es o f t h e L . d i s p a r

str a in designat ed ‘‘New J ersey St a nda rd’’(NJ S td) were

obta ined from th e US DA/AP HI S L a bora tory a t Ot is AirForce Base (MA). Egg masses were placed in environm en t a l g r ow t h ch a m b er s t o i n it i a t e h a t c hi n g. Th ela rv a e w e re t ra n s fe rred t o fres h 15 0-m l d iet cu psa pproximately 50 la rvae/cup, a nd reared to t he t hirdsta dium. Infections produced in NJ S td verifi ed infectivi t y o f t h e m ic ro s p o rid ia , a n d t h e p ro g re s s io n o f t h einfections in NJ St d wa s compar ed to th e progression oinfections in susceptible nont a rget species in each tria l

Acqui si ti on and r ear i ng of nont ar get species. Nontarget Lepidoptera tested for susceptibility to L . d i s p a r

microsporidia (Ta ble 2) were r eceived from th e following s ources.

Dr. Da le S chweitzer (P ort Norris , NJ ) provided 36species of nat ive forest -dw elling moth s a nd 3 species obutterfl ies for t his s tudy. Larva e produced by tra ppedw ild - m a t e d fe m a le s w e re re a re d in c a g e s w it h h o splant ma teria l or on sleeved host plant s until shipmento this laboratory. Egg masses of several species werea lso shipped. Identifi cations were made by Dr. Schweitzer ba sed on a dult female morphology. After a rriva l inou r la b ora t ory, la rv a e w e re re a re d on n a t u ra l foodpla nt f olia ge (Ta ble 2)i n 150-ml pla st ic food cups. Fresh

leaves were supplied every 1–2 days, and 2-in. dentawicks were provided and moistened daily to increasehumidity in the cups and t o serve as a source of wa terI n s om e c a s es , l a r v a e w e r e i n t h e t h ir d or f ou r t hsta dium upon a rrival a nd were held at 4°C for severaday s unt il bioassa yed. One or tw o individuals of eachspecies w ere rea red to the fi na l larva l s ta dium, photographed, and then placed in 70% alcohol as voucherspecimens.

Th e De p a rt m e n t of N a t u ra l R es ou rce s Ca n a d a a tS a u lt S t e . M a rie, On t a rio, p rov ide d fou r s pe cies onontarget forest lepidopterans. These insects were

TABLE 1

Microsporidia from Europea n P opulations of L y m a n t r i a dispar Bioassayed against Native North American Lepidop-tera

I sola t e Or igin C ollect or

Micr o sp o r id iu m sp .(MP)

P or t ug a l, 1985 J eff or ds a n d M a dd ox(IN HS )a

Micr o sp o r id iu m sp .

(MR)

R om a nia , 1993 D u bois a n d M on t -

gomery (USFS)b Micr o sp o r id iu m sp .

(MS)S lov a kia , 1994 M a dd ox (I NH S ) a n d

M cM anus (U S FS )

Nosema lymantr iae c

(NL)Czechoslovakia, 1985 Weiser (Acad. of Sci-

ences, Prague)

Endoreticulatus s p.

(EP)

P o rt u ga l , 1985 J eff or ds a n d M a dd ox

(IN HS )

a Illinois Nat ura l History Survey.b U nited S t at es For est S er vic e, N or theast For est Exper iment S t a-

ion.c No se ma lyma n tr ia e Weiser, 1957, will be placed in the genus

Va ir imo r p h a (Madd ox, persona l correspondence).

137H O S T S P E C I F I C I TY O F M I C R O S P O R I D I A


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TABLE 2

Native North American Forest Lepidoptera Challenged by Microsporidia Found in EuropeanP opulations of the G ypsy Moth

S pecies t est ed Loca t ion /sour ce D iet /h ost pla n t a

ArctiidaeArctiinae, Phaegopterini

Eu chaeti as egle Cha mpaign Co. , IL A. su l l iva n t i i Hyphantr ia cunea b U n iv. K Y, L exingt on Mer idic d iet c

Geometr id aeEnn ominae, Angeronini

Euchlaena am oenari a Dow ne Town ship, NJ Q. alba Ennominae, Boar miini

Cleora sublun ari a Cumberla nd Co., NJ Q. alba Ennominae, Our a pter y gini

Eutralepa clemataria S tr a f for d , PA and Cumber land Co. , N J A. r u b r u m Prochoerodes transversata W. Nott ingha m Town ship, PA Q. alba

Tetracis cachexiata Dow ne Town ship, NJ Q . r u b r a Oenochrominae

Alsophil a pometaria Eld or a, N J P. ser oti na Lasioc ampid ae

Lasioc ampinaeMalacosoma americanum Wyt he Co., VA M a l u s s p.

M alacosoma di sstr ia b,d F or est r y C a n a da , On t a rio Mer id ic d iet e

LycaenidaeTheclinae, St rymonini

Incisl ia henrisi f Eld or a, N J I . opaca Ly mantr i id ae

Ly mantr i iniL yma n tr i a d isp ar b ,f U S D A, Ot is AF B , MA Mer id ic d iet e

L y m a n t r i a m a t h u r a J apan g Meridic diet e

Orgyiinae, Orgyiini

Dasychir a obliquat a h Cumberland Co., NJ Q. alba Dasychir a pini cola b,d For estr y Canad a, Ontar io P. str obus O r g yia a n t ig u a b,d F or est r y C a n a da , On t a rio Mer id ic d iet e

Orgyia definita Cumberla nd Co., NJ Q. alba

Orgyia leucostigma Por t N or r is , N J ; U r ba na, IL Q. alba Orgyi a pseudotsugata b,d F or est r y C a n a da , On t a rio Mer id ic d iet e

NoctuidaeAmphipyrinae, Amphipyrini

Amp h ip yr a p yr a mid o id es f Cumberland Co., NJ P. ser oti na Catoc al inae

Catocala gracil is Cumberla nd Co., NJ V. cory m bosum Catocala i l ia C h a t s w or t h , N J Q. alba Catocala m icronympha Cumberla nd Co., NJ Q. alba

Catod ali inaeZaleaeruginosa Cumberla nd Co., NJ Q. alba, A. rubr um

Cuculliina e, Xylenini

Ch aetagl ea ser i cea i Cumberland Co., NJ P. ser oti na Eu p si l ia ci r r ip a lea Cumberla nd Co., NJ P. ser oti na Eu p si l ia v in u len ta Cumberla nd Co., NJ P. ser oti na Eu p si l ia sp. C um ber la n d C o., NJ Q . r u b r a Lithophanegrotei f Cumberla nd Co., NJ a nd Egg H ar bor Township, NJ A. r u b r u m

Lithophanequerquera Cumberla nd Co., NJ A. r u b r u m L i th o p h a n e u n imo d a Cumberla nd Co., NJ P. ser oti na Psaphida resumens Cumberla nd Co., NJ Q. alba Seri caglea signata Cumberla nd Co., NJ P. ser oti na Suni ra bi colorago Cumberla nd Co., NJ P. ser oti na Xylotypecapax i Cumberland Co., NJ P. ser oti na Xystopeplus (Jodia) rufago Cumberla nd Co., NJ Q. alba

Had eninae, Had enini

Eg ir a a l ter n a n s Cumberla nd Co., NJ Q. alba O r th o sia a lu r in a Cumberla nd Co., NJ P. ser oti na Orth osia hi bisci Cumberla nd Co., NJ P. ser oti na Orth osia r evicta Cumberla nd Co., NJ Q. alba, A. rubr um

Notodontidae

Da ta n a min istr a J ohnson Co., IL Q. alba

H eterocampa umbr ata Dow ne Town ship, NJ Q . r u b r a



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s h ip pe d a s e gg s a n d w e re re a re d o n t h e a p p rop ria t e

meridic diet or host plant foliage.L y m a n t r i a m a t h u r a wa s collected in J apa n a s egg

m a s s es a n d la rv a e w e re re a red a t t h e U S DA /AP H ISLaboratory (Otis AFB, MA). Third-instar larvae weretra nsferred to the US DA/US FS Quara ntine Laboratory(Ansonia , CT), wh ere they w ere bioa ssa yed.

Thr ee species of nont a rget hosts w ere collected fr omvegetat ion in central I ll inois as first or second instarlarva e and were reared on host plant foliage. BecauseSphingidae were not represented in field collections,M andu ca sexta la rv a e fro m t h e c o lo n y o f Dr . S u s a nFahrbach, University of Ill inois Entomology Depart-

ment, were reared on art ificial diet for bioassays. Dr.Arthur Zangerl of the University of Illinois EntomologyDepartment provided Pa pi l i o pol yxenes aster i us, wh ichwere reared for three generations in the laboratory. H y- phant ri a cunea were obta ined from the colony of Dr. Gera ldNordin a t the U niversity of Kentucky a nd w ere rear ed inthe laboratory on meridic diet. M alacosoma amer icanum la rva e were fi eld-collected in Wyt he Count y, Virginia .

Bioassays. B ioa ssa ys were conducted in four sets ofexperiments over a 3-year period. The EP, MP, and NLi s o l a t e s w e r e t e s t e d i n t h e fi r s t t h r e e s e t s , M R w a stested in the second a nd third sets , an d MS w a s tested

in t h e fi n a l s e t . F o r t h is re a s o n , a n d b e c a u s e n o t a l

nontarget host species were available for each set ofexperiments, not all biotypes of microsporidia weretested in all of the nontarget hosts . The numbers oindividua ls of each nonta rget lepidoptera n species testeddepended on the number received from the varioussources and our success with rea ring th e anima ls to thet h ird s t a d iu m p rio r t o t e s t in g . Ou r e x p e rie n c e w it hmicrosporidia n infections of na tur a l hosts in t he laborat o r y s u g g e s t s t h a t t h i r d i n s t a r l a r v a e a r e g e n e r a l l ysusceptible to infection but tha t prema ture morta litycaused when some species of microsporidia are fed ah igh d os es , occu rs les s f req u en t ly in la t e r t h a n in

earlier s tadia. Feeding spores during the third instaroffered th e best opport unit y t o observe th e full extent odisease in su sceptible host species. In ideal sit ua tionswe tested each microsporidian biotype in 40 larvae oeach nonta rget host, 20 lar vae a t a low concent ra tion ospores, and 20 larvae at a high concentrat ion. Whenfewer specimens of each nontarget species were availa ble, we reduced the number tested t o 10 larva e/sporeconcentr a tion/microsporidia n biotype. When t he n umbers of nonta rget hosts ava ilable were less tha n neededto test all microsporidian biotypes, the microsporidiawere ranked in order of interest as possible biologica

TABLE 2—Continued

S pecies t est ed Loca t ion /sour ce D iet /h ost pla n t a

N y mphalid aeA patur inae

Asterocampa celt is P ort Norris, NJ C. occidentali s Asterocampa clyton P ort Norris, NJ C. occidentali s

Papil ionid aePapil ioninae

Papil io polyxenes asteri us b

U niv. of IL, U r ba na, I L A. petr oseli num Psy c hid aeOiketicinae

Thyridopteryx ephemeraeformis d Cha mpaign Co. , IL Q. alba, A. rubr umS a t u r n i i d a e

Hemileucina e, H emileucini

Hemileuca maia Cumber land Co. , N J and S tr a f for d , PA Q. alba S atur niinae, S atur niini

Actia s lu n a Dow ne Town ship, NJ L . st yr aci flu a Antheraea polyphemus Cumberla nd Co., NJ Q . r u b r a

S phingid aeSphinginae, Sphingini

M and uca sexta b,d U n iv. of I L, U r ba n a , I L Mer id ic d iet j

Note. All larva e were exposed to microsporidian spores a t t hird inst ar except d , f , a n d i .

a Acer ru bru m, A piu m petr oseli num , Asclepias syriaca, Celt is occidentali s, Il ex opaca, L iqu id amber styr aciflua, M alu s sp., Pin us strobusPru nus seroti na, Quercus alba, Quercus rubr a, Vaccin iu m corymbosum .

b Reared from la boratory colony insects.c Yearian et al . (1966).d Exposed at second inst ar.e Bell et al . (1981).f Exposed at third and fourth instar.g Via U SD A, Otis La boratory, MA.h Accidenta l ma tin g of offspring of fi eld-collected female.i Exposed at t hir d , four th, and fi f th instar . j B ell a nd J oachim (1976).



6/16

control agents based on earlier s tudies of laboratoryhost range, and lower ranking species were not bioas-sayed. The order of ranking was as follows: MP, MR(tested only in the second sea son), NL, a nd E P. MS w a son ly t e s t ed in t h e fi n a l s ea s on a n d in n o n t a rg et h os tspecies in w hich the fi rst four microsporidia n biotypeshad been previously bioassayed, with the exception ofEupsi l ia v inulenta.

To feed microsporidian spores to larva e reared onhost plants, pieces of host plant foliage, cut to approxi-ma tely 900 mm2, were rinsed in tap wa ter and a gitat edi n e it h er a 103 or 105 spore/µl solution to coa t thes u rfa c e w it h s pore s. L ea v e s t h a t w e re h y d rop h ob icbecause of waxy or ha iry surfaces w ere dama ged w ithforceps so tha t the solutions w ould a dhere to the leafs u rfa c e . T h e t re a t e d p la n t m a t e ria l w a s p la c e d in t oempty 150-ml cups and larvae of one nontarget specieswere placed onto the ma teria l. Not more tha n 15 lar vaewere fed per cup. The la rva e were a llowed t o feed untilthe leaves were completely devoured or skeletonized,

typically 4 t o 36 hr. After feeding on t he tr eat ed leaves,fresh foliage wa s a dded every 1–2 days. F or L . d ispar larva e and the nontar get species tha t were fed meridicdiet, 40 µl of one of t he suspensions w a s sprea d on t hediet surfa ce with the bulb end of a P ast eur pipette a ndla rva e w ere pla ced in 30-ml cups, 10 la rva e/cup. After2 – 3 d a y s o f fe e d in g , la rv a e w e re t ra n s fe rre d t o u n -tr eat ed diet cups, 5 la rva e/cup.

Du rin g t h e c ou rs e of re a rin g, t re a t m e n t , a n d p os t -t re a t m e n t re a rin g , a l l in s ect s w e re m a in t a in ed on a nopen la bora tory bench w ith a consta nt tempera ture of24° 6 1° C. We did not a t tempt to a scertain t ha t a ll of

th e lar va e fed on the spore-coa ted lea ves, nor how muchof the mat erial wa s ea ten by individua l la rva e. Feedingbehavior was observed periodically and treated leaveswere eat en near ly completely, indicat ing t hat most ofthe larva e had fed.

B etween 10 a nd 20 la rva e of each nonta rget species,based on the number of individuals of each species thatw e re t re a t e d w it h s p o re s , w e re fe d c o m p a ra b le le a fpieces dipped in t a p wa ter or 40 µl ta p wa ter sprea d onthe surface of meridic diet. These individuals served ascontrols for naturally occurring diseases which mighthave been present in the nontarget species and pro-

vided a st a nda rd for the development ra te of uninfectedindividuals of ea ch nonta rget species under laborat oryconditions.

Tw o to 4 days a fter feeding spores, then a ga in a t 5 to10 days postfeeding, one or two larvae of each nontar-get species from t he tr eat ment gr oup fed 105 spor es /µlwere dissected a nd fresh tissue squa shes were mad e ina drop of invertebrat e saline of the midgut, Ma lpighia ntubules, salivary glands, fat body, and nervous tissues.The tissues were examined for the presence of primaryspores and other early developmental stages of micro-sporidia using 4003 pha se-cont ra st microscopy. When

extensive morta lity occurred in larva e fed h igh sporeconcent ra tions, a living lar va fed 103 spores/µl w a s a lsodissected and examined. All of the remaining larvaewere dissected prior to pupation, from 12 to 30 dayspostfeeding, an d t issues were examined microscopica l ly for t h e p res en ce of in fect ion . S e le ct e d t is su especimens showing typical and atypical responses tothe microsporidia were photographed under phase

contrast microscopy 31000. I n the fi rst set of experiments, Giemsa stains (Vavra and Maddox, 1976) werem a d e of t h e m id gu t t i ss ues of a l l d is sect ed l a r va eshowing early signs of infection (2 to 4 days postfeeding) to confirm the presence or the absence of earlydevelopmenta l forms of microsporidia . St a ins w ere alsoma de of the ta rget t issues for each microsporidiumbiotype in la te sta ge infections (.10 da ys postinfectionof all nonta rget hosts. Additionally, a ll larva e survivingup to 30 da ys w ith n o apparent infection were dissectedand t issue smears were stained and examined to confi rm t h a t m ic ro s p o rid ia h a d n o t in v a d e d t h e t is s u e s

For the remaining three sets of experiments, midguttissue sam ples of all t reat ed species were stain ed at 2 to4 days postfeeding. Giemsa stains were also made ofin fect e d t a rg et t is su es of rep res en t a t ive in d iv id u ala rv a e of e a ch h os t s pe cies . Th e re sp on s es of t h enontarget hosts to each microsporidian biotype werecompared with the progression of infection in L . d ispala rv a e .

RESULTS

Vegetative forms of all five biotypes of microsporidiaw ere detected in th e midgu t t issues of susceptible hosts48–60 hr postinfection w hen t issue smears w ere exa mined using pha se-cont ra st microscopy and G iemsa sta ining. Vegeta tive forms, prima ry spores, and germina tedp rim a ry s p o re s w e re s e e n in M P, M R , N L , a n d M Sinfections at 48–96 hr postexposure. Environmentaspores, wh en produced, were found in t issue squa shesbetween 4 an d 10 days postinfection. Cellular imm uner e sp on s es w e r e a l s o d e t ect e d i n t h e f r es h t i s su esqua shes of several nonta rget hosts.

We divided t he responses of the 49 nonta rget lepidopter a n host species to the microsporidia t ested into thr eebroa d ca tegories (Ta ble 3): (1) the host w a s refr a ctory(2) a typical infections occurred a nd few if a ny environmental spores were formed in t he nontarget host , a nd(3) heavy infections occurred in nontarget host . B eca u s e re sp on s es v a ried a m o n g in d iv id u a ls of e a chnonta rget species, th e response recorded for each hosspecies represents t he most a dva nced development of amicrosporidian biotype in one or more of the nontargethost larvae. Nontarget species in the refractory groupwere not infected (tissues examined under the microscope and stained with Giemsa contained no spores orvegetat ive stages), and the feeding behavior, growthand development of these larvae were comparable to



7/16

TABLE 3

Results of Exposure of Na tive North American L epidoptera ns t o Five Biotypes of MicrosporidiaFound in Gy psy Moth P opulat ions in E urope

Host species

M icrosp.(Por tugal)

(MP)

Microsp.(Romania)

(MR)

M icrosp.(Slovakia)

(MS)

N osema l y m a n t r i a e

(NL)

Endoret(Por tugal

( E P )

ArctiidaeE. egle R (30) — — — —H . cunea A (23) H (29) — H (39) H (47)

Geometr idaeEuc. amoenaria A (10) — — — —C. s ublunar ia R (9) — — — —Eut. c lem atar ia R (42) R (28) — A (41) H (24)P. transversata R (14) — R (30) R (9) R (6)T. cachexiat a R (15) — — — —A. pom etar i a A (16) — R (22) R (14) H (11)

LasiocampidaeM . am eric anum A (35) — A (38) H (17) —M . dis s tr ia A (42) A (34) — H (42) H (27)

LycaenidaeI . h enr i si R (7) R (11) — R (16) A (4)

Lymantr i idaeL. di s par N J S t d H (127) H (88) H (66) H (114) H (126)L . m a t h u r a A (13) H (8) — H (3) H (5)D. obl iquat a R (34) R (34) — H (27) R (21)D. pini c ola H (21) H (25) — H (29) H (26)

O. ant igua H (36) H (24) — H (37) R (36)O. definita A (34) H (21) — H (29) R (31)O. l eucosti gma H (49) H (30) H (33) H (43) R (44)O. pseudotsugata H (22) H (35) — H (37) H (27)

NoctuidaeA. py ram i doides A (44) R (61) R (22) A (31) H (27)C. grac i l i s R (15) A (11) — A (14) —C . i l i a R (22) — — — —C. m ic rony m pha R (12) — — — —Z. aerugi nosa R (6) — — — —Ch. sericea H (43) H (39) H (17) H (48) H (35)Eup. cir r i palea R (14) — — — —Eup. v inulenta — — R (5) — —E u p s i l i a sp. R (48) R (37) R (18) H (23) H (28)Li. grotei A (27) A (25) H (24) H (22) H (18)Li . querquera R (5) — — H (20) R (3)L i . u n i m o d a R (11) — — R (10) H (10)

Ps. resum ens A (8) — — — —

S. signata R (19) — R (38) R (15) R (16)Su. bicolorago H (15) — — H (17) R (12)X. capax A (49) H (26) H (33) H (31) H (26)Xy. rufago A (14) — — A (8) A (17)Eg. al ternan s H (13) H (11) H (27) A (25) H (18)O r. a l u r i n a A (16) — — H (17) R (11)Or. hi bisci A (47) H (29) A (30) H (32) R (32)Or. revicta H (46) H (28) H (30) H (45) H (56)

NotodontidaeD a . m i n i s t r a R (13) — — — —Het. um brata H (14) H (11) — A (9) R (7)

N ymphalidaeAs. celtis R (9) R (8) — A (2) —As. clyton R (41) — R (37) H (18) —

Papil ionidaeP. pol yxenes R (18) — — — —

PsychidaeT. ephemeraeformis R (16) — — R (16) R (18)

S a t u r n i i d a eHem . m aia H (23) H (22) H (20) H (29) H (33)Ac . lun a A (13) A (15) — H (17) H (15)An. polyphemus R (17) R (19) — H (16) R (16)

S phingidaeM . sexta A (35) A (31) — A (31) H (37)

Note. R 5 host wa s r efr actor y to the micr ospor idium t ested. A5 light or otherwise atypical infection occurred. H 5 heavy infection, envir onmental spor e

produced. —5 not test ed. (N)5Total nu mber individua ls recovered at t wo spore concentrat ions.

Tota l number of nont a rget host s pecies/cat egory

MP : R 5 22 A 5 16 H 5 10

MR: R 5 7 A 5 5 H 5 14

NL : R 5 6 A 5 8 H 5 23

MS : R 5 7 A 5 2 H 5 7

E P : R 5 13 A 5 2 H 5 18



8/16

those of contr ols. Host species d esigna ted ‘‘a typical’’were those in which infectious spores were not pro-d u ce d o r w e re p rod u ce d in v ery low n u m b ers (,10environm ent a l spores/4003 microscopic fi eld) com-pa red t o spore production in L . dispar. Many nontar gethost species also showed one or more of the followingeffects: developmental forms and spores were atypicalin a ppeara nce when compared w ith t he same forms in

L . dispar, death occurred within 2–7 days postinfection,a nd la rva e fed low doses showed str ong responses sucha s h ig h p e rc e n t a g e s o f in fe c t io n a n d h ig h m o rt a l i t yrates. Infections were considered heavy in nontargetspecies wh en more tha n 10 environmenta l spores w erepresent in a 4003 microscopic fi eld. Ta ble 3 does notdistinguish t wo cat egories of unusua l responses, thoseinsta nces in wh ich t he infections, a lthough heavy, werenot t ypical of those seen in L . d ispar a nd th ose in wh ichfew la rv a e b eca m e in fe ct e d , e v en a t t h e h igh s poreconcentrat ion. The percenta ges of larva e becominginfected in each bioassa y a re report ed in Ta bles 4–8,

not for st a tistical purposes, but t o indicat e large differ-ences in percentages of infection between nontargetinsects a nd the na tura l host .

Th e M P b iot y p e w a s t e st e d in 48 n o n t a rg e t h os tspecies, 22 of w hich w ere completely refra ctory to t hismicrosporidium. The responses of the remaining 26nonta rget species ar e recorded in Ta ble 4. Eight speciesdeveloped infections in which few or no environmentalspores were produced; in 6 of those species, few indi-vidua l la rva e developed infections. Ten n onta rget spe-cies developed heavy infections (Table 3), and in 2species, Heterocampa umbrata a n d Orgyia pseudotsu-

gata, infections w ere ty pica l of infections in L . d ispar (Table 4). Nevertheless, ma ny at ypical spore formsw ere produced in O. pseudotsugata in a ddit ion to largen u m b ers of m orp h olog ica l ly t y p ica l e n viron m e n t a lspores. In t he 8 species remaining, either morta lity w a shigh or few individuals became heavily infected. In 4species, lar ge numbers of at ypica l spores a nd develop-mental forms of the microsporidium were observed.Fa ilure to complete division into single spores w ascom m on a n d s pore s w e re of u n u s ua l s h a p es , of t enshort ened a nd rounded (Figs. 1A a nd 1B). Tw o speciesof n on t a rg et h os t s s h ow e d s t ron g ce llu la r im m un e

re sp on s es , w it h e n ca p s u la t ion of s pore s a n d h ea v ymelanization occurring in the midgut cells.

Twent y-six nonta rget h osts were fed t he MR biotypea nd 7 were refra ctory. Many of the infections observedwere similar to those seen in la rvae fed MP, a lthoughenvironmental spores were produced in more nontargetspecies (Ta ble 5). Nine of 14 species tha t developedheavy infections also showed responses tha t were nottypical of infection in L . d ispar. Atypical spores wereproduced in all 9 species (Figs. 1C an d 1D) and, in 5species of the nonta rget hosts , man y la rvae died ea rlyin t he cours e of infection.

The MR biotype produced more heav y infections th a nth e MP biotype in the 26 nonta rget hosts tha t w ere fedboth isolates (14 for MR, 9 for MP) and also producedmore typical, host-like infections (7 for MR, 2 for MP)All 9 hosts that were heavily infected by MP were alsoheavily infected by MR. Of the 7 nonta rget species thaw ere refra ctory t o MP a nd t he 7 host species refra ctoryto MR, 6 w ere the sa me species. Five host species tha

TABLE 4

Nonta rget H ost Responses to M i c r ospor i d i u m sp. (P ortugalIsolate) (MP)

%infected103 sp or es /µla (n )b

%infected105 sp ore s/µl (n )

Type ofinfection

Species showingaty pic alresponses

H . cunea 0.0 (12) 36.4 (11) b, fEuc. am oenari a 0.0 (4) 33.3 (6) a , b

A. pometari a 16.7 (6) 0.0 (10) a , b

M. americanum 0.0 (15) 10.0 (20) a , b

M . d isstr i a 9.1 (22) 40.0 (20) b, c, f

L . m a t h u r a 16.7 (6) 0.0 (7) a , bO. definita 6.3 (16) 5.6 (18) a , b, dA. pyrami doides 5.3 (19) 4.0 (25) a , b

L i. grotei 0.0 (14) 23.1 (13) a , b, c

Ps. resumens 33.3 (3) 40.0 (5) b, d, g

X. capax 4.5 (22) 18.5 (27) a , b, c, g

Xy. rufago 20.0 (5) 100.0 (9) b, c, eO r. a lu r i n a 33.6 (6) 100.0 (10)c b, cOr. hibi sci 34.8 (23) 66.7 (24) b, c, eAc. luna 0.0 (4) 11.1 (9) a , b

M . sexta 0.0 (14) 14.3 (21) a , bSpecies showing

typical (heavy )

responsesL. dispar NJ S t d . 43.5 (62) 83.1 (65) H ost

D. pinicola 0.0 (9) 25.0 (12) a

O. antigua 0.0 (18) 38.9 (18) a

O. l eucostigm a 4.8 (21) 10.7 (28) a

O. pseudotsugata 0.0 (7) 80.0 (15) f, hCh . ser icea 45.2 (31) 75.0 (12) c, fSu. bicolorago 0.0 (8) 100.0 (7) c, g

Eg. altern ans 100.0 (4) 66.7 (9) e

Or. revicta 29.6 (27) 100.0 (19) c, f

H et. u mb r a ta 33.3 (6) 37.5 (8) h

H em. ma ia 62.5 (16) 71.4 (7) c

Note. a 5 low num bers of individuals became infected compared t

the gy psy moth; b 5 few or no environmenta l spores were producedc 5 high ear ly mor tal i ty ; d 5 infections typical of those in the hosocc ur r ed in some la r vae (usually a t the lower c onc entr a t ion); ahigher spore concentra tions, ma ny la rvae died before infections w erfully developed; e 5 hypersensitivity to low doses (high infection

r a t e s , h i g h m o rt a l i t y ); f 5 a ty pical d evelopmenta l f or ms of thepathogen wer e pr od uc ed ; g 5 s tr ong host immune r esponses occurred; h 5 infections were similar to those produced in the gypsymoth host.

a Concentration of spore suspension used to dip leaves or spread, 40µl/cup, on mer idic d iet.

b Number of larva e recovered from rearing dishes.

c A l l l a r v a e d e a d b y D a y 7 . O n e l a r v a d i s s e c t e d b u t r e m a i n i n glarvae considered infected. All controls survived.



9/16

FIG.1. Micr o sp o r id iu m sp., P ortuga l biotype (MP ), typical primary spores and germina ted primar y spores in L . dispar midgut muscle (A)At y p ica l , r ou n d ed s por e f or m s (a r r o w h ea d s ) i n t h e m i d gu t e pi t h el ia l t i s su es o f t h e n o nt a r g e t h os t , Amp h ip yr a p yr a mid o id es (B)Micr o sp o r id iu m sp., Roman ia biotype (MR), typical prima ry spores and germina ted prima ry spores in L . dispar midgut tissues (C). Light MRnf ec tion with incomplete d ivision of pr imar y spor es (ar r owhead ) in the nontar get host Orgyia definita (D). Typica l prima ry spores a nd

germinated primary spores of Nosema lymantr iae (NL) in midgut tissues of L . d isp a r (E). Chains of incompletely formed primary spores(ar rowhea ds) were produced in Or thosia r evicta NL infections (F). ps, primar y spores; gs, germinat ed primar y spores. Ba rs, 5 µm.



10/16

developed light infections wh en trea ted w ith MP devel-oped heavy infections when tr eat ed with MR.

The host range of MS was similar to that of MP forthe host species tested and the responses of the hostswere similar (Ta ble 6). Nine species beca me infectedw it h t h is b iot y p e; s ev en w e re c a t e g oriz ed a s h e a v yinfections.

The physiological responses of nonta rget hosts to

in fe ct ion b y N L w e re s im ila r t o t h os e of in fe ct ion s

produced by the MP a nd MR isolat es but the host ra ngew a s broa der (Ta ble 7). S ix of 37 nont a rget species wererefra ctory to the microsporidium; 8 species developedatypical infections. Infections in 23 nontarget speciesw ere ra nked as heavy but in most heavy infections, thepathogen either produced many atypical spores (Figs1E and 1F) or produced environmental spores in lowpercentages of the nontarget host larvae, often resulting in high early mortality. Unlike the M icrospori d i umbiotypes, NL infected 2 species of butterflies, Asterocampa celtis a n d As. cl yton. Infections were a typical inAs. cel ti s but w ere typica l in As. clyton lar vae, althoughth ey occurr ed in low num bers of larva e. The NL biotypeproduced cellular immune responses in 5 nonta rgehost species, evidenced by heavy melanization aroundclusters of spores in the midgut a nd fa t body t issues.

Th e E P b iot y p e p rod u ce d re su lt s t h a t w e re v e rydifferen t fr om th ose of the other f our bioty pes (Ta ble 7)Twent y of 33 nonta rget host species beca me infectedwhen fed EP and the infections were nearly all heavyand typical of infections in L . d ispar. Prevalences oinfection w ere lower in 3 nonta rget species tha t devel

TABLE 5

Nonta rget H ost Responses to M i c r ospor i d i um sp .(Roma nia Isolat e) (MR)

%infected103 sp or es /µla

(n )b

%infected105 sp or es /µl

(n )Type of

infection

Species showing

aty pic alresponses

M . d isstr i a 82.4 (17) 82.4 (17) b, c, e, fC. gracil is 33.3 (6) 0.0 (5) a , b

Li. grotei 14.3 (14) 72.7 (11) b, c, f

Ac. luna 0.0 (8) 14.3 (7) a , b

M . sexta 0.0 (9) 9.1 (22) a , b, fSpecies showing

typical(heavy)responses

H . cunea 6.3 (16) 23.1 (13) a , d, fL. dispar NJ S t d 88.6 (44) 90.9 (44) H ostL . m a t h u r a — 25.0 (8) h

D. pinicola 63.6 (11) 64.3 (14) h

O. antigua 100.0 (10) 100.0 (14) d , f c

O. definita 63.6 (11) 70.0 (10) b, f

O. l eucostigm a 76.9 (13) 82.4 (17) f, h

O. pseudotsugata 100. 0 (16) 100. 0 (19) c, e, f d

Ch . ser i cea

3r d in st a r 82.4 (17) 100.0 (10) h5t h inst a r 0.0 (7) 0.0 (5) No in fect ion

X. capax 58.3 (12) 50.0 (14) h

Eg. alternan s 80.0 (5) 100.0 (6) f, hOr. hibi sci 71.4 (7) 95.6 (22) d, fOr. revicta 72.7 (11) 88.2 (17) c, f

H et. u mb r a ta 20.0 (5) 100.0 (6) h

H em. ma ia 100.0 (9) 92.3 (13) c, e, f

Note. a 5 low num bers of individuals became infected compared t ohe gy psy moth; b 5 few or n o environment al spores w ere produced;

c 5 high ear ly mor tal i ty ; d 5 infections typical of those in the hostocc ur r ed in some la r vae (usually a t the lower concentr at ion); a thigher spore concentra tions, ma ny la rva e died before infections w erefully developed; e 5 hypersensitivity to low doses (high infectionr a t e s , h i g h m or t a l i t y ); f 5 a ty pical d evelopmental f or ms of th epathogen wer e pr od uc ed ; g 5 s tr ong host immune r esponses oc-

curred; h 5 infections were similar to those produced in the gypsymoth host.

a Concentration of spore suspension used to dip leaves or spread, 40µl/cup, on m eri dic diet .

b Number of larva e recovered from rearing dishes.c 12 of 14 larvae infected when an infected, homogenized, nontarget

host w as fed to conspecifi c la rvae.d No infection in 10 larva e when a n infected, homogenized, nonta r-

get host w as fed to conspecific larva e.

TABLE 6

Nonta rget H ost Responses to M i cr ospor i d i u m sp.(Slova kia I solat e) (MS)


(n )b


(n )Type of

infection

Species showingaty pic al

responsesM. americanum 5. 6 (18) 100. 0 (20)c b, cOr. hibi sci 80.0 (10) 100.0 (20) b, c, e, f

Species showingtypical (heavy )responses

L. dispar NJ S t d 97.1 (34) 93.8 (32) H ostO. l eucostigm a 38.9 (18) 60.0 (15) hCh . ser icea 12.5 (8) 22.2 (9) a

L i. grotei 86.7 (15) 100.0 (9) h

Eg. altern ans 62.5 (16) 90.9 (11) d, f

Or. revicta 100.0 (15) 86.7 (15) d, f

X. capax 13.3 (15) 66.7 (18) d, fH em. ma ia 100.0 (2) 94.4 (18) d, e

Note. a 5 low num bers of individuals became infected compared tthe gy psy moth; b 5 few or no environmenta l spores were producedc 5 high ear ly mor tal i ty ; d 5 infections typical of those in the hosocc ur r ed in some la r vae (usually a t the lower c onc entr a t ion); ahigher spore concentra tions, ma ny la rva e died before infections w er

fully developed; e 5 hypersensitivity to low doses (high infectionr a t e s , h i g h m o rt a l i t y ); f 5 a ty pical d evelopmental f or ms of thepathogen wer e pr od uc ed ; g 5 s tr ong host immune r esponses occurred; h 5 infections were similar to those produced in the gypsymoth host.


b Number of larva e recovered from rearing dishes.c All lar vae d ead d a y 2 . Two lar va d issec ted but r emaining lar va e

considered infected. All controls sur vived.



11/16

oped heavy infections tha n is t ypica l for L. dispar. Tw o

species developed at ypical in fections; n o packeted environmental spores were produced in Inc is i la henris i , abutterfly, and vegetative forms were seen but very fewma tur e spores were present in X ystopepl us r uf ago.

We averaged the prevalence of infections in the NJS t d L . d ispar for a ll tria ls in t he four different sets obioassays in order to more easily compare the preva-lence of infection with those of the nonta rget hosts(Ta bles 4–8). Most of th e preva lences w ere compa ra blea cross tr ials but in one set of bioa ssa ys, the prevalenceof infection in L . d ispar was less than expected, especia l ly a t t h e low e r s pore con cen t ra t ion s of t h e fou r

TABLE 7

Nonta rget H ost Responses to Nosema lym antr iae (NL)


(n )b


(n )Type of

infection

Species showing

aty pic alresponses

Eut. clemataria 0.0 (21) 5.0 (20) a , b

A. pyrami doides 0.0 (16) 0.0 (15) a , b c

C. gracil is 25.0 (4) 70.0 (10) b, c, g

Xy. rufago 0.0 (2) 100.0 (6) b, c, f

Eg. alternan s 16.7 (6) 94.7 (19) b, c

H et. u mb r a ta 66.7 (3) 83.3 (6) b, c, eAs. celt is — 50.0 (2) b, f, g

M . sexta 20.0 (10) 19.0 (21) a , b, cSpecies showing

typical (heavy )

responsesH . cunea 68.4 (19) 85.0 (20) d, e, fM. americanum — 52.9 (17) f

M . d isstr i a 90.9 (22) 80.0 (20) c, e, f d

L. dispar N J S t a n -da r d 72.2 (54) 95.2 (60) h ost

L . m a t h u r a — 66.7 (3) f

D. obliquata 20.0 (10) 100.0 (17) dD. pinicola 0.0 (14) 80.0 (15) h

O. antigua 100.0 (15) 100.0 (22) d, e, f d

O. definita 31.3 (16) 84.6 (13) h

O. l eucostigm a 9.1 (22) 90.5 (21) h

O. pseudotsugata 89.5 (19) 100.0 (18) d, e, f

Ch. ser i cea 78.6 (28) 95.0 (20) c, e, f, gEu p si l ia sp. 0.0 (15) 62.5 (8) hLi. grotei 11.1 (9) 61.5 (13) a , d

Li . querquera 100.0 (10)e 100.0 (10)e c

Su. bicolorago 0.0 (9) 100.0 (8) d , f, gX. capax 71.4 (14) 70.6 (17) d , e, f

O r. a lu r i n a 100. 0 (7) 100. 0 (10)e d , eOr. hibi sci 91.7 (12) 95.0 (20) d , eOr. revicta 43.8 (16) 100.0 (29) d , f, g

As. clyton — 27.8 (18) a

H em. ma ia 20.0 (10) 84.2 (19) d

Ac. luna 36.4 (11) 0.0 (6) a , h

An. polyphemu s 0.0 (10) 33.3 (6) a

Note. a 5 low num bers of individuals became infected compared t o

he gy psy moth; b 5 few or n o environment al spores w ere produced;c 5 high ear ly mor tal i ty ; d 5 infections typical of those in the hostocc ur r ed in some la r vae (usually a t the lower c onc entr a t ion); a thigher spore concentra tions, ma ny la rva e died before infections w ere

fully developed; e 5 hypersensitivity to low doses (high infectionr a t e s , h i g h m or t a l i t y ); f 5 a ty pical d evelopmental f or ms of thepathogen wer e pr od uc ed ; g 5 s tr ong host immune r esponses oc -curred; h 5 infections were similar to those produced in the gypsymoth host.

a Concentration of spore suspension used to dip leaves or spread, 40µl/cup, on m eri dic diet .

b Number of larva e recovered from rearing dishes.c Infection produced only at 106 sp or es/µl.d 2 of 1 2 lar vae d eveloped inf ec tions when an inf ec ted , homog-

enized, nonta rget host w as fed to conspecific larva e.e Al l l a r v a e d e a d b y D a y 7. O n e l a r v a d i ss ect e d b u t r em a i n i n g

arva e considered infected; a ll contr ols survived.

TABLE 8

Nontar get Host R esponses to E ndor et i c ul at us s p.(P ortugal isolat e) (EP )


(n )b


(n )Type of

infection

Species showingaty pic al

responsesI . henri si 100.0 (1) 66.6 (3) b, fXy. rufago 0.0 (7) 33.3 (10) a , b

Species showingtypical (heavy )responses

H. cunea 4.8 (21) 34.6 (26) aEut. clemataria 58.3 (12) 83.3 (12) hA. pometari a 0.0 (4) 42.9 (7) h

M . d isstr i a 83.3 (18) 55.6 (9) h

L. dispar N J S t a n -d a r d 60.0 (50) 51.3 (76) h ost

L . m a t h u r a 0.0 (5) h c

D. pinicola 0.0 (13) 46.2 (13) hO. pseudotsugata 6.7 (15) 75.0 (12) h

A. pyrami doides 0.0 (13) 7.1 (14) a , h c

Ch . ser icea 70.6 (17) 44.4 (18) h

Eu p si l ia sp. 42.9 (14) 28.6 (14) h

Li. grotei 14.3 (7) 72.7 (11) hL i . u n i m od a 0.0 (4) 33.3 (6) hX. capax 0.0 (12) 42.9 (14) h

Eg. altern ans 57.1 (7) 45.5 (11) h

Or. revicta 55.6 (27) 82.8 (29) h

H em. ma ia 100.0 (16) 88.2 (17) h

Ac. luna 0.0 (7) 12.5 (8) a c

M . sexta 52.9 (17) 15.0 (20) h

Note. a 5 low num bers of individuals became infected compared tthe gy psy moth; b 5 few or no environmenta l spores were produced

c 5

high ear ly mor tal i ty ; d 5

infections typical of those in the hosocc ur r ed in some la r vae (usually a t the lower c onc entr a t ion); ahigher spore concentra tions, ma ny la rvae died before infections w er

fully developed; e 5 hypersensitivity to low doses (high in fectionr a t e s , h i g h m o rt a l i t y ); f 5 a ty pical d evelopmenta l f or ms of thepathogen wer e pr od uc ed ; g 5 s tr ong host immune r esponses occurred; h 5 infections were similar to those produced in the gypsymoth host.


b Number of larva e recovered from rearing dishes.c Typical in fection at 106 sp ore s/µl on ly.



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biotypes fed to nonta rget h osts. Two nonta rget h osts, I .henrisi a nd H emi leuca m aia, were tested in this set ofexperiments and the responses to the microsporidiawere not a ffected by th e less infective spores. In tr eat -ments for w hich t he nontarget host w as susceptible tothe microsporidium biotype, both concentra t ions ofspores produced infection; w hen n o infection w a s pro-duced by the lower concentration, neither was infection

produced by t he higher concentr a tion. In t he fi rst set ofexperiment s, th e 105/µl spore concent ra t ions of MP a ndNL produced the expected 100%infection in L . d ispar but no infections were produced a t the lower sporeconcentr a tions in L . d i spar. EP only infected L . d ispar la rv a e w h e n fed a t a con cen t ra t ion of 1 06 sp or es /µl.Neverth eless, positive responses to th e low er concentr a -tion of MP, NL, and E P were recorded for t he nonta rgetspecies found t o be susceptible at th e higher concentr a -tions and EP infections were produced in four nontar-get species a t 105 sp ores /µl.

We recorded infections for some nontarget host spe-

cies in w hich very few la rva e were ava ilable for exami-na tion, probably as a result of cannibalism of smallerlar va e (Schw eitzer, 1979) a nd of th ose tha t died due todisease or s tress. In four treatments, NL in Orthosia a l u r i n a , M P in O r . a l u r i n a , MS in M. americanum a tthe higher spore concentration, and NL in L i t h o ph a n e querquera at both concentrations, 100% mortality re-sulted between 2 and 7 days postfeeding. Because few o r n o c o n t r o l l a r v a e d i e d a n d t h e l a r v a e f e d l o w e rconcentrations in three of the treatments survived, wed id n o t d is s e c t a l l o f t h e d e a d la rv a e a n d , b a s e d o ne x a m in a t io n s o f o n e o r t w o la rv a e , w e re p o rt e d t h e

lar va e to be infected (Ta bles 4, 6, a nd 7). F or a ll othertreatments, we examined t issues of all reported indi-viduals. Although no conclusions can be made regard-ing prevalences of infection, the information on nontar-get susceptibility and types of infection is important,e ve n w h en col lect e d fro m v e ry few la rv a e , a n d w a sincluded in t he ta bles. We also included da ta on r efrac-tory species for 11 treatments in which less than 10larva e were retrieved (Table 3). We recognize thelimitat ions with such small sample sizes but wish toshow a ll possible pat terns of susceptibility w ithin hostfamilies.

In severa l experiments, higher percenta ges of lar va efed the low concentra t ion of spores became infectedthan those fed the high spore concentrat ions. Theseresults are probably due to a combination of factorsi ncl ud in g l ow n u m ber s of l a r v a e t es t ed , s t a g e a n dfeeding behavior of th e larva e, and low infectivity of themicrosporidia to the nonta rget hosts.

Ea ch of the microsporidian biotypes w e tested occurin specific t issues of L . d i s p a r a n d i n a pa r t i cu la rsequence du ring the course of infection. No infectionsoccu rred in t is su es of n on t a rg e t h os t s t h a t a re n otinfected in L . d i spar. Although several nontarget spe-

cies did not develop mature infections, the sequence oftissues infected always followed that of infection in Ldispar.

No L . d ispar contr ol lar va e or nonta rget host cont rolarvae were infected with microsporidia in the secondthird, and fourth sets of experiments. In the first set ofexperiments, one of four cups of Ch aeta glea ser i ceacontr ols cont a ined three lar va e tha t w ere infected wit h

a microsporidium . Spore morphology an d Giemsa s ta insindicated that the larvae were probably contaminatedwith MP. The results from t wo subsequent tria ls withuninfected controls confi rmed the responses of Chser i cea t o the microsporidia .

DISCUSSION

The responses of the nontarget hosts fed spores ofexotic L . d ispar microsporidia ranged from no infectionto infections typical of those in t he na tur a l host. I n moscases, not all individuals of each species tested devel

oped infections but , in those individuals tha t becameinfected by a part icular microsporidian biotype, thedisease followed a similar developmental course. In afew ca s e s, t h e d ise a s e p rog res s ed fu rt h er in s om eindividuals t ha n in others. The most extr eme situa tionwas always recorded. Although the responses we recorded represent a cont inuu m, we grouped the differenttypes and combinations of responses into three majorcategories t o guide our a ssessment of the phy siologicahost specificity studies: refractory responses, atypicainfections, a nd h eavy infections.

Refr actory nontar get hosts. N o n t a rg e t h o s t s w e renot infected an d t here is no possibility of horizonta l orv ert ica l t ra n s m is s ion w it h in t h e p op u la t ion of t h enonta rget species we ca tegorized as r efractory, wit h th epossible exception of those species in which fewer than10 la rv a e w e re re t r iev ed . We d id n ot m ea s u re t h egermin a tion of spores in the midgut lum en of refra ctorynonta rget hosts but if inva sion of midgut cells did ta keplace, no detecta ble merogony occurred in these cellsSpecies tha t w ere refra ctory to a microsporidium cont inu ed t o f eed a n d d ev el op a t t h e s a m e r a t e s a s t h econspecific control larvae that were treated with water

More of the nonta rget hosts w e tested w ere refra ctoryto the MP isolat e th a n to other isolat es, a lthough initiatest ing of the MS biotype indicat ed that i t ma y ha ve asimilar host range. The NL biotype is a more virulentmicrosporidium a nd w e found a broader host ra nge andfewer refractory nonta rget hosts.

Atypical in fect i ons. Th e r a n g e of r es pon s es r ecorded a s at ypical on Tables 4–8 wa s extensive a nda l w a y s i ncl ud ed t h e ca t e gor y of f ew or n o t y pi caenvironment a l spores produced. We do not believe thahorizonta l tr a nsmission is likely for microsporidia t hain fect n on t a rg e t s pe cies w e p la c ed in t h e a t y p ica



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category, excluding those species for which insufficientnumbers of individuals w ere retrieved. For example, anMP infection occurr ed in one Alsophi la pometar ia la rv ain which only one midgut muscle cell infected withv eg et a t iv e form s cou ld b e loca t e d. N o s pores w e ref or m ed , n o ot h er l a r v a e b eca m e i nf ect ed , a n d t h edevelopment of a ll treat ed individuals correspondedwit h t ha t of the contr ols. At t he other extreme, all Het.

u m b r a t a lar va e infected with NL a t both spore concen-trations died within 4 days after ingesting spores. Theon ly l a r v a t h a t s ur v iv ed i n t h i s t r ea t m e nt w a s n otinfected, a nd 5 of 12 cont rol larva e survived 15 da ys, a tw h ich t im e t h e y w e re d iss ect e d. N o e n viron m en t a lspores were produced in this species. It is possible thatvery low spore concentrations might eliminate prema-tu re morta lity bu t, becau se of th e scope of th ese experi-ments, we w ere unable to test a large number of sporecon ce n t ra t ion s t o d et e rm in e IC 50 a n d L D 50 values.There is a lso concern wit h a response of this t ype tha teven low concentrat ions of the microsporidium, a l-

though unable to cycle in the nontarget host popula-tion, could kill a ny individuals encount ering spores inthe environment.

H eavy i nf ecti ons. We scored tw o types of infectionsas heavy; both types produced environmental sporestha t a ppea red t o be mat ure. We ca tegorized infectionsas heavy if many atypical primary and environmentalspores were present but modera te or la rge numbers ofmorphologically typical environmental spores were alsoformed. ‘‘Host -like’’ infections a re t hose wh ich ar e indis-t inguishable from infection in L . d i s pa r. Nontargethosts with host-like infections may be at risk if ecologi-cal sympatry w ith L . d ispar occurs.

Severa l nonta rget host species developed heavy infec-tions that were strongly atypical, suggesting that themicrosporidia a re unlikely to be horizonta lly tra nsmit-ted w ith in th e conspecifi c nont a rget populat ion. To testthis supposition, we performed some limited tests onthe infectivity of microsporidia produced in nontargethosts to conspecific nontarget individuals. O. pseud otsu- gata larvae fed spores of MR developed infections and,in some individua ls, considerable n umbers of environ-mental spores w ere produced, but at ypica l responsesa lso occurred. We homogenized t he most heavily in-

fected O. pseudotsugata larva an d fed the homogena tet o e a rly in s t a r O. pseudotsugata larvae. No infectionsw ere produced in t his h ost/pa th ogen a ssocia tion (Ta ble5), but a s ma ll number of O. ant igua la rva e fed spores ofNL th at were produced in O. ant igua became infected(Ta bl e 7).

Some nonta rget species developed infections tha twere sufficiently typical of infection in L . d i s p a r t os u gg es t t h a t t h e se la b ora t ory h os t s m igh t p os s ib lyserve a s alterna te hosts in the field. Individua l la rva e ofO . a n t i g u a developed MR infections similar to thoseseen in L. dispar. Although a typical spores were pres-

e n t , M R w a s t ra n s m is s ib le t o O . a n t i g u a la rv a e fe dhomogenized infected O. ant igua tissues (Table 5).

Nont a rget species with intermedia te responses, sucha s light to modera te production of environment a l sporebut lit t le or no evidence of at ypica l forms or immunere sp on s e, a re t h e m os t d if ficu lt t o a s s es s a s t o t h elikelihood of horizontal or vert ical transmission in an on t a rg e t p op ula t ion . In p ub lish e d re p ort s t h a t a d

dressed this situation for microsporidian infections inmosquit o hosts, tr a nsm ission did not occur even th oughenvironment a l spores were produced (Andr ead is, 19891994).

We ha ve conducted a dditional studies in our la boratory w hich indica te t ha t, w hen ecologica l complexity isincrea sed (e.g., t ra nsmission from living infected nonta rget hosts to uninfected conspecific individuals inconfi ned a renas), tra nsmission of microsporidia between nontarget lepidopteran hosts usually does notoccur. When tra nsmission did occur, the ra tes weremuch lower compared to transmission in the natura

hosts tha n w hen larva e were fed purifi ed spores, evenwhen the infections appeared comparable to those inth e nat ura l host (ma nuscript submitt ed). Tra nsmissionof other pa thogen groups in nonta rget organ isms showsimilar patterns (Hajek et al., 1995, 1996; H a sa n et al.1992).

The EP biotype either produced heavy infectionswh ich occurred in tw o-th irds of the nonta rget h osts fedspores, or the nontarget hosts were refractory to thism icros p orid iu m . Th is w a s v ery d i ffere n t f rom responses to the other four microsporidian biotypes. TheEndoret iculatus group ty pica lly infects only th e midgu

of t h e h os t a n d n o ea r l y d ev el op men t a l cy cl es orprimary spores have been found in biotypes isolatedfrom Choristoneura fumiferana ( C a l i a n d E l G a r h y1991), Leptinotarsa decemlineata (Brooks et al., 1988Hyphantr ia cunea, a nd L . d i s p a r (personal observat i on s ). B e ca u s e on ly t h e m id gu t i s i n vol ved , i t i spossible that the E ndoret i cul atus microsporidia elicifewer t issue-level ba rriers to infection a nd the production of environmenta l spores. P ercenta ges of infectionw e re h igh er a n d m ore e nv iron m en t a l s pore s w e reproduced in many of the susceptible nontarget hoststha n is t ypica l for infected L . d ispar la rv a e .

Although it may be premature to establish a rankingof t he microsporidia we tested for safety to nonta rgeorganisms, the evaluation of these biotypes for purposes of release for biological control was one purpose oour experiments. Our ra nking is ba sed on t he results ohost specificity testing in the laboratory and uses onlythe criterion of physiologica l host ra nge. Other criteriasuch a s t a xonomic identifi cation, life cycle informat ionand the infectivity, virulence, and persistence of them icros porid ia in t h e n a t u ra l h os t p op u la t ion in t h ea re a of or igin a re im p ort a n t a n d cou ld c h a n g e t h isra nking. We suggest the followin g ra nking for sa fety o



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L . di spar microsporidia n biotypes to nonta rget Lepidop-tera (Ta ble 9): M P . MS . NL . M R . N L is ra n k e dabove MR because a smaller proportion of the nontar-get hosts developed infections t ha t a re t ypica l of infec-tion in L . d i s p a r a n d b eca u s e t h is b iot y p e is m oreinfective to L . d ispar and produces stronger effects oninfected L . d ispar lar vae in the laboratory (Onstad a ndM cM a n u s , 199 6). Th e E P b iot y p e a p pea rs t o b e ageneralist and would not be recommended for releaseoutside the ar ea of origin.

When a nonta rget host species is fed spores of amicrosporidium in t he labora tory, a nd infective environ-menta l spores a re produced, these questions ar ise: Is itl ikely t hat , under na tura l condit ions, a nonta rget hostwill ingest sufficient spores to become infected? If so,can the microsporidium be transmitted from the in-fected host to conspecific individuals and, thus, becomeesta blished in t he nonta rget populat ion? The na tur e ofinfections produced in la borat ory tests a nd t he complex-i t y a n d v a r ia b i li t y of t h e n a t u r a l en v ir on m en t a r eimport a nt considerat ions wh en using results of labora -tory studies to at tempt to predict the ecological hostrange of a microsporidium.

Most of the infections produced by MP, MR, NL, a ndMS in t he nonta rget species w ere sufficiently differentfrom infections in L . d i sp ar t o s u gg es t t h a t t h es e

m icros porid ia w o uld n ot b e t ra n s m it t e d w it h in t h enonta rget populat ions. H igh premat ure morta lity, forexam ple, w ould preclude vert ical tra nsmission of thepathogen, even if sufficient spores were produced toinfect a conspecifi c individual ora lly. Multiple at ypica lhost responses in add ition to high prema tur e mort a lity,s uch a s t h a t s een i n M P a n d N L i n fect i on s i n Ch .ser i cea (Ta bles 4 a nd 7), could fur ther limit t he a bilityof these microsporidia to be tra nsmitt ed in the nonta r-get popula tion. Microsporidia ma y slow th e develop-ment of infected imma tur e hosts (Solter et al., 1990). Ifthe rema inder of the nonta rget host populat ion contin-

ues to develop and la ter life sta ges of the nonta rget hosta re less susceptible t o infection (Milner, 1972; Wa tanabe, 1987), the conspecific population may develop tothe point where a small, dead, adventit iously infectedla rv a d oe s n ot s erv e a s a n a d e q u a t e in ocu lum in t h enont a rget popula tion (Anderson, 1982; Ma ddox, 1973)For example, both spore concentrations of MR infectedCh. ser i cea l a r v a e a t h ig h per cen t a g es w h e n t h i rd

in st a r la rv a e w e re t re a t e d. F i f t h in s t a r la rv a e fed t h esame spore concentrat ions did not become infected(Ta bl e 5).

The la bora tory experiments w e conducted r epresena maximum challenge situation in which larvae wereforced to feed on purified and concentrated infectivespores produced in t he na tur a l host. The fi eld situat ionpresents bar riers to cross-species tra nsmission t ha t a renot present in the laboratory. Because spores releasedfrom a cadaver or in the feces or silk of infected hostsare susceptible to degradation in freeze–thaw cycles(Maddox and Solter, 1996; Undeen and Solter, 1996

a nd to U V sunlight (Weiser, 1963; Maddox, 1973Undeen et al., 1984; Undeen and VanderMeer, 1990)maintenance of most microsporidian species in insecthost populat ions ma y depend on successful vert icat ra n s m is s ion in a d d it ion t o h oriz on t a l t ra n s m is s ion(Weiser, 1961; Anderson a nd Ma y, 1981; Anderson1982; J effords et al., 1989).

Th e s pa t ia l a n d t e m pora l ov erla p of s u sce pt ib lenontarget species with L . d i s p a r a l s o m u s t a l s o b econsidered. The population density of a nontarget hostma y be too low for tra nsmiss ion to occur (Anderson a ndMa y, 1981; Onsta d et al., 1990; Onstad and McManus

1996). Nontarget species feeding on host plants thaare not preferred foods for L . d ispar ha ve litt le conta cwith infected L . d ispar la rv a e , a n d t h e a v a i la b i li t y oth e pat hogen t o nont a rget species also depends on lifecy cle p a ra m e t ers . In a d d it ion , g iv en t h e e colog icacomplexities of th e fi eld situa tion, the sma ll number onont a rget species tha t developed host-like pat ent infections of MP, MR, MS, a nd NL in a ma ximum-challengesitua tion, a nd fi eld records indica ting th a t lepidoptera nspecies with well-known pathogen compliments ar ep a ra s i t iz ed b y l im it ed a rra y of a p p a ren t ly s pe ciesspecifi c microsporidia (Nordin et a l . , 1972; Ma ddox

1973; Andreadis et al ., 1983; And rea dis, 1989, 1994), wdo not believe that infection passed from one infectednontarget individual t o individuals of other nontargespecies is likely t o occur.

Host specifi city st udies contribute to our underst a nding of the evolutionar y a da pta bility of microsporidia tonew hosts in a ddition to providing host ra nge information necessary for obta ining regulatory approval formicrosporidia being considered for r elea se a s classicabiological control agents. Microsporidia must adapt toand evolve with their hosts in order to survive. Thegenetic relat edness of the MP, MR, NL, a nd MS isolat e

TABLE 9

Number of Tested Species Tha t Would B e Considered a tRisk if Evaluation Included Column (1) All Susceptible Spe-cies, C olumn (2) Species Ra nked a s Hea vily I nfected (MoreThan 10 Environmental Spores Formed per Microscopic Fieldin at L e ast On e L arva) ; I n clu d e s T h o se with A typ ical Re -sponses, or Column (3) Only Species That Developed Infec-tions Compara ble to L. di spar in Quality and P revalence

Microsporidian

biotype/no. nont a rgetspecies tested

No.nontar gets

(susceptible)

No.nontar gets

(heavyinfection)

No.nontar gets(host-likeinfection)

Micr o sp o r id iu m sp. (MP )/48 26 10 1

Micr o sp o r id iu m sp. (MR)/26 19 14 5

Micr o sp o r id iu m sp. (MS )/16 10 7 2

Nosema lymantr iae (NL )/37 31 23 4Endoreticulatus sp. (E P )/33 20 18 15



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s u gg es t s t h a t m orp h olog ica l a n d l ife cy cle ch a n g e soccur even in the same host species when host popula-tions ar e isolat ed and evolutionary pressures are differ-ent. Based on our observations, other laboratory stud-ies , a n d on m olecu la r d a t a s h ow in g re la t e d n es s ofmicrosporidia in different hosts , host shifts and hostra nge expa nsion undoubtedly occur in this group overevolutionary time. Many variables, however, influence

t h e ra t e o f e v o lu t io n a n d a d a p t a t io n a n d t h e re is n ocurrent evidence tha t host r a nge expansion by inocula-tively released exotic insect microsporidia will signifi-can tly ha rm indigenous nonta rget host species (Fe-derici a nd Ma ddox, 1996).

Our research supports the generally accepted hypoth-esis that physiological host specificity is broader thane colog ica l h os t s pe cifi c it y, a n d w e s u gg es t t h a t t h ephysiological host specificity of a microsporidium mustbe interpreted within an epidemiological and ecologicalcon t e xt in ord er t o es t im a t e e colog ica l h os t ra n g e(Federici an d Maddox, 1996; Onstad, 1993). In our

labora tory st udies of nonta rget h ost susceptibility, non-ta rget lepidopteran larva e cannot avoid exposure tohigh concentrations of fresh, infective microsporidianspores. E ven so, init ial infections do not inevitablyresult in pa tent infections; Ta bles 4–9 clea rly show t ha tmost susceptible nonta rget h osts did not support opti-ma l reproduction of L . d ispar mi crosporidia . We believetha t a typical development of microsporidia in a nonta r-get host is a l imit ing fa ctor in t he tra nsmission of thepathogen from an infected nontarget host to an unin-fected conspecific individual (unpublished data). Ourstudies of fi ve different lepidopteran microsporidia

representing at least three genera provide some basicinformation about the infection process in the nontar-get hosts a s w ell a s in t he nat ura l host . This informa -tion should a llow a better interpreta t ion of nonta rgetresponses an d more a ccura te predictions of the ecologi-cal host ra nge of microsporidia being considered forrelease as classical biological control agent.

ACKNOWLEDGMENTS

We thank S. Fahrbach, D. Onstad, and H. Robertson for commentson an ea rly dra ft. We also tha nk M. J effords, Illinois Natur al H istoryS ur vey, f or photogr aphic assistanc e; W. H off man and M . Kusha d ,

U niver si ty of I l l inois f or host plant mater ials ; and L. Car loy e, L.Ha nes, J . C at let t , and A. M c Car t er f or tec hnic al a ssistanc e. This

research is supported in part by the Illinois Natural History Survey,US DA Forest Service Coopera tive Agreement 23-829, C SRS /US DAAgreemen t 94-37312-0674, a nd th e Office of Resea rch/Illi nois Agri cul-u ra l Experiment St a tion P roject No. 65-309-S265.

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