Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1985 Compatability of parasitism by Bonnetia comta (Fallén), Lydella thompsoni Herting, and Macrocentrus grandii Goidanich, and a bacterial, viral, or microsporidian infection in larvae of Agrotis ipsilon (Hufnagel) and Ostrinia nubilalis (Hubner) Joan E. Cossentine Iowa State University
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Recommended Citation Cossentine, Joan E., "Compatability of parasitism by Bonnetia comta (Fallén), Lydella thompsoni Herting, and Macrocentrus grandii Goidanich, and a bacterial, viral, or microsporidian infection in larvae of Agrotis ipsilon (Hufnagel) and Ostrinia nubilalis (Hubner)" (1985). Retrospective Theses and Dissertations. 7833. https://lib.dr.iastate.edu/rtd/7833
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8614385
CoMtntin*,Joan E.
COMPATABILITY OF PARASITISM BY BONNETIA COMTA (FALLEN). LYOELLA THOMPSONI HERTINQ, AND MAWOCENTRUS QRANOil GOiOANICH. AND A BACTERIAL. VmAL, OR MICROSPORtOIAN INFECTION IN LARVAE OF AOR0TIS IPSILON (HUFNAOEL) AND OSTRINIA NUBILALI8 (HUWER)
lowê Stêt9 Ph.0. 1968
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University Microfilms International
CoiQpatâbility of parasitism by Bometia amta
(Fallln), LydêlZa thompaoni Harting, and Mùofcamtrua gwmàii Coidanlch» and a bacterial, viral, or mlcrosporidian infaction in larvaa of Agrotie ipaiîon (Kufnagal) and Oetrinia nubiîaîie (Kubnar)
by
Joan E. Cosiantina
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requlremnt# for the Degree of DOCTOR OF PHILOSOPHY
Department: Entomology Major; Entomology (Insect Pathology)
Approved
Signature was redacted for privacy.
Signature was redacted for privacy. T Departmei
Signature was redacted for privacy. For the Graduate College
lOTfa State University Ames, Iowa
1985 il
TABLE OF CONTENTS
Page
INTRODUCTION 1
Intact Pathogen and Paraaitoid Interactiona 2
Bacteriun-paraaitoid interactiona 2
Virua-paraaltoid interactiona S
Microaporidium-paraaitoid interactiona 6
Paraaitoida Studied 8
Bometia omta (Fallen) 8 Lydelîs tfwmpmni Her ting 10
Abexvwrtrw §pmtdii Coidanicb 13
PART l: A SURVEY OF THE PARASITOIOS OF THE BLACK CUTWRM, Ammis mmn (HUFNACEL) AM* THE EVROPEAR CORN BORER, osmnu mmjiAUS (HOBNIX) AT THE USDA. ARS. CORN INSECTS RESEARCH UNIT, ANKEM. IOWA I) INTRODUCTION 16 MATERIALS AND METHODS 17 RESULTS AND DISCUSSION 19 PART II; MAIMENANCE OF BOSmU COMTA (FAUZN). MCBCCSMNM GMSPII COIDANICH. AND LxmiLA fmmsom HERTING COLONIES 33 INTRODUCTION 34 MATERIALS AND METHODS 35 RESULTS AND DISCUSSION 39 ill
Page
PART III: l^tPACtOÊ VAimmRPM mCATRIX (KRAMER), VAmmoRPm SP.« AND MCIUVS mmisamtsis BERLINER SUBSPECIES KtmSTAXI ON BOmSTIA OMVT (FALLÊT) WITHIN AGROTIS IPSIWS (NUMCEL) TK^STS 64 INTRODUCTION 45
MATERIALS AM) MEMODS 47
RESULTS AND DISCUSSION 50
PART IVs IMPACT OP A NUCLEAR ^LYHEDROSIS VIRUS FROM BACUI^m QU (GUENB) ON BOmSTU COmA (FALLEN) tnTHlN AGROfîS ITSim (NUPNAGEL) LARVAE AND USE OP BOTN THE VIRUS AND THE PARASITOID TO CONTROL THIS HOST INSECT 66
INTRODUCTION 67
MATERIALS AND METHODS 69
RESULTS AND DISCUSSim 75
PART V: IMPACT OF mmtA FmUSTA (PAILLOT), mSiHA SP., AND A^NUCLEAR POLYHIDROSIS VIRUS FROM RACBIPIUSU OU (CUENEE) m imiLA fUCHPSOm HERTim. WITHIN INFECTED Qsmnu mmiMUS (HUBNER) HOSTS 85
INTROWCnON 86
MATERIALS AND METHODS 88
RESULTS AND DISCUSSICHf 91 PART VI; IMPACT OP VAmmRPm mCATRIX (KlUMER), SOSEM fUMumA (PAIILOT). mssm SP,. AND A NUCMI POLYHEDROSIS VIRUS FROM MCBIFWSU OU (GUENEE) ON mcmcsmuB cmsmi GOIDANICH oevuopuic WITHIN INPECTED OSFRISIA SUBIZAUS (HUBNER) LARVAE 103
INTRODUCTION 104
MATERIALS AND METHODS 106 RESULTS AND DISCUSSION 110 SUMMARY AND CONCLUSIONS
UTERATURE CITED
ACXNOWLEDCESŒNTS I
INTRODUCTION
ParMltolda «d pathogms play vary important rolaa in regulating inaact population#. In nature, interaction# occur between theee two
biological control agent# whenever they frequent a common insect ho#t.
Such interaction# have the potential to be Wvantageow or detrimental to
either agent» and conaequently, to the control of the ho#t.
It be advantageou# to the in#ect pathogen, and increaae ho#t
mortality, when the pathogen cm be tranaaitted on the paraaitoid'# body
(e.g. ovipoaitor), am* when the phyaical atre## caused by a paraaitoid
increaae# the host*# #u#ceptibility to a pathogenic infection (King and
Atkinaon, 1928; Jaques, 1961). A lethargic, diseased host my be
advantageous to a paraaitoid in that it nay not resist parasitism.
It hM alao bem apeculated that a patho^n infecting am inaect host
larva have one of several detrimental effects on a paraaitoid. The
pathogen may; (1) cause the adult paraaitoid to avoid ovipositing in the
infected host; (2) came the premature death of the hMt. thereby in
directly killing the developing paraaitoid within; (3) alter the host,
rendering it nutritionally or physiologically unsuitable for paraaitoid
development; or (4), the pathogw* indirectly infect the paraaitoid
tissues (Wigo md Tasasbiro, 1966).
Much research has been done on the individual roles of parasitoids
and pathogens in controlling insect pests. The interactions occurring
betwecm these biological «mtrol agents must also be studied to realis
tically assess their potential to control insect populations in natural
ecosystems, as well as in integrated pest management systems. 2
This dissertation studies potential pathogen-parasitoid interactions occurring between the major parasitoids of Agvotie ipeiton (Hufnagel) and Ostrinia nuHlaîie (Hubner); and, host pathogens currently used or with the potential for future use as e^crobial introductions or insecticides.
Insect Pathogen and Para*itoid Interactions
Bacterium-parasitold interactions
Most studies of the impact of insect bactarial pathogan* on associated parasitoid sp«cl«* have involved B€t&illue thuHn^meis Berliner. Dunbar and Johnson (1975) found that ingestion of B. thuHngimeis in sugar water by adult AmKwWZff nigriaepg Viereck (Braeonidae) resulted in a signifi cant decrease in post-treatment longevity of this tobacco budworw,
Belicthie vireaamm (fabricius) parasitoid. Similarly# Named (1979) found four hymenopterans: Biadema amillata (Gravenhorst)» Hmpta twHmeltae (Bouché) (Ichneumonidde). Agenimpie fueaiootlie Oalman (Encrytidae). and Tetvmtviokue evonymllae (Bouché) (Eulophidae) died when they took up spores of B. thuHn^endis with food sap. Diadecmt asmtllata males were sore sensitive to 3. thuHngfienHs than were the females.
In contrast, no detrimental effects from adult parasitoids ingesting
B, thztpin^enaia have been recorded in the following Hynenoptera and Diptera; Brophymna intermia (Nees) (Chalcidae); Camjpoletis 8onovmH& (Cameron) and Tviokiomtue sp. (Ichneumonidae); Chelonus blaokbumi (Cameron) and Meteovuz leoiventvm (Mesmael) (Braconidae); fviahogvarma oaooeda Marchai (Trichogrammatidae); and VoHa rumlis (Fallen) Beaaa
fugax (Bondani) and Zenilla dolosa (Heigen) (Tachinidae) (Hassan and 3
Krittg, 1975; Wilkinson et al., 1975; Hamed, 1979). In surveys of gypsy
moth, Lymantria dispar (L.) paraaitoida after field treatment with 5. thurinfftëneie it waa found that significantly fever adult Braahymria intûmgdia (Nees) (Chalcidae) 4pant«2«« mîaiWêewhtB (Rataeburg) (Brecon- idae), Paratêtigéna aiîVêBtria (Robineau-Deavoidy) and CompHlura ocnain- nata (Maigen) (Tachinidae) were found. However, mortality of these para-
aitoid apeciea cannot be attributed to the 5. except
indirectly through large acale hoat deatruction (Reardon et al., 1979).
Few atudiea have accurately meaaured the effect of the bacterium on
immature paraaitoida developing in f. infected hoata. Kaya
and Dunbar (1972) reported the larval atage elm apanworm, Emow méboignar^
iua (Hubner) paraaitoid, Telemms alaopkilm Viereck (Scelionidae) was unaffected by field applicationa of B, thuringimsis. However, the hoat larva# fed on B, theringiewis for 24 hours while paraaitoida within were
in the late larval or pupal atage, therefore minimising any deleterious
contact. Similarly. Biliotti (1956) showed that the bynenopterous para
aitoid of H&He bmeeicoê L., ApantaUo gtcmmtm L. (Braconidae) can survive B, thurinffiemiS''induced death of its host if the hoat is in the
last larval instar on the dn^ of treatment. The endoparaaitic life of the
4. gUmvatm is also significantly shorter in the B, tkuHngiemie"
infected hosts.
Hnel (1977) recorded significwt alteratioas in western spruce
budworm, ChoHstomum oooidentalie Freemai, parasitoid populations following #. tkupingien»i8 treatment. Three weeks after treatment, there were significantly nwre Apmtelee fimifevasnae Viereck (Braconidae) and 4
Glypta fUaifÊranat Vl«reck (IchntuaonidM), which paraslclze predlapauslng
first Instar budworos» and algnlflcMtly fwsr Phatogmtëê haeioîu» (Cresson) (Ichneunonldas) » Cêromcoia catriaaudata Towsend, and Madrmyia êaundêreii (Williams) (Tachlnldas) which psrssltlss Ists Instars snd pupss. Haosl attrlbutss the dlffsrentlsl psrssltlsm to a host response. Apantëîte fiméfêramu-' snd g. fi^riftramt-pêtaaitiMtâ budworms bscoms photonsgstlve snd lack feeding stimuli responslvsnsss, therefore probsbly avoiding contact with 3* thsàHnffimwië, The other parasltolds llksly were killed when the hwts dlsd from Ingsstlng B, thurinffi*nti§. In slmllsr studlss,
Morris <1977, 1983) found no dslstsrlous sffects of B, thynHt^eneia
applications on psrssltlsm rstss which could not be attributed to changes
In the spruce budworm populatlcm.
Little research has been done on the effect of other entomcpathogenlc
bacteria on parasltold species. Adult Apmtëîëe gîmëmtm L. (Braconldae)
and Ftevmalue pupaHum L. ((%alcldldae) were found to be nonsusceptlble to Bûoiîîm aeveue Frankland sad Frankland (Xsskovs* 1958). fipkia mrmlis Rohwer (Tlphlldae) larvae parasitising the Japmese beetle, Papilla ic^ctrdm Newman have been fotmd to contain milky disease, Baedltus papilliae Dutky spores (White, 1943). In soma cases, the spores were
voided with the meconium when the larvae spun cocoons. White (1943) con
cluded that the B, papilliae did indirectly affect T. vevnalis larval
mortality by causing the death of the host. King et al. (1975) also
reported that Usaphaga âiatvaeae (Tachinidae) is very susceptible to the
bacterium SermHa impcesoena within its host, the sugarcm*e borer, Diatvaea eaoehamlis (F.). 5
Virut-parasicoid interactions
Tha primary advaraa effect that insect viruses have on parasitoids is
the indirect effect of causing the premature death of their hosts. Vovia puralia (Fallin) (Tachinidae) parasitising nuclear polyhedrosis virus- (HPV) infected cabbage loopers, Triahoplusia ni (Kubner); Fhryaae mlgarie (Tachinidae) and Apmteîee gîmêmtua L. (Braconidae), parasitising granu losis virus (GV)-infected PieHa rapae L. larvae, and Apanteles margini-
ventrie (Cresson) parasitising NPV-infected lawn armyworms, Spodoptem irmtHtia aarûnyatûidee (Cuenie) were all unaffected by the diseases unless
they were unable to complete development before their hosts died (Kelsey,
1960; Harcourtt 1967; Laigo and Tamashiro. 1966; Vail, 1981). Kelsey
(1960) suggests that these underdeveloped parasitoids die of starvation.
Similarly, mortality of Ptewmîue pup&mm L. (Pteromalidae) occurred
when its host. F. v&pm died of a 0V infection before the parasitoid was
mature enough to pupate (Kelsey, 1960; Laigo and Paschke. 1968). Ftevmalm pupœntm which were able to reach the adult stage were smaller and gen
erally shorter lived than parasitoids from noninfected hosts (Laigo and
Paschke. 1968).
The survival of two ichneumonids. Campoletin sonovenHs (Cameron) in the tobacco budworm. Heliothia mveaaens (F.) and Ifyppaotef ^guae
(Viereck) in the cabbage looper increased with the time between host
parasitism and exposure of the host to a NPV (Irabagon and Brooks. 1974;
Beegle aid Oatman, 1975). Hypoaoter esiguas larvae required significantly
less time to develop in virus-Infected hosts than they did in noninfected
hosts (Beegle and Oatman. 1975). 6
Parasitlam before viral infection of a hoat may be what more coimnonly occurs in nature, as the parasitoids, ApantéZêe glcmmtuê (Braconidae) and Ptarmaîua pupartm (Pterosalidae), both avoid ovipositing in virus- infected caterpillars (Kelsey, 1960; Steinhaus, *954). The tachinid.
Varia mcealie hotrnver, did not avoid ovipositing in virus infected hosts in laboratory studies (Vail, I98t).
Laigo and Tamashiro (1966) and Laigo and Paschke (1968) histologi cally examined Aipmtalés mi'ffiwimntria (Braconidae) and Ptêrctmîue puparm (Pteromalidae) from severely diseased hosts and found no evidence of viral infections in the parasitoids. Polyhedra were found only in the midgut lumen of V, ntralie (Tachinidae), C, aonorens-i^ and H. exignae
(lehneuiMnidae) within infected host larvae (Irabagon and Brooks, 1974;
Beegle and Oatman, 1975; Vail, 1981). Polyhedra in the midguts of the
C. aon&penaie and the V, mmtlie were voided in the parasitoid meconium during pupation and the adults were free of the polyhedra and any syo^tome of viral infection (Irabagon and Brooks. 1974; Vail, 1981).
Kaya (1970) reported another potentially detrimental effect of a CV and a NPV on Apmtslee militceHs (Braconidae). When either a synergistic strain of GV or a hypertrophic strain of a KPV infect the am^orm.
Pamtdaletia imipmota, (Haworth). the viruses produce a toxic factor which will kill the braconid (Kaya and Tanada, 1973).
Hicrosporldium^parasttoid interactions
There are numerous records in the literature of microsporidian infec tions in parasitoids within diseased hosts. These citations include:
Soeema deatruotov Steinhaus and Hughes infecting Maavaoentvus ancylivovus 7
Rohwer (Braconidae)« a parasltold of the potato tuberwora» GnùrimoÉahêma opêrauîêîta (Zallar) (Allan and Brunaon» 1945); Soama pyraueta (Paillot) In Oiêîcmta amutipée dctmal, Maorcoéntrue gnmdii Goldanlch (Braconldaa) and Lydëîîa t^Kmpaoni Harting (Tachinidaa)t all paraaitoida of tha Europaan corn borar* Qetrinia mbilalia (Hubnar) (York* 1961); dceam mtmili (Paillot) infecting Hypoaotar akanime (Gravanhorat)(lchnauRoni- daa) Hmpla inatigat&p (F.) (Hoatounaky» 1970), Apmtalaa fuha&ula (Marah) (Bluncki 1958) and Apmtalaa glommttta (L.) (Braconldaa) (Tanada, 1955)
(aa wall aa paraaitoida of the laat), all paraaitoida or hyparparaaitoid# of tha cabbage worm, Haria popm (i.) and HaHa bmaaiaae (L.)» and
ApcHa dvataeffi (L.)ï and ^Iceem heîietshidee Luts and Splendore Infecting Cmpoletea scmrmaia (Cameron) (Ichneumonidae) and QaeMoohilee nigvio^pe Viereck (Braconidae), both paraaitoida of the corn earworn. Beliethia s^a
(Boddie) and the tobacco budworn» fi. iHvaaaem (f.) (Brooka and Cranford,
1972).
Uoema deatvuot&v in H, moylivcfm was ond of the first micro- eporidian infections in a parasitoid to be recognized, and it# pathology appears to be characteristic of others. The microsporidium infects the potato tuberwrm in which the parasitoid is reared (Allen and Brunson,
1947) and the MaoTcemtvtiS cannot maintain it in its populations without constant exposure to infected hosts (McCoy, 1947). When individuals of
M. cmoftflivGvixiB are infected with the Hosema, only 40-60 percent eclose as adults (McCoy, 1947). Similarly, the other above-mentioned micro- sporidia have the potential to cause mortality in the parasitoid#, should
their hosts be heavily infected (Tanada, 1955; York, 1961; Brooks and 8
Cranford» 1972). lnf«cc«d M. ano^livcitius adules which arc able co exie thalr cocoon* exhibit «vollen and malformed abdomens, are ahort lived, and have lowered reproductive potential# (Allen and Brunmon, 1943} McCoy,
1947; Allen, 1954). It appears that 4. glomêvatua females infected with U. immili do not have reduced reproductive potentials and at least some of their eggs are viable (Issi and Maslennikova, 1966).
Hostounsky (1970) has demonstrated that microsporidia may infect hymenopterous parasitoids in three ways. A paras!toid may become infected:
(1) by feeding on infected host tissue as in Pimpîa imeeHgatcv (F.)i
(2) by carrying the microsporidiun in the egg at oviposition. as in 4. glommtuei or (3) through places of contact with diseased host tissue, as when host spores pass through a respiratory cavity in the rectum of
4. gîmeratue larvae. Uaaema deatruotov spores have been found in the midgut, laemolymph, muscle, and other tissues of K cm&yliv&Hue larvae (Allen, 1954). The types of parasitoid tissues infected by memili appear to depend on the intensity of the host's infection, the age of the parasitoid. and the
Runner in which it becowe infected (Hostounsky, 1970),
Parasitoids Studied
Bonmtia aomta (Fallen) Bcmetia {plirmaemyia) eemta (Fallen) is a common tachinid para sitoid of noctuid larvae in Itorth America. Europe, and Asia. North
American noctuids which B. oomza has been found to parasitize include: AgroHs spp., Copablepiumm viridisparsa Dod, Euxoa spp., Peltia spp.. 9
Fm*iàPoma accuaia (Rubner>, Polia aautêmina (Smith)» and Spodopteva f)nigipêrda (J. E. Ss^ch)* as trail a# unldantlflad Koctuidaa. Scarabatld spaciaa of the Phyîîoph^a genua have also been found paraaitized by 5. amta (Brooks, I9A4).
Fecund B, &omta my individually oviposit over I,000 eggs (Wen ee al., 1965) primarily on plants, as well aa on soil surfaces. Oviposi- tion occurs in response to a kairomone in the host's feces and perhaps voMtus (Levine and Clement, 1981a). As this species is ovoviparous, the embryos are fully developed in the mother's body and the first instar maggots (planidia) eclose Immediately after oviposit ion. The fiiwll black planidia are covered with cuticular plates Wiich probably allow them to avoid desication. They attach themelves perpendicular to the sub strate by means of a small wW*ranows posterior cup (Allen,' 1929). The planidia may remain thus, motionless, for several days. Th#se first in- star Mggots react quickly to vibrations of the substrate. When disturbed, they swing their heads or leave the basal wmbrane and move rapidly over the leaf or soil surface, seeking a host.
Upon finding a host, the planidium generally penetrates the cuticle of the cutworm through the dorsum of the seg9»ntal me=*rane of the first abdominal segment. Entrance into the host's body requires less than five minutes (levine and Clement, 1981a). The puncture in the host formed by this act is maintained as a breathing pore. The parasitoid emerges from the host tissue in its last instar and pupates within six hours (Levine and Clement, 1981a). 10
Lkdêlla thomsoni Hatting
Among the firat parMltoid* fron Europe and tha Oriant iaportad to
{forth Mwriea to control Europan com borar, Oatrinia mtbitatio (Siibnar) (ECB) (wpulationa waa tha tachinid Lydêlla thcmpeimi {'ttcâmîam var griaaêftê (Roblncau-Daavoidy) Rarting. Adult L, thanptonit first ralaaaad
in tha Onitad Stataa in 1920, rapidly bacana aatabliahad aa a major
controlling factor of ECB (Bakar at al., 1949).
Adult It* thmfêoni ovariaa, with tha potential to produce lOOO eggs,
generally develop aome 180-470 eggs which are incubated in the uterus.
The larvae ecloae during larvipwition (Baker et al., 1949) which is stimu
lated by a kairomone-type attractmt in the fraas of the host (Sradlan
et al., 1983). Cmerally, such fraas is found in ECB timnels. The active
first instar I, tkanptoni maggots move down the com borer tunnel seeking
the hwt.
Opon finding a suitable host, the mouthpsrts of the I.
msggot en^le its rapid entrmca into the E(3 larva through its cuticle.
Entrance primarily occurs through m anterior intersegmental men^rane.
although the maggots have been observed to enter their host through
spiracles and the anus (Baker et al., 1949).
Once within the host, the parasitoid punctures in a main tracheal
tnmk of the EO larva, enabling it to respire through its posterior
stigmata. The host's tracheal epithelial cells form a sheath about the
maggot (Baker et al., 1949).
t^âelta thempsom has three larval instars. The first two instars
feed primarily cm host body fluids while the third feeds Juac before this leac Ineter exits* it conauoes moat of the hoat*a internal orgma. When the boat dlaa, the paraaltoid forma a new reapiratory hole in the cuticle of the dead boat. It e*ita through thia rupture and forma a pupariuo (Baker et al.» 1949). Thia paraaitoid overwintera aa second or rarely aa firat inatar mag- gota in the body of diapauaing ECB boata (Thompaon «td Thompaon» 1923). It has alao been obaerved to overwinter aa a puparitn outaide of the boat (Ytt-ahin. 1960). When L. thcu^emi waa firat introduced into the eaatem and central corn-growing atatea, it appeared to become the most effective of the five moat aucceaaful European paraaitoid introductiona in controlling ECS popu- latims. Sparks et al. (1963) reported 41.7 percent paraaitiam by this eachinid in m Iowa survey, and Brindley and Dicks (1963) found 75 percent psfssitism by £. thai^ami the sme year in a county in lllinoia. It was in the 1960s that there was a rapid decreaae in paraaitiam by •L. thompaonif and currently, few, if any, can be found in ECB in the mid- western Vnited Statea (Hill et al.. 1978: S«*dlan et al.. 1983). Various hypotheses for this previously very successful parasitoid's decline have been tested, Frmklin m*d Holdaw^ (1966) studied the effect of com variety on the I. parasitism of ECB. This tew found a marked preference of the parasitoids for a particular hybrid of com; however, this was when the tachinids were given a choice betwen two hybrids. In 1983, Sandlan et al, could not find a signific^mt difference in parasitism of the ECB by L. thmpeoni in four different com genotypes possessing different levels of resistance to ECB (U22xA73, Its5302, H14, and (*143). 12 The fliw «merging from ECB reared in the laboratory on these hybrids did not differ significantly in site» longevity, or fecundity. This research group did notice a ho#t density response by the parasitoid. I^ydêtUt ttwmpêoni has been found to parasitise other hosts. Alternate hosts include the common stalk borer, Papaipma nêbriê (Guenle) in which it is believed to pass its first generation where the ECB population is too immature to serve as hosts for the parasitoid (York et al., 1955). All but the first instar of ECB c«i be successfully paraaitimed by 6. tkcmptami in the laboratory, although fourth instar hwts sre said to have the highest percentage of parasitism (Baker et al., 1949). Others state that only third instar or older ECB larvae are suitable for I*, thempsmi parasitiiation in the field (Rsiao and Holdaway, 1966). Hsiao and Holdaway (1966) found L. thcmpaoni emerging from over wintering ECB larvae from early June until «Êd-July in Minnesota. Should only small ECB be available to the first generation of L, thompeoni, these authors found that most of the adult parasitoids would die before larvi- positing. Thwe few late emerging adults, finding larger ECB larvae, may maintain the parasitoid p<^ulatim*, resulting in three generations a year. Stalkborers are generally larger than ECBa early in the spring, mâ the parasitoid may carry Cole (1929) found L. thcmpaoni to parasitise Archman eubéamêa Kell, and Schaffner (1953) reported the tachlnld parasitising O^trinia pmtitalia Crote. In Europe, Ânehanara gminiptmcta Herorth appears to play a role aimilar to that of the common stalkborer in America, acting as an alternate host for the first generation of L, thompâord (Calichet and Radiason, 1976). Nyperparasitism of L. thcn^emi appears to be low. In a Russian study, only three percent of the parasitoid pupae were parasitised (BJegovic, 1970). Suptérmaluê duHua (Ashswsd) (Pteronalidse) was found to hyperparasitise srae relessed ttu^aord in Minnesota (Hsiso and Holdaway. 1966). Haarao«ntrue grmdii Coidanich Haarccmt^PM granâii Coidanich Ashmead) (Braconidae) is a B4jor parasitoid of the European com borer (ECB). Oatrinia màdlalis (Hubner) in North America, Europe, the Orirat, and Russia (Parker, 1931). The species is not native to W. Awrica. Haeopcomtvm granéii collected in ECB in 1926 fro# France «md in 1929 from Japan were mass-reared and released in the United States and Canada to reduce ECB populations. Releases of the parasitoid in Iowa occurred fron 1944 until 1951 (Blickenstaff et al., 1952) and currently the species is the predominant parasitoid of the ECB in this state, representing 9.56 percent of the ECB parasitised in 1980 (lewis. 1982). Each oviparous female gmnàii is enable of developing some 200- 300 eggs (Parker, 1931). Oviposition occurs through an elongate oviposi tor which the female imsheaths. loops between the two metatarsi and probes 14 where an ECB has hem feeding. Upon finding an ECB larva (preferably In the aecond or third Inatar), the female thrwta the tip of her ovlpoaltor mywhere In the hoat'a body and deposits one to two eggs. Maaroemtna grandii eggs are polyetabryonlc and approximately 20-40 Mies, or 16-18 female larvae may develop from a alngle egg (Parker, 1931; Wlshart, 1946). itore than one egg may develop in a alngle hoat. The first three Instars feed on host fat globulea and poaalbly on other organs and muscle tissue. At the completion of the third Instar, the parasltold larva chews through the host's cuticle, shedding its exuvlum as it emerges. As a fourth instar, it then feeds externally on the host, rests for approxi mately 24 hours, and then spins an individual silken cocoon (Parker, 1931). An oblong sha^ group cocoon is usually formed, parceling the parallel- lying cocoona of the prepupal parasitoids from a common host. Adult M, grmâùi from a single hcwt generally emerge simultaneously after chewing off nail disklike caps on the anterior of their cocoons (Parker. 1931). Mating occurs sow after adult eclosion. Females may be parthenogeretici unfertilised eggs develop into males, fertilized eggs into f«sales (Fl«iders, 1942). Haavaœntpm granMi overwinter as egg# in the adipose tissue of diapmising ECB larvae (Thompson and Parker, 1928). A percentile of natural ECB populations is infected by a micro- sporidian, Sceema pi^raueta (Paillot) (Steinbaus, 1954). gwmMi developing in S, p^rwgtg-infected ECB are also susceptible to infection. This pathogen has bem shown to reduce by 38 percent the M, gmnàii able to ecloee as adults and decreases the longevity of any infected adult survivors.(Andreadis, 1980). 15 PART LÎ A SimVEY OP TMI PARASITOIDS OP THE BLACK OmWRM. AGfiOTIS iVSIWlt (HVP^EI) AÎTO THE EUROPEAN CORN BORER, OSTRIftIA mBILALIS (80BNER) AT THE USBA, ARS» CORN INSECTS RESEARCH UNIT, ANKENT, IOWA 16 INTRODUCTION The predominant paraeitold of the black cutworm Gonid is capable of parasitising ten to 65 percent of BCWs in corn fields (Schoenbohm and Turpin, 1977). Other BCW parasitoids of lesser importance include the gregarious braconid Mi&rcplitia kswîêyi Muesebeck and the tachinid Bonmtia ocmta (Fallen). The European corn borer, (ECB) Qetvinia mbilalia (Mubner), has a more substantial number of parasitoids in Iowa. Parasitoids collected in Europe, the Orient, and Russia were mass reared and released in Iowa from 1943 until 1950 in an effort to combat the newly established ECB (Blickenstalf et al., 1952). ECB parasitoids which became established in low include Hcunpcomtm» gmndii Coidanich, a polyeo^ryonic braconid, Eribovtto terebrme (Cravenhorst), a solitary ichneumonid, and a larvi- parous tachinid. ùydelîa thempsoni Herting. From 1970 until 1980, para sitism of ECB larvae in Iowa by M. gvandii and S. tembvam has ranged from .3 to 56.2 percent, and from .04 to 8.9 percent, respectively (lewis, 1982). The number of L, tkmpsoni parasitising ECB in Iowa has been declining since the 19608, and currently, few. if any, can be found in ECB collected in Iowa (Lewis, 1982). In order to establish a research program to study parasitoid- pathogen interactions occurring between major parasitoids of BCWs and ECBs and pathogens of these hosts, local surveys were conducted to assess the predominant parasitoids of each host, and to identify any pathogenic microorganisms naturally interacting with these parasitoids. 17 MATERIALS AND METHODS In the mummer# of 1983 and 1984, three (#ix in 1984) thr##-m#t«r #quar# aluminum barrier#, .5 meter high, were embedded in com field# at the USDA, ARB Corn Insect# Reeearch Unit in Ankeny, Iowa. At the end of $ May a corn hybrid Pioneer 3537 wa# planted in each plot. One week after planting, each plot wa# infeated with approximately 100 laboratory reared third inatar BCU larvae (Coaaentine, 1982). Planting and infeatation wa# ataggered through the plot# so that there wa# alway# one (1983) or two (1984) plot# ready to be infeated with larvae each week throughout the #ummer#. In a ceparate study, in 1983, three row# of corn (Pioneer 3537) were marked off into group# of ten plants and labeled in a randomised complete block design. In 1984, six row# were eimilarly aubdivided. Each week, one group of ten plant# per row wa# infested with four pathogen- free ECB egg naaaes per plant from a laboratory-reared colony (Lewie and Lynch, 1969). Two week# after infestation, the SCW# from the barriers, and the ECB# from dissected com stalk# were retrieved, rinsed in a one-percent solution of phenylmrcurlc nitrate to kill any associated bacteria or fungi, and placed individually in 30-mi plastic cups of BCV or ECB diet. The cups were sealed with polyethylene-lined lids and incubated at 27'C and 70 percent relative humidity (RH). All cups were checked daily and any emerging paraeitoids were identified. Paraaitoids unable to ecloee as adults were fixed in 58'C alcoholic Bouin's fixative for an hour and 18 24 hours at room tamperacurc, and ambadded in paraplast. These spacimans vara than aactionad, mountad on glaas alidaa and atainad with Ciamaa colophonlua atain (Shortt and Coopar, 1948). Thua praparad, thaaa para- aitoida vara axaminad for infactioua microorganisaa. If mora than ona spaciman amargad from a aingla hoat inaact than ona waa alao praparad aa a vat mount for microscopic examination. 19 RESULTS AND DISCUSSION Except for an Ichneunonld which paras!eitad many of tha BCVs aarly In Juna of 1983 (Tabla 1), tha two major parasltoida of the BCU wara a gragarlous braconid, M§t«cnt8 lêvivêntrua (Waanaal) and a tachinld, Bonmtia oonrùa (Fallen). Paraaitiam by M. l«vimntvm bagan In Juna of aach yaar when larval BCW would be naturally present in the com fields (Fig. 1 and 2; Tables I and 2). Few of tha M. lêvivëntpue and only 17 of tha ichnatnonid larvaa wara able to pupate and eclose as adults after emerging frcns their hosts in the laboratory. In 1983, 104 of these non- eclosed K îevii/entruê larvaa and 12 ichneumon Id larvae were observed as fresh mounts as wall as stained serial sections under a cwxpwund micro scope. All larvaa unable to eclose as adults and thus examined, contained saprophytic bacterial roda, auggesting that the condition# in the 30-ml BCV diet cups war# t(w humid to allow th# larva# to d#v«lop further. Hn pathogenic microorganiws w#re found associated with the larvae of either parasitoid sp#ci##. Although a few 3. aomta were found parasitizing the ECVe in June of 1982, this specias primarily parasitized releasad later in #ach summer (figs. 1 and 2). BCUs are major pests early in the growing season when they feed at the base of seedling com plants* Bometia Qomta does not appear to pl^ a role in controlling this dmsaging first generation of BCW larvae. No pathogenic microorganisms were found associated with these parasitoids. Despite the large numbers of BCWs released weekly in the barriers, relatively low nmbers were recovered (Tables 1 and 2). The low recovery Table 1. Parasitoids emerging from released third instar Agmtia ipstitm (BCIf) larvae in the summer of 1983 Parasitoids Unidentified Total Ichneumonidae Collection BCWe B. Total t date collected Total % Total t Total z parasitism 6/6 48 0 0 20 41.7 41.7 6/13 58 2 ils 0 m m 5 8.6 12.1 6/20 37 7 18.9 3 8.1 0 27.0 7/1 16 6 37.5 1 6.3 0 22.6 7/5 26 22 84.6 0 . * 0 84.6 7/12 17 8 47.1 0 .* 1 5.9 52.9 7/18 7 0 « • 0 « # 0 0 7/25 5 0 0 • • 0 0 8/1 18 5 27.8 0 0 27.8 8/8 18 10 55.6 4 22.2 0 77.8 8/15 13 8 61.5 4 30.8 0 92.3 8/22 17 1 5.9 3 17.7 3 17.7 41.2 8/29 18 2 11.1 10 55.6 0 66.7 9/8 149 6 4.0 136 91.3 0 93.3 9/16 11 1 9.1 5 45.5 0 54.6 9/21 89 10 11.2 62 69.7 0 83.2 ^Excluding dead BQWs. Itfmwtitmfi, «ajariiy identified from ixmatwrif specimens. ^Bonmtia omUx, Fig. 1. Parasicoids emerging from released third inscar ipaiUm (Ifaifiiagel) larvae in the sumner of 1983 0 Heteorua leidmntrm (Weamael) S Bometia loomta (Fallan) 0 unlmmm ichiwuxmnid %PAM8ni8M SSSSSGASSL Fig. 2, Paraaitoide emerging frnai released third instar Agr&Ha ipsilan (Ihifnagel) larvae in the summer of 1984 B HaUtorm leidmtitms OWmael) 0 BonmHa omta (Fallen) DATE K MLON COLLECTED Table 2« Parasitolds emerging from released third instar Agmtia ipsil&n (BCU) larvae in the summer of 1984 Parasitoids Total b e Unidentified Collection BOIs »i_iê2«2_ % date collected Total X Total % Total % parasitism 6/6 25 1 4.0 0 • « 0 4.0 6/U 15 0 ,, 0 •. 0 0 6/18 137 39 28.0 1 1.0 0 29.0 6/25 37 4 10.8 0 ,. 0 10.8 7/2 19 1 5.3 0 . « 0 5.3 7/9 20 2 10.0 0 « « 0 10.0 7/16 44 22 50.0 5 11.4 0 61.4 7/24 20 9 45.0 10 52.6 0 95.0 7/30 15 2 13.3 12 80.0 0 93.3 8/6 7 1 14.3 ' 6 85.7 0 100.0 8/14 2 0 ,. 0 • • 0 0 8/20 2 2 100.0 0 • * 0 100.0 8/30 0 0 ,, 0 •• 0 0 9/12 3 I 33.3 1 33.3 0 66.7 ^Excluding dead tiOis, ^U&toGrus leoiventma, majority identified from larval specimens. ^Bonmtia aomta. 26 ratas appaarad to occur in tha pariods of axtrana haat and drought aach aummar, aapaclally in August of 1984. Each luamar, only one Erihcnts t*mbrem vas racovarad from tha col- lac tad ECB larvaa (Figs. 3 and 4; Tablaa 3 and 4). Thara waa only ona major parasitoid of tha ECB larvaa, a braconid, Maaroaentna Qvandiit which parasitiaad up to 80 parcant of tha collactad ECB in 1983 and 40 parcant in 1984 (Tablas 3 and 4). Parasitism of tha ECB was much lowar in 1984 ralativa to 1983. Ninaty-si* fi. gmnàii vara praparad both as wat mounts and stainad sactions for anamination with tha light microacopa. Of thasa parasitoids, 65.5 parcant vara found infactad with a microspori- diust prasumbly Itoeem pifvmtota, and 24 parcant with opportunistic bactaria. As %ws obsarvad in the collection of the BCWs, relatively few ECB larvae were recovered, despite the com being artifically infested with egg masses (Tables 3 and 4). The low recovery rates in August of 1984 were probably the result of extremely hot md dry field conditions. Fro» this small, local survey of the parasitoids of the BCW and ECB using released insects* one may conclude: that H, levivmtvue and B, aomta are the two major parasitoids of the BCW, neither of which appears to be associated with a pathogenic microorganism; and tâ, gmnàii is the primary parasitoid of the ECB, and a large proportion of this parasitoid's popu- latioo supports a microsporidiun, probably the S. pifvamta found in its host (Andreadis, 1980). Fig. 3. i*ar«slcoids emerging from OatHnia mMlalùi (#&bmer) larvae collected in (be wcr of 1983 00 Neu>r>9omtma gtwdHi Goidmicli 0 BHboma Wmbfww (Graveoiiorst) DATE O.MUMUUS COLLECTED Fig. 4. Parasicotds «nergins fr«wi 0@trini4i màdlàli^ (Hïïboer) collected lu the summer of 1984 B ikuaromntrm grmdii Ck>id«nich 0 Uimbpcma (CravWborgt) DATE O. NUHLAUS COLLECTED 31 Table 3. Parasltolda emerging from naturally occurring and introduced Ostrinia nuHtaîie (ECB) larvae in the summer of 1983 Paraaitoids Total Collection ECBs S. têT9bwma^ Total % date collected Total % Total % parasitism 7/\ 7 5 71.4 0 71.4 7/8 10 8 80.0 0 @0.0 7/14 96 28 29.2 0 29.2 7/21 16 7 43.8 0 43.8 7/27 42 3 7.1 0 7.1 8/3 30 4 13.3 0 13.3 8/11 21 12 57.1 1 4.8 61.9 8/18 246 93 37.8 0 •• 37.8 8/25 313 26 8.3 0 • • 8.3 8/31 108 9 8.3 0 » • 8.3 9/8 19 3 15.8 0 15.8 9/16 19 2 14.3 0 14.3 ^Excluding dead ECBe. gnmdii. ^Evihovm tevebrma. 32 Table 4. Parasltolds emerging fron naturally occurring and Introduced ûetpinia nuMtaHe (ECB) larvae In the summer of 1984 Parasltolds Total f. Collection ECBs tercbrane'^ Total % date collected Total X Total X parasitism 6/26 12 0 • I 0 0 7/2 34 3 8.8 0 8.8 7/9 61 6 9.8 0 9.8 7/16 34 1 2.9 0 2.9 7/23 SI 3 5,9 I 1.9 7.8 7/30 140 3 2.1 0 2.1 8/3 17 4 23.5 0 23.5 8/14 5 2 40.0 0 40.0 8/22 7 0 0 0 8/28 16 0 0 0 9/12 9 0 0 0 ^Excluding dead ECBe. ^Ma&r€0&n.tvu9 gvanMi, tevebvmw. 33 PART 11! MAlltTE8ANC£ OF BOmETIA COMTA (FAuIk) mcmcmms ammii GOZOANICH, AHD rnsuA mumoai HEmsc coiomis 34 iimKœucTMm Using small seals surveys of the local parasltold complexes of black cutworm (BCtt), Agrotio ipéiîcn (Hulnagel) and European com borer (ECB), Oetrinia mddîaîis (Hubner) populations at the Com Insects Unit Research Laboratory in Ankeny, Xova, 1 found Metsona levivmtrue Wesmael and S MaoiK MATERIALS AND METHODS In Cha mummer of 1963, BCW and ECB larvae were collected aa deacrlbed in Part I. 5. aomta and M, grandii emerging from collected BCW and ECB larvae, respectively, vera used to aatabllih laboratory colonies of theme apeciea. No Ù. ishompeoni tmergad from the field collected ECB. The L, thompeoni waa eatabliahed from pupae raceivad from R. Jonea.* Bormetia amtai The 5. oomta were placed in a 29 * 29 % 31 cm acreened wooden cage provided with cotton wicka, one soaked with a 30% honey solution airà a second with water. The cage was maintained in an incubator at 27*C, 70% RH, and continuous light. Levine and Clement (1981a) described a method of inducing femal# B, Qomta to ovipoait on fecea from BCW larvae fed a pinto bean diet. I fouw* that by soaking BCW facal material in water, the supernatant sprayed on filter paper in a plastic petri dish would alao induce ovipoeition. BCW larvae were parasitized manually. With the aid of a dissecting microscope, individual planidia were placed with a camel's hair brush on the dorsum of fourth instar BCW larvae, just posterior to the head capsule. The parasitized BCWs were individually sealed into 30-ml plastic cups of BCW diet. The cups were incubated at 27*C and 702 RH until the host of the parasitoid pupated. To determine the most efficacious manner of storing planidia over time, fecal supernatant-treated filter paper in petri dishes exposed to ^R. Jones, Department of Entfwology, University of Minnesota. St. Paul, Mn 55108. 36 f«Ml« B. oomta for six hours was collsctsd for sight consscutivs days. Each day ths planidia in sach dish wars counttd (rang# 68-232) and the dishss vers covsrsd, with ths lids, held in placs by a strip of masking taps. Four of thsss dishss wsrs placed in a 4.4*C eoolsr and ths other four, in a 27*C incubator. Every five days, the number of living plani dia was countsd. Ths filtsr papsr in OfO of ths dishss stored at each temperature was dnspsnsd with distillsd watsr every five days. To study the iafMict of multiple parasitism by the B. amta on the BCU larvae, five BCUs from sach instar (nsonats to sixth) were each p@r@- sitiasd with a single planidiua as described above. After placing the planidium on the host's body, the parasitoid was observed until tt pene trated the larval cuticle. Five additional hosts from each instar were each parasitised in a similar fashion with two planidia and another five such BCWs were left unparasitised. Each BCU was sealed individually into a 30-ml plastic cup containing BCU diet. All cups were incubated at 27*C and 70% RB. This experiment was replicated three times over days. All insects were inspected daily and larval death and/or host or parasitoid pupation was recorded. Parasitoid pupae were weighed eight days after pupation. Ma&poeentvm gvandiii The M, gremdii were maintained in tw 29 % 29 X 31 cm screened wooden cages supplied with cotton wicks, one soaked with a 30% hon^ solution and one with water. The cages were incubated in a 14 hour 21*C pbotophase, ten hour 16*C scotophase, and a KB of more than 702. Pathogen free, second to fourth instar ECB were presented 37 to th# paraaitoid* on ECB diet (without fomaldchyda and with mold inhibitora) (Lawia and Lynch, 1969) in a 100 x 15 am plaatic patri diah, tha aurfaca of which had baan acorad. Tha ECB wara than covarad with a aingla layar of chaaaa cloth which waa aacurad with maaking tapa. Tha fraaa which accumulatad on tha aurfaca of tha diat waa aufficiant to attract tha female grca^i to ovipoait in tha ECB through tha chaaaa cloth barrier. Tha ECB ware expoaed to the M. grmdii in auch a manner for 24 houra, after which time tha hoat larvae were individually placed into 30-ml cupa containing ECB diet. The cufw were incubated at 27*C and 70% RH until the hoat had pupated or tha H. gmnâii larvae had emerged. One or two gmndii larvae from each paraaitiied larva were aquaahed on a glaaa alida and checked under a compound microacope to confirm the preaenca or abaance of N, çyrmêta aporea. If the microaporldian aporea were preaent, any adulte emerging from that cup were placed in the aecond cage, leaving tha firat cage for the noninfected M, gvmàii adulte only, l^âella thon^eemii The Ù. thoff^&oni colony va» maintained in a IB % 18 % 20 cm acreened wooden cage under the same environmental condi tion* and supplied with the same food as the M, granMi. The female Ù, thampsmi were induced to larviposit on frass from ECBs fed seedling com. This frass was spread on moist filter paper in a 100 % 15 mm plastic petri dish which was placed in the parasitoid's cage for up to six hours. Mating and larviposition was stimulated by placing the cage in sunlight. With a camel's hair brush and the aid of a dissecting microscope. individual first instar I. thompsani maggots were placed on the dorsum of fourth instar pathogen-free ECB larvae, just posterior to the bead capsule. 38 Each parasitized ECB was placed on ECB diet in a 30-ml plastic cup. The cups were sealed with polyethylene-lined lids and incubated at 27*C and 70% RM until the host or the parasitoid pupated. 39 RESULTS AND DISCUSSION Bn#rg#nc# data collected on 895 Individual* frota the B. eomta colony ahowad that the naggota davaloping in th# BCUa hald at 27*C and 70% RM raqulrad alx to 19 daya to amarga from thair hoata (x • 10.63 daya S.E. t 1.60). Bomëtia aemtc pupaa held in aimilar conditiona ecloaad in aix to 13 daya (x • 9.99 S.E. t .96, n » 839). From the multiple paraaitian atudy, no two B. omta were able to aucceaafully pupate froa a aingle hoat. the day* required for the B. QcmtQ to develop to the pupal atage decreaaed aa the inatar of the hoat larva at the time of paraaitiaation increaaed (Table 5). These observa tion* agree with those of Levine and Clement (1981a). Only in the second inatar BCW hoat did the presence of two competing B. amta maggot* sig nificantly increaae the time required for one to develop to the pupal stage. Day* to B. omta pupal ecloaion generally increased with the age of the host larvae in which the maggot developed (Table 5). This trend was not significant, unlike the study reported by levine and Clement (1981a) where the pupal period wa* significantly (F < 0,1) longer for parasitoids reared from larvae parasitised as sixth instars (11.2 t 0.2 days n » 9) than for those reared from larvae parasitised as third instars (10.5 t 0.1 days n • 14). The pupal weights of the B. oomta increased with an increase in the instar of the BCW in which they developed (Table 5). Only the male puparia within the third instar BCVs showed a significant difference in the weights between the singly or dually parasitized hosts. Table S. Intact of single and dual parasltlsw by Bemusiia mmta (Fallen) on AgroHa iptdUm (BCM) (Wufnagel) Nuiri»er tiCW mortality t BCW B, &cmm pupatioo Days to Mean welgjit (mg) Instar of % Rays pupae % Bays B. eomta B. ganta pu paria B, oamta ecloslon Female Male i 0 15,0 a* 14.7 a 74.7 a 0 1 60.0 b 4.0 b 6.7 b 26.7 b 15.3 6.7 83^0 29.0 2 100.0 b 2.3 b 0 b 0 a » • .. .. .« 2 0 6.7 a 7.3 a 90.9 a 0 a 1 26.7 a 9.7 a 13.4 b 60.0 b 16.5 a 11.4 a .. 80.3 a 2 13.4 a a.o a 0 b 80.0 b 21.5 b 11.1 a 89.0 55.4 a 3 0 0 100.0 a 0 a •• •• .« I 0 ,« 6.7 b 93.0 b 14.3 a 11.7 a 105.8 a 78.3 a 2 0 • * 0 100.0 b 13.9 a 11.7 a 110.8 a 87.9 b 4 0 0 100.0 a 0 a •• I 0 •« 6.7 b 93.0 b 10.3 a 10.8 a 114.7 a 86.8 a 2 0 •• 0 b 100.0 b 9.9 a 10.8 a 114.7 a 88.6 a 5 0 0 100.0 a 0 a •• 1 0 •• 40.0 b 60.0 b 9.2 a 11.9 a 129.0 a 99.0 a 2 0 •• 20.0 b 80.0 b 9.4 a 11.3 a 128.8 a 96.2 a 6 0 7.0 9.0 100.0 a 0 a • e «• I 0 •• 33.4 b 60.0 b 9.1 a 11.6 a 123.5 a 110.5 a 2 0 26.7 b 73.3 b 8.7 a 11.9 a 130.8 a 102.7 a * Means (within columns within instars) followed by the same letter, are not significantly different (P < .OS) as determined by Duncan's multiple range test. 41 Both singl* and dual paraaitian had aubatantial (P < .OS) impact on first inatar hoat mortality. Thia mortality occurred very early after paraaitisation (2.3-4 daya) and therefore probably resulted from the actual penetration of the pianidia into the comparatively small hosts. Parasitism by the B. somta caused significant (P < 0.5) reduction in the percentage of BCW pupation in every hoat stage» whether by causing an early death of the host in the first two BCW instars, or by producing a B. acmta puparium from the older hoat instara. It seems possible from this laboratory experiment that B, acmta pianidia could be used to control any instar of BCW in the field, providing large numbers of pianidia could be easily made available. The technique of spraying filter paper with BCW frass supernatant allows the collection of large numbers of pianidia without the necessity of separating the maggots from the fecal pellets. Data trtm the cold storage study (Table 6) indicate that the pianidia can be stored on the original filter paper in a covered petri dish at 4.4*C for five days with minimal mortality. Repeated moistening of the paper was detrimental to pianidia! longevity. The K gvandii required 15.74 days S.E. t 2.46 (n • 195) to emerge from their ECB hosts in the N. pymueta free colony and 15.42 days S.E. t 1.64 (n » 107) in the S. pyvccueta infected colony. The braconid then required 10.19 days S.E. t 1.34 (n » 175) and 10.33 days S.E. t 1.61 (n * 101) to eclose as adults from the noninfeeted and the infected colonies,respectively. The presence of the i?. pifraueta in the colony did not significantly (P < .05) effect these ^ang. The microsporidium did 62 Table 6. Impact of storage at two temperatures» with and without water. on Bométia ocmta planldla Temp. +/- Mean X mortality* •c water Day 5 Day 10 Day 15 Day 20 4.4 + 13.8 a 100.0 a 100.0 a 100.0 a - 9.4 a 35.5 b 70.5 a 98.5 a 27 + 81.0 b tOO.O a 100.0 a 100.0 a - 93.7 b lOO.O a 100.0 a 100.0 a follmcd by the mm (within cach day) not significant ly different (P < .05) as determined by Duncan's multiple range test. appear to decrease the nu8*er of female adults able to eclose in the all female group only (Table 7). Lyâella thtmpaoni required an average of 9.2 days S.C. i 2.2 (n > 30) to emerge from their ECB hosts held at 27*C and 70% RH, and an additional 8.0 days S.E. t 1.9 (n • 45) to eclose as adults under the same condi tions. Ninety-four % of all L. thompsoni adult* were able to eclose. 43 Table 7. Eclosion of Haotoamtrus adules developing in Nosma pyrauota infected and i?. p^mm«ta-free ECB hosts Adult groups Mean number adults (±S.E.)/colony Soavna-tf Uonmm infected All female 8.2 (±6,9) a 5.6 (t3.7) b All male 8.7 (±6.2) a 7.7 (±6.0) a Mixed groups Females 4.4 (±3.7) a 3.6 (±3.1) a Males 4.0 (±3.6) a 6.2 (±5.3) b Both sexes 8.4 (±5.5) a 9.8 (±6.1) a Means (within rows) followed by the same letter do not differ significantly as determined by Student's t test (P < .05). 44 PART 111; IMPACT OF VAIRmBFm mCATBIX (KIUttOR), miBimBPM SP., AMD BÂCIUUS fUUBISGmSIS SERLINER SUBSPECIES imsTAMi m mirsfiA COMTA WITHIN AGBOTIS miicgl (WFNACm,) IK^TS 45 INTRODUCTION Thtt Blcroporldia, Vairimorpha ntoa^trix (Kramer) and VcdHmrpha «p., ar« pathoganic to tha black cutwrm (BCW), i^votiê ipeiîon (Hufnagal). &*poaure of aarly BCW inatara to V, maatrix and relatively higher con- centratlona of ^etivinwrpha ap., cm cauae Imandlate larval mortality. Expoaure to lower concentratlona of either apeclea, or exposure of later larval Inatara may Induce the development of a mlcroaporidioala condition, cauae the larvae to become aluggiah, and decreaae their feeding activity (Coaaentine and Lewia, 1984). Both Vairimrpha species, when incorporated into bran bait formula tions, significantly reduced first and third instar BCW larval feeding on seedling com under greenhouae and field conditiona (Coaaentine, 1982). These microsporidia have also been shown to be pathogenic to other lepidopterous corn pests (Maddox and Sprenkel, 1978; Lewis et al., 1982; Hamn and Ellie.^unpubliehed). Consequently, these microsporidia are strong candidates for development as biological control agents. It is therefore Important to assess their impact on or compatibility with potential parasitoids of these com insects, especially as there are numerous records in the literature of microsporidian infections in para sitoids within diseased hosts (Allen and Brunson, 1945; Tanada. 1955; Blunck, 1958; York, 1961; Brooks and Cranford, 1972; Hostounsky, 1970). In this study, I have examined the effect of both Vaivimovpha species on ^SDA, ASS, Insect Biology & Population Management Research Laboratory, Tiftcm, GA 31793. 46 the tachinld, Bometia aomta (Fallln) which» although It paraaltlxas lacond and third ganarationa of tha BCU In Iowa, haa tha potential to be introduced earlier in the aeaaon to protect seedling corn from firat gen eration BCW larvae (Part 1). Low mortality of neonate BCWa haa been observed when they were fed BaoiZtue tkuHn^enêie Berliner subspecies hapâtakii however, later instar» of the BCW do not seem to be susceptible to this bacterium (Ignoffo and Garcia, 1979). Other lepidopteroua corn peata are susceptible to 3. tkuHnçieneie, (Krieg and Langenbruch, 1981) and it is currently registered for use against the European corn borer on corn. BCWs may encounter and ingest 8, tehuHngimeis in the field and later become parasitized with 8, acma. Therefore, the impact of this bacterium on 8. amta was assessed. 47 MATERIALS AND METHODS Pathogen aaaays: Aaiaya for each pathogen were dealgned as factorial two x two randoalied complete block experiments. Two treat ment* were the presence or absence of an infection within the BCW hosts. These factors were crossed with the parasitism, or lack of parasitism by 8. aomta planidia. 1 2 i^airimrpfta sp. and V. m penicillin-G sodium was added to each 100 ml of microsporidian suspension, as wll as the control, to retard bacterial growth on the surface of the diet. B, thtringienaia subspecies kuntcki (HO-263)^ was propagated on nutrient agar plates from lypholised powder. The bacterium was harvested in sterile dH^O four days after plating and was applied to similar cup* 4 in .2 ml dWgO 3.4 x 10 spores/mm diet surface. After the diet sur face had dried, a single neonate BCW was placed in each of 50 cups/treat ment. the cups were sealed with polyethylene-lined lids and all cups were incubated at 27*C and 70% relative humidity (BH) for one week. BCW larvae incubated in a similar manner in 50, 30-ml cups of diet, surface treated with .2 ml dWgO were the controls. ^Originally obtained from J. Hans, USDA, ARS, Insect Biology & Popu lation Management Research laboratory, Tifton, GA 31793. ^Originally obtained from J. Maddox, Illinois Natural History Survey, Urbana, 111 61801. ^Originally obtained from H. Dulmage, USDA, ARS, Brownville TSC 78520. 48 A laboratory colony of B, oomta was induced to oviposit on filter paper soaked with the supernatant of frass from 6CW larvae fed artificial media, mixed with dHjO. A single B, acmta planidium was placed with a camel's hair brush on the dorsum of each of IS infected BCWs just posterior to the head capsule. The BCWs were then placed on fresh BCW diet (Cossentine* 1982) in 30-ml plastic cup*. Fifteen noninfected (con trol) BCWs were similarly parasitized with B, acmta planidia. An addi tional IS infected and IS control BCWs were placed on clean diet without exposure to 8, osmta planidia. All assays were incubated at 27*C and 70Z RH. The above assay, for each of the three pathogens, was replicated five tims over days. Each insect was examined daily for 30 days and parasitoid pupation and eclosion was recorded. Bonmtia atmta pupae were weighed eight days after pupation. A single BCW larva from each treatment was fixed in 58'C alcoholic Bouin's for one hour and an additional 24 hours at room temperature, three, five, and seven days after parasitization. Two larvae from each treatment were similarly fixed on day nine. These larvae were embedded in paraplast, sectioned, mounted on microscope slides, and stained with a modified Coodpasteur's stain (Kallory, 1938) (those exposed to B, thztyingiensie}, and a Giemsa colophonium stain (Shortt and Cooper. 1948) (those exposed to microsporidia), for histological examination. lOjQ studies: Vaivimovpha sp. and V, neaatvix spores were applied in .2 ml dH^O to the surface of 50 30-ml diet cups at concentrations of 50, 25, 12.5, 6.25, 3.13, and 0 spores Vaivimvpha sp./mm^ and 25, 12.5, 49 6.25» 3.13, 1.56» and 0 sporaa V. necatrix/mm 2 diet surface. One-half g of penlcillln-G aodium was added to each 100 ml of odcroaporldian suspen sion aa well aa to the control to retard bacterial growth on the surface of the diet. After the diet surfaces had dried, one neonate BCW/cup was incubated on the pathogen-treated diet for one week at 27*C and 7% RH. After one week, 15 BCWa from each microaporidian concentration and the control had a single B, aomta planidiua placed behind each head capsule. ^ additional 15 cupa from each concentration were left unparaaitiied. All 30 BCWs were transferred to clem 30-ml cups of diet. Each assay was incubated at 27* and 701 RH and was replicated five times over days, the insects were examined daily and data on days to parasitoid pupation, adult eclosion, and puparial weights were recorded. After % days, all hosts not yielding f. ocmta pupae were dissected. Any dead parasitoid larvae within these hosts were vigorously rinsed several times in dNgO with TWeen W to remove external spores. The mag gots were then weighed, homogenised, and the number of spores per mg of maggot weight was counted. A spore emmt within the host cadaver, or of the whole host when it was not parasitised, was similarly obtained. The ovaries from female adult 3. acmta pupating from V. waatWar-infected BCWs were dissected out, fixed, mâ prepared for histological examination in the manner described above. 50 RESULTS AMD DISCUSSION Pathogen a##ay#: V» nêoatrùe, Vairimorpha «p., and 5. thutingiénoie aubapacias hiœêtcki all appear to have aoma datrimantal affect on the davalopmnt of B. amtOt within BCW hoata. Baoilîu» thuHnffimtie ia not pathogenic to the BCW and alone did not cauae aignificant mortality (7%) in the host (Table 8). The presence of the bacterium did however* signifi cantly (P < .05) decrease the BCW mortality resulting from parasitism by the B. (Table 8). There were correspondingly, significantly (P • .02) fewer f. amta able to develop from these B. thuHingieneia containing BCW hosts (Table 9). The B. oemta adversely affected by the B. thuring- ienaië were nwst likely killed very early in their development as their BCW hosts did not die. Larvae prepared for histological examination exhibited bacterial rods only in the lumen of the BCW guts. Consequently» there would be no B, thuvingieneis available within the BCW tissue for the developing B. amta maggot to consume. Bcurillue thuHngieneie rod# were observed externally in the intersegmental mes^ranes of the BCWs cuticle, and it is possible that a planidium entering the host may encounter and ingest B, thuHngieneis spores and/or crystals during its period of pene tration. This may be particularly likely* considering that the paraeitoid is observed to usually enter its host through an intersegmental membrane where an accumulation of B, thuHngiemia may be found. This puncture in the host is maintained as a breathing pore and it is possible that B. thuringieneie passed through this entrance similar to the manner in which host microsporidian spores of Uos&na me&nili (Paillot) entered Apantelea glomeratiis (L.) (Braconidae) larvae through a respiratory cavity in their 51 Table 8. Effect of Baoitlua thurii^tnaie subapacies kuMtakit Vairimorpha moatrixt and yctirimrTpha ap. on AgroHs ipeiton (BCW) mortality, ttlth, and without the affect of paraaltlam by Bommtia ocmta X Mean BCW mortality* Pathogen With B. donta Without (control) B. thutin^meie 74 b* 7 a Control 96 c 5 a V, maatHs 99 b 98 b Control 94 b 3 a Vaivimrpha ap. 100 b 82 b Control 98 b 7 a Maana within each pathogen group followed by the sane letter are not significantly different (P < .05) aa determined by Duncan's multiple range teat. Table 9. Effect of Baaillm thuHn^iemis (#.&.) on Bemmtia mmta parasitising -treated Agrotig ipeilon larvae Means* Test variable Control % B,0, parasitism 70.0 a 97.0 b Z B.9, eclosion 20.0 a 21.0 a Days to pupation 11.9 a 12.7 a Days to eclosion 10.4 a 10.5 a Weight (mg) - female puparia 115.1 a 105,1 a - male puparia 89.2 a 83.8 a *Meana within row# followed by the letter are not significantly (P < .05) different as deteraioed by Student's t-test. 52 rcctrum (Hostounsky, 1970). Several dipteran maggots are readily killed when they ingest crystals of 5. thœingimBie (Kreig and Langenbruch» 1981) and B, oomta may be similarly susceptible to B, thuringimeis crystals. Bomêtia aomta able to develop in hosts exposed to 5. ihwfing- itmtia exhibited no significant puparial mortality or increase in days to pupation or adult eclosion, and there were no changes in puparial weights (Table 9). Three other tachinid parasitoids, VoHa mmlis (Fallen) Beasa fiAgaa: (Rondani), and Zénilla âotoâa (Meigen) have been recorded as exhibit ing no detrimental effects from ingesting 8. tUn^n^ênaisi however, they were administered the bacterium as adults (Wilkinson et al., 1975; Hamad, 1979). Both Vedrimrpha species are effective pathogens of BCV larva (Table 8). Parasitism by S. ^ma does not interfere with the near 100 percent control of the BCVs by the K mmivix or Vairùmrpha sp. at 25 and 30 spores per mst^ of diet surface, respectively. However, both micro- sporidia have detrimental effects on F, amta maggot* developing within infected hosts. Vai^'ùmppha maatrix significantly (P < .0001) decreased the number af B, ûmta able to pupate from BCW hosts (Table 10). The presence of the V. maatrinf significantly (P < .05) decreased the days required for adult paraeitoid eclosion and the weight of the male puparia (Table 10). Vaijnmorpha sp. also decreased the number of B, aomta able to pupate from new hosts from 91 to 53 percent (Table 11). As well, this micro- sporidium decreased the days to adult B. aomta eclosion and the weights of 53 Table 10. Efface of Vàirimrp^ ntaatrïx on Bomttia acmta (S»a.) paraaltlting infactad Agrûtie ipêiîon (BCW) larvaa Mean#* Test variable 7. maatrix Control % B.o. parasitism 6.0 a 96.0 b % B.q, acloaion 30.0 a 28.0 a Days to pupation 19.8 a 12.3 a Daya to acloaion 8.0 a 11.0 b Weight (o#) - female puparia ** 127.4 - male puparia 78.0 a 86.5 b *Maan# within row# followad by (ha tana latter ara not significantly différant (P < .OS) as determined by Student's t test A# No female 8, etmtû found in V, moatvix infected hosts. Table 11. Effect of VaiHmefvjpha sp. of amta parasitizing V, sp. treated Agmtie ifeilm larvae Means* Test variable y. sp. Control % B.a. parasitism 53.0 a 91.0 a % B,a, eclosion 23.0 a 15.0 a Days to pupation 12.4 a 11.2 a Days to eclosion 9.9 a 10.3 a Weight (b^) - female puparia lOl.l a 118.9 a - male puparia 79.8 a 87.4 a Means within rows followed by the sane letter are not significantly (P < .05) different as determined by Student's t-test. 54 the pup«ria; however, none of these effects were significant at the .05 level (Table 11). The lover percentage of parasltoid pupation from (Wr&wrp&i-infected hosts is probably not a result of the BCWs dying before parasitoid develop ment is cofi^lete, but Instead, is a direct effect of the microsporidia on the parasitoid. The LT^^ for BCW exposed as neonates to 25 and 50 spores 2 per m of diet surface of V, ma&trix and VedHmrpha sp. are both 14-21 days (Cossentine, 1982). Developmental time for the B. omta until pupa tion is 10-12 days. Therefore, the Bmrntia should have had tin» to develop and pupate prior to the death of the hosts. Histological examina tion of the 8. somta maggots within V, infected BCW hosts showed the spores primarily restricted to the gut (figs. 5 and 6). Bmmtia amta dissected from dead V, maatriX'inteeteû larval hosts contained the spores throughout their bodies. Within V(HHmrpha sp.-infected BCW hosts, spores were found within the gut of the B, amta maggot (Figs. 7 and 8). Ovaries from female adult B, aemta that pupated from V. neaatvix-ixitecteû BCWs did not contain any microsporidian spores and therefore the potential for transovarial transmission of the spores seem# negligible. The decrease in parasitoid puparial weights caused by the two micro sporidia is possibly a result of developing in smaller host larvae. Both VaiHmrpha species significantly (P < .05) reduce BCW larvae weights (Cossentine, 1982). iCjjj studies; To better understand the impact of both YaCHmovpha species on the development of B» aomta, the parasitoid was allowed to develop in BCW hosts, exposed to a gradation of spore concentrations (25 Fig. 5. Bomêtia oomta (Fallln) maggot tdehln a Vaiivimorpiui neaatrix (KraMr)-inl«ettd Agrctiê ipeiîcn (Hufnagal) larva <350X) Ic » B, acmta maggot: 1 • tnlactad hoat tiaaua Fig. 6» Vaivimcrpha moatvix iVLrmst} spores vitbin the alimentary emal of a Bametia samta (Fallm) maggot (14(K)X) al • allmm*tary canal: sp » V, mo€(tH9 spore Fig. 7. Bmrntia amta (ftdiin) maggot within a yairimcrpha #p. Infaetad Agrotie ipeiltm (Kufnagal) larva <875X) Be • 3. aemta maggot; i • infaetad hoat tiaaua Fig. 8. Vaipim&rpha ep. eporea within the alimentary canal of a Botmetia camta (Fallen) maggot (1400%) al » alimentary canal; sp « V. sp. spore 59 CO 0 and 50 to 0 sporas of 7. néoatrix and VaiHmrpha #p., raapactivaly par an2 of diat aurfaca). Tha concantratlon of V, nêoatrix raquirad to 2 kill SOX of tha BCUa in 30 daya waa 3.6 aporaa par mm of diat aurfaca confidanca interval (CI) • 6.22-1.81. Tha 5. a&ffta developing within aimilar BCWa expoaed to V. nêoatrix exhibited an of 2.1 aporaa per 2 mm of diat aurfaca while developing in infected BCWa which had been expoaed to concentration» of y. moatHx, 1.71 x lower than that concentration required to kill 301 of the BCW hoata thenaelvea. Thia aupporta the earlier auggeation that the Smmtia are killed by the V. nesatvix itaelf and not indirectly by the death of their hoata. The alopea of the two logarithmic regreasion lines for tha BCW and the 5. (Kmta expoaed to moûtrix wwre parallel. The V. maatvix apore counta par of infected tiaaue were con- aiatently loirer in the f. aomta maggot tiaaue than in the BCW hoat (Table 12). Thia awy alao indicate that the B. amta waa more auaceptible to theK moatvix than waa ita hoat. The weights of both the wle and the female B, acmta puparia decrease with an increase in the concentration of V. moatvix to which the BCW hosts had been exposed. This supports the earlier suggestion that the smaller size of the parasitoid puparium is a result of the smaller host in which the parasitoid is developing (Table 13). The concentration of Vaivimavpha sp. required to kill 50% of the BCWs after 30 days is .94 (CÏ 6.65-0.02) spores per diet surface. The for the Bcmnetia developing within these BCW hosts is 3.87 (CI 9.33-0.67) 60 Table 12. Vctipimrpha nêaatrix spores per mg of AQTOHQ ipaitm and Bcmêtia acmta (issue after development of B. ecmta within infected Agrotie ipeiîm hosts V. nêoatrùs Spores/ag host tissue (xlO^? spores/^ 4. ipeilm B. ocmta diet surface 25.0 20.5 a* 13.6 b 12.5 19.3 a 15.4 b 6.25 22.0 ab 16.1 b 3.13 23.6 a 11.6 b 1.56 19.2 ab 9.1 b 0 0 ni 0 ni Means followed by the sne letter are not significantly different (P < .05) both within colusms and across rows, as determined by Duncan's multiple range test, ni - not included in analysis. Table 13. Effect of Vedvimovpha meatvis within AçfPoHs ipHlm hosts on mem wights in ^ of Bomêtia oamta puparia, weighed eight days after pupation y, nemiHx Mean puparial weight (mg)* spores/m»2 Female Male Both diet surface 25.00 •• • f 37.0 a 12.50 # * 63.0 a 46.6 a 6.25 86.0 a 97.0 b 88.3 b 3.13 125.8 b 83.6 b 100.6 b 1.56 126.5 b 90.2 b 95.4 b 0 119.6 b 98.2 b 109.5 b *Heans (within columis) followed by the earn# letter are not signifi cantly different (P < .05) as determined by the Duncan's multiple range test. 61 •porc» per tmi2 diet surface. Therefore, SOX of the B. oomta died while developing in infected BCWs which had been exposed to concentrations of Vediriincrpha sp. .24% higher than the concentration required to kill 50% of the BCW hosts themselves. The slopes of the two logarithmic regression lines for the BCW (slope • 1.29) and the B. âcmta (slope • .73) exposed to VedHmrpha sp. were not parallel. V&iHmrpha maatrix has a more inmed- iate effect on the host, while the VaiHmrpJm sp. requires a longer time to kill the BCW. Therefore, although the Vairimrpha sp. LC^^ is signifi cantly lower for the BCW than for the 8, amta* it is unlikely that the death of the BCW is what is killing the parasitoid as the B. acmta can pupate in 10-12 days. The B, amta appears to be less susceptible to the VaiHmrpha sp. than it is to the V. maatrix. Again, the VedHmrpha sp. spore counts per mg of infected tissue were consistently lower in the B, amta maggot tissue than in the BCW host (Table 14), As in the B, amta exposed to V. neoatviXt the weights of the 8, o&mta puparia decreased with an increase in the concentration of Vaivimvpha sp. to which the BCW hosts had been exposed (Table 15). This effect is more pronounced in the male puparia. Again, this supports the suggestion that the size of the pupariu# is dependent on the size of the host which is in turn affected by the Vaivimffvpha infection. Both Vcdvimrpha species appeared to have a greater impact on female B. oomta than upon the male parasitoid (Figs. 9 and 10). In the V, mmtvix studies 59% of the f. ocmta developing from the BCWs with no microsporian infection were female, while only 41% were male. The average percentages of females and males from BCWs with V, neaatvix infections were 62 Table H. Vairimrpha sp. spores per og ot Agrotis ipsilon and Bometia acmta tissus aftsr development of B, ocmta within infected ÂgrotÎB ip&iîon hosts mrirnrpKo •!>. SpotM/.» ho.t (» 10«)« spores/oB^ A. ipeiîon B. acmta diet surface 50.00 9.4 be 6.9 a 25.00 12.4 a 7.0 a 12.50 to.6 ab 5 5 ab 6.25 6.0 c 2.3 b 3.13 7.9 be 4.9 ab 0 0 ni 0 ni MSARS followed by the same letter are not significantly different Table 15. Effect of VaiHtwvpha sp. within AgvoHe ip9il mrlmtrpha .p. W #por##/w#2 Ponol. Mole Both diet surface 50.00 99.00 ab 61.67 a 63.65 a 25.00 82.00 a 75,05 ab 67.77 ab 12.50 118.50 ab 85.76 be 87.90 be 6.25 102.60 ab 104.38 d 102.14 cd 3.13 114.84 ab 88.84 be 78.82 ab 0 129.96 b 98.76 cd 116.12 d Means (within columns) followed by the smme letter are not signifi cantly different (P < .05) as determined by the Duncan's multiple range test. Fig. 9. Ptrccne and ml* Bometïa a&mta after development in VedHmvpha leegtrùe-infeeted and control Agvotis ipsiton larvae 0 female • male Fig, 10, Percent female and male Bornietia omta after development in Vcdvimrpha ep,-infected and control Agvotia ipsiton larvae TOTAL BJOOMTA ADULTS TOTAL BuCOliTA ADULTS 5 # ( # 9 $ 9 $ 65 30 and 26» reapactlvcly. This sax effect is more evident in the Vairvnorpha sp. assay#. Again, 59% of the 5. acmta developing from hoses vith no adcro- sporidian infection were female and 61% were male. However, the average percentages of femalea and males from Vàirùwrpha sp.-infected BCWs were 26 and 47, respectively, and these percentages were significantly differ ent (F < .05). the greater impact of both Va£j*^wrpha species on the female S. acmta is probably a result of the larger sizes of the females trtiich would require more time and imre uninfected host tissue in which to develop. 66 PART IV: IMPACT OP A NUCLEAR POLYHEOROSXS VIRUS FROM RACHIPWSIA OU (CUENEE) ON BOmETU CCMTA (PALLDI) WITHIN AQBOflS miWB (HUFNAGEL) LARVAE AND USE OF BOTH THE VIRUS AND THE PARASITOID TO CONTROL THIS HOST INSECT 67 INTRODUCTION BonntHa amta (Fallln) la • cachlnld parMitoid primarily of #«cond and third ganaraeion black cutworma (BCWa), Agr Iowa. It appaara to ba a vary afficiant natural anaaqr, paraaitizing up to 91% of BCW larvaa collaetad in a amall aurvay in Ankany, Iowa (Part 1>« Tha paraaitoid ia oviparoua, laying agga which inmadiataly hatch into firae inatar oaggota (planidia). Tha planidia ara partially protactad from dal^dration by cuticular plataa. Larga nuabara of tha planidia cm ba aaaily raarad and atorad for aavaral d^a (Part II). Tha nuclaar polyhadroaia virtM iaolatad from a mint loopar. BaoMpîmia m (Cuania) (ScMfPV) haa provan to ba a potant pathogen of BCW larvaa (iawia md Adama, 1979). In graanhotiaa and fiald taata uaing RcMIFV^inpragnatad wheat bran the feeding on aeedling com by first and third inatar BCWa waa aignificmtly reduced (Johnaon and lewia. 1982; (kwaentine, 1982). RoifliPV ia therefore a potential biological control agent for BCW and it ia Important to assess the virus' impact on BCW paraaitoida such as B. oanta. There are several atudiea reported in the literature in which tachinid paraaitoida paraaitizing nuclear polyhedroaia" or granulosis virus- infected hosts have been unaffected by the diseases unless they were unable to complete development before their host* died (Kelsey. 1960; Laigo and Tamaehiro, 1966). In this study, the impact of WWV on B. ocmtia developing within infected BCW larvae was examined. In additicm, the potential of the 68 S. omta alone» as well as in combination with RotÇtfV to control BCW larvae in the greenhouse and field was tested. 69 MATERIALS AND METHODS Laboratory amaaym: In th# first laboratory study, th« Impact of parasitism by B» oomta on th# BCW was examlnad by looking at food utili zation by th# host. Chromic oxid# (CrgOy),an in#rt ingr«dl#nt used as an indicator in nutritional analyses, was incorporated Into S5*C liquid BCW diet (Cossentin#, 1982) such that it represented four percent of the diet by weight. The treated diet wis distributed among SO-M plastic cups. When the diet had cooled and solidified, a single third instar BCW was placed in each of 20 cups. Ten of these BCWs had a single f. amta planidium placed behind the head capsule. Ten of the larvae were left unparasitized. Every day after treatment, each larva was placed on fresh BCW diet con taining chromic oxide and the used diet in the cup was frozen. After all the larvae had pupated, the frass from each of the ten cups from each treatment was consolidated for every day until pupation. The fraee was dried in an oven at l(K>*C and stored in a vacuum desiccator until analysis. The techniques of McGinnis and (Casting (1964) were used to analyze the frass and unconsumed diet. From these analyses the amount of diet con sumed and the percent digestion of the consumed diet was determined. This study was replicated three times over days. The laboratory RoWPV assay was designed as a two % two factorial experiment in a randomized complete block design, replicated five tines over days. Two treatments were the infection or absence of an infection in BCW larvae by RoMSPV. The virus was propagated in cabbage looper, Tviahoplmia ni (Hubner) larvae and then semi-purified using a modification of the technique of Tompkins et aX. (1981). Viral polyhedral inclusion 70 bodies (PXBs) suspended In dHgO vere applied in .2-ml aliquocs Co the surface of BCU diet in 30-ml plastic cups such that 200 PlBs were present per an 2 of diet surface. Fifty 30-ml cups of BCW diet were similarly superficially treated with .2 mi of dHgO. One-half g of penlcillin-G sodium was added to each 100 ml of viral suspension and the control to prevent bacterial growth on the surface of the diet. All cups were allowed to dry before a single BCW neonate was sealed into each cup with a polyethylene-lined lid. All cups were Incubated at 27*C and 70% rela tive humidity (RH). The other two factors were the parasitism or lack of parasitism of these RoMNPV Infected or nonlnfected BCW larvae by B. aomza. Bcmetia amta females were Induced to oviposit on filter paper soaked in a supernatant of BCW frass. Alter the BCWs had incubated on the virus- created diet cups for six days» a single B, &€mia planldlum was placed behind the head capsule of 15 of the BCWs from both the RoMNPV and the control treatments. The BCWs were sealed into 30-ml cups containing fresh MW diet and were Incubated as before. The insects were examined daily for 30 days and any parasitism, mortality, or host pupation was recorded. Three, live, and seven days after parasltlzation, a single BCW from each treat8»nt was fixed in hot alcoholic Bouin's fixative for om hour and at room temperature for 24 hours. Two BCW from each treatment wre similarly fixed on day nine. All fixed larvae were dehydrated through alcohols, embedded in paraffin, sectioned at 6 um on a rotary microtome, mounted on microscope slides, and were stained with a modified Azan stain (Hamm, 1966). 71 Greenhouse Experiment: This experiment tested the ability of RoWPV, S. ùomta planidia, and RoMNPV plus B. acmta planidia, to control third instar BCW larval feeding on seedling com. The study was done in January» 1984, when the interior temperatures of the USDA Plant Introduc tion greenhouse on the Iowa State University campus ranged from 32* to 20* C from day to night. Solar radiation was the only source of light used. Sixteen 35.6 x SO.8 x 15.2 cm flats of soil were planted with two rows of ten corn seeds (Pioneer^ 3537). A 31 x 46 x 10 cm galvanised steel barrier was embedded around the planted seeds in each flat to pre vent larval escapes. Ten days after planting, the com seedlings were thinned to ten plants per flat and the soil surface was treated with bran baits. Twenty- five ml of R<^(PV, prepared at a concentration of 6 x 10^ piBa/ml of dH^O, was sprayed with a plant mister onto 25 g of autoclaved wheat bran rolling in a 3.8 1 jar on a plaster batch mixer. The treated bran was rolled an additional 30 minutes to ensure thorough mixing. Twenty-five ml of dH^O vsa sprayed on 25 g of autoclaved bran in a similar manner, constituting the control. The experiment was designed as a split plot, replicated four tines. The whole plots were the presence or absence of the virus on the wheat bran. The presence or the absence of B, oomta planidia on the wheat bran constituted the split plots. Approximately 20 0. aomta planidia were mixed into one g portions of the virus-treated and control bran. One-g portions of each treatment were sprinkled over the surface of four com 72 flats. Ten third instar BCW larva# wars than placed down the center of each flat. Seven and ten days after treatment and infestation, the damage caused by BCW feeding on each plant was rated on a scale of one to four. One was a cut plant; two, a plant which had been severely fed upon but not cut; three, a plant exhibiting minimal feeding; and four, a plane with no feeding damage. After ten days, as many BCWs as possible were retrieved from each flat, rinsed in one percent phenylmrcuric nitrate solution to kill my associated fungi or bacteria, and placed on diet in 30-ml plastic cup». The insects were observed for parasitoid emergence. Field studies: Two studies were carried out at the USDA Corn Insects Research Unit in Ankeny, Iowa. In the first experiment I studied the control of BCW larvae by adult and first instar B, amta. In the second experiment 1 studied the control of BCW larvae by Ro^PV, B, aamta planidia, and RoMWV + B. aomta planidia together. The first study warn conducted in August of 1983, The experiment was designed as a randomized complete block with three treatwnte and four replications. Pioneer 3537 com was planted in three rows of 15 hills with three seeds per hill. ,5 m apart. Alter the com spiked throui^ the ground. 15.S em diameter x 25 cm high tin cans with the ends removed were embedded in the ground around each group of three seedlings. %ven days after planting, the three treatments were applied to the com within the cans. The first treatment was one female and one male B. aamta adult placed in each of five cans in each replication. The second treatment was ten B. aomta 73 planldla in on« g of BCW fraas distributad batwaan fiva cana par rapll- eation. îha third eraatnant waa eha control. In aach can, including tha control traatmant, two third inatar BCW larvaa vara placed and all of tha cana vara covered vith tvo layera of cheese cloth fastened vith a band of thick rubber. Seven days after treatment, the damage to the plants vithin each can vare rated using the scale described above. Again, as many BCWs as pos sible vere retrieved, rinsed in phenylmacuric nitrate, and obaarved for parasitoid emergence. Tha com (Pioneer 3337) for the second field study vas plmted singly in hills .3 m apart in June 1984. The experiment vas designed as a randomized complete block of four treatments replicated four tiaws. Tha four treatments vera RoMWV, B. a&mta planldia, WKPV + B. aomta planidia, and the control. Seven days after planting,15.3 cm diameter % 25 cm high tin cans %%re embedded in the soil around each of the 15 corn seedlings per treatment, per replication. The treatments vere prepared and applied in the field nine days later. RoMXPV vas prepared at a Q concentration of 3.5 % 10 PÏ8» per ml of dH^O and 25 ml of this suspen sion vas applied to 25 g of autoclaved vheat bran in a manner similar to that used in the greenhouse study. Tventy-five g of vheat bran vas similarly treated vith 25 ml of dwyo to serve as the control. Both treat ments vere divided into one-g portions. Approximately 20 B. acmta planidia vhich bad been stored from one to seven days at 4,4°C vere placed vith a camel's hair brush into 15 of the one-g portions of Ro^PV treated vheat bran. Tventy 3. oomta planidia 74 v«rtt similarly mixed into IS one g portions of dH^O treated wheat bran. Each one-* portion of the four treatment# (RcMNPV, B, ocmta^ RoMiPV -f g. aomtat and the control) was distributed over the surface of the soil within five cans. Then two third-instar BCWs were placed with in each can. Seven and t4 days after treatment and infestation* the corn within each can was rated as described above, To check the viability of the organism* used in the field study, the following tests were conducted in the laboratory. Third instar BCWs were placed in cup* of each of the four treatments. Twenty-four hour* later. 25 larvae from each treatment were placed on BCW diet in 30-ml cup* and incubated at 27*C and 7Q% RH. The number of B, o&nta pupae and BCW death# twre recorded. 75 RESULTS AND DISCUSSION Tfi« chronic oxid* study rcvaaltd that 58% diet vaa consunad by the B. aomta-paraaitiaad as conparad to the nonparasititad BCWa. Othar raaaarchars working with plant material found that BCWa paraaitizad by the two braconids, Mêteoma îeviventma (Wasnwal) and (Horoplitie ketéiîeyi Muaaabeck cortaum lata corn foliage than do nonparaaitized BCWa, cutting 36.4% and 68% fewer corn seedlings, respectively (Schoenbohm and Turpin, 1977; Sajap et al., 1978). Lavine and Clanwnt (1981b) found B. acrrrta parasitized BCWa to cut 32 to 53% fewer corn seedlings than did nonpara- sitized BCWa. However, from the chromic oxide study it waa determined that this reduction in mg of dry matter consumed by parasitized larvae only occurs eight days alter initial parasitization (Fig. 11). Simi larly, the percentage utilization of the dry matter consumed by B. aomta parasitized-BCW larvae was similar to that of nonparasitized larvae (if not greater on the first two days) until day eight (Fig. 12). Therefore it appears that any reduction in feeding or food utilization by 5. oomta" parasitized BCW larvae, at least under laboratory conditions, does not occur until just one or two days before the parasitoid emerges from the host and pupates. The RoWPV infection within the BCW hosts apparently had no detri mental effect on the B&rmetia maggots developing within except in those instances where the EojHWP? killed the host before the parasitoid was able to emerge (Table 16). lAien the parasitoids within the BCW larvae were studied histologically, the RogMPV PIBs were found within the gut lumen (Figs. 13 and 14). This is similar to the study of Vovia vttmlia (Fallen). Fig. 11. Dry matter coneumed («%) by Bcmêtia omta (Fallén)- pareeitized Âfffctie ipeiîon Kufnagei larvae and nonparaaitiaed larvae 0 nonparaaitiaed larvae m paraaitiaed larvae Fig. 12. Percent utilization of dry matter coneumed by Bmmtia omta (FallenVparasitized Açr&tis ipeilm (Bufnagel) larvae and nonparasitized larvae X imuamON OF OW «WriEROOHBUMBD «a ORV UKTIER OOmUHED * 9 M $ * Fig. 13. Bomêtia oomta (Fallln> maggot within an AgroHs ipsilon (Hufnagol) larva Infcctcd with a RaohipluHa ou (Gucnic) nuclcar polyhcdrocla vlrua (3S(^) Be • B. oomta maggot ; i • infcctcd host tissue fig. 14, Roffhiflmia ou (Guenee) nuclear polyhedrosis virus polyhedral inclusion bodies within the alimentary canal of a Bometia oomta (Fallen) maggot (14(KH() al » alimentary canal; pib » viral polyhedral inclusion body 80 Tabl# 16. Effect of a nuclcar polyhadrosis virvis isolated from Raohiplusia ou (RoMIPV) on Bometia aomta parasitizing RdWPV treated Agrotie ipellon larvae " % Mean* Test variable RoSflPV Control X 3. omta parasitism 62.0 a 86.0 b t J. aomta ecloslon 26.0 a 37.0 a Days to B. ocmta pupation 12.2 a 12.1 a Days to B, aomta eclosion 10.6 a 10.0 a Weight (mg) - female puparla 127.3 a 113.5 b - male puparia 94.7 a 86.3 b *Mean# (within row#) followed by the eaae letter are not signifi cantly different (P < .OS) as determined by Student's t-test. a tachinid parasitising virus infected f. ni, in which the PlBs were found in only the midgut lumen of the parasitoid maggots (Irabagon and Brooke, 1974), Ro*#pV PlBs were also found in the fat body, and the associated muscle of some f. arnia maggots (Figs. U and 14). in these instances, the BCU hosts contained many PlBs, and it is probable that these hosts would have died of a viral infection before the parasitoids were able to pupate. Bonmtia omta which pirated from RcMW-infected hosts did not take significantly longer to pupate or to eclwe. nor did they exhibit higher puparial mortality (Table 16). Bcnmtia aomta pupating from infected BCW 81 hoses did however weigh more than those from noninfected hosts. This is quite likely a result of the smaller BCU larvae being more susceptible to infection by the R(40IPV. Weights of 5. emta puparla increase with m incresse in the weights of their host larvae (fart IX). The smaller BCU hosts probably were killed by the virus treatment before the B, oomta were able to pupate, while these smaller individuala were able to live and pro duce 5. Qcmta in the control treatment. From the laboratory studies it was evident that both an infection by the RoMIPV. parasitism by the f. ocmta planidia, and also the combination of these two organisms, had significant detrimental effects on the survival of the BCU hosts (Table 16). Since parasitism by B. amta almost elimi nates feeding by BCU by day nine (Fig. 9). and since the had no detrimental effect on the B, omta able to pupate from infected hosts (Table 17), the ability of the virus «nd the Bcmetia to control BCU larvae was tested in a greenhouse situation. By day seven there were no cut plants in «ty of the treatments (Table 18). The amount of severe feeding by the BCU larvae in all three treatments was reduced as compared to the control: however, only the g. amta alone m4 in cwAination with the virus reduced this feeding significantly (P < .05) (Table 18). By day ten, there was a reduction in the number of cut com seedlings in all three treatments compared to the control. Again, only the B. (fcmta alone, md in combination with the virus significantly (P < .05) reduced this cutting. The virus and i. ctmta also reduced the mount of severe feed ing. However, these effects were not significantly different from the cmitrol (P < .05). 82 Tabic 17. Efface of a nuclear polyhadrosls virus Isolated from Raeihiplwia ou (RolOIFV) on Agrotis ipoiîcn (BCW) mortality, with, and without Uia affect of parasitism by Bonrntia aomta Treatment X Mean BCW mortality* RoMfPV 30.0 b 5« acmta 88.0 a IWOMPV + 8, aomta 94.0 a Control 5.0 e *Mean# followed by the sate letter are not significantly different (P < .05) as determined by the Duncan's multiple range test In the field, the potential for using released B. ocmta adults opposed to the planldia was tested. Both the adult flies and the planldla reduced the number of cut plants, md the amount of severe feeding, as compared to the control treatments (Table 19). None of these controlling effects were signiflcmt at the .05 level. The cms treated with the 9. Q The secwd field study duplicated the greenhouse study in a more realistic environmmt. Seven d^s after treatment, all B. oomta and WMPV bran baits reduced the severe feeding damais by the BCWs to a per centage which was significantly lower than that in the control plots 83a Tabic 18. Greenhouse study of Agrotxe ipeiîon larvae feeding on corn treated with bran alone, or bran with a nuclear polyhedrosls virus Isolated from Raahiplueia ou (RoWîPV), Bommtia ocmta planidia, or RdffiiFV plus B. omta planidia % Plant damage* Treatment Cut Severe Minimal None Day 7 Control 0 a 25.0 a 48.0 a 28.0 a B. aomta 0 a 5.0 be 63.0 a 33.0 a m^ipv 0 a 18.0 ab 63.0 a 20.0 a B, aomta -¥ RoMfPV 0 a 3.0 c 60.0 a 38.0 a ly 10 Control 13.0 a 40.0 a 38.0 a 10.0 a B. aomta 3.0 b 15.0 a 58.0 a 20.0 a W«PV 5.0 ab 38.0 a 45.0 a 13.0 a B, aemta * RcMIPV 0 b 8.0 a 65.0 a 28.0 a Means (within colinms and days) followed by the same letter are not significantly different (f < .05) as determined by the Duncan's multiple range test. (P < ,05) (Table 20). Pourteen days after treatment, the B. oçmta and iUrf»PV in all treatments also reduced the percentage of cut plants. Bow- ever, only in the treatment co#ining the B. o these effects significmtly different from that of the control (P < .05). Prom these studies it may be seen that the combination of B, aomta planidia with a RoMNPV bran bait is an effective control of third instar 83b Table 19. Feeding damage by AgroHs ipeilon larvae on corn created with Bcnmtia aomta adults and 5. aomta planldia Plant damage* X X Treatment Cut Severe Minimal Rone Parasitism Control 23 a 46 a 19 a 13 a .0 a B. aomta adults 15 a 27 a 38 ab 19 a .0 a B. aomta planidia 13 a 19 a 54 b 14 a .1 b *Means (within columns) followed by the same letter are not signifi cantly different (P < .05) as determined by Duncan's multiple range test Table 20. Field study of A^rcHe ipeilon larval feeding on com treated with bran alone, or bran nixed with a nuclear polyhedrosis virus isolated from Baokiplmia &u (RoWPV), Bcmetia oonta planidia, or RclflfFV and f. aomta " % Plant damage* Treatment Cut Severe Minimal None Day 7 Bran control 10 a 65 a 11 a 13 a 3, aomta 17 a 45 b 28 a 10 a R«MfPV 13 a 40 b 20 a 27 a B, aomta + RoiOfPV 10 a 40 b 35 a 14 a Day 14 Bran control 52 a 13 a 18 a 17 a B. aomta 42 ab 12 a 20 ab 27 a WMPV 32 ab 13 a 27 ab 28 a B, aomta + RtrfWPV 22 b 22 a 43 b 13 a Means (within colinns and within days) followed by the same letter are not significantly different (P < .05) as determined by Duncan's multiple range test. 84 BCW on toadllng corn. Paraaitlni of BCW larva# by B. aomta alona will atop faadlng in nina daya. Ingaation of high eoncantrationa of RoMWV PlBa will kill tha BCW larva# in two to fiv# daya (Johnaon, 1980). Tha B, aomta ara not auacaptibla to infection by th# vim# and ara only killed by it indirectly when it cauaea the premature death of the BCW hoat. 85 fAKt V* IMPACT or mmm pmmTA (PAIUOT). mssm sp.. AND A NUCLEAR POLYHIDROSIS VIRUS PROM MCMIPWSIA QU (CUHtEE) 08 umiLA mmsmi MAMW, WITHIN IMTECTID mninu MMILAUS (RUINER) HOSTS 86 INTRODUCTION Lydêlta thaapeoni H#rtin# vas introihiccd into eh# central com growing scatM between 1944 and 1955» and rapidly became the moat auc- ceaaful of the paraaitoid introduction# in controlling Europe** com borer, OêtHnia mibiîatie 1963; Sparka et al., 1963). In the 19608 there wa# a rapid decreaae In ECB para#iti#s by L, thai^ëoni md currently, few, if any, can be fotmd in ECB in the aldweatem United Statea (Hill et al., 1978$ Sandlan et al., 1983). Nuaeroua theorie# have been propoaed to explain thi# once *uc- ceaaful paraaitoid*a decline. One auch hypothesis is that a micro- sporldiuB, pathogenic to I. thcmpemd, may have caused its sudden disappearance (LawIs, 1982). There are mas^ records in the literature of paraaitoids which are susceptible to the mlcroeporidian infections of their hosts (Brooks, 1973). ECB populations sustain indigenous infections of the micro- sporidia Soeema pyvmtsta and Hoeem sp. (Zimmack and Brindley, 1957; Maddox^, unpublished) and nicrosporldlan spores have been found in ù. thompsatd within S. -infected ECB hosts (York. 1961), Although the spores in the paraaitoid were not Identified, they were most likely H. pyrauata. A nuclear polyhedrosis vims. Isolated from a mine looper, Baokiflmia ou (Guenée) (RoWIPV), significantly reduced the nwmter of ECB larvae per ^Illinois Natural History Survey, Urbma, 111. 61801. 87 plant and plant damage when applied to corn under field conditions. The vims has been suggested as a nontoxic alternative for the control of ECB in com (Levis snd Johnson, 1982). The primary adverse effect that insect viruses have on parasitoids is the indirect effect of causing the pre mature death of their hosts (Kelsey, 1960; Harcourt, 1967; Laigo and Tamashiro» 1966; Vail, 1981). The impact of RoMfPV, p^rmeta» and atoBma sp. on L, thcmpgoni developing within infected ECB hosts were studied, to better understand any detrimental effects that these pathogens of the ECB have on this parasitoid. 88 MATERIALS AND METHODS A##ay# wcr* csrriad out with RoHKFV, SoBvm #p., and Soêêma pyrauata infactad EC6 larvaa paraaitiaad by I. thcmptoni larvaa. Tha bioaaaays vara daaignad aa factorial axparimanta in a randonisad coo^lata block daaign, raplicatad four timaa ovar daya. Tha aaaaya with H, pyvaueta vara raplicatad aavan timaa. Two factora of aach axparisant wara tha axpoaura of tha ECB larvaa to a pathogan or to no pathogan on tha aurfaca of tha diat. Fifty, SO-ml plaatic cupa containing aolidifiad ECB diat (without foroaldahyda and that included aa mold inhibitors 4.8 g mathy1 p-hydroxy-banzoata, 8.3 ml propionic acid, and 0.8 ml phoaphoric acid) (Lavis and Lynch, 1969) were surfaca-traatad with .2 ml of a virus or microsporidiun suspension in dWgO at a concentration which resulted in 200 RolifFV polyhedral inclusion 2 bodies (PIBs), 100 pyvausta spores, or 50 Soe&rm sp. spores per mm of diet surface. Fifty, 30-»l diet cups in the control treatment were similarly surface-treated with .2 ml of dH^O alone. One-half g of peni- cillin-C sodium was added to each 100 mis of suspended microbes in all treatments, as well as the control, to retard bacterial growth on the sur face of the cups. The litjuid was swirled to cover the surface of the diet and the cups were left on a flat surface to dry. When dry, a single neonate (second instar for the RoMfPV- assays) pathogen-free ECB larva was placed in each cup. The cups wre sealed with polyethylene-lined lid* and incubated at 27'C and 50% relative humidity (BB) and for six days (RolfitPV-assdys) or seven days (ffoaema-assays) at which time the majority of the larvae had reached the fourth instar. 89 Th# third and fourth factor# of th« exp#rimant vara tha paraaitita- eion and no paraaitiiation of tha pathogan or control traatad ECB larvaa by L* thcn^ami maggot#. Tha L. thompeoni colony warn originally obtained from Dr. R. Jonaa.^ Fraaa from ECBa feeding on aeedling com atalka waa placed on dau^ filter paper in an open 100 x 15 mm plastic petri diah inside the L* th&npemi cage, two to eix hour# later, a aingle first inatar Ù, thcmçBorvi maggot, larvipoaited onto filter paper, was picked up with a camel*a hair brush, under a diaaecting microecope. The maggot waa placed on the dorsum, JtMt posterior to the head capaule of each of 15, fourth instar ECB from both the pathogen and the control treatments. Each parasitised larva was observed until the paraaitoid appeared to penetrate the host's cuticle. Ail parasitised larvae were placed on fresh ECB diet in individual 30-nl plastic cups sealed with polyethylene-lined lids. Fifteen similar pathogen created, as well as 15 control larvae, were also placed in freah ECB diet cups. All cups were incubated at 27*C and 50% RN. Each insect was observed daily and all host and paraaitoid deaths, pupa tions, and adult ecloeions were recorded. On d^s three, five, and seven, a single larva from each treatment was fixed in 58*C alcoholic Bouin's fixative for one hour. After 24 additional hours in fixative at room temperature these larvae were dehydrated through a series of alcohols, infiltrated and embedded in paraplast. and sectioned at 6 wm on a rotary microtome. The sections Jcmes. Department of Entomolof^. University of Minnesota, St. Paul. Mn 55108. 90 wmre dried on albmln-coattd nlcroscopa alidas and rahydratad. Slidas of ECB infactad with aicroaporidla tmra acalnad %d,th a Glataaa colophonlun acain (Stiortt and Coopar, 1948) and alidaa of ECB infactad wieh RcANPV vara atainad with a aodifiad Aian atain (Baam, 1966). Tha alidaa vara flwuntad and «taninad with a light nicroacopa. 91 RESULTS AND DISCUSSION Sons of the Af. p^nm«ta-inftct«d ECB hosts contained spores in the Malpighlan tubules only, while others had additional ft. pyraueta infec tions of the muscle, fat body, and hypodertsal tissues. Noetma sp.-infected ECB larvae contained extensive infections in the Malpighian tubules as well as in muscle, and occaaionally gonadal, hypodermal and fat body tissues (Figs. 15 and 16). Lydatla thcpmaoni developing within ECB hosts have three larval in stars. In the first and second instara, the larva feeds primarily on the host's body fluids and not on the fat body tissue or internal organs (Baker et al., 1949). These t%fo instars require approximately three and tiro days to develop, respectively (Baker et al., 1949). Examined histo logically, L. thmpemi larvae within U, pyraueta as well as Hoeema sp.- infeeted ECB hosts, fixed on day five, were found to contain no Useem spores (Pigs. 17 and 18). This would be because they had not as yet con sumed infected internal host organs. The third instar of ù, tTwmpftoni requires approximately 2.7 days to develop, feeds on the fat body. and. before exiting its host, consums most of its internal organs (Baker et al., 1949). lyâetla thcmpsani emerging from both pyramta and Noaema sp.-infected hosts contained Soaema spores only in their alimentary canals. There are numerous records in the literature of microsporidian infec tions in braconid, ichneumonid, and tachinid parasitoids within diseased hosts. In each case, the microsporidia have the potential to cause Fig. 15. Sotma #p. #por## infceting mu#cl# ki##u# of «n Oêtvinia mûdUtîie (Rubnor) larva (2430X). mt • muacl* fibers; sp » ft. #p. apor# fig, 16. Scs&na sp. spores infeecing Halpigblsn cubules of an OetHnia nuMîalis (lubner) larva (140%). fb • fat body tissue; ne « Malpigblan tubule; sp " 9. sp. spore 93 Flf. 17. Ly^îîa tharpsoné Rcrting maggot within a Soe«ma sp.-inf«ctcd Qntrinia nvtilalia (Riibnar) larva (160%) i " infactad host tissue; Lt • L. thompeoni maggot fig. 18. Umma sp. spores of infected an Oetvinia mûdîalie (Hubner) larva adjacent to cuticle of ùydelta thmpemd Herting maggot (2450%) ht > host tissue; Lt • C. thompeoni maggot; sp > Ih sp. spore 95 96 paraslcoid mortality should their hosts be heavily infected (Tanada, 1955; York» 1961; Brook# and Cranford. 1972). A •IgnlClcanc (P < .05) percentage of pyreweta-infected ECB died, indirectly killing any L, thcxnpaoni developing within (Table 21). These were undoubtedly the hosts containing the It, pyrauêta infections in tis sues other than the Halpighian tubules. The number of Ù. thmpemi able to emerge and pupate af tar consuming the internal organis of pj^raueta- infected ECB larvae was not significantly different from the number pupating from noninfected hosts (Table 22). fewer of these parasitoids pupating from infected hosts were able to eclose (66%) than were the controls (1(X)%): however, this difference was not statistically significant (Table 22). CydeUa thcmpeml able to pupate and eclose from the p%f@w*Ae-infected ECB did not experience retarded development, and they appeared morphologically normal. With the limited I, thcmpemi mortality caused by pywoéetat its role in the disappearance of this parasitoid from the mid-western United States is questionable# E(^ mortality by day 30 due to Hoeem sp. infection was not as high as that caused by 4?, pyvmt^ta (Table 23). Fewer Ù, were able tp pupate from Sûeem sp.-infected hosts than from noninfected hosts. How ever, I. thompeoni pupating from Soeema sp.-infected hosts soon succumbed to the infection obtained in their development, and none were able to eclose as adults (Table 23). Comparing the pathologies of the two Boeema species in ECB and their effects on L, thompeoni, it seems logical to conclude that the intact of the microsporidia on the parasitoid is a dosage effect. Parasitoids developing within an il. pyrawa&a-infected ECB larva would have the 97 Table 21. ECB mortality caused by Sosma pyraustOt Nosama ap,, Rc^FV and parasitism by Lyàêtîa thcmpaoni (6.6.) Mean % ECB mortality bv dav 30* Pathogen With L,t, Without L.t. ft, p\fraueta 87 a 58 ab Control 47 be 10 d mtoarna sp. 40 a 29 ab Control 37 ab 14 b RoWIPV 43 a 22 a Control 30 a 6 a Means within each pathogen/control group followed by the same letter are iwt significantly different (P < .03) as determined by Duncsn's miltiple range test Table 22. Effect of Soama pyvmteta Cff.p.) on Ly^lla thcmpeani developing within infected QstHnia mbilalie larvae Means* Test variable Son- infected infected host host % l.t, pupation 20.6 a 29.3 a t l.t. eclosion 63.7 a 100.0 a Days to L,t. pupation 7.2 a 6.0 a Days to L,t. eclosion 15.3 a 15.7 a *Means within rows followed by the same letter are not significantly different (P < .05) as determined by Student's t-test. 98 Table 23. Effect of Hoema sp. on Lyàêlla thompeoni (£.&.) developing within infected Oetrinia nutilatie larvae * Mean# Test variable sp. Non- infected infected host host X I,t, pupation 19.0 a 30.5 a % eclosion 0.0 a iOO.O b Day* to L.t, pupation 6.5 a 6.9 a Day» to L,t> cclo»ion NA 15.3 *Mean# within row# followed by the MM letter are not aignificantly different (P < .OS) a# determined by Student*» t-te»t. KA: not applicable. potential to consume the spore» primarily in the Malpi#hian tubules as this is the first site of major infection by the microsporidian after the midgut cells in ECB. Hosts wre heavily infected with »wuld die before the parasitoid is able to complete its larval development. The ECB hosts with a lew Uofsem sp. infection did not die in the larval stage and therefore did not die while the parasitoid was still developing within it. if, thom^emi in Sosma sp.-infected hosts would therefore have the potential to consume more spores, as the infection of Noema sp. in these ECB is more intense than that of B, pyrmeta, and would be infecting the ECB muscle as wll as the Malpighian tubules and possibly, hypodermal and gonadal tissues. The Rol^V within infected, parasitised ECB hosts appeared to have no significant (P < .05) impact on L. thcmpsoni development at least until 99 after the parasitold adulta acloaad (Tabla 24). Thaaa data are similar to obaarvationa on tha Impact of host viruaaa on othar braconld and tach- Inid paraaltold# (Kalaay, 1960; Rarcowrt, 1967{ ialgo Md Twaahlro, 1966; Vail, 1981). Tha ràova authors found that tha primary datrlmantal affact that insact viruses have on parasitoids is the indirect effect of causing the premature death of their hosts* Sines the RdQtPV at 200 Pits per mm 2 diet surfsce cauaed minimal mortality when fed to second Instar ECB larvae (Table 21), this hsssrd to the I. thmpêmi WM of little conssquence. If the WCB had been eapoaed to a higher concentration of the RoMtPV, or to a more virulent isolata, possibly it would have had a more serious impact on the I. thempemi by killing the ECB before the parasitold's development was complete. When the sectioned, RcMiPV infected, and parasitized ECB larvae were stained and examined with a microscope, the viral PlBs were found infee ing the fat body, hypodermsl, Hslpighin) tiAules, and midgut tissues of the ECB larvse. The PlBs were found only in the gut lumen of the para sitold (Figs, 19 and 20). Ifuclear polyhedra were similarly found only in the midgut IWMW of VoHa rumlie (Fallen) (Tachinidae), soncreneis (Cameron) and Bypceoter exiguae (Viereck) (IchneuMmidae) with in infected host larvae (Irabagon and Brooks, 1974; Beegle mut Oatman, 1975; Vail, 1981). In its third instar, the 6. thompemi within such an RqMNPV-infected ECB host probably ingests mmy PIBs as it consumes most of the host's infected internal organs (Baker et al., 1949). As mmiy L. thompaoni pupated from RqMKPV-lnfected hosts as did from noninfected hosts (Table 24), and it appears that the parasitold is not susceptible to infection by the RoMSPV infecting the host. too Tabla 24. Efface of RoMfPV on t^dblîa thanpsoni (l.t») davaloping within infacted QotHnia mtHlalie Meana* Test variable RoMfPV Bom- infected infected hosts hosts % ù.t» pupation 24.0 a 14.5 a % ù.t. ecloaion 91.0 a 100.0 a Days to &.&. pupation 6.7 a 6.7 a Daya to &.6. ecloaion 15.5 a 15.3 a *Meana within rowa followed by the aane letter are not significantly different (P < .05) aa determined by Student's t-test. Fig. 19. l^^Xla thcmpecni B#rtlo# nagsoe within m Oëtrinia méilalis (Rubiicr) larva Infccead with « Raahipîutia ou (Guctila) nuclear polyhadroala vlru# (875X) 1 • Infactad host tlsaua; Lt • ù. thompëord maggot Fig. 20. Baehiplmia &u (Guenco) nwelear polyhedroal# virus polyhedral Inclusion bodies within the alimentary canal of a iydetîa theapsmi Bertlng maggot (2450%) al » alimentary canal: pib » polyhedral Inclusion body 103 PART VI: IMPACT OF VAIRMOBIM MCAFRIX (KXAMES), MSEMA pmusTA (PAiuoT), mssm sp., AND A HUOEAK POLYnSROSlS VIROS HUM MCUIMISU OU (CUBliE), OH MCROCBMSUS ONAMII GOIOAINCA DEVUOPHKÏ WIIRIIF MNCTED osmniiA mmiiAUs (WMMM) LARVAE 104 INTRODUCTION R#«##Tch#r# have found th# European com borar (ECB)» Oetrifiia mûdîalie (Hubnar) to ba auacapelbla to aavaral alcroaporldian pathogana (Kramar. 1959; Lawia at al,, 1982; Haddoi,^tmpubliahad). ttoema pyraueta (Paillot) ia found naturally aaaoclatad with ECB populatlona (Ziomack at al., 1954; Lawla, 1982). Thta pathogan primarily infacta tha Malpig- hlan tubulaa of larval and adult ECB, tha ailk glanda of larvaa and tha ganital tract of adult faaalaa (Kramar, 1959). Such an infaction ahortana tha ECB'a lifa apan and raducaa ita fecundity (ZioMck and Brindley. 1957; Krmar, 1959). A second Scaem has been found naturally associated with a small percentage of ECB (Naddox, unpublished). Vcdrimn^ha nemtHx (Kramer) has been found to be a highly virulent pathogen of ECB. This last microaporidian causes midgut disruption» septicemia, and aubaequent death of larval ECB or microaporidioais and ultimate pupal mortality (Lewis et al.. 1982). Th# ECB is also susceptible to the nuclear poly hedrosis virus isolated from a mint looper. Backiiplmla m (Guenee) (RfMMPV), has significantly reduced ECB larval infestations in field tests on com (lewis aW Johnson. 1982). All of the above patho gens are good natural or potential biological control agents of ECB. and as such, their impact on ECB parasitoids mist be considered in an inte grated pest management systea. There are nwerous records in the literature of microaporidian infections in parasitoids within diseased hosts (Allen and Brunson, 1945; ^Illinois natural History Survey, Urbana, 111. 61601. 105 Tanada» 1955; Blunck, 1958; York, 1961, Brooka and Cranford, 1972; Hoatounalqr, 1970). fiaarcatntrua grandii Goldmich (Braconidaa), tha pra- dominamt paraaitoid of ECB in lowa (Lawia, 1982) axhibita auch a auacapti- bili&y to H, pyrmtBta within infaetad ECB larvaa (Parkar, 1931; York, 1961; Andraadla, 1980). Soëêma pyrausta infactiona within M, grandii raduca tlM pareantaga ahla to acloaa aa adulta and dacraaaa tha longavity of any infaetad adult aurvivora (Andraadia, 1980). In contrast, moat inaact viruaaa do not appear to ba infective to their hoata* paraaitoida. The primary adverse effect of viruaaa on parasitoids developing within infected hoata ia that of cauaing the pre mature death of the latter (Kelaey, 1960; Harcourt, 1967; Laigo and Tamaahiro, 1966; Vail, 1981). In this study. I examined the impact of four natural or potential biological control agenta of ECB, V, neaeitrix, S, pyrauataf Hotema ap., and RirffSPV, on tf. grandii developing within in fected hosts. 106 MATERIALS AND METHODS All M. grmâii and ECB naonataa uaad in thaaa aaaaya wara fraa of any pactioganic infaction. Aaaaya «rara daaignad aa two x two factorial mdontaad complota block «cvariaanta raplicatad four timaa ovar daya. Two traatmanta in aach axpariaant wara tha infaction or lack of an infac tion of RoîttlPV, V. ntaatrix, ft. pj^reeuBta, or #oaaw@ ap. in tha ECS hoata. Thaaa traatmanta wara croaaad with axpoaura of tha hoata to paraaitiam by M. granàii or no axpoaura to H, granàii. At laaat 12 granàéi famalaa «id aix malaa wara placad in a 250-ol plaatic bavaraga cup convartad to form m ovipoaition caga, within 12 houra aftar thair acloaion, to allow thm to mata Tha inaacta war# auppliad with cotton wick#, on# #oak#d with a 3C^ hon#y solution #nd the othar with watar. Tha H, grmMi wara incubatad undar a 14 hour 21*C photophaaa, t«* hour 16*C acotophaaa, and a rolativa humidity (RH) of mor# th«i 7M». Four day# #ft#r #clo#ion. #ach famala grmâii wa# placed into an individual oviposition cage (five females per treatment). for these asaays, V, moatvia^ was propagated in black cutworm, Agvotia iipsilm (Rufnagel) larvae. fyvciuota and Scema ap,* were pro pagated in eo larvae, and RcMW was propagated in cabbage looper larvae, friohcplmia ni (Hubner). On the fourth day after K grandii eclosion, ECB larvae were exposed to a pathogen In the following manner. ^Originally obtained from J. Haddox, Illinois Natural Biatory Surv^, llrb^ia. 111. 61801. 107 Nicrosporldlan spores or RoMNPV polyhedral inclusion bodies (PXBs) were applied in .2 ml dBgO to the aurface of ECB diet (vlthout formaldehyde and that included aa mold inhibitora» 4.8 g methyl p-hydroxybensoate, 8.3 ml propionic acid, and 0.8 ml phoaphoric acid) (Lavia and Lynch, 1969) in 30-ml plaatic cupa, at 25, SO, and 100 aporea of y. nêoatrix, Noema ap., and pifrmtata, reapectively, and 20 R The liquid wa# awirlad to cover the aurface of the diet and the cupa were left on a flat aurface to dry before three ECB neonatea were aealed into each cup with a polyethylene-lined lid. All cupa were incubated at 27*C and 70% RH for four days. Eight days after adult paraaitoid ecloaion and four daya after exposure of the ECB larvaa to the pathogen, a aingle cup containing three second to third instar pathogen-treated E(3 waa placed in each of the five K grandii oviiiwsltion cagaa par treatment through a recloaable opening in the side of each cage. A single cup containing noninfected ECB larvae was similarly placed in each of the five ctmtrol oviposition cages. These cages were incubated under a 14 hour 21*C photophase, ten hour 16*C scotophase, and more than 70Z RH. Host larvae within each cage were replaced with similarly treated larvae every 24 hours during a four-day period. After 24 hours of exposure to a grandii female, ECB larvae from each cup were individually placed cm fresh ECB diet in 30-ml plastic cups, and incubated at 27"C and 70% RH. The larvae m ware examined daily for parasitoid emergence, host mortality or host or parasitoid eclosion. Any pathogen-treated or control ECBs not exposed to grmdii females were placed in individual 30-ml plastic ECB diet cups and mortality, pupation, eclosion, and percent infection of these insects were recorded. A study of the effect of the level of pyrmeta infection in the ECB hosts on M, §vmdii development was conducted, U, pyrmteta In .2-ml aliquots was applied to the surface of ECB diet at 200, lOO, SO, and 25 spores per mm 2 of diet surface. Cups treated similarly with .2-ml of dWgO only were the controls. Again, .5 penicilliun-G sodium was added to each 100 ml of suspended #. pymusta spores and the control to retard bacterial growth on the diet surface. ECB neonates were placed on the diet and four days later, a cup containing three larvae from each dosage was exposed for 24 hours to an eight-day-old K gvmdii female held in an ovipostion cage as described above. Host larvae were replaced three times and each ECB larva transferred to ECB diet in a 30-mi plastic cup and incubated at 27*C and 70% RH. All larvae were examined daily for parasitoid or host pupation and eclosion. Infected adult female H* grandit eclosing from any pathogen assay wre used to test transovarial transmission of the pathogen. In total, six V, neoatvix and 30 H, pyraueta infected M. grmdii females were each placed individually in oviposition cages and supplied with a 30% honey solution and water as described above. Eight days after eclosion, cups of ECB diet containing three, four-day-old second instar ECB larvae were placed in each cage for 24 hours. Host larvae were replaced daily 109 for six days. After 24 hours of exposure to parlsitization, each EC6 %m# placed In an individual 30-ml plastic cup of ECB diet and incubated at 27*C and 70% RH. All larvae %wre examined daily for host pupation or parasitoid emergence. Randomly samqpled EC# and emerged M, gvandii from all pathogen assays were fixed for one hour in 58*C alcoholic Bouin's and for an additional 24 hours at room temperature. They were then dehydrated in alcohol, embedded in paraffin, sectioned at 6 wm on a rotary microtome, and mounted on slides. Insects from microsporidian assays were stained with Giemsa colophonium (Shortt and Cooper. 1948} and insects from Ro#PV assays were stained with a modified Azan stain (Hanm. 1966) for histo logical examination. no RESULTS AND DISCUSSION ECB «xpoMd M n«on«tM to RoMNPV-tremted diet died m tacond or third instars bafor# M, gtwidii from any paraaitiiad hwts war* able to aoarga (Tabla 25)» This indirect effect of causing the premature death of the host has been the primry adverse effect that other insect viruses have had on parwitoids (Kelaay, 1960; Harcourt, 1967; Laigo and Tamaahiro, 1966; Vail, 1981). Not all RcMRPV-infacted ECB die in these early instars wider field conditions md it is possible the H. gwmàii may emerge from ECS infected with RoWfPV alter the first instar, aa other braconida have survived viral infections of their hosts (Kelsey, 1960; Laigo md Taaashiro, 1966). The impact that the three microsporidian species had on M. grandii development ap^ars to be closely related to tiMir pathologies in the infected ECB hcwts, and the developmental biology of the parasitoid. Of the three microsporidia, Sceem sp. had the greatest detrimental impact on ECB hosts and on the H, grandii developing within them. Forty-two Z of these ffoeemt sp.-infected ECB died as fourth or fifth instars of acute micrMporidiosis and only 14% of the 32% able to pupate, eclosed as 2 infected adults. When exposed to 50 spores of Hoeema sp. per BOB diet sur face. ECB hosts developed extensive infecticwis in the Malpighim tubules and muscle tissue and occasimtally in gonadal, hypodermal and fat body tissues as well. Parker (1931) fowid that first, second, and third instar W. gmndii consume primarily host fat body tissue. The parasitoid then migrates to in Tabla 25. Efface of Sosma pyrauata, Soama ap., Vcdrimrpha naeatrix, and a nuclaar polyhadroaia vima frra RaekiptuHa ou (RolfflFV) on (ktrinia mSiîatia mortality, with» and without, tha affect of paraaitiaa by fktarooantrM grandti (M.g.) % Mean mortality* Pathogen With M,g. Without Kg. 96 a 97 a Control 15 b 8 b #. pyrauêta SO a 46 a Control 18 a 9 a Hoama sp. 92 a 76 b Control 18 c 9 d y. mmtHx 76 a 84 a Control 15 b 18 b Meana within each pathogen group (rows and columns) followed by the aame latter are not aignificamtly different a# deteminad by Duncan's multiple range test. the exterior of the host and consumes the rest of the internal organs as an ectophage. Within Sosem sp,-infected ECB, H, gmnéti larvae would have little potential to consuw spores in the Halpi^ian tubules and muscles during the first three instars, but would then consume iwst of the body tissues as an ectophage. All M. gvcmdii emerging from Sosem sp.-infected ECB contained Soeem sp. spores, and these were primarily restricted to the intestinal tract (Table 26; Figs. 21 and 22). Only 25% of these M, gvandii emerging from So^ema sp.-infected hosts were able to eclose as adults. All of these eclosed adults were males. rigs. 21 and 22. fiaoNiomtme gvmàii Goldanlch larva after lea •aar^ca from a Itoaema ap.-infactad OetHnia mtitalis (Mubnar) larva al • allmantary canal; a • wall of aliaaneary cmal; ap » iV. ap. apora rig. 21: 875% rig. 22: 1400% 113 lU Tabic 26. Effect of Itoema ap. on Maarcomtrua granàii (^.gr.) para- aleiaing S. ap.-infacted (^tvCnia mûdîalie larvaa Mean* Teat variable Hoema ap. Control % Kg. paraaitian 21.3 a 14.3 a % aeloaion 23.3 a 97.0 a Day# to pupation 20.1 a 16.9 a Days to aeloaion 10.5 a 10.3 a % Nwt infection 96.0 a 0.0 b % M,$. infection 1(K).0 a 0.0 b *Meana (within rows) followed hy the same letter are not signifi cantly (P < .05) different as determined by Student's t-teat. Vairi^norpha maatHx at 25 spores per diet surface also had a very detrimental effect on the ECB. Forty-one % of these insects died as third to fifth instar ECB of aiicrosporidiosis. Only 30% of the 57% able to pupate, eclosed as infected adults. VaiHmvpha neaatvix infections within ECB were restricted primarily to the fat body tissue. Therefore, within y. infected ECB, M, gvcmdii larvae would have the potential to con sume infective spores in the fat body tissue during the first three instars of their development. Maavoaentvua grandii exiting from an ECB consume all but the bead capsule and some cuticle of the host and therefore it is not possible to determine if the host was infected after parasitoid emergence. Studied histologically, most M. grandii larvae exiting from V, neaatHx' 115 infected hosts contained spores only in their alimentary canal (Figs. 23 and 24). Ten and one-half % of y. nëoatrix treated ECB hosts escaped infection and it is probable that the six % M, ffrandii which developed in V, ntfootri»-treated ECt which were not infected, developed within these noninfected hosts (Table 27). Only 53% of M. gremdii emrging from V, ifoaWap-infected ECB were able to eclose as adults (Table 27), and all these adults contained V. nêoatrix spores. Six female M. gvtenàii with f. meotrix infections, when presented with noninfected ECB larvae, did not transovarially transmit the pathogen (n • 14). The females lived for 11 to 14 days. The effect of «V. pyraueta on M. gvmdii within infected ECB host# varied with the concentration to which the hosts were exposed. This sup ports the idea that the Impact that a given microsoridian species has on H. Qrcmdii development is related to its pathology in the ECB host. Uoema pifvausta at a low concentration infects primarily the Halpighian tubes and silk glands of ECB larvae (Kramer. 1959). At higher concentrations the exposed ECB develop d, infections in the fat body as well as muscle tissues. Only 33% of the H, gvmdii developing in the ECB exposed 2 to 100 spores of S, pyvaueta per m diet surface were able to eclose as adults (Table 28). Noeema py^raueta spores were found primarily in the ali mentary canal of M. gvandii larvae emerging from the infected hosts (Fig. 25). ^oeema pgraweta-infected female U. gvandii were able to live for up to 14 days but were unable to transovarially transmit the microspordium to their offspring. In contrast, Andreadis (1980) found U, pypaueta infected figs. 23 and 24. Maavoemtma grandii Coldanlch larva after it# emergence frwi a VedHmvpka maatrix (Krmer)- tnfeeced Oetvinia nukilalio (Hîîbner) larva ai • alimentary canals e • wall of alimentary canal; mp • V. neaatrix spore- Fig. 23: 3)0% Fig. 24; 1400% M U8 Tabla 27. Effect of Yaiptmrpha »êoati4x on l4euroa«ntrue grandii (W.gf.) paraaititing y. Maootrùe-infaccad Oetrinia nuHtatia larvaa Maan* Taat variable V. moaiHm Control % M.g. paraaielam 17.3 a 11.0 a % M.g. acloaion 52. S a 90.3 a Daya to pupation 20.2 a 17.2 a Day# to acloaion 8.0 a 10.3 a % boat infaction 89.) a 0.0 b % M.g. infaction 93.8 a 0.0 b *Maan# (within rowa) followed by the ewe letter are not aignifi- cantly different (P < .OS) aa determined by Student'a t-teat. Table 28. Effect of fim&m pymueta on HaovGomtvm gmuMi (M.g.} paraaitizin# N. f^rmwtariniwt^ Oetritda mtHlalis larvae Mean* Teat variable pymusta Control X H.g. pupation 8.0 a 6 3a % H.g. eclosion 38.7 a 100.0 a Days to pupation 19.3 a 16.2 3 Day# to ecloaion 10.6 a 6.7 a % boat infection 88.7 a 0.0 b X M.g. infection 77.7 a 0.0 b Meana (%rithin rowa) followed by the game letter are not eignifi- cantly (P < .05) different as determined by Student's t-test. Fig. 2). AllMneary cnuil cell# of a grmMi Goidonieh larv«, cmtainliit Schema pyrauêta (failloe) spores (1400X) e • wall of sllmncsiy carnal; sp • It. pifrmeta spore 120 121 f#m#le H, granàLi eo live only three to four deye et 25*C. The 7. nêeatri»' end S, p^rausto*Infected M, granàli in thie etudy were mein- teined et 16* to 21*C for et leeet ten edditionel d^e, end it ie poeeible thet the longevity of infected edulte would be ehorter under more etreee- ful conditions such es higher tei^eretures. Differences found in this reseerch compered to thet reported by Andreedis (1980) might elso be due to e difference in strein of M. ffpandii and/ot N, p^pouêta. 122 mMARY AND CONCLUSIONS This dimmertmeiom mCwIimm potcntlml pathogmn-parmmltoid incmractionm occurring bmcwmmn: chm major paramieoidm of AgroHa ipeiUm (Hufnagal) and Oêtrinia nuHîaîie (Rubnmr); and, home paetogtna currently umed, or with the potential for future we am microbial introductiona or inmecti- cidem. In murveym (in central Iowa}, over two mumMrm, uming releamed third-inatar black cutworm (KW) larvae it warn found that Metêorus lêvivêntrua (WWmmael) (Braconidae) and a tachind, Somtêtia aomta (Fallen) were the two major paraaitoida of the BCW. Paramitimm by M. levivmtvm appeared to be mynchronixed with the firmt «id damaging generation of BCW in com» while B. amta paraaitised KW later in the mummer. NeitMr of these two paraaitoida were found to be aamociated with a pathogenic micro- organic». In thim murvey (umii% releamed ECB larvae) Haovommtm» gmnêii Coidanich (Braconidae) warn the only paramitoid commonly collected. Of the M. gvmêii emerging from field-collected ECB, were infected with a microm^ridim, nomt likely Baeem pyramta (Paillot). Laboratory colonies of B. (scmta, M. grmdii» and Lydelta thompsoni Berting (Tachinidae) (also an ECB paraeitoid) were established aW uwtWds developed for the ease of their manipulation in pathogen-parasi toid inter action studies, Bometia amta planidia were found to remain viable after five days at 4.4*Ç which increased the potential for their mass production and possible introductimi into the field to control first-generation BCW larvae. 123 Baoilîus tfmHnffiênsia B#rlin#r aubspacltts hœataki appeared oe have a detrimental effect on the development of S. oomta in BCW hoata exposed to the B, thuringiêneie as neonatea* BCWa expoaed to B. thurir^meie exhibited bacterial roda only in their gut lumen and conaequently* there waa no B, thuHnffitneie available within BCW tiaaue for the B. oomta to conauma. However, paraaitism by B. acmta waa significantly lower (P < .05) in BCWa exposed to 8. thuringieneie. Baailîu» thuHngiensia rods were observed externally in the intersegmental regiona of the BCWa cuticle, and it ia poaaible that a 5. oomta planidium. entering the hoat through the cuticle, may encounter and ingest i. thsxpin^maia spores and/or crystals during its period of penetration. ^aiHmfipha moatvis (Kraawr) and Vairimrpha sp. in BCW; Nomm pynaata (Palllot) and Soama sp. in EC#; and y. mo&tvix, and Soaem sp. in ECB, all had detrimental effects on B. oomta, I, thompa&ni, and K gvméii, respectively. Vaivim&vçha moatvix and Vaivi' mrpha sp., both infected the B, oomta maggots and decreased the number of f. omta able to pupate from BCW hosts, the days required for adult ecle sion, and the weights of the puparia. These effects increased with an increase in the intensity of the BCW host's infection. Ovaries from adult g. oomta that pupated from V, neoatvix-'infecteû BCWs did not contain any spores and therefore the potential for trans- ovarial transmission of the pathogen seems negligible. Both microsporidia appeared to have a greater detrimental impact on female B, oomta, than they did on the males. M ttoe&m pywxueta and ^osêtm sp. reduced eclosion of adult L, thmpeoni infected ECB hoata by 36 and 100%$ reapectively. floema ap. cauaed excen- aiv# infection in the Malplghion tubolea «id ontacle and occaaionally in gonadal, «id hypodexnal tiaauea of the ECB hoata. Itoêêma pyrauêta infecta primarily the Malplghion tubulea and the ailk glmda of ECB larvae. Lyâêlîa than^êoni maggota conaume ooat of the hoat'a internal organa and would therefore ingeat a higher cmcentration of Soëtma ap. aporea than they vottld It, pyirau9ta, aporea, explaining the more aevere impact that the lto«ema ap. had on the paraaitoid. The limited L. thempgoni mortality cauaed by the pynmêia diminlahea thia microaporidim' a role in the rapid decline of thia paraaitoid in the midweatem United Statea. Vaivimcppha neoatriXf S, pifpouata, and Stmema ap. reduced adult M. grasnàii eclocim after developing in Infected ECB bo#ta, by 37, 61, *md 74%, reapectively. Again, the impact of the three nicroaporidia on the M. gnmàii depended on the pathology of the pathogen within the boat. Haspceentpua gponàii feed on the fat body in their firat three in#tara, and they feed on the rest of the ho#t in their fourth instar. Vaivimvpha mmtrix infects ECB fat body tissue while Seeems sp. and K pyrauêta only infect these tissues when the host has been exposed to a very high con centration of inoculum. Vaivimvpha neoatrix- mà H, infected female H, gvmdii were unable to trmsovarially tranmait these pathogens to their offspring. Maavocentrm gvmàii adults with either of these infections were able to survive for at least 14 d^s imder 14 hour 21*C phocophaae and ten hour 16*C seotophase. A nuclear polyhedrosis virus from a mint looper, Raohiplusia ou (Guenée)(RclfliPV) had no detrimental effect on B. oomta or L. thon^aoni 125 developing within RolffitPV-infccted hosts, exempt indirectly when the virua killed the hosts before the psrssitoids were able to emerge. When these paraaitoida within the RoMRPV-infected BCWa and EOa were atudied hiato- logically, the R Itanana of the paraaitoida' guta. In an experiment meaauring the food utilization by B. omta-para- aitixed BCW larvae. It was found that the mg of dry matter conaumed end % utilisation of the dry matter conaumed by paraaitimed BCW larvae were aiailar to thoae of nonparaaitizad larvae until day eight, juat one to two d«ya before the B. amta emergea. Becawe of the ability of 5. aamta to reduce hwt feeding, and the apparent compatability of the R^#PV and the f. greenhouae «d field studies were conducted to test the potential of theaa ^ biological control aganta to control BCW larvae feeding on com. In the greenhouae, both the f. ocmta planidia alone, as well aa in coa^inatim with RoMRPV on a bran bait, and in the field, the combination treatment, significantly (F < .05) reduced the percentage of seedling corn eut by third instar BCWs. 126 LITERATURE CITED Allen, H. W. 1926* Observations upon the early maggot stage of timcum^ia aomta Fall. (DipteraiTachinidae). Entomol. News 37: 283-286. Allen» H. W. 1934. Soeema disease of Qncvimeah§ma opêv&uîêîla (Zeller) and Maoroaentrua cmo\flivcruê Rohwr. Ann. Entomol. Soc. Am. 47: 407-424. Allen, H. W. and M. H. Brunson. 1945. A microsporidian in Maaroamèrue ano\fîivem8, J. Eton. Entomol. 36:393. Allen. H. W. and M. H. Brunson. 1947. 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Entoa»l. 47*641-645. 137 ACXNOWLESCEMENtS The author wlahcc to thank h«r major profeaaor. Dr. L. Lavis for his advice, encouragement, and friendahip. Thanka ia alao expreased to the iMinbers of her committee, Drs. D. Foster, P. Kartman, R. Levia, and J. Mutchfflor for their intereat and support. The technical aasistance of R. Gunnerson, L. Anderson, 0. Lang, D. Cunnarson, and M. Lodholz vaa very much appreciated. Financial support waa provided by the United Statea Department of Agriculture, Iowa State University, and Agriculture Canada. Moral support vas provided by Bob.