JOURNAL OF INVERTEBRATE PATHOLOGY 48, 344-354 (1986) Comparative Effects of Nosema epilachnae and Nosema varivestis on the Mexican Bean Beetle, Epilachna varivestis WAYNE M. BROOKS Department of Entomology, North Carolina State University, Raleigh, North Carolina 276957613 Received February 27. 1986; accepted May 16. 1986 In comparative tests with two species of microsporidia of the Mexican bean beetle, Epilachnu varivestis. Nosema epilachnue was decidedly more virulent than N. varivestis, with the lowest dosage rate of N. epilachnue (I x IO3 spores per larva) resulting in an adult emergence rate of only 50% in contrast to a 96.3%, survival rate of larvae exposed to the highest dosage rate of N. lwri- vestis (I x IO6 spores per larva)-a IOOO-fold difference in spore dosage rates. The adults infected with N. varivestis also survived an average of two to three times longer than those infected with N. epilachnue. Although both species produced significant reductions in adult longevity and fecun- dity. the test individuals were exposed to N. epiluchnue as either late-stage larvae or as newly emerged adults while those exposed to spores of N. vurivestis were inoculated as neonate larvae. Reductions in fecundity were manifested as lower average numbers of egg masses per adult and egg masses per adult per day; however, neither species reduced the average number of eggs per mass on a consistent basis. Reductions in adult longevity and fecundity by N. epiluchnue were related to adult age at exposure. Both species were also transmitted transovarially, but the average rate of transmission of N. vtrrivestis was low and did not increase in time. In contrast. the inci- dence of transovarian transmission of N. epiLx~/znue increased with time to levels approaching 100% and was accompanied by a dramatic decline in egg hatching rates. The highly virulent nature of N. epilachnae for both larvae and adults of the MBB indicates that further effort to evaluate its potential as a microbial control agent is warranted. ‘i 19X6 Academic Prc\\. Inc. KEY WORDS: Mexican bean beetle; Epiluchnrr rwril.e.stis; Nosemu epilachnrre; Nosemtr lwri- vestis; Microsporidia, comparative pathogenicity. INTRODUCTION formulations of B. thlrringiensis are not yet registered for use in the US. The Mexican bean beetle (MBB), Epi- The MBB has been considered to be vir- fachna varivestis, is a serious pest of bush tually free of naturally occurring pathogens beans and soybeans in the United States in the US (Aldred et al., 1980), but early (Aldred et al., 1980; Barrows and Hooker, reports of a few bacteria and fungi of un- 1981; Michels and Burkhardt, 1981) and certain taxonomic status and more recent has become the focus of increasing atten- reports of viruses and a fungus associated tion by workers interested in biological and with the MBB are briefly reviewed by microbial control. The eulophid parasite Brooks et al. (1985). In addition, two mi- Pediobius foveolatus is the primary factor crosporidia initially reported as pathogens around which several pest management of the MBB in a note (Brooks et al., 1980) programs for the MBB have been recently have been recently described as new structured (Stevens et al., 1975; Coulson, species of the genus Nosema (Brooks et 1976; Schroder, 1981), while microbial con- al., 1985). Their interrelationships with P. trol efforts have involved preparations of foveolatus have also been recently de- Bacitfus thuringiensis that contain a heat- scribed (Own and Brooks, 1986). stable exotoxin (Cantwell and Cantelo, The purpose of the present paper is to 1982; Cantwell et al., 1985). However, such describe the host-pathogen relationships 344 0022-2011186 $1.50 Copyright Q 1986 by Academic Presr. Inc. Ml rights of reproduction in any form reserved. EFFECTS OF Nosemn spp. ON E. varivestis 345 of these two microsporidia, Nosema epi- containing a thin layer of 2% agar, and al- lachnae and N. varivestis, with the MBB lowed to dry for ca. 1 hr prior to the addi- and to enhance awareness of the potential tion of the neonate larvae. Larvae were ex- of at least one of the species as a microbial posed in groups of five per disk for 48 hr at control agent for the MBB. 26.5”C. Since each larva ate about ‘/s of the treated disk, spore dosage was calculated MATERIALS AND METHODS on the basis of a further ‘/s dilution of the Isolates of the two microsporidia were original spore count per milliliter of stock derived from strains previously reported by suspension. The spore concentration in Brooks et al. (1980) that had been main- stock suspensions of each species was de- tained by frequent passage through larvae termined with a bacterial counter (Petroff- of E. varivestis. Suitable quantities of Hauser), and adjustments were made to spores of each species were produced peri- obtain the desired concentration per milli- odically by feeding neonate MBB larvae liter. In tests involving late-stage larvae or with disks cut from lima bean leaves inocu- adults, only one individual was placed on a lated with ca. 0.05 ml of stock spore sus- single leaf disk treated and handled in a pensions of each species. Spores were har- similar manner for a 24- to 48-hr exposure vested from heavily infected cadavers by period. Only individuals or groups of larvae masceration in distilled water, filtration that consumed more than 90% of the leaf through cheese cloth, and differential cen- disks within 48 hr were used in the various trifugation. Clean spore suspensions of treatments in each test. In tests with newly each species in distilled water were stored emerged adults, each individual was al- at 4°C for up to 1 month before utilization lowed to feed for 24 hr prior to exposure to in infectivity studies. the microsporidium and was mated imme- Mexican bean beetles were obtained as diately after removal from the exposure needed from a microsporidian-free stock chamber. After the exposure period, each culture maintained at North Carolina State larva or pair of mated adults was main- University on flats of 3- to j-week-old lima tained in a rearing room at 26.7”C, 60% beans (Fordhook 242). Samples of larvae RH, and 16L:8D photoperiod within sepa- and adults were checked routinely to as- rate plastic Petri dishes (15 x 100 mm) with sure their microsporidian-free status before fresh leaves added daily for the duration of use in various infectivity studies. each test. Of several options compared in The primary pathogenic effects of each preliminary tests, these conditions proved microsporidian species on the MBB were to be the most satisfactory for rearing indi- determined by exposing neonate larvae to vidual larvae to adults that were long lived varying spore dosages of each Nosema and reproductively active. species. One to three replicates of 25 larvae In tests designed to determine effects on each were exposed in each treatment; repli- adult longevity and fecundity, MBB were cates were combined for analysis. How- exposed either as late-stage larvae or as ever, because of the high virulence of N. adults to spores of N. epilachnae. The first epilachnae for the MBB, tests carried out test with this species involved the following to determine its influence on adult lon- treatments: infected females (I?) (exposed gevity and fecundity also involved the ex- as late-stage larvae) x healthy males (Hd); posure of late-stage larvae and newly IO (exposed as newly emerged adults) x emerged adults to spores of this species. Hc? ; HO x infected males (16) (exposed In the tests involving neonates, leaf disks as late-stage larvae; and H 0 x H 6. Since (21 mm in diameter) were inoculated with the infected adults died fairly rapidly in this either 0.05 or 0.025 ml of each spore sus- test (most within 3 weeks), the second test pension, placed in a 30-ml plastic jelly cup involved only females that were exposed as 346 WAYNE M. BROOKS newly emerged adults after intervals of 1 The data were analyzed using a one-way (T,), 4 (TJ, or 7 (T,) days post adult emer- analysis of variance and Tukey’s procedure gence. A recently emerged and healthy for comparing means of unequally repli- male was placed with each female immedi- cated treatments. In one test, means were ately after the exposure period. An appro- compared directly using Student’s t test. priate control was included in the test. Two tests with N. varivestis were also con- RESULTS ducted utilizing beetles inoculated only as As summarized in Table 1, N. epiluchnue neonate larvae. The tests differed in the was much more virulent for the MBB than spore dosage to which the neonate larvae N. vurivestis. Of the neonate larvae ex- were exposed and in the number of days posed to the highest dosage of spores of N. post adult emergence during which data varivestis, 96.3% of the infected individuals were obtained on ovipositional activities. emerged as adults while only 50% of those Because of the extensive effort required infected at the lowest dosage of N. epi- and often poor results obtained, only lim- luchnae successfully emerged as adults. In- ited efforts were directed at obtaining data fection rates for both species were directly on egg hatch and transovarian transmission related to spore dosage although there was of each microsporidian species. Individual little difference between the percentage in- egg masses attached to the leaf surface on fection with N. epiluchnae at the two which they had been oviposited were highest dosage rates. Larval mortality was transferred to a Petri dish which contained also directly related to spore dosage for N. a fresh lima bean leaf (periodically re- epiluchnue, but pupal mortality was the placed) to provide adequate atmospheric same for the two lowest spore dosage rates.