JOURNAL OF INVERTEBRATE PATHOLOGY 48, 344-354 (1986)

Comparative Effects of Nosema epilachnae and Nosema varivestis on the Mexican Bean , 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 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 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. moisture within the dish. These conditions There were no differences in the pupation proved to be the best of several techniques and adult emergence rates of larvae in- tried to obtain data on egg hatch. Attempts fected with N. vurivestis. The average to surface-sterilize the eggs or to provide larval and total developmental periods of moisture on filter paper or via cotton wicks individuals infected with N. epiluchnue in resulted in extremety poor egg hatching all three treatments were similar and signif- rates. Observations on transovarian trans- icantly longer than those infected with N. mission were obtained by dissecting ap- vurivestis or control beetles; however, only proximately 10 neonate larvae per egg mass the average pupal period of those infected or by examining the entire egg mass as a with the lowest dosage rate of N. epi- squash preparation when the eggs failed to luchnue was lengthened significantly. The hatch. average larval, pupal. and total develop- Records were maintained on the length mental periods of infected beetles in both of the larval and pupal period; date of treatments of N. vurivestis were similar larval, pupal or adult death; number of egg and not significantly different from those of masses per female; number of eggs per control beetles. The adults in all of the mass; and egg hatching rate. However. no treatments were held until death, with effort was made to relate the develop- some of the noninfected or control beetles mental period or adult longevity to the sex surviving for as long as 95 days. Adults in- of the adult, except in those tests designed fected with N. epiluchnue, however, were specifically to measure adult fecundity and short-lived (averaging only 5 to 10 days in longevity. All individuals were examined at the two treatments in which some adults death or at the end of each test for micro- successfully emerged), while those infected sporidian infection in wet-mount prepara- with N. vurivestis lived significantly longer tions of tissue smears by phase micros- (averaging 27 to 35 days). Only those in- copy, fected adults exposed to the highest spore TABLE 1 SUMMARY OF THE PATHOGENIC EFFECTS OF Nosetnrr epilnclme AND NO.WI?I~I~r’i~rstis ON THE MEXICAN BEAN BEETLE, Epilnchnrr wwivesti.P

No. Larval Pupal Developmental Adult Treatmentb larvae period No. (%) period period longevity (spores/ per No. (%) No. (96) (days) adult (days) (days) (days) larva) treatment infected pupation Y ” SD emergence S 2 SD .\- t SD F f SD

Nosema epilachnue 1 x 10’ 43 24 (55.81) 23 (95.83) 18.35 ~fr 3.7la 12 (50.00) 6.00 2 0.85a 22.00 k 2.70a 4.92 5 5.30a 1 x 104 69 60 (86.95) 36 (60.00) 17.58 2 2.74a 18 (50.00) 5.33 5 0.77b 21.11 * 1.99a 10.11 * 12.33a 1 x 105 23 22 (95.65) 3 (13.64) 18.00 2 l.O@a 1 ( 4.55) - Cl.0 Nosema wrivestis 1 x 104 67 36 (53.73) 35 (97.22) 13.86 r 0.85b 35 (95.24) 5.14 t- 0.36b 19.00 i 0.84b 35.12 2 13.7lb,c 1 x 106 57 54 (94.74) 53 (98.15) 14.33 -+ 0.89b 52 (96.30) 5.06 ” 0.57b 19.40 2 0.96b 26.95 k 12.59b Control 73 0 72 (98.63) 13.90 2 0.63b 69 (94.52) 5.14 2 0.52b 19.00 i 0.75b 42.54 -t 22.68c a Means followed by the same letter are not significantly different at the 5% level, as determined by Tukey’s procedure. b Exposed as neonate larvae. 348 WAYNE M. BROOKS concentration of N. varivestis lived for a microsporidium as late-stage larvae (H P x significantly shorter period than did the I??). The ovipositional activities of the fe- control adults. (Although not included in males infected by N. ‘epilachnae in all three Table 1, the data on the average larval, treatments were not significantly different, pupal, and total developmental periods as but in all treatments the average numbers well as adult longevity of the exposed but of egg masses per female and egg masses noninfected beetles in each treatment were per female per day were significantly less similar to each other and not significantly than those produced by control females. different from those of the control.) Infected females in two of the treatments The first experiment designed to deter- also produced significantly smaller average mine the influence of N. epilachnae on egg masses than did the controls. In addi- adult longevity and fecundity (Table 2) was tion, many infected females produced no concluded 31 days post adult emergence eggs in two of the treatments. due to the apparent mortality of most of the A more extensive effort was made in the infected females in each treatment. At this second experiment (Table 3) to determine time 20 female beetles in the control were the effects of N. epilachnae on adult fecun- still alive. In contrast, only 12 of the re- dity and to measure parameters indicative maining 39 females in the three treatments of transovarian transmission of the micro- exposed to N. epilachnae were found to be sporidium. When terminated at 46 days infected. Although male beetles in only one post adult emergence, only 5.8% (4 of 69) treatment (HP x 18) were exposed di- of the infected females in all three of the rectly to the microsporidium, many of the treatments were still alive in contrast to males in the other two treatments as well as 54.2% (13 of 24) of the control females. some of the unexposed females in this Many males confined with infected females treatment also become infected as a result also became infected and their average lon- of their having been confined with their in- gevity was slightly longer than their female fected mates. In these cases it was impos- counterparts. In this test the exposure of sible to determine exactly when these indi- females at 1, 4, and 7 days post adult emer- viduals became infected. In addition, not gence generally resulted in higher infection all of the beetles exposed per OS to the mi- rates than those exposed to the same spore crosporidium became infected; thus the dosage as larvae or newly emerged adults data included in Table 2 is based only on in the first test. In addition, most of the in- those individuals that were infected at fected females were reproductively active. death or upon dissection. Again, the data The data (not included in Table 3) from the (not included in Table 2) on the exposed but only treatment (T,) in which there were noninfected individuals were statistically many noninfected but exposed adults were similar to those of the control. found to be statistically similar to those of In this test (Table 2) female longevity was the control. significantly shorter only in the treatment The average longevities of the infected where they had been exposed to N. epi- males and females were statistically similar lachnae as late-stage larvae (IO x Hc?). but they were both significantly shorter The average longevity of the infected males than the control, except those in treat- in the same treatment was also signfkantly ment T7 where the females were exposed 7 shorter than that of the control males as days post adult emergence. Females in was that of the males exposed directly as treatment T, produced significantly fewer late-stage larvae (H 0 x 16). And although average numbers of egg masses per female not compared statistically, males lived and egg masses per female per day than did slightly longer than females, except where those in treatment T7 or the control. The the males had been exposed directly to the females in treatment T4, however, were in- TABLE 2 EFFECT OF No.~na epilachncw ON THE LONGEVITY AND FECUNDITY OL’ Eoi/crchncl ,wri\v.sti.s (EXPERIMENT IP Longevityd No. No. egg masses Males Females No. egg masses per female No. eggs females per female per day per mass Treatment No. .w 2 SD No. x- k SD ovipositing (.T k SD) (S ” SD) (2 k SD) I?* x Hd 16 21.56 2 8.02a 19 15.79 -+ 8.61a 7 5.00 k 3.92a 0.186 2 0.12a 43.43 i 7.96a,b IP’ x H6 18 27.72 it 2.35b 26 24.15 2 6.20b 22 2.73 t 1.98a 0.104 ‘-t 0.07a 33.80 2 11.17a HO x Id* 13 17.69 k 8.43a 13 23.08 t 7.92b 7 4.00 k 4.16a 0.160 2 0.15a 33.13 2 11.49a E 26 2 HP x H6 26 27.65 k 6.82b 27.12 7.02b 24 9.42 -’ 3.30b 0.318 + O.IOb 46.09 e 7.59b z a Means followed by the same letter are not significantly different at the 5% level, as determined by Tukey’s procedure. 2 * Exposed as late-stage larvae to 1 x IO4 spores/larva. c Exposed as neonate adults to 1 x lo4 spores/adult. g d Test concluded after 31 days. h3

TABLE 3 EFFECT OF Nosernu epilachnue ON THE LONGEVITY AND FECUNDITY OF Epilachna vnrivestis (EXPERIMENT IHa

Longevity’ No. Males Females No. egg masses No. egg females per female masses/? /day Treatmen@ No. Jr 2 SD No. .r -e SD ovipositing (X k SD) (If t SD) T, 17 32.24 2 4.48a 23 27.96 5 7.62a 21 5.19 k 2.04a 0.178 ? 0.06a T4 17 35.76 2 5.78a 22 32.77 k 7.22a 21 8.38 ? 3.23a.b 0.249 k 0.06a.b T, 5 35.40 k 0.89a.b 9 36.33 t 3.87a,b 9 11.78 2 3.56b 0.306 2 0.06b.c Control 25 41.12 k 6.44b 24 39.25 2 10.60b 22 16.14 2 5.76~ 0.371 -t O.llc n Means followed by the same letter are not significantly different at the 5%~level. as determined by Tukey’s procedure. * Female beetles were exposed I, 4, and 7 days post adult emergence to 1 x IO4 spores/adult and then mated with healthy male beetles. c Test concluded after 46 days. 350 WAYNE M. BROOKS termediate in their ovipositional activities egg masses produced by an infected female and thus the average egg mass per female hatched. These masses were consistently and egg mass per female per day were sta- found to be infected when the whole mass tistically similar to T, and T7 but were sig- was examined in a wet-mount, squash nificantly different from the control. These preparation. More specific data on the inci- ovipositional indices of treatment T7 were dence of transovarian transmission are pro- the same as those in T4 and the control. vided, for example, by the females in treat- Unlike the females in the first test (Table 2), ment T,. Based on the sampling procedure there were no significant differences in the used where 10 neonate larvae per mass average number of eggs per mass of the fe- were examined for infection, the preva- males in any of the treatments. There was, lence of infection ranged from 10 to 100% however, a significant difference in the within egg masses where some larve average preovipositional period between T, hatched and at least 1 larva was infected. and the control but the other exposure The average rate of transmission was timings produced no significant differences 56.5% in similar egg masses produced by from the control; T, was also the same all of the females in this treatment. How- as T,. ever, the average rate of infection in the Mean egg hatching rates for the infected first egg mass producing at least one in- females were lower than those of the con- fected larvae was 45.6% while the average trol but the differences were only signifi- rate in the last egg mass from which any cant for females in treatments T, and T4. larva was able to hatch was 66.7%. While not evident in the data means in- The average number of days to the pro- cluded in Table 3, data for individual fe- duction of the first infected egg mass per males of each treatment indicated trends in female and the average number of egg the prevalence of transovarian transmis- masses produced per female to the produc- sion and in the egg hatching rates asso- tion of the first infected egg mass were cor- ciated with microsporidian infection. Once related with the timing of adult exposure. an infected female began producing in- The differences between treatments T4 and fected egg masses, only rarely did she pro- T, were nonsignificant, but these param- duce a subsequent egg mass that was com- eters were significantly lower for the fe- pletely free of the microsporidium. Also, males in treatment T,. in contrast to the controls in which egg Because of the low virulence of N. vari- hatching rates were consistently high vestis for the MBB, only limited data were throughout the 6- to 7-week ovipositional obtained on its influence on adult fecundity period, the egg hatching rates among the (Table 4). Infection rates among the adult infected females exhibited a dramatic de- females were low even though they had cline once an infected egg mass was pro- been exposed to the microsporidium as duced. These trends were particularly evi- neonate larvae, and the generally low dent in the females from treatments T, and hatching rates of eggs produced by the T4 where high egg hatching rates were ob- control females limited the observations tained for the first several egg masses prior largely to the ovipositional activities of the to the production of the first infected egg females. In addition, egg production rates mass. Thereafter, the infection rate in by females in the second test were gener- newly hatching larvae usually increased ally lower than those in the first, although rapidly and often reached 100% in egg this difference is likely related to the higher masses from which some of the larvae were dosage of spores to which these females able to hatch. Simultaneously, the egg had been exposed as neonate larvae. hatching rate decreased, and generally In the first test, concluded 22 days post none of the eggs within the last two or three adult emergence, there was no significant EFFECTS OF Nosemrr spp. ON E. vnrivcstis 351

TABLE 3a EFFECT OF Nosemu epilachnne ON THE LONGEVITY AND FECUNDITY OF Epilachnu varivestis (EXPERIMENT IIla Preovipositional No. days to No.eggmasses No. eggs s Egg period first infected to first infected per mass hatch/P (days) egg mass egg mass 4 2 SD .u 2 SD i k SD .u t SD X + SD 42.20 2 6.46a 47.57 2 16.86a II.86 2 2.71a 16.74 k 3.66a 1.79 k 1.13a 42.60 t 7.08a 52.14 2 16.lOa 10.00 ? l.lSb 22.63 -t 6.47b 5.11 * 2.35b 43.40 k 4.25a 69.83 ? l0.38b 11.00 of- 1.32a.b 23.71 t 5.02b 6.71 rf- l.BOb 47.99 2 8.77a 72.55 + 12.80b 9.64 k 0.85b - -

(1Means followed by the same letter are not significantly different at the 5% level. as determined by Tukey’s procedure, difference in the average longevities of the one infected larva. ‘However, the average infected and control females. However, the rate of transmission, 24.6%, was low, and infected females produced significantly in only 3 of 32 egg masses did the rate of fewer average numbers of egg masses per transmission exceed 40%. Infection rates female and egg masses per female per day did not increase in time and no relationship than did the controls. These differences was apparent between microsporidian in- were also significant and even more pro- fection and low egg hatching rates. nounced in the second test that was con- cluded 46 days post adult emergence. The DISCUSSION average longevity of the infected females, however, was significantly lower in this Like most species of the entomophilic test than those of the control. In addition, protozoa, microsporidia generally produce the average number of eggs per mass of the chronic, sublethal infections in their infected and control females did not differ hosts characterized by such symptoms as significantly in either test. irregular growth, retarded larval develop- Observations on transovarian transmis- ment, incomplete metamorphosis, and re- sion of N. i>nYireStiS indicated that the inci- duced adult vigor, fecundity, and longevity dence of transmission ranged from 10 to (Brooks, 1974; Canning, 1981). And, while 80% in egg masses that produced at least most species are preceived as important

TABLE 4 EFFECT OF Nosema ~uri\Bestis ON THE LONGEVITY AND FECUNDITY OF Epilachna vcrrivestisa No. Female No. egg No. egg No. eggs females longevity masses/ 0 masses/P /day per mass Treatment ovipositing S t SD S t SD 4 ” SD X 2 SD (Test lb) Infected 13 17.92 2 3.64a 4.46 2 2.30a 0.238 5 O.lOa 40.1 I t- 9.54a Control 21 19.33 t 4.15a 7.67 2 3.15b 0.380 t 0.13b 42.75 5 8.12a (Test ?) Infected 16 20.19 2 8.40a 3.44 2 2.39a 0.158 2 0.06a 33.55 2 12.24a Control I4 32.28 ” 12.28b 11.00 t 3.84b 0.341 f 0.08b 36.04 2 13.lOa D Means within the columns for each test followed by the same letter are not significantly different, as deter- mined by Student’s t test. b Each adult female had been exposed to 1.9 x lo5 spores as a neonate larva; test concluded 22 days post adult emergence. ’ Each adult female had been exposed to 1.29 x IO6 spores as a neonate larva: test concluded 46 days post adult emergence. 352 WAYNE M. BROOKS

natural mortality factors but of only limited On the other hand, female beetles ex- value as candidates for short-term, micro- posed to N. epilachnae as either late-stage bial insecticides, several species have been larvae (Table 2, treatment I’? x Hd) or as evaluated for use in applied pest control newly emerged adults (Table 3, treatments programs (see reviews by McLaughlin, T, and T4), exhibited significant reductions 1971; Brooks, 1980; Henry, 1981; Canning, in both their longevity and fecundity as 1982). Of these, Nosema locustae has al- compared to the controls despite the lower ready been developed as a control agent for spore dosage rates used. The only treat- rangeland grasshoppers (Henry, 1981) and ment in which female longevity and fecun- Vairimorpha necatrix possesses high po- dity were not significantly lowered was in tential as a short-term control agent for use T, (Table 3) where the females were ex- against lepidopterous pests (Maddox et al., posed 7 days post adult emergence. The 1981). effect of N. epilachnae on adult longevity Of the two species involved in this study, is further indicated by the fact that the N. epilachae is decidedly more virulent for average longevity of the infected males was the MBB than is N. varivestis and appears generally less than that of the control males to possess the greater potential as a micro- even though they were not exposed di- bial control agent (Table 1). At relatively rectly to spores of the microsporidium. Al- low spore doses, larval mortality ranged though the males in each treatment gener- from 50 to 95% and most of the success- ally survived slightly longer than did their fully emerging adults die within an average respective female counterparts, this differ- of 10 days or less. The average develop- ence may have been due to delayed inno- mental period of these adults was also sig- culation from their indirect exposure to the nificantly lengthened over those infected microsporidium via spores liberated in the with N. varivestis or the controls. In con- feces of the infected females. trast more than 95% of the neonate larvae Despite the significant reductions in exposed to the highest dosage of N. vari- adult longevity and fecundity by both vestis emerged as adults, and the adults species of microsporidia, one parameter in- survived at least two to three times longer dicative of fecundity (number of eggs per on the average than did those infected with mass) was not consistently affected. There N. epilachnae. In fact, the only significant was no difference in the number of eggs per difference between the controls and those mass in the tests with N. varivestis (Table beetles infected with N. varivestis was in 4) or in the more extensive test with N. the average adult longevity of those ex- epilachnae (Table 3). Differences noted in posed to highest dose of spores. the other test with N. epilachnae (Table 2) Because the MBB is long-lived and feeds may have resulted from a small sample size extensively in both the larval and adult in two of the treatments or abnormal varia- stage, any potential microbial control agent tion in the data. Regardless, the general should also be effective against the adults lack of significant differences in the as well as the larvae. In this respect N. number of eggs per mass is consistent with varivestis is, again, decidedly less attrac- the results of Kitayama et al. (1979) who tive than N. epilachnae. Adult longevity found that this parameter for the MBB was was only reduced significantly by N. vari- not affected by host plant stage. They con- vestis when MBB were exposed as neo- cluded that a certain level of nutrients was nates to the highest spore dose (Table 4). required to produce an egg mass and that And, while egg production was also signifi- oviposition would not occur until that level cantly lower, it is highly unlikely that sim- was reached. ilar results would have been obtained had While N. epilachnae is obviously patho- the beetles been exposed as late-stage genic to adult MBB, the responses ob- larvae or as adults. tained in this study also indicate that they EFFECTS OF Nose/w spp. ON E. varivestis 353 were related to adult age at exposure (Table as a microbial control agent of the MBB are 3). Thus, the reductions in adult longevity warranted. This species is also infectious and fecundity were greatest in females ex- for the squash beetle, Epilachna borealis posed 1 day post adult emergence (treat- (Brooks et al., 1980), the Colorado potato ment T,) and the reductions noted in the beetle, Leptinotarsa decemlineata, and other two treatments (T, and T,) were ob- several species of Lepidoptera (Brooks et viously correlated, respectively, to the al., 1985). Preliminary results indicate that delays of 3 and 6 days before their expo- this microsporidium can be mass produced sure to the microsporidium as adults. This in larvae of the corn earworm, Hefiothis apparent increase in the resistance of fe- zea, and may be useful as a control agent of males with age might, however, be over- the Colorado potato beetle (unpublished come with higher spore dosage rates. observations). On the other hand, N. vuri- Higher spore rates might also considerably vestis is less attractive as a control agent lower adult fecundity and longevity. because of its low virulence for the MBB The eggs produced by adults infected and the difficulties involved in its propaga- with N. epilachnae also exhibited reduc- tion. However, both species are present at tions in their average hatching rates which low levels in natural populations of the were correlated with adult age at exposure. MBB and, at least under laboratory condi- Again, as the reductions were only signifi- tions, may pose serious problems in main- cant for the females exposed 1 and 4 days taining laboratory colonies of the MBB post adult emergence, reductions in the egg (Brooks et al., 1980) or the eulophid para- hatching rates for older females could prob- site, P. fo\veolatus (Own and Brooks, ably only be achieved through the use of 1986). higher spore dosage rates. Another attribute of N. epilachnae as a ACKNOWLEDGMENTS potential microbial control agent is its ca- I am grateful to Mr. J. D. Cranford for his technical pacity to be transmitted transovarially at assistance. This is Paper 10359 of the Journal Series of relatively high rates and its ultimate effect the North Carolina Agricultural Research Service. on egg hatching rates of the MBB. The Raleigh, N.C. 27695-7601. length of time and the number of egg REFERENCES masses to the production of the first in- fected egg mass were correlated, again, ALDRED. J. M., SHEPARD, M.. AND HOLMES. P. T. with adult age at exposure; however, once 1980. A stochastic model of parasitism of the Mex- ican bean beetle, Epilachna varivestis Mulsant (Co- an infected egg mass was produced, the in- leoptera: ), in soybeans by Pediobius cidence of infection generally increased in foveolatus (Crawford) (Hymenoptera: Eulophidae). subsequent masses accompanied by dra- Res. Popul. Ecol. 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