A Pathogenic Bacterium,Enterococcus faecalis,to the Beet Armyworm,Spodoptera exigua

A bacterial disease was found in the beet armyworm, Spodoptera exigua (Hübner). Blackened body of the infected larvae was a typical symptom of the epizootic disease especially at the intersegmental areas. We isolated the bacteria from the hemolymph of the infected 5th instar larvae and identified the isolate as a gram-positive bacterium, Enterococcus faecalis. When the 4th instar larvae were injected with the bacteria, half lethal dose of the bacteria was estimated as 22,593 colony-forming units (cfu) per larva and half lethal time of the bacteria was estimated as 2 days at 10⁷ cfu injection and 6 days at 10⁸ cfu injection. The bacteria were strongly resistant to each 1,000 ppm of ampicillin, kanamycin, and streptomycin. They were, however, relatively susceptible to mixture (1,000 ppm) of different combinations of the three antibiotics.     

Insights into How Longicorn Beetle Larvae Determine the Timing of Metamorphosis: Starvation-Induced Mechanism Revisited

Larvae of holometabolous insects must determine the timing of their metamorphosis. How they determine this timing has only been studied in detail for a few insect species. In a few species of Coleoptera, starvation is known to be a cue for metamorphosis, leading to the formation of smaller adults (starvation-induced pupation, SiP). We investigated the occurrence of SiP in the beetle Psacothea hilaris. When P. hilaris larvae were starved late in the feeding phase of the last (5th) instar, they exhibited typical SiP characterized by constancy of the time from food deprivation to pupation (TTP) irrespective of the body weight upon food deprivation or the length of prior feeding. In contrast, when larvae were starved early in the feeding phase, TTP decreased by roughly 1 day as the feeding became 1 day longer. The change in the response to starvation was estimated to occur on day 5.9 in the last instar. A series of refeeding experiments suggested that whereas SiP occurred readily in the larvae starved in the late feeding phase, activation of SiP was suspended until day 5.9 in the larvae starved early in the feeding phase. When P. hilaris larvae were fed continuously, they eventually ceased feeding spontaneously and pupated. The time length between spontaneous cessation of feeding and pupation was approximately equal to the TTP in SiP. This suggests that the same mechanism was activated by food deprivation in the late feeding phase and by spontaneous cessation of ad libitum feeding.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Effects of azadirachtin on the nutrition and development of the tobacco budworm,Heliothis virescens (Fabr.) (Noctuidae)

The plant chemical azadirachtin was administered, either in artificial diet or by oral injection, to fifth instar larvae of the tobacco budworm, Heliothis virescens (Fabr.). At a dietary concentration of 0.03125 ppm, azadirachtin significantly reduced the amount of diet consumed and the weight gained by the larvae. Higher dietary concentrations (0.25 and 0.5 ppm) were necessary to reduce the efficiency of larval conversion of digested and ingested food, respectively. However, the approximate digestibility increased at the dietary concentration of 0.25 ppm.Orally injected azadirachtin (0.25 and 0.5 μg) delayed moulting to the pupal stage, produced defective pupae or adults, and inhibited development to the adult stage. Higher doses (5.0 and 10.0 μg) reduced the pre-pupal weight loss normally associated with pupation, and completely inhibited pupation. At the critical dose of 1.0 μg (the minimal dose that disrupted development to the pupal stage), azadirachtin had less of an effect on older than on younger larvae. Larvae injected on the first day of the fifth instar failed to pupate, whereas approx 40% of those injected on subsequent days pupated.The results suggest that azadirachtin affects H. virescens in a manner similar to other tested species of insects. The significance of these results, especially regarding hormonal events in the insects, is discussed.     

A method for rearing the yellow-spotted longicorn beetle, <Emphasis Type="Italic">Psacothea hilaris</Emphasis> (Coleoptera: Cerambycidae), to stabilize the last larval instar

The larvae of Psacothea hilaris (Pascoe) grown continuously under a long day in the laboratory pupate after the 4th, 5th, or 6th instar. This developmental polymorphism has complicated studies on the control of metamorphosis in P. hilaris. Since pupation in P. hilaris is known to be suppressed under a short day, a change in the photoperiod from a short to a long day with appropriate timing may assist in obtaining physiologically homogeneous larvae that pupate at the next molt. When the photoperiod was changed from 12-h light:12-h dark to 15-h light:9-h dark at the beginning of the 5th instar, the pupation rate at the next (5th) molt reached 90%, which was significantly higher than the proportion of larvae that pupated after the 5th instar when the larvae were grown continuously under a long day (52%). This rearing technique will expedite studies on the control of metamorphosis in P. hilaris.     

Effect of Host Plant on the Infectivity of Sl MNPV to Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Larvae

The susceptibility of Spodoptera litura to SlMNPV infection was markedly affected by phyto-chemicals ingested during the acquisition of viral inoculum on foliage of tomato and cauliflower. The LD₅₀ values computed for second, third and fourth instar larvae assayed on tomato leaves were 254, 819 and 23395 PIBs/larva, respectively whereas, it was 326, 1719 and 43843 PIBs/larva for respective instars when assayed on cauliflower leaves. Thus LD₅₀ values for second, third and fourth instar larvae were 1.28-, 2.09- and 1.87- fold lower, respectively in tomato leaves. Similarly, LT₅₀ values for second, third and fourth instar larvae assayed on tomato leaves were 7.1 and 7.5 days, respectively at inoculum dose of 2.7×10⁴ PIBs/larva whereas, it was 7.7 and 8.0 days for respective instars when assayed on cauliflower leaves at same inoculum. This result also showed that the S. litura were more susceptible on tomato leaves in comparison to cauliflower leaves as the time required for mortality was lower in tomato leaves. The possible biochemical bases for differential level of mortality of S. litura larvae on tomato and cauliflower crops needs to be investigated.     

Effect of Secondary Metabolites <Emphasis Type="Italic">Juniperus sabina</Emphasis> L. (Cupressaceae) on the Survival and Nutrition of <Emphasis Type="Italic">Galleria mellonella</Emphasis> L. (Pyralidae)

The effect of alcohol extract and essential oil from Juniperus sabina L. (Cupressaceae) on the survival and nutrition of the larvae Galleria mellonella L. (Pyralidae) has been studied. It is found that the essential oil has insecticidal, antifeedant, deterrent, and repellent properties. The loss of larvae with topical application of 0.2 μL of essential oil to the dorsal part (contact action) reaches 64.0%. Feeding larvae with food treated with essential oil J. sabina (200 μL of essential oil per 1.5 g of feed) is reduced 2.1 times compared to the control. It is shown that larvae G. mellonella avoid feeding on oil-treated feed for 14 days and actively move in the opposite direction from the treated feed when they are placed in Petri dishes. It has been established that vapors of essential oil suppress the development of larvae G. mellonella even when fed with untreated food—there is a lag in body weight and a delay in molting in the next age. The essential oil J. sabina also exhibits moderate toxicity (20.0%) when the larvae eat the treated food, i.e., possess intestinal action. The alcohol extract of J. sabina at a concentration of 2.5% of active substances (on dry residue) has a negligible effect on the loss of larvae in the case of treated feed (10.0% contact action). With topical application to the dorsal part, the extract causes 20.0% insect death. An alcohol extract of J. sabina does not elicit any antifeedant or repellent properties against larvae; however, a low level of deterrental activity, expressed in suppressing the formation and development of pupae of Galleria mellonella, is noted. Essential oil reduces the percentage of pupated larvae and emitted adults 3 times and the mass of larvae 40.7–48.0% when compared with the control.     

Organization of protein digestion in adult Calosoma calidum (Coleoptera: Carabidae)

Organization of protein digestion was examined in adult male Calosoma calidum (Carabidae) fed either ground-beef or waxmoth larvae (Galleria mellonella). Although trypsin activities in the foregut are consistently higher than those in the midgut, the luminal contents of each region are in equilibrium. Movement of fluids between the midgut and foregut is brought about by muscular contractions of the proventriculus. A 42% fall in trypsin activity of the foregut after feeding on ground-beef is due largely to disgorged enzymes being left on the food. A far higher proportion of disgorged trypsin is recovered when C. calidum feed on waxmoth larvae; beetles ingest about 74% prey protein and yet avoid ingesting digestively refractory solids. The retention of undigested macromolecules in the midgut and foregut lumen was determined using [¹⁴C]inulin labelled waxmoth larvae. Nine per cent and 25% of the radiolabel was passed in the faeces in 2 and 4 days respectively, whilst 59 and 91% of the weight gained at feeding was lost in the same intervals. The role that the peritrophic membrane and pyloric valve play in this process, as well as its implications for enzyme conservation, is discussed.     

Photoperiodic response of larvae of the yellow-spotted longicorn beetle Psacothea hilaris after removal of the stemmata

The role of the stemmata in photoperiodism has been examined in holometabolic insects, but the only reliable results in Coleoptera have been obtained in Leptocarabus kumagaii (Carabidae), the larvae of which do not respond to photoperiod without stemmata. In the present study, photoperiodism was examined in another coleopteran, Psacothea hilaris (Pascoe) (Cerambycidae), after surgical removal of the stemmata. Larvae reared under short-day conditions and transferred to long-day conditions on day 2 of the 5th instar pupated without further larval molts, whereas those continuously reared under short-day conditions underwent supernumerary molts and did not pupate. When the stemmata were removed on day 2 of the 5th instar, the larvae pupated under long-day conditions but did not do so under short-day conditions. However, under long-day conditions some underwent supernumerary molts before pupation. Larvae from which the sensilla trichodeum were removed showed a similar response to that of stemmata-deficient larvae, and larvae from which stemmata were removed at a younger stage (day 2 of the 4th instar) responded to photoperiod similarly to intact larvae. Thus, supernumerary molts under long-day conditions after removal of the stemmata were attributed to injury due to surgery, rather than a change in photoperiodic photoreception. Therefore, we conclude that larvae of P. hilaris show a photoperiodic response after removal of stemmata, in contrast to larvae of L. kumagaii.     

Infection of a nucleopolyhedrovirus to Neodiprion zhejiangenis Zhou & Xiao (Hymenoptera: Diprionidae)

Neodiprion zhejiangenis Zhou &; Xiao 1981 (Hymenoptera: Diprionidae) nuclear polyhedrosis virus (NezhNPV) is a biocontrol factor with high development potential and application prospects. To study the insecticidal activity of NezhNPV against the larvae of N. zhejiangenis, the susceptibility of larvae at different instars to different NezhNPV concentrations was determined based on laboratory bioactivity. At 2.0?×?10⁷ polyhedron inclusion body (PIB)/mL, the median lethal time required to kill 50% of the instar 2 and 3 larvae was approximately 5 d, whereas the median lethal time of the instar 4 and 5 larvae was 8–9 d. Interestingly, at the NezhNPV concentrations of 2.0?×?10³ and 2.0?×?10⁴ PIB/mL, the feeding ability of the surviving larvae was decreased by 57.4% and 76.4%, respectively, compared with the controls; the pupal weight at both concentrations was decreased by approximately 27%, and the adult emergence rate was decreased by 27.4% and 50.9%, respectively, with a significantly higher proportion of males than of females. The results showed that younger instar larvae of N. zhejiangenis were more susceptible to NezhNPV infection than older instar larvae. The larval mortality rate was dependent on the larval instar and NezhNPV concentration.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

Transmission of Spiroplasma citri by the aster leafhopper Macrosteles fascifrons (Homoptera: Cicadellidae)

The aster leafhopper (Macrosteles fascifrons), injected with an isolate of Spiroplasma citri obtained from brittle root-diseased horseradish (Armoracia rusticana), transmitted the spiroplasma to horseradish and China aster (Callistephus chinensis.) After feeding on plants infected with S. citri, M. fascifrons transmitted the spiroplasma from aster to aster and horseradish, from yellow rocket (Barbarea vulgaris) to aster, and from turnip (Brassica rapa) to turnip. Symptoms in infected horseradish were chlorosis and stunting of newly formed leaves, discoloration of root phloem, and reduced plant growth typical of brittle root disease. Chlorosis, stunting, and asymmetry of young leaves occurred in affected aster and turnip. Flowers of infected aster were small and pale in colour and occasionally showed other symptoms including asymmetry, petal distortion, or light green petals. Spiroplasmas were isolated from all plants showing symptoms. Transmission rates by M. fascifrons which acquired S. citri by feeding on infected plants were very low, but injected leafhoppers transmitted more frequently. This is the first report of the transmission of S. citri from diseased to healthy plants by M. fascifrons.     

Studies on the biology of Chaoborus flavicans (Meigen) (Diptera: Chaoboridae) in a fish-free eutrophic pond,Japan

Population dynamics of Chaoborus flavicans larvae of various instars was studied from November 1986 to December 1987 in a eutrophic, fish-free pond, Japan. First and 2nd instar larvae were observed from late April to late October, indicating a reproductive period of about half a year. C. flavicans overwintered in the 4th instar larvae. In water column samples, total density of all instars was 680–23680 m^-2, and pupal density 0–2600 m^-2; larvae of the 1st, 2nd, and 3rd instars showed 5–6 density peaks in 1987, suggesting that 5–6 generations occur during a year (peaks of the 4th instar larvae were not clear, probably due to their longer development than those of younger instars). In sediment samples, no 1st and 2nd instar larvae were found, 3rd instar larvae were found occasionally but density of the 4th instar larvae was 280–18600 m^-2, and pupal density varied between 0–502 m^-2. Fouth instar larvae accumulated in sediment in the cold season and in the water column in the warm season; high temperature and low oxygen concentration were the most important factors limiting the distribution of larvae in the sediment in summer in the NIES pond. The dry weight of total C. flavicans larvae was 0.08–4.2 g m^-2 in sediment samples and 24–599 μg l^-1 (0.10–2.40 g m^-2) in water column samples. Comparisons of maximum densities in the NIES pond in different years and in waters of different trophic status show that density is generally higher in eutrophic than in oligotrophic habitats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Selection of plant parts by Depressaria multifidae (Lep., Oecophoridae) on its seasonally-restricted hostplant, Lomatium grayi (Umbelliferae)

ABSTRACT.

• 1 Depressaria multifidae Clarke feeds on a broader variety of Umbelliferae plant parts than other Depressaria species.
• 2 Early instar larvae feed in the sheaths surrounding floral buds and leaves. Later instar larvae feed in the sheaths and floral stems and on flowers and leaves.
• 3 Floral stems bored by larvae had significantly larger basal stem diameters than floral stems that were not bored. Smaller stems usually have umbels with only male flowers, and wither after flowering, too soon for larvae to complete development. In contrast, larger stems often have umbels with some hermaphroditic flowers, which remain green and erect long enough for larvae to complete development. Hence, selection may favour larvae that bore only in relatively large stems.
• 4 In the laboratory, larvae fed sheaths with enclosed floral buds, flowers, or leaves all pupated at the same weight, but larvae fed floral stems pupated at a significantly lower weight. Larval and pupal development time was the same on all plant parts.
• 5 In the field, larvae restricted to a single umbel throughout development pupated at the same weight as those restricted to a single leaf.
• 6 Unlike in other Depressaria species, nitrogen levels only partly correspond to the pattern of use of plant parts in D.multifidae. Nitrogen values varied as follows: floral buds > immature leaves ≥ flowers > floral stalks > sheaths excluding floral buds or leaves.
• 7 The broad variety of plant parts used by D.multifidae may result partly from the problem of feeding on a small, seasonally restricted hostplant; the greater use of sheaths and floral stems than in other Depressaria species may result from selection for safety from parasites or predators.
• 8 The results for D.multifidae indicate that the way in which an insect feeds on a plant species can vary broadly even at a single site.

     

Nuclear polyhedrosis virus as a biological control agent for Malacosoma americanum (Lepidoptera: Lasiocampidae)

In addition to damaging trees, the eastern tent caterpillar is implicated in early fetal loss and late‐term abortion in horses. In a field study, we evaluated the potential biological control of the caterpillar using eastern tent caterpillar nuclear polyhedrosis virus (ETNPV), a naturally occurring virus that is nearly species‐specific. Egg masses were hatched and second instar larvae were fed virus‐inoculated foliage to propagate the virus in vivo. Then, a viral pesticide was formulated at concentrations of 10⁴, 10⁶ and 10⁸ polyhedral inclusion bodies per ml. The pesticide was applied to foliage on which second, third and fourth instar caterpillars were feeding. When the majority of surviving larvae reached the sixth instar, colonies were collected and the surviving caterpillars counted. Mean numbers of surviving caterpillars per treatment were compared via 95% bootstrap confidence intervals. The data indicate second instar caterpillars were highly susceptible to the virus, but only at the highest concentration tested. Third instar caterpillars were also somewhat susceptible to high virus concentrations, while fourth instar caterpillars were fairly resistant. Our data provide the strongest evidence to date that ETNPV can be propagated, harvested and refined for formulation as a biological control agent for eastern tent caterpillar. Its use on this insect may be merited in circumstances where landowners and managers need to protect trees and horses.     

Laboratory studies of the biology and food requirements ofMacrorhaphis acuta [Hemiptera: Pentatomidae)

Macrorhaphis acuta were bred in the laboratory (25°C) and fed on larvae ofAscotis selenaria reciprocaria. The incubation period was 8.4 days and the mean hatch was 94.4%. There were 5 nymphal instars which occupied 3.3, 5.0, 5.6, 6.6 and 12.0 days, respectively, from the lst to the 5th. Each nymph consumed an average of 22.8 host larvae to complete development. Adults had a mean longevity of 74.9 days and each consumed an average of 67.5 host larvae. Significant reduction was noted in pupation of 5th instar host larvae sucked by adult predators for periods ranging from 4 to 10 mn. The food requirements of 3rd, 4th, and 5th instar nymphs fed on 3rd instar host larvae were also investigated. The conversion ratio varied with the weight of food consumed during the instar and later instars were more efficient than earlier ones. Predatory value did not vary significantly with successive instars.     

转Bt基因棉花对棉铃虫不同龄期幼虫的杀虫活性和抑制生长作用

以我国培育的转Bt基因棉花(简称Bt棉)为材料,系统测定了Bt棉叶片对棉铃虫 Helicoverpa armigera(Hubner)不同龄期幼虫的杀虫活性和抑制生长的作用。结果表明,Bt棉的杀虫活性随着龄期的增大而趋于降低,其中处理3d只对1龄幼虫有较高效果;从1～4龄开始连续取食Bt棉后均不能化蛹,5龄幼虫则能正常化蛹。3龄幼虫取食Bt棉叶3d后的体重与初始体重之比为0.94,而取食常规棉叶的相应比值为5.48,对幼虫生长的抑制作用明显。在28℃条件下用Bt棉饲养棉铃虫1～3龄幼虫3d,对幼虫的致死率显著高于25℃处理,对3龄幼虫抑制生长的作用也显著提高。研究结果可为确定棉铃虫对Bt棉的抗性监测与治理技术以及Bt棉的田间应用技术提供依据。     

Evidence for the presence of a threshold weight for entering diapause in the yellow-spotted longicorn beetle, Psacothea hilaris

Under long-day conditions larvae of Psacothea hilaris (Coleoptera: Cerambycidae) pupate after the 4th or 5th instar, while under short-day conditions they undergo 2-4 nonstationary supernumerary molts and eventually enter diapause. To explore the possibility of a threshold weight for entering diapause, P. hilaris larvae were deprived of food on days 0 (day of ecdysis), 4 or 8 of the 4th, 5th and 6th instars under short-day conditions. Within the first 40 days of starvation, 60% of the larvae starved starting on day 0 of the 4th instar died, but all the larvae starved at later stages survived. The incidence of diapause in these survivors was determined by the occurrence of pupation after a temporary chilling at 15 degrees C for 15 days. Diapause incidence increased as the onset of starvation was delayed; from 11% in the larvae starved on day 0 of the 5th instar to 100% in the larvae starved on day 4 and day 8 of the 6th instar. Analysis of the relationship between the initial weight of a respective larva at the onset of starvation and its pupation success revealed that none of the larvae weighing 690 mg did. This finding suggests the presence of a threshold weight (about 600 mg), below which larvae are incapable of entering diapause. We discuss these findings with reference to the life history of P. hilaris.     

Translocation and multiplication ofSpiroplasma citri in turnip (Brassica rapa)

Following inoculation of designated leaves of turnip plants withSpiroplasma citri byCirculifer tenellus, spiroplasmas were cultured first from roots (four days) and then from youngest leaves (eight days), but almost never from oldest leaves. In experiments using enzyme-linked immunosorbent assay to monitor changes in titer in turnip leaves during the course of plant infection,S. citri was detected seven days after inoculation and reached peak titers of 10¹⁰–10¹¹ colony-forming units/g 12–20 days after inoculation, declining thereafter. Spiroplasmas were detected 5–9 days before symptoms appeared.