Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.     

Daphnia vertical distribution and the presence of toxic cyanobacteria

Daphnia can alter its vertical position in the water column in response to chemical cues from predators. In this study we tested the hypothesis that a Daphnia pulex clone with little evolutionary exposure to cyanobacteria would move away from patches of cyanobacteria (Anabaena affinis and A. flos-aquae) which contain potent endotoxins. Daphnia was censused at 2 h intervals for 6 h in laboratory columns in which there was a steep vertical gradient of cyanobacteria. Data were analyzed by repeated-measures ANOVA. Control (no Anabaena) and experimental columns showed no significant differences in Daphnia distributions. Daphnia in experimental columns frequently moved into areas with dense concentrations of Anabaena and stayed there for long periods of time. Our results show that this D. pulex clone does not exhibit a rapid (within 6 h) avoidance response to toxic Anabaena.     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

颈盲蝽取食对薇甘菊叶片营养物质和防御酶的影响

【目的】薇甘菊是世界最具危害性的入侵杂草之一,对我国生态环境和农业、林业生产造成了严重的危害。颈盲蝽是控制薇甘菊的一种潜在的重要天敌昆虫。本研究旨在探讨薇甘菊被颈盲蝽取食后,叶片防御相关酶系活性、营养物质和叶绿素含量的变化,阐明颈盲蝽取食对薇甘菊生理功能的影响,为利用颈盲蝽防控薇甘菊提供依据。【方法】从云南瑞丽野外采集薇甘菊的本地天敌昆虫颈盲蝽,测定了颈盲蝽取食前及取食12、24、48、96 h后,薇甘菊叶片中过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性,以及可溶性糖、可溶性蛋白及叶绿素含量,以取食前的植株作为对照。【结果】与对照相比,颈盲蝽取食12 h时,薇甘菊叶片中的POD、CAT活性升高,SOD活性降低;之后POD、SOD活性上升,CAT活性降低,取食48 h时,POD和SOD活性达到最高值,CAT活性达到最低值;取食96 h时,POD与SOD活性降低,但仍高于对照,CAT活性与取食48 h时相近。颈盲蝽取食后,薇甘菊叶片中的可溶性糖含量明显上升,取食96 h达到最高值;可溶性蛋白和叶绿素含量显著降低,在96 h达到最低值,分别低于对照39.30%和69.94%。【结论】颈盲蝽取食严重破坏了薇甘菊叶片的正常生理功能,并最终导致其叶片萎蔫和坏疽。     

Role of symbiotic and non-symbiotic bacteria in carbon dioxide production from hosts infected with Steinernema riobrave

Entomopathogenic nematodes of the family Steinernematidae and their mutualistic bacteria (Xenorhabdus spp.) are lethal endoparasites of insects. We hypothesized that growth of the nematode’s mutualistic bacteria in the insect host may contribute to the production of cues used by the infective juveniles (IJs) in responding to potential hosts for infection. Specifically, we tested if patterns of bacterial growth could explain differences in CO₂ production over the course of host infection. Growth of Xenorhabdus cabanillasii isolated from Steinernema riobrave exhibited the characteristic exponential and stationary growth phases. Other non-nematode symbiotic bacteria were also found in infected hosts and exhibited similar growth patterns to X. cabanillasii. Galleria mellonella larvae infected with S. riobrave produced two distinct peaks of CO₂ occurring at 25.6–36 h and 105–161 h post-infection, whereas larvae injected with X. cabanillasii alone showed only one peak of CO₂, occurring at 22.8–36.2 h post-injection. Tenebrio molitor larvae infected with S. riobrave or injected with bacteria alone exhibited only one peak of CO₂ production, which occurred later during S. riobrave infection (41.4–64.4 h post-infection compared to 20.4–35.9 h post-injection). These results indicate a relationship between bacterial growth and the first peak of CO₂ in both host species, but not for the second peak exhibited in G. mellonella.     

An efficient protocol for transient transformation of intact fruit and transgene expression inCitrus

In this study, conditions were optimized for transient gene expression in Rough Lemon (Citrus jambhiri Lush.), a major rootstock used in the citrus growing regions of Pakistan.Agrobacterium tumefaciens carrying the binary vector p35GUSINT, containingNPT II andGUS genes, was used in the study. The transformation method was based on injection ofAgrobacterium intoCitrus fruits followed by histochemical assay ofGUS activity in different tissues. Different tissues of mature fruits exhibited significantly different percentages of transientGUS expression: in rind (76%), spongy tissue (92%), juice vesicles (0%) and seeds (83%) (P<0.01)., The incubation period after injecting theAgrobacterium culture also showed a significant (P<0.01) effect on the transient expression ofGUS in these tissues. An incubation period of 48 h was found to be the best (72%) for transformation of whole fruit, followed by 72 h (67%) and 96 h (49%). TransientGUS expression also varied significantly (P<0.01) in juice vesicles and seeds as fruit matured. Juice vesicles from mature fruits showed no transientGUS expression, while those from immature fruits showed 50% expression. Furthermore, transformation of seeds had no effect on their germination capability. Germinating seeds from mature fruits injected withAgrobacterium culture showed tolerance to kanamycin (100 mg/L), which varied with the incubation period (55% at 48 h, 25% at 72 h and 23% at 96 h). This report offers an easy protocol for transient expression studies of transgenes and has the potential to be used for stable transformation ofCitrus.     

Bacillus fermentation of soybeans

Kinema fermentations of Indian and Canadian No. 1 soybeans by Bacillus sp. DK-W1 and by mixed cultures of Bacillus sp. DK-W1 and Enterococcus faecium DK-C1 were essentially identical. The viable cell count of Bacillus increased from an initial 10⁵ to 10¹⁰ c.f.u./g wet wt after 48 h incubation at 37°C. The pH of the fermentation dropped from an initial 6.9 to about 6.4 after 8 h and then rose to 8.6 after 32 h, with a coincident increase in proteolytic activity and ammonia concentration. The fermentations containing E. faecium and Bacillus exhibited a greater initial pH decline and a slightly retarded subseqent increase in pH compared with fermentations with Bacillus only. The presence of E. faecium had no detectable effects on growth of the Bacillus, proteolytic activity, ammonia production or the final pH of the fermentations.P.K. Sarkar was and P.E. Cook and J.D. Owens are with the Food Microbial Interactions Laboratory, Department of Food Science and Technology, University of Reading, Reading RG6 2AP, UK; P.K. Sarkar is now with the Microbiology Laboratory, Centre for Life Sciences, University of North Bengal, Siliguri 734430, India.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Floral evolution and phylogeny in the tribeThelymitreae (Orchidaceae: Neottioideae)

The Australian orchid tribeThelymitreae, composed ofCalochilus, Epiblema, andThelymitra, is unique in theOrchidaceae because of the presence of a mitra or staminodal complex. Evidence from floral structure suggests thatEpiblema andThelymitra are sister genera and thatCalochilus is derived from aThelymitra ancestor. A Gene Pool Vortex model and a hypothetical phylogeny illustrate that introgressive hybridization, allopatry, and long distance dispersal have played a major role in the evolution of the tribe. Pollination and hybridization in the tribe are discussed with major emphasis on floral mimicry.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Protein enrichment of grain sorghum by submerged culture of the amylolytic yeastsSchwanniomyces occidentalis andLipomyces kononenkoae

Cultivation of aSchwanniomyces occidentalis derepressed mutant in a 10% (w/v) gelatinized grain sorghum slurry increased the crude protein content of the biomass from an initial value of 12% to 41% (dry) within 20 h, with no detectable residual starch. Co-cultivation ofCandida utilis with theS. occidentalis mutant improved the final crude protein content to 47% within 18 h, whereas a co-culture ofC. utilis with aLipomyces kononenkoae mutant resulted in a cultivation time of 50 h with a significantly lower protein content and a low final -amylase activity. In a 15% (w/v) grain sorghum slurry aC. utilis/S. occidentalis co-culture increased the protein content to about 44% within 30 h. Yeast cultivation increased the lysine and threonine content of the final biomass considerably.     

An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi

Summary A 9.35 kbp element with long terminal direct repeats (LTRs) of 2.4 kbp has been characterized from the large genome of Lilium henryi. The organization of the element, named del, was examined in 20 fragments from a genomic DNA library constructed using phage EMBL3. Five fragments apparently contained full 9.35 kbp elements while in 11 del was recovered in part. Two clones carried tandem arrangements of del, with single LTRs and internal sequences alternating, and one insert contained a solo LTR. Evidence suggests that these arrangements, which can arise by unequal crossing over, have a genomic rather than a cloning origin. Finally, one cloned fragment had a complex del arrangement yet to be fully defined. Limited sequencing of five LTRs (from a full del, a tandem arrangement and a solo LTR) indicates that the consensus termini are 5TGT ... ACA 3, with del sequences flanked by a 5 bp tandem repeat. Thus del shares properties with retrotransposons such as the copia family of Drosophila and Ty of yeast. However with more than 13000 copies per genome it seems del has been amplified more than is usual for such elements.     

Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila

Summary We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3 and 5 to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a housekeeping gene-rosy (ry), or Xdh. In contrast, DNA sequences 5 to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.     

Ontogenetic changes in the ecological function of the association behavior between jack mackerel <Emphasis Type="Italic">Trachurus japonicus</Emphasis> and jellyfish

Commensal behavior of jack mackerel Trachurus japonicus (Temminck & Schlegel) with jellyfishes has been widely observed but its ecological function is still unclear. The goal of the present research is to examine the function of association behavior with jellyfish in the laboratory and in field observations with an emphasis on ontogenetic changes. In the laboratory, jack mackerel juveniles (mean standard length (SL) = 11, 19, 38, and 55 mm) were placed in 500-l polycarbonate tanks with two live moon jellyfish, Aurelia aurita (Linné), and one artificial jellyfish made of silicon. Association behavior with either live or artificial jellyfish was visually observed under the following conditions: control, presence of a predator model, before and after feeding live Artemia, 1 h and 3 h after feeding, and at night. Jack mackerel at 11 mm SL associated with both the moon jellyfish and artificial jellyfish, unrelated to the presence of a predator model or feeding. Juveniles at 19 mm associated with moon jellyfish only in the presence of a predator model. Larger juveniles associated with moon jellyfish at 1 h and 3 h after feeding. Thus the ecological function of association was proposed to develop first from school formation, next as a hiding place from predators, and then as a food source. Underwater observations of jack mackerel associating with giant jellyfish Nemopilema nomurai (Kishinouye) in two different areas in the Sea of Japan supported this hypothesis. High predation pressure from benthic piscivorous fishes in the southern area (Tsushima) may encourage association with jellyfish, whereas pressure from pelagic predators in the northern area (Maizuru) may encourage settlement to rocky reef habitats in temperate waters. Thus the jellyfish may also function as a vehicle for the northward migration of this species. Guest editors: K. A. Pitt & J. E. Purcell Jellyfish Blooms: Causes, Consequences, and Recent Advances     

Control of green mold and sour rot of stored lemon by biofumigation with Muscodor albus

Control of postharvest lemon diseases by biofumigation with the volatile-producing fungus Muscodor albus was investigated. In vitro exposure to M. albus volatile compounds for 3 days killed Penicillium digitatum and Geotrichum citri-aurantii, causes of green mold and sour rot of lemons, respectively. Lemons were wound-inoculated with P. digitatum and placed in closed 11-L plastic boxes with rye grain cultures of M. albus at ambient temperature. There was no contact between the fungus and the fruit. Biofumigation for 24–72 h controlled green mold significantly, even when treatment began 24 h after inoculation. Effectiveness was related to the amount of M. albus present. In tests conducted inside a 11.7-m³ degreening room with 5 ppm ethylene at 20 °C, green mold incidence on lemons was reduced on average from 89.8 to 26.2% after exposure to M. albus for 48 h. Ethylene accelerates color development in harvested citrus fruit. M. albus had no effect on color development. Biofumigation in small boxes immediately after inoculation controlled sour rot, but was ineffective if applied 24 h later. G. citri-aurantii may be less sensitive to the volatile compounds than P. digitatum or escapes exposure within the fruit rind. Biofumigation with M. albus could control decay effectively in storage rooms or shipping packages.     

Influence of diet and photoperiod on development and reproduction of European populations of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae)

The current study examines the effect of photoperiod (16:08 or 12:12 h L:D) and diet (eggs of Ephestia kuehniella Zeller (Lepidoptera: Phycitidae) or the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae)) on the development and reproduction of the multicoloured Asian lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). A long-term laboratory population of H. axyridis (since 1998) and a melanic and non-melanic population originating from field collected individuals of H. axyridis in Belgium were used in this study. Long day conditions (16 h photoperiod) shortened development of the field populations with 2–3 days when compared with short day conditions (12 h photoperiod). Oviposition in the field populations was delayed by 1–3 months when reared at a 12 h photoperiod. Dissections indicated that the females were in reproductive diapause. As compared with live pea aphids, a diet consisting of E. kuehniella eggs yielded heavier adult body weights (up to 12%) and increased the number of egg laying days (by 45–169%) for both field populations at a 16 h photoperiod and lengthened adult life span (by 45–92%) under both light regimens. The morph types differed in their response to the foods offered in terms of developmental rate, pre-oviposition period and number of oviposition days. The laboratory and field strains responded differentially to regimens of food and photoperiod. The study indicated a greater nutritional plasticity of the non-melanic morphs which may offer them a competitive advantage that may in part explain the predominance of non-melanic morphs in newly colonized areas.     

Volatiles released from cotton plants in response to<Emphasis Type="Italic"> Helicoverpa zea</Emphasis> feeding damage on cotton flower buds

Feeding of Helicoverpa zea larvae on cotton (Gossypium hirsutum L.) flower buds (squares) for 24 or 48 h induced the release of a number of terpenes [(E)--ocimene, linalool, (E)--farnesene, (E,E)--farnesene, (E)-4,8-dimethyl-1,3,7-nonatriene, (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene], isomeric hexenyl butyrates, 2-methylbutyrates, indole and (Z)-3-hexenyl acetate. These compounds are not released in significant amounts from undamaged squares and freshly damaged squares. The release of inducible compounds was not limited to the damaged squares themselves. The compounds were also released systemically from the upper undamaged leaves of the same plant after 72 h. However, the composition of the blend of systemically released volatiles differed from the blend released by damaged squares. The compounds that were systemically released from undamaged leaves in response to feeding on the squares were (Z)-3-hexenyl acetate, (E)--ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, (E)--farnesene, (E,E)--farnesene, and indole. This study shows that insect damage inflicted to the reproductive parts of a plant causes a systemic emission of volatiles from its vegetative parts.     

Pathogenicity of Sporotrichum pruinosum and Cladosporium oxysporum,isolated from the bronchial secretions of a patient,for laboratory mice

In this study we have demonstrated the occurrence of Sporotrichum pruinosum and Cladosporium oxysporum in the bronchial secretions of a patient with a presumptive diagnosis of tuberculosis. This observation coupled with the ability of both fungi to cause infection and elicit tissue responses in experimentally infected mice supported a probable etiologic relationship with the patient which could not be confirmed in the absence of histologic evidence. In vitro some antimycotics were tested against S. pruinosum and C. oxysporum by the agar dilution method. Oxiconazole with a minimum inhibitory concentration of 0.1 g/ml^–1 after 72 h and amorolfine at a concentration of 0.001 g/ml^–1 after 72 h were the most active ones against S. pruinosum and C. oxysporum respectively. It is suggested that the isolation of S. pruinosum and C. oxysporum from patients with bronchopulmonary disorders should be viewed with caution. Clinical and laboratory evaluation of such patients should be done critically before arriving at a firm diagnosis.     

Phylogenetic relationships and generic affinity of Uncinula septata inferred from nuclear rDNA sequences

Based on 18S, 5.8S, and 28S rDNA sequences, the phylogenetic position of Uncinula septata within the Erysiphales has been inferred. Although appendages of the ascomata are uncinula like, i.e., unbranched with curved-coiled apices, U. septata is situated at the very base of the large Erysiphales cluster, far away from the pseudoidium clade (Erysiphe emend., including Microsphaera and Uncinula). Morphologically, U. septata differs from the species of Erysiphe sect. Uncinula (Uncinula) in having terminal, pluriseptate ascoma appendages, curved ascospores, and the absense of an anamorph. This species is a basal, tree-inhabiting powdery mildew with some additional ancestral characteristics, viz., uncinula-like appendages and 8-spored asci. The new genus Parauncinula with U. septata as the type species is proposed. Uncinula curvispora (U. septata var. curvispora) is tentatively maintained as a separate species, which is also assigned to Parauncinula.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.