Cryptic antifungal compounds active by synergism with polyene antibiotics.

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity.

High duty cycle pulses suppress orientation flights of crambid moths.

Bat-and-moth is a good model system for understanding predator-prey interactions resulting from interspecific coevolution. Night-flying insects have been under predation pressure from echolocating bats for 65Myr, pressuring vulnerable moths to evolve ultrasound detection and evasive maneuvers as counter tactics. Past studies of defensive behaviors against attacking bats have been biased toward noctuoid moth responses to short duration pulses of low-duty-cycle (LDC) bat calls. Depending on the region, however, moths have been exposed to predation pressure from high-duty-cycle (HDC) bats as well. Here, we reveal that long duration pulse of the sympatric HDC bat (e.g., greater horseshoe bat) is easily detected by the auditory nerve of Japanese crambid moths (yellow peach moth and Asian corn borer) and suppress both mate-finding flights of virgin males and host-finding flights of mated females. The hearing sensitivities for the duration of pulse stimuli significantly dropped non-linearly in both the two moth species as the pulse duration shortened. These hearing properties support the energy integrator model; however, the threshold reduction per doubling the duration has slightly larger than those of other moth species hitherto reported. And also, Asian corn borer showed a lower auditory sensitivity and a lower flight suppression to short duration pulse than yellow peach moth did. Therefore, flight disruption of moth might be more frequently achieved by the pulse structure of HDC calls. The combination of long pulses and inter-pulse intervals, which moths can readily continue detecting, will be useful for repelling moth pests.

Sticky Traps Baited with Synthetic Aggregation Pheromone Predict Fruit Orchard Infestations of Plautia stali (Hemiptera: Pentatomidae).

The brown-winged green bug, Plautia stali Scott, mainly reproduces on Japanese cedar or cypress cones in Japanese plantation forests during summer and autumn. It often depletes its food sources in forest habitats and moves to cultivated crops in large numbers. To establish an easy method for assessing the risk of fruit orchard infestation by P. stali, we conducted a 3-yr field survey that monitored the attraction of bugs to the synthetic P. stali aggregation pheromone using a sticky trap. We used a morphological indicator, variable body size depending on food intake, to estimate the nutritional status in nymphs, which showed that nymphs attracted to the synthetic pheromone were starving. Comparisons between increasing changes in the number of stylet sheaths left on the cones by P. stali and the number of trapped nymphs show that monitoring nymphs with the pheromone-baited sticky trap is useful for inferring conditions regarding food resources in forest habitats. The trend toward trapping second instars can provide a timely overview of resource competition for cones. Trapping middle-to-late (third-fifth) instars is a warning that the cones are finally depleted and that there is a high probability that adults will leave the forests and invade the orchards. In addition, trends in trapping adults suggest that there is a potential risk of orchard infestation by the pest and predict the intensity and period of the invasion. The pheromone-baited sticky trap is an easy but useful survey tool for predicting P. stali orchard infestations.

Double meaning of courtship song in a moth.

Males use courtship signals to inform a conspecific female of their presence and/or quality, or, alternatively, to 'cheat' females by imitating the cues of a prey or predator. These signals have the single function of advertising for mating. Here, we show the dual functions of the courtship song in the yellow peach moth, Conogethes punctiferalis, whose males generate a series of short pulses and a subsequent long pulse in a song bout. Repulsive short pulses mimic the echolocation calls of sympatric horseshoe bats and disrupt the approach of male rivals to a female. The attractive long pulse does not mimic bat calls and specifically induces mate acceptance in the female, who raises her wings to facilitate copulation. These results demonstrate that moths can evolve both attractive acoustic signals and repulsive ones from cues that were originally used to identify predators and non-predators, because the bat-like sounds disrupt rivals, and also support a hypothesis of signal evolution via receiver bias in moth acoustic communication that was driven by the initial evolution of hearing to perceive echolocating bat predators.

Cyclic lipodepsipeptides verlamelin A and B, isolated from entomopathogenic fungus Lecanicillium sp.

Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val). This is the first study that precisely analyzed the structure of verlamelin.

Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species.

Lack of genetic tools in entomopathogenic fungi, especially those for targeted homologous recombination, hindered the advance in this field. To facilitate the genetic study, we constructed a transformation system in entomopathogenic fungus Lecanicillium sp. strain HF627 using the uridine auxotrophic pyrG mutant strain as host and endogenous pyrG as marker. pUC19 harboring endogenous pyrG successfully restored the uridine auxotrophy of the host strain, and the integration of the vector DNA was confirmed by Southern hybridization. An autonomously replicating vector harboring an AMA1 sequence was constructed and applied to the constructed transformation system, which improved the transformation efficiency 16.7-fold. Southern hybridization revealed replication of the AMA1-harboring vector with an average copy number of 2.4. A ku80 knock-out strain was created to improve the efficiency of gene targeting. Deletion of the pyrG locus, which is homologous to the marker gene, from the ku80 knock-out strain achieved a targeting efficiency of 62.5 % against both trp1 and his3; the levels of these genes were 3.2- and 5-fold higher, respectively, than the ku80-intact strain. With the pyrG-deleted and ku80-inactivated strain constructed in this study, transformation and targeted homologous recombination were highly enhanced, by which genetic analysis in Lecanicillium spp. will be performed quickly and efficiently.

Production of antioomycete compounds active against the phytopathogens Phytophthora sojae and Aphanomyces cochlioides by clavicipitoid entomopathogenic fungi.

A total of 412 strains belonging to 14 genera of clavicipitoid entomopathogenic fungi (EPF) were screened for activities against two economically important plant pathogenic oomycetes, Phytophthora sojae and Aphanomyces cochlioides. To identify the antioomycete compounds produced by EPF, the extracts of 13 highly active EPF strains were characterized in detail by high performance liquid chromatography with diode array detection and high-resolution mass spectrometric detection and antioomycete assay. The antioomycete activity of several Metarhizium extracts was associated with previously isolated aurovertins, fungerin, N-(methyl-3-oxodec-6-enoyl)-2-pyrroline, and N-(methyl-3-oxodecanoyl)-2-pyrroline. The depsipeptide beauvericin was confirmed to be one of the active principles of three strains of Isaria tenuipes, which strongly inhibited mycelial growth of both P. sojae and A. cochlioides. Two known bioactive metabolites, paecilosetin and aranorosinol A, together with a novel and potent antioomycete compound, farinomalein, were isolated from the extracts of Isaria farinosa and all compounds were confirmed to have antioomycete activity. Identification of 8 antioomycete compounds from 13 clavicipitioid EPF demonstrated a new potential use of EPF as a source of compounds for the control of soil-borne plant pathogenic oomycetes.

Observation of the peach fruit moth, Carposina sasakii, larvae in young apple fruit by dedicated micro-magnetic resonance imaging.

Infestation of young apple fruits by the larvae of the peach fruit moth, Carposina sasakii Matsumura (Lepidoptera: Carposinidae), was studied by a small dedicated micro-magnetic resonance imaging (MRI) apparatus using the three-dimensional (3D) gradient-echo method and the two-dimensional (2D) and 3D spin-echo methods. Changes from a young larva at 1.8 mm in length to a mature one ready to leave the fruit were observed in relation to the progression of infestation of the fruit tissues. The trace of larva intrusion was demonstrated by a series of sliced images in the 3D image data of an infested fruit, where it entered from outside the calyx, and migrated to near the vasculature around the carpel through the core. The small, dedicated MRI device was proven useful for ecological studies of the growth and movement of insect larvae in their food fruits. It can also be applied to detect the infestation of small fruits by insect larvae.

Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae.

A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by 1H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis.

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 degrees C to 20 or 25 degrees C and chilled them again before incubation at 20 degrees C. With increasing warming duration at 20 degrees C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 degrees C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 degrees C after the first chilling were subsequently incubated at 15, 20, or 25 degrees C, then chilled at 5 degrees C before incubation at 20 degrees C. Diapause termination increased significantly after the larvae were treated at 15 or 20 degrees C but decreased significantly after they were treated at 25 degrees C. The intensification of prolonged diapause at 25 degrees C was reversed when the larvae were transferred to 20 degrees C. Diapause intensity in C. sikkimensis therefore decreases at 20 degrees C, increases at 25 degrees C, and can be reversed by alternately exposing diapause larvae to 20 and 25 degrees C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.

Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi.

Based on comparative amino-acid sequence alignment of geranylgeranyl diphosphate (GGPP) synthase from filamentous fungi, degenerated oligonucleotide primers were designed for searching GGPP synthase gene(s) in entomopathogenic fungi. Polymerase chain reaction with the designed primers amplified GGPP synthase homologues from five representative entomopathogenic fungi: Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Paecilomyces farinosus, and Nomuraea rileyi. Sequence comparison of the amplified of GGPP synthase homologue fragments revealed that M. anisopliae and B. bassiana have at least two different types of the GGPP synthase gene homologues. The first type (designated as ggs1), which is highly conserved among the five strains, has a unique Ser-rich region, SSXSSVSGSSS (X refers to L, A, V, or S), and is constitutively expressed throughout growth. In contrast, the second type of GGPP synthase gene homologue (ggs2) was discovered only in some strains, and genes of this type possessed high similarity to each other but showed relatively weak similarity to the ggs1 genes, with no detectable transcription under the cultivation conditions applied in this experiment. The ggs1 cloned from M. anisopliae, which encoded a putative protein of 359 amino acid residues, was heterologously expressed in E. coli. The recombinant protein showed activity to synthesize GGPP from farnesyl diphosphate and isopentenyl diphosphate. These results strongly suggested that the ggs1 gene encodes a GGPP synthase involved in primary metabolism.

Farinomalein, a maleimide-bearing compound from the entomopathogenic fungus Paecilomyces farinosus.

A new maleimide-bearing compound, farinomalein (1), was isolated from the entomopathogenic fungus Paecilomyces farinosus HF599. The structure was determined on the basis of spectroscopic analyses and chemical conversion. Compound 1 showed potent activity (5 mug/disk) against the plant pathogenic Phytophthora sojae P6497.

Identification of novel derivative of helvolic acid from Metarhizium anisopliae grown in medium with insect component.

Entomopathogenic fungi are unique owing to their versatile ability to produce many bioactive compounds and from the dependence of their morphological differentiation on the presence of insect-derived materials. An entomopathogenic fungus, Metarhizium anisopliae HF293, was found to show insect-material-dependent production of antibacterial compounds, which were purified to homogeneity from 10-d culture broth when the production reached maximum. Two compounds were isolated: the major compound was determined to be helvolic acid and the minor one was a novel derivative of helvolic acid (1,2-dihydrohelvolic acid). Discovery of a novel bioactive compound indicated that insect-derived material would be a useful factor for enhancing the diversity of compounds produced by entomopathogenic fungi.

Aurovertins F-H from the entomopathogenic fungus Metarhizium anisopliae.

Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to isolation of aurovertin D ( 1) and three new aurovertin-type metabolites, aurovertin F ( 2), aurovertin G ( 3), and aurovertin H ( 4). Their structures were determined on the basis of spectroscopic analyses and chemical conversions.

Active lactonizing lipase (LipL) efficiently overproduced by Pseudomonas strains as heterologous expression hosts.

Pseudomonas sp. strain 109 secretes lactonizing lipase (LipL), which catalyzes efficient intramolecular transesterification of omega-hydroxyfatty acid esters to form macrocyclic lactones. Because Escherichia coli was found to be unsuitable as an expression host due to the predominant formation of inactive LipL-inclusion bodies and a lack of proper secretion machinery which is also required for the formation of active LipL, Pseudomonas strains were surveyed as expression hosts. Pseudomonas sp. strain 109, an original LipL producer, showed a 7.1-fold higher level of active LipL when the lipL gene under the control of tac-lacUV5 tandem promoter was introduced together with a limL gene encoding a LipL-specific chaperon. Pseudomonas aeruginosa ADD 1976 containing a T7 RNA polymerase gene in the chromosome and plasmid-borne lipL-limL genes under the control of T7 promoter showed a 13-fold higher level of active LipL. Several combinations in the number of lipL and/or limL genes on the plasmid were investigated, and (lipL)3-limL was found to be most efficient, yielding a 67-fold greater production of active LipL than that obtained by the wild-type Pseudomonas sp. strain 109.

Cultivation of entomopathogenic fungi for the search of antibacterial compounds.

Entomopathogenic fungi are a rich source of natural bioactive compounds. To establish cultivation conditions which facilitate the production of bioactive compounds and to select good genera among entomopathogenic fungi as the producer, 47 typical entomopathogenic fungi were tested for their ability to produce antibiotic activity. Thirty-eight strains (81%) and 30 strains (64%) of these fungi produced either anti-Bacillus compounds or anti-Staphylococcus compounds, respectively, indicating that the majority of the entomopathogenic fungi tested possessed the ability to produce antibacterial compounds. Using 9 representative strains (Aschersonia sp. HF724, Beauveria bassiana HF338, Cordyceps ramosopulvinata HF746, Metarhizium anisopliae HF293, Metarhizium flavoviride HF698, Nomuraea rileyi HF588, Paecilomyces fumosoroseus HF254, Paecilomyces tenuipes HF419, and Verticillium lecanii HF238), the cultivation conditions in liquid medium were surveyed with respect to the cultivation procedure and medium composition, particularly in terms of the presence or absence of insect-derived materials. At 26 degrees C, M. anisopliae HF293, N. rileyi HF588, and V. lecanii HF238 strains produced clear antibiotic activity against Bacillus and Saccharomyces, but only in the presence of insect-derived materials, suggesting that the production of antibacterial/antifungal compounds by entomopathogenic fungi is triggered by the presence of insect-derived materials.