Alkamides from Echinacea disrupt the fungal cell wall-membrane complex.

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.

If a bird flies in the forest, does an insect hear it?

Birds are major predators of many eared insects including moths, butterflies, crickets and cicadas. We provide evidence supporting the hypothesis that insect ears can function as 'bird detectors'. First, we show that birds produce flight sounds while foraging. Eastern phoebes (Sayornis phoebe) and chickadees (Poecile atricapillus) generate broadband sounds composed of distinct repetitive elements (approx. 18 and 20 Hz, respectively) that correspond to cyclic wing beating. We estimate that insects can detect an approaching bird from distances of at least 2.5 m, based on insect hearing thresholds and sound level measurements of bird flight. Second, we show that insects with both high and low frequency hearing can hear bird flight sounds. Auditory nerve cells of noctuid moths (Trichoplusia ni) and nymphalid butterflies (Morpho peleides) responded in a bursting pattern to playbacks of an attacking bird. This is the first study to demonstrate that foraging birds generate flight sound cues that are detectable by eared insects. Whether insects exploit these sound cues, and alternatively, if birds have evolved sound-reducing foraging tactics to render them acoustically 'cryptic' to their prey, are tantalizing questions worthy of further investigation.

Vibration detection and discrimination in the masked birch caterpillar (Drepana arcuata).

Leaf-borne vibrations are potentially important to caterpillars for communication and risk assessment. Yet, little is known about the vibratory environment of caterpillars, or how they detect and discriminate between vibrations from relevant and non-relevant sources. We measured the vibratory 'landscape' of the territorial masked birch caterpillar Drepana arcuata (Drepanidae), and assessed its ability to detect and respond to vibrations generated by conspecific and predatory intruders, wind and rain. Residents of leaf shelters were shown to respond to low amplitude vibrations generated by a crawling conspecific intruder, since removal of the vibrations through leaf incision prevented the resident's response. Residents did not respond to large amplitude, low frequency disturbances caused by wind and rain alone, but did respond to approaching conspecifics under windy conditions, indicating an ability to discriminate between these sources. Residents also responded differently in the presence of vibrations generated by approaching predators (Podisus) and conspecifics. An analysis of vibration characteristics suggests that despite significant overlap between vibrations from different sources, there are differences in frequency and amplitude characteristics that caterpillars may use to discriminate between sources. Caterpillars live in a vibration-rich environment that we argue forms a prominent part of the sensory world of substrate bound holometabolous larvae.

Grunting for worms: seismic vibrations cause Diplocardia earthworms to emerge from the soil.

Harvesting earthworms by a practice called 'worm grunting' is a widespread and profitable business in the southeastern USA. Although a variety of techniques are used, most involve rhythmically scraping a wooden stake driven into the ground, with a flat metal object. A common assumption is that vibrations cause the worms to surface, but this phenomenon has not been studied experimentally. We demonstrate that Diplocardia earthworms emerge from the soil within minutes following the onset of grunting. Broadband low frequency (below 500 Hz) pulsed vibrations were present in the soil throughout the area where worms were harvested, and the number of worms emerging decreased as the seismic signal decayed over distance. The findings are discussed in relation to two hypotheses: that worms are escaping vibrations caused by digging foragers and that worms are surfacing in response to vibrations caused by falling rain.

Caterpillar talk: acoustically mediated territoriality in larval Lepidoptera.

We provide evidence for conspecific acoustic communication in caterpillars. Larvae of the common hook-tip moth, Drepana arcuata (Drepanoidea), defend silk nest sites from conspecifics by using ritualized acoustic displays. Sounds are produced by drumming the mandibles and scraping the mandibles and specialized anal "oars" against the leaf surface. Staged interactions between a resident and intruder resulted in escalated acoustic "duels" that were typically resolved within minutes, but sometimes extended for several hours. Resident caterpillars generally won territorial disputes, regardless of whether they had built the nest, but relatively large intruders occasionally displaced residents from their nests. All evidence is consistent with acoustic signaling serving a territorial function. As with many vertebrates, ritualized signaling appears to allow contestants to resolve contests without physical harm. Comparative evidence indicates that larval acoustic signaling may be widespread throughout the Lepidoptera, meriting consideration as a principal mode of communication for this important group of insects.

Sound production and hearing in the blue cracker butterfly Hamadryas feronia (Lepidoptera, nymphalidae) from Venezuela.

Certain species of Hamadryas butterflies are known to use sounds during interactions with conspecifics. We have observed the behaviour associated with sound production and report on the acoustic characteristics of these sounds and on the anatomy and physiology of the hearing organ in one species, Hamadryas feronia, from Venezuela. Our observations confirm previous reports that males of this species will take flight from their tree perch when they detect a passing conspecific (male or female) and, during the chase, produce clicking sounds. Our analyses of both hand-held males and those flying in the field show that the sounds are short (approximately 0.5 s) trains of intense (approximately 80-100 dB SPL at 10 cm) and brief (2-3 ms) double-component clicks, exhibiting a broad frequency spectrum with a peak energy around 13-15 kHz. Our preliminary results on the mechanism of sound production showed that males can produce clicks using only one wing, thus contradicting a previous hypothesis that it is a percussive mechanism. The organ of hearing is believed to be Vogel's organ, which is located at the base of the forewing subcostal and cubital veins. Vogel's organ consists of a thinned region of exoskeleton (the tympanum) bordered by a rigid chitinous ring; associated with its inner surface are three chordotonal sensory organs and enlarged tracheae. The largest chordotonal organ attaches to a sclerite positioned near the center of the eardrum and possesses more than 110 scolopidial units. The two smaller organs attach to the perimeter of the membrane. Extracellular recordings from the nerve branch innervating the largest chordotonal organ confirm auditory sensitivity with a threshold of 68 dB SPL at the best frequency of 1.75 kHz. Hence, the clicks with peak energy around 14 kHz are acoustically mismatched to the best frequencies of the ear. However, the clicks are broad-banded and even at 1-2 kHz, far from the peak frequency, the energy is sufficient such that the butterflies can easily hear each other at the close distances at which they interact (less than 30 cm). In H. feronia, Vogel's organ meets the anatomical and functional criteria for being recognized as a typical insect tympanal ear.

Janus Green B as a rapid, vital stain for peripheral nerves and chordotonal organs in insects.

Effective staining of peripheral nerves in live insects is achieved with the vital stain Janus Green B. A working solution of 0.02% Janus Green B in saline is briefly applied to the exposed peripheral nervous system. The stain is then decanted and the dissection flooded with fresh saline, resulting in whole nerves being stained dark blue in contrast to surrounding tissues. This simple and reliable technique is useful in describing the distribution of nerves to their peripheral innervation sites, and in locating small nerve branches for extracellular physiological recordings. The stain is also shown to be useful as a means of enhancing the contrast between scolopale caps and surrounding tissues in chordotonal organs, staining chordotonal organ attachment strands, and the crista acustica (tympanal organ) of crickets and katydids. The advantages of Janus Green B over traditional peripheral nerve strains, in addition to its shortcomings, are discussed.

The evolutionary biology of insect hearing.

Few areas of science have experienced such a blending of laboratory and field perspectives as the study of hearing. The disciplines of sensory ecology and neuroethology interpret the morphology and physiology of ears in the adaptive context in which this sense organ functions. Insects, with their enormous diversity, are valuable candidates for the study of how tympanal ears have evolved and how they operate today in different habitats.

A multiterminal stretch receptor, chordotonal organ, and hair plate at the wing-hinge of Manduca sexta: unravelling the mystery of the noctuid moth ear B cell.

The present study aims to shed light on the evolutionary origin of the B cell, a sensory element of unknown function in the noctuid moth ear. Peripheral projections of the metathoracic nerve IIIN1b1, homologue of the noctuid moth tympanic nerve, are described in the atympanate moth Manduca sexta on the basis of dissections with the aid of Janus Green B, and intracellular tracer dyes Lucifer yellow and cobalt lysine. A large multiterminal (Type II) neurone, attaching to membranous cuticle ventral to the hind wing axillary cord, was discovered. This cell appears to be homologous to the B cell in the noctuid moth ear. Recordings from the IIIN1b1 nerve in M. sexta reveal a continuous train of large, uniform spikes, presumed to originate from the multiterminal cell. This unit increases its rate of firing in response to hind wing elevation, suggesting that it functions as a stretch receptor monitoring wing movements during flight. Also identified in the tympanic nerve homologue, and closely associated with the multiterminal cell, were a chordotonal organ and hair plate. The chordotonal organ consists of a proximal scolopidial region and a distal strand that attaches to the sclerotized epimeron slightly medial to the multiterminal cell. This simple chordotonal organ, having three uniterminal (Type I) sensory cells, is homologous to the auditory cells of the noctuid moth ear. The significance of these receptors as proprioceptors in M. sexta, and as evolutionary precursors to the noctuid moth ear, is discussed.

The metathoracic wing-hinge chordotonal organ of an atympanate moth, Actias luna (Lepidoptera, Saturniidae): a light- and electron-microscopic study.

The structure of a simple chordotonal organ, the presumed homologue of the noctuoid moth tympanal organ, is described in the atympanate moth, Actias luna. The organ consists of a proximal scolopidial region and a distal strand, which attaches peripherally to the membraneous cuticle ventral to the hindwing alula. The strand is composed of elongate, microtubule-rich cells encased in an extracellular connective tissue sheath. The scolopidial region houses three mononematic, monodynal scolopidia, each comprised of a sensory cell, scolopale cell, and attachment cell. The dendritic apex is octagonally shaped in transverse section, its inner membrane lined by a laminated structure reminiscent of the noctuoid tympanal organ 'collar'. A 9 + 0-type cilium emerges from the dendritic apex, passes through both the scolopale lumen and cap, and terminates in an extracellular space distal to the latter. Proximal extensions of the attachment cell and distal prolongations of the scolopale cell surrounding the cap are joined by an elaborate desmosome, with which is associated an extensive electron-dense fibrillar plaque. Within the scolopale cell, this plaque constitutes the scolopale 'rod' material. The data are discussed in terms of both the organ's potential function, and its significance as the evolutionary prototype of the noctuoid moth ear.

The mechanoreceptive origin of insect tympanal organs: a comparative study of similar nerves in tympanate and atympanate moths.

A chordotonal organ occurring in the posterior metathorax of an atympanate moth, Actias luna (L.) (Bombycoidea: Saturniidae), appears to be homologous to the tympanal organ of the noctuoid moth. The peripheral anatomy of the metathoracic nerve branch, IIIN1b1 was examined in Actias luna with cobalt-lysine and Janus Green B, and compared to its counterpart, IIIN1b (the tympanal branch), in Feltia heralis (Grt.) (Noctuoidea: Noctuidae). The peripheral projections of IIIN1b1 were found to be similar in both species, dividing into three branches, the second (IIIN1b1b) ending as a chordotonal organ. The atympanate organ possesses three sensory cell bodies and three scolopales, and is anchored peripherally via an attachment strand to the undifferentiated membranous region underlying the hindwing alula, which corresponds to the tympanal region of the noctuoid metathorax. Extracellular recordings of the IIIN1b1 nerve in Actias luna revealed a large spontaneously active unit which fired in a regular pattern (corresponding to the noctuoid B cell) and smaller units (corresponding to the noctuoid acoustic A cells) which responded phasically to low frequency sounds (2 kHz) played at high intensities (83-96 dB, SPL) and also responded phasically to raising and lowering movements of the hindwing. We suggest that the chordotonal organ in Actias luna represents the evolutionary prototype to the noctuoid tympanal organ, and that it acts as a proprioceptor monitoring hindwing movements. This system, in its simplicity (consisting of only a few neurons) could be a useful model for examining the changes to the nervous system (both central and peripheral) that accompanied the evolutionary development of insect tympanal organs.