Variation in Morphology and Airborne Dispersal of the Urticating Apparatus of Ochrogaster lunifer (Lepidoptera: Notodontidae), an Australian Processionary Caterpillar, and Implications for Livestock and Humans.

True setae borne on the abdominal tergites of Ochrogaster lunifer Herrich-Schӓffer caterpillars are the agents of an irritating contact dermatitis, osteomyelitis, ophthalmia, and severe allergic reactions in humans, and are the cause of Equine Amnionitis and Fetal Loss in Australia. The setae are detached and readily dislodge from the integument whereby they disperse throughout the environment. To better understand the true setae of O. lunifer as agents of medical and veterinary concern, we studied their characteristics and distance dispersed. Whereas members of the European Thaumetopoeinae have been widely studied, their southern-hemisphere counterparts such as O. lunifer are not well known despite their harmfulness and known medical and veterinary importance. The caterpillar's investment in true setae increased with age and size, and two distinct size classes co-occurred in setae fields. A previously undescribed morphological type of true seta was found on the first abdominal segment. All true setae were calculated to travel long distances in the air even under light breeze conditions. Our results show there is a high risk of exposure to airborne urticating setae within 100 m of elevated caterpillar activity, and a likely risk of exposure for some kilometers in the direction of the prevailing breeze. This information should be used to inform management strategies in areas where urticating processionary caterpillars are active, and especially during periods of an outbreak.

Associations Between the Small Hive Beetle and the Yeast Kodamaea ohmeri Throughout the Host Life Cycle.

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a pest of colonies of social bees, including the honeybee Apis mellifera L. (Hymenoptera: Apidae). We investigated A. tumida oviposition behavior and development and found that it laid eggs in clutches that ranged in size (3-75 eggs per clutch) and that when fed on hive products in laboratory culture (27°C; RH 65%; 12:12 (L:D) h) it completed three larval instars before pupation. The yeast Kodamaea ohmeri (Etchells & Bell) Y. Yamada, T. Suzuki, M. Matsuda & K. Mikata (Ascomycota: Saccharomycotina) is associated with A. tumida, but the exact nature of this relationship is unknown. We examined the association in host eggs, larvae, pupae, and adults to establish its extent and potential specificity and determined the likely mechanism of vertical transmission. K. ohmeri was detected in egg mucilage and on host cuticle and from internal preparations of A. tumida at every stage of development. Based on colony forming unit (CFU) counts, the K. ohmeri densities varied significantly between developmental stages; the highest internal density was recorded in third instar larvae. Presence of K. ohmeri within adult A. tumida was not affected by contamination of the cuticle by the yeast during the larval and pupal stages nor by the mated status of the adult. This deepened understanding of A. tumida ovipositional behavior and larval development along with a better understanding of the relationship between K. ohmeri and its host is important for the development of management strategies for this important pest.

Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane Incorporating a Low-Adhesion, Nonwetting, Superhydrophobic, Bactericidal, and Biocompatible Surface.

Nature has produced many intriguing and spectacular surfaces at the micro- and nanoscales. These small surface decorations act for a singular or, in most cases, a range of functions. The minute landscape found on the lotus leaf is one such example, displaying antiwetting behavior and low adhesion with foreign particulate matter. Indeed the lotus leaf has often been considered the "benchmark" for such properties. One could expect that there are animal counterparts of this self-drying and self-cleaning surface system. In this study, we show that the planthopper insect wing (Desudaba danae) exhibits a remarkable architectural similarity to the lotus leaf surface. Not only does the wing demonstrate a topographical likeness, but some surface properties are also expressed, such as nonwetting behavior and low adhering forces with contaminants. In addition, the insect-wing cuticle exhibits an antibacterial property in which Gram-negative bacteria (Porphyromonas gingivalis) are killed over many consecutive waves of attacks over 7 days. In contrast, eukaryote cell associations, upon contact with the insect membrane, lead to a formation of integrated cell sheets (e.g., among human stem cells (SHED-MSC) and human dermal fibroblasts (HDF)). The multifunctional features of the insect membrane provide a potential natural template for man-made applications in which specific control of liquid, solid, and biological contacts is desired and required. Moreover, the planthopper wing cuticle provides a "new" natural surface with which numerous interfacial properties can be explored for a range of comparative studies with both natural and man-made materials.

Diversity of Cuticular Micro- and Nanostructures on Insects: Properties, Functions, and Potential Applications.

Insects exhibit a fascinating and diverse range of micro- and nanoarchitectures on their cuticle. Beyond the spectacular beauty of such minute structures lie surfaces evolutionarily modified to act as multifunctional interfaces that must contend with a hostile, challenging environment, driving adaption so that these can then become favorable. Numerous cuticular structures have been discovered this century; and of equal importance are the properties, functions, and potential applications that have been a key focus in many recent studies. The vast range of insect structuring, from the most simplistic topographies to the most elegant and geometrically complex forms, affords us with an exhaustive library of natural templates and free technologies to borrow, replicate, and employ for a range of applications. Of particular importance are structures that imbue cuticle with antiwetting properties, self-cleaning abilities, antireflection, enhanced color, adhesion, and antimicrobial and specific cell-attachment properties.

Contaminant adhesion (aerial/ground biofouling) on the skin of a gecko.

In this study, we have investigated the micro- and nano-structuring and contaminant adhesional forces of the outer skin layer of the ground dwelling gecko--Lucasium steindachneri. The lizard's skin displayed a high density of hairs with lengths up to 4 µm which were spherically capped with a radius of curvature typically less than 30 nm. The adhesion of artificial hydrophilic (silica) and hydrophobic (C18) spherical particles and natural pollen grains were measured by atomic force microscopy and demonstrated extremely low values comparable to those recorded on superhydrophobic insects. The lizard scales which exhibited a three-tier hierarchical architecture demonstrated higher adhesion than the trough regions between scales. The two-tier roughness of the troughs comprising folding of the skin (wrinkling) limits the number of contacting hairs with particles of the dimensions used in our study. The gecko skin architecture on both the dorsal and trough regions demonstrates an optimized topography for minimizing solid-solid and solid-liquid particle contact area, as well as facilitating a variety of particulate removal mechanisms including water-assisted processes. These contrasting skin topographies may also be optimized for other functions such as increased structural integrity, levels of wear protection and flexibility of skin for movement and growth. While single hair adhesion is low, contributions of many thousands of individual hairs (especially on the abdominal scale surface and if deformation occurs) may potentially aid in providing additional adhesional capabilities (sticking ability) for some gecko species when interacting with environmental substrates such as rocks, foliage and even man-made structuring.

Firing the sting: chemically induced discharge of cnidae reveals novel proteins and peptides from box jellyfish (Chironex fleckeri) venom.

Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.

Removal mechanisms of dew via self-propulsion off the gecko skin.

Condensation resulting in the formation of water films or droplets is an unavoidable process on the cuticle or skin of many organisms. This process generally occurs under humid conditions when the temperature drops below the dew point. In this study, we have investigated dew conditions on the skin of the gecko Lucasium steindachneri. When condensation occurs, we show that small dew drops, as opposed to a thin film, form on the lizard's scales. As the droplets grow in size and merge, they can undergo self-propulsion off the skin and in the process can be carried away a sufficient distance to freely engage with external forces. We show that factors such as gravity, wind and fog provide mechanisms to remove these small droplets off the gecko skin surface. The formation of small droplets and subsequent removal from the skin may aid in reducing microbial contact (e.g. bacteria, fungi) and limit conducive growth conditions under humid environments. As well as providing an inhospitable microclimate for microorganisms, the formation and removal of small droplets may also potentially aid in other areas such as reduction and cleaning of some surface contaminants consisting of single or multiple aggregates of particles.

A gecko skin micro/nano structure - A low adhesion, superhydrophobic, anti-wetting, self-cleaning, biocompatible, antibacterial surface.

Geckos, and specifically their feet, have attracted significant attention in recent times with the focus centred around their remarkable adhesional properties. Little attention however has been dedicated to the other remaining regions of the lizard body. In this paper we present preliminary investigations into a number of notable interfacial properties of the gecko skin focusing on solid and aqueous interactions. We show that the skin of the box-patterned gecko (Lucasium sp.) consists of dome shaped scales arranged in a hexagonal patterning. The scales comprise of spinules (hairs), from several hundred nanometres to several microns in length, with a sub-micron spacing and a small radius of curvature typically from 10 to 20 nm. This micro and nano structure of the skin exhibited ultralow adhesion with contaminating particles. The topography also provides a superhydrophobic, anti-wetting barrier which can self clean by the action of low velocity rolling or impacting droplets of various size ranges from microns to several millimetres. Water droplets which are sufficiently small (10-100 µm) can easily access valleys between the scales for efficient self-cleaning and due to their dimensions can self-propel off the surface enhancing their mobility and cleaning effect. In addition, we demonstrate that the gecko skin has an antibacterial action where Gram-negative bacteria (Porphyromonas gingivalis) are killed when exposed to the surface however eukaryotic cell compatibility (with human stem cells) is demonstrated. The multifunctional features of the gecko skin provide a potential natural template for man-made applications where specific control of liquid, solid and biological contacts is required.

Production and packaging of a biological arsenal: evolution of centipede venoms under morphological constraint.

Venom represents one of the most extreme manifestations of a chemical arms race. Venoms are complex biochemical arsenals, often containing hundreds to thousands of unique protein toxins. Despite their utility for prey capture, venoms are energetically expensive commodities, and consequently it is hypothesized that venom complexity is inversely related to the capacity of a venomous animal to physically subdue prey. Centipedes, one of the oldest yet least-studied venomous lineages, appear to defy this rule. Although scutigeromorph centipedes produce less complex venom than those secreted by scolopendrid centipedes, they appear to rely heavily on venom for prey capture. We show that the venom glands are large and well developed in both scutigerid and scolopendrid species, but that scutigerid forcipules lack the adaptations that allow scolopendrids to inflict physical damage on prey and predators. Moreover, we reveal that scolopendrid venom glands have evolved to accommodate a much larger number of secretory cells and, by using imaging mass spectrometry, we demonstrate that toxin production is heterogeneous across these secretory units. We propose that the differences in venom complexity between centipede orders are largely a result of morphological restrictions of the venom gland, and consequently there is a strong correlation between the morphological and biochemical complexity of this unique venom system. The current data add to the growing body of evidence that toxins are not expressed in a spatially homogenous manner within venom glands, and they suggest that the link between ecology and toxin evolution is more complex than previously thought.

The comparative morphology of epidermal glands in Pentatomoidea (Heteroptera).

The Heteroptera show a diversity of glands associated with the epidermis. They have multiple roles including the production of noxious scents. Here, we examine the cellular arrangement and cytoskeletal components of the scent glands of pentatomoid Heteroptera in three families, Pentatomidae (stink bugs), Tessaratomidae, and Scutelleridae (shield-backed bugs or jewel bugs). The glands are; (1) the dorsal abdominal glands, (2) the tubular glands of the composite metathoracic gland, and (3) the accessory gland component of the composite metathoracic gland. The dorsal abdominal glands are at their largest in nymphs and decrease in size in adults. The metathoracic gland is an adult-specific gland unit with a reservoir and multiple types of gland cells. The accessory gland is composed of many unicellular glands concentrated in a sinuous line across the reservoir wall. The lateral tubular gland is composed of two-cell units. The dorsal abdominal glands of nymphs are composed of three-cell units with a prominent cuticular component derived from the saccule cell sitting between the duct and receiving canal. The cuticular components that channel secretion from the microvilli of the secretory cell to the exterior differ in the three gland types. The significance of the numbers of cells comprising gland units is related to the role of cells in regenerating the cuticular components of the glands at moulting in nymphs.

Generalist insects behave in a jasmonate-dependent manner on their host plants, leaving induced areas quickly and staying longer on distant parts.

Plants are sessile, so have evolved sensitive ways to detect attacking herbivores and sophisticated strategies to effectively defend themselves. Insect herbivory induces synthesis of the phytohormone jasmonic acid which activates downstream metabolic pathways for various chemical defences such as toxins and digestion inhibitors. Insects are also sophisticated animals, and many have coevolved physiological adaptations that negate this induced plant defence. Insect behaviour has rarely been studied in the context of induced plant defence, although behavioural adaptation to induced plant chemistry may allow insects to bypass the host's defence system. By visualizing jasmonate-responsive gene expression within whole plants, we uncovered spatial and temporal limits to the systemic spread of plant chemical defence following herbivory. By carefully tracking insect movement, we found induced changes in plant chemistry were detected by generalist Helicoverpa armigera insects which then modified their behaviour in response, moving away from induced parts and staying longer on uninduced parts of the same plant. This study reveals that there are plant-wide signals rapidly generated following herbivory that allow insects to detect the heterogeneity of plant chemical defences. Some insects use these signals to move around the plant, avoiding localized sites of induction and staying ahead of induced toxic metabolites.

Comparing the secretory pathway in honeybee venom and hypopharyngeal glands.

We provide insights into the secretory pathway of arthropod gland systems by comparing the royal jelly-producing hypopharyngeal glands and the venom-producing glands of the honeybee, Apis mellifera. These glands have different functions and different product release characteristics, but both belong to the class 3 types of insect glands, each being composed of two cells, a secretory cell and a microduct-forming cell. The hypopharyngeal secretory cells possess an extremely elongate tubular invagination that is filled with a cuticular structure, the end-apparatus, anchored against the cell membrane by a conspicuous series of actin rings. In contrast, venom glands have no actin rings, but instead have an actin-rich brush border surrounding the comparatively short and narrow end-apparatus. We relate these cytoskeletal differences to the production system and utilisation of secretions; venom is stored in a reservoir whereas royal jelly and enzymes are produced on demand. Fluorescence-based characterisation of the actin cytoskeleton combined with scanning electron microscopy of the end-apparatus allows for detailed characterisation of the point of secretion release in insect class 3 glands.

Novel actin rings within the secretory cells of honeybee royal jelly glands.

We describe a novel cytoskeletal element within secretory cells of an arthropod gland system, the hypopharyngeal gland of the honeybee, Apis mellifera. The hypopharyngeal secretory cells are the source of royal jelly in nurse bees and enzymes in foragers. Each cell possesses an elongate invagination that is occupied by a tubular cuticular structure, the end-apparatus, that accumulates secretion and transfers it into a cuticular microtube and then into a collecting duct. Within the secretory cell, a conspicuous series of actin rings, about 3 µm in diameter, follows the same path as the end-apparatus, surrounding it at spaced intervals. Transmission electron microscopy confirmed that the actin rings lie within septa of the secretory cell that are closely juxtaposed to the end-apparatus at regularly spaced intervals. We speculate that the function of the actin rings is to hold the end apparatus in place as secretion swells the extracellular compartments between the end apparatus and the cell membrane. To our knowledge, no such cytoskeletal component has been described in animal cells. © 2012 Wiley Periodicals, Inc.

Fouling of nanostructured insect cuticle: adhesion of natural and artificial contaminants.

The adhesional properties of contaminating particles of scales of various lengths were investigated for a wide range of micro- and nanostructured insect wing cuticles. The contaminating particles consisted of artificial hydrophilic (silica) and spherical hydrophobic (C(18)) particles, and natural pollen grains. Insect wing cuticle architectures with an open micro-/nanostructure framework demonstrated topographies for minimising solid-solid and solid-liquid contact areas. Such structuring of the wing membranes allows for a variety of removal mechanisms to contend with particle contact, such as wind and self-cleaning droplet interactions. Cuticles exhibiting high contact angles showed considerably lower particle adhesional forces than more hydrophilic insect surfaces. Values as low as 3 nN were recorded in air for silica of ~28 nm in diameter and <25 nN for silica particles 30 µm in diameter. A similar adhesional trend was also observed for contact with pollen particles.

Contrasting micro/nano architecture on termite wings: two divergent strategies for optimising success of colonisation flights.

Many termite species typically fly during or shortly after rain periods. Local precipitation will ensure water will be present when establishing a new colony after the initial flight. Here we show how different species of termite utilise two distinct and contrasting strategies for optimising the success of the colonisation flight. Nasutitermes sp. and Microcerotermes sp. fly during rain periods and adopt hydrophobic structuring/'technologies' on their wings to contend with a moving canvas of droplets in daylight hours. Schedorhinotermes sp. fly after rain periods (typically at night) and thus do not come into contact with mobile droplets. These termites, in contrast, display hydrophilic structuring on their wings with a small scale roughness which is not dimensionally sufficient to introduce an increase in hydrophobicity. The lack of hydrophobicity allows the termite to be hydrophilicly captured at locations where water may be present in large quantities; sufficient for the initial colonization period. The high wettability of the termite cuticle (Schedorhinotermes sp.) indicates that the membrane has a high surface energy and thus will also have strong attractions with solid particles. To investigate this the termite wings were also interacted with both artificial and natural contaminants in the form of hydrophilic silicon beads of various sizes, 4 µm C(18) beads and three differently structured pollens. These were compared to the superhydrophobic surface of the planthopper (Desudaba psittacus) and a native Si wafer surface. The termite cuticle demonstrated higher adhesive interactions with all particles in comparison to those measured on the plant hopper.

A dual layer hair array of the brown lacewing: repelling water at different length scales.

Additional weight due to contamination (water and/or contaminating particles) can potentially have a detrimental effect on the flight capabilities of large winged insects such as butterflies and dragonflies. Insects where the wing surface area-body mass ratio is very high will be even more susceptible to these effects. Water droplets tend to move spontaneously off the wing surface of these insects. In the case of the brown lacewing, the drops effectively encounter a dual bed of hair springs with a topographical structure which aids in the hairs resisting penetration into water bodies. In this article, we demonstrate experimentally how this protective defense system employed by the brown lacewing (Micromus tasmaniae) aids in resisting contamination from water and how the micro- and nanostructures found on these hairs are responsible for quickly shedding water from the wing which demonstrates an active liquid-repelling surface.

Non-wetting wings and legs of the cranefly aided by fine structures of the cuticle.

Non-wetting surfaces are imperative to the survival of terrestrial and semi-aquatic insects as they afford resistance to wetting by rain and other liquid surfaces that insects may encounter. Thus, there is an evolutionary pay-off for these insects to adopt hydrophobic technologies, especially on contacting surfaces such as legs and wings. The cranefly is a weak flier, with many species typically found in wet/moist environments where they lay eggs. Water droplets placed on this insect's wings will spontaneously roll off the surface. In addition, the insect can stand on water bodies without its legs penetrating the water surface. The legs and wings of this insect possess thousands of tiny hairs with intricate surface topographies comprising a series of ridges running longitudinally along the long axis of the hair fibre. Here we demonstrate that this fine hair structure enhances the ability of the hairs to resist penetration into water bodies.

Experimental determination of the efficiency of nanostructuring on non-wetting legs of the water strider.

Water striders demonstrate an amazing talent which enables them to effectively "row" across water surfaces without immobilization. This ability has previously been ascribed to the wax-like chemistry of the small hairs (setae) found on the legs, and theoretically attributed to the nano/microscaled hierarchical architecture of individual seta using the Cassie-Baxter equations. Here we show experimentally the strength of the contribution of the seta surface architecture to superhydrophobicity by maintaining identical surface chemistry (thin and thick coating of the setae with polydimethylsiloxane). Atomic force microscopy-based force and adhesion measurements of single uncoated and coated seta interacting with water quantitatively demonstrate the efficiency of the topographical component of the setae for repelling water.

The role of micro/nano channel structuring in repelling water on cuticle arrays of the lacewing.

Non-wetting surfaces help in the survival of terrestrial and semi-aquatic insects. Some insects encounter wetting by rain, through contact with water collected on foliage, or in ponds, rivers or streams. There is an evolutionary pay-off for such insects to adopt hydrophobic structuring especially on regions that contact water, such as legs or large surface areas such as wings. Here we investigate lacewings which are good candidates for getting trapped to water because of a large wing surface area-to-body mass ratio. The lacewing utilises a variety of structures/mechanisms to contend with water contact. The first level involves small hairs (macrotrichia) that project from veins on the wings and collectively hold up droplets of water above the wing surface. This defence against wetting is aided by longitudinal ridges and troughs along the hair shaft. We show, by coating single hairs with a hydrophobic polymer (similar in hydrophobicity to insect cuticle), that the channels significantly contribute to repel water droplets. Beneath the array of hairs lies a dense netting on the cuticle wing surface which reduces contact with smaller droplets. The study has implications for both insect biology and biomimetic surfaces such as light weight superhydrophobic materials.