Horizontal gene transfer underlies the painful stings of asp caterpillars (Lepidoptera: Megalopygidae).

Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata. We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution.

Variable seed bed microsite conditions and light influence germination in Australian winter annuals.

Environmentally cued germination may play an important role in promoting coexistence in Mediterranean annual plant systems if it causes niche differentiation across heterogeneous microsite conditions. In this study, we tested how microsite conditions experienced by seeds in the field and light conditions in the laboratory influenced germination in 12 common annual plant species occurring in the understorey of the York gum-jam woodlands in southwest Western Australia. Specifically, we hypothesized that if germination promotes spatial niche differentiation, then we should observe species-specific germination responses to light. In addition, we hypothesized that species' laboratory germination response may depend on the microsite conditions experienced by seeds while buried. We tested the laboratory germination response of seeds under diurnally fluctuating light and complete darkness, which were collected from microsites spanning local-scale environmental gradients known to influence community structure in this system. We found that seeds of 6 out of the 12 focal species exhibited significant positive germination responses to light, but that the magnitude of these responses varied greatly with the relative light requirement for germination ranging from 0.51 to 0.86 for these species. In addition, germination increased significantly across a gradient of canopy cover for two species, but we found little evidence to suggest that species' relative light requirement for germination varied depending on seed bank microsite conditions. Our results suggest that variability in light availability may promote coexistence in this system and that the microsite conditions seeds experience in the intra-growing season period can further nuance species germination behaviour.

A global review of seed enhancement technology use to inform improved applications in restoration.

Seed-based restoration often experiences poor success due to a range of edaphic and biotic issues. Seed enhancement technologies (SETs) are a novel approach that can alleviate these pressures and improve restoration success. Broadly, SETs have been reviewed for agricultural and horticultural purposes, for specific types of SETs such as coating or priming, or for focal ecosystems. However, information is lacking for SETs within a restoration focused context, and how they are being used to alleviate certain barriers. This review aimed to synthesise the current literature on SETs to understand what SETs are being tested, in which sectors and locations they are being tested, what issues are faced within restoration using SETs, and how SETs are being used to approach these issues. Priming was highlighted as the main SET investigated. Inoculation, pesticide application and magnetic fields were also commonly tested (SETs we termed 'prospective techniques'). SET research mainly occurred in the agricultural sector. More recently, other sectors, such as restoration and rangeland management, have increased efforts into SET research. The restoration sector has focused on extruded pelleting and coating (with activated carbon), in combination with herbicide application, to overcome invasive species, and coating with certain additives to alleviate edaphic issues. Other sectors outside restoration were largely focused on evaluating priming for overcoming these barriers. The majority of priming research has been completed on crop species and differences between these species and ecosystems must be considered in future restoration efforts that focus on native seed use. Generally, SETs require further refinement, including identifying ideal additives and their optimum concentrations to target certain issues, refining formulations for coating and extruded pelleting and developing flash flaming. A bet-hedging approach using multiple SETs and/or combinations of SETs may be advantageous in overcoming a wide range of barriers in seed-based restoration.

Experimental seed sowing reveals seedling recruitment vulnerability to unseasonal fire.

Unseasonal fire occurrence is increasing globally, driven by climate change and other human activity. Changed timing of fire can inhibit postfire seedling recruitment through interactions with plant phenology (the timing of key processes, e.g., flower initiation, seed production, dispersal, germination), and therefore threaten the persistence of many plant species. Although empirical evidence from winter-rainfall ecosystems shows that optimal seedling recruitment is expected following summer and autumn (dry season) fires, we sought experimental evidence isolating the mechanisms of poor recruitment following unseasonal (wet season) fire. We implemented a seed-sowing experiment using nine species native to fire-prone, Mediterranean-climate woodlands in southwestern Australia to emulate the timing of postfire recruitment and test key mechanisms of fire seasonality effects. For seeds sown during months when fire is unseasonal (i.e., August-September: end of the wet winter season), seedling recruitment was reduced by up to 99% relative to seeds sown during seasonal fire months (i.e., May-June: end of the dry summer season) because of varying seed persistence, seedling emergence, and seedling survival. We found that up to 70 times more seedlings emerged when seeds were sown during seasonal fire months compared to when seeds were sown during unseasonal fire months. The few seedlings that emerged from unseasonal sowings all died with the onset of the dry season. Of the seeds that failed to germinate from unseasonal sowings, only 2% survived exposure on the soil surface over the ensuing hot and dry summer. Our experimental results demonstrate the potential for unseasonal fire to inhibit seedling recruitment via impacts on pregermination seed persistence and seedling establishment. As ongoing climate change lengthens fire seasons (i.e., unseasonal wildfires become more common) and managed fires are implemented further outside historically typical fire seasons, postfire seedling recruitment may become more vulnerable to failure, causing shifts in plant community composition towards those with fewer species solely dependent on seeds for regeneration.

Physical and Chemical Barriers in the Larval Midgut Confer Developmental Resistance to Virus Infection in Drosophila.

Insects can become lethally infected by the oral intake of a number of insect-specific viruses. Virus infection commonly occurs in larvae, given their active feeding behaviour; however, older larvae often become resistant to oral viral infections. To investigate mechanisms that contribute to resistance throughout the larval development, we orally challenged Drosophila larvae at different stages of their development with Drosophila C virus (DCV, Dicistroviridae). Here, we showed that DCV-induced mortality is highest when infection initiates early in larval development and decreases the later in development the infection occurs. We then evaluated the peritrophic matrix as an antiviral barrier within the gut using a Crystallin-deficient fly line (Crys-/-), whose PM is weakened and becomes more permeable to DCV-sized particles as the larva ages. This phenotype correlated with increasing mortality the later in development oral challenge occurred. Lastly, we tested in vitro the infectivity of DCV after incubation at pH conditions that may occur in the midgut. DCV virions were stable in a pH range between 3.0 and 10.5, but their infectivity decreased at least 100-fold below (1.0) and above (12.0) this range. We did not observe such acidic conditions in recently hatched larvae. We hypothesise that, in Drosophila larvae, the PM is essential for containing ingested virions separated from the gut epithelium, while highly acidic conditions inactivate the majority of the virions as they transit.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Environmental Factors Driving Seed Hydration Status of Soil Seed Banks and the Implications for Post-fire Recruitment.

Changes in fire regimes due to climate change and fire management practices are affecting the timing, length, and distribution of vegetation fires throughout the year. Plant species responses and tolerances to fire differ from season to season and are influenced by species-specific phenological processes. The ability of seeds to tolerate extreme temperatures associated with fire is one of these processes, with survival linked to seed moisture content at the time of exposure. As fire is more often occurring outside historic dry fire seasons, the probability of fire occurring when seeds are hydrated may also be increasing. In this study, we set out to understand the seasonal dynamics of seed hydration for seeds of Banksia woodland species, and how certain seed traits interact with environmental conditions to influence survival of high temperatures associated with fire. We measured the moisture content of seeds buried to 2 cm in the soil seed bank for four common native species and one invasive species on a weekly basis throughout 2017, along with soil moisture content and environmental correlates. We determined water sorption isotherms at 20°C for seeds of each species and used these functions to model weekly variation in seed water activity and predict when seeds are most sensitive to soil heating. Using Generalised additive models (GAMs), we were able to describe approximately 67% of the weekly variance in seed water activity and explored differences in seed hydration dynamics between species. Seed water activity was sufficiently high (i.e., ≥ 0.85 a w) so as to have created an increased risk of mortality if a fire had occurred during an almost continuous period between May and November in the study period (i.e., 2017). There were brief windows when seeds may have been in a dry state during early winter and late spring, and also when they may have been in a wet state during summer and late autumn. These data, and the associated analyses, provide an opportunity to develop approaches to minimize seed mortality during fire and maximize the seed bank response.

Hindgut microbiota reflects different digestive strategies in dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae).

Gut microbes play an important role in the biology and evolution of insects. Australian native dung beetles (Scarabaeinae) present an opportunity to study gut microbiota in an evolutionary context as they come from two distinct phylogenetic lineages and some species in each lineage have secondarily adapted to alternative or broader diets. In this study, we characterised the hindgut bacterial communities found in 21 species of dung beetles across two lineages using 16S rRNA sequencing. We found that gut microbial diversity was more dependent on host phylogeny and gut morphology than specific dietary preferences or environment. In particular, gut microbial diversity was highest in the endemic, flightless genus Cephalodesmius that feeds on a broad range of composted organic matter. The hindgut of Cephalodesmius harbours a highly conserved core set of bacteria suggesting that the bacteria are symbiotic. Symbiosis is supported by the persistence of the core microbiota across isolated beetle populations and between species in the genus. A co-evolutionary relationship is supported by the expansion of the hindgut to form a fermentation chamber and the fermentative nature of the core microbes. In contrast, Australian species of the widespread dung beetle genus Onthophagus, specialise on a single food resource such as dung or fungus, exhibit minimal food processing behaviour, have a short, narrow hindgut and a variable gut microbiota with relatively few core bacterial taxa. A conserved, complex gut microbiota is hypothesised to be unnecessary for this highly mobile genus.IMPORTANCE Dung beetles are a very important part of an ecosystem because of their role in the removal and decomposition of vertebrate dung. It has been suspected that symbiotic gut bacteria facilitate this role, a hypothesis that we have explored with high throughput barcoding. We found that differences in hindgut morphology had the greatest effect on the bacterial community composition. Species with a hindgut fermentation chamber harboured a distinctly different hindgut community compared to those species with a narrow, undifferentiated hindgut. Diet and phylogeny were also associated with differences in gut community. Further understanding of the relationships between dung beetles and their gut microbes will provide insights into the evolution of their behaviours and how gut communities contribute to their fitness.

Mechanisms of Fire Seasonality Effects on Plant Populations.

Altered fire regimes resulting from climate change and human activity threaten many terrestrial ecosystems. However, we lack a holistic and detailed understanding of the effects of altering one key fire regime component - season of fire. Altered fire seasonality can strongly affect post-fire recovery of plant populations through interactions with plant phenology. We identify seven key mechanisms of fire seasonality effects under a conceptual demographic framework and review evidence for these. We reveal negative impacts of altered fire seasonality and identify research gaps for mechanisms and climate types for future analyses of fire seasonality effects within the identified demographic framework. This framework and these mechanisms can inform critical decisions for conservation, land management, and fire management policy development globally.

Seed storage behaviour of tropical members of the aquatic basal angiosperm genus Nymphaea L. (Nymphaeaceae).

Eighteen native species of Nymphaea (waterlilies) inhabit a range of freshwater wetlands in northern Australia, which are threatened by increased development and the potential impacts of climate change. To investigate conservation seed banking of these vulnerable species, we aimed to characterize their seed storage physiology by determining (i) seed desiccation tolerance and (ii) the effects of moisture content and storage temperature on seed germination and viability. Seeds of N. immutabilis, N. lukei, N. macrosperma and N. violacea (including multiple collections of three species) were placed in experimental storage at a range of temperatures (25°C, 5°C, -20°C and -190°C) following pre-equilibration at different RHs (15%, 30%, 50%, 70% or 95%). Seeds were also experimentally aged at 60% RH and 45°C to assess comparative longevity. We found seeds of all species to be desiccation tolerant. However, the responses of seeds to experimental storage conditions were complex and variable between species and collections of the same species, and seeds of many species/collections were short-lived across many of the storage treatments. In many cases decreasing storage temperature did not increase longevity. Additional protocol development is necessary before we can have confidence that ex situ seed banking is a viable long-term germplasm conservation strategy for Nymphaea.

Inorganic soil amendments alter seedling performance of native plant species in post-mining arid zone rehabilitation.

Rehabilitation of degraded drylands is challenged by environmental and anthropogenic constraints, such as limited availability of locally-sourced topsoil and poor quality alternative soil substrates. Current rehabilitation practices, at times, utilise inorganic soil amendments to improve the physicochemical and biological characteristics of reconstructed soil profiles. These approaches may be appropriate for dryland rehabilitation, but there is limited research available regarding the benefits of using these amendments. Here, we present a study in the Pilbara region of Western Australia, an arid landscape subject to intensive mining that currently uses inorganic soil amendments (gypsum and urea) in post-mining rehabilitation. The aim of this study was to assess the effectiveness of these amendments to (1) promote seed germination, seedling emergence and seedling growth across five plant species and, (2) re-instate soil quality in mine waste substrates. A series of glasshouse experiments assessed eight application combinations of these amendments in two alternative substrates and compared these to unamended substrates and topsoil. Soil amendments had a limited influence on seed germination, were detrimental to seedling emergence and resulted in increased seedling mortality. Mortality in the waste ranged from 2 to 61% but increased to 7-92% in amended waste. Seedling growth improved with high doses of amendments in waste, with a 1.3-5.6-fold increase across all plant species. Soil quality was relatively unaffected by amendments with soil nitrogen ranging from 0.01 to 0.08%, organic carbon from 0.01 to 0.12% and soil microbial activity from 2.3 to 2.4 ppm-CO2 in the amended and unamended waste. The use of soil amendments in mine rehabilitation requires consideration of the trade-off between initial reductions in seedling recruitment and enhanced seedling development at later stages. Future rehabilitation should consider the timing of amendment application to avoid detrimental impacts on seedling recruitment and maximise the benefits to seedling growth.

Protecting Offspring Against Fire: Lessons From Banksia Seed Pods.

Wildfires are a natural component in many terrestrial ecosystems and often play a crucial role in maintaining biodiversity, particularly in the fire-prone regions of Australia. A prime example of plants that are able to persist in these regions is the genus Banksia. Most Banksia species that occur in fire-prone regions produce woody seed pods (follicles), which open during or soon after fire to release seeds into the post-fire environment. For population persistence, many Banksia species depend on recruitment from these canopy-stored seeds. Therefore, it is critical that their seeds are protected from heat and rapid oxidation during fire. Here, we show how different species of Banksia protect their seeds inside follicles while simultaneously opening up when experiencing fire. The ability of the follicles to protect seeds from heat is demonstrated by intense 180 s experimental burns, in which the maximum temperatures near the seeds ranged from ∼75°C for B. serrata to ∼90°C for B. prionotes and ∼95°C for B. candolleana, contrasting with the mean surface temperature of ∼450°C. Many seeds of native Australian plants, including those of Banksia, are able to survive these temperatures. Structural analysis of individual follicles from these three Banksia species demonstrates that all of them rely on a multicomponent system, consisting of two valves, a porous separator and a thin layer of air surrounding the seeds. The particular geometric arrangement of these components determines the rate of heat transfer more than the tissue properties alone, revealing that a strong embedment into the central rachis can compensate for thin follicle valves. Furthermore, we highlight the role of the separator as an important thermal insulator. Our study suggests that the genus Banksia employs a variety of combinations in terms of follicle size, valve thickness, composition and geometric arrangement to effectively protect canopy-stored seeds during fire.

Morphophysiological dormancy in the basal angiosperm order Nymphaeales.

Background and Aims: Substantial evidence supports the hypothesis that morphophysiological dormancy (MPD) is the basal kind of seed dormancy in the angiosperms. However, only physiological dormancy (PD) is reported in seeds of the ANA-grade genus Nymphaea. The primary aim of this study was to determine the kind of dormancy in seeds of six species of Nymphaea from the wet-dry tropics of Australia. Methods: The effects of temperature, light and germination stimulants on germination were tested on multiple collections of seeds of N. immutabilis, N. lukei, N. macrosperma, N. ondinea, N. pubescens and N. violacea. Embryo growth prior to hypocotyl emergence was monitored. Key Results: Germination was generally <10 % after 28 d in control treatments. Germination percentage was highest at 30 or 35 °C for seeds exposed to light and treated with ethylene or in anoxic conditions in sealed vials of water, and it differed significantly between collections of N. lukei, N. macrosperma and N. violacea. Seeds of N. pubescens did not germinate under any of the conditions. Embryo growth (8-37 % in length) occurred before hypocotyl emergence (germination) in seeds of the five species that germinated. Conclusions: Fresh seeds were dormant, and the amount of pregermination embryo growth in seeds of N. lukei and N. immutabilis was relatively small, while in seeds of N. macrosperma, N. ondinea and N. violacea it was relatively large. Thus, seeds of N. lukei and N. immutabilis had PD and those of N. macrosperma, N. ondinea and N. violacea had MPD. Overall, we found that seeds in the most phylogenetically derived clades within Nymphaea have MPD, suggesting that PD is the most likely basal trait within the Nymphaeales. This study also highlights the broad range of dormancy types and germination strategies in the ANA-grade angiosperms.

A research agenda for seed-trait functional ecology.

Trait-based approaches have improved our understanding of plant evolution, community assembly and ecosystem functioning. A major challenge for the upcoming decades is to understand the functions and evolution of early life-history traits, across levels of organization and ecological strategies. Although a variety of seed traits are critical for dispersal, persistence, germination timing and seedling establishment, only seed mass has been considered systematically. Here we suggest broadening the range of morphological, physiological and biochemical seed traits to add new understanding on plant niches, population dynamics and community assembly. The diversity of seed traits and functions provides an important challenge that will require international collaboration in three areas of research. First, we present a conceptual framework for a seed ecological spectrum that builds upon current understanding of plant niches. We then lay the foundation for a seed-trait functional network, the establishment of which will underpin and facilitate trait-based inferences. Finally, we anticipate novel insights and challenges associated with incorporating diverse seed traits into predictive evolutionary ecology, community ecology and applied ecology. If the community invests in standardized seed-trait collection and the implementation of rigorous databases, major strides can be made at this exciting frontier of functional ecology.

Seed dormancy regulates germination response to smoke and temperature in a rhizomatous evergreen perennial.

Seed dormancy status regulates the response of seeds to environmental cues that can trigger germination. Anigozanthos flavidus (Haemodoraceae) produces seeds with morphophysiological dormancy (MPD) that are known to germinate in response to smoke, but embryo growth dynamics and germination traits in response to temperatures and after-ripening have not been well characterized. Seeds of A. flavidus, after-ripened for 28 months at 15 °C/15 % relative humidity, were incubated on water agar, water agar containing 1 µM karrikinolide (KAR1) or 50 µM glyceronitrile at 5, 10, 15, 20, 25, 20/10 and 25/15 °C for 28 days. After incubation at 5, 10 and 25 °C for 28 days, seeds were transferred to 15 °C for another 28 days. Embryo growth dynamics were tested at 5, 10, 15 and 25 °C. Results demonstrated that fresh seeds of A. flavidus had MPD and the physiological dormancy (PD) component could be broken by either glyceronitrile or dry after-ripening. After-ripened seeds germinated to ≥80 % at 15-20 °C while no additional benefit of germination was observed in the presence of the KAR1 or glyceronitrile. Embryo length significantly increased at 10 °C, and only slightly increased at 5 °C, while growth did not occur at 25 °C. When un-germinated seeds were moved from 5-10 °C to 15 °C for a further 28 days, germination increased from 0 to >80 % in significantly less time indicating that cold stratification may play a key role in the germination process during winter and early spring in A. flavidus. The lower germination (<50 %) of seeds moved from 25 to 15 °C was produced by the induction of secondary dormancy. Induction of secondary dormancy in seeds exposed to warm stratification, a first report for Anigozanthos species, suggests that cycling of PD may be an important mechanism of controlling germination timing in the field.

Temperature-induced self-sealing capability of Banksia follicles.

Many plants in fire-prone regions retain their seeds in woody fruits in the plant canopy until the passage of a fire causes the fruit to open and release the seeds. To enable this function, suitable tissues are required that effectively store and protect seeds until they are released. Here, we show that three different species of the Australian genus Banksia incorporate waxes at the interface of the two valves of the follicle enclosing the seeds, which melt between 45°C and 55°C. Since the melting temperature of the waxes is lower than the opening temperatures of the follicles in all investigated species (B. candolleana, B. serrata, B. attenuata), we propose that melting of these waxes allows the sealing of micro-fissures at the interface of the two valves while they are still closed. Such a self-sealing mechanism likely contributes to the structural integrity of the seed pods, and benefits seed viability and persistence during storage on the plants. Furthermore, we show in a simplified, bioinspired model system that temperature treatments seal artificially applied surface cuts and restore the barrier properties.

Measuring metabolic rates of small terrestrial organisms by fluorescence-based closed-system respirometry.

We explore a recent, innovative variation of closed-system respirometry for terrestrial organisms, whereby oxygen partial pressure (PO2 ) is repeatedly measured fluorometrically in a constant-volume chamber over multiple time points. We outline a protocol that aligns this technology with the broader literature on aerial respirometry, including the calculations required to accurately convert O2 depletion to metabolic rate (MR). We identify a series of assumptions, and sources of error associated with this technique, including thresholds where O2 depletion becomes limiting, that impart errors to the calculation and interpretation of MR. Using these adjusted calculations, we found that the resting MR of five species of angiosperm seeds ranged from 0.011 to 0.640 ml g-1 h-1, consistent with published seed MR values. This innovative methodology greatly expands the lower size limit of terrestrial organisms that can be measured, and offers the potential for measuring MR changes over time as a result of physiological processes of the organism.