Seed germination and dormancy traits of forbs and shrubs important for restoration of North American dryland ecosystems.

In degraded dryland systems, native plant community re-establishment following disturbance is almost exclusively carried out using seeds, but these efforts commonly fail. Much of this failure can be attributed to the limited understanding of seed dormancy and germination traits. We undertook a systematic classification of seed dormancy of 26 species of annual and perennial forbs and shrubs that represent key, dominant genera used in restoration of the Great Basin ecosystem in the western United States. We examined germination across a wide thermal profile to depict species-specific characteristics and assessed the potential of gibberellic acid (GA3 ) and karrikinolide (KAR1 ) to expand the thermal germination envelope of fresh seeds. Of the tested species, 81% produce seeds that are dormant at maturity. The largest proportion (62%) exhibited physiological (PD), followed by physical (PY, 8%), combinational (PY + PD, 8%) and morphophysiological (MPD, 4%) dormancy classes. The effects of chemical stimulants were temperature- and species-mediated. In general, mean germination across the thermal profile was improved by GA3 and KAR1 for 11 and five species, respectively. We detected a strong germination response to temperature in freshly collected seeds of 20 species. Temperatures below 10 °C limited the germination of all except Agoseris heterophylla, suggesting that in their dormant state, the majority of these species are thermally restricted. Our findings demonstrate the utility of dormancy classification as a foundation for understanding the critical regenerative traits in these ecologically important species and highlight its importance in restoration planning.

Cyanobacteria inoculation enhances carbon sequestration in soil substrates used in dryland restoration.

Despite significant efforts to restore dryland ecosystems worldwide, the rate of success of restoration is extremely low in these areas. The role of cyanobacteria from soil biocrusts in reestablishing soil functions of degraded land has been highlighted in recent years. These organisms are capable of improving soil structure and promoting soil N and C fixation. Nevertheless, their application to restore functions of reconstructed soils in dryland restoration programs is yet to be harnessed. In this study, we used microcosms under laboratory conditions to analyse the effects of inoculating soil substrates used in post-mine restoration with a mixture of N-fixing cyanobacteria isolated from soil biocrust (Nostoc commune, Tolypothrix distorta and Scytonema hyalinum) on i) the recovery of the biocrust, and ii) the carbon sequestration and mineralisation rates of these substrates. Soils were collected from an active mine site in the mining-intensive biodiverse Pilbara region (north-west Western Australia) and consisted of previously stockpiled topsoil, overburden waste material, a mixture of both substrates, and a natural soil from an undisturbed area. Our results showed that cyanobacteria rapidly colonised the mine substrates, with biocrust coverage ranging from 23.8 to 52.2% and chlorophyll a concentrations of up to 12.2 µg g-1 three months after inoculation. Notably, soil organic C contents increased 3-fold (P < 0.001) in the mine waste substrate (from 0.6 g kg-1 to 1.9 g kg-1) during this period of time. Overall, our results showed that cyanobacteria inoculation can rapidly modify properties of reconstructed soil substrates, underpinning the potential key role of these organisms as bio-tools to initiate recovery of soil functions in infertile, reconstructed soil substrates.

Ecophysiology of seed dormancy in the Australian endemic species Acanthocarpus preissii (Dasypogonaceae).

BACKGROUND AND AIMS: Seedlings of Acanthocarpus preissii are needed for coastal sand dune restoration in Western Australia. However, seeds of this Western Australian endemic have proven to be very difficult to germinate. The aims of this study were to define a dormancy-breaking protocol, identify time of suitable conditions for dormancy-break in the field and classify the type of seed dormancy in this species. METHODS: Viability, water-uptake (imbibition) and seed and embryo characteristics were assessed for seeds collected in 2003 and in 2004 from two locations. The effects of GA(3), smoke-water, GA(3) + smoke-water and warm stratification were tested on seed dormancy-break. In a field study, soil temperature and the moisture content of soil and buried seeds were monitored for 1 year. KEY RESULTS: Viability of fresh seeds was >90 %, and they had a fully developed, curved-linear embryo. Fresh seeds imbibed water readily, with mass increasing approx. 52 % in 4 d. Non-treated fresh seeds and those exposed to 1000 ppm GA(3), 1 : 10 (v/v) smoke-water/water or 1000 ppm GA(3) + 1 : 10 (v/v) smoke-water/water germinated <8 %. Fresh seeds germinated to >80 % when warm-stratified for at least 7 weeks at 18/33 degrees C and then moved to 7/18 degrees C, whereas seeds incubated continuously at 7/18 degrees C germinated to <20 %. CONCLUSIONS: Seeds of A. preisii have non-deep physiological dormancy that is released by a period of warm stratification. Autumn (March/April) is the most likely time for warm stratification of seeds of this species in the field. This is the first report of the requirement for warm stratification for dormancy release in seeds of an Australian species.

The changing window of conditions that promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae).

BACKGROUND AND AIMS: Following a period of burial, more Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae) seeds germinate in smoke water. The main aim of this study was to determine whether these fire-ephemeral seeds exhibit annual dormancy cycling during burial. This study also aimed to determine the effect of dormancy alleviation on the range of light and temperature conditions at which seeds germinate, and the possible factors driving changes in seed dormancy during burial. METHODS: Seeds were collected in summer, buried in soil in mesh bags in autumn and exhumed every 6 months for 24 months. Germination of exhumed and laboratory-stored (15 degrees C) seeds was assessed at 20 degrees C in water or smoke water. Germination response to light or dark conditions, incubation temperature (10, 15, 20, 25 and 30 degrees C), nitrate and gibberellic acid were also examined following burial or laboratory storage for 24 months. In the laboratory seeds were also stored at various temperatures (5, 15, 37 and 20/50 degrees C) for 1, 2 and 3 months followed by germination testing in water or smoke water. KEY RESULTS: The two species exhibited dormancy cycling during soil burial, producing low levels of germination in response to smoke water when exhumed in spring and high levels of germination in autumn. In autumn, seeds germinated in both light and dark and at a broader range of temperatures than did laboratory-stored seeds, and some Actinotus leucocephalus seeds also germinated in water alone. Dormancy release of Actinotus leucocephalus was slow during dry storage at 15 degrees C and more rapid at higher temperatures (37 and 20/50 degrees C); weekly wet/dry cycles further accelerated the rate of dormancy release. Cold stratification (5 degrees C) induced secondary dormancy. By contrast, no Tersonia cyathiflora seeds germinated following any of the laboratory storage treatments. CONCLUSIONS: Temperature and moisture influence dormancy cycling in Actinotus leucocephalus seeds. These factors alone did not simulate dormancy cycling of Tersonia cyathiflora seeds under the conditions tested.

Survival of four accessions of Anigozanthos manglesii (haemodoraceae) seeds following exposure to liquid nitrogen.

This study investigated the survival of seeds from the prominent endemic Western Australian species Anigozanthos manglesii following exposure to liquid nitrogen (cryostorage). Seeds from four different accessions (collected in 1987, 1990, 1993 and 1998) adjusted to different water contents were tested for survival following cryostorage. Water content was a significant determining factor with survival of cryostored seeds declining rapidly at water contents above c. 18%. These water contents were deemed as critical water contents and were supported by DSC scans showing high endothermic peaks indicating ice crystallisation. In some instances, survival of cryostored seeds also declined at low water contents. Seeds from 1990 had a lower than expected survival compared to the other accessions. This may have resulted from the higher lipid content of seeds from this accession, or the reduced germination and vigour of these seeds prior to cryostorage.

Wolbachia density and virulence attenuation after transfer into a novel host.

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. melanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. melanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

Wolbachia-mediated sperm modification is dependent on the host genotype in Drosophila.

Estimates of Wolbachia density in the eggs, testes and whole flies of drosophilid hosts have been unable to predict the lack of cytoplasmic incompatibility (CI) expression in so-called mod(-) variants. Consequently, the working hypothesis has been that CI expression, although related to Wolbachia density, is also governed by unknown factors that are influenced by both host and bacterial genomes. Here, we compare the behaviour of the mod(-) over-replicating Wolbachia popcorn strain in its native Drosophila melanogaster host to the same strain transinfected into a novel host, namely Drosophila simulans. We report that (i) the popcorn strain is a close relative of other D. melanogaster infections, (ii) the mod(-) status of popcorn in D. melanogaster appears to result from its inability to colonize sperm bundles, (iii) popcorn is present in the bundles in D. simulans and induces strong CI expression, which demonstrates that the bacterial strain does not lack the genetic machinery for inducing CI and that there is host-species-specific control over Wolbachia tissue tropism, and (iv) infection of sperm bundles by the mod(-) D. simulans wCof strain indicates that there are several independent routes by which a strain can be a CI non-expressor.

Central projections of Drosophila sensory neurons in the transition from embryo to larva.

Sensory axons of different sensory modalities project into typical domains within insect ganglia. Tactile and gustatory axons project into a ventral layer of neuropil and proprioceptive afferents, including chordotonal axons, into an intermediate or dorsal layer. Here, we describe the central projections of sensory neurons in the first instar Drosophila larva, relating them to the projection of the same sensory afferents in the embryo and to sensory afferents of similar type in other insects. Several neurons show marked morphologic changes in their axon terminals in the transition between the embryo and larva. During a short morphogenetic period late in embryogenesis, the axon terminals of the dorsal bipolar dendrite stretch receptor change their shape and their distribution within the neuromere. In the larva, external sense organ neurons (es) project their axons into a ventral layer of neuropil. Chordotonal sensory neurons (ch) project into a slightly more dorsal region that is comparable to their projection in adults. The multiple dendrite (md) neurons show two distinctive classes of projection. One group of md neurons projects into the ventral-most neuropil region, the same region into which es neurons project. Members of this group are related by lineage to es neurons or share a requirement for expression of the same proneural gene during development. Other md neurons project into a more dorsal region. Sensory receptors projecting into dorsal neuropil possibly provide proprioceptive feedback from the periphery to central motorneurons and are candidates for future genetic and cellular analysis of simple neural circuitry.

In vivo dynamics of axon pathfinding in the Drosophilia CNS: a time-lapse study of an identified motorneuron.

We developed a system for time-lapse observation of identified neurons in the central nervous system (CNS) of the Drosophila embryo. Using this system, we characterize the dynamics of filopodia and axon growth of the motorneuron RP2 as it navigates anteriorly through the CNS and then laterally along the intersegmental nerve (ISN) into the periphery. We find that both axonal extension and turning occur primarily through the process of filopodial dilation. In addition, we used the GAL4-UAS system to express the fusion protein Tau-GFP in a subset of neurons, allowing us to correlate RP2's patterns of growth with a subset of axons in its environment. In particular, we show that RP2's sharp lateral turn is coincident with the nascent ISN.

Genetic basis of the formation and identity of type I and type II neurons in Drosophila embryos.

The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons. Here, we analyse the origin of the difference between type I and type II in the case of the neurons that depend on the proneural genes of the achaete-scute complex (ASC). We show that, in Notch- embryos, the type I neurons are missing while type II neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication. In contrast, both type I and type II neurons are absent in numb- embryos and after ubiquitous expression of tramtrack, indicating that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates. The analysis of string- embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate. We also report a new mutant phenotype where the ASC-dependent neurons are converted into type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate. Our results suggest that the same mechanism of type I/type II specification may operate at a late step of the ASC-dependent lineages, when multidendritic neurons arise as siblings of the external sense organ neurons and, at an early step, when other multidendritic neurons precursors arise as siblings of external sense organ precursors.

Transformation of external sensilla to chordotonal sensilla in the cut mutant of Drosophila assessed by single-cell marking in the embryo and larva.

The cut gene of Drosophila melanogaster is an identity selector gene that establishes the program of development and differentiation of external sense organs. Mutations in the cut gene cause a transformation of the external sense organs into chordotonal organs, originally assessed by the use of immunostaining methods [Bodmer et al. (1987): Cell, 51:293-307]. Because of evidence that axonal projections of the transformed neurons within the central nervous system are not completely switched in cut mutants, the transformation of the four cells making up a sense organ was reassessed using single-cell staining with fluorescent dye and differential interface contrast (DIC) microscopy of the embryo and larva. The results provide strong evidence that all cells of the sense organs are completely transformed, exhibiting the morphologies and organelles characteristic of chordotonal sense organs. A comparison of the structures of external sense organs and chordotonal organs indicates that a number of the differences could be due to the degree of development of common structures, and that cut or downstream genes modulate effector genes that are normally utilized in both receptor types. The possible derivation of insect chordotonal and external sense organs from a receptor type found in crustaceans is discussed in the light of arthropod phylogenetics and the molecular genetics of sense organ development.

Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function.

The peripheral nervous system (PNS) of the Drosophila embryo is especially suited for investigating the specification of neuronal identity: the PNS consists of a relatively simple but diverse set of individually identified sensory neurons; mutants, including embryonic lethals, can be readily generated and analyzed; and axon growth can potentially be followed from the earliest stages. We have developed a staining method to reveal the central projections of the full set of sensory neurons in the preterminal abdominal segments of the embryo. The sensory neurons exhibit modality-specific axonal projections in the CNS. The axons of external sense (es) organ neurons, primarily tactile in function, are restricted to a particular region within each neuromere and exhibit a somatotopic mapping within the CNS. The axons of stretch-receptive chordotonal (ch) organs project into a discrete longitudinal fascicle. Sensory neurons with multiple-branched dendrites (md neurons) project into a separate fascicle. A small number of md neurons have distinctive dorsal-projecting axonal processes in the CNS. A classification of sensory neurons based on their axon morphology correlates closely with the identity of the proneural gene responsible for their generation, suggesting that proneural genes play a central role in determining neuronal identity in the PNS of the embryo.

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system.

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell-cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity.

The role of the cut gene in the specification of central projections by sensory axons in Drosophila.

Mutations in the cut gene transform sense organs in Drosophila embryos from external sensory (es) receptors to chordotonal (ch) organs. We have investigated whether their central axonal projections are also transformed. Following Lucifer yellow injection of the sensory neuron, wild-type es and ch organs show characteristic, different projection patterns in the CNS. Transformed es neurons in cut embryos are variable in their projection patterns: some resemble wild-type es neurons, others ch neurons, while yet others are unlike either of these. We conclude that the cut gene influences axonal projections, although its action as a simple modality switch is open to question. Additional genes could be involved in the specification of the central axonal projection of the transformed neurons.

Projections of leg proprioceptors within the CNS of the fly Phormia in relation to the generalized insect ganglion.

We previously reported a modality-specific layering of leg sensory axons in the CNS of the flies Phormia regina and Drosophila melanogaster with tactile and gustatory axons projecting into a ventral layer and the proprioceptive hair plate axons into an intermediate layer. Here the description is expanded to include the afferent projections of campaniform sensilla on the legs and wings of Phormia. The leg campaniform sensilla produce a number of patterns of projections within an intermediate layer of their ganglion, some of which project intersegmentally into the other thoracic ganglia. One of these patterns is shared by the hair plate sense organs. Selected wing campaniform sensilla were also stained and showed that there is little or no overlap between the projections of leg and wing campaniform sensilla. Similarities with the arrangement of campaniform sensilla and their central processes in Drosophila melanogaster are discussed. To apply the results of this study to a broader range of insects we provide an atlas of the fly CNS and compare it with the locust, which has been the model for much insect neuroanatomy and neurophysiology.

From embryo to adult: anatomy and development of a leg sensory organ in Phormia regina Meigen (Insecta: Diptera). I. Anatomy and physiology of a larval "leg" sensory organ.

Neurons within the precursor of the adult leg, the imaginal disc, innervate a larval sense organ, Keilin's organ. Electron microscopical investigations of first instar larvae show that five dendrites end at the organ: three insert at the bases of the three hairs of the organ and two end against the cuticle, without any apparent cuticular specialization. In third instar larvae, the imaginal leg discs invaginate into the body cavity, and only four of the dendrites (the outer segments of which become greatly elongated) remain in contact with Keilin's organ. The axons of the neurons that supply Keilin's organ project into a ventral neuropile region of the central nervous system, with a pattern that resembles the projections of other larval sensilla. Electrical activity can be recorded from neurons of the imaginal disc in response to mechanical stimulation.

From embryo to adult: anatomy and development of a leg sensory organ in Phormia regina, Meigen (Insecta: Diptera). II. Development and persistence of sensory neurons.

The imaginal leg disc of Phormia regina contains eight neurons that arise during embryogenesis. Five of the neurons are associated with Keilin's organ, and of these five, two persist to the adult fly. Two new neurons arise at about the time of pupariation and flank each of these persisting neurons, forming two triplets of cells. Both triplets can be followed throughout metamorphosis; in the late pupa they are situated anteriorly and posteriorly at the tip of the fifth tarsomere. Two triplets of cuticular specializations are found at corresponding positions in the adult fly, each consisting of two campaniform sensilla and a trichoid hair. The central member of each set of sensilla, a campaniform sensillum, is associated with the persisting cell.

Modality-specific axonal projections in the CNS of the flies Phormia and Drosophila.

There is a rich history of behavioral and physiological studies on the leg sensory systems of flies. Here we examine the anatomy of the sensory axons of two species of fly and demonstrate that the location of the axonal projections in the CNS can be correlated with the modality they encode. We studied receptors associated with proprioceptive, tactile, and multimodal hairs. Proprioceptive hairs occur in clusters, called hair plates, and are situated near joints. The neuron innervating each proprioceptive hair has a large axon and coarse arborization in the intermediate neuropil. Tactile receptors have smaller arbors, which are located in a ventral region of the thoracic neuromere. Finally, the multimodal hairs are each innervated by one tactile and four chemosensory neurons. The single tactile neuron has a central arbor that is indistinguishable from those of the tactile hairs; the four chemosensory neurons project to yet a third region of neuropil near the ventral surface of each neuromere. Thus there is a clear modality-specific segregation of axonal arbors in the CNS. This organization is identical in Phormia and Drosophila and thus apparently highly conserved within the Diptera. We presume that, as in other insect sensory systems, this anatomical specificity is linked to synaptic specificity.

The cercal sensilla of the blowfly Lucilia cuprina. II. Structure of the enveloping cells and the basal regions of the sensory dendrites.

The gustatory, olfactory, touch and stress receptors on the cerci of Lucilia cuprina Wied. (Diptera: Calliphoridae) have either two or three enveloping cells. The gustatory and olfactory sensilla have three enveloping cells: a tormogen, trichogen and thecogen cell. The tormogen and trichogen cells contribute to a sub-cuticular sensillar lumen which divides into two lobes basally. The thecogen cell forms a lumen around the dendrites. Distally the dendrites lie in the contents of the thecogen lumen within the dendritic sheath. Proximally the dendrites embed in the thecogen cell which has an expanded, microlamellate lumen basally. The sensillar lumen of the mechanosensory (trichoid mechanoreceptors and campaniform) sensilla is formed by a single enveloping cell: the presumptive tormogen cell. In trichoid mechanoreceptors the thecogen lumen is restricted to the region of the transitional region of the dendrite whereas the thecogen lumen of campaniform sensilla extends proximally although it is not as well-developed as that of the chemoreceptive sensilla. The dendrites of all sensillum types on the cerci have a granular body in the transitional region: a situation which has not been previously reported in chemoreceptive sensilla although common in the mechanoreceptors of Calliphoridae and Sarcophagidae.