Invertebrate DNA metabarcoding reveals changes in communities across mine site restoration chronosequences

Invertebrate biomonitoring can reveal crucial information about the status of restoration projects; however, it is routinely underused because of the high level of taxonomic expertise and resources required. Invertebrate DNA metabarcoding has been used to characterize invertebrate biodiversity but its application in restoration remains untested. We use DNA metabarcoding, a new approach for restoration assessment, to explore the invertebrate composition from pitfall traps at two mine site restoration chronosequences in southwestern Australia. Invertebrates were profiled using two cytochrome oxidase subunit 1 assays to investigate invertebrate biodiversity. The data revealed differences between invertebrate communities at the two mines and between the different age plots of the chronosequences. Several characteristic taxa were identified for each age within the chronosequence, including springtails within the youngest sites (Order: Collembola) and millipedes within the oldest and reference sites (Order: Julida). This study facilitates development of a molecular “toolkit” for the monitoring of ecological restoration projects. We suggest that a metabarcoding approach shows promise in complementing current monitoring practices that rely on alpha taxonomy.     

Extended-X-ray-absorption-fine-structure investigations of zinc in 5-aminolaevulinate dehydratase.

The zinc co-ordination in 5-aminolaevulinate dehydratase (5-aminolaevulinate hydro-lyase, EC 4.2.1.24) was investigated by recording and interpreting the extended X-ray-absorption fine structure (e.x.a.f.s.) associated with the zinc K-edge. The enzyme has a molecular mass of 280 000 Da and consists of eight subunits of 35 000 Da each; the samples studied contained approx. 1 g-atom of zinc/mol of subunit. Four forms of the enzyme were investigated and details of the zinc environment were elucidated, as follows. In the native enzyme, zinc is considered to be co-ordinated to three sulphur atoms at 0.228(2)nm [2.28(2)A] and a lower-Z atom at 0.192(5)nm [1.92(5)A] (if nitrogen) or 0.189(5)nm [1.89(5)A] (if oxygen). Reaction of the enzyme with the inhibitor 2-bromo-3-(imidazol-5-yl)propionic acid produced significant changes in the e.x.a.f.s., the nature of which are consistent with co-ordination by about three sulphur atoms at 0.222(2)nm [2.22(2)A], a nitrogen atom at 0.193(5)nm [1.93(5)A] and a nitrogen atom from the inhibitor at 0.214(5)nm [2.14(5)A]. Inactivation of the enzyme by air-oxidation of essential thiol groups and binding of the substrate produce slight changes in the e.x.a.f.s. consistent with slight re-arrangement of ligands with additional lighter ligands (nitrogen or oxygen). These results, when combined with previous findings, are taken to indicate that zinc has a structural rather than a direct catalytic role in 5-aminolaevulinate dehydratase.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Reviewers of the Journal of Ethology for volume 27

Journal of Ethology -     

Phylogeny and taxonomy of the Prenolepis genus‐group of ants (Hymenoptera: Formicidae)

Abstract. We investigated the phylogeny and taxonomy of the Prenolepis genus‐group, a clade of ants we define within the subfamily Formicinae comprising the genera Euprenolepis, Nylanderia, gen. rev. , Paraparatrechina, gen. rev. & stat. nov. , Paratrechina, Prenolepis and Pseudolasius. We inferred a phylogeny of the Prenolepis genus‐group using DNA sequence data from five genes (CAD, EF1αF1, EF1αF2, wingless and COI) sampled from 50 taxa. Based on the results of this phylogeny the taxonomy of the Prenolepis genus‐group was re‐examined. Paratrechina (broad sense) species segregated into three distinct, robust clades. Paratrechina longicornis represents a distinct lineage, a result consistent with morphological evidence; because this is the type species for the genus, Paratrechina is redefined as a monotypic genus. Two formerly synonymized subgenera, Nylanderia and Paraparatrechina, are raised to generic status in order to provide names for the other two clades. The majority of taxa formerly placed in Paratrechina, 133 species and subspecies, are transferred to Nylanderia, and 28 species and subspecies are transferred to Paraparatrechina. In addition, two species are transferred from Pseudolasius to Paraparatrechina and one species of Pseudolasius is transferred to Nylanderia. A morphological diagnosis for the worker caste of all six genera is provided, with a discussion of the morphological characters used to define each genus. Two genera, Prenolepis and Pseudolasius, were not recovered as monophyletic by the molecular data, and the implications of this result are discussed. A worker‐based key to the genera of the Prenolepis genus‐group is provided.     

Divergence estimates and early evolutionary history of Figitidae (Hymenoptera: Cynipoidea)

We examine the phylogenetic relationships of Figitidae and discuss host use within this group in light of our own and previously published divergence time data. Our results suggest Figitidae, as currently defined, is not monophyletic. Furthermore, Mikeiinae and Pycnostigminae are sister‐groups, nested adjacent to Thrasorinae, Plectocynipinae and Euceroptrinae. The recovery of Pycnostigminae as sister‐group to Mikeiinae suggests two major patterns of evolution: (i) early Figitidae lineages demonstrate a Gondawanan origin (Plectocynipinae: Neotropical; Mikeiinae and Thrasorinae: Australia; Pycnostigminae: Africa); and (ii) based on host records for Mikeiinae, Thrasorinae and Plectocynipinae, Pycnostigminae are predicted to be parasitic on gall‐inducing Hymenoptera. The phylogenetic position of Parnips (Parnipinae) was unstable, and various analyses were conducted to determine the impact of this uncertainty on both the recovery of other clades and inferred divergence times; when Parnips was excluded from the total evidence analysis, Cynipidae was found to be sister‐group to [Euceroptrinae + (Plectocynipinae (Thrasorinae + (Mikeiinae + Pycnostigminae)))], with low support. Divergence dating analyses using BEAST indicate the stem‐group node of Figitidae to be c. 126 Ma; the dipteran parasitoids (Eucoilinae and Figitinae), were estimated to have a median age of 80 and 88 Ma, respectively; the neuropteran parasitoids (Anacharitinae), were estimated to have a median age of 97 Ma; sternorrhynchan hyperparasitoids (Charipinae), were estimated to have a median age of 110 Ma; the Hymenoptera‐parasitic subfamilies (Euceroptinae, Plectocynipinae, Trasorinae, Mikeiinae, Pycnostigminae, and Parnipinae), ranged in median ages from 48 to 108 Ma. Rapid radiation of Eucoilinae subclades appears chronologically synchronized with the origin of their hosts, Schizophora (Diptera). Overall, the exclusion of Parnips from the BEAST analysis did not result in significant changes to divergence estimates. Finally, though sparsely represented in the analysis, our data suggest Cynipidae have a median age of 54 Ma, which is somewhat older than the age of Quercus spp (30–50 Ma), their most common host.     

Sperm mobility: deduction of a model explaining phenotypic variation in roosters (Gallus domesticus)

In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca2+ content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca2+ -specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca2+ chelator. Thus, a relationship was established between a dynamic intracellular Ca2+ pool and sperm motility. Sperm N-methy-D-aspartic acid (NMDA) receptors were inferred by the action of D-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca2+ pump and thereby promotes Ca2+ efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. In summary, we propose that 1) glutamate enables Ca2+ uptake into sperm before ejaculation, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.