Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

The unholy trinity: taxonomy, species delimitation and DNA barcoding

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this 'DNA barcoding' initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the 'DNA barcoding' initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the 'DNA barcoding' initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings--Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of 'DNA barcoding'.     

Immunocompetence increases with larval body size in a phytophagous moth

Despite the obvious benefit of an immune system, its efficacy against pathogens and parasites may show great variation among individuals, populations and species. Understanding the causes of this variation is becoming a central theme in ecology. Many biotic and abiotic factors are known to influence immunocompetence (temperature, age, etc.). However, for a given age, size among individuals varies, probably as a result of accumulated resources. Thus, these variable resources could be allocated to immune defence and, consequently, body size may explain part of the variation in immune responsiveness. However, the influence of body size on immune defence is often overlooked. The present study investigates variations in haemocyte count and phenoloxidase activity in larvae of the phytophagous vine moth Eupoecilia ambiguella Hübner of the same age, although differing in body size. The measurements of immune function are made both when the insects are immunologically naïve and 24 h after a bacterial immune challenge. The base levels of these immune parameters do not covary with body size in naïve larvae. After the bacterial immune challenge, more haemocytes and phenoloxidase enzyme are mobilized, and the mobilization of these immune effectors is correlated positively with individual body size. Thus, larger larvae exhibit higher immunocompetence than smaller ones, suggesting that smaller larvae might be more vulnerable to infection. These results suggest that body size is probably an underestimated variable, which nevertheless modulates the insect immune system and should thus be considered as a covariate in insect immune system measurement. It is recommended therefore, that body size should be taken into account in ecological immunity studies with insects. © 2013 The Royal Entomological Society     

Genome‐wide search for leech antiplatelet proteins in the non‐blood‐feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

The genome of the non‐blood‐feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen‐stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross‐validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e‐values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal‐peptide toward the N‐terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeria‐derived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP‐tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non‐blood‐feeding leech has a hematophagous ancestry.     

Bacterial symbiont and salivary peptide evolution in the context of leech phylogeny

The evolutionary history of leeches is employed as a general framework for understanding more than merely the systematics of this charismatic group of annelid worms, and serves as a basis for understanding blood-feeding related correlates ranging from the specifics of gut-associated bacterial symbionts to salivary anticoagulant peptides. A variety of medicinal leech families were examined for intraluminal crop bacterial symbionts. Species of Aeromonas and Bacteroidetes were characterized with DNA gyrase B and 16S rDNA. Bacteroidetes isolates were found to be much more phylogenetically diverse and suggested stronger evidence of phylogenetic correlation than the gammaproteobacteria. Patterns that look like co-speciation with limited taxon sampling do not in the full context of phylogeny. Bioactive compounds that are expressed as gene products, like those in leech salivary glands, have 'passed the test' of evolutionary selection. We produced and bioinformatically mined salivary gland EST libraries across medicinal leech lineages to experimentally and statistically evaluate whether evolutionary selection on peptides can identify structure-function activities of known therapeutically relevant bioactive compounds like antithrombin, hirudin and antistasin. The combined information content of a well corroborated leech phylogeny and broad taxonomic coverage of expressed proteins leads to a rich understanding of evolution and function in leech history.     

Leech mycetome endosymbionts are a new lineage of alphaproteobacteria related to the Rhizobiaceae

Mycetomal organs attached to the esophagus of hematophagous leeches which are known to harbor endosymbiotic bacteria were removed from three species in the leech family Glossiphoniidae. Anatomical observations indicated that placobdellid mycetomes are paired and caecate, inserting into the esophagus posterior to the proboscis. Light and electron microscopy demonstrated that there is a single layer of mycetome epithelial cells harboring gram-negative rods and that these epithelial cells are ultrastructurally distinct from neighboring esophageal epithelial cells. Fluorescent in situ hybridization with eubacterial and alphaproteobacterial probes localized the bacteria solely to the mycetomes both in adult and in unfed juvenile leeches whereas a gammaproteobacterial probe did not yield a bound fluorescencent signal. DNA was isolated from these tissues and subjected to PCR amplification using bacteria-specific primers for 16S and 23S rDNA. Results from sequencing the amplification products and phylogenetic analysis with other Alphaproteobacteria revealed that the bacteria resident in these organs comprise a new genus of Alphaproteobacteria, Reichenowia n. gen., closely related to the nitrogen-fixing, nodule-forming Rhizobiaceae. The three bacterial strains, though different from each other were each other's closest relatives, suggesting a history of close coevolution with their leech hosts.