Uncovering species boundaries in the Neotropical ant complex Ectatomma ruidum (Ectatomminae) under the presence of nuclear mitochondrial paralogues

Nuclear mitochondrial (mt) paralogues (numts) are non‐functional fragments of mtDNA integrated into the nuclear genome that can overestimate the number of species in analyses based on mtDNA sequences. As numts have relatively slow mutation rates, they can pass undetected by conventional procedures such as inspecting for internal stop codons, indels or apparent polymorphism in chromatograms. Species boundaries based on mtDNA markers therefore require a thorough assessment of numts, especially in insects, where this phenomenon appears to be relatively frequent. Ectatomma ruidum is a widely distributed Neotropical ant species that is distributed from northern Mexico to northern Brazil. Previous behavioural and molecular evidence suggests that this species actually represents a composite taxon. Here we assessed the species boundaries in E. ruidum based on two mt (COI, cyt b) and one nuclear (H3) marker, as well as on external morphology. Ancient and recent mt paralogues were detected in several specimens, although pre‐PCR dilution of DNA template helped to recover most of the mt orthologues. Based on the congruence found between our species delineation obtained from the mt genealogies and the discriminated morphospecies, we propose that E. ruidum is actually composed of at least three species. Two of these species have a wide geographical distribution in the Neotropics, whereas the remaining one was restricted to localities situated near the Pacific coast in south‐east Mexico. We also found extensive intra‐ and interspecific variation in the barcoding locus. Moreover, the nuclear evidence suggests the existence of hybrids between two of these species in Oaxaca, south‐east Mexico. This study agrees with previous studies of other closely related animal taxa, which have revealed a complex evolutionary history and overlooked species diversity in the latter region.     

The taxonomic challenge posed by the Antarctic echinoids <Emphasis Type="Italic">Abatus bidens</Emphasis> and <Emphasis Type="Italic">Abatus cavernosus</Emphasis> (Schizasteridae,Echinoidea)

Cryptic species have been repeatedly described for two decades among the Antarctic fauna, challenging the classic model of Antarctic species with circumpolar distributions and leading to revisit the richness of the Antarctic fauna. No cryptic species had been so far recorded among Antarctic echinoids, which are, however, relatively well diversified in the Southern Ocean. The R/V Polarstern cruise PS81 (ANT XXIX/3) came across populations of Abatus bidens, a schizasterid so far known by few specimens that were found living in sympatry with the species Abatus cavernosus. The species A. cavernosus is reported to have a circum-Antarctic distribution, while A. bidens is only recorded with certainty in South Georgia and at the northern tip of the Antarctic Peninsula. Based on genetic and morphological analyses, our results clearly show that A. bidens and A. cavernosus are two distinct species. The analyzed specimens of A. bidens group together in two haplogroups separated from one another by 2.7 % of nucleotide differences. They are located in the Weddell Sea and in the Bransfield Strait. Specimens of A. cavernosus form one single haplogroup separated from haplogroups of A. bidens by 5 and 3.5 % of nucleotide differences, respectively. The species was collected in the Drake Passage and in the Bransfield Strait. Morphological analyses differentiate A. bidens from A. cavernosus. In contrast, the two genetic groups of A. bidens cannot be differentiated from one another based on morphology alone, suggesting that they may represent a case of cryptic species, common in many Antarctic taxa, but not yet reported in Antarctic echinoids. This needs to be confirmed by complementary analyses of independent genetic markers.     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae)

Potassium inward rectifier K_IR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the K_IR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of K_IR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. K_IR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian K_IR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of K_IR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed K_IR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle K_IR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 K_IR2.1 sequences showed that all disease causing K_IR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake K_IR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC₅₀ ∼6 μM) inward rectifier currents.     

Neuroendocrinology and neurobiology of sebaceous glands

The nervous system communicates with peripheral tissues through nerve fibres and the systemic release of hypothalamic and pituitary neurohormones. Communication between the nervous system and the largest human organ, skin, has traditionally received little attention. In particular, the neuro‐regulation of sebaceous glands (SGs), a major skin appendage, is rarely considered. Yet, it is clear that the SG is under stringent pituitary control, and forms a fascinating, clinically relevant peripheral target organ in which to study the neuroendocrine and neural regulation of epithelia. Sebum, the major secretory product of the SG, is composed of a complex mixture of lipids resulting from the holocrine secretion of specialised epithelial cells (sebocytes). It is indicative of a role of the neuroendocrine system in SG function that excess circulating levels of growth hormone, thyroxine or prolactin result in increased sebum production (seborrhoea). Conversely, growth hormone deficiency, hypothyroidism, and adrenal insufficiency result in reduced sebum production and dry skin. Furthermore, the androgen sensitivity of SGs appears to be under neuroendocrine control, as hypophysectomy (removal of the pituitary) renders SGs largely insensitive to stimulation by testosterone, which is crucial for maintaining SG homeostasis. However, several neurohormones, such as adrenocorticotropic hormone and α‐melanocyte‐stimulating hormone, can stimulate sebum production independently of either the testes or the adrenal glands, further underscoring the importance of neuroendocrine control in SG biology. Moreover, sebocytes synthesise several neurohormones and express their receptors, suggestive of the presence of neuro‐autocrine mechanisms of sebocyte modulation. Aside from the neuroendocrine system, it is conceivable that secretion of neuropeptides and neurotransmitters from cutaneous nerve endings may also act on sebocytes or their progenitors, given that the skin is richly innervated. However, to date, the neural controls of SG development and function remain poorly investigated and incompletely understood. Botulinum toxin‐mediated or facial paresis‐associated reduction of human sebum secretion suggests that cutaneous nerve‐derived substances modulate lipid and inflammatory cytokine synthesis by sebocytes, possibly implicating the nervous system in acne pathogenesis. Additionally, evidence suggests that cutaneous denervation in mice alters the expression of key regulators of SG homeostasis. In this review, we examine the current evidence regarding neuroendocrine and neurobiological regulation of human SG function in physiology and pathology. We further call attention to this line of research as an instructive model for probing and therapeutically manipulating the mechanistic links between the nervous system and mammalian skin.