Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA ß subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

Unveiling the Fecal Microbiota in Two Captive Mexican Wolf (Canis lupus baileyi) Populations Receiving Different Type of Diets.

The Mexican wolf (Canis lupus baileyi) was once distributed in southern United States and northern Mexico. It is an endangered subspecies detached from the gray wolf, and likely exemplifies one of the original migration waves of C. lupus into the new world. This is a canine whose individuals survive in specialized facilities, zoos, and museums as part of captive-breeding programs. In order to contribute to the improvement of the management of this species and favor its long-term conservation in Mexico, we aimed to evaluate the diversity and abundance of the fecal bacterial microbiota in two populations exposed to different types of diet: (1) Michilia (23° N, 104° W); kibble daily and raw meat sporadically, and (2) Ocotal (19° N, 99° W); raw meat daily and live animals periodically. Next generation sequencing (V3-V4 16S rRNA gene) by Illumina was implemented. The operational taxonomic units (OTUs) in Michilia resulted in 9 phyla, 19 classes, 34 orders, 61 families, 204 genera, and 316 species, while in Ocotal there were 12 phyla, 24 classes, 37 orders, 69 families, 232 genera, and 379 species. Higher estimated Chao1 richness, Shannon diversity, and core microbiota were observed in Ocotal. Differences (p < 0.05) between populations occurred according to the Bray-Curtis beta diversity index. In the Michilia, dominance of bacteria that degrade carbohydrates (Firmicutes, Lachnospiraceae, Blautia, Clostrodium, Eisenbergiella, Romboutsia, and Ruminococcus) was observed; they are abundant in kibble diets. In contrast, the Ocotal microbiota was dominated by protein-degrading bacteria (Fusobacteria, Fusobacteriaceae, and Fusobacteria), indicating a possible positive relation with a raw meat diet. The information generated in this study is fundamental to support the implementation of better management plans in the two populations considered here, as well as in different facilities of southern United States and Mexico, where this subspecies is kept in captivity for conservation purposes.

Identification of High Molecular Variation Loci in Complete Chloroplast Genomes of Mammillaria (Cactaceae, Caryophyllales).

In plants, partial DNA sequences of chloroplasts have been widely used in evolutionary studies. However, the Cactaceae family (1500-1800 species) lacks molecular markers that allow a phylogenetic resolution between species and genera. In order to identify sequences with high variation levels, we compared previously reported complete chloroplast genomes of seven species of Mammillaria. We identified repeated sequences (RSs) and two types of DNA variation: short sequence repeats (SSRs) and divergent homologous loci. The species with the highest number of RSs was M. solisioides (256), whereas M. pectinifera contained the highest amount of SSRs (84). In contrast, M. zephyranthoides contained the lowest number (35) of both RSs and SSRs. In addition, five of the SSRs were found in the seven species, but only three of them showed variation. A total of 180 homologous loci were identified among the seven species. Out of these, 20 loci showed a molecular variation of 5% to 31%, and 12 had a length within the range of 150 to 1000 bp. We conclude that the high levels of variation at the reported loci represent valuable knowledge that may help to resolve phylogenetic relationships and that may potentially be convenient as molecular markers for population genetics and phylogeographic studies.

General Microbiota of the Soft Tick Ornithodoros turicata Parasitizing the Bolson Tortoise (Gopherus flavomarginatus) in the Mapimi Biosphere Reserve, Mexico.

The general bacterial microbiota of the soft tick Ornithodoros turicata found on Bolson tortoises (Gopherus flavomarginatus) were analyzed using next generation sequencing. The main aims of the study were to establish the relative abundance of bacterial taxa in the tick, and to document the presence of potentially pathogenic species for this tortoise, other animals, and humans. The study was carried-out in the Mapimi Biosphere Reserve in the northern-arid part of Mexico. Bolson tortoises (n = 45) were inspected for the presence of soft ticks, from which 11 tortoises (24.4%) had ticks in low loads (1-3 ticks per individual). Tick pools (five adult ticks each) were analyzed through 16S rRNA V3-V4 region amplification in a MiSeq Illumina, using EzBioCloud as a taxonomical reference. The operational taxonomic units (OTUs) revealed 28 phyla, 84 classes, 165 orders, 342 families, 1013 genera, and 1326 species. The high number of taxa registered for O. turicata may be the result of the variety of hosts that this tick parasitizes as they live inside G. flavomarginatus burrows. While the most abundant phyla were Proteobacteria, Actinobacteria, and Firmicutes, the most abundant species were two endosymbionts of ticks (Midichloria-like and Coxiella-like). Two bacteria documented as pathogenic to Gopherus spp. were registered (Mycoplasma spp. and Pasteurella testudinis). The bovine and ovine tick-borne pathogens A. marginale and A. ovis, respectively, were recorded, as well as the zoonotic bacteria A. phagocytophilum,Coxiella burnetii, and Neoehrlichia sp. Tortoises parasitized with O. turicata did not show evident signs of disease, which could indicate a possible ecological role as a reservoir that has yet to be demonstrated. In fact, the defense mechanisms of this tortoise against the microorganisms transmitted by ticks during their feeding process are still unknown. Future studies on soft ticks should expand our knowledge about what components of the microbiota are notable across multiple host-microbe dynamics. Likewise, studies are required to better understand the host competence of this tortoise, considered the largest terrestrial reptile in North America distributed throughout the Chihuahuan Desert since the late Pleistocene.

De Novo Assembly Discovered Novel Structures in Genome of Plastids and Revealed Divergent Inverted Repeats in Mammillaria (Cactaceae, Caryophyllales).

The complete sequence of chloroplast genome (cpDNA) has been documented for single large columnar species of Cactaceae, lacking inverted repeats (IRs). We sequenced cpDNA for seven species of the short-globose cacti of Mammillaria and de novo assembly revealed three novel structures in land plants. These structures have a large single copy (LSC) that is 2.5 to 10 times larger than the small single copy (SSC), and two IRs that contain strong differences in length and gene composition. Structure 1 is distinguished by short IRs of <1 kb composed by rpl23-trnI-CAU-ycf2; with a total length of 110,189 bp and 113 genes. In structure 2, each IR is approximately 7.2 kb and is composed of 11 genes and one Intergenic Spacer-(psbK-trnQ)-trnQ-UUG-rps16-trnK-UUU-matK-trnK-UUU-psbA-trnH-GUG-rpl2-rpl23-trnI-CAU-ycf2; with a total size of 116,175 bp and 120 genes. Structure 3 has divergent IRs of approximately 14.1 kb, where IRA is composed of 20 genes: psbA-trnH-GUG-rpl23-trnI-CAU-ycf2-ndhB-rps7-rps12-trnV-GAC-rrn16-ycf68-trnI-GAU-trnA-AGC-rrn23-rrn4.5-rrn5-trnR-ACG-trnN-GUU-ndhF-rpl32; and IRB is identical to the IRA, but lacks rpl23. This structure has 131 genes and, by pseudogenization, it is shown to have the shortest cpDNA, of just 107,343 bp. Our findings show that Mammillaria bears an unusual structural diversity of cpDNA, which supports the elucidation of the evolutionary processes involved in cacti lineages.

Staurosporine rapidly commits 3T3-F442A cells to the formation of adipocytes by activation of GSK-3beta and mobilization of calcium.

Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3beta was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl(2), which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.