The Xenopus Lefty (xlefty) Prodomain Negatively Regulates Xlefty Activity And Is Necessary For Proper Xlefty Secretion

In vertebrates, the TGF #914; superfamily of secreted peptides are stringently regulated since they are responsible for multiple cell processes and behaviors that give rise to the proper patterning of embryonic tissues. Examples of members of the TGF #914; superfamily are Nodal and Lefty. Dysregulation of these proteins can lead to many diseases and developmental syndromes in humans. Lefty functions by antagonizing Nodal an essential organizer signal that patterns dorsal mesoderm and the embryonic axes. Loss of Lefty expression results in excess Nodal signaling which has been shown to cause several perturbations including metastatic cancer. Although studies have clearly shown that Lefty antagonizes Nodal signaling, there is a lack of understanding of the regulatory mechanism of Lefty itself. Previous studies have shown that proteolytic cleavage of the prodomain (PD) from mature Lefty (Mat-Lefty) is necessary for Lefty activity. Here we present studies carried out using the Xenopus laevis embryo that demonstrate that PD over-expression causes exogastrulation, a phenotype also resulting from a loss of Xenopus Lefty (Xlefty) function. Furthermore, when the PD and Xlefty are co-expressed, the effects of Xlefty over-expression are rescued. Our biochemical studies also showed that the PD interacts with Mat-Xlefty but a PD mutated (PDmut) molecule does not. The sites mutated in the PDmut are evolutionarily-conserved residues that mediate the interaction between the prodomain and the mature ligand in other TGF #914; proteins making our results consistent with previously observed behavior of TGF #914; prodomains but new to Lefty. Our study also shows that the PDmut is unable to rescue the effects of a Xlefty over-expression phenotype unlike the un-mutated PD. In addition we show here that mutations of the PD affect the secretion of these Xlefty-mutated derivatives. Taken together, these results suggest that the PD negatively regulates Xlefty activity by interacting with Mat-Xlefty and cleavage of the PD releases regulation allowing proper secretion and function. This new insight into the regulatory role of the Xlefty PD provides potential therapeutic value to address dysfunctional Nodal signaling. Furthermore, our secretion studies of the PD and Xlefty revealed that Xlefty is secreted but the PD is not. The Xlefty-mutated derivatives, including a PD Less, Xlefty, Xlefty cleavage mutants and the PDmut, resulted in no secreted products. These secretion results open a Pandora\u27s box and further studies are warranted to elucidate the mechanism of PD regulation of Xlefty and whether it occurs in an intracellular or extracellular context

The Digestive Composition And Physiology Of Water Mites

Water mites are a diverse group of arachnids that inhabit aquatic habitats and have been studied in the past for their biodiversity, unique lifecycle, bioindicator species use and for their impact as parasites on insects of human pathological significance such as the mosquito. Water mites are critical in their environment as possible apex predators however, their life cycle and morphological complexity has made taxonomy and description of water mites difficult. Although water mite species richness is estimated at over 6000 species described to date, descriptions of extant North American water mite species are estimated to be only 50% of the existing species. Water mite digestive physiology is also virtually unknown even though water mites are known to be efficient predators and parasites of dipteran pest such as chironomids. With the use of microscopic, biochemical and molecular genetic technologies this work aims to improve water mite knowledge in both digestive physiology and diversity of North American water mite populations. Water mites from Blue Heron Lagoon at Belle Isle, Detroit were collected and processed for assessment of both species diversity and gut molecular contents. Using genetic and morphological methods, water mites and their prey were identified. Water mites in different genera are observed to be generalists as we did not see any water mite genera feeding exclusively on only one type of prey. Gut molecular contents were assessed using primers targeting the COI gene that has been used for molecular barcoding. Dipteran “specific” primers (mLep) were used to elucidate what prey were being consumed. These sequences were obtained by Sanger Sequencing and by Next Generation Sequencing. These sequences were compared to a large database of chironomid species that were generated from the same biogeographic region. The conclusion is that Lebertia water mites are generalist and opportunistic predators who consume a large diversity of chironomids, including various species of Cricotopus, Chironumus, and Paratanytarsus. A novel finding of this study is that for some mites the nearest matches to the DNA sequences of gut-associated DNA were sequences from oligochaetes, albeit in most cases the percentage identity to any GenBank sequence of oligochaetes was in the range of 80 – 90%. Water mite diversity in the Blue Heron Lagoon is also reported here with a new record for Lebertia quinquemaculosa Marshall and the possibility of new Lebertia species descriptions from Blue Heron Lagoon. Scanning electron microscopy was used to verify morphological characters and to aid in describing the new Lebertia, which we are proposing to name L. davidcooki. L. quinquemaculosa and L. davidcooki were also studied to characterize the structures that facilitate digestive passage of ingested food. Fluorescein, a fluorescent metabolic product from fluorescein diacetate (FDA), was used to visualize the gut structures of the water mites by feeding them fluorescent chironomid larval prey that had been exposed to FDA. Water mites were also examined using confocal fluorescent microscopy to describe gut structures. Transmission electron (TE) microscopy was used to visualize the internal microstructures of L. quinquemaculosa and L. davidcooki for the first time. Digestive structures such as the excretory organ and mid-gut were observed from water mite dissections and further analyzed by toluidine blue staining of mite sagittal sections. This work represents the first ever digestive physiology experiments on Lebertia. The results of this work have also contributed new North American DNA barcode genetic representation of water mites, chironomids, and a morphological and molecular description of a Great Lakes invasive copepod Eurytemora carolleae to the public databases. The importance and contribution of water mites to aquatic ecosystems validates this study which begins to fill in knowledge gaps on water mite physiology and biodiversity

Mite Diet Sequences Obtained by High Throughput Sequencing of Gut Contents of Freshly Collected Water Mites

Cytochrome oxidase subunit I (COI) barcode sequences in this file were obtained from gut DNA extracted from 54 freshly collected water mites, comprising 21 Lebertia quinquemaculosa , 30 Lebertia davidcooki , 1 Limnesia , and 2 Arrenurus specimens. Methods and other details about these sequences are described in a paper by the same authors in a submitted publication (2021: URL to be given here when published). Data on collection locations, primers (mLep and LCOI), amino acid translations, etc. are included in corresponding sequences uploaded to GenBank. The right column below contains additional notes on naming the taxa of the sequences that were not included in the GenBank annotation. These notes include the highest percentage identity to a previous sequence in GenBank as determined by BLASTN in June 2018. The FASTA file name given here includes the Accession ID, followed by the best match taxon (at an appropriate taxonomic level, dependent on the percent identity, as described in the notes in the right-hand column), the phrase water mite diet isolate , a specific RamLab sequence identifier of the sequence, and then the COI gene description. Accession IDs of sequences uploaded to GenBank begin with MW; other sequences begin with RL and a RamLab sequence identifier. The RamLab sequence identifier in the FASTA name includes information as follows: RamLab ID number-location and date of collection with three location letters (e.g., BHL stands for Blue Heron Lagoon) and the date usually in a 6-character format of MMDDYY-information on the location of sequence on the Illunina sequencing plate-and a 4- to 6-character identifier of the mite species (Lq=L. quinquemaculosa ; Ldc=L. davidcooki ; Lim=Limnesia ; Arr=Arrenurus ) and the animal number in that series of experiments (2 digits)

The biodiversity of freshwater Crustaceans revealed by taxonomy and mitochondrial DNA barcodes

Cytochrome oxidase subunit I (COI) barcode sequences in this file were obtained from specimens collected by plankton net in western Lake Erie in 2012 & 2013, along with later specimens collected at various locations and times, including some collected in Belize in 2015. Methods and other details about these sequences are described in a paper by the same authors in a submitted publication (2021: URL to be given here when published). The right columns below contain additional notes on lengths of sequences, GenBank accession ID (when obtained), and annotation as to whether the sequence represents a new barcode for its genus or species taxon. According to our experience, a DNA identity of \u3e96.5% with previous GenBank barcodes is a reliable range for determining a species level barcode for that morpho species; a DNA identity of 90.5% to 96.5% with previous barcodes is sufficient to identify genus. DNA identities within these ranges are considered to be barcode confirmations. Conversely, DNA identities outside of these ranges are considered to be new barcodes for that species or genus, respectively. Contradictions with previous GenBank sequences are discussed in the manuscript. The submitted manuscript includes the highest percentage identity to a previous sequence in GenBank as determined by BLASTN in June2021. The FASTA file name given here begins with a Ram Lab ID number-location and date of collection with format varying somewhat between various collections/collectors but generally including several (usually three) location letters (e.g., BHL stands for Blue Heron Lagoon) and the date usually in a 6-character format of MMDDYY, and optionally a sample number for that date either preceding the location letters or following the date. Collection location abbreviations include the following: All sequences starting with PM, Toledo Harbor in western Lake Erie; LMUSK, Lake Muskoday, Belle Isle, Detroit; SCL, Saint Clair River; BHL, Blue Heron Lagoon, Belle Isle; LE, LakeErie; LSC, Lake St.Clair; MMLE; Metzgers Marsh, LakeErie; MM, Metzgers Marsh; LP, Leonard Preserve, Manchester, Michigan; HR, Huron River Drive, Ypsilanti, Michigan; LCL, Little Cedar Lake, Orion, MI; HLE, Harbor Lake Erie; LHLE, Lorain Harbor Lake Erie; BZEB1P, Cenote in Shipstern Reserve, Corozal, Belize, Central America