The current paradigm of gut evolution assumes that non-bilaterian metazoan lineages either lack a gut (Porifera and Placozoa) or have a sac-like gut (Ctenophora and Cnidaria) and that a through-gut originated within Bilateria [1-8]. An important group for understanding early metazoan evolution is Ctenophora (comb jellies), which diverged very early from the animal stem lineage [9-13]. The perception that ctenophores possess a sac-like blind gut with only one major opening remains a commonly held misconception [4, 5, 7, 14, 15]. Despite descriptions of the ctenophore digestive system dating to Agassiz [16] that identify two openings of the digestive system opposite of the mouth-called "excretory pores" by Chun [17], referred to as an "anus" by Main [18], and coined "anal pores" by Hyman [19]-contradictory reports, particularly prominent in recent literature, posit that waste products are primarily expelled via the mouth [4, 5, 7, 14, 19-23]. Here we demonstrate that ctenophores possess a unidirectional, functionally tripartite through-gut and provide an updated interpretation for the evolution of the metazoan through-gut. Our results resolve lingering questions regarding the functional anatomy of the ctenophore gut and long-standing misconceptions about waste removal in ctenophores. Moreover, our results present an intriguing evolutionary quandary that stands in stark contrast to the current paradigm of gut evolution: either (1) the through-gut has its origins very early in the metazoan stem lineage or (2) the ctenophore lineage has converged on an arrangement of organs functionally similar to the bilaterian through-gut.

The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R traffickinginstead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increaseonmutantreceptorrecycledbacktotheplasmamembrane

Ascidians (Chordata, Tunicata) are an important group for the study of invasive species biology due to rapid generation times, potential for biofouling, and role as filter feeders in an ecosystem. Phallusia nigra is a putative cosmopolitan ascidian that has been described as introduced or invasive in a number of regions in the Indo-Pacific Ocean (India, Japan, and Hawaii) and in the Mediterranean. The taxonomic description of P. nigra includes a striking smooth, black tunic and large size. However, there are at least two similar Phallusia species-P. philippinensis and P. fumigata-which also have dark black tunics and can be difficult to discern from P. nigra. The distribution of P. nigra broadly overlaps with P. philippinensis in the Indo-Pacific and P. fumigata in the Mediterranean. A morphological comparison of P. nigra from Japan, the Caribbean coast of Panama, and Brazil found that Atlantic and Pacific samples were different species and led us to investigate the range of P. nigra using morphological and molecular analyses. We sequenced 18S rDNA and cytochrome oxidase B of individual ascidians from the Red Sea, Greece, Singapore, Japan, Caribbean Panama, Florida, and Brazil. Our results show that identification of the disparate darkly pigmented species has been difficult, and that several reports of P. nigra are likely either P. fumigata or P. philippinensis. Here we include detailed taxonomic descriptions of the distinguishing features of these three species and sequences for molecular barcoding in an effort to have ranges and potential invasions corrected in the ascidian literature.

The cephalopod genus Nautilus is considered a “living fossil” with a contested number of extant and extinct species, and a benthic lifestyle that limits movement of animals between isolated seamounts and landmasses in the Indo‐Pacific. Nautiluses are fished for their shells, most heavily in the Philippines, and these fisheries have little monitoring or regulation. Here, we evaluate the hypothesis that multiple species of Nautilus (e.g., N. belauensis, N. repertus and N. stenomphalus) are in fact one species with a diverse phenotypic and geologic range. Using mitochondrial markers, we show that nautiluses from the Philippines, eastern Australia (Great Barrier Reef), Vanuatu, American Samoa, and Fiji fall into distinct geographical clades. For phylogenetic analysis of species complexes across the range of nautilus, we included sequences of Nautilus pompilius and other Nautilus species from GenBank from localities sampled in this study and others. We found that specimens from Western Australia cluster with samples from the Philippines, suggesting that interbreeding may be occurring between those locations, or that there is limited genetic drift due to large effective population sizes. Intriguingly, our data also show that nautilus identified in other studies as N. belauensis, N. stenomphalus, or N. repertus are likely N. pompilius displaying a diversity of morphological characters, suggesting that there is significant phenotypic plasticity within N. pompilius.

Innate immunity is an ancient physiological response critical for protecting metazoans from invading pathogens. It is the primary pathogen defense mechanism among invertebrates. While innate immunity has been studied extensively in diverse invertebrate taxa, including mollusks, crustaceans, and cnidarians, this system has not been well characterized in ctenophores. The ctenophores comprise an exclusively marine, non-bilaterian lineage that diverged early during metazoan diversification. The phylogenetic position of ctenophore lineage suggests that characterization of the ctenophore innate immune system will reveal important features associated with the early evolution of the metazoan innate immune system. Here, we review current understanding of the ctenophore immune repertoire and identify innate immunity genes recovered from three ctenophore species. We also isolate and characterize Mnemiopsis leidyi cells that display macrophage-like behavior when challenged with bacteria. Our results indicate that ctenophores possess cells capable of phagocytosing microbes and that two distantly related ctenophores, M. leidyi and Hormiphora californiensis, possess many candidate innate immunity proteins.

We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

The formation of extracellular DNA traps (ETosis) is a mechanism of first response by specific immune cells following pathogen encounters. Historically a defining behavior of vertebrate neutrophils, cells capable of ETosis were recently discovered in several invertebrate taxa. Using pathogen and drug stimuli, we report that ctenophores -thought to represent the earliestdiverging animal lineage -possess cell types capable of ETosis, suggesting that this cellular immune response behavior likely evolved early in the metazoan stem lineage.

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood (1,2,3). Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) (1,2) and precocious puberty (4). Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR. Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well. The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies (1,4,5,6,7,8). As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation (4) as well as to alter the rate of degradation of KISS1R (9). Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors (9). In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.