The microbial community inhabiting the root-soil contact interface, rhizobiomes, represent a critical link between plant, ecosystems, and geomorphic processes. In landslides where fresh silicate rocks are exposed, rhizobiomes ...

Plant-pollinator interactions provide a crucial ecosystem service for which many depend upon. These interactions can be threatened by many different anthropogenic and natural disturbances. While most studies on plant-pollinator ...

Palm crops worldwide are experiencing adverse effects in crop yields and survivorship from exposure to phytopathogens, including phytoplasmas (Ntushello et al., 2013). Phytoplasmas are plant-pathogenic bacteria that are associated with over 1000 plant diseases. (Duan et al., 2013; Ntushello et al., 2013). The 16SrIV group of phytoplasmas is associated with lethal yellowing diseases that affect at least 30 species of palms worldwide. Available information for Puerto Rico indicates the existence of 16SrIV phytoplasma group in some palm species, including the Royal palm Roystonea borinquena O. F. Cook, the Fishtail palm Caryota mitis Lour, the Carpentaria palm Carpentaria acuminata (H. Wendl. & Drude) Becc., and the Coconut palm Cocos nucifera L. The visual symptoms of the diseases include leaf discoloration (e.g., yellowing, bronzing), flower malformation, premature nuts drop, and death of the plant. This work aimed to identify and characterize palm species, phytoplasma, and associated Auchenorrhyncha occurrence in Puerto Rico. For this study, we collected samples from over 1,027 palms between July 2015 to May 2017 around the island of Puerto Rico. The samples comprise 40 palm species representing species native to the island and introduced ornamental ones. Sixty percent of the collected palms presented symptoms associated with phytoplasma diseases, mainly with premature leave yellowing and decay. We used the coding ribulose-bisphosphate carboxylase gene (rbcL) and maturase K gene (matK) to determine their power to distinguish between species in the palm family Arecaceae in Puerto Rico. Our study found that rbcL and matK place the species with 100% accuracy at the family and subfamily levels. However, additional markers may be needed to identify species within the Arecaceae. To amplify the phytoplasma DNA, we carried out direct and nested PCRs to a sub-sample of 192 palms from the initial sample (n=1,027). Forty-three percent of the samples tested showed amplification using specific primers in the nested PCR. Of these, only 1.5% of the samples were positive for phytoplasma 16SrIV-D associated with lethal bronzing disease of palms. The three confirmed sequences of phytoplasma were from positive samples: two from Dypsis lutescens and one Aiphanes minima, both collected at the UPR Botanical Garden. Phloem feeder insects are the vectors of phytoplasmas. The suborder Auchenorrhyncha contains phloem-feeder insects that are listed as vectors of phytoplasmas. We surveyed Auchenorrhyncha insects associated with palms in three localities of different altitudes between August 2016 to October 2018: the UPR Botanical Garden in San Juan, Punta Santiago Natural Reserve in Humacao, and El Verde Field Station (El Yunque) in Rio Grande. Thirty-three species of planthoppers and leafhoppers were sweep-collected from palms and associated weeds. Genetic analyses evaluated the mitochondrial gene region for cytochrome c oxidase subunit I (COI) to support morphological identification. A subsample of (n=120) insects was screened for hytoplasma by direct and nested PCR assays. We found seven samples that tested positive for phytoplasma. Three samples belonged to Hortensia similis (Walker) and four to Xyphon reticulatum (Signoret), all collected from C. nucifera in Humacao. To our knowledge, this is the first report of these Cicadellidae species associated with phytoplasma in Puerto Rico. The early detection and characterization of phytoplasma-related diseases and the identification of vector species can help reduce the spread of this organism and avoid the negative impact on crop yields in palm species of agricultural importance....

<strong>Chapter I abstract:</strong> The Caribbean region is expected to face an increase in frequency and intensity of climatic disturbances, such as hurricanes. Amphibians and reptiles are especially vulnerable to local abiotic regimes and their response to changes are limited by physiological constraints intrinsic to their evolutionary history. In September of 2017, El Yunque National Forest in Puerto Rico experienced extensive forest damage by the effect of hurricane Maria. In this study, we assessed the effect of hurricane Maria on the abundance, age-specific survivorship, detection probability, and expected gains of a well-studied high-elevation population of Eleutherodactylus coqui. We hypothesize that the population structure would change due to the canopy loss and consequent habitat disturbances caused by the storm blowout. To measure abundance estimates, we used a multi-state N-mixture model to analyze the changes in age-specific demographics on&nbsp;E. coqui population. We found that hurricane Maria had a negative impact on local expected gains and abundance estimates of E. coqui that caused a disruption in the typical demographic pattern as age classes transition through the warm-wet and cool-dry seasons in Puerto Rico. However, no drastic declines were observed. These results point to plasticity in the response of E. coqui to severe environmental events. Recording changes in high-elevation E. coqui population structure may offer insight to how other endangered sympatric species would respond to such phenomena. As stronger and more frequent tropical storms are predicted soon, this study provides baseline data that can help predict, compare, and evaluate future changes to biodiversity in critical areas. <strong>Chapter II abstract:</strong> Amphibians are declining drastically due, in part, to a pathogenic fungal parasite named Batrachochytrium dendrobatidis (Bd). In addition, extreme climatic events, like hurricanes, are increasing in frequency and intensity and this points to the need to understand the way they influence host-pathogen dynamics. Studies in the Americas have advanced our understanding on how environmental factors affect virulence and dispersion of Bd in wild populations. However, in some amphibian communities where Bd is enzootic, the dynamics of the disease may be driven by pathogen overdispersion. Under these circumstances, it is important to understand how Bd infection intensity and prevalence influences host survival and reproduction because this may explain the underlying mechanisms of persistence or extinction. In this study, we evaluate the effect of severe natural disturbances on the dynamics of Bd infections across seasons in a population of highland Eleutherodactylus coqui in Puerto Rico. We found significant differences in Bd infection intensities in the population of E. coqui after the hurricane, suggesting a hurricane effect on the vulnerability of hosts. In addition, a zero-inflated regression model distinguished the effects of the hurricane, seasons and their interaction on the population-level prevalence of Bd. Our work highlights the value of long-term field studies because they provide time-sensitive data necessary to evaluate population responses to global change. <strong>Chapter III abstract:</strong> Marking juveniles of terrestrial direct-developing frogs is challenging because of their small size (&lt; 18 mm) and fragility. This difficulty has limited studies on demography or population dynamics where empirical data on the survivorship of juveniles or their recruitment to adulthood are missing. In a controlled laboratory experiment we tested the survivorship of wild caught juvenile Eleutherodactylus coqui Thomas, 1966 to marking with a single colour Visual Internal Elastomer (VIE) in the thigh with and without additional ventral skin-swabbing for disease or microbiome monitoring. Results revealed 100 % survival in all groups, and all juveniles remained unharmed, moved freely, and fed actively during three days after treatments suggesting that this type of manipulation does not cause direct mortality. After 17 months of the experiment, we have recaptured 11 % of the marked juveniles as adults indicating that they can survive to recruitment age. We propose the use of a single VIE colour as a method to mark and follow date-specific cohorts of juvenile direct-developing frogs or young metamorphs until they reach older and larger age classes. This marking method can be used safely together with skin swabbing and provide valuable information for studies on population biology and age-specific response to environmental or disease stressors....

How sociality interacts with other behaviours is a long standing question in insect biology. Simultaneously, in chronobiology, there is an unanswered question concerning how sociality influences patterns of daily activity. ...

Echinoderms comprise a group of animals with the capability of replacing complex internal organs following injury or autotomy. In holothurians or sea cucumbers, cellular processes of intestinal regeneration have been extensively studied, however, the molecular machinery behind this faculty remains to be understood. In this study, a de novo transcriptome was assembled and annotated using RNA-seq data consisting of regenerating and non-regenerating intestinal tissues from the sea cucumber Holothuria glaberrima. Comparisons of differential expression were made using the mesentery as a reference against 24 hours and 3 days regenerating intestine, revealing numerous differentially expressed transcripts. Gene ontology and pathway enrichment analysis showed evidence of increasing transcriptional activity. Further analysis of transcripts associated with transcription factors revealed diverse expression patterns with developmental and cancer-related mechanisms linked with regenerative responses. This study demonstrates the diverse gene expression profile during the early stages of regeneration using the mesentery as a focal point....

The sea cucumber Holothuria glaberrima is a mighty model to study organ regeneration. Our group has described the cellular mechanisms underlying intestine re-growth in H. glaberrima and is exploring the signaling mechanisms involved in this process. One of the limitations to our studies has been the lack of suitable cell culture methodologies required to advance the regeneration studies. I have now established in vitro systems where individual cells or explants can be kept and studied. Cultured cells and explants were analyzed using various techniques that include light, fluorescence, and electron microscopy. Cells isolated by enzymatic dissociation from regenerating guts of H. glaberrima could be maintained in the right conditions for up to two weeks in vitro. Different phenotypes were identified using cellular markers established previously in our lab. However, no mature muscle cells or neurons were found, which is not suitable to perform studies about cell dedifferentiation/redifferentiation. Additionally, isolated cells showed deficient proliferative activity. For that reason, I characterized gut explants as an alternative. Remarkably, the histological characteristics, including the presence of mature muscle cells and the proliferative rate, in cultured explants resembled more accurately the in vivo conditions compared to the dissociated cultures. Thus, I decided to use gut explants to gain new insights about the signaling mechanisms associated with the intestine regeneration in H. glaberrima.<br /> <br /> Initial in vivo studies using small molecules that are putative disruptors of the Wnt pathway supported the involvement of the Wnt pathway on intestine regeneration. These studies showed that iCRT14, a Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a Wnt pathway activator, increased its size. The possible cellular mechanisms by which the signaling disruptors affected the gut rudiment size were further studied in vitro using additional pharmacological agents. Among them, those that inhibited the GSK-3 enzyme (a component of the Wnt pathway) were found to increase muscle cell dedifferentiation. However, these agents also induced a reduction in cell proliferation, suggesting that cell dedifferentiation can be decoupled from cell proliferation during intestinal regeneration.<br /> <br /> Interestingly, the drop of cell proliferation in explants was also caused by small molecules that block the Wnt pathway in other points different to GSK-3. For that reason, I propose that GSK-3 is the mediator of the cellular dedifferentiation response and that it takes place by a signaling pathway that is independent of Wnt. Differently, cellular proliferation appears to be controlled by the canonical Wnt pathway during intestine regeneration. My results open the door for future studies where the signaling pathways involved at each regeneration stage can be determined during intestine regeneration....

Transcription factors (TFs) are regulatory proteins that bind tightly to specific DNA sequences 15-20 base pairs long. Being sequence-specific DNA-binding proteins, TFs bear the key to the cellular state and control how organisms respond to different environmental stresses. OxyR, a LysR-type transcription factor, binds strongly to H₂O₂ and activates a set of genes whose main purpose is to protect bacteria against oxidative stress. The genome of Aliivibrio fischeri bacteria codes for two different OxyR proteins, OxyR1 and OxyR2. However, the collection of target genes of OxyR1 and OxyR2 in A. fischeri remains to be determined. This study aims to clone, overexpress, and purify OxyR1 and OxyR2 TFs to determine their DNA binding functionality throughout the assessment of their intrinsic DNA-binding preferences. Purified OxyR1 and OxyR2 TFs were used to determine their DNA binding specificity using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq)....

The hyperpolarization-activated cation current (I_h) is a determinant of intrinsic excitability in dopaminergic neurons (DA) of the ventral tegmental area (VTA). I_h is a slowly activating cation inward current triggered by hyperpolarization. When elicited I_h depolarizes the membrane to threshold for the generation of action potentials. Our laboratory has previously demonstrated that cocaine sensitization, a chronic cocaine administration model, significantly reduces I_h amplitude in VTA DA neurons (Arencibia-Albite et al., 2012). Despite this current reduction, the spontaneous firing of VTA DA cells remained similar to control animals. The role of I_h in controlling VTA DA excitability is poorly understood. Our hypothesis was that I_h reduction could play a role as a homeostatic controller which compensates for cocaine-induced change in excitability. Our main goal was to understand how I_h contributes to VTA DA neuronal excitability. In our first aim, we tried to elucidate if I_h reduction can alter basal VTA DA neuronal excitability. We blocked I_h and evaluated firing properties, such as rebound spiking, using whole-cell patch-clamp electrophysiology from na&iuml;ve rats. Rebound spiking is defined as the production of one or more action potentials in response to the cessation of a prolonged hyperpolarizing current step. We also determined the effect of I_h blockade on in vitro spontaneous excitability using cell-attached configuration from na&iuml;ve rats. We hypothesized that I_h blockade will reduce rebound and spontaneous firing of VTA DA cells. It was found that I_h blockade can decrease evoked and spontaneous firing activity of VTA DA neurons. In addition, we evaluated the effect of I_h blockade on spontaneous firing patterns using single-unit extracellular recordings from na&iuml;ve rats. We found that I_h blockade can decrease evoked and spontaneous firing activity, while increasing the interspike interval of VTA DA cells.<br /> <br /> To investigate how this current modulates cocaine-dependent excitability, we postulated that I_h reduction serves as a homeostatic regulator (or controller) to oppose cocaine-induced excitability. We measured I_h current using whole-cell patch-clamp electrophysiology, 2 and 24 hours after an acute cocaine injection. We found a progressive reduction of I_h starting 24 hours after acute cocaine administration. Thus, this progressive decline observed from acute to chronic cocaine administration could serve as a homeostatic mechanism to reduce cocaine enhanced excitability. In addition, we quantified the increase in rebound action potentials, 2 and 24 hours after an acute cocaine injection. We found that after 2, 24 hours or seven days of cocaine administration, there is a significant increase in rebound action potentials of VTA DA neurons. Interestingly, there is a higher number of rebound action potentials after 2 hours of cocaine in comparison to 24 hours. When ZD, an I_h blocker was perfused, there was a significant reduction in rebound action potentials. The progressive I_h reduction may be a key factor in the decrease of rebound action potentials found from 2 to 24 hours after cocaine injections. The effect of I_h blockade was also evaluated on in vitro spontaneous excitability using cell-attached configuration, 2 hours after an acute cocaine injection. When perfused with ZD, the spontaneous firing activity was also reduced, while significantly increasing the interspike interval providing further evidence in favor of an I_h acting as a homeostatic regulator of VTA DA cell excitability.<br /> <br /> The overall cocaine effect can be influenced by the environmental context in which the drug is experienced (Caprioli et al., 2007). One question we addressed is how exposure to cocaine in a novel context interferes with chronic alterations on synaptic potentiation and neuronal excitability in the mesolimbic system (MLS). To evaluate this question an acute cocaine injection was administered to subjects in both a novel context or their home-cage. We found a significant increase in the AMPA-to-NMDA receptor-mediated EPSC ratio of VTA DA neurons in both the novelty and the homecage groups. Cocaine experienced in a novel environment produced a higher degree of synaptic potentiation. In addition, we found that I_h blockade can also diminish the acute cocaine-induced increase in spontaneous firing activity in in vivo anesthetized rats. Again, the reduction in firing was correlated with an increase in the inter-spike interval.<br /> <br /> Lastly, we evaluated if I_h can function as a homeostatic regulator of intrinsic excitability and can be modulated when an altered firing activity in VTA DA cells is present. We enhanced VTA DA excitability pharmacologically and recorded changes in evoked rebound action potentials and I_h. VTA brain slices from naive rats were incubated with bicuculline methiodide (BIC) as a mechanism to enhance excitability and promote rebound excitation through GABAergic disinhibition. Our hypothesis was that I_h will be decreased as a consequence of the pharmacological enhancement of firing activity. We found that BIC-induced excitability facilitates a reduction in I_h conductance. Furthermore, I_h blockade can significantly reduce this enhanced BIC-induced excitability.<br /> <br /> It has been reported that high frequency stimulation of the ventral subiculum (HFSvSUB) elicits in vivo persistent hyperactivity of VTA DA neurons (Glangetas et al. 2015). We elucidated the effect of I_h blockade on HFSvSUB induced potentiation on in vivo spontaneous excitability. We hypothesized that I_h blockade will significantly decrease HFSvSUB-induced firing patterns on VTA DA cells. We demonstrated that I_h blockade significantly reduces firing activity after HFSvSUB. Additionally, I_h blockade also significantly increased the interspike interval. These results suggest that I_h is an intrinsic mechanism that can modulate the excitability of VTA DA neurons after an hyperexcitable state is present. <br /> <br /> The study of the regulatory role of this current can provide novel insights on how changes in intrinsic neuronal properties might establish cellular homeostasis in the VTA DA system and in conditions of altered excitability such as substance use disorder (SUD)....

Clinical studies provide strong evidence that stress is an environmental risk factor that can trigger the onset of several neuropsychiatric disorders such as anxiety and depression in humans. Pre-clinical evidence suggests ...