Biology subjects

Castillo, K. D.

Publications and source records attributed to Castillo, K. D..

8 recordsLinked to original sources

bioRxiv2026

Efficient permuted intron-exon RNA circularization system based on the LtrB group II Intron from Lactococcus lactis

Circular RNAs (circRNAs) have emerged as promising platforms for stable RNA expression and therapeutic applications, but methods for their generation remain limited. Here, we engineered the Lactococcus lactis LtrB group II intron into a permuted intron-exon (PIE) configuration to enable efficient circRNA formation (circLtrB). Among the tested split designs, LtrB domain IV cleavage yielded the highest circularization efficiency. Although LtrB encodes LtrA maturase required for efficient in vivo splicing, in vitro application of LtrA to the PIE constructs resulted in lower circularization efficiency than self-splicing conditions. We further examined circularization under distinct salt conditions and showed that NH4Cl- and KCl-based splicing buffers independently support high circularization efficiencies (>80%), while producing distinct splicing intermediates, indicating ion-specific mechanistic effects on circularization. Extending outer homology arms did not enhance circularization efficiency, demonstrating that efficient circRNA formation in the circLtrB platform is independent of extended exon pairing interactions. Using this system, we generated circRNAs across a range of coding sequence lengths, highlighting its payload flexibility. To optimize translation, we evaluated multiple internal ribosome entry sites (IRES), including Aichivirus, Crohivirus, CVB3, improved EMCV, HRV, and Salivirus, and found that the CVB3 IRES drove the highest translation efficiency. Linear NLuc showed faster decay kinetics, while circLtrB-NLuc exhibited more sustained expression over time. Transcriptome-wide RNA sequencing revealed comparable innate immune responses between circular and linear RNAs. Together, these results establish circLtrB as a versatile and efficient platform for circRNA production, providing mechanistic insight into group II intron-mediated circularization and expanding the toolkit for functional circRNA engineering.

molecular biology↗

bioRxiv2024

Cryptic diversity shapes coral symbioses, physiology, and response to thermal challenge

Coral persistence in the Anthropocene is shaped by interactions among holobiont partners (coral animals, microbial symbionts) and their environment. Cryptic coral lineages-genetically distinct yet morphologically similar groups-are critically important as they often exhibit functional diversity relevant to thermal tolerance. Additionally, environmental parameters such as thermal variability may promote tolerance, but how variability interacts with holobiont partners to shape responses to thermal challenge remains unclear. Here, we identified three cryptic lineages of Siderastrea siderea in Bocas del Toro, Panama that differ in distributions across inshore and offshore reefs, microbial associations, holobiont phenomes, and skeleton morphologies. A thermal variability experiment failed to increase thermal tolerance, but subsequent thermal challenge and recovery revealed one lineage maintained elevated energetic reserves, photochemical efficiency, and growth. Lastly, coral cores highlighted that this lineage also exhibited faster growth historically. Functional variation among cryptic lineages highlights their importance in predicting coral reef responses to climate change. TeaserCryptic host diversity drives coral phenotypes relevant to climate change.

evolutionary biology↗

bioRxiv2021

Global change differentially modulates coral physiology and suggests future shifts in Caribbean reef assemblages

Global change driven by anthropogenic carbon emissions is altering ecosystems at unprecedented rates, especially coral reefs, whose symbiosis with algal endosymbionts ise particularly vulnerable to increasing ocean temperatures and altered carbonate chemistry. Here, we assess the physiological responses of the coral holobiont (animal host + algal symbiont) of three Caribbean coral species from two reef environments after exposure to simulated ocean warming (28, 31 {degrees}C), acidification (300 - 3290 atm), and the combination of stressors for 93 days. We used multidimensional analyses to assess how multiple coral holobiont physiological parameters respond to ocean acidification and warming. Our results demonstrate significantly diminishing holobiont physiology in S. siderea and P. astreoides in response to projected ocean acidification, while future warming elicited severe declines in P. strigosa. Offshore S. siderea fragments exhibited higher physiological plasticity than inshore counterparts, suggesting that this offshore population has the capacity to modulate their physiology in response to changing conditions, but at a cost to the holobiont. Plasticity of P. strigosa and P. astreoides was not clearly different between natal reef environments, however, temperature evoked a greater plastic response in both species. Interestingly, while these species exhibit unique physiological responses to ocean acidification and warming, when data from all three species are modeled together, convergent stress responses to these conditions are observed, highlighting the overall sensitivities of tropical corals to these stressors. Our results demonstrate that while ocean warming is a severe acute stressor that will have dire consequences for coral reefs globally, chronic exposure to acidification may also impact coral physiology to a greater extent than previously assumed. The variety of responses to global change we observe across species will likely manifest in altered Caribbean reef assemblages in the future.

ecology↗

bioRxiv2021

Twenty years of change in benthic communities across the Belizean Barrier Reef

Disease, ocean warming, and pollution have caused catastrophic declines in the cover of living coral on reefs across the Caribbean. Subsequently, reef-building corals have been replaced by invertebrates and macroalgae, leading to changes in ecological functioning. We describe changes in benthic community composition and cover at 15 sites across the Belizean Barrier Reef (BBR) following numerous major disturbances--bleaching, storms, and disease outbreaks--over the 20-year period 1997-2016. We tested the role of potential drivers of change on coral reefs, including local human impacts and ocean temperature. From 1997 to 2016, mean coral cover significantly declined from 26.3% to 10.7%, while macroalgal cover significantly increased from 12.9% to 39.7%. We documented a significant decline over time of the reef-building corals Orbicella spp. and described a major shift in benthic composition between early sampling years (1997-2005) and later years (2009-2016). The covers of hard-coral taxa, including Acropora spp., M. cavernosa, Orbicella spp., and Porites spp., were negatively related to marine heatwave frequency. Only gorgonian cover was related, negatively, to our metric of the magnitude of local impacts (the Human Influence Index). Changes in benthic composition and cover were not associated with local protection or fishing. This result is concordant with studies throughout the Caribbean that have documented living coral decline and shifts in reef-community composition following disturbances, regardless of local fisheries restrictions. Our results suggest that benthic communities along the BBR have experienced disturbances that are beyond the capacity of the current management structure to mitigate. We recommend that managers devote greater resources and capacity to enforce and expand existing marine protected areas and that government, industry, and the public act to reduce global carbon emissions.

ecology↗

bioRxiv2020

Exposure duration modulates the response of Caribbean corals to global change stressors

Global change is threatening coral reefs, with rising temperatures leading to repeat bleaching events (dysbiosis of coral hosts and their symbiotic algae) and ocean acidification reducing net coral calcification. Although global-scale mass bleaching events are revealing fine-scale patterns of coral resistance and resilience, traits that lead to persistence under environmental stress remain elusive. Here, we conducted a 95-day controlled-laboratory experiment to investigate how duration of exposure to ocean warming (28, 31{degrees}C), acidification (pCO2 = 400-2800 atm), and their interaction influence the physiological responses of two Caribbean reef-building coral species (Siderastrea siderea, Pseudodiploria strigosa) from two reef zones of the Belize Mesoamerican Barrier Reef System. Every 30 days, calcification rate, total host protein and carbohydrate, chlorophyll a pigment concentration, and symbiont cell density were quantified for the same coral colony to characterize acclimatory responses of each genotype. Physiologies of the two species were differentially affected by these stressors, with exposure duration modulating responses. Siderastrea siderea was most affected by extreme pCO2 (~2800 atm), which resulted in reduced calcification rate, symbiont density, and chlorophyll a concentration. Siderastrea siderea calcification rate initially declined under extreme pCO2 but recovered by the final time point, and overall demonstrated resistance to next-century pCO2 and temperature stress. In contrast, P. strigosa was more negatively impacted by elevated temperature (31{degrees}C). Reductions in P. strigosa calcification rate and total carbohydrates were consistently observed over time regardless of pCO2 treatment, with the greatest reductions observed under elevated temperature. However, nearshore colonies of P. strigosa maintained calcification rates under elevated temperature throughout all exposure durations, suggesting individuals from this environment may be locally adapted to the warmer temperatures characterizing their natal reef zone. This experiment highlights how tracking individual coral colony physiology across broad exposure durations can capture acclimatory responses of corals to global change stressors.

ecology↗

bioRxiv2019

Eukaryotic plankton community stability across reef environments in Bocas del Toro Archipelago (Panama)

Variation in light and temperature can influence the genetic diversity and structure of marine plankton communities. While open ocean plankton communities receive much scientific attention, little is known about how environmental variation affects tropical coral reef plankton communities. Here, we characterize eukaryotic plankton communities on coral reefs across the Bocas del Toro Archipelago in Panama. Temperature loggers were deployed for one year and mid-day light levels were measured to quantify environmental differences across reef zones at four inner and four outer reef sites: Inner: Punta Donato, Smithsonian Tropical Research Institute (STRI) Point, Cristobal, Punta Laurel and Outer: Drago Mar, Bastimentos North, Bastimentos South, and Popa Island. Triplicate vertical plankton tows were collected mid-day and high-throughput 18S ribosomal DNA metabarcoding was leveraged to investigate the relationship between eukaryotic plankton community structure and reef zones. Plankton communities from STRI Point were additionally characterized in the morning ([~]08:00), mid-day ([~]12:00), and evening ([~]16:00) to quantify diel variation within a single site. We found that inshore reefs experienced higher average seawater temperatures, while offshore sites offered higher light levels, presumably associated with reduced water turbidity on reefs further from shore. However, these significant reef zone-specific environmental differences did not correlate with overall plankton community differences or changes in plankton genetic diversity. Instead, we found that time of day within a site and diel vertical migration played structuring roles within these plankton communities, and therefore conclude that the time of community sampling is an important consideration for future studies. Overall, plankton communities in the Bocas del Toro Archipelago appear relatively well mixed across space; however, follow-up studies focusing on more intensive sampling efforts across space and time coupled with techniques that can detect more subtle genetic differences between and within communities will more fully capture plankton dynamics in this region.

ecology↗

bioRxiv2017

Symbiodinium functional diversity and clade specificity under global change stressors

Coral bleaching episodes are increasing in frequency, demanding examination of the physiological and molecular responses of corals and their Symbiodinium to climate change. Here we quantify bleaching and Symbiodinium photosynthetic performance of Siderastrea siderea from two reef zones after long-term exposure to thermal and CO2-acidification stress. Molecular response of in hospite Symbiodinium to these stressors was interrogated with RNAseq. Elevated temperatures reduced photosynthetic efficiency, which was highly correlated with bleaching status. However, photosynthetic efficiencies of forereef symbionts were more negatively affected by thermal stress than nearshore symbionts, indicating greater thermal tolerance in nearshore corals. At control temperatures, CO2-acidification had little effect on symbiont physiology, although forereef symbionts exhibited greater photosynthetic efficiencies than nearshore symbionts. Transcriptome profiling revealed that S. siderea were dominated by clade C Symbiodinium, except under thermal stress, which caused shifts to thermotolerant clade D. Comparative transcriptomics of conserved genes across symbiotic partners revealed few differentially expressed Symbiodinium genes when compared to corals. Instead of responding to stress, clade C transcriptomes varied by reef zone, with forereef Symbiodinium exhibiting enrichment of genes associated with photosynthesis. Our findings suggest that functional variation in photosynthetic architecture exists between forereef and nearshore Symbiodinium populations.

ecology↗

bioRxiv2017

Coral Symbiodinium community composition across the Belize Mesoamerican Barrier Reef System is driven by host species and environmental variability

Reef-building corals maintain a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium and this symbiosis is vital for the survival of the coral holobiont. Symbiodinium community composition within the coral host has been shown to influence a corals ability to resist and recover from stress. A multitude of stressors including ocean warming, ocean acidification, and eutrophication have been linked to global scale decline in coral health and cover in recent decades. Three distinct thermal regimes (highTP, modTP, and lowTP) following an inshore-offshore gradient of declining average temperatures and thermal variation were identified on the Belize Mesoamerican Barrier Reef System (MBRS). Quantitative metabarcoding of the ITS-2 locus was employed to investigate differences and similarities in Symbiodinium genetic diversity of the Caribbean corals Siderastrea siderea, S. radians, and Pseudodiploria strigosa between the three thermal regimes. A total of ten Symbiodinium lineages were identified across the three coral host species. Siderastrea siderea associated with distinct Symbiodinium communities, however Symbiodinium communities of its congener, S. radians, and P. strigosa, were more similar to one another. Thermal regime played a role in defining Symbiodinium communities in S. siderea but not S. radians or P. strigosa. Against expectations, Symbiodinium trenchii, a symbiont known to confer thermal tolerance, was dominant only in S. siderea at one sampled offshore site and was rare inshore, suggesting that coral thermal tolerance in more thermally variable inshore habitats is achieved through alternative mechanisms. Overall, thermal parameters alone were likely not the only primary drivers of Symbiodinium community composition, suggesting that environmental variables unrelated to temperature (i.e., light availability, or nutrients) may play key roles in structuring coral-algal communities in Belize and that the relative importance of these environmental variables may vary by coral host species.

molecular biology↗