Glycans have emerged as critical determinants of immune maturation, microbial nutrition, and host health in diverse symbioses. In this study, we asked how cyclic delivery of a single host-derived glycan contributes to the dynamic stability of the mutualism between the squid Euprymna scolopes and its specific, bioluminescent symbiont, Vibrio fischeri. V. fischeri colonizes the crypts of a host organ that is used for behavioral light production. E. scolopes synthesizes the polymeric glycan chitin in macrophage-like immune cells called hemocytes. We show here that, just before dusk, hemocytes migrate from the vasculature into the symbiotic crypts, where they lyse and release particulate chitin, a behavior that is established only in the mature symbiosis. Diel transcriptional rhythms in both partners further indicate that the chitin is provided and metabolized only at night. A V. fischeri mutant defective in chitin catabolism was able to maintain a normal symbiont population level, but only until the symbiotic organ reached maturity (∼4 wk after colonization); this result provided a direct link between chitin utilization and symbiont persistence. Finally, catabolism of chitin by the symbionts was also specifically required for a periodic acidification of the adult crypts each night. This acidification, which increases the level of oxygen available to the symbionts, enhances their capacity to produce bioluminescence at night. We propose that other animal hosts may similarly regulate the activities of epithelium-associated microbial communities through the strategic provision of specific nutrients, whose catabolism modulates conditions like pH or anoxia in their symbionts' habitat.symbiosis | squid-vibrio | metabolism | chitin

We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal's symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid-vibrio partnership.

Vesicular stomatitis virus (VSV) emerges periodically from its focus of endemic transmission in southern Mexico to cause epizootics in livestock in the US. The ecology of VSV involves a diverse, but largely undefined, repertoire of potential reservoir hosts and invertebrate vectors. As part of a larger program to decipher VSV transmission, we conducted a study of the spatiotemporal dynamics of Simulium black flies, a known vector of VSV, along the Rio Grande in southern New Mexico, USA from March to December 2020. Serendipitously, the index case of VSV-Indiana (VSIV) in the USA in 2020 occurred at a central point of our study. Black flies appeared soon after the release of the Rio Grande’s water from an upstream dam in March 2020. Two-month and one-year lagged precipitation, maximum temperature, and vegetation greenness, measured as Normalized Difference Vegetation Index (NDVI), were associated with increased black fly abundance. We detected VSIV RNA in 11 pools comprising five black fly species using rRT-PCR; five pools yielded a VSIV sequence. To our knowledge, this is the first detection of VSV in the western US from vectors that were not collected on premises with infected domestic animals.

Assessing the applicability of theory to major adaptive radiations in deep time represents an extremely difficult problem in evolutionary biology. Neoaves, which includes 95% of living birds, is believed to have undergone a period of rapid diversification roughly coincident with the Cretaceous–Paleogene (K-Pg) boundary. We investigate whether basal neoavian lineages experienced an ecological release in response to ecological opportunity, as evidenced by density compensation. We estimated effective population sizes (Ne) of basal neoavian lineages by combining coalescent branch lengths (CBLs) and the numbers of generations between successive divergences. We used a modified version of Accurate Species TRee Algorithm (ASTRAL) to estimate CBLs directly from insertion–deletion (indel) data, as well as from gene trees using DNA sequence and/or indel data. We found that some divergences near the K-Pg boundary involved unexpectedly high gene tree discordance relative to the estimated number of generations between speciation events. The simplest explanation for this result is an increase in Ne, despite the caveats discussed herein. It appears that at least some early neoavian lineages, similar to the ancestor of the clade comprising doves, mesites, and sandgrouse, experienced ecological release near the time of the K-Pg mass extinction.

A long-term relationship between symbiotic partners is often characterized by development and maturation of host structures that harbor the symbiont cells over the host’s lifetime. To understand the mechanisms involved in symbiosis maintenance more fully, we studied the mature bobtail squid, whose light-emitting organ, under experimental conditions, can be transiently or persistently colonized by Vibrio fischeri or remain uncolonized. Superficial anatomical changes in the organ were largely independent of symbiosis. However, both the microanatomy of cells with which symbionts interact and the patterns of gene expression in the mature animal were due principally to the persistent interactions of host and symbiont cells rather than to a response to early colonization events. Further, the characteristic pronounced daily rhythm on the host transcriptome required persistent V. fischeri colonization of the organ. This experimental study provides a window into how persistent symbiotic colonization influences the form and function of host animal tissues.

Summary Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host‐associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host‐like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI‐MS to verify eptA as an ethanolamine transferase for the lipid‐A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H‐NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.

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