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Fin whale song is a ubiquitous low-frequency pulsing that has been recorded in every ocean basin. In recent years, the complexity of fin whale song has been realized. In the North Atlantic, this song is made up of two low-frequency note types (A and B notes) and one higher-frequency note type (upsweeps). The A and B notes are produced with both singlet and doublet inter-note intervals. These song patterns are interwoven throughout an individual’s song and have been observed to shift over time, both gradually and suddenly. Like many baleen whales, the population size of fin whales is poorly defined. Passive acoustic monitoring has been suggested as a way to estimate abundance if a cue rate or calling rate can be determined. In this study, we used recordings from 119 fin whale tracks from 2013–2023 generated from passive acoustic recorders in the North Atlantic to define fin whale song patterns in this ocean basin, identify changes in these song patterns over time, and calculate cue rates. The fin whales recorded sang in 4 distinct patterns. Inter-note intervals were 25 s for A-A singlets, 13 s for B-B singlets, 12/20 s for B-B doublets, and 10/11 s for A-B doublets. B-B doublet inter-note intervals significantly increased from May 2019–April 2023 and A-B inter-note intervals showed an intra-annual increasing trend that reset each summer. Fin whale song in the North Atlantic also often includes higher frequency upsweep notes immediately prior to B notes. While A and B note peak frequency was steady over these years, upsweep peak frequency significantly decreased, continuing a similar trend that has been observed over 30 years. Despite these changes, the dominant song pattern remained consistent and cue rate for actively singing whales was relatively stable, which is in contrast to patterns observed in the North Pacific. Many of these nuances in fin whale singing behavior are newly described herein, and these findings are vital for monitoring baseline behavior, behavioral trends over time, and ultimately estimating abundance of a poorly understood species.

A unique acoustic call type was identified and attributed to Bryde’s whales in the central North Pacific in 2015, but little is known about the distribution, calling behavior, or swimming behavior of Bryde’s whales in the region. Acoustic detections attributed to Bryde’s whales were used to localize and track individual whales on the U.S. Pacific Missile Range Facility (PMRF) in Kaua‘i, Hawai‘i. This study included 150 acoustically derived tracks from recordings spanning the years 2011–2022 with recording effort in nearly every month. Bryde’s whale movement was examined relative to calendar year, day of year, hour of day, wind speed, and acoustic calling rate. Hidden Markov models were used to identify two kinematic states (slower, less directional movement and faster, more directional movement). The findings indicate that Bryde’s whales were more likely to travel in a faster and more directional state during the daytime than at night and between May and August when compared to other times of year. The along-track acoustic cue rate was examined for 118 tracks, and the findings indicate a possible lengthening of the median inter-call interval over the duration of the study period. These results are an important first step in understanding more about behavior in Bryde’s whales, a relatively under-studied species.

Automatic algorithms for the detection and classification of sound are essential to the analysis of acoustic datasets with long duration. Metrics are needed to assess the performance characteristics of these algorithms. Four metrics for performance evaluation are discussed here: receiver-operating-characteristic (ROC) curves, detection-error-trade-off (DET) curves, precision-recall (PR) curves, and cost curves. These metrics were applied to the generalized power law detector for blue whale D calls [Helble, Ierley, D'Spain, Roch, and Hildebrand (2012). J. Acoust. Soc. Am. 131(4), 2682-2699] and the click-clustering neural-net algorithm for Cuvier's beaked whale echolocation click detection [Frasier, Roch, Soldevilla, Wiggins, Garrison, and Hildebrand (2017). PLoS Comp. Biol. 13(12), e1005823] using data prepared for the 2015 Detection, Classification, Localization and Density Estimation Workshop. Detection class imbalance, particularly the situation of rare occurrence, is common for long-term passive acoustic monitoring datasets and is a factor in the performance of ROC and DET curves with regard to the impact of false positive detections. PR curves overcome this shortcoming when calculated for individual detections and do not rely on the reporting of true negatives. Cost curves provide additional insight on the effective operating range for the detector based on the a priori probability of occurrence. Use of more than a single metric is helpful in understanding the performance of a detection algorithm.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, establishes a long-term infection and leads to disease manifestations that are the result of host immune responses to the pathogen. Inflammatory manifestations resolve spontaneously despite continued bacterial presence, suggesting inflammatory cells become less responsive over time. This is mimicked by in vitro repeated stimulations, resulting in tolerance, a phenotypic subset of innate immune memory. We performed comparative transcriptional analysis of macrophages in acute and memory states and identified sets of Tolerized, Hyper-Induced, Secondary-Induced and Hyper-Suppressed genes resulting from memory induction, revealing previously unexplored networks of genes affected by cellular re-programming. Tolerized gene families included inflammatory mediators and interferon related genes as would be predicted by the attenuation of inflammation over time. To better understand how cells mediate inflammatory hypo-responsiveness, we focused on genes that could mediate maintenance of suppression, such as Hyper-Induced genes which are up-regulated in memory states. These genes were notably enriched in stress pathways regulated by anti-inflammatory modulators. We examined one of the most highly expressed negative regulators of immune pathways during primary stimulation, Aconitate decarboxylase 1 (Acod1), and tested its effects during in vivo infection with Bb. As predicted by our in vitro model, we show its inflammation-suppressive downstream effects are sustained during in vivo long-term infection with Bb, with a specific role in Lyme carditis.

Summary: Lyme disease is caused by the bacterial pathogen Borrelia burgdorferi, which can be readily modeled in laboratory mice. In order to understand the cellular and transcriptional changes that occur during B. burgdorferi infection, we conducted single-cell RNA sequencing (scRNA-seq) of ankle joints of infected C57BL/6 mice over time. We found that macrophages/monocytes, T cells, synoviocytes and fibroblasts all showed significant differences in gene expression of both inflammatory and non-inflammatory genes that peaked early and returned to baseline before the typical resolution of arthritis. Predictions of cellular interactions showed that macrophages appear to communicate extensively between different clusters of macrophages as well as with fibroblasts and synoviocytes. Our data give unique insights into the interactions between B. burgdorferi and the murine immune system over time and allow for a better understanding of mechanisms by which the dysregulation of the immune response may lead to prolonged symptoms in some patients.

Over the last two decades, product development partnerships have proven to be a successful model of public-private partnership. Product development partnerships are not-for-profit research and development organizations, typically funded by [...]

Behavioral responses to sonar have been observed in a number of baleen whales, including minke whales (Balaenoptera acutorostrata). Previous studies used acoustic minke whale boing detections to localize and track individual whales on the U.S. Pacific Missile Range Facility (PMRF) in Kaua ‘i, Hawai‘i before, during, and after Navy training activities. These analyses showed significant changes in central North Pacific minke whale distribution and swimming behavior during Navy sonar events. For the purposes of contextualizing changes in animal movement relative to Navy sonar, we expanded on this research to examine the natural variation in minke whale movement when Navy sonar was not present. This study included 2,245 acoustically derived minke whale tracks spanning the years 2012–2017 over all months that minke whales were detected (October–May). Minke whale movement was examined relative to calling season, day of the year, hour of day, wind speed, calling state (nominal or rapid), and distance to the nearest calling conspecific. Hidden Markov models were used to identify two kinematic states (slower, less directional movement and faster, more directional movement). The findings indicate that minke whales were more likely to travel in a faster and more directional state when they were calling rapidly, when other vocalizing minke whales were nearby, during certain times of the day and calling seasons, and in windier conditions, but these changes in movement were less intense than the changes observed during exposure to Navy sonar, when swim speeds were the fastest. These results start to put behavioral responses to Navy sonar into an environmental context to understand the severity of responses relative to natural changes in behavior.

Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.

Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rβ/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.

The Lyme disease bacterial pathogen, Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. Following B. burgdorferi infection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+ T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.

North Pacific minke whale (Balaenoptera acutorostrata) boing calls are commonly detected in Hawaiian waters. When producing boing vocalizations, minke whales seem to be in one of two calling behavioral states. Most often minke whales produce boings with inter-call intervals of several minutes, but sometimes minke whales call rapidly with inter-call intervals of less than a minute. Since minke whales are difficult to detect visually, cue-rate-based density estimation using passive acoustic monitoring has been proposed. However, the variables that influence cue rate or calling rate are poorly understood in most whales, including minke whales. We collected passive acoustic recordings from 47 bottom-mounted hydrophones at the Pacific Missile Range Facility’s instrumented range off the coast of Kauaʻi, Hawaiʻi to test the hypothesis that minke whales call more rapidly when closer in proximity to other calling conspecifics. A total of 599 days of data were recorded between August 2012 and July 2017 and were automatically post-processed to detect, classify, and localize calls. Localized calls were grouped into tracks and manually validated, resulting in 509 individual tracks composed of 36,033 calls within a 16 x 39 km focal study area. Tracked minke whales exhibited a strong bimodal call rate with means of one call every 6.85 min (σ= 2.54 min) and 0.63 min (σ= 0.36 min). We ran hidden Markov models to quantify the relationship between call rate and the distance to the nearest calling conspecific. Overall, the probability of the higher call rate occurring increased as the distance to the nearest conspecific decreased, and the probability of the lower call rate occurring increased as the distance to the nearest conspecific increased. We also examined individual track data and found that minke whales may also exhibit other responses (i.e. increased speed, changes in heading, and cessation of calling) when calling conspecifics are nearby. These findings provide new information about minke whale calling behavior in what is likely a breeding area.

Eastern North Pacific gray whales make one of the longest annual migrations of any mammal, traveling from their summer feeding areas in the Bering and Chukchi Seas to their wintering areas in the lagoons of Baja California, Mexico. Although a significant body of knowledge on gray whale biology and behavior exists, little is known about their vocal behavior while migrating. In this study, we used a sparse hydrophone array deployed offshore of central California to investigate how gray whales behave and use sound while migrating. We detected, localized, and tracked whales for one full migration season, a first for gray whales. We verified and localized 10,644 gray whale M3 calls and grouped them into 280 tracks. Results confirm that gray whales are acoustically active while migrating and their swimming and acoustic behavior changes on daily and seasonal time scales. The seasonal timing of the calls verifies the gray whale migration timing determined using other methods such as counts conducted by visual observers. The total number of calls and the percentage of calls that were part of a track changed significantly over both seasonal and daily time scales. An average calling rate of 5.7 calls/whale/day was observed, which is significantly greater than previously reported migration calling rates. We measured a mean speed of 1.6 m/s and quantified heading, direction, and water depth where tracks were located. Mean speed and water depth remained constant between night and day, but these quantities had greater variation at night. Gray whales produce M3 calls with a root mean square source level of 156.9 dB re 1 μPa at 1 m. Quantities describing call characteristics were variable and dependent on site-specific propagation characteristics.

Fin whale song is a ubiquitous low-frequency pulsing that has been recorded in every ocean basin. In recent years, the complexity of fin whale song has been realized. In the North Atlantic, this song is made up of two low-frequency note types (A and B notes) and one higher-frequency note type (upsweeps). The A and B notes are produced with both singlet and doublet inter-note intervals. These song patterns are interwoven throughout an individual's song and have been observed to shift over time, both gradually and suddenly. Like many baleen whales, the population size of fin whales is poorly defined. Passive acoustic monitoring has been suggested as a way to estimate abundance if a cue rate or calling rate can be determined. In this study, we used recordings from 119 fin whale tracks from 2013-2023 generated from passive acoustic recorders in the North Atlantic to define fin whale song patterns in this ocean basin, identify changes in these song patterns over time, and calculate cue rates. The fin whales recorded sang in 4 distinct patterns. Inter-note intervals were 25 s for A-A singlets, 13 s for B-B singlets, 12/20 s for B-B doublets, and 10/11 s for A-B doublets. B-B doublet inter-note intervals significantly increased from May 2019-April 2023 and A-B inter-note intervals showed an intra-annual increasing trend that reset each summer. Fin whale song in the North Atlantic also often includes higher frequency upsweep notes immediately prior to B notes. While A and B note peak frequency was steady over these years, upsweep peak frequency significantly decreased, continuing a similar trend that has been observed over 30 years. Despite these changes, the dominant song pattern remained consistent and cue rate for actively singing whales was relatively stable, which is in contrast to patterns observed in the North Pacific. Many of these nuances in fin whale singing behavior are newly described herein, and these findings are vital for monitoring baseline behavior, behavioral trends over time, and ultimately estimating abundance of a poorly understood species. [ABSTRACT FROM AUTHOR]

Male fin whales sing using 20 Hz pulses produced in regular patterns of inter-note intervals, but little is known about fin whale swimming behavior while they are singing. Even less is known about fin whales in Hawaiian waters because they have rarely been sighted during surveys and passive acoustic monitoring has been limited to sparse hydrophone systems that do not have localization capabilities. We hypothesized that fin whale kinematics may be related to their singing behavior, or external variables such as time and sea state. To investigate this hypothesis, we analyzed 115 tracks containing 50,034 unique notes generated from passive acoustic recordings on an array of 14 hydrophones from 2011 to 2017 at the U.S. Navy Pacific Missile Range Facility off Kauai, Hawaii. Fin whales swam at an average speed of 1.1 m/s over relatively direct paths. We incorporated the whales' speed and turning angle into hidden Markov models to identify different behavioral states based on the whales' movements. We found that fin whale kinematic behavioral state was related to the vocalization rate (also known as cue rate) and time of day. When cue rate was higher, fin whales were more likely to swim slower and turn more than when cue rate was lower. During the night, fin whales were also more likely to swim slower and turn more than during the day. In addition, we examined whether the presence of singing fin whales was related to time and sea state using generalized additive models. Fin whale track presence was affected by day of the year and song season, and possibly also wind speed and wave height. Although the track kinematics from the fin whale tracks presented here are limited to a subset of whales that are acoustically active, they provide some of the only detailed movements of fin whales in the region and can be compared against fin whale swim speeds in other regions. Understanding how fin whale swimming behavior varies based on their vocalization patterns, time, and environmental factors will help us to contextualize potential changes in whale behavior during Navy training and testing on the range.

Many marine mammals rely on sound for foraging, maintaining group cohesion, navigation, finding mates, and avoiding predators. These behaviors are potentially disrupted by anthropogenic noise. Behavioral responses to sonar have been observed in a number of baleen whale species but relatively little is known about the responses of minke whales (Balaenoptera acutorostrata). Previous analyses demonstrated a spatial redistribution of localizations derived from passive acoustic detections in response to sonar activity, but the lack of a mechanism for associating localizations prevented discriminating between movement and cessation of calling as possible explanations for this redistribution. Here we extend previous analyses by including an association mechanism, allowing us to differentiate between movement responses and calling responses, and to provide direct evidence of horizontal avoidance responses by individual minke whales to sonar during U.S. Navy training activities. We fitted hidden Markov models to 627 tracks that were reconstructed from 3 years of minke whale (B. acutorostrata) vocalizations recorded before, during, and after naval training events at the U.S. Navy’s Pacific Missile Range Facility, Kauai, Hawaii. The fitted models were used to identify different movement behaviors and to investigate the effect of sonar activity on these behaviors. Movement was faster and more directed during sonar exposure than in baseline phases. The mean direction of movement differed during sonar exposure, and was consistent with movement away from sonar-producing ships. Animals were also more likely to cease calling during sonar. There was substantial individual variation in response. Our findings add large-sample support to previous demonstrations of horizontal avoidance responses by individual minke whales to sonar in controlled exposure experiments, and demonstrate the complex nature of behavioral responses to sonar activity: some, but not all, whales exhibited behavioral changes, which took the form of horizontal avoidance or ceasing to call.

Summary: The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion. : Molecular Mechanism of Behavior; Medical Microbiology; Methodology in Biological Sciences; Cell Biology; Host-pathogen Interactions; Molecular Microbiology; Genetic Engineering; Genetic Screens Subject Areas: Molecular Mechanism of Behavior, Medical Microbiology, Methodology in Biological Sciences, Cell Biology, Host-pathogen Interactions, Molecular Microbiology, Genetic Engineering, Genetic Screens

This paper demonstrates the importance of accounting for environmental effects on passive underwater acoustic monitoring results. The situation considered is the reduction in shipping off the California coast between 2008-2010 due to the recession and environmental legislation. The resulting variations in ocean noise change the probability of detecting marine mammal vocalizations. An acoustic model was used to calculate the time-varying probability of detecting humpback whale vocalizations under best-guess environmental conditions and varying noise. The uncorrected call counts suggest a diel pattern and an increase in calling over a two-year period; the corrected call counts show minimal evidence of these features.

Male fin whales sing by producing 20 Hz pulses in regular patterns of inter-note intervals. While singing, fin whales may also alternate the frequency ranges of their notes. Different song patterns have been observed in different regions of the world's oceans. New song patterns suddenly emerging in an area have been hypothesized to either be indicators of new groups of whales in the area or signs of cultural transmission between groups. Since the status of fin whales around Hawaii is unknown and visual surveys are expensive and difficult to conduct in offshore areas, passive acoustic monitoring has been proposed as a way to monitor these whales. We used passive acoustic recordings from an array of 14 hydrophones to analyze the song patterns of 115 fin whale encounters made up of 50,034 unique notes off Kauai, Hawaii from 2011 to 2017. Fin whale singing patterns were more complicated than previously described. Fin whales off Hawaii sang in five different patterns made of two 20 Hz note types and both singlet and doublet inter-note interval patterns. The inter-note intervals present in their songs were 28/33 s for the lower frequency doublet, 30 s for the lower frequency singlet, 17/24 s for the higher frequency doublet, 17 s for the higher frequency singlet, and 12/20 s for the doublet that alternated between both note types. Some of these song patterns were unique to these fin whales in Hawaiian waters, while others were similar to song patterns recorded from fin whales off the U.S. west coast. Individual fin whales often utilized several different song patterns which suggests that multiple song patterns are not necessarily indicators of different individuals or groups. The dominant song pattern also changed over these years. Cultural transmission may have occurred between fin whales in Hawaiian waters and off the U.S. west coast, which has resulted in similar songs being present at both locations but on lagged timescales. Alternatively, groups occupying the Hawaiian waters could shift over time resulting in different song patterns becoming dominant. This work has implications for the population structure and behavior of Hawaii fin whales.