Single Fish Research Articles

A Stage-Structured Mathematical Model for Fish Stock with Harvesting

In this paper, we propose a mathematical model for a single species fish stock with a three-stage structure: Juveniles, small adults, and large adults with two harvesting strategies for mature classes, maturity, and size selectivities. The purpose of the work is to investigate the dynamical behavior of the model and discuss the effect of harvesting. We identify the adult reproduction number $\mathcal{R}_A$ for the model; obtain the local and global stability of the trivial equilibrium when $\mathcal{R}_A 1$. Numerical simulations are provided to investigate the influence of harvesting functions, discuss the optimal harvesting rates, and explore the effect of periodic coefficients on the dynamical system.

871: Primary Placental Trophoblasts from Primate Species are Permissive for Zika Virus (ZIKV) Replication

We and others have recently shown that human placental trophoblasts are infected by ZIKV, and function as a viral reservoir. However, it is unknown if the capacity for productive infection is unique to humans or true of other primate species. This is important given the need for relevant clinical models, as well as furthering our understanding of congenital viral-host interactions. Our study aims were to look for common patterns of infectivity and gene expression in primate trophoblasts (e.g., marmoset, baboon and human) employing whole transcriptome RNA Sequencing (RNA Seq). A primary Brazilian ZIKV isolate from the current pandemic (ZIKV/H.sapiens/Brazil/SPH2015) was employed. Trophoblasts were isolated from non-infected, uncomplicated term marmoset and baboon pregnancies (n=5) following Cesarean delivery, and compared to n=25 human primary trophoblasts infected with recent other ZIKV strains. Trophoblasts were FACS analyzed for purity, and infected with 0.25ml of 1x105 pfu/ml of ZIKV or mock. Infectivity was analyzed by ZIKV-specific qRT-PCR, IHC & single molecule RNA FISH. RNA-Seq was carried out on the Illumina HiSeq platform with 150nt paired-end settings and analyzed by HTseq/edgrR; significance was determined after controlling for multiple comparisons. We observed log replication of ZIKV in pure isolate populations of marmoset and baboon primary trophoblasts; imaging confirmed intracytoplasmic replication (Fig. 1A,1B). >30 million reads/sample were generated, with 60% (+/-2%) mapped to the primate species-specific genome. For example, whole transcriptome gene expression patterns in the marmoset showed significant up-regulation (q<1x10-6) of interferon-associated pathways, with differential expression most prominent on days 4-7 following infection (dpi; Fig. 1C). This study demonstrates for the first time that placental trophoblasts from multiple primate species are permissive for ZIKV replication and likely serve as portals for infection. As ZIKV was first isolated from rhesus macaques in the Ugandan Zika forest in 1947 and human transcriptional pathways modify interferon responses, our findings suggest that ZIKV may share opportunistic pathogenesis in the primate placenta. We speculate that these studies are of likely significant mechanistic interest to ZIKV perinatal pathogenesis.

Solitude and its Language Manifestation in Ernest Hemingways the Old Man and the Sea: A Psycholinguistic Inspection

Santiago, the old man in Earnest Hemingway’s The Old Man and the Sea fishes alone in a small boat in the Gulf Stream and for almost three months he fails to catch a single fish, which entices him to embrace the challenge to go out too far not to be back until and unless he succeeds in catching a big fish. Solo he goes far out leaving the smell of the land behind and baits his hooks deep in the sea. His solitary waiting begins thereby and he watches birds as well as flying fishes. But the thing most needed to accompany him is his language, of which he makes a successful display through his unattended thoughts, expressions and utterances. He talks to himself, to the birds, to his hands, and to the fish he ultimately succeeds in hooking. The linguistic presentation that the old man demonstrates in his solitary days and nights while he struggles with Marlin, the fish he catches expresses a great deal regarding the conjunction between solitude and language. This article regards that the psychological standing the old man holds during his utter loneliness deep in the desolate sea has much to do with the kind of thoughts and language exposures he produces. Accordingly the researcher gives critical perusal to the novella and intends to delineate the prevalent inter-connection between solitude and language in the light of psycholinguistics.

Automatic quantification of juvenile zebrafish aggression

BackgroundAlthough aggression is a common symptom of psychiatric disorders the drugs available to treat it are non-specific and can have unwanted side effects. The zebrafish is an ideal model for aggression research. Zebrafish are small, amenable to genetic and pharmacological manipulation, and agonistic behaviour can be measured reliably. New methodIn this study we have established a novel setup to automatically quantify aggression and locomotion in one-month old juvenile zebrafish, a stage at which fish exhibit adult-like behaviour but are small so that one camera can film several animals. ResultsWe have validated our novel software by comparison to manual quantification of behaviour, characterised the aggression of one-month old fish, and demonstrated that we can detect alterations to aggression caused by mutation or drug application. Comparison with other methodsThe ability to record up to 12 juvenile fish allows us to speed up and standardise data acquisition compared to studies of single fish. ConclusionsThis setup appears to be suitable to screen for drugs that decrease zebrafish aggression as a first step toward developing novel treatments for this behaviour.

Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH.

Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step-by-step protocol that allows quantification of mRNA decay in single Saccharomyces cerevisiae using smFISH. Our approach relies on fluorescent labeling of single cytoplasmic mRNAs and nascent mRNAs found at active sites of transcription, coupled with mathematical modeling to derive mRNA half-lives. Commercially available, single-stranded smFISH DNA oligonucleotides (smFISH probes) are used to fluorescently label mRNAs followed by the quantification of cellular and nascent mRNAs using freely available spot detection algorithms. Our method enables quantification of mRNA decay of any mRNA in single, unperturbed yeast cells and can be implemented to quantify mRNA turnover in a variety of cell types as well as tissues.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

Isolation of mammalian stress granule cores for RNA-Seq analysis.

Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as "cores" that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.

Fish‐Microarray: A Miniaturized Platform for Single‐Embryo High‐Throughput Screenings

AbstractSmall molecule high‐throughput screenings are essential for the fields of drug discovery and toxicology. Such screenings performed on whole animals are more physiologically relevant leading to more predictive results. However, due to challenges in automation, high costs and absence of miniaturized solutions for animal‐based experiments, high throughput screenings based on animal models are still in its infancy. Here a platform for miniaturized high throughput whole‐organism screenings is presented. The new platform is based on patterns of hydrophilic spots separated by superhydrophobic borders. The difference in wettability of spots and borders generates the effect of discontinuous dewetting and allows for formation of arrays of microdroplets that incorporate single fish embryos. Due to the flat border‐less nature of the platform, the system is compatible with single‐step collection of embryos and pipetting‐free parallel addition of chemical libraries using the “sandwiching method.” The system is miniaturized and allows for incubation of embryos in volumes as low as 5 µL. Finally, the platform realizes surface tension based immobilization of single embryos inside of each microcompartment and permits high‐throughput microscopic analysis directly on the platform. Thus, this method combines the advantages of microarrays, such as high‐throughput and simplicity, with the power of in vivo experiments.

Conflict and cooperation in an age structured fishery

The literature on ‘fish wars', where agents engage in non-cooperative exploitation of single fish stocks or interacting fish stocks is well established, but age and stage structured models do not seem to have been handled within this literature. In this paper we study a game where two agents, or fishing fleets, compete for the same fish stock, which is divided into two harvestable age classes. The situation modelled here may be representative for many fisheries, such as the Norwegian North Atlantic cod fishery where the coastal fleet targets old mature fish while the trawler fleet targets young mature fish. We analyze the game under different assumptions about the underlying information available to each fleet and the actions of the agents. The outcomes of the games are compared to the optimal cooperative solution. The paper provides several results, which differ in many respects from what are found in biomass models. The analysis is supported by numerical examples.

Transcriptional Output Transiently Spikes Upon Mitotic Exit

The pulsatile nature of gene activity has recently emerged as a general property of the transcriptional process. It has been shown that the frequency and amplitude of transcriptional bursts can be subjected to extrinsic regulation. Here we have investigated if these parameters were constant throughout the cell cycle using the single molecule RNA FISH technique. We found evidence of transcriptional spikes upon mitotic exit in three different human cell lines. Recording of cell growth prior to hybridization and immuno-RNA FISH analysis revealed that these spikes were short-lived and subsided before completion of cytokinesis. The transient post-mitotic increase in transcriptional output was found to be the result of cells displaying a higher number of active alleles and/or an increased number of nascent transcripts per active allele, indicating that both the burst fraction and the amplitude of individual bursts can be increased upon mitotic exit. Our results further suggest that distinct regulatory mechanisms are at work shortly after mitotic exit and during the rest of interphase. We speculate that transcriptional spikes are associated with chromatin decondensation, a hallmark of post-mitotic cells that might alter the dynamics of transcriptional regulators and effectors.

Cryptic effects of biological invasions: Reduction of the aggressive behaviour of a native fish under the influence of an "invasive" biomolecule.

The invasive green alga Caulerpa cylindracea has become an important component of the diet of the Mediterranean white seabream Diplodus sargus. As a consequence of this “exotic diet”, the algal bisindolic alkaloid caulerpin accumulates in the fish tissues. Although the compound shows structural similarity to endogenous indolamines that modulate animal behaviour, the potential impact of caulerpin on fish behaviour still remains unexplored. In this report, behavioural experiments both on groups and on single fish responding towards a mirror were performed under different doses of dietary caulerpin. Differences between treated and control groups for each behaviour and for the overall aggressive pattern during the different experimental phases showed that the aggressiveness of D. sargus decreased with the administration of caulerpin. These results call the attention to a still unexplored potential ability of bioactive metabolites from marine invasive species, to alter the behaviour on native species, with putative negative effects on patterns of fish growth and population dynamics.

A comparison of the survival and migration of wild and F1-hatchery-reared brown trout (Salmo trutta) smolts traversing an artificial lake

Supplementing salmonid populations by stocking is a widely-used method to improve catch or to rehabilitate populations. Though, most studies found that survival and fitness of hatchery-reared salmonids is inferior to wild fish. We compared survival, emigration patterns, migration speed and return rates from the sea of wild and 1-year old F1-hatchery-reared brown trout smolts in a Danish lowland stream that contains an artificial lake using passive integrated transponder telemetry in the years 2011–2013 and 2016. The majority of hatchery-reared smolts descended within 72h after their release, whereas wild fish migration was mainly triggered by increased water discharge. Increased probability of a successful lake passage was found at higher discharge. Within years, the groups differed in lake passage time, but without a significant overall difference. Overall, there was no difference in lake survival (wild: 30%, hatchery-reared: 32%) between the two groups, but survival differed between years. Only a single fish (0.9%) of the hatchery-reared smolts tagged in 2011–2013 returned from the sea compared to 11 (6.4%) wild smolts tagged in that period, which questions the value of supplementary stocking of smolts for conservation purposes.

Group foraging increases foraging efficiency in a piscivorous diver, the African penguin.

Marine piscivores have evolved a variety of morphological and behavioural adaptations, including group foraging, to optimize foraging efficiency when targeting shoaling fish. For penguins that are known to associate at sea and feed on these prey resources, there is nonetheless a lack of empirical evidence to support improved foraging efficiency when foraging with conspecifics. We examined the hunting strategies and foraging performance of breeding African penguins equipped with animal-borne video recorders. Individuals pursued both solitary as well as schooling pelagic fish, and demonstrated independent as well as group foraging behaviour. The most profitable foraging involved herding of fish schools upwards during the ascent phase of a dive where most catches constituted depolarized fish. Catch-per-unit-effort was significantly improved when targeting fish schools as opposed to single fish, especially when foraging in groups. In contrast to more generalist penguin species, African penguins appear to have evolved specialist hunting strategies closely linked to their primary reliance on schooling pelagic fish. The specialist nature of the observed hunting strategies further limits the survival potential of this species if Allee effects reduce group size-related foraging efficiency. This is likely to be exacerbated by diminishing fish stocks due to resource competition and environmental change.

Geographic variation in host fish use and larval metamorphosis for the endangered dwarf wedgemussel

Abstract Host fishes play a crucial role in survival and dispersal of freshwater mussels (Unionoida), particularly rare unionids at conservation risk. Intraspecific variation in host use is not well understood for many mussels, including the endangered dwarf wedgemussel (Alasmidonta heterodon) in the USA. Host suitability of 33 fish species for dwarf wedgemussel glochidia (larvae) from the Delaware and Connecticut river basins was tested in laboratory experiments over 9 years. Relative suitability of three different populations of a single host fish, the tessellated darter (Etheostoma olmstedi), from locations in the Connecticut, Delaware, and Susquehanna river basins, was also tested. Connecticut River basin A. heterodon metamorphosed into juvenile mussels on tessellated darter, slimy sculpin (Cottus cognatus), and Atlantic salmon (Salmo salar) parr. Delaware River basin mussels metamorphosed using these three species, as well as brown trout (Salmo trutta), banded killifish (Fundulus diaphanus), mottled sculpin (Cottus bairdii), striped bass (Morone saxatilis), and shield darter (Percina peltata). Atlantic salmon, striped bass, and sculpins were highly effective hosts, frequently generating 5+ juveniles per fish (JPF) and metamorphosis success (MS; proportion of attaching larvae that successfully metamorphose) ≥ 0.4, and producing juveniles in repeated trials. In experiments on tessellated darters, mean JPF and MS values decreased as isolation between the mussel source (Connecticut River) and each fish source increased; mean JPF = 10.45, 6.85, 4.14, and mean MS = 0.50, 0.41, and 0.34 in Connecticut, Delaware, and Susquehanna river darters, respectively. Host suitability of individual darters was highly variable (JPF = 2–11; MS = 0.20–1.0). The results show that mussel–host fish compatibility in A. heterodon differs among Atlantic coastal rivers, and suggest that hosts including anadromous Atlantic salmon and striped bass may help sustain A. heterodon in parts of its range. Continued examination of host use variation, migratory host roles, and mussel–fish interactions in the wild is critical in conservation of A. heterodon and other vulnerable mussel species.

Estimating the Irish public's willingness to pay for more sustainable salmon produced by integrated multi-trophic aquaculture

Integrated Multi-Trophic Aquaculture (IMTA) has been put forward as a potential sustainable alternative to single fin fish species aquaculture. In IMTA, several species are combined in the production process. Integrating species has a conceivable dual advantage; the environmental impact can be lowered through nutrient cycling and from an economic perspective there is potential for increased efficiency, product diversification and a higher willingness to pay for more environmentally friendly produced salmon. This paper presents the results from a choice experiment which examines whether the Irish public is willing to pay a premium for “sustainably produced” farmed salmon from an IMTA process. Uniquely, an ecolabel was used in the design, based on familiar energy rating labels, to communicate the environmental pressure of fish farming to respondents. The experiment demonstrates that the Irish public has a willingness to pay a price premium for sustainability in salmon farming and for locally produced salmon.

Observations and first reports of saprolegniosis in Aanaakłiq, broad whitefish (Coregonus nasus), from the Colville River near Nuiqsut, Alaska

We report the first confirmed cases (2013–2016) of saprolegniosis caused by water mold from the genus Saprolegnia in Aanaakłiq, broad whitefish (Coregonus nasus), from the Colville River near Nuiqsut, Alaska. While this mold is known to be worldwide, these instances represent the first cases in Nuiqsut and only the second instance on a single fish on the North Slope, occurring in 1980. We describe the collaborative work on monitoring this emerging disease. Because fish constitute a critical component of the diet in Nuiqsut and fishing is an integral part of Inupiaq nutritional and cultural subsistence activities overall, individual subsistence fishers, local governmental entities, and Alaska Native organizations representing Nuiqsut requested an examination of affected fish and information on possible drivers of this emerging disease. The collaborative work described here ranges from recording fishermen observations, acquiring fish and mold specimens, histopathology, and molecular identification of the mold. This work, not currently grant-funded, begins with Native observation that incorporates western scientific methods and involves local, state, and federal departments as well as for-profit and non-profit organizations. Additionally, we report the more recent (2016) observation of this disease in a second species of whitefish, Pikuktuuq, humpback whitefish (Coregonus pidschain).

Implementation of reflexive genomic profiling in non-squamous NSCLC: Results of single center experience.

e13010 Background: Identification of oncogenic driver mutations (ODM) and targeted therapies have increased the number of treatment options for patients (pts) with non-squamous non-small cell lung cancer (ns-NSCLC). Different methods exist to detect ODM and there is no standard approach. This study examines the changes in detection of ODM over a 2-year period before and after implementing reflexive genomic testing using next generation sequencing (NGS) in January 2015. Methods: We retrospectively reviewed ns-NSCLC cases (stages I-IV) in 2014 and 2015 using the UVA Cancer Registry. We identified pts with pathologically-confirmed ns-NSCLC and reviewed any genotyping - single gene pyrosequencing, NGS, or FISH for gene rearrangements - within 90 days of pathologic confirmation. Comprehensive genomic profiling (CGP) was defined as both NGS and ALK/ROS1 FISH. Chi-square analyses were generated using SAS 9.4. Results: A total of 360 pts were included. 60% had genotyping while 19% underwent CGP. Mutations are summarized in the table below. From 2014 to 2015, genotyping increased from 50% to 69% (p&lt;0.001), with CGP increasing from 2% to 48% of testing performed (p&lt;0.001). Detection of oncogene driver mutations increased from 28% to 46% (p=0.009). Doubletons (pts with 2 driver mutations) increased from 0% to 5% (p=0.036). Conclusions: Reflexive CGP significantly increased the rate of detection of ODM at our center with results similar to previous studies. Previous studies detected low rates of doubleton mutations, perhaps due to lack of uniform NGS testing. Utilization of NGS may increase detection of doubleton mutations, for which further study is necessary to evaluate efficacy of targeted therapies. For centers without reflexive CGP, implementing this approach is likely to increase rates of ODM detection and expand treatment options. [Table: see text]

Growth and Tissue Elemental Composition Response of Spinach (Spinacia oleracea) to Hydroponic and Aquaponic Water Quality Conditions

Spinach (Spinacia oleracea cv. Carmel) was grown in a conventional glass greenhouse under three different nutrient solution treatments. Lighting and temperature conditions were identical. Six growing systems were used to provide a duplicate trough system for each of these three treatments. Six trials were harvested from each system over a two month time period. Two treatments received hydroponic nutrient inputs, with one treatment at pH 7.0 (referred to as H7) and the other at pH 5.8 (H5), and the third treatment was aquaponic (A7), receiving all of its nutrients from a single fish tank with koi (Cyprinus carpio) except for chelated iron. System pH was regulated by adding K2CO3 to aquaponic systems and KOH to hydroponic systems. Comparisons made between treatments were total yield, leaf surface area, tissue elemental content, and dry weight to fresh weight ratio. Dry weight biomass yield values were not different in pairwise comparisons between treatments (A7 vs. H5: p = 0.59 fresh weight, p = 0.42 dry weight). Similarly, surface area results were not different between treatments. The important comparison was that A7 achieved the same growth as H5, the conventional pH with a complete inorganic nutrient solution, despite unbalanced and less than “ideal” nutrient concentrations in the A7 condition.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction.

Nocturnal migration reduces exposure to micropredation in a coral reef fish

Tropical Atlantic reef fishes in the family Haemulidae (grunts) remain quiescent on reefs during the day and migrate to seagrass beds or sand flats at night. Hypothesized advantages of such nocturnal migrations are increased food availability and/or decreased predation risk. Here, we tested predictions of an alternative hypothesis that nocturnal migrations of French grunt, Haemulon flavolineatum (Desmarest, 1823), reduce exposure to blood-feeding gnathiid isopods. The departure of grunts from the reef coincides with increasing gnathiid activity. In field experiments, subadult fish placed in cages and deployed on the reef at night harbored significantly more gnathiids than those placed in the seagrass habitat. However, this was not the case during the day when gnathiid activity in all habitats is low. In another experiment, the timing of return to the reef was determined to coincide with the postdawn decrease in gnathiid activity. Estimates of cumulative gnathiid exposure at two sites revealed that grunts remaining in reef habitat at night would experience an average of 3 and 44 times more gnathiids than if they spent the night in the seagrass bed, and could reach more than 300 gnathiids on a single fish. In a final field experiment, even recently-settled (<2 cm) juvenile grunts were infested by gnathiids, supporting previous laboratory experiments showing that a single third-stage gnathiid will infest and kill grunts of this size. Combined, these findings suggest that nocturnal feeding migrations of French grunts and ecologically similar fishes result in reduced exposure to blood-feeding gnathiid isopods.