Immunohistochemical Characterization of the Development of Long Photoreceptor Cells in the Lamprey Retina.

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Non-ammocoete larvae of Palaeozoic stem lampreys.

Ammocoetes-the filter-feeding larvae of modern lampreys-have long influenced hypotheses of vertebrate ancestry1-7. The life history of modern lampreys, which develop from a superficially amphioxus-like ammocoete to a specialized predatory adult, appears to recapitulate widely accepted scenarios of vertebrate origin. However, no direct evidence has validated the evolutionary antiquity of ammocoetes, and their status as models of primitive vertebrate anatomy is uncertain. Here we report larval and juvenile forms of four stem lampreys from the Palaeozoic era (Hardistiella, Mayomyzon, Pipiscius, and Priscomyzon), including a hatchling-to-adult growth series of the genus Priscomyzon from Late Devonian Gondwana. Larvae of all four genera lack the defining traits of ammocoetes. They instead display features that are otherwise unique to adult modern lampreys, including prominent eyes, a cusped feeding apparatus, and posteriorly united branchial baskets. Notably, phylogenetic analyses find that these non-ammocoete larvae occur in at least three independent lineages of stem lamprey. This distribution strongly implies that ammocoetes are specializations of modern-lamprey life history rather than relics of vertebrate ancestry. These phylogenetic insights also suggest that the last common ancestor of hagfishes and lampreys was a macrophagous predator that did not have a filter-feeding larval phase. Thus, the armoured 'ostracoderms' that populate the cyclostome and gnathostome stems might serve as better proxies than living cyclostomes for the last common ancestor of all living vertebrates.

Cephalochordates: A window into vertebrate origins.

How vertebrates evolved from their invertebrate ancestors has long been a central topic of discussion in biology. Evolutionary developmental biology (evodevo) has provided a new tool-using gene expression patterns as phenotypic characters to infer homologies between body parts in distantly related organisms-to address this question. Combined with micro-anatomy and genomics, evodevo has provided convincing evidence that vertebrates evolved from an ancestral invertebrate chordate, in many respects resembling a modern amphioxus. The present review focuses on the role of evodevo in addressing two major questions of chordate evolution: (1) how the vertebrate brain evolved from the much simpler central nervous system (CNS) in of this ancestral chordate and (2) whether or not the head mesoderm of this ancestor was segmented.

Analysis of the lamprey genotype provides insights into caspase evolution and functional divergence.

The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.

Novel developmental bases for the evolution of hypobranchial muscles in vertebrates.

BACKGROUND: Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic. RESULTS: Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle. CONCLUSIONS: We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development.

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Predicting larval metamorphosis of Pacific lamprey Entosphenus tridentatus through measurements of length and mass.

The utility of length and mass measurements to predict the larval metamorphosis of Pacific lamprey Entosphenus tridentatus was evaluated. During 2004-2008, larval E. tridentatus were collected from Cedar Creek (Washington, USA) in either the spring or autumn, measured for total length and total mass, reared in captivity and monitored for metamorphosis. The minimum total length, total mass and condition factor of larvae that were observed to go through metamorphosis were 102 mm, 2.0 g and 1.52, respectively. Logistic models indicated that total length and condition factor in both spring and autumn were the most significant variables for predicting metamorphosis of Pacific lamprey during the subsequent summer. Mass in the autumn also appeared important to predict whether metamorphosis occurred in the subsequent summer. Collectively, all models using specific minimums of total length, total mass or condition factor of larvae as criteria for them to metamorphose were sometimes (5 of 14 cases) able to predict the percentage of larvae that would metamorphose but rarely (1 of 12 cases) able to predict which individual larvae would metamorphose. Similar to other anadromous species of lampreys, the size and condition of larval E. tridentatus have utility for predicting metamorphic fate.

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey.

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode-derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD-A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near-total loss of cranial sensory neurons. Taken together, our cell-lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.

Thrust generation during steady swimming and acceleration from rest in anguilliform swimmers.

Escape swimming is a crucial behavior by which undulatory swimmers evade potential threats. The hydrodynamics of escape swimming have not been well studied, particularly for anguilliform swimmers, such as the sea lamprey Petromyzon marinus For this study, we compared the kinematics and hydrodynamics of larval sea lampreys with those of lampreys accelerating from rest during escape swimming. We used experimentally derived velocity fields to calculate pressure fields and distributions of thrust and drag along the body. Lampreys initiated acceleration from rest with the formation of a high-amplitude body bend at approximately one-quarter body length posterior to the head. This deep body bend produced two high-pressure regions from which the majority of thrust for acceleration was derived. In contrast, steady swimming was characterized by shallower body bends and negative-pressure-derived thrust, which was strongest near the tail. The distinct mechanisms used for steady swimming and acceleration from rest may reflect the differing demands of the two behaviors. High-pressure-based mechanisms, such as the one used for acceleration from rest, could also be important for low-speed maneuvering during which drag-based turning mechanisms are less effective. The design of swimming robots may benefit from the incorporation of such insights from unsteady swimming.

Dual functionality of lamprey VLRB C-terminus (LC) for multimerization and cell surface display.

Lamprey, one of the living representatives of jawless vertebrates, uses variable lymphocyte receptors B (VLRB) for antigen recognition, rather than immunoglobulin (Ig) based receptors as used by higher vertebrates. The C-terminus of lamprey VLRB (LC) possess a glycosylphosphatidylinositol (GPI) signal sequence and seven cysteine residues providing dual functionality of the VLRB antibody in the form of a humoral agglutinin and cell membrane receptors. Here, we show that the LC can be either secreted or be membrane anchored as a heterologous fused protein in a multimeric form comprising of eight or ten monomeric units. Using serially truncated LC variants, we showed that the LC, in which the last three amino acid "RKR" were deleted, referred to as LC7, was the most suitable domain for multimeric construction, whereas, the intact LC is more tailored for applications involving membrane anchorage. We show that an antibody specific for viral hemorrhagic septicemia virus (VHSV) (VLR43), displayed on HEK-293F cells using a PiggyBac (PB) transposase system, exhibited a dose-dependent reaction with its antigen, verifying that the LC can be applied in antibody display technology. Therefore, the present report provides valuable insight into the structure of the lamprey VLRB and highlights its potential use as a novel fusion partner for multimerization and membrane anchorage of chimeric proteins.

Genetic variation and population structure among larval Lethenteron spp. within the Yukon River drainage, Alaska.

The absence of information on genetic variation and population structure of lampreys Lethenteron spp. in the eastern part of their distribution limits our understanding of the migration ecology and spatial population genetic structure of the species. We examined genetic variation within and among three aggregations of Lethenteron spp. larvae in the Yukon River drainage, Alaska, using microsatellite genotypes. A total of 120 larval lampreys were genotyped at eight microsatellite loci. Global FST was 0.053 (95% CI 0.021-0.086), while pairwise FST values ranged from 0.048-0.057. Model-based Bayesian clustering analyses with sample locality priors (LOCPRIOR) identified three distinct, but admixed, genetic clusters that corresponded with the three aggregations. Estimates of contemporary gene flow indicate substantial reciprocal migration among sites consistent with no or low-fidelity natal homing. These results are largely in agreement with previous reports of historic and contemporary gene flow among Lethenteron spp. in other parts of their geographic distribution.

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)-a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species-a hagfish, a lamprey and a shark-encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates.

Cloning and characterization of a second lamprey pituitary glycoprotein hormone, thyrostimulin (GpA2/GpB5).

A novel heterodimeric glycoprotein hormone (GpH) comprised of alpha (GpA2) and beta (GpB5) subunits was discovered in 2002 and called thyrostimulin for its ability to activate the TSH receptor in mammals, but its central function in vertebrates has not been firmly established. We report here the cloning and expression of lamprey (l)GpB5, and its ability to heterodimerize with lGpA2 to form a functional l-thyrostimulin. The full-length cDNA of lGpB5 encodes 174 amino acids with ten conserved cysteine residues and one glycosylation site that is conserved with other vertebrate GpB5 sequences. Phylogenetic and synteny analyses support that lGpB5 belongs to the vertebrate GpB5 clade. Heterodimerization of lGpB5 and lGpA2 was shown by nickel pull-down of histidine-tagged recombinant subunits. RNA transcripts of lGpB5 were detected in the pituitary of lampreys during both parasitic and adult life stages. Intraperitoneal injection with lGnRH-III (100 µg/kg) increased pituitary lGpA2, lGpB5, and lGpHß mRNA expression in sexually mature, adult female lampreys. A recombinant l-thyrostimulin produced by expression of a fusion gene in Pichia pastoris activated lamprey GpH receptors I and II as measured by cAMP enzymeimmunoassay. In contrast to jawed vertebrates that have pituitary LH, FSH, and TSH, our data support that lampreys only have two functional pituitary GpHs, lGpH and l-thyrostimulin, which consist of lGpA2 and unique beta subunits. It is hypothesized that lGpH and l-thyrostimulin differentially regulate reproductive and thyroid activities in some unknown way(s) in lampreys.

The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.

COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.

Lamprey metamorphosis: Thyroid hormone signaling in a basal vertebrate.

As one of the most basal living vertebrates, lampreys represent an excellent model system to study the evolution of thyroid hormone (TH) signaling. The lamprey hypothalamic-pituitary-thyroid and reproductive axes overlap functionally. Lampreys have 3 gonadotropin-releasing hormones and a single glycoprotein hormone from the hypothalamus and pituitary, respectively, that regulate both the reproductive and thyroid axes. TH synthesis in larval lampreys takes place in an endostyle that transforms into typical vertebrate thyroid tissue during metamorphosis; both the endostyle and follicular tissue have all the typical TH synthetic components found in other vertebrates. Furthermore, lampreys also have the vertebrate suite of peripheral regulators including TH distributor proteins (THDPs), deiodinases and TH receptors (TRs). Although at the molecular level the components of the lamprey thyroid system are ancestral to other vertebrates, their functions have been largely conserved. TH signaling as it relates to lamprey metamorphosis represents a particularly interesting phenomenon. Unlike other metamorphosing vertebrates, lamprey THs increase throughout the larval period, peak prior to metamorphosis and decline rapidly at the onset of metamorphosis; patterns of deiodinase activity are consistent with these increases and declines. Moreover, goitrogens (which suppress TH levels) initiate precocious metamorphosis, and exogenous TH treatment blocks goitrogen-induced metamorphosis and disrupts natural metamorphosis. Despite this clear physiological difference, TH action via TRs is consistent with higher vertebrates. Based on observations that TRs are upregulated in a tissue-specific fashion during morphogenesis and the finding that lamprey TRs upregulate genes via THs in a fashion similar to higher vertebrates, we propose the following hypothesis for further testing. THs have a dual role in lampreys where high TH levels promote larval feeding and growth and then at the onset of metamorphosis TH levels decrease rapidly; at this time the relatively low TH levels function via TRs in a fashion similar to that of other metamorphosing vertebrates.

Regulatory evolution of Tbx5 and the origin of paired appendages.

The diversification of paired appendages has been a major factor in the evolutionary radiation of vertebrates. Despite its importance, an understanding of the origin of paired appendages has remained elusive. To address this problem, we focused on T-box transcription factor 5 (Tbx5), a gene indispensable for pectoral appendage initiation and development. Comparison of gene expression in jawless and jawed vertebrates reveals that the Tbx5 expression in jawed vertebrates is derived in having an expression domain that extends caudal to the heart and gills. Chromatin profiling, phylogenetic footprinting, and functional assays enabled the identification of a Tbx5 fin enhancer associated with this apomorphic pattern of expression. Comparative functional analysis of reporter constructs reveals that this enhancer activity is evolutionarily conserved among jawed vertebrates and is able to rescue the finless phenotype of tbx5a mutant zebrafish. Taking paleontological evidence of early vertebrates into account, our results suggest that the gain of apomorphic patterns of Tbx5 expression and regulation likely contributed to the morphological transition from a finless to finned condition at the base of the vertebrate lineage.

Similarities and Differences for Swimming in Larval and Adult Lampreys.

The spinal locomotor networks controlling swimming behavior in larval and adult lampreys may have some important differences. As an initial step in comparing the locomotor systems in lampreys, in larval animals the relative timing of locomotor movements and muscle burst activity were determined and compared to those previously published for adults. In addition, the kinematics for free swimming in larval and adult lampreys was compared in detail for the first time. First, for swimming in larval animals, the neuromechanical phase lag between the onsets or terminations of muscle burst activity and maximum concave curvature of the body increased with increasing distance along the body, similar to that previously shown in adults. Second, in larval lampreys, but not adults, absolute swimming speed (U; mm s(-1)) increased with animal length (L). In contrast, normalized swimming speed (U'; body lengths [bl] s(-1)) did not increase with L in larval or adult animals. In both larval and adult lampreys, U' and normalized wave speed (V') increased with increasing tail-beat frequency. Wavelength and mechanical phase lag did not vary significantly with tail-beat frequency but were significantly different in larval and adult animals. Swimming in larval animals was characterized by a smaller U/V ratio, Froude efficiency, and Strouhal number than in adults, suggesting less efficient swimming for larval animals. In addition, during swimming in larval lampreys, normalized lateral head movements were larger and normalized lateral tail movements were smaller than for adults. Finally, larval animals had proportionally smaller lateral surface areas of the caudal body and fin areas than adults. These differences are well suited for larval sea lampreys that spend most of the time buried in mud/sand, in which swimming efficiency is not critical, compared to adults that would experience significant selection pressure to evolve higher-efficiency swimming to catch up to and attach to fish for feeding as well as engage in long-distance migration during spawning. Finally, the differences in swim efficiency for larval and adult lampreys are compared to other animals employing the anguilliform mode of swimming.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain.

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Pacific lamprey (Entosphenus tridentatus) ammocoetes exposed to contaminated Portland Harbor sediments: Method development and effects on survival, growth, and behavior.

Many anthropogenic disturbances have contributed to the decline of Pacific lampreys (Entosphenus tridentatus), but potential negative effects of contaminants on lampreys are unclear. Lamprey ammocoetes are the only detritivorous fish in the lower Willamette River, Oregon, USA, and have been observed in Portland Harbor sediments. Their long benthic larval stage places them at risk from the effects of contaminated sediment. The authors developed experimental methods to assess the effects of contaminated sediment on the growth and behavior of field-collected ammocoetes reared in a laboratory. Specifically, they developed methods to assess individual growth and burrowing behavior. Burrowing performance demonstrated high variability among contaminated sediments; however, ammocoetes presented with noncontaminated reference sediment initiated burrowing more rapidly and completed it faster. Ammocoete reemergence from contaminated sediments suggests avoidance of some chemical compounds. The authors conducted long-term exposure experiments on individually held ammocoetes using sediment collected from their native Siletz River, which included the following: contaminated sediments collected from 9 sites within Portland Harbor, 2 uncontaminated reference sediments collected upstream, 1 uncontaminated sediment with characteristics similar to Portland Harbor sediments, and clean sand. They determined that a 24-h depuration period was sufficient to evaluate weight changes and observed no mortality or growth effects in fish exposed to any of the contaminated sediments. However, the effect on burrowing behavior appeared to be a sensitive endpoint, with potentially significant implications for predator avoidance. Environ Toxicol Chem 2016;35:2092-2102. © 2016 SETAC.