Ichthyomyzon Unicuspis Research Articles

It's Beginning to Look a Lot like #25DaysofFishmas: Communicating Freshwater Biodiversity Using Social Media

As audiences increasingly turn to social media platforms for information about the environment, scientists can use a variety of techniques to engage with the public in meaningful and productive dialogues about “wicked” ecological problems such as the loss of global freshwater biodiversity. Using #25DaysofFishmas, an annual Twitter campaign that I created to highlight fish diversity in the Laurentian Great Lakes, I examined the relationship between tweet attributes and the number of audience impressions (i.e., views) and engagements (i.e., interactions such as retweets and likes). I hypothesized that audiences would be more likely to engage with tweets about familiar species such as those considered “sportfish.” However, the species that received the highest total impressions and engagements included nongame species like Silver Lamprey Ichthyomyzon unicuspis and Bowfin Amia calva. Using generalized linear mixed effects models, I found that the use of images consistently explained the number of impressions and engagements a tweet received, with other explanatory variables including the use of hashtags and timing of posts. This analysis illustrates how scientists can use social media not only as a megaphone to broadcast research, but as an interactive platform to directly engage broader audiences and foster appreciation for regional and global fish biodiversity.

Incidence of lamprey marks on Lake Sturgeon ( Acipenser fulvescens Rafinesque, 1817) in the St. Clair – Detroit River System: Implications for Sea Lamprey ( Petromyzon marinus Linnaeus, 1758) effects

Laurentian Great Lakes Lake Sturgeon (Acipenser fulvescens) are hosts to lamprey species, including native Silver Lamprey (Ichthyomyzon unicuspis) and invasive Sea Lamprey (Petromyzon marinus). Silver Lamprey coevolved with Lake Sturgeon and cause negligible mortality, but Sea Lamprey can negatively affect Lake Sturgeon populations. Sea Lamprey abundance in Lake Erie has been above targets set by resource managers, with the St. Clair – Detroit River System (SCDRS) suspected as a source of Sea Lamprey production into Lake Erie. This study summarizes lamprey marking on Lake Sturgeon captured during agency assessment surveys in the SCDRS since 1996 and provides insight on the potential for Sea Lamprey to negatively affect Lake Sturgeon in the SCDRS. Lamprey marks (any lamprey species) were noted on 48.2% of Lake Sturgeon (2.5 marks/fish) and 3.3% of Lake Sturgeon assumed to be susceptible to mortality by Sea Lamprey (<760 mm TL; 0.06 marks/fish). Silver Lamprey were the only lamprey species found attached to Lake Sturgeon and there was no difference between oral disc diameters of Silver Lamprey and marks measured on Lake Sturgeon in Lake St. Clair and the lower St. Clair River (p = .45). Based on logistic regression, probability of at least one lamprey mark increased with Lake Sturgeon total length and was highest in Lake St. Clair. The probability of observing at least one lamprey mark on a 760 mm Lake Sturgeon was 8.1% or less for each sampling location in the SCDRS aside from Lake St. Clair (28.1%). Results suggest that parasitism of Lake Sturgeon by Sea Lamprey in the SCDRS is rare, particularly for Lake Sturgeon <760 mm TL. Low incidence of lamprey marks on Lake Sturgeon assumed to be susceptible to mortality from Sea Lamprey parasitism and zero occurrence of Sea Lamprey being observed attached to a Lake Sturgeon suggest Sea Lamprey at their current abundance likely have little effect on the Lake Sturgeon population in the SCDRS. Caution should be taken when using mark size to assign marks to lamprey species as there is substantial overlap among species oral disc diameters, potentially inflating the perceived impact of Sea Lamprey on Lake Sturgeon in areas with native lampreys.

Changes in native lamprey populations in the Great Lakes since the onset of sea lamprey (Petromyzon marinus) control

The control of invasive sea lamprey in the Great Lakes basin has been highly successful, but has deleteriously affected native lamprey species. American brook, northern brook, silver and chestnut lampreys are all susceptible to lampricide treatments. Incidental catch from surveys that targeted sea lamprey were used to determine changes in distributions and catch rates of these native species within the Great Lakes basin since the onset of the sea lamprey control program. Over 2.4 million native lampreys were collected during sea lamprey control operations between 1947 and 2018. Native lampreys were no longer captured from many treated and untreated streams. Silver lamprey and northern brook lamprey distributions were reduced most substantially. These species were no longer found in about three quarters of the number of streams in which they were historically present. The number of streams inhabited by chestnut lamprey has diminished by half. American brook lamprey were still present in the majority of streams previously occupied. Catch rates declined in northern brook lamprey and Ichthyomyzon larvae in some Great Lakes basins, but changes in collection gear and methods made comparisons difficult. Trap and weir data show drastic declines in silver lamprey and chestnut lamprey. Streams that have not been treated with lampricide have suffered similar or greater declines in both the number of streams occupied and catch rates for all four species when compared to untreated streams. Therefore reductions in native lamprey populations were likely due to a number of environmental changes, and not exclusively from lampricide treatments.

Stream characteristics associated with feeding type in silver (Ichthyomyzon unicuspis) and northern brook (I. fossor) lampreys and tests for phenotypic plasticity

In most lamprey genera, “paired” species exist in which the larvae are morphologically indistinguishable but adult feeding type differs. The lack of diagnostic genetic differences in many pairs has led to suggestions that they constitute a single gene pool with environmentally influenced feeding types. To investigate whether stream characteristics are correlated with feeding type in the parasitic silver lamprey Ichthyomyzon unicuspis and non-parasitic northern brook lamprey I. fossor, eight variables (pH, alkalinity, conductivity, discharge, total dissolved solids, and density of larval sea lamprey Petromyzon marinus, Ichthyomyzon spp., and total larval density) were compared among eight streams with only silver lamprey, 10 with only northern brook lamprey, and 13 with both species, using classification tree analysis. The most parsimonious model had a 24% misclassification rate, with silver lamprey tending to inhabit streams with higher sea lamprey larval density and northern brook lamprey tending to inhabit streams with higher Ichthyomyzon larval density. We then conducted a pilot study investigating phenotypic plasticity in a lab-based common garden experiment and an in situ transplant experiment. These studies encountered myriad difficulties and were unable to demonstrate plasticity, but did identify challenges associated with culturing Ichthyomyzon larvae. Development of effective rearing procedures for Ichthyomyzon lampreys is essential for any future similar studies.

Intensive Sampling Reveals Underreported Use of Great-River Tributaries by Large-River Fishes in Missouri

Large tributaries may help sustain large-river fish populations by mitigating fish-habitat losses within the highly modified great rivers of the Mississippi River basin. These tributaries are likely most beneficial for fish species specializing on non-degraded large-river habitat for some portion of their life histories. Few great-river tributaries, however, have been surveyed using methods that comprehensively target all fish species, resulting in uncertainty or bias in the reported composition of many tributary fish assemblages. We report important distributional records, including 23 new accounts, for 12 large-river specialist fishes in Missouri—Alosa alabamae (Alabama Shad), Cycleptus elongatus (Blue Sucker), Pimephales vigilax (Bullhead Minnow), Notropis wickliffi (Channel Shiner), Polyodon spathula (Paddlefish), Hybognathus placitus (Plains Minnow), N. blennius (River Shiner), Macrhybopsis hyostoma (Shoal Chub), Scaphirhynchus platorynchus (Shovelnose Sturgeon), M. storeriana (Silver Chub), Ichthyomyzon unicuspis (Silver Lamprey), and Alosa chrysochloris (Skipjack Herring)—following 38 comprehensive fish surveys in tributaries of the Missouri and Mississippi rivers. New accounts collectively demonstrate tributaries support more large-river specialists than historically documented and thus may be currently undervalued sources of habitat for large-river fishes.

Body stiffness and damping depend sensitively on the timing of muscle activation in lampreys.

Unlike most manmade machines, animals move through their world using flexible bodies and appendages, which bend due to internal muscle and body forces, and also due to forces from the environment. Fishes in particular must cope with fluid dynamic forces that not only resist their overall swimming movements but also may have unsteady flow patterns, vortices, and turbulence, many of which occur more rapidly than what the nervous system can process. Has natural selection led to mechanical properties of fish bodies and their component tissues that can respond very quickly to environmental perturbations? Here, we focus on the mechanical properties of isolated muscle tissue and of the entire intact body in the silver lamprey, Ichthyomyzon unicuspis. We developed two modified work loop protocols to determine the effect of small perturbations on the whole body and on isolated segments of muscle as a function of muscle activation and phase within the swimming cycle. First, we examined how the mechanical properties of the whole lamprey body change depending on the timing of muscle activity. Relative to passive muscle, muscle activation can modulate the effective stiffness by about two-fold and modulate the effective damping by >10-fold depending on the activation phase. Next, we performed a standard work loop test on small sections of axial musculature while adding low-amplitude sinusoidal perturbations at specific frequencies. We modeled the data using a new system identification technique based on time-periodic system analysis and harmonic transfer functions (HTFs) and used the resulting models to predict muscle function under novel conditions. We found that the effective stiffness and damping of muscle varies during the swimming cycle, and that the timing of activation can alter both the magnitude and timing of peak stiffness and damping. Moreover, the response of the isolated muscle was highly nonlinear and length dependent, but the body's response was much more linear. We applied the resulting HTFs from our experiments to explore the effect of pairs of antagonistic muscles. The results suggest that when muscles work against each other as antagonists, the combined system has weaker nonlinearities than either muscle segment alone. Together, these results begin to provide an integrative understanding of how activation timing can tune the mechanical response properties of muscles, enabling fish to swim effectively in their complex and unpredictable environment.

Swimming rhythm generation in the caudal hindbrain of the lamprey.

The spinal cord has been well established as the site of generation of the locomotor rhythm in vertebrates, but studies have suggested that the caudal hindbrain in larval fish and amphibians can also generate locomotor rhythms. Here, we investigated whether the caudal hindbrain of the adult lamprey ( Petromyzon marinus and Ichthyomyzon unicuspis) has the ability to generate the swimming rhythm. The hindbrain-spinal cord transition zone of the lamprey contains a bilateral column of somatic motoneurons that project via the spino-occipital (S-O) nerves to several muscles of the head. In the brainstem-spinal cord-muscle preparation, these muscles were found to burst and contract rhythmically with a left-right alternation when swimming activity was evoked with a brief electrical stimulation of the spinal cord. In the absence of muscles, the isolated brainstem-spinal cord preparation also produced alternating left-right bursts in S-O nerves (i.e., fictive swimming), and the S-O nerve bursts preceded the bursts occurring in the first ipsilateral spinal ventral root. After physical isolation of the S-O region using transverse cuts of the nervous system, the S-O nerves still exhibited rhythmic bursting with left-right alternation when glutamate was added to the bathing solution. We conclude that the S-O region of the lamprey contains a swimming rhythm generator that produces the leading motor nerve bursts of each swimming cycle, which then propagate down the spinal cord to produce forward swimming. The S-O region of the hindbrain-spinal cord transition zone may play a role in regulating speed, turning, and head orientation during swimming in lamprey. NEW & NOTEWORTHY Although it has been well established that locomotor rhythm generation occurs in the spinal cord of vertebrates, it was unknown whether the hindbrain of the adult vertebrate nervous system can also generate the locomotor rhythm. Here, we show that the isolated hindbrain-spinal cord transition zone of adult lamprey can generate the swimming rhythm. In addition, the swimming bursts of the hindbrain lead the bursts occurring in the first segment of the spinal cord.

Characterization of the encoding properties of intraspinal mechanosensory neurons in the lamprey.

Proprioceptive sensory inputs are an integral part of the closed-loop system of locomotion. In the lamprey, a model organism for vertebrate locomotion, such sensory inputs come from intraspinal mechanosensory cells called "edge cells". These edge cells synapse directly onto interneurons in the spinal central pattern generator (CPG) circuit and allow the CPG to adjust the motor output according to how the body is bending. However, the encoding properties of the edge cells have never been fully characterized. To identify these properties and better understand edge cells' role in locomotion, we isolated spinal cords of silver lampreys (Ichthyomyzon unicuspis) and recorded extracellularly from the lateral tracts where edge cell axons are located. We identified cells that responded to mechanical stimuli and used standard spike sorting algorithms to identify separate units, then examined how the cells respond to bending rate and bending angle. Although some cells respond to the bending angle, as was previously known, the strongest and most common responses were to bending velocity. These encoding properties will help us better understand how lampreys and other basal vertebrates adapt their locomotor rhythms to different water flow patterns, perturbations, or other unexpected changes in their environments.

Implications of absence of seawater-type mitochondria-rich cells and results of molecular analyses for derivation of the non-parasitic Ukrainian brook lamprey Eudontomyzon mariae

The Ukrainian brook lamprey Eudontomyzon mariae is the most widespread lamprey species in eastern Europe. Although E. mariae is generally considered a derivative of Eudontomyzon danfordi, an exclusively freshwater parasitic species, it has alternatively been suggested that it was recently derived from a now extinct anadromous Black Sea ancestor. Several non-parasitic lampreys and the landlocked sea lamprey, which have recently evolved from anadromous ancestors, still develop a seawater-type mitochondria-rich cell (SW-MRC) in their gills. In contrast, this cell type is not present in the gills of either Lampetra aepyptera, a non-parasitic lamprey of ancient origin, or the parasitic Ichthyomyzon unicuspis and I. castaneus that likewise have long evolutionary histories in fresh water. Eudontomyzon mariae from the Vistula River in the Baltic River basin does not possess SW-MRC, which is inconsistent with a recent origin from an anadromous ancestor. Mitochondrial DNA sequence data were thus used to infer the relationship between different populations of E. mariae and E. danfordi, and to reconstruct the transition from anadromy to freshwater residency. The results suggest that E. mariae evolved independently in the Baltic, Black, and Caspian Sea basins, and not recently from an anadromous ancestor. Although E. mariae in the Danube River may have arisen relatively recently from E. danfordi (differing by 0.7–1.1% in cytochrome b gene sequence), other E. mariae populations (including in the Vistula River) are genetically closer (0.6%) to the hypothetical ancestor of both E. mariae and E. danfordi. That ancestor was probably a freshwater resident, since SW-MRCs are not rapidly lost following confinement in fresh water.

Detection and identification of lampreys in Great Lakes streams using environmental DNA

Abstract Control of sea lamprey Petromyzon marinus in the Great Lakes requires accurate assessment of the instream distribution of this pest species and the ability to distinguish it from the four lamprey species that are native to the Great Lakes (American brook lamprey Lethenteron appendix , chestnut lamprey Ichthyomyzon castaneus , northern brook lamprey Ichthyomyzon fossor , and silver lamprey Ichthyomyzon unicuspis ). We developed PCR-based environmental DNA (eDNA) assays to distinguish among the four “genetic species” of Great Lakes lampreys (silver and northern brook lampreys were genetically indistinguishable), tested them under laboratory conditions, and demonstrated in the field that both spawning and larval sea lamprey can be detected using eDNA. In the laboratory, mean detection frequency decreased with increasing flow rate, but was not significantly related to larval density or temperature over the range of relatively high densities tested. Proof-of-concept was demonstrated in the field when sea lamprey eDNA was detected during the spawning season (when large-bodied adults, their gametes, and later their carcasses were present); sampling effort involved collection of three 1–2 L water samples at each of four transects (0.4–1.0 km apart). Mean detection frequency remained high (81–97%) until spawning ceased at the end of June, but decreased thereafter, falling to 6% by mid-August. We also demonstrated that eDNA of smaller, burrowing larvae (in water collected after mid-August) was detectable in two of four streams with medium to high larval density (1–2 larvae/m 2 ), but not at low densities ( 2 ), potentially due to the exacerbating effect of high flow rates.

Complete mitochondrial genomes of paired species northern brook lamprey (Ichthyomyzon fossor) and silver lamprey (I. unicuspis)

The complete mitogenomes of paired species northern brook lamprey (Ichthyomyzon fossor) and silver lamprey (I. unicuspis) is reported. The two mitogenomes show a 13 bp length difference on the tRNA-Gly and two control regions. The gene order and contents are conserved in the two lampreys and identical to the lamprey mitogenomes published. Except for three indel polymorphic sites, there are 27 SNP sites which are all synonymous substitution sites and occurred on 9 protein-coding genes, two rRNAs and one tRNA. The control region1 contains six consecutive 39-nt repetitive strings in both lampreys. A 7-nt repetitive string in the control region2 is repeated 3 and 5 times in northern brook lamprey and silver lamprey, respectively. The observed level of similarity between nucleotide sequences (99.74%) is consistent with the hypothesis that northern brook lamprey and silver lamprey represent two ecotypes of one species.

Historical distribution records and new records confirm and extend the distribution of the Silver Lamprey, &lt;em&gt;Ichthyomyzon unicuspis&lt;/em&gt;, in the Hayes River, Hudson Bay watershed, Manitoba

We reviewed historical records and observations of the Silver Lamprey (Ichthyomyzon unicuspis) in the Hayes River in the Hudson Bay watershed. New records are presented from the Seeber River in the upper Hayes River system that extend the distribution of the Silver Lamprey in northern Manitoba. A new designatable unit for the Silver Lamprey, the Southern Hudson Bay–James Bay, is proposed.

Evidence for a receiver bias underlying female preference for a male mating pheromone in sea lamprey

Receiver bias models suggest that a male sexual signal became exaggerated to match a pre-existing sensory, perceptual or cognitive disposition of the female. Accordingly, these models predict that females of related taxa possessing the ancestral state of signalling evolved preference for the male trait in a non-sexual context. We postulated that female preference for the male-released bile alcohol mating pheromone, 3 keto petromyzonol sulfate (3kPZS), of the sea lamprey (Petromyzon marinus) evolved as a result of a receiver bias. In particular, we propose that migratory silver lamprey (Ichthyomyzon unicuspis), a basal member of the Petromyzontidae, evolved a preference for 3kPZS released by stream-resident larvae as a means of identifying productive habitat for offspring. Larval silver lamprey released 3kPZS at rates sufficient to be detected by migratory lampreys. Females responded to 3kPZS by exhibiting upstream movement behaviours relevant in a migratory context, but did not exhibit proximate behaviours important to mate search and spawning. Male silver lamprey did not release 3kPZS at rates sufficient to be detected by females in natural high-volume stream environments. We infer that female silver lamprey cue onto 3kPZS excreted by stream-resident larvae as a mechanism to locate habitat conducive to offspring survival and that males do not signal with 3kPZS. We suggest that this female preference for a male signal in a non-sexual context represents a bias leading to the sexual signalling observed in sea lamprey.

Functional and evolutionary implications of the cellular composition of the gill epithelium of feeding adults of a freshwater parasitic species of lamprey, Ichthyomyzon unicuspis

This paper provides the first description of the cellular composition of the gill epithelium of feeding adults of Ichthyomyzon unicuspis Hubbs and Trautman, 1937 (silver lamprey), a parasitic species of lamprey that is confined to fresh water. The surface layer of this epithelium consists solely of pavement cells and intercalated mitochondria-rich cells, which are the only cell types found in all freshwater stages of lampreys and thus considered responsible for the uptake of Na+ and Cl– in hypotonic environments. This epithelium does not contain, however, the chloride cells present during the marine parasitic phase of anadromous lamprey species, such as Petromyzon marinus L., 1758 (sea lamprey), and which are responsible for secreting excess Na+ and Cl–. The absence of this cell type in parasitic adults of I. unicuspis also differs from its presence in parasitic adults of landlocked P. marinus and metamorphosing individuals of the exclusively freshwater nonparasitic species Lethenteron appendix (DeKay, 1842) (American brook lamprey), and which thus reflects the retention of a cell type that was crucial for osmoregulation during the marine phase of their respective anadromous parasitic ancestors. The absence of chloride cells in I. unicuspis is consistent with the hypothesis that Ichthyomyzon, which is at or close to the base of the phylogenetic tree for Northern Hemisphere lampreys (Petromyzontidae), evolved in fresh water or has been confined to fresh water for a very long period.

Contemporary gene flow between “paired” silver (Ichthyomyzon unicuspis) and northern brook (I. fossor) lampreys: implications for conservation

In most lamprey genera, “paired” species exist in which the larvae are morphologically similar or indistinguishable but, following metamorphosis, one species becomes parasitic while the other bypasses the adult feeding phase and rapidly becomes sexually mature. Since DNA barcoding and similar studies using short segments of the mitochondrial genome do not provide sufficient resolution to distinguish between recent divergence and a lack thereof, the current study examined the relationship between the parasitic silver lamprey (Ichthyomyzon unicuspis) and nonparasitic northern brook lamprey (I. fossor) using up to 10,230 bp from the mitochondrial genome to make phylogenetic inferences and mitochondrial restriction fragment length polymorphism (RFLP) and microsatellite markers to test for significant differences in allele frequencies. We found that silver and northern brook lampreys are not reciprocally monophyletic; two lineages were observed but each occurred within both species. There also were no significant range-wide differences in RFLP haplotype or microsatellite allele frequencies between the species, nor were there significant differences where they occurred sympatrically within the Lake Huron basin. Significant genetic differentiation was found only within the Lake Michigan basin where the results were potentially confounded by geographic separation. Our results thus support suggestions that silver and northern brook lampreys represent ecotypes of a single species since, where they are sympatric, they appear to be experiencing ongoing gene flow. However, alternative life history strategies can be important for a species’ long-term persistence, and critical data are needed to decide whether these two feeding types should be managed as a single unit.

Wake structures behind a swimming robotic lamprey with a passively flexible tail.

A robotic lamprey, based on the silver lamprey, Ichthyomyzon unicuspis, was used to investigate the influence of passive tail flexibility on the wake structure and thrust production during anguilliform swimming. A programmable microcomputer actuated 11 servomotors that produce a traveling wave along the length of the lamprey body. The waveform was based on kinematic studies of living lamprey, and the shape of the tail was taken from a computer tomography scan of the silver lamprey. The tail was constructed of flexible PVC gel, and nylon inserts were used to change its degree of flexibility. Particle image velocimetry measurements using three different levels of passive flexibility show that the large-scale structure of the wake is dominated by the formation of two pairs of vortices per shedding cycle, as seen in the case of a tail that flexed actively according to a pre-defined kinematic pattern, and did not bend in response to fluid forces. When the tail is passively flexible, however, the large structures are composed of a number of smaller vortices, and the wake loses coherence as the degree of flexibility increases. Momentum balance calculations indicate that, at a given tailbeat frequency, increasing the tail flexibility yields less net force, but changing the cycle frequency to match the resonant frequency of the tail increases the force production.

First Record of the Silver Lamprey, Ichthyomyzon unicuspis (Petromyzontiformes: Petromyzontidae), from Arkansas

Abstract We report the Silver Lamprey (Ichthyomyzon unicupis) from Arkansas for the first time. Fifteen adult I. unicuspis attached to paddlefish (Polyodon spathula) were collected in Apr. 2005 from the White River near the confluence of the Black River, Independence and Jackson counties. Twelve (80%) of the I. unicuspis possessed 3 teeth (including the apex) in the anterior row, while the other 3 had 4 teeth in their anterior row; unicuspid teeth arrangement in the lateral row were 3 + 3 for 11 (73%) lampreys while 4 had 4 + 4 teeth in the lateral row. Two additional specimens collected in Apr. 1972 and May 1998 from the Buffalo and White rivers in Marion and Prairie counties, respectively, and originally identified as Chestnut Lampreys (Ichthyomyzon castaneus), were re-examined and identified as I. unicuspis. The addition of I unicuspis to the lamprey fauna of Arkansas brings to five the number of species currently occurring in the state.

Attachments by parasitic lampreys within the branchial cavities of their hosts

Silver Lamprey (Ichthyomyzon unicuspis) in the Wisconsin River attached within the branchial cavity of Paddlefish (Polyodon spathula) significantly more often (6.5% of 1,578 attachments) than would be expected by chance on the basis of its relative surface area (3.7%), with as many as four lampreys found together within the branchial cavity of the same Paddlefish. Similar behavior has occasionally been noted for lampreys in other systems, but the phenomenon may be underreported when the lampreys are concealed from view. As suggested previously for some parasitic trychomycterid catfishes, lampreys in the branchial cavity may benefit from accessing blood under pressure, especially in the ventral aorta. In addition, Silver Lamprey within the branchial cavities of Paddlefish may be relatively protected from any negative effects that might result from Paddlefish breaching.

Characterization of 14 microsatellite loci in the paired lamprey species Ichthyomyzon unicuspis and I. fossor and cross amplification in four other Ichthyomyzon species

We describe the isolation and development of 14 polymorphic microsatellite loci in silver lamprey (Ichthyomyzon unicuspis). The number of alleles ranged from 2 to 11 (with an average of 5.1 alleles per locus) in 23–37 lampreys and genetic diversity ranged from 0.036 to 0.837. All of these loci were also polymorphic (with 2–10 alleles, average 4.4) in the closely related northern brook lamprey (I. fossor), and at least eight were polymorphic in four other Ichthyomyzon species. These loci will aid in studying the population dynamics of these six Ichthyomyzon species.

Charged Amino Acid Residues 997–1000 of Human Apolipoprotein B100 Are Critical for the Initiation of Lipoprotein Assembly and the Formation of a Stable Lipidated Primordial Particle in McA-RH7777 Cells

We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpin-bridge mechanism. We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model. To identify the specific motif within residues 931 and 1000 that is critical for apoB particle assembly, apoB:956 and apoB:986 were produced. To test the hairpin-bridge hypothesis, the following mutations were made: 1) residues 997-1000 deletion (apoB:996), 2) residues 717-720 deletion (apoB:1000Delta717-720), and 3) substitution of charged residues 997-1000 with alanines (apoB:996 + 4Ala). Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. 1) ApoB:956 did not form stable particles and was secreted as large lipid-rich aggregates. 2) ApoB:986 formed both a lipidated particle that was denser than HDL(3) and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000Delta717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) In striking contrast to wild-type apoB:1000, (i) apoB:996 and apoB:996 + 4Ala were highly susceptible to intracellular degradation, (ii) only a small proportion of the secreted proteins formed stable HDL(3)-like lipoproteins, and (iii) a majority of the secreted proteins formed large lipid-rich aggregates. We conclude that the first 1000 amino acid residues of human apoB100 are required for the initiation of nascent apoB-containing lipoprotein assembly, and residues 717-720 and 997-1000 play key roles in this process, perhaps via a hairpin-bridge mechanism.