Red Abalones Research Articles

Species identification in Haliotis genus from the northeastern Pacific Ocean using genome-wide RAD-SNPs

Abalone is a marine gastropod mollusk that belongs to the genus Haliotis high-priced and with commercial importance worldwide. Because of its high demand, its products are susceptible to retail fraud by substituting lower-value products or protected species instead of higher-value ones. Therefore, species identification is a crucial traceability element, and restriction-site-associated DNA sequencing (RAD-seq) is a genomic tool that permits achieving this goal in non-model species with few developments in genetic data. This study analyzed five ecological and socio-economically important abalone species that inhabit the west coast and islands of the Baja California Peninsula (BCP), Mexico: green H. fulgens, pink H. corrugata, red H. rufescens, black H. cracherodii and threaded/pinto H. kamtschatkana abalones. The number of putative bi-allelic single nucleotide polymorphisms (SNPs) found across the five species was 150 326 loci. After filtering data, 1 127 SNPs (Total panel) were retained from 223 sequenced abalones. Subsequently, two criteria were used to identify the most informative SNPs markers: private alleles (species-specific) and FST outliers (adaptive markers). The reduced 66 Private SNPs allele panel performs similarly to the Total panel with a 100% correct assignment among the five species, whereas the 24 SNPs FST outlier panel could only correctly assign four out of five species, confusing the threaded/pinto and red abalones. Furthermore, both reduced panels successfully identify black abalone individuals, which has a commercial and conservation importance for seafood trade in Mexico. Therefore, all these markers can be used in seafood traceability and specimen identification in enforcing fishery management measures among these five abalone species.

Early Red Abalone Shell Middens, Human Subsistence, and Environmental Change on California's Northern Channel Islands

Red abalone (Haliots rufescens) shell middens, a distinctive site type on the California Coast dating primarily to the Middle Holocene, provide important insight into human-environmental interactions and climatic and environmental change. Despite decades of research and debate about why red abalone middens formed and how they relate to past human behavior and environmental change, significant questions persist about the nature of these sites and how they relate to broader trends in human diet and land use. Here, we present the results of excavation at three red abalone middens on the northern Channel Islands. Two of these middens, CA-SRI-26 on Santa Rosa Island and CA-SMI-692 on San Miguel Island, are among the older reported red abalone middens, at roughly 8000–7300 cal BP, and help fill a significant temporal gap in our understanding of the initial period of intensive red abalone harvest. The third midden, CA-SMI-172, dates to ~6300 cal BP and fills a spatial gap on eastern San Miguel Island. When placed in the context of other Channel Island red abalone middens, our analysis demonstrates that people at some of the earliest red abalone middens harvested more red abalone and larger sized individuals than later in time. These data suggest that people likely placed predation pressure on red abalones across the red abalone midden period (8500–3500 cal BP) and that human activities worked in tandem with other environmental changes to influence the disappearance of the red abalone midden phenomenon ~3500 years ago.

Withering syndrome susceptibility of northeastern Pacific abalones: A complex relationship with phylogeny and thermal experience.

Population declines in wild and cultured abalones (Haliotis spp.) due to a bacterial disease called withering syndrome (WS) have been documented along the northeastern Pacific Ocean. However, observed differences in species susceptibility to the disease are not well understood. Here, we examined the susceptibility of three temperate abalone species, the cool water (4-14 °C) pinto or northern abalone (Haliotis kamtschatkana), the intermediate water (8-18 °C) red abalone (H. rufescens), and the warm water (12-23 °C) pink abalone (H. corrugata), to experimental WS infection at temperatures facilitating disease proliferation. Mortality data paired with histological and molecular detection of the WS pathogen confirmed that these abalone species exhibit different levels of susceptibility to infection and resistance to WS development ranging from high susceptibility and low resistance in pinto abalone to moderate/low susceptibility and resistance in red and pink abalones. The temperature associated with WS induced mortalities also varied among species: pinto abalone died at the lowest experimental temperature (17.32 ± 0.09 °C), while red abalone died at an intermediate temperature (17.96 ± 0.16 °C), and pink abalone required the highest temperature (18.84 ± 0.16 °C). When data from the current and previous studies were examined, susceptibility to WS was inversely related to phylogenetic distance from white abalone (H. sorenseni), which had the highest susceptibility and lowest resistance of all abalone species tested prior to the current study. These results provide further evidence that an abalone's thermal optima and phylogenetic relationship can determine its susceptibility to WS; species with cool water evolutionary histories are most susceptible to WS and the most susceptible species appear to be closely related. Differences among the thermal ranges of abalone species have broad implications for WS disease dynamics and highlight the importance of understanding the mechanisms governing the abalone-WS relationship in order to properly manage declining abalone populations.

External Auditory Exostoses and its Relationship to Prehistoric Abalone Harvesting on Santa Rosa Island, California

AbstractBioarchaeological research among prehistoric Pacific Coast populations has shown that external auditory exostoses (EAE) are found in high frequencies. On California's northern Channel Islands archaeological research has demonstrated that there was an intense exploitation of red abalone (Haliotis rufescens), particularly between ~8000 and 3500 years ago. If the Island Chumash were free‐diving for red abalone in subtidal waters, EAE should be prevalent in prehistoric populations from Santa Rosa Island. We recorded the presence or absence, number, side and severity of EAE growths for 207 adult individuals from three time periods on Santa Rosa Island. Our results show that ~11% of the total population was affected, including 22.5% of males and ~3.4% of females. The incidence among females increases from 0% in the Early period, to 2.6% in the Middle period, and 6.8% in the Late period. Overall these are lower rates than those reported for other Channel Island and Pacific Coast sites. Given the relatively low percentage of Santa Rosa Islanders affected with EAE, red abalones may have been harvested primarily in shallow waters rather than the deeper subtidal zone. Our data suggest that gender differences in food procurement activities existed on Santa Rosa Island throughout prehistory, congruent with the argument that EAE is an activity‐induced pathology that occurs with prolonged exposure to cold water and wind conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Effect of LED light quality on the phototaxis and locomotion behaviour ofHaliotis discus hannai

Abalones were reared in a laboratory to determine the percentage response rate, response time, average crawling speed and the time taken to recover an upright posture under nine light-emitting diode light quality treatments (red, orange, yellow, green, cyan, blue, purple, white and grey) and a dark environment. Animals were placed in the centre of an experimental device, and the tropism of each animal was continuously monitored by video. The highest percentage response rate (80% in dark adapted abalones, 60% in light adapted abalones) was observed in the dark environment, followed by red and orange light (27% and 30% in dark adapted abalones, respectively, 22% and 24% in light adapted abalones). Two induction materials (substrate and brown algae Laminaria japonica) were used to assess the effect of light quality on the tropism of abalones, with the highest percentage response rate (76% in the L. japonica treatment, 22% in the substrate treatment) also observed in the dark environment, followed by red and orange light (both 25% in the L. japonica treatment, and 26% and 32%, respectively, in the substrate treatment). The tropism order of the abalones under dark, red, orange and yellow light was as follows: dark > orange > red, yellow, but fewer abalones chose to stay in blue, green, cyan and purple light. The response time (about 700 s) in the dark environment was significantly longer than for the other light quality treatments. Compared with the average crawling speed in the other light quality treatments, abalones were relatively slower (about 3.8 mm s−1) in red and orange light, and the dark environment. The mean time required for the recovery of an upright posture in red light and the dark environment was longer than in the other light quality treatments, with the average recovery time reaching a maximum of 60 s in the dark environment. The results demonstrate the phototaxis and locomotion behaviour of abalones, as well as confirming the necessity of a dark, orange or red environment for their management and aquaculture.

Central place foraging and shellfish processing on California’s Northern Channel Islands

We use a central place forager model for shellfish processing to understand Middle Holocene (7550–3600calBP) human settlement patterns on California’s Northern Channel Islands. This period was associated with increasing sedentism and special purpose sites. We examine the processing and transport costs of two high-ranked shellfish species collected during the Middle Holocene, red abalone (Haliotis rufescens) and California mussel (Mytilus californianus), and how these costs influence archaeological assemblages at coastal and interior settlements. Experimental data and the biology of these species suggest that red abalones are less likely than mussels to be transported long distances (∼2km) without field processing. Consistent with these expectations, coastal red abalone midden sites (CA-SRI-109 and -338) are dominated by large red abalone shells and California mussels are most abundant at contemporaneous inland sites (e.g., CA-SRI-50). Large coastal settlement sites (CA-SRI-5, -19, -116, and -821) had the highest overall shellfish diversity. A stable oxygen isotope study suggests that special purpose sites were occupied seasonally and large coastal settlements were more likely to be inhabited year-round. Our study suggests that transportation and processing costs of food resources were important variables in the development of early hunter–gatherer settlement patterns.

Characterization of the growth-related transcriptome in California red abalone (Haliotis rufescens) through RNA-Seq analysis

One of the largest detriments in the abalone aquaculture industry is the inherently low growth rate of this marine gastropod. In order to confront this issue, greater molecular knowledge is needed on growth traits. Therefore, transcriptome analyses were performed using RNA-Seq for groups of California red abalones (Haliotis rufescens) cultured under the same conditions, but with high growth rates (HGR) or low growth rates (LGR). De novo assembly generated 44312 contigs used as references for RNA-Seq analysis. Results showed a total of 1437 differentially expressed contigs, among which, 435 were up-regulated in the HGR group and 1002 in LGR individuals. Overall, LGR abalones evidenced a greater number of exclusive transcripts and differentially transcribed genes. These results provide a valuable resource of novel transcripts in this species and further understandings of the molecular bases regulating growth traits in H. rufescens.

Abalone withering syndrome: distribution, impacts, current diagnostic methods and new findings

Withering syndrome (WS) is a fatal disease of abalone caused by a Rickettsiales-like organism (WS-RLO). The causative agent, 'Candidatus Xenohaliotis californiensis', occurs along the eastern Pacific margin of North America in California, USA, and Baja California, Mexico. However, as infected abalones have been transported to Chile, China, Taiwan, Iceland, Ireland, Israel, Spain, Thailand and Japan, the geographical range of the etiological agent is suspected to be broad, especially where California red abalones Haliotis rufescens are cultured or in areas where native species have been exposed to this species. Susceptibility varies among species, with up to 99% losses of black abalone H. cracherodii in laboratory and field studies in the USA to no losses among the small abalone H. diversicolor supertexta in Thailand. Some populations that have suffered catastrophic losses due to WS have developed resistance to the disease. In addition, a newly identified phage hyperparasite of the WS-RLO may reduce pathogenicity and dampen associated losses. Diagnosis of WS requires the identification of infection with the pathogen (WS-RLO detected via in situ hybridization or histology coupled with PCR and sequence analysis) accompanied by morphological changes that characterize this disease (e.g. pedal and digestive gland atrophy, and digestive gland metaplasia). A quantitative PCR assay was developed and may be useful in quantifying pathogen DNA. Confirmation of infection cannot be done by PCR analysis alone but can be used as a proxy for infection in areas where the agent is established and is recommended for inclusion in health examinations. Avoidance of WS is best accomplished by the establishment of a health history and multiple health examinations prior to movement of animals.

Differential susceptibility to the Withering Syndrome agent and renal coccidia in juvenile Haliotis rufescens, Haliotis discus hannai and the interspecific hybrid

Withering Syndrome (WS) is a pathogenic chronic disease caused by the intracellular rickettsial-like bacterium “Candidatus Xenohaliotis californiensis” (WS-RLOs), which affects many abalone species. The renal coccidium (Margolisiella haliotis) has often been observed concurrent with WS infection. The red abalone Haliotis rufescens is a very susceptible species to WS and is also infected by the coccidium M. haliotis. In contrast, the Japanese abalone Haliotis discus hannai is not infected by these parasites. Interspecific hybridization is a method for improving important traits in animal husbandry. The objective of this study was to determine susceptibility to WS-RLO and M. haliotis infection in the hybrid generated from a cross between red and Japanese abalones. Juveniles from both species and the interspecific hybrid were challenged by exposure to effluent from red abalone adults infected with both parasites. The animals were analyzed by histology at 130days post-challenge. A 33% prevalence WS-RLOs was observed in the red abalone H. rufescens, whereas a 20% prevalence was observed in the hybrid. Infections were graded on a scale of 0–3. Of these red abalones infected, 53% presented grade 1 infection intensity, 10% had grade 2 infections, and 50% had grade 3 infections. However, the hybrids only presented intensities at the extremes of the scale; of those infected 33% showed grade 1 infections and 66% had grade 3 infections. The coccidium prevalence was 7% in red abalone individuals and 13% in the hybrid abalone. In contrast, the Japanese abalone did not present infections with either parasite. As with the prevalence, the infection intensities for the coccidium were higher in the hybrid abalone; of those infected 25% had grade 2 infections, and 75% had grade 3 infections, but the red abalone presented only grade 2 infection intensities. Therefore, the hybrid did not inherited non-susceptibility or resistance characteristics of the parental H. discus hannai and possessed biological conditions that could foster development of both parasites. Development of a culture based on this hybrid abalone should consider its susceptibility to infection by coccidian, WS-RLOs and the potential for developing the WS disease.

Stable Isotopes from Marine Shells, Ancient Environments, and Human Subsistence on Middle Holocene Santa Rosa Island, California, USA

ABSTRACT We report the analysis of stable isotopes on a suite of marine and estuarine shellfish, along with faunal data, from a Middle Holocene red abalone midden (CA-SRI-191) on Santa Rosa Island, California. Our research suggests that people harvested red abalones, mussels, and estuarine shellfish at about 6000 cal BP. Later in time around 4300 cal BP, people relied on California mussel and sea urchin with only trace amounts of red abalone and no estuarine taxa. Analyses of stable oxygen isotopes from California mussel, red and black abalone, and Venus clam shells indicate that a significant change in sea surface temperatures (SST; ∼2°C) between the two occupations is the primary cause for this discrepancy. Differences between the isotopic values of California mussel and black and red abalone shells from the same strata also suggest that people were probably diving in fairly shallow waters for red abalone rather than obtaining them from the intertidal zone. Our research and other studies in the region suggest that environmental and cultural variables (e.g., human harvest pressure) may have worked together to cause changes in the composition of local shellfish habitats. These data demonstrate the importance of conducting stable isotope analysis on a variety of different shellfish species (e.g., rocky coast, estuarine, and other taxa) in concert with traditional midden analysis to help understand ancient environmental, biological, and cultural developments in coastal, island, and other aquatic settings.

A 6,000 Year Old Red Abalone Midden from Otter Point, San Miguel Island, California

For decades, researchers have been interested in archaeological sites on California's Channel Islands that contain large concentrations of red abalone ( Haliotis rufescens) shells. Known as red abalone middens and dated to between about 7500 and 3500 years ago, these sites have been used to understand broad subsistence patterns, provide chrono-stratigraphic control, and investigate past environmental changes in the region. Most detailed studies have focused on red abalone middens from Santa Cruz Island, with comparatively limited data from other islands. Analysis of a red abalone midden sample excavated from a large dune site on San Miguel Island illustrates the diversity of the red abalone site type and associated human behaviors. Rapid changes in the primary midden constituents, from California mussels ( Mytilus californianus) and sea mammals to red abalones, demonstrate the complexities of differentiating between the effects of environmental change, human food choices, and ecological changes associated with human impacts on local environments.

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

AbstractMicrosatellite loci were isolated in Haliotis fulgens using a (CT)n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.

Restored Top Carnivores as Detriments to the Performance of Marine Protected Areas Intended for Fishery Sustainability: a Case Study with Red Abalones and Sea Otters

Abstract: Marine protected areas are possible solutions to the problems of protecting the integrity of marine ecosystems and of sustaining harvested marine populations. We report demographic data for red abalones ( Haliotis rufescens ) at nine sites along the California coast. Six of our sites are within marine protected areas, and four of those six sites are occupied by sea otters ( Enhydra lutris ). Sea otters are known abalone predators and are believed to have an important role in facilitating biodiversity within coastal kelp forest communities along the North Pacific Rim. We asked whether marine protected areas intended to conserve ecosystems are compatible with use of marine protected areas for abalone fishery sustainability. We found that both sea otters and recreational harvest alter the density, size distribution, and microhabitat distribution of red abalones in qualitatively similar ways. Red abalone populations in marine protected areas outside the current sea otter range have higher density, are composed of larger individuals, and occur in more‐open microhabitats compared with populations in locations lacking sea otters but subject to harvest and with populations in locations with sea otters. The effects of sea otters are stronger than the effects of harvest. Characterization of harvest effects on density may be confounded by other uncontrolled factors. We conclude that coastal marine protected areas off California cannot enhance abalone fisheries if, in the interest of ecosystem integrity, they also contain sea otters. Where restored top carnivores limit the sustainability of commodity harvest, it may be possible to resolve conflicts with two categories of spatially segregated, single‐use marine protected areas, one focusing on ecosystem restoration and the other on fishery development.

Polymorphism in abalone fertilization proteins is consistent with the neutral evolution of the egg's receptor for lysin (VERL) and positive darwinian selection of sperm lysin.

The evolution of species-specific fertilization in free-spawning marine invertebrates is important for reproductive isolation and may contribute to speciation. The biochemistry and evolution of proteins mediating species-specific fertilization have been extensively studied in the abalone (genus Haliotis). The nonenzymatic sperm protein lysin creates a hole in the egg vitelline envelope by species-specifically binding to its egg receptor, VERL. The divergence of lysin is promoted by positive Darwinian selection. In contrast, the evolution of VERL does not depart from neutrality. Here, we cloned a novel nonrepetitive region of VERL and performed an intraspecific polymorphism survey for red (Haliotis rufescens) and pink (Haliotis corrugata) abalones to explore the evolutionary forces affecting VERL. Six statistical tests showed that the evolution of VERL did not depart from neutrality. Interestingly, there was a subdivision in the VERL sequences in the pink abalone and a lack of heterozygous individuals between groups, suggesting that the evolution of assortative mating may be in progress. These results are consistent with a model which posits that egg VERL is neutrally evolving, perhaps due to its repetitive structure, while sperm lysin is subjected to positive Darwinian selection to maintain efficient interaction of the two proteins during sperm competition.

An Introduced Sabellid Polychaete Pest Infesting Cultured Abalones and Its Potential Spread to Other California Gastropods

In 1993 an unusual sabellid polychaete was brought to our attention. It grossly deformed the shells of cultured abalones in some of the California abalone mariculture facilities. This worm is not native to California and was subsequently found in rocky intertidal and subtidal habitats in southern Africa, where it had not previously been recognized. The worm is hermaphroditic and has benthic larvae that are competent to settle within 12 h and soon secrete a mucous sheath. Development of the tentacular crown occurs within a week and generation time can be short, about one month. The worm has a unique association with host gastropods. Unlike all other known shell-fouling organisms, the sabellid routinely settles inside the aperture at the growing edge of the shell. The host responds by secreting a layer of nacre over the mucous sheath to form a tube enclosing the worm, whose crown of tentacles extends through the opening of the tube to the outer surface of the shell. Heavy infestations cause the cessation of linear growth of the host as prismatic shell deposition cannot be resumed after repeated settlement of larvae. The sabellid is not very host specific; many other California native gastropods are readily infested. Bivalves do not appear to be susceptible. Efforts to find a native California predator of the adult worms were not successful. The sabellid has caused great economic damage to some facilities commercially culturing abalones. An established population of this worm has been detected in California, and further risk of establishment and spread of this worm is great. Its unique biology suggests that it may be a useful experimental probe for studies of molluscan shell deposition and may also serve to reveal how molluscs defend themselves against organisms attempting to settle in and foul their apertures. Additional key words: molluscan mantle, shell deposition, fouling, South Africa In October 1993, growers at some commercial abalone mariculture facilities in southern California brought an interesting sabellid polychaete to our attention. The worm infested shells of cultured red abalones, Haliotis rufescens. Heavily infested abalones ceased growth. Their shells exhibited an abnormal, beaked shape and often lacked respiratory holes. The worms appeared to bore in the shell. Because this worm had not previously been reported from California and only a few species of shell-boring sabellid in the genus Caobangia were known (Jones 1969), it was of obvious systematic and biological interest. It has been described as a new genus and species, Terebrasabella heterouncinata FITZHUGH & ROUSE 1999. It is evidently native to at least South Africa, as recognized by Ruck & Cook (1998). Several types of polychaetes infest mollusc shells. a Author for correspondence. E-mail: kuris@lifesci.ucsb.edu Spirorbids are sometimes abundant epibionts, often settling on the outer surfaces of molluscan shells (Gee & Knight-Jones 1962; Zuschin & Piller 1997). They also are sometimes very abundant on the shells of cultured abalones in California. Some serpulids are also epibionts. Spionid polychaetes, particularly species of Polydora and Boccardia, known as mudworms, infest molluscan shells. They settle on the outer surface and bore into the shell, often forming a U-shaped or flasklike burrow. The mechanisms by which spionids bore have received considerable attention (Dorsett 1961; Haigler 1969; Zottoli & Carriker 1974; Sato-Okoshi & Okoshi 1993), and these worms can cause damage to both cultured and wild populations of molluscs by weakening shells and increasing their susceptibility to predation, or by loss of energy expended in shell repair (Kent 1979; Bergman et al. 1982; Kojima & Imajima 1982; Mori et al. 1985; Sato-Okoshi and Nomura 1990; Ambariyanto & Seed 1991; Wargo & Ford This content downloaded from 207.46.13.129 on Sun, 26 Jun 2016 07:01:20 UTC All use subject to http://about.jstor.org/terms

Interactions of chemical and natural stresses in the abalone ( Haliotis rufescens) as measured by surface-Probe localized 31P NMR

Application of nuclear magnetic resonance spectroscopy (NMR) for elucidating the sublethal actions of toxicants in intact aquatic organisms, and the influence of natural stress factors, has recently been demonstrated. Using a flow-through chamber, red abalones ( Haliotis rufescens) were exposed (at 14 °C) to ambient (35%.), hyposaline (25%.), or hyper saline (4 5%.) seawater, with and without a sublethal concentration (1.2 mg l −1) of pentachlorophenol (PCP). They were then maintained in clean seawater (at the same salinity) to check both rate and degree of recovery; phosphagen changes in foot muscle were monitored by surface-probe localized 31P NMR. Whereas different water salinities alone did not produce measurable effects, upon PCP exposure phosphoarginine levels declined, inorganic monophosphate levels increased, and intracellular pH decreased. Whereas during recovery all biochemical endpoints returned to normal, both onset and intensity of effects were significantly influenced in an unexpected manner by changes in seawater salinity. Application of 31P NMR represents a powerful new tool for elucidating the influence of natural stress factors on pollutant actions in vivo, providing a more accurate assessment of environmental effects.

Critical Transitions in the Biofabrication of Abalone Shells and Flat Pearls

Analyses of biopolymer/calcium carbonate composites grown on inorganic abiotic substrates implanted between the shell and the shell-secreting epithelium of live red abalones (Haliotis rufescens) provide detailed spatial and temporal data on the in vivo assembly process that generates the shell. X-ray diffraction and scanning electron microscopy analyses of the growth of these flat pearl composites reveal that biomineralization is initiated by the deposition of an organic sheet on the implanted substrate, followed by the growth of a calcite layer with preferred {10.4} orientation and, finally, by the growth of nacreous aragonite. The calcite layer is structurally similar to the green organic/calcite heterolayer of native shell nacre. It comprises 0.2−2.0-μm-diameter elongated crystallites of typical geological habits in various aggregate arrangements. The shell also contains an external layer of (00.1)-oriented prismatic calcite, which is deposited on one edge of a flat pearl and has a morphology similar to that of the {10.4}-oriented calcite layer. The transition from {10.4}-oriented calcite to aragonite in both the shell and the flat pearl is abrupt. In vitro calcium carbonate growth experiments reveal that a similar calcite-to-aragonite transition is induced by the addition of soluble proteins isolated from the aragonitic nacre. The growth of flat pearls is highly sensitive to physical and chemical properties of the abiotic substrate. Either roughened or hydrophobic substrates result in abnormal arrangements of the basal calcite layer, which are corrected for by a reinitiation of the biomineralization process, beginning with the deposition of an organic sheet. Insertion of flat pearls as substrates, however, results in continued nacre growth without the deposition of an organic sheet and a calcite layer.

Interactive effects of pentachlorophenol and temperature in the abalone ( Haliotis rufescens) as measured by in vivo 31P-NMR spectroscopy

The influence of temperature on the effects of pentachlorophenol (PCP) was described in red abalones ( Haliotis rufescens) using in vivo 31P nuclear magnetic resonance spectroscopy (NMR). Specifically, two effects were measured: the exposure times required for onset of PCP effects; and both intensity of, and recovery from, such effects. Following acclimation to clean seawater, abalones were exposed to a sublethal water concentration of PCP (1.2 mg l −1) at either 9 or 19°C until the spectral peak area for inorganic monophosphate (P i) was one-half that of phosphoarginine (PA); temperatures represented the seasonal range in California. The endpoint served to compare the times required to reach equivalent metabolic condition, and insured that all individuals were metabolically equivalent at the onset of exposure to clean seawater; abalones were maintained in clean seawater for up to 16 h to monitor recovery. In foot muscle, PCP caused a decrease in concentration of PA and ATP, an increase in that of P i, and acidification of intracellular pH; effects were produced more rapidly at 19°C, as the endpoint was attained in less than half the time (2.4 h) as was that at 9°C (5.4 h; P < 0.05). Both the average response to, and the average extent of recovery from, the biocide were greater in abalones exposed at 19°C. Upon exposure to clean seawater, all effects generally reversed within 10 to 16 h, and control abalones maintained at the two temperatures alone exhibited no adverse effects. Finally, abalones exposed to 120 μg l −1 of PCP for 18 h at both 9 and 19°C exhibited no adverse effects, indicating that PCP does not impact abalones at current environmental concentrations. In conclusion, high temperature potentiates the time both to onset of and for recovery from the sublethal effects of PCP in the red abalone. Use of in vivo NMR allows measurement of multiple-stress interactions in live, intact aquatic organisms, facilitating the detailed elucidation of individual responses and the laboratory assessment of the environmental effects of pollutants.

Interactive effects of pentachlorophenol and hypoxia in the abalone ( Haliotis rufescens) as measured by in vivo 31P NMR spectroscopy

The effects of hypoxia and pentachlorophenol (PCP) were investigated in red abalones ( Haliotis rufescens) using flow-through exposure and in vivo 31P nuclear magnetic resonance spectroscopy (NMR). Following acclimation to clean seawater, three abalones were separately exposed to a 6-h sublethal concentration of PCP (1.2 mg 1 −1) until the spectral resonance for inorganic monophosphate (P i) was half-height to that of phosphoarginine (PA); the endpoint assured that all individuals were metabolically equivalent at the start of air exposure. They were exposed to clean seawater for 2 h to allow the effects from PCP to stabilize, then air for 45 min, and finally clean seawater for 15 h to check recovery. The effects of PCP in foot muscle included a decrease in the concentrations of PA and adenosine triphosphate ([PA] and [ATP], respectively), an increase in P i, and a decrease in intracellular pH (pH i). Upon air exposure, [PA] and pH i rapidly declined further, [P i] increased, and [ATP] did not change; the magnitudes were similar to those from emergence alone. After resubmergence, both [PA] and [P i] recovered rapidly from the effects of hypoxia, but slowly from those of PCP. While pH i initially declined further, both [ATP] and pH i recovered to near-initial levels after 15 h. In general, the metabolic effects of hypoxia did not change upon pretreatment with PCP; the biochemical interaction of the two factors was additive. Another three abalones were separately exposed to PCP at an environmentally relevant concentration of 120 μg 1 −1 for 6 h, air for 1 h, then the same PCP concentration for 2.5 h to check recovery. At the lower concentration, PCP produced no discernable effects, and those of hypoxia were similar to those without PCP. Since PCP produced no additional effects with or without hypoxia, it does not significantly impact red abalones at currently measured environmental concentrations. Use of in vivo NMR allows measurement of the interactive effects of multiple stress factors in live aquatic organisms, allowing assessment of the actual environmental effects of pollutants in the laboratory.

Sublethal effects of pentachlorophenol in the abalone (haliotis rufescens) as measured by in vivo 31P NMRSpectroscopy

The sublethal biochemical effects of pentachlorophenol (PCP) were investigated in live, intact red abalones (Haliotis rufescens), using a flow-through exposure system, by in vivo 31P NMR spectroscopy. Based on rangefinding tests (6-hr LC50 = 1.6 mg/L; 6-hr no-observable-effect-level (NOEL) = 0.8 mg/L), three abalones were separately exposed to a sublethal concentration (1.2 mg/L) for 5 hr, followed by a 13 hr recovery period. Effects in foot muscle included both a decrease in phosphoarginine and an increase in inorganic monophosphate concentrations ([PA] and [Pi], respectively); both foot muscle concentrations of adenosine triphosphate [ATP] and intracellular pH (pHi) also declined. Parallel in vitro experiments revealed that concentrations of glycerol 3-phosphate, lactate, citrate, succinate, malate, and alanine (Ala) all increased, while those of glyceraldehyde 3-phosphate and glutamine (Gln) remained stable. Also, these effects were not evident until 2 hr into exposure, possibly the time required for PCP to attain an effective concentration in foot muscle. During recovery, while Pi declined to pre-exposure levels, [PA] completely recovered in only one individual. Also, realkalinization of pHi was similar to recovery of [Pi], and ATP returned to near-initial levels, as did glycerol 3-phosphate, lactate, succinate, malate, and Ala; glyceraldehyde 3-phosphate, citrate, and Gln levels declined. Recovery responses corresponded to the time for PCP clearance from foot muscle. The effects of PCP were similar to those of hypoxia, fatigue, hypersalinity, and arginine kinase inhibitors, and so sublethal PCP concentrations may also inhibit electron transport and arginine kinase as well as uncouple mitochondrial oxidative phosphorylation in intact molluscs. Thus, the effects of pollutants on key biochemical processes may now be measured in intact aquatic organisms as they occur, improving our ability to accurately assess the environmental effects of pollutants in the laboratory.