Variation in the Transcriptome Response and Detoxification Gene Diversity Drives Pesticide Tolerance in Fishes.

Pesticides are critical for invasive species management but often have negative effects on nontarget native biota. Tolerance to pesticides should have an evolutionary basis, but this is poorly understood. Invasive sea lamprey (Petromyzon marinus) populations in North America have been controlled with a pesticide lethal to them at lower concentrations than native fishes. We addressed how interspecific variation in gene expression and detoxification gene diversity confer differential pesticide sensitivity in two fish species. We exposed sea lamprey and bluegill (Lepomis macrochirus), a tolerant native species, to 3-trifluoromethyl-4-nitrophenol (TFM), a pesticide commonly used in sea lamprey control. We then used whole-transcriptome sequencing of gill and liver to characterize the cellular response in both species. Comparatively, bluegill exhibited a larger number of detoxification genes expressed and a larger number of responsive transcripts overall, which likely contributes to greater tolerance to TFM. Understanding the genetic and physiological basis for pesticide tolerance is crucial for managing invasive species.

Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications.

Although confined to fresh water, non-parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from an adromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non-parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non-parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.

Differences in the transcriptome response in the gills of sea lamprey acutely exposed to 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide or a TFM:niclosamide mixture.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America makes use of two pesticides: 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide, which are often co-applied. Sea lamprey appear to be vulnerable to these agents resulting from a lack of detoxification responses with evidence suggesting that lampricide mixtures produce a synergistic effect. However, there is a lack of information pertaining to the physiological responses of sea lamprey to niclosamide and TFM:niclosamide mixtures. Here, we characterized the transcriptomic responses of the sea lamprey to TFM, niclosamide, and a TFM:niclosamide (1.5 %) mixture in the gill. Along with a control, larval sea lamprey were exposed to each treatment for 6 h, after which gill tissues were extracted for measuring whole-transcriptome responses using RNA sequencing. Differential gene expression patterns were summarized, which included identifying the broad roles of genes and common expression patterns among the treatments. While niclosamide treatment resulted in no differentially expressed genes, TFM- and mixture-treated fish had several differentially expressed genes that were associated with the cell cycle, DNA damage, metabolism, immune function, and detoxification. However, there was no common differential expression among treatments. For the first time, we characterized the transcriptomic response of sea lamprey to niclosamide and a TFM:niclosamide mixture and identified that these agents impact mRNA transcript abundance of genes associated with the cell cycle and cellular death, and immune function, which are likely mediated through mitochondrial dysregulation. These results may help to inform the production of more targeted and effective lampricides in sea lamprey control efforts.

Transcriptomic impacts and potential routes of detoxification in a lampricide-tolerant teleost exposed to TFM and niclosamide.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America often relies on the application of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide mixtures to kill larval sea lamprey. Selectivity of TFM against lampreys appears to be due to differential detoxification ability in these jawless fishes compared to bony fishes, particularly teleosts. However, the proximate mechanisms of tolerance to the TFM and niclosamide mixture and the mechanisms of niclosamide toxicity on its own are poorly understood, especially among non-target fishes. Here, we used RNA sequencing to identify specific mRNA transcripts and functional processes that responded to niclosamide or a TFM:niclosamide mixture in bluegill (Lepomis macrochirus). Bluegill were exposed to niclosamide or TFM:niclosamide mixture, along with a time-matched control group, and gill and liver tissues were sampled at 6, 12, and 24 h. We summarized the whole-transcriptome patterns through gene ontology (GO) term enrichment and through differential expression of detoxification genes. The niclosamide treatment resulted in an upregulation of several transcripts associated with detoxification (cyp, ugt, sult, gst), which may help explain the relatively high detoxification capacity in bluegill. Conversely, the TFM:niclosamide mixture resulted in an enrichment of processes related to arrested cell cycle and growth, and cell death alongside a diverse detoxification gene response. Detoxification of both lampricides likely involves the use of phase I and II biotransformation genes. Our findings strongly suggest that the unusually high tolerance of bluegill to lampricides is due to these animals having an inherently high capacity and flexible detoxification response to such compounds.

Genetic diversity, endemism and phylogeny of lampreys within the genus Lampetra sensu stricto (Petromyzontiformes: Petromyzontidae) in western North America.

Phylogenetic structure of four Lampetra species from the Pacific drainage of North America (western brook lamprey Lampetra richardsoni, Pacific brook lamprey Lampetra pacifica, river lamprey Lampetra ayresii and Kern brook lamprey Lampetra hubbsi) and unidentified Lampetra specimens (referred to as Lampetra sp.) from 36 locations was estimated using the mitochondrial cytochrome b gene. Maximum parsimony and Bayesian inferences did not correspond with any taxonomic scheme proposed to date. Rather, although L. richardsoni (from Alaska to California) and L. ayresii (from British Columbia to California) together constituted a well-supported clade distinct from several genetically divergent Lampetra populations in Oregon and California, these two species were not reciprocally monophyletic. The genetically divergent populations included L. pacifica (from the Columbia River basin) and L. hubbsi (from the Kern River basin) and four Lampetra sp. populations in Oregon (Siuslaw River and Fourmile Creek) and California (Kelsey and Mark West Creeks). These four Lampetra sp. populations showed genetic divergence between 2.3 and 5.7% from any known species (and up to 8.0% from each other), and may represent morphologically cryptic and thus previously undescribed species. A fifth population (from Paynes Creek, California) may represent a range extension of L. hubbsi into the Upper Sacramento River.

Characterization of 12 microsatellite loci for the Pacific lamprey, Entosphenus tridentatus (Petromyzontidae), and cross-amplification in five other lamprey species.

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish that is of conservation concern in North America and Asia. Data on Pacific lamprey population structure are scarce and conflicting, impeding conservation efforts. We optimized 12 polymorphic microsatellite loci for the Pacific lamprey. Three to 13 alleles per locus were observed in a sample of 51 fish collected from the West Fork Illinois River, Oregon. Observed heterozygosity ranged from 0.235 to 0.902 and expected heterozygosity ranged from 0.214 to 0.750. Cross-species amplification produced 8 to 12 polymorphic loci in four other Entosphenus species and in the western brook lamprey (Lampetra richardsoni). Two loci appear to be diagnostic for distinguishing Entosphenus from Lampetra. These markers will be valuable for evaluating population structure and making conservation decisions for E. tridentatus and other lamprey species.

Contrasting physiological responses between invasive sea lamprey and non-target bluegill in response to acute lampricide exposure.

Control of invasive sea lamprey (Petromyzon marinus) in the Laurentian Great Lakes of North America uses lampricides, which consist of 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide. Lampricides are thought to inhibit aerobic energy synthesis, with TFM having a relatively greater selective action against lampreys. While the toxicity and physiological effects of TFM are known, the impacts associated with exposure to niclosamide and TFM:niclosamide mixtures are poorly characterized in fishes. Therefore, focusing on energy metabolism, we quantified the physiological responses of larval sea lamprey and bluegill (Lepomis macrochirus), a non-target, native species. Exposures consisted of each lampricide alone (TFM at the species-specific 24 h LC10; niclosamide at 1.5% of the mixture's TFM concentration) or a mixture of the two (larval sea lamprey at TFM 24 h LC10 + 1.5% niclosamide; bluegill at sea lamprey's TFM 24 h LC99.9 + 1.5% niclosamide) for 24 h. Tissues (brain, skeletal muscle, and liver) were sampled at 6, 12, and 24 h of exposure and assayed for concentrations of ATP, phosphocreatine, glycogen, lactate, and glucose and tissue lampricide levels. In larval sea lamprey, TFM had little effect on brain and skeletal muscle, but niclosamide resulted in a depletion of high energy substrates in both tissues. Mixture-exposed lamprey showed depletion of high energy substrates, accumulation of lactate, and high mortality rates. Bluegill were largely unaffected by toxicant exposures. However, bluegill liver showed lower glycogen and lactate under all three toxicant exposures suggesting increased metabolic turnover. Bluegill also had lower concentrations of TFM and niclosamide in their tissues when compared to lamprey. Our results indicate that lampricide toxicity in sea lamprey larvae is mediated through a depletion of high energy substrates because of impaired aerobic ATP synthesis. We also confirmed that non-target bluegill showed high tolerance to lampricide exposure, an effect potentially mediated through a high detoxification capacity relative to lampreys.

Ultrasound follicle diameter measurement: an assessement of interobserver and intraobserver variation.

The intraobserver and interobserver variation of ultrasound measurement of Graafian follicles was assessed. Fourteen follicles from 10 to 30 mm in diameter were each measured three times in three orthogonal planes by three observers. The intraobserver standard deviation (SD) was 0.6 mm and interobserver SD, 1.2 mm, irrespective of follicle diameter. The pooled SD for both variables was 1.2 mm, giving 95% confidence limits of +/- 2.4 mm for any measurement.

Characteristics and incidence of dysfunctional ovulation patterns detected by ultrasound.

The nature and incidence of normal and abnormal spontaneous ovarian cycles, identified with ultrasound and endocrine tracking, were examined in 45 regularly cycling infertile women with no definitive cause and 15 women who were apparently normal and were receiving donor insemination because of clearly infertile partners. In 136 cycles, four apparently distinct abnormal patterns were detected. The total incidence in the infertile group was 58% compared with 23% in the donor insemination group (P less than 0.005). Twelve of 26 subjects who had at least three cycles tracked showed two different abnormalities, and 1 subject had three different abnormalities in five abnormal cycles. These results suggest that abnormal cycles are a significant factor in unexplained infertility and that diagnosis and treatment cannot be based on the study of a single cycle.

Follicular growth and endocrine profiles in spontaneous and induced conception cycles.

Twenty conception cycles were tracked with ultrasound ovarian scanning from the midfollicular to midluteal phase, and simultaneous hormone profiles were obtained in 18 of the cycles. Twelve cycles were spontaneous and 8 were induced with a variety of regimens. Two of the spontaneous cycles did not have endocrine data, and in one other no follicular growth was observed, despite repeated scanning. The induced cycles did not differ significantly from the spontaneous ones in any respect except the number of follicles and the consequent estradiol (E2) levels. E2 levels per unit total follicular surface area, however, were similar in both groups and remained relatively constant throughout the late follicular phase.

Ultrasound-enhanced diffusion into coupling gel during phonophoresis of 5-fluorouracil.

PURPOSE: To investigate the competitive transport across skin and back-diffusion of 5-fluorouracil into coupling gel under the influence of ultrasound, heat-alone and Azone enhancement. METHODS: The ultrasound effect on 5-fluorouracil penetration through whole rat skin was investigated in modified diffusion cells using a commercial ultrasound generator which was calibrated with a bilaminar membrane hydrophone. RESULTS: Ultrasonic dosimetry measurements demonstrated that the skin membrane was subjected to a complex and unpredictable standing wave field which induced physiologically acceptable heating of the tissue. Surprisingly, ultrasonication produced a decrease in percutaneous drug penetration. Quantification studies indicated that this effect was due to the diffusive loss of the hydrophilic substance 5-fluorouracil from the skin surface into the overlying volume of coupling gel. This phenomenon could be duplicated by the application of conductive heating, indicating that the thermal effects of ultrasound were probably responsible for accelerated 5-fluorouracil diffusion through the gel. CONCLUSION: This study acutely demonstrates how formulation design of the donor vehicle/coupling gel may radically affect therapeutic efficacy in phonophoretic systems.

A new species of Loma (Microsporea) in shiner perch (Cymatogaster aggregata).

Loma embiotocia n. sp. is described from the gills of shiner perch (Cymatogaster aggregata) from waters off Vancouver Island, British Columbia, Canada. Highest prevalence at a site was 15% and greatest intensity was 583 xenomas per fish. Xenomas averaged 0.13 mm in diameter (0.06-0.16 mm) and contained ovoid spores 4.8 x 2.6 (4.0-5.0 x 2.0-3.0) microns. Sporogonic stages were dispersed throughout the xenomas. The xenoma wall was smooth lacking invaginations into the cyto plasm: sporoblasts were not highly vacuolated, and the sporophorous vesicle formed before sporogony. In addition to differences in host and geographic location the new species is distinguished from Loma salmonae, the only other species in the genus known from British Columbia, by its internal transcribed spacer (ITS) ribosomal DNA sequence.

Body shape vs. colour associated initial divergence in the Telmatherina radiation in Lake Matano, Sulawesi, Indonesia.

Highly polymorphic colouration patterns are often associated with sexual selection in fish and can be the initial cause of divergence among closely related taxa. Here we use genetic, body colour and geometric morphometric data collected on 118 fish from Lake Matano, Sulawesi, Indonesia to test if colouration is the initial cause of divergence in the radiating Telmatherina genus. Results reveal that all Telmatherina previously described in this system can be categorized into three mitochondrial lineages and that colouration is only weakly associated with early divergence. Clade-specific body shapes, however, likely adapted to microenvironments are key to the initial divergence in this system. Data also show that although colourations were not likely instrumental in seeding divergence in these fish, they appear to have developed in parallel within each clade. Our results are consistent with an emerging pattern repeated in many vertebrate radiations, whereby divergence by colouration or other display traits is preceded by specialization to environmental adaptive peaks.

Invasion genetics of a freshwater mussel (Dreissena rostriformis bugensis) in eastern Europe: high gene flow and multiple introductions.

In recent years, the quagga mussel, Dreissena rostriformis bugensis, native to the Dnieper and Bug Limans of the northern Black Sea, has been dispersed by human activities across the basin, throughout much of the Volga River system, and to the Laurentian Great Lakes. We used six published microsatellite markers to survey populations throughout its native and introduced range to identify relationships among potential source populations and introduced ones. Mussels from 12 sites in Eurasia, including the central Caspian Sea and one in North America (Lake Erie), were sampled. Field surveys in the Volga River basin suggested that the species first colonized the middle reach of the river near Kubyshev Reservoir, and thereafter spread both upstream and downstream. Evidence of considerable gene flow among populations was observed and genetic diversity was consistent with a larger, metapopulation that has not experienced bottlenecks or founder effects. We propose that high gene flow, possibly due to multiple invasions, has facilitated establishment of quagga mussel populations in the Volga River system. The Caspian Sea population (D. rostriformis rostriformis (=distincta)) was genetically more distinct than other populations, a finding that may be related to habitat differences. The geographical pattern of genetic divergence is not characteristic of isolation-by-distance but, rather, of long-distance dispersal, most likely mediated by commercial ships' ballast water transfer.

Hybridization dynamics between sympatric species of trout: loss of reproductive isolation.

Although reinforcement should enhance reproductive barriers in sympatric species, sympatric trout species do hybridize. Using mitochondrial and nuclear species markers, we investigated hybridization directionality, hybrid mating biases, and selection against hybrids in 13 sympatric cut-throat and rainbow trout populations on Vancouver Island, Canada. Approximately 50% of the genotyped fish were hybrid (F1 or higher-order) and populations ranged from very recent (all F1 hybrids) to extremely advanced higher-order hybridization. Overall, interbreeding was reciprocal, although some populations showed directional hybridization. Pronounced cytonuclear disequilibrium in post-F1 hybrids indicated a remarkable mating bias not previously reported, which is most likely because of behavioural reproductive preferences. Selection against hybrids was observed in only two populations, indicative of extrinsic selection. Two populations were 'hybrid swarms', with a complete loss of reproductive isolation. The complex hybridization dynamics in this system represent a valuable natural experiment of the genetic and evolutionary implications of recent and on-going interspecific hybridization.

Doppler ultrasound monitoring technology.

Developments in the signal processing of Doppler ultrasound used for the detection of fetal heart rate (FHR) have improved the operation of cardiotocographs. These developments are reviewed and the advantages and disadvantages of the various Doppler and signal processing methods are compared.

Comparison between linear array real time ultrasonic scanning and conventional compound scanning in the measurement of the fetal biparietal diameter.

Ultrasonic measurements of fetal biparietal diameter (BPD) obtained by the Real Time scanning (Dynamic Section Scan) and conventional compound scanning (Static Section Scan) in twenty patients were compared. A comparison between two operators, one of whom was relatively inexperienced, was also made, both measuring the same BPDs. The mean variance of 20 groups-of-four 'blind' measurements was reduced using dynamic scanning, particularly for the less experienced operator. A significant difference was observed between operators using conventional scanning but not when using dynamic scanning. Both operators were able to obtain results in a shorter time with dynamic scanning, the faster operator averaging less than one minute for each observation. Apparent advantages of dynamic scanning are discussed. The terms 'Dynamic Section Scan' and 'Static Section Scan' are suggested to emphasize the difference between the two imaging systems.

Accurate screening for abdominal aortic aneurysm.

In Birmingham a screening programme has been set up to detect abdominal aortic aneurysm in the male population between the ages of 65 and 75 years. The men to be screened are invited to their own general practitioners surgery and offered an abdominal ultrasound. The accuracy with which abnormalities can be detected is of crucial importance to a screening programme. Repeatability has been assessed for the single operator involved and is found to be +/- 1.3 mm. The accuracy of a single scan has been calculated by comparison with the measured diameter from a CT scan of the patient, taken subsequently. This comparison indicates that 95% of the differences between the two measurements can be expected to be less than 3.5 mm.