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.

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.

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation.

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings.

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus).

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.

Study of the relationship between thyroid hormones and lipid metabolism during KClO4-induced metamorphosis of landlocked lamprey, Petromyzon marinus.

This study examines the role of thyroid hormones (TH) (thyroxine and triiodothyronine) in regulating lipid metabolism of landlocked larval sea lampreys, Petromyzon marinus. Larvae were treated with either thyroxine (0.5 or 1 mg l(-1) water) or triiodothyronine (0.25 or 1 mg l(-1) water) in the presence or absence of the goitrogen, potassium perchlorate (KClO4) (0.05% w/v), for 4, 8, and 16 weeks. Treatment with KClO4 alone, which induced metamorphosis after 8 weeks and lowered plasma TH levels, reduced hepatic and renal total lipid content after 8 weeks of treatment. KClO4-induced lipid depletion after the 8-week treatment was supported by an increased rate of hepatic lipolysis, as indicated by increased triacylglycerol lipase activity. Furthermore, reduced lipogenesis in the liver was indicated by decreased hepatic acetyl-CoA carboxylase and diacylglycerol acyltransferase (DGAT) activities, and by decreased renal DGAT activity following 8 weeks of KClO4 treatment. Treatment of larvae for 4 weeks with TH alone resulted in either no change or a slight increase of lipid in the liver and kidney. TH treatments in combination with KClO4 failed to induce metamorphosis and, after up to 8 weeks, several TH treatments blocked changes in lipid content and enzyme activity associated with KClO4-induced metamorphosis. These experimental results suggest that TH deficiency during metamorphosis may promote lipid catabolism, while the presence of TH tends to protect/promote lipid reserves, perhaps favoring the larval condition. The actions of TH and KClO4 on metamorphosis-associated lipid metabolism in sea lampreys may be direct, permissive, and/or indirect via other factors.

Temperature and KClO(4)-induced metamorphosis in the sea lamprey (Petromyzon marinus).

Larval sea lampreys (Petromyzon marinus) were exposed to either a warm (18 degrees C) or a cold (3 degrees C) water temperature and either with (treated) or without (untreated) the presence of potassium perchlorate (KClO4). After 23 weeks, larvae were examined for signs of metamorphosis and serum samples were collected to assay thyroxine (T4) and 3,5,3'-triiodothyronine (T3) concentrations. Water temperature did not significantly affect serum T4 or T3 concentrations in untreated larvae and no metamorphosis occurred in these groups. Serum T4 concentrations were not significantly different between the two temperature groups treated with KClO4. However, serum T3 concentrations were significantly higher in the cold water, KClO4-treated larvae (5.4 nmol/l) than in the warm water, KClO4-treated larvae (1.2 nmol/l). KClO4 treatment at a warm water temperature induced metamorphosis in all larvae and resulted in serum T4 and T3 concentrations which were 66 and 95% lower, respectively, than untreated larvae in warm water. Despite having significantly lower serum T4 and T3 concentrations (73 and 80%, respectively) than untreated cold water larvae, metamorphosis was not observed in cold water, KClO4-treated larvae. The results of this study indicate that warm water is a requirement for the successful induction of metamorphosis with KClO4, and provide further evidence of water temperature as an important factor in the metamorphosis of lampreys.

Blocking of KC1O4-induced metamorphosis in premetamorphic sea lampreys by exogenous thyroid hormones (TH); effects of KC1O4 and TH on serum TH concentrations and intestinal thyroxine outer-ring deiodination.

Immediately premetamorphic larval sea lampreys (Petromyzon marinus) (>/=120 mm in length) were treated for 4, 8, or 16 weeks with one of two concentrations of either exogenous thyroxine (T4; 1 or 0.5 mg/L) or 3,5,3'-triiodothyronine (T3; 1 or 0.25 mg/L) in the presence or absence of the goitrogen potassium perchlorate (KC1O4; 0.05%) as well as with KC1O4 alone. Larvae from all treatments were examined for signs of metamorphosis, changes in serum T4 and T3 concentrations (serum T4 and serum T3), and changes in intestinal T4 outer-ring (5') deiodination to T3 (T4ORD). KC1O4 depressed both serum T4 and T3 and induced metamorphosis in 80% of larvae treated for 8 weeks or longer. However, neither effect was observed in larvae exposed to KC1O4 combined with either thyroid hormone (TH). These data confirm previous suggestions that exogenous TH blocks KC1O4-induced metamorphosis by elevating serum TH concentrations, and provide evidence that declines in serum TH concentrations are mandatory for precocious metamorphosis. Serum T4, but not serum T3, was elevated following exogenous T4 treatment in the presence or absence of KC1O4. This maintenance of serum T3 at control levels, in the presence of a T4 challenge, was not due to decreases in intestinal T4ORD activity, since T4ORD activity was not affected by any treatments in the study. Exogenous T3 elevated both serum T4 and T3. However, serum T3 in T3-treated larvae decreased with time, suggesting a stringent T3 regulation. Elevation of serum T4 following T3 treatment may have been a result of either inhibition of T4 metabolism, or stimulation of T4 secretion by the endostyle. Based on these results, we conclude that (i) exogenous TH blocks KClO4-induced metamorphosis in sea lampreys and (ii) serum T3 is maintained at control levels despite elevations in serum T4 (its immediate precursor), but this does not involve any changes in intestinal T4ORD activity.

Effects of exogenous thyroxine (T4) and triiodothyronine (T3) on spontaneous metamorphosis and serum T4 and T3 levels in immediately premetamorphic sea lampreys, Petromyzon marinus.

The effect of exogenous thyroid hormones (TH), thyroxine (T4) or triiodothyronine (T3), on spontaneous metamorphosis and serum T4 and T3 levels was examined in immediately premetamorphic sea lampreys (Petromyzon marinus) from two populations. Size (> or = 120 mm in length and 3.0 g in weight) and a condition factor (CF) of > or = 1.50 were used to predict the number of larvae that were expected to metamorphose. The smallest size and lowest CF found in metamorphosing animals of each population (i.e., the minimum length, weight, and CF) were also used in our assessment. Untreated larvae from Putnam Creek metamorphosed at a larger size (minimums; 134 mm, 4.12 g) than anticipated, out the minimum CF (1.59) and the incidence of metamorphosis (4/5 based on minimums) were consistent with results from the Salmon River population (minimums: 121 mm, 3.15 g, 1.54 CF, 8/9 metamorphosing). In the two experiments, T4-treated animals showed the predicted incidence of metamorphosis (2/2, 10/10), but significantly fewer larvae metamorphosed in the T3-treated groups (1/5, 5/11) than predicted. It was concluded that exogenous T3 administration affected the incidence of metamorphosis. In the treatment groups, serum TH levels in most nonmetamorphosing and metamorphosing animals were significantly higher than controls. Metamorphosing animals exposed to either TH had lower serum TH concentrations than nonmetamorphosing animals. A decline in serum TH levels is an early feature of metamorphosis in lampreys, but the artificial maintenance of elevated serum levels of TH cannot inhibit the decline in spontaneous metamorphosis. If the depression of serum TH levels contributes to the initiation of metamorphic change, the magnitude of the decline is not a contributing factor.

The effects of exogenous thyroxine (T4) or triiodothyronine (T3), in the presence and absence of potassium perchlorate, on the incidence of metamorphosis and on serum T4 and T3 concentrations in larval sea lampreys (Petromyzon marinus L.).

Larval sea lampreys (Petromyzon marinus) measuring 100-119 mm in length were exposed to thyroxine (T4; 10 mg liter-1) or 3,5,3'-triiodothyronine (T3; 1 mg liter-1) in the presence and absence of the goitrogen potassium perchlorate (KClO4; 0.01%), for 4-24 weeks. Every 4 weeks, treated and untreated (control) groups of sea lampreys were examined for external signs of metamorphosis and serum was assayed for T4 and T3 concentrations. Precocious metamorphosis was observed following 8, 12, and 24 weeks of KClO4 treatment; however, metamorphosis was not observed in any control, or T4-, T3-, T4+KClO4-, and T3+KClO4-treated larvae. In addition, serum T4 and T3 concentrations were 62 and 72% lower in KClO4-treated individuals than in control animals, respectively. Treatment with exogenous thyroid hormones (TH), in the presence or absence of KClO4, resulted in serum T4 concentrations which were significantly greater (1.2- to 58-fold) than those of the controls in all sampling periods except one, but serum T3 concentrations were not significantly elevated in more than 50% of the cases. TH+KClO4 treatments produced serum T3 concentrations which were significantly greater than those of KClO4-treated animals and never less than those of controls. These data indicate that larval sea lampreys have a tremendous capacity to take up and store exogenous T4 in their serum, but the uptake and/or serum storage of T3 appears to be stringently regulated. Also, the absence of both metamorphosis and a decline in serum TH concentrations in TH+KClO4-treated animals suggests that a decline in serum TH concentrations may be an essential factor contributing to the induction of metamorphosis by KClO4.