Neuroendocrine regulation of carbonic anhydrase expression in the gills of the euryhaline green crab, Carcinus maenas

The relationship between branchial carbonic anhydrase (CA) activity, CA gene expression and salinity, and potential mechanisms of regulation, was investigated in the euryhaline green crab, Carcinus maenas, acclimated to 33 ppt and transferred to 10 ppt, and the stenohaline rock crab, Cancer irroratus, acclimated to 32 ppt and transferred to 18 ppt. CA activity in green crabs acclimated to high and low salinity was a function of CA mRNA expression, with low salinity exposure resulting in an increase in both CA expression and activity. Eyestalk ablation (ESA) in green crabs acclimated to high salinity resulted in an increase in CA expression in the posterior, ion-transporting gills, in the absence of the low salinity stimulus. There were no changes in CA activity or expression in the anterior, respiratory gills. ESA also potentiated low salinity-stimulated CA induction, again, only in posterior gills. There were no changes in CA activity in any gills of Cancer irroratus, in response to either ESA or low salinity. These results suggest that CA expression in euryhaline, osmoregulating species, is under inhibitory regulation by a putative repressor found in the eyestalk, and that this mechanism is absent in stenohaline, osmoconforming species. CA expression is maintained at low, baseline levels in crabs acclimated to high salinity by the presence and action of this compound. The effects of the repressor appear to be reduced upon exposure to low salinity, allowing CA induction to occur.     

Recovery of growth of Hyphochytrium catenoides after exposure to environmental stress

ABSTRACT. The survival of an isolate of Hyphochytrium catenoides collected from soil in the Blue Mountains in eastern New South Wales, Australia, was tested under extreme conditions in the laboratory. This isolate recovered growth after being subjected to drying on filter paper, to heat while desiccated, to hypersalinity, to strict anaerobic conditions, to freezing temperatures, and to a short period in solutions at pH 2.8–11.2. The capacity to survive under these conditions in the laboratory suggests adaptation to fluctuating conditions in the soil. The partial DNA sequence of the 28S ribosomal RNA gene in the isolate from New South Wales was 98% similar to that in an isolate from Arizona with a similar morphology.     

Domestication and the behavior-genetic analysis of captive populations

Captive environments are believed to produce behavioral changes in animal populations that may limit our ability to generalize back to natural populations. These behavioral changes are thought to be associated with one or both of the following: (a) changes in frequencies of genes or gene complexes due to the effects of inbreeding or to changes in selection pressure; (b) changes in development of the phenotype due to the effects of changes in environmental variables. Inbreeding leads to increase in homozygosity, that may result in developmental anomalies because of a breakdown in developmental homeostasis. Changes in selection pressure may disrupt coadapted gene complexes that have evolved in the wild. Often, domestication is believed to result in individuals that are “degenerate”; i.e. inferior to individuals in the wild. However, this notion has received no empirical support. In fact, if phenotype changes do occur under domestication, these are usually quantitative, not qualitative, in nature. We suggest that the study of the domestication process may reveal evolutionary principles that would be difficult to discover in other ways, and the zoological parks may be ideal situations for such research.     

Sporophytic response to pollen selection for Alachlor tolerance in maize

In order to assess the efficiency of male gametophytic selection (MGS) for crop improvement, pollen selection for tolerance to herbicide was applied in maize. The experiment was designed to test the parallel reactivity to Alachlor of pollen and plants grown in controlled conditions or in the field, the response to pollen selection in the sporophytic progeny, the response to a second cycle of MGS, and the transmission of the selected trait to the following generations. The results demonstrated that pollen assay can be used to predict Alachlor tolerance under field conditions and to monitor the response to selection. A positive response to selection applied to pollen in the sporophytic progeny was obtained in diverse genetic backgrounds, indicating that the technique can be generally included in standard breeding programs; the analysis of the data produced in a second selection cycle indicated that the selected trait is maintained in the next generation.     

稻虾共作水体浮游植物群落结构特征分析

为认识稻虾共作水体浮游植物群落结构特征, 于2016年4月至12月对江汉平原4处稻虾共作水体浮游植物和理化因子开展了逐月调查与分析。共鉴定出浮游植物7门124种, 其中绿藻78种、蓝藻16种、硅藻15种、裸藻3种、隐藻2种、甲藻7种、金藻3种。6—9月浮游植物的种类数、细胞密度、叶绿素a含量达到最大值, 最小值出现在12月份; 浮游植物细胞密度波动范围在1.37×105—2.93×108 cells/L, 叶绿素a含量的变化范围为0.15—208.60 μg/L。调查期间浮游植物的优势种共28种, 主要优势种有颤藻、蓝纤维藻、微囊藻、小球藻、隐藻等。浮游植物Shannon-Wiener多样性指数周年变化范围为0.64—6.3, 多样性指数最高出现在10月份, 最低出现在8月份。结果显示, 稻虾共作水体浮游植物群落结构较复杂, 细胞密度变化显示时空的一致性, 优势种组成以及优势度存在明显的空间差异(P<0.05), 稻田的浅水环境以及小龙虾的养殖行为显著影响浮游植物群落的结构。鉴于藻类作为初级生产者对于水生态环境和小龙虾健康具有重要作用, 关注种养结合水体中藻类群落的演变规律对于保障稻田种养的综合效益具有积极意义。     

Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS)

Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.     

湖北省褐飞虱对吡虫啉、噻嗪酮及氟虫腈的抗药性监测

采用稻茎浸渍法监测湖北省武穴、天门、监利、通城、孝感、公安及枣阳七地褐飞虱Nilaparvata lugens(Sta1)田间种群对吡虫啉、噻嗪酮、氟虫腈的抗药性。结果表明:2006、2007和2008年3年7地褐飞虱种群对吡虫啉的抗性分别为98.89～389.19倍、69.00～153.33倍和56.32～116.89倍,达高水平至极高水平抗性,但3年期间总体抗性水平呈现一定程度的下降趋势;2007和2008年2年7地褐飞虱对噻嗪酮的抗性分别为13.39～41.06倍和6.94～20.44倍,达低水平至高水平抗性;2007和2008年2年7地褐飞虱对氟虫腈抗性分别为0.97～2.64倍和0.63～1.42倍,为敏感阶段。此外,还对褐飞虱的治理进行讨论。     

The role of T56 in controlling the flexibility of the distal histidine in dehaloperoxidase-hemoglobin from Amphitrite ornata

The activation of dehaloperoxidase-hemoglobin (DHP) to form a ferryl intermediate requires the distal histidine, H55, to act as an acid base catalyst. The lack of ancillary amino acids in the distal pocket to assist in this process makes H55 even more important to the formation of active intermediates than in conventional peroxidases. Therefore, one can infer that the precise conformation H55 may greatly affect the enzymatic activity. Using site-direct mutagenesis at position T56, immediately adjacent to H55, we have confirmed that subtle changes in the conformation of H55 affect the catalytic efficiency of DHP. Mutating T56 to a smaller amino acid appears to permit H55 to rotate with relatively low barriers between conformations in the distal pocket, which may lead to an increase in catalytic activity. On the other hand, larger amino acids in the neighboring site appear to restrict the rotation of H55 due to the steric hindrance. In the case of T56V, which is an isosteric mutation, H55 appears less mobile, but forced to be closer to the heme iron than in wild type. Both proximity to the heme iron and flexibility of motion in some of the mutants can result in an increased catalytic rate, but can also lead to protein inactivation due to ligation of H55 to the heme iron, which is known as hemichrome formation. A balance of enzymatic rate and protein stability with respect to hemichrome formation appears to be optimum in wild type DHP (WT-DHP).     

Cell wall and protoplast isoperoxidases in tobacco plants in relation to mechanical injury and infection with tobacco mosaic virus

Leaves and pith of Turkish, Wisconsin 38, and Samsun NN tobacco (Nicotiana tabacum) varieties, which differ in their sensitivity to tobacco mosaic virus, showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and wall-free, ionically, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or leaf infection with tobacco mosaic virus. The distinctive isoperoxidases which reacted to infection were the same as those responsive to mechanical injury, confirming that the enzyme reaction to infection results from a nonspecific response to injury. The increase in peroxidase activity in response to infection or mechanical injury, or both, was greater in young tissue than in the older ones. The great increase in Samsun NN leaves and no increase in those of the two other varieties in response to infection may be due to differences in the degree to which the pathogen affected processes controlling the nonspecific peroxidase reaction to injury. Peroxidase development in the infected Samsun NN leaves was due to isoenzymes which form the wall-bound fraction in very young tissues, and to those which increase in activity with aging in the protoplast and wall-free fractions. In mechanically injured tissue, only the first group of isoenzymes increased in activity. In Samsun NN plants, the increased peroxidase activity in upper intact leaves above the infected ones was only due to isoenzymes whose activity increases with both normal and virus-accelerated senescence. Peroxidase reaction to challenge inoculation in these leaves was the same whether the lower ones were intact, infected and/or mechanically injured. Thus, the induced systemic resistance to tobacco mosaic virus may be due to other than peroxidase factors.     

‘Only Fathers Smoking’ Contributes the Most to Socioeconomic Inequalities: Changes in Socioeconomic Inequalities in Infants’ Exposure to Second Hand Smoke over Time in Japan

BackgroundExposure to second hand smoke (SHS) is one of the major causes of premature death and disease among children. While socioeconomic inequalities exist for adult smoking, such evidence is limited for SHS exposure in children. Thus, this study examined changes over time in socioeconomic inequalities in infants’ SHS exposure in Japan.MethodsThis is a repeated cross-sectional study of 41,833 infants born in 2001 and 32,120 infants born in 2010 in Japan from nationally representative surveys using questionnaires. The prevalence of infants’ SHS exposure was determined and related to household income and parental education level. The magnitudes of income and educational inequalities in infants’ SHS exposure were estimated in 2001 and 2010 using both absolute and relative inequality indices.ResultsThe prevalence of SHS exposure in infants declined from 2001 to 2010. The relative index of inequality increased from 0.85 (95% confidence interval [CI], 0.80 to 0.89) to 1.47 (95% CI, 1.37 to 1.56) based on income and from 1.22 (95% CI, 1.17 to 1.26) to 2.09 (95% CI, 2.00 to 2.17) based on education. In contrast, the slope index of inequality decreased from 30.9 (95% CI, 29.3 to 32.6) to 20.1 (95% CI, 18.7 to 21.5) based on income and from 44.6 (95% CI, 43.1 to 46.2) to 28.7 (95% CI, 27.3 to 30.0) based on education. Having only a father who smoked indoors was a major contributor to absolute income inequality in infants’ SHS exposure in 2010, which increased in importance from 45.1% in 2001 to 67.0% in 2010.ConclusionsThe socioeconomic inequalities in infants’ second hand smoke exposure increased in relative terms but decreased in absolute terms from 2001 to 2010. Further efforts are needed to encourage parents to quit smoking and protect infants from second hand smoke exposure, especially in low socioeconomic households that include non-smoking mothers.     

Macrolide resistance and <Emphasis Type="Italic">In Vitro</Emphasis> selection of resistance to antibiotics in <Emphasis Type="Italic">Lactobacillus</Emphasis> isolates

Spreading of resistance to antibiotics is of great concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria may transfer determinants of resistance to pathogens, studies on development of resistance should include also lactobacilli. Resistance to macrolides, penicillins and tetracycline was determined in 40 isolates of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus crispatus, and Lactobacillus casei isolated from faeces of apparently healthy volunteers. Frequency of mutation and changes in susceptibility after serial exposure to these antibiotics at concentrations of 4× and 8× MIC were evaluated in susceptible isolates. Acquired resistance was defined as an increment in MIC values of at least four times in respect to the pre-selection values. Resistance to macrolides and/or tetracycline was identified in 14 and 4 isolates, respectively. ermB gene and A2058G mutation in 23S rRNA were detected in macrolide resistant isolates. Frequencies of mutation of susceptible isolates (n=26) were lower for ampicillin and erythromycin than for tetracycline. Serial exposure to antibiotics led to selection of resistant mutants. However, acquired resistance was rather unstable and was lost after subcultures in antibiotic-free medium in most mutants. Resistance to erythromycin was associated to a A2058G mutation in 23S rRNA. In conclusion, results indicate that resistance to macrolides and tetracycline is present among intestinal lactobacilli. Decrease in susceptibility following serial exposure to antibiotics might occur in lactobacilli, in a strain- and antibiotic-dependent way. Since lactobacilli are often used as probiotics, their ability to acquire resistance should be evaluated for isolates candidate to be included in probiotics based products.     

Comparative ungulate dynamics: the devil is in the detail

Attempts to relate species differences in population dynamics to variation in life histories rely on the assumption that the causes of contrasts in demography are sufficiently simple to be derived from first principles. Here, we investigate the causes of contrasts in dynamics between two ungulate populations on Hebridean islands (red deer and Soay sheep) and show that differences in stability, as well as in the effects of variation in density and climate, are related to differences in timing of reproduction relative to seasonal variation in resource abundance. In both populations, attempts to predict changes in population size sufficiently accurately for the results to be useful for management purposes require a knowledge of the responses of different age and sex categories to changes in density and climate, as well as of population structure.     

Fingerling production and stock enhancement of Mahisefid (Rutilus frisii kutum) lessons for others in the south of Caspian Sea

Rutilus frisii kutum (Kamensky 1901) is one of the economically important fishes that migrate for spawning to rivers in the Caspian Sea. However, the fish populations have slowly decreased in recent years. The declining of these resources has resulted from some activities by the Iranian Fisheries Organization (IFO is responsible for stock enhancement) to catch some broodstocks of Rutilus frisii kutum from their natural spawning rivers. The broodstocks are caught for artificial propagation of the fish. Artificial propagations are carried out every year to produce fingerlings to be released into the rivers in the Caspian Sea. In recent years, total catch of this fish have greatly fluctuated due to the disruption of the natural spawning grounds and over fishing. The substantial reduction to 1,298 metric tons, the lowest total catch reported in 1984–1985, could be due to over-exploitation of the fishery resources. However, the total catch has increased after the fingerlings release programs started in 1979. The total numbers of Rutilus frisii kutum fingerlings released had increased from 12 million to 225 million in 2002, to 155 million pieces in 2003, to 179 million pieces in 2004, 229 million pieces in 2005, 174 million pieces in 2006, 262 million pieces in 2007 and 187.1 in 2008. The total catch was also increased from 6,417 metric ton to 8,984 metric ton, to 7,036 metric ton, to 9,631 metric ton and 16,117, 17,196, 14,835 in years 2002, 2003, 2004, 2005, 2006, 2007 and 2008, respectively.     

Brain size, innovative propensity and migratory behaviour in temperate Palaearctic birds

The evolution of migration in birds remains an outstanding, unresolved question in evolutionary ecology. A particularly intriguing question is why individuals in some species have been selected to migrate, whereas in other species they have been selected to be sedentary. In this paper, we suggest that this diverging selection might partially result from differences among species in the behavioural flexibility of their responses to seasonal changes in the environment. This hypothesis is supported in a comparative analysis of Palaearctic passerines. First, resident species tend to rely more on innovative feeding behaviours in winter, when food is harder to find, than in other seasons. Second, species with larger brains, relative to their body size, and a higher propensity for innovative behaviours tend to be resident, while less flexible species tend to be migratory. Residence also appears to be less likely in species that occur in more northerly regions, exploit temporally available food sources, inhabit non-buffered habitats and have smaller bodies. Yet, the role of behavioural flexibility as a response to seasonal environments is largely independent of these other factors. Therefore, species with greater foraging flexibility seem to be able to cope with seasonal environments better, while less flexible species are forced to become migratory.     

Effects of immobilization of a limb on the maturation of a peripheral nerve in kittens

Right or left hindlimbs of kittens were immobilized in a plaster cast, in the resting position. Three groups of animals belonged to the same age group, one to older, and another to younger. Kittens in the same age group were subjected to progressively increasing periods of immobilization, the older group to a shorter and the younger to the longest period. By virtue of this arrangement, it was found that the demonstrated changes in the peripheral nerve, which included a decrease in the myelinated nerve fibre population and a reduction in the mean nerve fibre diameter, have been proportionate among the animals in the same age group, more pronounced in younger, and less so in older ones, indicating that in younger animals longer periods of immobilization lead to a greater degree of harmful effects, although it was also evident that younger animals attempt to adapt to such adverse conditions.     

Strategies adopted by the mudskipper Boleophthalmus boddaerti to survive sulfide exposure in normoxia or hypoxia

The effects of sulfide on the energy metabolism of Boleophthalmus boddaerti in normoxia and hypoxia were examined. The 24-, 48-, and 96-h LC50 values of sulfide for B. boddaerti with body weight ranging from 11.6 to 14.2 g were 0.786, 0.567, and 0.467 mM, respectively. The tolerance of B. boddaerti to sulfide was not due to the presence of a sulfide-insensitive cytochrome c oxidase. There was no accumulation of lactate in the muscle and liver of specimens exposed to sulfide in normoxia. In addition, the levels of ATP, AMP, and energy charge in both the muscle and the liver were unaffected. These results indicate that B. boddaerti was able to sustain the energy supply required for its metabolic needs via mainly aerobic respiration when exposed to sulfide (up to 0.4 mM) in normoxia. Exposure of B. boddaerti simultaneously to hypoxia and 0.2 mM sulfide for 48 h resulted in decreases in the ATP levels in the muscle and liver. However, the energy charge in both tissues remained unchanged, and the level of lactate accumulated in the muscle was too low to have any major contribution to the energy budget of the fish. Our results reveal that B. boddaerti possesses inducible mechanisms to detoxify sulfide in an ample supply or a lack of O2. In normoxia, it detoxified sulfide to sulfate, sulfite, and thiosulfate. There were significant increases in the activities of sulfide oxidase in the muscle and liver of specimens exposed to sulfide, with that in the liver being >13-fold higher than that in the muscle. However, in hypoxia, sulfide oxidase activity in the liver was suppressed in response to environmental sulfide. In such conditions, there were significant increases in the activities of sulfane sulfur-forming enzyme(s) in the muscle and liver that were not observed in specimens exposed to sulfide in normoxia. Correspondingly, there were no changes in the levels of sulfate or sulfite in the muscle or liver. Instead, B. boddaerti detoxified sulfide mainly to sulfane sulfur in hypoxia. In conclusion, B. boddaerti was able to activate different mechanisms to detoxify sulfide, producing different types of detoxification products in normoxia and hypoxia.     

Active drag, useful mechanical power output and hydrodynamic force coefficient in different swimming strokes at maximal velocity.

By comparing the time of the same distance swum with and without an added resistance, under the assumption of an equal power output in both cases, the drag of 73 top swimmers was estimated. The active drag Fr(a.d.) at maximal swimming velocities varied considerably across strokes and individuals. In the females Fr(a.d.) ranged from 69.78 to 31.16 N in the front-crawl, from 83.04 to 37.78 N in dolphin, from 93.56 to 45.19 N in breaststroke, and from 65.51 to 37.79 N in back-stroke. In the males Fr(a.d.) ranged from 167.11 to 42.23 N in front-crawl, from 156.09 to 46.95 N in dolphin, from 176.87 to 55.61 N in breaststroke, and from 146.28 to 46.36 N in back-stroke. Also, the ratio of Fr(a.d.) to the passive drag Fr(a.d.) as determined for the analogical velocity in a tugging condition (in standard body position-front gliding) shows considerable individual variations. In the female swimmers variations in Fr(a.d.)/Fr(p.d.) ranged from 145.17 to 59.94% in front-crawl, from 192.39 to 85.57% in dolphin, from 298.03 to 124.50% in breaststroke, and from 162.87 to 85.61% in back-stroke. In the male swimmers variations in Fr(a.d.)/Fr(p.d.) ranged from 162.24 to 62.39% in front-crawl, from 191.70 to 70.38% in dolphin, from 295.57 to 102.83% in breaststroke, and from 198.82 to 74.48% in back-stroke. The main reason for such variations is found in the individual features of swimming technique and can be quantitatively estimated with the hydrodynamic force coefficient, which thus provides an adequate index of technique.     

Effects of estrogen-depletion on rat casein gene expression

Mammary tissue from rats that had been ovariectomized and adrenalectomized 4 weeks previously was compared to that from intact rats in terms of epithelial content and hormone-responsiveness in vitro. The endocrinectomy resulted in about a 30% enlargement of the gland, but led to a loss of only about 12% of the epithelium. This estrogen-depleted epithelium was able to acquire full responsiveness in vitro to insulin in terms of the accumulation of alpha-aminoisobutyric acid, and induction of glucose-6-phosphate and gluconate-6-phosphate dehydrogenases. It was also fully responsive to cortisol in relation to the induction of NADH-cytochrome C reductase, and to prolactin in terms of total RNA synthesis. However, estrogen-depletion led to an 82% loss in the ability of a unit amount of the epithelium to synthesize casein in response to these 3 hormones, and to a similar loss in relation to the accumulation of 25K casein mRNA. Estrogen administration in vivo could prevent and reverse the casein lesion. The disparity between constitutive and casein hormone-responsiveness in the absence of estrogen is discussed in relation to cell commitment.     

Trade-offs and seasonal variation in territorial defence and predator evasion in the European Robin Erithacus rubecula

The low incidence of intraspecific combat in territorial systems has traditionally been accounted for by theories that emphasize the bio-energetic advantages or the diminished risk of injury of threat display posture when compared with combat. Recently, however, it has been suggested that territory-holding passerines engaging in highly aggressive defensive behaviour are likely to pay a cost in terms of reduced vigilance for avian predators. To examine this further, two defensive options (combat and threat display) were evoked and observed in territorial European Robins Erithacus rubecula , in both winter and summer. It was found that Robins that engaged in combat during simulated territorial intrusion were significantly slower to react to a stuffed Sparrowhawk Accipiter nisus , than those eliciting threat display. Therefore, the decreased vigilance in escalated fighting may result in higher vulnerability to predation, and this might help, in part, to explain why threat display is favoured over direct combat. Moreover, the mean time to evade the predator was significantly longer in summer than in winter in threat-displaying Robins. In addition to this finding, a clear difference in the type of behaviour adopted between the seasons was observed, with lower incidence of combatative response during the winter months. This difference in response to the appearance of a predator is discussed in relation to reduced levels of safe cover, a reduction in the availability of alternative prey during the winter months and to seasonal fluctuations in plasma androgen levels.     

WHAT MOTIVATES WOMEN TO TAKE PART IN CLINICAL AND BASIC SCIENCE ENDOMETRIOSIS RESEARCH?*

BACKGROUND: The objective of this study was to identify factors motivating women to take part in endometriosis research and to determine if these factors differ for women participating in clinical versus basic science studies. METHODS: A consecutive series of 24 women volunteering for participation in endometriosis-related research were asked to indicate, in their own words, why they chose to volunteer. In addition, the women were asked to rate, on a scale of 0 to 10, sixteen potentially motivating factors. The information was gathered in the form of an anonymous self-administered questionnaire. RESULTS: Strong motivating factors (mean score > 8) included potential benefit to other women's health, improvement to one's own condition, and participation in scientific advancement. Weak motivating factors (mean score < 3) included financial compensation, making one's doctor happy, and use of 'natural' products. No difference was detected between clinical and basic science study participants. CONCLUSION: This study is the first study to specifically investigate the factors that motivate women to take part in endometriosis research. Understanding why women choose to take part in such research is important to the integrity of the informed consent process. The factors most strongly motivating women to participate in endometriosis research related to improving personal or public health; the weakest, to financial compensation and pleasing the doctor.