Detoxification and elimination of nicotine by nectar-feeding birds.

Many dilute nectars consumed by bird pollinators contain secondary metabolites, potentially toxic chemicals produced by plants as defences against herbivores. Consequently, nectar-feeding birds are challenged not only by frequent water excess, but also by the toxin content of their diet. High water turnover, however, could be advantageous to nectar consumers by enabling them to excrete secondary metabolites or their transformation products more easily. We investigated how the alkaloid nicotine, naturally present in nectar of Nicotiana species, influences osmoregulation in white-bellied sunbirds Cinnyris talatala and Cape white-eyes Zosterops virens. We also examined the metabolic fate of nicotine in these two species to shed more light on the post-ingestive mechanisms that allow nectar-feeding birds to tolerate nectar nicotine. A high concentration of nicotine (50 µM) decreased cloacal fluid output and increased its osmolality in both species, due to reduced food intake that led to dehydration. White-eyes excreted a higher proportion of the ingested nicotine-containing diet than sunbirds. However, sugar concentration did not affect nicotine detoxification and elimination. Both species metabolised nicotine, excreting very little unchanged nicotine. Cape white-eyes mainly metabolised nicotine through the cotinine metabolic pathway, with norcotinine being the most abundant metabolite in the excreta, while white-bellied sunbirds excreted mainly nornicotine. Both species also utilized phase II conjugation reactions to detoxify nicotine, with Cape white-eyes depending more on the mercapturic acid pathway to detoxify nicotine than white-bellied sunbirds. We found that sunbirds and white-eyes, despite having a similar nicotine tolerance, responded differently and used different nicotine-derived metabolites to excrete nicotine.

Effects of nicotine on the digestive performance of nectar-feeding birds reflect their relative tolerance to this alkaloid.

The paradox of secondary metabolites, toxic defence compounds produced by plants, in nectar and fruits is well known. Deterrence of feeding by nectarivorous and frugivorous birds is better understood than the effect of these chemicals on the digestive performance of birds. Digestive parameters such as transit time and sugar assimilation are important in assessing nutrient utilization and deterrence may be related to post-ingestive effects involving these parameters. Nectar and many fruits contain mainly sugars and water, and avian consumers compensate for low sugar content in their diet by increasing food intake: this may also increase their intake of secondary metabolites. We investigated how the alkaloid nicotine, naturally present in nectar of Nicotiana species, influences compensatory feeding and digestive performance of nectar-feeding birds. High nicotine concentration negatively affected compensatory feeding and apparent assimilation efficiency of white-bellied sunbirds Cinnyris talatala and Cape white-eyes Zosterops virens; but nicotine slowed gut transit time only in the latter species. In contrast, food intake and digestive performance of dark-capped bulbuls Pycnonotus tricolor was unaffected by nicotine up to a concentration of 50µM. Bulbuls are primarily frugivorous; hence, they are more exposed to secondary metabolites than sunbirds and possibly white-eyes. Because their diet is richer in toxins, frugivorous birds may have evolved more efficient detoxification strategies than those of specialist nectar-feeding birds.

Distinct requirements of Autophagy-related genes in programmed cell death.

Although most programmed cell death (PCD) during animal development occurs by caspase-dependent apoptosis, autophagy-dependent cell death is also important in specific contexts. In previous studies, we established that PCD of the obsolete Drosophila larval midgut tissue is dependent on autophagy and can occur in the absence of the main components of the apoptotic pathway. As autophagy is primarily a survival mechanism in response to stress such as starvation, it is currently unclear if the regulation and mechanism of autophagy as a pro-death pathway is distinct to that as pro-survival. To establish the requirement of the components of the autophagy pathway during cell death, we examined the effect of systematically knocking down components of the autophagy machinery on autophagy induction and timing of midgut PCD. We found that there is a distinct requirement of the individual components of the autophagy pathway in a pro-death context. Furthermore, we show that TORC1 is upstream of autophagy induction in the midgut indicating that while the machinery may be distinct the activation may occur similarly in PCD and during starvation-induced autophagy signalling. Our data reveal that while autophagy initiation occurs similarly in different cellular contexts, there is a tissue/function-specific requirement for the components of the autophagic machinery.

Resistance of developing honeybee larvae during chronic exposure to dietary nicotine.

The effects of pesticides on honeybee larvae are less understood than for adult bees, even though larvae are chronically exposed to pesticide residues that accumulate in comb and food stores in the hive. We investigated how exposure to a plant alkaloid, nicotine, affects survival, growth and body composition of honeybee larvae. Larvae of Apis mellifera scutellata were reared in vitro and fed throughout development on standard diets with nicotine included at concentrations from 0 to 1000µg/100g diet. Overall mortality across all nicotine treatments was low, averaging 9.8% at the prepupal stage and 18.1% at the white-eyed pupal stage, but survival was significantly reduced by nicotine. The mass of prepupae and white-eyed pupae was not affected by nicotine. In terms of body composition, nicotine affected water content but did not influence either protein or lipid stores of white-eyed pupae. We attribute the absence of consistent negative effects of dietary nicotine to detoxification mechanisms in developing honeybees, which enable them to resist both natural and synthetic xenobiotics.

Can I leave the theatre? A key to more reliable workplace-based assessment.

BACKGROUND: The value of workplace-based assessments such as the mini-clinical evaluation exercise (mini-CEX), and clinicians' confidence and engagement in the process, has been constrained by low reliability and limited capacity to identify underperforming trainees. We proposed that changing the way supervisors make judgements about trainees would improve score reliability and identification of underperformers. Anaesthetists regularly make decisions about the level of trainee independence with a case, based on how closely they need to supervise them. We therefore used this as the basis for a new scoring system. METHODS: We analysed 338 mini-CEXs where supervisors scored trainees using the conventional system, and also scored trainee independence, based on the need for direct, or more distant, supervision. As supervisory requirements depend on case difficulty, we then compared the actual trainee independence score and the expected trainee independence score obtained externally. RESULTS: Compared with the conventional scoring system used in previous studies, reliability was very substantially improved using a system based on a trainee's level of independence with a case. Reliability improved further when this score was corrected for case difficulty. Furthermore, the new scoring system overcame the previously identified problem of assessor leniency and identified a number of trainees performing below expectations. CONCLUSIONS: Supervisors' judgements on trainee independence with a case, based on the need for direct or more distant supervision, can generate reliable scores of trainee ability without the need for an onerous number of assessments, identify trainees performing below expectations, and track trainee progress towards independent specialist practice.

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila.

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.

Cell death by autophagy: facts and apparent artefacts.

Autophagy (the process of self-digestion by a cell through the action of enzymes originating within the lysosome of the same cell) is a catabolic process that is generally used by the cell as a mechanism for quality control and survival under nutrient stress conditions. As autophagy is often induced under conditions of stress that could also lead to cell death, there has been a propagation of the idea that autophagy can act as a cell death mechanism. Although there is growing evidence of cell death by autophagy, this type of cell death, often called autophagic cell death, remains poorly defined and somewhat controversial. Merely the presence of autophagic markers in a cell undergoing death does not necessarily equate to autophagic cell death. Nevertheless, studies involving genetic manipulation of autophagy in physiological settings provide evidence for a direct role of autophagy in specific scenarios. This article endeavours to summarise these physiological studies where autophagy has a clear role in mediating the death process and discusses the potential significance of cell death by autophagy.

Tricuspid valve dysplasia with severe tricuspid regurgitation: fetal pulmonary artery size predicts lung viability in the presence of small lung volumes.

Congenital tricuspid valve disease (Ebstein's anomaly, tricuspid valve dysplasia) with severe tricuspid regurgitation and cardiomegaly is associated with poor prognosis. Fetal echocardiography can accurately measure right atrial enlargement, which is associated with a poor prognosis in the fetus with tricuspid valve disease. Fetal lung volumetric assessments have been used in an attempt to predict viability of fetuses using ultrasonogram and prenatal MRI. We describe a fetus with tricuspid dysplasia, severe tricuspid regurgitation, right atrial enlargement and markedly reduced lung volumes. The early gestational onset of cardiomegaly with bilateral lung compression raised the possibility of severe lung hypoplasia with decreased broncho-alveolar development. Use of fetal echocardiography with measurement of pulmonary artery size combined with prenatal MRI scanning of lung volumes resulted in an improved understanding of this anomaly and directed the management strategy towards a successful Fontan circulation.

Nectar concentration preferences and sugar intake in the white-bellied sunbird, Cinnyris talatala (Nectariniidae).

Plant nectar is a simple food consumed by many different animals. Preferences regarding its components, especially sugars, have been studied for many species, but the preferences of nectar-feeding birds for different sugar concentrations are less well known than their sugar type preferences. The concentration preferences of white-bellied sunbirds, Cinnyris talatala, were examined using paired solutions of either sucrose or equicaloric 1:1 mixtures of glucose and fructose. Preferences were tested over a broad concentration range of 0.25-2.5 M sucrose equivalents (using 0.25 or 0.5 M differences between pairs). On both sucrose and hexose diets, the higher concentration was preferred up to 1 M, but there were no significant preferences above this concentration, except that birds preferred 1.5 to 2 M sucrose. As with other nectar-feeding vertebrates, the laboratory preferences of sunbirds do not explain the low concentration of their natural nectars. We recorded apparent excess sugar consumption during 6 h preference tests involving concentrated hexose diets; this could be due to digestive constraints or viscosity differences between sucrose and hexose solutions.

Parental perception of time and decision-making in neonatal research.

OBJECTIVE: To assess the impact of time on parental decision-making for research participation for neonates with congenital heart disease. STUDY DESIGN: Interviews were conducted with 37 parents of 19 neonates with congenital heart disease who were eligible for three different studies: genetic etiology of congenital heart disease, heart rate variability (HRV) and structural and functional cranial magnetic resonance imaging (MRI). All parents were asked the same questions: (1) 'Did you have adequate time to make a decision about research?' and (2) 'Why?' Differences between groups (reporting adequate and inadequate time) were evaluated using Fisher's exact tests; central themes were examined using qualitative analysis. RESULT: Of those parents who reported having adequate time to make their decision (22 of 37), the majority chose to participate when compared to those who reported inadequate time (genetics study, P<0.01; HRV, P=0.05; MRI, P<0.01). For the parents reporting inadequate time, consistent themes emerged: insufficient time to make an educated choice (n=10), consideration of study logistics (n=8), spouse not present at the time of decision (n=7) and insufficient time to discuss the studies (n=4). CONCLUSION: Parental perception of adequate time to decide about research participation was associated with parental willingness to enroll the child in research. Despite any time limitations, parents were satisfied with the decisions they made. Optimizing the time available for the parental permission process could enhance research participation in the neonatal period.

Changes in nectar concentration: how quickly do whitebellied sunbirds (Cinnyris talatala) adjust feeding patterns and food intake?

Nectarivorous birds encounter varying nectar concentrations while foraging on different food plants and must adjust their consumption to maintain constant energy intake. We determined how rapidly captive whitebellied sunbirds (Cinnyris talatala) adjust their volumetric intake and feeding patterns after changes in diet concentration. On four consecutive days, birds were fed sucrose diets alternating between a standard diet of 16% w/w and test diets of 2.5, 8.5, 16 or 30% w/w, respectively, for 1.5 h periods. Feeding events were recorded with an infrared photo-detection system and food intake and body mass were monitored continuously by electronic balances interfaced to a computer. Generally, birds demonstrated a measurable increase in feeding frequency and food intake within 10 min after a decrease in sucrose concentration. However, individuals responded differently to the most dilute diet (2.5%): while most increased their food intake, others stopped feeding for a short while, appearing to dislike this diet. Furthermore, the number and duration of feeding events increased in the first 5 min after the switch from 2.5% back to 16%, as the birds attempted to compensate for previous reduced sugar intake. Daily sugar intake was lower when birds alternated between 2.5 and 16% diets than on other test days, but birds were able to maintain body mass, presumably through behavioural adjustments.

Honeybee (Apis cerana) foraging responses to the toxic honey of Tripterygium hypoglaucum (Celastraceae): changing threshold of nectar acceptability.

To investigate honeybee foraging responses to toxic nectar, honey was collected from Apis cerana colonies in the Yaoan county of Yunnan Province, China, during June, when flowers of Tripterygium hypoglaucum were the main nectar source available. Pollen analysis confirmed the origin of the honey, and high-performance liquid chromatography showed the prominent component triptolide to be present at a concentration of 0.61 mug/g +/- 0.11 SD. In cage tests that used young adult worker bees, significantly more of those provided with a diet of T. hypoglaucum honey mixed with sugar powder (1:1) died within 6 d (68.3%) compared to control groups provided with normal honey mixed with sugar powder (15.8%). Honeybees were trained to visit feeders that contained honey of T. hypoglaucum (toxic honey) as the test group and honey of Vicia sativa or Elsholtzia ciliata as control groups (all honeys diluted 1:3 with water). Bees preferred the feeders with normal honey to those with toxic honey, as shown by significantly higher visiting frequencies and longer imbibition times. However, when the feeder of normal honey was removed, leaving only honey of T. hypoglaucum, the foraging bees returned to the toxic honey after a few seconds of hesitation, and both visiting frequency and imbibition time increased to values previously recorded for normal honey. Toxic honey thus became acceptable to the bees in the absence of other nectar sources.

Influence of pollen quality on ovarian development in honeybee workers (Apis mellifera scutellata).

Protein-rich diets are known to promote ovarian and egg development in workers of the honeybee, Apis mellifera, even in the presence of a queen. Since the main source of protein for honeybees is pollen, its quality and digestibility might be important dietary factors determining reproductive capacity. We have compared the effect of two types of pollen-sunflower, Helianthus annuus, and aloe, Aloe greatheadii var davyana-on ovarian development in A. mellifera scutellata workers. Under queenright conditions in the field, worker bees exhibited greater ovarian development when feeding on aloe pollen than on sunflower pollen. In their midgut, we observed higher extraction efficiency for aloe (80%) than for sunflower (69%) pollen. This may be attributed to the morphology and size of the two kinds of pollen grains and explains, together with the high protein content of aloe pollen (32% dry mass in bee-collected pollen) compared to sunflower pollen (15%), why aloe pollen promoted higher ovarian development. However, in the laboratory workers sustained on aloe pollen had significantly less-developed ovaries and higher mortality than those fed sunflower pollen. These detrimental effects may be due to an unbalanced protein:carbohydrate ratio. We discuss the effects of unbalanced diets on the physiology and ecology of honeybee reproduction.

Whitebellied sunbirds (Nectarinia talatala, Nectariniidae) do not prefer artificial nectar containing amino acids.

Amino acids are the most abundant class of compounds in nectar after sugars. Like its sugar concentration, the amino acid concentration of nectar has been linked to pollinator type, and it has been suggested that amino acid concentrations are high in the floral nectars of plant species pollinated by passerine birds compared to those pollinated by hummingbirds. We investigated the feeding response of whitebellied sunbirds (Nectarinia talatala) to the inclusion of amino acids in artificial nectar (0.63 M sucrose solution). The response to asparagine, glutamine, phenylalanine, proline, serine and valine, amino acids commonly found in floral nectars, was tested individually and using a mixture of all six amino acids, at two different concentrations (2 and 15 mM). Sunbirds showed no significant preference for amino acids in nectar, or avoided them, especially at the higher concentration. We discuss these findings in the light of the nitrogen requirements of nectarivorous birds and data on amino acids in floral nectars.

Beetle diuretic peptides: the response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella).

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH(31), has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH(37) in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH(46) and Dippu-DH(46) fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH(37) increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.

Short-term energy regulation of whitebellied sunbirds (Nectarinia talatala): effects of food concentration on feeding frequency and duration.

Avian nectarivores show compensatory feeding by adjusting their volumetric intake in response to variation in nectar concentration. This study used an infrared photo-detection system to investigate the short-term feeding patterns of whitebellied sunbirds (Nectarinia talatala) consuming three different sucrose concentrations (10, 20 and 30% w/w). Sunbirds increased their feeding frequency on the most dilute diet, but there was no change in feeding duration. Thus, the increase in total time spent feeding on the dilute diet was due to the increased feeding frequency. No difference in short-term feeding patterns was found between the 20% and 30% diets. Total time spent feeding was extremely short on all diets (96-144 s in every hour). Birds maintained the same steady increase in body mass over the course of the day on all three diets. Daily rhythms in feeding patterns were evident, with longer feeding duration and lower feeding frequency in the early morning and evening than during the rest of the day. Because ingestion rates on a particular diet may vary through the day, caution must be exercised in using feeding duration as a surrogate for meal size. Individual birds varied greatly in their feeding patterns irrespective of diet concentration.

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis).

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.

Dietary intake effects on arginine vasotocin and aldosterone in cloacal fluid of whitebellied sunbirds (Nectarinia talatala).

The main objective of this study was to determine whether or not the renal outputs of the osmoregulatory hormones arginine vasotocin (AVT) and aldosterone (ALDO) reflect the osmotic status of whitebellied sunbirds (Nectarinia talatala). The birds were fed a range of sucrose concentrations (from 0.07 to 2.5 mol/l, with osmolalities of 70 to approximately 5,800 mosM/kg), and adjusted their intakes so that they drank large volumes of dilute diets and small volumes of concentrated diets. Renal fluid outputs were appropriately regulated so that large volumes of cloacal fluid (CF) were voided on the dilute diets and small volumes on the concentrated diets. Accordingly, plasma AVT concentrations increased with increasing sugar concentration; however, AVT outputs in CF did not change in a similar manner, rather they decreased as dietary concentration increased. It was not possible to measure plasma ALDO concentrations in the small sunbirds because of insufficient blood samples available; however, ALDO outputs in CF did vary with the sucrose diets and renal function, being highest on the most concentrated diet. In addition ALDO output in CF fell markedly when sodium was added to the 0.5 mol/l sucrose diet. We conclude that in sunbirds fed increasingly concentrated sucrose solutions, changes in CF outputs of ALDO, but not AVT, appear to reflect the water flux and hydration state of these birds.

The effect of different oral antibiotics on the gastrointestinal microflora of a wild rodent (Aethomys namaquensis).

Gut sterilization via the oral administration of antibiotics facilitates physiological studies of the nutritionally important relationship between intestinal microflora and the host. However, the composition of gut flora is extremely variable, and as a result, the efficacy of antibiotics in achieving gut sterilization varies considerably between species. We tested the effectiveness of three antibiotic cocktails in sterilizing the gut of a rodent pollinator, the Namaqua rock mouse (Aethomys namaquensis). The cocktails were (1) streptomycin sulfate and bacitracin (previously used with domestic mice and rats), (2) chloramphenicol and bacitracin (based on antibiotic screening tests performed on faecal flora) and (3) Baytril 10% oral solution (a veterinary antimicrobial agent containing enrofloxacin). We tested for antibiotic inactivation by determining bacterial viability through fluorescence staining of faecal samples. We also tested techniques to maintain sterility during antibiotic treatment without the benefit of a laminar flow cabinet. Antibiotics were administered orally in food and water consumed ad libitum over 4 consecutive days. Antibiotic effectiveness was assessed by culturing anaerobic bacteria from faecal samples collected before and after each antibiotic treatment. Treatment with Baytril 10% oral solution eliminated or significantly reduced faecal flora, whereas other antibiotics did not. This study clearly demonstrates the importance of testing the effectiveness of antibiotics before their use in studies that involve antibiotic-treated subjects, particularly if these are species previously untested.

Osmoregulatory response to acute diet change in an avian nectarivore: rapid rehydration following water shortage.

Nectarivores may be required to switch between water conservation and water excretion as rapidly as they change food plants in nature. We examined the rehydration response in Whitebellied Sunbirds (Nectarinia talatala) that had been fed a concentrated sucrose diet (2.5 mol/l) for 2 days and then were switched to a diet 10 x less concentrated (0.25 mol/l) on the morning of the third day. We measured water gain as well as cloacal fluid (CF) volume hourly over 12 h, and analysed CF osmolality and calculated osmotic excretion. CF was also assayed for the osmoregulatory hormone aldosterone (ALDO). As in most water-deprived birds, whitebellied sunbirds cope with water shortage when fed a concentrated sugar diet by almost completely ceasing to void CF. Although osmolality of CF is high, volumes are not sufficient to maintain a steady rate of excretion and the birds retain osmolytes. Immediately upon switching to dilute diet, sunbirds produce copious volumes of CF and osmotic excretion is elevated and maintained at high levels over the first 6 h of rehydration. This stabilises by the afternoon at levels expected for hydrated birds. Some 2-3 h after peak osmotic excretion, there is a peak in the discrepancy between water intake and output, mirrored in an increase in ALDO output. These data suggest that excretion of retained osmolytes is undertaken as soon as water is available, with changes in the body fluid composition occurring subsequently. This study vindicates the use of CF to obtain repeated measurements of changes in the osmoregulatory steroid ALDO in small birds.