Retention of different-sized particles and derived gut fill estimate in tammar wallabies (Macropus eugenii): physiological and methodological considerations

The capacity of the digestive tract is an important parameter in understanding digestive adaptations, particularly in herbivores. Measures of this capacity (‘gut fill’) are commonly performed in killed animals, which has ethical and logistical implications. Alternatively, dry matter gut contents (DMC) can be estimated in live animals from food intake, digesta retention and digestibility, based on physical principles (Holleman and White, Can. J. Zool. 67, 488-494, 1989). Although this method has been used to some extent, it still awaits thorough validation. Here we estimated DMC in seven tammar wallabies during 5-day feeding trials and compared the results to those gained from dissections immediately after the trials. Calculated DMC exceeded that actually measured by 29 ± 22%. A closer inspection of the data suggested that this was partly due to the fact that DMC as measured by dissection is susceptible to short-term influences such as daily variation in food intake, whereas the calculated DMC represents an integrative measure over the whole period of the feeding trial. Correlations between both the measured digesta retention times, and the calculated DMC, with the measured wet contents mass suggest that it is particularly the DMC determined via dissection that needs to be measured with care. For a comparison of gut capacities, the calculated DMC therefore can be considered adequate, but should for a more widespread use be validated in further studies including more species and experimental regimes controlling food intake variation. Additionally, we tested whether very small (100-500 μm) and small (500-1000 μm) particles were retained differently in the tammar wallabies. There was no indication of such a difference. Whether the macropod forestomach selectively passes a certain particle fraction (that represents microbes) with the generally faster-passing fluids remains to be investigated with even smaller markers, e.g. labelled bacteria.     

Differential passage time of mistletoe fruits through the gut of honeyeaters and flowerpeckers: effects on seedling establishment

McKey's (1975) hypothesis that avian dispersers with a specialized gut provide higher quality seed dispersal than unspecialized frugivores was tested using grey mistletoe (Amyema quandang) fruits, and captive mistletoebirds (Dicaeum hirundinaceum) and spinycheeked honeyeaters (Acanthagenys refogularis) in arid South Australia. Mistletoebirds have a specialized gut, unlike spiny-cheeked honeyeaters. The gut passage time of A. quandang fruits through mistletoebirds was 820±29 s (mean±SE, n=188), compared to 2434±36 s (n=436) for honeyeaters. The seeds defecated by both bird species were deployed on twigs of host trees. Despite the longer retention time of fruit in the gut of honeyeaters, the germination percentage of seeds defecated by mistletoebirds (85% of 485 seeds) and honeyeaters (81% of 485 seeds) did not differ significantly 1 week after deployment. However, after 5 months, a significantly greater proportion of seedlings had established from seeds passed by mistletoebirds (42.7%) than from seeds defecated by honeyeaters (31.1%). The data support the notion that the more gentle treatment of seeds in the gut of specialized dispersers translates into higher seedling establishment.     

Parasites in space and time: a case study of haemosporidian spatiotemporal prevalence in urban birds

Prevalence responses to anthropic factors differ across hosts and parasite species. We here analyzed the spatiotemporal variation of avian haemosporidian prevalence in bird assemblages of the Mooswald forest (i.e., urban greenspace; Freiburg, Germany), in response to local environmental features (e.g., water sources, human presence (visited)/absence (unvisited)) and bird-level traits (e.g., body condition, age, sex) in 2?years. We used a nested PCR protocol (mitochondrial (mt)DNA cytochrome b (cyt b) gene) and microscopy to determine haemosporidian infections. Prevalence was analyzed using a general linear multi-model (glmulti) approach with Akaike information criterion corrected for small samples (AICc), with subsequent model inferences using a GLMM on the best selected model, considering bird species as a random factor. Analyses were conducted for the main understory bird species (Blackcap – Sylvia atricapilla, Chaffinch – Coereba flaveola, Great Tit – Parus major, Blue Tit – Cyanistes caeruleus, European Robin – Erithacus rubecula, Blackbird – Turdus merula, Song Thrush – Turdus philomelos). We further conducted spatial autocorrelation analyses for all haemosporidian infections, and classification and regression trees (CARTs) for focal species. We analyzed a total of 544 samples of seven bird species. In 2011 prevalence for Haemoproteus/Plasmodium was 25.8% and 11.7% for Leucocytozoon. In 2013 prevalence for Haemoproteus/Plasmodium was 26.5% and 35.5% for Leucocytozoon. Haemosporidian prevalence was significantly different between some focal species. There was a negative association between distance to the nearest water source and prevalence in the year 2011, and the opposite pattern for the year 2013. However, when analyzed for the six focal species separately, such a relationship could change from a negative to a positive one, or there could be no relationship at all. For Leucocytozoon there was higher prevalence in the section of the forest visited by humans. We did not find spatial autocorrelation for prevalence across the study site, but there were statistically significant local spatial clusters in the visited section. Although there were similar responses of prevalence to some factors, infection patterns were generally bird species-specific. Thus, prevalence is a labile epidemiological parameter, varying spatiotemporally in an idiosyncratic way.     

Time–frequency analysis of variabilities of heart rate, systolic blood pressure and pulse transit time before and after exercise using the recursive autoregressive model

Time–frequency (T–F) analysis is often used to study the non-stationary cardiovascular oscillations such as heart rate and blood pressure variabilities in dynamic situations. This study intends to use the T–F recursive autoregressive technique to investigate variability in pulse transit time (PTT), which is a cardiovascular parameter of emerging interest due to its potential to estimate blood pressure non-invasively, continuously and without a cuff. Recent studies suggest that PTT is not only related to systolic blood pressure (SBP) but also to heart rate. Therefore, in this study, variability of PTT is analyzed together with the variabilities of R–R interval (RRI) from electrocardiogram and beat-to-beat SBP on 9 normotensive subjects before and shortly after three successive bouts of treadmill exercise. The results showed that both low frequency (LF) and high frequency (HF) components were found in the spectra of RRI, SBP and PTT in the 5-min recordings collected before and after exercise. Compared to the baseline, a decrease in the power of the HF component of RRI followed by an increase in its LF component indicated firstly a vagal withdrawal and then sympathetic activity enhancement after successive bouts of exercise. On the other hand, although changes in the LF and HF components of PTT were more similar to those of SBP than of RRI, the LF/HF ratio of SBP was almost 4 times higher than that of PTT. Based on the results, it is therefore suggested that the relationship between SBP and PTT is frequency-dependent.     

Size scaling and stiffness of avian primary feathers: implications for the flight of Mesozoic birds

The primary feathers of birds are subject to cyclical forces in flight causing their shafts (rachises) to bend. The amount the feathers deflect during flight is dependent upon the flexural stiffness of the rachises. By quantifying scaling relationships between body mass and feather linear dimensions in a large data set of living birds, we show that both feather length and feather diameter scale much closer to predictions for geometric similarity than they do to elastic similarity. Scaling allometry also indicates that the primary feathers of larger birds are relatively shorter and their rachises relatively narrower, compared to those of smaller birds. Two-point bending tests indicated that larger birds have more flexible feathers than smaller species. Discriminant functional analyses (DFA) showed that body mass, primary feather length and rachis diameter can be used to differentiate between different magnitudes of feather bending stiffness, with primary feather length explaining 63% of variance in rachis stiffness. Adding fossil measurement data to our DFA showed that Archaeopteryx and Confuciusornis do not overlap with extant birds. This strongly suggests that the bending stiffness of their primary feathers was different to extant birds and provides further evidence for distinctive flight styles and likely limited flight ability in Archaeopteryx and Confuciusornis.     

Neuraminidase inhibitors for treatment of human and avian strain influenza: A comparative modeling study

Treatment of seasonal influenza viral infections using antivirals such as neuraminidase inhibitors (NAIs) has been proven effective if administered within 48 h post-infection. However, there is growing evidence that antiviral treatment of infections with avian-derived strains even as late as 6 days post-infection (dpi) can significantly reduce infection severity and duration. Using a mathematical model of in-host influenza viral infections which can capture the kinetics of both a short-lived, typical, seasonal infection and a severe infection exhibiting sustained viral titer, we explore differences in the effects of NAI treatment on both types of influenza viral infections. Comparison of our model's behavior against experimental data from patients naturally infected with avian strains yields estimates for the times at which patients were infected that are consistent with those reported by the patients, and estimates of drug efficacies that are lower for patients who died than for those who recovered. In addition, our model suggests that the sustained, high, viral titers often seen in more severe influenza virus infections are the reason why antiviral treatment delayed by as much as 6 dpi will still lead to reduced viral titers and shortened illness. We conclude that NAIs may be an effective and beneficial treatment strategy against more severe strains of influenza virus characterized by high, sustained, viral titers. We believe that our mathematical model will be an effective tool in guiding treatment of severe influenza viral infections with antivirals.     

Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes

Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.     

The effect of temperature on digestive and assimilation efficiency,gut passage time and appetite in an ambush foraging lizard,<Emphasis Type="Italic"> Cordylus melanotus melanotus</Emphasis>

In ectotherms, an increase in body temperature increases metabolic rate and may increase rates of digestive processes. We measured the thermal dependence of the apparent digestive and apparent assimilation efficiencies (ADE and AAE), gut passage time (GP) and appetite in Cordylus melanotus melanotus, a medium sized Crag Lizard, which is endemic to South Africa. Trials were conducted at 20, 22, 25, 30, 32 and 35 °C under controlled conditions. Trials lasted 14 days, during which, lizards were fed ca. 1 g mealworms per day. Glass beads were used as markers to determine GP at the beginning and end of trials. Faeces and urates were collected daily and oven dried at 50 °C. The energy content of egested matter was then measured using bomb calorimetry. ADE and AAE were not affected by temperature for either males or females. The mean±SE ADE and AAE were 94.4±0.3% and 87.2±0.6%, respectively. GP was not significantly different between males and females at any temperature, but decreased significantly with increasing temperature. Appetite was significantly different between the different temperatures measured. The decrease of gut passage time with increasing temperature was expected, since the digestive and assimilation efficiencies are similar over the range of temperatures tested. Lizards are thus assimilating a similar proportion of ingested energy, but at faster rates at higher temperatures. The results indicate that the digestive physiology of this species results in maximum energy gain per meal in environments where food is scarce.Abbreviations AAE apparent assimilation efficiency - ADE apparent digestive efficiency - AE assimilation efficiency - DE digestive efficiency - GP gut passage rate - NEA net energy absorbed through gut - NER net usable energy retained - SVL snout-vent length - T _b body temperature Communicated by G. Heldmaier     

Determining high value areas for steppe birds in Spain: hot spots,complementarity and the efficiency of protected areas

We have examined the distribution of 26 species of steppe birds in the Iberian Peninsula and the Balearic Islands within a grid of 5,070 10 × 10 km grid cells. The most valuable areas for steppe birds have been identified by selecting the upper 5% of the 10 × 10 km grid cells after a simple ranking based on the following criteria: species-richness, richness of rare species, rarity index, species vulnerability at Spanish, European and Global levels, and using an index combining the previous criteria. We have also used a heuristic algorithm to select those areas which offered most complementarity. The results have been analysed on a national scale and have been compared with those obtained by species assessments in previous status summaries. Finally, we have performed an analysis of the coverage afforded to locations which are valuable for steppe species by Natural Protected Areas (NPAs) and Special Protected Areas (SPAs), and an evaluation of the potential effects of temporal changes in species’ threat-status. The combined index was the most reliable criterion for defining hotspots, encompassing 15% of the species’ distributions within 5% of the total area considered. This index showed a high level of geographical concordance with the other criteria (nearly 70% of the selected grid cells coincided). Analysis of complementarity delivered poorer results than simple rank-scoring. The analysis of the efficiency of NPA network showed a very low coverage (less than 2%) of the hotspots selected according to the Combined Index. Coverage of the SPA network was higher (nearly 45%), although it diminished (to <35%) when only steppe-defined SPAs were included. The geographical concordance between high-value areas selected using current status summaries and those chosen using earlier ones was low for European threat status (53.4%), intermediate (63.2%) for Spanish threat status and high for SPEC status (78.1%). The Combined Index showed a high level of geographical concordance between the old and new data (76.6%). We conclude that automatic scoring methods (identifying hotspots) are useful for selecting valuable areas and for analysing the efficiency of the network of protected natural spaces, as well as for examining the potential effects of status changes on hotspot definition. Furthermore, the Spanish SPA network does not cover the most important areas for steppe birds adequately.     

Biochemical and immunochemical determination of (Na+ + K+)-ATPase content in subcellular fractions prepared from the avian salt gland: evidence for enzyme heterogeneity

Subcellular membrane fractions were prepared from the salt glands of osmotically-stressed ducklings. Two fractions were characterized biochemically with respect to (Na+ + K+)-ATPase, alkaline phosphodiesterase I, succinate dehydrogenase, esterase, and galactosyltransferase activities and immunochemically with respect to (Na+ + K+)-ATPase. The ratios of the estimates of the (Na+ + K+)-ATPase contents obtained biochemically and immunochemically from the two fractions differed by more than 2 X. The results are consistent with the presence of at least two molecular species of (Na+ + K+)-ATPase, unevenly distributed between the two fractions.     

A reevaluation of the logic of pilferage effects, predation risk, and environmental variability on avian energy regulation: the critical role of time budgets

We studied the effect of pilferage rates, variation in foodencounter rate, and predation risk on cache and fat-storageregulation using dynamic programming. Previous predictionsthat small birds facing increased pilferage rates should cacheless and store more body fat are not generally supported. Instead,cache investment (caching rate or percent of food cached) is predicted to be unimodal, peaking at intermediate pilferagerates. This pattern is determined, in part, by pilferage-inducedchanges in time budgets: at low pilferage rates, a marginalincrease in pilferage rates can be offset by an increase incache investment. However, increased caching increases time allocated to both caching and foraging. The increased foragingis caused by the energetic costs of caching and by the lossof energy from the cache. Increased time spent caching andforaging in turn decreases time spent resting under low predationrisk. Above some threshold pilferage rate, the marginal valueof resting exceeds the marginal value of caching, and cacheinvestment declines with further increasing pilferage rates.These patterns hold for three levels of variation in food encounterrate: time-invariant, between-day, and within-day variation;they also hold across different mean rates of food encounter.We show that previous predictions concerning decreased energy-storagelevels with increased food abundance are not supported when there is between-day variation in mean food encounter ratesand food abundance increases only on "good" days. Finally,predation risk affects the predictions described above in twoways. First, these trends assume that the birds can rest ina predator-free refuge. If the refuge is not available, birdsare predicted to cache less at higher pilferage rates irrespectiveof the absolute level of pilferage. With the refuge in place,levels of predation risk affect the skew in the pilferage-rate/cachingfunction. As a result, the relative effect of predation riskon caching intensity varies with pilfer rate. At very low pilferrates, lowered predation risk causes more caching, but loweredpredation risk under high pilferage rates can lower caching intensity, contrary to previous predictions. Surprisingly, predationrisk has an appreciable effect on body mass only when the birdis predicted to cease caching (i.e., at the highest pilferrates); otherwise a change of two orders of magnitude in theprobability of encountering predators has little effect on body mass. Our results suggest that the tradeoffs associatedwith the joint regulation of internal energy stores and externallycached stores are more complicated than previous literaturewould indicate. Our results also show that we have underestimatedthe role that time budgets play in patterns of energy regulation.     

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000–12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern‐East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000–67 000 ya using an average mammalian microsatellite mutation rate and at 4700–44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70–110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.     

Real time PCR: A rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines

In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (−3.324) and (−3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID₅₀ method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines.     

Evidence for association of chromosomal form and development time from complex clines and geographic races in the grasshopper Caledia captiva (Orthoptera: Acridadae)

Evidence for an association between chromosomal form and development time in the grasshopper Caledia captiva (F.) was obtained through comparison of two geographic taxa and analysis of a complex latitudinal cline within one of the taxa. Northern populations of the Moreton taxon possess a metacentric genome and are slow-developing. In contrast, the Torresian taxon, distributed throughout northern, coastal Australia, a region of pronounced seasonality in rainfall, and southern populations of the Moreton taxon, which inhabit a region of pronounced seasonality in temperature, Soth have an acrocentric genome and are fast-developing. The convergence of chromosomal form and development time between Torresian and southern Moreton populations appears to be driven by convergence in life history. Seasonality limits grasshoppers to one generation per year and favours fast development. The transition between relatively acrocentric southern Moreton populations and relatively metacentric northern Moreton populations is gradual but not monotonic. Instead, a shift to a bivoltine life history in the middle of the transect occurs and is associated with shifts in both development time and chromosomal form. These results imply an adaptive role for chromosomal form, although the causative link between chromosomal variation and variation in development time remains to be established.