The use of Raman microscopy to determine and localize vitamin E in biological samples

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Energetics reveals physiologically distinct castes in a eusocial mammal.

Eusociality, which occurs among mammals only in two species of African mole-rat, is characterized by division of labour between morphologically distinct 'castes'1. In Damaraland mole-rats (Cryptomys damarensis), colony labour is divided between 'infrequent worker' and 'frequent worker' castes2. Frequent workers are active year-round and together perform more than 95% of the total work of the colony, whereas infrequent workers typically perform less than 5% of the total work3. Anecdotal evidence suggests that infrequent workers may act as dispersers, with dispersal being limited to comparatively rare periods when the soil is softened by moisture4, 5. Here we show that infrequent workers and queens increase their daily energy expenditure after rainfall whereas frequent workers do not. Infrequent workers are also fatter than frequent workers. We suggest that infrequent workers constitute a physiologically distinct dispersing caste, the members of which, instead of contributing to the work of the colony and helping the queen to reproduce, build up their own body reserves in preparation for dispersal and reproduction when environmental conditions are suitable.

The energy costs of sexual dimorphism in mole-rats are morphological not behavioural.

Different reproductive strategies of males and females may lead to the evolution of differences in their energetic costs of reproduction, overall energetic requirements and physiological performances. Sexual dimorphism is often associated with costly behaviours (e.g. large males might have a competitive advantage in fighting, which is energetically expensive). However, few studies of mammals have directly compared the energy costs of reproductive activities between sexes. We compared the daily energy expenditure (DEE) and resting metabolic rate (RMR) of males and females of two species of mole-rat, Bathyergus janetta and Georychus capensis (the former is sexually dimorphic in body size and the latter is not) during a period of intense digging when males seek females. We hypothesized that large body size might be indicative of greater digging or fighting capabilities, and hence greater mass-independent DEE values in males of the sexually dimorphic species. In contrast to this prediction, although absolute values of DEE were greater in B. janetta males, mass-independent values were not. No differences were apparent between sexes in G. capensis. By comparison, although RMR values were greater in B. janetta than G. capensis, no differences were apparent between the sexes for either species. The energy cost of dimorphism is most likely to be the cost of maintenance of a large body size, and not the cost of behaviours performed when an individual is large.

Virgin birth in a hammerhead shark.

Parthenogenesis has been documented in all major jawed vertebrate lineages except mammals and cartilaginous fishes (Class Chondrichthyes: sharks, batoids, chimeras). Reports of captive female sharks giving birth despite being held in the extended absence of males have generally been ascribed to prior matings coupled with long-term sperm storage by the females. Here we provide the first genetic evidence for chondrichthyan parthenogenesis, involving a hammerhead shark. This finding also broadens the known occurrence of a specific type of asexual development (automictic parthenogenesis) among vertebrates, extending recently raised concerns about the potential negative effect of this type of facultative parthenogenesis on the genetic diversity of threatened vertebrate species.

Tracing early stages of species differentiation: Ecological, morphological and genetic divergence of Galapagos sea lion populations

Background: Oceans are high gene flow environments that are traditionally believed to hamper the build-up of genetic divergence. Despite this, divergence appears to occur occasionally at surprisingly small scales. The Galápagos archipelago provides an ideal opportunity to examine the evolutionary processes of local divergence in an isolated marine environment. Galápagos sea lions (Zalophus wollebaeki) are top predators in this unique setting and have an essentially unlimited dispersal capacity across the entire species range. In theory, this should oppose any genetic differentiation.
Results: We find significant ecological, morphological and genetic divergence between the western colonies and colonies from the central region of the archipelago that are exposed to different ecological conditions. Stable isotope analyses indicate that western animals use different food sources than those from the central area. This is likely due to niche partitioning with the second Galápagos eared seal species, the Galápagos fur seal (Arctocephalus galapagoensis) that exclusively dwells in the west. Stable isotope patterns correlate with significant differences in foraging-related skull morphology. Analyses of mitochondrial sequences as well as microsatellites reveal signs of initial genetic differentiation.
Conclusion: Our results suggest a key role of intra- as well as inter-specific niche segregation in the evolution of genetic structure among populations of a highly mobile species under conditions of free movement. Given the monophyletic arrival of the sea lions on the archipelago, our study challenges the view that geographical barriers are strictly needed for the build-up of genetic divergence. The study further raises the interesting prospect that in social, colonially breeding mammals additional forces, such as social structure or feeding traditions, might bear on the genetic partitioning of populations.

Body temperature daily rhythm adaptations in African savanna elephants (Loxodonta africana)

The savanna elephant is the largest extant mammal and often inhabits hot and and environments. Due to their large size, it might be expected that elephants have particular physiological adaptations, such as adjustments to the rhythms of their core body temperature (T-b) to deal with environmental challenges. This study describes for the first time the T-b daily rhythms in savanna elephants. Our results showed that elephants had lower mean T-b values (36.2 +/- 0.49 degrees C) than smaller ungulates inhabiting similar environments but did not have larger or smaller amplitudes of T-b variation (0.40 +/- 0.12 degrees C), as would be predicted by their exposure to large fluctuations in ambient temperature or their large size. No difference was found between the daily T-b rhythms measured under different conditions of water stress. Peak T-b's occurred late in the evening (22: 10) which is generally later than in other large mammals ranging in similar environmental conditions. (C) 2007 Elsevier Inc. All rights reserved.

Mole-rats from higher altitudes have greater thermoregulatory capabilities

Subterranean mammals (those that live and forage underground) inhabit a challenging microenvironment, with high levels of carbon dioxide and low levels of oxygen. Consequently, they have evolved specialised morphological and physiological adaptations. For small mammals that inhabit high altitudes, the effects of cold are compounded by low oxygen partial pressures. Hence, subterranean mammals living at high altitudes are faced with a uniquely demanding physiological environment, which presumably necessitates additional physiological adjustments. We examined the thermoregulatory capabilities of two populations of Lesotho mole-rat Cryptomys hottentotus mahali that inhabit a 'low' (1600 in) and a 'high' (3200 m) altitude. Mole-rats from the high altitude had a lower temperature of the lower critical point, a broader thermoneutral zone, a lower thermal conductance and greater regulatory non-shivering thermogenesis than animals from the lower altitude. However, minimum resting metabolic rate values were not significantly different between the populations and were low compared with allometric predictions. We suggest that thermoregulatory costs may in part be met by animals maintaining a low resting metabolic rate. High-altitude animals may adjust to their cooler, more oxygen-deficient environment by having an increased non-shivering thermogenesis whilst maintaining low thermal conductance. (c) 2006 Elsevier Inc. All rights reserved.

Seasonal acclimatization of non-shivering thermogenesis in common spiny mice (Acomys cahirinus) from different habitats

We compared non-shivering thermogenesis between two adjacent populations of freshly captured common spiny mice (Acomys cahirinus) during both winter and summer. Mice were captured from north- and south-facing slopes (NFS and SFS) of the same valley that represent 'Mediterranean' and 'Desert' habitats, respectively. Oxygen consumption and body temperature responses to an injection of exogenous noradrenaline (NA) were higher during the winter than during the summer. in addition, SFS mice had a lower body temperature response to NA during the summer than the other groups of mice. This suggests that heat dissipation is likely to have been greatest in SFS mice during the summer. Overall this study shows that seasonal acclimatization of NST mechanisms is an important trait for small mammals that inhabit the Mediterranean ecosystem. Differences in physiological capabilities can occur temporally within populations across seasons, and spatially between populations that are only a short distance (200-500 m) apart.

Seasonal energetics of the Hottentot golden mole at 1500m altitude

Winter is an energetically stressful period for small mammals as increasing demands for thermoregulation are often coupled with shortages of food supply. In sub-tropical savannah, Hottentot golden moles (Ambysomus hottentottus longiceps) forage throughout the year and for lone periods of each day. This may enable them to acquire sufficient resources from an insectivorous prey base that is both widely dispersed and energetically costly to obtain. However, they also inhabit much cooler regions; how their energy budgets are managed in these areas is unknown. We measured the daily energy expenditure (DEE), resting metabolic rate (RMR) and water turnover (WTO) of free-living golden moles during both winter and summer at high altitude (1500 m). We used measurements of deuterium dilution to estimate body fat during these two periods. DEE, WTO and body mass did not differ significantly between seasons. However, RMR values were higher during the winter than the summer and, in the latter case were also lower than allometric predictions. Body fat was also higher during the winter. Calculations show that during the winter they may restrict activity to shorter, more intense periods. This, together with an increase in thermal insulation, might enable them to survive the cold. (c) 2005 Elsevier Inc. All rights reserved.

The energetics of lactation in cooperatively breeding meerkats Suricata suricatta

Species may become obligate cooperative breeders when parents are unable to raise their offspring unassisted. We measured the daily energy expenditure of mothers, helpers and offspring during peak lactation in cooperatively breeding meerkats Suricata suricatta using the doubly labelled water technique. Lactating mothers expended more energy per day than allo-lactating subordinate females, non-lactating females or suckling offspring. Metabolizable energy intakes of lactating mothers were calculated from isotope-based estimates of offspring milk energy intake, and were not significantly different from the previously suggested maximal limit for mammals. Allo-lactating females were the only category of animals that lost weight during the period of study, probably because they spent more time babysitting than non-lactating females. Daily energy expenditure (DEE) of lactating mothers increased with litter size but decreased with the number of helpers. Calculations show that for every 10 helpers, even in the absence of allo-lactators, mothers are able to reduce their DEE during peak lactation by an amount equivalent to the energy cost of one pup. These results indicate that helpers have beneficial energetic consequences for lactating mothers in an obligate cooperatively breeding mammal.

Burrow architecture and digging activity in the Cape dune mole rat

While females are traditionally thought to invest more time and energy into parental care than males, males often invest more resources into searching and displaying for mates, obtaining mates and in male-male conflict. Solitary subterranean mammals perform these activities in a particularly challenging niche, necessitating energetically expensive burrowing to both search for mates and forage for food. This restriction presumably affects males more than females as the former are thought to dig longer tunnels that cover greater distances to search for females. We excavated burrow systems of male and female Cape dune mole rats Bathyergus suillus the, largest truly subterranean mammal, to investigate whether male burrows differ from those of females in ways that reflect mate searching by males. We consider burrow architecture (length, internal dimensions, fractal dimension of tunnel systems, number of nesting chambers and mole mounds on the surface) in relation to mating strategy. Males excavated significantly longer burrow systems with higher fractal dimensions and larger burrow areas than females. Male burrow systems were also significantly farther from one another than females were from other females' burrow systems. However, no sex differences were evident in tunnel cross-sectional area, mass of soil excavated per mound, number of mounds produced per unit burrow length or mass of soil excavated per burrow system. Hence, while males may use their habitat differently from females, they do not appear to differ in the dimensions of the tunnels they create. Thus, exploration and use of the habitat differs between the sexes, which may be a consequence of sex differences in mating behaviour and greater demands for food.

Signals of need in a cooperatively breeding mammal with mobile offspring

In many bird species with biparental care for young in the nest, hungry chicks beg repeatedly and parents adjust their feeding rate to the call rate of young. Repetitive calling also occurs in fledglings and in some mammals where offspring follow provisioners. It is not yet clear whether, in mobile systems with dispersed young where adults cannot compare the vocal behaviour of all young simultaneously, the calls represent a signal of need. We investigated repetitive begging by cooperatively reared meerkat, Suricata suricatta, pups that foraged with the group. Pups produced two types of begging calls: repeat calls over long periods and high-pitched calls mainly confined to feeding events. Food-deprived pups stayed closer to feeders, and begged for longer and more intensely by calling at a higher rate. Hungry pups increased both the rate of repeat calls, which were given continually, and the number of high-pitched bouts, but adults increased their food allocation only in relation to the rate of repeat calls. Our study indicates that hunger may lead to several changes in vocal behaviour, only some of which may be used by adults to assess need.

First record of minimum Irish hare (Lepus timidus hibernicus Bell 1837) longevity in the wild.

Average longevity of the mountain hare (Lepus timidus L., 1758) has been estimated at nine years in the wild (Macdonald D. and Barrett, P. 1993 Mammals of Britain and Europe. Harper Collins Publishers, London) with a maximum recorded age of 18 years for one marked animal (Angerbjörn, A. and Flux, J. E. C. 1995 Lepus timidus. Mammalian Species 495: 1–11). However, the longevity of the Irish hare (L. t. hibernicus Bell 1837) is entirely unknown. A total of 14 Irish hares was trapped and tagged at Belfast International Airport, Co. Antrim from February to April 2005. The sex, age (juvenile or adult) and weight of each animal were recorded. Adults were taken as those individuals >8-10 months old defined by the fusing of the notch between the apophysis and diaphysis of the tibia and humerus (Flux, J. E. C. 1970 Journal of Zoology 161: 75-123). Individual identification was made by a system of colourcoded ear tags (Roxan iD Ltd. Selkirk, Scotland) being inserted in the centre of the pinna of each ear. Each ear tag (6 × 34 mm) and puncture site was disinfected with 70 per cent ethanol prior to insertion. An adult male, #001/002 ‘Blue/Blue’, was tagged on 3 March 2005 weighing 3.8 kg and was sighted during a return site visit on 4 April 2007. An adult female, #026/003 ‘Green/Yellow’, was tagged on 15 April 2005 weighing 4.0 kg and was sighted during return visits on 25 March 2010 and 19 October 2010. The latest possible date of birth for both individuals was spring/summer 2004. Consequently, they were at least 3 years and 6.5 years old, respectively. This is the first record of minimum Irish hare longevity in the wild. These observations suggest that ear tagging does not compromise animal welfare and is an effective means of long-term monitoring. Future research may utilize capture-mark-recapture methods.

Effects of the amphibian skin-derived bradykinin B2 receptor antagonist peptide, kinestatin, on the proliferation of human prostate cancer cell lines

Bradykinin and related peptides are found in the defensive skin secretions of many frogs and toads. While the physiological roles of bradykinin-related peptides in sub-mammalian vertebrates remains obscure, in mammals, including humans, canonical bradykinin mediates a multitude of biological effects including the proliferation of many types of cancer cell. Here we have examined the effect of the bradykinin B2 receptor antagonist peptide, kinestatin, originally isolated by our group from the skin secretion of the giant fire-bellied toad, Bombina maxima, on the proliferation of the human prostate cancer cell lines, PC3, DU175 and LnCAP. The bradykinin receptor status of all cell lines investigated was established through PCR amplification of transcripts encoding both B1 and B2 receptor subtypes. Following this demonstration, all cell lines were grown in the presence or absence of kinestatin and several additional bradykinin receptor antagonists of amphibian skin origin and the effects on proliferation of the cell lines was investigated using the MTT assay and by counting of the cells in individual wells of 96-well plates. All of the amphibian skin secretion-derived bradykinin receptor antagonists inhibited proliferation of all of the prostate cancer lines investigated in a dose-dependent manner. In addition, following incubation of peptides with each cell line and analysis of catabolites by mass spectrometry, it was found that bradykinin was highly labile and each antagonist was highly stable under the conditions employed. Bradykinin signalling pathways are thus worthy of further investigation in human prostate cancer cell lines and the evidence presented here would suggest the testing of efficacy in animal models of prostate cancer as a positive outcome could lead to a drug development programme for the treatment of this disease.

Millimeter-wave stealth radio for special operations forces

For Special Operations Forces, an important attribute of any future radio will be the ability to conceal transmissions from the enemy while transmitting large amounts of data for situational awareness and communications. These requirements will mean that military wireless systems designers will need to consider operating frequencies in the mm-wave bands: The high data rates that are achievable at these frequencies and the propagation characteristics at this wavelength will provide many benefits for the implementation of 'stealth radio'. This article discusses some of the recent advances in RF front-end technology, alongside physical layer transmission schemes that could be employed for millimeter-wave soldier-mounted radio. The operation of a hypothetical millimeter-wave soldier-to-soldier communications system that makes use of smart antenna technology is also described.