Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.

Representing the acquisition and use of energy by individuals in agent-based models of animal populations

Summary 1. Agent-based models (ABMs) are widely used to predict how populations respond to changing environments. As the availability of food varies in space and time, individuals should have their own energy budgets, but there is no consensus as to how these should be modelled. Here, we use knowledge of physiological ecology to identify major issues confronting the modeller and to make recommendations about how energy budgets for use in ABMs should be constructed. 2. Our proposal is that modelled animals forage as necessary to supply their energy needs for maintenance, growth and reproduction. If there is sufficient energy intake, an animal allocates the energy obtained in the order: maintenance, growth, reproduction, energy storage, until its energy stores reach an optimal level. If there is a shortfall, the priorities for maintenance and growth/reproduction remain the same until reserves fall to a critical threshold below which all are allocated to maintenance. Rates of ingestion and allocation depend on body mass and temperature. We make suggestions for how each of these processes should be modelled mathematically. 3. Mortality rates vary with body mass and temperature according to known relationships, and these can be used to obtain estimates of background mortality rate. 4. If parameter values cannot be obtained directly, then values may provisionally be obtained by parameter borrowing, pattern-oriented modelling, artificial evolution or from allometric equations. 5. The development of ABMs incorporating individual energy budgets is essential for realistic modelling of populations affected by food availability. Such ABMs are already being used to guide conservation planning of nature reserves and shell fisheries, to assess environmental impacts of building proposals including wind farms and highways and to assess the effects on nontarget organisms of chemicals for the control of agricultural pests. Keywords: bioenergetics; energy budget; individual-based models; population dynamics.

Effect of forage conservation method on plasma lipids, mammary lipogenesis, and milk fatty acid composition in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio

The effects of forage conservation method on plasma lipids, mammary lipogenesis, and milk fat were examined in 2 complementary experiments. Treatments comprised fresh grass, hay, or untreated (UTS) or formic acid treated silage (FAS) prepared from the same grass sward. Preparation of conserved forages coincided with the collection of samples from cows fed fresh grass. In the first experiment, 5 multiparous Finnish Ayrshire cows (229 d in milk) were used to compare a diet based on fresh grass followed by hay during 2 consecutive 14-d periods, separated by a 5-d transition during which extensively wilted grass was fed. In the second experiment, 5 multiparous Finnish Ayrshire cows (53 d in milk) were assigned to 1 of 2 blocks and allocated treatments according to a replicated 3 × 3 Latin square design, with 14-d periods to compare hay, UTS, and FAS. Cows received 7 or 9 kg/d of the same concentrate in experiments 1 and 2, respectively. Arterial concentrations of triacylglycerol (TAG) and phospholipid were higher in cows fed fresh grass, UTS, and FAS compared with hay. Nonesterified fatty acid (NEFA) concentrations and the relative abundance of 18:2n-6 and 18:3n-3 in TAG of arterial blood were also higher in cows fed fresh grass than conserved forages. On all diets, TAG was the principle source of fatty acids (FA) for milk fat synthesis, whereas mammary extraction of NEFA was negligible, except during zero-grazing, which was associated with a lower, albeit positive calculated energy balance. Mammary FA uptake was higher and the synthesis of 16:0 lower in cows fed fresh grass than hay. Conservation of grass by drying or ensiling had no influence on mammary extraction of TAG and NEFA, despite an increase in milk fat secretion for silages compared with hay and for FAS than UTS. Relative to hay, milk fat from fresh grass contained lower 12:0, 14:0, and 16:0 and higher S3,R7,R11,15-tetramethyl-16:0, cis-9 18:1, trans-11 18:1, cis-9,trans-11 18:2, 18:2n-6, and 18:3n-3 concentrations. Even though conserved forages altered mammary lipogenesis, differences in milk FA composition were relatively minor, other than a higher enrichment of S3,R7,R11,15-tetramethyl-16:0 in milk from silages compared with hay. In conclusion, differences in milk fat composition on fresh grass relative to conserved forages were associated with a lower energy balance, increased uptake of preformed FA, and decreased synthesis of 16:0 de novo in the mammary glands, in the absence of alterations in stearoyl-coenzyme A desaturase activity.

'Nature concocts & expels': the agents and processes of recovery from disease in Early Modern England

The ‘golden saying’ in early modern medicine was ‘Nature is the healer of disease’. This article uncovers the meaning and significance of this forgotten axiom by investigating perceptions of the agents and physiological processes of recovery from illness in England, c.1580-1720. Drawing on sources such as medical texts and diaries, it shows that doctors and laypeople attributed recovery to three agents – God, Nature, and the practitioner. While scholars are familiar with the roles of providence and medicine, the vital agency of Nature has been overlooked. In theory, the agents operated in a hierarchy: Nature was ‘God’s instrument’, and the physician, ‘Nature’s servant’; but in practice the power balance was more ambivalent. Nature was depicted both as a housewife who cooked and cleaned the humours, and as a warrior, who defeated the disease. Through exploring these complex dynamics, the article sheds fresh light on concepts of gender, disease, and bodies.

The symbolic value of grafting in ancient Rome

Some scholars have read Virgil’s grafted tree (G. 2.78–82) as a sinister image, symptomatic of man’s perversion of nature. However, when it is placed within the long tradition of Roman accounts of grafting (in both prose and verse), it seems to reinforce a consistently positive view of the technique, its results, and its possibilities. Virgil’s treatment does represent a significant change from Republican to Imperial literature, whereby grafting went from mundane reality to utopian fantasy. This is reflected in responses to Virgil from Ovid, Columella, Calpurnius, Pliny the Elder, and Palladius (with Republican context from Cato, Varro, and Lucretius), and even in the postclassical transformation of Virgil’s biography into a magical folktale.

Redefinition of the kilogram, ampere, kelvin and mole: a proposed approach to implementing CIPM recommendation 1 (CI-2005)

The International System of Units (SI) is founded on seven base units, the metre, kilogram, second, ampere, kelvin, mole and candela corresponding to the seven base quantities of length, mass, time, electric current, thermodynamic temperature, amount of substance and luminous intensity. At its 94th meeting in October 2005, the International Committee for Weights and Measures (CIPM) adopted a recommendation on preparative steps towards redefining the kilogram, ampere, kelvin and mole so that these units are linked to exactly known values of fundamental constants. We propose here that these four base units should be given new definitions linking them to exactly defined values of the Planck constant h, elementary charge e, Boltzmann constant k and Avogadro constant NA, respectively. This would mean that six of the seven base units of the SI would be defined in terms of true invariants of nature. In addition, not only would these four fundamental constants have exactly defined values but also the uncertainties of many of the other fundamental constants of physics would be either eliminated or appreciably reduced. In this paper we present the background and discuss the merits of these proposed changes, and we also present possible wordings for the four new definitions. We also suggest a novel way to define the entire SI explicitly using such definitions without making any distinction between base units and derived units. We list a number of key points that should be addressed when the new definitions are adopted by the General Conference on Weights and Measures (CGPM), possibly by the 24th CGPM in 2011, and we discuss the implications of these changes for other aspects of metrology.

On defining base units in terms of fundamental constants

The definitions of the base units of the international system of units have been revised many times since the idea of such an international system was first conceived at the time of the French revolution. The objective today is to define all our units in terms of 'invariants of nature', i.e. by referencing our units to the fundamental constants of physics, or the properties of atoms, rather than the characteristics of our planet or of artefacts. This situation is reviewed, particularly in regard to finding a new definition of the kilogram to replace its present definition in terms of a prototype material artefact.

Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins

The cupin superfamily is a group of functionally diverse proteins that are found in all three kingdoms of life, Archaea, Eubacteria, and Eukaryota. These proteins have a characteristic signature domain comprising two histidine- containing motifs separated by an intermotif region of variable length. This domain consists of six beta strands within a conserved beta barrel structure. Most cupins, such as microbial phosphomannose isomerases (PMIs), AraC- type transcriptional regulators, and cereal oxalate oxidases (OXOs), contain only a single domain, whereas others, such as seed storage proteins and oxalate decarboxylases (OXDCs), are bi-cupins with two pairs of motifs. Although some cupins have known functions and have been characterized at the biochemical level, the majority are known only from gene cloning or sequencing projects. In this study, phylogenetic analyses were conducted on the conserved domain to investigate the evolution and structure/function relationships of cupins, with an emphasis on single- domain plant germin-like proteins (GLPs). An unrooted phylogeny of cupins from a wide spectrum of evolutionary lineages identified three main clusters, microbial PMIs, OXDCs, and plant GLPs. The sister group to the plant GLPs in the global analysis was then used to root a phylogeny of all available plant GLPs. The resulting phylogeny contained three main clades, classifying the GLPs into distinct subfamilies. It is suggested that these subfamilies correlate with functional categories, one of which contains the bifunctional barley germin that has both OXO and superoxide dismutase (SOD) activity. It is proposed that GLPs function primarily as SODs, enzymes that protect plants from the effects of oxidative stress. Closer inspection of the DNA sequence encoding the intermotif region in plant GLPs showed global conservation of thymine in the second codon position, a character associated with hydrophobic residues. Since many of these proteins are multimeric and enzymatically inactive in their monomeric state, this conservation of hydrophobicity is thought to be associated with the need to maintain the various monomer- monomer interactions. The type of structure-based predictive analysis presented in this paper is an important approach for understanding gene function and evolution in an era when genomes from a wide range of organisms are being sequenced at a rapid rate.

Sequence analysis of the cupin gene family in Synechocystis PCC6803

The recently described cupin superfamily of proteins includes the germin and germinlike proteins, of which the cereal oxalate oxidase is the best characterized. This superfamily also includes seed storage proteins, in addition to several microbial enzymes and proteins with unknown function. All these proteins are characterized by the conservation of two central motifs, usually containing two or three histidine residues presumed to be involved with metal binding in the catalytic active site. The present study on the coding regions of Synechocystis PCC6803 identifies a previously unknown group of 12 related cupins, each containing the characteristic two-motif signature. This group comprises 11 single-domain proteins, ranging in length from 104 to 289 residues, and includes two phosphomannose isomerases and two epimerases involved in cell wall synthesis, a member of the pirin group of nuclear proteins, a possible transcriptional regulator, and a close relative-of a cytochrome c551 from Rhodococcus. Additionally, there is a duplicated, two-domain protein that has close similarity to an oxalate decarboxylase from the fungus Collybia velutipes and that is a putative progenitor of the storage proteins of land plants.

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved beta-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the beta-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase.

Breeding distribution and population decline of globally threatened Grey-necked Picathartes Picathartes oreas in Mbam Minkom Mountain Forest, southern Cameroon

The Grey-necked Picathartes Picathartes oreas, considered 'Vulnerable', is an enigmatic ground-dwelling bird endemic to the central African equatorial rainforest and belongs to a family of only two species. Its distribution extends to the two Endemic Bird Areas within Cameroon (Guinea Congo forest biome and Cameroon mountain arc) and its population is thought to be in decline throughout its range due to increasing habitat fragmentation and disturbance. During March-April 2003 and June and October 2007 we surveyed Grey-necked Picathartes in the north-western region of the Mbam Minkom Mountain Forest. In January-March 2006 we surveyed the entire mountain range and found go breeding and 24 potential breeding sites, mostly located on the western slopes. From the complete survey, we estimated the population at 44 breeding individuals. Populations were highest in the north-west region but had apparently declined from 40 breeding individuals in 2003 to 20 in 2007. This region accounted for 41% of the entire population on the mountain range during the 2006 survey. The Mbam Minkom/Kala Important Bird Area was designated based on the presence of Grey-necked Picathartes but is under high pressure of imminent destruction from agricultural encroachment and illegal timber exploitation. These results have important implications for decision making in delimiting forest boundaries and core areas for protection in the development of management plans. We suggest possible remedial actions, appropriate repeatable methods for future monitoring and opportunities for community involvement in the management and conservation of the site.

Investigating the practicality of using radio tracking to determine home range and movements of Picathartidae

Radiotelemetry is an important tool used to aid the understanding and conservation of cryptic and rare birds. The two bird species of the family Picathartidae are little-known, secretive, forest-dwelling birds endemic to western and central Africa. In 2005, we conducted a radio-tracking trial of Grey-necked Picathartes Picathartes oreas in the Mbam Minkom Mountain Forest, southern Cameroon, using neck collar (two birds) and tail-mounted (four birds) transmitters to investigate the practicality of radio-tracking Picathartidae. Three birds with tail-mounted transmitters were successfully tracked with the fourth, though not relocated for radio tracking, resighted the following breeding season. Two of these were breeding birds that continued to provision young during radio tracking. One neck-collared bird was found dead three days after transmitter attachment and the other neither relocated nor resighted. As mortality in one bird was potentially caused by the neck collar transmitter we recommend tail-mounted transmitters in future radio-tracking studies of Picathartidae. Home ranges, shown using minimum convex polygon and kernel estimation methods, were generally small (<0.5 km(2)) and centred around breeding sites. A minimum of 60 fixes were found to be sufficient for home range estimation.

Seed survival in Chilean Nothofagus in response to desiccation and storage

Nothofagus alpina, N. obliqua, N. glauca, N. leonii, N. dombeyi and N. pumilio seeds exhibited consistent, albeit slight, sensitivity to extreme desiccation, but nevertheless maintained viability at low moisture contents and cool temperatures (-10 degrees to -20 degrees C) over 2 years. Nothofagus alpina, N. obliqua, N. glauca, N. leonii and N. dombeyi conformed to the seed viability equation of Ellis and Roberts; sensitivity of longevity to temperature was quantitatively similar to that of crop seeds, sensitivity to moisture was somewhat less, and a low-moisture-content limit to the equation was detected at 4.8% moisture content in hermetic storage at 65 degrees C, and possibly similar moisture contents at 30-40 degrees C. These five species show orthodox seed storage behaviour. Therefore, ex-situ conservation of these Nothofagus species in seed banks is possible, but the quality of seed lots collected requires attention. Seed storage behaviour was not defined in N. pumilio: initial seed quality was poor and loss of viability was detected over 2 years at 0 degrees, -10 degrees and -20 degrees C at 2.7% moisture content, but not at 5.2%. The results confirm that the economy of nature in seed storage physiology extends to forest tree seeds, but the repeated observation of reduced sensitivity of longevity to moisture in forest tree seeds requires further investigation.

Comparison of survival rates of captive-reared and wild-bred Mauritius kestrels (Falco punctatus) in a re-introduced population

Re-introduction is a technique widely used in the conservation of threatened bird species. With advances in aviculture the use of captive-produced individuals as the release stock is becoming more commonplace, and ideally, survival of captive-produced, released individuals should be no different from their wild-bred counterparts. During the late 1980s the Critically Endangered Mauritius kestrel (Falco punctatus) was successfully re-introduced into the Bambous mountain range, Mauritius, some 30 years after its local extinction. Between 1987 and 2001 the developing population was closely monitored enabling us to construct re-sighting histories for 88 released and 284 wild-bred kestrels. We used age-structured models in the survival analysis software program MARK to determine if an individual's origin influenced its subsequent survival. Our analysis indicated no compelling evidence for reduced survival among juvenile captive-reared and released individuals, relative to their wild-bred counterparts, across the majority of cohorts and only limited evidence of a cohort-specific effect. This study illustrates that despite the lack of a formal experimental approach it is still feasible to conduct an assessment of re-introduction outcomes and techniques.