Background Despite extensive research, many questions remain unanswered about common problems that impact dog welfare, particularly where there are multiple contributing factors that can occur months or years before the problem becomes apparent. The Generation Pup study is the first longitudinal study of dogs that recruits pure- and mixed-breed puppies, aiming to investigate the relative influence of environmental and genetic factors on a range of health and behaviour outcomes, (including separation related behaviour, aggression to familiar/unfamiliar people or dogs and obesity). This paper describes the study protocol in detail. Methods Prior to commencing recruitment of puppies, the study infrastructure was developed, and subject specialists were consulted to inform data collection methodology. Questionnaire content and timepoint(s) for data collection for outcomes and potential predictors were chosen with the aim of providing the best opportunity of achieving the aims of the study, subject to time and funding constraints. Recruitment of puppies (< 16 weeks, or < 21 weeks of age if entering the United Kingdom or Republic of Ireland through quarantine) is underway. By 23 January 2020, 3726 puppies had been registered, with registration continuing until 10,000 puppies are recruited. Data collection encompasses owner-completed questionnaires issued at set timepoints throughout the dog’s life, covering aspects such as training, diet, exercise, canine behaviour, preventative health care, clinical signs and veterinary intervention. Owners can elect to submit additional data (health cards completed by veterinary professionals, canine biological samples) and/or provide consent for access to veterinary clinical notes. Incidence and breed associations will be calculated for conditions for which there is currently limited information (e.g. separation related behaviour). Multivariable statistical analysis will be conducted on a range of outcomes that occur throughout different life stages, with the aim of identifying modifiable risk factors that can be used to improve canine health and welfare. Discussion The Generation Pup project is designed to identify associations between early-life environment, genotypic make-up and outcomes at different life stages. Modifiable risk factors can be used to improve canine health and welfare. Research collaboration with subject specialists is welcomed and already underway within the fields of orthopaedic research, epilepsy, epigenetics and canine impulsivity.

We have previously shown that the gas-vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33-RR) sandwiched between 18 N-terminal and 10 C-terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33-amino-acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33-RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33-amino-acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.

Filaments of Anabaenaflos-aquae lost over half of their gas vesicles when exposed to light of high irradiance (> 115 pmol m-2 s-l) for long periods (up to 23 h). An investigation using different irradiances showed that the major loss of gas vesicles occurred after prolonged exposure to irradiances exceeding 80 pmol m-2 s-I. In a timecourse experiment it was found that 56% of the gas vesicles were lost after 16 h exposure to an irradiance of 135 pmol m-* S -I . Most of this loss could be accounted for by collapse under turgor pressure, which rose by 0.24 MPa to 0.54 MPa after 16 h. A further unexplained loss of 15 % after 23 h exposure may indicate that prolonged high irradiance can cause weakening of gas vesicles in the cells. In this time-course experiment 98% of the filaments lost their buoyancy after 6 h exposure, before any gas vesicles were lost; this buoyancy change was quantitatively accounted for by the rise in cell carbohydrate. The relative importance of gas vesicle collapse and carbohydrate accumulation as mechanisms of buoyancy loss is discussed in relation to vertical movements of planktonic cyanobacteria in lakes.

Sleep is a vital behaviour that can reflect an animal’s adaptation to the environment and their welfare. However, a better understanding of normal age-specific sleep patterns is crucial. This study aims to provide population norms and descriptions of sleep-related behaviours for 16-week-old puppies and 12-month-old dogs living in domestic environments. Participants recruited to a longitudinal study answered questions relating to their dogs’ sleep behaviours in surveys issued to them when their dogs reached 16 weeks (n = 2332) and 12 months of age (n = 1091). For the statistical analysis, subpopulations of dogs with data regarding sleep duration at both timepoints were used. Owners of 16-week-old puppies perceived their dogs to sleep longer during the day and over a 24 h period, but for less time during the night than owners of 12-month-old dogs. At both timepoints, dogs were most commonly settled to sleep by being left in a room/area without human company. However, of dogs that had access to people overnight, 86.7% and 86.8% chose to be around people at 16 weeks and 12 months of age, respectively. The most common sleeping place was in a kennel/crate at 16 weeks (49.1%), and a dog bed at 12 months (31.7%). Future research within this longitudinal study will investigate how sleep duration and behaviours change with age and impact on a dog’s health and behaviour.

Only two gas vesicle genes have been previously identified in the cyanobacteria, gvpA and gvpC, both of which encode structural gas vesicle proteins. Analysis of the nucleotide sequence immediately downstream of gvpC in the cyanobacterium Anabaena flos-aquae has revealed the presence of 4 ORFs (open reading frames) the products of which share significant homology with a number of the gene products derived from halobacterial gvp gene clusters. In halobacteria the gas vesicle gene clusters consist of 14 genes involved in gas vesicle synthesis and assembly. The product of Anabaena ORF 1, located immediately downstream of gvpC is homologous to halobacterial GvpNs. For the remaining ORFs the predicted gene products show homology to both GvpJ and GvpA for ORF 2, to GvpK and GvpA for ORF 3, and to both GvpF and GvpL for ORF 4.