Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Anterior cruciate ligament transection alters the n-3/n-6 fatty acid balance in the lapine infrapatellar fat pad.

BACKGROUND: The infrapatellar fat pad (IFP) of the knee joint has received lots of attention recently due to its emerging role in the pathogenesis of osteoarthritis (OA), where it displays an inflammatory phenotype. The aim of the present study was to examine the infrapatellar fatty acid (FA) composition in a rabbit (Oryctolagus cuniculus) model of early OA created by anterior cruciate ligament transection (ACLT). METHODS: OA was induced randomly in the left or right knee joint of skeletally mature New Zealand White rabbits by ACLT, while the contralateral knee was left intact. A separate group of unoperated rabbits served as controls. The IFP of the ACLT, contralateral, and control knees were harvested following euthanasia 2 or 8 weeks post-ACLT and their FA composition was determined with gas chromatography-mass spectrometry. RESULTS: The n-3/n-6 polyunsaturated FA (PUFA) ratio shifted in a pro-inflammatory direction after ACLT, already observed 2 weeks after the operation (0.20 ± 0.008 vs. 0.18 ± 0.009). At 8 weeks, the FA profile of the ACLT group was characterized with increased percentages of 20:4n-6 (0.44 ± 0.064 vs. 0.98 ± 0.339 mol-%) and 22:6n-3 (0.03 ± 0.014 vs. 0.07 ± 0.015 mol-%) and with decreased monounsaturated FA (MUFA) sums (37.19 ± 1.586 vs. 33.20 ± 1.068 mol-%) and n-3/n-6 PUFA ratios (0.20 ± 0.008 vs. 0.17 ± 0.008). The FA signature of the contralateral knees resembled that of the unoperated controls in most aspects, but had increased proportions of total n-3 PUFA and reduced MUFA sums. CONCLUSIONS: These findings provide novel information on the effects of early OA on the infrapatellar FA profile in the rabbit ACLT model. The reduction in the n-3/n-6 PUFA ratio of the IFP is in concordance with the inflammation and cartilage degradation in early OA and could contribute to disease pathogenesis.

Abortion debates in Finland and the Republic of Ireland: textual analysis of experiential thinking and argumentation in parliamentary and layperson discussions.

BACKGROUND: The ethical discussion about abortion has been polarized in Finland and the Republic of Ireland, two European countries with very different abortion legislation (liberal vs. highly restrictive). The aim of the present study was to analyze experiential thinking patterns and argumentative strategies in political and layperson debates regarding induced abortion. METHODS: The content of Finnish and Irish texts (n = 493), consisting of transcripts of parliamentary debates and online texts, such as blogs, was analyzed systematically. The texts were investigated for the aspects of experiential thinking, for selected argumentative moves and for any differences in the prevalence of these features between countries or between political vs. layperson debates. RESULTS: The Finnish and Irish discussions about induced abortion relied heavily on experiential thinking patterns and emotionally laden arguments instead of objective research data. This was evident in the very high prevalence of testimonials, narratives, loaded language and appeals to emotion in both political and layperson debates regardless of the country or the debater's position on abortion issue. Research data that did not support the position of the debater were relatively often omitted by confirmation bias. The Irish debaters appealed to popularity more often than the Finnish ones, while magical/religious thinking was mainly observed in the Finnish layperson discussion. The national history and the prevailing cultural and religious atmosphere of the two countries could explain these differences. CONCLUSIONS: The abortion debate mostly reinforces the opinions of one's peer group rather than convinces the opposite party to change their position. The stalemate and continuation of the same arguments being repeated could be associated with experiential thinking and emotional argumentative strategies in both political and layperson debates.

Life stage-related differences in fatty acid composition of an obligate ectoparasite, the deer ked (Lipoptena cervi)-influence of blood meals and gender.

Metamorphosis and diet often influence fatty acid (FA) signatures (FAS) of insects. We investigated FAS in a hematophagous ectoparasite, the deer ked (Lipoptena cervi). Deer keds shed their wings upon attachment on the host and, thus, the FAS of an individual blood-fed imago/pupa in the fur of its host can be traced back to the blood FA profile of a single moose (Alces alces). Host blood and different life stages of deer keds were investigated for FA by gas chromatography. The FAS of life stages resembled each other more closely than the diet. Blood meals modified the FAS of both sexes but the FAS of the blood-fed females were closer to those of the prepupae/pupae. The parasitizing males had higher proportions of major saturated FA (SFA) and polyunsaturated FA (PUFA) than the females, which contained more monounsaturated FA (MUFA) with higher ratios of n-3/n-6 PUFA and unsaturated FA (UFA)/SFA. The proportions of 16:1n-7 were <1% in the blood but 18% (males) and 29% (females) in the blood-fed keds. Allocation of lipids to offspring by the females and possible accumulation of PUFA in male reproductive organs may have induced these sex-related differences. MUFA percentages and UFA/SFA ratios increased while SFA and many PUFA decreased from the reproducing females to the pupae. The diapausing pupae displayed lowered n-3/n-6 PUFA ratios and could have mobilized 16:0 and 18:3n-3 for the most fundamental metabolic processes. In conclusion, FAS are modified through the life stages of the deer ked possibly due to their different FA requirements.

Acute impacts of the deer ked (Lipoptena cervi) infestation on reindeer (Rangifer tarandus tarandus) behaviour.

Blood-sucking ectoparasites have often a strong impact on the behaviour of their hosts. The annual insect harassment of reindeer (Rangifer tarandus tarandus) has increased in the southern part of the Finnish reindeer herding area because of the recent invasion of a blood-feeding ectoparasitic louse-fly, the deer ked (Lipoptena cervi). We studied the impact of the deer ked on the behaviour of reindeer. Twelve reindeer were infested with a total of 300 keds/reindeer on six occasions in a 5-week period during the deer ked flight season in autumn, while six non-infested reindeer were used as controls. Behavioural patterns indicating potential stress were monitored by visual observation from August to December. The infested reindeer displayed more incidences of restless behaviour than the controls. Shaking and scratching were the most common forms of restless behaviour after infestation of deer keds. Increased grooming was also observed after the transplantation and also later, 1 month after the infestation. Based on the results, the deer ked infestation can cause acute behavioural disturbance in reindeer and, thus, could pose a potential threat to reindeer welfare. Antiparasitic treatment with, e.g. ivermectin, may increase the welfare of parasitized reindeer by reducing deer keds. If the deer ked infestation intensity on the reindeer herding area increases and restless behaviour of reindeer becomes more common, the present results can help in further evaluation of the duration and magnitude of behavioural changes.

Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

OBJECTIVE: Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. DESIGN: We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. RESULTS: The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. CONCLUSIONS: Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

Equine osteoarthritis modifies fatty acid signatures in synovial fluid and its extracellular vesicles.

BACKGROUND: Individual fatty acids (FAs) and their derivatives (lipid mediators) with pro-inflammatory or dual anti-inflammatory and pro-resolving properties have potential to influence the health of joint tissues. Osteoarthritis (OA) is an age-associated chronic joint disease that can be featured with altered FA composition in the synovial fluid (SF) of human patients. The counts and cargo of extracellular vesicles (EVs), membrane-bound particles released by synovial joint cells and transporting bioactive lipids, can also be modified by OA. The detailed FA signatures of SF and its EVs have remained unexplored in the horse - a well-recognized veterinary model for OA research. METHODS: The aim of the present study was to compare the FA profiles in equine SF and its ultracentrifuged EV fraction between control, contralateral, and OA metacarpophalangeal joints (n = 8/group). The FA profiles of total lipids were determined by gas chromatography and the data compared with univariate and multivariate analyses. RESULTS: The data revealed distinct FA profiles in SF and its EV-enriched pellet that were modified by naturally occurring equine OA. Regarding SFs, linoleic acid (generalized linear model, p = 0.0006), myristic acid (p = 0.003), palmitoleic acid (p < 0.0005), and n-3/n-6 polyunsaturated FA ratio (p < 0.0005) were among the important variables that separated OA from control samples. In EV-enriched pellets, saturated FAs palmitic acid (p = 0.020), stearic acid (p = 0.002), and behenic acid (p = 0.003) indicated OA. The observed FA modifications are potentially detrimental and could contribute to inflammatory processes and cartilage degradation in OA. CONCLUSIONS: Equine OA joints can be distinguished from normal joints based on their FA signatures in SF and its EV-enriched pellet. Clarifying the roles of SF and EV FA compositions in the pathogenesis of OA and their potential as joint disease biomarkers and therapeutic targets warrants future studies.

The effects of an abundant ectoparasite, the deer ked (Lipoptena cervi), on the health of moose (Alces alces) in Finland.

The deer ked (Lipoptena cervi, Diptera, Hippoboscidae) is a haematophagous parasitic fly of the moose (Alces alces) and other cervids, and it is very common in southern and central parts of Finland. The aim of this study was to determine how the intensive parasitism caused by the deer ked affects the health and welfare of the moose. Moose blood samples (n = 78) were collected from deer ked-infested and ked-free regions at 62-68° N and analysed for haematology and clinical chemistry. In addition, tissue samples of moose (n = 23) were collected from a deer ked-infested region at 62° N to determine how the parasite load correlates to several physiological variables of the host. The differences in the blood and plasma values between the deer ked-free and ked-infested animals were minor. In the infested regions, the moose had higher mean corpuscular haemoglobin concentrations unlikely to have been caused by the parasitism. The intensities of deer keds had no consistent correlations with the values of plasma clinical chemistry, endocrinology, amino acids, tissue enzyme activities or body energy stores. However, the hepatic percentages of several individual n-3 polyunsaturated fatty acids (PUFA) and the n-3 PUFA sum correlated inversely with the intensity and density of deer keds. Although a wide array of physiological variables was determined, only minor effects caused by the heavy deer ked parasitism could be detected, suggesting that the moose might tolerate this parasite relatively well.

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse-a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA-EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA-EV count, and HA-EV % (rs = - 0.264 to - 0.327, p = 0.012-0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA-EVs) for naturally occurring equine OA. The roles of HA-EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.

Metabolomics of Synovial Fluid and Infrapatellar Fat Pad in Patients with Osteoarthritis or Rheumatoid Arthritis.

Osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA) are inflammatory joint diseases with complex and insufficiently understood pathogeneses. Our objective was to characterize the metabolic fingerprints of synovial fluid (SF) and its adjacent infrapatellar fat pad (IFP) obtained during the same surgical operation from OA and RA knees. Non-targeted metabolite profiling was performed for 5 non-inflammatory trauma controls, 10 primary OA (pOA) patients, and 10 seropositive RA patients with high-resolution mass spectrometry-based techniques, and metabolites were matched with known metabolite identities. Groupwise differences in metabolic features were analyzed with the univariate Welch's t-test and the multivariate linear discriminant analysis (LDA) and principal component analysis (PCA). Significant discrimination of metabolite profiles was discovered by LDA for both SF and IFP and by PCA for SF based on diagnosis. In addition to a few drug-derived substances, there were 16 and 13 identified metabolites with significant differences between the diagnoses in SF and IFP, respectively. The pathways downregulated in RA included androgen, bile acid, amino acid, and histamine metabolism, and those upregulated included biotin metabolism in pOA and purine metabolism in RA and pOA. The RA-induced downregulation of androgen and bile acid metabolism was observed for both SF and IFP. The levels of 11 lipid metabolites, mostly glycerophospholipids and fatty acid amides, were also altered by these inflammatory conditions. The identified metabolic pathways could be utilized in the future to deepen our understanding of the pathogeneses of OA and RA and to develop not only biomarkers for their early diagnosis but also therapeutic targets.

Near-Infrared Spectroscopy for Mapping of Human Meniscus Biochemical Constituents.

Degenerative changes in meniscus are diagnosed during surgery by means of mechanical testing and visual evaluation. This method is qualitative and highly subjective, providing very little information on the internal state of the meniscus. Thus, there is need for novel quantitative methods that can support decision-making during arthroscopic surgery. In this study, we investigate the potential of near-infrared spectroscopy (NIRS) for mapping the biochemical constituents of human meniscus, including water, uronic acid, and hydroxyproline contents. Partial least squares regression models were developed using data from 115 measurement locations of menisci samples extracted from 7 cadavers and 11 surgery patient donors. Model performance was evaluated using an independent test set consisting of 55 measurement locations within a meniscus sample obtained from a separate cadaver. The correlation coefficient of calibration (ρtraining), test set (ρtest), and root-mean-squared error of test set (RMSEP) were as follows: water (ρtraining = 0.61, ρtest = 0.39, and RMSEP = 2.27 percentage points), uronic acid (ρtraining = 0.68, ρtest = 0.69, and RMSEP = 6.09 basis points), and hydroxyproline (ρtraining = 0.84, ρtest = 0.58, and error = 0.54 percentage points). In conclusion, the results suggest that NIRS could enable rapid arthroscopic mapping of changes in meniscus biochemical constituents, thus providing means for quantitative assessment of meniscus degeneration.

Synovial Fluid Fatty Acid Profiles Are Differently Altered by Inflammatory Joint Pathologies in the Shoulder and Knee Joints.

Anomalies of fatty acid (FA) metabolism characterize osteoarthritis (OA) and rheumatoid arthritis (RA) in the knee joint. No previous study has investigated the synovial fluid (SF) FA manifestations in these aging-related inflammatory diseases in the shoulder. The present experiment compared the FA alterations between the shoulder and knee joints in patients with end-stage OA or end-stage RA. SF samples were collected during glenohumeral or knee joint surgery from trauma controls and from OA and RA patients (n = 42). The FA composition of SF total lipids was analyzed by gas chromatography with flame ionization and mass spectrometric detection and compared across cohorts. The FA signatures of trauma controls were mostly uniform in both anatomical locations. RA shoulders were characterized by elevated percentages of 20:4n-6 and 22:6n-3 and with reduced proportions of 18:1n-9. The FA profiles of OA and RA knees were relatively uniform and displayed lower proportions of 18:2n-6, 22:6n-3 and total n-6 polyunsaturated FAs (PUFAs). The results indicate location- and disease-dependent differences in the SF FA composition. These alterations in FA profiles and their potential implications for the production of PUFA-derived lipid mediators may affect joint lubrication, synovial inflammation and pannus formation as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases.

Biomechanical, biochemical, and near infrared spectral data of bovine knee ligaments and patellar tendon.

Knee joint ligaments and patellar tendon are rope-like tissues that enable the proper function of the knee by connecting the bones that form the joint. A better understanding of ligament structure-function relationships is needed to develop objective and reliable diagnostic methods for ligaments. Recently, arthroscopic near infrared spectroscopy (NIR) has shown the potential to quantitatively evaluate the health of the cartilages and menisci of the knee. In this dataset, we present a unique combination of NIR spectral data, biomechanical properties, and biochemical composition of bovine primary knee ligaments and patellar tendon (10 knees, 50 tissue samples). NIR spectral data were measured at 5 locations in each sample, biomechanical properties were obtained with tensile testing, and biochemical composition was quantified using colorimetric biochemical methods. The data can be reused for investigations of structure-function relationships of knee ligaments and patellar tendon, for the development of NIR spectroscopic methods to quantify the health of these tissues, and to develop new computational models to describe ligament and tendon biomechanics.

Distinct fatty acid signatures in infrapatellar fat pad and synovial fluid of patients with osteoarthritis versus rheumatoid arthritis.

BACKGROUND: Infrapatellar fat pad (IFP) has recently emerged as a potential source of inflammation in knee arthropathies. It has been proposed to be one source of adipocytokines, fatty acids (FA), and FA-derived lipid mediators that could contribute to the pathophysiological processes in the knee joint. Alterations in synovial fluid (SF) lipid composition have been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA). The aim of the present study was to compare the FA signatures in the IFP and SF of RA and OA patients. METHODS: Pairs of IFP and SF samples were collected from the same knees of RA (n = 10) and OA patients (n = 10) undergoing total joint replacement surgery. Control SF samples (n = 6) were harvested during diagnostic or therapeutic arthroscopic knee surgery unrelated to RA or OA. The FA composition in the total lipids of IFP and SF was determined by gas chromatography with flame ionization and mass spectrometric detection. RESULTS: Arthropathies resulted in a significant reduction in the SF proportions of n-6 polyunsaturated FA (PUFA), more pronouncedly in OA than in RA. OA was also characterized with reduced percentages of 22:6n-3 and lower product/precursor ratios of n-3 PUFA. The proportions of total monounsaturated FA increased in both RA and OA SF. Regarding IFP, RA patients had lower proportions of 20:4n-6, total n-6 PUFA, and 22:6n-3, as well as lower product/precursor ratios of n-3 PUFA compared to OA patients. The average chain length of SF FA decreased in both diagnoses and the double bond index in OA. CONCLUSIONS: The observed complex alterations in the FA signatures could have both contributed to but also limited the inflammatory processes and cartilage destruction in the RA and OA knees.

Seasonal rhythms of body temperature in the free-ranging raccoon dog (Nyctereutes procyonoides) with special emphasis on winter sleep.

The raccoon dog (Nyctereutes procyonoides) is the only canid with passive overwintering in areas with cold winters, but the depth and rhythmicity of wintertime hypothermia in the wild raccoon dog are unknown. To study the seasonal rhythms of body temperature (T(b)), seven free-ranging animals were captured and implanted with intra-abdominal T(b) loggers and radio-tracked during years 2004-2006. The average size of the home ranges was 306+/-26 ha, and the average 24 h T(b) was 38.0+/-<0.01 degrees C during the snow-free period (May-November). The highest and lowest T(b) were usually recorded around midnight (21:00-02:00 h) and between 05:00-11:00 h, respectively, and the range of the 24 h oscillations was 1.2+/-0.01 degrees C. The animals lost approximately 43+/-6% of body mass in winter (December-April), when the average size of the home ranges was 372+/-108 ha. During the 2-9-wk periods of passivity in January-March, the average 24 h T(b) decreased by 1.4-2.1 degrees C compared to the snow-free period. The raccoon dogs were hypothermic for 5 h in the morning (06:00-11:00 h), whereas the highest T(b) values were recorded between 16:00-23:00 h. The range of the 24 h oscillations increased by approximately 0.6 degrees C, and the rhythmicity was more pronounced than in the snow-free period. The ambient temperature and depth of snow cover were important determinants of the seasonal T(b) rhythms. The overwintering strategy of the raccoon dog resembled the patterns of winter sleep in bears and badgers, but the wintertime passivity of the species was more intermittent and the decrease in the T(b) less pronounced.

Fatty acid modifications during autumnal cold-hardening in an obligatory ectoparasite, the deer ked (Lipoptena cervi).

Poikilothermic organisms often modify their tissue fatty acids (FA) in response to cold exposure by increased unsaturation. In insects, this has been found to be accompanied by increases in the activities or mRNA expression of desaturase enzymes. In the present study, the FA composition of an obligatory ectoparasite, the deer ked (Lipoptena cervi), was analyzed in August-November. In addition to studying the general FA profile of the species, the possible contribution of FA to autumnal cold-hardening was examined. The FA composition of the deer ked imago was relatively similar to previously studied dipteran species, with high percentages of monounsaturated FA (especially 18:1n-9 and 16:1n-7) and 16:0. The individuals caught later in autumn had significantly higher values for the ratio of unsaturated to saturated FA and, regarding individual FA, the percentages of 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 were higher but those of 16:0 and 16:1n-7 lower than in August. Potential selective use of particular FA for energy could not account for the large increase in the levels of polyunsaturated FA (PUFA). The observed increased degree of FA unsaturation may have resulted from cold-induced desaturation, as observed previously in other species, or increased survival of the keds with relatively large PUFA contents. The PUFA with low melting points probably allow lipid membranes to maintain sufficient fluidity required to maintain protein functions at low ambient temperatures.

Freezing tolerance and low molecular weight cryoprotectants in an invasive parasitic fly, the deer ked (Lipoptena cervi).

Insect cold hardiness is often mediated by low molecular weight cryoprotectants, such as sugars, polyols, and amino acids (AA). While many free-living northern insects must cope with extended periods of freezing ambient temperatures (Ta), the ectoparasitic deer ked Lipoptena cervi imago can encounter subfreezing Ta only during a short autumnal period between hatching and host location. Subsequently, it benefits from the body temperature of the cervid host for survival in winter. This study investigated the cold tolerance of the species by determining its lower lethal temperature (100% mortality, LLT100) during faster and slower cold acclimation, by determining the supercooling point (SCP) and by measuring the concentrations of potential low molecular weight cryoprotectants. The LLT100 of the deer ked was approximately -16 ° C, which would enable it to survive freezing nighttime Ta not only in its current area of distribution but also further north. The SCP was -7.8 ° C, clearly higher than the LLT100 , indicating that the deer ked displays freezing tolerance. The concentrations of free AA, especially nonessential AA, were higher in the cold-acclimated deer keds similar to several other insects. The concentrations of proline increased together with γ-aminobutyrate, arginine, asparagine, cystine, glutamate, glutamine, hydroxylysine, sarcosine, serine, and taurine. AA could be hypothesized to act as cryoprotectants by, e.g., protecting enzymes and lipid membranes from damage caused by cold.

Experimental infection of the deer ked (Lipoptena cervi) has no negative effects on the physiology of the captive reindeer (Rangifer tarandus tarandus).

The deer ked (Lipoptena cervi) is a haematophagous parasitic fly of cervids that spread to Finland in the early 1960's. Presently its northern distribution limit lies at approximately 65°N and it is gradually spreading northwards. In Finland the principal host species has been the moose (Alces alces), but the deer ked is about to establish contact with another potential host, the semi-domesticated reindeer (Rangifer tarandus tarandus) causing possible threats to reindeer health and management. The aim of this study was to investigate if the deer ked would have an influence on the welfare of the reindeer. Eighteen adult reindeer were divided into three experimental groups: the control group and two infected groups with 300 deer keds per reindeer introduced in August-September. One of the infected groups was treated with subcutaneous ivermectin in November. To gather comprehensive data on potential health hazards caused by the deer ked a wide array of physiological variables was measured during and at the end of the experiment in December. The keds caused no clear changes in the complete blood count, plasma clinical chemistry, amino acids, endocrinology, energy stores, enzyme activities or tissue fatty acid profiles of the host. The haematological, clinical chemical and endocrinological values displayed changes that could be related to the seasonal physiological adaptations of the species. In conclusion, at the duration and intensity of infection that were employed, the effects of the deer ked on the measured physiological variables of the reindeer were insignificant.

Adaptations to fasting in a terrestrial mustelid, the sable (Martes zibellina).

The aim of this study was to investigate whether the actively wintering Palearctic sable Martes zibellina has evolved physiological adaptations to tolerate nutritional scarcity. Sixteen farm-bred male sables were divided into a fed control group and an experimental group fasted for 4 days. The rate of weight loss in the sable was similar to other medium-sized mustelids. Fasting led to hypoglycaemia and to a decreased lymphocyte percentage. The sable derived metabolic energy from both subcutaneous and intraabdominal white adipose tissues and the relative decrease in fat mass was the largest for the retroperitoneal and subcutaneous depots. Metabolic energy derived partly from body proteins indicated by the increased plasma levels of urea, uric acid and total essential amino acids. Triacylglycerols accumulated in the livers of the fasted sables and the increased plasma aminotransferase activities suggested hepatic dysfunction. The decreased plasma insulin concentrations and the elevated cortisol levels probably contributed to stimulated lipolysis and protein catabolism. Moreover, fasting increased the plasma ghrelin concentrations of the sables and down-regulated the thyroid activity.