Background: The ability to perceive two tactile stimuli as asynchronous can be measured using the somatosensory temporal discrimination threshold (STDT). In healthy humans, the execution of a voluntary movement determines an increase in STDT values, while the integration of STDT and movement execution is abnormal in patients with basal ganglia disorders. Sensorimotor integration can be modulated using focal muscle vibration (fMV), a neurophysiological approach that selectively activates proprioceptive afferents from the vibrated muscle. Method: In this study, we investigated whether fMV was able to modulate STDT or STDT–movement integration in healthy subjects by measuring them before, during and after fMV applied over the first dorsalis interosseous, abductor pollicis brevis and flexor radialis carpi muscles. Results: The results showed that fMV modulated STDT–movement integration only when applied over the first dorsalis interosseous, namely, the muscle performing the motor task involved in STDT–movement integration. These changes occurred during and up to 10 min after fMV. Differently, fMV did not influence STDT at rest. We suggest that that fMV interferes with the STDT–movement task processing, possibly disrupting the physiological processing of sensory information. Conclusions: This study showed that FMV is able to modulate STDT–movement integration when applied over the muscle involved in the motor task. This result provides further information on the mechanisms underlying fMV, and has potential future implications in basal ganglia disorders characterized by altered sensorimotor integration. The ability to temporally discriminate tactile stimuli can be measured with the somatosensory temporal discrimination threshold (STDT) technique, defined as the shortest time interval necessary for two sequential tactile stimuli to be perceived as consecutive ,2,3. STDA neurophysiological approach that may potentially modulate sensorimotor integration is muscle vibration ,15,16,17Based on this evidence, it is reasonable to hypothesize that if fMV can modulate sensory afferents, motor outputs and their integration, then it may also induce changes in mechanisms of sensorimotor integration such as those involved in the STDT–movement integration task.Therefore, the aim of the present study was to assess whether fMV, applied over the first dorsalis interosseous (FDI) muscle, would modulate STDT values tested during and after voluntary movement execution in healthy subjects. It has been also tested whether fMV applied on FDI can induce changes in the STDT–movement integration. In order to better clarify whether the effect of fMV on STDT–movement integration depended on the vibrated muscle, in a control experiment, fMV was applied over the abductor pollicis brevis (APB) or the flexor radialis carpi (FRC) muscles. Finally, in order to investigate the effect of the amplitude of the vibratory stimulus on the STDT–movement integration changes, fMV was used to generate a different displacement in a separate control experiment. These data may clarify the mechanism of action of the fMV in physiological conditions, and whether fMV might have therapeutic applications in movement disorders that are known to underlie abnormal sensorimotor integration.In the following sections, the methodological approach used in the study, including details on the experimental procedure, will first be described; afterwards, results will be reported, explained and discussed in the following “results” and “discussion” sessions.The effects of FMV on STDT and STDT–movement integration were investigated in a sample of healthy volunteers. To follow, demographic features of the volunteers, inclusion and exclusion criteria and methodological details for each experimental procedure will be detailed.A total of 17 healthy volunteers were enrolled in the study .The sample size was based on an a priori analysis with G*Power 3.1 software, assuming an expected effect size (Cohen’s d) of 0.85 and a statistical power of 0.80 at a 0.05 alpha level, with the number of measurement set as 4 for experiment 1 and as 3 for experiments 2 and 3. This resulted in a total sample size of 17 subjects for experiment 1 and 15 subjects for experiments 2 and 3.Accordingly, all participants took part in experiment 1, while only fifteen of them took part in experiments 2 and 3.Exclusion criteria were a history of neuropsychiatric disorders or symptoms of focal upper limb nerve entrapment, cervical brachialgia, polyneuropathy or central nervous system (CNS)-acting drug intake at the time of the experiments. Volunteers were asked to avoid the intake of caffeine, nicotine or any other substances interfering with CNS at least 6 h before the experimental session.All participants except two were right-handed, as evaluated by the Edinburgh Handedness Inventory . All expSTDT–movement integration paradigm paragraph).EMG activity was recorded through a pair of surface electrodes placed over the FDI muscle of the dominant hand, in a belly–tendon configuration. The EMG signal was recorded (×1000) and filtered (5–5000 Hz) with a Digitimer D360 , and then analyzed offline with a personal computer through a 1401 plus analog–digital (A/D) laboratory interface  [STDT was investigated according to previous studies ,3,4, usiSimilarly to previous studies ,9, paireFocal muscle vibration was applied to the FDI muscle using a specific device consisting of an electromechanical transducer, a mechanical support and an electronic control .The instrument consisted of an electromechanical transducer, a specific mechanical support and an electronic control device. The transducer was positioned over the FDI muscle at a point approximately corresponding to its maximal transversal size. The FDI was chosen as it is feasible for the fMV protocol and it is involved in the motor task used for the STDT–movement integration paradigm.The vibration was applied by means of a small probe 10 mm in diameter directly over the FDI muscle belly. The transducer was driven to produce sinusoidally modulated forces. Soft tissues were compressed to ensure better transmission of the vibrations into the muscle ,24,25.The vibration frequency was set at 100 Hz and vibration amplitude ranged from 0.1 to 0.2 mm peak-to-peak, which was sufficient to drive Ia spindle afferents, to avoid muscle fiber injury and as a subthreshold for the tonic vibration reflex. Each instance of stimulation consisted of two blocks lasting 10 min (‘), with an interblock interval of 1 min.The design of the study included a main experiment (experiment 1) and two control experiments (experiments 2 and 3), which took place one week apart from the main experiment to avoid interference due to possible long term effects of each fMV session. The order of the experimental sessions was randomized across the participants.Experiment 1: Effects of fMV applied over the FDI muscle on STDT and STDT–movement integrationAll subjects participated at this experiment. STDT and the STDT–movement paradigm were tested prior to, during and after the application of fMV over the FDI muscle, which was involved in the motor task underlying STDT–movement integration. In particular, the STDT at rest and the STDT–movement paradigm were tested at the beginning of each experimental session (T0), during the second block of fMV (T1) and 5′ (T2) and 10′ (T3) after fMV. FMV was applied to generate a 0.1 mm peak-to-peak sinusoidal displacement. For the STDT–movement paradigm, the different intervals  between movement onset and the STDT testing were selected in a randomized order for each subject .Experiment 2 (fMV topographic specificity): effects of fMV applied over the abductor pollicis brevis (APB) or the flexor radialis carpi (FRC) muscles on STDT and STDT–movement integrationIn order to investigate whether the possible effects of fMV on STDT or STDT–movement changes were specific to the stimulated muscular district, STDT and STDT–movement integration were tested prior to, during and after the application of fMV on different muscles of the upper limbs that were not involved in the movement execution. To this aim, in 14 out of 17 participants enrolled in experiment 1, the STDT and the STDT–movement paradigm were tested “before”, “during” and “after” fMV was applied over the APB, as well as over the FRC, in separate experimental sessions. The stimulation parameters and time points were the same as those used for experiment 1.Experiment 3: Effects of fMV applied over the APB generating a 0.2 mm muscle displacementIn order to investigate whether possible effects of fMV on STDT or STDT–movement integration depended on vibration parameters, STDT and STDT–movement integration were tested prior to, during and after the application of fMV, which generated a 0.2 mm displacement of the APB muscle. To this aim, in 14 out of 17 participants enrolled in experiment 1, the STDT and the STDT–movement paradigm were tested “before”, “during” and “after” fMV was applied, generating a 0.2 mm displacement of the APB muscle. The stimulation parameters and time points were the same as those used for experiment 1.t-tests with Bonferroni’s correction for multiple comparisons. Compound symmetry was calculated with Mauchly’s test, and the Greenhouse–Geisser correction was applied when required. All variables were first tested for normality using the Kolmogorov–Smirnov test. Significance was set at p value < 0.05. Unless otherwise stated, values are expressed as means ± standard error of the mean (SEM). To quantify the magnitude of effect for the observed differences, the partial eta-squared effect size (ηp2) was computed.Statistical analysis was performed using SPSS 20 software . Repeated measures (RM) analysis of variance (ANOVA) and post hoc analyses were conducted by means of For all the experiments, to evaluate changes in STDT value due to fMV, a one-way RM-ANOVA was used, with time (before (T0), during (T1) and 5′ (T2) and 10′ (T3) after fMV) as a within-subject factor. For the STDT–movement integration, to evaluate changes in STDT values during movement execution as determined by fMV, a two-way RM-ANOVA was performed with time (before (T0), during (T1) and 5′ (T2) and 10′ after (T3) fMV for experiment 1; and only T0, T1 and T2 for experiments 2 and 3) and ISI  as within-subject factors.In the following section, the results obtained from each experimental session will be described. All the results are summarized in Experiment 1: Effects of fMV applied over the FDI muscle on STDT and STDT–movement integration3,39) = 1.239; p = 0.299, ηp2 = 0.072), showing no differences in STDT values measured at rest before, during or after fMV  = 57.739; p < 0.001, ηp2 = 0.783), time  = 34.894; p < 0.001, ηp2 = 0.686) and interaction between factors  = 8.69; p < 0.001, ηp2 = 0.352) , during  and after  fMV. Moreover, the post hoc analysis showed that STDT–movement integration at T1 (during fMV) significantly differed from that at T0 (before fMV) (p = 0.001). Similarly, STDT–movement at T2 (5 ‘ after fMV) (p < 0.001), as well as at T3 (10′ after fMV), was significantly different from that at T1 (during fMV) (p < 0.001). Finally, the post hoc analysis showed a significant effect of fMV on STDT modulation only “during” fMV at all the ISIs  after index finger abduction .The one-way RM-ANOVA performed to investigate changes in STDT values while fMV was applied over the FDI failed to detect a significant effect of the time factor (F(fter fMV . The two= 0.352) . First, Experiment 2: fMV topographic specificity effects of fMV applied over the APB or the FRC muscles on STDT–movement integration task = 2.199; p = 0.139, ηp2 = 0.136). The two-way RM-ANOVA performed to compare STDT–movement integration before, during and after fMV’s application over the FDI showed a significant effect of ISI  = 23.517; p < 0.001, ηp2 = 0.681), but a non-significant effect of time  = 1.885; p = 0.179, ηp2 = 0.146) and of interaction between factors  = 0.917; p = 0.459, ηp2 = 0.077). Post hoc analysis showed a significant difference between the baseline and 0 ms, 100 and 200 ms (p < 0.05). Similarly, in the experimental session in which fMV was applied over the FRC muscle, the one-way RM-ANOVA showed a non-significant effect of time  = 0.043; p = 0.956; ηp2 = 0.003) for STDT values. In addition, the two-way RM-ANOVA on STDT–movement integration showed a significant effect of ISI  = 30.851; p < 0.001, ηp2 = 0.720), but a non-significant effect of time  = 0.811; p = 0.411, ηp2 = 0.063) and of interaction between factors  = 0.517; p = 0.738, ηp2 = 0.041). Post hoc analysis still showed a difference between baseline, 0 ms and 100 ms .The one-way RM-ANOVA performed to investigate changes in STDT values at rest when fMV was applied over the FDI muscle showed a non-significant effect of time  = 2.199; p = 0.139, ηp2 = 0.136). The two-way RM-ANOVA performed to compare STDT–movement integration before, during and after the application of fMV, generating a 0.2 mm displacement of the APB muscle, showed a significant effect of ISI  = 23.517; p < 0.001, ηp2 = 0.681), but a non-significant effect of time  = 1.885; p = 0.179, ηp2 = 146) and of interaction between factors  = 0.917; p = 0.459, ηp2 = 0.077). Post hoc analysis showed significant differences between the baseline, 0 ms, 100 ms and 200 ms (p < 0.05).The one-way RM-ANOVA performed to investigate changes in STDT values at rest during the application of fMV, generating a 0.2 mm displacement of the APB muscle, showed a non-significant effect of time (FThe present study demonstrates that fMV does not influence STDT at rest, but significantly modulates STDT–motor integration. FMV-induced modulation was observed only during fMV administration, and was specific to the muscle involved in the motor task performed to evaluate STDT–motor integration.To rule out the possibility that movement-induced STDT modulation evaluated before, during and after fMV was due to changes in attention, the ISIs for the STDT–movement protocol were tested in a randomized order, except for the STDT measurement at rest, which was performed as the first assessment in all trials. In addition, the order of the experimental sessions was randomized across the participants. The fMV protocol used in our study was selected in order to obtain the highest muscle–tendon complex displacement without evoking reflex muscular contractions, i.e., the tonic vibration reflex, or selectively stimulating the Ia afferents . As the The observation that fMV modified STDT in healthy subjects only when tested during movement, but not at rest, suggests that the fMV is able to interfere with mechanisms of sensorimotor integration, but not with pure sensory processing in the temporal domain. The modulation of STDT–movement integration observed during fMV may possibly reflect an interaction between the neural circuits involved in the STDT–movement integration task and the proprioceptive circuits activated by fMV in physiological conditions.One possible explanation for our findings is that vibratory stimulation may affect cortical processing of sensorimotor information related to movement execution. However, the observation that fMV left STDT values unchanged tentatively rules out that changes in STDT–movement integration during fMV rely on inhibitory circuits in S1 ,5. MoreoAnother possible explanation is that fMV interferes with mechanisms of STDT–movement integration by altering M1 excitability. According to previous studies, fMV selectively increases the excitability of the representation of the vibrated muscle in M1 . Our obsStudies on monkeys showed that the putamen and globus pallidus process Ia afferent inputs ,30. MoreThe muscle spindle input activation determined by fMV, as well as subsequent changes in sensory gating at the subcortical level, could also potentially have an influence on the sensorimotor integration processes underlying STDT–movement integration at the cortical level . FMV-indFinally, a further relevant observation of this study was that the effects observed in the present study on STDT–movement integration only lasted for the duration of the stimulation, without inducing plasticity. This is apparently in contrast with a previous study which demonstrated that fMV can induce long-term depression-like plasticity in healthy subjects . HoweverWe acknowledge some limitations of this study. First, we did not measure kinematic features of movement, which would have clarified whether fMV induced changes in motor performance. A further limitation is that we did not test M1 excitability ; thus, cNevertheless, our results have potential implication for future studies aiming to investigate the effects of fMV in patients with dystonia, a movement disorder characterized by altered sensorimotor integration and gating of sensory processing ,35. In c


Although the prevalence of attention deficit hyperactivity disorder (ADHD) has remained stable, the number of patients diagnosed with ADHD has increased in recent years owing to increased awareness. Despite this increase, medical schemes in South Africa have not improved their funding models for this condition.The study aimed to provide an account of the funding that medical schemes provisioned for treating ADHD in South Africa during 2022.n = 72) and all their listed options were evaluated (n = 279).All the South African medical schemes that were registered with the Council of Medical Schemes during 2022  condition and therefore each medical scheme used its own approach to providing its beneficiaries with some or no benefits for ADHD. It was evident that ADHD was underfunded and lacked structured or standardised funding approaches.Attention deficit hyperactivity disorder is underfunded in the private healthcare sector in South Africa. Better funding models are needed or ADHD needs to be registered as a PMB condition.Findings from this study highlight the urgency for structured and sufficient ADHD-specific funding by medical schemes. Considerations based on these findings may be applied in the National Health Insurance and in other countries around the world. Attention deficit hyperactivity disorder (ADHD) is being treated as the orphan of mental health disorders in the private healthcare sector in South Africa. This study highlights the lack of proper funding models available for the treatment of this condition in South Africa.1 when Sir George Still described children with restlessness, inattention and impulsiveness. Attention deficit hyperactivity disorder affects between 2% and 16% of schoolgoing children. The notion that ADHD is only a childhood disorder was discredited in the 1990s with studies demonstrating that ADHD symptoms last well into adulthood.3 Research has revealed that 60% – 70% of children with ADHD continue to have symptoms throughout adulthood.4 The prevalence of adult ADHD is between 2.5% and 4.3% of the population.5The earliest and most accurate description of ADHD as we know it today came from 190212It is well known that the ADHD neurodevelopmental disorder causes patients to suffer from multiple mental health comorbidities, resulting in social, personal and interpersonal challenges negatively affecting the overall quality of life. In addition, patients with ADHD in the South African private healthcare sector have many out-of-pocket (OoP) expenses related to medication and the cost of consultations with a psychiatrist or paediatrician.1 The costs are divided into direct and indirect costs. Direct costs are the healthcare and OoP expenses that relate to consultations, medication, transportation, food and lodging incurred as a direct result of ADHD healthcare visits.13A major study concluded that ADHD runs a chronic and costly course, costing governments and patients with ADHD and their families hundreds of thousands of dollars annually.15 Some overheads of ADHD are intangible, such as educational and judicial system costs. Educational costs relate to special needs tuition, as required by those with learning disabilities, other health impairments and emotional disturbances. Judicial system costs relate to the costs of larceny, arrests, adjudication, robberies and similar misdemeanours.17Indirect costs are those resulting from the time of patients and their families lost because of ADHD, the losses in productivity, the cost of absenteeism and presenteeism, wage losses, the reduction in efficiency and the cost of other disabilities caused by ADHD.18 A 2017 South African study illustrated that patients were responsible for major OoP expenses relating to the cost of medication, consultations and accessing alternative therapies.10 The study further demonstrated that patients with ADHD paid 35.98% of the costs of psychiatric consultations, 41.17% of medicine costs and 62.20% of the costs for supportive therapies. Another finding from this study was that the presence of adult ADHD more than doubled all healthcare-associated expenses of the medical scheme beneficiaries. This study was the largest at the time and included 3 300 000 beneficiaries.10 The study calculated the funding availability of the beneficiaries to be only R534 ($62) per beneficiary per annum.Many studies conducted across the world during 2019 have determined the annual costs of ADHD per patient. Patients with ADHD had on average $1000 incremental costs per annum in the United States as compared with non-ADHD patients and in Germany the incremental costs were ¨ 2900 per patient per annum.10 The prevalence of ADHD and the high OoP expenses created the need to evaluate the current funding structures of the private healthcare sector in South Africa. Never before has the entire medical scheme population of South Africa been evaluated on its funding benefits for ADHD. This article gives an overview of the funding of ADHD for the 72 registered medical schemes and their options during 2022. This is a first for South Africa.19In South Africa, the treatment gap for mental health disorders in the private healthcare sector is 75%. This treatment gap reflects the number of medical scheme beneficiaries (as a percentage) that do not have access to a mental healthcare benefit. The ADHD population in 2017 was between 771 264  and 1 285 439 . This was calculated by multiplying the prevalence (as a percentage) with the population of South Africa during 2017. Using statistical inferential calculations, these figures stand between 1 635 707 and 2 727 168 for 2022.Insights into the funding of ADHD indicate where funding can be restructured and improved to match international treatment protocols.This naturalistic study examined the benefits for ADHD management in the private healthcare sector in South Africa during 2022. Secondary data desktop collection and analysis were used as the data were in the public domain. All the medical schemes that were registered under the Council of Medical Schemes (CMS) during 2022 were included.n = 72) that were registered in South Africa during 2022. The study sample included all the options of the 72 medical schemes for 2022 (n = 279). The total number of beneficiaries belonging to the schemes during 2022 were 8 938 872 members.19The study population was the medical schemes . Moreover, it was found that the Discovery Medical Scheme had no benefits for ADHD across its entire in-house medical scheme. The total number of beneficiaries under Discovery administration that had access to some form of ADHD-specific benefits was 223 845 out of 1 632 306 beneficiaries (treatment gap of 86%). The treatment gap indicates the percentage of beneficiaries in the study population that did not have access to an ADHD-specific benefit . Some ofMedscheme managed 12 schemes , represeMMI Holdings administered 10 schemes  at the tUniversal Health administered 11 schemes , with 88In South Africa, on November 2022, 8 938 872 people belonged to a medical scheme . At the time of the study, there were 16 open schemes and 56 restricted schemes in South Africa. The open schemes represented 4 830 000 beneficiaries  and the restricted schemes represented 4 110 000 beneficiaries . The number of beneficiaries who had access to some ADHD benefits in the open pool of schemes was 1 700 000 (38% coverage ratio) .The number of people who had access to ADHD funds in the restricted pool was 3 340 000 (87% coverage ratio) .The most common general mental health benefit was a monetary limit or value that applied to in-hospital benefits, out-of-hospital benefits and chronic medication benefits, or a combination of these. The number of scheme options that included a monetary benefit was 149. This represented 4 800 000 beneficiaries and a coverage ratio of 54%.n = 102), and 37% of the sample of scheme options (n = 279) had this benefit. This amounted to a total of 4 680 000 beneficiaries with a coverage ratio of 52%.The second most common general mental health benefit was the 21-day in-hospital admission benefit . Furthermore, 20% of medical scheme options only funded prescribed minimum benefit (PMB) conditions. This amounted to 1 380 000 beneficiaries and a coverage ratio of 15%. Also, 31% of medical scheme options had a 21-day drug rehabilitation admission benefit. This represented 3 910 000 beneficiaries and a coverage ratio of 44%. Only 20% of the schemes had the option of forfeiting the 21-day in-hospital admission programme for the benefit of 15 out-of-hospital consultations per annum with either a psychologist or a psychiatrist. This benefit covered 3 870 000 beneficiaries (43% coverage ratio).The study found that 9% of the medical scheme population had a major depression management and prevention benefit, available to 715 880 beneficiaries (92% treatment gap). A mere 5% of the options had an unlimited telephonic counselling benefit with a psychologist, representing 55 724 beneficiaries (treatment gap of 99%). Also, 5% of options had a 3-day admission benefit for alcohol detoxification, representing 155 038 beneficiaries (2% coverage).The study found that 9% of medical scheme options had a 3-day admission benefit for proven attempted suicide, available to 2 990 000 beneficiaries (33% coverage). Furthermore, 23% of medical scheme options had a general out-of-hospital consultation benefit with a mental health practitioner, available to 3 810 000 beneficiaries (coverage of 43%).The most common form of ADHD benefit was the registration of ADHD as a treatable condition on the additional disease list (ADL) of schemes or scheme options. This was the case for 7 590 000 beneficiaries (85% coverage ratio). The ADHD was listed as a chronic disease list (CDL) condition on eight of the options, benefitting 137 211 beneficiaries. The ADHD was listed as a non-PMB condition on 10 options, benefitting 134 977 beneficiaries.The most common ADL benefit was the chronic medicine benefit (7 650 000 beneficiaries), followed by consultation benefits (7 230 000 beneficiaries – 81% coverage ratio) and lastly childhood wellness benefits (1 250 000 beneficiaries). Consultation and childhood benefits are not discussed in this article.There were 13 options where ADHD benefits were limited to a specific age group, either for children between 2 and 12 years (28 117 beneficiaries), for children between 5 and 18 years (92 048 beneficiaries), for children between 6 and 18 years (138 291 beneficiaries), or for young adults below the age of 21 years (36 181 beneficiaries). There were no options where funding was specifically available for adults with ADHD.Correlation analysis was performed on the relationship between the presence of ADHD-specific benefits (chronic medicine benefits) and the premium paid per single beneficiary of the medical scheme options. The chronic medicine benefit was chosen for the analysis because it was the most common of all the ADHD-specific benefits .The correlation coefficient indicates a moderately strong relationship between an increase in premium paid and the presence of ADHD chronic medicine benefits (coefficient: 0.5).Compared with the open schemes, the restricted pool of schemes had a better ADHD funding coverage ratio. The administrator with the greatest treatment gap for ADHD was Discovery. It is important to observe, however, that an administrator is not responsible for the funding decisions of the individual schemes being administered by it.14 Further literature revealed, at the respective times of the studies, that 2.5% – 4.3% of adults struggled with prominent ADHD symptoms that impacted their daily functioning significantly,2 and that none of the medical schemes had adult-specific ADHD benefits. The closest to ADHD-specific benefits were 13 scheme options across all the schemes that dedicated either chronic medicine or consultations under their childhood wellness programmes for ADHD management. These programmes were however limited only to beneficiaries below the age of 21 years.There is currently a lack of scientific data regarding administrators of medical schemes in South Africa. Data regarding comparisons between all the medical schemes are also lacking. This is the first comparative study of its kind in South Africa. The study on MMI by Schoeman and De Klerk suggested that funding was insufficient in view of the excessive OoP expenses that patients were required to pay to obtain ADHD management and care.1 It is clear that current private funding for ADHD falls short when it comes to a structured approach to ADHD management. Of the 279 scheme options, only 66 had a chronic medicine benefit where ADHD was included as an ADL condition. The implications are that these benefits need to be shared among other mental health comorbidities that the patients might have.13 This sharing decreases funds available for the treatment of ADHD specifically.The funding benefits for ADHD at the time of recent studies were non-specific and non-standardised. According to the South African treatment guidelines for ADHD, a structured protocol is needed in order to manage and treat this condition effectively.13 The 21-day in-hospital admission  would not assist people with ADHD directly, but indirectly it could help them deal with comorbid conditions such as anxiety, depression or substance abuse, which is rife among ADHD patients.10The general mental healthcare benefits indirectly assist people with ADHD who have other mental health comorbidities.This study found that 15% of the medical scheme population only had access to treatment and management for PMB conditions. A PMB condition is a disease or ailment for which a medical scheme must pay in full, as required by PMB legislation. In South Africa there are 270 PMB conditions. In essence, a PMB is a defined set of benefits, which ensure that all members of medical schemes have access to minimum health services. The notion that only PMBs are to be covered should be reviewed, because – with the current omission of ADHD as a PMB condition – this policy automatically excludes 1 340 000 members from accessing some form of ADHD benefit. It is recommended that ADHD be registered as a PMB condition requiring structured funding benefits for all age groups affected and policies need to be implemented for this to materialise. There is a need for a cost–benefit study to determine the overall impact of registering ADHD as a PMB condition, as opposed to increasing and standardising ADHD funding benefits across the schemes.10 There was a 92% treatment gap for major depression management programmes, which is problematic owing to the high levels of comorbid depression among ADHD patients.There was a 56% treatment gap for the 21-day in-hospital drug rehabilitation programme, which is concerning because patients with ADHD often have comorbid substance abuse problems.18 In rand terms, this amounted to R49 532 on average per patient (using the July 2022 exchange rate). In South Africa, the burden rested on patients to pay OoP expenses of nearly 35.98% of the costs of psychiatric consultations, 41.17% of medication to treat ADHD and 62.20% of the costs of supportive and alternative therapies. This is a further indication of the lack of proper funding models for ADHD in South Africa.Previous research showed that the average incremental costs per patient with ADHD were around €2000 per patient with ADHD per annum.The findings also indicated that ADHD benefits were only available for the more expensive options of the schemes. This implies that only affluent members could benefit from ADHD funds and benefits, whereas the lower income groups would not have any benefits for ADHD.The main contribution of this study is the evidence that ADHD is only funded as a bundled benefit together with all the other conditions listed on the ADL, non-PMB and non-CDL of medical schemes. The fact that the benefits for ADHD need to be shared with other listed conditions implies that the funding figures are, in fact, lower than found, and this should be taken into account.Attention should be paid to the cost savings that would be derived from the better funding of ADHD if implemented by the schemes. Costs related to accidents, absenteeism and presenteeism, as well as the direct and indirect costs related to ADHD, could be greatly reduced if better funding is structured for ADHD. Cost savings will also result from ADHD beneficiaries requiring fewer admissions into mental healthcare institutions, fewer consultations and less drug rehabilitation support if these are better controlled and stabilised.A discussion between the council of schemes and the administrator that had minimal coverage ratios for ADHD (in this case Discovery) to make them aware of the need for better funding models and more benefits, specifically for ADHD, needs to take place. Even though an administrator is not directly responsible for the funding decisions of the individual schemes it administers, it could influence the schemes to create better funding models.The overall general mental healthcare package should be optimised to close the treatment gaps. Taking into account the results of this study, the two benefits that need to be improved are the 21-day in-hospital admission and the monetary benefit. The 16% of beneficiaries (1 400 000 people) that do not have any form of mental health benefits need to be reduced to zero. This 16% is considered to be insufficient because mental healthcare-related conditions are on the increase.It is expensive for patients to consult with psychiatrists. Now that it is clear that patients pay major OoP expenses for psychiatry consultations, owing to the limited funding available for ADHD, it would make sense to upskill the general practitioner population in order to raise the diagnostic threshold of ADHD. General practitioners are the custodians or gatekeepers of any healthcare ecosystem, and if they could become accredited by doing an ADHD diploma, they could be empowered to diagnose and initiate treatment for ADHD patients. This added expertise provided by an ADHD-specific accreditation will not only result in patients saving on psychiatrist consultation fees but will also alleviate pressure on the psychiatrist profession.The open pool of schemes should be targeted first when better funding models need to be implemented (the open pool had a treatment gap of 62%). The Council for Medical Schemes needs to become more aware and mindful of the need for ADHD to be structurally funded. A structured funding model could include standardised medications and treatment packages that are included across the board in all options of all schemes.A future study could look at the cost–benefit analysis of either creating specific structured funding models for ADHD as opposed to registering ADHD as a PMB condition with unlimited benefits. A discussion is also recommended regarding the steps needed for ADHD to be registered and recognised as a PMB condition in South Africa. If ADHD could be registered as a PMB condition, this would imply unlimited funding being made available by all medical schemes and would end the debate about requiring better funding models.20Efforts should be established to develop a well-structured clinical care pathway for ADHD in the private sector in South Africa. A clinical pathway will result in cost savings for patients, medical schemes and the government.21In addition, the recognition of ADHD as a PMB condition in South Africa might ensure that patients are eligible for unlimited chronic treatment and other benefits.The strength of this study is that it is the first to analyse ADHD benefits across every medical scheme in South Africa, laying the foundation for future ADHD funding-related studies. A limitation was that only the private healthcare sector was evaluated and not the public sector’s management of ADHD. The implication is that if the funding and management of ADHD in the private healthcare sector is scarce, the situation in the public sector could be assumed to be worse owing to a lack of resources and inadequate treatment guidelines.Other limitations were that there were no exact figures available for the numbers of beneficiaries for every option of every scheme for 2022. The treatment gaps were calculated on exact numbers of beneficiaries obtained from 2021 data. Another limitation is the fact that only public domain data were evaluated; thus, in-house scheme benefits that are available to patients after registration were not taken into account.This study gives a first-ever overview of the total funding available for the treatment of ADHD across all the medical schemes in the private healthcare sector during 2022 in South Africa. It is evident that in the time period under investigation, namely 2022, there were no structured or proper funding models for ADHD, as illustrated by the erratic funding for ADHD across the four largest scheme administrators and the individual schemes themselves. The most important finding was that the existing ADHD-specific benefits were insufficient and scantly distributed across the scheme population.Attention deficit hyperactivity disorder, as a recognised mental disorder, needs proper protocols and sufficient funding. This article provided evidence that ADHD is underfunded and that clear, well-structured funding benefits and models are recommended.


B. breve) or a vehicle from postnatal day 1 (P1) to P21. Splanchnic organs and skeletal muscles were evaluated at six weeks of age. Compared with the sham group, the LBW-vehicle group presented significant changes as follows: overgrowth from infancy to childhood; lighter weight of the liver, kidneys, and gastrocnemius and plantaris muscles; reduced height of villi in the ileum; and increased depth of crypts in the jejunum. Some of these changes were milder in the LBW-B.breve group. In conclusion, this rat model could be useful for investigating the mechanisms of how FGR-LBW leads to future health problems and for developing interventions for these problems. Supplementation with B. breve in early life may modestly attenuate these problems.Children with fetal growth restriction (FGR) and its resultant low birthweight (LBW) are at a higher risk of developing various health problems later in life, including renal diseases, metabolic syndrome, and sarcopenia. The mechanism through which LBW caused by intrauterine hypoperfusion leads to these health problems has not been properly investigated. Oral supplementation with probiotics is expected to reduce these risks in children. In the present study, rat pups born with FGR-LBW after mild intrauterine hypoperfusion were supplemented with either Bifidobacterium breve ( Epidemiological studies have shown that the intrauterine environment is crucial for the future health of each individual, i.e., a concept of DOHaD  . Such inPreviously, we developed a rat model of LBW due to FGR induced by mild intrauterine hypoperfusion ,9. In ouBifidobacterium longum is the most dominant species, followed by Bifidobacterium breve (B. breve) and Bifidobacterium bifidum [B. breve M-16V is a strain commonly used as a probiotic for infants, and its early oral supplementation has been shown to promote the acquisition of beneficial commensal bacteria in preterm LBW neonates [B. breve M-16V normalizes such differences [B. breve M-16V on late-onset sepsis and mortality in preterm infants [B. breve M-16V is suggested to regulate immune balance and inflammatory response [B. breve M-16V could alleviate the influence of LBW-FGR on splanchnic organs and skeletal muscles, i.e., the probiotic could ameliorate putative hypotrophy through promoting the acquisition of normal gut microbiota and modulating immune/inflammation. The second aim of the present study was to investigate the effects of the oral supplementation of B. breve M-16V during the neonatal and infantile periods on splanchnic organs and skeletal muscles in the rat model of LBW-FGR.The gut microbiota plays a crucial role in healthy development of not only gastrointestinal system but also many other organ systems . LBW inf bifidum . B. brevneonates ,22. Our ferences . A meta- infants . B. brevresponse . We hypon = 9) were used for this study. Dams were housed in a standard environment on a 12 h light/dark cycle (lights on at 7 a.m.) and provided ad libitum access to water and a regular diet . On gestational day 17 (G17), six dams were subjected to mild intrauterine hypoperfusion as previously described [Pregnant female Sprague Dawley rats  n =  were useB. breve-administered litter (LBW-B.breve group) or vehicle-administered litter (LBW-vehicle group). To prevent microbial transmission among littermates in the same cage, they were assigned to the same group. Three litters from dams without mild intrauterine hypoperfusion were assigned to the sham group.The dams were allowed to deliver spontaneously, and pups were born on E21 or E22. Only the males were used for this study, and females were used for a different study. Pups with birth weight less than 5.5 g from dams with mild intrauterine hypoperfusion were defined as LBW pups, since we had observed that a birth weight of 5.5 g was the –2 standard deviation level among normal control pups. The litter size was adjusted to 8–10 pups per litter. Six litters from dams with mild intrauterine hypoperfusion were randomly assigned to either the B. breve M-16V (5 × 108 CFU/mL) was orally administered to the pups in the LBW-B.breve group at 10 μL/g of body weight once daily from the next day of birth, i.e., postnatal day 1 (P1) up to weaning (P21)  inserted into the stomach. The dose of B. breve was determined according to previous animal and clinical studies showing its safety and efficacy for other conditions [B. breve M-16V was provided by Morinaga Milk Industry Co., Ltd. . The LBW-vehicle group and sham group received normal saline containing starch using the same protocol as that used for the LBW-B.breve group. After weaning, the pups were allowed access to water and a regular diet  ad libitum. All animals, except one in the LBW-B. breve group, survived up to six weeks of age. This rat died on P19, presumably due to the stress of daily needle insertion into the stomach, and not due to the adverse effects of B. breve, as some rats had died in our preliminary study with normal saline to test the feasibility of daily gavage administration into the stomach in LBW pups from P1. The animals used in this study were the same as those used in our previous report on behavioral and brain analyses [A solution containing ng (P21) . Gavage nditions ,27,28. Banalyses .At six weeks of age, which is considered equivalent to human adolescents, the animals were deeply anesthetized with intraperitoneal administration of butorphanol tartrate, medetomidine hydrochloride, and midazolam. The small and large intestines were removed, and their lengths were measured. The number of Peyer patches was counted macroscopically. The liver, spleen, kidneys, skeletal muscles of the hindlimb, and cecum were removed and weighed. These organs were placed in ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS). After fixation, the kidneys and small intestines were embedded in paraffin, and thin sections  were cut using a microtome. The sections were stained with hematoxylin and eosin (H&E). The number of nephrons in the entire coronal section of the kidney was counted. The number of Paneth cells in 10 randomly selected crypts was counted in the duodenum, jejunum, and ileum. Using ImageJ software version 1.53b , the heights and depths of 10 randomly selected, intact, well-oriented villi and crypts were measured in the duodenum, jejunum, and ileum. The average number of Paneth cells, height, and depth in each region of the small intestine were used for the statistical analyses. All analyses were performed by investigators who were blinded to the group allocation.p < 0.05. When the p value was = or >0.05 but <0.10, the exact p value is described in the Results section and shown in the figures. Data are expressed as means ± standard deviation.Statistical analyses were performed using Prism 8 software . We always compared the data of three groups: sham, LBW-vehicle, and LBW-B.breve. Temporal changes in body weight were assessed using two-way repeated-measures analysis of variance (ANOVA), followed by Tukey’s test. To assess the number of Peyer’s patches and Paneth cells, the Kruskal–Wallis test, followed by Dunn’s test, was used. For kidney weight, two-way ANOVA followed by Tukey’s test were used. All other analyses were performed using one-way ANOVA, followed by Tukey’s test. The Geisser–Greenhouse correction was used when variances were not equal. The significance level was set at n = 5) or LBW-B.breve group (n = 8). The mean birth weights of the LBW groups were significantly lower than those of the sham group  male pups were obtained at the time of allocation (P1); a few LBW pups died before allocation. LBW pups were allocated to either the LBW-vehicle   D. Howevep = 0.069 for LBW-vehicle and p = 0.12 for LBW-B.breve) (p = 0.086) (p = 0.053). Concerning villus height in the small intestine, both the LBW-vehicle and LBW-B.breve groups had significantly shorter villi in the ileum than the sham group (p = 0.095) and significantly deeper than that in the LBW-B.breve group. Apart from the villus height and crypt depth, there was no histologically significant difference among the three groups. The feces characteristics, such as softness, color, odor, and volume, did not differ among the groups, although we did not evaluate them quantitively. Taken together, LBW moderately altered the anatomy of the small intestine, and B. breve supplementation partially alleviated these alterations.The small intestine in the LBW groups was shorter than that in the sham group, but the differences were not statistically significant (B.breve) A. The leB.breve) B. The ce= 0.086) C. The nu= 0.086) D. Conceram group F,H–J. Coam group G,H–J. ImThe weights of the gastrocnemius and plantalis muscles in the LBW groups were significantly lower than those in the sham group A,C. TherB. breve M-16V supplementation during the neonatal and infantile periods. Most of the beneficial effects of supplementation were non-significant, but the amelioration of crypt depth in the small intestine was statistically significant.The present study showed that rats with LBW due to intrauterine hypoperfusion exhibited alterations in several organs, which may reproduce the conditions associated with health problems seen in people with LBW. Alterations observed in the LBW rats at adolescence were (1) rapid catch-up in body weight during infancy, (2) reduced liver weight, (3) reduced kidney weight, (4) reduced height of villi and increased depth of crypts in the small intestine, and (5) reduced skeletal muscle weight. These changes tended to be milder with B. breve supplementation attenuated such overgrowth in LBW pups; this suppressing effect on overgrowth is considered to be beneficial for human infants born with LBW in order to lower the risk of non-communicable diseases in later life.Individuals with LBW are inherently at risk for developing non-communicable diseases in later life ,2,29,30.In situations where intrauterine perfusion is reduced, fetal blood flow is preferentially distributed to the brain to secure the optimal growth of this key organ, thereby reducing distribution to other organs. A necropsy study on human neonates showed that small and large intestinal lengths are reduced in FGR infants compared with appropriately grown infants . The relConcerning the liver, a study in a rat model with nutritional restriction showed reduced liver weight when assessed up to P21 , while aFGR, preterm birth, and LBW are reportedly associated with reduced nephron numbers and consequently higher blood pressure in later life . Human nFew studies have assessed the small intestine in rodent LBW models. One such study in a food restriction model showed lower intestinal weight up to 4 weeks but not at 12 weeks of age compared with control rats . A studyLBW is associated with a higher likelihood of lower lean body mass and sarcopenia in human adults ,40. AlthB. breve induces extensive transcriptome regulation in murine intestinal epithelial cells, including the up-regulation of key genes linked with epithelial barrier function [B. breve supplementation in this study might have been mediated by its anti-inflammatory and modulatory effects on the gut microbiota. A limitation of this study is that the exact mechanism of action was not explored. The literature shows that B. breve M-16V downregulates levels of interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1 in porcine intestinal epithelial cells [Bifidobacteria are known to exert anti-inflammatory effects and support the intestinal barrier integrity through producing acetate ,47,48. Bfunction . We perffunction . There wal cells ; reducesal cells .B. breve supplementation in early life may alleviate some of these problems in infants with LBW-FGR. Similarly, the mechanisms underlying the beneficial effects of B. breve supplementation on each organ remain to be clarified.In summary, the rat model of LBW caused by mild intrauterine hypoperfusion reproduced several health problems that human infants with LBW-FGR may frequently suffer in later life; hence, it could be a useful model for studying the mechanisms of LBW-FGR’s harmful effects and for developing therapeutic interventions for these problems. The mechanisms through which LBW-FGR leads to alterations in each organ remain unclear.


Saccharomyces cerevisiae, combined or not with a sourdough starter including Limosilactobacillus fermentum, Weissella confusa and Weissella cibaria. LAB fermentation significantly increased total titrable acidity and dough rise regardless of the flour used. Targeted metagenomics revealed a strong impact of germination on the bacterial community profile of sprouted rye flour. Doughs made with germinated rye displayed higher levels of Latilactobacillus curvatus, while native rye doughs were associated with higher proportions of Lactoplantibacillus plantarum. The oligosaccharide profile of rye doughs indicated a lower carbohydrate content in native doughs as compared to the sprouted counterparts. Mixed fermentation promoted a consistent decrease in both monosaccharides and low-polymerization degree (PD)-oligosaccharides, but not in high-PD carbohydrates. Untargeted metabolomic analysis showed that native and germinated rye doughs differed in the relative abundance of phenolic compounds, terpenoids, and phospholipids. Sourdough fermentation promoted the accumulation of terpenoids, phenolic compounds and proteinogenic and non-proteinogenic amino acids. Present findings offer an integrated perspective on rye dough as a multi-constituent system and on cereal-sourced bioactive compounds potentially affecting the functional properties of derived food products.A multi-omics approach was adopted to investigate the impact of lactic acid fermentation and seed germination on the composition and physicochemical properties of rye doughs. Doughs were prepared with either native or germinated rye flour and fermented with  Secale cereale L.) is a valuable crop because of its resistance towards cold temperatures and northern climates [Rye bread is one of the most consumed cereal-based foods in northern Europe, China, and North America . In thesclimates .From a nutritional perspective, rye flour is gaining attention for its health-promoting potential considering its hypocholesterolemic, anti-diabetic, anti-inflammatory, and cardioprotective properties . Whole gPrevious research had shown that both germination of grains before fermentation and the type of fermentation markedly affected the structure and the potential bioactive properties of rye constituents ,10. HoweLimosibacillus fermentum UC3641, Weissella confusa UC4052, and Weissella cibaria UC405, previously isolated from sorghum sourdough [g × 10 min), washed twice with sterile saline solution (0.9% NaCl), and re-suspended in 5 mL of water. Saccharomyces cerevisiae was a commercial, compressed fresh baker’s yeast , reintegrated in water before use.ourdough , were usUnprocessed commercial rye grains (SU Bendix winter rye) were subjected to a preliminary sieving step. Kernel size fractions between 2 and 2.5 mm were obtained using an Octagon 200 test sieve shaker . Malting was performed on 100 g sieved seed batches with an Automatic Micromalting System  . The folw/v) of NaCl. The kneading process was preformed through a commercial kneading machine , initially without inoculum, at machine speed 1 for 2 min. As for fermentation, three different experimental conditions were tested: (i) S. cerevisiae fermentation, in which S. cerevisiae was inoculated at a final concentration of 2% (w/w), SC; (ii) mixed fermentation, in which a mix of the three LAB strains (109 CFU/mL) plus S. cerevisiae (2% w/w) was added to the dough, LAB + SC; and (iii) spontaneous fermentation, where non-inoculated doughs were prepared by replacing the inoculum with an equal volume of plain water, considered as the control. The initial concentration of LAB in each inoculated dough was between 1.89–3.00 × 107 cfu/g, as expected, whereas the initial concentration of S. cerevisiae in inoculated doughs was 1.30–1.68x106 cfu/g. After inoculation, the kneading process was carried out for 5 min at machine speed 3. All doughs were maturated for 24 h; for the mixed fermentation, an initial maturation with only inoculated LAB was performed for 8 h (30 °C and 60% relative humidity), after which yeast was added and the dough was left to leaven for the remaining 16 h (35 °C and 65% relative humidity). The same temperature and humidity conditions were applied for SC and control. Dough samples were prepared in duplicate and fermentations were repeated twice. All the doughs were analyzed for pH (pH meter Hanna Edge), total titratable acidity (TTA) [w) , dough rise, total yeast, and LAB count. Dough rise was determined as follows: after mixing, 20 g of the dough was transferred into a graduated cylinder. The height of the dough was measured before and after fermentation, and the dough increase in volume was calculated as previously reported [In brief, 250 g of native or sprouted rye flours were mixed with 250 mL of tap water and 1% (ty (TTA) , water a® Express Template Prep Kit 2.0 including 27F-1492R primers [® PB bead . Purified SMRTbell library from the pooled and barcoded samples was sequenced on a single PacBio Sequel cell using a SMRT Cell 1M v3 Tray. The resulting data were processed using the rDNATools pipeline [Microbial cells were harvested from doughs as previously described . Total D primers  along wipipeline . Ambiguopipeline , which pg for 10 min at 4 °C . The supernatants were collected, syringe filtered (0.22 µm pore size), and transferred into vials for the subsequent analysis. The experimental conditions applied for HPAEC-PAD analysis were previously described [−1 and temperature for both column and detector compartments were set at 27 °C, following a multi-gradient elution system: 0–10 min, 98% A; 10–35 min, 55% A; 65–75 min, 98% A. The amperometric detector was set in terms of several potentials and durations as follows: E1 = 0.10 V (t1 = 0.40 s); E2 = −2.00 V (t2 = 0.01 s); E3 = 0.60 V (t3 = 0.01 s); E4 = −0.10 V (t4 = 0.06 s). The semi-quantification of oligosaccharides was achieved according to their polymerization degree (PD), with respect to individualized standards that are representative of three different well-separated structural classes: monosaccharides, low-PD oligosaccharides, and high-PD oligosaccharides. Thus, xylose was applied as the reference standard for monosaccharides , arabinotriose was selected as the reference standard for low-PD oligosaccharides , and arabinooctaose was chosen as the reference standard for high-PD oligosaccharides . The results were expressed as carbohydrate content in mg g−1 of sourdough. The standards and reagents employed for oligosaccharide profiling were purchased from Sigma Aldrich®, Darmstadt, Germany (xylose) and Megazyme®, Bray, Ireland (arabinotriose and arabinooctaose).The oligosaccharide semi-quantification of the different rye doughs was performed by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) approach. All samples were subjected to a previous extraction, in which 1 g of sample was mixed with 10 mL of deionized water, and the resulting mixture was homogenized by a high-speed rotor (Polytron PT 1600-E) for 1 min and centrifuged at 8000× escribed . The equ2O:HCOOH  and the extraction was performed under the same conditions described earlier [2O binary mobile phase with a gradient elution: 6%–94% organic phase for a 33 min run, with a flow rate of 200 µL min−1. The analytical conditions for the QTOF performance were employed as follow: N2 as sheath gas with a flow of 10 L min−1 at 350 °C, drying gas was applied with a flow of 8 L min−1 at 330 °C, nebulizer pressure was set at 60 psi, nozzle voltage at 300 V, and capillary voltage at 3.5 kV. The mass spectrometer was adjusted in positive polarity and SCAN mode, with a detection range of 100–1200 m/z, with a nominal resolution of 40,000 FWHM. Moreover, quality control pooled samples were obtained and further analyzed through data-dependent MS/MS mode , applying different collision energies: 10, 20 and 40 eV. The acquired raw data were later processed by the Agilent MassHunter Profinder v.10.0 using the “find-by-formula” algorithm, through mass and retention time alignments . The annotation of the obtained chemical features was based on their accurate mass and isotopic patterns, given by the exact masses, relative abundance and m/z spacing, using the FooDB database (available at foodb.ca) to achieve their identification. Data reduction was achieved by applying the “filter-by-frequency” feature, exclusively retaining the features observed in all the replicates within the same treatment. As a result, the untargeted compound annotation obtained was in compliance with identification level 2 provided by the COSMOS Metabolomics Standard Initiative (putatively annotated compounds). To improve the confidence in the compound annotation raw data were processed by MS-DIAL software (v. 4.90), achieving the identification of chemical features through MS-MS spectral data, according to mass accuracy data, mass isotopic patterns, and spectral alignment matching. The parameters set for identification were: retention time range, 1–32 min; mass range, m/z 80–1200; mass tolerance, 0.05 Da. For data reduction, a filter step was performed, removing the identities that were not acquired in, at least the 80% of replicates. Finally, a score cut-off of 80% was selected to retain those compounds with the highest identification fidelity according to MS2 level. All chemicals used for extraction and chromatographic equipment were LC-MS grade, purchased from VWR .The untargeted metabolomic profiling of the different rye doughs was obtained through an ultra-high performance liquid chromatography instrument (Agilent 1200 series), presenting a binary pump and JetStream electrospray source, coupled to a quadrupole time-of-flight mass spectrometer . Before the analytical determination, 1 g of each rye doughs was mixed with 10 mL of the extraction solvent MeOH:H earlier . All ana earlier ,22. Brien = 6). The results for carbohydrate content of rye sourdoughs were statistically analyzed by one-way ANOVA followed by Duncan’s post hoc test, setting a significance value of α = 0.05, using the software SPSS 25 (IBM). The Agilent Mass Profile Professional v. 15.1 software analyzed the metabolomics data as previously indicated compounds were filtered by abundance, log2-transformed, and normalized at the 75th percentile [p < 0.05) and with fold-change values > 2 were considered [2X and R2Y), and goodness-of-prediction parameter (Q2Y). OPLS-DA predictive models were further statistically validated by cross-validation ANOVA (CV-ANOVA), and model overfitting was discarded through the development of permutation tests (n = 100). Such approach was followed by a variable importance in projection (VIP) analysis, providing insight into the compounds exhibiting the highest influence on the discrimination between treatments, known as VIP markers, according to their given VIP score [A two-way analysis of variance (ANOVA) was performed to analyze the effect of starter and germination on dough general parameters using GraphPad Prism version 8.0.0 for Windows . Metagenomic data were processed using the QIIME2 v.2020.2 platform for diversity analysis of dough microbial communities. Observed species, Chao1, Shannon and Gini-Simpson indices were calculated to assess within sample diversity; sequencing depth was characterized by Good’s coverage. Weighted and unweighted Unifrac distances were calculated, and Principal Coordinate Analysis (PCoA) was performed on the distance matrices to visualize community variation across samples. The OTU table was uploaded to the Microbiomeanalyst server for compositional profiling and comparative analysis, using 10% prevalence in samples for the low count filter, the default settings for other filtering and total sum scaling for data normalization . To testrcentile . The abuIP score .Key technological parameters of the different doughs after fermentation are presented in p = 0.002) in sprouted rye samples. Consistently, the application of LAB starters significantly increased total acid concentration in rye doughs as compared to either yeast or control . Notably, the TTA level of LAB + SC doughs made with sprouted rye flour was higher than that of the corresponding doughs produced from native rye flour . The inclusion of LAB as a starter for rye flour fermentation displayed no significant impact on dough aw. Significant differences were observed for yeast  versus control in native rye flour doughs . Concerning dough volume, LAB seemed to contribute to dough rise during leavening markedly. The volume increase was higher for LAB + SC vs. SC or control regardless of the type of rye flour used; differences were all statistically significant but for LAB + SC vs. SC in native rye dough . Analogously to aw, statistical analysis indicated that the type of fermentation had the same effect in sprouted and native rye doughs .As expected, the addition of LAB starters caused a decrease in the pH value to about 4 both in sprouted and native rye doughs, as compared to either SC or control. However, the difference between final pH values was significant only when comparing LAB + SC vs. SC . Clustering to 99% similarity yielded 5,657 distinct OTUs; the mean number of OTUs per sample was 353  in sprouted rye doughs, while Lacticaseibacillus paracasei and Latilactobacillus graminis were found at low relative abundance. Native rye doughs harbored a bacterial community consisting of Lactoplantibacillus plantarum (with an average of 42.6%) followed by Lacticaseibacillus paracasei (relative abundance > 16%); Lactobacillus curvatus was detected with a relative abundance over 4%, while W. confusa/cibaria accounted for 38.5% of total sequences in nDY . Conversely, L. plantarum was significantly higher in native rye samples to sprouted samples .Moreover, the species The oligosaccharide semi-quantification of rye flour doughs is shown in Thus, for native rye doughs, SC-mediated fermentation promoted a significant decrease of both monosaccharides and low-PD oligosaccharides that were further significantly decreased in the LAB + SC fermentation by whereas high-PD carbohydrates were not affected A. In tot2 spectral features  was performed. It was followed by a variable importance in projection (VIP) analysis, with the aim of discriminating the effect of germination and starters on the metabolome of these matrices, providing insight on the metabolic markers mostly involved in such discrimination (VIP markers). The 2X and R2Y coefficients, and predictability, given by the Q2Y coefficient  encoding for a NAD(P)H-dependent mannitol/alcohol dehydrogenase [This study explored the microbial, chemical, and technological profiles of rye doughs made with either native or sprouted flour and fermented with ucanases . Due to ucanases . Consideucanases . Analysiucanases ,29. Indeucanases . Interesnases [2 . In mixeeshments ,33,34,35eshments ,37. All  species . Consistlusively . Notablylusively . Consistrogenase .L. plantarum was typical of the microbiota of native rye doughs, whereas L. curvatus was significantly higher in doughs made with sprouted rye. As for L. plantarum, this species is known to metabolize a wide range of different carbohydrates of varying complexity, thanks to its rich repertoire of lytic enzymes [In addition to oligosaccharide semi-quantification, a metabolomics approach was employed to investigate the overall effect of germination and fermentation on the metabolome of rye sourdoughs. The unsupervised HCA analysis of doughs revealed that germination of rye grains played a major role on the metabolic profile than fermentation, which was also supported by the results from the OPLS-DA models. Germination has been previously assessed as a physiological process in which phytohormones can play a critical role on the development and metabolome of rye grains, which may affect further processing, including fermentation . Further enzymes . Furtherp-coumaric acids are the most prevalent phenolics attributed to rye, reaching a proportion of about the 95% of total phenolic compounds [p-coumaric acid and ferulic acid in their esterified derivatives. It is important to note that phenolic acids have been previously reported in their esterified forms with diverse biogenic amines [Concerning phenolics, the accumulation of flavonoids and phenolic acids was mostly modulated by fermentation, with yeast as the sole fermenting agent or in combination with LAB, which agrees with the previous study by Katina et al.  who repoompounds . Notablyompounds , suggestc amines  as reflec amines .L. fermentum is among the Lactobacillus species that can convert arginine to ornithine via the arginine-deiminase (ADI) pathway [Limosilactobacillus reuteri was attributed to strain-specific biosynthetic capabilities and consistently improved the sensory attributes of the resulting bread [S. cerevisiae alone. It is known that hydratases by sourdough lactobacilli can convert oleic acid, linoleic acid, and linolenic acid to hydroxy fatty acids.In the case of terpenoids, little is known about the effect of fermentation on biosynthesis of these compounds in rye sourdough  that wer pathway . Notablyng bread . Lipid mng bread . According bread  on the uS. cerevisiae and LAB promoted the accumulation of nutritionally important metabolites, such as polyphenols, terpenoids, hydroxy fatty acids, and peptides, which also contribute to the enhancement of the technological and sensorial properties associated with rye flour. Indeed, the integrated information provided by metagenomics and untargeted metabolomics offered new insights into the impact of processing technologies on dough quality, which can guide the design and development of novel, health-promoting rye foods.The results of the present study provide a comprehensive view of multiple compositional changes induced by germination and lactic fermentation in cereal flour, which may have implications for the nutritional value, sensory attributes, and functional characteristics of rye bakery products. Fermentation by selected sourdough lactic acid bacteria in addition to baker’s yeast resulted in lower levels of simple sugars and increased levels of mannitol in the dough system, and could thus represent a relevant strategy to reduce sugar in baked goods. Grain germination promoted the accumulation of maltooligosaccharides, a class of molecules displaying several potential biological capabilities. Overall, the combination of rye germination with the combined fermentation of


Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg2+-2+-OH− fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as 18O ~ 22‰, Δ17O ~ 6‰) were observed in shallow aquifer fluids, indicative of 18O of 53.7‰, Δ17O of 16.8‰) mixed with in situ through nitrification (estimated endmember δ18O and Δ17O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH3 (>100 μM) with little 3 in hyperalkaline fluids is a product of 18ε / 15ε) measured in Samail Ophiolite 18ε / 15ε ~ 1), which is consistent with dissimilatory narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.Nitrogen (N) is an essential element for life. N compounds such as ammonium ( These boreholes are situated in crustal gabbros and mantle peridotites in the Wadi Tayin block of the Samail Ophiolite. We additionally sampled borehole BA1A of the Oman Drilling Project multi-borehole observatory during the 2018 field season; the hydrological properties of this borehole are described extensively in Lods et al. . The lit15N of reduced N (Nred)  was deployed to sample discrete depth intervals in the borehole. A detailed description of sampling with the packer system is provided by Nothaft et al. .15N and δ17O + δ18O of A single rain event was sampled in 2017 for ~1 min of rainfall by holding an open, acid-washed 15-mL Falcon tube at ~5 ft over the ground. The tube was not opened until after the rain event had started in order to minimize the potential contamination of the sample with dust. The sample was immediately filtered through a 0.2-μm polycarbonate filter to remove cells and prevent the biological processing of N and then placed on ice in a cooler in the field. The sample was stored frozen at −20 °C until the analysis of δ2+-2+-OH− type compositions according to pH and concentrations of ∑Mg, ∑Ca, and ∑CO2 ] and USGS34  potassium nitrate standards at two concentrations (to correct for volumetric effects) every eight samples with analytical precision: 0.1‰ for δ15N and 0.3‰ for δ18O . Prior to analysis with the denitrifier method, ∑NH3 was oxidized to via the persulfate method using N-clean recrystallized potassium persulfate .The δ17O measurements of 2O)  using the USGS34 and USGS35  standards.The δIsotopic data are reported with conventional delta notation vs. the international reference scales  in per mil (‰):2O was determined from gas sampled by a modified bubble strip method (protocol available at: http://dx.doi.org/10.17504/protocols.io.2x5gfq6). The N2O was measured with an HNU GC 301 gas chromatograph that was equipped with a Porapak N column under P-5 carrier gas  at the USGS Water Mission Area Laboratories in Boulder as described in Repert et al. , low-quality bases were trimmed off read ends (< 15), and reads of < 100 bases were discarded using the AdapterRemoval v2  in an aquifer fluid can be calculated through a simple mass balance:The fraction of atmospheric endmember 17Omixed is the 17O difference of 17Oatm) and biogeochemical endmember 17Obiogeo) 17O. The Δ17Obiogeo can be assumed to equal 0 because biogeochemical processes follow mass-dependent fractionation. Accordingly, equation (5) simplifies to:where Δ17Oatm, we used the measured Δ17O of sampled rainwater. We recognize a single rainwater sample may not be entirely representative of the isotopic composition of mean annual rainfall since the Δ17O of dissolved NOx in rainwater has been documented to fluctuate ~15‰ seasonally  compared to the hyperalkaline pH (10–11.4) and high ∑Ca concentrations (0.43–7.8 mM) of Ca2+-OH− fluids .Mg− fluids . The Ca22+-OH− fluids with less reacted Mg2+-2+-OH− fluids sampled in this study displayed ∑Si concentrations as low as expected for chrysotile-brucite-calcite±diopside equilibrium, indicating some degree of mixing with Mg2+-2+-OH− fluids were mixed with < 10% Mg2+-2+-2+-To assess the degree of mixing of deep, reacted Cag et al.  approachg et al.  reactiong et al.  for the d mixing , most Ca2+-2+-OH− peridotite-hosted fluids comprised primarily of reduced N (∑NH3) (2+-2+-OH− fluids only contained up to 26 μM (2+-OH− fluids were enriched in NH3 (up to 114 μM), while ∑NH3 concentrations were ~5 μM in all Mg2+-The predominant dissolved N species was dependent on the fluid type, with alkaline MgN (∑NH3) . The Mg2to 26 μM . Converse fluids .2+-2+-2+-OH− type fluids had lower concentrations of 2O concentrations were analyzed contained detectable N2O, which varied in concentration from 5 to 177 nM, with the highest concentration observed in the packed-off interval 55–66 m in borehole BA1A  offers valuable information on potential sources and subsequent transformations of 15N and δ18O of 15N −2.2‰, δ18O 55.6‰)  , the Δ17O of measured NOx can be used to calculate the relative contribution of biogeochemical and atmospheric sources of 17O value of 0‰. We found that all measured fluids contained 17O above 0‰ (17O was highest in Mg2+-18O and concentration of 17O corresponded to estimated fractions of atmospheric endmember atm) ranging from 0.09 to 0.41, accounting for uncertainty in the endmember Δ17Oatm isotopic composition.To investigate atmospheric deposition as a potential source of above 0‰ . The Δ17d fluids  and gene18O biogeo, can be calculated  spanned a range of 30‰  observed for borehole WAB71 where the concentration of NH3 was greatest (114 μM).The δe of 30‰ . In most15N of peridotite samples varied from 3.5 to 6.7‰  involved in the reduction of To assess the possibility of microbial involvement in the cycling of N within Samail Ophiolite aquifers, we used metagenomic sequencing of groundwater fluid biomass to probe for genes known to be involved in N utilization or transformation . N-cycliIt is important to note that these methods only detect the presence of these genes in aquifer fluids, and do not indicate whether individual organisms possess all genes involved in any specific N-cycling pathway, or whether these genes are actively expressed or utilized. Nevertheless, the presence of gene homologs for N-cycling processes in Samail Ophiolite fluids suggests the potential for biological transformations.17O of 17O of 18O/16O becomes equally elevated as the ratio of 17O/16O, thus enriching both isotopes independent of their mass difference  which can be inherited by N-oxides during photochemical reaction   and the likely additional input of surface runoff due to the moderate relief of gabbro outcrops, both of which could explain the higher A partial atmospheric source for the measured l et al. , 50 mm o3 is oxidized to 3 oxidation, but only bacteria are known to carry out the 3 to 3 oxidation (amoA gene), with no differential abundance by fluid type, despite ∑NH3 concentration acting as a strong selector for NH3 oxidizing taxa in other environments , which encodes the key enzyme in anaerobic ammonia oxidation (anammox) for catalyzing hydrazine oxidation to N2 gas   . We can 3 concentration with increasing pH  or the membrane-bound cytosolic enzyme Nar  which have 15ε ranges of −11.4 to −39.8‰ and −6.6 to −31.6‰, respectively , mineral-facilitated reduction by Fe-bearing phases could occur. For example, the quantitative conversion of 80Fe20), although this reaction was highly temperature-dependent and proceeded almost negligibly at 22 °C   in sampled aquifer fluids  in aquifer fluids may indicate the potential for NR for assimilation into biomass instead of respiration   associated with ∑NH3 speciation, volatilization, and degassing , whereas Mg-15N composition of source We observed a large range in δtrations . The δ15stem see , which d3 in aquifer catchments such as remineralized organic matter, fertilizer, or wastewater .Common sources of ∑NH3 to Samail Ophiolite aquifers could be atmospheric deposition. Unfortunately, we did not acidify an aliquot of rainwater for analysis during the one rain event that coincided with our geochemical sampling, so the assessment of wet deposition was not possible. Whether through wet or dry deposition, atmospheric ∑NH3 could be effectively transported to the subsurface aquifer through rainfall. However, despite the apparent atmospheric source for a significant fraction of shallow aquifer 3 because the atmospheric deposition of reduced nitrogen species (NHx) is not necessarily correlated with the magnitude of N-oxide deposition. Sources of oxidized and reduced N in the atmosphere are quite different, with NHx primarily originating from agricultural pollution such as emissions from livestock and volatilization of fertilizers , their detection did not correlate with ∑NH3 concentrations. Alternatively, surficial ∑NH3 produced by diazotrophic biological soil crusts could be transferred to the subsurface via recharging rainfall. N fixation by soil crusts commonly occurs in arid ecosystems, including Oman , evidence for aerobic nitrification can be observed in some boreholes such as WAB71 where the δ18O of 3 oxidation could occur in the shallow aquifer where Ca2+-OH− fluids come in contact with the atmosphere or mix with Mg-sampling . Nitrifisampling , and thu2O or N2 gas, retains N in the subsurface aquifer, thus preventing N from acting as a limiting nutrient for biological growth. The oxidation of ∑NH3 produced by the reduction of atmospheric Overall, the reduction of 17O indicative of atmospheric deposition as a major source of oxidized N to the serpentinite-hosted aquifers. The Δ17O and δ18O of via nitrification. However, 15N. In part, this is due to the ~30‰ variation of reactant ∑NH3. Concentrations of ∑NH3 increased concomitantly with a decrease in the concentration of 3 detected in Ca2+-OH− fluids. The isotopic fractionation imparted by NR seemingly varied with 15 ~ −17.5‰) observed in shallow groundwaters, and less apparent fractionation (ε15 ~ −4.5‰) in deeper groundwaters where 18ε/15ε) during We employed dual N and O isotopic analysis of dissolved N species to probe the origin and subsequent cycling of https://github.com/KopfLab/OmanN_Cycling. Metagenomic sequence files are available from the MG-RAST database under accession numbers mgm4795805.3 to mgm4795809.3 and mgm4795811.3.Raw isotopic data presented in this study and source code used to produce the figures and data tables in this manuscript are available at AT, SK, JM, JS, and KR conceived the study. KR, DN, EK, JM, JS, and AT collected samples in the field. KR, DN, EK, CA, RDE, and SK analyzed samples and assisted in the data interpretation. KR wrote the manuscript. All authors critically revised the manuscript text and figures.


The number of recovered and surviving plantlets did not significantly differ among durations of air exposure. For recovered plantlets, the long-term viability and clonal reproduction of two duckweed species, Lemna minor and L. gibba, were confirmed following greater than eight months of growth. These data further amplify the key role of waterbirds as vectors for aquatic plant dispersal and demonstrate the internal transport (i.e. endozoochory) of metabolically active plantlets. These data suggest dispersal of vegetative plant propagules by avian vectors is likely to be a common occurrence, underpinning connectivity, range expansion and invasions of some aquatic plants.Avian vectors, such as ducks, swans and geese, are important dispersers of plant propagules. Until recently, it was thought that small vegetative propagules were reliant on adherence to vectors and are unlikely to survive passage through the avian digestive tract. Here, we conclusively demonstrate that metabolically active angiosperms can survive passage through the digestive tract of a large-bodied waterbird. In addition, we show that extended periods of air exposure for up to 7 days does not inhibit the survival of plantlets embedded in faecal matter. Following air exposure, plantlets ( IntriLemna, Spirodela, Landoltia, Wolffia and Wolfiella) [Duckweeds (i.e. Lemnaceae) are a family of floating freshwater monocotyledonous plants  that canlfiella) , severallfiella) . Dependilfiella) , while flfiella) ,16,17 Allfiella) ), epizoolfiella) . As ducket al. [Wolffia columbiana fronds from fresh faecal samples (n = 3) obtained from white-faced whistling ducks, Dendrocygna viduata, residing within a Brazilian wetland. This is an important finding as it is the first and only report of a whole duckweed surviving gut passage through the digestive tract of a bird. However, W. columbiana can assume two different physiological states: a rapidly growing state and a starch-rich survival state (i.e. a turion). Turions are considered to play a key role in survival during suboptimal conditions, as well as during the colonization of new habitats [W. columbiana appear deceptively similar, and quantification of starch content and/or histological examination are the only ways to unambiguously distinguish between these two life stages [et al. [Wolffia plantlets, with resemblance to live plants  were considered, this does not conclusively exclude the possibility that Wolffia plantlets were originally in a dormant, stress-resistant turion state when ingested by waterbirds. Thus, the question remains whether rapidly growing, metabolically active duckweed plants, and indeed metabolically active fragments of any macrophyte, can survive passage through the gut of a bird. Furthermore, W. columbiana fronds are particularly small  and it remains unknown whether the findings by Silva et al. [Recently, Silva et al.  reportedhabitats . Turionse stages . While S [et al.  state tha et al.  can be eFollowing gut passage, plantlets embedded within faecal matter may not immediately re-enter the aquatic environments, as waterbirds will frequently defecate in loafing, roosting and nesting sites immediately adjacent to waterbodies. Accordingly, it is possible that plantlets may remain embedded within faecal matter for several days prior to re-entering the waterbody via a chance physical disturbance (e.g. pushed or washed into a waterbody). Although it has been established that greater resistance to desiccation tends to increase survival and viability of vegetative plantlets and fragments , it remaLemna (i.e. L. minor and L. gibba) following the passage of fronds through the digestive tract of mute swans, Cygnus olor, coupled with extended periods of post-dispersal air exposure for up to 7 days. We hypothesized that air exposure would reduce the survival of duckweed plantlets embedded within swan faecal samples. Neither of these duckweed species produce true, stress-resistant turions [The present study reports the survival and long-term viability of two duckweed species belonging to the genus  turions . These d. 2Cygnus olor, faecal samples were collected between October and November 2020 from a single site located in central Ireland . Samples were collected from patches of grassland surrounding a multi-trophic aquaculture system used to produce rainbow trout, Oncorhynchus mykiss, and European perch, Perca fluvialis. The system included 16 canals that supported a mixed population of the duckweed species Lemna gibba (greater than 90%) and Lemna minor (less than 10%). This system is described in detail by Stejskal et al. [Cygnus olor and their cygnets foraging and apparently ingesting duckweed. A breeding pair of Cygnus olor has inhabited the canals during the breeding season for over a 3-year period.Mute swans, l et al. . SamplesIn total, 75 faecal samples were collected across 13 different sampling days . Each sa. 2.1In the laboratory, the faecal samples were carefully extracted from the plastic bags, weighed and placed on shallow plastic trays (1.5 cm in depth) for air exposure. To assess the out-of-water survival of any encapsulated duckweed, the samples were exposed to a constant temperature (18 ± 2°C) and relative humidity regime (40–50% RH) for up to 7 days. These conditions were used to mimic the moderate to warm but damp conditions that are generally experienced during Irish summer months (June–August). As duckweed tends to be most abundant over the summer period, it is inferred that any ingestion and passive dispersal of duckweed will similarly be more likely to occur during periods of greater abundance.−2 s−1 and a light to dark photoperiod of 14 h : 10 h. After 7 days, the number of fronds still surviving was recorded. Etiolated fronds were considered dead, while fronds that appeared green and healthy were counted, with Y(II) being assessed once more to confirm metabolic activity.Following air exposure, each faecal sample was individually placed in deeper (3.5 cm) plastic trays. Distilled water was then added to aid the gentle disintegration of each faecal sample. This process was carefully assisted with the aid of a stainless-steel micro-spatula, to tease apart the sample. When each sample had been turned into liquid suspension, duckweed fronds that maintained buoyancy on the surface were removed and counted. A single duckweed plantlet can be composed of one or more fronds . Only floating fronds were considered, so as to exclude the possibility of survival through starch accumulating ‘heavy fronds’ known to occasionally occur in several duckweed species, which show reduced photosynthetic activity and sink to the bottom of waterbodies . FurtherL. gibba to distinguish L. gibba from L. minor [L. minor and L. gibba can appear morphologically identical.Surviving fronds were maintained for approximately six months in magentas within the controlled growth room, with fresh Hutner's medium being changed on an ad hoc basis. In early May 2021, all duckweed biomass was placed in artificial, outdoor mesocosms . This was done to promote the expression of gibbosity  in L. minor . Under o. 2.2α < 0.05 (lmtest).Statistical analyses were conducted using R software . Similarly, following the 7-day assessment period for survival, the number of fronds surviving per gram of faecal sample did not significantly differ . Overall, approximately 93% of fronds originally recovered from samples had to some extent etiolated upon completion of the 7-day assessment period. However, most of the surviving fronds also displayed viability, as small, newly developed ‘daughter’ fronds were observed. Although Y(II) values were initially very low in fronds recovered from faecal samples (less than 0.04), indicating substantially impaired photosynthetic activity, values in surviving fronds bounced back to a value indicative of healthy fronds (greater than 0.6) . The number of fronds recovered per gram of sample did not vary significantly (GLM: han 0.6) . Duckwee. 4The present study unambiguously demonstrates that whole angiosperms can be dispersed by gut passage through waterbirds in field environments. In particular, the resumption of growth as well as long-term viability for two duckweed species is shown (greater than eight months), following gut passage and subsequent air exposure of the faecal samples for up to 7 days. These data agree with reports in the literature that indicate duckweed can survive in wet mud for several months , which sW. columbiana, with a frond size 0.5–1.0 mm in comparison with fronds of 8.0–10.0 mm for both L. minor and L. gibba, can survive gut passage. Following the recovery of 18 living W. columbiana, it has been suggested that the especially small size of W. columbiana fronds could be advantageous for gut passage survival [Anas platyrhynchos; body mass: 0.7–1.6 kg) has also previously been evidenced [Crassula helmsii  [Azolla filiculoides, Elodea spp., L. minuta and Myriophyllum spp. among others. It has recently been demonstrated by Coughlan et al. [C. helmsii, E. canadensis, and Lagarosiphon major can grow from small, vegetative fragments  even when cultivated under less-than-optimal conditions. Although endozoochory of seeds and even macroinvertebrates by waterbirds have been widely reported  . When tan et al.  that inved (e.g. ), there yganodes .C. olor, have been observed to contain a variety of macrophytes including duckweed spp., Elodea spp. and Myriophyllum spp. . Fur. FurC. op.  [Largely sedentary in many regions, with most regional dispersal distances tending to be less than 35 km , C. olohabitats . While Crish Sea . Furtherltic Sea . The cal10.8 kg) . However10.8 kg) ). Nevert10.8 kg) , with sm10.8 kg) ).Cygnus spp.), given their overall ecology it has been hypothesized that they may be exceptionally important vectors of plant dispersal [Cygnus spp. has only been considered for black swan, Cygnus atratus, which have been found to disperse a variety seeds and invertebrate propagules in Australia [While relatively little is known about zoochorous dispersal by true swans (i.e. ispersal . To dateustralia . The preustralia ,34. Furt