We aimed to embed silica optical fibers in composites  in order to further monitor the robustness of civil engineering structures (such as bridges). A simple system was implemented using two different silica optical fibers (F1—double coating of 172 μm diameter and F2—single coating of 101.8 μm diameter respectively). The optical fibers were dynamically tensile tested and Weibull plots were traced. Interfacial adhesion stress was determined using pull-out test and stress values were correlated to fracture mechanisms based on SEM observations. In the case of the optical fiber (OF) (F1)/resin system and OF (F1)/composite system, poor adhesion was reported that may be correlated to interface fracture at silica core level. Relevant applicable results were determined for OF (F2)/composite system. These materials with their high specific strength and stiffness are gradually replacing their metal counterparts due to reduced weight, improved wear and corrosion resistance, increased fatigue life, and the ability of the material and component to be formed at the same time. The wide ranges of structural composites are based on polymeric matrices with glass or carbon reinforcement .Optical fiber sensors have gained much attention in recent years for a variety of physical and chemical measurements. Intense research has been performed to develop “special fibers” for integration in smart structures in order to replace conventional sensors –4 and tod appears as a critical factor for structural robustness monitoring being given by:The efficiency of optical fiber sensors to continuously monitor structure deformation and reinforcement cracks depends mainly on strength transfer at the interface level between the embedded optical fiber and the composite structure ,8. In ordF is the maximum force corresponding to the linear zone of the force versus displacement curve; R is the core optical fiber radius and eL the optical fiber embedded length.where In the context of recently developed optical fibers for distributed sensors and other smart structures, the reliability of optical fibers appears as a critical factor. Testing procedures and damage mechanisms identified on a series of experimental studies are given elsewhere .The present study has the aim to manage a reliable fabrication process of embedding optical fiber in an epoxy/glass fiber composite and to obtain basic data on a simple system. In a further step, Bragg grating optical fiber is envisaged to be embedded in a similar composite structure in order to implement a smart structure sensitive to flexion stress.2.2.1.The polymer matrix used in this study, provided by the French company DFC-DCP Pultrusion , is a mixture of epoxy vinylester resin DERAKANE 470-36 and a catalytic system composed of Styrene, Perkadox 16 and Trigonox C. After homogenously stirring the epoxy resin, the powdered Perkadox 16 is firstly diluted in Styrene so as to form a homogeneous Styrene/Perkadox 16 system, and then the Trigonox C is added. For the experimental implementation, small matrix quantities were prepared, such as 30 g of epoxy resin and 0.66 g Styrene, 0.264 g Perkadox 16 and 0.108 g Trigonox C.3. A mass fraction of 63% glass fiber in epoxy matrix was envisaged, meaning an incorporation of 12 roving in the implemented composite sample. To calculate the roving number, the following relation is used:Composite samples were prepared, reinforcing the epoxy matrix with E-glass fibers Roving 4800 Tex having a density of 2.62 g/cmf is glass fiber volume fraction and comV the composite volume. Roving 4800 Tex means 1 roving = 0.0048 g/mm. For the samples of 50 mm × 5 mm × 10 mm, 12 roving correspond to a volume fraction of 40% and a mass fraction of 63%, respectively. The composite samples were weighted after fabrication and effective mass fraction ranging between 55% and 65% were obtained.where In order to characterize the interfacial adhesion between the optical fiber and the epoxy vinylester/glass fiber composite material (OF/C), a simple system composed of a single optical fiber embedded in epoxy vinylester resin (OF/R) was preliminarily studied. The system is a unidirectional composite, where glass fibers have the sample length direction and the optical fiber is centrally embedded in the reinforcement direction.Two different optical fibers elaborated by iX Fibers S.A.S. company  were used: firstly, the fiber, denoted by F1, having a 80 μm silica core and two acrylate polymer layers of 172 μm secondary coating diameter and secondly, the fiber, denoted by F2, having a 80 μm silica core, but a single acrylate polymer layer of 101.8 μm fiber coating diameter.As optical fiber surface has determined fracture to a large extent, external coating appears critical. This coating is polymeric in most cases, and modern optical fibers are coated by two different layers: a soft coating at glass surface and a hard coating at external surface. The coating makes a protection against scratches that occur in normal handling and it reduces water activity at glass surfaces. Polymeric coatings  are preferred in practice because they are more efficient at inhibiting surface defects. As one will notice later in our study, regarding MEB examinations (see Section 3.3), fiber F1 has the typical section with two layers, but fiber F2 was subjected to a thermal treatment and presents one layer surrounding the silica core.The thermal treatment consisted of exposure at 200 °C curing temperature for 100–200 h. The fiber behavior appears similar to polyimide, those polymers containing chemical groups that promote solubility without affecting significantly mechanical properties. Moreover, curing at 200 °C for 100 h, leads to the lost of 1.5% of the mechanical strength as compared to 9% lost in the case of polyimide fiber. Fiber parameters are given in The sample preparation consisted of incorporating the glass fiber in the resin into a mold and embedding the optical fiber centrally positioned into the sample. As seen in Then, curing in preheated oven at 80 °C temperature for 90 min was applied and samples were extracted after 60–90 min.2.2.−1, which corresponds to a strain rate of about 0.02 min−1. The de-bonding force F was considered as the maximum force preceding partial de-bonding. A scanning electron microscope (SEM)  was used to investigate the surface of optical fibers after pull-out tests.Following the procedure, series of F1 optical fiber embedded in resin and in composite, respectively and F2 optical fiber embedded in composite were prepared in order to perform pull-out tests. Using a LLOYD LR 50K tensile testing setup , a gripping force was applied, as seen in 2.3.dn-stress  corrosion factor was calculated.A tensile bench Lloyds Instruments LR 50K (maximum: 100 N) was used. In order to be dynamically tensile tested, sample fibers were three tours rolled on the cylinder pulley having 50 mm in diameter. The pulleys were covered with a powerful double face adhesive; the mechanical properties of the adhesive layer appeared to be important controlling factors. Despite the standard conditions, for economy and testing time reasons, sample testing free length was chosen to 200 mm. A testing strain rate of 20, 100, 200, respectively 500 mm/min was chosen. These rates were selected in order to correspond to 10, 50, 100, respectively 250%/min as compared to sample main length. In the case of the reference fiber, at least 20 samples were tested and Weibull plots were traced, then In order to briefly evidence several parameter influences  on optical fiber mechanical properties, series of F1 fiber were treated, then tensile tested and compared to as-received fiber.Results treatment is then usually based on Weibull plots, despite some opponents concerning the adequacy of Weibull distribution use in case of tensile experiments –14. Even3.3.1.−1 corresponding respectively to the tensile testing cross speed of 20, 100, 200 and 500 mm/min. For each tested sample, we determined the stress to fracture, and then the results were treated through a statistical approach using the Weibull theory. The classical Weibull plots of the logarithm function of the cumulative failure probability (kF) related to the logarithm of the stress to fracture (C) has allowed us to calculate and to compare the statistical parameters and the dn-stress corrosion parameter.Based on our previous experimental studies, the optical fiber was dynamically tensile tested for four different strain rates of 0.1, 0.5, 1 and 2.5 minKF of fiber fracture and the applied stress (C). The evolution of ln[−ln(1 − KF)] according to lnC is called Weibull diagram.It is assumed that fracture at the most critical flaw on a fiber leads to total failure. For brittle materials as silica optical fibers, strength results obtained from tensile tests present a significant scattering. Then, the statistical Weibull method is commonly used. This method leads us to obtain the mean stress value (strength at 50% fracture probability of the Weibull plot), the medium stress, the Weibull slope and the distribution of the critical flaw size in the sample. The statistical Weibull law gives a relationship between the probability p of the curve lnCversus ln (where is the testing rate in μm/s) is related to the dynamic stress corrosion parameter dn, a parameter characterizing the material capacity to resist to a stress. The accepted stress corrosion parameter is ~20 for high strength fiber.The slope Weibull plots corresponding to the fiber denoted by F1 (two polymer layer coating to protect the silica core) are given in dn = 33.8 appears adequate with a linear interpolation of 2R = 0.98.The medium values for the tensile stress correlated to the given strain rates were respectively 4034.3, 4183.7, 4328.5 and 4426.6 MPa. As compared to our previous dynamic tensile testing performed on Verrillon Inc.  and Alcatel optical fibers, one may conclude that F1 fiber presents quite broad dispersion and lower tensile stress. The stress corrosion factor Due to experimental reasons, the F2 fiber (single polymer layer coating) was tested for one speed (100 mm/min) and quite similar results are obtained, as seen in The fabrication parameters influence the optical fiber tensile strength as seen in 3.2.Le. The force (in N) versus displacement (in mm) curve was recorded and the maximum value was considered for comparison.The fabricated systems OF (F1)/Resin and OF /Composite were prepared as described. The notch at 8 mm length from the sample end was cut to control the optical fiber embedding length Fd was taken as the maximum force preceding partial debonding.The debonding force In the case of optical fibers embedded in resin, noted OF (F1)/Resin, a poor interface adhesion is reported for all samples with a maximum of 0.98 N. Using the linear model The optical fiber F1 embedded in the composite presented poor adhesion interface, too, slightly higher than the resin, but still low. A maximum value of 1.32 N see  was recoFinally, the optical fiber F2 embedded in the composite presented better adhesion at OF/composite interface .A maximum value of 7.1 N see  was recoThe decrease of the force seems to be controlled by friction over the entire embedded length. The composite rigidity is higher than that of the non-reinforced resin. On the other hand, for the composite sample reinforced with glass fibers, one may expect less friction at the interface between the optical fiber and the composite than for non-reinforced resin in contact with the optical fiber coating. Thus, in the case of the composite sample with glass fibers, the interfacial debonding can propagate with a small instability and the obtained curve appears quasi-continual .3.3.An overall image of the composite end is given in In the case of the optical fiber (F1—double polymer coating) embedded in the resin, The physico-chemical nature of interfacial bonds depends on both elaboration temperature, and the time maintained at this elaboration temperature. The kinetics of interfacial bonding creation can be connected to a diffusive mechanism. The curing time enhances the formation of chemical bonds, and improves the diffusion mechanism at the interface optical fiber/resin. The adhesion at the interface results from the interdiffusion of macromolecular chains between the two polymer surfaces (resin and coating), thus forming an interphase. This interdiffusion could originate from a good compatibility between the acrylate coating and the vinyl ester resin,  or it could be the result of a macromolecular mobility, which leads to a tangle by reptation ,16.At the silica/polymer coating interface the coherence appears affected, as seen in Despite attentive precautions and rigorous fabrication among composite prepared samples, one may conclude that, following the pull-out test, half of the samples presented as-stripped optical fiber core  or polymFor the F2 fiber embedded in the composite that has led to improved adhesion of the optical fiber, one may notice good cohesion at the silica core/polymer coating interface, see In certain zones, maybe due to stress concentration, polymer fracture and detachment along the optical fiber  was notiGlass fiber reinforcements may scratch the polymer coating and certain imprints are also visible , but thiSEM observations have confirmed that polymer detachment were more rare in the F2 fiber case than in the case of F1 fiber embedded similarly in the composite system.4.dn = 33.8). Based on previous experimental studies, polymeric coatings are efficient to inhibit surface defects at the silica core interface.As-received optical fibers F1 (double coating 172 μm diameter) and F2 (single coating 101.8 μm diameter) present similar mechanical strength with medium tensile strength between 4 and 4.4 GPa with broad dispersion and adequate stress corrosion factor  that leads to a behavior close to polyimide.As SEM observations exhibited, in the case of optical fiber with double layer polymer coating (F1), embedded in resin or composite, the soft polymer layer around the silica core retracted, probably due to the internal stresses following polymerization cycle of the resin matrix. Therefore, the interface has lost its coherence, leading to low interfacial adhesion stresses ranging from 0.4 to 0.66 MPa.Optical fibers heat treatment at curing parameters revealed a strength decrease, but the resin catalytic system (Trigonox C) appeared favorable; the strength slightly increased and presented steeper distribution. Water at environment temperature has a similar effect after one week of exposure of the optical fiber, observations that appeared coherent to our previous results on other silica optical fibersOne may conclude that fabrication parameters acted synergistically on the optical fiber interfacial adhesion in the resin matrix, but certain precautions and a rigorous procedure should be followed to manage a coherent interface. Glass reinforcements of 55%–65%, fraction in mass, acted in the favor of the interfacial strength, the composite case exhibiting slightly better interfacial adhesion stress than the non-reinforced matrix resin, which may be explained through the harmful effect of internal stresses concentration in composite systems.Finally, the optical fiber with a single layer coating (F2) subjected to a thermal treatment embedded in the composite system has allowed better quality at interface level. The polymer coated fiber appeared to be detached in certain zones following the pull-out test, scratched by reinforcements on its external surface and resin filaments. Drops were seen too, but the silica core/polymer coating revealed coherence and the interfacial adhesion stress is reported at 3.5 MPa.Our objective to manage a reliable fabrication process of embedding optical fiber in an epoxy/glass fiber composite has been attainted and subsequent testing of Bragg gratings optical fiber (using the F2 single coating) should follow.


Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for < 0.05% of the total read count of each site’s individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities. However, a precise estimation of species distribution is often difficult and inefficient, particularly in aquatic systems where most organisms are not visible owing to complex habitat topology and vegetation. Moreover, field monitoring sometimes appears to be destructive to the target species or ecosystem under study4. To overcome such limitations, there has been a need for alternative approaches.Knowledge of species distribution is essential for understanding community dynamics and biodiversity patterns, and for planning management and conservation of threatened and endangered species8. In contrast to conventional survey methods, eDNA offers the advantages of being noninvasive and potentially more sensitive at low population densities of target organisms11. In particular, aquatic environments are suitable for applying eDNA techniques, where eDNA is distributed more homogeneously in water than in soil or other sediments12. To date, the analysis of eDNA using gel electrophoresis or quantitative PCR (qPCR) has proven highly successful for targeted detection of one or a few species inhabiting various aquatic environments17. Although these PCR-based approaches are powerful tools for monitoring target species, they cannot be used for assessing community composition of organisms.Recently, environmental DNA (eDNA) techniques have been developed for species detection of aquatic macroorganisms19. Although several technical and methodological challenges remain , eDNA metabarcoding has great advantages in terms of speed, cost per sample, coverage, and independence of taxonomic expertise compared to conventional morphology-based surveys22.An alternative approach is eDNA metabarcoding, which involves parallel sequencing of whole communities of organisms, and thereby offers comprehensive and efficient tools for assessing total biodiversity and community composition21. These studies first focused on describing fish communities in tanks or aquaria, and thereby they confirmed that the method is highly sensitive for detecting rare species25. More recently, metabarcoding has been used successfully to assess biodiversity and community structure of various macroorganisms in natural settings, including freshwater fishes29, marine fishes33, amphibians34, mammals35, and freshwater invertebrates36. Moreover, eDNA metabarcoding approaches potentially can be extended to explore large-scale spatiotemporal variations of community structure in aquatic ecosystems, unravelling trends linked to environmental variables or to human impacts37.An increasing number of studies have performed eDNA metabarcoding for species detection, biodiversity assessment, and relative quantification of aquatic macroorganisms36 . Another labor-saving method is pooling of samples collected from multiple locations before DNA extraction. A strategy of pooling of eDNA samples has been used commonly to reduce sample numbers for evaluations of microbial community structure41. Importantly, pooling eDNA could dilute DNA of rare species, resulting in masking a significant portion of communities if communities are extremely complex 40. However, communities of aquatic macroorganisms are obviously much less complex than microbial communities in soil and, thus we hypothesized that pooling of eDNA samples potentially might be effective to assess biodiversity of freshwater fishes.To assess biodiversity and community composition of aquatic vertebrates using eDNA metabarcoding, the sampling design not only must be effective but also efficient, especially for a large-scale study of communities over space and time. Use of small sample sizes may lead to underestimation of biodiversity because recent eDNA metabarcoding studies indicated that eDNA in aquatic systems appears to be spatially autocorrelated at large spatial scalesAccordingly, we investigated the community composition of freshwater fishes in the satellite lakes of Lake Biwa, Japan, using eDNA metabarcoding. By comparing detected fish communities between the individual and pooled samples taken from the same sites, we tested whether pooling of eDNA samples (pooling of water samples) could reduce the effectiveness of eDNA metabarcoding with respect to the biodiversity assessment.In total, 9,083,566 MiSeq reads were obtained, of which 8,139,675 passed the quality control processes , but the correlation was not significant in the incidence-based index .The Adonis test for the individual samples indicated that geographic locations (latitudes) of water sampling had a significant effect on the abundance-based and incidence-based Jaccard community dissimilarities Table , indicatIn the individual samples, the total numbers of lineages detected from the Nodanuma, Sonenuma, Ibanaiko, and Nishinoko sites were 31, 22, 33, and 31, respectively. In the pooled samples, 30, 20, 29, and 27 lineages were observed from each of the four satellite lakes, respectively, with 15 PCR replicates. The number of lineages that were detected in the individual sample but not in the pooled sample increased with increasing the surface area of satellite lake  showed the most abundant sequence reads in most of the samples  were also frequent and were characterized by relatively abundant reads, but the latter was relatively rare in the Sonenuma samples , three , five , and five  for the Nodanuma, Sonenuma, Ibanaiko, and Nishinoko sites, respectively . The subsequent PERMDISP analysis further indicated that among-sample heterogeneity of fish communities within the same satellite lake was significantly different between the individual and pooled samples .Moreover, the relative read fractions of respective lineages were considerably different among samples within the individual samples, whereas they appeared to be highly similar among samples within the pooled samples Fig. . SimilarWe present the first attempt to compare the effectiveness of individual versus pooled samples to assess biodiversity of fish communities in freshwater using eDNA. Based on the results, we discuss potential limitations and applications of the pooling method for assessing biodiversity using eDNA metabarcoding.36, suggesting that not only sampling volume but also geographic distance between sampling locations are essential considerations for assessing biodiversity using eDNA metabarcoding. Although the spatial signals were somewhat obscured in the incidence samples , as reported previously38.The fish communities observed in the individual eDNA samples have implications for understanding spatial structure of eDNA in the satellite lakes. Our results indicated that increasing the number and spatial replication of samples leads to increasing sensitivity and reliability of estimating species richness of fishes in the satellite lakes using eDNA metabarcoding Fig. . Importaes Table ; Fig. 1,es Table . The pre43. Filtering a double water volume of the pooled samples slightly improved the detection rates  fish communities were combined. However, the number of fish lineages detected in the pooled samples nearly reached an asymptote with increasing number of PCR replicates . Because the major advantage of the pooling strategy is its efficiency, we do not suggest use of these alternative procedures.45. However, the risk of false-positives in the pooled samples seems to be similar to that in the individual samples in the present study, in which fish communities detected in the pooled samples were almost nested in those detected in the individual samples.False-positives are also more likely in pooled samples because greater numbers of PCR replicates increase PCR-induced artifacts and opportunities for contamination46. Although locally dominant but spatially rare phylotypes often become undetectable in the pooled samples for soil microbial communities40, such trends do not appear to be true for the present data  potentially serves to compare community structure (beta diversity) of fishes among sites. Small among-sample heterogeneity of fish communities detected in the pooled samples  is presumably crucial for this method, although the number of PCR replicates should not be increased unlimitedly to avoid the potential risk of false-positives45. Moreover, rates of dropouts in the pooled samples appeared to increase with increasing the surface areas of satellite lakes . Therefore, a sufficient number of PCR replicates  of Lake Biwa in Shiga Prefecture, Japan. Sampling locations from each satellite lake were located around the edges of the lakes, with one in the center  of the “individual” water samples were measured in the field using portable waterproof testers  in combination with a spin column . After removing the originally attached membrane of the spin column (EZ-10), the sample filter was placed in the spin column (EZ-10). The spin column with the sample filter was centrifuged at 6000 g for 1 minute and water remaining in the sample filter was removed (the filtration was discarded). After placing the spin column in a new 2-mL tube, the mixed solution of Milli-Q water (200 μL), proteinase K (10 μL), and Buffer AL (100 μL) was pipetted gently onto the filter in the spin column. The tube was then incubated at 56◦C for 30 minutes. After incubation, the liquid held in the filter was collected by centrifugation. To increase the yield of eDNA, 200 μL TE buffer was pipetted gently onto the filter and the spin column was centrifuged again at 6000 g for 1 minute. The collected DNA solution was purified using the DNeasy Blood and Tissue Kit following the manufacture’s protocol, except for a modification in the final step of elution of DNA from the filter column. We eluted DNA using 100 μL Buffer AE, though the original manual specified 200 μL.21, which were designed to contain Illumina sequencing primer regions and 6-mer Ns for improved“chastity”in Illumina sequencing , in which NNNNNN represents a 6–base pair (bp) random sequence. We used a KOD FX Neo polymerase  for the first PCR to facilitate amplifications of DNA from crude extracts. The first PCR was performed with a 12 μL reaction volume containing 1 × PCR Buffer for KOD FX Neo, 0.4 mM dNTP mix, 0.24 U KOD FX Neo polymerase, and 3.5 pmol of each primer. The thermal cycles of this step were as follows: initial denaturation at 94 °C for 2 minutes, followed by 35 cycles of denaturation at 98 °C for 10 seconds, annealing at 65 °C for 30 seconds, and elongation at 68 °C for 30 seconds, followed by final elongation at 68 °C for 5 minutes. The first PCR was replicated 3 and 15 times per sample for the individual and pooled samples, respectively. The PCR replicates of the individual samples were pooled to mitigate the false negatives (PCR dropouts), whereas those of the pooled samples were subjected to the second PCR, separately. The first PCR products were purified using Exo-SAPIT  according to the manufacturer’s instructions. The purified first PCR products were used as templates for the second PCR.A two-step PCR-procedure was used for library preparation. In the first step, a fragment of the mitochondrial 12S rRNA gene was amplified using the MiFish-U-F and MiFish-U-R primers49 were added in the subsequent PCR process using a forward and reverse fusion primer . The second step was done with a 12 μL reaction mixture containing 1 × KAPA HiFi HotStart ReadyMix , 3.5 pmol of each primer, and 1 μL of the PCR products. The thermal cycles of the second PCR were as follows: initial denaturation at 95 °C for 3 minutes, followed by eight cycles of denaturation at 98 °C for 20 seconds, annealing and elongation combined at 72 °C for 15 seconds, with a final elongation at 72 °C for 5 minutes.The Illumina sequencing adaptors plus the 8-bp identifier indicesThe indexed second PCR products were pooled in equal volumes and 25 μL of the pooled libraries were loaded on a 2% E-Gel SizeSelect  and a target size of the libraries (ca. 370 bp) was collected. The DNA concentrations were then estimated by a Qubit dsDNA HS assay kit and a Qubit fluorometer (Thermo Fisher Scientific). The amplicon libraries were sequenced by 2 × 150 bp paired-end sequencing on the MiSeq platform using the MiSeq v2 Reagent Kit according to the manufacturer’s instructions.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). After trimming low-quality tails from each read using DynamicTrim.pl from the SOLEXAQA software package50 with a cutoff threshold set at a Phred score of 10, the paired-end reads were assembled using the software FLASH with a minimum overlap of 10 bp51. The assembled reads were filtered further to remove forward and reverse primer positions, ambiguous sites (Ns), and sequences showing unusual lengths.The overall quality of the obtained sequence  was evaluated by the program FASTQC  were subjected to pairwise alignment using a “usearch global” command in UCLUST. If the latter sequences showed more than or equal to 99% identity with one of the former reads, they were considered operationally as identical and they were merged.52 against the comprehensive reference database of fish species that were established previously21, using an e-value cutoff of 10−5 and an identity cutoff of 99%. This procedure also works to removing erroneous reads because erroneous reads are expected never to match the reference sequences at ≥99% similarity by chance. Because some closely-related species shared identical sequences in the barcode region, those species were merged  before the BLAST searches (Supplementary Table Carassius spp. vs. Carassius cuvieri or Carassius spp. vs. Cyprinus carpio). These exceptional sequences were not included in the downstream analyses because they accounted for a small proportion of the total reads (0.0137%) and taxonomic assignment of them did not influence the presence/absence of fishes in each sample. After BLAST searches, sequences that were assigned to the same species (or species complex) were clustered, and we considered the clustered sequences as proxies for species . Because most of the sequence reads detected in 17th sample of the individual samples from the Nishinoko site  failed to be assigned to a lineage name, presumably due to failure in the PCR step, this sample was not used for the subsequent analyses.The processed reads were subjected to local BLAST searches53 of R 3.1.254 , only the latitude was included in the model.Firstly, we measured the spatial autocorrelation of fish communities in the individual samples. We evaluated the relative importance of geographic locations within each satellite lake (latitude and longitude) and water environmental variables  on the composition of fish species , with the “adonis” function of the vegan package, in which permutations were constrained within each satellite lake. For this analysis, single factor models were first tested and, thereafter, factors were added in the final model in order of their R statistic indicates the Pearson’s correlation between the multivariate fish community structures detected in samples and the geographic distance between samples.A Mantel test was also performed to test whether fish communities were clustered by geographic locations (“‘mantel” function of the vegan). For the analysis, permutations were constrained within each satellite lake using the “strata” argument. The Mantel test We compared the number of lineages detected in the individual samples to that in pooled samples. Sample-based species accumulation curves were calculated based on the sample-species matrix using the “specaccum” command in the vegan package of R, thereby testing whether numbers of sampling locations  and PCR replicates (the pooled samples) were large enough to allow measurement of species richness at each site using sample-based species accumulation curves. We also estimated the total number of lineages at each site using nonparametric first-order jackknife (Jack1) estimator and confidence interval (Jack1 ± 2 SE), implemented in the “specpool” function of the vegan. Moreover, the read fractions of respective fish lineages in each individual sample were plotted against the number of samples (PCR replicates) in the pooled samples where sequences of the same lineages were detected, to identify fish lineages that were detectable only in the individual samples.We also compared the fish community detected in eDNA metabarcoding between two sources . Primarily, the relative read fraction of individual fish lineage within each sample was converted into a heatmap using the function “pheatmap” as implemented in pheatmap v.1.0.8 package of R. In addition, differences in the community compositions were visualized using the two-dimensional nonmetric multidimensional scaling (NMDS) ordination with “metaMDS” function in the vegan package of R, where the program choose the best solution  from 100 separate runs of real data. For the NMDS, the community dissimilarity was calculated based on abundance-based (use of sequence reads) and incidence-based (presence/absence) Jaccard indices.We tested for variability in the community composition between two sources using a permutational multivariate analysis of variance  with 9999 permutations, based on abundance-based and incidence-based Jaccard dissimilarity indices. We also tested heterogeneity of dispersion between sources using a permutational analysis of multivariate dispersion . For those statistical analyses, permutations were constrained within each satellite-lake using the “strata” argument to account for nestedness.Supporting information


Metabolic syndrome, variously known also as syndrome X, insulin resistance, etc., is defined by WHO as a pathologic condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Though there is some variation in the definition by other health care organization, the differences are minor. With the successful conquest of communicable infectious diseases in most of the world, this new non-communicable disease (NCD) has become the major health hazard of modern world. Though it started in the Western world, with the spread of the Western lifestyle across the globe, it has become now a truly global problem. The prevalence of the metabolic syndrome is often more in the urban population of some developing countries than in its Western counterparts. The two basic forces spreading this malady are the increase in consumption of high calorie-low fiber fast food and the decrease in physical activity due to mechanized transportations and sedentary form of leisure time activities. The syndrome feeds into the spread of the diseases like type 2 diabetes, coronary diseases, stroke, and other disabilities. The total cost of the malady including the cost of health care and loss of potential economic activity is in trillions. The present trend is not sustainable unless a magic cure is found (unlikely) or concerted global/governmental/societal efforts are made to change the lifestyle that is promoting it. There are certainly some elements in the causation of the metabolic syndrome that cannot be changed but many are amenable for corrections and curtailments. For example, better urban planning to encourage active lifestyle, subsidizing consumption of whole grains and possible taxing high calorie snacks, restricting media advertisement of unhealthy food, etc. Revitalizing old fashion healthier lifestyle, promoting old-fashioned foods using healthy herbs rather than oil and sugar, and educating people about choosing healthy/wholesome food over junks are among the steps that can be considered. With the successful conquest of many of the old infectious diseases in the world, non-communicable diseases (NCD) have become the major cause of morbidity and mortality not only in the developed world but also in the underdeveloped countries. Among all these NCD, metabolic syndrome had been the real scourge globally.Metabolic syndrome (MetS), also variously known as syndrome X, Insulin resistance, etc. in the literature, is really not a single disease but a constellation of cardiovascular disease risk factors and had been defined slightly differently by various organizations. Three most popular definitions  used forHDL cholesterol < 0.9 mmol/L (35 mg/dl) in men, < 1.0 mmol/L (40 mg/dl) in womenTriglycerides > 1.7 mmol/L (150 mg/dl)2Waist/hip ratio > 0.9 (men) or > 0.85 (women) or BMI > 30 kg/mBlood pressure > 140/90 mmHgPresence of insulin resistance or glucose > 6.1 mmol/L (110 mg/dl), 2 h glucose > 7.8 mmol (140 mg/dl) (required) along with any two or more of the following:Blood glucose greater than 5.6 mmol/L (100 mg/dl) or drug treatment for elevated blood glucoseHDL cholesterol < 1.0 mmol/L (40 mg/dl) in men, < 1.3 mmol/L (50 mg/dl) in women or drug treatment for low HDL-CBlood triglycerides > 1.7 mmol/L (150 mg/dl) or drug treatment for elevated triglyceridesWaist > 102 cm (men) or > 88 cm (women)Blood pressure > 130/85 mmHg or drug treatment for hypertensionPresence of any three or more of the following:Blood glucose greater than 5.6 mmol/L (100 mg/dl) or diagnosed diabetesHDL cholesterol < 1.0 mmol/L (40 mg/dl) in men, < 1.3 mmol/L (50 mg/dl) in women or drug treatment for low HDL-CBlood triglycerides > 1.7 mmol/L (150 mg/dl) or drug treatment for elevated triglyceridesBlood pressure > 130/85 mmHg or drug treatment for hypertensionNCEP and IDF definition are very similar except in the waist parameter of 102 vs. 94 cm in men and 88 vs. 80 cm in women.Note: Waist > 94 cm (men) or > 80 cm (women) along with the presence of two or more of the following:Other organizations like the American Association of Clinical Endocrinologist (AACE) 2003 and the European Group for the Study of Insulin Resistance (EGIR) used slightly different definitions but they are not as commonly used.One quarter of these people (23.8%) were not aware of having diabetes. Incidence of T2DM increased with age, reaching a high of 25.2% among US seniors (65 years or older). Prevalence of prediabetes or MetS was about three times more. So, about one third of US adults have metabolic syndrome [The incidence of metabolic syndrome often parallels the incidence of obesity and incidence of type 2 diabetes (one of the outcome of MetS). According to NHNES data, during 1988–2010, average BMI in USA increased by 0.37% per year in both men and women and waist circumference (WC) increased by 0.37 and 0.27% per year in women, respectively. According to CDC data published in 2017, about 30.2 million adults aged 18 years or older or 12.2% of USA adults had type 2 diabetes (T2DM). Incidence of T2DM was even higher among certain ethnic groups—15% among American Indians but lower among Chinese Americans − 4.3%. South Asian Americans had a very high prevalence of metabolic syndrome along with higher incidence of abdominal obesity [In China, between 1992 and 2002, the prevalence of overweight and obesity increased from 14.6 to 21.8%—based on WHO criterion. Using the Chinese obesity definition, with lower BMI cutoff, the increase was from 20 to 29%. The incidence of MetS increased from 8 to 10.6% in urban areas and 4.9 to 5.3% in rural areas. Assuming the same rate of increase, the prevalence of MetS in China in 2017 would be about 15.5% [According to global survey of obesity in 195 countries, done in 2015, 604 million adults and 108 million children were obese. Since 1980, prevalence of obesity doubled in 73 countries and increased in most other countries. Of even greater concern was that the rate of increase was even higher in childhood obesity [Obesity also contributed to 120 million disability-adjusted life-years. The highest percentage change in age-standardized BMI related deaths and disability-adjusted life-years occurred in Bangladesh—one of the poorest country and the country where the present author comes from. On the other hand, age-standardized BMI-related morbidity and mortality decreased by 37.2 and 43.7%, respectively, in Turkey.According to this survey, obesity is no longer a disease of affluence. The highest increase in prevalence of obesity in young men (25–29 years) occurred in countries with low socio-economic index (SDI). In the last three decades, the prevalence went from 1.1% in 1980 to 3.85 in 2015. Between 1990 and 2015, global rate of death related to high BMI increased by 28.3%. Obesity, however, is not always synonymous with MetS. There are so-called metabolically healthy obese (MHO) individuals who have high level of insulin sensitivity and do not have hypertension and hyperlipidemia and other features of MetS. Epidemiological survey suggests that MHO may account for a significant percentage of obese population . AccordiAccording to IDF diabetes atlas , global In other words, over a billion people in the world are now affected with metabolic syndrome.We do not have similar global data on metabolic syndrome—which is harder to measure, but since MetS is about three times more common than diabetes, the global prevalence can be estimated to be about one quarter of the world population. The prevalence estimates vary, based on the criteria used for the definition of MetS. For example, a national survey in Iran in 2007 showed prevalence of MetS was about 34.7% based on ATP III criteria, 37.4% based on IDF definition, and 41.6% based on ATP III/AHA/NHLBI criteria. In another Middle Eastern country, Tunisia, prevalence was 45.5% based on IDF criteria but 24.3% based on ATP III criteria. But in all the Middle Eastern countries, prevalence was much higher among women than men .white adipocytes, brown adipocytes, and beige adipocytes. The brown and beige adipocytes are morphologically and functionally different than white adipocytes. These cells have more mitochondria in the cytoplasm enriched with more uncoupling protein 1 (UCP1) and able to produce more thermogenesis [During the last three decades, while prevalence of MetS increased, our understanding of the biology of the malady also increased. All fats are no longer considered the same. Adipocytes are now categorized as ogenesis .Rather than being an inert energy storage depot, adipocytes are now known to be metabolically active, secreting over a dozen of hormones affecting the appetite, satiety, and energy metabolism of the body. While first known adipocyte hormone leptin suppresses appetite and genetic absence of which causes massive obesity, other hormones like adiponectin have just the opposite effect . AdiponeThe macrophages residing in the fat tissue are a major player in energy metabolism. Pro-inflammatory (M1) macrophages promote hepatic steatosis and adipogenesis, while anti-inflammatory macrophages (M2) do the opposite. Macrophage-restricted deficiency of transcriptional regulator methyl-CpG-binding protein 2 (MECP2) also caused hepatic steatosis and obesity phenotype in a mice model .alphabeta1 integrin. The process is also dependent on expression of VCAM1 which encodes vascular cell adhesion molecules—the counter receptor for alpha-beta1 integrin. The retention and activation of M1 macrophages in fat tissue inhibit adipocyte UCP1 expression and beige adipocytes. The role of alphabeta1 integrin in obesity is particularly important since there is already an FDA-approved drug Natalizumab, a monoclonal antibody that blocks this integrin, used in the treatment of multiple sclerosis.In a recent study, Chug et al. reported that M1 macrophage retention in adipose tissue is mediated by IRE1alpha) activity. Deficiency of IRE1alpha prevented obesity, insulin resistance, and hepatic steatosis induced by HFD. IRE1alpha also suppresses the M2 macrophages in in adipose tissue [Endoplasmic reticulum (ER) stress is another important factor in the pathogenesis of metabolic syndrome. In mice experiment, Shan et al. showed that high fat diet (HFD)-induced ER stress is dependent on inositol-requiring enzyme 1 alpha  maintains glucose homeostasis by inducing insulin secretion and insulin sensitivity. LCN2 also inhibits food intake by suppressing melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial neurons of the hypothalamus and activates MC4R-dependent anorexigenic pathway [One of the phenotypic features of MHO is their strong skeletal built. Underlying physiology of this is now better understood. An  pathway .thyroid stimulating hormone (TSH)-releasing hormone (TRH)-positive neurons in the hypothalamus and stimulated TSH from the pituitary gland leading to increased secretion of thyroid hormone. Also, decreased expressions of perinatal deiodinase 2 in hepatocytes greatly reduce the susceptibility to diet induced steatosis and obesity [Thyroid hormones along with beta-adrenergic hormones are well-known factors regulating energy metabolism. Miao et al. recently described the interaction of thyroid hormones and liver x receptor (LXR) which serves as repressors of UCP1 in classic brown adipocytes. Depletion of LXR in mice activated  obesity .Akkermansia muciniphila, a mucin-degrading bacterium that resides in the mucus layer of gut epithelium. Feeding mice with perbiotic (oligofructose) normalized A. muciniphila abundance (increased by 100-fold) and corrected the metabolic syndrome in the obese mice. A. muciniphila administration increased the intestinal level of endocannabinoids that control the inflammation, gut barrier, and gut peptide secretion. This group now reported that use of membrane protein from A. muciniphila improves metabolic syndrome in mice [Gut microbiome has now become an important part of human biology in health and diseases. Healthy microbiome prevents many cardiovascular diseases as well as MeTS/diabetes. Everard et al. isolated one specific gut microbe  in mice , 18.PPARdelta agonist which when used in mice reduced the metabolic syndrome [In addition to these, other biologic mechanisms of MetS, recently discovered, have potential therapeutic implication. Kopec et al. showed that thrombin promotes diet-induced obesity through fibrin-driven inflammation and that treatment with thrombin inhibitor drug dabigatran—already in use for treatment of DVT—limited HFD-induced obesity and MetS . Maternasyndrome •.In all epidemiologic studies, prevalence of MetS increases with age. This is not surprising since there are many commonalities in biochemical changes of aging process and metabolic syndrome/diabetes .While there are some known genes associated with obesity and MetS, the epidemic growth of the malady in a short period makes genetic predisposition a minor component. A genome-wide association study and Metabochip meta-analysis of BMI done in 339,224 individuals identified 97 BMI-associated loci, 56 of which were novel. The 97 loci accounted for ~ 2.7% of BMI variation and common variation accounted for > 20% BMI variation .However, epigenetics seems to have a bigger role in promoting MetS. Parenteral obesity may cause obesity in the offspring through epigenetic changes in the spermatozoa or oocytes or more commonly in utero environment. Children born to obese mother or father who underwent bariatric surgery prior to conception of the children are less prone to obesity/MetS than children born before bariatric surgery .Epidemiological studies have shown strong association between intrauterine nutrition, patterns of postnatal nutrition, and growth and metabolic syndrome in adults. Mothers exposed to the Dutch famine of 1944/45 during the first two trimesters of the pregnancy had babies with low birth weights (LBW), but these babies had higher incidence of obesity in adult life. LBW infants, who had rapid catch up growth as infant, had the highest risk of developing obesity and MetS in adult life. A similar phenomenon was observed in China after the famine there in 1959–1961. Mechanistically, this phenomenon seems to occur via decreased DNA methylation of the imprinted IGF2 gene in the offspring and hypermethylation of two obesity-related genes—leptin and TNF. In rodent models of maternal under- or over-nutrition and newborn-specific nutritional modifications, epigenetic changes were seen that affected growth factors, adipogenesis, appetite control, and glucose homeostasis in the offspring .This pheIrisin with reversal of muscle insulin resistance, and reduction in postprandial hepatic lipogenesis [Physical activity (PA) and exercise are key components of energy expenditure and energy balance. But the benefit of exercise in preventing metabolic syndrome goes beyond the immediate benefit of caloric expenditure . With chogenesis .However, according to NHANES data during the period of 1988 and 2010, when the average BMI and WC increased in US adults, the proportion of adults who reported NO leisure lime physical activity (LTPA) increased from 19.1 to 51.7% in women and from 11.4 to 43.5% in men. According to this study, average caloric intake did not change during this time and BMI and WC trends were associated with LTPA level and not caloric intake .According to recent CDC data, 40.8% of adults in USA were physically inactive . While we do not have easy access to such physical activity data in the developing countries, it can be assumed that with the acceptance of Western lifestyle, increased use of automobiles and more time spent indoor watching TV or playing video games, the data trend will be very similar. Increased sedentary lifestyle is a major driving force for increased prevalence of MetS .extra virgin olive oil (EVOO) given as supplement to usual Western type diet reduced the incidence of MetS and hypertension [Predimed and other studies provided evidence supporting a beneficial role of traditional Mediterranean diet in preventing diabetes and metabolic syndrome. In the Predimed study, it was also observed that just an ounce of rtension .are olive oil, capsaicin, luteolin, curcumin, cinnamon, rosemary, etc.Several dietary factors are known to prevent MetS. Notable among these A systemic review on the effects of dietary polyphenols on metabolic syndrome done recently showed that, at relatively high doses, many polyphenols favorably influence different features of metabolic syndrome. Soy isoflavone, citrus products, hesperidin, and quercetin improved lipid metabolism, and cocoa supplement improved high blood pressure and blood glucose. Green tea significantly reduced BMI and waist circumference and improved lipid metabolism .Epidemiologic data show that consumption of foods high in capsaicin is associated with lower prevalence of obesity/MetS. Capsaicin present in hot pepper works on the TRPV1 and PPAR alpha receptors and reduces metabolic dysregulation in obese/diabetic mice by enhancing expression of adiponectin and its receptor , 32.Cheng et al. recently reported that low-calorie, low-protein, and low-carb but high fat fasting mimicking ketogenic diet for 4 days increased the generation of pancreatic beta cells and reversed both T1D and T2D phenotypes in mouse models •. induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing β cells, resembling that observed during pancreatic development.) FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 (Neurogenin 3) expression and insulin production.Nature that blocking follicle-stimulating hormone (FSH) induces thermogenic adipose tissue and reduces body fat [Liu et al. recently reported in body fat . A non-cbody fat .Of the traditional approved drug, the most effective medicine in preventing MetS is metformin which is a unique anti-diabetic medicine whose mechanism of action is not fully understood yet but closely mimics the effect of exercise , 37•, 38Carnosic acid is a major bioactive component in Rosemary extract and it is found to ameliorate HFD-induced obesity and metabolic syndrome in mice . RecentlGalantamine is a centrally active acetyl cholinesterase inhibitor with anti-inflammatory properties and is FDA-approved for treatment of dementia. According to a recent RCT done in Brazil, daily intake of 8–16 mg of galantamine orally for 4 weeks reduced plasma levels of TNF and leptin and increased level of adiponectin and IL-10 and significantly lowered plasma insulin and insulin HOMA-IR . If thesBut knowledge of special diet and use of different herbs/functional foods can only go so far. To really stop and control the epidemic of MetS, a societal/governmental/global approach is urgently needed.Civil societies, outside of the strict medical community, need to help disseminate the awareness of the huge impact of MetS on the overall health in the global community. Civil society can help disseminate the awareness and relevant issues to the common citizenry through discussion, debates/lectures, and effective use of mass media. Rather than viewing the obesity and leanness as an anthropometric and fashion issue, attention should be directed to the associated health issue with acknowledgement that all obese persons are not metabolically unfit and all lean people are not metabolically fit •.Producers of literatures on health should try to popularize the waist/hip ratio concept as better matrix of metabolic syndrome than BMI . BuildinNewer technology can be used to educate and create awareness. Use of Facebook and electronic apps may be used in promoting healthy lifestyle. Newer technology is also needed in creating better food quality supply. We know that eating whole grain product is less likely to cause MetS than refined carbohydrate . A simplGovernmental involvements at municipal levels to country level are very important. Sometime, a minor change in law can accomplish much more than many lectures and good advice. The small tax imposed on sugary drinks in New York City, Philadelphia, or at the country level like in Mexico was effective in lowering consumption of sugary drinks . But lawGovernment can help create life-long health impact by ensuring good minimal perinatal care and promotion of breast feeding for every newborn. It can also introduce good cooking lesson and basic nutritional knowledge in secondary schools. Generations of young people are now entering adulthood without knowing how to cook and thereby depending on fast foods for survival. If these young men and women discover the joy of cooking, promoting healthy diet among them will be easier.Governments can also ensure creation of more parks and pedestrian walkways during urban development plan. One Latin American city once banned its citizens from using any automobile one Sunday a month. The result was astonishing. Suddenly, there were swarms of kids and adults in the street playing football or other games and no one was driving. It was magical.And lastly, governments can lead in educating the health care provider about the enormous economic cost of the MetS epidemic and mandate some incentive for preventive care. But medical doctors are not well suited for preventive care and cadres of barefoot doctors may need to be used for this purpose. Since in many countries vaccination programs are used by such providers, they can be retrained now for the prevention of this new epidemic.WHO or similar organizations can work with country level governments to make country-specific plan to confront the epidemic, keeping in mind the cultural, economic, and educational matrix of individual country •. While And lastly, health care providers need to take leadership position in directing the society towards a healthy community that are not only free of infectious disease but are physically fit and enjoying disease-free long life. Most practicing clinicians do not talk to the patients about good eating habits and need for adequate physical activity. But few words of such reminders and encouragements during routine visit can go a long way in changing people’s habit.Lastly, many of the MetS are iatrogenic due to wide use of some drugs especially the new generation of anti-depression and anti-psychotic meds •. Many HThe metabolic syndrome is a complex pathophysiologic state that originates primarily from an imbalance of calorie intake and energy expenditure but also affected by genetic/epigenetic make up of individual, predominance of sedentary lifestyle over physical activity, and other factors like quality and composition of food and composition of gut microbes. No single remedy can be prescribed for its eradication or even curtailment.The epidemic did not happen suddenly and it cannot be controlled quickly, but if there is a societal will, it can be done. As in the control of other epidemics, education of the population about the health hazard of the metabolic syndrome will be very important. Since metabolic syndrome is a complex medical diagnosis requiring blood tests, simple anthropometric definition that can be easily used and accepted by populace is necessary. While exact measure to be taken can be debated, promoting the danger of increase in abdominal girth or waist/hip ratio can be a starting point. A slogan like, if your belt size increases by a notch, danger is lurking inside the belly can be promoted. Or “sweets are for weak-lings, hot peppers are for the machos.”Cooking skills should be taught to the students in high school/college. Without learning to cook healthy yet enjoyable food, the dependency on fast food cannot be controlled. Many commercial ventures in the USA that are helping young couples to cook are a good start but they are expensive and not widely adoptable.Governments need to stop subsidizing unhealthy food (farm support bill in the USA).Before embarking on any large-scale change, planners need to do pilot studies to see what works and what does not.Increase investment in finding pharmaceutical assistance by developing drugs like metformin.Nature medicine reported the biological importance of GDF15 and its receptor in maintaining energy balance [Understand the biology even better. Recently, three publications in  balance , 57•. ThThe present epidemic of MetS is also related to the wide acceptance of predominantly Western capitalistic economic development model. A better, more humane economic development plan can address the inherent health hazards of this capitalistic model where profits supercede long-term effects on the society. Better understanding of socio-economic plans that promote conditions favoring the MetS epidemic can help choose alternate development plan.Targeting the younger population to prevent MetS before it happens is a better plan than trying to cure it after it happens. While a dedicated group of public health planner can create a comprehensive plan for a given society or a given community, here are some suggestions:


Elephas maximus) and clinical EEHV infections account for the majority of their fatalities. The anti-herpesviral drug famciclovir (FCV) has been used routinely to treat viraemic at-risk elephants, but thus far without proven efficacy. This paper presents clinical and virological investigations of two EEHV-1A infected elephants treated with FCV, and discusses anti-herpesvirus therapies of viraemic elephants.Elephant Endotheliotropic Herpesviruses (EEHVs) can cause acute haemorrhagic disease in young Asian elephants (Two 1.5 year old male Asian elephants at a zoological collection in the UK developed clinical EEHV-1A infections. Case 1 showed signs of myalgia for the duration of 24 hours before returning back to normal. EEHV-1A DNAemia was confirmed on the day of clinical signs and continued to be present for 18 days in total. Trunk shedding of the virus commenced 10 days after detection of initial DNAemia. Case 2 tested positive for EEHV-1A DNAemia in a routine blood screening sample in the absence of clinical signs. The blood viral load increased exponentially leading up to fatal clinical disease seven days after initial detection of DNAemia. Both calves were treated with 15 mg/kg FCV per rectum on detection of DNAemia and penciclovir, the FCV metabolite, could be detected in the blood at assumed therapeutic levels. The early indicators for clinical disease were a marked absolute and relative drop in white blood cells, particularly monocytes prior to the detection of viraemia. The most prognostic haematological parameter at later stages of the disease was the platelet count showing a continuous sharp decline throughout, followed by a dramatic drop at the time of death.The EEHV-1A viraemic animals investigated here further highlight the ongoing threat posed by these viruses to juvenile Asian elephants. The findings call into question the efficacy of rectal FCV in clinical cases and direct towards the use of alternative anti-herpesvirus drugs and complementary treatments such as plasma infusions if no improvement in either viral load or the above-mentioned blood parameters are observed in the initial days of viraemia despite anti-herpesvirus therapy. Proboscivirus of the family Herpesviridae -1,3-propanediol diacetate, is a synthetic acyclic guanosine analogue that is rapidly de-acetylated and oxidized in the body to its metabolite penciclovir (PCV). Activation of PCV requires phosphorylation in the virally infected cells by viral and cellular kinases. The drug has a half-life of approximately three hours and its catabolite is excreted renally . PCV intThis paper describes the investigations into two EEHV-1A viraemic elephants from a UK zoological collection that were both treated with FCV. It also discusses findings of the investigations in relation to anti-herpes viral therapies in EEHV viraemic elephants.Blood samples of EDTA whole blood and blood swabs were collected under trained behaviour from an ear vein after aseptic preparation of the medial aspect of the ear pinna. For EDTA whole blood samples, the animal was placed in lateral recumbency and the lowermost ear was sampled, using a standard 21G 1-inch needle and a Vacutainer® blood collection system. For blood swabs, a dry 6-inch foam tipped swab  was used for collection of a drop of blood after puncture of an ear vein with an insulin needle, with the elephant in standing position. Blood samples were kept at room temperature before being shipped to the laboratory, where they were stored at 4–8 °C until being processed. Under trained behaviour, conjunctival swabs and trunk swabs were collected. Conjunctival swabs were collected using a sterile dry swab for each eye, running these on the inside of the lower palpebra. Trunk secretions from the mucosa of each naris were collected on a dry 6-inch foam tipped swab at a depth of approximately 8–10 cm, under trained behaviour, and stored at −20 °C prior to further testing. Blood smears were made of freshly collected EDTA blood samples, air dried and stained with Diff-Quick  for an in-house relative white blood cell (WBC) differential count.Nucleic acid was extracted from 140 μl EDTA whole blood using the QIAamp® Viral RNA Mini kit  according to the manufacturer’s protocol. For blood swabs, 560 μl buffer AVL from the kit were directly added to the foam tipped swabs, left for 15 minutes at room temperature, vortexed and spun briefly, and processed as per the manufacturer’s instructions. To process trunk swab samples for nucleic acid extraction, 300 μl PBS and 6–8 glass beads (3 mm in diameter) were added to the swab inside a 2 ml Eppendorf tube, vortexed briefly and incubated for 30 minutes at room temperature. The tube content was then vortexed and nucleic acid was extracted following the EDTA whole blood protocol using 140 μl of the cleared suspension.The qPCR used in this study was performed as described  using th6 to 101 copies per PCR test. The synthetic oligonucleotide dilutions were run in triplicate in the PCRs to create a standard curve for EEHV genome quantification.The DNA standard used was a HPLC purified 100 bases synthetic oligonucleotide , identical to the sequence of the qPCR target, diluted to provide serial copy numbers from 10Rectal fluids were provided after manual removal of all faeces from the rectum. Several litres of hand-warm water were then deposited into the distal colon at approximately one meter depth, through a standard garden hose pipe. Intravenous (i/v) access was acquired after antiseptic preparation of the medial aspect of the ear pinna, using an 18G 2.5-inch indwelling catheter  in an ear vein. The catheter was secured using Durapore® tape and superglue. Standard giving sets were used for i/v fluids and filtered giving sets for the administration of blood and blood components . Plasma was collected from adult herdmates under trained behaviour, in lateral recumbency from either an ear vein or a medial hind leg vein, in one litre citrate blood collection bags , stored for a minimum of eight hours at 5 °C and separated using manual pressure. The plasma was then stored at 5 °C and warmed to room temperature prior to administration.Anti-herpes viral treatment consisted of either FCV or GCV. FCV  250 and/or 500 mg tablets were crushed using a pestle and mortar and suspended in hand warm water. Using a 60 ml syringe and a meter long tube, the suspension was administered in the distal colon after removal of all faeces as described above. GCV  was diluted in one litre of physiological saline attached to a giving set and an i/v catheter, and administered slowly over a period of one hour.For measurement of plasma levels of GCV and PCV, the FCV metabolite, whole blood samples obtained approximately 6-10 hours following drugs administration, were centrifuged at 800 × g for 10 minutes and plasma was collected and stored at −20 °C for later analysis. Plasma samples were analysed at the College of Veterinary Medicine, University of Tennessee, USA using a reverse phase HPLC method with solid phase extraction (SPE). The compounds were separated on an Atlantis T3 (4.6 × 250 mm) column with a mobile phase of 10 mM ammonium phosphate pH 2.9 and acetonitrile (95:5). The flow rate was 1.2 ml/min and fluorescence was measured at excitation 253 nm and emission 360 nm. Standard curves for plasma analysis were prepared by fortifying untreated, pooled elephant plasma with the drug to produce a linear concentration range of 10-5000 ng/ml. Average recovery was 100 % while intra and inter-assay variability ranged from 0.5 to 5.1 %. The lower limit of quantification was 10 ng/ml.3 viral genome copies (vgc)/ml , but was normal (51 % of WBC) when tested three weeks later (Table 3 vgc/test reaction. No further samples were tested beyond Day 57.Treatment with 15 mg/kg FCV three times daily (TID) per rectum was instigated eight hours after these initial clinical signs. The first two treatments, six hours apart, consisted of ground FCV tablets suspended in ultrasound gel ; however, most of the drug was expelled at the next defaecation. Twelve hours after the initial presentation, the calf started to voluntarily bend his right foreleg again, and by the next morning, despite some stiffness in the shoulders, the legs were in normal use and its appetite had returned. The dose of FCV was therefore reduced to 12 mg/kg TID (Day 1); this time dissolved in hand warm water and placed 1.5 meters deep into the rectum. All clinical signs were resolved by Day 2, when the blood viral load peaked at 3.61*10er Table . No viruAs a pre-emptive measure, the calf’s two siblings, a 3-year old male and a 4-year old female, were treated simultaneously with 12 mg/kg FCV TID from Day 0 to Day 6, which was tapered to 8 mg/kg BID from Day 7 to Day 12, after which treatment ceased. Neither sibling was positive for EEHV-1 DNA in blood or bodily secretions throughout this period.Plasma PCV levels, measured at trough levels 8-10 hours following the latest FCV administrations in both Case 1 Fig.  and its 2 vgc/ml of blood. At this point monocytes were abundant  and the platelet count was 950*109/L.  and the platelet count almost halved to 560*109 /L, in the absence of clinical signs (Day -5). PCR results for this sample became available two days after the actual sampling date and treatment was immediately instigated at 15 mg/kg FCV TID per rectum. Blood samples were then taken regularly for EEHV-1 qPCR, viral quantification and haematology.In 2015, a blood swab collected during routine weekly sampling of 1.5 year old male calf ‘Max’ tested positive for EEHV-1 DNA by qPCR. This result was confirmed by a subsequent EDTA whole blood sample that tested qPCR positive at 5.0*10L. Table , Day -33Genomic analysis of this EEHV-1 by sequencing of PCR amplicons of partial viral POL, gL and gM genes identified the virus as EEHV-1A genotype. However, comparing these nucleotide sequences to those of Case 1, it was revealed that the virus was genomically slightly different, but both cases were more dissimilar to Riddle and Betts Fig. . Nucleot9/L (17 % of WBC), but platelet counts showed a continuous decline . Due to logistics, GCV (5 mg/ml) could not be administered until 16:00h that day, but achieved plasma levels of 2529 ng/ml following i/v injection. Clinical signs progressed despite treatment and the platelet count further reduced to 13*109/L and viral load increased to 1.9*107 vgc/ml of blood. The calf subsequently died at midnight, just into the next day as a result of hypoxaemia and hypovolaemia.The decision was made to start i/v fluids, consisting of Harmann’s  supplemented with Duphalyte , and complement the treatment from solely FCV per rectum to i/v plasma and GCV. The calf first displayed clinical signs of haemorrhagic disease six days into the DNAemia: oedema of the head and ventral abdomen, tongue petechiae and progressive signs of cyanosis. The platelet count had also reduced drastically at this point to 98*10The early clinical signs of EEHV-HD are non-pathognomonic and often manifested as lethargy, anorexia and lameness. As a consequence of generalized capillary endothelial cell damage, these early clinical signs are likely to be followed by more severe and generally terminal signs , 8, 12. 9/L for platelets [In addition to detection and quantification of viral DNA through real-time qPCR, drop in the monocyte/neutrophil ratio, and absolute reduction of the platelet count, make both the monocytes and platelets the most useful prognostic haematological parameters of EEHV-HD, although the exact physiological process for these changes is as yet unknown. It is possible that the monocytes are subjected to immediate assault of the virus , and thalatelets –35. MonoMonitoring kinetics of EEHV-1A load in the two elephants revealed rapid increase in viral load in the elephant that succumbed to the disease (Case 2) despite continuous anti-herpesviral treatment up to the EEHV-1 clinical signs. From at least three days prior to onset of clinical signs, PCV reached plasma levels suggested as therapeutic for treatment of diseases caused by alphaherpesviruses infections in humans , but herThe efficacy of FCV treatment to curb progression to EEHV-HD in clinical EEHV has always been debatable, as compromised cellular function at enteric level could affect absorption. This was indicated by the measured PCV level in Case 2, which dropped remarkably 48 hours prior to the onset of outwardly visible clinical signs. Factors such as drug expulsion and individual differences in drug metabolism are also considered possible causes of this drop . These fTaking into account the EEHV viraemia sequel following FCV administration in current and previous studies, the efficacy of FCV remains doubtful. Therefore the use of other, i/v administered and potentially more effective, anti- herpesviral therapies should be explored, especially in cases where pre-EEHV-HD viraemia, established through routine blood PCR monitoring, does not appear to be affected by FCV within the first days of treatment. In the absence of comprehensive data on a highly effective and safe anti-herpesviral drug, alternative drugs at present could include i/v administered nucleoside analogues such as GCV and ACV. The second-line drugs could be the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir, but these drugs all have their own safety issues. The most common adverse effect during GCV therapy in humans has been haematological toxicity, but this appears to be readily reversible on discontinuation of the drug . It shouComplementary therapies that are likely to be of value include supportive therapies e.g. maintaining hydration and actions to prevent DIC and shock, such as i/v plasma administration from EEHV antibody positive adult donors, as a source of both antibodies and platelets, and a broad spectrum antibiotic to combat potential concurrent bacterial infections that might possibly be at the root of triggering the viral replication, predisposing to the infection or arise as a secondary infection.www.eehvinfo.org). In elephants that have not attained this stage of training at the time of infection, repeated standing sedations using butorphanol and detomidine are possible, as used successfully in other institutions .Intravenous access appears to be of significant value for supportive treatment and anti-viral drug administration, and is straightforward in trained elephants that are used to being handled. The EEHV Advisory Group convened to debate this in 2014 and currently recommends minimum standards of care for all at-risk elephants irrespective of management systems, which include a section on Calf Training for intravenous access  about 9 months prior to the clinical signs and survived the viraemia . On the 3 vgc/ml of blood for Case 1 and 7.2*106 vgc/ml for Case 2. A previous report states that a threshold of around 1.0*104 vgc/ml of blood had coincided with apparent illness [In this study, a detectable EEHV-1 DNAemia was correlated with the disease symptoms only when the blood viral load was at around 8.16*10 illness . Clinica illness , 18, 19.The EEHV viraemic cases investigated here further highlight the ongoing threat posed by the virus to the juvenile Asian elephants, potentially leading to gradual disappearance of their free-living population and an inability to maintain a viable captive population to safeguard the species from extinction. Therefore, effective control of future EEHV infections is fundamental to improve the survival rate of affected animals. Routine EEHV testing combined with viral quantification, WBC, monocyte and platelet counts following confirmed DNAemia in at-risk elephants should deliver an early warning of the clinical disease and prognosis, providing enough time to evaluate treatment opportunities to avert a fatal outcome. This investigation further questions the efficacy of the rectally administered FCV in controlling the viraemia and urges towards the exploration of alternative therapies. These therapies could include preferably i/v administered anti-herpes viral drugs, complementary plasma transfusions and antibiotics against potential secondary infections.


Ucides cordatus is the main faunal modifier of microtopography establishing up to 2 m deep burrows. They process more than 70% of the leaf litter and propagule production, thus promoting microbial degradation of detritus and benefiting microbe-feeding fiddler crabs. The accelerated nutrient turn-over and increased sediment oxygenation mediated by U. cordatus may enhance mangrove tree growth. Such positive feed-back loop was tested in North Brazil through a one year crab removal experiment simulating increased harvesting rates in a mature Rhizophora mangle forest. Investigated response parameters were sediment salinity, organic matter content, CO2 efflux rates of the surface sediment, and reduction potential. We also determined stipule fall of the mangrove tree R. mangle as a proxy for tree growth. Three treatments were applied to twelve experimental plots (13 m × 13 m each): crab removal, disturbance control and control. Within one year, the number of U. cordatus burrows inside the four removal plots decreased on average to 52% of the initial number. Despite this distinct reduction in burrow density of this large bioturbator, none of the measured parameters differed between treatments. Instead, most parameters were clearly influenced by seasonal changes in precipitation. Hence, in the studied R. mangle forest, abiotic factors seem to be more important drivers of ecosystem processes than factors mediated by U. cordatus, at least within the studied timespan of one year.Mangrove crabs influence ecosystem processes through bioturbation and/or litter feeding. In Brazilian mangroves, the abundant and commercially important crab  Uca spp.) on the growth of mangrove seedlings, small scale experiments with plot sizes of 1 m × 1 m proved to be sufficient [2 plots, has been performed to date in Australia [Burrowing crabs are ecosystem engineers and their importance for sediment processes has been discussed for many years –5, alongfficient . AssessiExclusion of burrowing fiddler crabs from salt marshes led to an increase in meiofaunal density, probably due to reduced competition for food  on the sediment surface ,14. In oMost past research efforts to investigate the ecosystemic role of crabs have focussed on smaller burrowing species and small-scale experiments, and on crabs from the Indo-West-Pacific (IWP). Results from the IWP region are not necessarily representative for the Atlantic-east-Pacific (AEP) system, since flora and invertebrate fauna of the latter biogeographical area is much less diverse .Ucides cordatus (Ucididae). The species is an obligate mangrove dweller, living in up to 2 m deep burrows [U. cordatus occurs at average densities of 1.7 individuals m-2 [-2 [U. cordatus sustains its high biomass by processing more than two thirds of the annual mangrove litter and propagule production in the high intertidal N-Brazilian forest, most of which would otherwise be exported by the tides [U. cordatus assimilates only parts of the energy inherent in the food, a large percentage of the matter remains as leaf fragments, due to sloppy feeding, or as faeces, becoming available for decomposing bacteria.On the Atlantic side of the AEP, mangrove microtopography is dominated by the large burrowing crab  burrows . In Nortuals m-2 . Due to uals m-2 ), it prohe tides ,9. HowevU. cordatus. They further postulated a positive feedback effect on the primary production, since nutrients in the leaf litter are first retained by the feeding activity of U. cordatus and then re-mineralized by bacteria, thereby becoming available to the trees. The authors additionally hypothesized that a significant reduction or loss of one of the model's components would have a negative impact on the remaining components, consequently affecting primary production If, for example, crab numbers decreased significantly in an otherwise healthy mangrove forest, nutrients locked in leaf litter would no longer be retained in the system under this scenario. Mangrove trees would suffer from decreased bioturbation, since e.g. the crabs’ burrows facilitate sediment oxygenation, preventing the formation of phytotoxins such as H2S [2 efflux rates of the sediment, which in turn may drive changes in stipule production.Koch and Wolff  hypothesh as H2S . BurrowiU. cordatus in the mangrove ecosystem, given re-occurring crab population declines in North-eastern Brazil caused by a spreading fungal disease  and disease” . U. cordper year . Due to per year ,28 the sper year .U. cordatus removal experiment in the mangrove forest of the Northern Brazilian Caeté estuary to investigate possible ecosystemic effects of significantly decreased crab numbers, such as would result from a significantly increased fishing pressure. The following sediment parameters were monitored: salinity, organic matter content, CO2 efflux rate of the surface sediment  and reduction potential. To assess impacts on biota, tree leaf production using stipule fall as proxy [We have conducted a one year as proxy  were assU. cordatus leads to 1) increasing sediment salinity, 2) decreasing organic matter content of the sediment, 3) reduced CO2 efflux rates of the surface sediment due to a decrease in organic matter and 4) more reduced conditions in the sediment. Further, we predict that the reduction of U. cordatus will 5) decrease stipule production.We hypothesize that a reduction of the number of Rhizophora mangle L. (Rhizophoraceae). Other mangrove tree species are Avicennia germinans (L.) L. (Acanthaceae) and Laguncularia racemosa (L.) C. F. Gaertn. (Combretaceae) [The study was performed in a mangrove forest in the Caeté estuary, Pará state, North Brazil. Field work permission was granted by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBIO) and the Stakeholder Council of the Extrativist Reserve Caeté-Taperaçu, SISBIO Authorization number: 30007–1. The removal experiment was implemented near the tidal channel Furo Grande on the Ajuruteua peninsula (46°38’W 0°50’S). At the study site, the dominant mangrove tree species is The region has semidiurnal tides with amplitudes of 2 to 5 m ,32. MeanR. mangle trees , with a minimum distance of 15 m in-between them. To reduce variability, plots with similar inundation frequencies were chosen, as indicated by height range of algal growth on the trees' stems, and visually similar sediment characteristics. Each plot was placed around a central mature R. mangle tree. The diameter of central trees  ranged between 18 and 26.5 cm (mean ± standard error: 21.7 ± 0.8), central tree heights ranged between 10–14 m. All above ground stilt roots of the central tree, which roughly mirror the extension of below ground roots [Twelve experimental plots (13 m × 13 m) were established in the high intertidal zone containing exclusively nd roots , were wi2 efflux rates from the sediment) and one biotic parameter ; a detailed description of the respective procedures is given below. Three replicate sampling points were randomly chosen inside each plot during each sampling campaign, but in a distance of at least 15 cm away from U. cordatus burrow entrances and from spots where superficial R. mangle roots touched the sediment surface. This allowed the investigation of potential larger scale ecosystemic changes beyond the immediate neighbourhood of burrows or roots. The selected parameters were measured at all three points  or at a subset of two .Plots were randomly assigned to three treatments . Possible side effects of the crab removal procedure (see details below) were assessed by the disturbance control plots, where crab removal was only simulated, following the approach of Smith et al. . We adde2 efflux rate and reduction potential were measured during eight sampling campaigns. From November 2011 until January 2012 the sampling of the above parameters was conducted monthly, thereafter in intervals of six weeks until April 2012 and after that every two months until November 2012. Stipule fall was assessed by biweekly collection of material in litter traps .The experiment ran from 19/11/2011 until 04/11/2012. Sediment salinity, organic matter, COhttp://www.mar.mil.br, 2012, 80 km northwest from the study site). Salinity and temperature readings of tidal surface water were taken in the middle of the tidal channel Furo Grande, approximately 300 m away from the experimental plots. Readings were taken in the morning and late afternoon of each sampling day.Water pressure data loggers  were employed from 05/03/2012 to 15/03/2012 to determine water levels and calculate inundation frequencies. To extrapolate the data to the entire study period, the pressure data obtained for the 10 days were matched with the tide table for the nearest site from the Brazilian National Oceanographic Database  per sampling day. The capture technique was modified after a technique called “redinha” (tangle-netting), used illegally by crab fishermen in many other parts of Brazil [R. mangle aerial roots  inserted into the sediment in front a crab burrow. Cuttings were rinsed and dried before applying them to minimize leaching into the sediment. The nets were then slightly pushed into the burrow entrances. The following day, crabs entangled in the nets were counted and the carapace width (cm) of all or every second individual (if there were more than 10) recorded. Due to the activity of crab eating raccoons (Procyon cancrivorus) and other predators, captured crabs had frequently been consumed before the tangle-nets could be controlled, indicated by crab remains. These remains were also counted and carapace widths measured, if possible. All survivors were released sufficiently far away from the experimental plots to prevent re-immigration. During the application of nets, care was taken to minimize sediment disturbance, e.g. by using firm stilt roots as walkways. Crab removal started with the first sampling campaign in November 2011 and was conducted biweekly for 3–6 days  for each plot during neap tides over one year. Crab removal was conducted during neap tides, because crabs are more active then, and close their burrows less frequently than during spring tides [-1 net-1). Additional manual removal of crabs moving around freely inside the plots was necessary during mass mate searching events during spring tides [-2) was monitored every 4–5 weeks over the entire study period by counting closed and open burrows in always the same two 1 m × 13 m subplots per removal plot. For the disturbance control plots the applied capture technique was simulated by pushing the mangrove cuttings without nets into the ground, followed by removal of the cutting, and stressing the tree roots by walking over them to a similar extent as in the removal plots.The term “crab removal” rather than of “crab exclusion” is used since no fences or other artificial borders were applied around the experimental plots. This way likely side effects of fencing in this dynamic macrotidal environment, such as changes in sediment deposition and leaf export, were avoided ,13,19. Uf Brazil ,35. Eachng tides . Captureng tides . These e≤ 0°C until further processing. Samples were homogenized and divided into two portions. One portion was used for the gravimetrical determination of the water content of the sediment through weight loss by drying at 104°C. The organic matter content of the dry sample was obtained subsequently through weight loss by combustion at 450°C. The second portion was used to analyse sediment salinity. Two grams of the sediment were mixed with 10 ml of distilled water and shaken for 24 h on a mechanical shaker . Afterwards, the salinity of the sediment extract was measured with a WTW TetraCon 325 conductivity meter connected to a WTW multi-parameter instrument (340i). Sediment salinity was calculated based on the previously measured original water content of the respective subsample [Two sediment cores were taken per plot and sampling campaign with a peat sampler (Eijkelkamp) of 50 cm length and 6 cm diameter. Subsamples of the extracted sediment were taken at core depths of 1, 5, 10, 20, 30, 40, and 50 cm, filled into plastic vials and stored at temperatures ubsample .2 efflux rate measurements of the surface sediment were performed in each of the twelve plots at each sampling date. At each of the three sampling points (see above), two PVC collars of 20 cm diameter were inserted several centimetres into the sediment void of visible roots, U. cordatus burrows and mostly also void of burrows of other crab species. These two collars were handled as replicates for one sampling point. A distance of 40–50 cm was maintained between the two collars at each sampling point, large enough to ensure that the disturbance created by the insertion of one collar into the sediment would not affect the sediment of the other collar and small enough to represent the sampling point. To avoid any influence of CO2 release due to the insertion of the collars, measurements were started 1 h after the installation. An opaque respiration chamber was connected to a CO2/H2O infrared gas analyser  and fitted on top of the PVC collar. The CO2 concentration inside the chamber was recorded for 2 min. The measurement was repeated four times per collar. Between replicates, the chamber was opened for 25 s to release the accumulated CO2. Sediment temperature was measured outside the collar at 2 cm sediment depth by thermocouple (OMEGA Engineering). The CO2 efflux rates were calculated [2 concentration over time. A correction for changing sediment temperature was applied.Six COlculated , assuminThree sediment cores were taken in each plot per sampling date at the three sampling points. Redox potential (± 1.0 mV), pH (± 0.1) and temperature (± 0.1°C) were measured within the sediment cores immediately after their extraction at 1, 5, 10, 20, 30, 40, and 50 cm depth with a Sartorius ORP (redox) combination electrode and a WTW Sentix 41 pH-electrode connected to a WTW portable meter (Multi 340i), respectively.As indicator of the reduction force of a reduction system, the rH value was calculated including the redox potential, temperature and pH value of each measurement . rH valuR. mangle trees is related to the unfolding of new pairs of leaves. It can therefore be used as an indicator for the leaf production of these trees [Rhizophora is known to be influenced by sediment characteristics [Stipule fall of se trees –42. Stiperistics –42 and weristics .2 each) fixed to the stem at 50 cm horizontal distance in 5–7 m height to ensure autochthonous litter. Traps were emptied biweekly. Stipules were separated from other litter components and dried at 104°C to constant weight (g). Stipule dry matter from both collectors was pooled and stipule fall rates (g m-2 d-1) calculated.Litter of the central tree in each plot was sampled with two litter traps  in relation to different treatments, time and their interaction effect. In some models , sediment depth was considered as an additional covariate. Plot and sampling points within plots (when appropriate) were used as random terms to account for the nested structure of the experimental design. When trends over sediment depth or time were not linear, these covariates were set as categorical covariates. To find the optimal fixed terms, stepwise backward model selection was used based on the maximum likelihood ratio test (ML) and/or the Akaike Information Criterion (AIC). When an interaction was part of the final model, all participating main factors were automatically retained. The validity of the models was checked by examining diagnostic plots of residual versus fitted values and residuals versus covariates. Independence was examined by plotting residuals versus time. Final models were presented with the restricted maximum likelihood estimation method (REML).Carapace widths of the captured crabs at each plot were analysed for differences over time with a one-way ANOVA. Linear mixed-effects models (LME) and generalized additive mixed-effects models (GAMM) ,50,44,51Stipule data were analysed with a GAMM model for differences among treatments over time. GAMM's are non-parametric regression models and allow, in the case of the stipule data, for nonlinear trends over time with a smoothing function for the predictor variable time.All twelve experimental plots had similar inundation levels and were flooded during high tide on 131 days out of the 355 days of the study period. This corresponded to a flooding frequency of 14–19 days per month. Surface water salinity at the Furo Grande varied from 22.8 to 36.9 during the twelve months. Lowest salinities were recorded during periods of high rainfall . SurfaceU. cordatus burrows and, at six months of the experiment, around 90–100% of all U. cordatus burrows. In total, 4866 crabs were caught during the one year study, including live crabs (2872), and remains of dead crabs (1994). Additional crab parts scattered by predators within plots of further 1563 captured crabs were counted, but not included in the capture success calculation for a more conservative estimate. During mass mate searching events 844 crabs were additionally caught by hand inside the removal plots. Capture success varied over the year from 0 to 0.2 crabs d-1 net-1 with a higher success during the mass mate searching events between January and March 2012  and 3 , showing that the larger animals (the more efficient bioturbators) were constantly removed. However, it did not differ over the year in removal plot 2  and 4   .p-value = 0.01, No consistent difference between treatments in respect to sediment salinity across depth and time were detected. In months with significant rainfall, salinity was lowest near the sediment surface and increased gradually with depth ; control values were higher than those of the other two treatments for most sampling date/depth combinations  and due to the inclusion of a disturbance control treatment in our experimental design, we can exclude that the findings are procedural artefacts. In fact, here we show for the first time that the mangrove ecosystem of the North Brazilian Amazon may be resilient to reduced U. cordatus numbers within a time span of one year, at least regarding the measured parameters.None of the studied response parameters was affected by the decrease in h et al.  in AustrU. cordatus burrows would significantly desalinise the sediment was based on findings of studies of other species [Uca spp. burrows. However, in contrast to their study which was performed in relatively open plots with small mangrove seedlings, our study was performed in a mature, closed-canopy R. mangle forest , when burrow walls are more oxidized due to contact with atmospheric oxygen. Consequently, carbon oxidation is facilitated by the presence of burrows, resulting in diminishing sediment organic matter [Our hypothesis of decreased sediment organic matter storage due to removal of he tides ,9. In adhe tides ,61. A rec matter . A decrec matter . Lower c2 efflux rates we assumed that crab removal would lead to a decrease in this parameter. Sediment CO2 efflux rates reflect the activity of microbes, and a reduced crab feeding activity would lead to a decrease in substrate availability for these organisms. However, since no changes in the organic matter content of the sediment occurred, it is not surprising that sediment CO2 efflux rates did also not change.Regarding sediment COU. cordatus burrow walls. In a North-Eastern Brazilian area with burrow densities much higher than at our study site , U. cor burrows  in combi-2), also recorded substantial changes in the reduction potential for the upper sediment layer in bioturbated areas [Several other studies focusing on fiddler crabs, with manifold higher burrow densities  resulted in a distinct decrease in stipule fall rate during a one year study period in Australia [U. cordatus caught with nets, Australia: approximately 1500 crabs—mostly Sesarma messa and Sesarma semperi longicristatum—caught with pitfall traps), and our design included four replicate plots, compared to only three in the Australian study. One reason for this outcome could be that our plots contained 6–18 relatively large trees each, whereas the Australian plots contained 52–81 smaller trees. Younger trees with smaller root system extension may react faster to changes in sediment characteristics than more mature trees with high root biomass. Furthermore, the location of the study site along the tidal gradient differed between the two sites. Our study was conducted in the high intertidal, whereas Smith III et al. [Experimental removal of mostly sesarmid mangrove crabs  effects of reduced crab numbers. A different experimental outcome than ours could also be thinkable for areas with higher initial crab densities and/or less pronounced rainfall during the rainy season than in Amazonian. Comparative removal studies involving the same crab species in different environmental contexts would further improve our understanding of the relative importance (and plasticity) of abiotic versus biotic factors as drivers of mangrove ecosystem functioning.At our Amazonian mangrove study site, all measured parameters remained unaffected by the artificial removal of more than 4866 S1 Fig(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.