PDT has been recently proposed as a treatment modality for cancers. A histopathological examination of PDT showed that apparent internal hemorrhage and red blood cell (RBC) extravasation are commonly observed in malignant tumors after PDT. Tumor angiogenesis has been identified as one of the key targets in tumor treatments. In this study, in vivo chicken chorioallantoic membranes (CAMs) and a stimulated malignant oral lesions animal model were used. The velocity and structural images of in vivo microcirculation and the relation between the changes in intrinsic signal in blood components and histopathological responses during PDT were examined for more understanding. Our study demonstrated that PDT can target the microvasculatures within the tumor and cause tumor destruction. The optimization of PDT for squamous cell carcinomas of the oral cavity may depend on the ability to cause the selective destruction of only vascular-mediated events or cellular targets without the production of heat through nonthermal mechanisms.® (2.5 mg/kg) exposed to different laser power densities at 630 nm. The vascular changes, the velocity of the blood flow, the speckle flow index (SFI) of fluorescence changes, and ultrastructure damage in the microvasculature before and after PDT were recorded. The subcellular localization of Photofrin® revealed satisfactory uptake throughout the cytoplasm of human red blood cells at 10 s and 20 s before PDT. The mean blood-flow velocities of the veins and arteries were 500 ± 40 and 1500 ± 100 μm/s, respectively. A significant decrease in the velocities of the blood flow in the veins and arteries was detected in the CAM model after PDT. The veins and arteries of CAMs, exposed to the power densities of 80, 100, and 120 mW/cm2, had average blood-flow velocities of 100 ± 20, 60 ± 10, and 0 μm/s and 300 ± 50, 150 ± 30, and 0 μm/s, respectively. In the stimulated malignant oral lesions animal model, the treated tumors exhibited hemorrhage and red blood cell extravasation after PDT. The oxyhemoglobin and total hemoglobin levels decreased, which resulted in a decrease in tissue oxygen saturation, while the deoxyhemoglobin levels increased. PDT using Photofrin® has the ability to cause the destruction of the targeted microvasculature under nonthermal mechanisms selectively.The objective of this study is to use imaging and histopathological analysis to characterize and monitor microvascular responses to photodynamic therapy (PDT). In vivo chicken chorioallantoic membranes (CAMs) and a stimulated malignant oral lesions animal model were used to determine the blood flow and the biological activities of Photofrin Photodynamic therapy (PDT) has been recently proposed as a treatment modality for cutaneous malignancies, which may offer results that are more appealing than those obtained using conventional treatment modalities. The fundamental concept of PDT is that some molecules can function as photosensitizers. The presence of photosensitizers in biological tissue makes the tissue vulnerable to the wavelengths of light absorbed by the chromophore. The stoppage of the blood flow to tumors shortly after the initiation of PDT has been demonstrated; however, the complete cessation of tumor circulation is required to achieve complete eradication. A histopathological examination of PDT showed that apparent internal hemorrhage and red blood cell (RBC) extravasation are commonly observed in tumors after PDT ,2.Our previous study used hematopoietic progenitor antigen CD36 to characterize human dermal microvascular endothelial cells (MECs), and the rationale for extending the use of PDT is that PDT facilitates the destruction of targeted blood vessels within the tissue without producing heat [® containing porfimer sodium  is a partially purified preparation of the photoactive ingredients within hematoporphyrin derivative (HPD). Photofrin® obtained from Quadra Logic Technologies, Inc.  was stored in dark at 4 °C. Photofrin® powder was prepared with 5% dextrose to a final concentration of 2.5 mg/mL. An intraperitoneal (IP) injection of Photofrin® at a concentration of 2.5 mg/kg was administered to the embryo of CAM models [®, the CAM models were illuminated with a laser beam (λ = 630 nm). An intravenous injection of Photofrin® at a concentration of 2.5 mg/kg was set for the hamsters with stimulated malignant oral lesions. No photosensitivity or side effects were observed. Then, 3 h after intravenously injecting the hamsters with Photofrin®, they were treated with a laser beam (λ = 630 nm).PhotofrinLaser illumination was performed with a Coherent Innova 20 argon ion laser , pumping a Coherent 599-01 dye laser, and was tuned to emit a lower level laser at 630 nm for each experiment. The wavelength was verified using a Jobin Yvon 5/354 UV monochromator . Illumination was coupled with a 400 mm fused silica fiber optic with a Spectra-Physics Model 316 fiber optic coupler . The output end of the fiber had microlens at its terminal that focused the laser passing into a circular field of uniform light intensity. The laser emitted from the fiber was monitored using a Coherent Model 210 power meter before and after illumination.The chicken chorioallantoic membrane (CAM) is a fitly biological model in this research. Because the CAM microvasculature is located in a transparent matrix, direct viewing and noninvasive imaging of the blood vessels are possible ,6,7. The® at a concentration of 2.5 g/kg. Subsequently, 3 h after the IP injection of Photofrin®, the CAMs were illuminated with a laser beam (λ = 630 nm). Prior to PDT, the window of the CAM was covered with a metal shield with a circular hole (1.5 cm in diameter) that was to be exposed to illumination. The total laser energy density was 100 J/cm2 at power densities of 80, 100, and 120 mW/cm2. The control groups underwent Photofrin® or laser illumination treatment only. At the time of illumination, the CAM was illuminated with a cold white-light fiber-optic source  and placed under a stereomicroscope  equipped with a video camera , which provided a magnification of 70× on a color monitor .Twenty CAMs were given an IP embryo injection of PhotofrinRecently, laser speckle imaging (LSI), in which coherent light incident on a surface produces a reflected speckle pattern, is related to the underlying movement of optical scatterers, such as red blood cells, indicating blood flow. The corresponding image-processing algorithms can be applied to produce the speckle flow index (SFI) maps of the relative blood flow . In this® (2.5 mg/kg body weight). Of these 16 hamsters, 4 received photosensitizers without laser illumination (control group 2). The remaining 12 hamsters received Photofrin® with laser illumination. Then, 3 h after the injection of the photosensitizer, the hamsters were exposed to the laser beam (λ = 630 nm). General lurance-oxygen gas anesthesia was used for all invasive procedures. To increase the specificity of laser illumination on the tumor, a metal shield with a circular hole was used to expose an illuminated area corresponding to the size of each tumor. The total laser energy density was 100 J/cm2, with a power of 100 mW at power densities of 100 mW/cm2 for each tumor. The 12 hamsters were euthanized immediately (n = 3), 3 h (n = 3), 6 h (n = 3), and 12 h (n = 3) after PDT, and the tumors were processed for the evaluation of histopathological destruction by using hematoxylin–eosin staining. Here, five specimens of each tumor were processed.Twenty-four male Golden Syrian hamsters were included in our experiment. Their age was about 3 months, and body weights ranged between 90 g and 115 g, with a mean weight of 105 g. 0.5% D.M.B.A  was brushed onto the cheek pouches bilaterally. The stimulated wounds were accomplished using a camel-hair brush. In each animal, both pouches were painted daily for 2 weeks [2), deoxyhemoglobin (Hb), and total hemoglobin (HbT) concentrations, as well as tissue oxygen saturation (StO2), were analyzed in 4 out of the 24 hamsters during tumor growth and after receiving the photosensitizer with laser illumination for PDT. In this study, we applied a typical type of pO2 measurement device, the OxyLite system , to measure the partial oxygen pressure (pO2) in tumor sites, where this method has been applied successfully to many human tumors in the clinic. The animals were treated in accordance with the ARC guidelines stipulated by the Institutional Animal Care and Use Committee at the Chang Gung Memorial Hospital.The correlated intrinsic components such as the tumor oxyhemoglobin  expressed in arbitrary units (a.u.), the subcellular localization of Photofrinand 20 s A,B. The M system . Because® during the PDT of the tumor, respectively, where the tumor area is marked with the symbol (*). On the other hand, the histopathological findings and fluorescent images (100×) of the stimulated squamous cell carcinoma on the buccal mucosa of the hamsters were identified (n = 4) or the photosensitizer (n = 4) without laser treatment, the treated tumors exhibited minimal hemorrhage and red blood cell extravasation 3 h after PDT. Internal hemorrhage and red blood cell extravasation were observed 6 h after PDT. Severe internal hemorrhage and red blood cell extravasation with the destruction of stimulated squamous cell carcinoma (200×) were observed 12 h after PDT (2) and total hemoglobin (HbT) increased, whereas that of deoxyhemoglobin (Hb) decreased, which resulted in an increase in tissue oxygen saturation (StO2). The blood flow in the tumor increased with tumor size growth (2). After PDT, the treated tumors exhibited hemorrhage and red blood cell extravasation after PDT. The oxyhemoglobin and total hemoglobin levels decreased, which resulted in a decrease in tissue oxygen saturation, while the deoxyhemoglobin levels increased. Changes in the concentration of the intrinsic components based on time after PDT are shown in Based on OCT imaging, senior pathologists confirmed the histopathological findings of stimulated malignant oral lesions as squamous cell carcinoma through tissue evidence and the received PDT in the 24 hamsters. entified A,B. In tfter PDT C,D. As te growth . These r1O2). Illumination at the appropriate wavelength absorbed by the photosensitizer provides the energy to drive photodynamic reactions without the generation of heat. The provided incident power density is <100 mW/cm2. The phototoxic reaction is a local phenomenon that occurs within the same cell on a time scale of microseconds [Photosensitizers absorb photons of an appropriate wavelength and are promoted to an excited state. The excited photosensitizers subsequently react with a substrate, such as oxygen, to produce highly reactive singlet molecular oxygen that causes irreversible oxidative damage to biologically crucial molecules ,13,14. Toseconds . Optimaloseconds ,16.® application caused effective vascular photosensitization, which would possibly result from the cellular events of endothelial cells [® in CAMs [®, as a photosensitizer, was found primarily in the plasma membrane, which suggested that an apparently nonapoptotic mechanism of cell death was involved photochemically [® located throughout the cytoplasm was highly susceptible to functional inhibition via PDT. We, therefore, believe that PDT-induced damage to mitochondrial function and lysosomes could be the major factor responsible for the effectiveness of PDT [Photofrinal cells ,18,19. Hemically . Howevers of PDT ,23,24,25® in human RBCs for the analysis of the mean blood-flow velocities in the veins and arteries.In the evaluation of the response of microvasculatures, one of the findings observed in our study was the high vulnerability of arterioles to PDT injury. A possible explanation might be based on considerations of vascular anatomy. This occurred for the three vessel calibers. The arteriolar walls consist of three concentric layers: an endothelial tube, an intermediate layer of smooth muscle cells, and an outer coat of fibrous elements. The thickness of the arteriolar wall varies with vessel caliber and function. On the other hand, the walls of the venules are always thinner than those of arterioles of equal caliber. In PDT, we might anticipate less damage to the thicker, more resilient arteriolar walls. This seems to be consistent with reports of PDT-induced vasoconstriction followed by vasodilatation. However, notably, coagulated blood emboli consisting of agglutinated damaged RBCs can be transported downstream to reduce the blood flow. The occlusion can be in arterioles because blockage occurs before they reach the capillaries and result in a decrease in blood flow and tissue oxygen saturation (StO2) . PermaneOur previous study identified four PDT-induced vessel damage grades: 0, no observable damage; 1, slight damage, vasodilatation/constriction, and temporary occlusion; 2, moderate damage and permanent occlusion; 3, severe damage, capillary extravasation, and hemorrhage . Because2) and total hemoglobin (HbT) increased, while deoxyhemoglobin (Hb) decreased, which resulted in an increase in tissue oxygen saturation (StO2). These results implied that the tumor angiogenesis induced an increase in the blood flow and blood volume (HbT), mainly by increasing the arterial blood (HbO2). After PDT, marked PDT-induced destruction vasculature with hemorrhage and RBC extravasation was observed in our histopathological examinations. These findings were consistent with the changes in the concentration of intrinsic components based on time after PDT  primary mucosal malignancies of the upper aerodigestive tract (UADT) is surgery and/or radiotherapy. Recently, promising results have been reported using PDT on complicated malignant lesions. The in vivo stimulated malignant oral lesions model in our study demonstrated that the blood flow in the tumors increased as the tumors grew. As the tumors grew, oxyhemoglobin (HbOfter PDT . Our resfrom PDT . In addi® can target the microvasculature within the tumor and cause tumor destruction. Continuing improvement in treatment results will depend on the ability to cause the selective destruction of only vascular-mediated events or cellular targets without the production of heat through nonthermal mechanisms. The optimization of PDT for squamous cell carcinomas of the oral cavity may extend to more fields for the application of PDT to tumors in other anatomic areas or of other histopathological patterns.In conclusion, PDT using Photofrin


Volcanic activity provides a unique opportunity to study the ecological responses of organisms to catastrophic environmental destruction as an essential driver of biodiversity change on islands. However, despite this great scientific interest, no study of the biodiversity at an erupting volcano has yet been undertaken. On La Palma (Canary archipelago), we quantified the main species affected and their fate during the 85-day eruption (September–December 2021). Our main objective consisted of monitoring the biodiversity subjected to critical stress during this volcanic eruption. We found that all biodiversity within a 2.5 km radius was severely affected after the first two weeks. It is challenging to assess whether volcanism can drive evolutionary traits of insular organisms. Examples are the adaptation of an endemic conifer to high temperatures, selection of functional plant types—secondary woodiness—, effects of the disappearance of invertebrates and their influence in trophic nets and vertebrate trophic plasticity. However, our data suggest that such previous evolutionary changes might continue to favour their resilience during this eruption. Lastly, it is a very good opportunity to assess the extent to which these periodic volcanic catastrophes may constitute temporary windows of repeated opportunities for the evolution and speciation of oceanic island biota. Since then, volcanic eruptions of different magnitudes and duration have been key factors in extinctions and evolution at local and global scales. Repeatedly, ecosystems have been modified substantially, and species populations were selected, thus stimulating speciation3. Most of the seminal studies on “volcano ecosystems” (sensu3) have been focused on community succession after an eruptive event such as the cases of Islas Revillagigedo (San Benedicto), Krakatau, Long Island (Papua New Guinea), Mount St Helens and Surtsey (see5).Volcanism has long been operating on Earth3. Vegetation is affected by tephra fall, including foliage abrasion and loading, burial, and chemical toxicity. Later, coarse or fine-grained tephra deposits can cover or damage surviving vegetation and inhibit photosynthesis and the emergence of buried seed sprouts. Animals die when subjected to extremes of temperature, impact force or burial due to lava flows, pyroclastic density currents, lahars or deep tephra (> 50 cm), in almost all circumstances3.Research at iconic volcanoes, such as those mentioned, has influenced the understanding of ecosystem dynamics and ecological responses to eruptive events. Effusive eruptions of lava often result in the disappearance of pre-existing ecosystems due to burial and extreme temperatures. Volcanic disturbances rarely result in the complete disappearance of biota, since survival is common, ranging from the persistence of most biota to the survival of only a few individuals of some species in isolated refugia6. Some studies have been tremendously influential in understanding ecosystem dynamics, such as long-term documentation after the cataclysmic 1883 Krakatau eruption4. However, quantitative and systematic ecological research during a volcanic eruption has been lacking.Unlike other volcanic eruptions whose impact on ecosystems could not be studied because they were either too large/hazardous (Pinatubo) or continuously ongoing , La Palma in 2021 had the advantage of being a discrete time-limited event, which allowed it to be intimately monitored. Therefore, this recent eruption in the Canary archipelago offers a unique opportunity to investigate (i) fundamental processes directly connected with an effusive—moderately explosive eruption (VEI = 3) and (ii) their impacts on oceanic island ecosystems and their terrestrial biodiversity, characterized by their high degree of endemicity8.Here, we specifically study the impact of the recent (Sept–Dec 2021) eruption on the biodiversity of La Palma, an island declared a World Biosphere Reserve in 2002. This island harbours a rich biodiversity at a regional and national scale. Indeed, more than 5979 terrestrial species  have been listed in its territory, including at least 904 vascular plant species, 2741 arthropods, and 62 vertebrates3 in volume and 177 m high9. About 177 Mm3 of lava flows were also produced, covering 12.41 km2 , causing stress conditions in the surrounding ecosystems, for instance through the emission of approximately 1 Mt of sulphur dioxide between September and December 202112.On 19 September, 2021, a volcanic eruption began in the southwest of La Palma, in a zone called “Hoya de Tajogaite” on Cumbre Vieja volcanic ridge  Is the dominance of particular plant functional types on oceanic islands linked to or even favoured by volcanic activity? (4) How strongly affected was the invertebrate community, on which many saurians and small passerines depend? (5) How important was trophic plasticity for some vertebrates during this volcanic process? (6) Do body size and mobility capabilities control the responses of vertebrates? Finally, (7) to what extent can regular volcanic catastrophes constitute time-windows of opportunities for the evolution and speciation of oceanic island biota?This study was an excellent opportunity to evaluate the influence of an eruption on the rich legacy of insular biodiversity by examining the vicinity of craters and lava flows. Indeed, we put forward some interesting research questions that had not hitherto been tackled in real-time, listed as follows. (1) At what time and geographical scale (distance from crater) did specific groups of plants and their populations collapse? (2) To what extent are key insular species like the endemic Canary pine, Our activity consisted of monitoring the biodiversity subjected to critical stress due to the eruptive process. The main aims of this work comprised spatial and temporal assessments of (1) plant species cover and vitality, (2) trends in the abundance of invertebrates on soil and plants, (3) patterns in the density and behavioural responses of terrestrial vertebrates .10, 165 ha (13%) hosted vegetation classifiable as pine forest, 826 ha (67%) as thermophilous shrubland and 250 ha (20%) as xerophytic scrub. The prevailing winds were from the north-east, corresponding to the predominant trade winds  mostly impacted the pine forest, the ecosystem in which the volcano arose , 42 invertebrates 12 endemic) and 27 vertebrates (3 endemic). Of the total 1241 ha covered by lava flows2 endemicP. canariensis) habitat reached about 7 km southwards of the volcano and 1.5 km to the north (Table Usnea articulata) are normally abundant on Canary pines and resisted well until the 12th week of the eruption. In contrast to lichens, ferns (Pteridium aquilinum) were the first organism clearly affected during the first week.Impact on the endemic Canary pine  endemic to the pine forest habitat were severely affected during the fourth week, coinciding with the first emission of ultrafine tephra. While C. symphytifolius had its leaves perforated by the falling pyroclasts, Ch. prolifer lost its leaves, thus limiting photosynthesis to its green stems. Contrarily, the woody thermophilous vegetation  located within the 2.5 km closest to the crater appeared deeply affected after the seventh week. In the xerophytic scrub (succulent plants), many plant populations disappeared completely under lava flows, such as the archipelago endemic Euphorbia canariensis, and others showed severely reduced populations, like Euphorbia balsamifera or Sideritis barbellata.The eruption began at the end of the warm dry summer season, of importance to flowering plants because they were probably at their most vulnerable and delicate. In the nearest 200 m to the lava flows, plant cover was clearly higher in the pine forest (83.1%) compared to the other two main habitats (thermophilous shrubland: 20.7% and xerophytic scrub: 21.8%). In the same 200 m band, a high percentage (91%) of undergrowth plant species were affected during the first month of the eruption in pine forest plots located in the same altitudinal band as the crater. However, this effect gradually decreased when habitats and plants were situated further from the crater, in both thermophilous shrubland (79%) and xerophytic scrub (25%)  were more abundant, and they were frequently observed nearby or flying within the volcanic plume. Concerning the time sequence in the most abundant species, numbers clearly decreased during the first two weeks  but small passerine birds and Eurasian collared doves (Streptopelia decaocto) instead, with six attempts recorded during this period. Furthermore, it struck our attention that small passerines like Sylvia/Curruca spp. maintained their territories until the arrival of the lava flows. The only native mammal recorded was the endemic insectivorous bat Pipistrellus maderensis. Its presence in the 2.5 km area decreased after the first two weeks of volcanic activity, being relatively frequent during the whole eruption period only in the distant coastal banana plantations .Birds were also affected by falling pyroclasts and tephra during the eruption. Their numbers were halved within 2.5 km of the crater, affecting communities (mainly Passeriformes and Columbiformes) typical of the pine forest and partially those of the thermophilous habitat Fig. . By conteks Fig. . ParticuTo our knowledge, this is the only work done on the terrestrial biodiversity status in the direct vicinity of a limited duration volcanic eruption. In this contribution, we document and assess the impact on the main plant and animal groups within the ecosystems during a volcanic eruption Table . While s14, harbouring a unique ecosystem of oceanic island organisms (38% of the Canary archipelago endemicity). Islands indeed exhibit a disproportionate amount of the world’s biodiversity but unfortunately a high number of extinctions have also occurred there14. The biodiversity in the south of the island is poorer than in the north. This is probably explained in part by the relatively frequent volcanic activity featuring seven major eruptions since 1585, including this one in 2021 (see15), which led to alternating destruction and neo-colonization processes.This study is particularly important because of its location in a Mediterranean biodiversity hotspotConcerning the flora, the Canary pine forest was the most affected ecosystem and vegetation type, as it is dominant in the vicinity of the new volcanic vents. The southern slopes of this forest were the most disturbed area due to the location of the volcano, combined with the prevailing northeasterly trade winds Fig. . Tephra 12, and short episodes of acid rain. However, this conifer shows high resistance to temperature, confirming its great adaptation to volcanic events16, which is probably also one of the keys to its resistance to the more frequent present-day wildfires17. This pine species has evolved among volcanoes for the last 13 My16 and has adapted successfully to high temperatures. Moreover, thunderstorms with lightning occur in the Canaries together with abundant rainfall; consequently, wild forest fires should presumably not have been so frequent in the island’s past, before human colonization. In this habitat it is also remarkable that epiphytic lichens (U. articulata) apparently resisted on the pines until the 12th week, considering their high sensitivity to anthropogenic pollution18.The Canary Island pine was thus notably affected by tephra fall, sulphuric acid aerosol19. The nearest individuals to the crater were most directly affected by intense tephra falls and concentrated volcanic gases . However, plants located in the nearest 200 m to the lava flows but at more than 2 km from the crater were presumably more disturbed by the high temperature of the slow-cooling lava and its lesser gas emissions.The life cycle of flowering plants was drastically disrupted due to all the above factors, with great impact on foliage, photosynthesis, and growth. However, soil changes due to the deposition of tephra and its lixiviation by rain is one of the most dramatic factors affecting plants and a long-term impact of volcanic eruptions19). In the Hekla area (Iceland), most trees have thickened trunks, indicating that those trees that survive have had a long life subjected to frequent volcanic damage19. Secondary woodiness of island plants (sensu20) has been traditionally related to drought21, ecological shift22 or a counter-selection of inbreeding depression in founding island populations23. However, this adaptation also favours the resistance of many shrubby plants to high temperatures close to craters and lava flows but primarily their resistance to the intense tephra falls that affect a much larger area. In addition, plant and stem height plays a fundamental role in overcoming the deep layers of deposits. This latter effect was particularly important up to 2.5 km from the crater (tephra thickness > 30 cm) , Echium (E. brevirame), Euphorbia , Aeonium (A. davidbramwellii), Rubia (R. fruticosa), Schizogyne (S. sericea), Carlina  or Sonchus (S. hierrensis)  Table , which c28 and water loss due to tegument abrasion29. In this period, many insect pests  in banana plantations (farmers’ observations) were drastically reduced. This sudden decrease in insect populations affected the whole food web and probably caused part of the ecological collapse of saurian and some passerine communities30. In the case of lizards, smaller individuals seem to resist the adverse conditions better than large ones, as observed in other eruptions3. This could be linked to their lower food requirements and greater ease in finding refuges. Loss of body condition in lizards post-eruption has been recorded and negatively affects reproduction quality31. However, some lizards have shown a good ability to find food in the tephra substrate32. We found abundant tephra particles in some vertebrate droppings  during the eruption, probably involuntarily ingested. At least in bats, ingestion during feeding produces physiological stress that is likely related to baldness, high ectoparasite loads or possible mineral deficiencies33.With regard to the fauna, the invertebrate community collapsed during the first two weeks Table , probabl34). During the eruption, Sardinian warblers  maintained their territories until the imminent arrival of lava flows. Larger birds  were well able to continue flying in the areas surrounding the crater. Furthermore, some cases like F. tinnunculus showed great feeding plasticity in the first couple of weeks. At least six times, kestrels tried to catch birds , contrary to their usual diet based on abundant lizards and insects35. Widening of trophic niches in island organisms has traditionally been interpreted as linked to disharmony in island ecosystems38. However, this plasticity is tremendously beneficial in ecological catastrophes, where food becomes exceptionally scarce. In the case of bats, their flight is limited by the delicate structure of their patagium, which can be damaged by the frequent pyroclastic tephra fall. Furthermore, scarcity of insects in the first few kilometres from the crater probably led to their displacement to other more distant and richer food resource zones.As described in the Canary Islands, some passerines show high fidelity to their territories  to volcanism (see16), including extremely harsh ecological conditions. Other insular trends related to the prevalence of woodiness in insular flowering plants21, or the high trophic plasticity of some vertebrates on oceanic islands36, have not previously been associated with their potential evolution along with volcanic processes. However, such evolutionary adaptations most likely played an important role in the survival of plants and animals affected by the volcano. For this reason, it is worth considering and debating whether these previously mentioned evolutionary processes are in fact also linked to repeated volcanic episodes on oceanic islands.Lastly, during this destructive event on La Palma, we had the opportunity to increase our knowledge of how ecological-evolutionary adaptations have favoured the survival of insular organisms. Such responses are traditionally mentioned in the context of island biology. As already mentioned, one of the most interesting findings verifies the remarkable adaptation of Canary Island pine trees (2) with high  and very steep topography. The humid north-east trade winds influence its climate, altitude and orientation. These prevailing air-currents provide conditions for highly heterogeneous environments and bioclimatic vegetation series, from coastal xeric to high mountain shrubs, via thermophilous, laurel and pine forests39.La Palma is a medium-sized island , over which the subaerial edifice developed, mainly after 1.8 Ma41. The aerial shield suffered from substantial gravitational instability that culminated in major flank collapses at ~ 1.1 Ma and ~ 560 ka41. Since the end of the intense post-collapse Bejenado activity at ~ 500 ka42, the volcanic activity was along the new Cumbre Vieja ridge, with the oldest exposed rocks dated at ~ 150 ka42. More than 80 relatively recent volcanoes can clearly be distinguished in all this area, indeed a total of 6 eruptions are recorded in historical chronicles (since the fifteenth century)15.The new volcano erupted in the southwestern part of La Palma Fig. , at Hoya43, increasing with altitude. In general, biodiversity could be considered relatively poor there, due to previous recent intense eruption activity. Three main habitats are represented, 1) xerophytic scrub (0–250 m a.s.l.), 2) thermophilous shrub (300–750 m a.s.l.) and, 3) pine forest (750–1200 m a.s.l.) , about 12 breeding birds and one bat (Pipistrellus maderensis) endemic to the Canary and Madeira archipelagos. In addition to this native fauna, mammalian fauna introduced by humans inhabit the area, such as: mice, rats, rabbits, and feral cats.The affected area includes an altitudinal cline from 0 to 1200 m a.s.l. and an annual rainfall range between 300 and 1300 mmWe studied the most important events affecting biodiversity during the three months of this eruption. A total of 32 original sample stations were studied at the beginning of the eruptive process, from the immediate vicinity of the main crater to the sea (lava delta) see Fig. . To follPEVOLCA). Furthermore, we closely observed areas within 200 m of the volcanic lava flows, including their advancing front and the flanks. Maintaining this routine, 32 workstations surrounding the expanding terrestrial area of the eruption were censused and monitored, from the main crater down to the sea shore.Our inventory quantified the species of flora and fauna in those areas; flowering plants, lizards and birds were intensively monitored as the most important groups in biomass represented in the ecosystem. A more approximate count and follow-up of insects and bats was also carried out. We worked up to a proximity of 1 km from the crater, following the security guidelines set by the Volcanic Emergency Plan of the Canary Islands  from each contact and whether they were heard or seen in the first 3 h after sunrise. Lizards were counted from 3 h after sunrise until 3:00 p.m. in a 2 × 30 m band within the plot and assigned a body size according to snout-vent length . Both censuses were done in the same plots as plants. Invertebrates were inventoried and followed up by 30 min visual surveys in a 50 × 50 m plot covering all three habitats. The number of plots in the respective habitats corresponded to their relative surface area. Only invertebrates on plants and soils or in flight were recorded, because under-rock invertebrates disappeared below the lava flows and falling tephra. There was a previously available dataset for this animal group from this zone and season due to recent work by one author (RG). To compare these previous data  with those obtained during the eruption, we adopted the same criteria. At the same time, information was gathered on the natural history of the area, which allowed us to verify the changes that occurred in animal behaviour, the state of plants and each interaction between animals and plants.44. We compared the lizard abundance during each two-week period in both the zone up to 2.5 km from the crater and beyond this distance. The same methodology was applied to the six most abundant bird species.Categorical data analyses (Chi-square) were carried out using the statistical package R 4.0.2.12). This campaign was undertaken a few days before the end of the eruption, between 30 November and 5 December. The dispersion direction of the ash plume was provided daily by the PEVOLCA and is reported in the inverse wind rose (Fig. To collect further basic data to understand particular processes affecting biodiversity, more than 150 tephra thickness measurements were performed along and across the main propagation direction of the eruption plume (Weiser et al.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Faecalibacterium prausnitzii), axenic clinical isolates of proinflammatory bacteria from CD patients , and the consortia of mucosa-associated microbiota recovered from multiple Crohn’s disease patients. Bacterial growth of the axenic isolates was evaluated using a habitat-simulating medium supplemented with either sodium sulfite, aluminum silicate, carrageenan, carboxymethylcellulose, polysorbate 80, saccharin, sucralose, or aspartame, intended to approximate concentrations found in food. The microbial consortia recovered from post-operative CD patient mucosal biopsy samples were challenged with either carboxymethylcellulose and/or polysorbate 80, and the bacterial communities compared to unchallenged consortia by 16S rRNA gene amplicon profiling. Growth of all F. prausnitzii strains was arrested when either sodium sulfite or polysorbate 80 was added to cultures at baseline or mid-exponential phase of growth, and the inhibitory effects on the Gram-negative bacteria by sodium sulfite were conditional on oxygen availability. The effects from polysorbate 80, saccharin, carrageenan, and/or carboxymethylcellulose on these bacteria were strain-specific. In addition to their direct effects on bacterial growth, polysorbate 80 and/or carboxymethylcellulose can drive profound changes in the CD mucosa-associated microbiota via niche expansion of Proteus and/or Veillonellaceae – both implicated in early Crohn’s disease recurrence. These studies on the interaction of food additives with the enteric microbiota provide a basis for dietary management in Crohn’s disease.Food additives have been linked to the pro-inflammatory microbial dysbiosis associated with Crohn’s disease (CD) but the underlying ecological dynamics are unknown. Here, we examine how selection of food additives affects the growth of multiple strains of a key beneficial bacterium ( The bacterium has also been shown to produce other “anti-inflammatory” factors11–13 promoting epithelial cell integrity and immune tolerization. The expansion of the relative abundance of Proteobacteria-affiliated lineages in IBD has been associated with both host genetics14 and changes in nutritional ecology favoring the metabolic capabilities of at least some of these bacteria.15 In that context, we have used a novel ex vivo combination of microbe culture with metagenomic sequencing (MC-MGS) for use with tissue biopsy samples16 and recovered axenic isolates of Proteobacteria, including urease-positive isolates of Proteus mirabilis, Morganella morganii, and Klebsiella pneumoniae from post-operative CD subjects, which substantiates the identification of urea metabolism as a key guild within the dysbiotic microbiome associated with CD.15 Although longitudinal studies of IBD in the clinical setting are relatively scant, the presence of F. prausnitzii in post-operative CD patients is associated with remission at six months.17 We have also established that concomitant low F. prausnitzii abundance and detectable Proteus spp. is associated with a significantly heightened risk of disease recurrence.18 Indeed, Mondot et al.19 proposed that Proteus spp. – of the bacterial family Morganellaceae20 – be considered a biomarker for post-surgery CD recurrence and our recent studies have further validated that Proteus mirabilis can be a key bacterium driving CD inflammation.21 In that context, Morganella morganii can also be recovered from CD mucosa-associated microbiota22 and Machiels et al.23 have also recently reported that CD patients that suffer early post-operative recurrence possess elevated abundances of the Negativicutes and Fusobacteria on ileal mucosa. In summary, the gross imbalances in microbial taxa that typify CD dysbiosis are linked with genetic and/or ecological alterations that can manifest in disruptions to gut homeostasis, and key bacterial taxa affecting predisposition to CD remission or recurrence have been identified. However, the ecological drivers of these changes in microbial community dynamics are still largely unknown, and a key knowledge gap to improved CD treatment and management.The microbial dysbiosis considered a hallmark of IBD has long been recognized to be typified by reduced relative abundances of Gram-positive 24 highlighted how diet and dietary components needed greater consideration as a trigger as well as a treatment of digestive diseases and disorders. The industrialization of food production and its diversification has influenced consumer choices, reflected in “Western diets” with reduced amounts of whole grains, plant-based non-starch polysaccharides, and other phytochemicals all known to promote gut microbial diversity. Processed foods also utilize various chemistries to minimize spoilage and extend shelf life, to enhance flavor, texture, and sensory properties of foods25–27 and/or as a substitute for other allergenic dietary components 28. These chemistries are holistically referred to as food additives and their impacts on the physicochemical and ecological characteristics of the stool microbiota has emerged as a new facet in the study of diet × microbiota interactions in the context of IBD onset and recurrence.29–31 However, and given that IBD is a “disease of tissue”, virtually nothing is known of how food additives might impact those bacterial taxa resident on mucosal tissue, and particularly for subjects with Crohn’s disease.In that context, Levine et al.(F. prausnitzii) and clinical isolates of proinflammatory mucosa-associated bacteria  from CD subjects. We have also used the parent consortia CD mucosa-associated microbiota (CD-MAM) from our earlier study16 to further characterize how polymicrobial communities are affected by their cultivation in the presence of low concentrations (0.1%) of the two emulsifiers widely used in previous studies: polysorbate 80 and carboxymethylcellulose.Here, we present our findings of the effects from common food additives on the growth of axenic isolates representing protective F. prausnitzii strains during their culture with either M2G, or with M2G also containing 0.1% concentrations of the different food additives tested, are shown in F. prausnitzii strains were incapable of growth when glucose was omitted from the basal medium, suggesting none of the food additives alone could support growth (data not shown). For all three strains, both sodium sulfite and polysorbate 80 were found to strongly inhibit growth (F. prausnitzii strain A2-165 when cultured in the presence of the other food additives tested (p > .05). For F. prausnitzii strain AHMP21, the growth profiles suggest that saccharin and perhaps carrageenan impose some metabolic burden on this bacterium, reflected in the reductions in maximal yield compared to the other cultures, although the doubling times were not significantly different to those produced from the control cultures . Collectively, these results suggest that sodium sulfite and polysorbate 80 both have a profound inhibitory effect on F. prausnitzii growth, and while the other food additives alone cannot support the growth of these strains, the artificial sweetener saccharin may impose a metabolic burden on members of some F. prausnitzii phylogroups C (KLE-1255), reflected in the small but significant increases in its doubling times compared to the other F. prausnitzii strains examined.The growth patterns and calculated doubling times of the three t growth . Overalls tested , p > .05cultures . Overalls strain , p < .05F. prausnitzii strains growth were arrested for the monitoring period (8 hours) whereas those cultures receiving water alone or no additions showed continued growth over the same period  or saccharin (increased doubling time). Interestingly though, while the doubling time of the P. mirabilis ProJ1 was uniformly reduced under microaerobic conditions, there appeared to be no additional effects on the growth rate of the bacterium (positive or negative) from any of the food additives tested  by the addition of carrageenan and while the aerobic growth of strain CD33-7 appeared to be unaffected by the other food additives tested, polysorbate 80 did appear to stimulate  the aerobic growth of strain CD34-4. Under microaerobic conditions, saccharin, and sodium sulfite both negatively affected the growth of both strains; and carrageenan had a negative effect on the growth of strain CD34-4. Sucralose appeared to have no effects on the growth of both strains under aerobic or microaerobic conditions, nor did polysorbate 80 appear to affect the growth of either strain under microaerobic conditions  recovered from CD patients. To that end, the CD-MAM from five different CD subjects were challenged with either no additions, or 0.1%  of polysorbate 80 and/or carboxymethylcellulose, and the profiles of the communities following their cultivation are shown in Proteus and Morganella, unclassified Family_Enterobacteriaceae, and key members of the Negativicutes: unclassified Family_Veillonellaceae and Veillonella spp. Concomitantly, there were substantial reductions in the relative abundances of Streptococcus spp., and Escherichia-Shigella spp. in the CD34 consortium. Polysorbate 80 alone also resulted in a reduction  or expansion (CD34) in the relative abundances of members of the genus Bacteroides, which are linked with a concomitant expansion (CD12) or reduction (CD34) in members of the genus Streptococcus. Similarly, carboxymethylcellulose per se is associated with an expansion of the relative abundance of Parabacteroides spp. (in CD10 but not CD33), Enterococcus spp.  and unclassified Family_Enterobacteriaceae (CD33 and CD34).The relative abundance data (genus level) for the CD-MAM consortia following their culture in basal M2GSC medium (control), or in the presence of polysorbate 80 and/or carboxymethylcellulose, are shown in Veillonellaceae, Veillonella spp., Morganella spp., and Proteus spp. (CD34) can show a profound increase in the relative abundances of these bacterial taxa, in response to relatively small concentrations of polysorbate 80 and/or carboxymethylcellulose.Collectively, these findings suggest that in 4 of 5 CD subjects, the impacts from polysorbate 80 and/or carboxymethylcellulose were relatively modest, and the microbial consortia retained their patient specificity, rather than a convergence of the microbial communities toward a common collection of bacterial taxa in response to either or both food additives. However, and exceptionally, subjects possessing specific bacterial taxa can show profound changes in the composition of their mucosa-associated microbiota. In this specific example, it appears that the MAM communities from subjects with detectable Lachnospiraceae and Ruminococcaceae and a commensurate expansion in the relative abundance of bacteria affiliated with the Proteobacteria.32–34 Furthermore, a limited number of longitudinal studies have also shown restoration of F. prausnitzii in the colonic microbiota is associated with promoting disease remission.Faecalibacterium spp. combined with detectable P. mirabilis populations are now strongly associated with disease recurrence19 and the Negativicutes  as well as Fusobacteria are enriched in CD patients that show early post-operative endoscopic recurrence.23 However, the ecological dynamics giving rise to these observations remain largely unexplained, and thereby difficult to predict or manage. Diet has emerged as key factor affecting the nutritional and physiological ecology of the gastrointestinal tract and can function both as trigger and as a treatment option for chronic and idiopathic conditions such as CD.24 In that context, along with the plethora of processed foods now available to consumers, the diversity and consumption of food additives that enhance taste, flavor, and sensory perceptions have also increased. Evidence linking the consumption of food additives with host- and/or microbial alterations characteristic of disruptions to gut homeostasis and inflammation is mounting, but the actual mechanisms driving these observations are still lacking.Microbial dysbiosis is widely considered a hallmark of IBD, typified by a depletion of the relative abundance of bacteria affiliated with the F. prausnitzii and Proteobacteria strains with 0.1% final concentrations of these additives is considerably less than that used in some in vivo animal studies and in vitro studies.35 However, our choice is based on the strict regulatory limits on the amounts of specific food additives that can be included in human foods, their recommended average daily intakes where available37 and our estimation of their dilution within the luminal contents of the colonic environment. At this concentration, only sodium sulfite and polysorbate 80 elicited profound effects on the F. prausnitzii strains examined here, while the other food additives had variable (strain-specific) effects on the Proteobacteria strains tested, which were also affected by oxygen availability during growth. Interestingly, while both polysorbate 80 and sodium sulfite were inhibitory to nascent and actively growing cultures of multiple F. prausnitzii strains, polysorbate 80 appeared to have either little or a growth-promoting effect on the axenic isolates of Proteobacteria, and the effects from sodium sulfite on the growth of these strains were conditional oxygen availability. Sulfites have long been used as an antimicrobial agent to prevent browning and food spoilage38 but there are relatively few studies examining the potential antimicrobial effects of this additive on the “commensal” gut microbiota.39 While an acceptable daily intake (ADI: 10ng/kg) of sulfites has been in place for nearly 50 years, Irwin et al.38 suggested that an “average” Western diet can result in double the recommended ADI of sulfites; and will elicit bactericidal or bacteriostatic effects on four of the commonly used probiotic strains, at concentrations regarded as safe by the FDA. Interestingly, the results with the Proteobacteriaceae strains collectively show that the inhibitory effect of sodium sulfite is conditional on their growth under “microaerobic” conditions. Taken together, the consumption of foods/diets resulting in sulfite concentrations ~0.1% (w/v) in the gut milieu would at least transiently suppress F. prausnitzii growth within the colonic microbiota and reduce the growth rates of at least some strains of Proteobacteria, provided oxygen availability is limited, as it should be during periods of gut homeostasis.The food additives tested in this study represent four different classes  and chemistries . Our choice of challenging the F. prausnitzii40 our findings suggest the impacts from this additive are not direct, but rather indirect via niche contraction and/or occupation. The artificial sweetener saccharin did result in a metabolic burden placed on the growth of F. prausnitzii strain AHMP21, and a small but statistically significant reduction in the growth of F. prausnitzii strain KLE-1255, whereas sucralose had minimal effects on all three strains, relative to the control cultures  by mice promotes structural and microbiological changes leading to gut inflammation, this food additive has received greater investigation about its potential health impacts. For example, Singh et al.45 have also found that polysorbate 80 administered by gavage not only induced gut inflammation and liver dysfunction but also reduced fecal short-chain fatty acid concentrations in mice. Gerasimidis et al.30 and Naimi et al.46 both reported that polysorbate 80 added to mixed bacterial cultures produced from fecal samples of healthy adults reduces the relative abundance of F. prausnitzii. Here, our studies using multiple axenic isolates F. prausnitzii show the effect of 0.1% (v/v) polysorbate 80 (and sodium sulfite) is direct and likely bacteriostatic to this bacterium, at a concentration representing a “real-world” intake of this food additive. Conversely, polysorbate 80 appears to have little or no inhibitory effects toward the urease producing Proteobacteria examined here and indeed, appears stimulatory to the growth rate of axenic cultures of M. morganii and K. pneumoniae.Since the findings by Chassaing 47 but also not subject to the growth constraints imposed from digesta flow. As such, the use of fed batch cultures to examine these communities provides a realistic approach to examine the dynamics of these communities. Our results with the consortia recovered from five different CD patients show that the most profound effects from polysorbate 80 and/or carboxymethylcellulose was “personalized” toward the communities possessing P. mirabilis, M. morganii and/or Veillonella spp. In that context, polysorbates are well recognized to support the growth of Veillonella spp.48 which are a numerically predominant member of the Negativicutes lineage of gut bacteria. It is clinically salient to note that the relative abundance of Veillonella spp. in 16S rRNA gene profiles of mucosal tissue and stool metagenomic sequence data were positively associated with new onset, treatment-naive CD and more severe lesions47 and also in CD patients with early post-operative endoscopic recurrence of disease.23 With respect to P. mirabilis, in addition to our own studies and others showing the links between this bacterium and CD pathogenesis21 as well as disease recurrence,19 there is emerging evidence that expansion of Proteus spp. within the gastrointestinal microbiome is linked with heightened inflammation and/or tumorigenesis. For instance, recent studies showed that the addition of 1% (v/v) polysorbate 80 in drinking water resulted in an expansion of the relative abundance of “Enterobacteriaceae” in ileal samples of mice and potentiated their susceptibility to indole-methicin induced damage to the epithelium.49 Culture of ileal contents revealed that P. mirabilis counts were specifically and significantly increased in those mice consuming polysorbate 80. There is also an expansion of the relative abundance of Proteus spp. in association with the provision of a “Western diet” to a mouse model of tumor formation following colorectal surgery, which was potentiated by the emendment of the mouse gut microbiome with Enterococcus spp.50 The authors note the collagenolytic activity of these two genera as a key metabolic feature contributing to tumor formation, so it is interesting to also note that the relative abundances Enterococcus spp. were also found to increase in those patient-specific mucosa-associated consortia containing these lineages.The composition of the mucosa-associated microbiota is not only different to that present within the fecal stream and/or stool in CD subjectsF. prausnitzii and thereby, its contributions to the prevention of, and recovery from, inflammation. Interestingly, the magnitude of the effects from sodium sulfite and polysorbate 80 on the Proteobacteria isolates examined here appear to be contingent on oxygen availability. By using mucosa-associated microbial consortia recovered from the biopsies of five CD subjects, we have not only validated key findings using axenic isolates, but also show that two different emulsifiers (polysorbate 80 and carboxymethylcellulose) – either alone or in combination – can produce a “personalized” impact on the composition of these communities, including bacterial taxa such as Proteus spp. and Negativicutes, which are implicated in promoting CD recurrence. This study reveals how some of the key bacterial taxa now being identified as biomarkers of CD recurrence, or remission, will respond to different food additives, which is an important step toward diet formulation in CD therapy and clinical management.51In conclusion, we have examined the direct effect from four different classes of food additives commonly used in western foods on the growth of bacteria linked with Crohn’s disease remission or recurrence. The collective results with axenic isolates suggest that, with the exceptions of sodium sulfite and polysorbate 80, the effects from the other food additives examined here on bacterial growth rates appear to be strain-specific rather than universal. Our findings most clearly demonstrate that sulfites and polysorbate 80 at relatively small concentrations elicit a direct and profound bacteriostatic effect on multiple strains of Faecalibacterium prausnitzii strain A2-165 (Accession: SAMN07362387), KLE-1255 (Accession: SAMN00189147) and AHMP21 (Accession SAMN07414879) representing Phylogroups B, C, and E, respectively52 were routinely cultured with either M2GSC agar as described by Miyazaki et al53 or a basal broth (M2) medium with only glucose added, designated M2G. All bacterial transfers and growth were performed under anaerobic conditions, either within an anaerobic chamber on agar plates  or as broth cultures within Hungate tubes, with the same gas mixture used in the headspace.Proteobacteria from these consortia have been described in detail by Teh et al.16 Briefly, tissue was collected using standard forceps from 5 CD subjects during their post-operative endoscopy as part of the Post-Operative Crohn’s Disease Endoscopic Recurrence (POCER) study.54 The tissue was transferred to sterile and anaerobically prepared cryopreservative and the microbial consortia propagated from individual tissue samples by 24 h culture with M2GSC broth medium as previously described54 and stored as glycerol-preserved stocks. Urease-positive clinical isolates were recovered from serial dilutions of these glycerol stocks as individual colonies developing on either MacConkey or Cysteine-, Lactose-, Electrolyte deficient agar plates  under aerobic conditions. Urease-positivity was confirmed via color change surrounding colonies on Urea Agar Base plates with added Phenol Red and 33 isolated colonies were re-plated to produce axenic cultures. The taxonomic assignment of the axenic isolates was confirmed by 16S rRNA gene amplification and Sanger sequencing.16 Following preliminary testing, an isolate of P. mirabilis, and 2 strains each taxonomically classified as Morganella morganii, Escherichia fergusonii, and Klebsiella pneumoniae were selected for further study.The production of the microbial consortia from tissue biopsies and the subsequent isolation of urease positive 2 for an hour. The solutions were transferred to an anaerobic chamber  and transferred to glass serum bottles sealed with a rubber stopper and crimp sealed. These solutions were then autoclaved and stored at room temperature prior to use.The food additives tested in this study were purchased from Sigma-Aldrich . Solutions of carboxymethylcellulose sodium salt (21904), ι-carrageenan (C1138), saccharin , sucralose , sodium sulfite , Tween (polysorbate) 80 (polysorbate 8074), aluminum silicate (520179), and aspartame (A5139) were prepared at 1% (w/v or v/v) concentration in milli-Q water and flushed with NF. prausnitzii strains, the food additive solutions were used to supplement the basal M2G medium by aseptically transferring 1 ml of each stock solution to individual tubes, to provide final concentrations of 0.1% (w/v or v/v), which is intended to approximate concentrations found within the gut milieu. The same approach was used with the Proteobacteria strains with the exception that Luria Bertani (LB) broth was used as the basal medium.For the growth studies with the F. prausnitzii strains was transferred to M2G broth and incubated at 37°C overnight (14 − 16 hours). Aliquots (100 μl) of these cultures were used to inoculate Hungate tubes containing anaerobically prepared M2G broth medium supplemented with either the food additives, or additional sterile water, to provide a final volume of 10 ml. Microbial growth was assessed hourly by measuring the culture optical density at 600 nm (OD600). Based on the results, a second series of experiments with all three F. prausnitzii strains were performed. All three strains were cultured with M2G broth and growth monitored as described above. Once the cultures had reached mid-exponential phase of growth, either the sodium sulfite or polysorbate 80 stock solutions were added to provide a final concentration of 0.1% and bacterial growth monitored longitudinally for another 8 hours, as described above. As controls, separate cultures received a similar volume of sterile anaerobic water or were left untreated.The growth experiments were repeated at least twice and with three technical replicates for each treatment group. For initial growth experiments, a single colony of each Proteobacteria strains were conducted within a 96-well microtiter plate format. Here, the inoculating cultures were prepared by using a single colony of each strain transferred to 5 ml of LB broth within a 50 ml centrifuge tube  and incubated for 5 hours aerobically with shaking at 200 rpm at 37°C. Aliquots (0.135 ml) of either sterile LB broth, or LB broth prepared to contain 0.1% final concentration one of the food additives described above  were dispensed into individual wells, which were then inoculated with 0.015 ml of a 1:100 dilution (with LB) of the inoculating cultures described above. As controls, wells containing 0.15 ml of uninoculated broth (with and without food additives) were also prepared and positioned around the edge of the microtiter plates. The plates were covered with Breathe-Easy sealing membranes and placed within a Multiskan GO microplate reader  for measurements of aerobic growth. The plate reader was programmed to shake at 300 rpm and OD600 measurements were taken every 30 minutes for 17 hours. A parallel series of growth studies were also done with the 7 Proteobacteria strains using a 96-well microtiter plate format under “microaerobic” conditions. Here, the LB medium was prepared with an additional step of gassing the solution with a steady stream of nitrogen gas for 60 minutes, and the manipulations described above were performed within a Coy Anaerobic chamber filled with a mixture of CO2:H2:N2 (15:5:80). The “microaerobic” conditions resulted from the periodic introduction of surrounding atmosphere during interchange operations, which resulted in transient readings of ~300 ppm oxygen within the chamber atmosphere, before returning to zero. Bacterial growth was monitored by OD600 measurements using a FLUOStar Omega  plate reader permanently housed within the chamber using the same operational parameters described above.The growth studies with 16Overnight cultures of each consortium were used to inoculate the same M2GSC medium prepared to contain either 0.1% (w/v) carboxymethylcellulose, 0.1% (v/v) polysorbate 80, or both. Control cultures  were also prepared. After incubation for 24 hours, the resulting cultures were sampled, pooled, and metagenomic DNA extracted and initially used for 16S rRNA gene amplicon sequencing.600 measurements representing the exponential phase of growth from individual cultures  and no less than two rounds of growth studies. Multiple comparisons were performed using ANOVA with Dunnett’s comparison test in GraphPad Prism 9.1.0. Statistical analyses with p-values less than 0.05 were considered significant. For the consortia-based studies, the production of 16S rRNA gene amplicon libraries, DNA sequencing and data filtering were processed using the QIIME2 software, and the taxonomic profiles and comparisons were computed using various R packages, all as described by Teh et al.16 The DNA sequencing data (fastq files) produced as part of this study are uploaded in NCBI SRA as accession number PRJNA779743.For studies with axenic bacterial isolates, the doubling times were calculated using the Malthusian growth model, applied to OD


A complete randomized design (CRD) experiment was used with four levels of treatments, namely P0 (control), P1 (FMH 100 mL/kg), P2 (FMH 200 mL/kg) and P3 (FMH 300 mL/kg) of feed. The fish were kept in a farm in cages at 75 fish/m3 and fed with the experimental diets for 60 days. The results revealed that FMH (P2) dietary administration improved hematological and physiological profile of catfish, i.e total erythrocytes of 2.81 x 106 cells/mm3, hematocrit values of 39.00%, hemoglobin levels of 10.73 g/dL, total leukocytes of 11.41 x 104 cells/mm3, blood glucose 97.33 mg/dL, and total serum protein 4.10 mg/dL compared to controls with 1.89 x 106 cells/mm3, 32.33 %, g/dL, 9.67 x 104 cells/mm3, 67.33 mg/dL, and total serum protein of 3.10 mg/dL, respectively. Moreover, the diet improved special growth rate, feed conversion ratio, feed efficiency and the survival rate of catfish. The hematological and physiological profile of catfish improvement are considered to be due to the content of secondary metabolites of FMH, namely curcuminoids, vitamin C, essential oils, tannins, and flavonoids, which trigger immunostimulation. The presence of curcuminoids provide an antioxidant effect on cell membranes reducing erythrocyte cell membrane damage due to oxidation. Similarly, flavonoids are natural antioxidants, which are credited with the ability of reducing free radicals and anti-free radicals.This study sought to determine the effect of fermented medicinal herbs (FMH), Evidence of their value include inhibition towards pathogens of relevance to aquaculture, including bacteria,– viruses,– and protozoa.,Attention in aquaculture in developing countries has focused on the use of nonspecific immunostimulants and plant products, which could have a beneficial effect in fish disease control. Interest in medicinal plants for application to aquaculture follows their use in human medicine and agriculture as proven prophylactic and therapeutic agents., For example, Chinese herbs (Lonicera japonica andGanoderma lucidum) enhanced the non-specific immune response of tilapia (Oreochromis niloticus), leading to protection againstAeromonas hydrophila. Similarly, the dietary administration of rose hip and safflower stimulated growth performance, hematological, biochemical parameters and innate immune responses of beluga (Huso huso), The supplemented probiotic stimulated growth performance and feed utilization of keureling fishTor tambra. Medicinal herb has weaknesses, one of which is a strong aroma and bitter taste, so it is no liked by fish. Therefore, it is necessary to carry out fermentation. Against this background, fermented medicinal herbs (FMH) in combination with molasses and probiotic drink (Yakult) have been used successfully as dietary supplements on a small number of rural catfish farms in Sumatra, Indonesia, for approximately two years. Anecdotal evidence has suggested that fish grow better and faster, appear to be healthier, and the flesh tastes better after cooking. Fish, which have received these diets, have been examined for potential improvements to hematology and physiology.Some researchers have reported the impact of herbal supplemented diets on hematology and innate immunity of fish.This research was conducted on floating net cages in the Reservoir of the Faculty of Fisheries and Marine Affairs by using a completely randomized design with four treatments and three replications, namely P0 (control), P1 (FMH 100 mL/kg feed), P2 (FMH 200 mL/kg), and P3 (FMH 300 mL/kg). all steps in this experiment was carried out within the international ethical guidelines provided by ARRIVE guidelines.3. The cages were constructed of polyethylene nets with a mesh size of 7 × 7 mm. Water was free-flowing, and the temperature ranged 27.5–29.5°C.A total of 2700 fingerling catfish used had an average length of 9 cm and an average weight of 6 g. Catfish were obtained from local farmers in Kampar Regency and distributed randomly into cages measuring 1 × 1.5 × 1 m with a density of 75 individuals/mAll fish were acclimatized for seven days before use. The fish samples were selected based on their performance. The fish that showed active swimming, no wounds or external parasites. To measure the growth of the fish, every 10 days a random weighing of the fish was carried out.Kaempferia galanga), turmeric (Curcuma longa) and curcuma (Curcuma zanthorrhiza) together with molasses, probiotic drink (Yakult), fresh water and yeast (Saccharomyces cerevisiae) were used  consisting of cutchery  and stored at room temperature until use. The fish that have been blood drawn are then awakened by placing them in a container filled with aerated water. The fish were returned to the cages after they were seen breathing and swimming normally. Then, the number of erythrocytes was determined by use of a hemocytometer at a magnification of ×40 with calculation according to the formula of Blaxhall and Daisley. The method of Blaxhall and Daisley was used to estimate the total number and types  of leucocytes. For this, blood was dripped onto a hemocytometer slide, covered with a coverslip, and examined microscopically with a magnification of ×40. The total number of leukocytes was calculated using the following formula: ∑Leukocytes = ∑n × 50 cells/mm3, where ∑n = total number of leukocytes in four large boxes, and 50=dilution factor. Calculation of hemoglobin levels was carried out using Sahli’s method. To determine hematocrit level, blood was drawn into a hematocrit capillary tube the end of which was blocked with crystoseal. Then the capillary tube was centrifuged for 3 min at 11000 rpm in a microhematocrit centrifuge Model SH120-1. The hematocrit was measured as a percentage of the hematocrit value in the microhematocrit readerTo determine the number of erythrocytes, blood in 0.1-mL quantities was mixed thoroughly with 1.0 mL of Hayem solution comprising sodium sulfate, sodium chloride and mercuric chloride. Total serum protein was measured by the method of Anderson and Siwicki. Blood was centrifuged at 3,500 rpm for 15 min to completely separate the serum, which was transferred to a fresh microtube. Then, 20 μL of serum and 1000 μL of protein test reagent (Reiged Diagnostics) were added to each microtube, with mixing. After incubation for 15 min, the absorbance was read at λ 595–610 nm.Blood glucose was measured using GlucoDr  with a range of 20–600 mg/dL. Glucose testing was carried out in the morning before the fish were fed.a)AW = Wt − Wo, where AW = absolute weight (g), Wt = average weight at the end of the study (g), and Wo = average length at the beginning of the study (g).The absolute weight was measured using the formulab)SGR = Specific growth rates were calculated using the formula,c)Feed conversion was calculated by using the formula,d)FE = (Bt + Bm) − Bo)/F × 100, where FE = feed efficiency, ΣF = amount of feed fed during experiment (g), Bt = fish biomass at the end of experiment (g), Bm = biomass of fish that died during the study (g), and Bo = fish biomass at the beginning of the study (g).Feed efficiency was calculated by using formula,The experiments employed a completely randomized design (CRD) involving use of SPSS version 22.All animals are treated according to animal welfare guidelines that have been established and approved by the Dean of the Faculty of Fisheries and Marine Sciences, Riau University .p < 0.05). In addition, the provision of the supplements maintained the survival rate of catfish at 100%  compared to the experimental controls. Thus, total erythrocyte counts ranged between 2.45–2.81 × 106 cells/mm3, hematocrit values were from 35.67 to 39.00%, and hemoglobin levels ranged from 9.2–10.73 g/dL  compared with the controls. Thus, blood glucose levels and total serum protein increased from 78.67 to 97.33 mg/dL and 3.70 to 4.10 mg/dL; osmoregulation ranged from 288–327 mOsm/L H2O, absolute weight from 89.11–119.08 g, and the survival rate from 96–100%  fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. They noted that innate immunity was significantly enhanced by CBF–BS (CB fermented withBacillus subtilis) supplementation. This study indicated that the fermentation of CB with probiotic had beneficial effects on innate immunity and thereby increased disease resistance. Furthermore, it is also supported by 17 the use of probiotics from herbal ingredients can increase the growth performance and feed efficiency of Keureling.This study has supported the use of FHM feed supplements in Indonesian aquaculture, and provided evidence for the mode of action. Thus, there were notable improvements in the hematological and physiological profiles of catfish. Indeed, similar results have been reported by other researchers. For example, Lee The presence of curcuminoids provide an antioxidant effect on cell membranes reducing erythrocyte cell membrane damage due to oxidation. Similarly, flavonoids are natural antioxidants, which are credited with the ability of reducing free radicals and anti-free radicals.The important components of FMH are considered to be due to the content of secondary metabolites, namely curcuminoids, vitamin C, essential oils, tannins, and flavonoids, which trigger immunostimulation.et al., who reported that dietary application of the phytoimmunostimulant Persian hogweed (Heracleum persicum) improved growth significantly. Specifically, the final weight, weight gain, specific growth rate and feed conversion ratio were significantly improved in fish that received dietaryH. persicum at or above 5 g/kg.The benefit of FMH for improved growth performance mirrors previous work by HoseinifarClearly, this study has demonstrated that dietary FMH, particularly when dosed at 200 mL/kg of feed, improved the hematological and physiological profile of catfish. Also, there was benefit for specific growth rate, feed conversion ratio and survival.All data underlying the results are available as part of the article and no additional source data are required More background is required on the application of fermented plant-based diets in aquaculture. A simple paragraph or summary on the type of fish used in terms of importance, production, or normal distribution. "fermented medicinal herbs (FMH) in combination with molasses and probiotic drink (Yakult) have been used successfully as dietary supplements on a small number of rural catfish farms in Sumatra, Indonesia, for approximately two years." - Is there any data published? The chemical composition, especially the percentage of protein and lipid in the feed provided to fish, must be clarified.weight at the beginning of the study (g). Correct it. The absolute weight equation: W0 = averageSGR = Specific Growth RateLn = Natural logarithmWt = Weight of fish at the end of the experimental period (T)W0 = Weight of fish at the initial of the experimental periodt = Experimental period in days Does data meet the assumption of homogeneity of variances? Clarify if the authors ran a homogeneity test. How authors presented their results (Means ± SE or SD). SGR equation is not correct. The correct formula is SGR = (Ln Wt - Ln W0) / twhere: Feed utilization (FCR and feed efficiency) is not described in the result section. Glucose is an indicator of exposure to stress, as glucose level rises with the level of stress. How do the authors explain the increased glucose level with feed additives?Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?No source data requiredIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquaculture; Immunology; NutritionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Title: Pangasianodon spelling.CorrectAbstract:Write only significant results and no need to mention values of estimated parameters in this section.Introduction: Author needs to highlight the researchable problem with normal herbal immunostimulant, and why is the fermentation process required for preparing the product?Methodology: Fish acclimatization and standard procedure of FHM product preparation need to be mentioned with suitable references.Statistical analysis of data needs to be mentioned.Results:Why do the significant change in the control group observe in RBC of 30 days sampled fish from 60 days?Do the experimental periods have a significant effect on observed parameters?Similar changes are also observed in haemoglobin, hematocrite values...Discussion:The section is well written however the mechanism need to be illustrated.References: Very few recent references and authors need to cite the recent findings.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquatic Environment and Health  ManagementI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Some specific comments: " 1) Please revise title as  "Fermented medicinal herbs improve hematological and physiological profiles of Striped catfish 2) "Some researchers have reported the impact of herbal supplemented diets on hematology and innate immunity of fish" - so why were you combining molasses and probiotics? Please explain this in introduction section. 3) What were the probiotics cell numbers in different experimental diets?   Kaempferia galanga), turmeric (Curcuma longa) and curcuma (Curcuma zanthorrhiza) devoid of molasses, probiotic drink (Yakult) and yeast (Saccharomyces cerevisiae) would be interesting to determine the role of probiotics and prebiotics - the authors are requested to include it. 4) An additional experiment with medicinal herbs, i.e. cutchery (Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquaculture health and nutritionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Hibiscus sabdariffa L., Calendula officinalis L., Matricaria recutita L., Achillea millefolium L.). Retention time shifting, principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal projections to latent structures (OPLS) analysis were used to improve and analyze the obtained fingerprints. HPLC data detection at 270 nm was determined superior to 360 nm for the distinction of medicinal plants and used data alignment method significantly increased similarity between samples. Analyzed medicinal plant extracts formed separate, compact clusters in PCA, and the results of HCA correlated with the evolutionary relationships of the analyzed medicinal plants. Herbal fingerprinting using chromatographic analysis coupled with multivariate analysis has a great potential for the identification of medicinal plants as well as for the distinction of Latvian native medicinal plants. The growing market of herbal medicines, the increase in international trade in Latvia, and the lack of adequate analytical methods have raised the question of the potential use of herbal fingerprinting methods. In this study, high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) methods were developed for obtaining chromatographic fingerprints of four taxonomically and evolutionary different medicinal plants ( Although in the last hundred years the backbone of Western medicine has been a great diversity of chemically synthesized drugs, billions of people still use herbal medicine for its well-known wellness-inducing properties and as a prophylactic and therapeutic tool for both minor and chronic health problems ,2,3. WitThe chemical composition of medicinal plants is very complex, with hundreds or even thousands of chemical components, which can be affected by such factors as growth and storage conditions, genetic composition, soil components, time of harvest, processing methods, and others . All of Latvians have deep-rooted traditions and knowledge about medicinal plant use that are to this day still put in practice . Many mePharmacopeias and regulatory instances, like European Pharmacopoeia, mostly suggest that the identification and quantitative assay of herbal medicines should be performed using sensory inspections and a quantitative determination of a few key components of each herb ,15. HoweThe chemical fingerprinting method, approved by the World Health Organization (WHO), the Food and Drug Administration (FDA) of the USA, the European Medicines Agency (EMA), and others, can provide a comprehensive chemical description of herbal medicines ,20,21. Tr (−1 ≤ r ≤ 1) is used . F. F61]. FA correlation between the phylogenetic relationships and groupings of fingerprint datasets was also confirmed in HCA . LatvianMixed extracts of different combinations of the four analyzed medicinal plants were made, and HPLC, together with OPLS and HCA, was performed to see if we could identify the components of mixed extracts and to further investigate the possible application of the herbal fingerprinting method. OPLS has also been applied in previous studies to differentiate medicinal plants and their products ,64,65. TTwenty-five commercially available medicinal plant samples were collected from four different medicinal plants: roselle, chamomile, calendula, and yarrow. Chamomile, calendula, and yarrow were chosen because these medicinal plants are native to Latvia, are commonly used in Latvian traditional medicine, and all belong to the Asteraceae family. Roselle was chosen as a non-native, taxonomically different  medicinal plant to compare the chemical composition and fingerprints to the phylogenetic relationships of native and non-native medicinal plants, as well as to see if developed chromatographic methods can be used for a wider spectrum of medicinal plants. The detailed sample information is listed in Certified reference materials (CRM): caffeic acid (≥98%), apigenin (≥95%), and apigenin-7-glucoside were purchased from Sigma-Aldrich . Primary reference standards: rutin was purchased from PhytoLab , chlorogenic acid was purchased from the HWI group . All solvents used were of analytical or HPLC grade. Water was distilled and purified using the Stakpure GmpH water system .All medicinal plant samples were ground to a fine powder, and the powders were stored in airtight packaging at room temperature till extraction. v/v ethanol was added. This procedure was executed in triplicate. Three extractions from each medicinal plant sample were made to reduce the impact of sample preparation and short time stability, as well as to additionally control measurement quality. The material was extracted by maceration for 24 h at room temperature. The extracts were filtered through Sartorius smooth filter paper, grade 3-HW , and the supernatant was collected in an airtight container and stored at 4 °C until chromatographic analysis. To make a simple extract from only one medicinal plant 5.0 g of powdered medicinal plant sample was weighed and transferred to a 50 mL conical flask with a glass stopper, and 50 mL of 96% To test the application of our methods, mixed extracts from different combinations of analyzed medicinal plants were made. All combinations and their codes can be found in v/v) or three simple extracts (ratio 33:33:33 v/v) were taken and transferred into airtight containers and carefully mixed. This procedure was executed in duplicate for all mixed extracts to reduce the impact of sample preparation. These extracts also were stored at 4 °C until chromatographic analysis.In the first method, two simple extracts , and the supernatant was collected in an airtight container and stored at 4 °C until chromatographic analysis.In the second method, each needed medicinal plant sample was weighed and transferred to a 50 mL conical flask with a glass stopper, all dry samples were mixed together and ethanol 96% The same certified reference materials and medicinal plant samples were used for the HPLC and TLC methods.(v/v) (A) and acetonitrile (B) with the following gradient elution: 0 min—95% A, 45 min—55% A, 50 min—95% A. Before each sample analysis, a 4 min equilibration with 5% B was performed. The flow rate was set at 0.425 mL/min, the column temperature was 40 °C, injection volume was 1 μL, and the time of analysis was 50 min. The detection wavelength was set at 360 nm and 270 nm. Before HPLC analysis, extracts were put at room temperature for 2 h to reach room temperature and reduce the possibility of sample temperature influence on results. Before analysis, extracts were filtered through syringe filters with 0.45 μm pore size. Peaks with early retention time (<4 min) were omitted from the chemical profiles and further analysis due to the chance of errors and false signals not related to the composition of the sample. The average chromatogram for each medicinal plant was generated by combining all obtained chromatograms according to detection wavelengths. Since the quantitative concentrations of compounds were not investigated, all chromatograms were normalized beforehand. HPLC was performed on both the simple extracts made from one medicinal plant and mixed extracts.HPLC analysis was performed on a DIONEX UltiMate 3000 UHPLC+ focused system , which consists of an UltiMate 3000 pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment, UltiMate 3000 variable wavelength detector, and Chromeleon software. As a stationary phase, an Ascentis Express 90 Å AQ-C18 column  was used. The mobile phase was composed of 0.1% aqueous trichloroacetic acid v/v/v) for 30 min and then developed until 80 mm height. The same mobile phase has been used in two previous studies analyzing Salvia officinalis L. extracts and Artemisia species, and it is also stated as the TLC method for analyzing Calendula flowers by the European Pharmacopoeia [TLC analysis was performed on a CAMAG TLC system  and visionCATS software. HPTLC Silica gel 60 F254 glass plates 20 cm × 10 cm from Merck  were used as a stationary phase. The plates were activated at 60 °C for one hour before use. Before TLC analysis, extracts were put at room temperature for 2 h. Using the CAMAG Linomat 5 semi-automatic sampler, 7 μL samples were applied using a 100 μL syringe. Samples were applied with 8 mm bandwidth and 8 mm from the bottom of the plate. The number of samples on each plate differed between medicinal plants. The syringe was washed with purified water between each sample. The CAMAG ADC2 automatic developing chamber was saturated with the mobile phase ethyl acetate:water:formic acid 80:10:10  [All experiments were performed at least in triplicate with constant results. Differences among groups were considered significant at Germany) . SpecAliGermany) . PCA, HCThe herbal fingerprinting method has a great potential for the identification and characterization of herbal extracts. Research shows clear differences in chemical fingerprints of Latvian native medicinal plants  and roselle. TLC can be used as an initial screening tool of herbal extracts, while HPLC gives much more detailed information regarding the chemical composition of herbal extracts. HPLC fingerprint similarity can be greatly improved by adjusting retention times to the average chromatogram of each medicinal plant. In HPLC data, detection at 270 nm is superior to 360 nm and provides more detailed results of the chemical composition of herbal extracts and can be used to identify and differentiate medicinal plants. The correlation between the phylogenetic relationships and groupings of fingerprint datasets of analyzed medicinal plants was confirmed. The developed HPLC method in conjunction with chemometrics can also be used to identify components of mixed extracts containing different combinations of analyzed medicinal plants. Future research using other methods such as mass-spectrometry and larger sample sizes should be conducted to gain a deeper knowledge of the chemical composition of herbal extracts.