We also investigated the impact of gold shell morphology—spiky or bumpy—on SERS enhancements and on particle stability over time. We found that spiky shells lead to greater enhancements, however their high aspect ratio structures are less stable and morphological changes occur more quickly than observed with bumpy shells.Multifunctional micro- and nanoparticles have potential uses in advanced detection methods, such as the combined separation and detection of biomolecules. Combining multiple tasks is possible but requires the specific tailoring of these particles during synthesis or further functionalization. Here, we synthesized nanostructured gold shells on magnetic particle cores and demonstrated the use of them in surface-enhanced Raman scattering (SERS). To grow the gold shells, gold seeds were bound to silica-coated iron oxide aggregate particles. We explored different functional groups on the surface to achieve different interactions with gold seeds. Then, we used an aqueous cetyltrimethylammonium bromide (CTAB)-based strategy to grow the seeds into spikes. We investigated the influence of the surface chemistry on seed attachment and on further growth of spikes. We also explored different experimental conditions to achieve either spiky or bumpy plasmonic structures on the particles. We demonstrated that the particles showed SERS enhancement of a model Raman probe molecule, 2-mercaptopyrimidine, on the order of 10 Mul4–107) . SERS se4–107) .3O4 particles to be superparamagnetic [Magnetic particles for the sensitive and selective separation of biomolecules are unbeatable in terms of simplicity and low-cost, as only particles and a magnet are needed . Among tmagnetic ,32, but magnetic ,34. Usinmagnetic . Several strategies have been applied to assemble magnetic and plasmonic particles, including electrostatic interactions , microflWe recently showed that growing gold spikes on magnetic particles allows for the combined separation and detection of oligonucleotides without the use of an extrinsic tag or secondary hybridization step . In this4∙xH2O), cetyl trimethylammonium bromide (CTAB), sodium borohydride (NaBH4), silver nitrate (AgNO3), ascorbic acid, FeCl3·6H2O, sodium citrate dihydrate, polyacrylamide, tetraethoxysilane (TEOS), (3-aminopropyl)triethoxysilane (APTES), mercaptoundecanoic acid (MUA), (3-mercaptopropyl)trimethoxysilane (MPTMS), N-hydroxysuccinimide (NHS), and N-ethyl-N’-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), were purchased from Sigma Aldrich, . Millipore water and reagent grade ethanol (EtOH) were used.Gold(III) chloride hydrate  and heated at 200 °C for 12 h. The product was recovered magnetically and washed with deionized water, then by ethanol by magnetic decantation, and finally dried under nitrogen. Silica coating was performed in a second step. Briefly, the magnetite particle powder was dispersed into a solution of EtOH and Millipore deionized water by probe sonication. Ammonium hydroxide was then added to the dispersion, followed by the slow dropwise addition of TEOS in EtOH solution over 1 h under vigorous mechanical stirring. This mixture was then stirred at room temperature for 18 h, after which the product was recovered magnetically and washed with EtOH by magnetic decantation, then dried under nitrogen. Magnetic separation of samples in all steps was performed using either a small neodymium magnet or a rare earth homogenous magnetic separator . The samples were placed in or near the magnet and left to separate for 1–2 min. The supernatant was then gently removed using a pipette.Magnetite spheres were synthesized using a previously developed hydrothermal synthesis method . In briev/v solution of APTES in Millipore water was prepared. While mechanically stirring the particles in a 50 °C water bath, APTES solution was quickly added so that the final APTES concentration was 2% v/v. After 24 h of stirring at 50 °C, the particles in solution were magnetically separated and decanted, then washed three times in Millipore water by magnetic separation and dried under a stream of nitrogen.Amine functionalization: Silica-coated magnetite spheres were dispersed in 2:1 ethanol/ultrapure water for a final concentration of 5 mg/mL by bath sonication. Separately, a 20% v/v. The particles in solution were sealed in a vial and placed in a hybridization oven at 50 °C with gentle mixing. After 24 h, the particles in solution were magnetically separated and decanted, then washed once in toluene/ethanol and twice in ethanol by magnetic separation and dried under a stream of nitrogen.Short-chain thiol functionalization: Silica-coated magnetite spheres were dispersed in 1:1 toluene:ethanol for a final concentration of 5 mg/mL by bath sonication. After heating the solution to 50 °C, MPTMS was added to a final concentration of 2.5% Mixed amine/long-chain thiol functionalization: Amine-functionalized silica-coated magnetite spheres were dispersed in ethanol at 1 mg/mL by bath sonication. To activate the amine groups, 12 mM of NHS and 12 mM of EDC were added to the particles in ethanol and placed on a shaker for 90 min. The particles were magnetically decanted, redispersed in 10 mM MUA in ethanol, and placed on a shaker again for 90 min. The particles were then magnetically decanted and washed three times in ethanol by magnetic separation.4 to the vial while magnetically stirring. The bright yellowish-orange solution was stirred for 5 min. While still under magnetic stirring in the water bath, 0.3 mL of 0.01 M NaBH4 in Millipore water was added. The light brown solution was further stirred for 10 min. Gold seeds attachment: Silica-coated magnetite spheres were dispersed in ethanol at 2 mg/mL using a sonic bath for 20 min. Gold seeds were prepared by warming 5 mL of 0.2 M CTAB in Millipore water in a glass vial to 30 °C using a water bath, then by adding 0.125 mL of HAuClThe silica-coated magnetite spheres, dispersed in ethanol, were combined with equal parts Millipore water and gold seeds (1 mL of each) and mixed by gentle shaking. The mixture was placed on a gentle rotating mixer for 1 h. The particles were then magnetically decanted, washed three times using magnetic separation in 1 mM CTAB, and redispersed in the same volume of 1 mM CTAB as the ethanol that was first used to disperse particles at 2 mg/mL.4 and 0.1 mL of 0.01 M AgNO3 to 10 mM or 100 mM CTAB, then partially reducing the metal salts using 0.08 mL of 0.1 M ascorbic acid . When multiple samples were being synthesized, growth solution volume was scaled up accordingly. The solution was warmed to 30 °C using a water bath and magnetically stirred for 10 min. Following this, seeded particles were added to 10 mL of growth solution and mixed by inversion. For the first set of experiments, 100 μL of seeded particles were added to 10 mL of growth solution, and for the second set, after optimization, 400 μL of seeded particles were added to 20 mL of growth solution. The mixture was kept at 30 °C using either a water bath (without magnetic stirrer) or oven for 30 min. If growth occurred, the bath changed from a light brown to light grey (bound gold nanoparticles), dark blue (free gold nanoparticles), or a color in between. The particles in the bath were magnetically decanted, washed three times using magnetic separation in 1 mM CTAB, and redispersed in a volume of 1 mM CTAB equal to the volume of seeded particles used for storage.Gold growth on the seeds: Growth solution was prepared by adding 0.5 mL of 0.01 M HAuCl2O was used as the blank.UV-Vis spectroscopy: UV-Vis spectra of different suspensions were recorded in a plastic cuvette with a Cary 50 spectrophotometer . Spectral analysis of colloidal suspensions was performed in the range 300–800 nm. Milli-Q HElectron microscopy: Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100 plus LaB6  microscope with an acceleration voltage of 200 kV and an Orius 4 K CDD camera . The images were analyzed using ImageJ Software version 1.51j8, NIH, .Magnetic properties characterization: Magnetization curves were acquired by a superconducting quantum interference device (SQUID) magnetometer  at 300 K using particles dried in air. Magnetic separation times were acquired using the Sepmag .−10 Torr). XPS spectra were collected in a fixed analyser transmission mode at pass energy of 100 eV for the survey spectrum and 20 eV for O1s, N1s, C1s, S2s, Si2s, and Au4f core XPS levels. After data collection, the peak areas were determined after subtraction of a Shirley background. The atomic ratio calculations were performed after normalization using Scofield factors. Spectrum processing was carried out using the Casa XPS software package version 2.3.15 (www.casaxps.com).X-ray photoelectron spectroscopy measurements (XPS): XPS analysis were performed on a Scienta Omicron Argus X-ray photoelectron spectrometer , using a monochromated AlK (h = 1486.6 eV) radiation source having a 300 W electron beam power. The emission of photoelectrons from the sample was analyzed under ultra-high vacuum conditions  equipped with a high-powered near-IR laser diode working at 785 nm. Before spectra acquisition, an optical microscope was used to focus the laser beam into the MPym solution containing particles. The laser output power was 100 mW. For each spectrum, 10 acquisitions of 30 s were recorded to improve the signal-to-noise ratio. A detailed description of the experimental conditions adopted for SERS measurement is given in the SI section.Surface enhanced Raman scattering (SERS): Spectra were recorded in the 500-3400 cmWe grew gold spikes on silica-coated iron oxide spheres using a two-step method as depicted in 2), MPTMS-modified silica forming thiol-terminated (SH), and finally a mixed layer exhibiting long chain thiols and amine functions (referred to as SH-NH2) obtained by MUA reaction on APTES-modified silica.We looked at growth on four differently functionalized silica surfaces to determine how the interaction between the surface and gold nanoparticles affects spiky shell growth. The four surface chemistries are shown in 1s, C1s, and S2s are shown in The particles were thoroughly analyzed with XPS to confirm successful functionalization and relatively estimate the functional group coverage. The XPS spectra of N2 and SH-NH2 terminated surfaces in a similar amount, which was significantly larger than the trace signal of nitrogen on bare particles (likely due to the ammonium hydroxide used in synthesis). Carbon contamination was also present on bare particles but the C1s signal increased largely when APTES was grafted and further increased upon MUA reaction on APTES, confirming the successful silanization of the particles. For SH surfaces, obtained by MPTMS reaction, the change in C1s signal was barely noticeable, showing a modest grafting of this silane on the surface. The S2s peak was present as shown in 2 one. A similar result was previously observed on planar silica surfaces [2 particles, the S2s signal appeared confirming its successful grafting. The amount of bound MUA was estimated to be around 13% of the amine groups, which is lower than the results previously observed on planar surfaces (approx. 24%). Nevertheless, it resulted in a thiol density comparable to or even slightly higher than that on the short-chain SH particles.The survey spectra showed that all of the expected elements were present on the particles. Nitrogen groups were present on both NHsurfaces  and can Small CTAB-stabilized gold nanoparticles were utilized as seeds for further growth of gold spikes. The silica-iron oxide core particles with the four surface chemistries described above  were imp2-SH  were seen on bare A and on 2-SH D silica surfaces B. It sho2-SH, where the long thiol chains seem to overcome the barrier of electrostatic repulsion with amine groups. The small size of the seeds compared to that observed for SH terminated surface is possibly the result of the absence of electrostatic interaction with the underlying layer and the anchoring of the seeds solely to SH groups.The first hypothesis to explain these differences observed for seeds binding would be electrostatic. The CTAB-stabilized nanoparticles are positively charged, therefore a strong interaction will occur with silica surface which PZC (point of zero charge) is around 2 and silica surface strongly adsorbs positively charges species above this value ,61. AminA growth solution containing gold salt, CTAB, and silver nitrate was combined with the seeded particles and the gold structures were left to grow. These were then analyzed by TEM  and UV-V2 particle surfaces , which eventually coalesce into full shells surrounding the particles. UV-Vis results shown in ormation ,62.2 surfaces because of the previous discussion as well as the strong gold-thiol bond that may form. Few gold seeds bound to the amine-functionalized surface because of electrostatic repulsion, then further seed growth enhanced this low binding since growth involves CTAB as a stabilizing group as well. Some gold nanostructures are still seen around the amine particles, possibly because of the strong gold-amine bond that may form, despite electrostatic repulsion between the surface and stabilizing groups.Differences in spiky shell growth can be seen as a direct result of the differences in gold seed binding to the surface. More gold seeds bound to SH surfaces than to bare or NHThe idea behind using a magnetic core is that particles can be easily separated from a solution using a magnetic field. Use of the particles in practical applications, such as the separation of biomolecules, requires fast separation times and the ability to be redispersed before and after separation. The magnetic properties of the particles were studied before and after gold coating to determine whether or not the particles fit these requirements. Note that, for all steps in the synthesis, magnetic separation was used to wash the particles, and, during these steps, the particles were easily dispersed in solution and quickly separated out under the influence of a magnet which clearly indicated that they retained their magnetic properties. A SQUID magnetometer was used to obtain magnetization curves of the particles before and after gold coating . Gold sh authors ,64. In s3 higher than with the particles, added to demonstrate the enhancement by particles compared with the unenhanced signal. To study whether or not the nanostructures prepared above demonstrate Raman scattering enhancement, we used 2-mercaptopyrimidine (MPym) as a Raman probe molecule because of its high Raman activity and ability to strongly interact with the gold surface through S-Au bonds. The Raman spectra of 2-mercaptopyrimidine, at a concentration of 10 μM, are shown in 4, a value comparable with those seen in literature [All the particles with spiky gold shells demonstrated SERS enhancement on the order of 10terature . We also−1 corresponds the symmetric stretch of the trimethylammonium head group from, at 1441 cm−1 are observed the CH2 scissor modes and the amine-bound C-H symmetrical deformation, and finally, the bands in the 2840–3000 cm−1 are due to symmetric and antisymmetric stretching vibrations modes of CH2, terminal CH3, and N-CH3 [We also checked whether the Raman signal varied with MPym concentration. nd N-CH3 ,66.−1 ring breathing modes, at 1073 cm−1 in-plane stretching modes, at 1156 and 1375 cm−1 CH bending, and at 1539 and 1562 cm−1 ring stretching [−1 as well as that of CTAB characteristic bands as a function of MPym concentrations  of CTAB is approximately 1 mM, so, a possible interpretation of these data is that gold chloride ions in the bath solution are stabilized by micelles as long as the concentration of CTAB is above the CMC [In addition to the amount of bound gold, CTAB concentration also impacted the morphology of particles. Indeed, with a lower CTAB concentration (10 mM) the shells were less spiky and exhibited lower aspect ratio protrusions resulting in rather bumpy than spiky particles. Growth also occurred more quickly when a lower CTAB concentration was used but the time parameter did not affect the morphology of particles. We also tried an even lower CTAB concentration, 1 mM, (results not shown) that resulted in a spontaneous HAuCl the CMC . Since aFor both bumpy (10 mM CTAB) and spiky (100 mM CTAB) particles, the UV-Vis absorbance increased in the NIR region with increased growth time. This result fits with previous research showing that hybridization between the resonances of the individual gold nanostructures on the particle surface leads to a broad NIR absorption band that red shifts with increased coverage ,71. In w2p, O1s, Au4f, Br3d, and Ag3d for spiky and bumpy nanoparticles are gathered in Prior to SERS enhancement investigation, we assessed the elemental composition of the spiky and bumpy particles with the aim of determining the precise content for each element. This information is needed to establish whether the differences in SERS enhancement result from different amounts of gold, silver, or CTAB bound to the surfaces, or whether the morphology did in fact play a role. The atomic percentages for SiAs suggested by TEM images, more gold is reduced on bumpy particles than on spiky ones, and this is confirmed by both the atomic content in Au and the attenuation of Si and O coming from the underneath silica. Considering that XPS is a surface technique, calculating the relative contents based on the ratio to silicon is a more accurate way to estimate the elemental content of each atom as, in this case, the attenuation of the elements by the adsorbed layers is taken into consideration. When comparing the Au/Si ratio, it appears that bumpy particles accommodate three times more gold than the spiky ones. The same factor 3 is observed for silver and for bromine signal due to CTAB. The ratio Br/Au was therefore the same for both particles, i.e., 0.13. This value is not significant in terms of surface chemistry since both adsorbed and unbound CTAB can be present in the sample, however, it is relevant when it comes to comparing SERS CTAB signal on both particles. To summarize XPS data, bumpy particles contain three times more gold, silver and CTAB. Therefore, if SERS enhancement was to be dependent on the amount of substance in the samples, it should be three times higher for bumpy compared to spiky.We followed the CTAB signal to investigate how SERS enhancement varied across the two morphologies. TEM images of the particles and the Raman spectra taken of the particles in a solution of 167 µM CTAB along with the Raman spectra of unenhanced 0.2 M CTAB (for comparison) are shown in −1  to cS sample .Bumpy particles contain higher amount of substance for Au and Ag, 3 times higher as evidenced by a set of elements in XPS, yet the enhancement is higher for spiky particles. We can therefore claim that the enhancement is solely related to the morphology. Spiky particles morphology exhibits sharp tips, more protrusions and nanostructures, and consequently higher surface to volume ratio, which may impact the enhancement either through surface effects, i.e., greater surface area available for CTAB adsorption, or through electromagnetic effects due to the sharp tips and hot spots. The surface effect is not validated here as the ratio Br/Au was identical for both spiky and bumpy. Therefore, we can conclude here that the larger Raman enhancement on the spiky shell particles is due to increased electromagnetic enhancement caused by the spike shape.Using the particles in practical situations requires that they are stable over a period of time. We synthesized spiky and bumpy particles and measured their absorbance (UV-Vis), change in morphology (TEM), and SERS enhancement (Raman spectroscopy) after 1 week and 1 month. Particles were washed three times in 10 mM CTAB before each measurement. The TEM results together with UV-Vis and Raman spectra are shown in The particles remained fairly stable after one week, although some morphological changes did occur. After one month, the UV-Vis absorbance of the particles decreases overall, but especially large decreases occur in the NIR range. We can observe a resemblance between these curves and those seen after short growth times see , as lessAfter one week, the bumpy particles remained stable while the spiky particles showed a change in SERS signal. Surprisingly, the SERS signals increased over time, with both spiky and bumpy particles showing the strongest signals after particles had been left for one month.The morphological changes that occur over time are likely the result of Ostwald ripening, where the “spiky” areas with a small radius are less energetically stable and over time, the atoms migrate to areas with a larger radius that are more energetically stable. This phenomenon was previously observed experimentally and theoretically when monitoring the long-term stability of CTAB-stabilized gold nanostars . To overWe synthesized spiky gold-coated superparamagnetic particles that are magnetically separable and can be used for surface enhanced Raman scattering (SERS) detection of model molecules in a concentration-dependent manner. These nanostructured objects were prepared starting from silica coated magnetite particles that were functionalized to attach gold seeds, then grown into spiky structures. By controlling the surface chemistry applied to attach the seeds, we obtained different geometries and gold coverage. We also varied the growth conditions to engineer either spiky or bumpy particles. The morphology of the nanostructured shell plays a role in SERS enhancement, i.e., spiky shells led to a greater enhancement than bumpy shells, but the morphology also affects the stability of the particles over time, as bumpy shells are more stable than spiky shells, showing fewer morphology changes and less change in SERS enhancement over time. Our findings demonstrate a first step in the development of particles that can be used for combined separation and detection of biomolecules. Ongoing work on the further stabilization of spiky particles via a thin silica layer coating aims to overcome the stability issues and expand the applications of these nanomaterials.


Cardiovascular diseases are a main cause of mortality whose prevalence continues to increase worldwide. Long non‐coding RNAs (lncRNAs) regulate a variety of biological processes by modifying and regulating transcription of coding genes, directly binding to proteins and even coding proteins themselves. LncRNAs play key roles in the occurrence and development of myocardial infarction, heart failure, myocardial hypertrophy, arrhythmias and other pathological processes that significantly affect the prognosis and survival of patients with cardiovascular diseases. We here review the latest research on lncRNAs in cardiovascular diseases as a basis to formulate future research on prevention and treatment of cardiovascular diseases. However, it has been proved that lncRNAs regulate gene expression through a variety of mechanisms, including affecting the stability of mRNAs and translation initiation, acting as a competitive endogenous RNA and regulating post‐translational modification, among other functions.LncRNAs can be divided into antisense lncRNAs, bidirectional lncRNAs, intron lncRNAs and intergenic lncRNAs according to the position of lncRNAs in the genome.3Cells under stress in the cardiovascular system may undergo apoptosis, autophagy, necrosis, fibrosis, proliferation or migration, leading to the occurrence and development of cardiovascular diseases. The biological functions of LncRNAs can be classified into four categories:3.1MiRNAs inhibit the translation or promote the degradation of mRNAs by binding to the complementary sequence of their 3′ untranslated‐untranslated region (3′‐UTR), thus regulating gene expression,For example, Lu et al demonstrated that overexpression of lncRNA LOC100129973 improves the viability of endothelial cells, while its inhibition promotes endothelial cell apoptosis.3.2Cells in multicellular organisms communicate with nearby and remote cells through complex processes, which include paracrine signals and interactions with the extracellular matrix.2O2.It was reported that exosomes derived from cardiac cells contain abundant non‐coding RNAs, which significantly promote the repair of ischaemic heart and angiogenesis and inhibit apoptosis.3.3LncRNAs not only regulate neighbouring genes in cis‐conformation, but also play a role in trans‐regulation of expression of genes that are not closely related to their location.The most recognized example of lncRNAs in cis‐conformation is Xist. One of the X chromosomes in female mammals is transcriptionally silent during early embryonic development. Xist will make its sited X chromosome to relocate to the edge of the nucleus, inhibit the deposition of chromatin markers and eventually transcriptionally silence almost the entire chromosome through a series of events after being induced.3.42+ATP enzyme 2a of sarcoplasmic reticulum) directly and inhibits the activity of it in molecular level.In addition to affecting the expression level of proteins by regulating transcription, lncRNAs can also interact with proteins to regulate their functions directly. Lee et al proved that lncRNA NORAD, which is widely and abundantly expressed in mammals, binds to PUMILIO directly to maintain genome stability.LncRNAs perform a variety of biological functions in organisms not only through the lncRNA‐miRNA‐mRNA axis, but also by binding to proteins directly. In addition, lncRNAs participate in cis‐ and trans‐transcriptional regulation and play an important role in intercellular communication , a chronic inflammatory change of blood vessels, is characterized by lipid necrosis and vascular wall sclerosis manifested as lipid deposition and smooth muscle cell proliferation in the intima of the artery. LncRNAs play significant parts in the apoptosis and autophagy of vascular endothelial cells, proliferation of smooth muscle cells, formation of foam cells, lipid metabolism and other processes, which are closely connected with AS.4.1.1Endothelial cell dysfunction is an important cause of AS, and eNOS is considered to reflect endothelial cell dynamic balance. Miao et al revealed that lncRNA LEENE is coregulated with eNOS by the enhancer of eNOS.4.1.2The proliferation of vascular smooth muscle cells is another crucial process in the formation of atherosclerotic plaques. LncRNA p21, a p53‐induced lncRNA, was identified as a novel regulator of smooth muscle cell proliferation and progression of atherosclerosis. LncRNA p21 is significantly down‐regulated in atherosclerotic plaques of patients with AS and of ApoE knockout mice on a high‐fat diet.4.1.3Foam cells are the characteristic pathological cells in AS and arise from phagocytosis of lipids by macrophages or vascular smooth muscle cells because of abnormal lipid metabolism. LncRNA Mexis was reported to exert great influence on lipid metabolism through regulating ABCA1, which is critical for regulation of cholesterol efflux and generation of high‐density lipoprotein (HDL). Specifically, Mexis greatly increases with the activation of liver X receptors (LXRs), which are sterol‐activated nuclear receptors regulating expression of genes involved in reverse cholesterol transport, and the increase in Mexis then augments Abca1 expression and macrophage cholesterol efflux. Further study revealed that the stable overexpression of Mexis in macrophages increases the expression of ABCA1 to eliminate cholesterol more effectively, which significantly reduces the probability of AS in mice. Mice lacking Mexis gene show tissue‐selective decreased expression of ABCA1, and the probability of vascular occlusion is twice as high as that in normal mice.4.2Myocardial infarction (MI), a major contributor to mortality around the globe, entails acute or persistent coronary artery ischaemia and hypoxia leading to myocardial necrosis. Early prediction and diagnosis of MI are of great importance to improve the prognosis and survival of patients. Ishii et al were the first to report that lncRNA MIAT is associated with MI.Besides being early indicators of MI, lncRNAs play a crucial role in the pathogenesis of MI by controlling autophagy, apoptosis and other processes.4.2.1Autophagy is considered to be vital for renewing organelles and meeting metabolic needs under stress, both in physiological and pathological processes of the body. Although the exact effect of autophagy under different conditions has not been fully clarified, abnormal autophagy is associated with a variety of cardiovascular diseases.4.2.2The regulating effect of lncRNAs on apoptosis is also of great importance in alleviating MI injury. For example, knockout of lncRNA Mirt1, which is mainly expressed in cardiac fibroblasts, reduces apoptosis in cardiomyocytes and infiltration of inflammatory cells into the heart thereby improving cardiac function. The underlying mechanism involves inactivation of the NF‐ κB signal pathway induced by the lack of Mirt1.4.2.3Loss and inability to proliferate of adult cardiomyocytes partly underlie impaired cardiac function and heart failure after cardiac injury.Similar to Carel, lncRNA CPR (cardiomyocyte proliferation regulator) also plays an important regulatory role in the proliferation of cardiomyocyte.Besides, a cardiomyocyte regeneration‐related lncRNA named CRRL was proved to suppress cardiomyocyte regeneration by binding to miR‐199a‐3p.4.3Myocardial hypertrophy is an adaptive and protective response to overload stress; however, persistent pathological myocardial hypertrophy is often accompanied by maladaptive myocardial remodelling as manifested in various cardiovascular diseases such as hypertension and valvular disease. Pathological myocardial hypertrophy is also an independent risk factor for myocardial ischaemia, arrhythmia, chronic heart failure and sudden death.4.3.1Han et al identified a cardioprotective lncRNA named myosin heavy chain‐associated RNA transcripts (Mhrt), which is made up of antisense transcripts from Myh7 loci and confers protection from cardiac hypertrophy and subsequent heart failure.4.3.2Inhibiting the expression of a heart‐rich lncRNA named CHAER in the heart dramatically reduces myocardial hypertrophy and dysfunction.4.3.3LncRNA CHRF (cardiac hypertrophy related factor) acts as an endogenous sponge RNA inhibiting the activity of miR‐489 thereby precluding suppression of miR‐489 on MyD88 and the downstream molecule NF‐κB.4.3.4A recent study identified an important role for lncRNA Chast (cardiac hypertrophy associated transcript) in cardiac remodelling induced by pressure overload.4.4Heart failure (HF) is the disturbance of cardiac circulation caused by the dysfunction of systolic and/or diastolic function of the heart and characterized by impaired cardiac structure and function. Heart failure is not an independent disease, but the final stage of the development of various heart diseases, and lncRNAs play a vital role in its occurrence and progression.4.4.1Although BNP and NT‐pro BNP are recognized biomarkers of HF in clinical diagnosis, their specificity and accuracy still warrant further improvement. At present, the gradually discovered strong correlation between lncRNA and HF renders lncRNA an attractive biomarker for HF. Kumarswamy et al explored circulating lncRNAs in plasma in patients with HF and found that mitochondrial lncRNA LIPCAR was down‐regulated in the early stage after MI, up‐regulated in the late stage of ventricular remodelling and further up‐regulated in patients with chronic heart failure.A recent study analysing heart tissues from patients with HF and healthy donors identified that lncRNA COL1A1, which had been associated with fibrosis, was significantly associated with the progression of HF.4.4.2Myocardial fibrosis is an important part of almost all forms of HF, which destroys myocardial structure and accelerates the progression from the initial heart disease to HF.4.5Dilated cardiomyopathy (DCM), a primary myocardial disease of unknown origin, is characterized by impaired ventricular dilatation and systolic function usually leading to chronic heart failure. There currently are few treatments for dilated cardiomyopathy, rendering it necessary to explore the pathophysiological mechanisms underlying DCM and to elucidate new therapies. Genetic factors play an important role in the pathogenesis of DCM, and more than 20 gene mutations have been associated with DCM.A newly discovered lncRNA, Novlnc6, is significantly down‐regulated in DCM.Haas et al reported that structural genomic variant (SVs) contributed greatly to differential gene expression, including that of lncRNAs, in DCM. Among the identified 80,635 elements of the genome change caused by SVs, lncRNAs account for 3758 variations, which may underlie the basic mechanism of cardiac maladaptation in DCM.4.62+ treatment, spatial heterogeneity of the above characteristics and myocardial structure (gap and scar). Arrhythmia may occur when any of these six attributes is abnormal.The electrophysiological activity of the heart is disordered during arrhythmia, which is characterized by excitation conduction, membrane repolarization, automaticity, intracellular Ca4.6.1Drosophila, which has two small open reading frames encoding small peptides containing 28 and 29 amino acids, respectively. There were more arrhythmic contractions when pncr003:2L was silenced, which resolved when the peptide chains encoded by the two small open reading frames were introduced.2+ transport by inhibiting the activity of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) the function of which is to transport Ca2+ from the cytoplasm back to the sarcoplasmic reticulum. LncRNAs also play a negative role in regulating Ca2+ transport. Zhang et al found that by inhibiting the activity of SERCA2a, lncRNA ZFAS1 changed the calcium transient of cardiomyocytes, which would result in intracellular calcium overload and increase the tendency to delay late de‐grading and arrhythmias.Magny et al firstly discovered an arrhythmia‐related lncRNA, the putative non‐coding RNA 003in2L (pncr003:2L), in 4.6.2Cellular gap junctions are also an important target of lncRNAs in modulating the electrophysiological activity of the heart. Cx43 (Connexin 43) in the gap junction is abnormal in HF patients, resulting in slower conduction velocity and scattered pulse propagation, thereby increasing the risk of arrhythmias and even sudden cardiac death. LncRNA CCRR (cardiac conduction regulation RNA), a kind of antiarrhythmic lncRNA, was recently reported to improve the presence of Cx43 in gap junctions and thereby helping to maintain normal cardiac conduction.4.6.3Atrial fibrillation (AF) is the most common arrhythmias with complex pathogenesis, high rates of morbidity and mortality and current treatments that are not optimal. Cardiac autonomic nerve function plays a crucial role in the occurrence and maintenance of AF.4.6.4Shi et al analysed the RNA sequencing data of epicardial adipose tissue in patients with AF and discovered dysfunctional lncRNAs associated with AF pathogenesis.LncRNA plays an important role in the occurrence and development of cardiovascular diseases such as coronary atherosclerosis, myocardial infarction, heart failure and arrhythmia and is a potential target and biomarker for prevention and treatment ; Writing‐original draft . Yuerong Xu: Software ; Writing‐original draft . Runze Wang: Software ; Validation . Lang Hu: Software ; Supervision . Dong Guo: Software ; Supervision . Feng Xue: Investigation ; Validation . Wangang Guo: Investigation ; Validation . Dongwei Zhang: Investigation ; Validation . Jianqiang Hu: Software ; Supervision . Yan Li: Writing‐review & editing . Wei Zhang: Writing‐review & editing . Mingming Zhang: Funding acquisition (lead); Writing‐review & editing (lead).


We report for the first time a detailed description of benthic microhabitats , macrohabitats  and associated megafauna within the NDMP. Descriptions were based on analysis of fauna collected by trawling and ROV video observations from ~ 50 to 370 m depth. Rocky, coarse sand and silty sediment bottom habitats were observed at island slopes. In contrast, rocky and coarse sandy bottom habitats with a predominance of rhodoliths, thanatocoenosis, and other biogenic components were observed at seamounts. Mobile fauna and predators dominated the oceanic islands and nearby seamounts, whereas seamounts farther from the islands were dominated by sessile and hemisessile fauna that were mainly suspension and deposit feeders. Based on the register of 118 taxonomic units, our results provide an expanded and updated baseline for the benthic biodiversity of NDMP habitats, which seemed pristine, without evidence of trawling or anthropogenic debris.Seamounts and oceanic islands of the Chilean Exclusive Economic Zone at the intersection of the Nazca and Salas y Gómez ridges lie within one of the least explored areas in the world. The sparse information available, mainly for seamounts outside Chilean jurisdiction and shallow-water fauna of the Desventuradas Islands, suggests that the area is a hotspot of endemism. This apparent uniqueness of the fauna motivated the creation of the large Nazca-Desventuradas Marine Park (NDMP, ~ 300,000 km These ecosystems are considered vulnerable marine habitats because they are physically fragile or inherently rare4. Studied seamounts and oceanic islands are generally considered to play important roles by being highly productive locally, acting as regional centers of speciation or stepping-stones for dispersion. They comprise a diversity of substrates and thus habitats that support and provide refuge and feeding areas, not only for the benthic fauna but also for the associated pelagic and overlying surface water animals 7. Although seamounts are generally considered to play these important roles, each seamount experiences unique environmental conditions such as current characteristics and water-column environmental conditions  that differ with depth and current dynamics6. They also have unique characteristics such as height and morphology that result in unique physical dynamics that can result in increased nutrient availability relative to the surrounding area (reviewed in6). Such variability in environmental conditions results in variable ecological characteristics. Despite the above-mentioned importance of seamounts, only 4% of the ~ 40,000 reported have been sampled for scientific purposes10. The available faunistic information for seamounts is limited  and, for unstudied seamounts, usually comes from commercial fishing activities11.Seamounts are topographic elevations that rise more than 1000 m above the seafloor12. The oceanic islands and seamounts of the southeast Pacific lie within one of the most unexplored areas in the world. Most of the ~ 940 southeast Pacific seamounts are located along the Salas y Gómez (SGR) and Nazca (NR) ridges14, and only 24 (< 3%) of the SGR and NR seamounts have been studied. Most of these studies were conducted by expeditions of the former USSR between 1973 and 1987 in international waters from ~ 80°W to 101°W at 162 to 1900 m depth15. They reported unprecedented rates of endemism for benthic communities  that were similar to those rates observed for seamounts of the remote Norfolk Ridge, near New Caledonia16. Despite studies being conducted on SGR and NR seamounts19, only one of the many seamounts within the Chilean EEZ adjacent to the Desventuradas Islands (San Felix and San Ambrosio islands) has been investigated. In addition to this seamount, known as Stockman Guyot15, the Desventuradas Islands were studied on two expeditions: Marine Research Cruises in Remote Areas (CIMAR 6) conducted by the National Oceanographic Committee of Chile (CONA) in 2001 and the "Pristine Seas Expedition" carried out by the National Geographic Society and Oceana in 2013. These studies, which were conducted at 40 to 2300 m20, revealed that the fauna of these islands are unique and irreplaceable, granting these areas a high conservation value21. These considerations prompted the creation, in 2015, of the large Nazca Desventuradas Marine Park (NDMP), protecting an area of ~ 300,000 km2 including the Desventuradas Islands and seamounts located northwest of them22. This marine park provides enormous advances in safeguarding the unique biodiversity of actual or past threats such as pelagic fishing of tuna and swordfish, as well as bottom trawling7.The Pacific Ocean basin is characterized by the existence of several relatively long quasi-linear chains of oceanic islands and seamounts23, nor the role of the habitat at different scales from centimeters to meters. Because distinct environmental conditions, such as internal tides and heterogeneity of substrates and oceanographic conditions , can differ considerably among and within seamounts24, these and other variables can play a critical role in the distribution and behavior of megabenthos, operationally defined here as organisms with a body size > 1 cm. Differences in these environmental conditions can generate a mosaic of micro- and macrohabitats, with breaks in species distributions that can enhance biodiversity and drive changes in community composition within or among seamounts24.Of the available studies in the region, none has explored the importance of substrate type on species composition25. These organisms are called habitat-forming species and are characterized by being small-scale engineers of autogenous ecosystems. These organisms, and the biogenic structures they produce, contribute to increasing spatial complexity in soft-sediment benthic systems25, promoting spatial heterogeneity on seamounts26, but they are often easily overlooked because seamounts are sampled at much more general spatial scales26. In recent years, the importance of sampling at smaller scales has been accentuated with the understanding that the characteristics of the bottom and the presence of habitat-forming species increases the heterogeneity of the habitat and fulfill the function of a structural habitat27.The heterogeneity and topographic complexity of seamounts is affected by the presence of organisms that can change spatial conditions and, directly or indirectly, affect the availability of habitat and resources for other species30. Because many of these studies lacked a standard habitat classification scheme, these observations cannot be compared efficiently among ecosystems and studies. To explore the importance of seabed habitat, Greene et al.31 proposed a standardized classification, which divides the habitat according to depth, size and biogenic and abiotic variables, for in situ observations of the seabed. Within the size category are the subcategories of macrohabitat and microhabitat. Macrohabitats range in size from one to ten meters and include seafloor features such as boulders, crevices, cracks, caves, scarps, sinkholes and bedrock corals (solitary and reef-building). Meanwhile, microhabitats operate at centimeters and smaller scales and include seafloor features such as sand, silt, gravel, pebbles, small cracks, crevices and fractures32.Several studies of continental shelf, slope and submarine canyon habitats have specified relationships of megafauna with certain microtopographic (microhabitat) characteristics To explore relationships among micro- and macrohabitats and the fauna living on the summit of the seamounts and upper slope of oceanic islands of the NDMP, we describe the benthic habitats and associated fauna of the upper slope (50–370 m) of Desventuradas Islands and the summits and upper slopes (150–305 m) of seven nearby seamounts. We conducted remotely operated vehicle (ROV) surveys complemented with benthic fauna collections using an Agassiz trawl, aiming to achieve the following objectives: (1) to describe the benthic habitats of the oceanic islands and seamounts of the NDMP at different spatial scales , (2) to describe the composition and the diversity of the benthic megafauna on these oceanic islands and seamounts, and (3) to evaluate the relationships between macrohabitat and megafauna species diversity and composition as well as feeding mode and movement type of the benthic communities. To our knowledge, this study is the first to describe benthic habitats and associated fauna at seamounts and oceanic islands of the NDMP.33 that is ~ 100 km wide along the SGR34 and ~ 300 km wide along the NR35. The Desventuradas Islands, constituted by San Felix and San Ambrosio islands , are located ~ 970 km off the coast of the Atacama Region, northern Chile. Seamounts of the Desventuradas Islands region are located northwest of these islands at the intersection of SGR and NR  with a constant speed of ~ 3 knots at each station Table . The gea37.Environmental data  used in our analysis were obtained from the global three-dimensional CSIRO Atlas of Regional Seas (CARS) climatology (2009 version) as presented by Mecho et al.38. Habitats were classified at different spatial scales: (1) macrohabitats  and (2) microhabitats . The habitat types were described by geomorphology, including by slope , sediment type , texture  and "modifier" elements  following Greene et al.32. The sections of the videos in which the ROV was steadily moving between 30 cm and 1 m above the ground along the bottom were used for the description of the macrohabitats (field of view per frame ~ 3 m2), and the sections in which the ROV remained sitting on the bottom (field of view per frame ~ 0.05 m2) were used for the description of the microhabitats. The field of view per frame was estimated using the laser pointers of the ROV (positioned 10 cm apart).The oceanic islands (OI) and seamounts (SM) were considered as two different environments  and were referred to as subsystems, based on differences in physiography and depth. The characteristics of the seafloor habitats for each subsystem were described based on ROV data. Videos for each dive were analyzed in a time-lapse mode at half normal speed in VLC media Player 3.0.1145). Taxonomic assignments were further validated with resources such as the World Register of Marine Species  and the Ocean Biodiversity Information System .Specimens collected with the Agassiz trawl were preliminarily sorted, counted, and preserved onboard in 95% ethanol. Definitive counts and identification to the lowest possible taxonomic unit  were performed at Sala de Colecciones Biológicas Universidad Católica de Norte (SCBUCN), where specimens were then assigned an ID number and cataloged. The observed fauna was classified according to relevant literature and previous reports for the area  was established as the product of the trawling speed (Vi), effective trawl time (ti) and width of the mouth of the trawl (Ahi) and abundance was standardized to 10 m2 following Barriga et al.46:Semi-quantitative data of abundance for each OTU were calculated based on the swept area for each trawl. The swept area  to Universidad Católica del Norte.47 using the “vegan v2.5-6” package48. Because both trawl and ROV data were not available for all stations (Table H′) and Pielou’s evenness (J) since abundance data is required. OTU richness  of each station was estimated based on the combined trawl and ROV data . In all cases, only "living" organisms were considered  for the analyses. Species accumulation curves were constructed for each subsystem (OI and SM) and data gathering approaches (only trawl and combined trawl and ROV data) to estimate the rarified number of species and to assess sampling effort48. The OTU richness and diversity indexes of the two subsystems (OI and SM) were compared using an unpaired two-sample Wilcoxon test.All analyses were performed using the R software, version 4.0.3ns Table , the ana49. A canonical correspondence analysis (CCA) was applied using the R function “cca” to relate the set of environmental parameters to: (1) OTUs presence/absence data, (2) feeding mode composition, and (3) movement type. Environmental variables included water hydrographic variables (temperature and oxygen), type of bottom , depth, and geographic position (longitude and latitude). Salinity was not considered within the environmental variables in the nMDS and CCA because it co-varies with the oxygen. nMDS and CCA analysis were graphed using the library ggplot2 v3.3.2 in R50.To evaluate if there were differences in OTU composition among subsystems and stations a permutational analysis of variance (PERMANOVA), based on combined trawl and ROV data , was conducted. The routine “adonis2” on the Bray–Curtis index of similarity of the raw OTU presence/absence data  with subsystems (two levels: OI and SM) and maximum depth as factors was used. Patterns in the structure of benthic communities among subsystems and stations were visualized using a cluster analysis UPGMA hierarchical clustering (using the functions “desvest” and “hclust”) and non-metric multidimensional scaling  analyses based on Bray–Curtis dissimilarity elaborated from OTU presence/absence data. To analyze differences in functional diversity among sampling stations, OTUs were assigned to feeding modes  and movement type  based on functional traits and associated categories as proposed by Jones & FridCentrostephanus sylviae, Fig. Hormathia sp.)  were classified to one of three types Table : (1) cobp.) Fig. B, and (3p.) Fig. C.Table 1Lotella cf. fernandeziana, Helicolenus lengerichi and Scorpaena thomsoni) and large crustaceans  , except at SF2, consisted of coarse sand and rhodolith beds  joiProtoptilum sp. and Scleroptilum sp.) and colonies of anemones (Hormathia sp. and Cerianthid), except at SF2. Besides, we also reported biogenic structures on coarse sand beds such as coquina of pteropod  and green sponges, small patches of coarse sand were observed between the rocks with sea pens of the genus Scleroptilum , which included fields of sea pens  were collected by trawling and assigned to 95 OTUs , belonging to nine phyla Table . In termS data. Invertebrates were assigned to eight phyla: Arthropoda (23%), Mollusca (13%), Echinodermata (13%), Cnidaria (12%), Annelida (10%), Porifera (3%), Sipuncula (1%), Bryozoa (1%). Vertebrates (Chordata) were assigned to two classes of fishes . An additional 13 OTUs were identified just by shells or skeleton remains , but were not included in the analyses. Invertebrates represented 75% of the OTUs, whereas vertebrates represented 25%  for oceanic islands was highest at station ST20 (S = 35), representing almost half of the total OTUs reported at the oceanic islands, followed by station ST21 (S = 15). The lowest OTU richness at oceanic islands was observed for station ST18 (S = 10) (Table S = 50), which represents ~ 40% of the total OTUs reported for seamounts, followed by station SF7 (S = 35). The lowest OTU richness was observed at seamount stations SF8 (S = 8) and SFX (S = 13)  or diversity  were observed between subsystems. Stations at oceanic islands, in general, were less diverse  in comparison to the seamounts (H′ = 1.9). The diversity for oceanic islands was highest at station ST20 (H′ = 2.5) followed by station ST22 (H′ = 1.6). The lowest diversity at oceanic island stations was observed at ST21 (H′ = 1.3). The diversity at seamounts was highest at station SF7 (H′ = 2.5), followed by station SF5 (H′ = 2.2). The lowest diversity at seamounts was observed at station SFX (H′ = 1.3). The evenness values were similar between subsystems . The evenness for oceanic islands was highest at station ST17 (J = 1.0) followed by station ST22 (J = 0.9). The lowest evenness at oceanic island stations was observed at ST21 (J = 0.5). The evenness at seamounts was highest at stations SF7 and SF8 (J = 0.9). The lowest evenness at seamounts was observed at station SF9 (J = 0.5)  Table .Table 2O2 = 0.273, F = 3.234, p < 0.001) and depth  were significant variables in structuring the benthic communities of NDMP. The interaction between subsystem type and depth was not significant   Table .Figure 52 = 0.155, F = 1.511, p = 0.002; ROV: R2 = 0.205, F = 2.662, p = 0.001)  but not for trawl data . No significant interaction was observed between factors for either trawl or ROV data  , a similar grouping pattern was observed in the cluster and nMDS analyses. However, while oceanic islands and seamounts formed separate groups for only trawl data Fig. , one sea1) Table . Depth w6) Table .Chryseofusus kazdailisi, Calliostoma sp., Atrimitra isolata, Cryptogemma praesignis, the crustaceans Munida diritas, Heteronida sp., Paragiopagurus spp., Ebalia sculpta, and the irregular urchins Brissopsis sp. and Spatangus sp., were exclusively associated with seamounts F6, SF7 and SF9. Conversely, most of the fishes, including Tetronarce sp., Callyonimus sp., Parapercis cf. dockinsi, Seriola lalandi, Nemadactylus gayi, and Gonorynchus greyi, and a small portion of invertebrates , including the bivalve Arca cf. fernanandezensis, stomatopod Hemisquilla ensigera, snapping shrimp Alpheus cf. romensky, dollars Clypeaster spp. and the sea star Astrostole platei, were associated with stations around the islands (ST20 and ST21).The first two axes of the CCA represented ~ 38% of the total variance and two groups of stations were evident Fig. C. The poSqualus mitsukurii, Lotella cf. fernandeziana, Helicolenus lengerichi and Scorpaena thomsoni, and large crustaceans , whereas seamounts farther from the islands were dominated by sessile and hemisessile invertebrates (i.e. crawlers and burrowers/tube dwellers) that were mainly suspension and deposit feeders .Regarding functional traits of benthic communities, the first two axes of the CCA analysis, based on movement type Fig. A and feeThis study constitutes the most complete and updated description of benthic habitats and fauna for the upper slope of Desventuradas Islands and summits of nearby seamounts within the NDMP. ROV videos allowed the first description of benthic habitats of the Desventuradas Islands and their surrounding seamounts at a fine scale (meters to centimeters). Although we are aware of the limitations of the present study , we consider that our information presents a fundamental base-line knowledge of the benthic fauna and environment in this relatively understudied region of the Pacific Ocean, with a high value for conservation.51 at the Desventuradas Islands and reports of substrates on other seamounts of the SGR19 as well as of the eastern Pacific Ocean52, northeast Atlantic53 and equatorial Atlantic9. For example, Parin et al.15 reported, based on collections and observations made from a manned submersible, that the upper slopes of SGR seamounts located > 400 km NW of Desventuradas Islands had a soft bottom composed of biogenic sands derived from shells of local mollusks with some pelagic pteropods, foraminiferal material and fossil corals. We observed a change in the kind of sediment/ bottom type with predominantly coarse sands at the 150–180 m stations and predominantly fine sands at 220–370 m. This pattern of sediment change with depth has also been recorded in other deep-sea areas57.Of the three macrohabitats Fig. , Table 120. The macrohabitat of Lord Howe Island at a depth of 50 m is characterized by having a rocky bottom covered with encrusting red algae and being dominated by urchins (Tripneustes and Centrostephanus)30.The rocky macrohabitat observed on oceanic island ST18 Fig. A is simi15. This macrohabitat is characterized by the presence of a longitudinal trench in the cemented rock, which gives shelter to numerous species of fishes  and large crustaceans   observed at seamount SF2 58. Hence, Clark et al.59 suggested that the concept of seamounts as a single, relatively well-defined habitat type appears outdated, giving way to a growing recognition that within seamount variability can have different spatial scales. Although not statistically tested, our results suggest a higher faunal diversity associated with biogenic microhabitats compared to sites with few or no biogenic microhabitats , further supporting the importance of such small-scale differences in understanding the mechanisms involved in maintaining species diversity and the linkages between habitat-forming species and users of these habitats  to large (macrohabitat) scales and to provide suitable refuge and nursery zone for smaller mobile benthic organisms61. Consistent with this pattern, it appears that OTU richness may be higher where habitat-forming species, such as sea pens (Protoptilum and Scleroptilum), anemones (Hormathia sp.), tube-dwelling anemones and stylasterids corals were present (Table Hormathia sp.), tube-dwelling anemones (cerianthids) and sea pens (Protoptilum sp. and Scleroptilum sp.). A marked pattern was observed between substrate type (soft vs. hard) and the type of habitat-forming species; anemones and sea pens of the genus Protoptilum were observed frequently on soft bottoms, whereas sea pen of the genus Scleroptilum and stylasterids corals were reported on hard bottoms. Similar patterns have been described for deeper habitats (> 400 m depth) of NR and SGR seamounts and oceanic islands19 and also for New Caledonia, particularly on the Norfolk Ridge seamounts16.Until recently, most seamount research related to benthic habitat heterogeneity and complexity has focused on larger-scale variations (macrohabitats)ts Table . The prent Table . Microha16. For example, at NDMP seamounts, squat lobsters (Munida diritas and Phylladiorhynchus pusillus), crabs (Latreillia sp.) and juveniles of Caprodon longimanus were observed hidden among anemones . Another role of microhabitats is contributing to predator–prey dynamics; for example, the use of microhabitats not only plays a role in predator avoidance by providing shelter but can also play a role in ambush tactics by providing a focal site where the prey may not recognize the predator28. In our study, squat lobsters (Munida diritas) were observed hunting mysidaceans that foraged around anemone tentacles. In a recent description, Gallardo et al.62 mentioned that the white coloration of the squat lobster favors camouflage among coquina sediments. Similar observations have been reported by Auster et al.28, who documented some fauna  using microtopographic features and resources of microhabitat to block visual and acoustic  recognition of occupants in ambush tactics.Habitat-forming species were distributed as small patches that supported a higher concentration of fauna compared to the surrounding seafloor, providing a structure that can support more species than habitats without such complexitynes Fig. D. This t20. Previous studies have found that the Nazca-Desventuradas region has unique marine ecosystems that consist of a mixture of tropical, subtropical and temperate species with a strong affinity with the Indo-Pacific20. We report a total of 118 OTUs (131 if adding non-living specimens or shells), of which 50 invertebrates and 30 fishes were identifiable to species level (Table 63 that reported 50 species (23 invertebrates and 27 fishes) and the Chilean expeditions  that reported a total of 37 species. This increase in species number for the region is, most probably, due to the few previous scientific studies that focused on benthic megafauna64. Although the sampling effort deployed in the area is still far from being satisfactory, as indicated by the species accumulation curves ; seven genera and five species of polychaetes , bringing the total to 13 genera and five species66; one genus of Sipuncula (Aspidosiphon sp.); 14 genera and four species of gastropods , bringing the total to 40 genera and 31 species67; 17 genera and 15 species of crustaceans  . Fishes is the best documented group in the NDMP area, including reports by Sepulveda68, Pequeño & Lamilla40, Dyer & Wesneat39 and National Geographic & Oceana51 around the Desventuradas Islands from 5 to 2,215 m depth and the summit of the Stockman guyot (station SFX) at 200 to 370 m depth.The NR and SGR are considered biologically-unique because of their remarkably high endemismel Table . The numves Fig. , this stves Fig. , of whici) Table , bringinTherefore, it is probable that a greater sampling effort  will continue revealing substantial numbers of novelties for the region. Although rare invertebrate species and possibly new species were observed in the present study, none of the new records appear to be endemic to a particular seamount Table . However69 and subsequently by Parin et al.15 and Mecho et al.37 which divides the hard-bottom seamounts of south NR from the soft bottom seamounts of the east SGR. Parin et al.15 suggested a faunistic break at ~ 88°W. Mecho et al.70 observed remarkable turnovers of echinoderms at ∼101° and ∼86°W of the SGR, where assemblages tended to differ more across seamounts, suggesting the effects of physical barriers to dispersion  and habitat changes. Our data suggests a transition zone located at the intersection of SGR and NR (~ 82.5°W), as indicated by the separation among stations in the nMDS  and large predators such as the shark Squalus mitsukurii7. This predominance of fishes is likely the result of the islands having a biogenic structure that could support higher fish densities because of higher availability of nutrients, terrigenous contributions and eventual trophic subsidies by macroalgae 71. In contrast, the seamount fauna was characterized by having hemisessile and sessile fauna that are opportunistic suspension feeders  or deposit feeders . This pattern suggests that the seamounts of NDMP might have short food webs and low guild complexity, i.e., the majority of the animals in seamounts consume similar resources at a low trophic level23. A similar pattern was described for the New Caledonian seamounts, where it has been hypothesized that the length of the food chain in seamounts is shorter than in other aquatic systems16. These authors also found that although the food web in seamounts is short, it is still relatively complex.The CCAs Fig.  suggest This study constitutes a first descriptive addition to the knowledge of benthic micro- and macrohabitats of the slope of Desventuradas Islands and summits of nearby seamounts within the NDMP, providing the most complete description of their benthic megafauna to date. The benthic habitats of oceanic island slopes and seamounts within the NDMP are home to a large diversity of habitats, including rock, rhodoliths, sandy bottoms, silty bottoms, vertical walls and caves. Our study highlights that faunal composition differed with seafloor habitat. For example, higher values of diversity and OTU richness were observed in areas with a predominance of habitat-forming species, which points to the need to study seamounts at finer scales (centimeters to meters) to better understand the links of habitat-forming species and faunal communities and the potential effects that would result from the loss of macro- and microhabitat-forming species. Currently, these habitats within the NDMP are in an apparent pristine state . Thus, these seamounts, now under protection, provide a regional unique opportunity to monitor effects of climate change without the confounding factor of human activities, such as fishing, deep seabed mining or bottom trawl fishing, and could also be useful for comparisons with other places in the world where such closures have been implemented for seamounts in the Azores, SW Indian Ocean, New Zealand, Hawaiian Islands and Palau, to mention a few.Supplementary Information 1.Supplementary Legends.


The majority of the confirmed cases, 19 (63%), reported contact with livestock during slaughter and consumption of meat from sick animals. All confirmed cases had fever, 40% (12) presented with hemorrhagic syndrome, and 23% (7) presented with jaundice. Forty-three livestock herds with at least one suspect and/or confirmed animal case were identified. Death of young animals was reported in 93% (40) and abortions in 84% (36) of livestock herds. The outbreak is indicative of the emergence potential of RVF in traditionally high- and low-risk areas and the risk posed by zoonosis to livestock keepers.On the last week of May of 2018, a community-based syndromic surveillance system detected mass abortions and deaths of young livestock in northeastern Kenya. Two weeks later, Rift Valley fever (RVF) was confirmed in humans presenting with febrile illness and hemorrhagic syndrome in the same region. A joint animal and human response team carried out an investigation to characterize the outbreak and identify drivers of disease transmission. Here, we describe the outbreak investigation and findings. A total of 106 human cases were identified in the months of May and June 2018: 92% (98) and 8% (8) of these cases occurring in the northern and western regions of Kenya, respectively. Seventy-six (72%) were probable cases, and 30 (28%) were laboratory confirmed by ELISA and/or PCR. Among the confirmed cases, the median age was 27.5 years (interquartile range = 20), and 60% (18) were males. Overall, the case fatality rate was 7% ( Phlebovirus in the family Phenuiviridae. Globally, epidemics of RVF are most frequent in the horn of Africa’s Rift Valley region and the Arabian Peninsula.1 Outbreaks, however, have been reported in islands off the East African coast: Madagascar and French Island of Mayotte; parts of West, South, and North Africa; and in the Arabian Peninsula countries of Saudi Arabia and Yemen, an indication of the potential for spread and global health security importance of the virus.2 In Kenya, outbreaks are associated with heavy rainfall and flooding, which provides ideal conditions for mosquito vector multiplication and, consequently, disease emergence.4 As such, RVF epizootics are cyclic and periodic in nature, sometimes occurring as explosive outbreaks that cause significant morbidity and mortality in humans and animals.5 Mosquitoes of the Aedes species are considered to be the primary vectors, whereas the Culex and Anopheles species and other biting flies have been reported to be the main secondary, amplifying vectors that propagate transmission after emergence.7 Susceptible livestock, primarily sheep, goats, cattle, and camels, are infected through bites of infected mosquitos and through mechanical transmission by biting flies.7 On infection, the disease in animals is characterized by fever, abortion storms, and high mortality rate, especially among young livestock.9 Spillover of infection from animals to humans occurs through direct contact with infected animal fluids and tissues and consumption of contaminated animal products. Human infection through bites of infected mosquito, although rare, has been reported.10 Acute human infections present as a self-limiting febrile illness with nonspecific symptoms; however, a small proportion of cases proceed to a more severe disease that may present with ocular complications, encephalitis, hemorrhagic fever, jaundice, and other signs of liver malfunction.11Rift Valley fever (RVF) is a vector-borne zoonosis caused by 12 The last major outbreak in 2006–2007 resulted in approximately 340 human cases, 90 human deaths, and economic losses of more than US$32 million in direct livestock mortality and indirect losses, partly due to impediment to trade.13 As such, early detection and response to RVF outbreaks in animals before spillover to humans is a primary objective of the country’s animal health surveillance system.In Kenya, RVF is a priority zoonotic disease because of the high morbidity and mortality, frequency of outbreak events, and socioeconomic impacts during outbreak events.14 In the first week of May 2018, the Kenya Directorate of Veterinary Services (KDVS) activated a community-based, syndromic surveillance (SS) system to monitor occurrence of RVF-associated syndromes in livestock. This system had been previously used in 2015–2016 in 22 RVF high-risk counties in Kenya.15In May 2018, the Kenya Meteorological Department reported that some regions of the North Eastern region of Kenya had received three times the expected annual rainfall in the period between March and May 2018.On May 25, 2018, reports of mass abortions and mortality of young sheep, camels, and goats were reported to the KDVS by the animal health services in Eldas subcounty, Wajir County. By June 4, 2018, four human mortalities from the same subcounty, with a history of febrile and hemorrhagic illness, were reported to national human health services. On June 7, 2018, patient samples were submitted for laboratory testing at the Kenya Medical Research Institute (KEMRI), Nairobi. Two of three patients tested positive for RVF virus by reverse transcription–PCR (RT-PCR). A national outbreak of RVF was declared on June 7, 2018. And, consequently, a multidisciplinary field investigation team was deployed on June 13, 2018, to investigate the outbreak and assist with response and control efforts. Here, we report findings from the field investigation carried out among humans and livestock in Wajir, and subsequent outbreaks in humans in Marsabit and Siaya counties, Kenya.The study was conducted in Wajir and Marsabit counties in northeastern Kenya and Siaya County in southwestern Kenya from May to June 2018 .16 A suspect case of RVF was any person presenting in health facilities in any of the affected counties with a fever (> 37.5°C) or a 2- to 6-day history of fever of unknown origin and sudden onset of flu-like symptoms such as muscle pain, joint pains, and headache with or without ocular disease, meningoencephalitis, or hemorrhagic fever. A probable case was a suspect case with close contact with sick or dead livestock  at least 14 days before the onset of illness or a person who died with hemorrhagic-like signs between April and June 2018. Contact with livestock was defined as drinking of unpasteurized milk or any activity that results in exposure to animal blood and body tissues including and not limited to slaughtering, care of sick animals, veterinary procedures, disposal of products of abortions, and assisted animal birthing procedures.The investigation case definition was adapted from the WHO.A confirmed case was a suspect case with laboratory confirmation of the presence of anti-RVF virus IgM by ELISA or RVF RNA by RT-PCR.17 A suspect livestock case was any animal presenting or with a history of abortions or deaths of young animals (< 3 months old) or hemorrhagic syndrome in individual animals. A confirmed herd was a suspected herd with a laboratory confirmation of the presence in serum of anti-RVF virus IgM by ELISA in any individual animal within the herd. During the outbreak, livestock case finding was conducted in Wajir alone by tracing back human cases to their residence and sampling all animals the human cases had contact with 2 weeks preceding their illness. Additional livestock cases were traced through key informants who identified livestock herds presenting with RVF-like syndromes  in affected villages.We reviewed medical records for the 1-month period preceding the outbreak and conducted human active case finding using community-level key informants and door-to-door case ascertainment in affected areas in the three counties. Additional cases were identified through snowball sampling, that is, already identified suspected cases assisted with recruitment of other people with similar symptoms within the village. For livestock, the case definition was adapted from the World Organization for Animal Health (OIE) terrestrial manual.Structured questionnaires were administered to all human cases to collect data on demographics , travel history, and clinical (illness history and outcome) and exposure information. Proxy interviews (close relatives) were conducted to obtain information on mortalities linked to the outbreak and minors (children younger than 10 years) or critically ill cases.For livestock, a separate structured questionnaire was administered to owners to collect information about the herd characteristics  and clinical information .In each of the outbreak areas, 5 mL of blood was collected from suspected human cases, processed to serum, aliquoted into sterile bar-coded cryovials, and transported in liquid nitrogen dry shippers to the National Public Health laboratory and KEMRI viral hemorrhagic fever (VHF) laboratory for testing. Specimens were tested for the presence of Rift Valley Fever Virus (RVFV)-specific IgM antibodies using capture ELISA and for viral RNA using conventional RT-PCR as described using standard manufacturer operating procedures.18 Briefly, a commercial antihuman IgM antibody  was coated on 96-well Nunc immunoplates . Plates were then washed using a wash buffer  followed by addition of the RVF IgM positive control, negative control, and respective samples all diluted 1:100 in diluent buffer . Plates were incubated at 37°C for 1 hour, washed, and 100 μL of the RVF antigen (RVFV ZH 501 strain) solution added in one half of the test wells and a corresponding negative antigen (same dilution) added in the other half of the test wells. This was followed by a 1-hour incubation at 37°C. Plates were washed and 100 μL of RVF-specific detector antibodies (anti-RVF hyperimmune mouse ascitic fluid) added to each well and incubated for 1 hour at 37°C. Plates were washed and 100 μL of goat anti-mouse IgG, heavy and light chain specific conjugate  added in all the wells and incubated for 1 hour at 37°C. The plates were washed and 100 μL of the ABTS substrate  added followed by 30-minute incubation at 37°C. The optical density (OD) value was read with a spectrophotometer at 405 nm and the adjusted OD calculated by subtracting the OD of the negative/mock antigen–coated wells from the positive antigen–coated wells. The OD cutoff was calculated as the mean of the adjusted OD of the negative control sera plus three times the SDs. Any sample with OD ≥ 0.2 was considered positive.A laboratory-derived test provided by the Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases was used to detect the presence of IgM antibodies.® Q real-time PCR system (QIAGEN GmbH), reverse transcription was performed at 50°C for 30 minutes followed by Taq activation at 95°C for 15 minutes, and then PCR was conducted for 45 cycles at 94°C for 15 seconds and 60°C for 1 minute. Following PCR analysis, any sample with a cycle threshold ≤ 37 was considered positive. The primers and probes used for the real-time PCR for this study are shown in the Annex.Assays were performed using real-time RT-PCR. RNA was extracted from the whole blood specimens using QIAamp Viral Mini Kit following the manufacturer’s protocol . Primers and probes targeting the G2 gene of the virus were then used to amplify a 94-nucleotide fragment using a TaqMan real-time RT-PCR assay. The PCR amplification of targeted viral sequence was performed in a 25-μL reaction mix containing 12.5 μL of 2× RT-PCR buffer,1 μL of 25× RT PCR enzyme, 0.25 μL of forward primer (40 µM), 0.25 μL of reverse primer (40 µM), 0.25 μL of probe (40 µM), and 5.75 μL of nuclease-free water. Five microliter of RNA template was then added to the reaction mix. An RVFV-positive control (RNA extracted from RVFV positive sample) and a negative control were included in the PCR reaction setup. Amplification was performed using Rotor-GeneWhole blood samples were collected by jugular venipuncture using Vacutainer EDTA collection tubes; the serum was processed by centrifugation, aliquoted, and kept in cooler boxes before transportation to the Central Veterinary Laboratory in Nairobi for testing.® RVF IgM capture ) per the manufacturer’s recommendations. Briefly, 10 µL of test and control serum samples were added to a microplate precoated with anti–bovine–ovine–caprine IgM antibody. The plate was incubated for 1 hour at 37°C and washed three times. Fifty microliters of RVF nucleoprotein was added, and the plate was incubated for 1 hour at 37°C. Fifty microlitres conjugate (anti-RVF virus nucleoprotein conjugated to horse radish peroxidase) was added to all wells and incubated at 37°C for 1 hour. After washing, 100 µL substrate solution was added to each well and incubated in the dark for 15 minutes at room temperature followed by adding 100 µL stop solution. The plate was read at 450 nm, and the OD was recorded. The test was valid if the net OD of the positive control was > 0.350, and the ratio of the mean values of the net positive and negative control ODs was > 3. Samples with percent positivity of ≥ 50% were considered positive, ≤ 40% negative, whereas results between 40% and 50% were equivocal and repeated.Serum samples were tested for the presence of RVFV-specific IgM antibodies by capture ELISA using the IDvet diagnostic kit  and Microsoft Excel . We calculated means for continuous variables and proportions for categorical variables. Analysis was conducted only for laboratory-confirmed cases. Case fatality rates (CFRs) and attack rates (ARs) in humans by age-group, gender, and counties based on projections from national census data were calculated.This investigation was conducted as part of an outbreak response to a public health emergency and did not require an approval from institutional ethics review committee; however, permission was obtained from national and county government authorities responsible for human and animal health. Verbal consent was obtained from all study participants aged ≥ 18 years; minors (aged < 18 years) were interviewed after consent was provided by a legal guardian or parent. Confidentiality of the participants was ensured by replacing their names with unique identifiers. Case patients were given feedback on the results of their laboratory investigation through the nearest health facilities.n = 83), 14% in Marsabit (n = 15), and 8% (n = 8) in Siaya. Of 80 suspected cases whose specimens were collected, 30 (38%) were confirmed for RVF, 23 (77%) positive by RT-PCR, 6 (20%) by IgM ELISA, and 1 (3%) by both tests. Among the 30 laboratory-confirmed cases, 70% (n = 21) were from Wajir County, 27% (n = 8) from Marsabit, and 3% (n = 1) from Siaya. The median age of the confirmed cases was 27.5 years (interquartile range = 20), and 18 (60%) were male. All confirmed cases presented with fever, 73% with headache, 70% with arthralgia, 33% with hemorrhagic symptoms, and 13% with jaundice  were identified in three counties: 78% in Wajir  of the confirmed cases reported consumption of meat from a sick animal, 28% (n = 7) drinking raw milk, and 28% (n = 7) preparation and cooking of meat.Of 30 confirmed cases, there were seven deaths (CFR = 23%): 6 (86%) males and 1 (14%) female. Males had a CFR of 33%, whereas age-group 0–14 years had a CFR of 67%. Marsabit County had the highest CFR of 38% . Of 25 cKey informants reported livestock abortions were first observed in Eldas subcounty, Wajir County, on May 1, 2018, reaching the peak on May 14, 2018. The suspected human index case was reported on May 11, 2018, a day after the first peak in livestock abortion cases on May 10, 2018 .n = 1,078) had abortions, 17% (n = 986) died, and 9% (n = 509) had a variety of clinical signs reported including fever, general malaise, and staggered gait  of these sera samples were positive for anti-RVF IgM antibodies by IgM ELISA including 10/22 (46%) goats, 10/22 (46%) sheep, and 2/22 (9%) camels.21 Although this new detection is on a small geographical scale, it is worth noting that there is potential of spread of RVF at a national and regional scale. The occurrence in Siaya highlights the potential for spread and introduction into new ecological niches not previously classified as high risk.22 Once RVF virus is introduced into a new area, the virus can persist, and RVF outbreaks may be reported in subsequent years when climatic and environmental drivers are conducive for vector multiplication.24This study describes the detection of RVF in humans in two high-risk counties of Kenya: Wajir and Marsabit, and a first time detection in Siaya County, an area previously perceived as low risk for RVF occurrence.26 Like other previous RVF outbreaks in Kenya and elsewhere, we found a higher proportion of cases among males and livestock keepers.29 This is probably indicative of occupational risk and exposure to men who are primarily responsible for livestock herding, care of sick animals, and slaughter of livestock in pastoral communities; consequently, these men may be predisposed to close contact with blood and animal tissue from infected animals during outbreaks of RVF.30Our investigation established a 30-day delay between the detection of suspected first case and the first laboratory-confirmed case. This is likely linked to low suspicion index for RVF cases among health workers coupled with limited capacity for RVF differential diagnosis with other acute febrile illnesses in the affected regions. The delayed detection of human cases could be associated with the low sensitivity of passive surveillance systems and the mild, nonspecific presentation of RVF, thus resulting to missed opportunities for early diagnosis.27 This could be associated with poor health-seeking behavior, which resulted in few residents seeking clinical care and subsequent low diagnostic capacity.31The case fatality ratio in this outbreak (23%) was higher than that (16%) reported in previous outbreaks in Kenya but lower than that (46%) reported in Tanzania.32The outbreak was preceded by a period of prolonged heavy rainfall and resultant floodwater collections which creates conducive mosquitos-breeding habitats, the emerging potential for virus amplification and intensification of infections if the competent vectors are present.The first time that RVF was reported in Siaya may be due to autochthonous RVFV transmission by the presence of competent mosquito vectors. The epicenter of the outbreak in Siaya was suspected to be in villages around Lake Kanyaboli, a delta that drains into the expansive Yala Swamp. There is also high possibility of introduction of RVFV because of livestock trade and migration from other areas that may be at high risk of the disease. The lack of reported human and animal cases in the regions surrounding Siaya could be mainly attributed to weak surveillance characterized by low index of suspicion among healthcare workers , misdiagnosis, and low reporting rates as opposed to the lack of active RVF transmission. This study had some limitations. About a quarter of the suspect human cases did not submit their specimens for testing because they were identified retrospectively at the community. This likely underestimated the number of confirmed cases reported during this outbreak. In addition, the CFR reported here is likely an overestimate and biased toward severe cases because it was based on confirmed cases, who were likely severe cases, and did not include mild cases identified at the community level that we did not have their specimen.15 In our investigation, we noted a significant proportion of suspect cases did not seek health care even though they fit the suspect case definition. This not only could be attributed to mild febrile illness on infection but also shows the need to educate vulnerable pastoral communities on RVF high-risk periods and the need to seek health care and report the presence of any signs of infection in their communities.Most cases in this outbreak occurred among pastoralist livestock keepers with extensive animal contact. Public health education on risk reduction measures that target persons at high risk of exposure during outbreaks of RVF is critical to reduce infections. In addition, market closures and ban on slaughter of livestock at the community level may likely have significant impact in reducing human infections. The main interventions that were implemented as informed by the findings of the investigation included sensitization trainings with human and animal healthcare workers in outbreak areas on case detection and management, public outreach and surveillance, data sharing and infection, and prevention and control strategies. Overall, strengthening existing surveillance programs in all regions, irrespective of risk profile, for early detection of human and livestock cases, and rapid response is key to mitigate public health and socioeconomic impacts of RVF outbreaks. More specifically, strategies to improve disease reporting by pastoralists in high-risk areas through sensitization and equipping private and public frontline animal health workers with surveillance and reporting tools like risk assessment questionnaires and smartphones for electronic reporting were conducted. In under-resourced settings, innovative approaches such as the use of mobile phone–based SS to guide response have been identified as a useful complement to the traditional passive surveillance systems.Supplemental materials