Emergence of epizootic ulcerative syndrome in native fish of the Murray-Darling River System, Australia: hosts, distribution and possible vectors

Epizootic ulcerative syndrome (EUS) is a fish disease of international significance and reportable to the Office International des Epizootics. In June 2010, bony herring Nematalosa erebi, golden perch Macquaria ambigua, Murray cod Maccullochella peelii and spangled perch Leiopotherapon unicolor with severe ulcers were sampled from the Murray-Darling River System (MDRS) between Bourke and Brewarrina, New South Wales Australia. Histopathology and polymerase chain reaction identified the fungus-like oomycete Aphanomyces invadans, the causative agent of EUS. Apart from one previous record in N. erebi, EUS has been recorded in the wild only from coastal drainages in Australia. This study is the first published account of A. invadans in the wild fish populations of the MDRS, and is the first confirmed record of EUS in M. ambigua, M. peelii and L. unicolor. Ulcerated carp Cyprinus carpio collected at the time of the same epizootic were not found to be infected by EUS, supporting previous accounts of resistance against the disease by this species. The lack of previous clinical evidence, the large number of new hosts (n = 3), the geographic extent (200 km) of this epizootic, the severity of ulceration and apparent high pathogenicity suggest a relatively recent invasion by A. invadans. The epizootic and associated environmental factors are documented and discussed within the context of possible vectors for its entry into the MDRS and recommendations regarding continued surveillance, research and biosecurity are made.     

In vitro investigations on extracellular proteins secreted by <Emphasis Type="Italic">Aphanomyces invadans</Emphasis>, the causative agent of epizootic ulcerative syndrome

Background

Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.

Results

BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.

Conclusions

The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

     

Molecular Assays for Detecting Aphanomyces invadans in Ulcerative Mycotic Fish Lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.     

Immune responses and protection in catla (Catla catla) vaccinated against epizootic ulcerative syndrome

Three different antigenic preparations from the epizootic ulcerative syndrome (EUS) pathogen Aphanomyces invadans were evaluated as vaccine candidate in catla (Catla catla). Anti-catla enzyme immunoconjugate was prepared after isolating catla immunoglobulin and raising hyperimmune sera against it, in rabbit. Three antigens namely, fungal extract (FE), fungal extract mixed with Freund’s incomplete adjuvant (FIA) in a 1:1 (v/v) ratio (FE + A) and extra cellular product (ECP) were prepared and three groups of catla were vaccinated intramuscularly with all these antigens (200 μg/fish). Different cellular and humoral immune responses were measured for the entire vaccinated and control group on 0th, 5th, 15th and 25th day post vaccination. Thirty days after the vaccination, the fish were challenged with an A. invadans zoospore dose of 1 × 10⁵ ml⁻¹ and mortality and relative percent of survival (RPS) were recorded. Study of cellular immunological parameters including antigen-specific leukocyte proliferation, antigen-specific nitric oxide production and superoxide anion production showed significantly higher (p < 0.05) values, in general, on 5th and 15th day post vaccination than the 0th day. Among all the antigenic groups, FE + A showed most significant response compared to the other groups. Among the humoral immune responses, lysozyme activity showed almost similar trend like cellular parameters. Anti-Aphanomyces antibody production was measured by enzyme-linked immunosorbent assay (ELISA) and it was increased with increasing days of vaccination in all the vaccinated groups with the highest observed on 25th day. Among the antigens, FE + A showed the highest antibody production following vaccination. The result of the homologous pathogen challenge study showed reduction in mortality in all the vaccinated groups. However, this reduction was not statistically significant (p > 0.05). Increased immune responses and protection have important implications with regard to the control of EUS by vaccination.     

An upstream initiator caspase 10 of snakehead murrel Channa striatus,containing DED,p20 and p10 subunits: Molecular cloning,gene expression and proteolytic activity

Caspase 10 (CsCasp10) was identified from a constructed cDNA library of freshwater murrel (otherwise called snakehead) Channa striatus. The CsCasp10 is 1838 base pairs (bp) in length and it is encoding 549 amino acid (aa) residues. CsCasp10 amino acid contains two death effector domains (DED) in the N-terminal at 2–77 and 87–154 and it contains caspase family p20 domain (large subunit) and caspase family p10 domain (small subunit) in the C-terminal at 299–425 and 449–536 respectively. Pairwise analysis of CsCasp10 showed the highest sequence similarity (79%) with caspase 10 of Paralichthys olivaceus. Moreover, the phylogenetic analysis showed that CsCasp10 is clustered together with other fish caspase 10, formed a sister group with caspase 10 from other lower vertebrates including amphibian, reptile and birds and finally clustered together with higher vertebrates such as mammals. Significantly (P < 0.05) highest CsCasp10 gene expression was noticed in gills and lowest in intestine. Furthermore, the CsCasp10 gene expression in C. striatus was up-regulated in gills by fungus Aphanomyces invadans and bacteria Aeromonas hydrophila induction. The proteolytic activity was analyzed using the purified recombinant CsCasp10 protein. The results showed the proteolytic activity of CsCasp10 for caspase 10 substrate was 2.5 units per μg protein. Moreover, the proteolytic activities of CsCasp10 in kidney and spleen induced by A. invadans and A. hydrophila stimulation were analyzed by caspase 10 activity assay kit. All these results showed that CsCasp10 are participated in immunity of C. striatus against A. invadans and A. hydrophila infection.     

Identification and Characterization of Pathogenic Aeromonas veronii Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

Epizootic ulcerative syndrome (EUS) in fish: history and current status of understanding

Epizootic ulcerative syndrome (EUS) is one of the most important diseases affecting more than 100 species of wild and cultured finfish. EUS was first reported in farmed ayu (Plecoglossus altivelis) from Japan in 1971 and has since then spread across different countries of four continents including Asia, Australia, North America and Africa. The spread of the disease, especially in Asia–Pacific region and Africa has led to substantial damage to the fish resources and livelihood of the fish farmers. No reports are available confirming the outbreak of the disease from Europe and South America. The latest outbreak of EUS has been reported from Canada in a new susceptible species brown bullhead, Ameiurus nebulosus. It seems that the disease has potential to spread further, owing to the epizootic nature of the disease and broad susceptible host range. Considering the global importance of this disease, this review provides the current status of understanding about the etiology, process of diseases development, species affected, diagnostic methods as well as control and preventive measures, in light of the historical developments in those areas.     

Pathogenicity of animal and plant parasitic Aphanomyces spp and their economic impact on aquaculture and agriculture

Parasitic Aphanomyces species are a global threat to agri- and aquaculture, causing multimillion USD damage every year. Via the global trade, Aphanomyces has spread across all continents with exception of South America and Antarctica, and has become a major problem in pea, sugar beet, fish and crayfish production. The widespread A. euteiches and A. cochlioides induce root rot diseases in leguminous species and sugar beet respectively. The fish pathogen A. invadans is the causative agent of Epizootic Ulcerative Syndrome in various fish species whilst A. astaci infects crayfishes causing crayfish plague. Aphanomyces have developed an efficient transmission and infection mechanism which allows a rapid colonization and disruption of the host's infected tissues. This review presents an overview on the current research on Aphanomyces genus. We summarise the latest research efforts on four pathogenic Aphanomyces species, shedding light on the biology of these microorganisms, the pathogenicity factors of these parasites, the diseases which they cause, their distribution and finally the strategies to control the diseases.     

A murrel interferon regulatory factor-1: molecular characterization,gene expression and cell protection activity

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27 % α-helices (85 aa residues), 5.7 % β-sheets (19 aa residues) and 67.19 % random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (?284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.     

Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

Phylogenetic relationships among plant and animal parasites,and saprotrophs in Aphanomyces (Oomycetes)

Molecular phylogenetic relationships among 12 species of Aphanomyces de Bary (Oomycetes) were analyzed based on 108 ITS sequences of nuclear rDNA. Sequences used in the analyses belonged to the major species currently available in pure culture and GenBank. Bayesian, maximum likelihood, and maximum parsimony analyses support that Aphanomyces constitutes a monophyletic group. Three independent lineages were found: (i) plant parasitic, (ii) animal parasitic, and (iii) saprotrophic or opportunistic parasitic. Sexual reproduction appeared to be critical in plant parasites for survival in soil environments while asexual reproduction seemed to be advantageous for exploiting specialization in animal parasitism. Repeated zoospore emergence seems to be an advantageous property for both plant and animal parasitic modes of life. Growth in unspecific media was generally faster in saprotrophs compared with parasitic species. A number of strains and GenBank sequences were found to be misidentified. It was confirmed molecularly that Aphanomyces piscicida and Aphanomyces invadans appear to be conspecific, and found that Aphanomyces iridis and Aphanomyces euteiches are closely related, if not the same, species. This study has shown a clear evolutionary separation between Aphanomyces species that are plant parasites and those that parasitize animals. Saprotrophic or opportunistic species formed a separate evolutionary lineage except Aphanomyces stellatus whose evolutionary position has not yet been resolved.     

马鞍列岛多种生境中鱼类群聚的昼夜变化

为了解岛礁水域鱼类群聚的昼夜变化特征,以便更全面地设计采样方法和掌握采样的时间尺度,于2009年9月对马鞍列岛7种生境进行了共计24网次的刺网昼夜采样,结合排序和聚类方法,从种类组成、相对生物量和丰度、种类丰富度、多样性和相似性等方面对研究海域鱼类群聚特征的昼夜变化作了探讨.在采获的55种鱼类中,昼夜出现的分别为41和46种,数量差别不大,但其昼夜组成却随栖息水层的变化而不同,底层鱼类更趋向于夜间在硬相生境集群活动;近底层鱼类的昼夜集群随生境变化而变化,在同一生境中既有偏向白天也有趋向夜间的;中上层鱼类更多地出现在白天的人工生境(AH).AH白天的丰度渔获率显著大于晚上,而天然生境(NH)昼夜差别不大;生物量渔获率无论NH还是AH皆无显著昼夜差异.具体到种类,仅有小黄鱼Larimichthys polyactis和赤鼻棱鳗Thryssa kammalensis等少数种类的数量在AH有显著的昼夜差别,其他多数种类虽然昼夜的出现率大多有别,但渔获率昼夜差异皆不明显.多样性差异更多的表现在不同生境之间,而同一生境的昼夜差异往往不甚显著.各个生境中鱼类的昼夜种类交替现象非常明显,形成了以褐菖(鲐)Sebastiscus marmoratus和鳗鲇Plotosus anguillaris为代表的夜间优势类群为主的硬相生境群聚格局、以丝背细鳞鲀Stephanolepis cirrhifer和细刺鱼Microcanthus strigatus为代表的白天优势类群为主的硬相生境群聚格局以及缺乏底层优势类群、以石首鱼科鱼类为代表的近底层鱼类为绝对优势类群的软相生境群聚格局.因此,采用被动性渔具在近岸典型生境进行鱼类等相关生物调查时,应使采样时间覆盖昼夜两个时段,且至少保证24h.     

Variability in natural behaviour, and observed reactions to an ROV, by mid-slope fish species

The behaviour of eight large benthopelagic fish taxa was analysed using video records collected with an ROV on the mid-slope of the Bay of Biscay. The studied species were roundnose grenadier (Coryphaenoides rupestris), orange roughy (Hoplostethus atlanticus), deep-sea scorpionfish (Trachyscorpia cristulata echinata), and black scabbardfish (Aphanopus carbo), as well as individuals belonging to the families Alepocephalidae, Chimaeridae, and Scyliorhinidae, and to the order Squaliformes. Some of the observed fish were grouped at the family taxonomical level due to visual identification to species being unreliable. Assumed natural (undisturbed) behaviour was categorised in terms of (i) body position with respect to the bottom sea floor, (ii) locomotion and (iii) activity type. Reaction (disturbed) behaviour to the approaching ROV was categorised in terms of reaction type and distance. Environmental conditions (depth, temperature, current speed and direction) and observation conditions (ROV speed and altitude) were recorded simultaneously with fish observations in order to explain the variability in the observed reaction behaviour. A multivariate analysis identified three groups corresponding to a behaviour pattern of a sit and wait strategist (one species), an active bottom hunter (three taxa), and a group of species displaying little activity in their bottom habitat (three taxa). At species level the environmental and observation conditions had some explanatory power for individual behaviour variability. It is hypothesised that the varied behaviour of mid-slope benthopelagic fish contributes to maintain a high species diversity of large predators in an energy poor environment.     

Parasites with zoonotic potential found in commercially important fish in Tamaulipas,Northeastern Mexico

Human population is exposed to numerous parasitic ichthyozoonoses. Although Tamaulipas state (northeastern Mexico) is well known for its fishing and aquaculture industry, there are few reports of this type of zoonosis. Therefore, it is imperative to investigate whether the parasites that affect these fish may represent a zoonotic risk for the inhabitants of the area.The objective of this study was to identify molecular and/or morphologically muscle parasites of fish from coastal locations in Tamaulipas, Mexico, and assess the risk of infection for humans. Between 2017 and 2018, 764 individual fish belonging to 28 species were examined for parasites. Collected worms were processed for their identification using morphological characteristics. In addition, partial sequences of the large subunit (28S) ribosomal RNA gene were obtained from some species to corroborate their identity. Prevalence and mean intensity of all registered infections were calculated. A total of seven species of parasites were found: cestodes (Poecilancistrium caryophyllum), trematodes (Clinostomum tataxumui, Clinostomum cichlidorum), nematodes (Eustrongylides sp., Contracaecum sp.) and pentastomids (Sebekia purdieae, Sebekia sp.). Parasites infected 10 species belonging to different fish families (Ariidae, Centrarchidae, Centropomidae, Cichlidae, Eleotridae, Ictaluridae, Mugilidae and Sciaenidae). Congeneric species of parasites or related to those registered in this study have been identified as zoonotic agents in other regions of the world. Despite the low levels of infection (2.6–16.6% prevalence and 1–5.5 parasites per infected host), there is a latent risk of transmission to humans, so it is recommended to avoid eating raw or undercooked fish meat.     

Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh

Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.     

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh,Takeo, and Kandal Provinces,Cambodia

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia.     

Pennsylvanian brachiopod,fish and conodont faunas from the Caliza Masiva (San Emiliano Formation) at the Mina Profunda area,Cantabrian Zone,NW Spain

A rock sample obtained from the Caliza Masiva of the San Emiliano Formation (Bashkirian–early Moscovian) in the Mina Profunda area (NE Villamanín) of the Bodón Nappe (Cantabrian Zone, NW Spain) has yielded numerous brachiopods and fish remains not frequently represented in the fossil record. The brachiopod assemblage comprises 13 taxa and is characterized by phosphatic (Langella, Orbiculoidea) as well as exceptionally preserved silicified calcitic elements (a small chonetid, Composita, Crurithyris, Lambdarina, and two minute terebratulids) as the main faunal components. Of special importance is the record of the microbrachiopod Lambdarina winklerprinsi nov. sp., which reduces the large Viséan–Upper Permian gap in the stratigraphic record of this genus. Conodont elements recovered from the same insoluble residue are indicative of the upper Bashkirian Idiognathoides sulcatus parvus Zone. The accompanying fish remains consist of chondrichthyan teeth and scales, an acanthodian scale and osteichthyan tooth-bearing bones, isolated teeth and isolated scales, representing the first Pennsylvanian ichthyoliths analyzed from the Cantabrian Zone. The limestone beds with selective silicification in the San Emiliano Formation provide an exceptional opportunity to improve our knowledge on the patterns of life diversity over geological time.