Do Hydrothermal Shrimp Smell Vents?

Deep-sea species endemic to hydrothermal vents face the critical challenge of detecting active sites in a vast environment devoid of sunlight. This certainly requires specific sensory abilities, among which olfaction could be a relevant sensory modality, since chemical compounds in hydrothermal fluids or food odors could potentially serve as orientation cues. The temperature of the vent fluid might also be used for locating vent sites. The objective of this study is to observe the following key behaviors of olfaction in hydrothermal shrimp, which could provide an insight into their olfactory capacities: (1) grooming behavior; (2) attraction to environmental cues (food odors and fluid markers). We designed experiments at both deep-sea and atmospheric pressure to assess the behavior of the vent shrimp Rimicaris exoculata and Mirocaris fortunata, as well as of the coastal species Palaemon elegans and Palaemon serratus for comparison. Here, we show that hydrothermal shrimp groom their sensory appendages similarly to other crustaceans, but this does not clean the dense bacterial biofilm that covers the olfactory structures. These shrimp have previously been shown to possess functional sensory structures, and to detect the environmental olfactory signals tested, but we do not observe significant attraction behavior here. Only temperature, as a signature of vent fluids, clearly attracts vent shrimp and thus is confirmed to be a relevant signal for orientation in their environment.

Neuroanatomy of a hydrothermal vent shrimp provides insights into the evolution of crustacean integrative brain centers.

Alvinocaridid shrimps are emblematic representatives of the deep hydrothermal vent fauna at the Mid-Atlantic Ridge. They are adapted to a mostly aphotic habitat with extreme physicochemical conditions in the vicinity of the hydrothermal fluid emissions. Here, we investigated the brain architecture of the vent shrimp Rimicaris exoculata to understand possible adaptations of its nervous system to the hydrothermal sensory landscape. Its brain is modified from the crustacean brain ground pattern by featuring relatively small visual and olfactory neuropils that contrast with well-developed higher integrative centers, the hemiellipsoid bodies. We propose that these structures in vent shrimps may fulfill functions in addition to higher order sensory processing and suggest a role in place memory. Our study promotes vent shrimps as fascinating models to gain insights into sensory adaptations to peculiar environmental conditions, and the evolutionary transformation of specific brain areas in Crustacea.

Gill chamber and gut microbial communities of the hydrothermal shrimp Rimicaris chacei Williams and Rona 1986: A possible symbiosis.

Rimicaris chacei Williams and Rona 1986, formerly named as Chorocaris chacei, is a caridean shrimp living in deep-sea hydrothermal ecosystems. This shrimp is endemic to the Mid Atlantic Ridge (MAR) and lives at the periphery of aggregates of its well-known congeneric R. exoculata Williams and Rona 1986. Contrasting with the very dense and mobile clusters formed by R. exoculata, R. chacei lives in small groups of several individuals that are not very mobile. Although devoid of the characteristic hypertrophied cephalothorax of R. exoculata, which harbors the ectosymbionts, a microbial community has also been reported in the cephalothorax of R. chacei. Previous data on morphology, behavior and isotopic values indicate a diet based on a combination of feeding on its epibiotic bacteria and scavenging or occasional predation. In this study, our objective was to describe, for the first time, the distribution, morphology and phylogeny of the microbial communities associated with R. chacei. This species is significantly less studied than R. exoculata, but nevertheless represents the only other known example of symbiosis in crustaceans of MAR hydrothermal vent sites. Microbial communities have been observed at the same locations as in R. exoculata (mouthparts, branchiostegites and digestive tract). However, in R. chacei, the surfaces occupied by the bacteria are smaller. The main lineages are affiliated to Epsilon and Gammaproteobacteria in the cephalothorax and to Deferribacteres, Mollicutes, Epsilon and Gammaproteobacteria in the digestive tract. Comparison with the well-described bacterial communities of R. exoculata and hypotheses about the role of these communities in R. chacei are discussed.

Comparison of Chemoreceptive Abilities of the Hydrothermal Shrimp Mirocaris fortunata and the Coastal Shrimp Palaemon elegans.

Chemoreception might play an important role for endemic shrimp that inhabit deep and dark hydrothermal vents to find food sources and to locate active edifices that release specific chemicals. We compared the chemosensory abilities of the hydrothermal shrimp Mirocaris fortunata and the coastal related species, Palaemon elegans. The detection of diverse ecologically relevant chemical stimuli by the antennal appendages was measured with electroantennography. The 2 species can detect food-related odor and sulfide, a short-distance stimulus, via both their antennae and antennules. Neither iron nor manganese, considered as long-distance stimuli, was detected by the antennal appendages. Investigation of the ultrastructure of aesthetasc sensilla revealed no specific features of the hydrothermal species regarding innervation by olfactory sensory neurons. Pore-like structures occurring in the aesthetasc cuticle and dense bacterial covering seem to be unique to hydrothermal species, but their potential link to chemoreception remains elusive.

Comparative Study of Chemosensory Organs of Shrimp From Hydrothermal Vent and Coastal Environments.

The detection of chemical signals is involved in a variety of crustacean behaviors, such as social interactions, search and evaluation of food and navigation in the environment. At hydrothermal vents, endemic shrimp may use the chemical signature of vent fluids to locate active edifices, however little is known on their sensory perception in these remote deep-sea habitats. Here, we present the first comparative description of the sensilla on the antennules and antennae of 4 hydrothermal vent shrimp (Rimicaris exoculata, Mirocaris fortunata, Chorocaris chacei, and Alvinocaris markensis) and of a closely related coastal shrimp (Palaemon elegans). These observations revealed no specific adaptation regarding the size or number of aesthetascs (specialized unimodal olfactory sensilla) between hydrothermal and coastal species. We also identified partial sequences of the ionotropic receptor IR25a, a co-receptor putatively involved in olfaction, in 3 coastal and 4 hydrothermal shrimp species, and showed that it is mainly expressed in the lateral flagella of the antennules that bear the unimodal chemosensilla aesthetascs.

New electroantennography method on a marine shrimp in water.

Antennular chemoreception in aquatic decapods is well studied via the recording of single chemoreceptor neuron activity in the antennule, but global responses of the antennule (or antennae in insects) by electroantennography (EAG) has so far been mainly restricted to aerial conditions. We present here a well-established underwater EAG method to record the global antennule activity in the marine shrimp Palaemon elegans in natural (aqueous) conditions. EAG responses to food extracts, recorded as net positive deviations of the baseline, are reproducible, dose-dependent and exhibit sensory adaptation. This new EAG method opens a large field of possibilities for studying in vivo antennular chemoreception in aquatic decapods, in a global approach to supplement current, more specific techniques.

Biogeographical distribution of Rimicaris exoculata resident gut epibiont communities along the Mid-Atlantic Ridge hydrothermal vent sites.

Rimicaris exoculata is a deep-sea hydrothermal vent shrimp whose enlarged gill chamber houses a complex trophic epibiotic community. Its gut harbours an autochthonous and distinct microbial community. This species dominates hydrothermal ecosystem megafauna along the Mid-Atlantic Ridge, regardless of contrasting geochemical conditions prevailing in them. Here, the resident gut epibiont community at four contrasted hydrothermal vent sites (Rainbow, TAG, Logatchev and Ashadze) was analysed and compiled with previous data to evaluate the possible influence of site location, using 16S rRNA surveys and microscopic observations (transmission electron microscopy, scanning electron microscopy and fluorescence in situ hybridization analyses). Filamentous epibionts inserted between the epithelial cell microvilli were observed on all examined samples. Results confirmed resident gut community affiliation to Deferribacteres, Mollicutes, Epsilonproteobacteria and to a lesser extent Gammaproteobacteria lineages. Still a single Deferribacteres phylotype was retrieved at all sites. Four Mollicutes-related operational taxonomic units were distinguished, one being only identified on Rainbow specimens. The topology of ribotype median-joining networks illustrated a community diversification possibly following demographic expansions, suggesting a more ancient evolutionary history and/or a larger effective population size at Rainbow. Finally, the gill chamber community distribution was also analysed through ribotype networks based on sequences from R. exoculata collected at the Rainbow, Snake Pit, TAG, Logatchev and Ashadze sites. Results allow the refining of hypotheses on the epibiont role and transmission pathways.

Thermal limit for metazoan life in question: in vivo heat tolerance of the Pompeii worm.

The thermal limit for metazoan life, expected to be around 50°C, has been debated since the discovery of the Pompeii worm Alvinella pompejana, which colonizes black smoker chimney walls at deep-sea vents. While indirect evidence predicts body temperatures lower than 50°C, repeated in situ temperature measurements depict an animal thriving at temperatures of 60°C and more. This controversy was to remain as long as this species escaped in vivo investigations, due to irremediable mortalities upon non-isobaric sampling. Here we report from the first heat-exposure experiments with live A. pompejana, following isobaric sampling and subsequent transfer in a laboratory pressurized aquarium. A prolonged (2 hours) exposure in the 50-55°C range was lethal, inducing severe tissue damages, cell mortalities and triggering a heat stress response, therefore showing that Alvinella's upper thermal limit clearly is below 55°C. A comparison with hsp70 stress gene expressions of individuals analysed directly after sampling in situ confirms that Alvinella pompejana does not experience long-term exposures to temperature above 50°C in its natural environment. The thermal optimum is nevertheless beyond 42°C, which confirms that the Pompeii worm ranks among the most thermotolerant metazoans.

A tale of two chitons: is habitat specialisation linked to distinct associated bacterial communities?

Although most chitons (Mollusca: Polyplacophora) are shallow-water molluscs, diverse species also occur in deep-sea habitats. We investigated the feeding strategies of two species, Leptochiton boucheti and Nierstraszella lineata, recovered on sunken wood sampled in the western Pacific, close to the Vanuatu Islands. The two species display distinctly different associations with bacterial partners. Leptochiton boucheti harbours Mollicutes in regions of its gut epithelium and has no abundant bacterium associated with its gill. Nierstraszella lineata displays no dense gut-associated bacteria, but harbours bacterial filaments attached to its gill epithelium, related to the Deltaproteobacteria symbionts found in gills of the wood-eating limpet Pectinodonta sp. Stable carbon and nitrogen isotope signatures and an absence of cellulolytic activity give evidence against a direct wood-feeding diet; both species are secondary consumers within the wood food web. We suggest that the distinct associations with bacterial partners are linked to niche specialisations of the two species. Nierstraszella lineata is in a taxonomic family restricted to sunken wood and is possibly adapted to more anoxic conditions thanks to its gill-associated bacteria. Leptochiton boucheti is phylogenetically more proximate to an ancestral form not specialised on wood and may itself be more of a generalist; this observation is congruent with its association with Mollicutes, a bacterial clade comprising gut-associated bacteria occurring in several metazoan phyla.

Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata.

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont's chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH(13)CO(3) and NaH(14)CO(3)) in the presence of two different electron donors (Na(2)S(2)O(3) and Fe(2+)) and with radiolabelled organic compounds ((14)C-acetate and (3)H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.

Sunken woods on the ocean floor provide diverse specialized habitats for microorganisms.

Marine waterlogged woods on the ocean floor provide the foundation for an ecosystem resulting in high biomass and potentially high macrofaunal diversity, similarly to other large organic falls. However, the microorganisms forming the base of wood fall ecosystems remain poorly known. To study the microbial diversity and community structure of sunken woods, we analyzed over 2800 cloned archaeal and bacterial 16S rRNA gene sequences from samples with different geographic locations, depths, and immersion times. The microbial communities from different wood falls were diverse, suggesting that sunken woods provide wide-ranging niches for microorganisms. Microorganisms dwelling at sunken woods change with time of immersion most likely due to a change in chemistry of the wood. We demonstrate, for the first time in sunken woods, the co-occurrence of free-living sulfate-reducing bacteria and methanogens and the presence of sulfide oxidizers. These microorganisms were similar to those of other anaerobic chemoautotrophic environments suggesting that large organic falls can provide similar reduced habitats. Furthermore, quantification of phylogenetic patterns of microbial community assembly indicated that environmental forces (habitat filtering) determined sunken wood microbial community structure at all degradation phases of marine woodfalls. We also include a detailed discussion on novel archaeal and bacterial phylotypes in this newly explored biohabitat.

Adaptation to thermally variable environments: capacity for acclimation of thermal limit and heat shock response in the shrimp Palaemonetes varians.

In the context of climate change, there is a sustained interest in understanding better the functional mechanisms by which marine ectotherms maintain their physiological scope and define their ability to cope with thermal changes in their environment. Here, we present evidence that the variable shrimp Palaemonetes varians shows genuine acclimation capacities of both the thermal limit (CT(max)) and the heat shock response (hsp70 induction temperature). During cold acclimation to 10 °C, the time lag to adjust the stress gene expression to the current environmental temperature proved to exceed 1 week, thereby highlighting the importance of long-term experiments in evaluating the species' acclimation capacities. Cold and warm-acclimated specimens of P. varians can mobilise the heat shock response (HSR) at temperatures above those experienced in nature, which suggests that the species is potentially capable of expanding its upper thermal range. The shrimp also survived acute heat shock well above its thermal limit without subsequent induction of the HSR, which is discussed with regard to thermal adaptations required for life in highly variable environments.

Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata.

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first 'epibiotic' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.

Bacterial communities associated with the wood-feeding gastropod Pectinodonta sp. (Patellogastropoda, Mollusca).

Even though their occurrence was reported a long time ago, sunken wood ecosystems at the deep-sea floor have only recently received specific attention. Accumulations of wood fragments in the deep sea create niches for a diverse fauna, but the significance of the wood itself as a food source remains to be evaluated. Pectinodonta sp. is a patellogastropod that exclusively occurs on woody substrates, where individuals excavate deep depressions, and is thus a potential candidate for a wood-eating lifestyle. Several approaches were used on Pectinodonta sampled close to Tongoa island (Vanuatu) to investigate its dietary habits. Host carbon is most likely derived from the wood material based on stable isotopes analyses, and high cellulase activity was measured in the digestive mass. Electron microscopy and FISH revealed the occurrence of two distinct and dense bacterial communities, in the digestive gland and on the gill. Gland-associated 16S rRNA gene bacterial phylotypes, confirmed by in situ hybridization, included members of three divisions (Alpha- and Gammaproteobacteria, Bacteroidetes), and were moderately related (90-96% sequence identity) to polymer-degrading and denitrifying bacteria. Gill-associated phylotypes included representatives of the Delta- and Epsilonproteobacteria. The possible involvement of these two bacterial communities in wood utilization by Pectinodonta sp. is discussed.

Microbial diversity associated with the hydrothermal shrimp Rimicaris exoculata gut and occurrence of a resident microbial community.

Rimicaris exoculata dominates the megafauna of several Mid-Atlantic Ridge hydrothermal sites. Its gut is full of sulphides and iron-oxide particles and harbours microbial communities. Although a trophic symbiosis has been suggested, their role remains unclear. In vivo starvation experiments in pressurized vessels were performed on shrimps from Rainbow and Trans-Atlantic Geotraverse sites in order to expel the transient gut contents. Microbial communities associated with the gut of starved and reference shrimps were compared using 16S rRNA gene libraries and microscopic observations (light, transmission and scanning electron microscopy and FISH analyses). We show that the gut microbiota of shrimps from both sites included mainly Deferribacteres, Mollicutes, Epsilon- and Gammaproteobacteria. For the first time, we have observed filamentous bacteria, inserted between microvilli of gut epithelial cells. They remained after starvation periods in empty guts, suggesting the occurrence of a resident microbial community. The bacterial community composition was the same regardless of the site, except for Gammaproteobacteria retrieved only in Rainbow specimens. We observed a shift in the composition of the microbiota of long-starved specimens, from the dominance of Deferribacteres to the dominance of Gammaproteobacteria. These results reinforce the hypothesis of a symbiotic relationship between R. exoculata and its gut epibionts.

Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma- and epsilonproteobacteria at four Mid-Atlantic Ridge hydrothermal vent fields.

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.

Molecular and ultrastructural characterization of two ascomycetes found on sunken wood off Vanuatu islands in the deep Pacific ocean.

A new genus of a deep-sea ascomycete with one new species, Alisea longicolla, is described based on analyses of 18S and 28S rDNA sequences and morphological characters. A. longicolla was found together with Oceanitis scuticella, on small twigs and sugar cane debris trawled from the bottom of the Pacific Ocean off Vanuatu Islands. Molecular and morphological characters indicate that both fungi are members of Halosphaeriaceae. Within this family, O. scuticella is phylogenetically related to Ascosalsum and shares similar ascospore morphology and appendage ontogeny. The genus Ascosalsum is considered congeneric with Oceanitis and Ascosalsum cincinnatulum, Ascosalsum unicaudatum and Ascosalsum viscidulum are transferred to Oceanitis, an earlier generic name.

Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean.

Specimens of Lamellibrachia (Annelida: Siboglinidae) were recently discovered at cold seeps in the eastern Mediterranean. In this study, we have investigated the phylogeny and function of intracellular bacterial symbionts inhabiting the trophosome of specimens of Lamellibrachia sp. from the Amon mud volcano, as well as the bacterial assemblages associated with their tube. The dominant intracellular symbiont of Lamellibrachia sp. is a gammaproteobacterium closely related to other sulfide-oxidizing tubeworm symbionts. In vivo uptake experiments show that the tubeworm relies on sulfide for its metabolism, and does not utilize methane. Bacterial communities associated with the tube form biofilms and occur from the anterior to the posterior end of the tube. The diversity of 16S rRNA gene phylotypes includes representatives from the same divisions previously identified from the tube of the vent species Riftia pachyptila, and others commonly found at seeps and vents.

Highly similar prokaryotic communities of sunken wood at shallow and deep-sea sites across the oceans.

With an increased appreciation of the frequency of their occurrence, large organic falls such as sunken wood and whale carcasses have become important to consider in the ecology of the oceans. Organic-rich deep-sea falls may play a major role in the dispersal and evolution of chemoautotrophic communities at the ocean floor, and chemosynthetic symbiotic, free-living, and attached microorganisms may drive the primary production at these communities. However, little is known about the microbiota thriving in and around organic falls. Our aim was to investigate and compare free-living and attached communities of bacteria and archaea from artificially immersed and naturally sunken wood logs with varying characteristics at several sites in the deep sea and in shallow water to address basic questions on the microbial ecology of sunken wood. Multivariate indirect ordination analyses of capillary electrophoresis single-stranded conformation polymorphisms (CE-SSCP) fingerprinting profiles demonstrated high similarity of bacterial and archaeal assemblages present in timbers and logs situated at geographically distant sites and at different depths of immersion. This similarity implies that wood falls harbor a specialized microbiota as observed in other ecosystems when the same environmental conditions reoccur. Scanning and transmission electron microscopy observations combined with multivariate direct gradient analysis of Bacteria CE-SSCP profiles demonstrate that type of wood (hard vs. softwood), and time of immersion are important in structuring sunken wood bacterial communities. Archaeal populations were present only in samples with substantial signs of decay, which were also more similar in their bacterial assemblages, providing indirect evidence of temporal succession in the microbial communities that develop in and around wood falls.

Early steps in microbial colonization processes at deep-sea hydrothermal vents.

A pluri-disciplinary in situ colonization experiment was performed to study early stages of colonization in deep-sea vent Alvinella spp. worm habitats. Four colonization devices were deployed onto Alvinella spp. colonies of different chimneys of the East-Pacific Rise (EPR 13 degrees N), for two different periods: a short (less than a week) and a longer one (3 weeks). Video imagery and monitoring of the thermal and physico-chemical conditions were performed during the colonization experiments. Numerous microorganisms bearing specialized adhesion-appendages and/or high amounts of polymeric extracellular matrix were observed on devices, which may efficiently contribute to the colonization of new surfaces. The microbial cohorts preceding and accompanying Alvinella spp. settlement were identified. In all cases, Archaea could not be detected and the microbial mats were essentially composed of e-Proteobacteria. Within this group, one phylotype (AlviH2) was found to dominate the libraries of three colonization devices. Dominance of e-Proteobacteria in the libraries may reflect the wide physiological variety encountered within this group or an adaptability of these microorganisms towards their changing environment. Bacteria affiliated to the Cytophaga-Flavobacterium-Bacteroides group or to the e-Proteobacteria, that grow either chemo-organoheterotrophically by fermentation or chemolithoautotrophically with H2 as an electron donor and S degrees /S2O32- or NO3- as a terminal electron acceptor, were isolated from one of the microbial mat formed in 20 days.