Magnetic anaerobic granular sludge for sequestration and immobilization of Pb.

The development of magnetic adsorbents with high capacity to capture heavy metals has been the subject of intense research, but the process usually involves costive synthesis steps. Here, we propose a green approach to obtaining a magnetic biohybrid through in situ grown anaerobic granular sludge (AGS) with the help of magnetite, constituting a promising adsorbent for sequestration and immobilization of Pb in aqueous solutions and soils. The resultant magnetite-embedded AGS (M-AGS) was not only capable of promoting methane production but also conducive to Pb adsorption because of the large surface area and abundant function groups. The uptake of Pb on M-AGS followed the pseudo-second order, having a maximum adsorption capacity of 197.8 mg gDS-1 at pH 5.0, larger than 159.7, 170.3, and 178.1 mg gDS-1 in relation to AGS, F-AGS (ferrihydrite-mediated), and H-AGS (hematite-mediated), respectively. Mechanistic investigations showed that Pb binding to M-AGS proceeds via surface complexation, mineral precipitation, and lattice replacement, which promotes heavy metal capture and stabilization. This was evident from the increased proportion of structural Pb sequestrated from the aqueous solution and the enhanced percentage of the residual fraction of Pb extracted from the contaminated soils.

Prevalence and diversity of mycoviruses occurring in Chinese Lentinula edodes germplasm resource.

Incidence and banding patterns of virus-like dsRNA elements in 215 Chinese genetically diverse Lentinula edodes strains collected from wide geographic distribution (or producing areas) were first investigated, and 17 viruses were identified including eight novel viruses. The results revealed a 63.3% incidence of dsRNA elements in the cultivated strains and a 67.2% incidence in the wild strains. A total of 10 distinguishable dsRNAs ranging from 0.6 to 12 kbp and 12 different dsRNA patterns were detected in the positive strains. The molecular information of these dsRNA elements was characterized, and the molecular information of the other 12 different viral sequences with (+) ssRNA genome was revealed in four L. edodes strains with complex dsRNA banding patterns. RT-PCR was also done to verify the five dsRNA viruses and 12 (+) ssRNA ones. The results presented may enrich our understanding of L. edodes virus diversity, and will promote further research on virus-host interactions. IMPORTANCE: Viral infections involve complicated interactions including benign, harmful or possibly beneficial to hosts. Sometimes environment could lead to a transition in lifestyles from persistent to acute, resulting in a disease phenotype. The quality of spawn, such as the vulnerability to infection of viruses, is therefore important for mushroom production. Lentinula edodes, a wood rot basidiomycete fungus, was widely cultivated in the world for its edible and medicinal properties. In this study, the profile of dsRNA elements from Chinese genetically diverse L. edodes strains collected from wide geographic distribution or producing areas was first investigated. The molecular information of the dsRNA elements was characterized. Additionally, 12 different viral sequences with (+) ssRNA genome from four L. edodes strains with complex dsRNA banding patterns were identified. The results presented here will broaden our knowledge about mushroom viruses, and promote further studies of L. edodes production and the interaction between viruses and L. edodes.

First report on the regulation and function of carbon metabolism during large sclerotia formation in medicinal fungus Wolfiporia cocos.

The medicinal fungus Wolfiporia cocos colonizes and then grows on the wood of Pinus species, and utilizes a variety of Carbohydrate Active Enzymes (CAZymes) to degrades wood for the development of large sclerotia that is mostly built up of beta-glucans. Some differentially expressed CAZymes were revealed by comparisons between the mycelia cultured on potato dextrose agar (PDA) and sclerotia formed on pine logs in previous studies. Here, different profile of expressed CAZymes were revealed by comparisons between the mycelia colonization on pine logs (Myc.) and sclerotia (Scl.b). To further explore the regulation and function of carbon metabolism in the conversion of carbohydrates from Pine species by W. cocos, the transcript profile of core carbon metabolism was firstly analyzed, and it was characterized by the up-regulated expression of genes in the glycolysis pathway (EMP) and pentose phosphate pathway (PPP) in Scl.b, as well as high expression of genes in the tricarboxylic acid cycle (TCA) in both Myc. and Scl.b stages. The conversion between glucose and glycogen and between glucose and ß-glucan was firstly identified as the main carbon flow in the differentiation process of W. cocos sclerotia, with a gradual increase in the content of ß-glucan, trehalose and polysaccharide during this process. Additionally, gene functional analysis revealed that the two key genes (PGM and UGP1) may mediate the formation and development of W. cocos sclerotia possibly by regulating ß-glucan synthesis and hyphal branching. This study has shed light on the regulation and function of carbon metabolism during large W. cocos sclerotium formation and may facilitate its commercial production.

Morphological and transcriptomic analysis revealing morphological variations and genetic clues in one Lentinula edodes abnormal browning strain.

Strain abnormal browning is a common problem during cultivation of Lentinula edodes. In this study, the L. edodes strain mycelia isolated from Le-WB and cultured on MYG (Le-WP) isolated from an abnormal browning bag was compared with its normal control mycelia isolated from Le-BB and cultured on MYG (Le-BP). The aerial hyphae of Le-WP were white, and the hyphal growth was significantly reduced. Morphological observation of Le-WP under scanning electron microscope (SEM) and transmission electron microscopy (TEM) revealed abnormal organelle structures. Through transcriptomic analysis, more differentially expressed genes (DEGs) were expressed in the metabolic process and catalytic activity in Le-WP than Le-BP. Two Kyoto encyclopedia of genes and genomes (KEGG) pathways named pentose and glucorunate interconversions, and starch and sucrose metabolism were found to be enriched in Le-WP. The gene expression profiles involved in these two pathways were further analyzed and 12 key genes were selected to be verified by quantitative real-time PCR (qRT-PCR), and the results showed that most of these genes were upregulated in Le-WP. Additionally, the content of 1,3-beta-glucan in Le-WP was also significantly higher than in other samples. This research suggests that abnormal strains may be related to the abnormal synthesis of 1,3-beta-glucan, and it needs further research. This research exhibits possible morphological and genetic clues of Le-WP and lays the foundation for understanding the degeneration of L. edodes strains.

Preparation and Synergistic Anti-Tumor Effect of Iridium Oxide Nanocomposites under Microscope.

In order to solve the great difficulties in cancer prevention, diagnosis, and treatment, a preparation method of iridium oxide nanocomposites under the microscope was proposed in this paper. Through a retrospective analysis of an experiment, IrOx nanoparticles were prepared by direct hydrothermal hydrolysis and loaded with chemotherapy drug adriamycin to construct nanodrug-loaded complex IrOx@DOX. At the same time, IrOx, as a sound-sensitive agent, can produce ROS under US irradiation, amplify intracellular oxidative stress, accelerate tumor cell death, and finally achieve the effect of SDT chemotherapy synergistic therapy. The experimental results show that IrOx@DOX has the dual response of pH and US, and the inhibition rates are 27%, 57%, and 76%, respectively. At the same time, ultrasound not only can enhance the uptake of nanoparticles by cells but also can promote the release of DOX in cells, which provides a basis for subsequent SDT chemotherapy synergistic therapy. Conclusion. Iridium oxide nanocomposite DOX combined with SDT can obtain a good therapeutic effect, which has positive feedback on the efficacy of chemotherapy and the therapeutic effect of cancer surgery.

Metal oxide nanocage as drug delivery systems for Favipiravir, as an effective drug for the treatment of COVID-19: a computational study.

This paper is a summary of research that looks at the potential of fullerene-like (MO)12 nanoclusters (NCs) in drug-carrying systems using density functional theory. Favipiravir/Zn12O12 (- 34.80 kcal/mol), Favipiravir/Mg12O12 (- 34.98 kcal/mol), and Favipiravir/Be12O12 (- 30.22 kcal/mol) were rated in order of drug adsorption degrees. As a result, Favipiravir attachment to (MgO)12 and (ZnO)12 might be simple, increasing Favipiravir loading efficiency. In addition, the quantum theory of atoms in molecules (QTAIM) assessment was utilized to look at the interactions between molecules. The FMO, ESP, NBO, and Eads reactivity patterns were shown to be in excellent agreement with the QTAIM data. The electrostatic properties of the system with the biggest positive charge on the M atom and the largest Eads were shown to be the best. This system was shown to be the best attraction site for nucleophilic agents. The findings show that (MgO)12 and (ZnO)12 have great carrier potential and may be used in medication delivery.

Novel insights into the adsorption of organic contaminants by biochar: A review.

With rising concerns in the practical application of biochar for the remediation of environment influenced by various organic contaminants, a critical review to facilitate insights the crucial role that biochar has played in wastewater and polluted soil decontamination is urgently needed. This research therefore aimed to describe different intriguing dimensions of biochar interactions with organic contaminants, which including: (i) an introduction of biochar preparation and the related physicochemical properties, (ii) an overview of mechanisms and factors controlling the adsorption of organic contaminants onto biochar, and (iii) a summary of the challenges and an outlook of the further research needs in this issue. In the light of the survey consequences, the appearance of biochar indicates the potential in substituting the existing costly adsorbents, and it has been proved that biochar is one promising adsorbent for organic pollutants adsorption removal from water and soil. However, some research gaps, such as dynamic adsorption, potential environmental risks, interactions between biochar and soil microbes, novel modification techniques, need to be further investigated to facilitate its practical application. This research will be conductive to better understanding the adsorption removal of organic contaminants by biochar.

Curing two predominant viruses occurring in Lentinula edodes by chemotherapy and mycelial fragmentation methods.

Previous research has established that Lentinula edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1(LePV1) are major mycoviruses identified in L.edodes germplasm. In this paper, two different methods for curing these two dsRNA mycoviruses, ribavirin treatment and mycelial fragmentation, were evaluated for the first time. Mycelial fragmentation was found to resulted in LeV-HKB- and LePV1-cured fungal strains, whereas ribavirin treatment could eliminate LeV-HKB only. Although no LePV1-cured strain was obtained via ribavirin treatment by the end of the experiment, the relative LePV1 concentration in the eighth successive subcultures was lower than that of the untreated control. The culture features of several virus-cured strains had faster mycelial growth rate and higher colony density than the infected ones. It was also suggested that LeV-HKB infection may affect the pigmentation in plate- and bag-cultivated mycelia of L. edodes strain L135.

Manganese ferrite modified biochar from vinasse for enhanced adsorption of levofloxacin: Effects and mechanisms.

The primitive biochar (BC) and NiFe2O4/biochar composites (NFBC), biological adsorbents prepared from vinasse wastes, possess the environmental application in levofloxacin (LEV) removal. In this study, the efficient adsorption of LEV onto biochar synthesized by pyrolysis of vinasse wastes from aqueous environment was investigated. The influencing factors (i.e., pH, reaction time, and temperature) of adsorption process were also well studied. The results indicated that the maximum adsorption capacities of both BC and NFBC were occurred in mildly acidic condition (pH 6). In addition, the biochar adsorption capacities were obviously increased in higher temperature (25-45 °C). The chemistry adsorption and monolayer homogeneous dominated adsorption process of LEV onto BC and NFBC. The adsorption process was spontaneous and endothermic by thermodynamic analysis. The SEDA (site energy distribution analysis) explained that the adsorption effectivity increased by increasing site energy of biochar surface. The SEDA revealed the more energy heterogeneity in NFBC, fitting the characterization result of Fourier-transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The electron-donor-acceptor (EDA) interactions and hydrogen bonds is suggested as the major adsorption mechanism. And as for the adsorption of the various biowaste recycled synthetic, this study can be referred in discussion of performance analysis and optimal condition.

Mycoviral diversity and characteristics of a negative-stranded RNA virus LeNSRV1 in the edible mushroom Lentinula edodes.

Bioinformatics and RT-PCR analysis of RNA from four Lentinula edodes samples identified 22 different virus-like contigs comprising 15 novel and 3 previously reported viruses. We further investigated the Lentinula edodes negative-stranded RNA virus 1 (LeNSRV1) isolated from a symptomatic sample, whose virion is a filamentous particle with a diameter of ~15 nm and a length of ~1200 nm. RT-PCR analysis detected LeNSRV1 in 10 of the 56 Chinese L. edodes core collection strains and 6 of the 22 monokaryotic strains from the L. edodes strain HNZMD. Genetic variation analysis showed that the sequences encoding the nucleocapsid protein (ORF2) from all the aforementioned LeNSRV1 positive strains are very conservative. The results presented here may enrich our understanding of L. edodes virus diversity and the characteristics of LeNSRV1, and will promote further research on virus-host interaction in L. edodes.

The development of an efficient RNAi system based on Agrobacterium-mediated transformation approach for studying functional genomics in medical fungus Wolfiporia cocos.

Genetic transformation methods reported for Wolfiporia cocos are limited. In this study, we describe an efficient RNA interference (RNAi) system based on Agrobacterium-mediated transformation approach in W. cocos for the first time. Actively growing mycelial plugs were used as recipients for transformation using endogenous orotidine-5'-phosphate decarboxylase gene (URA3) as both a selective marker and a silencing gene, under the control of the dual promoters of Legpd and Leactin from Lentinula edodes and the single promoter of Wcgpd from W. cocos, respectively. The results showed that both the two kinds of promoters effectively drive the expression of URA3 gene, and the URA3-silenced transformants could be selected on CYM medium containing 5'-fluoroorotic acid. In addition, silencing URA3 gene has no effect on the growth of W. cocos hyphae. The incomplete silencing of the URA3 locus was also observed in this study. This study will promote further study on the mechanism of substrate degradation, sclerotial formation, and biosynthesis network of pharmacological compounds in W. cocos.

Characterization of Two Mitochondrial Genomes and Gene Expression Analysis Reveal Clues for Variations, Evolution, and Large-Sclerotium Formation in Medical Fungus Wolfiporia cocos.

Wolfiporia cocos, a precious mushroom with a long history as an edible food and Asian traditional medicine, remains unclear in the genetic mechanism underlying the formation of large sclerotia. Here, two complete circular mitogenomes (BL16, 135,686 bp and MD-104 SS10, 124,842 bp, respectively) were presented in detail first. The salient features in the mitogenomes of W. cocos include an intron in the tRNA (trnQ-UUG2), and an obvious gene rearrangement identified between the two mitogenomes from the widely geographically separated W. cocos strains. Genome comparison and phylogenetic analyses reveal some variations and evolutional characteristics in W. cocos. Whether the mitochondrion is functional in W. cocos sclerotium development was investigated by analyzing the mitogenome synteny of 10 sclerotium-forming fungi and mitochondrial gene expression patterns in different W. cocos sclerotium-developmental stages. Three common homologous genes identified across ten sclerotium-forming fungi were also found to exhibit significant differential expression levels during W. cocos sclerotium development. Most of the mitogenomic genes are not expressed in the mycelial stage but highly expressed in the sclerotium initial or developmental stage. These results indicate that some of mitochondrial genes may play a role in the development of sclerotium in W. cocos, which needs to be further elucidated in future studies. This study will stimulate new ideas on cytoplasmic inheritance of W. cocos and facilitate the research on the role of mitochondria in large sclerotium formation.

Fabrication of sustainable manganese ferrite modified biochar from vinasse for enhanced adsorption of fluoroquinolone antibiotics: Effects and mechanisms.

An effective adsorbent towards fluoroquinolone antibiotics was synthesized via a facile two-step approach, the co-precipitation of Fe, Mn with vinasse wastes and then pyrolysis under controlled conditions which denoted as FMB. Its adsorption behavior was examined based on a batch adsorption experiment of fluoroquinolone antibiotics pefloxacin (PEF) and ciprofloxacin (CIP). Experimental factors, such as pH, adsorbent dose, ionic strength, contact time and temperature have done a great deal to influence the adsorption of PEF and CIP. The FMB demonstrated excellent performance in reusability tests towards to both PEF and CIP, which showed that the recycling efficiency of PEF and CIP could remain ~55% and ~80% after five recycle cycles, respectively. The dominated adsorption mechanisms included pore filling effect, π-π stacking interaction, π-π EDA, hydrogen bonding and hydrophobicity. Overall, this work presented FMB was recognized as an effective, environmental-friendly and magnetically separable adsorbent for alleviating fluoroquinolone antibiotics contamination from water.

A sustainable ferromanganese biochar adsorbent for effective levofloxacin removal from aqueous medium.

This present study reported the synthesis and characterization of a low-cost, environment friendly and high efficient biochar, ferromanganese modified biochar (Fe/Mn-BC) for the removal of levofloxacin (LEV) from aqueous medium. Fe/Mn-BC was synthesized through the facile co-precipitation of Fe, Mn with vinasse wastes and then pyrolysis under controlled conditions. The characterization of Fe/MnBC was analyzed by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction patterns (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman. Some influencing factors (e.g., pH, Fe/Mn-BC dosage, initial LEV concentration, ionic strength, contact time and temperature) were comprehensively investigated. The results manifested that the adsorption process of LEV onto Fe/Mn-BC was high pH dependence and the maximum adsorption capacity was achieved at pH 5. Moreover, the adsorption capacity of LEV was increased with increasing ionic strength. To gain a clearer perspective on the adsorption behavior of LEV onto Fe/Mn-BC, the adsorption kinetics and isotherms were also performed, revealing pseudo-second-order and Freundlich model had a better fitting effect. Reusability experiments indicated that Fe/Mn-BC could maintain a certain adsorption capacity for LEV after 5 recycles. Overall, this work showed that Fe/Mn-BC was an effective and promising adsorbent for eliminating LEV from aqueous medium.

Carbon-based materials as adsorbent for antibiotics removal: Mechanisms and influencing factors.

With the development of the removal of organic pollutants in the soil and water environment, antibiotics have been considered as emerging pollutants and received considerable attention among the scientific community. Thus, there is a need for an effective, economical, fast, operational feasible and environmental-friendly technology to remove antibiotics. Adsorption technology would be one of the most promising option on the basis that it best meets the criteria we set out above. From the most primitive activated carbon to the most innovative modified biochar, carbon-based materials have played a significant role in the adsorption process of antibiotics all the time. This paper reviews the adsorption behavior of some representative antibiotics (e.g., chloramphenicols, sulfonamides, tetracyclines, flouroquinolones) over various carbonaceous materials (i.e., activated carbon, carbon nanotubes, graphene, and biochar). Nevertheless, in addition to the structural characteristics and adsorption capacities of carbon-based materials, a special emphasis was placed on the underlying adsorption mechanisms and roles of different influencing factors in the adsorption process. Moreover, the knowledge gaps and research challenges have been highlighted, including design and optimization of the carbonaceous materials for antibiotics adsorption.

Genomics and Development of Lentinus tigrinus: A White-Rot Wood-Decaying Mushroom with Dimorphic Fruiting Bodies.

Lentinus tigrinus is a species of wood-decaying fungi (Polyporales) that has an agaricoid form (a gilled mushroom) and a secotioid form (puffball-like, with enclosed spore-bearing structures). Previous studies suggested that the secotioid form is conferred by a recessive allele of a single locus. We sequenced the genomes of one agaricoid (Aga) strain and one secotioid (Sec) strain (39.53-39.88 Mb, with 15,581-15,380 genes, respectively). We mated the Sec and Aga monokaryons, genotyped the progeny, and performed bulked segregant analysis (BSA). We also fruited three Sec/Sec and three Aga/Aga dikaryons, and sampled transcriptomes at four developmental stages. Using BSA, we identified 105 top candidate genes with nonsynonymous SNPs that cosegregate with fruiting body phenotype. Transcriptome analyses of Sec/Sec versus Aga/Aga dikaryons identified 907 differentially expressed genes (DEGs) along four developmental stages. On the basis of BSA and DEGs, the top 25 candidate genes related to fruiting body development span 1.5 Mb (4% of the genome), possibly on a single chromosome, although the precise locus that controls the secotioid phenotype is unresolved. The top candidates include genes encoding a cytochrome P450 and an ATP-dependent RNA helicase, which may play a role in development, based on studies in other fungi.

Genetic variation and phylogenetic analyses reveal transmission clues of Lentinula edodes partitivirus 1 (LePV1) from the Chinese L. edodes core collection.

Lentinula edodes partitivirus 1 (LePV1), a new mycovirus possibly responsible for serious morphological deformities during L. edodes cultivation, is widespread in the Chinese L. edodes germplasm. Here, LePV1 isolates from the Chinese genetically-diverse L. edodes core collection were identified to be highly conserved and devoid of codivergence between virus and its hosts. Phylogenetic analysis showed that the LePV1 isolates could be grouped into two distinct clades (subtype I and subtype II), without geographical bias in the composition of this population. Compared with the other LePV1 isolates, one non-synonymous variation was observed in the LePV1 isolate from the symptomatic strain SX12. Purifying selection appears to be the main evolutionary force acting on LePV1 and it may be subject to negative selection. Based on the aforementioned results, the domestication history of L. edodes in China and the high frequency of virus incidence in basidiospores, we postulate that LePV1 may exist in nature and have had relationship with L. edodes wild strains since early times. Moreover, wind-blown spores carrying LePV1 may play an important role for the transmission of LePV1 in nature, while artificial activities such as vegetative propagation and hybridization breeding may also transmit virus from wild strains to cultivated ones.

Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes.

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

Genome Sequence of the Edible Cultivated Mushroom Lentinula edodes (Shiitake) Reveals Insights into Lignocellulose Degradation.

Lentinula edodes, one of the most popular, edible mushroom species with a high content of proteins and polysaccharides as well as unique aroma, is widely cultivated in many Asian countries, especially in China, Japan and Korea. As a white rot fungus with lignocellulose degradation ability, L. edodes has the potential for application in the utilization of agriculture straw resources. Here, we report its 41.8-Mb genome, encoding 14,889 predicted genes. Through a phylogenetic analysis with model species of fungi, the evolutionary divergence time of L. edodes and Gymnopus luxurians was estimated to be 39 MYA. The carbohydrate-active enzyme genes in L. edodes were compared with those of the other 25 fungal species, and 101 lignocellulolytic enzymes were identified in L. edodes, similar to other white rot fungi. Transcriptome analysis showed that the expression of genes encoding two cellulases and 16 transcription factor was up-regulated when mycelia were cultivated for 120 minutes in cellulose medium versus glucose medium. Our results will foster a better understanding of the molecular mechanism of lignocellulose degradation and provide the basis for partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

Development of crossbreeding high-yield-potential strains for commercial cultivation in the medicinal mushroom Wolfiporia cocos (Higher Basidiomycetes).

Wolfiporia cocos is a well-known medicinal mushroom, and its dried sclerotia has been widely used as a traditional medicine in China, Japan, and other Asian countries for centuries. However, long-term asexual reproduction of the breeding system in W. cocos results in a current universal degeneration of cultivated strains. To develop a W. cocos breeding program that will benefit commercial cultivation, we previously developed an optimum method for indoor induction of W. cocos fruiting bodies and clarified the nature of preponderant binuclear sexual basidiospores. In this paper, we first show that the majority of W. cocos single-spore isolates cannot form sclerotium in field cultivation. We then investigated the possibility of breeding new strains by crossbreeding. Three types of mating reactions were observed in both intra-strain pairings and inter-strain pairings, and a total of fifty-five hybrids were selected by antagonistic testing and allele-specific polymerase chain reaction (PCR). Field cultivation of hybrids demonstrated that some hybrids can form sclerotium via two cultivated methods. Two new high-yield strains were identified. This report will stimulate new thinking on W. cocos and promote further extensive studies on crossbreeding in W. cocos, a new topic related to the development of more efficient protocols for the discrimination of hybrids in W. cocos.