Scorpionicidal activity of secondary metabolites from Paecilomyces sp. CMAA1686 against Tityus serrulatus.

INTRODUCTION: Urban pests pose enormous risks to human health. Control initiatives are carried out in regions of high infestation and incidence of accidents caused by scorpions OBJECTIVE: In this study, we aimed to analyze the anti-scorpionic activity of fungal isolates obtained from a cemetery in Brazil. MATERIALS AND METHODS: A total of thirteen fungi were subjected to a bioassay test against Tityus serrulatus, and the two isolates with the highest scorpionicidal activity were selected for molecular identification through sequencing of the ITS DNA hypervariable region and large-scale cultivation on liquid medium for secondary metabolite extraction. The crude extracts were partitioned by solid-phase extraction, and the resulting purified extracts were tested for anti-scorpionic activity. The extracts from one of the isolates presented better results and were submitted to UPLC-MS/MS. The metabolomics data were submitted to GNPS website for Molecular Networking and MASST searches. We also performed a MolNetEnhancer analysis to identify the chemical classes of the molecules found in the samples. RESULTS: The most promising fungal isolate was identified as Paecilomyces sp. CMAA1686 which has 98% of similarity to Paecilomyces formosus. The sub-fractions C and D had the best activity against the scorpions (54 and 32% mortality, respectively). Molecular Networking and MolNetEnhancer revealed a range of molecular classes in our extracts that are known to include bioactive metabolites from Paecilomyces species. CONCLUSIONS: The scorpionicidal activity of Paecilomyces sp. CMAA1686 and its secondary metabolites may provide new alternative compounds for biological and chemical control of scorpions from the species T. serrulatus. Paecilomyces sp. CMAA1686 is an isolate that has great potential for isolation of secondary metabolites.

Infection With a Novel Rickettsiella Species in Emperor Scorpions (Pandinus imperator).

Rickettsiella infection was diagnosed in 4 adult emperor scorpions (Pandinus imperator) from 2 different collections over a 3-year period. One case had a 2-day history of weakness, failure to lift the tail, or respond to stimulation, with rapid progression to death. The other 3 cases were found dead. There were no gross lesions, but histologically the hemolymphatic vasculature and sinuses, presumed hematopoietic organ, heart, midgut and midgut diverticula, nerves, and skeletal muscle were infiltrated with phagocytic and granular hemocytes with necrosis. Phagocytic hemocytes contained abundant intracellular microorganisms that were Fite's acid-fast-positive, Macchiavello-positive, variably gram-positive or gram-negative, and Grocott's methenamine silver-negative. By transmission electron microscopy, hemocytes contained numerous phagocytic vacuoles with small dense bacterial forms (mean 0.603 × 0.163 µm) interspersed with large bacterial forms (mean 1.265 × 0.505 µm) and few intermediary forms with electron-dense nucleoids and membrane-bound crystalline arrays (average 4.72 µm). Transmission electron microscopy findings were consistent with bacteria of the family Coxiellaceae. Based on sequencing the 16S ribosomal RNA gene, the identity was confirmed as Rickettsiella, and phylogenetic analysis of protein-coding genes gidA, rspA, and sucB genes suggested the emperor scorpion pathogen as a new species. This study identifies a novel Rickettsiella causing infection in emperor scorpions and characterizes the unique pathological findings of this disease. We suggest this organism be provisionally named Rickettsiella scorpionisepticum.

Highly divergent Mollicutes symbionts coexist in the scorpion Androctonus australis.

Androctonus australis is one of the most ubiquitous and common scorpion species in desert and arid lands from North Africa to India and it has an important ecological role and social impact. The bacterial community associated to this arachnid is unknown and we aimed to dissect its species composition in the gut, gonads, and venom gland. A 16S rRNA gene culture-independent diversity analysis revealed, among six other taxonomic groups (Firmicutes, Betaproteobacteria, Gammaproteobacteria, Flavobacteria, Actinobacteria, and Cyanobacteria), a dominance of Mollicutes phylotypes recorded both in the digestive tract and the gonads. These related Mollicutes include two Spiroplasma phylotypes (12.5% of DGGE bands and 15% of clones), and a new Mycoplasma cluster (80% of clones) showing 16S rRNA sequence identities of 95 and 93% with Mollicutes detected in the Mexican scorpions Centruroides limpidus and Vaejovis smithi, respectively. Such scorpion-associated Mollicutes form a new lineage that share a distant ancestor with Mycoplasma hominis. The observed host specificity with the apparent phylogenetic divergence suggests a relatively long co-evolution of these symbionts with the scorpion hosts. From the ecological point of view, such association may play a beneficial role for the host fitness, especially during dormancy or molt periods.

Species-specific diversity of novel bacterial lineages and differential abundance of predicted pathways for toxic compound degradation in scorpion gut microbiota.

Scorpions are considered 'living fossils' that have conserved ancestral anatomical features and have adapted to numerous habitats. However, their gut microbiota diversity has not been studied. Here, we characterized the gut microbiota of two scorpion species, Vaejovis smithi and Centruroides limpidus. Our results indicate that scorpion gut microbiota is species-specific and that food deprivation reduces bacterial diversity. 16S rRNA gene phylogenetic analysis revealed novel bacterial lineages showing a low level of sequence identity to any known bacteria. Furthermore, these novel bacterial lineages were each restricted to a different scorpion species. Additionally, our results of the predicted metagenomic profiles revealed a core set of pathways that were highly abundant in both species, and mostly related to amino acid, carbohydrate, vitamin and cofactor metabolism. Notably, the food-deprived V. smithi shotgun metagenome matched almost completely the metabolic features of the prediction. Finally, comparisons among predicted metagenomic profiles showed that toxic compound degradation pathways were more abundant in recently captured C. limpidus scorpions. This study gives a first insight into the scorpion gut microbiota and provides a reference for future studies on the gut microbiota from other arachnid species.

A novel cysteine-free venom peptide with strong antimicrobial activity against antibiotics-resistant pathogens from the scorpion Opistophthalmus glabrifrons.

Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic-resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α-helical molecule. Protein sequence homology search revealed that Opisin shares 42.1-5.3% sequence identities to the 17/18-mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram-positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0-10.0 µM; in contrast, it possesses much lower activity against the tested Gram-negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin- and vancomycin-resistant pathogens with the MICs ranging from 2.0 to 4.0 µM and from 4.0 to 6.0 µM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin-resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic-resistant bacterial infections.

A first molecular characterization of the scorpion telson microbiota of Hadrurus arizonensis and Smeringurus mesaensis.

Scorpions represent an ancient lineage of arachnids that have radiated across the globe and are incredibly resilient-since some thrive in harsh environments and can exist on minimal and intermittent feedings. Given the emerging importance of microbiomes to an organism's health, it is intriguing to suggest that the long-term success of the scorpion bauplan may be linked to the microbiome. Little is known about scorpion microbiomes, and what is known, concentrates on the gut. The microbiome is not limited to the gut, rather it can be found within tissues, fluids and on external surfaces. We tested whether the scorpion telson, the venom-producing organ, of two species, Smeringurus mesaensis and Hadrurus arizonensis, contain bacteria. We isolated telson DNA from each species, amplified bacterial 16S rRNA genes, and identified the collection of bacteria present within each scorpion species. Our results show for the first time that telsons of non-buthid scorpion species do indeed contain bacteria. Interestingly, each scorpion species has a phylogenetically unique telson microbiome including Mollicutes symbionts. This study may change how we view scorpion biology and their venoms.

The divergent genome of Scorpion Group 1 (SG1) intracellular bacterium from the venom glands of Vaejovis smithi (Scorpiones: Vaejovidae).

Scorpions were among the first animals on land around 430 million years ago. Like many arachnids, scorpions have evolved complex venoms used to paralyze their prey and for self-defense. Here we sequenced and analyzed the metagenomic DNA from venom glands from Vaejovis smithi scorpions. A metagenome-assembled genome (MAG) of 624,025 bp was obtained corresponding to the previously reported Scorpion Group 1 (SG1). The SG1 genome from venom glands had a low GC content (25.8%) characteristic of reduced genomes, many hypothetical genes and genes from the reported minimal set of bacterial genes. Phylogenomic reconstructions placed the uncultured SG1 distant from other reported bacteria constituting a taxonomic novelty. By PCR we detected SG1 in all tested venom glands from 30 independent individuals. Microscopically, we observed SG1 inside epithelial cells from the venom glands using FISH and its presence in scorpion embryos suggested that SG1 is transferred from mother to offspring.

Cophylogenetic analysis suggests cospeciation between the Scorpion Mycoplasma Clade symbionts and their hosts.

Scorpions are predator arachnids of ancient origin and worldwide distribution. Two scorpion species, Vaejovis smithi and Centruroides limpidus, were found to harbor two different Mollicutes phylotypes: a Scorpion Mycoplasma Clade (SMC) and Scorpion Group 1 (SG1). Here we investigated, using a targeted gene sequencing strategy, whether these Mollicutes were present in 23 scorpion morphospecies belonging to the Vaejovidae, Carboctonidae, Euscorpiidae, Diplocentridae, and Buthidae families. Our results revealed that SMC is found in a species-specific association with Vaejovidae and Buthidae, whereas SG1 is uniquely found in Vaejovidae. SMC and SG1 co-occur only in Vaejovis smithi where 43% of the individuals host both phylotypes. A phylogenetic analysis of Mollicutes 16S rRNA showed that SMC and SG1 constitute well-delineated phylotypes. Additionally, we found that SMC and scorpion phylogenies are significantly congruent, supporting the observation that a cospeciation process may have occurred. This study highlights the phylogenetic diversity of the scorpion associated Mollicutes through different species revealing a possible cospeciation pattern.

Genome Analyses of a New Mycoplasma Species from the Scorpion Centruroides vittatus.

Arthropod Mycoplasma are little known endosymbionts in insects, primarily known as plant disease vectors. Mycoplasma in other arthropods such as arachnids are unknown. We report the first complete Mycoplasma genome sequenced, identified, and annotated from a scorpion, Centruroides vittatus, and designate it as Mycoplasma vittatus We find the genome is at least a 683,827 bp single circular chromosome with a GC content of 42.7% and with 987 protein-coding genes. The putative virulence determinants include 11 genes associated with the virulence operon associated with protein synthesis or DNA transcription and ten genes with antibiotic and toxic compound resistance. Comparative analysis revealed that the M. vittatus genome is smaller than other Mycoplasma genomes and exhibits a higher GC content. Phylogenetic analysis shows M. vittatus as part of the Hominis group of Mycoplasma As arthropod genomes accumulate, further novel Mycoplasma genomes may be identified and characterized.

Inducible antibacterial response of scorpion venom gland.

Innate immunity is the first line defense of multicellular organisms that rapidly operates to limit aggression upon exposure to pathogen microorganisms. Although the existence of some antibacterial peptides in scorpion venoms suggests that venom gland could be protected by these effector molecules, antibacterial activity of venom itself has not been assessed. In this study, we reported the antibacterial activity of the venom of Chinese scorpion Buthus martensii. Protease K digestion test indicated that it is venom peptide/protein components, as key players, which are involved in such antibacterial response. As the first step toward studying molecular mechanism of scorpion venom gland immunity, we established an infection model which supports inducible antibacterial response of scorpion venom gland. A known B. martensii antibacterial peptide gene BmKb1 was up-regulated at the transcriptional level after venom gland was challenged, suggesting its key defense role. This is further strengthened by the presence of several immune response elements in the BmKb1 promoter region. Our work thus provides the first evidence supporting the role of venom antibacterial peptides (ABPs) in controlling scorpion venom gland infection and lays a basis for characterizing related components involved in regulation of scorpion venom gland ABP gene expression.

[Microbial diversity in scorpion intestine (Buthus martensii Karsch)].

Scorpion is an important officinal animal, and has a high nutritional value. In this study, the culture-independent and culture-dependent methods were used to investigate the microbial diversity in the scorpion's intestine. Results based on culture-independent method showed the bacteria to be related to alpha, beta, gamma-proteobacteria. Bacteria isolated by the culture-dependent method were high G + C, gram-positive bacteria. The genera Enterobacter, Serratia and Ochrobactrum were detected by both methods. To sum up the results from the two methods, the bacteria in scorpion intestine belong to 23 genera, which are Enterobacter, Serratia, Pseudomonas, Acinetobacter, Aeromonas, Citrobacter, Pedobacter, Delftia, Ralstonia, Ochrobactrum, Sphingomonas, Exiguobacterium, Gordonia, Nocardia, Rhodococcus, Janibacte, Kocuria, Micrococcus, Agromyces, Microbacterium, Agrococcus, Deinococcus, Ornithinimicrobium, and some uncultured species. The two methods have both advantages and shortcomings. However, when used simultaneously, they complement each other.

Getting insight into the prevalence of antibiotic resistance genes in specimens of marketed edible insects.

This study was aimed at investigating the occurrence of 11 transferable antibiotic resistance (AR) genes [erm(A), erm(B), erm(C), vanA, vanB, tet(M), tet(O), tet(S), tet(K), mecA, blaZ] in 11 species of marketed edible insects (small crickets powder, small crickets, locusts, mealworm larvae, giant waterbugs, black ants, winged termite alates, rhino beetles, mole crickets, silkworm pupae, and black scorpions) in order to provide a first baseline for risk assessment. Among the AR genes under study, tet(K) occurred with the highest frequency, followed by erm(B), tet(S) and blaZ. A high variability was seen among the samples, in terms of occurrence of different AR determinants. Cluster Analysis and Principal Coordinates Analysis allowed the 11 samples to be grouped in two main clusters, one including all but one samples produced in Thailand and the other including those produced in the Netherlands.

[The hygienic examination and quality research of Chinese crude drug scorpion].

We selected the method of hygienic test to determinate the infection of colibacilli, salmonelli, mixed bacteria, mould fungus and yeasts on Chinese crude drug scorpion from 29 commerical samples in different storaged period, habitats and commerical standard. The results showed there were not colibacilli and salmonelli in all 29 samples, but infectious mixed bacteria rate is 100%, which is 2.1 times more than salty scorpion in 29 tested samples, and the infectious fungi rate is 72.4%. The fungi of salty scorpion is 15% more than fresh scorpion. The quantity of infected yeasts on salty scorpion is much more than scorpion. There are 4 species of fungi such as Alternaria neesex Wallroth, Aspergillus fumigatus Fresenius, Nocarcia sp. and Tricophyton violaceum Sabouraud. Because of infectious pathogenic bacteria rate is so high, we suggest to increase the item of hygienic test to control the quality of crude drug scorpion and strengthen the administration of commerical drugs, so as to reduce the contaminative condition.

Bacterial endosymbiont infections in 'living fossils': a case study of North American vaejovid scorpions.

Bacterial endosymbionts are common among arthropods, and maternally inherited forms can affect the reproductive and behavioural traits of their arthropod hosts. The prevalence of bacterial endosymbionts and their role in scorpion evolution have rarely been investigated. In this study, 61 samples from 40 species of scorpion in the family Vaejovidae were screened for the presence of the bacterial endosymbionts Cardinium, Rickettsia, Spiroplasma and Wolbachia. No samples were infected by these bacteria. However, one primer pair specifically designed to amplify Rickettsia amplified nontarget genes of other taxa. Similar off-target amplification using another endosymbiont-specific primer was also found during preliminary screenings. Results caution against the overreliance on previously published screening primers to detect bacterial endosymbionts in host taxa and suggest that primer specificity may be higher in primers targeting nuclear rather than mitochondrial genes.

Natural occurrence of the Fusarium solani on Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae).

Members of the Fusarium solani species complex are agents of human mycoses, also affecting plants and other animals. Nevertheless, this fungus has not been reported on scorpions. Ten specimens of Tityus stigmurus collected in the field and showing their surface covered by white mycelia were used to assess fungus presence in the animal after its death. Identification of the fungi was based upon the cultural and morphological characteristics. The fungus was isolated from chelicerae and intersegmental regions. Infected individuals had their behaviour modified by reducing feeding and locomotion. None of the infected individuals survived. It is likely that this fungus may have a role in the regulation of field scorpion populations.

Wolbachia are present in southern african scorpions and cluster with supergroup F.

The presence and distribution of the intracellular bacteria Wolbachia in the arthropod subphylum Chelicerata (including class Arachnida) has not been extensively explored. Here we report the discovery of Wolbachia in scorpions. Five strains found in host species of the genus Opistophthalmus (Southern African burrowing scorpions) have been characterized by Multilocus Sequence Typing and by Wolbachia Surface Protein. Phylogenetic analyses indicate clustering in the supergroup F and a high genetic relatedness among all scorpion strains as a result of a potential transmission within the host genus. The F-group is an uncommon lineage compared to the A and B supergroups, although it is present in a broad range of hosts (including insects, filarial nematodes, and now arachnids) and across a large geographical area (e.g., North America, Africa, Europe, and Australia). It also shows no evidence of recombination and has a significantly higher genetic diversity than supergroup A and B. Overall, this pattern suggests an older radiation of F-strains with respect to A and B-strains, followed by limited horizontal transmission across host genera and reduced genetic flux among strains. A more extensive sampling of supergroup F-strains is required to confirm this scenario.

Natural occurrence of the Fusarium solani on Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae) / Ocorrência natural de Fusarium solani em Tityus stigmurus (Thorell, 1876) (Scorpions: Buthidae)

Members of the Fusarium solani species complex are agents of human mycoses, also affecting plants and other animals. Nevertheless, this fungus has not been reported on scorpions. Ten specimens of Tityus stigmurus collected in the field and showing their surface covered by white mycelia were used to assess fungus presence in the animal after its death. Identification of the fungi was based upon the cultural and morphological characteristics. The fungus was isolated from chelicerae and intersegmental regions. Infected individuals had their behaviour modified by reducing feeding and locomotion. None of the infected individuals survived. It is likely that this fungus may have a role in the regulation of field scorpion populations.

Ocorrência natural de Fusarium solani em Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae). Membros do complexo de espécies de Fusarium solani são freqüentemente referidos como agentes de micoses humanas, podendo também afetar plantas e outros animais. Contudo, esse fungo ainda não foi registrado como causador de infecções em escorpiões. Dez espécimes de Tityus stigmurus coletados em campo e apresentando micélio branco cobrindo a superfície do corpo foram usados para analisar a presença de fungo após a sua morte. A identificação do fungo foi baseada nas características da cultura e morfológicas. O fungo foi isolado de quelíceras e regiões intersegmentais. Indivíduos infectados tiveram seu comportamento modificado, reduzindo sua alimentação e locomoção. Nenhum dos indivíduos infectados sobreviveu. É possível que este fungo possa ter um papel na regulação da população de campo desse escorpião.

[A cytoplasmic virus in the scorpion Buthus occitanus Amoreux]. / Un virus cytoplasmique chez le scorpion Buthus occitanus Amoreux

Isometric virus particles, 60 nm in diameter, have been isolated from diseased scorpions. The virus is pathogenic after ingestion. Its morphogenesis occurs in the cytoplasm of hepatopancreas cells which are totally destroyed at the end of the disease.

[Scorpion toxins and defensins]. / Toxines et défensines de scorpions.

The scorpion venoms possess many neurotoxic peptides which constitute a group of molecular families with a common architecture and a high degree of polymorphism. This architecture is found also in circulating antimicrobial peptides belonging to the defensins family, which are especially structurally related to the blocking potassium channels neurotoxins. The diversification in functions with a unique architectural scheme is discussed taking in account the biophysiological characteristics of the scorpion order.