RESUMO
In contrast to vertebrate immune systems, invertebrates lack an adaptive response and rely solely on innate immunity in which antimicrobial peptides (AMPs) play an essential role. Most of them are membrane active molecules that are typically unstructured in solution and adopt secondary/tertiary structures upon binding to phospholipid bilayers. This work presents the first characterization of a constitutive AMP from the hemolymph of an Opiliones order animal: the harvestman Acutisoma longipes. This peptide was named longipin. It presents 18 aminoacid residues (SGYLPGKEYVYKYKGKVF) and a positive net charge at neutral pH. No similarity with other AMPs was observed. However, high sequence similarity with heme-lipoproteins from ticks suggested that longipin might be a protein fragment. The synthetic peptide showed enhanced antifungal activity against Candida guilliermondii and C. tropicalis yeasts (MIC: 3.8-7.5 µM) and did not interfered with VERO cells line viability at all concentrations tested (200-0.1 µM). This selectivity against microbial cells is related to the highest affinity of longipin for anionic charged vesicles (POPG:POPC) compared to zwitterionic ones (POPC), once microbial plasma membrane are generally more negatively charged compared to mammalian cells membrane. Dye leakage from carboxyfluorescein-loaded POPG:POPC vesicles suggested that longipin is a membrane active antimicrobial peptide and FT-IR spectroscopy showed that the peptide chain is mainly unstructured in solution or in the presence of POPC vesicles. However, upon binding to POPG:POPC vesicles, the FT-IR spectrum showed bands related to ß-sheet and amyloid-like fibril conformations in agreement with thioflavin-T binding assays, indicating that longipin is an amyloid antimicrobial peptide.
Assuntos
Amiloide , Peptídeos Catiônicos Antimicrobianos , Aracnídeos , Proteínas de Artrópodes , Bactérias/crescimento & desenvolvimento , Candida/crescimento & desenvolvimento , Amiloide/química , Amiloide/genética , Amiloide/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aracnídeos/química , Aracnídeos/genética , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/farmacologia , Chlorocebus aethiops , Células VeroRESUMO
Natural selection shapes prey-predator relationships and their behavioral adaptations, which seek to maximize capture success in the predator and avoidance in the prey. We tested the ability of adults of the scorpion Bothriurus bonariensis (Bothriuridae) to prey on synchronous and sympatric adults harvestmen of Acanthopachylus aculeatus, Discocyrtus prospicuus, Parampheres bimaculatus and Pachyloides thorellii (Gonyleptidae). In 72.5% of the cases B. bonariensis tried to prey on the harvestmen. The most successful captures occurred in the trials against A. aculeatus and D. prospicuus. In all the successful attacks the scorpions stung the prey between the chelicerae and consumed them, starting by the anterior portion of their bodies. The harvestmen used different defensive strategies such as fleeing before or after contact with the predator, exudating of chemical substances or staying still at the scorpion's touch. When scorpions contacted the chemical substances secreted by the harvestmen, they immediately rubbed the affected appendix against the substrate. However, exudating of chemical substances did not prevent, in any case, predation on the harvestmen. This is the first study showing the ability of scorpions to prey on different species of harvestmen, as well as the capture and defensive behaviors used by the predator and the prey.
Assuntos
Comportamento Predatório/fisiologia , Escorpiões/fisiologia , Animais , Aracnídeos/química , Aracnídeos/fisiologia , Comportamento Animal , Secreções Corporais/metabolismo , Escorpiões/químicaRESUMO
Harvestmen have a pair of scent glands that open through ozopores. The literature suggests a link between the morphology of the ozopore area and the emission of a defensive secretion. A previous study on a species that aggregates in open areas, where individuals are probably more easily spotted by predators, showed that this defensive secretion causes conspecifics to flee. However, it is unknown whether this behavior occurs in species that aggregate in sheltered areas, where prey are harder to find. Herein, we describe the morphology of the ozopore area, the mode of emission of the defensive secretion, and its chemical composition in the harvestman Discocyrtus pectinifemur. We also tested if the defensive secretion is used as an alarm pheromone. We found that D. pectinifemur releases the defensive secretion in different ways, one of them being as a jet. Emission as a jet contrasts with that known for all congeners previously studied, and is in accord with the expected morphology of the ozopore. We found that the defensive secretion of D. pectinifemur does not function as an alarm pheromone. The composition of the defensive secretion, a mixture of quinones, is congruent with those already described for the clade that includes Discocyrtus. Our results support the link between the morphology of the scent glands area and the emission behavior of the defensive secretion, and they suggest that the alarm pheromone function in harvestmen may be dependent on ecological factors.
Assuntos
Aracnídeos/fisiologia , Animais , Aracnídeos/anatomia & histologia , Aracnídeos/química , Feminino , Masculino , Feromônios/análise , Glândulas Odoríferas/anatomia & histologia , Glândulas Odoríferas/química , Glândulas Odoríferas/fisiologiaRESUMO
Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation.
Assuntos
Músculo Liso/química , Cadeias Leves de Miosina/química , Dobramento de Proteína , Animais , Aracnídeos/química , Aracnídeos/metabolismo , Cálcio/metabolismo , Simulação de Dinâmica Molecular , Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Estrutura Secundária de ProteínaRESUMO
Benzoquinones are usually present in arthropod defence exudates. Here, we describe the chemical profiles of 12 harvestman species belonging to the neotropical family Gonyleptidae. Nine of the studied species produced benzoquinones, while three produced alkyl phenols. Two benzoquinones and one phenol exhibited biological activity against bacteria and fungi. We also studied the biosynthesis of 2-ethyl-1,4-benzoquinone by feeding Magnispina neptunus individuals with ¹³C-labelled precursors; the benzoquinones were biosynthesised through a polyketide pathway using acetate and propionate building blocks.
Assuntos
Aracnídeos/metabolismo , Benzoquinonas/química , Fenóis/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aracnídeos/química , Bacillus/efeitos dos fármacos , Benzoquinonas/metabolismo , Benzoquinonas/farmacologia , Vias Biossintéticas , Candida albicans/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Estrutura Molecular , Fenóis/metabolismo , Fenóis/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Four species of gonyleptid harvestmen, Acanthogonyleptes pulcher, Gonyleptes saprophilus (Gonyleptinae), Sodreana barbiellini, and Sodreana leprevosti (Sodreaninae), were examined by GC-MS and ¹³H NMR. All of these species release vinyl ketones, and three of them produce the corresponding pyranyl ketones, which are presumed hetero-Diels-Alder (HDA) dimers. The vinyl ketones 5-methyl-1-hexen-3-one, rac-4-methyl-1-hexen-3-one, and (S)-4-methyl-1-hexen-3-one were synthesized. Natural 4-methyl-1-hexen-3-one is present as a single stereoisomer and has the R-configuration. Vinyl ketone dimers (HDA dimers) were also observed in the scent gland exudate and characterized by HRMS, ¹³C NMR, and ¹H NMR chemical shifts of the pyranyl moiety.
Assuntos
Aracnídeos/química , Cetonas/química , Piranos/química , Glândulas Odoríferas/química , Compostos de Vinila/química , Animais , Aracnídeos/classificação , Aracnídeos/genética , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Glândulas Odoríferas/metabolismo , EstereoisomerismoRESUMO
The defensive secretions of five neotropical species of harvestmen (Opiliones: Gonyleptidae) from the Brazilian Atlantic Forest were analyzed and chemically characterized by GC-MS and NMR methods. Three of the species, Cobania picea, Roweria virescens, and Serracutisoma proximum, secrete a mixture of 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. The secretions produced by the other two species, Iporangaia pustulosa and Neosadocus maximus, contain 1-hepten-3-one, 5-methyl-1-hexen-3-one, and 1-(6-butyl-3,4-dihydro-2H-pyran-2-yl)pentanone (1) as major components, as well as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone as minor constituents. The dihydropyran 1-(6-butyl-3,4-dihydro-2H-pyran-2-yl)pentanone (1) is a new natural product, composed of two 1-hepten-3-one subunits formally linked in a hetero-Diels-Alder reaction. The natural product was proven to be racemic, and its biogenetic origin is discussed.
Assuntos
Aracnídeos/química , Piranos/isolamento & purificação , Animais , Brasil , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pentanonas , Piranos/química , Piranos/farmacologia , Glândulas Odoríferas/metabolismo , EstereoisomerismoRESUMO
The importance of midgut diverticula (M-diverticula) and hemolymph lipoproteins in the lipid homeostasis of Polybetes phythagoricus was studied. Radioactivity distribution in tissues and hemolymph was analyzed either after feeding or injecting [1-(14)C]-palmitate. In both experiments, radioactivity was mostly taken up by M-diverticula that synthesized diacylglycerols, triacylglycerols and phospholipids in a ratio close to its lipid class composition. M-diverticula total lipids represent 8.08% (by wt), mostly triacylglycerols (74%) and phosphatidylcholine (13%). Major fatty acids were (in decreasing order of abundance) 18:1n-9, 18:2n-6, 16:0, 16:1n-7, 18:0, 18:3n-3. Spider hemocyanin-containing lipoprotein (VHDL) transported 83% of the circulating label at short incubation times. After 24h, VHDL and HDL-1 (comparable to insect lipophorin) were found to be involved in the lipid uptake and release from M-diverticula, HDL-2 playing a negligible role. Lipoprotein's labelled lipid changed with time, phospholipids becoming the main circulating lipid after 24h. These results indicate that arachnid M-diverticula play a central role in lipid synthesis, storage and movilization, analogous to insect fat body or crustacean midgut gland. The relative contribution of HDL-1 and VHDL to lipid dynamics indicated that, unlike insects, spider VHDL significantly contributes to the lipid exchange between M-diverticula and hemolymph.
Assuntos
Aracnídeos/metabolismo , Proteínas de Insetos/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Animais , Aracnídeos/química , Transporte Biológico , Sistema Digestório/metabolismo , Lipídeos/químicaRESUMO
In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Ceratite por Acanthamoeba/tratamento farmacológico , Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Aracnídeos/química , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hemócitos/química , Estrutura Secundária de Proteína , Coelhos , Proteínas e Peptídeos Salivares/síntese química , Proteínas e Peptídeos Salivares/química , Inibidores de Serina Proteinase/farmacologia , Triatoma/química , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismoRESUMO
Two alkylated 1,4-benzoquinones were identified from the defensive secretion produced by the neotropical harvestman Goniosoma longipes (Gonyleptidae). They were characterized as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. We tested the effectiveness of these benzoquinone secretions against several predator types, including invertebrates and vertebrates. Different predators were exposed to the harvestmen's gland secretion or to distilled water in laboratory bioassays. Our results indicate that secretions containing the 1,4-benzoquinones released by G. longipes can be an effective defense against predation, and that the effectiveness of the secretion is dependent on the predator type. The scent gland secretion repelled seven ant species, two species of large wandering spiders, and one frog species, but was not an effective defense against an opossum. Our study also demonstrates that the scent gland secretion of G. longipes can work as a chemical shield preventing the approach of three large predatory ants for at least 10 min. The chemical shield may protect the harvestman against successive attacks of the same ant worker and also allow the harvestman to flee before massive ant recruitment. Our data support the suggestion that chemical defenses may increase survival with some but not all potential predators. This variation in defense effectiveness may result from many interacting factors, including the attack strategy, size, learning ability, and physiology of the predators, as well as the chemical nature of the defensive compounds, type of emission, and amount of effluent released by the prey.
Assuntos
Aracnídeos/química , Benzoquinonas/farmacologia , Comportamento Predatório/efeitos dos fármacos , Glândulas Odoríferas/metabolismo , Adaptação Fisiológica , Comunicação Animal , Animais , Benzoquinonas/isolamento & purificação , Cicloexenos , Invertebrados/efeitos dos fármacos , Glândulas Odoríferas/química , Fatores de Tempo , VertebradosRESUMO
In the isolated rat heart, Phoneutria nigriventer spider venom (10-100 microg) produced a dose-dependent and reversible rise in left ventricular developed pressure. A low dose (10 microg) of venom induced a short-lasting, positive inotropic effect (P < 0.05) with no change in heart rate or coronary flow. At a dose of 50 microg, the venom caused significant positive inotropic and chronotropic responses associated with occasional ventricular arrhythmia, whereas coronary flow was not significantly affected within 10 min after venom administration. The highest dose of venom (100 microg) caused bradycardia, transient cardiac arrest, rhythm disturbances and an increase in end diastolic pressure followed by a reduction in coronary flow. Hearts treated with the non-selective beta-adrenoceptor antagonist propranolol (3 microM) and the selective beta1-adrenoceptor antagonist CGP-20712A (10 microM) were protected against all the cardiac actions of the venom. The selective beta2-adrenoceptor antagonist butoxamine (10 microM) slightly reduced the cardiac response to 50 microg, but not to 100 microg of venom. Butoxamine also prevented the reduction in coronary flow induced by 100 microg of venom. Hearts from reserpine-treated rats (5 mg kg(-1) day(-1), i.p., for 2 days) showed a marked decrease in all venom (< or = 100 microg)-induced cardiac responses. The muscarinic receptor antagonist atropine (1 microM) slightly potentiated the response to 50 microg of venom but had little or no effect on the responses to 100 microg of venom. The cardiac responses to venom (50-100 microg) were unaltered in hearts from rats treated with 8-methyl N-vanillyl-6-nonenamide (capsaicin; 50 mg/kg, s.c.). These findings indicate that P. nigriventer venom releases norepinephrine from cardiac sympathetic nerve endings and this may explain the observed increase in contractile force and heart rate.