Loxosceles gaucho Venom-Induced Acute Kidney Injury – In Vivo and In Vitro Studies

Loxosceles (recluse or brown spider) is the most important spider genus causing human envenomation. In Brazil Loxosceles spiders were responsible for approximately 7,000 cases of spider envenomation per year. The brown spider accidents may cause late cutaneous necrosis at the bite site, intravascular hemolysis, rhabdomyolysis, coagulation system changes and acute kidney injury (AKI). Even patients with mild cutaneous lesion may develop severe hemolysis and AKI, which is the main cause of death after these accidents. The mechanisms causing kidney injury are poorly understood. In this manuscript we described a consistent rodent model of Loxosceles gaucho venom-induced AKI and studied some of the possible mechanisms of the renal lesion. The results of this research showed that kidney injury may occur independently of the cutaneous lesion and without changes in the systemic blood pressure. Kidney dysfunction occurred likely due to intra-renal vasoconstriction and rhabdomyolysis, although a direct toxic effect of the venom on the proximal tubules cannot be ruled out

Predatory behavior of three centipede species of the order Scolopendromorpha (Arthropoda: Myriapoda: Chilopoda)

ABSTRACT Studies related to centipede feeding and predatory behavior are rare in the literature, and are limited to observations made during fieldwork. Furthermore, they lack descriptions of prey capture. We conducted a laboratory experiment using South American specimens of Scolopendra viridicornis Newport, 1844 (n = 5), Otostigmus tibialis Brölemann, 1902 (n = 5), and Cryptops iheringi Brölemann, 1902 (n = 5), as well as 13 different kinds of prey, to map and describe their predatory behavior. The analysis of video images (65 hours of recordings) resulted in 15 behavioral categories that describe foraging, prey capture, feeding, and cleaning habits. Almost all observations (95%) concluded with the centipede killing the prey. Although we witnessed that a stimulus triggered the movement of the centipede toward the prey in all observation events (suggesting a sit-and-wait strategy), our experiments also showed that these arthropods actively forage to seek food. Field observations during the experiment allowed us to document that scolopendromorphs feed on plants when animal prey items are not available. Moreover, we observed that the size and aggressiveness of the prey determined the centipede capture process. Our results revealed that two behavioral categories were performed only by S. viridicornis , and thus might be genus or species-specific. These are: raising the first third of the body while the rest of the body remains adjacent to the substrate; and restraining the prey along the ventral region of the first third of the body with the aid of locomotory legs. We also observed some peculiar behaviors performed only by O. tibialis . Our results confirm that S. viridicornis , O. tibialis and C. iheringi hold prey between their ultimate pair of legs

<i>Bothrops jararaca</i> Venom Metalloproteinases Are Essential for Coagulopathy and Increase Plasma Tissue Factor Levels during Envenomation

<div><p>Background/Aims</p><p>Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF), resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a) whether TF and protein disulfide isomerase (PDI), an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b) the involvement and significance of snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) to hemostatic disturbances.</p><p>Methods/Principal Findings</p><p>Crude <i>Bothrops jararaca</i> venom (BjV) was preincubated with Na₂-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na₂-EDTA nor AEBSF could effectively abrogate this fall. However, Na₂-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na₂-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV.</p><p>Conclusions</p><p>SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in engendering bleeding manifestations in severely-envenomed patients.</p></div

Predatory behavior of three centipede species of the order Scolopendromorpha (Arthropoda: Myriapoda: Chilopoda)

ABSTRACT Studies related to centipede feeding and predatory behavior are rare in the literature, and are limited to observations made during fieldwork. Furthermore, they lack descriptions of prey capture. We conducted a laboratory experiment using South American specimens of Scolopendra viridicornis Newport, 1844 (n = 5), Otostigmus tibialis Brölemann, 1902 (n = 5), and Cryptops iheringi Brölemann, 1902 (n = 5), as well as 13 different kinds of prey, to map and describe their predatory behavior. The analysis of video images (65 hours of recordings) resulted in 15 behavioral categories that describe foraging, prey capture, feeding, and cleaning habits. Almost all observations (95%) concluded with the centipede killing the prey. Although we witnessed that a stimulus triggered the movement of the centipede toward the prey in all observation events (suggesting a sit-and-wait strategy), our experiments also showed that these arthropods actively forage to seek food. Field observations during the experiment allowed us to document that scolopendromorphs feed on plants when animal prey items are not available. Moreover, we observed that the size and aggressiveness of the prey determined the centipede capture process. Our results revealed that two behavioral categories were performed only by S. viridicornis , and thus might be genus or species-specific. These are: raising the first third of the body while the rest of the body remains adjacent to the substrate; and restraining the prey along the ventral region of the first third of the body with the aid of locomotory legs. We also observed some peculiar behaviors performed only by O. tibialis . Our results confirm that S. viridicornis , O. tibialis and C. iheringi hold prey between their ultimate pair of legs

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express

Platelet counts in rats 3 and 6 h after BjV administration.

<p>Rats were injected s.c. (1.6 mg/kg) or i.v. (100 µg/animal) with venom incubated with saline (<b>BjV+Saline</b>), 13 mM Na₂-EDTA (<b>BjV+Na₂-EDTA</b>) or 4 mM AEBSF (<b>BjV+AEBSF</b>). Control animals were treated under the same conditions, but were injected with saline (<b>Saline</b>). *p<0.002 compared with saline-treated rats (<b>Saline</b>). ^#p<0.001 compared with <b>BjV+Saline</b>. Data are expressed as mean ± s.e.m (n = 5–6/group).</p

Prothrombin time (a), factor VII coagulant activity (b) and plasma TF activity (c) in rats 3 and 6 h after BjV administration.

<p>Rats were injected s.c. (1.6 mg/kg) or i.v. (100 µg/animal) with venom incubated with saline (BjV+Saline), 13 mM Na₂-EDTA (BjV+Na₂-EDTA) or 4 mM AEBSF (BjV+AEBSF). Control animals were treated under the same conditions, but were injected with saline (Saline). *p<0.04 compared with saline-treated rats (Saline). ^#p<0.05 compared with BjV+Saline. Data are expressed as mean ± s.e.m (n = 5–6/group).</p

Hydrolysis of the chromogenic substrate BAPNA by <i>B. jararaca</i> venom, incubated or not with inhibitors of serine proteinase and metalloproteinases.

<p>*Specific activity  =  48.3±0.3 nmol p-nitroaniline/min/mg venom (triplicate determinations of two different experiments). ^†Since o-phe was diluted in ethanol, BjV was incubated with the same volume of vehicle to determine the extent to which BAPNA hydrolysis was inhibited by the solvent. Results are expressed as mean ± s.e.m.</p

Circulating venom levels (a), local hemorrhage (b), plasma fibrinogen levels (c), and serum FDP/fdp levels (d) in rats 3 and 6 h after BjV administration.

<p>Rats were injected s.c. (1.6 mg/kg) or i.v. (100 µg/animal) with venom incubated with saline (BjV+Saline), 13 mM Na₂-EDTA (BjV+Na₂-EDTA) or 4 mM AEBSF (BjV+AEBSF). Control animals were treated under the same conditions, but were injected with saline (Saline). *p<0.005 compared with saline-treated rats (Saline). ^#p<0.005 compared with BjV+Saline. Data are expressed as mean ± s.e.m (n = 5–6/group).</p

TF activity in skin and lung (a), and protein expression of TF (b) and PDI (c) in skin from rats 3 and 6 h after BjV administration.

<p>Rats were injected s.c. (1.6 mg/kg) or i.v. (100 µg/animal) with venom incubated with saline (BjV+Saline), 13 mM Na₂-EDTA (BjV+ Na₂-EDTA) or 4 mM AEBSF (BjV+AEBSF). Control animals were treated under the same conditions, but were injected with saline (Saline). *p<0.04 compared with saline-treated rats (Saline). Data are expressed as mean ± s.e.m (n = 5–6/group). (d) Typical results obtained from western blottting analysis of TF (47 kDa), PDI (57 kDa), and GAPDH (37 kDa, internal control) bands from skin homogenates at 3 and 6 h after venom or saline injection. The intensity of the bands was quantified by densitometry and the values obtained from the analysis of 5–6 individuals are shown in figures b and c.</p