“This land, Lord, it seems to me, from the southernmost point within my sight, to the northernmost point that can be seen from this port, is so vast that there must be 20 or 25 leagues of coast. Along the sea, in some parts there are great barriers, some red and others white, and the land above with plains covered in large trees. From one end to the other, it is all beaches..very level and most pleasant. From the sea, the remote and arid interior seems very large; as far as the eye can see there is tree-covered land–land which seems to us to be very extensive.” - Pero Vaz de Caminha

The Brazilian Wandering Spider

Genus Phoneutria

Phoneutria negriventer

Spiders belonging to the genus Phoneutria are common to the biodiversity of Brazil. Their venoms contain a wide variety of proteins and peptides including neurotoxins, which act on ion channels and chemical receptors of the neuro-muscular systems in mammals and insects. Their venoms have been described as particularly useful for future development and discovery of biologically active molecules that have potential applications in medicine and agriculture.

The two most toxic peptides are found in the venom of P. nigriventer. These peptides are referred to as Tx2-6 and Tx2-5, both of which cause death within 2-5 minutes in mice injected with this spider venom (RICHARDSON et al., 2006).

The Brazilian Wandering Spider

Phoneutria negriventer

The Brazilian wandering Spider, also known as the Banana spider is considered to be very dangerous and deadly. One bite from this particular spider can be fatal. The toxin carried by this spider is a very potent neurotoxin called PhTx3. This neurotoxin is attributed to loss of muscle control and breathing problems. After exposure to the venom, people usually experience intense pain and inflammation around the injection site (RICHARDSON et al., 2006). As discussed in class, venom production is very costly and the Brazilian wandering spider often engages in dry bites as a warning to predators.

In 2007, an article appeared in online at Lifescience.com that discussed a potential benefit for the use of Brazilian wandering spider venom. As described in the article a Brazilian spider delivers more than a painful bite because its venom is able to stimulate an hours-long erection. The erection is a side effect seen in men, that is a result of an increase in blood pressure due to exposure to their toxic venom.

Research conducted at John Hopkins University found that about 18 million men in the U.S. suffer from erectile dysfunction and that 1 in 3 men do not respond to Viagra or other ED medication (BRYNER, 2007). The active compound in the spider venom that stimulates erection was experimentally determined using male rats. This particular compound turned out to be a peptide named Tx2-6. The mechanism of action of the toxin Tx2-6 is different than traditional ED medications because it affects an earlier step in the erection process by increasing the amount of nitric oxide, a chemical that is released due to sensations of sexual arousal (BRYNER, 2007).

Scientists are currently working on creating a synthetic version of the toxin Tx2-6 found in the Brazilian Wandering Spider and finding ways to combine it with active components in common ED medication like Viagra to get a drug that would be more efficient in patients that don’t respond well to Viagra (BRYNER, 2007).

References:
Bryner, J. (2007). Natural Viagra: Spider bite causes erection. Retrieved from: www.livescience.com/4429-natural-viagra-spider-bite-erection.html.

Richardson, M., Pimenta, A.M.C., Bemquerer, M.P., Santoro, M.M., et al. 2006. Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria. Comparative Biochemistry and Physiology Part C Vol:142 (1) 173-187.

Africanized Honey Bees

Africanized Honey Bees:  Apis mellifera scutellata

In 1956, scientists in Brazil were looking for a way to increase honey production made by the local bees. As part of their experiment they imported honeybees from Africa and to their surprise some of the imported bees managed to escape and began mating with the local honeybees. The result was a hybrid cross between two species of bees, which were named the “Africanized honeybee”. These hybrid bees proved to be aggressive and dangerous. They were soon referred to as “Killer bees” because their stings had the ability to kill humans. Since 1990 scientists estimate that there have been at least 17 deaths in the United as a result of a Killer bee envenomation (KOLECKI, 1998). In another study involving a small group of patients the cause of death following honeybee stings was respiratory failure.

Clinical studies of Apis Mellifera have shown that many who encounter their potential fatal sting do not survive. The prevalence of this insect is rather high in Brazil as well as many other countries including the U.S. and due to their aggressive nature they are a concern for public safety. There are clinical cases of patients who have survived numerous Africanized bee stings, however they did suffer physically. Clinical manifestation of the bee toxin was characterized by diffuse and wide spread edema, a burning sensation in the skin, weakness, dizziness, and hypotension. Acute renal failure also developed and was attributed to hypotension, intravascular hemolysis and myoglobinuria due to the direct toxic effect of the massive quantity of injected venom (KOLECKI, 1998).

 It has also been observed that multiple Africanized bee stings can cause death in adults. Case reports of people who had been stung by the Africanized bee indicated that even healthy adolescents were likely to experience muscular weakness, diffuse edema, general paresthesia, nausea, vomiting and loss of consciousness (DAHER et al., 2003).

Clinical research has suggested that systematic toxic effects of bee venom are generally seen on patients who have at least 50 stings. The potentially lethal number of stings is estimated to be about 500, with death occurring due to a direct systemic effect of the venom due to a large amount of toxin flowing through the body (KOLECKI, 1998).

The bee's venom contains toxins such as mellitin, which if the main component and has hemolytic and vasoactive properties, it is analogs to the C9 component of the complement cascade and it is able to metabolize the arachidonic acid; phospholipase A2, it is the most active component within the animals venoms, it has myotoxic action; biogenics amines, it is involved with signalization of systems such as immune and nervous; hyaluronidase, degrades the extracellular matrix.  

It also contains apamin, a neurotoxin which could contribute to the lysis of red blood cells and leukocytes (ERLER et al., 2011). The Africanized bee stings are particularly interesting because even after the stinger is detached from the insect body it continues to inject venom because the long sting shaft remains inside the skin. This poses concern for the complete and effective removal of the stinger because it is possible for a portion of the stinger to remain inside the body and continue to release venom. Relative mortality rates from insect bites and stings have been estimated from several case studies and it is assumed to be between 15-25% (DAHER et al., 2003).




References:
Daher, E., Silvia-Junior, G., Bezerra, G., et al. 2003. Case Report: Acute Renal Failure After Massive Honeybee Stings. Rev. Inst. Med. Trop. Säo Paul. Vol:45 (1) 45-50.

Erler, S., Lommatzsch, S., and Lattorff, H.M.G. 2012. Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens in the key pollinators Apis mellifera and Bombus terrestris. Parasitology Research. Vol:110 (1) 1403-1410.

Kolecki, Paul. 1999. Delayed Toxic Reaction Following Massive Bee Envenomination. Annals of Emergency Medicine. Vol:33 (1) 114-118.

Ayahuasca

Ayahuasca (Banisteriopsis caapi and Psychotria viridis) 

Ayahuasca is a tea prepared from the Banisteriopsis caapi vine and the leaves of Psychotria viridis. Psychotria viridis leaves contain Dimethyltryptamine (DMT). DMT depending on the  consumed dose results can range from short-lived milder psychedelic states to powerful immersive experiences. Banisteriopsis caapi contains three main compounds that have physical effects: harmine, harmaline and tetrahydroharmine. Harmine and harmaline are Monoamine oxidase inhibitors (MAOI) they specifically inhibit MAO-A (CALLAWAY J.C. et al, 1999). The presence of these compounds in the tea prevents the metabolism of DMT in the stomach and intestines allowing it to reach the blood stream and the brain. Tetrahydroharmine is a serotonin uptake inhibitor (CALLAWAY J.C. et al, 1999).

Ayahuasca largely used for religious purposes but also has traditional medicinal applications. The ritual is a spiritual it is intended as a purification. Ayahuasca translates to “spirit vine” in Quechua. The harsh diarrhea and vomiting associated with is used to purge the body of parasitic worms. Harmala alkaloids themselves have been shown to be anthelmintic (ANDRITZKY, 1989).

It has been used in Amazonia for centuries. Christian missionaries encountered its use in the 16th century. They equated its use with Satan. The active compounds of Banisteriopsis caapi were isolated in the 20th Century. A number of modern religious movenments in Brazil extensively use Ayahuasca. Santo Daime, established in the 1930s integrates ayahuasca with Christianity. Since the 1990’s the movement has followers around the world.

Legally DMT is still listed as a schedule one substance. Due to their usage of ayahuasca as a sacrament and the spread of the religion, Santo Daime has found itself the center of Court battles and legal wrangling in various countries. In March 2009, there was court ruling that the utilization of ayahuasca for religious ceremonies was legal in the United States (ANDRITZKY, 1989).

References: 

Callaway, J.C., et al. (1999). "Pharmacokinetics of Hoasca alkaloids in healthy humans". Journal of Ethnopharmacology 65 (3): 243–256.

Andritzky, W. (1989). "Sociopsychotherapeutic functions of ayahuasca healing in Amazonia". J Psychoactive Drugs 21 (1): 77–89.

Andiroba

Andiroba (Carapa guianensis)

Andiroba (Carapa guianensis) is a large tree in the mahogany family that grows up to 40m. The tree is common in the rich soils of the Amazon Basin and surrounding area. The tree has a soft, durable wood that is sought for lumber. The tree produce a four corned nut about 7-10cm across. The nut contains kernels from which oil is harvested.

Andiroba oil has been harvested and utilized by indigenous people of the region for centuries. It has been used to treat skin parasites like ticks. The oil is applied to injuries, bites, rashes, boils. Brewed into a tea it is also used to treat fevers, worms, and ulcers. 


The oil is also burnt in lamps and is reported to repel mosquitoes. Andiroba is a potent insect repellent (MIOT et al, 2004). However, currently used throughout Brazil for many things. It is commercially manufactured into anti-inflammatory, antimicrobial, and insecticidal products

Andiroba oil is primarily composed of fatty acids including oleic, palmitic, stearic, and linoleic acids. Linoleic acid is an essential fatty acid required for many biological pathways. However, it cannot be synthesized by animals it is obtained through diet only. It serves in cell signaling and is positively linked to skin health.

Andiroba bark, oil, and leaves also contains limonoids, including a novel type andirobin (ROY et al, 2006). Limonoids are currently being investigated for a variety of therapeutic effects such as antiviral, antifungal, antibacterial, antibacterial, antimalarial and chemotherapy. Some are also utilized as an insecticide.

References: 

Miot, H. A., et al. “Comparative study of the topical effectiveness of the Andiroba oil (Carapa guianensis) and DEET 50% as repellent for Aedes sp.” Rev. Inst. Med. Trop. Sao Paulo. 2004 Sep-Oct; 46(5): 253-6.

Roy, A., et al. “Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biol. Pharm. Bull. 2006; 29(2): 191-201.

Yellow Scorpion

Tityus bahiensis

Yellow Scorpion (Tityus sp)

Scorpion stings are an important cause for concern in Brazil because they occur frequently and also have the ability to induce severe and often fatal reactions to their venom. Research conducted in a Brazilian clinical study included data from 4 patients who were suffering from scorpion stings and who had all developed heart failure and pulmonary edema.

Three of the 4 patients died within 24 hours of being stung. The autopsy results of some of the patients indicated that they also suffered from vascular congestion probably due to circulatory failure as a result of Yellow Scorpion venom being present in the blood which physician’s believe could have triggered death (CUPO et al., 1994).

One of the case studies examined a young boy who was stung by the Yellow Scorpion because he experienced intense pain with immediate and frequent vomiting occurring right after the sting. The initial evaluation of this patient revealed that he was severely dehydrated and was experiencing cardiac arrhythmias. Interestingly enough all of the patients in this clinical case report study suffered from cerebral edema and dilation of their cardiac chambers, all of which had visible blood clots in their heart (CUPO et al., 1994).

Tityus serrulatus

Additional studies were conducted in a laboratory setting using mice to test the toxic effects of scorpion venom. In this experiment the mice who were exposed to the venom indicated that Yellow Scorpion stings have the ability to induce seizures. Mice that were injected with Yellow Scorpion toxin were also subject to massive pyramidal neuronal loss in the dorsal hippocampal region of the brain (SANDOVAL and LEBRUN, 2003).

The researchers that examined these effects were interested in the possible connection to a condition in Humans known as Hippocampal sclerosis. Hippocampal sclerosis is described as a common type of neuropathological damage resulting from neuron cell loss primarily occurring in the hippocampus. A very long-standing question is whether hippocampal sclerosis is the consequence of repeated seizures, or whether it plays a role in the development of the epilepsy (JEFFERYS, 1999). The researchers who conducted the mice experiment have suggested the possible benefits of incorporating the Yellow Scorpion toxin in attempt to design a new effective antiepileptic drug (SANDOVAL and LEBRUN, 2003).

Between the most important and dangerous species occurring in Brazil are Tityus serrulatus, Tityus bahiensis, Tityus stigmurus and Tityus cambridgei.

The venom of these animals doesn't produce necrosis or hemorrhagic effect but it affects the permeability of neurons and muscles. Some of the most important toxins are:

Alpha toxin: decrease or block the inactivation of Na+ channels voltage-dependent resulting in constant depolarization of the cells.

Beta toxin: enable or facilitates the activation of Na+ channels voltage-dependent, causing the cell depolarization.

Blockers of K channels: block the K channels. It causes a retardation on cell polarization return for difficult the ion flux trough this channels.

Blockers of Cl channels: block the Cl channels. It prevents the cell polarization.

Tityus serrulatus attacking a cockroach during a lab experiment

References:

Becerril, B. et al, "Toxins and genes isolated from scorpions of the genus Tityus", Toxicon (1997) 35:821-835.

Cupo, P., Jurca, M., Azevedo-Marques, M., et al. 1994. Severe Scorpion Envenomation in Brazil: Clinical, Laboratory and Anatomopathological aspects. Rev. Inst. Med. Trop. Säo Paul. Vol:36 (1) 67-76.

Jefferys, John G. R. 1999. Editorial: Hippocampal sclerosis and temporal lobe epilepsy: cause or consequence? Brain: Oxford University Press. Vol:122 (1) 1007-1008.

Sandoval, M., and Lebrun, I. (2003) TsTx Toxin isolated from Tityus serrulatus sacorpion venom induces spontaneous recurrent seizures and mossy fiber sprouting. Epilepsia Vol:42 (7) 904-911.

Curare

Curare

Ilustration of a plant used to make curare

Throughout time humans have relied on Nature for their basic needs for the production of food-stuffs, shelters, clothing, means of transportation, fertilizers, flavors, fragrances and medicines.

Curare is a mixture of plants from South America and is used as a paralyzing poison by South American indigenous people, especially for hunting purposes. Arrows or darts shot at the prey are dipped in curare, which leads to asphyxiation owing to the inability of the victim's respiratory muscles to contract. The curare from eastern Amazonia comes from various species of Strychnos (family Loganiaceae) plants that contain chiefly quaternary alkaloids with neuromuscular blocking action. The use of curare by Indians makes a good example of the prelude of biotechnology using venoms. Indeed, in this sense, the Indians can be considered the precursors of toxicologists in South America (LIMA et al., 2010). Based on this effect, curare started to be study by many researches. 

Amazon indian using a narrow with curare to hunt

Curare is an example of a non-depolarizing muscle relaxant that blocks the nicotinic acetylcholine receptor (nAChR), one of the two types of acetylcholine (ACh) receptors (MANALIS et al.,1977).

In the late-1930s and early 1940s, the American pharmaceutical industry launched the first modern paralyzing drugs based on indigenous South-American arrow-poisons, curare.

Distribution map of the plant in the South America

Curare never became a therapeutic drug; instead, it became a valuable tool that facilitates medical interventions, just as it facilitated animal experimentation. Its entry to modern medicine took place in the early 1940s, and its most prized application today is in surgery, where curarizing drugs complement anesthesia by imposing an utter stillness on the patient, leaving the body passive and pliant. Abdominal muscles relax and yield, and involuntary movements and contractions disappear. Classified as "surgical muscle relaxants," these modern drugs have made surgery safer by reducing the need for dangerously high levels of general anesthetics to achieve the same end (TRAUTMANN, 1982).

Preparation of the Curare used by Huaorani in the Yasuni.



References:
Lima, M. E., Forte-Dias L., Carlini C. R. and Guimaraes J. A. Toxinology in Brazil: A big challenge for rich biodiverity. Toxicon, 1084-1091, 2010.

Manalis R. S. Voltage-dependent effect of curare at the frog neuromuscular junction. Nature, 267, 1977.

Trautmann, A. Curare can open and block ionic channels associated with cholinergic receptors. Nature, 298, 1982.

Jararaca

Jararaca (Bothrops jararaca)

Jararaca - Bothrops jararaca

The snakes from genus Bothrops are divided into 21 species that have a large distribution in Brazil. They are responsible for 90% of accidents caused by snakes bites and their venoms have a large variety of complex toxins that act on the hemostatic system. In a general way, these toxins can be separated in groups based on their activity in the hemostatic system. Therefore, there are toxins that act in the coagulation process; there are those that act in the plaquetes and cause lesions in the vascular system resulting in a hemorrhagic effect (ROSENFELD, 1971). Based on these facts, the Bothrops venom is largely studied, especially the toxins that can have therapeutic applications for various snake bites. 

Local pain, edema, local and systemic hemorrhages, and blood coagulation disturbances are usual symptoms observed in accidents inflicted by Bothrops snakes (ROSENFELD, 1971). In fact, edema is one of the local effects of Bothrops envenomation less efficiently neutralized by serum therapy (CARDOSO et al., 1993). The rapid manifestation of edema and its modulation by endogenous mediators decrease the efficacy of serum therapy (FRANCA et al, 2003). Once serum therapy has a low efficacy to treat the local edema of patients bitten by Bothrops snakes, other therapies have been investigated.

Damage of Jararaca bitten

Myotoxins and metalloproteinases present in Bothrops venoms can induce liberation of endogenous inflammatory mediators from affected tissues (TEIXEIRA et al., 2003; BJARNASON and FOX, 1994). Thus, associating anti-inflammatory drugs, such as dexamethasone or indomethacin, with antivenom should be a rational alternative to treat the local reaction provoked by Bothrops venoms. Studying this idea, many tests were made to get into the conclusion that the efficiency of serum therapy was improved when antivenom was administered in combination with dexamethasone or indomethacin (ARAUJO et al., 2007). Preliminary results point out to the efficacy of treatment of patients accidentally envenomed by Bothrops snakes with an association of dexamethasone and serum, especially for reducing edema in the first day after envenomation (SUSAKI et al., 2005).

Oral bleeding due to the toxic effect of Jararaca venom

Below are some important substances contained in the Bothrops venom that act in the coagulation system:

Trombin-like: serinoproteases that convert fibrinogen into fibrin non-stable. This makes with the clots be not stable.

Pro-coagulant: serinoproteases that activate the coagulate cascade. The most important are: Factor X activator, that convert factor X into Xa; Protrombim activator, that converts protrombin into trombones independently of phospholipids and factor Va and Trombocitin, activate factors V, VIII and XIII.

Anticoagulants: the most important are: Protein C activator, the protein C activation leads to the inhibition of factors Va and VIIIa; Botrojaracin, trombone inhibitor; Botrocetins, inhibitors of factor Xa, IX and X and Jararafibrases, fibrin and fibrinogen degradation.

Pro-aggregating platelets: proteases that activate platelets due to the secretion of ADP.

Anti-aggregating platelets: prevents that the platelets binds in the surface of the damaged tissue, blocking the integrins.

Metaloproteases: enzymes that degrade many kinds of protein and can cause hemorrhage.

Myotoxic Phospholipases: activites the phospholipase A.

Tissue damage after 24h from the bit 

Studies indicating how Bothrops venom affects the offspring were developed in mice. As a result of envenomation the researchers observed that the offspring presented a high incidence of skeletal anomalies such as vertebrae anomalies, sternebrae anomalies and incomplete skull ossification (BERNARDI et al., 2011).

Bothrops jarara

Other kinds of studies that have being developed utilizing the Bothrops venom are those that use the toxins for medical treatments, such the drug Captopril first developed from the jararaca venoms to treat hypertension. Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angio- tensin I-converting enzyme (ACE), which is a new class of therapeutic agents for the treatment of hypertension (SMITH and VANE, 2003). Studies about the Bj-PRO-10c, a oligopeptide from Bothrops jararaca, has shown that this oligopeptide evoked changes in arterial blood pressure that were followed by a significant reduction of heart rate (FERREIRA et al, 1970a and 1970b). Taking the results presented in the literature, the mechanism of action of Bj-PRO-10c  appears to provide evidence that Bj-PRO-10c induces transient increases of free intracellular calcium concentration in neuronal cells through a mechanism by activation of a yet unknown Gi/o-protein coupled receptor and promotes release of neurotransmitters in neuronal cells, namely GABA and glutamate, which may contribute to central cardiovascular effects exerted by Bj-PRO-10c (IANZER et al., 2007; LAMEU et al., 2010).

Botrhops jararaca attack 

References:
Araujo, S. D., Souza A., Nunes, F.P.B. and Goncalves, L.R.C. Effect of dexamethasone associated with serum theraphy on treatment of Bothrops jararaca venom-induced paw edema in mice. Inflamm. Res. 56, 2007.

Bernardi, M. M., Kirsten, T. B., Manetta, P. R., Harb, S.F., Macrini D.J. And Cury, Y. Maternal exposure to Bothrops jararaca snake venom: effects in mice offsprings. J Health Sci Inst. 2011.

Bjarnason JB, Fox JW. Hemorrhagic metalloproteinases from snake venoms. Pharmacol. Ther. 1994.  

Cardoso JL, Fan HW, França FO, Jorge MT, Leite RP, Nishioka SA et al. Rando- mized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (Bothrops Jararaca) in São Paulo. Q J Med., 86, 1993.

Ferreira SH, Greene LH, Alabaster VA, Bakhle YS, Vane JR. Activity of various fractions of bradykinin potentiating factor against angiotensin I converting enzyme. Nature, 1970.

Ferreira SH, Bartelt DC, Greene LJ. Isolation of bradykinin-potentiating peptides from Bothrops jararaca venom. Biochemistry, 1970.

França FOS, Málaque CMS. Acidente botrópico. In: Cardoso JLC, França FO, Fan HW, Málaque CMS, Haddad Jr V (eds.), Animais Peçonhentos no Brasil: Biologia, clínica e terapêutica dos aciden- tes. São Paulo: Sarvier, 2003.

Ianzer D, Santos RA, Etelvino GM, Xavier CH, de Almeida Santos J, Mendes EP, Machado LT, Prezoto BC, Dive V, de Camargo AC. Do the cardiovascular effects of angiotensin-converting enzyme (ACE) I involve ACE-independent mechanisms? New insights from proline-rich peptides of Bothrops jararaca. J Pharmacol Exp Ther, 2007.  

Lameu, C., Hayashi, M. A., Guerreiro, J. R., Oliveira, E. F., Lebrun, I., Pontieri, V., Morais, K. L. P., Camargo, A. C. M. and Ulrich H. The Central Nervous System as Target for Antihypertensive Actions of a Proline-rich Peptide from Bothrops jararaca Venom. Cytrometry Part A, 2010.

Rosenfeld G. Symptomathology, pathology and treatment of snakes bites in South America. In: Bucherl W, Buckley E,Editors. Venomous animals and their venoms. New York: Academic Press; 1971.

Smith CG, Vane JR. The discovery of captopril. FASEB J, 2003.

Susaki TT, Fan HW, Málaque CMS, Medeiros CR, Cardoso JLC, Ferrari RA et al. Effect of dexamethasone therapy on local edema after Bothrops accidents. Mem. Instituto Butantan 2005.

Teixeira CFP, Landucci ECT, Antunes E, Chacur M, Cury Y. In- flammatory effects of snake venom myotoxic phospholipase A2. Toxicon, 2003.

Brown Spider

Loxosceles sp (Brown Spider)

Venomous animals use their venom as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries (CHAIM et al, 2011).

The spiders Loxosceles intermedia (Li), Loxosceles laeta and Loxosceles gaucho belong to a group of arachnids (known as “brown spiders”), which are found in the South and South-East of Brazil and are responsible for most of the accidents by this genus (MOURA et al, 2011). Loxosceles spider bites have become an increasing public health problem in Brazil (ARAUJO et al, 2003).

Loxosceles gaucho

The color of spiders of this genus ranges from a fawn to dark brown. The brown spiders are sedentary, non-aggressive, have nocturnal habits and prefer to inhabit dark areas. In human habitats, brown spiders are often found behind furniture, pictures and associated with clothes (CARDOSO, 1997; CHAIM et al, 2011).

Loxoscelism and dermonecrotic arachnidism are two terms used to describe the cutaneous lesions and various clinical manifestations following bites by members of the Loxosceles genus. Systemic effects, such as thrombocytopenia, disseminated intravascular coagulation and renal failure, have also been reported. Indeed, the crude venom from Loxosceles spiders can degrade the extracellular matrix and induce endogenous responses, such as platelet aggregation, hemolysis, nephrotoxicity, hepatotoxicity  and cardiotoxicity (CHAIM et al, 2011; MOURA et al, 2011).

The Loxosceles Venoms

The venom of Loxosceles spiders is a complex mixture of protein and peptide toxins with a molecular mass profile ranging from 1 to 40 kDa. To date, several molecules in the Loxosceles spider crude venoms have been described, including alkaline phosphatase, 5‘-ribonucleotide phosphohydrolase, sulfated nucleosides, hyaluronidase , fosfolipases-D, metalloproteases, serine proteases and insecticide toxins (CHAIM et al, 2011).

A group of toxins from the Sphingomyelinases D (SMases D) or dermonecrotic factor (DNF) family have been characterized as responsible for most of the toxic effects of Loxosceles spider venoms. The venom is classically described as having a hemolytic action on red blood cells. This effect is calcium and complement-dependent but antibody-independent, and hemolysis is induced by native and recombinant dermonecrotic toxins (phospholipase-D).

The exact mechanism of action of the venom is not yet fully understood, thus hindering the development of effective medical treatments for loxoscelism (ARAUJO et al, 2003).

Damage after 12h from the accident with Loxosceles

Damage after 30h from the accident

Damage after 60h 

Biotechnological applications of brown spider venom toxins

The first biotechnological application of brown spider venom constituents is the product ARACHnase. ARACHnase is a  biotechnological product usefulness like a positive control for lupus anticoagulant testing (SENFF-RIBEIRO et al, 2008).

Recombinant toxins could be useful for biotechnology as positive controls for hemolytic assays as well as for analysis of the complement system and to study new molecular mechanisms involved in such events (SENFF-RIBEIRO et al, 2008).

Loxosceles gaucho

LPA receptors are potential targets for Loxosceles envenomation treatment. The possibilities for biotechnological applications in this area are enormous. Recombinant dermonecrotic toxins could be used as reagents to establish a new model to study the inflammatory response, as positive inducers of the inflammatory response and edema (CHAIM et al, 2011;  SENFF-RIBEIRO  et al, 2008). 

Several toxins from different sources have been studied as potential insecticidal bioactives with great biotechnological possible applications. Senff-Ribeiro et al (2008) investigated the presence of insecticide toxin in the venom of L. intermedia against Spodoptera frugiperda, an insect that has caused decreased corn production of Brazil. Three new potential insecticide toxins LiTx1, LiTx2 and LiTx3 were identified containing peptides that were active against Spodoptera frugiperda. These venom-derived products open a source of insecticide toxins that could be used as substitutes for chemical defensives and lead to a decrease in environmental problems.

Brown spider venom has the presence of hyaluronidases, that are known to be involved in physiological and pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of hyaluronic acid (HA) increases the permeability of connective tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, fertilization and cancer progression. Inhibition of HA degradation, therefore, may be crucial in reducing disease progression and the spread of venom/toxins and bacterial pathogens. Other possible applications are anti-tumor and, possibly, antibacterial and antivenom activities (SENFF-RIBEIRO  et al, 2008). 

Future perspectives

Research on the Loxosceles toxins has gained great attention. In the last five years, using molecular biology techniques, scientists have obtained different recombinant toxins and enough material to bring deeper insight into the molecular action of these toxins. 

Recombinant toxins have been expressed in bacteria, simple organisms that are easy to manipulate and cheap to work with (CHAIM et al, 2011).

The use of combinatorial data from proteomic and molecular biology techniques, such as mass spectrometry, transcriptome analysis and cDNA library constructions, will open possibilities for the discovery of novel toxins in complex venoms (CHAIM et al, 2011).

References:

Araujo, S. C. et al. (2003). Protection against dermonecrotic and lethal activities of Loxosceles intermedia spider venom by immunization with a fused recombinant protein. Toxicon 41, 261–267.

Cardoso, J. L. C.; Wen, F. H. (1997). Doenças infecciosas e parasitárias: Enfoque Amazonico. Cejup: UEPA: Instituto Evandro Chagas, 3-7.

Chaim, O. M. et al (2011). Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purpuses. Toxins, 3, 309-344.

Moura, J. et al (2011). Protection against the toxic effects of Loxosceles intermedia spider venom elicited by mimotope peptides. Vaccine, vol. 29, issue 45, 7992-8001.

Senff-Ribeiro, A. et al (2008). Biotechnological applications of brown spider (Loxosceles genus) venon toxins. Biotechnology Advances, vol. 26, issue 3, 210-218.

The Frog Vaccine

“The frog vaccine” (Phyllomedusa bicolor) 

The anuran skin displays great morphofunctional diversity adapted to a number of adverse factors present in the species habitat environment. (CALDERON, 2010). The skin of the neotropical and South American frogs contains large amounts of a wide range of biologically active peptides that are either identical or highly homologous to hormones or neurotransmitters of the nervous system and diffuse endocrine system of the higher vertebrates (LACOMBE et al, 2000).

Ultrastructural characterization of the Phyllomedusa species skin demonstrated that the profile of skin glands are composed by lipid, mucous, and serous glands that lie deep in the skin and subcutaneous connective tissue (LACOMBE et al. 2000; CALDERON et al., 2010). These glands produce a wide variety of noxious or toxic substances with various pharmacological effects on microorganisms, vertebrate, and invertebrate species (LACOMBE et al. 2000).

Some indigenous people in southwestern Amazonia use these secretions from P. bicolor for medicinal purposes. Indians of the language “Pano” on the border between Brazil and Peru are the ones who originally used this vaccine. In Acre, the Katukina Indians call these frogs of "Kampo" or "Kambô" and apply its secretions to remove the "panema" (a kind of weakness or bad luck), giving more force to hunters. While Indians use the "frog vaccine" to ward off "panema" and various ailments, making a ritual with spiritual meaning, non-Indian users tried this treatment for some specific problems (gastritis, rheumatism, diabetes, allergies, etc.) and curiosity (BERNARDE; SANTOS, 2009; DALY, 1992).

The method of application of the secretion of Phyllomedusa bicolor in humans is known as "frog vaccine", "frog injection" or "Kambô" (BERNARDE; SANTOS, 2009).

The skin secretion is mixed with saliva and introduced into a line of fresh burns on the arms or chest. This induces within minutes violent illness, including rapid pulse, incontinence and vomiting, after which the recipient lapses into a state of listlessness and, finally, into a state perhaps to be described as euphoric; he later claims to be a better hunter, with improved stamina and keener senses (BERNARDE; SANTOS, 2009; DALY, 1992).

The intensity of human reactions to frog secretion is doubtless dose-dependent. The period of intense illness (<1 hr) is followed by a state of listlessness and sleep lasting from one to several days (DALY, 1992).

"frog vaccine"

Phyllomedusa skin peptides

Phyllomedusa skin peptides include: dermaseptin, dermatoxin, distinctin, phylloseptin, phylloxin, plasticin and skin polypeptide YY.

These peptides are synthesized as prepropeptides that are processed into mature peptides after removal of the peptide signal and the acidic propiece. These are then stored in the granules (CALDERON et al., 2010).

The Phyllomedusa skin peptides are grouped in to three main groups according to their ‘‘primary’’ activity: antimicrobial peptides (AMPs); smooth muscle active peptides; and nervous system active peptides. The first group acts as a skin anti-infective passive defense barrier, the second and the third groups cause the disruption of the predator homeostasis balance (CALDERON et al., 2010). Also, skin extracts from this species have been previously studied and are known to contain a variety of vasoactive peptides, including high levels of phyllocaerulein, phyllokinin, and phyllomedusin and moderate levels of sauvagine. (DALY, 1992).

The biological significance of such a complex mixture of antibiotic peptides with different specificity and potency in Phyllomedusa skin is possibly related to a greater protection against a wide range of potential invaders at a minimum metabolic cost, e.g., dermaseptins exhibit synergy of action upon combination with other antibiotic molecules or AMPs, resulting in a 100-fold increase in antibiotic activity (CALDERON et al., 2010).

The ensuing effects depend on the antimicrobial peptide and the severity of the damage, and usually include dissipation of ionic gradients across the PM, leakage of nutrients and/or larger cytoplasmic components, and finally, a collapse of the parasite bioenergetics and osmotic lysis (CALDERON et al., 2010). This killing mechanism acts promptly by destroying their PM, promoting the reduction of log orders of pathogens in a few minutes. This mechanism is unlikely to induce antibiotic-resistance in microorganisms due to a great metabolic change in the PM composition. Two elements seem to be relevant to the antimicrobial action: the selectiveness, and the ability to destabilize PMs (CALDERON et al., 2010). 

Therapeutic peptide antibiotics

Many efforts have been carried out in order to use the AMPs in the development of new infection-fighting drugs applicable to new treatments of nosocomial infections and multidrug-resistant infections, due to the skill of the AMPs to kill drug resistant strains by a mechanism unlikely to induce antibiotic-resistance. The sources from the biodiversity, such as the skin of several frogs’ species, e.g., as Phyllomedusa and other vertebrate and invertebrate animals, plants, and microorganisms, have proved to be an inexorable source of antimicrobial molecules, with a broad spectra of activity, in which the AMPs have highlights in their potential therapeutical application. In order to develop new peptide antibiotics, synthetic changed peptides might offer significant advantages over native AMPs as therapeutical agents. Compared with conventional antibiotics, these bacteria-killing peptides are extremely rapid and attack multiple bacterial cellular targets.

Even with the expected advantages in the use of AMPs as antibiotics, several impediments to therapeutic peptides arise. The main problems at the present moment are the cost of manufacturing peptides, which is economically unfeasible for the amounts of AMPs needed compared to other antibiotics, preventing the widespread clinical use of AMPs as a common antibiotic, and the shortage of studies thoroughly examining systemic peptide  pharmacodynamic and pharmacokinetic issues, including peptide aggregation problems, the in vivo halflife of peptides (and particularly their susceptibility to mammalian proteases), and the required dosing frequency. Due to the specific characteristics of the AMPs, that differentiate them from other antibiotics, the development of new strategies for the therapeutic use of AMPs in medicine are necessary in order to reduce the amount of AMPs necessary to promote the therapeutic infection suppression effect, including the addition of striking affinity to specific targets, efficiency at very low concentrations and negligible toxicity. In this way, nanotechnology has become an efficient and viable alternative to promote the therapeutic application of AMPs. It is expected that in the forthcoming years nanotechnology will promote the emergence of new products for control and prevention of multidrug-resistance microbe infection arising from the identification and analysis of AMPs from South American frog biodiversity (CALDERON et al., 2010).

References: 

Bernarde, P. S.; Santos, R. A. (September-2009) Medicinal use of secretions (“the frog vaccine”) from the kambô frog (Phyllomedusa bicolor) by non-indigenous peoples in Rondônia, Brazil. Biotemas, 22 (3): 213-220.

Calderon L. A. et al. (2010) Antimicrobial peptides from Phyllomedusa frogs: from biomolecular diversity to potential nanotechnologic medical applications. Springer-Verlag, 3, 120-141.

Daly, J. W. (November 1992) Frog secretions and hunting magic in the upper Amazon: Identification of a peptide that interacts with an adenosine receptor. Proc. Nati. Acad. Sci. USA Vol. 89, pp. 10960-10963.

Lacombe, C. et al. (September-2000) Peptide secretion on the cutaneous glands of south American tree frog Phyllomedusa bicolor: an ultrastructural study. European Journal of Cell Biology, 79, 631-641.