Venom Profiling of the Insular Species Bothrops alcatraz: Characterization of Proteome, Glycoproteome, and N-Terminome Using Terminal Amine Isotopic Labeling of Substrates.

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

Venom profiling of the insular species Bothrops alcatraz: characterization of proteome, Glycoproteome, and N-Terminome using terminal amine isotopic labeling of substrates

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

Deworming protocols of island snakes kept in capitivity at the Butantan Institute

Bothrops insularis and B. alcatraz are critically endangered species inhabiting two different coastal islands of the state of São Paulo. Although they are closely related species, they show distinct morphological characteristics due to their isolation from the continent. There is a general lack of studies on conservation and captive husbandry of these species, including their parasites. Our aim was to evaluate the deworming protocols used in B. insularis and B. alcatraz in captivity. We evaluated three deworming protocols through fecal examination: (1) adult B. insularis were treated with ivermectin and praziquantel, (2) adult B. alcatraz were treated with ivermectin, and (3) juvenile B. insularis and B. alcatraz were treated with ivermectin. The snakes are kept in the Laboratory of Ecology and Evolution at the Instituto Butantan (São Paulo, Brazil) for conservation purposes. To evaluate the deworming protocols, we performed the Willis-Mollay (1921) technique. After treating with the first protocol, 19% of the animals showed only eggs of a pseudoparasite (Syphacia obvelata). After the second protocol, 75% of the animals showed cestodes (Ophiotaenia sp.). After the third protocol, 32% of the animals showed only the pseudoparasite, S. obvelata. The pseudoparasite was found only in B. insularis individuals that ate adult mice in captivity. However, cestode eggs were found only in those adult snakes of the second protocol that did not receive praziquantel. Based on the negative results for pathogenic parasites, we conclude that the first protocol was effective for adult snakes whereas the third protocol was effective for juveniles.

A new insular species of pitviper from Brazil, with comments on evolutionary biology and conservation of the Bothrops jararaca group ( Serpentes, Viperidae)

We describe a new pitviper species, Bothrops alcatraz, of the Bothrops jararaca group, from Alcatrazes Island, off the coast of São Paulo, southeastern Brazil. It differs from the mainland coastal populations of B. jararaca in southeastern Brazil mostly by its darker coloration; smaller size; lower number of ventrals, subcaudals, and infralabials; number and shape of anterior cephalic scales; shape of hemipenis spines; intense coagulant activity of venom; and three specific venom proteins. From Bothrops insularis, another island species from southeastern Brazil, the new species differs mainly by its color pattern, smaller size, lower number of subcaudals in males, and absence of hemiclitoris in females. Bothrops alcatraz presents some features that may be viewed as paedomorphic within the B. jararaca group, such as small adult size, proportionally large eyes,intense coagulant venom activity, and diet composed of centipedes and lizards. We postulate that the dwarfism and characteristics of venom in B. alcatraz may be related to its diet (similar to thatof juveniles of the mainland B. jararaca). Bothrops alcatraz and B. insularis may have originatedthrough the isolation of populations of a B. jararaca-like ancestor on the Alcatrazes and Queimada Grande islands, respectively. The new species is regarded as critically endangered due to its very small area of occurrence and the declining quality of its habitat.