Paralytic activity of lysophosphatidylcholine from saliva of the waterbugBelostoma anurum

Abstract

SUMMARYLysophosphatidylcholine (LPC) is a major bioactive lipid that is enzymatically generated by phospholipase A 2 (PLA 2 ). Previously, we showed that LPC is present in the saliva of the blood-sucking hemipteran Rhodnius prolixus and modulates cell-signaling pathways involved in vascular biology, which aids blood feeding. Here, we show that the saliva of the predator insect Belostoma anurum contains a large number of lipids with LPC accounting for 25% of the total phospholipids. A PLA 2 enzyme likely to be involved in LPC generation was characterized. The activity of this enzyme is 5-fold higher in Belostoma saliva than in other studied hemipterans, suggesting a close association with the predator feeding habits of this insect. Belostoma employs extra-oral digestion, which allows for ingestion of larger prey than itself, including small vertebrates such as amphibians and fish. Therefore, prey immobilization during digestion is essential, and we show here that Belostoma saliva and B. anurum saliva purified LPC have paralytic activity in zebrafish. This is the first evidence that lysophospholipids might play an important role in prey immobilization, in addition to contributing to blood feeding, and might have been an evolutionary acquisition that occurred long before the appearance of hematophagy in this animal group.Key words: Belostoma, extra-oral digestion, lysophosphatidylcholine, paralysis, phospholipase A2, saliva. THE JOURNAL OF EXPERIMENTAL BIOLOGY 33062003), and have described the dual anti-hemostatic activity of LPC that inhibits platelet aggregation and induces NO production in endothelial cells. Belostoma saliva enables different feeding strategies when compared with hematophagous hemipterans from the Reduviidae family. After catching prey, Belostoma inserts its piercing mouthparts to inject saliva and liquefy the prey tissues. Accordingly, Belostoma saliva must contain an array of digestive enzymes. Their saliva is white and viscous, indicating that it may contain an unusual amount of lipids. In the present study, we demonstrate for the first time the presence of lipids and bioactive lysophospholipids in the saliva of a heteropteran predator, B. anurum. We show that these compounds are toxic to neurons in culture, can block neurally evoked twitch contractions, and can paralyze and kill zebrafish (Dario rerio). MATERIALS AND METHODS Belostoma anurum salivaAdult specimens of Belostoma anurum were collected from Picinguaba (São Paulo, Brazil). The belostomatids were kept, under laboratory conditions, in tanks with 200 ml of non-chlorinated tap water and provided with synthetic floats. Aedes aegypti fifth instar larvae and Biomphalaria glabrata served as food. The saliva was obtained from the proboscis by means of electrical stimulation using an isolated pulse stimulator (Model 2100 A-M Systems, Sequim, WA, USA) at the insertion of the first pair of legs to the thorax (50-100V, 3-4Hz, and square pulses of 3ms duration), as previously described (Dan et al., 1993). Lipid analysisSamples of B. anurum s...

Lysophosphatidylcholine: A Novel Modulator ofTrypanosoma cruziTransmission

“…In this context, using the predator insect Belostoma anurum as model, we showed that the salivary LPC also has this property. Our hypothesis is that B. anurum uses lysophospholipids as a way to paralyze the prey while it feeds, since it makes an extraoral digestion [80]. We obtained similar results with LPC from R. prolixus , with less pronounced blockage of exocytosis.…”

Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

“…Finally, LPCs were recovered in the methanolic phase of the SPE column. PLA2 treatment was performed following the same procedure steps used for PLA1 treatment but using a different reaction buffer (100 mM Tris-HCl, 5 mM CaCl 2 , 100 mM NaCl, pH 8.0) and 5 units PLA2 [55]. The purified lipids (methanolic phase of the SPE) were analyzed by ESI-LIT-MS in 100% methanol containing 5 mM LiOH or 5 mM NaCl.…”

Venoms of Heteropteran Insects: A Treasure Trove of Diverse Pharmacological Toolkits

“…Most information regarding the venoms of the water-associated groups is from Belostomatidae, Nepidae and Notonectidae, while essentially nothing has been recorded of the venoms of Gerromorpha or Leptopodomorpha. Humans bitten by aquatic water bugs experience pronounced pain, swelling, vasodilation and sometimes numbness [ 7 ] while invertebrates and small vertebrates bitten by belostomatids or injected with their venom are typically paralysed after several minutes [ 24 , 68 ]. Venom gland extracts equivalent to 1% of the glands of the creeping water bug Naucoris cimicoides dissolved in 100 μL saline result in immediate cessation of beating of the cockroach heart-dorsum preparation [ 6 ].…”

“…The detailed composition of belostomatid venom is unknown, but electrophoresis of the venom reveals many protein components ranging from low molecular weight peptides (<5 kDa) up to proteins ~55 kDa in size [ 39 , 70 , 71 ]. Enzymatic assays ( Table 1 ) have revealed phospholipase A 2 , hyaluronidase, protease, amylase, esterase, α-glucosidase, glucosaminidase, invertase, lipase, nuclease, phosphatase and phosphohydrolase activities [ 39 , 68 , 71 , 72 ]. Of these, phospholipase A 2 and hyaluronidase are of particular interest because they have been convergently recruited into phylogenetically diverse animal venoms [ 11 ].…”

“…Water scorpion (Nepidae) venom has not been examined in terms of its protein content or bioactivity, but a transcriptomic study of salivary glands from Ranatra chinensis revealed many transcripts encoding proteins with homology to proteases, acid phosphatases, apyrases, dipeptidylpeptidases IV, hyaluronidases, and prophenyloxidases [ 76 ]. Aside from protein components, Silva-Cardoso and colleagues [ 68 ] reported a rich lipidic content of venom from the giant water bug B. anurum , which was found to contain 88% lipid and only 12% protein. These authors focused on the presence of lysophosphatidylcholine (lysPC) in water bug venom.…”