Exudate droplets incorporated on eggs by Raoiella indica Hirst female during oviposition may avoid the predation of Amblyseius largoensis (Muma).

Raoiella indica Hirst has rapidly and widely spread throughout the New World since 2004, primarily infesting coconut palms and interacting with the predator Amblyseius largoensis (Muma). Although A. largoensis feeds on R. indica at all stages of development, it cannot naturally reduce its population to levels that do not impact the host plant. Raoiella indica possesses dorsal setae that secrete exudates during all post-embryonic developmental stages, and females have a behavior that deliberately deposits droplets on the freshly laid egg, possibly as a defense strategy against predation in vulnerable stages. In this context, we analyzed whether the presence or absence of droplets in R. indica eggs affects predation using A. largoensis as a biological model. Thus, we evaluated whether some biological and behavioral characteristics of A. largoensis could be affected by the consumption of R. indica egg masses washed or unwashed with water. Also, we performed a chemical analysis of the droplets exuded by R. indica and provided a description of the oviposition behavior of R. indica. The predator showed a higher consumption rate and preference for washed eggs. The results suggest that the exudate droplets have defensive functions, which are incorporated by the female onto the egg during oviposition and subsequently during a patrolling behavior, as they lose their effect after being washed with water. Although the droplets do not prevent the predator from feeding, they reduce the number of R. indica eggs consumed without affecting the growth of A. largoensis.

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

A highly stable, non-digestible lectin from Pomacea diffusa unveils clade-related protection systems in apple snail eggs.

The acquisition of egg protection is vital for species survival. Poisonous eggs from Pomacea apple snails have defensive macromolecules for protection. Here we isolated and characterized a novel lectin called PdPV1 that is massively accumulated in the eggs of Pomacea diffusa and seems part of its protective cocktail. The native protein, an oligomer of ca 256 kDa, has high structural stability, withstanding 15 min boiling and denaturing by SDS. It resists in vitro proteinase digestion and displays structural stability between pH 2.0 and pH 12.0, and up to 85°C. These properties, as well as its subunit sequences, glycosylation pattern, presence of carotenoids, size and global shape resemble those of its orthologs from other Pomacea. Furthermore, like members of the canaliculata clade, PdPV1 is recovered unchanged in feces of mice ingesting it, supporting an anti-nutritive defensive function. PdPV1 also displays a strong hemagglutinating activity, specifically recognizing selected ganglioside motifs with high affinity. This activity is only shared with PsSC, a perivitelline from the same clade (bridgesii clade). As a whole, these results indicate that species in the genus Pomacea have diversified their egg defenses: those from the bridgesii clade are protected mostly by non-digestible lectins that lower the nutritional value of eggs, in contrast with protection by neurotoxins of other Pomacea clades, indicating that apple snail egg defensive strategies are clade specific. The harsh gastrointestinal environment of predators would have favored their appearance, extending by convergent evolution the presence of plant-like highly stable lectins, a strategy not reported in other animals.