Multimodal Aposematic Defenses Through the Predation Sequence

Aposematic organisms warn predators of their unprofitability using a combination of defenses, including visual warning signals, startling sounds, noxious odors, or aversive tastes. Using multiple lines of defense can help prey avoid predators by stimulating multiple senses and/or by acting at different stages of predation. We tested the efficacy of three lines of defense (color, smell, taste) during the predation sequence of aposematic wood tiger moths (Arctia plantaginis) using blue tit (Cyanistes caeruleus) predators. Moths with two hindwing phenotypes (genotypes: WW/Wy = white, yy = yellow) were manipulated to have defense fluid with aversive smell (methoxypyrazines), body tissues with aversive taste (pyrrolizidine alkaloids) or both. In early predation stages, moth color and smell had additive effects on bird approach latency and dropping the prey, with the strongest effect for moths of the white morph with defense fluids. Pyrrolizidine alkaloid sequestration was detrimental in early attack stages, suggesting a trade-off between pyrrolizidine alkaloid sequestration and investment in other defenses. In addition, pyrrolizidine alkaloid taste alone did not deter bird predators. Birds could only effectively discriminate toxic moths from non-toxic moths when neck fluids containing methoxypyrazines were present, at which point they abandoned attack at the consumption stage. As a result, moths of the white morph with an aversive methoxypyrazine smell and moths in the treatment with both chemical defenses had the greatest chance of survival. We suggest that methoxypyrazines act as context setting signals for warning colors and as attention alerting or “go-slow” signals for distasteful toxins, thereby mediating the relationship between warning signal and toxicity. Furthermore, we found that moths that were heterozygous for hindwing coloration had more effective defense fluids compared to other genotypes in terms of delaying approach and reducing the latency to drop the moth, suggesting a genetic link between coloration and defense that could help to explain the color polymorphism. Conclusively, these results indicate that color, smell, and taste constitute a multimodal warning signal that impedes predator attack and improves prey survival. This work highlights the importance of understanding the separate roles of color, smell and taste through the predation sequence and also within-species variation in chemical defenses.

Molecular physiology of chemical defenses in a poison frog

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well-documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the Little Devil Frog (Oophaga sylvatica) and compared wild caught chemically defended frogs to laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin, and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs.ResumenLas ranas venenosas obtienen moléculas lipofílicas a partir de su dieta de artrópodos que luego usan como una defensa química contra depredadores. Mientras que la acumulación de toxinas dietéticas ha sido bien documentada, el mecanismo fisiológico de obtención de alcaloides no ha sido investigado. En este estudio usamos secuenciación de RNA y proteómica para determinar cómo la presencia de alcaloides afecta la abundancia de mRNA y proteínas en ranas diablito (Oophaga sylvatica) silvestres con defensas químicas en comparación a ranas diablito criadas en laboratorio con una dieta sin alcaloides. Para entender cómo las ranas venenosas mueven los alcaloides de su dieta a las glándulas granulares en su piel, nos enfocamos en medir la expresión de genes en tres tejidos: intestinos, piel e hígado. En estos tejidos, encontramos varios transcriptomas regulados diferencialmente que tienen actividades involucradas con el transporte y metabolismo de pequeñas moléculas, además de canales de sodio y bombas de iones. Luego usamos métodos proteómicos para cuantificar proteínas en plasma, donde encontramos varias diferencias en abundancia de proteínas entre las ranas silvestres y de laboratorio, incluyendo la proteína anfibia de fijación de toxinas, saxifilina. Finalmente, debido a que muchas proteínas encontradas en la sangre se sintetizan en el hígado, usamos la técnica de perfilación proteómica termal para seleccionar imparcialmente las proteínas solubles que fijan el alcaloide decahydroquinolina. Usando este método, identificamos varias posibles proteínas que interactúan con este alcaloide, incluyendo saxifilina. Estos patrones de cambios en abundancia de transcriptomas y proteínas en ranas con y sin defensas químicas sugieren que la presencia de alcaloides influye en la fisiología de las ranas y que moléculas proteicas pequeñas de transporte podrían estar involucradas en la bioacumulación de toxinas en ranas venenosas dendrobátidos.Summary StatementChemically defended wild poison frogs have gene expression and protein abundance differences across several tissue systems compared to poison frogs reared on an alkaloid-free diet.

Changes in lipid profile during growth and senescence of Catharanthus roseus leaf

Various lipid classes and compounds were monitored during the period of leaf emergence to leaf drop of Catharanthus roseus. The expansion to early maturation phase was accompanied by cellular build-up of all major lipid classes, whilst aging and senescence were characterized by their substantial decline, except for the neutral lipids; the leaf monogalactosyl diglyceride/digalactosyl diglyceride ratio decreased from 4.3 (complete maturity) to 2.1 (abscised stage). The early maturation stage was the earliest stage when appreciable amounts of free sterols and fatty acids could be observed. Sterol/phospholipids ratios increased by 68-fold in the abscised leaf as compared to that at full maturity. The unsaturated/saturated fatty acid ratio was far lower in the senescent leaf as compared to that of the fully expanded leaf. The spatial alterations in lipid profiles may be suggestive of concomitant changes in membrane ultrastructure and functions, putatively leading to perturbation of indole alkaloid sequestration capability of the tissues of a species of pharmaceutical significance.

Sequestration of Dietary Alkaloids by the Spongivorous Marine Mollusc Tylodina perversa

Specimens of the spongivorous Mediterranean opisthobranch Tylodina perversa that had been collected while feeding on Aplysina aerophoba were shown to sequester the brominated isoxazoline alkaloids of their prey. Alkaloids were stored in the hepatopancreas, mantle tissues, and egg masses in an organ-specific manner. Surprisingly, the known sponge alkaloid aerothionin which is found only in A. cavernicola but not in A. aerophoba was also among the metabolites identified in wild caught specimens of T. perversa as well as in opisthobranchs with a documented feeding history on A. aerophoba. Mollusc derived aerothionin is postulated to be derived from a previous feeding encounter with A. cavernicola as T. perversa was found to freely feed on both Aplysina sponges in aquarium bioassays. The possible ecological significance of alkaloid sequestration by T. perversa is still unknown.

Alkaloid Sequestration by Papaver somniferum Latex

The 1000 x g organelles of Papaver somniferum latex have been shown to accumulate rapidly, large quantities of alkaloids (0.05- 1 mg/mg organelle protein), particularly morphine, thebaine, codeine and papaverine and have been found to exhibit a surprising degree of specificity for the accumulation of these alkaloids. The uptake of alkaloid is independent of temperature and does not show a requirement for ATP 10-3 ᴍ, Mg2+ 10-3 ᴍ, or KCl 10-3 ᴍ. It is unaffected by known ATPase inhibitors (chlormadinone acetate 2.5 × 10-5 ᴍ, N,N-dicyclohexylcarbo- diimide 5 × 10-4 ᴍ and sodium vanadate 10-4 ᴍ) but showed considerable increase in morphine accumulation in the presence of 4-chloromercumbenzoate 5 x 10-4 ᴍ, while N-methylmaleimide 5 × 10-4 ᴍ had no effect, and carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone 5 × 10-4 ᴍ caused a reduction (ca. 30%) of morphine uptake. An acid medium, pH 4.5-5.5, also results in decreased morphine uptake.