BackgroundUnderstanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach.ResultsProteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a β-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9–18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulhood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus.ConclusionsExisting snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112–125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.

The male genitalia of arthropods consistently show negative static allometry (the genitalia of small males of a species are disproportionally large, and those of large males are disproportionally small). We discuss relations between the ‘one‐size‐fits‐all’ hypothesis to explain this allometry and the regimes of selection that may be acting on genitalia. We focus on the contrasts between directional vs. stabilizing selection, and natural vs. sexual selection. In addition, we point out some common methodological problems in studies of genital allometry. One‐size‐fits‐all types of arguments for negative allometry imply net stabilizing selection, but the effects of stabilizing selection on allometry will be weaker when the correlation between body size and the trait size is weaker. One‐size‐fits‐all arguments can involve natural as well as sexual selection, and negative allometry can also result from directional selection. Several practical problems make direct tests of whether directional or stabilizing selection is acting difficult. One common methodological problem in previous studies has been concentration on absolute rather than relative values of the allometric slopes of genitalia; there are many reasons to doubt the usefulness of comparing absolute slopes with the usual reference value of 1.00. Another problem has been the failure to recognize that size and shape are independent traits of genitalia; rapid divergence in the shape of genitalia is thus not paradoxical with respect to the reduced variation in their sizes that is commonly associated with negative allometric scaling.

Abstract.-Hypotheses regarding the function of elaborate male genitalia were tested in a sample of insects and spiders by comparing their allometric values (slopes in log-log regressions on indicators of body size) with those of other body parts. Male genitalia consistently had lower slopes than other body parts. Perhaps as a consequence of this pattern, genitalic size also tended, though less consistently, to have lower coefficients of variation than did the size of other body parts. The morphological details of coupling between males and females in several species clearly indicated that selection favoring mechanical fit is not responsible for these trends. Sexual selection on male courtship structures that are brought into contact with females in precise ways may favor relatively low allometric values, in contrast to the high values seen in the other sexually selected characters (usually visual display devices) that have been studied previously, because a female's own size will influence her perception of the contact courtship devices of a male.Key lVords.-Allometry, genitalia, sexual selection.Received February 14, 1997. Accepted December 9, 1997 Animal genitalia show unusual patterns of evolution, and many hypotheses have been proposed to explain these patterns (summary Eberhard 1985; also Shapiro and Porter 1989; Alexander et al. 1997). Although male genitalia may often evolve under sexual selection by cryptic female choice (Eberhard 1985(Eberhard , 1996, they might also show positive allometric relationships to body size like many other sexually selected traits (e.g., Petrie 1988Petrie , 1992 Alatalo et al. 1988; Meller 1991;Green 1992;Burkhardt et al. 1994). This latter possibility has never been systematically studied.Genitalic allometries might be important for female choice in the following manner. Large male size may be associated with superior abilities to accumulate resources and to survive (Andersson 1995). According to a "good viability genes" model of male genitalic evolution, females might use male genitalia to evaluate overall male size, and choose sires with superior viability genes by favoring males with larger genitalia. This could result in selection that favors males with relatively large genitalia. Analysis from the female point of view yields a similar conclusion. A male's genitalia would be an especially useful cue to a female attempting to judge the male's overall size if the genitalia of larger males were disproportionately large. If, on the other hand, the slope of male genitalic size on body size is low, female attempts to judge male size would be expected to utilize other cues rather than the male's genitalia.Another hypothesis regarding genitalic evolution also predicts relatively high allometric values. Male genitalia may function as weapons in male-female conflicts over control of events associated with copulation (Lloyd 1979; Alexander et al. 1997). In as much as these conflicts are decided by force, then male genitalia should have relatively high allometric values, just as ...

Copulations of Pseudoxychila tarsalis Bates and Pseudoxychila bipustulata Latreille consisted of 1–20 intromissions. The male performed four different genitalic movements: prying, thrusts, small thrusts, and pulls. The male also courted the female during copulation by rubbing her with his middle legs. The observed male behavior does not fit previous hypotheses concerning the phases of copulation in tiger beetles. The female sometimes ejected 1 or 2 spermatophores during or after copulation. There were geographic differences in quantitative aspects of P. tarsalis male copulatory behavior, and more pronounced differences between P. tarsalis and P. bipustulata. This variation could result from sexual selection by cryptic female choice on male copulatory behavior.

No abstract