In vitro metabolic fate of nine LSD-based new psychoactive substances and their analytical detectability in different urinary screening procedures.

The market of new psychoactive substances (NPS) is characterized by a high turnover and thus provides several challenges for analytical toxicology. The analysis of urine samples often requires detailed knowledge about metabolism given that parent compounds either may be present only in small amounts or may not even be excreted. Hence, knowledge of the metabolism of NPS is a prerequisite for the development of reliable analytical methods. The main aim of this work was to elucidate for the first time the pooled human liver S9 fraction metabolism of the nine d-lysergic acid diethylamide (LSD) derivatives 1-acetyl-LSD (ALD-52), 1-propionyl-LSD (1P-LSD), 1-butyryl-LSD (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD), 1-propionyl-N6-ethyl-nor-LSD (1P-ETH-LAD), N6-allyl-nor-LSD (AL-LAD), N-ethyl-N-cyclopropyl lysergamide (ECPLA), (2'S,4'S)-lysergic acid 2,4-dimethylazetidide (LSZ), and lysergic acid morpholide (LSM-775) by means of liquid chromatography coupled to high-resolution tandem mass spectrometry. Identification of the monooxygenase enzymes involved in the initial metabolic steps was performed using recombinant human enzymes and their contribution confirmed by inhibition experiments. Overall, N-dealkylation and hydroxylation, as well as combinations of these steps predominantly catalyzed by CYP1A2 and CYP3A4, were found. For ALD-52, 1P-LSD, and 1B-LSD, deacylation to LSD was observed. The obtained mass spectral data of all metabolites are essential for reliable analytical detection particularly in urinalysis and for differentiation of the LSD-like compounds as biotransformations also led to structurally identical metabolites. However, in urine of rats after the administration of expected recreational doses and using standard urine screening approaches, parent drugs or metabolites could not be detected.

A reply to Nieberding and Holveck: beyond experimental design and proximate mechanisms - mate choice in the face of sexual conflict.

We summarise our work on male mating behaviour in the tropical butterfly Bicyclus anynana, responding to the commentary provided by Nieberding and Holveck. We acknowledge that our laboratory studies are not free of shortcomings and potential caveats, though we attempted to address or highlight these within each paper. The concerns raised seem to stem mainly from different notions with respect to the proximate basis of old male mating advantage, and specifically the relative importance of male behaviour versus pheromone blend. In our view, our experiments provided compelling evidence for a prominent role of male behaviour, while we were unable to obtain clear evidence for a major role of male sexual pheromones. In addition to the lack of evidence we argue that a preference of females for older males based on pheromone blend is unlikely, as pheromone titres do not seem to indicate male quality and, more importantly, females actually suffer a fitness cost when mating with older males. The latter suggests that old male mating advantage arises from sexual conflict rather than cooperation. We thus highlight the importance of considering both the proximate and the ultimate level for gaining an integrative understanding of complex behavioural patterns.

Young male mating success is associated with sperm number but not with male sex pheromone titres.

BACKGROUND: Intraspecific communication is of crucial importance throughout the animal kingdom and may involve a combination of visual, gustatory, olfactory and acoustic cues. Variation in male sex pheromone amount and composition may convey important information to female conspecifics, for instance on species identity or age. However, whether increased male pheromone titres are associated with fitness benefits for the female, thus indicating a role as an honest signal, is under debate. RESULTS: Against this background, we tested in the butterfly Bicyclus anynana (1) whether young males being successful or unsuccessful in gaining a mating differed in sex pheromone titres and (2) for associations between male pheromone titres and spermatophore mass, eupyrene sperm number, and a variety of female and offspring life-history traits. Successful and unsuccessful males did not differ in pheromone titres, however eupyrene sperm number was much higher in successful males. Pheromone titres were not associated with any fitness-related female or offspring trait measured in our study, though correlation analyses yielded evidence for trade-offs among specific traits. Patterns did not differ among control and olfaction-blocked females. CONCLUSION: Therefore, we suggest that in young B. anynana pheromone titres do not indicate male quality.

Old-male paternity advantage is a function of accumulating sperm and last-male precedence in a butterfly.

Old-male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double-mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2-3 days old) and afterwards an older virgin male (12-13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last-male precedence and the number of sperm transferred upon mating, both increasing paternity success.

Larval starvation reduces responsiveness to feeding stimuli and does not affect feeding preferences in a butterfly.

It is commonly assumed that holometabolic insects such as Lepidoptera rely primarily on larval storage reserves for reproduction. Recent studies though have documented a prominent role of adult-derived carbohydrates for butterfly reproduction. Moreover, a few studies have shown that adult butterflies may also benefit from adult-derived amino acids, at least when larval storage reserves are reduced. Given that in holometabolous insects larval deficiencies are carried over into the adult stage, reduced storage reserves have the potential to modulate adult feeding preferences and responses in order to allow for a successful compensation. We tested this hypothesis here in the fruit-feeding butterfly Bicyclus anynana using larval food stress to manipulate storage reserves. Alcohols (methanol, ethanol, butanol, propanol), sugars (maltose, glucose, fructose, sucrose), and acetic acid acted as feeding stimuli, while butterflies did not respond to other substances such as amino acids, yeast, salts, or vitamins. Contrary to expectations, stressed butterflies showed a weaker response than controls to several feeding stimuli. In preference tests, butterflies preferred sugar solutions containing proline, arginine, glutamic acid, acetic acid, or ethanol over plain sugar solutions, but discriminated against salts. However, there were no general differences among starved and control butterflies. We conclude that larval food-stress does not elicit compensatory feeding behavior such as a stronger preference for amino acids or other essential nutrients in B. anynana. Instead, the stress imposed by a period of starvation yielded negative effects.