The search for human pheromones: the lost decades and the necessity of returning to first principles

As humans are mammals, it is possible, perhaps even probable, that we have pheromones. However, there is no robust bioassay-led evidence for the widely published claims that four steroid molecules are human pheromones: androstenone, androstenol, androstadienone and estratetraenol. In the absence of sound reasons to test the molecules, positive results in studies need to be treated with scepticism as these are highly likely to be false positives. Common problems include small sample sizes, an overestimate of effect size (as no effect can be expected), positive publication bias and lack of replication. Instead, if we are to find human pheromones, we need to treat ourselves as if we were a newly discovered mammal, and use the rigorous methods already proven successful in pheromone research on other species. Establishing a pheromone relies on demonstration of an odour-mediated behavioural or physiological response, identification and synthesis of the bioactive molecule(s), followed by bioassay confirmation of activity. Likely sources include our sebaceous glands. Comparison of secretions from adult and pre-pubertal humans may highlight potential molecules involved in sexual behaviour. One of the most promising human pheromone leads is a nipple secretion from the areola glands produced by all lactating mothers, which stimulates suckling by any baby not just their own.     

Studies of gonadal sex differentiation

Gonadal differentiation has a determinative influence on sex development in human embryos. Disorders of sexual development (DSD) have been associated with persistent embryonal differentiation stages. Between 1998 and 2015, 139 female patients with various (DSD) underwent operations at the Scientific Center of Obstetrics, Gynaecology and Perynatology in Moscow, Russia. Clinical investigations included karyotyping, ultrasound imaging, hormonal measurement and investigations of gonadal morphology. The male characteristics in the embryo are imposed by testicular hormones. When these are absent or inactive, the fetus may be arrested at between developmental stages, or stay on indifferent stage and become phenotypically female. A systematic analysis of gonadal morphology in DSD patients and a literature review revealed some controversies and led us to formulate a new hypothesis about sex differentiation. Proliferation of the mesonephric system (tubules and corpuscles) in the gonads stimulates the masculinization of gonads to testis. Sustentacular Sertoli cells of the testes are derived from mesonephric excretory tubules, while interstitial Leydig cells are derived from the original mesenchyme of the mesonephros. According of the new hypothesis, the original mesonephric cells (tubules and corpuscles) potentially persist in the ovarian parenchyma. In female gonads, some mesonephric excretory tubules regress and lose the tubular structure, but form ovarian theca interna and externa, becoming analogous to the sustentacular Sertoli cells in the testis. The ovarian interstitial Leydig cells are derived from intertubal mesenchyme of the mesonephros, similar to what occurs in male gonads (testis). Surprisingly, the leading determinative factor in sexual differentiation of the gonads is the mesonephros, represented by the embryonic urinary system.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

Oviposition aggregation pheromone in the Simulium damnosum complex

Abstract. Communal oviposition by the Simulium damnosum complex of Afrotropical blackflies (Diptera: Simuliidae) was investigated under controlled laboratory conditions, using wild-caught flies in Sierra Leone. Volatile compounds emitted by Simulium eggs were trapped using a closed collection system, and their attractiveness to gravid flies was tested in a two-choice behavioural bioassay. Significantly more female blackflies oviposited on substrates baited with freshly laid eggs (100% chose the baited substrate), or with the volatiles collected from freshly laid eggs (85% chose the baited substrate), in preference to the relevant control substrates. Substrates baited with volatiles from 12-h-old eggs were not significantly more attractive than controls (only 31% chose the baited substrates; P = 0.33). Gas chromatographic analysis of the egg volatiles consistently showed two peaks emanating from fresh eggs, but significantly lower amounts from 12-h-old eggs ( P <0.05). A novel system for collecting the volatiles from this and other blackfly species, as they laid eggs on a substrate in flowing water, is described. Volatiles collected using this method showed identical gas chromatographic profiles to those of fresh eggs alone, indicating that the flies themselves produced no other volatile chemical signals during oviposition. Evidently communal oviposition by S. damnosum s.l . was mediated by a pheromone emanating from fresh eggs. The role of pheromone-mediated egg aggregation in blackfly ecology is discussed, and its possible manipulation is considered.     

Offspring allocation in structured populations with dimorphic males

Summary Many fig wasp species have dimorphic males. These males often mate in different localities; one typically disperses before mating whereas the other does not disperse. In 1979 a model was developed for offspring allocation in dimorphic fig wasps, but it assumed that females only lay a single egg per fig. This assumption is not realistic and precludes any effects local mate competition (LMC) may exert on morph abundance. I develop a model without these restrictions and show that the optimal proportions of each morph is determined by two parameters. Firstly, the proportion of the non-dispersing morph is affected by the mean number of females that oviposit in a patch. This effect is due to the negative correlation between LMC between brothers and the number of females that oviposit in a patch. Secondly, the proportions of both male morphs correlate with the expected proportion of females which will mate with each morph. The separation of the two parameters generalizes the model to any other species which is spatially structured and which has two male morphs or even two alternative mating strategies. A comparison of two models shows that parent—offspring conflict involving morph ratios will not have far reaching consequences. I test these models using the 1979 model's data and both models accurately predict the variation in morph ratios in six species of dimorphic fig wasps.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Influence of body size on fecundity and sperm management in the parasitoid wasp Anisopteromalus calandrae

A large body size is considered to be advantageous to the reproductive success of females as a result of several factors, such as the allocation of more resources to reproduction and the efficient management of sperm transferred by males. In the present study, the effects of female body size, female mating status and additional food availability on fecundity and the offspring sex ratio are investigated in the parasitoid wasp Anisopteromalus calandrae Howard (Hymenoptera: Pteromalidae). Because of haplodiploid sex determination, females must fertilize eggs to produce female offspring but not to produce male offspring. As predicted, female fecundity and the number of female offspring are positively correlated with body size. However, although the volume of the spermatheca increases with female body size, the amount of sperm stored in the spermatheca is relatively constant, irrespective of body size. Consequently, larger females produce a greater proportion of male offspring, especially at the end of the oviposition sequence, suggesting that larger females that possess more resources for reproduction and produce a larger number of offspring are more likely to suffer sperm depletion. The results of the present study also show that mated females have an increased fecundity compared with virgin females, although the opportunity to feed on honey along with host feeding has no impact upon fecundity or the sex ratio.     

Development of mating disruption for control of pine sawfly populations

Mating disruption of the pine sawflyNeodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) was strongly indicated by reduced male trap catches in pine plantations permeated with the sex phermone, (2S, 3S, 7S)-diprionly acetate. The trap catch reduction was 95 to near 100% when dispensers every 10 m were used, giving a total release of about 3 mg per hectare and day. Two mg of pheromone per cotton roll dispenser maintained low catches for the whole season (about 2 months) without any renewal of disruption dispensers. Anerythro-mixture was as effective as the pure pheromone isomer. The effects of the experiments on population density and sex ratio were not possible to investigate, due to a general collapse of the population, also outside experimental plots, the year after the experiments.