Thomas E. Goodwin

Insect Pheromones and Precursors in Female African Elephant Urine

Using automated solid-phase dynamic extraction and gas chromatography–mass spectrometry, our search for urinary chemical signals from ovulatory female African elephants (Loxodonta africana) has revealed the bark beetle aggregation pheromones frontalin, exo-brevicomin, and endo-brevicomin, as well as their precursors and the aphid alarm pheromones (E,E)-α-farnesene and (E)-β-farnesene. Enantiomeric ratios for brevicomins have been determined. Prior discovery of common insect/elephant pheromones in Asian elephants, namely, (Z)-7-dodecenyl acetate and frontalin, suggests that the present findings may yield valuable insights into chemical communication among African elephants.

Male African elephants, Loxodonta africana, can distinguish oestrous status via urinary signals

African elephants are a polygynous species that raise offspring in a matriarchal society. Unlike females, males disperse, spend time in male groups and search for mates when mature. Urinary chemical signals aid males in detecting reproductively active females. A preovulatory pheromone has been identified in Asian elephants, Elephas maximus, but has not yet been experimentally identified in African elephants. In this study, the goal was to determine whether adult captive male African elephants can distinguish between urine from conspecific females in luteal and periovulatory oestrous stages as an indication that a preovulatory pheromone is released in the urine. Urine was collected from seven different female African elephants during their luteal and periovulatory periods of oestrus. Bioassays were conducted with nine adult male elephants housed at six different facilities. Males were presented with the two urine types and a control sample once a day over 3 days to reduce sample novelty, which can result in misleadingly high responses. All males showed greater chemosensory responses to the periovulatory urine by trial 3 with the ability to distinguish the urines increasing over the 3 days. This is the first experimental behavioural evidence that African elephants release an oestrous pheromone in the urine. The ability of the captive male elephants to discern between the two urine types bolsters the hypothesis that there is a preovulatory pheromone in African elephants and encourages efforts to identify it.

The "secret" in secretions: methodological considerations in deciphering primate olfactory communication.

Olfactory communication in primates is gaining recognition; however, studies on the production and perception of primate scent signals are still scant. In general, there are five tasks to be accomplished when deciphering the chemical signals contained in excretions and secretions: (1) obtaining the appropriate samples; (2) extracting the target organic compounds from the biological matrix; (3) separating the extracted compounds from one another (by gas chromatography, GC or liquid chromatography, LC); (4) identifying the compounds (by mass spectrometry, MS and associated procedures); and (5) revealing biologically meaningful patterns in the data. Ultimately, because some of the compounds identified in odorants may not be relevant, associated steps in understanding signal function involve verifying the perception or biological activity of putative semiochemicals via (6) behavioral bioassays or (7) receptor response studies. This review will focus on the chemical analyses and behavioral bioassays of volatile, primate scent signals. Throughout, we highlight the potential pitfalls of working with highly complex, chemical matrices and suggest ways for minimizing problems. A recurring theme in this review is that multiple approaches and instrumentation are required to characterize the full range of information contained in the complex mixtures that typify primate or, indeed, many vertebrate olfactory cues. Only by integrating studies of signal production with those verifying signal perception will we better understand the function of olfactory communication. Am. J. Primatol. 75:621–642, 2013.

Night and day: the comparative study of strepsirrhine primates reveals socioecological and phylogenetic patterns in olfactory signals

Studies of chemical signals in vertebrates typically target single species; however, a broader understanding of olfactory communication may derive from comparative studies. We collected urine from 12 species representing most families of strepsirrhine primates – an excellent model clade because of variation in scent marking and socioecology. Using SPDE/GC–MS, we identified the volatile chemical composition of male and female urine from six ‘urine marking’ species and six glandular or ‘non‐urine marking’ species. We found no sex differences, but as predicted, urine markers expressed the most chemically complex and distinctive urine. More distantly related species had more dissimilar urinary profiles, suggesting gradual signal evolution. Reconstructing ancestral chemical profiles revealed different evolutionary trajectories for urine and non‐urine markers. We suggest that urine marking is an ancestral behaviour related to solitary, nocturnal living and that parallel evolutionary shifts towards greater reliance on derived glandular marking occurred in a family (Lemuridae) characterized by diurnality and sociality.

Chemical Signals of Elephant Musth: Temporal Aspects of Microbially-Mediated Modifications

Mature male African (Loxodonta africana) and Asian (Elephas maximus) elephants exhibit periodic episodes of musth, a state in which serum androgens are elevated, food intake typically decreases, aggressiveness often increases, and breeding success is enhanced. Urine is a common source of chemical signals in a variety of mammals. Elephants in musth dribble urine almost continuously for lengthy periods, suggesting that the chemicals in their urine may reveal their physiological condition to conspecifics. We investigated the volatile urinary chemicals in captive male elephants using automated solid phase dynamic extraction (SPDE) and gas chromatography–mass spectrometry (GC-MS). We found higher levels of alkan-2-ones, alkan-2-ols, and some aromatic compounds in urine from males in musth than in urine from non-musth males or from females. Levels of ketones and alcohols increased as the urine aged, likely due to microbial metabolism of fatty acids. Protein-derived aromatic metabolites also increased in abundance after urination, likely due to microbial hydrolysis of hydrophilic conjugates. We suggest that microbes may play an important role in timed release of urinary semiochemicals during elephant musth.

Intrasexual chemical communication and social responses of captive female African elephants, Loxodonta africana

In matrilineal societies, competition between females can occur within and between social units. Dominance hierarchies reduce costly conflicts when reliable cues of status are available, and reproductive condition may alter individual or group status. Female African elephants live in matriarchal groups with linear dominance hierarchies occurring within and between groups; elephants use chemical signals to mediate social interactions. If reproductive condition has important implications for inter- or intragroup behaviour, then females should discriminate between chemical signal sources that reveal reproductive condition. We examined whether trunk-tip contacts between females within a social group were related to phase of oestrus. Observations were conducted on 21 reproductively viable females at nine zoological facilities in North America. Females in the follicular phase received contacts to the urogenital region at a higher rate than did luteal phase females, and contacts increased with approaching ovulation. This supports the existence of an oestrous signal. We also examined whether an oestrous signal was evident by female investigation of urine collected from the luteal and follicular phases of unfamiliar conspecifics. Elephants responded to unfamiliar urine more than to the control, but response rates to the urine types did not differ. Females within a social unit detected differences in oestrus, but they did not show such discrimination to urinary signals from unfamiliar females. Further evaluation of the existence of a female-to-female oestrous pheromone requires assessing responses to urine from familiar individuals. Understanding the relationship between oestrous condition and dominance status can shed light on the adaptive value of sociality.

Unwinding of unnatural substrates by a DNA helicase.

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.

A simple procedure for solid-phase synthesis of peptide nucleic acids with N-terminal cysteine

Problems were encountered during attempts to prepare N-terminal cysteine-substituted peptide nucleic acids (PNAs) from commercially available, Fmoc-protected monomers. These problems have been surmounted by the use of an S-t-butylmercapto protecting group on the cysteine moiety. The solid-phase syntheses are carried out via a simplified procedure which should be generally useful for manual PNA synthesis.

Assessing chemical communication in elephants

The study of chemical communication in elephants has resulted in startling and exciting new discoveries in the past decade (Rasmussen and Schulte, 1998; Rasmussen et al., 2003). To date, the highlight of this research has been the identification of two compounds that serve as pheromones (as defined by Karlson and Luscher, 1959). Rasmussen et al. (1996, 1997) identified (Z)-7-dodecen-1-yl acetate (Z7-12:Ac) in female Asian elephant urine collected during the pre-ovulatory period. This estrous pheromone signals approaching ovulation to conspecific males but elicits little interest from female conspecifics. The compound is not unique to elephants; Z7-12:Ac is a component of the mating pheromones for numerous lepidopteran species. More recently, Rasmussen and Greenwood (2003) isolated frontalin, a known aggregation pheromone in bark beetles (Kinzer et al., 1969), from male Asian elephant temporal gland secretion. Male and female Asian elephants exhibit a range of behaviors when exposed to frontalin. The responses depend on the age, sex, and status of the receiver. Male Asian elephants release temporal gland secretion profusely only during musth (Jainudeen et al., 1972a,b), suggesting that frontalin carries a musth-alerting message. Thus, two single compounds, likely acting in conjunction with other chemical components such as proteins (Lazar et al., 2002), mediate sexual and social interactions among Asian elephants.

Sexual dimorphism in the performance of chemosensory investigatory behaviours by African elephants ( Loxodonta africana )

Sexual dimorphism in morphology can be accompanied by behavioural differences between the sexes. We examined if investigatory behaviour involving the trunk of African elephants showed sexual dimorphism. Males compete and search for females, but they have a lengthy period of development before they are socially viable mates. Receptive females are relatively rare. We hypothesized that males would display higher rates of chemosensory behaviour following puberty than females. Because males disperse, they were hypothesized to be more likely to contact elephants outside their kinship group. We observed the trunk tip, chemosensory behaviours of African elephants at Addo Elephant National Park, South Africa. For 208 elephants, we found no significant differences in state behaviours around waterholes by age or sex. Yet, older elephants were more likely to investigate the environment and elephant excrement than younger animals. Males were more likely to investigate urine and faeces than females. Only post-puberty animals contacted non-family with males investigating both sexes, while investigations by and to females only involved post-puberty males. Overall, the probability of performing chemosensory behaviours depended on age and sex. Male elephants appear more reliant than females on signals in urine and faeces with ensuing inspections of individuals through trunk tip contacts.