Behavioural and electrophysiological responses of dung beetles to livestock dung volatilomes and constituent compounds

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Dung beetles (Coleoptera: Scarabaeoidae) are a generalist group of coprophagous insects that feed mainly on vertebrate dung and several other food sources such as mushrooms and decaying animal and plant materials. Adult beetles typically arrive at a dung pat for feeding and/or breeding, causing the rapid breakdown of livestock dung accumulated on pastures. Most importantly, as ecosystem engineers the beetles provide critical ecosystem services. These include removing dung from the surface by feeding, tunnelling or rolling and shallow burying activity, nutrient cycling resulting in increased soil fertility, enhanced soil aeration and water transfer, increased water retention, secondary seed dispersal, and parasite and fly control. Despite over 500 Australian native dung beetle species, exotic species were introduced in the 1960s due to the lack of native dung beetle activity on the dung of introduced livestock. To date, out of more than ~60 species introduced, about 33 species are considered established in Australia.
Olfaction plays a pivotal role in attracting dung beetles to dung pats. In this case, dung emitted volatile organic compounds (VOCs) act as chemical cues that can be detected by the beetle antennae. Very few studies have been conducted on dung volatile-induced behavioural
responses of dung beetles. Therefore, this PhD project was designed under the Dung Beetle
Ecosystem Engineers (DBEE) program to investigate the olfactory ecology of dung beetles in
Initially, a passive cage olfactometer assay was deployed to evaluate the olfactory responses to fresh livestock dung volatiles using a well-established, autumn/winter-active species Bubas bison and a recently introduced spring/summer-active Onthophagus vacca as model species. Study results revealed the ability of adult B. bison to discriminate horse dung odours over sheep and cattle dung odours. Dung headspace volatiles were collected using a solid-phase
microextraction technique (SPME) and analysed using GC-MS/QToF. This untargeted metabolomic approach and subsequent chemometric analysis resulted in identification of volatilomes among the three different livestock dung types used in the bioassay. Numerous VOCs belonging to diverse chemical groups such as alkanes, alkenes, alkynes, alcohols, aldehydes, ketones, esters, phenols, and sulphurous compounds were identified during the analysis. Abundance and composition of the annotated VOCs varied with livestock dung type
and animal’s diet. Adult B. bison were more attracted to dung from pasture-fed horses than
lucerne hay-fed horses. Electroantennography revealed that a mixture of indole, butyric acid,
butanone, p-cresol, skatole, and phenol elicit responses in B. bison antennae. In addition, subsequent olfactometer studies provided evidence that these compounds were behaviourally active as a mixture and not as individual components.
Although beetles do not exhibit an exclusive preference, depending on the chemical cues
presented, they can select one dung type over another, a behaviour which can be termed as
‘choosy generalism’. In a similar olfactometer bioassay, female O. vacca showed significantly
greater attraction towards cattle dung odour than that of sheep and horse dung. Concurrent
volatilome analysis revealed diverse volatilomes among summer collected cattle, sheep, and
horse dung. Among the plethora of volatiles included in the dung headspace, dimethyl sulphide and phenol were identified as potential VOCs that may have been involved in the choosy generalistic behaviour of O. vacca beetles.
Similar to many other insects, dung beetles perceive chemical cues from the surrounding environment through olfactory receptors housed on the olfactory sensilla on the antennae. To
characterise these chemoreceptors, a scanning electron microscopy study was conducted to
examine the density distribution of major types of antennal sensilla in three introduced dung
beetle species introduced to Australia: Geotrupes spiniger, Bubas bison, and Onitis aygulus.
The gross morphology of the antennal sensilla of these three species is described for the first
time. No sexual dimorphism was observed in sensilla type or distribution among any of the
species examined. Notably, distinct patterns of sensilla trichodea, sensilla basiconica, and
sensilla chaetica were observed on the proximal and distal surfaces of the three lamellae in
their antennal clubs. Interestingly, similar distribution and density of sensilla basiconica were
observed on distal surfaces of lamella 1 (L1) and lamella 2 (L2) in both B. bison and G. spiniger, which belong to two different dung beetle families. Furthermore, the olfactory responses of these dung beetles to ten selected dung volatiles and mixtures of the same were investigated using electroantennography. These VOCs were phenol, skatole, indole, p-cresol, butanone, butyric acid, eucalyptol, dimethyl sulphide, dimethyl disulphide, and toluene. The test chemicals evoked differential antennal responses in all three test species. These results
indicate the possibility of using EAG-active compounds to lure dung beetles in the field.
Although many volatiles are present in the dung headspace, not all are detected and perceived by the dung beetle antenna during food searching behaviour. Only a subset of odour molecules is translated into behaviour. To determine the behavioural activity of the screened EAG-active compounds, a series of field studies were conducted covering all four seasons. The olfactory preference of the introduced dung beetles under Australian field conditions was evaluated for the first time. Cattle dung baits and six different combinations of synthetic baits (M1-M6) were used to compare the attraction to dung beetles. Volatiles evaluated were phenol, skatole, indole, p-cresol, butanone, butyric acid, eucalyptol, dimethyl sulphide, dimethyl disulphide, and toluene, all of which generated antennal responses in dung beetles.
A total of 1926 dung beetles belonging to 10 species were collected from field trapping performed over four successive sessions over the course of a year. Exotic species included Bubas bison, Onthophagus taurus, Euoniticellus pallipes, E. fulvus, E. africanus, Aphodius fimetarius, Onitis alexis, Digitonthophagus gazella, and two native species were Onthophagus pentacanthus and O. dandalu. The results show that warmer temperatures in the summer tend to enhance the volatilisation of chemicals, therefore in terms of abundance, synthetic lures caught more beetles than the dung baits. Despite a significant season effect, synthetic lures showed differential attractiveness, suggesting either a synergistic or antagonistic effect on dung beetles which requires further research to clarify.
Insights from this multidisciplinary PhD study provide solid evidence of volatile-mediated
dung attraction in dung beetles. When there are several options available, dung beetles can
discriminate among livestock dung types depending on the available airborne chemical cues.
Of the hundreds of compounds that can be available in the dung volatilome, only a subset of
volatiles elicited responses on the beetle’s antennae, suggesting potential roles as behaviourally active compounds. It is proposed that the screened, EAG-active compounds can be utilised as lures for dung beetles in the field. This knowledge will assist formulation of a synthetic lure that is consistent, standardised, and clean (no biosecurity risk), which can be used to monitor and trap dung beetles for redistribution in order to reduce the geographical gaps that currently exist in their distribution in Australia.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
  • Gurr, Geoff, Principal Supervisor
  • Barrow, Russ, Co-Supervisor
  • Weston, Leslie A, Co-Supervisor
  • Weston, Paul Adam, Co-Supervisor
Place of PublicationAustralia
Publication statusPublished - 2024


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