Nontoxic effects of thymol, carvacrol, cinnamaldehyde, and garlic oil on dung beetles: A potential alternative to ecotoxic anthelmintics.

The sustainability of the traditional extensive livestock sector will only be possible if healthy dung-decomposing insect communities are preserved. However, many current pharmaceutical anthelmintics are harmful to dung beetles, their presence can have a negative impact on biological systems. Phytochemical anthelmintics are an alternative to ecotoxic synthetic pharmaceutical anthelmintics, although ecotoxicological tests of their possible indirect effects on dung beetles are required to demonstrate their viability. In this study, the potential ecotoxicity of thymol, carvacrol, cinnamaldehyde and garlic oil (diallyl disulfide and diallyl trisulfide) were tested for the first time. Inhibition of antennal response was measured as a relevant parameter by obtaining relevant toxicity thresholds derived from concentration‒response curves, such as the IC50. All phytochemical compounds tested were demonstrated to be suitable alternative candidates to the highly ecotoxic compound ivermectin, considering their non-toxicity to nontarget organisms. Residues of the phytochemical antiparasitics found in cattle droppings were extremely low, even undetectable in the case of diallyl disulfide and diallyl trisulfide. Furthermore, our results showed that none of the phytochemical compounds have ecotoxic effects, even at extremely high concentrations, including those almost 1000 times higher than what is most likely to be found in dung susceptible to ingestion by dung beetles in the field. We can conclude that the four selected phytochemical compounds meet the requirements to be considered reliable alternatives to ecotoxic veterinary medicinal products, such as ivermectin.

Links Between Feeding Preferences and Electroantennogram Response Profiles in Dung Beetles: The Importance of Dung Odor Bouquets.

The detection of dung odors is a crucial step in the food-searching behavior of dung beetles (Coleoptera: Scarabaeoidea). Yet, whether certain compounds characteristic of a given dung type contribute to a 'choosy generalism' behavior proposed for this taxonomic group is unknown. To address this, we analyzed the chemical composition of three types of dung (cow, horse, and rabbit) and conducted behavioral and electroantennogram (EAG) bioassays on 15 species of dung beetles using 19 volatile organic compounds representing the three dung samples. Chemical analyses revealed substantial qualitative and quantitative differences among dung types. When offered these food options in an olfactometer, 14 species exhibited a feeding preference. Surprisingly, all 19 compounds used in the EAG assays elicited antennal responses, with species displaying different olfactory profiles. The relationship between behavioral preferences and electrophysiological profiles highlighted that species with different food preferences had differences in antennal responses. Moreover, a specific set of EAG-active compounds (nonanal, sabinene, acetophenone, ρ-cresol, 2-heptanone, 1H-indole, and 6-methyl-5-hepten-2-one) were the strongest drivers in the distinct sensory profiles of the trophic preference groups. Our results point to the importance of the whole bouquet of dung-emanating compounds in driving food-searching behavior, but specific volatiles could aid in determining highly marked trophic preferences in certain species.

First observation on the predation of a non-arthropod species by a dung beetle species: The case of Canthon chalybaeus and the snail Bulimulus apodemetes.

We described, for the first time, a case of predation of a non-arthropod species by a dung beetle species. Canthon chalybaeus Blanchard, 1843 kills healthy individuals of the terrestrial snail Bulimulus apodemetes (D'Orbigny, 1835) showing an evident pattern of physical aggressiveness in the attacks using the dentate clypeus and the anterior tibiae. The description of this predatory behaviour was complemented with the analysis of the chemical secretions of the pygidial glands of C. chalybaeus, highlighting those main chemical compounds that, due to their potential toxicity, could contribute to death of the snail. We observed a high frequency of predatory interactions reinforcing the idea that predation in dung beetles is not accidental and although it is opportunistic it involves a series of behavioural sophistications that suggest an evolutionary pattern within Deltochilini that should not only be better studied from a behavioural point of view but also phylogenetically.

Dung Beetle Assemblages Attracted to Cow and Horse Dung: The Importance of Mouthpart Traits, Body Size, and Nesting Behavior in the Community Assembly Process.

Dung beetles use excrement for feeding and reproductive purposes. Although they use a range of dung types, there have been several reports of dung beetles showing a preference for certain feces. However, exactly what determines dung preference in dung beetles remains controversial. In the present study, we investigated differences in dung beetle communities attracted to horse or cow dung from a functional diversity standpoint. Specifically, by examining 18 functional traits, we sought to understand if the dung beetle assembly process is mediated by particular traits in different dung types. Species specific dung preferences were recorded for eight species, two of which prefer horse dung and six of which prefer cow dung. Significant differences were found between the functional traits of the mouthparts of the dung beetles attracted to horse dung and those that were attracted to cow dung. Specifically, zygum development and the percentage of the molar area and the conjunctive area differed between horse and cow dung colonizing beetles. We propose that the quantitative differences in the mouthpart traits of the species attracted to horse and cow dung respectively could be related to the differential capacity of the beetles to filtrate and concentrate small particles from the dung. Hence, the dung preference of dung beetles could be related to their ability to exploit a specific dung type, which varies according to their mouthpart traits. Moreover, we found that larger and nester beetles preferred cow dung, whereas smaller and non-nester beetles preferred horse dung. This finding could be related to the tradeoff between fitness and parental investments, and to the suitability of the trophic resource according to the season and species phenology.

Onthophagus pilauco sp. nov. (Coleoptera, Scarabaeidae): evidence of beetle extinction in the Pleistocene-Holocene transition in Chilean Northern Patagonia.

The South American Pleistocene-Holocene transition has been characterized by drastic climatic and diversity changes. These rapid changes induced one of the largest and most recent extinctions in the megafauna at the continental scale. However, examples of the extinction of small animals (e.g., insects) are scarce, and the underlying causes of the extinction have been little studied. In this work, a new extinct dung beetle species is described from a late Pleistocene sequence (~15.2 k cal yr BP) at the paleoarcheological site Pilauco, Chilean Northern Patagonia. Based on morphological characters, this fossil is considered to belong to the genus Onthophagus Latreille, 1802 and named Onthophagus pilauco sp. nov. We carried out a comprehensive revision of related groups, and we analyzed the possible mechanism of diversification and extinction of this new species. We hypothesize that Onthophagus pilauco sp. nov. diversified as a member of the osculatii species-complex following migration processes related to the Great American Biotic Interchange (~3 Ma). The extinction of O. pilauco sp. nov. may be related to massive defaunation and climatic changes recorded in the Plesitocene-Holocene transition (12.8 k cal yr BP). This finding is the first record of this genus in Chile, and provides new evidence to support the collateral-extinction hypothesis related to the defaunation.

Thermal niche helps to explain the ability of dung beetles to exploit disturbed habitats.

In terrestrial ecosystems, insects face a wide range of temperatures among habitats and time; consequently, the thermal niche is one of the main determinants of habitat selection and temporal patterns of activity. The replacement of native forests changes micro-climatic conditions and reduces the diversity of dung beetles; however, the physiological mechanisms behind these changes are not clear. We explore the role of the thermal niche in dung beetles to explain the ability of native species to exploit human-created habitats. Using infrared thermography, we measured variables associated with the thermal niche in 17 native species and used linear mixed-effects model and ANOVAs to compare disturbed habitats and the native forest. Endothermy and body mass explained the ability of dung beetles to exploit human-created open habitats. Small and diurnal species with very low endothermy were able to exploit deforested open habitats; evening/nocturnal/crepuscular species showed similar body mass and high endothermy in all habitats. Regarding thermoregulation mechanisms, none of the species (except one) showed defined or efficient mechanisms of physiological thermoregulation. In view of the accelerated process of forest replacement and climate change, a more profound understanding of the physiological requirements of species is essential to predict and mitigate future extinctions.

Biomagnification and body distribution of ivermectin in dung beetles.

A terrestrial test system to investigate the biomagnification potential and tissue-specific distribution of ivermectin, a widely used parasiticide, in the non-target dung beetle Thorectes lusitanicus (Jekel) was developed and validated. Biomagnification kinetics of ivermectin in T. lusitanicus was investigated by following uptake, elimination, and distribution of the compound in dung beetles feeding on contaminated faeces. Results showed that ivermectin was biomagnified in adults of T. lusitanicus when exposed to non-lethal doses via food uptake. Ivermectin was quickly transferred from the gut to the haemolymph, generating a biomagnification factor (BMFk) three times higher in the haemolymph than in the gut after an uptake period of 12 days. The fat body appeared to exert a major role on the biomagnification of ivermectin in the insect body, showing a BMFk 1.6 times higher than in the haemolymph. The results of this study highlight that the biomagnification of ivermectin should be investigated from a global dung-based food web perspective and that the use of these antiparasitic substances should be monitored and controlled on a precautionary basis. Thus, we suggest that an additional effort be made in the development of standardised regulatory recommendations to guide biomagnification studies in terrestrial organisms, but also that it is necessary to adapt existing methods to assess the effects of such veterinary medical products.

Thermoregulatory syndromes of two sympatric dung beetles with low energy costs.

In heterotherm insects, endothermy implies a high energy cost due to the generation and regulation of body temperature during different activities such as flight, food location, fighting and even walking. We studied the thermoregulation process and the cost of the thermoregulation strategies in two sympatric dung beetles, Sulcophanaeus batesi and S. imperator under heat and cold stress conditions. We used a set of physiological variables to exemplify the capacity of thermolimit respirometry combined with infrared thermography to derive relevant variables capable of describing different thermoregulation syndromes. Habitat use and thermal niche differed notably between S. batesi and S. imperator, reflecting their contrasted thermal requirements. In S. imperator, thermal specialization for high temperatures was observed, being active mainly during the warmer period of the day. On the other hand, thermal adaptation in S. batesi allows its preference for cold exhibiting a morning activity periods, avoiding higher temperatures. The thermophilic strategy used by Sulcophanaeus imperator minimized the energy expenditure produced during the cooling of the body by respiration without thereby endangering higher thermal limits. In this case, S. batesi, the species with a preference for the coldest environments, presented the lowest thermal limits, although the energy cost needed to stay active during cooling was significantly lower than that in S. imperator. Sulcophanaeus imperator and S. batesi showed evident 'economizing' strategies associated with hot and cold environmental conditions, respectively. In contrast, if both species experience a deviation from their thermal optimum, a decrement in their performance could be produced.

Author Correction: First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

First assessment of the comparative toxicity of ivermectin and moxidectin in adult dung beetles: Sub-lethal symptoms and pre-lethal consequences.

Among macrocyclic lactones (ML), ivermectin (IVM) and moxidectin (MOX) potentially affect all Ecdysozoan species, with dung beetles being particularly sensitive. The comparative effects of IVM and MOX on adult dung beetles were assessed for the first time to determine both the physiological sub-lethal symptoms and pre-lethal consequences. Inhibition of antennal response and ataxia were tested as two intuitive and ecologically relevant parameters by obtaining the lowest observed effect concentration (LOEC) values and interpolating other relevant toxicity thresholds derived from concentration-response curves (IC50, as the concentration of each ML where the antennal response is inhibited by half; and pLC50, as the quantity of ingested ML where partial paralysis was observed by half of treated individuals) from concentration-response curves. Both sub-lethal and pre-lethal symptoms obtained in this study coincided in that IVM was six times more toxic than MOX for adult dung beetles. Values of LOEC, IC50 and pLC50 obtained for IVM and MOX evaluated in an environmental context indicate that MOX, despite needing more time for tis elimination in the faeces, would be twice as harmful to dung beetles as IVM. This approach will be valuable to clarify the real impact of MLs on dung beetle health and to avoid the subsequent environmental consequences.

Comparative thermoregulation between different species of dung beetles (Coleoptera: Geotrupinae).

Insects can use thermoregulation to keep their body temperatures within a certain range in response to thermal stress situations. The mechanisms to regulate internal temperature depend on whether species are endothermic or ectothermic species, i.e., if the heat source is internal and/or external. In this study, the thermal stress response due to excess heat was examined in individuals belonging to different species of the Geotrupinae subfamily by using a standardized protocol based on infrared thermography. All the measured heat stress variables allow discrimination among the considered species to a greater or lesser extent. The body heating rate in the heat stress range, the critical thermal maximum and the stress start temperature were the most important variables in discriminating between apterous and winged individuals (R2 = 52.5%, 51.1% and 50.0%, respectively). Examining the degree of association between the physiological similarity of individuals and some species traits suggest that flying capability and daily activity are related with different thermal responses. Based on the obtained results, the Geotrupinae subfamily can be divided into three ecophysiological groups: good, medium and non-thermoregulators. Within these groups, apterism appears mainly in those species with the ability to actively decrease their body temperature. Our results indicate that this ability may be partially due to the management of water loss related with evaporative cooling.

Ivermectin residues disrupt dung beetle diversity, soil properties and ecosystem functioning: An interdisciplinary field study.

Ivermectin is the most common endectocide used to control parasites affecting livestock. Short-term physiological and behavioural effects of ivermectin on dung beetles may have long-term consequences for beetle populations and ecosystem functioning. Long-term effects of the use of ivermectin can be estimated by comparing dung assemblages and ecosystem functions in areas with conventional ivermectin-treated livestock and environmentally similar areas in which livestock are not treated with veterinary medical products (organic farming). In this study, we investigated both short-term and long-term effects of the administration of ivermectin on the characteristics of dung beetle assemblages and the services they provided in a protected area (Doñana National Park, SW Spain). We examined short-term dung colonization, dwelling, relocation, and disaggregation rates and the associations between these processes and the key assemblage parameters of species richness, abundance, biomass and functional diversity. Furthermore, we analysed changes in soil physical-chemical properties and processes. Short-term differences were observed in the total amount of dung relocated by dung beetles at different colonization vs. emigration stages, suggesting that dung beetles in this area were affected by the recent treatments of livestock with ivermectin. Moreover, short-term effects could also be responsible for the significant differences in dung spreading rates between sites. Conventional use of ivermectin disrupted ecosystem functioning by affecting species richness, abundance and biomass. The decrease in diversity parameters was related to a reduction in the functional efficiency, which resulted in the long-term accumulation of dung on the ground and considerable changes in soil functionality.

Isolation and determination of ivermectin in post-mortem and in vivo tissues of dung beetles using a continuous solid phase extraction method followed by LC-ESI+-MS/MS.

A new analytical method based on solvent extraction, followed by continuous solid-phase extraction (SPE) clean-up using a polymeric sorbent, was demonstrated to be applicable for the detection of ivermectin in complex biological matrices of dung beetles (hemolymph, excreta or dry tissues) using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI+-MS/MS). Using a signal-to-noise ratio of 3:1, the limit of detection (LOD) in the insect matrices at trace levels was 0.01 ng g-1 and the limit of quantification (LOQ) was 0.1 ng g-1. The proposed method was successfully used to quantitatively determine the levels of ivermectin in the analysis of small samples in in vivo and post mortem samples, demonstrating the usefulness for quantitative analyses that are focused on future pharmacokinetic and bioavailability studies in insects and the establishment of a new protocol to study the impact of ivermectin on non-target arthropods such as dung beetles and other insects that are related with the "dung community". Because satisfactory precision and accuracy values were obtained in both in vivo matrices, we suggest that the method can be consistently used for quantitative determinations that are focused on future pharmacokinetic and bioavailability studies in insects. Furthermore, this new analytical method was successfully applied to biological samples of dead dung beetles from the field suggesting that the method can be used to establish a new routine analysis of ivermectin residues in insect carcasses that is applied to complement typical mortality tests.

Identification and evaluation of semiochemicals for the biological control of the beetle Omorgus suberosus (F.) (Coleoptera: Trogidae), a facultative predator of eggs of the sea turtle Lepidochelys olivacea (Eschscholtz).

The beetle Omorgus suberosus (F.) is a facultative predator of eggs of the olive ridley turtle Lepidochelys olivacea (Eschscholtz). Laboratory and field investigations were conducted in order to characterize volatile attractants of O. suberosus and to explore the potential for application of these volatiles in a selective mass trapping method. Headspace sorptive extraction (HSSE) coupled to thermo-desorption gas chromatography-mass spectrometry (TD-GC-MS) analysis of the volatile constituents from beetles or turtle nests revealed 24 potential compounds. However, electroantennographic (EAG) measurements revealed antennal sensitivity only to indole, linoleic acid, trimethylamine, dimethyl sulphide, dimethyl disulphide and ammonia. Behavioural tests showed that these compounds are highly attractive to O. suberosus. Field trapping experiments revealed that indole and ammonia were more attractive than the other volatile compounds and showed similar attractiveness to that produced by conventional baits (chicken feathers). The use of a combined bait of indole and NH3 would therefore be the most effective trap design. The data presented are the first to demonstrate effective massive capture of O. suberosus using an attractant-based trapping method. These findings have potential for the development of an efficient mass trapping method for control of this beetle as part of efforts towards conservation of L. olivacea at La Escobilla in Oaxaca, Mexico.

Effects of grazing intensity and the use of veterinary medical products on dung beetle biodiversity in the sub-mountainous landscape of Central Italy.

Grazing extensification and intensification are among the main problems affecting European grasslands. We analyze the impact of grazing intensity (low and moderate) and the use of veterinary medical products (VMPs) on the dung beetle community in the province of Pesaro-Urbino (Italy). Grazing intensity is a key factor in explaining the diversity of dung beetles. In the case of the alpha diversity component, sites with a low level of grazing activity-related in a previous step to the subsequent abandonment of traditional farming-is characterized by a loss of species richness (q = 0) and a reduction in alpha diversity at the levels q = 1 and q = 2. In the case of beta diversity, sites with a different grazing intensity show remarkable differences in terms of the composition of their species assemblages. The use of VMPs is another important factor in explaining changes in dung beetle diversity. In sites with a traditional use of VMPs, a significant loss of species richness and biomass is observed, as is a notable effect on beta diversity. In addition, the absence of indicator species in sites with a historical use of VMPs corroborates the hypothesis that these substances have a ubiquitous effect on dung beetles. However, the interaction between grazing activity and VMPs when it comes to explaining changes in dung beetle diversity is less significant (or is not significant) than the main effects (each factor separately) for alpha diversity, biomass and species composition. This may be explained if we consider that both factors affect the various species differently. In other words, the reduction in dung availability affects several larger species more than it does very small species, although this does not imply that the former are more susceptible to injury caused by the ingestion of dung contaminated with VMPs. Finally, in order to prevent negative consequences for dung beetle diversity, we propose the maintenance of a moderate grazing intensity and the rational use of VMPs. It is our view that organic management can prevent excessive extensification while providing an economic stimulus to the sector. Simultaneously, it can also prevent the abuse of VMPs.

Effect of wetland management: are lentic wetlands refuges of plant-species diversity in the Andean-Orinoco Piedmont of Colombia?

Accelerated degradation of the wetlands and fragmentation of surrounding vegetation in the Andean-Orinoco Piedmont are the main threats to diversity and ecological integrity of these ecosystems; however, information on this topic is of limited availability. In this region, we evaluated the value of 37 lentic wetlands as reservoirs of woody and aquatic plants and analyzed diversity and changes in species composition within and among groups defined according to management given by: (1) type (swamps, heronries, rice fields, semi-natural lakes, constructed lakes and fish farms) and (2) origins (natural, mixed and artificial). A total of 506 plant species were recorded: 80% woody and 20% aquatic. Of these, 411 species (81%) were considered species typical of the area (Meta Piedmont distribution). Diversity patterns seem to be driven by high landscape heterogeneity and wetland management. The fish farms presented the highest diversity of woody plants, while swamps ranked highest for aquatic plant diversity. Regarding wetland origin, the artificial systems were the most diverse, but natural wetlands presented the highest diversity of typical species and can therefore be considered representative ecosystems at the regional scale. Our results suggest that lentic wetlands act as refuges for native vegetation of Meta Piedmont forest, hosting 55% of the woody of Piedmont species and 29% of the aquatic species of Orinoco basin. The wetlands showed a high species turnover and the results indicated that small wetlands (mean ± SD: size = 11 ± 18.7 ha), with a small area of surrounding forest (10 ± 8.6 ha) supported high local and regional plant diversity. To ensure long-term conservation of lentic wetlands, it is necessary to develop management and conservation strategies that take both natural and created wetlands into account.

A protocol for analysing thermal stress in insects using infrared thermography.

The study of insect responses to thermal stress has involved a variety of protocols and methodologies that hamper the ability to compare results between studies. For that reason, the development of a protocol to standardize thermal assays is necessary. In this sense, infrared thermography solves some of the problems allowing us to take continuous temperature measurements without handling the individuals, an important fact in cold-blooded organisms like insects. Here, we present a working protocol based on infrared thermography to estimate both cold and heat thermal stress in insects. We analyse both the change in the body temperature of individuals and their behavioural response. In addition, we used partial least squares regression for the statistical analysis of our data, a technique that solves the problem of having a large number of variables and few individuals, allowing us to work with rare or endemic species. To test our protocol, we chose two species of congeneric, narrowly distributed dung beetles that are endemic to the southeastern part of the Iberian Peninsula. With our protocol we have obtained five variables in the response to cold and twelve in the response to heat. With this methodology we discriminate between the two flightless species of Jekelius through their thermal response. In response to cold, Jekelius hernandezi showed a higher rate of cooling and reached higher temperatures of stupor and haemolymph freezing than Jekelius punctatolineatus. Both species displayed similar thermoregulation ranges before reaching lethal body temperature with heat stress. Overall, we have demonstrated that infrared thermography is a suitable method to assess insect thermal responses with a high degree of sensitivity, allowing for the discrimination between closely related species.

The classification and phylogenetic status of Jekelius (Reitterius) punctulatus (Jekel, 1866) and Jekelius (Jekelius) brullei (Jekel, 1866) (Coleoptera: Geotrupidae) using molecular data.

The phylogenetic placement of Jekelius brullei (Jekel, 1866) and J. punctulatus (Jekel, 1866) (Coleoptera: Geotrupidae) was assessed using mitochondrial and nuclear molecular data to discern contrasting nomenclatural views provided by López-Colón (1996) and the Catalogue of Palaearctic Coleoptera (Löbl et al. 2006). Our results support both the monophyletic and classification status of the genus Jekelius López-Colón, 1989; and the splitting of the genus into the subgenera Jekelius López-Colón, 1989 and Reitterius López-Colón, 1996. The basal phylogenetic placement of these two species also suggests an oriental origin for Jekelius within the western Palaearctic region. Finally, we include a potential distributional map of Jekelius (Reitterius) punctulatus (Jekel, 1866) based on an exhaustive search of occurrence data.

Low doses of ivermectin cause sensory and locomotor disorders in dung beetles.

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin's effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung.

Culturable aerobic and facultative bacteria from the gut of the polyphagic dung beetle Thorectes lusitanicus.

Unlike other dung beetles, the Iberian geotrupid, Thorectes lusitanicus, exhibits polyphagous behavior; for example, it is able to eat acorns, fungi, fruits, and carrion in addition to the dung of different mammals. This adaptation to digest a wider diet has physiological and developmental advantages and requires key changes in the composition and diversity of the beetle's gut microbiota. In this study, we isolated aerobic, facultative anaerobic, and aerotolerant microbiota amenable to grow in culture from the gut contents of T. lusitanicus and resolved isolate identity to the species level by sequencing 16S rRNA gene fragments. Using BLAST similarity searches and maximum likelihood phylogenetic analyses, we were able to reveal that the analyzed fraction (culturable, aerobic, facultative anaerobic, and aerotolerant) of beetle gut microbiota is dominated by the phyla Proteobacteria, Firmicutes, and Actinobacteria. Among Proteobacteria, members of the order Enterobacteriales (Gammaproteobacteria) were the most abundant. The main functions associated with the bacteria found in the gut of T. lusitanicus would likely include nitrogen fixation, denitrification, detoxification, and diverse defensive roles against pathogens.