We found antiparasitic effects in Papaver rhoeas and Echium plantagineum, two plants strongly selected by great bustards during the mating season. From these plants we obtained two categories of active extracts: apolar (EO and Hex) and polar (alcoholic: EtOH / MeOH and aqueous infusion: IFD). We determined their chemical composition and tested their activity on a sample of common laboratory pathogens. Based on these results and a review of the antipathogenic properties of both plants, we infer that great bustards feed on them to reduce their load of pathogens.
Non-polar and polar extracts of aerial parts of P. rhoeas, especially flowers and capsules, showed activity against nematodes and trichomonads. Among non-polar extracts, the flower essential oil (EO) and the capsule hexane extract showed strong trichomonacidal effects, the effect of EO being particularly powerful. A study of P. rhoeas EO from aerial parts collected in Turkey showed phytol (52.8%), tricosane (7.8%), 2-pentadecanone (6%) and heneicosane (5.3%) as the major compounds , while the EO studied here was characterized by alkanes such as n-hexatriacontane and 1-eicosanol and the fatty acid palmitic acid. The non-polar extract (Hex) of the capsules mostly contained methyl / ethyl linoleate, 1-eicosanol and palmitic acid. Palmitic acid has been reported as being the main component of non-polar (Hex) extracts of P. rhoeas leaves .
Previous results have shown trichomonacidal effects on T. vaginalis of Nigella sativa seed oil containing fatty acids . However, this is the first report on trichomonacidal effects of P. rhoeas lipid extracts. Trichomonads are unable to biosynthesize fatty acids and cholesterol but can incorporate these compounds from the medium without further modification . Therefore, externally supplied fatty acids and sterols could interfere with Trichomonads lipid metabolism.
The bioactivity of P. rhoeas polar extracts correlated with their content in polar compounds (peaks with retention times of < 10 min). The alcoholic ones (L-EtOH, F-MeOH ) showed trichomonacidal effects, while the F-IFD showed potent trichomonacidal and nematicidal effects. Nematicidal activity has been previously reported for aqueous extracts (4%) of P. roheas leaves against M. javanica . These extracts contained molecular ions compatible with reported Papaver alkaloids , mostly concentrated in the dichloromethane partition of the flower infusion (F-IDCM), with rheagenine / criptopine, rhoeadine and hydroxy-N-methyl-coclaurine ions being the most abundant in the F-IDCM, F-MeOH and L-EtOH extracts. Since the alkaloid-rich F-IDCM partition was not active, the trichomonacidal /nematicidal effects of P. rhoeas polar extracts cannot be attributed to these alkaloids, indicating that more polar components of the extracts are responsible for the observed effects. Among P. rhoeas components, alkaloids are the most representative, especially (+)-rhoeadine, along with N-methylasimilobine, rhoeagenine, epiberberine and canadine, depending on the plant origin . Minor alkaloids included roemerine , with reported antibacterial, antifungal and anthelmintic activities [38, 39]. Furthermore, alkaloids such as allocryptopine, potopine and berberine were nematicidal against Strongyloides stercolaris larvae . P. rhoeas also contains flavonoids, phenols, organic acids and vitamin C [41–43]. Flavonoids may reduce the oxidative stress and enhance immunity, so they are selected by different bird species, presumably as a prophylactic drug  against pathogens. Polyphenols regulate immune and inflammatory responses during enteric bacterial and parasitic infections in livestock , and organic acids can significantly reduce microbial contamination in turkeys .
Corn poppy has been used since ancient times as a food ingredient and traditional remedy , but cases of poisoning with P. rhoeas in adults, children and animals have been described [47, 48]. Poppy poisoning in humans can cause nausea, vomiting, altered mental state, headache, convulsions, miotic pupils, lethargy and disorientation . Papaver species are actively toxic or narcotic and unpalatable to grazing animals. Animals are safe since the odour and taste of the plants render them obnoxious but there are reports of cattle and horses being poisoned by P. rhoeas . Nonetheless, great bustards include Papaver rhoeas in their diet throughout most of the year and, to our knowledge, they are not poisoned by corn poppies. The diet composition of great bustards and the activity of P. rhoeas extracts shown here support the hypothesis of a self-medication function for this plant species during the bird’s mating season .
Extracts of the aerial parts of E. plantagineum (non-polar and polar) also showed trichomonacidal and nematicidal effects. The flower essential oil (EO) of E. plantagineum, with moderate trichomonacidal effects, was characterized by alkanes such as n-hexatriacontane and related substances, and the fatty acid ester 2-monopalmitin.
Similarly to P. rhoeas extracts, the bioactivity of E. plantagineum polar extracts also correlated with their content in high polarity compounds. The alcoholic F-MeOH showed trichomonacidal effects, while the freeze-dried infusion (IFD) showed potent trichomonacidal and nematicidal effects. Several molecular ions compatible with Pas reported in E. plantagineum [28–31] were identified in the polar extracts, mostly concentrated in the organic fraction of the flowers’ infusion (IDCM). Echimidine molecular ions were the most abundant in all extracts. Since the PA-rich IDCM partition was not active, the trichomonacidal /nematicidal effects of E. plantagineum polar extracts cannot be attributed to these alkaloids. However, nematicidal effects of PAs on Meloidogyne incognita have been reported for heliotrine, lasiocarpine and monocrotaline, but these effects were dependent on the PA structure and the exposure period (168 h).
E. plantagineum produces different classes of secondary metabolites, including pyrrolizidine alkaloids (PA) in the aerial parts and seeds [29, 51, 52], echimidine and echiumine N-oxide being particularly abundant . Pas are easily reduced to free bases and are metabolized by the herbivorous cytochrome P-450 oxidases, which give rise to pyrrol alkylating intermediates. Reactive pyrroles damage cellular DNA and are dangerous to cattle, horses, sheep, pigs, and rats, affecting also humans [53–56]. Harmful effects on bird health have also been described as a result of PA consumption [57, 58].
Diet and health of great bustards
In this study we suggest that selection of P. rhoeas and E. plantagineum by great bustards could be based on the antipathogenic effects of these plants. The use of plants with active secondary metabolites for preventing or reducing parasite and pathogen loads (self-medication) has been described in invertebrates [59–61], mammals [62, 63] and birds [64–66]. As for great bustards, Bravo et al.  described for the first time a probable case of self-medication by ingestion of toxic insects. They found that bustards included two blister beetles of the family Meloidae in their diet. These beetles contain cantharidin, a highly toxic compound that can be even lethal for bustards if ingested in high doses . Bravo et al.  found a male-biased consumption of blíster beetles, and interpreted it as a way to enhance their attractiveness to females by reducing their parasite load. Before selecting a mate for copulation, a female bustard carefully examines the cloaca of the displaying male and usually pecks it, probably looking for parasites. Bravo et al.  suggested that a higher consumption of blister beetles by males could be a sexually-selected mechanism to enhance their mating success. The hypothesis of self-medication in bustards was supported by Withman et al. , who demonstrated antiparasitic effects of extracts from blister beetles (Berberomeloe majalis) against different models (protozoan, nematodes, ticks and insects). Heneberg  proposed that bustard males self-medicated seeking sexual arousal rather than antipathogenic effects.
Regardless of the ultimate function of blister beetle selection, here we propose that there are more species with similar properties in the diet of great bustardst, and we present the antiparasitic results for two plant species as an example. Although the toxicity of P. rhoeas and E. plantagineum differs, great bustards show a marked selection for both plants during the mating season (Fig. 1), and during that season we found higher amounts of P. rhoeas in male than in female fecal samples (Table 1). Why could males be more interested in this plant than females and why during the mating season? Courtship is strenuous for males in most polygynous species and particularly in great bustard males, who show the most strongly skewed mating success reported among lekking birds, suggesting an extreme intensity of sexual selection in this species . Males develop costly ornaments every spring and perform exhausting displays to attract females [19, 20]. It is known that physiological investment in sexually selected characters competes with investment in immune response , a trade-off which is not as demanding for females, with the consequence of smaller loads of parasites and pathogens in this sex compared to males . Great bustard males would hold higher load of parasites and other pathogens than females and still sire a number of descendants in the next generation if females chose to mate them, according to the Handicap Principle [71, 72]. Attracting females while keeping pathogens may be quite demanding, so polar components in P. rhoeas’ capsules and flowers would help males control pathogens, reduce fatigue [sensu 68], or both. Measuring these effects in vivo is beyond any feasible experimental setup, at least with current designs and legal restrictions on experimenting with this vulnerable species. But inferring the causal links is reasonable, so we put forward the challenging hypothesis that great bustard males disproportionately foraged on P. rhoeas during the mating season due to the effects of some non-nutritional compounds present in this plant.
Activity of E. plantagineum against pathogens tested in the present study was noticeably higher than that of P. rhoeas, but the proportion of Echium plantagineum dry weight in faeces of great bustards was about 50 times smaller than that of P. rhoeas during the mating season, and seven times smaller in the non-mating season. The harmful effects of pyrrolizidine alkaloids on bird health described in previous studies [57, 58] could explain the small amount of E. palntagineum in great bustard faeces, but not its higher proportional dry weight in males compared to females during the non-mating season, unless we admit that males would have a greater need of these compounds than females also outside the mating season. We cannot discard that males would also benefit more than females from the properties of E. plantagineum during the months we define as non-mating season (November-January, and July, see ). Males indeed start displaying and fighting to establish their dominance hierarchy in the male group in December-January , so these winter months could also be highly energy demanding. As for July, this is the hottest month of the year, when males suffer the debilitating effects of a much lower heat resistance compared to females , coinciding with the moult of the flight feathers.