Our study confirms previous findings [18, 19] that whole body extracts containing the defensive chemicals of ladybirds such as alkaloids may be lethal for the water flea D. magna and thus this standard toxicological assay can be used for comparative quantification of ladybird toxicity.
Toxicity and invasive success
We found that H. axyridis was more toxic to D. magna than the other ladybirds. This finding is similar to our previous results  where the lethal concentration (C50=0.06 mg/ml) was many times lower than that of other two conspicuous aposematic ladybirds A. bipunctata (0.6 mg/ml) and C. septempunctata (4 mg/ml). Our present results confirms the order of species in toxicity levels measured in the previous study, but not the magnitude of the interspecific differences. Age can be responsible for the differences (see below). It is believed that the high toxicity is one of the factors that can help the invasive alien species H. axyridis to be so successful in establishing in new areas .
Several studies have been conducted to characterize the ecological aspects, properties and biological functions of harmonine, the alkaloid of H. axyridis. Ants Myrmica rubra showed deterrence to harmonine at concentrations of 10−4 M revealing the protective function of harmonine against invertebrate predators . In our related study  we found only small differences between six species of ladybirds in repellence to ant Lasius niger. A recent study explored the presence of parasitic microsporidia in the haemolymph of H. axyridis . Although detrimental to other coccinellid species, these microsporidia do not affect H. axyridis  and thus harmonine protects H. axyridis from self-infection .
Aposematism and toxicity: interspecific comparison
We consider the brown-and-white C. quatuordecimguttata as moderately aposematic, while it was the least toxic ladybird among our species. Another study using bioassays on D. pulex with ladybird toxins extracts  included somewhat similar (orange-and-white) H. sedecimguttata which was the most toxic among their ladybird species analysed. Thus, toxicity of ladybirds with such type of pattern is quite unpredictable.
The cryptic ladybird C. impunctata that could be assumed less chemically defended against predators than the aposematically coloured species was in fact moderately toxic to water fleas in our study. In the study by Arenas et al. , the non-aposematic Aphidecta obliterata was the least toxic among the species used. Thus, cryptic species seem to show little to moderate toxicity.
In our study, aposematic A. bipunctata appeared to be more toxic than other species except H. axyridis, and it was also the second most toxic species in the study by Arenas et al. . They considered both melanic and typical morphs of this species very aposematic (according to contrast and colour saturation). In our study, A. bipunctata was more toxic than C. septempunctata, while Arenas et al.  did not study the latter species. Repellence by A. bipunctata for ants (expressed as concentration repelling half of individuals, C50) was also higher . These findings are in contrast to the toxicity of the two species for the blue tit C. caeruleus where only C. septempunctata killed the nestlings . It indicates lower efficacy of chemical protection in A. bipunctata against vertebrate predators, despite the presence of a higher concentration of alkaloids than in C. septempunctata . Thus, the standard toxicity test using Daphnia species need not show accurate differences between toxicity of ladybird species against bird predators.
The hypothesized positive relationship between the aposematism and toxicity in ladybirds (called signal honesty ) was not supported in our study. More species of ladybirds is needed in future studies to support or falsify possible hypotheses about the role of colouration, body size, food specificity and habitat preference on their toxicity level.
The invasive H. axyridis, apparently as much aposematic as A. bipunctata and C. septempunctata, was the most toxic in our study. The order of repellence to ants was different: A. bipunctata > H. axyridis > C. septempunctata . It is notable that dead individuals of C. septempunctata were less scavenged by invertebrates (more repellent) than otherwise highly toxic H. axyridis . Antimicrobial activity of haemolymph of H. axyridis against Escherichia coli was 4 times greater than that of C. septempunctata . We conclude that toxicity of individual species of ladybirds to diverse predators differs from repellence and from antimicrobial activity, the former probably being caused by alkaloids, the second by pyrazines, the third by alkaloids and peptides.
Aposematism and toxicity: intraspecific comparison
We observed no difference between melanic (spectabilis) and non-melanic (nevemdecimsignata) morphs of H. axyridis, thus showing that colour morphs may not differ in effects of their defensive chemicals. It is in accordance with the study by Arenas et al.  in which the extracts from melanic and non-melanic forms of A. bipunctata showed no differences in toxic effects on D. pulex. In other study , we compared phytotoxicity of 13 colour morphs of the polymorphic H. axyridis without consistent differences. Colour morphs of H. axyridis collected in the field in Czechia did not differ significantly in the parasitization rate by fungus Hesperomyces virescens and infection rate by Spiroplasma , while in wider center-European comparison, the melanic colour forms conspicua and spectabilis were less often parasitized than non-melanic form novemdecimsignata .
Fischer et al.  reported non-melanic H. axyridis with pale-orange colour possessing a higher content of harmonine than melanic individuals. Nevertheless, Sloggett  observed almost equal repellence to invertebrates by melanic and non-melanic H. axyridis mixed to food. Fischer et al.  observed lower production of methoxypyrazine by red individuals than other colour forms. The alkaloid level was negatively correlated with the extent of melanic pattern on the elytra of the non-melanic H. axyridis . Revealing the importance of methoxypyrazines as warning odours for repellence and toxicity, Fischer et al. [42, 44] accomplished no correlation between methoxypyrazine emission and harmonine content in H. axyridis. This confirms the above mentioned interspecific difference between repellence and toxicity.
Age and toxicity
Although we did not find difference in toxicity between ages 2 weeks and 4 weeks, the toxicity of much older adults (3 months) was 3.8 times higher. Similarly, phytotoxicity assay  showed much stronger effect caused by extracts from 3 months old adults than from 1 week old ones. The carotenoid pigment uses to accumulate throughout the life of a ladybird resulting in the darkening of elytra [23, 46]. However, there was no relationship between alkaloid content and either elytra redness or carotenoid pigment concentration in either sex of field collected H. axyridis . Younger orange individuals had higher number of body zones with thalli of the parasitic fungus H. virescens than red individuals , but older red individuals were not protected against H. virescens . We suggest that some inconsistency between various studies regarding the relationship between age and toxicity can be ascribed to differences between laboratory reared and field collected ladybirds, although Arenas et al.  report indistinguishable toxicity of bought and wild-caught individuals of A. bipunctata.