## 6 Biological Activity of the Hapalindole-Type Alkaloids
### Toxicity to Plants and Animals  
Finally, alongside pharmacological studies of the haplindole-type alkaloids, several studies have investigated their toxic effects in multicellular organismal systems. In some cases, these effects could be attributed to biochemical, molecular or cellular mechanisms. For example, inhibition of mitosis by ambiguine D isonitrile was linked to phytotoxic effects, and specifically inhibition of seedling germination [66]; the phytotoxic activity was, in fact, successfully employed for bioassay-guided fractionation and purification of the compound. However, in most cases, the connection between mechanism and toxicity remains to be elucidated. The haplindole-type alkaloids have, for example, been shown as toxic to invertebrate animals, and particularly insects. The larvae of the fly, _Chironomus triparius_, was used as a model system to isolate several haplindoles (_i.e._, C, E, J and L including 12-epimers) as insecticidal agents from a biofilm-forming species of _Fischerella_[32; 60]; in the same study, haplindole J (identified as a new variant) was also found to be lethal to the freshwater crustacean, _Thammoccephalus platyurus_. Moreover, recent studies identified apparent sodium channel modulating activity of these same compounds, as discussed above, and it was proposed that this activity might explain the observed insecticidal activity [59]. In fact, other studies have, likewise, reported the apparent insecticidal activity of the haplindole-type alkaloids. It was observed that welwitindolionnes, and particularly \(N\)-methylwellwitindolinone C isothiocyanate, isolated as the major alkaloid from _W. intricate_, were insecticidal against blowfly larvae [45]. More recently an evaluation of crude extracts, and purified alkaloids, from a freshwater _Fischerella_ isolate was shown to inhibit development of mosquito (_Aedes aegypti_) larvae [61]. Crude extracts, in this case, were lethal to larvae by day 4 of development, and larvae never transitioned beyond 1 st instar stages. Both increased mortality and delays in development were, likewise, observed for larvae exposed to 12-_epi_-hapalindole H isonitrile compared to control (untreated) larvae; specifically, larvae exposed to the alkaloid generally were arrested at 1 st instar stage after 5 days (whereas control larvae transitioned to 2 nd instar by day 2), and development was observably impaired for larvae (only one) which did transition to 2 nd instar (Figure 17). Figure 17: Inhibition of mosquito (_Aedes aegypti_) larval development by 12-_epi_-hapalindole H isonitrile. Shown are 2 nd instar larvae treated with the alkaloid (**a**) showing development abnormalities compared to untreated control larvae (**b**). Most larvae treated with 12-epi-hapalindole H, however, do not transition from 1 st to 2 nd instar. Most recently, the zebrafish (_Danio rerio_) embryo as a vertebrate model of teratogenicity (_i.e._, development toxicity) was used to identify, purify and characterize several, diverse alkaloids from a _Fischerella_ isolate [39; 41; 70]. Teratogenicity of the strain was first identified in screening studies, and initial characterization supported their identity as haplindole-type alkaloids [70]. Subsequent studies were able to utilize bioassay-guided fractionation, based on observed teratogenicity, to purify and chemically characterize several variants including haplindoles, ambiguines and fischerindoles, including new nitrile ambiguities [39; 41]. Toxicity to embryos included--in addition to mortality at the highest exposure concentrations--impairment of melanophores, leading to lack of pigmentation, as well as deformity of the body axis and severe edemas (Figure 18). The observed impairment of melanophore development, in particular, is notable as melanocytes are derived from neural crest cells of embryos, may thereby support, as discussed below (Section 8, Relevance as Harmful Algae), a role of these compounds in myelinopathies observed among waterfowl exposed to epiphytic signonematalean cyanobacteria [71].