The association of bacterial species with the production of the toxin is unequivocal – Lago and coworkers state, "ndocellular symbiotic bacteria have been proposed as a possible source of eukaryotic TTX by means of an exogenous pathway," and Chau and coworkers note that the "widespread occurrence of TTX in phylogenetically distinct organisms… strongly suggests that symbiotic bacteria play a role in TTX biosynthesis" – although the correlation has been extended to most but not all metazoans in which the toxin has been identified. To the contrary, there has been a failure in a single case, that of newts ("Taricha granulosa"), to detect TTX-producing bacteria in the tissues with highest toxin levels (skin, ovaries, muscle), using PCR methods, although technical concerns about the approach have been raised. Critically for the general argument, "Takifugu rubripes" puffers captured and raised in laboratory on controlled, TTX-free diets "lose toxicity over time," while cultured, TTX-free "Takifugu niphobles" puffers fed on TTX-containing diets saw TTX in the livers of the fishes increase to toxic levels. Hence, as bacterial species that produce TTX are broadly present in aquatic sediments, a strong case is made for ingestion of TTX and/or TTX-producing bacteria, with accumulation and possible subsequent colonization and production. Nevertheless, without clear biosynthetic pathways (not yet found in metazoans, but shown for bacteria), it remains uncertain whether it is simply via bacteria that each metazoan accumulates TTX; the question remains as to whether the quantities can be sufficiently explained by ingestion, ingestion plus colonization, or some other mechanism.