In this study, we assessed pirAB toxin transcription in (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: and . Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed transcription during the course of a natural infection; this is when the bacteria are delivered by nematodes. Transcript abundance in was higher than in at two of the observed time points: 15 and 18 h. Expression of reached above endogenous control levels at 22 h in but not in . Overall, transcripts were not as highly expressed in as in from 15 to 22 h. This is the first study to directly compare toxin transcript production considering different portals of entry.