Detection of phenobarbital in Chrysomya albiceps (Diptera: Calliphoridae) larvae by immunohistochemistry and its importance to the drugs metabolism study

Detection of drugs from fly larvae, puparia and adults can provide needed information to estimate the post-mortem interval. Recent research has demonstrated that the presence of drugs or toxins in decomposing tissues may alter the insect development rate when they using such tissues as food, thus potentially altering the post-mortem interval estimate. Blow flies (Diptera: Calliphoridae) are the most abundant insects associated with decomposing animal tissue. Phenobarbital is a barbituric that has sedative, hypnotic and anestesic effects, being commercially available under the brand names of Gardenal® and Luminal®. Chrysomya albiceps (Diptera: Calliphoridae) maggots were reared in an artificial diet with phenobarbital in the concentrations 1000 mg/kg (A), 500 mg/kg (B) and 150 mg/kg (C). Larvae reared without the presence of the drug were used as negative control. Third instar specimens were collected from the diets, washed in PBS buffer, fixed using buffered paraformaldehyde at 4% and embedded in parafine. The samples were then cut at 5 ìm and immunohistochemically treated using the peroxidase complex technique for phenobarbital. Positive specimens showed intense immunoreaction in the muscular fibers. Literature data show that usually drug accumulation in the immatures occurs in an area located at the limit between the exocuticle and the endocuticle. The concentrations B and C did not present significant immunoreaction in the experiments. These results constitute an evidence that phenobarbital accumulates in the muscular fibers of the larvae during larval development, showing that the concept of drug accumulation in the maggot cuticle, and consequently in the puparium, may not be always true.