# Effect of trimethoprim on Calliphora stygia (Diptera: Calliphoridae) larvae growth and detection via liquid chromatography–tandem mass spectrometry

## TL;DR

This study shows that the antibiotic trimethoprim can significantly increase the growth of blow fly larvae, which could impact forensic time-of-death estimations.

## Contribution

The study is the first to investigate the effects of trimethoprim on Calliphora stygia larvae and its detectability using HPLC–MS/MS.

## Key findings

- Trimethoprim-exposed larvae were significantly longer than controls by mid-late experiment (~3,000 accumulative degree hours).
- Trimethoprim was detected in all larvae reared on antibiotic-treated substrates using HPLC–MS/MS.
- The antibiotic affected larval instar development and larval mass, though with higher variability in mass.

## Abstract

Insects are crucial in the estimation of time-since-death of remains due to their predictable growth. Pharmaceuticals present in decomposing remains may alter insect growth and could affect postmortem interval calculations. Here we report on an investigation into the effects of trimethoprim, on the growth of Calliphora stygia (Fabricius, 1781) (Diptera; Calliphoridae) larvae, a common species of blow fly in Australia. We asked: (i) does trimethoprim influence larval growth, and (ii) can trimethoprim be detected in C. stygia larvae reared on trimethoprim? We found a significant increase in larval length in the trimethoprim-exposed group relative to the control group as accumulated degree hours increased. By mid-late experiment (~3,000 accumulative degree hours), larvae exposed to trimethoprim measured significantly longer than the control larvae; 15 to 18 mm (SD = 2.5 mm), representing a 164% increase compared to the control, which measured 11 to 13 mm (SD = 3.0 mm). We also observed a significant effect of trimethoprim on larval instar development and larval mass, however the latter was accompanied by higher variability. Additionally, trimethoprim was detected in all larvae (analyzed via HPLC–MS/MS) reared on antibiotic-treated substrates, indicating choice of growth models can be important when using larval length to estimate postmortem interval. Our findings highlight the potential for trimethoprim to affect larval growth of C. stygia, and likely other blow flies. This has implications for interpreting growth models used in postmortem interval estimations, and the need to expand knowledge of entomotoxicology into forensic investigations where data on the effects of pharmaceuticals on local insect species remains limited.