Preservation Methods Alter Carbon and Nitrogen Stable Isotope Values in Crickets (Orthoptera: Grylloidea)

Stable isotope analysis (SIA) is an important tool for investigation of animal dietary habits for determination of feeding niche. Ideally, fresh samples should be used for isotopic analysis, but logistics frequently demands preservation of organisms for analysis at a later time. The goal of this study was to establish the best methodology for preserving forest litter-dwelling crickets for later SIA analysis without altering results. We collected two cricket species, Phoremia sp. and Mellopsis doucasae, from which we prepared 70 samples per species, divided among seven treatments: (i) freshly processed (control); preserved in fuel ethanol for (ii) 15 and (iii) 60 days; preserved in commercial ethanol for (iv) 15 and (v) 60 days; fresh material frozen for (vi) 15 and (vii) 60 days. After oven drying, samples were analyzed for δ (15)N, δ (13)C values, N(%), C(%) and C/N atomic values using continuous flow isotope ratio mass spectrometry. All preservation methods tested, significantly impacted δ (13)C and δ (15)N and C/N atomic values. Chemical preservatives caused δ (13)C enrichment as great as 1.5‰, and δ (15)N enrichment as great as 0.9‰; the one exception was M. doucasae stored in ethanol for 15 days, which had δ (15)N depletion up to 1.8‰. Freezing depleted δ (13)C and δ (15)N by up to 0.7 and 2.2‰, respectively. C/N atomic values decreased when stored in ethanol, and increased when frozen for 60 days for both cricket species. Our results indicate that all preservation methods tested in this study altered at least one of the tested isotope values when compared to fresh material (controls). We conclude that only freshly processed material provides adequate SIA results for litter-dwelling crickets.