Comparing the accuracy of protein measures for arthropods

Measures of the protein content of animals are useful for examining their nutritional state as well as the nutritional content of their bodies for consumers and have been used in a variety of studies of arthropods (Wilder et al., 2013, Zaguri et al., 2021). Significant effort has been devoted to developing methods for protein quantification (Cuff et al., 2021, Zaguri et al., 2021). Unfortunately, all methods have limitations or biases and one of the most accurate methods, amino acid analysis, is time consuming, requires specialized equipment, and is prohibitively expensive for many studies (Mariotti et al., 2008, Mæhre et al., 2018, Cuff et al., 2021, Zaguri et al., 2021). For example, a kit capable of running 2,400 microplate Lowry assays costs approximately the same as analyzing 2 samples for amino acid content. It is important to understand which of the more commonly used measures of protein, or combinations of measures, are most closely related to the more accurate amino acid analysis to allow for accurate measures of arthropod protein content until such time that amino acid analysis is more feasible for widespread use.

One of the most commonly used estimates of protein is crude protein, which is nitrogen content multiplied by 6.25 (Jones 1941, Marriotti et al. 2008, Zaguri et al. 2021). Since the development of this metric in the 19th century, it has been widely applied (Jones 1941, Marriotti et al. 2008). The crude protein measure assumes that all protein is 16% nitrogen and that all nitrogen in an animal is present in protein (Jones 1941). Yet, the nitrogen content of amino acids varies widely from 8 – 32 % (Sterner and Elser 2002). In addition, there are a variety of nitrogen-containing compounds in organisms that vary in concentration among species, including nucleic acids and chitin (Imafidon and Sosulski, 1990, Sterner and Elser, 2002, Elser et al., 2003). Use of the 6.25 conversion factor often results in overestimation of protein content in samples (Marriotti et al. 2008, Mæhre et al., 2018, Zaguri et al., 2021). Recognition of these criticisms of crude protein has resulted in research to quantify nitrogen-to-protein conversion factors for a variety of organisms (Jones, 1941, Mariotti et al., 2008, Jonas-Levi and Martinez, 2017). As likely occurs with any method estimating protein, the factors for converting nitrogen-to-protein often differ among species and can also differ within a species depending on the diet and life stage of individuals, which casts doubt on the generalizability of using such conversion factors (Jones, 1941, Mariotti et al., 2008, Janssen et al., 2017, Jonas-Levi and Martinez, 2017, Boulos et al. 2020, Ritvanen et al., 2020, Smets et al., 2021, Zaguri et al., 2021).

Spectrophotometric methods are another commonly used means of estimating protein content (Cuff et al., 2021, Zaguri et al., 2021). Prior to conducting spectrophotometric methods, samples are often digested in a solution such as NaOH that solubilizes soft tissue protein in a sample but not exoskeleton (Babarino and Lourenco 2005, Zaguri et al. 2021). There are a variety of spectrophotometric methods commonly used including: the Bradford assay, Lowry assay, and Bicinchoninic acid (BCA) assay (Lowry et al., 1951, Bradford, 1976, Smith et al., 1985, Cuff et al., 2021, Zaguri et al., 2021). Each spectrophotometric method typically reacts most strongly with only a small subset of the amino acids present in a sample and different assays react with different subsets of amino acids (Cuff et al. 2021). Hence, variation among samples in amino acid composition can affect the accuracy of protein assays and different protein assays can result in quantitative and qualitative differences in results for both intraspecific and interspecific comparisons (Zaguri et al. 2021).

The study of protein content in arthropods is complicated due to their exoskeleton, which can vary widely as a percentange of dry mass among species (Lease and Wolf 2010). In arthropods, total protein can be broadly divided into two pools: protein in soft tissue and protein in the exoskeleton. The exoskeleton of arthropods is made of chitin, which is 7 % nitrogen, and protein, which can be up to 50 % of the weight of exoskeleton (Sterner and Elser, 2002, Klowden, 2007). Proteins that are bound within the chitinous matrix of the arthropod exoskeleton are: 1) not part of the metabolically-active tissue of an arthropod, and 2) indigestible to any consumer that is unable to digest chitin (Barnes et al., 2019, Wilder et al., 2019). The ability of most vertebrates to digest chitin appears limited, except for seabirds (Whitaker, 1995, Cohen, 1995, Weiser et al., 1997, Barnes et al., 2019; but see Jackson et al. 1992). Chitin digestibility also appears low among invertebrates, as the majority of predatory invertebrates fail to even ingest exoskeleton, as they feed using extraoral digestion (Cohen 1995). Exoskeleton solubility also differs among the chemicals used to prepare samples for different analyses. Spectrophotometric analyses typically use chemicals that do not dissolve exoskeleton while amino acid analysis typically prepares samples using acid that dissolve exoskeleton. Crude protein also measures total nitrogen in a sample regardless of whether it is in soft tissue or exoskeleton. Hence, when analyzing protein in arthropods, it is critical to define if measures are made on total protein (i.e., protein in both soft tissues and exoskeleton) or digestible protein (i.e., protein in the soft tissue or non-exoskeleton portion of arthropods).

The goal of this study was to analyze a diverse set of terrestrial arthropods to test which measures of protein (i.e., crude protein, Bradford, BCA, Lowry, and averages of the spectrophotometric assay) were most closely related to the digestible protein content of arthropods as measured using amino acid analysis, which is considered one of the most accurate measures of protein analysis. As spectrophotometric protein assays only measure digestible protein (i.e., due to the use of NaOH or similar chemicals in sample preparation), we compared assays with digestible protein content measured through amino acid analysis (i.e., total amino acid content – amino acids in exoskeleton). In addition to ensuring the measures are quantifying the same pool of protein, digestible protein may be more relevant for studies interested in the metabolically-active tissue in an invertebrate or the nutritional quality of an invertebrate for a predator.

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