The use of DNA-based studies for investigating genetic diversity, adaptive potential and fitness of threatened and endangered species has become increasingly popular in the field of conservation biology (1–3). Data gathered from these studies can be used to inform management strategies, allowing intervention when extinction due to loss of adaptive potential is imminent (4). Notably, adaptive potential is becoming increasingly important due to the rapidly appearing effects of human-induced climate uncertainty in addition to stochastic fluctuations of the environment (4, 5). Therefore, analysing levels of genetic diversity is considered to be integral in deciding which populations should be prioritised for protective action (2, 6, 7).
In order to analyse levels of genetic diversity, biological material such as blood, muscle, feathers, or faeces, which contain a reliable source of DNA, is required (8). There are various methods that can be used to obtain the chosen biological material with different DNA extraction techniques being more suited to certain materials than others. Recently, non-invasive sampling has become a preferred choice – particularly when studying endangered species as this limits further harm to the already stressed populations (9, 10). It should, however, be noted that there are some concerns when using non-invasive samples as a source of DNA due to the fact that they often yield low amounts and/or low quality DNA (11, 12). There are also authors who believe that the term non-invasive should be strictly reserved for samples which can be collected without having to catch or disturb the animal in any way (13). Under this definition, fish scales would not be considered non-invasive samples as they need to be physically removed from the fish (14). Regardless, for this study (as with many others), scale sample collection was considered to be non-invasive (10, 15, 16). While the removal of scales, similar to taking fin clippings, might cause a slight acute stress response in the fish, these sampling methods are non-lethal and long-term studies suggest that they have no significant effect on fish health (17, 18).
Although challenging, scale samples have been found to yield sufficient amounts of DNA to be used with a variety of molecular techniques making them extremely valuable for studying changes in populations (19). Dried scales are commonly stored by fishery authorities, and are often readily available to be used in research (16). Therefore, along with an optimised extraction technique, scales as a source of DNA might become a preferential DNA source (especially when studying ornamental or endangered fish) due to the non-invasive sampling method and ease of acquisition (16).
Teleost fish are the largest and most diverse vertebrate group and occupy nearly every ecological niche possible for an aquatic vertebrate (20, 21). Globally, teleost fish are becoming increasingly threatened at all levels of biological organisation which is proving to have significant impacts on many ecological processes (21). Consequently, the need to study and conserve the genetic diversity of these species is essential.
The scales that are commonly found on teleost fish are referred to as elasmoid scales (22, 23). Elasmoid scales consist of haphazardly arranged collagen fibres that are covered by a layer of acellular bone material (24). These scales lie within a papilla called the scale pocket which consists of scleroblasts and fibroblasts that surround the scale (25–27). Elasmoid scales are critical for the propulsion of the fish as they allow for a large range of motion which facilitate greater swim speed (22). Due to their characteristically hard texture, they also aid in the protection of the fish from threats such as abrasions (22). There are two subgroups of elasmoid scales, namely ctenoid and cycloid (28). The only distinction between the two subgroups is the serrations on the free field of ctenoid scales which are called ctenii (29).
Morphologically, these scales can be divided into three distinct regions: anterior, lateral and posterior (Fig. 1). While the posterior region (which lies directly below the epidermis) is exposed to the environment, the anterior and lateral regions are covered by surrounding scales with the anterior region embedded within the dermis (25).Within the anterior region are a series of radial grooves and circular ridges around the central ‘focus’ (22, 25).The dermis and epidermis entirely consist of living cells (27). Extracted DNA from dried scales would thus be sourced from dermal and epidermal cells that dried and collected within the ridges of the scale (30).
Kabeljou (Argyrosomus japonicus), the species used for this study, have elasmoid scales – specifically ctenoid scales (25).
This study aimed to optimise a protocol for DNA extraction from fish scales so that it may be applied to a limited number of dried archived scales. As previously mentioned, dried scales are commonly stored by fishing authorities. This is however often done for the purposes of short-term objectives and maintaining archived collections is therefore not a common occurrence (35).
Successfully extracting DNA from the archived samples will enable us to greatly improve our existing knowledge of species in general. Gathering population genetic data will enable the investigation of the potential effects that a stock collapse has had on genetic diversity within a population as an example. It will also contribute to determining if the conservation measures for a specific species have been an effective tool in protecting the population and allowing for genetic diversity to be gained over time.
In addition to the challenges posed to DNA extraction by the nature of fish scales in general, archived samples might have been washed in bleach as a cleaning strategy (32, 36). While a study by Kemp & Smith36 showed that endogenous DNA remains relatively stable following different bleaching treatments, the specifications of how exactly the archived samples might have been cleaned are unknown. It was therefore assumed that the DNA obtained from the scales would be highly degraded and fragmented. This further highlighted the need for a protocol that maximises DNA yield as bleach is known to lower the quantity and quality of DNA (38).
Since standard DNA extraction requires that samples be cut up/ground to a powder etc., Hematoxylin & Eosin (H&E) staining was used to first determine whether there were still DNA-containing cells from which successful extractions could be completed (39). This was done with the intention to prevent unnecessary destruction of archived samples which would likely result in unsuccessful DNA extraction but could possibly be used for future research. Hematoxylin is a basic stain that would stain acidic components of the cell. The presence of DNA, using this stain, would be indicated by a purple colour (39). The rest of the scale which is made up of collagen would be stained light pink by the eosin (39). It was previously thought that histochemical staining inhibits DNA amplification reactions, however a study by Morikawa et al.39 has proven that DNA tests such as amplification by PCR and gel electrophoresis are unaffected following H&E staining.
Two main DNA extraction methods were used in this study. These included phenol/chloroform DNA extractions and DNA extractions using the commercially available Qiagen DNeasy Blood & Tissue Kit. Variations of these extraction methods concerning incubation time and temperature were included in the methods to identify an optimized protocol for the isolation of high quantities of DNA. Lastly, in addition to the use of histochemical staining to prevent unnecessary destruction of samples, the scales were divided into three regions (Fig. 8) to determine if, on average, a certain region contained a higher concentration of DNA. If this held true, the particular region could be used when extracting DNA from the archived samples as this would likely lead to the most successful extractions while the remainder of the scale could be kept and possibly used in future research.