Forensic biological pursuits of exotic fish origins: piranha in Hawaii

Synopsis The otoliths of an adult red-bellied piranha, Pygocentrus nattereri, captured from a reservoir in Hawaii were scrutinized to determine the fish's origin and growth history. Sagittal otoliths of the piranha, P. nattereri, contained internal microincrements visible by Scanning Electron Microscopy. The medial cross sectional plane of the sagitta was resolved to provide counts for the most visible micro-increments. An opportune spawning of confiscated adults provided samples for verification of daily increment formation. Daily formation of microincrements was verified from hatched individuals, and confirmed the suitability of otoliths for revealing daily patterns in the age and growth of piranhas. The central area of the sagitta was diffuse in regards to otolith microstructure and indicated the fish was held in an unchanging environment (aquarium). Therefore, otoliths provide important life history or forensic information incorporated within their structural components. The visualization of daily microincrements in the otolith of a juvenile allowed the determination of age at and since release. Fish grew rapidly after being released into the wild. From otolith increments the date of release for an individual fish can be calculated with acceptable accuracy. As presented, otolith structural information can provide age and growth data which are essential to the management of introduced species.     

Otolith Microstructure of Larval Gymnocypris potanini Herzenstein from the Minjiang River in China

Synopsis Otolith microstructure of about 120 Gymnocypris potanini larvae from the Minjiang River in China was examined and analyzed. Larvae had multiple primordia in most lapilli and sagittae, while had only one primordium in a few specimens. There had only one nucleus in otoliths of the larvae, except for some few specimens with 2 nuclei. The transparence of many otoliths differed from center to edge, and part of them could be divided into inner low optically dense zone (LODZ) and outer optically dense zone (ODZ). Based on increment clarity, otoliths of this species could be classified into three types, which were otolith with subtile increments, otolith with almost identified increments, and otolith with fairly clear increments characterized by high contrast. The last two types of otolith accounted for 87.07% in lapilli and 94.46% in sagittae, respectively. Increment clarity of sagitta was higher than that of lapillus. Natural checks were identified in 32.50% lapilli and 48.33% sagittae. These checks primarily located in the first to sixth increment. According to the number of increments in otoliths, the age of this batch larvae was 14 – 22 days, birth date was on June 17 – 25, and average growth rate of body length was 0.8936 ± 0.08769 mm/d.     

Orange roughy otolith growth rates: a direct experimental test of the Romanek–Gauldie otolith growth model

Endolymph chemistry (Ca²⁺, pH, Na⁺) was measured in 17 orange roughy (Hoplostethus atlanticus; Teleostei; Trachichthyidae). Theoretical daily microincrement widths based on the Romanek and Gauldie model of otolith growth were calculated from the measure of otolith chemistry. Theoretical daily microincrement widths were well-correlated (r²=59%) with the width of the last daily microincrement growing at the posterior edge of otoliths taken from the sampled fish.     

Validation of daily otolith increments in larval and juvenile Chinese sucker, <Emphasis Type="Italic">Myxocyprinus asiaticus</Emphasis>

Otolith development was observed and the formation of daily growth increments in otoliths of Chinese sucker, Myxocyprinus asiaticus, was validated by monitoring known-age larvae and juveniles in the laboratory from 2003 to 2005. Otolith shape changed with larval and juvenile development, and there was an exponential relationship until a body length of 16 mm or so, and a linear relationship after a body length of 16 mm between otolith size and fish size. The first increment was identified in larvae 1 day after hatching. The regressed equations between daily age (D) and increment number in otoliths (N) were N = −0.64 + 0.96D in lapillus, and N = −0.31 + 0.98D in sagitta. The slopes were not significantly different than 1.0. This demonstrated that otolith increments in this species were formed daily and can be used for daily age determination.     

Ontogenetic pattern,morphological sexual and side dimorphism in the saccular otolith of a scaleless killifish Aphanius furcatus (Teleostei: Aphaniidae)

The relationships between ontogenetic and sexual factors with otolith morphology are poorly studied in killifish genus Aphanius Nardo, 1827. We studied the ontogeny, morphological sexual and side dimorphism in the saccular otolith of a scaleless killifish Aphanius furcatus inhabiting a high‐salinity environment in Southern Iran. The results highlighted growth‐dependent variability in the otolith of A. furcatus, which might be useful to define its certain developmental stages. We underlined those characters that were consistent during fish development, which could be valuable to identify the species. Considering the ontogenetic pattern of otolith in A. furcatus, it was found that the otoliths of young individuals have largely been influenced by sexes and that it was reduced during the fish development. Morphological side dimorphism in the otolith of A. furcatus tends to be increased during fish development. The outcomes of this study are essential since they have provided new information on ontogenetic changes and the sexual and side dimorphism of Aphanius otolith.     

Relationships between fish size and otolith size of four bathydemersal fish species from the south eastern Arabian Sea,India

The objective of this study was to estimate a prey body size from the hard parts (e.g. otoliths) of a fish species frequently found in the guts of predators. Length–weight relationships between otolith size (length, height, weight and aspect ratio) and fish size (total length and weight) were determined for four fish species captured in the Arabian Sea by bottom trawl (2015 survey on‐board FORV Sagar Sampada, 200–300 m depth), off the west coast of India: Psenopsis cyanea, Pterygotrigla hemisticta, Bembrops caudimacula and Hoplostethus rubellopterus. No significant differences were noted between the size of the left and right otoliths (t test) in any of the four species. The length–weight relationship of the otolith in all four species showed a negative allometric growth pattern (t test, p < .05). The data fitted well to the regression model for otolith length (OL), otolith height (OH) and otolith weight (OW) to total length (TL) and total weight (TW). Results showed that these relationships are a helpful tool in predicting fish size from the otoliths and in calculating the biomass of these less‐studied fish species during feeding studies and palaentology.     

Age and growth of <Emphasis Type="Italic">Schizothorax waltoni</Emphasis> in the Yarlung Tsangpo River in Tibet,China

Age and growth were studied for Schizothorax waltoni in the middle reaches of the Yarlung Tsangpo River in Tibet, southwest of China, from April 2004 to September 2006. A total of 201 specimens were collected ranging from 110 to 580 mm in standard length (SL). In contrast to other otoliths, sectioned lapillus showed a clear pattern of alternating opaque and hyaline zones. Marginal increment analyses showed that the increments, each composed of one opaque and one hyaline zone, are deposited annually. Opaque edges were prevalent from May to August. The von Bertalanffy growth parameters based on sectioned otolith data were L _t  = 689.8{1 − exp[0.051 (t + 3.275)]} for males, and L _t  = 691.1{1 − exp[0.056 (t + 2.466)]} for females. The slow growth and long life indicate that S. waltoni is vulnerable to overfishing and that harvesting strategies for the species should be conservative.     

Computed tomography scanning as a tool for linking the skeletal and otolith‐based fossil records of teleost fishes

Micro‐computed tomography (μCT) scanning now represents a standard tool for non‐destructive study of internal or concealed structure in fossils. Here we report on otoliths found in situ during routine μCT scanning of three‐dimensionally preserved skulls of Palaeogene and Cretaceous fishes. Comparisons are made with isolated otolith‐based taxa to attempt correlations between the body fossil and otolith fossil records. In situ otoliths previously extracted mechanically from specimens of Apogon macrolepis and Dentex laekeniensis match our μCT models. In some cases, we find a high degree of congruence between previously independent taxonomic placements for otolith and skeletal remains (Rhinocephalus, Osmeroides, Hoplopteryx). Unexpectedly, in situ otoliths of the aulopiform Apateodus match isolated otoliths of Late Cretaceous age previously interpreted as belonging to gempylids, a group of percomorph fishes that do not appear in the body fossil record until the Palaeogene. This striking example of convergence suggests constraints on otolith geometry in pelagic predators. The otoliths of Apateodus show a primitive geometry for aulopiforms and lack the derived features of Alepisauroidea, the lizardfish clade to which the genus is often attributed. In situ otoliths of Early Cretaceous fishes (Apsopelix and an unidentified taxon) are not well preserved, and we are unable to identify clear correlations with isolated otolith morphologies. We conclude that the preservation of otoliths suitable for μCT scanning appears to be intimately connected with the taphonomic history, lithological characteristics of surrounding matrix, and syn‐ and postdepositional diagenetic effects.     

Using otolith shape and morphometry to identify four Alburnus species (A. chalcoides,A. escherichii,A. mossulensis and A. tarichi) in Turkish inland waters

Asteriscus otolith shapes as well as their morphometry and shape contours were investigated in order to identify four allopatric Alburnus species: A. chalcoides (Güldenstädt, 1772) (Ordu), A. escherichii Steindachner, 1897 (Eski?ehir), A. mossulensis Heckel, 1843 (Tunceli), and A. tarichi (Güldenstädt, 1814) (Van) in Turkish inland waters. These were compared using the shape indices (form factor, roundness, circularity, ellipticity, rectangularity and aspect ratio), and the morphological characters [otolith weight (OWE), otolith length (OL), otolith width (OW), otolith perimeter (OP), and otolith area (OA)]. The overall canonical discriminant analysis (CDA) classification score was 93.8%, with the lowest score for A. escherichii (82.5%) and the highest for A. chalcoides (100%). The otolith shapes, morphology and shape contours of all sampled fish were a clear species differentiator, thereby demonstrating that the otolith shape is species‐specific. The current study presents for the first time comprehensive variation information on interspecific left‐right asteriscus otoliths in males and females of each Alburnus species: A. chalcoides from Ordu, A. escherichii from Eski?ehir, A. mossulensis from Tunceli and A. tarichi from Van, based on a total of 307 individuals. Scanning electron microscopy (SEM) images, shape contours and other otolith characters vary within the same genus; these differences should be investigated not only in other freshwater fish species or genera but also in the same species living in different habitats. In addition, further investigation is required not only with respect to the morphometry, biometry, shape, geometry, and shape contours of the otoliths, but also regarding the genetic methods for robust identification of various sympatric and allopatric fish populations.     

All in the ears: unlocking the early life history biology and spatial ecology of fishes

Obtaining biological and spatial information of the early life history (ELH) phases of fishes has been problematic, such that larval and juvenile phases are often referred to as the ‘black box’ of fish population biology and ecology. However, a potent source of life‐history data has been mined from the earstones (otoliths) of bony fishes. We systematically reviewed 476 empirical papers published between 2005 and 2012 (inclusive) that used otoliths to examine fish ELH phases, which has been an area of increasing attention over this period. We found that otolith‐based research during this period could be split into two broad themes according to whether studies examined: (i) biological objectives related to intrinsic processes such as larval and juvenile age, growth and mortality, and/or (ii) spatial objectives, such as habitat use, dispersal and migration. Surprisingly, just 24 studies (5%) explored a combined biological–spatial objective by simultaneously exploiting biological and spatial information from otoliths, suggesting much more scope for such integrated research objectives to be answered via the use of multiple otolith‐based techniques in a single study. Mapping otolith analytical techniques across these two approaches revealed that otolith structural analysis was mainly used to investigate biological processes, while otolith chemical analyses were most often applied to spatial questions. Heavy skew in research effort was apparent across biomes, with most (62%) publications specific to marine species, despite comparable levels of species richness and the importance of freshwater taxa (just 15% of papers). Indeed, around 1% (380 species) of a possible 31400+ extant species were examined in our surveyed papers, with a strong emphasis on temperate marine species of commercial value. Potential model species for otolith‐based ELH ecology research are arising, with the eel genus Anguilla (24 studies) and the European anchovy Engraulis encrasicolis (14 studies) attracting more research effort than most other taxa. While there is a preponderance of common techniques (e.g. daily otolith increment counts, increment widths), novel techniques such as transgenerational marking and computed X‐ray tomography, are increasingly being applied in published studies. The application of an integrative approach based on a combination of emerging techniques and traditional methods holds promise for major advances in our understanding of ELH fish ecology and to shine light into the ‘black box’ of fish ecology.     

Dietary histories of herbivorous loricariid catfishes: evidence from δ<Superscript>13</Superscript>C values of otoliths

The ecology of many Neotropical fishes is difficult or often impossible to study during rainy seasons. Thus, ecological studies of tropical fishes are usually performed on fish captured only during dry seasons. Because otoliths preserve a record of life history, this study evaluated the utility of otolith stable isotope values for the investigation of trophic ecology of Neotropical fishes (specifically herbivorous loricariid catfish) throughout their lives. Because plant dietary materials have δ¹³C values that are determined by their photosynthetic pathways, metabolism and environmental conditions, different plants may impart different isotope values on fish otoliths that reflect consumption of these plants. The δ¹³C_(otolith) values of xylophagous Panaque nigrolineatus captured in the field were significantly lower than those of algivorous Hypostomus regani from a nearby region. A laboratory experiment wherein Hypostomus sp. had δ¹³C_(otolith) values that reflected the δ¹³C values of their plant diet and additional evidence indicate that δ¹³C_(otolith) values in loricariid catfish otoliths can record dietary history.     

Otolith age determination for adult tilapia, Oreochromis niloticus L. from Lake Awassa (Ethiopian Rift Valley) by interpreting biannuli and differentiating biannual recruitment

Age of mature Oreochromis niloticus in Lake Awassa, Ethiopia, was estimated by analysing optical macrozones (translucent and opaque) in sagittal otoliths from fish sampled over a 12-month period. Seasonal record on the type of macrozone at the edge of otoliths suggested that two translucent macrozones associated with biannuli were formed each year; one during January and February and another during June and July. Formation of translucent macrozones coincided with minimum water temperature, spawning associated loss in condition and presumably with reduction in the quantity and quality of the food consumed by the fish. Relative marginal increment analysis showed that biannulus formation may be completed in March and in August each year. A concurrent study has confirmed a biannual recruitment in O. niloticus in Lake Awassa, from which mid-February and mid-August were taken as median hatch-dates. A procedure to assign otolith age is described which considers median hatch-dates and uses the number of biannuli in otoliths without discriminating between fish from the two recruitment cohorts. We also show a procedure to discriminate between the two cohorts by using the number of biannuli, conditions on the edge of the otolith and time of capture to assign age for the two recruitment cohorts separately.     

Biometric relation between body size and otolith size of seven commercial fish species of the south‐western Atlantic

Given the importance and applicability of these biometric relations, the present work aimed to verify the existence of correlations between the length of saggitae otoliths and the body size of seven south‐western Atlantic marine fish species and to generate equations to estimate the body size of these species through otolith measurements. Fifty otoliths of Centropomus parallelus, Centropomus undecimalis, Lutjanus analis, Lutjanus jocu, Lutjanus synagris, Chaetodipterus faber and Mugil curema were collected for analysis. Significant relations between otolith length and total length and otolith width and total length were found for all the species. The highest coefficient of determination was observed for Centropomus undecimalis and Lutjanus synagris, for both relations, and the lowest was observed for Mugil curema. The results show that estimates of body size of the species through biometric analyses of otoliths are reliable. Based on this, the equations generated to obtain the total length of the fish using biometric otolith data can be used in dietary studies of top predators and in paleontological recostructions of modern fish.     

The endangered cyprinodont <Emphasis Type="Italic">Aphanius ginaonis</Emphasis> (Holly, 1929) from southern Iran is a valid species: evidence from otolith morphology

Aphanius Nardo, 1827 (Actinopterygii, Cyprinodontidae) is a widely distributed genus in the Mediterranean and Persian Gulf area and includes several endangered species. The otolith morphology in Aphanius is known to represent a valuable tool for the taxonomy, and is also indicative for the genetic diversity of a particular population. The present study focuses on the otoliths of the endangered A. ginaonis (Holly, 1929), which is endemic to the Geno hot spring in southern Iran. The taxonomic status of A. ginaonis has repeatedly been questioned, and some scholars have argued that it merely represents a morphological variation of the widespread A. dispar. We present a comparison of the otolith morphology of A. ginaonis (52 specimens) with that of A. dispar (Rüppell, 1828) from the Mehran River Basin (southern Iran) (17 specimens) and an A. dispar population from the Persian Gulf coast of the United Arab Emirates (32 specimens). Our data obtained from SEM pictures, otolith morphometry and statistical analyses suggest that A. ginaonis represents a valid species. In A. ginaonis individuals with a standard length exceeding 23 mm, the otolith variables length–height and rostrum length represent useful complementary diagnostic characters discriminating this species from other Aphanius species. Besides ontogenetic variation, we found extremely high otolith form variability in A. ginaonis, including some otoliths with a morphology distinctly deviating from the basic morphology type. We hypothesize that these variations may be a result of the artificial introduction of A. dispar into the Geno hot spring during the last years and subsequent hybridisation.     

Sexual dimorphism in the otolith shape of shi drum,Umbrina cirrosa (L.), in the eastern Mediterranean Sea: Fish size–otolith size relationships

Little is known about possible differences in sagitta otolith size and shape between sexes of the shi drum, Umbrina cirrosa, and relationships between their body and otolith size. Thus, this study aimed to fill this knowledge gap via examination of 414 sagittal otoliths from 108 male (total length 13.8–26.8 cm) and 99 female (13.5–26.7 cm) U. cirrosa caught between May 2017 and April 2018 in gillnets set at a depth of ~15 m in Mersin Bay, Eastern Mediterranean Sea. No statistical differences were observed between the shape indices of the left-sided and right-sided sagitta. However, there were significant differences in the size and shape of otoliths between males and females. The slopes of allometric power functions from otolith width × fish sizes gave significant differences between males and females (ANCOVA, P < 0.05). The relationship for length × weight of otoliths from both males and females showed isometric growth, whereas the relationship of otolith width × otolith weight showed positive allometry. Negative allometric growth was observed for the relationship otolith length × otolith width. In summary, this study revealed the presence of sexual dimorphism in the otolith shape of U. cirrosa, and the data on regression relationships of fish-otolith sizes can be used to estimate fish size from U. cirrosa otolith sizes.     

Ontogenetic and environmental effects on otolith shape variability in three Mediterranean European eel (Anguilla anguilla, L.) local stocks

Otolith morphology is an efficient tool for the discrimination of fish stocks, populations and species when comparative genetic data are not available. Currently, the relationship between environmental factors and otolith shape is poorly characterized for the European eel (Anguilla anguilla), a highly migratory catadromous species constituting a single, randomly mating stock. The present study analyses the differences in otolith morphology between three Mediterranean eel local stocks from different environmental contexts (i.e. two brackish lagoons and one river). The relationship between otolith shape and otolith size was studied by means of Elliptic Fourier analysis and multivariate statistics. Otolith profile was digitally acquired and Cartesian coordinates were extracted. Partial Least Square (PLS) analysis pointed to continuous allometric growth in size and shape in otoliths from all three sites. In the three environments, shape variations occurred during growth as indicated by the presence of a significant and positive relationship between otolith size and the first PLS latent vector (i.e. which bears most of the information regarding otolith outline). Differences between smaller and larger sized otoliths were investigated using PLS Discriminant Analysis (PLSDA) and cluster analysis. Results indicate that otolith shape is highly uniform at smaller than at larger sizes. These shape differences apparently overlap the initial differentiation of the small otolith outlines acquired by eels during the growing phase as elvers in the marine environment. Data were discussed considering that the physical and chemical habitat variability in brackish lagoons and river could underlie a marked change in otolith shape during the animals' growth.     

Demographic characteristics of an intertidal bay goby (Lepidogobius lepidus)

Synopsis In a fourteen month study (May 1976 – June 1977) I examined the following characteristics of an intertidal bay goby (Lepidogobius lepidus) in Morro Bay, California, U.S.A.: annual and seasonal patterns of abundance, age composition and growth rates, survivorship and mortality patterns, and the reproductive cycle for female gobies. Fishes were collected with the aid of quinaldine and otoliths and ovaries removed. Age and growth rates were estimated from otolith annuli using a back calculation formula and a Brody-Bertalanffy growth curve. Mortality rates were derived using the methods of Heincke (1913), Robson & Chapman (1960), mean age, and a catch curve (Ricker 1975). A gonad index was used to describe the annual reproductive cycle. Results indicated that abundance fluctuated seasonally and that these fluctuations appeared to be caused by reproductive emigrations. Bay gobies reached an age of 7+ and a standard length of 87 mm. Growth was relatively constant (6 mm yr⁻¹) until age 5, at which point it began to decline. The mean rates of survivorship, mortality, and instantaneous mortality were 0.75, 0.25, and 0.29 respectively. Mortality rates for individual age classes ranged from 0.13 to 0.51 and increased with age. This stock appears to reproduce mainly during the winter.     

Saccular otolith mass asymmetry in adult flatfishes

A dimensionless measure of otolith mass asymmetry, χ, was calculated as the difference between the masses of the right and left paired otoliths divided by average otolith mass. Saccular otolith mass asymmetry was studied in eight flatfish species (110 otolith pairs) and compared with data from a previously published study on roundfishes. As in the case of symmetrical fishes, the absolute value of χin flatfishes does not depend on fish length and otolith growth rate, although otolith mass and the absolute value of otolith mass difference are correlated with fish length. The values of χwere between ?0·2 and +0·2 in 96·4% of flatfishes studied. The mean ±s .e . value of χin flatfishes was significantly larger than in standard bilaterally symmetrical marine fishes (‘roundfishes’), respectively 0·070 ± 0·006 and 0·040 ± 0·006. The most prominent distinction is the existence of downside prevalence of saccular otolith mass in flatfishes, which contrasts with no right–left prevalence in roundfishes found in a previous study. In the right‐eyed flatfishes (Soleidae), the left saccular otoliths are heavier than the right otoliths. In the left‐eyed flatfishes (Bothidae and Citharidae), the right saccular otoliths are heavier than the left otoliths. Not all flatfishes, however, fit in this design: 11·8% of flatfishes studied had the heavier saccular otoliths in the upside labyrinth and 5·4% of flatfishes had no otolith mass asymmetry (within the accuracy of the analysis). At the same time, the more mobile flatfishes (bothids and citharids) have more symmetrical and, hence, more precisely organized saccular otolith organs than the bottom‐associated flatfishes (soleids). It is possible to assume that the value of the otolith asymmetry is not only correlated with flatfish placement in a particular family, or position of eyes, but also may correlate with general aspects of their ecology. Mathematical modelling indicated that for most flatfishes one‐side saccular prevalence had no substantial significance for sound processing. On the other hand, calculations showed that 49% of flatfishes (but only 14·5% of roundfishes) have |χ| which exceed the critical level and, in principle, could sense the difference between the static displacement of the large and small paired otoliths. At that, the number of the soleids that could sense this difference is greater than the number of the bothids and citharids, 84 and 27%, respectively.     

Comparative morphology of the sagittal otolith in three species of south Caspian gobies

Sagittal otolith shapes were investigated in order to identify three sympatric species of south Caspian gobies (Caspian goby Neogobius caspius, deepwater goby Ponticola bathybius and bighead goby Ponticola gorlap). The sagittal otoliths in P. bathybius have a rectangular shape and are thick, whereas in N. caspius they are relatively round and thin. In P. gorlap, otoliths have an elongated shape and are relatively thick. The noticeable difference among the otoliths of the three species is the presence of one anterior and one posterior projection in the otoliths of N. caspius and P. gorlap. Among shape indices, form factor (irregularity of surface area), circularity, aspect ratio and rectangularity are the foremost that indicate interspecific variability. The canonical discriminant analysis correctly classifies 94·7% of the original group cases. The overall analyses show the relevance of applying otolith shape for interspecific distinction of the three species of Caspian gobies.     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.