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. 2020 Jun 24;237(5):979–987. doi: 10.1111/joa.13259

From yellow to silver: Transforming cranial morphology in European eel (Anguilla anguilla)

Jochem Baan 1,2,3, Jens De Meyer 1, Barbara De Kegel 1, Dominique Adriaens 1,✉
PMCID: PMC7542187  PMID: 32579740

ABSTRACT

The European eel (Anguilla anguilla) has been extensively studied, especially because of its highly specialized migratory behaviour associated with substantial phenotypic transformations. During this migration, one of those transformations the eel undergoes is from yellow to silver eel, a process known as silvering. Although the cranial morphology during the earlier glass, elver and yellow eel stages are well studied, little is known about actual morphological changes during the transformation process from the yellow to the silver eel stage. Yet, literature suggests drastic changes in musculoskeletal anatomy. Here, we investigated the cranial musculoskeletal morphology of 11 male European eels at different stages during silvering, resulting both from natural and artificial maturation. Using 3D‐reconstructed µCT data of the head, the skull and cranial muscles associated with jaw closing and respiration were studied. Eye size was used as a proxy for the silvering stage. Size‐adjusted jaw muscle volumes increased during silvering, although insignificantly. Accordingly, a near‐significant increase in bite force was observed. Respiratory muscles size did increase significantly during silvering, however. Considering the eel's long migration, which often includes deep and thus potentially oxygen‐poor environments, having a better performing respiratory system may facilitate efficient migration. Both overall skull dimensions and specifically orbit size increased with eye index, suggesting they play a role in accommodating the enlarging eyes during silvering. Finally, artificially matured eels had a wider and taller skull, as well as larger jaw muscles than wild silver eels. This could be caused (a) by different conditions experienced during the yellow eel stage, which are maintained in the silver eel stage, (b) by side effects of hormonal injections or (c) be part of the maturation process as artificially induced silver eels had a higher eye index than the wild silver eels.

Keywords: bite force, musculoskeletal system, myology, osteology, silvering, Anguilla

During the metamorphosis from yellow to silver eels (Anguilla anguilla), the cranial musculoskeletal system changes to prepare the eel for its 6500 km migration. Both the jaw and respiratory muscles increase in size, with the latter potentially being beneficial for swimming in deeper, and thus less oxygenated waters. Accordingly, the overall skull dimensions increase in size, most likely to accommodate the enlarging eyes and muscles

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1. INTRODUCTION

The European eel (Anguilla anguilla) is a thoroughly studied species, both for its role in aquaculture and its complicated lifecycle (Törlitz, 1922; Tesch, 2003). Anguilla comprises catadromous eel species that spend the majority of their lifecycle in freshwater but migrate back to the oceans for spawning only (Durif et al., 2009a). Hatching from their eggs in the Sargasso Sea, the Gulf Stream currents bring the larvae from their spawning grounds to the coastal waters of Europe and North Africa (van den Thillart et al., 2009). Reaching the continental shelf, the larvae metamorphose into unpigmented glass eels. Hereafter, during the elver eel stage, the onset of feeding and pigmentation occurs. Then, the eels migrate upstream and enter the yellow eel phase, which lasts for three up to fifteen years in male eels, or four up to twenty in female eels. Once the yellow eels mature during a process known as silvering, the skin colour changes (the belly becomes silvery white), the eyes become bigger, the pectoral fins become longer and the skin thickens (Durif et al., 2009b). These silver eels cease feeding and migrate across the North Atlantic ocean to reach the spawning grounds assumed to be in the Sargasso sea (Tesch and Wegner, 1990; van Ginneken and Maes, 2005). This cessation of feeding can have an impact on the morphology of the maturing eels, as dietary shifts are known to impact morphology in European eel (De Meyer et al., 2016), as well as in other fish species (Svanbäck et al., 2008; Svanbäck and Eklöv, 2002). The cranial morphology has been thoroughly studied at life stages preceding the silver eel stage (Bouilliart et al., 2015; De Meyer et al., 2018a, 2018b), and also bite force has previously been determined in these stages (De Meyer et al., 2018a, 2018b, 2018c). To fully understand the eel's morphology during ontogeny, we here want to determine the cranial changes associated with silvering.

Since the silvering eel undergoes physical changes (e.g. enlarged eyes), changes in behaviour (e.g. stops feeding and migrates) and the onset of bone degradation (Rolvien et al., 2016), musculoskeletal differences might be found in the cranium across a maturation gradient. An eye index, calculated from the relative eye diameter, has previously been proven to be a good indication for the eel's maturation process (Pankhurst, 1982a; Durif et al., 2005, 2009a). Accordingly, in the current study, cranial musculoskeletal morphology was studied in yellow eels and silver eels. Specifically, muscle volumes, osteology and bite force were compared along the eye index gradient. We first test the hypothesis that relative volumes of jaw muscles responsible for jaw closure (and therefore bite force) decrease during silvering, as silver eels stop feeding. Alternatively, relative volumes might remain similar during transformation and only start decreasing following feeding cessation. We also test the hypothesis that relative volumes of muscles associated with respiration increase during silvering: during migration, eels were found to swim at depths up to 800 m (Jellyman and Tsukamoto, 2005; Righton et al., 2016). At these depths, oxygen levels can be low; aerobic bacteria use oxygen for their metabolic processes, thus lowering the oxygen concentrations, while oxygen itself is either replenished at the surface or through deep, polar oxygen‐rich waters. Consequently, the lowest oxygen levels are typically found between 200 and 1000 m of depth (Lali and Parsons, 1993). Silver eels might thus exhibit enlarged respiratory musculature to deal with these more oxygen‐poor environments.

Additionally, eels have been successfully artificially matured from the yellow to silver eel stage (Palstra et al., 2005; Palstra and van den Thillart, 2009; Tanaka, 2015). It was found that these eels displayed more bone tissue degradation compared to wild silver eels (Rolvien et al., 2016). As such, we also compared cranial morphology between wild silver eels and artificially matured silver eels to test the hypothesis that artificially matured eels exhibit amplified morphological maturation effects. The insights in how the cranial musculoskeletal system changes during silvering obtained during this study can provide useful information about how and whether this transformation has an adaptive role during migration.

2. METHODS

2.1. Sample collection

Eleven male European eels (A. anguilla) were selected along a maturation gradient according to the eye index (EI) (Pankhurst, 1982a; Durif et al., 2009a):

EI=(MD/2)2×π/TL

where MD is the average of the horizontal and vertical eye diameter, and TL is the total length of the specimen. As defined by Durif et al. (2005), the male eels were classified along the EI gradient as such: EI < 7.9 are yellow eels (YE) and EI > 7.9 are silver eels (SE). The sample consisted of four yellow eels and seven silver eels (Table S1). Three yellow eels (YE1‐3) were captured in the Demer river in 2014 in fyke nets. Another three eels (YE4, SE1 & SE2) were captured in Nieuwpoort in 2001 using the same method. Five male silver eels (SE3‐7) were obtained from Leiden University in 2008, which were hormonally induced to stimulate the development from yellow to silver eel (Palstra et al., 2005), from here on referred to as artificial silver eels. Before decapitation, all specimens were anaesthetized with MS222, killed by an MS222 overdose and the total length (TL) was measured to the nearest millimetre. All eel heads were fixed in 10% formalin and preserved in 70% ethanol.

2.2. µCT scanning, 3D‐reconstructions and myology

All eel heads were µCT scanned, first untreated to visualize bone structure, and a second time after treatment with phosphomolybdic acid (2.5%) for 14 days to visualize soft tissues (Metscher, 2009). All µCT scans were performed at the Centre for X‐ray Tomography at Ghent University (UGCT) with the HECTOR scanner, built and developed in collaboration with the company X‐Ray Engineering. Scans were made using 60‐140 kV tube voltage with 1801 up to 2401 projections over 360°. The pixel pitch of the detector was 200 μm, with the reconstructed voxel sizes of the different specimens varying between 29.99 and 113.85 μm (specific scanner data per specimen can be found in Table S1).

3D reconstructions of cranial bones were generated using data from the first scan, and of cranial muscle groups with the second scan, using AMIRA 5.5.0 (Visage Imaging, San Diego, CA, USA). Skull reconstructions were used for measurements and 3D‐landmark placement. Following nomenclature of Winterbottom (1973), reconstructions were made and volumes were determined for the following muscle bundles: adductor mandibulae (A1, A2 & A3), adductor arcus palatini (AAP), levator arcus palatini (LAP), protractor hyoidei (PH), adductor operculi (AO), dilatator operculi (DO) and levator operculi (LO). Tendons of the A1 and A3 muscle bundles were also reconstructed. Additionally, the eyes were reconstructed to determine the horizontal and vertical eye diameter.

2.3. Osteology

Measurements on the skull were done in AMIRA 5.5.0 and were used to determine morphological changes in bone structure along the maturation gradient. Where applicable, measurements were averaged between the left and right side of the skull. Skull length (from tip of the snout to the most caudal tip of the skull), skull width (distance between the distal tips of the left and right sphenotic) and a proxy for skull height (distance between the lateral tip of the sphenotic and the caudal lower jaw tip) were determined to analyse general skull proportions (Figure 1a,b; lengths A, B and C, respectively). Dimensions of the orbit were measured as the sides of a triangular shape (Figure 1b; D, E and F), and orbit width was determined on the lower and upper caudal part of the orbit (Figure 1c; H and J, respectively). Snout length was determined from the tip of the upper jaw to the rostral corner of the orbit (Figure 1b; G). Jaw width was determined as the distance between the most caudal points on the left and right halves of the lower jaw.

FIGURE 1.

FIGURE 1

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Skull measurements and landmarks in dorsal view (a,d), lateral view (b,d) and zoomed in view of the caudal part of the orbit (c). The 3D‐measurements done are skull length (A), skull width (B), skull height (C), orbit height (D), orbit length (E,F), snout length (G) and orbit width (H,J). Letter ‘I’ was omitted due to poor visibility in the figure. Distance between caudal lower jaw tips is not visualized here.

In addition, a total of 37 landmarks were placed onto the 3D mesh, to capture the overall shape of the skull, lower and upper jaw, orbit and hyomandibula (Figure 1d,e). 3D‐Coordinates of landmarks were then imported into MorphoJ version 1.06d (Klingenberg, 2011). To account for variability in orientation, positioning and size among specimens, a Generalized Procrustes analysis was applied that rescales, translates and rotates the coordinate data. Subsequently, a principal component analysis (PCA) was performed on the transformed landmark data to determine and visualize the major axes of shape variation in the skull. The first four major axes principal component (PC) scores were exported for further analysis. These axes cumulatively explain over 80% of the shape variation and were the significant according to a broken stick analysis.

2.4. Bite force model

2.4.1. Input data collection

Volume, whole muscle length, tendon length and tendon cross‐sectional area (CSA) of the A1 and A3 muscle bundles were collected in AMIRA and used as input data for the bite force simulations. Tendon length and CSA of the A2 muscle bundle could not reliably be determined and was therefore deduced from other reconstructions of eels by De Meyer et al. (2018b). In the latter study, the A2 tendon length was on average 13.7% of the A2 total length, while CSA was on average (A2 total length × 0.045)2. The length and CSA of the A2 tendon in our specimens were calculated accordingly. Additionally, 3D landmarks were placed at positions of the origin and insertion points of A1, A2 and A3, at the proximal and distal bite points, at the jaw tip and at the left and right jaw joints. Muscle, tendon, bite point and jaw tip data were collected from the head‐side with the greatest scan quality. The 3D‐landmark coordinates were transformed and oriented in the same direction to have the jaw joints set at (0, 0, z) and (0, 0, −z), for the right and left jaw joint, respectively. The tip of the lower jaw was set at (x, 0, 0). Lastly, muscle fibre length of A1, A2 and A3 and pennation angle of A1 were determined. For A2 and A3, no pennation angle was determined as these are parallel fibred. After CT scanning, eel heads were dissected to expose the adductor mandibulae A1. A picture was taken to determine the pennation angle using ImageJ (version 1.52c) for ten measurements, after which the average was used. Afterwards, the muscle bundles of A1, A2 and A3 were removed and immersed in HNO3 (30%) for up to 72 hours to isolate individual muscle fibres. Individual fibres were then photographed (Olympus SZX‐ILL B200) and the average length of ten fibres of each muscle bundle was determined using ImageJ.

2.4.2. Determining bite force

Bite force was simulated using a bite model based upon the dynamic muscle‐tendon model of van Leeuwen (1992). The model was developed and written by Van Wassenbergh et al. (2005) in MATLAB Simulink version R2013b (8.2.0.701) using the SimMechanics toolbox (First Generation) and full details of the model and its variables can be found in De Meyer et al. (2018b) and Van Wassenbergh et al. (2005). The model assumes that the lower jaw forms a single rigid body, able to rotate around one axis through the left and right jaw joints. The adductor mandibulae, subdivided into three main bundles (A1, A2 & A3), are responsible for the mouth‐closing torque about this axis. According to the observed morphology, the A1 muscle bundle was modelled as a bipennate muscle, whereas A2 and A3 were modelled as unipennate muscle. Bite force was subsequently calculated at two bite points along the lower jaw, that is a proximal bite point at the most posterior tooth and a distal bite point at the most anterior tooth and this at gape intervals of 10°, from a nearly closed mouth (0°, i.e. how the specimens were µCT scanned) up to a gape angle of 30°. Maximum bite forces were obtained at a gape angle of 0° in 9 out of 11 specimens, and therefore, bite forces generated at this gape angle were used for further analysis.

2.5. Data analysis

First, the cube root of each muscle volume (expressed in mm3) was calculated. Subsequently, all skull measurements, the cube rooted muscle volumes and TL (expressed in mm) were log‐transformed. Next, each measurement was size‐corrected by dividing the log‐transformed measurement by log(TL). Log(TL) can be reliably used as there are no changes in the relative post‐cranial body length along the eye index gradient (Figure S1). For certainty, we also divided each log‐transformed measured by log(skull length) and repeated our analyses, which gave similar results as for log(TL). The results of these analyses can be found in Table S2. Bite force (BF) was size‐corrected as the square root of BF divided by TL (in cm). Data were checked for normality using Shapiro–Wilk tests along with visual inspection using density plots and quantile–quantile plots. Potential outliers were not removed since sample size was small already (n = 11). Subsequently, linear regression (Reduced major axis regressions) and Pearson correlation analysis was performed to determine (significant) changes in muscle volumes, skull measurements and bite forces with the eye index.

3. RESULTS

3.1. Myology: cranial muscle volumes

No apparent changes in origin and insertion of the different muscle bundles are observed among the eels, independent of life stage (see Figure S2 for reconstructed muscles and see Brocklehurst et al. (2019) and De Meyer et al. (2018b), for more details on the muscle morphology).

The size‐adjusted A1 and A3 showed an insignificant, positive increase with eye index (p = 0.11 and p = 0.06, respectively; Figure 2), while no changes are observed in the size‐adjusted A2 (p < 0.01). Size‐adjusted AO and DO, associated with closing and opening of the gills, respectively, on the contrary, showed a significantly positive correlation with eye index (p = 0.03 and p < 0.01, respectively; Figure 2), with a moderately strong fit for AO (r 2 = 0.45) and strong fit for DO (r 2 = 0.61). The size‐adjusted LO showed no significant correlation with eye index (p = 0.26, Figure 2). Although the size‐adjusted LAP showed a significant correlation with the eye index (p = 0.03), the fit of the linear model is moderately poor (r 2 = 0.39) and the slope is heavily influenced by a single outlier. The PH and AAP muscles showed no significant correlation in their volume with eye index (p = 0.24 and p = 0.10 for PH and AAP, respectively).

FIGURE 2.

FIGURE 2

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Plots of the size‐corrected muscle volumes and bite force against eye index. Specimens are categorized according to maturation stage. Yellow eel: circles, wild silver eels: diamonds and artificial silver eels: triangles). Dashed lines represent linear models, and r 2 and p values are given in each plot (n = 11). Lines for linear models are given irrespective of significance, to illustrate general trends along the maturation gradient. A1‐A3: Adductor mandibulae 1‐3; AO: Adductor operculi; DO: Dilatator operculi, LO: Levator operculi

3.2. Osteology: cranial skeletal morphology

PC1 (explaining 38% of the variation) distinguishes the wild silver eels from the artificial silver eels (Figure 3). The PC1 axis predominantly describes variation in orbit size, skull width and skull height. As such, the upper orbit length is shorter and the cranium is narrower and less tall in yellow and wild silver eels, compared to artificial silver eels (wireframes in Figure 3). PC2 (explaining 28% of the variation) shows shape variation between yellow and silver eels, although not unambiguously (one yellow eel plots among the silver eel range. Artificial silver eels have slightly higher PC2 scores than wild silver eels. As for PC1, PC2 also describes variation in orbit size, with yellow eels having a smaller, shorter orbit compared to the wild silver eels. PC2 also shows that yellow eels show a larger distance between the caudal tips of the lower jaw and a more abducted hyomandibula compared to the wild silver eels.

FIGURE 3.

FIGURE 3

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Results of the principal component analysis (PCA) on the landmark data. Middle: Plot of PC1 vs PC2. Specimens are categorized according to maturation stage: yellow eels (YE; circles), wild silver eels (SE; diamonds) and artificially matured silver eels (AM; triangles). Surrounding the graph: Wireframe graphs representing shape variation are given for low and high PC1‐ (right) and PC2‐scores (left). Both the dorsal view (top) and lateral view (bottom) are shown.

The correlation matrix shows that the eye index is positively correlated with relative skull length, skull width, skull height, orbit height and snout length (0.6 < r < 0.7), and more strongly with upper and lower orbit length (r = 0.87 for both; Table 1). Additionally, relative skull length shows a moderately strong to strong positive correlation with all size‐corrected measurements (0.6 < r < 0.9), except for orbit height.

Table 1.

Correlation matrix of the size‐adjusted measurements done on the skull, total length (TL) and eye index (EI), showing correlation coefficients (r) for the correlation of each pair of variables

Correl Skl SW SH OH UOL LOL SL LOW UOW CLJ
EI 0.64 0.71 0.66 0.68 0.87 0.87 0.60 0.17 0.37 0.43
SkL 0.81 0.83 0.25 0.81 0.81 0.89 0.63 0.71 0.76
Sw 0.77 0.15 0.87 0.65 0.79 0.52 0.80 0.88
SH 0.32 0.73 0.76 0.58 0.22 0.43 0.62
OH 0.45 0.72 0.16 −0.13 −0.27 −0.08
UOL 0.88 0.72 0.47 0.65 0.70
LOL 0.64 0.32 0.35 0.46
SL 0.75 0.80 0.74
LOW 0.69 0.55
UOW 0.90
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Abbreviations: CLJ, Caudal lower jaw distance; LOL, lower orbit length; LOW, lower orbit width; OH, orbit height; SH, skull height; SkL, skull length; SL, snout length; SW, skull width; UOL, upper orbit length; UOW, upper orbit width.

Regressing the skull measurements against the eye index shows that skull length, width and height show a significant positive correlation with eye index but that the fit was only moderately poor to moderately strong (p = 0.03 and r 2 = 0.41; p = 0.02 and r 2 = 0.51; p = 0.03 and r 2 = 0.43, respectively). Orbit height upper and lower orbit lengths and snout length also showed significant positive correlation with eye index, with a moderately poor fit for orbit height and snout length and a strong fit for upper and lower orbit length (p = 0.02 and r 2 = 0.47; p = 0.04 and r 2 = 0.36; p < 0.01 and r 2 = 0.76; p < 0.01 and r 2 = 0.75, respectively). Orbit width and distance between caudal lower jaw tips showed no significant correlation with the eye index according to the respective linear models (pUOW = 0.27; p LOW = 0.62; pCLJ = 0.18).

3.3. Bite force

The large A1 is responsible for most of the generated bite force. Cumulative bite forces on the proximal bite point ranged from 1.8 N up to 7.3 N, while bite forces on the distal bite point ranged from 0.9 N up to 3.5 N. Generally, the proximal bite force was 2 to 2.5 times higher compared to the distal bite force. Size‐adjusted bite forces (proximal and distal) appeared to have no clear relationship with the eye index (Figure 2). Although the positive correlation between distal bite force and eye index approximated significance, the fit of the linear model was moderately poor (p = 0.053 and r 2 = 0.35, Figure 2).

4. DISCUSSION

4.1. From yellow to silver eel

The aim of this study was to get a better understanding of how the cranial morphology in the European eel changes during the transformation from the yellow eel to the silver eel stage. We expected that during this transformation process, changes in the musculoskeletal system would occur in order to prepare the eels for their migration across the Atlantic Ocean. Additionally, with the inclusion of artificial silver eels, it was expected that these eels specifically would show amplified morphological changes during the transformation from yellow to silver eel, as was observed by Rolvien et al. (2016). As such, we investigated morphological changes in the skull, as well as cranial muscles involved in generating bite force and respiration, across a gradient describing the transformation from yellow to silver eel.

We found both an increase in the relative size of several skull features, such as relative skull length and skull width, as well as in relative orbit size, with increasing eye index. During silvering, the eyes substantially increase in size, both in absolute and relative terms (Durif et al., 2005). Both the overall increase in skull size as in orbit size might be crucial to accommodate these enlarging eyes. While the larger orbits most likely directly support the larger eyes during silvering, it might not be sufficient to properly accommodate them. An overall increase in skull dimensions could consequently be necessary to retain a functional architecture, that is when the eyes become too large relative to the skull, the eel might just not be able to physically support them.

As the silver eel stops feeding (van Ginneken and Maes, 2005), we expected the jaw muscles to decrease in relative volume during silvering. Instead, we observed no change in relative the jaw muscle volume of adductor mandibulae 2 and even an (insignificant) increase in the other adductor mandibulae‐muscle bundles during the transformation from yellow to silver eel. This increase in jaw muscle volume corresponds to a near‐significant increase in bite force along the eye index gradient. Both the increase in jaw muscle volume as well as bite force are unexpected, considering the cessation of feeding. One possible explanation is that the wild silver eels did not yet complete the maturation process. This process is known to be triggered by sustained swimming (Palstra et al., 2009; Palstra and van den Thillart, 2010). As the wild silver eels used were caught in river estuaries, thus still at the onset of their migration, it is possible that these eels are either still maturing or only recently ceased feeding, and thus no changes in jaw muscle volume can be observed yet. Another explanation for the absence of decreases in relative jaw muscle volume could be that these muscles act as an extra energy source during the long migration. However, eels are known to be very efficient swimmers, compared to other migrating semelparous fish species (Palstra and van den Thillart, 2010; van Ginneken and Maes, 2005), and accumulate high body fat percentages that should allow successful migration (van Ginneken and van den Thillart, 2000), making this scenario unlikely. A third explanation is that the increase in jaw muscle volume corresponds to an increase in body muscle volume. Pankhurst (1982b) observed that red body muscle volumes increase approx. 8% during sexual maturation due to an increase in fibre diameter. Still, the jaw muscles of fish mainly or exclusively consists of white muscle fibres (Baldwin et al., 1991; Maie et al., 2011). A similar increase in jaw muscle volume as in body muscle volume thus seems unlikely. Finally, it is possible that jaw muscle volume does not decrease at all in silver eels. To determine this, it would be necessary to evaluate the muscular anatomy of silver eels near the spawning site, not having fed during the migration.

As hypothesized, an increase in eye index is associated with larger respiratory muscle volumes. Silver eels can be found at great depths during their migration, as deep as 800 m (Jellyman & Tsukamoto 2010,2005; Righton et al., 2016). Greater depths are associated with lower oxygen levels, as oxygen is depleted by aerobic bacteria that use it to metabolize organic matter. In general, oxygen minima are obtained between 200 and 1000 m depending on the location (Lalli and Parsons, 1993). Yellow eels live in rivers, lakes and estuaries, which are shallow compared to the migratory depths of silver eels. Consequently, yellow eels will be rarely exposed to the same low oxygen levels silver eels experience. Hypertrophied respiratory muscles might thus serve as an adaptive trait in silver eels to deal with these circumstances. A common behaviour for fish to deal with low oxygen conditions is by increasing the water flow over the gills. Ram‐ventilating sharks (creating water flow over the gills by swimming with the mouth opened, Bone and Moore, 2008) swim faster in low oxygen conditions to increase the water flow, while three‐spined sticklebacks almost double the gill cover movement rate from 90 to 165 per minute when oxygen levels were dropped to half the normal level (Jones, 1952; Carlson and Parsons, 2001). Increased gill ventilation, facilitated by the hypertrophied respiratory muscles, might thus serve as a way for silver eels to deal with lower oxygen conditions, especially since alternatives such as decreased activity and anaerobic respiration (which results in toxic by‐products) do not coincide with a long, constant migration.

4.2. Wild vs. artificial silver eels

Over the last decades, successful hormonal induction of maturation from yellow eel to silver eel has been achieved in the Japanese eel (A. japonica; Kagawa et al., 2005; Tanaka, 2015) and European eel (Palstra and van den Thillart, 2009). However, this process also induces abnormalities, as for example a limited reproductive success and variations in yolk accumulation and egg membrane formation (Adachi et al., 2003; Kagawa et al., 2005). Moreover, it has been found that levels of bone degradation in artificially matured eels are higher compared to wild silver eels (Rolvien et al., 2016). Whether maturation from yellow to silver (artificial or natural) also impacts skull shape has not been evaluated yet. According to the 3D‐landmark analysis, silver eels obtain longer and taller orbits, corresponding to the larger eye size (Durif et al., 2009a). Yet, wild and artificial silver eels showed additional skull differences, such as differences in the height and width of the (neuro‐) cranium. Artificial silver eels exhibit a taller and wider cranium than wild silver eels. Two scenarios could explain this observation: First, morphological variation is often associated with variation in habitat and diet in fish (Svanbäck et al., 2008; Bartels et al., 2012; Faulks et al., 2015; Svanbäck & Eklöv 2002,2003). Accordingly, diet‐related morphological differences have been found in European eel, where eels fed on a hard diet developed wider heads (De Meyer et al., 2016). Thus, dietary differences in the yellow eel stage resulting in skull differences might have been maintained in the silver eel stage, potentially explaining the difference between artificial and wild silver eels. This could concur with the observation of the larger jaw muscles (even though insignificant) of the artificial silver eels, as De Meyer et al. (2018b) hypothesized that a broader, taller skull can allow the accommodation of larger jaw muscles. Alternatively, the difference in skull and also muscle volume might be caused by the weekly injections of human chorionic gonadotropin, which induces maturation (Palstra et al., 2005; Palstra and van den Thillart, 2009). This hormone elevates testosterone levels (Winters et al., 1972), which, in turn can increase muscle mass (Griggs et al., 1985). Consequently, the increase in jaw muscle volume, combined with a necessary broadening and heightening of the skull, might stem from elevated testosterone levels following hormonal injections. Whether such weekly hormonal injections inducing maturation correspond to the natural maturation process has not been evaluated yet. As such, we are currently unable to determine whether the musculoskeletal morphology of the artificial silver eels is unnaturally caused by the hormonal injections or whether the wild silver eels will obtain larger jaw muscles when they have reached the same eye index as the artificial silver eels. However, since silver eels fasten during migration, we hypothesize that the larger jaw muscle volumes are most likely side effects of hormonal stimulation, as such morphological changes are expected to be irrelevant during migration. The broader, taller skull might then either be necessary to accommodate the larger jaw muscles or help in supporting the enlarging eyes as discussed above.

5. CONCLUSIONS

In conclusion, several cranial musculoskeletal changes take place during the transformation from the yellow to the silver eel stage. An increase in relative orbit size, as well as in overall skull dimensions, might be necessary to accommodate the enlarging eyes. In addition, an increase in the respiratory muscles volumes might be adaptive for the eels during migration, swimming at greater depths with lower oxygen levels. We also found that artificial silver eels possess a larger skull and jaw muscles compared to wild silver eels. These differences might either result from dietary differences in the yellow eel stage that are retained in the silver eel stage or be a side effect of the hormonal injections. However, currently, we cannot confirm that artificial silver eels exhibit amplified changes in morphology during the transformation.

AUTHOR CONTRIBUTIONS

J. Baan was responsible for the data gathering (3D reconstructions, bite force modelling), data analysis, figure preparations and manuscript writing. J. De Meyer was responsible for the experimental design and helped with data interpretation, manuscript writing and revision of figures. B. De Kegel did the PMA staining and µCT scanning. D. Adriaens helped with critically revising the manuscript.

Supporting information

Supplementary Material

Click here for additional data file. (1.1MB, docx)

ACKNOWLEDGEMENTS

The authors would like to thank the Leiden University for providing the artificially matured silver eels and the INBO (Institute of Nature and Forest Research) for capturing the wild yellow and silver eels. The authors are indebted to B. De Kegel for scanning the eels. This research project was funded by the Special Research Fund (BOF; PDO.2017.001.301 Fund IV1).

Baan J, De Meyer J, De Kegel B, Adriaens D. From yellow to silver: Transforming cranial morphology in European eel (Anguilla anguilla). J. Anat. 2020;237:979–987. 10.1111/joa.13259

DATA AVAILABILITY STATEMENT

The data that support our results will be made publically available on Dryad upon manuscript acceptance.

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