Ontogenetic shifts in brain scaling reflect behavioral changes in the life cycle of the pouched lamprey Geotria australis

Abstract

Very few studies have described brain scaling in vertebrates throughout ontogeny and none in lampreys, one of the two surviving groups of the early agnathan (jawless) stage in vertebrate evolution. The life cycle of anadromous parasitic lampreys comprises two divergent trophic phases, firstly filter-feeding as larvae in freshwater and secondly parasitism as adults in the sea, with the transition marked by a radical metamorphosis. We characterized the growth of the brain during the life cycle of the pouched lamprey Geotria australis, an anadromous parasitic lamprey, focusing on the scaling between brain and body during ontogeny and testing the hypothesis that the vast transitions in behavior and environment are reflected in differences in the scaling and relative size of the major brain subdivisions throughout life. The body and brain mass and the volume of six brain structures of G. australis, representing six points of the life cycle, were recorded, ranging from the early larval stage to the final stage of spawning and death. Brain mass does not increase linearly with body mass during the ontogeny of G. australis. During metamorphosis, brain mass increases markedly, even though the body mass does not increase, reflecting an overall growth of the brain, with particularly large increases in the volume of the optic tectum and other visual areas of the brain and, to a lesser extent, the olfactory bulbs. These results are consistent with the conclusions that ammocoetes rely predominantly on non-visual and chemosensory signals, while adults rely on both visual and olfactory cues.

Full text

ORIGINAL RESEARCH
published: 28 July 2015
doi: 10.3389/fnins.2015.00251
Frontiers in Neuroscience | www.frontiersin.org 1July 2015 | Volume 9 | Article 251
Edited by:
Jorge Mpodozis,
Universidad de Chile, Chile
Reviewed by:
Andrew Iwaniuk,
University of Lethbridge, Canada
Thomas Lisney,
University of Western Australia,
Australia
*Correspondence:
Carlos A. Salas,
Neuroecology Group, School of
Animal Biology, The University of
Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia
carlos.salas.uwa@gmail.com
Specialty section:
This article was submitted to
Evolutionary Psychology and
Neuroscience,
a section of the journal
Frontiers in Neuroscience
Received: 17 April 2015
Accepted: 03 July 2015
Published: 28 July 2015
Citation:
Salas CA, Yopak KE, Warrington RE,
Hart NS, Potter IC and Collin SP
(2015) Ontogenetic shifts in brain
scaling reflect behavioral changes in
the life cycle of the pouched lamprey
Geotria australis.
Front. Neurosci. 9:251.
doi: 10.3389/fnins.2015.00251
Ontogenetic shifts in brain scaling
reflect behavioral changes in the life
cycle of the pouched lamprey Geotria
australis
Carlos A. Salas 1*, Kara E. Yopak1, Rachael E. Warrington 1, Nathan S. Hart 1,
Ian C. Potter 2and Shaun P. Collin1
1Neuroecology Group, School of Animal Biology and UWA Oceans Institute, The University of Western Australia, Crawley,
WA, Australia, 2Centre for Fish and Fisheries Research, School of Veterinary and Life Sciences, Murdoch University,
Murdoch, WA, Australia
Very few studies have described brain scaling in vertebrates throughout ontogeny and
none in lampreys, one of the two surviving groups of the early agnathan (jawless)
stage in vertebrate evolution. The life cycle of anadromous parasitic lampreys comprises
two divergent trophic phases, firstly filter-feeding as larvae in freshwater and secondly
parasitism as adults in the sea, with the transition marked by a radical metamorphosis.
We characterized the growth of the brain during the life cycle of the pouched lamprey
Geotria australis, an anadromous parasitic lamprey, focusing on the scaling between
brain and body during ontogeny and testing the hypothesis that the vast transitions in
behavior and environment are reflected in differences in the scaling and relative size of
the major brain subdivisions throughout life. The body and brain mass and the volume of
six brain structures of G. australis, representing six points of the life cycle, were recorded,
ranging from the early larval stage to the final stage of spawning and death. Brain mass
does not increase linearly with body mass during the ontogeny of G. australis. During
metamorphosis, brain mass increases markedly, even though the body mass does not
increase, reflecting an overall growth of the brain, with particularly large increases in the
volume of the optic tectum and other visual areas of the brain and, to a lesser extent, the
olfactory bulbs. These results are consistent with the conclusions that ammocoetes rely
predominantly on non-visual and chemosensory signals, while adults rely on both visual
and olfactory cues.
Keywords: growth, agnathan, lifestyle, filter feeder, heterochrony, jawless vertebrate, metamorphosis, parasite
Introduction
Lampreys are extant relatives of an early and diverse group of jawless vertebrates (Kumar and
Hedges, 1998; Heimberg et al., 2008; Janvier, 2008; Smith et al., 2013). The results of early
studies on the agnathan nervous system (Johnston, 1902; Heier, 1948; Nieuwenhuys, 1977)
have thus been used as an indicator of the ancestral condition of the vertebrate brain (Fritzsch
and Northcutt, 1993a; Butler and Hodos, 1996; Northcutt, 2002; Gilland and Baker, 2005;
Khonsari et al., 2009; Suárez et al., 2014). The design or bauplan of the vertebrate brain and the
developmental mechanisms that underlie their subdivisions are considered to be highly conserved

Salas et al. Brain scaling during ontogeny in lampreys
(Striedter, 2005; Ota and Kuratani, 2007; Guerin et al., 2009;
Charvet et al., 2011). However, it is expected that the various
sensory modalities and other neural specializations will evolve,
to a degree, in association with ecological niche, and that
this relationship will be reflected in adapted behaviors and/or
enhanced cognitive capabilities (Barton et al., 1995; Barton and
Harvey, 2000; De Winter and Oxnard, 2001). Indeed, brain size
and the relative development of major brain subdivisions vary at
intraspecific, interspecific, and ontogenetic levels across a range
of vertebrates (e.g., Kruska, 2005; Gonda et al., 2013) in relation
to factors such as life style, habitat, and behavior (e.g., Pollen
et al., 2007; Yopak and Montgomery, 2008; Barton and Capellini,
2011), as well as phylogenetic and developmental constraints
(e.g., Finlay and Darlington, 1995; Yopak et al., 2010).
The size of the brain relative to the body (scaling) has long
since been used in studies of brain development and evolution
(Ariëns Kapper, 1936; Gould, 1975; Deacon, 1990; Aboitiz,
1996), in which brain mass (E) is characterized as a function
of body mass (S) with Snell’s formula: E=k∗S∝or log E=∝
log S+k, where ∝= allometric slope or scaling power. It is a
common assumption that encephalization (a larger than expected
brain size for a given body size) reflects enhanced cognitive
capabilities (Jerison, 1977; Ebbesson, 1980; Lefebvre et al., 2004),
although this is still the subject of debate (Healy and Rowe,
2007; Herculano-Houzel, 2012). Previous studies have examined
encephalization of the brain of jawless fishes (Platel and Delfini,
1981; Ebinger et al., 1983; Platel and Vesselkin, 1989; Wicht,
1996) and have shown that agnathans, particularly lampreys,
possess a relatively small brain and some of the highest degrees
of intraspecific variation in brain and body mass when compared
to any other vertebrate group (Ebinger et al., 1983; Platel and
Delfini, 1986). However, these data have been collected from
very few species and no consideration has yet been given to
changes in encephalization and brain organization that may
occur throughout their life cycle. Indeed, ontogenetic studies of
diverse groups of vertebrates have shown that the brain grows at
different rates during their lifespan, with the rates being greatest
in the embryonic and early postnatal phases (Bauchot et al.,
1979; Gille and Salomon, 2000; Fu et al., 2013; Ngwenya et al.,
2013). Although some studies have shown shifts in ecology and
corresponding shifts in brain development occur in fishes (e.g.,
Brandstätter and Kotrschal, 1990; Wagner, 2003; Lisney et al.,
2007; Iribarne and Castelló, 2014), there are no data on the
pattern of encephalization or brain subdivision scaling during the
ontogeny of lampreys.
The life cycle of lampreys is very conserved (Chang et al., 2014;
Potter et al., 2015), consisting of a prolonged and sedentary larval
phase, followed by metamorphosis into the free-swimming adult
phase (Manzon et al., 2015), as illustrated in Figure 1. In the
pouched lamprey Geotria australis, which is widely distributed in
temperate regions of the southern hemisphere (Renaud, 2011),
the life cycle has an approximate duration of 8 years (Potter et al.,
1980, 1983; Potter and Hilliard, 1986).
After hatching, the larvae (ammocoetes) burrow in the
soft sediments of streams and rivers, filtering detritus, algae
and other organisms from the overlying water (Piavis, 1971;
Moore and Mallat, 1980; Richardson et al., 2010; Dawson
et al., 2015). Ammocoetes have rudimentary eyes with a largely
undifferentiated retina (Meyer-Rochow and Stewart, 1996; Villar-
Cheda et al., 2008), and also a well-developed non-visual
photoreceptive system, e.g., the pineal organ (García-Fernández
and Foster, 1994; Deliagina et al., 1995; Melendez-Ferro et al.,
2002; Vigh et al., 2002). In fact, they exhibit nocturnal habits with
synchronized, seasonal downstream movements (Gritzenko,
1968; Potter, 1980), which may be controlled by circadian
rhythms. An octaval lateral line system provide additional
mechano-, electro-, and photo-perception, with photoreception
being mediated by dermal non-visual photoreceptors located in
the tail (Ronan, 1988; Ronan and Bodznick, 1991; Deliagina
et al., 1995; Gelman et al., 2007). Ammocoetes also have well-
developed gustatory (Baatrup, 1985; Barreiro-Iglesias et al.,
2010) and olfactory (Vandenbossche et al., 1995; Zielinski et al.,
2005) systems, and behavioral evidence has revealed that rotting
potato haulms attracted ammocoetes when placed on the bed
of freshwater streams (Enequist, 1937; Hardisty and Potter,
1971), indicating that they may actively search for food using
chemosensory cues. Therefore, taste and olfaction are likely
important drivers of their behavior.
The metamorphosis of anadromous parasitic species of
lampreys, such as G. australis, involves major morphological
and physiological changes and the development of new sensory
and motor capabilities. These include the development of
image-forming eyes with the potential for pentachromacy in G.
australis (Meyer-Rochow and Stewart, 1996; Collin et al., 1999,
2003; Davies et al., 2007), a reduction of lateral line-mediated
negative phototaxis that marks a switch from non-visual to
visual perception (Binder et al., 2013), the rearrangement of the
gustatory and lateral line systems (Currie and Carlsen, 1988;
Jørgensen, 2005; Gelman et al., 2008; Barreiro-Iglesias et al.,
2010), and the development of a tooth-bearing suctorial disc
and “tongue-like” piston with the associated musculature and
trigeminal motor innervation (Homma, 1978; Lethbridge and
Potter, 1981). Metamorphosis also involves fundamental changes
in a number of internal organs, including the intestine and gills,
which enable the lamprey to osmoregulate in the sea (Youson
et al., 1977; Hilliard et al., 1983; Bartels and Potter, 2004; Reis-
Santos et al., 2008).
During the marine parasitic phase, G. australis swims toward
and attaches to a host, often a teleost fish, and feeds from its
flesh (Hilliard et al., 1985; Renaud et al., 2009), thereby increasing
in body size from approximately 100 mm and 0.75 g to 620 mm
and 220 g (Potter et al., 1980, 1983). There is strong evidence
that during its marine parasitic phase, G. australis occupies an
epipelagic niche in the sea and exhibits diurnal habits (Potter
et al., 1979; Cobley, 1996; Collin et al., 1999; Davies et al.,
2007). Following the completion of the parasitic phase, the
adult lamprey re-enters rivers cued mainly by pheromones that
are released by the ammocoetes (Vrieze and Sorensen, 2001;
Sorensen et al., 2005; Vrieze et al., 2010, 2011), where they
migrate upstream at night (Jellyman et al., 2002; Binder and
McDonald, 2007; Vrieze et al., 2011). Geotria australis does
not feed during its exceptionally long spawning run, using
body reserves accumulated during the marine phase to develop
secondary sexual characters and mature gonads (Potter et al.,
1983; Paton et al., 2011). The life cycle culminates in spawning
and subsequent death.
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Salas et al. Brain scaling during ontogeny in lampreys
FIGURE 1 | Life cycle of Geotria australis presenting anadromous
reproductive and feeding migrations. After hatching (bottom), the
larvae—also called ammocoetes—burrow in the sediments of rivers, becoming
a microphagous filter-feeder for approximately 4 years. The larval phase is
followed by a metamorphosis, a non-feeding transition to adult stage that lasts
for approximately 6 months (left), where there is a marked transformation in
most of the body systems. Animals at this stage start migrating downstream
and enter the sea, where they locate a teleost host and feed on its flesh using a
specialized buccal apparatus (top). G. australis return years later to the rivers,
where they start a long upstream migration, subsisting only on body reserves,
which are expended in developing secondary sexual characteristics and
reproductive behavior. Upstream migrants finally spawn and die (right).
During its life cycle, G. australis occupies different ecological
niches and encounters diverse environmental conditions, yet
there have been no comprehensive studies that have quantified
the changes in brain organization corresponding to these marked
changes in ecology and behavior. In this study, we assess
changes in relative brain size (encephalization) and in the
volume of six major brain structures (brain organization) at
different phases of the life cycle in G. australis. We hypothesize
that differences in brain size and organization will reflect
the pronounced environmental and physiological changes that
lampreys experience during ontogeny.
Methods
All the procedures described below were performed in
accordance with the ethical guidelines of The University of
Western Australia Animal Ethics Committee—Research Project
RA/3/100/917.
Data Collection
Forty specimens of G. australis were analyzed in this study,
representing six different points in their life cycle (ammocoetes
of second, third, and fourth age class, downstream migrants,
upstream migrants, and maturing adults). Specimens within a
stage had the same fixation and preservation methods, as shown
in Supplementary Table 1, and were captured in the same year
(ammocoetes and downstream migrants) or in different years
(upstream migrants and maturing adults). Morphometrics (body
mass, body length, sex) were collected for each individual when
possible. After a period of fixation, the brain was removed
from the chondrocranium. The meninges were removed and the
cranial nerves were cut to within 0.5 mm of the base. The brains
were blotted and weighed to the nearest 0.1 mg (ammocoetes
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Salas et al. Brain scaling during ontogeny in lampreys
and downstream migrants) or 1 mg (upstream migrants and
maturing adults). Neither brain nor body mass were corrected
for shrinkage due to fixation.
Photographs of the lateral and dorsal views of each brain
were taken using a Leica EC3 camera attached to a Nikon
SMZ-745T dissecting microscope. Brains were submerged in
a solution of 0.1 M phosphate buffer while photographed
to prevent volume distortions caused by dehydration of the
tissue. Measurements of length were taken for each of the six
brain structures as shown in Figure 2. Brain structures were
determined from previously published descriptions of the brain
and the cranial nerve distribution in lampreys (Nieuwenhuys and
Nicholson, 1998). The length (l), height (h), and width (w) of the
olfactory bulbs (OB), telencephalic hemispheres (Te), the pineal
organ (PO), the optic tectum (OT), the octaval-trigeminal area
(OCT; defined as the anterior region of the rhombencephalon
comprising the V–VIII nerves), and the gustatory area (GUS;
defined as the posterior region of the rhombencephalon
comprising the IX–XII nerves) were measured using ImageJ
(Rasband, 1997) as described previously (Huber et al., 1997;
Wagner, 2001; Yopak and Lisney, 2012). The pineal organ
was dissected out of the brain and photographed separately,
see Figure 2B.
Volumes of each major brain structure were estimated using
the ellipsoid method, which approximates the volume of a
structure by assuming it takes the shape of an idealized ellipsoid,
or a fraction of it as shown below (Huber et al., 1997; Wagner,
2001). The general formula of an ellipsoid is:
FIGURE 2 | Estimation of the volume of brain structures using the
ellipsoid method. Measurements of length (l), width (w), and height (h) of six
brain structures taken from a dorsal view (A, top) or lateral view (A, bottom) of
the brain of an upstream migrating G. australis. In the case of the olfactory
bulbs and the telencephalic vesicles, these were defined as parallel or
perpendicular lines to the Fissura circularis (fc), which is highlighted with a
discontinuous line in the telencephalon. The limit of the octavo-trigeminal and
gustatory areas was defined by a line running parallel to the posterior end of
the head of the eighth nerve (white arrow). (B) The same measurements were
performed in the pineal organ after it was dissected and separated from the
remainder of the brain. OB: olfactory bulbs, Te: telencephalic vesicles, PO:
pineal organ, OT: optic tectum, OCT: octavo-trigeminal area, Gus: gustatory
area. Scale bars =1 mm.
V=4
3πa b c
where a, b, c are the radii of the ellipsoid. Using the measurements
of length (l), height (h), and width (w) shown for each structure
in Figure 2, the volumes were defined as:
V=1
6πl h w
for the OB, Te, PO, and the OT, which were all modeled as half
ellipsoids,
V=1
3πl h w
while the volume of the OCT and GUS were modeled as a quarter
of an ellipsoid. In the case of bilateral structures (i.e., OB, Te, and
TO), the values of the volumes were doubled. Volume estimates
were not corrected for ventricular volume. Total brain volume
was calculated from total brain mass using the estimated density
of the brain tissue, d=1.036 mg/mm3(Stephan, 1960).
Age Determination
The approximate age of the ammocoete samples was
estimated from length-frequency histograms for larval and
metamorphosing representatives of G. australis (Potter et al.,
1980; Potter and Hilliard, 1986). Age of adult stages was
inferred from the timing of the upstream migration and sexual
maturation (Potter et al., 1983).
Data Analysis
All analyses were performed using the open source software R
(R Core Team, 2013). The complete dataset was divided into two
subsets, one containing body and brain mass (n=32) and the
other containing total brain and brain structure volume estimates
(n=39).
Linear Models
For brain and brain structure scaling analyses, each data set
was log10 transformed to improve normality prior to analysis,
after being multiplied by an arbitrary factor (10 and 1000,
respectively), in order to obtain positive values of the variables
following log10 transformation. We conducted similar analyses
on both datasets: we fitted least squares regressions within
and between stages, and performed analyses of covariance
(ANCOVA), with brain mass as the response variable, body
mass as the covariate, and stage as a factor for the brain and
body mass comparisons. In the case of the brain structures,
total brain structure volume was compared to total brain volume
minus total structure volume as a covariate. This was done to
account for the bias that exists when a brain subdivision is
scaled against total brain mass (which includes the subdivision
of interest) (Deacon, 1990; Iwaniuk et al., 2010). To control
for similarity within the larval or adult phases of the life
cycle, stages were combined in “stage 1” (no combination),
“stage 2” (all ammocoetes grouped together), “stage 3” (all
adults grouped together), “stage 4” (all ammocoetes grouped
together, downstream and upstream migrants grouped together),
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Salas et al. Brain scaling during ontogeny in lampreys
“stage 5” (all ammocoetes grouped together, upstream migrants
and maturing adults grouped together), and “stage 6” (all
ammocoetes grouped together, all adults grouped together) (See
Supplementary Table 2). Linear models were fitted to each of
these factors and the linear assumptions for each were checked
using the R package glvla (Pena and Slate, 2014); valid linear
models were then compared and selected using the second-order
Akaike Information Criterion (AICc); If the best model had a
AICc value indistinguishable from the following model(s), they
were averaged using multi-model inference methods contained
in the R package MuMIn (Barton, 2014), and the relative
importance of the factor in the resulting model was used as a
criterion for selection. Tukey Post-hoc tests were used to detect
differences between groups in the selected models.
Principal Component Analysis
We also used a multivariate approach to determine the clustering
of the samples in multidimensional space and characterize the
patterns of brain organization of G. australis at each point of the
life cycle. Principal component analysis (PCA) was performed
using relative volume of each structure, calculated as a fraction
of the sum of the volume of all six brain structures measured
within a specimen (Wagner, 2001; Lisney et al., 2007). Structure
proportions were normalized using the arcsine square root
transformation previous to analysis. PCA was run using the
autocovariance matrix and the singular value decomposition
method for better numerical accuracy.
Results
Brain Scaling
The brain of G. australis shared similar characteristics with
those of other species of lampreys (Figure 3) (Wicht, 1996;
Nieuwenhuys and Nicholson, 1998). Our analysis of the scaling
of brain and body mass in G. australis at successive stages of
development revealed that the brain and body have different
scaling patterns during ontogeny (Figure 4A). Body mass grows
at a higher rate than brain mass in both the adult phase
and the analyzed period of the larval phase, a trend that is
interrupted during metamorphosis (Figure 4A, arrows), where
body mass was similar between downstream migrants and the
latest ammocoete stage (Two-tailed Welch t-test, T=1.98,
p=0.201); however, brain mass was significantly higher in
downstream migrants as compared to ammocoetes IV (One-
tailed Welch t-test, T=7.8, p=0.037).
According to the second-order Akaike information criterion,
the best model of brain mass as a function of body mass
occurred when stage 2 was used as a factor, grouping all
ammocoetes together (Supplementary Table 3). We fitted stage-
specific (intraspecific) regressions to each of these groups, whose
slopes varied across ontogeny (Figure 4B); all groups showed
intraspecific negative allometry of brain mass with body mass.
The highest rate of brain growth was reached at the larval phase
(α=0.47), followed by downstream and upstream migrants
(Supplementary Table 4), while the period of regression of body
mass in the course of maturation was accompanied by a steep
reduction of brain mass (α=0.90). We also defined an
ontogenetic linear regression as the line of best fit between all
specimens, where most of the groups had large deviations from
the predicted values of brain mass (Figure 4C), indicating that
brain mass does not scale linearly with body mass at all stages
in the life cycle of G. australis. These two sets of regressions
were combined in an analysis of covariance (ANCOVA), the
results of which are illustrated in Figure 4D. These data show
that both stage 2 and body mass are significant when explaining
the observed variance of brain mass (ANCOVA, p<0.001), and
no significant interaction between factor (stage 2) and covariate
(body mass) is found, indicating no significant differences in the
FIGURE 3 | Brain of Geotria australis during ontogeny. A representative
brain of each stage studied is shown in a dorsal (top) and lateral view
(bottom): (A) second age class ammocoete, (B) third age class ammocoete,
(C) fourth age class ammocoete, (D) maturing adult, (E) upstream migrant,
and (F) downstream migrant. Note the marked difference between the brain
of a late ammocoete and a downstream migrant (C,F). Scale bars =1 mm.
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Salas et al. Brain scaling during ontogeny in lampreys
FIGURE 4 | Brain and body growth vary during the ontogeny
of Geotria australis. (A) Brain and body mass growth traced
over time. Arrows mark the period of metamorphosis. (B)
Intraspecific linear regressions, (C) Ontogenetic regressions, and (D)
Linear regressions fitted for each stage after an ANCOVA analysis.
For the values of the parameters of these regressions, refer to
Supplementary Table 4. amII, second year class ammocoetes; am
III, third year class ammocoetes; amIV, fourth year class
ammocoetes; ds, downstream migrants; us, upstream migrants; sa,
spawning adults.
slopes calculated for each group in the stage-specific regressions.
The ANCOVA calculated a common slope, with a similar value to
the slope obtained in the intraspecific regression of ammocoetes,
and different intercepts for each group (See Supplementary Table
4), which represent differences in relative brain mass between
groups. The Tukey Post-hoc test showed significant differences
between all groups of stage 2 (p<0.001); downstream migrants
had the highest intercept, demonstrating an increase in relative
brain mass at this stage.
Scaling of Brain Structures
The analyzed brain structures showed different patterns of
growth during the life cycle of G. australis. Ontogenetic
regressions of total structure volume against total brain volume
minus structure volume (hereafter referred to as brain volume)
were fitted to each of the structures analyzed and their parameters
are tabulated in Supplementary Table 5. A general trend between
these regressions was the large deviations from the expected
values shown by the downstream migrants, which were positive
for the telencephalon and the optic tectum, but negative in the
case of the pineal organ, the octavo-trigeminal area and the
gustatory area.
The olfactory bulb was the only structure where the observed
values fitted the expected values closely in all the stages,
supporting a linear scaling of this structure with total brain
throughout ontogeny (Figure 5A). Remarkably, the olfactory
bulbs showed the steepest hyperallometric growth reported in
this study (α=1.27), generating highly developed olfactory
bulbs in upstream migrants and maturing adults. The pineal
organ and the octavo-trigeminal area also showed a significant
linear fit with total brain volume, as shown in Supplementary
Table 5, although this was not the best model for these structures
(see below).
Similar to the olfactory bulbs, the telencephalic hemispheres
showed a close fit to brain volume in most stages, but because
of the high heteroscedasticity in the values of maturing adults,
the linear assumptions were violated in this case and in other
tested linear models of the telencephalic hemispheres (results
not shown). Nevertheless, we found that these assumptions were
valid when fitting the telencephalic volume with the volume of
the olfactory bulbs, and thus in this case total olfactory bulbs
volume was used as covariate in the ANCOVA analysis. The best
model for the telencephalic hemispheres included stage 6 as a
factor (Figure 5B). This structure showed linear growth with the
olfactory bulbs along the larval phase and an increase in size
after metamorphosis, which is maintained throughout the adult
phase of the life cycle. However, only a marginal difference was
detected between ammocoetes and adults (Tukey Post-hoc test,
p=0.091).
The best models for the pineal organ and the gustatory area
had stage 2 as factor, whereas for the octavo-trigeminal area it was
stage 1 and for the optic tectum it was stage 4 (Supplementary
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Salas et al. Brain scaling during ontogeny in lampreys
FIGURE 5 | Calculated regression lines after ANCOVA. Best linear
models are plotted for each structure, showing the differences in
scaling of each structure to the rest of the brain: (A) olfactory bulbs
(OB), (B) telencephalic hemispheres (Te), (C) pineal organ (PO),
(D) optic tectum (OT), (E) octavo-trigeminal area (OCT), and
(F) gustatory area (GUS). For the values of the parameters of these
models, refer to Supplementary Table 5. amII, second age class
ammocoetes; amIII, third age class ammocoetes; amIV, fourth age
class ammocoetes; ds, downstream migrants; us, upstream migrants;
sa, spawning adults.
Table 3). The calculated slope for the pineal organ in the
ANCOVA was higher than in the ontogenetic regression, and
ammocoetes had the highest intercept (Figure 5C). We found
no significant differences between ammocoetes, downstream and
upstream migrants, but the pineal organ in maturing adults
was significantly different from that of downstream migrants,
although only marginally different from upstream migrants
(Tukey Post-hoc test, p=0.017 and 0.053, respectively). The
corrected slope for the optic tectum showed two markedly slow
phases of growth, larval and adult, with a significant difference in
size between them (Tukey Post-hoc test, p<0.05; Figure 5D);
the optic tectum of maturing adults was significantly reduced
compared to downstream and upstream migrants (Tukey Post-
hoc test, p<0.05), and not different from the optic tectum of
ammocoetes (Tukey Post-hoc test, p=0.45).
The volume of the gustatory area of the downstream migrants
was significantly different to the other stages (Tukey Post-hoc
test, p<0.05), with a shallow slope (α=0.43). However,
considering the value of the calculated intercepts in the ANCOVA
of the gustatory area, the downstream migrants clustered with
ammocoetes, whereas upstream migrants and maturing adults
had higher values of intercepts (Figure 5E). This was also the case
for the octavo-trigeminal area, where the volume in downstream
migrants was different from all the other stages (Tukey Post-hoc
test, p<0.05) and their volume was closer to ammocoetes than
to adults although, in contrast to all other structures, we found
that in this area the ammocoetes were best fitted as separate
groups, where the second age class ammocoetes had a smaller
intercept than other larval stages (Tukey Post-hoc tests: amIII,
p=0.020; amIV, p=0.083; Figure 5F). Some maturing
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Salas et al. Brain scaling during ontogeny in lampreys
adults possessed a relatively higher octavo-trigeminal area than
upstream migrants, consistent with the modifications of the
oral disc and the appearance of the gular sac in this period
(Potter et al., 1983; Neira, 1984). However, we did not observed
significant differences between these groups. Our results also
showed no consistent differences between male and female
lampreys in any structure (results not shown).
Multivariate Analysis and Stage Clustering
The principal component analysis performed on the correlation
matrix of the relative size of the six brain structures measured in
this study provided a clear separation in the multidimensional
space of the two phases of the life cycle of G. australis. The
relative loadings of the first four components and their relative
importance are given in Table 1. The first two components
explained 93.3% of the overall variance and their scores are
plotted in Figure 6. The first component (PC1) reflects the high
loadings for the optic tectum and gustatory area, and secondarily
in the olfactory bulbs, separating larvae, which had a relatively
large gustatory area and pineal organ, from adults, which had
relatively larger optic tecta, olfactory bulbs and telencephalic
hemispheres. Similarly, the second component (PC2) separated
younger and older individuals within a phase, where older
individuals had relatively larger olfactory bulbs and octavo-
trigeminal areas than younger individuals in both phases of the
life cycle.
Discussion
Lampreys experience very different behavioral phases during the
life cycle, from a microphagous sedentary mode to an active
parasitic mode. This study characterized the growth of the brain
(encephalization) during the life cycle of G. australis, focusing on
the scaling between brain and body throughout ontogeny and
testing the hypotheses that the vast transitions in behavior and
environment are reflected in differences in both encephalization
and the relative development of major brain subdivisions.
The changes occurring in the nervous system of lampreys
during ontogeny have attracted the attention of many
comparative neurobiologists, who have shown extensive
TABLE 1 | Results of the principal component analysis for the first four
components.
Importance of components PC1 PC2 PC3 PC4
Standard deviation 0.189 0.093 0.046 0.031
Proportion of the variance 0.749 0.183 0.045 0.021
Cumulative proportion 0.749 0.933 0.978 0.998
RELATIVE LOADINGS
OB 0.313 0.523 −0.710 0.150
Te 0.125 −0.044 0.007 −0.868
PO −0.094 −0.089 0.084 −0.101
TO 0.666 −0.472 0.193 0.345
OCT −0.186 0.576 0.584 0.217
GUS −0.633 −0.403 −0.332 0.218
morphological and physiological modifications of the peripheral
and central nervous system, such as the development of the
visual system (Kennedy and Rubinson, 1977; Kosareva, 1980;
De Miguel and Anadon, 1987; Rubinson, 1990; Fritzsch and
Northcutt, 1993b; Pombal et al., 1994; Davies et al., 2007; Villar-
Cheda et al., 2008). However, in spite of the multiple studies
quantifying these changes throughout the life cycle (Tamotsu and
Morita, 1986; De Miguel and Anadon, 1987; Currie and Carlsen,
1988; Melendez-Ferro et al., 2003; Vidal Pizarro et al., 2004; Antri
et al., 2006), an overall view of the pattern of development of the
brain and its organization, including larval and adult phases, has
been absent until now.
Brain Scaling
The description of the changes in encephalization during the life
cycle of jawless fishes will improve our current understanding
brain development at multiple levels. Previous interspecific
studies in agnathans have differed on the scaling relationship
between brain size and body size of lampreys, ranging from 0.23
(Ebinger et al., 1983) to 0.56 (Platel and Vesselkin, 1988). In
addition to discrepancies in the value of the scaling exponent,
both studies suffered from low sample sizes, with data on only
three (Ebinger et al., 1983) and two (Platel and Vesselkin, 1988)
species, out of 41 currently recognized species of lampreys (Potter
et al., 2015). This discrepancy in the scaling exponent requires
improved resolution, as one value classifies lampreys as being
far less encephalized than other gnathostomes, with a slow rate
of growth of the brain in relation to the body (α=0.23),
FIGURE 6 | A scatterplot of principal components PC1 and PC2.
Principal component analysis, representing the major changes in the
composition of the brain during the life cycle. OB, olfactory bulbs; Te,
telencephalic hemispheres; PO, pineal organ; OT, optic tectum; OCT,
octaval-trigeminal area; GUS, gustatory area; amII, second age class
ammocoetes; amIII, third age class ammocoetes; amIV, fourth age class
ammocoetes; ds, downstream migrants; us, upstream migrants; sa, spawning
adults.
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Salas et al. Brain scaling during ontogeny in lampreys
while the other places this group within the known range of
the interspecific variation in the scaling exponent between most
vertebrate groups (α=0.56), which usually falls between 0.5 and
0.6 (Striedter, 2005). Similarly, no consensus has been reached
with regards to the intraspecific scaling exponent in the sea
lamprey Petromyzon marinus, which ranges from −0.04 (Ebinger
et al., 1983) to 0.56 (Platel and Delfini, 1986). However, given
the dramatic shifts that occur throughout the life history of
lampreys, these published values for brain scaling are likely to
be highly dependent on when in the life cycle the brains were
sampled. In fact, this study shows that, as lampreys advance in
their upstream migration, they lose both body and brain mass at
different rates, which is reflected in a higher intraspecific scaling
factor in maturing adults (Figure 4). This variation between early
and late upstream migrants may explain previously reported
discrepancies in the intraspecific allometric slope in P. marinus.
Nonetheless, it is possible that the observed differences in relative
brain mass may also be related to intraspecific variation between
separate populations (Gonda et al., 2011) or according to mating
strategies (Kolm et al., 2009), which have also been described in
lampreys (Hume et al., 2013).
The ontogenetic scaling of brain and body mass in other
basal vertebrate groups, such as teleost fishes, has shown that
the larvae of both metamorphic (Bauchot et al., 1979; Tomoda
and Uematsu, 1996; Wagner, 2003; Sala et al., 2005) and
non-metamorphic fishes (Iribarne and Castelló, 2014) exhibit
allometric scaling between brain and body size in the early
post-hatching development phase, which may be equivalent
to the linear phase of growth reported for ammocoetes in
this study. However, in the case of metamorphic fishes, such
as the rainbow trout Oncorhyncus mykiss or the Japanese eel
Anguilla japonica, there is no clear evidence of an increase of
encephalization associated with metamorphosis (Bauchot et al.,
1979; Tomoda and Uematsu, 1996), as our results suggest for
lampreys, but constitutes an interesting point that warrants
further investigation and should be an area of future study.
Teleost fishes possess continuous growth of both the body
and the nervous system throughout life (Bauchot et al., 1979;
Leyhausen et al., 1987), as opposed to amniotes where brain
growth plateaus before the animal reach its final body size
(reviewed in Striedter, 2005), although there are some exceptions
(Ngwenya et al., 2013). Yet in lampreys, our results and previous
records on P. marinus (Ebinger et al., 1983) suggest that, in early
upstream migrants (end of the parasitic phase), brain growth
may have actually reached a plateau, given the low intraspecific
scaling factor found at this point of the life cycle (Figure 4B:
α=0.09 for G. australis, α= −0.04 for P. marinus), although
these values were not statistically significant in either study. In
addition, we found evidence that a relative reduction in brain
mass occurs in parallel with the typical reduction of body mass
in maturing lampreys (Potter et al., 1983; Paton et al., 2011),
which has not been previously shown in other ontogenetic studies
of brain scaling in vertebrates. Even though complex behavior
is generally associated with larger brains (reviewed by Striedter,
2005), lampreys still exhibit sophisticated behaviors, such as nest
construction, in this period (Hardisty and Potter, 1971; Sousa
et al., 2012; Johnson et al., 2015).
Brain growth in vertebrates has been described as the result
of several processes, including cell growth and the addition and
elimination of cells (Pirlot and Bernier, 1991; Candal et al., 2005;
Bandeira et al., 2009; Fu et al., 2013; Boyd et al., 2015). In
lampreys, neuro- and glio-genesis are restricted to ventricular
proliferative zones in late embryos and early to mid larval stages
(Vidal Pizarro et al., 2004; Villar-Cheda et al., 2006; Guerin
et al., 2009) and, although adult neurogenesis is widespread
in other basal vertebrate groups (Kaslin et al., 2008), it is
considered mostly absent in lampreys (Villar-Cheda et al., 2006;
Kempermann, 2012). Taken together, these results suggest that
brain growth from late ammocoetes onwards is mainly due to
the addition of glia, cell growth, and the establishment of new
synapses that contribute to the formation of plexiform tissue or
neuropil, as suggested previously for lampreys (Rovainen, 1979,
1996).
Scaling of Brain Structures
Transitions in habitat and behavior are common during the
development of aquatic vertebrates, even if they do not undergo
a metamorphic stage, such as recruitment of fish larvae
(Kingsford et al., 2002; Kotrschal et al., 2012; McMenamin and
Parichy, 2013) and the use of nursery areas in sharks (e.g.,
Bethea et al., 2004; Heupel and Simpfendorfer, 2011). Usually
these transitions are accompanied by ad-hoc sensorimotor
specializations (Brandstätter and Kotrschal, 1990; Montgomery
and Sutherland, 1997; Lisney et al., 2007; Lecchini et al., 2014).
Similarly, adults of both bony and cartilaginous fishes, as well
as other vertebrates, possess well-developed adaptations to their
ecological niche, which are generally reflected in their nervous
system as a variation in the relative size of brain subdivisions
(Kotrschal and Palzenberger, 1992; Gonzalez-Voyer et al., 2009;
Gonzalez-Voyer and Kolm, 2010; Yopak, 2012). Surprisingly, the
relative size of these brain subdivisions appear to be constant
between species of parasitic lampreys, despite the diverse aquatic
niches in which they inhabit (Renaud, 2011; Potter et al., 2015).
We found that the optic tectum and olfactory bulbs in adults
of G. australis comprise similar proportions of the brain to that
of P. marinus (Platel and Delfini, 1986) and other species of
lampreys (Platel and Vesselkin, 1989), concordant with the lack
of appreciable neuroanatomical differences in the brain between
lamprey species, as reported previously (Platel and Vesselkin,
1989; Nieuwenhuys and Nicholson, 1998). However, we consider
that more species of lampreys needs to be examined, including
those with alternative life style strategies, such as parasitic
and non-parasitic paired species of lampreys, to have a wider
perspective of the diversity found in the nervous system of extant
agnathans.
Olfactory Bulbs
It has been suggested that the level of variation in the relative
size of the major brain subdivisions may occur in particular
structure in a modular or mosaic fashion (Barton and Harvey,
2000), or with a concerted pattern of allometric scaling (Finlay
and Darlington, 1995). It has recently been shown that most
major brain areas in cartilaginous fishes scale with a characteristic
slope that may be conserved across other vertebrates, including
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Salas et al. Brain scaling during ontogeny in lampreys
mammals (Yopak et al., 2010). One notable exception is found
in the olfactory bulbs, which maintain a level of statistical
independence from total brain size in a range of vertebrate groups
(Finlay and Darlington, 1995; Gonzalez-Voyer et al., 2009; Yopak
et al., 2010, 2015). At the ontogenetic level, however, our analysis
of the scaling of the olfactory bulbs shows the opposite pattern,
whereby the olfactory bulbs scale very tightly with total brain size,
with a highly hyperallometric growth (Figure 5A).
Multiple functional hypotheses have been proposed to explain
the relative size of the olfactory bulbs (reviewed in Yopak
et al., 2015), including the relationship of olfactory cues with
navigation, which may play an important role in lampreys while
finding a host or on their way back to rivers for the spawning
run (Siefkes et al., 2003; Johnson et al., 2005, 2009; Sorensen
et al., 2005; Wagner et al., 2009). The olfactory spatial hypothesis
predicts that the size of the olfactory bulbs should covary with
navigational ability, which is supported by the olfactory input
to the hippocampus (Jacobs, 2012). The statistical independence
of the olfactory bulbs is then substantiated by the fact that the
olfactory bulbs, the hippocampus, and other associated areas
of the telencephalon do not scale as tightly with brain size
as do other brain subdivisions (Finlay and Darlington, 1995;
Finlay et al., 2001; Gonzalez-Voyer et al., 2009; Yopak et al.,
2010) and can vary across mammalian taxa depending on the
influence of olfactory cues in their behavior (Reep et al., 2007).
If these theories can be applied in the context of the lamprey
life cycle, we would therefore expect that, should homologous
olfactory areas exist in the telencephalon of G. australis, they
would also scale isometrically with the rest of the brain in this
group during ontogeny. Early descriptions of the telencephalon
of the lamprey and later hodological evidence have suggested
the presence of a hippocampal primordium or medial pallium
(Johnston, 1912; Northcutt and Puzdrowski, 1988; Polenova and
Vesselkin, 1993; Northcutt and Wicht, 1997). However, scaling of
these telencephalic structures have not been studied in agnathans
at any level, and even the existence of a medial pallium is disputed
by neuroanatomical descriptions based on molecular markers
(Pombal and Puelles, 1999; Weigle and Northcutt, 1999; Pombal
et al., 2011). Considering that interspecific scaling of the olfactory
bulbs has not yet been described in jawless fishes, the available
evidence does not permit any definitive conclusions to be made
with regard to differences found in the scaling of the olfactory
bulbs between lampreys and other vertebrates.
An alternative explanation of the involvement of olfaction
in navigation in lampreys is the hypothesis of dual olfaction,
which assumes parallel processing of distinct sets of molecules
or environmental odors by the main olfactory system and
pheromones by the vomeronasal system, following independent
pathways in the brain, and acting synergistically in the regulation
of olfactory-guided behaviors (reviewed in Suárez et al., 2012).
In lampreys, two anatomically distinct sets of olfactory epithelia
have been described that show different patterns of central
projections, which suggests the existence of a precursor of the
vomeronasal system in this group (Ren et al., 2009; Chang et al.,
2013). This accessory olfactory system is tightly coupled to motor
areas of the brain, constituting an unusual motor system in
vertebrates, which is capable of eliciting swimming movements
after olfactory stimulation with both naturally occurring odors
and pheromones (Derjean et al., 2010). Since lampreys can
detect very low (subpicomolar) concentrations of pheromones
(Sorensen et al., 2005), this system may be employed in
navigation and other behaviors involving pheromone perception,
such as searching for a natal river environment to spawn (Siefkes
et al., 2003; Johnson et al., 2005, 2009; Sorensen et al., 2005;
Wagner et al., 2009). However, whether these differential central
projections vary interspecifically and affect the relative size of the
olfactory bulbs and/or a tight coupling between development of
the olfactory bulbs and motor areas in the brain is unknown and
requires further research.
The Telencephalic Hemispheres
Interspecific studies of the scaling of major brain subdivisions
have shown that areas of the brain associated with behavioral
and motor complexity, e.g., telencephalon and cerebellum,
enlarge disproportionately as brain size increases in a range of
vertebrates (Finlay and Darlington, 1995; Finlay et al., 2001;
Pollen et al., 2007; Yopak et al., 2010). In lampreys, the
everted portion of the telencephalon considered in this study
(the cerebral hemispheres or telencephalic hemispheres) can be
regarded as the multimodal sensorimotor integration center of
the telencephalon, providing a neural substrate for orientation
movements of the eyes, trunk, and oral movements, due to direct
efferent projections to brainstem motor centers and the optic
tectum, in a similar fashion to motor control systems of amniote
vertebrates (Ericsson et al., 2013; Grillner and Robertson, 2015;
Ocaña et al., 2015). The telencephalic hemispheres are also the
main target of secondary olfactory projections from the lateral
olfactory bulb, which, in turn, receives its primary afferents from
the main olfactory epithelium (Northcutt and Puzdrowski, 1988;
Northcutt and Wicht, 1997; Ren et al., 2009; Derjean et al.,
2010). Therefore, it is not surprising to find a tight scaling
relationship between this structure and the olfactory bulbs (R2=
0.987). In addition, this telencephalic area receives afferent fibers
from the dorsal thalamus, possibly relaying visual and other
sensory input that converge on this thalamic area (Polenova
and Vesselkin, 1993; Northcutt and Wicht, 1997). Although not
significant, there is some evidence of differences in the size of
the telencephalic hemispheres between larvae and adults (Tukey
Post-hoc test, p=0.091), which may be due to the increase of
secondary sensory fibers terminating in this area, as both the
primary olfactory system (Vandenbossche et al., 1995; Villar-
Cheda et al., 2006) and the primary visual projections to the
dorsal thalamus (Kennedy and Rubinson, 1977; Kosareva, 1980)
develop during metamorphosis. Despite the various studies on
the pallial telencephalon of lampreys, no consensus has been
achieved yet in relation to the homology of this area with the
pallium of other vertebrates (Northcutt and Puzdrowski, 1988;
Nieuwenhuys and Nicholson, 1998; Pombal et al., 2009).
The Pineal Organ
The pineal complex in lampreys is formed by the pineal and
the parapineal organs (Eddy and Strahan, 1970; Puzdrowski and
Northcutt, 1989; Pombal et al., 1999; Yáñez et al., 1999), which
participate in non-visual photo-perception and neuroendocrine
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Salas et al. Brain scaling during ontogeny in lampreys
control of the circadian rhythms in these animals, as it does in a
range of vertebrates (Ekström and Meissl, 1997, 2003; Vernadakis
et al., 1998). The pineal organ has also been documented in
extinct agnathans, where it was similar in relative size to that
of contemporary ammocoetes (Gai et al., 2011), suggesting that
non-visual light perception was also highly developed in these
extinct groups. The observed morphological and physiological
variability of this organ in tetrapods has been linked to latitudinal
distribution of the species (Ralph, 1975), nocturnality (Bhatnagar
et al., 1986; Haldar and Bishnupuri, 2001), and habitat depth
in demersal fishes (Wagner and Mattheus, 2002; Bowmaker and
Wagner, 2004), although none of these factors fully explained the
variability found in the size and morphology of this organ across
species.
The best model for the pineal organ described three distinctive
periods of growth in the life cycle of G. australis. First, there
was consistent hyperallometric growth throughout the larval
phase; in the second period, during early adult life, including
the marine parasitic phase, we observed that the growth of
this organ plateaus after metamorphosis, where the size of
the pineal organ of ammocoetes was not significantly different
to that of downstream or upstream migrants, opposite to
what was observed in the other brain structures; and third,
we found a relative increase in the size of the pineal organ
during sexual maturation. A similar pattern of growth has been
documented in the pineal organ of the arctic lamprey Lethenteron
camtschaticum (Tamotsu and Morita, 1986). The larval phase and
sexual maturation periods anticipate important milestones in the
ontogeny of lampreys, such as the onset of metamorphosis and
spawning, both of which likely depend on the timing of circadian
rhythms (Freamat and Sower, 2013). In this regard, it was shown
that metamorphosis was prevented with pinealoctomy in G.
australis and other species (Eddy and Strahan, 1968; Cole and
Youson, 1981), and maturation was delayed in adults of the river
lamprey Lampetra fluviatilis after the same procedure (Eddy,
1971).
The Optic Tectum
In lampreys and other non-mammalian vertebrates, the optic
tectum is the main primary visual center of the brain, receiving
extensive topographic retinal (retinotopic) projections to the
superficial layers (Butler and Hodos, 1996; Iwahori et al., 1999; De
Arriba and Pombal, 2007; Jones et al., 2009). Electroreceptive and
other sensory input also converge onto this tectal map (Bodznick
and Northcutt, 1981; Ronan and Northcutt, 1990; Robertson
et al., 2006), where the relevance of salient stimuli can be assessed,
as in other vertebrates (Karamian et al., 1966, 1984; Pombal et al.,
2001; Gruberg et al., 2006; Kardamakis et al., 2015), leading to
orienting movements of the eye, head and trunk (Saitoh et al.,
2007; Ocaña et al., 2015).
Ontogenetic comparisons of the relative size of the
optic tectum have been documented in several species
of elasmobranchs (Lisney et al., 2007) and teleost fishes
(Brandstätter and Kotrschal, 1990; Kotrschal et al., 1990;
Wagner, 2003), and have shown a shift from an initially well-
developed visual system, followed by a relative reduction in
the size of the optic tectum and a corresponding increase in
other sensory brain areas, such as those that process olfactory
or lateral line input, as the animal matures. This change in
brain organization has been associated with shifts in ecological
niche, from a well-lit environment in epipelagic fish larvae or
nurseries of juvenile elasmobranchs to a different primary habitat
as adults. In contrast to these groups, we report an opposite
shift in brain organization. In ammocoetes of G. australis, the
optic tectum underwent moderate growth with total brain size
(α=0.47; Figure 5D). In fact, this structure remains mostly
undifferentiated and poorly layered during most of the larval
phase in lampreys (Kennedy and Rubinson, 1977; De Miguel and
Anadon, 1987; De Miguel et al., 1990) and only the central retina
is differentiated (Meyer-Rochow and Stewart, 1996; Villar-Cheda
et al., 2008). The major growth of the optic tectum occurs in
conjunction with the development of the adult eye, in a rapid
process that starts at the end of the larval phase and continues
during the initial stages of metamorphosis (Potter et al., 1980;
De Miguel and Anadon, 1987). Indeed, it is only at the end of
the larval phase that the typical retinotopic projections found
in adults reach the optic tectum (Jones et al., 2009; Cornide-
Petronio et al., 2011). Soon after metamorphosis (downstream
migrants), the relative size of the optic tectum is more similar
to that of adults than ammocoetes (Supplementary Table 5,
Figure 5D).
This rapid development of the visual system explains the lack
of a linear fit of the optic tectum in the ontogenetic scaling of
this structure with the rest of the brain. We expect that this
fast switch from non-visual to visual perception will also affect
the scaling of other visual areas of the brain receiving primary
retinal input, such as the dorsal thalamus, and that it may be
less pronounced in non-visual areas receiving retinal projections,
such as the hypothalamus and pretectal area, which are already
developed in ammocoetes, where they participate, for example,
in non-visual reflexes (De Miguel and Anadon, 1987; Ullen et al.,
1995, 1997; Jones et al., 2009). Nevertheless, the scaling of these
visual and non-visual areas of the brain has yet to be studied.
Our results suggest that vision may be important during
the parasitic phase, reflected in the high development of the
optic tectum during metamorphosis. However, the significant
reduction in the size of the optic tectum in maturing adults,
which is corroborated with reports of eye degeneration during
the spawning run (Applegate, 1950), supports previous evidence
that vision is not important in lampreys during their upstream
migration (Binder and McDonald, 2007; Johnson et al., 2015).
Medulla Oblongata
Interspecies comparisons in gnathostomes and agnathans have
shown that the size of the rhombencephalon, i.e., the medulla
oblongata plus the cerebellum, is well-predicted from total
brain size in both groups (Ebinger et al., 1983; Yopak et al.,
2010), although in lampreys only cerebellum-like structures
can be identified (Weigle and Northcutt, 1998; Northcutt,
2002; Montgomery et al., 2012). When comparing brain
subdivisions, the medulla oblongata had the lowest scaling factor
in cartilaginous fishes (Yopak et al., 2010), whereas it was the
highest in agnathans (Ebinger et al., 1983). Indeed, the medulla
accounts for approximately half of the total brain size in adult
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Salas et al. Brain scaling during ontogeny in lampreys
lampreys (this study, Platel and Vesselkin, 1989), and even more
in early larvae (Scott, 1887), although this is not as obvious
in downstream migrants (see below). The medulla is the first
to develop cranial nerves in lampreys (Kuratani et al., 1997;
Barreiro-Iglesias et al., 2008) and maintains a relatively stable
scaling relationship with total brain size during the later larval
phase and even throughout metamorphosis (Figures 5E, F).
However, there was a significant difference in the size of the
octavo-trigeminal area between the second-age class ammocoetes
and older stages (see intercepts in Supplementary Table 5), which
may be related to the development of a number of the diverse
sensory and motor systems located in this brain structure, as
discussed previously.
The growth of the medulla oblongata during metamorphosis
maintains a tight scaling relationship with total brain size
in late ammocoetes, which supports previous findings that
the motoneurons of the trigeminal nucleus in lampreys are
conserved through metamorphosis, in spite of the massive
replacement of muscle in the head during this period (Homma,
1978; Rovainen, 1996). This has also been documented in other
metamorphic vertebrates, such as frogs (Alley and Omerza,
1998).
However, while several brain structures, e.g., the olfactory
bulbs and the optic tectum, exhibit greater rate of growth during
metamorphosis, both the octavo-trigeminal and gustatory areas
grow with a slower rate during this phase, which is expressed
as a lower proportion of this area compared to total brain
volume in downstream migrants. Nonetheless, our results show
a later growth phase of this subdivision during the parasitic
phase, particularly of the octavo-trigeminal area, which may
be associated with the development of the musculature of the
ventilatory branchial basket and the oropharyngeal region, and
to the scaling of other somatic and sensory functions as body
size enlarges during the marine parasitic phase (Aboitiz, 1996;
Rovainen, 1996; De Winter and Oxnard, 2001).
Neuroecology of the Life Cycle
Growth of the central nervous system in lampreys is a
discontinuous process, with a variable rate of growth of both
total brain and its subdivisions throughout life, which was
expressed in the relative size of diverse brain structures in
each phase of the life cycle (Figure 6). These patterns of brain
organization may be interpreted as “cerebrotypes” (Clark et al.,
2001; Iwaniuk and Hurd, 2005; Willemet, 2012, 2013), whereby
similar patterns of brain organization exist in species that share
certain lifestyle characteristics. In this case, different cerebrotypes
may in fact exist within a species at different phases of the life
cycle.
The ammocoetes of G. australis are less encephalized
compared to young adults (downstream migrants), with brains
that are characterized by a relatively large gustatory area
and a highly developed pineal organ (Figures 3,6). The
relative size of the octavo-trigeminal area is increased in
late ammocoetes (Figure 5E), whereas the olfactory bulbs,
telencephalic hemispheres and optic tectum were relatively
small during the whole larval phase (this study, Scott,
1887). It is possible that these characteristics are related to
the requirements of a sessile, burrower lifestyle and/or to
filter-feeding specializations in this group. Patterns of brain
organization of other filter-feeding vertebrates has been described
previously, such as the basking shark Cetorhinus maximus and
the whale shark Rhincodon typus (Kruska, 1988; Yopak and
Frank, 2009), and mobulid rays (Ari, 2011), which similarly
possess a relatively small telencephalon and mesencephalon
(Kruska, 1988; Yopak and Frank, 2009). However, given the
drastic differences in the ethology between filter feeding jawless
and cartilaginous fishes, it is impossible to draw parallels between
patterns of brain organization in these groups. Further research
is required to determine the existence of common characteristics
in brain organization associated with a filter-feeding lifestyle in
lampreys.
In contrast to ammocoetes, adult parasitic lampreys are active
swimmers who are highly encephalized and possess a battery of
well-developed sensory systems during the adult phase, including
vision and olfaction. Correspondingly, they also possess a
relatively large telencephalon and olfactory bulbs, structures that
may be important in navigation (Derjean et al., 2010; Ocaña
et al., 2015), and a relatively large optic tectum, which participates
in orientation movements and plays a role in visual processing
(Saitoh et al., 2007; Kardamakis et al., 2015). Interestingly, some
of these features, such high levels of encephalization and a well-
developed optic tectum, have also been observed in many coastal-
oceanic and pelagic species of both cartilaginous and bony fishes
(Lisney and Collin, 2006; Yopak, 2012; Yopak et al., 2015), which
may be related to the sensory requirements of the open water
habitat across both jawed and jawless fishes.
Conclusions
We have employed a widely-used volumetric approach (Huber
et al., 1997; Wagner, 2001; Gonzalez-Voyer et al., 2009; Yopak
and Lisney, 2012; Lecchini et al., 2014) to quantify differences in
the relative size of major brain structures during the ontogeny
of lampreys. Our results demonstrate shifts in encephalization
between larvae and adults, as well as considerable differences in
the relative size of brain subdivisions. Taken together, these shifts
in brain organization may reflect the sensory requirements of this
species at each stage of the life cycle. The inclusion of data of the
growth of the brain and its subdivisions in embryonic, prolarva,
and early larval stages of ammocoetes, metamorphic, as well as
individuals sampled during the parasitic phase, will provide a
more comprehensive insight of the growth of the brain and body
during the life cycle of lampreys and eventually allow the use
of alternative mathematical functions to describe the process of
growth in each phase (i.e., Gompertz models, e.g., Calabrese et al.,
2013).
It is yet to be determined whether this pattern of brain
development is conserved in other species of lampreys, but we
anticipate that it is, based on how conserved the life cycle is in
this group (Potter et al., 2015), which could explain the reported
homogeneity of the central nervous system between species of
lampreys. Further studies on the changes in the brain of lampreys
throughout ontogeny will contribute to the understanding of the
evolution of the brain in agnathans and across vertebrates.
Frontiers in Neuroscience | www.frontiersin.org 12 July 2015 | Volume 9 | Article 251

Salas et al. Brain scaling during ontogeny in lampreys
Author Contributions
SC, NH, IP, and CS contributed to the conception, RW and
CS acquired the data, KY and CS designed the analyses and
interpreted the data. CS drafted the article, and all authors
collaborated in its revision.
Acknowledgments
This work was supported by an Australian Research Council
Discovery Grant (DP120102327) to SC, UWA SIRF, UIS, and UIS
Top Up Scholarships to RW, and UWA SIRF and CONICYT
Becas Chile Scholarships to CS. We thank Dr. Maik Kschischo
from University of Applied Sciences Koblenz for his advice on
growth curves, and Dr. Evelyn Habit from the University of
Concepcion for kindly donating samples for this study. We also
thank Dr. Macarena Faunes from the University of Auckland
and Dr. Howard Gill from Murdoch University for their critical
review of the work, and the reviewers who helped to improve a
previous version of this article. We are also grateful to Mr. Helios
Lara for adapting the illustration on the life cycle of Geotria
australis.
Supplementary Material
The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fnins.
2015.00251
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Conflict of Interest Statement: The Reviewer Thomas Lisney declares that, despite
being affiliated to the same institution as authors Carlos A. Salas, Kara E. Yopak,
Rachael E. Warrington, Nathan S. Hart and Shaun P. Collin, the review process was
handled objectively and no conflict of interest exists. The authors declare that the
research was conducted in the absence of any commercial or financial relationships
that could be construed as a potential conflict of interest.
Copyright © 2015 Salas, Yopak, Warrington, Hart, Potter and Collin. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Frontiers in Neuroscience | www.frontiersin.org 18 July 2015 | Volume 9 | Article 251