Environmental biology of fishes, tập 95, số 2, 2012

Environ Biol Fish (2012) 95:185–189
DOI 10.1007/s10641-012-9981-9

BOOK CRITIQUE

Fish on glass. The science and art of histology
Fish histology: female reproductive systems, 2007. McMillan, D.B. Springer, 598
pp., $269.00
Kathleen S. Cole

Received: 27 October 2011 / Accepted: 16 January 2012 / Published online: 14 February 2012
# Springer Science+Business Media B.V. 2012

Histology, the study of minute structure of tissues
and organs of plants and animals in relation to
their function, is fundamental to our modern understanding of organismal biology. The expansion
of histology as a scientific discipline dates from
the formulation of cellular theory by Matthias
Jakob Schleiden (1838) and Theodor Schwann
(1839) who independently developed the concept
of the cell as the basic unit of life. This idea was
subsequently expanded by Rudolph Virchow
(1858) to the concept that all cells arise from
pre-existing cells. Concurrently, an improved optical compound microscope developed by Carl Zeiss
became commercially available and both the ongoing classification of tissues and the fledgling science of histopathology took a step forward
(Fournier et al. 2008). By the late 1800s, histology
was a well-established discipline that served as the
main driver for the development of new biological
concepts. As a reflection of the impact of this field
on Biology, two histologists, Camillo Golgi and

Santiago Ramon y Cajal, were awarded the 1904
Nobel Prize in Physiology or Medicine for their
study of the neural structure of the brain.

K. S. Cole (*)
Department of Zoology, University of Hawaii at Mānoa,
2538 McCarthy Mall, Edmondson 152,
Honolulu, HI 96822, USA
e-mail:

It is against this historical background that, with great
dismay, I must acknowledge a sad fact: the study and
practice of comparative histology is a dying discipline.
The technique is taught with less and less frequency in
undergraduate Biology curricula in universities. With
luck, an intrepid student may trek over to the campus
medical school and learn how the human body is constructed from a micro-anatomical perspective. Unfortunately, she or he is unlikely to learn much about
comparative organismal histology or of the composition
and architecture of non-human tissues and organs. Nor
will the student be able to develop the insights that a
broader treatment of the topic provides in order to recognize evolutionary patterns and innovations at the
micro-anatomical level. The fact that traditional histology is becoming an orphan discipline, reduced to a limited
presence in medically-oriented fields and pathology laboratories, represents a critical loss to our knowledge base
in the biological sciences.
Within this context, the publication of Fish Histology,
Female Reproductive Systems by Donald B. McMillan is
a much needed and welcome addition to the library of
histology resources. There are few other books currently
available that focus on fish histology, much less histology of the female reproductive system. The seminal atlas
by Takashima and Takashi (1995) and a book by Rocha

(2009), both of which cover the histology of fish systems
in a broad sense, are out of print. A volume by Genten et
al. (2009), Atlas of Fish Histology that is currently available provides limited coverage of all anatomical systems


186

in a volume less than half the length of the MacMillan
book. Sharks, Skates and Rays by Hamlett (1999)
includes a chapter on the female reproductive system
which is well-illustrated and detailed, but offers relatively little histological information. Consequently, the book
reviewed here provides a new and critically important
addition to fish research.
I was also delighted at the publication of this book for
a more personal reason. When I was a wide-eyed undergraduate, I was privileged to take a year-long course in
Histology with Don McMillan. Histology was certainly
not my primary interest (at that time I was far more
interested in animal behavior). And, I certainly had no
inkling of how important histology would be for my
future research. I remember spending hours looking
through a microscope, completely mesmerized by a
world of vibrantly colored cells tinted by various tissue
stains and arranged in astonishing patterns, a universe
onto themselves. I also remember the first time I saw
(and recognized!) a simple duct nestled within a crowded
field of tissues, with its neatly arranged cuboidal cells
organized like a ring of tightly packed Chiclets. Pattern
existed! As I became more experienced in reading slides,
I learned to recognize arrangement patterns of cells as
functionally distinct units that collectively built tissues

and organ systems. And by their morphologies, I could
infer cellular and tissue function. Identifying cell and
tissue functions by their arrangements and respective
morphologies, I was able to use histology as a diagnostic
tool to identify reproductive state within hermaphroditic
(and gonochore) fishes.
Over time, my sense of wonder for the microanatomical world has not diminished. In class and
laboratory, Don was an ideal mentor: patient, insightful and deeply knowledgeable. He would explain what we were looking at on our slides with
such highly informed enthusiasm that you wanted to
dive right back into that micro-sized world that
appeared to float on a glass slide. I didn’t fully
appreciate his extraordinary breadth of knowledge
until many years later when I found myself in the
unexpected position of teaching comparative vertebrate histology. I still had my detailed notes from his
class, and my old copy of Ham’s Histology (1969),
which proved invaluable. It was while developing
lectures and laboratories for the course that I realized
how really lucky I had been to have received my
own instruction from Don and, through his mentorship, to fall in love with the discipline.

Environ Biol Fish (2012) 95:185–189

The volume that Don McMillan has crafted reflects
both his vast knowledge and his attention to detail. The
topic is female reproductive systems of fishes. This
represents a tremendous undertaking, given the wide
diversity of fish taxa (62 orders, 515 families and an
estimated 32,500 species [Nelson 2006]) and the extraordinary variation of their reproductive modes. Any
attempt at a comprehensive review of fish reproductive
modes faces almost overwhelming challenges. In this,

Don McMillan’s book succeeds.
The book is subdivided into six chapters that comprise 560 pages (the book totals 598 pages, including
references and the index) and include 509 figures, the
majority half or full page size. The images are the
dominant component and central strength of this volume. They include histological sections of tissues which
provide cellular and tissue-level detail, SEMs which
detail cell surface features and TEMs that bring cell
components into the visual realm. In addition, a good
number of figures are schematic representations that
provide clear and simplified constructs to help the reader
better understand the photographed images.
In each chapter, the text portion leads, followed
by the chapter figures. The text serves as a research
review of the chapter topic, but is broken down into
easily digestible, well-written subsections. My only
disappointment is that much of the review information presented in the text is somewhat dated. Only
twenty-five percent of the references cited are from
the last two decades and of these, less than 2% are
from 2000 on, comprising two papers published in
2001. Some of this can be ascribed to the time lag
between completion of a manuscript and final publication (late 2007) and the even later date of this
review. However, the paucity of coverage of post2000 work limits the usefulness of this volume as a
current-state-of-knowledge resource.
The real value of this book is in its atlas-style, fully
captioned, imagery. Do you want to visualize how an
oocyte develops, and how this development may vary
from species to species? On pages 93–130 you will be
transported through a wide range of fish taxa, each
carefully detailing in figure(s) and legend the process
of oogenesis across a broad reach of systematic diversity. Do you want to understand how ovarian follicles

form? That is covered in exquisite illustrative detail
between pages 131 and 208. Equally stunning visual
treatments are provided for ovulation and for fertilization events. In the last chapter, on viviparity, illustrations


Environ Biol Fish (2012) 95:185–189

show how embryos are frequently associated with specialized portions of the maternal reproductive system. I
found this chapter among the most engaging, probably
because it relates most closely to the organismal level.
The first chapter, ‘Female genital systems of fish’,
provides a basic overview of female reproductive systems and the early development of the system components. It includes a brief outline of the early formation of
the gonadal primordium and the subsequent arrangement of primordial germ cells (PGCs), somatic cells
and the germinal epithelium. This is followed by a
comparison of reproductive morphology, mostly of the
ovary, drawing on studies spanning a wide variety of
fish species. Most frequently discussed include the Japanese ricefish or Medaka (Oryzias latipes), Atlantic
hagfish (Myxine glutinosa), Sea lamprey (Petromyzon
marinus), Gulf pipefish (Syngnathus scovelli), Zebrafish
(Danio rerio, referred to in the text by its older name
Brachydanio rerio]), Green swordtail (Xiphophorus hellerii), Mummichog (Fundulus heteroclitus) and Goldfish (Carassius auratus). Parts of the text could be
clearer. For example, information on different fish species is often drawn from various cited studies but the
species themselves are not identified. Presumably the
reader is expected to tease these out by checking the
references or, when included, figure legends. However,
the absence of such information within the text itself
makes it difficult to fully appreciate where information
reflects common patterns across taxa versus where it
corroborates existing information on a single species.
Much of the material in this chapter will provide a

good review for the non-specialist. Some statements
are outdated or incorrect, and this will likely be noticeable to readers who are well-informed in those
areas. For example, there is some doubt expressed by
the author as to whether the germinal epithelium is the
intra-gonadal source of PGCs and oogonia in all fishes
(pg.2). This argument has been settled: oogonia restricted to the germinal epithelium is the general condition of teleosts (see Grier and Taylor 1998; Grier
2000), with the notable exception of syngnathids (seahorses and pipefishes) in which PGCs and their next
developmental stage, oogonia, are confined to a germinal ridge within the ovary (Grier 2000). On page 3,
PGCs are stated to “arise early in development within
embryonic endoderm or mesoderm” and that their
origins are “extragonadal and, in some fishes, may
be extraembryonic”. In fact, PGCs have been shown
to arise as a germ cell line, the precursors of which are

187

identifiable as early as the 32-cell stage in Zebrafish,
Danio rerio (Yoon et al. 1997; Braat et al. 1999; Knaut
et al. 2000) and even earlier, at the 16-cell stage in two
species of goby (Saito et al. 2004; Miyake et al. 2006),
well before the formation of endoderm or mesoderm.
The germ cell line, which is initiated at the onset of
symmetric segregation of germ plasm-bearing cells,
also occurs early in development, anywhere from the
early blastula to late gastrula stage depending on the
species, and, therefore, before completion of germ
layer formation (see Knaut et al. 2000; Shinomiya et
al. 2000; Otani et al. 2002; Fujimoto et al. 2006).
Regarding where germ cells originate, in all fish taxa
so far studied the germ cell line originates outside the

embryo proper (Richards and Thompson 1921; Hamaguchi 1982; 1983; Weidinger et al. 1999; Shinomiya et
al. 2000; Kurokawa et al. 2006). Only after the formation
of the coelomic cavity and dorsal and ventral mesenteries
do the PGCs migrate into the embryo, either along the
splanchnic or somatic mesoderm, to the site of the future
gonadal primordium (Moore 1937; Gamo 1961;
Weidinger et al. 2002; Saito et al. 2002; 2004;
Reichman-Fried et al. 2004; Kurokawa et al. 2006), so
in all known cases the origin of PGCs is extraembryonic.
The section of the text on oviduct origins (pg.5),
which is described as being formed by the caudal extension of a peritoneal sheath surrounding the ovary,
presents a highly simplified scenario. Across teleosts,
the oviduct can have different, and frequently more
complex, origins. Depending on the species, oviduct
formation may result from either somatic cell proliferation from the posterior gonad, proliferation of mesenchymal cells surrounding the urethra, cavitation of a
solid cord of posteriorly located peritoneal cells, posterior tubular extensions of gonadal serosa, peritoneal folds,
dorsal mesentery folds, or by some combination of the
above (Essenberg 1923; Anteunis 1959; Takahashi and
Iwasaki 1973; Shimizu and Takahashi 1980; Suzuki and
Shibata 2004). However, as stated at the outset, this
chapter offers a good review of female fish genital systems for the non-specialist reader.
The second chapter is entitled ‘Ovarian follicles.’
For the uninitiated, the follicle is a compound structure
made up of a single oocyte and surrounding layers of
somatic cells which become organized through a series of induction events, following the formation of the
gonadal primordium. This section of the book provides detailed descriptions of the events that take place
during the development of the maturing oogonium


188

through various oocyte stages, and of ovarian follicle
development. The information in this chapter is both
detailed and comprehensive, with the figures providing a perfect complement to the text.
This and the following chapters also illuminate and
seek to rectify a persistent and problematic issue in
histology, that of confusing terminology. Descriptions
of reproductive histology are permeated with a bewildering array of terms that are often poorly, or incompletely,
defined. To address this issue, a series of workshops have
been chaired by Nancy J. Brown-Peterson of the Gulf
Coast Research Laboratory in an effort to streamline and
standardize terminology associated with reproductive
morphology and processes. The establishment of these
workshops attests to the need for a new and more critical
approach to reproductive terminology associated with
fishes and so far has resulted in a timely publication on
this topic (Brown-Peterson et al. 2011). In this light, this
and the succeeding chapters are valuable not only for the
detailed information they provide, but also for the inclusion of alternate terminology, which serves as an invaluable guide to the present terminological maze.
The next two chapters (3 and 4) cover ovulation and
fertilization-associated events and are rich in descriptive
detail and imagery. Early in Chapter 3 (entitled ‘Ovulation’) the processes involved in the exit of the ovum
from the ovary are discussed over a broad range of fish
taxa. Most of the text is devoted to the fate of the ovarian
follicle post-ovulation (or pre-ovulation in the case of
atretic follicles) and to changes associated with the
chorion or zona pellucida just prior to, and following,
ovulation. For this, the author draws on primary literature representing a wide range of fish taxa to construct a
step by step scenario of ovulation-associated events.
This information nicely sets up Chapter 4 (‘Events

associated with fertilization’) for describing the series
of events that are triggered by the penetration of the
ovum wall by a spermatozoon. Each step from first
contact through to the formation of the fertilization
membrane is discussed using information available
from a variety of fish taxa. Together, these two chapters
should be required reading for every upperclass student
majoring in Biology or Zoology. The information provided is succinct, clearly written, and provides good
introductory overviews of their respective topics.
The fifth chapter (‘Oviducts and oviparity’) is relatively short. Little is known regarding the ontogeny of
teleost oviducts (see Wake 1985, 1986 for an early
treatment and Cole 2010 for a review) and this section

Environ Biol Fish (2012) 95:185–189

of the chapter is understandably short. Much of the
chapter is devoted to the chondrichthyan reproductive
system, for which much more information is available,
and to differences associated with reproductive mode
(oviparity versus viviparity).
The sixth and final chapter, ‘Viviparity’, deals with
fish that have internal fertilization followed by partial or
complete embryonic development within the maternal
body. Associated with the retention of embryo(s) within
the maternal body are a variety of modifications to the
reproductive system, some of which are extensive, including features that facilitate the storage of sperm for
delayed insemination, the accommodation of one or
more embryos in different stages of development (superfetation), and the provision of a supportive environment
for the developing young. The development and final
morphology of these features can differ substantially

across viviparous fish taxa. The chapter is divided into
three sections. One section is devoted to viviparity
among elasmobranchs consisting of the sharks, skates
and rays (note that viviparity is unknown among the
other branch of chondrichthyans, the holocephalans, or
rabbitfishes). Another section reviews viviparity in teleosts and provides excellent coverage, both in text and
images, of the considerable diversity of morphological
adaptations found within this equally large and diverse
group. A brief third section is devoted to Latimeria
chalumnae, one of the two species of living the coelacanth. Not surprisingly, this section is quite short, reflecting the paucity of information on coelacanth reproductive
morphology. As a whole, this chapter will be of interest
not only to those with a histological background, but also
to a broader audience within ichthyological, morphological and vertebrate biology disciplines. Here the interested reader will find a solid micro-anatomical overview of
the extraordinary diversity of viviparous specializations
and adaptations found among fishes.
In summary, this book fills a critical need for the
coverage of female fish reproduction from a histological
perspective. This is a topic that has broad applications
but for which information has been relatively limited,
and somewhat scattered throughout the primary literature. As mentioned earlier, one notable drawback of this
volume is that it provides little information on recent
research developments in the field. Despite that, Fish
Histology, Female Reproductive Systems provides an
excellent overview of the topic, uses a broad comparative approach, and offers superb imagery that will make
it a classic and influential atlas-style reference.


Environ Biol Fish (2012) 95:185–189
Acknowledgements I am grateful to L.R. Parenti for insightful
comments on an earlier version of this review.

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Environ Biol Fish (2012) 95:191–194
DOI 10.1007/s10641-012-9979-3

Using energy dispersive x-ray fluorescence microchemistry
to infer migratory life history of Atlantic sturgeon
Matthew T. Balazik & Stephen P. McIninch &
Greg C. Garman & Michael L. Fine & Clint B. Smith

Received: 3 May 2011 / Accepted: 9 January 2012 / Published online: 26 January 2012
# Springer Science+Business Media B.V. 2012

Abstract Atlantic sturgeon migrate between ocean
and freshwater habitats to spawn, and juveniles spend
several years in fresh/brackish water before returning
to the ocean. Because strontium/calcium (Sr/Ca) ratios
are diagnostic for freshwater and marine environments, we examined the utility of energy-dispersive
x-ray fluorescence (EDXRF) to quantify Sr/Ca ratios

of Atlantic sturgeon pectoral fin spines. Atlantic sturgeon spines from wild adults and experimental juveniles were analyzed along a linear transect from the
primordium to the outermost point. To verify the technique hatchery juvenile Atlantic sturgeon were held in
experimental tanks at <0.5, 13–15, or 33–35‰ and
sampled after 5 months. Sr/Ca ratios of experimental
hatchery fish increased with salinity, and Sr/Ca ratios
M. T. Balazik (*) : S. P. McIninch : G. C. Garman
Center for Environmental Studies,
Virginia Commonwealth University,
1000 West Cary Street,
Richmond, VA 23284, USA
e-mail:
M. L. Fine
Department if Biology, Virginia Commonwealth University,
1000 West Cary Street,
Richmond, VA 23284, USA
C. B. Smith
U.S. Army Engineer Research and Development Center
Geospatial Research and Engineering Division Alexandria,
7701 Telegraph Road,
Alexandria, VA 22315, USA

in wild adults varied predictably along the measurement transect. However, the ratio decreased in the
outermost region of the spine in mature fish collected
during a return to freshwater for spawning. Therefore
EDXRF is a useful tool to track individual movements
of Atlantic sturgeons and other diadromous fish.
Keywords Atlantic sturgeon . Sturgeon spine . Sr/Ca
ratio . Diadromy . EDXRF

Introduction

Strontium/calcium (Sr/Ca) ratios of selected biogenic
tissues (e.g. otoliths) are used to infer migration patterns between marine and freshwater environments for
many fish species using laser ablation or wavelength
dispersive microprobe analysis (Limburg 1995; Secor
et al. 1995; Arai and Tsukamoto 1998; Allen et al.
2009). Elemental composition of fish tissues may be
used to discriminate between marine and freshwater
populations (or life history stages), determine links
between natal rivers or nursery areas and adult stocks,
and assess population structure in marine fishes (Sauer
and Watabe 1989; Secor and Rooker 2000; Kraus and
Secor 2004; Limburg et al. 2007). Energy dispersive
x-ray fluorescence (EDXRF) is proposed as an alternative method that allows non-destructive and accurate determination of elemental composition of
biogenic materials (Paiva et al. 1997; Lundblad et al.
2008). An EDXRF system works by detecting the


192

x-ray energy released due to electrons changing shell
layers of an atom after it has been excited by an x-ray
laser. A silicon detector is used to determine the
amount of energy released which is known for most
elements and therefore percent mass of elements in an
area can be quantified.
Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus)
is an anadromous, long-lived (60+ y), iteroparous, historically fishery-targeted species, making it a good candidate for Sr/Ca analysis. Atlantic sturgeon populations
are depleted in the United States (Atlantic Sturgeon
Status Review Team 2007). A better understanding of
migration patterns and life history may aid in management and recovery efforts.

We evaluated the use of EDXRF analysis of pectoral fin spines from Atlantic sturgeon to potentially
provide migratory and life history information. The
ratio of Sr to Ca is positively correlated with environmental salinity (Limburg 1995); we quantified this
ratio as distance increases from the primordium in
juveniles held under experimental salinity regimes
and in wild adults during freshwater residence (Arai
and Tsukamoto 1998; Secor and Rooker 2000).

Methods
We used 2 year old hatchery juvenile Atlantic sturgeon
(295–340 mm fork length) to verify the instrument
could detect variations of Sr/Ca ratios in the fish
spines. The hatchery Atlantic sturgeon were acquired
from the Maryland Department of Natural Resources
Fisheries Division and held at the aquatics facility of
Virginia Commonwealth University (VCU). The
hatchery fish were maintained in freshwater prior to
being moved to VCU and held at VCU for 3 months
prior to salinity treatment. Richmond city water was
filtered to remove chloramines, and salinity regimes
were prepared with Instant Ocean© sea salt. Salinity
was monitored using a refractometer (Bath et al.
2000). The ion ratio of Instant Ocean© sea salt mimics
typical saltwater by 98.5% (US Aquatics Consumer
Support). The three treatment tanks were maintained
at identical temperature (16–18°C) and fed an identical diet (Zeigler Bros Inc. product # 306540-18-44)
because both affect Sr/Ca uptake (Fowler et al. 1995;
Secor et al. 1995; Gallahar and Kingsford 1996; Bath
et al. 2000).

Environ Biol Fish (2012) 95:191–194

A section from the left leading fin spine was taken
from each fish for pre-treatment analysis. The following day we measured fish for fork length and placed
them in experimental tanks. The fish were separated
into three treatment groups with three fish per treatment, freshwater (<0.5‰), brackish (13–15‰) or saltwater (33–35‰) tanks for 5 months. Fish were
measured, and the right leading pectoral spine was
removed (VCU IACUC AT20127) at the termination
of the experiment.
Pectoral fin spines were removed from carcasses of
13 recently killed wild adult male Atlantic sturgeon
found in September, 2008 and 2009 during a putative
spawning period in the freshwater portion of the James
River, Virginia. The carcasses were found by researchers examining the shoreline for Atlantic sturgeon mortalities. These fish were confirmed as adult males due
to fork length measurement and having fully developed gonads.
A 2 mm thick section of the leading fin spine
was cut within 1 cm of the articulation point with
an isomet saw. A section from the left spine was
used when available; however, the right spine was
used if the left spine was not present. Extreme care was
taken to insure the sample section was cut orthogonally. Soft tissue on the spine was removed with a
fine brush. The spine was then rinsed with deionized
water and air-dried.
Samples were analyzed for elemental analysis on a
Horiba X-Ray Guide Tube XGT-7000V EDXRF microscope with 50 kVof energy at 1 mA with a 100 μm probe
held under vacuum. Each sample point was ~100 μm in
diameter. The machine was calibrated using protocols
and samples provided by the microscope’s manufacturer.
To support the sample with minimal background, plastic
wrap (Fisher Scientific) was placed on a flat stage with a

5 cm×5 cm hole in the middle of the stage. Two millimeter thick spine samples were attached to the plastic
wrap using double sided tape. Analysis with a copper
plate backing indicated 1.9 mm thick samples were
sufficient to block the copper signature, i.e. laser excitations are restricted to the spine sample. Sampling points
were measured equidistant along a linear transect across
the spine section for 30 s per point, and each point
measurement was repeated to verify precision (Fig. 1).
For all samples extreme care was taken to ensure the
most peripheral portion of the spine section was sampled. The Sr/Ca ratio from both transects was averaged
for each point.


Environ Biol Fish (2012) 95:191–194

193

Fig. 1 Photograph of Sr/Ca analysis points on an Atlantic
sturgeon pectoral spine. The red circles show loci where Sr/Ca
was analyzed, and the yellow number (0–60 in this case) is the
point number along the transect. Point one is at the primordium
and point 60 is at the spine edge. Each point was measured twice
and the average was used for analysis

Results
The salt and brackish water tanks had one fatality
each leaving an n02 for these treatments. After
5 months in the aquatic center, the average fork
length of the hatchery fish increased 31 mm (23–
50 mm). An ANOVA (F01.62, p00.13) indicated
no significant difference in the Sr/Ca ratio among

the pre-treatment samples (Fig. 2a). The ratios of
freshwater-control fish stayed flat between 0.2×
10 −3 and 0.3 × 10 −3 (Fig. 2b). In the salt and
brackish treatments Sr/Ca values increased toward
the edges of the spine and leveled off. The brackish water treatment maximum ratio was 0.9×10−3
(greater than 3× freshwater values), and the saltwater treatment maximum was 1.7×10−3 (a further
doubling compared to brackish water). The lack of
overlap in Sr/Ca ratios between treatments is a
strong result and additional statistics are not necessary (Yoccuz 1991). Variation in the replicate
point runs averaged 3.7% and ranged between 0
and 6% indicating reasonable precision.
The spines of all 13 wild fish had similar
patterns, and transects from three representative
individuals are shown in Fig. 2c. Mean Sr/Ca

Fig. 2 The percent mass of Sr/Ca ratios at different distances from the primordium of Atlantic sturgeon spines. a.
The percent mass of Sr/Ca ratios of experimental juveniles
prior to salinity level treatment. b. The percent mass of Sr/
Ca ratios of experimental juveniles maintained at different
salinity levels. c. The percent mass of Sr/Ca ratios of three
representative wild adult male Atlantic sturgeon captured in
the James River

ratios in wild Atlantic sturgeon increased from
0.3×10−3 at the spine primordium to 1.5×10−3 at
the periphery (paired t12 0−16.0949, p<0.0005). Ratios decreased at the outermost portion of the spine
consistent with a return to a freshwater environment
(Fig. 2c).

194

Discussion
This study successfully demonstrates the ability of
EDXRF to indicate an ontogenetic change in Sr/Ca
ratios of Atlantic sturgeon consistent with migration
across an environmental salinity gradient. It has an
advantage over other methods because it is nondestructive to the sample. However, similar results
have been found in green sturgeon (A. medirostris)
using laser ablation on spines and Russian sturgeon
(A. guldenstadi) using wavelength dispersive x-ray
electron microprobe analysis on otoliths (Arai and
Miyazaki 2001; Allen et al. 2009). Ratios in experimental juveniles in this study increased with salinity
indicating that 5 months and 23 mm of growth are
sufficient for a salinity signature to imprint on an
Atlantic sturgeon spine. By comparing annuli (Balazik
et al. 2010) of the 13 wild fish with the position of
increased Sr/Ca ratios our data suggest Atlantic sturgeon out migrate from natal rivers between 1 and
4 years of age. The ratio increased by age 4 for the
14 years old, by age 3 for the 16 years old, and by age
2 for the 19 years old (Fig. 2c). These findings agree
with previous catch data on Atlantic sturgeon outmigration (Bain 1997; Kynard and Horgan 2001). Sr/Ca
ratio decreased at the spine periphery in the three adult
male samples collected in freshwater, indicating that
water chemistry can be imprinted on the spine during
inward migration. With increased sampling point density this new technique could be used to determine
natal immigration to brackish environments, further
movement from brackish to ocean environments and
perhaps even spawning events.
Acknowledgements We thank the Virginia Atlantic sturgeon

restoration team and the US Army Engineer Research and
Development Center (ERDC) Geospatial Research and Engineering Division – Photonics Imaging and Spectroscopy Laboratory located at George Mason University. We thank Brianna
Langford for laboratory assistance. This is VCU Rice Center
contribution number 20.

References
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SI (2009) Using trace elements in pectoral fin rays to assess
life history movements in sturgeon: estimating age at initial
seawater entry in Klamath River green sturgeon. Trans Am
Fish Soc 138:240–250

Environ Biol Fish (2012) 95:191–194
Arai T, Miyazaki N (2001) Use of otolith microchemistry to
estimate the migratory history of the Russian sturgeon,
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Arai T, Tsukamoto K (1998) Application of otolith Sr:Ca ratios
to estimate the migratory history of masu salmon, Oncorhynchus masou. Ichthyol Res 45:309–313
Atlantic Sturgeon Status Review Team (2007) Status review of
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(2000) Strontium and barium uptake in aragonitic otoliths
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element maps: new approaches with synchrotron microbeam x-ray fluorescence. X Ray Spectrom 36:336–342
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fluorescence (EDXRF): effects of post-depositioinal chemical weathering and sample size on analytical precision.
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JMS (1997) Elemental distribution in a sediment deposit
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aspects of zinc accumulation by fish scales. Tissue Cell
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life cycles in estuarine fishes? Fish Res 46:359–371

Secor DH, Henderson-Arzapalo A, Piccoli PM (1995) Can
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72:106–111


Environ Biol Fish (2012) 95:195–200
DOI 10.1007/s10641-012-9982-8

Melanization of the alimentary tract in lizardfishes
(Teleostei, Aulopiformes, Synodontidae)
Lev Fishelson & Daniel Golani & Barry Russell &
Bella Galil & Menachem Goren

Received: 6 March 2011 / Accepted: 17 January 2012 / Published online: 26 January 2012
# Springer Science+Business Media B.V. 2012

Abstract Investigating the alimentary tract in several
species of lizardfishes (Synodontidae, Teleostei) of the
genera Saurida, Synodus and Trachinocephalus, from
various sites of their distribution, revealed melanization of the tract wall. This phenomenon was observed
in several species of the genus Saurida, but not in the
other two genera. This melanization is caused by
layers of melanosomes rich in melanin granules and
deposited within the connective tissue of the submucosa, between the muscular wall and the muscularis
submucosa. From this site this black submucosa
extends into the folds of the mucosa. In S. tumbil
and S. filamentosa, the entire alimentary tract is black,

whereas in S. macrolepis only the stomach is partially
L. Fishelson (*) : M. Goren
Department of Zoology,
George S. Wise Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel
e-mail:
D. Golani
Department of Evolution, Ecology and Behavior,
Hebrew University,
Jerusalem 91904, Israel
B. Russell
School of Environmental and Life Sciences,
Charles Darwin University,
Darwin, NT 0801, Australia
B. Galil
National Institute of Oceanography,
Shikmona, Haifa, Israel

or entirely black. This phenomenon and its possible
importance are discussed.
Keywords Lizardfishes . Alimentary tract . Saurida .
Submucosal melanization

In numerous species of bony fish, the peritoneal
mesothelium that envelopes the alimentary tract
(serosa) is rich in melanocytes and melanosomes,
which are laden with dark pigment granules that consequently blacken the exterior of the gut. However, the
formation of a melanized layer within the intestinal
wall of fishes is a rare phenomenon, described until
now in moray eels (Böhlke 1989; Fishelson 1994),

cardinal fishes (Fishelson et al. 1997), some Antarctic
Nothoteniids (Eastman and DeVries 1997) and in the
scalloped ribbonfish, Zu cristatus (Bottaro et al.
2005). It was suggested (Fishelson 1994; Fishelson
et al. 1997) that this dark cover within the gut shields
the light of possibly bioluminescent food items hunted
and engulf by these night-active predators. In close
related and shallow-water species such melanization
was not observed.
The present study compares the morphology and
melanization of the alimentary tract in lizardfishes
(Synodontidae) of the order Aulopiformes (Euteleostei),
focusing on a group of species belonging to three major
genera of the family: Saurida, Synodus and Trachinocephalus (Table 1). The lizardfish studied reach up to


196

Environ Biol Fish (2012) 95:195–200

Table 1 Data on the studied lizardfishes and their alimentary tracts (lengths in mm)
Species

Origin

No. of
specimens

SL

Stomach
length

Stomach
color

Intestine
color

Saurida macrolepis (Tanaka, 1917)

RS, EM

20

45–320

20–85

pale

pale

Saurida macrolepis

EM

4a

80–287

40–120

partly black

pale

Saurida argentea (Macleay, 1881)

AU (GBR)

2

182,230

40, 70

pale

pale

Saurida filamentosa Ogilby, 1910

AU (GBR)

3

180–210

75–100

black

black

Saurida undosquamis (Richardson, 1848)

AU (GBR)

2

325,360

96,110

pale

pale

Saurida tumbil (Bloch, 1975)

SRS, SA, GA

10

35–470

25–125

black

blackc

Saurida gracilis (Quoy & Gaimard, 1924)

Taiwan

5

128–109

60–65

black

pale

Synodus ariegates (Lacepede, 1803)

GA, RS, SA

12

60–145

42–60

pale

pale

Synodus kaianus (Günther, 1880)

Hawaii

2

110,120

70–78

paleb

pale

Synodus saurus (L., 1758)

MS

4

110–165

68–72

pale

pale

Trachinocephalus myops (Forster, 1801)

GA

10

86–115

30–36

pale

pale

AU Australia; GBR Great Barrier Reef; RS northern Red Sea; SRS southern Red Sea; SA South Africa; GA Gulf of Aqaba; EM East
Mediterranean; SL standard length (in mm);a , 60–100 m deep; b , peritoneum black; c , in some fish of this species the intestine is pale,
collections

600 mm standard length (SL), are epi-benthic, and predominantly found in the tropical and subtropical marine
littoral and on the continental shelves of the Indo-Pacific
and Atlantic, though some species, like Saurida undosquamis, have expanded into the cooler waters of the
Mediterranean (Golani 1993). The bathymetric range
of some species extends from the shallow waters to
about 800 m deep. Sections of the gut disclosed that
all are mainly piscivorous, feeding on a variety of fish
(small groupers, anchovy, sardines, sparids, smaller
lizardfishes and ophiichthids), but feed also on crustaceans and cephalopods.
The studied lizardfishes were collected along the
Israeli part of the Eastern Mediterranean, the Red Sea,
and off the Great Barrier Reef (GBR) of Australia.
Specimens well preserved in ethanol were also donated by various museums (Table 1); such preservation

does not affect the melanization found in the body
(Fishelson, personal observations). Freshly collected
fishes were preserved on ice until processing. Following the removal of food items, the guts was dissected
out and fixed in 3.5% glutaraldehyde+1% formaldehyde for electron microscopy (EM), and in Bouins
solution for light microscopy (LM) (More about the
methods see Fishelson et al. (2010a, b)).
In all the studied species, the esophagus is short and
wide, opening at its distal end into the cardiac part of
the stomach, adjacent to the opening of the pyloric part
of the intestine bearing the ceca (Fig. 1a, b). The

stomach (ST) is fusiform with thick walls. Its length
differs among the species, and in general increases
with increase in fish length. In adult specimens the
empty stomach is 6.0–8.0 mm thick externally at the
apical part, widening to about 10.0 mm in the middle,
and narrowing to 2.0–3.0 mm at the tapering caudal
end (Fig. 1a). The stomach wall is strong, enveloped
by a delicate serosa, and mainly formed by a 350–
500 μm thick muscularis consisting of smooth muscle
fibers. The submucosa, rich in blood capillaries, is
formed by a wide outer layer of collagen fibers and a
more inner layer of loose connective tissue rich in
elastic fibers. Adjacent to the mucosal epithelium is
the delicate ring of muscularis submucosa, located
adjacent to the lamina propria (Fig. 1b, c). The distal,
tapered (“vermiform”) part of the stomach is partly
separated by a muscular sphincter from the main
stomach. The wall of this part is very strong, especially
toward the apex, and its inner folds are lined by a

mucosal epithelium prominent in dense and elongated
villi. The present study complements the more general
study of the alimentary tract of lizardfishes by Fishelson
et al. (2011).
The black appearance of the alimentary tract was
observed in several species of the genus Saurida
(Table 1); collected by us and by colleagues (Tables 2
and 3). Dark parts of stomachs in some lizardfishes
were also noted by Dutt and Vidya Sagar (1981). This
phenomenon was found here to be most prominent in


Environ Biol Fish (2012) 95:195–200

197

Fig. 1 The black gut of
Saurida tumbil. a, Total
length of the gut; b,
Cross-section of the stomach
at the pylorus, SEM; c, ibid
in the mid-stomach; gb,
gall-bladder; i, intestine; l,
lumen of the stomach; li,
liver; mc, melanized ceca;
mv, folds of mucosa; po,
opening of pylorus; s,
stomach; sp, spleen; sw,
stomach wall; v, distal
tapering end of stomach;

arrowhead, melanin in the
submucosa, between folds
and along the villi

Table 2 Additional data on lizardfish from museum collection dissected for microscopy
Species

Museum

Lot number

N. of fish

Collection site

Saurida tumbil

HUJ

6434

6

S. Red Sea

S. tumbil

HUJ

6666

2

Gulf of Aqaba

S. tumbil

SAIAB

52851

2

Near Madagascar

S. macrolepis

ASI

6241

2

Taiwan

S. elongatus

ASI

61980

2

Taiwan

S. filamentosa

CRM

016955

3

GBR Australia

S. undosquamis

ASI

61690

2

Taiwan

S. undosquamis

ICTAU

P 3425

3

Mediterranean

S. argentea

CRM

007128

2

GBR Australia

Synodus variegatus

ICTAU

P9396

4

Gulf of Aqaba

Sy. variegatus

SAIAB

40292

2

KwaZulu, SA

Sy. saurus

ICTAU

P2724

4

Mediterranean

Sy. kaianus

BPBM

24256

2

Malokai, Hawaii

Trachinocephalus myops

ICTAU

P9975

3

Gulf of Aqaba

ASI academia sinica ichthyological collection; BPBM bishop museum ichthyologic collection; CRM site of collection by Barry Russell;
HUJ Hebrew University, Jerusalem; ICTAU ichthyologic collection, Tel Aviv University; SAIAB South African institute of biodiversity;
SA South Africa


198
Table 3 Stomach and intestine
color in additional species of
lizardfish

a

Dutt and Vidya Sagar (1981)
claim that the specimens seen by
them had a black stomach
b

Posterior intestine pale in all
these species

Environ Biol Fish (2012) 95:195–200

Species name

Stomach colorb

Informed by:

Saurida brasiliensis

pale

Ofer Gon, Barry Russell

Saurida carribbaea

pale

Barry Russell

Saurida elongates

pale

Barry Russell

Saurida gracilis

pale

“

Saurida isurankurai

black

Hsun-Ching Ho, Barry Russell

Saurida longimanus

black

Dutt and Sagar, Barry Russell

Saurida micropectoralis

pale

Hsun-Ching Ho, Barry Russell

Saurida nebulosa

pale

“ ”

Saurida normani

pale

Barry Russell

Saurida parri

pale

Barry Russell

Saurida pseudotumbil

black

Dutt and Sagara

Saurida undosquamis

pale

Ofer Gon, Barry Russell

Saurida wanieso

black

Hsun-Ching Ho, Barry Russell

Saurida tumbil of the Red Sea (Fig. 1a) and S. filamentosa from the GBR, in which the black appearance
extends along the entire alimentary tract and the pyloric caeca, with only the distal narrow end of the
stomach remaining pale. Cross-sections revealed that,
from this layer, melanosome-containing extensions of
the submucosa continue within the folds of the mucosa epithelium (Figs. 1c and 2d). This is in contrasts
with the pale color of the abdominal peritoneum. This
black color is formed by a dense layer of large melanosomes in the submucosa, laden with granules of
Fig. 2 Pigmentation in
the alimentary tract of

Saurida tumbil. a. Exposed
melanosomes with
melanin granules from the
submucosa, SEM; b. ibid in
LM; c, Strands of melanin
between the submucosa
fibers, LM; d, Extension of
the melanized layer into a
gastric fold, LM. ct, fibers of
submucosa connective
tissue; ex, extension of
submucosa between the
folds; gg, gastric glands;
m1-m4, melanosomes
(periphery marked by
white); mg, melanin
granules; mt, melanin
along connective tissue
fibers; sm, submucosa

melanin (Fig. 2a, b). The aggregates of melanosomes
form rows between the fibers of the submucosa
(Fig. 2c), and on some sites, such as the anterior part
of the stomach, they color the entire layer black, also
extending around the blood vessels and nerves.
In some species, such as in the East-Mediterranean
Saurida macrolepis, fish captured in shallow water,
down to 20 m, posses a pale alimentary tract; however,
in fish collected from the depth of 40–100 m the
stomach is black while the posterior part of the gut is

pale. This phenomenon was also seen in one of the


Environ Biol Fish (2012) 95:195–200

studied S. filamentosa stomachs, in which a ring of
black pigment occurred in the wall without prominent
extensions into the folds. In these latter species the
density of melanin granules in the stomach gradually
declines posterior, fading posterior towards the apex.
In juvenile fish the initial stages of melanization can
be observed in the gut of 65–75 mm SL fishes, beginning with the anterior part of the stomach.
The occurrence of a black submucosa in the walls
of the alimentary tract of several species of the genus
Saurida, not observed in Synodus species and Trachinocephalus, is remarkable. A dark appearance of the
alimentary tract in fishes has mostly been described
from the dark serosa, a part of the melanised dark
peritoneum that envelopes the viscera. Melanization
in the sub-muscular connective tissue derives from an
entirely different source and has to date been observed
in a few fish families only, e.g. moray eels (Böhlke
1989; Fishelson 1994), cardinal fishes (Fishelson et al.
1997), Nototheniid species (Eastman and DeVries
1997) and the trachipterid ribbonfish (Bottaro et al.
2005). Bottaro et al. (2005) situated the black layer in
the mucosa; whereas in fact, as in other fish with black
alimentary tracts, in the ribbonfish too it is also located
in the submucosal connective tissue. Of the studied
lizardfishes, the submucosal melanin deposition along
the entire alimentary tract was observed in Saurida

tumbil, a species found in relatively deeper waters, and
in the studied specimens of S. filamentosa. Depth
related tract melanization was also observed in deepwater moray eels (Fishelson 1994). However, a
studied specimen of S. tumbil collected in the shallow
waters of the Persian Gulf had a pale alimentary tract.
This interesting difference indicates the need for a
taxonomic revision of the S. tumbil populations; or
the “black gut” and “pale gut” groups are of a single
stock, but each represents a morphotype adapted to a
different diet or different bathymetric habitat. Partial
melanization of the stomach only was found in the
studied Saurida macrolepis and some related species
(see Table 2). Such species–specific melanization can
possibly be used as a marker in future phylogenetic
studies of the species-rich genus Saurida, and contribute to the ecomorphological classification of this
family of fishes (Norton et al. 1995; Davis 2010).
As in moray eels (Fishelson 1994), in the studied
species of lizardfishes too, the melanosomes, containing 1.5–2.0 μm melanin granules, are dispersed
among the fibers of the connective tissue, being

199

especially dense close to the internal layer of the
muscular wall. The extensions of this submucosa into
the folds of the alimentary tract also places the melanosomes near the mucosal epithelium. The questions
that still remain open are: What is the role of the
fishes’ internal black layer, between the muscularis
and mucosal epithelium; what is the possible function
of melanin in the fish physiology; and what environmental factors may have induced the formation of
such a cover inside the gut wall? Fishelson (1994),

Fishelson et al. (1997) and Eastman and DeVries
(1997) found a correlation between melanization of
the alimentary tract and nocturnal activity/deep-water
habitat, including a diet of bioluminescent prey. As
this luminescence does not cease after the bioluminescent fish or crustacean has been swallowed, the melanized cover may dampen the bioluminescense of the
consumed prey. Additionally, melanin and its various
forms are recognized as effective neutralizers (antioxidants) of the free radicals formed during lipid peroxidation and are also involved in iron regulation (Edelstein
1971). In parallel, it is recognized that environmental
stress induces the production of alpha—MSH (melanin
stimulating hormone) (Thody 1999). Is melanin deposition in the submucosa of fishes, as described for some of
the nocturnal or deep-water species, a response to stress,
for example to the low levels of illumination or darkness
during their activity? Or is melanin a form of ironstorage that enables the recycling of this element, so
important for blood-cell production? These very interesting questions should be addressed in the future.
Acknowledgments Thanks are due to L. O’Hara and A.
Suzumoto of the Bernice Bishop Museum (Hawaii), and O.
Gon of the South African Institute of Aquatic Biodiversity,
Grahamstown (South Africa), for samples of fishes from their
museums. Thanks are also extended to N. Paz for editorial help,
and I. Brickner and Y. Delarea for assistance in the histological
work and electron microscopy, and to V. Wexler for help in the
artwork. The authors are thankful to the anonymous reviewers
for their constructive remarks to the MS.

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Environ Biol Fish (2012) 95:201–212
DOI 10.1007/s10641-012-9977-5

Habitat use of the Rio Grande silvery minnow (Hybognathus
amarus) during a long-term flood pulse in the Middle Rio
Grande, New Mexico
Hugo A. Magaña

Received: 20 August 2010 / Accepted: 9 January 2012 / Published online: 5 February 2012
# Springer Science+Business Media B.V. (outside the USA) 2012

Abstract The Middle Rio Grande (MRG) of New
Mexico has been influenced by man for over
500 years. Native Americans began diverting water
to irrigate agricultural crops in the floodplain in the

14th century. The Spanish followed and increased
agricultural irrigation to over 125 000 acres. Frequent
flooding of the MRG valley in the 19th century led to
many engineering projects in the early 20th century to
control flooding. A series of impoundment dams, diversion dams, and levees were constructed. The loss of
floodplain habitats throughout the MRG Valley has
altered the riparian community and caused the demise
of many fish species. A controlled flood pulse from
Cochiti Reservoir, New Mexico was initiated in April
2005 to support the recovery of the endangered Rio
Grande silvery minnow, Hybognathus amarus. This
study documents habitat selection by larval fishes in a
restored floodplain in the Rio Grande, NM. Larval fish
light traps captured 394 larvae representing four cyprinid species (Pimephales promelas, H. amarus, Cyprinella lutrensis and Cyprinus carpio). Results for
CCA indicate that Hybognathus amarus prefer shallow, low velocity habitats. Results from Chao-Jaccard
similarity index indicated that relative contribution
was highest in P. promelas at 64% followed by H.
H. A. Magaña (*)
U.S.D.A. Forest Service,
Rocky Mountain Research Station,
333 Broadway Blvd SE #115,
Albuquerque, NM 87102, USA
e-mail:

amarus 33%. Results from (dis)similarity analysis reveal that species composition between habitat orientation and date was highest in H. amarus at 42%
followed by P. promelas 40%. Cyprinella lutrensis
and C. carpio represented 9.5 and 8.5%, respectively.
A general linear model indicated that only depth and
velocity were significantly different (p00.02 and p0
0.03 respectively).

Keywords Floodplain . Flooding . Rio Grande .
Hybognathus amarus . Fishes

Introduction
It has been said “understanding how fauna respond to
flooding in floodplain rivers is the holy grail of river
ecologists” (Humphries, P. pers. comm., 2011). The
present study was intended to elucidate the response of
Rio Grande ichthyofauna to a long-term flood pulse in
the Middle Rio Grande (MRG) of New Mexico. Few
large alluvial rivers of the southwestern U.S. have
been studied and documented as well as the MRG of
New Mexico (Richard 2001). However, little is known
about floodplain habitat use by Rio Grande ichthyofauna during floods in the MRG. The MRG is defined
as the reach from Cochiti Dam to Elephant Butte Dam
in southern NM, a distance of 289 km.
For the past 5 million years, the Rio Grande has
flowed south through its valley from its origins in the
San Juan Mountains of southern Colorado to the Gulf


202

of Mexico (Crawford et al. 1993). The Rio Grande is
the fourth largest river in North America, totaling
more than 1900 miles in length (USGS 2011). Unlike
typical river valleys, the river did not create the valley
it flows through. Instead, the Rio Grande flows
through the Rio Grande rift at the lowest point of the
trough (Scurlock 1998). For millions of years the river

would freely migrate across the floodplain (Molles et
al. 1998) which varied in width from less than 1.5 km
to approximately 10 km and bound on the east and
west by raised landforms and mountains of varying
geological origin (Crawford et al. 1993). The typical
hydrological pattern of the Rio Grande’s bed would be
to aggrade over time, and in response to a flood or series
of floods, would leave its elevated channel to lower
elevation in the valley and establish a new course
through a process known as ‘avulsion’ (Crawford et al.
1993). The historical Rio Grande is unique among other
rivers described in the literature because of the high
frequency of channel migration and avulsion (Mack
and Leeder 1998). Richard (2001) used more than
74 years of hydraulic, topographic, sediment, and photographic data to quantify lateral migration of the Middle Rio Grande in the Cochiti reach. Since 1918, the
channel moved toward a more stable state as peak discharges decreased prior to and following construction of
Cochiti Dam, and shifting from a multi-thread to a more
single-thread pattern (Richard 2001). This pattern has
led to an incised channel, which rarely overtops its
banks except in the lower sections leading to Elephant
Butte Reservoir (Crawford et al. 1993).
The severity and frequency of major flooding along
the MRG began in the late 19th and early 20th centuries (Wozniak 1987). Between 1849 and 1942, a total
of 50 moderate to severe floods were recorded along
the MRG reach with an average occurrence of approximately every 1.9 years (Scurlock 1998). The demand
for water in this highly restricted and physically altered river led to the enactment of International, Federal and state laws to allocate water to the states of
Colorado, New Mexico, Texas, and the Republic of
Mexico (Crawford et al. 1993). Continued flooding
led to the formation of the Middle Rio Grande Conservancy District (MRGCD) in 1925, whose emphasis
was to increase the efficient use of river water (Scurlock

1998). By 1935 the MRGCD had constructed 555 km of
drainage canals, 290 km of river levees, 400 km of main
irrigation ditches, and almost 640 km of old irrigation
ditches (Crawford et al. 1993).

Environ Biol Fish (2012) 95:201–212

The most significant ecological effect of Cochiti
Dam was to diminish the river’s historic flood regime
(Crawford et al. 1996; Dahm et al. 2003). As a result
of dam operations, the MRG no longer has a predictable flood regime; rather it has a ‘naturalized flood
regime’ (Bayley 1995) in the form of annual hypolimnetic releases from Cochiti Reservoir. Flow in the
MRG is confined to the area between levees
(Crawford et al. 1996; Molles et al. 1998; Massong
and Slaugh 2002), where much of the floodplain has
become disconnected and abandoned through degradation and aggradation of the channel bed (Valett et al.
2005; Massong et al. 2006). Most floodplains in the
MRG remain isolated from flooding while few are still
regularly inundated by the flood pulse (Valett et al.
2005). Loss of connectivity between the river and floodplains in the MRG is due to flow regulation and has
shifted the flood regime to longer inter-flood intervals
(Valett et al. 2005). Presently, the riverbanks along the
MRG are generally 1.2 to 1.5 m high and the incision of
the river channel makes it very unlikely that controlled
discharges from Cochiti Dam will overtop the riverbanks under present reservoir management practices
(Crawford et al. 1993).
Floodplain inundation in rivers is thought to enhance fish recruitment by providing a suitable environment and abundant food and habitat for larvae
(King et al. 2003). Existing literature indicates that
fish yields are higher in river-floodplains, including
individual temperate floodplain lakes that are connected

to the river (Bayley 1995), particularly those with predictable annual flood pulse (Balcombe et al. 2007). The
MRG lacks flood pulses of sufficient duration to provide
adequate time for spawning, nursery, and recruitment of
native ichthyofauna.
Virtually the entire endemic native fish fauna in the
southwestern U.S. is listed as threatened or endangered under the Endangered Species Act, largely as a
consequence of water withdrawal, flow stabilization,
and exotic species proliferation (Poff et al. 1997).
The target species of this study, the Rio Grande
silvery minnow (Hybognathus amarus) was at one
time the most abundant fish in the Rio Grande and
Pecos River occupying approximately 3800 river km
(2400 mi) in New Mexico and Texas to the Gulf of
Mexico (Bestgen and Platania 1991), but was listed as
endangered by the U.S. Fish and Wildlife Service in
1994 (USFWS 1994). Officially, H. amarus only
occurs in the Middle Rio Grande of New Mexico, a


Environ Biol Fish (2012) 95:201–212

280 km (174 mi) stretch of river that runs from Cochiti
Dam to the headwaters of Elephant Butte Reservoir or
approximately seven percent (7%) of its former range.
However, H. amarus is confined to an even smaller
reach between Angostura diversion dam and south of
San Acacia dam, a distance of 141 km, or approximately 3.7% of H. amarus former range.
Studies of contemporary habitat use by H. amarus
are very limited (Dudley and Platania 1997; Pease et
al. 2006) therefore, the present study is important to

understanding habitat use by H. amarus and associated
ichthyofauna in a restored floodplain especially since it
occurred during prolonged flood pulse. Hybognathus
amarus uses only a small portion of the available aquatic
habitat since channelization of the MRG has reduced or
eliminated most backwaters, edge areas, and slow-water
refugia which are typical habitat (Bestgen and Platania
1991; Dudley and Platania 1997). The present study was
the first investigation of habitat use by Rio Grande ichthyofauna during a prolonged flood pulse in the MRG. In
general, H. amarus most often uses silt substrates in areas
of low or moderate water velocity (e.g., eddies formed by
debris piles, pools, and backwaters) (Dudley and Platania
1997; Pease et al. 2006). Hybognathus amarus is rarely
found in habitats with high water velocities, such as main
channel runs, which are often deep and swift (Dudley and
Platania 1997; USFWS 2001). The objectives of this
study were; 1) Assess patterns of habitat utilization of
restored floodplain by H. amarus, and Rio Grande ichthyofauna, and 2) Measure physical and chemical parameters as they relate larval fish diversity to habitat
conditions.

Methods
The Los Lunas, NM, Habitat Restoration Project is
located at approximately river kilometer 252, on the
west bank of the Rio Grande adjacent to Mid Valley
Airpark, Los Lunas, NM (Fig. 1). The restored overbank area is approximately 1800 x 100 m along the
existing riverbank, encompassing an area of approximately 16 hectares that is bounded on the west by a
two meter high earthen and rootwad berm. In 2001,
the U.S. Fish and Wildlife Service (USFWS 2001)
concluded that current management practices in the
MRG would likely jeopardize the continued existence

of H. amarus. Funded through an interagency collaborative program, the Middle Rio Grande Endangered

203

Species Act Collaborative Program, the Los Lunas,
New Mexico Habitat Restoration Project was initiated
in 2002 to improve habitat conditions for H. amarus
(Slaugh 2003). The project was designed to mechanically widen the active river channel and improve
adjacent riparian habitats by moving over 53 518 m3
of material within the former floodplain to produce a
heterogeneous topography with goals to produce inundation of the floodplain at flows of greater than or
equal to 70 m3 s-1 and to ensure some inundation at a
wide range of flows less than 70 m3 s-1 (Slaugh 2003).
Specific areas within the site were revegetated by the
USCOE using seed, potted shrubs, or cottonwood
(Populus deltoids) and willow poles (Salix exigua
Nutt). Other features of floodplain modification included a network of variable depth side and transverse
channels designed to aid in minnow egg retention and
provide shallow water/low velocity rearing habitat
(USBOR 2007). These alterations within the historic
floodplain were intended to produce a variety of additional shallow water/low velocity egg-retention and
nursery habitats for H. amarus during spring spawning
flows. Spawning habits of H. amarus are unknown but
are believed to occur in spring and summer in still
waters over sandy-silt substrates (Sublette et al. 1990;
Dudley and Platania 1997). Substrate at the Los Lunas
Restoration site consisted mainly of sand and silt.
This study took place during a “wet” year in New
Mexico where snowpack in northern New Mexico
mountains was higher than normal levels (NOAA

2007) indicating a higher than normal spring runoff.
Discharge data for this study was taken from U.S.
Geological Survey (USGS) (Gage station 08330000)
at Central Bridge in Albuquerque, NM.
Bayley (1995) stated that annual primary and secondary production in many in temperate systems may
depend more on mechanisms occurring during drawdown than those occurring when the water is rising,
therefore, this study was initiated at peak discharge
(198 m3 s-1) on 24 May 2005 during a prolonged
hypolimnetic release from Cochiti Reservoir (8 April17 July 2005) and continued during the 44 days of the
descending limb of the hydrograph.
At peak flow (24 May 2005), six larval fish light
traps (Aquatic Research Instruments Inc., ARII 2007)
illuminated with chemical light sticks (Cyalume, Omniglow Corp) were deployed at six sampling sites representing three habitat orientations; perpendicular to flow
(LT1 and LT4), parallel to flow (LT2 and LT5), and


204

Environ Biol Fish (2012) 95:201–212

Fig. 1 Middle Rio Grande,
NM. Inset: Los Lunas
Habitat Restoration Project.
White area (center-left) is
the restored floodplain
showing area of inundation
at >71 m3 s-1. Gray areas
indicate areas of inundation
at <71 m3 s-1. Center of
picture is the Rio Grande

(flowing south). Dots indicate locations of light traps
on the floodplain numbered
1-6 north to south

leeward side of islands (LT3 and LT6). No light trap
controls were used in the main channel margin since H.
amarus is rarely collected in this most abundant habitat
(Dudley and Platania 1997). All habitat orientations had
varying degrees of water velocity throughout the study.
The water velocities at sites perpendicular to river flow
where the river entered the floodplain (6.0 cm s-1 vs.
main channel 80 cm s-1), parallel to river (~11.0 cm s-1)
flow, or leeward side of islands (~1.0 cm s-1). Light traps
were deployed weekly at dusk and retrieved at dawn on
24 May, 1 June, 8, 14, 21, and 28 at six permanent
habitat locations. On each sampling date, light traps
were deployed and anchored to a metal post in water
less than 1 m deep (39 cm±1.9 SE). Upon retrieval,
larval fish were removed from cod-end of light trap and
placed into 250 ml polycarbonate bottles of ice water
and Alka-Seltzer® tablets were added to euthanize fish

via CO2 narcosis (Wall 1993). Euthanized fish were
placed in 5% buffered formalin for 48 h, transferred to
35% ETOH for 7 days, and transferred to 70% ETOH
for long-term preservation (Wall 1993, Pease et al.
2006).
Water samples (60 ml) were collected during light
trap deployments, placed on ice, and delivered to the lab
for analysis. Water samples were analyzed for dissolved

nitrate (NO3-N), soluble reactive phosphorus (PO4-P)
and ammonium (NH4-N) (Magaña 2009). A YSI 556
multi-probe meter (Yellow Springs Inc.) was used at
light trap deployment to measure water quality parameters (temperature (°C), conductivity (μSiemens cm-1),
dissolved oxygen (mg L-1), percent saturation dissolved
oxygen (%DO), and pH adjacent to the light traps.
Depth was measured to the nearest tenth of a meter
using a stadia rod (Crain Enterprises Inc. model #


Environ Biol Fish (2012) 95:201–212

205

Table 1 Los Lunas environmental parameters measured during
24 May to 28 June 2005
Variable
Temp (C°)
Conductivity (μS cm-1)
DO (mg/L)
DO (% saturation)
pH
Depth (cm)
Velocity (cm/s)

Mean (±SD)

Range

22.6 (1.95)

20.72–27.45

231.1 (80.9)

201–356

6.9 (1.0)

2.35–9.29

79.5 (13.6)

27.9–117.8

8.1 (0.3)

6.69–8.76

39.1 (10.9)

17–69

6.3 (13.1)

0–77

NO3-N (μg/L)

138.9 (60.1)

5–298

PO4-P (μg/L)

59.2 (41.5)

0–150

NH4-N (μg/L)

67.5 (95.2)

0–430

90370). Water velocity was measured at six-tenths total
depth using a flow pressure sensor (Marsh-McBirney
Model 2000). Light quanta were measured using a LiCor quantum meter (Li-Cor Biosciences model Li-1000
and a 4π quantum sensor model Li-193SA).
A Canonical Correspondence Analysis (CCA) was
used to provide visual representation of the data for
physical and chemical characteristics of habitats occupied by fish across temporal and spatial scales. The
CCA is a multivariate analysis technique that directly
relates community composition to known variation in
the environment (ter Braak 1986). Two data sets are
used, one on the occurrence or abundance of a number
of species at a series of sites, and data on a number of
environmental variables measured at the same sites

(ter Braak 1986). The technique generates an ordination diagram, where species and light trap orientation

are represented by points and numbers, respectively,
and environmental variables are represented by arrows
(ter Braak 1986). The CCA analysis allows for a quick
appraisal of how community composition varies with
the environment (ter Braak 1986). By looking at the
signs and relative magnitude of the intraset correlations we may infer the relative importance of
each environmental variable for predicting the
community composition (ter Braak 1986). For example, the arrow referring to “pH” on a CCA diagram allows us to infer which species largely occur at
sites with highest and lowest pH. Two separate CCA
triplots were produced for the data obtained. One triplot
represents data for chemical site characteristics (e.g.
NO3-N, NH4-N, PO4-P, pH, and DO) while the other
triplot represents data for physical site characteristics
(e.g. Depth, velocity, temperature, and light quanta).
Since DO and %DO were highly correlated (0.9799)
only DO was used in the analyses (p<0.0001).
Two similarity indices were used to quantitatively
compare fish species composition and trap light orientation. The two indices employed were the Chao-Jaccard
index, a Jaccard coefficient weighted by abundance
(Chao et al. 2005) used for assessing compositional similarity of assemblages based on the presence/absence of
species in paired assemblages, and the (dis) similarity
index (Dyer 1978) designed for data sets which involve
both multiple species and multiple environmental variables. The total species dissimilarity is divided into

80

Fig. 2 Larval fish captures
at Los Lunas Restoration
site during sampling period

Individuals

60

C. lutrensis
C. carpio
H. amarus
P. promelas

40

20

0
05/23/2005 05/30/2005 06/06/2005 06/13/2005 06/20/2005 06/27/2005 07/04/2005


206

components with one (1) component being assigned to
each environmental variable or interaction of environmental variables. This similarity index provides a versatile and convenient tool for quantitatively comparing the
species composition of one (1) multispecies sample with
another (Dyer 1978).
A generalized linear mixed model (SAS ver. 9.3,
GLIMMIX procedure, SAS Institute Inc. Cary, NC)
was used to determine the relationship between fish,
environmental variables, and light trap orientation. A
scatterplot was generated for habitat orientation versus
each candidate explanatory variable. Then a linear
regression between habitat orientations versus each

explanatory variable was computed with fish species
included as a class variable and as an interaction with
the explanatory variable.

Results
During the descending limb of the hydrograph (MayJune, 2005) deployed larval fish light traps captured
Fig. 3 The distribution of
four Cyprinidae species
captured in larval fish light
traps as they relate to chemical site characteristics.
Definitions as follows;
cyp-car 0 Cyprinus carpio,
hyb-ama 0 Hybognathus
amarus, pim-pro 0 Pimephales promelas, and
cyp-lut 0 Cyprinella lutrensis. The labels po4 0 phosphate, do 0 dissolved
oxygen, nh4 0 ammonium.
Numbers 1–6 relate to light
trap and location

Environ Biol Fish (2012) 95:201–212

394 individuals representing four fish species from the
Family Cyprinidae (Pimephales promelas (n0228,
59%), Hybognathus amarus (n0123, 32%), Cyprinella lutrensis (n027, 7%) and Cyprinus carpio (n0
16, 2%) (Table 1 and Fig. 2). The highest captures
were obtained in leeward habitats (46%) followed by
perpendicular habitats (33%) and parallel habitats
(21%). While larval fish light traps only captured four
cyprinid species during the flood pulse a total of
nine (9) species were captured with seines during

drawdown. The larval fish light trap may be biased
towards Cyprinidae species, but may be due to
color of the light source (Kissick 1993; Marchetti
et al. 2004).
Results from the chemical site characteristics CCA
(Table 1 and Fig. 3) showed that PO4-P, DO, and NH4-N
were positively correlated and NO3-N was negatively
correlated. Cyprinella lutrensis was positively associated
with DO, PO4-P, NH4-N, and LT3. Pimephales promelas,
C. lutrensis, LT1 and LT3 were positively associated with
pH. Pimephales promelas, LT6, LT1, LT4, and LT2 were
positively associated with NO3-N. Results from the


Environ Biol Fish (2012) 95:201–212

physical site characteristics CCA (Fig. 4) showed that
I100 and I170 (light quanta) were positively correlated.
Cyprinella lutrensis, H. amarus, LT3 and LT5 were
positively associated with light.
Comparison of depth among LT locations showed
that LT4 was significantly different from LT2 (p0
0.005), LT5 (p00.02), and LT6 (p00.001) (Fig. 5).
Results from the general linear mixed model indicated
that only depth and velocity were significantly different among the environmental variables. Comparisons
of velocity among LT locations showed that LT5 was
significantly different from LT1 (p00.03) and LT3
(p00.03). Pimephales promelas, C. lutrensis, LT1
and LT3 were positively associated with depth. Cyprinus carpio, H. amarus, LT2, LT4-LT6 were positively associated with velocity. Cyprinella lutrensis and
LT3 were positively associated with temperature.

Results from Chao-Jaccard similarity index (Chao
et al. 2005) indicate that compositional similarity,
weighted on abundance, was highest in P. promelas
at 64% followed by H. amarus 33% C. lutrensis and

Fig. 4 The distribution of
four Cyprinidae species
captured in larval fish light
traps as they relate to physical site characteristics. Definitions as follows; cyp-car 0
Cyprinus carpio, hyb-ama 0
Hybognathus amarus, pimpro 0 Pimephales promelas,
and cyp-lut 0 Cyprinella
lutrensis. The labels I100
and I170 represent light
quanta at depths of 100 mm
and 170 mm respectively.
Numbers 1–6 relate to light
trap and location

207

C. carpio played a lesser role at 2.6 and 0.1% respectively. Results from (dis)similarity analysis (Dyer
1978) reveal that species composition between habitat
orientation and date was highest in H. amarus at 42%
followed by P. promelas 40% C. lutrensis and C. carpio
represented 9.5 and 8.5% respectively (Table 2).
Nutrient concentrations at the Los Lunas study
site varied considerably during the flood pulse (Table 2
and Fig. 5) and increased noticeably during final
stages of dewatering of the floodplain. These

results are similar to those reported by Valett et
al. (2005) who found that NO3-N and PO4-P concentrations increased dramatically during the initial
stages of flooding. Dissolved inorganic nitrate increased throughout the study ranging from 63 to
178 μg L-1; however, no significant differences
were observed. Mean ammonium concentration ranged
from 39 to 190 μg L-1, but no significant differences
were observed. Results only indicate significant differences in PO4-P concentrations between LT3 and LT6
(p00.05).


208

Environ Biol Fish (2012) 95:201–212
180

Fig. 5 Los Lunas floodplain nutrient concentrations during sampling period

160

NO3-N (mg/L)

140
120
100
80
60
40
20
0
180

160

PO4-P(mg/L)

140
120
100
80
60
40
20
0
180
160

NH4-N (mg/L)

140
120
100
80
60
40
20
0
05/23/05

Discussion
King et al. (2003) reported that few studies have
recorded larvae or juveniles using temporary floodplain habitats. However, results from the Los Lunas

site provide evidence that Hybognathus amarus as
Table 2 Captured cyprinid
fish at the restored Los Lunas
floodplain. Genus, species,
quantity, and similarity indices
for larval fish collected
during sampling period
(24 May- 28 June, 2005)

05/30/05

06/06/05

06/13/05

06/20/05

06/27/05

well as other ichthyofauna in the MRG do make use
of temporary floodplains where habitats are shallow
with lower water velocities. Since flood pulses in the
MRG are too short lived (5–10 days) (M. Porter, pers.
comm., 2009) and the naturalized flood pulses are
probably not favorable for successful spawning for

Species

n

%

(%) Chao-Jaccard
contribution

(%) Similarity
contribution

P. promelas

228

59

64

40.7

H. amarus

123

32

33.3

42.1

C.lutrensis

27

7

3.6

6.4

C. carpio

16

2

0.1

7.4

394

100


Environ Biol Fish (2012) 95:201–212

209

temperature sensitive species (Bayley 1995) combined, may be the leading causes for the decline in
recruitment of ichthyofauna in the MRG (Thorp et al.
1998). In semiarid and arid-zone rivers, hydrological

connectivity, unpredictable flooding combined with
low flows governs the "boom and bust" ecology of
these systems (Bunn et al. 2006) which ultimately
influences food availability for fish and other consumers (Balcombe et al. 2007). The importance of the
timing and duration of floods on the floodplain may
modulate water temperatures appropriate for spawning
of native fish species and may dictate the strength of
biotic responses to the flood (King et al. 2003).
27

Temperature (Co)

26
25
24
23
22
21
20
55
50

Depth (cm)

45
40
35
30
25
20

10

8

Velocity (cm/s)

Fig. 6 Los Lunas, NM
environmental variables
during sampling
period

Controlled flooding has occurred previously in the
MRG, and floods of similar magnitude and duration
have occurred prior to 2005 (e.g. 1993). However, yearly total surface area inundated and yearly overbank
surface area inundated in 2005 exceeded that of 1993
(USCOE 2010). While there have been 18 major floods
of similar duration and magnitude to that of 2005 between 1942 and 2009 at the USGS station 08330000
these floods were unable to access the floodplain due to
channelization and incision of the channel even at high
flows throughout the MRG (Figs. 6, 7 and 8).
The U.S. Fish and Wildlife Service (USFWS) initiated rescue and salvage operations beginning in 2001

6

4

2

0
05/23/05

05/30/05

06/06/05

06/13/05

06/20/05

06/27/05