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VetBooks.ir

Manuel Yúfera Editor

Emerging
Issues in Fish
Larvae Research
 Emerging Issues in Fish Larvae Research
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VetBooks.ir

Gilthead seabream larva (42 days after hatching). Photo: Bernd Ueberschär
 Manuel Yúfera
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Editor

Emerging Issues in Fish

Larvae Research

123
 Editor
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Manuel Yúfera
Instituto de Ciencias Marinas de Andalucía,
(ICMAN-CSIC), Campus Universitario
Río San Pedro s/n
Puerto Real, Cádiz
Spain

ISBN 978-3-319-73243-5 ISBN 978-3-319-73244-2 (eBook)

https://doi.org/10.1007/978-3-319-73244-2
Library of Congress Control Number: 2017962104

© Springer International Publishing AG 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made. The publisher remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Printed on acid-free paper

This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
 Preface
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Fish larvae are amazing organisms. They are among the smallest free-living forms
of vertebrates and exhibit fascinating growth potential. In fish aquaculture, all of the
phases of the life cycle from the fertilized egg to the adults are essential, and if one
of them fails, the whole production cycle fails. The larval phase, however, has
traditionally been considered the most sensitive period both in the wild and for
cultured fish. The vulnerability at this stage has been a powerful driving force for
fundamental and applied research. In this research line, the horizon is to know what
is necessary to allow the fish larvae to grow and develop properly and to become
healthy. The development of basic larviculture procedures based on feeding with
rotifers and Artemia in the 1970s and 1980s allowed a constant supply of fry to
support the increasing fish farming production during the last decades. Research on
fish larvae biology has continued since then and has been aimed at increasing our
basic knowledge and improving rearing methodologies. However, there are still
large knowledge gaps and the rearing process is far from optimal. For years, sci-
entific research has focused on relevant limiting factors seriously affecting survival
and growth, in a sequential manner. New species have been introduced by applying
similar protocols, and only some aspects related to feeding and nutrition have
received scientific attention. This simplistic empirical approach is insufficient for
understanding mechanisms for development and interrelations with the surrounding
rearing water. During the last decade, new analytical tools have opened up new
avenues for research in both physiology and the influence of environmental con-
ditions. In the past few years, books and specialized journals have published
reviews on the different disciplines related to fish larvae biology and aquaculture,
providing wide descriptions of main topics like development, pathologies, nutrition,
feeding, and physiology. These publications have increased our knowledge from a
textbook perspective. It may thus be considered that there is overall, good
knowledge on larval fish biology and on the current reality of larval rearing. Most
of this information, however, is based on a small number of species, and knowledge
in larval biology is advancing fast. Nowadays, there are new research ideas that
have implications not only for fish larvae but also for other vertebrates, and there
are new approaches to old problems, using omics methodologies, for example.

v
 vi Preface

The research effort on larvae is now moving beyond the strict interest for growing
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fish; it is also focusing on using them as model organisms for fundamental research.
Moreover, the numerous species being studied within the context of aquaculture
will help to uncover both the variability and the similarities within this diverse
group of vertebrates.
Instead of providing broad overviews covering general topics in larval fish
biology, this book presents examples of novel research in fish larvae with very
specific objectives. It refers to current advances derived from recent projects and a
doctoral thesis targeted at filling specific gaps in our knowledge. By representing
alternative points of view, the different chapters show how rearing and environ-
mental conditions affect physiology and developmental processes from a molecular
basis, and how these factors influence the final characteristics of late larvae and
juveniles. Overall, this book will point to recent findings on the importance of
environmental cycles, some specific nutrients, and the microbial environment on
developmental processes. There are more emerging topics of interest, but with these
few examples we hope to illustrate the dynamism of current research within
this field. These are exciting times for biologists and the discipline of biology—
especially when the target of the research is fish larvae.

Puerto Real, Spain Manuel Yúfera

 Contents
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1 Investigating Fish Larvae-Microbe Interactions in the 21st

Century: Old Questions Studied with New Tools . . . . . . . . . . . . . . 1
Ragnhild I. Vestrum, Birgit Luef, Torunn Forberg, Ingrid Bakke
and Olav Vadstein
2 Environmental Cycles and Biological Rhythms During Early
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Francisco Javier Sánchez-Vázquez and José Fernando López-Olmeda
3 The Digestive Function in Developing Fish Larvae and Fry. From
Molecular Gene Expression to Enzymatic Activity . . . . . . . . . . . . . 51
Manuel Yúfera, Francisco J. Moyano
and Gonzalo Martínez-Rodríguez
4 Variability in Digestive Enzyme Capacity in Early Stages of
Marine Fish Larvae: Ontogenetic Variations, Biorhythms,
Hormonal Control and Nutrient Sensing Mechanisms . . . . . . . . . . 87
Bernd Ueberschär, Carmen Navarro-Guillén, Ana Gomes,
Ivar Rønnestad, Carlos Rojas-Garcia, Inken Hanke,
Dagh Sommerfeld and Robert Tillner
5 Phospholipids in Marine Larval Rearing . . . . . . . . . . . . . . . . . . . . 131
Keshuai Li, Rolf Erik Olsen, Yang Jin and Yngvar Olsen
6 Fat-Soluble Vitamins in Fish: A Transcriptional Tissue-Specific
Crosstalk that Remains to be Unveiled and Characterized . . . . . . . 159
Ignacio Fernández, Paulo Gavaia, Maria J. Darias and Enric Gisbert
7 Nutritional Modulation of Marine Fish Larvae Performance . . . . . 209
Sofia Engrola, Cláudia Aragão, Luisa M. P. Valente
and Luís E. C. Conceição

vii
 viii Contents

8 Fish Pigmentation. A Key Issue for the Sustainable Development

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of Fish Farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Laura Cal, Paula Suarez-Bregua, Paloma Moran,
José Miguel Cerdá-Reverter and Josep Rotllant
9 Novel Aspects of Phosphate Endocrine Control: A Key Element
for the Long-Term Sustainability of Finfish Aquaculture . . . . . . . . 253
Paula Suarez-Bregua, Laura Cal, Pedro M. Guerreiro
and Josep Rotllant
10 Feeding and Development of Warm Water Marine Fish Larvae in
Early Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Jing Hu, Yibing Liu, Zhenhua Ma and Jian G. Qin
 Chapter 1
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Investigating Fish Larvae-Microbe

Interactions in the 21st Century:
Old Questions Studied with New Tools

Ragnhild I. Vestrum, Birgit Luef, Torunn Forberg, Ingrid Bakke

and Olav Vadstein

Abstract All animals need a mutualistic interaction with their microbiota for
proper development and functioning. Also for the fish-microbiota interaction con-
siderable research has been done, and especially for reared fish larvae this inter-
action is crucial for their viability. However, during the 1980s and 1990s a number
of findings revealed at that time current methods were not suitable for studying the
total microbial community and that data on composition of microbiota was biased.
Several recent methodological revolutions have boosted the possibilities for
addressing questions related to fish larvae-microbiota interactions that previously
lacked suitable tools for proper evaluation. These methodological achievements
include the development of experimental rearing systems including gnotobiotic
systems for fish, new visualization tools, and molecular “omics” tools for charac-
terizing the response of the host on a variety of levels and for characterizing both
composition and activity of fish microbiota. We present and review these tools and
give examples on how they have been used to improve our understanding of fish
larvae-microbiota interactions. With respect to understanding, this includes in
particular how the microbiota is established and maintained, what the functionality
of the microbiota is and how it affects fish health, and finally how we can apply this
knowledge for management of a healthy and beneficial microbiota in aquaculture
settings.

Keywords Microbiome Germ-free model systems
Imaging


Molecular methods Omics

R. I. Vestrum
B. Luef
T. Forberg
I. Bakke
O. Vadstein (&)

Department of Biotechnology and Food Science, NTNU Norwegian
University of Science and Technology, 7491 Trondheim, Norway
e-mail: [email protected]
Present Address:
T. Forberg
BioMar AS, Pirsenteret, 7010 Trondheim, Norway

© Springer International Publishing AG 2018 1

M. Yúfera (ed.), Emerging Issues in Fish Larvae Research,
https://doi.org/10.1007/978-3-319-73244-2_1
 2 R. I. Vestrum et al.

1.1 Introduction
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Mariculture has the possibility of being a major contributor to feeding a growing

human population, but this will require solutions to significant challenges (Duarte
et al. 2009). A major biological challenge will be to overcome bottlenecks with
regards to producing high quality juveniles suitable for the intensive cultivation
environment. We know that e.g. egg quality, nutrition and physiochemical condi-
tions contribute to the observed problems, but the well-known observation of poor
reproducibility between fully replicated tanks with equal genetic variability (e.g.
full sibling groups) cannot be explained by such factors (Vadstein et al. 1993). Even
though this is a complex problem with multiple dimensions, it is now demonstrated
beyond any doubt, that detrimental host-microbe interactions are a major cause of
these problems (Vadstein et al. 1993, 2013).
Since Robert Kock and Louis Pasteur’s major discoveries in the 1880s, the
relationship between man and their microbes has had primary focus on disease.
However, during the last 15 years a paradigm shift has occurred, as substantial
effort has been devoted to better understanding the role of our microbiota for health
and normal development. The last decennium has revealed a multitude of ways in
which the microbiota affects the normal development of animals, including fish
(Kanther and Rawls 2010; Sekirov et al. 2010). Most of this research has been
motivated by human health and therefore most studies have been done in man and
mammalian model organisms. It is now important to verify the generality of these
findings with other vertebrates and invertebrates, including fish as they have a core
role in the evolution of vertebrates.
Until 15 years ago quantitative and qualitative studies of microbes in aquacul-
ture systems were performed by culture-based techniques, and until the 60s it was a
general consensus that the knowledge of microbes associated with animals was
fairly well understood. Culture-dependent techniques mean that samples are serially
diluted and spread on so-called non-selective and selective medium on agar plates.
Quantitative estimates of densities are then given as colony forming units (CFU).
Since the late 1970s it has been shown that the discrepancy between total counts in
epifluorescence microscope and CFU is huge—typically a factor of 1000 for natural
waters. This is the first problem for culture-dependent methods. For some time it
was claimed that this difference was due to dead microbes, but it is now well
established that this can only account for a limited part of the discrepancy (e.g.
Karner and Fuhrman 1997). For man-made systems, like aquaculture systems, the
culturability can be considerably higher than in natural systems, but the difference
in the estimated abundance of microbes between the two methods is normally still
one order of magnitude. Studies during the last two decades have shown that the
difference between CFU and total counts is due to the selectivity of non-selective
agar—i.e. the majority of the microbes are still not possible to culture at laboratory
conditions (Hugenholtz et al. 1998). This is known as the “great plate anomaly”
(Hugenholtz 2002) and points to a second problem with culture-based methods; for
qualitative analysis of the composition of the microbes, the culture-based results are
 1 Investigating Fish Larvae-Microbe Interactions … 3

highly biased, as the culturability is dependent on phylogeny (Hugenholtz et al.

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1998). A third drawback of culture-based methods is that it is time consuming. Pure

cultures have to be established from colonies through several steps of sub-culturing,
and finally the pure cultures must be identified through a substantial number of
tests. For appropriate identification of species composition more than 100 pure
cultures should be characterized per sample (1% resolution = 1 isolate represents
1% of the population). In reality, few studies have characterized that many pure
cultures per sample, which is a clear indication of the labor involved. For one of the
authors of this chapter (O.V.) these methodological limitations felt overwhelming in
the early 90s. However, since then the revolution in method development has
caused another type of paradigm shift. This shift is largely due to the rapid
development in molecular biology and imaging methods. Method development,
possibilities, and applications are covered in detail below.
Based on the knowledge of the bias in culture-based studies one question is
unpleasant but impossible to disregard: Do we have to do it all over again? Or more
specific, is the cultivation-based knowledge we have on host-microbe interactions
in fish so biased that it counteracts the progress in our knowledge and under-
standing? The answers to these questions are not straight forward. First, all types of
“old” knowledge is probably not equally biased. Second, even though results from
high-throughput sequencing studies of fish microbiota are accumulating at a decent
speed, the amount of data is still too limited to do a proper evaluation of “old” data.
The severity of the problem is exemplified by the study of Fjellheim et al. (2012)
who found a negative correlation between quantification of bacteria associated with
larvae by plating on agar plates versus by quantitative PCR of rDNA. Thus we are
convinced that we have to do a lot of the work over again.
The aim of this chapter is to give an overview of new methods and how they can
be used to speed up and improve our understanding of fish-microbiota interactions.
With respect to understanding, this includes in particular how the microbiota is
established and maintained, what the functionality of the microbiota is and how it
affects fish health, and finally how we can apply this knowledge for management of
a healthy and beneficial microbiota in aquaculture settings. We will describe the
methods with focus on applications without going into technical details, and give
examples of applications for studying host-microbiota interactions.

1.2 Experimental Designs for Studying Fish-Microbiota

Interactions

Traditionally, experiments with fish are run in cultivation tanks. These are normally
just a downscaling of aquaculture production systems, and therefore realistic sys-
tems from an aquaculture perspective. However, the degree of experimental control
for fish-microbe studies is limited. Below we will describe different experimental
systems, going from high relevance/low control to low relevance/high control.
 4 R. I. Vestrum et al.

We want to stress that it is important to keep this range of experimental systems, as

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high control systems can be used to study mechanisms and hypotheses which then
can be verified in systems where the fish live a more normal life, i.e. low control
systems. We will try to highlight pros and cons within the different systems.

1.2.1 Traditional Rearing Tanks

Traditional rearing tanks can have variable volumes and can be run in traditional
flow-through systems (FTS) or as recirculating aquaculture systems (RAS), where
the water is reused after removal of waste and toxic substances. Tank volumes
depend on the size of the fish and the population size needed for experimental
reasons. From an ethical point of view and due to legislation the number of animals
used in an experiment should be as low as possible, but especially in applied
research the experimental systems should be relevant for a practical setting. This
might create a conflict between ethics and the practical relevance of new knowl-
edge. The use of FTS versus RAS has clear implications for fish-microbe studies, as
for RAS there will also be a recirculation of microbes. In some situations this can be
an advantage (maintaining the effect of the fish on the microbiota), whereas in other
cases a disadvantage (a high background concentration of microbes). FTS versus
RAS is therefore an important part of the experimental design. Traditional rearing
tanks have limited possibilities to control the microbiota in the rearing water. This
includes import and export of microbes, but particularly microbial growth in the
rearing tanks. For example, in studies with probiotics, it might be difficult to predict
the probiotic microbes’ ability to establish in the system as the competitive situation
with background microbes will vary depending on the species present and system
design. However, we have shown that it is possible to control and stabilize the
microbiota in rearing tanks through a selection regime against some microbes, and
favoring others (r- and K-selection) (Attramadal et al. 2014).

1.2.2 Systems with Rearing of Single Individuals

One of the drawbacks of using normal rearing tanks is that you have limited
possibilities for controlling import and export of microbes. Another problem is that
one fish may affect the other individuals in the same tank. This is important as
moribund individuals may infect healthy individuals, and that there is a continuous
sharing of microbes by defecation and re-ingestion (Reitan et al. 1998). A way out
of this problem is to rear single individuals in small units. Size and type of units
may vary from e.g. 40 ml in plastic cups (Forberg et al. 2016) to 2 ml in
24-multiwell plates (Fjellheim et al. 2010; Sandlund and Bergh 2008). Size of the
system may vary dependent on species and the length of the experiment. These
types of systems can be run without water exchange for yolk sac stages and early
 1 Investigating Fish Larvae-Microbe Interactions … 5
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Fig. 1.1 Survival of yolk-sac larvae of Atlantic cod (Gadus morhua) incubated together with
different bacteria, including background bacteria (positive control), a pathogen (negative control)
and three probiotics candidates, versus time. Each treatment included 72 individuals. Data from
Fjellheim et al. (2010)

first feeding (Fjellheim et al. 2010; Sandlund and Bergh 2008) or with an irregular
water exchange for longer experiments (Forberg et al. 2016). In systems with water
exchange, there are possibilities for maintaining some degree of control of the
microbes entering the system by e.g. adding a defined or described microbiota. It is,
however, important to keep in mind that the way by which the renewal of the water
takes place may create different types of selection regimes for the microbiota in the
rearing unit (e.g. continuous versus periodic dilution rate).
An example of the use of 24-multiwell plates with single individuals in 2 ml of
water is shown in Fig. 1.1. In this experiment, unfed yolk-sac larvae were exposed
to either probiotic candidates, a pathogen (negative control) or only background
bacteria (positive control). As shown in the figure, you get reliable results with a
limited number of individuals (in this case 72 individuals per treatment), and the
reproducibility is very good (unpublished results). This simple system easily
revealed that one probiotic candidate actually was pathogenic (ID 4-29). In systems
with rearing of single individuals, it is possible to reduce the number of individuals
used for the experiment to a minimum, and confidence intervals for the survival can
be calculated from surviving individuals and the total number of individuals based
on binomial statistics (Box et al. 1978). Moreover, chi-square and exact tests can be
used for statistical analysis.
The pros of these systems should be evident based on the information above.
The down side is mainly the simplicity of the system and for some species the
difficulty of rearing the fish for sufficiently long periods. The simplicity includes
lack of interactions between individuals of fish, which is an advantage for some
 6 R. I. Vestrum et al.

types of experiments. This clearly emphasizes the advantage of having a possibility

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for doing experiments in different types of systems, and selecting the systems based
on the aim of the study.

1.2.3 Germ-Free and Gnotobiotic Systems

If traditional rearing tanks is on one end of the experimental system scale, germ-free
and gnotobiotic systems are on the other end. A germ-free system has no microbes
at all, and in a gnotobiotic system the biota in the system is known (= gnotos) and
predefined. This can only be achieved by first generating germ-free test animals or
fish, which are then deliberately colonized by one or several bacterial strains known
to the researcher. Gnotobiotic systems were first developed for mammals such as
rats, but also for fish several gnotobiotic systems have been published (Table 1.1).
In general, the procedure of creating germ-free fish involves a first step with surface
disinfection of the eggs and subsequent hatching in an environment without
microbes. The subsequent rearing under germ-free or gnotobiotic conditions may be
in the presence or absence of antibiotics. When rearing the fish with antibiotics, you
can only use antibiotic resistant bacteria and there is also a possibility of negative
effects on larvae due to long term exposure to antibiotics (Moullan et al. 2015). As
documented in Table 1.1, many different techniques have been used to surface
disinfect the eggs, and gnotobiotic systems have been attempted for several fish
species.
The main advantage of using these systems is the high degree of control of the
microbial environment. This is appealing for many types of experiments with a
reductionistic approach—a strategy with a long history of success in natural sci-
ence. The main disadvantage of germ-free and gnotobiotic systems is the com-
plexity in performing these experiments. A reproducible method for achieving
germ-free larvae is needed, and avoiding contamination during the experiment is
also crucial (i.e. maintain them germ-free or gnotobiotic). In the cases were the fish
is fed live prey, the prey also needs to be germ-free. When using Artemia as live
feed it is possible to hatch them from disinfected cysts, and this makes the process
more straightforward. However, when using e.g. rotifers a germ-free culture has to
be established, they have to be reared germ-free, and therefore must be fed
germ-free feed (microalgae or yeast). Moreover, this production line has to be kept
throughout the germ-free or gnotobiotic fish experiment. One problem not given
much attention in the literature, is the maintenance of the gnotobiotic condition.
When only one microbe is added it is a presence/absence problem, but when several
species are added in a given ratio it is not straight forward to maintain this ratio.
This has to do with the selection regimes in the rearing unit and due to renewal of
water (see also Sect. 1.2).
Results generated from one fish species may not be easily transferrable to
another, as germ-free zebrafish (Danio rerio) seem to be less developed than
conventional fish (Rawls et al. 2004), while germ-free sea bass larvae have more
Another random document with
no related content on Scribd:
 Crotophaga ani. Linn.
Petit Bout-de-Petun, pl. enl. 102. f. 2.

Rio de Janeiro. May. The stomach of several specimens contained

remains of numerous Orthopterous, and some Coleopterous insects.
Order GYRATONES. Bonap.
 1. Columba Fitzroyii. King.
Columba Fitzroyii, King., in Proc. of Zool. Soc. part 1, 1830, p. 14.
Columba denisea, Temm. pl. col. 502.
Columba araucana, Less. Voy. de Coqu. pl. 40.?

Peninsula of Tres Montes. Lat. 46° S. January. Captain King’s

specimens were obtained at Chiloe, three degrees northward. I
procured other specimens near Valparaiso. This bird therefore
frequents dry rocky land, and damp impervious forests.
 Birds. Pl. 46.

Zenaida Galapagoensis.
2. Columba loricata. Licht. Vög. Verz. s. 67.
Columba gymnophthalmus, Temm., Pig. i. 18.
—— leucoptera, Pr. Max. Reise, 2, p. 242.
—— picazuro, Temm. Pig. p. 111.
Picazuro, Azara, Voy. No. 317.

Frequents in large flocks the fields of Indian corn in the

neighbourhood of Maldonado. Legs dull “carmine red.” This,
probably, is the representative on the eastern side of the Andes of the
foregoing or Chilian species.
 1. Zenaida aurita. G. R. Gray.
Columba aurita, Temm. Pig. p. 60. Wagl. sp. 70.

I procured specimens of this bird at Maldonado (where it was very

abundant) in La Plata, and at Valparaiso in Chile.
 2. Zenaida Galapagoensis. Gould.
Plate XLVI.
Z. vertice, cervice, dorso caudæque tegminibus obscurè fuscis
vinaceo-tinctis; dorso nigro-guttato; alarum tegminibus fuscis,
plumâ singulâ pallidè vinaceo-fusco terminatâ, pogonii
utriusque margine, maculâ oblongâ magnâ nigrâ, lineâ albâ
separatâ; remigibus primariis et secundariis nigrescenti-fuscis,
cinerascenti-albo angustè marginatis; caudâ fuscescenti cinereo
ad apicem fasciâ latâ irregulari nigra; loris lineâque angustâ
supra et infra oculari nigris pallidè fusco mixtis; gulâ
pectoreque vinaceis, colli lateribus ærato tinctis; crisso,
caudæque tegminibus inferioribus cinerascentibus, rostro nigro,
pedibus rufescenti aurantiacis.
Long. tot. 8½ unc.; alæ, 5¼; caudæ, 3¼; tarsi, ⅞; rostri, 1.
Crown of the head and back of the neck, dark chocolate brown,
with a vinous tinge; back and tail-coverts the same, the former
spotted with black; wing-coverts brown, each feather having a
large oblong spot of black on the margin of either web, separated
by a line of white, and tipped with light vinous brown, the white
predominating on the larger coverts, primaries and secondaries
blackish brown, finely edged with greyish white; tail brownish
grey, crossed near the extremity with a broad irregular band of
black; lores and a narrow line above and beneath the eye black,
interrupted with light brown: throat and chest rich vinous,
glossed on the sides of the neck with metallic bronze, and fading
into greyish on the vent and under tail-coverts; bill black; feet
reddish orange.
Habitat, Galapagos Archipelago. (Sept. and Oct.)
This species may at once be distinguished from the Z. aurita, by
the redder tint of its breast,—the greater number of black marks on
the wing coverts and back—the outer half of some of the feathers on
the wing coverts being white—the marks on the under side of the tail
being grey (instead of white as in the Z. aurita) and by the larger size
of its beak.
This dove is one of the most abundant birds in the Archipelago. It
frequents the dry rocky soil of the low country, and often feeds in the
same flock with the several species of Geospiza. It is exceedingly
tame, and may be killed in numbers. Formerly it appears to have
been much tamer than at present. Cowley,[21] in 1684, says that the
“Turtle doves were so tame that they would often alight upon our
hats and arms, so as that we could take them alive: they not fearing
man, until such time as some of our company did fire at them,
whereby they were rendered more shy.” Dampier[22] (in the same
year) also says that a man in a morning’s walk might kill six or seven
dozen of these birds. At the present time, although certainly very
tame, they do not alight on people’s arms; nor do they suffer
themselves to be killed in such numbers. It is surprising that the
change has not been greater;—for these islands during the last
hundred and fifty years, have been frequented by buccaneers and
whalers; and the sailors, wandering through the woods in search of
tortoises, take delight in knocking down the little birds.
 3. Zenaida Boliviana. G. R. Gray.
Columba Boliviana, D’Orb. & Lafr. Mag. de Zool. 1836. Ois. p. 33. pl. 75.

My specimen was obtained (end of August) at Valparaiso.

 1. Columbina strepitans. Spix.
(Av. pl. 75, f. 1.)

I procured specimens at Maldonado (where it was not common),

on the banks of the Plata, and at Rio Negro, in Northern Patagonia.
 2. Columbina talpacoti. G. R. Gray.
Columba Talpacoti, Temm. Pig. p. 22. t. 12.
Columbina Cabocolo, Spix, Av. pl. 75a. f. 1.
Le Pigeon rougeatre, Azara, No. 323.

My specimens were obtained at Rio de Janeiro.

 1. Attagis Falklandica. G. R. Gray.
Tetrao Falklandicus, Gmelin, Syst. 1. 762.
La Caille des Isles Malouines, Buff. pl. enl. 222.
Coturnix Falklandica, Bonn. Ency. Meth. Orn. 220.
Perdix Falklandica, Lath. Ind. Orn. 11, 652.
Ortyx Falklandica, Steph. Shaw’s Zool. xi. 386.

This bird is not uncommon on the mountains in the extreme

southern parts of Tierra del Fuego. It frequents, either in pairs or
small coveys, the zone of alpine plants above the region of forest. It is
not very wild, and lies very close on the bare ground.
 2. Attagis gayii. Less.
Attagis Gayii, Less. Cent. Zool. pl. 47, p. 155.

A specimen was given me, which was shot on the lofty Cordillera of
Coquimbo, only a little below the snow-line. At a similar height, on
the Andes, behind Copiapo, which appear so entirely destitute of
vegetation, that any one would have thought that no living creature
could have found subsistence there, I saw a covey. Five birds rose
together, and uttered noisy cries; they flew like grouse, and were very
wild. I was told that this species never descends to the lower
Cordillera. These two species, in their respective countries, occupy
the place of the ptarmigan of the northern hemisphere.
 Tinochorus rumicivorus. Eschsch.
Thinocorus rumicivorus, Eschsch. Zool. Atl. pl. 2.
Tinochorus Eschscholtzii, Less. Cent. Zool. pl. 50.

This very singular bird, which in its habits and appearance

partakes of the character both of a wader and one of the gallinaceous
order, is found wherever there are sterile plains, or open dry pasture
land, in southern South America. We saw it as far south as the inland
plains of Patagonia at Santa Cruz, in lat. 50°. On the western side of
the Cordillera, near Concepcion, where the forest land changes into
an open country, I saw this bird, but did not procure a specimen of it:
from that point throughout Chile, as far as Copiapo, it frequents the
most desolate places, where scarcely another living creature can
exist: it thus ranges over at least twenty-three degrees of latitude. It
is found either in pairs or in small flocks of five or six; but near the
Sierra Ventana I saw as many as thirty and forty together. Upon
being approached they lie close, and then are very difficult to be
distinguished from the ground; so that they often rise quite
unexpectedly. When feeding they walk rather slowly, with their legs
wide apart. They dust themselves in roads and sandy places. They
frequent particular spots, and may be found there day after day.
When a pair are together, if one is shot, the other seldom rises; for
these birds, like partridges, only take wing in a flock. In all these
respects, in the muscular gizzard adapted for vegetable food, in the
arched beak and fleshy nostrils, short legs, and form of foot, the
Tinochorus has a close affinity with quails. But directly the bird is
seen flying, one’s opinion is changed; the long pointed wings, so
different from those in the gallinaceous order, the high irregular
flight, and plaintive cry uttered at the moment of rising, recall the
idea of a snipe. Occasionally they soar like partridges when on the
wing in a flock. The sportsmen of the Beagle unanimously called it
the short-billed snipe. To this genus, or rather to that of the
sandpiper, it approaches, as Mr. Gould informs me, in the shape of
its wing, the length of the scapulars, the form of the tail, which
closely resembles that of Tringa hypoleucos, and in the general
colour of the plumage. The male bird, however, has a black mark on
its breast, in the form of a yoke, which may be compared to the red
horseshoe on the breast of the English partridge. Its nest is said to be
placed on the borders of lakes, although the bird itself is an
inhabitant of the parched desert. I was told that the female lays five
or six white eggs, spotted with red. I opened the stomachs of many
specimens at Maldonado, and found only vegetable matter, which
consisted of chopped pieces of a thick rushy grass, and leaves of
some plant, mixed with grains of quartz. The contents of the
intestine and the dung were of a very bright green colour. At another
season of the year, and further south, I found the craw of one full of
small seeds and a single ant. Those which I shot were exceedingly fat,
and had a strong offensive game odour; but they are said to be very
good eating, when cooked. Pointers will stand to them. In the
Appendix Mr. Eyton has given an anatomical description of this bird,
which partly confirms that affinity both to the Grallatores and
Razores, which is so remarkable in its habits and external
appearance.
 Chionis albâ. Forst.
Shaw’s Nat. Miscel. pl. 481.

I opened the stomach of a specimen killed at the Falkland Islands,

and found in it small shells, chiefly Patellæ, pieces of sea-weed, and
several pebbles. The contents of the stomach and body smelt most
offensively. Forster remarked this circumstance; but since his time,
other observers, namely, Anderson, Quoy, Gaimard, and Lesson
(Manuel d’Ornithologie, tom. ii, p. 342) have found that this is not
always the case, and they state that they have actually eaten the
Chionis. I was not aware of these observations, but independently
was much surprised at the extraordinary odour exhaled. We, like
other voyagers in the Antarctic seas, were struck at the great distance
from land, at which this bird is found in the open ocean. Its feet are
not webbed, its flight is not like that of other pelagic birds, and the
contents of its stomach, and structure of legs, show that it is a coast-
feeder. Does it frequent the floating icebergs of the Antarctic ocean,
on which sea-weed and other refuse is sometimes cast?
 1. Nothura major. Wagl.
Nothura major, Wagl. Syst. Av. p. sp. 4.
Tinamus major, Spix, Av. pl. 80.

These birds are very common on the northern shores of the Plata.
They do not rise in coveys, but generally by pairs. They do not
conceal themselves nearly so closely as the English partridge, and
hence great numbers may be seen in riding across the open grassy
plains. Note, a shrill whistle. It appears a very silly bird: a man on
horseback, by riding round and round in a circle, or rather in a spire,
so as to approach closer each time, may knock on the head almost as
many as he pleases. The more common method is to catch them with
a running noose, or little lazo, made of the stem of an ostrich’s
feather, fastened to the end of a long stick.[23] A boy on a quiet old
horse will frequently thus catch thirty or forty in a day. The flesh of
this bird, when cooked, is most delicately white, but rather tasteless.
The egg of this species, I believe, closely resembles that of the two
following.
 2. Nothura minor. Wagl.
Nothura minor, Wagl. Syst. Av. p. sp. 4.
Tinamus minor, Spix, Av. Br. pl. 82.

I procured a specimen of this bird at Bahia Blanca, in northern

Patagonia, where it frequented the sand-dunes and the surrounding
sterile plains. Its habits appear similar to those of the N. major, but
it lies closer and does not so readily take to the wing. It is the
smallest of the species mentioned in this work, and its plumage is
less distinctly spotted. The egg of this bird is described below. Spix’s
specimens were obtained at Tijuco in Brazil. The figure in his work
on the Birds of Brazil, differs slightly from mine, in being less
marked on the breast.
 3. Nothura perdicaria. G. R. Gray.
Crypturus perdicarius, Kittlitz, Vögel von Chili.

This species closely resembles, in its general appearance and

habits, the N. major, of which probably it is the analogue on the
western side of the Cordillera. It is larger and has a considerably
longer beak than the N. major; its breast is not spotted, and its
abdomen has a less fulvous tinge. The N. perdicarius runs on the
open ground, generally a pair together, in the same unconcealed
manner, as its analogue, and does not readily lie close. Flight similar,
but on rising it utters a shriller whistle, of a different tone. It does not
appear to be so easily caught as the Plata species. It is tolerably
abundant in all parts of Chile, as far north as the valley of Guasco;
but I was assured, that it has never been seen in the valley of
Copiapo, although only seventy miles north of Guasco, and of a
similar character. The egg is very glossy and of a peculiar colour,
which, according to Werner’s nomenclature, is a palish chocolate
red: length in longer axis 2·07 of an inch; shorter axis 1·495 of an
inch. The egg of the N. minor is of a similar colour, but a shade paler,
and rather smaller; its length being 1·815, and its transverse
diameter 1·3 of an inch.
 Rhynchotus rufescens. Wagl.
Rhynchotus rufescens, Wagl. Av. Syst.
Tinamus rufescens. Temm. Gall. iii. p. 552.
Rhynchotus fasciatus. Spix, Av. Br. pl. 76.
Cryptura Guaza. Vieill.
Crypturus rufescens. Licht. Vög. Verz. s. 67.

My specimens were procured at Maldonado, where it is a much

rarer bird than the Nothura major; I met with it also in the sterile
country near Bahia Blanca. At Maldonado it frequented swampy
thickets on the borders of lakes. It lies very close, and is unwilling to
rise, but often utters, whilst on the ground, a very shrill whistle.
When on the wing, it flies to a considerable distance. Several are
generally found together, but they do not rise at the same instant,
like a covey of partridges. Flesh, when cooked, perfectly white. Spix’s
specimens were procured in the country between St. Paul’s and
Minas Geraes; so that this bird, as well as the Nothura minor, has a
considerable range.
Order—CURSORES. Temm.
 1. Rhea Americana. Lath.
This bird is well known to abound on the plains of La Plata. To the
north it is found, according to Azara, in Paraguay, where, however, it
is not common; to the south its limit appears to be from 42° to 43°. It
has not crossed the Cordillera; but I have seen it within the first
range of mountains on the Uspallata plain, elevated between six and
seven thousand feet. The ordinary habits of the ostrich are well
known. They feed on vegetable matter, such as roots and grass; but
at Bahia Blanca, I have repeatedly seen three or four come down at
low water to the extensive mud-banks which are then dry, for the
sake, as the Gauchos say, of catching small fish. Although the ostrich
in its habits is so shy, wary, and solitary, and although so fleet in its
pace, it falls a prey, without much difficulty, to the Indian or Gaucho
armed with the bolas. When several horsemen appear in a semicircle,
it becomes confounded, and does not know which way to escape.
They generally prefer running against the wind; yet at the first start
they expand their wings, and like a vessel make all sail. On one fine
hot day I saw several ostriches enter a bed of tall rushes, where they
squatted concealed, till quite closely approached. It is not generally
known that ostriches readily take to the water. Mr. King informs me
that in Patagonia, at the Bay of San Blas and at Port Valdes, he saw
these birds swimming several times from island to island. They ran
into the water, both when driven down to a point, and likewise of
their own accord, when not frightened: the distance crossed was
about 200 yards. When swimming, very little of their bodies appear
above water, and their necks are extended a little forward: their
progress is slow. On two occasions, I saw some ostriches swimming
across the Santa Cruz river, where it was about four hundred yards
wide, and the stream rapid. Captain Sturt,[24] when descending the
Murrumbidgee, in Australia, saw two emus in the act of swimming.
The inhabitants who live in the country readily distinguish, even at
a distance, the male bird from the female. The former is larger and
darker coloured,[25] and has a larger head. The ostrich, I believe the
cock, emits a singular, deep-toned, hissing note. When first I heard
it, standing in the midst of some sand-hillocks, I thought it was made
by some wild beast, for it is a sound that one cannot tell whence it
comes, or from how far distant. When we were at Bahia Blanca in the
months of September and October, the eggs were found, in
extraordinary numbers, all over the country. They either lie scattered
single, in which case they are never hatched, and are called by the
Spaniards, huachos, or they are collected together into a shallow
excavation, which forms the nest. Out of the four nests which I saw,
three contained twenty-two eggs each, and the fourth twenty-seven.
In one day’s hunting on horseback sixty-four eggs were found; forty-
four of these were in two nests, and the remaining twenty scattered
huachos. The Gauchos unanimously affirm, and there is no reason to
doubt their statement, that the male bird alone hatches the eggs, and
for some time afterwards accompanies the young. The cock when on
the nest lies very close; I have myself almost ridden over one. It is
asserted that at such times they are occasionally fierce, and even
dangerous, and that they have been known to attack a man on
horseback, trying to kick and leap on him. My informer pointed out
to me an old man, whom he had seen much terrified by one chasing
him. I observe, in Burchell’s Travels in South Africa, that he remarks,
“having killed a male ostrich, and the feathers being dirty, it was said
by the Hottentots to be a nest bird.” I understand that the male emu,
in the Zoological Gardens, takes care of the nest: this habit therefore
is common to the family.[26]
The Gauchos unanimously affirm that several females lay in one
nest. I have been positively told, that four or five hen birds have been
actually watched and seen to go, in the middle of the day, one after
the other, to the same nest. I may add, also, that it is believed in
Africa, that two or more females lay in one nest.[27] Although this
habit at first appears very strange, I think the cause may be
explained in a simple manner. The number of eggs in the nest varies
from twenty to forty, and even to fifty; and according to Azara to
seventy or eighty. Now although it is most probable, from the
number of eggs found in one district being so extraordinarily great,
in proportion to that of the parent birds, and likewise from the state
of the ovarium of the hen, that she may in the course of the season
lay a large number, yet the time required must be very long. Azara
states,[28] that a female in a state of domestication laid seventeen
eggs, each at the interval of three days one from another. If the hen
were obliged to hatch her own eggs, before the last was laid, the first
probably would be addled; but if each laid a few eggs at successive
periods, in different nests, and several hens, as is stated to be the
case, combined together, then the eggs in one collection would be
nearly of the same age. If the number of eggs in one of these nests is,
as I believe, not greater on an average than the number laid by one
female in the season, then there must be as many nests as females,
and each cock bird will have its fair share of the labour of incubation;
and this during a period when the females probably could not sit, on
account of not having finished laying.[29] I have before mentioned the
great numbers of huachos, or scattered eggs; so that in one day’s
hunting the third part found were in this state. It appears odd that so
many should be wasted. Does it not arise from some difficulty in
several females associating together, and in finding a male ready to
undertake the office of incubation? It is evident that there must at
first be some degree of association, between at least two females;
otherwise the eggs would remain scattered at distances far too great
to allow of the male collecting them into one nest. Some authors
believe that the scattered eggs are deposited for the young birds to
feed on. This can hardly be the case in America, because the huachos,
although often found addled and putrid, are generally whole.
 Birds. Pl. 47.

Rhea Darwinii.
 2. Rhea Darwinii. Gould.
Plate XLVII.
Gould, in Proceedings of Zoological Soc. 1837, p 35.

R. pallide fusca, plumâ singulâ distinctâ semilunari notâ candidâ

terminatâ; capite collo, femoribusque pallidioribus: rostri
culmine augusti, ad apicem latiore, frontes plumis parvis setosis
anticè directis et supra nares extensis; tarsi lateribus in
dimidiam partem plumis parvis mollibus tectis; tarso ⅔ anticè
posticeque toto, squamis reticulatis tecto.
Long. tot. 52 unc.; alæ, 30; tarsi, 11; rostri, 2.
The whole of the plumage light brown, each feather with a decided
crescent-shaped mark of pure white at the extremity; head, neck,
and thighs lighter; base of the neck blackish; culmen of the bill
narrow, becoming a little broader towards apex; front with small
bristly feathers, pointing forwards and reaching over the nostrils.
Tarsus with small downy feathers on sides, extending half way
downwards; upper two-thirds of front of tarsus, and whole
hinder side, with reticulated scales.
Habitat, Eastern Patagonia (Lat. 40° S. to 54° S.)
This species, which Mr. Gould, in briefly characterizing it at a
meeting of the Zoological Society, has done me the honour of calling
after my name, differs in many respects from the Rhea Americana. It
is smaller, and the general tinge of the plumage is a light brown in
place of grey; each feather being conspicuously tipped with white.
The bill is considerably smaller, and especially less broad at its base;
the culmen is less than half as wide, and becomes slightly broader
towards the apex, whereas in the R. Americana it becomes slightly
narrower; the extremity, however, of both the upper and the lower
mandible, is more tumid in the latter, than in the R. Darwinii.
 R. R.
Darwinii. Americana,
inches inches
Length of beak, from edge of membrane at
base to the apex 2 2⁶⁄₈
Length, from anterior margin of eye to apex 3⁴⁄₁₂ 5⁶⁄₁₂
Width of upper mandible, measured across
middle of nostrils 1¹⁄₂₀ 1⁶⁄₂₀

The skin round and in front of the eyes is less bare in R. Darwinii;
and small bristly feathers, directed forwards, reach over the nostrils.
The feet and tarsi are nearly of the same size in the two species. In
the R. Darwinii, short plumose feathers extend downwards in a
point on the sides of the tarsus, for about half its length. The upper
two-thirds of the tarsus, in front, is covered with reticulated scales in
place of the broad transverse band-like scales of the R. Americana;
and the scales of the lower third are not so large as in the latter. In
the R. Darwinii the entire length of the back of the tarsus is covered
with reticulated scales, which increase in size from the heel upwards:
in the common Rhea, the scales on the hinder side of the tarsus are
reticulated only on the heel, and about an inch above it; all the upper
part consisting of transverse bands, similar to those in front.
The first notice I received of this species was at the Rio Negro, in
Northern Patagonia, where I repeatedly heard the Gauchos talking of
a very rare bird, called Avestruz Petise. They described it as being
less than the common ostrich (which is there abundant), but with a
very close general resemblance. They said its colour was dark and
mottled, and that its legs were shorter, and feathered lower down
than those of the common ostrich. It is more easily caught by the
bolas than the other species. The few inhabitants who had seen both
kinds, affirmed that they could distinguish them apart, from a long
distance. The eggs, however, of the small species appeared more
generally known, and it was remarked with surprise, that they were
very little less than those of the common Rhea, but of a slightly
different form, and with a tinge of pale blue. Some eggs which I
picked up on the plains of Patagonia, agree pretty well with this
description; and I do not doubt are those of the Petise. This species
occurs most rarely in the neighbourhood of the Rio Negro; but about
a degree and a half further south they are tolerably abundant. One
Gaucho, however, told me he distinctly recollected having seen one,
many years before, near the mouth of the Rio Colorado, which is
north of the Rio Negro. They are said to prefer the plains near the
sea. When at Port Desire in Patagonia (Lat. 48°), Mr. Martens shot
an ostrich; I looked at it, and from most unfortunately forgetting at
the moment, the whole subject of the Petises, thought it was a two-
third grown one of the common sort. The bird was skinned and
cooked before my memory returned. But the head, neck, legs, wings,
many of the larger feathers, and a large part of the skin, had been
preserved. From these a very nearly perfect specimen has been put
together, and is now exhibited in the museum of the Zoological
Society. M. A. D’Orbigny, a distinguished French naturalist, when at
the Rio Negro, made great exertions to procure this bird, but had not
the good fortune to succeed. He mentions it in his Travels (vol. ii. p.
76.) and proposes (in case, I presume, of his obtaining a specimen at
some future time, and thus being able to characterize it,) to call it
Rhea pennata. A notice of this species was given long since (A.D.
1749) by Dobrizhoffer, in his account of the Abipones (vol. i. Eng.
Trans. p. 314). He says, “You must know, moreover, that Emus differ
in size and habits in different tracts of land; for those that inhabit the
plains of Buenos Ayres and Tucuman are larger, and have black,
white, and grey feathers; those near to the Strait of Magellan are
smaller, and more beautiful, for their white feathers are tipped with
black at the extremity, and their black ones in like manner terminate
in white.”
Among the Patagonian Indians in the Strait of Magellan, we found
a half-bred Indian, who had lived some years with this tribe, but had
been born in the northern provinces. I asked him if he had ever
heard of the Avestruz Petise? He answered by saying, “Why there are
none others in these southern countries.” He informed me that the
number of eggs in the nest of the Petise is considerably less than with
the other kind, namely, not more than fifteen on an average; but he
asserted that more than one female deposited them. At Santa Cruz
we saw several of these birds. They were excessively wary: I think
they could see a person approaching, when he was so far off as not to
distinguish the ostrich. In ascending the river few were seen; but in
our quiet and rapid descent, many, in pairs and by fours or fives,
were observed. It was remarked by some of the officers, and I think
with truth, that this bird did not expand its wings, when first starting
at full speed, after the manner of the northern kind. The fact of these
ostriches swimming across the river has been mentioned. In
conclusion, I may repeat that the R. Americana inhabits the eastern
plains of S. America as far as a little south of the Rio Negro, in lat.
41°, and that the R. Darwinii takes its place in Southern Patagonia;
the part about the Rio Negro being neutral territory. Wallis saw
ostriches at Bachelor’s river (lat 53° 54′), in the Strait of Magellan,
which must be the extreme southern possible range of the Petise.
Order—GRALLATORES.