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First published online August 3, 2006
Journal of Experimental Biology 209, 3055-3061 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02336
Effect of parental age and associated size on fecundity, growth and survival in the yellow seahorse Hippocampus kuda
1 Institute for Problems of Cryobiology and Cryomedicine of the National
Academy of Sciences of the Ukraine, 23 Pereyaslavskaya Street, Kharkov 310015,
Ukraine
2 Institute of Zoology, Zoological Society of London, Regent's
3 Park, London NW1 4RY, UK and 3Zoological Society of London,
Regent's Park, London NW1 4RY, UK
* Author for correspondence (e-mail: Bill.holt{at}ioz.ac.uk)
Accepted 16 May 2006
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Key words: yellow seahorse, Hippocampus kuda, post-natal growth, attachment site, pouch, embryo, Barker hypothesis, foetal origins hypothesis
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Introduction |
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). Much current
data support the hypothesis that conditions in the uterus effectively
`programme' postnatal growth dynamics and the efficiency of fundamentally
important physiological systems such as cardiovascular and pancreatic function
(Barker, 1999;
Kwong et al., 2000). Research
in humans and experimental mammalian species has linked the prenatal and
postnatal effects within a theoretical framework known as the `foetal origins'
or Barker's hypothesis. Here we present a preliminary investigation of a
radically different, but analogous, reproductive system to see whether any
parallels are discernible. We consider the possibility that similar principles
might apply to seahorses that are famous for their elaborate paternal care
system, in which embryonic growth and development occurs exclusively within
the brood pouch of the male. In this study we show that there is an effect of
age and associated size on fecundity in the yellow seahorse, Hippocampus
kuda and suggest that this effect is related to aspects of embryonic
development.
Even though Syngnathidae (seahorses and pipefishes) embryos possess a yolk
sac and are nutritionally independent of the male
(Azzarello, 1991), aspects of
pouch physiology and size may still influence the future growth and
development of offspring. In seahorses the female deposits eggs inside the
brood pouch during mating, the pouch opening closes immediately after mating
and the fertilised eggs develop to term within an enclosed environment. Pouch
physiology and its interaction with embryos bear some resemblance to placental
function in mammals; the embryos become embedded within depressions of the
interior lining of the brood pouch
(Carcupino et al., 1997). Given
that the pouch is sealed during gestation, it has been suggested that embryos
depend upon pouch function for gaseous exchange, removal of waste products
(Carcupino et al., 1997) and
osmoregulation (Linton and Soloff,
1964; Quast and Howe,
1980).
In this study we examine the effects of several sources of variation on the
early postnatal growth characteristics of seahorses and experimentally
investigate the hypothesis that age-associated size of parents is a
significant determinant of offspring growth and viability. We propose that
variability in the efficacy of the embryo-pouch interactions may lead to
differences that affect future growth and development, thereby causing
embryonic heterogeneity within single broods. This is in addition to other
sources of variation in embryonic size that can arise from differences in the
size of eggs [usually positively correlated with female size
(Berglund et al., 1986b) and
pouch size, which is related to the density of competing embryos
(Ahnesjo, 1992;
Ahnesjo, 1996;
Watanabe and Watanabe,
2002)].
Seahorses present several experimental limitations that unavoidably
confound studies of this type. We were unable to disentangle male size from
the effects of female size because the seahorses prefer to mate with
individuals of their own size (Foster and
Vincent, 2004) and because age and size are strongly correlated,
it is also difficult to separate size effects from age effect. Nevertheless,
there are also some advantages: as seahorses produce large broods it is also
possible to examine heterogeneity of growth within, as well as between,
broods. While heterogeneity may reflect differences in egg quality, we also
consider the possibility that it may also arise through differences in the
positions in the pouch.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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)
suggests that the name H. kuda has often been used to describe any
Indo-Pacific seahorse that could not readily be identified. For the purpose of
this ex situ study, however, the use of this population offered a
convenient means of generating animals of known ages and sizes.
Culture and breeding of seahorses
Yellow seahorses, H. kuda, were housed in two 560 l seawater
aquaria (to keep the two age groups separate; see below); each tank was
separated into compartments by fine mesh dividers. Water was maintained at a
constant temperature (26°C) and tanks were maintained under a 12 h:12 h
L:D photoperiod. This photoperiod regime was adopted because this population
of H. kuda has been shown to breed throughout the year; this
indicated that photoperiod is not an important determinant of breeding
activity. Furthermore, the Seahorse Manual
(Bull and Mitchell, 2002)
recommends this photoperiod for six other seahorse species. Adults were fed
four times per day with live and frozen food (Artemia and
Mysis). Although embryonic seahorses possess a yolk sac while in the
pouch, this normally disappears within 1 day of birth and juveniles are
dependent on external nutritional sources. Juveniles were therefore fed four
times per day with Artemia nauplii enriched with algae
(Nannachloropsis and Spirulina). Normal feeding behaviour
was observed in all groups of offspring from 1 day after birth.
To prevent overcrowding in the tanks, the number offspring populations within any single tank had to be reduced to a maximum of 50 individuals at about 4 weeks of age. This population reduction step only affected the old-couples cohort; the young-couples cohort never exceeded 50 individuals after 4 weeks.
Experimental design
Two groups of breeding pairs were established using a captive-bred line of
H. kuda. One group consisted of adults that had already bred more
than once and were over 8 months old [N=6 `old couples' (OC)], the
other group [N=6 `young couples' (YC)] consisted of 3- to 4-month-old
animals that had never previously bred. Mass measurements of the adults were
made immediately after the birth of each cohort; postnatal body mass (mean
± s.d.) of OC males and females was 8.43±2.62 g and
6.00±1.85 g, respectively, and of YC males and females,
3.76±1.10 g and 2.46±0.87 g, respectively. The Zoological
Society of London Ethics Committee approved the experimental design, handling
and measurement techniques.
Measurement techniques
The number of offspring produced by each male was recorded, and the numbers
surviving were recorded daily for 7 weeks thereafter. Offspring taken out at
the population reduction step were counted and included in the statistical
analysis as `censored' cases. For 7 weeks, body length and height of all
surviving offspring were measured from standardized video recordings of
free-swimming individuals.
Individual newborn and juvenile seahorses were captured in a wide-ended
clear plastic pipette. The pipette was placed vertically near the inside front
of the tank and image sequences of the captured seahorse, which could move
freely within the pipette, were recorded using a video camcorder (Sony, Hi-8).
Measurements of height (tip of coronet to tip of tail, following the contours
of the dorsal surface) and body length (tip of coronet to base of the dorsal
fin) were made manually from the still images (Image Pro Plus, Media
Cybernetics, Silver Spring, MD, USA). These measurements differed slightly
from standard length or height (Lourie,
2003) as they were adapted to provide a non-invasive method of
measuring the length of very small seahorse fry. Although body mass is usually
preferred for this type of study (Koops et
al., 2004) it was impossible to handle these tiny offspring for
direct length and mass measurement without causing damage and stress. Mass of
the adults (parents) was measured by placing them in a container of water on a
balance.
The relationship between body mass and height was studied in a subsample of newborn seahorses that were sacrificed for management purposes and did not form part of the experiment. Body mass and height of newborn and juvenile seahorses were highly correlated (N=89; r=0.96; P<0.0001) but the relationship was non-linear. Logarithmic transformation of both variables produced a linear (r=0.98; P<0.0001) scatterplot (Fig. 1), thereby revealing that the variables were related by a power dependency of the form logMb=a+k(logH), where Mb=body mass, H=height; `a' and `k' are intercept and coefficient, respectively. This calibration showed that height is a good surrogate for mass; however, for the sake of clarity in presentation within this paper the height data has not been transformed.
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Statistical analysis
Data were analysed using Statistica V6.1 (Statsoft UK, Letchworth, Herts,
UK). Height and body length data from juveniles were used for calculating
cohort means from each breeding couple; these values were log-transformed
(Zar, 1984) and used to
represent the OC or YC replicate results in ANOVAs. Data were treated in this
way to avoid pseudoreplication; this would have artificially increased the
sensitivity of the experiment if individual values for juvenile height and
body length had been used as basic statistical units. Specific contrasts in
the ANOVAs were examined using independent orthogonal contrasts. Survival of
newborns from OC and YC was compared using the Cox-Mantel test, taking account
of artificially removed (censored) individuals. Homogeneity of variances was
analysed using Levene's test.
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Results |
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The higher mortality was not attributable to deficient feeding ability, as might be expected if the offspring were significantly smaller than their counterparts. No significant differences in height and body length measurements were detectable in the first week after birth. The overall heights calculated using mean data from each brood were (±s.e.m.): OC vs YC, 12.92±0.47 mm and 12.38±0.46 mm, respectively; P=0.343. However, juveniles produced by OC showed significantly higher average growth rate over the first 3 weeks of life (P<0.001, F2,32=133.6; Fig. 3). Newborn juveniles from the YC group also exhibited significantly greater variance in height than the OC (P<0.005). Mean height of the OC group was approximately 10% higher than the YC group by the seventh week (Fig. 3; OC vs YC group mean (±s.e.m.) heights were 31.22±1.29 and 27.92±1.05 mm, respectively; P=0.028, F1,39=6.45).
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The population derived from the OC showed no statistical skew after week 1 (Fig. 4C), but conversely the population derived from the YC showed significant left-skewing between weeks 2 and 4 (Fig. 4D). This emphasises the unexpectedly high frequency of slower growing individuals from the YC during the first few weeks after release from the pouch.
Effects of parental size on heterogeneity of early postnatal growth rates
When heights of individual newborn seahorses within any single cohort were
plotted against age, the average growth curves could be modelled by a
logarithmic relationship of the form (y=q[log(x)]+b) where
x was age in weeks, q was a coefficient of growth rate and b was the
intercept. Two examples of such curves, which represent offspring from one
large and one small male, are shown in Fig.
5A; scatter around the two fitted lines is also shown. In these
two examples there is considerable separation of individual heights during
weeks 1-3; by week 4 the points overlap, an effect likely to be caused by
selective mortality of the smaller individuals.
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Magnetic resonance imaging of pregnant males and examination of embryo attachment sites in pouch
Transverse scans through different levels of the pouch
(Fig. 6A,B) revealed that
embryos were distributed around the periphery and that the pouch lumen was
devoid of embryos. This suggested that embryos were directly attached to the
walls of the pouch, a finding confirmed by direct examination of embryos in
the pouch after dissection. However, the nature of the embryonic attachment
differed considerably between different regions of the pouch. Although not
formally quantified, it was apparent that whereas dorsally located embryos
were deeply embedded within individual compartments of the pouch wall
(Fig. 6C), those located
ventrally were merely attached to the wall in shallow depressions
(Fig. 6D).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Effects of parental age on growth rates and mortality
Egg size, concentration of eggs within the pouch, and embryonic mass have
previously been shown to depend on male and female size in syngnathids,
especially in pipefishes (Ahnesjo,
1992; Ahnesjo,
1996; Berglund et al.,
1986a; Watanabe and Watanabe,
2002). Here we present evidence of the relationship between
age-associated size of parents and newborn postnatal growth. Our results, in
combination with existing knowledge of syngnathid reproduction, support the
speculative argument that relationships between pre- and post-natal
development in seahorses share some features with those of mammalian
development (Barker, 1995).
The slower initial embryonic growth rates from younger (smaller) parents were accompanied by significantly higher embryo mortality during the first 7 weeks of life. Statistical techniques for mortality analysis, or failure analysis, take account of individuals that were deliberately removed from the experimental groups (`censored') to prevent overcrowding. The high mortality rate is therefore not a statistical artefact, but neither is it caused by a feeding inability of embryos from the YC group. In our study there was no evidence that newborns from the two groups differed in average size at birth and therefore their respective abilities to catch and consume Artemia nauplii are likely to have been similar. The younger, or smaller, parents produced broods with higher than expected frequencies of small newborns and greater variance within the newborn cohort. These features are apparent from the flat-topped or left-skewed body height histograms that were a feature of the YC offspring during the first 4 weeks of life. Inspection of data from within individual broods showed that the unusually small individuals had mostly disappeared by the fourth week after birth. Although we were unable to trace these newborns individually, the combination of mortality and size data strongly suggests that the high mortality was mainly due to their poor growth and survival. In this experiment the main factors determining offspring survival and growth rates were the age and size of both parents.
The observed negative correlation between number of newborns and growth coefficients for cohorts produced by the OC, and the absence of this correlation for YC, may be explained by differences in embryonic development, which might arise either from differences in egg size, or from topological heterogeneity within the pouch, and possibly both.
Effects of embryo-pouch interactions on growth
Although the reproductive biology of seahorses involves a complex
combination of male and female influences on postnatal growth, we have tried
to consider separately the extent to which the males and females contribute to
the significant effects detected. In agreement with other studies of
syngnathid embryo development (Carcupino
et al., 2002) our observations showed that, during pregnancy in
the male, embryos were distributed around the inner walls of the pouch and
individually located within specialised attachment sites. Within an individual
pouch it was also apparent that the size and complexity of individual
attachment sites was variable, and ranged from simple depressions in the pouch
surface to deep cavities into which the embryos were inserted. The hypothesis
that these sites are important for the nutrition of embryos has been shown to
be unsustainable (Azzarello,
1991), but it is plausible that they control other functions.
Histological and ultrastructural characteristics of the brood pouch attachment
sites in seahorses (Carcupino et al.,
2002), and in pipefishes
(Carcupino et al., 1997;
Carcupino et al., 2002) that
are closely related to the seahorses, were interpreted as indicative of
osmoregulatory and gaseous exchange functions. The interfaces between pouch
and embryo are therefore important for the control of respiration, as had
previously been suggested (Nikolsky,
1963). Moreover, the increased metabolic rate of gravid males
(Berglund et al., 1986a) has
also been considered indicative of paternal energy investment in
embryogenesis.
These observations suggest that, within the pouch of the older and more
mature males, the location of embryonic attachment, namely whether within a
shallow or deep site, may influence and possibly limit the quality of
respiratory support obtained by individual embryos. Within the OC group, the
significant negative correlation between the number of embryos within a brood
and the subsequent growth rate may therefore be interpreted in terms of
competition for the highest quality attachment sites within the pouch. When
small numbers of embryos are present, they are able to occupy the most
functionally advantageous sites; conversely, however, when embryos are present
in large numbers they are forced to occupy all available sites. This result is
similar to previous observations in pipefish
(Ahnesjo, 1992;
Ahnesjo, 1996) that superior
juveniles are produced when fewer newborns are present in the pouch. Other
observations in pipefishes, that larger males with decreased density of
embryos in the pouch are associated with better embryonic growth, also support
the belief that paternal influence on embryonic growth is exerted through
pouch function. The same principle of a parental effect on offspring growth
has also been demonstrated recently in the oviparous eelpout, Zoarces
viviparus, in which embryos grow within ovarian follicles
(Vetemaa et al., 2006). In
this species, females with higher relative fecundity had significantly smaller
average length, suggesting that `growth is limited by competition for maternal
energy supplies'. In Ahnesjö's investigations
(Ahnesjo, 1992;
Ahnesjo, 1996) only the dry
mass of newborns was studied and the question about future growth was never
investigated. In our study it is notable that no evidence of a significant
negative correlation between paternal size and embryonic growth rate was
detected when the smaller (YC) seahorse group was considered. We speculate
that the pouches of the smaller males used in the present study, although
obviously functional, were small and still insufficiently mature for the
effects of attachment site heterogeneity to confer significant advantage upon
any cohort of embryos.
The data presented here underline the importance of the prenatal conditions experienced by embryos in the pouch upon their future development and survival. This parallels the situation in mammals where the concept of foetal programming is widely accepted, although still controversial. The phenomenon has been attributed to poor body condition and nutrition, prior to, and during pregnancy, and epidemiological studies have reported effects that persist into adult life. The evidence presented here mimics the effects of foetal programming in two different ways. Individual seahorse embryos within a pouch may experience different degrees of respiratory and osmoregulatory support. Embryos embedded in deep pouch cavities are likely to be immersed in their own individual microenvironment, whereas others in the shallow depressions may receive less optimal support. The present data also suggest that the reproductive success of individual male seahorses is likely to be correlated with their own body size as this is reflected in their pouch size and physiological condition. As the young male seahorses produced a higher proportion of newborns with poor growth and life expectancy, this mimics the situation in which poor embryonic support results in poor fecundity. As our study considered only the first 7 weeks of life we are, as yet, unable to determine whether the effects persist into adult life, as seen in mammals.
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Acknowledgments |
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