NATIONAL SCIENCE FOUNDATION
TOKYO REGIONAL OFFICE
The National Science Foundation's (NSF) Tokyo Regional Office periodically
receives and disseminates reports on research developments in Japan that are
related to the Foundation's mission. It also provide occasional reports on
developments in other East Asian Countries (http://www.twics.com/~nsfasia/as-reports.htm).
These reports present information for the use of NSF program officers and
policy makers; they are not statements of NSF policy..
Special
Scientific Report #01-09
(December
10,
2001)
INVESTIGATING
THE COMPOSITION OF THE EXOSKELETON OF MACROBRACHIUM
ROSENBERGII
The
following report was prepared by Mr. Andrew J Wigginton, a graduate student at
the T.H. Morgan School of Biology at the University of Kentucky.
Mr. Wigginton was a participant in the 2001 Summer Programs in Japan,
co-sponsored by the National Science Foundation and the Japanese Ministry of
Education and Science (Monbukagakusho). While
in Japan he conducted his research under the guidance of Dr. Marcy Wilder,
Fisheries Division, Japan International Center for Agricultural Research in
Tsukuba. Mr. Wigginton may be
reached at RumicWorld@aol.com.
My previous work has concerned the
toxicity of cadmium to freshwater crayfish. Cadmium is considered one of the
most toxic metals in part because it has a very high binding affinity for the
amino acid cysteine, allowing it to alter the activity of a variety of proteins.
Cadmium is particularly interesting as a study subject for its potential
effects upon calcium metabolism. This is potentially a very significant effect
upon crustaceans since they must periodically shed their exoskeleton. This
requires a considerable amount of ion transport both in removing materials from
the old exoskeleton prior to molting and in hardening the new exoskeleton. Since
cadmium is know to affect various membrane bound calcium transporters, and
calcium carbonate is a major constituent of the exoskeleton, then crustaceans
may be especially vulnerable to cadmium toxicity during molting.
Sodium-potassium ATPase (Na/K ATPase) is
perhaps the most important ion regulating enzyme in most osmoregulating aquatic
animals. It has considerable effect upon the transport of other ions, including
calcium, because sodium and potassium concentration gradients drive certain
other ion transport proteins. The laboratory of Dr. Marcy Wilder, at the Japan
International Research Center for Agricultural Science (JIRCAS), has
considerable expertise measuring the activity of Na/K ATPase. The chance to work
in her laboratory provided an excellent opportunity to learn an assay that will
be important in my own research. Additionally, I was able to collaborate with
one of Dr. Wilder’s co-workers, Dr. Hatta Tamao, a geologist, in developing a
novel technique for assessing the chemical composition of crustacean
exoskeletons using Scanning Electron Microscopy with Energy Dispersion
Spectroscopy. This technique would almost certainly prove useful in
environmental toxicology for examining the effects of cadmium on calcium
dynamics in the molt cycle.
One of the prime foci of Dr. Wilder’s
laboratory is elucidating the basic biology of the Giant Freshwater Prawn, Macrobrachium
rosenbergii, to support the efforts of JIRCAS to improve the prawn’s
aquiculture in developing countries, in this case Vietnam. The prawn lives much
of its adult life in fresh water. However, it must move to brackish water to
spawn because the newly hatched larvae are not capable of osmoregulating in
fresh water. Only after the larvae metamorphose into juveniles can they begin
returning to fresh water.
To determine their osmoregulatory
capabilities under various conditions of salinity, larvae resulting from the
eggs of five individuals were reared in water of 12 ppT salinity for 0, 5, 10,
15, 20 and 25 days, then transferred to water of 0, 6, or 12 ppT salinity. After
5 days, the individuals were collected and frozen at
–80°C until analysis. The preceding work had been conducted by other
researchers prior to my arrival, so my involvement was limited to the analysis
of these samples using methods modified from McCormick and Bern, 1989. While
sample processing has been completed, data analysis is still proceeding.
Currently, Dr. Wilder, other researchers, and I are working to prepare a
manuscript on this research for publication.
SEM-EDS (Scanning Electron Microscopy
with Energy Dispersion Spectroscopy) is a tool often used by geologists and
engineers to assess the surface composition of objects. This technique can also
be used to examine biological samples. While soft tissues are relatively
difficult to prepare, calcified materials, such as the exoskeleton of
crustaceans, can be observed fairly easily. The exoskeleton of Macrobrachium
rosenbergii was examined at different stages of the molt cycle to observe
the dynamics of calcium mobilization and deposition. Unfortunately, I was only
able to begin work on this project by conducting preliminary analysis and
establishing a study plan.
Males
in three stages of development are to be sampled, as well as shed exoskeletons.
Newly molted individuals (Stage A), intermolt (Stage C0 ) and late
premolt (Stage D3) individuals will be dissected to remove their
exoskeleton. Additionally shed exoskeletons, preferably those belonging to the
newly molted individuals, will be taken for analysis. The carapace, pleuron,
uropod, rostrum, and antenna will be specifically selected for detailed analysis
for changes in composition during the molt cycle.
Several
different methods of analysis are available with SEM-EDS. Secondary electron
imaging (SEI) provides the typical images associated with SEM. Back scattering
electron imaging (BEIW) allows both the topography of a sample to be seen, as
well as a general assessment of sample composition, since elements with a higher
atomic number return a brighter image than lighter elements. EDS allows a much
more detailed assessment of composition. Area analysis will yield an average
percent wise elemental composition of the whole area under observation. Mapping
analysis, as the name implies, generates a map of the relative abundance of
selected elements in the area of observation. In point analysis, the percent
composition of points selected on the field of observation is measured. This
allows structures of interest to be examined in greater detail and enables the
comparison of very specific areas under observation.
Preliminary
work using SEM-EDS focused on the examination of general features of the
exoskeleton, and the establishment of a research plan. Initially, samples from a
shed exoskeleton, including the carapace, pleuron, antenna, and uropod were
examined using SEM-EDS. From these, it was observed, using mapping EDS, that the
external surfaces were typically very even in their distribution of calcium,
oxygen, and carbon. Specific surface features, such as joints and sensory hairs,
may differ from this general condition, possessing less calcium than surrounding
areas. However, it can be difficult to make determinations about percent
composition on curved surfaces, or objects protruding from a flat surface, as
the angle of electron impact can affect how well an element is reported.
Additionally, protruding objects can block the path of the electron beam, thus
creating shadows across the surface. In another case, an area possessed a
scratch, which perhaps penetrated the first layer or two of the exoskeleton.
This allowed a glimpse of the composition of the underlying layer(s). The area
of the scratch clearly showed higher calcium content than the surrounding areas.
The inner surface, in general, showed considerably greater variation. Areas that
appeared to be crystalline in structure (appearing lightly colored in the SEM
pictures) possessed much more calcium than other areas (which appeared dark in
color). More specific point analysis of these features lent support to this
apparent trend. Perhaps this indicates that, when shedding its exoskeleton. Macrobrachium is somewhat inefficient at removing calcium from its
exoskeleton before ecdysis. Thus, perhaps the lighter areas were portions of the
endocuticle that had not been reabsorbed.
In
the establishment of the sampling plan, SEI has been used to find and photograph
likely areas for analysis. BEIW has been used to determine that samples are
mounted as flatly as possible, and to examine areas along fractures caused by
sample preparation. This is of interest because some fractures reveal the
pattern of layers in the cuticle. Individual layers can be seen under SEI, but
the composition of these layers can be seen to abruptly change from primarily
lighter elements to include heavier elements as one progresses from the
innermost surface towards the middle layers of the exoskeleton. This phenomenon
can also be observed using mapping EDS analysis. In this case, it can be seen
that the innermost layers of the cuticle are calcium poor and relatively rich in
carbon, while deeper towards the middle of the exoskeleton, the layers can be
seen to be more rich in calcium and carbon is less abundant. For example,
between two closely spaced layers at a particular location along a fracture, the
atomic abundance of calcium changed from 0.68% to 5.54% and carbon abundance
changed from 48.71% to 69.35%. In the very outermost layers, the trend reverses
and carbon once again becomes relatively more rich while calcium becomes
relatively less abundant. For more quantitative analysis of changes in elemental
composition during the molt cycle, representative locations on the inner surface
of the exoskeleton were chosen and standardized for all individuals and will be
sampled using EDS point analysis.
In
summary, the 2001 Summer Institute in Japan Program provided me an opportunity
to meet and work with a number of exceptional scientists. Currently, Dr. Wilder,
other researchers, and I are writing a manuscript, which includes the Na/K
ATPase activity research, for submission in the near future. A second manuscript
is possible in the future upon the completion of the SEM-EDS research. This,
however, is not the limit of possible collaboration, as Dr. Wilder and the
leadership of JIRCAS were both very positive about the possibility of future
research visits, a very interesting prospect.
Acknowledgements
I
would especially like to thank Dr. Marcy Wilder for hosting me while in Japan.
She was generous with her time and forgiving of my mistakes. I would like to
thank Dr. Inoue Takahiro, Dr. Maeda Masachika, and Dr. Ishitani Takasuke for
their warm welcome and assistance. My lab mates, Dr. Vidya Jayasankar, Dr. Safia
Jasmani, and Dr. Saido-Sakanaka Hisako, deserve many thanks as well for all the
friendship and day-to-day help they provided. Additionally, I would like to
thank the staff at JISTEC and NSF for their tremendous efforts to make the
program run so smoothly. Finally, I would like to thank my graduate studies
advisor, Dr. Wesley J. Birge, for his encouragement and support.
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