Download Astyanax altiparanae - Sistema de Eventos

Aplication of fluorescence spectroscopy in Astyanax altiparanae scales for
differentiation of populations.
1º SCRN
23 a 26 de maio de 2017 – Dourados/MS
LIMA, D.M.V.; ALMEIDA, F.S.A.; SANTANA, C.A..
Programa de Pós-graduação em Recursos Naturais, Universidade Estadual de Mato Grosso do Sul – UEMS, C.P. 351, 79804-970,
Dourados, MS, Brazil.
Introduction
Astyanax altiparanae (Figure 1) belongs to the subfamily
Tetragonopterinae, which in the most diverse and largely
distributed of Characidae. These fish have a small size,
whose total length reach up to 150mm, and its diet revels a
specie generalist and opportunist, which is highly adaptable
to the physical environment changes with elevated plasticity
in response to resource availability. It is the most common
fish found in Upper Paraná river, occupying diferente types
and sizes of environments (streams, lakes and rivers)
(SÚAREZ et al., 2011).
Statistical analysis
The existence of some inter-population difference was
verified using a discriminant function analysis, in wich the
selected spectral peak intensities were used as variables.
This is a highly efficient method and is used to find a linear
combination of variables that best explain the differentiation
between analyzed populations.
The geographical distance was obtained through of
cartographic base (1:1000000) and the distance between
sites was measured using the river flow. The environmental
descriptors, after standardizations (mean=0 and standard
deviation=1) were used to produce a matrix of
environmental distance among habitats using a Euclidean
distance method.
The partial Mantel test was used to quantify the influence of
limnology and geographical distance on populations
differentiation.
explaining 80.3% of the data variation (figure 6). For the
external face, Wilk's Lambda = 0.073; F=15.35; p <0.001
were obtained, with the first canonical root explaining 76.1%
of the data variation.
Results and Discussion
Despite of greater application of spectroscopy techniques in
many biological compounds (CHENG et al., 2013), the
objective of this study is to analyze if Astyanax altiparanae
scales exhibit fluorescence behavior to differentiate the
chemical composition of the scale of different populations.
Methodology
Collection
The fish were sampled in ten streams from the Ivinhema
river Basin, Upper Paraná river, Brazil (Figure 2).
Wavelenght (nm)
450
500
400
550
1800
Fluorescence Intensity (arb. u.)
Figure 1. Astyanax altiparanae
Figure 3 below show the emission spectrum under excitation
at =360nm. This spectrum can be decomposed in four
peaks. The hydroxiapatite appear at 18000 and 24500 cm-1,
and the collagen at 20500 and 23000 cm-1 (Figure 3).
600
Figure 5. Scatterplot of scales fluorescence spectroscopy, excitation at
=360 nm, internal face.
650
B
576 nm: HAp
490 nm: Collagen
440 nm: Collagen
424 nm: HAp
Cumulative Fit Peak
1500
1200
900
600
300
0
26000
24000
22000
20000
18000
16000
Figure 6. Scatterplot of scales fluorescence spectroscopy, excitation at
=405 nm, internal face.
-1
Wavenumber ( cm )
Figure 3. Fluorescence under excitation at =360 nm of scale A.
altiparanae.
The excitation spectrum at =405nm show three peaks. The
hydroxiapatite appear at 18000 cm-1, and the collagen at 20100
and 22500 cm-1(Figure 4).
Wavelength (nm)
450
500
550
600
650
700 750
Figure 2. Map of Ivinhema river basin, Upper Paraná basin with
location of sampled streams.
Immediately after being collected, the fish were fixed in 10%
formaldehyde to be transfer to the laboratory, where they
were transfered to bottles containing 70% alcohol. From each
stream, with five to ten portion of each fish. After the scales
were dried in vacum for at least 12 hours to remove the
moisture. For each stream portion a set of environmental
descriptors were obtained.
Fluorescence
Fluorescence were perfomed at two excitation wavelengths:
360 e 405nm. For 360nm was used Ar+ laser using a
Coherente Innova 308C and 405nm was a diode laser. A
bifurcated optical fiber was used to conduct the excitation
light and capture the emission signal of the sample and lead
it to the portable Ocean Optics HR4000 spectrometer, where
the fluorescence spectrum was detected and stored in a
microcomputer. After analysis, a deconvolution process was
made to find that the best adjusted Gaussian spectrum. The
intensities of these data were collected to build the statistical
matrix.
Fluorescence Intensity (arb. u.)
300
B
490 nm: Collagen
576 nm: HAp
440 nm: Collagen
Cumulative Fit Peak
250
200
The result of the Mahalanobis distance using the
limnological data streams and data from fluorescence
analysis proved to be not significant for the differentiation
of populations (r = 0.06; p = ns). The results using the
geographical distance data together with the data of the
fluorescence analysis is also shown no significant (r = 0.23;
p = ns). These results indicate that the fluorescence has
potential to find differences between populations through
the statistics.
Conclusion
50
The results indicate that although each face of the scale has
a different chemical composition, it was not observed
strong difference in the luminescence curve shapes, and
both excitation wavelengths are capable to analyze the
scales A. altiparanae, providing meaningful and
enlightening results for the differentiation of populations.
0
References
150
100
22000
20000
18000
-1
Wavenumber (cm )
16000
14000
Figure 4. Fluorescence with excitation at =405 nm of scale A.
altiparanae.
The discriminant analysis demonstrated a significantly
differentiation among analyzed populations. Under excitation
at =360 nm, in the internal face, the statistical analysis
provided a Wilk’s Lambda = 0.145; F= 10.66; p < 0.001. The
first root explain 71% data variation (Figure 5). The external
face provided Wilk's Lambda = 0.143; F = 10.77; p <0.001,
exhibiting a result similar to the internal dispersion. The first
canonical root explained 74.5% of the data variation. For
excitation in =405 nm, the internal face provided Wilk's
Lambda = 0.106; F= 13.54; p < 0.001, showing significant
difference between the streams, with the first canonical root
SÚAREZ, Y. R., SOUZA, M. M., FERREIRA, F. S., PEREIRA,
M. J., SILVA, E. A., XIMENES, L. Q. L., AZEVEDO, L. G.,
MARTINS, O. C., LIMA-JUNIOR, S. E. Patterns of species
richness and composition of fish assemblages in streams of the
Ivinhema River basin, Upper Paraná River. Acta Limnologia
Brasiliensia, v. 23, n. 2, p. 177-188, 2011.
CHENG, J.; DAI, Q.; SUN, D.; ZENG, X.; LIU, D.; PU, H.
Appçications of non-destructive spectroscopy techniques for fish
quality and safety evaluation and inpection. Trends in Food
Science & Technology, v. 34. p. 18-31, 2013.
Acknowledgments