Color Variations of Immature Nypa Worms in The Kapuas Estuary, West Kalimantan
COLOR VARIATIONS OF IMMATURE NYPA WORMS IN THE
KAPUAS ESTUARY, WEST KALIMANTAN
RIYANDI
POST GRADUATE SCHOOL
BOGOR AGRICULTURE UNIVERSITY
BOGOR
2013
PERNYATAAN MENGENAI TESIS DAN
SUMBER INFORMASI SERTA PELIMPAHAN HAK CIPTA*
Dengan ini saya menyatakan bahwa Tesis berjudul Color Variations of
Immature Nypa Worms in The Kapuas Estuary, West Kalimantan adalah benar
karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam
bentuk apa pun kepada perguruan tinggi mana pun. Sumber informasi yang
berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari
penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di
bagian akhir tesis ini.
Dengan ini saya melimpahkan hak cipta dari karya tulis saya kepada Institut
Pertanian Bogor.
Bogor, Agustus 2013
Riyandi
NRP G352110051
* Pelimpahan hak cipta atas karya tulis dari penelitian kerja sama dengan pihak
luar IPB harus didasarkan pada perjanjian kerja sama yang terkait.
SUMMARY
RIYANDI. Color Variations of Immature Nypa Worms in The Kapuas Estuary,
West Kalimantan. Supervised by BAMBANG SURYOBROTO, TRI
ATMOWIDI and IIN INAYAT AL HAKIM
Nypa worm (Namalycastis rhodochorde) previously described to the new
species, included in Namalycastis abiuma species group with had an unusual
shape and had large body. Color variation in N. rhodochorde was obtained in
mature. N. rhodochorde had normal color when life was bright pink, while mature
were turned red (females) and green (males). But until now there was no report
color variation in immature N. rhodochorde. This study had examined color
variations of nypa worms in Kapuas Estuary, West Kalimantan.
Photogrammetry is one of the methods used to study the variations in color.
This method could be measure color through photos. Samples were photographed
on spiral labyrinth glass to limit the movement of the worm and Strobist Macro
Photo Studio for light stabilization. Digital images of nypa worm was recorded in
RAW format then converted to 8-bit TIFF. Determination of the measured area is
by selecting the whole dorsal body seen in TIFF files as ROI. Therefore the values
of RGB colour spaces was converted to the corresponding values of CIE Lab
colour space. Beside digital photogrammetry, color was described by the eye of
author.
Histological analysis was performed observed the anatomical difference
between the color groups. This was due to morphological and anatomical changes
occurring during maturation N. rhodochorde. Epidermal cells was observed due to
contains of secretory cells that function as mucus secretion. Mucus plays an
important role in the life of polychaeta. Epidermal preparation of nypa worm was
done using paraffin method.
Based on the analysis using photogrammetry, there is no significant
difference between males and females colors. By eye, obtained color variations in
immature worms categorized as bright pink, dark pink and brown. Most of the
worms were bright pink and the least is brown. Bright pink worm was immature
whereas dark pink worm was submature. These suggest that color variation
related to gametogenesis. From histology of epidermis we obtained three forms of
secretory cell within each group. Dark pink had more closed cell, it was suspected
that cell more active so that more of mucus in cell. This was coused the nypa
worm prepared to maturity. Different with brown worms that was immature had
larger cells, it was suspected to be an adaptation to the environment. In this study
was not found mature samples. It was suspected when sampling was not a of
spawning time. Polychaeta spawning time is strongly influenced by the physical
and biological environment conditions.
Keywords : color variation, estuary,immature, Namalycastis rhodochorde, nypa
worm
RINGKASAN
RIYANDI. Variasi Warna Pada Cacing Nipah yang Belum Matang Di Muara
Kapuas, Kalimantan Barat. Dibimbing oleh BAMBANG SURYOBROTO, TRI
ATMOWIDI dan IIN INAYAT AL HAKIM
Cacing nipah (Namalycastis rhodochorde) sebelum dideskripsikan menjadi
jenis baru adalah termasuk dalam kelompok jenis Namalycastis abiuma yang
memiliki bentuk yang tidak biasa dan tubuh yang panjang. Variasi warna pada N.
rhodochorde didapatkan pada individu yang sudah matang. N. rhodochorde
memiliki warna normal merah muda cerah, namun pada waktu matang akan
berubah menjadi merah (betina) dan hijau (jantan). Sampai saat ini belum ada
laporan mengenai variasi warna N. rhodochorde yang belum matang. Penelitian
ini bertujuan mengkaji variasi warna pada cacing nipah yang belum matang di
muara Sungai Kapuas, Kalimantan Barat.
Fotogrametri adalah salah satu metode untuk mempelajari variasi warna.
Metode ini memungkinkan mengukur warna melalui foto. Sampel difoto di atas
spiral labirin kaca untuk membatasi gerak cacing dan Strobist Macro Photo Studio
untuk mendapat kondisi cahaya yang stabil. Foto digital cacing nipah diambil
dengan menggunakan bentuk RAW yang dikonversi ke bentuk TIFF 8-bit. Bagian
yang diukur (ROI) adalah seluruh bagian dorsal yang dipilih dari berkas TIFF.
Kemudian nilai RGB dikonversi ke ruang warna CIE Lab. Selain dengan metode
fotogrametri, warna juga dideskripsikan menggunakan mata oleh penulis.
Analisis histologis dilakukan untuk melihat perbedaan anatomi dari
beberapa kelompok warna. Hal ini disebabkan selama proses kematangan N.
rhodochorde akan terjadi perubahan morfologi dan anatomi. Sel epidermis dikaji
karena memiliki sel sekresi yang berfungsi menghasilkan lendir. Lendir beperan
sangat penting dalam kehidupan polychaeta. Preparat epidermis disiapkan
menggunakan metode parafin.
Hasil analisis fotogrametri, tidak didapatkan perbedaan warna nyata antara
jantan dan betina. Variasi warna cacing yang belum matang yang didapatkan
dengan mata, dapat dikelompokkan ke warna merah muda cerah, merah muda
gelap dan coklat. Sebagian besar berwarna merah muda cerah dan paling sedikit
adalah coklat. Cacing merah muda cerah termasuk yang belum matang
(immature) dan cacing merah muda gelap termasuk yang setengah matang
(submature). Variasi warna ini disebabkan oleh proses gametogenesis. Dari
histologi epidermis didapatkan tiga bentuk sel sekresi dari masing-masing
kelompok warna. Kelompok warna merah muda gelap memiliki sel rapat, ini
diduga bahwa selnya lebih aktif untuk menghasilkan lendir. Hal ini disebabkan
cacing sedang mempersiapkan proses kematangan. Berbeda dengan cacing coklat
yang termasuk dalam cacing yang belum matang dimana memiliki sel lebih besar,
hal ini diduga disebabkan oleh proses adaptasi terhadap lingkungan. Polychaeta
menghasilkan lendir sebagai proses adaptasi terhadap lingkungan. Hal ini diduga
pada waktu pengambilan sampel bukan merupakan waktu pemijahan. Pemijahan
polychaeta sangat dipengaruhi oleh kondisi lingkungan baik fisik maupun biologi.
Kata Kunci : belum matang, cacing nipa, muara, Namalycastis rhodochorde,
variasi warna.
© Hak
Cipta Milik IPB, Tahun 2013
Hak Cipta Dilindungi Undang-Undang
Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan
atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan,
penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau
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IPB
Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis
ini dalam bentuk apa pun tanpa izin IPB
COLOR VARIATIONS OF IMMATURE NYPA WORMS IN
THE KAPUAS ESTUARY, WEST KALIMANTAN
RIYANDI
Thesis
As partial fulfillment of the requirements for Master Degree in
Animal Biosciences
POST GRADUATE SCHOOL
BOGOR AGRICULTURE UNIVERSITY
BOGOR
2013
Extra-committee examiner : Dr. Ir. Isdradjad Setyobudiandini, M.Sc
Title
: Color Variations of Immature Nypa Worms in The Kapuas
Estuary, West Kalimantan
Name
: Riyandi
Registration number : G352110051
Certified by,
Advisory Committee
Dr. Bambang Suryobroto
Chairman
Dr. Tri Atmowidi
Member
Dra. Iin Inayat Al Hakim, M.Si.
Member
Acknowledged by
Coordinator
Major of Animal Biosciences
Dean of Postgraduate School
Dr. Ir. R.R. Dyah Perwitasari, MSc
Dr. Ir Dahrul Syah, MScAgr
Date of Examination:
Date of Graduation:
PREFACE
Praise and gratitude writer prayed to Allah Subhanahu wa ta'ala for all gifts
so that this thesis was completed. Selected titles in a study is the Color Variations
of Immature Nypa Worms in the Kapuas Estuary, West Kalimantan.
Authors say thanks to Dr. Bambang Suryobroto, Dr. Tri Atmowidi and Dra.
Iin Inayat Al Hakim, M.Si as supervisors, as well as Junardi M.Si that has a lot to
give suggestions during this study. Thanks also to Mr. Yuliadi Zamroni M.Si and
Andy Darmawan M.Si who have been willing to take the time and suggestions
during the study. I also would like to thank Dikti Depdiknas for Postgraduate
Scholarships (BU).
In addition, the authors also say thank you to friends BSH 2011 who are
always provides motivation and inspiration in completing the study. Also to the
“zoo corner” friends who have been willing to sharing during the author
completed this studies at BSH. Thanks are also due to the father and mother who
always pray and support the whole family. Thanks also to my beloved wife and
daughter for their support to author complete this study. Thank you to all who
could not mention one by one for the prayers and support. Hopefully this paper
useful for all.
Bogor, August 2013
Riyandi
CONTENTS
LIST OF TABLE
vi
LIST OF FIGURE
vi
1. INTRODUCTION
1
2. MATERIAL AND METHOD
2
Study Site and Subjects
Identification
Colecting Digital Photos
Color Analysis
Histology
3. RESULT
Identification
Correspondences of Color Determination by Eye and Photogrammetry
Sexual Dimorphism in Color Variation
Histology of Epidermis
2
2
4
5
5
6
6
9
9
4. DISCUSSION
10
5. CONCLUSION
12
6. REFERENCES
12
LIST OF TABLE
1. Number of individual with brown, dark pink or bright pink with eye
2. Coefficients of linear discriminants in each function
3. Statistical Analysis of L* a * b * and chroma for each sex
7
7
8
LIST OF FIGURE
1. (A) Mangrove forest in Kapuas Estuary. (B) Sample collection by
manual digging (hand collection)
2. Research Map in Kapuas Estuary (Pulau Panjang). Located in the
western of Pontianak city (West Kalimantan) and faces Karimata
Strait.
3. Spiral labyrinth glass (A) and Strobist Macro Photo Studio (B)
4. (A) Normal color was bright pink (Glasby et al. 2007); (B) Dark
pink pattern was color with almost dark in all part of body; (C)
Brown pattern was having patch of dark color (like black).
5. Dorsal (A) and lateral (B) view of Namalycastis rhodochorde.
antenae (an); prostomium (pro); peristomium (pr); peristomial cirrus
(Pc); palps (P)
6. Lateral view (A) showed that sub-biramus type of 9th, 10th and 11th
parapodia. (B) showed heterogomph falciger chaetae. cirrophore (c);
chaetae (ct); blade (b)
7. Gametes of nypa worm. (A) male gametes (cluster of
spermatogonia). (B) female gametes (oocyte)
8. Visualization the log eyes vs photogrammetry data on the first two
discriminant axes. Individual scores are plotted on the first
(horizontal) and second (vertical) discriminant axes. Bright pink (1),
dark pink (2) and brown (3). Proportion of trace first function
comprises 96.8% and the second function comprises 3.2% of the
variance
9. Longitudinal section of epidermis of N. rhodochorde. (A) Bright
pink worm. (B) Dark Pink Worm. (C) Brown worm. (cu) cuticle;
(ec) epidermis cell; (cm) circular muscle; (cf) collagen fibre; (sc)
secretory cell.
2
3
3
4
6
7
7
8
10
1. INTRODUCTION
Polychaeta is a diverse class (McHugh 2005; Heuer et al. 2010). The high
diversity of Polychaeta because it has a high level of evolutionary plasticity
(Heuer et al. 2010). Nypa worm (Namalycastis rhodochorde) was include in
Family Nereididae Sub-Family Namanereidinae (Glasby et al. 2007). Nypa worm
before described to the new species, included in Namalycastis abiuma species
group with had an unusual shape and had large body (Glasby et al. 2007). It was
indicated that the nypa worm had a unique morphology.
Nypa worm distributed in South-east Asia (Sunda shelf only) including
Mekong Delta (Vietnam), West Kalimantan (Indonesia) and Sabah (Malaysia)
(Glasby et al. 2007). Nypa worm had a smaller range of habitats which in Nypa
frutican forest (Junardi 2008), so there was also possible to be obtained on the
area of mangrove forests in the dominance of nypa. Nypa worm is living on a
muddy estuary (Glasby et al. 2007; Junardi 2008). This may cause the nypa worm
had a high risk to environmental changes. Dynamics of estuarine environments
that was given effect for polychaeta such as environment and development
(Hamzavi et al. 2012).
Nypa worm is gonochoristic who have one organ sex on the individual.
Reproductive pattern of nypa worm is monotelik that spawn only once in the life
cycle. In spawning time, nypa worm released sperm into water and fertilize
similarly released eggs (ect-aquasperm) (Junardi et al. 2010). After fertilization,
the embryo of Nereididae develops in the egg then undergoes cleavage until
become larvae. The larvae develop into juvenile to mature individuals and
evolved freely in their habitat (Fischer et al. 2010).
Color variation in N. rhodochorde was obtained in mature. N. rhodochorde
generally when life was bright pink, while mature were turned red (females) and
green (males) (Glasby et al. 2007). N. rhodochorde in Glasby et al. (2007) was
photographed in mangroves of the Kota Kinabalu Wetland Center (Sabah,
Malaysia) and there was no color recorded in West Kalimantan. Moreover, until
now there was no report color variation in immature N. rhodochorde.
This study had examined color variations of nypa worms in Kapuas Estuary,
West Kalimantan. Photogrammetry is one of the methods used to study the
variations in color. This method could be measure color through photos.
Interpretation of color data through CIE Lab color space (Koyabu et al. 2008).
Images capture using Strobist Macro Photo Studio to light stabilization. Digital
images were taken at the time of worm still alive to avoid any discoloration of the
worm. By photogrammetry, there was no significant color differences between
males and females. Used eye of author obtained 3 color groups (bright pink, dark
pink and brown). Canonical discriminant obtained was red gived a high influence
on the grouping. From histologic of epidermis obtained three forms of secretory
cell of each group. This was suspect caused by adaptation to gametogenesis and
environmental changes
2
2. MATERIAL AND METHOD
Study Site and Subjects
Research has been carried out in December 2012 to May 2013. The
sampling method was purposive random sampling. Determined of sampling
location by purposive that was from the mangrove forest (Figure 1A) at the
intertidal zone of the Pulau Panjang in Kapuas Estuary (West Kalimantan
Province, Indonesia) (Figure 2).
Samples were taken randomly in mangrove areas at low tide by manual
digging (hand collection) (Figure 2B).
2B) Samples were stored in box filled with the
soil and midrib of nypa tree from the original habitat. Soil moisture in the box was
maintained by spraying brackish water (Junardi 2008).
20
All images of life worm
were taken at the Laboratory of Zoology,, Faculty of Mathematics and Natural
Sciences Tanjungpura University.
Identification
dentification and analysis were carried out at the Laboratory of
Worm identification
Section of Biosystematic and Ecological
E
of Animal,, Bogor Agricultural
University and Oceanographic Research Center Indonesia LIPI. Sampel was
preserved using alcohol 70% (Wu
(
et al. 1985).
Nypa worms were identified in reference to Wu et al.. (1985) and Glasby
(1999). Identification used characters that are commonly used in the assessment oof
the phylogeny such as prostomium,
prostomium peristomium, eversible pharynx,, parapodia,
and setae (Fauchald 1977). Sub-Family Namanereidinae
manereidinae is characterized by
having parapodia that uniramous
uniramou or sub-biramous,
ramous, notosetae and notosetae lobe
absent but having 2 aciculae (Wu et al. 1985). Genus identification was done by
observing the shape and length of the antenna and the form of pygidium (Glasby
1999). Description of N. rhodochorde followed Glasby et al. (2007).
Figures 1
(A) Mangrove forest in Kapuas Estuary. (B) Sample
Sample collection
by manual digging (hand collection)
3
Figure 2
Research Map in Kapuas Estuary (Pulau Panjang). Located in
the western of Pontianak city (West Kalimantan) and faces
Karimata Strait.
Strait
Figure 3 Spiral labyrinth glass (A) and Strobist Macro Photo Studio (B)
4
Figure 4 (A) Normal color was bright pink (Glasby et al. 2007); (B) Dark pink
pattern was color with almost dark in all part of body; (C) Brown
pattern was having patch of dark color (like black).
Object of research
arch is 73 individual (38 males and 35 females). Nypa worm
is extraovarian polychaeta ((Eckelbarger 2005; Junardi 2010) so male
ale and female
worms were distinguished by the type of gametes in the coelomic fluids.
Coelomic fluid were taken using a glass capillary tube inserted between the nypa
worm's body segments. Individual males were characterized by sperm
sperms while
females by the presence of eggs. Immature male was determined by th
the form of
clusters of spermatogonia,, whereas females based on clusters of oocytes (Junardi
2008; Junardi et al. 2010). Length of individual sampel ranged from 23--157.3 cm.
Beside digital photogrammetry, color was determined by the eye of author.
Colors for all individuals were observed directly in the field and by looking at
digital images. Individuals were classified into several groups:: brown, dark pink
and bright pink (Figure 4).. Brown color if the body of nypa worm had a band of
dark color (mostly black) in dark or bright pink specimen.. When the dark band
mostly wider than pink, it appears dark pink. Bright pink if it show completely
saturated pink without any pattern. The proportion of sexual differences judged by
eye has been examined by the chi square test.
test
Colecting Digital Photos
hotogrammetry was used to analyze color. Photogrammetry provides a
Photogrammetry
method to give quantitative data (Linder 2006). Samples were photographed on
spiral labyrinth glass to limit the movement of the worm (Figure 3A)
A). Images
were taken while the worm was alive to avoid any discoloration of the worm.
Image capture was done using Strobist Macro Photo Studio with external flash for
light stabilization using 4 external flash YN 460-II
460 Speedlight (ShenZhen
YongNuo Photographic Equipment Co. Ltd) (Figure 3B).
B). Ambient light condition
was adjusted with 18% reflective gray card (Mennon, China). Color
olor temperature
were set to 6000 K to adapt the ambient light condition at shooting time.
Digital images of nypa worm was recorded with a Canon EOS 1100 D
digital SLR camera (12.2 megapixel, fitted with a Canon EF
EF-S 18-55mm
55mm f/3.5
f/3.5-5.6
IS lens)) (Canon Inc, Taiwan) in RAW format. Camera RAW files is a near
near-to-thesubject recording enabling work to be done at the pixel level without in camera
processing occurring (Perez 2007).
20
RAW files were converted to 8-bit
bit TIFF files
using UFRAW (http://ufraw.sourceforge.net/
http://ufraw.sourceforge.net/).
5
Color Analysis
Measured area is the dorsal area, which is darker than other parts of the
body. Determination of the measured area is by selecting the whole dorsal body
seen in TIFF files as ROI (Regions of Interest). Data were processed with ImageJ
(http://imagej.nih.gov). Data was average from all pixels measured inside ROI.
The output from the camera (in this case EOS 1100D using CMOS sensor)
is interpreted in RGB values. RGB color space is not designed for human vision,
but for the physical display devices, for instance computer monitor (Baldevbhai
and Anand 2012). CIE L*a*b* color space was recommended by the CIE
(Commission Internationale de l'Eclairage) as a standardized interpretattion based
on color matching experiments using principle of trichromatic theory of color
vision (Perez 2007). CIE Lab color space describes the human colour vision and
independent of the device used as a reference. It give a good interpretation of the
color segmentation and most sensitive component to changes in practical
imaging conditions (Baldevbhai and Anand 2012). Therefore the values of RGB
colour spaces was converted to the corresponding values of CIE colour space.
CIE color space is a 3-dimensional hypothetical space with 3 color axes
(Perez 2007). Color is expressed via 3 parameters: L*, a*, and b*. The L* value,
represents the lightness of color responses, L* ranges from 0-100 (black to white);
the a* value from -60 (green) to +60 (red), represents the hue degree of green to
red; and b* value was -60 (blue) to +60 (yellow), represents the hue degree blue to
yellow. If the a* and b* values further away from 0 then the colors would be
brighter. Further, one can calculated the chroma value, which is a plain indicator
for the degree of color hue, from the a* and b* values as follows (Perez 2007). The
chroma value, ranging 0-85 (from square root of minimum and maximum square
of a* and b* value) :
Chroma = √
∗
+
∗
The sexual differences between males and females has been examined via
the Wilcoxon test (Koyabu et al. 2008). The relationship between the combination
of color values and the color determined by eye was using linear discriminant
analysis on L*, a*, and b* data. Calculation was done using R 2.11.0.
Histology
Histological analysis was performed to observed the anatomical difference
between the color groups. This was due to morphological and anatomical changes
occurring during maturation N. rhodochorde. Maturation is characterized by
changing of body color (red in females and green in males) and having softer
body than immature (Junardi 2012). Epidermis of polychaeta is composed of
pseudostratified and covered by a cuticle. Collagenous cuticle produced by the
epidermis covers the body wall of polychaeta. Epidermal cells consists of
secretory and non-secretory cells. Secretory cells play a role to produce mucus in
nypa worm (Hausen 2005; Mastrodonato et al. 2006).
Epidermal preparation of nypa worm was done using paraffin method.
Epidermal tissue samples were taken from each color group (bright pink, dark
pink and brown). Samples were fixed using FAAC (Formaldehyde Acetic Acid
6
ample was
Calcium Chloride) solution for ±3 days at room temperature. Sample
dehydrated with graded alcohol treatment and cleared using xylol. Paraffin
araffin was
used for infiltration and embedding.
embedding Tissue was cut using a rotary microtome
microtome.
Sample was stained using hematoxylin
h
and eosin.
3. RESULT
Identification
The identification of specimens showed that samples were indeed N.
rhodochorde (n=73). Characters
haracters used were body shape and colouration;
prostomium; peristomium and pharynx;
phary
parapodia; chaetae. Body shape was
almost uniformly rounded from the anterior to mid
mid-body but
ut slightly flattened
towards the posterior. Prostomium cleft anteriorly and the shape of antenae
relatively short subconical ((Figure 5A).
A). Palps biarticulate with compact
palpophores and palpostyles spherical (Figure 5B). The eye was not visible inside
peristomium.. Having 4 pairs short peristomial cirrus (Figure 5A-B). Pharynx was
had
invisible inside proboscis. Parapodia type was sub-biramous.. Each parapodia ha
dorsal and ventral cirrus.. D
Dorsal cirri anteriorly with cylindrical cirrophores,
posteriorly cirrophores were flattened (leaf-like) (Figure 6A). Notoacicula
otoacicula and
neuroacicula was present but had only neorochaetae
neorochaetae. Having sesquigomph
spiniger in supraacicular position.
position Lacking spiniger chaetae in subacicular
chaetae in supraacicular with smooth
position. Having heterogomph falcigerous
falci
teeth blade (Figure 6B).
Identification of males and females was using coelomic fluid to observed
gamets. The observation of coelomic fluid was found that they were mostly of
stages 2 or immature to sub-mature
sub
in gamet maturity (gamete sizes was ranged
from 25μm - 60μm) (Figure7).
(Figure7) The result showed that there was no mature worms
in all samples. This may be because samples were collected in December January, while according to Glasby (2007) spawning time in S
SeptemberNovember. There were 38 males and 35 females.
Figure 5 Dorsal (A) and lateral (B) view of Namalycastis rhodochorde
rhodochorde. antenae
(an); prostomium (pro); peristomium (pr); peristomial cirrus (Pc); palps
(P)
7
Figure 6 Laterall view (A) showed that sub-biramus
biramus type of 9th, 10th and 11th
parapodia.. (B) showed heterogomph falciger chaetae. ccirrophore (c);
chaetae (ct); blade (b)
Figure 7
Table 1
Gametes
ametes of nypa worm. (A) male gametes (cluster of
spermatogonia
atogonia). (B) female gametes (oocyte)
Number of individual with brown, dark pink or bright pink with eye
Sex
Male
N
Chroma
Female N
Chroma
Table 2
Brown
1
19.11
2
14.29-17.35
Dark Pink
4
15.94-19.79
4
16.43-19.41
Bright Pink
33
18.24-26.8
29
18.95-27.92
Coefficients of linear discriminants in each function
Function
First Function
Second Function
L*
-0.165
0.034
a*
-0.319
0.369
b*
-0.146
-0.650
8
Figure 8
Visualization the log eyes vs photogrammetry data on the first two
discriminant axes. Individual scores are plotted on the first
(horizontal) and second (vertical) discriminant axes. Bright pink
(1), dark pink (2) and brown (3). Proportion of trace first function
comprises 96.8% and the second function comprises 3.2% of the
variance
Table 3
Statistical Analysis of L* a * b * and chroma for each sex
Sex
n
Male
38
35
Female
*
*
*
L*
Mean
a*
b*
Chroma
SD
CV
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
50.9
5.58
11
15.7
2.72
17.3
15.8
1.9
12
22.4
2.85
12.7
50.7
4.77
9.42
15.6
3.24
20.8
15.4
1.83
11.9
21.9
3.27
14.9
L a b represent luminance, hue degree of red-greeness, hue degree of yellowblueness. Chroma is a plain indicator of color hue calculated as follows: Chroma
= √a∗ + b ∗ . n = number samples; SD; standar deviation; CV = coefficient of
variation
9
Correspondences of Color Determination by Eye and Photogrammetry
Color of living worms mostly bright pink to dark pink, after preservation
turned to pale yellow (Figure 5). Table 1 shows that, using the eye, the bright pink
was the dominant color in males and females while the least is brown. The
chroma values showed that bright pink was more vivid than dark pink and brown.
Based on the analysis using the chi square test ( p > 0.05), there was no difference
in proportion of different colors between males and females. Based on the
analysis using the Wilcoxon test (p >0.05), there is also no significant difference
between males and females colors judged by photogrammetry (Table 3).
The correspondence between color values from photogrammetry and color
determined by eye was assesed using discriminant analysis. Coefficients of linear
discriminants are in Table 2. Color hues of a* and b* rather than the L* luminance
value were strong determinants of color differentiation. Color hues of a* was the
highest value or the greatest influence in the grouping on discriminant analysis in
first function; b* on second function. Figure 8 show that most variation in samples
was grouped by first function (96.8%). In the first function, bright pink and dark
pink-brown were separated; the second function (3.2%) separate the groups dark
pink and brown.
Sexual Dimorphism in Color Variation
Statisitical analysis for males and females could be seen in the Table 3. The
L* value of 50.9 in males and 50.7 in females showed that they had neutral color
or grey. Value of a* in males was 15.7 and females 15.6, so the color was reddish.
The b* values in males was 15.8 and females 15.4, so they had a yellowish color.
Chroma describes the vividness or dullness of a color. Table 3 showed that
chroma value was 22.4 in males and 21.9 in females, it mean the chroma showed
the color were dull. All parametric value of male tended to have on average higher
than the females. Based on Tabel 3 males were more vivid than females.
Canonical discriminant analysis showed that the chroma is an important
determinant of color differentiation. Value of a* had the highest value so that red
has a big influence in the grouping.
Histology of Epidermis
Epidermis of N. rhodochorde was cellular monolayer covered by cuticle.
Cuticle were composed of fine fillamentous matrix that usually inhabits collagen
fibrils (Figure 9A1-B1-C1). Under epidermis there was circular muscular. Figure
4 showed the muscular fibres. Cuticle and muscle in all of types showed the same
composition.
Epidermis was pseudostratified by the presence of secretory and nonsecretory cells. Secretory cells were randomly distributed in the epidermis. There
was no difference between epidermal structure and their content in the dorsal and
ventral region of the body wall.Light microscopy showed that there were 3 types
of secretory cells based on color group. The first type (bright pink worm) showed
that composition of secretory cell were smaller and sparse (Figure 9A). The
second (dark pink worm) of secretory cell was closely spaced than bright pink
worm (Figure 9B). The third (brown worm) of secretory cell was bigger than
other type (Figure 9C).
10
Figure 9
Longitudinal section of epidermis of N. rhodochorde.. (A) Bright
pink worm. (B) Dark Pink Worm.
Worm (C) Brown worm. (cu) cuticle;
(ec) epidermis cell; (cm) circular muscle; (cf) collagen fibre; (sc)
secretory cell.
4. DISCUSSION
Glasby et al.. (2007) described that immature N. rhodochorde had bright
pink coloration which turned red (females)
(
and green (males) when matured
matured.
There is no reports on color variations in immature worms. Present study obtained
color variations in immature worms categorized as bright
right pink, dark pink and
brown. Most of the worms were bright pink and the least is brown. There was no
size differences between colors because the ranges
r
of body length were almost the
same. Oocyte size of bright pink worm were small ranging from 25µm - 35µm
(immature),
), while the dark pink were bigger ranging from 35µm - 50
50µm (submature) and
brown worm ranging from 25µm - 30µm (immature).
Spermatogenesis of bright pink and brown were in stage of the spermatogonia
cluster (immature). Dark pi
pink worms were found in the stage of spermato
spermatogonia
clusters but it was in smaller amounts. It was suspected that reduction
eduction of the
11
number of cells in the clusters was a step toward maturity (Junardi 2010). These
suggest that color variation related to gametogenesis.
Secretory cells have diverse function in polychaeta, for instances they
secrete mucus to help the worm to eat (Costa et al. 2006) and to move (Pardo and
Amaral 2004). In Syllidae, they used mucus to brood egg (Martin 2005).
Namanereis littoralis member of the same sub-family Namanereidinae as N.
rhodochorde, produce abundant secretion to ensure viability of sex products and
successful fertilization out of the water environment and protecting the animals
and their clutches from desiccation (Ezhova 2011).
From histology of epidermis we obtained three forms of secretory cell
within each group. Dark pink had more closed cell, it was suspected that cell more
active so that more of mucus in cell. Solid composition of the cell caused the cell
density also increased that make the color more darker. In females, this was
caused by the dark pink were a color that had toward to maturity (Junardi 2008)
which mean the worm was preparing mucus to keep the eggs. Dark pink also
found in males. This coused the males also was prepare to maturity.
Different with brown worms that had larger cells, it was suspected to be an
adaptation to the environment. This was caused nypa worm had a smaller range of
habitats (Junardi 2008). Polychaeta could been secrete mucus for adaptation to
environment. Laeonereis acuta (Nereididae) produced mucus to antioxidant
defence system of the worm against environmental (Moraes et al. 2006). But on
the contrary if there was exposure to pollutant (such heavy metal) it would be
interfere with the production of mucus such as Laeonereis acuta that had chronic
or acutely exposed to copper, mucus was absence on their body wall (Geracitano
et al. 2004).
In this study was not found mature samples. It was suspected when
sampling was not a of spawning time. Polychaeta spawning time is strongly
influenced by the physical environment conditions such as temperature, day
length, and lunar cycles (Belal 2012). Phytoplankton bloom is one of the
biological events that may cause the spawning mechanism as in Platynereis
dumerilii (Watson et al 2003). It was also suspected of sampling sites residing on
a single location because microhabitat conditions greatly affect the maturity.
Individual populations can breed at different conditions, according to local
conditions. In addition Nereidae can inhibit the maturation of gametes by
promoting growth and regeneration through hormonal mechanisms (Lawrence and
Soame 2004; Rouhi et al. 2008).
Nypa worm is one Nereididae Family and living on a muddy estuary
(Glasby et al. 2007; Junardi 2008). Nypa worm immersed himself into the ground
(infaunal), so we could not found nypa worms that exist on the surface. This may
cause the nypa worm had a high risk to environmental changes. Polychaeta was a
group that is often used as an indicator species because lived in sediment (Dean
2008; Alam et al. 2010). It was supported by the limited habitat choice of nypa
worm, because it could be at the nypa palm root. Nypa worm had a muddy-clay
habitat preferences and have a high organic carbon with low salinity and
temperature (Junardi 2008).
12
5. CONCLUSION
All samples included in immature to sub-mature category. Color analysis
showed no significant color differences between males and females in immature
worm. Through the eyes, obtained variations of color (bright pink, dark pink and
brown) with the dominant red in the grouping. In each group were also found
variations in secretions cell.
6. REFERENCES
Alam MA, Gomes A, Sarkar SK, Shuvaeva OV, Vishnevetskaya NS, Gustaytis
MA, Bhattacharya BD, Godhantaraman N. 2010. Trace Metal
Bioaccumulation by Soft-bottom Polychaetes (Annelida) of Sundarban
Mangrove Wetland, India and Their Potential Use as Contamination
Indicator. Bull Environ Contam Toxicol. 85:492–496
Baldevbhai PJ, Anand RS. 2012. Color Image Segmentation for Medical Images
Using L*a*b* Color Space. IOSRJECE. 1: 24-45
Belal AAM. 2012. Oogenesis and Spawning of Pomatoleious kraussii (Baired,
1865) (Polychaeta: Serpulidae) in Suez Bay. Egypt J Aquati Res. 38:
119–124
Costa PFE, Oliveira RF, Fonseca LCD. 2006. Feeding Ecology of Nereis
diversicolor (O.F. Müller) (Annelida, Polychaeta) on Estuarine and
Lagoon Environments in the Southwest Coast of Portugal. Panam J
Aquat . 1:114-126
Dean HK. 2008. The Use of Polychaetes (Annelida) as Indicator Species of
Marine Pollution: A Review. Rev Biol Trop. 56: 11-38
Eckelbarger KJ. 2005. Oogenesis and Oocytes. Hydrobiologia. 535/536:179-198
Ezhova EE. 2011. Spawning and Early Ontogenesis of the Littoral Polychaete
Namanereis littoralis (Grube, 1876) (Nereididae, Namanereidinae). Russ
J Dev Biol.42: 160–167
Fauchald K. 1977. The Polychaete Worm: Definition And Keys to the Orders,
Famili and Genera. Los Angeles (US): Natural History Museum of Los
Angeles County
Fischer AHL, Henrich T, Arendt D. 2010. The Normal Development of
Platynereis dumerilii (Nereididae, Annelida). FRONT ZOOL. 7:31
Geracitano LA, Bocchetti R, Monserrat JM, Regoli F, Bianchini A. 2004.
Oxidative Stress Responses in Two Populations of Laeonereis acuta
(Polychaeta, Nereididae) after Acute and Chronic Exposure to Copper.
Mar Environ Res. 58: 1–17
Glasby CJ. 1999. The Namanereidinae (Polychaeta: Nereididae): Part 1,
taxonomy and phylogeny. Rec Aust Mus, Supplement 25: 1–129
Glasby CJ, Miura T, Nishi E, Junardi. 2007. A New Species of Namalycastis
(Polychaeta: Nereididae: Namanereidinae) from The Shores of Southeast Asia. The Beagle, Records of Museum Art and Gallery of the
Northern Territory. 23: 21-27
13
Hamzavi SF, Kamrani E, Salarzadeh A, Salarpouri A. 2012. Study of Polychaete
Seasonal Changes (Ecological Indices) in Basatyn Estuary Nay band Bay
of Bushehr. J Basic Appl Sci Res. 8: 8466-8470
Hausen H. 2005. Comparative Structure of the Epidermis in Polychaetes
(Annelida). Hydrobiologia. 535/536: 25-35
Heuer CM, Muller CHG, Todt C, Loesel R. 2010. Comparative Neuroanatomy
Suggests Repeated Reduction of Neuroarchitectural Complexity in
Annelida. Front Zool. 7:13
Junardi. 2008. Karakteristik Morfologi dan Habitat Cacing Nipah Namalycastis
rhodochorde (Polychaeta: Nereididae: Namanereidinae) di Hutan
Mangrove Sungai Kakap Kalimantan Barat. Jurnal Sains MIPA. 14: 8589
Junardi, Setyawati TR, Yuwono E. 2010. Gametogenesis Cacing Nipah
Namalycastis rhodochorde (Polychaeta: Nereididae). Jurnal Ilmu
Dasar.11: 39–44
Koyabu DB, Malaivijitnond S, Hamada Y. 2008. Pelage Color Variation of
Macaca arctoides and Its Evolutionary Implications. Int J Primatol
29:531–541
Lawrence AJ, Soame JM. 2004. The Effects of Climate Change on the
Reproduction of Coastal Invertebrates. Ibis. 146: 29–39
Linder W. 2006. Digital Photogrammetry: A Practical Course. Second Edition.
New York (US): Springer.
Martin GS. 2005. Exogoninae (Polychaeta: Syllidae) from Australia With the
Description of a New Genus and Twenty-two New Species. Rec Aust
Mus. 57: 39–152
Mastrodonato M, Gherardi M, Todisco G, Sciscioli M, Lepore E. 2006. The
Epidermis of Timarete filigera (Polychaeta, Cirratulidae): Histochemical
and Ultrastructural Analysis of the Gland Cells. Tissue Cell. 38: 279–284
McHugh D. 2005. Molecular Systematic Of Polychaetes (Annelida).
Hydrobiologia 535/536: 309-318
Moraes TB, Ferreira JLR, Rosa CEd, Sandrini JZ, Votto AP, Trindade GS,
Geracitano LA, Abreu PC, Monserrat JM. 2006. Antioxidant Properties
of the Mucus Secreted by Laeonereis acuta (Polychaeta, Nereididae): A
Defense Against Environmental Pro-Oxidants?. Comp Biochem Physiol.
142: 293–300
Pardo Ev, Amaral ACZ. 2004. Feeding Behavior of the Cirratulid Cirriformia
filigera (Delle Chiaje, 1825) (Annelida: Polychaeta). Braz J Biol. 64:
283-288
Perez MP. 2007. Focal Encyclopedia Of Photography: Fourth Edition. Oxford
(US): Elsevier Inc
Rouhi A, Sif J, Gillet P, Deutsch B. 2008. Reproduction and Population Dynamics
of Perinereis cultrifera (Polychaeta: Nereididae) of the Atlantic Coast, El
Jadida, Morocco. Cah Biol Mar. 49: 151-160
Watson GJ, Bentley MG, Gaudron SM, Hardege JD. 2003. The Role of Chemical
Signals in the Spawning Induction of Polychaete Worms and other
Marine Invertebrates. J. Exp Mar Biol Ecol. 294: 169–187
Wu B, Sun R, Yang DJ. 1985. The Nereidae (Polycahetous Annelids) of The
Chinese Coast. China Ocean Press: China
14
CURRICULUM VITAE
Author was born in Sambas on June 18th, 1986 and as the first child of
Herwadi and Musfayati. Authors school at SMUN 1 Sambas in 2001-2004.
Subsequently continued his studies in Biology Faculty of Mathematics and
Natural Sciences at Tanjungpura University Pontianak 2004-2009. During the
lecture the author active in the activities of the Department of Biology Student
Association.
KAPUAS ESTUARY, WEST KALIMANTAN
RIYANDI
POST GRADUATE SCHOOL
BOGOR AGRICULTURE UNIVERSITY
BOGOR
2013
PERNYATAAN MENGENAI TESIS DAN
SUMBER INFORMASI SERTA PELIMPAHAN HAK CIPTA*
Dengan ini saya menyatakan bahwa Tesis berjudul Color Variations of
Immature Nypa Worms in The Kapuas Estuary, West Kalimantan adalah benar
karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam
bentuk apa pun kepada perguruan tinggi mana pun. Sumber informasi yang
berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari
penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di
bagian akhir tesis ini.
Dengan ini saya melimpahkan hak cipta dari karya tulis saya kepada Institut
Pertanian Bogor.
Bogor, Agustus 2013
Riyandi
NRP G352110051
* Pelimpahan hak cipta atas karya tulis dari penelitian kerja sama dengan pihak
luar IPB harus didasarkan pada perjanjian kerja sama yang terkait.
SUMMARY
RIYANDI. Color Variations of Immature Nypa Worms in The Kapuas Estuary,
West Kalimantan. Supervised by BAMBANG SURYOBROTO, TRI
ATMOWIDI and IIN INAYAT AL HAKIM
Nypa worm (Namalycastis rhodochorde) previously described to the new
species, included in Namalycastis abiuma species group with had an unusual
shape and had large body. Color variation in N. rhodochorde was obtained in
mature. N. rhodochorde had normal color when life was bright pink, while mature
were turned red (females) and green (males). But until now there was no report
color variation in immature N. rhodochorde. This study had examined color
variations of nypa worms in Kapuas Estuary, West Kalimantan.
Photogrammetry is one of the methods used to study the variations in color.
This method could be measure color through photos. Samples were photographed
on spiral labyrinth glass to limit the movement of the worm and Strobist Macro
Photo Studio for light stabilization. Digital images of nypa worm was recorded in
RAW format then converted to 8-bit TIFF. Determination of the measured area is
by selecting the whole dorsal body seen in TIFF files as ROI. Therefore the values
of RGB colour spaces was converted to the corresponding values of CIE Lab
colour space. Beside digital photogrammetry, color was described by the eye of
author.
Histological analysis was performed observed the anatomical difference
between the color groups. This was due to morphological and anatomical changes
occurring during maturation N. rhodochorde. Epidermal cells was observed due to
contains of secretory cells that function as mucus secretion. Mucus plays an
important role in the life of polychaeta. Epidermal preparation of nypa worm was
done using paraffin method.
Based on the analysis using photogrammetry, there is no significant
difference between males and females colors. By eye, obtained color variations in
immature worms categorized as bright pink, dark pink and brown. Most of the
worms were bright pink and the least is brown. Bright pink worm was immature
whereas dark pink worm was submature. These suggest that color variation
related to gametogenesis. From histology of epidermis we obtained three forms of
secretory cell within each group. Dark pink had more closed cell, it was suspected
that cell more active so that more of mucus in cell. This was coused the nypa
worm prepared to maturity. Different with brown worms that was immature had
larger cells, it was suspected to be an adaptation to the environment. In this study
was not found mature samples. It was suspected when sampling was not a of
spawning time. Polychaeta spawning time is strongly influenced by the physical
and biological environment conditions.
Keywords : color variation, estuary,immature, Namalycastis rhodochorde, nypa
worm
RINGKASAN
RIYANDI. Variasi Warna Pada Cacing Nipah yang Belum Matang Di Muara
Kapuas, Kalimantan Barat. Dibimbing oleh BAMBANG SURYOBROTO, TRI
ATMOWIDI dan IIN INAYAT AL HAKIM
Cacing nipah (Namalycastis rhodochorde) sebelum dideskripsikan menjadi
jenis baru adalah termasuk dalam kelompok jenis Namalycastis abiuma yang
memiliki bentuk yang tidak biasa dan tubuh yang panjang. Variasi warna pada N.
rhodochorde didapatkan pada individu yang sudah matang. N. rhodochorde
memiliki warna normal merah muda cerah, namun pada waktu matang akan
berubah menjadi merah (betina) dan hijau (jantan). Sampai saat ini belum ada
laporan mengenai variasi warna N. rhodochorde yang belum matang. Penelitian
ini bertujuan mengkaji variasi warna pada cacing nipah yang belum matang di
muara Sungai Kapuas, Kalimantan Barat.
Fotogrametri adalah salah satu metode untuk mempelajari variasi warna.
Metode ini memungkinkan mengukur warna melalui foto. Sampel difoto di atas
spiral labirin kaca untuk membatasi gerak cacing dan Strobist Macro Photo Studio
untuk mendapat kondisi cahaya yang stabil. Foto digital cacing nipah diambil
dengan menggunakan bentuk RAW yang dikonversi ke bentuk TIFF 8-bit. Bagian
yang diukur (ROI) adalah seluruh bagian dorsal yang dipilih dari berkas TIFF.
Kemudian nilai RGB dikonversi ke ruang warna CIE Lab. Selain dengan metode
fotogrametri, warna juga dideskripsikan menggunakan mata oleh penulis.
Analisis histologis dilakukan untuk melihat perbedaan anatomi dari
beberapa kelompok warna. Hal ini disebabkan selama proses kematangan N.
rhodochorde akan terjadi perubahan morfologi dan anatomi. Sel epidermis dikaji
karena memiliki sel sekresi yang berfungsi menghasilkan lendir. Lendir beperan
sangat penting dalam kehidupan polychaeta. Preparat epidermis disiapkan
menggunakan metode parafin.
Hasil analisis fotogrametri, tidak didapatkan perbedaan warna nyata antara
jantan dan betina. Variasi warna cacing yang belum matang yang didapatkan
dengan mata, dapat dikelompokkan ke warna merah muda cerah, merah muda
gelap dan coklat. Sebagian besar berwarna merah muda cerah dan paling sedikit
adalah coklat. Cacing merah muda cerah termasuk yang belum matang
(immature) dan cacing merah muda gelap termasuk yang setengah matang
(submature). Variasi warna ini disebabkan oleh proses gametogenesis. Dari
histologi epidermis didapatkan tiga bentuk sel sekresi dari masing-masing
kelompok warna. Kelompok warna merah muda gelap memiliki sel rapat, ini
diduga bahwa selnya lebih aktif untuk menghasilkan lendir. Hal ini disebabkan
cacing sedang mempersiapkan proses kematangan. Berbeda dengan cacing coklat
yang termasuk dalam cacing yang belum matang dimana memiliki sel lebih besar,
hal ini diduga disebabkan oleh proses adaptasi terhadap lingkungan. Polychaeta
menghasilkan lendir sebagai proses adaptasi terhadap lingkungan. Hal ini diduga
pada waktu pengambilan sampel bukan merupakan waktu pemijahan. Pemijahan
polychaeta sangat dipengaruhi oleh kondisi lingkungan baik fisik maupun biologi.
Kata Kunci : belum matang, cacing nipa, muara, Namalycastis rhodochorde,
variasi warna.
© Hak
Cipta Milik IPB, Tahun 2013
Hak Cipta Dilindungi Undang-Undang
Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan
atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan,
penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau
tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan
IPB
Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis
ini dalam bentuk apa pun tanpa izin IPB
COLOR VARIATIONS OF IMMATURE NYPA WORMS IN
THE KAPUAS ESTUARY, WEST KALIMANTAN
RIYANDI
Thesis
As partial fulfillment of the requirements for Master Degree in
Animal Biosciences
POST GRADUATE SCHOOL
BOGOR AGRICULTURE UNIVERSITY
BOGOR
2013
Extra-committee examiner : Dr. Ir. Isdradjad Setyobudiandini, M.Sc
Title
: Color Variations of Immature Nypa Worms in The Kapuas
Estuary, West Kalimantan
Name
: Riyandi
Registration number : G352110051
Certified by,
Advisory Committee
Dr. Bambang Suryobroto
Chairman
Dr. Tri Atmowidi
Member
Dra. Iin Inayat Al Hakim, M.Si.
Member
Acknowledged by
Coordinator
Major of Animal Biosciences
Dean of Postgraduate School
Dr. Ir. R.R. Dyah Perwitasari, MSc
Dr. Ir Dahrul Syah, MScAgr
Date of Examination:
Date of Graduation:
PREFACE
Praise and gratitude writer prayed to Allah Subhanahu wa ta'ala for all gifts
so that this thesis was completed. Selected titles in a study is the Color Variations
of Immature Nypa Worms in the Kapuas Estuary, West Kalimantan.
Authors say thanks to Dr. Bambang Suryobroto, Dr. Tri Atmowidi and Dra.
Iin Inayat Al Hakim, M.Si as supervisors, as well as Junardi M.Si that has a lot to
give suggestions during this study. Thanks also to Mr. Yuliadi Zamroni M.Si and
Andy Darmawan M.Si who have been willing to take the time and suggestions
during the study. I also would like to thank Dikti Depdiknas for Postgraduate
Scholarships (BU).
In addition, the authors also say thank you to friends BSH 2011 who are
always provides motivation and inspiration in completing the study. Also to the
“zoo corner” friends who have been willing to sharing during the author
completed this studies at BSH. Thanks are also due to the father and mother who
always pray and support the whole family. Thanks also to my beloved wife and
daughter for their support to author complete this study. Thank you to all who
could not mention one by one for the prayers and support. Hopefully this paper
useful for all.
Bogor, August 2013
Riyandi
CONTENTS
LIST OF TABLE
vi
LIST OF FIGURE
vi
1. INTRODUCTION
1
2. MATERIAL AND METHOD
2
Study Site and Subjects
Identification
Colecting Digital Photos
Color Analysis
Histology
3. RESULT
Identification
Correspondences of Color Determination by Eye and Photogrammetry
Sexual Dimorphism in Color Variation
Histology of Epidermis
2
2
4
5
5
6
6
9
9
4. DISCUSSION
10
5. CONCLUSION
12
6. REFERENCES
12
LIST OF TABLE
1. Number of individual with brown, dark pink or bright pink with eye
2. Coefficients of linear discriminants in each function
3. Statistical Analysis of L* a * b * and chroma for each sex
7
7
8
LIST OF FIGURE
1. (A) Mangrove forest in Kapuas Estuary. (B) Sample collection by
manual digging (hand collection)
2. Research Map in Kapuas Estuary (Pulau Panjang). Located in the
western of Pontianak city (West Kalimantan) and faces Karimata
Strait.
3. Spiral labyrinth glass (A) and Strobist Macro Photo Studio (B)
4. (A) Normal color was bright pink (Glasby et al. 2007); (B) Dark
pink pattern was color with almost dark in all part of body; (C)
Brown pattern was having patch of dark color (like black).
5. Dorsal (A) and lateral (B) view of Namalycastis rhodochorde.
antenae (an); prostomium (pro); peristomium (pr); peristomial cirrus
(Pc); palps (P)
6. Lateral view (A) showed that sub-biramus type of 9th, 10th and 11th
parapodia. (B) showed heterogomph falciger chaetae. cirrophore (c);
chaetae (ct); blade (b)
7. Gametes of nypa worm. (A) male gametes (cluster of
spermatogonia). (B) female gametes (oocyte)
8. Visualization the log eyes vs photogrammetry data on the first two
discriminant axes. Individual scores are plotted on the first
(horizontal) and second (vertical) discriminant axes. Bright pink (1),
dark pink (2) and brown (3). Proportion of trace first function
comprises 96.8% and the second function comprises 3.2% of the
variance
9. Longitudinal section of epidermis of N. rhodochorde. (A) Bright
pink worm. (B) Dark Pink Worm. (C) Brown worm. (cu) cuticle;
(ec) epidermis cell; (cm) circular muscle; (cf) collagen fibre; (sc)
secretory cell.
2
3
3
4
6
7
7
8
10
1. INTRODUCTION
Polychaeta is a diverse class (McHugh 2005; Heuer et al. 2010). The high
diversity of Polychaeta because it has a high level of evolutionary plasticity
(Heuer et al. 2010). Nypa worm (Namalycastis rhodochorde) was include in
Family Nereididae Sub-Family Namanereidinae (Glasby et al. 2007). Nypa worm
before described to the new species, included in Namalycastis abiuma species
group with had an unusual shape and had large body (Glasby et al. 2007). It was
indicated that the nypa worm had a unique morphology.
Nypa worm distributed in South-east Asia (Sunda shelf only) including
Mekong Delta (Vietnam), West Kalimantan (Indonesia) and Sabah (Malaysia)
(Glasby et al. 2007). Nypa worm had a smaller range of habitats which in Nypa
frutican forest (Junardi 2008), so there was also possible to be obtained on the
area of mangrove forests in the dominance of nypa. Nypa worm is living on a
muddy estuary (Glasby et al. 2007; Junardi 2008). This may cause the nypa worm
had a high risk to environmental changes. Dynamics of estuarine environments
that was given effect for polychaeta such as environment and development
(Hamzavi et al. 2012).
Nypa worm is gonochoristic who have one organ sex on the individual.
Reproductive pattern of nypa worm is monotelik that spawn only once in the life
cycle. In spawning time, nypa worm released sperm into water and fertilize
similarly released eggs (ect-aquasperm) (Junardi et al. 2010). After fertilization,
the embryo of Nereididae develops in the egg then undergoes cleavage until
become larvae. The larvae develop into juvenile to mature individuals and
evolved freely in their habitat (Fischer et al. 2010).
Color variation in N. rhodochorde was obtained in mature. N. rhodochorde
generally when life was bright pink, while mature were turned red (females) and
green (males) (Glasby et al. 2007). N. rhodochorde in Glasby et al. (2007) was
photographed in mangroves of the Kota Kinabalu Wetland Center (Sabah,
Malaysia) and there was no color recorded in West Kalimantan. Moreover, until
now there was no report color variation in immature N. rhodochorde.
This study had examined color variations of nypa worms in Kapuas Estuary,
West Kalimantan. Photogrammetry is one of the methods used to study the
variations in color. This method could be measure color through photos.
Interpretation of color data through CIE Lab color space (Koyabu et al. 2008).
Images capture using Strobist Macro Photo Studio to light stabilization. Digital
images were taken at the time of worm still alive to avoid any discoloration of the
worm. By photogrammetry, there was no significant color differences between
males and females. Used eye of author obtained 3 color groups (bright pink, dark
pink and brown). Canonical discriminant obtained was red gived a high influence
on the grouping. From histologic of epidermis obtained three forms of secretory
cell of each group. This was suspect caused by adaptation to gametogenesis and
environmental changes
2
2. MATERIAL AND METHOD
Study Site and Subjects
Research has been carried out in December 2012 to May 2013. The
sampling method was purposive random sampling. Determined of sampling
location by purposive that was from the mangrove forest (Figure 1A) at the
intertidal zone of the Pulau Panjang in Kapuas Estuary (West Kalimantan
Province, Indonesia) (Figure 2).
Samples were taken randomly in mangrove areas at low tide by manual
digging (hand collection) (Figure 2B).
2B) Samples were stored in box filled with the
soil and midrib of nypa tree from the original habitat. Soil moisture in the box was
maintained by spraying brackish water (Junardi 2008).
20
All images of life worm
were taken at the Laboratory of Zoology,, Faculty of Mathematics and Natural
Sciences Tanjungpura University.
Identification
dentification and analysis were carried out at the Laboratory of
Worm identification
Section of Biosystematic and Ecological
E
of Animal,, Bogor Agricultural
University and Oceanographic Research Center Indonesia LIPI. Sampel was
preserved using alcohol 70% (Wu
(
et al. 1985).
Nypa worms were identified in reference to Wu et al.. (1985) and Glasby
(1999). Identification used characters that are commonly used in the assessment oof
the phylogeny such as prostomium,
prostomium peristomium, eversible pharynx,, parapodia,
and setae (Fauchald 1977). Sub-Family Namanereidinae
manereidinae is characterized by
having parapodia that uniramous
uniramou or sub-biramous,
ramous, notosetae and notosetae lobe
absent but having 2 aciculae (Wu et al. 1985). Genus identification was done by
observing the shape and length of the antenna and the form of pygidium (Glasby
1999). Description of N. rhodochorde followed Glasby et al. (2007).
Figures 1
(A) Mangrove forest in Kapuas Estuary. (B) Sample
Sample collection
by manual digging (hand collection)
3
Figure 2
Research Map in Kapuas Estuary (Pulau Panjang). Located in
the western of Pontianak city (West Kalimantan) and faces
Karimata Strait.
Strait
Figure 3 Spiral labyrinth glass (A) and Strobist Macro Photo Studio (B)
4
Figure 4 (A) Normal color was bright pink (Glasby et al. 2007); (B) Dark pink
pattern was color with almost dark in all part of body; (C) Brown
pattern was having patch of dark color (like black).
Object of research
arch is 73 individual (38 males and 35 females). Nypa worm
is extraovarian polychaeta ((Eckelbarger 2005; Junardi 2010) so male
ale and female
worms were distinguished by the type of gametes in the coelomic fluids.
Coelomic fluid were taken using a glass capillary tube inserted between the nypa
worm's body segments. Individual males were characterized by sperm
sperms while
females by the presence of eggs. Immature male was determined by th
the form of
clusters of spermatogonia,, whereas females based on clusters of oocytes (Junardi
2008; Junardi et al. 2010). Length of individual sampel ranged from 23--157.3 cm.
Beside digital photogrammetry, color was determined by the eye of author.
Colors for all individuals were observed directly in the field and by looking at
digital images. Individuals were classified into several groups:: brown, dark pink
and bright pink (Figure 4).. Brown color if the body of nypa worm had a band of
dark color (mostly black) in dark or bright pink specimen.. When the dark band
mostly wider than pink, it appears dark pink. Bright pink if it show completely
saturated pink without any pattern. The proportion of sexual differences judged by
eye has been examined by the chi square test.
test
Colecting Digital Photos
hotogrammetry was used to analyze color. Photogrammetry provides a
Photogrammetry
method to give quantitative data (Linder 2006). Samples were photographed on
spiral labyrinth glass to limit the movement of the worm (Figure 3A)
A). Images
were taken while the worm was alive to avoid any discoloration of the worm.
Image capture was done using Strobist Macro Photo Studio with external flash for
light stabilization using 4 external flash YN 460-II
460 Speedlight (ShenZhen
YongNuo Photographic Equipment Co. Ltd) (Figure 3B).
B). Ambient light condition
was adjusted with 18% reflective gray card (Mennon, China). Color
olor temperature
were set to 6000 K to adapt the ambient light condition at shooting time.
Digital images of nypa worm was recorded with a Canon EOS 1100 D
digital SLR camera (12.2 megapixel, fitted with a Canon EF
EF-S 18-55mm
55mm f/3.5
f/3.5-5.6
IS lens)) (Canon Inc, Taiwan) in RAW format. Camera RAW files is a near
near-to-thesubject recording enabling work to be done at the pixel level without in camera
processing occurring (Perez 2007).
20
RAW files were converted to 8-bit
bit TIFF files
using UFRAW (http://ufraw.sourceforge.net/
http://ufraw.sourceforge.net/).
5
Color Analysis
Measured area is the dorsal area, which is darker than other parts of the
body. Determination of the measured area is by selecting the whole dorsal body
seen in TIFF files as ROI (Regions of Interest). Data were processed with ImageJ
(http://imagej.nih.gov). Data was average from all pixels measured inside ROI.
The output from the camera (in this case EOS 1100D using CMOS sensor)
is interpreted in RGB values. RGB color space is not designed for human vision,
but for the physical display devices, for instance computer monitor (Baldevbhai
and Anand 2012). CIE L*a*b* color space was recommended by the CIE
(Commission Internationale de l'Eclairage) as a standardized interpretattion based
on color matching experiments using principle of trichromatic theory of color
vision (Perez 2007). CIE Lab color space describes the human colour vision and
independent of the device used as a reference. It give a good interpretation of the
color segmentation and most sensitive component to changes in practical
imaging conditions (Baldevbhai and Anand 2012). Therefore the values of RGB
colour spaces was converted to the corresponding values of CIE colour space.
CIE color space is a 3-dimensional hypothetical space with 3 color axes
(Perez 2007). Color is expressed via 3 parameters: L*, a*, and b*. The L* value,
represents the lightness of color responses, L* ranges from 0-100 (black to white);
the a* value from -60 (green) to +60 (red), represents the hue degree of green to
red; and b* value was -60 (blue) to +60 (yellow), represents the hue degree blue to
yellow. If the a* and b* values further away from 0 then the colors would be
brighter. Further, one can calculated the chroma value, which is a plain indicator
for the degree of color hue, from the a* and b* values as follows (Perez 2007). The
chroma value, ranging 0-85 (from square root of minimum and maximum square
of a* and b* value) :
Chroma = √
∗
+
∗
The sexual differences between males and females has been examined via
the Wilcoxon test (Koyabu et al. 2008). The relationship between the combination
of color values and the color determined by eye was using linear discriminant
analysis on L*, a*, and b* data. Calculation was done using R 2.11.0.
Histology
Histological analysis was performed to observed the anatomical difference
between the color groups. This was due to morphological and anatomical changes
occurring during maturation N. rhodochorde. Maturation is characterized by
changing of body color (red in females and green in males) and having softer
body than immature (Junardi 2012). Epidermis of polychaeta is composed of
pseudostratified and covered by a cuticle. Collagenous cuticle produced by the
epidermis covers the body wall of polychaeta. Epidermal cells consists of
secretory and non-secretory cells. Secretory cells play a role to produce mucus in
nypa worm (Hausen 2005; Mastrodonato et al. 2006).
Epidermal preparation of nypa worm was done using paraffin method.
Epidermal tissue samples were taken from each color group (bright pink, dark
pink and brown). Samples were fixed using FAAC (Formaldehyde Acetic Acid
6
ample was
Calcium Chloride) solution for ±3 days at room temperature. Sample
dehydrated with graded alcohol treatment and cleared using xylol. Paraffin
araffin was
used for infiltration and embedding.
embedding Tissue was cut using a rotary microtome
microtome.
Sample was stained using hematoxylin
h
and eosin.
3. RESULT
Identification
The identification of specimens showed that samples were indeed N.
rhodochorde (n=73). Characters
haracters used were body shape and colouration;
prostomium; peristomium and pharynx;
phary
parapodia; chaetae. Body shape was
almost uniformly rounded from the anterior to mid
mid-body but
ut slightly flattened
towards the posterior. Prostomium cleft anteriorly and the shape of antenae
relatively short subconical ((Figure 5A).
A). Palps biarticulate with compact
palpophores and palpostyles spherical (Figure 5B). The eye was not visible inside
peristomium.. Having 4 pairs short peristomial cirrus (Figure 5A-B). Pharynx was
had
invisible inside proboscis. Parapodia type was sub-biramous.. Each parapodia ha
dorsal and ventral cirrus.. D
Dorsal cirri anteriorly with cylindrical cirrophores,
posteriorly cirrophores were flattened (leaf-like) (Figure 6A). Notoacicula
otoacicula and
neuroacicula was present but had only neorochaetae
neorochaetae. Having sesquigomph
spiniger in supraacicular position.
position Lacking spiniger chaetae in subacicular
chaetae in supraacicular with smooth
position. Having heterogomph falcigerous
falci
teeth blade (Figure 6B).
Identification of males and females was using coelomic fluid to observed
gamets. The observation of coelomic fluid was found that they were mostly of
stages 2 or immature to sub-mature
sub
in gamet maturity (gamete sizes was ranged
from 25μm - 60μm) (Figure7).
(Figure7) The result showed that there was no mature worms
in all samples. This may be because samples were collected in December January, while according to Glasby (2007) spawning time in S
SeptemberNovember. There were 38 males and 35 females.
Figure 5 Dorsal (A) and lateral (B) view of Namalycastis rhodochorde
rhodochorde. antenae
(an); prostomium (pro); peristomium (pr); peristomial cirrus (Pc); palps
(P)
7
Figure 6 Laterall view (A) showed that sub-biramus
biramus type of 9th, 10th and 11th
parapodia.. (B) showed heterogomph falciger chaetae. ccirrophore (c);
chaetae (ct); blade (b)
Figure 7
Table 1
Gametes
ametes of nypa worm. (A) male gametes (cluster of
spermatogonia
atogonia). (B) female gametes (oocyte)
Number of individual with brown, dark pink or bright pink with eye
Sex
Male
N
Chroma
Female N
Chroma
Table 2
Brown
1
19.11
2
14.29-17.35
Dark Pink
4
15.94-19.79
4
16.43-19.41
Bright Pink
33
18.24-26.8
29
18.95-27.92
Coefficients of linear discriminants in each function
Function
First Function
Second Function
L*
-0.165
0.034
a*
-0.319
0.369
b*
-0.146
-0.650
8
Figure 8
Visualization the log eyes vs photogrammetry data on the first two
discriminant axes. Individual scores are plotted on the first
(horizontal) and second (vertical) discriminant axes. Bright pink
(1), dark pink (2) and brown (3). Proportion of trace first function
comprises 96.8% and the second function comprises 3.2% of the
variance
Table 3
Statistical Analysis of L* a * b * and chroma for each sex
Sex
n
Male
38
35
Female
*
*
*
L*
Mean
a*
b*
Chroma
SD
CV
Mean
SD
CV
Mean
SD
CV
Mean
SD
CV
50.9
5.58
11
15.7
2.72
17.3
15.8
1.9
12
22.4
2.85
12.7
50.7
4.77
9.42
15.6
3.24
20.8
15.4
1.83
11.9
21.9
3.27
14.9
L a b represent luminance, hue degree of red-greeness, hue degree of yellowblueness. Chroma is a plain indicator of color hue calculated as follows: Chroma
= √a∗ + b ∗ . n = number samples; SD; standar deviation; CV = coefficient of
variation
9
Correspondences of Color Determination by Eye and Photogrammetry
Color of living worms mostly bright pink to dark pink, after preservation
turned to pale yellow (Figure 5). Table 1 shows that, using the eye, the bright pink
was the dominant color in males and females while the least is brown. The
chroma values showed that bright pink was more vivid than dark pink and brown.
Based on the analysis using the chi square test ( p > 0.05), there was no difference
in proportion of different colors between males and females. Based on the
analysis using the Wilcoxon test (p >0.05), there is also no significant difference
between males and females colors judged by photogrammetry (Table 3).
The correspondence between color values from photogrammetry and color
determined by eye was assesed using discriminant analysis. Coefficients of linear
discriminants are in Table 2. Color hues of a* and b* rather than the L* luminance
value were strong determinants of color differentiation. Color hues of a* was the
highest value or the greatest influence in the grouping on discriminant analysis in
first function; b* on second function. Figure 8 show that most variation in samples
was grouped by first function (96.8%). In the first function, bright pink and dark
pink-brown were separated; the second function (3.2%) separate the groups dark
pink and brown.
Sexual Dimorphism in Color Variation
Statisitical analysis for males and females could be seen in the Table 3. The
L* value of 50.9 in males and 50.7 in females showed that they had neutral color
or grey. Value of a* in males was 15.7 and females 15.6, so the color was reddish.
The b* values in males was 15.8 and females 15.4, so they had a yellowish color.
Chroma describes the vividness or dullness of a color. Table 3 showed that
chroma value was 22.4 in males and 21.9 in females, it mean the chroma showed
the color were dull. All parametric value of male tended to have on average higher
than the females. Based on Tabel 3 males were more vivid than females.
Canonical discriminant analysis showed that the chroma is an important
determinant of color differentiation. Value of a* had the highest value so that red
has a big influence in the grouping.
Histology of Epidermis
Epidermis of N. rhodochorde was cellular monolayer covered by cuticle.
Cuticle were composed of fine fillamentous matrix that usually inhabits collagen
fibrils (Figure 9A1-B1-C1). Under epidermis there was circular muscular. Figure
4 showed the muscular fibres. Cuticle and muscle in all of types showed the same
composition.
Epidermis was pseudostratified by the presence of secretory and nonsecretory cells. Secretory cells were randomly distributed in the epidermis. There
was no difference between epidermal structure and their content in the dorsal and
ventral region of the body wall.Light microscopy showed that there were 3 types
of secretory cells based on color group. The first type (bright pink worm) showed
that composition of secretory cell were smaller and sparse (Figure 9A). The
second (dark pink worm) of secretory cell was closely spaced than bright pink
worm (Figure 9B). The third (brown worm) of secretory cell was bigger than
other type (Figure 9C).
10
Figure 9
Longitudinal section of epidermis of N. rhodochorde.. (A) Bright
pink worm. (B) Dark Pink Worm.
Worm (C) Brown worm. (cu) cuticle;
(ec) epidermis cell; (cm) circular muscle; (cf) collagen fibre; (sc)
secretory cell.
4. DISCUSSION
Glasby et al.. (2007) described that immature N. rhodochorde had bright
pink coloration which turned red (females)
(
and green (males) when matured
matured.
There is no reports on color variations in immature worms. Present study obtained
color variations in immature worms categorized as bright
right pink, dark pink and
brown. Most of the worms were bright pink and the least is brown. There was no
size differences between colors because the ranges
r
of body length were almost the
same. Oocyte size of bright pink worm were small ranging from 25µm - 35µm
(immature),
), while the dark pink were bigger ranging from 35µm - 50
50µm (submature) and
brown worm ranging from 25µm - 30µm (immature).
Spermatogenesis of bright pink and brown were in stage of the spermatogonia
cluster (immature). Dark pi
pink worms were found in the stage of spermato
spermatogonia
clusters but it was in smaller amounts. It was suspected that reduction
eduction of the
11
number of cells in the clusters was a step toward maturity (Junardi 2010). These
suggest that color variation related to gametogenesis.
Secretory cells have diverse function in polychaeta, for instances they
secrete mucus to help the worm to eat (Costa et al. 2006) and to move (Pardo and
Amaral 2004). In Syllidae, they used mucus to brood egg (Martin 2005).
Namanereis littoralis member of the same sub-family Namanereidinae as N.
rhodochorde, produce abundant secretion to ensure viability of sex products and
successful fertilization out of the water environment and protecting the animals
and their clutches from desiccation (Ezhova 2011).
From histology of epidermis we obtained three forms of secretory cell
within each group. Dark pink had more closed cell, it was suspected that cell more
active so that more of mucus in cell. Solid composition of the cell caused the cell
density also increased that make the color more darker. In females, this was
caused by the dark pink were a color that had toward to maturity (Junardi 2008)
which mean the worm was preparing mucus to keep the eggs. Dark pink also
found in males. This coused the males also was prepare to maturity.
Different with brown worms that had larger cells, it was suspected to be an
adaptation to the environment. This was caused nypa worm had a smaller range of
habitats (Junardi 2008). Polychaeta could been secrete mucus for adaptation to
environment. Laeonereis acuta (Nereididae) produced mucus to antioxidant
defence system of the worm against environmental (Moraes et al. 2006). But on
the contrary if there was exposure to pollutant (such heavy metal) it would be
interfere with the production of mucus such as Laeonereis acuta that had chronic
or acutely exposed to copper, mucus was absence on their body wall (Geracitano
et al. 2004).
In this study was not found mature samples. It was suspected when
sampling was not a of spawning time. Polychaeta spawning time is strongly
influenced by the physical environment conditions such as temperature, day
length, and lunar cycles (Belal 2012). Phytoplankton bloom is one of the
biological events that may cause the spawning mechanism as in Platynereis
dumerilii (Watson et al 2003). It was also suspected of sampling sites residing on
a single location because microhabitat conditions greatly affect the maturity.
Individual populations can breed at different conditions, according to local
conditions. In addition Nereidae can inhibit the maturation of gametes by
promoting growth and regeneration through hormonal mechanisms (Lawrence and
Soame 2004; Rouhi et al. 2008).
Nypa worm is one Nereididae Family and living on a muddy estuary
(Glasby et al. 2007; Junardi 2008). Nypa worm immersed himself into the ground
(infaunal), so we could not found nypa worms that exist on the surface. This may
cause the nypa worm had a high risk to environmental changes. Polychaeta was a
group that is often used as an indicator species because lived in sediment (Dean
2008; Alam et al. 2010). It was supported by the limited habitat choice of nypa
worm, because it could be at the nypa palm root. Nypa worm had a muddy-clay
habitat preferences and have a high organic carbon with low salinity and
temperature (Junardi 2008).
12
5. CONCLUSION
All samples included in immature to sub-mature category. Color analysis
showed no significant color differences between males and females in immature
worm. Through the eyes, obtained variations of color (bright pink, dark pink and
brown) with the dominant red in the grouping. In each group were also found
variations in secretions cell.
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14
CURRICULUM VITAE
Author was born in Sambas on June 18th, 1986 and as the first child of
Herwadi and Musfayati. Authors school at SMUN 1 Sambas in 2001-2004.
Subsequently continued his studies in Biology Faculty of Mathematics and
Natural Sciences at Tanjungpura University Pontianak 2004-2009. During the
lecture the author active in the activities of the Department of Biology Student
Association.