Journal of
Fisheries and
Aquatic Science
ISSN 1816-4927
www.academicjournals.com
Journal of Fisheries and Aquatic Science 11 (1): 85-92, 2016
ISSN 1816-4927 / DOI: 10.3923/jfas.2016.85.92
© 2016 Academic Journals Inc.
Spawn and Early Larval Development of Spanish Dancer
Nudibranch Hexabranchus sanguineus (Rüppell and Leuckart, 1828)
(Gastropoda: Nudibranchia)
Mostafa A.M. Mahmoud and Mahmoud Raafat
National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada, Red Sea, Egypt
Corresponding Author: Mostafa A.M. Mahmoud, National Institute of Oceanography and Fisheries, Red Sea Branch,
Hurghada, Red Sea, Egypt Tel: +201094576510
ABSTRACT
Hexabranchus sanguineus is best known as the Spanish dancer was found predominantly on
the shallow fringing reef platforms all around Hurghada. Egg ribbons, fecundity and growth of
larval stages of four specimens of H. sanguineus were studied in the laboratory. It lays a rose shape
egg ribbons, attached to suitable hard substrate, varied in lengths and breadths. The egg ribbons
lengths ranged from 19.68-20.28 mm. Eggs are clustered together in clear and transparent spawn
jelly capsules. Eggs are spherical in shape with 100-113 microns in diameter. The number of eggs
in the capsules was not the same in all ribbons, ranged between 8-34 eggs/capsule. The estimated
total fecundity of H. sanguineus ranged from 1.5×106-3.6×106 eggs. The small, translucent, slow
rotary movement trochophore larva was developed within capsules on the 6th day and reached
120-150 μm. The active mobile veliger larvae were released successively into the surrounding
medium like red fumes with 150-190 μm from 8-9 days. The shell possesses only a single spire. It
measures 170-200 μm in length and 130-150 μm in height.
Key words: Spanish dancer, nudibranchia, hexabranchus sanguineus, larval development
INTRODUCTION
Global distribution of opisthobranch (third large group of snail) is a specialized group of phylum
mollusk (Bouchet and Rocroi, 2005). Marine habitat of opisthobranch is illustrated by two pairs of
tentacles and a single gill located behind the heart. Morphologically diverse group of opisthobranch
represents over 6000 species in all over the world (Yonow, 2008) and engage the great variety of
ecological niches (Kristof and Klussmann-Kolb, 2010). As a defense mechanism it secretes strong
acids or toxins and follows the camouflage characters (Gosliner, 1987; Wagele and Klussmann-Kolb,
2005; Dhivya et al., 2012).
The nudibranchs are amongst the most beautiful and flamboyant of the marine gastropod
mollusks as well as the most highly evolved. Because of their attractive color many of them are
offered for sale in pet shops (Kasamesiri et al., 2014). They lack a shell as an adult although many
planktotrophic species hatch with a larval shell, which is lost at metamorphosis when the animal
assumes its adult body form. All species have lost the shell as adults (Yonow, 2008; Sreeraj et al.,
2012). Some nudibranch species have secondary metabolites which can be used as an antitumor,
antimicrobial and inhibitor for barnacle larval settling (He et al., 2014).
Hexabranchidae is a monotypic family of colorful nudibranchs (often called “Sea slugs”) which
contains only a single genus hexabranchus, with two species, Hexabranchus morsomus and
Hexabranchus sanguineus and has no subfamilies (Valdes, 2002).
85
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
(a)
(b)
R
R
2 cm
Fig. 1(a-b): (a) Dorsal view of Spanish dancer Hexabranchus sanguineus and (b) Rhinophores of the
animals
Hexabranchus sanguineus (Rüppell and Leuckart 1828) is best known as the Spanish dancer
and is endemic to the red sea, it has a deep red colour with white markings just inside the mantle
edge, which is held curled up when the animal is crawling. The rhinophores (Fig. 1b) are deep red
and the gills are paler with white axes. Owing to its large size, extraordinary colors and striking
forms, it is particularly conspicuous and easily obtained (Gohar and Soliman, 1963; Valdes, 2002).
The Spanish dancer nudibranch, H. sanguineus provides a well-documented example, reviewed
in Pawlik (1993) of sequestration of chemical defenses by a specialist consumer. It feeds on sponges
in the genus Halichondria that contain oxazole macrolides that deter predators. The nudibranch
modifies these compounds somewhat and concentrates them in its dorsal mantle and in its egg
masses where they serve as defenses against predators. Concentrations of the macrolides are little
in the sponge, higher in the nudibranch and highest in the egg masses but even the lowest natural
concentrations result in strong suppression of feeding by fish (Hay and Fenical, 1996; Debelius,
1998).
Hexabranchus sanguineus is hermaphrodite, the gonad is located on the right-hand side of the
body and the gonopore, where exchange of sperm is effected. Individuals are capable of fertilizing
each other after fertilization, the animals separate and crawl away to lay their spawn on suitable
substrata (Ramakrishna et al., 2010).
The breeding season extends the whole year round but mainly from April to November and
attains its climax in June and July. This activity gradually declines with the onset of the cold
months of December and January, though it does not cease entirely (Gohar and Soliman, 1963;
Yonow, 2008).
Little work has been done on the biology or ecology of hexabranchus as the comprehensive study
is that of Gohar and Soliman (1963) on Red Sea, Hurghada and Francis (1980) on Tongatapu
Island.
The main aim of this study is to determine and document the spawning habits, egg ribbons,
fecundity and growth of larval stages of H. sanguineus under laboratory conditions.
MATERIALS AND METHODS
Hexabranchus sanguineus (Fig. 1) were found predominantly on the shallow fringing reef
platforms all around Hurghada such as Abu-Galawa and Abu-Sadaf reefs all over the year but they
tend to be less active during the winter season. Eight mature specimens of H. sanguineus were
collected by snorkeling at depths up to 2 m from Abu-Sadaf reefs 900 m from the seashore in
86
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
front of National Institute of Oceanography and Fisheries (NIOF), Red Sea Branch, Hurghada, Red
Sea (latitude 27°17'37"N, longitude 33°47'10E), during May 2014.
Collected specimens were taken immediately to the wet laboratory at NIOF, Red Sea Branch
and placed in a 1.0 m3 capacity cylindrical fiberglass tank then in continuous observation in rearing
glass aquaria. Continuous flow of well aerated sea water was used.
During the first few days after hatching, the water in the tanks was partially changed daily but
when the larvae showed positive geotaxis, the still living ones were daily removed by a pipette to
new aquaria with fresh filtered sea water.
Although, the aerated sea water was used water temperature was controlled in the tanks using
heaters. Water temperature (°C), salinity (%), pH and dissolved oxygen were measured daily inside
the tanks using multiparameter to prevent over-stress. Developmental stages from egg to hatching
larvae were observed daily with a stereoscopic microscope.
Animals and egg ribbons dimensions were measured in millimeters using 30 cm soft plastic
ruler to prevent tissues damage. Two aspects of the spawns were observed, size of egg ribbons and
number and size of eggs. Eggs, pre-larval and larval stages of development were examined and
photographed alive with USB camera linked to a binocular stereoscopic microscope and their
dimensions were measured in microns using micrometer. Standard Deviations (SD) are given in
parentheses.
For the study of fecundity in H. sanguineus, the number of eggs per millimeter was counted
with an ocular micrometer under an optical microscope. The total number of eggs were calculated
based on 30 parts with 1 mm2 were taken randomly from the initial, middle and last portion for
each ribbon related to the ribbon dimension.
RESULTS
The collected animals of H. sanguineus (Fig. 1) had a large body in size, dorso-ventrally
compressed, especially when they extended, with generally oval outline. The dimensions body was
17.99 cm (±1.87) and 12.19 cm (±1.41) in length and breadth, respectively.
Immediately after transportation of the animals to the aquarium, they showed their remarkable
mating activities that will last for long periods may be several hours or even several days.
Four specimens only laid their spawn ribbons, rose shape in the laboratory (Fig. 2) after
bringing from the sea, the 1st and 2nd animals laid their ribbons in the wall of the fiber glass tank
within one day, the 3rd animals laid the ribbon in the middle tube which used for exchange water
after two days and the 4th animals laid the ribbon in the small glass aquaria after five days.
Water temperature (°C), salinity (‰) and pH were measured daily inside the tanks using
multiparameter, where the water temperature was controlled in the tanks using heaters and the
water temperature was 24°C, salinity was 41.1‰ and the pH was 7.9.
The four egg ribbons shape as the same, generally, spirally coiled in anticlockwise direction into
up to 4 unequal rounds and its dimensions varied from animal to another depends on the animal
size, the average length was 731.5 mm (±85.5) and the average breadth was 21.9 mm (±2.3), for the
four ribbons, as shown in Table 1. The color of the newly deposited egg ribbon ranged from reddish
orange to rose-red or dark red (Fig. 2) but by time the color been dark orange as a result of eggs
growth and metamorphosis process.
Eggs are clustered together in clear and transparent spawn jelly capsules which varied in shape
and dimensions (Fig. 2). Generally, eggs are spherical in shape, with 100-113 microns in diameter
and the red yolk globules are concentrated at the vegetative pole (Table 1 and Fig. 3). The number
of eggs in the capsules was not the same in all ribbons, ranged between 8-44 eggs/capsule according
87
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
(a)
(b)
(c )
(d)
Fig. 2(a-d): Four spawn ribbons of the four Hexabranchus sanguineus which laid their spawn
ribbons in the laboratory (a, b) Two specimens laid their ribbons in the wall of the
fiberglass tank within one day, (c) One specimen laid the ribbon in the middle tube
which used for exchange water after two days and (d) One specimen laid the ribbon in
the small glass aquaria after five days
(c)
(a)
( b)
350 µm
5 mm
1.0 mm
Fig. 3(a-c): (a) Part of egg ribbon (b) Eggs capsules and (c) Eggs from a portion of egg ribbon of
Hexabranchus sanguineus in different magnification
Table 1: Average size of specimens, egg ribbons and egg diameters
Animal dimensions
Egg ribbon
----------------------------------------------------------------------------------------------Samples
Length (cm)
Width (cm)
Length (mm)
Width (mm)
1st
15.6
10.8
652
19.5
2nd
18.8
12.3
703
21
3rd
19.3
13.1
715
22
4th
19.9
13.8
852
25
Average
18.4±2.0
12.4±1.5
730.5±85.5
21.9±2.3
88
Egg diameters
-----------------------------------------Length (μm)
Width (μm)
110±2.65
102±4.03
105±5.45
101±3.99
112±1.02
102±2.87
111±1.09
099±5.69
110±3.12
102±1.61
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
to the size of the animal and the position of the capsule in the beginning or in the last portion in
egg ribbon. The estimated total fecundity of H. sanguineus based on number of eggs per one square
millimeter ranged between 1,510,423-3,621,000 eggs (Table 2).
Development begins immediately after oviposition within egg ribbon up to veliger larvae in
about eight days. Hatching starts along the whole ribbon almost at the same time because the
embryos of one spawn (same ribbon) are nearly of the same age and due to the short period of
oviposition, the embryo (Fig. 3) reached 100-130 μm on 4th day after hatching within capsules. The
small, translucent, slow rotary movement trochophore larva was developed within capsules on the
6th day and reached 120-150 μm.
From 2-3 days before hatching Ribbon loses its flexibility and cohesion and become flaccid and
soft moreover the colour of the ribbon loses its brightness and become dirty red blessed. The
developmental larvae become in active circling movement within their capsules, searching for an
exit from the ribbon. The water movement effects on larvae liberation from egg ribbons
(Fig. 4b). From 8-9 days the active mobile veliger larvae (Fig. 4) were released successively into the
Table 2: Average numbers of egg ribbons, egg clusters and eggs in each animal
No. of capsules
-----------------------------------------Samples
1.0 mm2
Ribbon
No. eggs per capsule
1st
6.6±1.3
83,912
18.2±9.6
2nd
7.1±1.5
104,817
16.7±7.8
3rd
6.1±2.2
95,953
22.6±4.4
4th
6.8±1.9
144,840
25.1±2.9
Average
(a)
(b)
No. of eggs (mmG2)
120.2±10.8
118.6±8.7
137.9±15.6
170.1±20.2
(c)
Fecundity
1,510,423
1,677,076
2,110,966
3,621,000
2,229,866
C
C
C
AG
v
AG
Sh
M
F
Sh
25 µm
(d)
(e)
Rc
Mc
St
M
F
Lh
Op
AG
Sh
Vs
Fig. 4(a-e): Showing larval development stages of Hexabranchus sanguineus (a) Dorsal view of
6 day old embryo, (b) Ventral view of 8 day old embryo, (c) Ventral view, (d) Lateral
view of newly hatched veliger larvae with 10 day old showed two nearly 8 shaped lobes
with markedly long and active cilia and (e) Colorless larvae shell which appeared
in the larval stages only, Mc: Mantel cavity, Lh: Larval heart, AG: Anal gland,
Rc: Retractile cilia, St: Statocyst, M: Mouth, F: Foot, Op: Operculum, Sh: Shell,
Vs: Visceral mass, C: Cilia, V: Velum
89
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
surrounding medium like red fumes (Fig. 4c and d) with 150-190 μm and swim actively upwards
then it moves down towards the bottom at the corner of the aquarium. In the newly hatched veliger
larvae, the mantle, operculum and shell cavity were observed which were in a reddish color.
The shell (Fig. 4e) possesses only a single spire. It measures 170-200 μm in length and
130-150 μm in height. It is nearly colorless and growth lines are moderately visible.
DISCUSSION
Nudibranchia is the largest group in opisthobranchia with more than 3500 described species.
The gills can be used for identification. Some species wave their gills as they move or feed.
Nudibranchs live in marine environments from Antarctica to the tropics and dwell at virtually
all depths of sea but reach their greatest size and variation in warm, shallow waters
(Ramakrishna et al., 2010).
Nudibranchs lay their eggs in flat ribbons attached to rocks or other objects (dorids) or in
tangled masses attached either to the sea bottom or to algae and other objects (aeolids). The eggs,
often multiply enclosed in capsules (as opposed to singly enclosed) are embedded in a mucous
matrix that both supports them and protects them (Goddard and Green, 2013). Hexabranchus
sanguineus as a dorids, laid their eggs in flat ribbons attached to the wall of the fiberglass tank,
in the middle tube which used for exchange water and in the small glass aquaria in the laboratory.
Nudibranch egg masses can be identified by placement, color, dimensions, number of eggs
per capsule and capsule spacing (Hurst, 1966). The color of the newly deposited egg ribbon of
H. sanguineus ranged from reddish orange to rose-red or dark red as mentioned in Gohar and
Soliman (1963).
The number of eggs is depending on the availability of food and the environmental influences,
which may be considered as controlling factors such as water temperature, salinity and pH
(Mahmoud et al., 2013) and also dependent on size ranging from 3×106-140×106 (Hadfield and
Switzer-Dunlap, 1984). The present study was agreed with the study of Gohar and Soliman (1963),
which showed that the period which the embryos spend within the capsules before hatching
depends mainly upon the temperature of the surrounding water while it lasts only 6 days at 27°C,
it extends up to 12 days at 22°C. In the present work the period extended to 10 days at 24°C. The
number of eggs ranged between 1.5×106-3.6×106 eggs was same as that of Gohar and Soliman
(1963) in the same species sizes (1.9×106-4.0×106 eggs).
Spawn is characteristic for each species in nudibranch species, in shape and form, colour size
and arrangement of eggs within. The eggs hatch either into planktonic veligers, a swimming larval
phase which then settles and begins benthic life or into a minute crawling juvenile (Yonow, 2008;
Mahmoud et al., 2013). Most nudibranch species produce planktonic larvae that remain in their
larval phase for periods of minutes to months, sometimes dispersing over great distances before
settling on suitable substrata (Todd et al., 2001). The larvae of H. sanguineus hatches as a
swimming veliger and metamorphosis will occur after a few days in the plankton.
Various life-cycle stages of opisthobranch mollusks have served for research in such diverse
areas as behavior, development and ecology (Faucci et al., 2007). In particular, adult
opisthobranchs have become premier models for neurobiological investigations, because neurons
in their central nervous system are large and easily identifiable and manipulated (Cohen et al.,
2006; Schlesinger et al., 2009).
Hexabranchus sanguineus veliger larvae as nudibranch veligers are all relatively similar
anatomically. Some features do different color of digestive gland, possession of eyes, behavior
90
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
within egg capsule, behavior after hatching and dimensions of the shell and sculptural patterns of
the shell (Thompson, 1966). The hatching veligers swim upwards after hatching and after a few
hours they begin to swim randomly over the bottom. This corresponds to the two phases of veliger
behavior found for the nudibranchs studied by Hadfield (1963) and the same species by Gohar and
Soliman (1963).
CONCLUSION
The present data showed that the perfect conditions to spawn H. sanguineus in the laboratory
must be in suitable conditions; 24oC for water temperature, 41.1‰ for salinity and 7.9 for pH.
Additional food is not required up to veliger larvae. Water renew is very important for larval
survival. The information obtained from this study may help to support enhancements in
nudibranch ecological and commercial culture for future utilization and conservation.
REFERENCES
Bouchet, P. and J.P. Rocroi, 2005. Classification and nomenclator of gastropod families,
Malacologia, 47: 1-397.
Cohen, A., J. Shappir, S. Yitzchaik and M.E. Spira, 2006. Experimental and theoretical analysis
of neuron-transistor hybrid electrical coupling: The relationships between the electro-anatomy
of cultured Aplysia neurons and the recorded field potentials. Biosens. Bioelectron., 22: 656-663.
Debelius, H., 1998. Red Sea Reef Guide. IKAN, Germany, Pages: 321.
Dhivya, P., V. Sachithanandam and P.M. Mohan, 2012. New records on the opisthobranch fauna
of the Andaman Islands, India. Indian J. Geo Mar. Sci., 41: 215-217.
Faucci, A., R.J. Toonen and M.G. Hadfield, 2007. Host shift and speciation in a coral-feeding
nudibranch. Proc. R. Soc. London B, 274: 111-119.
Francis, M.P., 1980. Habitat, food and reproductive activity of the Nudibranch Hexabranchus
sanguineus on Tongatapu Island. Veliger, 22: 252-258.
Goddard, J.H.R. and B. Green, 2013. Developmental mode in opisthobranch molluscs from the
northeast pacific ocean: Additional species from southern California and supplemental data.
Bull. Southern California Acad. Sci., 112: 49-62.
Gohar, H.A.F. and G.N. Soliman, 1963. The biology and development of Hexabranchus sanguineus
(Ruppell & Leuckart) (Gastropoda, Nudibranchiata). Publ. Mar. Biol. Station Al-Ghardaqa
Egypt, 12: 219-247.
Gosliner, T., 1987. Nudibranchs of Southern Africa: A guide to opisthobranch Molluscs of Southern
Africa. Sea Challengers, California, ISBN-13: 978-0930118136, Pages: 136.
Hadfield, M.G. and M. Switzer-Dunlap, 1984. The Mollusca. Academic Press, New York, USA.
Hadfield, M.G., 1963. The biology of nudibranch larvae. Oikos, 14: 85-95.
Hay, M.E. and W. Fenical, 1996. Chemical ecology and marine biodiversity: Insights and products
from the sea. Oceanography, 9: 10-20.
He, W.F., Y. Li, M.T. Feng, M. Gavagnin, E. Mollo, S.C. Mao and Y.W. Guo, 2014. New
isoquinolinequinone alkaloids from the South China sea nudibranch Jorunna funebris and its
possible sponge-prey Xestospongia sp. Fitoterapia, 96: 109-114.
Hurst, A., 1966. The egg masses and veligers of opisthobranchs. Annual Reports for 1966 of the
American Malacological Union, USA., pp: 64-65.
Kasamesiri, P., S. Meksumpun and C. Meksumpun, 2014. Embryonic development of nudibranch
species (Mollusca: Opisthobranchia) in the Gulf of Thailand. J. Coastal Life Med., 2: 931-939.
91
J. Fish. Aquat. Sci., 11 (1): 85-92, 2016
Kristof, A. and A.K. Klussmann-Kolb, 2010. Neuromuscular development of Aeolidiella stephanieae
Valdez, 2005 (Mollusca, Gastropoda, Nudibranchia). Front. Zool., Vol. 7.
Mahmoud, M.A.M., T.A.A. Mohammed and M.H. Yassien, 2013. Spawning frequency, larval
development and growth of Muricid gastropod Chicoreus ramosus (Linnaeus, 1758) in the
Laboratory at Hurghada, Northern Red Sea, Egypt. Egypt. J. Aquat. Res., 39: 125-131.
Pawlik, J.R., 1993. Marine invertebrate chemical defenses. Chem. Rev., 93: 1911-1922.
Ramakrishna, C.R., C. Sreeraj, C. Raghunathan, J.S. Sivaperuman and Y. Kumar et al., 2010.
Guide to Opisthobranchs of Andaman and Nicobar Islands. Zoological Survey of India, India,
pp: 1-196.
Schlesinger, A., R. Goldshmid, M.G. Hadfield, E. Kramarsky-Winter and Y. Loya, 2009. Laboratory
culture of the aeolid nudibranch Spurilla neapolitana (Mollusca, Opisthobranchia): Life history
aspects. Mar. Biol., 156: 753-761.
Sreeraj, C.R., C. Sivaperuman and C. Raghunathan, 2012. Addition to the opisthobranchiate
(Opisthobranchia, Mollusca) fauna of Andaman and Nicobar Islands, India. Galaxea J. Coral
Reef Stud., 14: 105-113.
Thompson, T.E., 1966. Development and life history of Archidoris pseudoargus. Malacologia,
5: 83-84.
Todd, C.D., W.J. Lambert and J. Daviee, 2001. Some perspectives on the biology and ecology of
nudibranch molluscs: Generalisations and variations on the theme that prove the rule.
Bollettino Malacol., 37: 105-120.
Valdes, A., 2002. How many species of Hexabranchus (Opisthobranchia: Dorididae) are there?
Molluscan Res., 22: 289-301.
Wagele, H. and A. Klussmann-Kolb, 2005. Opisthobranchia (Mollusca, Gastropoda)-more than
just slimy slugs. Shell reduction and its implications on defence and foraging. Front. Zool.,
Vol. 2. 10.1186/1742-9994-2-3
Yonow, N., 2008. Sea Slugs of the Red Sea. Coronet Books Incorporated, Bulgaria, ISBN-13:
9789546423276, Pages: 304.
92