Observations on embryonic development of black-spot Jorunna, Jorunna funebris (Kelaart, 1859) (Gastropoda: Nudibranchia).
Article Type: Report
Subject: Embryonic development (Physiological aspects)
Gastropoda (Physiological aspects)
Authors: Kasamesiri, Pattira
Meksumpun, Shettapong
Meksumpun, Charumas
Pub Date: 04/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: April, 2012 Source Volume: 31 Source Issue: 1
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Thailand Geographic Code: 9THAI Thailand
Accession Number: 288172743
Full Text: ABSTRACT Jorunna funebris is one of the nudibranch species with the largest distribution worldwide, but there is lack of information about its life history. To provide more information on embryonic development, 19 adult nudibranchs ranging in length from 1.3-6.5 cm were collected from the Gulf of Thailand for examination of embryonic development under laboratory conditions. The seawater conditions in the broodstock aquaria were as follows: temperature range, 22.3-26.3[degrees]C; salinity, 27.9-34.0 psu; dissolved oxygen, 7.0-8.3 mg/L; and pH, 7.2-8.9. Each nudibranch laid only I egg mass after culturing for 1-3 days. The egg masses of J. funebris ranged from 17.1-48.0 cm in length, and between 0.4 cm and 0.7 cm in width. The density of egg capsules varied from 27-64 egg capsules/[mm.sup.2] of egg mass (n = 4), and each egg capsule had 1-4 larvae inside (n = 9). The embryonic period (expressed as the mean [+ or -] SD) was 7.3 [+ or -] 1.0 days (n = 7 egg masses). Based on the results of uncleaved embryonic diameter, embryonic period, and shell pattern, the development mode of J. funebris could be defined as "planktotrophic development."

KEY WORDS: nudibranch, Jorunna funebris, Dorididae, embryonic development, veliger, Gulf of Thailand

INTRODUCTION

The nudibranch, Jorunna funebris (Kelaart, 1859) is widely distributed along the coastline of the tropical Indo-Pacific (Sittithaweepat 2001, Camacho-Garcia & Gosliner 2008). The spatial distribution of J. funebris is driven largely by food supply. J. funebris is a prey-specific nudibranch feeding only on blue sponge Xestospongia sp. (Fontana et al. 2001; Duramas et al. 2007, Chavanich et al. 2008, Viyakam et al. 2008). Kasamesiri (2010) reported that more than 65% of colonies of this sponge were inhabited by J. funebris. The distribution of J. funebris was in clumped and/or spatial dispersion patterns. Aggregation behavior during spawning of this nudibranch has not been reported, unlike with other gastropods (Kulikova & Omel'yanenko 2000), such as the large numbers of aplysiids (Stylochelilus striatus) (Switzer-Dunlop 1978) and dorids (Onchidoris bilamellata) (Claverie & Kamenos 2008) that can be found copulating together (breeding aggregation), which provides a greater fertilization efficiency, and large populations of adults will be found in close proximity to each other (Rudman & Willan 1998).

There have been many studies conducted on the biochemistry, taxonomy, and distribution of nudibranchs, but few works have studied the embryonic development of J. funebris (Sittithaweepat 2001, Saito et al. 2004, Wahidullah et al. 2006, Camacho-Garcia & Gosliner 2008). The embryonic development of nudibranchs have been divided into three major types--namely, planktotrophy, lecithotrophy, and the direct mode (Goddard 2004). The studies of nudibranch embryonic development has been limited to the temperate zone and include Rostanga pulchra (Chia & Koss 1978), Doridella steinbrege (Bickell & Chia 1979), Hermissenda crassicornis (Avila et al. 1997), and Cadlina flarornaculata (Goddard 2004). To date, there have been no reports on the embryonic development of J. funebris in Thai waters. LI. funebris is under threat because its features and attractive colors make it a popular aquarium acquisition, and many individuals are offered for sale in pet shops (Rudman 2002, Tserda 2005). For successful conservation measures to be implemented, the life history and ecology of the species need to be clarified, especially its embryonic development.

There have been no previous reports on the successful culture of J.funebris from the veliger to the juvenile stage under laboratory conditions. This may be a result of the fact that the veligers of this nudibranch require a long period (at least 4 days) in planktotrophic development (Kasamesiri 2010), and they are attracted by a chemical cue from their diet to enter metamorphosis and settle down (Avila 1998). The main objective of the current study was the characterization of the egg masses and the embryonic development of J.funebris. This information can be used to assist in nudibranch culture development and conservation planning.

MATERIALS AND METHODS

Study Site

Nudibranchs, Jorunna funebris (longer than 1 cm) were collected at depths of 3-10 m from coral reefs on the eastern coast of the Gulf of Thailand, near Krok Island, Pattaya (12[degrees]56'33.42" N, 100[degrees]47'32.48" E) in December 2006 and January 2008, and near Mannai Island, Rayong province (12[degrees]36'34" N, 101[degrees]41'13" E) in December 2008 (Fig. 1). Both collection sites were on flat reefs dominated by massive hard corals (mainly of the genus Porites), table corals (genus Acropora), and other sessile animals, such as sponges, sea anemones, and macroalgae. The nudibranchs were collected using scuba-diving equipment during the daytime (from 11 AM-2 PM). The environmental conditions (temperature, salinity, dissolved, oxygen and pH) at the study sites were measured during nudibranch collection using a MultiParameter Water Quality Sonde model YSI6600 (YSI Incorporated, Yellow Springs, OH).

[FIGURE 1 OMITTED]

Culture Conditions

After collection (5 nudibranchs for December 2006, 10 nudibranchs for January 2008, 4 nudibranchs for December 2008), the adult nudibranchs were placed into separate aquaria (60 x 40 x 45 cm) and supplied with aerated natural seawater. Conditions in the laboratory aquaria at the Department of Marine Science, Kasetsart University were controlled so that the seawater temperature was in the range of 23.0-26.0[degrees]C and salinity was 30.0-32.0 psu, with seawater monitoring conducted twice a week to adjust the salinity to compensate for evaporation of seawater from the tanks. As soon as possible after copulation and spawning, egg masses were removed to 250-mL Erlenmeyer flasks containing sterilized seawater (natural seawater from the study site was autoclaved at 121.0[degrees]C for at least 15 min before placing it in an incubation chamber). The egg masses were maintained in an incubation chamber at 25.0-26.0[degrees]C, with a cycle of 12 h light and 12 h darkness. The culture seawater was changed every 2 days until hatching.

After hatching, veligers were transferred to small tanks (40 x 20 x 25 cm) with very gentle aeration under the same environmental conditions as the broodstock tanks. The flasks containing veligers were placed in the tanks and veligers were released underwater to prevent any mortality of veligers from air-water interface entrapment. The density of veligers was kept to 300--450 individuals per 100 mL (Bickell & Kempf 1983). Veligers were fed on Tetraselmis sp. at the rate of 1 x [10.sup.4] cells/mL once daily. The salinity in the veliger tanks was adjusted to about 32.0 psu every 2 days.

[FIGURE 2 OMITTED]

Egg Mass Characteristics and Embryonic Development Observation

The egg masses were observed and categorized by morphological descriptors such as shape, color, rotation form, and extracapsular yolk according to a scheme introduced by Hurst (1967) and Wilson (2002). Hurst (1967) suggested 4 categories of egg masses: coiled ribbons, egg cords, ovoid or globular jelly masses, and saclike masses. Egg masses of the family Chromodorididae can be classified into 3 types; type A is laid flat on the substratum, type B is laid upright and may slope inward, and type C is laid upright, slopes outward, and may be crenulated (Wilson 2002). The density of egg capsules was counted under a light microscope (Olympus CX41) by cutting a piece of egg mass (approximately 4 [mm.sup.2]) from 4 egg masses (observed in 3 replicates from each egg mass), and 9 egg masses were observed to determine the number of embryos per egg capsule (observed in 3 replicates from each egg mass).

Embryonic development was monitored using a light microscope with a digital camera (Olympus DP12) by observing a piece of egg mass (approximately 4 [mm.sup.2]) instead of the whole egg mass. The whole egg mass was kept in an incubator, with the temperature controlled at all times to reduce the impact of interference that might occur from the microscope light. A second piece of egg mass was examined to compare with the first piece to ensure that both samples were at a similar stage of development and had hatched during the same period. Every day until hatching, diameters of the embryo and egg capsule (outermost encapsulating structure of the egg) were measured from a random sample of at least 10 embryos and egg capsules per egg mass using an ocular micrometer, and embryonic development was monitored at the same time. The 9 stages of development (oviposition, first division of cleavage, 4-cell stage, 8-cell stage, morula, blastula, gastrula, motion of larvae by velar rudiment, and early veliger stage) proposed by Chia and Koss (1978) and Buckland-Nicks et al. (2006) were used to identify progress. Embryo diameter was measured using an ocular micrometer every hour on the first day and then every 24 h until hatching occurred. The appearance of larval structures, such as the velum, statocyst, left and right digestive diverticula, eyespots, and propodium was recorded. The shell patterns were categorized according to Thompson (1961). Veliger feeding behavior was observed every day after hatching until death (approximately 4 day).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

RESULTS

Study Site and Environmental Conditions

Generally, J.funebris (Kelaart, 1858) was found on the coral reefs of Krok and Mannai islands. This nudibranch was found in association with the blue sponge (Neopetosia sp.). The environmental conditions (expressed as the mean [+ or -] SD) near Krok and Mannai islands inhabited by J. funebris during nudibranch collection in 2006 and 2008 were as follows: temperature, 25.7 [+ or -] 1.6[degrees]C; salinity, 30.4 [+ or -] 0.5 psu; dissolved oxygen, 7.3 [+ or -] 1.7 mg/L; and pH, 8.8 [+ or -] 0.2.

Broodstock and Egg Mass Characteristics

J. funebris was observed to be a nudibranch that reproduced hermaphroditically, with sperm exchanged between mating partners during copulation. The penis of each partner is located on the right-hand side of the body, so that one of the partners is upside down when sperm is transferred simultaneously between them (Fig. 2A). The copulation period ranged from 5 to 120+ min. Although there were 19 nudibranchs in the current study (Fig. 3), ranging between 1.3 cm and 6.5 cm in length, only 9 nudibranchs (size range, 3.5-6.5 cm) laid egg masses under laboratory conditions. The ranges in environmental conditions were as follows: temperature, 22.3-26.3[degrees]C; salinity, 27.9-34.0 psu; dissolved oxygen, 7.0-8.3 mg/L; and pH, 7.2-8.9. Oviposition took about 4 h for each egg mass, which ranged in length between 17.1 cm and 48.0 cm, and in width between 0.4 cm and 0.7 cm. The mean density [+ or -] SD of egg capsules varied from 27 [+ or -] 1.5 (n = 3) to 60 [+ or -] 4.7 (n = 3) egg capsules/[mm.sup.2] of egg mass (Table 1).

The egg masses of J. funebris can be described as being type A opisthobranch, which is typical of dorid nudibranchs (Hurst 1967), with thin ribbons attached along the length of one edge to the substratum. The ribbons are upright or slope outward, and are crenulated or curved (Fig. 2B, C). The egg masses were white (from the color of the zygotes) at oviposition, but with subsequent development they changed to purple (the shell color changed from transparent to purple; Table 2).

[FIGURE 5 OMITTED]

Embryonic Development

The number of embryos inside the egg capsules of J. funebris ranged from 1 to 4 embryos per egg capsule (number of egg masses, 9; 10 egg capsules per egg mass). It was found that most egg capsules contained 1 to 2 embryos per egg capsule, and only nudibranchs with a mean body length greater than 4.0 cm could produce 4 embryos per capsule. The average diameter [+ or -] SD of the uncleaved embryos was 112 [+ or -] 11 [micro]m (n = 27; Fig. 4A). The embryonic development stages within the egg capsule are summarized in Table 2. The cleavage stage in the early embryo occurred within 5-6 h after oviposition (Fig. 4B), and all embryos showed asynchronous cell division. The embryos were the same size as the original zygotes or slightly larger, depending on the axis of the blastomeres, which caused some variability in measurements. The asymmetric cell division became apparent at the 8-cell stage (Fig. 4C), with 4 cells of macromeres at the vegetal pole (lower hemisphere) and 4 cells of micromeres at the animal pole (upper hemisphere). The morula stage appeared 16 h after oviposition (Fig. 4D) and remained at this stage for more than 10 h. The blastula stage then followed. The blastocoels were observed and developed quickly to the gastrula stage within 2 days. Then, 3 days after oviposition, the embryos began to move by using cilia on the velar rudiment (Fig. 4E). Thereafter, they developed their velum, shell, and visceral organs to the veliger stage (Fig. 4F). The total embryonic period was 6-8 days after spawning, with an average [+ or -] SD of 7.3 [+ or -] 1.0 days (n = 7 egg masses) with a temperature range of 25-26[degrees]C.

The current study indicates that egg capsules of J. funebris can enlarge to support embryonic development until hatching. The capsule and embryo diameter relationship (Fig. 6) is given by Eq (1) (n = 80, [R.sup.2] = 0.9782):

Capsule length ([micro]m) = 2.152 (Embryo length ([micro]m)) - 14.036 (1)

Hatched Veliger Characteristics

The larval shells of J. funebris, which were thin and transparent, can be described as type 1, following Thompson (1961). There was a small amount of stored food and cilia inside the digestive tract of a newly hatched veliger. The newly hatched veligers were observed to be positively phototactic. Veligers moved toward the water surface and were trapped by the air-water interface. This behavior caused mass mortality during the veliger stage (usually within 2-3 days after hatching).

The total 99 veligers were observed for their feeding behavior. After feeding on Tetraselmis sp., veligers transferred their food through the mouth, esophagus, and mid gut, respectively. The mid gut was composed of a stomach and 2 digestive glands, (with the left usually bigger than the right). Enlargement of the left digestive diverticulum (from 35-47 [micro]m) was observed in veligers after feeding with Tetraselmis sp. (Fig. 5). A greenish pigment was observed in the left digestive gland of the veligers before transfer to the end of intestine after digestion.

DISCUSSION

The percentage of J. funebris spawning in the laboratory was only 47%, which may have been caused by inappropriate environmental conditions and the immaturity of the small nudibranchs (length, <2.0 cm). Kasamesiri (2010) and Rudman (2002, 2008) reported that small nudibranchs could not lay their egg masses under laboratory conditions, which was a finding consistent with the current study in which nudibranch size was more than 3.5 cm for egg masses laid in the laboratory. The results of the current study indicated that 4 embryos could fit inside an egg capsule of an adult J. funebris with a mean body length greater than 4.0 cm. This was probably a result of the maturity of adults, which was related to their body length.

[FIGURE 6 OMITTED]

Nudibranchs had the ability to spawn readily in captivity, perhaps in response to the stress resulting from capture and handing (Hadfield & Switzer-Dunlap 1984). The other reason why the percentage of spawning was low could have been a result of the specimens laying their egg masses before they were captured and brought back to the laboratory.

Fertilized embryo size has been used to categorize the type of development (Kolbin & Kulikova 2008). Based on the conclusions by Thompson (1967), the embryo size of J.funebris can be classified in the small-embryo (30-170 [micro]m) group that usually develops into planktotrophic larvae. After being considered together with other factors, the mode of development of J. funebris should be via planktotrophic larvae, because of the rapid larvae development from small embryos without the propodium and eyespots, with an embryo diameter mean [+ or -] SD of 86.2 [+ or -] 1.7 [micro]m (Goddard 2004).

In the current study, the larvae of J. funebris in the egg capsules at the edge of egg masses usually hatched earlier than those at the inner sites of the egg masses. This was probably a result of differences in oxygen diffusion through the egg mass (Chaffee & Strathmann 1984). The culture condition of the egg mass may also influence the time required for prehatching development (Chia & Koss 1978). Furthermore, the development rate has been reported to depend on such parameters as water temperature and the quantity and quality of food (Kulikova & Omel'yanenko 2000). Differences in the temperature of the culture water have pervasive effects on oxygen consumption and transport within the egg mass (Moran & Woods 2007). Because the egg masses of J. funebris in the current study were incubated at 25.0-26.0[degrees]C under a closed system, the prehatching development period of embryos was almost identical.

Chester (1999) suggested that food stored for embryonic development in the digestive tract was different between the modes of development. The lecithotrophic species contained much more stored food inside the digestive tract than the planktotrophic species. Thus, a small amount of stored food inside the digestive tract of J. funebris could be used to define this species as a planktotrophic species. The other major difference between lecithotrophic and planktotrophic species was considered by Bickell and Chia (1979) to be in the structure of the larval mid gut as both development types have complete digestive tract formation, but the gut of planktotrophic species is more complex, involving a ciliated tract composed of more differentiated cells than in lecithotrophic species. J. funebris veligers also have cilia inside, which is one of the characteristics of planktotrophic veliger.

Enlargement of the left digestive diverticulum in veligers after feeding with Tetraselmis sp. confirmed the development mode of J. funebris is planktotrophic development. In the case of Berghia verrucicornis, Carroll and Kempf (1990) suggested that the stomach and the left and right digestive diverticula were similar during the initial phase, but that the right digestive diverticulum decreased in size during development because yolk reserves inside were reduced, and the left digestive diverticulum of lecithotrophic larvae remained unchanged. For planktotrophic larvae, such as J. funebris, feeding larvae will enlarge their left digestive diverticulum after they consume phytoplankton during the veliger stage. The left digestive gland of Doridella steinbergae also continues to enlarge as embryonic development proceeds (Bickell & Chia 1979). Overall, the results from the current study provided more information on the embryonic development of J. funebris, and confirmed that J. funebris has planktotrophic development.

ACKNOWLEDGMENTS

This study was supported by a research scholarship from the Graduate School, Kasetsart University. We gratefully acknowledge the assistance of associate professors Seangtein Aujjimangkul and Jantra Srisomwong. We give special thanks to Dr. Juliana M. Harding and Prof. Sandra E. Shumway for editing the manuscript and for their helpful comments. We also thank the staff of the Marine Environmental Laboratory and the Fishery Resources Laboratory, Kasetsart University, for ensuring the success of the field operations.

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PATTIRA KASAMESIRI, (1) SHETTAPONG MEKSUMPUN (1,3) * AND CHARUMAS MEKSUMPUN (2)

(1) Department of Marine Science, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand;

(2) Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand;

(3) Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

* Corresponding author. E-mail: ffisspm@ku.ac.th

DOI: 10.2983/035.031.0114
TABLE 1.
Mean embryonic production from 4 adults 1 day after capture
and reared in the aquarium.

Egg    Width   Length           No. of
Mass   (cm)    (cm)       Embryos/[mm.sup.2]

1      0.65    48.00    27 [+ or -] 1.5 (n = 3)
2      0.45    26.50    42 [+ or -] 2.5 (n = 3)
3      0.50    17.10    60 [+ or -] 4.7 (n = 3)
4      0.40    18.10    34 [+ or -] 2.9 (n = 3)

                  No. of
Egg             Embryos/Egg
Mass               Mass

1      84,240 [+ or -] 2,207 (n = 3)
2      50,085 [+ or -] 1,687 (n = 3)
3      51,300 [+ or -] 1,209 (n = 3)
4      24,616 [+ or -] 2,090 (n = 3)

Average body length, 4-.3 cm; temperature, 25-26[degrees]C. Data
are mean [+ or -] SD.

TABLE 2.
Embryonic development stages of Jorunna funebris within the
egg capsule.

Developmental Stage                             Time(h or days)

Oviposition                                     0 h
First division                                  6 [+ or -] 1 h
  of cleavage
Four-cell stage                                 8 h
Eight-cell stage                                11 h
Morula                                          16 h
Blastula, appearance of blastocoel              2 days
Gastrula                                        2 days
Motion of larvae by velar rudiment              3 days
Early veliger stage, velum visible, viscera     4 days
  differentiated in stomach, left and right
  digestive diverticulum, shell color
  changed to purple
Hatched                                         6 days

No. egg masses, 3; temperature, 25.0-26.0[degrees]C.
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