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LARVICIDAL EFFECTS OF KOREAN SEAWEED EXTRACTS ON BRINE SHRIMP ARTEMIA SALINA.

Byline: Young Dae Kim and Jae-Suk Choi

ABSTRACT

A total of 38 methanol and water extracts of macroalgal species (18 brown, 14 red, and 6 green) collected from the Korean coast were investigated for their lethality to brine shrimp larvae. Of the solutions tested, methanol extracts of six species (15.79%) showed larvicidal effects against Artemia salina. Aqueous extracts of two species (5.26%) were lethal to A. salina. The brown algae Dictyota dichotoma, Ecklonia kurome, Ishige okamurae, Sargassum sagamianum, and Pachydictyon coriaceum, and the green algae Enteromorpha linza and Ulva pertusa, displayed significant larvicidal activity in 2.5% methanol extracts. None of the methanol extracts of the 14 red algae species tested had larvicidal effects. The brown alga Colpomenia bullosa and the red alga Lomentaria catenata displayed significant larvicidal activity in 2.5% extracts. No larvicidal effects were observed from extracts of green algae. These results offer insight into the larvicidal potential of Korean seaweed extracts.

Keywords: Seaweed extract, Larvicidal effect, Artemia salina, Enteromorpha linza, Gelidium amansii.

INTRODUCTION

Over the past several decades, seaweeds (marine macroalgae) and their extracts have generated great interest in the pharmaceutical industry as a source of bioactive compounds with immense medicinal potential. Seaweeds produce a great variety of secondary metabolites with a wide range of functions, making them potentially promising sources of pharmaceutical agents (Mayer and Hamann, 2002; Newman et al., 2003). Compounds with antiviral, antihelminthic, antifungal, antibacterial, antioxidant, anti-inflammatory, and anti-allergenic properties have been found in green, brown, and red algae (Newman et al., 2003; Wijesinghe and Jeon, 2011).

There are about 50 known species of edible seaweed in Korea (Kang 1968; Sohn 1998; Oh et al., 1990). Several Korean seaweeds are also used for medicinal purposes (Oh et al., 1990). Seaweeds may also contain toxic compounds including bromophenol (Wang et al., 2005; Choi et al., 2011), iodine (Mussig, 2009), arsenic (Sakurai et al., 2006; Almela et al., 2006), terpenoid (Ricci et al., 1999), lead, and cadmium (Almela et al., 2006; Khan et al., 2015). These compounds can be used to produce mosquito larvicide (Ali et al., 2013; Bianco et al., 2013), spermicidal compounds (Prakash et al., 2014), and biologics for killing ciliate protists (Ricci et al., 1999). However, indigenous Korean seaweeds have not yet been investigated for their larvicidal capabilities.

Brine shrimp lethality bioassays have been used to determine the cytotoxic activities of many seaweed species (Sukoso et al., 2012; Devi et al., 1998; Ara et al., 1999; Ayesha et al., 2010). However, the larvicidal activity of indigenous Korean seaweeds has not been researched until now.

Therefore, we conducted a brine shrimp lethality assay to determine the larvicidal activity of methanol and water extracts of 38 indigenous Korean seaweed species. Our results provide insight into the larvicidal potential of Korean seaweed extracts.

MATERIALS AND METHODS

Seaweed extracts: A total of 38 species of seaweed were collected from various locations in South Korea between November 2013 and April 2014. Seaweed tissues were washed with tap water to remove salt, epiphytes, and sand, then dried for 1 day at room temperature using an electric fan. A mill (Sinil Co., Seoul, Korea) was used for 5 min to grind the dried tissues into a powder, which was then passed through a 500 mesh sieve. To extract methanol-soluble components, 1 L methanol was added for 1 day to 20 g sieved powder. This process was repeated three times, and the combined extracts were evaporated until completely dry. Then distilled water (1 L) was added to the remaining powdered tissue to extract water-soluble components. Stock solutions were prepared by adding 1 mL methanol or distilled water to 100 mg dried extract and filtering through a 0.22 um filter. Stock solutions were stored at -20degC until needed (Choi et al., 2011).

Artemia salina hatching: Dried brine shrimp eggs (Artemia salina; SWORM, Seoul, Korea) were hatched in 0.22 um filter-sterilized seawater (1 g eggs per L) at 20degC, with mild aeration, under constant illumination (light intensity: 40 umol/m2/s). The eggs were incubated in a glass flask (250 mL) with a water height of 20 cm. These hatching conditions correspond to the natural environment of A. salina. Shrimp larvae were used for the experimental bioassay 24 h after hatching (Milhem et al., 2008). At this stage, nauplii larvae are still nourished by yolk sacs and we supplied no further food during the experimental period. The living larvae are highly mobile and phototaxic, making them easy to collect near a light source. Larvae were collected with a Pasteur pipette and concentrated in a small vial (Zhang et al., 2012).

Brine shrimp lethality assay: An assay was conducted to determine the toxic effects of seaweed extracts on Artemia salina nauplii larvae using the methods described in Milhem et al. (2008) and Vinayak et al. (2011), with minor modifications. Briefly, 100 uL aliquots of 0.22 um filter-sterilized seawater were pipetted into a 24-well plate. Each experimental condition was accompanied by positive and negative controls in separate wells, so that a total of three wells were used per condition. Brine shrimp larvae solution (50 uL, containing ca. 20 larvae) was added to each well along with individual seaweed extracts at varying concentrations (0.25%, 0.5%, and 2.5%). Sterilized seawater was added immediately to bring the final volume of wells to 200 uL.

Toxicity was determined after 2, 4 6, 12, 24, and 24 h of exposure by counting the number of survivors under a stereomicroscope (Olympus, Tokyo, Japan) and calculating the mortality rate. Solutions (50, 100, and 500 ug/mL) of potassium dichromate (K2Cr2O7; Sigma-Aldrich 207802) were used as a positive control, and 2.5% methanol and 0.22 um filter-sterilized seawater were used as a vehicle control and a negative control, respectively. The results of these assays are expressed as the means +- SDs from at least three separate experiments.

Mortality was evaluated using the criteria established by Solis et al. (1993), with slight modifications. Larvae were considered dead if they did not exhibit any internal or external movement during the observation period. Extracts with a mortality rate below 50% were considered non-larvicidal. Those with a rate higher than 50% but below 75% were considered mildly larvicidal. Those with a rate higher than 75% were considered highly larvicidal, and if 100% of larvae died, the extract was considered extremely larvicidal.

Statistics: Each experiment was repeated at least three times for each independent assay. Mean values of indices were compared to controls using Student's t-test.

RESULTS AND DISCUSSION

Larvicidal effects of methanol and potassium dichromate: Methanol at a concentration of 2.5%, used as a vehicle control, caused very little mortality among Artemia salina nauplii larvae (Fig. 1). Therefore, methanol seaweed extracts were always added to assay cultures in concentrations that resulted in less than 2.5% methanol. The survival rates in 50, 100, and 500 ug/mL potassium dichromate (K2Cr2O7), used as a positive control, were 97.6%, 80.0%, and 60.2% at 12h, and 91.7%, 38.9%, and 5.0% at 24 h, respectively.

Larvicidal effects of methanol extracts of six species of Chlorophyta: The larvicidal effects of methanol extracts of six species of Chlorophyta against Artemia salina nauplii larvae are shown in Table 1. The survival rate of A. salina exposed to a 2.5% concentration of Enteromorpha linza methanol extract was 83.3% at 4 h, 29.4% at 6 h, and 0.0% thereafter; thus, this extract was classified as extremely larvicidal. The survival rates after exposure to 2.5% Enteromorpha compressa and Ulva pertusa methanol extracts were 4.2% and 1.8% at 48 h, respectively; these extracts were classified as highly larvicidal. The rates for 2.5% Capsosiphon fulvescens and Monostroma nitidum methanol extracts were 37.0% and 33.3% at 48 h, respectively; these were classified as mildly larvicidal. The rates for 0.25%, 0.5%, and 2.5% concentrations of Codium fragile methanol extract were [greater than or equal to]79.4% at 12, 24, and 48 h; this extract was considered non-larvicidal (Table 1).

Larvicidal effects of methanol extracts of 18 species of Phaeophyta: The larvicidal effects of methanol extracts of 18 species of Phaeophyta have been examined. Among them, 9 species showed larvicidal activities on Artemia salina nauplii larvae (Table 2). Seaweed extracts considered extremely larvicidal included those from Pachydictyon coriaceum, Dictyota dichotoma, and Ecklonia kurome. The survival rates after exposure to 0.5% and 2.5% concentrations of P. coriaceum methanol extract were 0.0% at 24 and 48 h, and those for 2.5% D. dichotoma and E. kurome methanol extracts were 0.0% at 48 h. The rates for 2.5% Ishige sinicola, Saccharina japonica, and Undaria pinnatifida methanol extracts were 48.1%, 46.4%, and 47.2% at 48 h, respectively; these were classified as mildly larvicidal.

The rates for 0.25%, 0.5%, and 2.5% Colpomenia bullosa, Costaria costata, Ecklonia cava, Eisenia bicyclis, Hizikia fusiformis, Myelophycus simplex, Petalonia binghamiae, Sargassum micracanthum, and Sargassum thunbergii methanol extracts were [greater than or equal to]69.7% at 12, 24, and 48 h; these were classified as non-larvicidal (data not shown).

Larvicidal effects of methanol extracts of 14 species

Rhodophyta: The larvicidal effects of methanol extracts of 14 species of Rhodophyta have been examined. Among them, 4 species showed larvicidal activities on Artemia salina nauplii larvae (Table 3). The survival rate after exposure to 2.5% Gelidium amansii methanol extract was 0.0% at 12, 24, and 48 h; this extract was classified as extremely larvicidal. The rate for 2.5% Chondracanthus intermedia methanol extract was 5.0% at 48 h; this extract was classified as highly larvicidal. Those for 2.5% Chondracanthus tenella and Gracilaria verrucosa methanol extracts were 26.6% and 33.3% at 48 h, respectively; these were classified as mildly larvicidal.

Those for 0.25%, 0.5%, and 2.5% Ahnfeltiopsis flabelliformis, Chondaria crassicaulis, Chondrus ocellatus, Corallina pilulifera, Grateloupia elliptica, Hypnea charoides, Lomentaria catenata, Meristotheca papulosa, Prionitis cornea, and Symphyocladia latiuscula methanol extracts were [greater than or equal to]52.0% at 12, 24, and 48 h; these were classified as non-larvicidal (data not shown).

Larvicidal effects of aqueous extracts of six species of Chlorophyta: The larvicidal effects of aqueous extracts of six species of Chlorophyta have been examined. Among them, 3 species showed larvicidal activities on Artemia salina nauplii larvae (Table 4). The survival rates after exposure to 2.5% Enteromorpha compressa, Enteromorpha linza, and Ulva pertusa extracts were 27.3%, 48.1%, and 49.9% at 48 h; these were classified as mildly larvicidal. The rates for 0.25%, 0.5%, and 2.5% Capsosiphon fulvescens, Codium fragile, and Monostroma nitidum extracts were [greater than or equal to]64.8% at 12, 24, and 48 h; these were classified as non-larvicidal (data not shown).

Larvicidal effects of aqueous extracts of 18 species of Phaeophyta: The larvicidal effects of aqueous extracts of 18 species of Phaeophyta have been examined. Among them, 13 species showed larvicidal activities on Artemia salina nauplii are shown in Table 5.

The survival rate after exposure to 2.5% Colpomenia bullosa extract was 0.0% at 24 and 48 h; this extract was classified as extremely larvicidal. The rates for 2.5% Costaria costata, Dictyota dichotoma, Ecklonia kurome, Eisenia bicyclis, Ishige okamurae, Ishige sinicola, and Sargassum micracanthum extracts were 17.8%, 22.5%, 15.8%, 5.1%, 18.3%, 24.1%, and 3.1% at 48 h, respectively; these were classified as highly larvicidal. The rates for 2.5% Ecklonia cava, Hizikia fusiformis, Myelophycus simplex, Sargassum sagamianum, and Scytosiphon lomentaria extracts were 33.8%, 29.4%, 45.6%, 38.6%, and 33.3% at 48 h, respectively; these were classified as mildly larvicidal. The rates for 0.25%, 0.5%, and 2.5% Pachydictyon coriaceum, Petalonia binghamiae, Saccharina japonica, Sargassum thunbergii, and Undaria pinnatifida extracts were [greater than or equal to]64.8% at 12, 24, and 48 h; these were classified as non-larvicidal (data not shown).

Larvicidal effects of aqueous extract of six species of Rhodophyta: The larvicidal effects of aqueous extracts of six species of Rhodophyta have been examined. Among them, 3 species showed larvicidal activities against Artemia salina nauplii larvae (Table 6). The survival rate after exposure to 2.5% Lomentaria catenata extract was 0.0% at 24 and 48 h; this extract was classified as extremely larvicidal. The rates for 2.5% Chondracanthus intermedia and Prionitis cornea extracts were 46.4% and 42.5% at 48 h, respectively; these were classified as mildly larvicidal. The survival rates for 0.25%, 0.5%, and 2.5% Ahnfeltiopsis flabelliformis, Chondracanthus tenella, Chondaria crassicaulis, Chondrus ocellatus, Corallina pilulifera, Gelidium amansii, Gracilaria verrucosa, Grateloupia elliptica, Hypnea charoides, Meristotheca papulosa, and Symphyocladia latiuscula extracts were [greater than or equal to]64.1% at 12, 24, and 48 h; these were classified as non-larvicidal (data not shown).

Marine organisms are increasingly being studied with a view toward isolating biologically active compounds. Seaweeds are considered very attractive subjects for this type of research, due to their huge diversity and their long history of being safely consumed in traditional foods and herbal medicines in Asia (although it must be noted that some macroalgae are toxic).

Brine shrimp lethality bioassays have been used to determine the cytotoxic activities of many seaweed species (Sukoso et al., 2012; Devi et al., 1998; Ara et al., 1999; Ayesha et al., 2010). Sukoso et al. (2012), for instance, showed that extracts of Porphyra spp. were cytotoxic in brine shrimp. In another study (Ara et al., 1999), 22 ethanol extracts of seaweed species collected from the Karachi coast in Pakistan were investigated for their cytotoxicity in brine shrimp. Only six of these species, the brown algae Stoechospermum marginatum, Sargassum swartzii, S. binderi, Spatoglossum asperum, and Stokeyia indica, and the green alga Caulerpa racemosa, showed significant cytotoxic activity (Ara et al., 1999).

A third study, also of seaweeds growing near the Karachi coast, used brine shrimp lethality assays to screen ethanol extracts of the seaweeds Dictyota dichotoma var. velutricata, D. hauckiana, D. indica, Iyengaria stellata, Jolyna laminarioides, Melanothamnus afaqhusainii, Sargassum ilicifolium, S. lanceolatum and Ulva fasciata. The extracts of eight species (all but S. ilicifolium) showed significant cytotoxicity (Ayesha et al., 2010). Devi et al. (1998) found that Acanthophora muscoides and Microdictyon pseudohapteron collected from India were toxic to Artemia larvae.

In this study, we investigated the larvicidal activity of indigenous Korean seaweeds, which has not been researched until now.

Among the Chlorophyta, we found a 2.5% concentration of Enteromorpha linza methanol extract to be extremely larvicidal. In Phaeophyta, 0.5% and 2.5% Pachydictyon coriaceum methanol extract and 2.5% Colpomenia bullosa extract were also extremely larvicidal, as was a methanol extract of Rhodophyta species Gelidium amansii at 2.5%. Artemia larvae exposed to these solutions had a 0.0% survival rate after 24 h. However, two of these seaweed species (Enteromorpha linza and Gelidium amansii) are known to be edible to humans and are commonly used as food and animal feed in Korea and Japan (Kang 1968).

The red alga Symphyocladia latiuscula contains high concentrations of bromophenols (Wang et al., 2005), some of which can be toxic to bacteria and other living organisms (Hetu et al., 1983; Calza et al., 2008). Methanol extracts of S. latiuscula can also have cytotoxic effects on eukaryotic cells (Choi et al., 2011). However, in this study, Artemia larvae had a 52.05% survival rate after 48 h in 2.5% Symphyocladia latiuscula methanol extract, indicating that this extract is non-larvicidal (data not shown).

It is apparent from these results that seaweed may have different toxicities for humans and brine shrimp larvae. This is important to note as medicinal chemicals are generally tested in animal models, but pharmacokinetics such as absorption, action, and excretion of medicines and chemicals may be drastically different between different animal species (Aubets et al., 2006; Christensen et al., 1990).

These preliminary results indicate that Enteromorpha linza and Gelidium amansii may be sources of larvicidal compounds that do not adversely affect humans, and future studies should seek to isolate the compound or compounds responsible for these larvicidal effects.

Table 1. The larvicidal effects of methanol extracts of six species of Chlorophyta on Artemia salina nauplii.

Chlorophyta###MeOH###Incubation time (h)

###extract###0###2###4###6###12###24###48

Capsosiphon###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###66.1+-47.9###95.0+-7.1

fulvescens###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.2###97.8+-3.2###95.9+-0.5

###2.5%###100.0+-0.0###100.0+-0.0###64.0+-36.2###100.0+-0.0###81.3+-26.5###44.0+-18.0###37.0+-7.6

Codium fragile###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###94.1+-8.3

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.8+-8.8

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.0+-2.8###88.0+-17.0###79.4+-21.7

Enteromorpha###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0

compressa###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-00###100.0+-0.0###94.0+-1.3

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###90.8+-7.2###51.2+-57.3###22.6+-26.9###4.2+-5.9

Enteromorpha###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.3+-2.4###90.9+-1.2

linza###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.5+-3.5###86.4+-9.1

###2.5%###100.0+-0.0###100.0+-0.0###83.3+-23.6###29.4+-7.4###0.0+-0.0###0.0+-0.0###0.0+-0.0

Monostroma###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.1###94.5+-1.7###92.3+-1.4

nitidum###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.5+-6.4###95.5+-6.4###95.5+-6.4###95.5+-6.4

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.2+-2.6###65.8+-43.1###49.2+-61.3###33.3+-47.1

Ulva pertusa###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###94.5+-79.0

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.1###97.8+-3.1###97.8+-3.1###90.4+-4.8

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###90.8+-2.1###52.1+-24.1###15.1+-11.###1.8+-2.5

Table 2. The larvicidal effects of methanol extracts of 9 species of Phaeophyta on Artemia salina nauplii.

Phaeophyta###MeOH###Incubation time (h)

###extract###0###2###4###6###12###24###48

Dictyota###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###87.5+-17###70.8+-17.7

dichotoma###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.7+-6.2###27.6+-2.2###19.2+-3.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###63.8+-7.7###36.2+-7.7###6.3+-8.8###0.0+-0.0

Ecklonia kurome###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.7+-4.8###96.7+-4.7

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.8+-0.8###64.9+-4.2###53.0+-0.8

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.75+-8.8###68.8+-26.5###40.6+-22.1###0.0+-0.0

Ishige okamurae###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.0+-0.7###92.2+-4.6###87.6+-1.8

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.2+-2.5###95.8+-0.8###64.9+-4.2###53.0+-0.8

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.9+-3.0###37.8+-45.6###5.0+-7.1###0.0+-0.0

Ishige sinicola###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.2+-5.4###90.1+-7.8

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.4+-5.1###96.4+-5.1###94.5+-2.3###92.6+-0.4

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###80.6+-27.5###75.3+-27.9###48.1+-52.3

Pachydictyon###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###61.5+-37.8###11.8+-16.6###2.9+-4.16

coriaceum###0.5%###100.0+-0.0###100.0+-0.0###90.0+-14.1###95.7+-6.2###22.2+-12.9###0.0+-0.0###0.0+-0.0

###2.5%###100.0+-0.0###100.0+-0.0###38.3+-24.4###44.7+-16.0###16.2+-11.1###0.0+-0.0###0.0+-0.0

Saccharina###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.1###97.8+-3.1###97.8+-3.1###96.0 +-0.5

japonica###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.7+-3.2###97.7+-3.2###97.7+-3.2###97.3+-3.2

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###86.0+-11.6###66.2+-12.9###46.4+-17.2

Sargassum###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.1+-2.7###92.3+-10.9###75.1+-13.5

sagamianum###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###91.7+-1.7###76.9+-27.9

###2.5%###100.0+-0.0###100.0+-0.0###93.7+-2.4###70.4+-3.3###44.8+-23.1###30.4+-15.0###0.0+-0.0

Scytosiphon###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.3+-2.9

lomentaria###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.7+-4.7###96.7+-4.7###90.0+-4.7

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.6+-4.9###82.3+-15.3###49.1+-21.5###27.7+-8.8

Undaria###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.1+-4.2###97.1+-4.2

pinnatifida###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.5+-6.4###95.5+-6.4###95.5+-6.4###90.5+-0.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.9+-0.8###70.0+-28.3###52.8+-19.7###47.2+-27.5

Table 3. The larvicidal effects of methanol extracts of 4 species of Rhodophyta on Artemia salina nauplii.

Rhodophyta###MeOH###Incubation time (h)

###extract###0###2###4###6###12###24###48

Chondracanthus###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.9+-4.4###96.9+-4.4###93.8+-8.8

intermedia###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-00###100.0+-0.0###96.3+-5.3###95.0+-3.0

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.7+-3.2###60.5+-49.4###51.2+-62.6###5.0+-7.0

Chondracanthus###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.7+-3.2###95.6+-0.3###93.4+-3.5

tenella###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.5+-6.4###95.5+-6.4###79.8+-2.9

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###83.3+-23.6###29.0+-39.8###26.6+-36.5

Gelidium###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.6+-3.4

amansii###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.0+-4.3###78.2+-22.2###78.2+-22.2###78.2+-22.2

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###52.1+-67.8###0.0+-0.0###0.0+-0.0###0.0+-0.0

Gracilaria###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.4+-7.5

verrucosa###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.4+-7.5

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.3+-9.3###83.3+-4.7###60.0+-0.0###33.3+-9.4

Table 4. The larvicidal effects of aqueous extracts of 3 species of Chlorophyta on Artemia salina nauplii.

Chlorophyta###Water###Incubation time (h)

###extract###0###2###4###6###12###24###48

Enteromorpha###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.9+-4.4

compressa###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.1+-4.2###91.5+-4.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.3+-5.2###83.5+-13.3###67.3+-4.3###27.3+-1.9

Enteromorpha###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.5+-0.9###73.3+-12.8

linza###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.2+-5.4###89.0+-6.1

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.7+-3.2###77.7+-19.8###48.1+-9.1###48.1+-9.1

Ulva pertusa###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###91.7+-11.8###86.7+-4.7

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.5+-3.5###87.5+-17.7###73.3+-18.8

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.7+-6.2###75.9+-8.9###67.9+-10.0###49.9+-15.3

Table 5. The larvicidal effects of aqueous extract of 13 species of Phaeophyta on Artemia salina nauplii.

Rhaeophyta###Water###Incubation time (h)

###extract###0###2###4###6###12###24###48

Colpomenia###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###82.7+-2.0

bullosa###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###92.5+-10.6###84.7+-13.7

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###94.7+-7.5###50.2+-11.5###0.0+-0.0###0.0+-0.0

Costaria costata###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.5+-6.4###92.5+-2.3###80.5+-11.0

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###85.9+-3.6###65.0+-10.3

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###85.0+-21.2###51.1+-12.7###28.0+-9.9###17.8+-10.2

Dictyota###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###95.8+-5.9###95.8+-5.9

dichotoma###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.4+-3.7

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###75.0+-35.4###51.1+-33.8###38.6+-16.1###22.5+-12.5

Ecklonia cava###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.2+-5.4###96.2+-5.4###96.2+-5.4###91.2+-1.6

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.3+-0.0###90.0+-4.7

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###87.5+-17.7###67.5+-24.8###41.9+-2.7###33.8+-5.3

Ecklonia kurome###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.9+-4.4

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.1+-4.2###90.6+-3.3

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###71.1+-40.9###50.0+-18.6###23.7+-3.7###15.8+-7.4

Eisenia bicyclis###0.25%###100.0+-0.0###100.0+-0.0###90.3+-0.4###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.9+-3.0###94.4+-2.1

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.5+-3.5###94.9+-0.2###35.7+-13.2###5.1+-0.2

Hizikia###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.9+-7.0

fusiformis###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.0+-2.8###95.2+-1.1###84.4+-6.2###78.9+-1.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###88.2+-16.6###70.6+-0.0###67.7+-4.2###29.4+-8.3

Ishige okamurae###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###86.1+-11.8

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.9+-4.4###93.5+-0.3###77.3+-5.6###77.3+-5.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###84.2+-22.3###58.3+-11.8###26.7+-9.4###18.3+-2.4

Ishige sinicola###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###91.2+-2.4

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.7+-4.7###84.6+-2.8###81.3+-1.9

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###90.9+-12.9###60.2+-46.6###33.2+-21.3###24.1+-14.7

Myelophycus###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###96.4+-5.1

simplex###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.0+-2.8###96.0+-5.7###94.2+-3.1

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###71.9+-18.5###63.1+-31.0###45.6+-6.2

Sargassum###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###89.6+-7.6

micracanthum###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###91.7+-11.8###86.1+-19.7###81.1+-12.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###82.2+-25.3###13.4+-1.3###3.1+-4.4

Sargassum###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###93.1+-0.3

sagamianum###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.1###91.5+-12.1

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###88.6+-16.1###75.4+-23.0###40.9+-12.9###38.6+-16.1

Scytosiphon###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###98.3+-2.4###92.9+-0.6###89.4+-0.9

lomentaria###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.8+-3.0###95.7+-6.5

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###88.6+-16.1###76.0+-23.9###39.1+-10.3###33.3+-8.5

Table 6. The larvicidal effects of aqueous extracts of 3species of Rhodophyta on Artemia salina nauplii.

Rhodophyta###Water###Incubation time (h)

###extract###0###2###4###6###12###24###48

Chondracanthus###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###94.2+-1.2###90.8+-5.9

intermedia###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###89.3+-15.2###74.4+-24.4

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###90.6+-13.3###67.4+-15.8###46.4+-30.3

Lomentaria###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###97.1+-4.2###94.1+-8.3###87.0+-1.8###77.5+-1.5

catenata###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###65.6+-41.3###61.0+-47.7

###2.5%###100.0+-0.0###100.0+-0.0###62.3+-14.7###62.8+-5.6###27.7+-10.8###0.0+-0.0###0.0+-0.0

Prionitis cornea###0.25%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###85.4+-3.0

###0.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###94.5+-7.9###86.1+-19.6###83.3+-23.6

###2.5%###100.0+-0.0###100.0+-0.0###100.0+-0.0###100.0+-0.0###89.6+-8.8###62.1+-12.4###42.5+-22.4

Acknowledgments: This study was partially conducted with the assistance of National Institute of Fisheries Science in Korea (Studies on the sea cucumber poly-culture below the hanging culture systems of Pacific oysters and sea squirt). We also are grateful to Ms. S. H. Choi, Ms. I. H. Kim, and Ms. Y. W. Lee for their excellent technical assistance throughout this work.

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