Searching for Radical Solutions against Fouling ...

Saad et al., J Mar Biol Oceanogr 2016, 5:3 DOI: 10.4172/2324-8661.1000162

Journal of Marine Biology & Oceanography

Research Article

Searching for Radical Solutions against Fouling Communities Inhabiting the Arabian Gulf in Saudi Arabia Saad GA*, Hussein AB and Abu El-saad AM

Abstract Sea squirts produce pelagic larvae that spend few days in the plankton before settling and metamorphosing into their adult sessile forms. Attachment mechanism developed usually relies on highly viscous or solid adhesive secretions, which all contain specialized proteins and glycogen. Adult sea squirts of Ciona intestinalis, Molgula manhattensis, Ascidella aspersa and Phallusia nigra were sampled for 12 months from three estuarine localities of the Arabian Gulf (Ad-Dammam; Northern Khobar and Southern Khobar). Seasonal variation of some analyzed abiotic factors revealed that the population densities of ascidians and the feasibility of larval dispersal were variable not only according to seasons but also according to the study locality where ascidians predominate Southern Khobar˃Northern Khobar ≈ Ad-Dammam and during spring and summer populations are ˃˃ those of autumn < those of winter. Procurement of gametes has been carried out by potassium chloride injection or/and artificial heterologous fertilization. Larvae were cultured and three larval stages were identified. Immunocytochemical approach has been applied to investigate larval metamorphosis under the influence of applied bioactive inducers which had been claimed to induce metamorphosis or involved in signaling systems or serve as neurotransmitters or control morphogenetic and behavioral reactions or interact with members of several signal transduction proteins pathways. The transformation of the larval stage with long tail to the next larval stage with remarkable tail resorption activity is accelerated by NH4Cl 2.5 mM, C8 1 µM, Acetyl choline 1 mM, NOS 1 mM and cGMP 1 µM. To investigate whether NO and HSP 90 play a role in larval metamorphosis, larvae with long tail, partially metamorphosed larvae with short tail and metamorphosing youngs were treated for whole-mount immunohistochemistry towards the green fluorescent protein (GFP). High-pressure chromatographic separation has been carried out to identify amino acids in the adhesive secretions of the larval stage. Western blotting routine technique (Immune blotting) using Bradford assay has been applied to identify proteins of adhesives. SDS-PAGE revealed that the adhesives contained about 11 different proteins. Their apparent molecular masses were approximately 300, 280, 250, 150, 100, 75, 50, 35, 25, 20, and 10 kDa. Total lipids were extracted using capillary gas chromatography and total carbohydrates of adhesives were analyzed by the Phenolsulphuric acid method. This study concluded that lipids of adhesives is secreted first and represented by C14:0; C18:2 V-32; C20:1 V-9; C18:1 V-7; C16:1 V-6 and C18:1 V-5. A post-translational modification of amino acids mainly Phenylalanine, Methionine Threonine, Valine and Serine with glycogen for adhesive and cohesive function occurs. The carbohydrate moiety of adhesives is a polymerization of monosaccharides. All data obtained were subjected to analysis of variance (ANOVA) means significant difference P>0.05 or P> those of autumn0.05 and -5.915 to 8.915 between Northern Khobar vs Ad-Dammam at P>0.05. The highest value of water Temperature was observed during July-August in the three study localities (37-41ºC). Tukey’s Multiple Comparison Test showed the loSouthern Mean Dif between Northern Khobar vs Southern Khobar -9.165 to 5.665 at P>0.05. Figure 13: Phase contrast photomicrograph of a whole mount of a partially metamorphosed larval stage

Ascidella aspersa GFP staining. Note, brain vesicle showed lesser illumination than the nerve cord. Heat Figure 13: Phase contrast photomicrograph of a whole mount of a partially shock protein 90 is present in a little amount inside the brain vesicle, the nerve cord and all neurons supplying the different parts ofstage the larval Ascidella body with long and short tails.GFP staining. Note, brain metamorphosed larval aspersa vesicle showed lesser illumination than the nerve cord. Heat shock protein 90 is present in a little amount inside the brain vesicle, the nerve cord and all neurons supplying the different parts of the larval body with long and short tails.

beach during December and this beach showed always the highest value all the year round. The water transparency in Northern Khobar was more or less parallel with that of Southern Khobar exceptionally it showed higher transparency during March and July with a reflex during November and December. Southern Khobar showed lesser transparency all the year round. One-way analysis of variance showed means signif. Different between study localities at P0.001.

Chlorides The Chlorides values of marine water are given in Histogram 7 for marine habitats in different seasons of the year. The lowest value of Chlorides was found in Ad-Dammam and Northern Khobar whereas the highest value was measured in Southern Khobar. Low Chlorides in water was found in Ad-Dammam during the months of JanuaryNovember and Northern Khobar during November-December (290-

Total alkalinity The Total Alkalinity values of marine water are given in Histogram 4 for marine habitats in different seasons of the year. The lowest value of Total Alkalinity was found in Ad-Dammam whereas the highest value was measured in Southern Khobar. Northern Khobar showed always a moderate value. Low Total Alkalinity in Ad-Dammam water during April–May and October–November (170-180 mg/l). One-way analysis of variance showed means significant difference at P0.001. The highest value of Total Alkalinity was observed during December in Southern Khobar (540 mg/l) and (470-490 mg/l) February–October. Tukey’s Multiple Comparison Test showed the loSouthern Mean Dif between Southern Khobar vs Ad-Dammam 231.1 to 309.4 at P>0.001

Histogram 3

Total hardness The Total Hardness values of marine water are given in Histogram 5 for marine habitats in different seasons of the year. The lowest value of Total Hardness was found in Ad-Dammam whereas the highest value was measured in Southern Khobar. Northern Khobar showed always a moderate value. Low Total Hardness in water was found in Ad-Dammam during the months of January-August (250-300 mg/l). One-way analysis of variance showed means significant difference at P0.001. The highest value of Total Hardness was observed during the year round in Southern Khobar (900-989 mg/l). Tukey’s Multiple Comparison Test showed the loSouthern Mean Dif between Southern Khobar vs. AdDammam 594.8 to 685.2 at P>0.001.

Histogram 4

Salinity The Salinity values of marine water are given in Histogram 6 for marine habitats in different seasons of the year. The lowest value of Salinity was found in Ad-Dammam whereas the highest value was measured in Southern Khobar. Northern Khobar showed always a moderate value. Low Salinity in water was found in Ad-Dammam during the year round (2,90-3,80 g/l). One-way analysis of variance showed means significant difference at P0.001. The highest value of Salinity was observed during almost all months in Southern Khobar (7,20-7,90 Volume 5 • Issue 3 • 1000162

Histogram 5

• Page 9 of 20 •

Citation: Saad GA, Hussein AB and Abu El-saad AM (2016) Searching for Radical Solutions against Fouling Communities Inhabiting the Arabian Gulf in Saudi Arabia. J Mar Biol Oceanogr 5:3.

doi: 10.4172/2324-8661.1000162 tin TBT in water during June was found in Ad-Dammam (320 μgg1). One-way analysis of variance showed means significant difference at P0.001. The highest value of Tributyl tin TBT was observed during almost all months in Southern Khobar (460-520 μgg-1).

Testing the effect of bioactive inducers on larval metamorphosis

Histogram 6

Histogram 7

Hatched Ciona intestinalis larvae were collected from the culture, transferred to wells of tissue culture plates, incubated in selected bioactive inducers as described in the methods section. All samples were maintained at a constant temperature of 20ºC, and inspected and scored for progress of metamorphosis at intervals of 24 hr for a total of 120 hr. Three different metamorphic stages were considered: Stage I: the hatched, larva showed all untransformed larval characteristics, exhibited a long tail, and strong movements. Stage2: Larva with a short tail. The larva showed significant retraction and coiling of the axial structures of the tail; degraded cells assemble at the posterior part of the trunk. The pigmented sensory organs within the brain vesicle were still prominent. Stage 3: Metamorphosed larva in the form of larval trunk portion with tail structures fully resorbed. Degrading sensory pigments visible, tunic layer present. Rotation of the alimentary tract and branchial basket anlagen completed growth and further differentiation of the juvenile ascidian underway. It was observed that NH4Cl 2.5 mM, C8 1 µM, Acetyl choline 1 mM, NOS 1 mM and cGMP 1 µ M enhanced and accelerated the transformation of the larval stage with long tail to the next larval tail with remarkable tail resorption activity. Tukey’s Multiple Comparison

390 mg/l). One-way analysis of variance showed means significant difference at P0.001 at lowest record. The highest value of Chlorides was observed during almost all months in Southern Khobar (710-790 mg/l).

Total nitrogen The Total Nitrogen values of marine water are given in Histogram 8 for marine habitats in different seasons of the year. The lowest value of Total Nitrogen was found in Ad-Dammam and Northern Khobar whereas the highest value was measured in Southern Khobar. Low Total Nitrogen in water was found in Ad-Dammam during September (1,00 mg/l). Oneway analysis of variance showed means significant difference at P0.05.

Histogram 8

The highest value of Total Nitrogen was observed during August in Southern Khobar (3,9- 5,4 mg/l) in 2012 and (5,30 mg/l). Tukey’s Multiple Comparison Test showed the loSouthern Mean difference between Southern Khobar vs Ad-Dammam 1.607 to 2.677 at P>0.001.

The Tributyl tin (TBT) The Tributyl tin TBT values of marine water are given in Histogram 9 for marine habitats in different seasons of the year. The lowest value of TBT was found in Ad-Dammam and Northern Khobar whereas the highest value was measured in Southern Khobar. Low Tributyl Volume 5 • Issue 3 • 1000162

Histogram 9

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doi: 10.4172/2324-8661.1000162 Table 1: Effect of bioactive inducers on larval metamorphosis in Ciona intestinalis.

Bioactive inducers

Sea water

ABS

Concentration

.

-

2.5 mM NH4Cl

300 µM

10 µ M

C8

1µM

0.1 µ M

Serotonine

10 µg/ml

10 mM

Acetyl choline

1 mM

0.5 mM

RF-amid

LW-amid

30 µ g/ml

30 µ g/ml

After 24 hrs.

No. of tested larvae

Stage obtained

19* 16** 17***

After 48 hrs.

After 72 hrs.

After 96 hrs.

After 120 hrs.

Ist trial 2nd trial

3rd trial

Ist trial

2nd trial

3rd trial

Ist trial

2nd trial

3rd trial

Ist trial

2nd trial

3rd trial

Ist trial

2nd trial

3rd trial

l st m d

19 -

16 -

17 -

16 3 -

14 2 -

16 1 -

16 3 -

9 4 3 -

11 2 4 -

2 8 9 -

4 7 5 -

5 4 7 1

0 6 13 -

2 5 9 -

2 5 10 -

l st m d

14 1 -

16 2 -

11 3 -

12 3 -

13 5 -

12 2 -

10 5 -

9 9 -

8 6 -

7 7 1 -

8 7 3 -

6 6 2 -

0 11 4 -

0 13 5 -

1 11 1 -

13* 19** 20***

l st m d

10 3 -

14 5 -

14 6 -

7 6 -

5 12 2 -

8 12 -

2 9 2 -

4 13 2 -

3 11 6 -

4 11 4 -

1 2 17 -

0 4 9 -

0 2 17 -

1 1 18 -

22* 21** 19***

l st m d

21 1 -

19 2 -

16 3 -

17 5 -

18 3 -

16 3 -

14 8 -

12 9 -

14 5 -

10 12 -

5 10 6 -

3 7 9 -

0 5 16 -

3 8 10 -

1 4 14 -

19* 14** 17

l st m d

17 2 -

11 3 -

16 1 -

15 4 -

10 4 -

12 5 -

12 7 -

10 4 -

10 7 -

9 9 1 -

6 8 -

7 9 1 -

1 15 3 -

0 7 7 -

0 8 9 -

18* 15** 16***

l st m d

17 1 -

13 2 -

14 2 -

17 1 -

13 7 -

14 2 -

12 6 -

11 4 -

13 3 -

10 8 -

7 8 -

9 7 -

1 0 17 -

18* 13** 12***

l st m d

15 3 -

10 3 -

8 4 -

11 7 -

6 7 -

4 8 -

7 6 5 -

3 7 3 -

5 4 3 -

3 7 8 -

2 7 4 -

1 8 3 -

0 1 17 -

0 0 13 -

0 2 10 -

l st m d

13 2 -

14 3 -

15 3 -

13 2 -

11 6 6 -

13 5 -

10 6 -

8 9 -

8 9 1 -

8 7 -

7 7 3 -

6 6 6 -

0 0 15 -

0 0 17 -

0 0 17 1

12* 19** 11***

l st m d

10 2 -

16 3 -

9 2 -

9 3 -

11 8 -

8 3 -

6 5 1 -

7 8 4 -

8 2 1 -

2 9 1 -

7 9 3 -

5 5 1 -

0 1 11 -

0 1 18 -

0 0 11 -

17* 14** 18

l st m d

14 3 -

11 3 -

16 2 -

9 4 4 -

5 7 2 -

5 6 7 -

1 7 9 -

2 6 6 -

3 6 9 -

0 5 12 -

0 3 11 -

0 8 10 -

0 1 16 -

0 2 12 -

0 2 16 -

12* 17** 14***

l st m d

11 1 -

15 2 -

13 1 -

11 1 -

10 6 1 -

11 3 -

8 4 -

9 8 -

11 3 -

1 11 -

3 14 -

4 9 1 -

0 0 12 -

0 0 17 -

1 1 12 -

18* 17** 12***

l st m d

17 1 -

15 2 -

10 2 -

15 3 -

14 3 -

11 1 -

10 6 2 -

11 6 -

10 2 -

9 8 1 -

6 7 4 -

5 5 2 -

0 1 16 1

0 0 17 -

0 0 12 -

18* 19** 13***

l st m d

15 3 -

16 3 -

11 2 -

14 4 -

13 6 1

10 3 -

11 7 -

9 8 1 -

7 5 1 -

6 6 2 -

5 7 6 -

4 6 3 -

1 1 16 -

0 0 18 -

0 0 13 -

15* 18** 14

15* 17** 18***

Volume 5 • Issue 3 • 1000162

2 2 9 -

0 2 13 -

0 2 14 -

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doi: 10.4172/2324-8661.1000162

21* 23** 22***

l st m d

17 4 -

20 3 -

18 4 -

15 6 -

14 7 2 -

11 10 1 -

10 7 4 -

9 7 7 -

6 9 7 -

5 6 10 -

3 7 13 -

4 6 12 -

21 -

23 -

22 -

1.5 mM

20* 21** 18***

l st m d

19 1 -

20 1 -

16 2 -

18 2 -

19 2 -

16 4 -

17 3 1 -

15 3 -

16 4 -

12 8 -

14 6 1 -

12 6 -

19 -

120 -

17 1

2.5 mM

20* 23** 21***

l st m d

20 3 -

20 1 -

19 1 -

20 3 -

19 1 -

8 12 -

9 11 -

2 8 10 -

4 6 13 -

4 6 10 -

2 18 -

1 4 18 -

1 1 18 -

1µM

22* 18** 20***

l st m d

15 3 -

15 5 -

10 7 4 1

8 6 4

9 8 3

5 10 6

3 7 10

2 6 13

4 5 9

3 9 8

1 3 17

0 5 13

1 2 17

1 mM

NOS

cGMP

*= No. of tested larvae in the first trial

19 1 -

18 4 -

10 13 -

4 6 8

l = larva with long tail

**= No. of tested larvae in the second trial

s t = larva with shortened tail

***= No. of tested larvae in the third trial

m = newly metamorphosed ascidian d = died larva of any stage

Test showed at P>0.05 Mean Diff. -28,06; 2,7; -5,86; -17,3 and -23,98 sea water versus those bioactive inducers respectively whereas it showed -11,34 and -12,4 at P>0.05 respectively for Serotonine 10 µg/ ml and Acetyl choline 10 mM with lesser activity. Treatment (between columns) were SS 9547, 83630 & 93180; df 16, 68 & 84 ; MS 596,7 & 1230. All other bioactive inducers did not show any role and have about the same results as control (sea water and antibiotic sea water). For more detail see Histogram 10 a,b for data analysis. Previously the effect of bioactive inducers on the larval metamorphosis of Molgula manhatensis has been tried (Saad, 2015). In this study, the author concluded that NH4Cl (2.5 mM), C8 (1 µM), NOS (2.5 mM), seratonine (10 µg/ml) and Acetyl choline (0.5 mM) accelerated the transition of larval stage from stage I with long tail to a metamorphosing stage with short tail (Table 1).

Testing the activity of Nitric Oxide and Heat Shock Protein 90 during metamorphic and settlement stages of ascidian larvae Nitric oxide synthase (NOS) activity of the neurosecretory cells of the brain vesicle catalyzed the formation of nitric oxide (NO) by converting arginine to citrulline with the concomitant release of NO. The production of NO by NOS has been associated with a wide range of important physiological functions including neuronal signaling in early embryonic development. In the present study, the three stages of larval development, namely larva with long tail, partially metamorphosed larva with short tail and metamorphosed larval stage were examined to test if HSP 90 is present in the nervous complex or any part of the body using green fluorescent protein (GFP). This test revealed that the heat shock protein 90 is present in the brain vesicle; the nerve cord and all neurons supplying the different parts of the larval body with long and short tails (Figures 11-13). Gradually this protein diminished from the brain vesicle as the larva lost almost its tail and if this protein presents, negligible amount. Newly metamorphosed larva showed fluorescence without illumination in its external boundary (Figure 14). This means that the brain vesicle has given a signal to the different body parts to enhance Volume 5 • Issue 3 • 1000162

or to activate the process of retrogressive development and metamorphosis of the larva to a newly squirt. In other words, HSP 90 may function in emitting signals necessary for development of the different body organs and to change the brain vesicle of the larval stage to the nervous complex of the adult stage. Meanwhile it may function in apoptosis of the tail region with its structures of notochord and nerve cord. NOS activity was present in neurons of larval tissues. The NADPH diaphorase histochemical assay was applied. Larvae were stained for diaphorase activity. These sites of NADPH diaphorase activity most likely represented sites of NOS activity. Diaphorase activity was found in a variety of structures. Stained cells were found within the larval brain vesicle. Stained cells often have a neuronal appearance. Larvae were stained with anti-NOS antibodies to see whether sites of NADPH activity were coincident with the location of NOS. The production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduced the production of NO, and consequently cGMP, to initiate metamorphosis. This experiment showed that NO accelerated larval metamorphosis and cGMP is a second enhancing factor for NO. Nos and cGMP were tested previously as external bioactive inducers of metamorphosis, see Histogram 10 for data analysis.

Amino acid composition of adhesives The chemical analysis of adhesives in larvae of the four studied ascidians (Ciona intestinalis, Molgula manhatensis, Ascidiella aspersa and Phallusia nigra) indicated that their adhesives are closely related (Histogram 11 a,b). The four species are rich in essential and nonessential amino acids. The essential amino acids predominated the adhesives especially Serine, Methionine and Phenylalanine, Threonine and Valine. Nineteen amino acids were identified. The most predominant amino acids were Asparagine, Glutamine, Glycine, Leucine, Methionine, Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine and Valine. Glycine ranged from 21.91 % to 25.70 % and Serine ranged from 12.78 % to 14.30 % of the amino acid content of the adhesives of the four studied ascidian larvae. There • Page 12 of 20 •


doi: 10.4172/2324-8661.1000162

1800

cGMP NOS3

1600

NOS2

percentage of metamorphosis

1400

NOS LW-amid

1200

RF-amid

1000

Acetyl choline3

800

Acetyl choline2 Acetyl choline

600

Serotonine

400

C8 0.1M

200 0

C8 1M C8 10 M 24 48 72 96 120 hrs

NH4Cl 300 M

Effect of bioactive inducers on larval metamorphosis

NH4Cl 2.5 mM ABS sea water

Histogram 10

majority of the resolved protein spots of the four larval species had pI values between pH 4 and pH 12 and the molecular size of the pattern ranged from 300 kDa to less than 10 kDa. Approximate 80 % of the proteins ranged from 100 kDa to 20 kDa. Around 250-50 spots were detected in the four gels (Figures 15-18), which were compared for their intensity to identify differentially expressed spots. After their separation by SDS-PAGE, proteins of the four ascidian larvae were blotted onto PVDF membranes and probed for phosphor proteins using the monoclonal anti-phosphorylated amino acid antibodies. Several labeled bands were present in the adhesives from the four species. Among them, the most strongly labeled are a protein of 100 kDa and a protein located just above the migration front.

Lipids analysis Total lipids were extracted using capillary gas chromatography. This test revealed that the lipid contents of adhesives of the four species studied were roughly similar but in different amounts. It was found that lipids of adhesives were represented by C14:0; C18:2 V-32; C20:1 V-9; C18:1 V-7; C16:1 V-6 and C18:1 V-5. The rest of lipid contents of adhesives are considered negligible (Histogram 12 a,b). Tukey’s Multiple Comparison Test at P>0.05 Mean Diff. showed -1,300, -0, 3200, 1,085, -5, 895, 0, 2900, 1,153 for C14:0; C18:2 V-32; C20:1 V-9; C18:1 V-7; C16:1 V-6 and C18:1 V-5 respectively. Treatment (between columns) were SS 1520, 51& 29, 8; df 36,67, 156 & 0,235; MS 1556 & 207.

Carbohydrate analysis Glycogen is the main carbohydrate constituent, representing about 50% of total carbohydrates. It is a preferred form of energy reserve particularly in settlement process of ascidian larvae because

Histogram 11

were differences in the percentage of amino acids in the four species studied with the exception of Cysteine, Lysine and Tryptophan, no significant differences. Dunnett’s Multiple Comparison Test at P>0.05 showed Mean Diff. 5,978, -16,57, 3,695, -11,77, -17,02, -16,82, 8,105, 5,018, 5,858 & 1,833 for Asparagine, Glutamine, Glycine, Leucine, Methionine, Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine and Valine versus Alanine respectively. Treatment (between columns) were SS 5679, 8 & 315,5; df 191,3, 57 & 3,357; MS 5871&75.

Protein analysis Proteins in different samples of the four studied ascidian larvae were resolved by SDS-PAGE and digested with trypsin. A peptide with the same sequence was obtained from protein bands of different apparent molecular masses. This analysis revealed that the adhesives contained about 11 different proteins. Their apparent molecular masses were approximately 300, 280, 250, 150, 100, 75, 50, 35, 25, 20, and 10 kDa. There was some variability between the four protein extracts of the four studied larval species, and little variability of protein contents were found within the same species. Two proteins were especially inconsistent in extraction, the 20-kDa and the 280kDa proteins. There was also a component that was too large to enter the running gel ˃500-kDa. The proteins that were analyzed in the four larval species had similar amino acid compositions. They were all rich in glycine (19-24%) and in acidic residues (16-21%). The Volume 5 • Issue 3 • 1000162

Figure 15: Immunoblotting showing protein analyses of adhesives in larvae of Ciona intestinalis.

Figure 15: Immunoblotting showing protein analyses of adhesives in larvae of Ciona intestinalis.

Figure 16 : Immunoblotting showing protein analyses of adhesives in larvae of Molgula manhattensis.

Figure 16: Immunoblotting showing protein analyses of adhesives in larvae of Molgula manhattensis.

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Figure 17: Immunoblotting showing protein analyses of adhesives in larvae of Ascidella aspersa.

Figure 17: Immunoblotting showing protein analyses of adhesives in larvae of Ascidella aspersa.

Figure 18: Immunoblotting showing protein analyses of adhesives in larvae of Phallusia nigra.

Figure 18: Immunoblotting showing protein analyses of adhesives in larvae of Phallusia nigra.

Fatty acids analysis 120

100

percentage

80

60

40

20

0

Ciona

Molgula

Ascidella

Phallusia

Column1

C10:0

C11:0

C12:0

C13:0

C14:0

C15:0

C16:0

C17:0

C18:0

C19:0

C20:0

C21:0

C22:0

C23:0

C24:0

SFAs

C14:1 v- 3

C14:1 v- 5

C14:1 v- 7

C15:1 v- 6

C16:1 v- 5

C16:1 v- 6

C16:1 v- 7

C16:1 v- 9

C17:1 v- 7

C17:1 v- 8

C18:1 v- 5

C18:1 v- 7

C18:1 v- 9

C19:1 v- 8

C20:1 v- 5

C20:1 v- 6

C20:1 v- 7

C20:1 v-9

C22:1 v- 7

C22:1 v- 9

C24:1 v- 3

C24:1 v- 6

C24:1

MUFAs

C16:2 v- 6

C18:2 v- 3

C18:2 v- 6

C18:2 v- 32

C18:2 v- 6

C18:2 v- 33

C19:2- v- 6

C20:2 v- 6

C20:3 v- 6

C20:4 v- 6

C20:5 v- 3

C20:5 v-3

Histogram 12

Volume 5 • Issue 3 • 1000162

of fast glycogen catabolism that provide instant energy under hypoxic or anoxic conditions. The varying amounts of carbohydrates were apparently dependent partly on larval stage and partly on the species. In the adhesives the following percentage amounts were found: larvae with short tail of Ciona intestinalis, 0.064 to 1.67 mg carbohydrates g-1 wet weight; larvae with short tail of Molgula manhattensis 0.001-1.6 mg carbohydrates g-1 wet weight; larvae with short tail of Ascidella aspersa 0-0.18 mg carbohydrates g-1 wet weight; larvae with short tail of Phallusia nigra 0 to 1.9 mg carbohydrates g-1 wet weight (Histograms 13a,b and 14). Newman-Keuls Multiple Comparison Test at P>0.05 did not show variances differ signif where Mean Diff between Ciona vs Ascidella -0,2361; Ciona vs Phallusia -0,1735 ; Ciona vs Molgula -0,1384 ; Molgula vs Ascidella -0,09769 ; Molgula vs Phallusia -0,03508 and Phallusia vs Ascidella -0,06261. Treatment (between columns) were SS 0,3591, 3 & 0,1197; df 14,88, 44 & 0,3382; MS 15,24 & 47.

Discussion Physicochemical parameters and quantity of nutrients play a significant role in the distributional patterns and species composition in aquatic ecosystem [81-83]. Depending on temperature fluctuations, the various aquatic species thrive and grow in different months. In summer season increasing temperature enhances the rate of decomposition due to which the water becomes nutrient rich, similarly due to concentration followed by evaporation. Fluctuation in marine population is a general phenomenon in the aquatic impoundments [84-86]. Factors contributed to its variations are rainfall, depth, silting and other physicochemical parameters (transparency (cm); water temperature (°C); total dissolved solids (mg/l); total alkalinity (mg/l); total hardness (mg/l); salinity (g/l); chlorides (mg/l); total Nitrogen (mg/l) and Tributyl tin (μgg-1). The presence of a species depends on its environmental tolerance, but the resources available would determine their abundance. Water temperature measurement is useful to indicate the trend of various chemical, biochemical and biological activities. A rise in temperature leads to the fast chemical and biochemical reactions. The light in water is a factor of profound importance for its role in the photosynthetic processes of all chlorophyll bearing aquatic plants and thus for the primary production. It is often a limiting factor in the distribution of organisms in water particularly. The capacity of water to neutralize a strong acid is known as alkalinity and is characterized by the presence of hydrogen ions; most of the alkalinity present in water is due to dissolution of carbonates [87]. Hardness of the natural water is mainly caused by the carbonates, bicarbonates, sulphates and chlorides of calcium and magnesium. Other cations which affect the hardness are iron and manganese. Natural water generally contains low concentration of chloride. Its higher amount always comes from the contamination of sewage. Salinity is one of the important factors that limit the abundance of many aquatic organisms directly or indirectly. It affects organisms mainly through changes in osmotic pressure and density of the water. The range of tolerance varies among different species of organisms. The majority of freshwater plants and animals have a low tolerance to any increase in salinity. This study concluded that water quality analysis indicated a range of parameters in three estuarine beaches under investigation during the study period (March/December 2014 and January/ February 2015). Transparency of water (34-99 cm), Temperature of surface water (20-41°C), Total Dissolved Solids (TDS) (1220-5786 mg/l), Total alkalinity (250-490 mg/l), Total hardness (390-990 mg/l), Salinity (2,9-7,9 g/l ), Chlorides (900-1520 mg/l), Total nitrogen (15,3 mg/l), Tributyl tin (390- 520 μgg-1). Water quality of Arabian Gulf has deteriorated. Most of the physicochemical parameters are beyond • Page 14 of 20 •


doi: 10.4172/2324-8661.1000162

Histogram 13

Histogram 14

the limits suggested by WHO. The Arabian Gulf has become saline and eutrophic [88].

This study aimed to understand and compare the spatial organization and the nature of the adhesive secreted by late larval stages and newly metamorphosed juveniles of Ciona intestinalis, Molgula manhattensis, Ascidella aspersa and Phallusia nigra. Immunocytochemical approach has been applied on hatched Ciona intestinalis larvae to investigate larval metamorphosis under the influence of applied bioactive inducers. It was observed that NH4Cl 2.5 mM, C8 1 µM, Acetyl choline 1 mM, NOS 1 mM and cGMP 1 µM enhanced and accelerated the transformation of the larval stage with long tail to the next larval tail with remarkable tail resorption activity. Serotonine 10 µg/ml and Acetyl choline 10 mM showed lesser activity. All other bioactive inducers did not show any role and have about the same results as control (sea water and antibiotic sea water). Previously the effect of bioactive inducers on the larval metamorphosis of Molgula manhatensis has been tried (Saad, 2015). In this study, the author concluded that NH4Cl (2.5 mM), C8 (1 µM), NOS (2.5 mM), seratonine (10 µg/ml) and Acetyl choline (0.5 mM) accelerated the transition of larval stage from stage I with long tail to a metamorphosing stage with short tail. Yamamoto et al. [95] concluded that dopamine and serotonin influence in larval attachment of the barnacle, Balanus amphitrite. In the present study immunohistochemical approach towards the green fluorescent protein (GFP) revealed that the heat shock protein 90 is present in the brain vesicle, the nerve cord and all neurons supplying the different parts of the larval body with long and short tails. Gradually this protein diminished from the brain vesicle as the larva lost almost its tail and if this protein presents, negligible amount. Newly metamorphosed larva showed fluorescence without illumination in its external boundary. This means that the brain vesicle has given a signal to the different body parts to enhance or to activate the process of retrogressive development and metamorphosis

Marine biofouling is an undesirable accumulation of organisms on immersed substrata that is formed due to the special adherence to the surface. The highly adapted processes for temporary or permanent surface attachment that these organisms use [51] are usually referred to as bioadhesion. Biofouling occurs naturally in a wide range of surfaces, either natural or man-made [89], and plays a vital role in organisms‘life cycles and survival [51,90]. Viscous substances, secreted by marine biofoulers, enable permanent or temporary adhesion, the mechanisms of which are not yet entirely understood. It is believed though, that two main steps determine the interaction between the adhesive, produced by biofouling organisms, and the surface [89]. The first step is wetting of the surface by the adhesive. Walley, Walker, Phang et al. [91-93] mentioned that lipids, secreted first, possibly displace water from the surface interface and create a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands of the barnacle Balanus amphitrite, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings [3]. However, a great number of parameters may influence the degree of biofouling. Among all the factors temperature can be put into the first place. Even though extremely severe biofouling majorly takes place in tropical areas, any warm waters and especially near-surface substrata, heated by the sun, are potential areas for fouling development. At present more than 4000 [1,94] biofouling species are known but their favorable conditions for settlement differ substantially. Volume 5 • Issue 3 • 1000162

Figure 19: Google earth map and land picture showing the three study estuarine beaches. (1) Ad-dammam estuarine beach. (2) North Khobar estuarine beach. (3) South Khobar estuarine beach.

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doi: 10.4172/2324-8661.1000162 of the larva to a newly squirt. HSP 90 may function in emitting signals necessary for development of the different body organs and to change the brain vesicle of the larval stage to the nervous complex of the adult stage. It may function in apoptosis of the tail region with its structures of notochord and nerve cord. NOS activity was present in neurons of larval tissues. The NADPH diaphorase histochemical assay was applied. Larvae were stained for diaphorase activity. These sites of NADPH diaphorase activity most likely represented sites of NOS activity. Diaphorase activity was found in a variety of structures. Stained cells were found within the larval brain vesicle. Stained cells often have a neuronal appearance. Larvae were stained with anti-NOS antibodies to see whether sites of NADPH activity were coincident with the location of NOS. The production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduced the production of NO, and consequently cGMP, to initiate metamorphosis. This experiment showed that NO accelerated larval metamorphosis and cGMP is a second enhancing factor for NO. Bishop and Brandhorst [77] concluded that Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod. Evidence for a similar role in the sea urchin Lytechinus pictus was provided. NO commonly signals via soluble guanylyl cyclase (sGC). Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in ascidian larvae as well. This study concluded that larvae in stage I had a considerable quantity of HSP90 in their neurons in all tissues of the trunk and tail regions. High-pressure chromatographic separation has been carried out to identify amino acids in the adhesive secretions of the larval stage. The four species are rich in essential and non-essential amino acids. The essential amino acids predominated the adhesives especially Serine, Methionine and Phenylalanine, Threonine and Valine. Nineteen amino acids were identified. The most predominant amino acids were Asparagine, Glutamine, Glycine, Leucine, Methionine, Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine and Valine. Glycine ranged from 21.91 % to 25.70 % and Serine ranged from 12.78 % to 14.30 % of the amino acid content of the adhesives of the four studied ascidian larvae. There were differences in the percentage of amino acids in the four species studied with the exception of Cysteine, Lysine and Tryptophan, no significant differences. Immunoblotting using Bradford assay has been applied to identify proteins of adhesives. Proteins in different samples of the four studied ascidian larvae were resolved by SDS-PAGE and digested with trypsin. A peptide with the same sequence was obtained from protein bands of different apparent molecular masses. This analysis revealed that the adhesives contained about 11 different proteins. Their apparent molecular masses were approximately 300, 280, 250, 150, 100, 75, 50, 35, 25, 20, and 10 kDa. There was some variability between the four protein extracts of the four studied larval species, and little variability of protein contents were found within the same species. Two proteins were especially inconsistent in extraction, the 20 kDa and the 280 kDa proteins. There was also a component that was too large to enter the running gel >500 -kDa. The proteins that were analyzed in the four larval species had similar amino acid compositions. They were all rich in glycine (19-24%) and in acidic residues (16-21%). The majority of the resolved protein spots of the four larval species had pI values between pH 4 and pH 12 and the molecular size of the pattern ranged from 300 kDa to less than 10 kDa. Approximate 80% of the proteins ranged from 100 kDa to 20 kDa. Around 250-50 spots were detected in the four gels, which were compared for their intensity to identify differentially expressed spots. In the available literature, it was mentioned that the mucus produced Volume 5 • Issue 3 • 1000162

by many marine organisms like Calliosioma zizyphinhtm [96] and Mytilus sp [97] is a complex mixture of proteins and polysaccharides forming a weak watery gel. It is essential for vital processes including locomotion, navigation, structural support, heterotrophic feeding and defense against a multitude of environmental stresses, predators, parasites, and pathogens. Gel-forming glycoproteins and proteoglycans are giant molecules consisting of a protein with attached carbohydrate chains. They are so large that they form gels solely by entangling [98-100]. Total carbohydrates of adhesives were analyzed by the Phenol-sulphuric acid method. Glycogen is the main carbohydrate constituent, representing about 50% of total carbohydrates [100,101]. Glycogen is a preferred form of energy reserve particularly in settlement process of ascidian larvae because of fast glycogen catabolism that provide instant energy under hypoxic or anoxic conditions [102-104]. Total lipids were extracted using capillary gas chromatography. This test revealed that the lipid contents of adhesives of the four species studied were roughly similar but in different amounts. It was found that lipids of adhesives were represented by C14:0; C18:2 V-32; C20:1 V-9; C18:1 V-7; C16:1 V-6 and C18:1 V-5. The rest of lipid contents of adhesives are considered negligible. The varying amounts of carbohydrates were apparently dependent partly on larval stage and partly on the species. In the adhesives the following percentage amounts were found: larvae with short tail of C. intestinalis, 0.064 to 1.67 mg carbohydrates g-1 wet weight; larvae with short tail of Molgula manhattensis 0.001-1.6 mg carbohydrates g-1 wet weight; larvae with short tail of Ascidella aspersa 0-0.18 mg carbohydrates g-1 wet weight; larvae with short tail of Phallusia nigra 0 to 1.9 mg carbohydrates g-1 wet weight. The result of this study provided information on the chemical composition and morphological structure of adhesive in the four species studied. It was concluded that newly metamorphosed youngs of Ciona intestinalis,Molgula manhattensis, Ascidella aspersa and Phallusia nigra attached and settled to a wide range of substrata (iron or/and wood objects), and are considered as the most notorious biofoulers. Wet adhesion of sea squirts relied on lipids followed by the post-translational modifications of amino acids mainly phenylalanine, methionine threonine, valine and serine with glycogen for adhesive and cohesive function. The adhesive system of sea squirts appeared to be unique compared to the wet adhesion of other sessile biofoulers. The goal of this study was to describe the adhesive with emphasis on finding out a radical solution. Molecular convergences have been recognized, and some adhesive motifs have been found to be shared by phylogenetically different animals [105-107]. DOPA has long been known as one such motif [43,108,109]. Dopa was found in adhesive of Bdelloura candida [110] and in the blood fluke Schistosoma mansoni [111]. Dopa-containing protein has identified from the blood cells of the stolidobranch ascidian Pyura stolonifera [112]. Using organic extraction protocols, a class of compounds known as tunichromes (tripeptides derived from Dopa and 3,4,5-trihydroxyphenylalanine ;Topa) have been isolated from the blood cells of ascidians belonging to the Phlebobranch suborder [113115]. Dopa-containing peptides have been isolated from the blood cells as the tunichrome-like peptides called halocyamines from the ascidian Halocynthia roretzi [116,117]. All these polymers consist of Michael-type cross-links, dehydro Dopa and Topa units, and metal binding sites, and thus can become an intractable quinone-tanned matrix, ideal for the repair of wounds or forming a strong bond with the ascidian’s substratum [118]. Another modified amino acid, phosphoserine (pSer), is emerging as an important motif in biological adhesives [119]. Protein phosphorylation occurs in marine adhesives • Page 16 of 20 •


doi: 10.4172/2324-8661.1000162 in the form of serine phosphorylation and has been described in two mussel (Mytilus edulis & Mytilus galloprovincialis) adhesive proteins namely and in one tube-worm (Phragmatopoma californica) cement protein [120-124]. The use of anti-phosphoserine antibodies on Cuvierian tubules revealed that their adhesive is rich in phosphoserine residues, a strong immunolabeling being detected in the granular cells from the tubules of three different species (B. subrubra, H. forskali, and P. graeffei) [125]. Finally, lectins (i.e., proteins that specifically bind to oligosaccharidic structures) were used to characterize carbohydrate distribution in the Cuvierian tubules of H. forskali [119]. Seven lectins specific of neutral sugars (Con A, LCA, and PSA specific of mannose containing oligosaccharides; GSL I, PNA, and RCA specific of galactose containing oligosaccharides; and UEA I specific of fucose containing oligosaccharides were identified [126,127]. Several management methods have been used for the control of sessile organisms, which can be classified into three broad categories: chemical, mechanical and biological (see [128] for review). 1. Chemical methods involve the use of chemical compounds such as bleach, vinegar, lime, freshwater, sodium hydroxide, and others to kill the target species [129,130]. 2. Mechanical methods involve the deployment of physical barriers with the aim of promoting unsuitable conditions for the survival of the target species (e.g. low oxygen concentrations) or physical removal (e.g. hand picking) from the fouled surface [131-135]. 3. Biological methods involve the use of a control agent (i.e. another organism) with the aim of decreasing the abundance of the target species. However, this method of control is poorly understood and tested for colonial ascidians [29,132,136,137].

Conclusions This study concluded that lipids were secreted first possibly to displace water from the surface interface and protect the nascent adhesive plaque from biodegradation. It was found that lipids of adhesives were represented by C14:0 ; C18:2 V-32 ; C20:1 V-9 ; C18:1 V-7 ; C16:1 V-6 and C18:1 V-5. Fatty acids contain double bonds, hydroxyl groups, or other functional groups. Searching for a benign chemical, to be added in paint as antifouling agent that dissolve this lipid moiety and thus settlement will fail to occur. The following reactions 1-3 can convert fatty acids into long-chain diols. These are usually alpha, omega-diols or diols whose hydroxyl groups lie far apart. 1- Suspension Hydrogenation: adding a fine powdery copper catalyst. The excess hydrogen serves to circulate the reaction mixture. The product mixture splits into a gas phase and a liquid phase. 2Bashkirov Oxidation: adding of boric acid esters which scavenge the hydroperoxides that are formed as intermediates. Hydrolysis leads to a statistical distribution of secondary alcohols in which the hydroxyl function may occupy any position on the carbon chain. 3- Industrial oxidation: using a nitrogen- air mixture containing approx. 3.5 % of oxygen. Searching for a methodology to prevent post-translational modifications of amino acids mainly Phenylalanine, Methionine Threonine, Valine and Serine with glycogen for adhesive and cohesive function. Didecyl polyoxyethyl ammonium borate (DPAB), also known as Polymeric Betaine with high protein resistance can be applied as marine coatings to resist attachment of marine organisms. Betaine-based polymer brushes are supposed to be among the most nonfouling surfaces to resist protein adsorption. Sulfobetaine polymer brushes were show strong resistance to the attachment of fouling organisms. Betaine polymers can be designed based on the main chains or the backbones of the polymers, the linking groups that Volume 5 • Issue 3 • 1000162

connect betaine moieties to the backbones. The transformation of the larval stage with long tail to the next larval tail with remarkable tail resorption activity is accelerated by NH4Cl 2.5 mM, C8 1 µM, Acetyl choline 1 mM, NOS 1 mM and cGMP 1 µM. Most of these bioactive inducers are neurosecretions during embryonic stages. So, a bioactive chemical acts against these inducers is necessary to be added in the paint. The carbohydrate moiety of adhesives was observed as glycogen. So, searching for a benign chemical that disintegrate this carbohydrate moiety or preventing the polymerization of monosaccharide’s and thus settlement will fail to occur. The investigated carbohydrate polymers do not seem to be major primary adhesives products; rather they appear to be formed by the DE hydrolysis of monosaccharaides. The distribution pattern of individual carbohydrates reflects the various stages of adhesives secretions, and the biological and abiotic processes controlling the formation and alteration of dissolved organic matter by adhesive papillae. Abiotic processes, like isomerization of monoto polysaccharides, are potentially useful in determining the age of marine dissolved organic matter. Acknowledgement Authors would like to express their gratitude and sincere to the Deanship of Scientific Research; Ministry of Higher Education at the University of Dammam. Especial thanks to Professor Badaruddin Abbasi for his guidance and support. This research was conducted under the project number 2015099 in the framework of an environmental monitoring program against fouling organisms inhabiting the Arabian Gulf in Saudi Arabia. We acknowledge the financial support of the University of Dammam for this research. We thank the referees for their helpful discussions, and for the critical revision and valuable commenting on the manuscript. The co-authors would like to express their special gratitude for the PI for his invaluable help regarding the localization of presumable type specimens in the collections of the sea squirts and providing important data on them. The authors would like to add that there was no conflict in this work.

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