Liosina Paradoxa Thiele, 1899 from Sandspit Backwater Mangroves at Karachi Coast, Pakistan Abstract 16 2.1

Home , Bubarida, Homotrema, Tethyspira

“He is the one who has set free the two kinds of water, one sweet and palatable, and the other salty and bitter. And He has made between them a barrier and a forbidding partition.” ﴿Al-Qur’an, 25:53﴾ MARINE SPONGES: ECOLOGY, ASSOCIATED COMMUNITIES AND BIOCHEMICAL ASSESSMENT, INHABITING MANGROVE OF SANDSPIT BACKWATER, KARACHI COAST

Thesis submitted to the University of Karachi in partial fulfillment of the requirement for the Degree of Doctor of Philosophy in Marine Biology

By Hina Jabeen M.Sc. (Zoology)

Centre of Excellence in Marine Biology University of Karachi 2018 CERTIFICATE

Certified that the PhD thesis entitled “Marine sponges: ecology, associated communities and biochemical assessment, inhabiting mangrove of Sandspit backwater, Karachi coast” is a record of research work carried by Ms. Hina Jabeen is satisfactory for partial fulfillment of the requirement for the degree of Doctor of Philosophy in Marine Biology and further evaluated by the external examiners and recommended her for confirmation of degree of Doctor of Philosophy in Marine Biology by the Centre of Excellence in Marine Biology, University of Karachi. The similar contents and form of this thesis has not presented for any other degree or diploma earlier elsewhere.

Research Supervisor: ______

Dr. Seema Shafique Assistant Professor Centre of Excellence in Marine Biology, University of Karachi Karachi 75270, Pakistan.

Research Co-Supervisor: ______

Dr. Munawwer Rasheed Associate Professor Centre of Excellence in Marine Biology, University of Karachi Karachi 75270, Pakistan.

External Examiner: ______

Director: ______Centre of Excellence in Marine Biology, University of Karachi Karachi 75270, Pakistan.

DEDICATIONS

I dedicate my PhD dissertation to my beloved Prophet and my first inspiration Muhammad (S.A.W.W.). My special thanks to my strong, loving and caring mother Mrs. Husna Jabeen who taught me to believe on Allah and hard work, my great supportive father, my first teacher Mr. Qazi Matloob Azam and my right hand, my brother Mr. Qazi Umair Azam.

A special feeling of gratitude to my beloved uncle Mr. Ali Ahmed Siddiqui (Late) for his always precious love and care. My loving aunt Mrs. Fateh-un-Nisa (Late), for her support, sincere love and care for every step of my life. May Allah forgive them and may their souls

آنیم !rest in peace

I also dedicate this thesis to my all inspiring teachers of Federal Urdu University (Zoology Department), specially Dr. Syed Kamaluddin Ahmed. My all beloved and caring primary and high school teachers, specially Mrs. Arshia, Mrs. Shumaila, Ms. Sualeha, and Prof. Masood Ahmed, who are meticulous to me and supported to brought up my personality.

I dedicate this work and give special thanks to my childhood and school friends Ms. Madiha Aslam, Mrs. Kausar Aleem, Ms. Sehrish Naeem Alvi and Mrs. Shafaq Yasir. My university friends and fellows Ms. Musarrat-ul-Ain, Mrs. Parveen Kamran and Mrs. Nazia Arif. At last, my best friend, my backbone and my precious diamond, Ms. Syeda Tatheer Fatima.

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ACKNOWLEDGEMENTS

,Who gave me strength ,ﷻFirst and foremost, my all heartily gratitude to ALMIGHTY ALLAH love, care, support and everything which I just wished and He accepted my each pray and gifted beautiful things and relations in my life.

Then subsequently, I want to express my deepest thanks to Director, Centre of Excellence in Marine Biology, University of Karachi. Prof. Dr. Pirzada Jamaluddin Ahmed Siddiqui, Former Director of Centre of Excellence in Marine Biology, University of Karachi. My loving research supervisor Dr. Seema Shafique, (Assistant Professor) for sparking my passion for marine sponges. Her always positive attitude and trust encouraged me and strengthened together with my growing tasks strongly. I am sincerely thankful to my research co-supervisor Dr. Munawwer Rasheed, (Associate Professor) and Madam Zaib-un-Nisa Burhan (Lecturer), who dedicated me her time, ideas and moral support in my research work, for being as an accompanied supervisor at Centre of Excellence in Marine Biology, University of Karachi.

Dr. John N.A. Hooper, Head of Natural Environments Program, Queensland Museum, Australia, who really supported and guided me in taxonomic work of sponges, also encourage me to do advanced aspects of sponge work in Pakistan. Dr. Rob W. M. Van Soest, Research Associate, Naturalis Biodiversity Centre, Leiden, The Netherlands, who guided me in sponge systematics.

Dr. Nasira Kazi, National Nematological Research Center, University of Karachi for their guidance regarding systematics of nematodes. Ms. Nazish George (Ph.D. student), Centre of Excellence in Marine Biology, University of Karachi, for assisted me in histological work of sponges. Dr. Amir Ahmed, Pharmaceutical Research Center, Pakistan Council for Scientific and Industrial Research, Karachi Laboratory Complex, Karachi Dr. Kehkashan Khan and Mrs. Javeria Zuhair, Department of Chemistry, University of Karachi for their cooperation in analyzation and interpretation of sponge extracted compounds.

My impressive seniors and fellows, who guided me in every step of my research experiments and stand with me all the time in field collections and in laboratory, Dr. Pervaiz Iqbal, Dr. Yasmeen Zamir Ahmed, Rukhsana Akhter, Saira Nasim, Sundas Awais, Fouzia Bibi and Sana Noman. In the last, I am thankful to field attendants, lab attendants and drivers of the Centre.

TABLE OF CONTENTS

Page #

LIST OF FIGURES VIII LIST OF TABLES XIII

Abstract ...... XVII Abstract in Urdu ...... XVIII

I. MANGROVES AND THEIR ECOLOGICAL PERSPECTIVES WITH MARINE SPONGES

Chapter 1: General Introduction 1.1. Mangroves 1 1.1.1. Distribution and diversity of mangroves 1 1.1.2. Mangroves of Pakistan 3 1.2. Impacts of environmental stressors on mangrove 3 1.2.1. Temperature 5 1.2.2. Salinity 5 1.2.3. Gases fluxion 5 1.2.4. Siltation and sedimentation 6 1.2.5. Zonation and tidal action 6 1.3. Significance of mangroves 7 1.3.1. Commercial role of mangroves 7 1.3.2. Ecological role of mangroves 9 1.3.3. Biological role of mangroves 10 1.3.3.1. Associated communities of mangroves 10 1.4. Mangrove associated sponges (Phylum Porifera) 12 1.4.1. Morphology 12 1.4.2. Sponge associated communities 13 1.4.3. Diversity and distribution of sponges 14

1.5. Study objectives 15

II. SYSTEMATIC STUDY OF MARINE SPONGES

(PORIFERA) IN PAKISTAN

Chapter 2: Marine Sponge (Porifera: Demospongiae) Liosina paradoxa Thiele, 1899 from Sandspit backwater mangroves at Karachi coast, Pakistan Abstract 16 2.1. Introduction 17 2.1.1. Classification of mangrove sponges of Pakistan 17 2.2. Materials and methods 18 2.3. Results 22 2.4. Discussion 26

Chapter 3: Two new records of marine sponges (Demospongiae: Haplosclerida)

from the coast of Karachi, Pakistan Abstract 27 3.1. Introduction 28 3.2. Materials and methods 31 3.3. Results 34 3.4. Discussion 41

III. GROWTH AND ABUNDANCE OF MARINE SPONGE

AND THEIR ASSOCIATED COMMUNITIES

Chapter 4: In situ variation in growth and abundance of marine sponge Liosina

paradoxa Thiele, 1899 at Sandspit backwater mangroves Abstract 43 4.1. Introduction 44 4.1.1. Environmental factors that affecting the sponge growth 45 4.1.2. Nutrients productivity and its effect on sponge growth 46 4.2. Materials and methods 48 4.3. Results 56

4.4. Discussion 77

Chapter 5: Phytoplankton community associated with marine sponge Liosina

paradoxa Thiele, 1899 at Sandspit, Karachi Abstract 80 5.1. Introduction 81 5.2. Materials and methods 83 5.3. Results 84 5.4. Discussion 98

Chapter 6: Seasonal diversity of benthic foraminifera associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwaters, Karachi Abstract 100 6.1. Introduction 101 6.2. Materials and methods 102 6.3. Results 103 6.4. Discussion 113

Chapter 7: The community composition of meso-zooplankton and association with Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves, Karachi Abstract 115 7.1. Introduction 116 7.2. Materials and methods 118 7.3. Results 119 7.4. Discussion 141

IV. GCMS PROFILE OF MARINE SPONGE LIOSINA PARADOXA THIELE, 1899

Chapter 8: Identification of secondary metabolites from marine sponge Liosina

paradoxa Thiele, 1899 in mangroves at Sandspit backwater, Karachi Abstract 143 8.1. Introduction 144 8.1.1. Marine natural products from invertebrates 144 8.1.2. Marine natural products from sponges 144 8.1.3. Bioactivity of marine sponges 146 8.1.4. Identification of secondary metabolites from marine sponges through GC-MS 147 8.1.5. Natural products from marine sponges of Pakistan 150 8.2. Materials and methods 151 8.3. Results 157 8.4. Discussion 160

V. Conclusion …………………………………………………………...... 165

VI. References …………………………………………………………………. 169

VII. Appendix …………………………………………………………………. 224

VII

LIST OF FIGURES

Fig. 1 Map of Pakistan coastline shows mangrove regions. 4

Fig. 2 (A) Study site of Sandspit backwater mangroves dominated by 8 Avicennia marina, (B) Channel water during high tide, (C) and (D) Browsing and grazing of camels on A. marina at study site.

Fig. 3 Internal and external morphological features of sponge. 13

Fig. 4 Map of Pakistan shows the collection site. Sandspit, Karachi. 19

Fig. 5 (A) Light microscope (Olympus, IX 51) and (B) Scanning Electron 21 Microscope (JEOL JSM-6380A).

Fig. 6 (A) and (B) Specimen (Liosina paradoxa Thiele, 1899) on 24 pneumatophores of Avicennia marina, (C) Collected specimens of L. paradoxa, (D) Light microscopic image of ectosomal skeleton showing hexagonal surface structure (scale = 20 µm), (E) Light microscopic image of spicules (scale = 100 µm) and (F) Scanning electron microscopic image of spicules (scale = 50 µm).

Fig. 7 Map of Karachi coast shows the collection site of Churna Island and 33 Buleji.

Fig. 8 (A) Specimen of Callyspongia (Cladochalina) fibrosa Ridley and 38 Dendy, 1886 (B) Light microscopic image of tangential skeleton shows tufted spongin network (scale = 20 µm), (C) Light microscopic image of microtome-section shows spiculo-fibres and (D) Scanning electron microscopic image of siliceous spicules (scale = 5 µm).

Fig. 9 (A) Specimen of Haliclona (Soestella) hornelli (Dendy, 1916) in rock 39 pool, (B) Preserved specimen of H. (S.) hornelli, (C). Light microscopic image of internal skeletal structure, (D) Scanning electron microscopic image of oxeas (scale = 20 µm), (E). Histological section of ectosomal

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and choanosomal skeleton (scale = 500 µm) and (F). Spicules (oxeas) in tangential section.

Fig. 10 Map of Karachi coast shows four collection transects {first transect 50

(TR1), second transect (TR2), third transect (TR3) and fourth transect

(TR4)} at Sandspit, Karachi.

Fig. 11 (A) Study site of Sandspit backwater, Karachi, (B) Liosina paradoxa 55 attached with pneumatophores, (C) Measurement of L. paradoxa in transect and (D) Growth size of sponge specimens in laboratory.

Fig. 12 Annual growth rate (days-1) of L. paradoxa on pneumatophores of 60

Avicennia marina at four transects (A - TR1; B - TR2; C - TR3 and D -

TR4) of Sandspit backwater, Karachi.

Fig. 13 (A) Annual volume (cm3) and (B) total abundance (%) of L. paradoxa 61

on pneumatophores of Avicennia marina at four transects (TR1, TR2,

TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 14 (A) Annual concentration of physicochemical parameters (temperature 62 °C, salinity PSU, pH and dissolved oxygen mg L-1) and (B) Nutrients - - + -3 (NO3 , NO2 , NH4 and PO4 ) in channel water of Sandspit backwater mangroves, Karachi.

Fig. 15 Moisture volume (g%) in mangrove sediment cores from January to 63

December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Fig.16 The amount of inorganic content (µg/g) in mangrove sediment cores 64

from January to December (A to L) at four transects (TR1, TR2, TR3 and

TR4) of Sandspit backwater, Karachi.

Fig.17 The amount of organic content (µg/g) in mangrove sediment cores from 65

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 18 The concentration of total organic carbon (mg/g) in mangrove sediment 66

cores from January to December (A to L) at four transects (TR1, TR2,

TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 19 Chlorophyll ‘a’ concentration (µg/g) in mangrove sediment cores from 67

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 20 Chlorophyll ‘b’ concentration (µg/g) in mangrove sediment cores from 68

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 21 The concentration of carotenoids (µg/g) in mangrove sediment cores 69

from January to December (A to L) at four transects (TR1, TR2, TR3 and

TR4) of Sandspit backwater, Karachi.

Fig. 22 The concentration of phaeo-pigments (µg/g) in mangrove sediment 70

cores from January to December (A to L) at four transects (TR1, TR2,

TR3 and TR4) of Sandspit backwater, Karachi.

Fig. 23 The cumulative dominance curves (%) for sponge abundance rank at 71 four transects of Sandspit backwater, Karachi.

Fig. 24 The cumulative dominance curves (%) for sponge biomass rank at four 71 transects of Sandspit backwater, Karachi.

Fig.25 Light microscopic images of species belong to Cyanophyceae; scale = 88 10 µm, (A) Oscillitoria princeps, (B) O. perornata, (C) O. tenuis and (D) Phormidium tenue.

Fig. 26 Light microscopic images of Bacillariophyceae species, (A) Diploneis 90 smithii (scale = 10 µm), (B) Gyrosigma sp. (scale = 10 µm), (C) Halamphora coeffoeformis (scale = 5 µm), (D) H. proteus (scale = 10 µm), (E) Navicula sp. (scale = 5 µm), (F) Nitzschia palae (scale = 10 µm), (G) N. sigma (scale = 15 µm), (H) Pinnularia sp. (scale = 10 µm),

(I) Surirella fastuosa (scale = 5 µm) and (J) S. ovata (scale = 5 µm).

Fig. 27 Light microscopic images of Chlorophyceae species (scale = 10 µm), 91 (A) Rhizoclonium tortosum and (B) Ulothrix tenuissima.

Fig. 28 Percent composition of phytoplankton groups associated with Liosina 92 paradoxa at Sandspit backwater, Karachi.

Fig. 29 Seasonal composition of phytoplankton groups associated with Liosina 93 paradoxa at Sandspit backwater, Karachi.

Fig. 30 Seasonal variation in physicochemical parameters (temperature °C, 93 salinity PSU, pH and dissolved oxygen mg L-1) at Sandspit backwater, Karachi.

Fig. 31 The K dominance curves (cumulative dominance) for seasonal 94 abundance of phytoplankton community associated with Liosina paradoxa at Sandspit backwater, Karachi.

Fig. 32 Light (LM; scale = 10 µm) and scanning electron (SEM; scale = 50 µm) 107 microscopic images of sponges associated foraminifera from Sandspit backwater mangroves, Karachi coast. (A) Ammonia beccarii (i: Distal and ii: Proximal view), (B) Brizalina subspinescens, (i: LM and ii: SEM image), (C) Rosalina globularis (i: Distal and ii: Proximal view), (D) Ammotium cassis (i: LM and ii: SEM image), (E) Entzia macrescens, (F) Trochammina inflata and (G) Quinqueloculina laevigata.

Fig. 33 Cluster analysis of foraminifera species associated with marine sponge 108 Liosina paradoxa at Sandspit, Karachi.

Fig. 34 The K dominance curves (cumulative dominance) for foraminifera 109 species abundance in four transects at Sandspit backwater, Karachi.

Fig. 35 Light microscopic images of Polychaeta species (scale: 20 µm), (A) 122 Lopadorhynchus henseni, (B) Harmothoë imbricata, (C) Branchiomma cingulata and (D) Sphaerosyllis sp.

Fig. 36 Light microscopic images of Nematoda species, (A) Paracanthonchus 125 sp. (entire, scale: 20 µm), (B) P. hawaiiensis (entire, scale: 20 µm), (C) P. hawaiiensis (i. head, scale: 5 µm; ii. tail, scale: 5 µm), (D) P. sandspitensis (i. entire, scale: 20 µm; ii. head, scale: 10 µm), (E) Paracyatholaimus sp. (i. head, scale: 5 µm; ii. tail, scale: 10 µm), (F) Desmodora sp. (i. entire and ii. tail, scale: 10 µm), (G) Dracograllus sp. (i. head and ii. tail, scale: 5 µm), (H) Enoplus sp. (entire, scale: 20 µm), (I) Eleutherolaimus inglisi (entire, scale: 20 µm), (J) E. inglisi (i. head and ii. tail, scale: 5 µm), (K) Monhystera marina (i. head and ii. tail, scale: 10 µm), (L) Monhystrella sp. (entire, scale: 5 µm), (M) Adoncholaimus sp. (entire, scale: 10 µm), (N) Halichoanolaimus balochiensis (head and tail, scale: 10 µm) and (O) Tricotheristus sp. (head, scale: 20 µm).

Fig. 37 Light microscopic images of crustacean larvae (scale: 5 µm). 126

Fig. 38 Light microscopic images of Crustacea species (scale: 20 µm), (A) 127 Copepoda, Cyclopoidea (Oithona sp. i. male and ii. female), (B) Amphipoda, (i. Urothoe sp. and ii. Corophium sp.), (C) Isopoda (Sphaeroma terebrans) and (D) Cirripede (Balanus amphitrite).

Fig. 39 Light microscopic images of minor phyla, (A) Rotifera, i. Philodina 128 roseola (scale: 20 µm) and ii. P. nitida (scale = 10 µm). (B) Platyhelminthes, Lehardyia sp. (i. scale: 20 µm and ii. scale: 10 µm). (C) Hemichordata, Saccoglossus sp. (i. entire, scale: 40 µm and ii. visible notochord, scale: 20 µm).

Fig. 40 Seasonal percent species composition of faunal communities associated 134 with Liosina paradoxa at Sandspit backwater mangroves, Karachi.

Fig. 41. Chemical structures of identified compounds from marine sponge 158 Liosina paradoxa.

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LIST OF TABLES

Table I. Distribution, diversity and occurrence of mangroves in the 2 countries bordering the Indian Ocean.

Table II. The worldwide geographical distribution of Liosina paradoxa 25 Thiele, 1899.

Table III. The worldwide geographical distribution of Callyspongia 40 (Cladochalina) fibrosa Ridley and Dendy, 1886.

Table IV. Pearson correlation of growth rate (days-1) of Liosina paradoxa 72

at four transects (TR1, TR2, TR3 and TR4) with physicochemical parameters (temperature °C, salinity PSU, pH and dissolved -1 - - + -3 oxygen mg L ) and nutrients (NO2 , NO3 , NH4 and PO4 ) at Sandspit, Karachi.

Table V. Pearson correlation of abundance (%) of Liosina paradoxa at 73

four transects (TR1, TR2, TR3 and TR4) with physicochemical parameters (temperature °C, salinity PSU, pH and dissolved -1 - - + -3 oxygen mg L ) and nutrients (NO2 , NO3 , NH4 and PO4 ) at Sandspit backwater, Karachi.

Table VI. Pearson correlation of growth rate (days-1) and abundance (%) of 74

Liosina paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Table VII. Pearson correlation of growth rate (days-1) and volume (%) of 75

Liosina paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Table VIII. Pearson correlation of abundance (%) and volume (cm3) of 76

Liosina paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

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Table IX. The seasonal occurrence of phytoplankton species associated 95 with Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves, Karachi.

Table X. Seasonal variation in diversity indices of phytoplankton species 96 at Sandspit backwater, Karachi.

Table XI. Pearson correlation coefficient matrix between phytoplankton 97 community and physicochemical parameters at Sandspit backwater, Karachi.

Table XII. Variation in species number and percent abundance of 110 foraminifera associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XIII. The occurrence of foraminifera species associated with Liosina 111 paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XIV. Diversity indices of foraminifera species associated with Liosina 112 paradoxa at four transects of Sandspit backwater, Karachi.

Table XV. The occurrence of polychaetes species associated with Liosina 129 paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XVI. The occurrence of nematodes species associated with Liosina 130 paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XVII. The occurrence of crustacean species associated with Liosina 132 paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

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Table XVIII. The occurrence of other associated groups with Liosina 133 paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XIX. Variation in species number and percent abundance of 135 polychaetes associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XX. Variation in species number and percent abundance of 136 nematodes associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XXI. Variation in species number and percent abundance of 137 crustaceans associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XXII. Variation in species number and percent abundance of other 138 groups associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Table XXIII. Seasonal variation in diversity indices of associated communities 139 of Liosina paradoxa at Sandspit backwater, Karachi.

Table XXIV. Pearson correlation coefficient matrix between associated 140 community with Liosina paradoxa and physicochemical parameters at Sandspit backwater, Karachi.

Table XXV. Compounds isolated from marine sponges. 146

Table XXVI. GC-MS profiling of marine sponges (Demospongiae) from 149

different regions of Indian Ocean.

Table XXVII. Chemical constituents identified from Liosina paradoxa through 150 GC-MS analysis.

Table XXVIII. Identified chemical constituents from marine sponge Liosina 159 paradoxa through GC-MS studies in Pakistan.

Table XXIX. Importance and natural occurrence of identified chemical 162 constituents from marine sponge Liosina paradoxa.

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ABSTRACT

Mangroves are salt tolerant coastal vegetation that inhabits tropical and sub-tropical regions and consider as one of the most productive ecosystem of the world. About 95% of Avicennia marina is distributed along the Indus delta at South west of Karachi. They provide a distinct habitat for sheltering and nourishing wide variety of fauna and flora. Among benthic fauna, largely diversified communities of marine sponges have found but taxonomically no data has recorded from Pakistan. Liosina paradoxa (family Dictyonellidae) is the dominant sponge species found on pneumatophores of A. marina. This species is systematically identified and widely distributed at Sandspit backwater mangroves, Karachi coast. Other marine sponge species, Callyspongia (Cladochalina) fibrosa and Haliclona (Soestella) hornelli belonged to order Haplosclerida has also taxonomically recognized and first recorded from Churna Island and Buleji rocky ledges, respectively. The growth rate and abundance of dominating mangrove sponge L. paradoxa was showing highest growth rate in July in TR4 and maximum abundance 3 was observed in TR3. The sponge volume was observed maximum in July (127.53 – 313.87 cm ) and minimum in December (6.24 – 24.48 cm3) in all four transects. During study, physicochemical parameters such as temperature was ranged between 19 - 32 °C, salinity was 35 – 41 PSU, dissolved oxygen was 0.11 – 3.44 mg L-1 and pH was 7.04 to 7.69. In addition to - -1 - + nutrients, NO3 concentration was maximum in June (5.64 µg L ), NO2 and NH4 concentrations were high in February (0.28 and 7.50 µg L-1, respectively) and the concentration -3 -1 of PO4 was maximum in April (2.95 µg L ). Growth and abundance of sponge showed positive correlation with temperature, oxygen, pH, nitrite and ammonium ions. Among the flora, diatoms (90%) were the dominant group associated with L. paradoxa. Including the fauna, Foraminifera has showed maximum diversity (42%) in TR2 during monsoon season. Nematoda (41%) were the most dominant sponge associated community followed by Crustacea (38%) and Polychaeta (20%). For the identification of secondary metabolites, sponge (L. paradoxa) sample were soaked in four different solvents (n-hexane, ethyl acetate, dichloromethane and methanol). Eleven compounds were identified through GCMS and structure elucidation from their methyl ester derivatives which mostly result as fatty acids. The data obtained from the present study provides the information regarding systematic distribution, growth, abundance and associated communities of marine sponge L. paradoxa along the coastal region of Karachi.

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الخہص

رمت ایکمنت روا اسیلح وپدے ںیہ وج احری اور مین احری العوقں ںیم پاےئ اجےت ںیہ اور داین ےک سب ےس ز پادہ دیپاواری اظنم

ںیم ےس ا یب ےھجمس اجےت ںیہ۔ رقت ًیا Avicennia marina ۹۵٪ ا ڈنس ڈاٹلی یک اج نب رکایچ ےک ونجب رغمب ںیم واعق

ںیہ۔ ہی وعیس امیپےن رپ فلتخم ااسقم ےک ابنپات و ویحاپات یک انپہ اورذغا ےکےئل وصخمص اموحل فرامہ رکےت ںیہ۔ رعقی ویحاپات ںیم

دنمسری اجنفس یک ونتمع آپا د پاں یتلم ںیہ نج اک اایمسیت ادعادوامشر اب یب پااتسکن ےک اسلح ےس راکیرڈ ںیہن ایک ایگ۔ .L (

paradoxaاخدنان Dictyonellidae) یک ڑج ںیم پایئ اجےن وایل ا یب اغ لب ونع ےہ۔ ہی ونع اایمسیت اابتعر ےس انشخب یک یئگ وج ڈنیسز نب ٬رکایچ ےک رمت ںیم وتعس ےس پایئ اجیت ےہ۔درگی رحبی اجنفس یک اوناع ںیم Callyspongia

Cladochalina) fibrosa) اور Haliclona (Soestella) hornelli شالم ںیہ وج فصی لہ ب لی Haplosclerida ےس قلعت ریتھک ںیہ٬ اںیہن یھب رچپا اور جی ےس یلہپ رمہبت اایمسیت وطر رپ راکیرڈ ایک ایگ۔ رمت ںیم .L paradoxa یک ومن و رثکت اک نیعت سب ےس ز پادہ وجالیئ ںیم پا رتلبیت رسیتے (TR3) اور وچےھتعلقے (TR4) ںیم ایک ایگ۔ اجنفس اک مجح س ےس ز پادہ وجالیئ )cm3 313.87-127.53(ںیم اور مک ےس مک دربمس )cm3 24.48-6.24( ںیم امتم

علقو ں ںیم پا پا ایگ۔ دورا نب زجتہی٬ پاین ںیم وموجدیعبط وایمیکیئ انعرصیک رجنی ےسیج٬ درہج رحارت; C°32-٬19 وشر نب 35-41 ; ٬PSU لیلحت دشہ آنجیسک; mg L-1 3.44-0.11 اور pH; 7.69-7.04 ےک درایمن ریہ۔ ذغایئ انعرص یک - -1 - پاین ںیم وموجد رجنی اک اشمدہہ ایک ایگ ےسیج٬ NO3 یک سب ےس ز پادہ دقمار وجن ( µg L 5.64) ںیم ٬ NO2 اور -1 -3 -1 + NH4فروری (0.28 اور µg L 7.5) ںیم اور PO4 یک سب ےس ز پادہ دقمار ارپلی ( µg L 2.95)

ںیم دیھکی یئگ۔ اجنفس یک ومن و رثکت ےن درہج رحارت٬ آنجیسک ٬ ٬pH پارٹئا ئب اور اوممین آنئ ےس تبثم قلعت اظ ہبر ایک۔ .L

paradoxaےس کلسنم ابنپات ےک امنیب diatoms (٪۲۵ ) ز پادہ پاےئ ےئگ۔ ومشبل ویحا اپت، وفرا مب ریفایک ریثک اوناع ( س TR2 ( ۴۲٪ ںیم ومن وسن ںیم پایئ یئگ۔ امینوٹڈا (٪۴۱(یک ریثک اوناع ومشبل رک یی شی ا (٪۳۸( اور وپل یکی ی ا (٪۲۰( اجنفس ےس

کلسنم پایئ ںیئگ۔ پاایمیت رمابکت یک اجچن ےئلیکاجنفس وک اچر فلتخم للحمںیم ڈاال ایگ۔ L. paradoxa ےس لک ایگرہ رم ّ اکت٬ شج GCMSاور اےکن اموخذاز methyl esters یک وتحیض اسوتخں ےک ذرےعی احلص ےیک ےئگ٬ وج ز پادہ تر می ترےش ںیہ۔ احہیل

قیقحت رکایچ ےک اسیلح ےطخ ںیم L. paradoxa یک اایمسیت وتعس٬ ومنورثکت اور کلسنم آپا دویں یک ولعمامت رفامہ رکیت ےہ۔

XVIII

PART – I

MANGROVES AND THEIR ECOLOGICAL PERSPECTIVES WITH MARINE SPONGES

CHAPTER - 1

GENERAL INTRODUCTION

Part: I Chapter - 1

GENERAL INTRODUCTION

1.1. Mangroves

Mangroves are salt tolerant coastal and intertidal vegetation (Duke, Ball and Ellison, 1998; Alongi, 2008) that inhabits in tropical and sub-tropical regions (Holguin, Vazquez and Bashan, 2001; Krauss, Lovelock, McKee, López-Hoffman, Ewe and Sousa, 2008; Andreote, Jiménez, Chaves, Dias, Luvizotto, Dini-Andreote and de Melo, 2012). They have ability to survive in harsh environmental conditions such as extreme temperature, salinity and tides (Alongi, 2009). Mangrove ecosystem considered as one of the most productive ecosystem of the world (Alongi, Tirendi and Clough, 2000; Holguin, Vazquez and Bashan, 2001; Lee, 2008; Noor, Batool, Mazhar and Ilyas, 2015).

1.1.1. Distribution and diversity of mangroves

Worldwide mangrove covered an area about 110,000 to 240,000 km2 (Wilkie and Fortune, 2003; FAO, 2010) of which 42% in Asian countries (Giri, Ochieng, Tieszen, Zhu, Singh, Loveland and Duke, 2011). Among Southeast Asian countries bordering Indian Ocean, mangrove forests covered an area of about 84,984.56 km2 specifically in Indonesia, Sundarbans (India and Bangladesh) and Malaysia, the rich growth of mangrove reported (Ahamada, 1997) where as in Pakistan, Arabian Gulf and Gujarat (India) has less growth due to semi-arid climate, high salinity ranges, less nutrients availability and mostly sandy sedimentation (Kathiresan and Rajendran, 2005).

There are 70 species of mangroves found worldwide (Blasco, Aizpuru and Gers, 2001; Polidoro, Carpenter, Collins, Duke, Ellison, Ellison and Livingstone, 2010), from which approximately 50% species recorded from Southeast Asia (Polidoro, Carpenter, Collins, Duke, Ellison, Ellison and Livingstone, 2010); 55 from Indian Ocean (Kathiresan and Bingham, 2001), 17 from Africa (Spalding, Blasco and Field, 1997) and 10 species from

America (IUCN, 2005). Country-wise distribution and diversity range of mangroves bordering Indian Ocean (Ahamada, 1997; Kathiresan and Rajendran, 2005) is set out in Table I.

Table I. Distribution, diversity and occurrence of mangroves in the countries bordering the Indian Ocean. Region Area coverage (km2) Species Source Indonesia 32,000 41 Santoso, 2004 Myanmar 6,950 28 Blasco and Aizpuru, 2002 Malaysia 5,650 42 FAO. 2007 India 4,871 39 Kathiresan and Rajendran, 2005 Northwest Australia 4,513 28 Duke, 1992 Bangladesh 4,500 22 Ellison et al., 2000 Madagascar 3,400 9 Rakotomavo and Fromard, 2010 Mozambique 5,211 8 Taylor et al., 2003 Pakistan 2,600 4 Siddiqui et al., 2008 Thailand 1,900 34 Aksornkoae et al., 1993 Tanzania 1,335 9 Wang et al., 2003 Kenya 500 8 Kairo et al., 2002 Sri Lanka 120 22 Jayatissa and Koedam, 2002 Singapore 18 31 Ng et al., 1999 South Africa 11 6 Diops et al., 2002 Maldives 4.18 13 Jagtap and Untawale, 1999 Mauritius 2 3 Gaudian et al., 1995

In countries of Arabian Peninsula (Arab Emirates, Abu Dhabi, Oman, Yemen and Iran) bordering Red Sea, Persian and Arabian Gulf have low biodiversity and monospecific (Avicennia marina) mangrove except Saudi Arabia which includes two species (Avicennia marina and Rhizophora mucronata) of mangrove due to arid environment, high salinity (low input of freshwater) and high sedimentation (ElAmry, 1998; Vannucci,

2002; Kumar, Asif Khan and Muqtadir, 2010; Polidoro, Carpenter, Collins, Duke, Ellison, Ellison and Livingstone, 2010).

1.1.2. Mangroves of Pakistan

The coastline of Pakistan encompasses about 1050 km with 250 km of Sindh and 800 km of Balochistan coasts (Fig. 1) (Saifullah and Rasool, 2002). The estimation of mangroves cover along Indus Delta is 2494.86 km2 and 0.2 km2 along the Makran coast of Balochistan (Tariq, Dawar, Mehdi and Zaki, 2006), while the total mangroves cover in Balochistan is 73.4 km2 (Saifullah, Ismail, Khan and Saleem, 2004; Tariq, Dawar, Mehdi and Zaki, 2006). According to floral taxa of Pakistan, 8 species of mangroves were reported (Shah and Baig, 2001; IUCN, 2005; Memon, 2012), but due to various factors e.g. environmental condition, deforestation and over exploitation etc. has limited these taxa into only four species including the dominating species Avicennia marina followed by Rhizophora mucronata, Ceriops tagal and Aegiceras corniculatum (Siddiqui, Farooq, Shafique and Farooqi, 2008). 95% of black mangroves (Avicennia marina) are widely distributed along the Indus deltaic region at South west of Karachi, constitute about 867.27 km2 (IUCN, 2005) and few pockets of Balochistan coasts (Saifullah and Rasool, 1995; Khan and Aziz, 2001; Barkati and Rahman, 2005).

1.2. Impacts of environmental factors on mangroves

Mangroves are well adapted to grow between land and sea thus have affected by various environmental stressors for example fluctuations in temperature, salinity, siltation and drought conditions effects adversely on growth and reproduction of mangrove plants.

Figure 1. Map of Pakistan coastline shows mangrove regions (Google Earth Pro 7.1.4.1529 and Snazzy Maps).

1.2.1. Temperature

Mangroves (Avicennia marina) are well adapted to semi-arid and arid zones thus they show good resistance with high temperature and effect of sunlight (ElAmry, 1998), however excessive temperature and sunlight may result in declining of photosynthetic rate (Field, 1995; Cheeseman and Lovelock, 2004). Air and water temperature both regulates the development of mangroves. A little increase in temperature (i.e., >35°C) directly affects the transpiration within stomata, photosynthesis, associated mangrove fauna (molluscs, fishes, crabs, shrimps and other crustaceans) and may cause desiccation for some gastropod species (Kjerfve and Macintosh, 1997; Alongi, 2002; Alongi, 2008). Mean air temperature in mangroves of Pakistan ranges between 20-28 °C (Saifullah and Rasool, 2002).

1.2.2. Salinity

Mangroves are adapted with physiological process of salt excretion through transpiration consequently they may mount up cytoplasmic solutes by osmoregulation, featuring as they are facultative halophytes (Aziz and Khan, 2001; Krauss and Allen, 2003; Wang, Yan, You, Zhang, Chen and Lin, 2011). The best growth of mangroves takes place in upper tidal areas where continuous freshwater flows which reduces salinity and increases soil osmotic potential that uptake from plant roots (IUCN, 2005). During high salinity fluctuations (as Avicennia marina can tolerate the salinity range between 8-75‰), they avoid salt accumulation from sea water due to salt glands present on their leaves (Hogarth, 2007; Krauss, Lovelock, McKee, López-Hoffman, Ewe and Sousa, 2008). Plant pigments (Carotenoids and Chlorophyll) also significantly increases with the increase of salt concentration, although in 50‰ the chlorophyll a/b concentration decreases consequently effects on the rate of photosynthesis (Parida and Jha, 2010). Average salinity ranges in Pakistan mangroves between 28-41‰ (IUCN, 2005).

1.2.3. Gases fluxion

Mangroves go through in frequent oxygen stress and does not retain oxygen pressure under hypoxic conditions, as a result, they showed various morphological and

5 physiological adaptations (McKee, Topa, Rygiewicz and Cumming, 1996; Skelton and Allaway, 1996). For instance, under gaseous pressure, they use aerial roots (pneumatophores, prop roots) for rapid air influx during low tide (Lu, Wong, Tam, Ye, Cui and Lin, 1998). High concentration of carbondioxide enhances the reproductive and photosynthetic rate, depends on physical conditions, species and habitat (Ellison, 1994; Ball, Cochrane and Rawson, 1997; Snedaker and Araújo, 1998).

1.2.4. Siltation and sedimentation:

Deposition of suspended solids in water causes formation of sediment layer on former upper layer of mangrove swamps. These suspended solids contain sulfides, phosphorus, carbon, iron and silicon which are closely packed with each other and when they filtered out from water, gives a positive effect to mineralization and assimilation (Alongi, 1996; Middelburg, Nieuwenhuize, Slim and Ohowa, 1996). High sedimentation causes increase of soil erosion and decrease the amount of water and oxygen resulting siltation which leads to negative impact on growth of mangrove trees specifically their seedlings (Duke, Ball and Ellison, 1998; Thampanya, Vermaat and Duarte, 2002; Vaiphasa, DeBoer, Panitchart, Vaiphasa, Bamrongrugsa and Santitamnont, 2007).

1.2.5. Zonation and tidal action

The pattern of zonation is extremely variable and distinct in mangroves as their growth occurs in inland shallow water zone at different tidal heights (Bunt, 1996). Topographical, hydrological, physicochemical changes, tidal current dynamics, riverine flow and over-flooding effects on mangroves production rate (Jennerjahn and Ittekkot, 2002; Kathiresan and Rajendran, 2005). The rise of sea-level also causes loss of habitat (specifically mangrove associated communities), changing in mangroves species composition and degradation (Christian, Stasavich, Thomas and Brinson, 2002; Cohen, Souza Filho, Lara, Behling and Angulo, 2005; Gilman, Ellison and Coleman, 2007; Erwin, 2009).

1.3. Significance of mangroves

Mangroves are unique and most beneficial ecosystem, thus have commercial, ecological, and biological importance for variety of purposes in human resources and marine systems.

1.3.1. Commercial role of mangroves

Mangrove ecosystem are commercially important and have multifarious resources for native income through direct and indirect ways. By direct means, they are sustainable source of commercial fisheries, shrimp farming, crab farming, aquaculture, agriculture and forestry (Dahdouh-Guebas, Jayatissa, di Nitto, Bosire, Seen and Koedam, 2005; Blaber, 2007), also minor supply of fuel wood, charcoal, timber, honey, medicine, poles and fodder crops up (Bann, 1997; Barbier, 2007). Portunid Crabs (Scylla serata), shrimps (Penaeus spp. and Metapenaeus spp.) are quality harvests from mangrove swamps to raise the economic rate of Pakistan (Qureshi, 2011; Abbas, Khan and Ahmed, 2013). Chiefly, Pakistan‘s commercial fishery depends upon mangrove ecosystem that earns annually around US $100 million. The annual merely shrimp catch was 25,541 metric tons in last three decades from mangrove areas of Pakistan (Hasan, 2000; Mukhtar and Hannan, 2012). Mostly camels, buffalos and other cattle grazes on quite nutritive leaves of Avicennia marina when they move on near urban areas of Karachi coast (Fig. 2) or its foliage is lopped to the cattle which does not access this fodder (IUCN, 2005; Mukhtar and Hannan, 2012). Some mangroves species have high concentration of tannin in their barks which has polyphenolic agents against fouling organisms and useful for dyeing the fishing nets (IUCN, 2005; Maie and Jaffe, 2006; Hunting, Soest, Geest, Vos and Debrot, 2008). Limited salt yields from sea water by forming salt pans near mangroves to consume for local products and foreign exportation. Mangrove environment attracts the eco-tourists, birdwatchers and hunters (Mahmood and Ali, 2000). These are sensitive reserved areas near Indus delta for wildlife watchers of migrating sea birds, mangrove jackals and dolphins (Memon, 2002). Indirect sources are stabilizing shoreline sediment and prevent from erosion (Bann, 1997; Sathirathai, 1998), act as natural barriers by reducing the influence of hurricanes, wave energies, tsunamis, cyclones and storm surges (Badola and Hussain 2005; Chong, 2005; Aziz and Khan, 2014).

A B

C D

Figure 2. (A) Study site of Sandspit backwater mangroves dominated by Avicennia marina, (B) Channel water during high tide, (C) and (D) Browsing and grazing of camels on A. marina at study site.

1.3.2. Ecological role of mangroves

Mangrove forests can produce rich supply of organic matter through litter decomposition to trophic levels. Litter decomposition and detritus breakdown in mangroves ecosystem depends on temperature, rainfall and tidal height, accelerated by microbial activities and foraging of invertebrates which produces large amount of organic matter and nutrients (Mackey and Smail, 1996; Woitchik, Ohowa, Kazungu, Rao, Goeyens and Dehairs, 1997; Skov and Hartnoll, 2002; Nordhaus, Wolff and Diele, 2006; Mukhtar and Hanan, 2012; Nazim, Ahmed, Shaukat and Khan, 2013; Shafique, Siddiqui, Aziz and Shoaib, 2013; Aziz and Khan, 2014; Farooqui, Siddiqui and Rasheed, 2014). These microbes (including bacteria and fungi) decompose detritus and remove nutrients in which potassium and carbohydrates leached out in a very short time span (Rajendran and Kathiresan, 2000; Bosire, Dahdouh-Guebas, Kairo, Kazungu, Dehairs and Koedam, 2005; Tariq, Dawar, Mehdi and Zaki, 2006; Siddiqui, Farooq, Shafique and Farooqi, 2008; Nazim, Ahmed, Shaukat and Khan, 2013). As degradation increases due to bacterial activity, tannins, carbon and nitrogen also removed rapidly (Dittmar and Lara, 2001). In Avicennia marina, as the nitrogen content increases, there is a sudden downfall of carbon and nitrogen ratio takes place due to nitrogen fixing azotobacters (Bashan and Holguin, 2002; Holmer and Olsen, 2002; Tam, Guo, Yau and Wong, 2002; Davis, Corronado-Molina, Childers and Day, 2003; Alongi, 2005).

Mangrove sediment retain nitrogen and microbes efficiently assimilate them in the form of ammonia to export thus habitat provides sufficient nutrient, carbon, nitrogen and energy fluxes to adjacent communities (Rivera-Monroy, Twilley, Boustany, Day and Vera-Herrera, 1995; Alongi, 1998; Bano and Siddiqui, 2004; Shafique, Siddiqui, Aziz and Shoaib, 2013; Farooqui, Siddiqui and Rasheed, 2014). Net primary production of photosynthetic carbon and energy flux in mangrove ecosystem can reach up to 40% which accumulates in sediment and transferred to the offshore waters (Lee, 1995; Duarte and Cebrian, 1996; Alongi, 2002; Bouillon, Koedam, Raman and Dehairs, 2002). This dynamic process strongly influenced on coastal food web and form a trophic association to benthic communities (Loneragan, Bunn and Kellaway, 1997; Chong, Low and Ichikawa, 2001; Saifullah and Ahmed, 2007).

1.3.3. Biological role of mangroves

Mangroves habitat provide shelter, feeding, breeding and nurturing grounds (Alongi, 2002; Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton and Somerfield, 2008; Walters, Rönnbäck, Kovacs, Crona, Hussain, Badola and Dahdouh-Guebas, 2009; Jabeen, Imran and Khatoon, 2014; Amjad, Rasheed and Baig, 2016) for offshore fisheries and several aquatic and terrestrial species to complete their life cycles, larval or juvenile stages (Fleck and Fitt 1999; Fleck, Fitt and Hahn, 1999). Mangroves ecosystem promotes highly extensive food chains to sustain various organisms (Alongi, 2005; Naidoo, Steinke, Mann, Bhatt and Gairola, 2008) and thus the diversity and biomass per unit area in mangroves swamps and mud flats are very productive (Granek and Ruttenberg, 2008; Sheaves, Baker, Nagelkerken and Connolly, 2015). Various edible fishes, shrimps and crabs have been harvested from mangrove area and exported abroad or locally consumed (Barbier, 2000; Mukhtar and Hanan, 2012).

1.3.3.1. Associated communities of mangroves

Mangrove forests create unique ecological environment and support high variety of fauna and flora due to rich source of nutrients and high primary productivity (Alongi, 1990; Barkati and Rahman, 2005; Farooqui, Shafique, Khan, Ali, Iqbal and Siddiqui, 2012; Holguin, Vazquez and Bashan, 2001; Shafique, Siddiqui, Aziz, Burhan and Mansoor, 2010). They facilitate the habitat to support various organisms as they provide a stable ground to protect them from variability and harshness of environmental factors such as extreme sunlight exposure, temperature, salinity, tidal action and sedimentation etc. (Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton and Somerfield, 2008). Their submerged roots in shallow water provide a definite environment to different marine communities including meiofauna, macrofauna (infauna, epifauna) and other benthos (Rützler, 1995; Morrisey, Swales, Dittmann, Morrison, Lovelock and Beard, 2010).

The microflora associated with mangroves is found on surface layer of sediment and roots for example microbial mats dominated by different species of cyanobacteria, diatoms and other microbial communities (Bano and Siddiqui, 2004; Saifullah and Ahmed, 2007). Other macroalgal species (Enteromorpha spp., Ulva spp., Vaucheria spp.,

Chaetomorpha spp., Polysiphonia spp., Rhizoclonium spp., Hydrocoleum spp. and Lyngbya spp.) also reported from mangroves area (Saifullah and Taj, 1995; Saifullah, Nizamuddin and Gul, 2003; Saifullah and Ahmed, 2007; Ichihara, Shimada and Miyaji, 2016).

The fauna associated with mangroves is diverse, including crustaceans, gastropods, bivalves, insects, sponges, ascidians, hydrozoans, bryozoans, fishes, birds, mammals and reptiles, etc. (Diaz and Rützler, 2001; Rützler, Duran and Piantoni, 2007; Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton and Somerfield, 2008).

Other than planktonic, several benthic fauna also reported from of mangroves swamps in backwaters area of Karachi coast comprises of foraminifera, polychaetes, oligochaetes (Qureshi, Naz and Saher, 2015), nematodes (Kamran, Nasira and Shahina, 2009), isopods, copepods, amphipods, cyprids, mysids, shrimps, barnacles and molluscs which are abundantly found on marshes and attached with the roots of mangroves (Barkati and Rahman, 2005; Qureshi, Naz and Saher, 2015). The vertebrate fauna containing different species of fishes (Pereiformes: 46 species, Clupeiformes: 15 species, Cypriniformes: 6 species, Mugiliformes and Pleuronectiformes) reported from mangrove swamps (IUCN, 2005). Other vertebrate fauna for example, mudskippers, amphibians (Bufo), reptiles including various species of sea snakes (Pelamis platurns, Microcephalophis gracilis, Hydrophis cyanocinctus, H. mamillaris, H. caerulescens and Ephydrina schistosa), lizards (Stenodactyhes orientalis and Acanthodagtylus cantoris) and various species of migratory birds (aquatic, terrestrial) also reported form backwater area of Karachi (IUCN, 2005).

Among benthic communities of marine ecosystem after coral reef, sponges are the dominating group (Diaz, 2012). From all over the world there are about 8,818 species of marine and non-marine sponges belonging to 20,000 taxon reports (Van Soest, Boury- Esnault, Hooper, Rützler, de Voogd, Alvarez de Glasby, Hajdu, Pisera, Manconi, Schoenberg, Klautau, Picton, Kelly, Vacelet, Dohrmann, Díaz, Cárdenas and Carballo, 2017).

1.4. Mangrove associated sponges (Phylum Porifera)

Sponges are diversified epibionts on mangrove roots due to availability of suitable substrate for attachment (Rützler and Feller, 1996; Rützler, Diaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000; Wulff, 2000; Diaz, Smith and Rützler, 2004). Abundance of sponges in mangrove habitat depends upon various physical and biological factors such as light exposure, availability of nutrients, tidal action, freshwater input and predation, which ultimately effects on their growth, distribution and diversity (Rützler, 1995; Farnsworth and Ellison, 1996; Rützler, Diaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000; Wulff, 2000; Engel and Pawlik, 2005). Although some species of mangrove sponges can survive in very harsh (expose) condition of low tidal cycle (Barnes, 1999; Rützler, 1995).

1.4.1. Morphology

Sponges are the most ancient (evolved about 600 million years ago) sessile, and incipient type of metazoans that lacks tissue system organization (Müller, Belikov, Tremel, Perry, Gieskes, Boreiko and Schröder, 2006; Gazave, Lapébie, Renard, Vacelet, Rocher, Ereskovsky and Borchiellini, 2010). The body is simple and diploblastic, consists of different types of cells (such as amoebocytes, choanocytes, sclerocytes, pinacocytes, porocytes, myocytes, etc.) which performs respective functions in the body of animal (Kotpal, 1994; Hooper, 2000; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012). Body contain numerous incurrent pore (ostia) and a large excurrent pore (osculum) (Fig. 3), principally there are three type of canal system present in sponges (ascon, sycon, and leucon type) which perform various functions such as, providing nutrition, carried out excretion, respiration and reproduction etc. (Kotpal, 1994; Hooper, 2000). Skeletal framework consists of either proteinaceous fibres or spicules which are of different types (monaxon, diaxon, triaxon, tetraxon, polyaxon, etc.) and their arrangement depends on structure of body and habitat type (Boute, Exposito, Boury‐ Esnault, Vacelet, Noro, Miyazaki and Garrone, 1996; Uriz, Turon, Becerro and Agell, 2003; Fernàndez-Busquets, 2008; Pallela, Koigoora, Gopal Gunda, Sakunthala Sunkara

12 and Janapala, 2011; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012).

Figure 3. Internal and external morphological features of sponge (Buchsbaum, Buchsbaum, Pearse and Pearse, 2013).

1.4.2. Sponges association communities

One of the more interesting characteristic of sponges are able to establish a great diversity of relationships (mutualism, commensalism and parasitism) with unicellular and multicellular organisms (Wulff, 2006). They contain various symbionts including microbes, cyanobacteria, algae, worms, crustaceans and diatoms that inhabits over its porous body structure (Taylor, Radax, Steger and Wagner, 2007; Usher, 2008). Sponges are the most dominating epibionts of mangrove roots (Rützler, Diaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000) and are adapted to this habitat. Although specific fauna, associated with

13 mangrove not only different with location but also varies with individual mangrove root and species (Farnsworth and Ellison, 1996; Diaz, Smith and Rützler, 2004; Wulff, 2004).

The associated communities with sponge is protected from potential predators and other unfortunate effect of abiotic factors (Dalby, 1996; Huang, McClintock, Amsler and Huang, 2008; Fiore and Jutte, 2010). They provide feeding habitat and source of food for varieties of organisms including microbes, nematodes, polychaetes, crustaceans (isopods, amphipods, copepods, mysids, larvae of decapods) and ophiuroides etc. (Duarte and Nalesso, 1996; Ribeiro, Omena and Muricy, 2003; Stofel, Canton, Antunes and Eutrópio, 2008; Fiore and Jutte, 2010; Morgado and Tanaka, 2001; Abdo, 2007).

The earlier work done on sponges mostly deal with their environmental factors (both biotic and abiotic), ecological role of sponges and mangrove-sponge associations (Engel and Pawlik, 2000; Pawlik, McMurray and Henkel, 2007; Wulff, 2005).

Marine organisms are the rich source of novel chemical compounds which are biologically active in nature (Faulkner, 2001). Among them marine sponges are the main producers of biologically active substances and used as drug leads or biomedical tools (Newbold, Jensen, Fenical and Pawlik, 1999; Brady, Simmons, Kim and Schmidt, 2009). These bioactive secondary metabolites have been found in all orders of demosponges and the number of new compounds isolated from sponges (Blunt, Copp, Hu, Munro, Northcote and Prinsep, 2008; Garson, 2001; Hunting, van der Geest, Krieg, van Mierlo and Van Soest, 2010).

1.4.3. Diversity and Distribution of Sponges

More than 5000 species identified and reported in taxa divided into four main classes in which Demospongiae is the largest class consists about 80% species. They found in form of colonies but some species are solitary, dominantly benthic from shallow to deepest regions of the ocean, even recorded from abyssal and hadal zones. Most of the marine sponge related studied have been recorded from Great Barrier Reef of Australia, Caribbean and Mediterranean. From Australian region, about 1400 species described and considered as the highly diversified region which contains about more than 5000

14 undescribed taxa (Hooper, 2000; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012). Although Micronesia, Indo-Malaya, Indonesia, Japan, Sri Lanka, Singapore, Thailand, Burma, Adaman Sea, Southern China, Philippines and Vietnam has comparatively different and moderate faunal diversity and some literature are promoted from old expeditions of early 1900s data (Hooper, 2000; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012). From past 20 years, New Zealand, Papua New Guinea and Madagascar have under explored by which 1200 species reported while further considerations revealed that these regions may contain about 4000-5000 species which requires comprehensive documentation (Hooper, 2000; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012; Wulff, 2012; Pallela and Janapala, 2013).

Within the Indian Ocean, demosponges are widely distributed over Gulf of Mannar and Western Indian Ocean (Pallela and Janapala, 2013; Sivaleela, 2014). In Pakistan, there is no information available on the taxonomy, distribution, associated communities and chemical ecology of marine sponges, even though some collected specimen were taken from the coast of Karachi (Sonahri and Buleji beach) during past 4 to 5 years for the assessment of antimicrobial activities (Nazim, Sherwani, Khan, Kausar and Rizvi, 2014). Due to lack of data on sponge fauna from Pakistan, the present study emphasizes to explore sponge communities.

1.5. Study Objectives

Therefore, the principle objectives of this study are:

1. To identify the species of marine sponges.

2. To calculate in situ variation in growth and abundance of marine sponges

3. To analyze the various communities associated with sponge species.

4. To isolate secondary metabolites from marine sponge.

PART – II

SYSTEMATIC STUDY OF MARINE SPONGES (PORIFERA) IN PAKISTAN

CHAPTER - 2: Marine Sponge (Porifera: Demospongiae) Liosina paradoxa Thiele, 1899 from Sandspit backwater mangroves at Karachi coast, Pakistan. CHAPTER - 3: Two new records of marine sponges (Demospongiae: Haplosclerida) from the coast of Karachi, Pakistan.

Part: II Chapter - 2

Marine Sponge (Porifera: Demospongiae) Liosina paradoxa Thiele, 1899 from Sandspit backwater mangroves at Karachi coast, Pakistan

Abstract

Marine sponge Liosina paradoxa was collected from pneumatophore of Avicennia marina at Sandspit backwater (66°54' E, 24°49' N), Karachi coast in May 2015. The identification of specimen was based on the structure of siliceous spicules scattered irregularly in mesohyl observed under light microscope and scanning electron microscope. Spicules are megascleres, entirely smooth, strongyle (length = 310-451 ± 59.65 µm, width = 5-8 ± 1.8 µm), microscleres absent. The result has been shown that the species is Liosina paradoxa (Family Dictyonellidae) first time reported from coastal area of Pakistan.

2.1. Introduction

Mangroves are salt tolerant vegetation that inhabits tropical and sub-tropical coastal regions and are considered among the world‘s most productive ecosystems (Farooqui, Shafique, Khan, Ali, Iqbal and Siddiqui, 2012), which provide food and shelter for a wide variety of organisms (Ellison, Fransworth and Twilley, 1996). Fungi, algal (micro and macro) communities and many other invertebrates (sponges, polychaetes, bryozoans, barnacles and molluscs) are the most abundant epibionts of mangrove habitats (Rützler, 1995; Wulff, 2000; Engel and Pawlik, 2005; Wulff, 2005; Pawlik, McMurray and Henkel, 2007; Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton and Somerfield, 2008). Both sponges and mangrove are beneficial to each other and showed facultative mutualism (Ellison, Fransworth and Twilley, 1996; IUCN, 2005; Duckworth and Wolff, 2011).

2.1.1. Classification of mangrove sponges of Pakistan

Demospongiae Sollas, 1885 is the largest class among four different classes (other three classes are Archaeocyatha, Calcarea and Hexactinellida) of phylum Porifera, comprises an estimation about 15,000 species (marine and freshwater) in which 7000 species reported worldwide (Hooper and Lévi, 1994; Hooper and Van Soest, 2002; Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012; Morrow and Cárdenas, 2015). This class is distinguished by siliceous skeletal composition (monactines, diactine and tetractine type of spicules) and spongin fibers (Van Soest, Boury-Esnault, Vacelet, Dohrmann, Erpenbeck, De Voogd and Hooper, 2012).

On the basis of reproductive features and larval forms, class Demospongiae is divided into three sub-classes; Heteroscleromorpha, Keratosa and Verongimorpha (Morrow and Cárdenas, 2015). Heteroscleromorpha is diversified sub-class having 16 orders and 70 families, recently added in class Demospongiae after cladistic analysis and its molecular phylogeny (Cárdenas, Perez and Boury-Esnault, 2012; Morrow and Cárdenas, 2015).

Within Heteroscleromorpha, order Bubarida Morrow and Cárdenas, 2015 has some incorporated characteristics such as, encrusting sponges has hispid surface where

17 ectosomal skeleton not found (Hooper and Van Soest, 2002). Choanosomal skeleton alienated into different layers that cover sinuous, flexuous or vermicular type of spicules (monactines and diactines), confine to axial or basal skeleton of sponges. Three families (Bubaridae, Desmanthidae and Dictyonellidae) and 188 species retained in order Bubarida.

The distinguishing features of family Dictyonellidae Van Soest, Diaz and Pomponi, 1990 are fleshy and smooth surface having thick ectosomal layer which lacks surface spicules but choanosomal skeleton and spicules directed towards the surface; spicules are mostly styles, strongyles, oxeas and its derivatives. Spicule arrangement is irregular, dispersed and disorganized (Hooper, 2000; Hooper and Van Soest, 2002). Most species of Dictyonellidae found in warm waters. The following ten genera have been included in this family; Liosina, Acanthella, Rhaphoxya, Lipastrotethya, Tethyspira, Scopalina, Dictyonella, Phakettia, Svenzea and Stylissa (Van Soest, Diaz and Pomponi, 1990; Hooper and Van Soest, 2002; Morrow and Cárdenas, 2015).

2.2. Materials and methods

2.2.1. Study site

Sandspit (66°54' E, 24°49' N) is located towards southwest of Karachi coast (Fig. 4) lies deltaic region between Hawks Bay and Manora channel. Backwater has dense mangrove belt dominant by grey mangroves Avicennia marina covers area range about 1056 hectares. Fewer studies have been done on mangroves biology, growth and ecology and its associated communities at Sandspit backwater area. There is no data available on any aspect of sponge from Pakistan therefore, the objective of the present study is to focus on the sponge fauna associated with mangrove (Avicennia marina). This is the first report on the presence of sponge from Karachi coast. Further study is in process to highlight their importance in several aspects.

Figure 4. Map of Pakistan shows the collection site. Sandspit, Karachi (Google Earth Pro: version 7.1.4.1529).

2.2.2. Material collection, fixation and preservation

Sponge samples were collected from pneumatophores of Avicennia marina at Sandspit backwater during low tide in May 2015. Specimens of sponges on pneumatophores were photographed with digital camera (Olympus VR-310) in situ for habitat characterization, removed from pneumatophores and washed thoroughly with seawater to separate all exotic substance and debris. The samples were fixed in 4% buffered formalin after brought to the laboratory and transferred in 85% ethanol after 24 hours for preservation and identification.

2.2.3. Spicule isolation

2.2.3.1. Material digestion a. Material treated with bleach

Small fragments from the ectosomal and inner part of the preserved specimen were bleached for few minutes in 5% sodium hypochlorite (commercial bleach) until stop off bubbling process and washed with Milli-Q water. b. Material treated with acid

After bleach treating, suspension digested in concentrated H2SO4 / HNO3 (4:1 v/v) mixture for 24 hours. The suspension centrifuged at 2000 rpm for 1 minute and acid decanted. The residue washed repeatedly (3 times) after centrifugation with Milli-Q water and rinsed with ethanol.

2.2.3.2. Microscopic analysis i. Evaluation of spicules in light microscope

Few drops of the cleaned spicule suspension were placed on glass cover slips, the ethanol allowed to evaporate and mounted to observe under light microscope (Olympus, IX 51) (Fig. 5A) at 40x, 60x and 100x magnifications. Minimum 40 spicules were measured for size verification.

20 ii. Spicule evaluation under scanning electron microscope (SEM)

For observing under Scanning Electron Microscope (Model: JEOL JSM-6380A, Japan) (Fig. 5B), 2-3 drops of cleaned suspension fixed on a glass cover slip and vaporized the alcohol for few minutes. A layer of 300°A with gold coated on the cover-glass slip by using auto ion sputtering coater (JEOL JFC-1500) for 5 minutes and viewed at 10 kV under scanning electron microscope.

2.2.3.3. Systematic documentation

Most of the sponge taxonomy carried out by interpretation of foremost morphological features (shape, size, colour, skeletal and choanosomal structure, texture and consistency) of specimen, taken from ‗Sponguide‘ by Hooper (2000) and through the description of specimen given in the site of ‗SpongeMaps‘ (Hall and Hooper, 2014). Classification reviewed through the literature given for each species data for Porifera in ‗World Porifera Database‘ (WPD) (Van Soest, Boury-Esnault, Hooper, Rützler, De Voogd, Alvarez de Glasby and Janussen, 2015) and ‗World Register for Marine Species‘ (WoRMS) (Mees, Boxshall, Costello, Hernandez, Bailly and Zeidler, 2015).

A B

Figure 5. (A) Light microscope (Olympus, IX 51) and (B) Scanning Electron Microscope (JEOL JSM-6380A).

2.3. Results

2.3.1. Systematics

Phylum – Porifera Class - Demospongiae Sollas, 1885 Order - Bubarida Morrow and Cárdenas, 2015 Family - Dictyonellidae Van Soest, Diaz and Pomponi, 1990 Genus - Liosina Thiele, 1899 Liosina paradoxa Thiele, 1899 (Fig. 6A-F)

2.3.2. Synonymized names

Auletta bia de Laubenfels, 1954 Migas porphyrion Sollas, 1908 Milene porphyrion de Laubenfels, 1954

2.3.3. Material examined

CEMB-POR-01; collected by Shafique S. Zaib-un-Nisa B. and Jabeen H.; May, 2015; Sandspit, Karachi. Material deposited in Centre of Excellence in Marine Biology, University of Karachi. Karachi, Pakistan.

2.3.4. Material description a. External morphology

Exposed, directly attached to substrate; massive; thinly encrusting (1 mm thick), irregular, soft, flexible, compressible, easily torn; brownish velvety hue in live coloration, greyish beige after preservation; unornamented rough muddy surface. Oscules small and distributed all over the surface while ostia not visible. b. Skeletal structure

Ectosomal skeleton consists of thick pigmented granules; choanosomal structure is densely muddy having spicules in criss-cross pattern (―halichondroid‖ type); siliceous spicules scattered irregularly in mesohyl forming hexagonal pattern.

22 c. Spicule

Spicules are megascleres, disarranged in loose bundles, entirely smooth with rounded ends, strongyle and oxeote form (length = 310 - 451 ± 59.65 µm, width = 5 - 8 ± 1.8 µm). Microscleres absent. d. Habitat

The specimen found on pneumatophores of grey mangroves Avicennia marina in muddy shallow water of intertidal zone at Sandspit backwater, Karachi. e. Distribution

Liosina paradoxa Thiele, 1899 is widely spread in the western Indo-Pacific region. The worldwide distribution of studied species is given in Table II. f. Remarks

The outer surface of studied specimen is rough, bushy, bears large amount of organic material, greyish beige in colour and well adapted to mangrove ecosystem, which is different from the appearance of Auletta bia de Laubenfels, 1954 from Micronesia, that is orange in water and grey in exposed condition. Also grooves and linings which form polygonal shapes on surface are missing on the outer surface of sponge specimen of this study. Megascleres of L. paradoxa reported in the present study are distinctly smaller than the spicules of the holotype (size range: 360 - 900 × 8 - 20 µm) in oxeas and style form (Hooper and Van Soest, 2002; Hooper, Hall, Ekins, Erpenbeck, Worheide and Jolley-Rogers, 2013; Hall and Hooper, 2014).

A B

C D

E F

Figure 6. (A) and (B) Specimen (Liosina paradoxa Thiele, 1899) on pneumatophores of Avicennia marina, (C) Collected specimens of L. paradoxa, (D) Light microscopic image of ectosomal skeleton showing hexagonal surface structure (scale = 20 µm), (E) Light microscopic image of spicules (scale = 100 µm) and (F) Scanning electron microscopic image of spicules (scale = 50 µm).

Table II. The worldwide geographical distribution of Liosina paradoxa Thiele, 1899. (―MEOW‖ is the abbreviation used for ―Marine Ecoregion of the World‖, ―EEZ‖ for ―Exclusive Economic Zone‖ and ―WPD‖ for ―World Porifera Database‖).

Taxon Synonymy Region Source

Central and South Great Barrier Liosina paradoxa - Reef Hooper, 2008

Liosina paradoxa - Coral Sea, Great Barrier Reef WPD

Liosina paradoxa - Delagoa (MEOW) WPD

East African Coral Coast Liosina paradoxa - (MEOW) Richmond, 1997

Liosina paradoxa Milene porphyrion Mozambique WPD

Liosina paradoxa - Seychelles (MEOW) Van Soest, 1994

Sulawesi Sea / Liosina paradoxa - Makassar Strait (MEOW) Thiele, 1899

Liosina paradoxa - Chumbe Island Marshall, 2009

Liosina paradoxa - Tanzania Richmond, 1997

Hooper & Van Soest, Liosina paradoxa - Indonesian EEZ 2002

Hooper & Van Soest, Liosina paradoxa Auletta bia Micronesian EEZ 2002

Hooper & Van Soest, Liosina paradoxa Migas porphyrion Mozambican EEZ 2002

Liosina paradoxa - Andaman Islands, India Sankar et al., 2016

2.4. Discussion

In the present study, Liosina paradoxa Thiele, 1899 is being reported in detail and described for the first time from the backwaters of Karachi, Pakistan, where it was found attached to pneumatophores of Avicennia marina.

The genus Liosina Thiele 1899, is widely distributed in Indo-Pacific region where it is common in shallow lagoons, reefs and bays. It is largely characterized by its muddy surface organization (Hooper, 2000; Hooper and Van Soest, 2002). Spicules may scatter irregularly near the surface in the form of bundles within spongin, mostly monactines and diactines, microscleres are not reported from this genus (Hooper and Van Soest, 2002). It contains four valid species (L. arenosa, L. blastifera, L. granularis and L. paradoxa) (Hooper, 2000; Hooper and Van Soest, 2002). The external surface of L. arenosa is greyish orange when alive and preserved specimen is brown in alcohol. Circular and hexagonal pores occur with muddy appearance and about 5 mm thick with a soft texture consisting of choanosome beams, spicules are tylostyles about 625 - 1000 × 6 - 10 µm in size range (Vacelet and Vasseur, 1971).

L. blastifera is distinguished by its muddy surface with groove-like depressions, dichotomous and pale yellow in colour. Massive, encrusting, irregular lobate with cylindrical digitations about 30 mm in length and 5 mm thick and spicules are straight oxeas, rarely styles (Vacelet, Bitar, Carteron, Zibrowius and Perez, 2007), 150 - 940 × 2 - 10 µm in size (Hall and Hooper, 2014).

Liosina granularis is relatively similar to L. paradoxa: encrusting, massive, branching and foliose, ectosome is white whereas choanosome is orange-brown. Megascleres are strongyles with oxeote forms in size range between 270 - 775 × 5 - 22 µm (Kelly-Borges and Bergquist, 1988).

The studied specimen has morphological affinities with L. blastifera due to its muddy surface and massive organization however it is appearing comparable to L. granularis for its skeletal structure, spicule size and strongyle form. The hexagonal structure in ectosomal region is a characteristic feature of this specimen.

Part: II Chapter - 3

Two new records of marine sponges (Demospongiae: Haplosclerida) from the coast of Karachi, Pakistan

Abstract

Both sponge taxa belonged to class Demospongiae, order Haplosclerida has been first time recorded from Karachi coast, Pakistan (North Arabian Sea). Callyspongia (Cladochalina) fibrosa of family Callyspongiidae has collected from Churna Island (66°36'48"E, 24°53'93"N) by SCUBA diving in February 2013 and Haliclona (Soestella) hornelli of family Chalinidae has collected from the rock pool of Buleji rocky ledge (64°50'21"E, 24°49'03"N) in January 2016. Both species were fixed in 4% buffered formalin solution and preserved in 85% ethanol. The skeletal structure observed under light microscope and scanning electron microscope. Spicules of C. (C.) fibrosa was smooth and densely packed with thick fibres (spicule size: 66-84 × 2-3 µm). The species H. (S.) hornelli is here assigned for the first time to the subgenus Soestella of the genus Haliclona. The choanosomal skeleton is sub-anisotropic, forming a reticulation of meshes with unispicular and paucispicular lines. Spicules are slender megascleres, smooth oxeas (size range: 80-148 × 5-7 µm) and microscleres were absent. Result showed that Callyspongia (Cladochalina) fibrosa and Haliclona (Soestella) hornelli are the two new records from the coastal region of Karachi.

3.1. Introduction

The sponges (Porifera) are the prevaling and most substantial biota among the benthic communities of coral reef ecosystem (Schmahl, 1990; Duckworth and Wolff, 2007). Their distribution and abundance were prominently influenced by various factors for example nutrient cycling, bio-erosion, cementation and physicochemical adaptation (Wilkinson and Cheshire, 1990; Díaz and Rützler, 2001; Rützler, 2004). Marine sponges also favor other associated communities (symbiotic association) in their habitat and form complex food chain and thus maintain high biodiversity (Díaz and Rützler, 2001). The rocky-sandy shallow water condition stabilizes the environmental stress and construct a community framework for micro and macrobenthic algae, sponges, corals, crustaceans, annelids, gastropods, bivalves and their associates (Rützler, 2004).

The sponge species of class Demospongiae Sollas, 1885 are most abundant and colonized in bathymetric substrata of coral reef and common shallow water habitats (Barnes, 1999; Díaz, 2012). The demosponges with an isodictyal, isotropic or anisotropic skeleton, diactinal spicule forms, fall into the order Haplosclerida Topsent, 1928 included in the sub-class Heteroscleromorpha Cárdenas, Pérez and Boury-Esnault, 2012 (Morrow and Cárdenas, 2015). Order Haplosclerida and Poecilosclerida Topsent, 1928 are found common in reef ecosystems (Díaz, 2012).

Order Haplosclerida Topsent, 1928 generally characterized by brittle, soft compressible or hard incompressible consistencies, which possess isodictyal isotropic or anisotropic skeleton with thick reticulation of spicules and spongin fibres (Hooper and Van Soest, 2002). According to the revised classification by Morrow and Cárdenas (2015), 6 marine sponge families (Callyspongiidae de Laubenfels, 1936, Chalinidae Gray, 1867, Niphatidae Van Soest, 1980, Calcifibrospongiidae Hartman, 1979, Petrosiidae Van Soest, 1980 and Phloedictyidae Carter, 1882) and a sub-order Spongillida Manconi and Pronzato, 2002 (contains 7 families of freshwater sponges) confined in this order.

Family Callyspongiidae de Laubenfels, 1936 characterized by surface ridges, conules or spikes. Skeleton consists of spiculo-fibre meshwork reticulation which sculptured tangentially towards the surface (Hooper and Van Soest, 2002). Twenty-three genera are

28 included in this family of which four genera (Arenosclera Pulitzer-Finali, 1982, Callyspongia Duchassaing and Michelotti, 1864, Dactylia Carter, 1885 and Siphonochalina Schmidt, 1868) are valid, distributed in temperate, cold and tropical waters (Hooper, 2000; Hooper and Van Soest, 2002). Spiny ectosomal layer with dense spongin on periphery is the characteristic feature of genus Callyspongia. It contains five subgenera; subgenus Cavochalina Carter, 1885d has fine choanosomal spongin sheath. The subgenus Cladochalina Schmidt, 1870 is visibly distinguished by fine conulose or spines on surface and absence of microscleres. Three surface layers present in subgenus Euplacella Lendenfeld, 1887, the ectosomal region contains loose spicules and primary lines of choanosomal skeleton are paucispicular (Desqueyroux-Faúndez and Valentine, 2002). The subgenus Toxochalina Ridley, 1884 has thick conulose on surface, choanosomal region contains tertiary spongin fibres network and toxas microscleres. The fibres of subgenus Callyspongia Duchassaing and Michelotti, 1864 are subdivided into parallel, multispicular and secondary fibres (Hooper, 2000; Hooper and Van Soest, 2002).

In order Haplosclerida, cushion-shaped sponges with oscular chimneys are signified in the family Chalinidae (Hooper and Van Soest, 2002). The skeleton in that family is composed of a delicate hexagonal tangential reticulation with unispicular, paucispicular or multispicular primary lines which are joined regularly with unispicular or paucispicular secondary lines (De Weerdt, 2000; Hooper, 2000; Hooper and Van Soest, 2002). According to De Weerdt (1986), based on a cladistic analysis of morphological features of Haplosclerida, most genera of the family Chalinidae are to be merged in a single genus Haliclona Grant, 1841.

Within the Family Chalinidae Gray, 1867, 27 genera recognized, of which four are currently considered valid including; Chalinula Schmidt, 1862, Cladocroce Topsent, 1892, Dendroxea Griessinger, 1971 and Haliclona Grant, 1841 (Hooper and Van Soest, 2002). The genus Haliclona has regular choanosomal reticulation of interconnected primary and secondary spiculofibres. Spicules are mostly oxeas and microscleres may be present in the form of sigmas and toxas (De Weerdt, 2000). It contains seven subgenera, Haliclona Grant, 1836, Reniera, Schmidt, 1862, Halichoclona de Laubenfels, 1932, Soestella De Weerdt, 2000, Gellius Gray, 1867, Rhizoniera Griessinger, 1971 and

Flagellia Van Soest, 2017. The subgenus Soestella De Weerdt, 2000 has a choanosomal skeleton consisting of a sub-anisotropic, paucispicular reticulation of primary lines irregularly interconnected by secondary unispicular lines. Smooth, slender oxeas with spongin at their nodes with tendency to form rounded meshes. (De Weerdt and Van Soest, 1986; De Weerdt, 2000).

The genus Haliclona Grant, 1841 has been reported worldwide 56 species have been recorded from the regions bordering the Indian Ocean and among them 29 species have not been assigned to subgenera (World Porifera Database, 2017). Thirteen species of subgenus Gellius Gray, 1867 are distributed along the regions Red Sea (Row, 1911; Lévi, 1958), Kenya (Pulitzer-Finali, 1993), Andaman and Nicobar Islands of Indian (Burton, 1928), Madagascar (Lévi, 1956; Vacelet and Vasseur, 1971) and East African coral coast (Richmond, 1997). Four species of the subgenus Halichoclona de Laubenfels, 1932 have been recorded from Indo-Pacific regions including Singapore (Lim, De Voogd and Tan, 2008), Madagascar (Lévi, 1956; Vacelet, Vasseur and Lévi, 1976) and Gulf of Tadjoura (Topsent, 1893). Three species of the subgenus Haliclona Grant, 1836 have been found in the Red Sea (Keller, 1883), Seychelles (Thomas, 1973), Indonesia (Topsent, 1897), Myanmar and Andaman (Carter, 1887). The subgenus Reniera has 7 species recorded from Zanzibar (Steindler, Beer and Ilan, 2002), East African coral coast (Pulitzer-Finali, 1993), Kenya (Barnes and Bell, 2002), Tanzania (Richmond, 1997), Arabian Sea, Seychelles (Thomas, 1981) in Indian Ocean. No species belonging to subgenera Rhizoniera and Soestella have been recorded from coastal waters of the Indian Ocean (World Porifera Database, 2017). Most species of the subgenus Soestella De Weerdt, 2000 have been recorded from the Mediterranean (Griessinger, 1971; Harmelin, Boury- Esnault, Fichez, Vacelet and Zibrowius, 2003; Bertolino, Bo, Canese, Bavestrello and Pansini, 2015), Brazil (Muricy, Lopes, Hajdu, Carvalho, Moraes, Klautau Menegola and Pinheiro, 2011; Sandes, Bispo and Pinheiro, 2014; Bispo, Correia and Hajdu, 2016), the Caribbean (Van Soest, 1980; De Weerdt, 2000; Rützler, Díaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000; Miloslavich, Díaz, Klein, Alvarado, Díaz, Gobin, Escobar- briones, Cruz-motta, Weil, Cortés, Bastidas, Robertson, Zapata, Martín, Castillo, Kazandjian and Ortiz, 2010; Rützler, Piantoni, Van Soest and Díaz, 2014; Pérez, Díaz, Ruiz, Cóndor-Luján, Klautau, Hajdu, Lobo-Hajdu, Zea, Pomponi, Thacker, Carteron, 30

Tollu, Pouget-Cuvelier, Thélamon, Marechal, Thomas, Ereskovsky, Vacelet, and Boury- Esnault, 2017), the North Atlantic (Van Soest, 2001) and North Pacific Ocean (Eldredge and Smith, 2001).

Very little information is available on marine sponges with reference to their presence along Karachi coast (Nazim, Sherwani, Khan, Kausar and Rizvi, 2014).

The chief objective of this study is to record sponge fauna and its distribution along Pakistan coast. To date, no published record available regarding taxonomy of marine sponges in this region. Thus, it is dire need to study sponge systematics, their reginal distribution pattern and related ecology to facilitate strong taxonomic database in Pakistan.

3.2. Materials and methods

3.2.1. Study sites

The study covers two sites of Karachi coast having rocky-sandy shorelines, Churna Island and Buleji rocky ledge. Churna Island (66°36' E, 24°53' N) is located about 40 km away towards northwest of Karachi between borderline of Sindh and Balochistan coasts (Fig. 7). This region inhabits rich coral colonies and reef-living associated communities. Buleji rocky ledge (66°49' E, 24°50' N) is situated at 30 km distance from Karachi. This coastal region has rocky shelves and rock pools that harbor abundant and diversified array of exposed and submerged faunal and floral communities.

3.2.2. Collection and preservation

The sponge sample of Callyspongia (Cladochalina) fibrosa Ridley and Dendy, 1886 were collected from the west coast of Churna Island (66°36'48"E, 24°53'93"N) from 7 m depth through SCUBA diving in February 2013. The collection of Haliclona (Soestella) hornelli (Dendy, 1916) was made from rock pool of Buleji rocky ledge (64°50'21"E, 24°49'03"N) in January 2016. Samples were directly removed from the rocks, collected in separate polythene bags, washed with seawater to remove extra debris and sediment, and fixed in 4% buffered formalin solution for 24 hours. Specimens were preserved in 85% ethyl alcohol in laboratory.

3.2.3. Histological examination of sponges

Microtome-sectioning of sponge

A small section cut from preserved specimen and passed through the series of dehydration, cleared in xylene for 2 hours and soaked in mixture of 50% (v/v) wax and xylene for 5 minutes. Cleared section embedded in wax block for 48 hours. Sections trimmed (7 µm) from the wax blocks from center to outer side. Trimmed floated sections placed on slides and dried at 60°C. Sections dewaxed in xylol for 5 minutes, passed through alcohol series and stained for 5 minutes in Hematoxylin and Eosin. After staining, sections dehydrated again, cleared in xylol and mounted with Canada balsam (Hooper and Van Soest, 2002).

3.2.4. Isolation of spicules

Description of material treated with bleach, evaluation of spicules under microscope and systematic documentation has given in Chapter 2 (section 2.2.3 page 20).

Figure 7. Map of Karachi coast shows the collection site of Churna Island and Buleji (Google Earth Pro: version 7.1.4.1529).

3.3. Results

3.3.1. Callyspongia (Cladochalina) fibrosa Ridley and Dendy, 1886 (Fig. 8A)

Phylum Porifera Grant, 1836 Class Demospongiae Sollas, 1885 Subclass Heteroscleromorpha Cárdenas, Pérez and Boury-Esnault, 2012 Order Haplosclerida Topsent, 1928 Family Callyspongiidae de Laubenfels, 1936 Genus Callyspongia Duchassaing and Michelotti, 1864 Sub-genus Cladochalina Schmidt, 1870

3.3.1.1. Synonymy

Pachychalina fibrosa (Ridley and Dendy, 1886); Dasychalina fibrosa (Ridley and Dendy, 1886).

3.3.1.2. Material examined

CEMB-POR-02; collected by Ali A.; 9 February 2013; Churna Island, Karachi; 7-10 m. Material deposited in Centre of Excellence in Marine Biology, University of Karachi. Karachi, Pakistan.

3.3.1.3. Specimen description a. Morphology:

Erect, irregular, branched; stout branches, coarsely fibrous, aculeated, elastic, brittle, hard and compressible. Each branch bears on one side large circular shallow osculum. Length of tubular branch ranges 20-50 mm from base to osculum, width is 35 mm and 110 mm in diameter. Canal length from inside the tubular branch is about 35 mm, narrower towards the base.

34 b. Color

Pale yellow alive and yellowish white in alcohol. c. Skeleton

Ectosomal skeleton is thin, spiny, distinct and translucent. Spines length ranges 3-5 mm. Choanosomal skeleton is irregular coarsely reticulation of stout spiculo-fibres (Fig. 8B and C). d. Spicules

Densely packed in spongin fibres in the form of reticulate bundles about the thickness of 0.1 mm. They are small, smooth slender with blunt ends (size range of length is 66-84 µm and width 2-3 µm) (Fig. 8D). e. Ecology

Shallow intertidal rocky shelf of about depth 7 m. f. Distribution

The worldwide distribution of Callyspongia (Cladochalina) fibrosa is given in Table III. g. Remarks

The specimen is certainly comparable with the Pachychalina fibrosa and Dasychalina fibrosa (Ridley and Dendy, 1886) with some exemptions as the size of branches larger than the original specimen. Color of specimen after preservation is not greyish yellow, but whitish yellow. Spicules although occurs in same size, not oxea while with blunt ends.

3.3.2. Haliclona (Soestella) hornelli (Dendy, 1916) (Fig. 9B)

Family Chalinidae Gray, 1867 Genus Haliclona Grant, 1841 Subgenus Soestella De Weerdt, 2000

3.3.2.1. Synonymy

Reniera hornelli Dendy, 1916; 110, plate II, figure 11.

3.3.2.2. Material examined

CEMB-POR-03; collected by Shafique, S. and Zaib-un-Nisa, B.; 26 January 2016; 1 to 1.5 m (rock pool), Buleji, Karachi. Material deposited in Centre of Excellence in Marine Biology, University of Karachi. Karachi, Pakistan.

3.3.2.3. Specimen description a. Morphology

Cushion shaped, spread over surface of rocks, firmly attached in the form of thickly encrusting sheets. b. Surface

Smooth and even, small chimney like volcano elevations on surface with 0.8 - 1 cm height, inner tube length was 0.4 - 0.8 cm and width was 0.6 cm. The oscules (diameter 0.2 - 0.4 cm) are irregular scattered over surface. c. Consistency

Soft, delicate and fragile. d. Color

Turquoise green in live and preserved specimen is greyish beige in alcohol.

36 e. Ectosomal skeleton

Unispicular, tangentially irregular reticulation of fibres. f. Choanosomal skeleton

Delicate with sub-anisotropic, unispicular and paucispicular reticulation irregularly and tangentially connected with ectosomal unispicular surface lines forming the meshes. g. Spicules

Oxeas, size range: length 80-148 µm and width 5-7 µm (135 × 7 µm), with pointed ends, loosely attached with nodal spongin (Fig. 9C and D). h. Ecology

Rock pool at shallow intertidal rocky shelf; depth 1.2 m (Fig. 9A). i. Distribution

Beyt Island, West and South shelf (71°43'8.4" E, 20°28'25.7" N), India; Buleji ledge, Karachi (64°50'21"E, 24°49'03"N), Pakistan (present study). j. Remarks

Haliclona hornelli (Dendy, 1916) was originally described as Reniera hornelli Dendy 1916 with skeletal features of isodictyal choanosomal skeleton, the multispicular lines interconnected irregularly with unispicular secondary lines. De Weerdt (2000) described the characteristic features of the subgenus Reniera as having a choanosomal skeleton consisting of a regular unispicular isotropic reticulation, unlike the presently studied specimen‘s features. Our specimen and Dendy‘s (1916) material has a sub-anisotropic choanosomal skeleton, ascending paucispicular lines that are irregularly connected to secondary unispicular lines. The ectosomal skeleton is formed by rounded meshes. This combination of characters indicates that the species has to be placed in the subgenus Haliclona (Soestella).

A B

C D

Figure 8. (A) Specimen of Callyspongia (Cladochalina) fibrosa Ridley and Dendy, 1886 (B) Light microscopic image of tangential skeleton shows tufted spongin network (scale = 20 µm), (C) Light microscopic image of microtome-section shows spiculo-fibres and (D) Scanning electron microscopic image of siliceous spicules (scale = 5 µm).

A B

C D

E F

Figure 9. (A) Specimen of Haliclona (Soestella) hornelli (Dendy, 1916) in rock pool, (B) Preserved specimen of H. (S.) hornelli, (C) Light microscopic image of internal skeletal structure, (D) Scanning electron microscopic image of oxeas (scale = 20 µm), (E) Histological section of ectosomal and choanosomal skeleton (scale = 500 µm) and (F) Spicules (oxeas) in tangential section.

Table III. The worldwide geographical distribution of Callyspongia (Cladochalina) fibrosa Ridley and Dendy, 1886. (―MEOW‖ is the abbreviation used for ―Marine Ecoregion of the World‖ and ―WPD‖ for ―World Porifera Database‖).

Taxon Synonymy Region Source

Eastern Philippines Ridley & Dendy, Callyspongia (C.) fibrosa - (MEOW) 1887

Callyspongia (C.) fibrosa Dasychalina fibrosa Philippines WPD, 2017

Callyspongia (C.) fibrosa Pachychalina fibrosa Philippines WPD, 2017

Callyspongia (C.) fibrosa - Southern Vietnam (MEOW) Lévi, 1961

Callyspongia (C.) fibrosa - Australia Thomas, 1979; 1981

Callyspongia (C.) fibrosa - Indian Ocean Thomas, 1979

Callyspongia (C.) fibrosa - Mahe Island Thomas, 1981

Callyspongia (C.) fibrosa - Paradise Islands Thomas, 1979

Callyspongia (C.) fibrosa Dasychalina fibrosa Paradise Islands Thomas, 1979

Inhaca Islands, Callyspongia (C.) fibrosa - Mozambique Thomas, 1979

Mambone Islands, Callyspongia (C.) fibrosa - Mozambique Thomas, 1979

Mambone Islands, Callyspongia (C.) fibrosa Dasychalina fibrosa Mozambique Thomas, 1979

Callyspongia (C.) fibrosa - Atlantic (General Sea Area) Thomas, 1979; 1981

Callyspongia (C.) fibrosa - Pacific Ocean Thomas, 1979

3.4. Discussion

This is the first work on taxonomic illustrations of marine sponges at Churna Island and Buleji, Karachi coast, in which two new records of order Haplosclerida has systematically described. Callyspongia (Cladochalina) fibrosa is commonly known from Gulf of Mannar, Indian Ocean (Pallela and Janapala, 2013). This species has considerably brittle consistency with reticulate meshes of tufted fibres, cemented with rocky ledge (Hooper and Van Soest, 2002). Churna Island has rich productive ecosystem due to nutrient rich water that supports high symbiotic and strong competitive association with diverse coral communities (Kazmi and Kazmi, 1997). The rocky shelf (for benthic organisms) and coral association flourishes the sponge fauna in this region (Ali, Ormond, Leujak and Siddiqui, 2014; Raza, Shaukat, Perveen and Hussain, 2014). The identification of Haliclona (Soestella) hornelli (order Haplosclerida, family Chalinidae) is primarily based on morphological features and skeletal framework. This is typically difficult to analyze, as external morphology may diverge due to change of environmental conditions and habitat from the type localities of species (Qu, Song, Cao and Zhang, 2012; Muricy, Esteves, Monteiro, Rodrigues and Albano, 2015).

This species is closely resembled to H. (Gellius) digitata (Koltun, 1958), H. semifibrosa (Dendy, 1916) and H. tenuiramosa (Burton, 1930), but small variations in these species indicated different morphological and skeletal features. The color pattern and spicules size of H. (G.) digitata (Koltun, 1958) similar but outer morphology (specimen shape and tube length) varied with the studied specimen. According to Dendy (1916), the outer structure and skeleton of H. semifibrosa revealed similar features but conflicting by the presence of dermal and sub-dermal reticulation of spiculofibres. Also H. tenuiramosa (Burton, 1930) has similar color and morphological features with H. (S.) hornelli but skeletal structure is far most different. Here, the morphology, entire surface structure (shape and size of surface elevations), oscules, skeletal features and spicules dimension of H. hornelli given by Dendy (1916), certainly resembles with our specimen. Although, live color of the specimen and some of the skeletal features of subgenus Haliclona (Soestella)has not mentioned in the original description but comprehensive assessment of

41 skeleton which consist of multispicular, discontinuous and irregular reticulation of spicules, spicules size and least spongin on ectosomal membrane consigns this species.

The species H. hornelli Dendy, 1916 was originally described from Beyt Island, India and around coastal margins of Western India, as Reniera hornelli by Dendy, 1916 (World Porifera Database, 2017) in which subgenus was not described. Even though, the species was originally described in the genus Reniera, due to spicular morphology and skeletal arrangement of existing sponge specimen, it is here proposed to place it in the subgenus Haliclona (Soestella). This subgenus so far has not been recognized in the Arabian Sea and is being reported for the first time from Pakistan.

This is the first work on the taxonomy of a marine sponge at Buleji, on the Karachi coast. This sandy-rocky beach has numerous tidal and rock pools which contain a rich variety of fauna and flora (Nazim, Ahmed, Abbas and Khan, 2012). However, no attention has been paid to the sponge fauna from Pakistan and due to its taxonomic and ecological perspective and potentially active pharmaceutical significance, there is a dire need to investigate in regions of the northern Arabian Sea.

PART - III

GROWTH AND ABUNDANCE OF MARINE SPONGE AND ASSOCIATED COMMUNITIES

CHAPTER - 4: In situ variation in growth and abundance of marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves. CHAPTER - 5: Phytoplankton community associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit, Karachi. CHAPTER - 6: Seasonal diversity of benthic foraminifera associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwaters, Karachi. CHAPTER - 7: The community composition of meso- zooplankton associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves, Karachi.

Part: III Chapter - 4

In situ variation in growth and abundance of marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves

Abstract

Among sessile benthic communities, sponges are essential and dominant component in mangrove ecosystem that showed limited growth in intertidal region. The present study has designed to evaluate the growth and abundance of dominating marine sponge Liosina paradoxa on pneumatophores of Avicennia marina with respect to changes in environmental factors in four transects of Sandspit backwater. Annual growth rate was observed quadrantile in live specimens in relation to physicochemical parameters. Results shown the growth rate of L. paradoxa was remarkably high in July in transect 4 (0.036 days-1) and maximum abundance was observed in TR1. The volume of specimen was going increase from January to July (22 - 313 cm3) and then started declining by shrinkage. Water temperature has ranged between 19 - 32 °C, salinity range was 35-41 PSU, dissolved oxygen was 0.11-3.44 mg L-1 - -1 and pH range was 7.04-7.69. The concentration of NO3 was maximum in June (5.64 µg L ) -1 - -1 and lowest in October (0.38 µg L ), while, NO2 concentration was low in June (0.03 µg L ). + -1 - Similarly, NH4 concentration was high in January (7.75 µg L ) and low in April (0.08 µg L 1 -3 -1 ) and PO4 concentration was high in April (2.95 µg L ). Pigment concentrations was frequently high in summer and after rainfall in monsoon season. Organic and carbon content was shown high amount in bottom layer of mangrove sediment late after monsoon season (0.11-0.37 µg/g and 1.13-1.99 µg/g, respectively) and lowest concentration of organic content recorded in March (0.04-0.10 µg/g) and carbon in October (0.10-0.34 µg/g), while inorganic content has inverse profile indicated high values in upper sediment layer in summer (May; 3.15 µg/g) and winter (November - February; 2.78-3.64 µg/g). The results suggested that sponges play crucial role in biogeochemical cycling and their growth and diversity pattern favorably affected by them. 43

4.1. Introduction

Among benthic communities of mangroves ecosystem, sponges are the most essential and abundant organisms (Wilkinson, 1983, 1987; Wilkinson and Cheshire, 1990). They play vital role in mangrove ecosystem as space competitors, primary producers, bioeroders, nitrifiers and as a food source for crabs and fishes (Diaz and Rützler, 2001). The sponge species at intertidal region grows very slow due to seasonal variations of their living history patterns, but their specific growth rate is constant in suitable season (Fell, 1976; Fell and Lewandrowski, 1981; Ayling, 1980). The existing data regarding in situ growth of sponges is mostly limited and somewhat not reliable (Reiswig, 1973; Sebens, 1987; Wilkinson and Cheshire, 1988; McMurray, Blum and Pawlik, 2008). Mainly intertidal and shallow water communities affected on the sponge growth as they feed on about 65- 93% small organisms and different particulate matter, to increase up their size and growth, this tidal effect influences on their distribution and abundance in their actual habitat (Lesser, 2006).

On the basis of sponge morphology (irregular, unsymmetrical and flexible structure), growth pattern is not uniform. They show variation in their growth dynamics in comparison with other groups of organisms due to their respective capabilities (as regeneration and competition) in environment to survive for a long time period (Sarà, 1970; Reiswig, 1973; Dayton, Robilliard, Paine and Paine, 1974; Dayton, 1979; Ayling, 1983; Pansini and Pronzato, 1990). Growth in sponge were measured by the accumulative area of biomass and their extended value of growth (Garrabou and Zabala, 2001) through statistical method given by Turon, Tarjuelo and Uriz (1998) and McMurray, Blum and Pawlik (2008) as follows;

-1 GRm = (Am - Am ) (equation 1.1) -1 Am

Where ‗GRm‘ is the monthly growth rate shows monthly change in area of sponges, -1 related to area at starting month interval (Am) and area of previous month interval (Am ). This correlation factor is suitable to relate ‗the change in area over time‘ (equation 1.1).

Volume of sponge could be calculated by using the geometric model equation for frustum of cone, which basically applies for measuring the cylindrical branch of thickly encrusting sponges.

V = 1πh (od2 + (od)(bd) + bd2) (equation 1.2) 2

Where ‗V‘ is the volume of sponge (in cm3), ‗h‘ is the ‗height of sponge‘ (in cm), ‗od‘ is osculum diameter (in cm) and ‗bd‘ is base diameter (in cm) (equation 1.2).

The specific growth rate could certainly assess by calculating the volume of sponge from the above mentioned factor (i.e., from equation 1.2).

G = (Vf - Vi) (equation 1.3)

Vi 3 Where, ‗G‘ is the specific growth rate, ‗Vi‘ is initial volume (cm ) and ‗Vf‘ is final volume (cm3) (equation 1.3).

In field study, better results of growth rates can be obtained by taking surface area measurements of sponges along its dimensions (Koopmans and Wijffels, 2008). To estimate the growth rate (increase or decrease), other environmental factor such as temperature, salinity, food availability, nutrients dynamics also measured (Koopmans and Wijffels, 2008). Growth may occur unequally throughout the organism in natural condition, as some part may grow, and shrinkage occurs at another point, this is ‗growth independent rates‘ in growth dynamics (Garrabou, 1998). This is non-destructive method to provide good observation besides determining wet weight and ash-free dry weight (Garrabuo and Zabala, 2001; de Caralt, Uriz and Wijffels, 2008; Koopmans and Wijffels, 2008).

4.1.1. Environmental factors that affecting the sponge growth

Ecological factors mainly effect on the shape, size and growth of an organism in a community (Peters, 1983; Werner and Gilliam, 1984). The size and growth patterns of the organism may vary according to different environmental conditions (Blueweiss, Fox, Kudzma, Nakashima, Peters and Sams, 1978). Many species cannot accede into hardy

45 ecological states, but mangroves sponge species have adapted themselves to tolerate in these environment (McMurray, Blum and Pawlik, 2008).

Sponges grows in the environment where suitable physical and chemical conditions, solid substratum (required for their settlement), space for their growth, moderate water flow for feeding etc. available (Rützler, Diaz, Van Soest, Zea, Smith, Al Varez and Wulff, 2000).

Short-term abundance could be assessed by biological processes such as interspecific epibionts competition and predation while long-term abundance is limited under seasonal and environmental changes as precipitation, effect of tides, waves and currents which creates fluctuation in community growth and abundance (Bingham and Young, 1995; Ellison, Farnsworth and Twilley, 1996; Wulff, 2000, 2004, 2005; Engel and Pawlik, 2005). It is demonstrated by Granek, Compton and Phillips (2009) that dissolved organic matter in mangroves consist of tannin and polyphenolic compounds which is mainly carbon resource for mangrove sponges, although bacterial community also play a vital role in organic degradation and assimilation in mangrove habitat (Taylor, Radax, Steger and Wagner, 2007). Mainly highest growth of sponges observed during high concentration of nutrients, algal biomass (chlorophyll) and temperature (Koopmans and Wijffels, 2008). Particulate and dissolved organic carbon (POC and DOC), dissolved inorganic carbon (DIC), dissolved organic and inorganic nitrogen (DON and DIN) in water column contribute as a food source for the growth of mangrove sponges (Ribes, Coma and Gili, 1999; Diaz and Rützler, 2001; Yahel, Sharp, Marie, Häse and Genin, 2003).

4.1.2. Nutrients productivity and its effect on sponge growth

The effect of internal waves, tidal bores and their upwelling intensity on intertidal benthic habitats is significantly high through nutrients transport and plankton abundance (Witman, Patterson and Genovese, 2004; Leichter, Wing, Miller and Denny, 1996; Leichter, Stewart and Miller, 2003; Gili and Coma, 1998; Nielsen and Navarrete, 2004; Lesser, 2006). Sponges have ability to filter large volume of water which effected on nutrient fluxes in channel water (Kahn, Yahel, Chu, Tunnicliffe and Leys, 2015; Archer, Stevens, Rossi, Matterson and Layman, 2017). The theoretical work of interpretation of

46 this study is nutrient/productivity model (N/PM) where food webs complexity increases with primary production and predation controls the lower trophic level (Menge, 2000). The coastal upwelling of nutrients effects on local sponge distribution more than biological components (Wilkinson and Cheshire, 1989; Alcolado, 1990; Alvarez, Diaz and Laughlin, 1990; Diaz, Alvarez and Laughlin, 1990; Schmahl, 1990; Witman and Sebens, 1990; Farooqui, Siddiqui, Shafique and Rasheed, 2013). The nutrient dynamics predicts the distributional variation of sponges (through its growth) in response of nitrogen and phosphate enrichment (Feller, Whigham, McKee and Lovelock, 2003).

In Pakistan, there is no study regarding the growth and ecology of marine sponges. Therefore, the present study has been focused on the annual growth and abundance of Liosina paradoxa and the effect of ecological factors on its growth, which is the first study from Sandspit backwater mangroves Karachi coast, Pakistan.

4.2. Materials and methods

4.2.1. Study site description

Four transects (of 0.5 m quadrate size each with a distance of 5-7 meters) at Sandspit backwater mangrove were selected to survey the distribution and abundance and growth rate of Liosina paradoxa during January to December 2013 (Fig. 10). First transect (TR1) is located at 24°49'19.83"N, 66°56'19.82"E and completely submerged in water under shady area in mangrove channel. Second transect (TR2) is rather exposed area with sunlight, distant 5 meters away from first transect (24°49'19.49"N, 66°56'19.81"E) where intertidal water covers the area two times a day. Third transect (TR3) is moderately exposed with rich biota (24°49'19.14"N, 66°56'20.01"E) and fourth transect (TR4) is also completely submerged shady area (24°49'19.00"N, 66°56'20.61"E) situated at a distance of 7 meters from third transect.

4.2.2. Field assessment

For growth measurement, samples of L. paradoxa were collected from pneumatophores of Avicennia marina in separate polythene bags from four transects of Sandspit backwater, Karachi. Quadrates (0.5 m2) were placed in these transects where sponges grow on pneumatophores, to evaluate quadrantile abundance. Total percent abundance of sponges was counted by number of sponge individual per unit area transect divided in total number of pneumatophores in quadrate area. A measurement scale of 30 cm was used to take length of large (>10 cm), medium (5-10 cm) and small (<5 cm) live sponges each month (Fig. 11). Water temperature, sediment temperature, salinity and pH were measured in situ through hand-held thermometer, refractometer (Atago, Thailand) and pH meter (El-Metron CP-401, Poland), respectively. Duplicate sediment cores (10 cm length and 10 cm diameter) was recovered aseptically from each transect to determine vertical distribution of water content, organic matter, inorganic matter, organic carbon, chlorophyll ‗a‘, ‗b‘ and phaeo-pigments in sediment.

4.2.3. Laboratory assessment

4.2.3.1. Morphometric analysis of sponge

Annual per day growth rate was calculated by measuring the volume of large, medium and small sized sponges from each transect.

ν = 0.25π {l × (w)2}

Where, ‗ν‘ is the sponge volume (in cm3), ‗l‘ is the sponge length (in cm) and ‗w‘ is sponge width (in cm).

The change in sponge volume was statistically analyzed by the equations given below, where the growth rate determined between two points exponentially (Koopmans and Wijffels, 2008).

Growth rate (days-1) =

Where, ‗ν‘ is the current sponge volume, ‗ν-1‘ is the previous sponge volume and ‗d‘ is the days between two data point readings.

The percent abundance cover of sponge species was measured by substituting the number of sponge in quadrate over the number of pneumatophores.

4.2.3.2. Physicochemical variables a. Dissolved oxygen

Dissolved oxygen in channel water was analyzed by modified ‗Spectrophotometric Winkler Method (Parsons, Maita and Lalli, 1984).

Arabian Sea

Figure 10. Map of Karachi coast shows four collection transects {first transect (TR ), second transect (TR ), third 1 2 transect (TR3) and fourth transect (TR4)} at Sandspit, Karachi (Google Earth Pro version 2017).

Sample preparation

Triplicate water samples (70 ml each) were fixed in situ during collection by adding 1 ml manganese sulfate (MnSO4) and 1 ml potassium iodide (KI) solution, mixed well, kept in dark and transported to laboratory. In the laboratory, 1 ml of concentrated sulfuric acid

(H2SO4) added and shake well to dissolve precipitate. Extinctions for dissolved oxygen were recorded at 287.5 nm by spectrophotometer (UV-1800). Reagents used in this experiment has given in Appendix I (page 224).

Calibration

Temperature and salinity of seawater were noted down, aerated for 30 minutes and re- measured the temperature and salinity. Seawater (70 ml) was fixed and followed the similar procedure mentioned above. For determination of blank, distilled water is used in place of seawater and followed same method.

Dissolved oxygen in sample was calculated by using given formula,

-1 Dissolved oxygen concentration (mg L ) = Fs (ER - EB)

Where, ‗Fs‘ is the factor which related to the temperature and salinity points of seawater and should close to 16 (Strickland and Parsons, 1972). ‗ER‘ is sample extinction and ‗EB‘ is extinction reading of blank. b. Nutrient analysis

For nutrient analysis water samples (triplicate) were collected from mangrove channels, in pre-acid washed polyethylene bottles, 2 to 4 drops of chloroform added to stop the microbial activity and kept in ice box till transferred to laboratory. Concentration of + Ammonium ion (NH4 ) was analyzed through Automated Phenate method, Nitrite ion - (NO2 ) by diazotizing with C6H8N2O2S coupling N-(l- naphthy1)-ethylenediamine - -3 method, Nitrate (NO3 ) by Cu-Cd column method and Phosphate (PO4 ) was determined by complex-reagent method, by using standard UV spectrophotometric method (Parsons, Maita and Lalli, 1984). The chemical reagents which were used in this experiment has

51 given in Appendix II (page 224-225). c. Analysis of total organic carbon (TOC)

Total organic carbon (TOC) was assessed through wet oxidation by acid dichromic method described by Parsons, Maita and Lalli (1984). The reagents which were used in this experiment has given in Appendix III (page 226).

Sample preparation

Sediment sub-samples were oven dried at 70°C for 48 hours and pulverized. Taken 1 gram of sample in a test tube and added 70% phosphoric acid (1 ml) and distilled water (1 ml). Heated at 100 -110 °C in sand bath for 30 minutes, followed by the addition of 10 ml sulfuric acid dichromate oxidant and glucose solution (4 ml). The sample was heated again at same temperature for further 60 minutes. Cool the sample and volume was made up to 50 ml with distilled water. Absorbance was recorded at 440 nm by UV spectrophotometer against reagent blank.

Calibration

All reagents and solutions were used in equal volume at similar temperature and time in five empty test tubes to calibrate the sample values.

Calculation

The total organic carbon (mg/g) was calculated using given formula,

TOC (mg/g) = E × F × V W

Where ‗E‘ is the correct extinction value of blank, ‗F‘ is the factor, ‗V‘ is the volume of oxidant used and ‗W‘ is the weight of sample

Carbon (mg/g) = (Sample extinction × Factor value × Oxidant volume) ÷ Sample weight

d. Water content, inorganic and organic matter

Water content was recorded in sediment sub-samples by taken difference between sediment wet weight and dried weight (in gram %). Organic and inorganic matter was determined gravimetrically by igniting the known amount of sediment sample in muffle furnace at 450°C for 8 hours. The lost ash weight (in µg/g) was considered as organic content and remaining ash-free dry weight was inorganic matter (Shafique, 2006).

e. Pigment analysis i. Chlorophyll ‘a’ and ‘b’ in sediment

Wet sediment samples from 10 cm sediment cores (10 cm diameter) sectioned vertically in five layers. Each layer (of 2 cm thick) sub-sampled (in triplicate), soaked in 90% acetone (10 ml) in brown vials and stored at 4 °C in dark for 48 hours. The samples were centrifuged at 2000 rpm for 5 minutes and supernatant was analyzed through spectrophotometer (UV-1800-Shimadzu) at extinctions of 630, 647, 664 and 750 nm. Concentration of chlorophyll ‗a‘ and ‗b‘ were calculated by the formulae given in Strickland and Parson (1972) as below,

Ca (Chlorophyll ‗a‘) = (11.85 × E664) - (1.54 × E647) - (0.08 × E630)

Cb (Chlorophyll ‗b‘) = (21.03 × E647) - (5.43 × E664) - (2.66 × E630)

Where, ‗E‘ is corrected extinctions at given wave length (in subscript)

The calculated value of Ca or Cb were put in the given formula for calculation of Chlorophyll ‗a‘ and ‗b‘ given,

Chlorophyll (µg/g) = (Ca or Cb) × ν V × 10

Where, ‗ν‘ is the volume of acetone used per sample (in ml) and ‗V‘ is the volume of water filtered (in ml or liter) and 10 is the path length of cuvette.

53 ii. Carotenoids in sediment

The procedure to estimate the carotenoids in sediment is similar as described for chlorophyll but the extinction (wave length) for carotenoids was 480, 510 and 750 nm. These extinctions corrected by subtracting the values of 750 nm from readings taken at 480 nm and 510 nm. The amount of carotenoids was estimated through the given equation;

Cp (Carotenoids) = 7.6 (E480 – (1.49 × E510)

Where, ‗Cp‘ is the amount of carotenoids, ‗E480‘ and ‗E510‘ are the extinctions noted at 480 nm and 510 nm. Further calculation was substituted by the formula used for

chlorophyll (where Cp was replaced by Ca and Cb).

iii. Phaeo-pigments in sediment

Phaeopigments was extracted using similar procedure as describe above for chlorophyll ‗a‘ and ‗b‘. Optical density (OD) of the extract was measured at 665 and 750 (after acidification) and then 2 drops of 1N HCl was added and read again at 665 and 750 (before acidification). Reading were corrected by subtracting optical density at 750 from corresponding OD at 665. Following equation was used to calculate the concentration of phaeopigment in the sample describe by Strickland and Parson (1972) using given formula:

Phaeopigment (µg/g) = 26.7 [(1.7 × 665a) - 6650] ν W × 10

Where, ‗665a‘ is the extinction at 665 nm after acidification, ‗6650‘ is the extinction noted at 665 nm before acidification, ‗ν‘ is the volume of acetone (in ml) and ‗W‘ is the sample weight in grams.

4.2.4. Statistical analysis

The relationship between sponge growth rate with physicochemical parameters were analyzed through PAST version 2.13 (Hammer, Harper and Ryan, 2001) and Minitab statistical software (version 17.1.0, 2013).

A B

C D

Figure 11 (A). Study site of Sandspit backwater, Karachi, (B) Liosina paradoxa attached with pneumatophores, (C) Measurement of L. paradoxa in transect and (D) Growth size of sponge specimens in laboratory.

4.3. Results

4.3.1. Growth and abundance of Liosina paradoxa Thiele, 1899

The size measurement (length and width) of marine sponge Liosina paradoxa showed variable results with reference to seasons as pre-monsoon (March to June), monsoon (July to October) and post monsoon (November to February), and transect locations in the study area for example, TR1 (sub-merged), TR2 (exposed), TR3 (semi-exposed in open area) and TR4 (semi-exposed in shady area). The volume and dimensions (length, width and height) of sponge were used to interpret the growth rate. Throughout the study the maximum values of growth rate were observed in TR1 (0.015 ± 0.02) and TR4 (0.036

± 0.03) in January whereas, TR2 (0.018 ± 0.02) in June and TR3 (0.029 ± 0.02) in July, respectively. Although negative or no growth was observed in December in all four transects (TR1; -0.077, TR2; -0.06, TR3; -0.059 and TR4; -0.1), respectively (Fig. 12).

The increasing trend in sponge volume recorded in January to July with highest values 3 3 3 3 detected in July for TR1 (274 cm ), TR2 (127 cm ), TR3 (313 cm ) and TR4 (307 cm ) and then declining from August to December in all four transects (Fig. 13A). The least values 3 3 3 were observed in all transects in December (TR1; 24 cm , TR2; 15 cm , TR3;18 cm and 3 TR4; 6 cm ), respectively (Fig. 13A). In case of abundance, the highest values were recorded in February and September for TR1 (98%), in January for TR2 (97%), in July for

TR3 (99%) and in October for TR4 (98%) and minimum percent abundance observed in

TR1 (August), TR2 (February), TR3 (March and November) and TR4 (April), (Fig. 13B).

4.3.2. Physicochemical variables

Water parameters such as temperature ranged between 24 - 32 °C with highest value in June (32 °C) and lowest in November to January (19 °C) and reverse is true in case of salinity (Fig. 14A). Maximum value of dissolved oxygen was recorded in June (3.44 mg L-1) and minimum in February and October (0.11 mg L-1) (Fig. 14A). Concentration of pH was ranged between 7.0 - 7.7 with a slight increase in January and June (7.6 and 7.7) and decrease in September (7.0), respectively (Fig. 14A).

4.3.3. Nutrients

Seasonal variation in nutrient ion concentrations (Nitrite, Nitrate, Ammonium and Phosphate ions) were observed during the study. Mean concentrations of nutrients were 0.10 ± 0.06 µg L-1 (nitrite ion), 2.26 ± 1.77 µg L-1 (nitrate ion), 4.31 ± 2.36 µg L-1 (ammonium ion) and 1.66 ± 0.74 µg L-1 (phosphate ion), respectively (Fig. 14B).

- -1 Nitrite ion (NO2 ) concentration was high in February (0.28 µg L ) and low in June (0.03 -1 - -1 µg L ) whereas nitrate ion (NO3 ) concentration was maximum (5.64 µg L ) in June + (Fig. 14B). The highest value of ammonium ion (NH4 ) concentration was recorded in -1 -1 -3 February (7.50 µg L ) and lowest in April (0.08 µg L ). In case of phosphate ion (PO4 ) concentration was high in April (2.95 µg L-1) and low in July (0.56 µg L-1), (Fig. 14B).

4.3.4. Sediment analysis

Sediment parameters (moisture, inorganic, organic content, total organic carbon, chlorophyll ‗a‘, ‗b‘, carotenoids and phaeopigments) were also observed throughout the study period in all four transects.

4.3.4.1. Moisture, inorganic and organic content

Moisture content in sediment was increasing with depth and ranged between 0.57-3.55 g% in TR1 0.83-2.97 g% in TR2, 0.31-2.57 g% in TR3 and 0.67-2.67 g% in TR4 (Fig. 15).

Highest values recorded in January; TR1 (3.55 g%), June; TR2 (2.97 g%), September;

TR3 (2.57 g%) and in July; TR4 (2.67 g%) whereas, minimum values observed in March for TR1 (0.57 g%), TR2 (0.84 g%) and TR3 (0.31 g%) whereas in November for TR4 (0.67 g%) (Fig. 15).

Inorganic profile indicated decreasing trend from top to bottom sediment layers.

Maximum values observed in November in TR1 (3.64 µg/g), February in TR2 (2.78

µg/g), September in TR3 (2.36 µg/g) and February in TR4 (2.05 µg/g). Lowest values recorded in June for TR1 (0.52 µg/g) and TR3 (0.59 µg/g) whereas in March for TR2

(0.55 µg/g) and in November for TR4 (0.51 µg/g) (Fig. 16). Whereas, reverse is true for organic content which was increase with depth. The values ranged between 0.06-0.372

µg/g (TR1), 0.076-0.362 µg/g (TR2), 0.048-0.311 µg/g (TR3) and 0.089-0.334 µg/g (TR4) (Fig. 17). Maximum values of organic matter were recorded in all four transects in October (0.372 µg/g), June (0.362 µg/g), February (0.311 µg/g) and July (0.334 µg/g) while minimum during May (0.06 µg/g), November (0.07 µg/g), March (0.05 µg/g) and August (0.09 µg/g), respectively (Fig. 21). The highest value of organic content was observed in TR1 during October and lowest in TR3 in March (Fig. 17).

4.3.4.2. Analysis of total organic carbon (TOC)

Total organic carbon showed decreasing trend from top to bottom in mangrove sediment and is ranged between 0.22-1.98 mg/g (TR1), 0.10-1.99 mg/g (TR2), 0.19-1.99 mg/g

(TR3) and 0.16-1.98 mg/g (TR4). Highest values of TOC observed in TR1 in June (1.99 mg/g), TR2 in April, TR3 in January and in TR4 in May whereas, lowest concentrations were recorded in October; TR1 (0.22 mg/g), November; TR2 (0.10 mg/g) and August;

TR3 (0.19 mg/g) and TR4 (0.24 mg/g). The maximum value observed in January (1.996 mg/g) in TR3 and minimum value in November (0.10 mg/g) in TR2 (Fig. 18).

4.3.4.3. Pigment analysis

Vertical profile of pigments in sediment layers indicated decreasing trend from top to bottom in all four transects. Chlorophyll ‗a‘ concentration showed maximum value (8.97

µg/g) in June, April (7.88), August (9.08 and 9.03 µg/g) in all transects TR1 TR2 TR3 and

TR4, respectively (Fig. 19).

Chlorophyll ‗b‘ results showed highest concentration in top sediment layer of TR4 in

August (3.29 µg/g) and lowest value recorded in May; TR1 (0.02 µg/g) in the bottom layer of sediment. Maximum values recorded in April (1.97 µg/g) and June (2.40 µg/g) in

TR1, July (1.84 µg/g) and December (1.87 µg/g) in TR2 and August; TR3 (1.53 µg/g) (Fig. 20).

Carotenoids concentration were ranged between 0.03-0.32 µg/g in May whereas, maximum values were ranged between 4.21-4.99 µg/g (Fig. 21).

Phaeo-pigment (degrading product of chlorophyll) also recorded and showed highest concentrations in September (9.90 µg/g) in TR1, December; (9.86 µg/g) in TR2, and

August (9.85 µg/g and 9.15 µg/g) in TR3 and TR4 within top layers of sediment (Fig. 22).

The cumulative dominance (K dominance curves) of sponge abundance and biomass in four transects at Sandspit backwater shown variation as the highest abundance and biomass of sponges attached with pneumatophores were found in TR3 followed by TR4,

TR1 and TR2 (Figs. 23 and 24).

A highly significant correlation was observed between growth rate of Liosina paradoxa in all four transects with water parameters. Growth rate of sponge significantly - - -3 correlated in TR1 with temperature, dissolved oxygen, NO2 , NO3 and PO4 , TR2 with -3 - + -3 - -3 PO4 , TR3 with NO2 , NH4 and PO4 and TR4 with pH, NO3 and PO4 , respectively

(Table IV). Salinity was indicated positive correlation with growth rate of sponge in TR1 and TR2 (Table IV). In case of abundance, significant positive correlation pointed - between temperature, salinity, dissolved oxygen, pH and NO3 in TR1, dissolved oxygen, - + -3 - + -3 pH, NO2 and NH4 in TR2, PO4 in TR3 and NO3 , NH4 , PO4 in TR4, respectively, - whereas NO2 in TR1 and salinity in TR2 indicated positive insignificant (p > 0.5) correlation (Table V). The growth rate and abundance has indicated significant positive correlation were present between TR1 with TR2 and TR3, TR2 with TR3 and TR3 with all transects while TR4 indicated insignificant positive relation with TR1 and TR3 (Table VI). The growth rate and volume shown positive correlation among all transects (Table VII). In case of abundance and volume, TR1 and TR3 indicated positive correlation with all transects and significant correlation observed between all transects with TR4 whereas,

TR2 shown negative correlation with all transects (Table VIII).

0.04 0.04

) 1

- 0 0

-0.04 -0.04

-0.08 -0.08 Growth Growth rate (days

-0.12 -0.12 F M A M J J A S O N D J F M A M J J A S O N D J A B

0.04 0.04

) 1

- 0 0

-0.04 -0.04

-0.08 -0.08 Growth Growth rate (days

-0.12 -0.12 F M A M J J A S O N D J F M A M J J A S O N D J Month Month C D

Figure 12. Annual growth rate (days-1) of L. paradoxa on pneumatophores of Avicennia

marina at four transects (A - TR1; B - TR2; C - TR3 and D - TR4) of Sandspit backwater, Karachi.

TR1 TR2 TR3 TR4 400 350 350 300

300

) 250 3 250 200 200 150 150 Volume (cm Volume 100 100 50 50 0 0 J F M A M J J A S O N D J A

120 120

100 100

80 80

60 60

40 40 Abundance (%) Abundance 20 20

0 0 B F M A M J J A S O N D J Month

Figure 13 (A). Annual volume (cm3) and (B) total abundance (%) of L. paradoxa on

pneumatophores of Avicennia marina at four transects (TR1, TR2, TR3 and

TR4) of Sandspit backwater, Karachi.

Temperature Salinity pH Dissolved oxygen 45 4

40 3.5

35 3 30

2.5 1 25 - 2

20 mg mg L Concentration 1.5 15 10 1 5 0.5 0 0 A F M A M J J A S O N D J

Nitrate Ammonia Phosphate Nitrite

) 0.5 10 1 - 9 0.4 8 7 0.3 6 5 0.2 4 Nutriens (µg (µg L Nutriens 3 0.1 2 1 0 0 F M A M J J A S O N D J B Month

Figure 14 (A). Annual concentration of physicochemical parameters (temperature °C, -1 - - salinity PSU, pH and dissolved oxygen mg L ) and (B) nutrients (NO3 , NO2 , + -3 NH4 and PO4 ) in channel water of Sandspit backwater mangroves, Karachi.

g % g % g % 0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 15. Moisture volume (g%) in mangrove sediment cores from January to December

(A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 16. The amount of inorganic content (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 0.2 0.4 0 0.2 0.4 0 0.2 0.4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 0.2 0.4 0 0.2 0.4 0 0.2 0.4 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 17. The amount of organic content (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

mg/g mg/g mg/g 0 1 2 0 1 2 0 1 2 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 1 2 0 1 2 0 1 2 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 1 2 0 1 2 0 1 2 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 1 2 0 1 2 0 1 2 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 18. The concentration of total organic carbon (mg/g) in mangrove sediment cores

from January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 5 10 0 5 10 0 5 10 0 0 0 2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 5 10 0 5 10 0 5 10 0 0 0 2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 19. Chlorophyll ‗a‘ concentration (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 2 4 0 2 4 0 2 4 0 0 0

2 2 2 4 4 4 6 6 6

TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 20. Chlorophyll ‗b‘ concentration (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 5 0 5 0 5 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 5 0 5 0 5 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 5 0 5 0 5 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 5 0 5 0 5 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 21. The concentration of carotenoids (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

µg/g µg/g µg/g 0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 A 10 B 10 C 10

0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 D 10 E 10 F 10

0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6

Depth (cm) 8 8 8 G 10 H 10 I 10

0 5 10 0 5 10 0 5 10 0 0 0

2 2 2 4 4 4 6 6 6 TR1 Depth (cm) 8 8 8 TR2 TR3 10 10 10 J K L TR4

Figure 22. The concentration of phaeo-pigments (µg/g) in mangrove sediment cores from

January to December (A to L) at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

100 TR1 TR2 TR3 TR4

c n

a 60

t a

l 40

u C

0 1 10 100 Abundance rank

Figure 23. The cumulative dominance curves (%) for sponge abundance rank at four transects of Sandspit backwater, Karachi.

100 TR1 TR2 TR3 TR4

c n

a 60

t a

l 40

u C

0 1 10 100 Biomass rank

Figure 24. The cumulative dominance curves (%) for sponge biomass rank at four transects of Sandspit backwater, Karachi.

-1 Table IV. Pearson correlation of growth rate (days ) of L. paradoxa at four transects (TR1, TR2, TR3 and TR4) with physicochemical -1 - - + -3 parameters (temperature °C, salinity PSU, pH and dissolved oxygen mg L ) and nutrients (NO2 , NO3 , NH4 and PO4 ) at Sandspit backwater, Karachi.

- - + TR1 TR2 TR3 TR4 Temperature Salinity Oxygen pH NO2 NO3 NH4 Temperature ***0.888 Salinity *0.584 **0.690 Oxygen ***0.908 0.220 0.292 pH 0.237 **0.618 0.127 **0.770 - NO2 ***0.844 0.237 **0.664 0.460 0.342 - NO3 **0.799 **0.643 0.319 ***0.853 0.391 0.052 + NH4 0.444 *0.546 ***0.982 0.286 **0.610 0.088 **0.797 -3 PO4 ***0.945 **0.737 ***0.907 ***0.908 0.342 0.354 **0.784 *0.582

TR4 0.001 **0.643 ***0.989 ***0.835 0.144 0.202 0.303 0.482 *0.550

TR3 0.006 0.003 0.404 0.501 0.031 **0.693 0.067 0.090 *0.559 0.223

TR2 0.002 0.015 0.008 ***0.835 ***0.817 0.202 ***0.999 0.030 0.132 0.178 0.003

(p > 0.5) *Significant value, **more significant value and ***Highly significant value

Table V. Pearson correlation of abundance (%) of L. paradoxa at four transects (TR1, TR2, TR3 and TR4) with physicochemical parameters -1 - - + -3 (temperature °C, salinity PSU, pH and dissolved oxygen mg L ) and nutrients (NO2 , NO3 , NH4 and PO4 ) at Sandspit backwater, Karachi.

- - + TR1 TR2 TR3 TR4 Temperature Salinity Oxygen pH NO2 NO3 NH4 Temperature ***0.883 Salinity **0.743 **0.665 Oxygen ***0.963 ***0.943 0.056 pH ***0.853 ***0.817 0.024 0.046

- NO2 *0.511 ***0.926 0.143 0.202 0.342

- NO3 ***0.944 0.256 0.409 ***0.889 0.391 0.052

+ NH4 0.464 ***0.889 0.493 ***0.871 **0.61 0.088 **0.797

-3 PO4 0.450 0.487 ***0.898 **0.763 0.342 0.354 **0.784 *0.582

TR4 0.318 **0.693 0.443 0.354 0.144 0.202 0.330 0.482 *0.550

TR3 0.052 **0.763 0.390 0.144 0.031 **0.693 0.067 0.090 *0.559 0.223

TR2 **0.617 *0.523 0.486 *0.531 ***0.817 0.202 0.100 0.297 0.132 0.178 0.003

(p > 0.5) *Significant value, **more significant value and ***Highly significant value

-1 Table VI. Pearson correlation of growth rate (days ) and abundance (%) of L. paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Growth rate Abundance

TR1 TR2 TR3 TR4 TR1 TR2 TR3

TR4 0.103

TR3 0.011 *0.537

TR2 0.066 **0.846 0.007 Growth rate Growth

TR1 0.092 ***0.903 0.003 ***0.884

TR4 ***0.898 **0.826 0.004 *0.690 0.001

TR3 0.004 ***0.979 0.012 ***0.885 0.006 0.003

Abundance TR2 ***0.808 *0.618 ***0.940 **0.842 0.002 0.015 0.008 (p > 0.5) *Significant value, **more significant value and ***Highly significant value

-1 Table VII. Pearson correlation of growth rate (days ) and volume (%) of L. paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Growth rate Volume

TR1 TR2 TR3 TR4 TR1 TR2 TR3

TR4 ***0.889

TR3 0.366 ***0.926

TR2 **0.799 0.391 ***0.963 Growth rate Growth TR1 **0.763 **0.745 0.307 **0.835

TR4 0.001 **0.781 *0.659 0.237 0.001

TR3 0.001 0.001 **0.853 *0.610 0.006 0.003 Volume TR2 0.001 0.001 0.001 ***0.963 0.002 0.015 0.008

(p > 0.5) *Significant value, **more significant value and ***Highly significant value

3 Table VIII. Pearson correlation of abundance (%) and volume (cm ) of L. paradoxa at four transects (TR1, TR2, TR3 and TR4) of Sandspit backwater, Karachi.

Abundance Volume

TR1 TR2 TR3 TR4 TR1 TR2 TR3

TR4 0.078

TR3 0.058 0.095

TR2 ***0.963 0.065 0.078 Abundance

TR1 ***0.908 **0.835 0.050 0.086

TR4 0.001 **0.843 ***1.000 0.038 0.318

TR3 0.001 0.001 **0.825 ***0.963 0.052 **0.763 Volume TR2 0.001 0.001 0.001 ***0.954 *0.617 *0.523 0.486

(p > 0.5) *Significant value, **more significant value and ***Highly significant value

4.4. Discussion

Annual growth rate and abundance of marine sponge Liosina paradoxa in intertidal region reveals variation with the environmental factors (temperature, salinity, oxygen, pH and nutrients) in four transects at Sandspit backwater. L. paradoxa proliferates on the pneumatophores of Avicennia marina, and therefore considered to evaluate its growth and abundance in natural (non-destructive condition) habitat (Garrabou and Zabala, 2001). Few studies have been taken to observed in situ growth of sponges in natural habitat (Ayling, 1983; Turon, Tarjuelo and Uriz, 1998) which gives more specific seasonal patterns (with effect of temperature and nutrients) other than laboratory cage culture or land-based systems (Koopmans and Wijffels, 2008; Sankar, Chadha, Roy, Banerjee, Saharan and Krishnan, 2016). The larvae of Liosina paradoxa shown confined movement in mangrove ecosystem as compare to other locations and therefore its growth and reproduction patterns formed patchiness on pneumatophores (Ayling, 1983).

The exposed roots provide habitat to sponges as primary substrates for their attachment and accede their settlement in extensive area (Rützler, Diaz, Van Soest, Zea, Smith, Al Varez and Wulff, 2000; Diaz, Smith and Rützler, 2004). In this area, light intensities, tidal fluctuation, channel water depth and moderate turbidity support their existence and presence of organic particulate matter and plankton communities fulfill the requirement of their feeding (Rützler, Diaz, Van Soest, Zea, Smith, Al Varez and Wulff, 2000). Growth of sponges has prominently affected by various factor for example water quality, temperature, dissolved oxygen etc. The growth rate increased in the area having low water salinity and optimum temperature, while suppressed in light exposing area as we observed in TR2, possibly due to higher algal biomass and their associated communities (micro and meso-crustaceans (copepods and small crabs e.g., Uca sp.) which covered sponges and damaged them showed low growth (Turon, Tarjuelo and Uriz, 1998; Appadoo, Beepat and Marie, 2011).

In the present study, overall TR1 showed maximum seasonal abundance (in average 80%) of sponge and found settlement of species larvae and their survival on pneumatophores of A. marina in all seasons. Whereas, maximum growth of L. paradoxa was observed in TR4 during post-monsoon and minimum in TR1 during pre-monsoon. The major difference in

77 values of growth and abundance according to transect locations may indicate the habitat variation likewise the TR1 is completely submerged area however TR4 is semi-exposed where channel water flow is moderately low. Thus, it could be stated that the sponge abundance was high due to larvae settlement on pneumatophores through water flow while growth was high in moderate exposed condition (Diaz and Rützler, 2001; McGill, Etienne, Gray, Alonso, Anderson, Benecha, Dornelas, Enquist, Green, He, Hurlbert, Magurran, Marquet, Maurer, Ostling, Soykan, Ugland and White, 2007). In size differences, the volume was remarkably high during monsoon when freshwater run off, nutrients availability, sedimentation and dissolved oxygen in channel water were high while salinity was low and showed negative correlation with other parameters, which supports good quality growth of sponges in mangroves (Diaz, Smith and Rützler, 2004).

Temperature has significantly influenced on growth of L. paradoxa as it increases in shallow- water during low tide and showed positive correlation with growth rate in all transects, while noticeable community abundance found in shady habitat (Turon, Tarjuelo and Uriz, 1998; Bell and Smith, 2004). Dissolved oxygen is the important factor which support sponge growth in mangrove ecosystem, the channel water has high concentration of dissolved oxygen during monsoon and post monsoon season may be due to photosynthetic activities of phytoplankton and freshwater runoff) and during the same period (monsoon and post- monsoon) maximum growth of sponges observed (Rützler, 1995; Bell and Smith, 2004; Bell, 2008; Hunting, Van Soest, Van der Geest, Vos, Debrot, 2008).

Salinity and nutrient ion concentration were also essential parameters and varied seasonally. The growth rate of sponge in mangrove habitat shown decreasing trend with the increase of salinity as reported earlier (Rützler, 1995; Barnes, 1999; Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton, Meynecke, Pawlik, Penrose, Sasekumar and Somerfield, 2008; Sankar, Chadha, Roy, Banerjee, Saharan and Krishnan, 2016). Nutrients have significant influence on sponge growth specifically high concentration of nitrite and ammonium ions in monsoon and low during pre- and post-monsoon support sponge growth (Dittmar and Lara, - -3 2001; Feller, Whigham, Mckee and Lovelock, 2003). The concentration of NO3 and PO4 was lowest could be suggesting due to influence of domestic sewage and industrial effluents drained through Lyari river and high accumulation by algal communities in sediment (Feller,

Whigham, Mckee and Lovelock, 2003; Naz, Burhan, Munir and Siddiqui, 2012; Farooqui, -3 Siddiqui, Shafique and Rasheed, 2013). The phosphate solubilizing bacteria releases PO4 from soluble organic and inorganic phosphate in mangrove sediment which contributes with the rise of phosphorus pool and indicates the significant influence of microbial community on mangrove sponges (Kumar and Ramanathan, 2013).

Marine sponges obtain their nutrition mainly through heterotrophy of micro-organisms (Lesser, 2006). During study, the micro-algal communities or sponge photosymbionts (like cyanobacteria and bacillariophytes) inhabiting on L. paradoxa supplied organic energy for sponge growth. Higher concentration of photosymbionts produces maximum (average) concentration of chlorophyll content which enhances the antioxidant enzymes activity in sponges (Steindler, Beer and Ilan, 2002). Results shown distinct overview of chlorophyll in different transects, which was higher after rainfall (monsoon) that ultimately was the result of phytoplankton production in mangrove channel water and decreased with depth due to non- availability of solar radiances (Steindler, Beer and Ilan, 2002; Farooq and Siddiqui, 2011). The microalgae on sponges have high carbon content, thus dependency of organic carbon versus pigment is highly variable (Yahel, Sharp, Marie, Häse and Genin, 2003). Additionally, sponges are potential consumers of organic matter through symbiotic bacteria in mangrove sediment which responsible for support sponge growth rate. Meanwhile, organic and inorganic matter in mangrove sediment shown reciprocal inverse pattern with each other. Organic matter increases during decomposition of mangrove litter in upper sediment layer and accumulated in sub surface sediment under semi-anaerobic condition (Pawlik, McMurray and Henkel, 2007; Shafique, Siddiqui, Aziz, Shaukat and Farooqui, 2015). Some other factors were also involved on sponge growth for example predation, aerial exposure and sedimentation (Witman, Patterson and Genovese, 2004; Lesser, 2006). The distribution of L. paradoxa on pneumatophores determines the nutritional quality of channel water. The community structure contributes in nutrient fluxion and outwelling of tidal particulate inorganic and organic matter with carbon and nitrogen fixation in Sandspit mangroves which provides a balance in sedimentation and water flow to the open sea. (Dittmar and Lara, 2001; Diaz, Smith and Rützler, 2004; Shafique, Siddiqui, Aziz, Burhan and Mansoor, 2010; Shafique, Siddiqui, Aziz and Shoaib, 2013).

Chapter - 5

Phytoplankton community associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit, Karachi

Abstract

The phytoplankton community associated with marine sponges was studied seasonally at Sandspit backwater during January to December 2013. The sponge samples were collected from pneumatophores of Avicennia marina and thoroughly washed with seawater. The samples were retained in 200 ml plastic bottles and 4% formalin added as a preservative. The phytoplankton communities were sorted from the samples and their members identified using light microscopy. Twenty species, representing three classes were found and highest diversity (14 species) observed in Bacillariophyceae while four species belonged to Cyanophyceae and two to Chlorophyceae. The highest number of individuals was recorded for Bacillariophyceae (157 individuals) which indicated Nitzschia palea (13%), Surirella ovata (17%) and Pinnularia spp. (20%) were the most dominant genera whereas minimum abundance recorded for Chlorophyceae (6 individuals). Highest numerical abundance was observed in summer and lowest during winter season. Physicochemical parameters of water recorded were temperature (27-35 ± 4.6°C), salinity (35-39 ± 1.47 PSU), dissolved oxygen (0.11-3.44 ± 1.15 mgL-1) and pH (7.04-7.69 ± 0.19). The results indicated that phytoplankton diversity is greatly influenced by environmental factors. This is the first study of the phytoplankton community associated with marine sponges in Pakistan. Further study is needed to determine the communities associated with Liosina paradoxa in mangrove area and other sponge species from different regions to understand the interaction between host-sponge and its inhabitants from coastal waters of Pakistan.

5.1. Introduction

Mangrove forests create distinct ecological environment to supports highly diverse biological communities (Shafique, Siddiqui, Aziz, Burhan and Mansoor, 2010; Farooqui, Shafique, Khan, Ali, Iqbal and Siddiqui, 2012). The forest provides a nurturing and stable ground that protects its habitants from variability and harshness in environmental factors such as sunlight, temperature, salinity, tidal action and sedimentation etc. (Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton and Somerfield, 2008). The presence of submerged mangrove roots in shallow water enables marine benthic communities with diverse meiofauna, macrofauna (infauna, epifauna) and planktonic species to become embellished (Morrisey, Swales, Dittmann, Morrison, Lovelock and Beard, 2010). Among benthic communities of marine ecosystems, sponges are dominant and most diversified epibionts on mangrove roots because of the availability of suitable substrate for attachment (Rützler, Diaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000; Wulff, 2000; Diaz, Smith and Rützler, 2004; Diaz, 2012). The association of sponges with bacteria, cyanobacteria, unicellular and multicellular algae (like diatoms and dinoflagellates) and facultative anaerobes in mangroves occurs at both intra and extra-cellular levels (Althoff, Schütt, Krasko, Steffen, Batel and Müller, 1998). Marine sponge benefits from supplemental provision of photosymbiotic organisms, obtaining more than 50% of their respiratory oxygen output by photosynthetic oxygen production (Steindler, Beer and Ilan, 2002). They harbor variety of organisms including unicellular or filamentous endosymbiotic cyanobacteria and eukaryotic micro-organisms (diatoms, dinoflagellates, fungi, zoochlorellae and some cryptomonads) either inside their vacuoles or extracellularly beneath their outer surface of sponge (Haygood, Schmidt, Davidson and Faulkner, 1999). The photosynthetic species provide oxygen, shade and protection from the damaging effects of light while the other organisms, such as bacteria and facultative anaerobic symbionts, provide with nutrients (such as glycerol, nitrogen) from (Becerro and Paul, 2004; Giamate, 2007). They maintain nitrogenase activity and involve in nitrogen fixation (Carpenter and Foster, 2002).

Marine cyanobacterial as symbionts make up about as much as 40% volume of sponge tissue (Vacelet, 1975). This allows optimum light reception for photosynthetic symbionts

(Wilkinson, 1992). Common sponge specific cyanobacteria genera are Proclorococcus, Synechocystis, Oscillatoria, Phormidium and Cyanobacterium. (Carpenter and Foster, 2002; Taylor, Radax, Steger and Wagner, 2007) are reported from other part of the world. Many species of micro and pico-plankton used as food source by sponges through active suspension feeding (Savarese, Patterson, Chernykh and Fialkov, 1997).

A number of studies have been done on different aspects of backwater mangroves in Pakistan but little information available on microbial (phytoplankton) communities including diatoms and dinoflagellates (Shoaib, Burhan, Shafique, Jabeen and Siddique, 2017; Gul, Saifullah and Nawaz, 2018). Ahmed, Shafique, Burhan and Siddique, (2016) reported dominant cyanobacteria (Phormidium tenue, Oscillatoria brevis, O. subbrevis, O. limosa, O. princeps and Spirulina labyrinthiformis). Several diatom species have also been reported from coastal water (Manora channel) of Karachi (Naz, Burhan, Munir and Siddiqui, 2010; 2012; 2014). There are no data available on the phytoplankton community associated with marine sponges in Pakistan. Therefore, the goal of present study was to explore the phytoplankton community structure concomitant with marine sponge Liosina paradoxa Thiele, 1899 that attaches to pneumatophores of Avicennia marina at Sandspit backwater, Karachi coast.

5.2. Materials and methods

5.2.1. Samples collection

Marine sponge Liosina paradoxa Thiele, 1899 was collected from pneumatophores of Avicennia marina from intertidal region of Sandspit backwater, Karachi coast in 2013. The collected samples were washed thoroughly with seawater, retained (200 ml) triplicate water subsamples in screw tap polythene bottles and preserved with 4% buffered formalin solution. During collection, physicochemical parameters of channel water (temperature, salinity, pH and dissolved oxygen) were recorded. The sponge specimen was identified through morphological features (shape, texture and color and skeletal framework) illustrated in previous taxonomic literature (Hooper, 2000; Hooper and Van Soest, 2002; Morrow and Cárdenas, 2015; Jabeen, Shafique, Burhan and Siddiqui, 2018) and World Porifera Database (Van Soest, Boury-Esnault, Hooper, Rützler, de Voogd, Alvarez de Glasby, Hajdu, Pisera, Manconi, Schoenberg, Klautau, Picton, Kelly, Vacelet, Dohrmann, Díaz, Cárdenas and Carballo, 2017).

5.2.2. Laboratory assessment

For qualitative assessment of phytoplankton, the preserved samples were sieved through plankton net (45 µm mesh size) and 1 ml from of the residue put onto a glass slide, mounted with canada balsam and photographed under light microscope (Nikon Eclipse 50i, Japan). The phytoplankton species were identified using literature provided via AlgaeBase (Guiry and Guiry, 2017). Statistical analyses were conducted using PRIMER version 7.0 (Lambshead, Platt and Shaw, 1983; Clarke & Gorley, 2015), PAST version 2.13 (Hammer, Harper and Ryan, 2001) and MINITAB version 17.1.0 (2013). The complete procedure for sponge specimen identification with its taxonomic illustration has given in Part-II, Chapter 2 (page 16-26).

5.3. Results

A total of twenty species, representing 11 genera and three classes of phytoplankton (Cyanophyceae, Bacillariophyceae and Chlorophyceae) were found in association with Liosina paradoxa.

5.3.1. Morphological features a. Cyanophyceae Schaffner, 1909

Oscillatoria perornata Skuja 1949: 47 (Guiry and Guiry, 2017). Trichomes erect, with attenuated and curved apices, well constricted at cross wall. Cells broad and granular with depressed ends. Size of filament: about 65 × 5 µm (Fig. 25A).

O. princeps Vaucher ex Gomont 1892: 206, pl. VI/6: fig. 9 (Guiry and Guiry, 2017; Ahmed, Shafique, Burhan and Siddique, 2016). Trichomes straight and shorter than wide cylindrical cells attenuated at their ends. Unbranched and smooth filaments. Akinetes and heterocysts are absent. Filament size: about 55 × 15 µm (Fig. 25B).

O. tenuis Agardh ex Gomont 1892: 220, pl. VII/7: figs. 2, 3 (Guiry and Guiry, 2017). Trichomes are thin, straight, curved at ends, not capitate and not attenuated at apices. Filaments are thin, well-branched, elongated. Cells are hemispherical. The size of filament: about 55 × 02 µm (Fig. 25C).

Phormidum tenue Gomont 1892: 169, pl. IV/4: figs. 23-25 (Guiry and Guiry, 2017; Ahmed, Shafique, Burhan and Siddique, 2016). Filaments long, solitary, finely thin and coiled consisting of thin cylindrical trichomes slightly attenuated at the ends and with apical rounded cells. Cells cylindrical, longer than wide without akinetes, heterocytes and calyptras. The filament size: about 110 × 1.5 µm (Fig. 25D). b. Bacillariophyceae Haeckel, 1878

Diploneis smithii (Brébisson) Cleve, 1894: 96, pl. 5 (Guiry and Guiry, 2017). Elliptical with small central nodule and terminal nodules close to end. Furrows narrow, costae

84 indistinctly punctate, 8 to 10 in 10 µm, alternating with alveoli rows arranged in oblique lines. Size: about 25 × 18 µm (Fig. 26A).

Gyrosigma sp. Hassall, 1845 (Guiry and Guiry, 2017). Elongated and sigmoid valves with small central nodule. The ends of median line directed contrary. Central and axial areas small, not distinct. Areolae arranged in longitudinal rows. Size: about 55 × 09 µm (Fig. 26B).

Gyrosigma wansbeckii (Donkin) Cleve, 1894: 119, pl. 5 (Guiry and Guiry, 2017). Linear and slightly curved cell with tapered and oblique rounded ends. Median line is sigmoid and eccentric. Size: about 45 × 06 µm.

Halamphora coffeaeformis (Agardh) Kützing, 1844: 108, pl. 5: fig. 37 (Guiry and Guiry, 2017). Valves lanceolate, delicate, obtuse rounded ends with strong marginal longitudinal lines. Size: about 20 × 07 µm (Fig. 26C).

Halamphora proteus Gregory, 1857: 518, pl. 13: fig. 81 (Guiry and Guiry, 2017). This is elliptical, truncate, narrow and long. Acute valves with obtuse ends, inner lines are curved and nodules are distinct. The cell size is about 25 × 10 µm. The striations in inner lines are longitudinally arranged while transverse striations are fine moniliform (Fig. 26D).

Navicula sp. Bory, 1822 (Guiry and Guiry, 2017). Oblong cell with median longitudinal line. Frustules are free, valves are convex and nodules present at center. Striations arranged in circular dots. Cell size: about 10 × 1.5 µm (Fig. 26E).

Navicula derasa Grunow, 1880: 39, pl. 2, fig. 46 (Guiry and Guiry, 2017). The striations on both sides of median line separated by a linear area, which is not strongly transverse. Cell size: about 35 × 05 µm.

Nitzschia longissima (Brébisson) Ralfs in Pritchard, 1861: 783, pl. 4: fig. 23 (Guiry and Guiry, 2017). Cell is much elongated, slightly attenuated with capitate edges and valves have striations. Size: about 35 × 2.3 µm.

Nitzschia palea (Kützing) Smith, 1856: 89 (Guiry and Guiry, 2017). This is linear, valves are linear-lanceolate with acute edges. Size: about 40 × 05 µm (Fig. 26F).

Nitzschia sigma (Kützing) Smith, 1853: 39, pl. 13: fig. 108 (Guiry and Guiry, 2017). Lanceolate and linear cell with acute, blunt edges and striations of keel present in a double row. Cell size: about 65 × 07 µm (Fig. 26G).

Pinnularia sp. Ehrenberg, 1843 (Guiry and Guiry, 2017). Cell is lanceolate and ribbed with distinct costa, not aligned into striate. Frustules are free, valves are convex with median line and nodules present at center and ends. Cell size: about 40 × 12 µm (Fig. 26H).

Surirella fastuosa (Ehrenberg) Ehrenberg, 1843: 388 (Guiry and Guiry, 2017). Ovate cell with small alae, few canaliculi, turgid median line and inflated towards margin. Cell size: about 18 × 10 µm (Fig. 26I).

Surirella linearis Smith, 1853: 31, pl. 8: fig. 58 (Guiry and Guiry, 2017). Cell is ovate with distinct canaliculi. Frustules are free, valves with longitudinal median line and margins produced into linear, parallel and acuminated alae. Cell size: about 15 × 11 µm.

Surirella ovata Kützing, 1844: 62, pl. 7: figs 1-4 (Guiry and Guiry, 2017). The cell is minute, ovate with small alae and marginal canaliculi. Cell size: about 25 × 16 (Fig. 26J). c. Chlorophyceae Wille, 1884

Rhizoclonium tortuosum (Dillwyn) Kützing, 1845: 205 (Guiry and Guiry, 2017). Thalli, soft, composed of a cluster of unbranched filaments, green, cells, barrel-shaped, not constricted at nodes, rhizoids absent; chloroplast parietal. Filament length: about 150 µm, cell size: about 15 × 03 µm (Fig. 27A).

Ulothrix tenuissima Kützing, 1833: 518 (Guiry and Guiry, 2017). Cylindrical barrel- shaped cells have distinct pyrenoids which distinguished by surrounding chloroplast. Curved and unbranched filaments in old stages contains uniseriate cells with girdle- shaped parietal chloroplast. Filament length: about 110 µm, cell size: about 10 × 04 µm (Fig. 27B).

5.3.2. Seasonal diversity

The seasonal diversity of Cyanophyceae was similar during pre-monsoon and monsoon and representative of Bacillariophyceae was maximum in monsoon while Chlorophyceae in post-monsoon (Table IX). The percent species composition of diatoms was dominated by 14 species (90%) among all other communities, along with 4 species of cyanobacteria (6%) and 2 species of green algae (3%) (Fig. 28). The seasonal variation between cyanophytes, bacillariophytes and chlorophytes were observed during pre-monsoon, monsoon and post monsoon periods and overall highest density of cyanophytes (11%) observed in monsoon, bacillariophytes (94%) in pre-monsoon, whereas, chlorophytes only observed during pre- and post-monsoon seasons, among all groups bacillariophytes were most abundant during all seasons (Fig. 29).

5.3.3. Physicochemical variables

The physicochemical factors of water in mangroves shown variation with season. Temperature shown decreasing point seasonally from pre-monsoon to post monsoon period while salinity value was found minimum (35 PSU) during monsoon (Fig. 30). The concentration of pH was observed alkaline throughout study whereas, the trend line of dissolved oxygen in water indicated decrease from pre-monsoon (3.45 mgL-1) to post monsoon (0.11 mgL-1) (Fig. 30). The range of temperature was 27-35 ± 4.6°C, salinity was 35-39 ± 1.47 PSU and pH was 7.04-7.69 ± 0.19, respectively.

5.3.4. Statistical analysis

The cumulative dominance (K dominance curve) of phytoplankton community shown highest species rank during monsoon followed by pre-monsoon whereas slight lowest rank observed during post-monsoon (Fig. 31). The maximum species richness was observed 0.53 Margalef (R1) index and 0.46 Menhinick (R2) index and species diversity was 0.25 Simpson (λ) index and 0.48 Shannon-Weiner (H') index during post monsoon. The range of species evenness (J') was 0.42-0.70 in which maximum value was observed during monsoon. The maximum dominance (D) was recorded 0.89 during pre-monsoon (Table X).

Pearson correlation coefficient between phytoplankton communities associated with L. paradoxa and physicochemical variables in mangroves at Sandspit backwater depicted in Table XI. The significant positive correlation was indicated between Cyanophyceae and Bacillariophyceae. Temperature, pH and dissolved oxygen were observed significantly positive correlation with Bacillariophyceae and salinity with Chlorophyceae.

A B

C D

Figure 25. Light microscopic images of species belong to Cyanophyceae; scale = 10 µm, (A) Oscillitoria princeps, (B) O. perornata, (C) O. tenuis and (D) Phormidium tenue.

A B

C D

E F

G H

I J

Figure 26. Light microscopic images of Bacillariophyceae species, (A) Diploneis smithii (scale = 10 µm), (B) Gyrosigma sp. (scale = 10 µm), (C) Halamphora coeffoeformis (scale = 5 µm), (D) H. proteus (scale = 10 µm), (E) Navicula sp. (scale = 5 µm), (F) Nitzschia palae (scale = 10 µm), (G) N. sigma (scale = 15 µm), (H) Pinnularia sp. (scale = 10 µm), (I) Surirella fastuosa (scale = 5 µm) and (J) S. ovata (scale = 5 µm).

A-i A-ii

B-i B-ii

Figure 27. Light microscopic images of Chlorophyceae species (scale = 10 µm), (A) Rhizoclonium tortosum and (B) Ulothrix tenuissima.

3% 6%

Cyanophyceae Bacillariophyceae Chlorophyceae

90%

Figure 28. Percent composition of phytoplankton groups associated with Liosina paradoxa at Sandspit backwater, Karachi.

Cyanophyceae* Chlorophyceae* Bacillariophyceae**

8 80 7 70

6 60 5 50 4 40 3 30

% composition% 2 20 1 10 0 0 Pre-monsoon Monsoon Post-monsoon Season *Primary axis, **Secondary axis

Figure 29. Seasonal composition of phytoplankton groups associated with Liosina paradoxa at Sandspit backwater, Karachi.

Temperature Salinity pH Dissolved Oxygen

45 4.0 40 3.5 35 3.0 30

2.5

1 25 - 2.0

20 mgL 1.5

15 Concentration 10 1.0 5 0.5 0 0.0 Pre-monsoon Monsoon Post-monsoon Physicochemical factor

Figure 30. Seasonal variation in physicochemical parameters (temperature °C, salinity, PSU, pH and dissolved oxygen mg L-1) at Sandspit backwater, Karachi.

100 Pre-monsoon

Monsoon %

e Post-monsoon

c 80

m o

D 60

a l

u 40

u C

20 1 10 Species rank Figure 31. The K dominance curves (cumulative dominance) for seasonal abundance of phytoplankton community associated with L. paradoxa at Sandspit backwater, Karachi.

Table IX. The seasonal occurrence of phytoplankton species associated with Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves, Karachi.

Seasons Species Pre-monsoon Monsoon Post monsoon Cyanophyceae Oscillitoria priceps - + + O. perornata - + - O. tenuis + - - Phormidium Pepperitima + - - Bacillariophyceae Diploneis smithii + + - Gyrosigma sp. + + + Gyrosigma wansbeckii + + + Halamphora coeffoeformis + - - H. proteus + + - Navicula sp. + + + Nitzschia sp. - + + Nitzschia palae + + + N. sigma + + + N. longissima - + - Pinnularia sp. + + + Surirella fastuosa + + + S. linearis + + + S. ovata + + + Chlorophyceae Rhizoclonium tortosum + - + Ulothrix tenuissima - - + + presence of species, - absence of species

Table X. Seasonal variation in diversity indices of phytoplankton species at Sandspit backwater, Karachi.

Indices Pre-Monsoon Monsoon Post-Monsoon

Margalef (R1) 0.471 0.241 0.539

Menhinick (R2) 0.359 0.252 0.469

Simpson (λ) 0.109 0.198 0.256

Shannon-Weiner (H') 0.251 0.349 0.482

Evenness (J') 0.429 0.709 0.540

Dominance (D) 0.891 0.803 0.744

Table XI. Pearson correlation coefficient matrix between phytoplankton community and physicochemical parameters at Sandspit backwater, Karachi.

Cyanophyceae Bacillariophyceae Chlorophyceae Temperature Salinity pH

Bacillariophyceae *0.510

Chlorophyceae -0.866 -0.872

Temperature -0.075 *0.819 -0.434

Salinity -0.996 -0.430 *0.817 0.165

Ph 0.287 **0.970 -0.727 **0.934 -0.198

Dissolved Oxygen 0.172 **0.935 -0.641 **0.970 -0.081 **0.993 *= significant, **=highly significant at p value >0.05

5.4. Discussion

Mangrove sponges are usually considered to live in submerged habitat due to their sensitiveness with air and light exposure (Osinga, Tramper and Wijffels, 1999). In submerged condition, their mode of nutrition is filter feeding where they capture food particles (Steindler, Beer and Ilan, 2002). In intertidal area of Sandspit backwater, the only reported sponge species Liosina paradoxa attached with pneumatophores of Avicennia marina have shown strong association with photosynthetic symbionts and their filtering capability may interrupt during exposure at low tide (Steindler, Huchon, Avni and Ilan, 2005; Jabeen, Shafique, Burhan and Siddiqui, 2018). Photosymbionts of marine sponges, specifically cyanobacteria produce mycosporine-like amino acids which provides protection from UV light for both host and symbiont (Gröniger, Sinha, Klisch and Häder, 2000), therefore, photosymbionts are additional source of nutrition during air exposure for intertidal sponges which may related to cellular adaptation and response for desiccation (Rützler, 1995). Several phytoplankton species such as Oscillatoria princeps, Navicula sp., Nitzschia sp., Nitzschia longissima, Gyrosigma sp. and Halamphora sp. have recorded earlier from mangrove region at Sandspit backwater and coastal waters ((Naz, Burhan, Munir and Siddiqui, 2010; 2012; 2014; Ahmed, Shafique, Burhan and Siddique, 2016; Shoaib, Burhan, Shafique, Jabeen and Siddique, 2017), but this study represents the first exploration of phytoplankton community assemblage with marine sponge L. paradoxa from A. marina in Pakistan, North Arabian Sea The results shown both diatoms and cyanophytes abundance in sponge mesohyl extracellularly. Among diatoms pennate were abundant as compare to centric while in cyanobacteria, particularly Oscillatoria spp. and Phormidium tenue shown symbiotic association with sponge as they are largely temperature tolerant and may resist to high temperature than other species (Usher, 2008). They are faster overgrowing and space competitive organisms among other benthic communities within host sponge (Diaz, Thacker, Rützler and Piantoni Dietrich, 2007). + -3 Sponge provides large access to nutrient concentration (NH4 and PO4 ions) and more likely shelter from predation of zooplanktons and zoobenthos to photosymbionts that proliferate within host tissue or grow as free-living species in mangrove habitat (Furnas and Crosbie, 1999; Partensky, Hess and Vaulot, 1999). Alternately, these photosymbionts are

98 responsible for nitrogen fixation, particularly, Oscillatoria sp. fixes nitrogen by utilizing ammonia for sponges, shelters the sponge body to give protection from sunlight exposure and extreme temperature, provides high oxygen level to sponges through photorespiration and produce toxic biologically active secondary metabolites for their defensive purpose (Erwin and Thacker 2007). Thus, the autotrophic and heterotrophic mutualistic combination maximizes the benefits of photosynthetic output and acceptable environment to grow (Usher, 2008). The freshwater species of Chlorophyceae (Rhizoclonium tortuosum and Ulothrix tenuissima) in mangrove region with sponges may proliferate with subject to high nutrient availability at study site through effluents of Lyari river (Shoaib, Burhan, Shafique, Jabeen and Siddique, 2017). During monsoon, rainfall is another chief factor that influence on diversity and abundance of phytoplankton community in mangrove habitat. The excessive precipitation during this season causes rising of pH level and decreasing salinity range which ultimately creates a phytoplankton bloom in channel water (Ahmed, Shafique, Burhan and Siddique, 2016).

The diversity of phytoplankton has varied with the season which shown maximum values during monsoon. The inter-relation of phytoplankton density with season may attribute to increase in temperature during pre-monsoon with respect to low salinity and high concentration of dissolved oxygen and other nutrients in channel water. The minimum density of phytoplankton has found in post-monsoon which may attribute to low temperature. The diversity indices, particularly Shannon-Weiner (H') and Simpson (λ) with and evenness (J') have indicated maximum values for Bacillariophyceae whereas, the values of species richness (Margalef and Menhinick index) have found to be high for Cyanophyceae which clearly indicated the pollution status in mangrove channel water (Brraich and Kaur, 2015). From the above estimated results, it can be concluded that the present study is the first assessment of phytoplankton community from marine sponge L. paradoxa which is first recorded sponge species found attached with pneumatophores of A. marina at Sandspit backwater, Karachi coast, Pakistan. The dominance order in terms of phytoplankton diversity in the present study was Bacillariophyceae > Cyanophyceae > Chlorophyceae. The physicochemical factors were highly influenced on their diversity pattern. They are active bio-indicators of water quality in dense contaminated environment in Sandspit mangrove ecosystem. 99

Chapter - 6

Seasonal diversity of benthic foraminifera associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwaters, Karachi

Abstract

Seasonal diversity and distribution of seven benthic foraminifera species belonging to 6 families in association with marine sponge Liosina paradoxa attached with pneumatophores of Avicennia marina has studied in four sampling transects at Sandspit backwaters, Karachi coast during 2013. The calcareous species Ammonia beccarii and Quinqueloculina laevigata were recorded in all transects while the agglutinated species Ammotium cassis and Entzia macrescens were most dominating species throughout the study. The maximum dominance (42 individuals) was found in second transect (TR2) from exposed are during monsoon season while minimum abundance of 12 individuals recorded in sheltered transect (TR3) during post-monsoon season. The diversity indices shown variation spatially as Shannon-Weiner (H') index was maximum (1.81) in TR2,

Simpson (λ) index was high (0.21) in TR1 whereas, evenness (J') and dominance (D) was high (0.93 and 2.08, respectively) in TR3. The present exploration is first study of foraminiferal assemblage with L. paradoxa in mangrove region of Sandspit backwaters which remarkably influenced by habitat and environmental conditions.

100

6.1. Introduction

Sponges are well-adapted and dominant group which attached with submerged roots and support mangrove community composition (Wulff, 2004; Hunting, van der Geest, Krieg, van Mierlo and Van Soest, 2010). They have tendency to harbor variety of biota for example micro-organisms (bacteria, cyanobacteria, diatom and foraminifera), meso- organisms (larvae of polychaetes, nematodes, cirripedes, copepods, isopods and amphipods) and macrofauna (hydrozoan, molluscs and echinoderms) (Bieler, 2004; Henkel and Pawlik, 2005; Thakur and Muller, 2005). They are efficient suspension feeders and filters large volume of water and acts as baffles to trap suspended sediment particles from tidal water (Cleary and De Voogd, 2007). During this process, the microfauna (<1 mm) such as foraminifera are also retaining in sponge crumbs (Guilbault, Krautter, Conway and Barrie, 2006).

Foraminifers are proliferating inhabitants of mangrove benthic communities but occasionally associated with sponges (Alongi and Sasekumar, 1992; Murgese and De Deckker, 2005; Senowbari-Daryan, Rashidi and Torabi, 2010). They are either entangled extracellularly, trapped inside the sponges or intimately coiled around the surface of ostia (Bromley and Nordmann, 1971; Guilbault, Krautter, Conway and Barrie, 2006). There are number of foraminifera species associated with sponges reported from various part of the world for example, Homotrema rubra reported as associated with Cliona sp. from Bermuda (Javaux and Scott, 2003), Planulina ariminensis reported in Pheronema carpenteri from Portugal (Lutze and Thiel, 1989), Bathysiphon sp., Hyperammina sp., Rhabdammina sp. and Pelosina sp., were reported in Hexactinellida sponge (Aphrocallistes vastus and Heterochone calyx) from northeast Pacific (Cook, 2005) and in Demospongiae (Bubaris sarayi) from Mediterranean (Buhl‐ Mortensen, Vanreusel, Gooday, Levin, Priede, Buhl‐ Mortensen, Gheerardyn, King and Raes, 2010), respectively. The species Telammina sp. and Pelosina sp. has reported in association with bryozoans from Pakistan coastal waters (Gooday and Bowser, 2005; Buhl‐ Mortensen, Vanreusel, Gooday, Levin, Priede, Buhl‐ Mortensen, Gheerardyn, King and Raes, 2010).

101

Mainly, foraminifers acquainted within two groups (agglutinated and calcareous test forms) which are extremely sensitive to environmental conditions (Zachos, Röhl, Schellenberg, Sluijs, Hodell, Kelly and McCarren, 2005; Drinia, 2009). Therefore, their diversity, association and distributional pattern on mangrove substrate frequently influenced by seasonality, sedimentation, temperature, salinity, dissolved oxygen, freshwater intrusion, food availability and water turbidity (Leckie and Olson, 2003; Zachos, Röhl, Schellenberg, Sluijs, Hodell, Kelly and McCarren, 2005; Drinia, 2009; Gandhi, Jisha and Rao, 2014; Yahya, Shuib, Minhat, Ahmad and Talib, 2014).

Several studies have been undertaken on various associated fauna and flora or organism with mangrove ecosystem including microbial communities (Siddiqui, Mansoor, Zaib-un- Nisa, Hameed, Shafique, Farooq, Aziz and Saeed, 2000; Mansoor, Siddiqui, Bano and Zaib-un-Nisa, 2000; Ahmed, Shafique, Burhan and Siddique, 2016; Shoaib, Zaib-un- Nisa, Shafique, Jabeen and Siddique, 2017), but foraminifers are the neglected group and have never been focused in mangrove region of Pakistan. Earlier work regarding marine foraminifera at continental margin off Karachi coast in Pakistan (northern Arabian Sea) has been carried out by Jannink, Zachariasse and Van der Zwaan (1998), Gooday and Bowser (2005) and Schumacher, Jorissen, Dissard, Larkin and Gooday (2007). This is the first study that aimed to observe foraminiferal assemblage with sponge Liosina paradoxa and their spatial structure in-correlation with environmental conditions from mangrove ecosystem at Sandspit backwaters, Karachi.

6.2. Materials and methods

6.2.1. Study area

The entire description of studied locality and transects has given in Chapter 4, section 4.2.1 (page 48).

6.2.2. Collection and preservation

The samples collection methodology has described in Chapter 5, section 5.2.1 (page 83).

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6.2.3. Laboratory analyzation

In the laboratory, the triplicate sub-sample fractions were retained by sieving from 50 µm and 100 µm mesh size sieves and preserved in 50 ml glass bottles by diluting the dense residual material up to 50 ml with pre-filtered seawater. Samples were carefully examined under stereomicroscope (Nikon SMZ800, Japan) at 2x and 4x magnifications. Sponge species was analyzed and identified through morphological and skeletal structure (Jabeen, Shafique, Burhan and Siddiqui, 2018). The specimens of foraminifera were analyzed, and total cell counts were obtained from each 50-ml sample and photographed under light microscope (Nikon Eclipse 50i, Japan). Systematic identification of foraminifera was made through previously described taxonomic illustrations (Rao, Geetha and Shanmhugavel, 2012; Ghosh, Biswas and Barman, 2014; Jeyabal, Muralidharan, Ramasamy, Rao, Kumar and Parthasarathy, 2016; Sen, Ghosh, Khanderao, Das, Chowdhury, Sarkar, Saha and Bhadury, 2016) and World Foraminifera Database (Hayward, Le Coze and Gross, 2017) to the smallest possible taxonomic level. Statistical analysis was computed through PRIMER-E version 7.0 (Clarke and Gorley, 2015), PAST statistical software version 2.13 (Hammer, Harper and Ryan, 2001) and MINITAB version 17.1.0 (2013).

6.3. Results

A total of 7 species belonging 6 families of benthic foraminifera were found associated with marine sponge Liosina paradoxa. The marine sponge L. paradoxa Thiele, 1899 (Demospongiae, Dictyonellidae) is the first recorded species found attached with pneumatophores of Avicennia marina at Sandspit, Karachi coast (Jabeen, Shafique, Burhan and Siddiqui, 2018). On the basis of test form, four species of foraminifers (Ammonia becarii, Brizalina subspinescens, Rosalina globularis, and Quinqueloculina laevigata) were identified as calcareous and other three (Ammotium cassis, Entzia macrescens and Trochammina inflata) were agglutinated (Table XII).

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6.3.1. Morphological description

Ammonia beccarii (Linnaeus, 1758): The test form is calcareous, slightly trochospiral coiled and biconvex. Surface is smooth with fine and dense perforations on both sides. Inflated and sub-globular chambers, sutures are thick on spiral side while incised and radially curved on umbilical side. The test aperture is arch shaped with an interio- marginal, extra-umbilical opening (Hayward, Le Coze and Gross, 2018). The average cell size was 33 × 25 ± 2 µm (Fig. 32A).

Brizalina subspinescens (Cushman, 1922): The test form is calcareous, elongated (lateral side), perforated and hyaline. The test chambers are inflated, slightly coiled and biserially arranged, sutures are compressed with sub-rounded periphery. The aperture surrounded by projecting lip and ovate in shape (Hayward, Le Coze and Gross, 2018). The cell size was 45 × 18 ± 2 µm (Fig. 32B).

Rosalina globularis d'Orbigny, 1826: The test form is calcareous, smooth, densely perforated and hyaline. The distal side is convex, the umbilical or proximal side is flattened with depressed umbilical segment. About five flattened chambers are apparent in final whorl on distal side while subtriangular on proximal side. Sutures are compressed and curved on distal side and radially oblique on proximal side. The aperture is elongated slit which interio-marginally and extra-umbilically extended into umbilicus opening (Hayward, Le Coze and Gross, 2018). The mean cell size was 25 × 20 ± 2 µm (Fig. 32C).

Ammotium cassis (Parker, 1870): The test structure is agglutinated, initially plano- spirally coiled and later chambers turned to uncoil, which forming an extended uniserial form with slanting. The suture lines are well-defined (Hayward, Le Coze and Gross, 2018). The average cell size was 60 × 35 ± 4 µm (Fig. 32D).

Entzia macrescens (Brady, 1870): The test form is agglutinated, perforated, inflated trochospiral tends to plano-spiral. Chambers gradually increasing in size as they further added. Sutures radial and slightly curved while periphery is rounded. The aperture is interio-marginal equatorial slit, with supplementary openings in lower portion that

104 surrounded by a lip (Hayward, Le Coze and Gross, 2018). The cell size was 50 × 35 ± 3 µm (Fig. 32E).

Trochammina inflata (Montagu, 1808): The test is agglutinated, smooth and trochospirally coiled. The flattened chambers gradually increasing in size. The aperture is a low arch with a surrounding lip (Hayward, Le Coze and Gross, 2018). The cell size was 50 × 40 ± 2 µm (Fig. 32F).

Quinqueloculina laevigata d'Orbigny, 1839: The test is calcareous, smooth, imperforated, laterally elongated and peripherally sub-ovate. The chamber pattern is "quinqueloculine." The three chambers are exteriorly visible. The ovate aperture provided by a long and slender tooth (Hayward, Le Coze and Gross, 2018). The mean cell size was 43 × 20 ± 3 µm (Fig. 32G).

6.3.2. Seasonal distribution and diversity

The distribution of foraminifera species in four transects were observed throughout sampling period and showed high diversity in second transect (TR2) during all seasons.

Total number of foraminifera species varied between each transects; TR1 (18 ± 2.6), TR2

(42 ± 3.3), TR3 (12 ± 1.4) and TR4 (20 ± 2.0), respectively (Table XIII). In case of relative abundance, A. cassis was most dominant species (30.4 %) and major contributor in each transect, followed by E. macrescens (19.6%) and Q. laevigata (17.4%) whereas, A. beccarii and T. inflata shown lowest (6.5%) abundance (Table XII). With respect to the seasonal pattern in Pakistan, from February to May described as pre-monsoon, June to September as monsoon and October to January as post-monsoon (Latif, Ayub and Siddiqui, 2013). All species of foraminifera were also revealed seasonal variation in abundance and were dominating in monsoon as compared to pre-monsoon and post- monsoon seasons (Table XIII).

6.3.3. Physicochemical factors

The physicochemical variables (temperature, salinity, oxygen and pH) of channel water have been observed and demonstrated in Chapter 5, section 5.3.3 (page 88).

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6.3.4. Statistical analysis

Hierarchical cluster analysis of foraminifera species shown that T. inflata was dominated by 95% of foraminifera species with composed similarity levels of sub-dominant B. subspinescens, (92%) and co-dominant R. globularis (83%), A. cassis (77%), E. macrescens (74%), A. beccarii (49%) and Q. laevigata (25%) (Fig. 33). The cumulative dominance (K dominance curves) of abundance of foraminifera species in four transects of at Sandspit backwaters shown variation as TR1 followed by TR3 and TR4 were ranked high whereas, TR2 indicated low rank (Fig. 34).

Spatial variation in species diversity were also estimated through indices in four transects. Shannon-Weiner (H') index varied from 1.64 (TR1) to 1.81 (TR2). Simpson (λ) index shown minor variation in all transects (0.17 to 0.21) which recorded maximum in

TR1, whereas variation in species dominance (D) was ranged between 1.62 to 2.08 in TR2 and TR3, and evenness (J') was ranged between 0.91 to 0.93 in TR1 and TR3 (Table XIV).

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A-i A-ii B-i B-ii

C-i C-ii D-i D-ii

E F G

Figure 32. Light (LM; scale = 10 µm) and scanning electron (SEM; scale = 50 µm) microscopic images of sponges associated foraminifera from Sandspit backwater mangroves, Karachi coast. (A) Ammonia beccarii (i: Distal and ii: Proximal view), (B) Brizalina subspinescens, (i: LM and ii: SEM image), (C) Rosalina globularis (i: Distal and ii: Proximal view), (D) Ammotium cassis (i: LM and ii: SEM image), (E) Entzia macrescens, (F) Trochammina inflata and (G) Quinqueloculina laevigata.

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Figure 33. Cluster analysis of foraminifera species associated with marine sponge Liosina paradoxa at Sandspit, Karachi.

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Figure 34. The K dominance curves (cumulative dominance) for foraminifera species abundance in four transects at Sandspit backwater, Karachi.

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Table XII. Variation in species number and percent abundance of foraminifera associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Number of individuals % Class Order Family Species (Relative abundance %) Range Abundance TR1 TR2 TR3 TR4 Ammoniidae Ammonia beccarii 1 (6.7) 3 (7.5) 1 (9.0) 1 (5) 1-3 6.5 Brizalina Rotaliida Bolivinitidae subspinescens 2 (13) 6 (15) 1 (9.1) - 1-6 9.8

Globothalamea Rosalinidae Rosalina globularis - 5 (12.5) 1 (9.1) 3 (15) 1-5 9.8 Lituolidae Ammotium cassis 8 (53) 10 (25) 4 (36.3) 6 (30) 4-10 30.4 Lituolida Entzia macrescens 3 (20) 9 (22.5) 2 (18) 4 (20) 2-9 19.6 Trochamminidae Trochammina inflata 1 (6.6) 1 (2.5) - 4 (20) 1-4 6.5 Quinqueloculina Miliolida Hauerinidae Tubothalamea laevigata 3 (20) 8 (20) 3 (27.2) 2 (10) 2-8 17.4 Total ± SD 18 ± 2.6 42 ± 3.3 12 ± 1.4 20 ± 2.0

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Table XIII. The occurrence of foraminifera species associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Seasons Species Pre-monsoon Monsoon Post monsoon 1 - + - 2 + + + Ammonia beccarii 3 - + - 4 + - + 1 - + + 2 - + + Ammotium cassis 3 + - + 4 + + + 1 + + - 2 + + + Brizalina subspinescens 3 + - - 4 - - -

1 - - + 2 + + + Entzia macrescens 3 - + +

Transects 4 + + + 1 - - - 2 + + + Rosalina globularis 3 + - - 4 - + + 1 - + - 2 + - - Trochammina inflata 3 - - - 4 + - + 1 - + - 2 + - + Quinqueloculina laevigata 3 + + - 4 - + - + presence of species, - absence of species

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Table XIV. Diversity indices of foraminifera species associated with L. paradoxa at four transects of Sandspit backwater, Karachi.

Indices Index TR1 TR2 TR3 TR4

Species number N0 6 7 6 6 Shannon-Weiner H' 1.640 1.810 1.673 1.670 Simpson Λ 0.218 0.175 0.207 0.205 Evenness J' 0.915 0.930 0.934 0.932 Dominance D 1.846 1.627 2.085 1.669

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6.4. Discussion

The marine sponge Liosina paradoxa has found on pneumatophores of Avicennia marina and recorded first time from Pakistan (Jabeen, Shafique, Burhan and Siddiqui, 2018). This study is the first account to describe foraminifera species associated with L. paradoxa at Sandspit backwaters, Karachi coast. Foraminifers are common in brackish water and abundantly found in tidal salt marshes, lagoons and estuaries (Woodroffe, Horton, Larcombe and Whittaker, 2005; Gandhi, Jisha and Rao, 2014; Sen, Ghosh, Khanderao, Das, Chowdhury, Sarkar, Saha and Bhadury, 2016). In the present study, three species such as Ammonia beccarii, Trochammina inflata and Entzia macrescens are considered as the diagnostic species of salt marshes reported earlier from other regions of the world (Jones, 1987; Leckie and Olson, 2003; Woodroffe, Horton, Larcombe and Whittaker, 2005; Gandhi, Jisha and Rao, 2014; Satyanarayana, Husain, Ibrahim, Ibrahim and Dahdouh-Guebas, 2014). Some species of foraminifers with large tubular structure (such as Brizalina subspinescens), occupies on exterior surface of sponges whereas other species inhabits within sponge body (Guilbault, Krautter, Conway and Barrie, 2006; Burone, de Sousa, de Mahiques, Valente, Ciotti and Yamashita, 2011; Khare, Nigam, Mayenkar and Saraswat, 2017). The species of foraminifera like T. inflata have well- adapted by entangled firmly with sponge meshwork and deformed as flattened and depressed whorls from its umbilical side (Guilbault, Krautter, Conway and Barrie, 2006) while A. beccarii is epipelic species which either suspend and form a complex interaction between epiphytic algal mats on sponge surface or may change its habitat by penetrating inside the sponge tissues through extruding its pseudopodial network in trochospiral test (Debenay, Bénéteau, Zhang, Stouff, Geslin, Redois and Fernandez-Gonzalez, 1998). These sessile foraminifers can either be colonized with other organisms (such as bacteria and cyanobacteria mats) on sponge surface or intergrow endo-symbiotically within sponge tissues (Gooday, Levin, da Silva, Bett, Cowie, Dissard, Gage, Hughes, Jeffreys, Lamont, Larkin, Murty, Schumacher, Whitcraft, Woulds, 2009; Buhl-Mortensen, Vanreusel, Gooday, Levin, Priede, Buhl‐ Mortensen, Gheerardyn, King and Raes, 2010).

The distributional pattern of foraminifera on mangrove substrate frequently influenced by seasonality, sedimentation, temperature, salinity, dissolved oxygen, food availability and

113 water turbidity (Debenay, Guial and Parra, 2002; Leckie and Olson, 2003). Wang (1992) concluded that salinity and pH are dominant variables in estuarine foraminiferal distribution which is true for the present study as the species prefer slight alkaline (pH >7) condition (Webster, Negri, Flores, Humphrey, Soo, Botte, Vogel, and Uthicke, 2013). The presence of calcareous foraminifera in the intertidal area has been associated to pH (Debenay, Guial and Parra, 2002; Berkeley, Perry, Smithers and Horton, 2008).

Although foraminifera are abundant at low salinity and minimum oxygen conditions in backwater mangroves area and similar condition is observed in the present study (Scott and Medioli, 1978; Laut and Barbosa, 1999). For instance, A. cassis was dominant species and abundantly observed with sponge present in high tidal region or on muddy substrate (partially submerged) in all four transects at Sandspit backwaters. However, A. beccarii proliferates in sponge present at submerged area (TR1) while T. inflata and E. macrescens were observed in the marginal area of semi-exposed transect (TR4) of mangrove. The composition of high diversity of foraminifera occurred in pre-monsoon season while less diversity observed in post monsoon which may suggests the distressed environment as A. beccarii and B. subspinescens was not observed during post monsoon. Variation in diversity of foraminifera species has corresponded with monsoon season as reported earlier (Hayward and Hollis, 1994), in increasing trend of oxygen, pH and temperature whereas decreasing salinity range. These foraminifers were recorded to increase in exposed area (TR2) as compared to sub-merged areas (TR1 and TR3) in mangroves habitat.

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Chapter - 7

The community composition of meso-zooplankton associated with marine sponge Liosina paradoxa Thiele, 1899 at Sandspit backwater mangroves, Karachi

Abstract

Marine sponge Liosina paradoxa harbors one of the diversified and productive communities in mangroves of tropical and sub-tropical regions. The sponge samples were taken from four transects of Sandspit backwater in 2013. A total of 29 species (840 individuals) of six taxonomic assemblages were recorded from associated communities of L. paradoxa, included dominating community of Nematoda (41%) followed by Crustacea (38%) and Polychaeta (20%) whereas, other minor groups as Platyhelminthes (1.3%), Hemichordata (0.5%) and Rotifera (0.2%) were found in lower diversity. Among nematodes, the most abundant species was Paracanthonchus sp. (27%) distributed among all transects, followed Paracyatholaimus sp. (17%) and Eleutherolaimus inglisi (16%). Crustaceans were the second large abundant group with 317 individuals. Oithona sp. (52%) and cypris larvae of Balanus sp. (42%) found abundantly in all transects. Among polychaetes, Sphaerosyllis sp. was the most dominant species in all transects (40.7%) and the relative abundance was indicated maximum in TR2 (51 ± 4). Lehardyia sp. (Platyhelminthes) was dominating among minor groups (64.7%) and found in all transects while rotifers and hemichordates found in less number. According to diversity indices, species richness (Margalef and Menhinick) and Shannon-Weiner index was maximum during post monsoon (R1 = 0.535; R2 = 0.242; H' = 1.140, respectively), while Simpson index was highest during monsoon (λ = 0.665). Dominance and evenness was observed maximum during pre-monsoon (D = 0.374) and post monsoon (J' = 0.782), respectively.

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7.1. Introduction

Mangrove largely forms a protective environment for variety of organisms that occur in high density and determines the mangrove significance by separating its ecological structure from other estuarine and shallow-water habitats (Alongi, 2002; Manson, Loneragan, Skilleter and Phinn, 2005; Nagelkerken Blaber, Bouillon, Green, Haywood, Kirton, Meynecke, Pawlik, Penrose, Sasekumar and Somerfield, 2008). The faunal composition of mangrove ecosystem is largely diverse which includes both aquatic and terrestrial organisms (Nagelkerken, Blaber, Bouillon, Green, Haywood, Kirton, Meynecke, Pawlik, Penrose, Sasekumar and Somerfield, 2008). The sandy-muddy sediment of mangrove provides a habitat for infaunal, meiofaunal, epifaunal and epibenthic organisms. Channel water within the mangrove ecosystem mainly supports the planktonic communities and fishes (Kathiresan and Rajendran, 2005).

The filter or suspension feeding invertebrates, specifically sponges are essential organizers of epibenthic ecosystem and food web in mangrove habitat by providing potential source to intensify secondary productivity (Gili and Coma, 1998). The fauna associated with mangrove sponges is diversified with distinct symbiotic relation (Henkel and Pawlik, 2005; Huang, McClintock, Amsler and Huang, 2008). They are seemly adequate microhabitats that primarily associated with bacteria, cyanobacteria, microalgae, protozoans, nematodes, annelids and bryozoans as well as several groups of crustaceans which shows the sponge community composition (Lesser, 2006; Huang, McClintock, Amsler and Huang, 2008). Some organisms live inside the canals of sponge body as obligatory sponge-dwellers, whereas some organisms (like copepods) move freely inside and outside of the sponge (Westinga and Hoetjes, 1981). The suspension feeding capability of sponges at intertidal and subtidal habitat supports the interaction of communities and predation intensity (Menge, 2000; Schiel, 2004; Lesser, 2006). During their feeding mode, the efficient filtration of seawater provides habitat for numerous mangrove species including fishes and shrimps (Diaz, Smith and Rützler, 2004). Morphogenetically, sponge cells characterized by intercellular specificity, in which they provide two distinctive systems for xenogeneic cells. The first system inhibits cell invasion from other species while second system supports the symbiotic association.

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Significantly, most sponges live in microbial symbiosis which occur either extracellularly in their mesohyl or intracellularly in vacuoles (Sarma, Daum and Müller, 1993). The host sponge provides shelter to specific associated groups in terms of morphological and physiological modifications (Hultgren and Duffy, 2010).

Many sponge contains phototrophic symbionts that are capable to produce oxygen for sponges and alternatively photosynthates (like glucose and glycerol) which translocates to these photoautotrophic organisms through consumption of host sponge‘s metabolic carbon (Taylor, Radax, Steger and Wagner, 2007). Some nitrifying, denitrifying (Pseudo- vibrio sp.), nitrite-oxidizing (Nitrospira sp.) and ammonia-oxidizing (Nitrosomonas sp.) bacteria also associated with sponges which indicates the availability and bioaccumulation of nitrite and ammonia in host sponge tissues with estimated rate of nitrification for other mangrove benthic communities (Taylor, Radax, Steger and Wagner, 2007). Sponge filter feeding activity in providing nitrogen is evidence the uptake of denitrifying bacteria that can produce sufficient particulate organic nitrogen to support both sponge and algal symbionts (Diaz, Akob and Cary, 2004). Similarly, endosymbiotic sulfur-oxidizing and reducing bacteria play a key role in sulfur metabolism of host sponge by oxidizing and reducing sulfur compounds in mangrove ecosystem (Hoffmann, Larsen, Thiel, Rapp, Pape, Michaelis and Reitner. 2005; Hoffmann, Rapp and Reitner, 2006).

This study was aimed to report the associated communities of Liosina paradoxa Thiele, 1899 attached with pneumatophores of Avicennia marina at Sandspit backwater mangroves, Karachi coast.

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7.2. Materials and methods

7.2.1. Study area

Sandspit backwater (66°54' E, 24°49' N) has dense mangrove belt dominant by Avicennia marina covers area range about 1056 hectares, located towards southwest of Karachi coast. Site description is given in Chapter 4 (section 4.2.1, page 48).

7.2.2. Collection, preservation and systematic analyzation a. Field assessment

Sampling was made along four transects (5-7 m distance each apart) at Sandspit backwater. The live exposed sponge samples were collected, attached with pneumatophores of Avicennia marina during low tide from January to December 2013. Physicochemical parameters (temperature, salinity and pH) were measured in situ through hand-held thermometer, refractometer (Atago, Thailand) and pH meter (El- - - Metron CP-401, Poland), respectively. Dissolved oxygen and nutrients (NO2 , NO3 , + -3 NH4 and PO4 ) in water were analyzed through method described in the Chapter 4 (section 4.2.3.2a and b; page 49 and 51). The sampling procedure has described in detail in Chapter 5 (section, 5.2.1, page 83). b. Laboratory assessment

The triplicate preserved sub-samples filtered through 45 µm and 0.5 mm mesh, retained in separate vials, fixed in 85% ethanol and preserved in 4% buffered formalin solution. The specimens sorted out with micropipette, place in a petri plate and observed under stereomicroscope (at 4x to 40x magnifications). For accurate assessment, specimens transferred in to glass slide, pour a drop of water, gently covered the slide with cover glass and observed in light microscope at 40 and 60x magnifications. c. Systematic identification

Identification of rotifers was investigated through Fontaneto, de Smet and Ricci (2006a), Fontaneto, Ficetola, Ambrosini and Ricci (2006b), Wallace, Snell, Riici and Nogrady

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(2006), Fontaneto, Smet and Melone (2008) and ‗The Rotifer World Catalog‘ website (Jersabek and Leitner, 2013), polychaetes were assessed by Lattig and Martin (2009) and Amaral, Nallin, Steiner, Forroni and Gomes-Filho (2013). Nematodes were identified to species level where possible (Maqbool and Nasira, 1999; Nasira and Shahina, 2007; Nasira, Shahina, Kamran and Ali, 2010; Guilini, Bezerra, Eisendle-Flöckner, Deprez, Fonseca, Holovachov, Leduc, Miljutin, Moens, Sharma, Smol, Tchesunov, Mokievsky, Vanaverbeke, Vanreusel, Venekey and Vincx, 2017). The identification of crustaceans and other groups were evaluated through corresponding literature (Poore, Watson, de Nys, Lowry and Steinberg, 2000; Yamani, Skryabin, Gubanova, Khvorov and Prusova, 2011).

7.2.3. Statistical analysis

Diversity indices (Dominance D, Evenness J, Simpson Λ and Shannon-Wiener H') were calculated by using PAST statistical software version 2.13 (Hammer, Harper and Ryan, 2001). Pearson correlation computed by Minitab software (version 17.1.0, 2013).

7.3. Results

The present study was carried out to assess the seasonal variation in faunal diversity associated with marine sponge Liosina paradoxa at Sandspit backwater, Karachi. The estimated results of physicochemical parameters (temperature, salinity, dissolved oxygen, pH) and nutrients (nitrite, nitrate, ammonia and phosphate ions) in channel water have described in Chapter 4 (section 4.3.2 and 4.3.3, page 58). Overall, six taxonomic assemblages with the total 29 species (840 individuals) belonging to 22 families were recorded corresponding to associated communities with L. paradoxa from mangrove region.

7.3.1. Associated communities with marine sponge Liosina paradoxa Thiele, 1899 i. Polychaeta

Four species of polychaetes associated with L. paradoxa belonging to 4 families: Lopadorhynchus henseni (family Lopadorrhynchidae), Harmothoë imbricata (family

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Polynoidae), Branchiomma cingulata (family Sabellidae) and Sphaerosyllis sp. (family Syllidae) were recorded (Fig. 35). Sphaerosyllis sp. was represented maximum average relative abundance (40%) in all transects. Annually, Sphaerosyllis sp. and B. cingulata were the most dominating species distributed in all transects, while L. henseni was found in less number (12%) (Table XV). The number of individuals diversified according to transects, TR1 (41 ± 5.5), TR2 (51 ± 4), TR3 (35 ± 3.4) and TR4 (38 ± 7) and highest value recorded in second transect (Table XIX). ii. Nematoda

Diversity of nematodes were dominant with total 13 species (341 individuals) belonging to 9 families among overall faunal communities, recorded as Paracanthonchus sp., P. hawaiiensis, P. sandspitensis, Paracyatholaimus sp. (Cyatholaimidae), Desmodora sp. (Desmodoridae), Dracograllus sp. (Draconematidae), Enoplus sp. (Enoplidae), Eleutherolaimus inglisi (Linhomoeidae), Monhystera marina, Monhystrella sp. (Monhysteridae), Adoncholaimus sp. (Oncholaimidae), Halichoanolaimus balochiensis (Selachinematidae), Tricotheristus sp. (Xyalidae) (Fig. 36). The highest diversity was recorded during post monsoon (Table XVI). The individuals in relation with transect showed variation, as maximum values observed in TR1 (113 ± 7) and minimum in TR4 (56 ± 4) (Table XX). Annually, the most abundant species was Paracanthonchus sp. (27%) followed by Eleutherolaimus inglisi (17%) distributed along all transects whereas Dracograllus sp. and Tricotheristus sp. were found rarely (Table XX). iii. Crustacea

The species of Amphipoda (Urothoe sp. and Corophium sp.) and Isopoda (Sphaeroma terebrans) were found occasionally in TR1, TR2 and TR3, respectively (Table XVII). Among copepods, Oithona sp. (family Oithonidae) was observed with highest abundance (52%) in all transects followed by cypris larva of Balanus sp. (42%) (Table XXI). Larval forms (2%) observed largely during pre-monsoon season (Figs. 37 and 38).

120 iv. Others

Group rotifers, helminthes and lower chordates observed frequently (Fig. 39). Among rotifers, overall 0.2% species (Philodina roseola and P. nitida) were found in TR4 during pre-monsoon and monsoon seasons, respectively (Table XVIII). Platyhelminthes (Lehardyia sp.) has maximum abundance (1.3%) and distributed in all transects (Table XVIII). Two species of Hemichordata (Saccoglossus sp. and Rhabdopleura sp.) were recorded from TR1, TR3 and TR4 during pre-monsoon and monsoon seasons (Table XXII).

7.3.2. Seasonal diversity

Nematodes were recorded with high diversity among associated symbiotic fauna contained 13 species (41%). The species composition of other major groups of associated fauna (Fig. 40) comprises, Crustacea (38%) including species of copepods, cirripedes, isopods and amphipods, Polychaeta (20%), Platyhelminthes (1.3%), Rotifera (0.2%), and Hemichordata (0.5%). The diversity indices of associated fauna with L. paradoxa showed variation according to season. The species richness (Margalef = 0.535 and Menhinick = 0.242) and Shannon-Weiner (H′ = 1.140) were maximum during post-monsoon season, whereas Simpson index was observed highest (λ = 0.665) in monsoon season. The dominance (D = 0.374) and evenness (J' = 0.782) was high during pre-monsoon and post- monsoon seasons, respectively (Table XXIII).

Pearson correlation was analyzed between associated fauna and physicochemical parameters which shown significant positive correlation with Crustacea. Salinity indicated positive correlation with Nematoda and other minor assemblages (Table XXIV).

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A B

C D

Figure 35. Light microscopic images of Polychaeta species (scale: 20 µm), (A) Lopadorhynchus henseni, (B) Harmothoë imbricata, (C) Branchiomma cingulata and (D) Sphaerosyllis sp.

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A B

C-i C-ii

D-i D-ii

E-i E-ii

123

F-i F-ii

G-i G-ii

H I

J-i J-ii

124

K-i K-ii

L M

N O

Figure 36. Light microscopic images of Nematoda species, (A) Paracanthonchus sp. (entire, scale: 20 µm), (B) P. hawaiiensis (entire, scale: 20 µm), (C) P. hawaiiensis (i. head, scale: 5 µm; ii. tail, scale: 5 µm), (D) P. sandspitensis (i. entire, scale: 20 µm; ii. head, scale: 10 µm), (E) Paracyatholaimus sp. (i. head, scale: 5 µm; ii. tail, scale: 10 µm), (F) Desmodora sp. (i. entire and ii. tail, scale: 10 µm), (G) Dracograllus sp. (i. head and ii. tail, scale: 5 µm), (H) Enoplus sp. (entire, scale: 20 µm), (I) Eleutherolaimus inglisi (entire, scale: 20 µm), (J) E. inglisi (i. head and ii. tail, scale: 5 µm), (K) Monhystera marina (i. head and ii. tail, scale: 10 µm), (L) Monhystrella sp. (entire, scale: 5 µm), (M) Adoncholaimus sp. (entire, scale: 10 µm), (N) Halichoanolaimus balochiensis

(head and tail, scale: 10 µm) and (O) Tricotheristus sp. (head, scale: 20 µm).

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A B

C D

Figure 37. Light microscopic images of crustacean larvae (scale: 5 µm).

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A-i A-ii

B-i B-ii

C D

Figure 38. Light microscopic images of Crustacea species (scale: 20 µm), (A) Copepoda, Cyclopoidea (Oithona sp. i. male and ii. female), (B) Amphipoda, (i. Urothoe sp. and ii. Corophium sp.), (C) Isopoda (Sphaeroma terebrans) and (D) Cypris (Balanus sp.).

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A-i A-ii

B-i B-ii

C-i C-ii

Figure 39. Light microscopic images of minor phyla, (A) Rotifera, i. Philodina roseola (scale: 20 µm) and ii. P. nitida (scale = 10 µm). (B) Platyhelminthes, Lehardyia sp. (i. scale: 20 µm and ii. scale: 10 µm). (C) Hemichordata, Saccoglossus sp. (i. entire, scale: 40 µm and ii. visible notochord, scale: 20 µm).

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Table XV. The occurrence of polychaetes species associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Seasons Species Pre-monsoon Monsoon Post monsoon

1 + + +

2 + + + Lopadorhynchus henseni 3 + + +

4 + + +

1 - + +

2 + + + Harmothoë imbricate

3 + + +

4 - + -

1 + + + Transects 2 + + + Branchiomma cingulate 3 - + +

4 + + +

1 + + +

2 + + + Sphaerosyllis sp. 3 + + +

4 + + + + presence of species, - absence of species

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Table XVI. The occurrence of nematodes species associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi. Seasons Species Pre- Post Monsoon monsoon monsoon 1 + + + 2 + + + Paracanthonchus sp. 3 + + + 4 + + + 1 + + + 2 + - + P. hawaiiensis 3 - - + 4 - - + 1 - + + 2 - + - P. sandspitensis 3 - - - 4 + + - 1 + + + 2 + + + Paracyatholaimus sp. 3 - + + 4 - + +

1 - + + Transects 2 + - - Desmodora sp. 3 - - - 4 - + - 1 + - - 2 - - - Dracograllus sp. 3 - - - 4 - - - 1 - - + 2 + + - Enoplus sp. 3 - - - 4 - + + 1 + + + Eleutherolaimus inglisi 2 + + + 3 + + +

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4 + + + 1 + + + 2 + + + Monhystera marina 3 + + + 4 + + + 1 - - + 2 - - - Monhystrella sp. 3 + - + 4 - - + 1 + + + 2 + - - Adoncholaimus sp. 3 + + + 4 + + + 1 + + - 2 + + + Halichoanolaimus balochiensis 3 + + + 4 + - - 1 - - - 2 + - - Tricotheristus sp. 3 - - - 4 - - - + presence of species, - absence of species

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Table XVII. The occurrence of crustacean species associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi. Seasons Species Pre-monsoon Monsoon Post monsoon 1 + + + Cypris 2 + + + (Balanus sp.) 3 + + + 4 + + + 1 + - + 2 + + - Crab larva 3 - + - 4 - - - 1 + - + 2 - + - Nauplius larva 3 - - - 4 - - -

1 - - - 2 - + - Corophium sp. 3 - - -

Transects 4 - - - 1 + + + 2 + + + Oithona sp. 3 + + + 4 + + + 1 - - - 2 - - - Sphaeroma terebrans 3 + - + 4 - - - 1 - - + 2 - - - Urothoe sp. 3 + - - 4 - - - + presence of species, - absence of species

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Table XVIII. The occurrence of other associated groups with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Seasons Species Pre-monsoon Monsoon Post monsoon 1 - - - 2 - - - Philodina roseola 3 - - - 4 + - - 1 - - - 2 - - - P. nitida 3 - - - 4 - + -

1 - + + 2 - + - Lehardyia sp. 3 + + -

Transects 4 - + - 1 - - - 2 - - + Saccoglossus sp. 3 - - - 4 - - - 1 - - - 2 - - - Rhabdopleura sp. 3 - + - 4 + - - + presence of species, - absence of species

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Polychaeta Nematoda Crustacea Rotifera Platyhelminthes Hemichordata

38% 0.2%

1.3%

41% 20% 0.5%

Figure 40. Seasonal percent species composition of faunal communities associated with L. paradoxa at Sandspit backwater mangroves, Karachi.

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Table XIX. Variation in species number and percent abundance of polychaetes associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Number of individuals (Relative abundance %) Size % Family Species Range (µm) Abundance TR1 TR2 TR3 TR4

Lopadorrhynchidae Lopadorrhynchus henseni 05 (12.2) 08 (15.6) 04 (11.4) 03 (7.9) 105 × 20 3-8 12.1

Polynoidae Harmothoë imbricate 06 (14.6) 11 (21.5) 07 (20.0) 02 (5.2) 86 × 38 2-11 15.7

Sabellidae Branchiomma cingulate 11 (26.8) 13 (25.5) 12 (34.2) 16 (42.1) 125 × 30 1-5 31.5

Syllidae Sphaerosyllis sp. 19 (46.3) 19 (37.2) 12 (34.2) 17 (44.7) 90 × 15 11-16 40.6

Total ± SD 41 ± 5.5 51 ± 4 35 ± 3.4 38 ± 7

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Table XX. Variation in species number and percent abundance of nematodes associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Number of individuals (Relative abundance %) Family Species Size (µm) Range % Abundance TR1 TR2 TR3 TR4 Cyatholaimidae Paracanthonchus sp. 28 (24.7) 28 (26.4) 24 (36.3) 14 (25.0) 180 × 04 14-28 27.5 Cyatholaimidae P. hawaiiensis 06 (5.3) 05 (4.7) 01 (1.5) 01 (1.7) 120 × 05 1-6 3.8 Cyatholaimidae P. sandspitensis 06 (5.3) 01 (0.9) - 04 (7.1) 240 × 05 1-6 3.2 Cyatholaimidae Paracyatholaimus sp. 18 (15.9) 27 (25.4) 06 (9.0) 07 (12.5) 135 × 09 6-27 17.0 Desmodoridae Desmodora sp. 06 (5.3) 01 (0.9) 01 (1.5) 01 (1.7) 190 × 12 1-6 2.6 Draconematidae Dracograllus sp. 01 (0.8) - - - 150 × 14 0-1 0.3 Enoplidae Enoplus sp. 03 (2.6) 02 (1.8) - 03 (5.3) 210 × 10 2-3 2.3 Linhomoeidae Eleutherolaimus inglisi 14 (12.3) 20 (18.8) 11 (16.6) 11 (19.6) 620 × 08 11-20 16.4 Monhysteridae Monhystera marina 12 (10.6) 09 (8.4) 09 (13.6) 07 (12.5) 52 × 10 7-12 10.8 Monhysteridae Monhystrella sp. 03 (2.6) 01 (0.9) 02 (3.0) 02 (3.5) 40 × 03 1-3 2.3 Oncholaimidae Adoncholaimus sp. 07 (6.1) 04 (3.7) 08 (12.1) 04 (7.1) 270 × 12 4-8 6.7 Selachinematidae Halichoanolaimus balochiensis 09 (8.0) 07 (6.6) 04 (6.0) 02 (3.5) 120 × 12 2-9 6.4 Xyalidae Trichotheristus sp. - 01 (0.9) - - 1100 × 10 0-1 0.3 Total ± SD 113 ± 7.4 106 ± 9.7 66 ± 6.5 56 ± 4.1

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Table XXI. Variation in species number and percent abundance of crustaceans associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Number of individuals (Relative abundance %) Family Species Size (µm) Range % Abundance TR1 TR2 TR3 TR4

Balanidae Cypris 43 (39.4) 36 (40.0) 30 (46.8) 25 (46.3) 100 × 70 25-43 42.3 Balanidae Crab larva 02 (1.8) 03 (3.3) 01 (1.5) - 15 × 08 0-3 1.9 Balanidae Nauplius 03 (2.7) 02 (2.2) - - 22 × 19 0-3 1.6 Corophiidae Corophium sp. - 01 (1.1) - - 1200 × 65 0-1 0.3 Oithonidae Oithona sp. 60 (55.0) 48 (53.3) 30 (46.8) 29 (53.7) 115 × 22 29-60 52.6 Sphaeromatidae Sphaeroma terebrans - - 02 (3.1) - 170 × 50 0-2 0.6 Urothoidae Urothoe sp. 01 (0.9) - 01 (1.5) - 120 × 30 0-1 0.6

Total ± SD 109 ± 23 90 ± 18.7 64 ± 13.2 54 ± 12.2

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Table XXII. Variation in species number and percent abundance of other groups associated with Liosina paradoxa Thiele, 1899 distributed at four transects of Sandspit backwater mangroves, Karachi.

Number of individuals (Relative abundance %) Group Species Size (µm) Range % Abundance TR1 TR2 TR3 TR4

Rotifera Philodina roseola - - - 01 (16.6) 100 × 30 0-1 5.8

Rotifera P. nitida - - - 01 (16.6) 60 × 30 0-1 5.8

Platyhelminthes Lehardyia sp. 03 (75) 02 (100) 04 (80) 02 (33.3) 118 × 10 2-4 64.7

Hemichordata Saccoglossus sp. 01 (25) - - - 280 × 30 0-1 5.8

Hemichordata Rhabdopleura sp. - - 01 (20) 02 (33.3) 85 × 10 0-2 17.6

Total ± SD 04 ± 1.1 02 ± 0.8 05 ± 1.5 06 ± 0.7

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Table XXIII. Seasonal variation in diversity indices of associated communities of Liosina paradoxa at Sandspit backwater, Karachi.

Indices Pre-monsoon Monsoon Post monsoon

Margalef (R1) 0.530 0.533 0.535

Menhinick (R2) 0.236 0.240 0.242

Simpson (λ) 0.626 0.665 0.648

Shannon-Weiner (H') 1.077 1.133 1.140

Evenness (J') 0.734 0.776 0.782

Dominance (D) 0.374 0.335 0.352

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Table XXIV. Pearson correlation coefficient matrix between aasociated community with Liosina paradoxa and physicochemical parameters at Sandspit backwater, Karachi.

Polychaeta Nematoda Crustacea Other Temperature Salinity pH

Nematoda -0.068

Crustacea -0.812 -0.526

Other -0.705 *0.756 0.159

Temperature -0.465 -0.851 **0.894 -0.300

Salinity -0.950 0.376 *0.590 **0.891 0.165

Ph -0.118 -0.983 *0.674 -0.622 **0.934 -0.198

Dissolved Oxygen -0.234 -0.954 *0.757 -0.525 **0.970 -0.081 **0.993

*= significant, **=highly significant at p value >0.05

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7.4. Discussion

The present study has designed in the first account of association of communities with mangrove sponge L. paradoxa along four transects at Sandspit backwater, Karachi coast. Results provided a total of 6 taxonomic fauna groups (polychaetes, rotifers, helminths, nematods, copepods, cirripedes, amphipods, isopods and lower chordates) were identified.

Sponges comprise one of the rich and productive biotopes after coral reefs in tropical and sub-tropical waters (Duarte and Nalesso, 1996). Mutualism is the common factor between sponges and mangrove habitat (Rützler, Diaz, Van Soest, Zea, Smith, Alvarez and Wulff, 2000). The pneumatophores provide good food source and substratum for attachment to sponges and community associated, and nutrient cycling provides good source for their growth (Diaz, Smith and Rützler, 2004). In mangrove ecosystem, Liosina paradoxa harbors diversified communities which possibly less stressful may be due to influence of ecological factors and freshwater input from Lyari river to backwater channel at Sandspit.

Mostly juveniles and larval forms of polychaetes and nematodes were found as endobiont of L. paradoxa which suggests that they occur as sponge endosymbionts during their early life stages whereas other phases completely laid outside in the sediment or water column. Polychaetes dominance and assemblage within sponges has previously reported which presented an aggregate pattern of occurrence inside the sponge canals (Duarte and Nalesso, 1996; Klitgaard, 1998; Neves and Omena, 2003). Sphaerosyllis sp. is widespread in Indian Ocean (Red Sea, Arabian Sea, Persian Gulf and Iranian coast) and common symbiont of host sponge L. paradoxa (Neves and Omena, 2003; Lattig and Martin, 2009). Results indicated that overall nematodes are the dominant community with maximum number of species among associated fauna but due to their juvenile stages, it was difficult to identify them up to species level. In sub-tropical habitat, sediment temperature and salinity largely influence on nematodes community. Their population growth maintained around 30°C and rapid exceed during warm conditions, however their eggs are resistant and adapt encystment in extreme environmental fluctuations (Alongi, 1990). Many dominant species (Monhystera marina,

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Eleutherolaimus inglisi, Paracanthonchus sp. and Paracyatholaimus sp.) are frequent inhabitants of intertidal mangrove sediment and eurytolerant in extreme environmental fluctuations (Alongi, 1990).

Crustaceans are the second most abundant group after nematodes with less number of species. The number of individuals of Cyclopoidea (Oithona sp.) and cypris (Balanus sp.) were the highest in all transects throughout the year. Although, the adult sessile barnacles attached with pneumatophores releases large amount of eggs which produces a rich quantity of cypris larva results cirripedes to grow in the water column or either go through their metamorphosis stage inside the sponge that correspondingly attached with the pneumatophores. The percent species composition of amphipods and isopods were found in low frequency. The perspective of amphipods association with sponges invoked as predation due to their herbivorous mode of feeding and for this purpose, they feed over algae provided by the host-sponge (Poore, Watson, De Nys, Lowry and Steinberg, 2000). Rotifers (Philodina sp.) are not sponge dwellers and commonly rely on water column, thus found in less quantity. Among hemichordates, Saccoglossus sp. is significantly inhabitants of mangrove sediment while Rhabdopleura sp. is sedentary and found attached with sponges in colonial form. Mostly branched coenoecium of Rhabdopleura sp. has observed attached with L. paradoxa.

The diversity of associated communities in sponge samples indicated that it is greatly influenced by environmental conditions. A large number of species inhabiting L. paradoxa on pneumatophores found in bottom rich detritus and their distribution across different transects revealed that the community composition and association specifically dependent on habitat and locality. Furthermore, the interaction between host-sponge and its inhabitants emphasizes the insufficiency of reliable data and information and require further detailed study in Pakistan.

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PART - IV

GC-MS PROFILE OF LIOSINA PARADOXA Thiele, 1899

CHAPTER - 8: Identification of secondary metabolites from marine sponge Liosina paradoxa Thiele, 1899 in mangroves at Sandspit backwater, Karachi.

Part: IV

Chapter - 8

Identification of secondary metabolites from marine sponge Liosina paradoxa Thiele, 1899 in mangroves at Sandspit backwater, Karachi

Abstract

Medicinally important mangrove sponge is a growing field of interest for researchers these days. Their diverse range of bioactive secondary metabolites including phospholipids and fatty acids, help in the phylogenetic characterization of an organism and are considered as biogeochemical markers. Present study reports chemical profile of n-hexane extract of marine sponge Liosina paradoxa through gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) analyses. The sponge specimen was collected from mangroves of Sandspit backwater, Karachi coast. GC-FID and GC-MS analyses resulted in identification of eleven metabolites. These included a hydrocarbon (HC); n-tricosane (11), three free fatty acids (FFA); two saturated (SFA), hexadecanoic (5) and octadecanoic acids (8), and a monounsaturated fatty acid (MUFA), 9-hexadecenoic acid (4), six fatty acid methyl esters (FAME); three saturated (SFAME), tetradecanoic acid, methyl ester (1), hexadecanoic acid, methyl ester (3), and octadecanoic acid, methyl ester (7), two monounsaturated fatty acid methyl esters (MUFAME), hexadec-9Z-enoic acid, methyl ester (2) and octadec-9Z-enoic acid, methyl ester (6), a polyunsaturated fatty acid methyl ester (PUFAME), 5,8,11,14-eicosatetraenoic acid, methyl ester, (all-Z)- (9), along with a phthalic acid ester 1,2-benzenedicarboxylic acid, ditridecyl ester (10). To the best of our knowledge, this is a first report of nine, six and three chemical constituents from the newly recorded marine sponge L. paradoxa, family Dictyonellidae, and order Bubarida, respectively, from Pakistan through GC-MS analysis.

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8.1. Introduction

Marine environment is a unique biological reservoir of diverse range of flora and fauna. To date, chemical diversity and pharmacology of marine micro- and macro-algae are least explored (Plaza, Herrero, Cifuentes and Ibañez, 2009). However, a comprehensive study on mangrove habitat reflects pharmacological development in terms of discovery of different classes of new bioactive secondary metabolites (Blunt, Copp, Keyzers, Munro and Prinsep, 2012; 2015; Blunt, Carroll, Copp, Davis, Keyzers and Prinsep, 2018).

8.1.1. Marine natural products from invertebrates

Marine invertebrates, for instance, exoskeleton of crustaceans (crab, shrimp or krill) are a rich source of astaxanthin and β-carotene (Félix-Valenzuela, Higuera-Ciaparai, Goycoolea-Valencia and Argüelles‐Monal, 2001). Similarly, visceral mass of squids and gonads of sea urchin (Strongylocentrotus nudus) are reported for their polyunsaturated fatty acids (PUFAs) (Kang, Ahn, Wilkinson and Chun, 2005). Moreover, glutathione peroxidase and superoxide dismutase are extracted from soft corals (Shahbudin, Deny, Zakirun, Haziyamin, John and Taher, 2011) and defatted pyloric caeca of starfish is used to purify phospholipase A2 (Chun, Kishimura, Kanzawa, Klomklao, Nalinanon, Benjakul and Ando, 2010).

Amongst marine sessile invertebrates, sponge (Porifera) releases excessive amount of bioactive compounds which are of economic importance for pharmaceutical industries (Ajuk, Alfian, Nunuk and Ahyar, 2014; Hutagalung, Karjadidjaja, Prasasty and Mulyono, 2014). These released allelochemicals help in space competition, chemical deterrence and self-defense (Uriz, Martin and Rosell, 1992), and ecologically interact with other organisms, for instance, soft corals and algal community and cause necrotic effect (Engel and Pawlik, 2000).

8.1.2. Marine natural products from sponges

Sponges are one of the greatest source for the production of bioactive compounds as the experimentation is relatively easy. However, massive amount is required for drug

144 development which may eventually lead to species extinction (Anderson, 1995; Munro, Blunt, Dumdei, Hickford, Lill, Li, Battershill and Duckworth, 1999). Hence, sponge farming and growing is an important field to be focused in order to increase drug development research (De voogd, 2005; Erpenbeck and Van Soest, 2007). Annually, out of ~ 5000 naturally occurring metabolites, 200-300 novel compounds have derived from marine sponges (Blunt, Copp, Keyzers, Munro and Prinsep, 2013; Turk, Ambrožič, Batista, Strugar, Kosmina, Čivovič, Janussen, Kauferstein, Mebs and Sepčič, 2013). However, in some cases, genetical, biological (inter- or intraspecific symbiotic association, predation or space competition), environmental or abiotic factors (such as variation in temperature, light intensity, salinity, nutrients availability, pH or pollution) may affect and hinder the production and isolation of compounds from marine sponges. Moreover, technical problems associated with transplanting, harvesting and cultivation of massive amount of sponges may also be a source of interfering agents (Agell, Uriz, Cebrian and Martí, 2001; Turon, Martí and Uriz, 2009; Hutagalung, Karjadidjaja, Prasasty and Mulyono, 2014; Anjum, Abbas, Shah, Akhter, Batool and Hassan, 2016).

Diverse classes of bioactive chemical constituents are reported from class Demospongiae of marine sponges. These include nucleosides, amino acids, macrolides, terpenoids, porphyrins, aliphatic cyclic peroxides, phospholipid fatty acids, long chain hydrocarbons

(C23 to C34), and sterols (Barnathan, Genin, Velosaotsy, Kornprobst, Al-Lihaibi, Al- Sofyani and Nongonierma, 2003; Thakur and Müller, 2004; Thomas, Kavlekar and Loka Bharathi, 2010). Furthermore, alkaloids, terpenoids and steroids are also reported from L. paradoxa (Sapar, Noor, Soekamto and Ahmad, 2014). Comparatively, marine sponges possess structurally diversified membrane sterols, particularly cholesterol (De Rosa, Iodice, Nechev, Stefanov and Popov, 2003; Santalova, Makarieva, Gorshkova, Dmitrenok, Krasokhin and Stonik, 2004). These sterols come from the sponge food system which comprises of various microorganisms, planktons and organic detritus (De Rosa, Iodice, Nechev, Stefanov and Popov, 2003). A brief literature survey showing compounds or class of compounds isolated or identified from marine sponges is summarized in Table XXV.

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Table XXV. Compounds isolated from marine sponges.

Compound/ Source Reference Class of Compound

Microcolin A Discodermia sp. Longely, Caddigan, Harmody, Gunasekera and Gunasekera, 1991

Manoalide Luffariella variabilis De Silva and Scheuer, 1980

Latrunculin S Fasciospongia rimosa Jun-ichi, Higa, Bernardinelli and Jefford, 1996

5α,8α-Epidioxy-24(S)- Biemna triraphis Ajuk, Alfian, Nunuk and Ahyar, ethylcholest-6-en-3β-ol 2014

Heolysin Tetchya lincurum Boobathy, Soundarapandian, Subasri, Vembu and Gunasundari, 2009

Suberitin Suberites douncula Hutagalung, Victor, Karjadidjaja, Prasasty, and Mulyono, 2014

1,5-Diazacyclohenicosane Mycale sp. Coello, Martín and Reyes, 2009

Halichondrin B Halichondria okadai Hirata and Uemura, 1986

Ara-A and Ara-C Cryptotethya cripta Sipkema, Franssen, Osinga, Tramper and Wijffels, 2005

Hydroxylated hepta-, octa- Sarcotragus spinosulus Abed, Legrave, Dufies, Robert, and Guérineau, Vacelet, Auberger, nonaprenylhydroquinones Amade and Mehiri, 2011

Peptides Jaspis sp. Ebada, Wray, de Voogd, Deng, Lin and Proksch, 2009

Terpenoids, alkaloids and Liosina paradoxa, Niphates Ajuk, Alfian, Nunuk and Ahyar, steroids sp. and Haliclona (Reniera) 2014 fascigera

8.1.3. Bioactivity of marine sponges

More or less all demosponges exhibit various similar bioactivities, however, temporal, seasonal and ecological deviations detected for the same species and communities in different habitat, may be a source of minor variations (Garson, 2001; Turon, Martí and Uriz, 2009). Some sponge micro-organisms (bacteria and fungi) are potential

146 endosymbionts (Koziol, Borojevic, Steffen and Müller, 1998). Hence, chemical synthesis and production of bioactive compounds from marine sponges may be similar to associated micro-organisms (Müller, Böhm, Batel, De Rosa, Tommonaro, Müller and Schröder, 2000).

Sponges accumulate microalgae through filter feeding and utilize their compounds for defensive purpose. Several anti-feedant, repellent, pesticidal or insecticidal compounds, earlier reported from marine algae, are also identified in marine sponges. (De Rosa, Iodice, Nechev, Stefanov and Popov, 2003). Other bioactive compounds from marine sponge associated micro-organisms showed remarkable chemical diversity and their antibacterial, anti-fungal, anti-viral, anti-inflammatory, cytotoxic and immunosuppressive activities are known (Anjum, Abbas, Shah, Akhter, Batool and Hassan, 2016). Moreover, isolated natural products from marine sponges are also investigated for their cytotoxic, anti-microbial and anti-inflammatory potential (Abdelgawwad, 2004). For instance, alkaloid dercitin from Dercitus sp. exhibited cytotoxic and anti-tumor activities (Gunawardana, Kohmoto, Gunasekera, McConnell and Koehn, 1988). Several sponge species (Halichondria sp., Geodia gibbes and Agelas wiedenmeyeri) extracts promote growth of other sponge species, however, a few (Dysidea etheria, Amphemidon compressa, Phorbas amaranthus, Aplysina cauliformis, Aplysilla longispina and Ectyoplasia ferox) are found to be growth inhibitors (Engel and Pawlik, 2000). Similarly, Aeroplysinin and kuanoniamine C and D from Aplysina aerophoba and Oceanapia sp. protect against predation (Ebel, Brenzinger, Kunze, Gross and Proksch, 1997; Schupp, Eder, Paul and Proksch, 1999; Sjögren, Johnson, Hedner, Dahlström, Göransson, Shirani, Bergman, Jonsson and Bohlin, 2006).

8.1.4. Identification of secondary metabolites from marine sponges through GC- MS

Gas chromatography (GC) is an analytical technique, frequently used for the quantification and identification of compounds in a mixture. Like other chromatographic techniques, it also requires mobile (inert gases as helium, nitrogen or argon) and stationary phases (coated in small diameter capillary column) (Pavia, Lampman and Kriz,

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1996). Separation of a compound depends on its different interactive strength with the stationary phase, hence, longer interaction results with larger retention time (Pavia, Lampman and Kriz, 1996). Moreover, polarity of compound and stationary phase, temperature and length of the column, flow rate of carrier gas, and the quantity of injected material, all affect component separation (Pavia, Lampman and Kriz, 1996).

Mass spectrometer (MS) is a common and good combination with gas chromatograph instrument. This combination helps in compound identification on the basis of its fragmentation pattern (Goyal and Agarwal, 2017). Usually the highest peak in mass spectrum shows the molecular ion [M]+ peak. High-energy bombarding electrons, not only convert a molecule into a molecular ion [M]+, but the energy absorbed during electronic collision may break the molecule into its characteristic fragments too (Pavia, Lampman and Kriz, 1996). Comparatively, the retention time of GC, infrared (IR), ultraviolet (UV) and nuclear magnetic resonance (NMR) spectra are less reliable than mass spectra (Goyal and Agarwal, 2017). Other GC detectors include flame ionization detector (FID), thermal conductivity detector (TCD) and electron capture detector (ECD) (Pavia, Lampman and Kriz, 1996; Skoog, Holler and Crouch, 2017).

Several Demospongiae species of marine sponges from different regions of the Indian Ocean have already been investigated for their chemical profile through GC-MS analysis (Table XXVI). Moreover, secondary metabolites identified from L. paradoxa through GC-MS studies are summarized in Table XXVII.

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Table XXVI. GC-MS profiling of marine sponges (Demospongiae) from different regions of Indian Ocean.

Order Family Species Region Reference

Axinellida Heteroxyidae Myrmekioderma granulate Orissa coast Mishra et al., 2009 Acanthella cavernosa Orissa coast Si et al., 2001

Bubarida Dictyonellidae A. dendyi Orissa coast Si et al., 2001 A. elongate Orissa coast Si et al., 2001

Liosina paradoxa Andaman Islands Patro, 2012

Spheciospongia inconstans Orissa coast Si et al., 2001 Clionaida Clionaidae S. vagabunda Orissa coast Si et al., 2001 Callyspongiidae Callyspongia sp. Orissa coast Si et al., 2001

Chalinula saudiensis Red Sea Velosaotsy et al., 2004 Chalinidae Haplosclerida Haliclona sp. Orissa coast Mishra and Sree, 2011 Petrosia spheroida Indian Ocean Gauvin et al., 1998 Petrosiidae Xetospongia testudinaria Orissa coast Si et al., 2001

Scopalinidae Stylissa carteri Red Sea Velosaotsy et al., 2004

Iotrochota baculifera Toticorin coast Muralidhar et al., 2003 Poecilosclerida Iotrochotidae I. purpurea Indonesia Ibrahim et al., 2009

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Table XXVII. Chemical constituents identified from L. paradoxa through GC-MS analysis Compounds* Piperidine 1-methyl Hexadecanoic acid 3-hydroxyl methyl ester 3-Isopropenyl-5-(4-methoxyphenyl) [1, 2, 4] Oxadiazole 4-Methyl-1H-pyranolo [4,3- C] pyridine Pentacosanoic acid methyl ester 9-Octadecanoic acid methyl ester Levulinamide 2- (methylamino)- 4- piperidinoquinoline- carbonitrile (E) 6-(2- methylbutyl)- 2, 5- dimethyl- 3- (3- methyl- 1- hydroxyl butyl) pyrazine 1,2- Benzenedicarboxylic acid bis (2- ethylhexyl) ester N-[2-[4-(benzyloxy)-3-methoxyphenyl] ethyl]-5-[(3-(benzyloxy)-4- methoxyphenyl)methylidene]-2-pyrrolidinone 6,7-dimethoxy-1-(2-amino-phenyl)-3, 4-dihydroisoquinoline *Patro, 2012

8.1.5. Natural products from marine sponges of Pakistan

In Pakistan, marine sponges are collected from Sunhari beach, Karachi. Qualitative biochemical analysis of marine sponges resulted in identification of bioactive metabolites such as flavonoids, alkaloids, phenols, saponins, etc. (Senevirathne, Kim, Siriwardhana, Ha, Lee and Jeon, 2006; Govinden-Soulange, Marie, Kauroo, Beesoo and Ramanjooloo, 2014; Nazim, Sherwani, Khan, Kausar and Rizvi, 2014). Similarly, another group of workers investigated antibacterial activity and minimum inhibitory concentration (MIC) of isolated compounds through microdilution method on gram-positive (Corynebacterium diptheriae, C. hofmanii, C. xerosis and Mycobacterium smegmatis) and gram-negative bacteria (Klebsiella pneumonia, Acinetobacter baumanii, Serratia marcesens and Vibrio cholera) (Nazim, Sherwani, Khan, Kausar and Rizvi, 2014).

Conclusively, so far chemical and pharmacological diversity of marine sponges of Pakistan are least explored. Accordingly, the purpose of present study is to identify natural bioactive compounds exploiting GC-MS from marine sponge L. paradoxa collected from Sandspit backwater of Karachi coast.

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8.2. Materials and methods

8.2.1. Sample collection

Fresh samples of marine sponge L. paradoxa Thiele, 1899 were collected twice during low tide from the pneumatophores of Avicennia marina at Sandspit, Karachi coast in March and May, 2015. The samples were washed thoroughly with seawater to remove all sediment and debris, and blot dried. The species was identified by the standard method of classification illustrated by Hooper and Van Soest (2002); based on morphological characters (outer and inner cellular structure and skeletal framework) and ecological conditions. The complete procedure for sponge specimen identification with its taxonomic illustration has already been discussed (vide Part-II, Chapter 2, page 16-26).

8.2.2. Extraction and identification of compounds from L. paradoxa

Sampling was done twice; first in the month of March (MRLP, 1.3 kg) and the second (MYLP, 1.25 kg) in May 2015. Both pre-weighed sponge collections; MRLP and MYLP were ground in mortar and pestle and treated differently with several combinations of organic solvents so as to get maximum volatiles (Schemes A and B; page 153 and 154, respectively).

MRLP was divided into two parts; A (800 g) and B (500 g). This unequal division of MRLP was adopted so as to get maximum extract in different polarity solvents; more sample for non-polar and less for moderately polar solvent systems. Both sample A and B were soaked thrice in 1.5 L n-hexane:ethyl acetate (7:3) and dicholoromethane: methanol (2:1), respectively, for a month. The three collections of both extracts were filtered and evaporated in vacuo to get residues material 1 (3.18 g) and material 2 (2.25 g).

Material 1 was soaked thrice for 3 days in 1.5 L n-hexane and filtered to get n-hexane soluble (HS) and insoluble (HIS) portions. The three HS obtained were combined and evaporated in vacuo to get residue a (0.64 g). HIS was treated thrice with 1.5 L ethyl acetate for 3 days and filtered. This yielded ethyl acetate soluble (EAS) and insoluble

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(EAI) portions. The three EAS were combined and evaporated in vacuo to furnish residue b (0.51 g). Similarly, EAI was treated thrice with 1.5 L dichloromethane for 3 days and filtered to give dichloromethane soluble (DCMS) and insoluble (DCMI) parts. The three DCMS fractions were combined and evaporated in vacuo to yield residue c (1.56 g). Lastly, DCMI was treated thrice with 70% methanol for 3 days and filtered. This provided methanol soluble (MS) and marc. The three MS were combined and evaporated in vacuo to give residue d (0.14 g). Marc was discarded. Similar protocol was repeated with material 2 and 3 (Scheme A, page 153).

8.2.2.1. Pigments removal from extracted material

It is not possible to completely remove chlorophyll pigments from crude extract, hence, a trace amount of these are retained in a sample. However, solid-phase extraction (SPE), liquid-liquid extraction (LLE) and gel filtration are used for sample purification purpose. Moreover, dual-column solid-phase extraction prevents major loss of crude sample (Ibañez, Elena, Herrero, Mendiola, and Castro-Puyana, 2012; Kool and Niessen, 2015).

In order to remove pigments, residue a, aa, and aaa were combined to afford residue A (0.66 g). Residue A was re-dissolved in n-hexane and treated with 80% aqueous acetone. The two layers were partitioned through liquid-liquid extraction (LLE) into n-hexane soluble (HL) and acetone soluble layer. HL was dried with Na2SO4, filtered and evaporated in vacuo to give HSG. Acetone layer was discarded. Similarly, residue b, bb, and bbb were combined to yield residue B (0.57 g). Residue B was separately treated with a few mL n-hexane at 60°C for 10-15 min on water bath (sample flask tightly covered to avoid moisture) and decanted. This was repeated thrice and the three layers obtained were combined and washed with aqueous acetone as described for residue A (vide supra). This procedure resulted with EASG (Scheme B, page 154). HSG and EASG were submitted for GC/GC-MS analyses.

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Liosina paradoxa Liosina paradoxa Liosina pedunculata Liosina pedunculata (MRLP,1.3 kg) (MYLP,1.25 kg) Material 3

A (800 g) B 500 g 1) Hexane:EtOAc, 7:3 (1.5 L x 3, 30 days) 1) DCM:MeOH, 2:1 (1.5 L x 3, 30 days) 2) Filtered 2) Filtered 3) Evap. in vacuo 3) Evap. in vacuo ResidueResidue ((3.18 g) g) ResidueResidue (2.25( g) g) MaterialMaterial 11 MaterialMaterial 22 1) n-Hexane (1.5 L x 3, 3 days) 2) Filtered Residue aa Residue aaa n-Hexane soluble (HS) n-Hexane insoluble (HIS) Residue bb Residue bbb Evap. in vacuo 1) Ethyl acetate (1.5 L x 3, 3 days) 2) Filtered Residue cc Residue ccc ResidueResidu ea a(0.64 ( g) g) Residue dd Ethyl acetate soluble (EAS) Ethyl acetate insoluble (EAI) Residue ddd 1) Dichloromethane (1.5 L x 3, 3 days) Evap. in vacuo 2) Filtered

ResidueResid bue (0.51 b ( gg)) Dichloromethane soluble (DCMS) Dichloromethane insoluble (DCMI) Evap. in vacuo 1) 70% Methanol (1.5 L x 3, 3 days) 2) Filtered ResidueResid cu e(1.56 c ( gg)) Methanol soluble (MS) Marc discarded Evap. in vacuo

ResidueResidue d ((0.14 g) g) Scheme A: Extraction and fractionation of marine sponge LL.. p paradoxaeduncula ta

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Material 1 Material 2 Material 3

Residue a Residue aa Residue aaa Residue b Residue bb Residue bbb Residue c Residue cc Residue ccc Residue d Residue dd Residue ddd

a + aa + aaa =Residue A ((0.66 g) g) b + bb + bbb =Residue B (( 0.57 g)g) c + cc + ccc =Residue C (( 1.61 g) g ) d + dd + ddd =Residue D ( ( 0.24 g)g)

Pigment removal Residue A ( (0.66 g)g) Hexane + 80% Aq. Acetone (1:1)

Hexane layer (HL) Aqueous layer 1. Dried with Na2SO4 2. Filtered 3. Evap. in vacuo Discarded HSG GC/GC-MS analysis

Residue B (( 0.57 g) g ) 1. Hexane (few mL) 2. in water bath (60 oC, 10-15 min) 3. Decanted

Hexane layer Residue 1. Hexane (few mL) 2. Decanted

Hexane layer Residue Washed with aq. acetone 1. Hexane (few mL) (as described for hexane 2. Decanted extract, vide supra) Hexane layer Residue

EASG Analyzed for GC/GC-MS analysis pigments

Scheme B: Pigment removal from LL.. p eparadoxadunculat a extract

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8.2.2.2. Gas chromatography-Mass spectrometry (GC-MS)

Initial chemical profiling of n-hexane (HSG and EASG) extract of L. paradoxa was performed using gas chromatography-flame ionization detection (GC-FID). It was installed on Shimadzu GC-17A (Shimadzu Corp., Kyoto, Japan) and equipped with SPB- 5® capillary column with the dimensions of 30 m length, 0.25 mm internal diameter and 0.25 μm film thickness. Flow rate of 1 mL/min was maintained for helium as a carrier gas. Initially the oven was set to 70 °C for 5 min, programmed at a rate of 5 °C/min to 260 °C, and finally set isothermally for next 20 min. Injector temperature was maintained at 280 °C with the split ratio of 1:30. GC-MS analysis of the sample was performed on gas chromatograph (Hewlett-Packard 5890, USA) coupled to mass spectrometer (Jeol JMS HX-110, Japan). The capillary column HP-5® was of 25 m length, 0.22 mm internal diameter, and 0.25 μm film thickness. The temperature and voltage of electron ionization source were fixed at 270 °C and 70 eV, respectively. Other parameters were the same as described for GC-FID (vide supra). The obtained retention times through GC were converted into constant retention indices through Kováts Retention Index and identification of respective peak was carried out by comparing the obtained retention indices with available retention indices in NIST (2005) library.

8.2.2.3. Gas chromatographic electron impact mass spectral (GC-EIMS) data of identified chemical constituents from L. paradoxa

Mass fragmentation pattern with relative abundance (%) of the identified chemical constituents (vide infra) and their details are summarized in Table-XXVIII. Moreover, the same (fragmentation pattern with %) is given for some of the unidentified constituents in section 8.2.2.4 (page 156). The mass spectra of identified chemical constituents are given in Appendix-IV (page 227).

+ n-Tetradecanoic acid, methyl ester (1): RT, 32.49; m/z (%): 242 (2) [M ; C15H30O2], 199 (10), 157 (3), 143 (18), 101 (4), 74 (100), 43 (39).

+ Hexadec-9Z-enoic acid, methyl ester (2): RT, 36.25; m/z: 268 (2) [M ; C17H32O2], 236 (4), 194 (6), 152 (6), 123 (7), 97 (38), 69 (67), 55 (100).

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+ n-Hexadecanoic acid, methyl ester (3): RT, 36.71; m/z: 270 (2) [M ; C17H34O2], 227 (10), 171 (3), 143 (16), 97 (8), 74 (100), 43 (42).

+ 9-Hexadecenoic acid (4): RT, 37.01; m/z: 254 (2), [M ; C16H30O2], 208 (15), 207 (50), 133 (10), 97 (32), 69 (67), 55 (100).

+ n-Hexadecanoic acid (5): RT, 37.40; m/z: 256 (10) [M ; C16H32O2], 213 (15), 185 (12), 157 (15), 129 (34), 97 (22), 73 (87), 43 (100).

+ Octadec-9Z-enoic acid, methyl ester (6): RT, 40.03; m/z: 296 (2), [M ; C19H36O2], 264 (11), 222 (5), 166 (6), 123 (15), 97 (42), 69 (60), 55 (100).

+ n-Octadecanoic acid, methyl ester (7): RT, 40.53; m/z: 298 (5) [M ; C19H38O2], 255 (10), 199 (10), 143 (19), 97 (9), 74 (100), 43 (48).

+ n-Octadecanoic acid (8): RT, 41.87; m/z: 284 (2) [M ; C18H36O2], 281 (17), 208 (25), 207 (100), 177 (10), 133 (16), 96 (29), 71 (59), 57 (90).

5,8,11,14-Eicosatetraenoic acid, methyl ester, (all -Z)- (9): RT, 45.99; m/z: 318 (3) + [M ; C21H34O2], 281 (18), 267 (4), 180 (5), 159 (7), 131 (19), 93 (46), 79 (100), 41 (62).

1,2-Benzenedicarboxylic acid, ditridecyl ester (10): RT, 47.28; m/z: 530 (2) [M+;

C34H58O4], 472 (1), 405 (1), 341 (2), 309 (2), 281 (8), 208 (12), 207 (68), 167 (41), 149 (100), 113 (12), 71 (37), 57 (55).

+ n-Tricosane (11): RT, 48.39; m/z: 324 (5), [M ; C23H48], 281 (19), 208 (25), 207 (100), 177 (8), 133 (17), 96 (24), 71 (60), 57 (89).

8.2.2.4. GC-EIMS data of unidentified (UI) chemical constituents from L. paradoxa

UI-1: 281 (9), 265 (4), 208 (22), 207 (100), 177 (8), 133 (17), 96 (28), 83 (59), 55 (63).

UI-2: 296 (3), 281 (12), 249 (2), 208 (21), 207 (83), 177 (4), 133 (7), 97 (22), 71 (68), 57 (100).

UI-3: 341 (3), 319 (2), 281 (18), 265 (3), 208 (28), 207 (100), 163 (10), 133 (22), 96 (25), 79 (48), 44 (60).

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UI-4: 341 (5), 281 (22), 265 (4), 208 (26), 207 (100), 177 (10), 133 (18), 96 (20), 85 (32), 57 (47).

UI-5: 386 (12), 341 (6), 281 (23), 255 (14), 208 (28), 207 (100), 163 (20), 133 (31), 95 (32), 73 (42), 43 (85).

8.3. Results

Marine sponge Liosina paradoxa Thiele, 1899 of family Dictyonellidae (Demospongiae, Heteroscleromorpha, Bubarida), collected from pneumatophores of Avicennia marina at Sandspit, Karachi coast, is the first recorded sponge species of Pakistan from North Arabian Sea. The comprehensive systematic description of the studied sponge specimen is given in chapter 2 (vide section 2.4).

The chemical profiling of n-hexane of marine sponge L. paradoxa was performed using GC-FID and GC-MS analyses. The two sample residues (HSG, EASG) for GC/MS analysis were assessed separately and the extracted compounds were resulted similar in their respective retention times therefore, it has not separately described in order to avoid repetition. The calculated retention indices (RI) of the identified compounds were compared with the reported RI values of NIST library (2005) (Table XXIX). In total, 11 compounds were identified (Fig. 41, Table XXIX), included a hydrocarbon (HC); n- tricosane (11), three free fatty acids (FFA); two saturated (SFA), hexadecanoic (5) and octadecanoic acids (8), and a monounsaturated fatty acid (MUFA), 9-hexadecenoic acid (4), six fatty acid methyl esters (FAME); three saturated (SFAME), tetradecanoic acid, methyl ester (1), hexadecanoic acid, methyl ester (3), and octadecanoic acid, methyl ester (7), two monounsaturated fatty acid methyl esters (MUFAME), hexadec-9Z-enoic acid, methyl ester (2) and octadec-9Z-enoic acid, methyl ester (6), a polyunsaturated fatty acid methyl ester (PUFAME), 5,8,11,14-eicosatetraenoic acid, methyl ester, (all-Z)- (9), along with a phthalic acid ester 1,2-benzenedicarboxylic acid, ditridecyl ester (10).

To the best of our knowledge, this is a first report of nine, six and three chemical constituents from the newly recorded marine sponge L. paradoxa, family Dictyonellidae, and order Bubarida, respectively, from Pakistan through GC-MS analysis.

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H3C CH2 CH3 19

n-Tricosane (11), C23H48

HO n O

n =14, n-Hexadecanoic acid (palmitic acid, 5), C16H32O2

n =16, n-Octadecanoic acid (stearic acid, 8), C18H36O2

O n O

n =12, n-Tetradecanoic acid, methyl ester (myristic acid, methyl ester, 1), C15H30O2

n =14, n-Hexadecanoic acid, methyl ester (palmitic acid, methyl ester, 3), C17H34O2

n =16, n-Octadecanoic acid, methyl ester (stearic acid, methyl ester, 7), C19H38O2 O

O 11 O 11 O

1,2-Benzenedicarboxylic acid, ditridecyl ester (10), C34H58O4

HO 5 4 O

9-Hexadecenoic acid (4), C16H30O2

O n O

n=1, Hexadec-9Z-enoic acid, methyl ester (palmitoleic acid, methyl ester, 2), C17H32O2

n=3, Octadec-9Z-enoic acid, methyl ester (oleic acid, methyl ester, 6), C19H36O2 O 3 O

5,8,11,14-Eicosatetraenoic acid, methyl ester, (all-Z)- (9) C21H34O2

Figure 41. Chemical structures of identified compounds from marine sponge L. paradox

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Table XXVIII. Identified chemical constituents from marine sponge L. paradoxa through GC-MS studies.

Molecular Molecular RI RI No Identified chemical constituents RT Reference formula weight (Calculated) (NIST)

C H O 1 Myristic acid, methyl estera (n-tetradecanoic acid, methyl ester) (1) 32.49 15 30 242 1675 1706 Wu et al., 2007 2 Palmitoleic acid, methyl estera,b,c (hexadec-9Z-enoic acid, methyl C H O Pattiram et al., 2 36.25 17 32 268 1839 1879 ester) (2) 2 2011 Palmitic acid, methyl estera,b,c (n-hexadecanoic acid, methyl ester) C H O 3 36.71 17 34 270 1861 1894 Ning et al., 2008 (3) 2 C H O Da Silva et al., 4 9-Hexadecenoic acida,b (4) 37.01 16 30 254 1870 1898 2 1999 C H O 5 Palmitic acida,b (n-hexadecanoic acid) (5) 37.40 16 32 256 1890 1931 Kim et al., 2006 2 C H O Asuming et al., 6 Palmitic acida,b (n-hexadecanoic acid) (5) 38.65 16 32 256 1943 1964 2 2005 C H O 7 Oleic acid, methyl ester (octadec-9Z-enoic acid, methyl ester) (6) 40.03 19 36 296 2017 2062 Ning et al., 2008 2 C H O 8 Oleic acid, methyl ester (octadec-9Z-enoic acid, methyl ester) (6) 40.13 19 36 296 2023 2062 Ning et al., 2008 2 C H O Vedernikov et al., 9 Stearic acid, methyl estera (n-octadecanoic acid methyl ester) (7) 40.53 19 38 298 2040 2106 2 2011 1 C H O Stearic acida (n-octadecanoic acid) (8) 41.87 18 36 284 2110 2157 Wu et al., 2005 0 2 1 Arachidonic acid methyl estera,b (5,8,11,14-eicosatetraenoic acid, C H O Golovnya and 42.90 21 34 318 2155 2231 1 methyl ester, (all-Z)- (9) 2 Kuzmenko, 1977 1 Arachidonic acid methyl estera,b (5,8,11,14-eicosatetraenoic acid, C H O 45.99 21 34 318 2233 2274 Tret'yakov, 2007 2 methyl ester, (all-Z)- (9) 2 1 Arachidonic acid methyl estera,b (5,8,11,14-eicosatetraenoic acid, C H O 46.23 21 34 318 2239 2274 Tret'yakov, 2007 3 methyl ester, (all-Z)- (9) 2 1 Phthalic acid, ditridecyl ester (1,2-benzenedicarboxylic acid, C H O 47.28 34 58 530 2267 4 ditridecyl ester) (10) 4 - 1 n-Tricosanea,b,c (11) 48.39 C H 324 2293 2300 5 23 48 - (RT = Retention time, RI = Retention index) a = new from L. paradoxa, b = new from family Dictyonellidae, c = new from order Bubarida 159

8.4. Discussion

Present study reports fatty acids and fatty acid methyl esters composition of marine demosponge L. paradoxa through GC-MS analysis. This species is identified and recorded for the first time from pneumatophores of Avicennia marina at Sandspit, Karachi coast, Pakistan (Jabeen, Shafique, Burhan and Siddiqui, 2018).

Marine sponges are a rich source of structurally complex bioactive volatile compounds (such as dimethyl sulfide), the synthesis of which is metabolically and ecologically beneficial for the organism itself as biofouling agent (Pawlik, McFall and Zea, 2002). Moreover, these bioactive metabolites also help in the phylogenetic classification of an organism (Erpenbeck and Van Soest, 2007). Naturally occurring organic metabolites mainly consist of hydrocarbons and carboxylic acids. Long-chain monocarboxylic acids are commonly known as fatty acids as these are obtained from the hydrolysis of animal and vegetable fats (Bahl, 1976). These may be saturated or unsaturated and branched or unbranched (Morrison and Smith, 1964). The saturated fatty acids, (palmitic and stearic acids) and unsaturated fatty acids (oleic acid) occur as glyceryl esters in animal tissues (Bahl, 1976). Importance and natural occurrence of identified chemical constituents from marine sponge L. paradoxa is summarized in Table XXIX.

Mass fragmentation pattern of straight chain hydrocarbons, fatty acids and fatty acid methyl esters are relatively predictable. Straight chain hydrocarbons show intense molecular ion peak [M]+ with a cluster of fragment ion peaks which are 14 mass units separated from each other (Pavia, Lampman and Kriz, 1996). On the other hand, straight-chain carboxylic acids, show weak [M]+ and α-cleavage of short chain acids furnish [M-17]+ and [M-45]+ showing a loss of -OH and -COOH group, respectively. Similarly, a diagnostic peak at m/z 74 appears in the mass spectrum of straight chain methyl esters (Pavia, Lampman and Kriz, 1996).

Saturated fatty acid methyl esters of the present study (1, 3 and 7) include even numbered carbon atoms. The only monounsaturated fatty acid of the present investigation was identified as 9-hexadecenoic acid (4). Unsaturated fatty acid methyl esters of the current study include two monounsaturated fatty acid methyl esters (2 and 6) and one polyunsaturated fatty acid methyl ester (9). Conclusively, except 6 and 10, all the identified

160 compounds of the present study are being reported for the first time from L. paradoxa of Pakistan.

Most of the fatty acids and their methyl esters exhibit antimicrobial and anticancer properties. Many mangrove sponges release antifouling substances which inhibit the growth of microbial and algal community on mangrove roots. Hence, these antifouling substances develop defensive mechanism against root epibionts (Iyapparaj, Revathi, Ramasubburayan, Prakash, Anantharaman, Immanuel, Palavesam, 2013). For instance, 1, 5 and 10 exhibit antifouling effect against predatory organisms which include small crustaceans (copepods or amphipods, sponge tissue grazers), spongivorous crabs and fish (Poore, Watson, De Nys, Lowry and Steinberg, 2000; Agoramoorthy, Chandrasekaran, Venkatesalu and Hsu, 2007; Sivakumar, Jebanesan, Govindarajan and Rajasekar, 2011; Patro, 2012). Moreover, larvicidal repellent activity also inhibits the growth of epibionts, particularly, algae, fungi, bryozoans and larvae of crustaceans (Balanus sp.) on pneumatophores (Sjögren, Johnson, Hedner, Dahlström, Göransson, Shirani, Bergman, Jonsson and Bohlin, 2006). Sponge also release some odor-producing volatiles which the metabolic waste products may be produced by the digestion of endosymbiotic bacteria, microbes, phyto or zooplanktons (Pawlik, McFall and Zea, 2002; Sjögren, Johnson, Hedner, Dahlström, Göransson, Shirani, Bergman, Jonsson and Bohlin, 2006; Iyapparaj, Revathi, Ramasubburayan, Prakash, Anantharaman, Immanuel, Palavesam, 2013).

Sponges primarily feed on the planktonic cells (>5µm), usually ultraplanktons or picoplanktons through suspension feeding (Steindler, Beer and Ilan, 2002). Some associated microbes such as bacteria and cyanobacteria, invaded in sponge cells and mesohyl as endosymbionts, also synthesizes active secondary metabolites in host species and produce antimicrobial compounds (Giamate, 2007). For example, metabolites 6, 8 and 11 collectively exhibit antibacterial and antifungal effect and are helpful to consume planktons in water column. These are also the suitable agents for safe biocontrol utilization (Taylor, Radax, Steger and Wagner, 2007; Huang, McClintock, Amsler and Huang, 2008; Fernández, Fernández, Thomas and Martínez, 2013). Hence, the identified chemical constituents of the present investigation are reported beneficial agents.

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Table XXIX. Importance and natural occurrence of identified chemical constituents from marine sponge L. paradoxa.

Identified Reported medicinal Natural Reference Reference compounds importance occurrence Hydrocarbons (HC) n-Tricosane (11) Antimicrobial and Urzua and Mendoza, Amphimedon Gold, O'reilly, Watson, antioxidant 2003; Fitsiou, Tzakou, queenslandica Degnan, Degnan, Krömer Hancianu and Poiata, and Summons, 2017 2007; Güleç, Yayli, Yesilgil, Terzioglu and Calyx podatypa Carballeira, Pagán and Yayli, 2007; Yassa, Rodríguez, 1998 Masoomi, Rohani and Hadjiakhoondi, 2009 Free fatty acids (FFA)

Saturated FFA n-Hexadecanoic acid Antitumor and Harada, Yamashita, Petromica Rodríguez, Osorno, (5) cytotoxic Kurihara, Fukushi, (Chaladesma) Ramos, Duque and Zea, Kawabata and Kamei, ciocalyptoides 2010 2002; Fernández, Fernández, Thomas and Martínez, 2013 Antimicrobial and Thomas, Kavlekar and anticancer LokaBharathi, 2010 n-Octadecanoic acid Antimicrobial and Fernández, Fernández, Acanthella dendyi Si, Sree, Bapuji, Gupta (8) DNA polymerase Thomas and Martínez, A. cavernosa and Siddiqui, 2001 inhibitor 2013

Unsaturated FFA 9-Hexadecenoic acid Petromica Rodríguez, Osorno, (4) (Chaladesma) Ramos, Duque and Zea, ciocalyptoides 2010

Fatty acid esters (FAE)

Saturated FAE n-Hexadecanoic Antimicrobial and Thomas, Kavlekar and acid, methyl ester (3) anticancer LokaBharathi, 2010 n-Octadecanoic acid Acanthella dendyi Si, Sree, Bapuji, Gupta methyl ester (7) A. cavernosa and Siddiqui, 2001 n-Tetradecanoic acid Antimicrobial and Agoramoorthy, Acanthella dendyi Si, Sree, Bapuji, Gupta methyl ester (1) larvicidal Chandrasekaran, A. cavernosa and Siddiqui, 2001 Venkatesalu and Hsu, 2007; Sivakumar, Jebanesan, Govindarajan and Rajasekar, 2011

Unsaturated FAE Octadec-9Z-enoic Antibacterial Agoramoorthy, Liosina paradoxa Patro, 2012 acid methyl ester Chandrasekaran, (6) Venkatesalu and Hsu, 2007; Abou-Elela, Elnaby, Ibrahim and Okbah, 2009; 162

Identified Reported medicinal Natural Reference Reference compounds importance occurrence

Fernández, Fernández, Thomas and Martínez, 2013 Antimicrobial and Thomas, Kavlekar and anticancer LokaBharathi, 2010 Hexadec-9Z-enoic Antimicrobial Huang et al., 2010 Amphimedon Gold, O'reilly, Watson, acid methyl ester (2) queenslandica Degnan, Degnan, Krömer and Summons, 2017 Calyx podatypa Carballeira, Pagán and Rodríguez, 1998 5,8,11,14- Liboxygenase Fernández, Fernández, Petromica Rodríguez, Osorno, Eicosatetraenoic inhibitor (mainly Thomas and Martínez, (Chaladesma) Ramos, Duque and Zea, acid, methyl ester, responsible for 2013 ciocalyptoides 2010 (all-Z)- (9) cancer, arteriosclerosis and Alzheimer diseases) Anticancer and Mayer and Lehmann, antitumor 2000; Khan, Firdous, Ahmad, Fayyaz, Nadir, Rasheed and Faizi, 2016 1,2-Benzene Antifouling Patro, 2012 Liosina paradoxa Patro, 2012 dicarboxylic acid, ditridecyl ester (10)

Biogenetic correlation amongst identified chemical constituents supports their natural existence from the species. For instance, n-hexadecanoic acid (5), upon chain elongation of two carbon units through acetate pathway, may be converted into n-octadecanoic acid (8). Similarly, 5 may also be desaturated to furnish 9-hexadecenoic acid (4). Conversely, 5 and 8, both may undergo esterification to yield n-hexadecanoic acid, methyl ester (3) and n-octadecanoic acid, methyl ester (7), respectively. Moreover, metabolites 3 and 7 upon desaturation, may give hexadec-9Z-enoic acid, methyl ester (2) and octadec-9Z-enoic acid, methyl ester (6), respectively (Scheme C, page 164).

It is noteworthy that identified fatty acid composition of the present investigation may constitute phylogenetic characterization of the L. paradoxa in family Dictyonellidae of order Bubarida. Present findings are in good agreement with the sponge taxonomy, symbiotic host relationship and chemosystematics with species distribution as previously reported (Patro, 2012; Sapar, Noor, Soekamto and Ahmad, 2014). To the best of our knowledge, the chemical profile of L. paradoxa through GCMS analysis is a new report from mangroves of Sandspit backwater, Karachi coast. Further investigations are needed to explore the biological activities of identified compounds from marine sponges of Pakistan.

163

HO desaturation HO chain elongation HO 5 4 14 16 O O O 9-Hexadecenoic acid (4) n-Hexadecanoic acid (5) n-Octadecanoic acid (8)

esterification esterification

O O 14 16 O O n-Hexadecanoic acid, methyl ester (3) n-Octadecanoic acid, methyl ester (7)

desaturation desaturation

O O 5 4 5 6 O O Hexadec-9Z-enoic acid, methyl ester (2) Octadec-9Z-enoic acid, methyl ester (6)

Scheme C: Biogenetic relationship between identified fatty acids and fatty acid methyl esters of LL.. p paradoxaeduncula ta

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PART – V

CONCLUSION

Part: V

CONCLUSION

Mangrove major communities along Karachi coast and Indus delta are mainly influenced by the provision of fresh water run-off from Indus river and heavy rain fall during monsoon seasons. The potential communities of Avicennia marina resides in this region holds commercially important invertebrate and vertebrate species. Although among benthic invertebrates, sponges are the main component of mangrove ecosystem and global biodiversity of sponges have previously reviewed and developed an online database system through which Porifera species distribution from all over the world has equipped to reserve taxonomic data, but they are not appeared to produce diversity or ecological information in Pakistan and several species are appealing with proper investigation to explore taxa and ecology of sponges.

Liosina paradoxa Thiele, 1899 is the most dominant and first recorded species from mangrove ecosystem at Sandspit backwater. The species was thickly encrusting attached with the pneumatophores of A. marina. These mangrove roots provide the principal habitat for nourishment of L. paradoxa, but their distribution was not uniform. The presence of this species in mangrove habitat reveals its significance as biological indicator. In previous literature, L. paradoxa has mostly reported from different regions of Indian Ocean mainly from Andaman Island, Cebu Island in Philippines and Kapoposang Island in Indonesia but it has reported first time from Pakistan.

Other common sponge species of Indian Ocean, Callyspongia (Cladochalina) fibrosa Ridley and Dendy, 1886 and Haliclona (Soestella) hornelli (Dendy, 1916) has also first recorded and taxonomically illustrated from Churna Island and Buleji rocky ledge, respectively. The species H. (S.) hornelli has first time assigned in subgenus Soestella, whereas its characteristic features was earlier described as subgenus Reniera of genus Haliclona (Dendy, 1916; De Weerdt, 2000).

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Mangrove indicates an essential source of rich biodiversity and supports sponge community but their species composition in open reef habitat is more diverse, well- adapted and colonized than the mangrove ecosystem. The single dominant species of L. paradoxa was found abundantly and well-flourished on pneumatophores of A. marina at Sandspit throughout the year, so the measurement of its growth and abundance according to its distribution was effortless and accessible. Indus delta supplies a water flow in the form of drainage system towards Northern Arabian Sea specifically Sindh coastal regions. During monsoon season, by the result of low rainfall in Indus Delta, there has been annually reduction in freshwater runoff which effect on mangroves community. Sponges in this region are also play a role as bio-indicators. They are strongly interrelated with abiotic factors and sensitive with ecological stress. Therefore, the annual distribution of L. paradoxa at four transects of Sandspit indicated that this species could be determined as the bio-indicator in oligotrophic water, and it could also possibly be concluded about the other open reef species C. (C.) fibrosa and H. (S.) hornelli, that they are bio-indicators of pollution-free water from Churna Island and Buleji.

The intertidal mangrove region of Sandspit is characterized by extreme environmental factors such as less salinity, temperature, light exposure, high turbidity and sedimentation. Sponges in these habitats may show less growth but high abundance in lower mangrove roots. The dominant species L. paradoxa was not consistently abundant in all sites and observations showed high abundance in light exposed areas and less abundant in shady environment, whereas its growth indicated inverse pattern from abundance and found in patch forms on pneumatophores. Also, it has a life span and reproduction frequency which is not remain restricted to a single locality. The results suggested that this encrusting sponge species is highly successful competitor in mangrove community.

By comparing the measurement of growth rate of L. paradoxa at four sites of Sandspit mangroves was not same, although it has measured in similar procedure. The growth was considerably low in extreme temperatures (as in extreme high temperature during summer and low temperature during winter), but by coinciding with other growth studies, its growth was increasing as the water temperature has increased. Negative growth rate of

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L. paradoxa could be the result of extreme light exposure (well-illuminated habitat), predation (low strategy of physical and chemical defense) and space competition with algae and other sessile organisms.

The abundance estimation of L. paradoxa by area coverage on pneumatophores has varied with physicochemical factors and geomorphological changes. The trend was increased in the areas of high light intensity and low sediment exposure, also declined where the space competition with algae and predation rate was possibly maximum. Presumably, the sponge species which are viviparous in their mode of reproduction, have short life span and their post-larval forms like to move another location mostly in low tide shallow water area for survival, which may be a defensive mechanism. The mangrove roots that extended from lowest low water zone entangled over tidal channels where shallow water becomes stagnant and soft sediment substratum supports the sponge larvae for their settlement.

In addition to sponge symbiosis with other communities in mangroves, L. paradoxa formed a mutualistic relationship with microalgae and diatoms were found the most dominant community among other phytoplanktons (as cyanobacteria and chlorophytes). Most species of cyanobacteria identified such as Oscillitoria and Phormidium, that lived extracellularly on L. paradoxa and it showed total dependence on these photosynthetic metabolites. Other intracellular phototrophs which either live in sponge amoebocytes (cyanocytes) or engulfed through phagocytosis, could not be identified, but they assist in providing nitrogen by N2 fixation, glycerol by carbon fixation and defensive compounds by toxic secondary metabolites. The dinoflagellates were not found but occasionally they found in the bloom of Noctiluca scintallins during winter which is not symbiotic phototrophs with sponges. Few individuals of radiolarian of triangular shaped have also been found, but not identified during study.

The zooplanktons and zoobenthos (25 µm to 1.2 mm in size) associated with L. paradoxa were found about 0.2 – 44.4% in relative abundance. Like other sponges, L. paradoxa has not any mucous on its surface which assist the water-column community to enter in inhalant canals without difficulty which either develop a symbiotic association or

167 contribute heterotrophic food source for sponge. The agglutinated foraminiferas mostly found inside the hollow spaces inside the sponge chambers but did not penetrated into the sponge cavities. Their globe-like structure enables them to anchor on the substratum and irregularly shaped sponge skeletal framework provides a tract to stab their pseudopodia. Nematodes were the highest communities in sponge and their larvae get shelter and nutrition in the sponge chambers, thus their juvenile forms were abundantly found. In the similar way, polychaetes and crustaceans larvae were obtained from sponge body. The polychaete species Haplosyllis is common inhabitant of L. paradoxa in Indian Ocean. This species is the chief predator of L. paradoxa and showed adaptation to consume sponge tissues by strategy to avoid sponge spicules. In addition, some copepod and amphipod species also showed parasitism on sponge. On the other hand, a variety of benthic dwellers micro-crustaceans abundantly occur as associates of sponge which accumulate organic particles from concentrated water flow inside the sponge canal and also reduce oxygen level through respiration. Principally, the sponge symbionts displayed host-specificity by suitable markers of secondary metabolites. Nevertheless, it is difficult to analyze the extracted compound either related to sponge or its symbiont.

This is the first study to provide sponge systematic assessment with its communities, ecology and secondary metabolites in Pakistan. Sponge associated communities and its secondary metabolites are the key factors in determining the sponge fauna. It seems more detailed study is needed regarding sponge diversity and distribution in region.

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PART – VI

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PART – VII

APPENDIX

Part: VII

APPENDIX

I. Chemical reagents for analyzation of dissolved oxygen

1. Manganese Sulfate (MnSO4) 60 g of MnSO4 was dissolved in distilled water and volume was made up to 100 ml. 2. Potassium Iodide (KI) 60 g of KI was dissolved in distilled water and volume made up to 100 ml. Allowed it to cool at room temperature. 3. Hydrochloric acid (HCl) 28 ml of HCl (conc.) was slowly added in 50 ml of distilled water with stirrer, allowed it to cool at room temperature and its volume was made up to 100 ml.

II. Chemicals for nutrients analyzation

1. Standard nitrite solution

0.0345 g anhydrous sodium nitrite (NaNO2) was dissolved in 100 ml of distilled water and 0.5 ml of this solution to calibrate 50 ml sample. 2. Standard nitrate solution

0.1 g potassium nitrate (KNO3) was dissolved in 100 ml of distilled water and 0.1 ml of this solution was used to calibrate 50 ml sample. 3. Standard ammonia solution

0.01 g ammonium sulfate [(NH3)2SO4] was dissolved in 100 ml de-ionized water and 0.1 ml of this solution was volume made up to 50 ml ammonia-free seawater. 4. Anhydrous potassium dihydrogen phosphate

0.0816 g of anhydrous potassium dihydrogen phosphate (KH2PO4) was dissolved in 100 ml distilled water and 0.5 ml of this standard solution was used in 50 ml distilled water for calibration. 224

5. Ammonium Chloride (NH4Cl)

25 g of Ammonium Chloride (NH4Cl) was dissolved in 100 ml distilled water and 1 ml of this solution used for 50 ml sample. 6. Sulfanilamide solution 1 g of sulfanilamide dissolved in the mixture of 10 ml HCl (conc.) and 60 ml distilled water and cooled. This solution diluted to 100 ml distilled water. 7. N-(1-naphthyl)-ethylenediamine dihydrochloride 0.1 g of N-(1-naphthyl)-ethylenediamine dihydrochloride was dissolved in 100 ml distilled water and 1 ml of this solution was used for 50 ml sample. 8. Synthetic seawater 31 g of analytical grade sodium chloride (NaCl), 10 g of magnesium sulfate (MgSO4) and 0.5 g of sodium bicarbonate (NaHCO3) was dissolved in 1 liter of distilled water. 9. Sodium nitroprusside solution 0.5 g of analytical grade sodium nitroprusside was dissolved in 100 ml of de- ionized water and 2 ml of this solution was used for 50 ml sample.

10. Oxidizing reagent 10 g of sodium citrate and 1 g of sodium hydroxide was dissolved in 50 ml de- ionized water to make alkaline reagent, 10 ml of this alkaline reagent was mixed with 2.5 ml sodium hypochlorite (fresh washing bleach) and 5 ml was used for 50 ml sample. 11. Mixing reagent 1 ml ammonium molybdate, 2.5 ml diluted sulfuric acid, 1 ml abscorbic acid and 0.5 ml potassium antimonyl tartarate were mixed together and used for 50 ml sample.

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III. Chemical reagents for the assessment of total organic carbon (TOC)

1. Sulfuric acid dichromate oxidant

4.8 g of potassium dichromate (K2Cr2O7) was dissolved in 20 ml distilled water

and 500 ml sulfuric acid (H2SO4) was slowly added in this solution and volume made up to 1 liter.

2. Phosphoric acid

70 ml phosphoric acid (H2PO4) was slowly mixed with 30 ml distilled water to make 70% solution.

3. Standard glucose solution

7.5 g glucose and few crystals of mercuric chloride was dissolved in 100 ml distilled water and 10 ml of this standard solution was used by volume made up to 1 liter with distilled water.

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IV. Mass spectra of identified compounds with their retention times from marine sponge Liosina paradoxa Thiele, 1899

(x10,000) 1.0 74

0.5 43

143 596 101 157 199 0.0 242 295 353 420441 472 517 549 574 100 200 300 400 500 600 RT: 32.49; n-Tetradecanoic acid, methyl ester (1)

(x10,000) 1.0 55

69 0.5 97 597 123 152 194 236 0.0 268 315 343364 424 466 537 564 100 200 300 400 500 600 RT: 36.25; Hexadec-9Z-enoic acid, methyl ester (2)

(x10,000) 1.0 74

0.5 43

143 97 171 227 270 0.0 300 341 402 429 505 549570 100 200 300 400 500 600 RT: 36.71; n-Hexadecanoic acid, methyl ester (3)

(x10,000) 1.0 55 69 0.5 207 97 208 133 254 281 342 371 418 0.0 482505 545 589 100 200 300 400 500 600 RT: 37.01; 9-Hexadecenoic acid (4)

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(x10,000) 1.0 43 73

0.5 129 97 157 185 213 256 436 460 497 527 0.0 289 316 348 387 584 100 200 300 400 500 600 RT: 37.40; Hexadecanoic acid (5)

(x10,000) 1.0 55

69 0.5 97

123 591 166 222 264 332 0.0 296 373 414 451 501 542 565 100 200 300 400 500 RT: 40.03; Octadec-9Z-enoic acid, methyl ester (6)

(x10,000) 1.0 74

0.5 43

143 97 157 199 255 298 0.0 338 372 402 433 493 533 578 100 200 300 400 500 600 RT: 40.53; n-Octadecanoic acid, methyl ester (7)

(x10,000) 1.0 57 207

71 0.5 96 208 133 281 177 265 308 341 385 429 458 482 530 564 591 0.0 100 200 300 400 500 600 RT: 41.87; n-Octadecanoic acid (8)

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(x10,000) 1.0 79

41 0.5 93

131 159180 211 267 361 424 580 0.0 303 336 384 451 500 551 100 200 300 400 500 600 RT: 45.99; 5,8,11,14-Eicosatetraenoic acid, methyl ester, (all-Z)- (9)

(x10,000) 1.0 149

57 0.5 71 167 595 113 221 279 325 353 415 463 0.0 180 501 539 583 100 200 300 400 500 RT: 47.28; 1,2-Benzenedicarboxylic acid, ditridecyl ester (10)

(x10,000) 1.0 57 207

71 0.5 96 208 133 281 592 177 249 302 341 378 430453 494 522 563 0.0 100 200 300 400 500 600 RT: 48.39; n-Tricosane (11)

229