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 Biology and Ecology of
Pharmaceutical Marine Tunicates
 Biology and Ecology of Marine Life
Series Lead Author
Ramasamy Santhanam
Former dean, Fisheries College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University,
Thoothukudi, India

Biology and Ecology of Pharmaceutical Marine Plants

Ramasamy Santhanam, Santhanam Ramesh & Hafiz Ansar Rasul Suleria

Biology and Ecology of Pharmaceutical Marine Sponges

Ramasamy Santhanam, Santhanam Ramesh & Anbu Jeba Sunilson

Biology and Ecology of Pharmaceutical Marine Cnidarians

Ramasamy Santhanam, Santhanam Ramesh & Gubbiyappa Shivakumar

Biology and Ecology of Pharmaceutical Marine Mollusks

Ramasamy Santhanam, Manavalan Gobinath & Santhanam Ramesh

Biology and Ecology of Pharmaceutical Marine Life: Echinoderms

Ramasamy Santhanam, Santhanam Ramesh & Sheba Rani Nakka David

Biology and Ecology of Pharmaceutical Marine Tunicates

Ramasamy Santhanam & Santhanam Ramesh

Biology and Culture of Portunid Crabs of World Seas

Ramasamy Santhanam

Biology and Ecology of Edible Marine Bivalve Molluscs

Ramasamy Santhanam

Biology and Ecology of Edible Marine Gastropod Molluscs

Ramasamy Santhanam

Biology and Ecology of Venomous Marine Snails

Ramasamy Santhanam

Biology and Ecology of Venomous Stingrays

Ramasamy Santhanam

Biology and Ecology of Toxic Pufferfish

Ramasamy Santhanam

For more information, please visit the Series Page https://www.crcpress.com/Biology-and-Ecology-of-Marine-Life/book-series/

AAPBIOECOMAR
 Biology and Ecology of
Pharmaceutical Marine Tunicates

Ramasamy Santhanam
Santhanam Ramesh
CRC Press
Taylor & Francis Group
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© 2020 by Taylor & Francis Group, LLC

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Contents
Preface.......................................................................................................................................................................................... ix
Authors.......................................................................................................................................................................................... xi

Chapter 1 Introduction.............................................................................................................................................................. 1

Chapter 2 Biology and Ecology of Marine Tunicates............................................................................................................... 5

Chapter 3 Pharmaceutical Compounds and Activities of Marine Ascidians......................................................................... 11

Class: Ascidiacea
A. Order: Aplousobranchia
Family 1. Clavelinidae............................................................................................................................................ 11
Clavelina cylindrica........................................................................................................................................... 11
Clavelina lepadiformis ...................................................................................................................................... 12
Clavelina moluccensis ....................................................................................................................................... 13
Clavelina oblonga ............................................................................................................................................. 13
Clavelina picta .................................................................................................................................................. 14
Clavelina sp. ...................................................................................................................................................... 15
Pycnoclavella kottae .......................................................................................................................................... 15
Family 2. Diazonidae............................................................................................................................................. 16
Diazona angulata............................................................................................................................................... 16
Diazona formosa ............................................................................................................................................... 16
Diazona sp. .������������������������������������������������������������������������������������������������������������������������������������������������������ 17
Rhopalaea birkelandi ........................................................................................................................................ 17
Tylobranchion speciosum .................................................................................................................................. 18
Family 3. Didemnidae............................................................................................................................................ 18
Atriolum robustum ............................................................................................................................................. 18
Didemnum albopunctatum ................................................................................................................................ 19
Didemnum candidum ......................................................................................................................................... 20
Didemnum chartaceum ...................................................................................................................................... 20
Didemnum cuculliferum .................................................................................................................................... 21
Didemnum fragile .............................................................................................................................................. 22
Didemnum granulatum ...................................................................................................................................... 23
Didemnum guttatum .......................................................................................................................................... 24
Didemnum lahillei ............................................................................................................................................. 24
Didemnum molle ............................................................................................................................................... 25
Didemnum moseleyi .......................................................................................................................................... 27
Didemnum obscurum ......................................................................................................................................... 28
Didemnum psammathodes ................................................................................................................................. 29
Didemnum rubeum ............................................................................................................................................ 31
Didemnum sp. .................................................................................................................................................... 32
Diplosoma virens................................................................................................................................................ 35
Diplosoma sp...................................................................................................................................................... 36
Leptoclinides dubius .......................................................................................................................................... 36
Leptoclinides durus ........................................................................................................................................... 37
Leptoclinides kingi ............................................................................................................................................ 38
Leptoclinides sp. ................................................................................................................................................ 38
Lissoclinum badium ........................................................................................................................................... 39
Lissoclinum bistratum ....................................................................................................................................... 41
Lissoclinum fragile ............................................................................................................................................ 44
Lissoclinum mandelai ........................................................................................................................................ 44

v
vi Contents

Lissoclinum notti ............................................................................................................................................... 45

Lissoclinum patella ........................................................................................................................................... 46
Lissoclinum timorense........................................................................................................................................ 49
Polysyncraton cuculliferum................................................................................................................................ 52
Polysyncraton lithostrotum................................................................................................................................. 52
Polysyncranton trivolutum.................................................................................................................................. 53
Trididemnum cereum.......................................................................................................................................... 53
Trididemnum cyanophorum................................................................................................................................ 54
Trididemnum cyclops.......................................................................................................................................... 54
Trididemnum solidum......................................................................................................................................... 55
Family 4. Holozoidae............................................................................................................................................. 56
Distaplia nathensis............................................................................................................................................. 56
Distaplia regina.................................................................................................................................................. 57
Distaplia skoogi.................................................................................................................................................. 57
Family 5. Polycitoridae.......................................................................................................................................... 58
Cystodytes dellechiajei....................................................................................................................................... 58
Cystodytes violatinctus ...................................................................................................................................... 60
Cystodytes sp. .................................................................................................................................................... 61
Eucoelium mariae ............................................................................................................................................. 61
Eudistoma album ............................................................................................................................................... 62
Eudistoma gilboviride........................................................................................................................................ 62
Eudistoma maculosum........................................................................................................................................ 63
Eudistoma obscuratum....................................................................................................................................... 64
Eudistoma olivaceum......................................................................................................................................... 64
Eudistoma reginum............................................................................................................................................. 65
Eudistoma rigidum............................................................................................................................................. 65
Eudistoma toealensis.......................................................................................................................................... 66
Eudistoma vannamei.......................................................................................................................................... 67
Eudistoma viride................................................................................................................................................ 68
Eudistoma sp...................................................................................................................................................... 69
Polycitor adriaticus............................................................................................................................................ 72
Polycitor africanus ............................................................................................................................................ 72
Polycitor sp. ....................................................................................................................................................... 72
Family 6. Polyclinidae............................................................................................................................................ 73
Aplidiopsis confluata.......................................................................................................................................... 73
Aplidiopsis ocellatus ......................................................................................................................................... 73
Aplidium albicans............................................................................................................................................... 74
Aplidium altarium ............................................................................................................................................. 74
Aplidium amphibolum........................................................................................................................................ 75
Aplidium antillense............................................................................................................................................. 75
Aplidium caelestis ............................................................................................................................................. 76
Aplidium californicum........................................................................................................................................ 76
Aplidium conicum............................................................................................................................................... 77
Aplidium constellatum........................................................................................................................................ 80
Aplidium crateriferum........................................................................................................................................ 80
Aplidium cyaneum ............................................................................................................................................. 81
Aplidium densum................................................................................................................................................ 82
Aplidium elegans................................................................................................................................................ 82
Aplidium falklandicum....................................................................................................................................... 83
Aplidium fuegiense............................................................................................................................................. 84
Aplidium glabrum............................................................................................................................................... 84
Aplidium haouarianum....................................................................................................................................... 86
Aplidium longithorax.......................................................................................................................................... 86
Aplidium meridianum......................................................................................................................................... 87
Aplidium multiplicatum...................................................................................................................................... 89
Aplidium nordmanni........................................................................................................................................... 90
Aplidium orthium................................................................................................................................................ 90
Contents vii

Aplidium pantherinum........................................................................................................................................ 91
Aplidium pliciferum ........................................................................................................................................... 92
Aplidium savignyi .............................................................................................................................................. 92
Aplidium scabellum............................................................................................................................................ 92
Aplidium tabascum............................................................................................................................................. 93
Aplidium turbinatum.......................................................................................................................................... 94
Aplidium undulatum........................................................................................................................................... 95
Aplidium uouo ................................................................................................................................................... 96
Aplidium sp. ....................................................................................................................................................... 96
Polyclinum madrasensis..................................................................................................................................... 97
Synoicum adareanum......................................................................................................................................... 98
Synoicum blochmanni......................................................................................................................................... 99
Synoicum globosum.......................................................................................................................................... 100
Synoicum macroglossum.................................................................................................................................. 100
Synoicum pulmonaria........................................................................................................................................101
Synoicum sp. .................................................................................................................................................... 102
Family 7. Pseudodistomidae................................................................................................................................. 104
Pseudodistoma antinboja................................................................................................................................. 104
Pseudodistoma arborescens............................................................................................................................. 105
Pseudodistoma aureum.................................................................................................................................... 105
Pseudodistoma cereum..................................................................................................................................... 106
Pseudodistoma crucigaster.............................................................................................................................. 107
Pseudodistoma kanoko..................................................................................................................................... 108
Pseudodistoma megalarva ............................................................................................................................... 109
Pseudodistoma obscurum................................................................................................................................. 109
Pseudodistoma opacum.....................................................................................................................................110
Pseudodistoma sp. ............................................................................................................................................111
Family 8. Ritterellidae...........................................................................................................................................111
Ritterella rubra..................................................................................................................................................111
Ritterella sigillinoides.......................................................................................................................................112
Ritterella tokioka...............................................................................................................................................113
Family 9: Stomozoidae..........................................................................................................................................115
Stomozoa roseola...............................................................................................................................................115
B. Order: Phlebobranchia
Family 1. Ascidiidae..............................................................................................................................................115
Ascidia mentula.................................................................................................................................................115
Ascidia sydneiensis............................................................................................................................................116
Ascidia virginea.................................................................................................................................................116
Phallusia arabica..............................................................................................................................................117
Phallusia fumigata............................................................................................................................................118
Phallusia mammillata.......................................................................................................................................118
Phallusia nigra..................................................................................................................................................119
Family 2. Cionidae............................................................................................................................................... 120
Ciona edwardsi................................................................................................................................................. 120
Ciona intestinalis.............................................................................................................................................. 121
Ciona savignyi.................................................................................................................................................. 122
Family 3. Corellidae............................................................................................................................................. 122
Corella parallelogramma................................................................................................................................. 122
Family 4. Perophoridae........................................................................................................................................ 123
Ecteinascidia turbinata.................................................................................................................................... 123
Perophora namei.............................................................................................................................................. 124
C. Order Stolidobranchia
Family 1. Molgulidae........................................................................................................................................... 124
Molgula enodis................................................................................................................................................. 124
Molgula manhattensis...................................................................................................................................... 125
viii Contents

Family 2. Pyuridae............................................................................................................................................... 125

Halocynthia aurantium..................................................................................................................................... 125
Halocynthia papillosa...................................................................................................................................... 126
Halocynthia pyriformis.................................................................................................................................... 126
Halocynthia roretzi........................................................................................................................................... 127
Herdmania momus........................................................................................................................................... 129
Herdmania pallida............................................................................................................................................131
Microcosmus curvus..........................................................................................................................................131
Microcosmus exasperatus................................................................................................................................. 132
Microcosmus helleri ........................................................................................................................................ 133
Microcosmus vulgaris ..................................................................................................................................... 133
Pyura sacciformis ............................................................................................................................................ 134
Family 3. Styelidae............................................................................................................................................... 135
Botrylloides leachii .......................................................................................................................................... 135
Botrylloides perspicuus ................................................................................................................................... 136
Botrylloides tyreum ......................................................................................................................................... 136
Botryllus schlosseri ......................................................................................................................................... 137
Botryllus tuberatus .......................................................................................................................................... 138
Botryllus sp. ..................................................................................................................................................... 139
Cnemidocarpa bicornuta ................................................................................................................................. 140
Cnemidocarpa irene ........................................................................................................................................ 140
Cnemidocarpa stolonifera ...............................................................................................................................141
Dendrodoa aggregata .......................................................................................................................................141
Dendrodoa grossularia .................................................................................................................................... 142
Eusynstyela latericius .......................................................................................................................................143
Eusynstyela misakiensis .................................................................................................................................. 144
Polyandrocarpa zorritensis.............................................................................................................................. 144
Polycarpa aurata ............................................................................................................................................. 145
Polycarpa clavata .............................................................................................................................................147
Stolonica socialis ............................................................................................................................................. 148
Stolonica sp. .................................................................................................................................................... 148
Styela clava ..................................................................................................................................................... 148
Styela plicata ................................................................................................................................................... 150
Symplegma rubra ............................................................................................................................................. 151
Symplegma sp................................................................................................................................................... 152
Syncarpa oviformis .......................................................................................................................................... 152
Class: Thaliacea (salps)
Order: Salpida
Family 1. Salpidae................................................................................................................................................ 152
Ihlea racovitzai ................................................................................................................................................ 152
Salpa thompsoni .............................................................................................................................................. 153
Unidentified tunicate spp. (1–10)..................................................................................................................... 154
Compounds............................................................................................................................................................................... 159
References................................................................................................................................................................................. 165
Index������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������175
Preface
The ocean, sometimes called the “mother of origin of
life,” is also the source of unique natural products that are
mainly accumulated in living organisms. The search for new
metabolites from these organisms has resulted in the isola-
tion of approximately 10,000 metabolites. Several of these
compounds show pharmacological activities and are helpful
for the invention and discovery of new drugs, primarily for
deadly diseases such as cancer, acquired immunodeficiency
syndrome (AIDS), arthritis, etc. Among the marine inverte-
brates, sponges are better known for their secondary metab-
olites. Other invertebrates such as cnidarians, tunicates,
molluscs and bryozoans that may have potential in producing
bioactive compounds have been much less studied compared
to sponges. The study of marine natural products is becoming
increasingly sophisticated, and collaborative efforts between
marine biologists, biochemists and pharmacologists are the
need of the hour.
The tunicates or sea squirts (Phylum, Chordata;
Subphylum, Urochordata) that comprise about 4000 species
have been receiving greater attention and are considered to
be one of the most important potential sources of bioactive
compounds of pharmaceutical value. The secondary metabo-
lites such as alkaloids isolated from these organisms have led
to the discovery of very interesting therapeutic candidates,
which include an approved antitumor drug, and many others
are potent drug leads. The drug trabectedin (Yondelis), which
is approved by the European Medicines Agency (EMEA),
has been derived from an ascidian Ecteinascidia turbinata
and is presently used in the treatment of advanced soft tis-
sue sarcoma; the Therapeutic Goods Administration (TGA)
approved drug plitidepsin (Aplidin), obtained from another
species of ascidian, Aplidium albicans, is currently used for
multiple myeloma; and the drug lurbinectedin (PM01183) of
Ecteinascidia turbinata is under clinical trial phase II for the
treatment of solid tumor. Likewise, many secondary metab-
olites isolated from the different species of tunicates are in
clinical trials. Intensive research on the bioactive compounds
of marine tunicates and a fair knowledge of the identifica-
tion and biology of such contributing species are very much
needed. Keeping this in consideration, this title has been
proposed.
Though a few books are presently available on marine
natural products, a comprehensive book on the biology and
ecology of pharmaceutical marine tunicates has not so far
been published. This publication, the first of its kind contrib-
uted by scientists of both Marine Biology and Pharmacy dis-
ciplines, would answer this long felt need. It deals with the
taxonomy, common name, global distribution, habitat, diag-
nostic features, and pharmaceutical compounds (along with
their activities) of 175 species of tunicates. It is hoped that
the present publication when brought out would be of great
use as a standard text-cum-reference for researchers, teach-
ers and students of various disciplines such as Pharmaceutical
Sciences, Fisheries Science, Marine Biology, Biochemistry,
and Biotechnology; libraries of colleges and universities; and
as a valuable guide for pharmaceutical companies involved in
the development of new drugs from marine tunicates.
We are highly indebted to Dr. K. Venkataramanujam, former
Dean, Fisheries College and Research Institute, Tamil Nadu
Veterinary and Animal Sciences University, Thoothukudi,
India, for his valued comments and suggestions on the manu-
script. We sincerely thank all our international friends who
were very kind to collect and send certain species of marine
tunicates for the present purpose. The services viz. photography
and secretarial assistance rendered by Mrs. Albin Panimalar
Ramesh are also gratefully acknowledged.
ix
Authors
Ramasamy Santhanam, PhD, is the former dean of the
Fisheries College and Research Institute, Tamil Nadu
Veterinary and Animal Sciences University, India. His fields
of specialization are fisheries environment and marine biol-
ogy. He presently serves as a fisheries expert for various gov-
ernmental and nongovernmental organizations in India. Dr.
Santhanam has published 27 books and 70 research papers.
He was a member of the American Fisheries Society, United
States; the World Aquaculture Society, United States; the
Global Fisheries Ecosystem Management Network (GFEMN),
United States; and the International Union for Conservation
of Nature’s (IUCN) Commission on Ecosystem Management,
Switzerland.
Santhanam Ramesh, PhD, P  rofessor, Department of Phar­
maceutics, Ratnam Institute of Pharmacy, Nellore, AP,
India, earned his PhD from Jawaharlal Nehru Technological
University, Hyderabad, India, in 2011. His fields of specializa-
tion are biopharmaceutical products, pharmaceutical nano-
technology, and novel drug delivery systems. Dr. Ramesh has
14 years of teaching/overseas experience. He has authored
seven books viz. Marine Pharmaceutical Compounds,
Freshwater Phytopharmaceutical Compounds, Biology and
Ecology of Pharmaceutical Marine Plants, Biology and
Ecology of Pharmaceutical Cnidarians, Biology and Ecology
of Pharmaceutical Marine Mollusks, Biology and Ecology of
Pharmaceutical Marine Life: Echinoderms and A Textbook
of Novel Drug Delivery Systems; and 10 research papers
published in nationally and internationally reputed journals.
He presently serves as an associate member of the Academy
Pharmacy Group, Royal Pharmaceutical Society, Great
Britain, and as a Fellow of the Institution of Chemists, India.
xi
 1 Introduction
The tunicates (Phylum, Chordata: Subphylum, Tunicata or Cultured Species
Urochordata), the evolutionarily advanced invertebrate marine
Species Culturing Countries
organisms, are said to be 500 million years old. This subphylum
comprises about 3000 species that are exclusively marine, and it Halocynthia aurantium Japan
has three classes, viz. Ascidiacea (benthic and sessile tunicates), Halocynthia roretzi Japan, Korea
Thaliacea and Larvacea (or Appendicularia) (pelagic tunicates). Styela clava Korea
Among these classes of tunicates, the class Ascidiacea assumes Styela plicata Korea
greater importance, nutritionally and pharmaceutically. Source: Lambert, G. et al. 2016. Manag. Biol.
Invasion., 7: 59–66.
1.1 ECOLOGY OF ASCIDIANS
Ascidians are exclusively marine, abundant in harbors, and 1.4 ROLE OF ASCIDIANS IN POLLUTION
can be found all over the world from near the surface to great MONITORING
depths. They exist below low-tide levels in protected areas
Many species of ascidians can tolerate and accumulate heavy
with good water movement. The three orders within the class
metals. They therefore serve as useful indicators of water
Ascidiacea, based on the structure of the adult branchial
quality in bioassays for pollutants (Watters, 2018). Further,
sac, are (1) Aplousobranchia (almost exclusively colonial),
these ascidians have been reported to help measure plastic
(2) Phlebobranchia and (3) Stolidobranchia (in both solitary
pollution as they filter tiny particles from the ocean and store
and colonial forms).
them in their soft tissue (Anon., https://www.ynetnews.com/
articles/0,7340,L-5466866,00.html).
1.2 DIETARY VALUES OF ASCIDIANS
In parts of the world where ascidians are treated as sea- 1.5 OTHER USES OF TUNICATES
food, the species eaten are all large—in fact, more than
10 cm long—and are easily collected in large quantities. The use of tunicates as a source of biofuel is being researched.
The solitary stolidobranch ascidian species Pyura chilensis The cellulose body wall of certain species can be broken down
and Pyura praeputialis, which may reach lengths of 30 cm, and converted into ethanol, and other parts of the animal are
are sold commercially both fresh and canned. In Europe, protein-rich and can be converted into fish feed. Some tunicates
Microcosmus spp. are eaten raw and are sold in markets all are used as model organisms. The ascidian species Ciona intes-
over Mediterranean France, Spain, Italy, and Greece. The tinalis and Ciona savignyi have been used for developmental
Japanese cook the bright red Halocynthia roretzi, and occa- studies. Both species’ mitochondrial and nuclear genomes have
sionally they eat the stalked Styela clava, which is also much been sequenced. The nuclear genome of the appendicularian
appreciated in Korea (Monniot et al., 1991). Oikopleura dioica appears to be one of the smallest among
metazoans, and this species has been used to study gene regu-
lation and the evolution and development of chordates.
1.3 HARVEST AND AQUACULTURE
OF ASCIDIANS
1.6 PHARMACEUTICAL VALUES
Several species of stolidobranchs are farmed for food in some OF MARINE LIFE
parts of the world, mainly Japan and Korea. The species of
ascidians that are wild-harvested and cultured are given in Marine organisms can be considered the most recent source of
the following table. bioactive natural products in relation to terrestrial plants and non-
marine microorganisms. Approximately 28,500 marine natural
Wild-harvested Species products have been isolated and identified as of 2016. Cytotoxic
and anticancer properties account for the majority of biological
Species Harvesting Countries
activity reported for these natural products. Among the different
Boltenia ovifera Russia components of marine life, marine invertebrates have been the
Halocynthia aurantium Korea, Russia
Microcosmus hartmeyeri Japan
source of the majority of bioactive marine natural products.
Microcosmus sabatieri Mediterranean
Microcosmus vulgaris Mediterranean 1.6.1 Distribution of Bioactive Compounds
Polycarpa pomaria Mediterranean in Marine Life
Pyura chilensis Chile
Pyura pachydermatina New Zealand Among the different phyla of marine organisms, the phyla
Pyura praeputialis Australia, Chile Porifera and Cnidaria have been reported to contribute more
Pyura vittata Japan, Korea
than 50% of the compounds as detailed in the following table.
1
2 Biology and Ecology of Pharmaceutical Marine Tunicates

Percentage Contribution of Bioactive Compounds 1.7.1 Natural Products of Ascidians

by Different Groups of Marine Organisms A total of 572 bioactive compounds with antibacterial, anti-
Sponges 37 inflammatory, antiviral, antidiabetic, antiproliferative and
Cnidarians 21 antiparasitic activities have been isolated from the various spe-
Microbes 18 cies of ascidians during 1994–2014 (Palanisamy et al., 2017).
Macroalgae 9
Among the different families of the class Ascidiacea, the
Echinoderms 6
Tunicates 6
families Didemnidae (32%), Polyclinidae (22%) and Styelidae
Molluscs 2 (12%) have been reported to be the most prolific producers
Bryozoans 1 of bioactive compounds with diverse activities and potential
Source: Pereira, R.C. Bioactive compounds from marine for development as therapeutic drugs for a wide variety of
organisms: How to prospect and preserve this diseases. There has already been some notable success with
potential? http://www.fapesp.br/eventos/2010/09/ two ascidian natural products marketed for cancer treatment.
Marine-Biodiversity/Renato_Crespo.pdf Ecteinascidin (ET-743, trabectedin) from Ecteinascidia turbi-
nata is approved by the U.S. Food and Drug Administration
1.6.2 Chemistry Class of Bioactive Compounds FDA and marketed under the trade name Yondelis. Aplidin
of Marine Life
(dehyrodidemnin B, plitidepsin)—first isolated from Aplidium
albicans—has attained orphan drug status. Both are marketed
Among the bioactive compounds produced by the marine by PharmaMar (Madrid, Spain) (Watters, 2018).
organisms, alkaloids such as indole alkaloids, p­ yrrolizidine
alkaloids and β-carboline alkaloids contributed much with Percentage of Natural Products Isolated from the Different
74% followed by peptides (4%), alkyl sulfates (3%), polysac- Families of Ascidiacea During 1994–2014
charides (2%), polyketides (2%) and steroids (2%), in that Didemnidae 32
order (Palanisamy et al., 2017). Polyclinidae 22
Styelidae 12
1.6.3 Activities of Bioactive Compounds Polycitoridae 11
of Marine Life Pyuridae 5
Clavelinidae 5
Among the bioactive compounds isolated so far from the Cionidae 4
marine organisms, antitumor/anticancer compounds ranked Pseudodistomidae 4
first with 64% distribution as detailed in the following table. Perophoridae 2
Ascidiidae 1
Percentage Distribution of Drug Classes of Bioactive Compounds Holozoidae 1
Antitumor/anticancer 64 Molgulidae 1
Antibacterial 14 Source: Palanisamy, S.K. et al. 2017. Nat. Prod. Bioprospect., 7: 1–111.
Antimalarial 6
Antifungal 3
Anti-inflammatory 3
Anti-HIV 3
1.7.2 Drug Classes of Natural Products
Central nervous system (CNS) 3 from Ascidians
Antioxidant 2
Antiviral 1
With regard to the distribution of chemistry class of ascidian nat-
Antidiabetic 1 ural products, anticancer compounds dominated (64%), followed
Source: Palanisamy, S.K. et al. 2017. Nat. Prod. Bioprospect., 7: 1–111.
by antibacterial (14%) and others as shown in the f­ ollowing table.

Distribution of Drug Classes of Ascidian Natural Products (%)

1.7 TUNICATES AND THEIR PHARMACEUTICAL
(Isolated During 1994–2014) with High Biomedical
VALUES Potential Applications
The tunicates are considered to be important next to sponges Anticancer 64
and cnidarians as far as the diversity and percentage contribu- Antibacterial 14
tion of bioactive compounds. It is important to note that exten- Antimalarial 6
sive works on the pharmaceutical aspect are only available Antifungal 3
in the ascidians (Class, Ascidiacea), and no works have been Anti-HIV 3
reported from the larvaceans (Class, Appendicularia). Ascidians Anti-inflammatory 3
produce a rich variety of bioactive compounds (natural products CNS disorders 3
or secondary metabolites), including cyclic peptides and depsi- Antioxidant 2
Antiviral 1
peptides, and many different types of aromatic alkaloids. Many
Antidiabetic 1
of these metabolites are produced not only by the ascidian them-
selves but also by their endosymbiotic microorganisms. Source: Palanisamy, S.K. et al. 2017. Nat. Prod. Bioprospect., 7: 1–111.
Introduction 3

1.7.3 Therapeutic Values of Ascidians

The bioactive compounds isolated from these tunicates have led to the discovery of very interesting therapeutic candidates as
detailed in the following table.

Marine Pharmaceutical Clinical Pipeline: 2004–2018

Compound Name (Trademark) NP*/Derivative Original NP/Source Organism Therapeutic Area Status
Trabectedin (Yondelis) NP Ecteinascidin 743/Ecteinascidia Cancer (advanced soft tissue EMEA approved
turbinata sarcoma)
Pliditepsin (Aplidin) NP Ascidian/Aplidium albicans Cancer (multiple myeloma) TGA approved
Lurbinectedin (PM01183) NP derivative Ecteinascidins/Ecteinascidia Cancer (solid tumor) Phase II
turbinata
Source: Martins, A. et al. 2014. Mar. Drugs, 12: 1066–1101; PharmaMar, https://www.pharmamar.com/wp-content/uploads/2018/12/PR_Approval-
APLIDIN-Australia.pdf
Note: EMEA, European Medicines Agency; NP*, natural product; TGA, Therapeutic Goods Administration (Australian regulatory agency).

Among the extant species of tunicates, barely 2% of the tunicates and their taxonomy and identification. This calls for
animals have been examined for their natural products of further intensive research on this group, and a joint venture
pharmaceutical importance. This large gap may possibly be between experts in the disciplines of Marine Biology and
due to difficulties and lack of expertise in the collection of Pharmacy would be of great help in this regard.
 2 Biology and Ecology of Marine Tunicates

Like other chordates, the tunicates have a characteristic noto-
chord during their early development, but it is lost by the
time they have completed their metamorphosis. These ani-
mals begin their life in a mobile larval stage that resembles
a tadpole. However, the species of the class Larvacea (or
Appendicularia) retain the general larval form throughout
life. But the species of ascidians (Class, Ascidiacea) very rap-
idly settle down and attach themselves to a suitable surface,
later developing into a barrel-like and usually sedentary adult
form. The thaliaceans (Class, Thaliacea) are pelagic through-
out their lives and may have complex life cycles.
2.1 DISTRIBUTION AND HABITAT
The tunicates are found distributed in ocean waters from
the polar regions to the tropics. While the thaliaceans (pyro-
somes, salps and doliolids) are free-swimming (planktonic),
the sessile forms (ascidians) grow mainly on solid surfaces
such as wharf piles, ship hulls, rocks, and the shells of various
sea creatures. These ascidians that are found both in shallow
and in very deep waters may also grow on rocks, coral rubble,
jetty pilings, or even on sea grasses, and algae.
2.3.2 Solitary and Colonial Ascidians
Ascidians occur as solitary or colonial (or compound) spe-
cies. Solitary species are individual animals with an inhal-
ant siphon and an exhalent siphon, often with a thick leathery
test that encloses the body of the animal. Colonial species
are groups of small animals (zooids) that are embedded in a
gelatinous test as a colony. Zooids can be arranged in circular
or linear systems, sharing common exhalent canals and aper-
tures. Other types can have zooids opening independently or
on stalks (stem-like structures called stolons) connected to a
common basal test. The colony can range from a few centi-
meters in diameter to a meter or more and can be up to several
centimeters thick. Colonial ascidians may grow as slimy lay-
ers and blobs on rocks, jetty pilings and other hard surfaces.

2.2 CHARACTERISTICS OF TUNICATES

All members of the phylum Chordata possess four character-
istic anatomical structures as embryos: a flexible body-length
rod (the notochord) that provides resistance against muscular
contractions and allows for more efficient movement; a dor-
sal, hollow, nerve cord that forms the central nervous system;
slits in the beginning of the digestive tract (the pharynx) that
allow filter feeding and gas exchange; and a post-anal tail.
The subphylum Urochordata differs from other chordate sub-
phyla (Cephalochordata and Vertebrata) in that the adult form
A solitary (simple) ascidian (Phallusia mammillata).
has no notochord, nerve cord or tail.

2.3 ASCIDIANS (CLASS, ASCIDIACEA)

2.3.1 Species Diversity
It is the largest class of tunicates with 2815 described spe-
cies with sizes ranging from tiny ones 1 mm long, to those
more than 10 cm. The highest number of species and fami-
lies is found in the order Aplousobranchia. Didemnidae and
Styelidae families have the highest number of species with
more than 500 within each group. Approximately 60% of
described species are colonial. Species richness of ascidians
is highest in tropical regions, where colonial species predomi-
nate. In higher latitudes, solitary species gradually contribute
more to the total species richness (Shenkar and Swalla, 2011). A colonial (compound) ascidian (Atriolum marinense).

5
6 Biology and Ecology of Pharmaceutical Marine Tunicates

2.3.3 Body Structure
The entire animal of an ascidian is encased in a little bag
(“Askidion” comes from the Greek word for “bladder” or
“little bag”). Some ascidians have a sturdy outer coat called
the “tunic.” The tunic supports and protects them. As the
ascidian grows bigger, the tunic also grows with it. Unlike
other creatures with a tough outer coat, tunicates do not
have to moult to get bigger. The tunic is made of protein
and a substance called tunicin that closely resembles cel-
lulose. Some solitary ascidians have bands of muscles along
their body. When these muscles contract, water squirts out
of the animal. So they are sometimes also called sea squirts.
They may do this exercise to get rid of something in them,
or when they are disturbed. Two openings are found in the
body wall of the ascidian. They are the buccal siphon at
the top through which water flows into the interior, and the
atrial siphon on the ventral side through which it is expelled.
A large pharynx occupies most of the interior of the body.
It has a ciliated groove known as an endostyle on its ven-
tral surface, and this secretes a mucous net that is used in
the collection of food. The gullet, at the lower end of the
pharynx, links it to a loop of gut that terminates near the
atrial siphon. The walls of the pharynx are perforated by
several bands of slits called stigmata, through which water
escapes into the surrounding water-filled cavity, the atrium.
An ascidian is a complex animal. It usually has a circulatory
system, a digestive system, a heart and other organs. It gen-
erates a one-way current through its body. A part of the gut
is modified to filter out plankton from this water flow. A tiny
specimen only a few centimeters long may pump a hundred (A) Generalized body structure of a solitary ascidian. (B) Zooid
of a colonial ascidian; 1, branchial aperture; 2, atrial aperture;
liters of water in a span of 24 hours.
3, endostyle; 4, branchial sac with stigmata; 5, esophagus; 6, gut
loop; 7, gonads; 8, anus; 9, heart.

The pyrosomes are bioluminous colonial tunicates with a hol-

low cylindrical structure. In these animals, the buccal siphons
are on the outside and the atrial siphons inside. There are
about 10 species of pyrosomes, and all are found in the trop-
ics. The 40 species of salps are also small, under 4 cm long,
and found in the surface waters of both warm and cold seas.
They often form long chains by budding off new individuals.
The 23 species of doliolids are small, mostly under 2 cm long.
They are solitary with two siphons located at opposite ends of
their barrel-shaped bodies.

Internal anatomy of a typical ascidian. (Courtesy of Jon

Houseman.)

2.3.4 Thaliacea
This class includes pyrosomes, salps and doliolids, which are
all free-swimming, pelagic individuals. They are all filter
feeders using a pharyngeal mucous net to catch their prey. Pyrosoma sp.
Biology and Ecology of Marine Tunicates
Salpa sp.
Doliolum sp.
2.3.5 Larvacea (or Appendicularia)
It is the only group of tunicates to retain their chordate char-
acteristics in the adult state. The 70 species of larvaceans
superficially resemble the tadpole larvae of amphibians,
although the tail is at right angles to the body. The noto-
chord is retained, and these animals are mostly under 1 cm
long. They secrete an external mucous net known as a house,
which may completely surround them and is very efficient at
­trapping planktonic particles.
Oikopleura sp.
2.4 INTERNAL FEATURES
2.4.1 Heart and Circulatory System
Tunicates have a well-developed heart and circulatory s­ ystem.
The heart is a double U-shaped tube situated just below the gut.
The blood vessels are simple connective tissue tubes, and their
7
blood has several types of corpuscles. The blood may appear
pale green, but this is not due to any respiratory pigments,
and oxygen is transported dissolved in the plasma. Tunicate
blood has unique features. Some species of Ascidiidae and
Perophoridae contain high concentrations of vanadium and
vanadium-associated proteins in vacuoles in blood cells
known as vanadocytes. It is interesting to mention here that
some tunicates concentrate vanadium up to 10 ­million times
that of the surrounding seawater. Other species of tunicates
concentrate lithium, iron, niobium and tantalum.
2.4.2 Skeleton, Tissues and Muscles
In the tunicates, the tunic functions as an external skeleton
that supports and protects the body. Additional support, how-
ever, is provided by their body fluids and connective tissue.
Firm proteinaceous rods may also support the branchial appa-
ratus. Although musculature is poorly developed in tunicates,
there are muscles that retract the body and constrict the atrial
cavity, allowing it to eject water. In doliolids and salps, these
muscles have become modified so as to produce jet propulsion.
2.4.3 Nervous System and Organs of Sensation
In the tadpole larvae and appendicularians, the dorsal nerve
cord is well developed. At the anterior end, there are usually
sensory structures, which detect light and orient the animal to
gravity. Similar sensory structures can be found in adult thali-
aceans. Special organs of sense are otherwise poorly developed.
When the larva metamorphoses into an adult, the original ner-
vous system and sensory organs degenerate, leaving a single
ganglion between the oral and atrial openings. Nerves grow to
the various organs of the body from this ganglion.
2.5 DIGESTION, NUTRITION AND EXCRETION
In ascidians and thaliaceans, the beating action of pharyngeal
cilia creates a water current. As the water is driven from the
branchial sac into the atrial cavity, a sheet of mucus, secreted
by the endostyle, traps a variety of very small food organ-
isms like phytoplankton suspended in the water current. The
mucus is rolled into a cord and then conveyed to the intestine,
where it is digested and absorbed. A stomach and digestive
glands may be present. The intestine ends as an anus in the
atrium below the atrial aperture. Wastes are ejected through
this aperture in a stream of water. Metabolic wastes, such as
the breakdown products of protein, are normally excreted
at various parts of the body, including the surfaces of the
gills and the intestine. In many cases, the wastes are stored as
solid deposits.
2.6 RESPIRATION
In tunicates, the gas exchange occurs across the gill and also
across various other body surfaces, such as the lining of the
atrium.
8 Biology and Ecology of Pharmaceutical Marine Tunicates
2.7 LOCOMOTION
Among the tunicates, the tadpole larvae and appendicularians
swim by undulating the tail, which contains a stiff notochord.
Despite their sessile lifestyle, some adult ascidians can move
by attaching with one area of the body and letting go with
another. Movement of colonies up to 1.5 cm per day has been
recorded. In thaliaceans, an exhalant current of water, which
in doliolids and salps is combined with a strong muscular con-
traction, creates a jet stream that propels the animal forward.
2.8 FOOD AND FEEDING
2.8.1 Filter Feeding
Almost all tunicates are suspension feeders, capturing plank-
tonic particles by filtering seawater through their bodies.
During feeding, water is drawn into the body through the buc-
cal siphon by the action of cilia lining the gill slits. Water is
drawn through a net lining the pharynx. The net is made of
sticky mucus threads with holes that can trap planktonic par-
ticles including bacteria. The net is rolled up on the dorsal side
of the pharynx, and the trapped particles are drawn into the
esophagus. The gut is U-shaped and also ciliated to move the
food particles along. The stomach is an enlarged region at the
lowest part of the U-bend. After digestion, the food is moved
on through the intestine, where absorption takes place, and
the rectum, where undigested remains are formed into fecal
pellets or strings. The anus opens into the dorsal or cloacal
part of the peribranchial cavity near the atrial siphon.
2.8.2 Other Feeding Habits
Some ascidians that live on soft sediments are detritivores.
A few deepwater species, such as Megalodicopia hians, are
sit-and-wait predators, trapping tiny crustaceans, nematodes
and other small invertebrates with the muscular lobes that
surround their buccal siphons. Certain species of the fam-
ily Didemnidae have symbiotic green algae or cyanobacteria
in their tunics, and one of these symbionts, Prochloron, is
unique to tunicates.
2.9 REPRODUCTION
2.9.1 Ascidians
Ascidians are almost hermaphrodites, and each has a single
ovary and testis. In some solitary species, sperm and eggs
are shed into the sea and the larvae lead a planktonic life.
In others, especially colonial species, sperm is released into
the water and drawn into the atria of other individuals with
the incoming water current. Fertilization takes place here,
and the eggs are brooded through their early developmental
stages. Some larval forms appear very much like primitive
chordates with a notochord, and superficially they resemble
small tadpoles. When sufficiently developed, the larvae of
the sessile species find a suitable substratum and metamor-
phose into adults. In some classes, the adults remain pelagic
(swimming or drifting in the open sea), although their larvae
undergo similar metamorphosis to a higher or lower degree.
Colonial forms also increase the size of the colony by budding
off new individuals to share the same tunic.
Internal anatomy of an ascidian tadpole. (Courtesy of Jon
Houseman.)
2.9.2 Pyrosomes
The pyrosome colonies grow by budding off new zooids near
the posterior end of the colony. Sexual reproduction starts
within a zooid with an internally fertilized egg. This develops
directly into an oozooid without any intervening larval form.
2.9.3 Doliolids
These animals have a very complex life cycle that includes
various zooids with different functions. The sexually repro-
ducing members of the colony are known as gonozooids.
Each one is a hermaphrodite with the eggs fertilized by sperm
from another individual. The gonozooid is viviparous, and at
first, the developing embryo feeds on its yolk sac before being
released into the sea as a free-swimming, tadpole-like larva.
This undergoes metamorphosis in the water column into an
oozooid. This is known as a “nurse” as it develops a tail of
zooids produced by budding asexually. Some of these are
known as trophozooids, have a nutritional function, and are
arranged in lateral rows. Others are phorozooids, have a trans-
port function, and are arranged in a single central row. The
asexual phase in the life cycle allows the doliolid to multiply
very rapidly when conditions are favorable.
2.9.4 Salps
These animals also have a complex life cycle with an alter-
nation of generations. In the solitary life history phase, an
oozoid reproduces asexually, producing a chain of tens or
hundreds of individual zooids by budding along the length of
a stolon. The chain of salps is the “aggregate” portion of the
life cycle. The aggregate individuals, known as blastozooids,
remain attached while swimming and growing larger. The
blastozooids are sequential hermaphrodites. An egg in each
is fertilized internally by a sperm from another colony. The
egg develops in a brood sac inside the blastozooid and has a
placental connection to the circulating blood of its “nurse.”
When it fills the blastozooid’s body, it is released to start the
independent life of an oozooid.
Biology and Ecology of Marine Tunicates
2.9.5 Larvaceans
These animals only reproduce sexually. They are protandrous
hermaphrodites, except for Oikopleura dioica, which is gono-
choric, and a larva resembles the tadpole larva of ascidians.
Development is very rapid and only takes 7 hours for a zygote
to develop into a house-building juvenile.
2.10 ASSOCIATED LIFE
A whole biocenosis may live around ascidians. Foraminifers
(Phylum, Protozoa) are found encrusted on the tunic, and
algae can settle there. Lamellibranchs (Phylum, Mollusca)
can inhabit their gills. The symbionts and parasites can also
occupy various organs of the ascidians. The symbiotic shrimp
9
Ascidonia flavomaculata lives in the tunicate Ascidia men-
tula. Invasive sea squirts such as the vase tunicate, Ciona
intestinalis, colonize mussel (Mytilus edulis) socks and rap-
idly become the dominant species in terms of abundance
and biomass. The colonial tunicates Didemnum vexillum
and Botrylloides violaceus have been found in association
with a colonial bryozoan (Schizoporella unicornis), a colo-
nial hydroid (Ectopleura crocea), a tube-building amphipod
(Jassa marmorata) and an unidentified barnacle species.
2.11 PREDATORS
Fish (sharks and skates), flatworms, periwinkles, sea urchins
and sea stars are the primary predators of tunicates.
 3 Pharmaceutical Compounds and
Activities of Marine Ascidians

in the larvae. Budding occurs at the terminal ampullae of the

Class: Ascidiacea basal vascular stolons.
A. Order: Aplousobranchia
Compounds and Activities
FAMILY 1. CLAVELINIDAE Cytotoxicity: It is the source of 11 tricyclic alkaloids viz.
Clavelina cylindrica (Quoy & Gaimard, 1834) cylindricines A–K. Among them, cylindricines A, B have
shown modest cytotoxicity (Blackman et al., 1993; Li and
Blackman, 1994, 1995).
Antitumor activity: Li and Blackman (1995) reported on the
antitumor activity of cylindricine C isolated from this species.
According to Murali and Chandrasekhar (2013), cylindricines
inhibit the growth of murine leukemia and human solid tumor
cell lines.

Cylindrocine G
R=OH Cylindrocine C
R=OMe Cylindrocine D
R=OAC Cylindrocine E
R=SCH Cylindrocine F
R=CI Cylindrocine A Cylindrocine H

Common name(s): Grape ascidian

Global distribution: Subtropical, temperate: Indo-West
Pacific: Australia
Ecology: This benthic, sessile species inhabits shallow
waters; depth range 20–22 m. Cylindrocine B Cylindrocine K

Biology
Description: Transparent zooids of this species are elongated
and are with pigmented lines along the endostyle and the peri-
pharyngeal groove. The pharynx has 8–20 rows of stigmata.
The body is divided into the thorax and abdomen; the latter
is elongated. Gonads are present in the intestinal loop; fertil-
Cylindrocine J
ization occurs in the end of the expanded oviduct, forming
Cylindrocine I
an incubation pouch inside the atrial cavity where numerous
larvae are brooded. Statocyte and ocellus are always present

11
12 Biology and Ecology of Pharmaceutical Marine Tunicates

Clavelina lepadiformis (Müller, 1776)
Hatching occurs in about 10 days. The free-swimming larvae
settle after about 3 hours and metamorphose into adults.
Parasites: The parasitizing copepod Enterocola hessei inhab-
its the zooids of this species.
Human uses: This species is often used as a model for the
morphology and physiology of all ascidians. Further, it may
also be useful as a water quality indicator due to its capability
of concentrating toxic substances, such as heavy metals and
hydrocarbons, within their tissues.

Compounds and Activities

nAChR blocking activity: Quinolizidine and decahydro-
quinoline alkaloids, pictamine and lepadins A, B have been
isolated from this species (Palanisamy et al., 2017; Steffan,
1992). Among them, pictamine and lepadin B are potent block-
ers at neuronal nicotinic acetylcholine receptors (nAChRs)
that perform a multitude of functions from conducting nerve-
muscle transmission to participating in the different cogni-
tive processes and regulation of the inflammatory response
(Tsuneki et al., 2005).
Grosso et al. (2014) reported that the compound lepadins
A, B served as nAChR antagonists with IC50 values of 0.7 and
0.9 µM, respectively.

Common name(s): Light-bulb tunicate

Global distribution: Atlantic waters; native from the Shet-
land Islands and Bergen, Norway, to the Bay of Biscay, the
Mediterranean, and the Adriatic
Ecology: It normally inhabits shallow littoral areas down to
50 m and grows on natural or artificial hard substrates, includ-
ing rocks, wharf pilings, docks, and boat hulls. It can also tol-
erate a wide range of habitats, from open coasts that are very
exposed, to estuaries and enclosed bays that are extremely
sheltered. These light-bulb tunicates have also been reported
to tolerate a wide range of temperatures, salinity (18%–40%)
and oxygen levels.
Pictamine Lepadin B
Biology
Description: It is a colonial, transparent ascidian with loosely
attached individual zooids reaching 20 mm in length and 5 mm
in diameter. Zooids are clear with white, yellow or pink bands
around the endostyle, dorsal lamina and oral siphon, giving the
species its light-bulb appearance. Individual zooids are housed
in a common structure made of tunicin, a cellulose-like sub-
stance. Each zooid is joined to the tunic (or test) at the base.
Food and feeding: It feeds on suspended particulate mat-
ter by actively pumping water through a mucus net lin-
ing the branchial basket. Water is pumped in through the
inhalant siphon using cilia, and the particles trapped in the
mucus net are rolled into a food string and drawn into the
Lepadin A
esophagus.
Reproduction: Like most colonial ascidians, the light-bulb
tunicate is a hermaphrodite that reproduces sexually and Cytotoxicity: The pyrroloperhydroquinoleine alkaloid,
asexually. Sexual reproduction occurs with internal fertiliza- lepadiformine 1 isolated from this species showed cytotoxic-
tion, allowing 50–70 embryos in the atrial cavity at one time. ity (Biard et al., 1994b).
Pharmaceutical Compounds and Activities of Marine Ascidians 13

Compounds and Activities

Cardiovascular effects and antiarrhythmic properties:
The alkaloids, lepadiformines A, B, C isolated from this spe-
cies have shown antiarrhythmic (abnormal rhythms of the
heart) properties (Jugé et al., 2001; Palanisamy et al., 2017).

Lepadiformine 1

Clavelina moluccensis (Sluiter, 1904)

Lepadiformines A, B, C

Clavelina oblonga (Herdman, 1880) (= Clavelina

phlegraea)

Common name(s): Bluebell tunicate or blue sea squirt

Global distribution: Tropical Indo-West Pacific: Australia,
Indonesia, Papua New Guinea, Mariana Islands, Philippines,
Singapore, and Malaysia
Ecology: These sessile animals grow in clusters and are
found attached to dead coral or other hard substrates, nor-
mally under overhangs; the depth range is 3–113 m.

Biology
Description: This colonial species is 0.5–2.5 cm long. Zooid
is oval or egg-shaped and sits on a thick stalk, with translu-
cent walls. Colonies are pale blue, violet or darker blue in
color.
Food and feeding: These animals are filter feeders.
Reproduction: It is a hermaphroditic species. Eggs develop
into lecithotrophic larva before metamorphosing into benthic
adults.
Common name(s): Speckled sea squirt
Global distribution: Temperate; Northeast Atlantic and the
Mediterranean: Europe.
Ecology: This sessile species lives in hard substrates like
ropes, wrecks and rocks. Normally in sediment-loaded shallow
waters (between 1 and 10 m depth) and preferably in mangrove
roots, port areas, lagoons, littoral ponds and bay bottoms.
14 Biology and Ecology of Pharmaceutical Marine Tunicates

Biology
Description: Consistency of this fixed, colonial ascidian is
cartilaginous and glairy to the touch. Colonies appear as a
tight mass of small cartilaginous beads which are almost
transparent. Individuals (zooids) are 1–3 cm long and are Clavaminol A
grouped into often very spherical bunches 10–20 cm in diam-
eter. Each bouquet consists of several tens to hundreds of
zooids. Individuals are united by a common base called sto-
lon. Each zoid, shaped like a long-necked club, is gelatinous,
translucent and slightly milky. Its branchial sac contains at
least 15 rows of gill slits (stigmas); the thorax occupies about Clavaminol B
half of the body and is followed by a ciliated gutter spotted
with white, leading to the esophagus. At the top is the mouth
siphon, while the cloacal siphon is lateral and slightly lower.
Food and feeding: It is an active filter feeder on phyto-
plankton, bacteria and suspended organic matter. The water Clavaminol C
is sucked by the inhalant siphon or mouth siphon and is fil-
tered in a branchial pharynx that is riddled with small slits.
The filtered water then passes through a large peribranchial
cavity called the atrium before emerging through the exhal-
ant siphon or cloacal siphon. On the medioventral side of the
pharynx, there is a ciliated and glandular gutter, the endostyle, Clavaminol D
while the mediodorsal surface bears a prominent row of hairy
tongues, the raphe. Mucosal secretions of endostyle engulf the
food particles that water brings into the pharynx. The latter
accumulate in the dorsal raphe and are driven by the ciliary
beat to the stomach. The waste is discharged through the anus Clavaminol E
located in the cloacal siphon.
Reproduction: It has an annual cycle of growth beginning in
the spring, when new zooids develop from larvae that formed
in winter. Larval production occurs from June to September,
followed by disappearance of zooids. This animal also devel-
ops by asexual multiplication. Self-fertilization is also pos- Clavaminol F
sible in this species.

Compounds and Activities

Antifungal activity: The compound, (2S,3R)-2-Aminodo- Clavaminols G,H,I
decan-3-ol isolated from this species showed antifungal
activity against Candida albicans and C. glabrata with mini-
mum inhibitory concentration (MIC) values of 0.7 µg/mL and
Clavaminols L, M, N
30 µg/mL, respectively (Palanisamy et al., 2017).

Clavelina picta (Verrill, 1900)

(2S,3R)-2-Aminododecan-3-ol

Cytotoxicity: The 2-amino-3-alkanol products, clavaminols

A–F and sphingoids, clavaminols G–N have been isolated
from this species. Among them, clavaminol A showed mod-
erate cytotoxicity against atypical glandular cells (AGC) (IC50
5 µg/mL), and compound clavaminol B showed less cytotox-
icity (Aiello et al., 2009; Palaisamy et al., 2017).
Pharmaceutical Compounds and Activities of Marine Ascidians
Common name(s): Painted tunicate
Global distribution: Western Atlantic: Florida, Bahamas,
and Caribbean
Ecology: This sessile species often occurs in large clusters
attached to black coral, sponges and gorgonians; the depth
range is 0–35 m.
Biology
Description: Colonies of this species may contain hundreds
of individuals. Zooids are up to 2 cm, transparent and are
often shaded with white, red or purple. Siphon rims and inter-
nal body parts are typically carmine to purple. Zooids are
jointed at their bases through stolons.
Food and feeding: It is a filter feeder on phytoplankton.
Compounds and Activities
Cytotoxicity: The alkaloids clavepictines A, B isolated from
this species have shown cytotoxicity (Blunden, 2001).
Clavepictines A, B
nAChR blocking activity: Quinolizidine alkaloid, pictamine
isolated from this species has shown to be a potent blocker
at the neuronal nicotinic acetylcholine receptor (nAChR) that
performs a multitude of functions from conducting nerve-
muscle transmission to participating in the different cognitive
processes and regulation of the inflammatory response (Mayer
et al., 2009; Palanisamy et al., 2017; Tsuneki et al., 2005).
Grosso et al. (2014) reported that the compounds lepadins
A, B served as nAChR antagonists with IC50 values of 1.3 and
1.5 µM, respectively.
15
Mosquito repellent activity: The methanol extracts of this
species showed mosquito repellent activity against four vec-
tor mosquitoes, viz. Anopheles maculatus, Culex quinque-
fasciatus, Aedes aegypti and Aedes albopictus, with median
effective dosage (ED50) values of 0.0055, 0.0093, 0.0746,
0.0712 mg cm−2 (Hussein et al., 2002).
Clavelina sp.
Compounds and Activities
Cardiovascular effects and K+ current inhibition activity:
The compounds lepadiformines A and B isolated from this
unidentified species have shown cardiac inward rectifying
K+ current inhibition activity with IC50 values ranging from
1.4 to 1.6 µM (Mayer et al., 2009).
Pycnoclavella kottae (Millar, 1960)
Common name(s): Stalked ascidian
Global distribution: Temperate; Southwest Pacific: New Zea-
land
Ecology: This sessile species is found occasionally on walls
and the edges of overhangs in areas of high exposure, where it
can be locally abundant.
Biology
Description: It is a distinctive colonial ascidian that has numer-
ous pod-like heads arising from a narrow basal mat. Stalks are
16
long and slender. Heads have an oral (inhalant) opening halfway
down and an atrial (exhalent) opening at the top of the colony
head. Color in life is gold and slightly translucent on the siphons.
Compounds and Activities
Anti-inflammatory, antimetabolic and antiproliferative
activities: The 2,2,5-trisubstituted imidazolone-containing
alkaloids, kottamides A–E have been isolated from this spe-
cies. Among them, kottamide D exhibited potent antimeta-
bolic activity (IC50, 6–10 µM) with both antiproliferative and
anti-inflammatory activity in the 2–200 µM range. All four
kottamides exhibited moderate antileukemia activity in a
P388 cell line (Appleton et al., 2002; Mollica et al., 2012).
FAMILY 2. DIAZONIDAE
Diazona angulata (Monniot F. & Monniot C., 1996)
Image not available
Common name(s): Not designated
Global distribution: Tropical Western Central Pacific: Phil-
ippines
Ecology: This sessile species is normally found in overhangs;
depth range 10–30 m.
Biology
Description: Colonial ascidian with the anterior part of
zooids free, but posterior part embedded inside the tunic. The
colonies are massive and can be as large as 40 cm in diam-
eter and 20 cm high, with zooids up to 5 cm long. Both oral
and atrial siphons in each zooid with strong sphincters. Oral
siphon does not have a velum.
Biology and Ecology of Pharmaceutical Marine Tunicates
Compounds and Activities
Anticancer activity: The cytotoxic peptide diazonamide
A isolated from this species showed anticancer activity by
potently disrupting tubulin polymerization in different cancer
cells (Ruiz-Torres et al., 2017; Zheng et al., 2011).
(-)-Diazonamide A
Diazona formosa (Monniot F. & Monniot C., 1996)
Common name(s): Encrusting ascidian
Global distribution: Tropical Western Central Pacific: Indo-
nesia, Philippines and Micronesia
Ecology: This sessile species that is found on offshore sand
clay prefers coral-rich reefs with current, in a depth range of
20–60 m.
Biology
Description: It is a beautiful, colony-forming ascidian grow-
ing up to 2.5 cm. It is translucent with fine white lines and six
white spots on both siphons.
Food and feeding: These organisms are filter feeders. By
pumping water through their siphons, they can sift out food
particles from the water.
Reproduction: It is hermaphroditic and is able to both self-
and cross-fertilize. An egg develops into a lecithotrophic
larva before settling to the bottom and transforming into its
adult form.
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and resourceful brain he was doubtless planning his campaign,
determining the best method of exploding his bombshell in Deanery
Street.
He paused at last in his restless pacing and turned to his
lieutenant, who knew the man too well to put any direct questions.
“Well, Sellars, we have drawn a blank with Alma Buckley, through
no fault of yours. You couldn’t have done more than you have. We
shall have to precipitate matters, and blow up Clayton-Brookes and
that young impostor, whom the world takes for his nephew, in the
process.”
Sellars would have dearly liked to have an actual inkling of what
his astute leader was planning, but he knew it was useless asking.
Lane never revealed his coups beforehand. When they were
accomplished, he was as frank as he had previously been reticent,
and would explain with perfect candour the processes by which he
had engineered them.
“Well, good-bye, Lane. Sorry the result wasn’t satisfactory. Better
luck next time. Can I get on to any other portion of the job?”
The detective thought not, at the moment; what was left he was
going to take into his own hands. But he praised his able young
lieutenant very highly for the work he had done down at Brinkstone,
the foundation on which the superstructure of the subsequent
investigations had been built.
In the meantime, while Lane was preparing his coup, Rupert
Morrice had been stealthily pursuing his line of investigation.
A passionate man by nature, he had experienced the greatest
difficulty in restraining himself on his return from the jeweller who
had told him that the supposed “birthday” necklace was a worthless
imitation. When his wife returned about five o’clock unconscious of
the tragic happenings during her brief absence, his first impulse was
to follow her up to her room, tell her what he had learned and wring
from her a confession.
But he held himself in by a great exercise of self-control. He
wanted more evidence, he wished to make sure if this was an
isolated instance or one of a series of similar transactions.
As it happened, fortune was adverse to the wrong-doer, and in the
banker’s favour. Mrs. Morrice’s friend was very unwell, and the lady
drove down to her on the two following days to cheer her up, leaving
early in the morning and returning about the same time in the
afternoon. As on the previous occasion, the maid was given a
holiday during the few hours of her mistress’s absence.
The coast therefore was quite clear for Morrice, and he took
advantage of his unique opportunities with grim determination.
Rosabelle alone in the house had an idea that something was going
on from noting the fact that she met him in the hall on one of the
mornings, carrying a small bag and wearing a very grim expression,
as if he were engaged on some urgent but disagreeable business.
In all he took some ten very valuable pieces of jewellery to the
same man for examination. The result in each case was similar, they
were all cleverly executed imitations of the original gifts he had
presented to her. That was enough for him. She had a pretty large
collection, and it might be that a great many of them were not
substitutes; that she had not so far made use of them for her secret
purposes. On those of which he was quite certain from the expert’s
evidence, he reckoned that, even selling at a greatly depreciated
price, she must have realized several thousands of pounds.
On the afternoon of the third day he was pacing his room about
five o’clock like a caged lion, feverishly awaiting his wife’s return,
waiting to confront her with the anonymous letter, and reveal to her
his verification of the charges it contained.
The clock on the mantelpiece struck five, the quarter, and the half-
hour. His face grew darker and darker, as the tide of his righteous
wrath swelled. Six o’clock struck, and no sign of Mrs. Morrice. Then
ten minutes later a telegram was brought to him which after reading
he cast angrily on the floor. It explained that her friend was very
unwell, that she was stopping the night at her house, and would
return home at lunch time to-morrow.
 The storm could not burst to-day on the devoted head of the
woman who had played so foolishly with her husband’s trust in her.
The unexpected delay incensed further the unfortunate financier,
against whom of late fate seemed to have a special grudge.
Rosabelle came in while he was fuming, to ask him for a small
cheque in anticipation of her quarter’s allowance. So preoccupied
was he with his bitter thoughts of the gross way in which he had
been deceived that he wrote the cheque like a man in a dream, and
the girl noticed that his hand trembled. When he looked up to give it
to her, she saw that his face was as black as night.
“Uncle dear, whatever is the matter?” she cried impetuously. For
some little time past she had had an uneasy feeling, one of those
presentiments which occur so often to sensitive people, that there
was trouble of some sort brewing in this household.
“Nothing the matter, my child,” he answered evasively, passing his
hand wearily across his forehead. Much as he loved his pretty niece,
much as he trusted her, he could not as yet reveal to her the cause
of his trouble, betray the woman in whom he had believed—who
bore his honoured name.
But the girl persisted. “But, dearest uncle, you are hiding
something from me. You look so strange, I am sure you are very
much moved. Have you had disturbing news?”
For a little time the unhappy man refrained from answering that
question, inspired by no spirit of girlish curiosity, but by the sincerest
and most loyal affection.
“Yes, my child, I have had bad news, very bad news, I am afraid I
am a poor dissembler,” he said at length. “Later on, under the strict
seal of secrecy, I may tell you the cause of my trouble. But not now,
not now. Run away, my precious little girl, and leave me to my black
mood.”
She dared not worry him further, although her heart was aching for
him. Nobody knew better than she the kind, tender nature underlying
that rather stern exterior. Before she obeyed him, she put her arms
round his neck and kissed him affectionately.
 “Tell me when you please, dear, in your own good time, and your
poor little Rosabelle, to whom you have always been so kind and
generous, will do her best to comfort you.”
“I know you will, you precious, warm-hearted girl.” He clasped her
hand almost convulsively. What he had found out had wounded him
to the core. Nothing hurt this strong, proud man so much as the
discovery that his confidence had been misplaced in those near to
him, that his trust in them had been abused.
“Thank heaven, I have one dear little friend in the world, one dear,
loyal little friend who has never given me a moment’s uneasiness,
who I am confident never will. But run away now, my darling. I
cannot speak yet, even to you, of what is troubling me.”
She obeyed him, and left the room wondering. The words he had
spoken had been very vague, but her quick instinct had prompted
certain suspicions of the cause of his deep perturbation. She was
confident that Mrs. Morrice was at the bottom of it. Had he found out
something to her discredit, and if so, what? Was it possible that Lane
had conceived it to be his duty to report to him that conversation
between aunt and nephew which she had overheard?
They dined alone that night, and she was sure that his deep gloom
must have been noticed by the servants who waited on them. And
she was sure it was not business matters that troubled him. He had
always boasted that he never brought home his office worries with
him, had expressed his contempt for men who did so, who had no
power of detachment. “When a man comes back to his home it is his
duty to make his family happy, and leave his business behind him,”
had been a favourite dictum of his, and to do him justice he had
always acted up to it.
After dinner they went up to the drawing-room, but he made no
pretence of being cheerful. Rosabelle asked if the piano would
disturb him. He shook his head, and she played very softly a few of
her favourite pieces. Suddenly Morrice rose, went to her, and kissed
her.
 “I am wretched company to-night, my little girl,” he said; his face
still wore its hard gloomy expression, but there was a sadness in his
voice that went to the girl’s heart. “You stay here and amuse yourself
as best you can. I am going to my study, and shall not see you again
this evening. Good-night, dear.”
Rosabelle clung to him. “Oh, uncle, can I do nothing to help you?”
He gave her a grateful smile, but shook his head obstinately, and
left the room. She played on a little after he had gone, but she was
full of troubled thoughts, and hardly knew what she was doing.
And Rupert Morrice, the great financier, the successful man of
business, respected by all who knew him, envied by many, sat alone
in his room, devoured by bitter and revengeful thoughts. What had
his wealth done for him, if it failed to buy loyalty from those who were
near to him, on whom he had lavished such kindness and
generosity?
It was only a little past eight o’clock, they had dined early as was
often their custom when they had no company. Would the weary
evening ever come to a close? But when it did, and he went to his
room, he knew he would not be able to sleep.
Suddenly the telephone bell rang. Glad of the momentary
diversion, he crossed to the instrument and unhooked the receiver.
It was Lane’s voice that was speaking. The detective was late at
his office, and it had occurred to him to ring up on the chance of
finding Morrice in and making an appointment for to-morrow
morning. He had that day, after much reflection, judged that it was
time to precipitate matters—to launch his coup.
“Ah, good-evening, Mr. Morrice. I have something of the utmost
importance to communicate to you, and the sooner the better. Can I
see you to-morrow?”
The financier’s deep voice came back through the telephone. “To-
morrow, certainly, any time you please, preferably in the morning.
But, if convenient to you, come round at once. Mrs. Morrice is away;
I am here alone.”
 Lane was rather glad to hear it. He answered that he would come
at once. What he was about to tell Morrice was bound to produce a
violent explosion, but it would not occur while he was in the house.
A few moments later the detective stood in the financier’s private
room, in a mood almost as serious as that of Morrice himself.
 CHAPTER XXI
ROSABELLE HAS A GRIEVANCE

“Y OU have something of importance to communicate to me, Mr.

Lane,” were Morrice’s first words. “Take a seat, please.”
“Something of the greatest importance, and also, I am very sorry
to say, of a most unpleasant nature. You must be prepared to receive
a great shock, Mr. Morrice.”
A grim smile fleeted across the financier’s gloomy countenance.
He had already received a very startling shock, in time he would get
inured to them.
“It concerns a young man named Archibald Brookes who, I
understand, is a frequent visitor at your house, also a member of
your family, the alleged nephew of your wife and also of Sir George
Clayton-Brookes, supposed to be her brother-in-law by the marriage
of his brother Archibald, who died in Australia, to her sister.”
At the two ominous words “alleged” and “supposed,” Morrice
looked keenly at his visitor, but he made no comment. He knew this
was a man who did not speak at random, who carefully weighed his
utterances. What was he going to hear now? Well, nothing would
surprise him after what he had already discovered for himself.
Duplicity came naturally to some temperaments.
The detective went on in his calm, even voice. “It is one of the
disagreeable duties of our profession to make unpleasant
disclosures. I made certain discoveries after taking up this case for
Mr. Richard Croxton which up to the present I have withheld from
you, out of consideration for your feelings. The time is come when
you ought to know the truth. Sir George’s family consisted of himself
and two brothers, there were no sisters. Both of these brothers died
unmarried. Therefore Sir George can have no nephew. Mrs. Morrice
was the only child of a not very successful artist; her mother lost her
life in giving her birth. Therefore the same remark applies to her,
young Archibald Brookes is no more her nephew than he is Sir
George’s. And, of course, it follows that there was no marriage
between her sister and his brother.”
Morrice’s face went very white. “You have satisfied yourself that
there is no flaw in your evidence—that it is quite reliable?”
“Unquestionably,” was the detective’s answer. “My evidence with
regard to your wife is her father’s statement made frequently in the
hearing of several persons. As to Sir George’s brother, a colleague
of mine in Australia made exhaustive inquiries on my behalf and
found that Archibald Brookes senior had never married. I have also
got further evidence from an old friend of mine at Scotland Yard who
has had Sir George and his supposed nephew under observation for
some time; that the young man was brought up under the charge of
a woman named Alma Buckley, a not very prominent member of the
music-hall profession, up to the period when Sir George adopted him
and put about this story. Further, that at the time of his adoption
young Archie Brookes was occupying an insignificant commercial
post in the city of London. Of course, you know nothing of all this?”
The words were not put in the form of a question, but rather
conveyed the assumption that it was impossible the financier could
have any knowledge of such a gross deception.
But they brought to the surface at once that fiery temper which up
to the present he had kept in check.
“What do you take me for, sir? My greatest enemy can never say
of me that I have been guilty of a mean or dishonourable action. Do
you think for a moment, from any motives whatever, even from a
desire to shield one so closely related to me, I would be a party to
such a shameful fraud?”
Lane hastened to pour oil on the troubled waters. “Pardon me, Mr.
Morrice, I did not hint at such a thing. I said that, as a matter of
course, you knew nothing about it.”
“It was almost unnecessary that you should say even as much as
that,” growled Morrice, only half appeased. His mind was quick
enough when he chose to exercise it. This man had been rendered
suspicious and distrustful of everybody by his calling, and the sinister
secrets he discovered in the pursuit of it. He had half suspected, or
at any rate thought it within the bounds of possibility, that Morrice
might have some inkling of what had been going on, and he had
chosen this way of provoking a definite disclaimer.
“There are other things it is my duty to tell you,” went on the
detective smoothly; he was not going to take any further notice of
that angry outburst. “For some long time past Mrs. Morrice has been
in the habit of supplying the young man with money. I cannot
estimate the amount that has passed into his hands, but judging
from his extravagant habits, I should say it must be a considerable
sum, much more than the lady could afford if she were to maintain
her position as the wife of a wealthy man.”
A lightning inspiration came to the unfortunate financier. “Am I not
right in saying that you sent me an anonymous letter on this very
subject?”
Lane felt it was useless to prevaricate. “I did. I may be wrong, but I
felt it was the best way to set you on the track. I thought it would be
very painful for you to be warned in a more open and direct way. I
trust that the suspicion I threw out was not justified.”
He said this with a very good show of concern, although he was
certain he had not fired that shot at random. Mrs. Morrice’s avowal
that she had been half ruined, and that it could not go on, had
convinced him that her assistance to young Brookes had not been
confined to a few hundreds out of her annual allowance—these
would have gone no way with such a determined prodigal.
For the first time in his life, Rupert Morrice’s proud head drooped
in deep humiliation. It was terribly degrading to him to listen to the
detective’s merciless recital, to know that the treachery of the woman
who bore his name, to whom he had given an honoured and assured
position, was, as it were, the common property of others.
“Alas,” he said, in a voice from which every trace of anger had
fled, which only expressed feelings of the most unutterable sadness.
“Your suspicions have been fully justified. From whence did you get
all this information that enabled you to make such an accurate
diagnosis of what was happening?”
But Lane was very staunch, and as high-minded as a man could
be in the trying circumstances of such a profession. He would
certainly not give Rosabelle away, for if he did Morrice would be sure
to think she should have come to her uncle first and discussed with
him the propriety of going to Lane at all. He had in a manner rather
stolen a march upon her, but she should not suffer.
“You must excuse me, Mr. Morrice, if I am unable to answer that
very natural question. I always like to be as frank as possible with my
clients, but there are times when, from motives perfectly satisfactory
to myself, I am unable to reveal the means by which I obtain our
information.”
Morrice made no reply. He would have dearly loved to know, but
he was fair-minded enough to appreciate the detective’s excuse.
Probably he had obtained his knowledge from some prying servant
in the house who had kept a close watch upon his wife. Lane was
not the man to despise the assistance of any instrument, however
humble. Not for one moment did it occur to Morrice that his niece
was implicated in the matter.
“And now, Mr. Morrice, I don’t wish to ask you more than I can
help, for I can fully understand how you must be suffering, and how
painful it must be for you to talk over these things with a stranger.
But you say that my suspicions are confirmed—in short, you have
made your investigations and found what I surmised, that a
considerable number of jewels have been realized, and imitations
put in their place. Am I right in saying that it means a large sum?”
“Several thousands of pounds, even taking into account the
depreciated price which could be obtained for them,” was Morrice’s
answer.
“I guessed it. But I doubt if it has all gone into the pockets of young
Brookes. Mind you, I have no actual evidence of what I am going to
say—it is, if you like, absolute theory—but Sir George is in this game
and has engineered it from the beginning. They are in this together,
depend upon it. Which gets the better share I cannot say; I should
fancy the older and more experienced rogue.”
“I daresay you are right,” said Morrice wearily. “We know him to be
a rogue from his being a party to this nephew fraud. And yet he
poses as a rich man, although Mrs. Morrice has more than once
dropped a hint that he is fast dissipating his money at the gaming-
table.”
So that was his vice attributed to him by one who knew too well,
thought the detective. That accounted for his being well-off one day
and a pauper the next.
After exacting from Morrice a promise that he would not use the
information in any way, Lane told him what he had picked up from
his friend at Scotland Yard, viz. that Sir George was strongly
suspected of being in league with high-class crooks.
The unhappy financier sat crushed and humbled by all these
terrible revelations. His world seemed falling about his ears—his
wife, of whose integrity he had never entertained the slightest
suspicion, the friend and confidant, the associate in a vile deception,
of a man of good birth and position strongly suspected of being
engaged in criminal enterprises. He had never taken kindly to Sir
George; he was too plausible and artificial for his liking. For the
supposed nephew he had entertained a good-natured contempt. But
he had never harboured the faintest idea that they were a couple of
base scoundrels.
Lane rose to go. Later on he would have to say more to Mr.
Morrice, but to-night he had said enough.
“I think you told me over the telephone that your wife was away. I
suppose you have said nothing to her yet?”
“Nothing,” answered Morrice, with a face like granite. “I have not
had time. It was only to-day that I got the full amount of proof I
wanted. If it had only concerned itself with one article of jewellery, or
a couple at the outside, I might have thought she had sold them to
defray some gambling debt, some bills that she was ashamed to tell
me about.”
“Quite so, Mr. Morrice. But I take it when your wife returns you will
confront her and extort a confession.”
Nothing could have been grimmer than the husband’s expression
as he answered. It was easy to see he would be as hard as flint
when his righteous wrath was aroused—pitiless, unforgiving.
“Of course. And please, Mr. Lane, do not speak of her as my wife.
The law, I know, will not sever the tie for such a cause as this, but so
far as I am concerned that tie is already severed. She returns to-
morrow, and in another twenty-four hours the same roof will not
shelter us. I shall not leave her to starve; I shall make her a decent
allowance, and she can live out the rest of her shameful life in the
society of friends congenial to her—this scoundrel Clayton-Brookes
and the rascal whose aunt she pretends to be—perhaps the woman
Alma Buckley, of whom I have never heard.”
“And whom she visits secretly,” interposed Lane. “I have had her
watched and know that for a fact.”
“Ah, I am not surprised; in fact, nothing would surprise me now.
Mark you, I shall not publish to the world the story of her treachery.
Why should I fill the mouths of curious fools? It would not undo my
wrongs nor alleviate my bitter humiliation. I shall agree with her to
concoct some tale of incompatibility extending over many years and
culminating in a separation absolutely necessary for the peace of
mind of both. The truth will be known for certain to two people, you
and myself, perhaps a third—my niece Rosabelle Sheldon. You, I am
convinced, Mr. Lane, are a man of discretion and will keep your
knowledge to yourself.”
Lane assured him that the secrets of all his clients were sacred to
him. One last question he put before he left.
“You will make her confess who this so-called Archie Brookes
really is?”
And Morrice’s voice was as hard as iron as he answered: “You
may rely upon me to do my best. Good-night, sir. What I have
learned through your masterly activities has been inexpressibly
painful, but thank heaven I know at last the foes in my own
household. I shall no longer live in a fool’s paradise.”
Shortly after Lane’s departure he went to his room, but try as he
would, sleep refused her kindly solace. The man had been shaken to
the very foundation of his being.
On his way out Lane found Rosabelle waiting for him in the hall as
on a previous occasion; she had heard of his visit from one of the
servants.
“Why are you here to-night?” she whispered. “Has anything of
importance happened?”
“A great deal,” Lane whispered back. “It was not till the last
moment I made up my mind to come, but certain things happened
which rendered it necessary to hasten matters. I have not time to tell
you now, it would take too long. Slip down to my office to-morrow
morning as early as you can.”
Much wondering, the girl promised she would be there as near ten
o’clock as possible.
“And just one last word, Miss Sheldon. I have told your uncle that
young Brookes has been sponging on Mrs. Morrice, and much has
been found out. But your name has not been brought in. Forget all
about that conversation you told me of. Best, if your uncle should
question you to-night or to-morrow, to dismiss it from your mind, to
appear surprised as you would have been if you had never
overheard it. I will explain to-morrow. Good-night. I will not stop a
second longer; he might come out any moment and surprise us.”
Restless and impatient for that to-morrow, the girl’s sleep was little
less broken than her uncle’s. What was Lane going to tell her? Was
he going to be perfectly frank after all?
She was there a little before the time appointed, but Lane was
disengaged and saw her at once. He made a clean breast of it this
time, and told her everything that had happened from the beginning
of his investigations.
 “I may as well tell you that I went over to Mr. Croxton the other day
and told him all that I knew. And I am afraid you will never forgive
me, Miss Sheldon, when you know that I made it a condition of my
confidence that he should keep it to himself till I removed the
embargo. But I had my reasons, reasons which I can’t very well
explain and which, I am sure, would be unconvincing to you.”
Rosabelle was very shocked at her aunt’s duplicity and disgusted
when she learned the truth about Archie Brookes. But she was not
so preoccupied with the emotions to which his recital gave rise as
not to be more than a little hurt that Lane had kept her in the dark
longer than anybody else.
“I suppose the truth is you have a contempt for women, and place
no trust in them?” she said resentfully.
The detective made the most diplomatic answer he could in the
circumstances, apparently with a satisfactory result. Anyway, they
parted good friends.
 CHAPTER XXII
HUSBAND AND WIFE

M ORRICE stayed in the next day waiting for the return of his wife
from her country visit. She was to arrive home in time for lunch.
About twelve o’clock Rosabelle came into his room; she had just
returned from her visit to Lane.
“Oh, uncle, there is a strange young man in the hall with a letter for
auntie. He says his instructions are to give it into her own hands. He
was told that she would be back before lunch-time, and he said he
would wait. He seems rather mysterious. Would you like to see
him?”
Morrice nodded his head and strode into the hall, where he found
standing a sallow-faced young fellow, quite a youth, with a tall
footman mounting guard over him, as it were, on the look-out for
felonious attempts.
“What is it you’re wanting, my man?” he asked roughly. He did not,
any more than his servant, like the appearance of the fellow, who
seemed a furtive kind of creature with a shifty expression.
The furtive one explained hesitatingly in a strong cockney accent:
“A letter for Mrs. Morrice, sir. I was to be sure and give it into no
hands but her own.”
Something very suspicious about this, certainly. Morrice thought a
moment, pondering as to the best way to proceed with this rather
unprepossessing specimen of humanity. He had a common and
unintelligent kind of face, but he looked as if he possessed a fair
share of low cunning.
A week ago Morrice would have thought nothing of such an
incident; he would have told the man to come later when his wife
would have returned. But recent events had developed certain
faculties and made him anxious to probe everything to the bottom, to
scent mystery in every trifling act.
“Who sent you with the letter, and gave you such precise
instructions, my man?”
The answer came back: “Mrs. Macdonald, sir.”
Morrice’s brows contracted. He was as sure as he could be of
anything that the man was telling a lie.
“Mrs. Macdonald, eh? Where does she live?” was the next
question.
This time the answer did not come as readily; there was a
perceptible hesitation. Morrice guessed the reason as rapidly as
Lane himself would have done. The sender of the letter had primed
the messenger with a false address. Out of loyalty to his employer,
he had been cudgelling his rather slow brains to invent one.
“Number 16 Belle-Vue Mansions, Hogarth Road, Putney,” he said,
speaking after that slight hesitation with a certain glibness that was
likely to carry conviction.
Morrice did not know of any woman of the name of Macdonald
amongst his wife’s acquaintances. Still, that might mean nothing; it
might be a begging letter which the writer had taken these unusual
means of getting to her.
“Let me have a look at the envelope,” demanded Morrice.
The shabby, furtive-looking young fellow began to appear a bit
uneasy, with the dictatorial master of the house regarding him with
anything but a favourable eye, the young girl standing in the
background who seemed no more friendly, and the tall footman
standing before the door, barring a sudden exit.
“Beg pardon, sir, but my orders was most precise to only give it
into the hands of the lady herself.”
Morrice saw that he must change his tactics. He took from his
pocket a couple of treasury-notes which made a pleasant crackle as
he flourished them before the youth’s face.
 “You see these, don’t you? I take it you haven’t got too much
money. They are yours if you let me see the envelope, only the
envelope. I don’t want to take your letter,” he added with a cunning
that was quite a recent development of his character. “As soon as
I’ve seen that you can go out and come back in an hour when Mrs.
Morrice will have returned home.”
The youth fell into the trap. Slowly he produced from his pocket
the letter which he held gingerly between his finger and thumb for
the inspection of the superscription on the envelope. Quick as
lightning, Morrice snatched at it and put his hand behind his back,
throwing at him with his disengaged hand the treasury-notes he had
promised.
“Now get out of this, my fine fellow, and never dare to come to this
house again with such an impudent message. Tell Mrs. Macdonald
of Putney, or whoever it may be that sent you, that Mr. Morrice
insisted on having that letter, and that it will be given to Mrs. Morrice
on her return.”
The furtive creature slunk away; after that drastic action he had no
more fight in him. Morrice remembered the waiting footman whose
impassive countenance did not betray any surprise at this rather
extraordinary scene over what seemed a trifle, and turned to his
niece with a smile that was decidedly forced.
“Never heard of such cheek in my life. Some impudent mendicant,
I expect. By gad, they are up to all sorts of dodges nowadays.”
He marched back into his own room, and Rosabelle went to hers
to think over what this action of her uncle’s meant. It was evident he
attached considerable significance to that letter which was only to be
delivered into Mrs. Morrice’s hands. What was he going to do with it?
Well, it did not much matter. He knew enough now, and in a very
short time the bolt would fall, according to what Lane had told her.
Morrice had made up his mind what to do with it. Never in his life
had he opened correspondence not intended for his perusal; never
again, he hoped, would he be forced to resort to such a mean action.
But everything was fair now; it was justifiable to meet cunning with
cunning, duplicity with corresponding duplicity.
He opened that letter with the sure instinct that it would be of help
to him, and he was not deceived. There was no address and no
signature. Evidently the handwriting was too well known to Mrs.
Morrice to require either. It was very brief; but even if he had not
known what he already did, it would have revealed to him a great
portion of what he had lately learned.
“A young man has been to see me, says he is not a
professional detective, and doesn’t look like one, but very
keen. Wanted to get out of me all about your early life. Of
course, he got nothing. The worst is he seems to know
something about Archie, knows that I brought him up. Be
on your guard; I am afraid trouble is brewing.”
He put this damaging missive in his pocket along with the
anonymous letter, and presently went up to his wife’s room to await
her return to the home which, he had resolved, should no longer
shelter a woman who had deceived him so grossly. He guessed at
once the writer of this warning note—it could be none other than
Alma Buckley, the friend of her youth. The reference to her having
brought up the man known as Archie Brookes proved that beyond
the possibility of doubt.
How long it seemed before the minutes passed and the door
opened to admit the familiar figure! Preoccupied with her own
thoughts, Mrs. Morrice hardly looked at her husband as she
advanced to give him the perfunctory kiss which is one of the
courtesies of a placid and unemotional married life.
But when he drew back with a gesture of something like
repugnance from the proffered caress, she noted for the first time the
terrible expression on his face, and was overcome with a deadly
fear.
“What is the matter? Why are you looking like that?” she gasped in
a trembling voice.
 Consumed inwardly with fury as Morrice was, he exercised great
control over himself. He knew that he would put himself at a
disadvantage if he stormed and raged; he must overwhelm this
wretched woman with the pitiless logic of the facts he had
accumulated. He must act the part of the pitiless judge rather than
that of the impassioned advocate.
He advanced to the door and turned the key, then came back to
her and pointed to a chair. There was a cold and studied deliberation
about his movements that filled her guilty soul with a fearful terror.
“Sit there while I speak to you,” he said in a harsh and grating
voice. “You have much to account to me for. Read that.”
He drew the anonymous letter from his pocket and flung it in her
lap.
Like one dazed, she drew it from the envelope with trembling
fingers, and very slowly, for her thoughts were in terrible confusion,
mastered its accusing contents. Then she looked up at him with a
face from which all the colour had fled, leaving it ghastly to look at.
“It is a lie,” she stammered in a voice scarcely above a whisper.
“It is the truth,” he thundered, “and you are as shameless in the
hour of your detection as you have been in your career of fraud and
deceit.”
“Prove it,” she cried faintly, still feebly trying to oppose his
gathering anger.
“You have lived with me a good many years,” he said witheringly,
“and yet you know so little of me as to think I should speak like that if
I were not sure I was on firm ground. And yet perhaps you have
some excuse. I have been a blind fool so long that you were justified
in your hopes I should continue blind to the end. Well, that letter
opened my eyes. Your fortunate absence gave me facilities that it
might have been difficult to create. I have taken several of the most
valuable articles in your collection and had them examined. Need I
tell you the result? Your guilty face shows plainly enough that you
need no telling.”
 And then her faint efforts at bravado broke down.
“Forgive me,” she moaned. “I yielded in a moment of temptation.
Many women have done the same; they were my own property after
all,” she added with a feeble effort at self-justification.
That answer only provoked him the more. “A moment of
temptation,” he repeated with scornful emphasis. “Rather many
moments of temptation. This has been going on for years; these
things were realized piece by piece. And now tell me—for I will have
the truth out of you before you leave this room—where have these
thousands gone, what have you got to show for them?”
It was a long time before she could steady her trembling lips to
speak, and when she did the words were so low that he could only
just catch them.
“Nothing. I have been a terribly extravagant woman. I have lost
large sums of money at cards. You never guessed that I was a
secret gambler—there is not a year in which I have not overstepped
my allowance, generous as it was. I was afraid to come to you.”
He silenced her with a scornful wave of the hand. “Lies, lies, every
word you have uttered! You have done none of these things you
pretend; it is an excuse you have invented in your desperation.”
He drew himself up to his full height and pointed a menacing finger
at the stricken woman. “Will you tell me where these thousands have
gone? No, you are silent. Well then, I will tell you—not in gambling
debts, not in unnecessary personal luxuries—no, if it were so I would
be readier to forgive. They have gone to support the extravagance of
that wretched idler and spendthrift who is known by the name of
Archie Brookes. Do you dare to deny it?”
She recognized that he knew too much, that further prevarication
was useless. “I do not deny it,” she answered in a moaning voice.
And after a little pause he proceeded with his denunciation.
“It is as well that you do not, seeing I know everything. Well, bad
as that is, there is worse behind. I have learned more; I know that
you, in conjunction with that smooth scoundrel Clayton-Brookes,