Debunking an urban legend of the Deep Sea: The Queen of the Abyss
and her contribution to Ceratioid Anglerfish biology.
Toshiro Saruwatari
Ocean Research Institute, The University of Tokyo
1-15-1 Minamidai, Nakano, Tokyo 164-8639
JapanE-mail: tsaruwat@ori.u-tokyo.ac.jp

We inhabit a watery planet. The sea covers more than 70 % of the earth's surface. The average
depth of the world's ocean is 3,800 m. So, if Mt. Fuji, with an elevation of 3,776 m, were placed on thebottom of the ocean, its summit would not break the sea surface at the ocean's average depth. The depth of
4,000 m is the divide between the bathypelagic and the abyssopelagic zone, so the major part of the world'socean consists of the abyss, or deep sea, one of mankind's few remaining frontiers. This vast expanse of theearth's aquatic environment is filled with fish. About 29,000 species of fish are currently recognized, andthey come in all sorts of shapes, sizes and colors (Nelson, 2006). Deep-sea fishes are no exception. Ask any
boy or girl,‘Do you know any deep sea fishes?’ The chances are nine to one that they will answer‘Anglerfish!! They have lanterns that light up dangling right in front of their mouths! They use that lantern
to attract and eat little fish!! Shrimp too!’ Anglerfishes, Ceratioid Anglerfishes to be more precise, are by
far THE most popular deep-sea organisms inhabiting the vast abyss of the world's oceans. Not only do
Ceratioid Anglerfishes play an important role as high- ranking predators in the deep sea ecosystem, but

example of such a mode of reproduction found among the vertebrates. This article is an account of myonce-in-a-lifetime encounter with the Queen of the Abyss, which literally jump-started my ichthyologicalresearch on Ceratioid Anglerfish biology. It is by pure coincidence, a stroke of luck, that I now find myself
looking into another human frontier, the abyss, through a unique looking glass, Ceratioid Anglerfishes. I

Toshiro Saruwatarialso, their unique mode of reproduction, males adhering to the body surface of the female, is the soleconsider myself a very lucky individual to be blessed with such an opportunity, and that is a grossunderstatement.

A short primer on Ceratioid Anglerfish biology.
Among the 29,000 species of fish recognized to date, no group of Teleost stands out in its
uniqueness, in both morphology and ecology, as the Ceratioid Anglerfishes. Currently 11 families, 35
genera and 162 species are recognized, making it one of the most specious groups of Teleosts in the deepsea. (Pietsch 2005. Pietsch and Orr, 2007). Attracting its prey with its bioluminescent light organ called the128

Fig. 1: Female Ceratias holboelli, Ceratiidae. HUMZ95300. Arrow indicates poiasitic male.

hanging from the tip of the illicium, a modified dorsal fin ray, Ceratioid Anglerfishes are the top ranking
carnivore of the deep sea (Shimazaki and Nakaya, 2004) (Fig. 1). There even exists an interesting video
taken from an underwater submersible showing a Gigantactinid Anglerfish fishing the ocean floor with its
esca, swimming inverted (Moore, 2002).

Not only is the esca important as an apparctus for predation, it isalso the most important morphological diagnostic character of Ceratioid Anglerfishes.
Another remarkably unique feature of Ceratioid Anglerfish is its mode of reproduction. Males
attach themselves to the female's body surface
and become parasitic male, forming an extreme
example of symbiosis in the abyss. (Fig. 2). 5families, 10 genera and 23 species of Ceratioid
Anglerfishes are known to possess this uniquemode of reproduction (Pietsch, 2005). Pelagicmales, when they find a female, bite onto herskin and make a permanent attachment to thefemale body. This unique and elaborate mode

Fig. 2: Male attached to female C. holboelli of Fig.1.
HUMZ95300.

of reproduction was first reported by an Icelandic ichthyologist, Bjarni Saemundsson in 1922
Toshiro Saruwatari(Saemundsson, 1922). Later, Regan and Trewavas (1932) made a first comprehensive review of Ceratioid
Anglerfish biology, followed by Bertelsen (1951), and Pietsch (1976, 2005). In species whose males attach
to the female, this attachment seems to be obligatory for the individual male's subsequent survival andsuccessful reproduction． Males which have failed to find and attach to a female perish. For example, thelargest known pelagic male Cryptopsaras couseii is 14.3 mm TL, smaller than most parasitic males
reported (Bertelsen, 1951). A rather bizarre and extreme example of parasitic male is reported in Pietschand Nafpaktitis (1971). A male Melanocetus johnsoni was found attached to a female Centrophryne
spimulosa. The two species belong not only to different genera, but they are of different families,
Melanocetidae and Centrophrynidae, respectively. Although parasitic male found in Ceratioid Anglerfish iswell known, fundamental aspects of the unique mode of reproduction, such as how males find females inthe abyss, how nutrients are passed over to the male from the female, how the immune system issuppressed so male tissue is not rejected by the female, are not known, and currently does not reach beyond

129the educated guess of researchers. The following is my small contribution to Ceratioid Anglerfish biology,
conducted with the help of numerous friends and colleagues, as well as from the Queen of the Abyss andher eight little dwarfs.

The Queen of the Abyss enters the stage!
My encounter with The Queen occurred unexpectedly. It was during research cruise KH00-2 of RV
Hakuhou-maru. One of the graduate students carrying a large bucket containing the samples taken with
4m-frame trawl net exclaimed, ‘There's something big in here!’ Big and beautiful indeed. It was a truly
beautiful specimen of Cryptopsaras couseii, 316.5 mm in standard length (SL)(Figs. 3,4) It was at thatmoment, there and then, that The Queen of the Abyss departed her abyssal palace and entered a new realm,that of mankind and more specifically, of ichthyology.

Toshiro SaruwatariFig. 3: The Queen of the Abyss. A female Cryptopsaras couseii,, Ceratiidae, 316 mm SL. Right side of the body.
bod
Number indicate the number of male described in text.

Fig. 4: The Queen of the Abyss. A female C. couseii 316 mm SL. Left side of the body.
Number indicate the number of male described in text.

The Queen of the Abyss in all her charm.
Gleaming in black, ‘The Queen’ was the most beautiful and intact specimen of an anglerfish one
would ever see, in an almost pristine condition, A rarity in deed considering the fact that most Ceratioid
Anglerfish are caught with torn and ruptured skin. Her spinulous skin was intact, not ruptured and torn
anywhere. A closer examination of this black beauty revealed even more surprising findings.

130

When touched, the three knob like appendages located at the anterior base of the dorsal fin secreted
light bluish fluid. At first, these knob-like appendages and fluid were thought to be parasitic males andsperm, but were later confirmed to be carancules and bioluminescent fluid. But the largest surprise(s?)
were to follow. A total of EIGHT! males were attached to her body. Yes,

not one, two or three, but eight.

One male on the right side of her body, and seven more males on her left side! Until then, the maximum
number of males attached to a female Ceratioid Anglerfish was six, so she was indeed The Queen of theAbyss, having eight little dwarfs attached to her body. One for the Guinness Book of World Records!

The eight little dwarfs.
A brief description of each parasitic male is provided below. Size of attached males ranged from35.0 to 56.0 mm in total length (TL). It should be noted that eye, dorsal, anal, pectoral and caudal fin rays,and mouth opening were still present, and also, the degree of development of the skin flap varied between
males. The skin flap is part of the skin around the snout of males, which expand after attaching to a female.
Male No. 1: 46.5 mm TL. Sole male attached to the right side of the female, in the branchial region. (Fig. 5)
Skin flap, where the male adheres to the female body was round in shape.
Male No. 2: 35.0 mm TL. One of twomales attached to the base of the leftpectoral fin. This individual is locatedmore dorsally, above male No. 3 (Fig. 6).
This individual is attached with an uprightbody orientation. Both upper and lower
skin flaps are well developed, fused toeach other as well as to that of male No. 3.
But an opening still exists between the twoskin flaps. One notable feature of how thisToshiro Saruwatarimale attaches to the female is that the skinFig. 5: Male No.1. A male C. couseii attached to the right sideof the female. 46.5 mmTL.

flap extending from the lower jaw islocated above the upper jaw skin flap ofmale No. 3.
Male No. 3: 56.0 mm TL. Attached to theleft side of the female, below male No. 2(Fig. 6). This male is the largest of theeight attached males. This male is also
attached with an upright body orientation.
Upper-jaw skin flap is partially covered bylower-jaw skin flap of male No. 2.

131

Fig. 6: Males No. 2 and 3, attached to the left side of the female.
35.0, 56.0 mm TL respectively.

lower-jaw skin flap of male No. 2.
Male No. 4: Attached to the female below the left pectoral fin, and below male No.3 (Fig.7). In Fig. 9,caudal fin of male No. 4 reaches the cephalicregion of male No. 8. Upper and lower jaw
skin flaps are not fused to each other, and an
opening exist between the two skin flaps.
Male No. 5: 47.0 mm TL. Attached below
male No.4, on the left abdominal region of thefemale (Fig. 7). Upper jaw skin flap is welldeveloped.

Mouth opening is still present.

This male is attached with an upright bodyorientation. After initial observation, this malewas surgically removed from the female andwas fixed in 10 % buffered formalin. It swam
in the formalin, vigorously moving its wholebody for a few seconds.

Fig. 7: Males No. 4, 5, and 6 attached to the left side ofthe female. Male 5 and 6 are 47.0, 43.3 mm
TL respectively.

Male No. 6: 43.3 mm TL. Attached to thefemale below male No. 4 and behind male No.
5 (Fig.7). This male is attached to the female
with an upright body orientation.
Male No. 7: 37.4 mm TL. Attached to the left
dorsal region of the female, below thecarancules, anterior to the dorsal fin origin(Fig. 8). Upper and lower jaw skin flaps arean opening still exists between the upper andFig. 8: Male No. 7, attached to the left side of the female.
37.4 mm TL.

lower skin flaps.
Male No. 8: 44.0 mm TL. Attached to the left
abdominal region of the female, posterior to

Toshiro Saruwatariwell developed, fused to each other. However,males Nos. 4, 5, 6 and below male No. 7 (Fig.
9).

Caudal fin of male No. 4 reaches thecephalic region of male No.8. Skin flaps ofboth upper and lower jaws are well developed,
the two fused to each other anteriory.
However, an opening still exists between thetwo skin flaps. This male is attached to thefemale with an upright body orientation.

Fig. 9: Male No. 8, attached ot the left side of the female.
44.0 mm TL.

132

After making initial observations and photographing the specimen, I put my head to work in full gear,
deciding what to do to make the best possible use of this specimen(s?). Fortunately, I had with me onboard RV Hakuhou-maru a JEOL rapid freezing apparatus and two thermos containers full of liquidnitrogen. Vials, ethanol and formalin were abundant. So, decided to make some samples for histologicalstudy using the rapid-freezing apparatus for freeze-substitution fixation, as well as to preserve tissue
samples from the males and female for molecular study.

Sibling analysis and electron microscopeobservations were later made utilizing these samples.

Sibling analysis between female and parasitic males.
Muscle tissues were taken from the dorsal region of female and male Nos. 2, 3, 5, 6 and 8 and werepreserved in absolute ethanol. Sibling analysis was conducted by RAPD-PCR fingerprinting using Operon
10-mer kit A set. Clear bands were obtained from primer nos. 3, 4, 5, 7, 9, 11, 13, 15 and 17 (Fig. 10). Theband patterns obtained denied sibling relationships between female and attached males as well as between
males (Saruwatari et al., 2001). The queen and the dwarfs did not share a common bloodline.

Toshiro SaruwatariFig. 10: Results of RAPD-PCR fingerprint method. Numbers above each image depict primer No. of Operon
10-mer kit A. Letters and numbers below are, MK: marker. F: female. Other numbers represent malenumbers described in text. Modified from Saruwatari et al., 2001.

After hatching, males of C. couseii have a pelagic larval stage in the sea prior to attaching to afemale. Therefore, individuals undergo a great deal of oceanic dispersal. causing the mixing of the genepool in the sea, resulting in the lack of sibling relationships between the female and the attached males aswell as between the attached males. Denial of sibling relationships reinforces another question on parasitic
males. If male and female do not possess sibling relationships, then why are males not rejected from thefemale body surface? It there some type of mechanism to suppress the female and male immune system?
Answers to these questions will have to wait, until an immunologist shows interest in Ceratioid
Anglerfishes and goes out to sea to collect new and fresh material, another Ceratioid Anglerfish female

133with male(s) attached to her body. An immunologial study on male attachment has great medical andpharmaceutical potential. It may be possible to apply the results of such studies to human organ transplantresearch, to aid in development of immune suppression agents.

Ultra microstructure of sperm.
After initial examination of the queen and her eight little dwarfs, testes and seminal fluid were
sampled from males Nos. 5 and 6. Samples were frozen rapidly using JEOL rapid freezing apparatus andwere preserved in liquid nitrogen. Samples were then transferred into freeze substitution fluid consisting of
2% osmium in acetone, and then into acetone frozen with dry-ice for 3 days at -79, -20 for 3 days andpreserved at 4. After freeze substitution, samples were embedded in Epon resin. Ultra-thin sections
were prepared using a Nanotome diamond knife attached to a LKB-V Ultra microtome and observed with aPhilips CM-10TEM. This is the first TEM observation made on the sperm of a Ceratioid anglerfish to date.
The spermatozoon possessed a large spherical head, numerous mitochondria and a single flagellum

Fig. 11: Electron micrograph of C. couseii spermatozoon. F: flagellum; M: mitochondria; N:nucleus; PC:proximal centriole. Modified from Saruwatari and Sakai, 2006.

Toshiro Saruwatari(Fig.11). Transverse section of the tail revealed a 9 + 2 flagellar pattern.

Debunking an urban legend of the abyss.
There exist an urban legend of the abyss. It has been believed for some time now, that after
attaching to a female, male Ceratioid Anglerfish lose their fins, are absorbed by the female and turn into a
simple knob like structure. And, that the male testes is the only remaining organ which mature and become
functional! Apparently, this is not the case, as the queen and her little dwarfs have shown. The sizes ofattached males ranged from 35 to 56.0 mm TL. Differences in the sizes of parasitic males are indicative ofdifferent and multiple incidences of attachment to the female at different times. As mentioned earlier, the134

largest known pelagic male C. couseii is 14.3 mm TL (Bertelsen, 1951). Males that fail to find a femaleand attach to her body perish. Male No.3 at 56.0 mm TL still possesses fin rays on all the fins, as did allother males. Degree of somatic development of the attached males is more advanced than pelagic malelarvae prior to attachment. When removed from the female and placed in a vial filled with formalin, maleNo.5 swam in the preservation fluid. This shows that males are able to move their muscles to some extent,even after attaching to a female. These observations show that after attaching to a female, somatic growthand development of parasitic males continue, utilizing nutrients received from the female for growthinstead of consuming internal nutritional reserves or through self-degeneration and absorption of their owntissue. How nutrients are transferred from the female to the attached male is yet to be revealed.
Debunking a long standing urban legend of the deep, that male Ceratioid Anglerfish degenerate and turninto a knob-like appendage after attaching to a female, is perhaps the greatest contribution of the Queen of
the Abyss and her eight little dwarfs to ichthyology so far.
My studies on Ceratioid Anglerfish biology are still ongoing, with aid from the Queen as well as frommuseum specimens. Efforts are being made to obtain fresh new material from the sea by going out on
research cruises…a vast frontier of research in Ceratioid anglerfish biology still remains to be explored!

Acknowledgements.
I am greatly indebted to Senzo Uchida, Keiichi Sato and Minoru Toda, as well as the curators andstaff of the National Okinawa Churaumi Aquarium for providing me with this opportunity to present a talkat their International Symposium as well as providing me with the opportunity to present this manuscript.
Sincere thanks goes to researchers and crew on board cruise KH-00-2 of RC Hakuhou-maru, especially
Teruhisa Komatsu, Kenichi Ishida, Tadashi Inagaki and Chiaki Igarashi. Thanks are also extended toTheodore W. Pietsch, Andrew Shedlock, Yoshiro Watanabe, Takanori Kobayashi and Toshio Sakai for their
help in the experiments and advice.

Toshiro Saruwatari

Literature cited
Bertelsen, E. 1951. The Ceratioid fishes. Ontogeny, taxonomy, distribution and biology. Dana Rep No. 39.
276 pp.
Moore, J.A. 2002. Upside-down swimming behavior in a Whipnose Anglerfish (Teleostei: Certioidei:Gigantactinidae). Copeia, 2002:1144-1146.
Nelson J.H. 2006. Fishes of the World. Fourth Edition. John Wiley & Sons, Inc. New York. Xx+ 601pp.
Pietsch, T.W. 1976. Dimorphism, parasitism and sex: Reproductive strategies among deep-sea Ceratioid
Anglerfishes. Copeia, 1976: 781-793.
Pietsch, T.W. 2005. Dimorphism, parasitism, and sex revisited: Modes of reproduction among deep-sea
ceratioid anglerfishes (Teleostei: Lophiiformes). Ichthyological Research, 52: 207-236.

135

Pietsch, T.W. and B.G. Nafpaktitis. 1971. A male Melanocetus johnsoni attached to a female Centrophryne
spinulosa (PISCES: Ceratioidea). Copeia: 1971: 322-324.
Pietsch, T.W. and J.W. Orr. 2007. Phylogentic relationships of deep-sea Anglerfishes of the suborder
Ceratioidei (Teleostei: Lophiformes) based on morphology. Copeia, 2007: 1-34.
Regan, C.T. 1925. Dwarfed males parasitic on the females in oceanic anglerfishes (Pediculati Ceratioidea).
Proc. Roy. Soc, B, 97:386-400.
Regan, C.T. and E. Trewavas, 1932. Deep-sea angler-fishes (Ceratoidea). Dana Rep No. 2. 113 pp., Xplates.
Shimazaki, M. and K. Nakaya. 2004. Functional anatomy of the luring apparatus of the deep-sea ceratioid
anglerfish Cryptopsaras couesii (Lophiiformes: Ceratiidae). Ichthyological Research. 51: 33-37.
Saemundsson, B. 1922. Zooliogiske meddelelser fra Island. XIV. 11 Fiske, nye for Island, og supplerendeom andre, tidligere kendte. Vidensk Medd Dan Nathist Foren 75:159-201.(Not read but cited fromRegan 1925)
Saruwatari T., T.W. Pietsch, A. Shedlock, I. Oohara, K. Itaya, T. Chiyotani, M. Abe, K. Ishihara, A. Sakai,A. Sugawara, T. Inagaki, K. Ishida, T. Komatsu, T. Sakai and T. Kobayashi. 2001. Sibling analysis
between female and paraitic males of Cryptopsaras couesi (Ceratiidae, Teleost). DNA
Polymorphism, official publication of the Japanese Society for DNA Polymorphsim Research.

Toshiro Saruwatari

9:82-85.(in Japanese)

136