Laboratory Manual on Fundamental Ichthyology
Edited by Hirokazu Kishimoto, Nobuhiro Suzuki and Izumi Akagawa
This selected English version is translated & edited by Benjamin F. Muto

Tokai University Press

Laboratory Manual on Fundamental Ichthyology
Edited by Hirokazu Kishimoto, Nobuhiro Suzuki and Izumi Akagawa
This selected English version is translated & edited by Benjamin F. Muto
Tokai University Press, 2017.
This publication is supported by Research Institute for Humanity and Nature (RIHN, Project No.14200061),
and School of Marine Science and Technology, Tokai University.

Contents

I. Observation of external morphology ―――――――――――――――― 1
1. General guidelines for sketching fish

Tanaka, Yoichi

2

2. External morphology of sharks (Chondrichthyes)

Tanaka, Sho

5

3. External morphology of rays (Chondrichthyes)

Tanaka, Sho

8

Kishimoto, Hirokazu

12

5. Scales and lateral line canals

Suzuki, Nobuhiro

16

6. Luminescent organ

Kubota, Tadashi

23

4. External morphology of Teleostei

II. Fish measurement ―――――――――――――――――――――――― 27
1. Measuring equipment

Kishimoto, Hirokazu

28

2. How to measure Chondrichthyes; sharks

Tanaka, Sho30

3. How to measure Chondrichthyes; skates and rays

Tanaka, Sho

33

Kishimoto, Hirokazu

35

4. How to measure Teleostei

III. Observation of internal morphology ―――――――――――――――― 41
1. Formation of viscera

Kishimoto, Hirokazu

42

2. Observation of gills

Kishimoto, Hirokazu

45

3. Names and classification of skeletons

Kishimoto, Hirokazu

48

4. How to observe skeletons

Kishimoto, Hirokazu

51

5. Observation of the splanchnocranium, pectoral girdle, and pelvic girdle
		
Kishimoto, Hirokazu & Aoki, Mitsuyoshi

54

6. Observation of the neurocranium

Kishimoto, Hirokazu & Aoki, Mitsuyoshi

58

7. Observation of the vertebral column and vertical fins		Kishimoto, Hirokazu & Aoki, Mitsuyoshi

60

8. Observation of the muscular system

Kishimoto, Hirokazu & Aoki, Mitsuyoshi

64

IV. Related field of experimental ichthyology ―――――――――――――― 69
1. Sketching eggs, larvae and juveniles

Tanaka, Yoichi

70

2. Morphological/ecological characteristics of fish eggs by type and developmental stage
		Tanaka, Yoichi

74

I
Observation of external morphology

I

1 General guidelines for sketching fish

Unlike landscape painting and the preparatory drawing we learned in high school and earlier,sketches in ichthyology experiments are not ones for which artistic quality is demanded. They are
rather understood as being similar to architectural and machinery designs in engineering, and “whatyou see” rather than “good-looking” works are demanded. In other words, sketching is a method
to accurately depict the morphological characteristics of subject fish in detail. Demonstration of theratio and/or the position of body parts will require observations from the front and rear, right and left,
and top and bottom. Basically, sketching uses only sharp lines and stippling, but not techniques of“gradation”, “shading”, and the like. In other words, structures are represented by solid lines, whereas
three-dimensional parts (unevenness) and characteristic markings are indicated by stippling.

1. Materials required
When sketching, ensure that you have a B-2B pencil with a soft lead for outlining the wholeshape in the first rough sketch, and an H-3H pencil with a hard lead that can draw sharp lines in
the finishing sketch. In addition, arrange for an eraser and a knife or a pencil sharpener to keep the
pencil leads constantly sharp. Be sure to have a vernier caliper for the measurement of body parts. An
approximately 30-cm ruler is also convenient to have for the measurement of larger material. It is also
convenient if you have a simple calculator for scaling.
As for the paper you use for sketching, prepare at least 1 sheet of A4-size Kent paper with asmooth surface per fish species. It is preferable to prepare 2 sheets or more at all times because thisquantity may be necessary depending on the contents of the experiment.

2. Before sketching
Depending on the fish species and the specimen condition, the fin rays may be folded insidea groove, and the opercle, preopercle, etc., may be stuck tightly in the opercular part, making theopercles unclear and undistinguishable at a glance in some cases. Therefore, confirm the borders
between parts of the material given to you by not only looking at it but also by opening and closing theparts by touching them with your hand. It will help your sketching later if you determine whether thefin rays are spines or soft rays on this occasion. Ideally, specimens to be drawn will have a regular bodyform, be tagged with a specimen number, and be sorted by individual or species. However, if many
specimens are handled, like in a student experiment, it is not always possible to use an ideal specimen.
It is not uncommon that the specimen itself is deformed, such as being bent. It is difficult in mostcases to correct the body form if specimens are preserved in formalin or alcohol. In the case of raw
specimens, fix the form by closing the mouth, closing the opercular widening, and by other means. For
fins, etc., take measures for extending them, including the use of specimen needles such as setting pins.
Usually in fish sketching, the left side is drawn by placing the fish so that it faces left. However,if the left side is heavily damaged, replace the specimen, or if no extra specimen is available, you may
draw the right side. For extremely depressed species, such as rays, where it is difficult to show theoverall shape of the fish if drawn laterally, sketch them from the top.

2

3. For accurate drawing
Regarding the sketch size of the fish body, draw the fish so that it fills approximately 70−90% of
the width of the Kent paper in the horizontal direction. Be careful not to draw the fish body small justbecause the specimen is small. For drawing accurate lines and dots as you intend, pay attention to thefollowing points as basic actions. Briefly, always keep your elbow to wrist touching the desk and theKent paper, and move your hand as if you are lightly rubbing the desk and paper with the whole arm
using your elbow as the basal point in drawing. If your wrist is above the Kent paper surface, you
cannot draw accurate lines and dots as you intend. In addition, in the case of stippling in particular,
maintain the condition in which the part from your little finger to wrist touches the Kent paper at all
times, and move your thumb, index finger, and middle finger positioning the pencil up and down like
you are putting a cap on it. It is common that you hear a loud “tap” sound while sketching. This is asign that your hand is above the Kent paper (desk), with which you cannot draw a true dot (round), anda short drifted line will be made. Because a “line” is originally consecutive dots, it is not so difficult todraw lines by stippling if you follow the basic actions mentioned above.

4. Sketching procedure
1) Set the scale ratio of the fish to sketch so that it will occupy 70−90% of the whole Kent paper
and is roughly at the center. Next, using projection (see the column), locate and measure the main
proportions, such as the fin size, in the body parts including the head. Mark lines and points on thepaper based on the sizes multiplied by the set scale ratio, and balance them on the whole (Figure
1A).
2) Draw a thin line along the marks to set the whole outline. In this case, the line does not need to becontinuous but may be chopped if it is easier to set the shape (Figure 1B).
3) Once the outlines are set, draw the rays in the fins. In this case, draw them not only by discriminating
spines and soft rays but also accurately in the number. In addition, for species with spines on thehead such as scorpionfish and squirrelfish, you need to accurately draw these as well. Because
these spines and soft rays are used as criteria for species identification in many cases, pay particularattention to their number and position (Figure 1C).
		 As 1)−3) above are only the steps of rough sketching, use a B-2B pencil with a soft lead so thatthe drawing can be easily rubbed out with an eraser.
4) Using an H-3H pencil with a hard lead, draw each outline with a clear solid line along the line(s)drawn in 3). Next, lightly tap the lines with an eraser to rub out the lines in the rough sketch part
(Figure 1D).
5) Next, represent the parts of concentrated pigments including unevenness and characteristic mottles
and spots by stippling. Adjust the pigment intensity by dot density, and never use any solid lines(Figure 1E).
6) Lastly, enter the names of the parts. At this point, consider the position of the lines indicating theparts so that they do not cross each other if possible (Figure 1F).

I. Observation of external morphology    3

A

D

B

E
dorsal fin

pectoral fineyepreopercle
opercleC

F

Fig. 1 Example of sketch of general fish.

4

caudal fin
anal finpelvic fin

2 External morphology of sharks (Chondrichthyes)

I

The skeleton of Chondrichthyes is formed of cartilage. Although the degree of calcification of
the cartilages varies considerably depending on the species, the cartilaginous skeleton sufficiently
protects the brain and supports the body and fins. The body (1) shows various morphologies basedon this skeleton. Generally, sharks and rays are distinguished by having gill slits (external gill slits)(13) on the lateral sides and the ventral side, respectively. The body form of many sharks is fusiform
or fusiform and elongated, but a few species are compressiform. The body (1) is covered by placoid
scales and consists of the head (2), trunk (3), tail (4), and fins (15−23). The trunk accommodates the
major digestive organs and urinary and reproductive organs. In sharks, mature females have higher
ratios of trunk length to total length than males in some cases because they form large eggs and/or
1
2

4

3
5

6

19

18
23

12

9

20

21

24

23-2

11

7

22

13
15

A

14

16

23-1

5

17
25

11

9

13
8
7
10

B

C

Fig. 1 Sketch of a fictional fish, “Spiny Porbeagle” (male). A, lateral view; B, ventral view of head; C, eye.
1. Body
2. Head
3. Trunk
4. Tail
5. Precaudal tail6. Snout
7. Mouth

8. Labial-furrow
9. Eye10. Nictitating eyelid
11. Nostril
12. Spiracle
13. Gill slits
14. Cloaca

15. Pectoral fin
16. Pelvic fin
17. Clasper
18. First dorsal fin
19. First dorsal fin spine
20. Second dorsal fin
21. Second dorsal fin spine

22. Anal fin
23. Caudal fin
23-1 Lower lobe23-2 Upper lobe
24. Precaudal pit
25. Caudal keel

I. Observation of external morphology    5become pregnant. Because the posterior terminus of the vertebral column curves dorsally entering thecaudal fin (23) in Chondrichthyes, the caudal fins are included in the tail. This structure of the caudal
fins is called the heterocercal type. In sharks belonging to the genus Lamna that swim at a high speed,
the lower lobe of the caudal fins (23-1) is developed, whereas in bottom-dwelling sharks, the lower
lobe is not extended because they swim around immediately above the seafloor. In sharks belonging
to Alopias, the upper lobe (23-2) is especially developed, accounting for approximately half of thetotal length. In Lamna sharks, keels (25) also seen in tunas are developed from the precaudal tail (5)to the caudal fin to stabilize the tail action without resistance during high-speed swimming. Some
species possess precaudal pits (24) at the base of the caudal fin. The pectoral fins (15) are locatedposterior to the head and form a clear border from the lateral side. Some pelagic shark species areseen to submerge to nearly 500 m depth in a gliding condition in which they use their relatively large
pectoral fins like the main wings of gliders and do not move their caudal fin. The dorsal fin (18, 20) is
single in sharks of the order Hexanchiformes (Chlamydoselachiformes, Hexanchiformes) and double
in other sharks. The tawny nurse shark Nebrius ferrugineus usually has two dorsal fins, but individuals
with a single dorsal fin are uncommonly witnessed. There are also sharks with the two dorsal fins
positioned posterior to the pelvic fins (16). Sharks belonging to Heterodontidae and Squaliformes(Dalatiiformes, Centrophoriformes, and Squaliformes), excluding a few genera, possess spines (19,21) on the anterior margin of the dorsal fin. A cloaca (14) is located between the right and left pelvic
fins. In males, the pterygiophores of pelvic fins are specialized and extended to be a copulatory organ(phallus) (17). The anal fin (22) is missing in sharks of the order Squaliformes (Echinorhiniformes,
Dalatiiformes, Centrophoriformes, Squaliformes), Squatiniformes, and Pristiophoriformes. The gillslits (13) are located anterior to the pectoral fins, and at least a part of them are on the lateral side
without exception. Sharks of the order Hexanchiformes (Chlamydoselachiformes, Hexanchiformes)
and a species in Pristiophoridae have 6 or 7 pairs of gill slits, whereas other sharks possess 5 pairs. In
sharks of the order Squatiniformes, the gill slits are hidden by the pectoral fins. The eyes (9) are on thelateral sides of the head. Some species have nictitating membranes (10) protecting the eyes. In many
sharks, there is a spiracle (12) immediately behind the eye. There is a pair of nostrils (11) anterior to themouth (7). The mouth of many sharks has labial furrows (8) on the edges. On the snout (6), ampullae
of Lorenzini (Figure 2) are scattered as electroreceptors.

Fig. 2 Dorsal view of head of a shark, Starspotted Smooth-hound Mustelus manazo.

6

* The classification to the rank of order is according to Compagno et al. 2005 (Sharks of the World,
Princeton Univ. Press). However, the order names given within parentheses are according to
Fishes of Japan with Pictorial Keys to the Species, Second Edition (edited by Tetsuji Nakabo,
Tokai University Press, 2000).

• Column: Two different measurement methods

(Hirokazu Kishimoto)

The measurement of parts of fish bodies is an important and basic requirement in morphologicalexamination. Nevertheless, a difference exists between Chondrichthyes researchers who habitually use
projection and Teleostei researchers who measure the shortest distance between two points. Either
method gives the same value or nearly the same value in measurements along the body axis such as of
the total length. However, the difference in measurement by the two methods is significant in the caseof the head length and the snout length of depressed fish, and the predorsal length of compressedand deep fish. An example of the measurement of the head length of a shark is as follows: In projection,
because the length is measured between the snout tip A and b which is the distance between A andB, the posterior terminus of the last gill slit, projected on the body axis, a ruler is practically placed
at A vertically to the body axis to measure the distance from A to b (hl). On the other hand, in themeasurement of the shortest distance between two points, the tips of a vernier caliper are placed
directly on A and B to measure the distance obliquely to the body axis (HL). It is clear that hl is shorter
than HL. Therefore, we must be fully aware that the head length may become consistent with thevalue of another species if a wrong method is chosen. Both methods have some advantages anddisadvantages. Projection is an essential method for sketching, in which the ratios between the bodyparts are reflected as you see the material. However, it is likely to cause errors in measurement in thestep of placing a vertical line to the body axis. On the other hand, distance measurement between two
points causes little measurement error, but is not suitable for sketching due to the difference between
the numerical and apparent ratios.

a

B

A

A

hl

b

Body Axis

HL

a

B

I. Observation of external morphology    73 External morphology of rays (Chondrichthyes)

I

The body (1) of rays, Chondrichthyes, is depressed in all species. The head (3) and the pectoral
fins (17) are fused and no border is present in most species. The anal fin is missing. In the case of rays,
the head is from the snout tip to the posterior terminus of the last gill slit (external gill slit) (15), thetrunk (4) is from the posterior terminus of the last gill slit to the center of the cloaca (16), and the tail(5) is from the center of the cloaca to the posterior terminus of the caudal fin (23), or the terminus of
the whiptail if there is no caudal fin. The external morphology of rays is continuous and diverse—from
1
2
6

A
10

21

7
17

8

B

9

23

18

18
17

15

14

16

13
11

21

19

12

5

20

21
C

3

4
2

22

Fig. 1 A, dorsal view of female Sepia Stingray Urolophus aurantiacus; B, Ventral view of male
Red Stingray Dasyatis akajei. C, Lateral view of whiptail of Red Stingray.

1. Body
2. Disc
3. Head4. Trunk
5. Tail
6. Snout

8

7. Interorbital space
8. Shoulder
9. Eye10. Spiracle
11. Mouth
12. Nasal groove

13. Nasal flap
14. Nostril
15. Gill slits
16. Cloaca
17. Pectoral fin
18. Pelvic fin

19. Clasper
20. Dorsal fin
21. Caudal spine
22. Ventral fold
23. Caudal fin

Pristoidei and Rhychobatoidei species that have a shark type body form, two dorsal fins (20), and aclear caudal fin, to the Dasyatidae and Myliobatidae species that have a whiptail instead of a caudal
fin. In the species without a shark-type form, the part where the head and pectoral fins are fused iscalled the body disc (2). These species have thorns on the mid-dorsal line of the body surface, between
the eyes (7), in the pectoral girdle part (8), etc., or a tail spine (21) on the dorsal surface of the tail insome cases. Among the species without a caudal fin, there are species that possess a cutaneous fold(21) on the mid-ventral line of the whiptail. There are 0−2 dorsal fins, and they are small in many
species. The pelvic fins (18) are clearly distinguished from the pectoral fins in many species. In raysin the superfamily Rajoidea, the pelvic fins are divided into the anterior and posterior lobes. In males,
the pterygiophores of the pelvic fins differentiate and extend to form a copulatory organ (phallus) (19).
These species have 5 pairs of gill slits on the ventral surface of the head, with only the sixgill stingray
Hexatrygon bickelli (6) being an exception. The eyes (9) are on the lateral surfaces of the head in rays
in Myliobatidae, and on the dorsal surface of the head in other rays. The spiracles (10) are present
immediately posterior to the eyes without exception and are well developed in many species. Except
for the giant oceanic manta ray Manta birostris, in which the mouth (11) opens in the anterior marginof the head, the mouth is located on the ventral surface of the head. The nostrils (14) are immediately
anterior to the mouth, and a nasal flap (13) covers the nostril groove (12). The ampullae of Lorenzini
are developed in the snout (6). In rays in Rajidae, the ampullae develop all over the ventral surface ofthe head and a part of the ventral surface of the trunk in some cases.

I. Observation of external morphology    9Nelson (1994)

PiscesOsteichthyes

Phylum Chordata*Subphylum Calcichordata
Subphylum Urochordata
Subphylum Cephalochordata*Subphylum Conodontophorida
Subphylum VertebrataSupercalss Agnatha
Class 1 Myxini*Class 2 Pteraspidomorphi
Class 3 Cephalaspidomorph
Supercalss Gnathostomata*Class 4 PlacodermiClass 5 Chondrichthyes
Subclass Holocephali
Subclass Elasmobranchii*Class 6 Acanthodii
Class 8 Actinopterygii
Subclass Chondrostei
Subclass Neopterygii
Other Division
Division Teleostei
Class 7 Sarcopterygii*Subclass position uncertain
Subclass Coelacanthimorpha
Subclass unnamed (Dipnoi and etc.)*Subclass Rhizodontimorpha*Subclass Osteolepimorpha
Subclass Tetrapoda

reference: N elson, J. S. (1994) Fishes of the World. 3rd edition* Academic Press. 600 p.
*4th edition .....

10• Column: The reason why “Osteichthyes” disappeared(Hirokazu Kishimoto)Thus far, in a class on ichthyology or related matters, it has been explained that the extant fish on
the earth include Chondrichthyes and Osteichthyes, and in more detail, a group called Cyclostomeslacking a bone in the jaw is also dealt with in ichthyology. However, as used in this book, a name,
Teleostei, similar to but distinct from Osteichthyes, has recently become remarkable. This is solely
because many ichthyologists follow the taxonomy in Fishes of the World, Version 3 by Nelson (1994)
that aggressively adopts recent study results. In his system, most of the commonly seen fish, including
Japanese eel Anguilla japonica, common carp Cyprinus carpio, tunas, Japanese sea bass Lateolabrax
japonicus, righteye flounders, and puffers, are grouped in Teleostei. If ancient fish such as gars andbowfin Amia calva are added to this, it is a group called Neopterygii. Moreover, if sturgeons andPolypteridae are added, it is called Actinopterygii. There is a group called Sarcopterygii, which is on
the same level as Actinopterygii, and it includes those in the shape of a fish but with lobed pectoraland pelvic fins, such as coelacanths and lungfishes, as well as Tetrapoda including up to humans andmonkeys. In other words, coelacanths and lungfishes are dealt with as a group more closely related to
Tetrapoda than common fish. In such a case, it is impossible to group them in a taxon “Osteichthyes”, aspreviously done by excluding only Tetrapoda from Sarcopterygii. For the same reason, it is impossible
to broadly classify Vertebrata into fish excluding coelacanths and Tetrapoda including coelacanths;
therefore, the taxonomic unit “fish” cannot be used in a strict sense.
In this book also, in compliance with the way of thinking by Nelson, the use of Teleostei for thoseother than Chondrichthyes serves its purpose in most cases. Use of the taxonomic name Actinopterygii,
including sturgeons, would only make it difficult to gain understanding. Moreover, mentioning thoseby including coelacanths and lungfishes as a wider taxon would make it impossible to find a name forthe taxon.
On the other hand, fish researchers generally drive coelacanths and lungfishes away into a groupof Tetrapoda by thinking that they are animals far from fish. However, does this ever convince Tetrapoda
researchers? I cannot help but feel that Coelacanths and lungfishes are in the shape of a fish rather
than Tetrapoda. Thus far, in this book, I have ignored taxonomic ranks due to their complexity andused fuzzy “groups” instead. I also have a question regarding how I should deal with the groups. In thetaxonomy proposed by Nelson, Tetrapoda as a whole is dealt with as a subclass in Sarcopterygii. Inline with this, when looking at how the breakdown is provided into subordinate ranks, the conclusion
is postponed. In all likelihood, if it is the detailed classification inside a subclass, they are supposed to
be an amphibian superorder, reptilian superorder (including birds), and mammalian superorder. The
question, “Is it acceptable to make them such a low taxon?” rises in me, accustomed as I am to theprevious taxonomy. There is something I find difficult to believe when I think of the balance between
fish and the larger taxonomic picture.

I. Observation of external morphology    11

4 External morphology of Teleostei

I

For the identification of diverse species of fish, it is necessary to accurately understand theircomplicated morphology. Detailed expression of behaviors of fish through observation of their ecology
requires an accurate understanding of their body forms and correct expression of the orientations.
Therefore, for structural observations, it is suitable if the material is a fresh specimen so that the joints
can be moved. However, fish can not keep their freshness during long-time observation, causing aputrid odor, and their surface dries. For advanced studies wherein you may need to observe valuablespecimens that require permanent preservation, you should prepare the specimens to set to hold anormal body form for materials according to the method in Motomura and Ishikawa (2013, http://
www.museum.kagoshima-u.ac.jp/staff/motomura/CollectionManual_lowres.pdf). If the specimen
is preserved in formalin, water-rinsing starting from the day before the observation can reduce theirritating odor. However, rinsing for any longer is harmful for specimens because the decalcificationof the skeleton occurs in freshwater. In the case of specimens preserved in ethyl alcohol, you shouldobserve them as soon as you have taken them out while paying close attention to their rapid drying,
because it is not adequate to soak them in water. You do not have to be worried about the odor of ethyl
alcohol, even though it is intense, because it is not harmful. On the other hand, you need to handle
formalin with caution.
A fish body is usually composed of the following four parts.
Head: From the snout tip to the posterior margin of the operculum (A−B in the figure)
Trunk: From the posterior margin of the operculum to the anus (B−C in the figure)
Tail:	From the anus to the base of the caudal fin (tail includes the caudal fin in some cases) (C−Din the figure)

10-1

6
1
3

2

3-1 3-2

10-3
4
5-1 5-4 5-2 5-3

10-2

5
9-1

7

8

9-2

A

B

C

Fig. 1 Red Pandora Pagellus belottii, from Africa.

12DFins: All fins (9 and 10 in the figure)
The parts are further composed of a variety of respective organs. The name of the body parts is asfollows in an example of a fish in Sparidae, Teleostei.
1. Eye: Although the eyes cannot be closed due to the lack of the eyelids, they are not significantly
different from human eyes. Fish in Mugilidae and Carangidae have a cover called the adipose
eyelid that covers the eye from the anterior and posterior sides, but it is difficult to identify due to
its transparency in most cases.
2. Nostril: In Teleostei, it is common that there is a pair of anterior nostrils and a posterior nostrilon each side. However, there are also species with only one nostril, such as Pomacentridae andsticklebacks.
3. Upper jaw: This is formed by three types of bones, premaxillary (3-1), maxillary (3-2), andsupramaxillary, which exist in varying numbers, from many to none, depending on the fish
species. In primitive fish, such as Clupeidae, the premaxillary is small and the oral margin of theupper jaw is occupied mainly by the maxillary. In many advanced fish, including Perciformes andScorpaeniformes, the premaxillary is large, occupying all margins of the upper jaw, whereas the
maxillary is recessive.
4. Lower jaw: The dentary is the only bone involved in the oral margin of the lower jaw. The dentary
and teeth on the premaxillary have variable morphologies related to the feeding habit.
5. Operculum (gill cover): This is composed of four types of bones, the preopercle (5-1), the opercle(5-3), the subopercle (5-2), and the interopercle (5-4). The size and/or number of spines aroundthe preopercle, and the presence or absence of any spine on the posterior margin of the opercle
among these bones are frequently used for species identification because they can be observed
from outside (for details, see III-6). The branchiostegals hidden by the posterior inferior margin of
the operculum, which support the branchiostegal membrane, look like the frame of an umbrella.
The number and shape are characteristic of each species. In a functional aspect, it significantly
contributes to gill ventilation by cooperating with the operculum.
6. Lateral line: This is a characteristic sensory organ most fish have, whereas only a few othervertebrate animals, such as amphibian larvae, have this organ. Normally, it is located a little
superior to the mid-lateral line and looks like a dotted line. This is because the scales, each ofwhich has a pore (pored lateral line scale, see Figure 4 in I-5), form a line. The double-structured
tunnel from the opening on the exposed external surface of the scale connects to the branch of
the subcutaneous lateral-line organ running longitudinally along the body at the opening of theinternal surface of the covered part. In some fish, the body surface has no scales and the lateralscales are covered by the skin, exposing only their pores, which form a line on the surface. Someother fish lack a lateral line. In addition, lateral lines are remarkably variable. Some fish haveseveral lateral lines on one side, a winding lateral line, or lateral lines running like a net.
7. Anus: This is a terminal opening of the digestive tract and is located in conjunction with the
Adipose fin

Finle

ts

ets

Finl

Fig. 2 Finlets of scombrid fish.

Fig. 3 Adipose fin of Ayu Plecoglossus altrivelis altivelis.
I. Observation of external morphology    13urogenital pore immediately anterior to the anal fin. However, there are exceptions, including
firefly-fish Acropoma japonicum, in which the anus is located in the vicinity of the pelvic fins.
8. Caudal peduncle: This is a part from the posterior terminus of the anal fin base to the caudal finbase or the terminus of the vertebral column.
9. Paired fins: These are left-to-right pairs of fins of two types, pectoral fins (9-1) and pelvic fins(9-2). The former is composed of only soft rays. However, pectoral fins of fish in Siluriformes
have the spiny soft ray in the superior margin. The latter is composed of 6 or more soft rays in theprimitive fish group, whereas in the advanced fish group, many fish have 5 soft rays or fewer anda spine at the most lateral side (anterior margin) (only fish in Siganidae have a spine each in the
medial and lateral (anterior and posterior) margins) (see Figure 4 in II-2). These are considered theorgans homologous to the anterior limb and the posterior limb of Tetrapoda, respectively.
10. Vertical fins = unpaired fins = median fins: These are the fins that are located at the median of thebody’s outline, do not form a left-to-right pair, and open and close vertically.
10-1. Dorsal fin: This is equipped with rays with the pterygiophores, and is arranged on the middorsal line from the trunk to the caudal peduncle. If it is divided into two or three parts
longitudinally by a clear indentation(s), they are distinguished as the first dorsal fin, second
d. f., and third d. f. in order posteriorly, and represented by the abbreviations D1, D2, andD3, respectively. This is composed of only soft rays in the primitive fish group, whereas theadvanced fish group is equipped with spines in the anterior part (all of the first dorsal fin)in many cases. Moreover, when this is followed by individually separated soft rays, like in
Scombridae and Carangidae, they are called finlets (Figure 2). In addition, when a small fin
without rays lies posterior to the dorsal fin, as seen in Salmonidae, it is called an adipose fin(Figure 3).
10-2. Anal fin: This is the same as the dorsal fin except that it is arranged on the mid-ventral linein the tail.
10-3. Caudal fin: This is a fin located at the posterior terminus of the body, and composed of only
soft rays. It is common that the part located in the upper and lower halves of the median of
the body is called the upper and lower lobes of the caudal fin, respectively. Because this is
Right
Dorsal / UpUp
Anterior

Left
Anterior

PosteriorDorsal / Up

Posterior

Ventral / Down

Yellow Tail
Seriola quinqueradiata

Blackmouth Angler
Lophiomus setigerus

Ventral /Anterior

Left

Left

Longitudinal
Dorsal

Dorsal /PosteriorDorsalTransverse

Transverse
Longitudinal
Anterior

Posterior

Left / Up

Anterior

Right / Up

Down

VentralBastard Halibut
Paralichthys olivaceus

VentralPacific Halibut
Hippoglossus stenolepis

Fig. 4 Direction of fishes. (Illustrations from Nakabo (2000)

14

Hippocampus
coronatus

the source of thrust in swimming, the shape varies depending on the swimming ecology.
The expression of orientation for the fish body is based on the following guidelines (Figure 4).
Dorsal side: The half of the body to the vertebral column, in which the spinal cord is arranged.
Ventral side: The half of the body to the vertebral column, in which the digestive tract is arranged
Left/right: In most fish, the surface appearing on the top is left and the surface coming in contact withthe chopping board is right when the fish is placed on the board so that the ventral side faces yourside (down) and the head is on the left.
You will understand and better relate to the above-described orientations if you imagine yourself
swimming with your face pointing down.
Anterior/posterior: The direction where the head lies is anterior (front), whereas the opposite
direction, where the tail lies, is posterior (rear). Exceptionally, in the sea horse, the ventral side isanterior and the dorsal side is posterior.
Longitudinal/transverse: The orientation from the head to the tail is longitudinal whereas theorientation from the dorsal side to the ventral side is transverse. Therefore, in most fish, thehorizontal orientation is longitudinal whereas the vertical orientation is transverse. Beware thatthese are the reverse for sea horses and humans.
Top/bottom, up/down: The orientation toward the core of the earth is down whereas the orientation
toward the sky is up. Therefore, generally, the dorsal side is the top and the ventral side is thebottom.
In the same position, if the fish is flattened from both the right and left sides, like a porgy (Figure5), it is called compressiform, whereas if the body form is flattened from the top (dorsal side), such as
monkfish and rays, it is called depressiform. However, the fish in Pleuronectiformes are not depressed,
but extremely compressed, because they are rotated sideways with their colored side with eyes (leftside in bastard halibut Paralychthis olivaceus and right side in halibut, = eyed side) up and theiropposite side, white and without eyes (right side in bastard halibut and left side in halibut, = eyelessside), down.
The midlines of a fish body running from the head to the tail (longitudinally) come in 5 types
below. Although these are substituted by median with an adjunctive in some cases, it is preferable todistinguish these as follows.
A : axis or axial line: the midline of a fish body (axial part)L : mid-lateral line: the midline of the lateral surface (right and left)
D : mid-dorsal line: the midline in the dorsal side
V : mid-ventral line: the midline in the ventral sideA

Right LD

D

L and A

Left LV

V

Fig. 5 Medial lines of fishes. (Illustrations from Nakabo (2000)
I. Observation of external morphology    15I5 Scales and lateral line canals

1. Scales of Teleostei
Except for contemporary Agnatha (Cyclostomata), many fish are equipped with scales on theirskin. Scales are derived from the dermis. Because scale morphology is variable among species andnearly constant within species, it is a taxonomic character. Scale patterns appearing on the scale surface
allows us to estimate the age, stock, etc. Cosmoid scales, which are covered by thick layers of harddentine, are seen in many fossil species and coelacanths, etc., and are regarded as primitive scales.
Hence, fish scales tend to become thinner as fish evolve. The scales of contemporary sturgeons, etc.
are called ganoid scales. The scales characteristic to Chondrichthyes are called placoid scales and arecovered by a relatively hard enamel layer. They are also called dermal denticles in some cases because
their structure is similar to that of teeth. In sharks, the body surface is densely covered by placoid
scales forming a so-called sharkskin. On the other hand, in rays, the placoid scales are as degenerative
as being scattered on the surface, making the skin relatively smooth. The scales of Teleostei are calledbony scales, and they are arranged as if the body surface is tiled. Scales of the Japanese sardine
Sardinops melanostictus (Figure 1C), chum salmon Oncorhynchus keta, cherry salmon O. masou(Figure 1A), common carp Cyprinus carpio, Carassius, dotted gizzard shad Konosirus punctatus(Figure 4B), etc. are cycloid scales, which are smooth when exposed on the body surface (Figure 1A).
Scales of red seabream Pagrus major (Figure 1D), Japanese sea bass Lateolabrax japonicus, sabre
squirrelfish Sargocentron spiniferum (Figure 4A), etc. are ctenoid scales equipped with small spines

A

B

C

D

Fig. 1 Scales of teleosts.
A, Cycloid scale of Masu Salmon Oncorhynchus masou masou; B, Regenerated cycloid scale of Masu Salmon
Oncorhynchus masou masou; C, Cycloid scale of Japanese Pilchard Saldinops melanosticus. Left, normal scale.
Right, regenerated scale; D, Ctenoid scale of Red Sea Bream Pagurus major.

16

A

B

Fig. 2 Scutes.

A, Japanese Jack Mackerel Trachurus japonicas (Left, from trunk. Right, from caudal peduncle);
B, Dotted Gizzard Shad Konosirus punctatus (From ventral).

in the part exposed on the body surface. In addition, there are plate-like scales with bony spines or
keels on the lateral line of jack mackerel and the ventral body margin of sardine, which are called
scutes by comparison of them to mountain ridges (Figure 2). Figure 3 shows the name of scales of the
spotbreast angelfish Genicanthus melanospilos. Figure 4 shows the name of the lateral side scales ofsabre squirrelfish Sargocentron spiniferum and spotted gizzard shad Konosirus punctatus. On the bonyscales, various scale patterns are seen in the layer of bony plate containing calcium. A scale pattern isformed by the array of the grooves arranged radially from the focus or longitudinally and by the ridges
arranged like rings or transversally. As the fish grows, an interval is formed between the ridges. Theinterval forms a wide growth zone when growth is rapid, whereas it forms a narrow resting zone whengrowth is slower. Because the resting zone is formed with a cycle of approximately one year, it is used
as a guide for determining the age by estimating that this is an annual ring (Figures 4 and 5). Generally,
the formation of the annual ring is due to the yearly appearance of the difference in growth speed on
the scale. However, it should be noted that because growth is influenced by changes in the environment
such as water temperature and physiological changes such as those for spawning, the annual rings
become unclear in artificially reared fish, and a pseudo annual ring (also called a spawning mark) is
formed when a fish has spawned. In addition, scales come off when a physical force is applied to themfrom outside in some cases. A scale is regenerated in the place where the previous scale came off,
and this is called the regenerated scale (Figure 1). Because no scale pattern in the process of growth
before the drop off is formed on the regenerated scale, it is not eligible for use in age determination. In
addition, lateral scales on the lateral line are also not eligible because it is difficult to see their centerand ridges.

(Observation and preparation of specimens)
1. Collect several scales from Japanese sardine Sardinops melanostictus (T. & S.), cherry salmon
Oncorhynchus masou (Brevoort), and red seabream Pagrus major (T. & S.). At this time, exclude
I. Observation of external morphology    17Annual Ring

Ridge
Focus

GrooveCteni

Covered Portion

Exposed Portion

Fig. 3 Parts names of ctenoid scale of Spotbrest angelfish Genicanthus melanospilos.
Sketch by Katsunori Nakamura.

ridge

cteni

A
groovepore oflatelal line

focus
annual ringcovered portion exposed
portionB

Fig. 4 Parts names of scales.
A, Sabre Squirrelfish Sargocentron spiniferum (Left, pored lateral line scale);
B, Dotted Gizzard Shad Konosirus punctatus.

1

2

3

Fig. 5 Annual rings of Masu Salmon Oncorhynchus masou masou.

18

regenerated scales, lateral scales, and scutes. At the time of the collection, lightly pinch the scaleusing forceps, etc. However, beware that pulling it too hard may cause a crack on the cover of thescale or damage small spines. If you can pinch the scale to some extent with your fingers, you may
remove the scale by pulling with the fingertips.
2. Wash the scales in tap water with gentle rubbing with your fingertips to remove the slime andstain.
3. Place the three types of scales separately on a glass slide in the same orientation. At this time, wipeaway the moisture sufficiently.
4. Overlap the glass slide on which the scales are placed with another glass slide before the scales
become dry and curled, and seal both ends of the glass slides with paper tags. At this time, be
careful not to make the scales dry. Enter your student No. and full name on the tags.
5. Sketch the three types of scales on Kent paper while paying attention to the ridges, grooves,
growth zones, resting zones, and scale periphery. As required, observe the scales by magnifying
them using a stereomicroscope, etc. At that time, observe the detail of the scales using reflective
light and transmission light properly. In addition, though beginner students frequently attempt to
express resting zones by writing them more densely than growth zones, this is wrong. Correct
sketching is to express it by writing the interval narrower because the interval of the scale pattern
becomes narrower in the resting zones.
6. Enter the experimental theme, data of the material fish, etc. on the Kent paper.
7. Determine the age using the resting zones of the scales.
8. Referring to Figure 3, enter the name of the parts of the scales (write both Japanese and English
names).

2. Lateral line canals in head
The lateral line is an organ present in both Chondrichthyes and Teleostei, and it functions not
only as a receptor of mechanical stimuli, including water flow and pressure, but also as a chemical
receptor for univalent ions. In many fish, the lateral line on the lateral side runs longitudinally under
the skin of the lateral side, and connects to the body surface through a pore or a canal pore at nearly
every segment. In fish species in which scales are developed, the pores of the lateral line are exposedon the scales in some cases, and these are called the pored lateral line scales. In many fish species,
the lateral line on the lateral side is a canal that goes from the upper terminus of the operculum tothe caudal peduncle on both sides of the body and runs nearly along median part of the lateral side
longitudinally. However, it is wavy or runs along the ventral side of the body in some species. In
addition, the number of lateral lines varies from 5, as in fat greenling Hexagrammos otakii, to 2, as inJapanese jack mackerel Trachurus japonicus, to none, as in Japanese sardine Sardinops melanostictus.
The lateral line canal organs in the head are partly or completely embedded under the dermalbone of the head and connect to the external environment through canals opening on the surfaceof the dermal bone. The lateral line canal organs in the head of Teleostei are broadly classified into
three elements, the supraorbital canal (SOC), the infraorbital canal (IOC), and the operculomandibular
canal. Moreover, the operculomandibular canals are further classified into the mandibular canal (MC)and the preopercular canal (PC). Because the auditory vesicle exists in the connection part fromthe infraorbital canal to the temporal canal, the lateral line canals in this connection part are further
classified into the otic canal and the postotic canal in some cases. The major canals, the supraorbital
canal, infraorbital canal, and operculomandibular canal, branch to the supratemporal canal and thetrunk canal in the temporal region via the otic and postotic canals to link to the lateral line canal on
the lateral side. Thus, there are seven left-to-right pairs of canal organs at the maximum on the head
I. Observation of external morphology    19of Teleostei. However, some of these elements are missing depending on the fish species or in thedevelopmental stages of individuals even in the same species in some cases. Each sensory cell of theselateral lines is the terminus of the nerve, such as the nervus buccalis, nervus glossopharyngeus, nervus
mandibularis, nervus opercularis, and nervus agus. Figure 7 shows the lateral line canals on the headof adult (over 40 mm total length) Tanakia lanceolata. In the experimental observation, observe thelateral line canals on the head of a bitterling, sketch them by paying attention to the number of poresof each lateral line canal organ opening on the surface, and enter each name.

• Column (Nobuhiro Suzuki)

In addition to the fact that fish scales are diverse morphologically, in fish with a very slimy surface
such as eels, degenerative small scales are embedded in the skin, and there are also fish species
without scales. Scale morphology also differs depending on the body part. For example, in bastard
halibut Paralichthys olivaceus, the colored surface of the side with eyes is covered by ctenoid scales,
whereas the white surface on the side without eyes has cycloid scales. In cultured bastard halibut, it
is often seen that the white surface on the eyeless side is black-pigmented. On the body surface of
this part, the cycloid scales develop small spines to change into scales resembling ctenoid scales insome cases. Scales are not present on larvae immediately after hatching, but appear in the juvenilestage. Even in fish species equipped with ctenoid scales, the scales close to their appearance (scales inearly development) are small and round, and as the individual grows they develop small spines on the
exposed part (Figure 6).

5.0 mm

5.0 mm

5.0 mm

A

10.0 mmB

10.0 mmC

10.0 mmE

F

0.97 mm

0.38 mm0.25 mmG

D

H

I

Fig. 6 Area expansion of distribution (A-F), and morphological change (G-I) of first scales of Yellowfin
Goby Acanthogobius flavimanus. G, first scale; H, developping scale; I, developed adult scale.

20

(Observation and preparation of specimens)
1. Prepare a specimen sufficiently fixed by 10% formalin and preserved in 70% ethyl alcohol(hereinafter referred to as simply alcohol). Because skin pigments of the specimen cause trouble
in observations, decolorize (blanch) the pigments first. To blanch, remove alcohol with running
tap water and then immerse the specimen in 5% H2O2 to sufficiently decolorize the pigments.
2. Remove H2O2 from the specimen with running tap water and immerse it in 2% KOH to make ittransparent until you can see through the muscle.
3. Remove KOH with running tap water and immerse the specimen in 70% alcohol solution.
4. For observation of the lateral line canals on the head, drop a staining solution prepared bydissolving Suminol Cyanine 5% Extra to saturation in 70% alcohol in a pore of the lateral line
using something pointed, such as a toothpick, to stain the inside of the lateral line canal (wiping
away alcohol remaining inside the lateral line canal with a gauze beforehand helps the staining
solution to go into the canal). Observe this under a stereomicroscope. The use of incident lighthelps the observation. Because the lateral line canals on the head are formed along with the growthof the individual, an adult fish (an individual with a total length over 40 mm or developing asecondary sexual character such as tubercles and an ovipositor) needs to be used for observationof completed lateral line canals on the head of bitterling. (Because the stain is decolorized once thespecimen is placed in 70% alcohol after observation, it can be reused as a specimen many times.
Preserve the specimen in 70% alcohol again.)

STC
SOC

TC

PC

IOC
MC

Fig. 7 Cephalic sensory system of adult acheilognathid fish

Tanakia lanceolata.
IOC, infraorbital canal; MC, mandibular canal; PC, preopercular
canal; SOC, supraorbital canal; STC, supratemporal canal.
I. Observation of external morphology    21

• Column (Nobuhiro Suzuki)

A sensory cell called a neuromast is found at the individual terminus of a lateral line pore. Thoseon the surface not being embedded in the skin are called free neuromasts, whereas those locatedat the bottom of the canal organ embedded in the skin are called canal neuromasts. A neuromast
contains many hair cells. The hair cell is composed of a long kinocilium and dozens of stereocilia,
which are arranged with polarity in a neuromast (Figure 8A). Upon stimulation, the kinocilium tail, andthe stimulus is amplified by the stereocilia to sense the direction and intensity of the stimulus in themechanism. The neuromast is in a plate-like or rod-like form if it is covered by a jelly-like substance, andis called the cupula (Figure 8B). There are already neuromasts on the epidermis of larvae immediately
after hatching. At this time, they take the form of free neuromasts, and they change to the cupulas byincreasing in number as the individual grows. Figure 9 shows the position of the free neuromasts (black
circles) and the direction of a stimulus (arrow) sensed by each neuromast on the surface of a larva ofredspotted grouper Epinephelus akaara in the pelagic stage.

A

B

Fig. 8 A: Free neuromast of larva of Hong Kong Grouper Epinephelus akaara.
B: Cupula of Japanese Rockfish Sebastes inermis.

Fig. 9 Distribution of cupula on pelagic larva of Hong Kong Grouper Epinephelus akaara.

22

I

6 Luminescent organ

Many fish species have luminescent organs, which can have a wide variety of structures. However,
the organs can be broadly grouped into those formed in the integument and those formed throughdifferentiation from the digestive tract. In addition, the luminescence forms also have several types.
In a broad grouping, there are the type that emits light by means of luminescent bacteria forming
a symbiosis in the luminescent organ, and another type that emits light due to a chemical reaction
between luciferin and luciferase occurring inside the luminescent organ. The latter luminescent organ
is rich in variation in the morphology, arrangement form, etc. depending fish species. As mentioned
above, fish species with luminescent organs include many species treated as deep-sea fish. Currently,
those are broadly classified into two groups based on their adaptation process*.
Particularly, on the body of meso- and bathypelagic deep-sea fish (apart from ancient deep-seafish), such as fish in Myctophidae, Neoscopelidae, Gonostomatidae, Sternoptychidae, Chauliodontidae,
and Stomiidae, spherical or oval luminescent organs are arranged from the lateral surface to the ventral
surface, and the number and arrangement form are important taxonomic characters. Although thestructure of these luminescent organs varies depending on the fish species, basically, a lens, an emitter,a reflection layer, chromatophores, etc. are arranged from the body surface to the inside of the body. In
all cases, it is spontaneous and intracellular luminescence.
Here, we deal with (easily obtainable) fish in Myctophidae distributed universally in the mesoand bathypelagic waters among the fish groups above.
Myctophidae: In addition to the spherical luminescent organs on the lateral side, there are large
luminous glands on the top and bottom of the snout and/or the caudal peduncle. These luminescentorgan groups form a small group as shown in Figure 1, which is important as a taxonomic character.
Approximately 250 species are known from the seas in the world. The scales easily come off in manyof the species. This group is the small deep-sea fish distributed in the meso- and bathypelagic waters.
Their habitat depth differs between the daytime and the nighttime because they practice diel verticalmigration in which the unit is a day. Additionally, they are foraged by many marine animals including
skipjack/tuna, salmon/trout, dolphin, and other sea animals.
Myctophidae is divided into the following three groups based on their distribution form at night.
1) Surface migrants (there are luminous glands on the upper or lower part of the caudal peduncle):This group ascends to the surface layer of less than 10 m depth, mainly 0−1 m, at night. For
that reason, they can be collected by a surface horizontal tow using a juvenile net at night. They
descend to a depth of 200−400 m in the daytime and inhabit the layer with little light. Myctophum,
Symbolophorus, Centrobranchus, etc. are the major genera.
2) Midwater migrants: This group inhabits mainly depths of 400−700 m in the daytime, but ascends
to 100−200 m depth. However, they do not ascend to immediately below the surface. This group
includes the fish ascending to the deeper layer and the fish ascending to the shallower layer.
* 2 large types in deep sea fishes.
1. Ancient type (or primary or true deep sea fishes): appeared from old geological (early Cenozoic or before) era,
and adapting to the deep-sea by relatively long period. Such as Lanternfishes (or Myctohphidae), Swallowers(Saccopharhynx spp.), and Footballfishes (Himantolophus spp.).
2. New type (or secondary or continental shelf type): advanced into deep-sea in glacial ages of Quaternary period
or later, from inland waters, coastal areas, or continental shelves. Thus, new type deep-sea fishes have related
species with coastal areas. Such as Eelpouts (or Zoarcidae), Sculpins (Cottidae), Snailfishes (Liparidae), Cusk-eels(Ophidiidae), or Rattails (Macrouridae), etc.
I. Observation of external morphology    23Suo Cp

Ce

SUGL

Ant

PLO

VLO

Bu

Dn

2

2

1

Vn

1
So

Br

Op

SAO
1 23

Pol

PVO
1 2 34
Prc

1

2

PO

3 45

1 2 3 4VO5

1 2 3 4 5 6 1 2 3 4 5 INGL
AOa
AOp
AO

Fig. 1 Distribution of luminescent organs of myctophid fish (modified from Fujii (1084)).
Ant, antorbital organ; AOa, anterior anal organ; AOp, posterior anal organ; Br branchiostegal organ; Bu, buccal organ;
Ce, cervical organ; Cp, cheek photophore; Dn, dorsonasal organ; INGL, infracaudal luminous gland; Op, opercularorgan; PLO, suprapectoral organ; PO, pectoral organ; Pol, posterolateral organ; Prc, precaudal organ; PVO, subpectoral organ; SAO, supraanal organ; So, suborbital organ; SUGL, supracaudal luminous gland; Suo, supraorbitalorgan; VLO, supraventral organ; Vn, ventronasal organ; VO, ventral organ.

Diaphus, Diogenichthys, Lampanyctus, Benthosem, etc. are the major genera.
3) Non-migrants: The habitat depth changes little between the daytime and the nighttime.
Stenobrachius is included.

Observation points
1. Arrangement of luminescent organs
The schematic diagram of the arrangement of luminescent organs shown above is of the groupbelonging to the abovementioned surface migrants. The luminescent organs of Myctophidae gather
in small groups that are named individually. The number of the groups and positional relationships
among the groups differ depending on the species.

2. Identification of species
Because the arrangement and number of luminescent organ groups rarely differ between the rightand left, those on the left side should be used for observation and counting in principle. I advise you
to focus on the following luminescent organ groups and scales in the specimens of Myctophidae
belonging to the abovementioned surface migrants.
a. Are the 3 luminescent organs (1−3) of SAO in a straight line or a bent line?
b. Positional relationships among SAO1−3, VO1−5, and VLO.
c. Number of AOa.
d. Number of AOp.
e. Total number of AOa + AOp.
f. Relative positional relationship between Pol and the adipose fin.
g. Which type of scales, cycloid scales or ctenoid scales (several species included in Myctophum
have ctenoid scales)?

3. Determination of the sex (Figures 2 and 3)
Among Myctophidae, in the species belonging to the surface migrants, males have a large SUGL
that is, however, missing in the lower part of the caudal peduncle. Females have a small INGL that is,24

Fig. 2 Pearly Lanternfish Myctophum nitidulum.
Upper, male with SUGL; Lower, female with INGL.

Fig. 3 Prickly Lanternfish Myctophum asperum.
Upper, dorsal view of male with SUGL; Lower, ventral view of female with INGL.

however, missing in the upper part of the caudal peduncle. Exceptionally, in Tarletonbeania, males
have it in the both upper and lower parts, whereas females lack it in the both parts. The appearance of
this luminescent gland is an effective character for the determination of sex only in adults in which it
is completely developed as a secondary sexual characteristic.
These differences between males and females are said to help them to identify the other sex intheir spawning season, when males swim in the lower layer whereas females swim in the upper layer.

4. Development of the caudal luminous gland (Figure 4)
With regard to the outline of the caudal luminous gland of adults, the length and width differ
depending on the sex. In females, small plates of the same size are arranged, whereas in males, platesof slightly varying sizes overlap each other in the structure. The number and shape of these plates are
effective taxonomic characters of each species because these are nearly constant in each species.
In the case of Myctophum nitidulum in Figure 4 below, the number of plates of adults is 3−4 infemales and 6−8 in males, showing a higher number in males. After their appearance, this caudal
luminous gland becomes larger in shape and increases in number to completion as the fish grows,
I. Observation of external morphology    25and the whole shape becomes unique to the species. For that reason, the sex of an individual can be
identified from the appearance even if the luminescent gland is incomplete (see the intermediate typein Figure 4).
This gland appears once the body length reaches around 35 mm in female individuals, andcompletes its development when the length reaches around 65 mm. On the other hand, in males, itappears once the body length reaches around 28−29 mm, and completes its development when the
body length reaches around 60 mm, earlier than in females. In addition, because the caudal luminous
gland is a secondary sexual characteristic, the developmental process and development of the gonad(ovary or testis) progress almost simultaneously. Therefore, I advise you to observe them in parallel.
However, you may miss the testis of immature males unless you pay attention because it is small, eventhough it leads to the anus.
Undeveloped

Intermediate

Completed

SUGL
（♂）

INGL
（♀）

Fig. 4 Ontogenic change of SUGL (upper) and INGL (lower) of Pearly Lanternfish Myctophum nitidulum.
Scales indicate 1 mm.

26

II
Fish measurements

1 Measuring equipment

II

Equipment for measuring the size of parts of a fish body varies depending on the size of the fish,and each type of equipment has a distinct precision. For measuring huge fish such as sharks, a tapemeasure is used, whereas fish larger than 30 cm are usually measured using a straightedge (wooden
ruler) in millimeters. If the size is <30 cm, a vernier caliper is used to accurately measure the size down
to the first decimal point (in tenths of millimeters). Vernier calipers include dial calipers and digital
calipers, the latter of which have a variety of types, including ones to which recording equipment
can be attached. Every caliper can read down to the second decimal point. However, precision to thefirst decimal point is sufficient for the measurement of organisms. In the case of a microscopic size
such as the case of larvae and juveniles, a micrometer is used. However, because the measurements
use projection (see the column) in most cases, it is inevitable that the measurement angle of each part
becomes a little inaccurate.

Division reading and principle of vernier calipers
You need to carefully understand the method and principle of vernier calipers because an error in
the reading of divisions of a vernier caliper frequently used in measurement of fish bodies can lead toa serious mistake.

The ways to divide scales on vernier calipers
Normally, a vernier has n divisions that are equal to n−1 divisions on the main scale. Dependingon the precision, there are two forms for the divisions, as shown in the table below.
The minimum division on themain scale (mm)(1) 1(2) 0.5

28

The way to divide the vernier
19 mm are equally divided by 20 (Fig. 1)
39 mm are equally divided by 20
12 mm are equally divided by 25
24.5 mm are equally divided by 25
49 mm are equally divided by 50

The minimum read value(mm)
1/20=0.05
1/50=0.02

measuring faces
for insidemeasurementsinternal jaw of main scaleinternal jaw of slider
locking screw
main scaleretainerbasal
outside facebasal face for depth

division linesof main scale

depth probe

division lines of vernierslideroutside jaw of slider
outside jaw ofmain scalemeasuring faces for outside measurements

Fig. 1 Legends of vernier caliper.

How to read divisions on vernier calipers(1) Principle: In the case of Figure 2, because the division line 0 on the vernier is over 7 on the main
scale and the 4th division line coincides with a division line (regardless of the position) on themain scale, the size is as follows:	  7 + (0.05 × 4) = 7 mm + 0.2 mm = 7.2 mm
		 Convenient method: Add (the division line on the vernier coinciding with a division line on themain scale) × 0.1 to the division line on the main scale over which the division line 0 on the vernierlies. That is, read it as follows:
7 + (2 × 0.1) = 7 mm + 0.2 mm = 7.2 mm
main scalevernierFig. 2(2) Principle: In the case of Figure 3, because the division line 0 on the vernier is over 4.5 on the main
scale and the 11th division line coincides with a division line (regardless of the position) on themain scale, the size is as follows:	  4.5 + (0.02 × 11) = 4.72 mm
		 Convenient method: Add (the division line on the vernier coinciding with a division line on themain scale) × 0.1 to the division line on the main scale over which the division line 0 on the vernierlies. That is, read it as follows:	  4.5 + (2.2 × 0.1) = 4.72 mm

main scalevernierFig. 3

II. Fish measurements    29

2 How to measure Chondrichthyes; sharks

II

Measurements of Chondrichthyes are generally done on projection on lateral axis. These customs
are different from those on Teleostei, done on point-to-point lengths.

1
2
6
4

5

18

3

19

9
11

21

17

27

22
20
26

7
13

8

25
28

A

24

23

29

24

15

14

16

10
B

C

12

Ｄ

29

Fig. 1 Common measurements on sharks.
A, lateral view; B, ventral view of head; C, dorsal view of head; D, ventral view of pelvic fin.
See text for numbers on the figures. Drawn by Yumi Kawai.

30

1. Total Length; TL
		 Length from anterior-most point of body to posterior-most point of caudal fin. Anterior mostpoint has both cases on upper and lower jaws. Generally this length has taken to the endpoint ofupper lobe of caudal fin with normal position, but sometimes taken with most end point by pulling
anterior direction. In some species the end of upper lobe is not be able to detect, and precaudal
lengs as below is used instead of total length.
2. Precaudal Length; PCL		 Length from anterior-most point of body to origin of upper lobe of caudal fin, or if present, todeepest point of precaudal pit, incision between caudal peduncle and upper lobe of caudal fin.
3. Head Length; HL		 Length from anterior most of body to posterior most point of last gill slit.
4. Trunk Length		 Length from anterior-most point of body to midpoint of insertion of vent (cloaca).
5. Tail Length		 Length from midpoint of insertion of vent to origin of upper lobe of caudal fin, or if present, todeepest point of precaudal pit.
6. Snout-vent Length		 Length from anterior-most point of body to midpoint of insertion of vent.
7. Body Depth; BD
		 Depth on deeper most part of body, without fin parts.
8. Depth of Caudal Peduncle.
		 Depth of lower most part of caudal peduncle. Generally measured on origin of caudal fin.
9. Preorbital Length		 Length between anterior-most points of body and orbit.
10. Preoral Length		 Length between anterior-most points of body and mouth.
11. Prespiacular Length		 Length between anterior-most points of body and spiracle (if exists).
12. Orbit Diameter
		 Horizontal diameter of orbit.
13. First Gill Slit Length		 Length between upper and lower-most points of first gill slit.
14. Interorbital Width.
		 Shorter-most width between right and left orbits, from dorsal view.
15. Internarial width
		 Shorter-most width between right and left nasal pores, from ventral view.
16. Mouth Width
		 Width between right and left ends of mouth.
17. First Dorsal-fin Length		 Length between origin and posterior most point of first dorsal-fin.
18. First dorsal-fin Spine Length		 Length of exposed part of first dorsal-fin spine from proximal origin to tip. Record in cases ofbroken and/or worn down.
19. First Dorsal-fin Hight.
		 From base to distal most point of first dorsal-fin.
20. Second Dorsal-fin Hight.
		 From base to distal most point of second dorsal-fin.
II. Fish measurements    31

21. Second Dorsal-fin Spine Length		 Length of exposed part of second dorsal-fin spine from proximal origin to tip. Record in casesof broken and/or worn down.
22. Second Dorsal-fin Hight
		 From base to distal most point of first dorsal-fin.
23. Pectoral-fin Length
		 From base to tip of pectoral-fin.
24. Pelvic-fin Length
		 From anterior origin to posterior tip of pelvic-fin.
25. Anal-fin Length
		 From anterior origin to posterior tip of anal-fin.
26. Anal-fin Hight
		 From base to higher-most point of anal-fin.
27. Dorsal caudal-fin length
		 From origin to posterior most point of upper lobe of caudal.
28. Preventral caudal-fin Length
		 From origin to posterior most point of lower lobe of caudal-fin.
29. Clasper Length
		 From anterior most point of insertion of vent (cloaca) to tip of clasper.

32

II

3 How to measure Chondrichthyes; skates and rays

Measurements of Chondrichthyes are generally done on projection on lateral axis. These customs
are different from those on Teleostei, done on point-to-point lengths.

1
2

6
A
8

16

7
9

B

14

13
11 12

15

10

2
4

5
1

Fig. 1 Common measurements on rays and skates.
A, dorsal view of Sepia Stingray Urolophus aurantiacus; B, ventral view of Wip Stingray Dasyatis akajei.
See text for numbers on the figures. Drawn by Yumi Kawai.

II. Fish measurements    33

3

1. Total Length; TL
		 Length from anterior most part of body to posterior end of caudal-fin or tail. Rays of theSuperfamily Dasyatoidea generally do not have caudal-fin, but with tail like a wip. Remind thatsuch tale sometimes lost their tips.
2. Disc Length; DL
		 Length from anterior-most point of body to posterior end of pectoral-fin. Posterior end of
pectoral fin is decided by point of left or right side of fin, or intersection of line between both endof pectoral fin and body medial axis.
3. Disc Width; DW
		 Distance between distal ends of both pectoral fin.
4. Snout-vent Length		 Length from anterior-most point of body to midpoint of insertion of vent.
5. Tail Length		 Length from midpoint of insertion of vent to posterior most of caudal-fin or tail.
6. Preorbital Length		 Length between anterior-most points of body and orbit.
7. Interorbital Width.
		 Shorter-most width between right and left orbits.
8. Orbit Diameter
		 Horizontal diameter of orbit (soft part) as sharks or teleosts.
9. Spiracle Length		 Length between anterior and posterior most points of spiracle.
10. Preoral Length		 Length between anterior-most points of body and mouth.
11. Mouth Width
		 Width between right and left ends of mouth.
12. Snout-last gill slit length (= Head Length; HL)		 Anterior most point to intersection of lines between both end of right and left last gill slit andbody medial axis.
13. Inter-first Gill Slit Width
		 Distance between right and left first gill slits,
14. Pelvic-fin Length		 Length from anterior origin to posterior tip of pelvic-fin. In some rajid skates, pelvic-fin has
anterior and posterior lobes, and should be measure both of them.
15. Clasper Length		 Length from anterior most points of insertion of vent (cloaca) to tip of clasper.
16. Caudal Spine Length		 Length of exposed part of caudal spine from proximal origin to tip. Record in cases of broken
and/or worn down.

34

II

4 How to measure Teleostei

Teleostei have many bones, many of which can be outlined from the body surface. Fins areconstituted of many foldable bony fin rays. The body surface is covered by bony scales in general.
Because many of these structures become traits for counting and measurement, Teleostei have many
more measurement parameters, particularly counting parameters, than do Chondrichthyes.
Measurement method: As opposed to measurements of Chondrichthyes, which generally use
projection, in measurements of Teleostei, the distance between two points is measured by directly
applying a vernier caliper to both ends of the measurement site, as shown in the figure below. Therefore,
the axis of measurements is independent of the body axis, and oblique measurements are frequently
used. However, this is only the method for making measurements; therefore, it is understood thatsketching uses projection.
To compare measurement values between species or individuals, it is typical to calculate andexamine the ratio or percentage of body parts to the standard length or the head length. In addition,
the percentage of one body part to another body part, for example, the eye diameter to the upper
jaw length, is also a valid examination parameter. When a graph is prepared with the vertical axis
representing such a ratio or percentage of the length of a body part and the horizontal axis representing
the body length, the plots are distributed horizontally if the species shows no change during growth,whereas an inclined distribution means that the rate changes with growth. If any difference is seenin the distribution range of the plots in a comparison of graphs of the same parameter between fish
species, it is a valid taxonomic character, and the less the overlap is, the higher the validity is estimated.

2
1

3
10

TRa

5

LLp

7
4

TRb

8

9
11
6

Fig. 1 Common counts and measurements on teleosts.
See text for numbers on the figures.

II. Fish measurements    35

1. Total length (TL)		 This is the length from the most anterior terminus of a body to the most posterior terminus of
the caudal fin. The anterior terminus may be on either the upper or lower jaw as long as it is themost anterior. For the posterior terminus of the measurement, use the most posterior terminus ofeither the upper or lower lobe of the caudal fin, including a thread-like extension. The official
definition prescribes to measure it by compressing the caudal fin if it is a fresh fish, the caudal finof which can be opened and closed. However, in the case of a specimen with fixed fins or of caudal
fins that cannot be opened and closed, like those of fish in Scombridae, use the posterior terminusof the caudal fin extended in the normal form. Although this is a measurement valued in market
and fisheries science, it is not valued very much in ichthyology because the posterior terminus of
caudal fins is damaged in many cases.
2. Standard length (SL) or body length (BL)		 This is the length from the snout tip to the terminus of the vertebral column (posterior marginof the caudal skeleton) (see Figure 4 in III-7). Because the most anterior terminus of the headvaries depending on the fish species, it is not necessarily consistent with the snout tip. Even if thelower jaw protrudes more than the upper jaw (superior mouth), as in Scorpaeniformes, Serranidae,
and Hemiramphidae, the lower jaw should not be included. When the edges of both jaws are at
the same position (terminal mouth) or the upper jaw is at the most anterior terminus (subterminal
mouth) as in Carangidae and Sparidae, measure from the most anterior terminus of the upper lip.
If the mouth is located on the ventral surface (inferior mouth) as in Engraulidae and Polynemidae,
measure from the snout tip located superior to the upper lip. Because the terminus of the vertebral
column, the posterior terminus of the measurement, is also the base of the caudal fin, use thefolding line made by bending the caudal fin hard as the terminus of the measurement. This is theparameter valued most in ichthyology as the measurement representing the size of fish.
3. Fork length (FL)		 This is the length from the snout tip to the point where the posterior margin of the upper andlower lobes of the caudal fin meet. This is used for the measurement of fish with a crescent or fork
type of caudal fin. The start point of measurement is the same as that of the standard length in 2.
The posterior terminus is the most concave part at the center of the posterior margin of the caudal
fin. This is regarded as an important measurement parameter as an alternative for the standard
length in fish in Carangidae and Scombridae in which joint parts of the caudal fin are so rigid that
it is difficult to bend the fin.
4. Head length (HL)		 This is the length from the snout tip to the most posterior margin of the branchiostegal

15, 16

12

14

Fig. 3

13

Fig. 236

membrane. In some cases, the measurement terminus is the posterior terminus of the skeleton
constituting the operculum. However, because it is difficult to ascertain in many cases, measure
this length by including the membranous part.
5. Predorsal length (PDL)		 This is the length from the snout tip to the start point of the dorsal fin or the first dorsal fin.
6. Preanal length (PAL)		 This is the length from the snout tip to the center of the anus. Alternatively, there is also amethod that measures the length to the start point of the anal fin.
7. Body depth (BD) or body height (BH)
		 This is the height at the highest part of the body. Measure this by excluding the fleshy part andscales belonging to fins, and additionally note the measurement point by the ray number of thedorsal fin, etc. Because the position of the highest part varies among individuals, or depending on
the physiological condition even in the same individual, the measurement site is often prescribed,
such as the start point of the first dorsal fin and the position of the median spine of the dorsal fin.
8. Depth of caudal peduncle
		 This is the minimum height of the caudal peduncle (from the posterior terminus of the anal finbase to the caudal fin base).
9. Length of caudal peduncle
		 This is the length from the posterior terminus of the anal fin base to the center of the caudal finbase.
10. Basal length of dorsal fin
		 This is the length from the start point of the dorsal fin (anterior terminus of the base of the firstray) to the posterior terminus of the base (posterior terminus of the base of the last ray). The finmembrane part following this should not be included. If the dorsal fin is separated into two or threeparts, measure the size of each of them.
11. Basal length of anal fin
		 This is the length from the start point of the anal fin to the posterior terminus of the base. The
notes for this length are the same as above.
12. Snout length
		 This is the length from the snout tip to the anterior margin of the orbit.
13. Maxillary length or length of upper jaw
		 This is the length from the most anterior point of the upper lip (premaxillary) to the posterior
terminus of the maxillary. Note that this is not the length of only the maxillary.
14. Interorbital width
		 This is the minimum width of the bony part between the orbits when viewed dorsally. The
minimum width including the fleshy part is measured in some cases.
15. Orbit diameter (OD) ≒ eye diameter		 This is the maximum diameter of the fleshy orbit, measured horizontally where possible. The
bony orbit diameter is measured by applying a vernier caliper hard in some cases. The orbit refers
to a groove in which an eyeball is located.
16. Eye diameter (ED)		 This is the maximum horizontal diameter of the eyeball across the cornea. This is equal to thefleshy orbit diameter in many fish.
17. Length of fin ray
		 Normally, the maximum ray length of each fin is measured. However, a ray is specified using
an ordinal number in some cases. However, in the case of the caudal fin, the soft ray at the medianis usually measured.
II. Fish measurements    37		 In the case of spines, a thread-like part attaching to the edge and a part extending like a soft
ray should not be included. In the case of soft rays, measure the length to the tip including these
extensions. Enter the number of the ray which was measured in ( ).

Counting method:
1. Number of fins		 If the dorsal fin (D) and the anal fin (A) among the vertical fins are divided longitudinally, countthem as the first dorsal (anal) fin, the second dorsal (anal) fin, etc.
2. Number of fin rays
		 The number of rays constituting each fin is the most basic and important countable trait for theidentification of the species. At the time of the counting, you need to have sufficient knowledge ofthe basic structure.
		 There are two kinds of Teleostei fin rays formed by any type of bony material, the non-paired
spine at the median of the fin (Figure 4A) and the left-to-right paired soft rays (Figure 4B and
C). Moreover, another difference between the types is that the soft rays have many transverse
segments whereas spines lack them. In addition, when the soft rays are observed from the side,
branched soft rays (Figure 4B) originating from a base and ramifying to several branches in
the middle, and unbranched soft rays (Figure 4C) that have no branching to the tip (Figure 4C)
are found. To distinguish such ray types on small specimens, you need to observe them undera stereomicroscope with transmission light. Even in the case of a large specimen, it is better toobserve them with a transmission light using a fluorescent lamp or sunlight. In the case of evenlarger specimens, the fins of which are covered by a thick fin membrane and/or have many black
pigments making ray identification difficult unless modified, compress them hard with your finger
pad, or if this does not work, somewhat peel off the fin membrane.
		 When counting fin rays, their bases are supposed to be counted. However, the counting of
branched soft rays requires particular care. An important rule is that the last two soft rays adjacentat the base should be regarded as a branched soft ray in the dorsal and anal fins. This is based on
the fact that such apparently two soft rays are supported by only one bone (pterygiophore). Do
not add the number of the rudimentary ray located at the start of the dorsal fin and top and bottomAanteriorview

Blateralview

anteriorviewClateralview

anteriorview

lateralview

Fig. 4 Skeletal fin rays of teleosts.
A, spine; B, unbranched soft ray; C, branched soft rays.

38

of the caudal fin base to the number of rays. In the caudal fin (see III-7-5 and Figure 4 in III-7),
only the number of all branched rays is described separately in the upper and lower lobes in somecases, and in other cases, soft rays of both the upper and lower lobes reaching nearly the posterior
terminus are counted and mentioned as the number of principal rays separately in the upper andlower lobes. Generally, the number of principal rays is equal to the number of branched soft rays
plus an unbranched soft ray in the upper and lower lobes.
		 The counting of rays of paired fins uses fins on the left side except in the case of damage and thelike. Because the starting part of the pectoral fin (P1) has a rudimentary ray and/or an unbranched
ray in some cases, not only the number but also the difference in the condition is important in somecases. Regarding pelvic fins (pelvic fin; P2), because a spine is close to the first soft ray in somecases, you must count it by separating them using a needle, etc.
		 The number of rays counted according to the criteria above is represented as follows:		 Represent spines by capital Roman numerals (I, II, …V, … X, see p. 40), unbranched soft raysor rudimentary rays by lower case Roman numerals (i, ii, … v, …), and branched soft rays by
Arabic numerals (1, 2, 3, …). In addition, represent the intervals between multiple fins separated
in a vertical fin by a hyphen (-), and the border between spines and soft rays in a fin by a comma(,). The ordinal number of a fin and the number of spines or soft rays of a fin are expressed by thefollowing fin formulas.
a) If a vertical fin composed of only soft rays is clearly separated:			 D. 13-18-16; A. 21-27 (Figure 5A-example of Pacific cod Gadus macrocephalus)b) If the dorsal fin is completely or nearly completely separated, and any vertical fin composed ofboth spines and soft rays is included:
			 D. XIII-1, 11; A. III, 7; P2. I, 5 (Figure 5B-example of Korean rockfish Sebastes schlegeli)
3. Number of scales in the lateral line (LL)		 All scales arranged on the lateral line should be counted. However, if any scales are on thecaudal fin, exclude them.
4. Number of pored scales in the lateral line (LLp)
		 Count all pored scales by excluding the poreless scales among the scales arranged on the lateralline. Exclude scales on the caudal fin.
5. Scales in a longitudinal series
		 In cases of lateral line not exit, running far from mid sagittal line, or breaking, scales in alongitudinal series, excluding on caudal fin, are counted, from pectoral girdle to the end ofvertebrae.
6. Number of transverse scales; TR  a) 
Scales above lateral line; TRa: vertical number of scales on a line from base of dorsal fin tolateral line. Lateral line scale is not included. In some cases this count starts from medial spineof dorsal fin etc. with special definition.

A

B

Fig. 5 Skematic figures of teleosts.
A, Pacific Cod Gadus macrocephalus. B, Korean Rockfish Sebastes schlegelii.
II. Fish measurements    39  b) Scales below lateral line; TRb: vertical number of scales on a line from base of anal fin tolateral line. Lateral line scale is not included. 
Lateral line scales and transverse scales are expressed by scale formula as below.
				
Scales: 7+70+11
		  This formula means TRa is 7, LL is 70, and TRb is 11.
7. Predorsal scales
		 Count of scales on mid dorsal line from anterior most of dorsal fin base to front.
8. Number of gill rakers; GR, see Figure 1 in III-2.
		 Gill rakers are aligned in two lows, and the outer low is longer. Generally the count is takenfrom the outer side row of 1st gill arch, but sometimes count only on lower limb of arch withremarks.
		Counts on upper limb and lower limb are joint by “+” and generally the raker on the joint iscounted on lower raker as 8+12. However sometimes counted and expressed as 8+1+11 as for“Figure 2 in III-2”.
		 Rakers on upper limb have basal structure for fordable to ventral angle, and those of lower limb
have such to dorsal angle. Also the raker on the angle is fordable to both angles.
9. Number of branchiostegals; Br
		 Number of limb-like bones under head, appearing when mouth closing.
10. Number of pyloric caeca
		 Count number of all tips, except in cases of branching.

correspondence of Arabian and Roman Numbers
Arabian Numbers
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

40

Roman Numbers
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
XX

Arabian Numbers
21
22
...

Roman Numbers
XXI
XXII
...

28
29
30
40
50
60
70
80
90
98
99
100
101
126
200
300
400
500

XXVIII
XXIX
XXX
XL
L
LX
LXX
LXXX
XC
IIC
XCIX
C
CI
CXXVI
CC
CCC
CD
D

III
Observation of internal morphology

1 Formation of viscera

III

Fresh fish viscera (sg. viscus) have peculiar solidity and touch. These characters are not available
from preserved specimens, and need fresh specimens to observe them. The positions of viscera inabdominal cavity are often dislocated by dissection in fresh specimens, so need formalin fixed (andethanol preserved) are used for such observations. In this chapter digestive organs and other viscera ofBlue Mackerel Scomber australasicus are shown in line figure (Fig. 1) and color photographs (Fig. 2).
Operation starts from cutting off opercle, by cutting from lower most part of gill membrane(widely united with throat in some species) to both side of joint of lower jaw. Lifting up opercle andcut off the joint with neurocranium, and continuously cut off postorbital region to both joints of upper
jaw. Or, just cut off suborbital region by heavy-duty scissors (= bone scissors).
The next is removing lateral muscles, by inexperienced students, cutting from anus (16) to ventral
medial line by scissors. Experienced students start cutting around anus by surgical knives without

8
12-1
5-1

4

1

17

10

7

18

6

2
3

5-4
5-2
5-3
11

9

12-2

19
16

12-3
15
13

14

Fig. 1 Viscera of Blue Mackerel Scomber australasicus.
1. mouth (jaws)

5-3 ventricle

11. gall bladder

15. rectum

2. oral cavity

5-4 arterial bulb

12. stomach

16. anus (=vent)

3. gill raker

6. head kidney12-1 cardia

17. gonad

4. gill filament

7. body kidney12-2 blind sac	  ovary

5. heart

12-3 pylorus	  testis

5-1 venosus sinus

9. liver13. pyloric caeca

18. urocystis

5-2 auricle

10. spleen

14. intestine

19. urogenital orifice42

8. air (gas) bladderdamaging digestive organs, and next, with scissors, continue cutting forward. Further, cut with scissors
from anus by dorso-anterior angle of left side to near pectoral fin base. Carefully remove kidneys (7)one by one, hidden under lateral process of vertebrae. Before doing this, swim bladder (absent in
Atlantic Mackerel S. scombrus) appears but is easily broken in dissecting process, and hard to observeor draw.

Fig. 2 Anatomical steps of Blue Mackerel Scomber australasicus.
Upper, removing opercula and lateral muscles; Middle, removing pectoral girdle and base of ribs;
Lower, Pulling off of gastrointestinal tract.
III. Observation of internal morphology    43The last of this serial dissection is removing firm bones of pectoral girdle (Fig. 2, middle). This
step should be very careful to avoid injure soft parts connected nearby underside of the bone, such ashead kidney (6) or sinus venosus (5-1). Cut off the connection of upper (=dorsal) side of pectoral girdle
and neurocranium. Also cut off lower (=ventral) side connection of both side of cleithrum.
Pull down viscera in abdominal cavity for observation and drawing (Fig. 2). To avoid damage by
dissecting instruments on viscera, this step should be done by hands, and carefully cut peritoneum by
fingers, and keep attaching of start and end of digestive organs with body.

Fig. 3 Dissecting Blue Mackrel Scomber australasicus.
Left upper: ventral view of head and thorax. Left middle: dissection of ventral view of head and thorax. Left lower:
lateral view of dissected head and thorax.
Right upper: dorsal view of head and parietal region. Right middle: Dissection of head and parietal region. Rightlower: inside of opercula, showing parabranchia.

44

III

2 Observation of gills

Gills are common in fish, occupy wide space under neurocranium, and are easily recognized
red organs by opening gill slit. One set of gill, the main part is thin curved bone, or gill arch, withtubercular or rodlike process on anterior part, or gill rakers. The posterior part is with numerous soft
red flaps, or gill filaments.
One gill arch consist of > shaped upper limb, and lower limb. The stem of upper limb is epibranchial(No.60), and the stem of lower limb consists of two bones, hypobranchial (No. 57) for anterior part,and ceratobranchial (No. 58) for posterior part. Both left and right sides of gill arch are connected by alined several bashibranchials (No. 56), and its anterior tip is homologous with human tang, including
basyhyal (No. 48) in hyoid arch. The anterior tip of upper limb is hanged down from neurocranium bysuprapharyngobranchial (No.61). On the way of evolution, original 5 pairs of gill arches modified inrecent fishes, as upper surface of lower branch of 5th gill arch with lower pharyngeal teeth, and called
as lower pharyngeal (No. 59). Also upper branch of 4th and both branch of 5th arches are modified
to upper pharyngeal (No. 63) with lower pharyngeal teeth on the lower surface. Pharyngeal teeth offishes have rich morphological variation, and used important taxonomic keys for some taxon such as
the order Cypriniformes and family Scaridae.
Generally there are gill rakers on the anterior surface of 4 pairs of gill arch, generally those on
the 1st (most outside, most anterior) are conspicuously developed. Each of gill rakers is covered with
densely minute spines on surface, and the epithelium has taste buds. Gill rakers are filtering organs for
aspirated water, and plankton feeder has many long rakers, but those of swallowing carnivores are lostor rudimental. The shape or number of rakers related with feeding habitat, and are used for importanttaxonomic key characters. Count of gill rakers is done on the outside row of 1st gill arch (see II-2).

upper limb
(epibranchial)(No. 60)

mouth
gill rakers(hypobranchial)(No. 57)(ceratobranchial)(No. 58)

gill filament

lower limb

Fig. 1 Anterior most (= external most) gill arch of Red Sea Bream Pagrus major.
Numbers on figure indicate names of skeletons, III-3. Dotted lines mean joints.

III. Observation of internal morphology    45Gill filaments are gas exchanging organs of oxygen and carbon dioxide between brad andenvironmental water, and also keep salinity balance of body. There are 2 rows of gill filaments on
posterior end of 1st to 4th gill arches. In elasmobranchs, 2 rows on each gill arches are divided byinterbranchial septum, and 5 – 7 pairs of external gill slits are opened. In teleosts, interbranchial
septum are degenerated and lost, and gill filaments are arranged in parallel. Each gill filaments has
many vertical (secondary) gill lamella on upper and lower sides, and such expanded surface area is
effective for gas and mineral exchanges. Length and number of gill filaments are related with fish
activity. Pelagic fishes have developed long filament, and benthic and slow swimming fishes have
undeveloped filaments. Thus, relative length of the filaments is a material to consider fish activities.

Anterior

Posterior

58
A

57

Anterior

60
59
B

48

61

56

62
63

Fig. 2 Gill arch of Japanese Sea Bass Lateolabrax japonicus.
A, lower half. B, upper half. From Mastsubara et al. (1979). See III-3 for names and numbers of bones.

46

A

C

B

D

E

Fig. 3 Gills of various fishes.
A, pelagic fish, Frigate Tuna Auxis thazard; B, carnivorous fish, Humpback Red Snapper Lutjanus gibbus; C, plankton
feeder, Midnight Snapper Macolor macularis; D, mesopelagic fish, Long Snouted Lancetfish Alepisaurus ferox; E,
benthic fish, Yellowfin Stargazer Uranoscopus chinensis.
III. Observation of internal morphology    47III3 Names and classification of skeletons
Numbers of text indicate those on figures of III-2~III-7.
⎧ Neurocranium ⎪⎪

Nos. 1-23

Protecting central nerve systems and sensory organs⎧ Cranium ⎨ splanchnocranium 
24-63⎪
Supporting and protecting both jaws, hyoid arch,⎪branchial arch, palate, and gills⎧ Axial Skeleton⎨⎩⎪⎪ Nothochord⎪⎪ Running below spinal cord, and supporting it
⎪
⎪
⎪
64-86
Column ⎪ Vertebral
Endoskeleton ⎨Covering and protecting spinal cord and notochord⎩⎪⎧ Pectoral Girdle 
⎪
87-96
⎪
⎪ Supporting pectoral fin⎪
97⎩ Appendicular ⎪ Pelvic Girdle ⎨ Supporting pelvic fin
skeleton⎪ Pterigiophore ⎪ Supporting rays of each fins

Exoskeletin ⎩⎧ Fin Ray ⎪⎨⎪⎩ ScaleNo.
1–23
1 (III-6, Fig. 1)
2 (III-6, Fig. 1)
3 (III-6, Fig. 1)
4 (–)
5 (–)
6 (III-6, Fig. 1)
7 (III-5, Fig. 1; III-6, Fig. 1)
8 (III-6, Fig. 1)
9 (III-5, Fig. 1)
10 (III-6, Fig. 1)
11 (III-6, Fig. 1)
12 (III-6, Fig. 1)
13 (–)
14 (III-6, Fig. 1)
15 (III-6, Fig. 1)
16 (III-6, Fig. 1)
17 (III-6, Fig. 1)
18 (III-6, Fig. 1)
19 (III-6, Fig. 1)
20 (III-6, Fig. 1)

48

98-104105-112English Names
neurocranium (= cranium)
ethmoidethmoid cartilage
lateral ethmoid (= prefrontal)
preethmoid
supraethmoid (= mesethmoid)
prevomer
nasal
rostral (cartilage)
sclerotic
frontalpterosphenoidbasisphenoid
orbitosphenoid
sphenotic
pterotic
epiotic
prootic
exoccipitalsupraoccipitalparietalNo.
21 (–)
22 (III-6, Fig. 1)
23 (III-6, Fig. 1)
24–63
24 (–)
25 (III-5, Fig. 1)
26 (III-5, Fig. 1)
27 (–)
28 (III-5, Fig. 1)
29 (III-5, Fig. 1)
30 (–)
31 (III-5, Fig. 1)
32 (III-5, Fig. 1)
33 (III-5, Fig. 1)
34 (–)
35 (–)
36 (III-5, Fig. 1)
37 (III-5, Fig. 1)
38 (III-5, Fig. 1)
39 (III-5, Fig. 1)
40 (III-5, Fig. 1)
41 (III-5, Fig. 1)
42 (III-5, Fig. 1)
43 (III-5, Fig. 1)
44 (III-5, Fig. 1)
45 (III-5, Fig. 1)
46 (III-5, Fig. 1)
47 (III-5, Fig. 1)
48 (III-2, Fig. 2; III-5, Fig. 1)
49 (III-5, Fig. 1)
50 (III-5, Fig. 1)
51 (III-5, Fig. 1)
52 (III-5, Fig. 1)
53 (III-5, Fig. 1)
54 (–)
55 (III-5, Fig. 1)
56 (III-2, Fig. 2)
57 (III-2, Fig. 1; III-2, Fig. 2)
58 (III-2, Fig. 1; III-2, Fig. 2)
59 (III-2, Fig. 2)
60 (III-2, Fig. 1; III-2, Fig. 2)
61 (III-2, Fig. 2)
62 (III-2, Fig. 2)
63 (III-2, Fig. 2)
64–86
64 (III-7, Fig. 1)
65 (III-7, Fig. 1; III-7, Fig. 4)
66 (III-7, Fig. 4)

English Names
intercalar (= opisthotic)
basioccipitalparasphenoid
splanchnocranium (= visceral skeleton)
supraorbital
lachrymal (= preorbital)
infraorbital
suborbital
premaxillarymaxillarysupramaxillarydentary
angularretroarticular
articular
coronomeckelian
mentomeckelian cartilage
palatineectopterygoid (= pterygoid)
endopterygoid (= mesopterygoid)
metapterygoid
quadrate
symplectic
hyomandibular
preopercle
operclesubopercle
interopercle
basihyal (= glossohyal)
upper hypohyal
lower hypohyal
ceratohyal
epihyalinterhyalbranchiostegal
gular plateurohyalbasibranchial
hypobranchialceratobranchial
lower pharyngeal
epibranchialsuprapharyngobranchial (= suspensory pharyngeal)
infrapharyngobranchial (= pharyngobranchial)
upper pharyngeal
vertebral column
abdominal vertebra
caudal vertebra
preuralcentrum 2, 3
III. Observation of internal morphology    49No.
67 (III-7, Fig. 4)
68 (III-7, Fig. 4)
69 (III-7, Fig. 4)
70 (III-7, Fig. 4)
71 (III-7, Fig. 4)
72 (III-7, Fig. 3)
73 (III-7, Fig. 3; III-7, Fig. 4)
74 (III-7, Fig. 3)
75 (III-7, Fig. 3)
76 (III-7, Fig. 3)
77 (III-7, Fig. 3)
78 (III-7, Fig. 3)
79 (III-7, Fig. 3; III-7, Fig. 4)
80 (III-7, Fig. 3)
81 (III-7, Fig. 3)
82 (III-7, Fig. 3)
83 (–)
84 (–)
85 (III-7, Fig. 3)
86 (–)
87–96
87 (III-5, Fig. 1)
88 (III-5, Fig. 1)
89 (III-5, Fig. 1)
90 (III-5, Fig. 1)
91 (III-5, Fig. 1)
92 (–)
93 (III-5, Fig. 1)
94 (III-5, Fig. 1)
95 (–)
96 (III-5, Fig. 1)
97
97 (III-5, Fig. 1)
98–10498 (III-7, Fig. 1)
99 (III-7, Fig. 1; III-7, Fig. 2)
99, 102 (III-7, Fig. 1; III-7, Fig. 2)100, 103 (III-7, Fig. 2)
101, 104 (III-7, Fig. 2)102 (III-7, Fig. 1; III-7, Fig. 2)
105–112105 (III-7, Fig. 1)
106 (III-7, Fig. 1; III-7, Fig. 2)
107 (III-7, Fig. 1)
108 (III-7, Fig. 1; III-7, Fig. 2)
109 (III-7, Fig. 1; III-7, Fig. 4)110 (III-5, Fig. 1)
111 (III-5, Fig. 1)
112 (III-5, Fig. 1)

50

English Names
urostyle (= preural centrum 1 + ural vertebra)
hypuraluroneural
epuralParhypural
neural spine
neural archneural zygapophysis
centrum
hemal zygapophysis
hemal arch
parapophysis (= transverse process)hemal spine
neural canalhemal canal
pleural rib (= rib)
epipleural
epineural
epicentral
myorhabdoi (= myorabdoi)
pectoral girdle
supratemporal (= extrascapular)
posttemporalsupracleithrum
cleithrum
scapulamesocoracoid
coracoid
postcleithrumclavicle
actinost
pelvic girdle
basipterygium
pterygiophore
free interneural spine (= predorsal bone)interneural spine
proximal pterygiophore
median pterygiophore
distal pterygiophoreinterhemal spine
exoskeleton
dorsal spine
dorsal soft ray
anal spine
anal soft ray
caudal soft ray
pelvic spine
pelvic soft ray
pectoral soft ray

III

4 How to observe skeletons

1. Studies on skeletons are called osteology. Osteology is peculiar to Vertebrata and is essential forstudies on their phylogenic evolution and species identification. This is because osteology has thefollowing advantages.
1) Highly objective data can be obtained because a stable and consistent result can be obtained
regardless of when or who observes and measures a skeletal trait, due to its very low deformation
in long-term storage.
2) Because bones, in particular, are likely to be preserved as fossils among skeletons, osteology
allows us to directly compare and examine historical taxa. In other words, in terms of the
traceability of evolutionary history, this is an advantage over studies on muscles, visceralorgans, chromosomes, genes, proteins, etc. from which no data can be obtained unless the
specimen is contemporary, due to the difficulty in preservation.
2. How to study skeletons of contemporary fish
1) X-ray photography: This has the advantage that individual variation in the counted values,
including the number of the vertebrae and the number of the pterygiophores, can be reduced
because many specimens can be relatively easily examined non-destructively if the specimens
are unpaired skeletons (vertebral column, vertical fins, etc.). However, disadvantages include
that the outline of cartilages and bone pieces with a thin periphery are not clear, and that it is
impossible to understand the shape of paired skeletons due to overlap in the image.
2) Separation of skeletons: Separate the skeleton from the body by boiling the raw or salted
specimen or letting insects (Dermestidae, etc.) eat the fleshy parts. Preservation by dehydrationis convenient for studies on the shape of individual bones and using the skeletons as educational
materials due to ease of handling, but cartilages can be so deformed that their original shapeis not preserved, and even bones will be a little deformed. Preservation of picked skeletons inalcohol can preserve the shape, but it has the disadvantage that the linkage between bones is notpreserved because the bones will eventually disassemble.
3) Preparation of double-stained transparent specimens: Stain the bones of specimens in red
with alizarin red and their cartilages in blue by alcian blue, make the skin and the musclesof the specimens transparent using potassium hydroxide and trypsin, and observe the bones
and cartilages. This method allows us to observe the best condition because the shape of the
fish body is maintained unchanged, preserving the linkages between the bones and causing
no deformation of bones and cartilages. However, it takes days to prepare a specimen, andthe protocol requires skill. In addition, the handling of specimens is a little troublesome
because they are preserved in glycerin. I have omitted this preparation method in this bookbecause many informative books that have been recently published discuss this. (References:
Osamu Fukuhara, Masaru Tanaka. 1987. Techniques for studies on the early life history ofmarine fish 1 - How to stain hard tissues of larvae and juveniles. Aquabiology, 9 (2): 97-99;
Koichi Kawamura, Kazumi Hosoya. 1991. A modified double staining technique for making atransparent fish-skeletal specimen. Bulletin of Natural Research Institute of Aquaculture, (20):
11-18)
3. Experimental method according to 2) above
1) Wrap the specimen with a sheet of gauze to prevent the loss of small bones and cartilages.
2)	Boil the specimen in dilute potassium hydroxide (KOH, below 0.1%) aqueous solution. Stop
III. Observation of internal morphology    51the boiling at the point when a little of the redness of blood remains in the skeleton when you
peel off the muscle in the vicinity of the most anterior part of the vertebral column. Excessive
boiling can cause the neurocranial members to be dislocated at the joints. Once dislocated, they
can never be restored. Although KOH makes it easy to remove flesh, excessive use is harmful
because bones, particularly soft rays, will be fragile.
3) Collect the external skeletons in the vicinity except scales (I postpone this to another occasion)in the following order in general. Because most of the bones except for the axial skeletal arein a right-to-left pair, preserve those on the left side, and put those on the right side aside forsupplementation.
4) Find the name of each bone, and note the mutual relationship through a joint by sketching thebones in a way that indicates their top and back as well as four quarters.
5) Draw an outline of the fish on another Kent paper prepared beforehand, and temporarily place
the picked bones on a place equivalent to their in vivo location. Avoid paying too much attention
to cleaning in this step and complete the procedure soon. Because the skeletons are arranged
three-dimensionally, you need to be creative in arranging the bones two-dimensionally.
6) First, pick up the nasal bone (No. 7), the circumorbital [there are the lacrimal bone (No. 25),
the infraorbital bone (No. 26), and, depending on the species, the supraorbital bone (No. 24)
and the subinfraorbital bone (No. 27)], and the supratemporal bone (No. 87). Because thesupratemporal bone is very likely to be lost, be sure to pick it up in this step. (A→B in thefoldout figure.)
7) Remove the muscles for moving the jaws and the muscles at the pectoral fin base, and then pickup the bones forming the upper jaw [there are the premaxillary (No. 28), the maxillary (No. 29),
and, depending on the species, the supramaxillary (No. 30)]. (B→C in the foldout figure)8) After picking up the dentary (No. 31) and the angular bone (No. 32) forming the lower jaw,pick up the palatine arch (Nos. 32–43) and the opercular arch (Nos. 44–47) together anddisassemble them later. (C→D in the foldout figure)9) Pick up the basihyal (No. 48, only one at the center) from the position equivalent to the human
tongue, and then the following hyoid arches (Nos. 48–53) together, and disassemble them later.
(D→E in the foldout figure)10) After picking up the urohyal (No. 55, only one at the center) which was between the bilateralhyoid arches, pick up all the branchial arches from both the right and left sides together, anddisassemble them later. (No foldout figure. See the branchial arches of Japanese sea Lateolabrax
japonicus in Figure 2 in III-2)
11) Disassemble and pick up the pectoral girdle (Nos. 88–96) and the pelvic girdle (No. 97)
inferiorly in order. At this time, combine the soft rays together because they are fragile.
12) Remove the upper part of the dorsal muscle, and then pick up the dorsal-fin pterygiophores(Nos. 99–101) and rays (Nos. 105–106) by recording the positional relationships with theneural spine (No. 72). (See Figure 1 on p. 58.)
13) Collapse the contact plane (horizontal septum) between the myomeres of the dorsal and ventral
muscles, record the position of the joints between the vertebra (Nos. 64–81) and the rib (No.
82) as well as the intermuscular bone (Nos. 83–86), and pick up the set except the vertebra.
Repeat this for the following sets of myomeres.
14) Remove the lower half of the ventral muscle, and then pick up the anal-fin pterygiophores(Nos. 102–104) and rays (Nos. 107–108) by recording the positional relationships with thehemal spine (No. 79). (See Figure 1 on p. 58.)
15) Finish this procedure by picking up the neural cranium (Nos. 1–23) and the vertebral column(Nos. 64–81) together.

5216) Carefully remove muscles, blood vessels, bone marrow, etc. from each of the picked skeletal
elements. Then, clean the element and arrange and tape it on thick paper or in a wooden box,
or preserve it in 70% ethyl alcohol solution.
17) You need to be extremely careful not to disassemble the caudal skeleton at the terminus of thevertebral column.

III. Observation of internal morphology    535 Observation of the splanchnocranium, pectoral girdle, and pelvic girdle

III

The major skeletal arranged in the axial part of a fish body is called the axial skeleton. The axial
skeleton is composed of the cranium or the skull (neurocranium and splanchnocranium) followedby the notochord and the vertebral column to which the pleural ribs are attached, and a variety ofintermuscular bones.
Because many fish crania are exposed medially and/or laterally, their morphological characteristics
are often seen in descriptions of fish species. Therefore, to avoid missing bones that can be examined in
species identification, it is important to know the site and morphological characteristics of individualbones.
It is not the case that all of the bones presented in the table in III-3 are present in a species usedas the experimental material, and that the figures on p. 45–62 show all bones. In addition, you should
pay close attention to the collection of individual bones because bones may have become soft andcartilages may have become hard.
Splanchnocranium; Nos. 24–63 (= visceral skeleton): This is present in the periphery of theneurocranium and composed of many of the very slender bones. When picking these up, refer to thefoldout figure to determine from where to pick them up. Temporarily place the removed bones on asheet of paper by determining their arrangement based on Figure 1 below, and enter the name. Clean

87

9

26

25

89
44

7

8

88

43

45

37
90

39
28

46

94

91

29
38
40
32
33
31

42
41
49 50

47
93
51

96
112

52
111

36
48
55

97
53

Fig. 1 Splanchnocranium of Red Sea Bream Pagrus major.
Large dots indicate cartilages. See table of III-3 for bone names and numbers.

54

110

the bones individually after observation and sketching, tape them to their original position, and makea clean copy of the names. Be careful to avoid an error in the anterior–posterior, top–bottom, and top–back orientations of each bone. Also, pay attention to the bones through which the lateral line canals
on the head run (lacrimal bone; No. 25, infraorbital bone; No. 26, dentary; No. 31, premaxillary; No.
44, etc.) and the route, opening, and arrangement of tunnels through which vessels or nerves run. In
addition, because there are not only the two jawbones but also the bones on which teeth are present(palatine: No. 37, etc.), follow the procedures below with careful observation.
Circumorbital; Nos. 24-27: The orbit of some splanchnocrania is constituted of a large lacrimalbone (No. 25) anterior to the eye and a series of infraorbital bones (No. 26) surrounding the posterior
margin of the eye from immediately posterior to the lacrimal bone via the infraorbital part. The
splanchnocranium of ancient fish has a supraorbital bone (No. 24) fringing the upper margin of the eye
in some cases. Naturally, these are in a left-to-right pair. The inner margin contacting the eye is thickbecause sensory canals pass through it, and it has scattered openings for branched canals on the outersurface. In fish belonging to Perciformes, etc., the first and second anterior infraorbital bones have an
overhang facing medially like it is supporting the eyeball, which is called the suborbital shelf. The
position and number of the suborbital shelf are a clue for investigating relationships. Scorpaeniformes
fish show a common peculiar specific trait that the posterior margin of the second infraorbital boneextends to form a joint with the preopercle (No. 44) on the surface. [In the anterior margin of the snout,
there is a pair of thin plate-like nasal bones (No. 7) inside the right and left nostrils. This is free fromthe neurocranium, but considered a part of neurocranium instead of the splanchnocranium.]
Upper jaw; Nos. 28–30: Inside the nasal bone, there is an ascending process of the premaxillary(No. 28), and inferior (posterior) to the processes of the right and left bones, there is an oval or
rice-ball-shaped rostoral bone (No. 8) (unpaired). After taking out the bones forming the upper jaw
in conjunction with the premaxillary, the bone adjacent superiorly is the maxillary (No. 29), and,depending on the species, 1–2 slender supramaxillary bones (No. 30) run in parallel in its posterior
dorsal part. On the inferior margin of the premaxillary, there are a variety of small to large teeth
depending on the species. In ancient fish including Clupeidae, the premaxillary is small and the oral
margin is formed by the maxillary, etc., where some species such as Amia, gars, and deep-sea fish inArgentiniformes and Stomiidae have teeth. In many of the advanced fish groups, such as Perciformes
and Scorpaeniformes, the mouth is formed by only the premaxillary extending highly to the posterior
direction, and the maxillary is recessive and does not face the oral margin. The developmental level ofthe ascending processes in the anterior superior part of the premaxillary varies remarkably dependingon the species, from those in which it moves like it is rotating using the short rostoral bone (No. 8)
immediately inferior as a fulcrum to the species in which the upper jaw can greatly protrude by sliding
the long process.
After removing the upper jaw, removal of the muscle, etc. of the cheek allows you to observe a
series of bones linked from the lower jaw to the posterior terminus of the operculum. Cut these bones,
keeping the joints where possible off the hyoid arch inside (below of observation angle) them, cut theleft-to-right jointing part of the lower jaw and temporarily place the whole piece longitudinally. Aftersuch preparation, disassemble and clean them step-by-step as follows.
Lower jaw, Nos. 31–36: The dentary (No. 31) located at the most anterior part of the selectedbones is the only bone constituting the oral margin of the lower jaw. Because the dentary and teeth on
the premaxillary have various morphologies associated with their feeding habit, you need to observe
them sufficiently. The arrowhead-like bone sticking into the deep notch of this dentary anteriorly iscalled the angular bone (No. 32). Inside the dentary and angular, there is the slender Meckel’s cartilage(No. 36) connecting the two bones. Because this will accompany either one when the bones are pulled
apart, do not further disassemble it. The minute bone called the coronomeckelian (No. 35) at theIII. Observation of internal morphology    55base of this cartilage on the angular side has been gaining phylogenic attention recently. Do not cut
off a small bone called the retroarticular bone (No. 33), either, which is seen at the lateral side of theposterior inferior part of the angular.
Suspensorium, Nos. 37–47: These are a series of bones suspending the mandibular arch fromthe neurocranium, and composed of the opercular arch (Nos. 44–47), commonly called the gill cover,and the palatine arch (Nos. 37–43) constituting the inner wall of the oral cavity. The opercular archis the main organ at the time of gill ventilation for gas exchange. In addition, because the four typesof membrane bones constituting the arch can be observed from the surface, these are also valued as
taxonomic characters. The large membrane bone at the upper right is called the opercle (No. 45). This
forms a joint with the hyomandibular bone (mentioned below) at the anterior supra corner, and thepresence or absence of any spine at the posterior margin is an important characteristic. The V-shaped
thin subopercle (No. 46) contacts the opercle such that it supports it from the bottom. There is a large
semilunar preopercle (No. 44) widely contacting the anterior margin of the opercle. The interopercle(No. 47) contacts the inferior side of both the preopercle and subopercle. The anterior margin of thepreopercle forms a joint with the palatine arch. It is particularly frequent that the shape of the lateral
margin of the preopercle is used as a taxonomic character.
The palatine arch is little exposed to the outside. However, if it has teeth, the teeth are exposedto the inner wall of the oral cavity, and are used as a taxonomic character. The somewhat T-shaped
firm bone forming a joint with the upper part of the preopercle posteriorly is called the hyomandibularbone (No. 43). The hyomandibular forms a joint with the neurocranium at the upper condyle, and alsowith the opercle at the posterior condyle. Between the inferior terminus of the hyomandibular and thequadrate mentioned below is the slender rod-like symplectic (No. 42). There is a very thin, plate-like
metapterygoid (No. 40) forming a joint with the anterior inferior part of the hyomandibular bone andthe endopterygoid (No. 39) anterior to it. The fan-shaped quadrate (No. 41) forms a joint with theinferior part of the metapterygoid, endopterygoid, and symplectic. The quadrate forms a joint with theangular of the lower jaw at the condyle on the part equivalent to the fulcrum of a fan. At the anterior
margin of the angular and the endopterygoid, there is the ectopterygoid (No. 38). There is the palatinearch (No. 37), the anterior tip of which is like a hook, attaching to the anterior half of this ectopterygoid
and the endopterygoid. The hook-like part is exposed in the form of an inverted V beside the prevomer,
the anterior terminus of the neurocranium, on the ceiling of the oral cavity. The presence of any tooth
there and the shape of the tooth are important taxonomic characters.
Hyoid arch; Nos. 48–55: There are several curved, sword-like slender branchiostegals (No.
53) inside the lower part of the opercular arch. The bones supporting them are composed of therelatively wide bone, the ceratohyal (No. 50), with a large hole at the center or in a saddle shape;
the epihyal (No. 51) sutured to the ceratohyal anteriorly; and the hypohyal (No. 49; composed of top
and bottom ones) sutured to the ceratohyal posteriorly. These three types of four bones cannot be
restored once disassembled. Because the number and shape of the branchiostegals and the arrangementof the attachment to the inferior margin of the ceratohyal and the epihyal vary greatly among fish
species, these are important taxonomic characters and powerful factors for considering phylogenic
relationships. There is a small, rod-shaped interhyal (No. 52) superior to the posterior terminus of the
epihyal, which helps to form a joint with the symplectic in the palatine arch from the inner surfaceof the operculum. All of the above are left-to-right paired. A sheet of the triangular urohyal (No. 55)
inferior to the most anterior basibranchial bone (No. 56 in the branchial arch below) sandwiched by
the bilateral hypohyals, and the unpaired baseball-bat-shaped basihyal (No. 48) forming a joint withthe anterior terminus of the basibranchial bone are the hyoid arch. The basihyal is the part equivalent
to the human tongue. Some fish have teeth here as well.
Branchial arch; Nos. 56–63 (see Figure 2 in III-2): After removal of the upper and lower jaws,

56the suspensorium, and the hyoid arch, the branchial arch appears. Pick these all up together, thenremove the arch individually from the lateral side in order on both the right and left sides, clean them,and place them in a row symmetrically on the paper. In the arrangement, I advise you to extend themin a shape that is formed in a widely opened mouth if seen posteriorly. Lastly, arrange the multiple
basibranchial bones (No. 56) in the middle of the lower branches of the branchial arch. The structureof each branchial arch is mentioned in detail in “III-2. Observation of gills”.
Appendicular skeleton: This refers to the skeletons supporting the rays of the dorsal fin, anal fin,and paired fins from inside of the body. Here we can observe the pectoral girdle supporting the pectoral
fin and the pelvic girdle supporting the pelvic fin, both of which are paired fins.
Pectoral girdle; No. 87–96: Removal of the skin and the muscle from the lateral surface of theoccipital region to the start point of the pectoral fin will let you find one or two slender supratemporalbones (No. 87) in some species but none in other species. This bone has a groove where the lateralline runs, which in the posterior part branches in a Y-shape. In the fish in Sparidae, this bone looks
like a rain gutter and is easily lost because it is smaller than a scale. Depending on the fish species, it
is treated as a part of the neurocranium because it firmly fastens itself to the cranium. Following the
supratemporal bone, the crab-claw-like posttemporal bone (No. 88) is seen. An oval supracleithrum(No. 89) forms a joint with the inferior part of the posttemporal bone from inside. After collecting
these, remove them up to the most anterior cleithrum (No. 90) with the accompanying soft rays of thepectoral fin (No. 112) in a group without collapse. Consequently, the two relatively thin postcleithra(No. 94) remain at the most anterior part of the ventral muscle.
Next, remove the muscle at the base of the picked pectoral fin and remove all rays of the pectoralfin (No. 112) from the attachment base in a group. In the remaining attachment base, four hand-drumshaped actinosts (No. 96) are generally seen, but these are easily lost because they are very small insome species. The actinosts are supported by two thin, plate-like bones. The one occupying the upper
half is the scapula (No. 91), with a hole near the center, whereas the lower half is occupied by thehammer-shaped coracoid (No. 93). Both bones form a joint with the posterior margin of the cleithrum(No. 90), a very large and rigid bone covering the entire area from the upper to the lower terminus of
the posterior margin of the gill slit. In the ancient fish groups, such as Clupeiformes and Cypriniformes,
there is a special bone connecting the cleithrum and the coracoid called the mesocoracoid (No. 92).
Pelvic girdle; No. 97: Pick up the basopterygium (No. 97) supporting the pelvic fin in conjunction
with the pelvic fin rays (No. 111 and 112). The pelvic girdle is composed of only a pair of basopterygia
in many of the advanced fish, and directly supports 1 spine and 5 soft rays. However, in the ancient fishgroups such as Salmoniformes, it only supports many soft rays through the radials.

III. Observation of internal morphology    57III6 Observation of the neurocranium

Head of Agnatha is only a cluster of several cartilages. The neurocranium of extant advanced
Chondrichthyes is called the chondrocranium and is composed of a mass of cube-type cartilages. The
neurocranium of Teleostei is composed primarily of many cartilaginous bones and membranous bones(= membrane bones) sutured together, and a minor cartilaginous part. Such a neurocranium is called anosteocranium. The neurocranium protects the brain, the most anterior part of the spinal cord.
At the time of the observation, remove the left half of the splanchnocranium, pectoral and pelvic
girdles, etc. in the vicinity of the head you observed earlier, cutting the remaining neurocranium (called
the skull in some cases) off the joint with the first vertebra to pick it up. If possible, I advise you to use
two individuals of the same species in the observation of the neurocranium to disassemble one andsketch the other in parallel. Once it is disassembled, it can never be restored.
As the arrangement of bones constituting the neurocranium is three-dimensional and complicated,
it is difficult to represent all parts unless you sketch the observation results not only from the lateral sides
but also from five directions, including the lateral, dorsal, ventral, anterior, and posterior directions.
At the center of the apex of the neurocranium is a prevomer (No. 6), the apex of which has ahorseshoe shape in its inferior surface. This horseshoe-like part is located immediately posterior to thejoint part of the right and left premaxillaries (No. 28) among the already observed splanchnocranium,
and can be identified without anatomy. The presence or absence of teeth in this part and, if there areteeth, the number and shape are important taxonomic characters and helpful for predicting the feeding
habit. You can see that, from both sides, the palatine arch (No. 37) among the splanchnocranium
connects to the prevomer anteriorly. There is a slender, plate-like parasphenoid (No. 23) covering
nearly all of the central part of the cranial base following the prevomer. This is located at the centerof the ceiling of the oral cavity. Ancient fish groups are also equipped with teeth here in some cases.
The basioccipital (No. 22) at the posterior terminus of the cranial base forms a joint with the vertebral
column. Being surrounded by four bones, basioccipital, exoccipitals (No. 18) dorsal bilateral to thebasioccipital, and dorsal supraoccipital (No. 19), the foramen magnum is formed at the posterior
terminus of the cranium through which the central nerve runs. This foramen magnum connects to theneural canal of the vertebra.
An ethmoid (No. 1) is sutured to the posterior dorsal part of the prevomer. The posterior terminusof the ethmoid, which is like a cartilage and penetrates the inter-orbital part, is called the ethmoidal
cartilage (No. 2). A rostoral bone (No. 8) forms a joint with it posteriorly, and a pair of slice-like nasal
bones (No. 7) forms a joint with the rostoral posteriorly. Because these are free from the main body,they are picked up at the time of observation of the splanchnocranium. A pair of lateral ethmoids (No.
3) overhangs on both lateral sides of the ethmoid. On the dorsal surface, a pair of longitudinally longplate-like frontals (No. 10) hangs on the dorsal surface of the binocular part. In fish species with largeeyes, this bone is highly indented from the lateral surface to be narrow. Between the right and left
frontals, a supraoccipital (No. 19) is inserted anteriorly, and the mid-dorsal line bulges like a plate. On
the lateral sides of the supraoccipital, the parietal (No. 20), the epiotic (No. 16), and the exoccipital(No. 18) lie on a line in this order posteriorly, and are sutured to the supraoccipital.
The largely overhanging autopterotic (No. 15) is sutured to the lateral side of each parietal, andthe autosphenoid (No. 14) is sutured to the anterior. [Because the supratemporal bone (No. 87) forms ajoint with or is adjacent in the vicinity of the posterior part of the autopterotic, the supratemporal bone
is treated as a part of the neurocranium in some cases. However, in this book, it was mentioned above58

as a part of the pectoral girdle.]
Between the posterior lateral side of the parasphenoid and the exoccipital, the prootic (No. 17) issutured, and the auditory and static organs, including the otolith, are protected inside. There is a small
opisthotic (No. 21) posterior superior to the prootic and superior to the exoccipital, but its presencecannot be shown unless you sketch it from the posterior surface.
There is a basisphenoid (No. 12) that is sandwiched by the right and left prootics and vertically
protruding to the posterior inferior part between the eyes. On the superior part, the foramen magnum
sandwiched by the right and left autosphenoids on the anterior surface of the cerebral ventricle is
the route of the optic nerve and eye-moving muscle. There is a pterosphenoid (No. 11) dorsal to thebasisphenoid.

A

15
7

6 1

3

10

14

19

20

16

8

B

11
14
12

6

3

2

23

10

14 17

15

18 22

19
C
16

Fig. 1 Neurocranium of Red Sea Bream Pagrus major. A, dorsal view; B, left lateral view; C, ventral view.
Large dots indicate cartilages. See table of III-3 for bone names and numbers.

III. Observation of internal morphology    59III7 Observation of the vertebral column and vertical fins

In the axial skeleton of Agnatha, the notochord is developed in the median, being accompanied bya very small number of cartilage pieces on the dorsal side, and several clusters of cartilages that only
gather in the head. In extant advanced Chondrichthyes and Teleostei, the notochord in the embryonicstage similar to that of Agnatha is subsequently replaced by the vertebra that has developed from thecircumference, and consequently remains rudimentarily at the joint part of each vertebra as the fish
grows. A series of vertebrae that are consecutive in a segmental manner from immediately posterior tothe cranium, called the vertebral column (Nos. 64–86), is attached by a variety of intermuscular bones,
including ribs and myorhabdoids.
For observation, after the lifting of the pectoral and pelvic girdles, remove the left lateral muscle
to expose the vertebral column. Individual detachment of the sarcomeres from the anterior will let
you find curved, sword-like intermuscular bones running along the horizontal septum separating thelateral muscle top and bottom. These are called the supraneural bones (No. 85). The one at the anterior
terminus attaches to the transverse process (No. 78) of the first centrum (No. 75). The more posterior,the lower the attachment position becomes, such as near the rib base, or, depending on the species, thelateral side of the inferior part of the centrum or even the hemal arch (No. 77). The sword-like firmbone that forms a joint with the transverse process of the abdominal vertebra (No. 64) in the vicinityof the third centrum or posterior and runs along the peritoneum as if wrapping the viscera is called the

105

99
98

10

6

72

109

79

64

85

10

8

82
102
107

65

Fig. 1 Vertebrae and vertical fins of Red Sea Bream Pagrus major.
See table of III-3 for bone names and numbers.

60rib (No. 82). In more ancient fish groups, apart from these, there are intermuscular bones including the
epipleural ribs (No. 83) and the epicentrum (No. 84) running from the ribs and the centrum laterally.
Moreover, the slender bones called the myorhabdoids (No. 86) are added to the inside of the diaphragmin some cases without attaching to the axial skeleton. When there are many types and a high numberof bones like this, it is referred to as “bony”.
After recording the positional relationships above in a rough sketch, expose the vertebral column.
At the same time, expose the pterygiophores (Figure 2) of the dorsal and anal fins as well, and sketch
the positional relationships between these bones and the neural spines/hemal spines of vertebrae inparticular by outline. Thereafter, pick up the free interneuron spine at the start of the dorsal fin (No.
98) from the posterior of the neurocranium existing to the base, then pick up the interneuron spines(Nos. 99–101) in order posteriorly in combination with the ray (No. 105 or 106) supported by thespine, and clean them. Similarly, pick up the interhemal spines (Nos. 102–104) in combination witha ray (No. 107 or 108) in the anal fin. Each of the interneuron/interhemal spines is composed of threeparts, the proximal pterygiophore (Nos. 99 and 102), median pterygiophore (Nos. 100 and 103), anddistal pterygiophore (Nos. 101 and 104), in order posteriorly. All proximal pterygiophores are ossified.
The median pterygiophores are also ossified and distinct in the anterior, but the ones in the posterior
are difficult to find because they are cartilaginous. The distal pterygiophores are even more difficult
to find because they are all small cartilages. In addition, it is impossible to completely pick up the soft

103 104
100 101

102
99

108
106

Fig. 2 Dorsal and anal fins and their supporting bones of teleost. From Bond (1979).

Frontal view

Lateral view

Frontal view

Lateral view

Frontal view

72
72

72

73

73

80

74

75
76

78

78

80

75

75
76
77

85
A

73
74

80

81

79

82

C
B

Fig. 3 Vertebrae of teleost.
A: 1st ventral vertebra. B: 2nd and posterior ventral vertebra. C: caudal vertebra. From Bond (1979).
See table of III-3 for bone names and numbers.
III. Observation of internal morphology    61rays (Nos. 106 and 108) because they are slender and fragile. Do not disassemble the soft rays of thecaudal fin (No. 109) because the rays form a direct joint with the terminus of the vertebral column.
Lastly, cut the vertebral column at the joint parts to observe the individual vertebrae (Figure 3).
1) The vertebral column is composed of a series of vertebrae, and broadly classified into theabdominal vertebrae (No. 64) in the trunk and the caudal vertebrae (No. 65) lining the tail.
2) The drum-shaped part forming the central part of each vertebra is called the centrum (No. 75).
All centra have the neural spine (No. 72) (called neurapophysis in Chondrichthyes) on the dorsal
side. The caudal vertebrae (No. 65) have the hemal spine (No. 79) (hemopophysis likewise) on
the ventral side. In each basal part, the neural canal (No. 80) and the hemal canal (No. 81), holes
through which the spinal cord or the blood vessel runs, are formed by the neural arch (No. 73) andthe hemal arch (No. 77), respectively. Some of the anterior vertebrae sometimes form a joint withthe centrum without fusion of their neural arch. The number is generally one in fish of Perciformes,
but multiple in the ancient fish groups.
3) Each centrum has 8 zygapophyses (Nos. 74, 76) at a maximum paired between right and left on theanterior, posterior, dorsal, and ventral sides. However, missing items vary depending on the placeand species.
4) The abdominal vertebrae (No. 64), except for several anterior ones, have the transverse process(No. 70) on both the right and left ventral sides, where the rib (No. 82) forms a joint. Each rib
is attached by the supraneural bone (No. 85). The transverse processes of the anterior vertebrae
protrude nearly laterally, whereas the more posterior the position is, the more inferiorly theyprocurrent caudal rays

65

69

66
66
67
（3rd）
（2nd）
（1st）

70

principal caudal rays

73

branched caudal rays

72

77

79

71

68

Fig. 4 Caudal skeletons of Red Sea Bream Pagrus major.
Large dots indicate cartilages. See table of III-3 for bone names and numbers.

62

109

protrude, and eventually the right and left transverse processes fuse, making a hollow. This fusedskeletal part is the hemal arch (No. 77), and the hollow part is the hemal canal (No. 81). Even if
there is a process in both sides inferior to the hemal canal or the processes are fused to form a shortspine, it is still considered an abdominal vertebra (No. 64). However, the clearly elongated spine
is judged to be a caudal vertebra (No. 65).
5) The caudal vertebrae near the posterior terminus are remarkably deformed/fused to support thecaudal fin. The vertebrae in this vicinity are generically named the caudal skeleton (Figure 4).
Because the difference in these fusing conditions varies depending on the evolutionary level of thespecies, it is valued for determining phylogenic relationships. Because the variation is remarkableeven in extant Teleostei, I explain it by focusing on red seabream Pagrus major here.
		 The centrum of the terminal vertebra is called the urostyle (No. 67), and it plays a central
role in the caudal skeleton. This is a small triangle if seen laterally, but a thick round bone if
seen posteriorly. Therefore, it can be distinguished from the surrounding plate-like bones. The
plate-like bone that forms a joint with the bottom of the urostyle and is equipped with a wire-likeprocess bilaterally on the base is the parhypural (No. 71). There are two hemal spines specialized
to a similar thickness immediately anterior to it, but they are not fused with the centrum (No. 79).
The hemal spines with a normal thickness located more anterior are fused with each centrum.
		 A large and a small uroneural bone (No. 69) are seen to contact the dorsal side of the urostyle.
These are left-to-right paired, distinguishing them from others. Between them and the last neuralspine (No. 73), there is a boomerang-shaped thin bone followed by the 2 epurals, for a total of
3 epurals (No. 70). Because the epurals come off by adhering to the caudal fin rays when lifted,
removal of the rays requires care.
		 Posterior to the urostyle, there are 5 flat bones named hypurals (No. 68) that are extended in a
fan shape. This is composed of several fused hemal spines. Those in red sea bream exhibit typicalmorphology of fish in Perciformes. The hypurals are further fused with each other or with otherbones in the vicinity depending on the species.
		 All of the branched caudal rays are supported by the parhypural and 5 hypurals (3 in the upper
half and 2 in the lower half). Superior to them, there is an unbranched soft ray that nearly reaches
the posterior terminus of the caudal fin and is supported by the fifth hypural. Also inferior to therays, there is an unbranched soft ray that reaches nearly to the posterior terminus of the caudal fin
and is supported by the parhypural. These two unbranched soft rays and all branched soft rays arecollectively called the principal caudal rays. The rays located more anterior (superior and inferior)
to the principal caudal rays are not included in the number of caudal fin rays in usual counting
because the anterior ones are embedded beneath the skin and become small rudimentarily (seethe counting method in II-5). However, these are valued in phylogenic studies, and counted usingtransparent specimens in which the skeletons are double-stained.
6) The number of fish vertebrae is an important taxonomic character expressed by the formula: total
number of vertebrae (V) = number of abdominal vertebrae (AV) + number of caudal vertebrae(CV). It is counted by estimating the urostyle as the last caudal vertebra. When discussing theshape of vertebrae, it is occasionally convenient if we count the bones anteriorly, like preural
centrum 1, 2, …, n, in addition to expressing the bones by the posterior ordinary number of theabdominal vertebrae and caudal vertebrae separately. At this time, the urostyle is estimated as
the first preural centrum. This is based on the concept that the ural vertebra and the first preural
centrum separately seen in the primitive fish are fused in the advanced fish groups.

III. Observation of internal morphology    63

8 Observation of the muscular system

III

The muscular system of fish controls their various life activities, including the movement ofvarious organs, swimming, respiration, and the ingestion and digestion of food. The muscles arebroadly classified into striated muscle and smooth muscle based on their cellular morphology. Anotherclassification is based on the organ where the muscles are arranged, which broadly divides the muscles
into three types. The striated muscle constituting the heart wall is called the cardiac muscle. The striatedmuscles moving fins and bones are skeletal muscles. The smooth muscles constituting the visceralorgans are called visceral muscles. Moreover, there are also physiological categories of involuntarymuscle and voluntary muscle. The cardiac muscle and the visceral muscle are involuntary muscles thatare unconsciously governed by the autonomic nervous system to act at all times. The skeletal muscle
is the voluntary muscle governed by the brain to consciously adjust activity. The skeletal muscles aredescribed below.
The cells constituting the skeletal muscle are called muscle fibers because they have a slendershape. These are multinucleated cells in which a large number of myofibrils are assembled.
Lateral muscle: The lateral muscle is an assembly of muscle fascicles arranged regularly on bothsides of the trunk and tail. Extension and shrinkage of the lateral muscle give the fish the power to swim.
In many cases, the lateral muscle is composed of W-shaped myomeres (Figure 1-7, 8) subcutaneously
lining the lateral side. The anterior and posterior myomeres are separated by the diaphragm (Figure1-6). In addition, in Chondrichthyes and Teleostei, each myomere is divided into a right and left
6

15

5

7

9

4

12

3
2
1

17

11
18

19

10

8

13

14

16

Fig. 1 Muscle system of Red Sea Bream Pagrus major.
1. adductor mandibulae; 2. adductor arcus palatine; 3. levator arcus palatine; 4. levator operculi; 5. horizontal septum;
6. myoseptum; 7. epaxialia; 8. hypaxialis; 9. supracarinalis posterior; 10. infracarinalis medium; 11. infracarinalis
posterior; 12. flexor dorsalis; 13. flexor ventralis; 14. intermedialis; 15. inclinator dorsales; 16. inclinator anales; 17.
abductor superficialis; 18. aductor profundus; 19. arrector ventralis

64side by the medium septum or the vertical septum, composed of a tendon-like membrane connecting
vertebra, and by the abdominal cavity, and divided into the dorsal muscle (Figure 1-7) and the ventral
muscle (Figure 1-8) by the horizontal septum (Figure 1-5) lying between the vertebrae and the bodysurface. No horizontal septum is seen in Agnatha.
When observing the lateral muscle of Chondrichthyes and Teleostei, in many cases, each myomere
is W-shaped on the body surface but has complicated morphology in the deep layer because it isequipped with two anterior muscle cones protruding anteriorly (Figure 2-5) and a posterior muscle
cone protruding posteriorly (Figure 2-4), separately on the top and bottom of the centra. In sharks, theW-shaped myomeres are bent even more posteriorly in the dorsal part, and another anterior muscle
cone develops in the deep layer of this part. In Agnatha, the shape of each myomere has a dull bending
part, relatively simple and arched, and lacks the anterior muscle cone and the posterior muscle cone.
In the surface of the lateral muscle, muscular fascicles have a reddish brown color due to theabundant myoglobin and dense distribution of capillary vessels running longitudinally in a zonal
manner along the horizontal septum (Figure 2-6). This is called the surface dark muscle (Figure 2-1),
and its developmental level varies considerably depending on the species. The surface dark muscle
is generally well developed in surface swimmers such as Japanese sardine Sardinops melanostictus,
flying fish, Pacific saury Cololabis saira, mackerel, and Japanese Spanish mackerel Scomberomorus
niphonius, and poorly developed in bottom-dwellers including wrasse, sargassum fish Histrio histrio,
lizardfish Saurida sp., Platycephalidae, and sea robin Chelidonichthys spinosus.
Moreover, highly migratory surface swimmers such as skipjack/tuna, shortfin mako shark Isurus
oxyrinchus, and salmon shark Lamna ditropis have paired red muscle fascicles running bilaterally
along the vertebral column. This is called the true dark muscle (Figure 3-4). The true dark muscle
is the part medial to the anterior muscle cone of the lateral muscle, and in the shortfin mako shark
Isurus oxyrinchus it is apart from the vertebrae and embedded in the lateral muscle. In frigate/bullet
tuna Auxis (Figure 3) and skipjack Katsuwonus pelamis, the dark muscle is remarkably developed andaccounts for around 1/5 of the ordinary muscles.

4
7

2

5
1

3

6
5
8

4

Fig. 2 One myoseptum of lateral muscle of a salmonid fish, King Salmon Oncorhynchus tschawytscha.
1, superficial dark muscle or lateralis superficialis; 2, epaxial; 3, hypaxialis; 4, posterior cone; 5, anterior cone; 6,
horizontal septum; 7, neural spine & medium septum; 8, hemal spine & medium septum. From Winterbottom (1974).
III. Observation of internal morphology    65

A

8

B

C

D
4

4
10

2

2

11

10
1

12

1

3

5

3
5

12

A

B
6
8

4

1

9
7
C

5

D

Fig. 3 Cross section of muscle of Frigate Tuna Auxis thazard. A-D on upper figure indicate section place of lower figures.
1, superficial dark muscle; 2, epaxial; 3, hypaxialis; 4, true dark muscle; 5, abdominal cavity; 6, infracarinalis medius;
7, horizontal septum & epineural; 8, medium (vertical) septum & neural spine; 9,medium septum & hemal spine; 10,
lateral line canal; 11, dorsal cutaneous vessel; 12, ventral cutaneous vessel.

66

Jaw muscles and opercular muscles: The most remarkable muscle among the muscles forclosing the mouth is the adductor mandibulae (Figure 1-1). Normally, this muscle is broadly divided
into three muscles that arise from the palatine arch centering the quadrate and connect the medial andposterior surfaces of the dentary and the upper jaw, and the muscle fascicles lying in the inner cavityof the lower jaw, a total of four groups. The developmental status of the muscle fascicles of theseparts varies depending on the species. The major muscles to open the lower jaw include the protractor
hyoidei. This is a pair of slender muscle fascicles that arise from the medial surface near the joint
between the right and left dentaries and reaches nearly all of the hyoid arch (excluding the urophyal
and the interhyal) including the branchiostega.
Dorsal posterior to the adductor mandibulae, there is the dilator operculus connecting verticallytoward the posterior part of the orbit of the neurocranium and the dorsal surface of the anterior partof the opercle. Posterior to this is the levator operculi (Figure 1-4) connecting vertically toward thedorsal surface of the posterior part of the opercle. In addition, there is the adductor operculi connecting
horizontally the medial lateral wall of the neurocranium and the medial surface of the dorsal part of theopercle. All of these control opercular movement.
Muscles to move fins: The muscles for moving the dorsal fin and the anal fin among vertical finsare symmetrically arranged and share a basic structure, whereas the muscles for moving the caudal fin
are very distinct.
Dorsal fin: The inclinator dorsalis (Figure 4-1) arises bilaterally from each ray base toward
the medial surface of the skin. This acts to inclinate the ray right and left. In puffers, this muscle isremarkably developed and wraps the lateral muscle to act not only as an inclinator but also as a flexorand extensor for the body. This appears to have been developed in relation to the fact that a puffer

5

6

1
3

1

2

3

2
3

4
7

8

9

Fig. 4 Muscles to move dorsal fin and related bones of Red Sea Bream Pagrus major.
1, incinator dorsales; 2, depressor dorsales; 3, erector dorsales; 4, epaxialis; 5, dorsal fin spine; 6, free neural spine;
7, proximal pterigiophores; 8, neural spine; 9, abdominal vertebrae.
III. Observation of internal morphology    67

moves forward by the movement of the dorsal and anal fins. Inside the lateral muscle (Figure 4-4) are
the erector dorsalis (Figure 4-3), running from the anterior terminus of the base of each ray (Figure4-5) toward the distal pterygiophore (Figure 4-7) immediately anterior; the neural spine (Figure 4-8),
or the medium septum; and the suppressor dorsalis (Figure 4-2), running from the lateral surface of theposterior terminus of the base toward the distal pterygiophore immediately inferior, the neural spine,or the medium septum. The former functions to erect the ray and the latter functions to fold the rayposteriorly, and both are left-to-right paired. In addition, these are fixed posteriorly and anteriorly by
the supracarinalis anterior connecting the neurocranium and the most anterior pterygiophore and thesupracarinalis posterior connecting the last neural spine and the last pterygiophore.
Caudal fin (see the foldout figure): The posterior terminus of the vertebral column has complicatedmuscles involved in the movement of the caudal fin. The major ones include the flexor dorsalis andthe flexor ventralis, which arise from a wide range of the lateral surface of the caudal skeletal and arearranged radically mainly at the base of the branched soft rays of the caudal fin, the adductor caudalisconnecting the lower central soft rays of the caudal fin and the urostyle as well as the hypurals, andthe interradialis caudalis between the caudal rays. Among them, the muscles with the most importantfunction are the flexor dorsalis and the flexor ventralis, which are separately composed of the muscle
fascicles that are further divided into, again, top and bottom.
Pectoral fin (see the foldout figure): The muscles for moving the pectoral fin also include many
types of muscles that contract medially, laterally, superiorly, or inferiorly. Major ones include thetriangle abductor superficialis that lies on the posterior and ventral surfaces of the cleithrum andattaches to the anterior surface of the lateral side of the ray base through its posterior terminus, and theabductor profundus that lies on the medial lateral surface of the coracoid and attaches to the posterior
inferior process of the base of all soft rays.
Pelvic fin: In the pelvic fin, the major muscles are the abductor profundus pelvicus, which islocated on the ventral surface of the pelvic girdle and is associated with the pelvic fin ray throughits posterior terminus, the arrector ventralis pelvicus, which arises from the ventral surface of thelateral side of the pelvic girdle to connect to the lateral surface of the first pelvic ray, and the adductor
profundus pelvicus, which lies on the dorsal side of the pelvic girdle and adheres to the dorsal surfaceof the pelvic ray base.
A detailed explanatory review of fish muscle that is highly recommended is: Winterbottom, R.
(1974) A descriptive synonymy of the striated muscles of the Teleostei. Proc. Acad. Nat. Sci. Philad.,125 (12): 225-317.

68

IV
Related field of experimental ichthyology

IV

1 Sketching eggs, larvae and juveniles

The observation and description of the shape of fish eggs, egg development, and morphologicalchanges of newly hatched larvae accompanying growth not only clarify relationships by similarity anddissimilarity but also are important in egg and fry production for healthy fry growth, and are essential
for promoting these studies. Sketching these materials by means of sharp lines and dots uses nearly thesame basic technique as that of adult fish. However, because the subjects are small and eggs and fishin the larval stage are transparent and easily damaged, the equipment and materials used are diverse
and the procedure is also distinct.

1. Equipment and materials used
These include microscopes, drawing apparatuses, microscope photography equipment, and profile
projectors. These tools have developed recently as represented by popularly used digital cameras anddigital photography equipment. The microscopes include the binocular stereomicroscope and thetrinocular biological microscope in addition to the monocular biological microscope. Choose a proper
model according to the purpose. Briefly, in the case of sketching by observing through a microscope,
use a monocular biological microscope or a binocular stereomicroscope. For drawing equipment, usea monocular microscope (this can project clearer images than binocular microscopes) compatible tothe equipment. For photography, use a binocular or a trinocular microscope with a cylinder compatible
with a camera. In addition, to measure the size of the subject, one of the binocular or trinocular cylinders
should be equipped with a micrometer. The microscopic observation of eggs or larvae requires a glass
slide on which to place these materials. Such glass slides include excavated slides with a cavity. In
addition, glass slides specifically for larvae and juveniles (with a pool-like water-retainable well whereorganisms can be accommodated) have recently become available. Regarding illumination, in addition
to the transmission illumination accompanying the microscopes, the use of a fiber type light sourceof incident light provides brighter images (Be very careful to avoid the evaporation of moisture anddryness of the sample from the heat generated by the illumination. Cool light illuminators that avoid
heat generation are on the market.). The sketching should be done with an H-2H pencil, but use a penin the case of article submission, etc.

2. Sketching egg development
First, collect 5–10 eggs for sketching from the rearing tank and keep them in an approximately
1-liter beaker already filled with the rearing water for all observations. Float the beaker in water in a
5–10 liter container with a thermometer (water-bath method). Place an airstone in the beaker for weakaeration.
In the case of recording the developmental process of eggs, sketches are often made from theviewpoint of the top of the blastodisc to help understand the manner of cleavage. However, after
embryonic formation, it is preferable to view it from the right side so that the embryo is positioned
right or left of the yolk sac (sketching embryos from the back, front, or halfway makes the wholeappearance unclear). However, in the case of a pelagic egg, it is difficult to fix it in the intendedorientation on a glass slide retaining water because it rotates. In this case, accommodate multiple eggs
so that they stick to each other, making it difficult for them to move, and then rotate the egg using a

70

A

B

F

C

G

D

H

E

I

J

K

L

M

Fig. 1 Oogenesis and larvae of Nagasaki Damsel Pomacentrus nagasakiensis.
A, 2-cell stage, 39 minutes after fertilization; B, early morula stage, 5 hour 4 minutes after fertilization; C, organizationof embryo, 16 hour 6 minutes after fertilization; D, 4-myomere stage, optic vesicle & Kupffer vesicle are organized, 19hour 16 minutes; E, 13-myomere stage, otic vesicle (otosyst) is organized, melanophores appear on yolk sac, 24 hour
36 minutes; F, 20-myomere stage, lens is organized, 31 hours; G, 25-myomere stage, tail is detached from surfaceof yolk sac, 31 hour 33 minutes; H, 27-myomere stage, granule melanophores appear on eye, 47 hours 15 minutes;
I, just before hatch, 112 hour 46 minutes; J, hatched larva, 3.08 mm TL; K, prolarva, 24 hour after hatching, 3.08 mm
TL; L, postlarva, 3 days after hatching, 3.59 mm TL; M, 5 days after hatching, 3.68 mm TL. Scales indicate 1 mm.

mounted needle to set it in the intended orientation for observation. On the other hand, a demersal egg
is relatively easy to fix in an intended orientation because it does not rotate. Figures 1 and 2 show an
example of a sketch of the manner of egg development and larval/juvenile growth of Nagasaki damsel
Pomacentrus nagasakiensis. The developmental stages of the egg in the sketch are after blastodisc
bulging, the 2-cell stage, the first cleavage, and immediately before hatch, each of which is a time point
when a characteristic change is seen. The size measurement should be the diameter in the case of roundeggs, and the major and minor axes in other cases, as well as the oil globule diameter (large, medium,
V. Related field of experimental ichthyology    71

A

B

C

D

E

F

Fig. 2 Larvae and juvenile of Nagasaki Damsel Pomacentrus nagasakiensis.
A, postlarva, 8 days after hatching, 2.42 mm TL; B, 9 days after hatching, 5.02 mm TL; C, 12 days after hatching, 5.26
mm TL; D, 15 days after hatching, 7.19 mm TL; E, juvenile stage, 16 days after hatching, 8.01 mm TL; F, 19 days after
hatching, 12.0 mm TL. Scales indicate 1 mm.

and small separately if multiple globules of different sizes exist).
Because egg development is quick in the case of pelagic eggs, and hatching happens mostly within
24 hours, continuous observation is relatively easy. In the case of demersal eggs, which are slow tohatch, work can be done with pauses in the observation and the occasional check of the developmental
stage after embryonic formation.

3. Sketching larvae and juveniles
To sketch a newly hatched larva, collect around 5 larvae from the rearing tank and accommodate
them in a beaker in the same way as the case of sketching egg development, and use them for72

observation. Collect a larva together with rearing water using a dropping pipette, place it on a glass
slide dedicated for larval observation, and repeat. Then, arrest the motion using an anesthetic for coldblooded animals. At this time, dissolve a small amount of the anesthetic in a stepwise manner in water
until you confirm the arrest because concentrated anesthetic can shrink or kill the sample. In addition,observe the sample promptly after the arrest, and replace it with a new one when the transparent
body starts becoming white. Because in many hatched larvae, pelvic fins and vertical fins (dorsal,
anal, caudal fins) are not differentiated and are all like thin membranous fins, be very careful to avoid
damage or folding. Usually during sketching, the sample is placed so that the head is on the left andthe left lateral side faces up. At this point, you need to correct the body using a mounted needle sothat it has no body twist and folding of the fins/membranes and is perfectly sideways. Regarding themagnification in the microscope, first, set it low so that you can observe the whole body and sketch thebasic morphology including the outline. After this, increase the magnification and observe the partsin detail for additional or new description. As for the light source, for basic sketching, illumination
solely with transmission light usually works. However, even in that case, adjust the light level to allow
clear observation. In addition, combination with incident light from above allows even more detailed
observation because it provides shadows. During the early larval stage from immediately after hatch tocomplete absorption of yolk sac, measure the total length (from the snout tip to the posterior terminusof the caudal fin membrane), body length (from the snout tip to the terminus of the notochord), yolk
length (major and minor axes), oil globule diameter (major and minor axes in the case of the sizeof elongated eggs), and preanus length (from the snout tip to the anus), and count the number ofmyomeres (the number in the ventral and tail parts bordering the anus, and the total number). From thelate larval stage after yolk sac absorption to immediately before the juvenile stage when the numberof rays of each fin reaches the fixed number of the species, measure the total length, body length, andpreanus length, and count the number of spines and soft rays and record the emergence and change
in mottles and spots (Recording by a camera is convenient for body color/mottles and spots. I advise
adding the details of mottles and spots, etc. to the sketch based on this record).

V. Related field of experimental ichthyology    73IV2 Morphological/ecological characteristics of fish eggs by type and developmental stage

1. Classification of fish eggs
Fish reproduction is broadly classified into two types, viviparity and oviparity. First, in the caseof in vivo fertilization by copulation (sexual intercourse) between males and females, females of somefish spawn large eggs wrapped in a hard sheath (Japanese bullhead shark Heterodontus japonicus,
skate), or give birth to larvae and juveniles (Scorpaenidae, Sebastes) or juveniles that have grown to thesame morphology as that of the parent fish (surfperch Ditrema temmincki, banded houndshark Triakis
scyllium, red stingray Dasyatis akajei). Among them, those that spawn eggs are called “oviparous,”and others are called “viviparous” (all of those using in vivo fertilization are called viviparous insome cases). On the other hand, as seen in many fish species, the type of ex vivo fertilization inwhich unfertilized eggs released by a female are mixed with sperm released by a male(s) is called“viviparity.” The group of sharks and rays includes both “oviparous” species that spawn eggs and“viviparous” species that give birth to juveniles/fry.
Eggs of Teleostei are divided into “demersal eggs”, which have high specific gravity and sink inwater, and “pelagic eggs”, which float in water (Figure 1). Among them, demersal eggs are divided
into “non-adhesive eggs”, “adhesive eggs”, and “entangling eggs” based on the adhesion manner ofthe surface of the egg membrane. Among these three types, non-adhesive eggs such as the eggs ofsalmon/trout and Japanese eel catfish Plotosus japonicus have no adhesion apparatus and adhesivenesson the surface of the egg membrane and therefore are spawned in a scattered manner on the bottom ofrivers, lakes, or the sea. “Adhesive eggs” have adhesive structures such as attachment filaments and anadhesion apparatus, and/or adhesiveness on the egg surface, and adhere to other eggs or objects. They
are further divided into “adhesive eggs” with adhesive structures, such as the eggs of goby, pearl-spot
chromis Chromis notata, ayu Plecoglossus altivelis, and Spirinchus lanceolatus and “tightly adhesiveeggs” with tight adhesiveness on the egg membrane surface, such as the eggs of Cyprinidae, puffer,sculpin, etc. “Entangling eggs” of the Pacific saury Cololabis saira, flying fish, and medaka Oryzias
latipes have long filamentous structures on both poles of the egg that entangle the egg on seaweeds,
water plants, etc. The pelagic eggs are divided into “agglutinated pelagic eggs”, where there are many
Non-adhesive Egg
Tightly Adhesive Egg
Demarsal Eggs

Adhesive Eggs
Entangling Eggs

Adhesive Eggs

Egg Ribbon
Agglutinated Pelagic Eggs

Egg Balloon
Egg Mass

Pelagic Eggs

Isolated Pelagic Eggs

Fig. 1 Types of teleosts eggs. Modified from Ikeda & Mito (1988).

74

eggs contained in a gelatin-like substance, and “isolated pelagic eggs” that separately float in water.
Moreover, “agglutinated pelagic eggs” are divided based on the shape of the sack wrapping the egg
into a ribbon-like “egg ribbon”, including eggs of frogfish and monkfish; a sack-like “egg balloon”,
including eggs of luna lionfish Pterois lunulata and Carapidae; and a bulky “egg mass”, including eggs
of mangrove dragonet Callionymus enneactis.
Isolated pelagic eggs are classified based on the number of oil globules, structure of the egg
membrane, breadth of the perivitelline space, and the presence or absence of any segment on the yolk,etc.
The number and size of eggs/larvae and juveniles produced by a female fish differ depending on
the manner of reproduction. Briefly, the number tends to be lowest in viviparous cartilaginous fishthat give birth to larvae and juveniles, and increases in the order cartilaginous fish spawning demersal
eggs, teleost fish spawning demersal eggs, teleost fish of Scorpaeniformes giving birth to larvae, andteleost fish spawning pelagic eggs. On the other hand, the trend is size is opposite that of the number,from the smallest in fish spawning pelagic eggs to the largest in viviparous cartilaginous fish giving
birth to larvae and juveniles. Generally, the relationship between the number and the size of eggs is thatspecies spawning larger eggs produce a smaller number and species spawning smaller eggs produce alarger number. Therefore, it is called “a small number of large eggs and a large number of small eggs”.
In addition, the larvae at the point of hatching are large in viviparity and demersal eggs, and at in thestage in which organs, including the eyes, mouth, digestive organs, and fins, have developed. On theother hand, larvae from pelagic eggs are small and their organs are undeveloped. This is considered to
be a possible “reproduction strategy” to leave as many offspring as possible. In other words, it is notproblematic that the number of larvae of viviparity and larvae hatched from demersal eggs is small
because they are large and their organs are in a developed stage, making the probability of subsequent
survival higher. On the other hand, because larvae hatched from pelagic eggs are small and their organs
are undeveloped in many cases, their survival rates are low.

2. Characteristics of Teleostei by growth stage
Many researchers have proposed distinct classifications of the developmental stages of Teleostei
according to various criteria. Here, I divide the life cycle into egg, early larval, late larval, juvenile,
young, immature, adult, and old stages according to Watanabe and Hattori (1971), and overview themorphological and ecological characteristics of these stages individually below.
Egg stage: This is the stage from fertilization to immediately before hatching, in which fish growthdepends on the nutrition (yolk) inside the egg membrane provided by the parent. The growth rate isstrongly influenced by the environmental water temperature, and varies depending on the spawningseason in the habitat of parent fish and species. The time required for hatching is generally longer indemersal eggs and large eggs, and shorter in pelagic eggs and small eggs. In addition, it is longer at
low water temperature and shorter at high water temperature.
Early larval stage: This is the stage from immediately after hatching to the complete absorption ofyolk. In succession from the egg stage, fish growth depends on the nutrition provided by the parent fish.
However, it different from the egg stage because fish are not protected by an egg membrane and growby contacting the outer environment directly. For this reason, adaptation to the outer environment,
including water temperature and flow, is more necessary than in the egg stage, and management byphysiological changes influences survival. Generally, in fish species spawning pelagic eggs, larvae
immediately after hatch have insufficient organogenesis, represented by not yet melanized eyes and an
unopened mouth and anus. On the other hand, in fish species spawning demersal eggs, organs are moredeveloped before hatching, as indicated by the fact that eyes are already melanized and the mouth and
V. Related field of experimental ichthyology    75

anus are formed and opened inside the egg membrane during the egg development. Therefore, initial
depletion is higher in fish spawning pelagic eggs than in fish spawning demersal eggs.
Late larval stage: This is the stage from after the absorption of yolk to the time the number of
fin rays reaches that particular to the species. This stage shows the most remarkable changes bothmorphologically and physiologically. “Transformation” also occurs in this stage. Fins as swimmingorgans differentiate and their spines and soft rays are formed in this stage. Ecological changes due
to increased swimming ability are seen. In larvae hatched from pelagic eggs, the eyes are melanized
and functional, the mouth and anus are opened, and fish start consuming food from the external
environment upon entering this stage. On the other hand, in larvae hatched from demersal eggs, theeyes are completely melanized and functional, and fish look for and eat food aggressively of their own
will.
Juvenile stage: The spines and soft rays of fins are already in the number particular to the species,
but not all of the body form elements that adult fish possess have been prepared, and the body form
is in the early stage of development. Because the swimming ability is greatly improved, fish swim ina school and ingest food more actively than earlier. In bottom-dwellers and species inhabiting rocky
shores, coral reefs, etc., fish settle themselves on the bottom in this stage, and, depending on thespecies, reach a stage of acquiring cannibalism and/or territory.
Young stage: The morphological characteristics are very similar to those of adult fish. However,fish are in the middle of development because body color, mottles, or spots peculiar to the species have
not yet appeared. This is equivalent to the stage in which the growth is most active. The generally
mentioned “fry stage” is equivalent to this stage.
Immature stage: Size, apparent morphology, body color, mottles, and spots are nearly similar to
those of adult fish. However, fish are in a sexually immature stage.
Adult stage: This is the stage in which fish are sexually mature and functional. “Biologicalminimum form” is equivalent to the first stage of this period.
Old stage: This is the stage in which viability and fertility deteriorate.
Growth stages are classified as above in general. However, although the late larval stage andjuvenile stage can be relatively clearly classified from appearance, it is difficult to clearly classify
other stages.

76

38

C

31

42

32

37

neurocranium

28

7

39

41

A

8

31

40

47

32

25

neurocranium

44

43

88

26

45

96

46

44

47

87

93

46

90

89

91

88

45
90

89

D

48

49

neurocranium

B

53

28

8

50

49

31

29

32

37

neurocrocranium

51

52

88

96

47

44

93

91

88

90

89

46

45
90

89

E

99

55

neurocranium

88

110

97

111

93

90

89

91

105

96

94

94

Sequential dissections to show bone distributions of Red Sea
Bream Pagrus major.
A, specimen without scale, skin and sclerotic (No. 9).
B, specimens without nasal (No. 7), supratemporal (No. 87),
lachrymal (No. 25), infraorbital (No. 26) and surface of muscles
to move bones and pectoral fin.
C, specimen without maxillary (No. 29), premaxillary (No. 28),
rostral cartilage (No. 8), cheek muscles, and remained muscles
to move pectoral fin.
D, specimens without bones of lower jaw (Nos. 31-36), palatinearch (Nos. 37-43), opercular arch (Nos. 44-47), and gill arch
(Nos. 56-73).
E, specimens without hyoid arch (Nos. 48-55) and pectoral softrays (No. 112).