dorsal hump morphology in pink salmon (oncorhynchus gorbuscha)

Dorsal Hump Morphology in Pink Salmon(Oncorhynchus gorbuscha)

Kenta Susuki,1,2 Masaki Ichimura,1,3 Yosuke Koshino,1 Masahide Kaeriyama,1 Yasuaki Takagi,2

Shinji Adachi,2 and Hideaki Kudo1*

1Laboratory of Studies on Marine Bioresources Conservation and Management, Faculty of Fisheries Sciences,Hokkaido University, Hakodate, Hokkaido, Japan2Laboratory of Aquaculture Biology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan3Shibetsu Salmon Museum, Shibetsu, Hokkaido, Japan

ABSTRACT Mature male Pacific salmon (Genus Onco-rhynchus) develop a dorsal hump, as a secondary male sex-ual characteristic, during the spawning period. Previousgross anatomical studies have indicated that the dorsalhumps of salmon are mainly composed of cartilaginous tis-sue (Davidson [1935] J Morphol 57:169–183.) However, thehistological and biochemical characteristics of such humpsare poorly understood. In this study, the detailed micro-structures and components of the dorsal humps of pinksalmon were analyzed using histochemical techniques andelectrophoresis. In mature males, free interneural spinesand neural spines were located in a line near to the medianseptum of the dorsal hump. No cartilaginous tissue wasdetected within the dorsal hump. Fibrous and mucous con-nective tissues were mainly found in three regions of thedorsal hump: i) the median septum, ii) the distal region,and iii) the crescent-shaped region. Both the median sep-tum and distal region consisted of connective tissue with ahigh water content, which contained elastic fibers andhyaluronic acid. It was also demonstrated that the lipidcontent of the dorsal hump connective tissue was markedlydecreased in the mature males compared with the imma-ture and maturing males. Although, the crescent-shapedregion of the hump consisted of connective tissue, it did notcontain elastic fibers, hyaluronic acid, or lipids. In anultrastructural examination, it was found that all of theconnective tissues in the dorsal hump were composed ofcollagen fibers. Gel electrophoresis of collagen extractsfrom these tissues found that the collagen in the dorsalhump is composed of Type I collagen, as is the case insalmon skin. These results indicate that in male pinksalmon the dorsal hump is formed as a result of anincrease in the amount of connective tissue, rather thancartilage, and the growth of free interneural spines andneural spines. J. Morphol. 275:514–527, 2014. VC 2013 Wiley

Periodicals, Inc.

KEY WORDS: connective tissue; collagen; secondarysexual characteristic; histochemistry

INTRODUCTION

At maturity, male anadromous Pacific salmon,Oncorhynchus spp., develop a prominent dorsalhump, as a secondary sexual characteristic (David-son, 1935; Groot and Margolis, 1991). Larger dor-sal humps are associated with enhanced mating

success during intrasexual competition. In addi-tion, Schroder (1981) reported that the dorsalhump plays a role in preventing attacks fromother males in chum salmon (O. keta). Studies onpink and sockeye salmon have also suggested thatdeveloping a larger dorsal hump might increase amale’s competitiveness (Keenleyside and Dupuis,1988; Quinn and Foote, 1994). Among Pacificsalmon species, male pink salmon (Oncorhynchusgorbuscha) and sockeye salmon (O. nerka) developthe most exaggerated dorsal humps (Vladykov,1962; Burgner, 1991; Heard, 1991).

Previous gross anatomical studies of maturemale pink salmon (Davidson, 1935 in Journal ofMorphology) and lacustrine sockeye salmon (Naka-mura, 1942) reported that the dorsal hump ismainly composed of cartilage. These studiesreported that the amount of cartilaginous tissueincreases around the dorsal median septum, abovethe dorsal posterior cones, and around the supra-carinalis anterior, when the salmon enter rivers.On the basis of these historical gross anatomicalstudies, many researchers believe that the dorsalhump is composed of cartilage (e.g., Fleming andReynolds, 2004; Butts et al., 2012). A corollary ofthis assumption is that the fast growth of cartilageoccurs by processes similar to those observed dur-ing the development of a chondrosarcoma.

Contract grant sponsors: Akiyama Life Science FoundationResearch Grant, Environmental Research Projects, The SumitomoFoundation, and The Clark Memorial Foundation at HokkaidoUniversity.

*Correspondence to: Hideaki Kudo, Laboratory of Studies onMarine Bioresources Conservation and Management, Faculty ofFisheries Sciences, Hokkaido University, Japan.E-mail: [email protected]

Received 17 June 2013; Revised 1 October 2013;Accepted 16 November 2013.

Published online 10 December 2013 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20234

VC 2013 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 275:514–527 (2014)

https://www.researchgate.net/publication/230152569_The_development_of_secondary_sexual_characters_in_the_pink_salmon_Oncorhynchus_gorbuscha?el=1_x_8&enrichId=rgreq-646653c8-280d-45da-be6e-276c0893b950&enrichSource=Y292ZXJQYWdlOzI1OTI1NDQyMjtBUzoxMDg4NTg4OTg5MTUzMjhAMTQwMjk2NTM5MzYyMA==

However, this is pure speculation because thedetailed structure and histological properties ofthe dorsal hump have never been confirmed.Therefore, we consider it necessary to reveal moredetails about the microstructure of salmon dorsalhumps and potentially the process responsible fortheir formation.

An osteological study of Oncorhynchus speciesdemonstrated the presence of both neural spinesand free interneural spines in the anterior dorsalregion (Hikita, 1962). However, the roles played byfree interneural spines and neural spines in theformation of the dorsal hump are unclear. It isassumed that these bone tissues grow during dor-sal hump development, but the growth patterns ofthese structures are largely unknown. Further-more, it has been reported that the water contentof the dorsal hump increases and its lipid contentdecreases as sexual maturity progresses (Robinsonand Mead, 1970; Hendry and Berg, 1999).Although cartilaginous tissue is probably responsi-ble for the high water content of the dorsal hump,the biochemical features of dorsal hump areunclear, e.g., it remains to be confirmed whether itcontains mucopolysaccharides.

Upriver-migrating salmon consume lipids astheir primary energy source, so reductions in theirlipid stores during maturation would not be sur-prising, as described in various biochemical stud-ies (e.g., Hendry and Berg, 1999; Crossin et al.,2003; Bower et al., 2011). However, most of thesestudies focused on the muscle tissue in the dorsalhump as a source of lipids, rather than cartilagi-nous tissue. Accordingly, there are various ques-tions regarding the histological reason for thedecrease in the lipid content of the dorsal hump aswell as whether the abovementioned increase inits water content is related to the development ofcartilage.

The main goal of the present study is to exam-ine the microstructures of the dorsal humps ofsalmon and clarify the changes that occur inthese structures during sexual maturation. Forthis purpose, we performed microscopic anatomi-cal analyses of the anterior dorsal tissues ofpink salmon at different stages of maturation. Inaddition, to biochemically characterize the dorsalhump, we examined collagen extracts fromvarious regions in the dorsal hump byelectrophoresis.

Fig. 1. O. gorbuscha, differences in the external morphologies at different stages of secondarysexual characteristic development. (A) Immature male, (B) immature female, (C) maturing male,(D) mature male, and (E) mature female. Scale bars: 100 mm.

TABLE 1. Data sets of the fish in this study. Values of fork length (mm) and body weight (g) are expressed as mean 6 SD

Fork length(mm)

Body weight(g)

Number ofsamples Sampling site

Immature males 392 6 15.5 614 6 71.1 n 5 12 Northwest Pacific Ocean (43�N, 155�E)Immature females 394 6 21.6 631 6 112 n 5 8 Northwest Pacific Ocean (43�N, 155�E)Maturing males 532 6 38.2 1763 6 473 n 5 5 Coast of Nemuro, eastern HokkaidoMature males 597 6 33.9 2598 6 395 n 5 11 Shibetsu RiverMature females 518 6 25.4 1668 6 276 n 5 5 Shibetsu River

515DORSAL HUMP MORPHOLOGY IN O. gorbuscha

Journal of Morphology

MATERIALS AND METHODSAnimals

We examined the anterior dorsal tissue of pink salmon,O. gorbuscha (Walbaum, 1792), at different stages of secondarysexual characteristic development; that is, immature, maturing,and mature salmon. The external morphology of the fish and

their sampling sites are shown in Figure 1 and Table 1. Theimmature fish were collected from the Northwest Pacific Ocean(43�N, 155�E) by anglers aboard the training ship Oshoro Maru(Hokkaido University) in May 2010. In September 2010, Matur-ing fish were captured by set nets in the coastal waters off Shi-betsu, eastern Hokkaido, and landed at Shibetsu fishing port.Mature spawning fish from the Shibetsu River were providedby the Nemuro Salmon Propagation Association in September2010 and September 2012. Using these fishes, we microscopi-cally investigated the differences in dorsal tissue structurebetween the sexes and each stage of maturation.

We measured the body weight and length (fork length: FL)of each fish and determined their sex via a visual inspection ofthe gonads. We isolated anterior dorsal tissue and used it inthe anatomical analyses. For the water content and collagenanalyses, dorsal tissue was also dissected from the maturemales and stored at 230�C until use.

Macroscopic Anatomy

To visualize the bony tissues in the dorsal hump, clearedand double-stained skeletal specimens were prepared accordingto the method of Dingerkus and Uhler (1977) with slight modi-fications: dorsal tissue samples from pink salmon were fixed in33 g/l formaldehyde in distilled water for more than 1 week,rinsed in tap water for 1 day, and then immersed in Alcianblue solution [0.1 g/l Alcian blue 8GX (Wako, Osaka, Japan) in760 ml/l ethanol and 200 ml/l glacial acetic acid] for 2 days.The specimens were then hydrated in a descending ethanolseries and distilled water for 1 day, before being cleared in asolution of 10 g/l trypsin (EC 3.4.21.4, Difco, Franklin Lakes,NJ) in sodium tetraborate solution (7.8 g/l sodium tetraboratein distilled water) for 1–2 weeks. Next, the specimens wereimmersed in a solution of 1 g/l alizarin red S in 5 g/l potassiumhydroxide for 2 days, dehydrated in a graded 5 g/l potassiumhydroxide: glycerol series (3:1, 1:1, and 1:3), and then stored inpure glycerol containing 0.05 g/l thymol powder to aid tissuepreservation.

Basic Histology

Dorsal tissue samples were fixed in Bouin’s solution for 72 h,dehydrated using a graded ethanol series, and embedded inparaffin (Histosec; Merck, Darmstadt, Germany) for cross sec-tioning. Then, tissue sections (approximate thickness: 8 lm)were prepared on a rotary microtome (Leica RM2125 RTS;Leica, Nussloch, Germany) and mounted on glass slides.

For hematoxylin and eosin (H&E) staining, deparaffinizedsections were stained with Delafield’s hematoxylin for 20 min.After being washed in distilled water, the sections were thenstained with 5 g/l aqueous eosin for 10 min, before being dehy-drated in a graded alcohol series and xylene and then mountedwith Entellan new (Merck). All of the light microscopic observa-tions including the histochemical examinations were performedusing a microscope (Eclipse 80i, Nikon, Tokyo, Japan) equippedwith a digital camera (EOS 5D, Canon, Tokyo, Japan).

For Masson’s trichrome staining, deparaffinized sectionswere stained with Mayer’s hematoxylin for 6 min. Subse-quently, the sections were treated with 10 g/l aqueous

Fig. 2. O. gorbuscha, cleared and double-stained skeletal speci-mens of the dorsal regions at different stages of secondary sexualcharacteristic development. Arrows and arrowheads indicate neu-ral spines and free interneural spines, respectively. (A) Immaturemale. In the immature males, the neural spines took the form of apair of spines in the dorsal region [arrows in (A)]. (B) Maturingmale. (C) Mature male. The blue region represents Alcian blue-positive connective tissue. (D) Mature female. Scale bars: 10 mm.

TABLE 2. Comparison of the length, diameter, and angle to the body axis of free interneural spines among groups in cleared skele-tal specimens of pink salmon

Immature males Maturing males Mature males Mature females

Length (mm) 16.8 6 1.17 24.6 6 3.13 41.0 6 5.57 *** 21.9 6 0.61Diameter (mm) 0.70 6 0.04 0.87 6 0.10 1.40 6 0.22 *** 0.73 6 0.05Angle (�) to body axis 66.0 6 1.79 79.4 6 4.22 91.0 6 2.29 65.0 6 2.00Number of samples n 5 5 n 5 5 n 5 8 n 5 5

Length and diameter are expressed. ***, P<0.005 (Scheffe’s test).

516 K. SUSUKI ET AL.


phosphotungstic acid and 10 g/l aqueous sodium citrate for30 s each and then stained with 10 g/l acetic acid containing10 g/l Biebrich scarlet for 30 s. Next, the sections weretreated with aqueous mordant phosphotungstic acid andphosphomolybdic acid (each 25 g/l) for 2 min each and thenstained with aniline blue solution (25 g/l acetic acid contain-ing 25 g/l aniline blue) for 4 min. Finally, the sections wererinsed in 10 ml/l acetic acid, dehydrated with ethanol andxylene, and mounted with Entellan new. This staining proce-dure resulted in collagen fibers being stained a bright bluecolor by the aniline blue.

To detect elastic fibers in the dorsal tissue, elastica van Gie-son staining was performed according to the following standardprocedure: deparaffinized sections were stained with resorcin-fuchsin solution (Muto, Tokyo, Japan) for 90 min, differentiatedin ethanol, and washed with tap water. The sections were thenstained in Weigert’s iron hematoxylin for 10 min, washed withdistilled water, and differentiated in acid alcohol. Finally, thesections were stained in van Gieson solution for 3 min, dehy-drated with ethanol and xylene, and mounted with Entellannew. The elastica van Gieson staining resulted in the elasticfibers being stained black by the resorcin-fuchsin.

To identify the main cell types present in each dorsal humpcomponent, we examined the dorsal hump tissue samples with atransmission electron microscope (TEM). Ultrastructural sam-ples were prepared as follows: small dorsal tissue samples werefixed in a mixture of 20 g/l paraformaldehyde and 25 g/l glutaral-dehyde in 0.1 mol l21 phosphate buffer at 4�C. They were thenpostfixed in a solution of 10 g/l osmium tetroxide in the samebuffer for 2 h at 4�C before being dehydrated in a graded series ofacetone and finally embedded in epoxy resin. Semithin sectionsof dorsal tissue were also prepared on an ultramicrotome (LeicaEM UC7 RT, Leica, Vienna, Austria) and stained with a mixtureof 5 g/l methylene blue, 5 g/l azure II, and 5 g/l borax in distilledwater. These sections were examined using light microscopy.Ultrathin sections of the dorsal region were also prepared on theabovementioned ultramicrotome and stained with 25 g/l samar-ium acetate and 26.6 g/l lead citrate. Then, they were examinedwith a JEM-1011 TEM (JEOL, Tokyo, Japan) equipped with adigital camera (iTEM, Olympus, M€unster, Germany).

Histochemical Examinations of the Bony andConnective Tissue

Dorsal tissue samples were fixed in 33 g/l formaldehyde indistilled water and embedded in Histosec. The samples werethen decalcified with 50 g/l formic acid if necessary, before

being sectioned into 8 lm thick slices and mounted on glassslides. Pre-embedding osmium tetroxide staining (modifiedCiaccio method; Exbrayat, 2013) was performed to detect lip-ids in the dorsal tissue: tissue samples were rinsed in distilledwater and 0.1 mol l21 phosphate buffer at 4�C, incubatedin 10 g/l osmium tetroxide and 50 g/l potassium dichromatesolution for 16 h at 4�C, and embedded in Histosec (Merck).Then, the sections were subjected to H&E staining, asdescribed above.

To detect acid mucopolysaccharides (including hyaluronicacid) in dorsal tissue, Alcian blue and hematoxylin stainingwas performed at pH 2.5: deparaffinized sections were treatedwith 30 ml/l acetic acid for 3 min, before being stained withAlcian blue solution (30 ml/l acetic acid containing 10 g/l Alcianblue, pH 2.5) for 30 min and rinsed in 30 ml/l acetic acid for 3min. Next, the sections were stained with Mayer’s hematoxylinfor 10 min, dehydrated with an absolute ethanol and xyleneseries, and mounted with Entellan new. Alcian blue-hematoxylin staining was also performed at pH 1.0, using 0.1N HCl instead of 30 ml/l acetic acid (Myers et al., 2008). Toassess the specificity of this method for detecting hyaluronicacid, adjacent sections were digested with 250 U/ml Streptomy-ces hyaluronidase (EC 3.2.1.35; Merck Millipore, MA) in 100mmol l21 acetate buffer for 2 h at 60�C before Alcian blue andhematoxylin staining was performed at pH 2.5.

Osteoid staining was performed to differentiate betweenosteoid tissue and mineralized bone matrix tissue, according tothe method described by Ralis and Watkins (1992).

Analysis of Tissue Water Content

Samples from the three major tissues that comprise the dor-sal hump (muscle, skin, and loose connective tissue [distalregion; wet weight: 5–10 g]) were dissected from mature males(n 5 3). Water content was determined gravimetrically by dry-ing samples at 103�C for 24 h and measuring their water loss(AOAC, 1990).

Determination of the Type of Collagen inDorsal Hump Tissue

To determine the type of collagen in the dorsal hump, biochem-ical analyses of collagen extracts from dorsal hump tissue wereperformed by electrophoresis. All crude collagen extraction pro-cedures were performed at 4�C. Three regions of nonskeletal andnonmuscular tissue were dissected from the dorsal humps ofmature pink salmon (the median septum, the distal region, andthe crescent-shaped region). Skin from mature male pink salmonwas also dissected as a Type I collagen-containing control tissue,as reported in Oncorhynchus species previously (Matsui et al.,1991). Each tissue was washed with distilled water and cut intosmall pieces (5 3 5 mm2). Fat was removed using ethanol threetimes. After being washed with distilled water, the tissues werecontinuously stirred in HCl (pH 2.0) containing 1 g/l porcine pep-sin (EC 3.4.23.1; 1:10,000, Wako Pure Chemical Industries,Osaka, Japan). Then, the mixtures were centrifuged at 2,200g at4�C for 1 h, and the resultant supernatants were collected. Thecrude collagen in each supernatant was precipitated by addingNaCl to a final concentration of 1 mol l21 and then centrifuged at2,200g for 90 min. The resultant precipitates were dissolved inHCl (pH 2.0). This process was repeated three times to purify thecollagen. Each collagen-containing solution was dialyzed againsta 50 times volume of distilled water for 24 h, and the resultantdialysates were lyophilized and stored at 230�C until use. Todetermine collagen subtype, 7.5% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) was performedaccording to the method of Laemmli (1970).

Statistical Analysis

Statistical analyses were performed with IBM SPSS Statis-tics 20.0 (IBM Japan, Tokyo, Japan). ANOVA and Scheffe’s F-

Fig. 3. O. gorbuscha, photograph (A) and schematic illustra-tion (B) of a cross-section of the dorsal hump of a mature male.In the cross-sections observed in this study, the internal struc-ture of the dorsal hump was found to be as shown in (B). CR,crescent-shaped region; CT, connective tissue; DR, distal region;DPC, dorsal posterior cone; FIS, free interneural spine; Int,integument; MS, median septum; NS, neural spine; SA, supra-carinalis anterior. Scale bar: 10 mm.



test were used for intergroup comparisons of the length anddiameter of the free interneural spines, as well as comparisonsof the water contents of the samples of the three dorsal humptissue types obtained from the mature males. Body length (FL),the length and diameter of the free interneural spines, theangle between each interneural spine and the body axis, andthe water content of each dorsal hump component areexpressed as mean and standard deviation values.

RESULTSMacroscopic Anatomy

The dorsa of the cleared and double-stainedskeletal specimens contained two bone tissues,free interneural spines, and neural spines (Fig.2). It was demonstrated that in males the free

Fig. 4. O. gorbuscha, low magnification photomicrographs of cross-sections of dorsal tissue stained with H&E. Immature male (A),maturing male (B), mature male [(C), (D), and (E)], and mature female (F). Arrows indicate neural spines. The arrowhead indicatesa free interneural spine. Asterisks indicate dorsal posterior cones (muscle tissue), except for the asterisk in (C), which indicates thesupracarinalis anterior (another muscle tissue). (A) Dorsal median septum of an immature male fish. A small amount of connectivetissue is present. (B) Median septum of a maturing male. This figure shows a slight increase in the amount of connective tissuearound the neural spines. (C) Connective tissue in the distal region around the supracarinalis anterior of a mature male. Adipose-like tissue was observed in some of the connective tissue. (D) Connective tissue in the median septum of a mature male. (E)Crescent-shaped region located above the posterior cones in a mature male. (F) Median septum of a mature female. A slightlyincreased amount of connective tissue was observed compared with the immature period, although the females still possessed muchless connective tissue than the mature males. Scale bars: 200 lm.



interneural spines extended distally and verti-cally relative to the body axis during maturation.In the mature males, the length and diameter ofthe longest and thickest free interneural spineswere significantly larger than those of the othergroups (Scheffe’s F-test; df 5 3, 19; P<0.001;Table 2 and Fig. 2C). However, the free interneu-ral spines of the female fish did not elongate dur-

ing sexual maturation (Fig. 2D). Neural spinesalso seemed to be involved in the development ofthe dorsal hump, but the elongation pattern ofthese spines varied greatly among individualmales. In the immature period, the neural spinestook the form of two symmetrical spine bones(Fig. 2A), and no longitudinal splitting of the freeinterneural spines was observed.

Fig. 5. O. gorbuscha, high magnification photomicrographs of cross-sections of dorsal tissue stained with H&E. (A) Dorsal medianseptum of an immature male fish. (B) Median septum of a maturing male. (C) Connective tissue in the distal region of a maturemale. (D) Connective tissue in the median septum of a mature male. (E) Crescent-shaped region above the posterior cones of amature male. It was demonstrated that the fiber orientation of this connective tissue differed from those of the other connective tis-sues. (F) Median septum of a mature female. No cells in these tissues displayed the typical features of chondrocytes, such as arounded shape and the presence of a territorial matrix. Typical fibroblasts (arrows) and blood vessels (arrowheads) were observed inthese tissues, suggesting that they were not cartilaginous. Asterisks indicate adipose-like tissue. Scale bars: 100 lm.



Microstructure of the Salmon Dorsal Hump

The gross anatomical observations of the inter-nal structure of the dorsal hump obtained in thisstudy were similar to those described in previousreports (Davidson, 1935; Nakamura, 1942; Fig. 3).However, cartilaginous tissue was not observed inthe dorsal tissue of any fish during light micros-copy (Figs. 4 and 5). Instead, connective tissue,including a large amount of adipose-like tissue,was observed in the corresponding regions. In the

immature and maturing males, a small amount ofcollagen fibers were detected in these connectivetissues by Masson’s trichrome staining (Fig. 6A,B).In the mature males, connective tissue, includingadipose-like tissue, was observed throughout thedistal region and median septum (Figs. 4C,D and5C,D). In addition, in the crescent-shaped regionconnective tissue was observed above the dorsalposterior cones (Figs. 4E and 5E). It was demon-strated that these connective tissues were mainly

Fig. 6. O. gorbuscha, photomicrographs of cross-sections of dorsal tissue stained with Masson’s trichrome stain. The aniline blue inMasson’s trichrome stain colored collagen fibers bright blue. (A) Median septum of an immature male fish. (B) Median septum of amaturing male. (C) The distal region of a mature male. (D) The median septum of a mature male. (E) Crescent-shaped region abovethe posterior cones of a mature male. (F) Median septum of a mature female. Arrowheads indicate free interneural spines. In maturemales, the connective tissues in the dorsal hump were mainly composed of collagen fibers. Arrows indicate neural spines. Asterisksindicate muscle tissue, as the supracarinalis anterior in (C) and the dorsal posterior cones in other panels. Scale bars: 200 lm.



composed of collagen fibers (Fig. 6C–E). Further-

more, elastica van Gieson staining detected elastic

fibers in the median septum and distal region

(Fig. 7B–D and F), as well as in blood vessels

(Fig. 7A). However, no elastic fibers were present

in the crescent-shaped region (Fig. 7E).These connective tissues did not exhibit the

characteristics of cartilage (e.g., a territorialmatrix, avascular tissue, and rounded chondro-cytes) at the light microscopic level. In the mature

females, connective tissue similar to that observedin the mature males was found in the medianseptum and the distal region, but the females pos-sessed much less of this tissue than the maturemales (Figs. 4F, 5F, 6F, and 7F). In lightmicroscopic observations of semithin sections,fat-containing adipocytes were indicated byosmium-black formation, and blood vessels wereobserved in the connective tissues in the dorsalhump (Fig. 8A), but no evidence of cartilage wasdetected, which agrees with the other staining

Fig. 7. O. gorbuscha, photomicrographs of cross-sections of dorsal tissue stained with elastica-van Gieson stain. The resorcin-fuchsin in elastica-van Gieson stain colored the elastic fibers black (arrows). (A) Median septum of an immature male. Elastic fiberswere clearly detected in the tunica intima and media of blood vessels. (B) Median septum of an immature male. (C) Connective tis-sue in the distal part of a mature male. A small number of elastic fibers were detected in this connective tissue. (D) Connective tis-sue in the median septum of a mature male. As in the other regions, elastic fibers were also observed in this connective tissue. (E)Crescent-shaped region above the posterior cones of a mature male. Elastic fibers were not detected in this region of connective tis-sue. (F) Median septum of a mature female. Scale bars: 50 lm.



results. As for the ultrastructure of the dorsalhump, collagen fibers and typical fibroblasts wereobserved (Fig. 8B).

Characterization of the Connective Tissuesin the Dorsal Hump

The lipid content of adipose tissue wasassessed in wide visual fields of connective tissueaccording to pre-embedding osmium-black forma-tion in paraffin-embedded sections (Fig. 9). As aresult, it was demonstrated that the connectivetissue of the median septum and the distal regionwas rich in lipids in the immature and maturingperiods (Fig. 9A). However, the adipocytes inthe corresponding regions of the mature malescontained few or no lipids (Fig. 9B,C) althoughhigh-magnification examinations found thatthe adipocytes in other regions did contain fat(Fig. 7A). The lipid content of the dorsal connec-tive tissue decreased during maturation. Itshould also be noted that the crescent-shapedregion did not contain any adipose tissue(Fig. 9D). In the mature female fish, the lipidcontent of connective tissue in the median sep-tum also decreased during maturation; however,the females did not exhibit as marked a reduc-tion as the male fish (Fig. 9E).

Acid mucopolysaccharides were not detected inthe dorsal connective tissue of the immature ormaturing fish. In contrast, in mature males theconnective tissue in the distal region and themedian septum of the dorsal hump contained acid

mucopolysaccharides (Fig. 10A,B), while that inthe crescent-shaped region did not. Hyaluronidasedigestion effectively reduced Alcian blue-reactivityin the connective tissue in the distal region andthe median septum of the dorsal hump, demon-strating that hyaluronic acid is a major mucopoly-saccharide in both regions (Fig. 10C,D). Inaddition, the mature females also developed con-nective tissue that contained hyaluronic acid inthe median septum and the distal region, althoughthey possessed much less of this tissue than themature males (data not shown). Conversely, sul-fated mucopolysaccharides, which are a character-istic component of the extracellular matrixes ofcartilaginous tissues, were not detected by Alcianblue and hematoxylin staining at pH 1.0 in theconnective tissue of the dorsal hump (data notshown). These histochemical results are summar-ized in Table 3.

The water contents of the three different tissuesthat comprise the dorsal hump (muscle, skin, andconnective tissue) were determined. The connec-tive tissue exhibited a significantly higher watercontent (95.5 6 0.18%) than the other componentsof the dorsal hump (muscle, 81.9 6 1.72%; skin,81.8 6 1.75%; Scheffe’s F-test; df 5 2, 6; P< 0.001).

Using SDS-PAGE, two distinct a chains, a1 anda2, which had molecular masses of approximately120 and 100 kDa, respectively, and are typical compo-nents of Type I collagen (Kimura et al., 1987), weredetected in collagen extracts from the skin. All of thecollagen samples extracted from the three compo-nents of the dorsal hump exhibited similar band

Fig. 8. O. gorbuscha, photomicrograph of a semithin section (A) and an electron micrograph of an ultrathin section (B) of the distalregion of a mature male. (A) Scattered fibroblasts [arrows in (A)] and vascular vessels [arrowheads in (A)] were observed in thisregion. The lipids in adipocytes were stained by osmium-black [asterisks in (A)]. Scale bar in (A): 50 lm. (B) Ultrastructure of thedorsal region. Typical fibroblasts [arrow in (B)] and collagen fibers [asterisks in (B)] were observed in the connective tissue, but nochondrocytes were present. Scale bar in (B): 5 lm.



patterns to those obtained from the skin (Fig. 11).However, Type II collagen, which is a typical compo-nent of cartilaginous tissue, was not detected in theconnective tissue of the dorsal hump.

Bony Tissue in the Dorsal Hump

Osteoid staining successfully distinguishedbetween the well-mineralized bone matrix (red)and less-mineralized matrix tissue (blue) in thedorsal tissues (Fig. 12). Free interneural spinesand neural spines were observed as well-mineralized bones in all three developmentalstages (Fig. 12A–C). In the mature males, theproximal and distal parts of the free interneuralspines were surrounded by osteoblasts and osteoidtissue (less-mineralized bone matrix tissue; Fig.12B,C). Although the free interneural spines dis-played prominent osteoblasts at both their distal

and proximal tips, macroscopic observations sug-gested that they grew distally. Less-mineralizedbone matrix tissue was much more common in theneural spines of the mature males (Fig. 12D).

DISCUSSION

Analysis of the dorsal endoskeleton showed thatnot only the neural spines reported by Davidson(1935) and Nakamura (1942) but also free interneu-ral spines, are involved in the formation of the dor-sal hump (Fig. 2). In mature males, free interneuralspines were present in the median septum of thedorsal hump and protruded distally and verticallyrelative to the body axis (Fig. 2C and Table 2). Inaddition, light microscopic observations alsodetected evidence of rapid bone growth in the freeinterneural spines (Fig. 12B,C). These results sug-gest that the vertical elongation pattern and rapid

Fig. 9. O. gorbuscha, photomicrographs of cross-sections of dorsal tissue stained with pre-embedding osmium staining. This stainingprocedure detected lipids via osmium-black formation. (A) Median septum of an immature male fish. The arrowhead indicates a freeinterneural spine. (B) Connective tissue in the distal region of a mature male. (C) Connective tissue in the median septum of a maturemale. (D) Crescent-shaped region above the posterior cones of a mature male. (E) Median septum of a mature female. Scale bars: 100 lm.



growth of free interneural spines facilitate thedevelopment of a larger dorsal hump. However,although there was histological evidence to indicatethat the neural spines exhibit rapid growth (Fig.12D), we were unable to evaluate the elongation of

these spines at the macroscopic level. Thus, a moreadvanced osteological analysis of neural spinedevelopment during salmon maturation is neces-sary. Conversely, neural spines were found to takethe form of two symmetrical spinal bones

Fig. 10. O. gorbuscha, photomicrographs of cross-sections of dorsal tissue stained with Alcian blue and hematoxylin at pH 2.5. (A)Connective tissue (asterisks) in the distal region of a mature male [(A), (C)] and the median septum of a mature male [(B), (D)]. Thebright blue signals representing acid mucopolysaccharides that were seen in [(A) and (B)] were absent from adjacent sections thathad been predigested with hyaluronidase [HAase; (C) and (D), respectively]. Scale bars: 100 lm.



protruding from a single vertebra in all specimens(e.g., Fig. 2A), and neither the neural spines norfree interneural spines showed signs of the longitu-dinal splitting described by Davidson (1935) andNakamura (1942). Previous osteological studies ofSalmonidae (e.g., Totland et al., 2011) have alsoindicated that neural spines originally protrude dis-tally as two symmetrical spines from a single verte-bra, although they might be observed as singlespinal structures at the macroscopic level in theimmature period.

Histochemical and biochemical analyses demon-strated that the dorsal humps of pink salmon arecomposed of connective tissue, rather than cartilage.It is widely considered that the dorsal humps of Sal-monidae are mainly composed of cartilaginous tissue,especially in the median septum, the cartilaginouscrescent above the dorsal posterior cones, and thecartilaginous mass around the supracarinalis muscle(Davidson, 1935; Nakamura, 1942). The general his-tological characteristics of cartilage have beenreported to be as follows: it is found in articular tis-sue, avascular, and composed of rounded chondro-cytes (Benjamin, 1990; Benjamin and Ralphs, 2004;Witten et al., 2010). Biochemically, cartilage containslarge amounts of sulfated mucopolysaccharides andType II collagen as the main molecules of the extrac-ellular matrix (Witten et al., 2010). However, thesemolecules were not detected in the connective tissueof the dorsal hump (Fig. 11), and it is clear that theconnective tissue in the dorsal humps of pink salmonis mainly produced by fibroblasts (Fig. 8B), ratherthan chondrocytes. Thus, we concluded that connec-tive tissue, rather than cartilage, participates in thedevelopment of the dorsal hump in pink salmon. Inparticular, the connective tissues in the median sep-tum and the distal region of the dorsal hump were

found to contain hyaluronic acid (Fig. 10). Thesehyaluronic acid-rich connective tissues might be simi-lar to gelatinous tissues such as that found in themammalian umbilical cord (Wharton’s jelly; Hadi-dian and Pirie, 1948; Catini and Gheri, 1983). Previ-ous studies (Robinson and Mead, 1970; Bower et al.,2011) reported that the dorsal humps of pink salmonhave high water contents although these studiesreferred to the whole dorsal hump or fillet, ratherthan to dissected connective tissues. The presentstudy revealed that the high water content of the dor-sal hump is mainly derived from the development ofconnective tissue. It is generally accepted that hyal-uronic acid exhibits strong water retention ability(Nakamura et al., 1993), and the loose and/or less-organized collagen fibers of water-retaining connec-tive tissue are highly suited to the acquisition of alarger dorsal hump. The kype, i.e., a hooked nose, isanother secondary sexual characteristic of male sal-monids (Tchernavin, 1938). As the kype might be aspecialized feature for attacking other males (Gross,1984; Hutchings and Myers, 1987), its structurerequires physical strength. Hence, the kype is mainlycomposed of skeletal tissue, which arises as aspongiosa-like meshwork, rather than a solid bonemass, and this structure suggests that the bony tis-sue of the kype is composed of the least amount ofmaterials possible and provides the maximum levelof mechanical stability (Witten and Hall, 2002, 2003).In contrast, the dorsal hump might serve as a shieldagainst attacks from other males (Schroder, 1981),act as a status indicator (Keenleyside and Dupuis,1988; Quinn and Foote, 1994), and be useful for pre-venting other males (i.e., competitors) from gainingaccess to spawning females (Fleming and Gross,1994). These studies suggest that developing a largerdorsal hump increases male competitiveness. Ourresults indicate that the development of the dorsalhump strongly depends on increasing the amount of

TABLE 3. Results of histochemical observations of dorsal tissuefrom pink salmon at different stages of maturation

Immaturemales

Maturingmales

Maturemales

Maturefemales

Distal regionConnective tissue 1 1 11 1

Elastic fibers 1 1 1 1

Lipids 11 11 1/2 1/2Hyaluronic acid 2 2 1 1

Median septumConnective tissue 1 1 11 1


Lipids 11 11 1/2 1/2Hyaluronic acid 2 2 1 1

Crescent-shapedregionConnective tissue 2 2 11 2


Lipids 2 2 2 2

Hyaluronic acid 2 2 2 2

Collagen fibers 1 1 11 1

Blood vessels 1 1 11 1

2: not present, 1/2: scarcely present, 1: slightly present, 11:abundant.

Fig. 11. SDS-PAGE of crude collagen extracts obtained from theconnective tissue in the median septum (lane 2), crescent-shapedregion (lane 3), or distal region (lane 4) or the skin (lane 5) of thedorsal hump of a mature male pink salmon. The arrow and arrow-head indicate a1(I) and a2(I) chains, respectively. The positions ofthe molecular mass markers (lane 1), which are expressed in kilo-daltons, are indicated on the left side of the figure.



connective tissue with high water content. Hence,the presence of hyaluronic acid-containing connectivetissue in the dorsal humps of pink salmon facilitatesthe development of a larger dorsal hump due to thestrong water retention ability of hyaluronic acid.Such humps might be particularly useful during thespawning period, when salmon stop feeding.

The connective tissues in the median septumand distal region of the dorsal hump containedhyaluronic acid (Fig. 10), lipids (Figs. 8A and 9),and elastic fibers (Fig. 7C,D), while that in thecrescent-shaped region did not. Accordingly, weassume that the connective tissue in the crescent-shaped region differs from those in the distalregion and median septum. Moreover, both maturemales and mature females possessed hyaluronicacid-containing connective tissue in the distalregion and median septum of their dorsal tissue,although the females only possessed an extremelysmall amount of this tissue. This suggests that thedevelopment of such connective tissue during sex-ual maturation is not limited to males, but onlymales are able to induce prominent dorsal develop-ment. The present study also obtained histologicalevidence that suggested that the adipose tissue inthe dorsal hump is replaced by fibrous and mucousconnective tissue in male fish during maturation(Figs. 6, 9, and 10). This process might be due tolipid consumption, i.e., the consumption of anenergy store during upriver migration in the

spawning period (Hendry and Berg, 1999; Kinni-son et al., 2003). In mature females, the medianseptum was also found to contain lipids; however,the females did not develop dorsal humps eventhough they developed similar connective tissue(containing hyaluronic acid and collagen fibers).Therefore, energy mobilization in dorsal tissuemight differ between the sexes, and lipid consump-tion in dorsal tissue might be associated with dor-sal hump formation. Further studies are requiredto elucidate the mechanism regulating the forma-tion of the dorsal hump in salmon, as well as othersalmonid species.

In conclusion, this study provided evidence thatthe dorsal humps of male pink salmon develop viathe elongation of bony tissues in the median septumand increases in the amount of connective tissue witha high water content, i.e., connective tissue composedof Type I collagen and hyaluronic acid. It was alsoclearly demonstrated that in pink salmon the dorsalhump is not composed of cartilaginous tissue.

ACKNOWLEDGMENTS

The authors are grateful to Captain S. Takagi andthe crew of the training ship Oshoro Maru of Hok-kaido University for providing immature pinksalmon from the Pacific Ocean, and also thank theNemuro Salmon Propagation Association for provid-ing mature fish from the Shibetsu River. The authors

Fig. 12. O. gorbuscha, photomicrographs of cross-sections of dorsal tissue stained with osteoid staining (Ralis and Watkins, 1992).Well- and less-mineralized bone matrixes were stained red and blue, respectively, by this process. (A) Free interneural spine of animmature male. (B) Proximal region of a free interneural spine in the dorsal hump of a mature male. (C) Distal region of a freeinterneural spine in the dorsal hump of a mature male. (D) Neural spine in the dorsal hump of a mature male. Arrows indicateosteoblasts located around bony tissues. Arrowheads indicate osteoblasts enclosed in osteoid tissue. Scale bars: 100 lm.



thank Ms. X. Zhang of Hokkaido University for hersupport with the collagen biochemistry experiments.

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dorsal hump morphology in pink salmon (oncorhynchus gorbuscha)

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