Changes in Tissue Biochemical Composition and Energy Reserves Associated With Sexual Maturation in the Ommastrephid Squids Illex coindetii and Todaropsis eblanae

2005·5.4 MB·

by  R Rosa

Reference: Bio/. Bull. 208: 100-113. (April 2005)
2005 Marine Biological Laboratory
Changes in Tissue Biochemical Composition and
Energy Reserves Associated With Sexual Maturation
and
in the Ommastrephid Squids Illex coindetii
eblanae
Todaropsis
R. ROSA1'*, P. R. COSTA2, N. BANDARRA1, AND M. L. NUNES1
1 Departamento de Inovafao Tecnologica e Valorizafao dos Produtos da Pesca, IPIMAR, Avenida de
Brasilia, 1449-006 Lisboa, Portugal; and ~ Departamento de Ambiente Aqudtico, IPIMAR, Avenida de
Brasilia, 1449-006 Lisboa, Portugal
Abstract. The aim of this study was to investigate the predators and important members of food webs (Clarke,
biochemical changes that occur during sexual maturation of 1966). Though these cephalopod species are commercially
the squids Illex coindetii and Todaropsis eblanae. In both important by-catches ofcommercial demersal trawls in the
species, amino acids and protein content increased in the east Atlantic (Lordan et at.. 1998; Robin et ai, 2002),
gonad throughout maturation, but the allocation of these several aspects of the biology and ecology remain poorly
nitrogen compounds from the digestive gland and muscle studied, including the reproductive biology (Laptikhovsky
was not evident. A significant (P < 0.05) increase in the and Nigmatullin, 1999; Hernandez-Garci'a, 2002). However,
content of lipids and fatty acids was observed in the gonad according to Rocha et al. (2001). both these squids seem to
and digestive gland. It seems that both species take energy have an intermittent terminal spawning; i.e., the spawning is
for egg production directly from food, rather than from monocyclic and eggs are laid in separate batches during the
stored products. Analyses for cholesterol revealed a signif- relatively long spawning period. According to Calow
icant (P < 0.05) increase in the gonad, and the lipid content (1973), terminal spawning species (semelparous strategy)
differences between species are potentially related to differ- invest more in their single spawning event than do multiple
ent feeding ecologies. The glycogen reserves in the gonad spawning species. In fact, the completededication ofenergy
<
increased significantly (P 0.05), suggesting that glycogen to reproduction results in a terminal spawning event; in
has an important role in the maturation process. It was contrast, a partial cost both before and during reproduction
evident that sexual maturation had a significant effect upon allows individuals to spawn repeatedly throughout their
tinhethgeondaigdesetnievregyglcaonndteannt,d bmuutscbleecawuasse tnhoenseingenirfgiycavnatri(aPtio>n aadsupltecliiefse.iMsoirneeoxtvreirc,abtlhyelrienpkreoddtuoctiitsvesosutrrcaeteogyfreexphriboidtuecdtibvye
0.05), there was no evidence that storage reserves are trans- energy (feeding or storage) (Jackson et al., 2004).
ferred from tissue to tissue. Sexual maturation and reproduction influence the status
of a number of physiological processes and consequently
Introduction
the animal's ecology and behavior. Clarification of the
Illex coindetii (Verany, 1839) and Todaropsis eblanae energy storage and consumption patterns of these squids
(Ball, 1841) are neritic ommastrephid squids that are caught will shed light on the interaction between organism and
near the bottom on the continental shelf and slope of the environment. Cephalopods have a protein-based metabo-
Atlantic Ocean, where they are voracious opportunistic lism (Lee, 1994), and the direct use ofprotein as an energy
reserve may account for the lack of major reserves of
R*eTcoeivwehdo2m0Jcuolyrr2e0s0p4o;ndaecncceeptesdho3u0ldNobveemabdedrres2s0e0d4.. E-mail: rrosa@ g1l9y8c4;ogSetnoraenydanldipiSdtoriney,ce1p9h8a3l)o.pTohdetliispsiudess i(nOt'hDeodrigeetstailv.e,
ipimar.pt gland have also been hypothesized to be possible substrates
100
BIOCHEMISTRY AND MATURATION OF SQUIDS 101
and a site for energy storage in cephalopods (O'Dor and el nl. ( 1951), using the Bio-Rad protein assay (BIO-RAD).
Webber, 1986; O'Dor and Wells, 1978: Moltschaniwskyj Bovine gamma globulin (BIO-RAD) was used as a stan-
and Semmens, 2000). dard.
Here, we focus on the changes in biochemical composi- Todeterminate the total amino acid profile, proteins were
tion that take place in the gonad, digestive gland, and hydrolyzed with 6 N hydrochloric acid (containing 0.1%
muscle of Illex coindetii and Todaropsis eblanae during phenol) in a MLS-1200 Mega Microwave System (Mile-
sexual maturation. Specifically, we examine the content of stone), at 800 W, 160 C for 10 min. The hydrolysis was
lipid, glycogen, cholesterol, and energy in these tissues, as performed under inert and anaerobic conditions to prevent
well as the profiles of total amino acid and fatty acid. oxidative degradation of amino acids. The hydrolysates
were filtered and dissolved in sodium citrate buffer, pH 2.2.
Materials and Methods Amino acids were separated by ion exchange liquid chro-
Samples matography in an automatic analyzer Biochrom 20 (Amer-
sham Biosciences), equipped with a column filled with a
Specimens of Illex coindetii and Todaropsis ehlanae polysulfonated resin (250 X 4.6 mm), using three sodium
were collected off the Portuguese west coast (Peniche) by citrate buffers pH 3.20. 4.25. and 6.45 (Amersham Bio-
commercial trawlers and off the south coast (Algarve) on sciences) and three temperatures (50 C, 58 C, and 95
several cruises aboard the R/V Nornega and R/V Capricor- C). The detection of amino acids was done at 440 nm and
nio of Institute de Investigate das Pescas e do Mar (IPI- 570 nm after reaction with ninhydrin (Amersham Bio-
MAR) in February, March. April, and June of 2002. For sciences). Amino acids were identified by comparison of
each animal, the following parameters were recorded: man- theirretention time with those ofspecific standards (Sigma)
tle length, total weight, gonad weight, digestive gland (also andquantifiedwiththesoftware EZChromChromatography
called "liver" in some biochemical literature) weight, and Data System, vers. 6.7 (Scientific Software Inc.) using nor-
depth range of catch (Table 1 ). For both species, maturity leucine (Sigma) as internal standard.
stages were determined following Lipinski (1979). and the
specimens were classified as immature (stages 1 and 2) or Total lipids andfatty acid analvses
mature (stage 5). Gonadosomatic index (GSI gonad wet
weight/body wet weight, X100) and digestive gland index Total lipids were extracted by the method of Bligh and
(Dgl digestive gland weight/ body wet weight, XlOO) Dyer(1959). The fatty acidprofile was determined using the
were also determined. Each of the tissue types collected experimental procedure ofLepage and Roy (1986) as mod-
(gonad, digestive gland, and muscle) were pooled, after ified by Cohen et ul. (1988). The fatty acid methyl esters
freeze-drying in a Savant VP100. The biochemical anal- were analyzed in a Varian 3400 gas chromatograph
yses were performed in triplicate in these tissues. equipped with an auto-sampler and fitted with a flame
ionization detector. The separation was carried out with
Protein and amino acid analyses ChehlrioummpaacskcaCrPrSiielr/8g8as(5in0 ma fXuse0d.32silmicma ciadp),ilplarroygrcaomlmuemnd
wetPrtoitsesiune)coonnctehnetwraatsihoendwTaCsAdeprteecrimpiintaetde(swoilutbhil1i0z0edming\oMf fCr.oman1d80heaCtetdot2o02010CCatf4orC14m.i5nm~in',. hweiltdhfaorde1tecmtionraatt220500
sodium hydroxide (NaOH) for 24 h as described by Lowry C. A split injector ( 100:1 ) at 250 C was used. Fatty acid
Table 1
Summary ofbiologicaldatafor Illex coindetti andTodaropsis eblanae
Maturation
Collection
Species Sex stage n ML (mm) TW (g) GW (g) DgW (g) GSI HSI
Illex
102 R. ROSA ET AL
methyl esters were identified by comparing their retention matic index increased through the oogenesis and spermat-
times with those ofchromatographic Sigma standards. Peak ogenesis of both the species, but the digestive gland index
areas were determined using the Varian software. showed an unclear variation trend.
Protein and amino acid content
Cholesterol analyses
The of cholesterol content was based on The protein content in the gonad, digestive gland, and
tmhoediefxipeqeduraibnmyteinfOtieachlaltieponrnsoccheldaugreero(f20N00a)e.mmThieetchoall.est(e1r9o9l5)waass mseunstceldeinofFiIglulreex 1c.oiBnodtehtisipeacnidesTsohdoawroepdsainsienbclraeansaeeoifsthperes-e
analyzed in a Hewlett Packard 5890 gas chromatograph. nitrogen compounds in the gonad throughout sexual matu-
The separation was carried out with helium as carrier gas in ration, the highest values being obtained during oogenesis
an HP5 column (30 m X 0.5 mm id). The temperatures of
the oven, injector, and detector were 280 C, 285 C. and Gonad
300 C, respectively. Cholesterol was identified and quan-
tified by comparison with standards (Sigma) from which a
standard curve was prepared.
60 -
Glycogen analysis and bioenergetic calculation
Glycogen concentrations were determined according to
the method described by Viles and Silverman (1949). Tis-
sue samples were boiled with 1 ml of 33% potassium
hydroxide for 15 min. After cooling, 50 ;ul of a saturate
sodium sulfate solution and 2 ml of 96% ethanol were
added. Samples were placed in an ice bath for precipitation
(-30 min). Following centrifugation, the precipitate was
dissolved in 0.5 ml of distilled water, again precipitated
with 1 ml of ethanol, and redissolved in 0.4 ml of distilled
water. Glycogen was then measured by the anthrone-re-
agent method (72 ml of concentrated sulfuric acid was
added to 28 ml of distilled water, 0.05 g of anthrone, and
0.05 g of thiourea; the mixture was heated at 90 C for 20
min) andthe absorbanceread at620nm. Acalibration curve
was prepared with a glycogen (Sigma) standard.
The energy content was estimated according to Winberg
(1971), using factors of 12.6, 12.1. and 39.3 J mg~' for
protein, carbohydrate, and lipid, respectively. It is worth
noting that the carbohydrate fraction was underestimated
since it included only the glycogen content.
Statistical analysis
Data were analyzed using an ANOVA. Previously, nor-
mality and homogeneity ofvariances were verified by Kol-
mogorov-Smirnov and Bartlett tests, respectively. Having
demonstrated a significant difference somewhere among the
groups with ANOVA, we applied the Tukey test to find out 60
where those differences were (Zar, 1996).
55
Results Im.-males Mat.-males Im.-females Mat.-females
BIllexcoindelii o Todaropsiseblanae
Biological data
Figure1. Proteincontent(%dryweight)inthegonad,digestivegland,
The mean mantle length, total weight, gonadosomatic and muscleofIllexcoindetiiandTodaropsiseblanaemalesandfemalesat
index, and digestive gland index of Illex coindetii and different stagesofgonaddevelopment. Means SD withdifferentletters
Todaropsis eblanae are shown in Table 1. The gonadoso- represent significant differences (P < 0.05). Im, immature; Mat. mature.
BIOCHEMISTRY AND MATURATION OF SQUIDS 103
(from 55.26% to 61.54% dry weight in /. coindetii and from
59.64% to 62.69% dryweight in T. eblanae) (F7-16 = 1 1.23,
Tukey testP < 0.05). The amino acidanalysescorroborated
these findings. The total (protein bound + free) amino acid
composition in the gonad is shown in Appendix Table 1.
The majoressential amino acids were arginine, leucine, and
lysine. The major nonessential amino acids were glutamic
acid, aspartic acid, and alanine. In the digestive gland, the
protein contentdecreased significantly only during spermat-
ogenesis of /. coindetii (F7 ,6 = 24.39, Tukey test P <
0.05). The major essential amino acids were also arginine,
leucine,andlysine; the majornonessential aminoacids were
glutamic acid, aspartic acid, and alanine (Appendix Table
2). In the muscle, both protein and amino acid analyses
revealed an unclear variation throughout the maturation
process ofboth species. The major essential and nonessen-
tial aminoacids inthe muscle werethe same found in gonad
and digestive gland (Appendix Table 3).
Lipid andfatty acid content
The lipid contents in the gonad, digestive gland, and
muscle of Illex coindetii and Todaropsis eblanae are
shown in Figure 2. A significant increasing trend was
observed in the gonad during the maturation of both
species (mainly during oogenesis), and between species,
higher values were always attained in males and females
of T. eblanae (F7J6 = 32.22, Tukey test P < 0.05).
Similar results were obtained with the fatty acid analyses
(Appendix Table 4). Most of the saturated fatty acid
content was represented by 16:0 and 18:0, monounsatu-
rated fatty acid content by 18:1 and 20:1. and polyunsat-
urated fatty acid content as arachidonic acid (20:4n-6).
eicosapentaenoic acid (20:5n-3), and docosahexaenoic
acid (22:6n-3). Between the different tissues analyzed,
the highest lipid and fatty acid levels were obtained in the
digestive gland and the lowest in the muscle. In the Im.-males Mai.-males Im.-females Mai.-females
digestive gland, a significant increase in the lipid (Fig. 2) Illexcoindetii o Todaropsiseblanae
and fatty acid (Appendix Table 5) content was observed
dfautrtiyngacmiadtsu:raFt7iJo6n,=pri2m1a.r7i9lyadnudri2n9g.6o7o,genTeuskiesy(ltiepsitdsPan<d dainfdFfiemrgueunsrtcelse2t.aogfesILliolpfeixdgcoocnioannddteetdnietiv(eal%nodpdmTreoydnwater.iogMphsetia)snisenbltahenSagDeonmwaaidlt.ehsddiiagfnefdsetrfieevnetmaglleletastnedra,st
0.05). In the muscle, the lipid content showed an unclear represent significant differences (P < 0.05). Im. immature; Mat. mature.
trend during maturation (Fig. 2) and, as in the other
tissues, the major fatty acids in the muscle were 16:0, P <
18:0, 18:1, 20:1, 20:4n-6, 20:5n-3, and 22:6n-3 (Appen- Tukey test 0.05). In the digestive gland, although there
dix Table 6). were significant differences between immature and mature
males ofboth species, the differences throughout oogenesis
Cholesterol, glycogen, and energv content were not significant. The muscle presented lower choles-
terol values inrelation to othertissues and revealed noclear
Thecholesterolcontent in the gonad, digestivegland, and pattern ofvariation throughout spermatogenesis and oogen-
muscle of Illex coindetii and Todaropsis eblanae is shown esis. The glycogen content in the different tissues of /.
in Figure 3. In the gonad, the differences during sexual coindetii and T. eblanae is shown in Figure 4. In the gonad,
maturation were not significant, with the highest values the glycogen content increased during maturation of both
being attained by females. Between species, T. eblanae species. Trends of variation in the digestive gland and
always had the highest values in both sexes (F-, )6 = 42.36, muscle were unclear. The energy content in the gonad.
104 R. ROSA ET AL
0.80 Gonad Discussion
In the present study, we observed a clear increase in the
gonadosomatic index, but there was not a concomitant de-
0.60 crease in the digital gland index, which suggests that the
digestive gland resources are not depleted and if resources
are mobilized from this organ, then these resources seem to
be compensated by those gained from feeding. The diges-
tive gland may continue to accumulate substantial reserves
of energy throughout sexual maturation, with no evidence
0.20 -
0,10
Im.-males Mat-males Im.-females Mat.-females
BItlexcoindetii 3 Todaropsiseblanae
Figure 3. Cholesterol content (<7c dry weight) in the gonad. digestive
gland, and muscle of Illex coindetii and Todaropsis eblanae males and
females at different stages of gonad development. Means = SD with
differentlettersrepresentsignificantdifferences(P<0.05).Im,immature:
Mat. mature.
digestive gland, and muscle ofI. coindetii and T. eblanae is
shown in Figure 5. There was a significant increase in the
gonad energy content during sexual maturation of both Im.-males Mat.-males Im.-females Mat-females
species (F-, 16 = 2.36, Tukey test P < 0.05). In the diges- IIllexcoindetii c Todaropsiseblanae
tive gland, the energy contentdifferences did not seemto be
0r.e0l5a)t.edItno stehxeualmumsactluerattihoerne(Fw-,asI6 =no26s.i1g8n.ifTiucaknetyvtae=rsitaPtio<n gfleamFnaidlg,eusraenadt4.mdiufsfGcellryeecntoogfsetInallgceexosnctooefinntdgeo(t<niaiddarndydevwTeeolidogaphrtmo)epnstiin.stMheeebalgnaonsnaaed~.madSliDegesswtaiintvdhe
betweenspecies, sexes, andmaturation stages (F-, 16 2.52, differentlettersrepresentsignificantdifferences(P<0.05).Im.immature:
P > 0.05). Mat. mature.
BIOCHEMISTRY AND MATURATION OF SQUIDS 105
before spawning. More recently, in a study ofthe deepwater
squid Moroteuthis ingens. Jackson et al. (2004) found that
water tended to increase in the mantle, while protein, lipid.
and carbohydrate all decreased with maturity, suggesting a
transfer ofenergy from the muscle tissue during reproduc-
tive maturation. Additionally, it is noteworthy that in some
gonatid squids, the proportions ofthe body and the integrity
ofthe mantle do notchange in males during maturation, but
do change in females. As an adaptation for a deepwater
bathypelagic "brooding" of the negatively buoyant egg.
some ofthe female's muscletissue degenerates (gelatinizes)
(Arkhipkin and Bjorke. 1999). According to Seibel et al.
(2000). the high lipid content of the digestive gland in
females provides fuel to support the extended "brooding"
period of these gonatids.
Because we used only specimens in stages 1. 2 (imma-
ture) and 5 (mature) in the presentstudy, wecould not apply
a size-independent method such as geometric mean regres-
sion (see McGrath andJackson. 2002: Jackson etal.. 2004),
to determine how somatic condition changed with maturity.
Studies on captive ommastrephids will also be needed to
resolve the important life-history question of whether so-
matic reserves are converted into gametes.
Both squid species showed an increase of amino acids
20 and protein content in the gonad throughout sexual matu-
_,
ration (mainly in oogenesis). but the allocation of these
nitrogen compounds from the muscle (protein catabolism)
15 . was not evident. In addition, there was little evidence that
accumulated lipid storage products were being used foresg
production.
10 . A
significant increasing trend in the lipid and fatty acid
contents in the gonad and digestive gland was observed,
mainly in oogenesis. which suggests that, for successful
reproduction, females require higher energetic resources
directed towards gonad development. Moreover, it is worth
noting that sex-related biochemical differences can be due
Im.-males Mat--males Im.-females Mat.-females
to distinct feeding ecologies. Female ommastrephids are
Illexcoindetii o Todaropsiseblanae
typically larger than males and are perhaps able to feed on
Figure5. Energy content iJ mg~'I in thegonad. digestivegland,and different prey: that is. they may take more fast-swimming,
nuscle of Illex coindetii and Todaropsis eblanae males and females at oily fish, while males may take slower crustaceans with
rdiefpfreerseennttsstiaggneisfiocfangtodniafdfedreevnceelsop(mPen<t.0.M0e5a).nsIm, iSmDmawtiutrhe:diMffaetr.enmtatleutrtee.rs lower lipid content.
Both Illex coindetii and Todaropsis eblanae seem to use
energy for egg production directly from food, rather than
for the utilization ofeitherdigestive gland or mantle tissues from stored products. This direct acquisition has also been
to supply energy for gonads. In fact, other studies have proposedby otherauthorsfortemperate (GuerraandCastro.
shown that ommastrephids and other species maintain their 1994: Collins et al.. 1995) and tropical (Moltschaniwskyj
condition, continue to growth, and fuel their reproduction and Semmens. 2000) squids. Some lipid classes, namely
from feeding ratherthan energy stores (Harman etal., 1989: phospholipids. have an important role in the composition of
Rodhouse and Hatfield. 1990: Clarke et al.. 1994: Gabr et the gametes (Pollero and Iribarne. 1988). because they are
a/.. 1999: McGrath and Jackson. 2002). probably involved in the synthesis of the \itellus. which is
Being fast-swimming organisms, squids may not be able a yolk phospholipoprotein (Fujii. 1960). Squid gonad fatty
;o utilize muscle tissue to fuel the production of gametes. acids were dominated by polyunsaturated fatty acids and
However, according to Moltschaniwskyj and Semmens highly unsaturatedfatty acids from the n-3 series (Sargentet
'2000). some species may utilize mantle tissue immediately al.. 1999). Eicosapentaenoic acid and arachidonic acid are
106 R. ROSA ET AL.
important as structural components of cell membranes and significantly. As it is in other marine organisms, glycogen
as precursors of prostaglandins (Lilly and Bottino, 1981). may be important for maturation and embryogenesis. Car-
Docosahexaenoic acid may be important for the correct bohydrates are precursors of metabolic intermediates in the
development and survivaloffast-growingphospholipid-rich production ofenergy and nonessential amino acids and are
cephalopod paralarvae (Navarro and Villanueva, 2000, acomponentofdecapodovarian pigments (Harrison, 1990).
2003). Lipid functions in the squid life cycle must include Summing up the findings of this study, it is evident that
supply of essential fatty acids and membrane components sexual maturation had a significant effect upon the energy
like phospholipids and cholesterol to cells. content of the gonads of Illex coindetii and Todaropsis
Levels of lipid, fatty acid, and cholesterol were signifi- eblanae, because it is associated with an increase in the
cantly higher in digestive gland than in gonad and muscle. products ofbiosynthesis that will support the lecithotrophic
The digestive gland is a site of digestive absorption and strategy ofparalarvae. The energy variation in the digestive
intracellular digestion (Boucher-Rodoni et ai, 1987; Sem- gland and muscle was nonsignificant, and because the di-
mens, 2002), and the differences between the two squid gestive gland, gonad, and muscle constituents varied inde-
fsapcetc,iecsommpaaryisboenrbeeltatweedentostdoifmfaecrhentconfteeendtinagndecloilpoigdieasn.alyI-n preesnedrevnetslywoefreonteraannsoftehrerre,drfevreoamleodrngoanevtiodeonrcgeant.haTthsetorbaigoe-
sis confirms that lipid from digestive gland is very likely to chemical compositionofdigestive glandand muscle may be
derive from the diet with little or no modification prior to influenced not by sexual maturation but instead by other
deposition (Hayashi et ai. 1990, Phillips et ai, 2001). bioticfactors, such as feeding activity, food availability, and
Moreover, recent findings for the southern pygmy squid spawning.
Idiosepius notoides have shown the presence, in the diges-
tive gland and cecum, of intracellular lipidic droplets that Acknowledgments
derive from that squid's field diet (Eyster and van Camp, The Portuguese Foundation for Science and Technology
2003). (FCT) supported this study through a doctoral grant to the
Voogt (1973) reported that cephalopods are able to syn- first author. Gratitude is due to Pedro da Conceicao for his
thesize sterols. although molluscs in general apparently can technical assistance and to Dr. Joao Pereira for his help
only carry out this biosynthesis slowly (Goad, 1978). Ac- obtaining the specimens.
cording to Kanazawa (2001), cephalopods seem to incorpo-
rate acetate and mevalonate into sterols poorly and then Literature Cited
rseurqvuiivrael.adSdqiutiidonaarledaicettiavreycsaornuirvcoerseso,fasntderiofltfhoeirrgcroomwptohneanntd Apsiilmloenc,ebrJo.sWus.,(MaonldlusDc.a,J.CBeuprhnaellolp.od1a9)8.0.CamSpt.eroBlisocfhreomm.tPhheyssiqouli.d,BIl6l5e:x
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Appendix
Supplementary data on composition ofIllex coindetii and Todaropsis eblanae males and females
at different stages of gonad development
Appendix Table 1. T<
BIOCHEMISTRY AND MATURATION OF SQUIDS 109
Appendix Table 2. Total(protein bound +free) amino acidcomposition (% dry weight) in the digestivegland
Amino acids
(7f dw)