Histological indices of the nutritional condition of sardine, Sardina pilchardus (Walbaum) larvae off the north coast of Spain

LJournal of Experimental Marine Biology and Ecology,212 (1997) 239–258

Histological indices of the nutritional condition of sardine,Sardina pilchardus (Walbaum) larvae off the north coast of

Spain

*I.R.B. McFadzen , S.H. Coombs, N.C. HallidayNatural Environment Research Council, Plymouth Marine Laboratory, Prospect Place, West Hoe,

Plymouth, PL1 3DH, UK

Received 25 January 1996; revised 27 September 1996; accepted 10 October 1996

Abstract

Larvae of sardine (Sardina pilchardus) were sampled off the north coast of Spain from March toJune 1992 as part of a programme to investigate factors affecting subsequent recruitment.Histological analyses of the larvae were carried out as a measure of nutritional condition forcomparison with environmental variables and subsequent survival at the juvenile stage. Tissueswere grouped into short-term indicators of nutritional condition, including the digestive tract, liverand pancreas, and long-term indicators represented by cartilage, notochord and muscle tissue.Long-term indices showed little difference between months, most specimens being in relativelygood condition. Short-term indices showed greater variation because larvae sampled in March andMay were in significantly poorer condition than those in April and June. A third measure ofcondition, the presence of irreversibly damaged tissue indicated that larvae sampled in March werein the poorest condition. This measure of condition was suggested as being the most objectiveassessment with respect to potential larval mortality. It was not possible to compare larvalcondition with survival to the juvenile stage because only an insignificant number of larvae fromspawning along the north coast of Spain recruited to the nursery area; most juveniles were derivedfrom spawning further to the south. It was suggested that offshore transport associated withupwelling off the north-west coast was responsible for the loss of larvae from the north coast andmay be a more significant factor in determining recruitment than nutritional condition. 1997Elsevier Science B.V.

Keywords: Histological analyses; Nutritional condition; Sardina pilchardus

*Corresponding author.

0022-0981/97/$17.00 1997 Elsevier Science B.V. All rights reservedPII S0022-0981( 96 )02755-4

240 I.R.B. McFadzen et al. / J. Exp. Mar. Biol. Ecol. 212 (1997) 239 –258

1. Introduction

The concept of a critical period in the early life-history of fish larvae is wellestablished, with variability in larval mortality being recognised as an important factoraffecting subsequent recruitment (May, 1974; Heath, 1992). Low food availability is asignificant determinant of larval mortality (Hunter, 1976) and is also reflected in lownutritional condition (Theilacker and Watanabe, 1989). Measures of larval condition can,

˚therefore, be used as indicators of survival and potential recruitment (Hakanson, 1993).Various analyses of nutritional condition of fish larvae have been reported (e.g.,

˚elemental composition, Ehrlich, 1974; histology, O’Connell, 1980; lipids, Hakanson,1989; morphometry, Frank and McRuer, 1989; RNA:DNA ratio, Clemmesen, 1987;otolith growth rate, Campana and Neilson, 1985). In the present study a set ofhistological criteria, as originally outlined by O’Connell (1976) for determiningcondition in anchovy larvae (Engraulis mordax), were used to investigate changes incondition of sardine (Sardina pilchardus) larvae off the north and north-west coasts ofSpain. Advantages of the histological approach are the ability to grade a number oftissues in an individual larva and to differentiate between short-term and long-term fooddeprivation (O’Connell, 1976).

The present study forms part of a European SARP programme (Sardine AnchovyRecruitment Project; IOC, 1989) in which larval abundance (Fig. 1) is compared withsubsequent juvenile recruitment and related to environmental conditions during the

´larval phase (Lopez-Jamar et al., in press). The nutritional condition of larvae describedin the present paper, based on histological criteria, was also measured for comparisonwith juvenile recruitment and food availability during the larval phase.

2. Materials and methods

2.1. Larval sampling

Sampling for sardine larvae was carried out on a series of five cruises along the northánd north-west coasts of Spain between March and July 1992 (Lopez-Jamar et al., in

press). Plankton sampling was by means of oblique slow speed ( | 2.5 knot) 50 cmdiameter bongo net tows (200 mm mesh aperture) to 100 m depth, or within 10 m of thebottom. Station positions were based on 8 nautical mile spacings extending out from thecoast (Fig. 2). For collection of microzooplankton, a fine mesh (53 mm mesh aperture)10 cm diameter bongo net was mounted above the main nets. Flowmeters were fitted toone side of each set of nets to measure the volume of water filtered. At all stations CTDdepth profiles were obtained. Few larvae were available on the final cruise (16–22 July;Fig. 1b) thus restricting the present study to specimens taken on the first four cruisesbetween March and June (Table 1).

On completion of a haul ( | 10 min duration), the nets were briefly rinsed beforeremoval of the cod-ends for preservation of the 53 mm mesh samples (4% formaldehydesolution) and preliminary sorting of the 200 mm mesh samples for sardine larvae.Specimens for histological analyses were measured (standard length) to the nearest 0.1

I.R.B. McFadzen et al. / J. Exp. Mar. Biol. Ecol. 212 (1997) 239 –258 241

Fig. 1. (a) The mean distribution and abundance of S. pilchardus larvae , 10.5 mm length taken on the fourcruises over the period March–June 1992 (see Table 1). (b) Larval abundance curve over the entire survey areafor larvae , 10.5 mm length plotted at the mid-date of each cruise.

mm and preserved in cold (48C) Baker’s formol calcium, this being completed withinapproximately 6 min of haul recovery in order to minimise post-mortem deterioration ofthe tissues. Depending on larval abundance, and the requirements for larvae for othercondition analyses, typically 5–10 sardine larvae were preserved from each station forhistological studies. Prior to analysis, a subsample of stations was selected (Table 1) togive representative samples along inshore /offshore transects in areas of larval abun-

´dance (essentially off Santander, between Gijon and Ortegal and off Vigo, see Fig. 1aánd Lopez-Jamar et al., in press) and in the area of potential upwelling from Finisterre to

Cape Ortegal (Fig. 2). Larvae for analysis were selected from stations sampled duringdaylight hours and, in order to minimise size specific differences in tissue organisation,were selected from a similar size range in all months (Table 1). All larvae were beyondyolk-sac stage and no specimens showed evidence of parasitic infection.


Fig. 2. Station positions for the four cruises off northern Spain. Dots indicate station positions and the adjacentnumbers indicate the number of months in which specimens were taken for histological grading.

The remainder of the 200 mm sample was preserved in 4% formaldehyde solution for´subsequent analysis of the remaining sardine larvae (Lopez-Jamar et al., in press). The

53 mm microzooplankton were analysed for the prey items selected by sardine larvae, asdetermined from gut content analysis, essentially these being copepod eggs, nauplii andcopepodite stages (Conway et al., 1991), to give food concentrations by area and cruise(Conway et al., 1994).

2.2. Histology

Subsequent to the cruise the preserved larvae were processed for methacrylateembedding (McFadzen et al., 1994). Larvae were then serially sectioned at 1–3 mmTable 1Sample information

Cruise Dates (1992) No. of No. of No. of Standard lengthstations larvae larvae of larvae inselected examined graded for which all tissuesfor analysis histologically all tissues were graded

Mean (mm)6S.D.

BO Cornide de Saavedra 6 March–20 March 11 46 37 5.9961.66MarchBO Cornide de Saavedra 31 March–15 April 13 70 64 7.0361.81AprilFS Valdivia 10 May–25 May 11 51 39 6.3561.08MayRRS Challenger 28 May–8 June 15 73 67 6.4161.94June


intervals in the sagittal plane, using Ralph glass knives and stained in Lee’s methyleneblue–basic fuchsin (Bennett et al., 1976). The plane of sectioning was approximate andno attempt was made to straighten larvae. Glycogen activity in the hepatic vacuoles andoesophageal goblet cells was determined from sections stained in periodic acid–Schiff(PAS) with an amylase control and coverslips mounted in Canada balsam (Bancroft,1967).

2.3. Histological grading

The nutritional condition of larvae was based on the histological grading of thecellular condition of selected tissues, as outlined below, based on criteria used byO’Connell (1976) for anchovy (Engraulis mordax) larvae. Tissues were graded from therelatively labile digestive tract and associated glands (Table 2) with rapid response timesto food deprivation (McFadzen et al., 1994), to the more resistant tissues of the cartilage,notochord and muscle (Table 3). These latter tissues are relatively robust as laboratorystudies have demonstrated that damage occurs only after prolonged periods of fooddeprivation (Theilacker, 1986; McFadzen et al., 1994). The histological variables used todetermine the extent of nutritional stress show growth and differentiation during normaldevelopment, but are not capable of demonstrating major changes in morphologicalorganisation between the different sizes of larvae studied.

Initial inspection of the tissues was carried out to reject specimens with evidence ofautolytic degradation (Hibiya, 1982). Examination of the remaining larvae was carried

öut with a Wild M20 microscope using standard Kholer illumination. Before assigningthe nutritional grade to a particular tissue, multiple sections taken throughout each larvawere examined for a representative assessment of the overall degree of tissue conditionwithin the specimen. The classification of a tissue grade was based on the poorestobserved condition of each specific tissue. It was not unusual for a specimen to have asuperficially healthy tissue condition, but when additional sections of the same tissuewere viewed, they were found to have regions of degenerative necrosis. Such foci ofnecrosis are irreversible and hence indicative of poorest possible condition.

Multiple sections were examined to assess the degree of tissue alteration throughoutthe specimen and each selected tissue variable assigned one of three grades (see Tables 2and 3) from healthy (scoring 1) to degraded (scoring 3) based on the poorest observedcondition in each specific tissue. The nutritional condition of each larva was thencharacterised as the mean score for all variables in all tissues indicative of eithershort-term (Table 2) or long-term (Table 3) food deprivation. The results used were onlythose for larvae in which all tissues could be assessed. A modification of O’Connell’s(O’Connell, 1976) method was to grade the foregut rather than the hindgut region of thedigestive tract. O’Connell (1976) stated that the condition of anchovy larvae could beevaluated from histological grading that does not include the status of the hindgutinclusions; therefore, this tissue characteristic was not graded in the present study. Theforegut was chosen for grading primarily due to the close physical association of theforegut to the hepato–pancreatic tissues and the ease of locating this tissue followingsectioning of any larvae with strong lateral bends. An additional record was kept oflarvae which had one or more extensively necrotic tissues, a condition comparable to the


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Table 3Histological criteria for long-term indices of condition of S. pilchardus larvae

Tissue Grade

1 (Healthy) 2 (Intermediate) 3 (Degraded)

Cartilage Chondrocyte nuclei Nuclei in variable Condensed nuclei,round and granular, degree of condensation, cytoplasm reduced,capsular space filled cytoplasm often collapse ofwith cytoplasm. contracted to occupy chondrocytes within

only a portion of the capsular matrix shell.capsular space.

Notochord No separation of the Isolated regions of Extensive shrinkageturgid notochord from notochord shrinkage. away from the trunktrunk muscle. muscle.

Trunk muscle Parallel fibres, Slight separation of Pronounced fibremyofibrils striated, fibres, loss of myofibril separation, highlyabundant basophilic striation, variable reduced myofibrilinterfibril tissue. nuclear staining, striations, dark

reduced basophilic indistinct nuclei.tissue.

concept of the point of no return (PNR) introduced by Blaxter and Hempel (1963), thisindex giving a third measure of nutritional condition. The degree of necrotic degenera-tion of these tissues was severe and deemed to be irreversible (Hibiya, 1982). If necroticlarvae were to encounter and ingest prey, effective digestion and assimilation of foodwould be unlikely, and a point of irreversible starvation reached (May, 1974; McGurk,1984).

Fig. 3. Ileo–ceacal constriction of S. pilchardus larva between the mid- and hindgut regions stained with Lee’smethylene blue /basic fuchsin. 3 mm section in the sagittal plane. Note the villi of the hindgut and thenumerous supranuclear vacuoles within the columnar epithelium. ic, ileo–caecal constriction; H, hindgut; M,midgut; mv, microvilli; sn, supranuclear vacuoles. Scale bar 15 mm.


2.3.1. Digestive tractTwo morphologically distinct regions of the digestive tract, the foregut and midgut,

were assessed. Sardine larvae possess a long straight gut, with the oesophagus markingthe posterior boundary of the buccal cavity. Oesophageal tissue is readily identified bythe presence of large PAS positive goblet cells anterior to the opening of the foregut.

The foregut mucosa is composed of simple cuboidal cells, terminating at the inlets ofthe pancreatic and bile ducts and leads into the highly differentiated region of the midgutwith the ileo–caecal constriction forming a common boundary between the mid- andhindgut regions (Fig. 3). The midgut mucosa is characterised by a compact highlyconvoluted columnar epithelium with distinct basal nuclei and by a continuous

Fig. 4. Midgut of S. pilchardus larvae stained with Lee’s methylene blue–basic fuchsin 3 mm sections in thesagittal plane with histological grades described in Table 2. (a) Healthy, Grade 1. (b) Severe localised necrosis,Grade 3. de, dorsal epithelium; is, intercellular space; lu, lumen; sc, sloughed cell; ve, ventral epithelium; vp,villi projection. Scale bar 10 mm.


microvillous border (Fig. 4a).Vacuolation of the midgut was prominent, care being takento distinguish vacuolisation as a result of degenerative processes from digestive activitydue to lipid digestion. Intracellular lipid vacuoles are characterised by loss of cyto-plasmic staining, nuclear displacement, do not stain positively with PAS and have a

Fig. 5. Liver and foregut of S. pilchardus larvae stained with Lee’s methylene blue–basic fuchsin 3 mmsections in the sagittal plane with histological grades described in Table 2. (a) Healthy, Grade 1, note numerouslarge intracellular glycogen vacuoles in the hepatocytes of the liver and the regular luminal surface of theforegut. (b) Degraded liver, Grade 3, note the region of necrotic tissue and the absence of cell structure.Although healthy tissue is present, the liver is assigned a low grade of condition (Grade 3) based on thecondition of the focus of necrosis in the posterior region. The foregut was graded as intermediate. Pyknoticnuclei are common in the remaining hepatocytes. a; anterior direction, ce, cuboidal epithelium; d, dermis; F,foregut lumen; gv, glycogen vacuole; hs; hepatic sinusoid; L, liver; n, nucleus; nt, necrotic tissue; p; posteriordirection, pn, pyknotic nucleus. Scale bar 12 mm.


general spherical appearance, whereas degenerative vacuolisation is associated withreduced cell size, necrosis and increased eosinophilic staining properties (Cowey andRoberts, 1978; Roberts, 1978; Hibiya, 1982). The relatively short hindgut extends to theanus. Hindgut epithelium is composed of compact columnar cells, covered on theirluminal surfaces by microvilli. These cells have large distinct basal nuclei, and arecharacterised by eosinophilic inclusion bodies in the supranuclear cytoplasm (Fig. 3)these being indicative of past feeding. The size and number of the supranuclear inclusionbodies is variable, and their presence is determined by feeding intensity and gut passagerates (Iwai and Tanaka, 1968).

2.3.2. Liver and pancreasThe liver is compact and elongate, lying ventral to the foregut. It is composed of

hepatocytes, containing pale staining regular nuclei (Fig. 5). Liver hepatocyte condition,particularly the relative amount of intracellular vacuolation associated with glycogenstorage, has been shown to be responsive to food availability (O’Connell, 1976).

Fig. 6. Cartilage of the buccal cavity of S. pilchardus larvae stained with Lee’s methylene blue–basic fuchsin 3mm sections in the sagittal plane with histological grades described in Table 3. (a) Healthy, Grade 1 with fullnuclei in the chondrocytes, forming regular isogenous groups. (b) Poor, Grade 3 with collapsed matrix andcondensed chondrocytes. cb, ceratobranchial cartilage; cc, condensed chondrocyte; ch, chondrocyte; cx,collapsed matrix; x, matrix. Scale bar 5 mm.


Hepatocyte cytoplasm, intracellular vacuolation and nuclear condition are variable(O’Connell, 1976; Theilacker, 1986) and thus were graded separately (Table 2), inaccordance with previous studies (O’Connell, 1976; Theilacker, 1986).

The pancreas is a compact basophilic gland of mixed endocrine and exocrine function,lying in close association with the liver and foregut. Healthy pancreatic acinar cells arepyramidal, forming distinct circular acini which contain varying numbers of smalleosinophilic storage granules (zymogen) in the cell apex above a distinct nucleus.Distinctive regular ducts containing numerous zymogen granules are found throughout a

Fig. 7. Notochord and trunk muscle of S. pilchardus larvae stained in Lee’s methylene blue–basic fuchsin 3mm sections in the sagittal plane with histological grades described in Table 3. (a) Healthy, Grade 1, note theturgid cells of the notochord in close association with the trunk muscle parallel myofibrils with basophilicconnective tissue between fibrils and, in particular, the striated appearance of the myofibrils (arrowed). (b)Degraded, Grade 3, note the loss of turgidity of the notochord, absence of striated myofibrils, irregularity ofthe fibres and loss of interfibril tissue (arrow heads). no, notochord; sm, striated myofibril; tm, trunk muscle.Scale bar 10 mm.


healthy pancreas (Table 2). Larvae classified as starving showed a loss of acinarsymmetry, with degradation of acinar cells and indistinct nuclei. Similarly, larvae inpoor condition showed evidence of collapse of luminal integrity and loss of granularcontent in the sinusoids of the pancreatic duct (Table 2).

2.3.3. Cartilage, notochord and trunk muscle tissueThe main areas of cartilage examined were the cranio–branchial elements and the

pelvic fin region. Healthy hyaline cartilage is composed of distinct elliptical chon-drocytes surrounded by a granular capsular space (Fig. 6a), particular care beingrequired to differentiate cellular degeneration from ontogenetic changes. Healthychondrocytes (Grade 1, see Table 3) are found in isogenous groups (clusteredappearance) evenly embedded within an amorphous matrix (Fig. 6a). This matrix is inturn surrounded by the perichondrium, a layer of dense connective tissue rich infibroblasts. Notochord and trunk muscle tissue was examined in sections along the dorsalmidline of the larvae (i.e., sections between the eyes) from the main body trunk andcaudal regions (Fig. 7).

3. Results

3.1. Short-term indicators of condition

The digestive tract and the associated glands (pancreas and liver) are short-termindicators of nutritional condition (Table 2; O’Connell, 1976; Theilacker, 1986;McFadzen et al., 1994). In all larvae, regardless of their nutritional classification, therewas evidence of supranuclear inclusion bodies in the hindgut which is indicative of apast feeding history (O’Connell, 1980). These bodies were, at times, in a degeneratedstate, and this condition was observed most often in specimens taken on the May cruise.

3.1.1. Digestive tractIn healthy larvae, the long, relatively undifferentiated foregut was composed of a

simple cuboidal epithelium beneath a smooth luminal surface (Fig. 5a). Occasionallarvae from each cruise showed degradation of the foregut epithelium. This was seen asincreased irregularity of the luminal surface, the presence of cell debris in the gut lumen,a reduction in cell size and the presence of pyknotic nuclei (Fig. 5b).

The midgut varied considerably in appearance (Fig. 4a,b), both within and betweencruises. Healthy tissue (Grade 1, see Table 2) being characterised by a compactcolumnal epithelium with distinct basal nuclei, supporting a regular microvillous borderon the apical surface (Fig. 4a). Intracellular vacuolation of the midgut was also highlyvariable, ranging from healthy intact columnar cells with no vacuolation (Fig. 4a) toextensive vacuolation and sloughing of cells into the enlarged lumen (Fig. 4b). Extensivevacuolation was invariably found associated with sloughed cells, areas of extensivenecrosis (Fig. 4b) and reduction of cell height and villi projection. Such severe localisednecrosis was found only in samples taken on the May and June cruises.


3.1.2. Liver and pancreasThe condition of the liver was variable (Fig. 5a,b). Liver tissue of healthy larvae was

homogeneous throughout, whereas larvae with livers graded as starving had distinct fociof colloid degeneration (Hibiya, 1982) or necrosis, of variable size and number.Hepatocyte cytoplasm ranged from slightly granular, with abundant large glycogenvacuoles (Fig. 5a), to dark staining collapsed cells without intracellular vacuolation.Healthy hepatocytes had pale staining nuclei (Fig. 5a) laterally displaced by intracellularvacuoles (PAS positive), and were present throughout the entire liver of those graded ashealthy. Hepatocytes lacking PAS positive vacuoles had dark staining collapsed nuclei(often pyknotic) and enlarged sinusoids (Fig. 5b). Pancreatic degeneration was character-ised by variable amounts of zymogen droplets in the acinar cells and by irregularities ofthe duct lumen. On all cruises pancreatic acinar degradation and pancreatic duct sinusoidcollapse (i.e., in larvae in poor condition) were invariably found together with liverdegradation.

3.2. Long-term indicators of condition

Long-term indicators of condition (Table 3) include the cartilage, notochord and trunkmuscle which are relatively less responsive to short periods of food deprivation than thedigestive tract and associated glands (Theilacker, 1978; Sieg, 1993; McFadzen et al.,1994).

3.2.1. Cartilage, notochord and trunk muscle tissueCartilage condition was relatively conservative between cruises, with only slight

variations in appearance. Degraded cartilage was rare, but when it occurred the nuclei ofthe chondrocytes were condensed and indistinct, with collapse of entire chondrocytesinto the surrounding matrix (Fig. 6b). Degradation of cartilage was noted in associationwith low scores (Grade 3) for tissues of the digestive tract, liver and pancreas, and wasevident in all months except March.

The notochord, similarly as for hyaline cartilage, was relatively stable in conditionduring the survey period. Healthy notochord maintained turgid cells and remained inclose association with the surrounding muscle (Fig. 7a). Severely degraded larvae hadcollapsed notochords (Fig. 7b) and extensive areas where the notochord was dis-associated from the trunk muscle, but such larvae were rare on all cruises.

Trunk muscle showed the least variation in cellular condition. In healthy tissue,muscle fibres and myofibrils were compact, with parallel fibres, distinctly striatedmyofibrils and abundant basophilic tissue between muscle fibre layers (Fig. 7a). In thedegraded condition there was pronounced fibre separation, reduced myofibril striationand minimal interfibril tissue (Fig. 7b).

3.3. Additional tissues

Further to the indices outlined in Tables 2 and 3, there was a general atrophy anddisintegration of cells of other tissues in poor grade larvae. In particular, renal tubulesshowed a loss of symmetry and luminal integrity. Eyes were prone to degeneration and


Fig. 8. Frequency distributions of histological tissue grades for S. pilchardus larvae on each cruise. (a)Short-term indices, based on scores for the liver, pancreas, fore and midgut. (b) Long-term indices, based onscores for the notochord, cartilage and trunk muscle.


Fig. 9. (a) The percentage occurrence of S. pilchardus larvae identified as severely necrotic on each cruise. (b)Mean food availability by cruise as the combined abundance of nauplii, copepodite stages and copepod eggs(from Conway et al., 1994).

lesions occurred in the brain. Mitotic activity was also reduced in starving larvae, butonly in those larvae which had a general decline in tissue condition, rather than oneisolated, but severely necrotic tissue. Swim bladders also showed enlargement and wasoften observed together with enlarged liver sinusoids.

3.4. Condition by cruise

The frequency distributions of histological grades for short-term condition indiceswere similar for the March and May cruises with a relatively low occurrence of larvae atthe lower grades (good condition). In contrast, there was a relatively high representationof larvae in good condition in April and June (Fig. 8a). However, larvae in the poorestcondition (high grades) were most evident in June, with little difference between theother months. The frequency distributions of long-term indices of condition were allbiased towards low values representative of larvae in good condition (Fig. 8b) but,nevertheless, showed similar patterns to the short-term indices, i.e., these were therelatively low occurrence of larvae with the lowest scores (good condition) in Marchand, to a lesser extent, in May and the prevalence of a few larvae scoring at the poorestcondition grades in June.


An additional, more stringent, measure of larval condition is plotted in Fig. 9a as thepercentage of larvae in which any single tissue was necrotic to the extent that recoveryof the larvae would have been improbable, irrespective of subsequent feeding level (pastPNR). For this variable, larvae in March were scored in the best condition (20.0% withnecrotic tissue), in May in the poorest condition (30.8%) and at intermediate levels inApril and June (25.0% and 25.0% respectively).

4. Discussion

Assessments of larval condition, as indices of survival potential, are most usefullybased on processes operating on longer time-scales than the feeding rhythmicity of fish

˚larvae (Coombs and Hakanson, 1991). The benefits of histological methods for suchmeasures are that a range of tissues responsive to both short-term and long-term feedinghistory can be simultaneously examined and some assessment can be made of theaetiology of the stressor, i.e., the source of tissue degeneration ascribed to foodavailability (Theilacker, 1986; Theilacker and Watanabe, 1989; Margulies, 1993),contaminant exposure (Wester and Canton, 1991; Johnson and Bergman, 1984), orparasitic /disease effects (Lom, 1970). Further benefits of the histological approachutilising methacrylate embedding, as in the present study, are that it provides thinsections with minimal processing artefact and gives a high degree of tissue resolution todistinguish environmentally derived stress effects from autolytic change. Inevitably,there will have been some influence on tissue condition during capture and sorting of thelarvae. However, the effects of mechanical damage will have been similar betweencruises since there was no essential change in the sampling protocol, and the lack of anyclear relationship between tissue condition (Fig. 8), and the seasonal change in water

´temperature (Lopez-Jamar et al., in press) does not suggest that temperature effects haveintroduced any serious bias in the results.

In the present study, the condition of sardine larvae was based on the histologicalgrading of the cellular properties of selected tissues, based principally on criteria used byO’Connell (1976). A number of other studies (e.g., Ehrlich et al., 1976; O’Connell,1976, 1980; Theilacker, 1978, 1986; Theilacker and Watanabe, 1989; Sieg, 1993;Margulies, 1993) have shown that histopathological responses to low food availabilityare common to many teleost larvae and that the fundamental cellular mechanisms ofatrophy and degeneration are not species specific (Roberts, 1978; Hibiya, 1982;Margulies, 1993). All tissues within an individual specimen are responsive to fooddeprivation so that the selection of suitable diagnostic criteria (O’Connell, 1976) enablesthe various alterations in cellular structure, organisation, shape, size and histochemicalproperty to be recognised. Such cellular alterations are caused by deficiency in the localsupply of nutrients derived from inadequate feeding (Hibiya, 1982), and do notnecessarily reflect the role of the tissue in food assimilation or digestive activity.

Although laboratory experiments to rear sardine larvae were an unsuccessful part ofthe present work, and therefore there is no specific validation of the histological indices,the criteria used in the present paper are sufficiently generalised to be applicable to mostfish species and, to some extent, throughout most vertebrates (Johnson and Bergman,


1984). However, some conditions, such as contraction and localised degeneration of themidgut epithelium, have been described as symptomatic of disease as well as starvation(Roberts, 1978). Since there was no evidence of parasitic infection or localised oedemaor haemorrhaging, the present observations are likely to relate entirely to variations infeeding. Similarly, Lom (1970) reported cellular alterations due to protozoan infectionsbut no signs of such infection were observed in sardine larvae of the present study.

Both short-term and long-term indices of condition showed similar relative changes intissue grade between cruises (Fig. 8). However, there was a noticeable difference in theshape of the frequency distributions of short-term and long-term indices. Long-termgrades were biased towards low values (good condition) whereas short-term indicesshowed a broader spread of values with the distributions extending to values . 2, thisbeing indicative of relatively poor condition. It is, perhaps, not unexpected thatlong-term indices give a consistent indication of larvae in good condition, sinceindividuals with any accumulation of poor short-term condition might be expected tohave been removed by some combination of starvation and predation mortality. Similarobservations of the generally healthy status of field-caught larvae have been reportedelsewhere (O’Connell, 1980; Pitcher and Hart, 1982; Theilacker, 1986; Owen et al.,1989). Conversely, short-term indices, which represent the more immediate effects offood availability, may be too closely associated with transient events to be a goodanalogue of survival. Since the necrotic classification (Fig. 9a) represents both a fixedcriterion, itself dependent on food availability, and unequivocally identifies larvae whichare not destined to survive (May, 1974; McGurk, 1984), this may be the most preciseindicator of potential survival (Blaxter and Hempel, 1963). However, although thenecrotic tissue classification showed larvae to be in relatively poor condition in May, inaccord with the short-term and long-term indices, there was no substantial agreement forthe other months. Laboratory rearing experiments are perhaps required to resolve suchapparent discrepancies, in particular by establishing the stability of both short- andlong-term indices with variation in food availability.

There was little indication of any significant truncation of values at the poor conditionend of the distribution of short-term condition values (Fig. 8a) which might indicatepreferential predation on larvae in low condition (Buckley, 1984). In contrast, there wasnoticeable truncation of the lower limb (good condition) of the distribution of short-termindices in March and May. It is unlikely that predation pressure would be greater forlarvae in good condition, thus the low representation of larvae at the better grades mayindicate relatively poor feeding conditions during these cruises; relatively low foodavailability during May (Fig. 9b) supports this conclusion. However, the lack of anyoverall correlation between food availability and histological condition does not supportany close relationship between these variables. At the same time it should be recognisedthat the measures of food availability in the present study are derived from integratedtows down the water column (Conway et al., 1994), while vertical stratification can lead

´to markedly different concentrations at discrete depths (Coombs et al., 1983; Fernandez-Puelles et al., 1993). Thus, depth-integrated plankton tows are not necessarily the mostprecise estimates of the actual prey field experienced by the larvae.

In the present study, the primary reason for measuring the condition of larvae was forcomparisons with subsequent survival to the juvenile stage. The hypothesis under test


being that condition indices, as a measure of incipient mortality, are an analogue orpredictor of subsequent survival. In the context of the SARP programme in which thestudy was based, larval survival was to be determined from a comparison of thebirth-date distribution of surviving juveniles, derived from back-calculation of their agedistribution from otolith daily growth-ring counts, with the larval abundance curve (Fig.1b). It is clear from the foregoing results that larval condition, as measured byhistological methods, grades larvae from the May cruise in consistently poor condition.

Ćomplementary findings are summarised by Lopez-Jamar et al. (in press) for measure-ments of larval condition on the same cruises from carbon, RNA/DNA and enzymeanalyses. However, observations on the birth-date distribution of sardine juveniles takenduring the summer and autumn of 1992 at the main nursery grounds off Galicia foundnegligible survival of larvae from spawning between March and June, i.e., the entireperiod during which measures of larval condition were taken. The majority of survivinglarvae were from January birth-dates, before the time at which spawning commencesalong the north coast of Spain; the conclusion was that the surviving juveniles had

´recruited from spawning in Portuguese waters further to the south (Lopez-Jamar et al., inpress). It is possible that larvae from spawning along the north coast were recruited asjuveniles elsewhere than off Galicia, but neither oceanographic nor information fromjuvenile surveys suggests that this takes place generally (Robles et al., 1991; Porteiro etal., 1993).

Thus, although relatively consistent measures of larval condition were derived, whichmight relate to some index of survival at the time the larvae were sampled, it wouldappear that subsequent mortality obscured any value of these indices for prediction ofjuvenile recruitment. One possible source of such mortality, in the particular situation ofsardine spawning along the north coast of Spain, is the effect of upwelling and offshoretransport in the Finesterre /Cape Ortegal region (Chesney and Alonso-Noval, 1989).Larvae are required to traverse this region to pass from the spawning areas along thenorth coast to the juvenile nursery areas off Galicia (Chesney and Alonso-Noval, 1989;Cabanas et al., 1992). The offshore directed tail of larval distribution north of CapeOrtegal (Fig. 1a) does gives some indication that offshore transport associated withupwelling, characteristic of that region in the summer months (Chesney and Alonso-

Ńoval, 1989; Lopez-Jamar et al., in press), may have resulted in the failure of larvaefrom the north coast to reach the nursery grounds off Galicia in 1992. In other years,starvation mortality, as suggested, may have occurred for many of the larvae during theMay cruise, thus having a more dominant effect on juvenile recruitment strength.

Acknowledgments

This work forms part of the programme of the Strategic Research Project 1(Productivity and Physical Structure In Pelagic Ecosystems) of the Plymouth MarineLaboratory, a component institute of the UK Natural Environment Research Council.Finance was provided, in part, by the Commission of the European Communities (DGXIV) under contract M.A. 1. 96. and by the UK Department of the Environment (DOE)under contract PECD 7/7/359. The authors gratefully acknowledge the assistance of theofficers, crew and all participants on the cruises.


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Histological indices of the nutritional condition of sardine, Sardina pilchardus (Walbaum) larvae off the north coast of Spain