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ICES WKIDCLUP REPORT 2014 SCICOM STEERING GROUP ON ECOSYSTEM SURVEYS SCIENCE AND TECHNOLOGY

ICES CM 2014/SSGESST:04

Report of the Workshop on the identification of clupeoid larvae (WKIDCLUP)

1-5 September 2014

Hamburg, Germany

International Council for the Exploration of the Sea Conseil International pour l’Exploration de la Mer

H. C. Andersens Boulevard 44–46 DK-1553 Copenhagen V Denmark Telephone (+45) 33 38 67 00 Telefax (+45) 33 93 42 15 www.ices.dk [email protected]

Recommended format for purposes of citation:

ICES. 2014. Report of the Workshop on the identification of clupeoid larvae (WKIDCLUP), 1-5 September 2014, Hamburg, Germany. ICES CM 2014/SSGESST:04. 36 pp.

For permission to reproduce material from this publication, please apply to the Gen-eral Secretary.

The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council.

© 2014 International Council for the Exploration of the Sea

ICES WKIDCLUP REPORT 2014 | i

Contents

Executive summary ................................................................................................................ 1

1 Opening of the meeting ................................................................................................ 2

2 Adoption of the agenda ................................................................................................ 2

3 Clupeid Larvae identification and description ........................................................ 3

3.1 Herring Clupea harengus .................................................................................... 6

3.2 Sprat Sprattus sprattus ....................................................................................... 10

3.3 Sardine Sardine pilchardus ............................................................................... 12 3.4 Anchovy Engraulis encrasicolus....................................................................... 15

3.5 Sardinella Sardinella aurita and S. maderensis .............................................. 17

4 Larvae identification results ...................................................................................... 19

4.1 Methods for larvae identification trials ........................................................... 19 4.2 Results of larvae identification trials ................................................................ 19

4.3 Sources of misidentification of larvae .............................................................. 23

5 Preservation methods of fish larvae ......................................................................... 24

6 References ..................................................................................................................... 28

Annex 1: List of participants............................................................................................... 32

Annex 2: Agenda ................................................................................................................... 34

Annex 3: Recommendations ............................................................................................... 36

ii | ICES WKIDCLUP REPORT 2014

WKIDCLUP in Hamburg, Germany, 1-5 September 2014

Maik Tiedemann Dorothee

Moll

Ineke Pennock

Jérémy Denis

Bastian Huwer

Lynette Ritchie

Andreis Makarchouck

Enda O’Calla-ghan

Marie Leiditz

Christophe Loots

Sakis Kroupis

Elisabete Hen-riques

Cindy van Damme

Birgit Suer

Anne-Marie Palmén Bratt

Matthias Kloppmann

ICES WKIDCLUP REPORT 2014 | 1

Executive summary

The Workshop on the Identification of Clupeoid larvae (WKIDCLUP) met from 1 to 5 Sep-tember 2014 in Hamburg, Germany, to calibrate clupeoid larvae identification. The meeting was chaired by Cindy van Damme, The Netherlands, and Matthias Kloppmann, Germany. In total 18 persons, representing 11 institutes from nine countries participated in the work-shop.

The majority of the time at the workshop was spent identifying fish larvae. The results promoted discussion and highlighted specific problem areas. These discussions led to the further development of standard keys and larval identification characteristic tables. There was a slight increase to 56% in the overall agreement in the larval identification. However, there was a decrease in herring larvae agreement, from 68% to 66% from the 1st to the 2nd round. Also for sardine larvae, the percentage agreement decreased, from 37% to 19%. For sprat and anchovy larvae percentage agreement increased, from 62% to 67% and 27% to 51% respectively. However, despite these results, it was encouraging to note that in the 1st round participants were mostly only using myotomes counts and thus confusing all larval species, while in the second round also developmental characteristics at length were taken into account and only herring and sardine were confused. In results of the 2nd round were probably due to the low quality of the available larvae and the rapid deterioration. For sprat, sardine and anchovy only limited numbers of larvae were available for the workshop and most of these were badly preserved.

From the results, it became clear that the more experienced clupeoid larvae identifiers showed high agreement with the actual species. Thus, it is crucial for institutes to ensure a continuity of staff and constant training for the larval surveys and identification to guaran-tee and improve the quality of the data derived from these surveys.

However, the results from the larval identification trials clearly show that there are still some uncertainties in the identification of clupeoid larvae.

For herring larval indices are used for the assessment of the spawning stock. Thus, the cor-rect identification of the clupeoid larvae is important for the management of these stocks. The results of this workshop show that there is still room for improvement in herring larval identification. Clupeoid larval identification workshop should be conducted regularly (i.e. every 5 years) to increase agreement in identification as quality assurance of the larval iden-tification of the surveys used in the assessments.

Different preservation methods are available for the preservation of fish larvae. Partici-pants agreed that it is most important that fish larvae are put in preservative immediately after the catch in order to preserve the larval characteristics. Formaldehyde based preserv-atives create lower shrinkage in larvae compared to ethanol solutions.

2 | Report of the Workshop on the identification of clupeoid larvae (WKIDCLUP)

1 Opening of the meeting

The meeting started at Monday 1 September 2014. In total, 11 institutes were represented from nine countries (see Table 1.1). In total, 18 participants joined the meeting (Annex 1).

Table 1.1. Represented countries and institutes during WKIDFL 2011.

COUNTRY INSTITUTE

D e nm a r k D T U- Aq u a

Fr a nc e I f r e m e r

Ge r m a ny T I ( H a m bu r g a nd Ro s to c k) , I H F

La tv i a BI O R

N e the r l a nds I MARE S

P o r tu ga l I P MA

Swe de n I MR

UK - N o r the r n I r e l a nd AFBI

UK - Sc o t l a nd MS

2 Adoption of the agenda

The terms of reference for this meeting were:

a ) Carry out comparative larvae identification trials following the pattern of trial – analysis – retrial;

b ) Review available information on the identification of clupeoid larvae on the Northeast Atlantic Shelf, under special consideration of larvae’s appearance with ongoing development;

c ) Identify sources of misidentification of larvae; d ) Standardize sample processing and data analysis of clupeoid larvae surveys.

An agenda was sent round prior to the workshop. The adopted agenda can be found in Annex 2.


3 Clupeid Larvae identification and description

WKIDCLUP compiled an overview of the reference literature used and the characteristics for identification of the different clupeoid larvae. The most used references for identifica-tion of fish larvae in the Northeastern Atlantic and Mediterranean are: (Ehrenbaum, 1909; Russell, 1976; Fahay, 1983; Moser et al., 1984; Munk and Nielsen, 2005).

Before identification of fish larvae, some background information is needed. To get ac-quainted to major fish larvae description literature, the introduction chapter of Russell (1976) gives all the background information on larvae characteristics and different devel-opment stages and should be read by anyone who wants to identify fish larvae. In addition, information on the timing of sampling and the area where samples were collected should be available. Based on this, larval identification information on the possible fish species spawning at the sampling time in the area should be collected.

A fish larva is the active immature form of a fish that differs greatly from the adult and is the stage between egg and metamorphosis. Larvae are the stage from hatching to attain-ment of characters that allow for identification according to descriptions of adult speci-mens. The larval stage is characterized by progressive changes throughout its duration:

• Organs develop and become functional • Pigmentation changes and becomes stronger • Fins develop and often change position. Most conspicuous is the development

of the caudal fin with flexion of the urostyle.

The above characteristics can and should all be used for the species identification of fish larvae.

The characteristics change with the different development stages of the larvae. The yolk sac stage is the transitional stage between the egg and larval stage. The larvae lack functional eyes and mouth and the fins are not developed. The characteristics known from the eggs are retained during this stage (e.g. yolk segmentation, oil globule). During this stage the characteristic pigmentation develops. Yolk-sac larvae from demersal eggs generally hatch at a further advanced development stage compared to larvae from pelagic eggs.

Most fish larvae live and have to adapt to a completely different environment compared to their adult conspecifics. They develop typical larval characters that can be used for identi-fication. Several larval characters, which need to be utilized for identification, are:

• Body shape • Fins and fin fold • Eyes • Spines • Fin rays and fin ray counts • Body proportions • Myotome counts • Pigmentation patterns

Four major groups can be identified from the shape of the body:

• Long, slender and elongated: clupeoids • Laterally compressed and dorsal-ventrally high: flatfish • More typically fish like forms: gadoids


• Some conspicuously aberrant forms: others

Clupeoid larvae general characteristics:

• Tubular shape of body • Number of myotomes in the trunk • The body proportion changes during development thus the anus moves forward

and the myotome count decreases with age • The difference between clupeoids and sandeel is the position of the anus. In

sandeel the anus is halfway the body, in clupeoids the guts is much longer and the anus is positioned close to the tail.

Table 3.1. Primary characteristics of clupeoids (from (Russell, 1976).

DEVELOPMENT

STAGE (TOTAL

LENGTH)

HERRING SPRAT PILCHARD/SARDINE ANCHOVY

Yolk sac Yolk not segmented

Yolk segmented Yolk segmented Yolk segmented, oblong shape

< 10 mm

No. myotomes in trunk

47 37 41–42

10–20 mm


46–47 35–37 41–42

Position pelvic fin Not appeared yet

Appears at 17.5–20 mm, 4–5 myotomes behind the pylorus

Appear at 18–20 mm, level with the pylorus

Dorsal fin Rear edge of dorsal fin overlaps with the anal fin

20–40 mm


41–46 31–35 36–41

Position pelvic fin 7–8 myotomes behind the pylorus

4–5 myotomes behind the pylorus

Level with the pylorus

Length of tail from anus to base of caudal fin

Greater than 6 times in total length

Less than 6 times in total length

Secondary characteristics:

Herring is always bigger at any developmental stage compared to the other species. Her-ring have pigmented eyes at hatching while other species do not gain pigment until later (5mm). Herring attain flexion stage later (17 mm) than other species so larvae at 11–13 mm with flexion will not be herring (Munk and Nielsen, 2005). The head of anchovy is bigger


compared to the other species (head measurements are however not very useful to separate the species).

Note: In southern Iberia also the clupeoids Sardinella aurita and S. maderensis can be found.

It is important that myotome counting is done correctly (i.e. start and end are well identi-fied). For myotome count in the larvae trunk (from the back of the head to anus), see Figures 3.1 to 3.3 (read also description in Russell, 1976).

Figure 3.1. Myotome counting in clupeoids: Start and endpoint of number of myotomes in the trunk.

Figure 3.2. Myotome counting in clupeoids: The first myotome after the head.


Figure 3.3. Myotome counting in clupeoids: The last myotome at the anus.

3.1 Herring Clupea harengus

Distribution of adults

Herring is a comparatively large pelagic and planktivorous clupeoid species with a Sub-arctic/boreal to temperate distribution pattern. On the eastern side of the North Atlantic, Herring occurs between the Barents Sea in the Northeast and the Bay of Biscay in the South. It is divided into a number of stocks with specific spawning sites and spawning time. It ranges from Iceland and southern Greenland southward to the northern Bay of Biscay and eastward to Spitsbergen and Novaya Zemlya in Russia, including the North Sea and Baltic Sea (Whitehead et al., 1985). In the western North Atlantic, herring occurs from southwest-ern Greenland and Labrador southward to South Carolina, USA.


Figure 3.1.2. Spatial distribution of herring stocks in the Northeastern Atlantic (von Dorrien et al., 2013).

Temporal distribution of spawning

Herring spawn at almost any time of the year, and there is possibly no month of the year at which none of the different herring stocks is spawning (see Russell 1976, Sinclair 1988). The spawning seasons of the major herring stocks is summarized in Table 3.1.1.


Table 3.1.1. The spawning seasons of different herring stocks (References: Jakobsson et al., 1969; Sinclair 1988; Holst et al., 2004; modified and extended pers. comm. by Enda O’Callaghan, Birgit Suer, Dorothee Moll).

Area Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

English Channel

North Sea

Norwegian Coast

Iceland

Buchan / Shetland

Central North Sea

Clyde Sea

Irish Sea

Celtic Sea

West of Ireland

Western

Baltic Sea

Central Baltic

Bothnian Sea

Recent spawning in the Baltic Sea:

During recent years (2011–2013), herring larvae have been observed during ichthyoplank-ton surveys conducted in the Bornholm Basin in November, which may indicate increased spawning activity and an increased stock size of the autumn spawning herring (Bastian Huwer, pers. comm.). Preliminary results show that larval abundance increased in the vi-cinity of Bornholm/Christiansø, while larval sizes are decreasing, indicating that larvae are


hatching in the vicinity of Bornholm Island. DTU Aqua is planning to conduct genetic anal-yses on these larvae to determine which stock component they belong to, and is planning to conduct a more detailed investigation of the spawning time and area during two research cruises in September and November 2015.

Eggs

• demersal eggs attached to substrates like gravel, broken shells or submerged aquatic vegetation (Baltic Sea)

• 0.9–1.5 mm in diameter, size is variable, depending on time of spawning and spawning groups

Larvae

Primary characteristics:

• newly hatched larvae: length 5–9 mm

• dorsal fin is developing at: 12 mm and is complete at 28–29 mm

• anal fin is developing at: 16 mm

• pelvic fin appears at: 22–23 mm

• notochord is straight at: 17 mm – 18 mm and completely turned up at 21 mm

• position of pelvic fin is 7–8 myotomes behind the pylorus

• the pylorus is found at myosept 21

• herring has, compared to sprat and sardines, the largest number of myotomes in the trunk. Number of myotomes in trunk: <10 mm: 47 myotomes 10–20 mm: 47–47 myotomes 20–40 mm: 41–46 myotomes

Secondary characteristics:

• until development of pelvic fins, herring larvae are always larger at any develop-mental stage than other clupeoid species

• eyes are fully pigmented at hatching, other clupeoid larvae hatch with unpig-mented eyes

• yolk sac is totally absorbed at a body length of 9–12 mm depending on stock

• 19 fin rays in the dorsal fin (17–21 fin rays)

• 17 fin rays in the anal fin (15–19 variation)

• 7–8 myotomes between the anal fin and the last ray of the dorsal fin

• the hindmost fin ray in the dorsal fin is formed at 18–19 mm


Figure 3.1.1. Developmental stages of herring larvae (from Russell, 1976)

3.2 Sprat Sprattus sprattus

Distribution of adults

The European sprat is a small planktivorous pelagic clupeoid species with a wide distribu-tion in shelf areas of the Northeast Atlantic, covering the coasts of Norway, the North Sea, Irish Sea, Bay of Biscay, the western coast of the Iberian peninsula down to Morocco, the northern parts of the Mediterranean, the Black Sea, and the Baltic Sea (see Haslob, 2011 and references therein). Sprat is able to tolerate salinities as low as 4 psu and especially juveniles are known to enter estuaries (Whitehead, 1985). In the Baltic Sea, sprat is located at its northern limit of geographic distribution (Muus and Nielsen, 1999). It is distributed throughout the western and eastern parts of the Baltic, up to the Gulf of Finland in the north.

Spawning

As many other clupeoid fish, sprat is an indeterminate batch spawner, i.e. it has indetermi-nate oocyte recruitment and is releasing several batches of pelagic eggs over a prolonged spawning season, and intra- and interannual variability is expected in spawning season length, batch fecundity, and batch frequency in all regions (Heidrich, 1925; Alheit, 1988). Based upon the timing of spawning at different latitudes, spawning occurs between 6 and 15 C (Peck et al., 2011). In northern European waters (North and Baltic Seas), spawning occurs from January to August with peaks in spring and early summer (e.g. Petrova, 1960; Wahl and Alheit, 1988; Munk and Nielsen 2005; Haslob et al., 2012; Voss et al., 2011) when water temperatures are commonly between 8 and 15 C. In southern European waters (Adri-atic Sea), sprat generally spawns during the cooler time of the year (October-April) with peak spawning in winter (November to December) at water temperatures between 9 and 14 C (Dulĉić, 1998). In all regions, however, the onset and duration of spawning may vary


due to temperature and feeding conditions. See Table 3.3.1 for a more detailed overview of spawning times in different areas.

Sprat spawns pelagic eggs that are buoyant at different water depths in different systems due to salinity effects on ambient density. In marine waters such as the North and Medi-terranean Seas, eggs remain in surface layers but in the Baltic, eggs sink below the low salinity (5–7 psu) surface waters through the thermocline to the halocline (6–15 psu) located at intermediate water depths of 30–60 m (Wieland and Zuzarte, 1991). Due to this particular hydrographic situation in the Baltic with strong vertical stratification of salinity and tem-perature, the main spawning areas are located in the deeper areas of the central Baltic, i.e. the Arkona Basin, Bornholm Basin, the Gdańsk Deep and the Gotland Basin (e.g. Aro, 1989; Parmanne et al., 1994; Ojaveer and Kalejs 2010; Köster et al., 2003). However, spawning is also observed in the Western Baltic, e.g. in the Kiel Bight, but a detailed mapping of spawn-ing areas in this region is lacking (Haslob pers. comm., Heidrich, 1925). In the most north-ern parts of the Baltic, sprat spawning occurs and sprat eggs can be found in the plankton, but no larvae (Sjöblom and Parmanne, 1980). In the central Baltic, spawning has been ob-served from February to August with a peak in spring, but differences in spawning time are possible due to temperature, salinity and potentially feeding conditions for adults (e.g. Haslob 2012; Voss et al., 2011; Ojaveer and Kalejs, 2010; Wahl and Alheit, 1988; Petrova, 1960). In 2002, a second spawning event was observed in autumn, which was explained by the inflow of unusual warm-water masses into the central Baltic (Kraus et al., 2003).

In other areas outside the Baltic, sprat eggs can be observed in almost all areas where adult sprat are distributed (Milligan, 1986), but areas with high concentrations of spawning adults are e.g. found in the inner German Bight, the English Channel, the southern North Sea, northeast of England, north and west of Scotland, as well as in Skagerrak and Kattegat (Knijn et al., 1993; Bailey and Braes 1976; Torstensen and Gjøsæter 1995; Worsøe et al., 2002; Warnar et al., 2011).

NOTE: INFORMATION ON SPAWNING AREAS IN OTHER REGIONS MISSING: Irish Sea, Bay of Biscay, the western coast of the Iberian peninsula down to Morocco, the north-ern parts of the Mediterranean, the Black Sea.

Eggs and larvae

Egg characteristics for sprat are given in Russell (1976), who describes the eggs as pelagic, large, spherical, without large perivitelline space, without oil globule, with segmented yolk and 0.8–1.3 mm in diameter. The sprat eggs come within the size range of many other fish, but are immediately recognizable by the segmented yolk which does not occur in eggs of comparable size without oil globules of other fish.

Ré and Meneses (2009) provide the following information on sprat larvae: Hatching length - 3.0–3.6 mm; Yolk-sac absorption - 5.0–6.0 mm; Flexion length - 11 mm; Transformation length - 32–41 mm; Pigmentation - yolk-sac: small scattered melanophores in head and dor-sal region (visible in the embryo).

Diagnostic features - newly hatched larva tube-like (typical clupeid form). Prominent sense organs (6) on each side of the body. Pigmented eyes at the end of yolk-sac absorption. Dor-sal fin formation (28th myomere) at 8 mm. Formation of pelvic fins 4 to 5 myomeres behind pylorus 17–20 mm. Number of preanal myomeres 35–37. Tail length less the six times into total length

Ehrenbaum (1936) reports a size at hatching of ca. 4 mm, with larvae being less developed than herring, and expressing weak pigmentation and no pigmented eyes at hatch. He fur-ther reports the period until yolk-sac absorption to be ca. 8 days, during which the larva


grows to ca. 5 mm. At 13–15 mm all fins are developed except for the pelvic fins which develop at ca. 18 mm. Metamorphosis (silvery appearance) occurs at 25 mm.

Laboratory studies on Baltic sprat revealed standard length (SL)-at-hatch was 3.3–3.5 mm and relatively similar at all temperatures <17 °C as was the SL at yolk-sac absorption (4.9–5.6-mm SL; Alshut, 1988b; Kanstinger, 2007; Petereit et al., 2008). Depending upon water temperature, sprat eye pigmentation occurs between 3 and 16 d post-hatch (dph) and jaw development and mouth opening occur ca. 48 and 72 h later (Nissling, 2004; Kanstinger, 2007). At constant temperatures between 5 and 13°C, the combined data of four studies on eggs and yolk-sac larvae (Thompson et al., 1981; Nissling, 2004; Kanstinger, 2007; Petereit et al., 2008) indicated that the duration of the endogenous feeding period is 135 ± 3 degree-days (°C d) after which the larva is ca. 5.5 mm SL and must initiate feeding. For Baltic Sea sprat, Peck et al. (2011) defined six life stages or life-history events that occur after the egg and yolk-sac larval phases, based on changes in growth allocation between mass and length and inferences from field observations: (i) exogenously feeding but non-schooling larvae from 5 to 14 mm SL, (ii) likely onset of schooling behaviour from 14 to 18 mm SL, (iii) a ‘‘transitional-larval’’ life stage from 18 to 35 mm SL, (iv) a period of late-larval/juvenile metamorphosis occurring at 35 to 55 mm SL, (v) a juvenile growth phase from 55 to 90 mm SL, and (vi) adult fish that exhibit seasonal energy allocation to somatic and gonadal growth starting at 100 mm SL.

Figure 3.2.1 Developmental stages of sprat larvae (from Russell 1976)

See Table 3.1 for an overview of larvae characteristics.

3.3 Sardine Sardine pilchardus

Adult characteristics and biology

Sardine is a small pelagic species characterized by an elongate and compressed body with large silvery scales of which about 30 can be counted in the lateral line. The mouth does not reach the posterior edge of the eye and the gill cover has radial striations. The pelvic fins are placed posterior to the origin of the dorsal fin.


It reaches 25cm and 15 years of age, lives pelagically in large schools and feeds mainly on zooplankton. It has an atlanto-mediterranean distribution pattern and ranges in the North-east Atlantic from Iceland (where it is rare) south to Senegal. It is common in the Mediter-ranean and the Adriatic Sea, the Sea of Marmara and the Black Sea (Parrish et al., 1989).

Its habitat is coastal and pelagic at 25 – 50m by day and 15 - 35m by night. It is shoaling and migratory. Sexual maturity is attained 2 – 3 years of age and spawning takes place in June to august in the southernmost North Sea.

Life History

The sardine spawns throughout the year off the Iberian Peninsula with peak periods during spring and autumn/early winter. Where it spawns in the North Sea it is further offshore as it requires lower temperatures and higher salinities (Alheit et al., 2007; Bils et al., 2012) peak-ing in late June, early July.

Table 3.3.1. The major spawning months for sardine in European waters.

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec North Sea English channel

Bay of Biscay

Iberia Mediter-ranean

Adriatic Sea

Description of the larval stages

Figure 3.3.1 Yolk-sack larva of S. pilchardus. From Ré and Meneses 2009.

• Hatching length 3.2 - 4.0 mm

• Newly hatched larva tube-like (typical clupeid form)

• Easily distinguishable from other clupeids by the presence of an oil globule in the yolk sac

• Yolk-sac absorption at 4.0 - 5.5 mm

• Mouth and jaws undeveloped and un-pigmented eyes at hatching


Primary identifying characteristics in larvae less than 20mm

• At 10mm there are 41–42 pre-anal (back of head to anus) myotomes compared to 47 in herring and 37 in sprat.

• Between 10 – 20 mm there are still 41 – 42 myotomes in the trunk compared to 47 in herring and 35–37 in sprat.

Figure 3.3.2 Larva of S. pilchardus. From Ré and Meneses, 2009.

Secondary (informative) characteristics in larvae less than 20 mm

• Notochord flexion occurs at 11 - 12.5 mm • Dorsal fin formation (31 myotomes) 7.5 mm • Typical larval pigmentation develops around 5 - 6 mm • Gas bladder formation at 10 mm

Primary Characteristics in larvae greater than 20mm

• Number of pre-anal myotomes: 41 reducing to 36 as larvae develops • Formation of pelvic fins (level with pylorus) at 20–22 mm

Figure 3.3.3. Late larva of S. pilchardus. From Ré and Meneses 2009.

Secondary characteristics (informative) in larvae greater than 20 mm

• Complete dorsal fin formation at 26 mm • Complete anal fin formation at 28 mm • Transformation length at 40–50 mm


3.4 Anchovy Engraulis encrasicolus

Figure 3.4.1. Geographical Distribution of adult Anchovy (FAO 2014).

Adult characteristics and biology

The Anchovy (Engraulis encrasicolus) is a small coastal marine fish species that forms large schools and feeds predominantly on zooplankton. It tolerates a large range of salinities of S = 5–41 and in some areas enters estuaries and lagoons, especially during spawning. Tends to move further north and into surface waters in summer, retreating and descending in winter. Temperature is the major factor influencing the distribution of the thermophilic anchovy.

Geographical Distribution

Anchovy occur in the eastern Atlantic between Bergen, Norway and Angola. It also occurs in the Mediterranean, the Black and Azov Seas (Figure 3.4.1). European Anchovy were also confirmed to occur in the Baltic, in the Bornholm Basin and the waters of the Gulf of Gdańsk (to the east of 18°20’E; Draganik and Wyszynski, 2004). [Anchovy were found even further east during the Baltic-Survey (BITS)].

Spawning season

Spawns from March to November with peaks usually in summer. Eggs are mainly found in warm waters, between 17 and 23°C (Palomera et al., 2007). North Sea spawning period from June until the end of September (Munk and Nielsen, 2005).

Anchovy are oviparous, and lay ellipsoid planktonic eggs.


Description of eggs and larvae

Figure 3.4.1 Egg and larval stages of anchovy (Engraulis encrasicolus) from Russell (1976).

Eggs

• Ellipsoid – therefore anchovy eggs can be almost immediately told from other fish eggs.

• Segmented yolk

Yolk-sac larvae

• Elongated yolk sac which stretches nearly to the anus

• Eyes are not pigmented until yolk is absorbed at a length of about 5 mm

Preflexion stage

• Head more than five times into total length, dorsal fin opposite to anus.

• The earliest stage E.encrasicolus is distinguishable from S.pilchardus, S. sprattus, C. harengus and A. fallax by the different pigmentation (a few groups of melanophores vs. rows of melanophores).

Post Flexion Stage

• The head has a characteristic rounded shape, and the lower jaw ends behind the upper jaw.

• The length of the tail is one-third of total length, and the dorsal and anal fins are overlapping. There are 46–48 vertebrae.

• Pigmentation does not appear to be quite typical.


Obvious distinguishing characters

• The posterior position of the dorsal fin overlapping the anal fin. • The long head which at this stage is about one-fifth of the body length. • The early appearance of the swimbladder (at around 11 mm TL). • The short gut and longer tail relative to all other clupeoid larvae of the area.

The characteristic lengths of the different developmental stage of anchovy are given in Ta-ble 3.4.1.

Table 3.4.1. Typical total lengths (TL) at the various developmental stages of anchovy larvae.

HATCHING LENGTH 3.0–4.0 MM

Yolk-sac absorption 5.0 mm

Dorsal fin starts development 6.0 mm

Urochord flexion 9.0–10.0 mm

Caudal and anal fin development starts 9.0mm

Swimbladder develops 11 mm

Pelvic fin development level of pylorus 15 mm

Transformation length around 25 mm

3.5 Sardinella Sardinella aurita and S. maderensis

Both sardinella species, Sardinella aurita and S. maderensis, are small pelagic fish with a broadly subtropical distribution pattern. They occur chiefly in the southern Mediterranean and in the waters west of Africa. Larvae are, therefore, more or less unlikely to occur in significant numbers in European waters. However, with ongoing climate warming, it may become possible that also larvae of these species become more abundant in European wa-ters, particularly around the Iberian Peninsula and in the northern Mediterranean. Brief summaries of descriptions of the two species are, therefore, given below.

Figure 3.5.1 Sardinella aurita larvae description from Ditty et al. 1994.


Egg and Larvae Description (from Fahay, 1983)

Eggs

• Pelagic, spherical • Diameter: 0.94–1.40 mm • Shell: Smooth, transparent and thin • Yolk: segmented 0.59 mm (0.54 – 0.62 mm) • Perivitelline space: moderate • Oil globules: 1 of 0.12–0.18 mm diameter

Larvae

• Body elongate with long straight gut • Vent posterior to dorsal fin • Dorsal pigment absent from notochord tip, but ventral pigment present • Pre-anal myomeres 37 – 41 (mainly 39 – 40) best characterist

from Sardinella maderensis (pre-anal myomeres mainly 37–38) - [for 5 – 15 mm TL specimens]

• Pelvic fins occur at about 13 – 14 mm TL, 2–3 myomeres behind the pylorus • Last 2 anal rays become elongate in larger larvae

Meristics

• Myomeres total: 45 - 48 • Pre-anal myomeres:

• At TL 5 – 10 mm: 40 - 39 • At TL 11 – 16 mm: 39 - 37

• Dorsal fin rays: (15) 16 - 19 (20) • Anal fins rays: (14) 16 - 17 (19) • Pelvic fin rays: 8 - 10

Table 3.5.1. Sardinella larvae development stages at total length.

HATCHING LENGTH 2.5 - 3 MM

Flexion starts at about 7.5 - 11 mm TL,

Metamorphosis about 16 - 25 mm TL


4 Larvae identification results

4.1 Methods for larvae identification trials

Trays containing 6 specimens of different fish larvae were used for the larvae identification exercises. There were 14 microscopes available for this workshop, all were identical and all contained eyepiece graticules for larval measurements. Each microscope had a transmitted and top light. No polarization filters were installed on the microscopes. As each participant moved from microscope to microscope they were asked to provide a species identification for each larva (herring, sprat, sardine, anchovy plus an “other” category). The ‘other’ cate-gory included the clupeoid sardinella, which might co-occur in the Mediterranean and off the coast of southern Spain and Portugal, but also other groups, like sandeel, which occur in the same samples from the larval surveys in the northern European areas. During the first trial round there were 2 trays with fish larvae per each of 14 microscopes while during the second trial round only 1 tray per each of 14 microscopes were examined. Because of the different levels of expertise in fish larvae identification not all participants were able to identify the major part of the larvae during the first trial. Therefore it was decided to reduce the number of specimens during the second round.

Descriptions of the major characteristics of the clupeoids were provided during an intro-ductory presentation. During the trials participants were allowed to utilize own as well as provided identification literature.

The results of the first round of larvae identifications were collated and input into spread-sheets. The results were presented and some larvae of all 4 species were displayed on a large screen and discussed in the group. Major characteristics of those specimens were thor-oughly discussed.

4.2 Results of larvae identification trials

The original assessment of species identification for each larva, by each participant, was put into a primary result table (not presented here). Once the results were available from every participant these were analysed. The results were compared with the validated or predetermined (identifications done by experts) species. For herring, some larvae were available from rearing experiments. Those larvae were, therefore, validated. For the other specimens only predetermined identifications were available, but these were considered to be correct.

Summaries of the results from the both rounds on clupeoid larval species determination are presented in Tables 4.1 and 4.2. Each of these tables is divided into three sub-tables labelled A-D, where the performance of each participant is judged against the actual correct species identification.

Sub-table A shows the number of larvae at each species that were assessed by each partic-ipant (i.e. the number of larvae which the participant should actually have found per spe-cies). The numbers at each species will therefore be the same for all participants that read all the larvae.

Sub-table B shows the numbers of larvae of each species as actually assessed by each par-ticipant.

Sub-table C shows the over- or underestimation of each participant per species.

Sub-table D shows the percentage agreement in species identification between the assess-ment of each participant and the actual species.


The results show a slight improvement in the allocation of larvae to the correctly deter-mined species, from the first to the second round of analysis. They also highlight the diffi-culties in being able to positively identify larvae where there are only few distinguishing features or myotome counting was limited.

During the first round of identification, larvae with low agreement were assigned to all possible species. Consequently, there was some discussion on the features which aided fish larvae identification. Larvae of all clupeoid species were shown on a big screen and identi-fication characteristics were shown. Some references and criteria were produced (see sec-tion 3) to help with the identification. Larval features change with size and after the discussion; it became clear that not all participants were measuring the larval length, alt-hough eyepiece graticules were available in all microscopes.

Analysis of the second round results showed that overall agreement increased from 55% to 56%. For herring larvae, the percentage agreement decreased from 68% to 66%. Also for sardine larvae, the percentage agreement decreased from 37% to 19%. For sprat and an-chovy larvae percentage agreement increased, from 62% to 67% and 27% to 51% respec-tively, from the first to the second round. At the individual larva level, it was clear that participants were confusing herring and sardine larvae, both small and larger larvae. How-ever, these species should be easily distinguished from each other since until appearance of the pelvic fins, development occurs earlier (at shorter length) in sardine than in herring (see section 3). In addition, the quality of almost all larvae was less than during the first round, and it was often difficult to see whether fins were present. The guts were often partly to completely detached from the trunk, making it difficult to distinguish the pylorus, let alone counting pre-anal myotomes. This drawback was partly due to the small number of larval samples in sardine, sprat and in particular anchovy. Clupeoid larvae are rather deli-cate, particularly in the earlier stages, and very prone to damage already during catch. In-appropriate treatment like e.g. delayed preservation or imposed mechanical stress may further deteriorate the larvae. Therefore, the proportion of damaged specimens is always higher in clupeoid than in other fish larvae. Given the rather limited amount of sample material for the course, it was inevitable that poor quality larvae had to be used.

However, despite that poor quality of the larvae during the 2nd round, the observed dis-crepancies in clupeoid larvae identification between determined and actual species clearly show that there still are some uncertainties in their identification. Results also showed that experts in larval identification had a higher agreement with the actual species compared to less experienced participants. This shows that constant training is imperative to maintain a high quality in fish larvae identification but also underscores the importance for institutes to ensure continuity of staff involved in the ichthyoplankton surveys.


Table 4.1. Species identification 1st round. The species compositions based on actual species reflecting the best estimates based on only those larvae that were used for species identification by the participant (A), the species compositions as obtained per participant (B), the percentage over- or underestimation (C) and the percentages agreement with actual species (D) are shown per species by participant and for the whole group that took part in the species identification exercise on fish larvae. A weighted mean percent agreement is given by person and all persons combined.

AReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 TOTAL

Herr ing 1 71 71 65 72 72 70 39 71 66 71 54 39 71 72 71 27 1002Sprat 2 51 51 40 51 51 51 28 51 40 51 29 21 51 51 50 28 695

Sardine 3 26 24 19 26 26 26 15 26 24 26 15 5 26 26 26 15 351Anchovy 4 10 10 5 10 9 10 6 10 10 6 5 1 10 10 9 1 122

Other 5 9 9 9 8 9 8 3 9 9 9 3 1 9 9 9 4 117Total 1-4 167 165 138 167 167 165 91 167 149 163 106 67 167 168 165 75 2287

BSpecies Reader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 TOTAL

Herr ing 1 72 30 45 83 52 66 44 97 47 53 68 24 77 86 98 26 968Sprat 2 43 89 43 41 45 50 31 21 80 48 30 15 50 62 21 27 696

Sardine 3 19 40 25 32 59 34 10 30 17 48 5 17 36 11 42 18 443Anchovy 4 8 2 3 2 2 6 2 16 1 5 - 10 3 8 2 2 72

Other 5 25 4 22 9 9 9 4 3 4 9 3 1 1 1 2 2 108Total 1-4 142 161 116 158 158 156 87 164 145 154 103 66 166 167 163 73 2179

CReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 ALL

Herr ing 1 1% -58% -31% 15% -28% -6% 13% 37% -29% -25% 26% -38% 8% 19% 38% -4% -3%Sprat 2 -16% 75% 8% -20% -12% -2% 11% -59% 100% -6% 3% -29% -2% 22% -58% -4% 0%

Sardine 3 -27% 67% 32% 23% 127% 31% -33% 15% -29% 85% -67% 240% 38% -58% 62% 20% 26%Anchovy 4 -20% -80% -40% -80% -78% -40% -67% 60% -90% -17% 900% -70% -20% -78% 100% -41%

Other 5 178% -56% 144% 13% 0% 13% 33% -67% -56% 0% 0% 0% -89% -89% -78% -50% -8%

DReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 ALL

Herr ing 1 99% 37% 49% 71% 60% 86% 100% 63% 50% 59% 83% 49% 62% 74% 82% 63% 68%Sprat 2 76% 78% 80% 61% 67% 86% 93% 14% 80% 59% 62% 19% 43% 71% 24% 75% 62%

Sardine 3 58% 21% 47% 19% 73% 62% 53% 23% 13% 42% 13% 40% 23% 27% 46% 33% 37%Anchovy 4 80% 20% 0% 20% 0% 60% 33% 40% 0% 17% 0% 0% 10% 60% 0% 100% 27%

1-4 79.0% 44.2% 52.9% 53.3% 57.5% 76.4% 82.4% 37.1% 45.6% 51.5% 61.3% 37.3% 43.7% 60.7% 49.7% 58.7%RANKING 2 13 9 8 7 3 1 16 12 10 4 15 14 5 11 6

Species compositions using actual speciesActual species

55.5%

Percentage overestimation / underestimation

Species compositions as estimated per participant and whole group

Actual species

Actual species

Percentage agreement in species identification per species


Table 4.2. Species identification 2nd round. The species compositions based on actual species reflecting the best estimates based on only those larvae that were used for species identification by the participant (A), the species compositions as obtained per participant (B), the percentage over- or underestimation (C) and the percentages agreement with actual species (D) are shown per species by participant and for the whole group that took part in the species identification exercise on fish larvae. A weighted mean percent agreement is given by person and all persons combined.

AReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 TOTAL

Herr ing 1 39 39 39 39 39 38 39 39 39 39 37 39 39 39 39 39 621Sprat 2 20 20 20 20 20 19 20 20 20 20 19 20 20 20 20 20 318

Sardine 3 12 12 8 12 12 10 12 12 12 12 9 7 12 9 12 12 175Anchovy 4 9 9 - 10 9 8 9 9 9 9 5 2 10 9 8 6 121

Other 5 3 3 2 3 3 1 3 3 3 3 1 2 3 3 3 3 42Total 1-4 83 83 69 84 83 76 83 83 83 83 71 70 84 80 82 80 1277

BSpecies Reader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 TOTAL

Herr ing 1 38 25 36 46 42 34 37 54 41 23 34 16 39 45 47 42 599Sprat 2 15 29 19 22 14 20 21 16 32 13 25 18 32 16 19 24 335

Sardine 3 4 17 12 12 21 13 13 9 6 32 6 33 9 7 5 10 209Anchovy 4 9 11 1 - 3 8 9 4 3 2 5 3 3 9 11 3 84

Other 5 17 1 1 4 3 1 3 - 1 13 1 - 1 3 - 1 50Total 1-4 66 82 68 80 80 75 80 83 82 70 70 70 83 77 82 79 1227

CReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 ALL

Herr ing 1 -3% -36% -8% 18% 8% -11% -5% 38% 5% -41% -8% -59% 0% 15% 21% 8% -4%Sprat 2 -25% 45% -5% 10% -30% 5% 5% -20% 60% -35% 32% -10% 60% -20% -5% 20% 5%

Sardine 3 -67% 42% 50% 0% 75% 30% 8% -25% -50% 167% -33% 371% -25% -22% -58% -17% 19%Anchovy 4 0% 22% - -67% 0% 0% -56% -67% -78% 0% 50% -70% 0% 38% -50% -31%

Other 5 467% -67% -50% 33% 0% 0% 0% -67% 333% 0% -67% 0% -67% 19%

DReader 1 Reader 2 Reader 3 Reader 4 Reader 5 Reader 6 Reader 7 Reader 8 Reader 9 Reader 10 Reader 11 Reader 12 Reader 13 Reader 14 Reader 15 Reader 16 ALL

Herr ing 1 97% 46% 74% 49% 67% 66% 74% 87% 69% 41% 81% 3% 62% 92% 85% 62% 66%Sprat 2 70% 85% 70% 75% 65% 79% 85% 25% 75% 60% 79% 45% 40% 70% 70% 75% 67%

Sardine 3 33% 17% 25% 8% 33% 0% 25% 0% 8% 58% 22% 0% 0% 44% 0% 25% 19%Anchovy 4 100% 100% - 0% 33% 100% 100% 0% 11% 11% 60% 0% 10% 100% 100% 17% 51%

1-4 78.3% 55.4% 65.2% 41.7% 55.4% 63.2% 69.9% 47.0% 53.0% 43.4% 70.4% 14.3% 39.3% 78.8% 67.1% 53.8%RANKING 2 8 6 14 8 7 4 12 11 13 3 16 15 1 5 10

Species compositions using actual speciesActual species

56.1%

Percentage overestimation / underestimation

Species compositions as estimated per participant and whole group

Actual species

Actual species

Percentage agreement in species identification per species


4.3 Sources of misidentification of larvae

Despite the discussion after the 1st round and the lower quality of the larvae in the 2nd round, there still appear to be difficulties in correctly identifying clupeoid species. These difficulties occurred in the first round mainly because participants were only using one characteristics, the number of myotomes, and not the developmental stage of the larvae at length, i.e. length at hatch, size at start of fin development and length of the gut. Thus, the major source of misidentification in the 1st round lay in the diffi-culties to count myotomes, particularly in determining where to start and to end count-ing. Furthermore, without the aid of polarization equipment it is often difficult to clearly identify single myotomes close to the head as well as in small larvae in general. Larvae handling with needles and tweezers (needed for positioning of the individual larvae for myotome counting) produced damage that introduced some counting errors towards the end of the microscopic analyses.

In the 2nd round participants tried to use the characteristics besides the myotome count, but the lower quality and deterioration of the larvae made it difficult to distin-guish the presence of fins and because the guts were often torn from the larvae it was difficult to decide the position of the pylorus.

24 | ICES WKIDCLUP REPORT 2014

5 Preservation methods of fish larvae

In general, fish larvae – clupeoid larvae in particular – are very delicate creatures. Most clupeoid larvae already die during catch, and as hyposmotic teleosts, they start shrink-ing immediately after their osmoregulatory system stopped functioning. Shrinkage carries on and is even increased once they arrive in the warm environment of the ship’s lab. Therefore, it is important that fish larvae be preserved in a fixative as soon as pos-sible after catching. If larvae cannot be preserved at once, they should at least be kept on ice and at lab temperatures that are at least equal or, even better, a few degrees less than their marine environment. However, even then serious shrinkage of the larvae, causing distortion of the vertebral column (Figure 5.1) can occur after catching if larvae are kept too long without preservation. Thus, if the larvae are not being used for histo-logical, biochemical or genetic analysis, they should be preserved immediately after catch.

Figure 5.1. Distorted vertebral column in a fish larva which was sorted in a cool environment from the catch before preservation in 4% buffered formaldehyde.

Different methods of preservation are available for fish larvae (e.g. (Steedman, 1976). Fixation in 4% formaldehyde solution is still the most widely used preservation method. The formaldehyde solution needs to be buffered at pH 7. Alkaline or acidic formaldehyde solutions cause loss of pigmentation or of calceus structure in the larvae, respectively. Borax and sodiumacetate-trihydrate are often used to buffer the formal-dehyde solutions. Sodiumacetate-trihydrate is a more stable buffer compared to Borax and Borax is also likely to produce a too high, alkaline pH. Formaldehyde causes a slight but constant shrinkage in the fish larvae (Fox, 1996, Santos et al., 2009), but larvae characteristics are well preserved, though some of the pigmentation is lost (Figure 5.2).


Figure 5.2. Fish larvae immediately after catching preserved in 4% buffered formaldehyde. Notice the undistorted vertebral column.

Due to the known health hazards of formaldehyde, other fixatives such as ethanol are used for preservation. Ethanol has the advantage that larvae can be used for genetics (although expensive, denatured ethanol would be preferable) and otoliths from the larvae can still be used for analysis. However, shrinkage in ethanol preserved larvae is larger compared to formaldehyde preserved larvae (Santos et al., 2009; Fox, 1996) and pigmentation is also lost in ethanol. When due to stricter health and safety rules a switch from formaldehyde contained fixative to ethanol is necessary, be aware of the larger shrinkage of the larvae.

DNA analysis of larvae can be done on ethanol preserved samples. However, recently methods have been developed to carry out DNA analysis on formaldehyde or Battaglia solution fixed ichthyoplankton as well (Goodsir et al., 2008; Lelievre et al., 2010). Larvae can be fixed for 12 to 24 hours in 4% buffered formaldehyde or Battaglia solutions be-fore transferring to ethanol.

Pigmentation of the larvae, especially the yellow and red pigments, disappear in both formaldehyde and ethanol solutions due to pigment oxidation. The Battaglia solution (Mastail and Battaglia, 1978; Bigot, 1979), in which the larval pigmentation is better preserved (Table 5.1), is described below.


Table 5.1. Recipe for Battaglia solution (10 litres).

INGREDIENT QUANTITY CAS NUMBER

Formaldehyde 36% (39% w/v), HCHO, MW: 30.03 g/mol

4 litre 50–00–0

Propanediol-1,2, CH3-CHOH-CH2OH, MW: 76,1 g/mol

2 litre 57–55–6

Distilled water 4 litre

EDTA: Ethylene Diamine Tetra acetic Acid disodium salt dehydrate, MW: 372.24 g/mol

40 g 6381–92–6, white crystals

BHA: Butyl Hydroxyl Anisole C11H16O2, 2-tert-Butyl-4-methoxyphenol

16 g 121–00–6, orange-brown crystals

L(+) Ascorbic acid (vitamin C) 4 g 50–81–7, yellow crystals

Sodium glycerophosphate hydrate (glycerol), C3H5(OH)2PO4,xH2O, MW: 216.04 g/mol

120 to 300 g 5507, white crystals, 3–41–1

Preparation of Battaglia solution (note: you need to stir vigorously and for a long pe-riod for the chemicals to dissolve!):

1 ) Dissolve 40 g EDTA in 1 litre distilled water with magnetic stirrer. Buffer to pH 7 with glycerol.

2 ) Dissolve 16 g BHA in 1 litre propanediol and stir with magnetic stirrer. 3 ) Mix into a 10 litre beaker with a magnetic stirrer 4 litre formaldehyde with

40 to 100 gr glycerol and buffer to pH 7. 4 ) Add EDTA dissolved in distilled water. Stir well! 5 ) Add BHA dissolved in propanediol. Stir well! 6 ) Add the remaining 1 litre of propanediol. Stir well! 7 ) Add ascorbic acid. Stir well! 8 ) Buffer to pH 7 with 20 to 50 gr glycerol. 9 ) Add remaining 3 litre distilled water. The mixture may become whitish for

a moment. 10 ) Buffer to pH 7 with 20 to 50 gr glycerol. Let it stir for about 30 minutes. 11 ) For storage, use a bottle with a double-seal, leak-resistant closure. 12 ) Wait for 10 days before using. Keep the solution at >15°C (not in the fridge!).

A thermal shock may induce polymerisation of formalin. This does not af-fect the solution’s properties. Filter the solution (but it is not easy!) or depol-ymerise with sodium carbonate (it is not easy too!).

13 ) Finally, the samples are preserved in seawater using 6% of the Bataglia so-lution (which is enough when plankton account for ¼ of the sample vol-ume). The resulting concentration of formalin in the sample is less than 1%.

14 ) If samples contain a high amount of diatoms or euphausids it is advised to use a higher percentage of Battaglia solution to ensure the fish larvae are well preserved.


Lugol iodine solutions (Steedman, 1976) can also be used for preservation of fish lar-vae. However due to the iodine the larvae are stained yellow-orange. Before species identification, the stain needs to be removed by putting the larvae in another chemical.

After thorough fixation in 4% buffered formaldehyde or Battaglia solution the larvae samples can be transferred to Steedman’s solution containing propylene phenoxetol and propane-1,2-diol (Steedman, 1976) only. These chemicals also fix most of the for-maldehyde fumes from the fixed larvae. The larvae can be kept in this solution for a couple of months to a few years at room temperature before deteriorating (Steedman, 1976). For long-term storage Steedman solution with the addition of 2% buffered for

Long-term storage of fish larvae samples should be done in dark and cooled storage rooms to prevent deterioration of the ichthyoplankton and loss of pigmentation. The pH should be kept at 7 and needs to be checked regularly.


6 References

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Annex 1: List of participants

Name Address Phone/Fax E-mail

Franziska Bils University of Hamburg, Institute for Hydrobiology and Fisheries, Science Olbersweg 24, 22767 Hamburg, Germany

+49 40 428386708 [email protected]

Cindy van Damme (Chair)

IMARES, Haringkade 1, 1976 CP Ĳmuiden, The Netherlands

+31317487078 +31317487326

[email protected]

Jérémy Denis Ifremer 150 Quai Gambetta. 62200 Bou-logne sur Mer, France

[email protected]

Elisabete Henriques

[email protected]

Bastian Huwer DTU Aqua National Institute of Aquatic Resources Kavalergården 6 DK-2920 Charlottelund Denmark

[email protected]

Annemarie Jetter TI-OF Alter Hafen Süd 2 18069 Rostock Germany

[email protected]

Matthias Kloppmann (Chair)

TI-SF, Palmaille 9, 22767 Hamburg, Germany

+494038905196 [email protected]

Sakis Kroupis TI-SF, Palmaille 9, 22767 Hamburg, Germany

[email protected]

Marie Leiditz Swedish University of Agricultural Sciences, Department of Aquatic Resources, Institute of Marine Research, Turistgatan 5, Box 45330, SE-453 21 Lysekil, Sweden

[email protected]

Christophe Loots Ifremer, 150 Quai Gambetta. 62200 Bou-logne sur Mer, France


mailto:[email protected]











Name Address Phone/Fax E-mail Andrejs Makarchouk

[email protected]

Dorothee Moll TI-OF Alter Hafen Süd 2 18069 Rostock Germany

[email protected]

Enda O’Callaghan AFBI, 18 Newforge Lane, Belfast, BT9 5PX, Northern Ireland

+442890255500 enda.o'[email protected]

Anne-Marie Palmén Bratt

Swedish University of Agricultural Sciences, Department of Aquatic Resources, Institute of Marine Research, Turistgatan 5, Box 45330, SE-453 21 Lysekil, Sweden

[email protected]

Ineke Pennock IMARES, Haringkade 1, 1976 CP Ĳmuiden, The Netherlands


Lynette Ritchie MSS 375 Victoria Rd, Aberdeen UK


Birgit Suer TI-SF, Palmaille 9, 22767 Hamburg Germany


Maik Tiedemann TI-SF, Palmaille 9, 22767 Hamburg, Germany

[email protected]



mailto:enda.o'[email protected]







Annex 2: Agenda

Monday 1 September

9:00 Preparations for the workshop (Matthias, Sakis, Birgit, Cindy)

12:30 Lunch

13:30 Start Workshop

13:30 Welcome and general announcements

13:40 Introduction round

13:50 Introduction to clupeid larvae identification (presentation Matthias)

15:00 Break

15:15 1st individual larvae identification trial

17:30 End of the day

Tuesday 2 September

9:00 Continue 1st individual larvae identification trial

12:30 Lunch

15:00 Break

15:15 Review available information on clupeid larvae identification and spatial and temporal distribution (break up in groups)


Wednesday 3 September

9:00 Discuss results of the 1st individual larvae identification trial

Identify sources of misidentification

11:00 Break

11:15 2nd individual larvae identification trial

12:30 Lunch

13:30 Continue 2nd individual larvae identification trial

Review available information on clupeid larvae identification and spatial and temporal distribution (presentations of groups)


Thursday 4 September

9:00 Review available information on larvae identification (break out in groups)

10:00 Discuss results of the 2nd individual larvae identification trial

Identify sources of misidentification

Establish and agree on a clupeid larvae identification key (presentations of groups)

12:30 Lunch


13:30 Establish and agree on a clupeid larvae identification key (presentations of groups)

Compile overview of methods of clupeid larvae sampling and sample processing, preservation used and agree on an overview of suggested future methods for different survey demands

15:00 Break

15:15 Review available information on larvae identification (break out in groups)


Friday 5 September

9:00 Report writing: discussion conclusions, recommendations and future

11:00 Break

11:15 Final discussions

12:00 End of the workshop

13:00 Finalize report and clear up of the workshop (Birgit, Sakis, Cindy, and Matthias)


Annex 3: Recommendations

RECOMMENDATION ADRESSED TO

1. Based on the low agreements during the workshop, it is clear that the identification of clupeoid larvae is difficult and identification should be improved. WKIDCLUP therefore recommends that workshops on fish larvae identification are held regularly (every 5 years) to exchange knowlegde and to increase agreement on sample processing and identification of fish larvae. Especially when conducting ecosystem wide surveys it is important to standardize methods and larvae identification.

SSGIEOM, SCICOM, PGDATA, WGBIOP, WGALES

2. WKIDCLUP recommends to investigate the effect of the low agreement in clupeoid larval identification on the herring assessment.

WGIPS, IBTSWG, HAWG, PGDATA, WGBIOP, WGALES

3. WKIDCLUP recommends to use validated larvae for future clupeoid larvae identification workshops, collected from incubation of eggs. WKIDCLUP also recommends to collect and preserve seperately clupeoid larvae from survey samples for use in future identification workshops.

WGIPS, IBTSWG, WGACEGG, PGDATA, WGBIOP

4.Experienced persons showed a much higher agreement in species identification compared to less experienced. WKIDCLUP recommends that institutes ensure the continuity of staff for fish larvae identification to increase the quality of larval identification in survey samples.

WGALES, WGIPS, IBTSWG, HAWG

5. Based on the experiences at the workshop it is recommended that a binocular microscope should have the following features:

Options for a black or white stage plate for use with incident (top) light.

A transparent stage plate for transmitted (bottom) light.

Dark field illumination for contrast.

Adjustable brightness.

Magnification with click stops.

Magnification should be at least 1.6x.

A choice of 10x and 20x eyepieces.

Adjustable binocular head and ergonomic design to allow flexibility of movement.

Adjustable focus on all eyepieces.

Calibrated eyepiece graticules.

Double (fibre optic) cold light source, with adjustable focus, to avoid shadows.

Polarization equipment.

Mechanical stages to position samples easily in the field of view and to hold the samples firmly.

All participants, Chairs of future larvae identification workshops

Documents

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