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Ingestion and digestion of micro-algaeconcentrates by veliger larvae of the giant clam,Tridacna noaeSouthgate, Paul C; Braley, Richard D; Militz, Thane Ahttps://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12126677780002621?l#13126871390002621

Southgate, Braley, R. D., & Militz, T. A. (2017). Ingestion and digestion of micro-algae concentrates byveliger larvae of the giant clam, Tridacna noae. Aquaculture, 473, 443–448.https://doi.org/10.1016/j.aquaculture.2017.02.032

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Accepted Manuscript

Ingestion and digestion of micro-algae concentrates by veligerlarvae of the giant clam, Tridacna noae

Paul C. Southgate, Richard D. Braley, Thane A. Militz

PII: S0044-8486(16)31136-XDOI: doi: 10.1016/j.aquaculture.2017.02.032Reference: AQUA 632545

To appear in: aquaculture

Received date: 4 December 2016Revised date: 6 February 2017Accepted date: 27 February 2017

Please cite this article as: Paul C. Southgate, Richard D. Braley, Thane A. Militz , Ingestionand digestion of micro-algae concentrates by veliger larvae of the giant clam, Tridacnanoae. The address for the corresponding author was captured as affiliation for all authors.Please check if appropriate. Aqua(2017), doi: 10.1016/j.aquaculture.2017.02.032

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Ingestion and digestion of micro-algae concentrates by veliger larvae of the

giant clam, Tridacna noae.

Paul C. Southgate1,*

, Richard D. Braley1,2

, Thane A. Militz1

1Australian Centre for Pacific Islands Research and Faculty of Science, Health, Education

and Engineering, University of the Sunshine Coast, Maroochydore, Queensland 4556,

Australia

2Aquasearch, 6-10 Elena Street, Nelly Bay, Magnetic Island, Queensland 4819, Australia

*Corresponding author. Email: psouthgate@usc.edu.au

Key words: Tridacna noae, larvae, ingestion, digestion, micro-algae, hatchery culture.

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Abstract

Knowledge of ingestion and digestion of micro-algae by bivalve larvae is critical for

provision of appropriate larval nutrition supporting maximal growth and survival. However,

little is known about the ingestion and digestion of micro-algae by giant clam larvae. This

study determined the rates of ingestion and digestion of commercially available micro-algae

concentrates by Tridacna noae larvae of different ages using epifluorescence microscopy.

The micro-algae used were Isochrysis sp. (Isochrysis 1800®), Pavlova sp. (Pavlova 1800®),

Tetraselmis sp. (Tetraselmis 3600®) and Thalassiosira weissflogii (TW 1200®). None of the

four micro-algal concentrates were ingested by T. noae larvae at 24 h post-fertilisation, but all

were ingested at 48 h and 72 h post-fertilisation, at different frequencies. At 48 h post-

fertilisation, Isochrysis sp. and Pavlova sp. were ingested by 77% and 70% of veligers,

respectively, while T. weissflogii and Tetraselmis sp. were ingested by 10% and 30% of

veligers, respectively. Similar rates of ingestion were observed for each micro-alga by larvae

at 72 h post-fertilisation. Larvae capable of ingesting micro-algae concentrates were

significantly larger than those that were empty and the minimum antero-posterior shell length

of T. noae larvae capable of ingesting Pavlova sp. and Isochrysis sp. was 141 µm and 132

µm, respectively. Digestion of micro-algae by 48 h-veligers was observed 2 h after the start

of feeding in 26.1% and 14.3% of larvae that had ingested Isochrysis sp. and Pavlova sp.,

respectively, but digestion of Tetraselmis sp. and T. weissflogii was not observed until 4 h

and 8 h after the start of feeding, respectively. Complete digestion of Pavlova sp. and

Isochrysis sp. took up to 12 hours in both 48 h and 72 h post-fertilisation. Our results provide

a basis for developing a more nutritionally informed approach to hatchery culture of T. noae.

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1. Introduction

Depletion of giant clam (Tridacninae) populations throughout the tropical Indo-Pacific has

resulted from exploitation for human consumption (Lucas, 1994; Kinch, 2002), shells and

curios (Usher, 1984; Heslinga, 1996), and the marine aquarium market (Wabnitz et al., 2003;

Kinch and Teitelbaum, 2010). Population declines prompted the listing of Tridacninae in

Appendix II of the Convention on International Trade in Endangered Species (CITES) since

1985 (UNEP-WCMC, 2016a, 2016b), and on the International Union for Conservation of

Nature (IUCN) Red List since 1986 (IUCN, 2016). As a result, there has been substantial

interest in the captive culture of giant clams for wild-stock replenishment and to supply

commercial markets (Tisdell and Menz, 1992; Lucas, 1994; Kinch and Teitelbaum, 2010).

Hatchery culture methods for giant clams were developed in the 1980s and 1990s and are

well documented (Heslinga et al., 1984; Crawford et al., 1986; Alcazar, 1988; Bell and

Pernetta, 1988; Braley, 1992).

Although giant clam larvae can be cultured to metamorphosis in some locations without an

external food source (Fitt et al., 1984; Heslinga et al., 1984; Alcazar, 1988), in other locations

this is not the case and a particulate food source must be supplied (Braley et al., 1988; Ellis,

1997). Feeding of giant clam larvae generally involves provision of cultured live micro-algae

(Bell and Pernetta, 1988; Braley et al., 1988; Braley, 1992; Ellis, 1997) that is usually

cultured on-site at the hatchery. However, provision of a reliable and consistent supply of

cultured live micro-algae requires considerable technical resources and skilled personnel

(Coutteau and Sorgeloos, 1992) that are often unavailable in the developing island nations

(Southgate et al., 2016a) that account for the majority of giant clam production (Kinch and

Teitelbaum, 2010). Furthermore, a considerable proportion of the infrastructure and cost

associated with establishing regional hatcheries is attributed to micro-algae culture (Ito,

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1999). Lack of technical resources can be addressed to some degree through simplification of

hatchery procedures. One such option is to minimise or eliminate the requirement for live

micro-algae culture. Total replacement of live micro-algae with commercially-available

micro-algae concentrates has been reported for sea cucumber (Holothuria scabra) larvae

(Duy et al., 2016a, 2016b) and for pearl oyster (Pteria penguin) larvae (Southgate et al.,

2016a; Wassnig and Southgate, 2016) and routine hatchery culture of both species now

occurs without live micro-algae.

A generic ‘intensive’ method for giant clam hatchery culture (Braley et al., 1988), including

provision of micro-algae concentrates as a larval food source, was recently shown to be

successful for culturing Tridacna noae (Southgate et al., 2016b). The micro-algae

concentrates used were Isochrysis sp. (Haptophyceae) (Isochrysis 1800®) and Pavlova sp.

(Haptophyceae) (Pavlova 1800®) (Instant Algae

®, Reed Mariculture Inc.), and successful

production of juveniles showed that these products can be used to support T. noae through

larval development and metamorphosis. However, it is unclear whether the larvae ingested

and digested the micro-algae concentrate cells that were introduced into larval cultures.

Given that a number of studies have reported successful hatchery production of giant clam

larvae without provision of micro-algae or particulate foods (Fitt et al., 1984; Heslinga et al.,

1984; Alcazar, 1988), successful production of T. noae coincident with provision of micro-

algae concentrates, is insufficient to confirm ingestion and digestion of these products. Fitt et

al. (1984) reported that T. gigas and Hippopus hippopus larvae were selective in ingesting

live micro-algal species, with some species not being consumed. This suggests the potential

for giant clam larvae to be discriminatory towards ingestion of certain micro-algae

concentrates.

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Little is known about digestion of micro-algae by giant clam larvae. It is apparent from

studies with other bivalves that ingestion does not necessary guarantee digestion and that

digestion may occur at different rates for a particular micro-algal species (Lora-Vilchis and

Maeda-Martínez, 1997; Martínez-Fernández et al., 2004). In giant clams, some single celled

micro-algae, such as dinoflagellate zooxanthellae, are ingested by larvae and retained in the

stomach without digestion, subsequently establishing symbiosis in early juvenile stage clams

(Fitt and Trench, 1981). The extent to which tridacnid larvae digest different species of

micro-algae commonly provided as a food source during culture (for both live and

concentrate forms) is unknown. Such knowledge is critical for providing optimal larval

nutrition, particularly when digestibility can be one of the main factors influencing larval

growth and survival (Ewart and Epifanio, 1981; Albentosa et al., 1993).

This study determined the suitability of commercially available micro-algae concentrates for

veliger larvae of T. noae on the basis of larval capacity to ingest and digest the micro-algae

cells. We examine variations in ingestion and digestion of multiple micro-algal species

available as concentrates for veliger larvae at different ages in culture. Our results provide a

basis for developing a more nutritionally informed approach to hatchery culture of tridacnids

and evaluation of the feasibility of simplified hatchery procedures for T. noae, without live

micro-algae culture, that are more appropriate for developing island nations.

2. Materials and Methods

Ingestion and digestion of micro-algae were determined directly using epifluorescence

microscopy which has been used extensively in similar studies with larval molluscs (e.g.

Aldana-Aranda et al., 1997; Lora-Vilchis and Maeda-Martinez, 1997; Martínez-Fernández et

al., 2004; Patino-Suarez et al., 2004). This method can be more accurate than cell counting

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using a high power microscopy since the photosynthesizing pigments of algal cells fluoresce

under blue light illumination close to the excitation maximum at 490 nm (long wave-length

blue) which can be seen at different intensities indicating various stages of ingestion and

digestion.

2.1 Broodstock and larvae

Broodstock T. noae were sourced from fringing reefs around the outer barrier islands of the

Kavieng lagoonal system (2° 36′S, 150° 46′E) of New Ireland Province in Papua New

Guinea, where T. noae occurs at relatively high densities (Militz et al., 2015). Collection

involved removing an individual clam and a portion of substrate to which its byssal threads

were anchored. Clams were held in insulated containers containing seawater and transported

by boat to the National Fisheries Authority (NFA) Nago Island Mariculture and Research

Facility (NIMRF). Here they were held in raceways for a week prior to spawning. Clams

were held in 2,000 L raceways provided with continuous aeration and continuous flow-

through of 5 µm filtered seawater sourced from the fringing reef surrounding the island

facility. Broodstock clams were fed once-daily with a 6,000 cells mL-1

ration of the micro-

algae concentrate Shellfish Diet® (Instant Algae®, Reed Mariculture, Campbell, CA 95008,

USA), consisting of a mixture of Isochrysis sp., Pavlova sp., Tetraselmis sp., Chaetoceros

calcitrans, Thalassiosira weissfloggi, and Thalassiosira pseudonana cells. Water temperature

was maintained at 28.1 ± 0.5 oC and salinity at 36 g L

-1. For spawning induction, heat stress

was applied to the broodstock by removing them from the water and laying them on their

sides in full sun for 20-30 min. Clams were then returned to the raceway and monitored for

release of spermatozoa or spawning of eggs. Lack of response to heat stress was the impetus

to induce spawning by injection of 0.7-1.0 mL of a 2 mM serotonin (5-hydroxytryptomine

creatinine sulphate complex) solution into the gonad by hypodermic needle insertion through

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the mantle (Braley, 1985; Crawford et al., 1986). Four clams (shell lengths of 22.0, 21.5,

19.0, and 17.0 cm), produced both sperm and eggs within 5 min of serotonin injection. A

total of 11.4 million eggs were collected, with individual clams contributing between 9.7%

and 42.6% of the total. Gametes from individual clams were collected separately to avoid

self-fertilisation and eggs from each individual were fertilised within 10 min of spawning.

Eggs were fertilised at densities between 22 and 55 eggs mL-1

with a small aliquot of sperm

suspension and hatched in 2,000 L tanks filled with 1 µm filtered seawater (FSW) at a density

of 2 eggs mL-1

. Seawater in egg incubation tanks was not UV-treated and no antibiotics were

used. Tanks were provided with continuous aeration in the centre of the tank. Water

temperature was 27.9 oC and salinity 36 g L

-1 when eggs were stocked for hatching. A

fertilisation rate of 81% was determined 4 h post-fertilisation, using three replicate counts of

100 eggs/embryos examined using Sedgewick-Rafter counters and a compound microscope.

Successful fertilisation was determined by the number of embryos at or beyond the 2-cell

stage compared to the number of undeveloped eggs observed.

Larvae were reared ‘extensively’ according to the methods of Braley (1992) without water

exchange or addition of food. Southgate et al. (2016) reported that swimming veliger larvae

of T. noae persist between 24 h and 72 h post-fertilisation and that by 96 h post-fertilisation,

98% of larvae had settled to the tank bottom for transition to the pediveliger stage. On this

basis veliger larvae were removed from the main larval culture tanks for use in experiments

at 24 h, 48 h, and 72 h post-fertilisation. Larvae were retained on a 60 µm mesh screen and

washed gently with FSW prior to transfer to five 20 L cylindrical aquaria supplied with

gentle aeration used for feeding trials where they were established at a density of 1 larva mL-

1. Larvae at 24 h and 48 h post-fertilisation were fed rations of 4,000 cells mL

-1 while larvae

at 72 h post-fertilisation were fed a ration of 8,000 cells mL-1

during the experiments. An

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unfed control was also maintained. Water temperature was maintained at 27.89 ± 0.11°C over

the course of the experiment.

2.2 Micro-algae concentrates

Commercially available micro-algae concentrates (Instant Algae®, Reed Mariculture Inc.)

purchased from an Australian distributor of the products were used in this study. Four Instant

Algae® products were used: (1) mono-cultured Isochrysis sp. (Haptophyceae) (Isochrysis

1800®); (2) mono-cultured Pavlova sp. (Haptophyceae) (Pavlova 1800®); (3) mono-cultured

Tetraselmis sp. (Chlorophycophyceae) (Tetraselmis 3600®); (4) mono-cultured Thalassiosira

weissflogii (Bacillariophyceae) (TW 1200®). Concentrates were stored in their original

bottles in a refrigerator at 4°C for the duration of the study. Prior to use, a 1 mL aliquot of

each concentrate was added to approximately 500 mL of FSW in a clean plastic bottle and

gently hand-shaken to disperse the micro-algae cells. The cell density in each micro-algae

stock suspension was determined using a haemocytometer and the volume needed to obtain

the required cell ration in each aquarium was calculated. The micro-algae used in this study

and their characteristics are shown in Table 1.

2.3 Assessing ingestion and digestion

To evaluate ingestion and digestion of the tested micro-algae concentrates, larvae were fed

separately in their 20 L aquaria with each diet for 2 h. Larvae were then retained on a 60 µm

mesh screen, washed gently with FSW, and transferred to new aquaria without food. They

were examined to evaluate ingestion and digestion of micro-algae after 2, 4, 8, 12, and 24 h

from the start of feeding. Because larvae were maintained in experimental treatments for only

24 h, and only 150 of the 20,000 larvae initially stocked into each aquarium were removed

for analysis, no effort was invested to maintain larval density over this period.

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To assess ingestion and digestion of each micro-alga using epifluorescence microscopy, 30

larvae from each aquarium were captured on a 60 µm mesh screen and fixed with 2%

formaldehyde in seawater (buffered) solution prior to examination. Each larva was

photographed and the digital imaging software ToupView (Version 3.7.2270) was used to

determine antero-posterior measurement (APM) as a measure of larvae size. Micro-algae

cells in the larval gut were identified and qualitatively assessed using a method similar to that

used by Duy et al. (2015) and Martínez-Fernández et al. (2004). The criteria used to assess

the degree of micro-algae ingestion and digestion in this study are shown in Table 2 and

illustrated in Fig. 1.

2.4 Statistical Analysis

The proportion of larvae having ingested each micro-algae concentrate was determined as the

proportion of both Stage II and Stage III larvae (Table 2) at 2 h from the start of feeding

compared to the total number of larvae sampled. The frequencies of larvae observed to have

ingested or digested each micro-algae concentrate were compared using χ2 contingency tests

using the statistical package S-Plus® (Version 8.0). The data from 24 h-, 48 h-, and 72 h-

veligers was pooled for each diet to assess whether larval size (i.e. APM) influenced

ingestion of the diet. Size data was assessed for normality using a Shapiro-Wilk test before

conducting two-sample t-tests, variances assumed equal when appropriate, for each micro-

algal concentrate. An ANOVA was used to compare the mean size of larvae capable of

ingesting the different micro-algae concentrates. Statistical significance was accepted at P =

0.05 for all analyses.

3. Results

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None of the four micro-algal concentrates were ingested by T. noae larvae at 24 h post-

fertilisation, however, at 48 h and 72 h post-fertilisation all four micro-algal concentrates

were ingested, albeit, at different frequencies (Fig. 2). At 48 h post-fertilisation, Isochrysis sp.

and Pavlova sp. were the most frequently ingested micro-algae, ingestion observed in 77%

and 70% of veligers, respectively (χ2 = 0.34, P = 0.56). The ingestion of both Isochrysis sp.

and Pavlova sp. was observed for significantly more larvae than for those fed T. weissflogii

(10% of veligers) and Tetraselmis sp. (30% of veligers; Fig. 2A). At 72 h post-fertilisation,

Isochrysis sp. and Pavlova sp. were also the most frequently ingested micro-algae, with

ingestion observed in 80.0% and 73.3% of veligers, respectively (χ2 = 0.37, P = 0.54). The

ingestion of both Isochrysis sp. and Pavlova sp. at 72 h post-fertilisation was observed for

significantly more larvae than for those fed T. weissflogii (10.0% of larvae) and Tetraselmis

sp. (26.7% of larvae; Fig. 2B). The frequency of both Pavlova sp. and Isochrysis sp. ingestion

increased by 3.3% from 48 h- to 72 h-veligers, this increase being nonsignificant (Pavlova

sp., χ2 = 0.08, P = 0.77; Isochrysis sp., χ

2 = 0.10, P = 0.75). In contrast, the frequency of T.

weissflogii ingestion remained unchanged while ingestion of Tetraselmis sp. decreased by

3.3% in veligers from 48 h to 72 h (χ2 = 0.08, P = 0.77).

Digestion of micro-algae by 48 h-veligers was observed 2 h after the start of feeding in

26.1% and 14.3% of larvae that had ingested Isochrysis sp. and Pavlova sp., respectively (χ2

= 0.62, P = 0.43). In comparison, digestion of Tetraselmis sp. was not observed in veligers

until 4 h after the start of feeding and digestion of T. weissflogii was not observed until 8 h

after the start of feeding (Fig. 2A). At 4 h after the start of feeding it was apparent that a

portion of larvae that had initially ingested Isochrysis sp. and Pavlova sp. had completed

digestion as the number of larvae at both Stage II and Stage III of ingestion/digestion had

decreased by 43.5% and 47.6%, respectively, compared to the Stage II and Stage III larvae

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observed 2 h prior. However, for 21.7% and 23.8% of larvae that initially ingested Isochrysis

sp. and Pavlova sp., respectively, digestion took over 12 h to complete (χ2 = 0.02, P = 0.90);

some individual larvae proving to be incapable of fully digesting these concentrates even

after 24 h (Fig. 2A).

Digestion of micro-algae by 72 h-veligers was observed 2 h after the start of feeding in

50.0% and 22.7% of larvae that had ingested Isochrysis sp. and Pavlova sp., respectively (χ2

= 1.72, P = 0.19). Digestion of T. weissfloggii and Tetraselmis sp. was also observed to occur

within 2 h for 72 h-veligers, although these observations came from only a single larva for

both concentrates (Fig. 2B). The proportion of larvae ingesting Isochrysis sp. and Pavlova sp.

that had completed digestion 4 h after the start of feeding was similarly approximated by the

decrease in Stage II and Stage III larvae initially observed, and found to be 70.8% and 59.1%

of larvae, respectively (χ2 = 0.34, P = 0.56). However, for 12.5% and 27.3% of larvae that

initially ingested Isochrysis sp. and Pavlova sp., respectively, digestion took over 12 h to

complete (χ2 = 1.07, P = 0.30). Some larvae (6.7%) proved to be incapable of digesting

Pavlova sp. even after 24 h, but all larvae fed Isochrysis sp. had completed digestion at this

point (Fig. 2B).

Larvae capable of ingesting (i.e. Stage II and III) cells of Pavlova sp., Isochrysis sp., and

Tetraselmis sp. were found to be significantly larger in size than larvae that did not ingest

cells of the same micro-algae (i.e. Stage I) (Fig. 3). Given the low level of ingestion observed

for the TW 1200® concentrate, it was not possible to assess differences in ingestion for this

diet. Larvae capable of ingesting Pavlova sp. had a mean APM (156.65 ± 1.01 µm) that was

significantly greater than that of empty larvae (146.02 ± 1.49 µm) 2 h after the onset of

feeding (t(2)88 = 5.79, P < 0.01; Fig. 3A). Size was also significantly higher among larvae

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capable of ingesting Isochrysis sp. (154.15 ± 1.29 µm) than empty larvae (144.88 ± 1.35 µm)

2 h after the onset of feeding (t(2)88 = 4.96, P < 0.01; Fig. 3B). Larvae capable of ingesting

Tetraselmis sp. had a mean APM of 155.06 ± 1.28 µm while empty larvae fed this diet had a

mean APM of 149.59 ± 1.14 µm (t(2)45 = 3.20, P < 0.01; Fig. 3C). The mean APM of larvae

capable of ingesting Pavlova sp., Isochrysis sp., and Tetraselmis sp. were similar (F(2,104) =

1.25, P = 0.29). Comparing the APM of larvae that failed to ingest the different micro-algae

concentrates, showed that those that failed to ingest T. weissfloggii had a greater mean APM

(149.6 ± 1.0 µm) than those that failed to ingest Isochrysis sp. (144.9 ± 1.4 µm) (F(3,243) =

3.60, P = 0.01). All other comparisons showed no differences between mean APM.

4. Discussion

This is the first study to quantitatively investigate the rates of relative ingestion and digestion

of micro-algae by tridacnine larvae and the first to report on any aspect of the larval feeding

behaviour of Tridacna noae. Although embryonic and larval development of T. noae were

recently described (Southgate et al., 2016b) details of the development of larval anatomy are

not yet available for this species. Prior research with the larvae of T. maxima and T.

squamosa reported that, for both species, the oesophagus and stomach are open and ciliated

in prodissoconch I veligers (48 h post-fertilisation) but that the intestine does not develop a

lumen until 72 h post-fertilisation (LaBarbera, 1975). Unfortunately, a food source was not

provided by LaBarbera (1975) and so the point at which ingestion began in T. maxima and T.

squamosa larvae was not noted. The results of the present study however, show that none of

the four micro-algal concentrates were ingested by T. noae larvae at 24 h post-fertilisation,

but that micro-algae ingestion had begun by 48 h indicating development of the oesophagus

and stomach between 24 h and 48 h post-fertilisation in T. noae. This suggests that there is

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limited value in feeding larvae within the first 24 h of hatchery culture and that first-feeding

of T. noae larvae should begin between 24 h and 48 h post-fertilisation.

Previous research on the hatchery culture of giant clams has identified that larval mortality in

these clams is high (Heslinga et al., 1984; Braley et al., 1988; Alcazar, 1988), but there has

been limited research effort to optimize hatchery culture conditions and larval feeding

protocols. Our study identified differential rates of ingestion and digestion of the cells of

micro-algae concentrates by T. noae larvae. The golden-brown flagellates Isochrysis sp. and

Pavlova sp. were the most readily ingested of the four micro-algae provided, regardless of the

age of the larvae. Both species have a relatively small cell size that is particularly suitable for

ingestion by early bivalve veligers. Isochrysis sp. and Pavlova sp. form a basis for hatchery

production of a broad range of bivalves (e.g. Helm and Bourne, 2004; O’Connor et al., 2008;

Southgate, 2008; Brown and Blackburn, 2013), with larger-celled species, such as

Tetraselmis sp., generally being added to the diets of older larvae. The frequency of

Isochrysis sp. and Pavlova sp. ingestion increased with increasing age of the larvae.

Tetraselmis sp. (10-12 µm) and T. weissflogii (7-20 µm) have much larger cell sizes than

Isochrysis sp. (5-7 µm) and Pavlova sp. (4-7 µm), and the frequency of ingestion of these

species by Tridacna noae veligers was significantly lower than those for Isochrysis sp. and

Pavlova sp. regardless of larval age.

These findings are similar to those reported for other bivalve larvae. Among 10 species of

live micro-algae trialled, only Isochrysis aff. galbana, Pavlova lutheri, and Nannochloris sp.

were ingested by 5, 10, and 22 day-old Pteria sterna (Martínez-Fernández et al., 2004). All

diatoms fed to P. sterna larvae, including Thalassiosira weisflogii, failed to be ingested along

with the larger (8-6 µm) Chlorophyta species: Dunaliella salina, Tetraselmis tetrahele, and T.

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suecica. While some T. noae larvae did demonstrate the capacity to ingest Tetraselmis sp.

and T. weissflogii in concentrate form in our study, the frequency of ingestion was very low

(≤ 30%) compared to the ingestion rates observed for Isochrysis sp. and Pavlova sp.

Results indicate that T. noae larvae may have selectively avoided ingestion of Tetraselmis sp.

and T. weissflogii. For example, larvae that were presented with Tetraselmis sp. but

had empty stomachs, were of similar size (APM), or even larger, than those that ingested

Tetraselmis sp. cells. This suggests that a large number of larvae were of suitable size to

ingest Tetraselmis sp. but still failed to do so. Whether this indicates selective avoidance or

the presence of a physiological barrier other than larval size preventing ingestion of this

micro-alga remains unresolved and merits further research. In contrast larval size was a

significant factor in determining whether Isochrysis sp. and Pavlova sp. would be ingested.

Where development rates of larval tridacnids differ due to water temperature or location, use

of larval size as an indication of when to begin first-feeding may be of greater use than larval

age. This study found that the minimum APM of T. noae larvae capable of ingesting Pavlova

sp. and Isochrysis sp. was 141.0 µm and 132.0 µm, respectively.

The larvae of T. noae also showed considerable differences in their ability to digest the four

micro-algae presented to them. Digestion of Isochrysis sp. and Pavlova sp. was evident

within 2 h of the start of feeding of 48 h-veligers, but this was not seen until 4 h after the start

of feeding in veligers fed Tetraselmis sp., and more than 8 h after the start of feeding in

veligers fed T. weisflogii. The digestive capacity of T. noae larvae improved with age with

digestion of both Tetraselmis sp. and T. weisflogii evident within 2 h after the start of feeding

for 72 h-veligers; however, these observations were limited to a single larva and most

digestion occurred after this point in time for these species. This suggests that T. noae larvae

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have limited capability to digest larger Bacillariophyta and Chlorophyta species. Similarly,

where larvae of the pearl oyster Pteria sterna were observed to ingest the Chlorophyte

Nannochloris sp., they were unable to digest this micro-alga (Martínez-Fernández et al.,

2004). Poor digestion of micro-algae is generally related to cell wall structure and

composition, particularly the presence of relatively thick cellulose cell walls, and it is notable

that Chlorophytes are not routinely used as foods for bivalve larvae in contrast to

Bacillariophytes, Prymesionphytes and Prasinophytes (Brown and Blackburn, 2013).

While we acknowledge that giant clam larvae can be cultured to metamorphosis in some

locations without an external food source (Heslinga et al., 1984; Alcazar, 1988), studies have

found that provision of a food source can greatly improve survival during the larval period

(Fitt et al., 1984; Ellis, 1997). Information on tissue composition and larval energetics of

giant clams is sparse, however, the larvae of Hippopus hippopus, like many other bivalves

(Gallager et al., 1986), utilise lipid as the principal energy source when food is not available

and during the non-feeding period associated with metamorphosis (Southgate, 1988). While a

proportion of unfed larvae are able to meet the energetic requirements of metamorphosis

from remaining tissue lipid reserves, if food is available to them, a greater proportion of

larvae are able to retain sufficient tissue reserves to successfully complete metamorphosis

(Fitt et al., 1984). Our results show that both Isochrysis sp. and Pavlova sp. micro-algae

concentrates are ingested and digested by T. noae larvae. Supplemental feeding with these

products during hatchery culture could support greater accumulation of tissue lipid reserves,

or reduced utilisation of existing reserves, that may influence subsequent post-larvae

performance. This aspect, as well as changes in larval tissue composition would be a logical

progression of future research following this study.

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4.1 Conclusion

The use of epiflouresence microscopy to investigate ingestion and digestion of various micro-

algae by T. noae larvae has shown that micro-algae varied in their rates of ingestion and

digestion and that both were influenced by larval age. Further research is required into the

relative benefits of providing a food source to T. noae larvae during hatchery culture and the

energetic basis of this. The influence of food provision on larval and post-larval success, the

nutrient compositions of micro-algae, rates of ingestion and digestion, and the influence of

diet composition on accumulation or retention of larval nutrient reserves will be important in

defining an optimal larval diet for tridacnids. Our results provide a basis for selection of more

appropriate micro-algae concentrates with potential in hatchery culture of T. noae, and

confirm the potential of these products as a replacement for live micro-algae. Further research

in this laboratory will aim to fine-tune hatchery culture methods for T. noae to facilitate

simpler protocols that are better suited to developing nations.

Acknowledgements

This study was supported by the Australian Centre for International Agriculture Research

(ACIAR) and the National Fisheries Authority (NFA) within ACIAR project FIS/2010/054

“Mariculture Development in New Ireland, Papua New Guinea” led by PCS at the University

of the Sunshine Coast. We are particularly grateful to Mr. Jeff Kinch and staff at the NFA

Nago Island Mariculture and Research Facility for in-country support and for facilitating this

research.

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Table 1: Live micro-algae concentrates (Instant Algae®, Reed Mariculture Inc.) used in this

study.

Group

(division)

Algae species

(diet)

Cell size (µm)

Golden-brown flagellates

(Haptophyta)

Isochrysis sp. (Isochrysis 1800®)

Pavlova sp. (Pavlova 1800®)

5-7

4-7

Green flagellates

(Chlorophyta)

Tetraselmis sp. (Tetraselmis 3600®) 10-12

Diatoms

(Bacillariophyta)

Thalassiosira weissflogii (TW 1200®) 7-20

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Table 2: Criteria used to assess the degree of micro-algae ingestion and digestion in this

study. Adapted from Duy et al. (2015) and Martínez-Fernández et al. (2004).

Stage Fluorescence Characteristics

(I) Empty No fluorescence Empty stomach; no micro-algae cells present; no

ingestion or digestion completed

(II) Ingestion Red Whole algal cells visible in the stomach.

(III) Digestion Pink, orange Whole and lysed algal cells mixed in the

stomach or lysed algae only

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Figure 1: Photosynthesizing pigments of micro-algae cells in the stomach of Tridacna noae

veligers fluorescing under blue-light illumination showing the difference between ingestion

and digestion: (A) red fluorescence of ingested but undigested, whole micro-algae cells in the

stomach (Stage II); (B) Pink and orange fluorescence of lysed micro-algae cells in the

stomach indicating digestion (Stage III). Arrow identifies fresh micro-algae concentrate for

comparison. Scale bar 50 µm.

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Figure 2: Frequency (%) of Tridacna noae larvae at (A) 48 h post-fertilisation and (B) 72 h

post-fertilisation observed at Stage II (shaded) and Stage III (unshaded) at different times

from the start of feeding. Different superscripts denote statistically different frequencies of

ingestion.

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Figure 3: Distribution of mean antero-posterior measurements (APM) for larvae observed to

ingest micro-algae concentrates: (A) Pavlova 1800®; (B) Isochrysis 1800®; (C) Tetraselmis

3600®. The distribution of APM for larvae fed micro-algae concentrates and observed to

remain empty (i.e. did not ingest) is also reported.

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Highlights:

We assessed ingestion/digestion of micro-algae by giant clam (Tridacna noae) larvae

Ingestion of micro-algae commenced between 24 h and 48 h post-fertilisation

Flagellates were ingested more readily than Tetraselmis sp. or Thalassiosira

weissflogii

Digestion of flagellates began 2 h after feeding began and completed within 12 h

Results provide a basis for nutritionally informed hatchery culture methods for T.

noae.

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