eftapWt 2

SUBSTITUTION OF BRINE SHRIMP NAUPLII WITH

TETRASElMIS CHYlI IN THE LARVICULTURE OF

MACROBRACHIUM ROSENBERGII

Su6stitution ofbrine shrimp naupliiwitli 'T.cftuii in tfte Iarviculture of'.M. rosen6ergii

2.1 Introduction

Microalgae are an essential food source in commercial rearing of

various animals including larval stages of many crustacean species. They

are an indispensable component in the feeding of penaeid larvae. In

addition, microalgae are used in the mass culture of several zooplankters

which in turn serve as food for larval and early juvenile stages of

crustaceans and fish.

Tetraselmis sp is a motile Parsinophyte, moving very actively in the

rearing medium. They are widely used for feeding penaeid larvae, bivalve

mollusc larvae and post larvae, Artemia and marine rotifers. Tetraselmis

chuii is an alga extensively used in the feeding of penaeid larvae. It is

having a protein content of 31 % on a dry weight basis (Lavens and

Sorgeloos, 1996). Macrobrachium rosenbergii larvae do not actively search

for food (New, 2002) and capture food by accidental collision (Moller,

1978). Gulbrandsen et al. (1996) suggested the use of larger micro algae

like Tetraselmis sp, which is ingested by larval fish, may provide critical

micronutrients. However, the use of T. chuii as a feed in the larviculture of

giant fresh water prawn has never been evaluated. Against these

backgrounds, a preliminary study was conducted to assess the

effectiveness of the alga T. cnuii (which is motile, nutritious and widely

used in the larviculture of many crustacean and fish species) in replacing

the highly priced and sometimes scarce Artemia nauplii which is considered

as an indispensable component in the larval rearing of the giant fresh water

prawn.

36

SuGstitution ofbrine shrimp naupliiwitli 'Lchuii in the Iarviculture 0/5\1. rosen6ergii

The objectives of the present experiment were to

1. Examine the usefullness of alga Tetraselmis chuii in fully or partially

replacing Artemia nauplii in the larviculture of the giant fresh water

prawn.

2. Reduce the cost and increase economic viability of post larval

production by reducing the quantity of brine shrimp nauplii.

2.2 Materials and Methods

The experiment was carried out following completely randomised

design with five overnight feeding regimes (including the control) with three

replicates each (Table 2.1).

2.2.1 Brood stock management

Berried prawns with greyish black eggs were procured from the wild

and transported to the hatchery in wide mouthed black plastic cans of 50 I

capacity. The brooders were disinfected (New and Singholka, 1985) and kept

in well aerated brackish water having a salinity of 5 ppt in 150 I FRP tanks.

Feeding was not done as the berried prawns with black eggs invariably

hatched by the next day morning.

2.2.2 Algal culture and hatching of Artemia Cysts

The alga Tetraselmis chuii was grown using the Modified Walne's

medium (Lavens and Sorgeloos, 1996), the composition of which is given in

Table 2.2. The stock culture was grown at a salinity range of 20 - 25 ppt in 3

Iiter Hoffkins flask. Before feeding the prawn larvae, the salinity was gradually

reduced to the range of 12 - 14 ppt in mass culture media maintained outside.

37

Su6stitutionofbrine shrimp nauplii witn T.chui: in tlie Iarvicuiture of:M. rosen6ergii

For Artemi« nauplii (also mentioned as BSN) production, OSI brand

Arlem;a cysts were used. The required quantity of cysts were weighed,

hydrated, decapsulated using commercial grade sodium hypochlorite and kept

in 25 ppt saline water with vigorous aeration for hatching (approximately I g

cystJ I sea water). Artificial light was also provided for hatching of the cysts.

The nauplii hatched in 18 - 24 hours. They were harvested and washed

thoroughly before feeding.

2.2.3 Larval rearing

The experiment was carried out using clear water method of larval

rearing in 100 I FRP tanks with 50 I rearing water. The water used for the

experiment was treated following New (2002). Larvae were stocked at the rate

of 100 numbers / I. They were fed with Artemia nauplii or algae (depending on

the treatment) on the second day of hatching in the morning and evening.

From the third day onwards feeding with live feed was done only in the

evening (5 PM). The inert feed used in the experiment was egg custard, the

composition of which is given in Table 2.3 (Kurup, 2003). The ingredients

were mixed well in an electric mixer and steam cooked for about 20 minutes.

Aftercooling it was stored in the refrigerator and used whenever required. Egg

custard was fed ad libitum to the larvae by dispersing it in the rearing water

evenly using dropper, starting on the third day when it was done only once in

the morning. On the fourth day the feeding frequency was increased to two

times and there after to four times daily at 8 30 AM, 10 30 AM, 12 30 PM and

3 PM after passing through the sieves to get the required particle size (Kurup,

2003, Table 2.4). Before dispersing the custard feed, the aeration was turned

38

Substitutionofbrine shrimp naupliiwitn 'T.chuiiin the Iarviculture of;M.rosen6erfJii

off in order to enable surfacing of the larvae. Artemia was given at the rate of

4 numbers/ ml (100 %) in the control and the alga T. chuii was given at the

rate of 1 lakh cells / ml in the treatment with 100 % alga (T4). The quantity of

live feed supplied in the other treatments were adjusted accordingly.

Siphoning of the waste and water exchange (20 - 50 %) was done daily before

the live feed was given in the evening.

2.2.4 Water quality and evaluation of various treatments

The daily water quality parameters measured included salinity and

temperature which was measured during the morning hours using Atago

refractometer and mercury thermometer respectively. pH and dissolved

oxygen were estimated twice weekly using pH meter (Eutech Cyberscan

model 510) and Winklers method (APHA, 1995) respectively.

The final evaluation of experimental diets was done based on the post

larval production, time required for the larvae to metamorphose to subsequent

stages and their relative survival. Mean larval stage was used to find out the

development of larvae. Mean larval stage was calculated using the formula,

ML8 = 2:(8 x Ps) where MLS is the mean larval stage, S is the larval stage

number and Ps is the proportion of larvae at stage S (Lovett and Felder,

1988).The larval stages were identified following Uno and Kwon (1969). The

larval progression and relative survival of larvae in each tank was estimated

every fifth day. The estimation of percentage composition of the different larval

stages and survival continued till 25th day when considerable number of post

larvae started appearing in all the tanks and it was difficult to draw uniform

samples (Alam et al., 1993a). In the mean time, daily observations were made

39

Su6stitution ofbrine shrimp naupfiiwitli T.cfiuii in tfie iaroiculture of:M. rosenberqii

in the tanks to assess the settlement of post larvae from zo" day of the

experiment and the appearance of first post larva in each tank was noted

down. On completion of rearing cycle (when more than 95 % of larvae

metamorphosed to PL), total length (from the tip of the rostrum to the end of

the telson) and wet weight of 50 post larvae which were randomly taken from

each tank was also measured as an additional parameter for assessing the

efficiency of different feeding regimes.

2.2.5 Statistical analysis

Data analysis was done by one-way ANOVA using SPSS 16.

Significant differences between the treatments were determined using

Duncan's multiple range test (DMRT) (P < 0.05). Data expressed in

percentages were normalised by arcsine transformation (Zar, 1984). However

non transformed data is given in the table (Table 2.7).

2.3 Results

The water quality parameters observed in all the experimental tanks

(Table 2.5) were well within the acceptable range (New and Singholka, 1985;

New, 2002) for the larval rearing of M. rosenbergii. Temperature was found be

to fluctuating between 27 to 30De in the present experiment. The optimum

temperature for larval rearing of M. rosenbergii is in the range of 28 to 31°C

(New, 2002). pH values ranged between 7.64 and 8.16. Salinity fluctuated

between 12 and 14 ppt while dissolved oxygen varied between 6.49 to 7.39

ppm during the larval rearing period. Slightly alkaline waters (pH 7 to 8.5) is

suggested by Valenti and Daniels (2000)) and salinity around 12 ppt and

dissolved oxygen near saturation level have been suggested as optimal by

40

Su6stitutionof6rine shrimp nauplii witfi 'I.cfiuii in tfie Iaroicuiture of:M. rosen6ergii

New (2002) for the larval rearing of M. rosenbergii. As recommended by the

above authors, the physico chemical parameters of the rearing water was

maintained stable without fluctuation through out the rearing period.

The highest mean survival of 24.4 ± 0.87 % was recorded in the control

followed by the treatment T1 wherein the mean survival was found to be 22.6

±1.5 %. No significant difference was observed among the control and T1 with

respect to mean survival.

The treatment T4 in which there was a complete substitution of Brine

shrimp nauplii (BSN) by the algae, no larvae were found to progress beyond

3rd larval stage and most of them were found dead by 3rd and 4th days with a

complete mortality on the 5th day of the experiment. The larvae were found to

be generally weak by the third larval rearing day. Hence, for convenience of

interpretation, data pertaining to this treatment was omitted from the graphs

and final production tables.

Although the duration for settlement of 95 % of post larva was observed

as 31 days in both control and T1 and the MLS showed significant variation

(P<O.05) among them only on the 5th day of the experiment, the appearance

of the first post larva was delayed in T1 (average of 26 days) and it took

significantly more number of days than in the control where it was 24 days.

The final mean length and wet weight of the larvae were also significantly

lower (P<O.05) in T1 than in the control. The MLS recorded in the different

treatments during the experimental period is presented in Table 2.6 and the

final percentage survival, duration of the larval rearing period, mean length

and the wet weight of the larvae are depicted in Table 2.7.

41

Substitutionofbrine shrimp naupliiwitn 'Echuii in the Iarvicuiture of:M,rosen6ergii

The treatments T2 and T3 with 50 % and 75 % replacement of BSN

respectively recorded average survival of 17.96 % and 15 % which was

significantly lower (P<0.05) than T1 and the control. The duration of the

experiment (settlement of more than 95 % post larvae) was longer and took

an average of 32 and 34 days in treatments T2 and T3 respectively. The

appearance of the post larva was also delayed (28 days in T2 and 29 days in

T3) and was significantly different from that of the control and T1. The total

length of the post larvae in T2 and T3 was also significantly lower than in the

control, but was not different from that in T1. The wet weight of the post larvae

recorded in T2 was comparable with in T1 and significantly lower than in the

control. Where as, the wet weight of post larvae in T3 was significantly lower

than T1 and the control (P<O.05).

2.4 Discussion

As seen from Tables 2.6 and 2.7 and Fig. 2.1 and Fig. 2.2, the control

supplied with 100 % brine shrimp nauplii (BSN) recorded significantly faster

growth and better survival than all the other treatments. This is indicative of

the poor performance of all other treatments due to the reduction in the BSN

supplied in various treatments. The survival and MLS of larvae in T1 was not

significantly different from the control, however it was definitely lower in T1

when compared to the control.

Microalgae has been widely used as a feed in the larval rearing of

fishes crustaceans and molluscs. Tetraselmis sp. is used as a feed for

penaeid larvae, bivalve mollusc larvae and post larvae, abalone larvae and

zooplankton like BSN and marine rotifers (Lavens and Soegeloos, 1996).

42

Su6stitutionof6rine shrimp nauphiwitfi T.cliuii in tlie iarviculture of:M. rosen6ergii

However, generally the larvae of M. rosenbergii larvae is judged primarily as

carnivorous as indicated by the higher levels of enzymes trypsin and esterase

in the early larval stages (Kamaruddin et al., 1994). Jones et al. (1993) also

reported the first feeding prawn larvae as carnivorous rather than herbivorous

based on enzyme studies. Cohen et al. (1976) concluded that M. rosenbergii

larvae do not feed directly on algae (Tetraselmis sp) and that the role of algae

in larviculture is primarily to purify the medium by assimilating ammonia.

However, the authors conducted the experiment by introducing prawn of

particular age into 14e labeled algal cultures. Cook and deBaissac (1994) also

reported the inability of the prawn larvae to derive nutrition from the algae by

investigating the fatty acid composition of larvae reared in green and clear

water. No significant difference in the fatty acid profiles was detected in the

larvae reared in the different rearing media and it was concluded by the

authors that phytoplankton contributed little to larval energy metabolism.

Joseph (1977) was also not able to perceive any fatty acid assimilation by the

larvae from algal populations in M. rosenbergii rearing tanks. Contrary to this,

Thresiamma et al. (2006) has stated that the micro alga Isochrysis galbana

was probably acting as a convenient feed to both prawn larvae and BSN

which resulted in a higher survival in the treatments which used the micro alga

when compared to the control in which no alga was used.

However, based on the results of the present experiment (all the larvae

perished in the treatment T4), it can be concluded that the prawn larvae were

notable to derive considerable nutrition from the micro algae especially during

the early stages. The prawn larvae might have ingested the T. chuii cells, but

43

Su6stitutionofbrine sfirimp naupliiwitfi 'T.cfiuii in the Iaroiculture of:Mo rosen6ergii

they were not able to assimilate considerable nutrition from the algae and as a

result, all of them died in spite of feeding the larvae with egg custard from the

third day of larval rearing. The surviving larvae in the treatment without BSN

(T4) were found to be generally weak by the third day followed by

considerable mortality leading to 100 % mortality on the fifth day. This result of

the present study is concurring with that of Sick and Beaty (1975) who could

not demonstrate a nutritional effect of phytoplankton added to the fresh water

prawn larval rearing systems.

While conducting a study on the larval rearing of green back flounder,

Rhombosolea tapirina using turbid green water constituted by Tetraselmis

suecice, Shaw et al. (2006) reported higher rotifer consumption rates than

clearwater particularly at low prey densities enabling reduction in the feeding

ration. Barahona- Fernandes (1982) reported a two fold increase in the

survival of larvae of marine fish, Dicentrarchus labrax when the same micro

alga (T. suecica) was added to the larval rearing medium. This was observed

in spite of daily water exchange which considerably reduced the algal

densities. According to the author, T. suecica has the added advantage of

avoiding algal settlement in green water culture systems during times of low

aeration and reduced algal densities due to its natural motility. While studying

the relationship between algae and larval nutrition, Maddox and Manzi (1976)

reported that all the seven algal species used for the larviculture of M.

rosenbergii were useful in enhancing the post larval production, among them,

the diatom Phaeodactylum tricornutum was found to be the most effective

one.

44

Su6stitution ofbrine shrimp nauplii witfi 'T.cliuii in ttie Iaroicuhure of:M..rosen6ergii

On the contrary, no such beneficial effect of the algae could be

demonstrated in the present experiment. Since the larval rearing of M.

rosenbergii was carried out in the present experiment using the clear water

system with regular siphoning of waste and water exchange, the algae were

not found grow to make the water green. The concentration of the algae used

in the present study in treatment T4 was comparable to that in penaeid larval

culture tanks where a density of 80,000 to 100000 cells of Tetraselmis sp. /ml

was maintained (FAO, 2007). In another feeding regime for penaeid larvae

using a combination of Chaetoceros neogracile and T. chuii (Lavens and

Sorgeloos, 1996), although, the concentration of the latter used as feed for

mysis stages (M1 to M3) varied from 20,000 to 30,000 cells/ml, Chaetoceros

neogracile was used at a higher density of 50,000 to 1 lakh cells / ml,

increasing the total number of algae supplied to the penaid mysis above I lakh

cells/ml. Cohen et al. (1976) used a concentration of 106 cells/ ml for 14C

labelled algal medium, where as the algal density was maintained between

1.1 to 1.4 x 105 cells fml by Thresiamma et al. (2006) in the larviculture of M.

rosenbergii. Compared to this the density of algal cells fed to the prawn larvae

in the present study was on the lower side (25,000, 50,000 and 75,000 cells

Irnl of rearing medium in T1, T2 and T3 respectively). In addition some of the

algal cells might have been lost while conducting cleaning and water

exchange, still lowering the available number of algae.

Moreover, the results of the present study fails to establish various

advantages of algae in the larval rearing of fishes and crustaceans like

enhanced survival and growth, resistance to diseases, enrichment of live feed

45

Su6stitutionofbrine shrimp naupliiwitli 'I. chuii in the Iartnculture of:M. rosenberqii

as claimed by many authors (Barahona-Fernandes, 1982; Naas et al., 1992;

Austin et al., 1992; Reghunathan and Wesley, 2004; Thresiamma et al., 2006;

Palmer et al., 2007). Most of all, the controlled production of micro-algae is a

complex and expensive procedure (Lavens and Sorgeloos, 1996).

It would thus appear that M. rosenbergii is unable to utilize algae as

food especially during the early larval stages and the lower density at which

the algae were supplied to the prawn larvae. It is worth reporting that even the

comparatively larger size and the natural motility of the alga T. chuii was not

utilized by the larvae of M. rosenbergii in the present experiment due to the

reasons discussed above.

2.5 Conclusion

The results of the present study showed that the M. rosenbergii larvae

were not able to derive considerable nutrition from the alga T. chuii, especially

during the early larval stages and when it is fed at lower densities. Though not

significantly different from the control, a lower post larval production was

recorded in TI (with 75 % Artemia nauplii) without considerable reduction in

the cost of production since T chuii is also a live feed and additional facilities

are required for maintaining its culture. Hence the use of the alga T. chuii as

feed for the M. rosenbergii larvae is not recommended.

46

Su6stitulion of6rine slirimp nauplii wttli 'T.cliuii in tlie tarvicu{turt of:M.rose n6eroii

Table 2.1 Feeding regimes in different treatments in the larvicul!ureof M. roseaberat!

~Treatments':' .v "'};;£" ' '; '.' j<..., :%,-o:tJ, 'Ii~!J:·;,~;I,-""eJ ' ·:<:f ';:'3, ;:-~: l ive feed "',',",A ,,'. ~ 'j;jf !··~=i<"ti.

T1 75 %A+25 % T

T2 50 % A + 50 % T

T3 25 % A + 75 % T

T4 Tetraselmis cnuii only

Control Artemia nauptf only

d'fW IdcT bla e 2.2 ornposition an I preparation 0 a nes me iurnConstituents I Quantities

Solution A (at 1 ml per IIter of culture)Ferric chloride (FeCI3) 0.8 q'lManoanoua chloride (MnCI2• 4H2D) 0.4 0Boric acid (H]B03) 33 .6 0EDTAI"), di-sodium salt 45.0 0Sodium di-hydrogen orthophosphate 20.0 9(NaH,PO•. 2H,O lSodium nitrate (NaND 3 ) 100 .0 0Solution B 1.0 ml

Make up to 1 litre with fresh water's Heat to dissolve

Solution BZinc chloride (ZnCI2) 2.1 0Cobaltous chloride (CoCI2,6 H2O) 2.0 0Ammonium molvbdate ((NH4)f\Mo,D 24, 4H, O) 0.9 0Cupric sulchate (CUS0 4, 5H2O) 2.00Concentrated HCI 10,0 ml

Make up to 100 rn! fresh water " Heal to dissolve

Solution C 'at 0.1 rnl er Iiter o f culture)

Vitamin B, 0.2 0Solution E 25.0ml

Make UD to 200 ml with fresh water"SolutIon D (for cu lture of diatoms-used in addition to solutions A and C. at 2 rnl per

Iiter of cu lture)

Sodium metasilicate (Na2Si03, 5H2O) I 40.00Make UDto 1 litre with fresh water" I Shake to dissolve

Solution E

Vitamin Bl ? I 0 1 0Make UP to 250 ml with fresh water') ISolution F (for cu lture of Chroomonas saUna - used in addition to so lutions A and C.

at 1 rnl per liter of culture)

Sodium nitrate (NaND ] ) I 200 .0 0Make up to 1 litre with fresh water" ) I

(a) Use 2.0 Q for culture o f Chaetoceros calcitran s in filtered sea wate r;

Cb) Ethvlen e dia m ine tetra acetic ac id ; I'c) Use dist illed wa ter if po ssib le. I

Su6stitutionof6rine shrimp naupliiwitli 'T.cliuii in tfie Iarviculture of'M. rosenberqii

Table 2.3 Composition of egg custard fed to larvae of M rosenbergii

",i~t; ::~:?~~~):)y.~)'<;;'

.,'.-.-- ..

Milk powder

Egg yolk

lecithin

.

15 9

1

1%

Squid oil + cod liver oil

Vitamin C

Red colour

Distilled water

1.5%

100 mg/kg

1 pinch

3-4 ml

Table 2.4 The particle size of custard feed given to the M. rosenbergii

larvae

Mesh size of the sieve (JJ) Larval stage

200-300 11 - IV

300 - 400 IV-V

400 - 500 VI-VIII

500 - 600 IX-X

-650 -1000 XI--_.

Table 2.5 Average ±s.d of various water quality parameters

recorded during the experimental period in the

larviculture of M rosenbergii.

~;Treatments

Parameters T1 T2 T3 Ck.Temperature ( UC) 28.01 ± 1 28.06 ± 0.96 28 ± 0.98 28.04 ± 0.94

pH 7.99 ± 0.13 8 ± 0.11 7.87 ± 0.09 7.89 ± 0.12

Salinity (ppt) 13.41 ±0.55 13.54 ± 0.56 13.57 ± 0.57 13.34 ± 0.54

Dissolved oxygen (mg/I) 7.03 ± 0.18 7.06 ± 0.16 6.99 ± 0.16 7.01 ± 0.21

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