Lal et al. SpringerPlus 2014, 3:568
http://www.springerplus.com/content/3/1/568
a SpringerOpen Journal
RESEARCH
Open Access
Complete larval development of the Monkey
River Prawn Macrobrachium lar (Palaemonidae)
using a novel greenwater technique
Monal M Lal1,2,3*, Johnson Seeto3 and Timothy D Pickering4
Abstract
This study documents the complete larval development of the Monkey River Prawn Macrobrachium lar using a
new greenwater rearing technique. Approximately 6,000 larvae were reared for 110 days at an initial stocking
density of 1 ind./6 L. Salinity at hatch was 10 ± 2 ppt and progressively increased to 30 ± 2 ppt until decapodids
had metamorphosed. Temperature was maintained at 28 ± 0.5°C, pH at 7.8 ± 0.2, DO2 > 6.5 mg/L and NH4+ and
NH3 ≤ 1.5 and ≤0.1 ppm respectively throughout the culture period. Larval development was extended and
occurred through 13 zoeal stages, with the first decapodid measuring 6.2 ± 0.63 mm in total length observed
after 77 days. 5 decapodids in total were produced, and overall survival to this stage was 0.08%. Overall, the
pattern of larval growth shares similarities with those of other Macrobrachium spp. that have a prolonged/normal
type of development, and it is likely that larvae underwent mark time moulting which contributed to the lengthened
development duration. While this study represents a significant breakthrough in efforts to domesticate M. lar,
improvement of larval survival rates and decreased time till metamorphosis are required before it can become fully
viable for commercial scale aquaculture.
Keywords: Biofloc; Greenwater technique; Macrobrachium lar; Zoea; Larvae; Decapodid; Larval development
Introduction
The Monkey River Prawn Macrobrachium lar is a large
palaemonid freshwater prawn indigenous to a number of
regions across the Indo-West Pacific, and of importance
to artisanal and subsistence fisheries in almost all areas
where it occurs (Holthuis 1950, 1980; Nandlal 2005, 2010;
Barbier et al. 2006; Ponia 2010).
Because of its size, relatively fast growth rates and a
number of other favourable culture characteristics, it
appears to have good potential for aquaculture except
for one major constraint; the availability of seed stock
for grow-out is severely limited by difficulties in rearing
the larvae from hatch until metamorphosis into the decapodid. Shokita et al. (1984) in their report on inland
water prawns present in Fiji identified 3 species (M. lar,
M. australe and M. equidens) which appeared to have
* Correspondence: monal.lal@my.jcu.edu.au
1
Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook
University, Townsville Campus, Townsville, Queensland, Australia
2
College of Marine and Environmental Sciences, James Cook University,
Townsville Campus, Townsville, Queensland, Australia
Full list of author information is available at the end of the article
potential for aquaculture in the country, and went on to
state that mass artificial seed production of any had yet
to succeed. Of the three species discussed, M. lar was
put forth as the leading candidate owing to the large size
it attains relative to the other two species.
The state of knowledge on the larval development of
M. lar is fragmentary, largely due to difficulties encountered by previous researchers in rearing larvae to
metamorphosis. The first attempt at completing larval
development in captivity was by Kubota (1972), who
was able to rear larvae till the fifth zoeal stage. Further
attempts were made by Atkinson (1973, 1977), Muranaka
in Hanson and Goodwin (1977), Takano (1987), Nandlal
(2010), Sethi and Roy in Kutty and Valenti (2010) and
Sethi et al. (2011), however all experienced total larval
mortality before metamorphosis to the decapodid. There
is also a distinct lack of further information resulting from
the work of M. S. Muranaka (in Hanson and Goodwin
1977), who had reportedly managed to produce decapodids, however it appears no further publications resulted
from that study.
© 2014 Lal et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
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These studies provide some indications why larval rearing so far has proved to be unsuccessful, and include conditions of improper salinity, temperature, disease (Kubota
1972), as well as nutrition (Kubota 1972; Atkinson 1973,
1977). The most recent investigation into the role of salinity and temperature on larval development found that
newly-emerged larvae are able to tolerate fresh or brackish
water of approximately 10 ppt, but require gradually increasing salinities post-hatch reaching 30–35 ppt, which
needs to be maintained until metamorphosis into the decapodid (Lal et al. 2012). The information obtained from
these investigations was utilised in an attempt to close the
lifecycle for this species, and to concurrently better understand its early life history.
A summary of developmental characteristics and rearing
condition requirements of Macrobrachium spp. whose
larviculture has been investigated is provided in Shokita
(1985) and Willführ-Nast et al. (1993). Among species
which exhibit the typical or prolonged/normal type of
development, there are comparatively few which like M.
lar, require oceanic salinities for successful development. This paper describes the morphological larval development of M. lar which was successfully completed
in the laboratory using a novel greenwater rearing technique, with the specific objective of describing larvae in
a simple manner through all larval developmental stages
until metamorphosis into the decapodid, in order to facilitate further rearing efforts for aquaculture.
Methods
Broodstock collection and maintenance
8 adult male and 19 female M. lar broodstock were collected at 2 sites in Waisere Creek, Vugalei District, Tailevu
Province, Viti Levu, Fiji (17° 56’ 42.14” S; 178° 33’ 11.31” E
and 17° 56’ 43.42” S; 178° 32’ 55.81” E). All prawns were
maintained in single 2500 L rectangular Fibre-Reinforced
Plastic (FRP) and square 1000 L polyethylene tanks, filled
to a depth of 500 mm with 50 μm filtered freshwater
maintained at 26 ± 0.5°C. Aeration was provided via four
air diffusers at ~200 mL/sec at each outlet. Water parameters remained at DO2 > 6.5 mg/L and pH 7.2-7.6. Females
bearing mature grey coloured eggs were transferred to
hatching tanks.
Hatch tank preparation
Circular 1000 L flat-bottomed polyethylene tanks for
hatching larvae doubled as larval rearing tanks (LRTs).
Approximately 300 L brackish water (10 ± 0.5 ppt at
28 ± 0.5°C) which had been filtered to 50 μm was prepared in a dedicated 1000 L polyethylene water preparation tank. Other water parameters were as follows;
pH 7.8 ± 0.2, DO2 > 6.5 mg/L and average NH4+ and
NH3 ≤ 1.5 and ≤0.1 ppm respectively. Gentle aeration
was provided at ~15–30 mL/sec via four air diffusers. A
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fluorescent light tube fitting with a 1.2 m Osram 36 W
‘warm white’ tube and 1.2 m Eurolux 36 W ‘cool white’
tube was suspended over each tank providing ~6700 lx at
the surface of the water. These lights were kept off during
hatching and turned on the morning after.
Rearing of larvae
Larvae were reared in a mass culture trial over 110 days.
5 ovigerous females at the grey egg stage were introduced
into 3 separate LRTs and maintained for 48 h until they
had spawned. Females were not fed during this period. An
initial hatch estimate was made after 24 h, and the tank
volume increased by 100 L using brackish water mixed
to 20 ± 0.5 ppt. This strategy was employed to slowly
increase the salinity of the culture medium, and tank
volume was increased by 100 L without any water exchanges every 24 h until 800 L was reached. This approach was developed after Lal et al. (2012). After 800 L
was reached, 12-25% (~100-200 L) was exchanged daily
with 50 μm filtered seawater at 30 ± 0.5 ppt and 28 ±
0.5°C, depending on requirements. The target salinity
when mixing replacement water was set at either 20 or
32 ppt. During the first few days of culture, replacement
water was mixed to 20 ppt, to progressively increase salinity with successive daily water exchanges. Because larvae were hatched at 10 ppt, ~20 ppt was attained by day
7 of culture.
Following this, all subsequent water exchanges for each
tank were mixed to 32 ppt, attaining ~30 ppt by day 30
of culture. This salinity was maintained until decapodids
were produced, after which point all water exchanges
used treated freshwater to progressively reduce salinity
to 0 ppt. Because larvae were reared using a greenwater
technique, at 2–3 day intervals the replacement water
for water exchanges was sourced from cultures of either
marine or freshwater microalgae or both. Both sets of
cultures were of mixed species of varying concentrations, as it was found to be much easier and less timeconsuming to mass culture using this method than to
maintain monospecific/axenic cultures of different species due to the volumes required.
In the marine microalgae cultures referred to as ‘brown
water’ (Imamura et al. 2009), various species of diatoms were
found to dominate, with the genera Nitzschia, Navicula
and Skeletonema being the most common. Cells of
these diatoms were naturally present in the seawater used
for the cultures, and were seedstock for the starter cultures. Likewise with the freshwater microalgae cultures
which were also of mixed species and referred to as ‘green
water’, a single Desmodesmus sp. and several Chlorella
spp. were dominant. The source of the stock green water
was a series of three 5000 L tanks containing Nile Tilapia
Oreochromis niloticus. By using a mixture of brown water
and green water mixed to the required target salinity, the
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Larvae were observed consuming a number of other
live feed items apart from Artemia nauplii and metanauplii. The majority of these comprised of biofloc,
and biofloc-associated microorganisms. A number of
the biofloc-associated microorganisms included various
types of rotifers, the most abundant of which was a Colurella sp. (Family Brachionidae), together with various nematodes and protozoans.
It proved to be difficult to quantify biofloc volumes,
however a general guide established was to maintain
concentrations of 1500–2500 pieces of biofloc/L. This
proved to be an apparently optimal density based on
qualitative observations of larval feeding behaviour. A
single piece of biofloc was loosely defined as any aggregation of biofloc material up to 5 mm. At times between
scheduled feeding intervals if larvae were observed to
have consumed all feed from the previous offering, they
were encouraged to feed on biofloc present in the tank
by stirring settled material on the tank floor.
formation of biofloc particles was encouraged in the tanks
which larvae were seen to actively feed on. Further descriptions of methods used to culture the brown and
greenwater are documented by Imamura et al. (2009).
Aeration volume was progressively increased as the larvae
developed. Gentle aeration was employed at ~15–30 mL/sec
at each air diffuser for early stage larvae e.g. zoea I, so as
not to damage them by excessive turbulence. This rate
was increased to ~150–200 mL/sec for mid and late stage
larvae (zoeae V to X), in order to keep feed and biofloc
particles in suspension where they could be accessed. This
also prevented circulating matter from settling on the tank
floor which would have lead to accelerated decomposition
and poor water quality.
Feeding
Larvae were fed ad lib. at 2-hourly intervals, from 0700
to 1900 daily with 3 different types of steamed custard
feeds (Table 1). These were offered by pressing the custard
particles through sieves of varying mesh sizes as outlined
in Figure 1, with Artemia nauplii being offered once daily
in the afternoon. The sieve mesh sizes were selected according to the development stage of larvae in individual
tanks, to ensure they were able to capture and feed on
particles of an appropriate size in relation to their body
and mouth sizes. The feeding sieve mesh sizes are detailed
in Table 2.
When larvae had developed to zoea XIII, crushed formulated prawn pellet (32% crude protein, Crest Chicken
Limited, Fiji) was offered to complement the custard
feeds. Algamac 3050 flake (Aquafauna Biomarine Inc,
Hawthorne, USA) was also used to supplement the custard feeds and to enrich Artemia nauplii offered to the
larvae. For direct feeding, the Algamac 3050 flake was
weighed according to the daily feed ration measured for
each tank and either screened through the appropriately
sized feeding mesh for zoeae I to IX larvae, or added
directly to the water for zoea X onwards.
Larval microscopy
All microscopy was carried out using a binocular compound microscope (Olympus CH-2) fitted with a calibrated
eyepiece graticule. All photomicroscopy was carried out
using a digital camera (Nikon Coolpix E995) mounted on
one of the microscope eyepieces. Larvae were routinely examined at 0900 daily, using a cavity slide without a coverslip to avoid squashing the specimens. All observations
and photographs were of live individuals and specimens
were either preserved immediately afterwards in 80%
ethanol for larval staging work, or returned to the tank.
Description of larval development in Macrobrachium lar
A select number of larval morphological features were
examined and changes in these recorded and used for
characterising larval development. These morphological
features included carapace armature (rostrum, supraorbital spines and pterygostomian spines), as well as developments of the tail fan (telson and uropods), pereiopods
Table 1 Prepared custard feed ingredients modified from Imamura et al. (2009)
Egg custard
Squid custard
b
Prawn custard
1 egg yolk (approx. 20 g in weight)
25 g whole squid flesh (including viscera)
25 g whole prawnsc
20 g milk powder
25 g whole prawns
1 EPA capsule (1000 mg)a
15 mL water
1 whole egg
1 multivitamin capsule
1 EPA capsule (1000 mg)a
1 egg yolk (approx. 20 g in weight)
10 mL soyabean cooking oil
1 multivitamin capsule
1 EPA capsule (1000 mg)a
15 g Algamac 3050 flake
10 mL soybean cooking oil
1 Multi-vitamin capsule
10 mL soybean cooking oil
1 lecithin capsule (1200 mg)
a
EPA 1000 mg capsules included 180 mg EPA and 120 mg DHA.
Whole Boston Squid Loligo pealei (Lund's Seafood Inc.).
c
Palaemon concinnus, P. debilis, Macrobrachium grandimanus and M. equidens.
b
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Figure 1 Larval feeding schedule. The feeds offered were Egg custard (EC), squid custard (SC), shrimp custard (ShC), Algamac 3050 flake (AM),
Artemia nauplii (Art.), Artemia meta-nauplii (mArt.) and commercial formulated prawn pellet (CFPP).
(walking legs), pleopods (swimmerets) and antennules and
antennae. Larvae were also measured to determine their
total and carapace lengths.
10 individuals of the same apparent morphological developmental stage and age were sampled and used in
making determinations of larval stage and average size.
All larvae were thoroughly examined to ensure the morphological features being recorded were consistent between the individuals sampled. Once determinations of
stage had been made, representative specimens were
lodged at the Marine Reference Collection of the School
of Marine Studies, Faculty of Science, Technology and
Environment, University of the South Pacific, Suva, Fiji
Islands under Catalogue Number 5940.
Descriptions of the morphological development of M.
lar larvae provided here have intentionally been kept
simple, for the purpose of easily identifying developmental
stages. Rather than adopting the approach of providing an
exhaustive description of larval morphological features,
descriptions are from a more practical perspective and
have used a few, major and easily detectable features rather than minor morphological changes. The rationale
behind this was to provide a means of simply and rapidly
Table 2 Feeding sieve mesh sizes used for particular
larval stages
Larval stages
Feeding sieve number
Feeding sieve
mesh size (μm2)
1
150
zoea I to zoea III
2
400
zoea II to zoea V
3
750
zoea VI to zoea X
4
1000
zoea X to decapodid
identifying live specimens for any future larviculture work
aimed at mass production of decapodids in a hatchery
system, involving this or a similar species.
Specimen drawings
Simple line diagrams of specimens showing the body
outlines without internal structures e.g. organs, musculature and external chromatophore patterns were produced
with the aid of photographs of live specimens. All diagrams were then outlined in black ink before being
scanned at 600 dpi and processed using Adobe Photoshop
version 7.0 software.
Results
Larval rearing
Larvae developed through 13 zoeal stages before metamorphosing (Table 3), with 5 decapodids produced after
77, 78, 85, 101 and 110 days of culture respectively. Survival to this stage was 0.08%, and 0.27% to zoea XII/XIII.
Mortality proved to be very high, especially during the
first few days of culture (Figure 2).
Patterns of growth were regular until zoea V was
reached (Figure 3). Development through zoeae I to IV
occurred consistently with average intermoult durations
of ~3 days, ~8 days from V to VIII and ~12 days from
IX to XI. From this point, development was irregular
with durations of 21 and 63 days for zoeae XII and XIII
respectively. Metamorphosis into the decapodid was also
prolonged, taking 34 days from the time of metamorphosis
of the first till last individuals. Salinity and temperature
variations over the culture period did not vary outside the
desired limits (Figure 2).
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Table 3 Age and size ranges of M. lar larvae
24 h, before salinity was reduced to 0 ppt in 5 ppt steps
each day by exchanging 20–25% of the tank volume
(Figure 2). This procedure was also carried out for the
next 4 decapodids collected from the tank.
Stage
Age (Day of first
appearance)
Carapace length
(mm)
Total length
(mm)
Zoea I
1
0.25 ± 0.20
0.8 ± 0.21
Zoea II
3
0.35 ± 0.10
1.1 ± 0.32
Zoea III
7
0.40 ± 0.10
1.2 ± 0.25
Descriptions of larval development stages
Zoea IV
9
0.45 ± 0.15
1.3 ± 0.38
Zoea V
11
0.50 ± 0.20
1.5 ± 0.28
Zoea VI
16
0.70 ± 0.15
2.7 ± 0.36
Zoea VII
20
0.65 ± 0.25
2.9 ± 0.42
Zoea VIII
23
0.80 ± 0.22
3.2 ± 0.31
Zoea IX
26
1.18 ± 0.12
4.15 ± 0.29
Zoea X
31
1.20 ± 0.15
4.2 ± 0.32
Zoea XI
39
1.20 ± 0.18
4.4 ± 0.28
Zoea XII
45
1.65 ± 0.26
5.0 ± 0.37
Zoea XIII
48
1.80 ± 0.32
5.4 ± 0.41
Decapodid
77
2.25 ± 0.38
6.2 ± 0.63
A summary of larval stages and their developmental features is presented in Table 4, and the appearance of each
larval stage is reproduced in line diagrams in Figure 4.
Photomicrographs showing specific larval staging features
for each zoeal and the decapodid stage are presented in
Additional file 1: Figure S1, Additional file 2: Figure S2,
Additional file 3: Figure S3, Additional file 4: Figure S4,
Additional file 5: Figure S5, Additional file 6: Figure S6,
Additional file 7: Figure S7, Additional file 8: Figure S8,
Additional file 9: Figure S9, Additional file 10: Figure S10,
Additional file 11: Figure S11, Additional file 12: Figure S12,
Additional file 13: Figure S13, Additional file 14: Figure S14,
Additional file 15: Figure S15.
Metamorphosis
Zoea I
Due to poor larval survival (10 individuals remaining by
day 65), it became impractical to rear survivors in the
single 1000 L tank which had not encountered total
mortality. All larvae were then transferred to a 60 L
cylindro-conical fibreglass LRT and the trial continued.
After the first decapodid was observed on day 77, salinity
was reduced from 30 to 24.3 ppt over days 80–81, with
further gradual reductions carried out until 0 ppt was
reached by day 96.
The first decapodid was removed and transferred to a
separate 60 L cylindro-conical fibreglass LRT, which was
maintained at 28.8 ppt and 28 ± 0.5°C for a period of
Zoea I larvae of M. lar possess a short, straight rostrum
which is not toothed. The telson does not possess uropods, is roughly heart-shaped and doesn't articulate, forming a solid join with the sixth abdominal somite. This
stage does not possess fully formed walking legs (pereiopods) with the first two pairs present as buds only. The
first three maxillipeds pairs are present, the eyes are sessile
and located on the anterior half of the cephalothorax. The
body is highly transparent and lipid globules can be seen
in the foregut and midgut regions (Additional files 1:
Figure S1). Most zoea I larvae were not observed to feed
immediately after hatch, however some individuals seized
Figure 2 Larval survivorship, salinity and temperature data recorded over the culture period. The increase in larval population over days 1
to 6 is due to continued input of larvae from spawning broodstock. Broodstock were removed on day 6. Following metamorphosis of the first
decapodid, the water parameters displayed here were recorded in the tank containing the remaining larvae.
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Figure 3 Day of first appearance of larval stages and their intermoult durations. Values indicated are ± Standard Deviation (SD).
and fed on egg custard and biofloc particles. Most individuals were seen feeding on the second day post-hatch.
with chromatophores distributed over various parts of
the body.
Zoea II
Zoea V
The rostrum remains largely unchanged from zoea I, however the carapace now has pairs of supra-orbital and pterygostomian spines. The most noticeable feature of this
stage is the presence of stalked eyes. A join starts forming
between the sixth abdominal somite and telson allowing
partial articulation, and within the telson, rudimentary
uropod exopods may be seen forming which will appear
in the next stage (Additional file 2: Figure S2). The antennal flagella are present but not segmented. The first two
pereiopods have developed and appear similar to the third
maxilliped.
4 and 1 segments are present in the antennal and antennular flagellae respectively. All pereiopods are present
now, with the fifth pereiopod becoming fully developed.
The second tooth on the dorsal carina of the rostrum remains. The telson has also gradually changed shape, becoming noticeably rectangular from its previous triangular
outline (Additional file 5: Figure S5).
Zoea III
The first tooth on the rostrum appears, located immediately behind the eyes on the dorsal carina and the
pterygostomian spine develops 2 obvious points. The
antennal flagellum becomes divided, and now contains
3 segments. Uropod exopods emerge, and rudimentary
uropod endopods can be seen developing inside the telson
(Additional file 3: Figure S3). All maxillipeds and pereiopods are better developed in this stage, with the
fourth pereiopod appearing as a biramous bud.
Zoea IV
The second tooth on the rostrum appears, appearing in
front of the first tooth. The fifth pereiopod appears as a uniramous bud, while the fourth pereiopod is no longer a
bud and possesses all segments. The uropod endopods
now emerge, making the tail fan complete (Additional file 4:
Figure S4). This stage is often noticeably pigmented,
Zoea VI
The rostrum remains unchanged, with the exception of
the appearance of 2 or more setae in front of the second
tooth. 5 and 1 segments are present in the antennal and
antennular flagellae respectively. Pleopod buds now appear,
but usually only for the third and fourth, and occasionally
fifth pairs of pleopods (Additional file 6: Figure S6).
Zoea VII
This stage is often the first point at which mark-time
moulting may be encountered, and variability in morphological development between individuals was observed.
The rostral tooth count can vary between 2–3 teeth on
the dorsal carina. Upon reaching zoea III, some individuals had their first rostral tooth emerging almost parallel
to the supra-orbital spine, thus the tooth was located well
behind the eye (termed the post-orbital tooth here). In
other individuals, their first rostral tooth emerged immediately behind or parallel to the eye (Additional file 7:
Figure S7).
For individuals which had their first rostral tooth well
behind the eye, they possessed a total of 3 rostral teeth
Stage
Eyes
Rostrum
(Teeth)
Antennal
flagellum
(Segments)
Antennal
flagellum
(Segments)
Uropod
Telson
Pleopods
I
Sessile
0
0
0
II
Stalked 0
0
0
Simple
exopods
present
Partially articulating
join with 6th
abdominal somite
III
1
3
0
Exopods
emerge
Fully articulating join
with 6th abdominal
somite
IV
2
3-4
0
Endopods
emerge
V
2
4
1
VI*
2
5
1
Buds for 3rd and 4th pair emerge, with 5th pair also
present on some individuals
VII**
2-3
6-8
1
Buds for 2nd and 5th pair emerge, already
emergent buds for 3rd and 4th pair elongate
VIII*
3
8
2
3rd and 4th pleopod pairs now biramous, with 5th
pair also biramous on some individuals. Buds for 1st
pair emerge
IX*
3-4
9
3
All pleopods now biramous and possess setae. Buds
of appendices internae seen on 3rd and 4th
pleopods
X*
4-5
10
4
XI**
5-6 or 6-7 14-18
6-8
XII*
7-8
15-20
9-12
XIII*
8-9
29+
14+
Decapodid
8-9 (v.c.#) 40+
+ 1 (d.c.#)
16+
Pereiopods
Non-articulating join
with 6th abdominal
somite
Shape changes from a
fan-like to rectangular
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Table 4 Summary of readily discernible features characterising the larvae and decapodid of M. lar
5th pair emerge
Chelae on 2nd pair visible
Appendices internae well developed on 3rd and 4th Chelae on 2nd pair larger.
pleopods
Chelae on 1st pair visible
Exopods with natatory setae absent or
greatly reduced. Chelae on 2nd pair
prominent
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*Indicates stages which may have at least 2–3 instars and **indicates stages which may have 3–4 instars. #V.c. refers to the ventral carina and d.c. the dorsal carina of the rostrum.
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Figure 4 Larval development stages of M. lar.
by the time they moulted to stage VI, whereas others
had only 2. Those individuals which had only 2 teeth occasionally developed a protrusion in front of the second
dorsal tooth where the next tooth would emerge (see
Additional file 11: Figure S11 for an example of this in a
Zoea X larva). 6–8 segments are present in the antennal
flagellum and 1 segment remains in the antennular flagellum. The pleopod buds have also become more developed, as the third and fourth pairs have elongated and
buds for the second and fifth pairs emerged. In individuals which already possessed a fifth pleopod bud, it elongated along with the third and fourth buds.
Zoea VIII
The rostral tooth count is usually 3 teeth along the dorsal carina, however some individuals may still possess 2
teeth, as described for zoea VII. 8 and 2 segments are
present in the antennal and antennular flagellae respectively. All pleopods which had elongated during the previous stage are biramous now and possess natatory
setae. In most individuals, the second pleopod bud elongates, and the first pair of pleopods emerges as a simple
bud (Additional file 8: Figure S8).
Zoea IX
This larval stage possesses 3–4 teeth along the dorsal
carina of the rostrum, and individuals which possessed
only 3 teeth did not have a post-orbital rostral tooth. 9
and 3 segments are present in the antennal and antennular flagellae respectively. All pleopods are biramous and
possess setae, making their development almost complete.
Now that the endopods of the third and fourth pleopods
are fully formed, buds of the appendices internae begin
to appear along their inner margins (Additional file 9:
Figure S9).
Zoea X
An additional tooth is added to the rostrum, bringing
the tooth count up to 4–5 teeth along the dorsal carina.
Individuals which possessed only 3–4 teeth did not have
a post-orbital rostral tooth (Additional file 11: Figure S11).
10 and 4 segments are present in the antennal and antennular flagellae respectively. Chelae appear, forming at the
ends of the second pair of pereiopods (Additional file 10:
Figure S10).
Zoea XI
The number of rostral teeth is 6–7, or 5–6 along the
dorsal carina. Individuals which possessed 5–6 teeth did
not have a post-orbital rostral tooth. 14–18 and 6–8 segments are present in the antennal and antennular flagellae respectively. All pleopods are now fully formed, with
complete development of the appendices internae. Chelae on the second pair of pereiopods are now larger, and
used by the larva in feeding. It was difficult to ascertain
whether chelae on the first pair of pereiopods had developed at this stage on the live specimens examined. The
basal segment of the fifth pair of pleopods now begins to
develop setae on its rear margin, with 4 present at first
appearance (Additional file 12: Figure S12).
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Zoea XII
The rostral tooth count is 7–8 teeth along the dorsal
carina, and individuals which possessed 7 teeth did not
have a post-orbital rostral tooth. 15–20 and 9–14 segments are present in the antennal and antennular flagellae respectively. Chelae on the second pair of pereiopods
(second chelipeds) further enlarge from the previous
stage, and have developed more setae along the pollex
and dactylus. Chelae are now evident on the first pair of
pereiopods (first chelipeds). The basal segment of the fifth
pair of pleopods possesses 8 setae on its rear margin
(Additional file 13: Figure S13).
Zoea XIII
This was the final zoeal stage observed, with a rostral
tooth count of 8–9 teeth along the dorsal carina. Individuals which possessed 8 teeth did not have a post-orbital
rostral tooth. >29 segments are present in the antennal flagellum which is twice the length of the scaphocerite, with
14 in the antennular flagellum. The second chelipeds have
enlarged and the larva can be seen capturing Artemia
nauplii with these while feeding. Although smaller in comparison, the first chelipeds are also noticeable when used
during feeding. The basal segment of the fifth pair of pleopods possesses 11 setae along its rear margin (Additional
file 14: Figure S14). A characteristic habit of this larval
stage not noticed in the earlier stages was to sit on the
bottom of the tank and walk for short distances using the
pereiopod endopods.
Decapodid
The rostral tooth count for this stage is 8–9 teeth along
the dorsal carina. Individuals which possessed 8 teeth on
the dorsal carina did not have a post-orbital rostral tooth,
and the ventral carina now bears a single tooth a short
distance from the rostral apex. This stage possessed >40
and >16 segments in the antennal and antennular flagellae
respectively. The telson now appears triangular from
above, with the rear margin coming to a point and resembling that of the adult.
The second chelipeds are now greatly enlarged and are
the largest pair of legs. In 2 of the 5 individuals observed,
rudimentary natatory exopods were visible after the moult
to decapodid had been completed (Additional file 15:
Figure S15). This feature, along with the benthic behaviour
characteristic of decapodids of other Macrobrachium spp.
was confirmation that this was indeed the decapodid stage
and not yet another zoeal stage. 3 of the 5 decapodids
produced were maintained in isolation after having been
acclimated to freshwater, and found to moult into the
first juvenile after a period of 5 days from metamorphosis. As mass cultures were used for rearing in this
Page 9 of 13
study, it was not possible to distinguish the exact number of instars the zoea larvae passed through before
metamorphosis.
Two particular stages viz. zoea VII and XI, may include
more than 2 and up to an estimated 4 instars, as some individuals showed nearly identical morphological features
but had increased in size relative to other larvae within
the same stage. Any changes noted in morphology were
subtle and very minor, e.g. additional setae on the antennal
scale, pleopods and pereiopod exopods. This was interpreted as possible evidence of mark time moulting, and in
the case of some zoea XI larvae, terminally additive staging. By taking these observations into consideration, the
larvae of M. lar may moult through a minimum of 22 and
maximum of 31 instars before being ready to metamorphose into the decapodid.
Discussion
Although larvae were morphologically similar to other
Macrobrachium spp. which display a 'prolonged/normal'
(Alekhnovich and Kulesh 2001; Jalihal et al. 1993) development pattern, there were important differences in behaviour, growth and feed preferences.
Larval behaviour
The larvae displayed a more benthic habit (even in the
presence of aeration), unlike M. rosenbergii where healthy
larvae without aeration remain near the water surface
(Valenti et al. 2010). This agrees with Atkinson (1973),
who mentions that larvae occupied the upper portion
of the water column but were not directly associated
with the surface. This may be related to predator
avoidance and use of sub-surface currents for larval
transport out of coastal waters during dispersal, and
could be important for providing feed where larvae are
able to easily access it in culture. Cannibalism was not
observed during this study although it cannot be conclusively ruled out, whereas this has been documented
for M. rosenbergii (Valenti et al. 2010). Our observations
differ with Nandlal (2010), who reported that M. lar larvae
did cannibalise.
Growth and development
Several Macrobrachium spp. produce larvae with requirements for oceanic salinity conditions (30–35 ppt).
The number of stages described for these ranges from
9 (M. grandimanus; Shokita 1985), 10 (M. equidens;
Ngoc-Ho 1976 and M. intermedium; Williamson 1971)
and up to 12 (M. sp.; Ngoc-Ho 1976 and M. olfersii,
Dugger and Dobkin 1975), compared to the 13 described
here for M. lar.
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The most obvious trends (Figure 3) are increasing
intermoult periods from averages of 4–8 and 12.6 days
during zoeal stages I, V and IX respectively, with corresponding shifts in moults into subsequent larval stages.
It is well known that Macrobrachium spp. exhibit plasticity in the number of instars, morphological development
stages and developmental pathways before metamorphosis, as responses to unfavourable environmental
conditions, inappropriate nutrition and the presence/
absence of settlement cues (Anger 2001).
It is possible that larvae in this study underwent marktime moulting due to either unfavourable environmental
conditions, inappropriate nutrition or both, and further
investigation is required to determine this. Nevertheless, it
appears that conditions were sufficient to allow 5 larvae to
metamorphose into decapodids. Evidence of mark time
moulting has also been reported in other Macrobrachium
spp., particularly those which inhabit marine or partlymarine conditions as adults, viz. M. equidens (Ngoc-Ho
1976), M. rosenbergii (Gomez Diaz and Kasahara 1987;
Valenti et al. 2010) and M. vollenhovenii (Müller et al.
2003).
Observations of inherent developmental plasticity have
been related to the wide marine dispersal capacity of
some species (Shokita 1985), and is an important factor
to consider in developing commercial hatchery operations, as extended larval development duration increases
operating costs. Other Macrobrachium spp. which share
a wide Indo-Pacific distribution with M. lar and exhibit
developmental variability include M. grandimanus (Shokita
1985) and M. equidens (Ngoc-Ho 1976). Studies of the
population structure of M. lar using mitochondrial DNA
markers in Japan (Imai et al. 2007) and in Pacific Island
countries (Mather et al. 2006; Nandlal 2010) have shown
high genetic diversity over large geographic scales. This
implies substantial gene flow between widely separated
habitats, and indicates long-lived pelagic larvae able to
colonise habitats far removed from their place of hatch.
Feeds and feeding
The observation that the larvae may have different feed
preferences to other Macrobrachium spp. requires further
investigation. The larvae of most Macrobrachium spp. are
omnivorous, with carnivorous tendencies. This has been
demonstrated for M. rosenbergii up to zoea VII, after
which they become more omnivorous (Dhont et al. 2010).
Reasons stated for this include the larvae remaining primitive during early development, with only partially developed systems for digestion, sight and chemoreception.
The gut remains poorly developed until larval stages V
and VI, resulting in a low digestive capacity and hence
the early stages are reliant on highly digestible live feeds
(eg. zooplankton), which provide exogenous prey enzymes
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to begin the proper processes of digestion (Dhont et al.
2010).
The primary feed used for most other species including M. rosenbergii (Ling 1961, 1962; Uno and Kwon
1969), M. vollenhovenii (Willführ-Nast et al. 1993), M.
carcinus (Choudhury 1971b, 1971c), M. novaehollandiae (Greenwood et al. 1976), M. americanum (Monaco
1975; Holtschmit and Pfeiler 1984), M. equidens (Ngoc-Ho
1976) and M. acanthurus (Choudhury 1970, 1971a) is
the nauplii of Artemia spp. If larval M. lar are proven to
show a preference for plant-based feeds, this may imply
lower feed-associated costs as they are generally cheaper
to obtain.
Previous studies which reared M. lar had used Artemia
nauplii as the staple feed with varying results, and all failed
to reach the decapodid stage (Kubota 1972; Atkinson
1973, 1977; Nandlal 2010). Supplementary feeds utilised
included ox liver particles (Nandlal 2010), Melon Fly Bactrocera (Dacus) cucurbitae larvae along with a prepared
feed incorporating shrimp meal (20%; Atkinson 1973,
1977).
The production of decapodids in this study may be
partly attributed to a more suitable larval diet. The specific feeds which may have met larval nutritional needs
were the prepared custard feeds and biofloc. It is likely
that the custard feeds supplied dietary energy requirements during the later zoeal stages when they were easier to metabolise, with biofloc being important earlier
during development. Avnimelech (2009) mentions that
suspended biofloc is eaten and contributes significantly
to the protein requirements of species reared in Biofloc
Technology systems including Tilapia, various Carp and
the marine shrimp Litopenaeus vannamei and Penaeus
monodon.
Larval rearing and survival
The overall survival rate from hatch till metamorphosis
was very low (0.08%), similar to likely rates in the wild
of <0.1% (Bagenal 1967; Jennings et al. 2006), and dependant on temperature, salinity, food availability and
development/settlement cues (Willführ-Nast et al. 1993;
Anger 2001). In crustacean species for which larviculture
techniques are being developed, larval survival rates in
early trials are not much better than those inferred for
wild larvae. As an example, initial research on the Mud
Crab Scylla sp. in Indonesia produced survival rates till
metamorphosis of 0.07–0.19 and 0.5–3.2% (Cholik
1999).
Survival rates reported for other Macrobrachium spp.
have been comparatively low during initial attempts, but
have improved with continued refinement of culture techniques. Perhaps the best example of this is M. rosenbergii.
When decapodids for this species were first produced, the
survival rate till metamorphosis was 16–17% (Ling 1961,
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1962). Today, survival rates are 40–50% in flow-through
hatchery systems, 60–80% in Thai backyard hatcheries
and 60–80% in experimental and commercial recirculation systems, with development durations of 29–35 days
(Valenti et al. 2010). It can thus be expected that there will
be room for improvement in M. lar larviculture performance as a result of further research.
Survival rates reported for other Macrobrachium spp.
assessed for culture potential are varied. Survival till
metamorphosis was 12% for M. vollenhovenii (WillführNast et al. 1993), 21% and 2.5% for M. acanthurus and
M. carcinus respectively (Dobkin et al. 1974), 9% for M.
acanthurus (Choudhury 1971a), >90% for M. amazonicum
(Anger et al. 2009), 20% for M. americanum (Holtschmit
and Pfeiler 1984) and ~59% for M. nipponense (MacLean
and Brown 1991). It is difficult to compare these rates
with those of M. lar in this study, as some of the species
do not have larvae which require fully marine conditions for development.
Those species which have a requirement for >20 ppt
include M. vollenhovenii (16–24 ppt), M. acanthurus
(<20 ppt) and M. americanum (20–30 ppt for early larval stages only; Choudhury 1971a; Dobkin et al. 1974;
Holtschmit and Pfeiler 1984 and Willführ-Nast et al.
1993). Although a more detailed discussion of the salinity requirements for M. lar is provided in Lal et al.
(2012), salinity tolerance investigations indicated that
survival and development of newly-emerged larvae was
highest in entirely fresh or slightly brackish water, increasing to full-strength seawater by the mid-point of
larval development. Past this point, salinities >30 ppt
were critical for larvae to progress past stages VII and
VIII (Lal et al. 2012). Larvae were also unable to survive
in freshwater beyond a period of 4 days, confirming that
this species has a truly oceanic larval dispersal phase
(Kubota 1972; Maciolek 1972; Mather et al. 2006;
Nandlal 2010).
Another consideration is that survival for extended periods may be genetically 'hard wired' due to the prolonged
larval dispersal phase. Despite this, optimising culture
methods can be expected to shorten development time, as
the first decapodid was produced on day 77 of culture
here, whereas Atkinson's (1973, 1977) study reached zoea
XI on day 89 before all larvae died.
Culture system
There has been considerable debate over whether greenwater or clearwater culture systems are better suited for
rearing larvae of Macrobrachium spp. While both systems
have their merits, clearwater systems have been proven
easier to manage (Valenti et al. 2010). During this study,
the propagation of biofloc in the LRTs was likely to have
provided adequate nutrition to some larvae, and responsible
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at least in part for enabling completion of development
and metamorphosis of decapodids. While it remains unclear if larvae derived benefits from feeding directly on
the microalgal particles bound up in the biofloc or on
biofloc-associated biota, if the simple greenwater system
used here can be adapted for mass seed production, the
potential to realise cost, time and labour savings by
using non-monospecific microalgal cultures is substantial, especially in resource-poor regions where hatchery
production of M. lar is a priority.
Several studies have examined the role that microalgae
play in the larviculture of Macrobrachium spp.. Lober
and Zeng (2009) found higher survival and shorter development duration in M. rosenbergii reared at higher
vs. lower microalgal concentrations, while reduced ammonia levels (Cohen et al. 1976) and enhanced survival
and metamorphosis rates were seen when larvae were
cultured with 7 species of unicellular algae (Manzi and
Maddox 1977; Manzi et al. 1977).
Although larval Macrobrachium spp. are known to be
visual, particulate feeders (Atkinson 1973, 1977), and do
not feed directly on algal cells except via accidental ingestion in negligible amounts (Cohen et al. 1976), the
benefits of microalgal enrichment of Artemia nauplii
have been well documented (Dhont et al. 2010; Valenti
et al. 2010). Cohen et al. (1976) report the presence of
algae facilitate growth only indirectly by removing toxic
material e.g. ammonia, however when considering the
incorporation of microalgal cells into biofloc particles in
the current study, they would offer similar benefits as
enriched Artemia. Further mention is made that when
incorporating microalgae, the balance of the ecological
system in the LRT is more complicated, as more trophic
levels exist and less control can be exercised over the
whole system. Contrary to this, M. rosenbergii has been
reared using no intensive hatchery techniques in a
greenwater system operated completely without water
exchange (Cheah and Ang 1979). LRTs were topped up
with greenwater to counter evaporative losses under
two salinity regimes of 6–8 ppt and 12–14 ppt. Results
showed no significant difference in survival rates to the
decapodid of 39.6% and 36.9% for the two regimes
respectively.
Conclusions
Based on the results of this study, commercial-scale
hatchery operations for M. lar require further research
into improvement of larval survival and reduction in
development duration to ensure feasibility. Nonetheless, our research provides a record of the first ever
complete larval development of M. lar with accompanied morphological descriptions, both of which are key
tools for successful larviculture for it and potentially
other related species.
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Additional files
Additional file 1: Figure S1. Zoea I. Lateral view (a), dorso-lateral view
of carapace showing sessile eyes (b) and non-articulating telson with
sixth abdominal somite join (c).
Additional file 2: Figure S2. Zoea II. Dorsal view (a) and lateral view
(b). Rudimentary uropod exopod development within telson (arrows) (c),
formation of join between telson and sixth abdominal somite (arrow) (d)
and supra-orbital spine (s.o.s.) (e).
Additional file 3: Figure S3. Zoea III. Dorsal view (a) and lateral view
(b) of larva. Antennal flagellum containing three segments (c), emergent
uropod exopods and rudimentary uropod endopods visible within the
telson (d). First rostral tooth on the dorsal carina (e).
Additional file 4: Figure S4. Zoea IV. Dorsal view (a) and lateral view
(b). Second rostral tooth on the dorsal carina (c), uniramous buds which
are the undeveloped fifth pereiopods (arrows) (d) and complete tail fan
development with the emergence of the uropod endopods (e).
Additional file 5: Figure S5. Zoea V. Dorsal view (a) and lateral view
(b). Telson almost rectangular (c), two teeth still present on the dorsal
carina (d) and the fully developed fifth pereiopod (e).
Additional file 6: Figure S6. Zoea VI. Lateral view (a). Emergent buds
for the third, fourth and fifth pairs of pleopods (b). Two setae present in
front of the second rostral tooth (c).
Additional file 7: Figure S7. Zoea VII. Lateral view (a). Elongated third
and fourth pleopod bud pairs (b) and 6 – 8 segments in the antennal
flagellum (c). Two setae still present in front of the second rostral tooth (d).
Additional file 8: Figure S8. Zoea VIII. Lateral view (a). Third rostral
tooth on the dorsal carina (b) and further pleopod development (c). 2
segments present in the antennular flagellum (arrows; d) and 8 segments
in the antennal flagellum (e).
Additional file 9: Figure S9. Zoea IX. Lateral view of larva (a). 3
segments present in the antennular flagellum (arrow; b) and 9 segments
in the antennal flagellum (c). Fourth rostral tooth on the dorsal carina
(obscured by eye but position indicated by arrow; d) and all pleopods
now biramous with setae (e). Buds of the appendices internae are visible
developing along the inner margins of the third and fourth pleopod pair
endopods (arrows).
Additional file 10: Figure S10. Zoea X. Lateral view (a). Four segments
present in the antennular flagellum (arrows; b) and fifth rostral tooth present
on the dorsal carina (c). Ten segments in the antennal flagellum (d) and
rudimentary chelae present on the second pair of pereiopods (arrow; e).
Additional file 11: Figure S11. Zoea X showing variable rostral
dentition. Individual with a post-orbital tooth (a) and an individual without a
post-orbital tooth displaying a protrusion of the carapace (arrow; b).
Additional file 12: Figure S12. Zoea XI. Lateral view (a). Chelae present
on the second pair of pereiopods are now larger (arrow; b) and appendix
interna development is complete on all pleopods (arrows; c). This
individual has 5 teeth on the dorsal carina (d). 14 – 18 segments present
in the antennal flagellum (e).
Additional file 13: Figure S13. Zoea XII. Lateral view (a). This individual
has 8 teeth on the dorsal carina (b). 9 segments present in the
antennular flagellum (c) and ~20 segments in the antennal flagellum (d).
8 setae present on the rear margin of the basal segment of the fifth pair
of pleopods (e).
Additional file 14: Figure S14. Zoea XIII. Lateral view (a). This
individual has 9 teeth on the dorsal carina (b). Chelae present on the
second pair of pereiopods are now further enlarged (c) and 11 setae
present on the rear margin of the basal segment of the fifth pair of
pleopods (d). 14 segments present in the antennular flagellum (e).
Additional file 15: Figure S15. Decapodid. Lateral view (a). This
individual has 8 teeth on the dorsal carina (b). The first rostral tooth on
the ventral carina (arrow; c) and greatly enlarged second pair of
pereiopods and chelae (d). Rudimentary natatory pereiopod exopodites
(arrows; e) and triangular telson (f). 16+ segments present in the
antennular flagellum (g).
Page 12 of 13
Abbreviations
ACIAR: Australian Centre for International Agricultural Research;
DO2: Dissolved oxygen; FRP: Fibre-reinforced plastic; LRT: Larval rearing tank;
mg/L: Milligrams per litre; mm: Millimetres; ppt: Parts per thousand.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MML carried out all larval rearing trials, participated in the investigation
design, developed the rearing protocol and drafted the manuscript. JS
participated in the investigation design, provided advice on larval rearing
and edited the manuscript. TDP initiated and conceptualised the project,
provided project funding, statistical advice and technical input on
investigation design. He also developed the rearing protocol and edited the
manuscript. All authors read and approved the manuscript.
Acknowledgements
This project was funded by the Australian Centre for International
Agricultural Research (ACIAR) DABL Mini-Project MS0808. The authors wish to
thank Cathy Hair for assistance with project administration, Maika Ciqo for
broodstock collection and Tomohiro Imamura for technical assistance in
developing a larval rearing technique for M. lar. We would also like to
acknowledge the assistance of Mere Brown in processing the larval
photomicrographs. The research was carried out during a USP-ACIAR
Scholarship awarded to MML and it forms a part of his MSc thesis.
Author details
1
Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook
University, Townsville Campus, Townsville, Queensland, Australia. 2College of
Marine and Environmental Sciences, James Cook University, Townsville
Campus, Townsville, Queensland, Australia. 3School of Marine Studies, Faculty
of Science, Technology and Environment, University of the South Pacific,
Laucala Campus, Suva, Fiji Islands. 4Coastal Fisheries Programme, Aquaculture
Section, Secretariat of the Pacific Community, Suva Regional Office, Nabua,
Suva, Fiji Islands.
Received: 17 June 2014 Accepted: 23 September 2014
Published: 30 September 2014
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doi:10.1186/2193-1801-3-568
Cite this article as: Lal et al.: Complete larval development of the
Monkey River Prawn Macrobrachium lar (Palaemonidae) using a novel
greenwater technique. SpringerPlus 2014 3:568.