Dynamics of glucose in the haemolymph of female giant freshwater prawn, Macrobrachium rosenbergii, influences reproductive and non-reproductive moulting cycles

Dynamics of glucose in the haemolymph of female

giant freshwater prawn, Macrobrachium rosenbergii,

influences reproductive and non-reproductive

moulting cycles

Noor Azlina Kamaruding1, Noraznawati Ismail2, Safiah Jasmani3, Marcy N Wilder4 &

Mhd Ikhwanuddin3

1School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia2Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia3Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Kuala Terengganu, Malaysia4Japan International Research Center for Agricultural Science, Tsukuba, Ibaraki Prefecture, Japan

Correspondence: N Ismail, Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu,

Malaysia. E-mail: [email protected]

Abstract

Glucose, when measured in haemolymph, has

been found to reflect a useful predictor of energetic

investment. This study evaluated the pattern of

glucose in the haemolymph, with an attempt to

gain a better insight into the role of glucose as

nutritional source of ovarian development and

energy reserves during reproductive and non-

reproductive moulting cycles. The haemolymph of

female giant freshwater prawn, Macrobrachium

rosenbergii, was obtained at eight different moult-

ing stages, and levels of glucose were determined

using an enzymatic colorimetric glucose-oxidase

method in parallel with a histological examination

of ovarian development. Glucose levels were rela-

tively low (0.15 � 0.02 mg mL�1) at D0 stage, an

abrupt increase (0.52 � 0.13 mg mL�1) during

premoult D1 stage and declined (0.32 � 0.10 and

0.31 � 0.09 mg mL�1) during premoult D2 and

D3 stages, respectively; thereafter, a slight increase

(0.43 � 0.09 mg mL�1) occurred at post-moult A

stage. The progression of ovarian growth, marked

by an increasing gonadosomatic index (GSI) pat-

tern during the reproductive moulting cycle (C0–D3 stages), was directly proportionate to fluctua-

tions in glucose levels. GSI was significantly posi-

tively correlated with glucose (R = 0.40;

P < 0.05). In contrast, glucose was notably higher

at post-moult A and premoult D2 stages during

non-reproductive moulting cycle, the period during

which glucose is crucial for exoskeletal chitin syn-

thesis. At this particular stage, a negative correla-

tion between body weight and glucose

(R = �0.36; P < 0.05) was observed. The dynam-

ics of glucose in the haemolymph of female

M. rosenbergii correlated with ovarian growth,

which signify that glucose as nutritional source

for vitellogenesis, and affects the body weight of

this species.

Keywords: Macrobrachium rosenbergii, glucose

metabolism, vitellogenesis, moulting, haemolymph

Introduction

The metabolic processes of energy usage during

moulting, or ecdysis, are important topics in aqua-

culture, due to the numerous physiological, bio-

chemical and behavioural changes that occur

during the course of each moulting cycle (Chang

1995). Knowledge of energy utilization can poten-

tially benefit hatchery operations and farm man-

agement, especially to assist aquaculture

practitioners in the selection of female broodstock

with quality traits, in order to eliminate unproduc-

tive females and non-viable spawns and produce

high-quality gametes (Emerenciano, Cuzon, Mas-

caro, Ar�evalo, Nore�na-Barroso, Jerŏnimo, Racotta

& Gaxiola 2012). It is also helpful for husbandry

protocols in development of feeds (Simon, Fitzgib-

bon, Battison, Carter & Battaglene 2015) as well

© 2016 John Wiley & Sons Ltd 1

Aquaculture Research, 2016, 1–10 doi:10.1111/are.13176

as the management of predation (Matsumasa &

Murai 2005).

In this study, we investigated glucose, the main

monosaccharide in crustacean haemolymph, which

serves several purposes such as synthesis of

mucopolysaccharides, chitin, nicotinamide adenine

dinucleotide phosphate (NADPH), glycogen and the

formation of pyruvate. In haemolymph, glucose

comes either from the direct absorption of dietary

glucose through hepatopancreatic and intestinal

epithelial cells or from hepatopancreas (Rosas,

Cuzon, Gaxiola, Arena, Lemaire, Soyez & Van

Wormhoudt 2000; Radford, Marsden, Davison &

Taylor 2005; Simon & Jeffs 2013), where it is stored

as glycogen or synthesized by the gluconeogenic

pathway. Glucose homoeostasis is tightly regulated

by crustacean hyperglycaemic hormone (CHH) pro-

duced by sinus gland (X-organ complex) of the eye-

stalks (Santos & Keller 1993; Nagaraju & Reddy

2002; Reddy, Nagaraju & Reddy 2004; Nagaraju,

Prasad & Reddy 2005; Nagaraju, Reddy & Reddy

2006; Nagaraju, Kumari, Prasad, Rajitha, Meenu,

Rao & Naik 2009; Chung, Zmora, Katayama &

Tsutsui 2010; Nagaraju 2011; Nagaraju, Kumari,

Prasad, Naik & Borst 2011; Pei-Chen, Su-Hua,

Nagaraju, Wei-Shiun & Chi-Ying 2013), in the bal-

ance between anabolic (gluconeogenesis and glyco-

genesis) and catabolic (glycogenolysis and

glycolysis) processes and rates of uptake from gas-

trointestinal tract (Verri, Mandal, Zilli, Bossa, Man-

dal, Ingrosso, Zonno, Vilella, Ahern & Storelli 2001;

Oliveira, Eichler, Rossi & Da Silva 2004). Energy in

the form of adenosine triphosphate (ATP) is pro-

duced from free glucose occurs in cells, whereby it

is rapidly converted to glucose-6-phosphate by hex-

okinase (HK). Then, glucose-6-phosphate follows

one of three primary fates: glycogenesis, glycolysis

or the pentose-phosphate pathway (Santos & Keller

1993; Oliveira et al. 2004).

Vitellogenesis is known as the biosynthesis of

yolk proteins that was secreted as vitellogenin (Vg)

in the haemolymph, transported into ovary and

accumulated in the oocytes as vitellin (Subramo-

niam 2011). Vg, the precursor of yolk protein,

acts to supply both energy and building blocks to

support embryonic growth, which has been

reviewed by Wilder, Okumura and Tsutsui (2010).

To date, extensive work has been done on mea-

surement of vitellogenin in the haemolymph as

predictor of gonad maturity (Okumura & Aida

2000; Arcos, Ibarra & Racotta 2010), but there is

a very limited study concerning the significance

functional of carbohydrate component of Vg with

specific focus on the free sugar (glucose). Accord-

ing to Tirumalai and Subramoniam (2001), glu-

cose to a lesser extent is one of the constituents of

egg yolk, the major source of nutrients for the

developing embryo.

Chitin (poly-ß-1,4-N-acetyl-D-glucosamine) has

been demonstrated widely in nature including in

the exoskeletal of most invertebrates (Cauchie

2002). In hepatopancreas, carbohydrate, which is

the complex macromolecule of sugar, is stored pri-

marily in the form of glycogen, converted into glu-

cose and later into glucosamine and

acetylglucosamine; the latter is polymerized to

form chitin (Merzendorfer & Zimoch 2003).

Although existing information on fundamental

biology stated an involvement of glucose in the

metabolic processes, but the precise nature of its

role in the giant freshwater prawn, Macrobrachium

rosenbergii has not been fully elucidated. As such,

this study was undertaken with the aim of gaining

a better insight into the role of glucose as the

nutritional source of energy during reproductive

and non-reproductive moulting cycles of female

M. rosenbergii. Another aim of this study was to

evaluate the pattern of glucose throughout the

moulting cycle to function as a predictive indicator

of future spawning capabilities and growth capaci-

ties in this species.

Materials and methods

Experimental animals and their maintenance

Eighty females M. rosenbergii with a mean body

weight of 31.03 � 0.75 g were obtained from a

commercial source in Thailand. Prior to their use

in the experiment, the broodstock was acclimated

to the laboratory conditions at Japan International

Research Center for Agricultural Sciences (JIR-

CAS), Tsukuba, Japan, for at least 2 weeks in a

five-ton circular polyvinyl chloride (PVC) matura-

tion tank equipped with a centre drain. Water

entering the centre drain of the tank flowed by

gravity into a common filtration system housed in

an adjacent room. The tank was connected to a

biological filter in a closed recirculating system.

Lighting to maintain a 12-L: 12-D photoperiod

over the PVC maturation tank was controlled

using a programmable timer. Substrate in the form

of horizontal strips of polyethylene was provided to

increase the submerged surface area and shelter

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–102

Glucose as biomarker of reproduction and growth N A Kamaruding et al. Aquaculture Research, 2016, 1–10

subordinate and newly moulted individuals. Water

quality was monitored weekly by checking ammo-

nium (≤0.03 ppm), nitrite (≤1 ppm) and nitrate

(≤60 ppm) levels. The pH value was maintained at

a range of 7.0–8.3. Food was offered in excess,

and the leftover food particles were removed by

siphoning the bottom of the tank. After the two-

week acclimation period, the individual prawns

were transferred to 59 9 34 cm2 Perspex aquaria,

each divided into two compartments by perforated

plastic, for ecdysis observation. Ecdysis, marked by

the presence of exuviae from the animal’s body,

was observed daily. As ecdysis occurs at night, the

incidence was recorded the following morning.

The temperature of the aquarium was maintained

at 28°C using a centralized-electronic control sys-

tem. Food was offered once per day ad libitum. A

total of 0.3 g of commercial finisher pellets (42%

protein) for bottom-feeding invertebrates was given

per animal.

Moulting staging

Moulting stages were determined based on obser-

vations of setagenesis occurring in the setae of the

pleopods. The general characteristic of moulting

stage is shown in Fig. 1. The distal fifth of the left-

side of each animal was excised, and the extent of

setagenesis was observed using a digital image sys-

tem connected to a computer (Olympus U-CMAD3,

Tokyo, Japan). Each moulting cycle was classified

into eight substages: post-moult (stages A and B),

intermoult (stages C0 and C1) and premoult (stages

D0, D1, D2 and D3).

Haemolymph sampling

Five prawns were collected for each representative

moulting stage. The animals were anesthetized by

placing them in an ice bucket for approximately

5–10 min, and 100 lL of haemolymph was

withdrawn through the pericardial cavity. The

haemolymph samples were plunged into liquid

nitrogen (�196°C) and stored at �80°C until

glucose analysis.

Determination of Gonadosomatic Index (GSI) and

Hepatosomatic Index (HSI)

Body weight was determined, which the ovaries

and hepatopancreas were quickly excised and

weighed. GSI and HSI were calculated using the

following formulas as the procedure outlined by

Zhang, Zuo, Chen, Zhao, Hu and Wang (2007):

GSI ð%Þ ¼ ovary weight ðgÞ=body weight ðgÞ� 100

HSI ð%Þ ¼ hepatopancreas weight ðgÞ=body weight ðgÞ � 100

Examination of the progress of vitellogenesis

during ovarian development

For microscopic observation of the ovarian devel-

opment, the prawns were killed at eight different

moulting stages. A small ovary tissues sample was

taken for histological examination to confirm the

extent of vitellogenesis. Briefly, the ovary tissues

were fixed in Bouin’s solution, embedded in paraf-

fin and sectioned into 5 lm pieces. The sections

were stained with haematoxylin and counter-

stained with eosin for histological observation.

Ovary development stage was classified based on

oocyte diameter and the relative abundance of

oocytes as described by Meeratana & Sobhon

(2007).

Quantification of haemolymph glucose

concentration

Haemolymph glucose concentration was assayed

with glucose-oxidase in a 96-well microplate for-

mat, using a commercial test kit (Merck kGaA,

Darmstadt, Germany). The analysis was performed

according to the manufacturer’s protocol. Briefly,

varying concentrations of 2, 4, 6, 8 and 10 nmol

of glucose standard per well were prepared by add-

ing the glucose standard in a concentration of

1 nmol mL�1. A total of 5 lL of haemolymph was

added to each well. Next, the volume of standard

or sample in each well was adjusted with glucose

assay buffer provided by the manufacturer to a

final volume of 50 lL. A total of 100 lL, contain-ing 50 lL of reaction mix and a 50 lL mixture of

sample or standard with glucose assay buffer, was

prepared in each well. The reaction was incubated

for 30 min at 37°C to allow for colour develop-

ment, after which the absorbance was measured

at 570 nm using a Multiskan Microplate Photome-

ter (Thermo-Scientific, Waltham, MA, USA). The

unknown haemolymph concentration of glucose

© 2016 John Wiley & Sons Ltd, Aquaculture Research, 1–10 3

Aquaculture Research, 2016, 1–10 Glucose as biomarker of reproduction and growth N A Kamaruding et al.

was determined from the standard curve con-

structed for glucose.

Statistical analysis

All data were subjected to analysis of variance fol-

lowed by comparison of means using Duncan’s

multiple range test which was significant at

P < 0.05. The statistical analysis was performed

using Statistical Package for Social Sciences (SPSS)

for Windows, version 20.0 (IBM, Armonk, NY,

USA). Pearson’s correlation was used to evaluate

the significance of the relationships among body

weight, GSI, HSI and glucose concentration.

Results and discussion

Figure 2a shows interactions among GSI, HSI and

haemolymph glucose levels during the reproduc-

tive moulting cycle, showing that development of

the ovary was synchronous with the moulting

stages. A synchronization of ovarian development

and moulting in M. rosenbergii was also reported

by Wilder, Okumura and Aida (1991) and Oku-

mura and Aida (2000). GSI was recorded at

0.57 � 0.02% at the initial stage of C0. A histo-

logical section of the ovary at the C0 stage, as

shown in Fig. 3a, shows oil globules surrounding

the nucleus, which mark the oocytes’ progress to

the endogenous vitellogenic stage. A slight incre-

ment in GSI was observed at the C1 stage

(1.21 � 0.14%), with oocytes completely filled

with yolk globules, indicating secondary vitelloge-

nesis, as shown in Fig. 3b. GSI was sustained at

the D0 stage (1.69 � 0.18%), and oocytes were

seen progressing to the late vitellogenic phase, as

shown by histological section in Fig. 3c. This stage

was followed by a marked increase in GSI at the

D1 stage (2.64 � 0.35%), with the nucleus posi-

tion at the centre of the oocytes in the maturation

stage, as shown in Fig. 3d. A steeper increased in

GSI was clearly seen later, at the D2 stage

(4.29 � 0.19%), and it continued to increase until

the D3 stage (6.28 � 0.83%). Both the D2 and D3

stages showed oocytes at the maturation stage,

based on the accumulation of yolk granules

(Fig. 3e, f). Breakdown of the germinal vesicle was

further seen at the D3 stage. Following moulting,

GSI reached 9.58 � 0.41% at Stage A, with the

oocytes achieving maximum diameter (ranging

300–450 lm) at the maturation stage as shown

in Fig. 3g. This stage was followed by spawning,

after which GSI value returned to the basal levels

of 0.54 � 0.06% at post-reproductive moulting

B stageDevelopment

of internal matrix

C0 stageInternal cones

(IC)

C1 stage Retraction of matrix in the

IC

D0 stageRetraction of

epidermis

D1 stageDeveloping new setae

D2 stageFormation of

barbules in the new setae

D3 stageFolding of new setae

A stage Clear setae

matrix

C0 C1 D0 B D1 D2 D3A

Soft & slippery SoftDegree of hardening increasing

Degree of carapace hardness:

Characteristics of setagenesis

Moulting stage:

Figure 1 General characteristics

of moulting stage with reference to

the characteristics of setagenesis

and degree of carapace hardness in

the giant freshwater prawn,

M. rosenbergii.



Stage B. The mature oocytes were released during

spawning, leaving the empty lobes and primary

oocytes in the ovarian pouch, as indicated by his-

tology (Fig. 3h).

Increase in oocytes diameter is associated with

vitellogenesis, which contributes to the rapid

development of oocytes; it conceptually involves

the synthesis of vitellogenin and the uptake of

vitellogenin from the haemolymph into the

oocytes, accompanied by an accumulation of yolk

globules (Okumura & Aida 2000). The oocytes

were found to undergo second vitellogenesis dur-

ing the C1 stage as the oocytes took up vitel-

logenin by endocytosis and lipid droplets appeared

at the periphery of the ooplasm and invaded it.

This process was followed by the early premoult

D0 stage, the germinal vesicle, which generally

contains one nucleolus that occupies a central

position in the oocyte. As the moulting progressed

to the late premoult D3 stage, the nucleolus began

to regress as the chromosomes condensed; this

phenomenon marks germinal vesicle breakdown

(GVBD), which begins 4–5 days before exuviation

(Cledon 1986).

Contrary to this finding, GSI displayed a consis-

tent trend during the non-reproductive moulting

cycle, ranging from 0.36 � 0.09 to

0.48 � 0.02%; the difference was not statistically

significant (P > 0.05) (Fig. 2b). During this entire

cycle, the oocytes remained in a previtellogenic

stage, as the cytoplasm of the oocytes was baso-

philic, and the nucleoli were clearly visible within

the nucleus, as shown by histology in Fig. 3i, indi-

cating that moulting is independent of ovarian

development. Only somatic growth continued. This

finding is consistent with those of O’Donovan,

aab

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C0 (5) C1 (5) D0 (5) D1 (5) D2 (5) D3 (5) A (5) B (5)

haemolym

ph glucose concentration(m

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olymph glucose concentration

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Reproductive molting cycle

a a a a a a a

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0.10

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C0 (5) C1 (5) D0 (5) D1 (5) D2 (5) D3 (3) A (5)

Non-reproductive molting cycle

Gonadosomatic index Hepatosomatic index Glucose

PO (immature oocytes)C0-A

Ovary development Oc3 Oc4 mOc pOcOocyte diameter(in range) (µm)

(50–150) (50–200) (100–500) (30–100)

C0 C1-D0 D1-A B (spawned)

(a)

(b)Figure 2 Interactions of GSI and

HSI with haemolymph glucose

concentration (mean � SEM) dur-

ing (a) reproductive and (b) non-

reproductive moulting cycles in

laboratory-maintained broodstock

of female giant freshwater prawn,

M. rosenbergii. mOc, mature

oocyte; pOc, primary oocyte; Oc3,

early vitellogenic oocyte (endoge-

nous); Oc4, late vitellogenic oocyte

(exogenous); PO, primary oocyte.

Numbers in parentheses represent

individual prawns sampled. Differ-

ent letters at each point indicate

significantly difference (P < 0.05).



Abraham and Cohen (1984), Wilder et al. (1991)

and Okumura and Aida (2000).

The highest significant HSI value was found at

the C1 stage during the reproductive moulting

cycle (6.18 � 0.81%; P < 0.05), compared to

post-moult (stages A and B) (3.82 � 0.33 and

3.78 � 0.39%, respectively) and intermoult stage

C0 (4.35 � 0.28%). There were no significant dif-

ferences in HSI values during the premoult stage

(4.89 � 0.29 to 5.37 � 0.71%; P > 0.05). Simi-

larly, there were no significant differences in HSI

during the non-reproductive moulting cycle

(3.80 � 0.62 to 5.15 � 0.23%; P > 0.05). It is

interesting to note that HSI trend indicated no

clear relation of hepatopancreas’s mobilization in

the secretion of new cuticle during moulting per-

iod or vitellogenesis, in spite of these events

generated competitive resource in the hepatopan-

creas (Subramoniam 2000; Magalhaes, Mossilin &

Mantelatto 2012).

Only significantly the highest value of HSI corre-

sponded to Stage C1 which could be associated

with active food consumption; however, this

assumption should be further assessed.

There were no significant fluctuations in haemo-

lymph glucose concentrations during the repro-

ductive moulting cycle in the female M. rosenbergii

among the intermoult, premoult D0, and post-

moult B stages (0.12 � 0.01 to 0.26 � 0.06 mg

mL�1; P > 0.05). An abrupt rise in glucose level

to 0.52 � 0.13 mg mL�1 was observed during

the early premoult D1 stage. However, the level

then declined to 0.32 � 0.10 mg mL�1 at pre-

moult D2 and stayed at 0.31 � 0.09 mg mL�1 at

(g)

mOc

(h)

(a)

Oc3

(b)

Oc4

PO

(c)

nuc

Oc4

Fc

(d)

mOcnuc

(e)

mOc(f)

mOc

GVBD

(i)

pOc

PO

Figure 3 Histological section of ovary (H&E staining) showing reproductive development stages synchronized with

moulting stage in laboratory-maintained broodstock of female giant freshwater prawn, M. rosenbergii (magnification

1009). (a) C0 stage: early vitellogenic oocyte (endogenous) (Oc3); (b) C1 stage: late vitellogenic oocyte (exogenous)

(Oc4); (c) D0 stage: late vitellogenic oocyte (Oc4) with nucleus (nuc) surrounded by follicle cells (Fc); (d) D1 stage:

mature oocyte (mOc); (e) D2 stage: mature oocyte (mOc); (f) D3 stage: mature oocyte (mOc) with condensed

nucleus, germinal vesicle breakdown (GVBD); (g) A stage: mature oocyte (mOc); (h) B stage: post-spawning with

primary oocyte (pOc) remaining in ovarian pouch; and (i) non-reproductive moulting cycle: previtellogenic oocytes

(PO). Scale bar: 100 lm.



premoult D3, followed by a slight increase to

0.43 � 0.09 mg mL�1 at the post-moult A stage.

It is noticeable that significantly elevated glucose

levels found at the premoult D1 and post-moult A

stages during the reproductive moulting cycle

were associated with the high demand for glucose

as an energy reserves for the maturation process

of oocytes. In this context, higher levels of glucose

are required to generate energy reserves in the

synthesis of vitellogenin, the precursor of the

major yolk protein, vitellin, and its uptake into the

oocytes. The rapid increase in oocyte diameter and

size (100–500 lm), which is obviously energy-

intensive, during this particular moulting stage, is

the result of yolk protein accumulation (Arcos

et al. 2010). Our data were demonstrated that a

significant moderate positive correlation was

observed between GSI and glucose concentration

(R = 0.40; P < 0.05) (Table 1), but we did not

find significant relationships (P > 0.05) between

body weight and glucose; body weight and GSI,

body weight and HSI, GSI and HSI, and HSI and

glucose. As GSI increased, glucose level increased

as indicated by linear regression: glucose

= 0.19 + 0.03*GSI (R2 = 0.16). This finding is in

agreement with Nagabhushanam and Kulkarni

(1980), who reported a direct relationship between

blood glucose and ovarian development in spear

shrimp, Parapenaeopsis hardwickii. In their study,

Nagabhushanam and Kulkarni (1980) classified

the development of ovary based on oocyte diame-

ter into five stages: Stage I (30–50 lm), Stage II

(169–190 lm), Stage III (235–260 lm), Stage IV

(290–235 lm) and Stage V (spawned). Their

oocytes ‘staging slightly differs to our staging, and

we classified ovarian development using GSI: Stage

A (9.58 � 0.41%), Stage B (0.54 � 0.06%,

spawned), Stage C0 (0.57 � 0.02%), Stage C1(1.21 � 0.14%), Stage D0 (1.69 � 0.18%), Stage

D1 (2.64 � 0.35%), Stage D2 (4.29 � 0.19%) and

Stage D3 (6.28 � 0.83%). In that study, blood

glucose level was at its minimum (2.8 � 0.26 mg

per 100 mL) in Stage I, increased (3.5 � 0.18

and 4.2 � 0.24 mg per 100 mL, respectively)

through stages II and III and reached its maxi-

mum (5.7 � 0.32 mg per 100 mL) in Stage IV,

after which it decreased severely (3.0 � 0.16 mg

per 100 mL) after spawning, Stage V. It was sug-

gested further this result could be due to a mobi-

lization of energy reserves to provide nutrients for

the eggs.

During the non-reproductive moulting cycle,

higher glucose levels were observed at the post-

moult A (0.36 � 0.07 mg mL�1) and late pre-

moult D2 (0.35 � 0.16 mg mL�1) stages. In con-

trast, there were no significant differences in

glucose level (range: 0.09 � 0.03 to

0.19 � 0.04 mg mL�1; P > 0.05) among the

intermoult (stages C0 and C1), early premoult

(stages D0 and D1) and late premoult D3 stage.

The raised levels of glucose during non-reproduc-

tive A and D2 moulting stages could be possibly

due to the moulting being energy-intensive, result-

ing in the mobilization of large quantities of sugar

from the blood in preparation for chitin synthesis

through the rapid breakdown of glycogen into glu-

cose (Galindo, Gaxiola, Cuzon & Chiappa-Carrara

2009). This findings was supported by Hornung

and Stevenson (1971), which their studies evi-

denced that using incorporation of 14C-glucose as

an indication of the rate of chitin biosynthesis in

crayfish, Orconectes obscurus, was found increasing

at stages D2 (197 mg chitin min�1) and D3

(388 mg chitin min�1) reached a peak at Stage B

(after ecdysis) (51 646 mg chitin min�1) and then

declined until stage C4 (21 mg chitin min�1).

According to Rocha, Garcia-Carre�no, Muhlia-

Almazan, Peregrino-Uriarte, Y�epiz-Plascencia and

Cŏrdova-Murueta (2012), greater availability of

glucose in the haemolymph could be served as

substrate in triggering up-regulation activity of

chitin-synthase to assemble monomers of N-acetyl-

glucosamine into chitin polymers during post-

moult A stage. Furthermore, we found a moderate

negative correlation between body weight and

haemolymph glucose concentrations (R = �0.36;

P < 0.05) (Table 2), indicated by linear regression:

glucose = 0.52 + 0.01*body weight (R2 = 0.13),

but no significant relationships (P > 0.05) were

shown between body weight and GSI, body weight

and HSI, GSI and HSI, GSI and glucose, and HSI

Table 1 Correlations matrix of parameters during the

reproductive molting cycle in laboratory maintained

adult female giant freshwater prawn, M. rosenbergii

BW GSI HSI Glucose

BW 1 0.02 �0.25 �0.12

GSI – 1 �0.15 0.40*

hsI – – 1 �0.14

Glucose – – – 1

Total number of prawns sampled, N = 40.

*Pearson’s correlation was significant (P < 0.05).



and glucose. Unlike insects which are the same

phylum of arthropoda, exoskeletal chitin scales iso-

metrically with dry body mass regardless of body

size (Lease & Wolf 2010). We believe that because

chitin accounts for a major fraction of cuticle and

is crucial to the cuticle’s structural integrity, thus,

it is suggested that evaluation of chitin mass is

needed to measure of exoskeletal investment.

Conclusion

Glucose is mobilized for different reasons during

reproductive and non-reproductive moulting cycles

in M. rosenbergii. Elevated level of glucose was

seen during D1 and A stages of reproductive

moulting cycle indicating that the period of which

glucose is crucial for vitellogenesis which is the

central process for maturation of oocytes. An

upsurge level of glucose was seen during A stage

of non-reproductive moulting cycle indicating

glucose is mobilized in preparation for chitin syn-

thesis. In future, it is recommended that further

study should be carried out to assess the metabolic

investment of glucose to assist aquaculture practi-

tioners in the management and development of

cost-effective feed in meeting the nutritional

requirement of females in order to improve

reproductive performance and spawn quality of

this species.

Acknowledgments

We would like to thank Ms. Yoko Furusawa and

Ms. Ooyama, who helped with the daily manage-

ment and maintenance of the experimental ani-

mals. We are also grateful to Dr. Tomoyuki

Okutsu, Dr. Kang Bung Jun and Dr. Bae Sun Hye

(researchers at JIRCAS) for their kind assistance

with histology techniques. The hatchery facilities

and laboratory were provided by the Fisheries

Division, JIRCAS, Ministry of Agriculture, Forestry

and Fisheries, Japan, in collaboration with the

Institute of Tropical Aquaculture and Institute of

Marine Biotechnology, both at the Universiti

Malaysia Terengganu.

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Dynamics of glucose in the haemolymph of female giant freshwater prawn, Macrobrachium rosenbergii, influences reproductive and non-reproductive moulting cycles