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The tetrapeptide APGW-amide induces somatic growth in Haliotis asinina linnaeus.

ABSTRACT APOW-amide is a well-known neurohormone modulator in several molluscs, and is involved in motor activities, feeding, and sexual behavior. In this report we show that injections of APGW-amide into 4-too-old juvenile Haliotis asinina stimulate growth of body weight and, to a lesser degree, shell length. The injections were given at 0 (control), 20, and 200 ng/g body weight (BW), at 1-wk intervals for 14 wk. BW and shell length (SL) were measured every week, and growth rates were calculated. When compared with control animals, there was an approximate 2-fold increase in body growth rates of animals given 20 ng/g BW and 200 ng/g BW APGW-amide (P [less than or equal to] 0.05), whereas only 20 ng/g BW APGW-amide produced significantly greater SL than controls (P [less than or equal to] 0.05), with an approximate 1.2-fold increase. Using an immunoperoxidase technique, we showed the presence of APGW-amide in neuronal cells of the cerebral ganglia and nerve fibers. Overall, these data indicate that APGW-amide is an important neurohormone/neuromodulator in the nervous system of H. asinina and plays a role in controlling the body growth of H. asinina.

KEY WORDS: abalone, APGW-amide, Haliotis asinina, neural ganglia, growth


The amidated tetrapeptide Ala-Pro-Gly-Trp-N[H.sub.2] (APGW-amide) acts as a neurotransmitter or neuromodulator in many mollusc species, including Lymnaea stagnalis (Croll & Van Minnen 1992, De Boer et al. 1997, McCrohan & Croll 1997), Aplysia californica (Fan et al. 1997), Mytilus edulis (Ohtania et al. 2000), Sepia officinalis, and Euhadra congenita (Henry & Zatylny 2002). The complementary DNA encoding this tetrapeptide has been cloned from Lymnaea stagnalis (Stair et al. 1992), M. edulis (Favrel & Mathieu 1996), and A. californica (Fan et al. 1997). Antibodies produced to the tetrapeptide have been used in immunolocalization techniques, resulting in the detection of APGW-amide in neurons within the central nervous system of a wide range of molluscs. Examples include the common garden snail Helix aspersa (Li & Chase 1995), the sea scallop Placopecten magellanicus (Smith et al. 1997), the California sea hare A. californica (Xuemo et al. 1997), and the pond snail L. stagnalis (De Boer et al. 1997).

In regard to function, APGW-amide has been reported to stimulate hormones that regulate mollusc reproduction (Croll & Van Minnen 1992, De Boer et al. 1997) and feeding behavior (De Boer et al. 1997). In Aplysia, movement of the radula during feeding has been shown to be under the control of the neurons containing APGW-amide (Jing & Weiss 2001, Morgan et al. 2002). In addition, it has been found that APGW-amide in the neural ganglia may be involved in controlling growth in L. stagnalis, possibly by the release of an insulin like growth hormone (Geraerts 1976, Tol-Steye et al. 1999). In this study we report that APGW-amide is present in neural tissue of H. asinina, and that injections of APGW-amide into juveniles accelerate body growth.



Four-month-old Haliotis asinina juveniles (n = 600), of shell length (SL) approximately 1.5 cm and body weight (BW) of approximately 0.62 g, were used in the tests. They were maintained in outdoor shaded tanks (each measured 2 x 3 [m.sup.2] in floor space and 0.5 m in depth) with shelters, running seawater, and a natural photoperiod. They were fed an excess amount of seaweed (Gracilaria sp.) daily.

Injections of A PG W-A mide

The animals were randomly divided into 3 groups (n = 200 per group). The APGW-amide (Sigma-Aldrich, St. Louis, MO) was freshly prepared in Ringer's solution (containing 13 g HEPES, 25.66 g NaC1, 0.82 g KCl, 1.69 g Ca[Cl.sub.2], 10.17 g Mg[Cl.sub.2], 2.56 g [Na.sub.2]S[O.sub.4], and 1 K deionized [H.sub.2]O at a pH of 7.2). It was then injected intramuscularly through the sole of the foot in a 20-[micro]L volume using the method of Liu et al. (1991). Each group received a total of 14 intramuscular injections at 1-wk intervals. The experimental groups were summarized as follows: group 1, Ringer's solution injection (control); group 2, 20 ng/g BW of APGW-amide in Ringer's solution; and group 3,200 ng/ g BW of APGW-amide in Ringer's solution. Each experiment was repeated twice.


SL (in millimeters) and BW (in milligrams) were measured at 2-wk intervals. For data analysis, an analysis of variance and multiple comparisons were used to test for significant differences between the control and experimental groups (P [less than or equal to] 0.05).

Immunolocalization of APGW-Amide in the Neural Ganglia

To identify the existence of APGW-amide in neural tissues, an immunoperoxidase technique was used (Chansela et al. 2008). Paraffin sections of cerebral ganglia that had been fixed in Bouin's solution were deparaffinized, and nonspecific binding sites were blocked for 2 h with 2% normal goat serum and 4% bovine serum albumin in PBST (0.05 M phosphate-buffered saline containing 0.4% Triton X-100). A primary antibody incubation with anti-APGW-amide (diluted 1:200 in blocking solution) was performed overnight at room temperature, and a secondary antibody incubation with biotinylated goat antirabbit immunoglobulin G (Sigma-Aldrich; diluted 1:200 in blocking solution) was performed for 45 min. After 4 washes, the sections were incubated with HRP-conjugated streptavidin (diluted 1:200 in PBST), for 45 min, and a color reaction was developed by Nova Red (Vector). Cellular details and immunoreactivity were observed under a Nikon eclipse E600 microscope, and photographs were taken with a Nikon digital camera (DXM 1200).



Effect of APGW-Amide on Juvenile Growth

The changes in BW and SL of the control and experimental groups during 14 wk are shown in Figure 1A and B, respectively. The BW of juvenile abalone in the control group at wk 14 was 1.99 [+ or -] 0.45 g, whereas those that received APGW-amide at the doses of 20 ng/g BW and 200 ng/g BW were 3.94 [+ or -] 0.41 g and 3.52 [+ or -] 0.38 g, respectively. During the same period, the SL of the control abalone increased to 2.43 [+ or -] 0.20 cm, whereas those of the groups that received 20 ng/g BW and 200 ng/g BW APGW-amide injections were 2.88 [+ or -] 0.23 cm and 2.70 [+ or -] 0.22 cm, respectively. Compared with the controls, BW of both groups of animals treated with APGW-amide were significantly different at 14 wk (P < 0.05).

Table 1 summarizes the increases in BW and SL during the 14-wk period, and also gives growth rates. There was an approximate 2-fold increase in BW for the groups treated with 20 ng/g BW and 200 ng/g BW of APGW-amide, but this was not shown in SL. Growth rates for BW over 14 wk were 32.7 mg/day (control), 64.8 mg/day for 20 ng/g BW APGW-amide, and 57.9 mg/day for 200 ng/g BW APGW-amide. These were significantly different from the control (P < 0.05). Growth rates for SL over the 14 wk were 0.16 mm/day (control), 0.16 mm/day for 20 ng/g BW APGW-amide, and 0.18 mm/day for 200 ng/g BW APGW-amide. Only the group injected with 20 ng/g BW APGW-amide showed a significant increase in SL (P < 0.05).

Localization of APGW-Amide in the Cerebral Ganglia

APGW-amide was identified in the cerebral ganglia of H. asinina (Fig. 2). The cells containing APGW-amide were neurons, and these were distributed through the cortex of the cerebral ganglia. In particular, the neurons were concentrated at the dorsolateral edges of the cerebral ganglion, but a few immunoreactive cells were scattered widely along the medial and ventral edges (Fig. 2B). In addition, a dense network of positively stained nerve fibers projected throughout the middle part (neuropil) of the ganglia (Fig. 2B).



Our study has shown APGW-amide to be present in H. asinina, and that exogenous administration at doses of 20 ng/g and 200 ng/g BW APGW-amide significantly stimulates body growth in juveniles (P < 0.05; Figs. 1 and 2). The increase in BW was approximately 2-fold during the 14-wk study period, and the lower dose appeared to have a greater, but not significant, stimulatory effect on growth than the higher dose. However, SL did not show the same increases, and only the 20-ng/g BW APGW-amide treatment produced a significant 1.2-fold increase (P < 0.05). This is not unexpected, because H. asinina does not produce a large shell like those of other abalone species, and so BW was expected to increase at a greater rate than SL.

The SL growth of H. asinina control juveniles obtained in our study was 0.16 mg/day. This value is similar to that of previous H. asinina growth studies. One study, on H. asinina cultured in Thailand, reported growth of approximately 0.10 mm/day on artificial diets (Kruatrachue et al. 2004), a similar study in the Philippines reported 0.13 mm/day (Fermin & Buen 2002), and 2 additional Thailand studies reported 0.15 mm/day using varying densities in cages and 0.18 mm/day on an algal diet (Nuurai et al. 2010).

Apart from APGW-amide being a neurotransmitter and neuromodulator, it has been directly involved in oocyte transport in the cuttlefish Sepia officinalis by triggering the contraction of adductor muscle (Zatylny et al. 2000), release of oocytes in oysters (Bernay et al. 2006), and induction of sex change in female mud snails to ones that exhibit male accessory sex organs, thereby producing effects similar to testosterone (Koene et al. 2000). Recently, we reported that APGW-amide is present in the male gonad of H. asinina, but it was not in female ovaries, and we noted that it could induce more than 80% of ripe males to release sperm by injection of APGW-amide directly into the foot muscle (Chansela et al. 2008). We have not tested the long-term effect of APGW-amide on juveniles, and it is possible that the stimulation of body growth we have observed could also be accompanied by an induction of sex change toward all males.

In our current report, we show that APGW-amide can also accelerate body growth of the juvenile abalone. This could result from 2 main reasons. First, many studies have reported that APGW-amide can stimulate feeding behavior of molluscs.

For example, a study in L. stagnalis indicated that the cerebrobuccal cells that contain APGW-amide are involved in feeding (McCrohan & Croll 1997). In addition, in A. californica, ingestion and egestion feeding behavior are 2 types of motor activities shown to be controlled by APGW-amide, both of which are modulated by 2 sets of radula movements: protraction retraction and opening-closing movements (Jing & Weiss 2001). We have not, as yet, examined whether APGW-amide-stimulated H. asinina juveniles feed more than controls, as this may account for the increase in body growth. Second, it is possible that the APGW-amide present in neural ganglia of H. asinina could have a neuromodulatory role like that observed in L. stagnalis, in which isolated cells in the cerebral ganglia (i.e., those stained by Light Green dye in fixed sections), could be induced to release an insulinlike growth hormone through the action of APGW-amide (Geraerts 1976, Tol-Steye et al. 1999). This hypothesis also needs to be tested.

We have shown that injections of 20 ng/g and 200 ng/g APGW-amide into juvenile abalone induce body growth, but more testing is required to determine the lowest effective dose. This dose may be considerably less than 20 ng/g APGW-amide. We have also presented evidence that APGW-amide is located in certain neurons of the cerebral ganglia, and these could also be involved in the control of growth. The cellular and molecular mechanism of APGW-amide in this aspect needs to be studied further, particularly in relation to stimulation of feeding.


This research was supported by the Thailand Research Fund (TRF), the Commission on Higher Education, and Mahidol University (P. S.). We thank the Coastal Aquaculture Research and Development Center, Department of Fisheries, Prachaubkirikun Province, Thailand, for providing abalone specimens.


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(1) Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; (2) Department of Preclinical Science, Faculty of Medicine, Thammasat University, Palhurnthani 12121, Thailand; (3) Faculty of Science and Technology, Deakin University, Geelong, Victoria 3217, Australia; (4) The Coastal Aquaculture Research and Development Center, Department of Fisheries, Klongwan, Prachaubkirikhan 77000, Thailand

* Corresponding author. E-mail:
Changes in body weight (BW) and shell length (SL) of each
treatment group over 14 wk, together with calculated
growth rates.

                                                           Growth Rat
                   BW at Wk 0           BW at Wk 14        (mg/day)

Control        0.62 [+ or -] 0.11   1.99 [+ or -] 0.45      32.7
APGW20         0.62 [+ or -] 0.21   3.94 [+ or -] 0.41 *    64.8
APGW200        0.62 [+ or -] 0.28   3.52 [+ or -] 0.38 *    57.9

                                                           Growth Rate
Treatments         SL at Wk 0           SL at Wk 14         (mg/day)

Control        1.50 [+ or -] 0.12   2.43 [+ or -] 0.20      0.16
APGW20         1.50 [+ or -] 0.16   2.88 [+ or -] 0.23 *    0.19 *
APGW200        1.50 [+ or -] 0.19   2.70 [+ or -] 0.22      0.18

* Significant difference at P [less than or equal to] 0.05.
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Article Details
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Author:Chansela, Piyachat; Saitongdee, Porncharn; Stewart, Praphaporn; Soonklang, Nantawan; Hanna, Peter J.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9THAI
Date:Nov 1, 2010
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