Changes in mRNA expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are key links to nutritional condition and growth regulation in teleost. To understand the endocrine mechanism of growth regulation in grouper, we cloned the cDNAs for grouper GH and IGF-I and examined their mRNA expression during different nutritional status. Grouper GH cDNA is 936 base pairs (bp) long excluding the poly-A tail. It contained untranslated regions of 85 and 231bp in the 5'- and 3'-ends, respectively. It has an open reading frame of 612bp coding for a signal peptide of 17 amino acids (aa) and a mature hormone of 187aa residues. Based on the aa sequence of the mature hormone, grouper GH shows higher sequence identity (>76%) to GHs of perciforms than to GHs of cyprinids and salmonids (53-69%). Grouper preproIGF-I cDNA consisted of 558bp, which codes for 186aa. This is composed of 44aa for the signal peptide, 68aa for the mature peptide comprising B, C, A, and D domains, and 74aa for the E domain. Mature grouper IGF-I shows very high sequence identity to IGF-I of teleost fishes (84-97%) compared to advanced groups of vertebrates such as chicken, pig, and human (=<80%). Using DNA primers specific for grouper GH and IGF-I, the changes in mRNA levels of pituitary GH and hepatic IGF-I in response to starvation and refeeding were examined by a semi-quantitative RT-PCR. Significant elevation of GH mRNA level was observed after 2 weeks of food deprivation, and increased further after 3 and 4 weeks of starvation. GH mRNA level in fed-controls did not change significantly during the same period. Hepatic IGF-I mRNA level decreased significantly starting after 1 week of starvation until the 4th week. There was no significant change in IGF-I mRNA levels in fed-controls. One week of refeeding can restore the GH and IGF-I mRNA back to its normal levels. Deprivation of food for 1-4 weeks also resulted in cessation of growth and decrease in condition factor.We thank Jossette Bangcaya and Josephine Nocillado for providing us degenerate IGF-I reverse primer, Mr. Roman Sanares for the statistical analyses, Dr. Ebonia Seraspe for her valuable advice and help. This study was funded by SEAFDEC AQD to EGT de Jesus-Ayson (Nr-01-F2001T) and by a grant from USAID to FG Ayson (Grant No. TA-MOU-99-C19-001, Program in US-Israel Cooperative Development Research, Economic Growth)

Local Vibrio isolates exhibit molecular characteristics distinct from reference V. harveyi and V. campbellii strains

Six Vibrio isolates identified biochemically as Vibrio campbellii from Southeast Asian Fisheries Development Center (SEAFDEC) in Tigbauan, Iloilo, were characterized by 16 rDNA sequence, total protein profile, and DNA profile analyses. Genomic DNA from the isolates were subjected to PCR using four sets of primers targeting gene fragments of hemolysin and toxR based on sequences from reference Vibrio harveyi (IFO15634), V. campbellii (IFO1563), and local isolates identified as V. harveyi. Total protein profile could not distinguish the isolates from one another and from the reference V. harveyi (IFO15634) and V. campbellii (IFO15631). Analysis of 16s rDNA sequences revealed high degree of sequence similarity (96% - 99%) of the six local isolates with other Vibrio species including V. campbellii and V. parahaemolyticus, indicating that this analysis will not be useful in resolving their identity. All six isolates exhibited characteristic reference V. harveyi PCR profile when a primer set designed to amplify a 308-bp fragment of hemolysin gene in that species was used. However, no amplicons were generated for these isolates using primers that amplify toxR gene fragments in V. harveyi. This suggests that the six isolates were not bonafide V. harveyi strains. The isolates also did not exhibit V. campbellii characteristics since the primer designed to target the toxR gene in V. campbellii could not amplify DNA from any of the six isolates, suggesting that they were not bonafide V. campbellii strains either. The toxR gene from the six isolates could be amplified using a primer based on toxR gene sequences from a SEAFDEC isolate previously identified as V. harveyi (PN-9801). These data suggest that the six isolates previously identified as V. campbellii as well as PN-9801 may be classified in one group separate from bonafide reference V. harveyi and reference V. campbellii strains, based on the identical results in the molecular analyses performed in this study