Off farm commercial culture, amino acid composition and COI and 16S rDNA sequence based identification of fish live food tubificid worms

Abstract

Tubificides (Tubificidae) are one of the best quality live aquaculture foods widely used in the nursing of spawn to fry and rearing of fry to fingerling. Tubificid worms also used as live food in aquarium / ornamental fish. Few studies have been undertaken on the on farm culture of fish life food tubificid worms. However, off farm commercial culture at farmer level has never been studied. Worms are not identified at species level by using molecular techniques particularly COI gene and 16S rDNA sequence found in Bangladesh. This study, therefore, was designed to test the effects of three wet mediums i.e., cattle blood, rice gruel and subsurface clean water on the yield, amino acid composition and COI gene and 16S rDNA sequence based identification of the fish live food tubificid worms found in Bangladesh. Tubificid worms were brought from the local collectors and cultured commercially in newly constructed culture raceways at Maa Fatema Fish Hatchery, Dalmil Jessore. Tubificid worms were also collected from Dhaka and Mymensingh to identify at species level. In the present study mixture of 30% Soybean meal, 40% mustard oil cake and 30% mud were used as media ingredients and cattle blood, rice gruel and subsurface clean water were used as the wet mediums. Amino acid profiles of the wild and cultured worms, media ingredient wetted in cattle blood, rice gruel and subsurface water was also determined in this study. Besides culture techniques, molecular techniques has been performed by using COI and 16S rDNA sequencing to identify tubificid worms at species level. The highest yield of tubificid worms (683.68 ± 3.86 mg cm-2) was harvested from the treatment where media ingredients were wetted in cattle blood while the lowest yield (584. 38 ± 1.41 mg cm-2) was found in the treatment in which the media ingredients were wetted in subsurface clean water (Figure 1). Rice gruel wetted media resulted in the yield of worms (615.63 ± 3.66 mg cm-2) that was significantly different from other two treatments. Worms raised in the media wetted in blood had nearly 58% protein followed by rice gruel (55%) and water (53%). Similarly highest level of fat (13%) was detected in the worms produced in the media wetted in blood followed by rice gruel (12%) and water (11%). Among 8 essential amino acids (EAA) detected in the worms, lysine, arginine and leucine were found highest level in the worms raised in the media ingredients wetted in blood followed by water and rice gruel. Among non-essential amino acids (NEAA), worms raised in the media wetted in blood had the highest level of glycine followed by wild worms. Level of glycine was similar in the worms raised in the media wetted in rice gruel. Alanine and glutamic acid followed similar trends in occurrence. Three samples of tubificid worms, denoted as TD, TM, TJ collected from Dhaka, Mymensingh and Jessore (cultured), respectively, selected for sequencing by COI and 16S rDNA gene. COI gene identified tubificid worms TD sample as Tubifex tubifex, TM as Tubifextubifex and TJ as Branchiura sowerbyi while 16S rDNA gene identified tubificid worms as Limnodrilus hoffmeisteri for all 3 samples. Multiple sequence alignment was performed to find out the polymorphic sites among the sequenced worms. After comparing the obtained sequence of COI gene it is observed that 154 of 630 sites are polymorphic and dissimilarities among 3 tubificid sample (TD,TM,TJ) 24.22%. From the multiple sequence alignment of 16S rDNA gene sequencing, is was observed that, total 49 of 363 sites were polymorphic and dissimilarities among 3 tubificid sample (TD, TM, TJ) were 13.50%. Through this study, identification of tubificid worms at species level and off farm commercial culture of tubificid worms has been conducted for the first time in Bangladesh.