Early Development And Mechanism Of Ammonia Tolerance In The Chinese Loach Paramisgurnus Dabryanus

Chinese laoch, Paramisgurnus dabryanus, a freshwater omnivorous fish, is one of the most commercially important cultured species in East Asia, especially in China and Korea. The culture of Chinese loach become more and more popular as the gradually increasing market demand in China especially in recent years. In the present study, we clarified the growth pattern, changes in nucleic acids and digestive enzymes activity and the occurrence of skeletal deformity during the larval stage and the mechanism of ammonia tolerance in the Chinese loach based on the previous results. Main results are shown as follows.(1) Growth pattern of larval and juvenile Chinese loachThe length-weight relationship and allometric growth patterns of hatchery-reared Chinese laoch, Paramisgurnus dabryanus(Dabry de Thiersant, 1872), were conducted from hatching to 60 days after hatching(DAH). A best power model(BW=0.025×TL2.649,R2=0.996) was observed for the length-weight relationship of Chinese loach during the early life stage. Allometrically positive growth of head segment, trunk length, tail length and eye diameter was found in early life stage of Chinese loach, while a negative coefficient for body depth, tail depth, tail fin length, pectoral fin length and barbel length.Subsequently, the growth coefficients showed a common evident tendency to isometry for all the measured body proportions during the early developmental stage. The allometric growth changes in Chinese loach during early stage likely result from selective organogenesis directed towards survival priorities.(2) Ontogenetic changes in RNA, DNA and protein contents of Chinese loach larvae and juvenilesThe changes in nucleic acid-based indices and protein variables of Chinese loach larvae and juveniles from hatching to 60 DAH were conducted to assess its growth potential. The nucleic acid contents were analysed using a UV-based method(n=3,rearing temperature 24.4±0.4 ℃, dissolve oxygen 7.1±0.5 mg L-1, p H 7.9±0.4).Ribonucleic acid(RNA) concentration significantly decreased from 2 to 5 DAH, then rapidly increased until 10 DAH and declined slightly thereafter. Deoxyribonucleic acid(DNA) concentration increased from 2 to 5 DAH, decreased until 9 DAH, slightly increased again around 26 DAH, and then declined to a relatively stable level. Both RNA-DNA and protein-DNA ratios showed a statistically obvious relationship with growth rates. Significant positive relationship was found between RNA-DNA ratio and growth rates during the early life stage of Chinese loach. According to the results, growth of Chinese loach is characterized by rapid hyperplasia from hatching through completion of the yolk-sac stage followed by continued rapid hyperplasia combined with increasing hypertrophy after feeding commences. And, the stage before 17 DAH of early life of Chinese loach supposed to be critical for the survival and growth at 24 ℃.(3) Changes in digestive enzyme activities during larval development of Chinese loachThe digestive physiology of Chinese loach was studied by assessing the specific and total activity of different pancreatic(trypsin, chymotrypsin, amylase and lipase), gastric(pepsin) and intestinal(alkaline phosphatase and Leucine-aminopeptidase) enzymes from hatching to 40 DAH. Larvae were reared at 24.4±0.4 ℃ and fed with rotifers from mouth opening(4 DAH) to 15 DAH, from 10-35 DAH with cladocera and from 30-40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH. While, a gradual increase of specific Leucine-aminopeptidase activity was observed until the end of the experiment.Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase of pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.(4) Osteological deformities in early stage of hatchery-reared Chinese loachThe specimens were stained with Alizarin red for bone and Alcian blue for cartilage for assessing the occurrence of skeletal deformities in larval and juvenile Chinese loach from hatching to 60 DAH. The main regions where abnormalities were found are the four regions of vertebral column(cephalic, pre-haemal, haemal and caudal regions), dorsal fin,anal fin and caudal fin, and total 14 osteological deformities were observed. The distribution of skeletal abnormalities in larvae and juvenile Chines loach were different at the different developmental stages. No vertebra deformities were found in present study,neural spine abnormalities reach minimum at stage A(TL<12 mm) and maximum at Stage D(TL>50 mm); haemal spine abnormalities reach minimum at stage B(TL 12-30mm) and maximum at stage C(TL 30-50 mm); fin abnormalites reach minimum at stage C(TL 30-50 mm) and maximum at stage B(TL 12-30 mm). Most common skeletal deformities in Chinese laoch larvae included malformed ray of fin, neural spine and haemal spine abnormalites. High percentage of skeletal abnormalities occurred during the larval stage possibly due to body shape transformation or degeneration-proliferation developmental events of tissues. The specific deformities with highest frequency were fin malformations, bifurcated neural spine and fusion of haemal spines, revealing the development of these structures may be the most susceptible to rearing conditions. No vertebra deformities were found until the end of the experiment, suggesting the occurrence of abnormalities in vertebra was quite late(after 60 DAH).(5) Accumulation of ammonia in the body during aerial exposure and ammonia loading in Chinese loachThe Chinese loach was exposed to 30 mmol L-1 NH4 Cl solution and air to assessing the change of body ammonia and urea contents and the activities of alanine aminotransferase(ALT) and aspartate transaminase(AST) during ammonia loading and aerial exposure. During the 30 mmol L-1 NH4 Cl solution exposure, ammonia contents in plasma and brain increased with the increasing of exposure time, while a slight increase of ammonia contents in liver and muscle during the first 24 h and then a marked increase after 48 h was observed. In aerial exposure, a slight increase of ammonia contents in plasma, brain, liver and muscle during the first 24 h and then a marked increase after 48 h was observed, and the ammonia contents in plasma, brain, liver and muscle were 2.2-fold,3.3-fold, 2.5-fold and 2.9-fold, respectively, those of the control values after 48 h of air exposure. The urea contents in plasma, liver and muscle of Chinese loach were only slightly affected by aerial and 30 mmol L-1 NH4 Cl exposure. Similarly, the ALT activity in plasma of Chinese loach was only slightly affected by 30 mmol L-1 NH4 Cl exposure.While, it was significantly affected by different time of aerial exposure(P<0.05), and its value increased markedly after 48 h of exposure. However, the AST activity in plasma of Chinese loach was not affected by the exposure time regardless of aerial exposure and ammonia exposure. Our results suggested that Chinese loach could accumulate high concentrations of ammonia in body because of the high ammonia tolerance in it cells and tissues, and it might volatilized some internal ammonia as NH3 as a strategy to defend against ammonia toxicity. The urea contents in plasma, liver and muscle of Chinese loach were only slightly affected by aerial and ammonia exposure, revealing that urea synthesis may be not an applicable strategy for Chinese loach to deal with ammonia issues. The significant increase of ALT activity in plasma during aerial exposure, indicating that alanine synthesis through partial amino acid catabolism would exist in Chinese loach.(6) The role of glutamine in response to ammonia and aerial exposure in Chinese loachThe Chinese loach was exposed to 30 mmol L-1 NH4 Cl solution and air to assessing the change of body glutamine content, glutamine synthetase(GS) and glutamate dehydrogenase(GDH) activities during ammonia loading and aerial exposure. The obvious accumulation of glutamine contents in liver and muscle was observed after ammonia exposure and aerial exposure with the increasing of exposure time. The significant increase of GS activity in brain, liver and intestine of Chinese suggested that Chinese loach could deal with the internal ammonia increase through the accumulation of body glutamine. And, the GS could stimulate glutamine formation pathway, and then converted ammonia into non-toxic glutamine. The GDH activities in brain and intestine of Chinese loach were significantly affected by 30 mmol L-1 NH4 Cl solution exposure.While, the GDH activity in liver of Chinese loach was not affected by ammonia exposure.The marked increase of GDH activity in intestine of Chinese loach demonstrated that intestinal GDH had a more important role than intestinal GS in the overall defense against ammonia toxicity in Chinese laoch. The change in GDH activity in liver of Chinese loach was not affected by ammonia exposure and aerial exposure and not associated by a concomitant increase of GS activity and glutamine content throughout the whole exposure period, probably because of and efficient replenishment of glutamine catalyzed by transaminase enzymes.(7) NH3 volatilization and body surface alkalization as response to aerial exposure and ammonia loading in Chinese loachThis study aimed to determine whether Paramisgurnus dabryanus could excrete NH3 by volatilization during air exposure or ammonia loading. After 24 h NH4 Cl exposure at different concentrations, the amounts of ammonia recovered from the acid in the presence of P. dabryanus were significantly higher than that of the control without a fish. In aerial exposure condition, it led to a marked increase in the amount of NH3 volatilized. The results suggested that P. dabryanus was able to excrete some ammonia in the gaseous form as the internal ammonia levels increased. The amount of NH3 volatilized increased with the time of exposure and with increasing temperature. In P. dabryanus, the posterior intestine become quiet more alkaline during ammonia loading and aerial exposure. The results indicated that the posterior intestine should be a site of NH3 volatilization during aerial exposure or ammonia loading. The skin, which also became more alkaline during air exposure, may also be a site of NH3 volatilization in air exposed fish. Briefly, P.dabryanus was capable of excreting some ammonia in gaseous(30-40%) form during ammonia loading or aerial exposure and high temperature facilitated the ammonia volatilized into air rather than excreted into surrounding water. The alkalization of suface of posterior intestine and skin suggested the two sites of of NH3 volatilization in P.dabryanus.