BIOCHEMICAL EFFECTS OF FLUORIDE ON HAEMOLYMPH OF THE SILKWORM, BOMBYX MORI L.

Transcription

1 Fluoride Vol. 37 No Research Report 117 BIOCHEMICAL EFFECTS OF FLUORIDE ON HAEMOLYMPH OF THE SILKWORM, BOMBYX MORI L. Yun-gen Miao, a Li-jun Jiang, a D. Bharathi b Hangzhou, PR China, and Andhra Pradesh, India SUMMARY: Changes in the Ca 2+ and Mg 2+ ion concentrations, catalase activity, and protein metabolism in the haemolymph of four different strains of silkworms fed a special diet supplemented with fluoride were investigated. Fluoride exposure caused decreases in the concentrations of the two ions, with significant differences between strains in their tolerance to fluoride. Catalase activity in the haemolymph of fluoride-exposed silkworm larvae of the Hang 8 strain was over 24 times higher than in the controls at its peak in the middle of the 4 th instar. In contrast, glutamate dehydrogenase (GDH) activity and the levels of protein and free amino acids in the haemolymph were significantly lowered by fluoride treatment in all strains. These results suggest that biochemical changes in the haemolymph can be used as biomarkers to evaluate the adverse health effects and the economic impact of fluoride exposure on the silkworm, Bombyx mori L. Keywords: Bombyx mori L., Calcium ions; Magnesium ions; Catalase; Fluoride exposure; Glutamate dehydrogenase (GDH); Silkworm haemolymph. INTRODUCTION Atmospheric pollution by fluoride is one of the main environmental problems in China. 1 In the Hang-Jia-Hu Plain of Zhejiang Province, the most important district for silk production, the sericulture (silkworm) ecosystem has been seriously affected economically by fluoride pollution, due mainly to the widespread use since the 1970s of brickkilns, which emit fluoride from clay soils in the form of gaseous HF and SiF 4 during the firing process. 2-5 These fluoride pollutants are taken up by mulberry leaves inducing toxicity to both the leaves and silkworm larvae. 6 Airborne fluoride enters the stoma and deposits on the inner part of the leaves, disrupting chlorophyll synthesis and inhibiting cellular metabolism. 7 As a consequence, the fluoride-polluted leaves are toxic to the silkworm larvae. Consequently, various studies have been conducted on the harmful effects of fluoride on silk production as well as the mechanism of fluoride action on the larvae Histological and histochemical changes involving structural damage to the midgut and fatty body tissues from the larvae feeding on the polluted leaves have also been investigated. 6 a For Correspondence: Yun-gen Miao, PhD, Department of Biological Resource Sciences, College of Animal Sciences, Zhejiang University, Hangzhou , China. miaoyg@zju.edu.cn; b Department of Sericulture, Sri Padmavathi Mahila University, Tirupathi , Andhra Pradesh, India.

2 118 Miao, Jiang, Bharathi Theoretically, such damage by fluoride should also cause biochemical changes in the haemolymph (worm blood) in silkworms. Previous investigations have reported results of preliminary investigations of fluoride effects on haemolymph biochemistry of the silkworm, Bombyx mori L The aim of this work was to conduct a further biochemical examination of haemolymph of silkworms exposed to fluoride, to explore the mechanism of the toxicity, and to provide a biochemical index for evaluating the health of fluoride-poisoned silkworms. MATERIALS AND METHODS Experimental colonies: Disease-free silkworm eggs were used to rear four strains of silkworms under standard conditions at 262 C, 70-85% RH, and a 12-hr L:12-hr D photoperiod. An artificial diet 13 consisting mulberry leaf powder, potato starch, soybean powder, sucrose, agar, mineral salts, and vitamin B complex was used as food for once-a-day feeding. After the third ecdysis, larvae were divided into groups and rearing was continued under these same conditions. Administration of fluoride: Fluoride (NaF) was added to the artificial diet at 8, 16, and 32 mg F/kg dry feed, which is equivalent to about 24, 48, and 96 mg F/kg, respectively, in dried mulberry leaves. The silkworm larvae were fed the fluoride-supplemented artificial diet successively from the 4 th instar b and 5 th instar, respectively. Each experiment was replicated three times with 200 larvae in each replication. Determination of Ca 2+ and Mg 2+ ions: Haemolymph (worm blood) was collected by cutting through the abdominal leg of 5 th instar larvae. One sample was taken from 20 larvae randomly. The concentrations of Ca 2+ and Mg 2+ ions were determined using the methods described by Lin. 14 Total proteins in haemolymph: Total protein content in the haemolymph was determined by the Folin phenol method 15 with a spectrophotometer (Beckman DU-600, Beckman, California, USA) at 500 nm. Determination of free amino acids: Collected haemolymph was centrifuged at 12,000 x g at 4 C for 30 min, and the resulting supernatant was subjected to vapor-phase 6N HCl automated hydrolysis (Applied Biosystems 420A). Amino acid analysis was conducted using standard protocols for the 420A system as described by West and Crabb. 16 Glutamate dehydrogenase activity: The activity of GDH in the haemolymph was determined by the method of Ruiz. 17 Enzyme activity was measured b The term instar refers to successive molting stages of the larvae. Newly hatched larvae are in the first instar. The second instar begins after the first molting, the third after the second molting, and the fourth after the third molting. After about 7 or 8 days into the fifth instar the larvae are matured and begin to spin cocoons.

3 Effects of fluoride on silkworm haemolymph biochemistry 119 spectrophotometrically at 340 nm and 72 C in a reaction mixture containing 50 mm Tris-HCl buffer (ph 8), 2 mm EDTA, 100 mm L-glutamate, and 3 mm NAD. Following equilibration at 72 C for 5 min, the reaction was initiated by the addition of NAD. Uric acid content: The haemolymph was added to water and made up to a final volume of 1 ml. Uric acid was determined by the method of Van Handel. 18 Assay of catalase activity: The haemolymph was collected according to the method described above from larvae in the 4 th and 5 th instar, respectively. Catalase activity was determined using hydrogen peroxide as a substrate, as described by Zhou and Chen. 19 RESULTS Changes in Mg 2+ and Ca 2+ ion concentrations: Table 1 shows the changes in Mg 2+ concentrations in haemolymph of four different strains of silkworms fed the artificial diet containing 16 mg F/kg dry feed over a five-day period in the 5 th instar. Although there were differences among the strains, generally they all appeared to have reduced concentrations of Mg 2+ compared to their respective controls. Haemolymph of the Zhenong 1 strain, which is fairly tolerant to fluoride, showed only a small reduction in Mg 2+ concentration by the 5 th day. On the other hand, haemolymph of the more sensitive, less tolerant Haoyue strain displayed the greatest over-all reduction in Mg 2+ concentration. Table 1. Change of Mg 2+ concentration (mg/100ml) in haemolymph of silkworms exposed to 16 mg F/kg dry artificial diet beginning in the 5 th instar Strains Treatment 1 st day 2 nd day 3 rd day 4 th day 5 th day Zhenong 1 Fluoride Control Su12 Fluoride Control Hang 8 Fluoride Control Haoyue Fluoride Control As seen in Table 2, the change in Ca 2+ concentration in the haemolymph of silkworms feeding on the 16 mg F/kg dry feed in the 5 th instar was similar

4 120 Miao, Jiang, Bharathi to that of Mg 2+, except for the Zhenong 1 strain, which showed an increase in Ca 2+ compared to its control on all but the 2 nd day. Haemolymph of the other three strains all had lower Ca 2+ concentrations on all five days of the 5 th instar than their controls. Table 2. Change of Ca 2+ concentration (mg/100ml) in haemolymph of silkworms exposed to 16 mg F/kg dry artificial diet beginning in the 5 th instar Strains Treatment 1 st day 2 nd day 3 rd day 4 th day 5 th day Zhenong 1 Fluoride Control Su 12 Fluoride Control Hang 8 Fluoride Control Haoyue Fluoride Control Effect of fluoride on protein metabolism in silkworm haemolymph: Table 3 shows a number of changes in protein metabolism on the 5 th day in the 5 th instar in the haemolymph of silkworms fed the artificial diet containing 16 mg F/kg dry feed. Glutamate dehydrogenase activity and the levels of protein and free amino acids were significantly lower compared to those of the controls. However, the uric acid content in the haemolymph of the fluoride-exposed larvae was higher than that of the control. Table 3. Some changes of protein metabolism in haemolymph of silkworms exposed to 16 mg F/kg dry artificial diet beginning in the 5 th instar with assays made on the 5 th day of the 5 th instar Item Treated Control T /C(%) GDH activity (U/100 ml) Protein content (mg/100 ml) Free amino acids (mg/100 ml) Uric acid content (mg/100 ml)

5 Effects of fluoride on silkworm haemolymph biochemistry 121 Effect of fluoride on catalase activity in silkworm haemolymph: Figure 1 shows the effects of 32 mg F/kg dry artificial diet on catalase activity in haemolymph of Hang 8 strain silkworms during the 4 th and 5 th instar. The catalase activity of silkworm haemolymph is normally low in the early stages of the 4 th and 5 th instar, increasing somewhat as the larvae grow and again becoming low before molting. On the other hand, the catalase activity in the haemolymph of Hang 8 silkworms fed the 32 mg F/kg dry artificial diet was much higher than that of the controls for both the 4 th and 5 th instar, reaching a 24-fold greater peak about 42 hr into the 4 th instar and a 21-fold higher peak about 62 hr into the 5 th instar. Ratio of catalase activity increase Hours after beginning of fluoride treatment 4th instar 5th instar Figure 1. Effect of fluoride on catalase activity in haemolymph in the 4 th and 5 th instar of Hang 8 strain silkworm larvae fed a 32 mg F/kg dry artificial diet successively during the 4 th and 5th instar, respectively. The bars show the ratio of catalase activity increase over controls after fluoride treatment. The effect of fluoride concentration on catalase activity in the haemolymph of 4 th instar larvae is shown in Figure 2. The activity was not affected when the silkworms were fed fluoride at 8 mg F/kg dry feed successively from the 4 th instar, indicating the worms were tolerant to this level of fluoride. But when the fluoride level reached 16 or 32 mg F/kg dry feed, the catalase activity of the haemolymph was significantly higher than that of the controls. The maximum increase of 17-fold occurred at the 84th hr of the 5 th instar at 32 mg F/kg dry feed.

6 122 Miao, Jiang, Bharathi Catalase activity(mg decomposed H 2 O 2 / ml haemolymph) Hours after fluoride treatment control 8 mg F/Kg 16 mg F/Kg 32 mg F/Kg Figure 2. Effect of fluoride concentration on catalase activity of haemolymph in the 4 th instar of Hang 8 strain silkworm larvae fed with 8, 16, or 32 mg F/kg dry artificial diet beginning with the 4th instar. DISCUSSION Field studies of a wide variety of invertebrate taxa invariably indicate that samples taken from areas near fluoride pollution sources have greatly elevated fluoride loads However, many of the toxicological effects of these fluoride loads on invertebrates are largely unknown. In China, during the 1980s, when considerable rural economic development began, many small town and village fluoride-releasing manufacturing operations and industries rapidly emerged. Meanwhile, pollution from increased use of agricultural chemicals also became more serious. In the spring of 1982 and again in the spring of 1986, incidents of fluoride pollution occurred over large areas and caused substantial declines in silk production. The economic loss to silkworm producers during those two seasons was estimated at about US$1.21 and 1.57 million, respectively. 5 There is much evidence that the widely distributed brickkilns were the main fluoride pollution source. 6 The biochemical characteristics of fluoride-poisoned silkworm haemolymph would be expected to reveal toxicological mechanisms and

7 Effects of fluoride on silkworm haemolymph biochemistry 123 serve as indexes for evaluating the health status of fluoride-polluted larvae. Chen and Jia 11 evaluated the activity of alkaline phosphatase (ALP) in midgut tissue and acid phosphatase (ACP) in haemolymph after feeding silkworms an artificial fluoride-containing diet. The results showed that excessive ingestion of fluoride could inhibit activity of ACP in the haemolymph and ALP in the midgut tissue, respectively. Furthermore, their study confirmed that oral administration of Ca(OH) 2 to the larvae improved ACP and AKP activities in the haemolymph of fluoride-poisoned larvae, suggesting that Ca 2+ could partly relieve silkworm fluoride intoxication. 11 In our work, we have investigated the general decrease in concentration of Ca 2+ and Mg 2+ ions in haemolymph of silkworms exposed to fluoride under controlled conditions. The results may imply that the Ca 2+ and Mg 2+ ions in haemolymph combine with fluoride to form relatively non-toxic MgF 2 and CaF 2 and excrete them in the faeces. The results also provided the possibility of supplying calcium, as in the form of lime, to reduce the toxicity of fluoride to silkworms. Catalases have evolved as a characteristic group of enzymes that efficiently decompose toxic peroxides to prevent their accumulation in the cell. It is generally accepted that damage occurs early during the oxygen reperfusion phase, due to the formation of reactive oxygen species (ROS) from various sources, and that antioxidant systems are critical for the removal of ROS to prevent subsequent damage from their activity. 23 The catalase activity in haemolymph of silkworms fed a fluoride-supplemented artificial diet was significantly higher than that of controls in both the 4 th or 5 th instar as illustrated with the Hang 8 strain in Figures 1 and 2. These results suggest that this enzyme acted defensively to protect against fluoride intoxication. On the other hand, glutamate dehydrogenase (GDH) activity and the levels of protein and free amino acids in haemolymph were lower after fluoride treatment of the larvae compared to those of the controls. However, the uric acid content haemolymph on the 5 th day loaf 5 th instar larvae was higher than that of the control. These results thus indicate that fluoride contamination also affected protein metabolism and harmed the growth and development of silkworm larvae. During rearing, the larvae exhibited serious poisoning symptoms such as decreased appetite, retarded growth, and lack of molting. Moreover, they suggest that some biochemical profiles of haemolymph may be useful as an index to evaluate the health status and economic prospects from fluoride exposure of the silkworm, Bombyx mori L. REFERENCES 1 China Environmental Monitoring General Station. The Background Values of Elements in Soils in China. Beijing: China Environmental Science Press; p [in Chinese].

8 124 Miao, Jiang, Bharathi 2 Xie ZM, Wu WH, Xu JM. Study on fluoride emission from soils at high temperature related to brick-making process. Chemosphere 2003;50: Shahied LS. Fluoride emissions from brick and tile raw materials. In abstracts to Bish DL, Los Alamos National Laboratory; p Fu LS, Liu C, Wu FZ. Main airborne fluoride sources and their treatment in Hangjiahu sericulture district. Rural Eco-Environ 1993;4:26-8. [in Chinese]. 5 Ma XK, Xu QY. A discussion on fluoride pollution of mulberry and silkworm and control strategy in Hang-jia-hu. Agro-Environ Pollut 1988;7(3):30-2. [in Chinese]. 6 Tong ZL. An approach to several aspects of silkworm toxicosis caused by fluoride pollution in the air in Hangzhow. Agro-Environ Protect 1988;7(5):46-8. [in Chinese]. 7 Tang LY, Liu C, Wu FZ. Quantity studies of fluoride compounds deposited in mulberry and silkworm, Bombyx mori L. Acta Sericologica Sinica 1984;10(3): [in Chinese]. 8 Chen YY, Wu YC. Fluoride loading and kinetics in different tissues of larvae of fluorosis silkworm (Bombyx mori L.). Sericologia 1995;35(1): Chen YY. Differences in fluoride effects on fecundity among varieties of the silkworm Bombyx mori. Fluoride 2003;36(3): Chen YY, Wu YC. Study on the effect of fluoride on the growth, development and economic parameters of silkworm Bombyx mori L. Sericologia 1994;34(4): Chen ZW, Jia XY. Effects of fluoride on activities of phosphatase in haemolymph and midgut tissues of silkworm. Agro-Environ Protect 2002;21(2): [in Chinese]. 12 Chen ZW, Jia XY, Lin MS. Effects of fluoride on catalase activity in silkworm. Rural Eco- Environ 2002;18(1):31-4. [in Chinese]. 13 Miao YG, Wu CX, Xu JL. Studies on the artificial diet of silkworm Bombyx mori L. Journal of Zhejiang Agric Univ 1999;25(3): [in Chinese]. 14 Lin QS. Clinic Chemical Assay. Press of Beijing University; [in Chinese]. 15 Lowry OH, Rosebrough NJ, Farr, AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193: West KA, Crabb JW. Automatic hydrolysis and PTC amino acid analysis: a progress report. In Techniques in Protein Chemistry, T. E. Huhli, editor. New York: Academic Press; p Ruiz J, Ferrer J, Camacho M, Bonete MJ. NAD-specific GDH from Thermus thermophilus HB8: purification and enzymatic properties. FEMS Microbiol Lett 1998; 159(1): Van HE. Direct determination of uric acid in fecal material. Biochem Med 1975;12: Zhou LW, Chen XC. Test of detoxicant selection. Jiangsu Sericulture 1994;15(4):80-4. [in Chinese]. 20 Garrec JP, Plebin R. Accumulation du fluor dans les vers de terre dans les sols contamines. Environ Pollut, Ser B 1984;7: Buse A. Fluoride accumulation in invertebrates near an aluminium reduction plant in Wales. Environ Pollut, Ser A 1986;27: Walton KC. Factors determining amounts of fluoride in woodlice Oniscus asellus and Porcellio scaber, litter and soil near an aluminium reduction plant. Environ Pollut, Ser A 1986;42: Marcel ZA, Mocky A, Franz K. Understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis. Progr Biophys Mol Biol 1999;72: Published by the International Society for Fluoride Research Editorial Office: 727 Brighton Road, Ocean View, Dunedin 9051, New Zealand