World J Microbiol Biotechnol (2011) 27:529–533 DOI 10.1007/s11274-010-0486-4
ORIGINAL PAPER
Effects on growth and digestive enzyme activities of the Hepialus gonggaensis larvae caused by introducing probiotics Youping Yin • Dongdong Mu • Shijiang Chen Li Liu • Zhongkang Wang
•
Received: 21 February 2010 / Accepted: 17 June 2010 / Published online: 4 July 2010 Ó Springer Science+Business Media B.V. 2010
Abstract A bacteria strain Hg4-03 of Carnobacterium sp., isolated from the intestine of Hepialus gonggaensis larvae, was fed back to the fourth instars larvae as probiotics to evaluate its impact on the growth performance and digestive enzymes. The larvae were reared in the lab with a natural diet treated with different concentrations of bacterial fermentation and heating killed bacteria, respectively. Compared with the control group, results showed that the growth rates significantly increased and the insect mortality rate decreased significantly after feeding with live probiotics. Meanwhile, the activities of protease, total amylase and trehalase rose significantly in intestinal fluid of the group fed with live probiotics compared with the control treatment. These findings demonstrated that the intestinal bacteria Hg4-03 play an important role for the growth of H. gonggaensis larvae. The bacteria community can improve the growth of H. gonggaensis larvae, indicating that intestinal bacteria may probably be one of the most important factors impacting H. gonggaensis larvae reared in control conditions. Keywords Probiotics Digestive enzymes Growth Hepialus gonggaensis larvae Interaction
Y. Yin D. Mu L. Liu Z. Wang (&) Chongqing Engineering Research Center for Fungal Insecticides, Key Laboratory of Genetic Function and Regulation, Bioengineering college of Chongqing University, 174 Shazheng Street, 400030 Shapingba Dist., Chongqing, China e-mail:
[email protected];
[email protected] S. Chen Chongqing Academy of Chinese Materia Medica, 34 Nanshan Street, 400065, Nan’an Dist., Chongqing, China
Introduction The swift moth Hepialus gonggaensis is a predominant host of Cordyceps sinensis. Its larvae parasitized by fungi forms a fungi-insect complex called Cordyceps or Dongchongxiacao, a valuable traditional Chinese medicine that can be used as a general tonic for protecting and improving lung and kidney function and enhancing physical performance (Li et al. 2007; Zhu et al. 1998). Wild Dongchongxiacao is one of the secondary grades of conservational organisms in China and is very rare because the host insect distributes only in a limited area and collection can take decades. Artificial culture in control conditions or hemi-wild rearing might be the best way to solve this source shortage problem and match the increasing demand (Leung et al. 2009). H. gonggaensis larvae live on the roots of special weeds in meadows in cold areas of high altitude (Fu et al. 1991). Their developmental period of the immature stages from egg to pupa takes about 4–5 years at natural condition (Li et al. 2007; Wang 2002). During this long post-embryo developmental period, mortality is very high due to a lack of nutrition, disease, hunted by natural enemy, changes of climate and pollution, etc. Many efforts have been made to improve the rearing of the insects in controlled conditions (Wang 2002), but little progress has been achieved. Probiotics is usually defined as microbial supplements that benefit the host (Fuller 1989) in terms of a stockbreeding aquatic product and even for people. Some research on using probiotics for environmentally friendly sustainable aquaculture has been reported (Gatesoupe et al. 1989; Wache´ et al. 2006; Wang 2007; Wang and Xu 2006; Ziaei-Nejad et al. 2006). In those studies, lactic acid bacteria and other bacteria were used to improve the host’s growth and feed efficiency, prevent intestinal disorders (Einar 1998;
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Va´zquez and Murado 2008), pre-digest anti-nutritional factors in the food and increase the immune response to infection (Robertson et al. 2000; Wang 2007; Wang and Xu 2006). Previous researches have shown that there is an abundance of microorganisms in the gut of H. gonggaensis larvae, and that the larvae collected from the natural habitat and the larvae reared in control conditions have different intestinal microflora (Liu et al. 2008a; Yu et al. 2008). The digestive enzyme activities of fourth instar H. gonggaensis larvae were reduced after intestinal bacteria were interfered by feeding with antibiotics (Liu et al. 2008b). However, what is the function of the microflora in H. gonggaensis larvae? How does the microflora effect the H. gonggaensis larvae growth and nutrition? Are the dominant bacteria important for larvae growth and development? All these questions still remain unanswered. In our study, we evaluate the effect of bacteria Hg4-03 (Carnobacterium sp.), the most dominant bacteria in the gut of H. gonggaensis larvae, on the growth and digestive enzyme activity in intestinal fluid, and hope to provide another pathway for artificial rearing of Dongchongxiacao.
Materials and methods
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Treatment 1 (TA-1): 20 g cells of live Hg4-03 bacteria were added to 1,000 g diet roots (20 g Kg-1) to feed the larvae. Treatment 2 (TA-2): 10 g cells of live bacteria were added to 1,000 g diet roots (10 g Kg-1). Two control groups were set. One group was fed with the diet of 10 g Kg-1 cells of Hg4-03 heating killed bacteria (CD) and the other control group was fed with diet roots without adding bacteria (C). The diet roots and bacteria were mixed immediately before feeding by surface coating. All larvae fed with different treated roots were reared at the conditions above-described. After 14 days, 12 larvae of each treatment group were subject to digestive enzyme assay, whereas the rest of the population (another 12 larvae of each group) were subject to weighing after 28 days and had their digestive enzymes tested. Mortality was also checked. All measurements were carried out in triplicate.The weight increase was registered at the beginning and ending of the experiment to calculate absolute growth rate as expressed in the formula. The growth rate (%) was expressed as: Finalweight Initialweight 100% Initial weight Enzymatic activity assays
Bacteria Crude enzymatic extracts The bacteria strain Hg4-03 (Carnobacterium sp.) was isolated from the gut of H. gonggaensis larvae by Liu and Yin (2008a) and stored with 30% glycerin at -80°C. Insects rearing H. gonggaensis larvae were supplied by Kangding Cordyceps Cultural Station, and wild larvae collected from Yajiageng Mountain, Kangding city, Sichuan province. The insects were reared in glass jars (a 60 mm 9 80 mm) filled with sterilized soil (115 ml per jar) and fed with roots of their natural diet, Polygonum viviparum L., at 15°C under scattered light. Containers and soil were changed and sterilized every 3 days, at the same time, to renew the fresh diet. Healthy fourth instars larvae were used in the experiment and weighed before the trial. Diet treatment and experimental design One colony of the bacteria strain Hg4-03 was picked from Luria–Bertani (LB) plated and then cultured in LB at 15°C, 250 rpm for about 60 h. The cells were harvested by centrifugation (20 000 g for 20 min) and wet weight was recorded. Two different trials and two controls were carried out. Each treatment included 14 H. gonggaensis larvae and three replicates.
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12 larvae of each treatment were selected randomly and starved for about 16 h and used for the digestive enzyme extraction at the end of both 14 days and 28 days. Crude enzymatic extracts was prepared as follows. The total intestine including content was obtained by dissecting at 4°C, and homogenized in 10 volumes v/w of cold distilled water. The mixture was centrifuged at 4,000g for 10 min at 4°C, and the supernatant was used as a crude enzymatic extract for digestive enzymatic assays within 24 h of extraction. The concentration of total protein in the supernatant was assayed using bovine albumin as a standard according to Bradford (1976).
Detecting enzymatic activities Protease activity was measured using a Folin phenol reagent according to Lowry et al. (1951) with the casein as the substrate. The activities of total amylase, trehalase and cellulase were determined with 3, 5-dinitrosalicylic acid according to Miller (1959) using the starch (10 mg/ml), trehalose (10 mg/ml) and sodium carboxymethyl cellulose (1 mg/ml) as substrates respectively. Enzyme activities were all expressed as a specific activity (U mg-1 protein). All measurements were carried out in triplicate.
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Statistical analysis The effects of probiotics on growth rate and digestive enzyme activities were analyzed using ANOVA one-way model. A significance level of P \ 0.05 was used.
Results Effect of bacteria supplement on growth and mortality of larvae The growth changes of H. gonggaensis larvae were measured after 14 and 28 days of bacteria supplement. The results in Table 1 showed that, 28 days after rearing, the mean weight of both treatment groups (TA-1 and TA-2) was significantly higher (P \ 0.05) than that of control groups CD and C, but there was no significant difference between the two live bacteria treatment groups. Meanwhile, there was also no marked difference between the mean weight of CD and C. After rearing, the live bacteria supplied groups of H. gonggaensis showed a lower mortality compared with CD and C. The count was 15.63, 18.75, 32.26 and 28.13% for TA-1, TA-2, CD and C, respectively (Table 1). Interestingly, the growth rate in the CD group feeding with heating killed bacteria had the lowest livability. Effect of bacteria supplement on digestive enzyme activity Adding live bacteria to the diets impacted the digestion of H. gonggaensis larvae. The activities of three of the four main digestive enzymes—protease, total amylase and trehalase—in the larvae intestine were significantly different between the different treatment groups (P \ 0.05) (Figs. 1 and 2). In all tested digestive enzymes, the activity of total amylase in the live bacteria treated groups improved most highly compared with the CD and control groups, which was 54.60 (±1.83) U mg-1 protein for TA-1, 50.25 (±1.57) U mg-1 protein for TA-2, 30.95 (±1.58) U mg-1 protein for CD and 31.19 (±2.12) U mg-1 protein) for C
Fig. 1 Activity of intestinal digestive enzymes from larvae fed diets containing different concentrations of probiotics after 14 days of culture. Means with different superscripts are significantly different (P \ 0.05). TA-1: TA-1 group, TA-2: TA-2 group, CD: CD group and C: control group
Fig. 2 Activity of intestinal digestive enzymes from larvae fed diets containing different concentrations of probiotics after 28 days of culture. Means with different superscripts are significantly different (P \ 0.05). TA-1: TA-1 group, TA-2: TA-2 group, CD: CD group and C: control group
after 14 days (Fig. 1). But there was no significant difference between TA-1 and TA-2. After 28 days (Fig. 2), the same phenomenon was observed but the value of total amylase activity in TA-2 (55.50 (±2.84) U mg-1 protein) was significant compared with the first testing (50.25 (±1.57) U mg-1 protein). Protease activity in the live bacteria supplied groups was also enhanced compared with the other two groups. After rearing for 14 days, protease activity in the intestine of larvae was 22.29 (±2.45) U for TA-1, 23.89 (±1.11) U for
Table 1 Effect of diet supplemented with different concentrations of probiotics (A-1, A-2) on the growth performance of larvae TA-1 Initial weight (g) Final weight (g)
TA-2
0.285 ± 0.053a
0.282 ± 0.048a
a
0.5206 ± 0.0816
Mortality (%)
15.63
0.5153 ± 0.0741 18.75
0.290 ± 0.055a
c
0.435 ± 0.060 a
Control
0.295 ± 0.049a
ab
0.442 ± 0.047
Growth rates
CD
0.397 ± 0.062bc
0.387 ± 0.042 a
b
0.3929 ± 0.0657
0.3634 ± 0.0774b
32.26
28.13
Initial weight, final weight and growth rates are presented as mean ± SE of triplicate observations. Means in the same row with different superscripts are significantly different (P \ 0.05). TA-1: TA-1 group, TA-2: TA-2 group, CD: CD group and Control: control group
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TA-2, 16.76 (±1.69) U for CD and 16.79 (±1.02) U for C. There were no significant differences between the two groups of live bacteria (Fig. 1). After 28 days, the activity of protease in TA-1 was remarkably higher than that in the first period (Fig. 2). Trehalase activity in the intestine of H. gonggaensis larvae was low, and showed little change in the experiment periods. The highest trehalase-specific activity was detected after 28 days in TA-1 and TA-2 as 11.88 (±0.44) U and 11.82 (±0.83) U. But on first detection (14 days), no remarkable difference was found in the TA-2, CD and control groups. There was no difference among the four values of CD and C in two periods (Figs. 1 and 2). Cellulase activity was very low in the intestine of H. gonggaensis larvae, and showed no significant change between groups (Figs. 1 and 2).
Discussion Lactic acid bacteria used as probiotics have demonstrated their ability to improve the growth rates and inhibit potential pathogens in farmed aquaculture (Hekmat et al. 2008; Ruiz-Moyano et al. 2008). Carnobacteria are part of the indigenous intestinal bacteria in a wide range of animals, especially in fish. Some studies demonstrated that most Carnobacterium species are harmless to the host (Balca´zar et al. 2007; Hassni et al. 2005; Robertson et al. 2000). H. gonggaensis lives in special conditions and has special intestinal microflora that can only be cultured in temperatures lower than 15°C (Liu et al. 2008a). Our study indicated that intestinal microflora could affect H. gonggaensis larvae growth and development. When larvae were reared in controlled conditions, the balance of the intestinal microflora was disordered (Mu et al. 2009), which was probably the main reason for the high mortality in H. gonggaensis larvae culture. The bacterial strain Carnobacterium sp. Hg4-03 is the most important permanent bacteria for H. gonggaensis larvae development. Introducing Hg4-03 improved the larvae’s digestive ability, especially protein and total amylase activities, which possibly led to the improvement of growth when supplemented with Hg4-03 in larvae diet. Low cellulase and trehalase activities were found in the intestine of all tested group, indicating that cellulose and trehalose are not efficient nutrient materials for H. gonggaensis larvae. Health is the most important factor of anti-adversity for H. gonggaensis larvae. Introducing Hg4-03 improved nutrition and adjusted the balance of intestinal microflora, directly resulting in lower larvae mortality. Growth rate and livability were unaffected by the initial concentration of the bacteria cells, while killed cell had no function, indicating that the bacteria strain can settle down in the
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intestine of H. gonggaensis larvae and play roles for long time. This phenomenon confirmed that well-balanced microflora is important to H. gonggaensis larvae and that interactions occurred between microflora and insect hosts. As previously reported, the possible reasons for the cause of anti-diseases include the following: 1. probiotics consume large amounts of oxygen and inhibit pathogen in intestine; 2. probiotics adhere to intestinal mucus, meaning that other bacteria including pathogens have insufficient space to adhere. Kim and Austin (2006) demonstrated that Carnobacterium sp. was able to survive in the digestive tract of rainbow trout; 3. probiotics produce substances that could inhibit the growth of pathogens (Einar 1998); and 4. the host’s immune system is stimulated by probiotics to protect the larvae against some pathogens (Kim and Austin 2006). No evidence could prove that whether or not the bacteria strain Carnobacterium sp Hg4-03 demonstrated any anti-pathogen actions in the intestine of H. gonggaensis, but we can see that the health of larvae improved after Hg4-03 was introduced, which might generate a stronger resistance for the larvae to pathogen infection.
Conclusion Adding bacteria strain Carnobacterium sp Hg4-03 to the diet of H. gonggaensis larvae can improve the activity of intestinal digestive enzymes and growth of H. gonggaensis larvae. The strain can settle in the intestine of larvae and could be an important probiotics as a feed additive for the host of Dongchongxiacao rearing. Acknowledgments The study was supported by the National Natural Science Foundation of China (grant no. 30572325).
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