Background
Earthworms, with their diverse utilization in waste management, sustainable agriculture and fisheries, become promising soil-macroinvertebrate species over the world. The role of earthworm species in vermicomposting (Ismail, 1997; Sinha et al., 2002; Edwards and Arancon, 2004; Gupta et al., 2007; Suthar and Singh, 2008), organic agriculture (Padmavathiamma et al., 2008; Suthar, 2009; Lazcano and Domĺnguez, 2011), and to feed chicken, pig, rabbit and fish species (Guerrero et al., 1984; Mason et al., 1992; Kostecka and Pączka, 2006; Chakrabarty, 2008) was well reported. It is not only live food for fish but also can be potential ingredient for supplementing nutrition in fish-feed (Guerrero, 1983; Akiyama et al., 1984; Stafford and Tacon, 1985; Hasanuzzaman et al., 2010). All such utilization of earthworms exacerbates their global demand day-by-day, but the supply of earthworms from natural habitat does not meet the global requirement for earthworms. Moreover, the availability of earthworm from natural habitat is seasonal and even when available, earthworm has high tendency to bioaccumulate toxic organic residues including pesticides, herbicides and antibiotics and heavy metals including cadmium, nickel, lead and zinc into their tissue (Ramu, 2001). There is likelihood of magnification of those toxicants in livestock, fishes, and ultimately in humans through wild earthworms if used. Therefore, earthworm rearing has been increasingly becoming popular in order to have earthworms all years and to avoid the biomagnifications of these hazardous pollutants.

Among various earthworm species; Eisenia fetida, Eisenia andrei, Eudrilus eugeniae and Perionyx excavatus are reported as the most promising earthworm species used for vermicomposting (Garg et al., 2005), and P. excavatus (Oligochaeta), an Asian native species, is a suitably potential species for vermicomposting and for live food for roosters and fishes (Ali, 2002). The life cycle of P. excavatus, its reproductive biology, its efficacy in waste decomposition and its rearing using organic wastes is substantially reported (Kale et al., 1982; Hallatt et al., 1990; Reinecke et al., 1992; Edwards et al., 1998; Suthar, 2007b, 2009) but the impact of food composition on nutritional value of this species is still few studied. To determine the changes in nutritional profile in response to food composition is essential while this earthworm species can be used as animal protein source. The aim of this study is to determine the influence of four different local and low cost food sources on the growth, the reproduction and the nutritional composition of P. excavatus reared in subtropical environment.

1 Results
1.1 Physico-chemical parameters
Before earthworms’ inoculation, the physico-chemical parameters of the four food sources were determined. The results were presented in Table 1. The average pH and temperature of the compositions were around to 7.0 (buffer level) and 29℃, respectively. The lowest moisture percentage was detected in control (65.85%) while T₃ held the highest moisture level (76.40%). Before inoculation of earthworms, the greatest and least OC/TN was recorded in C and T₂ respectively, while the ratio was highest in T₃ and lowest in C after earthworm activities.

Table 1 Physico-chemical parameters of four food sources

 
1.2 Growth rate of P. excavatus
Figure 1 shows the growth curve of P. excavatus larvae reared in different organic food sources. For all treatment, the growth of larvae increased over the period of rearing. Maximum biomass production (1.36 g /worm) and relative growth rate (0.16 g·worm^-1·week^-1) were found in T₂, which made significant difference (P < 0.05) compared to T₁ and T₃ but not significantly different (P > 0.05) from C (Table 2). Minimum biomass production (0.81 g /worm) and relative growth rate (0.09 g·worm^-1·week^-1) were observed in T₃.

Figure 1 Growth curve of P. excavatus reared in different food sources

Table 2 Growth rate of P. excavatus fed with 4 different food sources

 
1.3 Reproduction performance of P. excavatus
After 120 days of earthworm (P. excavatus) rearing in four different organic food sources, the results demonstrated that the food source T₃ had a net reproduction rate (2.508 ± 0.75 larvae /week) significantly higher than the other food sources (Table 3). The lowest larval production rate was found in the control (0.227 ± 0.18 larvae /week) while others demonstrated marginally better performance than control and all were distinctly significantly varied from T₃ (P < 0.05).

Table 3 Production performance of P. excavatus fed organic food with different compositions

 
1.4 Nutritional composition
The results on nutritional compositions of the earthworms fed with different organic food sources showed that the highest protein and moisture percentage were recorded in T₂, where T₃ contained the lowest protein percentage and the highest lipid and ash percentage (Table 4). But there were no significant difference (P > 0.05) in nutritional compositions among the different groups.

Table 4 Nutritional compositions of P. excavatus fed with four different organic food sources

 
2 Discussion
In the present study, the reproduction rate and biomass production of P. excavatus were found varied with the organic food compositions (Table 2 and Table 3). The higher growth rate was estimated in T₂ and the lower in T₃. On the contrary, the highest reproduction rate was observed in T₃ and the lowest rate was in C. Suthar (2007a) reported maximum reproduction rate of P. excavatus when worms fed on decomposed food of kitchen wastes and leaf litter but higher biomass reported in case of farmyard manure. According to Chaudhuri and Bhattacharjee (2002), both growth rate and reproduction rate of P. excavatus was minimum in the diet of mixture with kitchen waste and maximum was in the diet of mixture with straw and bamboo leaf litter. The varied effect of kitchen waste on growth and reproduction of P. excavatus could be associated with different compositions of kitchen waste. Most of the experiments showed maximum bio-potential of kitchen waste in earthworm culture. Different compositions of food thus affect the earthworm culture (Fayolle, 1997).

Though the reproduction rate was higher in diet rich with kitchen waste but the growth of new hatched worms was quiet low. Probably in this study, the higher proportion of kitchen waste and lower proportion of water hyacinth and cow dung in T₃ affected the growth and reproduction rate. The explanation could be related with the materials (discarded vegetables, vegetable peel off) used for kitchen waste composition were degraded soon in T₃ which cause food limitation and hamper the growth of the worms but stimulate their reproduction potential. The higher proportion of water hyacinth and cow dung in T₂ was not potential media for reproduction but it remains long time serving as good food and facilitates the growth.

Apart from the influence of different food sources, the physico-chemical parameters in different culture beds also affect production and nutrition of earthworm species. The effect of moisture, pH, temperature, organic carbon and nitrogen content in culture substrates on the abundance of earthworms has been reported in previous studies (Edwards et al., 1998; Karmegam and Daniel, 2007; Pattnaik and Reddy, 2010). The reproductive potentiality of earthworms is also influenced by the seasonal changes (Giraddi et al., 2008). The mean pH value in all culture beds of the present study was found near to 7.0, which was in accordance with the findings of Pattnaik and Reddy (2010), and in the range of 6.4~7.4 reported by Bhattacharjee and Chaudhuri (2002) for P. excavatus. According to Singh et al. (2005), this earthworm species can perform very well in a wide range of substrate pH (4.3~8.2). The moisture percentage in this experiment was slightly higher than the level (65%~67%) reported by Parthasarathi (2007) but lower than the moisture content of 80%~90% in organic compost supporting best growth of earthworm species (Edwards and Arancon, 2004). However, the moisture level in the present study did not have significant effects on the growth and reproduction of P. excavatus. The present study recorded the temperature ranged between 27℃ and 30℃, which was not significantly different from the range of 20~28℃ reported by Bhattacharjee and Chaudhuri (2002). Edwards et al. (1998) reported the highest reproduction rate of P. excavatus at 25℃, and maximal growth at 30℃. According to Manivannan et al. (2004), the optimum temperature and moisture percentage is 30 ± 2℃ and 60%~70% for maximum cocoon production of P. excavatus.

In the current study, the OC:TN ratio was declined over the 120 days of earthworm culture period. This indicates that the species utilized organic matter for their biomass production, and added nitrogenous excretory materials in the vermicompost. According to Sinha et al. (2002) earthworms secrete different enzymes such as protease, lipase, amylase, cellulase and chitinases which enhance the decomposition process and help to convert them into worm tissues. The mineralization of organic matter (Kaushik and Garg, 2003) and conversion of ammonium nitrogen into nitrate (Atiyeh et al., 2000) causes enrichment of TN in vermicompost.

Declined C:N ratio in the culture substrates over the culture period after earthworm inoculation is well reported (Vinceslas-Apka and Loquet, 1997; Kale, 1998; Chaudhuri et al., 2000; Gupta and Garg, 2008). The reduction of OC:TN ratio over time is an important index to assess the efficiency and maturity of the vermicomposting process (Sen and Chandra, 2007). Suthar (2007a) also reported negative correlation between the production cycle and C:N ratio of culture bed for P. excavatus.

The proximate analysis in the present study reported that there was no significant difference in major nutritional compositions of earthworms among the different treatments. The moisture content of P. excavatus ranged from 77.26% to 81.85%, the lipid content was 3.06% to 4.82% and the protein percentage was between 39.65% and 48.36%. According to the proximate composition analysis by Hasanuzzman et al. (2010), 71.53% moisture content and 46.57% protein percentage was recorded for wild earthworm P. excavatus but the lipid content was found 8.03% which was highly varied with the present study. Moderate dissimilarity for protein content was found from Guerrero (1983) experiment, where it was 69.8% for the same earthworm species. The ash content in all treatments was found lower than 24.26% mentioned by Hasanuzzman et al. (2010). This variation in nutritional composition in the reared earthworm species may be associated with the variation in proportional chemical compositions of different organic food composites.

3 Materials and Methods
3.1 Collection and identification of P. excavatus
A number of epigeic earthworms were collected from wild muddy soil and the species was identified as P. excavatus by observing some key features of external characteristics according to Vargo (1999).

3.2 Measurement of physic-chemical parameters
The physico-chemical characteristics of culture substrates: pH, temperature and moisture percentage of culture beds were recorded fortnightly. To find out the changes in organic carbon (OC) and total nitrogen (TN) content ratio in the culture composition, the OC and TN were measured by Walkley- Black method and AOAC (2000) respectively before and after earthworm rearing.

3.3 Preparation of rearing media, experimental set-up and sampling
Four different food sources, namely control (C), Treatment-1 (T₁), Treatment-2 (T₂), Treatment-3 (T₃), were prepared using different percentages of various organic materials (Table 5), and kept 5~6 weeks for fermentation. The fermented mixtures were put up to 20 cm covering 5 cm bricks and 5~7 cm sand layer above in the cemented ring tanks (0.5 m height and 0.28 m diameter) with two replications of each food source. Five adult earthworms (individual mean weight ranging from 4.05 to 4.41 g) were stocked in each treatment and reared for 120 days under a shed covering with jute bag. Wetting was done twice a day and fed was provided at 10% of the worm’s body weight (Dynes, 2003) twice a week with the respective fermented food mixture.

Table 5 Composition (%) of different culture media of P. excavatus

 
To determine the growth rate, a distinct experiment was conducted for 60 days where five newly hatched larvae (individual mean larval weight of 0.12~0.17 g) were collected from each treatment and stocked in earthen tub separately with respective rearing media. The biomass gain of newly hatched earthworms in each treatment was observed fortnightly from growth test experiment and observation was continued till four fortnights. The growth (g) was calculated in each fortnight for individual treatment to compare the differences and from that data the relative growth rate (RGR) (g·worm^-1·week^-1) was projected. After the culture experiment, the total number of worms was counted to calculate net reproduction rate (larvae/week) and their overall weight was quantified for each composition. The proximate compositions of newly hatched worms of each treatment were analyzed separately for the content of moisture, crude protein, lipid and ash according to the methods of AOAC (2000).

3.4 Statistical analysis
All data were organized, assembled and analyzed using Statistical Package for the Social Sciences (SPSS), version 12.0. The average values of the parameters were compared by one-way ANOVA and Tukey’s post hoc test was performed at 5% level of significance to reveal the significant differences among the treatments.

4 Conclusion
The performance analysis of earthworms in context of the reproduction, growth and nutritional profiles suggested that T₃ composition could be assessed as good medium for reproduction but T₂ composition could be a better food composite for rearing the earthworm (P. excavatus). Further study is needed to find out the effect of different kitchen waste compositions on growth and production performance of P. excavatus as well as other earthworm species. A comprehensive research works are also recommended in the face of nutritional, environmental parameters requirement and culture bed optimization in different stages of life cycle of P. excavatus as well as the feasibility of using this species as live feed for fish, and fishmeal alternative.

Authors’ Contributions
AFMH, the first author, has the principal contribution to the experimental design, data collection, analysis, data interpretation, and preparing as well as revising this manuscript. MD was actively involved in setting the experiment, collecting data from the experimental field, and preparing the manuscript. GMZR worked intensively for experimental set up and data collection. KAH has significantly contributed to developing this article through continuous supervision and criticism. All authors have read and approved the final manuscript.

Acknowledgments
The authors would like to acknowledge the fund received from the Khulna University Research Cell, Khulna University, Bangladesh. We are also thankful to Fisheries and Marine Resource Technology Discipline for providing logistic supports.

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