Vol 12 (1): 14-25, June 2021 eISSN: 2536-8400 © Faculty of Science http://doi.org/10.4038/rjs.v12i1.97 University of Ruhuna SRI LANKA © Faculty of Science, University of Ruhuna Sri Lanka 14 Mass production of the nematode Acrobeloides longiuterus using Tribolium castaneum and artificial solid media *N. Thiruchchelvan, G. Thirukkumaran and G. Mikunthan Department of Agricultural Biology, Faculty of Agriculture, University of Jaffna, Ariviyal Nagar, Kilinochchi 44000, Sri Lanka *Correspondence: thiruchchelvann@univ.jfn.ac.lk; ORCID: https://orcid.org/0000-0003-3800-7104 Received: 12th February, 2021, Revised: 30th May, 2021, Accepted: 11th June, 2020 Abstract. Free-living nematode Acrobeloides longiuterus (Rhabditida: Cephalobidae) exhibits a potential to kill some insect pests. Mass production of this species is a requirement for use it in pest management programs. Tribolium castaneum has been used as a primary host for this nematode as an alternative for Galleria mellonella. Use of artificial media is another option for mass culturing and such recipes based on soy flour are available. Production of A. longiuterus using cost effective method and easily available insect host is important in setting up of small-scale production unit. Therefore, this study has the objectives of evaluating the production feasibility of A. longiuterus on T. castaneum larvae, pupae and adults as in vivo production method. Further, feasibility of using different solid media such as soy flour, palmyra tuber flour, corn flour, black gram flour and dhal flour with other basic ingredients as in vitro conditions system was evaluated. Results revealed that pupa of T. castaneum yielded the highest number of infective juveniles (36112 IJs/ pupa) compared to other life stages tested. In vitro production of A. longiuterus on soy flour and black gram flour media yielded 21530 and 16538 IJs/20g, respectively. Pathogenicity against T. castaneum was shown up to 93% by the infective juveniles produced from the in vitro cultures. In conclusion, T. castaneum is an alternative insect that can be used as a host to produce the A. longiuterus. In addition, soy flour and black gram flour can be used as the sources for this nematode production without losing their entomopathogenicity. Keywords: Entomopathogenic nematodes, free-living nematode, insect host, flour media, red flour beetle. 1 Introduction Application of entomopathogenic nematodes (EPNs) in pest management is ecologically safe and has a quick response. EPNs have been formulated as bio- https://rjs.ruh.ac.lk/index.php/rjs/index https://rjs.ruh.ac.lk/index.php/rjs/index http://doi.org/10.4038/rjs.v12i1.97 https://creativecommons.org/licenses/by-nc/4.0/ mailto:thiruchchelvann@univ.jfn.ac.lk https://orcid.org/0000-0003-3800-7104 N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 15 pesticides and are commercially available to be used in pest management programs (Ehlers and Shapiro-Ilan 2005). Free-living nematode Acrobeloides longiuterus (Rhabditida: Cephalobidae) which is not a true member of entomopathogenic genera such as Steinernema and Heterorhabditis has shown a potential to kill economically important insect pests (Thiruchchelvan et al. 2021). This nematode species causes mortality of the insect host within 48-72 h of application (Thiruchchelvan et al. 2019, 2021), which is a quick knock down effect similar to the classical EPNs. Therefore, A. longiuterus could be used as an additional insect management strategy in integrated pest management. This strategy is environmentally friendly as well as safe for non-target organisms. In order to use this nematode as a biological control agent, it is necessary to produce it in bulk at a reasonable cost. This can be achieved through in vivo and in vitro production using insect hosts and artificial solid media, respectively. Glaser (1940) introduced a method of large-scale in vitro production of EPNs using solid media. Then, Friedman (1990) took it into the next level of in vitro liquid fermentation production method from the three-dimensional in vitro production by Bedding (1981, 1984). Meanwhile, farmers are benefited from both in vivo and in vitro production of nematodes if the production process utilizes substrates generated from their farms (Ehlers and Shapiro-Ilan 2005). In vivo production is a simple process of culturing EPNs in live insect hosts. In vivo production is based on the White’s trap method, which involves the natural migration of infective juveniles (IJs) from the infected host cadaver into a surrounding water layer, from where it can be harvested. White (1927) invented a method, later it was modified by several researchers (Dutky et al. 1964, Poinar, 1979, Woodring and Kaya 1988, Lindegren et al. 1993, Abdel-Razek and Abd-elgawad 2007). The selection of the insect host totally depends on the susceptibility of a particular host for the nematode infestation. In addition, the host must be easily culturable using cost effective materials (Shapiro-Ilan and McCoy 2000, Ehlers and Shapiro-Ilan 2005). Diets for the insect host rearing should be carefully selected, as the diet may influence on juvenile yields (Nunchanart 2002). Generally, last instar larva of the wax moth (Galleria mellonella) is the conventional host used for in vivo multiplication of EPNs (Shapiro-Ilan and McCoy 2000, Shapiro-Ilan et al. 2002, Ehlers and Shapiro-Ilan 2005, Costa et al. 2007). Galleria mellonella is naturally found in beehives and can be reared using artificial diets containing cereals, wax, yeast and glycerol; however, these ingredients are relatively expensive (Costa et al. 2007). Use of other insect hosts is an option for in vivo production to G. mellonella larva. Such hosts are Tenebrio molitor (Blinova and Ivanova 1987, Shapiro-Ilan et al. 2002, Shapiro-Ilan and Gaugler 2002), Diaprepes abbreviates (Shapiro-Ilan and McCoy 2000), Corcyra cephalonica (Blinova and Ivanova 1987, Karunakar et al. 1999, Ganguly and Singh 2000, Shapiro-Ilan and Gaugler 2002, Raj Kumar et al. 2003, Singh and Gupta 2006, Khan et al. 2007, Ali et al. 2008, Shapiro-Ilan et al. 2012), Diatraea saccharalis (Folegatti et al. 1988), Achroia grisella, Bombyx mori (Saenz and Luque 2000, Zaki et al. 2000), Chilo sacchariphagus indicas (Karunakar N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 16 et al. 1999), Hellicovepa armigera (Subramanian 2003, Ali et al. 2008, Rishi and Prasad 2012), Spodoptera litura (Ali et al. 2008, Gupta et al. 2008), Plutella xylostella (Rishi and Prasad 2012), Odontotermes obesus (Devi et al. 2018) and Capnodis tenebrionis (Morton and Gracia-del-Pino 2009). In vitro EPN production is the practice of culturing nematodes on a non-living, nutritive medium containing pre-cultured symbiotic bacteria. Culture media should be sterilized to avoid any microbial contamination and should facilitate the EPN- specific bacterial growth. Sterilized medium is inoculated with bacteria followed by the inoculation of EPNs. Infective juveniles could be collected from the second week after inoculation of EPNs and collected IJs could be stored in water (Devi 2018). House et al. (1965) developed a dog food based medium for commercial scale production of EPNs. Hara et al. (1981) reported the production of 125 million IJs/100 g of dog food agar within a week. Animal protein-fat based media were developed by Bedding (1981, 1984) viz, Pig’s kidney-beef fat homogenate on shredded polyether polyurethane sponge (Bedding 1981). Then, Bedding (1984) developed another animal protein-fat based media for EPN production, such as homogenate of chicken offal for steinernematids, homogenate of chicken offal with 10% beef fats for heterorhabditds, absorbed on shredded polyether polyurethane sponge. Similar chicken offal medium has been used for EPN production by Tabassum and Shahina (2004) in Pakistan. Solid culture method is economically feasible up to a production level of approximately 10×1012 nematodes/month (Friedman et al. 1989, Ramakuwela et al. 2016). However, in in vitro production of EPNs, solid media compositions and culturing environment of the culture conditions should be considered. In addition, proper selection of the nutrients should be considered because it has an impact on the nematode virulence (Ehlers and Shapiro-Ilan 2005). Therefore, the objective of this study was to evaluate the production efficiency of Acrobeloides longiuterus using larva, pupa and adult stages of Tribolium castaneum and artificial solid nutrient media under in vitro conditions. 2 Materials and Methods The research was carried out at the Laboratory of Department of Agricultural Biology, Faculty of Agriculture (FoA), University of Jaffna (UoJ), Sri Lanka during 2017 to 2018. The Coleopteran insect species T. castaneum and the nematode A. longiuterus obtained from the Entomology Laboratory, Department of Agricultural Biology, (FoA, UOJ, Sri Lanka) were used for this study. All the experiments were conducted under the room temperature (27 ± 2 oC). N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 17 2.1 In vivo production of Acrobeloides longiuterus using Tribolium castaneum Red flour beetle Tribolium castaneum (Herbst) larvae, pupae and adults were assessed for their suitability to be used in mass culturing of nematodes following the procedure described by Shapiro-Ilan et al. (2002). For each life stage, ten specimens were put in a moisture chamber (9 cm diameter Petri dish containing moistened filter paper). Infective juveniles (IJs) were pipetted directly onto the insects at the concentrations of 50, 100 and 150 IJs/1 mL water in moisture chamber. There were four replicate dishes per each concentration and hence, 40 insects were in total per treatment. The moisture chambers were maintained at 27 ± 2 oC in darkness for 10 days. Insects were deemed dead if they did not move following gentle prodding with a needle. Dead insects were transferred into the White’s trap separately and IJs were collected 10 days after inoculation (DAI) in water, and the final volume of the IJs suspension was adjusted to 100 mL. Nematodes were counted using a counting dish under a stereo microscope (X 40 magnification). This was done by taking 1 mL nematode sample using a pipette and placing it on a counting dish. Total nematode counts were taken five times. 2.2 In vitro production of Acrobeloides longiuterus using flour media Acrobeloides longiuterus culture was produced in vitro using artificial nutrient media containing different flour with other common ingredients. Five culture media were prepared using following ratios. Soy flour medium was prepared using 75 g of soy flour, 4.5 g of nutrient broth, 1.5 g of yeast extract, and 49.5 mL of corn oil mixed with 100 mL of distilled water (Salma and Shahina 2012). Four other media were prepared replacing soy flour by 75 g of either palmyra tuber flour, corn flour, black gram flour or dhal flour. All the ingredients of a medium were mixed thoroughly and the 20 g of each was made absorbed to 5 pieces of polyurethane sponge (1.5 mm3). Five sponge pieces were added into a polyethylene bag (10x15 cm2), plugged with cotton, and was replicated five times. All the bags were sterilized using an autoclave at 121oC, 1.054 kg/cm3 for 20 min. Thereafter, 2 mL of bacterial suspension isolated from the nematodes was added per bag and incubated over three days under the room temperature at 27±2 oC. Subsequently, 100 IJs were released into each bag. Produced IJs were collected using White’s trap technique 21 DAI. Bacterial isolation was done as described by Upadhyay et al. (2015); A. longiutreus (400-500 IJs/mL) were pipetted directly onto the larvae of T. castaneum in a moisture chamber and incubated at 27±2 oC for 72 hours in the dark. Then, infected dead larva were surface sterilized by dipping them in 70% ethanol for 1-2 seconds and the cadavers of dead larvae were aseptically dissected and a loop full of haemolymph was streaked on nutrient agar plates. Bacterial culture plates were incubated in an N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 18 incubator at the temperature of 27±1 oC for 48 hours. Bacterial colony was washed off into a 100 mL volumetric flask and final volume of bacterial suspension was adjusted to a 100 mL suspension. 2.3 Quality testing of Acrobeloides longiuterus produced from the in vitro culture media against Tribolium castaneum larvae and pupae A moisture chamber assay was used to test the quality of A. longiuterus from in vitro production method against T. castaneum larvae and pupae as described by Kaya and Stock (1997). For each life stage, 10 specimens were put into a moisture chamber. Infective juveniles were pipetted directly onto the insects at concentrations of 50, 100, 150 and 200 IJs/dish in 1 mL water. Control insects were only treated with 1 mL of double distilled water. There were four dishes per treatment; hence, there were 40 insects in total per treatment. The dishes were maintained at 27±2oC in darkness. Mortality of pupae and larvae stages was recorded 48 and 72 hours after inoculation, respectively. LC50 and LC90 values were calculated based on the mortality data obtained by this study. The experiment was arranged in a completely randomized design. 2.4 Statistical analysis All the experiments were arranged in a complete randomized design (CRD) and the data of in vivo experiment were analyzed using the ANOVA- General Linear Model (GLM) with two factor and comparison among the means were done using the Fisher test at 95% confidence interval (CI). Data from the in vitro production and quality testing were analyzed using one way-ANOVA and mean separation were done as per the Fisher test at 95% CI and the Probit analysis was used to calculate the LC50 and LC90 using Minitab ver. 17. 3 Results 3.1 In vivo production of Acrobeloides longiuterus using Tribolium castaneum life stages Figure 1 illustrates in vivo production of A. longiuterus using different life stages of T. castaneum at three different concentrations of infective juveniles (IJs). Number of IJs was significantly different among the concentrations (F(2,54) = 422.48, P<0.05), different life stages (F(5,54) = 87.52; P < 0.05) and the interaction of concentration x life stage (F(10,54) =10.30, P<0.05). Significantly highest yield of IJ (36,112.5 ±14.5 IJs/pupa) was obtained with the pupal stage inoculated with 150 IJs/mL. This was followed by seventh and sixth instars larvae of T. castaneum with 150 IJs/mL N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 19 inoculation producing 31,031±9.1 and 25,425±19.2 IJs/larva, respectively. The use of T. castaneum adults as the host yielded 21,031±47 IJs/adult. Generally, pupa yielded the highest number of IJs/mL at each concentration compared to the other insect stages tested. Fig.1: Production of Acrobeloides longiuterus with respect to six life stages of Tribolium castaneum at three different concentrations of IJs. 3.2 In vitro production of Acrobeloides longiuterus using flour media Production of A. longiuterus from the different types of flour-based media is shown in Table 1. Nematode production was significantly different among the media (F(4,20) = 111.51, P<0.05). Soy flour medium yielded the highest IJs as 21530/20 g of medium whereas the lowest yield was observed in corn flour medium (977/20 g of medium). Table 1: Mean number of Acrobeloides longiuterus produced under in vitro conditions. Medium Number of IJs ±SD/20 g* Soy flour 75 g + Ing. 21530 ± 143 a Corn flour 75 g + Ing. 977 ± 51 e Black gram flour 75 g + Ing. 16538 ± 380 b Dhal flour 75 g + Ing. 12531 ± 148 c Palmyra flour 75 g + Ing. 1533.8 ± 122 d Ing. - Nutrient broth 4.5 g, Yeast extract 1.5g, Corn oil 49.5 mL and Distilled water 100 mL *Values having the same letter were not significantly different (Fisher test at 95% confidence level) N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 20 3.3 Quality testing of Acrobeloides longiuterus produced from the in vitro culture media against Tribolium castaneum larvae and pupae Mortality of the larvae and pupae stages of T. castaneum at different concentrations are given in Table 2. Mortality of larvae at all concentrations of IJs/mL were significantly different from the untreated control (F(4,15) = 149.25, P<0.05). The highest mortalities of larvae and pupae were recorded as 92.5 % (9.25 ± 0.50 mean mortality) at the concentration of 200 IJs/mL. LC50 and LC90 of larvae were calculated as 48.55 and 210.42 IJs/mL, respectively. Mortalities of T. castaneum pupa were significantly different from the untreated control at all four concentrations (IJs/mL) tested (F(4,15) = 203.2, P<0.05). Mortalities of pupa were recorded as 65, 75 and 90 % at the concentrations of 50, 100, 150 IJs/mL, respectively. LC50 and LC90 of the pupae were 32. 94 and 173.97 IJs/mL, respectively. Table 2: Bio-efficacy of Acrobeloides longiuterus produced from the in vitro culture media against Tribolium castaneum larvae and pupae. Concentrations IJs/ mL Mean ±SE Mortality* Larva Pupa 0 0.25 ± 0.500 d 0.5 ± 0.577 d 50 5.25 ± 0.500 c 6.5 ± 0.577 c 100 7.25 ± 0.500 b 7.5 ± 0.577 b 150 8.00 ± 0.816 b 9.0 ± 0.0 a 200 9.25 ± 0.500 a 9.25 ± 0.5 a LC50 48.55 32.94 LC90 210.42 173.97 * Values having the same letter in a column were not significantly different according to the Fisher test at 95% confidence level 4. Discussion Determination of multiplication and production potential of any new nematode isolate using in vivo production in live insect hosts, as well as using in vitro production in solid culture media are the most important steps before either initiating mass production or formulation and commercialization of EPNs (Shapiro-Ilan and Ehlers 2002, Ehlers and Shapiro-Ilan 2005). Thiruchchelvan et al. (2021) reported first that Acrobeloides longiutreus isolated from Sri Lanka showed entomopathogenic characteristics. Therefore, it is important to study the mass production ability for the future studies and experiments. Shapiro-Ilan et al. (2002) stated that in vivo production of EPNs could be used for the laboratory use and small-scale field experiments or applications. In vivo production of EPNs appears to be the suitable N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 21 method for niche markets and small growers. In addition, it requires a minimum investment cost and technical skills for the initial start-up. However, labour cost and availability on production of the insect hosts are the difficulties for in vivo production (Ehlers and Shapiro-Ilan 2005). There are many lepidopteron, coleopteran and dipteran insect hosts that have been used for in vivo production of EPNs (Devi 2018). The host used in this study was T. castaneum, a member of the order coleoptera and the same family of Tenebrio molitor (Tenibrionidae), which were used by many researchers previously (Ehlers and Shapiro-Ilan 2005, Devi 2018) for in vivo EPNs production. The size of the insect host plays a major role in EPNs production (Flanders et al. 1996, Kaya and Stock 1997). This has been confirmed with the use of G. mellonella in EPN production. Larger larvae give a better yield. For instance, G. mellonella larvae (20- 22 mm length) yielded the highest of EPN/larva (Flanders et al. 1996). In this study, availability of insect host, cost effective and convenient rearing of the host insect are considerations in selecting the host insect, T. castaneum. The efficiency of in vivo production correspondingly depends on the quality of insect hosts and their life stages used for the production (Morales-Ramos et al. 2011). However, the quality of insect hosts and their nutritional characteristics depend on the media where they are reared and the life stages of insects (Shapiro- Ilan 2008). In this study T. castaneum was reared in wheat flour, where they are normally crowding and completing their life cycle. Therefore, the IJs production differences with respect to different stages mainly depend on the nutritional properties in the particular life stage, anatomical features and the movement of the insect host. Another factor that influences on IJs yield is the inoculum doses (Shapiro-Ilan et al. 2002). Nematode concentrations in this study had a positive effect on the IJs production, and Boff et al. (2000) and Shapiro-Ilan et al. (2002) obtained similar results. Moreover, we selected intermediate IJs doses (50, 100 and 150 IJ/mL) since the best yields are achieved with intermediate inoculum dosage because higher doses create lower yield due to competition for nutrients (Shapiro-Ilan et al. 2002). Environmental factors including temperature, aeration, and moisture can affect yield of EPNs (Grewal et al. 1994, Dolinski et al. 2007, Shapiro-Ilan et al. 2012). Optimum production temperatures lie between 18 and 28°C for different EPN species (Karagoz et al. 2009, Morton and Gracía-del-Pino 2009). Production of EPNs for the commercial use or for international markets, in vitro liquid culture is considered the most cost-effective process while in vitro solid culture is generally considered intermediate between in vivo and liquid culture (Shapiro-Ilan et al. 2012). Soya flour-based medium yielded the highest production of IJs (21500 IJs/20 g) compared to the other four flour-based media. Similar results were reported by Banu and Meena (2015) who recorded the yield of the nematodes ranging from 3.22 to 3.87 x 103 with highest multiplication rate of 3.87 x 103 in medium-I which has soya flour and corn oil as important ingredients. In addition, Cao et al. (2013) stated that soya flour has high protein content which is important to build new tissue N. Thiruchchelvan et al. Mass production of the nematode Acrobeloides longiuterus Ruhuna Journal of Science Vol 12 (1): 14-25, June 2021 22 in EPNs population that were cultivated in various in vitro media. Results of this study also showed a higher nematode production with increasing protein and fat contents. Soybean powder contained 40.5% of protein, 20.5% of fat, and 22.2% of carbohydrate (Indriyanti and Muharromah 2016). While other flours contain lower amount of protein and fat contents, such as black gram and dhal (protein 25% Fat 6% carbohydrate 53%) (Mazmanyan 2020), palmyra flour (protein 4% and 1% fat) (Vengaiah et al. 2013) and Corn flour (14% protein and 5% fat) (Anonymous 2021). Thus, nematode production has a positive correlation with protein and fat contents of the media or the nutrition content of ingredients. However further experiments with other flour types along with their proximate analysis will provide more information. 5 Conclusions Acrobeloides longiuterus was successfully cultured in vivo using different life stages: larva, pupa and adult of T. castaneum. Of them, T. castaneum pupa was found as the best stage to produce IJs. Among the in vitro media tested, the soy flour-based medium produced the highest yield of IJs compared to black gram, dhal, palmyra and corn flour media. The results show that mass production of A. longiuterus is feasible using the tested in vivo and in vitro methods. Acknowledgements Authors wish to acknowledge Sri Lanka Council for Agriculture Research Policy (SLCARP), Ministry of Agriculture funded the project of National Agricultural Research Policy [NARP/12/UJ/AG/01] for the funding of this research work. Further, we extend our heart-felt gratitude to the reviewers who have made the valuable comments on the initial draft of the manuscript. 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