Correspondence: Yeon Ho Je, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea. Tel.: +82 2 880 4706; fax: +82 2 873 2319; e-mail: firstname.lastname@example.org
Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.
The ever increasing meat production and consumption, rising concerns over meat quality and safety, and recent livestock disease outbreaks are some of the major factors driving higher growth trajectory of the global feed industry seen in the last several years (Castanon, 2007; Windisch et al., 2008). This growth is also high due to increasing levels of country income and rising per capita meat consumption.
The global feed additive market has a significant need for antibiotics and amino acids due to the domestic and export demand for meat (Wenk 2003a, b; Castanon, 2007). Enzymes and probiotics that increase food digestibility and/or suppress harmful microorganisms, organic acids that reduce gut pH for bacterial control, and edible vaccines for disease control (DeFoliart, 1995; Windisch et al., 2008) are also being purchased. Efforts are underway to develop more effective and safer products for supplementing meat production.
We hypothesize that entomopathogenic fungi can be practical expression platforms of functional genes within insects which have been used as animal feed additives. Functional proteins can be produced in insects by the infection of genetically engineered entomopathogenic fungi. Finally, the mycotized insects can be used as raw materials for feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by a highly infective Beauveria bassiana isolate.
Herein, we first tested seven entomopathogenic Beauveria bassiana isolates to select the most feasible one for the construction of the efficient fungal expression platform. In the selection, consideration was given to conidial infectivity against yellow mealworms in the laboratory. Conidial productivity on one-quarter strength Sabouraud dextrose agar (SDA/4) medium was also determined. The selected isolate ERL1170 was used as a fungal platform, and a plasmid with enhanced green fluorescent protein (egfp) gene was integrated into the fungus by a restriction enzyme-mediated integration method to investigate whether enhanced green fluorescence protein (EGFP) could be produced in the fungus–mealworm complex.
Materials and methods
Microbial strains and culture media
The wild-type strains, B.bassiana ERL1050, 1170, 1578, 1575, 1576, 836 and GHA (Kim et al., 2010), were maintained on SDA/4 in darkness at 25 °C for colony growth. For conidial production, 100 μL of each isolate was plated on SDA/4 at (1 × 107 conidia per mL) per Petri dish (60 mm diameter). After incubation in darkness at 25 °C for 10 days, conidia were harvested and suspended in 0.03% (v/v) siloxane solution (Silwet L-77®) as a wetting agent. Hyphae were removed by filtration and the solution adjusted to 1 × 107 conidia per mL. Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) were used for DNA manipulation and cultured on Luria–Bertani (LB) medium containing 50 g mL−1 ampicillin.
Yellow mealworms, Tenebrio molitor L., were used for fungal infection and supplied by the Department of Agricultural Biology, National Academy of Agricultural Science, Republic of Korea. They were reared in plastic boxes on wheat bran according to the methods of Weaver et al. (1989). A piece of Chinese cabbage leaf was placed on the wheat bran as a water source. For fungal application, ten 3rd instar mealworms were placed in a 60-mm diameter Petri dish, where 2 g of wheat bran was added. Petri dishes were then placed at 4 °C for 20 min to reduce mealworm mobility (Butt & Goettel, 2000). Then at room temperature, 10 mL of conidial suspension (1 × 107 conidia per mL) was sprayed onto each Petri dish using a microsprayer. Petri dishes were covered with lids. Nontreated insects served as the control group. Each treatment was replicated three times and held in an incubator at 25 °C and in a 16:8 hours light/dark cycle. Petri dishes were not stacked to keep excess moisture from forming inside of the dishes. The number of dead mealworms was counted at 1, 2, and 5 days post-treatment. This experiment was repeated twice using the different batches of conidia on different days.
The fungal transformation vector pABeG containing egfp gene (provided by Dr. Ming-Guang Feng, Zhejiang University, Hangzhou, Zhejiang 310058), was used as a plasmid for fungal transformation. The fungal transformation vector, pABeG, has phosphinothricin (PPT) resistant bar and egfp genes, and they are expressed under the control of gpdA promoter in the same transcriptional direction. The plasmid, linearized by cutting with HindIII, was integrated into the genomic DNA of ERL1170 by the restriction enzyme-mediated integration method based on blastospores (Ying & Feng, 2006). Transformants were collected on Czapek's agar solution containing 400 μg mL−1 PPT. They were sequentially grown on 600 μg mL−1 PPT+ Czapek's agar solution. Putative transformants were subcultured twice on SDA/4 at 25 °C. Genomic DNAs were extracted from 5-day-old fungal mycelial masses by using the quick fungal genomic DNA extraction method (Chi et al., 2009), and the presence of bar and egfp was examined by PCR with primers Bar-F and Bar-R (5′-AGT CGA CCG TGT ACG TCT CC-3′ and 5′-GAA GTC CAG CTG CCA GAA AC-3′) and primers egfp-F and egfp-R (5′-ATG GTG AGC AAG GGC G-3′ and 5′- CTT GTA CAG CTC GTC C-3′).
Verification of expression
Transcription of egfp in the putative transformants was examined by the extraction of RNAs from 5-day-old fungal mycelial masses using the TRIZOL (Invitrogen) method and the reverse transcription PCR (RT-PCR) with the primers egfp-F and egfp-R. The expression of egfp gene in the selected transformant (Bb-egfp#3) was observed under fluorescence at 100× magnification.
Production of EGFP in mealworms
The production of EGFP in mealworms was examined by the infection of Bb-egfp#3 as previously described in the mortality test and the observation under fluorescence. Wild-type treatment served as a control. At 7-days post-treatment, infected mealworms were cut using a sterile mesh, and the production of EGFP was observed under fluorescence at 100× magnification. Next, dead mealworms were placed in 90-mm diameter Petri dishes containing filter paper moistened with 1 mL distilled water. They were incubated for 3 days to observe mycelial outgrowth on the surface of the dead mealworms.
Data on the percentage of dead mealworms were analyzed by the general linear model (GLM), followed by Tukey's honestly significant difference (HSD) for multiple comparisons. The GLM analysis had three variables as follows: experimental repletion, treatment in each experiment, and observation time (1, 2, and 5 days) in each treatment. The analysis was conducted using spss ver. 17.1 (SPSS Inc. 2009) at the 0.05 (α) level of significance.
Infectivity of the wild-type B.bassiana isolates
Among the test isolates, ERL1170 had the highest infectivity of mealworms (F7,48 = 632.1, P < 0.001) (Fig. 1). It had 57.1% and 98.3% mortality of mealworms 2 and 5 days post-treatment, respectively. Nontreated control had < 10% mealworm mortality in 5 days. Dead mealworms turned dark brown compared to the light brown live mealworms in the nontreated control. No mycelial outgrowth was observed on the surface of the dead mealworms at 5 days.
Generation of Bb-egfp transformants
Several ERL1170 transformants expressing the egfp gene were generated by the fungal transformation, and Bb-egfp#3 showed the highest level of fluorescence (Fig. 2). No significant difference in morphology between the wild-type ERL1170 and Bb-egfp#3 was observed. Bb-egfp#3 showed high similarity to the wild type in conidial germination, mycelial growth, conidiogenesis, and conidial productivity (c. 2.0 × 108 conidia per cm2) 7 days post-treatment (Fig. 2a). RT-PCR of Bb-egfp#3 with the primers egfp-F and egfp-R detected 716-bp target fragment, but not in wild-type ERL1170 (Fig. 2b). As controls, 18s rRNA gene fragments (180 bp) were detected in both wild-type ERL1170 and Bb-egfp#3. The highest level of fluorescence was observed in Bb-egfp#3, but not in the wild-type culture (Fig. 2c).
Production of EGFP in mealworms
Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-day mealworm sections showed a strong fluorescence (Fig. 3). Wild-type and Bb-egfp#3-treated mealworms turned dark brown 4 days after the fungal infection, but no pathogenesis was found in the nontreated control (Fig. 3a). No significant difference in infectivity (mortality) between wild type and Bb-egfp#3 (c. 95% in 7 days) was observed. Bb-egfp#3-treated mealworms had a strong fluorescence, but not in the wild-type-treated mealworms (Fig. 3b). When the dead mealworms were placed in moisturized Petri dishes and incubated for 3 days, fungal outgrowth was observed on the surface of wild-type and Bb-egfp#3-treated mealworms (Fig. 3c).
The genetically engineered fungus can be used in two ways. The fungus itself can be worked under fermentation to produce functional proteins. But we considered that the mealworms would be used as animal feed additives for health management. The fungus-mediated expression of functional genes in the mealworms enabled them to acquire additional benefits. The mealworm-based fungal expression platform consists of (1) infection of an entomopathogenic fungus containing functional genes, (2) hyphal growth and production of functional proteins in insects, and (3) use of infected mealworms as animal feed additives (Fig. 4).
In this mealworm-based fungal expression platform, not only egfp gene but also other functional genes can be expressed, such as antimicrobial peptides (AMP), edible vaccines, or digestive enzymes. The AMPs (also called host defense peptides), an alternative to antibiotics, are small genes encoding 1–5 kDa of peptides that have a broad range of activity against Gram-positive and Gram-negative bacteria, fungi, and mycobacteria (Zasloff, 2002). Some AMPs include lactoferrin, lactoferricin, lysozyme, neutrophil peptides, and purothionin. Secondly, animal vaccines can be produced in this expression platform although many efforts have been given to plant-based vaccine production (Gleba et al., 2005). Several functional genes on digestive enzymes, proteases, peptidases, lipases, and carbohydrases can be integrated into mealworm-pathogenic fungi and be possibly expressed in this mealworm-based fungal expression platform.
There are several advantages of using the mealworm-based fungal expression platform to produce functional proteins for feed additives. Firstly, entomopathogenic fungi, growing in insects, produce their own functional secondary metabolites, which contain large amounts of proteases and antimicrobial metabolites for bacterial control during hyphal penetration into proteinaceous cuticles and the hemocoel of insects (Roberts & Hajek, 1992; Charnley, 2003). Proteases are produced to degrade several layers of insect cuticles (during the molting process), and these exist in the form of a chitin-protein matrix. In entomopathogenic fungi, antimicrobial metabolites suppress the growth of other bacteria which could possibly hinder fungal colonization in insects. Secondly, the final mealworm-fungal complex has high levels of nutrition coming from the host insect (mealworms). Generally, insects have high levels of nutritional resources, especially proteins, lipids, minerals, and vitamins. In particular, the amino acid content in insects is significantly higher than that in other plant legumes (Chavunduka, 1975; Ramos-Elorduy et al., 1997). Mealworms in their larval and pupal stages are rich in protein and are easy to rear (Ghaly & Alkoaik, 2009). Thirdly, proteins from insects are clean and safe to animals and have little chance of being infected by other harmful microorganisms or diseases (DeFoliart, 1995, 1999). Lastly, mass production of entomopathogenic fungi as inocula for host insects has been well developed and is cost-effective (Kim et al., 2011).
Theoretically, there are many strategies to produce useful proteins in mealworms. One possible idea is the generation of transgenic mealworms (Wimmer, 2003). Another strategy is the use of entomopathogenic bacteria, virus, or fungi to express foreign genes in insects. Unfortunately, little information on mealworm-specific bacteria and baculovirus (Bharadwaj & Stafford, 2011) has been reported. Entomopathogenic bacteria, particularly Bacillus thuringiensis is mostly pathogenic to lepidopteran insects, not to coleopteran pests, such as mealworms. However, some entomopathogenic fungi, such as B.bassiana and/or Metarhizium anisopliae, are pathogenic to mealworms as shown in this work and possibly contribute to the production of functional proteins in insects as feed additives. In agricultural pest control, integration of insecticidal genes in entomopathogenic fungi for enhancing control efficacy was successfully conducted (St. Leger et al., 1996; Wang & St. Leger, 2007).
The final mealworm–fungal complex (products) can be used as animal feed additives, and its application possibly expands to human healthcare products. Integration of AMPs, edible vaccines, and useful amino acids to Cordyceps species could significantly improve their biological activities. Several species of Cordyceps are considered as fungal medicines in classical Asian pharmacologies such as that of traditional Chinese medicine. Some Cordyceps species are sources of biochemicals with interesting biological and pharmacological properties, like cordycepin and ciclosporin which are helpful in human organ transplants by suppressing the immune system (Holliday et al., 2004).
In this work, Bb-egfp#3 successfully produced EGFP in mealworms for animal feed additives. We for the first time suggest that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the feed additive industry.
We are grateful to Dr. Ming-Guang Feng in Zhejiang University for providing the transformation vector. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008036), Rural Development Administration, Republic of Korea.