Properties of Enhanced Calcium-alginate Beads as a Formulation for Disseminating the Entomopathogenic Nematodes Heterorhabditis bacteriophora, Steinernema carpocapase, and Steinernema feltiae

Entomopathogenic nematodes (EPNs) from the families Steinernematidae and Heterorhabditidae are insect-parasitic organisms widely utilized as bioinsecticides (Glazer, 1992; Grewal, 2002; Karimi et al, 2018; Okram et al., 2025). They infect a wide range of insect hosts. Application of EPNs for control of soil-borne pests is effective because nematodes are protected against ultraviolet (UV) radiation (Kaya, 1990); However, environmental stress to EPNs applied against foliar pests remains a constraint (Glazer, 1992). EPNs have apparent potential for application as biopesticides, but their low survival in vitro at room temperature further constrains their practical use (Grewal, 2002). The third larval stage of EPNs, the infective juvenile (IJ) stage, is formulated as a product for field application. The IJs are free-living and searching for their eventual host, which is killed after infection. EPNs are suspended in the water and sprayed onto the surface of plants or soil. Commercial successes of EPN application are rare because IJs are very susceptible to UV (Gaugler et al., 1997) and spraying them onto the surface of plants results in high EPN mortality and/or loss (Shapiro-Ilan et al., 2006).

Given the constraints related application of EPNs as biopesticides in the field, recent studies have focused on improving formulations and application methods (Georgis and Kaya, 1998; Shapiro-Ilan et al., 2006; Aquino-Bolaños et al., 2019; Platt et al., 2019; Nxitywa and Malan, 2021; Fallet et al., 2024; Waweru et al., 2025). Suspensions of nematodes must be stirred continuously to achieve uniform mixtures. Additionally, IJs require high oxygen levels and are sensitive to elevated temperatures (Nxitywa and Malan, 2021). Moreover, formulations stored at room temperature are highly desirable, as this facilitates the transfer and application of nematodes in the field (Grzywacz et al., 2014; Ruiz-Vega et al., 2018). Nematodes should be formulated immediately after production, as dead nematodes can interfere with storage (Ehlers, 2007).

Recent research has focused on development of low-cost formulation capable of maintaining nematode viability at ambient temperatures (Ruiz-Vega et al., 2018). For long-term storage and transport, EPNs can be formulated using various carrier materials. Several studies have investigated the impact of different substrates on the longevity and pathogenicity of EPNs. Among these, sponge and vermiculite-based formulations are commonly employed for storing small quantities of nematodes. However, sponge formulations are not suitable for high nematode densities because IJs may migrate out of the sponge matrix. Additionally, IJs remain metabolically active within the sponge, leading to the depletion of stored energy reserves such as carbohydrates (Grewal, 2002; Grewal and Peters, 2005). A primary objective of EPN formulations is to reduce IJ activity and conserve their energy during storage. Both sponge and vermiculite formulations typically require refrigeration during storage and transportation to maintain nematode viability (Grewal, 2002). Alternative formulation approaches include the use of polyacrylamide gels, wettable powders, water-dispersible granules, alginate beads, clay, and activated charcoal (Bedding, 1988; Grewal, 2002; Chen and Glazer, 2005; Nxitywa and Malan, 2021).

Silver et al. (1995) developed granules composed of diatomaceous earth, hydroxyethyl cellulose, amorphous silica, fumed hydrophobic silica, lignosulfonate, starch, pregelatinized starch, and pre-gelled attapulgite clay, achieving a nematode survival rate of 90% after six weeks of storage at 25°C. Leite et al. (2018) evaluated the survival of Steinernema feltiae formulated in polyacrylamide gel and vermiculite at 25°C and 35°C, reporting higher survival rates of IJs in these substrates compared to water. Similarly, Grewal (2000) demonstrated that formulating IJs in water-dispersible granules reduces desiccation and enhances survival for up to three months at ambient temperature, outperforming aqueous formulations. Navon et al. (2002) encapsulated S. carpocapsae in an edible gel suitable for insect ingestion, achieving over 95% mortality of Helicoverpa armigera Hübner and Spodoptera littoralis Boisduval. Matadamas-Ortiz et al. (2014) encapsulated S. glaseri in diatomaceous earth pellets, reporting a 56% survival rate after 14 days at room temperature. Cruz-Martínez et al. (2017) observed that EPNs formulated and applied as infected insect cadavers exhibited higher pest control efficacy than those applied in aqueous suspension.

Formulations that suppress the metabolic activity of IJs enhance nematode survival. In certain formulations, IJs experience a reduction in water content, which leads to decreased activity (Kondo and Ishibashi, 1989). Alginate bead formulations have demonstrated the ability to limit nematode mobility (Georgis, 1990; Kaya and Nelson, 1985; Kaya et al., 1987). Alginate, a polysaccharide derived from brown algae (Yabur et al., 2007), was first utilized by Kaya and Nelson (1985) to encapsulate Heterorhabditis heliothidis and Steinernema feltiae. In this formulation, EPNs became immobile within the beads but were able to escape once the beads softened after several days—an attribute considered unfavorable for commercial applications (Hiltpold et al., 2012; Kim et al., 2015). To address this limitation, Kim et al. (2021) enhanced the calcium alginate formulation by incorporating 18% glycerol, which effectively induced a quiescent state in EPNs.

Calcium alginate beads are water-insoluble. The aim of this study was to develop a formulation that is water-soluble and stable at room temperature while also preserving the pathogenicity of nematodes over time. In this research, we investigated the potential for creating calcium alginate beads that could dissolve in water. Subsequently we assessed the survival and infectivity of IJs of three species of nematodes, Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema feltiae encapsulated within these beads and in sterile water. Our hypothesis was that nematodes enclosed in alginate beads would exhibit higher survival rates compared to those in sterile water. Additionally, we examined the survival of nematodes to address the issue of transferring nematodes to a cooling device. Furthermore, we analyzed the impact of varying sodium alginate concentrations on bead hardness and the release rate of IJs. Lastly, we investigated the pathogenicity and reproduction rate of nematodes throughout the formulation period, the pathogenicity and reproductive rate of IJs suspended within the formulated alginate beads, recognizing the significance of maintaining these traits throughout the formulation period.

EPNs can be formulated in various ways to promote long-term storage and transportation to the field as biopesticides (Nxitywa and Malan, 2021). Ehlers (2007) suggested that the formulation of nematode IJs could help reduce nematode mortality after emergence. Furthermore, the physical attributes of a formulation and abiotic conditions during storage, such as temperature, can be controlled to facilitate the duration of storage and successful delivery to the field (Grzywacz et al., 2014; Ruiz-Vega et al., 2018).

Sponges are commonly used to store and transport EPN IJs in small volumes. However, sponges are not an optimal substrate for maintaining high densities of IJs because the nematodes are capable of escaping, given their mobility. Excessive nematode movement causes depletion of energy and desiccation (Grewal and Peters, 2005). Additionally, use of sponges is costly and requires refrigeration during storage and transportation (Grewal, 2002; Cruz-Martínez et al., 2017). Andalo et al. (2011) reported that the survival rate of S. carpocapsae in sponge formulations at 16°C was 89.3% after 90 days and 57.5% at 180 days post-formulation. Similarly, Grewal (2002) reported survival durations of EPNs between 1–3 months in sponges at 5ºC −10°C. A further disadvantage of using sponges is the complexity of extracting and isolating IJs from the sponge matrix, which can hinder downstream applications.

In order to be effective, an ideal formulation should possess high quality and efficacy. Moreover, it should be easily transportable and applicable in field settings. Recent research has focused on developing IJ formulations that can be stored at high concentrations for extended periods without a decline in pathogenicity (Shapiro-Ilan et al., 2012). Various formulations have been developed, including wettable powders, water-dispersible granules, surfactants, oil emulsions, and alginate beads (Grewal, 2002; Beck et al., 2013; Noosidum et al., 2016; Aquino et al., 2019; Platt et al., 2019; Fallet et al., 2024; Nxitywa and Malan, 2021). Alginate formulations are particularly noteworthy due to their environmentally benign nature, making them suitable for field use (Nxitywa and Malan, 2021). Furthermore, their use to formulate EPNs is cost-effective and they are easy to apply (Vemmer and Patel, 2013).

Herein, we investigated an alginate bead formation using various concentrations of sodium alginate and CaCl₂. The escape rate, longevity, and pathogenicity of IJs were monitored over time. Our findings indicate that the escape rate of IJs from alginate beads can be reduced for extended periods by manipulating the formulation to circumvent the challenges of long-term storage (Kagimu and Malan, 2019). IJs were observed escaping from softer formulations of alginate beads, consistent with the results of Hiltpold et al. (2012). Specifically, IJs escaped prematurely from beads produced with 0.5% or 1% sodium alginate and 10, 15, or 20 mM CaCl₂ prior to transfer into water. Conversely, beads produced with 1.5% and 2% sodium alginate retained IJs more effectively, with the nematodes escaping only upon transfer to water. This outcome is in agreement with previous findings by Hiltpold et al. (2012). The concentration of alginate has a direct impact on bead hardness, with increasing alginate concentration resulting in harder beads. As bead hardness increases, the number of nematodes escaping after transfer to water decreases. Furthermore, beads formed with 2% sodium alginate exhibited an irregular shape, which hindered IJ escape.

Calcium chloride concentration also had an effect on bead hardness, consistent with previous findings (Kim et al., 2015). Beads formed in 10 mM CaCl₂ and 15 mM CaCl₂ solutions were fragile and exhibited irregular morphologies, readily deforming under gentle pressure. Conversely, beads formed at higher concentrations of CaCl₂ (20 and 30 mM) demonstrated increased structural complexity and regular shapes, resisting deformation under equivalent pressure. Notably, IJs encapsulated in these beads exhibited rapid escape upon transfer to water, with all individuals escaping within 24 hours. In contrast, beads formed at higher concentrations of CaCl₂ (50 and 100 mM) were tougher and resistant to IJ escape, with no individuals escaping within the same time frame. These findings suggest that a CaCl₂ concentration of 30 mM is optimal for the current formulation. In contrast to our results, Nxitywa and Malan (2021) found that while varying alginate concentration significantly affected the number of IJs escaping from beads, Ca²⁺ concentration and hardening time did not yield significant differences. For the currently described formulation, beads were prepared with 1.5–1.75% sodium alginate and 50–100 mM CaCl₂, and allowed 1-minute to harden, effectively retaining IJs until immersion in water.

Previous studies have demonstrated that the storage temperature of encapsulated EPNs significantly affects their viability and reproductive potential. Kagimu and Malan (2019) reported that beads stored at 25°C retained a greater number of IJs than those stored at lower temperatures (8°C and 14°C). Our results show that EPNs stored at 25°C can maintain viability for up to 180 days, exceeding the longevity observed at 8°C. This suggests that refrigeration may not be necessary for the storage of EPNs, potentially reducing storage-related costs. The reproductive potential of H. bacteriophora was lower at 8°C compared to 25°C, while the opposite trend was observed with S. carpocapsae and S. feltiae. This is in agreement with previous studies, which have reported varying survival rates for different EPN species at different storage temperatures (Ruiz-Vega et al., 2018; Kary et al., 2018; Hussein and Abdel-Aty, 2012). For example, Ruiz-Vega et al. (2018) reported survival times of 35, 28, and 14 days for H. bacteriophora, S. carpocapsae, and S. feltiae, respectively, formulated in alginate beads stored at room temperature (25°C). Similarly, Kary et al. (2018) found that glycerol-dehydrated H. bacteriophora stored in sodium alginate beads had a 40% survival rate after 60 days, which decreased to 10% after 90 days. In contrast, Nxitywa and Malan (2023) reported high survival rates for IJs of Steinernema yirgalemense stored in agar formulations at 25°C, with 77% and 82% survival rates recorded after 6 weeks for 1% and 1.5% agar formulations, respectively.

Temperature is a critical factor affecting the survival of IJs (Stuart et al., 2015; Kagimu et al., 2017). The optimal storage temperature for EPNs is a topic of ongoing debate. According to Poinar (1979), Steinernema species are cold-tolerant, with an optimal storage temperature of 5-9°C, while the preferred storage temperature for Heterorhabditis species is 12°C (Bedding, 1981). However, our findings suggest that EPNs can be stored at 25°C for up to 180 days without significant loss of viability. In the current study, we surveyed the survival rate of EPNs for up to 6 months at 25°C. The survival rate for H. bacteriophora and S. carpocapsae was 60% and 50% within 3 months after formulation, respectively, while S. feltiae had a 30% survival rate in the third month. Understanding the effects of temperature on the survival and reproductive potential of EPNs will be essential for optimizing storage conditions and improving the efficacious use of these beneficial organisms in biological control programs.

Our study demonstrates that beads formulated with 1.5% sodium alginate are a suitable matrix for storing EPNs at room temperature for extended periods. Notably, at least 50% of the nematodes remained viable and capable of escaping from beads within 24 hours after transfer to water, even after three months of storage. The dissolution of calcium alginate beads in water facilitates the escape of EPNs from the bead matrix. Upon transfer to water, the beads absorb water, soften, swell, and cleave, resulting in the formation of slits that allow the nematodes to escape. This characteristic makes calcium alginate beads a versatile formulation for both aquatic and terrestrial applications. The applicability of calcium alginate beads to soil has also been explored in preliminary tests in which EPNs escaped from beads after transfer to soil followed by irrigation. This suggests that these beads could be applied to soil during seed planting, offering a novel approach for the delivery of beneficial nematodes to agricultural fields. Our results provide a foundation for further research on optimization of bead alginate formulations. Future work is needed to investigate the effects of varying sodium alginate concentrations, bead sizes, and hardening times on the survival rate and infectivity of EPNs. Additionally, more replication across a larger number of EPN species is necessary to ensure generalizability of the current results.

In this research, our hypothesis that nematodes survive longer in the formulation than in sterile water was confirmed. We identified two compounds that acted as absorbents when added to the sodium alginate suspension. These compounds absorb water and cause the beads to swell, creating gaps in the beads that facilitate the emergence of the IJs. The results showed that alginate beads were able to maintain EPNs at room temperature for three months without reducing their pathogenicity. Therefore, there is no need to use a refrigerator to transport nematodes to the field or greenhouse. The survival of nematodes at room temperature and maintaining their ability to be pathogenic within a formulation is important and this was achieved with this formulation. However, this formulation also has limitations.

Large-scale production of alginate beads is currently limited, and further research is also needed on mass production methods to meet the demand for these beneficial organisms. Research efforts should focus on increasing the survival of IJs over time at room temperature and optimizing the number of IJs per bead to maximize their efficacy in biological control programs. In line with previous studies (Ruiz-Vega et al., 2018), the survival rate and infectivity of EPNs may be related to the fitness of each EPN species tested. Therefore, further research is also needed to understand the factors influencing the survival and infectivity of EPNs deployed using beads within different environments.