Mass rearing protocol and density trials of Lilioceris egena (Coleoptera: Chrysomelidae), a biological control agent of air potato

Insect mass rearing protocols are essential in classical biological control programs. These protocols enable the production and release of thousands of biological control agents, increasing their impact and rate of establishment in the field, thus reducing the abundance of the targeted pest (Moran et al. 2023; Parra and Coelho 2022). However, in some cases, protocols may have to be adjusted based on the quantity of control agents required, number of staff available, and size of the rearing facility (Kraus et al. 2022).

In the last two decades, the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS-DPI), has played a critical role in multiple biological control projects targeting invasive insect and plant pests. Its efforts include developing or improving mass rearing protocols of biological control agents. For instance, FDACS-DPI has successfully reared and released various agents such as Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) against the Asian citrus psyllid (Patt and Rohrig 2017), Apanteles opuntiarum Martínez and Berta (Hymenoptera: Braconidae) for the Argentine cactus moth (Varone et al. 2024), Gratiana boliviana Spaeth (Coleoptera: Chrysomelidae) to control tropical soda apple (Overholt et al. 2009), and Pseudophilothrips ichini (Hood) (Thysanoptera: Phlaeothripidae) against Brazilian peppertree (Wheeler et al. 2022). However, one of the most successful projects has been the air potato biological control program (Kraus et al. 2022; Manrique et al. 2023; Overholt et al. 2016). Its success is due to the biological control agent’s high degree of specificity and conspicuous impact on the vine, the environmental and economic benefits, the long-term effectiveness, and finally, the strong community involvement. In this regard, the program actively involves community members in releases across infested areas.

Air potato (Dioscorea bulbifera L., Dioscoreales: Dioscoreaceae) is an invasive perennial vine originally from Asia, Africa, and northern Australia, which can climb tree canopies, displace native vegetation, and disrupt ecological functions (Croxton et al. 2011; Pemberton and Witkus 2010). Currently, air potato is present in several southeastern states, including Florida (Manrique et al. 2023). Air potato reproduces asexually, generating aerial potato-like structures (i.e., bulbils), which fall off as the vine senesces. Previous methods of control included herbicide applications and mechanical removal of bulbils; however, these approaches were proven to be costly, labor-intensive, time-consuming, and only provided a temporary solution (Kraus and Murray 2021; Overholt et al. 2014; Wheeler et al. 2007).

Biological control was considered the best, cost-effective management tactic against air potato. Between 2011 and 2021, the leaf-feeding beetle, Lilioceris cheni Gressitt and Kimoto (Coleoptera: Chrysomelidae) was mass produced and over one million beetles were released as a biocontrol agent of air potato by multiple state and federal agencies (Kraus et al. 2022; Rayamajhi and Dray 2022). Although L. cheni successfully reduced vine coverage and propagule pressure of air potato (Rayamajhi et al. 2019), the production of bulbils, particularly in South Florida, remains relatively high compared to North and Central Florida (Kraus et al. 2022; Manrique et al. 2017, 2023]; Overholt et al. 2016; Rayamajhi and Dray 2022; Wheeler et al. 2020). To overcome this issue, an additional biological control agent of air potato was federally approved to be released across Florida in 2021. Lilioceris egena (Weise) is an air potato feeding specialist, where both larvae and adults feed primarily on bulbils (Dray and Goldstein 2019; Dray et al. 2023). The bulbil damage caused by L. egena paired with the defoliation by L. cheni is expected to further reduce the dominance of air potato in agricultural and natural ecosystems.

Here, we present an efficient protocol to mass rear L. egena in laboratory settings. Furthermore, we document how the number of mating pairs and the condition of bulbils (whole or bisected) used in the mass rearing system influence the number of adults produced. This study aimed to optimize L. egena adult production, particularly when the number of bulbils in the rearing system is limited.

The following protocol yielded approximately 675 beetles per month from each rearing colony cage. Mass rearing of L. egena was conducted in a walk-in rearing room under controlled conditions at 25 ± 2 °C, 65 ± 5 % relative humidity (RH), and 14L:10D (Kraus et al. 2021). For one L. egena colony, an open 4.5 L translucent plastic container (24 × 18 × 15 cm) filled with approximately 100 g of dry sterilized grade A-1 vermiculite was placed into a 40 × 40 × 60 cm polyvinyl chloride cage (Bug Dorm, Catalog No. B09GRTB95S). Two whole large bulbils (250 ± 50 g) were placed in the container along with a 37 mL plastic cup filled with reverse osmosis water. The plastic cup lid had a 1.5 cm hole to insert a 5 cm long braided cotton wick. Subsequently, 17 L. egena active and vigorous mating pairs, all collected within 1–2 weeks post-emergence from laboratory stock colonies, were released into the cage, and allowed to oviposit freely. After four days, adults were gently separated from bulbils. The container was covered with a lid that had a 6-cm plastic screen vent, and then removed from the cage. Subsequently, dead beetles were replaced, and a new container with bulbils was placed into the cage to repeat the cycle. Every two months, cages, mating pairs, and containers were replaced entirely to maintain productivity and prevent frass build up and potential pests and pathogens.

Containers with infested bulbils were monitored daily to remove frass and deliquesced bulbils. Daily monitoring and sanitation prevent pest infestations like mites or diseases caused by fungi and bacterial growth. When bulbils build up too much frass or become overcrowded, second and third instar larvae tended to exit the bulbils and search for fresh food (Figure 1). Consequently, extra bulbils were added as needed. Neonate larvae could not penetrate the rough skin of new bulbils; thus, new bulbils were cut in half to facilitate their boring and feeding activities (Figure 1). Once mature, fourth instar larvae exited the bulbils and burrowed into the vermiculite to pupate. When larvae have pupated (approximately 86 %, Menocal et al. unpublished), the remaining bulbils were removed, and the containers were left undisturbed for approximately 12 days until adults began to emerge. A period of 12 days was used based on L. egena pupal developmental time at 24 °C (Dray et al. 2023). Emerging adults were collected daily and placed into vented 4.5 L containers of one hundred adults with bulbil slices and air potato leaves. These containers were replaced three times a week until the beetles were used for research or field releases.

Figure 1:

Damage caused by Lilioceris egena larvae to air potato bulbils. Note larvae crawling around due to overcrowding and bulbils cut in half to facilitate boring activities.

To determine the impact of adult beetle density (3F:3M, 5F:5M, 10F:10M, 20F:20M, and 30F:30M; where F = female and M = male) and the condition of bulbils (whole or bisected) on the number of beetles produced, we conducted a 5 × 2 full factorial experimental design. Each treatment was replicated 10 times, except the 10F:10M whole and 10F:10M bisected treatments, which were replicated 20 times. The experiment followed the aforementioned protocol with some minor modifications. Briefly, medium-sized bulbils (150 ± 50 g), whether whole or bisected, were individually placed in terraria (33 × 18.5 × 21 cm) set up with a trifold paper towel at the bottom. Recently emerged (>3 days old) mating pairs selected from stock colonies were released into each terrarium according to the desired adult beetle density to be tested. Beetles oviposited for 72 h. Then, infested bulbils (whole or bisected) were individually transferred to plastic containers with vermiculite. The containers were left undisturbed until adults emerged, at which point the adults were removed and counted.

Data were analyzed using JMP Pro. 16.0 (SAS Institute, Cary, North Carolina, USA). A two-way analysis of variance (ANOVA) was used to determine the effect of adult beetle density, condition of bulbils (whole or bisected), and their interaction on the total number of adults produced. To determine the optimal beetle density, data were analyzed in R (version 4.1.3.) using the Ricker function (Pinheiro et al. 2024), which calculates density-dependent reproduction rate (Brännström and Sumpter 2005). Results are presented as the mean ± standard error of the mean (SEM) unless otherwise indicated; results were considered significant at a critical level of α = 0.05.

The interaction of adult beetle density and condition of bulbils was not statistically significant (F = 0.0570, p = 0.9939). The number of adults produced was significantly influenced by beetle density (F = 6.9298, p < 0.0001) but not by the condition of bulbils offered (F = 0.5364, p = 0.4655). A beetle density of 20F:20M produced more beetles (90.45 ± 8.60) than densities of 10F:10M (76.65 ± 5.56) and 30F:30M (74.25 ± 10.39). However, adult production among these three densities was not significantly different. Densities of 3F:3M and 5F:5M produced the least number of beetles, 35.90 ± 4.37 and 59.30 ± 6.99, respectively. The Ricker function revealed an optimal adult beetle density of 17F:17M beetles with an estimated adult production of 90.30 (Figure 2).

Figure 2:

Total number of Lilioceris egena adults produced at five beetle densities. Each data point represents raw data for each treatment (beetle density and bulbil condition). The Ricker model shows the estimated optimal beetle density (X _opt; 95 % CI) and the estimated adult production (Y _max; 95 % CI) at the optimal beetle density.

In mass-rearing biological control programs, a factor that limits the production of biocontrol agents is host availability (Kraus et al. 2022). The air potato program at FDACS-DPI relies on bulbils collected from state and national parks in Florida. However, acquiring these bulbils involves a significant amount of travel, time, and labor. Therefore, it is critical to maximize the number of beetles produced per bulbil in each rearing cycle. Our results indicate that 17 mating pairs per bulbil is an optimal beetle density to maximize beetle production and the use of bulbils. Although higher beetle density did not significantly increase the number of beetles produced; it increased the amount of frass, which led to more frequent cleaning of the rearing units. Contamination and overcrowding are known to weaken insect colonies (Maciel-Vergara et al. 2021; Mahavidanage et al. 2023; Morales-Ramos and Rojas 2015). Thus, good sanitary practices are highly recommended to maintain healthy colonies. These practices include the disposal of dead insects, frass, used rearing substrate, and old adults at the end of rearing cycles (Eilenberg et al. 2015). Additionally, it is critical to maintain several colonies that are isolated to prevent disease or pest transmission among colonies thereby protecting against entire colony collapse.

Insect mass-rearing protocols require a thorough understanding of the biology and ecology of the intended biological control agent and host. Additionally, a well-designed protocol ensures consistent production of thousands of active and vigorous biological control agents for research and field applications while maintaining efficiency and affordability. The rearing method proposed here has consistently proven to be highly effective at producing large quantities of healthy beetles while utilizing low-cost rearing equipment. However, we note that slight deviations from our protocol would likely still result in conditions conducive to mass rearing. For instance, if controlled environmental conditions are unavailable, room temperature and ambient humidity should be sufficient for insect production. This protocol can easily be adopted by integrated pest management (IPM) and master gardener programs, universities, and regulatory agencies in other southeastern states currently facing air potato infestations. These protocols should be reviewed periodically to incorporate the most up-to-date information derived from additional empirical research.