Introduction

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), a polyphagous defoliating pest native to Canada, USA, and Mexico, has been reported invading in Europe (over 15 countries), Eurasia (Russia and Turkey), and Asia (Azerbaijan, Georgia, Iran, China, Korea, and Japan)1. This pest was first discovered in Liaoning Province, China in 1979, and has quickly spread to Shandong, Shaanxi and Hebei provinces, and to Tianjin Municipality as well. Hyphantria cunea can feed on and damage a total of 175 plant species in 49 families and 108 genera in China2. Such invasive pests could cause important yield losses in key crops in invaded countries3,4,5,6, and prompt for developing sustainable management methods to prevent overuse of insecticides and potential associated environmental side effects of such chemicals7,8,9.

Chouioia cunea Yang (Hymenoptera: Eulophidae), an indigenous pupal endoparasitoid of the fall webworm, was first documented in China10, subsequently reported in Italy11 and Turkey1 and is currently used to control H. cunea in a biological control program. Field releases of C. cunea resulted in higher percentages of parasitism of H. cunea, reaching a parasitism rate of 88%, than that in nonrelease control plots with a 4.7–12.9% parasitism rate12. In China, the mass production of C. cunea is primarily via the Chinese oak silkworm, also known as the tussah silkworm, Antheraea pernyi (Guerin-Meneville) (Lepidoptera: Saturniidae)2,13, and approximately 325.54 billion C. cunea adults have been released to two-thirds of the H. cunea infested area (235,000 ha) in China in biological control programs from 1986 to 201212. Compared with other hosts, A. pernyi has various advantages14 and has also been largely used for mass production of Trichogramma parasitoids for biological control of corn borers in northeastern China15,16. The mass rearing of C. cunea on A. pernyi, as well as its field release to suppress H. cunea, have been recognized as major biocontrol successes in China2,17.

Although A. pernyi is a good alternative host for rearing C. cunea, some challenges remain limiting its application. For example, in areas unsuitable for A. pernyi production, the transportation and storage of A. pernyi can be costly. In addition, bacterial diseases infecting A. pernyi pupae have posed severe threats to C. cunea mass rearing18. Therefore, it is essential to seek alternative host species for mass production and field application of C. cunea.

Previous research highlights that C. cunea can be reared on pupae of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae)19, Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae)20, and the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae)21. Tenebrio molitor can be easily reared with low cost, and the larvae are currently recognized as one of the most common foods for captive insectivore mammals, birds, reptiles, and amphibians22,23. Tenebrio molitor has also become popular as human food in some countries or regions due to its high nutritional value24,25, yielding many research efforts on food processing and safety concerns26,27.

A previous study reports that 75% of T. molitor pupae can be successfully parasitized by C. cunea, including deceased T. molitor pupae21, and 167.3 progeny per pupa are reported from treatment with a parasitoid:pupa ratio of 3:1, which indicate that T. molitor pupae are a promising alternative host for mass rearing C. cunea. However, little study has been conducted to optimize the rearing conditions such as determining host age and other factors of T. molitor pupae for mass rearing of C. cunea. Notably, host age is reported to have generaly a substantial influence on the development of parasitoids in their hosts28,29,30.

In this study, we investigated the parasitism and development of C. cunea parasitoids on T. molitor pupae at different ages (i.e., 0, 1, 2, 3 and 4-day-old pupae) and the effects of pupa age on fertility of C. cunea females. This study was expected to provide useful information to optimize the mass rearing of C. cunea on T. molitor.

Results

Effect of host age on parasitism of C. cunea

When 0, 1, 2, 3 and 4-day-old pupae of T. molitor were provided to C. cunea, no significant differences in percentage of parasitized pupae were found (F4,15 = 1.95, P = 0.154) (Fig. 1). Significant differences in percentage of deformed T. molitor (presumably caused by parasitoid host feeding at T. molitor pupae stage) were detected among different host ages (F4,15 = 3.44, P = 0.035). The highest percentage of deformed T. molitor adults emerged from 4-day-old hosts (30.0%), followed by 0 and 3-day-old pupae, and no deformed T. molitor adults emerged from 1 and 2-day-old pupae.

Figure 1
figure 1

Mean (±SE) percentage of parasitized T. molitor pupae at different ages and percentage of deformed T. molitor adults. Above each bar, different lowercase letters indicate significant differences (ANOVA, Tukey’s HSD test, P < 0.05).

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Effect of host age on C. cunea development

The results showed that host pupae of all ages were parasitized and that C. cunea emerged successfully. However, significant differences occurred in the total number of C. cunea obtained per host (Nc) among host ages (F5,15 = 8.84, P = 0.001). One-day-old pupae produced the highest number of parasitoids (154.7), followed by 0, 2, and 3-day-old pupae, and 4-day-old pupae produced the lowest number of parasitoids (72.1) (Table 1).

Table 1 Suitability of T. molitor pupae of different ages for C. cunea.
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The highest percentage of unemerged C. cunea per pupae (Pue) was observed in 4-day-old pupae (10.3%), followed by 0 and 1-day-old pupae. The lowest percentage of unemerged parasitoids was observed in 2 and 3-day-old pupae (1.2–1.4%) (F5,15 = 3.40, P = 0.036). Although no significant difference occurred in the percentage of adult females reared per pupa (Pf) among different host ages (F5,15 = 0.49, P = 0.746), the host age significantly affected the number of emerged female adults per host (Nfe) (F5,15 = 5.90, P = 0.005). No significant differences in Nfe were found between 1-day-old pupae and 0, 2 and 3-day-old pupae; however, the value was significantly higher than that from 4-day-old pupae. No significant differences were found in C. cunea developmental time among all host ages (F4,15 = 2.91, P = 0.058) (Table 1).

Effect of host age on fertility of female C. cunea

Significant differences were detected in number of all eggs in ovarioles per C. cunea female among all host ages (F4, 15 = 10.30, P < 0.001). Generally, C. cunea females emerging from 2-day-old pupae carried the largest number of eggs (258.2), followed by those from 3 and 4-day-old pupae. The number of eggs carried per female was the lowest in parasitoids emerged from 0 and 1-day-old pupae (178.4–178.9) (Fig. 2).

Figure 2
figure 2

Fertility of C. cunea emerged from pupae of different ages. Mean (±SE) number of eggs carried per C. cunea female. Above bars, different letters indicate significant differences (ANOVA, Tukey’s HSD test, P < 0.05).

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Discussion

The results of the current study indicated the great potential of T. molitor pupae as alternative hosts for mass rearing of C. cunea. We highlight that C. cunea preferred parasitizing younger T. molitor pupae. When older pupae were provided, the percentages of unsuccessfully parasitized hosts and unemerged C. cunea remaining in hosts were higher, whereas more female adult parasitoids emerged from hosts when C. cunea females parasitized younger hosts. Our results showed that 95% of 1-day-old and 90% of 2-day-old pupae of T. molitor were successfully parasitized by C. cunea. This result consolidates the earlier research by Yang & Li21, where only 75% of T. molitor pupae were successfully parasitized by C. cunea without host optimization reported. Overall, 1 and 2-day-old pupae of T. molitor can be considered as highly suitable hosts for the mass rearing of C. cunea. In addition, C. cunea females emerging from 2-day-old host pupae showed the greatest fertility compared with that from the other host ages.

Compared with A. pernyi, T. molitor showed many advantages as host for C. cunea. For example, A. pernyi overwinters as diapausing pupae and generally takes six months to complete the cycle31, whereas T. molitor can breed continuously throughout the year and provide fresh pupae uninterrupted. Thus, T. molitor as host can help to reduce the storage cost for C. cunea mass rearing. Furthermore, other hosts such as A. pernyi and B. mori are economically important insects and can only be cultured in limited regions and seasons, which has limited the potential of improving mass production of C. cunea via A. pernyi and B. mori. Other hosts, such as O. furnacalis pupae, show potential for C. cunea mass production20, but for O. furnacalis, strict management is required in the field since it can be a major agricultural pest during mass production.

Our results also showed that approximately 30% of beetles emerged deformed from 4-day-old parasitized pupae, indicating the unsuccessful parasitism of C. cunea. Yang & Li also found that the percentage of deformed T. molitor beetles reached 50% when C. cunea parasitoid and host pupa were at the ratio of 1:321. Deformed T. molitor beetles usually showed a pair of incomplete forewings but could still crawl and forage. The secretions from the venom gland of C. cunea may play a major role in causing deformities of parasitized beetles32. Similarly, previous research showed that 10% venom sac extract from Tetrastichus sp. (Hymenoptera: Eulophidae) artificially injected in O. furnicalis pupae caused 8.2% deformed moths with defects of spreading wings and that soon died33. In addition, the deformed beetles may be explained by contributions from other factors such as host resistance to parasitism and host feeding by C. cunea. Indeed, Yang & Xie also found that C. cunea adult females display host feeding after oviposition34. Zhu et al. indicated that the expression of 74 unigenes involved in T. molitor immune response was significantly altered after T. molitor pupae were parasitized by Scleroderma guani35. Although our study reported significant differences in the percentages of deformed T. molitor beetles among different host ages, further studies to elucidate the physiological mechanisms leading to these deformities in T. molitor are urgently needed.

The host age is reported to significantly affect the number of parasitoids per host, while a similar number of C. cunea can be reared on 0 and 2-day-old hosts. When C. cunea adults and their hosts were tested at a ratio of 2:1, the total number of C. cunea reared per host pupa reached 181.7 parasitoids21, a number higher than the 154.7 C. cunea reared on 1-day-old pupae in our study. Moreover, the developmental period of C. cunea in all host ages ranged from 21.8 to 23.6 days at 25 °C, which is longer than that reported by Yang & Li (17.2 to 22.9 days at 28 °C)21. Generally, our results indicated that the pupal age had no effect on developmental times of C. cunea. However, pupal age significantly affected the number of adult females emerging per host. The number of adult females emerged from 0 to 2-day-old pupae was approximately 2-fold of that emerged from 4-day-old pupae. The number of eggs carried by female parasitoids is a key index for quality evaluation and reflects the fertility of a parasitoid in biological control programs36. Our results indicated that the C. cunea females emerging from 2-day-old pupae carried more eggs than those that emerged from 0, 1, and 3-day-old pupae. Notably, Sun et al. reported a positive linear relationship between egg load and female body size of C. cunea37. However, our results indicated a balance occurred between the number of eggs carried and the number of C. cunea reared on pupae of different ages.

As T. molitor beetles can be easily reared worldwide with fewer limitations and ecological risks, the beetle can be considered as a promising host to enhance mass rearing of C. cunea. Overall, our study showed that 2-day-old pupae were the most suitable age for rearing C. cunea. This research adds valuable information to optimize the mass rearing of C. cunea on its alternative host T. molitor. Further studies are needed to develop efficient field release operations of C. cunea in biological control programs, as well as the technology for long-term storage of C. cunea parasitoids.

Methods

Hosts

Tenebrio molitor larvae were initially obtained from the Aquaculture Co., Ltd. (Wudi County, Xinchong Aquaculture Co., Ltd., China) in 2016 and maintained on artificial diet (85% wheat bran, 10% Chinese cabbage and 5% cucumber) in an insectary at 26 ± 1 °C and 55 ± 3% R.H. with a 10:14 (L:D) h photoperiod using 450 ± 50 lux white LED light conditions38 for more than 5 generations. Newly pupated T. molitor beetles with similar size (mean weight 0.159–0.165 g) were selected for the subsequent experiments. Tests started at 8:00 a.m. Since the pupae need an average of six days to emerge (Zang et al. unpublished data), 0, 1, 2, 3, and 4-day-old pupae of T. molitor were selected as experimental host ages.

Parasitoids

Chouioia cunea was initially obtained from the North Greening Center (Changchun, China) in 2017. The colony of C. cunea was continuously maintained in the laboratory on pupae of A. pernyi at 25 ± 1 °C and 65 ± 5% R.H. for over 4 generations39. Newly emerged C. cunea parasitoids (<5-h-old) reared on A. pernyi pupae were used for the experiments.

Effect of host age on C. cunea parasitism

The experiment was conducted at 25 ± 1 °C, 65 ± 5% R.H. and in complete darkness in an incubator (versatile environmental test chamber, MLR-351H, SANYO Electric Co., Ltd., Japan). Previous research shows that the adult females of C. cunea mate before emerging from the host pupa of A. pernyi34 and that the females oviposit 51.7% of their total eggs in the first day after emergence37. Therefore, during the experiment, two newly emerged (<5 h) and mated C. cunea adult females were introduced into a glass tube (diameter: 3.5 cm, length: 10 cm) with one T. molitor pupa (1, 2, 3, or 4-day-old) to allow parasitism21. After 24 hours, the parasitoids were removed, and the pupa from each age treatment was monitored daily to document the parasitoid emergence. The number of emerging T. molitor adults, deformed T. molitor adults (presumably caused by host feeding) and parasitized pupae were recorded. The numbers of parasitoids emerged and unemerged parasitoids from each host were recorded, and sexed. Parasitoid larvae remaining in each host pupa were also counted. The developmental time of C. cunea was documented from parasitism to adult emergence. Four replicates were conducted for each pupal age, and 10 pupae were examined per replicate; thus, a total of 40 pupae were tested at each pupal age.

Effect of host age on fertility of female C. cunea

A previous study indicated that the eggs of newly emerged females of C. cunea parasitizing Chinese oak silkworm pupae were nearly matured37. Therefore, the fertility of C. cunea females was evaluated by assessing the number of all eggs carried by each adult female immediately after emergence. For all tested ages of parasitized T. molitor pupae, 10 newly emerged C. cunea adult females were randomly selected for each replicate. The collected parasitoids were then dissected under a stereomicroscope (SMZ-168 series, Motic, China) to count the number of eggs per C. cunea female40. Each treatment was replicated 4 times. A total of 40 adult females emerged from each host age were examined.

Statistical analyses

For each biological parameter described above, data were analyzed using one-way ANOVA, and means were compared using Tukey’s HSD test at P < 0.05. All data were subjected to a normality test (Shapiro–Wilk test) prior to ANOVA. Female progeny (%) and unemerged C. cunea (%) data were arcsine square root transformed, while count data were logarithm-transformed prior to the normality test. All the statistical analyses were performed using the SAS statistical software package (SAS Institute, Cary, NC, USA). The figures were plotted using OriginPro 2017 SR2.

Number of C. cunea adults reared per host was calculated based on the equation below:

$${N}_{a}={N}_{fe}+{N}_{me}+{N}_{fue}+{N}_{mue}$$

where Na is the total number of C. cunea adults per host, Nfe is the number of emerged adult females per host, Nme is the number of emerged adult males per host, Nfue is the number of unemerged adult females per host, and Nmue is the number of unemerged adult males per host.

The total number of C. cunea reared per host was calculated based on the equation below:

$${N}_{c}={N}_{a}+{N}_{ll}$$

where Nc is the total number of C. cunea reared per host, Na is the number of C. cunea adults reared per host, and Nll is the number of larvae per host.

Percentage of unemerged C. cunea reared per host was calculated based on the equation below:

$${P}_{ue}( \% )=[({N}_{fue}+{N}_{mue}+{N}_{ll})/{N}_{c}]\times 100$$

where Pue is the percentage of unemerged C. cunea reared per host, Nfue is the number of unemerged adult females per host, Nmue is the number of unemerged adult males per host, Nll is the number of larvae remained per host, and Nc is the total number of C. cunea reared per host.

Percentage of adult females reared per host pupa was calculated based on equation below:

$${P}_{f}( \% )=[({N}_{fe}+{N}_{fue})/{N}_{a}]\times 100$$

where Pf is the percentage of adult females reared per host, Nfe is the number of adult females emerged per host, Nfue is the number of unemerged adult females per host, and Na is the number of C. cunea adults reared per host.