Shuo Tian · Tianzi Gu · Cong Chen ·Xudong Zhao · Pengcheng Liu · Dejun Hao

Abstract Brachymeria lasus Walker is a solitary endoparasitoid that attacks the pupae of a wide range oflepidopteran hosts, including an important invasive species, the fall webworm (Hyphantria cunea Drury). We studied the relationship between temperature and development of B. lasus from egg to adult hatching. The results show a decrease in parasitoid development time from 34.4 days at 18 °C to 10.6 days at 32 °C. The minimum threshold temperature of B. lasus was 13.2 °C ± 1.7 °C, and the effective accumulated temperature was 210.3 ± 28.7 degree days. These results provide a basis for optimizing the production of this parasitoid.In addition, the effects of host size on offspring performance of B. lasus were investigated under laboratory conditions.Offspring longevity, size, and percentage of females were positively correlated with host size. Female offspring are larger and live longer than males. Furthermore, this research showed that parasitoid adults successfully emerged from approximately 27.9% of pupae. However, eclosion or hatching of H. cunea decreased dramatically, which may be due to damage caused by female B. lasus when testing hosts with their ovipositors or by feeding on them. The results suggest that B. lasus has the potential to become an efficient natural enemy for controlling H. cunea.

Keywords Hyphantria cunea · Brachymeria lasus ·Parasitoid · Temperature · Development · Fitness

Introduction

There have been 37 natural enemy species reported in China (Yang et al. 2006; Luo et al. 2018). Among them,Brachymeria lasus (Walker) (Hymenoptera: Chalcididae)is a solitary pupal endoparasitoid of H. cunea, with a host range of 104 species of Lepidoptera, Hymenoptera, and Diptera. It has been utilized as a biological control agent for a number of pest insects, including the gypsy moth,Lymantria dispar L. (Simser and Coppel 1980), the oriental tea tortix moth, Homona magnanima Diakonoff (Mao and Kunimi 1991), and the pine caterpillar, Dendrolimus punctatus Walker (Yan et al. 1989). Field investigation indicated that B. lasus was a predominant parasitoid species for summer pupae of fall webworm in Xuzhou City,Jiangsu Province, which suggests that it may be a promising parasitoid of H. cunea.

Ambient temperature is one of the main factors affecting the development, reproduction, and behavior of insects (Atkinson 1994; Wang et al. 1999; Golizadeh et al.2009; Torqueti et al. 2013; Yu et al. 2013). Therefore,investigating the influence of temperature on the development and growth of natural insect enemies is essential for optimising biocontrol release strategies (Pilkington and Hoddle 2006) and for confirming temperature conditions for mass rearing of B. lasus in an insectary (Qiu et al. 2012). Delineating temperature parameters, including developmental thresholds and effective accumulated temperature, is important for predicating natural enemy establishment in target habitats (Pilkington et al. 2014).The influence of temperature on the development and parasitism of B. lasus on H. magnanima (Lepidoptera: Tortricidae) has been previously reported (Mao and Kunimi 1994). However, the effect of temperature on the development and parasitism of B. lasus on H. cunea remains unknown.

In Hymenopteran parasitoids, host size-dependent selection is common because host quality is often determined by size (Charnov and Skinner 1984; Goubault et al.2004; Liu et al. 2011). Host size influences parasitoid selection, development, reproductive success, percentage of female offspring, and these biological parameters play an important role in parasitoid life history and biology.

In this study, the relationship between temperature and developmental duration from egg to adult was examined to determine the optimum temperature range for B. lasus and to calculate accumulated degree days using the minimum developmental threshold. Under laboratory conditions, the performance of the parasitoid offspring (longevity and size), and the percentage of females emerging from hosts of different sizes was investigated. The relationship between parasitoid fitness and host size was then evaluated by regression analysis.

Materials and methods

Host and parasitoid sampling and preparation

Pupae of H. cunea were collected from a Populus plantation in Xuzhou City (117° 24′ 47″ E, 34° 8′ 55″ N), Jiangsu Province August 2016. They were individually transferred into polyethylene tubes with openings covered with cotton,and maintained in environmental chambers at 28 °C, relative humidity of 70%, and a photoperiod of 14 h light:10 h dark. The numbers of emerged parasitoids and adult fall webworms and the dates of emergence were checked and recorded daily.

When adult H. cunea emerged from the pupae, they were paired in a plastic cuboid container for mating. There were paper cards in the container for female oviposition. Once the eggs hatched, the larvae were reared on a standard artificial diet (Cao et al. 2014) at a constant temperature of 26 °C),relative humidity of 65% and the photoperiod as previously described (Wu et al. 2012). After pupating, the pupae were individually transferred into small plastic vials and stored at 4 °C until needed.

Adult B. lasus were collected from parasitised pupae and continuously reared on H. cunea pupae in the laboratory.Newly emerged females and males were paired in plastic containers for mating. Cotton balls saturated with diluted 30% honey were placed in the containers as a food source and replaced every 2 days. Male parasitoids were removed after 2-4 days. This treatment was sufficient to ensure that all female parasitoids were fertilised. The containers were kept in environment chambers at 28 °C, 70% relative humidity and the photoperiod noted previously.

Effect of temperature on the development and survival of immature B. lasus

The duration of development of B. lasus under different temperature treatments was determined from egg to adult hatching as this wasp is an endoparasitoid and its immature stages cannot be observed without dissecting the host body.Virgin females and males were placed in a cylindrical container until mating was observed and were then separated into similar cylindrical containers at 28 °C, 70-75% relative humidity and a photoperiod of 14 light/10 dark hours.When mated females were seven days old, host pupae were supplied for oviposition. After 24 h, the parasitoids and host pupae were removed, and the pupae weighed using an electronic balance and put into covered polyethylene tubes.Pupae were randomly divided into five groups at different developmental temperatures of 18, 22, 25, 28, and 32 °C(70-75% relative humidity and photoperiod as noted previously. The sample sizes of the five treatments were 30 individuals comprising three replicates. Control groups without B. lasus were established. The host pupae and emerging B.lasus progeny of each sex were checked daily as well as the number of B. lasus and H. cunea.

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Effects of host size on longevity, body size,and percentage of females of B. lasus offspring

To determine the importance of size for host selection, the correlations between H. cunea size (represented by weight)and longevity, body size, and percentage of females were investigated. Adults from the different temperature treatments were randomly selected, kept individually in a container and reared at 28 °C, 70-75% relative humidity, and a photoperiod as noted previously. Longevity of B. lasus adults was considered to be the time from emergence to death. Hind tibia length were measured under a stereoscopic microscope and used as an index of offspring size (Liu and Li 2006; Liu et al. 2017).

The B. lasus progeny were divided into three groups of 30 consisting of three replicates: small (< 110 mg), medium(110-129 mg), and large (> 129 mg) (Supplementary Table S1). The relationship between percentage of female offspring and host size was determined using the percentage of emerged female parasitoids to the total number of emerged offspring. Females were counted owing to the maternal control of the percentage of females upon fertilization. Longevity and body size were compared between females and males.

Data analysis

A correlation analysis was performed in SPSS (Chicago,IL, USA) to analyse differences in developmental duration and developmental rate of B. lasus at different temperatures,host sizes, and parasitoid size/longevity. A Student’s t test was used to analyse the differences among hind tibia lengths between females and males. A chi-squared test analysed differences in rate of hatching among the three host groups at different temperatures. Number of survival days for both females and males was analysed using a Wilcoxon rank sum test. The overall differences in developmental duration of offspring under the five temperature treatments were compared using a one-way analysis of variance, ANOVA (Li et al. 2013).

The traditional regression line method was used for statistical analysis according to the recorded data, i.e., using the least square method of computing approximate value of higher mathematics calculus to calculate development starting temperatures (C) and effective accumulated temperatures(K) (Ding 1994).

The formula was

where C and K and standard error S c and S k are calculated using the least square method according to the following:

Results

Development and survival of immature B. lasus parasitizing H. cunea

The results show that B. lasus can successfully develop at different temperatures from 18-32 °C. However, temperature significantly affected the period of development from egg to adult hatching. Parasitoid adults successfully emerged from 27.9% of pupae at 18-32 °C temperatures. However, temperature had no significant effect on the hatching or eclosion ratio(χ2= 3, df = 4,P> 0.05; Table 1). In approximately 53.4% of pupae, no wasps or moths emerged. Approximately 18.2% of moths parasitised by B. lasus successfully emerged as adults which was significantly lower than the percentage of moth pupae not parasitised that emerged as adults (90.6%; Table 2).

Table 1 Mean developmental duration from egg to adult eclosion and hatching rate of Brachymeria lasus reared in Hyphantria cunea at different temperatures

Table 2 Percentage of Hyphantria cunea pupae with and without Brachymeria lasus parasitism that underwent eclosion at different temperatures

Fig. 1 Relationships among development duration, development rate,and treatment temperature of Brachymeria lasus

Fig. 2 Relationship between host weight and Brachymeria lasus size

There was a significant positive relationship between temperature and development rate from egg to adult (Fig. 1).The developmental duration of B. lasus was on average 34.4 days from egg to the adult stage at 18 °C. At higher temperatures, (32 °C), the developmental duration decreased to approximately 10.6 day on average. The developmental starting temperature was approximately 13.2 °C and the effective accumulated temperature was 210.3 ± 28.7 degree days. The regression equation of temperature and developmental rate was T = 13.2 + 210.3 V (r = 0.95). The number of degree days for completion of the immature development of a single generation was calculated as 210.3 (averaged across temperature treatments) above the theoretical lower developmental threshold of 13.2 °C.

Effects of host size on longevity, body size,and percentage of females of B. lasus offspring

The average hind tibia length of parasitoids from the 67-mg pupa was 1.2 mm, (longevity 4 days), while parasitoids that emerged from the 154-mg pupa was 2.0 mm (longevity 60 days). By correlation analysis, it was found that the larger the pupae, the larger the parasitoids that emerge from it and the longer they live (Figs. 2, 3).

The development duration of females was significantly longer than that of males at all temperatures in the egg-adult stages (Table 1). Females were usually larger larger (Fig. 4)and lived longer (Fig. 5). More B. lasus males emerged from smaller hosts and more females from larger hosts (Fig. 6).

Discussion

Fig. 3 Relationship between host weight and Brachymeria lasus longevity

Fig. 4 Differences between female and male body size of Brachymeria lasus (different letters represent significant difference between the sexes with independent t test (P < 0.01).)

Temperature is the most important factor affecting the range, development, reproduction, and survival of insects(Hagstrum and Milliken 1988; Keena and Moore 2010; Qiu et al. 2012). Our results show that B. lasus can successfully develop at temperatures from 18 to 32 °C. The number of degree-days required for development from oviposition to adult emergence was 210.3 above a threshold of 13.2 °C.This is similar to the results of Mao and Kunimi (1994). In addition, our results indicate that parasitoid adults successfully emerged from about 27.9% of pupae at temperatures from 18 to 32 °C. Temperature had no effect on eclosion ratios (Table 1) but parasitoid presence did. The eclosion ratio of H. cunea pupae parasitised by B. lasus was significantly lower than that of pupae without B. lasus (Table 2).In approximately 54.2% of H. cunea pupae, no wasps or moths emerged. A possible explanation is that the pupae might have been damaged by the female B. lasus when testing the hosts with her ovipositor but without laying eggs or feeding on them (Vinson 1985). Alternatively, during the developmental period within the host, the egg may die naturally. However, B. lasus had a considerable effect on the emergence success of H. cunea and therefore has potential as a biocontrol agent for this species.

Fig. 5 Differences between female and male longevity of Brachymeria lasus

Fig. 6 Percentage of females among different host sizes (letters represent significant differences among groups with chi-square test(P < 0.01).)

In Hymenopteran parasitoids, host size-dependent selection is common because body size is a determinant of host quality (Charnov and Skinner 1984; Goubault et al. 2004;Liu et al. 2011). Host size determines parasitoid size that affects their reproductive success (King and Hurlbutt 1987;Ueno 1999; Heimpel and Lundgren 2000; Henter 2004;Wang and Messing 2004; Traynor and Mayhew 2005; Liu et al. 2011). However, our results suggest that B. lasus can parasitize different sizes of hosts and that the larvae can successfully develop in both small and large hosts (Table 1;Fig. 2). This suggests that the suitability of H. cunea as a host does not depend on its size (Fig. 2). Moreover, host size was positively related to the size of B. lasus offspring(Fig. 2) which is possibly due to higher levels of nutrients in larger hosts for larvae development (King and Hurlbutt 1987; Ueno 1998, 1999, 2015).

Large hosts are generally more suitable for larval development, contributing to lower mortality and more large offspring with higher fitness (e.g., fecundity and longevity)(King and Hurlbutt 1987; Quicke 1997). Our results indicated that the longevity of emerged parasitoids increased with increasing host weight. In addition, a higher proportion of female offspring emerged from larger hosts, while more males emerged from smaller hosts (Fig. 6). This has been widely reported among Hymenopteran parasitoid species(Holdaway and Smith 1935; Napoleon and King 1999; West and Sheldon 2002; Henter 2004; Wang and Messing 2004;Traynor and Mayhew 2005; Liu et al. 2011; Ueno 2015). The theory of host quality in Hymenopteran parasitoids indicates that more males emerged from smaller hosts, while more females emerged from larger ones (Holdaway and Smith 1935; Napoleon and King 1999; West and Sheldon 2002)and is also verified in B. lasus. However, additional research is required to determine whether host age affects parasitoid sex ratios.

B. lasus is a solitary parasitoid species with a wide range oflepidopteron hosts, and has been applied as a biocontrol agent for insects such as the Oriental tea tortrix moth, H.magnanima (Mao and Kunimi 1991) and the gypsy moth, L.dispar (Weseloh and Anderson 1982). Previous field investigations indicated that the percentage of H. cunea pupae parasitised by B. lasus was as high as 3.4% (Yang et al.2001). In our study, we found that B. lasus could significantly reduce the eclosion ratio of H. cunea in the laboratory. Thus, B. lasus has potential as a biocontrol agent for controlling H. cunea populations.

This study provides information pertaining to the development of B. lasus, which will facilitate further investigations into mass rearing of this important parasitoid. Knowledge of the effects of host quality on parasitoid wasps provides relevant information to develop parasitoids for biological control. Large hosts should be selected to produce higher numbers of females, resulting in increased oviposition, and thus more effective biological control. In this study,B. lasus parasitism did significantly reduced the eclosion ratio of H. cunea in the laboratory and has potential to be developed as a biocontrol agent for suppressing populations of H. cunea. There is still a requirement to find substitute hosts and optimise reproductive procedures for mass rearing before B. lasus can be employed in the field as a biocontrol agent of H. cunea.

Conclusions

As determined through 11 subcultures of Taxus cuspidata callus, the growth in FW and level of Taxol production were stable by the ninth generation. FW increased 2.77-fold, and Taxol production was 259.14 μg g ?1 DW. So the callus from the ninth generation could be used for selecting high Taxolproducing cell lines. After comparing normal plate culture,conditioned culture, and nursing culture, conditioned culture was used for single-cell cloning and an initial cell density 3 × 10 3 cells mL ?1 was appropriate. After a stability analysis of 10 high-yielding cell lines, lines CL5, CL12, and CL21 were selected; Taxol contents were 0.0448%, 0.0477%, and 0.0428% DW, respectively. The proliferation increase of CL5, CL12, and CL21 was 346.3%, 382.5%, and 409.2%,respectively. These three stable, high yielding cell lines are therefore candidate sources for the mass producing Taxol and promoting commercial production using cell culture. For obtaining a more stable, higher yield from such cells, selection and cultures methods will continue to be investigated in future research. This study on selection methods for high Taxol-producing cell lines of T. cuspidata lays a foundation for selection of other plant cells. Further research, such the optimising the medium and culturing conditions, is now needed to move beyond the laboratory stage into full-scale production.

AcknowledgementsThanks go to Professor Liangjian Qu of the Chinese Academy of Forestry and Tongbin Guo of the Xuzhou Forest Protection Station for their valuable help.