Effects of Wolbachia Infection, Parasitic Order, And Time Interval On The Outcome of Inter-Strain Intrinsic Competition Between Bisexual Strain And Wolbachia-Induced Thelytokous Strain of Egg Parasitoid, Trichogramma Dendrolimi

Parasitoids may deposit a second clutch of eggs on a host that has been parasitized by the same parasitoid species. This is termed “superparasitism”. Wolbachia infection increases the superparasitism frequency of Trichogramma females. We investigated the outcome and fitness cost of inter-strain intrinsic competition between Wolbachia-infected thelytokous strain (TDW) and uninfected bisexual strain (TDB) of Trichogramma dendrolimi. To determine the developmental rate of both strains, the size of immature stages of T. dendrolimi offspring at different times after parasitization was measured in single parasitism and superparasitism conditions. The results showed increased superparasitism by Wolbachia-infected females compared to uninfected females, regardless of the time interval between oviposition; Trichogramma females were unable to distinguish between host eggs previously parasitized by TDB or TDW females. When the first oviposition was performed by TDB females, the TDB offspring outcompeted TDW offspring deposited later. However, although the TDW offspring was deposited 8 h earlier than the TDB offspring, it gained no advantage over TDB offspring. Regardless of parasitism conditions, the differences in development rate and time between TDW and TDB offspring were not significant. This study revealed that albeit TDW females presented a higher tendency of superparasitism than TDB females, TDB offspring outcompeted TDW offspring even when the latter was deposited 8 h earlier. The negative effects of Wolbachia infection on the competitive ability of TDW offspring inside the host eggs were due to offspring’s low fitness rather than delayed development.

Trichogramma ostriniae Is More Effective Than Trichogramma dendrolimi As a Biocontrol Agent of the Asian Corn Borer, Ostrinia furnacalis

The Asian corn borer (ACB), Ostrinia furnicalis, is a serious corn pest in south-east Asia, causing huge economic losses every year. Trichogramma dendrolimi and Trichogramma ostriniae, two egg parasitoids, have previously been identified as key biological control agents. To determine the age impact of ACB eggs on their effective biocontrol potential, herein we compared the biological parameters (i.e., number of parasitized eggs, emergence, developmental time, and sex ratio) of both parasitoids on ACB eggs of various ages (i.e., 0–4, 4–8, 8–12, 12–16, 16–24, 24–36, and 36–48 h old), respectively. Our results showed that the age of ACB eggs had a significant impact on the parasitization activity of T. dendrolimi in both choice and no-choice conditions. Trichogramma dendrolimi preferred to parasitize 0–8-h-old ACB eggs, and its parasitization dramatically declined on ACB eggs older than 8 h under choice and no-choice conditions. On the other hand, T. ostriniae showed high preference to parasitize all tested ACB egg ages. The age of ACB eggs had no significant impact on the parasitization of T. ostriniae under choice and no-choice conditions. Furthermore, the female progeny of T. dendrolimi decreased as the age of ACB increased, while no differences were found in female progeny of T. ostriniae. Trichogramma ostriniae also developed faster on each ACB egg age group in comparison with T. dendrolimi. Overall, the age of ACB eggs had a significant impact on T. dendrolimi performance, leading us to conclude that T. ostriniae is more effective than T. dendrolimi as a biocontrol agent of the ACB.

Effects of Thelytokous Parthenogenesis-Inducing Wolbachia on the Fitness of Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) in Superparasitised and Single-Parasitised Hosts

Thelytokous Wolbachia-infected Trichogramma species have long been considered as biological control agents against lepidopteran pests in agriculture and forestry. Wolbachia has been suggested to increase the probability of the superparasitism of Trichogramma, but the fate of infected offspring in the superparasitised host is still unknown. The present study aimed to evaluate the fitness of thelytokous Wolbachia-infected (TDW) and bisexual Wolbachia-free (TD) Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) lines in superparasitised or single-parasitised hosts. The results showed that irrespective of whether Trichogramma wasps were developed from superparasitised or single-parasitised hosts, the TDW line was characterized by reduced fitness, including lower fecundity, shorter longevity, and smaller body size of F1 offspring, and lower emergence rate of F2 offspring than the TD line. This was not true for the survival rate and developmental time of F1 offspring. Additionally, the fitness parameters of T. dendrolimi that developed from superparasitised hosts were lower compared with that of T. dendrolimi that developed from single-parasitised hosts. Interestingly, Wolbachia-infected females had higher dispersal capacity than bisexual females when they developed from superparasitised hosts. The results indicated that Wolbachia negatively affects fitness of T. dendrolimi, but enhance dispersal capacity of T. dendrolimi females in superparasitism condition. Further studies need to be carried out to select the best line that will allow Wolbachia and their host Trichogramma to be better adapted to one another.

Selection and evaluation of qPCR reference genes for expression analysis in the tiny egg parasitoid wasp, Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae)

The egg parasitoid Trichogramma spp. is an important biological control agent used against multiple species of Lepidopteran pest in forestry and agriculture. Due to the importance of Trichogramma spp. in biocontrol programs, its biological characteristics have been studied in detail, and current investigations should focus on the molecular biology of these tiny parasitoids. Real-time quantitative PCR (qPCR) is considered as the standard method for quantifying the gene expression of organisms. Surprisingly, the appropriate reference genes to ensure robust qPCR have not been documented at all for the Trichogramma genus. This study aimed to identify suitable reference genes for use in qPCR procedure of Trichogramma dendrolimi . Nine candidate housekeeping genes, namely glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), forkhead box O ( FOXO ), superoxide dismutase ( SOD ), beta-actin ( ACTIN ), ribosomal protein L10a ( RPL10a ), L18 ( RPL18 ), L28 ( RPL28 ), S13 ( RPS13 ), and S15 ( RPS15 ), were tested for their suitability as reference genes for developmental stage (3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , and 10 th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17?, 25?, and 32?). According to the GeNorm analysis, robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperature, respectively. According to the RelFinder method and by assessing the integrated values from using the ?Ct method, GeNorm, NormFinder, and BestKeeper, we identified the developmental stage-specific reference genes SOD , GAPDH , and ACTIN ; tissue-specific reference genes RPL18 and RPS15 ; sex-specific reference genes SOD and RPL18 ; and temperature-specific reference genes RPL18 and RPL10 . When testing the use of stable vs. unstable reference genes, the substantial differences were observed in the estimation expression of a hypothetical target gene, HSP90 , in response to temperature. The present study provides a robust method for the measurement of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Background: The parasitoid wasp, Trichogramma dendrolimi, can enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi. Previous studies on diapause have mainly focused on ecological characteristics, so the molecular basis of diapause in T. dendrolimi is unknown. We compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in diapause development.Results: Transcriptome sequencing was performed on diapause prepupae, pupae after diapause, non-diapause prepupae, and pupae. Analysis yielded a total of 87,022 transcripts with an average length of 1,604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7,593 sequences with an average length of 3,351 bp. Among them, 5,702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using quantitative real-time PCR (qPCR). The changes showed the same trend as between the qPCR and RNA-Seq results. Several genes were identified as involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase, Glutathione-S-transferase, p53, and DNA damage-regulated gene 1 (pdrg1). Genes related to lipid metabolism were also included.Conclusions: We generated a large amount of transcriptome data from T. dendrolimi, providing a resource for future gene function research. The diapause-related genes identified help reveal the molecular mechanisms of diapause, in T. dendrolimi, and other insect species.

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Background The parasitoid wasp, Trichogramma dendrolimi, can enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi. Previous studies on diapause have mainly focused on ecological characteristics, so the molecular basis of diapause in T. dendrolimi is unknown. We compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in diapause development. Results Transcriptome sequencing was performed on diapause prepupae, pupae after diapause, non-diapause prepupae, and pupae. Analysis yielded a total of 87,022 transcripts with an average length of 1604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7593 sequences with an average length of 3351 bp. Among them, 5702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using quantitative real-time PCR (qPCR). The changes showed the same trend as between the qPCR and RNA-Seq results. Several genes were identified as involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase, Glutathione-S-transferase, p53, and DNA damage-regulated gene 1 (pdrg1). Genes related to lipid metabolism were also included. Conclusions We generated a large amount of transcriptome data from T. dendrolimi, providing a resource for future gene function research. The diapause-related genes identified help reveal the molecular mechanisms of diapause, in T. dendrolimi, and other insect species.

Gene Characterization and Enzymatic Activities Related to Trehalose Metabolism of In Vitro Reared Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) under Sustained Cold Stress

Trichogramma spp. is an important egg parasitoid wasp for biocontrol of agriculture and forestry insect pests. Trehalose serves as an energy source or stress protectant for insects. To study the potential role of trehalose in cold resistance on an egg parasitoid, cDNA for trehalose-6-phosphate synthase (TPS) and soluble trehalase (TRE) from Trichogramma dendrolimi were cloned and characterized. Gene expressions and enzyme activities of TdTPS and TdTRE were determined in larvae, prepupae, pupae, and adults at sustained low temperatures, 13 °C and 16 °C. TdTPS and TdTRE expressions had similar patterns with higher levels in prepupae at 13 °C and 16 °C. TdTPS enzyme activities increased with a decrease of temperature, and TdTRE activity in prepupae decreased sharply at these two low temperatures. In vitro reared T. dendrolimi could complete entire development above 13 °C, and the development period was prolonged without cold injury. Results indicated trehalose might regulate growth and the metabolic process of cold tolerance. Moreover, 13 °C is the cold tolerance threshold temperature and the prepupal stage is a critical developmental period for in vitro reared T. dendrolimi. These findings identify a low cost, prolonged development rearing method, and the cold tolerance for T. dendrolimi, which will facilitate improved mass rearing methods for biocontrol.

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Background: The parasitoid wasp, Trichogramma dendrolimi, can enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi. Previous studies on diapause have mainly focused on ecological characteristics, so the molecular basis of diapause in T. dendrolimi is unknown. We compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in diapause development.Results: Transcriptome sequencing was performed on diapause prepupae, pupae after diapause, non-diapause prepupae, and pupae. Analysis yielded a total of 87,022 transcripts with an average length of 1,604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7,593 sequences with an average length of 3,351 bp. Among them, 5,702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using quantitative real-time PCR (qPCR). The changes showed the same trend as between the qPCR and RNA-Seq results. Several genes were identified as involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase, Glutathione-S-transferase, p53, and DNA damage-regulated gene 1 (pdrg1). Genes related to lipid metabolism were also included.Conclusions: We generated a large amount of transcriptome data from T. dendrolimi, providing a resource for future gene function research. The diapause-related genes identified help reveal the molecular mechanisms of diapause, in T. dendrolimi, and other insect species.

High-throughput profiling of diapause regulated genes from Trichogramma dendrolimi, an important egg parasitoid

Background: The parasitoid wasp Trichogramma dendrolimi can successfully enter diapause at the prepupal stage. Thus, diapause is an efficient preservation method during the mass production of T. dendrolimi . Previous studies on diapause have focused largely on ecological characteristics, so the molecular mechanism of diapause in T. dendrolimi is mostly unknown. In this study, we compared transcriptomes of diapause and non-diapause T. dendrolimi to identify key genes and pathways involved in the development of diapause. Results: Transcriptome sequencing was performed using different samples, including diapause prepupae, pupae after diapause, normal prepupae, and pupae. Initially, it yielded a total of 87,022 unigenes with an average length of 1,604 bp. By removing redundant sequences and those without significant BLAST hits, a non-redundant dataset was generated, containing 7,593 sequences with an average length of 3,351 bp. Among them, 5,702 genes were differentially expressed. The result of Gene Ontology (GO) enrichment analysis revealed that regulation of transcription, DNA-templated, oxidation-reduction process, and signal transduction were significantly affected. Ten genes were selected for validation using a quantitative real-time PCR (qPCR). The changes showed the same trend between the qPCR and RNA-Seq results. Based on our data, several genes were identified to be involved in diapause, including ribosomal proteins, zinc finger proteins, homeobox proteins, forkhead box proteins, UDP-glucuronosyltransferase , Glutathione-S-transferase , p53 , DNA damage-regulated gene 1 ( pdrg1 ), and genes related to lipid metabolism were also included. Conclusions: In this study, we generated a great amount of transcriptome data from T. dendrolimi , providing a resource for gene function research. The diapause-related genes that we identified establish a valuable basis for future studies on the molecular mechanisms of diapause, not only for T. dendrolimi , but for other species as well.