extracting-venom-from-parasitoid-wasp-trichogramma-dendrolimi-using

Extracting Venom from the Parasitoid Wasp <em>Trichogramma dendrolimi</em> Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

Nanjing Agricultural University

Ministry of Agriculture

Most female herbivores ensure to lay eggs where their offspring can develop successfully. The oviposition preferences of females affect strategies in pest management. In this study, the performance of two cohorts of Trichoplusia ni larvae on cabbage and cotton (after they had been transferred from their original host plants) were investigated. The preferences of female moth ovipositing and larval feeding on these two host plants were observed. The results indicated that plants significantly affected oviposition preference of the female adults and development and survival of larvae of T. ni. All females preferred to lay eggs on cabbage than cotton regardless from which host they originated. The detrimental effects of cotton on the development and survival of T. ni larvae originated from cabbage (CaTn) increased with the increase of the larval age when they were transferred. In addition, the host plant change did not significantly affect the development and survival of larvae of T. ni originating from cotton (CoTn). Larvae of CaTn preferred cabbage plants as compared to cotton plants, whereas larvae of CoTn did not show a significant choice. Although the adult females preferred laying eggs on cabbage, they did not show preferences between cotton and cabbage in a Y-tube olfactometer test. The hypothesis of oviposition preference and performance of larvae was supported by the results of CaTn, whereas they not supported by those from CoTn. Based on these results, the strategy to manage this serious pest was discussed.

Oviposition preference, larval performance and adaptation of Trichoplusia ni on cabbage and cotton.

Bemisia tabaci is an important agricultural pest with worldwide distribution and host preference. Therefore, understanding the biology of this pest is important to devise specific pest control strategies. The antennae of herbivorous insects play an important role in the identification of hosts using plant volatiles. To understand the features of antennae in B. tabaci MEAM 1(formerly known as biotype 'B') and MED (formerly known as biotype 'Q'), the morphology and distribution of the antennal sensilla were examined using scanning electron micrographs. The results showed that the average antennae length in MEAM 1 was longer than MED. No differences were observed in the number and distribution of antennal sensilla in MEAM 1 and MED antennae; each antenna had nine different types of sensilla. Both cryptic species possessed Microtrichia, Grooved surface trichodea sensilla, Chaetae sensilla, Coeloconic sensillaⅠandⅡ, Basiconic sensilla Ⅰ, Ⅱ and Ⅲ and Finger-like sensilla. This is the first report of Grooved surface trichodea sensilla and Basiconic sensilla Ⅱ on B. tabaci flies. The numbers of Chaetae sensilla were different in the females and males of MEAM 1 and MED, which females having 5 and males containing 7. The surface structure of Basiconic sensilla Ⅰ was different with MEAM 1 showing a multiple-pitted linen surface and MED showing a multiple-pitted pocking surface. Basiconic sensillaⅡ were double in one socket with the longer one having a multiple-pitted surface and the shorter one with a smooth surface. Basiconic Ⅲ and Finger-like sensillae were longer in MEAM 1 antennae than in MED antennae. Our results are expected to further the studies that link morphological characteristics to insect behavior and help devise strategies to control insect pests.

Comparison of the antennal sensilla ultrastructure of two cryptic species in Bemisia tabaci.

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a globally distributed pest. One of the key endosymbionts in B. tabaci is Wolbachia, an α-proteobacterium implicated in many important biological processes. Previous studies indicated that the infection frequency of Wolbachia in Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED) varied greatly among populations in different areas. However, little is known about the factors that influence the prevalence of Wolbachia in B. tabaci. In this paper, 25 field populations were collected from different locations in China, and 1,161 individuals were screened for the presence of Wolbachia using a nested polymerase chain reaction (PCR)-based method, which targets the wsp gene, to confirm Wolbachia infection status. The prevalence of Wolbachia ranged from 1.54 to 66.67% within the 25 field populations, and the infection frequency of Wolbachia was affected significantly by the putative species of B. tabaci. The infection frequency (51.55%) of Wolbachia was significantly greater in native species than in the MED (25.65%) and MEAM1 (14.37%). With the exception of host plant, all factors, including putative species, geographic location, and the sex of the host, affected the Wolbachia infection frequency in whiteflies. Six Wolbachia strains were found and clustered into four distinct clades upon phylogenetic analyses. Furthermore, Wolbachia in B. tabaci have close relationships with those from other host species, including Liriomyza trifolii (Burgess), Sogatella furcifera (Horvath), Nilaparvata lugens (Stål), and Culex pipiens L. The results demonstrated the variation and diversity of Wolbachia in B. tabaci field populations, and that the application of nested PCR extended our knowledge of Wolbachia infection in B. tabaci, especially in invasive whiteflies.

Effects of Host Sex, Plant Species, and Putative Host Species on the Prevalence of Wolbachia in Natural Populations of Bemisia tabaci (Hemiptera: Aleyrodidae): A Modified Nested PCR Study.

Wolbachia pipientis are intracellular endosymbionts that induce parthenogenesis in the parasitoid Encarsia formosa. Previous studies that focused on effects of Wolbachia on the wasp usually used tetracycline to remove Wolbachia without concern for the joint influences of tetracycline and Wolbachia. Here we treated the wasps (F0 lines) with tetracycline to produce offspring (F1 lines) which were not fed tetracycline to avoid antibiotic influence. The quantitative data and fluorescence in situ hybridization showed that Wolbachia titers were reduced but not totally removed. The Wolbachia that infected the male offspring were unpredictably detected. Low dose tetracycline enhanced the fertility of 2-day-old F0 wasps after 24 h of treatment; however, compared with controls, the oocyte load of 3- to 6-day-old tetracycline-treated wasps decreased day by day, and tetracycline reduced the longevity of the wasps. The fecundity of controls was significantly higher than that of the treated F1-10 and F1-20 generations. Gene expression of vitellogenin reflected the same trend as that of wasp fecundities in both F0 and F1 lines. Moreover, female offspring proportions of F0 and F1 lines were related to the titer of infected Wolbachia, demonstrating that Wolbachia titer affected the sex determination of E. formosa.

Incomplete removal of Wolbachia with tetracycline has two-edged reproductive effects in the thelytokous wasp Encarsia formosa (Hymenoptera: Aphelinidae).

Temperature plays an important role in the growth, development, and geographic distribution of insects. There is convincing evidence that heat-shock proteins (HSPs) play important roles in helping organisms adapt to thermal stress. To better understand the physiological and ecological influence of thermal stress on the different development stages of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Mediterranean species (MED), nymphs and adults were shocked with temperatures of 35, 38, and 41℃ for 1 and 2 h, respectively, and the survival rate, fecundity, and developmental duration were investigated in the laboratory. The expression levels of the hsp40, hsp70, and hsp90 genes were assessed using real-time PCR. The results indicate that the survival rates of the nymphs and adults decreased with increased temperature. A 2-h heat shock at 41℃ induced a significant reduction in fecundity in adults and an increase in developmental duration in young nymphs. Hsp90 showed higher temperature responses to thermal stress than hsp40 or hsp70. The expression levels of the hsps in the adults were significantly down-regulated by a 2-h heat shock at 41℃ compared with that by a 1-h treatment. A significant decrease in the expression levels of the hsps also occurred in the adults when the temperature increased from 38 to 41℃ for the 2-h treatment, whereas no significant decrease occurred in the nymphs. Compared with previous studies, we provide some evidence indicating that MED has the potential to adapt to a wider temperature range than the Middle East-Asia Minor 1 species.

Thermotolerance and Heat-Shock Protein Gene Expression Patterns in Bemisia tabaci (Hemiptera: Aleyrodidae) Mediterranean in Relation to Developmental Stage.

The identification of Trichogramma (Hymenoptera: Trichogrammatidae) species is problematic due to their small size and lack of distinct morphological characters. In this study, we combined morphological characters of the male genitalia and molecular methods using the mitochondrial cytochrome oxidase I (COI) gene as a molecular marker to identify eight species from 16 geographic populations: T. evanescens Westwood, T. cacoeciae Marchal, T. ostriniae Pang et Chen, T. chilonis Ishii, T. japonicum Ashmead, T. brassicae Bezdenko, T. bilingensis He et Pang, and T. dendrolimi Matsumura. We developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method targeting the mitochondrial COI to distinguish the eight Trichogramma species using three restriction enzymes. We further analyzed 109 COI fragments from 18 Trichogramma species and found that the PCR-RFLP method could distinguish both intra- and inter-specific genetic variation among most of the species using four additional restriction enzymes.

Inter- and Intra-Specific Differentiation of Trichogramma (Hymenoptera: Trichogrammatidae) Species Using PCR-RFLP Targeting COI.

Animal mitochondrial genomes usually exhibit conserved gene arrangement across major lineages, while those in the Hymenoptera are known to possess frequent rearrangements, as are those of several other orders of insects. Here, we sequenced two complete mitochondrial genomes of Trichogramma japonicum and Trichogramma ostriniae (Hymenoptera: Chalcidoidea: Trichogrammatidae). In total, 37 mitochondrial genes were identified in both species. The same gene arrangement pattern was found in the two species, with extensive gene rearrangement compared with the ancestral insect mitochondrial genome. Most tRNA genes and all protein-coding genes were encoded on the minority strand. In total, 15 tRNA genes and seven protein-coding genes were rearranged. The rearrangements of cox1 and nad2 as well as most tRNA genes were novel. Phylogenetic analysis based on nucleotide sequences of protein-coding genes and on gene arrangement patterns produced identical topologies that support the relationship of (Agaonidae + Pteromalidae) + Trichogrammatidae in Chalcidoidea. CREx analysis revealed eight rearrangement operations occurred from presumed ancestral gene order of Chalcidoidea to form the derived gene order of Trichogramma. Our study shows that gene rearrangement information in Chalcidoidea can potentially contribute to the phylogeny of Chalcidoidea when more mitochondrial genome sequences are available.

Extensive gene rearrangements in the mitochondrial genomes of two egg parasitoids, Trichogramma japonicum and Trichogramma ostriniae (Hymenoptera: Chalcidoidea: Trichogrammatidae).

Endosymbionts are widely distributed among insects via intraspecific vertical transmission and interspecific horizontal transmission. Parasitoids have attracted considerable interest due to their possible role in the horizontal transmission of endosymbionts. Horizontal transmission of endosymbionts between whiteflies via parasitoids has been revealed in the laboratory. However, whether this occurs under field conditions remains unknown. Here, the diversity and phylogenetic relationships of endosymbionts in 1,350 whiteflies and 36 parasitoids that emerged from whitefly nymphs collected from three locations in Jiangsu Province of China were investigated. Only Rickettsia and Wolbachia were identified in both whiteflies and parasitoids, with an overall infection frequency of 22.67% in whiteflies and 16.67% in parasitoids for Wolbachia and of 12.15% in whiteflies and 25% in parasitoids for Rickettsia. Despite the distant relationship between whiteflies and their parasitoids, phylogenetic analyses revealed that the Rickettsia and Wolbachia individuals collected from the two types of organisms were grouped together. Furthermore, shared haplotypes were also identified, which was consistent with the horizontal transmission of endosymbionts between parasitoids and whiteflies. In addition, a parasitoid resistance-related symbiont, Hamiltonella, was detected in whiteflies at a 100% infection frequency, probably accounting for the relatively low parasitism of the whiteflies in the field. The factors affecting the infection frequency of the four secondary endosymbionts in whiteflies were also examined.

Diversity and Phylogenetic Analyses Reveal Horizontal Transmission of Endosymbionts Between Whiteflies and Their Parasitoids.

The taxonomic status and phylogenetic affinities of Mymaridae and Scelionidae are controversial, based on similarities between these families in the characteristics of adults, larvae, and eggs. In this study, we sequenced the mitochondrial (mt) genomes of representatives from these two families and found that the derived secondary structure of tRNA-Arg was the same in each family due to the absence of the D-stem. The segment of "cox1 trnLcox2 trnK trnD atp8 atp6 cox3" in Gonatocerus sp. (Mymaridae) is conserved and distinct from those of four other species of Chalcidoidea but similar to that in Proctotrupoidea and Platygastroidea. However, phylogenetic analysis indicated that Gonatocerus sp. was sister group to other species of Chalcidoidea. Comparisons based on complete gene orders may be more useful in a phylogenetic and systematic context, as different branches may exhibit partially homoplastic gene orders.

Information from the mitochondrial genomes of two egg parasitoids, Gonatocerus sp. and Telenomus sp., reveals a controversial phylogenetic relationship between Mymaridae and Scelionidae.

Insects perceive dangerous or attractive chemicals in the environment (such as the presence of predators, food or mates) through their olfaction and gustation. This leads to host searching, mate finding and other behaviors that are critical for insect survival. These vital activities are mediated by many chemosensory receptor proteins, including odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs). Trichogramma japonicum Ashmead (Hymenoptera: Trichogrammatidae) is a commonly used egg parasitoid for controlling lepidopteran pests in rice fields. To reveal the roles of its olfactory and gustatory systems in biological control, we systematically analyzed major chemosensory receptor genes from T. japonicum. Through transcriptomic analyses of male and female heads, we found that the differentially expressed genes (DEGs) were mainly related to chemosensory functions, and there were more chemosensory-related genes expressed in female heads than male heads, which may be related to the need for females to search for their hosts or sense male pheromone. Furthermore, we identified 66 chemosensory receptor genes, including 51 ORs, seven IRs and eight GRs, that were most closely relate to those of other hymenopteran species according to the results of both Blast best-hit and phylogenetic analyses. The tissue expression profile showed that 65 of the 66 chemosensory receptors were highly expressed in the heads, suggesting their putative roles in olfaction and gustation. In addition, the sex-specific expression patterns suggested their potential functions in host-seeking or mate sensing behaviors. This study may provide base for further understanding the olfactory and gustatory systems of T. japonicum and increasing its efficiency in pest control in the future.

Transcriptomic analyses of chemosensory genes in Trichogramma japonicum (Hymenoptera: Trichogrammatidae).

Despite its substantial costs, sexual reproduction dominates in animals. One popular explanation for the paradox of sex is that asexual reproduction is more likely to accumulate deleterious mutations than sexual reproduction. To test this hypothesis, we compared the mitogenomes of two asexual wasp strains,  and  to their sexual relatives. These two asexual strains represent two different transition mechanisms in  from sexual to asexual reproduction. Asexual  is induced by , while  presumably originated from interspecific hybridization. We sequenced and assembled complete mitochondrial genomes of asexual  and . Compared to four sexual relatives, we found no evidence of higher mutation accumulation in asexual  mitogenomes than in their sexual relatives. We also did not detect any relaxed selection in asexual  mitogenomes. In contrast, the intensified selection was detected in Nad1 and Nad4 of the asexual  mitogenome, suggesting more purifying selection. In summary, no higher mitochondrial mutation accumulation was detected in these two asexual  strains. This study provides a basis for further investigating mitochondrial evolution and asexual reproduction in .

Mitochondrial Genomes of Two Asexual  (Hymenoptera: Trichogrammatidae) Strains and Comparison with Their Sexual Relatives.

Parasitoid wasps are invaluable agents in pest biological control. Early detection and identification of parasitoid immatures are vital in characterizing parasitoid-host interactions and for evaluating parasitism rates accurately in the field. Trichogramma is the most widely used parasitoid wasp, and several studies have been performed for its molecular identification. However, those studies were mainly focused on Trichogramma adults and rarely on immatures. Here, we report a method to detect and identify Trichogramma larvae in their host eggs. We designed a pair of Trichogramma-specific primers that amplified Trichogramma mtCOI sequences from Corcyra cephalonica (Stainton) eggs parasitized by any of eight Trichogramma species tested but not from nonparasitized eggs of four lepidopteran hosts. This PCR method reliably detected Trichogramma immatures in parasitized eggs as early as 1 h after parasitism. We further developed an RFLP (restriction fragment length polymorphism) assay using restriction enzymes SspI and VspI to differentiate eight Trichogramma species at their immature stage. Overall, we developed a sensitive and reliable PCR-RFLP method to detect and identify immature-stage Trichogramma in their lepidopteran hosts. This method shows promise for conveniently identifying Trichogramma in insectaries and accurately evaluating parasitism rates in the field.

Early Detection and Identification of Parasitoid Wasps Trichogramma Westwood (Hymenoptera: Trichogrammatidae) in Their Host Eggs Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Article	Year
Oviposition preference, larval performance and adaptation of Trichoplusia ni on cabbage and cotton. Insect science\| PubMed ID: 24431263	2015
Comparison of the antennal sensilla ultrastructure of two cryptic species in Bemisia tabaci. PloS one\| PubMed ID: 25822843	2015
Effects of Host Sex, Plant Species, and Putative Host Species on the Prevalence of Wolbachia in Natural Populations of Bemisia tabaci (Hemiptera: Aleyrodidae): A Modified Nested PCR Study. Journal of economic entomology\| PubMed ID: 26470122	2015
Incomplete removal of Wolbachia with tetracycline has two-edged reproductive effects in the thelytokous wasp Encarsia formosa (Hymenoptera: Aphelinidae). Scientific reports\| PubMed ID: 28266601	2017
Thermotolerance and Heat-Shock Protein Gene Expression Patterns in Bemisia tabaci (Hemiptera: Aleyrodidae) Mediterranean in Relation to Developmental Stage. Journal of economic entomology\| PubMed ID: 28961720	2017
Inter- and Intra-Specific Differentiation of Trichogramma (Hymenoptera: Trichogrammatidae) Species Using PCR-RFLP Targeting COI. Journal of economic entomology\| PubMed ID: 29718401	2018
Extensive gene rearrangements in the mitochondrial genomes of two egg parasitoids, Trichogramma japonicum and Trichogramma ostriniae (Hymenoptera: Chalcidoidea: Trichogrammatidae). Scientific reports\| PubMed ID: 29728615	2018
Diversity and Phylogenetic Analyses Reveal Horizontal Transmission of Endosymbionts Between Whiteflies and Their Parasitoids. Journal of economic entomology\| PubMed ID: 30535231	2019
Information from the mitochondrial genomes of two egg parasitoids, Gonatocerus sp. and Telenomus sp., reveals a controversial phylogenetic relationship between Mymaridae and Scelionidae. Genomics\| PubMed ID: 31533898	2019
Transcriptomic analyses of chemosensory genes in Trichogramma japonicum (Hymenoptera: Trichogrammatidae). Comparative biochemistry and physiology. Part D, Genomics & proteomics\| PubMed ID: 33166853	2021
Mitochondrial Genomes of Two Asexual (Hymenoptera: Trichogrammatidae) Strains and Comparison with Their Sexual Relatives. Insects\| PubMed ID: 35735886	2022
Early Detection and Identification of Parasitoid Wasps Trichogramma Westwood (Hymenoptera: Trichogrammatidae) in Their Host Eggs Using Polymerase Chain Reaction-Restriction Fragment Length P... Journal of economic entomology\| PubMed ID: 35746894	2022

Yuan-xi Li

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