The complete mitochondrial genome and gene rearrangements in a gall wasp species, Dryocosmus liui (Hymenoptera: Cynipoidea: Cynipidae).

Mitochondrial genomes (mitogenomes) have been widely used in comparative and evolutionary genomics, molecular evolution, phylogenetics, and population genetics, but very limited information is available for the family Cynipidae. In this report, we describe the mitogenome of Dryocosmus liui Pang, Su et Zhu, providing the first complete mitogenomic data for a cynipid gall wasp species. The mitogenome of D. liui is 16,819 bp in length, and contains the typical set of 37 genes. Two control regions were detected, with the second being a perfect inverted repeat of the major portion of the first. Gene rearrangements were found in transfer RNA (tRNA) genes, protein-coding genes (PCGs) and ribosomal RNA (rRNA) genes, compared with the putative ancestral mitogenome. Similar to two other Cynipidae species with mitogenome data available, D. liui has a novel tRNA gene cluster trnL1-trnI-trnL2-trnW-trnM-trnQ between nad1 and nad2. Phylogenetic analysis based on sequences of PCGs and rRNA genes with D. liui included obtained topologies identical to previous studies supporting the a relationship of (Cynipoidea , (Platygastroidea, Proctotrupoidea)) within the monophyletic Proctotrupomorpha and (Cynipidae, Figitidae), Ibaliidae) within the Cynipoidea.

Pangenomic analysis of Wolbachia provides insight into the evolution of host adaptation and cytoplasmic incompatibility factor genes.

Introduction: The genus Wolbachia provides a typical example of intracellular bacteria that infect the germline of arthropods and filarial nematodes worldwide. Their importance as biological regulators of invertebrates, so it is particularly important to study the evolution, divergence and host adaptation of these bacteria at the genome-wide level. Methods: Here, we used publicly available Wolbachia genomes to reconstruct their evolutionary history and explore their adaptation under host selection. Results: Our findings indicate that segmental and single-gene duplications, such as DNA methylase, bZIP transcription factor, heat shock protein 90, in single monophyletic Wolbachia lineages (including supergroups A and B) may be responsible for improving the ability to adapt to a broad host range in arthropod-infecting strains. In contrast to A strains, high genetic diversity and rapidly evolving gene families occur in B strains, which may promote the ability of supergroup B strains to adapt to new hosts and their large-scale spreading. In addition, we hypothesize that there might have been two independent horizontal transfer events of cif genes in two sublineages of supergroup A strains. Interestingly, during the independent evolution of supergroup A and B strains, the rapid evolution of cif genes in supergroup B strains resulted in the loss of their functional domain, reflected in a possible decrease in the proportion of induced cytoplasmic incompatibility (CI) strains. Discussion: This present study highlights for reconstructing of evolutionary history, addressing host adaptation-related evolution and exploring the origin and divergence of CI genes in each Wolbachia supergroup. Our results thus not only provide a basis for further exploring the evolutionary history of Wolbachia adaptation under host selection but also reveal a new research direction for studying the molecular regulation of Wolbachia- induced cytoplasmic incompatibility.

Effects of Flight on Reproductive Development in Long-Winged Female Crickets (Velarifictorus aspersus Walker; Orthoptera: Gryllidae) with Differences in Flight Behavior.

A trade-off between the capacity for flight and reproduction has been documented extensively in wing polymorphic female insects, thereby supporting the possible fitness gain due to flightlessness. However, most of these studies were conducted without considering the effect of flight behavior. In the present study, we assessed the flight duration by long-winged (LW) females in the cricket species Velarifictorus aspersus on different days after adult emergence and examined the effect of flight on ovarian development in LW females with different flight capacities. Our results showed that the flight capacity increased with age and peaked after 5 days. In addition, the flight capacity varied among individuals, where most LW females could only take short flights (sustained flight time < 10 min) and only a few individuals could take long flights (sustained flight time > 20 min). In LW female crickets demonstrating only short flights, repeated flying for 30 or 60 min significantly promoted reproductive development. However, in those capable of long flights, reproductive development was affected only after a flight of 60 min. The flight muscles degraded after the start of rapid reproduction in those with both short and long flights. Our results indicated that the critical flight time for switching from flight to reproduction varies among LW V. aspersus female crickets with polymorphic flight behavior.

Physiological and biochemical differences in diapause and non-diapause pupae of Sericinus montelus (Lepidoptera: Papilionidae).

The swallowtail butterfly, Sericinus montelus Gray, is endemic to East Asia, has high ornamental value but faces an increased risk of extinction. To understand the overwintering strategies of this species, the dynamic changes in supercooling point (SCP) and water and biochemical contents of diapause-destined and non-diapause S. montelus pupae were investigated. The SCP of laboratory-reared diapause pupae was as low as -26°C compared to -24°C in diapause pupae in the field. Although there was no significant difference in total water content between diapause-destined and non-diapause pupae, the free water of diapause-destined pupae was significantly lower, and the bound water was significantly higher, than that of non-diapause pupae. Lipid, glycogen, and protein contents of diapause-destined pupae showed a downward trend, whereas the total sugar content showed the opposite trend after pupation. The glycogen content decreased rapidly during the initial stage of pupation, whereas the lipid content decreased significantly after 30 days of pupation, suggesting that diapause-destined pupae deplete glycogen stores during the pre-diapause period and then switch to using lipids during the diapause maintenance phase. Trehalose levels in diapause-destined pupae increased significantly and remained high after pupation. Meanwhile, the trehalose content of overwintering pupae during the diapause maintenance period was significantly higher than that of diapause termination pupae in the field. These results suggest that trehalose is the main cryoprotectant for overwintering pupae. Thus, diapausing S. montelus pupae appear to be freeze avoidant, accumulate trehalose as a cryoprotectant, and reduce the free water content to decrease the SCP, enhancing their cold tolerance.

High Levels of Multiple Phage WO Infections and Its Evolutionary Dynamics Associated With Wolbachia-Infected Butterflies.

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Phage WO is the only bacteriophage known to infect Wolbachia, and may provide benefit to its host or arthropods. We screened for the presence of phage WO in Wolbachia-infected butterfly species for the first time, to investigate their diversity and evolutionary dynamics. All Wolbachia-infected butterfly species, including members of the families Hesperiidae, Lycaenidae, Nymphalidae, Papilionidae, and Pieridae, were found to harbor phage WO. Interestingly, 84% of 19 butterfly species, which were infected with a single Wolbachia strain harbored high levels of multiple phage types (ranging from 3 to 17 types), another three species harbored one or two phage types. For Wolbachia strains (ST-41, ST-19, ST-125 and ST-374) shared among various butterfly species, their host insects all harbored multiple phage types, while two Wolbachia strains (ST-297 and ST-wPcau) were found to infect one butterfly species, whose insect hosts harbored a single phage type, suggesting that horizontal transfer of Wolbachia between insects increased the likelihood of exposure to phages, resulting in increased phage genetic diversity. Twelve horizontal transmission events of phage WO were found, which shared common phage WO types among different Wolbachia strains associated with butterflies. Most horizontal transfer events involved different Wolbachia supergroups (A and B). Horizontal acquisition of phage WO might also occur between eukaryotes without Wolbachia transfer. Furthermore, 22 putative recombination events were identified in 13 of 16 butterfly species which harbored multiple phage types. These results showed that horizontal transfer of Wolbachia caused it to be exposed to the phage gene pool, and that horizontal transmission of phage WO, as well as intragenic recombination were important dynamics for phage WO genome evolution, which effectively promoted the high level of phage WO diversity associated with butterflies.

A new species of Andricus Hartig, 1840 (Hymenoptera, Cynipidae) from China, with references to DNA taxonomy and Wolbachia infection.

In the present paper, a new species of cynipid gall wasp, Andricuselodeoides Liu & Pang, is described from several provinces in southern China. The new species is closely related to the recently redescribed A.mairei (Kieffer, 1906). In addition to differences in adult and gall morphology, the new species is also readily separated by COI sequences, with a 6.2-8.9% genetic distance between populations of the new species and those of A.mairei. A contrasting difference in sex ratios was also observed between the two species, with A.elodeoides extremely female-biased (95.5-97.8% female) while A.mairei male-biased to more balanced (5.4-43.5% female). PCR screening for Wolbachia infection further revealed contrasting infection rates between populations of A.elodeoides and A.mairei: the Wolbachia infection rate was 0% in A.elodeoides and 100% in A.mairei. Cytoplasmic incompatibility induced by Wolbachia is proposed as a potential mechanism of speciation of the sympatric A.elodeoides and A.mairei.

A new species of Plagiotrochus from China (Hymenoptera: Cynipidae: Cynipini) galling on Quercus glauce (Fagaceae).

A new gall wasp species, Plagiotrochus hepingensis sp. n. is described from Hunan Province, China that forms integral galls on the central vein on the abaxial leaf blade surface of Quercus (Cyclobalanopsis) glauca (Fagaceae). The morphological characters of the new species are described and photographed and data on the diagnosis, distribution, and biology are given. Additionally, a specific region of the COI gene was amplified using the primers HCO-2198 and LCO-1490 and compared for both the new species and Plagiotrochus masudai Ide, Wachi Abe.

Comparison of Auxin and Cytokinins Concentrations, and the Structure of Bacterial Community between Host Twigs and Lithosaphonecrus arcoverticus Galls.

Insect galls are the abnormal growth of plant tissues induced by a wide variety of galling insects and characterized by high concentrations of auxins and cytokinins. It remains unclear whether the auxins and cytokinins affect the bacterial community structure of insect galls. We determined the concentrations of indoleacetic acid (IAA) as an example of auxin, trans-zeatin riboside (tZR) and isopentenyladenine (iP) as cytokinins in Lithosaphonecrus arcoverticus (Hymenoptera: Cynipidae) galls and the galled twigs of Lithocarpus glaber (Fagaceae) using liquid chromatography-tandem mass spectrometry. Moreover, for the first time, we compared the bacterial community structure of L. arcoverticus galls and galled twigs by high-throughput sequencing, and calculated the Spearman correlation and associated degree of significance between the IAA, tZR and iP concentrations and the bacterial community structure. Our results indicated the concentrations of IAA, tZR and iP were higher in L. arcoverticus galls than in galled twigs, and positively correlated with the bacterial community structure of L. arcoverticus galls. We suggest the high concentrations of IAA, tZR and iP may affect the bacterial community structure of L. arcoverticus galls.

Design and Testing of Effective Primers for Amplification of the orf7 Gene of Phage WO Associated with Andricus hakonensis.

Phage WO was first characterized in Wolbachia, an obligate intracellular Rickettsiales known for its ability to regulate the reproduction of arthropod hosts. In this paper, we focus on the study of virus diversity in Andricus hakonensis and the development of highly effective primers. Based on the existing Wolbachia genome sequence, we designed primers (WO-TF and WO-TR) to amplify the full-length orf7 gene of phage WO. Surprisingly, sequencing results showed a high abundance of other phage WO groups in A. hakonensis, in addition to the four groups previously identified. The results also showed that A. hakonensis contained most of the known types of orf7 genes (I, III, IV, V and VI) and the level of diversity of harbored phage WO was very high. Therefore, we speculated that existing primers were not specific enough and that new primers for the detection of phage WO were needed. Based on the existing orf7 gene sequence, we designed specific detection primers (WO-SUF and WO-SUR). Sequencing results showed that the primers effectively amplified all known types of phage WO. In addition to amplifying most of the known sequences, we also detected some new genotypes in A. hakonensis using the new primers. Importantly, all phage WO groups could be efficiently detected. Combined with the results of previous studies, our results suggest that A. hakonensis contains the largest number of phage types (up to 36 types). This study is novel in that it provides practical molecular evidence supporting base deletions, in addition to gene mutations and genetic recombination, as an important cause of phage WO diversity.

A Case of Intragenic Recombination Dramatically Impacting the Phage WO Genetic Diversity in Gall Wasps.

The phage WO was characterized in Wolbachia, a strictly intracellular bacterium causing several reproductive alterations in its arthropod hosts. This study aimed to screen the presence of Wolbachia and phage WO in 15 gall wasp species from six provinces of southern China to investigate their diversity and prevalence patterns. A high incidence of Wolbachia infection was determined in the gall wasp species, with an infection rate of 86.7% (13/15). Moreover, seven species had double or multiple infections. All Wolbachia-infected gall wasp species were found to harbor phage WO. The gall wasp species infected with a single Wolbachia strain were found to harbor a single phage WO type. On the contrary, almost all species with double or multiple Wolbachia infections harbored a high level of phage WO diversity (ranging from three to 27 types). Six horizontal transfer events of phage WO in Wolbachia were found to be associated with gall wasps, which shared identical orf7 sequences among their respective accomplices. The transfer potentially took place through gall inducers and associated inquilines infected with or without Wolbachia. Furthermore, 10 putative recombination events were identified from Andricus hakonensis and Andricus sp2, which harbored multiple phage WO types, suggesting that intragenic recombination was the important evolutionary force, which effectively promoted the high level of phage WO diversity associated with gall wasps.

The Diversity and Dynamics of Fungi in Dryocosmus kuriphilus Community.

Dryocosmus kuriphilus (Hymenoptera: Cynipidae) is a gall wasp that induces insect galls on chestnut trees and results in massive yield losses worldwide. Fungi can cause the necrosis of chestnut trees and the death of gall wasps. The aim of this research was to investigate the potential role of D. kuriphilus in the transmission of fungi. We sequenced the ribosomal RNA internal transcribed spacer region 1 of fungi in D. kuriphilus adults, associated insect galls and the galled twigs of Castanea mollissima, using high-throughput sequencing. We compared the species richness, α-diversity and community structure of fungi in D. kuriphilus adults, insect galls and the galled twigs. We provide the first evidence that D. kuriphilus adults shared most fungal species with associated insect galls and the galled twigs, and were dominated by Botryosphaeria sp., Aspergillus sp. and Diaporthe sp. We suggest D. kuriphilus adults may be potential vectors of plant pathogens and may facilitate the transmission of fungi between chestnut trees. Furthermore, the fungi may horizontally transmit among D. kuriphilus adults, associated insect galls and the galled twigs.

A new species of Periclistus Foerster, 1869 from China and review of the tribe Diastrophini (Hymenoptera, Cynipoidea, Cynipidae).

A new species of cynipid gall wasps, Periclistus orientalis Pang, Liu & Zhu, sp. nov., is herein described from Hunan, China in the tribe Diastrophini (Hymenoptera: Cynipoidea: Cynipidae). The phylogenetic relationship between Periclistus and all the other Diastrophini genera, except the recently described XestophanopsisPujade-Villar et al., 2019, was analyzed using a fragment of the mitochondrial COI gene and a fragment of the nuclear 28S gene. A taxonomic key to the known genera of Diastrophini and an updated taxonomic key to the known Eastern Palearctic species of Periclistus were provided. In addition, an updated checklist of the known species of the genus from the world is given.

Transcriptome Profile Changes Associated With Heat Shock Reaction in the Entomopathogenic Nematode, Steinernema carpocapsae.

The entomopathogenic nematode Steinernema carpocapsae has been used for control of soil insects. However, S. carpocapse is sensitive to environmental factors, particularly temperature. We studied an S. carpocapse group that was shocked with high temperature. We also studied the transcriptome-level responses associated with temperature stress using a BGIseq sequencing platform. We de novo assembled the reads from the treatment and control groups into one transcriptome consisting of 43.9 and 42.9 million clean reads, respectively. Based on the genome database, we aligned the clean reads to the Nr, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and analyzed the differentially expressed genes (DEGs). Compared with the control, the heat-shocked group had significant differential expression of the heat shock protein (HSP) family, antioxidase [glutathione S-transferases (GSTs) and superoxide dismutase (SOD)], monooxygenase (P450), and transcription factor genes (DAF-16 and DAF-2). These DEGs were demonstrated to be part of the Longevity pathway and insulin/insulin-like signaling pathway. The results revealed the potential mechanisms, at the transcriptional level, of S. carpocapsae under thermal stress.

Fight outcome briefly affects the reproductive fitness of male crickets.

Sexual selection allows male individuals to adopt different evolutionary strategies in mating system. In this study, we determined whether dominance affected reproductive fitness of male crickets Velarifictorus aspersus during both pre-copulatory and post-copulatory selection when we excluded male-male competition. The results showed that females mated more often with male winners only during the first 2 h after a fight when male winners were more likely to produce courtship songs than losers. However, females did not retain the attached spermatophores of male winners longer than those of male losers, and the fecundity and fertilization success also did not differ significantly between females mated different times with male winners and losers. Instead, the fertilization success was positively correlated with male body weight. These results suggest that a recent wining experience increases reproductive fitness of males during pre-copulatory selection, but females may prefer larger males rather than winners during post-copulatory selection. The incoordination between pre- and post-copulatory selection may allow males to adopt different evolutionary strategies in mating system.

Discovery of a new species of Dryocosmus Giraud, 1859 (Hymenoptera: Cynipidae: Cynipini) on Castanopsis tibetana from China.

A new gall wasp species, Dryocosmus liui Pang, Su et Zhu n. sp., is described from Hunan Province, China. The new species forms integral galls on branches of Castanopsis tibetana Hance. The results of morphological comparison, pair-wise genetic distance of mtDNA COI, and phylogenetic analysis based on COI gene sequences did not support treating Chilaspis and Dryocosmus as separate genera. Our results also provided new evidence supporting the notion that the Nearctic Dryocosmus species may represent a lineage distinct from the Old World species of the genus.

New gall wasp species attacking chestnut trees: Dryocosmus zhuili n. sp. (Hymenoptera: Cynipidae) on Castanea henryi from southeastern China.

A new gall wasp species, Dryocosmus zhuili Liu et Zhu, is herein described from the southeastern Fujian province of China. The new species induces galls on trees of Henry's chestnut, Castanea henryi, which is also a native host for the notorious Oriental chestnut gall wasp (OCGW, Dryocosmus kuriphilus Yasumatsu). D. zhuili overlaps with OCGW in emergence time and induces galls morphologically similar to that of OCGW on similar plant parts. In a previous study, we reported considerable divergence between mtDNA CO1 (mitochondrial DNA Cytochrome c oxidase subunit I) sequences of these wasps and the true OCGW wasps and suggested the existence of a cryptic species. Herein, we confirm the identity of the new species based on morphological and biological differences and provide a formal description. Although the new species is relatively easily separated from OCGW on basis of morphology, field identification involving the two species can still be problematic because of their small body size, highly similar gall morphology, and other life history traits. We further discussed the potential of the new species to be a pest for the chestnut industry and the consequences of accidental introduction of this species into nonnative areas, especially with regard to the bisexual reproduction mode of the new species in contrast to the parthenogenetic reproduction mode of OCGW.

Effects of environmental factors and appendage injury on the wing variation in the cricket Velarifictorus ornatus.

The effects of environmental factors and appendage injury on the wing variation in Velarifictorus ornatus (Shiraki) (Orthoptera: Gryllidae) were investigated. The percentage of micropters was more than 95% when the nymphs were reared at constant photoperiods, and changing photoperiod did not affect wing variation in V. ornatus at 25 or 30°C. In the crowding experiment, the percentage of macropters was only 11.2% when the nymphs were reared separately at 25°C. In contrast, the percentage of macropters was significantly higher when the rearing density was increased to two nymphs per container and lower when the rearing density was increased to five or 10 nymphs per container. These results indicate that low and high rearing densities induce micropters, but intermediate rearing density stimulates the formation of macropters. Meanwhile, severance of appendages, such as antennae, femora, and tibiae, in the nymph stage exerted a micropterizing effect. The period sensitive to such stresses ranged from 35 to 60 days of nymph development.

High levels of multiple infections, recombination and horizontal transmission of Wolbachia in the Andricus mukaigawae (Hymenoptera; Cynipidae) communities.

Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W8, W2 and W6 apparently have derived from W3 and W5a, W6 and W7, W4 and W9, respectively. W8 and W2 and their respective parental strains were detected in S. japonicus. W6 was detected with only one parent (W4) in S. japonicus; W9 was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.

Adaptive strategies of overwintering adults: reproductive diapause and mating behavior in a grasshopper, Stenocatantops splendens (Orthoptera: Catantopidae).

To understand the adaptive strategies of the overwintering adults of Stenocatantops splendens, the mechanism of maintenance and termination of the reproductive diapause, the variation in mortality between overwintering females and males, and the mating strategy of the males were investigated. The results indicated that the adult reproductive diapause in natural conditions was mainly regulated by photoperiod in the fall - long photoperiods promoted reproductive development and short photoperiods maintained reproductive diapause, and the sensitivity of the overwintering adults to photoperiod was over before the end of the winter. When transferred from natural conditions to controlled laboratory conditions on dates from September through February, pre-oviposition became increasingly shorter with increasingly deferred transfer dates regardless of photoperiod conditions. The adults treated with low temperature for 30 days in September through November had significantly shorter pre-oviposition, suggesting that low temperatures in winter had an important role in the termination of reproductive diapause. The female had a significantly lower supercooling point than the male, which was related to their lower mortality after winter. In addition, observations of wild populations of the species indicated that mating behavior prior to winter and the duration of pre-mating period were not affected by photoperiod; mating and sperm transfer were mostly completed by November. Compared with females only mating before winter, females mating in the spring had shorter life span, longer pre-oviposition, lower hatching rate and laid fewer egg pods while showing no significant difference with regard to ovipositional interval, per pod number of eggs and nymph dry weight.

The dark-color inducing neuropeptide, [His(7)]-corazonin, causes a shift in morphometic characteristics towards the gregarious phase in isolated-reared (solitarious) Locusta migratoria.

The neurohormone, [His(7)]-corazonin is known to induce dark color in the cuticle and epidermis of Locusta migratoria. In the present study, we examined the effects of this hormone on development and morphometrics in two strains, albino and normal, of this locust under isolated conditions. Injection of [His(7)]-corazonin induced dark color in both strains. In either strain, [His(7)]-corazonin injected at the second and third instars did not affect duration of nymphal development or the number of nymphal instars. The shape of the pronotum was more convex in isolated-reared animals than in crowd-reared ones, and injection of [His(7)]-corazonin caused isolated-reared animals to develop a less convex pronotum in the normal strain injected at a high dose (1 nmolx2) but not in the albino strain injected at a low dose (50 pmolx2). [His(7)]-corazonin injected into isolated-reared nymphs caused a shift in classical morphometric ratios (F/C and E/F; F=length of the hind femur, C=maximum width of the head, E=length of the fore wings) towards values typical for crowd-reared (gregarious) individuals of the two strains. This study demonstrated for the first time that [His(7)]-corazonin affected morphometric characteristics in L. migratoria.