A chromosome-level genome assembly of the forestry pest Coronaproctus castanopsis.

As an important forestry pest, Coronaproctus castanopsis (Monophlebidae) has caused serious damage to the globally valuable Gutianshan ecosystem, China. In this study, we assembled the first chromosome-level genome of the female specimen of C. castanopsis by merging BGI reads, HiFi long reads and Hi-C data. The assembled genome size is 700.81 Mb, with a scaffold N50 size of 273.84 Mb and a contig N50 size of 12.37 Mb. Hi-C scaffolding assigned 98.32% (689.03 Mb) of C. Castanopsis genome to three chromosomes. The BUSCO analysis (n = 1,367) showed a completeness of 91.2%, comprising 89.2% of single-copy BUSCOs and 2.0% of multicopy BUSCOs. The mapping ratio of BGI, second-generation RNA, third-generation RNA and HiFi reads are 97.84%, 96.15%, 97.96%, and 99.33%, respectively. We also identified 64.97% (455.3 Mb) repetitive elements, 1,373 non-coding RNAs and 10,542 protein-coding genes. This study assembled a high-quality genome of C. castanopsis, which accumulated valuable molecular data for scale insects.

Characterization of the Complete Mitochondrial Genome of Eight Diurnal Hawkmoths (Lepidoptera: Sphingidae): New Insights into the Origin and Evolution of Diurnalism in Sphingids.

In this study, the mitochondrial genomes of 22 species from three subfamilies in the Sphingidae were sequenced, assembled, and annotated. Eight diurnal hawkmoths were included, of which six were newly sequenced (Hemaris radians, Macroglossum bombylans, M. fritzei, M. pyrrhosticta, Neogurelca himachala, and Sataspes xylocoparis) and two were previously published (Cephonodes hylas and Macroglossum stellatarum). The mitochondrial genomes of these eight diurnal hawkmoths were comparatively analyzed in terms of sequence length, nucleotide composition, relative synonymous codon usage, non-synonymous/synonymous substitution ratio, gene spacing, and repeat sequences. The mitogenomes of the eight species, ranging in length from 15,201 to 15,461 bp, encode the complete set of 37 genes usually found in animal mitogenomes. The base composition of the mitochondrial genomes showed A+T bias. The most commonly used codons were UUA (Leu), AUU (Ile), UUU (Phe), AUA (Met), and AAU (Asn), whereas GCG (Ala) and CCG (Pro) were rarely used. A phylogenetic tree of Sphingidae was constructed based on both maximum likelihood and Bayesian methods. We verified the monophyly of the four current subfamilies of Sphingidae, all of which had high support. In addition, we performed divergence time estimation and ancestral character reconstruction analyses. Diurnal behavior in hawkmoths originated 29.19 million years ago (Mya). It may have been influenced by the combination of herbaceous flourishing, which occurred 26-28 Mya, the uplift of the Tibetan Plateau, and the large-scale evolution of bats in the Oligocene to Pre-Miocene. Moreover, diurnalism in hawkmoths had multiple independent origins in Sphingidae.

Characterization of male-biased fatty acid desaturase in the jewel wasp Nasonia vitripennis and its role in pheromone production.

Linoleic acid is the material for biosynthesis of sex attracting and blocking (postmating) pheromones in Nasonia vitripennis, it is synthesized from oleic acid by a male-biased fatty acid desaturase (SCD5a). In this study, we developed a specific antibody and further characterized the expression patterns of SCD5a in males at different mating stages by western blot. SCD5a was mainly expressed in male heads rather than in abdomens. Along with the aging process (from Day 1 to Day 3), SCD5a increased significantly. Compared with virgin males, mated males showed higher levels of SCD5a. Likewise, abdomen dipping frequency, during which males release attracting pheromone, increased with age and mating. Moreover, real-time quantitative PCR revealed that genes responsible for the first three steps of attracting pheromone biosynthesis were more highly expressed in head than in abdomen, but the final gene for transformation of attracting pheromone was more highly expressed in abdomen than in head. These results suggest that linoleic acid for biosynthesis of attracting pheromones may also originate from the head rather than only synthesized at the rectal vesicles.

Novel gene rearrangement pattern in mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994: new gene order in Encyrtidae (Hymenoptera, Chalcidoidea).

Studies of mitochondrial genomes have a wide range of applications in phylogeny, population genetics, and evolutionary biology. In this study, we sequenced and analyzed the mitochondrial genome of Ooencyrtusplautus Huang & Noyes, 1994 (Hymenoptera, Encyrtidae). The nearly complete mitogenome of O.plautus was 15,730 bp in size, including 13 PCGs (protein-coding genes), 22 tRNAs, 2 rRNAs, and a nearly complete control region. The nucleotide composition was significantly biased toward adenine and thymine, with an A + T content of 84.6%. We used the reference sequence of Chouioiacunea and calculated the Ka/Ks ratio for each set of PCGs. The highest value of the Ka/Ks ratio within 13 PCGs was found in nad2 with 1.1, suggesting that they were subjected to positive selection. This phenomenon was first discovered in Encyrtidae. Compared with other encyrtid mitogenomes, a translocation of trnW was found in O.plautus, which was the first of its kind to be reported in Encyrtidae. Comparing with ancestral arrangement pattern, wasps reflect extensive gene rearrangements. Although these insects have a high frequency of gene rearrangement, species from the same family and genus tend to have similar gene sequences. As the number of sequenced mitochondrial genomes in Chalcidoidea increases, we summarize some of the rules of gene rearrangement in Chalcidoidea, that is four gene clusters with frequent gene rearrangements. Ten mitogenomes were included to reconstruct the phylogenetic trees of Encyrtidae based on both 13 PCGs (nucleotides of protein coding genes) and AA matrix (amino acids of protein coding genes) using the maximum likelihood and Bayesian inference methods. The phylogenetic tree reconstructed by Bayesian inference based on AA data set showed that Aenasiusarizonensis and Metaphycuseriococci formed a clade representing Tetracneminae. The remaining six species formed a monophyletic clade representing Encyrtinae. In Encyrtinae, Encyrtus forms a monophyletic clade as a sister group to the clade formed by O.plautus and Diaphorencyrtusaligarhensis. Encyrtussasakii and Encyrtusrhodooccisiae were most closely related species in this monophyletic clade. In addition, gene rearrangements can provide a valuable information for molecular phylogenetic reconstruction. These results enhance our understanding of phylogenetic relationships among Encyrtidae.

Complete mitochondrial genome of a parasitoid, Trichogramma chilonis (Hymenoptera: Chalcidoidea: Trichogrammatidae) and phylogenetic analysis.

Trichogramma chilonis is a kind of ovoid parasitic wasp, which has important application value in the biological control of pests. In this study, we sequenced and analyzed the complete mitogenome T. chilonis to compare mitogenomic structures and reconstruct phylogenetic relationships. The complete mitogenome sequence of T. chilonis is circular, 16,176 bp in size and encodes 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), and a control region (CR). Nucleotide composition is highly biased toward A + T nucleotides (85.2%). All 13 protein-coding genes (PCGs) initiate with the standard start codon of ATN and terminate with the typical stop codon TAA/TAG. Phylogenetic analyses were performed using amino acids of 13 PCGs showed that T. chilonis is closely related to Trichogramma ostriniae.

Sexual dimorphism and sex-biased gene expression in an egg parasitoid species, Anastatus disparis.

BACKGROUND: Differences in the expression of genes present in both sexes are assumed to contribute to sex differences including behavioural, physiological and morphological dimorphisms. For enriching our knowledge of gender differences in an important egg parasitoid wasp, Anastatus disparis (Hymenoptera: Eupelmidae), sex-biased differences in gene expression were investigated using Illumina-based transcriptomic analysis. RESULTS: A total of 15,812 resulting unigenes were annotated, and a large set of genes accounting for 50.09% of the total showed sex-biased expression and included 630 sex-specific genes. Gene Ontology (GO) enrichment analyses showed that the functional categories associated with sex-biased genes were mainly related to reproduction. In addition, the transcriptome data provided evidence that sex pheromones in A. disparis are produced by the female, and activity of Δ12-desaturases appear to have been replaced by Δ9-desaturases playing roles in sex pheromone production. The large set of sex-biased genes identified in this study provide a molecular background for sexually dimorphic traits such as flyability, longevity, and aggression in this species and suggests candidate venom proteins expressed only in females that could be used for biological control. CONCLUSIONS: This study provides comprehensive insight into sexually dimorphic traits of a parasitoid wasp and can inform future research into the molecular mechanisms underlying such traits and the application of parasitoids to the biological control of pest species.

Effect of Winning Experience on Aggression Involving Dangerous Fighting Behavior in Anastatus disparis (Hymenoptera: Eupelmidae).

Aggressive behavior is widely observed in animal species for acquiring important resources and usually includes both dangerous and nondangerous fighting patterns. Only a few species show dangerous fighting patterns that are defined by fights ending with contestants being severely injured or killed. Prior experience, an important factor in many species, has been demonstrated to affect a contestant's subsequent fighting behavior. Few studies have focused on the effect of experience on aggression involving dangerous fighting patterns. Here, an egg parasitoid wasp, Anastatus disparis, which shows extreme and dangerous fighting behavior to acquire mating opportunities, was used as an experimental model. Our results showed that the fighting intensity of the winning males significantly decreased subsequent fighting behavior, which was inconsistent with general predictions. Transcriptomic analyses showed that many genes related to energy metabolism were downregulated in winners, and winners increased their fighting intensity after dietary supplementation. Our study suggested that fighting in A. disparis is a tremendous drain on energy. Thus, although males won at combat, significant reductions in available energy constrained the intensity of subsequent fights and influenced strategic decisions. In addition, winners might improve their fighting skills and abilities from previous contests, and their fighting intensity after dietary supplementation was significantly higher than that of males without any fighting experience. Generally, in A. disparis, although winners increased their fighting ability with previous experience, the available energy in winners was likely to be a crucial factor affecting the intensity and strategic decisions in subsequent fights.

Comparative transcriptomics analysis of the river pufferfish (Takifugu obscurus) by tributyltin exposure: Clues for revealing its toxic injury mechanism.

TBT residual in water had become a noticeable ecological problem for aquatic ecosystems. The river pufferfish (Takifugu obscurus) is a kind of an anadromous fish species and widely distributed in the East China Sea and the Yellow Sea. Because of the water contamination, the pufferfish wild resource had a sudden decline in recent years. Therefore, the study on the response of pufferfish to the TBT exposure may contribute to reveal toxic injury mechanism of T. obscurus under TBT exposure. In this study, the transcriptional library of T. obscurus liver and gill was constructed and sequenced by an improved Illumina HiseqX10 high-throughput sequencing platform under different concentrations of TBT acute stress. The blood cell numbers distinctly decreased after TBT exposure, showing the adverse effects of TBT invasion and self-adjusting ability of the pufferfish. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT. The obtained data were compared with the genome data of Takifugu rubripes and transcriptional resource database. On this basis, gene function annotation, analysis and classification were carried out by bioinformatics method, and differential genes related to toxic injury function were screened out. Meanwhile, new toxic related genes and related signal pathways were sought to provide new theoretical guidance for the pathogenesis of T. obscurus exposed to TBT. This study not only enriched the transcriptome data of T. obscurus under environmental stress, but also provided a new research method for the response mechanism of T. obscurus under the stimulation of environmental factors.

Hyperparasitism in a Generalist Ectoparasitic Pupal Parasitoid, Pachycrepoideus vindemmiae (Hymenoptera: Pteromalidae), on Its Own Conspecifics: When the Lack of Resource Lead to Cannibalism.

Hyperparasitism is a normal behavior of parasitoids, which often happens among species. Conspecific hyperparasitism, such as some kinds of heteronomous hyperparasitic behaviors, has been only reported in some species belonging to Aphelinidae. In this article, the conspecific hyperparasitism of Pachycrepoideus vindemmiae (Pteromalidae) is reported, with Drosophila puparia as hosts. Hosts were exposed to P. vindemmiae females twice to parasitism with nine, twelve, and fifteen day intervals between the two exposures. None of the infested hosts emerged more than one offspring, and emergence of parasitoid offspring occurred in two obvious events, synchronously with the exposure time intervals, which suggested that offspring emerging during the first and second events would come from the primary and secondary parasitoids, respectively, and the inference with the developmental duration of offspring also indicated this. With two P. vindemmiae strains that could be identified by a simple sequence repeat marker, the above speculation of the origin of those offspring emerging during the two events was confirmed. Dissection of hosts exposed twice revealed a cannibalism behavior of larvae from the secondary foundresses on the primary conspecific pupae. Our results suggested a conspecific hyperparasitism behavior of the secondary parasitoids on the primary conspecifics. Measures showed a reduced body size for the adults from the conspecific hyperparasitism. Foundresses from the conspecific hyperparasitism had less fitness variables than those from primary parasitism, with shorter longevity, less life time fecundity, lower values of infestation degree, and lower success rate of parasitism. However, when the parasitoids from the conspecific hyperparasitism met healthy Drosophila puparia, their offspring would recover to normal size. Frequency of the conspecific hyperparasitism behavior enhanced with the decreasing of proportion of healthy hosts in the oviposition patch. The conspecific hyperparasitism of P. vindemmiae on the primary conspecifics would be helpful to last the population when healthy hosts are absent in the oviposition patch.

Pollinating fig wasp Ceratosolen solmsi adjusts the offspring sex ratio to other foundresses.

Local mate competition theory predicts that offspring sex ratio in pollinating fig wasps is female-biased when there is only one foundress, and increased foundress density results in increased offspring sex ratio. Information of other foundresses and clutch size have been suggested to be the main proximate explanations for sex ratio adjustment under local mate competition. Our focus was to show the mechanism of sex ratio adjustment in a pollinating fig wasp, Ceratosolen solmsi Mayr, an obligate pollinator of the functionally dioecious fig, Ficus hispida Linn., with controlled experiments in the field. First, we obtained offspring from one pollinator and offspring at different oviposition sequences, and found that offspring sex ratio decreased with clutch size, and pollinators produced most of their male offspring at the start of bouts, followed by mostly females. Second, we found that offspring sex ratio increased with foundress density, and pollinators did adjust their offspring sex ratio to other females in the oviposition patches. We suggest that when oviposition sites are not limited, pollinators will mainly adjust their offspring sex ratio to other foundresses independent of clutch size changes, whereas adjusting clutch size may be used to adjust sex ratio when oviposition sites are limited.

[Effects of different treatment methods of housefly pupae for the reproduction of Pachycrepoideus vindemmiae Rondani].

It is an important way to massively rear parasitoid wasps by using appropriate methods to treat the wasps' hosts and preserve them for a long duration. Pachycrepoideus vindemmiae Rondani is a pupal parasitoid of several dipteral pests, being of significance for the biological control of the pests. In this paper, housefly pupae were frozen at -20 degrees C, cold storage-preserved at 6 degrees C, and CO2-asphyxiated for 1-, 3-, and 30 days, respectively, and some pupae were heat-killed and cold storage-preserved for 30 days, aimed to approach the effects of these treatment methods on the reproduction of P. vindemmiae on the pupae. The results showed that P. vindemmiae could reproduce on the pupae treated with the above-mentioned methods, and the tibia length of the offspring had less difference with that on the fresh pupae. However, the reproduction of P. vindemmiae on the pupae treated with the above-mentioned methods except frozen decreased with the increasing preserving duration of the pupae. At the prerequisite of preserving for 30 days, frozen pupae had the highest P. vindemmiae offspring reproduction, suggesting that P. vindemmiae could be massively reared when the housefly pupae were treated by frozen and cold storage-preserved.

The integrative taxonomic approach reveals host specific species in an encyrtid parasitoid species complex.

Integrated taxonomy uses evidence from a number of different character types to delimit species and other natural groupings. While this approach has been advocated recently, and should be of particular utility in the case of diminutive insect parasitoids, there are relatively few examples of its application in these taxa. Here, we use an integrated framework to delimit independent lineages in Encyrtus sasakii (Hymenoptera: Chalcidoidea: Encyrtidae), a parasitoid morphospecies previously considered a host generalist. Sequence variation at the DNA barcode (cytochrome c oxidase I, COI) and nuclear 28S rDNA loci were compared to morphometric recordings and mating compatibility tests, among samples of this species complex collected from its four scale insect hosts, covering a broad geographic range of northern and central China. Our results reveal that Encyrtus sasakii comprises three lineages that, while sharing a similar morphology, are highly divergent at the molecular level. At the barcode locus, the median K2P molecular distance between individuals from three primary populations was found to be 11.3%, well outside the divergence usually observed between Chalcidoidea conspecifics (0.5%). Corroborative evidence that the genetic lineages represent independent species was found from mating tests, where compatibility was observed only within populations, and morphometric analysis, which found that despite apparent morphological homogeneity, populations clustered according to forewing shape. The independent lineages defined by the integrated analysis correspond to the three scale insect hosts, suggesting the presence of host specific cryptic species. The finding of hidden host specificity in this species complex demonstrates the critical role that DNA barcoding will increasingly play in revealing hidden biodiversity in taxa that present difficulties for traditional taxonomic approaches.

Effects of female diet and age on offspring sex ratio of the solitary parasitoid Pachycrepoideus vindemmiae (Rondani) (Hymenoptera, Pteromalidae) / Efeitos da dieta e idade da fêmea em relação à prole e à razão sexual do parasitoide solitário Pachycrepoideus vindemmiae (Rondani) (Hymenoptera, Pteromalidae)

Effects of female diet and age on offspring sex ratio of the solitary parasitoid Pachycrepoideus vindemmiae (Rondani) (Hymenoptera, Pteromalidae). Theories predict that females of parasitoid wasps would adjust the offspring sex ratio to environmental conditions in the oviposition patch, but the diet and age of females would also affect the sex ratio adjustment. Our focus was to test the effects of female diet and age on offspring sex ratio of the solitary parasitoid wasp, Pachycrepoideus vindemmiae (Rondani, 1875). Our results showed that females fed with honey had significantly less female biased offspring sex ratio than those fed only with water. Offspring sex ratio (male percentage) decreased with female age or female longevity at the beginning of oviposition but increased at the end. There should be a sperm limitation in P. vindemmiae females at the end of oviposition, and a higher frequency of unfertilized eggs were laid then. Females also laid more unfertilized eggs at the beginning of oviposition, which would be necessary to insure the mating among offspring. Male offspring developed faster and emerged earlier, which would also reduce the risk of virginity in offspring with female-biased sex ratio.

Efeitos da dieta e idade da fêmea em relação à prole e à razão sexual do parasitoide solitário Pachycrepoideus vindemmiae (Rondani) (Hymenoptera, Pteromalidae). As teorias predizem que as fêmeas parasitoides de vespas ajustam a relação razão sexual dos descendentes, de acordo com condições ambientais, em cada oviposição. Entretanto, a dieta e idade das fêmeas também podem afetar o ajuste da razão sexual. Nosso foco foi testar os efeitos da dieta e idade da fêmeas em relação a razão sexual da prole da vespa parasitoide Pachycrepoideus vindemmiae (Rondani, 1875). Nossos resultados mostraram que as fêmeas alimentadas com mel apresentaram uma razão sexual significativa menor de fêmeas, do que aquelas alimentadas apenas com água. A razão sexual (percentagem dos machos) diminuiu com a idade ou longevidade das fêmeas no início da oviposição, mas com aumento no final. Deve haver uma limitação de esperma nas fêmeas de P. vindemmiae no final da oviposição e uma maior frequência de posturas de ovos infertilizados. As fêmeas também fazem posturas de um número maior de ovos infertilizados no início da oviposição, que seria necessário para assegurar o acasalamento entre os descendentes. A prole masculina desenvolverá mais rapidamente e emergirá mais cedo, o que também reduziria o risco de virgindade da prole, com a tendenciosa proporção de razão sexual maior das fêmeas.

DNA barcoding of six Ceroplastes species (Hemiptera: Coccoidea: Coccidae) from China.

Ceroplastes Gray (wax scales) is one of the genera of Coccidae, most species of which are considered to be serious economic pests. However, identification of Ceroplastes species is always difficult owing to the shortage of easily distinguishable morphological characters. Mitochondrial cytochrome c oxidase I (COI) sequences (or DNA barcodes) and the D2 expansion segments of the large subunit ribosomal RNA gene 28S were used for accurate identification of six Ceroplastes species (C. floridensis Comstock, C. japonicus Green, C. ceriferus (Fabricius), C. pseudoceriferus Green, C. rubens Maskell and C. kunmingensis Tang et Xie) from 20 different locations in China. For COI data, low G·C content was found in all species, averaging about 20.4%. Sequence divergences (K2P) between congeneric species averaged 12.19%, while intra-specific divergences averaged 0.42%. All 112 samples fell into six reciprocally monophyletic clades in the COI neighbour-joining (NJ) tree. The NJ tree inferred from 28S showed almost same results, but samples of two closely related species, C. ceriferus and C. pseudoceriferus, were clustered together. This research indicates that the standard barcode region of COI can efficiently identify similar Ceroplastes species. This study provides an example of the usefulness of barcoding for Ceroplastes identification.

Molecular approaches identify known species, reveal cryptic species and verify host specificity of Chinese Philotrypesis (Hymenoptera: Pteromalidae).

Philotrypesis, a major component of the fig wasp community (Hymenoptera: Pteromalidae), is a model taxon for studying male fighting and mating behaviour. Its extreme sexual dimorphism and male polymorphism render species identification uncertain and in-depth research on its ecology, behaviour and other evolutionary topics challenging. The fig wasps' enclosed habitat within the syconia makes their mating behaviour inaccessible, to the extent of matching conspecific females and males. In this study, we combine morphological and molecular analyses to identify species of Philotrypesis sampled from south China and to associate their extraordinarily dimorphic genders and labile male morphologies. Morphological evaluations of females identify 22 species and 28 male morphs. The mitochondrial cytochrome c oxidase I and nuclear internal transcribed spacer 2 data detect 21 species using females, and 15 species among the males. Most of the males match the species as delimited by females. Both markers reveal cryptic species in P. quadrisetosa on Ficus vasculosa. Most species of wasps live on one species of fig but three species co-occur in two hosts (F. microcarpa and F. benjamina), which indicates host switching.

Molecular approaches to identify cryptic species and polymorphic species within a complex community of fig wasps.

Cryptic and polymorphic species can complicate traditional taxonomic research and both of these concerns are common in fig wasp communities. Species identification is very difficult, despite great effort and the ecological importance of fig wasps. Herein, we try to identify all chalcidoid wasp species hosted by one species of fig, using both morphological and molecular methods. We compare the efficiency of four different DNA regions and find that ITS2 is highly effective for species identification, while mitochondrial COI and Cytb regions appear less reliable, possibly due to the interference signals from either nuclear copies of mtDNA, i.e. NUMTs, or the effects of Wolbachia infections. The analyses suggest that combining multiple markers is the best choice for inferring species identifications as any one marker may be unsuitable in a given case.