Large proportion of genes in one cryptic WO prophage genome are actively and sex-specifically transcribed in a fig wasp species.

BACKGROUND: Cryptic prophages are genetically defective in their induction and propagation, and are simply regarded as genetic remnants. There are several putative cryptic WO prophages in the sequenced Wolbachia genomes. Whether they are lytic is unclear and their functions are poorly understood. Only three open reading frames (ORFs) in cryptic WO prophages have been reported to be actively transcribed. RESULTS: In this study, we comprehensively examined the transcription of the only cryptic WO prophage (WOSol) in a Wolbachia strain that infects a fig wasp, Ceratosolen solmsi (Agaonidae, Chalcidoidea). By analyzing the transcriptions of all the ORFs of WOSol in both sexes of C. solmsi, using qualitative and quantitative methods, we demonstrated that i) a high percentage of ORFs are actively transcribed (59%, 17/29); ii) the expression of these ORFs is highly sex-specific, with a strong male bias (three in females and 15 in males); iii) an ank (ankyrin-domain-containing) gene actively transcribed in both wasp sexes is more highly expressed in males. CONCLUSIONS: A large proportion of the genes in the cryptic WO prophage WOSol are expressed, which overturns the concept that cryptic prophages are simply genetically defective. The highly sex-specific expression patterns of these genes in the host suggest that they play important roles in Wolbachia biology and its reproductive manipulation of its insect host, particularly through the males.

Evidence for the circadian gene period as a proximate mechanism of protandry in a pollinating fig wasp.

Protandry in insects is the tendency for adult males to emerge before females and usually results from intra-sexual selection. However, the genetic basis of this common phenomenon is poorly understood. Pollinating fig wasp (Agaonidae) larvae develop in galled flowers within the enclosed inflorescences ('figs') of fig trees. Upon emergence, males locate and mate with the still galled females. After mating, males release females from their galls to enable dispersal. Females cannot exit galls or disperse from a fig without male assistance. We sampled male and female Ceratosolen solmsi (the pollinator of Ficus hispida) every 3 h over a 24 h emergence period, and then measured the expression of five circadian genes: period (per), clock (clk), cycle (cyc), pigment-dispersing factor (pdf) and clockwork orange (cwo). We found significant male-biased sexual dimorphism in the expression of all five genes. per showed the greatest divergence between the sexes and was the only gene rhythmically expressed. Expression of per correlated closely with emergence rates at specific time intervals in both male and female wasps. We suggest that this rhythmical expression of per may be a proximate mechanism of protandry in this species.

Delimitation and description of the immature stages of a pollinating fig wasp, Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae).

The mutualism between fig trees and their wasp pollinators is a model system for many ecological and evolutionary studies. However, the immature stages of pollinating fig wasps have rarely been studied. We monitored developing fig wasps of known ages and performed a series of dissections at 24 h intervals to identify key developmental traits of Ceratosolen solmsi marchali Mayr (Hymenoptera: Agaonidae), a pollinator of Ficus hispida L. (Moraceae). We identified where in the Ficus ovary eggs were deposited and time to hatch. We were also able to identify the timing and key underlying characters of five larval instars, three sub-pupal stages, and a single prepupal stage. We provide detailed morphological descriptions for the key stages and report some behavioral observations of the wasps in the several developmental stages we recorded. Scanning electron microscope images were taken.

Identification, characterization and hypolipidemic effect of novel peptides in protein hydrolysate from Protaetia brevitarsis larvae.

The proteins were mainly derived from Protaetia brevitarsis larval extracts obtained using two empty intestine methods (traditional static method: TSM or salt immersion stress method: SISM) and extraction solvents (water: W or 50 % water-ethanol: W:E), and the proteins were used as objects to investigate the effect of emptying intestine methods on hypolipidemic peptides. The results revealed that the F-2 fractions of protein hydrolysate had stronger in vitro hypolipidemic activity, with the peptides obtained by SISM possessing a stronger cholesterol micelle solubility inhibition rate, especially in SISM-W:E-P. Moreover, a total of 106 peptides were tentatively identified, among which SISM identified more peptides with an amino acid number < 8. Meanwhile, five novel peptides (YPPFH, YPGFGK, KYPF, SPLPGPR and VPPP) exhibited good hypolipidemic activity in vitro and in vivo, among which YPPFH, VPPP and KYPF had strong inhibitory activities on pancreatic lipase (PL) and cholesteryl esterase (CE), and KYPF, SPLPGPR and VPPP could significantly reduce the TG content in Caenorhabditis elegans. Thus, P. brevitarsis can be developed as a naturally derived hypolipidemic component for the development and application in functional foods.

High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) for determination of a highly degenerated prophage WO genome in a Wolbachia strain infecting a fig wasp species.

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.

Adaptive evolution of vertebrate-type cryptochrome in the ancestors of Hymenoptera.

One of the most mysterious aspects of insect clock mechanisms is that some insects, including Hymenoptera and Tribolium, only express a vertebrate-type cryptochrome (cry2). It is unknown whether or not cry2 underwent adaptive evolution in these insects. In the present study, we cloned and sequenced the full-length cry2 from a fig pollinator species, Ceratosolen solmsi (Hymenoptera: Chalcidoidea: Agaonidae), and examined the molecular evolution and daily expression of this gene. Our results suggest that cry2 underwent positive selection in the branch leading to hymenopteran insects. The function of CRY2 might have been fixed since undergoing natural selection in the ancestor of Hymenoptera. Male pollinators showed stronger rhythmicity in the host figs, which reflect an adaptation to their life cycles.

Characterization of bioactives and in vitro biological activity from Protaetia brevitarsis larval extracts obtained by different pretreatment extractions.

Intestinal contents affect the characterization of edible insect bioactive compounds. Two empty intestine methods, namely, traditional static method (TSM) or salt immersion stress method (SISM), associated with extraction solvents water (W), 50 % water-ethanol (W:E) or 100 % ethanol (E), were used to obtain six Protaetia brevitarsis larval extracts. The total flavonoid content (TFC) in the W:E extracts was significantly higher than that in the W and E extracts, with TSM-W:E the highest (p < 0.05). The relative contents of 132 bioactive compounds, especially p-hydroxyphenylacetic acid, citric acid, and dehydroepiandrosterone, were different between TSM-W and SISM-W. TSM-W:E had significantly higher 2,2-diphenyl-1-picrylhydroxy· (DPPH) scavenging and pancreatic lipase (PL) inhibitory activity than SISM-W:E (p < 0.05). DPPH scavenging and PL inhibitory activities were highly correlated with TFC and carbohydrates, respectively. Thus, bioactive compounds in P. brevitarsis extracts can be obtained selectively using pretreatment methods, which might be beneficial for high-value utilization of P. brevitarsis.

Evolutionary timescale of chalcidoid wasps inferred from over one hundred mitochondrial genomes.

Chalcidoidea is one of the most biologically diverse groups among Hymenoptera. Members are characterized by extraordinary parasitic lifestyles and extensive host ranges, among which several species attack plants or serve as pollinators. However, higher-level chalcidoid relationships remain controversial. Here, we performed mitochondrial phylogenomic analyses for major clades (18 out of 25 families) of Chalcidoidea based on 139 mitochondrial genomes. The compositional heterogeneity and conflicting backbone relationships in Chalcidoidea were assessed using various datasets and tree inferences. Our phylogenetic results supported the monophyly of 16 families and polyphyly of Aphelinidae and Pteromalidae. Our preferred topology recovered the relationship (Mymaridae+(Signiphoridae+Leucospidae)+(Chalcididae+((Perilampidae+Eucharitidae)+ remaining Chalcidoidea)))). The monophyly of Agaonidae and Sycophaginae was rejected, while the gall-associated ((Megastigmidae+Ormyridae)+(Ormocerinae+Eurytomidae)) relationship was supported in most results. A six-gene inversion may be a synapomorphy for most families, whereas other derived gene orders may introduce confusion in phylogenetic signals at deeper nodes. Dating estimates suggested that Chalcidoidea arose near the Jurassic/Cretaceous boundary and that two dynamic shifts in diversification occurred during the evolution of Chalcidoidea. We hypothesized that the potential codiversification between chalcidoids and their hosts may be crucial for accelerating the diversification of Chalcidoidea. Ancestral state reconstruction analyses supported the hypothesis that gall-inducers were mainly derived from parasitoids of gall-inducers, while other gall-inducers were derived from phytophagous groups. Taken together, these findings advance our understanding of mitochondrial genome evolution in the major interfamilial phylogeny of Chalcidoidea.

Impact of mitotype diversity on metabarcoding biodiversity estimations in Insecta and Arachnida using different sample preparation strategies.

DNA barcoding and metabarcoding have been increasingly used in species delimitation and species diversity assessment, respectively, and the molecular markers used in animals are mainly derived from mitochondrial DNA. It is well known that the phenomenon of multiple mitochondrial haplotypes within the same specimen (hereafter referred to as "mitotype diversity") may have a negative impact on the proper assessment of biodiversity by metabarcoding. However, few studies have focused on the incidence of this phenomenon and its effects on metabarcoding results using different sample preparation strategies, such as mock community construction using pooled high-throughput sequencing (HTS) data, DNA-pooling and Tissue-pooling. In this study, we investigated mitotype diversity and its influence on metabarcoding based on 398 specimens from 66 species of Insecta and 82 specimens from 16 species of Arachnida by HTS of the mitochondrial cox1 gene fragment. The results revealed that mitotype diversity was common in the studied taxa and significantly increased the number of operational taxonomic units (OTUs) using the three sample preparation strategies. The results also showed that the bioinformatics pipeline based on authentic amplicon sequence variants was more reliable than the pipeline based on OTUs. Regarding the sample preparation strategies of DNA-pooling and Tissue-pooling commonly used in metabarcoding, our results revealed that their results of metabarcoding were quite similar, and the Tissue-pooling strategy was therefore preferred because of its simplicity. Our study calls for additional attention to the interference of mitotype diversity on the results of DNA metabarcoding in biodiversity assessment.

The gender-specific impact of starvation on mitotypes diversity in adults of Drosophila melanogaster.

In animals, starvation can increase the level of reactive oxygen species (ROS) in some tissues. Mitochondrial DNA (mtDNA) is more vulnerable to being attacked by ROS due to the lack of histone protection, leading to oxidative damage. However, whether starvation is associated with the genetic diversity of mtDNA remains unclear. Here, by using adult individuals of Drosophila melanogaster under three different feeding treatments (starvation, with the provision of only water, and normal feeding), based on the high-throughput sequencing results of the PCR amplicons of the partial sequences of the mitochondrial gene cytochrome c oxidase subunit I (mt-cox1), no significant difference in the mean number of mitochondrial haplotypes and the mean genetic distance of haplotypes within individuals were identified between the three treatment groups. Coupled with the low proportion of heterogeneous mt-cox1 sequences within each individual, it suggested that starvation had a limited impact on mitotype genetic diversity and mitochondrial function. Nevertheless, starvation could significantly increase the sequence number of haplotypes containing specific mutations, and for males with higher levels of mitochondrial heteroplasmy than females in the normal feeding group, starvation could further increase their mitochondrial heteroplasmy.

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity.

BACKGROUND: Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. RESULTS: We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. CONCLUSIONS: Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA.

A possibly new Rickettsia-like genus symbiont is found in Chinese wheat pest aphid, Sitobion miscanthi (Hemiptera: Aphididae).

In this study, we investigated Rickettsia infection in Chinese wheat pest aphid (Sitobion miscanthi), moreover detected a possibly new Rickettsia-like symbiont, provisionally named as SMLS(1) (S. miscanthi L type symbiont). The sequence of SMLS 16S rRNA gene is 94% similar to that of its presumed closest relative, Orientia tsutsugamushi. If levels of divergence indicate taxonomic distinctiveness, SMLS probably represents a new genus in the family Rickettsiaceae. SMLS occurs in most populations of S. miscanthi, and with divergent infection frequencies, from 5.0% to 93.8%.

Transcriptomic Analysis Reveals the Sexually Divergent Host-Wolbachia Interaction Patterns in a Fig Wasp.

Wolbachia are widely distributed in arthropods and nematodes, acquiring nutrients from the hosts, and inducing remarkable reproductive modulations on the hosts. To investigate the interaction of Wolbachia and insects, Wolbachia are often artificially eliminated from Wolbachia-infected hosts, which may produce negative effects of antibiotics. In the present study, based on the transcriptomic data of a fig wasp species Ceratosolen solmsi with two sibling lineages, one natively infected and the other noninfected with Wolbachia, we investigated the expression patterns of genes. The comparison results of differently expressed genes (DEGs) between Wolbachia infected and noninfected samples show that males have many more DEGs than females. The male unique upregulated genes are enriched in biological processes mainly related to biosynthesis, transport, positive regulation of I-kappaB kinase/NF-kappaB signaling, MAPK cascade, and pathogenesis; the male unique downregulated genes are enriched in biological processes mainly related to transport, oxidation-reduction, cellular responses to oxidative stress, lipid oxidation, cytoskeleton organization, actin filament-based process, and localization. In addition, for the Wolbachia's gene expression, the number of genes up-regulated in males is higher than that in females. The results revealed divergent patterns of the host-Wolbachia interactions between males and females in the fig wasp species.

Genome-Wide Analysis of Gene Families of Pattern Recognition Receptors in Fig Wasps (Hymenoptera, Chalcidoidea).

Pattern recognition receptors (PRRs) play important roles in detecting pathogens and initiating the innate immune response. Different evolutionary histories of pollinators and non-pollinators may result in different immune recognition systems. A previous study had reported that there were significant differences in peptidoglycan recognition proteins (PGRPs) between pollinators and non-pollinators in gene number and lineage of specific genes. In this study, based on the genomic data of 12 fig wasp species, with seven pollinators and five non-pollinators, we investigated the evolution patterns of PRRs, such as Gram-negative bacteria-binding proteins (GNBPs), C-type lectins (CTLs), scavenger receptors class B (SCRBs), fibrinogen-related proteins (FREPs), galectins, and thioester-containing proteins (TEPs). Our results showed that pollinators had no GNBP, but non-pollinators all had two gene members, which were clustered into two different clades in the phylogenetic tree, with each clade having specific domain and motif characteristics. The analysis of CTL and SCRB gene families also showed that there were lineage-specific genes and specific expansion in non-pollinators. Our results showed that there were significant differences in immune recognition between pollinators and non-pollinators, and we concluded that they had undergone flexible adaptive evolution in different environments. Our study can provide more molecular evidence for future functional studies on the immune system of fig wasps.

A Cretaceous bug with exaggerated antennae might be a double-edged sword in evolution.

Sexual selection can favor production of exaggerated features, but the high cost of such features in terms of energy consumption and enemy avoidance makes them go to extinction under the influence of natural selection. However, fossils preserved with specialized features are very rare. Here, we report a new nymph from Burmese amber, Magnusantena wuae Du & Chen gen. et sp. nov., which has exaggerated leaf-like expanded antennae. Such bizarre antennae indicate that sensitive and delicate sensory system and magnificent appearance in Hemiptera have been already established in mid-Cretaceous. Our findings may provide evidence for Darwin's view that sensory organs play an important role in sexual selection. This nymph with the leaf-like antennae may also represents a new camouflage pattern. However, the oversized antennae are costly to develop and maintain, increasing the risks from predators. Such unparalleled expanded antennae might be the key factor for the evolutionary fate of the coreid.

Distribution and Evolution of the Bacteriophage WO and Its Antagonism With Wolbachia.

The symbiosis system comprising eukaryotic hosts, intracellular bacterium Wolbachia, and temperate bacteriophages WO is widely spread through nearly half the number of arthropod species. The relationships between the three components of the system are extremely intricate. Even though the bacteriophage WO can have diverse influences on the ecology and evolution of Wolbachia, little is known about the distribution and evolution of the phages. To the best of our knowledge, this study is the first to report that in infected fig wasps (Ceratosolen solmsi, Kradibia gibbosae, and Wiebesia pumilae), the genomes of all the Wolbachia strains had only one cryptic WO prophage, which contained defects in the genomic structural modules. This phenomenon was contrary to the widely accepted understanding that Wolbachia with cryptic prophages usually possesses at least one intact WO prophage consisting of gene sequences of the head, baseplate, and tail modules, through which the prophage could form intact virions. In addition to the genetic structure features, the phylogenetic relationships of WO and Wolbachia also revealed that bacteriophage WO can horizontally spread among a certain genus or a group of insect hosts, nearly free from the restriction of the affiliation of Wolbachia. Combined with the vertical transmission along with Wolbachia, the wide spread of WO phages can be explained. Furthermore, the gender preference and functional module preference for transcriptional activity of the genes in cryptic WOs implied the antagonized coevolutionary pattern between WO prophages and their Wolbachia hosts.

Tracking the Distribution and Burst of Nuclear Mitochondrial DNA Sequences (NUMTs) in Fig Wasp Genomes.

Mitochondrial DNA sequences can be transferred into the nuclear genome, giving rise to nuclear mitochondrial DNA sequences (NUMTs). NUMTs have been described in numerous eukaryotes. However, the studies on the distribution of NUMTs and its influencing factors are still inadequate and even controversial. Previous studies have suggested that Hymenoptera may be a group rich in NUMTs, in which we selected 11 species of fig wasps (Chalcidoidea, Hymenoptera) to analyze the distribution and evolution of NUMTs at the genomic level. The results showed that the contents of NUMTs varied greatly in these species, and bursts of NUMTs existed in some species or lineages. Further detailed analyses showed that the large number of NUMTs might be related to the large genomes; NUMTs tended to be inserted into unstable regions of the genomes; and the inserted NUMTs might also be affected by transposable elements (TEs) in the neighbors, leading to fragmentations and duplications, followed by bursts of NUMTs. In summary, our results suggest that a variety of genomic environmental factors can determine the insertion and post-insertion fate of NUMTs, resulting in their species- or lineage-specific distribution patterns, and that studying the evolution of NUMTs can provide good evidence and theoretical basis for exploring the dynamics of exogenous DNA entering into the nuclear genome.

Transcriptome analysis of the male polymorphisms of fig wasp species Philotrypesis tridentata.

Intraspecific male polymorphism exhibiting extreme differences in morphology, behavior and life history presents good opportunities to explore adaptation mechanisms to different environments. In this study, we examined the transcriptomic differences between wingless and winged morphs of a fig wasp species Philotrypesis tridentata to investigate molecular basis to maintain polymorphisms. The winged male adults fly outside fig syconia to mate, while the wingless only stay and mate inside fig syconia where they have developed. We identified 2,391 differentially expressed genes (DEGs) with 1,396 highly expressed in winged morphs and 995 in wingless morphs. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses on the DEGs and differential alternative splicing genes and analyzed the top ten DEGs with the highest differential expression in each morph. The results showed that genes related to biosynthesis processes, lipid metabolism, energy production, flight and defense of the complex environments outside fig syconia were up-regulated in winged morphs. Genes involved in substance and energy metabolism and chemical reception were up-regulated in wingless morphs which might relate to their living inside fig syconia. The differences in highly expressed genes between two morphs prove adaptation of P. tridentata male polymorphism to different living environments.

Genome-Wide Analysis of Peptidoglycan Recognition Protein Genes in Fig Wasps (Hymenoptera, Chalcidoidea).

The innate immunity is the most important defense against pathogen of insects, and the peptidoglycan recognition proteins (PGRPs) play an important role in the processes of immune recognition and initiation of Toll, IMD and other signal pathways. In fig wasps, pollinators and non-pollinators present different evolutionary histories and lifestyles, even though both are closely associated with fig syconia, which may indicate their different patterns in the evolution of PGRPs. By manual annotation, we got all the PGRP genes of 12 fig wasp species, containing seven pollinators and five non-pollinators, and investigated their putative different evolutionary patterns. We found that the number of PGRP genes in pollinators was significantly lower than in non-pollinators, and the number of catalytic PGRP presented a declining trend in pollinators. More importantly, PGRP-SA is associated with initiating the Toll pathway, as well as gram-negative bacteria-binding proteins (GNBPs), which were completely lost in pollinators, which led us to speculate that the initiation of Toll pathway was simpler in pollinators than in non-pollinators. We concluded that fig pollinators owned a more streamlined innate immune recognition system than non-pollinators. Our results provide molecular evidence for the adaptive evolution of innate immunity in insects of host specificity.

Author Correction: The first amber caridean shrimp from Mexico reveals the ancient adaptation of the Palaemon to the mangrove estuary environment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.