Rapid spread of a vertically transmitted symbiont induces drastic shifts in butterfly sex ratio.

The causes and consequences of sex-ratio dynamics constitutes a pivotal subject in evolutionary biology1. Under conditions of evolutionary equilibrium, the male-to-female ratio tends to be approximately 1:1; however, this equilibrium is susceptible to distortion by selfish genetic elements exemplified by driving sex chromosomes and cytoplasmic elements2,3. Although previous studies have documented instances of these genetic elements distorting the sex ratio, studies specifically tracking the process with which these distorters spread within populations, leading to a transition from balanced parity to a skewed, female-biased state, are notably lacking. Herein, we present compelling evidence documenting the rapid spread of the cytoplasmic endosymbiont Wolbachia within a localized population of the pierid butterfly Eurema hecabe (Figure 1A). This spread resulted in a shift in the sex ratio from near parity to an exceedingly skewed state overwhelmingly biased toward females, reaching 93.1% within a remarkably brief period of 4 years.

Genomic insights into Spiroplasma endosymbionts that induce male-killing and protective phenotypes in the pea aphid.

The endosymbiotic bacteria Spiroplasma (Mollicutes) infect diverse plants and arthropods, and some of which induce male killing, where male hosts are killed during development. Male-killing Spiroplasma strains belong to either the phylogenetically distant Citri-Poulsonii or Ixodetis groups. In Drosophila flies, Spiroplasma poulsonii induces male killing via the Spaid toxin. While Spiroplasma ixodetis infects a wide range of insects and arachnids, little is known about the genetic basis of S. ixodetis-induced male killing. Here, we analyzed the genome of S. ixodetis strains in the pea aphid Acyrthosiphon pisum (Aphididae, Hemiptera). Genome sequencing constructed a complete genome of a male-killing strain, sAp269, consisting of a 1.5 Mb circular chromosome and an 80 Kb plasmid. sAp269 encoded putative virulence factors containing either ankyrin repeat, ovarian tumor-like deubiquitinase, or ribosome inactivating protein domains, but lacked the Spaid toxin. Further comparative genomics of Spiroplasma strains in A. pisum biotypes adapted to different host plants revealed their phylogenetic associations and the diversity of putative virulence factors. Although the mechanisms of S. ixodetis-induced male killing in pea aphids remain elusive, this study underlines the dynamic genome evolution of S. ixodetis and proposes independent acquisition events of male-killing mechanisms in insects.

Pectoralis Major Muscle Flap Utilization in Salvage Esophagectomy Including Great Vessel Resection Reconstructed by Prosthetic Grafts.

BACKGROUND: Salvage surgery is a therapeutic option for recurrent or residual esophageal cancer after definitive chemoradiation therapy. This report aimed to describe the procedure of reconstruction after salvage esophagectomy involving great vessel resection using prosthetic grafts, a pectoralis major muscle (PM) flap, and free jejunal transfer, if required. To the best of our knowledge, no previous report has described the reconstruction of the defect after combined esophageal and great vessel resection. PATIENTS AND METHODS: From January 2017 to December 2022, 4 patients underwent salvage esophagectomy with excision of the great vessels and reconstruction with prosthetic grafts, as well as a PM flap placement in a single center. We retrospectively investigated the patients' clinical data. The patients were all men, with a median age of 70 (range, 67-77) years. Regarding neoadjuvant therapy, 2 patients received chemoradiation therapy, 1 patient received radiotherapy only due to drug-induced pneumonia, and 1 patient received chemotherapy with adjuvant radiotherapy. RESULTS: Alimentary tract reconstruction was performed by free jejunal transfer in 2 cases, direct suture in 1 case, and stomach roll in 1 case. In all cases, a vascular bypass was established before tumor resection. We created mediastinal tracheostoma in 2 cases. A PM flap was inserted to cover the prosthetic grafts and approximate the tracheal mucosa. With regard to major complications, leakage from the jejunal esophageal anastomotic site was observed in 2 cases. The leakage improved with conservative treatment without graft removal or replacement in both cases. CONCLUSIONS: In cases of locally recurrent or residual tumors after definitive chemoradiation therapy, salvage esophagectomy along with great vessel resection, followed by reconstruction using prosthetic grafts, PM flaps, and free jejunal transfer, if necessary, is a useful option.

The Ischemic Tolerance up to Four Hours of Free Jejunum Flap: A Retrospective Cohort Study.

BACKGROUND: While free jejunum transfer (FJT) following total pharyngo-laryngo-esophagectomy (TPLE) is a reliable reconstruction technique, the jejunum flap is viewed as more susceptible to ischemia than a standard free flap. Animal studies have indicated that the jejunum can tolerate ischemia for as little as 2 to 3 hours. Clinical studies also reported increased complications after the FJT with more than 3 hours of ischemia. Traditionally, our institution has carried out FJT with an initial intestinal anastomosis, followed by a vascular anastomosis, which often results in extended jejunal ischemia time. In this study, we retrospectively examined the actual tolerance of the jejunum to ischemia, considering perioperative complications and postoperative dysphagia. METHODS: We retrospectively studied 402 consecutive cases involving TPLE + FJT. Patients were divided into five groups based on jejunum ischemia time (∼119 minutes, 120∼149 minutes, 150∼179 minutes, 180∼209 minutes, 210 minutes∼), with each variable and result item compared between the groups. Univariate and multivariate analyses were conducted to identify independent factors influencing the four results: three perioperative complications (pedicle thrombosis, anastomotic leak, surgical site infection) and dysphagia at 6 months postoperatively. RESULTS: The mean jejunal ischemia time was 164.6 ± 28.4 (90-259) minutes. When comparing groups divided by jejunal ischemia time, we found no significant differences in overall outcomes or complications. Our multivariate analyses indicated that jejunal ischemia time did not impact the three perioperative complications and postoperative dysphagia. CONCLUSION: In TPLE + FJT, a jejunal ischemia time of up to 4 hours had no effect on perioperative complications or postoperative dysphagia. The TPLE + FJT technique, involving a jejunal anastomosis first followed by vascular anastomosis, benefits from an easier jejunal anastomosis but suffers from a longer jejunal ischemia time. However, we found that ischemia time does not pose significant problems, although we have not evaluated the effects of jejunal ischemia extending beyond 4 hours.

Two male-killing Wolbachia from Drosophila birauraia that are closely related but distinct in genome structure.

Insects harbour diverse maternally inherited bacteria and viruses, some of which have evolved to kill the male progeny of their hosts (male killing: MK). The fly species Drosophila biauraria carries a maternally transmitted MK-inducing partiti-like virus, but it was unknown if it carries other MK-inducing endosymbionts. Here, we identified two male-killing Wolbachia strains (wBiau1 and wBiau2) from D. biauraria and compared their genomes to elucidate their evolutionary processes. The two strains were genetically closely related but had exceptionally different genome structures with considerable rearrangements compared with combinations of other Wolbachia strains. Despite substantial changes in the genome structure, the two Wolbachia strains did not experience gene losses that would disrupt the male-killing expression or persistence in the host population. The two Wolbachia-infected matrilines carried distinct mitochondrial haplotypes, suggesting that wBiau1 and wBiau2 have invaded D. biauraria independently and undergone considerable genome changes owing to unknown selective pressures in evolutionary history. This study demonstrated the presence of three male-killers from two distinct origins in one fly species and highlighted the diverse and rapid genome evolution of MK Wolbachia in the host.

Male-killing virus in a noctuid moth Spodoptera litura.

A female-biased sex ratio is considered advantageous for the cytoplasmic elements that inhabit sexually reproducing organisms. There are numerous examples of bacterial symbionts in the arthropod cytoplasm that bias the host sex ratio toward females through various means, including feminization and male killing. Recently, maternally inherited RNA viruses belonging to the family Partitiviridae were found to cause male killing in moths and flies, but it was unknown whether male-killing viruses were restricted to Partitiviridae or could be found in other taxa. Here, we provide compelling evidence that a maternally inherited RNA virus, Spodoptera litura male-killing virus (SlMKV), selectively kills male embryos of the tobacco caterpillar Spodoptera litura, resulting in all-female broods. SlMKV injected into uninfected S. litura can also be inherited maternally and causes male killing. SlMKV has five genomic segments encoding seven open reading frames, has no homolog of known male-killing genes, and belongs to an unclassified group of arthropod-specific viruses closely related to Tolivirales. When transinfected into larvae, both male and female recipients allow SlMKV to proliferate, but only males die at the pupal stage. The viral RNA levels in embryonic and pupal male killing suggest that the mechanism of male killing involves the constitutive expression of viral products that are specifically lethal to males, rather than the male-specific expression of viral products. Our results, together with recent findings on male-killing partiti-like viruses, suggest that diverse viruses in arthropods tend to acquire male killing independently and that such viruses may be important components of intragenomic conflict in arthropods.

Microbiome of Zoophytophagous Biological Control Agent Nesidiocoris tenuis.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

GENETIC VARIATION OF LEPTOTROMBIDIUM (ACARI: TROMBICULIDAE) MITES CARRYING ORIENTIA TSUTSUGAMUSHI, THE BACTERIAL PATHOGEN CAUSING SCRUB TYPHUS.

Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626-0.1732, whereas the distances within each subcluster were 0.003-0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010-0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality.

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

Soft Tissue Reconstruction Using the Free Forearm Fillet Flap in the Salvage of Forequarter Amputations for Recurrent Sarcoma: A Report of 2 Cases.

ABSTRACT: Reconstruction after radical forequarter amputation for recurrent proximal extremity sarcoma is challenging because the defect is significant, and the axillary or subclavian vessels are resected with the tumor, indicating that the pedicle of nearby flaps is often cut off. Free flaps are commonly used to cover the defect, but the donor site morbidity is problematic. Another problem with resecting the axillary or subclavian vessels is difficulty obtaining recipient vessels with matching calibers for another free flap. The authors presented 2 cases that successfully solved all these problems by covering the defects by the forearm fillet flaps.The forearm fillet flap has the advantage of using a portion that is usually discarded, so there is no donor site morbidity. Moreover, using the brachial artery as the pedicle of the flap allows the anastomosis to the stump of the resected axillary or subclavian artery because differences in caliber are slight.The use of the forearm fillet flaps has been reported less frequently than the fillet flaps in the lower extremities, and most cases are posttraumatic. In cases of trauma, complications are reported to occur in about 1 in 4 patients, but in cases after tumor resection, ischemic time can be controlled, and there are no risks of contamination or unnoticed forearm damage, so more stable results can be expected as in this report.Using the free flap from the distal portion of the resected upper limb, especially in cases with a short prognosis, is a viable option that effectively minimizes damage to the donor site and maintains quality of life.

A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

BCOR-CCNB3 sarcoma arising in the pharynx.

Reports on BCOR-CCNB3 sarcoma in the head and neck region are scarce, given their unknown etiology. An 18-year-old male patient presented a rapidly enlarging tumor extending from the right nasopharynx to the oropharynx. Histological examination showed a spindle cell sarcoma with BCOR-CCNB3 fusion detected by fluorescence in situ hybridization, and BCOR-CCNB3 was diagnosed. After three courses of alternating VDC-IE therapy, the patient underwent tumor resection based on the original tumor range with a minimal margin, using the mandibular swing technique. Radiation therapy (50.4 Gy) was administered postoperatively, followed by three additional courses of alternating VDC-IE therapy. The patient survived and showed no evidence of disease at 12 months postoperatively. BCOR-CCNB3 sarcoma is a chemotherapy-sensitive sarcoma, and conservative resection with a minimal margin that does not interfere with the treatment flow is preferable.

Male-killing mechanisms vary between Spiroplasma species.

Male-killing, a male-specific death of arthropod hosts during development, is induced by Spiroplasma (Mollicutes) endosymbionts of the Citri-Poulsonii and the Ixodetis groups, which are phylogenetically distant groups. Spiroplasma poulsonii induces male-killing in Drosophila melanogaster (Diptera) using the Spaid toxin that harbors ankyrin repeats, whereas little is known about the origin and mechanisms of male-killing induced by Spiroplasma ixodetis. Here, we analyzed the genome and the biological characteristics of a male-killing S. ixodetis strain sHm in the moth Homona magnanima (Tortricidae, Lepidoptera). Strain sHm harbored a 2.1 Mb chromosome and two potential plasmids encoding Type IV effectors, putatively involved in virulence and host-symbiont interactions. Moreover, sHm did not harbor the spaid gene but harbored 10 ankyrin genes that were homologous to those in other S. ixodetis strains. In contrast to the predominant existence of S. poulsonii in hemolymph, our quantitative PCR assays revealed a systemic distribution of strain sHm in H. magnanima, with particularly high titers in Malpighian tubules but low titers in hemolymph. Furthermore, transinfection assays confirmed that strain sHm can infect cultured cells derived from distantly related insects, namely Aedes albopictus (Diptera) and Bombyx mori (Lepidoptera). These results suggest different origins and characteristics of S. ixodetis- and S. poulsonii-induced male-killing.

Genetic variations and microbiome of the poultry red mite Dermanyssus gallinae.

The poultry red mite Dermanyssus gallinae poses a significant threat to the health of hens and poultry production. A comprehensive understanding of D. gallinae is necessary to develop sustainable and efficacious control methods. Here we examined 144 D. gallinae collected from 18 poultry farms throughout the Japanese Archipelago for their genetic variations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences, and microbiome variations based on amplicon sequencing of the 16S ribosomal RNA gene. According to COI sequencing, the Japanese samples were categorized into three haplogroups, which did not reflect the geographical distribution. Microbiome analyses found that the major bacteria associated with D. gallinae were Bartonella, Cardinium, Wolbachia, and Tsukamurella, with Bartonella being most predominant. Among 144 individual mites, all possessed one of the two major types of Bartonella (Bartonella sp. A), while 140 mites possessed the other type (Bartonella sp. B). The presence of the two strains of Bartonella was also confirmed by a single copy gene, rpoB. The presence of Bartonella in laid eggs suggested transovarial vertical transmission. Given that obligate blood-feeding arthropods generally require a supply of B vitamins from symbiotic bacteria, Bartonella may play an important role in mite survival. Rickettsiella, a major symbiont in European D. gallinae populations, and suggested to be an important symbiont by genomic data, was rarely found in Japanese populations. Cardinium detected from D. gallinae fell into a major clade found widely in arthropods, whereas Wolbachia detected in Japanese D. gallinae appear to be a new lineage, located at the base of Wolbachia phylogeny. Of the mitochondrial phylogeny, infection patterns of Cardinium and Wolbachia were strongly correlated, possibly suggesting one or both of the symbionts induce reproductive manipulations and increase spread in the host populations.

A new antimicrobial peptide, Pentatomicin, from the stinkbug Plautia stali.

Antimicrobial peptides (AMPs) play crucial roles in the innate immunity of diverse organisms, which exhibit remarkable diversity in size, structural property and antimicrobial spectrum. Here, we describe a new AMP, named Pentatomicin, from the stinkbug Plautia stali (Hemiptera: Pentatomidae). Orthologous nucleotide sequences of Pentatomicin were present in stinkbugs and beetles but not in other insect groups. Notably, orthologous sequences were also detected from a horseshoe crab, cyanobacteria and proteobacteria, suggesting the possibility of inter-domain horizontal gene transfers of Pentatomicin and allied protein genes. The recombinant protein of Pentatomicin was effective against an array of Gram-positive bacteria but not against Gram-negative bacteria. Upon septic shock, the expression of Pentatomicin drastically increased in a manner similar to other AMPs. On the other hand, unlike other AMPs, mock and saline injections increased the expression of Pentatomicin. RNAi-mediated downregulation of Imd pathway genes (Imd and Relish) and Toll pathway genes (MyD88 and Dorsal) revealed that the expression of Pentatomicin is under the control of Toll pathway. Being consistent with in vitro effectiveness of the recombinant protein, adult insects injected with dsRNA of Pentatomicin exhibited higher vulnerability to Gram-positive Staphylococcus aureus than to Gram-negative Escherichia coli. We discovered high levels of Pentatomicin expression in eggs, which is atypical of other AMPs and suggestive of its biological functioning in eggs. Contrary to the expectation, however, RNAi-mediated downregulation of Pentatomicin did not affect normal embryonic development of P. stali. Moreover, the downregulation of Pentatomicin in eggs did not affect vertical symbiont transmission to the offspring even under heavily contaminated conditions, which refuted our expectation that the antimicrobial activity of Pentatomicin may contribute to egg surface-mediated symbiont transmission by suppressing microbial contaminants.

Sex determination systems as the interface between male-killing bacteria and their hosts.

Arthropods host a range of sex-ratio-distorting selfish elements, including diverse maternally inherited endosymbionts that solely kill infected males. Male-killing heritable microbes are common, reach high frequency, but until recently have been poorly understood in terms of the host-microbe interaction. Additionally, while male killing should generate strong selection for host resistance, evidence of this has been scant. The interface of the microbe with host sex determination is integral to the understanding of how death is sex limited and how hosts can evolve evasion of male killing. We first review current knowledge of the mechanisms diverse endosymbionts use to induce male-specific death. We then examine recent evidence that these agents do produce intense selection for host nuclear suppressor elements. We argue, from our understanding of male-killing mechanisms, that suppression will commonly involve evolution of the host sex determination pathways and that the host's response to male-killing microbes thus represents an unrecognized driver of the diversity of arthropod sex determination. Further work is required to identify the genes and mechanisms responsible for male-killing suppression, which will both determine the components of sex determination (or other) systems associated with suppressor evolution, and allow insight into the mechanism of male killing itself.

Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali.

Development of a reliable method for RNA interference (RNAi) by orally-delivered double-stranded RNA (dsRNA) is potentially promising for crop protection. Considering that RNAi efficiency considerably varies among different insect species, it is important to seek for the practical conditions under which dsRNA-mediated RNAi effectively works against each pest insect. Here we investigated RNAi efficiency in the brown-winged green stinkbug Plautia stali, which is notorious for infesting various fruits and crop plants. Microinjection of dsRNA into P. stali revealed high RNAi efficiency-injection of only 30 ng dsRNA into last-instar nymphs was sufficient to knockdown target genes as manifested by their phenotypes, and injection of 300 ng dsRNA suppressed the gene expression levels by 80% to 99.9%. Knockdown experiments by dsRNA injection showed that multicopper oxidase 2 (MCO2), vacuolar ATPase (vATPase), inhibitor of apoptosis (IAP), and vacuolar-sorting protein Snf7 are essential for survival of P. stali, as has been demonstrated in other insects. By contrast, P. stali exhibited very low RNAi efficiency when dsRNA was orally administered. When 1000 ng/µL of dsRNA solution was orally provided to first-instar nymphs, no obvious phenotypes were observed. Consistent with this, RT-qPCR showed that the gene expression levels were not affected. A higher concentration of dsRNA (5000 ng/µL) induced mortality in some cohorts, and the gene expression levels were reduced to nearly 50%. Simultaneous oral administration of dsRNA against potential RNAi blocker genes did not improve the RNAi efficiency of the target genes. In conclusion, P. stali shows high sensitivity to RNAi with injected dsRNA but, unlike the allied pest stinkbugs Halyomorpha halys and Nezara viridula, very low sensitivity to RNAi with orally-delivered dsRNA, which highlights the varied sensitivity to RNAi across different species and limits the applicability of the molecular tool for controlling this specific insect pest.

Combined use of ipsilateral latissimus dorsi flap and anterolateral thigh flap to reconstruct extensive trunk defects.

Reconstruction of a full-thickness trunk defect is challenging because of the complex nature of such defects, which include the chest wall, abdominal wall, and diaphragm. We herein describe three patients in whom extensive trunk defects after sarcoma resection were reconstructed with a latissimus dorsi flap and an anterolateral thigh flap. In two patients, the defect included both the chest wall and the abdominal wall. The other patient had an extensive full-thickness chest wall defect. The size of the anterolateral thigh flap for each patient was 34 × 10 cm, 26 × 15 cm, and 23 × 5 cm, respectively. Although one patient required take-back for additional venous drainage, all wounds healed with no other complications. No respiratory dysfunction or abdominal wall hernia occurred in any patients. The combined use of a latissimus dorsi flap and an anterolateral thigh flap may provide reliable coverage of an extensive trunk defect and robust support of the chest and abdominal walls. Additionally, the availability of a two-team approach without a positional change makes this combination a versatile reconstructive option.

Wolbachia have made it twice: Hybrid introgression between two sister species of Eurema butterflies.

Wolbachia, cytoplasmically inherited endosymbionts of arthropods, are known to hijack their host reproduction in various ways to increase their own vertical transmission. This may lead to the selective sweep of associated mitochondria, which can have a large impact on the evolution of mitochondrial lineages. In Japan, two different Wolbacahia strains (wCI and wFem) are found in two sister species of pierid butterflies, Eurema mandarina and Eurema hecabe. In both species, females infected with wCI (C females) produce offspring with a nearly 1:1 sex ratio, while females infected with both wCI and wFem (CF females) produce all-female offspring. Previous studies have suggested the historical occurrence of hybrid introgression in C individuals between the two species. Furthermore, hybrid introgression in CF individuals is suggested by the distinct mitochondrial lineages between C females and CF females of E. mandarina. In this study, we performed phylogenetic analyses based on nuclear DNA and mitochondrial DNA markers of E. hecabe with previously published data on E. mandarina. We found that the nuclear DNA of this species significantly diverged from that of E. mandarina. By contrast, mitochondrial DNA haplotypes comprised two clades, mostly reflecting Wolbachia infection status rather than the individual species. Collectively, our results support the previously suggested occurrence of two independent historical events wherein the cytoplasms of CF females and C females moved between E. hecabe and E. mandarina through hybrid introgression.