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.

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.

FNAIT pathogenesis determined by serial analysis of three subsequent pregnancies of a woman with severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) with anti-HPA-4b and anti-HPA-5b alloantibodies in the first sibling.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by anti-HPA alloantibody, and anti-HPA-4b is the most common cause in Japanese. Anti-HPA-5b is frequently detected in pregnant women, but it is still controversial whether anti-HPA-5b causes severe FNAIT. CASE PRESENTATION: A Japanese woman with anti-HPA-4b and anti-HPA-5b alloantibodies delivered a baby with severe FNAIT who was both HPA-4b and HPA-5b incompatible. We carefully monitored the patient's following three pregnancies (the second and the fourth siblings were HPA-4b incompatible and HPA-5b compatible; the third sibling was both HPA-4b and HPA-5b compatible). FNAIT was not observed in all three siblings, although a modest decrease in cord blood platelet count was observed in the HPA-4b incompatible siblings compared to the HPA-4b compatible sibling. Serial monitoring of anti-HPA titer showed that anti-HPA-4b markedly decreased in late pregnancy and recovered after delivery of the HPA-4b incompatible siblings, but these decreases were not observed during the mother's pregnancy with the HPA-4b compatible sibling. In contrast, anti-HPA-5b remained at a high titer during pregnancy with all three siblings. CONCLUSION: Our data indicate that dynamic changes of anti-HPA-4b occur during pregnancy and strongly suggest that anti-HPA-5b was mainly responsible for severe FNAIT in this case.

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.

Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library.

Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

Microhabitat Variation in Egg Diapause Incidence in Summer Within a Local Population: An Adaptation to Decline in Host-Plant Suitability in Trigonotylus caelestialium (Hemiptera: Miridae).

Diapause induction in multivoltine insects is an ecophysiological event that is generally triggered by seasonal cues such as photoperiod and temperature. The rice leaf bug, Trigonotylus caelestialium (Kirkaldy), feeds on various Poaceae grasses and produces several generations a year. Previous studies have shown that adults produce diapause and nondiapause eggs under short-day and long-day conditions, respectively. However, there is a distinct mid-summer peak in diapause incidence before an autumnal increase in diapause incidence in the field, which cannot be explained by the laboratory results. The present study was performed to examine the environmental factors affecting the diapause incidence in mid-summer and the adaptive significance of this phenomenon. Seasonal trends in diapause incidence differed significantly among three sites located 150-400 m apart from each other and with different host plants. The suitability of host plants differs depending on species and seasonally. Therefore, the microhabitat difference in diapause trend is believed to be due to the difference in host plants. When field-collected female adults laying diapause eggs in late June were fed a seasonally deteriorating host (the orange foxtail, Alopecurus aequalis Sobol. [Poales: Poaceae]), they kept laying diapause eggs, whereas when fed a suitable host (the wheat, Triticum aestivum L. [Poales: Poaceae]) for 5 d, they changed oviposition mode to lay nondiapause eggs. These results indicate that host-plant suitability affects the oviposition mode of T. caelestialium. Diapause-egg oviposition in mid-summer in T. caelestialium has adaptive significance as a bet-hedging strategy against unpredictable dietary conditions.

Life-History Traits of a Subtropical Cerambycid Beetle, Ropica honesta (Coleoptera: Cerambycidae).

Ropica honesta Pascoe is a small-sized cerambycid that has been recorded in tropical to subtropical Asia. In this study, life-history traits were examined for a local population collected from Iriomote Island (24.3°N, 123.8°E), Okinawa, Japan, by rearing insects on artificial diet as larval food. The egg period was 5.9 ± 0.3 d at 25°C. There was no significant difference in the duration of the larval, pupal, and adult preoviposition periods between long-day (14:10 [L:D]) and short-day (12:12 [L:D]) photoperiods at both 20 and 25°C. These periods at 25°C (14:10 [L:D]) were 28.5 ± 1.4, 8.4 ± 0.5, and 9.6 ± 1.9 d (mean ± SD), respectively. The relationship between the developmental rate and temperature followed the law of total effective temperature; thus, the developmental threshold temperature and thermal constant were estimated based on these data. Together with the finding that R. honesta may not have diapause in the egg stage, it is suggested that this beetle does not have diapause in the life cycle. Furthermore, when larvae were reared on natural food (dead twigs of hardwoods) adults emerged from the twigs 47.6 ± 2.9 d after oviposition, and this value was close to the total duration of the egg to pupal periods. Together with the data for annual temperature of the habitat and the fact that food resources for the species (dead twigs and leaves of hardwoods) are available throughout the year, we conclude that R. honesta develops and reproduces all year round, with five generations at maximum.

Rearing Theretra oldenlandiae (Lepidoptera: Sphingidae) Larvae on an Artificial Diet.

The hawk moth Theretra oldenlandiae (Fabricius) is an important insect pest because in the larval stage it feeds on agricultural crops and ornamental plants such as the eddoe and garden balsam. In this study, we established methods for rearing T. oldenlandiae in the laboratory using an artificial diet containing dry powder of a wild grass Cayratia japonica (Thunb.) Gagnep. Several artificial diets were tested with different ratios of a commercial diet, Insecta LFM, and the dry leaf powder, and including different antibiotics, and the composition of the standard diet on which larvae performed best was determined. The standard diet contains 20 g of Insecta LFM, 4 g of leaf powder, 100 ml of water, 75 mg of chloramphenicol, and 200 µl of propionic acid. Larvae reared on the standard diet became larger pupae than those reared on C. japonica leaves. This result suggests that the larvae have growth potential that is masked on C. japonica leaves, and that C. japonica may not be the most suitable host species for T. oldenlandiae larvae in terms of nutrient level.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method).

BACKGROUND: There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive. STUDY DESIGN AND METHODS: To develop a new method to negate the DARA interference, the effects of various concentrations of dithiothreitol (DTT) on RBC CD38 and Kell antigenicity in combination with an automatic blood cell washing centrifuge were compared with the AABB standard procedure in parallel. Moreover, direct antiglobulin tests (DATs) for RBCs in DARA-treated MM patients were examined. RESULTS: A quantity of 0.01 mol/L DTT as well as the AABB procedure (equivalent to 0.15 mol/L DTT in our procedure) markedly reduced the reactivity of phycoerythrin-mouse anti-CD38 MoAb and DARA with RBCs. In sharp contrast to the AABB procedure, 0.01 mol/L DTT partially preserved K antigenicity and allowed the determination of phenotype of K antigen even in the presence of the DARA interference. In contrast, DAT for RBCs obtained from MM patients showed a weak positive or negative reaction. Immunoblotting further indicated that DARA induced loss of CD38 in vivo. CONCLUSION: A simple and reliable method to negate the DARA interference with partially preserving Kell antigenicity is proposed (Osaka method). CD38 antigenicity is susceptible to 0.01 mol/L DTT treatment even in the presence of DARA. Our data also demonstrate distinct effects of DARA on IAT in vitro and DAT in vivo.

Immature platelet fraction (IPF) as a predictive value for thrombopoietic recovery after allogeneic stem cell transplantation.

We consecutively examined the utility of measurements of percentage of immature platelet fraction (IPF%) and absolute IPF number (A-IPF) in predicting thrombopoietic recovery in 15 adult patients who underwent allogeneic hematopoietic stem cell transplantation (allo-SCT). Four patients were excluded from the evaluation due to insufficient data. Platelet count and IPF were measured by Sysmex XN-1000 (XN), a newer generation analyzer. First, we confirmed that platelet count measured by XN was more accurate than by XE-2100 (XE). IPF measurement was effective to predict the recovery in 7 of the 11 patients examined. Moreover, IPF measurement, especially IPF% measurement, suggested accelerated platelet turnover in two patients who failed to achieve platelet recovery by day 60. In addition to IPF%, A-IPF showed a complementary role on the prediction of thrombopoietic recovery. The increase in IPF% was only transient, while A-IPF values showed lasting increase during platelet recovery. In two patients (cases 6 and 7) an increase in A-IPF, but not in IPF%, was observed during platelet recovery. Our data suggest that IPF% and A-IPF measured by XN are useful for the prediction of thrombopoietic recovery and the assessment of pathogenesis of thrombocytopenia in patients after allo-SCT.

Severe Disseminated Mycobacterium avium Infection in a Patient with a Positive Serum Autoantibody to Interferon-γ.

We herein report a case of disseminated Mycobacterium avium infection that involved both optic nerves, the conjunctiva, the right lower lung, and multiple skin lesions, including a thoracic nodule. The patient was a 65-year-old man without any significant medical history. The pathogen was detected in the patient's eye discharge, sputum, bronchial lavage fluid, and thoracic nodule. Anti-mycobacterial chemotherapy, including clarithromycin, rifampicin, and ethambutol, was administered, and the thoracic nodule was resected. An autoantibody to interferon-γ was detected in the patient's serum. Bilateral swelling of his optic nerves and facial dermatitis improved after initiating anti-mycobacterial chemotherapy.

Insights into How Longicorn Beetle Larvae Determine the Timing of Metamorphosis: Starvation-Induced Mechanism Revisited.

Larvae of holometabolous insects must determine the timing of their metamorphosis. How they determine this timing has only been studied in detail for a few insect species. In a few species of Coleoptera, starvation is known to be a cue for metamorphosis, leading to the formation of smaller adults (starvation-induced pupation, SiP). We investigated the occurrence of SiP in the beetle Psacothea hilaris. When P. hilaris larvae were starved late in the feeding phase of the last (5th) instar, they exhibited typical SiP characterized by constancy of the time from food deprivation to pupation (TTP) irrespective of the body weight upon food deprivation or the length of prior feeding. In contrast, when larvae were starved early in the feeding phase, TTP decreased by roughly 1 day as the feeding became 1 day longer. The change in the response to starvation was estimated to occur on day 5.9 in the last instar. A series of refeeding experiments suggested that whereas SiP occurred readily in the larvae starved in the late feeding phase, activation of SiP was suspended until day 5.9 in the larvae starved early in the feeding phase. When P. hilaris larvae were fed continuously, they eventually ceased feeding spontaneously and pupated. The time length between spontaneous cessation of feeding and pupation was approximately equal to the TTP in SiP. This suggests that the same mechanism was activated by food deprivation in the late feeding phase and by spontaneous cessation of ad libitum feeding.

[A Case of Brucellosis with Intermittent Fever in a Patient Returning from France].

We herein report on a 62-year-old man who presented with symptoms of intermittent fever that persisted after returning from a trip to France. During his trip, he had eaten natural cheese. Although no bacteria could be isolated from blood culture, the serum agglutination test showed a positive antibody titer of 1 : 160 for Brucella canis. The patient responded well to combination antibiotic therapy consisting of gentamicin, rifampicin, and doxycycline, and his symptoms improved. He became antibody-negative after antibiotic therapy. Although the present case may have been a case of B. canis infection, considering the epidemiology of brucellosis in France, serological cross-reactivity with Brucella melitensis infection is also a possibility. Concerns regarding the reemergence of brucellosis have recently been reported in France, and most cases are caused by B. melitensis. Clinicians should be aware of the fact that blood cultures must be incubated for ≥ 21 days for isolation of Brucella and that in Japan, antibody measurement of B. melitensis cannot be performed on a commercial basis.

Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene.

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.

Clinical significance of IPF% or RP% measurement in distinguishing primary immune thrombocytopenia from aplastic thrombocytopenic disorders.

The diagnosis of primary immune thrombocytopenia (ITP) is based on differential diagnosis. Although the measurement of percentages of reticulated platelets (RP%) by flow cytometry is useful as a supportive diagnostic test, this method is nonetheless a time-consuming, laboratory-based assay. To identify alternative assays that are useful in daily practice, we compared three methods in parallel, IPF% measured by XE-2100 [IPF% (XE), Sysmex Corp.], IPF% measured by new XN-1000 [IPF% (XN)], and RP%. We examined 47 patients with primary ITP, 28 patients with aplastic thrombocytopenia (18 aplastic anemia and 10 chemotherapy-induced thrombocytopenia) and 80 healthy controls. In a selected experiment, we examined 16 patients with paroxysmal nocturnal hemoglobinuria (PNH) to examine the effect of hemolysis. As compared with IPF% (XE), IPF% (XN) showed better within-run reproducibility. The sensitivity and specificity for the diagnosis of ITP were 83.0 and 75.0 % for IPF% (XE), 85.1 and 89.3 % for IPF% (XN), and 93.6 and 89.3 % for RP%, respectively. Examination of PNH patients revealed that hemolysis and/or red blood cell fragments interfered with IPF% (XE) values, but not with IFP % (XN) values. Our results suggest that IPF% measured by XN-1000 may be of comparable value with RP% as a supportive diagnostic test for ITP.

Cloning, phylogeny, and expression analysis of the Broad-Complex gene in the longicorn beetle Psacothea hilaris.

Seven isoforms of Broad-Complex (PhBR-C), in which the sequence of the zinc finger domain differed (referred to as Z1, Z2, Z3, Z2/Z3, Z4, Z5/Z6, and Z6, respectively), were cloned from the yellow-spotted longicorn beetle Psacothea hilaris. The Z1-Z4 sequences were highly conserved among insect species. The Z5/Z6 isoform was aberrant in that it contained a premature stop codon. Z6 had previously only been detected in a hemimetabola, the German cockroach Blattella germanica. The presence of Z6 in P. hilaris, and not in other holometabolous model insects such as Drosophila melanogaster or Tribolium castaneum, suggests that Z6 was lost multiple times in holometabolous insects during the course of evolution. PhBR-C expression levels in the brain, salivary gland, and epidermis of larvae grown under different feeding regimens were subsequently investigated. PhBR-C expression levels increased in every tissue examined after the gut purge, and high expression levels were observed in prepupae. A low level of PhBR-C expression was continuously observed in the brain. An increase was noted in PhBR-C expression levels in the epidermis when 4th instar larvae were starved after 4 days of feeding, which induced precocious pupation. No significant changes were observed in expression levels in any tissues of larvae starved immediately after ecdysis into 4th instar, which did not grow and eventually died.

A case of neonatal alloimmune thrombocytopenia in the presence of both anti-HPA-4b and anti-HPA-5b antibody: clinical and serological analysis of the subsequent pregnancy.

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies raised against fetal platelet antigens inherited from the paternal parent. In contrast to Caucasians, in Asians, predominantly in Japanese, most frequently detected antibodies in NAIT are anti-HPA-4b and anti-HPA-5b. In some NAIT cases multiple alloantibodies are detected. In such cases it is very difficult to determine which antibody is the dominant antibody in NAIT. In this case report, we describe a NAIT case (first sibling) with severe thrombocytopenia and cephalhematoma in the presence of both anti-HPA-4b and anti-HPA-5b antibodies in the maternal serum. We carefully examined titers of anti-HPA antibodies during the subsequent pregnancy with HPA-4b-positive and HPA-5b-negative fetus determined by amniocentesis at gestational week 16. We administered IVIG (1 g/kg/w) to the mother from gestational week 32 to 35. The mother subsequently delivered a second sibling with normal platelet count by cesarean section. Although we could not completely rule out the involvement of anti-HPA-4b, our findings suggested that anti-HPA-5b was implicated in the NAIT in the first sibling.

Fetal and neonatal anemia associated with anti-Jr(a) : a case report showing a poorly hemolytic mechanism.

Although recently published case reports suggest the significance of Jr(a) alloimmunization in the obstetric setting, the involved mechanism still remains unclear. Here we report a case of severe fetal and neonatal anemia associated with anti-Jr(a) alloimmunization, which was successfully managed using Doppler assessment of peak systolic velocity of the fetal middle cerebral artery (MCA-PSV). A Japanese woman with anti-Jr(a) (titer 1024) was referred to our department at 20 weeks' gestation. As fetal MCA-PSV exceeded 1.5 multiple of median, labor was induced and a female neonate of 1998 g was delivered vaginally at 33 weeks and 5 days of gestation. The infant's hematocrit and hemoglobin levels were 25.4% and 82 g/L, respectively, but her total bilirubin level (15 µmol/L; 0.9 mg/dL) and reticulocyte counts (4.5%) were low. During the course, the infant showed no apparent signs of hemolysis. Jr(a) alloimmunization should be recognized as a possible cause of fetal anemia with no direct hemolytic process.