E93 is indispensable for reproduction in ametabolous and hemimetabolous insects.

Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. Although E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discover that, in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNA is broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we reveal that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis that is necessary to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects, and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.

Juvenile hormone signaling is indispensable for late embryogenesis in ametabolous and hemimetabolous insects.

BACKGROUND: Juvenile hormone (JH) is an insect-exclusive hormone involved in regulating diverse aspects of insect physiology, and the evolution of its diverse function is widely interesting. Studying embryogenesis in basal wingless insects is important to understand the functional evolution of JH; however, experimental studies in this regard are scarce. In this study, we conducted CRISPR/Cas9-mediated knockout (KO) of genes involved in JH biosynthesis and signaling cascades in the ametabolous firebrat, Thermobia domestica. Additionally, we investigated whether the primitive action of JH is conserved in the hemimetabolous cricket, Gryllus bimaculatus. RESULTS: We observed that KO of JHAMT, CYP15A1, Met, and Kr-h1 resulted in embryonic lethality in T. domestica. Deprivation of JH or JH signaling arrested the progression of extraembryonic fluid resorption after dorsal closure and hatching, which is consistent with the gene expression pattern showing high Kr-h1 expression in the late embryos of T. domestica. The embryos deficient in JH signaling displayed wrinkled and weak legs. Comparative transcriptome analysis revealed that JH signaling promotes embryonic leg maturation through inducing energy supply and muscle activity, as validated by transmission electron microscopy (TEM). In addition, JH signaling exhibited similar embryonic effects in G. bimaculatus. CONCLUSIONS: This study reveals the indispensable role of JH signaling in facilitating the maturation of terminal tissues during late embryogenesis, as demonstrated by the regulation of leg development, in ametabolous and hemimetabolous insects. These findings further indicate that the embryonic functions of JH evolved earlier than its anti-metamorphic functions during postembryonic development.

Convergent Adaptation of Ootheca Formation as a Reproductive Strategy in Polyneoptera.

Insects have evolved numerous adaptations and colonized diverse terrestrial environments. Several polyneopterans, including dictyopterans (cockroaches and mantids) and locusts, have developed oothecae, but little is known about the molecular mechanism, physiological function, and evolutionary significance of ootheca formation. Here, we demonstrate that the cockroach asymmetric colleterial glands produce vitellogenins, proline-rich protein, and glycine-rich protein as major ootheca structural proteins (OSPs) that undergo sclerotization and melanization for ootheca formation through the cooperative protocatechuic acid pathway and dopachrome and dopaminechrome subpathway. Functionally, OSP sclerotization and melanization prevent eggs from losing water at warm and dry conditions, and thus effectively maintain embryo viability. Dictyopterans and locusts convergently evolved vitellogenins, apolipoprotein D, and laminins as OSPs, whereas within Dictyoptera, cockroaches and mantids independently developed glycine-rich protein and fibroins as OSPs. Highlighting the ecological-evolutionary importance, convergent ootheca formation represents a successful reproductive strategy in Polyneoptera that promoted the radiation and establishment of cockroaches, mantids, and locusts.

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate.

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.

Evolution of the Cholesterol Biosynthesis Pathway in Animals.

Cholesterol plays essential roles in animal development and disease progression. Here, we characterize the evolutionary pattern of the canonical cholesterol biosynthesis pathway (CBP) in the animal kingdom using both genome-wide analyses and functional experiments. CBP genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of both the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might be due to the geologic events during the Ediacaran period, such as the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation. Meanwhile, the indispensable utilization processes of cholesterol potentially strengthened the maintenance of the complete set of CBP genes in vertebrates. These results strengthen both biotic and abiotic roles in the macroevolution of a biosynthesis pathway in animals.

Ben-JNK signaling is required for host mortality during Periplaneta fuliginosa densovirus infection.

BACKGROUND: Cockroaches are widely acknowledged as significant vectors of pathogenic microorganisms. The Periplaneta fuliginosa densovirus (PfDNV) infects the smoky-brown cockroach P. fuliginosa and causes host mortality, which identifies the PfDNV as a species-specific and environmentally friendly biopesticide. However, although the biochemical characterization of PfDNV has been extensively studied, the immune response against PfDNV remains largely unclear. RESULTS: Here, we investigated the replication of PfDNV and its associated pathological phenotype in the foregut and hindgut. Consequently, we dissected and performed transcriptome sequencing on the foregut, midgut, and hindgut separately. We revealed the up-regulation of immune response signaling pathway c-Jun N-terminal kinase (JNK) and apoptosis in response to viral infection. Furthermore, knockdown of the JNK upstream gene Ben resulted in a decrease in virus titer and delayed host mortality. CONCLUSION: Taken together, our findings provide evidence that the Ben-JNK signaling plays a crucial role in PfDNV infection, leading to excessive apoptosis in intestinal tissues and ultimately resulting in the death of the host. Our results indicated that the host response to PfDNV fosters viral infection, thereby increasing host lethality. This underscores the potential of PfDNV as a viable, environmentally friendly biopesticide. © 2024 Society of Chemical Industry.

Two transcriptional cascades orchestrate cockroach leg regeneration.

The mystery of appendage regeneration has fascinated humans for centuries, while the underlying regulatory mechanisms remain unclear. In this study, we establish a transcriptional landscape of regenerating leg in the American cockroach, Periplaneta americana, an ideal model in appendage regeneration studies showing remarkable regeneration capacity. Through a large-scale in vivo screening, we identify multiple signaling pathways and transcription factors controlling leg regeneration. Specifically, zfh-2 and bowl contribute to blastema cell proliferation and morphogenesis in two transcriptional cascades: bone morphogenetic protein (BMP)/JAK-STAT-zfh-2-B-H2 and Notch-drm/bowl-bab1. Notably, we find zfh-2 is working as a direct target of BMP signaling to promote cell proliferation in the blastema. These mechanisms might be conserved in the appendage regeneration of vertebrates from an evolutionary perspective. Overall, our findings reveal that two crucial transcriptional cascades orchestrate distinct cockroach leg regeneration processes, significantly advancing the comprehension of molecular mechanism in appendage regeneration.

ERK-activated CK-2 triggers blastema formation during appendage regeneration.

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal-regulated kinase (ERK)-activated casein kinase 2 (CK-2), which has not been previously implicated in appendage regeneration, triggers blastema formation during leg regeneration in the American cockroach, Periplaneta americana. After amputation, CK-2 undergoes rapid activation through ERK-induced phosphorylation within blastema cells. RNAi knockdown of CK-2 severely impairs blastema formation by repressing cell proliferation through down-regulating mitosis-related genes. Evolutionarily, the regenerative role of CK-2 is conserved in zebrafish caudal fin regeneration via promoting blastema cell proliferation. Together, we find and demonstrate that the ERK-activated CK-2 triggers blastema formation in both cockroach and zebrafish, helping explore initiation factors during appendage regeneration.

Two sex pheromone receptors for sexual communication in the American cockroach.

Volatile sex pheromones are vital for sexual communication between males and females. Females of the American cockroach, Periplaneta americana, produce and emit two sex pheromone components, periplanone-A (PA) and periplanone-B (PB). Although PB is the major sex attractant and can attract males, how it interacts with PA in regulating sexual behaviors is still unknown. In this study, we found that in male cockroaches, PA counteracted PB attraction. We identified two odorant receptors (ORs), OR53 and OR100, as PB/PA and PA receptors, respectively. OR53 and OR100 were predominantly expressed in the antennae of sexually mature males, and their expression levels were regulated by the sex differentiation pathway and nutrition-responsive signals. Cellular localization of OR53 and OR100 in male antennae further revealed that two types of sensilla coordinate a complex two-pheromone-two-receptor pathway in regulating cockroach sexual behaviors. These findings indicate distinct functions of the two sex pheromone components, identify their receptors and possible regulatory mechanisms underlying the male-specific and age-dependent sexual behaviors, and can guide novel strategies for pest management.

Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects.

Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. Here, we found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction.

5mC modification orchestrates choriogenesis and fertilization by preventing prolonged ftz-f1 expression.

DNA methylation at the fifth position of cytosine (5-methylcytosine, 5mC) is a crucial epigenetic modification for regulating gene expression, but little is known about how it regulates gene expression in insects. Here, we pursue the detailed molecular mechanism by which DNMT1-mediated 5mC maintenance regulates female reproduction in the German cockroach, Blattella germanica. Our results show that Dnmt1 knockdown decreases the level of 5mC in the ovary, upregulating numerous genes during choriogenesis, especially the transcription factor ftz-f1. The hypomethylation at the ftz-f1 promoter region increases and prolongs ftz-f1 expression in ovarian follicle cells during choriogenesis, which consequently causes aberrantly high levels of 20-hydroxyecdysone and excessively upregulates the extracellular matrix remodeling gene Mmp1. These changes further impair choriogenesis and disrupt fertilization by causing anoikis of the follicle cells, a shortage of chorion proteins, and malformation of the sponge-like bodies. This study significantly advances our understanding of how DNA 5mC modification regulates female reproduction in insects.

[Construction and characterization of aroA deletion mutant of Pasteurella multocida strain C51-17].

OBJECTIVE: We constructed Pasteurella multocida strain C51-17 aroA mutant and characterized its virulence. METHODS: Suicide recombinant plasmid of pBC-aroA-Tn903 was constructed with kanamycin gene and transformed into Pasteurella multocida by electroporation. aroA mutant strain of P. multocida was screened by kanamycin resistance and chloromycetin sensitivity and identified by PCR. Virulence of aroA mutant strain was tested in mice by intraperitoneal injection. RESULTS: The aroA mutant strain of P. multocida C51-17 was successfully constructed. Growth rate of aroA mutant strain was slower within the first 6 hour and similar to the wild-type strain in vitro. The virulence test indicated that aroA mutant strain was significant attenuated and no death was found in mice even inoculated at a dosage of 1.0 x 10(6) CFU. CONCLUSION: The virulence of aroA mutant strain of P. multocida was highly attenuated in mice. The results of this study will contribute to a better understanding of pathogenesis of P. multocida.

Juvenile hormone membrane signaling phosphorylates USP and thus potentiates 20-hydroxyecdysone action in Drosophila.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

Isolation and Characterization of a Porcine Transmissible Gastroenteritis Coronavirus in Northeast China.

Transmissible gastroenteritis virus (TGEV) is a coronavirus (CoV) that is a major pathogenity of viral enteritis and diarrhea in suckling piglets, causing high morbidity and mortality. In this study, a TGEV strain HQ2016 was isolated from northeast China and characterized its genome sequence and pathogenicity. The phylogenetic analysis indicated that the TGEV HQ2016 strain was more similar to the TGEV Purdue cluster than to the Miller cluster. Both recombination and phylogenetic analysis based on each structural and non-structural gene revealed no recombination event in the HQ2016 strain. Experimental infection study using colostrum-deprived newborn piglets successfully showed that the HQ2016 can cause clinical symptoms including anorexia and yellow-to-whitish watery diarrhea, which are characteristics of TGE, in the inoculated piglets 48 h post-inoculation. These results provide valuable information about the evolution of the porcine CoVs.

The steroid-induced microRNA let-7 regulates developmental growth by targeting cdc7 in the Drosophila fat body.

In insects, 20-hydroxyecdysone (20E) limits systemic growth by triggering developmental transitions. Previous studies have shown that 20E-induced let-7 exhibits crosstalk with the cell cycle. Here, we examined the underlying molecular mechanisms and physiological functions of 20E-induced let-7 in the fat body, an organ for energy storage and nutrient mobilization which plays a critical role in the larval growth. First, the overexpression of let-7 decreased the body size and led to the reduction of both nucleolus and cell sizes in the larval fat body. In contrast, the overexpression of let-7-Sponge increased the nucleolus and cell sizes. Moreover, we found that cdc7, encoding a conserved protein kinase that controls the endocycle, is a target of let-7. Notably, the mutation of cdc7 in the fat body resulted in growth defects. Overall, our findings revealed a novel role of let-7 in the control of endoreduplication-related growth during larval-prepupal transition in Drosophila.

A molecular epidemiological investigation of PEDV in China: Characterization of co-infection and genetic diversity of S1-based genes.

Porcine epidemic diarrhoea virus (PEDV) is an emerging and re-emerging epizootic virus of swine that causes substantial economic losses to the pig industry in China and other countries. The variations in the virus, and its co-infections with other enteric viruses, have contributed to the poor control of PEDV infection. In the current study, a broad epidemiological investigation of PEDV was carried out in 22 provinces or municipalities of China during 2015-2018. The enteric viruses causing co-infection with PEDV and the genetic diversity of the PEDV S1 gene were also analysed. The results indicated that, of the 543 diarrhoea samples, 66.85% (363/543) were positive for PEDV, and co-infection rates of PEDV with 13 enteric viruses ranged from 3.58% (13/363) to 81.55% (296/363). Among these enteric viruses, the signs of diarrhoea induced by PEDV were potentially associated with co-infections with porcine enterovirus 9/10 (PEV) and torque teno sus virus 2 (TTSuV-2) (p < .05). The 147 PEDV strains identified in our study belong to Chinese pandemic strains and exhibited genetic diversity. The virulence-determining S1 proteins of PEDV pandemic strains were undergoing amino acid mutations, in which S58_S58insQGVN-N135dup-D158_I159del-like mutations were common patterns (97.28%, 143/147). When compared with 2011-2014 PEDV strains, the amino acid mutations of PEDV pandemic strains were mainly located in the N-terminal domain of S1 (S1-NTD), and 21 novel mutations occurred in 2017 and 2018. Furthermore, protein homology modelling showed that the mutations in pattern of insertion and deletion mutations of the S1 protein of PEDV pandemic strains may have caused structural changes on the surface of the S1 protein. These data provide a better understanding of the co-infection and genetic evolution of PEDV in China.

Helicobacter pylori promotes hepatic fibrosis in the animal model.

Helicobacter pylori infection has been reported to be very common in patients with chronic liver diseases, including cirrhosis. To elucidate the pathological effect of H. pylori infection on the progression of hepatic fibrosis, C57BL/6 mice and Sprague-Dawley rats were orally inoculated with H. pylori, and hepatic fibrosis was induced with carbon tetrachloride (CCl(4)) administration. We observed the histopathological changes and the presence of H. pylori genes by PCR in the liver. Significant increase in the fibrotic score as well as in serum alanine aminotransferase and aspartate aminotransferase levels was shown in the CCl(4)+H. pylori group compared with that in the CCl(4)-treated group. Compared with the CCl(4)-treated group, alpha-smooth muscle actin and transforming growth factor-beta1 were enhanced; however, senescence marker protein-30, a multifunctional protein protecting hepatocytes against oxidative stress and apoptosis, was suppressed in the CCl(4)+H. pylori group. The 16S rRNA (400 bp) was demonstrated by PCR for H. pylori genes from genomic DNA extracted from the liver, and H. pylori-infected mice showed 93.8% (15 of 16) seropositivity by contrast with seronegativity in all H. pylori-noninfected mice. In addition, immunohistochemical study against H. pylori showed positive antigen fragments in the liver of the infected groups. Consequently, our data suggest that H. pylori infection could be an important contributing infectious factor to the development of liver cirrhosis.

Multiple urogenital abnormalities in a Persian cat.

A 1.5-year-old female Persian cat was presented for inappetence and azotemia. Ultrasonography and urography revealed multiple abnormalities involving the genitourinary tract, including agenesis of the right kidney and ureter. Gross examination of the abnormal uterus revealed segmental aplasia of right caudal uterine horn causing cranial distension with fluid, a normal left uterine horn, and both normal ovaries. Microscopically, endometrial glands of the right uterine horn were markedly decreased in number. The right uterine horn was hemorrhagic suggesting estrus. This is the first report of this combination of urinary and uterus abnormalities in the veterinary literature.

Magnetostrictive Properties of the Grain-Oriented Silicon Steel Sheet under DC-Biased and Multisinusoidal Magnetizations.

As an intrinsic property, elasticity of soft material is affected significantly by the externally applied alternating magnetic field. Magnetostrictive properties of the grain-oriented (GO) silicon steel under DC-biased and multisinusoidal magnetizations are measured by using a laser-based measuring system. Magnetostriction curves of the GO silicon steel sheet under different magnetizations are obtained and the influence of frequency and DC bias on the magnetostrictive property is observed and analyzed based on the measured data. In addition, the spectrum of magnetostriction under harmonic magnetization is obtained, and the acoustic noise level of the GO silicon steel sheet represented by the A-weighted decibel value caused by magnetostriction is measured under DC-biased and multisinusoidal magnetizations. The measurement results are applied to the simulation of the three-limb laminated core model, and the effects of DC bias and harmonics on magnetic flux density and displacement are analyzed.

Complete mitochondrial genome of two Thitarodes species (Lepidoptera, Hepialidae), the host moths of Ophiocordyceps sinensis and phylogenetic implications.

Thitarodes (Lepidoptera, Hepialidae) is the only genus that hosts to the Ophiocordyceps sinensis, a traditional Chinese medicine considered as a powerful medicinal supplement. In this study, the complete mitochondrial genomes (mitogenomes) of two species, T. damxungensis and T. pui, have been sequenced, which are 15,928 bp and 15,362 bp in size respectively, and both contain 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs and an AT-rich region. Like other hepialoids, the gene arrangement of the mitogenomes of T. damxungensis and T. pui is identical to the ancestral arrangement but differs from those of other lepidopteran species on account of the different arrangements of trnM, trnI, and trnQ. The size of AT-rich region is 545 bp in T. damxungensis and 1030 bp in T. pui. Tandem repetition in the AT-rich region is responsible for the length difference of the A + T-rich region in both species. In Hepialidae, the phylogenetic study based on the dataset of the sequences that combined the protein-coding genes and RNA genes suggested that the species T. yunnanensis should still belong to the genus Thitarodes rather than Ahamns, which is different from the results based on the traditional phylogeny.