A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

A cell line derived from the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae).

Insect cell lines are effective tools used in industry and academia. For example, they are used in screening potential insecticides, in making certain proteins for biomedical applications, and in basic research into insect biology. So far, there are no cell lines derived from the black soldier fly, Hermetia illucens (BSF). This may become an issue because BSFs are employed in a range of industrial and household processes. BSFs are used in producing biodiesel, in developing cosmetics and skin creams, and in the production of some medicines and animal feeds. BSF larvae process waste streams from a variety of sources into food for some animals and are also used in household composting. Our BSF cell line, designated BCIRL-HiE0122021-SGS, was developed from eggs using the medium CLG#2 (50% L-15 + 50% EX-CELL 420, with 9% FBS and antibiotics), with many other media being tested. This cell line consists of attached cells with a variety of morphologies and its identity was authenticated using CO1 barcoding. A growth curve was generated and the resulting doubling time was 118 h. We quantified the fatty acid methyl esters (FAMES) and recorded the expected range of saturated, monounsaturated, and polyunsaturated FAMEs, with only trace levels of lauric acid being noted. The BSF cell line is available free of charge by request.

A content analysis of counseling psychology literature: Resilience against oppression among people of color.

The researchers analyzed articles from two flagship counseling psychology journals (i.e., Journal of Counseling Psychology and The Counseling Psychologist) to examine current understandings of resilience. There were 54 articles included in the final analysis that spanned the years 1997-2022. The researchers conducted a content analysis to identify, analyze, and report patterns across counseling psychology journals with regard to how resilience has been defined, the racial/ethnic groups that were of focus, and the forms of oppression that were studied or addressed. Five themes were generated that are a direct representation of the topics within previous literature on resilience in the field of counseling psychology. Those topics included: (a) ways of coping, (b) ethnic and racial identity, (c) advocacy/collective action, (d) personality factors, and (e) ability to endure stress and thrive. Implications for future research, clinical applications, mentoring, and advocacy are discussed. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

Regulatory network in heat stress response in parasitoid wasp focusing on Xap5 heat stress regulator.

Insects are susceptible to elevated temperatures, resulting in impaired fertility, and shortened lifespan. This study investigated the genetic mechanisms underlying heat stress effects. We conducted RNA sequencing on Pteromalus puparum exposed to 25°C and 35°C, revealing transcriptional signatures. Weighted Gene Co-expression Network Analysis uncovered heat stress-associated modules, forming a regulatory network of 113 genes. The network is naturally divided into two subgroups, one linked to acute heat stress, including heat shock proteins (HSPs), and the other to chronic heat stress, involving lipogenesis genes. We identified an Xap5 Heat Shock Regulator (XHSR) gene as a crucial network component, validated through RNA interference and quantitative PCR assays. XHSR knockdown reduced wasps' lifespan while directly inducing HSPs and mediating lipogenesis gene induction. CRISPR/Cas9-mediated knockout of the Drosophila XHSR homolog reduced mutants' survival, highlighting its conserved role. This research sheds light on thermal tolerance mechanisms, offering potential applications in pest control amid global warming.

Trade-Offs among Immune Mechanisms: Bacterial-Challenged Spodoptera frugiperda Larvae Reduce Nodulation Reactions during Behavioral Fever.

Insect innate immunity is composed of cellular and humoral reactions, the former acting via circulating hemocytes and the latter via immune signaling that lead to the production of antimicrobial peptides and phenol oxidase-driven melanization. Cellular immunity involves direct interactions between circulating hemocytes and invaders; it includes internalization and killing microbes (phagocytosis) and formation of bacterial-laden microaggregates which coalesce into nodules that are melanized and attached to body walls or organs. Nodulation can entail investing millions of hemocytes which must be replaced. We hypothesized that biologically costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae of fall armyworms, Spodoptera frugiperda, that were allowed to fever. We tested our hypothesis by infecting larvae with the Gram-negative bacterium, Serratia marcescens, placing them in thermal gradients (TGs) and recording their selected body temperatures. While control larvae selected about 30 °C, the experimental larvae selected up 41 °C. We found that 4 h fevers, but not 2, 6 or 24 h fevers, led to increased larval survival. Co-injections of S. marcescens with the prostaglandin (PG) biosynthesis inhibitor indomethacin (INDO) blocked the fevers, which was reversed after co-injections of SM+INDO+Arachidonic acid, a precursor to PG biosynthesis, confirming that PGs mediate fever reactions. These and other experimental outcomes support our hypothesis that costly hemocyte-based immunity is traded off for behavioral fevers in infected larvae under appropriate conditions.

Identification and characterization of ecdysis-related neuropeptides in the lone star tick Amblyomma americanum.

Introduction: The lone star tick, Amblyomma americanum, is an important ectoparasite known for transmitting diseases to humans and animals. Ecdysis-related neuropeptides (ERNs) control behaviors crucial for arthropods to shed exoskeletons. However, ERN identification and characterization in A. americanum remain incomplete. Methods: We investigated ERNs in A. americanum, assessing their evolutionary relationships, protein properties, and functions. Phylogeny, sequence alignment, and domain structures of ERNs were analyzed. ERN functionality was explored using enrichment analysis, and developmental and tissue-specific ERN expression profiles were examined using qPCR and RNAi experiments. Results and discussion: The study shows that ERN catalogs (i.e., eclosion hormone, corazonin, and bursicon) are found in most arachnids, and these ERNs in A. americanum have high evolutionary relatedness with other tick species. Protein modeling analysis indicates that ERNs primarily consist of secondary structures and protein stabilizing forces (i.e., hydrophobic clusters, hydrogen bond networks, and salt bridges). Gene functional analysis shows that ENRs are involved in many ecdysis-related functions, including ecdysis-triggering hormone activity, neuropeptide signaling pathway, and corazonin receptor binding. Bursicon proteins have functions in chitin binding and G protein-coupled receptor activity and strong interactions with leucine-rich repeat-containing G-protein coupled receptor 5. ERNs were expressed in higher levels in newly molted adults and synganglia. RNAi-mediated knockdown of burs α and burs ß expression led to a significant decrease in the expression of an antimicrobial peptide, defensin, suggesting they might act in signaling or regulatory pathways that control the expression of immune-related genes. Arthropods are vulnerable immediately after molting because new cuticles are soft and susceptible to injury and pathogen infections. Bursicon homodimers act in prophylactic immunity during this vulnerable period by increasing the synthesis of transcripts encoding antimicrobial peptides to protect them from microbial invasion. Collectively, the expression pattern and characterization of ERNs in this study contribute to a deeper understanding of the physiological processes in A. americanum.

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum.

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. Here, we report on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis we identified a set of immune-related genes that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, we identified a novel AMP, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-κB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. We conducted Ten3LP RNAi, synthesized recombinant Ten3LP protein for microbial inhibition assays and functionally characterized Ten3LP as an AMP specific for fungi and Gram-positive bacteria. We demonstrate that expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.

HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection.

Tomato spotted wilt virus (TSWV) causes a serious plant disease and is transmitted by specific thrips including the western flower thrips, Frankliniella occidentalis. The persistent and circulative virus transmission suggests an induction of immune defenses in the thrips. We investigated the immune responses of F. occidentalis to TSWV infection. Immunofluorescence assay demonstrated viral infection in the larval midguts at early stage and subsequent propagation to the salivary gland in adults. In the larval midgut, TSWV infection led to the release of DSP1, a damage-associated molecular pattern, from the gut epithelium into the hemolymph. DSP1 up-regulated PLA2 activity, which would lead to biosynthesis of eicosanoids that activate cellular and humoral immune responses. Phenoloxidase (PO) activity was enhanced following induction of PO and its activating protease gene expressions. Antimicrobial peptide genes and dual oxidase, which produces reactive oxygen species, were induced by the viral infection. Expression of four caspase genes increased and TUNEL assay confirmed apoptosis in the larval midgut after the virus infection. These immune responses to viral infection were significantly suppressed by the inhibition of DSP1 release. We infer that TSWV infection induces F. occidentalis immune responses, which are activated by the release of DSP1 from the infection foci within midguts.

Beyond Cellular Immunity: On the Biological Significance of Insect Hemocytes.

Insect immunity is assorted into humoral and cellular immune reactions. Humoral reactions involve the regulated production of anti-microbial peptides, which directly kill microbial invaders at the membrane and intracellular levels. In cellular immune reactions, millions of hemocytes are mobilized to sites of infection and replaced by hematopoiesis at a high biological cost after the immune defense. Here, we considered that the high biological costs of maintaining and replacing hemocytes would be a better investment if hemocytes carried out meaningful biological actions unrelated to cellular immunity. This idea allows us to treat a set of 10 hemocyte actions that are not directly involved in immunity, some of which, so far, are known only in Drosophila melanogaster. These include (1) their actions in molting and development, (2) in surviving severe hypoxia, (3) producing phenoloxidase precursor and its actions beyond immunity, (4) producing vitellogenin in a leafhopper, (5) recognition and responses to cancer in Drosophila, (6) non-immune actions in Drosophila, (7) clearing apoptotic cells during development of the central nervous system, (8) developing hematopoietic niches in Drosophila, (9) synthesis and transport of a lipoprotein, and (10) hemocyte roles in iron transport. We propose that the biological significance of hemocytes extends considerably beyond immunity.

Two Alimentary Canal Proteins, Fo-GN and Fo-Cyp1, Act in Western Flower Thrips, Frankliniella occidentalis TSWV Infection.

Tomato spotted wilt virus (TSWV) is a plant virus that causes massive economic damage to high-valued crops. This virus is transmitted by specific thrips, including the western flower thrips, Frankliniella occidentalis. TSWV is acquired by the young larvae during feeding on infected host plants. TSWV infects the gut epithelium through hypothetical receptor(s) and multiplies within the cells for subsequent horizontal transmission to other plant hosts via the salivary glands during feeding. Two alimentary canal proteins, glycoprotein (Fo-GN) and cyclophilin (Fo-Cyp1), are thought to be associated with the TSWV entry into the gut epithelium of F. occidentalis. Fo-GN possesses a chitin-binding domain, and its transcript was localized on the larval gut epithelium by fluorescence in situ hybridization (FISH) analysis. Phylogenetic analysis indicated that F. occidentalis encodes six cyclophilins, in which Fo-Cyp1 is closely related to a human cyclophilin A, an immune modulator. The Fo-Cyp1 transcript was also detected in the larval gut epithelium. Expression of these two genes was suppressed by feeding their cognate RNA interference (RNAi) to young larvae. The RNAi efficiencies were confirmed by the disappearance of the target gene transcripts from the gut epithelium by FISH analyses. The RNAi treatments directed to Fo-GN or Fo-Cyp1 prevented the typical TSWV titer increase after the virus feeding, compared to control RNAi treatment. Our immunofluorescence assay using a specific antibody to TSWV documented the reduction of TSWV in the larval gut and adult salivary gland after the RNAi treatments. These results support our hypothesis that the candidate proteins Fo-GN and Fo-Cyp1 act in TSWV entry and multiplication in F. occidentalis.

Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing.

Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20th compared to conventional Illumina methods.

The clock gene, period, influences migratory flight and reproduction of the oriental armyworm, Mythimna separata (Walker).

The oriental armyworm, Mythimna separata, is a major long-distance migratory insect pest of grain crops in China and other Asian countries. Migratory flights and reproductive behavior usually occur at night, regulated by a circadian rhythm. However, knowledge about the linkages between adult flight, reproduction, and clock genes is still incomplete. To fill this important gap in our knowledge, a clock gene (designated Msper) was identified and phylogenetic analysis indicated that the encoded protein (MsPER) was highly similar to PER proteins from other insect species. Quantitative RT-PCR assays demonstrated that significantly different spatiotemporal and circadian rhythmic accumulations of mRNA encoding MsPER occurred during development under steady 14 h : 10 h light : dark conditions. The highest mRNA accumulation occurred in adult antennae and the lowest in larvae. Msper was expressed rhythmically in adult antennae, relatively less in photophase and more entering scotophase. Injecting small interference RNA (siRNA) into adult heads effectively knocked down Msper mRNA levels within 72 h. Most siRNA-injected adults reduced their evening flight activity significantly and did not exhibit a normal evening peak of flight activity. They also failed to mate and lay eggs within 72 h. Adult mating behavior was restored to control levels by 72 h post injection. We infer that Msper is a prominent clock gene that acts in regulating adult migratory flight and mating behaviors of M. separata. Because of its influence on migration and mating, Msper may be a valuable gene to target for effective management of this migratory insect.

Why can insects not biosynthesize cholesterol?

Two aspects of insect lipid biochemistry differ from the mammalian background. In one aspect, nearly a hundred years ago scientists demonstrated that the polyunsaturated fatty acid (PUFAs), linoleic acid (LA; 18:2n-6) is an essential nutrient in the diets of all mammals that have been studied in that regard. An unknown number of insect species are able to biosynthesize LA de novo. Some species take the biosynthesized LA into fatty acid elongation/desaturation pathways to produce other PUFAs, 18:3n-6, 20:3n-6 and 20:4n-6. A couple of species use the de novo produced LA to biosynthesize prostaglandins and other eicosanoids, short-lived signal moieties that mediate important physiological actions in immunity and reproduction. Insects differ from mammals, also, in their lack of genes that encode enzymes acting in biosynthesis of cholesterol. Insects require dietary cholesterol to meet their cellular, physiological, developmental, and reproductive needs. Looking at a broader view of invertebrate biochemistry, most protostomes lost all or most genes involved in cholesterol biosynthesis. The massive gene loss occurred during the Ediacaran Period, which lasted 96 million years, from the end of the Cryogenian Period (635 million years ago; MYA) to the beginning of the Cambrian Period (538.6 MYA). The key point here is that the inability to biosynthesize cholesterol is not limited to insects; it occured in most protostomes. We address the protostome need and benefits of acquiring exogenous sterols.

What is in a model?

After reading contradictory claims of model status for some insect species, we feel a brief discussion of the topic may be useful. Here, we document a few examples where clarity on model status seems to be lacking, briefly review work on widely recognized models, and offer criteria for including any given species as a model organism.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea.

Cytochrome P450 monooxygenases (CYPs) are present in almost all areas of the tree of life. As one of the largest and most diverse superfamilies of multifunctional enzymes, they play important roles in the metabolism of xenobiotics and biosynthesis of endogenous compounds, shaping the success of insects. In this study, the CYPome (an omics term for all the CYP genes in a genome) diversification was examined in the four Tenebrionidea species through genome-wide analysis. A total of 483 CYP genes were identified, of which 103, 157, 122, and 101 were respectively deciphered from the genomes of Tebebrio molitor, Asbolus verucosus, Hycleus cichorii and Hycleus phaleratus. These CYPs were classified into four major clans (mitochondrial, CYP2, CYP3, and CYP4), and clans CYP3 and CYP4 are most diverse. Phylogenetic analysis showed that most CYPs of these Tenebrionidea beetles from each clan had a very close 1:1 orthology to each other, suggesting that they originate closely and have evolutionally conserved function. Expression analysis at different developmental stages and in various tissues showed the life stage-, gut-, salivary gland-, fat body-, Malpighian tubule-, antennae-, ovary- and testis-specific expression patterns of T. molitor CYP genes, implying their various potential roles in development, detoxification, immune response, digestion, olfaction, and reproduction. Our studies provide a platform to understand the evolution of Tenebrionidea CYP gene superfamily, and a basis for further functional investigation of the T. molitor CYPs involved in various biological processes.