Insect Biochemistry and Molecular Biology最新文献

Insect prophenoloxidases (proPO) are activated during immune responses by a proPO activating protease (PAP) in the presence of a high molecular weight cofactor assembled from serine protease homologs (SPH) that lack proteolytic activity. PAPs and the SPHs have a similar architecture, with an amino-terminal clip domain and a carboxyl-terminal protease domain. The SPHs belong to CLIPA subfamily of SP-related proteins. In Manduca sexta, a well characterized biochemical model system for insect immunity, the functional SPH cofactor contains one molecule each from two SPH subfamilies, SPH-I and SPH-II. In Anopheles gambiae, three SPHI-SPHII pairs (CLIPs A4-A6, A4-A7Δ, and A4-A12) were previously reported as cofactors for CLIPB9-mediated activation of proPO2 and proPO7. In this study, we produced recombinant proteins for two splicing variants of CLIPA7, proCLIPA7s (s for short), proCLIPA7f (f for full-length) and proCLIPA14. We cleaved each along with proCLIPA4 using M. sexta PAP3 and found that the CLIPA pairs A4-A7s and A4-A14 are better than A4-A7f in generating highly active PO2 or PO7. CLIPA7f and CLIPA7s, products of alternative splicing, have different strengths as cofactors in combination with CLIPA4. Because mRNA for CLIPA7f is expressed at a significantly higher level than CLIPA7s, cofactors with the weaker combination A4-A7f may predominate in hemolymph, resulting in a potential dampening effect on proPO activation as a regulatory mechanism for altering the strength of the melanization response. A. gambiae CLIPB10x_a is involved in proPO activation but its role as a PAP was not established using mosquito proPOs. Here we showed that factor Xa-treated proCLIPB10_Xa activated proCLIPs A7s, A7f, A14, A4 (poorly), and proPO2. At higher concentrations, CLIPB10x_a efficiently activated proPO2 in the absence of a cofactor, but at low concentrations it required a CLIPA cofactor, suggesting that highly active PO2 can be generated at low concentration of CLIPB10 in cooperation with an SPH cofactor in vivo. Using cofactors generated by PAP3, we demonstrated the order of efficacy for proPO2 activation by B10_Xa is A4-A6 > A4-A14 or A4-A7s > A4-A7f > A4-A12. This agrees with their relative strengths as cofactors for proPO2 and proPO7 activation by M. sexta PAP3. In summary, we further developed an in vitro assay system to elucidate biochemical details of the complex process of proPO activation in A. gambiae.

The sterile insect technique (SIT) is a well-established and environmentally benign method for population control. Identifying genes that regulate insect fertility while preserving growth and development is crucial for implementing a novel SIT-based pest management approach utilizing CRISPR/Cas9 to target these genes for genetic manipulation. Tektin (TEKT), an essential alpha-helical protein pivotal in sperm formation due to its role in cilia and flagella assembly, has garnered attention. In this study, we identified 7 TEKT genes in the testis of Cydia pomonella, a globally invasive fruit pest. Notably, Tektin4-like (TEKT4L) displayed the highest expression level in male adult especially the testes, suggesting its significance in reproductive processes. By utilizing CRISPR/Cas9 technology to knockout TEKT4L, male sterility was induced, showcasing dominant inherited. When wild-type (WT) females mated with TEKT4L^-/- males, eggs laying proceeded normally, but the hatching rate was dramatically reduced, with only 15.49% progressing to the eyespot stage and 68.86% failing to develop normally. The reproductive fitness of TEKT4L^-/- males was robust enough to facilitate the transmission of genetic modifications efficiently within the C.pomonella population, yielding a small number of viable offspring. Subsequent cage trials demonstrated the effectiveness of this population in suppressing laboratory populations of C.pomonella, achieving notable results with a relatively low release ratio (TEKT4L^-/-♂ : WT♂: WT♀=5:1:5). Consequently, the targeted disruption of the TEKT4L gene holds promise as a fundamental element in a novel pest control strategy against C. pomonella.

Insect parasitoids have evolved sophisticated strategies to evade or modulate host immunity for parasitic infections. The precise mechanisms by which parasitoids counteract host anti-parasitic responses are poorly defined. Here we report a novel immune evasion strategy employed by the parasitoid Exorista sorbillans (Diptera: Tachinidae) to establish infection. We find that E. sorbillans larva construct a respiratory funnel that gradually increases in size as development progresses. This respiratory funnel, which connect to the parasitoid invasion aperture on the host silkworm epidermis, proves essential for E. sorbillans development, as sealing the invasion aperture results in complete mortality of larval parasitoids. Our investigation reveals that E. sorbillans infection reduces both host silkworms' hemocyte counts and the expression of hemocyte-specific genes, while simultaneously inducing varying degrees of host silkworm encapsulation at different parasitic stages. Nevertheless, more complete inhibition of host silkworm encapsulation through RNAi leads to parasitoid's defective respiratory funnel formation and increased mortality rates of the parasitoid. Further observations demonstrate that this suppressed encapsulation response triggers an enhanced activation of Toll/IMD pathways in the host silkworm. Take together, we show that E. sorbillans may utilize host silkworm encapsulation to construct a respiratory funnel for both respiration and immune evasion. Our findings provide new insights into the evasion tactics employed by parasitoids win out in the ongoing parasite-host evolutionary arms race.

Controlling Aedes aegypti mosquitoes is crucial for managing mosquito-transmitted diseases like dengue, zika, chikungunya, and yellow fever. One of the efficient methods to control mosquitoes is to block their progression from the larval to the adult stage. Juvenile hormones (JH) maintain the larval stage and ensure proper developmental timing for transitioning from larval-pupal-adult stages. Our previous studies showed that histone deacetylases (HDACs) regulate JH signaling and metamorphosis in the red flour beetle Tribolium castaneum. However, the role of HDACs in regulating JH signaling in Ae. aegypti mosquito is unknown. To investigate the role of HDACs in JH signaling, we knockdown each HDAC coding gene in Aag-2 cells derived from Ae. aegypti. Knockdown of HDAC1, HDAC4, and HDAC11 increased the expression of the JH primary response gene, Krüppel homolog 1 (Kr-h1), which represses the larval-pupal metamorphosis. Moreover, the simultaneous knockdown of these three HDACs synergistically increased the Kr-h1 promoter activity and its expression, mimicking JH action in inducing Kr-h1. Nevertheless, each HDAC regulates the transcription of different sets of genes, except for a few common genes involved in JH signaling. Furthermore, the knockdown of these HDACs in Ae. aegypti larvae caused different phenotypes apart from delayed pupation: HDAC1 knockdown caused larval growth retardation, body shrinkage, and eventual death; HDAC4 knockdown led to incomplete head capsule shedding after metamorphosis; and HDAC11 knockdown caused higher pupal mortality. Our data demonstrates functional overlap and distinct functions for HDAC1, HDAC4, and HDAC11 in modulating JH signaling, with each HDAC having a unique role in mosquito development.

In animals, sexual maturation coincides with the development of sexual behaviors and reproductive system. These developmental events are influenced by diet and governed by endocrine signals. Here, for the first time in insects, we explored functional links between nutrition and juvenile hormone (JH) in the male reproductive physiology through the insulin signaling pathway (ISP) acting as a transducer of nutritional signals. We turned to the male moth Agrotis ipsilon for which sexual maturation, including accessory sex glands (ASGs) development concomitantly with antennal lobes (ALs) maturation for female sex pheromone processing and display of sexual behavior, is known to be JH- and diet-dependent. Indeed, a diet rich in sugars with sodium was previously shown to accelerate sexual maturation, which was achieved from the third day of adult life. In this study, we demonstrated that such a diet raised i) the expression of JH signaling actors (Methoprene-tolerant, Taiman, and Krüppel homolog 1) in ALs and ASGs, ii) the biosynthesis and circulating levels of JH, and iii) the expression of both insulin receptor (InR) and insulin-like peptides (ILPs) in corpora allata (CAs) and brain respectively. Insulin injection raised JH biosynthesis following increased HMG-CoA reductase expression in CAs; opposite effects were induced in InR-deficient males. Thus, we highlighted that promoting effects of a diet composed of sugars with sodium on male sexual maturation results from an early induction of ISP causing an increase in JH biosynthesis followed by a potentiation of JH actions on the development of ASGs and ALs in A. ipsilon.

The brown planthoppers (BPH, Nilaparvata lugens), white backed planthopper (WBPH, Sogatella furcifera) and small brown planthopper (SBPH, Laodelphax striatellus) are widely distributed rice insect pests, causing huge annual yield loss of rice production. Though these three planthoppers belong to the same family, Delphacidae of Hemiptera, their genome sizes (GS) are very different, ranging from 541 to 1088 Mb. To uncover the main factors driving GS changes of three planthoppers, we first estimated the GS of their ancestor Fulgoroidea, to be 794.33 Mb, indicating GS expansion in BPH but contraction in SBPH and WBPH. Next, we identified repetitive sequences and compared the TE landscapes, showed that three types of transposon superfamilies, hAT, Tc1-Mariner and Gypsy, expanded within 25 Mya in BPH. In addition, BPH kept ancient TEs of Fulgoroidea dated back to 175 Mya, while SBPH and WBPH have lost most of these ancient TEs. Here, we present evidence that the gain of recently expanded TEs driving the GS expansion and loss of ancient TEs leading to the GS contraction, providing new insights into the mechanism of GS variation.

Nilaparvata lugens, the brown planthopper (BPH), is a notorious pest threatening rice production across Asia. The heavy reliance on synthetic insecticides for control has led to resistance and raised ecological concerns. Substrate-borne vibrational communication, integral to species-specific mate recognition systems in insects, presents a potential avenue for pest management through mating disruption. However, the molecular mechanisms regulating vibrational signals in BPH remain poorly understood. In this study, we cloned and analyzed the clock gene period from BPH. The open reading frame of Nlper is 3708 bp, encoding a 1235-amino acid protein with two conserved domains: the Per-ARNT-Sim domain and the Period protein 2/3C-terminal region. It shares a closer evolutionary relationship with Laodelphax striatellus and Frankliniella occidentalis. Spatiotemporal expression analysis showed that Nlper was consistently expressed across all life stages and adult tissues, with the highest levels in macropterous males and male head, respectively. Rhythmic expression exhibited significant circadian oscillations under both light-dark and constant darkness conditions, peaking at 00:00 and reaching a trough at 12:00, with fold changes ranging from 2.47 to 3.39. Moreover, after dsRNA injection, Nlper expression decreased by 77.21%-84.26% from day 2 to day 5, disrupting the circadian oscillation of female vibrational signals (FVS) and causing a significant peak shift, along with a 30.56% reduction in FVS frequency on day 5. These findings underscore the essential role of Nlper in regulating the circadian rhythm of courtship vibrational signals, deepening our understanding of the genetic basis of insect communication and opening new possibilities for innovative pest management approaches.

The evolution of insect metamorphosis has profoundly influenced their successful adaptation and diversification. Two key physiological processes during insect metamorphosis are notable: wing maturation and prothoracic gland (PG) histolysis. The ecdysone-induced protein 93 (E93) is a transcription factor indispensable for metamorphosis. While it has been established that both wing maturation and PG histolysis are dependent on E93, the molecular mechanisms through which E93 regulates these seemingly 'opposing' events remain poorly understood. In this study, time-course transcriptome profiles were generated for wing pads and PGs during metamorphosis in Blattella germanica, a hemimetabolous model insect. Comparative transcriptomic analyses demonstrated that E93 exerts predominant control over extensive gene transcription during wing morphogenesis and PG histolysis. During wing morphogenesis, E93 selectively enhances the expression of genes associated with cell proliferation, energy supply, signal transduction, actin cytoskeleton organization, and cell adhesion, etc. Additionally, E93 activates the transcription of the majority of genes within the wing gene network that are crucial for wing development in B. germanica. During PG histolysis, E93 preferentially promotes the expression of genes related to endocytosis, focal adhesion, the AMPK signaling pathway, adipocytokine signaling pathway, Toll and Imd signaling pathways, and autophagy, etc. The key genes involved in the aforementioned pathways were subsequently confirmed to contribute to the E93-dependent degeneration of the PG in B. germanica. In summary, our results reveal that E93 functions as a master transcriptional regulator orchestrating both tissue morphogenesis and histolysis during insect metamorphosis. These findings contribute to a deeper understanding of the genetic underpinnings of insect metamorphosis.

The olfactory system of insects plays a pivotal role in multiple, essential activities including feeding, mating, egg laying, and host localization. The capacity of odorant receptors to recognize odor molecules relies on odorant receptor co-receptors forming heterodimers. Here we report the successful engineering a homozygous mutant strain of diamondback moth (Plutella xylostella) in which the odorant receptor co-receptor PxOrco was silenced using CRISPR/Cas9. This insect is a globally important crop pest for which novel control methods are urgently required. Behavioral assays demonstrated that PxOrco knockout males exhibited abolished courtship behaviors, inability to mate, and loss of selective preference for P. xylostella's key sex pheromone components. Whilst female mating behavior and fecundity remained unaffected by PxOrco knockout, oviposition response to leaf alcohol, a key cue for normal oviposition behavior, was lost. Electroantennography revealed drastically reduced responses to sex pheromones and plant volatiles in PxOrco-deficient adults but food location by larvae was unaffected. Moreover, expression analysis of PxOrco-deficient pheromone receptors (PRs) indicated varied regulation patterns, with down-regulation observed in several PRs in both sexes. These findings underscore the critical role of PxOrco in regulating multiple olfactory aspects in P. xylostella, including feeding, mating, and host location. Our study identifies the potential of disrupting the Orco gene in this and other pest species to provide novel avenues for future pest control.

At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.