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The pathogenicity of avian pathogenic Escherichia coli (APEC) can be reliably determined using a biological model with 1-day-old chicks. However, public concerns regarding the use of vertebrates in research have led to the exploration of other biological models. Alternative infection models, including Galleria mellonella larvae, have also been studied. This study aimed to evaluate whether G. mellonella larvae are a viable in vivo model for evaluating the pathogenicity of APEC isolates and to determine the best protocol for this model. First, the pathogenicity index (PI) in vivo was determined for 40 APEC isolates from 1-day-old chicks. The same isolates were then used to establish the best protocol for the determination of PI in larvae of G. mellonella by determining the LD50, melanization, cytotoxicity test, total haemocyte count, mortality rate, construction of survival curves, and larval disease score. Cytotoxicity tests and total haemocyte counts were not suitable for distinguishing the pathogenicity groups. LD50 was a useful tool for differentiating between high and low pathogenicity but not for intermediate pathogenicity. Similarly, melanization could be used as a variable to characterize the pathogenicity of APEC isolates but should be associated with other variables. Mortality rates and survival curves were useful tools for characterizing nonpathogenic and highly pathogenic isolates. In this study, G. mellonella larvae were a viable model, and the larval disease score was the main variable that determined the pathogenicity of APEC isolates in the G. mellonella model. RESEARCH HIGHLIGHTS: Galleria mellonella larvae are a viable model for determining APEC pathogenicity. Larval disease score is the main variable for determining APEC pathogenicity. Response variables should be evaluated up to 24 h post-inoculation. [ABSTRACT FROM AUTHOR]

Silver oxide nanoparticles (Ag2O NPs) find enormous applications in electronics, industry, biomedical, pharmaceutical field, etc. In the present study, we have focused on yet unrevealed property of these nanoparticles to exert melanin modulatory effects in Drosophila. Silver nanoparticles (AgNPs) are known for its toxicity and alteration in body pigmentation by various mechanisms such as by binding with precursor amines (tyrosine, dopa) and by making phenol oxidase (PO) non-functional when applied in high concentration. This raises a concern whether its oxide will have the same biological effects as that of the original metallic form or not. To address this, the impact of Ag2O NPs on Drosophila was studied focusing on its melanizing modulatory potential. The present study highlights the quasi-reversal of depigmentation when silver is present in its oxide form. Surprisingly, when silver is in its oxide form, it interacts unusually with the components of the melanization pathway producing an altered phenotype. As revealed in the present study, phenol oxidase activity and circulating levels of tyrosine (which are targeted by elemental silver) were found to be unaffected upon treatment with Ag2O NPs. FTIR studies revealed the minimal interaction of silver oxide with tyrosine due to which it was available for further reactions of melanin production and restored the full enzymatic activity of phenol oxidase. However, dopa which is another target for elemental silver was found to be interacting strongly with Ag2O NPs, and hence, it was not available for melanization. The availability of one amine (tyrosine) but the unavailability of the second (dopa) led to partial de-melanization in flies. Such unusual interplay of an oxide form of silver with the melanization pathway inducing reversal of completely de-pigmented phenotype to partially de-pigmented phenotype has not been reported earlier. Considering the multifarious role of melanin in the fitness and survival of insects in particular, the implication of Ag2O as a melanin modulator is vindicated. [ABSTRACT FROM AUTHOR]

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye‐staining properties were used to characterize hemocyte types from T. castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2 h), then increased over time (at 4 and 6 h) and eventually returned to the naive state by 24 h post‐injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T. castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph in vivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes in vitro, forming capsules to encapsulate and melanize nickel–nitrilotriacetic acid (Ni–NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T. castaneum larvae, offering valuable insights into cell‐mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T. castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T. castaneum hemocytes in combating pathogens. [ABSTRACT FROM AUTHOR]

Nowadays, particularly metallic, and polymeric nanoparticles (NPs) are widely produced and used in many fields. Due to the increase in both their usage and diversity, their release and accumulation in the environment are also accelerating. Therefore, their interactions with cells, especially immune cells, and their health risks are not fully understood. The impacts of metallic alumina (Al) NPs and polystyrene (PS) NPs obtained after the polymerization of carcinogenic styrene on living organisms have not yet been elucidated. Galleria mellonella larvae can biodegrade plastics. While biodegradation and solving the waste problem have attracted much attention, the interactions of this distinctive property of G. mellonella larvae in the immune system and ecosystem are not yet completely understood. Al and PS NPs were applied to G. mellonella separately. Al NPs were purchased and PS NPs were prepared from PS by single-emulsion technique and characterized. Then LC50 values of these NPs on G. mellonella were determined. The interactions of these NPs with encapsulation, melanization, and phenoloxidase activity, which express innate immune responses in G. mellonella larvae, were revealed. NP exposure resulted in suppression of the immune response, probably because it affects the functions of hemocytes such as enzymatic activation, hemocyte division, and populations. In this context, our data suggest that Al and PS NPs induce toxic impacts and negatively alter the physiological status of G. mellonella. It is also shown that G. mellonella has the potential to be an impactful alternative model for biosafety and nanotoxicology studies. [ABSTRACT FROM AUTHOR]

The primary function of the skin is to form a mechanical, permeability, antimicrobial, and ultraviolet radiation barrier, which is essential for maintaining physiological homeostasis. Our previous studies demonstrated that cutaneous pigmentation could promote skin barrier function in addition to providing anti‐ultraviolet irradiation defense. The present study aimed to develop a new regimen that enhances skin barrier function by regulating skin pigmentation using low‐concentration imiquimod. Results showed that topical application of low‐concentration imiquimod effectively induced skin hyperpigmentation in the dorsal skin and external ear of mice without inducing inflammatory cell infiltration. An in vitro study also revealed that low‐concentration imiquimod did not induce any cytotoxic effects on melanoma cells but triggered excessive melanin synthesis. In coculture systems, low‐concentration imiquimod was noted to increase tyrosinase activity in a broader cellular context, revealing the potential role of neighboring cells in melanin production. The next‐generation sequencing result indicated that PKCη and Dnm3 might regulate melanin synthesis and release during imiquimod treatment. Overall, our study presents new insights into the regulation of melanin production by low‐concentration imiquimod, both in a mice model and cultured cells. Furthermore, our study highlights the potential benefits of imiquimod in promoting melanin synthesis without causing skin disruptions or inducing inflammation, validating its potential to serve as a method for enhancing skin barrier functions by regulating the epidermal melanization reaction. [ABSTRACT FROM AUTHOR]

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers selfproperties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanizationbased immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only latestage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes frommelanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGNdegrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms. [ABSTRACT FROM AUTHOR]

Lichens are extremophilic symbiotic associations possessing phenomenal resistance to abiotic stress-factors. In this regard, melanization of thalli in response to UV is one of the mechanisms protecting lichens from excessive insolation. However, microstructure and biochemical properties of the melanized thalli are still poorly investigated. In the present study, morphological, nanomechanical, and physiological, and biochemical traits of naturally melanized thalli of the Cetraria islandica (L.) Ach. lichen were examined. In the upper cortex of its thallus, the nature of the pigment layer was verified using typical qualitative reactions for melanins. It was found that melanization leads to changes in microstructure of the upper cortex of the mycobiont, in particular, thickening of the cell walls and extension of the interhyphal space. The melanized and pale (nonmelanized) thalli were found to differ from each other in their nanomechanical properties, including the parameters of adhesion and rigidity. This implies the possible formation of complex associates of melanin with cell wall components in the melanized mycobiont. In addition, higher antioxidant activity and lower respiratory activity were found in the melanized thalli of C. islandica in comparison with the pale thalli. Presumably, the found modifications in the microstructure and nanomechanical, physiological, and biochemical properties of thalli occurring in the course of melanization make lichens more resistant to intense insolation. [ABSTRACT FROM AUTHOR]

Medically, the most dangerous genera are Anopheles, Aedes and Culex due to their ability to transmit mosquito-borne human diseases, such as malaria, yellow fever, chikungunya, lymphatic filariasis, dengue fever, hemorrhagic fever and encephalitis. Mosquito control is a prerequisite work for fighting the increasing mosquito populations and mosquito-borne diseases. Generally, chemical insecticides have been widely used to suppress mosquito populations but these chemicals were evidenced to be less-than-perfect solutions for the long-lasting control of mosquitoes due to the development of insecticide resistance in all mosquito species and different hazards all over the world. Therefore, the search for alternative agents is urgent to avoid or minimize the insecticidal hazards on ecosystem components. One of the potential alternatives is biological control which received great research attention in the world. One of these biocontrol agents is Entomopathogenic Nematodes (EPNs). Therefore, the objective of the present article was primarily to review the current knowledge focusing on the use of EPNs for controlling mosquito vectors. It highlighted the global attention to EPN application as a novel strategy for combating mosquito vectors. Also, this review discussed the pathogenic efficiencies of three EPNs, Mermithidae, Steinernematidae and Heterorhabditidae. Other important issues had been reviewed, such as diverse susceptibility of mosquito larvae and variation of EPN virulence, Key parameters and environmental conditions affecting the virulence of EPNs against mosquito larvae, EPN-symbiotic bacteria and their potential role for the control of mosquito vectors and Suppressive activity of EPN/symbiotic bacteria complex against innate immune defences of mosquito larvae. This review provided, also, some future prospects for the EPN application for controlling mosquito vectors. In conclusion, EPNs can be equal to chemical pesticides against human-disease-transmitting mosquitoes if they are applied in critical concentration under suitable environmental conditions and precautions for application. [ABSTRACT FROM AUTHOR]

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth‐blocking peptide (GBP) is a member of the stress‐responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity‐related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens. [ABSTRACT FROM AUTHOR]

Background: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. Methods and results: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. Conclusions: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection. [ABSTRACT FROM AUTHOR]

Objective: This study aims to assess the clinical characteristics of sporotrichosis in low‐endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. Methods: Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. In vitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. Results: In our low‐endemic region for sporotrichosis, the majority of cases (23) were observed in middle‐aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose‐dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower in vitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. Conclusion: Given the predominance of elderly women engaged in agricultural labour in our region, which is a low‐epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first‐line antifungal options for treating S.globosa infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect’s innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Abstract Background Tomato leaf miner (Tuta absoluta) is a quarantined pest that damages Solanaceae crops worldwide. The overuse of traditional pesticides negatively affects both human health and the environment. RNA interference (RNAi), based on double-stranded RNA (dsRNA) induction, can be beneficial in the control of Tuta absoluta; one of the key points of using this technique is the selection of target genes. Exploring the ecdysone receptors (EcR) associated with the growth and development of tomato leaf miners is an important research topic and the primary aim of this study. Methods In this study, RNA extraction, cDNA synthesis, gene cloning, bioinformatics analysis, and quantitative real-time polymerase chain reaction were used to obtain the full length, conserved domain, and relative expression levels of the EcR. RNAi was used to explore the effects of EcR on larval growth and development, pupal weight, and emergence rate. Results The full-length cDNA of T. absoluta TaEcR was 1859 bp, and the coding region including the ZnF_C4 and HOLI domains was relatively conserved. The relative expression of EcR in the early pupal stage was substantially higher than that in the other instars. Approximately 70% of TaEcR RNAi larvae died or pupated abnormally. In the few successful pupations, the pupa weights were substantially lower (36.44%) than those of the control group. The color of the pupae was abnormal, and they did not enter their normal black state; the emergence rate of pupae was reduced by 43.45% compared to that of the control group. Conclusion These results indicate that TaEcR inhibition can affect larval metamorphosis, pupation, melanism, eclosion abnormalities, and, ultimately, lead to death. The results of this study suggest that TaEcR may be a candidate factor for developing environmentally-friendly RNAi pesticides that have practical value in field control.

Simple Summary: Endoparasitic wasps are nature's pest controllers, targeting various harmful insects. They lay their eggs inside these pests and release special substances (like viruses or venoms) that change the way the pest's immune system works. However, we are still uncovering exactly how these changes happen. Our research looks into how certain wasps, which do not use these viruses, can still affect their host's immune system. We studied this using a specific wasp–beetle pair as an example. We found that these wasps can control a crucial part of the beetle's immune response through a process that modifies the beetle's genetic material, specifically by adjusting certain markers on the genes that are important for immunity. This discovery sheds light on the sophisticated ways wasps can suppress the immune systems of their hosts, making them more effective at controlling pest populations. Endoparasitoids are insects that develop within other insects, employing unique strategies to enhance their offspring's survival. They inject polydnavirus and/or venom into their hosts along with eggs, effectively suppressing the host's immune system. Polydnavirus from Braconidae and Ichneumonidae wasps can integrate into the host's genome to express viral genes using the host's transcription systems. However, the ability of parasitoids without polydnavirus to manipulate host gene expression remains unclear. Lysine acetylation (LysAc), a post-translational modification critical for gene regulation, is hypothesized to be used by endoparasitoids lacking polydnavirus. We utilized the Chalcidoidea wasp Tetrastichus brontispae, which lacks polydnavirus, as an idiobiont endoparasitoid model to test this hypothesis, with pupae of the nipa palm hispid beetle Octodonta nipae as the host. Parasitism by T. brontispae resulted in the reduced expression of histone deacetylase Rpd3 and elevated levels of LysAc modification at histones H3.3K9 and H3.3K14 through proteomics and LysAc modification omics. The knockdown of Rpd3 increased the expression level of OnPPAF1 and OnPPO involved in the phenoloxidase cascade, leading to melanization in the host body whereby it resembled a mummified parasitized pupa and ultimately causing pupa death. This study enhances our understanding of how endoparasitoids employ histone acetylation to regulate immunity-related genes, offering valuable insights into their survival strategies. [ABSTRACT FROM AUTHOR]

Spatial transcriptomics is a technique that provides insight into gene expression profiles in tissue sections while retaining structural information. We have employed this method to study the pathological conditions related to red and melanized focal changes in farmed Atlantic salmon (Salmo salar). Our findings support a model where similar molecular mechanisms are involved in both red and melanized filet discolorations and genes associated with several relevant pathways show distinct expression patterns in both sample types. Interestingly, there appears to be significant cellular heterogeneity in the foci investigated when looking at gene expression patterns. Some of the genes that show differential spatial expression are involved in cellular processes such as hypoxia and immune responses, providing new insight into the nature of muscle melanization in Atlantic salmon. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play a pivotal role in important biological processes by regulating post-transcriptional gene expression and exhibit differential expression patterns during development, immune responses, and stress challenges. The diamondback moth causes significant economic damage to crops worldwide. Despite substantial advancements in understanding the molecular biology of this pest, our knowledge regarding the role of miRNAs in regulating key immunity-related genes remains limited. In this study, we leveraged whole transcriptome resequencing data from Plutella xylostella infected with Metarhizium anisopliae to identify specific miRNAs targeting the prophenoloxidase-activating protease1 (PAP1) gene and regulate phenoloxidase (PO) cascade during melanization. Seven miRNAs (pxy-miR-375-5p, pxy-miR-4448-3p, pxy-miR-279a-3p, pxy-miR-3286-3p, pxy-miR-965-5p, pxy-miR-8799-3p, and pxy-miR-14b-5p) were screened. Luciferase reporter assays confirmed that pxy-miR-279a-3p binds to the open reading frame (ORF) and pxy-miR-965-5p to the 3′ untranslated region (3′ UTR) of PAP1. Our experiments demonstrated that a pxy-miR-965-5p mimic significantly reduced PAP1 expression in P. xylostella larvae, suppressed PO activity, and increased larval mortality rate. Conversely, the injection of pxy-miR-965-5p inhibitor could increase PAP1 expression and PO activity while decreasing larval mortality rate. Furthermore, we identified four LncRNAs (MSTRG.32910.1, MSTRG.7100.1, MSTRG.6802.1, and MSTRG.22113.1) that potentially interact with pxy-miR-965-5p. Interference assays using antisense oligonucleotides (ASOs) revealed that silencing MSTRG.7100.1 and MSTRG.22113.1 increased the expression of pxy-miR-965-5p. These findings shed light on the potential role of pxy-miR-965-5p in the immune response of P. xylostella to M. anisopliae infection and provide a theoretical basis for biological control strategies targeting the immune system of this pest. [ABSTRACT FROM AUTHOR]

Abstract Background Patinopecten yessoensis, a large and old molluscan group, has been one of the most important aquaculture shellfish in Asian countries because of its high economic value. However, the aquaculture of the species has recently been seriously affected by the frequent outbreaks of Polydora disease, causing great economic losses. Long non-coding RNAs (lncRNAs) exhibit exhibit crucial effects on diverse biological processes, but still remain poorly studied in scallops, limiting our understanding of the molecular regulatory mechanism of P. yessoensis in response to Polydora infestation. Results In this study, a high-throughput transcriptome analysis was conducted in the mantles of healthy and Polydora-infected P. yessoensis by RNA sequencing. A total of 19,133 lncRNAs with 2,203 known and 16,930 novel were identified. The genomic characterizations of lncRNAs showed shorter sequence and open reading frame (ORF) length, fewer number of exons and lower expression levels in comparison with mRNAs. There were separately 2280 and 1636 differentially expressed mRNAs and lncRNAs (DEGs and DELs) detected in diseased individuals. The target genes of DELs were determined by both co-location and co-expression analyses. Functional enrichment analysis revealed that DEGs involved in melanization and biomineralization were significantly upregulated; further, obviously increased melanin granules were observed in epithelial cells of the edge mantle in diseased scallops by histological and TEM study, indicating the crucial role of melanizaiton and biomineralization in P. yessoensis to resist against Polydora infestation. Moreover, many key genes, such as Tyrs, Frizzled, Wnts, calmodulins, Pifs, perlucin, laccase, shell matrix protein, mucins and chitins, were targeted by DELs. Finally, a core lncRNA-mRNA interactive network involved in melanization and biomineralization was constructed and validated by qRT-PCR. Conclusions This work provides valuable resources for studies of lncRNAs in scallops, and adds a new insight into the molecular regulatory mechanisms of P. yessoensis defending against Polydora infestation, which will contribute to Polydora disease control and breeding of disease-resistant varieties in molluscs.

Abstract Background Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti, coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. Results In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. Conclusions While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Cellular encapsulation associated with melanization is a crucial component of the immune response in insects, particularly against larger pathogens. The infection of a Drosophila larva by parasitoid wasps, like Leptopilina boulardi, is the most extensively studied example. In this case, the encapsulation and melanization of the parasitoid embryo is linked to the activation of plasmatocytes that attach to the surface of the parasitoid. Additionally, the differentiation of lamellocytes that encapsulate the parasitoid, along with crystal cells, is accountable for the melanization process. Encapsulation and melanization lead to the production of toxic molecules that are concentrated in the capsule around the parasitoid and, at the same time, protect the host from this toxic immune response. Thus, cellular encapsulation and melanization represent primarily a metabolic process involving the metabolism of immune cell activation and differentiation, the production of toxic radicals, but also the production of melanin and antioxidants. As such, it has significant implications for host physiology and systemic metabolism. Proper regulation of metabolism within immune cells, as well as at the level of the entire organism, is therefore essential for an efficient immune response and also impacts the health and overall fitness of the organism that survives. The purpose of this "perspective" article is to map what we know about the metabolism of this type of immune response, place it in the context of possible implications for host physiology, and highlight open questions related to the metabolism of this important insect immune response. [ABSTRACT FROM AUTHOR]

Insect immune‐associated phospholipase A2 (PLA2) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA2 on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA2 gene (BmsPLA2), and its protein had characteristic features of secreted PLA2 (sPLA2). After injection of bacteria, BmsPLA2 expression and sPLA2 activity in hemolymph significantly increased. BmsPLA2 fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA2 was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA2 by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA2 overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA2 inhibitor dosage. These results demonstrated that BmsPLA2 plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA2 after injection of bacteria, implying that BmsPLA2 was activated prior to BmDDC. Moreover, the alteration of BmsPLA2 by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA2 regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects. [ABSTRACT FROM AUTHOR]

Background: Patinopecten yessoensis, a large and old molluscan group, has been one of the most important aquaculture shellfish in Asian countries because of its high economic value. However, the aquaculture of the species has recently been seriously affected by the frequent outbreaks of Polydora disease, causing great economic losses. Long non-coding RNAs (lncRNAs) exhibit exhibit crucial effects on diverse biological processes, but still remain poorly studied in scallops, limiting our understanding of the molecular regulatory mechanism of P. yessoensis in response to Polydora infestation. Results: In this study, a high-throughput transcriptome analysis was conducted in the mantles of healthy and Polydora-infected P. yessoensis by RNA sequencing. A total of 19,133 lncRNAs with 2,203 known and 16,930 novel were identified. The genomic characterizations of lncRNAs showed shorter sequence and open reading frame (ORF) length, fewer number of exons and lower expression levels in comparison with mRNAs. There were separately 2280 and 1636 differentially expressed mRNAs and lncRNAs (DEGs and DELs) detected in diseased individuals. The target genes of DELs were determined by both co-location and co-expression analyses. Functional enrichment analysis revealed that DEGs involved in melanization and biomineralization were significantly upregulated; further, obviously increased melanin granules were observed in epithelial cells of the edge mantle in diseased scallops by histological and TEM study, indicating the crucial role of melanizaiton and biomineralization in P. yessoensis to resist against Polydora infestation. Moreover, many key genes, such as Tyrs, Frizzled, Wnts, calmodulins, Pifs, perlucin, laccase, shell matrix protein, mucins and chitins, were targeted by DELs. Finally, a core lncRNA-mRNA interactive network involved in melanization and biomineralization was constructed and validated by qRT-PCR. Conclusions: This work provides valuable resources for studies of lncRNAs in scallops, and adds a new insight into the molecular regulatory mechanisms of P. yessoensis defending against Polydora infestation, which will contribute to Polydora disease control and breeding of disease-resistant varieties in molluscs. [ABSTRACT FROM AUTHOR]

In this study, we examined the effects of different doses (100, 500, 1000, 3000, and 5000 ppm) of zinc oxide nanoparticles (ZnO NPs) on the total hemocyte count and hemocyte-mediated immune responses of the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae). The results showed that NPs caused a decrease in hemocyte count at 1000, 3000, and 5000 ppm doses. To investigate the effects of ZnO NPs on the encapsulation and melanization response of G. mellonella, the pre-dyed Sephadex chromatography beads were injected into the hemolymph of each last-instar larva. Larvae were dissected in the 4th and 24th hours after the injection. The level of the encapsulation response and melanization status around the beads were determined under microscopy. The analyses of the beads injected into the insects as encapsulation targets revealed that the number of weakly encapsulated beads increased significantly at 100, 1000, 3000, and 5000 ppm doses when compared to the control group after a short (4-h) post-injection. The number of melanized beads increased significantly at 100, 1000, and 3000 ppm doses in comparison to the control group after the short (4-h) post-injection. Finally, the number of melanized beads decreased significantly at 1000 and 5000 ppm doses when compared to the control group after the long-term (24-h) post-injection. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) appear to be ubiquitous in almost all living organisms, with a conserved structure and varying functions. Serpins can modulate immune responses by negatively regulating serine protease activities strictly and precisely. The codling moth, Cydia pomonella (L.), a major invasive pest in China, can cause serious economic losses. However, knowledge of serpin genes in this insect remain largely unknown. In this study, we performed a systematic analysis of the serpin genes in C. pomonella, obtaining 26 serpins from the C. pomonella genome. Subsequently, their sequence features, evolutionary relationship, and expression pattern were characterized. Comparative analysis revealed the evolution of a number of serpin genes in Lepidoptera. Importantly, the evolutionary relationship and putative roles of serpin genes in C. pomonella were revealed. Additionally, selective pressure analysis found amino acid sites with strong evidence of positive selection. Interestingly, the serpin1 gene possessed at least six splicing isoforms with distinct reactive-center loops, and these isoforms were experimentally validated. Furthermore, we observed a subclade expansion of serpins, and these genes showed high expression in multiple tissues, suggesting their important roles in C. pomonella. Overall, this study will enrich our knowledge of the immunity of C. pomonella and help to elucidate the role of serpins in the immune response. [ABSTRACT FROM AUTHOR]

Marine-capture shrimp is a general term for a type of shrimp that is different from marine-aquaculture shrimp. It is rich in protein, trace elements, and essential amino acids. It is an aquatic product with delicious meat and high nutritional and economic value. Most marine-capture shrimp are consumed fresh, with a small proportion subjected to processing. However, marine-capture shrimp is a highly perishable aquatic product, and hence, extensive preservation and processing methods have been developed to extend its shelf life. This review presents the current status of marine-capture shrimp in the world and the key issues (spoilage and melanization inhibition) regarding marine-capture shrimp preservation. Low-temperature preservation remains the predominant strategy for preserving marine-capture shrimp. Different preservation strategies combined with low temperature would maintain the quality of marine-capture shrimp more effectively, thereby extending the shelf life of the shrimp. The advantages and disadvantages of various preservation technologies are comprehensively considered and compared, as well as their scope of application. New processing technologies should be introduced, and various preservation technologies with complementary advantages should be organically combined to achieve the best preservation effect on marine-capture shrimp. This review provides a useful theoretical reference and a practical basis for future research on the shelf life extension of marine-capture shrimp. [ABSTRACT FROM AUTHOR]

Background: Up to 40% of the world population live in areas where mosquitoes capable of transmitting the dengue virus, including Aedes aegypti, coexist with humans. Understanding how mosquito egg development and oviposition are regulated at the molecular level may provide new insights into novel mosquito control strategies. Previously, we identified a protein named eggshell organizing factor 1 (EOF1) that when knocked down with RNA interference (RNAi) resulted in non-melanized and fragile eggs that did not contain viable embryos. Results: In this current study, we performed a comprehensive RNAi screen of putative A. aegypti eggshell proteins to identify additional proteins that interact with intracellular EOF1. We identified several proteins essential for eggshell formation in A. aegypti and characterized their phenotypes through a combination of molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel messenger RNA (mRNA) expression is highly upregulated in response to blood feeding. Furthermore, we identified four additional secreted eggshell enzymes that regulated mosquito eggshell formation and melanization. These enzymes included three dopachrome-converting enzymes (DCEs) and one cysteine protease. All eight of these eggshell proteins were essential for proper eggshell formation. Interestingly, their eggshell surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1, suggesting that additional mechanisms may influence how EOF1 regulates eggshell formation and melanization. Conclusions: While our studies did not identify a definitive regulator of EOF1, we did identify eight additional proteins involved in mosquito eggshell formation that may be leveraged for future control strategies. [ABSTRACT FROM AUTHOR]

DNA vaccine, melanization, pancreas disease, salmonid alphavirus, side-effect, vaccination (d) Retraction of the dnv to allow entrance of the next fish for vaccination. gl During vaccination with automatic vaccination machines, Clynav® is often administered simultaneously with i.p. administered oil-based vaccines. Keywords: DNA vaccine; melanization; pancreas disease; salmonid alphavirus; side-effect; vaccination EN DNA vaccine melanization pancreas disease salmonid alphavirus side-effect vaccination 1157 1161 5 09/15/23 20231001 NES 231001 Pancreas disease (PD) is a severe infectious disease affecting farmed salmonids in Norway and other European countries, caused by different genotypes of salmonid alphavirus (SAV) (World Organization for Animal Health, [21]). To the authors' knowledge, this is the first description of this phenomenon following vaccination with dual vaccination in Atlantic salmon. [Extracted from the article]

Abstract Background As one of the components of visual photopigments in photoreceptor cells, opsin exhibits different spectral peaks and plays crucial roles in visual function. Besides, it is discovered to evolve other functions despite color vision. However, research on its unconventional function is limited nowadays. With the increase in genome database numbers, various numbers and types of opsins have been identified in insects due to gene duplications or losses. The Nilaparvata lugens (Hemiptera) is a rice pest known for its long-distance migration capability. In this study, opsins were identified in N. lugens and characterized by genome and transcriptome analyses. Meanwhile, RNA interference (RNAi) was carried out to investigate the functions of opsins, and then the Illumina Novaseq 6000 platform-based transcriptome sequencing was performed to reveal gene expression patterns. Results Four opsins belonging to G protein-coupled receptors were identified in the N. lugens genome, including one long-sensitive opsin (Nllw) together with two ultraviolet-sensitive opsins (NlUV1/2) and an additional new opsin with hypothesized UV peak sensitivity (NlUV3-like). A tandem array of NlUV1/2 on the chromosome suggested the presence of a gene duplication event, with similar exons distribution. Moreover, as revealed by spatiotemporal expression, the four opsins were highly expressed in eyes with age-different expression levels. Besides, RNAi targeting each of the four opsins did not significantly affect the survival of N. lugens in phytotron, but the silencing of Nllw resulted in the melanization of body color. Further transcriptome analysis revealed that silencing of Nllw resulted in up-regulation of a tyrosine hydroxylase gene (NlTH) and down-regulation of an arylalkylamine-N-acetyltransferases gene (NlaaNAT) in N. lugens, demonstrating that Nllw is involved in body color plastic development via the tyrosine-mediated melanism pathway. Conclusions This study provides the first evidence in a Hemipteran insect that an opsin (Nllw) takes part in the regulation of cuticle melanization, confirming a cross-talk between the gene pathways underlying the visual system and the morphological differentiation in insects.

Cellular encapsulation associated with melanization is a crucial component of the immune response in insects, particularly against larger pathogens. The infection of a Drosophila larva by parasitoid wasps, like Leptopilina boulardi, is the most extensively studied example. In this case, the encapsulation and melanization of the parasitoid embryo is linked to the activation of plasmatocytes that attach to the surface of the parasitoid. Additionally, the differentiation of lamellocytes that encapsulate the parasitoid, along with crystal cells, is accountable for the melanization process. Encapsulation and melanization lead to the production of toxic molecules that are concentrated in the capsule around the parasitoid and, at the same time, protect the host from this toxic immune response. Thus, cellular encapsulation and melanization represent primarily a metabolic process involving the metabolism of immune cell activation and differentiation, the production of toxic radicals, but also the production of melanin and antioxidants. As such, it has significant implications for host physiology and systemic metabolism. Proper regulation of metabolism within immune cells, as well as at the level of the entire organism, is therefore essential for an efficient immune response and also impacts the health and overall fitness of the organism that survives. The purpose of this “perspective” article is to map what we know about the metabolism of this type of immune response, place it in the context of possible implications for host physiology, and highlight open questions related to the metabolism of this important insect immune response.

Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high Mr cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. In this study, we produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high Mr complexes of the cSPHs, PO at a high specific activity of 260 U/mg was generated in vitro. To complement the in vitro analysis, we measured hemolymph PO activity levels in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/ml) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2- mediated PPO1 activation, which are indispensable for early melanization in adults. [ABSTRACT FROM AUTHOR]

Catecholamine metabolites are not only involved in primary metabolism, but also in secondary metabolism, serving a diverse array of physiologically and biochemically important functions. Melanin, which originates from dopa and dopamine, found in the hair, eye, and skin of all animals, is an important biopolymeric pigment. It provides protection against damaging solar radiation to animals. N-Acetyldopamine and N-β-alanyldopamine play a crucial role in the hardening of the exoskeletons of all insects. In addition, insects and other arthropods utilize the melanogenic process as a key component of their defense systems. Many marine organisms utilize dopyl peptides and proteins as bonding materials to adhere to various substrata. Moreover, the complex dopa derivatives that are precursors to the formation of the exoskeletons of numerous marine organisms also exhibit antibiotic properties. The biochemistry and mechanistic transformations of different catecholamine derivatives to produce various biomaterials with antioxidant, antibiotic, crosslinking, and gluing capabilities are highlighted. These reactivities are exhibited through the transient and highly reactive quinones, quinone methides, and quinone methide imine amide intermediates, as well as chelation to metal ions. A careful consideration of the reactivities summarized in this review will inspire numerous strategies for synthesizing novel biomaterials for future medical and industrial use. [ABSTRACT FROM AUTHOR]

Candida auris, a multidrug-resistant fungal pathogen, significantly threatens global public health. Recent studies have identified melanin production, a key virulence factor in many pathogenic fungi that protects against external threats like reactive oxygen species, in C. auris. However, the melanin regulation mechanism remains elusive. This study explores the role of the Ras/cAMP/PKA signaling pathway in C. auris melanization. It reveals that the catalytic subunits Tpk1 and Tpk2 of protein kinase A (PKA) are essential, whereas Ras1, Gpr1, Gpa2, and Cyr1 are not. Under melanin-promoting conditions, the tpk1Δ tpk2Δ strain formed melanin granules in the supernatant akin to the wild-type strain but failed to adhere them properly to the cell wall. This discrepancy is likely due to a decreased expression of chitin-synthesis-related genes. Our findings also show that Tpk1 primarily drives melanization, with Tpk2 having a lesser impact. To corroborate this, we found that C. auris must deploy Tpk1-dependent melanin deposition as a defensive mechanism against antioxidant exposure. Moreover, we confirmed that deletion mutants of multicopper oxidase and ferroxidase genes, previously assumed to influence C. auris melanization, do not directly contribute to the process. Overall, this study sheds light on the role of PKA in C. auris melanization and enhances our understanding of the pathogenicity mechanisms of this emerging fungal pathogen. [ABSTRACT FROM AUTHOR]

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We found that increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: The tachinid fly Drino inconspicuoides parasitizes various lepidopteran larvae, including Noctuidae and Nymphalidae. The adult female oviposits the eggs on the surface of the host, and the hatched larvae immediately penetrate into the host hemocoel and develop there during the larval stage. The caterpillar possesses immune defense mechanisms to eliminate such invaders, but the tachinid larva can avoid these defenses. Within 24 h, the parasitizing larva is surrounded by a black pigmented structure called the "funnel", and the larva and the funnel are wrapped with a cottony structure called the "cloak". The funnel is a physically rigid structure that penetrates the epidermis of the host, allowing the larva to breathe through contact with the outside air. This study aimed to clarify the function of the cloak, which has not previously been elucidated. Our findings suggest that formation of the funnel generates ROS, but that these are detoxified in the cloak to enhance the survival of the tachinid larva. The funnel and cloak are composed of host cells. Our study provides insight into the parasitic strategy of a tachinid fly by through which it ingeniously diverts host cells. Drino inconspicuoides (Diptera: Tachinidae) is an endoparasitoid that develops inside the lepidopteran host. When the larva of D. inconspicuoides penetrates into the host, Mythimna separata (Lepidoptera: Noctuidae), the larva creates a cap-like structure, called the funnel, by using host hemocytes, forming a respiratory attachment to permit efficient respiration. A newly described cloudy and cottony structure, called the "cloak", is formed outside the funnel within 24 h of parasitism. The cloak contains the host fat body and hemocytes. In this study, we aimed to clarify the function of the cloak, which has to date remained unknown. We hypothesized that the funnel generates reactive oxygen species (ROS) through melanization, and that the cloak detoxifies them. We confirmed that the black pigments of the funnel were caused by melanization, which inevitably generates ROS that are potentially harmful to the D. inconspicuoides larva inside the funnel. The cloak showed high activities of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase. These results suggest that the cloak scavenged the ROS from the melanized funnel through the diversion of antioxidant enzymes in the fat body, thereby protecting the D. inconspicuoides larva from oxidative damage. [ABSTRACT FROM AUTHOR]

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.

Clip domain serine proteases and clip domain serine protease homologs (cSPHs) are key components of serine protease cascades that drive the melanization response. Despite lacking catalytic activity, cSPHs play essential roles in regulating melanization, but the spectrum of functions they catalyze within and outside these cascades is not fully understood. Aside from their classical role as cofactors for PPO activation, we have previously revealed an unprecedented complexity in the function and molecular organization of these cSPHs in the immune response of the malaria vector Anopheles gambiae. Here, we add yet another dimension to the complex roles underpinning the contributions of cSPHs to mosquito immunity by showing that CLIPA7, a member of the expanded cSPH family, defines a novel branch within the cSPH network that is essential for the melanization of Escherichia coli but not Plasmodium ookinetes or Gram-positive bacteria. Despite its dispensability for the melanization of Gram-positive bacteria, we show that CLIPA7 is required for the clearance of systemic infections with Staphylococcus aureus. CLIPA7 is produced by hemocytes and associates with the surfaces of live E. coli and S. aureus cells in vivo as well as with those of melanized cells. Based on its RNAi phenotypes and its unique domain architecture among A. gambiae cSPHs including the presence of an RGD motif, we propose that CLIPA7 exhibits pleiotropic roles in mosquito immunity that extend beyond the regulation of melanization to microbial clearance.

Background: As one of the components of visual photopigments in photoreceptor cells, opsin exhibits different spectral peaks and plays crucial roles in visual function. Besides, it is discovered to evolve other functions despite color vision. However, research on its unconventional function is limited nowadays. With the increase in genome database numbers, various numbers and types of opsins have been identified in insects due to gene duplications or losses. The Nilaparvata lugens (Hemiptera) is a rice pest known for its long-distance migration capability. In this study, opsins were identified in N. lugens and characterized by genome and transcriptome analyses. Meanwhile, RNA interference (RNAi) was carried out to investigate the functions of opsins, and then the Illumina Novaseq 6000 platform-based transcriptome sequencing was performed to reveal gene expression patterns. Results: Four opsins belonging to G protein-coupled receptors were identified in the N. lugens genome, including one long-sensitive opsin (Nllw) together with two ultraviolet-sensitive opsins (NlUV1/2) and an additional new opsin with hypothesized UV peak sensitivity (NlUV3-like). A tandem array of NlUV1/2 on the chromosome suggested the presence of a gene duplication event, with similar exons distribution. Moreover, as revealed by spatiotemporal expression, the four opsins were highly expressed in eyes with age-different expression levels. Besides, RNAi targeting each of the four opsins did not significantly affect the survival of N. lugens in phytotron, but the silencing of Nllw resulted in the melanization of body color. Further transcriptome analysis revealed that silencing of Nllw resulted in up-regulation of a tyrosine hydroxylase gene (NlTH) and down-regulation of an arylalkylamine-N-acetyltransferases gene (NlaaNAT) in N. lugens, demonstrating that Nllw is involved in body color plastic development via the tyrosine-mediated melanism pathway. Conclusions: This study provides the first evidence in a Hemipteran insect that an opsin (Nllw) takes part in the regulation of cuticle melanization, confirming a cross-talk between the gene pathways underlying the visual system and the morphological differentiation in insects. [ABSTRACT FROM AUTHOR]

Insect herbivores simultaneously experience bottom‐up effects of plant defensive chemistry and the top‐down effects of natural enemies. At the intersection of these effects are herbivore immune systems, herbivore traits that have largely been overlooked in studies of plant‐insect interactions. Most previous studies have demonstrated compromised immunity of herbivores that feed on plants with higher defensive chemistry. Many studies have used embedded microfilaments or silica beads as proxies for parasitoid eggs. Yet, parasitoids may evade or suppress host immune responses by injecting venom and calyx fluid, or through modifications of their egg surface structure, necessitating studies that include all three trophic levels to obtain a complete picture of how plant traits may modulate herbivore immunity.Here, we examined the effect of host plant species that differ in glucosinolate (anti‐herbivore compounds produced by plants in the Brassicaceae) concentrations on the immune status of an herbivore and its consequences for two species of parasitoids with different life history traits.We found that larvae of the butterfly Pieris rapae that fed on field mustard Brassica rapa, which contain 52‐fold higher glucosinolate concentrations than collards B. oleracea, attained lower body weights and experienced prolonged development to adulthood.Yet, caterpillars that fed on B. rapa had enhanced cellular immunity, as measured by total and differential haemocyte counts as well as melanization capacity, compared to larvae that fed on B. oleracea.In turn, the likelihood that at least some eggs in clutches of the gregarious endoparasitoid Cotesia glomerata would be encapsulated, leading to a reduction in brood size, were three times greater when their host caterpillars fed on B. rapa compared to B. oleracea.Interestingly, eggs of the solitary endoparasitoid Cotesia rubecula were rarely encapsulated irrespective of the host plant on which their host caterpillar fed. Therefore, our results suggest that plant defence metabolites can influence the expression of herbivore immunity, but the effectiveness of this response strongly depends on the identity of the parasitoid and its ability to evade the caterpillar immune response, and possibly the evolution of these trophic interactions in non‐native systems. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Endoparasitoids are insects that develop within other insects, employing unique strategies to enhance their offspring’s survival. They inject polydnavirus and/or venom into their hosts along with eggs, effectively suppressing the host’s immune system. Polydnavirus from Braconidae and Ichneumonidae wasps can integrate into the host’s genome to express viral genes using the host’s transcription systems. However, the ability of parasitoids without polydnavirus to manipulate host gene expression remains unclear. Lysine acetylation (LysAc), a post-translational modification critical for gene regulation, is hypothesized to be used by endoparasitoids lacking polydnavirus. We utilized the Chalcidoidea wasp Tetrastichus brontispae, which lacks polydnavirus, as an idiobiont endoparasitoid model to test this hypothesis, with pupae of the nipa palm hispid beetle Octodonta nipae as the host. Parasitism by T. brontispae resulted in the reduced expression of histone deacetylase Rpd3 and elevated levels of LysAc modification at histones H3.3K9 and H3.3K14 through proteomics and LysAc modification omics. The knockdown of Rpd3 increased the expression level of OnPPAF1 and OnPPO involved in the phenoloxidase cascade, leading to melanization in the host body whereby it resembled a mummified parasitized pupa and ultimately causing pupa death. This study enhances our understanding of how endoparasitoids employ histone acetylation to regulate immunity-related genes, offering valuable insights into their survival strategies.

Aims To investigate the relationship between microbial community profiles and gill pathology during a production cycle of Atlantic salmon in two commercial hatcheries. Methods and Results Relationships between gill histology, environmental conditions, and microbiome were determined using high-throughput data, including 16S rDNA amplicon sequencing data, histopathology data, and water quality parameters. Hatchery A used riverine water and operated a mixed system of recirculation aquaculture system (RAS) and flowthrough. Hatchery B was used bore water and operated a RAS. Melanin deposits, hyperplastic, and inflammatory lesions were observed histologically in the gills. A higher prevalence of melanin deposits was detected and correlated to a change in beta diversity of bacterial communities in early time points (fingerling and parr stages). High abundance of Sphaerotilus sp. Pseudomonas sp. Nitrospira sp. Exiguobacterium sp. Deinococcus sp. and Comamonas sp. was correlated with a high prevalence of melanin in filaments. Bacterial diversity increased as the fish cohort transitioned from RAS to flowthrough in hatchery A. Conclusions Under commercial conditions, the commensal community of gill bacteria was related to melanin prevalence. [ABSTRACT FROM AUTHOR]

Insect life on earth is greatly diversified despite being exposed to several infectious agents due to their diverse habitats and ecological niche. One of the major factors responsible for their successful establishment is having a powerful innate immune system. The most common and effective method used by insects in recognizing pathogen and non-self-substances is the melanization process among others. The key enzyme involved in melanin biosynthesis is the copper containing humoral defense enzyme, phenoloxidase (PO). This review focused on understanding about PO and that had been in research for nearly a century. The review elaborates about evolutionary significance of PO in arthropods, its relationship with mammalian tyrosinases, various substrates, activators and inhibitors involved in the activation of phenoloxidase cascade, as it requires an integrated system of activation that vary among insect species. The enzyme also plays a vital role in insect immunity by involving in several other immune functions like sclerotization, wound healing, opsonization, encapsulation and nodule formation. Further, gene knock down or knock out of PO genes and inhibition of PO-melanization cascade by several mechanisms can also be considered as promising future alternative to control serious pests by making them highly susceptible to any targeted attack. [ABSTRACT FROM AUTHOR]

Abstract Background In arboviral disease systems where the virus can be transmitted from male to female vectors and from one generation to the next, targeting the female (especially when she is gravid) can help alter the persistence of the virus in nature and its transmission. A typical example is Aedes aegypti, which has become unmanageable due to the development of insecticide resistance. Despite evidence that monomolecular surface films prevent the selection of genetic resistance, their potential in Aedes vector control remains largely unexplored. Methods We examined the oviposition, egg retention, oocyte melanization, and female mortality of the Cayman Islands strain of Ae. aegypti, using choice (balanced and unbalanced) and no-choice bioassays involving Aquatain® Mosquito Formulation (AMF; Aquatain Products Pty Ltd.), a polydimethylsiloxane–based liquid used for mosquito control. Results When presented with similar opportunities to oviposit in two sites treated with AMF and two other sites with untreated water (control), egg deposition rates were significantly higher in the untreated water sites than in the AMF-treated sites (P

Leaf-cutting ant queens mate with multiple males during a single nuptial flight and store sperm for up to two decades. During mating, males transfer sperm from their accessory testes to the queen bursa copulatrix from where it enters the spermatheca, an insect sperm storage organ that has become highly specialized in long-lived ant queens who never re-mate later in life. Long-term storage without the possibility to obtain new sperm creates an immune defence dilemma, because recognition of non-self cells eliminates infections but may also target irreplaceable sperm and reduce lifetime reproductive success. We therefore hypothesized that non-specific immune responses, like pathogen melanization, should be silenced in the spermatheca, because they rely on general non-self recognition, and that specific responses such as antimicrobial peptides are activated instead as they specifically target pathogenic bacteria and/or fungi. The maintenance of uninfected sperm cells by males before mating is not constrained by non-self recognition, meaning immune regulation might be more liberal in male reproductive organs. To test this hypothesis, we measured gene expression of two antimicrobial peptides, abaecin and defensin, and prophenoloxidase, an important enzyme of the melanization pathway, in male accessory glands and testes and in queen bursae copulatrix and spermathecae of Acromyrmex echinatior and Atta colombica leaf-cutting ants. As expected, prophenoloxidase expression was low in reproductive organs that sustain prolonged contact with sperm, whereas antimicrobial peptides showed average to high expression, indicating that leaf-cutting ants invest in specific rather than generalist immune defences for pathogen protection in organs that store sperm.

Maternally transmitted microbes are ubiquitous. In insects, maternal microbes can play a role in mediating the insect immune response. Less is known about how ecological factors, such as resource use, interact with maternal microbes to affect immunity.In the context of a recent colonization of a novel host plant by the Melissa blue butterfly Lycaeides melissa, we investigated the interaction between host plant use and vertically transmitted, extracellular egg‐associated microbes in determining the strength of the insect immune response.We reared larvae on two different host plant species: a native host Astragalus canadensis and a novel host Medicago sativa. Egg‐associated microbes were removed through a series of antimicrobial egg washes prior to hatching. Immune response was measured through three assays: standing phenoloxidase (PO), total PO and melanization.We detected strong effects of microbial removal. Egg washing resulted in larvae with an increased immune response as measured by total PO—contrary to reports from other taxa. The effect of washing was especially strong for larvae consuming the native host plant.This result may explain why consumption of the egg casing is not a universal behaviour in insects, due to negative effects on larval immunity. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Long-term exposure to infections can exert serious pressure on the host's immune system and influence its capacity to react adequately when challenged by another parasite. Rather than reacting readily to the attack of a secondary pathogen the immune response might be weaker because of the costs imposed by resisting the primary infection. Rickia wasmannii is an ectoparasitic fungus, which attaches to the outer layer of Myrmica ants without killing the host, but causing different modifications on individual and social level. We examined the immune competence of M. scabrinodis workers of different age categories either coming from infected colonies or from healthy control colonies using encapsulation assays. Infection and age did not affect the encapsulation response separately, but their interaction had a considerable effect: the response was stronger in young, infected workers. Smaller individuals also had stronger responses. Encapsulation response was not influenced by fat content of workers. Immune facilitation might indeed work in infected colonies, although not for each worker category. This emphasizes the importance of within-colony diversity when considering colony level response to parasitic attacks. [ABSTRACT FROM AUTHOR]

Understanding Cryptococcus neoformans pathogenesis requires a detailed analysis of the various virulence factors that contribute to its ability to cause disease. Cyclosporine, calcineurin inhibitor, impairs C. neoformans production of a polysaccharide capsule and secretion of urease, which are critical for cryptococcal pathogenesis. Two particularly important virulence factors are the production of cell wall melanin and formation of biofilm. In this study, we investigated cyclosporine's effects on melanin production and biofilm formation in C. neoformans. Initially, we examined melanin production in planktonic cells treated with cyclosporine using an L-DOPA containing melanin-inducing medium. Visual inspection and optical microscopy revealed a notable reduction in the characteristic dark coloration of cultures treated with cyclosporine, which indicate decreased melanin production in daughter cells compared to mother cells. Spectrophotometric analysis also demonstrated a significantly altered ultraviolet-visible (UV/vis) absorption spectra in cyclosporine-treated yeast cells, indicative of structural changes in melanin. Additionally, cyclosporine-treated cells exhibited reduced conductance (P-value < 0.0001), suggesting altered cellular ionic properties. The impact of cyclosporine on biofilm formation and mature biofilm disruption was also assessed. Despite cyclosporine's efficacy in modifying virulence factors during planktonic growth, cyclosporine did not inhibit biofilm formation or melanization under biofilm growth conditions, nor did it disrupt mature biofilms in terms of biomass or metabolic activity. However, there was a significant reduction in extracellular matrix production in cyclosporine-treated non-melanized biofilms. Our findings underscore the complex interplay between cyclosporine and C. neoformans, highlighting its differential effects on melanization and biofilm dynamics, which provides new insights into the shortcomings of cyclosporin for combatting cryptococcosis and informs pathways for future therapeutic strategies against cryptococcosis., Competing Interests: Declaration of Competing Interest ☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Rodrigo Almeida-Paes reports financial support was provided by National Council for Scientific and Technological Development. Rodrigo Almeida-Paes and Susana Frases reports financial support was provided by Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State. Iara Bastos de Andrade reports financial support was provided by Coordination of Higher Education Personnel Improvement. Joshua Nosanchuk reports financial support was provided by National Institutes of Health. Joshua Nosanchuk reports financial support was provided by US Department of Defense. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)