The plasticity of immune memory in invertebrates.

Whether specific immune protection after initial pathogen exposure (immune memory) occurs in invertebrates has long been uncertain. The absence of antibodies, B-cells and T-cells, and the short lifespans of invertebrates led to the hypothesis that immune memory does not occur in these organisms. However, research in the past two decades has supported the existence of immune memory in several invertebrate groups, including Ctenophora, Cnidaria, Nematoda, Mollusca and Arthropoda. Interestingly, some studies have demonstrated immune memory that is specific to the parasite strain. Nonetheless, other work does not provide support for immune memory in invertebrates or offers only partial support. Moreover, the expected biphasic immune response, a characteristic of adaptive immune memory in vertebrates, varies within and between invertebrate species. This variation may be attributed to the influence of biotic or abiotic factors, particularly parasites, on the outcome of immune memory. Despite its critical importance for survival, the role of phenotypic plasticity in immune memory has not been systematically examined in the past two decades. Additionally, the features of immune responses occurring in diverse environments have yet to be fully characterized.

Evaluation and Characterization of the Insecticidal Activity and Synergistic Effects of Different GroEL Proteins from Bacteria Associated with Entomopathogenic Nematodes on Galleria mellonella.

GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin.

Arbovirus surveillance on the Mexico-USA border: West Nile virus identification in various species of field mosquitoes.

West Nile virus (WNV) has been documented in human and/or mosquito samples near the border with Mexico in El Paso, Texas, and Doña Ana County, New Mexico. However, on the Mexican side of the border, particularly in the State of Chihuahua, no such cases of WNV-infected mosquitoes have been documented. We tested 367 mosquitoes of four species (Culex quinquefasciatus, Cx. tarsalis, Aedes aegypti, and Aedes (Ochlerotatus) epactius) and found a high rate of WNV-positivity, including the first record of Ae. (Ochlerotatus) epactius infection with WNV. These results call for intensifying WNV surveillance efforts on the border between the United States and Mexico, with particular emphasis on vector control and monitoring of the species included in this study.

The honey bees immune memory.

Invertebrates' immune priming or innate immune memory is an analogous response to the vertebrates' adaptive memory. We investigated if honey bees have immune memory. We compared survival and immune response between bees that were: 1) manipulated (Naïve), 2) challenged twice with the same pathogen Escherichia coli (Memory), 3) challenged twice with different pathogens (Staphylococcus aureus versus E. coli, Micrococcus lysodeikticus versus E. coli), or 4) with PBS (the diluent of bacteria) versus E. coli (heterologous challenge; Control). Results indicate better survival in the Memory than the Control group, and the Memory group showed a similar survival than Naïve insects. The Memory group had higher lytic activity but lower prophenoloxidase, phenoloxidase activity, and hemocyte count than the Control and Naïve groups. No differences were found in relative expression of defensin-1. This first demonstration of immune memory opens the questions about its molecular mechanisms and whether, immune memory could be used against natural parasites that affect honey bees, hence, if they could be "vaccinated" against some natural parasites.

The costs of transgenerational immune priming for homologous and heterologous infections with different serotypes of dengue virus in Aedes aegypti mosquitoes.

The immune system is a network of molecules, signaling pathways, transcription, and effector modulation that controls, mitigates, or eradicates agents that may affect the integrity of the host. In mosquitoes, the innate immune system is highly efficient at combating foreign organisms but has the capacity to tolerate vector-borne diseases. These implications lead to replication, dissemination, and ultimately the transmission of pathogenic organisms when feeding on a host. In recent years, it has been discovered that the innate immune response of mosquitoes can trigger an enhanced immunity response to the stimulus of a previously encountered pathogen. This phenomenon, called immune priming, is characterized by a molecular response that prevents the replication of viruses, parasites, or bacteria in the body. It has been documented that immune priming can be stimulated through homologous organisms or molecules, although it has also been documented that closely related pathogens can generate an enhanced immune response to a second stimulus with a related organism. However, the cost involved in this immune response has not been characterized through the transmission of the immunological experience from parents to offspring by transgenerational immune priming (TGIP) in mosquitoes. Here, we address the impact on the rates of oviposition, hatching, development, and immune response in Aedes aegypti mosquitoes, the mothers of which were stimulated with dengue virus serotypes 2 and/or 4, having found a cost of TGIP on the development time of the progeny of mothers with heterologous infections, with respect to mothers with homologous infections. Our results showed a significant effect on the sex ratio, with females being more abundant than males. We found a decrease in transcripts of the siRNA pathway in daughters of mothers who had been exposed to an immune challenge with DV. Our research demonstrates that there are costs and benefits associated with TGIP in Aedes aegypti mosquitoes exposed to DV. Specifically, priming results in a lower viral load in the offspring of mothers who have previously been infected with the virus. Although some results from tests of two dengue virus serotypes show similarities, such as the percentage of pupae emergence, there are differences in the percentage of adult emergence, indicating differences in TGIP costs even within the same virus with different serotypes. This finding has crucial implications in the context of dengue virus transmission in endemic areas where multiple serotypes circulate simultaneously.

Plasmodium exposure alters midgut epithelial cell dynamics during the immune memory in Anopheles albimanus.

Immunological priming in insects is defined as a previous contact with non-virulent pathogens, which induces protection after a second virulent infection. The mechanism of this process is not well understood. We have observed midgut DNA synthesis (endoreplication) in Plasmodium berghei exposure mosquitoes (primed) and after the immune challenge, which could be an essential component of the priming response in the mosquito. Endoreplication requires cell cycle components re-direction to make multiple DNA copies. Therefore, it is fundamental to understand the role of cell cycle components in priming. Here, we analyzed the expression of the cyclins A, B, E, and AurkA, and the endoreplication components NOTCH and HNT in the mosquito Anopheles albimanus; after priming with non-infective Plasmodium berghei and challenged with an infective P. berghei. The overexpression of cell cycle elements occurred seven days after priming with a quick reduction 24 h after the challenge. Hnt and NOTCH overexpression occurred 24 h after priming. Antimicrobial peptide cecropin is quickly overexpressed after 24 h in primed mosquitoes, then is downregulated at day seven and overexpressed again after parasite challenge. We also found that DNA synthesis occurs in cells with different nuclear sizes, suggesting a change in midgut epithelial dynamics after Plasmodium exposure. Inhibition of DNA synthesis via cisplatin revealed that DNA synthesis is required for priming to limit Plasmodium infection. Our results indicate the importance of cell cycle components on DNA synthesis and Notch pathway during priming response in An. albimanus mosquitoes.

Detection of NS1 protein from dengue virus in excreta and homogenates of wild-caught Aedes aegypti mosquitoes using monoclonal antibodies.

Dengue fever is one of the most devastating infectious diseases worldwide. Development of methods for dengue virus (DENV) detection in mosquitoes to assess prevalence as a preliminary screen for entomological surveillance in endemic regions of DENV will certainly contribute to the control of the disease. A monoclonal antibody against the NS1 (nonstructural protein 1) viral protein was generated using recombinant NS1 protein and used to detect and analyze DENV in both excreta and total homogenates from Aedes aegypti mosquitoes. Results demonstrated expression of NS1 in excreta of DENV laboratory-infected mosquitoes and homogenates from field mosquitoes infected with DENV. The immunodetection method reported here represents a first-line strategy for assessing the prevalence of DENV in mosquitoes, for entomological surveillance in endemic regions of dengue. Detection of DENV prevalence in field mosquitoes could have an impact on vector surveillance measures to interrupt dengue transmission.

Innate immune memory in invertebrates: Concept and potential mechanisms.

Invertebrates are the protagonists of a recent paradigm shift because they now show that vertebrates are not the only group with immune memory. This review discusses the concept of immune priming, its characteristics, and differences with trained immunity and immune enhancement. We include an update of the current status of immune priming within generations in different groups of invertebrates which now include work in 5 Phyla: Ctenophora, Cnidaria, Mollusca, Nematoda, and Arthropoda. Clearly, few Phyla have been studied. We also resume and discuss the effector mechanism related to immune memory, including integrating viral elements into the genome, endoreplication, and epigenetics. The roles of other elements are incorporated, such as hemocytes, immune pathways, and metabolisms. We conclude that taking care of the experimental procedure will discern if results provide or do not support the invertebrates' immune memory and that regarding mechanisms, indeed, there are no studies on the immune memory mechanisms, this is how specificity is reached, and how and where the immune memory is stored and how is recall upon subsequent encounters. Finally, we discuss the possibility of having more than one mechanism working in different groups of invertebrates depending on the environmental conditions.

Heat Shock Causes Lower Plasmodium Infection Rates in Anopheles albimanus.

The immune response of Anopheles mosquitoes to Plasmodium invasion has been extensively studied and shown to be mediated mainly by the nitric oxide synthase (NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity. Here, we studied the correlation between a heat shock insult, transcription of immune response genes, and subsequent susceptibility to Plasmodium berghei infection in Anopheles albimanus. We found that transcript levels of many immune genes were drastically affected by the thermal stress, either positively or negatively. Furthermore, the transcription of genes associated with modifications of nucleic acid methylation was affected, suggesting an increment in both DNA and RNA methylation. The heat shock increased PO and NOS activity in the hemolymph, as well as the transcription of several immune genes. As consequence, we observed that heat shock increased the resistance of mosquitoes to Plasmodium invasion. The data provided here could help the understanding of infection transmission under the ever more common heat waves.

Adult damselflies as possible regulators of mosquito populations in urban areas.

BACKGROUND: Dragonfly and damselfly larvae have been considered as possible biocontrol agents against young instars of mosquito vectors in urban environments. Yet our knowledge about adult odonate predation against mosquito adults is scarce. We quantified daily and annual predation rates, consumption rates and prey preferences of adult Hetaerina vulnerata male damselflies in an urban park. A focus on predation of mosquito species was provided, quantified their arbovirus (dengue, chikungunya and Zika) infection rates and biting activity. RESULTS: Foraging times of H. vulnerata overlapped with those of the maximum activity of hematophagous mosquitoes. The most consumed preys were Diptera and Hymenoptera and, in lower quantities, Hemiptera, Coleoptera, Trichoptera, Psocoptera and Neuroptera. Of note, 7% of the diet was represented by hematophagous dipterans, with 2.4% being Aedes aegypti and Aedes albopictus. Prey abundance in the diet coincided with that of the same species in the environment. The arboviral infection rate (dengue, chikungunya and Zika) was 1.6% for A. aegypti and A. albopictus. The total biting rate of these mosquito vectors was 16 bites per person per day, while the annual rate of infectious bites was 93.4. CONCLUSION: Although 2.4% for both Aedes species seems a low consumption, considering the presence of 12 odonate species at the park, it can be argued that adult odonates may play a relevant role as mosquito vector regulators, therefore impacting the spread of mosquito-borne diseases. Our study outlines the need for further research on the topic of the possible role of adult odonates for mosquito biocontrol. © 2021 Society of Chemical Industry.

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection.

Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.

Dietary and Plasmodium challenge effects on the cuticular hydrocarbon profile of Anopheles albimanus.

The cuticular hydrocarbon (CHC) profile reflects the insects' physiological states. These include age, sex, reproductive stage, and gravidity. Environmental factors such as diet, relative humidity or exposure to insecticides also affect the CHC composition in mosquitoes. In this work, the CHC profile was analyzed in two Anopheles albimanus phenotypes with different degrees of susceptibility to Plasmodium, the susceptible-White and resistant-Brown phenotypes, in response to the two dietary regimes of mosquitoes: a carbon-rich diet (sugar) and a protein-rich diet (blood) alone or containing Plasmodium ookinetes. The CHCs were analyzed by gas chromatography coupled to mass spectrometry or flame ionization detection, identifying 19 CHCs with chain lengths ranging from 20 to 37 carbons. Qualitative and quantitative changes in CHCs composition were dependent on diet, a parasite challenge, and, to a lesser extent, the phenotype. Blood-feeding caused up to a 40% reduction in the total CHC content compared to sugar-feeding. If blood contained ookinetes, further changes in the CHC profile were observed depending on the Plasmodium susceptibility of the phenotypes. Higher infection prevalence caused greater changes in the CHC profile. These dietary and infection-associated modifications in the CHCs could have multiple effects on mosquito fitness, impacts on disease transmission, and tolerance to insecticides.

Transcriptome analysis uncover differential regulation in cell cycle, immunity, and metabolism in Anopheles albimanus during immune priming with Plasmodium berghei.

In invertebrates, "immunological priming" is considered as the ability to acquire a protective (adaptive) immune response against a pathogen due to previous exposure to the same organism. To date, the mechanism by which this type of adaptive immune response originates in insects is not well understood. In the Anopheles albimanus - Plasmodium berghei model, a DNA synthesis that probably indicates an endoreplication process during priming induction has been evidenced. This work aimed to know the transcriptomic profile in the midguts of An. albimanus after priming induction. Our analysis indicates the participation of regulatory elements of the cell cycle in the immunological priming and points out the importance of the cell cycle regulation in the mosquito midgut.

Prediction of neuropeptide precursors and differential expression of adipokinetic hormone/corazonin-related peptide, hugin and corazonin in the brain of malaria vector Nyssorhynchus albimanus during a Plasmodium berghei infection.

Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.

Affinity purification of Plasmodium ookinetes from in vitro cultures using extracellular matrix gel.

Malaria transmission depends on the parasites' successful invasion of the mosquito. This is achieved by the ookinete, a motile zygote that forms in the blood bolus after the mosquito takes an infectious blood meal. The ookinete invades the midgut epithelium and strongly attaches to the basal lamina, differentiating into an oocyst that produces the vertebrate-invasive sporozoites. Despite their importance, the ookinete and the oocyst are the least studied stages of the parasite. Much of what we know about the ookinete comes from in vitro experiments, which are hindered by the concomitant contamination with blood cells and other parasite stages. Although methods to purify them exist, they vary in terms of yield, costs, and difficulty to perform. A method for ookinete purification taking advantage of their adhesive properties was herein developed. The method consists of covering any culture-suitable surface with extracellular matrix gel, after which the ookinete culture is incubated on the gel to allow for ookinete attachment. The contaminant cells are then simply washed away. This procedure results in purer and less stressed ookinete preparations, which, by the nature of the method, are ready for oocyst production. Furthermore, it allows for micro-purifications using only 1 µl of blood, opening the possibility to make axenic ookinete cultures without sacrificing mice.

DNA synthesis increases during the first hours post-emergence in Anopheles albimanus mosquito midgut.

In hematophagous insects, the midgut is a fundamental barrier against infections and limits the development and transmission of pathogens. However, in mosquitoes, cell differentiation, proliferation, and cell cycle process in the midgut have not been characterized. Here we provide evidence of how cell cycle progression occurs in the newly emerged Anopheles albimanus mosquito midgut and describing cyclins expression as mediators of the cell cycle. The cell cycle at different post-emergence times was evaluated in disaggregated cells from midgut tissue using flow cytometry. Also, cyclins A, B, and E were identified by bioinformatics tools. These cyclins were used to analyze cell cycle progression. Flow cytometry data and the expression-pattern of the cyclins by qRT-PCR supported a polyploidy process, besides mitosis marker was marginally detected and only in newly emerged mosquitoes. Our results suggest that DNA increment in midguts occurs by polyploidy during the first hours post-emergence.

Immune priming with inactive dengue virus during the larval stage of Aedes aegypti protects against the infection in adult mosquitoes.

Several studies have observed that the immune response in insects can be conserved, a phenomenon known as immune priming, which has been mostly tested in adult stages. However, it is unknown if induction of immune priming in larval stages protects against dengue virus (DENV) infections in adult mosquitoes. In this work, we primed larval instar 3rd of Aedes aegypti with inactive dengue virus, producing adult mosquitoes with i) an enhanced antiviral-immune response; ii) a reduction in the load and replication of RNA of dengue virus (DENV); iii) a decline in viral infective particles production. Adult mosquitoes previously primed during larval stages over-expressed RNA interference (RNAi) markers Argonaute-2 (AGO-2) and Dicer-2 (DCR-2). We also observed inter-individual variations of DENV infection in adult mosquitoes, indicating a heterogeneous response to DENV infection in the same mosquito strain. However, mosquitoes primed during larval stages appear to control the infection, reducing the viral load. The over-expression of interferon-like factors (VAGO) and AGO-2 in the pupa stage suggests a fast activation of antiviral mechanisms after immune priming in larvae, creating a condition in which adult mosquitoes are resistant to the pathogen in the posterior exposure.

Induction of innate immune response in whiteleg shrimp (Litopenaeus vannamei) embryos.

The immune response of commercially relevant marine invertebrates has been extensively studied, in search of new disease-control strategies. Immune training is considered a novel approach that could help improve resistance to different pathogens. Here, we stimulated the white shrimp (Litopenaeus vannamei) during embryo development by exposure to heat-killed bacteria and evaluated their effect on hatching, larval development, and the expression of immune-related genes. In addition, we evaluated its impact on the response of shrimp nauplii during a challenge with Vibrio parahaemolyticus. We observed that the percentage of hatching and the resistance to bacterial infection increased due to the treatment of embryos with heat-killed cells of Vibrio and Bacillus. Apparently different stimuli could generate a differential pattern of gene expression, e.g., Vibrio induced a strong effector immune response whereas Bacillus elicited a protective immune profile. In addition, each response was triggered by molecular patterns detected in the environment. The results obtained in this study provide new insights for immune training to improve shrimp farming.

DNA Methylation in Anopheles albimanus Modulates the Midgut Immune Response Against Plasmodium berghei.

Epigenetic mechanisms such as DNA methylation and histone post-translational modifications are fundamental for the phenotypic plasticity of insects during their interaction with the environment. In response to environmental cues, the methylation pattern in DNA is dynamically remodeled to achieve an epigenetic control of gene expression. DNA methylation is the focus of study in insects for its evolutionarily conserved character; however, there is scant knowledge about the epigenetic regulation in vector mosquitoes, especially during their infection by parasites. The aim of the present study was to evaluate the participation of DNA methylation in the immune response of Anopheles albimanus to a Plasmodium infection. For this, we first investigated the presence of a fully functional DNA methylation system in A. albimanus by assessing its potential role in larval development. Subsequently, we evaluated the transcriptional response to Plasmodium berghei of two mosquito phenotypes with different degrees of susceptibility to the parasite, in a scenario where their global DNA methylation had been pharmacologically inhibited. Our study revealed that A. albimanus has a functional DNA methylation system that is essential to larval viability, and that is also responsive to feeding and parasite challenges. The pharmacological erasure of the methylome with azacytidine or decitabine abolished the divergent responses of both mosquito phenotypes, leading to a transcriptionally similar response upon parasite challenge. This response was more specific, and the infection load in both phenotypes was lowered. Our findings suggest that DNA methylation may constitute a key factor in vector competence, and a promising target for preventing malaria transmission.

DNA Synthesis Is Activated in Mosquitoes and Human Monocytes During the Induction of Innate Immune Memory.

Endoreplication is a cell cycle program in which cells replicate their genomes without undergoing mitosis and cytokinesis. For the normal development of many organisms (from fungi to humans) and the formation of their organs, endoreplication is indispensable. The aim of the present study was to explore whether endoreplication and DNA synthesis are relevant processes during the induction of trained innate immunity in human monocytes and in the Anopheles albimanus mosquito cell line. During the induction of trained immunity in both models, endoreplication markers were overexpressed and we observed an increase in DNA synthesis with an augmented copy number of genes essential for trained immunity. Blocking DNA synthesis prevented trained immunity from being established. Overall, these findings suggest that DNA synthesis and endoreplication are important mechanisms involved in inducing innate immune memory. They have probably been conserved throughout evolution from invertebrates to humans.