Midgut Tissue Research Articles

Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect.

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects' ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, Belgica antarctica, is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against non-lethal freezing injury. Larvae of B. antarctica were exposed to control (2°C), direct freezing (-9°C for 24 h) or RCH (-5°C for 2 h followed by -9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had a significantly higher metabolic rate during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts (hsp60 and hsp90), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly fine-tune their performance in thermally variable environments.

Longevity of Apis mellifera workers fed on a diet incorporating entomopathogens

ABSTRACT: The present study had the objective of evaluating the longevity of A. mellifera workers fed on a diet incorporating commercial entomopathogens, Beauveria bassiana, and Bacillus thuringiensis. It also aimed at verifying possible morphological alterations in the midgut. To this purpose, the entomopathogens used were B. bassiana (Product A) (5.0 × 1011 viable conidia.kg-1), B. thuringiensis (Product B) (2.5 × 109 viable spores.g-1), and B. thuringiensis (Product C) (1.0 × 109 viable spores.g-1); and two controls: T1: sterilized distilled water, and T2: sterilized distilled water + Tween 80® (0.01%). For the bioassays, 2 mL of each treatment were incorporated into Candy paste. For each treatment, 80 bees were individually in flat bottom glass tubes (2.5 cm Ø) covered with voile, containing a piece of cotton soaked in water and Candy paste. These tubes were stored in a B.O.D (30 ± 2°C, R.H 70% ± 10%, 12 h), and mortality was evaluated every six hours, for 10 days. Soon after verifying mortality, two bees per treatment were selected for the removal of their midgut. Midgut samples were processed using standard methodology for Scanning Electron Microscopy (SEM). It was verified that products A, B, and C reduced the longevity of bees when compared to T1 and T2 controls. In the qualitative analyses carried out using SEM, it was not possible to observe external or internal morphological alterations to midgut tissues. Although products A, B, and C cause a reduction in longevity, their presence was not verified when tissues were analyzed using SEM.

Identification of a cytochrome P450 CYP6AB60 gene associated with tolerance to multi-plant allelochemicals from a polyphagous caterpillar tobacco cutworm (Spodoptera litura)

Generalist phytophagous insects adapt to adventurous chemical environment in a wide variety of host plants by extraordinary detoxifying metabolic abilities. However, how polyphagous insect cope with the diversity of plant defenses remains largely unknown and only a few counter-defense genes detoxifying a wide range of toxic secondary metabolites have been well characterized. Here, we identify a cytochrome P450 gene (CYP6AB60) from tobacco cutworm (Spodoptera litura) in response to three different plant's defense metabolites. After being exposed to artificial diet supplemented with coumarin (COU), xanthotoxin (XAN) or tomatine (TOM), activities of P450 and CYP6AB60 transcript levels in both midgut and fat body tissues were significantly increased. Developmental expression analysis revealed that CYP6AB60 was expressed highly during the larval stages, and tissue distribution analysis showed that CYP6AB60 was expressed extremely high in the midgut, which correspond to the physiological role of CYP6AB60 from S. litura larvae in response to plant allelochemicals. Furthermore, when larvae are injected with double-stranded RNA (dsRNA) specific to CYP6AB60, levels of this transcript in the midgut and fatbody decrease and the negative effect of plant's defense metabolites on larval growth is magnified. These data demonstrate that the generalist insect S. litura might take advantage of an individual detoxificative gene CYP6AB60 to toxic secondary metabolites from different host plants. The CYP6AB60 can be a potential gene to carry out RNAi-mediated crop protection against the major polyphagous pest S. litura in the future.

Isolation, characterization and mode of action of a larvicidal compound, 22-hydroxyhopane from Adiantum latifolium Lam. against Oryctes rhinoceros Linn.

Oryctes rhinoceros Linn. is one of the most serious pests of coconuts and other palms. Following bioassay guided method, a larvicidal compound, 22-hydroxyhopane has been isolated for the first time from methanol extract of leaves of Adiantum latifolium Lam. against the pest (LC50 value 20.81 μg/g). It is a hopanoid triterpene with molecular mass of 442.42 g/mol. The compound exhibited antibacterial activity against symbiotic gut bacteria, caused histolysis of midgut tissues and inhibited secretion of digestive enzymes such as protease, amylase and trehalase resulting in weight loss of larvae. Enzyme immunoassay showed an elevation of 20-hydroxyecdysone level in haemolymph causing disruption of metamorphosis of larvae.

Parameters of oxidative stress, cholinesterase activity, Cd bioaccumulation in the brain and midgut of Lymantria dispar (Lepidoptera: Lymantriidae) caterpillars from unpolluted and polluted forests

Cadmium (Cd) can display a variety of different effects on living organisms. The objectives of the present study were to investigate Cd bioaccumulation and differences in parameters of oxidative stress (activities of the enzymes: superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase, glutathione S-transferase, and amounts of non-enzymatic free sulfhydryl groups and total glutathione) and cholinesterase activity in larval brain and midgut tissues of the polyphagous forest insect Lymantria dispar collected from unpolluted and polluted oak forests. Fourth instar L. dispar caterpillars from the unpolluted forest had higher body mass but accumulated more Cd in comparison to caterpillars from the polluted forest. In both populations the midgut was more sensitive than the brain to the prooxidative effects of Cd. Enzyme activities and amounts of non-enzymatic parameters tended to be greater in midgut tissues than in the brain, except for cholinesterase activity. Parameters of oxidative stress had higher values in caterpillar tissues from the polluted than from the unpolluted oak forest. The observed differences between the two natural populations point to the importance of knowing the history of population exposure to environmental pollution when monitoring forest ecosystems.

Global Analysis of Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Gene Expression in the Midgut of the Lepidopteran Host Trichoplusia ni.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.

Effects of coumaphos and imidacloprid on honey bee (Hymenoptera: Apidae) lifespan and antioxidant gene regulations in laboratory experiments

The main objective of this study was to test comparatively the effects of two common insecticides on honey bee Apis mellifera worker’s lifespan, food consumption, mortality, and expression of antioxidant genes. Newly emerged worker bees were exposed to organophosphate insecticide coumaphos, a neonicotinoid imidacloprid, and their mixtures. Toxicity tests were conducted along with bee midgut immunohistological TUNEL analyses. RT-qPCR assessed the regulation of 10 bee antioxidant genes linked to pesticide toxicity. We tested coumaphos at 92,600 ppb concentration, in combination with 5 and 20 ppb imidacloprid. Coumaphos induced significantly higher bee mortality, which was associated with down regulation of catalase compared to coumaphos and imidacloprid (5/20 ppb) mixtures, whereas, both imidacloprid concentrations independently had no effect on bee mortality. Mixture of coumaphos and imidacloprid reduced daily bee consumption of a control food patty to 10 mg from a coumaphos intake of 14.3 mg and 18.4 and 13.7 mg for imidacloprid (5 and 20) ppb, respectively. While coumaphos and imidacloprid mixtures induced down-regulation of antioxidant genes with noticeable midgut tissue damage, imidacloprid induced intensive gene up-regulations with less midgut apoptosis.

Metabolic Activity of Cytochrome P450s Towards Four Pyrethroids in Midgut Tissue From Locusta migratoria (Orthoptera: Acrididae).

Cytochrome P450 monooxygenases (P450s) play important roles in metabolizing various insecticides and often contribute to the development of insecticide resistance in insects and other arthropod species. The objective of this study was to compare the metabolism of four commonly used pyrethroids including deltamethrin, fluvalinate, fenvalerate, and permethrin in the midgut tissue of Locusta migratoria Linnaeus (Orthoptera: Acrididae) by using synergism bioassay and ultra-performance liquid chromatography (UPLC)-mass spectrometer (MS) analyses. Our study showed that piperonyl butoxide (PBO, P450 enzyme inhibitor) can significantly synergize the toxicity of deltamethrin, fluvalinate, and fenvalerate with synergism ratios ranging from 1.30 to 1.70 folds. Preincubations of the midgut tissue with PBO followed by incubations with each of the four pyrethroids resulted in significantly higher amounts of unmetabolized deltamethrin and fluvalinate than those in the control (preincubation without PBO) as well as preincubations with other two detoxification enzyme inhibitors. These results indicate that P450s play important roles in metabolizing deltamethrin and fluvalinate in the midgut tissue. Our further study using deltamethrin as a representative pyrethroid and UPLC-MS techniques confirmed that the reduced amount of deltamethrin in the control (preincubation without PBO) was due to the metabolism of deltamethrin to yield hydroxydeltamethrin which is a major metabolite produced by P450-mediated aromatic hydroxylation of deltamethrim. These results provide new insights into differential metabolic activity of P450s towards different pyrethroids in the midgut tissue of L. migratoria.

Characterization of Sphingomyelin Phosphodiesterase Expression in Bumblebee (Bombus lantschouensis).

Sphingomyelin phosphodiesterase (SMPD) is a hydrolase that plays a major role in metabolic reactions involving sphingomyelin. Here, we describe an analysis of the cDNA sequence and gene structure of SMPD in bumblebee (Bombus lantschouensis). The expression of SMPD in different tissues and at different developmental stages and reproductive statuses was examined by real-time polymerase chain reaction (PCR). The results showed that the SMPD cDNA has a length of 2000 bp and contains an open reading frame (ORF) of 1,801 nucleotides that encodes a polypeptide of 599 amino acids. The full-length SMPD gene is 4228 bp and contains eight exons and seven introns. A comparative analysis revealed that the SMPD gene sequence in B. lantschouensis shares high sequence identity with those in other Bombus species. The SMPD gene is expressed broadly in various tissues and presents higher transcript levels in the ovary, midgut, and epidermis and thoracic tissues. Among the different developmental stages, the highest expression of SMPD was detected at the Pw pupal stage (pupae with an unpigmented body cuticle and white eyes), and the expression of this gene decreased from the Pp (pupae with pink eyes) to the Pdd (dark-eye pupae with a dark-pigmented cuticle) stages. In addition, SMPD expression was significantly upregulated after female egg laying. In conclusion, our results show that the bumblebee SMPD gene might play a key role at the Pw developmental stage and in female oviposition.

Ontogeny, sex and adult tissues influence activities of detoxification enzymes in the Japanese beetle (Popillia japonicaNewman)

AbstractThe detoxification enzymes superfamilies of cytochrome P450 (P450), glutathioneS‐transferase (GST) and carboxylesterase (CoE) play important roles in the metabolism of xenobiotics and endogenous substrates. Despite their importance in insect physiology and ecology, the relative activities of these enzyme systems among developmental stages, sexes and tissues of insect species have not been studied extensively. In the present study, we determine the relative activities of these enzymes in eggs, larval stages, adults of each sex and different tissues of the Japanese beetle (Popillia japonicaNewman) in response to model exogenous substrates. The effect of food deprivation on enzyme activities is also examined in adult gut tissue. In this scarab species, ontogeny influences enzyme activities and catalytic efficiency, with eggs exhibiting only GST activity, first‐instar larvae exhibiting GST and CoE activities, and other life stages exhibiting P450 7‐ethoxycoumarinO‐deethylation (ECOD), GST and CoE activities. Second‐instar larvae have the highest GST activity, whereas third‐instar larvae have the highest P450 and CoE activities. Male and female adults show differences in P450 ECOD activity but not in CoE activity. Adult tissues vary in enzyme activities, with midgut tissues having the greatest P450 ECOD and CoE activities. A feeding bout of 24 h induces the optimal activities of P450 ECOD, GST and CoE in the gut of adult beetles. Interestingly, activity of GST is induced upon prolonged starvation. The findings of the present study highlight the apparent importance and contribution of detoxification enzymes in the physiology and ecology of this economically‐important pest.

EXPERIMENTAL INFECTIONS OF MILKY HEMOLYMPH DISEASE IN SPINY LOBSTER Panulirus homarus

Milky hemolymph disease of spiny lobster (MHD-SL) is categorized as the most destructive disease in farming spiny lobster. Therefore, it is required to investigate the routes of milky disease infection in spiny lobster as a basic knowledge in order to prevent milky disease transmission. The aim of the present study was to perform an experimental infection of milky disease in spiny lobster Panulirus homarus. Experimental infection of milky disease was carried out by several modes of infection which were injection, immersion and per os exposure. Injection of each 0.2 mL undiluted and diluted hemolymph from the diseased lobster resulted in a cumulative mortality of 100% at 15 days post-infection (dpi), and 75% at 16 dpi, respectively. Experimental infection through water immersion caused in a cumulative mortality of 50% at 7 dpi. In contrast, no mortality was observed in per os exposure as well as in control groups. Results of this experimental study provided evidence for horizontal transmission of MHD-SL among P. homarus. Histopathological analysis exhibited that there were masses of Rickettsia-like bacteria (RLB) in the connective tissues of the gill, hepatopancreas, gonad, midgut, and muscle tissues of the affected lobsters. Mass of RLB was not only found in the moribund lobsters but also in the surviving lobsters with milky hemolymph appearance.

Mosquito Innate Immunity

Mosquitoes live under the endless threat of infections from different kinds of pathogens such as bacteria, parasites, and viruses. The mosquito defends itself by employing both physical and physiological barriers that resist the entry of the pathogen and the subsequent establishment of the pathogen within the mosquito. However, if the pathogen does gain entry into the insect, the insect mounts a vigorous innate cellular and humoral immune response against the pathogen, thereby limiting the pathogen’s propagation to nonpathogenic levels. This happens through three major mechanisms: phagocytosis, melanization, and lysis. During these processes, various signaling pathways that engage intense mosquito–pathogen interactions are activated. A critical overview of the mosquito immune system and latest information about the interaction between mosquitoes and pathogens are provided in this review. The conserved, innate immune pathways and specific anti-pathogenic strategies in mosquito midgut, hemolymph, salivary gland, and neural tissues for the control of pathogen propagation are discussed in detail.

Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1

In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. Here, we have examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, we examine the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, in the regulation of DEOM histolysis. We find that loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. Herein, we also find that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, our results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death.

Effects of Vip3AcAa+Cry1Ac Cotton on Midgut Tissue in Helicoverpa armigera (Lepidoptera: Noctuidae).

To determine cellular changes caused by the chimeric protein Vip3AcAa against Helicoverpa armigera, we used transmission electron microscopy to examine ultrastructural changes in midgut cells of third-instar larvae of Cry1Ac-susceptible H. armigera after feeding on an artificial diet containing the Vip3AcAa toxin. Midgut epithelial cells of Cry1Ac-resistant H. armigera larvae that had fed on an artificial diet containing Vip3AcAa or on Bt cotton expressing Vip3AcAa+Cry1Ac were also examined using optical microscopy and hematoxylin–eosin staining. In the midgut cells of H. armigera larvae fed with Vip3AcAa, microvilli were swollen and broken; inner cristae of the mitochondria were indistinct and vacuolated; endoplasmic reticulum was swollen, fractured, and disordered; boundaries of karyotheca in the nucleus were indistinct and chromatin underwent pyknosis and was pressed close to the karyotheca. Histopathological changes and the time of onset in midgut tissues of H. armigera larvae fed on Vip3AcAa or Cry1Ac were similar. Vip3AcAa and transgenic cotton expressing Vip3AcAa+Cry1Ac caused the goblet cell cavity and microvilli pathological changes in the midgut epithelial cells of the Cry1Ac-susceptible and Cry1Ac-resistant H. armigera larvae that eventually killed the larvae.

Host plant-derived allelochemicals and metal components are associated with oxidative predominance and antioxidant plasticity in the larval tissues of silkworm, Antheraea mylitta: Further evidence of joint effects hypothesis.

Tasar silkworm, Antheraea mylitta is a polyphagous insect that primarily feeds on Terminalia arjuna, Terminalia tomentosa and Shorea robusta. However, larval rearing on S. robusta results in poor performance for the reasons unexplored. Oxidative burden imposed by host plants is presumed to be a determining factor for larval fitness. With this hypothesis we have analyzed the foliar constituents of the respective host plants, the levels of oxidative stress and antioxidant protection in the larval tissues in response to their altered feeding on different host plants for different durations (2 and 10 days). Results indicate that S. robusta leaves contain the highest amount of tannin and redox active metals compared to those of other host plants. Consequently, hemolymph and midgut tissues of the larvae shifted to S. robusta exhibited oxidative predominance. Increased activities of superoxide dismutase, catalase and glutathione S-transferase in the larval tissues indicated an adaptive response to host plant driven oxidative assault. Our in vitro study also strongly supplements the in vivo findings indicating S. robusta foliages as a strong inducer of lipid peroxidation (LPx). Copper and Iron were found to be more potent in inducing LPx in the midgut tissues of the larvae compared to Zinc and Manganese. This study for the first time demonstrates the combined implications of host plant derived allelochemicals and elements on oxidative stress and antioxidant plasticity in this insect. The overall findings also brace up the newly emerging concept on joint effects hypothesis (organic and elemental defence) for enhanced plant defence.

Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti.

Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We have shown that infection of vertebrate cells by SINV, chikungunya virus (CHIKV), and Ross River virus (RRV) produces two subpopulations of virus particles separable based on density. In contrast, a single population of viral particles is produced by mosquito cells. Previous studies demonstrated that the denser vertebrate-derived particles and the mosquito-derived particles contain components of the small subunit of the host cell ribosome, whereas the less dense vertebrate-derived particles do not. Infection of mice with RRV showed that both particle subpopulations are produced in an infected vertebrate, but in a tissue specific manner with serum containing only the less dense version of the virus particles. Previous infectivity studies using SINV particles have shown that the denser particles (SINVHeavy) and mosquito derived particles SINVC6/36 are significantly more infectious in vertebrate cells than the less dense vertebrate derived particles (SINVLight). The current study shows that SINVLight particles, initiate the infection of the mosquito midgut more efficiently than SINVHeavy particles and that this enhanced infectivity is associated with an exacerbated immune response to SINVLight infection in midgut tissues. The enhanced infection of SINVLight is specific to the midgut as intrathoracically injected virus do not exhibit the same fitness advantage. Together, our data indicate a biologically significant role for the SINVLight subpopulation in the efficient transmission from infected vertebrates to the mosquito vector.

Transmission of Severe Fever with Thrombocytopenia Syndrome Virus by Haemaphysalis longicornis Ticks, China.

We demonstrate maintenance and transmission of severe fever with thrombocytopenia syndrome virus by Haemaphysalis longicornis ticks in the larva, nymph, and adult stages with dissemination in salivary gland, midgut, and ovarian tissues. The H. longicornis tick is a competent vector to transmit this virus in both transovarial and transstadial modes.

De Novo DNA Synthesis in Aedes aegypti Midgut Cells as a Complementary Strategy to Limit Dengue Viral Replication.

Aedes aegypti is the main vector of Dengue Virus, carrying the virus during the whole mosquito life post-infection. Few mosquito fitness costs have been associated to the virus infection, thereby allowing for a swift dissemination. In order to diminish the mosquito population, public health agency use persistent chemicals with environmental impact for disease control. Most countries barely use biological controls, if at all. With the purpose of developing novel Dengue control strategies, a detailed understanding of the unexplored virus-vector interactions is urgently needed. Damage induced (through tissue injury or bacterial invasion) DNA duplication (endoreplication) has been described in insects during epithelial cells renewal. Here, we delved into the mosquito midgut tissue ability to synthesize DNA de novo; postulating that Dengue virus infection could trigger a protective endoreplication mechanism in some mosquito cells. We hypothesized that the Aedes aegypti orthologue of the Drosophila melanogaster hindsight gene (not previously annotated in Aedes aegypti transcriptome/genome) is part of the Delta-Notch pathway. The activation of this transcriptional cascade leads to genomic DNA endoreplication. The amplification of the genomic copies of specific genes ultimately limits the viral spreading during infection. Conversely, inhibiting DNA synthesis capacity, hence endoreplication, leads to a higher viral replication.

PiRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues.

The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2) in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR) methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24–30 nts) were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A) in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs). However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were identified in Ae. albopictus; however, only AalPiwi5–7 and AalAgo3(1–2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut.

Ultrastructural Alterations of Midgut Epithelium, But Not Greater Wing Fluctuating Asymmetry, in Paper Wasps (Polistes dominula) from Urban Environments.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.