Armigeres subalbatus prophenoloxidase III: Cloning,characterization and potential role in morphogenesis

It has long been suggested that phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning and melanotic encapsulation of pathogens. Here we report that a mosquito PO, designated Armigeres subalbatus prophenoloxidase III (As-pro-PO III), is likely involved in the morphogenesis in mosquito. Expression profile analysis found that As-pro-PO III mRNA is persistently expressed in adult mosquitoes and is not significantly affected by blood feeding, microfilariae inoculation, or Escherichia coli inoculation, but expression levels of As-pro-PO III fluctuated in larval and pupal stages. Knockdown of As-pro-PO III expression in pupae using double-stranded RNA resulted in high pupal mortality and deformed adults that subsequently died following emergence. Promoter activity analyses by electrophoretic mobility-shift assays and transfection assays suggest that the As-pro-PO III gene is positively regulated by a putative Zeste motif, a developmental regulatory element. These results suggest that As-pro-PO III is associated with morphogenesis of mosquitoes.     

Intracellular melanization of the larvae of Dirofilaria immitis in the malpighian tubules of the mosquito, Aedes sollicitans

Frequent melanization of larvae of the nematode Dirofilaria immitis parasitizing the Malpighian tubules of the mosquito, Aedes sollicitans, has been observed. Melanized and nonmelanized larvae in the Malpighian tubules were examined using light and electron microscopy. The results indicate that the pattern of melanin deposition and the ultrastructural characteristics of the pigment around the worms are identical to that observed on nematodes which have undergone humoral melanization in other dipteran insects. In the Malpighian tubules, no contact between the intracellular melanized nematodes and the hemolymph or hemocytes was observed. The results suggest that the Malpighian tubules of this species of mosquito are capable of inducing a melanotic response to invading nematode parasites. It is proposed that this is an example of “humoral” melanization at an intracellular site.     

Catecholamine metabolism and in vitro induction of premature cuticle melanization in wild type and pigmentation mutants of Drosophila melanogaster

The major pathway leading to adult cuticle melanization in Drosophila melanogaster has been investigated by a combination of biochemical and genetic approaches. By comparing catecholamine pools in newly emerged flies and in frass (excreta) collected 1 to 4 days after eclosion from wild type with those obtained from several pigmentation mutants, the major flow of catecholamines through the pathway to an unidentified final catabolite was determined. We also demonstrate that incubation with dopamine in vitro induces premature melanization in wild type unpigmented pharate adults several hours before the developmentally programmed onset of melanization, supporting the hypothesis that the availability of catecholamines may be the limiting factor determining the onset of melanization and that the major enzymatic activities that act downstream of dopa decarboxylase in the pathway are deposited into the cuticle before pigmentation begins. In vitro melanization studies with various pigmentation mutants that are associated with critical enzymatic steps in Drosophila catecholamine metabolism are consistent with their proposed function and suggest a central role of N-β-alanyldopamine in adult cuticle pigmentation. © 1996 Wiley-Liss, Inc.     

Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora

Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culex pipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts' defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed.     

Hormonal control of lutein incorporation into pupal cuticle of the butterfly Inachis and the pupal melanization reducing factor

Abstract. . Morphological colour adaptation of pupae of the butterfly Inachis io L. (Lepidoptera: Nymphalidae) is controlled by a factor which reduces cuticular melanization (Biickmann & Maisch, 1987). This so-called pupal melanization reducing factor (PMRF) is located throughout the entire central nervous system of prepupae (Stamecker et al. , 1994).
Extracts of abdominal ganglia also stimulated dose-dependently lutein incorporation into pupal cuticle. In the bioassay higher doses were required to increase cuticular lutein content than to reduce melanization. Ligatures during the prepupal stage demonstrated two different critical periods for these pigmentation effects: an early one for melanization reduction and a late one for lutein incorporation.
An initial chromatographic purification yielded only two adjacent fractions which contained both the PMRF and the stimulation of lutein incorporation activity. Therefore it is assumed that only one hormone with a dual function may be responsible for pupal pigmentation.
Lutein content was found in gut, fat body, epidermis and haemolymph of I.io. Lutein incorporation into cuticle occurred within 1.5 days of the pupal moult when the cuticle was not yet fully sclerotized. Lutein content is significantly higher in cuticle of yellow pupae than of black ones.     

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.     

Body melanization and its adaptive role in thermoregulation and tolerance against desiccating conditions in drosophilids

Melanism seems to have evolved independently through diverse mechanisms in various taxa and different ecological factors could be responsible for selective responses. Increased body melanization at higher altitudes as well as latitudes is generally considered to be adaptive for thermoregulation. Physiological traits such as body melanization and desiccation resistance have been investigated independently in diverse insect taxa at three levels: within populations, between populations and among species. A substantial number of Drosophila studies have reported clinal variations in both these traits along latitude. A possible link between these traits had remained unexplored in wild and laboratory populations of ectothermic insect taxa, including drosophilids, to date. Simultaneous analysis of these traits in assorted darker and lighter phenotypes in each population in the present study showed parallel changes for body melanization and desiccation resistance. The mechanistic basis of evolving desiccation resistance was explained on the basis of differential rates of water loss per hour in darker versus lighter phenotypes in six populations of Drosophila melanogaster from adjacent localities differing substantially in altitude all along the Indian subcontinent. Data on cuticular impermeability suggest a possible role of melanization in desiccation tolerance. However, substantial gaps remain in extending these results to other insect taxa and further exploring the physiological and molecular changes involved in melanization for conferring desiccation resistance.     

Wound reactions and Aphanomyces astaci growth in crayfish cuticle

Superficial wounding of crayfish soft cuticle by removal of the epicuticle caused hemocyte aggregation beneath the wound. The aggregation seemed to be induced by osmotic factors. Subsequently, melanin was only formed in parts of the wound region. Swimming zoospores of the fungal parasite Aphanomyces astaci preferentially encysted in the near vicinity of fresh superficial wounds. Cysts attached onto the naked endocuticle became encapsulated by melanin and were mostly killed during the process. The induced melanization reactions may have antifungal effects. The intermolt cuticle of a highly resistant crayfish species, Pacifastacus leniusculus, was easily penetrated by the fungus only via wounds. The cell walls of the penetrating hyphae became heavily melanized, growth was visibly disturbed, and they grew sparsely in the wound region. As soon as they reached outside the wound region they grew profusely. In susceptible crayfish Astacus astacus melanization was slower and the wound reactions prevented fungal growth much less as evidenced by microscopy. The preferential direction of hyphal growth is parallel to the chitin fibrils in both the resistant crayfish P. leniusculus and the susceptible A. astacus.     

Isolation and amino terminal sequence of melanization and reddish coloration hormone (MRCH) from the silkworm,Bombyx mori

Cuticular melanization associated with the gregarious phase of the common armyworm larvae, Leucania separata, is caused by a neurohormone, melanization and reddish coloration hormone (MRCH). Two molecular species of MRCH were isolated from 211,000 heads of adult Bombyx mori with conventional column chromatography and reversed-phase high performance liquid chromatography. As little as 6 ng of purified MRCH elicited marked melanization in the cuticle of an L. separata larva. Automated Edman degradation confirmed 16 residues of N-terminal amino acid sequences of the purified MRCHs; these showed homology with each other.     

Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Induction of Fas mediated caspase-8 independent apoptosis in immune cells by Armigeres subalbatus saliva

Background

It is widely recognized that the introduction of saliva of bloodsucking arthropods at the site of pathogen transmission might play a central role in vector-borne infections. However, how the interaction between salivary components and the host immune system takes place and which physiological processes this leads to has yet to be investigated. Armigeres subalbatus is one of the prominent types of mosquitoes involved in the transmission of parasitic and viral diseases in humans and animals.

Methodology/Principal Findings

Using murine peritoneal macrophages and lymphocytes, and human peripheral mononuclear cells (PBMCs), this study shows that saliva of the female Ar. subalbatus induces apoptosis via interaction with the Fas receptor within a few hours but without activating caspase-8. The process further activates downstream p38 MAPK signaling, a cascade that leads to the induction of apoptosis in capase-3 dependent manner. We further illustrate that Ar. subalbatus saliva suppresses proinflammatory cytokines without changing IL-10 levels, which might happen as a result of apoptosis.

Conclusions

Our study shows for the first time that saliva-induced apoptosis is the leading phenomenon exerted by Ar. subalbatus that impede immune cells leading to the suppression of their effecter mechanism.     

Filarial worms reduce Plasmodium infectivity in mosquitoes

Background

Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness).

Methodology/Principal Findings

Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections.

Conclusions/Significance

These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.     

Effects of U.V. light and temperature on the melanization and the formation of daily growth layers of Sarcophaga falculata.

Adult Sarcophaga flies, immediately after eclosion, were subjected to different temperature régimes or irradiated with u.v. light. The effect of the treatment on cuticular melanization was studied by comparison with specimens of control series or by comparing the areas of cuticle on the thoracic phragma of the same specimen that were deposited at different times under different conditions. The cuticle of flies that were tanned at 15 or 31°C was less melanized than that of control flies at 26°C. Irradiation with long wave u.v. light suppressed mainly the melanization whereas both the melanization and sclerotization processes were inhibited by short wave u.v. A decrease in adult melanization was caused also by exposure to u.v. of the heads of pharate adults 24 hr before eclosion.     

Insect cytokine growth-blocking peptide may regulate density-dependent phase trait of cuticular melanization in the larval armyworm,Mythimna separata

Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities such as larval growth regulation, paralysis induction, cell proliferation and stimulation of immune cells. Density-dependent phase polyphenism is a phenomenon of phenotypic plasticity in which the expression of a variety of traits can be affected by local population density. In the present study, the armyworm Mythimna separata larvae with four rearing densities (1 larva/vial, 2 larvae/vial, 4 larvae/vial and 6 larvae/vial) were tested for cuticular melanization and body weight throughout the third-fifth instar, and the functional role of GBP in regulating the changes was investigated. The results indicated that when reared at high densities, the larvae exhibited less body weight and more degree of cuticular melanization than larvae reared at low densities. The gene expression of GBP in armyworm larvae showed an initial rise and then decline trend with increased rearing densities in the third to fifth instar. Compared with control, more degree of cuticular melanization was observed in GBP-injected larvae (500 ng/larva in volume 50 μL) than that in Ringer’s solution-injected counterparts. Furthermore, the gene expression level of dopa decarboxylase and prophenoloxidase increased significantly in GBP-injected fifth instar larvae from 6 h to 12 h after injection, suggesting the role of GBP in modulating density-dependent phase trait of armyworm cuticular melanization.     

Predator–prey interactions and the cannibalism of larvae of Armigeres subalbatus (Diptera: Culicidae)

Predation and cannibalism can affect the co-existence of mosquito species or the assemblage of mosquito species (i.e., community structure). In this study, predatory feeding patterns and cannibalism of larvae of Armigeres subalbatus mosquitoes were quantified under laboratory conditions using Aedes albopictus and Culex uniformis larvae as prey organisms. Rate of consumption of prey larvae and the rate of cannibalism of 1st to 4th instar larvae of Armigeres subalbatus were reported at 24 h intervals with and without alternative food supplement. Both the 3rd and 4th instar larvae of Ar. subalbatus showed a substantial predation on the larvae of Ae. albopictus (33.6 ± 4.4) and Cx uniformis (47.3 ± 7.6). The cannibalism of the predatory larvae was strongly correlated to the larval density (r = 0.9). The predator and the prey density and the given food type were significant factors that determined the rate of pupation, death and the emergence as adults. A significant relationship was shown for the co-occurrence of predatory larvae and the prey larvae at the 85 natural breeding habitats observed at the field (χ² = 74.4, p < 0.001). Nine breeding sites (10.6%) were positive for both prey and predatory larvae. Our study showed robust information about the predatory and cannibalistic behavior of Ar. subalbatus larvae emphasizing their role in managing the population structure of mosquito communities.     

Parasitism of Kenyan Mosquito Larvae (Diptera: Culicidae) by Romanomermis culicivorax (Nematoda: Mermithidae)

The ability of Romanomermis culicivorax to infect, develop, and emerge from Kenyan mosquito hosts was evaluated in the laboratory. Host species tested were Aedes aegypti, Ae. dentatus, Ae. hirsutus, Anopheles arabiensis, An. coustani, An. funestus, An. gambiae, An. pharoensis, Culex duttoni, Cu. ethiopicus, Cu. poicilipes, Cu. quinquefasciatus, Cu. tigripes, Cu. univittatus, Coquillettidia metallica, Mansonia africana, Ma. uniformis, Mimomyia splendens, Mi. uniformis, Toxorhyncites brevipalpis, and Uranotaenia balfouri. R. culicivorax penetrated all the host species tested and developed and emerged from most of the hosts. Both penetration and some development, but not nematode emergence, were observed from all instars of Ma. uniformis. T. brevipalpis exhibited signs of resistance in the form of melanization of R. culicivorax within 48 hours of infection in all four instar stages. Nematode melanization, especially in older hosts, was observed in Ae. dentatus, Ae. hirsutus, Cu. duttoni, Cu. tigripes, and Mi. splendens. When melanization occurred, the melanized carcass of the nematode was passed on from instar to instar. The implications for field release of R. culicivorax in Kenya are still good, especially in habitats where different mosquito species occupy the same niche at different times, which would allow for nematode recycling.     

家蚕血淋巴体外黑化观察及相关黑色素合成催化酶基因

观察不同生长期家蚕幼虫血淋巴在体外的黑化速度和对大肠杆菌生长的影响结果显示,随食桑生长幼虫血淋巴的黑化速度逐渐变快,对大肠杆菌生长的抑制作用逐渐增强。RT-PCR实验显示,黑色素合成催化酶Bm Tan、Bm Po-1、Bm Yellow-f和Bm Ddc等的基因在家蚕5 L 3 d血淋巴中表达量高,Bm Black、Bm Yellow和Bm Pah等的基因也有明显表达。q PCR分析显示,黑化病蚕中Bmtan、Bmddc、Bmyellow、Bmebony和Bmblack,尤其Bmddc表达发生了显著上调。与对照相比,Ddc酶的抑制剂能显著抑制脂多糖对血淋巴的诱导黑化作用。用大肠杆菌注射家蚕幼虫,血淋巴中多巴和多巴胺的含量明显上升。这些表明家蚕幼虫血淋巴黑化与防御免疫有关,Bmddc很可能在幼虫血淋巴的免疫黑化中发挥作用。     

The influence of phenylthiourea on encapsulation,melanization, and survival in larvae of the mosquito Aedes aegypti parasitized by the nematode Neoaplectana carpocapsae

Aedes aegypti larvae, treated with phenylthiourea (PTU) and untreated, were exposed to concentrations of 1000–4000 Neoaplectana carpocapsae juveniles/ml for 1 hr. During the first 9 hr after exposure, the PTU-treated larvae had a significantly lower mortality than the untreated larvae. The rate of encapsulation or melanization of N. carpocapsae juveniles within the hemocoels of the mosquito larvae was investigated by gross dissections and histological techniques. The amount of melanization occurring in the PTU-treated larvae was found to be significantly reduced. Possible explanations were given for the observed differences in mortality. One possibility was that PTU may inactivate the phenol-oxidase system in mosquito larvae, thereby inhibiting the formation of toxic melanin intermediates. Another possibility is that PTU may inactivate a toxin emitted by the nematode or in some way have an effect on its associated bacterium, Achromobacter nematophilus.     

Effect of adult nutrition on the melanization immune response of the malaria vector Anopheles stephensi

Two dietary resources - blood and sugar - were assessed for effects on the melanization immune response of the mosquito Anopheles stephensi Liston (Diptera: Culicidae) towards inoculated Sephadex beads (negatively charged C-25). This melanization is conferred by genetic factors capable of making the mosquito refractory to malaria parasites. If An. stephensi females had obtained a bloodmeal one day before inoculation with a bead, the efficacy of their immune response increased with the concentration of sugar ingested. At the highest sugar concentration (6%) tested, 38% of the mosquitoes completely melanized their bead, whereas at the lowest sugar concentration (2%), none of the mosquitoes were able to melanize their bead completely. Among mosquitoes not having a bloodmeal, the immuno-competence was low (c. 9% of the mosquitoes completely melanized their bead) and independent of sugar concentration. The observed interaction between these two resources indicates that both resources are required for the Anopheles female to develop an effective melanization immune response.     

Hemolymph melanization and alterations in hemocyte numbers in Lymantria dispar larvae following infections with different entomopathogenic microsporidia

Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae can be infected in the laboratory with a variety of entomopathogenic microsporidia. In many cases, however, L. dispar is only a semi‐permissive host for such infections. In this study, we analyzed changes in the melanization of hemolymph and hemocyte numbers in L. dispar larvae after inoculation with various entomopathogenic microsporidia. We compared the infections produced by microsporidia isolated from L. dispar and infections produced by isolates from other Lepidoptera to which L. dispar is only a semi‐permissive host. Microsporidiosis induced a significant activation of the prophenoloxidase system leading to melanization; activation was highest when the pathogen caused heavy infections of the fat body, which was the case with two microsporidia originally isolated from L. dispar. Infection of only the silk glands or light infection of the fat body by two Vairimorpha spp. from other lepidopteran hosts elicited a lower response. Very light infections caused by a microsporidium isolated from Malacosoma americanum were not accompanied by elevated hemolymph melanization activity. Heavy infections by Endoreticulatus spec. that remained restricted to the gut tissue likewise did not elicit melanization. One Vairimorpha spec. from L. dispar induced a significant increase in total hemocyte numbers; the other infections led to temporarily decreased numbers. Microscopic examinations showed that parts of infected tissue were encapsulated by hemocytes. We conclude that measured alterations in hemolymph melanization and hemocyte numbers were likely to be induced by the damaging effects of heavy infections. Observed defense responses did not prevent the progression of infections.